# Patent

## Title
Drug-eluting intravascular prostheses and methods of use

## Abstract

The present invention provides intravascular prostheses and methods of production and use. An implantable device for treating a vascular disease or disorder includes an intravascular prosthesis containing an inhibitor of smooth muscle cell proliferation and a growth factor. The device can be coated with a biodegradable drug-eluting polymer that is impregnated with the inhibitor of smooth muscle cell proliferation and the growth factor. The device is useful for treating or preventing a vascular disease or disorder such as restenosis, by simultaneously inhibiting vessel blockage and enhancing recovery of the vessel wall following an intravascular intervention.

## Background
RELATED APPLICATIONS
This application is a continuation-in-part of U.S. Ser. No. 10/163,387, filed Jun. 4, 2002 (now U.S. Pat. No. 7,364,725), and U.S. Ser. No. 10/164,219, filed Jun. 4, 2002 (now U.S. Pat. No. 7,175,658), each of which is a continuation-in-part of U.S. Ser. No. 09/620,227, filed Jul. 20, 2000 (abandoned). Each of these applications is incorporated by reference, including drawings, as if fully set forth herein.


FIELD OF THE INVENTION
The present invention relates to the fields of chemistry, biochemistry, cellular biology, genetic engineering, medical devices and medicine. In particular, the invention relates to intravascular prostheses coated with cells genetically altered to express factors that improve the performance of the prostheses and that prevent narrowing of the lumen.
BACKGROUND OF THE INVENTION
Vascular diseases affect a large part of the world's population. Bypass surgery, whereby a conduit, either artificial or autologous, is grafted into an existing vessel to circumvent a diseased portion of the vessel or to restore blood flow around a blocked or damaged blood vessel, is one of the most common treatments for such diseases. It is estimated that over 1 million such procedures are performed annually.
Atherosclerosis is the most common form of vascular disease and leads to insufficient blood supply to critical body organs, resulting in heart attack, stroke, and kidney failure. Additionally, atherosclerosis causes major complications in those suffering from hypertension and diabetes, as well as tobacco smokers. Atherosclerosis is a form of chronic vascular injury in which some of the normal vascular smooth muscle cells (“VSMC”) in the artery wall, which ordinarily control vascular tone regulating blood flow, change their nature and develop “cancer-like” behavior. These VSMC become abnormally proliferative, secreting substances (growth factors, tissue-degradation enzymes and other proteins) which enable them to invade and spread into the inner vessel lining, blocking blood flow and making that vessel abnormally susceptible to being completely blocked by local blood clotting, resulting in the death of the tissue served by that artery.
Restenosis, the recurrence of stenosis or artery stricture after corrective surgery, is an accelerated form of atherosclerosis. Recent evidence has supported a unifying hypothesis of vascular injury in which coronary artery restenosis along with coronary vein graft and cardia allograft atherosclerosis can be considered to represent a much accelerated form of the same pathogenic process that results in spontaneous atherosclerosis. Restenosis is due to a complex series of fibroproliferative responses to vascular injury involving potent growth-regulatory molecules, including platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF), also common to the later stages in atherosclerosis lesions, resulting in vascular smooth muscle cell proliferation, migration and neointimal accumulation.
Restenosis occurs after coronary artery bypass surgery (CAB), endarterectomy, and heart transplantation, and particularly after heart balloon angioplasty, atherectomy, laser ablation or endovascular stenting (in each of which one-third of patients redevelop artery-blockage (restenosis) by 6 months), and is responsible for recurrence of symptoms (or death), often requiring repeat revascularization surgery. Despite over a decade of research and significant improvements in the primary success rate of the various medical and surgical treatment of atherosclerosis disease, including angioplasty, bypass grafting and endarterectomy, secondary failure due to late restenosis continues to occur in 30-50% of patients.
During angioplasty, intraarterial balloon catheter inflation and stent deployment results in deendothelialization, disruption of the internal elastic lamina, and injury to medial smooth muscle cells. A significant number of patients have a biological reaction of the arterial wall characterized by smooth muscle cell proliferation that results in luminal narrowing. While restenosis likely results from the interdependent actions of the ensuing inflammation, thrombosis, and smooth muscle cell accumulation, the final common pathway evolves as a result of medial VSMC dedifferentiation from a contractile to a secretory phenotype. This involves, principally, VSMC secretion of matrix metalloproteinases degrading the surrounding basement membrane, proliferation and chemotactic migration into the intima, and secretion of a large extracellular matrix, forming the neointimal fibroproliferative lesion. Much of the VSMC phenotypic dedifferentiation after arterial injury mimics that of neoplastic cells (i.e., abnormal proliferation, growth-regulatory molecule and protease secretion, migration and basement invasion).
To overcome this phenomenon, and in an attempt to prevent restenosis, several companies have developed stents that are permanently implanted in coronary or peripheral vessels and release (elute) drugs that inhibit or prevent cell proliferation and therefore prevent the narrowing created by smooth muscle proliferation. However, while drug eluting stents reduce smooth muscle cell proliferation and restenosis on one hand, they also prevent recovery of endothelial cell monolayer for a very long period of time, thereby making the vessel wall thrombogenic for an extended period. The resulting clinical outcome is late clotting of drug eluting stents. To prevent stent thrombosis, patients are recommended to take anticoagulant and antiplatelet drugs (e.g., aspirin and Plavix) for at least one year, assuming that endothelial cell recovery and vessel healing will happen within this one year window.
Vascular grafts are also used as entry sites in dialysis patients. The graft connects an artery to a vein in the patient's body. A needle is inserted into the graft, blood is withdrawn, passed through a hemodialysis machine and returned to the patient through a second needle inserted in the graft.
Small caliber synthetic vascular grafts have high failure rate in the long term. Thirty to 50 percent of by-pass grafts fail within 5 to 7 years. The average life-span for hemodialysis grafts is even shorter, often less than two years. A primary cause of graft failure is the closing of the graft due to tissue in-growth and eventually thrombosis formation. The smaller the diameter of a graft, the higher rate of failure. Numerous approaches to improving the performance of vascular grafts have been proposed. One such approach is the use of more biocompatible and durable synthetic materials in artificial grafts.
Among the synthetic materials that have been used in vascular grafts is polytetrafluoroethylene (PTFE, Teflon®), which has high durability complemented by good biocompatibility. However, as with intraluminal stents, PTFE and similar materials are still susceptible to thrombosis formation, which limits their utility. To counter this, the interior walls of PTFE grafts and intraluminal stents have been seeded with autologous endothelial cells (ECs) before implantation. Not only do ECs provide an excellent biocompatible surface, they also have substantial thrombolytic activity. In addition, ECs prevent neointimal proliferation and inflammatory reaction in the graft. However, EC-seeded grafts and stents suffer from incomplete endothelialization and detachment of the endothelial cells from the surface of the graft due to the shear force of flowing blood.
To improve endothelialization, ECs have been genetically altered to express or over-express vascular endothelial growth factor (VEGF, U.S. Pat. No. 5,785,965). Not only can VEGF reduce the time from cell harvesting to seeding, it also permits use of lower initial graft seeding densities since rapid proliferation leads to faster graft coverage.
VEGF has advantages over other less EC-specific growth factors that can enhance endothelialization due to its reduced impact on other vascular cells, in particular smooth muscle cells (SMCs), and, as such, its reduced potential for causing adverse stimulatory effects. For instance, VEGF will recruit ECs, but not SMCs, from anastomosis sites.
While genetically altered ECs over-expressing VEGF resolve to a large extent the problem of incomplete endothelialization, detachment of cells from the interior surface of a graft under the shear stress of flowing blood still remains a problem. Furthermore, the drug eluting stents that inhibit or prevent cell proliferation also prevent recovery of endothelial cell monolayer, thereby making the vessel wall thrombogenic for an extended period.
One approach to dealing with the detachment problem has been to precoat the interior surface of a graft with an adhesive matrix to more solidly fix the cells to the surface. Also, exposing the cells seeded on the wall of the graft to continuous flow conditions during proliferation to simulate blood flow has been reported. While occurring at a slower rate, ECs still detach from the walls of grafts prepared using these techniques and thus the useful life span of the grafts remains sub-optimal. In addition, the detached cells leave extracellular matrix, which is highly thrombogenic, on the grafts after detachment.
Thus, there remains a need for endothelialized vascular grafts in which the ECs can withstand the shear force of flowing blood for a longer time. There is also a need for intravascular prostheses with improved long-term patency, which reduces graft thrombosis and neointima formation in vivo. The present invention provides such intravascular devices and methods for their preparation and their use.
SUMMARY OF THE INVENTION
Thus, in one aspect, the present invention relates to an artificial vascular graft comprising a synthetic tubular element having an exterior surface and an interior surface that describes a lumen. The tube has a plurality of cells seeded and cultured on its interior surface. The cells are either all endothelial cells or a mixture of endothelial and smooth muscle cells. If only endothelial cells are used, at least a portion of them is genetically altered to express or over-express one or more cell adhesion factor(s). If both endothelial cells and smooth muscle cells are used, at least a portion of the endothelial cells, at least a portion of the smooth muscle cells or at least a portion of both is genetically altered to express or over-express one or more cell adhesion factor(s).
In another aspect this invention relates to an artificial vascular graft comprising a synthetic tubular element comprising an exterior surface and an interior surface that describes a lumen. A plurality of smooth muscle cells is seeded and cultured on the interior surface of the tube. At least a portion of the smooth muscle cells is genetically altered to express or over-express one or more cell adhesion factor(s).
In another aspect, this invention relates to a method of producing an artificial vascular graft. First, a synthetic tubular element is obtained. The tube has an exterior surface and an interior surface that describes a lumen. A plurality of cells is then seeded on the interior surface of the tube and cultured there. The cells are either all endothelial cells or a mixture of endothelial cells and smooth muscle cells. Again, if only endothelial cells are used, at least a portion of them is genetically altered to express or over-express one or more cell adhesion factor(s). Likewise, if both endothelial cells and smooth muscle cells are used, at least a portion of the endothelial cells, at least a portion of the smooth muscle cells or at least a portion of both is genetically altered to express or over-express one or more cell adhesion factor(s).
In an aspect of this invention, the above method further comprises applying a fluidic shear force at the interior surface of the tubular element during culturing of the cells.
An aspect of this invention is a method of bypassing a portion of a vascular vessel in a patient. The method comprises using a synthetic tubular element having a first end, a second end, an exterior surface and an interior surface that describes a lumen. A plurality of cells is seeded and cultured on the interior surface of the tubular element. The cells comprise only endothelial cells or a mixture of endothelial cells and smooth muscle cells. If only endothelial cells are used, at least a portion of them is genetically altered to express or over-express one or more cell adhesion factor(s). If both endothelial cells and smooth muscle cells are used, at least a portion of the endothelial cells, at least a portion of the smooth muscle cells or at least a portion of both is genetically altered to express or over-express one or more cell adhesion factor(s). The first end of the tube is grafted into the vessel proximal to the portion to be bypassed and the second end of the tube is grafted into the vessel distal to the portion to be bypassed. As a result, fluidic continuity is established in the vessel from the site of the proximal graft, through the lumen of the tubular element, and back into the vessel at the site of the distal graft.
An aspect of this invention is a method of performing hemodialysis on a patient. A synthetic tubular element comprising an exterior surface, an interior surface that describes a lumen, a first end and a second end is used. the tube is implanted under the skin of the patient with its first end grafted into an artery and its second end grafted into a vein such that fluidic continuity is established from the artery through the lumen of the tubular element and into the vein. The lumen of the tube is connected to a hemodialysis filtration unit such that blood can be withdrawn from the patient, sent through the hemodialysis filtration unit where it is filtered and then can be returned to the patient through the lumen. The synthetic tubular element comprises a plurality of cells seeded and cultured on its interior surface. the cells comprises all endothelial cells or a mixture of endothelial and smooth muscle cells. If only endothelial cells are used, at least a portion of them are genetically altered to express or over-express one or more cell adhesion factors. If both endothelial cells and smooth muscle cells are used, at least a portion of the endothelial cells, at least a portion of the smooth muscle cells or at least a portion of both is genetically altered to express or over-express one or more cell adhesion factor(s).
In an aspect of this invention, only a plurality of endothelial cells is seeded and cultured on the interior surface of the tubular element.
In an aspect of this invention, both endothelial cells and smooth muscle cells are seeded and cultured on the interior surface of the tubular element.
In an aspect of this invention, when both endothelial and smooth muscle cells are used in the above methods, at least a portion of the endothelial cells is genetically altered to express or over-express a cell adhesion factor.
In an aspect of this invention, when both endothelial and smooth muscle cells are used in the above methods, at least a portion of the smooth muscle cells is genetically altered to express or over-express a cell adhesion factor.
In an aspect of this invention, when both endothelial and smooth muscle cells are used and at least a portion of the smooth muscle cells is genetically altered to express or over-express a cell adhesion factor, at least a portion of the endothelial cells is genetically altered to express or over-express a cell proliferation growth factor.
An aspect of this invention is an endothelial or a smooth muscle cell that has been genetically altered to express or over-express one or more cell adhesion factor(s).
In an aspect of this invention, the above cell is further genetically altered to express or over-express one or more cell proliferation growth factor(s). In an aspect of this invention, the above cell is further genetically altered to express or over-express one or more marker polypeptides.
The marker polypeptide is a selection marker or a reporter marker in an aspect of this invention.
An aspect of this invention is a nucleic acid expression construct, comprising a first polynucleotide sequence encoding a cell proliferation growth factor and a second polynucleotide sequence encoding a cell adhesion factor.
In an aspect of this invention, the above nucleic acid expression construct further comprises a promoter sequence that directs expression of both the first and second polynucleotide sequences.
In an aspect of this invention, the above nucleic acid expression construct comprises two promoter sequences, one of which directs expression of the first polynucleotide sequence and the other of which directs expression of the second polynucleotide sequence.
In an aspect of this invention, the above nucleic acid construct of further comprising a linker sequence interposed between the first and the second polynucleotide segments.
The linker sequence comprises IRES or a protease cleavage recognition site in an aspect of this invention.
In an aspect of this invention, the promoter sequence(s) is/are independently selected from the group consisting of a constitutive promoter, an inducible promoter and a tissue specific promoter sequence.
In an aspect of this invention, the first and second promoter sequences are both inducible promoter sequences.
The first and second inducible promoter sequences are regulated by effector molecules in an aspect of this invention.
In an aspect of this invention, the first and second inducible promoter sequences are regulated by the same effector molecule.
In an aspect of this invention, the above nucleic acid expression construct further comprises a third polynucleotide sequence encoding a marker polypeptide.
The marker polypeptide is selected from the group consisting of a selection marker and a reporter marker in an aspect of this invention.
In an aspect of this invention, the polynucleotide sequence encoding the marker polypeptide is transcriptionally linked to the first or the second polynucleotide sequence.
In an aspect of this invention the transcriptional link comprises IRES or a protease cleavage recognition site.
In an aspect of this invention, the polynucleotide sequence encoding the marker polypeptide is translationally fused to the first or the second polynucleotide segment.
An aspect of this invention is a nucleic acid expression construct system comprising a first nucleic acid expression construct comprising a first polynucleotide sequence encoding a cell proliferation growth factor and a second nucleic acid expression construct comprising a second polynucleotide sequence encoding a cell adhesion factor. In an aspect of this invention, in the above construct, the first or the second nucleic acid expression construct further comprises an additional polynucleotide sequence encoding a marker polypeptide.
In an aspect of this invention, in the above construct, the marker polypeptide is selected from the group consisting of a selection marker and a reporter marker.
In an aspect of this invention, the above construct further comprises a promoter sequence that directs expression of the first and the second polynucleotide sequence.
In an aspect of this invention, the above construct comprises two promoter sequences, one of which directs expression of the first polynucleotide sequence and the other of which directs expression of the second polynucleotide sequence.
In an aspect of this invention, in the above construct, the promoter sequence(s) are independently selected from the group consisting of a constitutive promoter, an inducible promoter and a tissue specific promoter.
In an aspect of this invention, in the above construct, the polynucleotide sequence encoding the marker polypeptide is transcriptionally linked to the first or the second polynucleotide sequence.
In an aspect of this invention, the cultured endothelial cells form a confluent monolayer.
In an aspect of this invention, the cell adhesion factor is selected from the group consisting of UP50, vitronectin, albumin, elastin, tropoelastin, E-cadherins, collagen I, collagen IV, Ang-1, fibronectin and laminin.
In a presently preferred aspect of this invention, the cell adhesion factor is UP50.
In an aspect of this invention, the interior surface of the tubular element comprises polytetrafluoroethylene (PTFE, TeflonTM), expanded polytetrafluoroethylene (ePTFE), polyester fiber, polyethylene terephthalate (DacronTM), polyurethane, a collagen protein, an elastin protein or a processed human or animal blood vessel.
In an aspect of this invention, the interior surface of the tubular element is coated with an adhesion matrix prior to seeding with the genetically altered endothelial cells.
In an aspect of this invention, the adhesion matrix is selected from the group consisting of fibronectin, collagen, elastin tropoelastin and smooth muscle cell-conditioned growth medium or any combination thereof.
In an aspect of this invention, at least a portion of the endothelial cells is genetically altered to express or over-express one or more cell proliferation growth factors.
In an aspect of this invention, the cell proliferation growth factor is selected from the group consisting of the VEGF family of proteins, acidic FGF, basic FGF and HGF.
In a presently preferred aspect of this invention, the cell proliferation growth factor is VEGF-A.
In an aspect of this invention, the same endothelial cells that are genetically altered to express or over-express the cell adhesion factor(s) are also genetically altered to express or over-express the cell proliferation growth factor(s).
In an aspect of this invention, a first portion of the endothelial cells is genetically altered to express or over-express the cell adhesion factor(s) and a second portion of the cells is genetically altered to express or over-express the cell proliferation growth factor(s).
In a presently preferred aspect of this invention, when both a cell adhesion factor and a cell proliferation growth factor are being expressed or over-expressed, the cell adhesion factor is UP50 and the cell proliferation growth factor is VEGF-A.
In an aspect of this invention, smooth muscle cells are seeded and cultured on the exterior surface of the tubular element.
In an aspect of this invention, at least a portion of the smooth muscle cells that are seeded and cultured on the exterior of the tube is genetically altered to express or over-express a cell adhesion factor.
In an aspect of this invention, at least a portion of the smooth muscle cells is genetically altered to express or over-express a cell proliferation growth factor.
In an aspect of this invention, the same smooth muscle cells that are genetically altered to express or over-express the cell adhesion factor are also genetically altered to express or over-express the cell proliferation growth factor.
In an aspect of this invention, a first portion of the smooth muscle cells is genetically altered to express or over-express the cell adhesion factor and a second portion of the cells is genetically altered to express or over-express the cell proliferation growth factor.
In an aspect of this invention, the lumen of the tubular element has a cross-sectional area substantially equivalent to a cross-sectional area of a lumen of a vessel to which the tubular element is grafted.
In an aspect of this invention, the cross-sectional area of the lumen is from about 7 to about 700 mm2.
In an aspect of this invention, the endothelial cells are obtained from a source selected from the group consisting of a vein, an artery and circulating endothelial cells or are derived from a source selected from bone marrow progenitor cells, peripheral blood stem cells and embryonic stem cells.
In an aspect of this invention, the smooth muscle cells are obtained from a vein or an artery or from bone marrow progenitor cells, peripheral blood stem cells and embryonic stem cells.
In an aspect of this invention, the endothelial and smooth muscle cells are obtained from a human or a non-human mammal.
In an aspect of this invention, the human is a patient who is to receive the vascular graft.
An aspect of this invention is a method of producing an artificial vascular graft, comprising providing a synthetic tubular element having a first end, a second end, an exterior surface and an interior surface that describes a lumen. A plurality of cells is seeded and cultured on the interior surface of the tubular element. The plurality of cells comprises a plurality of endothelial cells, a plurality of smooth muscle cells or a plurality of endothelial cells and a plurality of smooth muscle cells. If only endothelial cells are used, at least a portion of them is genetically altered to express or over-express one or more cell adhesion factor(s). If only smooth muscle cells are used, at least a portion of them is genetically altered to express or over-express one or more cell adhesion factor(s). If both endothelial cells and smooth muscle cells are used, at least a portion of the endothelial cells, at least a portion of the smooth muscle cells or at least a portion of both the endothelial cells and the smooth muscle cells is genetically altered to express or over-express one or more cell adhesion factor(s).
In an aspect of this invention, in the above method a fluidic shear force is applied at the interior surface of the tubular element during culturing of the cells.
An aspect of this invention relates to a method of bypassing a portion of a vascular vessel in a patient using an artificial vascular graft of this invention. One end of the artificial graft is grafted into the vessel proximal to the portion to be bypassed and the other end of the artificial graft is grafted into the vessel distal to the portion to be bypassed, whereby fluidic continuity is established from the site of the proximal graft, through the lumen of the tubular element, to the site of the distal graft.
A further aspect of this invention relates to a method of performing hemodialysis on a patient using an artificial vascular graft of this invention. The graft is implanted under the skin of the patient with one end of it grafted into an artery and the other end grafted into a vein whereby fluidic continuity is established from the artery through the lumen of the tubular element and into the vein. The lumen of the graft is connected to a hemodialysis filtration unit such that blood can be diverted from the lumen into the hemodialysis filtration unit, filtered, and then returned into the lumen.
In one embodiment, the invention provides an implantable device for treating a vascular disease or disorder that includes an intravascular device and a biodegradable drug-eluting polymer disposed on and/or within the prosthesis. The polymer can be impregnated with an inhibitor of smooth muscle cell proliferation/migration, and can also be impregnated with a growth factor. In one aspect the intravascular prosthesis is a stent, a vascular graft, an artificial heart, or an artificial valve. In another aspect, the inhibitor of smooth muscle cell proliferation/migration can be fibulin5 (UP50), and the growth factor can be VEGF. The vascular disease or disorder to be treated can include, for example, stenosis, restenosis, atherosclerosis, cardiac arrest, stroke, thrombosis, or atherectomy, or injuries caused by intravascular interventions used to treat these diseases or disorders.
In another aspect, the implantable device can include a substrate coated with endothelial cells that are genetically altered to express or over-express fibulin-5, with or without VEGF. The substrate can be disposed on and/or within the prosthesis.
In another embodiment, the invention provides methods of treating or preventing a vascular disease or disorder by simultaneously inhibiting vessel blockage and enhancing recovery of the vessel wall following an intravascular intervention by inserting an intravascular device coated with a biodegradable drug-eluting polymer impregnated with an inhibitor of smooth muscle cell proliferation/migration and a growth factor, within a vessel of a subject in need thereof, and eluting the inhibitor and the growth factor from the polymer into the vessel, thereby inhibiting smooth muscle cell proliferation and enhancing endothelial cell proliferation.
In yet another embodiment, the invention provides methods of preventing neointima formation of smooth muscle cells following an intravascular intervention by delivering a plurality of vectors containing a polynucleotide sequence encoding an inhibitor of smooth muscle cell proliferation/migration to a site of vascular injury.



BRIEF DESCRIPTION OF THE DRAWINGS
The drawings herein are provided solely as visual aids to the understanding the present invention. They are not intended, nor should they be construed, to limit the scope of this invention in any manner whatsoever.
FIG. 1 shows a vascular graft of the present invention.
FIG. 2 is a flowchart of the strategy for constructing a hybrid biological-synthetic vascular grafts of the present invention.
FIGS. 3a-b depict adenoviral (3a) and retroviral (3b) constructs that express VEGF-GFP.
FIGS. 4a-d depict adenoviral vectors (4a and 4b) and retroviral vectors (4c and 4d) that express UP50 and UP50-GFP.
FIG. 5 is a histogram showing the proliferation of ECs transfected with adenoviral vectors encoding UP50-GFP, VEGF-GFP or GFP.
FIG. 6 is a histogram derived from an adhesion assay of Ad.UP50 transfected (“up” legend), Ad.GFP transfected (“gfp” legend) or non-transfected (“cont” (control) legend) ECs. Two different experiments, performed in triplicate, are represented.
FIG. 7 is a histogram derived from an adhesion assay of ECs to UP50-containing extra-cellular matrix (ECM). Results are presented as corrected OD values after subtraction of background.
FIG. 8 shows the adhesion of ECs over-expressing UP50 exposed to continuous shear stress. FIG. 8 is a histogram of the percentage of cells remaining after rocking. Results are shown as a percentage based on the ratio of the number of cells remaining after rocking to the number of cells present before rocking.
FIG. 9 shows the system used to test cell retention under laminar flow conditions in vitro. FIG. 9 is a schematic of the system.
FIG. 10 shows improved retention on ePTFE grafts of endothelial cells over-expressing fibulin-5 or co-expressing fibulin-5 and VEGF165. Endothelial cells (EC) were seeded on ePTFE grafts and were exposed to pulsatile flow conditions in an in-vitro flow apparatus. Non-transduced, naive EC, GFP transduced EC, VEGF165 transduced EC, fibulin-5 transduced EC and dual transduced EC with fibulin-5 and VEGF165 were seeded on ePTFE grafts. Results are presented as mean percentage of cell retention ± standard deviation. Retention of EC expressing VEGF was similar to retention of cell expressing GFP or naive cells while EC expressing VEGF and fibulin-5 demonstrated improved adhesion.
FIG. 11 In vitro studies of endothelial cell and smooth muscle cell proliferation in the presence of fibulin-5: (A) Endothelial cells are inhibited in presence of fibulin-5 alone (-bFGF), and then proliferate rapidly when a growth factor is added; (B) Smooth muscle cell proliferation is retarded or inhibited in presence of fibulin-5 and growth factor, as compared to endothelial cells.
FIG. 12: Biodistribution PCR results of one rabbit of therapeutic dose, 24 weeks sacrifice. DNA samples from select tissues and organs, and appropriate controls were analyzed for the presence of vector sequences by PCR (A, B and C). A positive result is indicated by the presence of a single, 140 bp PCR product. (*) indicates that the sample had been spiked with 50 copies of vector plasmid. M indicates a DNA molecular size marker.
FIG. 13: DNA integrity by β-actin. Tissue samples that were analyzed for the presence of vector sequences were concurrently tested for DNA integrity by the presence of a 106 bp band indicating an intact rabbit β-actin gene.
FIG. 14: Effect of Fibulin-5 and VEGF165 on ERK activation in Human Saphenous Venous Endothelial Cells (HSVEC) and Smooth muscle cells. 4×105 naive HSVEC or HSVSMC were seeded in 6 wells plates for 24 h in 10% serum. The medium was then replaced with 1 ml conditioned media (CM) of HSVECs-naïve or overexpressed Fibulin-5 without or with VEGF165 for 90 min. Addition of exogenous VEGF165 (50 ng/ml), which was previously demonstrated to induce the activation of ERK phosphorylation, for 30 min served as positive control for this assay. Western blot analysis of the ERK and pERK proteins in 10 μg total protein from cell lysates was performed. Shown is representative experiment which was repeated at list once with the same results.
FIG. 15: Time-dependent effect of VEGF165 on p38 activation in Human Saphenous Venous Endothelial Cells (HSVEC). 4×105 naive HSVEC were seeded in 6 wells plates for 24 h in 10% serum. The medium was then replaced with 1 ml conditioned media (CM) of HSVECs-naïve or overexpressed VEGF165 for 3-30 min. Western blot analysis of the phospho-p38 and β-actin proteins in 10 μg total protein from cell lysates was performed. Shown is representative experiment which was repeated at list once with the same results.
FIG. 16: Effect of Fibulin-5 and VEGF165 on p38 activation in Human Saphenous Venous Endothelial Cells (HSVEC). 4×105 naive HSVEC were seeded in 6 wells plates for 24 h in 10% serum. The medium was then replaced with 1 ml conditioned media (CM) of HSVECs-naïve or overexpressing Fibulin-5 without or with VEGF165 for 30 min. Western blot analysis of the phospho-p38 and β-actin proteins in 10 μg total protein from cell lysates was performed. Shown is representative experiment which was repeated at list once with the same results.
FIG. 17: Effect of VEGF on rabbit EC proliferation. 1×104 human and rabbit naive EC (human saphenous vein endothelial cells (HSVEC)) and rabbit EC (Rb. EC) respectively) were seeded on fibronectin-coated 12 wells plates. The medium was then replaced to medium containing 2% FBS with or without the addition of 20 and 50 ng/ml VEGF for 72 h. Cells were then counted in a Coulter cell Counter. The assay was preformed in duplicates.
FIG. 18: Over-expression of fibulin-5 and VEGF165 in sheep endothelial cells. Sheep endothelial cells were transduced with retroviral vector encoding fibulin-5, followed by transduction with VEGF165 encoding retroviral vector. Transduced cells were incubated with serum-free growth medium for 48 hr, and conditioned medium samples were collected and analyzed for fibulin-5 (A) and VEGF165 (B) expression by Western blot.
FIG. 19: In vivo model of neointima formation by smooth muscle cells—rat carotid artery model: (A) Neointimal formation with saline (control) and with fibulin-5 alone: Immunostaining shows that fibulin-5 is exclusively expressed after gene transfer in the arteries exposed to adenoviral vectors expressing fibulin-5. Staining with KI-67 to detect the number of proliferating cells shows that proliferation was reduced after fibulin-5 transfer, and was present after saline transfer; (B) Summary of the amount of neointima of smooth muscle cells using the intima media ratio as an indicator.
FIG. 20: Angiography of implanted grafts seeded with naive ECs and ECs over-expressing both fibulin-5 and VEGF165. Note that the naïve graft in 20A is filled with a blood clot and that the bare graft is completely occluded as demonstrated by selective angiography. The graft with dual gene expression is patent and demonstrates good flow. The table in 20B summarizes the patency data.



DETAILED DESCRIPTION OF THE INVENTION
Each year, numerous people lose the ability to deliver sufficient amounts of blood to various organs and limbs. The most well-known of these maladies is the coronary occlusion, the blockage of one or more of the arteries leading to the heart. However, hundreds of thousands of people also suffer loss of blood flow to the limbs. If the loss of flow is significant, tissue at the extremity becomes ischemic and eventually dies. Such loss of peripheral blood flow can result from injury but most often it is the result of disease such as atherosclerosis or diabetes complicated by accelerated atherosclerosis. To remedy these situations, surgeons often turn to vascular grafts to circumvent the injured or diseased portion of a blood vessel and restore blood flow.
Vascular grafts are generally classified as either biological or synthetic (or, synonymously, artificial). Examples of biological grafts include autografts and allografts. An autograft is taken from another site in a patient's body. For instance, in peripheral vascular surgery, the most common graft comprises the long saphenous vein in which the valves have been surgically removed with an intraluminal cutting valvutome.
An allograft, on the other hand, is a biological graft taken from another animal or the same or different species.
Synthetic or artificial grafts are made of non-biological materials such as, without limitation, polytetrafluoroethylene (PTFE Teflon®), expanded PTFE (ePTFE), polyester, polyurethane, polyethylene terephthalate (Dacron®) and the like. Dacron grafts are commonly used in aortic and aorto-iliac surgery. Presently, below the inguinal ligament, results with synthetic grafts are considered inferior to biological (venous) grafts. However, when a suitable vein is not available, PTFE is most often the graft material of choice. Also, using a synthetic graft results in a shorter operation and spares veins for future procedures. Artificial grafts are not yet used extensively in heart bypass procedures. One of the limitations of these grafts (and some biological grafts as well) is the lack of long-term patency, that is, the ability to remain open to blood flow for extended periods. This is particularly problematic with regard to small blood vessels such as those related to below inguinal peripheral blood vessels and the coronary arteries. While large and medium diameter blood vessel replacement with a Dacron® or Teflon® graft may have a patency of 10 years or more, the results with small blood vessels have been markedly poorer. The problem is that the lumens of these grafts tend to occlude due to tissue in-growth and thrombosis, i.e., formation of blood clots. Endothelial cells (ECs) are sometimes used to line the lumen of synthetic grafts. The cells enhance performance of the grafts due to their thrombolytic activity (Dichek, et al., Circulation, 1996, 93:301; Gillis-Haegerstrand, et al., J. Vasc. Surg., 1996, 24:226) and their ability to prevent neointimal proliferation (tissue and extracellular in-growth) and inflammatory reactions in the graft (Pasic, et al., Circulation, 1995, 92:2605). Unfortunately, the cells often cannot withstand the shear force of flowing blood and eventually detach from the surface of the graft, thus negating their utility. The synthetic vascular grafts of this invention address this situation.
A graft of this invention comprises a tubular element manufactured from a completely synthetic material such as, without limitation, PTFE (Teflon®), ePTFE, polyethylene terephthalate (Dacron®), polyester or polyurethane. While rigid-walled grafts may be used in the circulatory system, they are not preferred due to their tendency to detrimentally effect blood wave propagation and local field velocity, thus acting in essence as “low pass filters” that damp out higher harmonics and introduce phase distortion. Thus, artificial grafts are most often manufactured in a textile motif, that is, they are usually fibrous materials that are woven or knitted although polyurethane grafts may be extruded. A “synthetic” graft of this invention may also comprise a processed animal or human blood vessel.
A typical synthetic graft of this invention is shown in FIG. 1. Graft 10 is comprised of a synthetic tubular element 12 having an outer surface 15 and an interior surface 14 that describes a lumen 13. Synthetic tubular element 12 has an inner cross-sectional area that is substantially equivalent to the inner cross-sectional area of the vessel to which it is grafted. In a presently preferred embodiment of this invention, the inner cross-sectional area is about 7 to 700 mm2. Interior surface 14 is constructed of a material such as, without limitation, PTFE, ePTFE, polyester fiber, collage fiber, elastin fibers, polyurethane, Dacron® or processed blood vessels obtained from an animal or human. Interior surface 14 preferably has a structure that facilitates cell seeding such as, without limitation, pits and/or projections.
In a presently preferred embodiment of this invention, interior surface 14 is coated with ECs and/or SMCs 16, at least a portion of which are genetically altered to express one or more cell proliferation growth factor(s) and a portion of which are altered to express one or more cell adhesion factor(s).
Cell proliferation growth factors include HGF (hepatocyte growth factor), EGF (epidermal growth factor), Epo (erythropoietin), FGF's (fibroblast growth factors), IGF (insulin-like growth factor), IL (interleukins), platelet derived growth factor (PDGF), transforming growth factor (TGF) and vascular endothelial growth factor (VEGF). While any of these may be used in the devices and methods of this invention, VEGF, which is a member of the PDGF family, is presently preferred because it is a very specific stimulator of the vascular endothelium.
The VEGF family at present consists of VEGF-A, VEGF-B, VEGF-C, VEGF-D and the most recently discovered VEGF-E. PIGF (placenta growth factor) is closely related to VEGF-A and is often considered a pseudo-VEGF family factor. Any of these may be, in fact, are presently preferred to be, used in the grafts and methods of this invention.
Cell adhesion factors useful in the grafts and methods of this invention include, without limitation, those factors that are considered part of the extracellular matrix (ECM) that connect the cell membrane to the ECM.
In a presently preferred embodiment of this invention, interior surface 14 is coated with ECs and/or SMCs, wherein the same cells are genetically altered to express both a cell proliferation growth factor and a cell adhesion factor.
In addition, external surface 15 of tubular element 12 can be coated with altered or unaltered smooth muscle cells to improve graft acceptance as well as to aid in graft durability.
Endothelial cells (ECs) are those cells that cover the interior or luminal surface of blood vessels. They serve numerous purposes, one of the most important of which with regard to the present invention is the prevention of thrombosis, i.e., blood clot formation, in the vessel as well as prevention of tissue in-growth and undesirable production of extracellular matrix. ECs useful in the synthetic grafts of this invention include, without limitation, arterial and venous ECs such as human coronary artery endothelial cells (HCAEC), human aortic endothelial cells (HAAEC), human pulmonary artery endothelial cells (HPAEC), dermal microvascular endothelial cells (DMEC), human umbilical vein endothelial cells (HUVEC), human umbilical artery endothelial cells (HUAEC), human saphenous vein endothelial cells (HSVEC), human jugular vein endothelial cells (HJVEC), human radial artery endothelial cells (HRAEC), and human internal mammary artery endothelial cells (HIMAEC). Useful ECs can also be obtained from circulating endothelial cells and endothelial cell precursors such as bone marrow progenitor cells, peripheral blood stem cells and embryonic stem cells.
Smooth muscle cells encircle the endothelial cells in a vessel and regulate the vessel's diameter by expanding and contracting. Most importantly for the purposes of this invention, smooth muscle cells are responsible for the secretion of most of the extracellular matrix. Smooth muscle cells useful in the grafts of this invention include, without limitation, human aortic smooth muscle cells (HAMC), human umbilical artery smooth muscle cells (HUASMC), human pulmonary artery smooth muscle cells (HPASMC), human coronary artery smooth muscle cells (HCASMC), human bronchial smooth muscle cells (HBSMC), human radial artery smooth muscle cells (HRASMC), and human saphenous or jugular vein smooth muscle cells.
The extracellular matrix (ECM) is a complex material that surrounds and supports cells in mammalian tissue. It is commonly referred to as the connective tissue. The ECM is composed of three major classes of biomolecules: structural proteins (collagen, elastin), specialized proteins (fibrillin, fibronectin, laminin) and proteoglycans (protein cores to which are attached repeating disaccharides called glycosaminoglycans).
Collagens comprise the major proteins of the ECM. In fact, they are the most abundant proteins found in the animal kingdom. There are at least 20 types of collagen. Collagen types I II and III are the most abundant and form fibrils of similar structure. Type IV forms a two-dimensional reticulum and is a major component of the basal lamina. Collagens are predominantly synthesized by fibroblasts in the natural state although epithelial cells also synthesize some collagen.
Fibronectin's role in the ECM is to attach cells to a variety of extracellular matrices. For example, fibronectin has been shown to attach cells to collagen I-, II- and III-containing ECMs.
Fibronectin does not attach cells to collagen IV-containing ECMs. In this case, laminin is the adhesive molecule.
Other cell adhesion factors include elastin and its precursor tropoelastin. Elastin is extremely insoluble due to extensive cross-linking of tropoelastin, which prior to cross-linking is quite soluble. Elastin and tropoelastin, are synthesized naturally by both smooth muscle and endothelial cells.
Endothelial cadherins (E-cadherins) are calcium dependent adhesion molecules. They tend to bind in a homophilic manner, that is, one cadherin binds to another cadherin in the extracellular space. The connections occur at specialized junctions.
Vitronectin, also known as S-protein, serum spreading factor and epibolin, is present in the extracellular matrix of many tissues. Along with fibronectin it is the major adhesive protein in plasma and serum. Interaction of vitronectin with other ECM components is mediated primarily by its collagen-binding domain. Used as a pre-coating on surfaces, vitronectin promotes cell attachment, spreading, proliferation and differentiation of many different types of cells.
The recently discovered protein, UP50, also know as fibulin-5 or DANCE (Developing Arteries and Neural Crest, EFG-like), has also been found in the ECM. UP50 has been implicated in the generation and organization of elastic fibers, which are essential to various organs that require elasticity, such as the lungs, large arteries and skin. This protein has an RGD motif that interacts with cell surface integrins and promote cell to matrix adhesion.
Many of the above factors are naturally expressed by ECs and SMCs. These cells can be genetically altered to over-express the factors to improve the performance of the cells as coatings on the interior surface of artificial grafts, in particular with regard to resistance to shear stress. If, on the other hand, a desired factor is not naturally expressed, the cells can likewise be genetically altered to express it.
While expression of cellular adherence factor(s) by the seeded cells themselves results in substantially improved cell-to-cell and cell-to-graft adhesion, it is also an aspect of this invention to pre-coat the interior surface of a graft with one or more ECM proteins such as, without limitation, fibronectin, prior to seeding with genetically altered ECs or SMCs to enhance adhesion even more. The proteins can be harvested from the cell cultures used to initially grow the ECs and SMCs, or can be isolated from the blood. It is also an aspect of this invention to use the cell culture medium itself after culturing of the cells, in which case the medium is termed a “conditioned medium.” A presently preferred conditioned medium is that obtained from cultures of altered or unaltered SMCs.
The above cells are genetically altered such that a portion of them express one or more cellular proliferation growth factors and a portion of them express one or more of the above ECM cellular adhesion factors. It is, however, a presently preferred embodiment of this invention that the same cells are genetically altered to express both a cellular proliferation factor and a cellular adhesion factor.
In a presently preferred embodiment of this invention the above, cells are seeded onto the interior surface of the graft and cultured to confluence.
It is noteworthy that improved adhesion conferred by the expression of a cellular adhesion factor does not come at the expense of cell proliferation, which has been found to proceed normally.
FIG. 2 is a flow chart that lays out the experimental design used to prepare and evaluate the artificial grafts of this invention.
First, vectors must be developed that reliably transfect cells to express a proliferation growth factor, an adhesion factor or both. In the present case, either one of two types of viral vectors was employed to transfer genes into vascular cells. The first was a recombinant adenoviral vector that gave high levels of transgene expression. Such vectors have the advantage of being less difficult to prepare than adeno-associated vectors (AAV) and lentiviral-based vectors. Furthermore, unlike other viral vector systems, adenoviral vectors may be employed after cell seeding of grafts because cell division is not essential for transgene expression. In contrast, retroviral vectors requires cell division, which, in the present invention, is generally carried out to a great extent on the tissue culture plate and less so on the graft.
The second vector was a retroviral vector pseudo-typed with GALV (Gibbon ape leukemia virus) glycoprotein. Pseudo-typed vectors have a high affinity for human ECs and SMCs (Cosset, F.-L. and Russell, S. J., Gene Therapy, 1996, 3:946-56) and, unlike adenoviral vectors, transduction with retroviral vectors leads to stable transgene expression and transmission of gene expression in daughter cells and to less immunogenic reaction in vivo.
The vectors listed in Table 1 were used to transfer UP50 and VEGF165 genes into ECs and SMCs. FIGS. 3a, 4a and 4b depict the adenoviral VEGF165-GFP, UP50 and UP50-GFP expression vectors and FIGS. 3b, 4c and 4d depict the retroviral VEGF165-GFP, UP50 and UP50-GFP vectors used.











TABLE 1






Packaging
Gene(s)




Viral system
cell line
encoded
Designation
Titer







Adenovirus

GFP
Ad.GFP
1010 pfu/ml


Adenovirus

VEGF-IRES-GFP
Ad.VEGF-GFP
1010 pfu/ml


Adenovirus

UP50-GFP
Ad.UP50-GFP
5 × 1010 pfu/ml


Adenovirus

UP50
Ad.UP50
pending


Pseudo-typed
TEFLYGA
GFP
RetroGFP
106 ffu


Retrovirus


Pseudo-typed
TEFLYGA
VEGF-IRES-GFP
RetroVEGF-GFP
5 × 106 ffu


Retrovirus


Pseudo-typed
293FLYGA
UP50-IRES-GPF
RetroUP50-GFP
106 ffu


Retrovirus


Pseudotyped
293FLY10A
UP50-IRES-GPF
RetroUP50-GFP
106 ffu


Retrovirus


Pseudo-typed
293FLYGA
UP50
RetroUP50
106 ffu


Retrovirus









ePTFE grafts seeded with human ECs transfected with Ad.UP50-GFP were found to express GFP as observed by fluorescent microscopy. ePTFE grafts seeded with human ECs transfected with Ad.VEGF-GFP were likewise found to express the transgene. Transgene expression was analyzed 24 hours following infection.
ePTFE grafts seeded with retrovirally-transduced human ECs over-expressing UP50-GFP, were found to express the transgene, also by fluorescent microscopy detection of GFP expressing cells since the UP50 is situated upstream in the expression cassette (if GFP is expressed UP50 must be expressed). Grafts seeded with retrovirally-transduced human ECs over-expressing VEGF-GFP were similarly found to express the transgene. Transgene expression was analyzed 48 hours following seeding.
Human EC identity was verified by immunohistochemical staining for CD31 (PECAM).
Transfection of ECs and SMCs by recombinant adenovirus encoding UP50-GFP also resulted in transgene expression. Transduction of ECs and SMCs by retroviral vector encoding UP50-GFP similarly resulted in production of GFP.
Transcription of UP50 mRNA was detected by RT-PCR analysis of Ad.UP50-GFP transfected ECs and SMCs and in ECs and SMCs genetically altered with retroviral vector encoding UP50-GFP.
UP50 (60 kD) expression was detected by Western blot analysis following transfection of ECs and SMCs with adenoviral and transduction with retroviral vectors encoding UP50-GFP.
UP50 was detected in Ad.UP50-transfected ECs and SMCs, by immunohistochemical analysis. Cytoplasmic staining occurred in Ad.UP50-GFP transfected cells but not in Ad.GFP transfected cells, indicating high level cytoplasmic expression of UP50.
Confocal microscopy was used to determine the sub-cellular location of UP50 within transfected ECs. UP50 was detected in Ad.UP50-GFP transfected endothelial cells in both the cytoplasm and in the cell membrane. It was also detected as filamentous structures in confluent cells.
The presence of UP50 in the ECM was detected by immunohistochemical analysis of ECs transfected with an adenoviral vector encoding UP50-GFP.
Co-cultures of ECs and/or SMCs, a portion of which were transfected with Ad.VEGF-GFP and a portion of which were transfected with Ad.UP50-GFP were found to express significant levels of VEGF and UP50. Likewise, analysis of co-cultures of SMCs and ECs, a portion of which were retrovirally transduced to express UP50 and a portion of which were retrovirally transduced to express VEGF were found to co-express significant levels of the factors by Western blot analysis.
The above infections-transductions produced almost 100% transgene-expressing cells, as detected by cytoplasmic GFP expression. Transgene over-expression of UP50 had no inhibitory effect on cell growth and proliferation (FIG. 5). Although the values measured in the adenovirus-transfected cells (Ad.GFP, Ad.UP50-GFP) were slightly higher than the non-infected control group, the effect of UP50 expression was not significant. There was no significant difference between ECs transfected with Ad.GFP and ECs transfected with Ad.UP50-GFP. Transfection with Ad.VEGF165-GFP induced significant proliferation compared to other factors.
The above demonstrates the viability of using co-cultures of cells some of which express a proliferation growth factor and some of which express a cell adherence factor. It is, however, a presently preferred embodiment of this invention to have the same cells express both factors. Therefore, ECs transduced with retroviral vector encoding UP50-GFP or GFP were incubated for 48 hrs and then infected with Ad.VEGF-GFP or Ad.GFP. The cells were incubated for an additional 24 hours in virus-containing medium. The virus medium was then replaced with serum-free medium and the cells were incubated for an additional 24 hours. Samples of the growth medium (30 μl) were separated on 10% SDS polyacrylamide gel, electroblotted onto a nitrocellulose membrane and incubated with either anti-VEGF or anti-UP50 antibody. Following exposure to a peroxidase-conjugated secondary antibody, the blots were developed with ECL reagents and exposed to X-ray film. The ECs retrovirally transduced to express UP50-GFP and subsequently transfected with adenoviral vector encoding VEGF-GFP displayed higher levels of GFP expression than cells infected with retroviral vector encoding UP50-GFP only, as determined by fluorescence microscopy. Western blot analysis further confirmed that the cells co-expressed VEGF and UP50 protein. Next, the use of ECs genetically altered to express both mitogeic and adhesion factors to produce synthetic vascular grafts possessing long-term biocompatibility and patency was investigated.
Human saphenous vein ECs were retrovirally transduced with two separate viral vectors, VEGF-GFP and UP50-GFP. Following transduction, the cells were seeded on ePTFE grafts. Fluorescence microscopy observation of endothelial cells in a cell culture dish after being transduced first with a retroviral vector encoding UP50-GFP and, 72-96 hours later, with a retroviral vector encoding VEGF-GFP showed that the cells could survive dual gene transduction and maintain normal morphology. A western blot revealed that the twice-transduced cells in fact express both genes.
To regulate the expression of transgene in ECs, an effector-regulated expression system may be used. For example VEGF expression can be up-regulated and UP50 expression down-regulated by using promoters that are themselves up-regulated and down-regulated by tetracycline. In this manner, cells can be made to express or over-express a cell adhesion factor, e.g., UP50, in the first week following bypass surgery when cell adhesion is a priority. Then, tetracycline can be administered to down-regulate the cellular adhesion factor expression while simultaneously up-regulating expression of a cell proliferation factor, e.g., VEGF, to effect enhanced coverage of the graft. Alternatively, cells can be retrovirally transduced to stably express or over-express UP50 and then transfected with Ad.VEGF in such a manner that expression of VEGF is transient. (Example 12).
Transgene expression having been verified, the effect of expression on cell physiology was next investigated. To accomplish this, in vitro angiogenesis in collagen gels was examined using adenovirus-infected EC spheroids. The generation of spheroids is described in the Examples section. The spheroids of Ad.GFP or AdUP50-GFP transfected ECs were found to have a low baseline sprouting activity. Sprouting was strongly stimulated by addition of exogenous VEGF to either. Likewise, spheroids of Ad.GFP or Ad.UP50-GFP transfected ECs showed a low baseline sprouting activity. Sprouting activity was stimulated in 50% co-cultures with Ad.VEGF-GFP infected ECs. The highest sprouting levels were observed in a co-culture of Ad.UP50-GFP and Ad.VEGF-GFP transfected ECs.
In a different set of experiments, which tested cell adherence (=retention), expression of UP50 following recombinant adenoviral transfection was shown to significantly increase cell retention (60%) compared to Ad.GFP infected cells (40%) or control, non-transfected cells (20%) (FIG. 6). It can be concluded, therefore, that co-over-expression of UP50 with VEGF increases sprouting of EC compared to that mediated by VEGF alone. These results demonstrate that ECs expressing such a combination of factors exhibit an enhanced proliferative and adhesive capacity.
In a presently preferred embodiment of this invention, the proliferation growth factor is VEGF (GenBank Accession Number AB021221), acidic or basic FGF (GenBank Accession Numbers S67291 and M27968) or HGF (GenBank Accession Number D14012). In a presently preferred embodiment if this invention, the cellular adherence factor is UP50 (SEQ ID NO. 1), fibulin-5/DANCE (GenBank Accession Number AF112152), vitronectin (GenBank Accession Number NM000638), albumin (GenBank Accession Number NM000477), collagen I (GenBank Accession Number J00114), collagen IV (GenBank Accession Number M15524), fibronectin (GenBank Accession Number X02761), laminin (GenBank Accession Number NM005560), tropoelastin (GenBank Accession Number XM065759, or VE cadherin (GeneBank Accession Number NM001795).
Other cellular adhesion factors that ECs or SMCs can be engineered to express or over-express will become apparent to those skilled in the art based on the disclosure herein. All such adhesion factors are within the scope of this invention.
Preferably, when both ECs and SMCs are used, the cell proliferation growth factor promotes the proliferation specifically of the ECs so as to avoid unwanted proliferation of SMCs. That is, SMCs are desirable where they can assist in providing a better surface for the adherence of the endothelial cells, which is the manner in which they are employed in the present invention. Thus, the use of SMCs on the exterior (abluminal) surface of a graft of this invention can result in the migration of extracellular matrix produced by the SMCs through the graft and onto the interior surface where the matrix will provide an improved surface for the ECs. Likewise, the use of SMCs on the interior surface of the graft is intended to produce a monolayer of SMCs on the interior surface to mimic the structure of vascular vessels in which such a layer naturally occurs beneath the layer of ECs.
To produce graft 10, cells 16 are seeded, that is, Multiple cells or colonies of endothelial cells are placed on interior surface 14 of element 12 and then the cells are cultured so that they will adhere and when needed grow and proliferate until a sufficient degree of coating of interior surface 14 is achieved. Preferably, cells 16 are cultured under conditions that result in a confluent monolayer of cells on interior surface 14. By a “confluent monolayer” is meant the stage in the proliferation of the endothelial cells at which they all come in contact with each other to form a continuous, uniform coating on the surface. At this point, normal cells stop proliferating due the phenomenon of contact inhibition.
Prior to seeding the interior surface may be coated with substances that aid in the adhesion, growth and proliferation of the endothelial cells. These substances may include, without limitation, amino acids, nucleotides, serum proteins, salts, vitamins, a supplemental serum such as human serum (FCS). Exogenous ECM substances may also be included to enhance adhesion of the cells to the surface.
Several seeding approaches can be used to coat interior surface 14 of graft 10. When both ECs and SMSc are used, the cells may be seeded simultaneously or sequentially on interior surface 14. Sequential seeding—first SMCs, then ECs—is presently preferred. That is, altered or unaltered SMCs are preferably seeded first followed by seeding with altered ECs. This predisposes the cells to their normal position, that is, SMCs beneath and between the ECs and the interior surface of the graft. When simultaneous seeding is employed, the cells may migrate to their desired locations, that is the SMCs migrate toward the surface of the graft and the ECs migrate so as to be on top of the SMCs. When sequential seeding is employed the time between seeding can vary but the presently preferred time lapse between seeding with SMCs and seeding with ECs is 24-96 hours. Since it is known that ECs have a natural affinity for SMCs, it is possible to seed a graft with SMCs, preferably genetically altered to over-express an adhesion factor to enhance the effect even more, and then allow seeded or circulating endothelial cells to adhere to the SMCs.
Cells 16 can be altered so that the same cells express both the cell proliferating growth factor and the cellular adhesion factor. In the alternative, one portion of the cells can be altered to express the cell proliferating growth factor and another portion can be altered to express the cellular adhesion factor. In general, when different cells are used to express or over-express a cell proliferation growth factor and an adhesion factor, it is preferred that a greater proportion of cells that express or over-express the cell adhesion factor are used. It is presently preferred that from about 60% to about 90% of the cells express or over-express the cell adhesion factor. Most preferred are cells that express or over-express both a cell proliferation growth factor and a cell adhesion factor, in which case, of course, the proportion is 1:1. Such cells were found to adhere better to a graft that native cells or cells expressing or over-expressing VEGF or UP50 alone.
ECs and SMCs can be obtained from various mammalian tissue sources. For example, ECs can be obtained from, without limitation, a segment of a vein, a segment of an artery, bone marrow progenitor cells, peripheral blood stem cells, embryonic stem cells or circulating endothelial cells. Smooth muscle cells can be obtained from, without limitation, human saphenous veins, left internal mammary arteries, the radial artery, bone marrow progenitor cells, embryonic stem cells, and peripheral blood stem cells.
In a presently preferred embodiment of this invention, ECs and SMCs are obtained from tissues of the intended recipient of the graft or a syngeneic donor.
Of course, it is possible to obtain ECs and SMCs that can be used in the devices and methods of this invention from xenogeneic tissue providing measures are taken to avoid cell rejection. These measures include, without limitation, use of transgenic animal tissues that express the human decay accelerating factor or that do not express the a-Gal epitope. Also, well-known immune suppressive drugs are often used. These and numerous other methods for reducing the risk of rejection are well-known in the art. Those skilled in the art will know which of these techniques would be best employed in a given situation. All such measures are within the scope of this invention.
The proliferation and adhesion factors can be encoded by polynucleotide sequences derived from human or other mammalian cells provided the factors expressed by the sequences are functional in ECs and SMCs.
The proliferation and adhesion factors can be endogenous or xenogenous to the EC or SMC cells used. If they are endogenous, that is if some or all of them are already expressed by the cells, the cells can be genetically altered to over-express one or more or of them. The cells can also be genetically altered to express desirable xenogenous factors.
As used herein, the terms “over-express,” “over-expressed,” or “over-expression” refer to expression levels that exceed those normally produced by a cell. Over-expression can be induced by introducing additional copies of an endogenous gene into a cell, which results in a higher level of expression of the factor. Over-expression can also be induced by introducing enhancer sequences into the cellular genetic material that up-regulate the transcription or translation of the endogenous genes. The latter can be accomplished by, for example, gene “knock-in” techniques, which are well-known in the art. It also can be achieved by introducing factors that will reduce level of RNA degradation or that will stabilize RNA of the relevant gene. These and other procedures that result in over-expression of genes and that will be useful with regard to the present invention will become apparent to those skilled in the art based on the disclosures herein. All such techniques are within the scope of this invention.
As used herein the phrase “genetically alter” refers to the introduction of one or more exogenous polynucleotide sequences into a cell. The sequences may be duplicates of sequences already in the cell's genetic material as might be the case where over-expression is the goal. Or, the sequences may be entirely xenogenous, such as would be the case of the cell does not normally express the factor encoded by the sequence. The sequences may integrate into the genome of the cell, thus becoming a permanent part thereof, or they may remain as separate, transient entities in the nucleus or cytoplasm of the cell. As described elsewhere herein, both stable and transitory expression of factors may, under certain circumstances be useful in carrying out the methods of this invention.
Another aspect of the present invention is a nucleic acid expression construct for genetically altering ECs and SMCs for use in the methods herein. As used herein, a “nucleic acid construct” refers to one or more polynucleotide sequences that encode for one or more of proliferation and/or adhesion factors. In a presently preferred embodiment, the construct comprises two sequences, one that encodes a cell proliferation growth factor and one that encodes a cell adhesion factor. A “polynucleotide sequence” refers to a linear array of nucleotide residues that encodes the expression of a particular factor. In a presently preferred embodiment, the construct also comprises one or more promoter sequences for directing the expression of the polynucleotide sequences. A promoter is a DNA sequence that facilitates the binding of RNA polymerase to a template and initiates replication. A promoter initiates transcription only of the gene or genes physically connected to it on the same stretch of DNA, that is, the promoter must be “in cis” with the gene it affects. A promoter may be constitutive, that is, always “on” and capable of initiating transcription at any time. It may be tissue specific and only initiate transcription in certain tissue environs. Or it may be inducible, in which case another molecule, known as an effector, or some other external influence such as, without limitation, temperature, light, shear stress, pH, pressure, etc., is needed to “induce” the promoter to operate. Any of these types of promoters may be used in the constructs of this invention and are within its scope.
As is further described in the Examples section, the cell proliferating growth factor and the cellular adherence factor may be expressed in different temporal patterns. That is, if desired, the expression of the genes can be controlled such that expression or over-expression of the cell adhesion factor can occur first and then, at a later time, expression or over-expression of the cell proliferation growth factor can be up-regulated. If desired, expression of the cell adhesion factor can be down-regulated when expression of the cell proliferation growth factor is up-regulated. However, it is presently preferred that expression or over-expression of the cell adhesion factor is simply maintained when the expression or over-expression of the cell proliferation growth factor is up-regulated.
In a presently preferred embodiment, the nucleic acid expression construct comprises two promoter sequences, each directing the expression of one of the polynucleotide sequences. It is further presently preferred that the promoters be inducible and that they are regulated by the same effector molecule. It is also presently preferred that the promoter sequences are selected such that one promoter is up-regulated and, at the same time, the other promoter is down-regulated by the effector.
Suitable inducible promoters include, without limitation, chemically (effector) induced promoters such as those used in the Tet-OnTM and Tet-OffTM gene expression systems commercially available from Clontech. Another example is shear stress induced promoters.
In the alternative, a single promoter sequence can be used to regulate both polynucleotide sequences provided that they are transcriptionally linked and that an internal ribosome entry site (IRES) is included for directing the translation of the second sequence of the polycistronic message.
The two polynucleotide sequences can also be translationally fused provided a protease cleavage site is inserted between the sequences so that cleavage and separation of the two polypeptides can occur in expressing cells.
If desired, the two polynucleotide sequences can be provided as separate nucleic acid constructs that are co-introduced into the cells.
Another aspect of the present invention is a nucleic acid construct system for genetically altering cells of this invention. A construct system, as the term is used herein, comprises two nucleic acid expression constructs as described above. One would encode for the cell proliferating growth factor and the other for the cellular adherence factor.
In a presently preferred embodiment, the expression construct or construct system includes additional polynucleotide sequences that code for reporter markers, selection markers and the like. Selection markers are used to assist in determining which cells have been genetically altered cells and isolating those cells. A common selection marker is antibiotic resistance. That is, a resistance gene is inserted into the cell along with the desired factor gene. After the cells have been treated with a vector, those that were successfully infected will survive exposure to an antibiotic and can be isolated while those that were not infected will die. Reporter markers are used to monitor the expression of cell proliferating growth factor(s) and cellular adherence factor(s). Examples of reporter markers include, without limitation, beta-galactosidase, luciferase and green fluorescent protein (GFP). Other selection and reporter markers that would be useful in the production of the genetically altered cells herein will become apparent to those skilled in the art based on the disclosures herein and are within the scope of this invention.
To monitor expression of the cell proliferation growth factor and the cellular adherence factor, the reporter marker gene can be transcriptionally linked or translationally fused to the polynucleotide sequence encoding the factor. Or, it can be placed under the transcriptional control of a promoter sequence identical to that directing the transcription of the factor.
The polynucleotide sequences encoding the cell proliferating growth factor and the cellular adherence factor can be ligated into a commercially available expression vector system suitable for transforming mammalian cells and for directing the expression of the factors in the cells. Such commercial vector systems can easily be modified by recombinant techniques well known in the art to replace, duplicate or mutate existing promoter or enhancer sequences or to introduce additional polynucleotide sequences.
Suitable mammalian expression vectors include, without limitation, pcDNA3, pcDNA3.1(+/−), pZeoSV2(+/−), pSecTag2, pDisplay, pEF/myc/cyto, pCMV/myc/cyto, pCR3.1 (Invitrogen); pCI (Promega); pBK-RSV and pBK-CMV (Stratagene) and pTRES (Clontech), and derivatives thereof.
A nucleic acid expression construct or construct system useful herein to up-regulate a factor may comprise transcriptional regulatory sequences in cis to endogenous sequences encoding the cell proliferation growth factor or the cellular adherence factor. By “in cis” is meant that the regulatory sequence is on the same DNA molecule as the sequence it is regulating. Alternatively, an expression construct or construct system useful to up-regulate a factor may comprise translational regulatory sequences in trans to endogenous sequences encoding the cell proliferating growth factor or the cellular adherence factor. By “in trans” is meant that the regulatory sequence is present on a different molecule of DNA than the sequence it is regulating.
Gene “knock-in” techniques well-known in the art can be used to introduce cis acting transcriptional regulatory sequences into the genome of the Ecs or SMCs (U.S. Pat. Nos. 5,487,992, 5,464,764, 5,387,742, 5,360,735, 5,347,075, 5,298,422, 5,288,846, 5,221,778, 5,175,385, 5,175,384, 5,175,383 and 4,736,866, each of which is incorporated by reference, including any drawings, as if fully set forth herein. See also, International publications WO 94/23049, WO93/14200, WO 94/06908 and WO 94/28123. For additional general information on the technique, see Burke and Olson, Methods in Enzymology, 194:251-270, 1991; Capecchi, Science 244:1288-1292, 1989; Davies et al., Nucleic Acids Research, 20 (11) 2693-2698, 1992; Dickinson et al., Human Molecular Genetics, 2(8):1299-1302, 1993; Duff and Lincoln, “Insertion of a pathogenic mutation into a yeast artificial chromosome containing the human APP gene and expression in ES cells”, Research Advances in Alzheimer's Disease and Related Disorders, 1995; Huxley et al., Genomics, 9:742-750 1991; Jakobovits et al., Nature, 362:255-261 1993; Lamb et al., Nature Genetics, 5: 22-29, 1993; Pearson and Choi, Proc. Natl. Acad. Sci. USA, 1993, 90:10578-82; Rothstein, Methods in Enzymology, 194:281-301, 1991; Schedl et al., Nature, 362: 258-261, 1993 and Strauss et al., Science, 259:1904-1907, 1993.
The nucleic acid expression constructs of the present invention can be introduced into ECs and SMCs using any of a number of methods including, but not limited to, direct microinjection of DNA, protoplast fusion, diethylaminoethyldextran and calcium phosphate-mediated transfection, electroporation, lipofection, adenoviral transfection, retroviral transduction and others. Such methods are well-known in the art and any of them are within the scope of this invention.
To assess the stability and adhesion of vascular ECs and SMCs to an ePTFE graft in vivo, an artificial pulsatile flow device, which can simulate a variety of mechanical and hemodynamic forces resembling in vivo conditions was developed. The device can also be used for quality assurance prior to implanting a biosynthetic product.
The device (shown schematically in FIG. 9) comprises a pulsatile blood pump (Harvard apparatus #1421, USA), rigid stainless steel 316 L tubes (OD of 12 mm and 6 mm), flexible Teflon tubes (OD of 12 mm and 6 mm), stainless steel 316 L connectors and valves (Hamlet, Israel), and glass made compensation tanks. The device is assembled in an incubator (37° C., 5% CO2) and is monitored by means of both pressure (#742 Mennen Medical, USA) and flow monitors (#T106 Transonic Systems Inc., USA). The data obtained is analyzed by a data acquisition system, which presents the calculated values of the shear forces produced inside the grafts on-line.
Once the device is assembled (clean and sterile before each experiment), the pressure and flow connectors are connected to the system and to the monitors, which are calibrated. The data acquisition software is activated and the system is filled with warm (37° C.) growth medium (M199, Penicillin (200 unit/ml)-Streptomycin (0.2 mg/ml), Amphothericin (0.5 microgram/ml) and fetal calf serum (FCS), 20%). Two PTFE grafts are then fitted onto the stainless steel connectors and secured in place with silicone strings. Pulsatile flow of medium in the system is slowly increased through adjustment of the stroke volume and flow rate of the pump. The fluid runs in the system in stainless steel and Teflon® tubing. A physiological pulse wave is generated using tank A. Control of the fluid wave is achieved with valve A. Valves B and C provide control of the fluid flow through the grafts. Pressure in the system is controlled by valve D. Equilibration of the system with the atmosphere in the incubator is achieved by compensation tank B, which also serves as a fluid reservoir.
Endothelial cells retention on an e-PTFE graft was examined following EC transduction with retroviruses encoding for the UP50, VEGF-GFP genes and native EC. Grafts seeded with EC over-expressing UP50 were tested against grafts seeded with EC expressing GFP. The results show that ECs over-expressing UP50 are retained much better than cells that express GFP when exposed to arterial-like flow and shear stress. No difference was observed when EC seeded grafts transduced with GFP were compared to graft seeded with native ECs. Retention of grafts seeded with EC over-expressing UP50 were also compared to grafts seeded with ECs over-expressing VEGF. The ECs over-expressing UP50 showed superior retention to those over-expressing VEGF. Retention of cells on a graft seeded with EC over-expressing UP50 and VEGF was compared to that on a graft seeded with EC over-expressing only VEGF. The cells over-expressing both factors showed substantially better retention than cells over-expressing VEGF alone.
To examine whether the above in vitro results would be duplicated in vivo, grafts seeded with sheep autologous ECs over-expressing cell proliferation growth factor and cell adhesion factor were implanted in donor sheep arteries (Example 26). The results showed that grafts seeded with cells over-expressing either UP50-GFP or VEGF-GFP had a higher number of cells adhering to the grafts following exposure to in vivo blood flow compared to cells over-expressing GFP only. In addition, grafts seeded with cells over-expressing UP50 displayed higher number of adherent cells than the grafts seeded with cells over-expressing GFP or VEGF.
Artificial vascular grafts of this invention may be used in place of any current by-pass or shunting graft, either natural or artificial, in any application. Thus, they may be used for, without limitation, arterial by-pass, both of the cardiac variety and that used to treat peripheral arterial disease (PAD). An artificial graft of this invention may also be used as a replacement or substitute for a fistula created for use in hemodialysis. Also the synthetic artificial vascular graft of the present invention can be used to replace a damaged blood vessel such as traumatically damaged limb arteries.
A presently preferred application for a graft of the present invention is an artificial arteriovenous shunt for use by dialysis patients.
In hemodyalysis, a patient's blood is “cleansed” by passing it through a dialyzer, which consists of two chambers separated by a thin membrane. Blood passes through the chamber on one side of the membrane and dialysis fluid circulates on the other. Waste materials in the blood pass through the membrane into the dialysis fluid, which is discarded, and the “clean” blood is re-circulated into the blood stream. Access to the bloodstream can be external or internal. External access involves two catheters, one placed in an artery and one in a vein. More frequently, and preferably, internal access is provided. This is accomplished either by an artriovenous fistula or an AV graft. An AV fistula involves the surgical joining of an artery and a vein under the skin. The increased blood volume stretches the elastic vein to allow for a larger volume of blood flow. Needles are placed in the fistula so that blood can be withdrawn for dialysis and then the blood is returned through the dilated vein.
An AV graft may be used for people whose veins, for one reason or another, are unsuitable for an AV fistula. An AV graft involves surgically grafting a donor vein from the patient's own saphenous vein, a carotid artery from a cow or a synthetic graft from an artery to a vein of the patient. One of the major complications with a synthetic AV graft is thrombosis and neointimal cell proliferation that cause closure of the graft.
To counter thrombosis and neointimal proliferation, grafts have been seeded with a patient's own endothelial cells. However, the high rate of blood flow through these grafts together with the damage caused by the incursion of needles through the layer of cells often results in the detachment of the ECs from the walls of the graft. The grafts of the present invention overcome this deficiency.
In the first place, the genetically altered ECs of this invention, which over-express UP50 and VEGF, are more capable of remaining attached to the graft at the site of puncture thus minimizing damage caused by the needle. Furthermore, the altered cells proliferate more rapidly than native ECs and thus cover the puncture site more quickly and completely. This reduces exposure of the ECM, other substances used to enhance the performance of the graft and the graft material itself to blood, which would be expected to reduce the occurrence of thrombosis at site of puncture. Rapid regeneration of the EC layer should also reduce SMC proliferation at site of anastamosis and will thus improve patency of the graft in the shunt. This is demonstrated in Example 27.
Thus, cells genetically altered to express or over-express endothelial proliferation growth factor and cell adhesion factor are substantially more resistant to the shear forces of blood flow and have a higher capacity to cover completely grafts even after mechanical damage or shear stress induced detachment. In addition, it has been found that cells that have been genetically altered to express or over-express VEGF and UP50 appear to cover and repair the damage caused by punctures much more rapidly than cells that do not express these factors. Thus, grafts of the present invention should also have a lower occurrence of thrombosis at the site of needle invasion into the graft or at a bare surface of the graft. These factors should result in substantially greater patency than current grafts and a longer useful lifetime in a patient, as demonstrated by Examples 27-29.
In another embodiment, devices and methods are provided for the treatment of pathologies associated with vascular injury, and particularly in relation to angioplasty and stent deployment, or atherectomy. See Example 29. Of particular interest is the injury referred to as restenosis, which results from the migration and proliferation of vascular smooth muscle cells into the intima of the vessel, as well as accretions associated with atherosclerosis. An additional advantage of using vascular prostheses coated with fibulin-5 (UP50) is that fibulin-5 also partially inhibits smooth muscle cell migration and proliferation. Unlike some drugs used on vascular prostheses such as stents, fibulin-5 does not completely suppress smooth muscle cell proliferation. Since some smooth muscle cell proliferation is needed for vessel wall healing after intervention, fibulin-5 has an advantage over current drugs used on stents, which completely inhibit all cell proliferation, including the beneficial endothelial cells. While use of fibulin-5 alone partially inhibits both smooth muscle cells and endothelial cell migration and proliferation, the addition of a growth factor such as VEGF promotes rapid proliferation of endothelial cells over proliferation of smooth muscle cells, which remain partially inhibited due to the presence of fibulin-5. Accordingly, there is a synergistic effect on endothelial cell proliferation and migration of using fibulin-5 and VEGF, than seen with VEGF alone.
The long term benefit of intravascular intervention due to treatment of various cardiovascular syndromes, e.g., coronary balloon angioplasty and atherectomy, is limited by the considerably high occurrence of symptomatic restenosis (40-50%) 3 to 6 months following the procedure. Restenosis is in part due to myointimal hyperplasia, a process that narrows the vessel lumen and which is characterized by vascular smooth muscle cell migration and proliferation. Medical therapies to prevent restenosis have been uniformly unsuccessful. Intravascular stents have been successfully used to achieve optimal lumen gain, and to prevent significant remodeling. However, intimal thickening still plays a significant role in stent restenosis. Thus, intravascular prostheses eluting fibulin-5 alone, or in combination with VEGF, will partially inhibit smooth muscle cell proliferation and migration, thereby preventing the consequent restenosis. Prostheses eluting fibulin-5 in conjunction with VEGF will simultaneously enhance recovery of the endothelial cell layer by enhancing endothelial cell proliferation, migration, and adhesion, and thus, will prevent clotting of the prostheses.
The subject methodology is employed with hosts who have suffered vascular injury, as caused by angioplasty or atherectomies. The time for the administration of the therapeutic mixture may be varied widely, providing a single administration or multiple administrations over a relatively short time period in relation to the time of injury. Accordingly, in one embodiment the invention provides introducing to or into the vessel walls at the site of injury a therapeutic mixture of fibulin-5 and VEGF which results in the enhancement of endothelial cell proliferation and migration at the site of injury. The two proteins can be delivered directly to the site of vascular injury (such as by injection), or can be incorporated into the prostheses as proteins, or can be incorporated into polymers that will cover the prostheses and will be released slowly by degradation of the polymer. Various delivery systems may be employed which result in the therapeutic mixture infusing into the vessel wall, and being available to the endothelial and smooth muscle cells. Devices which may be employed include drug delivery balloons, e.g., porous, sonophoretic, and iontophoretic balloons, as exemplified by the devices depicted in WO92/11895, WO95/05866 and WO96/08286, which are incorporated herein by reference thereto. Also, stents may be employed where the stent carries the therapeutic mixture. Preferably, the stent is conveniently introduced with a catheter, so that both short and long term delivery of the fibulin-5 and VEGF proteins can be provided for enhanced protection against blockage.
In conjunction with the intraluminal or intramural delivery of the therapeutic mixture by the catheter, a stent may be introduced at the site of vascular injury. The stent may be biodegradable or non-biodegradable, may be prepared from various materials, such as metals, ceramics, plastics or combinations thereof. Biodegradable plastics, such as polyesters of hydroxycarboxylic acids, are of particular interest. Numerous stents have been reported in the literature and have found commercial acceptance. An example of the type of stent which may be modified to deliver a fibulin-5 and VEGF type mixture is disclosed in U.S. Pat. Nos. 5,591,227; 5,733,327; 5,899,935; 6,364,856; 6,403,635; 6,425,881; 6,716,242; and 6,918,929, each of which is incorporated herein by reference in its entirety.
Any suitable biodegradable drug-polymer coatings, or other means by which to release the therapeutic mixture of fibulin-5, with or without VEGF, known to those skilled in the art may be used. Exemplary methods of using such polymers or delivery systems are also provided by U.S. Pat. Nos. 5,591,227; 5,733,327; 5,899,935; 6,364,856; 6,403,635; 6,425,881; 6,716,242; 6,918,929; and 6,939,376, each of which is incorporated herein by reference in its entirety. Depending on the nature of the stent, the stent may have the therapeutic mixture incorporated in the body of the stent or coated thereon. For incorporation, normally a biodegradable plastic stent will be used which will release the therapeutic mixture while supporting the vessel and protecting against restenosis. In the fabrication of the stent, the biodegradable matrix may be formed by any convenient means known in the art. Alternatively, the stent may be coated with the therapeutic mixture, using an adhesive or coating which will allow for controlled release of the therapeutic mixture of fibulin-5, with or without VEGF. The stent may also be comprised of fibulin-5 with simultaneous or consecutive administration of VEGF or another suitable growth factor. The stent may be dipped, sprayed or otherwise coated with a composition containing the therapeutic mixture and a matrix, such as the biodegradable polymers described above, a physiologically acceptable adhesive, proteins, polysaccharides or the like. By appropriate choice of the material for the stent and/or the coating comprising the therapeutic mixture, a physiologically active amount of the therapeutic mixture of fibulin-5, with or without VEGF or another suitable growth factor, may be maintained at the site of the vascular injury, usually at least one day and up to a week or more.
Accordingly, in one embodiment the invention provides an implantable device for treating a vascular disease or disorder that includes an intravascular device and a biodegradable drug-eluting polymer disposed on and/or within the prosthesis. The polymer can be impregnated with an inhibitor of smooth muscle cell proliferation/migration, and can also be impregnated with a growth factor. In one aspect the intravascular prosthesis is a stent, a vascular graft, an artificial heart, or an artificial valve. In another aspect, the inhibitor of smooth muscle cell proliferation/migration can be fibulin-5 (UP50), and the growth factor can be VEGF. The vascular disease or disorder to be treated can include, for example, stenosis, restenosis, atherosclerosis, cardiac arrest, stroke, thrombosis, or atherectomy, or injuries caused by intravascular interventions used to treat these diseases or disorders.
In another aspect, the implantable device can include a substrate coated with endothelial cells that are genetically altered to express or over-express fibulin-5, with or without VEGF. The substrate can be disposed on and/or within the prosthesis.
In another embodiment, the invention provides methods of treating or preventing a vascular disease or disorder by simultaneously inhibiting vessel blockage and enhancing recovery of the vessel wall following an intravascular intervention by inserting an intravascular device coated with a biodegradable drug-eluting polymer impregnated with an inhibitor of smooth muscle cell proliferation/migration and a growth factor, within a vessel of a subject in need thereof, and eluting the inhibitor and the growth factor from the polymer into the vessel, thereby inhibiting smooth muscle cell proliferation and enhancing endothelial cell proliferation.
In one aspect, the intravascular device can be a stent. In another aspect, the device can be a vascular graft (of any caliber). In another aspect the inhibitor of smooth muscle cell proliferation/migration is fibulin-5, and the growth factor is VEGF. The vascular disease or disorder can include, for example, restenosis, thrombosis, atherosclerosis, atherectomy, or intravascular injuries related to treating various cardiovascular conditions. In still another aspect, the device can include a substrate coated with endothelial cells that are genetically altered to express or over-express an inhibitor of smooth muscle cell proliferation and a growth factor.
In yet another embodiment, the invention provides methods of preventing neointima formation of smooth muscle cells following an intravascular intervention by delivering a plurality of vectors containing a polynucleotide sequence encoding an inhibitor of smooth muscle cell proliferation/migration to a site of vascular injury. In another aspect, the vectors can additionally include a polynucleotide sequence encoding one or more growth factors, such as VEGF.
In one aspect, the inhibitor of smooth muscle cell proliferation/migration is fibulin-5 (UP50). The delivery step can be performed by stent delivery, or local delivery, such as by injection. (See Examples 28-29).
EXAMPLES
The following examples are provided solely to illustrate various aspects of the present invention. They are not intended, nor are they to be construed, to limit the scope of the invention in any manner whatsoever.
The examples that follow employ nomenclature and procedures used generally in the molecular, biochemical, microbiological and recombinant DNA arts. See, for example, “Molecular Cloning: A laboratory Manual,” Sambrook et al. (1989); “Current Protocols in Molecular Biology,” Volumes I-III, Ausubel, R. M., ed. (1994); Ausubel, et al., “Current Protocols in Molecular Biology”, John Wiley and Sons, Baltimore, Md. (1989); Perbal, “A Practical Guide to Molecular Cloning”, John Wiley & Sons, New York (1988); Watson et al., “Recombinant DNA”, Scientific American Books, New York; Birren et al. (eds) “Genome Analysis: A Laboratory Manual Series”, Vols. 1-4, Cold Spring Harbor Laboratory Press, New York (1998); “Cell Biology: A Laboratory Handbook”, Volumes I-III Cellis, J. E., ed. (1994); “Current Protocols in Immunology” Volumes I-III Coligan J. E., ed. (1994); Stites et al. (eds), “Basic and Clinical Immunology” (8th Edition), Appleton & Lange, Norwalk, Conn. (1994); Mishell and Shiigi (eds), “Selected Methods in Cellular Immunology”, W. H. Freeman and Co., New York (1980); “Oligonucleotide Synthesis” Gait, M. J., ed. (1984); “Nucleic Acid Hybridization” Hames, B. D., and Higgins S. J., eds. (1985); “Transcription and Translation” Hames, B. D., and Higgins S. J., eds. (1984); “Animal Cell Culture” Freshney, R. I., ed. (1986); “Immobilized Cells and Enzymes” IRL Press, (1986); “A Practical Guide to Molecular Cloning” Perbal, B., (1984) and “Methods in Enzymology” Vol. 1-317, Academic Press; “PCR Protocols: A Guide To Methods And Applications”, Academic Press, San Diego, Calif. (1990); Marshak et al., “Strategies for Protein Purification and Characterization—A Laboratory Course Manual” CSHL Press (1996) and U.S. Pat. Nos. 4,666,828; 4,683,202; 4,801,531; 5,192,659 and 5,272,057; 3,791,932; 3,839,153; 3,850,752; 3,850,578; 3,853,987; 3,867,517; 3,879,262; 3,901,654; 3,935,074; 3,984,533; 3,996,345; 4,034,074; 4,098,876; 4,879,219; 5,011,771 and 5,281,521. The U.S. patents are incorporated by reference, including any drawing, as if fully set forth herein.
Example 1
Generation of Recombinant Adenoviral Vectors Encoding the LacZ Gene
A 3700 bp HindIII-BamHI fragment containing the bacterial β-galactosidase gene LacZ (Clontech, CA, USA) was inserted into plasmid pCA3 for constitutive expression under the control of the CMV immediate-early promoter. The resultant plasmid was co-transfected with plasmid pJM17 into “293” cells that constitutively express the adenoviral E1 gene. Plasmid pJM17 contains the adenovirus genome, excluding the E3 region, and including an insert (pBRX) in the E1 region of the virus. Homologous recombination between pCA3 encoding β-galactosidase and pJM17 following transfection replaced the E1 region of pJM17 with the CMV-β-galactosidase expression cassette from pCA3. Plaque formation occurred 2 to 4 weeks following co-transfection, after which individual plaques were isolated and viral extracts were amplified therefrom by infection of “293” cells. The titer of each viral stock was determined by plaque assay in “293” cells. Viral titers of  ̃1010 pfu/ml were obtained. Expression of β-galactosidase by infected cells was confirmed by X-gal staining.
Example 2
Generation of Recombinant Bicistronic Adenoviral Vectors Encoding the VEGF and GFP Genes
A 600 bp BamHI fragment containing the human VEGF165 cDNA (Genbank Accession number AB021221), including the signal sequence for secretion (gift of Dr. J. Abraham, Scios Nova, Mountain View, Calif.) was inserted into the BglII site of shuttle vector pQBI-CMV5-GFP (QBI, Canada), thereby generating shuttle vector CMV5-VEGF165-IRES-EGFP (FIG. 3a). The expression plasmid pQBI-CMV5-GFP contains the left arm (16%) of the Ad5 genome with a deletion in the E1 region containing a hCMV5 insert. Shuttle vector CMV5-VEGF165-IRES-EGFP was co-transfected with plasmid pJM17 into “293” cells constitutively expressing the E1 gene. The pJM17 plasmid contains the adenovirus genome, excluding the E3 region, and including an insert (pBRX) in the E1 region of the virus. Homologous recombination between CMV5-IRES-VEGF165-GFP and pJM17 following transfection replaced the E1 region and pBRX insert with the expression cassette from CMV5-VEGF165-IRES-GFP. Plaque formation occurred 2 to 4 weeks following co-transfection after which individual plaques were isolated and viral extracts were amplified by infection of “293” cells. The titer of each viral stock was determined by plaque assay in “293” cells. Titers of  ̃1010 pfu/ml were obtained. Transgene expression was confirmed by Western blot analysis of infected, cell-conditioned medium.
Example 3
Generation of Pseudotyped Retroviral Vectors Encoding Human VEGF and GFP
Recombinant retroviral vectors encoding the GFP and human VEGF165 genes were constructed by cloning into plasmid pLXSN (# K1060-B Clontech, USA) in two steps. First, a 600 bp BamHI fragment encoding VEGF165 (Genbank Accession number AB021221) was inserted into the BamHI site of plasmid pIRES2-EGFP (#6029-1 Clontech). Then, a 2.0 kB EcoRI-MunI fragment containing the VEGF165, IRES and EGFP encoding sequences was cloned into the EcoRI restriction site in pLXSN resulting in vector LXSN-VEGF165-IRES-EGFP (FIG. 3b). For retroviral vector production, 293E3 ecotropic packaging cells were transiently transfected with LXSN-VEGF165-EGFP. After 48 hours, the supernatant from confluent cultures of G418-resistant producer cells was collected, filtered (0.45 μm) and used to transduce PA317 amphotropic packaging cells. Transduced PA317 cells were grown under G418 (Gibco, BRL USA) selection (300 mg/ml) and after 48 hours the supernatant was collected and used to transduce TEFLYGA packaging cells which express GALV envelope glycoprotein to generate pseudotyped virus capable of transducing ECs and SMCs with high efficiency. After G418 selection (400 μg/ml) of transduced TEFLYGA cells, individual colonies were collected and screened for EGFP and VEGF165 expression. Viral titers of each colony were determined by TE671 cell transduction and were found to range from 105 to 106 pfu/ml. The highest-titer producing colonies were selected and freshly collected supernatants were employed for transduction.
Example 4
Generation of Recombinant Adenoviral Vectors Encoding the UP50 Gene
Recombinant adenoviral vector expressing the human UP50 gene (obtained from Y. Shaul, Weizmann Institute) was constructed as described above. A 1361 bp BglII fragment containing the human UP50 cDNA (SEQ. ID No. 1) was inserted into plasmid pCA3. Transgene-containing plasmid pCA3 was co-transfected with plasmid pJM17 into “293” cells. Homologous recombination between the expression plasmid and pJM17 following transfection replaced the E1 region with the expression cassette from the pCA3 plasmid thereby generating shuttle vector CMV5-UP50 (FIG. 4a). Plaque formation occurred 2-4 weeks following co-transfection. Individual plaques were isolated and viral extracts were amplified by infection of 293 cells. Titers of viral stock of  ̃1011 pfu/ml were obtained. Transgene expression was confirmed by Western blot analysis of infected cell-conditioned medium.
Example 5
Generation of Recombinant Adenoviral Vectors Encoding Both the UP50 and GFP Genes
A recombinant adenoviral vector co-expressing human UP50 and GFP genes was constructed via a modified AdEasy protocol (28). A 1361 bp BglII fragment of UP50 cDNA was inserted into the BglII site pAdTrack-CMV shuttle vector under the control of the CMV promoter. The shuttle vector encodes GFP under the control of an additional CMV promoter downstream to the transgene. Insert-containing shuttle vector was linearized by PmeI digestion and purified using a Qiaquick gel extraction kit (Qiagen, Germany). Competent BJ5183 cells were co-transfected with insert-containing shuttle vector and pAdEasy-1 by electroporation and positive clones containing recombinant adenoviral vector encoding UP50-GFP (FIG. 4b) were selected by PCR and restriction map analysis. Recombinant adenoviral plasmids were linearized by PacI digestion, purified and transfected into “293” cells using Lipofectamine 2000 (Gibco BRL, USA). Seven days following transfection, cytopathic effect occurred and 100% of the cells were found to express GFP. The cells were harvested and viral extracts were further amplified in “293” cells. The titer of each viral stock was determined by serial dilution assay in “293” cells and the titers of 1011 pfu/ml were obtained. Expression of transgene was confirmed by Western blot analysis of infected cell-conditioned medium.
Example 6
Construction of Retroviral Vectors for Expression of UP50 or Co-Expression of UP50 and EGFP
Recombinant retroviral vector LXSN-UP50 encoding the human UP50 gene (FIG. 4c) was constructed by inserting the human UP50 cDNA 1361 bp BglII fragment into the BamHI site of plasmid pLXSN (# K1060-B Clontech, USA) under the control of Mo-MULV 5′ long terminal repeat (LTR).
A bicistronic recombinant retroviral vector encoding both the UP50 and EGFP genes was cloned into plasmid pLXSN in two steps. First, a 1400 bp IRES-EGFP EcoRI-HpaI fragment excised from pIRES2-EGFP (Clontech, #6029-1) was inserted into EcoRI-HpaI-digested pLXSN for construction of the control plasmid pLXSN-IRES-EGFP. Next, pLXSN-UP50-IRES-EGFP (FIG. 4d) was constructed by cloning human UP50 EcoRI fragment (1361 bp) into the EcoRI site of pLXSN-IRES-EGFP. Gene expression in these constructs is regulated by Mo-MULV 5′ long terminal repeat (LTR).
Example 7
Generation of Pseudo-Typed Recombinant Retroviral Vectors Encoding UP50
For retroviral vector production, vector pLXSN-UP50-EGFP or pLXSN-UP50 was transfected into 293FLYA packaging cells using Lipofectamine (Gibco BRL, USA). After 48 hours, supernatant from confluent cultures of viral producer cells was collected, filtered (0.45 μm) and added to 293FLY10A or 293 FLYGALV packaging cells. Transduced cells were grown under G418 selection (400 μg/ml) and individual colonies were collected and screened for EGFP expression using an inverted fluorescent microscope. They were also screened for UP50 expression by Western blot analysis of transduced cell-conditioned medium. The viral titer of each colony was determined via transduction of TE671 cells and titers of  ̃106 ffu/ml were obtained. Supernatant from colonies with the highest-titers was collected freshly for transduction of EC and SMC.
Example 8
Tissue Culture of Primary Vascular Cells
Human saphenous vein ECs (HSVEC), human radial artery ECs (HRAEC) and human left internal mammary artery ECs (HLAEC) were harvested from 5 cm-long vascular segments by collagenase digestion, as previously described (29). Isolated ECs were cultured on gelatin-coated dishes containing M199 medium (Gibco BRL, USA) supplemented with 20% fetal calf serum (hyclone, USA), 2 mM L-glutamine (Biological Industries, Israel), 100 units/ml penicillin (Biological Industries, Israel), and 0.1 mg/ml streptomycin (Biological Industries, Israel), 100 μg/ml heparin (Sigma, USA) and 2 ng/ml bFGF (obtained from Prof. Neufeld). Cells from passages 3-9 were collected to ensure phenotypic stability, which was monitored on the basis of cellular morphology and by immunohistochemical staining for von Willebrand factor and CD31. Human SMCs were cultured by ex-plant outgrowth from human saphenous veins, human radial artery (HRASMC) and left internal mammary arteries (HLSMC). Cells were cultured in Dulbecco's Modified Eagles Medium (DMEM) (Gibco BRL, USA) supplemented with 10% pooled human serum, 2 mM L-glutamine, 100 units/ml penicillin, 0.1 mg/ml streptomycin and 2 ng/ml bFGF. SMCs were identified by immunohistochemical staining for smooth muscle α-actin (Dako, USA). Cells were routinely tested for sterility, endotoxins and mycoplasma infection. Animal veins were excised from minipigs and sheeps using the same procedures as with human vascular segments. The ECs and SMCs from both pigs and sheep were likewise harvested in the same manner as human ECs and SMCs.
Example 9
Tissue Culture of Cell Lines
The packaging cell lines 293-FLYA, 293-FLY10A, 293-FLYGALV and TEFLYGA (obtained from Dr. F. L. Cosset-Lion, France) were grown in DMEM supplemented with 10% FCS (Biological Industries, Israel), 2 mM L-glutamine, 100 units/ml penicillin, 0.1 mg/ml streptomycin, 6 μg/ml blasticidin (Sigma, USA) and 6 μg/ml phleomicin (Sigma, USA).
The packaging cell lines PA317, 293E3 (obtained from Dr. J. Exelrod, Haddasa, Jerusalem) were grown in DMEM supplemented with 10% FCS, 2 mM L-glutamine, 100 units/ml penicillin and 0.1 mg/ml streptomycin. Cells were routinely tested for sterility, endotoxins, and mycoplasma contamination.
Example 10
Infection of EC and Vascular SMC with Recombinant Adenoviral Vectors
Cells were seeded at 70% confluence on fibronectin (Sigma, USA) pre-coated plates (4.5 μg/ml) 20 hours prior to infection and grown in complete medium (M20). On the day of infection, the culture medium was replaced with fresh serum-free M199 medium and recombinant virus was added at a multiplicity of infection (MOI) of 3000 (i.e., 3000 viral particles/cell). The cells were incubated for 90 minutes with gentle tilting every 20 minutes after which the virus-containing medium was replaced with complete medium (M20). The infection rate was monitored by visualization of GFP expression using a fluorescent inverted microscope (TE200 Nikon, Japan) equipped with a fluorescent GFP filter (GFP-LP, Nikon).
Example 11
Transduction of EC and SMC with Recombinant Retroviral Vectors
EC or SMC (passage 4-9) were seeded at 105 cells/35-mm well in plates coated with 4.5 μg/ml fibronectin and grown in complete medium for 24 hours. The cells were pre-conditioned one hour prior to transduction by replacement of the culture medium with serum-free M199 medium containing 0.1 mg/ml DEAE-dextran (Sigma, USA). Following preconditioning, the cells were washed three times with phosphate-buffered saline (PBS). Transduction was performed by a 4 h incubation of the cells with supernatant freshly collected and filtered (0.45 μl) from virus-producing packaging cells. At the end of the incubation the virus-containing medium was replaced with M20 medium.
Example 12
Seeding of PTFE Grafts with EC
A PTFE graft (Gore co. USA) was aseptically cut to the required length, washed three times in PBS (30 min at room temperature) and incubated in fibronectin (20 μg/ml in PBS overnight, 37° C.). Two connectors (Teflon or stainless steel) were inserted, one into each side of the graft. The connectors were secured by silicon strings tied in three double knots. Each graft end is corked with a rubber cap.
Cells were trypsinized and centrifuged at 1200 rpm, for 5 min. The pellet was re-suspended in growth medium at a cell density equivalent to 400,000 cells/cm2 of graft. HEPES buffer 10 mM pH 7.3 was added to the cell suspension.
Grafts were aseptically mounted on the central axis of the seeding tube and filled with the cell suspension using a Pasteur pipette. The grafts were examined for leaks and the central axis was then inserted into the surrounding tube, which was filled with growth medium containing HEPES buffer 10 mM. The tube was sealed and placed in an incubator (5% CO2, 37° C.) where it was rotated at 1⁄6 rpm for 2 hr. Then, the rubber corks were aseptically removed and rotation continued for an additional 2 hours. The grafts were transferred from the seeding device to culture plates containing growth medium supplemented with bFGF 2 ng/ml and incubated (5% CO2, 37° C.) for 48 hours. The efficiency of graft seeding was evaluated by fluorescence microscopy detection of GFP production by seeded cells or by histochemical hematoxylin-eosin staining.
Example 13
Endothelial Cell Quantitative Morphometry
At the end of the incubation period, the grafts were fixed in 4% paraformaldehide for 1 hour. Following fixation, the grafts were cut longitudinally, examined under fluorescent microscope, photographed and the images processed by an image analysis system (Image pro Plus, Media cybernetics USA). Morphometric evaluation of endothelial seeded grafts was performed by computerized image analysis to evaluate surface covering of the graft by the endothelial cells. The image analysis system consists of a digital video camera (DXM1200 Nikon, Japan) installed on a fluorescent inverted microscope (TE200 Nikon, Japan). The data is digitized and transferred to an image analysis system (Image Pro Plus 4 image analysis software). At least ten fields were selected by random movement of the graft under the microscope (at ×100 magnification) of the graft surface were analyzed. The analysis included determination of the ratio of endothelial cell coverage area to whole field area (Per-area) as using the image analysis software. Each field was divided to 12 equal quadrants and the same ratio was determined for each quadrant. In addition, a subjective assessment was performed by scoring the observed homogeneity (scale of 1-3) and density (scale of 1-5) of coverage.
Example 14
Detection of Transgene Expression in Genetically Modified Vascular Cells
Total RNA was isolated from ECs and SMCs 48 hours after transformation with UP50-encoding adenoviral and retroviral vectors, using a PURESCRIPT RNA isolation kit (Gentra systems, USA). RNA concentration was calculated from the absorbance at 260 nm.
For cDNA synthesis, 1 μg of RNA was mixed with 500 ng random hexamers, the mixture was heated at 70° C. for 10 minutes and then cooled on ice. A mixture of 0.4 mM dNTPs, 5 units AMV-reverse transcriptase (RT) (Promega), 5 mM DTT, 32 units RNAse-out (Gibco BRL) and RT buffer (Promega) was added to the RNA. The reaction mixture was then incubated for 2 hours at 38° C. followed by 15 minutes at 95° C. PCR was performed in a volume of 50 μl with 7 μl of reverse transcriptase (RT) reaction mix, 20 pmole of sense primer:










(SEQ ID NO:2)











5′-GAAGATCTTGACATGCCAGGAATAAAAAGGATACTC-3′,







20 pmol of anti-sense primer:









(SEQ ID NO:3)









5′-



GAAGATCTTCAGAATGGGTACTGCGACACATATATCCGCAGTCG-3′,






240 mmol dNTPs, 1 U Ex-Taq DNA polymerase (Takara, Japan) and reaction buffer (Takara). The PCR cycling protocol was: 94° C. for 2 minutes followed by 10 cycles of 94° C./30 sec→50° C./30 seconds→72° C./60 sec. This was followed by 21 cycles of: 94° C./30 seconds→60° C./30 seconds→72° C./60 seconds +5 seconds/cycle→72° C./10 min. RT-PCR products were analyzed by electrophoresis on 1% agarose gel.

Example 15
Western Immunoblot Analysis of UP50 and VEGF Protein Expression
Expression of UP50 or VEGF protein by adenovirally or retrovirally altered ECs and SMCs was detected by Western blot analysis of altered cell-conditioned medium. Culture medium was replaced with serum-free medium 24 hours post-alteration and the cells were cultured for an additional 24 hours. Samples of the altered-cell conditioned medium (CM) (30 μl) were separated by electrophoresis in 10% SDS polyacrylamide gel under reducing conditions. Separated protein was electroblotted onto a nitrocellulose membrane (Schleicher & Schuell). The blots were blocked with incubation blocking solution (TBS containing 0.1% skim milk and 0.3% Tween-20 (TBST)) for 1 hour at room temperature with gentle agitation. Afterwards, the blots were incubated with primary antibody diluted in blocking solution for 2 hours at room temperature. Affinity purified polyclonal rabbit anti-UP50 antibody (#9855, custom made, Sigma, Israel) (1:5000) was used for UP50 detection, and polyclonal rabbit anti-VEGF165 antibody (#SC 152 Santa-Cruz, USA) (1:700) was used for VEGF detection.
Following incubation with primary antibody, the blots were washed three times with TBST and incubated with anti-rabbit peroxidase-conjugate secondary antibody (Sigma, USA) diluted with TBST for 1 hour at room temperature. After three washes with TBST, specific protein was visualized by development of blots with ECL reagents (Sigma, USA) and exposure to X-ray film.
Example 16
Immunohistochemical Analysis of UP50 Expression
Adenovirally-infected EC and SMC were seeded on chamber slides (Lab-Tek, USA) pre-coated with fibronectin (4.5 μg/ml), and cultured for 24 hours. Cells were fixed 48 hours following adenoviral infection by incubation at room temperature for 20 minutes in 4% paraformaldehyde followed by two washes with PBS. The cells were denatured by heating the slides with 1 mM EDTA, pH 8.0, in a microwave oven for 5 minutes. The samples were blocked using blocking solution supplied with the Histostain—Plus kit (Zymed, USA) according to manufacturer's instructions. After blocking, the cells were incubated with affinity-purified anti-UP50 (1:50) for 1 hour at room temperature. The samples were washed 3 times with PBS-T (PBS containing 0.3% Tween-20) and incubated for one hour with rhodamine-conjugated goat anti-rabbit IgG antibody (#SC 2091, Santa Cruz, USA) diluted 1:400 in PBS-T. The cells were washed 3 times with PBS-T and covered with mounting medium (H-1000, Vector laboratories, USA). Samples were then visualized by fluorescence scanning confocal (MRC-1024, BioRad) microscopic detection of GFP and rhodamine in the cells.
For immunohistochemical analysis of UP50 in the ECM, ECs infected with recombinant adenoviral vector encoding the UP50 gene were induced to generate ECM by addition of dextran to the growth medium. After denudation of the EC layer using 20 mM NH4OH solution, the ECM was subjected to rhodamine-based immunostaining using anti-UP50 antibody.
Example 17
UP50 and VEGF Protein Expression by Co-Cultures of Adenovirally-Infected ECs and SMCs
Adenovirally-infected ECs or mixes of adenovirally-infected ECs and SMCs were cultured for 24 hours following infection in serum-supplemented medium followed by an additional 24 hours in serum-free medium. Supernatant proteins were separated electrophoretically on 10% SDS polyacrylamide gel and the separated proteins were electroblotted onto nitrocellulose membranes. Blots were incubated with anti-VEGF or anti-UP50 antibodies. Following exposure to peroxidase-conjugated secondary antibody, the blots were developed with ECL reagents and exposed to X-ray film.
Example 18
Western Blot Analysis of VEGF and UP50 Protein Expression by Co-Cultures of Retrovirally-Infected EC and SMC
ECs infected with different retroviruses or mixtures of infected ECs and SMCs were cultured in serum-supplemented medium for 24 hours following infection after which the cells were cultured in serum-free medium for an additional 24 hours. Samples of the growth medium (30 μl) were separated on a 10% SDS polyacrylamide gel and electroblotted onto a nitrocellulose membrane. Blots were incubated with either anti-VEGF or anti-UP50 antibodies. Following exposure to a peroxidase-conjugated secondary antibody the blots were developed with ECL reagents and exposed to X-ray film.
Example 19
Functional Analysis of Over-Expressed Recombinant VEGF and UP50 in Vascular Cells
Endothelial cells (passages 5-11) were seeded at 30% confluence (104 cells/well) in 24-well plates pre-coated with 4.5 μg/ml fibronectin 24 hours prior to adenoviral infection. The cells were infected with Ad.UP50-GFP, Ad.GFP or Ad.VEGF165-GFP. Following 90 minutes of exposure to adenoviral vectors at 37° C., serum-containing medium was added and 16-18 hours later the medium was substituted with M199 medium containing 2% human serum and 2 ng/ml bFGF. Assays were performed in triplicate and proliferation was measured by XTT colorimetric assay on day 7 following infection.
Example 20
Adhesion of EC to ECM Generated by UP50-Over-Expressing EC
ECs were seeded on fibronectin pre-coated 48 well plates (104 cells/well) 24 hours before infection. The cells were infected with Ad.UP50-GFP or Ad.GFP, as previously described. After infection the cells were grown in M20 medium supplemented with 4% dextran 42000 (Sigma USA) for 7 days. Following this infection period, the culture medium was aspirated from the infected cells and the matrix producing cells were denuded by contact with 20 mM NH4OH for approximately 5 minutes. After cell lysis ECM-coated wells were washed three times with PBS and stored in PBS at 4° C.
ECs were detached by incubation in 10 mM EDTA solution, the matrix was washed with M199 medium and incubated in culture medium (CM) from Ad.UP50-GFP infected, Ad.GFP infected, or non-infected ECs for 15 minutes. Following incubation, ECs were seeded on the ECM coated wells (2×104 cell per well). The cells seeded on ECM generated by Ad.UP50-GFP infected EC were incubated in CM from Ad.UP50-GFP infected cells. Cells seeded on ECM generated by Ad.GFP infected cells were incubated with CM collected from Ad.GFP infected cells and the control group of cells seeded on ECM generated by non-infected cells were incubated with non-infected cell-conditioned medium. The cells were incubated for an additional 30 minutes to allow cell adhesion and were then washed with PBS. Quantitation of the remaining adherent cells was performed via XTT colorimetric assay.
Similar experiments were performed utilizing ECM generated from retroUP50-GFP transduced EC.
Example 21
Effect of UP50 on EC Growth in Three-Dimensional Collagen Culture—in Vitro Angiogenesis
In vitro angiogenesis in collagen gels was quantitated using adenovirus infected EC spheroids. The generation of EC spheroids was performed as previously described (33). Endothelial cells were suspended in culture medium containing 0.25% (w/v) carboxymethylcellulose and cultured in non-tissue culture-treated round-bottomed 96-well plates (Nunc, Denmark) for 24 h at 37° C., 5% CO2, during which time the suspended cells formed a single spheroid per well of defined size and cell number ( ̃750 cells/spheroid). The spheroids were then embedded in collagen gels. Collagen stock solution was prepared prior to use by mixing 8 volumes of acidic rat tail collagen extract (equilibrated to 2 mg/ml, 4° C.), 1 volume 10×M199 (Gibco BRL, USA). The pH was adjusted to 7.4 by addition of 0.34N NaOH. To prevent sedimentation of spheroids before polymerization of the collagen gel, 1 volume of collagen stock solution was mixed with 1 volume of room temperature M199 medium containing 40% human serum and 0.5% (w/v) carboxymethylcellulose. Spheroid-containing gels (20-30 spheroids/gel) were rapidly transferred into pre-warmed 24-well plates and allowed to polymerize. The gels were incubated at 37° C. with 5% CO2 and proliferation of ECs in the gels was documented by photomicrography using a digital video camera (DXM1200 Nikon, Japan). The sprouting of at least 40 spheroids from each group was analyzed.
Example 22
Effect of UP50 Over-Expression on Adhesion of In Vitro-Cultured EC Following Trypsinization
Endothelial cells were seeded at 70-80% confluence (6-7.5×104 cells/well) in 12 well plates, 24 hours prior to adenoviral infection. The cells were infected (3×103 pfu per cell), as previously described, with Ad.UP50-GFP or Ad.GFP. Non-infected cells served as control. After infection the cells were grown in M20 medium for 4 days. The cells were washed with PBS and adhesion assay was performed by trypsinization of the cells using 0.0025% trypsin containing 0.001% EDTA. After 3 minutes incubation at room temperature, trypsin was neutralized by addition of complete medium (M20). The cells were washed three times with PBS and then M20 (300 μl) medium was added to the cells. Quantitation of the remaining adherent cells, as percent of control non-trypsinized cells, was determined via colorimetric XTT assay.
Example 23
Effect of UP50 Over-Expression on Cell Adhesion to ECM
ECs were detached by 10 mM EDTA solution, washed with M199 medium containing 0.1% BSA (Sigma, USA), 10 mM HEPES, and incubated with the culture medium (CM) from Ad.UP50-GFP or Ad.GFP infected or from non-infected EC for 15 minutes. After incubation, the cells were seeded in the ECM coated wells (2×104 cell per well). The cells seeded on ECM generated by Ad.UP50-GFP infected EC, were incubated in CM from Ad.UP50-GFP infected cells. Cells seeded on ECM generated by Ad.GFP infected cells were incubated with CM collected from Ad.GFP infected cells and the control group of cells seeded on ECM generated by non-infected cells were incubated with non-infected cell-conditioned medium. The cells were incubated for additional 30 minutes to allow cell adhesion and were then washed with PBS. Quantitation of the remaining adherent cells was performed by colorimetric XTT assay.
Exposure to secreted UP50 and to ECM-bound UP50 resulted in >30% increased adhesion of EC in comparison to the control group (FIG. 7).
Example 24
Effect of UP50 Over-Expression on Retention of EC Under Continuous Shear Stress
Human saphenous vein ECs (passage 8-11) were seeded (105 cells per 35 mm well) and grown in M20 up to 60-80% confluence. The cells were infected with Ad.UP50-GFP or Ad.GFP (MOI 3000) as described previously. Non-infected cells served as the control. The cells were grown for 30 hours to ensure transgene expression before exposure to shear stress. Prior to the experiment, two wells from each group (Ad.UP50-GFP, Ad.GFP, Control) were harvested using trypsin-EDTA and cells were counted. To cover the cells completely during rocking, 5 ml of M20 medium were added to each well. The plates were placed on a rocking table inside a CO2 incubator and incubated for 20-24 hours with intense rocking (approximately 140 cycles/minute). Following rocking, the cells were washed 5 times with PBS. Cells were harvested using trypsin-EDTA and were counted by hemacytometer. The assay was also performed with cells transduced using retroviral vectors respectively.
The results presented in FIG. 8 demonstrate that UP50 overexpression by retroviral-transduction results in increased adherence of EC exposed to continuous shear stress. Ninety-eight percent of the UP50-expressing cells 98% remained adhered to the plate in contrast to the GFP-expressing (72%±1%) and non-transduced ECs (69±2%). Similar results were obtained with EC adenovirally-infected to express UP50.
Example 25
Retention of Human EC on ePTFE Grafts Under Pulsatile Flow
Grafts were seeded as described above.
Each graft is subjected to a steady pressure of 120/80 mmHg and flow of 300 ml/minute for 2 hours. The grafts are then removed from the device and cell stability and adhesion to the graft are evaluated as described.
Seeded grafts were exposed to laminar flow conditions. The flow was adjusted to reach physiological blood pressure of 120/75 (mean of 90 mm Hg) for two hours at flow rate of 300 ml/min using 60 pump strokes per minute. The grafts were evaluated by microscopy and morphometric analysis. After exposure to flow conditions, evaluation of cell density and homogeneity on the graft inner surface was performed. The grafts were fixed in freshly prepared 4% paraformaldehyde in PBS at room temperature for 1 hour. The grafts were cut open, flattened between two glass slides, viewed under a fluorescent microscope and analyzed using morphometric analysis as previously described. Complementarily, the grafts were stained with hematoxylin-eosin and analysed as previously mentioned.
Example 26
Retention of Genetically Modified EC Seeded onto Artificial Vascular Grafts Under In Vivo Flow Conditions; Short-Term Implantation of ePTFE Grafts Coated with Genetically Modified Sheep EC in Sheep Arteries
Small caliber ePTFE grafts (6 mm) were seeded with sheep EC that were genetically modified by retroviral transduction to over-express GFP, UP50-GFP or VEGF165-GFP 36 hours prior to implantation.
Fasting (12 hours) adult sheep were pre-medicated with 10 mg diazepam injected intramuscularly and 500-600 mg of intravenously administered sodium pentobarbital. They were then intubated and anesthesia was maintained with inhaled 1%-2% isoflurane. Aspirin (600 mg) was administered preoperatively. The monitoring system during the experiment included blood pressure measurement, pulse oxymetry, and ECG. Heparin (300 U/kg) was injected intravenously for systemic anticoagulation following exposure and preparation of arteries for graft implantation. Blood samples were taken during the procedure every 30 minutes to assess the efficacy of heparinization by measuring partial thromboplastin time (PTT).
The seeded grafts were then implanted bilaterally end to side in sheep carotid and femoral arteries by an expert cardiac surgeon. On one side of the femoral artery the implanted graft was seeded with retroGFP transduced ECs and on the other side the implanted graft was seeded with retroUP50-GFP transduced ECs (femoral arteries). In the femoral artery on one side the implanted graft was seeded with retroVEGF165-GFP transduced EC and on the other side the implanted graft was seeded with retroUP50-GFP transduced EC. Patency of the implanted grafts was assessed 30 minutes following exposure of the implanted grafts to blood flow and prior to graft harvesting by direct palpation, flow measurements using a Doppler flow meter (Transonic Animal Research Flowmeter, NY, USA) and by performing selective angiography.
Flow rates through the femoral grafts were similar on both sides ( ̃50 ml/min, 38% of femoral blood flow). The flow through the carotid grafts, which was higher at the beginning of the experiment, was bilaterally diminished at the end of two hours due to local thrombosis at the anastomosis site (secondary to surgical complications). The femoral and carotid grafts were harvested two hours following implantation. Both grafts were then harvested and cellular retention on the interior surfaces of the grafts was analyzed by fluorescence microscopy.
Following graft removal, sheep were sacrificed by intravenous potassium chloride administration. All experiments were performed according to animal care and experimentation laws of the Technion ITT, Haifa, Israel.
Example 27
Recovery of Genetically Modified EC Over-Expressing UP50 and VEGF Seeded onto Artificial Vascular Grafts Subjected to Dialysis Needle Puncture
ePTFE grafts were seeded with genetically modified ECs over-expressing UP50 and VEGF and ECs over-expressing GFP. Twenty-four hours after seeding, the grafts were mounted on the outer surface with Matrigel (2 mg/ml) and then were punctured with a 14 G needle used in dialysis. The grafts were incubated for 24 hours and then visualized using fluorescent microscopy. Grafts seeded with UP50 and VEGF showed enhanced proliferation and bridging across punctures of endothelial cells closing the gap in the needle track. Grafts seeded with ECs over-expressing GFP did not exhibit this effect. Closure of the gap produced by the needle provide a biocompatible surface to the graft and hence reduce local thrombosis.
Example 28
Fibulin-5 Alone Inhibits Both Smooth Muscle Cell and Endothelial Cell Proliferation/Migration
In vitro studies demonstrate that fibulin-5 alone partially inhibits both smooth muscle cell and endothelial cell proliferation/migration. Upon addition of a growth factor such as VEGF, endothelial cells proliferate rapidly, whereas smooth muscle cells remain partially inhibited. Thus, in the presence of a growth factor, fibulin-5 has a differential effect on endothelial cell proliferation/migration over smooth muscle cell proliferation/migration. FIGS. 11A and 11B summarize the results.
Effect of UP50 on Endothelial Cell Proliferation and Reversal of the Effect with Addition of VEGF:
Primary endothelial cells were isolated from human saphenous vein. Cells were retrovirally transduced using a LXSN based viral vector encoding UP-50. Additional study groups included—naive EC, VEGF165 (a specific mitogen of endothelial cells) expressing EC, GFP expressing EC, UP-50 and VEGF165 expressing EC, UP-50 expressing EC supplemented with exogenous VEGF165. For proliferation assays 20,000 cell/well in a 24 wells plate pre-coated with gelatin were seeded. Cells were counted using a coulter counter at day 0, day 2, day 4, and day 5 (FIG. 11A).
Effect of UP50 on Smooth Muscle Cell Proliferation and No Reversal of the Effect with Addition of bFGF:
Primary smooth muscle cells were isolated from human saphenous vein. Cells were retrovirally transduced using a LXSN based viral vector encoding UP-50. For control, cells transduced with GFP were used. For the assay, 20,000 cell/well in a 24 wells plate pre-coated with gelatin were seeded. Cells were counted using a coulter counter at baseline, day 2, day 6, and day 9. The cells were grown with bFGF (a strong mitogen of smooth muscle cells). FIG. 11B.
Example 29
Co-Expression of Fibulin-5 and VEGF by Endothelial Cells Seeded onto Synthetic Grafts Improves Long-Term Patency in a Sheep Model, and Reduces Neointimal Formation after Vascular Injury in a Rat Carotid Artery Injury Model
The effect of fibulin-5 and VEGF on endothelial and smooth muscle cell proliferation in vitro, and on neointimal formation in the rat carotid artery injury model to explore the mechanisms by which these two genes can improve graft patency were studied. Long-term patency, at 3 and 6 months, of end-to-side implanted, 15-18 cm long, 6 mm ePTFE grafts, seeded with autologous EC over-expressing fibulin-5 and VEGF, was also studied.
Autologous EC from short venous segments were isolated, and the endothelial cells (EC) were transduced with retroviral vectors expressing fibulin-5 and VEGF165. The cells were then seeded on 6 mm ePTFE grafts (treated grafts). The grafts were implanted in the sheep carotid artery model (15-18 cm graft) (described below). For control, bare ePTFE grafts and grafts seeded with naive EC were used. At 3 months, all treated grafts were patent using selective angiography (6/6), only 33% of the control groups were patent (2/6 in the bare grafts and 2/6 in the grafts seeded with naive EC). At 6 months 5 out of 6 of the treated grafts were patent compare to 1 out of 6 in the bare grafts and 2 out of 6 in the grafts seeded with naive EC.
To demonstrate the mechanism of these findings, it was determined whether fibulin-5 inhibits smooth muscle proliferation and neointima formation in rat carotid artery model of vascular injury. Fibulin-5 was transferred to the cells of the arterial wall of rat carotid artery after inducing vascular injury. Neointimal formation 14 days after injury was tested. At 14 days neointimal formation was completely suppressed, as well as SMC proliferation, in rats treated with adenoviral vectors expressing fibulin-5. Fibulin-5 expression was verified by immunohistochemistry.
Seeding PTFE vascular grafts with EC over-expressing Fibulin-5 and VEGF165 improved long term patency of small caliber ePTFE grafts. This phenomenon is due to inhibition of neointimal formation by fibulin-5. The methods and results are further described below.
Methods
EC Isolation and Culture
For the in vitro experiments, autologous endothelial cells were isolated by collagenase digestion of short segments of remnants from human veins used for aorto-coronary bypass surgery. Use of vein remnants was approved by the IRB of Carmel Medical Center. Sheep endothelial cells were isolated from the hind limb lateral saphenous vein using similar methods. Animal protocols were reviewed and approved by the “Animal handling and care committee”, Technion, Haifa, Israel.
EC were cultured on gelatin-coated dishes in M-199 medium (Biological Industries, Israel) supplemented with 20% fetal calf serum (Hyclone, USA), 100 units/ml penicillin (Biological Industries, Israel), 0.1 mg/ml streptomycin (Biological Industries, Israel), 2.5 μg/ml amphotericin B (Biological Industries, Israel), 2 mM L-glutamate (Biological Industries, Israel), 100 μg/mL heparin (Sigma, USA), and 2 ng/mL basic fibroblast growth factor (kind gift of Prof. G. Neufeld). Human EC were identified by their typical cobblestone morphology and by immunohistochemistry (IHC) using anti-CD31 antibodies (Santa Cruz, USA). Sheep EC were identified by their typical cobblestone morphology and by IHC using anti-eNOS antibodies (Santa Cruz, USA).
Retroviral Vector Production
These vectors were used for the in vitro experiments and for the sheep experiments.
Generation of Pseudo-Typed Recombinant Retroviral Vectors Encoding Fibulin-5
The recombinant retroviral vector encoding fibulin-5 was constructed by inserting the 1361 bp fibulin-5 cDNA fragment (Genebank accession: #NM 006329) into the BamHI site of pLXSN plasmid (# K1060-B Clontech, USA) under the control of Mo-MULV 5′-long terminal repeat (LTR) and the SV40 poly A signal. For retroviral vectors production, 10 μg of pLXSN-fibulin-5 plasmids were transfected into 293FLYA packaging cells using Lipofectamine (Gibco BRL, USA). After 48 hours the supernatant from confluent cultures of viral producer cells were collected, filtered (0.45 μm) and added to 293FLY10A or 293 FLYGALV packaging cells. The transduced cells were grown under G418 selection (400 μg/ml) and individual colonies were collected and screened for fibulin-5 expression by western blot analysis of conditioned medium samples. Viral titers of each colony were determined by transduction of TE671 cells and the titers ranged around 106 pfu/ml.
Generation of Pseudo-Typed Retroviral Vector Encoding VEGF165 and GFP
The VEGF165 600 bp BamH1 cDNA fragment was cut from pcDNA-VEGF165 (a kind gift of Dr. J. Abraham, Scions Nova, Mountain View Calif.). A recombinant retroviral vector encoding human VEGF165 gene was constructed by inserting the 600 bp VEGF165 BamHI fragment into the BamHI site of a pLXSN plasmid.
The recombinant retroviral vector pLXSN-IRES-EGFP was cloned into a pLXSN plasmid in a similar process as described above. A 1400 bp IRES-EGFP EcoRI-HpaI fragment was excised from pIRES2-EGFP (Clontech, #6029-1) and inserted into EcoRI-HpaI-digested pLXSN for construction of the pLXSN-IRES-EGFP plasmid. A similar way to the fibulin-5 viral stock production described above, was used for retroviral vectors stock production of GFP, and VEGF165.
Adenoviral Vector Preparation
These vectors were used for the rat carotid model of vascular injury.
Generation of Recombinant Bicistronic Adenoviral Vector Encoding the VEGF165-GFP and GFP Genes
The recombinant adenoviral vector expressing the human VEGF165 and the GFP genes were constructed in several steps, consisting of routine prokaryotic cloning into a shuttle vector and a homologous recombination procedure in the 293-cell line. A 600 bp BamHI fragment containing the human VEGF165 cDNA, including the signal sequence for secretion (gift of Dr. J. Abraham, Scios Nova, Mountain View Calif.) was inserted into BglII site in pQBI-CMV5-GFP shuttle vector (QBI, Canada). The expression plasmid pQBI-CMV5-GFP also contained the left arm (16%) of the Ad5 genome with a deletion in the E1 region into which hCMV was inserted. The shuttle vector pQBI-VEGF-IRES-EGFP was co-transfected with the pJM17 plasmid into 293 cells, which constitutively expressed the E1 gene. The pJM17 plasmid contained the adenovirus genome, excluding the E3 region, and including an insert (pBRX) in the E1 region of the virus. Homologous recombination between the expression plasmid and pJM17 following transfection replaced the E1 region and pBRX insert with the expression cassette from pCA3 plasmid. Plaque formation occurred between 2 to 4 weeks after co-transfection. Individual plaques were isolated and the viral extracts were amplified by infection of 293 cells. The titer of each viral stock was determined by plaque assay in 293 cells and the titers ranged-1010-1011 pfu/ml. Transgene expression was confirmed by western analysis of infected cells conditioned medium. GFP viral stock production was performed in a similar way.
Generation of Recombinant Adenoviral Vectors Encoding Fibulin-5-GFP Genes
The recombinant adenoviral vector expressing the human fibulin-5 and GFP genes was constructed by a modified AdEasy protocol (Vogelstein B. PNAS 1998). A 1361 bp BglII fragment of fibulin-5 cDNA was inserted separately into the BglII site in the pAdTrack-CMV shuttle vector under the control of the CMV promoter. The shuttle vector contains GFP driven by additional CMV promoter downstream to the transgene. The shuttle vectors were linearized by PmeI digestion and purified by Qiaquick gel extraction kit. The shuttle vector and pAdEasy-1 were co-transformed by electroporation into competent BJ5183 cells. Positive clones containing the recombinant adenoviral vectors were selected according to PCR and restriction map analysis. The recombinant adenoviral plasmids were linearized by PacI digestion, purified and transfected into 293 cells using Lipofectamine 2000 (Gibco BRL, USA). Seven days after transfection cytopathic effect occurred and 100% of the cells expressed GFP. The cells were harvested and viral extracts were further amplified in 293 cells. The titer of each viral stock was determined by serial dilution assay in 293 cells and the titers ranged 1010-10 μl pfu/ml. The expression of the transgene was confirmed by western analysis of the infected cells conditioned media.
Endothelial Cell Transduction
4×105 EC were seeded on 60 mm fibronectin-coated plates (Biological Industries, Israel) 24 h prior to transduction. Cells were incubated with DEAE-dextran (Sigma, USA) for 1 h and then exposed to the retroviral vector for 4 hrs at 37° C. The viral vector stock was diluted in M199 medium to achieve a ratio of 5-8 viral particles per cell.
Dual transduction of EC was achieved by a two-step process: (1) initial transduction with the retroviral vector encoding fibulin-5, followed by selection with G418 (0.5 mg/ml, Calbiochem) until >80% of the EC population was expressing fibulin-5; (2) transduction of this fibulin-5 expressing EC population with VEGF165 encoding retroviral vector. EC were cultured in the presence of G418 and trans-genes expression was monitored by Western blot, ELISA and by IHC for the relevant trans-gene. The resulting dually transduced EC population comprised cells expressing fibulin-5 (>80%) and a sub-population of these cells that additionally expressed VEGF165 (15-30% of cells).
Endothelial Cell Seeding of ePTFE Grafts
ePTFE grafts (6 mm, Gore co., USA) were pre-incubated with fibronectin (45 μg/ml, Sigma USA) prior to seeding. EC seeding was performed using a rotation device to allow homogenous, gravity independent graft seeding. The vascular grafts were filled with an EC suspension, which contained 4.5×105 cells/cm2 graft surface area in EC medium. The grafts seeding was performed in a 5% CO2, 37° C. environment. This procedure yielded PTFE grafts coated with a confluent EC monolayer.
At the end of seeding procedure, 48 hr after of seeding, the graft edge was cut, fixed in 2.5% Gluteraldehyde for seeding quality evaluation. Nucleus staining using DAPI containing VECTA-SHIELD mounting solution (VECTOR, USA) was performed. Grafts sections were tested for coverage by seeded cells in 5 random fields at a magnification of ×200 using inverted fluorescent microscope.
Western Blot
A Western blot was performed to verify transgene expression in the transduced cells. Conditioned medium from the transduced cells was collected and was separated by 10% SDS-PAGE. The proteins were electro-transferred to nitrocellulose paper which was blocked with 10% low-fat milk and incubated at a 1:500 dilution with rabbit polyclonal antibodies directed against a synthetic peptide derived from human fibulin-5 sequence (custom made, Sigma Israel). Horseradish peroxidase labeled goat anti-rabbit antibodies (1:7000) were used to visualize bound antibody using the ECL detection system. VEGF165 detection was performed using at 1:500 dilution of goat anti human-VEGF antibody (Santa-Cruz, USA). Horseradish peroxidase labeled donkey anti-goat antibodies (1:10000) were used to visualize bound antibody using the ECL detection system.
Immunohistochemistry for Transgene Expression
Tissue culture: Immunohistochemistry was used for identification of transgene expression in the transduced cells. Cells were seeded on 4 well tissue culture slides (Nunc) for 24 h and then fixed with 4% PFA (paraformaldehyde). For antigen retrieval, slides were microwave-treated in 1 mM EDTA buffer (pH 8.0) for 15 min.
For ecNOS staining slides were incubated with CAS block (Zymed, USA) and then immunostained with rabbit anti human NOS3 antibodies (1:100, Santa Cruz, USA) in CAS block. Secondary antibody was Envision-rabbit peroxidase conjugate (1:1, Dako, Denmark). Bound antibody detection was performed with AEC substrate (Dako, Denmark).
For αSMC-actin staining slides were incubated with CAS block (Zymed, USA) and then immunostained with mouse anti human SMC actin antibodies (1:50, DAKO, Denmark). Secondary biotin-conjugated goat anti mouse (1:1000, Chemicon, USA). Bound antibody detection was performed with Horseradish Peroxidase conjugated streptavidin (Chemicon, USA).
For VEGF165 staining slides were incubated with CAS block (Zymed, USA) and then immunostained with goat anti human VEGF antibodies (1:50, R&D USA). Secondary biotin-conjugated donkey anti goat (1:2000, Chemicon, USA). Bound antibody detection was performed with Horseradish Peroxidase conjugated streptavidin (Chemicon, USA).
For Fibulin-5 staining, slides were incubated with 70% CAS block (Zymed, USA) and 30% goat serum (Zymed, USA) and then immunostained with rabbit anti human antibody (1:200, custom made, Sigma, Israel) in blocking solution supplemented with 2% tween20. Secondary antibody was Envision-rabbit peroxidase conjugate (1:2, Dako, Denmark). Bound antibody detection was performed with AEC substrate (Dako, Denmark).
Grafts: grafts were fixed with formalin 4% for 24 h and then embedded in paraffin. De-paraffinized serial sections were microwave-treated in 1 mM EDTA buffer (pH 8.0) for 20 min.
For vWF and αSMC actin staining, graft sections were incubated with rabbit anti-human vWF antibody (1:100, Dako, Denmark) and mouse anti human αSMC actin (1:50, Dako, Denmark). For the vWF antibody the secondary antibody was FITC goat anti rabbit conjugate (1:50, Zymed, USA). For the αSMC antibody the secondary antibody was Rhodamin Goat anti mouse (1:50, Jackson ImmunoResearch, USA).
Tissues: tissues were fixed with formalin 4% for 48 h and then embedded in paraffin.
For fibulin-5 staining: De-paraffinized serial sections were incubated in endogenous peroxidase inactivation solution. Slides were then microwave-treated in 1 mM EDTA buffer (pH 8.0) for 20 min. slides were then incubated with 70% CAS block and 30% goat serum (Zymed, USA) and then immunostained with rabbit anti human antibody (1:50, custom made, Sigma, Israel) in blocking solution supplemented with 2% tween20. Secondary antibody was Envision-rabbit peroxidase conjugate (1:2, Dako, Denmark). Bound antibody detection was performed with AEC substrate (Dako, Denmark).
For Ki-67 staining, slides were incubated in endogenous peroxidase inactivation solution. Slides were then microwave-treated in 10 mM sodium citrate buffer (pH 6.0). Slides were then incubated with 20% rabbit serum supplemented with 0.3% triton. Tissues were immunostained with rat anti human Ki-67 antibody (1:50, Serotec) in 3% supplemented with 0.1% triton. Secondary antibody was rabbit anti rat(Vectrashield). Bound antibody detection was performed with ABC reagent (Vectrashield) and DAB substrate.
ELISA for Transgene Expression
Serum free condition medium of 5×105 cells was collected after 24-48 hr, centrifuge at 2000 rpm for 2 min for cell centrifugation and was frozen at −80° C. until used.
For VEGF165 commercial kit, human VEGF immunoassay kit, was used according to manufacturer instructions (R&D, USA).
For Fibulin-5 protein level was measured using an ELISA that was developed by our lab. Standards and samples were distributed into a non-antibody-coated 96-well plate. Rabbit anti-human fibulin-5 custom made specific antibody (1:1000, Sigma, Israel) is added and binds to fibulin-5 present in the well. Secondary antibody was Envision-rabbit peroxidase conjugate (1:50, Dako, Denmark). TMB/E solution (Chemicon, USA) was used as a substrate for color reaction. The reaction was stopped using a 0.25M HCl stop solution. The plate was read in ELISA reader at 450/540 nm wave length.
Biodistribution in the Rabbit and Sheep Toxicity Model
General Description
Bio-distribution of gene-modified endothelial cells used for MultiGeneGraft seeding was tested in the rabbit/sheep toxicity model. Biodistribution studies were conducted to test the distribution of MultiGeneGraft cells in case they detach from the graft surface. Rabbits were injected with therapeutic or high dose of dual-transduced endothelial cells formulated for MultiGeneGraft and sacrificed after 3 or 24 weeks.
Sheep were implanted with bare, naive EC-seeded, or MultiGeneGraft bypass conduits and were sacrificed after 24 weeks. Eight sheep, were analyzed for biodistribution of vector-specific sequences. Biodistribution was examined in major organs and tissues, including testes and ovaries using PCR, as follows:
DNA Sample Preparation
Duplicate samples of rabbit/sheep tissues were obtained from freshly sacrificed animals and stored at −80° C. until DNA isolation. Aliquots of 50-70 mg were prepared from tissue samples. In addition, leukocytes (WBC) samples were prepared, each from 5 ml fresh whole blood samples. RBC were lysed and WBC were collected after centrifugation, washed in saline and frozen at −70° C. until DNA isolation.
Extraction of genomic DNA from tissue and WBC was performed using the QIAmp DNeasy according to protocol. RNase A was added to samples to eliminate co-extraction of RNA.
In order to identify the presence of vector-specific sequences in sheep tissues, DNA samples were extracted from the following tissues: peripheral blood cells, eye, kidney, spleen, lung, testes/ovaries, liver, heart, bone marrow, skeletal muscle [mastication muscle—the muscle supplied by the MultiGeneGraft and the RT quadriceps—control muscle] and brain, as well as graft samples that were obtained from the distal, medial and proximal parts of the implanted graft.
The following rabbit tissues were tested: WBC, eye, liver, spleen, lung, bone marrow, testes/ovaries, kidney, heart, skeletal non-ischemic muscle [LT quadriceps], skeletal ischemic muscle [RT. quadriceps], skeletal ischemic muscle [RT gastrocnemious], and brain.
Preparation of PCR Reaction.
A nested PCR assay was performed for the detection of vector specific sequences from the SV40 promotor (SV40) region and a sequence from the Neomycin resistance gene (Neo) region found in the retroviral vector (pLXSN, gene bank accession #: M28248). The primers chosen led to the amplification of a single PCR amplification product of 140 bp.
Triplicate PCR was performed for each tissue, each containing  ̃1 μg tissue DNA. One of the triplicates was spiked with 50 copies of vector DNA.
PCR Assay Controls
Negative Reagent Control (NRC)— consists of PCR reaction mix without nucleic acids.
Sentinel Control (SC)— consists of the reaction mix without template nucleic acid. These tubes were left open at specific times, the first during tissue harvest (at post mortem) and the second during preparation of tissue samples.
Positive Specificity Control (PSC)— consists of DNA prepared from recombinant vector plasmid (pLXSN-VEGF). Positive specificity control included three concentrations of 500, 50 and 10 vector plasmid DNA copies.
Positive Tissue Control (PTC)— consists of three samples of 1 μg DNA extracted from vector free tissue. Each sample is spiked with 3 separate concentrations (500, 50, and 10 copies) of vector DNA.
PCR was performed according to protocol (MGVS, in file). A PCR result was considered positive when a specific single amplification band of 140 bp was observed and controls conformed as expected.
Assessment of DNA Integrity
The integrity of the DNA extracted in the tissue samples was assessed by an additional PCR specific for the housekeeping gene β-actin (with primers specific to rabbit β-actin and primers specific to sheep β-actin). Rabbit sample DNA (0.1 μg) yielding a single specific PCR amplification product of 106 bp was considered affirmative and PCR amplification product of 182 bp was considered affirmative for the sheep sample DNA.
Results of the Biodistribution Study in Rabbits
Representative results of one rabbit of the therapeutic dose—24 weeks sacrifice group are displayed in FIG. 12. PCR results of other rabbits tested to date are summarized in Table 2. The integrity of the DNA extracted from the tissue samples was assessed by a PCR reaction specific for the rabbit housekeeping gene β-actin. Seven rabbits were tested and no vector specific sequences were detected in any of the tissues examined (see Table 2).







TABLE 2







Summary of biodistribution results of rabbits tested to date.








Allocation group
Number of rabbits tested












Control
 3 weeks sacrifice
2



24 weeks sacrifice
1


Theraputic dose
 3 weeks sacrifice
1



24 weeks sacrifice
2


High dose
24 weeks sacrifice
1









A single positive PCR product of 140 bp was observed in all spiked tissues and positive controls. No amplification signals were observed for sentinel and negative controls. All tissues were found to be negative for vector sequences. The sensitivity of this PCR assay is 10 copies of vector DNA in 1 μg of DNA.
The integrity of the DNA extracted from all tissues was assessed by a PCR specific for the rabbit housekeeping gene β-actin. The presence of a 106 bp amplification product for all samples and no product for the negative control (FIG. 13) was indicative of DNA integrity.
Conclusion for the Rabbit Biodistribution Study
Biodistribution studies were conducted to test the distribution of MultiGeneGraft cells in case they detach from the graft surface. No evidence of vector-specific sequences was detected in any of the tissues tested of the rabbit model of direct intra-arterial injection of MultiGeneGraft cells.
Results of the Biodistribution Study in Sheep
PCR result summary of the sheep tested so far is presented in Table 3. It is important to note that one out of the three tested sheep implanted with MultiGeneGraft was found negative for the presence of vector-specific sequence in all tissues tested.
One sheep of the implanted with the MultiGeneGraft-24 weeks sacrifice group was similarly tested. PCR results of other sheep tested to date are summarized in Table 3.







TABLE 3







Biodistribution result summary of sheep, sacrificed 24 weeks after graft


implantation.









weeks sacrifice 24












Bare ePTFE graft .1



Number of animals tested
3 = n


Vector sequence detected in the
Not detected


implanted graft


Vector sequence detected in other
Not detected


tissues or organs


seeded graft-Naive EC .2


Number of animals tested
2 = n


Vector sequence detected in the
Not detected


implanted graft


Vector sequence detected in other
Not detected


tissues or organs


MultiGeneGraft .3


Number of animals tested
3 = n


Vector sequence detected in the
Positive signal in the proximal


implanted graft
2medial and distal graft segments in



animals 3out of


Vector sequence detected in other
Positive signal in the eye of one


tissues or organs
marrow of-animal and in the bone



another animal









Discussion and Conclusions for the Sheep Biodistribution Study
1. Vector Sequence Detected in the Implanted Graft at Autopsy, 24 Weeks after Implantation

    
    
        Vector-specific sequence was detected in MultiGeneGraft, but not in naive EC-seeded or bare ePTFE grafts, which supports the validity of our PCR-based biodistribution assay system.
        Vector-specific sequence was detected in MultiGeneGraft in 2 out of 3 tested sheep 24 weeks after implantation. Thus, gene-modified cells will continue to adhere to the graft surface for at least 24 weeks.
        Vector-specific sequence was detected in the proximal, medial and distal graft segments of 2 out of 3 MultiGeneGrafts tested. This implies a homogenous pattern of cell adherence and retention throughout the graft surface for at least 24 weeks.
        Grafts were evaluated for lumenal patency and EC coverage of the graft's lumenal surface using morphometric analysis of proximal, median and distal graft sections. Serial 6 μm thick slides were stained by Hematoxylin-Eosin. The staining revealed that the coverage of the graft's lumen was negative for biodistribution.
    
    


2. Vector Sequence Detected in Other Tissues or Organs, 24 Weeks after Implantation
As summarized in Table 3, the biodistribution of vector-specific sequence was tested in 3 sheep implanted with MultiGeneGraft, 24 weeks after implantation. Vector-specific sequence was detected in eye tissue of one sheep and in bone marrow of another sheep. No evidence of vector containing cells was found in other tissues or organs.
The blood supply to the eye is down stream to the implanted graft (carotid artery). Therefore cell detach from the graft surface may reach the eye. In the proposed clinical setting the graft will be implanted in the limbs and in this scenario detached cell will not reach the eye. In addition, vector-specific sequence was detected in 1 out of 3 animals, in 2 out of 5 independent DNA extractions. These results may indicate that the amount of gene-modified cells in the eye is small and represent a “model related result” which may not be relevant to the proposed indication of leg bypass.
The positive signal from the bone marrow was found in 1 out of 3 animals, in 1 out of 3 independent DNA extractions. It may be an indication for the presence of a small amount of gene-modified DNA in bone-marrow or a “false positive” resulting from contamination during the tissue collection procedure.
Amplification of vector-specific sequence was observed in spiked tissues and positive controls but not in negative controls. Amplification of β-actin gene sequence in all tissues demonstrated the integrity of the DNA extracted.
Biological Activity Assay
Transgene Activity.
High efficiency gene transfer of fibulin-5 and VEGF165 by retroviral vectors to endothelial cells was shown to be feasible. EC were shown to express the transgenes and in this section in vitro evidence that the trans-proteins are biologically active on human and rabbit cells used for toxicity study is provided.
Transgenes Effect on Intracellular Signal Transduction
Fibulin-5 is extracellular matrix protein that have an important role in the regulation of cell adhesion, proliferation and motility. The involvement of Fibulin-5 in these pathways is still not fully understood. In a previous study it was found that Fibulin-5 enhances the activation of the MAP kinases ERK and p38 in epithelial cells. Activation of these kinases, also upregulated by several growth factors and cytokines, amongst them VEGF165, occurs through the phosphorilation of threonine and tyrosine by a single upstream MAP kinase kinase (MEK). Here, we examined whether Fibulin-5 and VEGF165 can activate ERK and p38 phosphorilation in isolated human saphenous derived endothelial (HSVEC) and smooth muscle cells (HSVSMC). In brief, naive EC and SMC were cultured in 6 well plates for 24 hours. The medium was then replaced with conditioned media of HSVEC overexpressing Fibulin-5, VEGF165 or both genes. Exogenous VEGF165 (50 ng/ml) was also added to cultures in some of the experiments. Afterward, cells were washed once with cold PBS and lysed with lysis buffer. Evaluation of ERK and p38 activation in whole cell extract was evaluated by Western blot analysis with anti-phospho-ERK or anti-phospho-p38 MAPK monoclonal antibodies. The differences in protein loading were monitored by re-probing stripped membranes with either anti ERK or anti-β-actin antibodies.
Results:


    
    
        1. Exposure of naïve HSVEC, to preconditioned media from Fibulin-5 expressing cells resulted in a significant enhancement of ERK phosphorilation with little or no effect on the inactive form. This elevation was even more pronounced in the naïve HSVSMC transduction of the cells with VEGF165 in addition to Fibulin-5 led to a slight further increase of ERK phosphorilation while not affecting its activation in HSVSMC (FIG. 14).
        2. It is shown in FIG. 15, that addition of preconditioned media from VEGF165 expressing cells to the naïve HSVECs led to a time-dependent upregulation of phospho-p38 reaching to a maximum after 30 min of exposure.
        3. Phosphorylation of p38 was not induced by overexpression of Fibulin-5 and elevation in its phosphorylation was detected just when the cells overexpressed Fibulin-5 together with VEGF165. (FIG. 16).
    
    


SUMMARY
Both Fibulin-5 and VEGF165 transgene expression were found to have a biological effect on Human EC. Fibulin-5 overexpression led to ERK activation, while VEGF165 enhanced both ERK and p38 phosphorilation. There are conflicting data regarding the functional involvement of ERK and p38 MAPKs in biological processes such as cell proliferation, differentiation, migration and survival. It is possible that the ability of Fibulin-5 to reduce proliferation of HSVEC is mediated through the enhancement of ERK activation. In addition, our observation of HSVEC proliferation induced by VEGF, could also implicate a role for ERK and p38 MAPK in this process.
Transgene Activity on Rabbit Endothelial Cells—Used for Toxicity Study
The biological effect of human VEGF165 protein on rabbit venous derived EC was assessed by proliferation assay and compared to human venous derived EC. EC (1×104 cells per well) were seeded on 12 wells plates pre-coated with fibronectin. EC were cultured in growth medium supplemented with 20% fetal bovine serum (FBS). On the next day the medium was changed to growth medium containing 2% FBS supplemented with 2 concentrations of recombinant human VEGF165 (rhVEGF, R&D Systems) for 72 h. The effect of VEGF on cell proliferation was evaluated by counting the cells with a Coulter cell Counter (FIG. 17).
Results:
This assay demonstrated that human VEGF have a biological effect on rabbit's endothelial cells. Human VEGF supplementation to rabbit EC from two donors resulted in 20-30% increase in cell proliferation at 72 hours.
Cell Proliferation Assay
Endothelial cells—Human saphenous vein endothelial cells (2×104 cells/well) were seeded in 24 wells culture dishes pre-coated with fibronectin (20 μg/ml). EC were harvested with trypsin and counted using a cell counter (Coulter, USA) 4 hr after seeding, and at days 2 (48 hrs), 4 (96 hrs), 5 (120 hrs). Each experiment was performed in triplicates and was repeated in 3 different vein donors (primary cell lines). Proliferation rate was tested in non-transduced EC (naive), retrovirally transduced EC over-expressing GFP (green florescent protein) retrovirally transduced EC over-expressing fibulin-5, and retrovirally transduced EC co-expressing VEGF165 and fibulin-5. The assay was performed with and without supplementation of bFGF (2 ng/mL).
Smooth muscle cells—Human saphenous vein smooth cells (2×104 cells/well) were seeded in 24 wells culture dishes pre-coated with PBS-Gelatin. SMC were harvested with trypsin and counted using a cell counter (Coulter, USA) 4 hr after seeding, and at 3 time points. Each experiment was performed in triplicates and was repeated twice in 3 different vein donors (primary cell lines). Proliferation rate was tested in non-transduced SMC (naive), retrovirally transduced SMC over-expressing GFP and retrovirally transduced SMC over-expressing fibulin-5. The assay was performed with and without supplementation of bFGF (2 ng/mL).
Rat Carotid Artery Injury Model
The rat carotid injury model, whereby the endothelial cells are denuded from the internal carotid artery by an over-inflated Fogarty balloon, is the most common animal model for testing the phenomenon of restenosis. This procedure induces vascular injury similar to that caused in human arteries after balloon dilation and stent placement. In response to the injury, smooth muscle cells proliferate and migrate to the intimal layer and create a neo-intimal layer, which protrudes to the arterial lumen.
Animal model—Animal protocols was reviewed and approved by the “Animal handling and care committee”, Technion, Haifa, Israel. Twenty five male Sprague-Dowley rats weighting 400-450 g were anesthetized with ketamine (100 mg/kg) and xylazine (10 mg/kg) and Acepromazine 0.25 mg/kg injected intramuscularly. Heparin (100 U) was injected at this stage subcutaneously and 50U were injected intramuscularly. The distal segment of the right external carotid was ligated and an arteriotomy was performed. A 2F Fogarty balloon catheter was introduced through arteriotomy and was advanced to the origin of the right common carotid artery. The balloon was inflated sufficiently to generate slight resistance (0.3 ml) and the filled balloon was withdrawn three times to produce endothelial denudation of the entire length of the right common carotid artery.
After induction of injury, a 24 G catheter was introduced via an arteriotomy into the injured segment, and 0.15 ml of the following solutions were injected to five rats: adenoviral vector suspension (1010 infecting particles) encoding fibulin-5-GFP, adenoviral vectors encoding GFP, adenoviral vectors encoding VEGF-GFP or adenoviral mixture of vectors encoding fibulin-5 and VEGF (5×109 infecting particle from each vector in total volume of 0.15 ml). Five rats were injected with normal saline. Using two ligatures the common carotid artery was isolated from the aorta and the distal artery for twenty minutes while allowing the injected solution to be maintained in the isolated segment. After removal of the catheter, the external right carotid artery was ligated proximally to the arteriotomy and blood flow in the injured fragment was restored allowing blood flow from the aorta through the right internal carotid artery. Core temperature was maintained between 36-37° C., keeping an animal on the dry electrical warming blanket.
The rats were anasthetised as described at day 14. Pressure—Perfusion Fixation was performed for the carotid arteries. The thoracic cage was opened and 18 G intravenous catheter was introduced into the ascending aorta. The rats were killed with lethal dose of sodium pentobarbital (75 mg/kg). Bleeding was induced via the two jugular arteries, and 100 ml of 4% formaldehyde solution was infused over 10 min at 120 mmHg. After pressure perfusion-fixation, the entire right common carotid arterys were retrieved and immersed in formalin for 24 hours and finally embedded in paraffin blocks. The left common carotid artery was also excised and used as control.
Evaluation—The injured arterial segment artery was evaluated for the presence of neointimal formation and luminal patency, using morphometric analysis of 2 cross sections from the mid and distal parts. Slides were stained by Hematoxylin-Eosin for morphometrical analaysis. EC coverage, intra-luminal thrombus area and the ratio of thrombus area to graft lumen area were calculated, using digital image analysis (Image Pro Plus 4, Media Cybernetics, USA). Cross-sectional areas inside the external elastic membrane (EELM), inside the internal elastic membrane (IELM), and arterial lumen were measured. The area of media and intima were calculated by subtraction of IELM area from EELM area and lumen area from IELM area accordingly. The degree of arterial stenosis was determined by the ratio of neointimal area to lumen area (I/L) and the ratio of the neointimal area to the medial area (I/M). Slides were also immunostained for the presence of fibulin-5 in the tissue. In addition slides were stained for Ki-67 for the presence of proliferating cell.
In Vivo Sheep Model
The experimental procedure consisted of the following steps: Lateral saphenous veins were stripped from 18 adult sheep. Endothelial cells (EC) from each of those veins were isolated and expanded to 50×106 cells over a time period of 20-24 days. EC were identified using immunohistochemical staining for CD31, while fibroblast and smooth muscle cell contamination was ruled out using immunostaining for α-smooth muscle actin. EC were retrovirally transduced with fibulin-5 and VEGF165 encoding genes. Expression of Fibulin-5 and VEGF165 after gene transfer and selection was verified by Immunohistochemical staining and ELISA. Following gene transfer and cell expansion, transduced EC were seeded onto 25 cm long, 6 mm caliber ePTFE grafts. Six grafts were seeded with autologous EC over-expressing Fibulin-5 (>90% of cells) and VEGF165 (15-25% of cell), 6 grafts were seeded with autologous non-transduced EC (naive), 6 bare grafts were implanted as control. The seeded grafts were implanted in the right carotid artery of the EC donor sheep. The graft was implanted end to side to the carotid artery, and the carotid artery between the anastamoses was occluded to maximize blood flow through the graft. At the end of surgery graft patency was verified by selective angiography and carotid artery blood flow was measured (Doppler flow probe, #T-106; Transonic System Inc. NY). Three months following implantation the sheep was anesthetized and selective angiography was performed to study graft patency. At 6 months, selective angiography was repeated as were measurements of blood flow through the carotid artery, and the sheep were sacrificed by KCl injections. The implanted grafts were selectively perfused using 1 liter of normal saline at 100 mmHG. The carotid artery with the graft was then dissected, serial sections from the proximal, and distal anastomoses and from the medial section of the graft were fixed in 4% formalin for additional 24 hours, and then paraffin embedded. Slides were stained by H&E staining to assess intraluminal thrombosis and endothelial cell coverage. Patent grafts were evaluated for the presence of luminal thrombus, using morphometric analysis of all 3 cross sections, namely, proximal and distal anastomoses, and mid-graft section. Serial 6 μm thick slides were stained by Hematoxylin-Eosin. EC coverage, intra-luminal thrombus area and the ratio of thrombus area to graft lumen area were calculated, using digital image analysis (Image Pro Plus 4, Media cybernetics, USA).
Graft sections were also immunostained for vWF and αSMC actin to assess the nature of luminal cells.
Graft sections were also evaluated by PCR for the Neo+ resistance gene for the presence of the transduced cells.
Statistical Methods
All the data was calculated as mean±SD. Comparison of the proliferation index between multiple non-parametric groups was performed using the Kruskal-Wallis ANOVA followed by the Dunn's post hoc test. Two non-parametric groups were compared using the Mann-Whithney U test. Comparison of the blood flow and morphometric variables between two groups was done using the unpaired Student T test. Two tailed P values of 0.05 or less were considered to be statistically significant.
Kruskal-Wallis test was used to compare EC retention of ePTFE grafts following exposure to 2 hrs of flow in an in-vitro flow apparatus. Chi-square test was used to compare thrombus area in the graft segments. Wilcoxon Rank Sum Test was used to assess the differences in EC coating (Statistix 8, USA).
Results
In Vitro Proliferation of EC and SMC:
See discussion in Example 28
Neointimal Formation: Rat Carotid Artery Injury Model: Over-Expression of Fibulin-5 and VEGF165 in Endothelial Cells
Following transduction with fibulin-5 encoding retroviral vector, 50-70% of the transduced cells expressed the transgene. Transgene expression was improved to 85-100% cells after 5-7 days of culturing under G418 selection. Following a second transduction with the VEGF165 encoding vector, 20-30% of the EC over-expressing fibulin-5 additionally over-expressed VEGF165. Fibulin-5 and VEGF165 over-expression was verified by Western blot analysis (FIGS. 18A and 18B). Immunohistochemical staining of these cells demonstrated dual gene expression. The expression of dual genes was stable and maintained through passage 16.
In this experiment, adenoviral vectors encoding i) fibulin-5, ii) fibulin-5 and VEGF, iii) GFP or saline (controls) were delivered to a vascular injury site. The amount of neointima formation at 14 days after injury was observed. Results show that fibulin-5 expression alone prevents neointimal formation, whereas the addition of VEGF allowed some, but not statistically significant, neointimal formation. FIG. 19A shows the neointimal formation with saline (control) and with fibulin-5. Using immunostaining, it is further shown that fibulin-5 is expressed after gene transger exclusively in the arteries exposed to adenoviral vectors expressing fibulin-5. Staining with KI-67 to detect the number of proliferating smooth muscle cells showed that proliferation was reduced after fibulin-5 transfer, and was present after saline transfer. FIG. 19B summarizes the amount of neointima using the intima media ratio as an indicatior. It is apparent that fibulin-5 transfer reduced the formation of neointima in the rat carotid artery.
EC Proliferation Assay
Fibulin-5 over-expression by EC had inhibitory effect on EC proliferation (FIGS. 11A and 11B). This inhibitory effect was observed in repeated human saphenous vein EC proliferation assays. The presence of exogenous growth factors such as bFGF inhibited this effect. Over-expression of VEGF165 in the same cells abolished this inhibitory effect and in the presence of bFGF had proliferation advantage over non-transduced EC or fibulin-5 transduced EC.
EC Seeded ePTFE Grafts
EC were seeded on ePTFE grafts as described in methods section. Grafts were coated with a confluent monolayer of EC, as demonstrated by green florescent protein expression, and scanning electron microscope. Seeded EC formed a single monolayer on the graft surface as shown by Hematoxillin & Eosin staining. Seeded cells were also stained with immunohistochemical staining for CD-31.
Improved Patency of ePTFE Grafts Seeded with EC Expressing Fibulin-5 and VEGF165 
Grafts were implanted in sheep carotid arteries bilaterally, and the animals were observed for 2 weeks. Grafts seeded with EC over-expressing both genes (n=8) were compared to a control group, which included bare ePTFE grafts (n=4), naive EC seeded grafts (n=2), and EC over-expressing GFP seeded grafts (n=4). Expression of Fibulin-5 and VEGF165 was verified by GFP expression, by Western blot and by immunohistochemistry for the relevant trans-gene. In two of the dual gene transduced EC seeded grafts, VEGF165 expression levels were not detected in Western blot analysis.
All animals completed the experimental protocol; one animal suffered from wound infection at the site of the surgery. Flow measurements and angiographic evaluation revealed that all implanted grafts were patent at the completion of implantation surgery.
Two weeks later, angiography was repeated, the animals were euthanized, and the reconstructed arteries dissected for examination. Thirty percent of the implanted control grafts were totally occluded (FIGS. 20A and 20B). In contrast, all of the implanted grafts, which were seeded with dual gene transduced EC, were patent and demonstrated good flow.
Morphometric analysis of all graft sections (proximal, mid and distal sections) revealed that graft occlusion was related to thrombosis. The data obtained from morphometric thrombus analysis of graft sections is presented in table 4. Segments of grafts seeded with ECs over-expressing Fibulin-5 and VEGF165 showed a significant reduction in laminating thrombus formation as compared to the graft segment from the control group: 55% (16/29) of control graft segments demonstrated thrombus area of more than 3%, as opposed to only 4% in the dual gene graft sections (p value<0.008).







TABLE 4







Patency rates of grafts










Patency at 3 month
Patency at 6 month



angiography
angiography













Bare Grafts
1/6
0/6


Grafts seeded with naive EC
1/6
1/6


Grafts seeded with endothelial
6/6
5/6


cells over expressing fibulin-5


and VEGF









Over-Expression of Fibulin-5 and VEGF165 Improves EC Retention on ePTFE Grafts
EC coverage of the graft was assessed as the percentage of the total graft area that remained coated with cells. All graft sections (proximal, mid and distal) were evaluated for EC coating. Summary of graft EC coating results are shown in table 4. The results demonstrated that dual expression of Fibulin-5 and VEGF165 significantly improved EC adherence to ePTFE grafts as compared with control EC coated grafts (73.4±22.4% vs. 29.6±24.8%; p value<0.004).
Discussion
Example 29 shows that co-expression of fibulin-5 and VEGF by endothelial cells seeded onto synthetic grafts improve long-term patency of small caliber synthetic grafts in a sheep model. Evidence that co-expression of the two genes reduced neointimal formation after vascular injury in a rat carotid artery injury model is also provided.
Small caliber synthetic grafts seeded with either naive endothelial cells, or cells transduced to co-express fibulin-5 and VEGF, and bare grafts were used to test the hypothesis that co-expression of fibulin-5 and VEGF by endothelial cells seeded onto synthetic grafts improves long-term patency of the graft. The sheep carotid artery model was employed and 15-20 cm long grafts were implanted using end-to-side anastamoses, as the size of the carotid artery in adult sheep is similar to the size of femoral artery in humans, providing surgical field similar to the field in femoral popliteal bypass surgery. Catheterization at the end of surgery was used to role out surgical failure. The time points of 3 and 6 months for selective angiography were used based on the assumption that anastamosis neointimal formation will be completed at 6 months.
As shown in Table 4, graft patency was significantly higher using grafts seeded with EC expressing the two genes. The patency rate of the seeded grafts is highly improved in comparison to reported rates in the sheep carotid artery (Ortenwall et al. (1988) Surgery, 103:199-205. Long term (>130 days) patency was reported in a sheep carotid interposition, short (4-5 cm) grafts of decellularized porcine ileac arteries pre-seeded with autologous EPC's. In that model, the authors used an approach different from the one described herein, to improve cell adhesion to the graft. As described herein, the adhesive capacity of fibulin-5 is used to improve seeded cell adhesion, while Kaushal et al, exposed the grafts to shear stress for 4 days prior to graft implantation. Graft coverage by circulating EPC's or EC from neighboring arterial section occur frequently in young animals and short graphs. 70% coverage of graft luminal surface by seeded grafts is observed, while only 30% of seeded naive EC were found after two weeks.
Neointimal formation at site of anastamoses reduces blood flow through the grafts and can eventually lead to graft failure. Neo-intimal formation as a result of vascular injury is a consequent of inflammatory and thrombotic processes derived smooth muscle cell proliferation and extracellular matrix production. Drug eluting stents efficiently inhibit smooth muscle cell proliferation and prevent neointimal formation in coronary arteries. Fibulin-5 inhibited both proliferation of EC and SMC, but the inhibition of EC proliferation was reversed by the addition of VEGF and basic FGF. The inhibitory effect of fibulin-5 on SMC was not reversed by supplementation of FGF. Furthermore, we showed that local expression of fibulin-5 after vascular injury completely abolished the formation of neo-intima. Co-expression of fibulin-5 and VEGF resulted in formation of minimal sized neo-intima. The concept endorsed by the in vitro proliferation assays, and the carotid artery in vivo experiments is that by using the combination of fibulin-5 and VEGF we selectively inhibit SMC proliferation and at the same time stimulate EC proliferation. Gene transfer with retroviral-based vectors secure transgene expression for at least several weeks in vivo. Apparently, this is the time period needed for inhibition of injury related cellular and molecular events. Localized narrowing at 3 months and near closure at 6 months of 1 out of the 6 grafts seeded with cells co-expressing the two genes may be related to the challenging animal model, or to a localized region of unseeded surface. It may also be related to the handling of the graft during surgery such as use of surgical bulldogs, and graft manipulation with mechanical removal of the seeded cells. The fact that narrowing at 3 months was observed in only one segment of the 6 grafts seeded with dual gene expressing cells, supports our hypothesis that seeding EC co-expressing fibulin-5 and VEGF prevents local thrombosis and neointimal formation.
In respect to the feasibility of graft seeding within clinically applicable time point, the average time from vein striping to graft implantation was 22 days. The viral vectors used were pseudo-typed to improve transduction rates in human and sheep vascular cells (ref the stent paper). Transduction of cells with two retroviral based vectors can increase cell propensity for malignant transformation as reported in bone marrow-derived cells (ref). To address this issue, telomerase activity of EC co-expressing the two genes was studied. It was found that activity was very low and comparable to control naive cells at same passage. The number of viral genome copies in cells transduced with the two different retroviral vectors (encoding fibulin-5 and VEGF) was also tested. The average number of copies in three primary EC lines was <10 copies in dually transduced cells. Lower than 10 viral genome copies in transduced cells was reported to be associated with lower rate of malignant cell transformation.
In summary, this Example shows that seeding small caliber ePTFE grafts with EC expressing fibulin-5 and VEGF improved long term graft patency. The mechanism by which patency rates were improved, based on this data, is attributable to the synergistic effect of fibulin-5 and VEGF on vascular cells.
OTHER EMBODIMENTS
From the foregoing detailed description of the specific embodiments of the invention, it should be apparent that unique devices and methods for treating various cardiovascular conditions have been described. Although particular embodiments have been disclosed herein in detail, this has been done by way of example for purposes of illustration only, and is not intended to be limiting with respect to the scope of the appended claims that follow. In particular, it is contemplated by the inventor that various substitutions, alterations, and modifications are a matter of routine for a person of ordinary skill in the art with knowledge of the embodiments described herein, and may be made to the invention without departing from the spirit and scope of the invention as defined by the claims.

## Claims

1. An implantable device for treating a vascular disease or disorder comprising an intravascular prosthesis, wherein said device is coated with a biodegradable drug-eluting polymer that is impregnated with an inhibitor of smooth muscle cell proliferation and a growth factor for enhancing endothelial cell proliferation, wherein said inhibitor of smooth muscle cell proliferation is fibulin-5 polypeptide or a nucleic acid encoding fibulin-5 polypeptide, and wherein said growth factor for enhancing endothelial cell proliferation is VEGF polypeptide or a nucleic acid encoding VEGF polypeptide.

2. The device of claim 1, wherein the intravascular prosthesis is a stent.

3. The device of claim 1, wherein the vascular disease is restenosis.

4. The device of claim 1, wherein said fibulin-5 and said VEGF are released slowly by degradation of said polymer.

5. An implantable device for treating a vascular disease or disorder comprising an intravascular prosthesis, wherein said device is coated with a substrate comprising endothelial cells that are genetically altered to express or over-express an inhibitor of smooth muscle cell proliferation and a growth factor for enhancing endothelial cell proliferation, wherein said inhibitor of smooth muscle cell proliferation is fibulin-5 polypeptide, and wherein said growth factor for enhancing endothelial cell proliferation is VEGF polypeptide.

6. The device of claim 5, wherein said intravascular prosthesis is a stent.

7. The device of claim 5, wherein said vascular disease is restenosis.