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Vascular tissue engineering: structure versus function
1268
JOURNAL OF VASCULARSURGERY lune 2000
Lifeline Research Meeting Abstracts
autologous in vitro endothelialization ofinfrainguinal ePTFE grafts in 100 patients: a 9-year experience. Surgery 1999;126:847-55. 12. Schense JC, Hubbell IA. Crossqinking exogenous bifunctional peptides into fibrin gels with factor XIIIa. Bioconjug Chem I999;I0:75-81. i3. Schima H, Tsangaris S, Zilla P, Kadletz M, Wolner E. Mechanical simulation of shear stress on the walls of peripheral arteries. J Biomech 1990;23:845-51.
VASCULAR TISSUE ENGINEERING: STRUCTURE VERSUS F U N C T I O N Elazar R. Edelman, MD, PhD Helen M. Nugent, MD MassachusettsInstitute of Technology Cambridge, Mass
The blood vessel is a structure of extraordinary sophistication. The physical continuity of vascular cells and tissues that make up the blood vessel wall contribute to its structural integrity while also maintaining homeostasis through biochemical regulation. The endothelial monolayer that lines the normal blood vessel serves as a twofold regulator of vascular physiology. The endothelium provides structural integrity to the blood vessel by forming a continuous, selectively permeable, thromboresistant bartier between drcnlating blood and the arterial wall. At the same time, it also produces and supplies products that control blood flow, vessel tone, thrombosis, platelet activation, adhesion, aggregation, leukocyte adhesion, monocyte infiltration, and smooth muscle cell migration and proliferation. Endothelial injury, such as occurs after angioplasty, not only removes the physical barrier but also interferes with the biochemical regulatory potential of the blood vessel and sets into motion a sequence of events that leads to the proliferation and migration of smooth muscle cells resulting in obstructive arterial lesions. This process of restenosis leads to critical narrowings in significant numbers of patients after angioplasty, bypass grafting, and organ transplantation. Tissue engineering enables the development of biological substitutes that restore, maintain, or improve tissue function 1 while also providing substrates by which to examine structure-function relationsbips for specific tissues or organs. Cells may be implanted at sites distant or in different configurations from their original state, enabling examination of any added potential benefit of cell secretory function to regulation of tissue biology above that imposed by preservation of tissue architecture alone. This might be especially important in vascular biology where the autocrine, paracrine, and endocrine function of the endothelium is rapidly emerging. Innovative studies
have attempted to recreate the structure of the blood vessel by autologous endothelial cell transplantation, implantation of endothelial cell-seeded interposition grafts, or endovascular stents. 2-6 Yet, the question remains as to whether reestablishing biochemical control ofvascnlar homeostasis also requires reestablishing the ordered architecture of the blood vessel. We have demonstrated, through the use of tissue-engineered endothelial cells, that the biologic effect of these cells on blood vessel regulation is maintained even when they are implanted at a distance from the lumen. Engrafted endothelial cells on three-dimensional polymer matrices, implanted in the perivascular space of injured rat carotid arteries, reduced intimal thickening by 88%.7 In contrast, the isolated infusion of heparin, an endothelial cell product analog and potent smooth muscle cell inhibitor, only reduced intimal hyperplasia by 30%. These experiments supported the hypotheses that endothelial control over vascular repair is derived from the sum of a number of secreted endothelial cell-based products and need not emanate from the luminal surface. 8 We recently addressed two important questions relating to the biologic effects of perivascular endothelial cell implantation. First, we examined whether allotransplantation of endothelial cell grafts was effective in controlling vascular repair in a porcine carotid artery model of vascular injury, thought to be less responsive to growth-regulatory agents than simpler animal models. Second, we explored whether xenotransplantation, a central issue in developing safe and practical clinical strategies, was more or less effective than allotransplantatlon. We now report that perivascular tissue-engineered endothelial cell implants exert profound control over intimal growth after arterial injury in pigs. Furthermore, despite an increased immtme response to cross-species transplantation, beneficial control of vascular repair was maintained. These results provide insight into how the endothellum controls vascular homeostasis and are a further step toward the development of clinically viable strategies for modulating vascular repair after injury. Further exploration of how tissue-engineered endothelial cell grafts control vascular repair, particularly in settings of more chronic vascular injury, will afford insight into the structure and function of the blood vessel wall and into how experimentally effective techniques may be brought to clinical fruition. REFERENCES
1. Langer R, Vacanti JP. Tissue engineering. Science 1993;260:920-5. 2. Nabel EG, Plautz G, Boyce FM, Stanley JC, Nabel GJ.
JOURNAL OF VASCULARSURGERY Volume31, Number6
3.
4.
5.
6.
7.
8.
Recombinant gene expression in vivo within endothelial cells of the arterial wall. Science 1989;244:1342-4. Messina LM, Podrazik RM, Whitehill TA, Ekhterae D, Brothers TE, Wilson JM, et al. Adhesion and incorporation of lacZ-transduced endothelial cells into the intact capillary wall in the rat. Proc Nat] Acad Sci U S A 1992;89:12018-22. Conte MS, Birinyi LK, Miyata T, F~allon JT, Gold HK, Whitemore AD, et al. Efficient repopulation of denuded rab~ bit arteries with autologous genetically modified endothelial cells. Circulation 1994;23:2161-9. Wilson JV¢, Birinyi LK, Salomon RN, Libby P, Callow AD, Mulligan RC. Implantation of vascular grafts lined with genetically modified endothelial cells. Science 1989;244:1344-6. Dichek DA, Neville RF, Zwiebel JA, Freeman SM, Leon MB, Anderson WF. Seeding ofintravascular stents with genetically engineered endothelial cells. Circulation 1989;80:134753, Nathan A, Nugent MA, Edelman ER. Tissue engineered perivascular endothelial cell implants regulate vascularinjury. Proc Nat] Acad Sci U S A 1995;92:8130-4. Nugent HM, Rogers C, Edelman ER. Endothelial implants inhibit intimal hyperplasia after porcine angioplasty.Circ Res 1998;84:384-91.
G E N E T I C M A N I P U L A T I O N S OB CELLS AND TISSUES F O R T H E T R E A T M E N T O F RESTENOSIS
Michael]-. Mann, MD Brigham and Women's Hospital~Harvard Medical School Boston, Mass
Recurrent narrowing of arteries following percutaneous angioplasty, atherectomy, or other disobliterative technique is a common clinical problem that severely limits the durability of these procedures for patients with atherosclerotic occlusive diseases. In the case of balloon angioplasty, restenosis occurs in approximatcly 30% to 40% of treated coronary lesions and 30% to 50% of superficial femoral artery lesions within the first year. Intravascular stents may reduce the restenosis rates. However, the incidence remains high, and long-term data are limited, Despite technological advances in the development of minimally invasive and endovascular approaches to treat arterial occlusions, the full benefit of these gains awaits the solution of this fundamental biologic problem. Restenosis is an attractive target for gene therapy not only because of its frequency but also because it is a local tissue reaction that develops precisely at a site of intervention to which access has already been achieved. A potential advantage of the genetic approach over more conventional pharmacotherapies is that a single dose o f a gene therapy agent may have a prolonged biological effect. The appropriate genetic modification, performed locally at the time of angioplasty, could induce a long-term benefit in patency by altering the healing response, The poten-
Lifeline Research Meeting Abstracts
1269
tial role for gene therapy in the prevention of restenosis will depend on the identification of an appropriate molecular target, a suitable vector system for efficiently targeting vessel wall cells, and methods of achieving local delivery without producing undue damage or distal tissue ischemia. Presently, considerable hurdles remain despite significant progress in each of these areas. Restenosis is composed of a contraction and fibrosis of the vessel wall known as remodeling, and an active growth of a fibrocellular lesion composed primarily of VSMCs and extracellular matrix. The latter process, known as neointimal hyperplasia, involves the stimulation of the normally "quiescent" VSMCs in: the arterial media into the "activated" state characterized by rapid proliferation and migration. A number of growth factors are believed to play a role in the stimulation of VSMCs during neointimal hyperplasia, including platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF[3), and angiotensin II. Activated VSMCs have also been found to produce a variety of enzymes, cytokines, adhesion molecules, and other proteins that not only enhance the inflammatory response within the vessel wall but also stimulate further vascular cell abnormality.1 Although it is now thought that remodeling may account for the majority of late lumen loss after balloon dilation of atherosclerotic vessels, proliferation has been the predominant target of experimental genetic interventions. There have been two general approaches: "cytostatic," in which cells are prevented from progressing through the cell cycle to mitosis, and cytotoxic, in which cell death is induced. A group of molecules known as cell cycle regulatory proteins acts at different points along the cell cycle, mediating progression toward division. It has been hypothesized that by blocking expression of the genes for one or more of these proteins, one could prevent the progression of VSMCs through the cell cycle and inhibit neointimal hyperplasia. Morishita et al 2 demonstrated near complete inhibition of neointimal hyperplasia after carotid balloon injury, through HVJ-liposome-mediated transfection of the vessel wall with a combination of antisense O D N against cell cycle regulatory genes. Arrest of the cell cycle through antisense blockade of either of two proto-oncogenes, c-mybor c-myc,has been found to inhibit neointimal hyperplasia in models of arterial balloon injury, a,4 although the specific antisense mechanism of the O D N used in these studies has subsequently been questioned. 5
JOURNAL OF VASCULARSURGERY lune 2000
Lifeline Research Meeting Abstracts
autologous in vitro endothelialization ofinfrainguinal ePTFE grafts in 100 patients: a 9-year experience. Surgery 1999;126:847-55. 12. Schense JC, Hubbell IA. Crossqinking exogenous bifunctional peptides into fibrin gels with factor XIIIa. Bioconjug Chem I999;I0:75-81. i3. Schima H, Tsangaris S, Zilla P, Kadletz M, Wolner E. Mechanical simulation of shear stress on the walls of peripheral arteries. J Biomech 1990;23:845-51.
VASCULAR TISSUE ENGINEERING: STRUCTURE VERSUS F U N C T I O N Elazar R. Edelman, MD, PhD Helen M. Nugent, MD MassachusettsInstitute of Technology Cambridge, Mass
The blood vessel is a structure of extraordinary sophistication. The physical continuity of vascular cells and tissues that make up the blood vessel wall contribute to its structural integrity while also maintaining homeostasis through biochemical regulation. The endothelial monolayer that lines the normal blood vessel serves as a twofold regulator of vascular physiology. The endothelium provides structural integrity to the blood vessel by forming a continuous, selectively permeable, thromboresistant bartier between drcnlating blood and the arterial wall. At the same time, it also produces and supplies products that control blood flow, vessel tone, thrombosis, platelet activation, adhesion, aggregation, leukocyte adhesion, monocyte infiltration, and smooth muscle cell migration and proliferation. Endothelial injury, such as occurs after angioplasty, not only removes the physical barrier but also interferes with the biochemical regulatory potential of the blood vessel and sets into motion a sequence of events that leads to the proliferation and migration of smooth muscle cells resulting in obstructive arterial lesions. This process of restenosis leads to critical narrowings in significant numbers of patients after angioplasty, bypass grafting, and organ transplantation. Tissue engineering enables the development of biological substitutes that restore, maintain, or improve tissue function 1 while also providing substrates by which to examine structure-function relationsbips for specific tissues or organs. Cells may be implanted at sites distant or in different configurations from their original state, enabling examination of any added potential benefit of cell secretory function to regulation of tissue biology above that imposed by preservation of tissue architecture alone. This might be especially important in vascular biology where the autocrine, paracrine, and endocrine function of the endothelium is rapidly emerging. Innovative studies
have attempted to recreate the structure of the blood vessel by autologous endothelial cell transplantation, implantation of endothelial cell-seeded interposition grafts, or endovascular stents. 2-6 Yet, the question remains as to whether reestablishing biochemical control ofvascnlar homeostasis also requires reestablishing the ordered architecture of the blood vessel. We have demonstrated, through the use of tissue-engineered endothelial cells, that the biologic effect of these cells on blood vessel regulation is maintained even when they are implanted at a distance from the lumen. Engrafted endothelial cells on three-dimensional polymer matrices, implanted in the perivascular space of injured rat carotid arteries, reduced intimal thickening by 88%.7 In contrast, the isolated infusion of heparin, an endothelial cell product analog and potent smooth muscle cell inhibitor, only reduced intimal hyperplasia by 30%. These experiments supported the hypotheses that endothelial control over vascular repair is derived from the sum of a number of secreted endothelial cell-based products and need not emanate from the luminal surface. 8 We recently addressed two important questions relating to the biologic effects of perivascular endothelial cell implantation. First, we examined whether allotransplantation of endothelial cell grafts was effective in controlling vascular repair in a porcine carotid artery model of vascular injury, thought to be less responsive to growth-regulatory agents than simpler animal models. Second, we explored whether xenotransplantation, a central issue in developing safe and practical clinical strategies, was more or less effective than allotransplantatlon. We now report that perivascular tissue-engineered endothelial cell implants exert profound control over intimal growth after arterial injury in pigs. Furthermore, despite an increased immtme response to cross-species transplantation, beneficial control of vascular repair was maintained. These results provide insight into how the endothellum controls vascular homeostasis and are a further step toward the development of clinically viable strategies for modulating vascular repair after injury. Further exploration of how tissue-engineered endothelial cell grafts control vascular repair, particularly in settings of more chronic vascular injury, will afford insight into the structure and function of the blood vessel wall and into how experimentally effective techniques may be brought to clinical fruition. REFERENCES
1. Langer R, Vacanti JP. Tissue engineering. Science 1993;260:920-5. 2. Nabel EG, Plautz G, Boyce FM, Stanley JC, Nabel GJ.
JOURNAL OF VASCULARSURGERY Volume31, Number6
3.
4.
5.
6.
7.
8.
Recombinant gene expression in vivo within endothelial cells of the arterial wall. Science 1989;244:1342-4. Messina LM, Podrazik RM, Whitehill TA, Ekhterae D, Brothers TE, Wilson JM, et al. Adhesion and incorporation of lacZ-transduced endothelial cells into the intact capillary wall in the rat. Proc Nat] Acad Sci U S A 1992;89:12018-22. Conte MS, Birinyi LK, Miyata T, F~allon JT, Gold HK, Whitemore AD, et al. Efficient repopulation of denuded rab~ bit arteries with autologous genetically modified endothelial cells. Circulation 1994;23:2161-9. Wilson JV¢, Birinyi LK, Salomon RN, Libby P, Callow AD, Mulligan RC. Implantation of vascular grafts lined with genetically modified endothelial cells. Science 1989;244:1344-6. Dichek DA, Neville RF, Zwiebel JA, Freeman SM, Leon MB, Anderson WF. Seeding ofintravascular stents with genetically engineered endothelial cells. Circulation 1989;80:134753, Nathan A, Nugent MA, Edelman ER. Tissue engineered perivascular endothelial cell implants regulate vascularinjury. Proc Nat] Acad Sci U S A 1995;92:8130-4. Nugent HM, Rogers C, Edelman ER. Endothelial implants inhibit intimal hyperplasia after porcine angioplasty.Circ Res 1998;84:384-91.
G E N E T I C M A N I P U L A T I O N S OB CELLS AND TISSUES F O R T H E T R E A T M E N T O F RESTENOSIS
Michael]-. Mann, MD Brigham and Women's Hospital~Harvard Medical School Boston, Mass
Recurrent narrowing of arteries following percutaneous angioplasty, atherectomy, or other disobliterative technique is a common clinical problem that severely limits the durability of these procedures for patients with atherosclerotic occlusive diseases. In the case of balloon angioplasty, restenosis occurs in approximatcly 30% to 40% of treated coronary lesions and 30% to 50% of superficial femoral artery lesions within the first year. Intravascular stents may reduce the restenosis rates. However, the incidence remains high, and long-term data are limited, Despite technological advances in the development of minimally invasive and endovascular approaches to treat arterial occlusions, the full benefit of these gains awaits the solution of this fundamental biologic problem. Restenosis is an attractive target for gene therapy not only because of its frequency but also because it is a local tissue reaction that develops precisely at a site of intervention to which access has already been achieved. A potential advantage of the genetic approach over more conventional pharmacotherapies is that a single dose o f a gene therapy agent may have a prolonged biological effect. The appropriate genetic modification, performed locally at the time of angioplasty, could induce a long-term benefit in patency by altering the healing response, The poten-
Lifeline Research Meeting Abstracts
1269
tial role for gene therapy in the prevention of restenosis will depend on the identification of an appropriate molecular target, a suitable vector system for efficiently targeting vessel wall cells, and methods of achieving local delivery without producing undue damage or distal tissue ischemia. Presently, considerable hurdles remain despite significant progress in each of these areas. Restenosis is composed of a contraction and fibrosis of the vessel wall known as remodeling, and an active growth of a fibrocellular lesion composed primarily of VSMCs and extracellular matrix. The latter process, known as neointimal hyperplasia, involves the stimulation of the normally "quiescent" VSMCs in: the arterial media into the "activated" state characterized by rapid proliferation and migration. A number of growth factors are believed to play a role in the stimulation of VSMCs during neointimal hyperplasia, including platelet-derived growth factor (PDGF), basic fibroblast growth factor (bFGF), transforming growth factor beta (TGF[3), and angiotensin II. Activated VSMCs have also been found to produce a variety of enzymes, cytokines, adhesion molecules, and other proteins that not only enhance the inflammatory response within the vessel wall but also stimulate further vascular cell abnormality.1 Although it is now thought that remodeling may account for the majority of late lumen loss after balloon dilation of atherosclerotic vessels, proliferation has been the predominant target of experimental genetic interventions. There have been two general approaches: "cytostatic," in which cells are prevented from progressing through the cell cycle to mitosis, and cytotoxic, in which cell death is induced. A group of molecules known as cell cycle regulatory proteins acts at different points along the cell cycle, mediating progression toward division. It has been hypothesized that by blocking expression of the genes for one or more of these proteins, one could prevent the progression of VSMCs through the cell cycle and inhibit neointimal hyperplasia. Morishita et al 2 demonstrated near complete inhibition of neointimal hyperplasia after carotid balloon injury, through HVJ-liposome-mediated transfection of the vessel wall with a combination of antisense O D N against cell cycle regulatory genes. Arrest of the cell cycle through antisense blockade of either of two proto-oncogenes, c-mybor c-myc,has been found to inhibit neointimal hyperplasia in models of arterial balloon injury, a,4 although the specific antisense mechanism of the O D N used in these studies has subsequently been questioned. 5