Regulation of smooth muscle profileration in hyperplastic arterial lesions

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endothelial cells occur, platelets may adhere and can also serve as sources of numerous growth factors, including PDGF. Platelet-derived growth factor probably plays a key role in both the chemotactic and proliferative smooth muscle events that lead to the advanced lesions of atherosclerosis. Platelet-derived growth factor can be present in three different isoforms, each of which consists of the different possible combinations of the A and B chains of PDGF, respectively. It has been shown that the responsiveness of cells to PDGF is dependent on the relative numbers of the different phenotypes of the PDGF receptor that are possible. There are two PDGF receptor subunits, termed alpha and beta, that can make up three different phenotypes of the PDGF receptor. The A chain of PDGF can bind to only one of the receptor subunits, the alpha subtmit, whereas the B chain of PDGF can bind to either the alpha subtmit or the beta subtmit. Thus the AA homodimer of PDGF can bind to only a single phenotype of the PDGF receptor (alpha-alpha), whereas the BB homodimer can bind to all three phenotypes (alpha-alpha, alpha-beta, and beta-beta). The AB heterodimer of PDGF, found principally in platelets, can bind to either alpha-alpha or alphabeta phenotypes of the PDGF receptor. Consequently, the relative mitogenicity of the different isoforms of PDGF is dependent not only on the amount of PDGF present but also on the relative numbers of receptor subunits found on a given cell type, and thus both ligands and receptor availability are responsible for the differential susceptibility of different cells to PDGF. Smooth muscle cells derived from lesions of atherosclerosis, in contrast to cells from the underlying normal media, can synthesize PDGF AA, and thus they are able to respond in an autocrine fashion to this isoform of PDGF. 4,s As a consequence of these observations, the interactions among cells present in lesions of atherosclerosis, that is, monocyte/macrophages, T-lymphocytes (which suggest the probability of an immune response in atherogenesis), endothelial cells, and smooth muscle cells, represent a complex interplay among cytokines that can be released by these different cell types and growth factors, in particular PDGF. Other factors, such as transforming growth factor-beta and gamma-interferon, could play inhibitory roles, unlike growth factors such as PDGF, and the balance between the effects of these different growth factors, inhibitors, and cytokines will determine the net resultant proliferative response within the intima of the affected arteries. 1-3

Russell Ross, PhD Department of Pathology University of Washington Seattle, Washington REFERENCES

1. Ross R, Glomset 1A. The pathogenesis of atherosclerosis. N Engl J Med 1976;295:369-77. 2. Ross R, Glomset JA. The pathogenesis of atherosclerosis. N Engl J Med 1976;295:420-5. 3. Ross R. The pathogenesis of atherosderosis--an update. N Engl J Med 1986;314:488-500.

4. Ross R, Raines EW, Bowen-Pope DF. The biology"ofplateletderived growth factor. Cell 1986;46:155-69. 5. Ross R. Platelet-derived growth factor. Ann Key Med 1987; 38:71-9. R E G U L A T I O N OF S M O O T H M U S C L E PROLIFERATION IN HYPERPLASTIC ARTERIAL LESIONS Effective therapy of occlusive disease of small-caliber arteries remains a challenge for modern practitioners of cardiovascular surgery and medicine. Advances in surgical technique and in biomaterial science now permit the insertion of grafts constructed of autogeneous vein or prosthetic conduits into the peripheral circulation. However, the development of a hyperplastic tissue response in the recipient artery often limits the mid- and long-term success of these approaches. Such lesions characteristically occur at the site of the distal anastomosis of the graft to the native artery; hence the use of the term anastomotic hyperplasia to describe this process. This problem can lead to failure even of grafts placed in arteries with focal disease and with good runoff and constructed with optimal surgical technique. Anastomotic hyperplasia that complicates small vessel arterial grafts is but one special case of a general phenomenon of tissue responsiveness that frustrates a number of modern "high technology" approaches to the n-eatment of arterial disease. A variety of signals might initiate this tissue response. Differences in compliance between the engrafted and native artery or other focal hydrodynamic perturbations may provoke this tissue reaction. Whatever the inciting factors, the final common pathogenic pathway involves abnormal proliferation of mesenchymal elements in the vessel wall, presumably vascular smooth muscle cells. In this respect anastomotic hyperplasia probably shares pathogenetic features with atherosclerosis, restenosis after percutaneous angioplasty or atherectomy, and the accelerated form of arteriosclerosis that develops in the arteries of allografted organs. In fact, inappropriate hyperplasia of mesenchyrnal cells including smooth muscle and tibroblasts provides a common thread in a variety of fibrotic diseases that affect other organs including the lung, kidney, liver, and skin, Understanding the pathogenesis of anastomotic hyperplasia will require identification of the proximate signal for the abnormal proliferation of vascular smooth muscle cells. Platelet-derived growth factor (PDGF) was an early candidate for the biochemical mediator of smooth muscle hyperplasia in vascular diseases? Originally thought to be elaborated only by platelets, recent work has established that a number of cells likely to be present at sites of healing vascular injury can elaborate this mediator. For example, endothelial cells, smooth muscle cells, and mononuclear phagocytes can all express genes for PDGF and may do so in situ in abnormal vessels? Arterial grafts excised from experimental animals can secrete substances that resemble PDGF? Adult human endothelial cells can express PDGF genes. 4,s Furthermore, smooth muscle cells grown from anastomotic hyperplastic lesions from tailed human arterial

Volume 10 Number 5 November 1989

" grafts can elaborate a PDGF-like mimgen and transcribe genes that encode the PDGF A chain (but not the B chain) and a receptor for PDGF. 6These various observations support a possible local autocrine pathway of growth stimulation in this situation that involves PDGF. Thus this mediator remains high on the list of possible pathogenic mediators of anastomotic hyperplasia. Recent work has suggested other mediators that may contribute to the formation of vascular hyperplastic lesions. Intrinsic vessel wall cells or infikrating leukocytes not only secrete PDGF but can also transcribe genes for and secrete biologically active transforming growth factor-alpha, acidic fibroblast growth factor (also known as heparin binding growth factor-I), and transforming growth factorbeta, a peptide that under some circumstances can stimulate smooth muscle proliferation. Activated mononuclear phagocytes, endothelial cells, and vascular smooth muscle cells can also elaborate the inflammatory mediator interleukin-1. The myriad biologic effects of this mediator include the ability to stimulate the proliferation of smooth muscle cells.7 This effect may be indirect, a result of interleukin-l-induced "autocrine" PDGF-A chain secretion (Ross, R. Personal communication). Further work will doubtless add candidates to this catalog of possible physiologically relevant promoters of smooth muscle cell growth. In addition to positive stimulation of .smooth muscle proliferation, reduced inhibition of growth of these cells could also account for the net increase in cell mass characteristic of the hyperplastic lesion. Clowes and Karnovsky8 found that heparin could retard smooth muscle proliferation in vivo. Subsequent in vitro experiments revealed that heparin sulfate glycosaminoglycans synthesized by endogenous vascular wall cells themselves can limit smooth muscle proliferation. Other locally produced mediators can also inhibit smooth muscle growth. For example, transforming growth factor-beta can either limit or promote smooth muscle proliferation depending on cell density.? Other mediators that can inhibit smooth muscle growth include prostaglandin E2 and interferon ~. The identification in cell culture experiments of these various positive and negative regulators of smooth muscle proliferation suggests a more complex scheme for the control of smooth muscle growth than traditionally acknowledged. At any given point in space and time proliferation of smooth muscle cells depends on a balance between stimulatory and inhibitory stimuli. The finding that some of the mediators that enhance smooth muscle proliferation can also favor elaboration by intrinsic vascular wall cells of further growth stimulators or growth inhibitors heightens awareness of the possible interplay between these factors. Because anastomotic hyperplasia generally occurs slowly, formation of lesions might result from only a slight imbalance in positive and negative growth stimuli over months or years. The previous discussion highlights some of the recent progress in understanding mechanisms that control smooth muscle proliferation based largely on cell culture experiments. This work actually represents only the first steps

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toward comprehension of the fimdarnental mechanisms that underlie the pathogenesis of vascular hyperplasia. One challenge for future research is to move out of the culture dish into the intact animal or at least into in situ analysis of actual hyperplastic lesions. Although the cell culture approach has proved very productive for formulating notions regarding the pathogenesis of these lesions, these hypotheses await testing in vivo. The complexities of growth control alluded to herein render it unlikely that in vitro experiments or computer modeling will be able to sort out which signals actually contribute significantly to formation of lesions in the intact organism. Another remaining challenge is to unravel the mechanisms of the interaction of risk factors and formation of lesions. Practitioners of cardiovascular surgery and medicine are struck by the almost invariable association of tobacco abuse or diabetes mellitus or both with severe peripheral vascular disease. Many other examples illustrate the interaction of risk factors with lesion formation. Another fimdamental problem that remains unsolved is the mechanism of transduction of signal between mechanical or hemodynamic stimuli and biochemical or molecular changes. The discovery of stretch-regulated ion channels in vascular endothelial cells provides one potentially productive model for fiarther experimentation in this regard.i° The role of phagocytic leukocytes in the foreign body response in the pathogenesis of hyperplasia is another intriguing possibility that merits further investigation. Observational or descriptive studies in vitro or in vivo will continue to help to identify candidate mediators that could contribute to formation of anastomotic hyperplastic lesions or other vascular diseases. Such experiments will aid thinking and generation of hypotheses regarding pathogenesis. However, the real challenge will be to test such hypotheses critically in vivo. Several points bear mention in this regard. First, this further level of analysis will probably require animal experimentation and the development of a practical model of clinical anastomotic hyperplasia. Second, novel selective inhibitors of various cytokines or growth factors, some emerging from the biotechnology industry, should aid this effort. (This pathway may also lead eventually to clinically useful therapeutic interventions.) Finally, the unambiguous definition of the roles of various growth factors, cytokines, or other inflammatory mediators in the formation of vascular lesions will probably emerge from the application of newer genetic approaches such as transgenic animals or other mutational strategies~ Modern practitioners of cardiovascular surgery and medicine alike have come to appreciate that technically superb interventions and state-of-the-art biomaterials or interventional devices provide only part of the solution to human arterial diseases. The "high technology" procedures and devices that we have brought to bear on this important clinical problem have so far proved inadequate as longterm therapeutic solutions in many instances. Until we are able to incorporate the biologic reactivity of the arterial wall into our thinking, and control cellular responses to these interventions, effective and enduring therapy for diseases of muscular arteries will remain elusive. We now have

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at hand file intellectual framework and certain methodologic tools appropriate for understanding the responses of the arterial wall during the pathogenesis of arterial diseases and the vessel's reaction to our attempts to treat these problems. It is likely that during the next decade, major inroads to the solutions of these persistent clinical problems will involve an amalgam of basic cell and molecular biology with applied vascular research. Peter Libby, 21419 Departments of~4edicine (Cardiology) and Physiology Tufts UniversitySchoolofA/ledicine and New England Medical Center Boston, Massachusetts REFERENCES

1. Ross R. The pathogenesis of atherosderosis--an update. N Engl J Med 1986;314:488-500. 2. Wilcox IN, Smith KM, Williams LT, Schwartz SM, Gordon D. Platelet-derived growth factor nRNA detection in human atherosclerotic plaques by in situ hybridization. 1 Clin Invest 1988;82:1134-43. 3. Golden MA, Au YPT, Kirkman TR, Raines EW, Ross R, Clowes AW. Platelet-derived growth factor (PDGF) activity and mRNA expression in healing vascular grafts. FASEB 1 1989;3:A611. 4. Libby P, Birinyi LK. The dynamic nature of vascular endothelial functions. In: Zilla P, Fasol R, Deutsch M, eds. Endothelialization of vascular grafts. Basel: Karger, 1987:80-99. 5. Limanni A, Fleming T, Molina R, et al. Expression of genes for platelet-derived growth factor in adult human venous endothelium. A possible non-platelet-dependent cause of intireal hyperplasia in vein grafts and perianastomotic areas of vascular prostheses. J"VAsc SUV,G 1988;7:10-20. 6. Birinyi LK, Warner SIC, Salomon RN, Callow AD, Libby P. Observations on smooth muscle cell cultures from hyperplastic lesions of prosthetic bypass grafts: production of platelet-derived growth factor-like mitogen and expression of a gene for a PDGF-receptor, a preliminary study. J VAsc SURGERY1989;10:157-65. 7. Libby P, Warner SIC, Friedman GD. Interleukin-l: a mitogen for human vascular smooth muscle cells that induces the release of growth-inhibitory prostanoids. J Clin Invest 1988;88:487-98. 8. Clowes AW, Karnovsky MJ. Suppression by heparin of injury-induced myointimal thickening. J Surg Res 1978; 24:161-8. 9. Majack RA. Beta-type transforming growth factor specifies organizational behavior in vascular smooth muscle cell culrares. I Cell Biol 1987;105:465-71. 10. Lansman lB. Endothelial mechanosensors--going with the flow. Nature 1988;331:481-2.

T H E R O L E OF T H E M A C R O P H A G E I N

INTIMAL HYPERPLASIA Proliferation of vascular smooth muscle cells with their concomitant phenotypic modulation and elaboration of extracellular matrix components plays a central role in intimal hyperplasia, atherogenesis, and the foreign body reactions to a number of biomaterials. The pathogenesis of each of these processes includes the early infiltration of

!ournal of VASCULAR SURGERY

monocyte-derived macrophages. In addition to their scavenger fimctions, activated macrophages secrete many ellector proteins, including growth factors, and may thereby mediate processes in vascular healing. Anastomotic intimal hyperplasia develops in an area of a highly complex injury in which an area of abnormal endothelium resides adjacent to an area devoid of endothelium but containing a platelet-rich fibrin coagulum on a foreign body. This perianastomotic area is subjected to complicated biomechanical abnormalities induced by the biomechanical characteristics of the vascular prosthesis, the tissue reactions, and the anastomosis. Early monocyte recruitment may be a response to the foreign body or to the plasma insudation. In addition, platelet-derived growth factor (PDGF) has been shown to be chemotactic for monocytes, ~ and DiCorleto and de la Motte 2 showed preferential adhesion of monocytes to areas of injured or regenerating endothelial cells in culture. In vivo angiogenesis assays and in vitro tissue culture studies from several laboratories have shown the activity of macrophage-derived growth factor (MDGF) released from activated macrophages. Activation follows a phagocytic challenge of a test substance. Martin et al.13 showed that activation of mouse peritoneal macrophages in culture led to their release into the culture media of factor stimulating deoxyribonucleic acid synthesis in quiescent bovine aortic endothelial cells, bovine aortic smooth muscle cells, and BALB/c 3T3 mouse embryonic fibroblasts. Until recently the identity of this MDGF was unknown. In 1985 Shimokado et al.4 showed a significant portion of MDGF activity to be PDGF. Platelet-derived growth factor is a potent mitogen for smooth muscle cells and has smooth muscle cell chemoattractant properties as well. This dual action makes PDGF a suspect as mediator of the smooth muscle cell migration and proliferation characteristics of intimal hyperplasia. However, the primary source of PDGF in vivo remains speculative inasmuch as this growth factor is produced in experimental conditions by macrophages, platelets, endothelial cells, and by smooth muscle cells themselves. Although future in situ hybridization studies may suggest primary sources for PDGF production in experimental models, it is quite likely that multiple sources, including the macrophage, may play roles under different conditions determined by local biochemical, biomechanical, and surface thrombogenicity characteristics. However, the early appearance of the macrophage after prosthetic implantation and the cells' likely activation by foreign bodies and components of plasma insudates suggest that macrophages may be an early source of mitogenic stimulation. Baird et al? showed that macrophages also produce basic tibroblast growth factor (basic FGF). Basic FGF is a cationic heparin-binding growth factor capable of stimulating endothelial cell proliferation but without mitogenic activity for vascular smooth muscle cells. However, macrophage-derived basic FGF may secondarily affect intimal hyperplasia. Endothelial cells are known to produce PDGF. In the perianastomotic zone in humans, endothelial cell ingrowth over currently available biomaterials proceeds