Cytotechnology 10: 189-204, 1992. 9 1992KluwerAcademic Publishers. Printed in the Netherlands.

Tissue engineering in the vascular graft Stephen P. Massia 1,2 and Jeffrey A. Hubbell 1 1Department of Chemical Engineering and 2Division of Biological Sciences, University of Texas at Austin, Austin, TX 78712-1062, USA Received 7 February 1992; acceptedin revised form 28 April 1992

Key words: vascular grafts, tissue engineering, endothelial cells, cell adhesion, adhesion peptides

Introduction The attachment of cells to solid substrates is an important phenomenon in the field of biotechnology. In biomedical technology, cell attachment to solid substrates is a critical and controlling series of events in the response of tissues to biomedical implants. The control of these adhesive responses is a major concern in the fledgling area of tissue engineering. One example where cell adhesion is desirable in tissue engineering applications is the endothelialization of vascular grafts to obtain hybrid artificial organs with enhanced hemocompatibility. In this review we will discuss current methods employed to promote endothelialization of vascular grafts, approaches to obtain cell typeselective attachment to synthetic vascular graft materials, promotion and control of cell growth on synthetic grafts, and the exploitation of the endothelial cell as a mode for gene therapy in the vascular wall. Cell adhesion to both natural and synthetic substrates is mediated by cell adhesion proteins which are secreted by cells in tissues and are present in the extracellular matrix in vivo. Examples of this family of adhesive proteins include fibronectin (FN), vitronectin (VN), collagens, thrombospondin, von Willebrand factor (vWF), elastin, and laminin (LN). The interaction of cell-surface receptors with cell adhesion proteins

promotes cell attachment to extracellular matrices as well as synthetic substrates. Some cell adhesion receptors recognize and bind to a select group of adhesion proteins whereas other more promiscuous receptors bind to a wide variety of adhesion proteins. The integrin superfamily is an important and well characterized group of cell-surface receptors for both cell-substrate and cell-cell adhesion (reviewed in: Ruoslahti, 1988; Albelda and Buck, 1990; Humphries, 1990; Akiyama et al., 1990; Ruoslahti, 1990). Integrins are characteristically membrane-spanning heterodimeric protein complexes consisting of an ct subunit and a 13subunit. 12 distinct a subunits and 7 distinct ~ subunits have currently been isolated and identified, and several ct[3 combinations have been observed. Integrin complexes containing ~1 and [33 subunits generally are involved in cell adhesion to the extracellular matrix, while the ~2 integrins are involved in cell-cell adhesion, The complement of integrins expressed by different cell types varies greatly. Mammalian cells can express from two to ten different integrins, depending on the cell type (Humphries, 1990). Vascular endothelial cells express the [31 group integrins cq[31, ~2~1, Ot3~l, 0~5~1 and (X6~I, which promote adhesion to collagens ((z3~i: Cheng and Kramer, 1989; Albelda et al., 1989), LN (t~2131: Cheng and Kramer, 1989; Albelda et al., 1989; Kramer et al., 1990;

190 Languino et al., 1989; Kirchhofer et al., 1990; c~6131: Languino et al., 1989), and FN (czs[31: Dejana et al. 1988a; Cheng and Kramer, 1989; Albelda et al., 1989; Conforti et al., 1989). The [33 group integrin CXv[33which is present on endothelial cells recognizes RGD ligands in VN, vWF, fibrinogen, thrombospondin, and FN (Dejana et al., 1988a; Dejana et al., 1989; Dejana et al., 1985; Dejana et al., 1987; Cheng and Kramer, 1989; Albelda et al., 1989; Dejana et al., 1988b; Charo et al., 1987; Cheresh, 1987; Lawler et al., 1988; Cheng et al., 1991). Integrins typically bind to cell adhesion proteins via the rather highly conserved sequence Arg-Gly-Asp-X (RGDX), where X is variant depending on the particular cell adhesion protein. This flanking variant residue has been reported to influence the affinities of particular integrins, however this variant residue does not invoke selectivity of the RGDX ligand for particular integrins (Pierschbacher and Ruoslahti, 1987). This is one of the reasons why there is considerable overlap between receptors and ligands. Furthermore, this is why many cell types, including the endothelial cell, have multiple integrins for the same cell adhesion protein and single integrins which bind to several cell adhesion proteins. Fibronectins contain additional RGDX-Iike sequences, namely Arg-Glu-Asp-Val (REDV) from the CS5 fragment of the type III connecting segment of certain fibronectin types (Humphries et al., 1986; Mould et al., 1991); and Leu-AspVal (LDV) from the CS 1 fragment of the type III connecting segment (Komoriya et al., 1991). Integrin o~4131 binds to these domains to promote attachment and spreading of a select group of cell types (Mould et al., 1991; Mould et al., 1990). Some adhesion receptor-binding domains in cell adhesion proteins are not derived from the RGDX sequence. Laminin has several of these sequences including the well characterized TyrIle-Gly-Ser-Arg (YIGSR) sequence (Graf et al., 1987). A monomeric 67 kD receptor has been reported to recognize this sequence and promote the adhesion, but not spreading, of several cell types including the vascular endothelial cell (Yannariello-Brown et al., 1988). Pro-Asp-Ser-

Gly-Arg (PDSGR) is another laminin-based adhesion ligand related to YIGSR which promotes cell adhesion via an unknown receptor (Kleinman et al., 1989). Endothelial cell adhesion to synthetic vascular graft materials may he beneficial for vascular grafts since the endothelium provides many anticoagulant activities necessary for maintaining patency in the vascular system. Anticoagulant functions are mediated by several products expressed by the endothelial cell. Cell surface-associated heparin-like glycosaminoglycans can inhibit clot formation by binding antithrombin III to facilitate deactivation of thrombin via this inhibitor (Marcum et al., 1984; Rosenberg, 1985). Thrombomodulin is expressed on the luminal surface of endothelial ceil and inhibits thrombin by binding and deactivating prothrombin and activating the thrombin inhibitor protein C (Esmon et al., 1982a; Esmon et al., 1982b). The endothelial cell can suppress platelet activation by secreting the potent platelet inhibitor prostacyclin (Weksler et al., 1977; Cazenave et al., 1979) and by hydrolysis and deactivation of the platelet agonist ADP via a membrane-bound ecto-ATPase (Dosne et al., 1979; Pearson and Gordon, 1979). Endothelial cells can inhibit thrombus formation and growth by producing the fibrinolytic agent tissuetype plasminogen activator (t-PA) (Levin and Loskutoff, 1982). In response to injury, endothelial cells can produce procoagulant factors such as platelet factor, tissue factor, thromboxane A2, clotting factor V, von Wiltebrand factor, and plasminogen activator inhibitor (Fajardo, 1989). The endothelium in vivo maintains a nonthrombogenic luminal surface in the vessel wall and is a net effect of the above described anticoagulant and procoagulant functions of the endothelial cell (Gimbrone, 1986).

Promotion of endothelial cell adhesion to synthetic materials via proteins

Cell adhesion to synthetic materials and extracellular matrices of tissues is mediated by cell adhesion proteins (Grinnell, 1978; Horbett and

191 Schway, 1988). Cell adhesion proteins are present on synthetic materials either by adsorption from the contacting fluid phase, e.g. blood or interstitial fluids, or by endogenous adhesion proteins which cells synthesize and secrete to produce their own extracellular matrix. In the absence of extracellular proteins, cell adhesion proceeds by the direct adsorption of cell-surface proteins to the substrate, followed by replacement of these proteins on the surface by cellularly secreted adhesion proteins. Polyethyleneterephthalate (PET), the base polymer for the woven Dacron vascular graft, and polytetrafluoroethylene (PTFE), the base polymer for the expanded PTFE (ePTFE) vascular graft, are conventional polymeric materials used for the manufacture of vascular prostheses. When seeded with endothelial cells, these materials either lose cells rapidly in a physiologically relevant shear environment (in the case of PET), or are poor substrates for attachment of cells in a stagnant fluid environment (in the case of PTFE) (Kesler et al., 1986). Coating these materials with cell adhesion proteins such as fibronectin has been shown to enhance the strength (Kesler et al., 1986; Seeger and Klingman, 1985,1988; Pratt et al., 1988) and kinetics (Seeger and Klingman, 1988; Pratt et al., 1988) of endothelial cell attachment. Precoating graft materials with platelet-rich plasma, anmiotic proteins, fibrin glue, gelatin, and combinations of collagens and laminin or fibronectin all have been shown to improve endothelial cell attachment (Anderson et al., 1987; Kaehler et al., 1989; Lindblad et al., 1986; Radomski et al., 1987). Although adsorbed cell adhesion proteins enhance short-term attachment of endothelial ceils, platelet attachment typically increases with these coatings (Seeger and Klingman, 1985). Therefore, coating materials with adhesion proteins could lead to increased thrombogenicity. Plasma discharge surface modifications of PET have been shown to enhance short-term retention of endothelial ceils under shear stress when pretreated with fibronectin or exposed to serum proteins during seeding (Pratt et al., 1989). This methodology is advantageous over coating the native polymers with adhesion proteins since the

surface chemistry can be modified to favor adhesion protein adsorption and to stabilize this adsorbed protein layer against desorption and displacement by nonadhesive proteins. However, the surface chemistry produced by plasma discharge methodology is not well defined and optimal conditions for producing adhesive substrates are determined empirically. Furthermore, selective adsorption of specific adhesion proteins is not optimal using this approach, since displacement or exchange of adsorbed proteins can occur, particularly in the presence of plasma proteins. It is quite difficult to control the adhesive and functional responses of a cell to a surface, since these phenomena rely upon the adsorption of cell adhesion proteins. Protein adsorption is an exceedingly complex phenomenon, and, correspondingly, it is very difficult to control. The main difficulty is in controlling the specificity of protein adsorption, e.g. it is difficult to control the retention of selectively adsorbed, specific cell adhesion proteins in the presence of a protein mixture such as blood plasma. Due to this lack of control of adhesion protein retention, it is possible that adsorbed cell adhesion proteins do not provide a finn anchor for the retention of attached cells which are subjected to shear forces present in a vascular graft. Moreover, due to receptor overlap among various cell-types and the general lack of selectivity of adhesion receptors for specific cell adhesion proteins, it is exceedingly difficult to promote the selective attachment of one cell type from a mixed population of cell types. A second difficulty with the adsorbed protein layer is its lack of stability to denaturation and proteolysis, which may affect the long-term behavior of cell adhesive biomedical materials. These difficulties are encountered in tissue engineering and are particularly severe when an endothelialized vascular graft is desired (Yeager and Callow, 1988). Here the adhesion, proliferation, and normal function of endothelial cells on synthetic materials is desired to obtain a hybrid artificial vascular graft with a living and biologically active endothelium. However, materials which support endothelial cell attachment also permit platelet attachment, leading to thrombosis

192 and clotting; and smooth muscle attachment, leading to pseudointimal thickening, the uncontrolled proliferation of smooth muscle cells from the wall of the adjoining native vessel. Endothelial cellselective attachment is desirable for vascular grafts, since thrombosis and pseudointimal thickening are a direct result of the lack of cell typeselective attachment in the vascular graft and lead to the eventual failure of most grafts. Specifically, if platelet attachment is prevented, then mural thrombosis cannot occur on the graft. If smooth muscle cell and fibroblast attachment is prevented, then proliferation and multilayer formation of these cells is prevented. Selective coverage of a graft with an endothelial monolayer should prevent pseudointimal thickening, since endothelial cells do not form multilayers even under conditions where anastomotic hyperplasia and pseudointimal thickening occurs (Callow 1990).

Promotion of endothelial cell adhesion to synthetic materials via peptide adhesion ligands Newer approaches for improved endothelialization of synthetic vascular graft materials have used covalently attached cell adhesion peptides, such as the sequences RGDX and YIGSR described in the introduction section, to promote cell adhesion to synthetic materials (Massia and Hubbell, 1990, 1991b). This new methodology for the promotion of cell adhesion to synthetic materials circumvents the requirement of protein adsorption for cell adhesion. Therefore the difficulties encountered in controlling protein adsorption are avoided. Furthermore long-term stability of adhesive materials is enhanced when adhesive peptides are employed, since these smaller molecules are more stable to denaturation and proteolytic degradation than the larger cell adhesion proteins. Although some peptide sequences are more susceptible to proteolysis than others, we have observed that substrates containing surfacegrafted GRGDY or GYIGSRY are resistant to cellular proteolysis for periods of at least 12 weeks in cell culture (Massia and Hubbell,

1991b). A further advantage of this approach, which could be critical for the success of endothelialized synthetic vascular grafts, is that single types of adhesive ligands can be grafted to material surfaces for potentially cell type-selective adhesion to materials (Hubbell et al., 1991). To accomplish this, the base material must first be rendered very resistant to protein adsorption, for example by the grafting of hydrophilic, nonionic polyethylene oxide chains (Desai and Hubbell, 1991a, 1991b). The subsequently grafted adhesion peptide to this material would provide the only cell adhesion signal, because there are no adsorbed proteins with potentially interfering adhesive signals. Under these conditions, cell type-selective adhesion could be achieved based on the particular peptide ligand employed for grafting, its flanking residues, its surface concentration, the spacer arm between the substrate and peptide, or the orientation of the immobilized peptide. Adhesive peptide ligands were grafted to a poorly adhesive, silylated glass (glycophase glass) (Massia and Hubbell, 1990), and two polymers commonly used in the fabrication of vascular grafts, PET and PTFE (Massia and Hubbell, 1991b). Synthetic peptides were constructed in the general structural design, G-(adhesive ligand sequence)-Y and were covalently coupled to material surfaces by the N-terminal primary amine using the glycyl residue (G) as a spacer. The C-terminal tyrosyl residue (Y) provided a site for radioiodination to determine the amount of surface-bound peptide on materials. When radiolabeled GRGDY was coupled to glycophase glass, the maximum surface concentration of peptide obtainable was determined to be 12 pmol/cm 2 on this substrate (Massia and Hubbell, 1990). Maximum concentrations for PET and PTFE were determined to be 0.139 and 0.031 pmol/cm 2 respectively with the chemical derivatization schemes used (Massia and Hubbell, 1991b). Using the glycophase substrate, it was determined that peptide coupling reaction conditions were quantitative when input peptide concentrations were below the saturated surface concentration level of 12 pmol/cm 2. Therefore precise

193 control of peptide surface density could be obtained by manipulation of the peptide concentration in the reaction mixture (Massia and Hubbell, 1990). By varying the peptide surface concentration on GRGDY-grafted glass substrates, it was shown that a minimal peptide surface density of 10 fmol/cm 2 was required to promote maximal spreading, stress fiber, and focal contact formation of human foreskin fibroblasts (HFFs). This peptide surface concentration corresponds to a peptide spacing of about 140 nm, or approximately 105 ligands per cell. Specific anti-integrin antibodies demonstrated that fibroblast adhesion to GRGDY-grafted glass substrates was mediated solely by the integrin r (Massia and Hubbell, 1991a). We utilized adhesion peptide grafting and examined several cell adhesion ligands to obtain selective attachment of endothelial cells from among blood cells and nonendotheliai vessel wall cells (Hubbell et al., 1991). Cultured human umbilical vein endothelial cells (HUVECs) were used as the endothelial cell prototype. Whole blood, platelet-rich plasma (PRP), PRP prestimulated with 5 I.tM ADP, HFFs, and human vascular smooth muscle cells (HVSMCs) were examined as prototypes of blood-derived and nonendothelial blood vessel wall cells. All cell types were seeded on glycophase glass containing covalently grafted GRGDY, GYIGSRY, GPDSGRY, and GREDVY. The adhesion ligand sequences within these synthetic peptides were described previously in the introductory section. GRGDY, GYIGSRY, and GPDSGRY supported attachment and spreading of HFFs, HVSMCs, and HUVECs. It was a surprising result to observe no spreading of platelets on GRGDY-grafted glass, even though the platelet expresses several integrins. The platelet fibrinogen receptor integrin (Xllb~3 (gp Ilb-IIIa) affinity is high only on activated platelets and can positively modulate the affinities of other platelet integrins (Du, Plow, Ginsberg et al., 1991). Platelets activated with 5 ~tM ADP, which is sufficient to trigger high affinity binding of 0~iib133to RGD, were still observed not to spread on N-terminally grafted GRGDY (Hub-

bell et al., 1991). When GRGDY was grafted by the C-terminus on aminosilane-modified glass, nearly 100% of the exposed substrate surface was covered with a monolayer of spread platelets, even without ADP preactivation. This demonstrated that the orientation of the adhesion ligand with respect to a substrate surface can affect the affinity of the adhesion receptor for the ligand. This orientation requirement for ligand binding seemed to be specific for the platelet integrin CZnb133, since HFFs and HUVECs do not express this integrin and were observed to spread equally well on both C-terminally and N-terminally grafted GRGDY (Hubbell et al., 1992). Surface-grafted GREDVY on glass and polymeric substrates was observed to be highly selective for endothelial cells, since these substrates supported HUVEC spreading but not HFF, HVSMC, or platelet spreading. HUVEC spreading on these substrates was a complete response, since focal contact and stress fiber formation was observed. Specific binding of GREDVY via a single class of receptors was observed in HUVECs with 5.8 • 106 binding sites per cell and a dissociation constant of 2.2 x 10-6 M (Hubbell et al., 1991). Further investigation identified the endothelial cell adhesion receptor for REDV as integrin cz4131. Antifunctional antibodies directed against integrin subunits cz4 and 131 inhibited cell adhesion on GREDVY-grafted substrates and the cz4 subunit localized into fibrillar structures within spread cells on the GREDVY-grafted substrates, but not within spread cells on RGD-grafted substrates. The o~4 subunit was shown to be expressed upon HUVEC membranes by whole-cell ELISA (Massia and Hubbell, 1992). Two proteins (M r 144 and 120 kD) were isolated from HUVEC membrane extracts by REDV ligand affinity chromatography and were demonstrated by immunoprecipitation to be the integrin subunits (24 (144 kD) and 131 (120 kD); furthermore these analyses demonstrated that the subunits formed a complex. Western blotting analysis confirmed the identification of the 144 kD band as the o~4 subunit and the 120 kD band as the

194 ~l subunit (Massia and Hubbell, 1992). These studies identified a new integrin receptor expressed by endothelial cells and provided more evidence that REDV is a cell type-selective ligand for integrin ot4131. Other workers have utilized adhesive peptide sequences to promote endothelial cell attachment to synthetic polymers. Matsuda et al. observed endothelial cell attachment to GRGDSP-grafted polyvinyl alcohol in the absence of serum-derived adhesion proteins (Matsuda et al., 1989). Urry et al. constructed synthetic elastic polymers based on the repeating amino acid sequence Gly-ValGly-Val-Pro (GVGVP) of the elastomeric protein elastin. They formed elastomeric materials by crosslinking GVGVP to form a polypentameric matrix with elastic characteristics similar to those of the vascular wall. This material could be formed into tubular shapes and was rendered cell adhesive by copolymerization of adhesive protein sequences into the matrix. These materials could support the attachment of endothelial cells in the absence of cell adhesion proteins when the percentage incorporation of the peptide GRGDSP into the matrix was 1% (Nicol et al., 1991).

Promotion of bioactive synthetic vascular grafts by the addition of endothelial cells Synthetic vascular grafts have only been successful as large vessel substitutes, whereas small diameter (3-4 nun) vascular grafts rapidly fail in humans by occlusion due to thrombosis and neointimal thickening within the lumen of the graft (Yeager and Callow, 1988; Callow, 1987). Blood flow is reduced in small diameter bypass grafts, resulting in the formation of a highly thrombogenic, typically acellular pseudointimal layer of compacted fibrin (Berger et al., 1972). In almost all animal models, migration of endothelial cells from the anastomosing native vessel tissue (pannus ingrowth) progresses until complete endothelial coverage of the thrombogenic acellular pseudointima of the synthetic graft occurs (Sauvage et al., 1974; Sottiurai et al., 1983). Pannus ingrowth of endothelium is a spontaneous event in most

nonhuman species, however endothelial cell growth in humans is limited to one centimeter pannus ingrowth at the anastomosis (Berger et al., 1972; Sauvage et al., 1974). Since an endothelial cell lining does not completely develop on synthetic graft surfaces in humans and a thrombogenic acellular pseudointima rapidly develops on the luminal surface of the graft, many efforts have focused on developing endothelial cell seeding techniques to promote the growth of endothelial cellson the luminal surface of the synthetic graft (Mosquera and Goldman, 1991; Yeager and Callow, 1988). Endothelial cell seeding, in general, is the transplantation of endothelial cells to the luminal surfaces of synthetic vascular grafts prior to implantation of the graft to promote the formation of a continuous nonthrombogenic endothelial lining on the graft lumen. Successful endothelialization of vascular grafts depends upon efficient harvesting of large numbers of viable endothelial cells from donor tissues, efficient attachment and retention of endothelial cells to the synthetic graft, and adequate replication of cells to completely cover the graft luminal surface. The harvesting method of choice for the isolation of endothelial cells from blood vessels is enzymatic, since mechanical scrape harvesting techniques, used in the earliest cell seeding trials, lead to extensive cell damage and smooth muscle cell contamination (Mosquera and Goldman, 1991). With enzymatic harvesting, the endothelium of vascular tissue is bathed in collagenase to separate endothelial cells from the extracellular matrix and basement membrane. Graham et al. were the first to isolate pure canine endothelial cells enzymatically and use them to seed vascular grafts. They demonstrated that the seeded grafts were endothelialized 4 weeks after implantation. With Dacron grafts, they observed no thrombus formation in 86% of the seeded grafts and 40% of the unseeded control grafts (Graham et al., 1980) 4 weeks after implantation in the canine model. 91% of the seeded ePTFE grafts examined by this group in the canine model were thrombus-free in comparison to only 4% of the unseeded controls (Graham et al., 1982). After 16 weeks, 73% of the

195 seeded ePTFE grafts remained patent compared with 27% of the unseeded controls (Stanley et al., 1982). Although large vessel endothelial cells have been successfully used to seed vascular grafts in dogs to form confluent luminal monolayers and increase graft patency, similar trials in humans have been inconclusive (Herring et al., 1987b; Herring et al., 1984; 0rtenwall et al., 1986; Silla et al., 1987). It was demonstrated by Kent et al. that there was significant species variation in endothelial cell harvesting, attachment, and growth. They observed that endothelial cells derived from long human saphenous veins produced a significantly lower yield of viable cells with a longer average doubling time than the values obtained with ceils derived from nonhuman large vessel endothelium. They concluded that poor harvests from human large vessels resulted in limited numbers of cells available for seeding and that growth requirements for the seeded cells were too stringent for successful endothelialization of grafts (Kent et al., 1989). Jarrell, Williams, et al. developed techniques for seeding vascular grafts with human microvascular endothelial cells derived from fat tissue. Sufficient amounts of viable cells were obtained from this tissue within a 1 hour time period for high-density graft seeding and improved endothelialization of grafts. Jarrell et al. observed rapid attachment of microvascular endothelial cells to Dacron coated with plasma. These cells were exceptionally resistant to physiological shear stresses after allowing only 1 hour for attachment (Jarrell et al., 1986). This research group observed in a more quantitative study that human microvascular endothelial cells attached to polymeric substrates maximally and demonstrated maximum retention 30 minutes after seeding (Pratt et al., 1988). This group observed further improvements in endothelialization rates, resistance to shear stress, and response to growth factors when cells were sodded rather than seeded on vascular grafts. Cell seeding was defined as preclotting graft materials with endothelial cells in platelet-rich plasma, whereas cell sodding was the transplantation of endothelial cells at or above

confluence onto preclotted material (Rupnick et al., 1989). Limited clinical trials have been undertaken in recent years to evaluate the performance of seeded vascular grafts in humans. Typically the results in these studies have been inconclusive because of the variabilities in harvesting yields and cell survival, and the small number of available participants for these trials. Another problem with clinical trials is the inability to directly observe the development of endothelium on seeded grafts. Therefore the primary indicators of success or failure in clinical trials are patency, platelet deposition, and thrombogenicity index (Mosquera and Goldman, 1991). Some early clinical trials demonstrated that endothelium can develop on seeded grafts in humans (Herring et al., 1985; Walker et al., 1988), however this was not a consistent finding (Herring et al., 1987a). The earliest clinical trial with enzymatically harvested large vessel endothelial cells demonstrated cumulative patency rates after 3 months of 93% for seeded and 84% for unseeded femoropopliteal ePTFE grafts. After 1 year the patency rates were significantly different, with 81% for seeded and 31% for unseeded grafts (Herring et al., 1987b). This success was demonstrated in a small population (n = 17) and similar results have not been repeated. {}rtenwall et al. evaluated the performance of seeded Dacron grafts in 22 patients undergoing reconstruction of the infrarenal artery. In each patient, one limb of aortic Dacron bifurcation graft was seeded with autologous endothelial cells derived from the saphenous vein and the other limb was sham-seeded with culture medium only. They observed an early decrease in platelet accumulation on the seeded graft limb (0rtenwall et al., 1986) and showed that platelet deposition on the seeded limb remained reduced over a period of 12 months ((3rtenwall et al., 1990). In other studies, this group evaluated seeded versus unseeded segments in preclotted femoropopliteal ePTFE grafts. They observed a reduction of platelet deposition in seeded segments at 1 and 6 months in 18 of 23 participants in the trial (Ortenwall et al., 1989).

196

Fasol et al. evaluated platelet deposition and viability in seeded and unseeded 6 mm ePTFE femoropopliteal and femorocrural grafts coated with fibrin glue. They observed no differences in platelet accumulation or platelet survival in the seeded group versus the unseeded group after 1 year of follow-up (Fasol et al., 1989). These results suggested that endothelialization did not occur or was incomplete in the vascular graft. Without direct assessment of endothelialization of the grafts, it was not conclusive that endothelialization did occur. The initial clinical studies of Jarrell et al. observed the effect of seeded ePTFE hemodialysis access grafts in patients with chronic renal failure who had demonstrated early failure of the routinely unseeded grafts. This limited study was undertaken with four patients who had a mean graft patency until first thrombosis of 1.4 + 0.6 months in untreated grafts. When grafts were sodded with microvascular endothelial cells and implanted, mean graft patency until first thrombosis increased to 5.8 + 3.8 months (Jarrell and Williams, 1991). In a case study, this group directly demonstrated complete endothelialization of a sodded Dacron mesoatrial venous graft which was retrieved 9 months after implantation. They observed an endothelial cell monolayer on the luminal surface of the graft, a subendothelial layer of extracellular matrix with spindle-shaped cells, and granulation tissue around the Dacron fabric (Park et al., 1990). Short harvesting time requirement and rapid attachment to substrates are properties which make microvascular endothelial cells advantageous for the success of seeded or sodded vascular grafts in humans. Furthermore donor adipose tissue for isolation of these ceils can be easily removed with minimal risk during surgery in large quantities sufficient to obtain confluently seeded vascular grafts at the time of implantation (Jarrell and Williams, 1986). Treatment of grafts with microvascular endothelial cells in clinical trials consistently improved short-term patencies, however further studies are necessary to evaluate the long-term performance of these grafts with respect to luminal surface thrombogenicity and modulation of anastomotic hyperplasia.

Endothelialization of synthetic vascular grafts by vascular healing mechanisms As previously mentioned, pannus ingrowth of endothelium and the underlying smooth muscle cell layer is not sufficient in humans to obtain complete luminal coverage in synthetic vascular grafts. The central portions of unseeded grafts in humans never develop an endothelial layer and remain covered with compacted fibrin and perhaps are thrombogenic. Although endothelial cell seeding has recently been successful in enhancing endothelialization of grafts and improving shortterm patencies in humans, Clowes et al. have investigated an alternate method to provide endothelial coverage within vascular grafts. Their approach for endothelialization relies upon healing responses of the host tissue, namely transanastomotic ingrowth (from the anastomosed vascular wall tissue) and transmural capillary ingrowth (through the wall of the graft from extra-graft granulation tissue), to obtain luminal coverage of porous grafts with endothelial and smooth muscle cells (Clowes and Kohler, 1991). The initial studies of Clowes et al. examined healing of ePTFE grafts (4 mm diameter, 30 ~tm internodal distance) implanted in the aortoiliac circulation of juvenile baboons (Clowes et al., 1985, 1986a). They observed smooth muscle cell proliferation underneath but not ahead of the advancing endothelial monolayer. Complete coverage with this luminal layer of endothelial cells and intimal layer of smooth muscle cells was observed at 12 months in 60% of the grafts (7 to 9 cm in length) examined (Clowes et al., 1986a). When 4 mm ePTFE grafts with higher porosity (60 l.tm intemodal distance) were implanted in baboons, endothelialization of the graft was complete within 2 weeks. Microscopy studies revealed that microvessels in the granulation tissue surrounding the graft would rapidly grow into the lumen of the graft through pores in multiple sites to provide endothelial cell monolayers at the luminal surface of the graft. Pericytes (capillary smooth muscle cells) comigrated with capillary endothelial cells and proliferated under the luminal endothelium to form an intimal layer, which

197 was indistinguishable from intima derived from large vessel smooth muscle cells. As in the less porous grafts, smooth muscle cells proliferated only in the presence of endothelial cells. This study demonstrated that capillary-derived endothelial and smooth muscle cells could transmurally co-migrate into grafts with a permissible porosity and rapidly form a large vessel-like endothelium and intimal smooth muscle cell layer (Clowes et al., 1986b). A more recent study by Clowes et al. further investigated the effects of graft porosity on vascular healing in the baboon arterial graft model. They examined explanted ePTFE grafts of varying porosity (between 10 and 90 ktm internodal distance) after 1 and 3 months. They observed that endothelialization was incomplete at both time points and healing occurred solely via ingrowth from the adjacent artery in low porosity 10 and 30 btm grafts. High porosity grafts (60 and 90 lam intemodal distances) developed complete luminal endothelial coverage by 1 month. By 3 months, the highest porosity graft (90 I.tm) developed focal regions where endothelial cells were lost. This group concluded from this study that a 60 ~tm internodal distance was near the optimal porosity for stable and complete endothelial cell coverage in the ePTFE graft (Golden et al., 1990b). Although Clowes et al. observed porosity effects on healing of ePTFE grafts in baboons, they recognized that Dacron grafts are sufficiently porous for capillary ingrowth but fail to heal in humans. The 60 ~tm ePTFE graft was not available for clinical use at that time, therefore it was not known if healing of grafts with these properties would occur in humans. The lack of spontaneous endothelialization of vascular grafts in humans had traditionally been attributed to a wound healing deficiency in humans. Clowes et al. proposed that Dacron itself may inhibit vascular healing by an unknown mechanism. They tested this hypothesis by comparing the healing of 4 mm porous Dacron grafts with that of 4 mm 60 ~tm porous ePTFE grafts in the baboon arterial graft model. Explants were examined at 2, 4, and 12 weeks. Endothelialization was complete in all of

the ePTFE grafts at all three time points, however healing was complete in only one of five Dacron grafts at 12 weeks (Zacharias et al., 1987). This result supported the theory of inhibition of vascular healing by Dacron itself and was in agreement with a similar effect observed by Greisler et al. in a rabbit aorta model (Greisler et al., 1986). Recent clinical trials with composite 30 and 60 ~tm ePTFE grafts by Clowes et al. in patients undergoing femoropopliteal bypass resulted in no difference in platelet uptake in the 30 and 60 ktm segments (Clowes and Kohler, 1991). One explanation for this result was that older humans could not heal porous grafts by an angiogenic mechanism. Since the grafts were not retrieved, which is the typical protocol in clinical trials, endothelialization was not determined directly and platelet deposition studies may not be a sensitive indicator for endothelialization. The grafts used in this study were wrapped with a porous PTFE film, whereas unwrapped grafts were used in the earlier baboon studies. Therefore the earlier baboon studies did not precisely parallel this clinical trial. Clowes et al. subsequently implanted wrapped grafts in baboons and observed transmural capillary ingrowth and endothelialization. However this response was retarded in comparison to that of unwrapped grafts (Clowes and Kohler, 1991). In light of these results, it was inconclusive why healing did not occur in the porous graft segments in humans. Although healing of vascular grafts progresses to completion in baboons and other animal species, this response has not yet been demonstrated in humans. Until the healing response can be enhanced in humans, endothelialization of vascular grafts by angiogenic mechanisms is not a feasible method for improving patencies in synthetic vascular grafts for clinical application.

The effects of growth factors on implanted vascular grafts In all synthetic vascular grafts, the biological response occurs rapidly and is initiated by the adsorption of proteins, including clotting factors,

198 onto the luminal surface of the graft. The conventional polymeric materials for vascular grafts, namely PET and PTFE, rapidly adsorb circulating proteins from the bloodstream to form a fibrinogen-rich, highly thrombogenic protein layer. Subsequently platelet deposition and mural thrombosis occurs on the graft lumen (LoGerfo, 1991). If this activity is not sufficient to occlude the graft, the thrombotic layer degrades to an acellular pseudointimal layer of compacted fibrin. Smooth muscle cells, fibroblasts, and perhaps endothelial cells migrate into the synthetic graft in a pannus or sheet from the adjacent native blood vessel. This pannus ingrowth rarely progresses in humans until complete luminal coverage by endothelial cells is obtained. Continued proliferation of the smooth muscle cell layer and secretion of a fibrous matrix by these cells at the anastomosis (anastomotic hyperplasia) typically occurs whether endothelialization is complete or not. Histological examination of hyperplastic lesions at anastomotic sites in the vascular graft demonstrated that the unique pathological feature in neointimal hyperplasia was the change in smooth muscle function from a contractile nature to predominantly secretory activity. Subsequent thickening of the neointimal layer, due to smooth muscle cell proliferation and secretion of vessel wall-active factors and extracellular matrix proteins, causes anastomotic narrowing and eventual occlusion of the graft (Sottiurai et al., 1983). The cellular response to the synthetic vascular graft can be thought of as a healing response where the wound is the graft itself and growth factor-mediated cellular proliferation is a consequence of the wound healing process (Clowes et al., 1985). The platelet was first thought to be an important mediator of cellular proliferation in the healing graft since platelet adherence and degranulation precede pannus ingrowth and smooth muscle cell proliferation. Ross et al. demonstrated that platelet granules contain potent mitogens for cultured smooth muscle cells and suggested that a high localization of growth factors (predominantly platelet-derived growth factor; PDGF) from degranulating platelets in a vessel injury site could induce smooth muscle cell pro-

liferation (Ross et al., 1974; Ross and Glomsett, 1976a, 1976b). Although platelet activation and PDGF release could play a major role in stimulating anastomotic hyperplasia, it is well known that anastomotic hyperplasia develops and progresses long after the peak in platelet activation and deposition on the vascular graft (Graham and Fox, 1991). Furthermore studies with antiplatelet agents suggest that platelet inhibition alone is not sufficient to prevent anastomotic hyperplasia (Clowes and Reidy, 1991). These observations suggest that factors other than platelet activation may play a role in the progression of anastomotic hyperplasia. When Clowes et al. began studies with the 4 mm ePTFE vascular graft in the baboon model, they observed migration of smooth muscle cells and endothelium from the cut edge of the artery. These cells were observed to continue to proliferate at the site of the anastomosis despite complete endothelial cell coverage. The intimal layer was observed to be consistently thicker at the anastomotic site than at adjacent regions in the graft. Clowes et al. hypothesized that anastomotic stenosis created flow abnormalities and induced chronic nondenuding endothelial cell injury. They initially proposed that thrombosis would occur on the injured endothelium and platelet release of PDGF was the major cause of continued smooth muscle cell proliferation (Clowes et al., 1985). Further investigation by this group indicated that platelet accumulation did not occur on endothelialized grafts, however smooth muscle cell proliferation continued (Reidy et al., 1986). Studies by Fox et al. demonstrated that injured endothelial cells in vitro produce more PDGF-like mitogens than uninjured cells (Fox and DiCorleto, 1984). Based on this knowledge and the observations that smooth muscle cell proliferation occurs exclusively in association with endothelium, Clowes et al. hypothesized that injured endothelial cells and smooth muscle cells may be secreting a smooth muscle cell mitogen (Clowes et al., 1986a; Reidy et al., 1986). To test this hypothesis, these workers retrieved ePTFE grafts from the baboon model, perfused these grafts and examined mitogenic activity of the

199 perfusates in cell culture assays. They observed markedly increased mitogenic activity in graft perfusates when compared to perfusates from arteries of equal length (Zacharias et al., 1988). To further identify mitogens in vascular grafts, Birinyi et al. cultured smooth muscle cells derived from hyperplastic lesions of patients with failed lower extremity vascular grafts. They demonstrated that these cells express genes for PDGF A chain (PDGF-A) and a PDGF receptor. Furthermore these cultures were observed to secrete biologically active PDGF-like molecules. It was concluded from these results that anastomotic stenosis in failing vascular grafts occurs by an autocrine growth effect, where the proliferating cells may stimulate their own growth by secreting and responding to PDGF-Iike mitogens (Birinyi et al., 1989). In the baboon ePTFE arterial graft model, Golden et al. observed that graft intimal cells produced more PDGF-A than intimal cells derived from aorta (Golden et al., 1990a). In situ hybridization studies by this group demonstrated that PDGF-A chain is expressed by endothelial cells and in limited amounts by smooth muscle cells in healed vascular grafts (Golden et al., 1991). These results are further evidence of an association between PDGF-A chain expression by neointimal cells and smooth muscle cell proliferation. It is not clear however that PDGF-A has a direct effect on smooth muscle cell proliferation. Cultured endothelial cells have been shown to produce PDGF-A exclusively on the abluminal side (Zerwes and Risau, 1991) suggesting the possibility of abluminal secretion of PDGF-A by endothelial cells in vivo. However, abluminal secretion of mitogens by endothelial cells in vivo cannot be monitored directly. Furthermore potent specific inhibitors for PDGF-A are not available to directly prove that PDGFoA promotes smooth muscle proliferation in the vascular graft (Clowes and Kohler, 1991). Therefore direct evidence for abluminal secretion of PDGF-A by endothelial cells in vivo cannot be demonstrated. It is also possible that other growth factors not detected intraluminally could be secreted abluminally and have major effects on smooth muscle cell proliferation.

The endothelial cell as a vehicle for gene therapy in the vascular graft

Although endothelial cell seeding of vascular grafts has resulted in short-term improvements in graft patency, long-term mechanisms of failure such as anastomotic hyperplasia have not been overcome in the small diameter vascular graft. Genetic modification of endothelial cells has recently been investigated as a possible means to influence both luminal and abluminal activities which contribute to graft failure. Zwiebel et al. demonstrated that efficient gene transfer was feasible in rabbit endothelial cells in vitro. They used three retrovirus vectors to transfer genes encoding for neomycin resistance, human adenosine deaminase, and rat growth hormone. Expression of all three genes was confirmed in these studies (Zwiebel et al., 1989). Another in vitro study by Dichek et al. used retroviral-mediated gene transfer to promote the expression of genes of 13-galactosidase and human tissue-type plasminogen activator into cultured sheep endothelial cells. The transduced cells were seeded onto intravascular stents and the stents were expanded by in vitro balloon inflation. They observed that most of the cells remained on the stent after expansion and continued to produce transferred gene products (Dichek et al., 1989). Based on their in vitro results, Dichek et al. suggested that genetically engineered endothelial cells seeded on intravascular stents could survive implantation and balloon catheter expansion in vivo and could be utilized to improve stent function through localized delivery of anticoagulant, thrombolytic, or antiproliferative agents. Nabel et al. developed a method to introduce genetically modified endothelial cells in vivo onto denuded segments of mini-pig arteries. They used a double balloon catheter to introduce genetically altered endothelial cells into discrete segments of the arteries. The genetically altered cells expressed 13-galactosidase, and examination of arterial explants 2 to 4 weeks after transfer demonstrated the presence of ~-galactosidase-producing cells on lumen of the vessel segments (Nabel et al., 1989). Further investigation by Nabel et al.

200 with a similar animal model and catheter delivery s y s t e m demonstrated that genes could be introduced directly to cells in the vascular wall. Gene expression was observed within all three layers of the arterial wall but was absent in the lung, kidney, liver, and spleen. This demonstrated that D N A delivery and transfection was limited to the arterial wall (Nabel e t al., 1990). Wilson e t al. seeded Dacron carotid interposition grafts with genetically modified [3-galactosidase-producing endothelial cells in dogs. 5 weeks after implantation, they observed genetically modified cells lining the luminal surface of the graft (Wilson e t al., 1989). These studies demonstrated the feasibility of using the endothelial cell as a vehicle for gene therapy at sites of vascular lesions and in endothelialized vascular grafts. Genetic modification of endothelial cells for vascular graft applications could include modifications to increase endothelial cell secretion of products which effectively inhibit thrombosis and neointimal hyperplasia.

Future outlook in vascular graft tissue engineering Unless synthetic vascular graft materials can be rendered completely nonthrombogenic, it will be necessary to establish a healthy endothelial layer on the luminal surfaces o f synthetic vascular grafts. It is unclear whether endothelialization of porous grafts in humans can occur via transmural capillary ingrowth, therefore material modifications to p r o m o t e either transmural migration or endothelial cell seeding are necessary to p r o m o t e cell coverage. With the availability o f microvascular endothelial cells f r o m adipose tissue for confluent seeding of synthetic grafts prior to implantation, it is possible to immediately reduce thrombogenicity of the implant and improve short-term patencies. W h e t h e r vascular healing occurs via induced capillary ingrowth or is enhanced by seeding, ultimately the small diameter vascular graft m a y fail via anastomotic hyperplasia. As the molecular basis for neointimal thickening is elucidated, the

application o f exogenous factors or genetic manipulation of the endothelial or smooth muscle cell m a y be efficacious in controlling anastomotic hyperplasia in the graft. Adhesion peptide-grafted materials that promote endothelial cell-selective attachment m a y effectively p r o m o t e transmural capillary migration and prevent smooth muscle cell and fibroblast ingrowth f r o m the adjacent native vessel. Without these latter cells forming the neointima beneath the endothelial layer in the seeded vascular graft, anastomotic hyperplasia m a y be avoided and perhaps long-term patencies could be obtained.

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Address for offprints: Jeffrey A. Hubbell, Department of Chemical Engineering, University of Texas at Austin, Austin, TX 78712-1062, USA, Telephone (512)471-1690, Fax (512) 4717963