Volume 13 Number May
5
1991
ment Program, still in the early stages, has attracted a number of qualified minority investigators and students into the research areas supported by the NHLBI. The Institute has also recently announced a program to support short-term research training experiences for minority students at the undergraduate and graduate level. This program is designed to attract minority students to careers in biomedical science. Research training and career development programs supported by the NHLBI are varied and provide ample opportunities for those interested in research careers. Moreover, new opportunities are available for attracting and supporting careers in biomedical research for underrepresented minority individuals. Although budgetary limitations have affected these programs, support of training and development programs continues to be at significant levels. Investigators interested in researchtraining, particularly in the area of vascular biology and medicine, are encouraged to consider the array of support mechanisms available from the NHLBI. John L. Fakundin., PhD National Heart, Lug, and Blood Institute Bethesda,Md.
BIOMATERIALS-RELATED PROGRAMS AND INITIATIVES AT THE NATIONAL HEART, LUNG, AND BLOOD INSTITUTE National Institutes of Health (NIH) supported research related to biomaterials is administered by 10 different institutes; the National Heart, Lung, and Blood Institute (NHLBI) funds the largest fraction, followed by the National Institute of Dental Research (NIDR) and National Institute of Arthritis and Musculoskeletal and Skin Diseases(NIAMS) . In NHLBI, biomaterials research is organized into a discrete program within the Devicesand Technology Branch. Currently this program comprises 68 grants and 1 contract. Topic areasand number of grants in each area are: biomaterials and biocompatibility, 30; vascular grafts and vascular healing, 25; rheology, 11; and calcification, 3. In 1987 a Request for Applications (RFA) on “Vascular Healing: Cell and Rheologic Factors” was released. Of 40 applications, 8 were funded in 1988 and are currently active. In August of 1990 an RFA on “Mechanisms of Damage Caused by Cardiopulmonary Bypass” was released.Funding of awards from this RFA is anticipated to begin in the summer of 1991. The RFA will be administered jointly by the Division of Heart and Vascular Diseases and the Division of Blood Diseases and Resources of NHLBI. In November of 1990 an RFA on “Cardiovascular Device-Centered Infections” was released. This RFA is unique in being a joint endeavor of NHLBI and the National ScienceFoundation (NSF), and in requiring both biomedical scienceand bioengineering components in the applications for them to be judged responsive. Funding of awards from this RFA is also anticipated to begin in the summer of 1991.
Special communication
729
An initiative currently under discussion is entitled “Genetically Enhanced Cardiovascular Implants.” The objective of this initiative would be to develop techniques for gene modification, regulation, or expression in endothelial or other cells growing on biomaterial substrates or prosthetic devices,to enhance healing, promote hemostasis,and prevent thrombosis and infection or beneficially modify the course of other disorders including hypertension, atherosclerosis,and diabetes. Cells may reach the surface by seeding, receptor-specific binding of circulating cells, or tissue ingrowth. Cells may be engineered in situ or in vitro. Examples of applications of such techniques include delivery of increased levelsof tPA locally to stems or vasculargrafts to prevent thrombosis, and delivery into the systemiccirculation of various recombinant DNA products from an “organoid,” a synthetic organ composed of a polymeric network seeded with cells genetically altered to secrete a desired product, such as vasodilators or angiogenic factors. Paul Didisbeim, MD National Heart, Llmg, and Blood Institute Bethesda, M2.
MECHANICAL SENSING MECHANISMS: SHEAR STRBSS AND ENDOTHELIAL CELLS Mechanical forces associatedwith blood flow (hemodynamic forces) play an important role in the regulation of vascular tone,’ vascular remodeling,’ and the focal development of atherosclerotic lesions.3 Hemodynamic forces can be resolved into two principal components; shearstress, the tangential frictional force acting at the endothelial cell surface in the direction of flow, and pressure-stretch, acting perpendicular to the vascular wall. The endothelial cell is the recipient of most of the shear stress,whereas both the endothelial and underlying smooth muscle cells, together with the extracellular matrix, are subjected to stretch. A variety of cellular responses to precisely applied shear stresseshave recently provided evidence of two broadly defined mechanisms by which flow stimulates the cell. One of these mechanisms is the direct action of the forces on the cell structure leading to activation of ion channels,* mobilization of phosphatidyl inositide metabolism and calcium, the release of prostaglandins,5 and vasorelaxing factors. The location in the cell of these membraneassociated events and the structures and mechanisms responsible for transducing the force to elicit biochemical responsesare unknown. The spectrum of investigations of the effects of flow on endothelial biology are still quite limited considering its multifunctional capabilities. Endothelium can be considered as a barrier, both structural and metabolic, between the blood and the rest of the vesselwall. A large body of experimental evidence strongly implicates endothelium asa key regulator in the passageof substancesbetween blood and artery in both directions and in the prevention of thrombosis in the vessellumen. Although the nature of many of the biochemicals synthesized and released from endothelium in maintaining its antithrombotic properties
730
Journal of VASCULAR SURGERY
Special uvnmunication
have been characterized, virtually nothing is known concerning the local concentrations of these molecules adjacent to the cell surface when the cell is subjected to blood flow, and only recently have investigations of mechanical forces on the synthesis and secretion of anticoagulants and procoagulants been initiated.6 A fnrther property of endothelium is its interactions with other cells, particularly circulating monocytes and polymorphonuclear leukocytes which, under certain pathologic conditions, can recognize specific receptors on the endothelial cell surface resulting in leukocyte adhesion and migration into the subendothelial space. It is unclear how flow may influence such interactions . A number of devices for studying hemodynamic force effects on endothelium have been used, including parallel plate flow channels, cone-plate Couette apparatus, and capillary flow tubes3 These afford study of cells exposed to unidirectional shear stress in steady and pulsatile laminar flow at amplitudes up to nearly 100 dynes/cm.’ What is the sensing mechanism that tranduces the mechanical force to a cellular response? It has been well established for a number of years that the shape of arterial endothelial cells correlates in vivo with regionally predicted flow patterns, and that cells will realign in the direction of flow and change their axial orientation in vitro. Such changes clearly involve major redistribution of the cytoskeleton, implying that the biochemistry of cytoskeletal proteins must be a major mechanism for these responses, generally detectable after several hours of exposure to the flow. Preceding cytoskeletal changes, however, are alterations of messenger RNA for a number of endothelial secretion products,” and preceding these in turn are a series ofvery rapid responses to the shear stress. It seems probable that the earliest responses are associated with a putative flow sensor at the endothelial surface. The fastest endothelial response to shear stress reported to date is activation of a potassium ion channel that regulates the transmembrane flux of K’ as a function of the shear stresses acting on the cell surface.4 Ion channel activation results in hyperpolarization of the cell (halfmaximal effect 0.7 dynes/cm*; range of activation 0.2-20-dynes/cm*; saturation of the effect was noted above 15 dynes/cm*). Activation of a flow-sensitive potassium channel could occur directly, that is, the channel itself being the mechanosensor, or the channel may be just downstream of mechanoreceptor activation.’ Efforts to isolate and clone inwardly rectifying K’ channels from endothelial cells, including the putative mechanosensing channel, are currently underway. Recent studies from this laboratory have demonstrated that at least part of the endothelial response to shear stress is mediated by the local concentration of metabolites in the diffusion boundary layer of the endothelial cell.” Agonistinduced intracellular calcium mobilization was exquisitely sensitive to the local shear stress characteristics. Changes in flow altered the mass transport of agonist/metabolite/ligand in the diffusion boundary layer thereby influencing its availability to the endothelial receptors. Thus for
example, local release of adenosine diphosphate from platelets will have an effect on the P2 purinoceptor of endothelial cells only to an extent dictated by the local flow and mass transport characteristics. These experiments, however, also clearly indicated an additional direct effect of the tlow upon the cell, that is, supported the existence of a discrete flow sensor associated with the endothelial cell. We therefore consider that the mechanisms responsible for endothelial reaction to flow involve both a mass transport component and a direct effect on the cell membrane. The location of a putative flow sensor in the endothelial cell could be either apical, lateral (associated with intercellular junctions), or basal, or a combination of all three locations interconnected by the cytoskeleton. At the basal side, the cell is adherent to the subendothelial basal membrane connective tissue. Cell-matrix contact is not uniform, however; there are regions of focal contacts between the basal plasma membrane and the underlying matrix. When physical forces are applied to the luminal side of the cell, there is a displacement of the focal adhesions that may elicit a mechanosensor response in the cell. Recent studies in this laboratory have modeled the focal adhesion responses to flow in endothelial cells by use of a combined structural biology/molecular approach. Using confocal image analysis of focal adhesions,’ we have been able to quantitate the relative adhesion of endothelial cells to a glass surface. It is surprising to note that spontaneous as well as mechanically directed remodeling of focal adhesion sites were observed and measured in the confluent endothelial monolayer. Under conditions of acute hemodynamic shear stress, there was very rapid (less than 30 seconds) directional remodeling of focal adhesion sites suggesting that these important structures, which are linked directly to actin stress fibers and hence to other parts of the cell, are rapidly responsive to the external shear stress forces. Furthermore, the techniques lend themselves to studies of adhesion in living cells as a function of the matrix composition.
Future research Future research in endothelial hemodynamic stimulusresponse coupling will undoubtedly continue to include documentation of the effects of defined shear stress on many of the known synthetic capabilities of endothelial cells; most of these can now be studied at the level of gene expression. The question of identifying a membrane associated mechanical sensor in endothelial cells is being addressed by modern molecular biologic techniques of differential hybridization and the cloning of potential candidates, for example, potassium ion channels. Fluid dynamic models of mass transport and boundary layer diffusion mechanics are already being applied to the flow environment; much will be learned very quickly concerning the regulation of cell responses mediated via mass transport mechanisms. Confocal image analysis of focal adhesion sites provides a measurement of relative cell adhesion at the
Volume 13 Number 5 May 1991
Specialcommunication
level of a single focal adhesion, groups of adhesion plaques, a single cell, or groups of cells. It should be possible to manipulate the composition of the extracellular matrix to quantitate the effects on cell adhesion. It should also be possible to use competitive binding studies of adhesion proteins, as well as selective blocking of domains of adhesion proteins by use of specific antibodies, to obtain insights concerning the molecular mechanisms of cell adhesion. Focal adhesions in endothelial cells may represent a complex of proteins capable of signaling the effects of external forces such as shear stress to the interior of the cell. It would be of interest to investigate whether such structures and their associated proteins play a regulatory role in the responses of endothelial cells to external physical forces. Such experiments are critical to an understanding of the adhesion of endothelial cells on vascular grafts where cells must resist detachment by flow and where the presence of an endothelial lining is likely to assist graft patency and survival. Peteev F. Davies, PhD Pritekw School of Medicine The University of Chicgo Chicago, Ill.
REFERENCES 1. Holtz J, Forstermann U, Pohl V, Giesler M, Bassenge EJ. Flow-dependent, endothelium-mediated dilation of epicardial coronary arteries in conscious dogs: effects of cycle-oxygenase inhibition. J Cardiovasc Pharmacol 1984;6:1161-9. 2. Langille BL, O’Donnell F. Reductions in arterial diameter produced by chronic decreases in blood flow are endothelialdependent. Science 1986;231:405-8. 3. Davies PF. Endothelial cells, hemodynamic forces, and the localization of atherosclerosis. In: Ryan US., ed. Endothelial Cells, Vol. II. Boca Baton, Florida: CRC Press, 1988: 123-39. 4. Olesen SP, Clapham DE, Davies PF. Hemodynamic shear stressactivates a K + current in vascular endothelial cells. Nature 1988;331:168-170. 5. Frangos JA, Eskin SE, McIntyre LV, Ives CL Flow effects on prostacyclin production by cultured human endothelial cells. Science 1985;227:1477-9. Diamond SL, McIntyre LV, Share&in JB, Dieffenbach C, Scott KF, Eskin SG. J Cell Physiol (In press). Davies PF. How do vascular endothelial cells respond to flow? News in Physiol Sci 1989;4:22-6. Dull RO, Davies PF. Hemodynamic shear stress modulates signal-response coupling in vascular endothelial cells (In press). Paddock SW. Tandem scanning reflected-light microscopy of cell-substratum adhesions and stress fibres in Swiss 3T3 cells. J Cell Sci 1989;93: 142-6.
ENDOTHELIAL
CELL SEEDING
Endothelial cell seeding is the transplantation of vascular endothelial cells to denuded vascular surfaces. Endothelium is a crucial cell in the modulation of clotting and inflammatory responses. Intuitively, if endothelial cells could be seeded on denuded vascular surfaces or on prostheses, the rate of thrombosis might be reduced.
73 1
Endothelium has a broad repertoire of clot modulating functions. It limits clot formation largely by deactivating thrombin via the antithrombin III and thrombomodulin systems. It suppresses platelet activation by elaborating prostacyclin and ecto-ATPase. Plasminogen activators and plasminogen activator inhibitors are products of endothelium. Therefore endothelium also exerts control over the dissolution of clot. During a response to injury, endothelium can produce clot promoting substances, such as platelet activating and thromboxane A, factor, tissue thromboplastin (TX&). Clotting factors V, von Willebrand’s factor, and plasminogen activator inhibitor are also made by unperturbed cells. It is widely believed that the patency of the grafts at the sites of vascular repair will be improved if endothelial clot promoting functions can be suppressed. Because endothelium inhibits and reverses clotting, endothelial linings inhibit experimental graft thromboses. Purely thrombotic occlusions tend to occur within a few weeks of grafting. Nevertheless, a very common event leading to later failure is neointimal fibrous hyperplasia. The development of hyperplasia begins with thrombin which sensitizes smooth muscle cells and fibroblasts to the effects of circulating growth factors. After complete endothelial resurfacing, the subjacent smooth muscle cells are no longer exposed to thrombin, nor are they directly exposed to serum-borne growth factors. Cellular growth factors are elaborated by endothelium, platelets, smooth muscle cells, and macrophages. Each of these cell types will remain in close contact with the vascular smooth muscle cells. Therefore rapid resurfacing of an injured vascular surface with endothelium is expected to reduce, but not eliminate, the hyperplastic response. Despite the theory and encouragement from laboratory animal implants, the reported clinical trials of endothelial seeding resulted in improvements in patency ranging from none to modest.“’ Is the theory wrong? The theory may be incomplete but not wrong. Three encouraging pieces of information have emerged from the clinical experience. Two pieces correlate with results in experimental animals: (1) adult human endothelium can be transplanted onto vascular prosthese?; (2) seeded human prostheses attract fewer platelets than unseeded ones.4 The third observation, although not particularly clear in animal models, is clinically glaring: (3) virtually all of the failures of seeded prostheses are caused by anastomotic neointimal fibrous hyperplasia.3 At the very root of the clinical results is the problem with massive inefficiency. Inefficient endothelial seeding means that fewer cells on the flow surface must replicate many more times to create an intact monolayer. The effect of prolonged exposure to thrombin and circulating growth factors on smooth muscle cells is obvious; however, replicating endothelium deposits its own growth factor until replication stops. Generally, replication stops when the monolayer of endothelium is complete.
5
1991
ment Program, still in the early stages, has attracted a number of qualified minority investigators and students into the research areas supported by the NHLBI. The Institute has also recently announced a program to support short-term research training experiences for minority students at the undergraduate and graduate level. This program is designed to attract minority students to careers in biomedical science. Research training and career development programs supported by the NHLBI are varied and provide ample opportunities for those interested in research careers. Moreover, new opportunities are available for attracting and supporting careers in biomedical research for underrepresented minority individuals. Although budgetary limitations have affected these programs, support of training and development programs continues to be at significant levels. Investigators interested in researchtraining, particularly in the area of vascular biology and medicine, are encouraged to consider the array of support mechanisms available from the NHLBI. John L. Fakundin., PhD National Heart, Lug, and Blood Institute Bethesda,Md.
BIOMATERIALS-RELATED PROGRAMS AND INITIATIVES AT THE NATIONAL HEART, LUNG, AND BLOOD INSTITUTE National Institutes of Health (NIH) supported research related to biomaterials is administered by 10 different institutes; the National Heart, Lung, and Blood Institute (NHLBI) funds the largest fraction, followed by the National Institute of Dental Research (NIDR) and National Institute of Arthritis and Musculoskeletal and Skin Diseases(NIAMS) . In NHLBI, biomaterials research is organized into a discrete program within the Devicesand Technology Branch. Currently this program comprises 68 grants and 1 contract. Topic areasand number of grants in each area are: biomaterials and biocompatibility, 30; vascular grafts and vascular healing, 25; rheology, 11; and calcification, 3. In 1987 a Request for Applications (RFA) on “Vascular Healing: Cell and Rheologic Factors” was released. Of 40 applications, 8 were funded in 1988 and are currently active. In August of 1990 an RFA on “Mechanisms of Damage Caused by Cardiopulmonary Bypass” was released.Funding of awards from this RFA is anticipated to begin in the summer of 1991. The RFA will be administered jointly by the Division of Heart and Vascular Diseases and the Division of Blood Diseases and Resources of NHLBI. In November of 1990 an RFA on “Cardiovascular Device-Centered Infections” was released. This RFA is unique in being a joint endeavor of NHLBI and the National ScienceFoundation (NSF), and in requiring both biomedical scienceand bioengineering components in the applications for them to be judged responsive. Funding of awards from this RFA is also anticipated to begin in the summer of 1991.
Special communication
729
An initiative currently under discussion is entitled “Genetically Enhanced Cardiovascular Implants.” The objective of this initiative would be to develop techniques for gene modification, regulation, or expression in endothelial or other cells growing on biomaterial substrates or prosthetic devices,to enhance healing, promote hemostasis,and prevent thrombosis and infection or beneficially modify the course of other disorders including hypertension, atherosclerosis,and diabetes. Cells may reach the surface by seeding, receptor-specific binding of circulating cells, or tissue ingrowth. Cells may be engineered in situ or in vitro. Examples of applications of such techniques include delivery of increased levelsof tPA locally to stems or vasculargrafts to prevent thrombosis, and delivery into the systemiccirculation of various recombinant DNA products from an “organoid,” a synthetic organ composed of a polymeric network seeded with cells genetically altered to secrete a desired product, such as vasodilators or angiogenic factors. Paul Didisbeim, MD National Heart, Llmg, and Blood Institute Bethesda, M2.
MECHANICAL SENSING MECHANISMS: SHEAR STRBSS AND ENDOTHELIAL CELLS Mechanical forces associatedwith blood flow (hemodynamic forces) play an important role in the regulation of vascular tone,’ vascular remodeling,’ and the focal development of atherosclerotic lesions.3 Hemodynamic forces can be resolved into two principal components; shearstress, the tangential frictional force acting at the endothelial cell surface in the direction of flow, and pressure-stretch, acting perpendicular to the vascular wall. The endothelial cell is the recipient of most of the shear stress,whereas both the endothelial and underlying smooth muscle cells, together with the extracellular matrix, are subjected to stretch. A variety of cellular responses to precisely applied shear stresseshave recently provided evidence of two broadly defined mechanisms by which flow stimulates the cell. One of these mechanisms is the direct action of the forces on the cell structure leading to activation of ion channels,* mobilization of phosphatidyl inositide metabolism and calcium, the release of prostaglandins,5 and vasorelaxing factors. The location in the cell of these membraneassociated events and the structures and mechanisms responsible for transducing the force to elicit biochemical responsesare unknown. The spectrum of investigations of the effects of flow on endothelial biology are still quite limited considering its multifunctional capabilities. Endothelium can be considered as a barrier, both structural and metabolic, between the blood and the rest of the vesselwall. A large body of experimental evidence strongly implicates endothelium asa key regulator in the passageof substancesbetween blood and artery in both directions and in the prevention of thrombosis in the vessellumen. Although the nature of many of the biochemicals synthesized and released from endothelium in maintaining its antithrombotic properties
730
Journal of VASCULAR SURGERY
Special uvnmunication
have been characterized, virtually nothing is known concerning the local concentrations of these molecules adjacent to the cell surface when the cell is subjected to blood flow, and only recently have investigations of mechanical forces on the synthesis and secretion of anticoagulants and procoagulants been initiated.6 A fnrther property of endothelium is its interactions with other cells, particularly circulating monocytes and polymorphonuclear leukocytes which, under certain pathologic conditions, can recognize specific receptors on the endothelial cell surface resulting in leukocyte adhesion and migration into the subendothelial space. It is unclear how flow may influence such interactions . A number of devices for studying hemodynamic force effects on endothelium have been used, including parallel plate flow channels, cone-plate Couette apparatus, and capillary flow tubes3 These afford study of cells exposed to unidirectional shear stress in steady and pulsatile laminar flow at amplitudes up to nearly 100 dynes/cm.’ What is the sensing mechanism that tranduces the mechanical force to a cellular response? It has been well established for a number of years that the shape of arterial endothelial cells correlates in vivo with regionally predicted flow patterns, and that cells will realign in the direction of flow and change their axial orientation in vitro. Such changes clearly involve major redistribution of the cytoskeleton, implying that the biochemistry of cytoskeletal proteins must be a major mechanism for these responses, generally detectable after several hours of exposure to the flow. Preceding cytoskeletal changes, however, are alterations of messenger RNA for a number of endothelial secretion products,” and preceding these in turn are a series ofvery rapid responses to the shear stress. It seems probable that the earliest responses are associated with a putative flow sensor at the endothelial surface. The fastest endothelial response to shear stress reported to date is activation of a potassium ion channel that regulates the transmembrane flux of K’ as a function of the shear stresses acting on the cell surface.4 Ion channel activation results in hyperpolarization of the cell (halfmaximal effect 0.7 dynes/cm*; range of activation 0.2-20-dynes/cm*; saturation of the effect was noted above 15 dynes/cm*). Activation of a flow-sensitive potassium channel could occur directly, that is, the channel itself being the mechanosensor, or the channel may be just downstream of mechanoreceptor activation.’ Efforts to isolate and clone inwardly rectifying K’ channels from endothelial cells, including the putative mechanosensing channel, are currently underway. Recent studies from this laboratory have demonstrated that at least part of the endothelial response to shear stress is mediated by the local concentration of metabolites in the diffusion boundary layer of the endothelial cell.” Agonistinduced intracellular calcium mobilization was exquisitely sensitive to the local shear stress characteristics. Changes in flow altered the mass transport of agonist/metabolite/ligand in the diffusion boundary layer thereby influencing its availability to the endothelial receptors. Thus for
example, local release of adenosine diphosphate from platelets will have an effect on the P2 purinoceptor of endothelial cells only to an extent dictated by the local flow and mass transport characteristics. These experiments, however, also clearly indicated an additional direct effect of the tlow upon the cell, that is, supported the existence of a discrete flow sensor associated with the endothelial cell. We therefore consider that the mechanisms responsible for endothelial reaction to flow involve both a mass transport component and a direct effect on the cell membrane. The location of a putative flow sensor in the endothelial cell could be either apical, lateral (associated with intercellular junctions), or basal, or a combination of all three locations interconnected by the cytoskeleton. At the basal side, the cell is adherent to the subendothelial basal membrane connective tissue. Cell-matrix contact is not uniform, however; there are regions of focal contacts between the basal plasma membrane and the underlying matrix. When physical forces are applied to the luminal side of the cell, there is a displacement of the focal adhesions that may elicit a mechanosensor response in the cell. Recent studies in this laboratory have modeled the focal adhesion responses to flow in endothelial cells by use of a combined structural biology/molecular approach. Using confocal image analysis of focal adhesions,’ we have been able to quantitate the relative adhesion of endothelial cells to a glass surface. It is surprising to note that spontaneous as well as mechanically directed remodeling of focal adhesion sites were observed and measured in the confluent endothelial monolayer. Under conditions of acute hemodynamic shear stress, there was very rapid (less than 30 seconds) directional remodeling of focal adhesion sites suggesting that these important structures, which are linked directly to actin stress fibers and hence to other parts of the cell, are rapidly responsive to the external shear stress forces. Furthermore, the techniques lend themselves to studies of adhesion in living cells as a function of the matrix composition.
Future research Future research in endothelial hemodynamic stimulusresponse coupling will undoubtedly continue to include documentation of the effects of defined shear stress on many of the known synthetic capabilities of endothelial cells; most of these can now be studied at the level of gene expression. The question of identifying a membrane associated mechanical sensor in endothelial cells is being addressed by modern molecular biologic techniques of differential hybridization and the cloning of potential candidates, for example, potassium ion channels. Fluid dynamic models of mass transport and boundary layer diffusion mechanics are already being applied to the flow environment; much will be learned very quickly concerning the regulation of cell responses mediated via mass transport mechanisms. Confocal image analysis of focal adhesion sites provides a measurement of relative cell adhesion at the
Volume 13 Number 5 May 1991
Specialcommunication
level of a single focal adhesion, groups of adhesion plaques, a single cell, or groups of cells. It should be possible to manipulate the composition of the extracellular matrix to quantitate the effects on cell adhesion. It should also be possible to use competitive binding studies of adhesion proteins, as well as selective blocking of domains of adhesion proteins by use of specific antibodies, to obtain insights concerning the molecular mechanisms of cell adhesion. Focal adhesions in endothelial cells may represent a complex of proteins capable of signaling the effects of external forces such as shear stress to the interior of the cell. It would be of interest to investigate whether such structures and their associated proteins play a regulatory role in the responses of endothelial cells to external physical forces. Such experiments are critical to an understanding of the adhesion of endothelial cells on vascular grafts where cells must resist detachment by flow and where the presence of an endothelial lining is likely to assist graft patency and survival. Peteev F. Davies, PhD Pritekw School of Medicine The University of Chicgo Chicago, Ill.
REFERENCES 1. Holtz J, Forstermann U, Pohl V, Giesler M, Bassenge EJ. Flow-dependent, endothelium-mediated dilation of epicardial coronary arteries in conscious dogs: effects of cycle-oxygenase inhibition. J Cardiovasc Pharmacol 1984;6:1161-9. 2. Langille BL, O’Donnell F. Reductions in arterial diameter produced by chronic decreases in blood flow are endothelialdependent. Science 1986;231:405-8. 3. Davies PF. Endothelial cells, hemodynamic forces, and the localization of atherosclerosis. In: Ryan US., ed. Endothelial Cells, Vol. II. Boca Baton, Florida: CRC Press, 1988: 123-39. 4. Olesen SP, Clapham DE, Davies PF. Hemodynamic shear stressactivates a K + current in vascular endothelial cells. Nature 1988;331:168-170. 5. Frangos JA, Eskin SE, McIntyre LV, Ives CL Flow effects on prostacyclin production by cultured human endothelial cells. Science 1985;227:1477-9. Diamond SL, McIntyre LV, Share&in JB, Dieffenbach C, Scott KF, Eskin SG. J Cell Physiol (In press). Davies PF. How do vascular endothelial cells respond to flow? News in Physiol Sci 1989;4:22-6. Dull RO, Davies PF. Hemodynamic shear stress modulates signal-response coupling in vascular endothelial cells (In press). Paddock SW. Tandem scanning reflected-light microscopy of cell-substratum adhesions and stress fibres in Swiss 3T3 cells. J Cell Sci 1989;93: 142-6.
ENDOTHELIAL
CELL SEEDING
Endothelial cell seeding is the transplantation of vascular endothelial cells to denuded vascular surfaces. Endothelium is a crucial cell in the modulation of clotting and inflammatory responses. Intuitively, if endothelial cells could be seeded on denuded vascular surfaces or on prostheses, the rate of thrombosis might be reduced.
73 1
Endothelium has a broad repertoire of clot modulating functions. It limits clot formation largely by deactivating thrombin via the antithrombin III and thrombomodulin systems. It suppresses platelet activation by elaborating prostacyclin and ecto-ATPase. Plasminogen activators and plasminogen activator inhibitors are products of endothelium. Therefore endothelium also exerts control over the dissolution of clot. During a response to injury, endothelium can produce clot promoting substances, such as platelet activating and thromboxane A, factor, tissue thromboplastin (TX&). Clotting factors V, von Willebrand’s factor, and plasminogen activator inhibitor are also made by unperturbed cells. It is widely believed that the patency of the grafts at the sites of vascular repair will be improved if endothelial clot promoting functions can be suppressed. Because endothelium inhibits and reverses clotting, endothelial linings inhibit experimental graft thromboses. Purely thrombotic occlusions tend to occur within a few weeks of grafting. Nevertheless, a very common event leading to later failure is neointimal fibrous hyperplasia. The development of hyperplasia begins with thrombin which sensitizes smooth muscle cells and fibroblasts to the effects of circulating growth factors. After complete endothelial resurfacing, the subjacent smooth muscle cells are no longer exposed to thrombin, nor are they directly exposed to serum-borne growth factors. Cellular growth factors are elaborated by endothelium, platelets, smooth muscle cells, and macrophages. Each of these cell types will remain in close contact with the vascular smooth muscle cells. Therefore rapid resurfacing of an injured vascular surface with endothelium is expected to reduce, but not eliminate, the hyperplastic response. Despite the theory and encouragement from laboratory animal implants, the reported clinical trials of endothelial seeding resulted in improvements in patency ranging from none to modest.“’ Is the theory wrong? The theory may be incomplete but not wrong. Three encouraging pieces of information have emerged from the clinical experience. Two pieces correlate with results in experimental animals: (1) adult human endothelium can be transplanted onto vascular prosthese?; (2) seeded human prostheses attract fewer platelets than unseeded ones.4 The third observation, although not particularly clear in animal models, is clinically glaring: (3) virtually all of the failures of seeded prostheses are caused by anastomotic neointimal fibrous hyperplasia.3 At the very root of the clinical results is the problem with massive inefficiency. Inefficient endothelial seeding means that fewer cells on the flow surface must replicate many more times to create an intact monolayer. The effect of prolonged exposure to thrombin and circulating growth factors on smooth muscle cells is obvious; however, replicating endothelium deposits its own growth factor until replication stops. Generally, replication stops when the monolayer of endothelium is complete.
