Method of reendothelializing vascular linings

A method for treating the vascular passage of a patient, damaged by procedures such as an endarterectomy which denude portions of the vascular passages of their endothelial cell linings, is disclosed. In this method, endothelial cells are isolated from the patient's own microvessels, the flow of blood through the patient's damaged vascular passage is interrupted, the endothelial cells isolated from the patient's microvessels are applied to the surface of the denuded portion of the patient's vascular passage in a density sufficient to provide covereage of at least about 50% of said denuded portion, and interruption of blood flow through the vascular passage is maintained for a period of time sufficient to allow the sodded cells to form an attachment to the vascular lining sufficient to withstand the shear created by resumed blood flow through the vascular passage.

BACKGROUND OF THE INVENTION 
1. Field of the Invention 
This invention relates to a method of reendothelializing the vascular 
passage of a patient, the lining of which has been substantially denuded 
of endothelial cells by virtue, for example, of procedures such as 
endarterectomy. 
2. Related Applications 
U.S. Ser. No. 742,086, filed June 6, 1985, by Williams and Jarrell, 
discloses a method for treating a synthetic or naturally occurring implant 
intended for implantation in a human patient comprising the steps of 
obtaining human microvascular endothelial cell rich tissue from that 
patient, separating microvascular endothelial cells from that tissue; and 
applying said microvascular endothelial cells onto said implant to provide 
at least about 50% or greater confluence of said cells on the surface of 
said implant to be treated. 
U.S. Ser. No. 848,453, filed on Apr. 4, 1986 as a continuation- in-part of 
U.S. Ser. No. 742,086, discloses a method of treating an implant intended 
for implantation in a human patient, comprising the steps of providing a 
synthetic substrate material and treating that material with Type IV/V 
collagen to improve human endothelial cell adhesion, proliferation and 
morphology. In the preferred embodiment, the endothelial cells are derived 
from the endothelial cell rich tissue of the patient undergoing 
implantation. 
U.S. Ser. No. 927,745, filed by Jarrell and Williams on Nov. 6, 1986, 
discloses a method of determining endothelial cell coverage on a 
prosthetic surface. 
The disclosures of these three applications are hereby incorporated by 
reference. 
3. Description of the Art 
Atherosclerotic vascular disease remains the leading cause of death among 
Americans. As medical science has become more sophisticated, increasing 
use of invasive vascular procedures are being applied to obstructed 
vessels in the absence of effective preventive or therapeutic drug 
modalities. For example, the use of arterial endarterectomy as well as 
percutaneous balloon dilatation of vessels for pathologic stenosis have 
become routine hospital procedures. Although these and other procedures 
are often successful, a common complication after the procedures is the 
occurrence of vessel wall abnormalities. These abnormalities include 
recurrent stenosis due to atherosclerosis, smooth muscle proliferation, 
loss of vessel wall integrity as a result of fibrosis and thrombosis of 
the vessel. Injury to or removal of the endothelial cells lining the blood 
vessels is one of several common denominators inherent to vascular 
procedures, and current data suggest that spontaneous reendothelialization 
of these injuries may occur slowly, partially, or not at all. 
The endothelial lining of blood vessels is a highly complex, 
multi-functional cell surface. These cells interact with both the blood 
and the underlying vessel wall components to maintain a physiological 
homeostasis. The effects of endothelial injury have been studied in 
several experimental models mostly designed to study the development of 
biological mechanisms. After endothelial injury, the vessel wall loses its 
non-thrombogenic properties. One of the first events to occur is platelet 
adherence to the vessel surface, which is extensive over the first several 
days but diminishes over the following week. Steele, P., Chesebro, J., 
Stanson, A., Holmes, D., Dewanjee, M., Badimon, L., Fuster, V., Circ. Res. 
57, No. 1:105-112 (1985). Platelets adhere to the subendothelium and 
undergo a release reaction, inducing further activation of the plasma 
coagulation system. Osterud, B. et al., J. Proc. Natl. Acad. Sci. U.S.A., 
74, p. 5260 (977). One of the released products is platelet derived growth 
factor (PDGF), which is mitogenic for vascular smooth muscle cells grown 
in tissue culture. It has been postulated that local release of this 
factor may play a role in the genesis of intimal hyperplasia and 
atherosclerosis. Harker, L. et al., J. Clin. Invest., 58, 731 (1976); 
Friedman, R. et al., J. Clin. Invest. 60, 1191 (1977). Other substances 
released from platelets include heparitinase and platelet factor 4. The 
latter protein has high affinity for heparin and has been shown to 
penetrate into the vascular media after de-endothelialization. Goldgerg, 
J. D. et al., J. Science. 209, 611 (1980). Macrophages, which are also a 
rich source of SMC mitogens, are frequently present in the injured area. 
Gimbrone, M. A. Jr., In: Jaffe, E. A., Editor, Biology of Endothelial 
Cells, Martinus Nijhoff Publishers, pp. 97-107 (1984). The final response 
of the injured arterial wall, independent of whether the injury is 
chemical, mechanical or biological, is characterized by proliferation of 
cells in the intima to form a fibro-musculo-elastic plaque. Hoff, H., 
Thromb. Haemostas., 40, 121 (1970). 
Clearly, the endothelial cell plays a key role in the etiology of blood 
vessel dysfunction. It is anticipated that restoration of intact 
endothelium immediately following injury might reduce or alter the events 
occurring immediately after injury. Therefore, it is an object of this 
invention to provide a method for reendothelializing the linings of 
vascular passages which have been substantially denuded of endothelial 
cells. 
Recent years have seen refinements made in the isolation of endothelial 
cells (EC) and their growth in culture. The addition of endothelial cell 
growth factor (ECFG) and heparin to culture medium has allowed human adult 
large vessel EC to remain in culture for greater than 50 population 
doublings. Jaffe, E. A. et al., J. Clin. Invest., 52:2745 (1973); Maciag, 
T., et al., J. Cell Biol., 91:420 (1981); Thornton, S. C. et al., Science, 
222:623-624 (1983); Jarrell, B. E., et al., J. Vasc. Surg., 1:757-764 
(1984). Human microvessel EC have also been routinely isolated in large 
quantities using collagenase digestion and Percoll gradient purification 
followed by long term cultivation in heparin - ECFG supplemented medium. 
Jarrell, B. E. et al., Surgery, Vol. 100, No. 2, pp. 392-399 (August 
1986). 
These advances in EC isolation and culture have been used to better 
understand the interactions between these EC and prosthetic vascular 
grafts. Watkins, M. T. et al., J. Surg. Res., 36:588-596 (1984); Williams, 
S. K. et al., J. Surg. Res., 38:618-629 (1985); Baker, K. S. et al., Am. 
J. Surg., 150:197-200 (1985). In these studies it has been noted that 
human EC possess the ability to firmly adhere to both plasma coated 
surfaces and human amnion type IV/V collagen after a ten to thirty minute 
incubation period. Jarrell, B. E. et al., Ann. Surg., Vol. 203, No. 6, pp. 
671-678 (June 1986). Another observation was that freshly isolated human 
microvessel EC obtained from fat tissue also possessed this property and 
could be isolated in quantities of 10.sup.6 EC per gram of fat. Radomski, 
J., et al., J. Surg. Res., 42, 133-140 (1987); Jarrell, B. E. et al., 
Surgery, Vol. 100, No. 2, pp. 392- 399 (August 1986). 
At least one study has examined the utility of treating the neointimal 
hyperplasia developed after endarterectomy of a normal artery by 
endothelial cell sodding. Bush, Jr., Harry L., Jakubowski, Joseph A., 
Sentissi, Joanna M., Curl, Richard G., Hayes, John A., and Deykin, Daniel, 
"Neointimal Hyperplasia Occurring After Carotid Endarterectomy in a Canine 
Model: Effect of Endothelial Cell Seeding vs. Perioperative Aspirin, 
Journal of Vascular Surgery, Vol. 3, No. 1, pp. 118-125 (January 1987). In 
this study, endothelial cells were harvested from the veins of dogs 
selected to undergo treatment. The cells were suspended in sterile 
autogenous serum, and this suspension was injected into the 
endarterectomized segment of the dog's artery. From this work, the authors 
concluded that sodding the endarterectomized surface with autogenous 
endothelial cells did minimize proliferative lesion in the artery. 
SUMMARY OF THE INVENTION 
This invention relates to a method of reendothelializing the vascular 
passage of a patient, the lining of which has been substantially denuded 
of endothelial cells, using that patient's own endothelial cells. In this 
method, endothelial cells are isolated from the patient's own 
microvessels, the flow of blood through the patient's damaged vascular 
passage is interrupted, the endothelial cells isolated from the patient's 
microvessels are applied to the surface of the denuded portion of the 
patient's vascular passage in a density sufficient to provide coverage of 
at least about 50% of said denuded portion, and interruption of blood flow 
through the vascular passage is maintained for a period of time sufficient 
to allow the cells to form an attachment to the vascular lining sufficient 
to withstand the shear created by resumed blood flow through the vascular 
passage. 
DETAILED DESCRIPTION OF THE INVENTION 
The patients who may benefit from the method of this invention are those 
who have been subjected to procedures which damage the endothelial cell 
linings of the vascular passages, e.g., percutaneous transluminal 
angioplasty. By substantially denuded of endothelial cells, as that phrase 
is used herein, we mean a vascular passage the endothelial cell lining of 
which has been injured or removed to an extent likely to cause adverse 
side effects to the patient. Such injuries can be classified as level I, 
II or III injuries, level I injury being one that exposes principally 
basement membrane, level II injury being one that exposes primarily 
sub-basement membrane, interstitial collagen and the internal elastic 
lamina, and a level III injury being one that exposes the deeper layers 
including the media and smooth muscle cells in areas of internal elastic 
lamina fracture. 
Endothelial cells for use in the method of this invention are preferably 
obtained from the patient undergoing the vascular treatment; however, it 
should be possible to obtain human perinephric fat from brain-dead but 
heart-beating cadaver donors or from donors other than the patient during 
the donor's surgery. Microvascular endothelial cells, that is, cells which 
are derived from capillaries, arterioles and venules, will function 
suitably in place of large vessel cells even though there are 
morphological and functional differences between large vessel endothelial 
cells and microvascular endothelial cells in their native tissues. 
Microvascular endothelial cells are present in an abundant supply in body 
tissue, and are therefore the preferred source of endothelial cells for 
use in this invention. Although endothelial cells may be isolated from 
tissues such as brain, lung, retina, adrenal glands, liver and muscle 
tissue, the use of fat tissue as the source for the cells is preferred due 
to its abundance and availability, and due to the fact that its removal 
should not adversely affect the patient being treated. 
To obtain microvascular endothelial cells from the patient, the source 
tissue, such as fat tissue, is removed from the patient after sterile 
conditions have been established. Microvascular endothelial cells in that 
fat tissue are then quickly separated from their related tissue by 
enzymatic digestion and centrifugation and may be used to treat the 
surface of the damaged vascular linings of the patient during the latter 
stages of the same operation. This procedure obviates any need to culture 
adult endothelial cells to increase their numbers, and permits a patient 
to receive treatment with his own fresh, "healthy" endothelial cells.

The procedure useful for isolating large quantities of endothelial cells 
without the need for tissue culturing may be readily performed in an 
operating room and is described in its preferred embodiment in greater 
detail as follows. The fat tissue retrieved from the patient or donated 
from another source is immediately transferred to ice cold buffered saline 
(pH 7.4) wherein the buffering agent is preferably a phosphate, i.e., a 
phosphate buffered saline (PBS). The tissue is minced with fine scissors 
and the buffer decanted. Alternatively, fat tissue obtained by liposuction 
may be used as the source of endothelial cells. The proteolytic enzyme 
collagenase, containing caseanase and trypsin, is added to the tissue and 
incubated at 37.degree. C. until the tissue mass disperses. This digestion 
occurs within thirty minutes and generally should occur in less than 
twenty minutes. The digest is transferred to a sterile test tube and 
centrifuged at low speed (700.times.g) in a table top centrifuge for five 
minutes at room temperature. The pellet of cells thus formed consists of 
greater than ninety-five percent (95%) endothelial cells. These 
endothelial cells are described herein as microvascular endothelial cells 
since they originate from the arterioles, capillaries and venules, all 
elements of the microvasculature. This microvascular endothelial cell 
pellet is washed one time by centrifugation with a buffer and can be used 
directly without further purification for application to the injured 
vascular lining of the patient. Suitable buffers include buffered saline 
such as PBS as well as intravenous infusion solutions and peritoneal 
dialysis solutions. 
Alternatively, the microvascular endothelial cells may be further purified 
by centrifuging the cells with a continuous gradient. This gradient can be 
formed from a number of large molecular weight solutes, including albumin, 
dextran, or commercially available density gradient materials, such as 
Percoll (Pharmacia Inc., Piscataway, N.J.) or Nycodenz (Nyegaard and 
Company, Norway). Gradient centrifugation is used to remove red cells, 
white cells and smooth muscle cells. A forty-five percent (45%) solution 
of Percoll has routinely been used in the studies reported herein. Cells 
are layered on the surface of the Percoll solution and centrifuged at 
13,000.times.g for twenty minutes. Alternatively, cells are layered on a 
preformed Percoll gradient and centrifuged at 400.times.g for five minutes 
at room temperature. A thick band of endothelial cells results at the 
upper end of the gradient. These cells are removed with a pipette and 
washed one time by centrifugation with phosphate-buffered saline. 
The endothelial cells isolated as described above may be used directly to 
treat the injured vascular lining of the patient. They may be mixed with 
blood or plasma and used to seed the surface of the patient's vascular 
lining or may be mixed with a non-clotting medium such as a buffered 
saline and used to sod the surface. The term "seeding" as it is used 
herein refers to the procedure which entails mixing cells with a matrix 
followed by placement of that mixture onto the surface to be seeded, e.g., 
the vascular lining. The matrix may be any gel or clot-forming substance, 
such as blood or plasma, that may be used as a vehicle in which to suspend 
and trap the cells. This endothelial cell matrix mixture adheres to the 
surface and gels, "trapping" the cells within the matrix until they are 
able to multiply and grow out over the surface. The term "sodding", on the 
other hand, is used herein to refer to the procedure which entails mixing 
cells in a simple medium such as a buffer solution that does not gel under 
the ambient conditions of the sodding procedure (e.g., a temperature of 
from about body temperature to about 37.degree. C.) and applying that 
mixture to cover a surface. In the sodding process, cells approach the 
surface to be treated due to gravity and attach directly to the surface, 
rather than being "trapped" within a portion of the mixture as in the 
seeding process. 
In a preferred embodiment, the cells are pelletized by centrifugation 
(200.times.g) and the pellet is then resuspended with protein-containing 
buffer solution. This resuspension should be performed at a ratio of 
approximately 1:5 to 1:15 or, preferably about 1:10 volumes of packed 
microvascular endothelial cells to buffer solution. This resuspension may 
then be used to sod the lining of the injured vascular passageway. Prior 
to sodding, certain agents may be added to the suspension in an effort to 
aid cell adhesion and spreading, including fibronectin, platelet poor 
plasma, albumin, Dextran 40 or Dextran 70, endothelial cell growth factor 
and heparin sulfate. 
The goal in this procedure, of course, is to create a confluent layer of 
endothelial cells on the vascular surface, or to restore the vascular 
surface to its pre-injury, "healthy" state. To achieve this goal, the 
initial adherence of cells to the vascular lining surface should 
preferably be sufficient to provide at least about fifty percent (50%) 
initial surface coverage. Application at fifty percent (50%) confluence 
requires the cells to duplicate one time to create a confluent cell layer. 
Since, as discussed below, it will be necessary to restrict the flow of 
blood through the vascular passageway during the seeding or sodding 
process, since such flow restriction should be maintained for as short a 
time as possible for the benefit of the patient, and since resumed blood 
flow through the passageway may hinder cell duplication, it is obviously 
desirable to apply as many endothelial cells to the vascular lining 
surface as possible. Most preferably, the cells are seeded or sodded onto 
the vascular lining surface at a density equivalent to confluence, i.e., 
greater than about 105 cells per cm.sup.2 surface area. It is necessary to 
interrupt or reduce the flow of blood through the vascular passageway 
during the time of seeding or sodding for a period of time sufficient to 
permit adhesion of the cells to the lining of that passageway. More 
preferably, flow is interrupted or reduced for a period of time sufficient 
for adhesion of a confluent monolayer of cells. For certain arteries, it 
may be necessary to use a temporary shunt around the affected portion to 
sufficiently reduce blood flow. In other situations, the artery may simply 
be clamped or constricted upstream to accomplish the necessary flow 
reduction. It is preferable, especially in the sodding procedure, to apply 
pressure to force the cells against the vessel wall. It is believed that 
the optimal temperature for inducing attachment of the cells to the vessel 
wall is about 37.degree. C. Once the endothelial cells have established an 
attachment sufficient to withstand the shear created by the blood flow 
through the vascular passageway, flow may be reestablished therethrough, 
whereupon the previously denuded portion of the passageway will be 
protected by a "natural" antithrombogenic surface. 
Studies on human basement membrane surfaces obtained from amnion have 
suggested that type IV/V collagen exhibits properties which support rapid 
adherence and spreading of endothelial cells after sodding. Conversely, 
human plasma derived clot and human type I/III interstitial collagen have 
been found to support less rapid cell spreading when examined under 
similar conditions. An injured vessel will most likely expose collagen 
I/III smooth muscle cells or become coated with plasma proteins, providing 
a suboptimal surface for endothelial cell attachment and spreading. 
Pre-treatment of the residual injured vessel wall may enhance adherence 
and spreading qualities and allow a more satisfactory surface to form. 
Thus, the vessel wall may be pretreated with one or more of the following: 
fibronectin 
laminin 
plasma, prepared with EDTA and clotted onto surface 
solubilized collagen IV/V 
platelets 
red blood cells 
Dextran 40 or Dextran 70 
heparin sulfate 
endothelial cell growth factor 
serum 
serum albumin 
thrombospondin 
heparan 
heparan sulfate