Abstract:
Systems and methods directed to sampling endothelial cells within a pulmonary vasculature are provided. The system and method may include a cell sampling device having an elongated sheath extending from a proximal end to a distal end of the cell sampling device and defining an access lumen. An endovascular brush includes brush elements coupled to a shaft. The endovascular brush is dimensioned to be received in the access lumen of the elongated sheath. An actuation control is coupled to the shaft of the endovascular brush to axially translate the endovascular brush between a retracted position and an expanded position. Upon translation of the endovascular brush from the retracted position to the expanded position, the brush elements moves from within the access lumen to the pulmonary vasculature to obtain endothelial cells from a target artery of the pulmonary vasculature.

Description:
CROSS-REFERENCE TO RELATED APPLICATIONS 
       [0001]    This application is based on, claims the benefit of, and incorporates herein by reference, U.S. Provisional Patent Application Ser. No. 61/990,754, filed on May 9, 2014 and entitled “Vascular Cytology Brush.” 
     
    
     STATEMENT REGARDING FEDERALLY SPONSORED RESEARCH 
       [0002]    NA 
       BACKGROUND 
       [0003]    The present disclosure relates generally to systems and methods for an endovascular cytology brush for use in the pulmonary vasculature to sample endothelial cells. More particularly, the disclosure relates to systems and methods for an endovascular cytology brush adapted to the pulmonary vasculature and used to isolate pulmonary vascular endothelial cells for diagnosis, prognosis, selecting a therapy, or evaluating a response therapy. 
         [0004]    Pulmonary arterial hypertension (PAH) is an insidious disease associated with pulmonary vascular remodeling that promotes right heart failure and premature death. In PAH, pulmonary endothelial dysfunction is identified as a critical mediator of distal pulmonary arteriole remodeling to induce pulmonary hypertension. To date, the spectrum of pulmonary endothelial dysfunction that is present in pre-clinical, clinically-relevant, and drug-treated pulmonary hypertension remains unknown, as does the relationship between endothelial dysfunction and increased pulmonary pressures, pulmonary vascular resistance, and right ventricular remodeling. Similarly, while contemporary pharmacotherapies for PAH may improve symptoms by replacing deficient endothelial-derived products (e.g., nitric oxide, prostacyclin, etc.) to the pulmonary vasculature, strategies for monitoring dynamic changes in the pulmonary endothelial phenotype in patients are not available. 
         [0005]    Although the etiology of PAH may vary between patients, the cardiac and pulmonary vascular pathophenotypes are remarkably similar. PAH is characterized by aberrant vascular remodeling with distal pulmonary artery endothelial dysfunction and dysregulated vascular smooth muscle cell proliferation leading to inflammation, neointimal formation, thrombosis, and luminal obliteration. The formation of plexiform lesions is progressive and contributes to impaired vascular reactivity, elevated pulmonary artery pressure, and increased pulmonary vascular resistance, which, in turn, promotes maladaptive right ventricular remodeling. The burden of structural and functional disruption of the pulmonary vascular-right ventricular circuit ultimately leads to right ventricular failure and death. 
         [0006]    PAH is associated with high rates of morbidity and mortality with approximately 45% mortality within 3 years of diagnosis, despite optimal medical therapy. To date, there are no peripheral biomarkers or diagnostic tests that identify preclinical PAH; provide prognostic information based on indices of pulmonary vascular remodeling; allow for the selection of tailored therapy based on genetic profiling, similar to cancer therapeutics; or monitor current disease status and response to medications. This deficit has occurred owing to the inaccessibility of the pulmonary vascular compartment for tissue or vascular cell sampling of endothelial cells. The vascular remodeling changes and endothelial dysfunction described in PAH have also been described, in part, for other forms of pulmonary hypertension. 
         [0007]    The endothelium, the cells that line the luminal surface of blood vessels, sense the micro-milieu of flowing blood and regulate important functions of blood vessels. For example, endothelial cells synthesize potent vasodilators and constrictors to alter the vascular tone in order to control blood pressure and blood flow. Endothelial cells also produce cytokines that mediate inflammatory responses in the vessel wall, which underscores the pathogenesis of many vascular diseases, such as atherosclerosis. Accordingly, a number of drugs have been developed for treating vascular diseases which target the endothelium. Endothelial gene expression changes in response to drug therapy long before anatomical changes or clinical symptoms occur. It is therefore possible to use endothelial gene expression patterns as a surrogate indicator of drug efficacy to facilitate drug design and testing. 
         [0008]    A number of brushes have been adopted in the art for minimally invasive cell sampling, such as the biopsy brushes designed for use in obtaining biopsy samples from the urinary, tracheal, bronchial, and gastrointestinal tracts. However, these brushes are not suitable for endovascular use, particularly with respect to relatively small blood vessels. Additionally, the brushes are often not very effective in obtaining cell samples. 
         [0009]    Accordingly, there is a need for an endovascular brush capable of being used in endovascular procedures. There is also a need for an endovascular brush that is capable of being used for endovascular cell sampling, including in-vivo endovascular cell sampling. 
       SUMMARY 
       [0010]    The present disclosure overcomes the aforementioned drawbacks by providing a system and method for an endovascular cytology brush capable of being utilized for repeated sampling of pulmonary artery endothelial cells for endothelial phenotypic profiling in PAH and other forms of pulmonary hypertension (World Health Organization Groups 1-5). The endothelial phenotypic profiling may use gene expression microarrays to reveal endothelial pathophenotypes that can serve as a biomarker of PAH disease severity. These profiles may also be exploited for the selection of drug therapies or to identify druggable targets. Thus, characterization of the cells may identify endothelial disease-related profiles that can provide diagnostic and prognostic information. Use of the disclosed endovascular cytology brush has broad applicability to patients with other pulmonary diseases where knowledge of the endothelial cell phenotype aides in prognosis, diagnosis, or therapeutic choices, such as following pulmonary transplant. In addition, the endovascular cytology brush may be applied to patients with lung pathologies, such as rejection in lung transplantation or inflammatory lung diseases. 
         [0011]    In accordance with one aspect of the disclosure, a cell sampling device for sampling endothelial cells within a pulmonary vasculature is provided. The cell sampling device includes an elongated sheath extending from a proximal end to a distal end of the cell sampling device and defining an access lumen. An endovascular brush includes brush elements coupled to a shaft. The endovascular brush is dimensioned to be received in the access lumen of the elongated sheath. An actuation control is coupled to the shaft of the endovascular brush to axially translate the endovascular brush between a retracted position and an expanded position. Upon translation of the endovascular brush from the retracted position to the expanded position, the brush elements moves from within the access lumen to the pulmonary vasculature to obtain endothelial cells from a target artery of the pulmonary vasculature. 
         [0012]    In accordance with another aspect of the disclosure, a method for sampling endothelial cells within a pulmonary vasculature, is provided. The method includes inserting an elongated sheath into the pulmonary vasculature of a subject, the elongated sheath defining an access lumen. An endovascular brush is inserted into the access lumen of the elongated sheath. The endovascular brush includes brush elements coupled to a shaft. An actuation control coupled to the shaft of the endovascular brush is used to axially translate the endovascular brush from a retracted position within the access lumen to an expanded position in a target artery of the pulmonary vasculature. Using the brush elements of the endovascular brush, endothelial cells from the target artery are obtained to determine a state of the pulmonary vasculature. 
         [0013]    The foregoing and other aspects and advantages of the disclosure will appear from the following description. In the description, reference is made to the accompanying drawings which form a part hereof, and in which there is shown by way of illustration a preferred embodiment of the disclosure. Such embodiment does not necessarily represent the full scope of the disclosure, however, and reference is made therefore to the claims and herein for interpreting the scope of the disclosure. 
     
    
     
       BRIEF DESCRIPTION OF THE DRAWINGS 
         [0014]      FIG. 1A  is a side view of a cell sampling device according to one embodiment of the present disclosure. 
           [0015]      FIG. 1B  is an enlarged view of an endovascular brush of the cell sampling device of  FIG. 1A . 
           [0016]      FIG. 2  is a side cross-sectional view of the endovascular brush in a retracted position being introduced into a sheath of the cell sampling device taken along line  2 - 2  of  FIG. 1A . 
           [0017]      FIG. 3  is a side cross-sectional view of the endovascular brush in a retracted position prior to entering a target artery. 
           [0018]      FIG. 4  is a side cross-sectional view of the endovascular brush in an expanded position after entering the target artery to collect endothelial cells. 
           [0019]      FIG. 5  is a flow chart setting forth the steps of a method for sampling endothelial cells within a pulmonary vasculature. 
       
    
    
     DETAILED DESCRIPTION 
       [0020]    Referring to  FIGS. 1A and 1B , a cell sampling device  10  according to one embodiment of the present disclosure is shown. The cell sampling device  10  may be utilized to sample pulmonary artery endothelial cells for endothelial phenotypic profiling in PAH and other forms of pulmonary hypertension. The cell sampling device  10  includes forceps  12  adapted for use internally of the body, for example in connection with vascular procedures. The forceps  12  include an elongated sheath  14  for introduction into the body and navigation to an area of interest. The forceps  12  include a control handle  16  at a proximal end  18  coupled to the elongated sheath  14  which extends over the main length of the cell sampling device  10 . At a distal end  20 , an endovascular brush  22  may extend from the elongated sheath  14  in an expanded position, as will be described in further detail below. 
         [0021]    As shown in  FIG. 2 , the elongated sheath  14  may be a tubular member that defines an access lumen  24  extending from the proximal end  18  to the distal end  20 . The elongated sheath  14  may receive at least a portion of the endovascular brush  22 , which may be inserted into the elongated sheath  14  in preparation for catheterization. The elongated sheath  14  may vary dimensionally to accommodate various endovascular brush sizes. In one embodiment, the elongated sheath  14  is approximately 120 cm long in order to cover the entire length of the endovascular brush  22 , and has an inner diameter of about 1.8 mm. In one non-limiting example, the elongated sheath  14  may be constructed of a transparent or semitransparent material so that the endovascular brush is visible through the elongated sheath  14 . In addition, the elongated sheath  14  may be small enough such that it can be inserted into a catheter  26 , such as a  5 F or  6 F coronary artery guiding catheter, a  7 F myocardial biopsy sheath, or any suitable guiding sheath to allow the elongated sheath  14  to be delivered to a pulmonary vasculature  28 . 
         [0022]    Referring again to  FIG. 1A , the forceps  12  include the control handle  16  and an actuation control  30 . The actuation control  30  may be coupled to a shaft  32  of the endovascular brush  22 , as shown in  FIG. 2 , such that movement of the actuation control  30  results in movement of the endovascular brush  22  within the access lumen  24 . The actuation control  30  can include any type of actuating mechanism capable of imparting bidirectional axial movement to the shaft  32  within the elongated sheath  14 . Loops  33  are provided in the control handle  16  and the actuation control  30  to form finger holes useful in grasping and manipulating the forceps  12 . 
         [0023]    Referring now to  FIG. 1B , the endovascular brush  22  includes a brush segment  34 , at a distal end of the endovascular brush  22 , and the shaft  32  extending from the brush segment  34  to the proximal end of the endovascular brush  22 . The brush segment  34  includes a plurality of brush elements  36  configured to retain sampled cells on the brush segment  34 . The plurality of brush elements  36  may be abrasive elements that allow a user to remove tissue, such as endothelial cells, from the pulmonary vasculature  28  by rubbing or grazing an intravascular surface with the plurality of brush elements  36 . Various types of brush elements  36  may be used to provide such functionality, including fibers, bristles, ridges, corrugations, and the like. 
         [0024]    The endovascular brush  22  may further include a rounded tip  38 , such as a spherical or semispherical tip, to inhibit injury to the vascular tissue during catheterization and/or sampling of endothelial cells. In one non-limiting example, the rounded tip  38  may include one or more radiopaque markers  40  at the distal end, or be made of a radiopaque material, allowing at least a portion of the endovascular brush  22  to be imaged for intraoperative image guidance. For example, the radiopaque markers  40  may be used to mark the intravascular location of the endovascular brush  22  of the cell sampling device  10  with fluoroscopy. 
         [0025]    With continued reference to  FIG. 1B , the plurality of brush elements  36  may be configured about the brush segment  34  in a variety of ways and still achieve the desired functionality. In one non-limiting example, the plurality of brush elements  36  may be disposed radially about the shaft  32  to form a conical or tapered shape. The tapered shape of the brush elements  36  may allow the endovascular brush  22  to extend into vessels with progressively narrower diameters and to ensure contact with the vessel wall, for example. The tapered brush elements  36  may have a distal diameter D 1  ranging from about 1 mm to about 3 mm, and a proximal diameter D 2  ranging from about 2.5 mm to about 5 mm. Other alternatives for the shape of the brush elements  36  include, but are not limited to, the incorporation of a curve in the brush, flat, one side flat or curved with a cylindrical inflatable balloon attached to the other side, helical, fan, oval, square, triangular, octagonal, or rectangular. 
         [0026]    Rapid blood flow in human arteries can easily wash away the endothelial or other target cells that have been dislodged from the vessel wall by the cell sampling device  10 . Accordingly, the plurality of brush elements  36  may be configured to retain the sampled endothelial cells in the presence of the moving fluid. In one embodiment, the size, shape, surface texture, spacing of the brush elements (e.g., the fibers or bristles) or a combination thereof are configured to provide a space or spaces where focal blood flow is relatively slow, which reduces the likelihood of target cells from being washed away in the fluid flow and correspondingly increases the likelihood for dislodged cells to remain on the brush elements  36 . The brush elements  36 , including fibers, bristles, or other elements may be modified to incorporate cell-specific antibodies to capture and retain cells or coated with a biological that will facilitate cell adhesion. 
         [0027]    Additionally, thrombogenicity may be tailored or reduced in various ways. For example, the brush elements  36  may be constructed from a non-thrombogenic material, such as platinum, or may be formed from a thrombogenic material coated or impregnated with a non- or antithrombogenic substance or agent, such as heparin, hirudin, and the like, to reduce the material&#39;s thrombogenicity. This aspect reduces thrombus formation as a result of contact between the brush elements  36  and blood. The brush elements  36  may be constructed of made of polyurethane, polyester, polyglycolic acid, PFTE, polypropylene, polyethylene, nylon, rayon, or Dacron, for example, to provide a softer bristle that is less likely to cause intimal dissection, vessel perforation or other type of vascular injury. In some embodiments, the configuration of the brush elements  36  may also vary and can be arranged in a homogeneous manner or contain open or closed areas, be wavy, bent, straight, or folded in different patterns, for example. 
         [0028]    In one non-limiting example, the brush segment  34  may be relatively flexible and elastic such that the brush segment  34  may be flexed, bent, turned, bowed, twisted, stretched, compressed, or otherwise deformed, from an original shape, without breaking, and capable of recovering the original shape from the deformed shape. This flexibility of the brush segment  34  may be achieved, for example, by forming the brush segment  34  from a material having suitable flexibility and elasticity so that the brush segment  34  returns to the original shape upon removal of the deforming forces. 
         [0029]    With continued reference to  FIG. 1B , the shaft  32  of the endovascular brush  22  may be made of various materials and in various lengths. The shaft  32  should generally be of sufficient length to access the pulmonary vasculature  28  from a selected point of entry. In one embodiment, the shaft  32  may be constructed of a stainless steel alloy, titanium, nitinol, or nickel alloys, for example. 
         [0030]    Referring now to  FIG. 5 , a flow chart setting forth exemplary steps  100  for sampling endothelial cells from arteries and veins within a subject&#39;s pulmonary vasculature  28  is provided. To start the process, at process block  102 , the elongated sheath  14  may be inserted into the guiding catheter  26  that has been inserted into the a target artery  42  (see  FIG. 2 ), proximal to the intended site of interest, such as at a vascular site have a lesion thereon. The elongated sheath  14  may be inserted into the guiding catheter  26  until the distal end  20  of the elongated sheath  14  engages a distal end of the catheter  26 , as shown in  FIG. 2 . Next, at process block  104 , a user may insert the endovascular brush  22  into the access lumen  24  of the elongated sheath  14 , such that the endovascular brush  22  is in a retracted position, as shown in  FIG. 2 . 
         [0031]    At process block  106 , the actuation control  30  of the forceps  12  may be used to axially translate the endovascular brush  22  from the retracted position to the expanded position, as shown in  FIG. 4 . Activation of the actuation control  30  may push the shaft  32  linearly through the access lumen  24  of the elongated sheath  14 , thereby advancing the brush elements  36  into the elongated sheath  14  toward the target artery  42 , as shown in  FIG. 3 . In one non-limiting example, the endovascular brush  22  may be inserted through the subject&#39;s right ventricular apex and advanced to the pulmonary artery under direct visualization, such as through biplane fluoroscopic guidance and Ultravist  370  contrast imaging. 
         [0032]    As shown in  FIG. 4 , the actuation control  30  may be used to push the brush elements  36  out from the elongated sheath  14  to expose the brush elements  36  in the target artery  42 . Next, at process block  108 , the brush elements  36  may be used to obtain endothelial cells  44  from the target artery  42 . Once inserted into the target artery  42 , the brush elements  36  may expand and provide the necessary pressure against the target artery  42  to remove endothelial cells  44  there from. In one non-limiting example, the location of the brush elements  36  in relation to the target artery  42  may be confirmed by imaging the site of interest, for example, with x-ray imaging, fluoroscopy, and/or contrast injection if necessary, to ensure that the brush elements  36  come into contact or overlap the intended vascular segment or lesion. 
         [0033]    The endothelial cells  44  may then be sampled from the target artery  42  by pulling the actuation control  30  in a swift motion, which causes the shaft  32  to retract, and the brush elements  36  to rub against or scrape the target artery  42 , and capture the endothelial cells  44  there from. Then, at process block  110 , the endovascular brush  22  may be retracted into the catheter  26  and into the access lumen  24  of the elongated sheath  14 , as shown in  FIG. 2 . In some embodiments, at process block  112 , endothelial phenotypic profiling may be applied to the captured endothelial cells  44  to determine a state of the pulmonary vasculature at process block  114 . For example, the state of the pulmonary vasculature may indicate the presence of PAH. 
         [0034]    Additionally, or alternatively, once the cell sampling device  10  is removed from the subject, the brush elements  36  of the endovascular brush  22  may be immersed in dissociation medium to collect the cells. The dissociation medium may then be centrifuged and the isolated endothelial cells  44  may be plated on a chamber slide and allowed to adhere. The cells may then be assessed for endothelial origin by immunofluorescent staining for CD31, or allowed to expand in culture. 
         [0035]    In one non-limiting example, to evaluate the efficacy of the cell sampling device  10  to sample pulmonary artery endothelial cells for phenotypic analysis by genome array expression profiling, the endothelial cells retrieved may be quantified using a hemocytometer. A 10 μl aliquot of suspended cells may be quantified by the hemocytometer and a total number determined by the average count per square multiplied by the dilution factor and multiplied by 10 4 . If the number of cells retrieved is insufficient to obtain enough RNA for microarray analysis, the cells may be plated in a P25 tissue culture dish, for example, and expanded. Briefly, cells may be placed in endothelial cell medium (Lonza) with 2.5% fetal bovine serum. Once the plate is confluent, the cells may be harvested for RNA isolation. The percentage of cells that are true endothelial cells may be determined using immunofluorescence immunohistochemistry to detect CD31 expression. 
         [0036]    To characterize endothelial gene expression, RNA may be isolated from cells using, for example, an RNEasy Minikit (Qiagen) and endothelial phenotype may be analyzed using a gene expression microarray that contains approximately 43,603 oligonucleotide probes. cDNA may be labeled using cyanine-3-CTP and hybridized with the microarray slide at approximately 65° C. for about 17 hours. The slides may then be washed according the manufacturer&#39;s instructions and scanned with, for example, an Agilent DNA Microarray Scanner. The images may be analyzed using Agilent Feature Extraction Software, and data may be normalized and functional gene categories may be assigned using the annotation, visualization and integrated discovery (DAVID) bioinformatics database. 
         [0037]    For each time-point that the endothelial cells are harvested, an endothelial phenotypic profile may be defined by a panel of up to 5 genes that show the greatest differential expression between a sham and pulmonary hypertension subjects. These genes may be validated using standard qRT-PCR methodology (ABI Biosystems). Preference may be given to genes that are also differentially expressed over time in the pulmonary hypertension model. The endothelial (patho) phenotypes may then be compared with the hemodynamic variables to identify patterns of gene expression that predict or correlate with hemodynamic indices of pulmonary hypertension. 
         [0038]    In yet another non-limiting example, the endovascular brush  22  may be by evaluated by assessing the pulmonary artery using angiography, histology, and electron microscopy to verify injury and/or damage to the pulmonary artery was minimal. After user of the endovascular brush  22 , selective pulmonary angiography may be used to determine if there has been a perforation (e.g., leakage of contrast dye), a dissection (e.g., “hang-up” of contrast dye outside the vessel), thrombus formation in situ (e.g., intravascular dye stain with filling defect), or vessel occlusion (e.g., abrupt cut-off with dye “hang-up”). 
         [0039]    To perform the angiogram, the endovascular brush  22  may be removed from the guiding catheter  26  and a SF pigtail catheter, for example, may be advanced over a wire to the recently sampled pulmonary artery and placement confirmed using biplane angiography. A 20 cc injection of Ultravist 370 contrast may be performed using a power injector and recorded as a cine angiogram. Angiograms may be reviewed by one or more operators to verify the absence of any vascular injury patterns. 
         [0040]    The present disclosure has been described in terms of one or more preferred embodiments, and it should be appreciated that many equivalents, alternatives, variations, and modifications, aside from those expressly stated, are possible and within the scope of the disclosure.