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Age-related changes in the signaling and function of vascular smooth muscle cells
Experimental Gerontology 34 (1999) 549 –557
Age-related changes in the signaling and function of vascular smooth muscle cells Martha S. Lundberg, Michael T. Crow* Vascular Biology Unit, Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA Received 22 March 1999; accepted 29 March 1999
Abstract Aging is an independent risk factor for the development of atheroscelrosis, a vascular abnormality that plays a significant role in the development of many cardiovascular disorders. Animal experiments have demonstrated that aging predisposes the vasculature to advanced atherosclerotic disease and vessel injury and that this predisposition is a function of age-associated changes in the vessel wall itself. Because vascular smooth muscle cells play important roles in the pathogenesis of many vascular disorders, identifying age-associated differences in the way these cells respond to extracellular clues has been an area of active research. Currently, the most remarkable differences in intracellular signaling between vascular smooth muscle cells isolated from young and old animals are related to the control of cell migration through the CamKII pathways and the accelerated transition of older vascular smooth muscle cells from the contractile to the synthetic phenotype. These differences may be due to alternative signaling pathways revealed by the inability of older cells to respond to inhibitors, such as transforming growth factor (TGF)-1, or to altered interactions with the extracellular matrix resulting from ageassociated shifts in integrin expression or changes in the matrix composition of blood vessels. The exact role that these alterations have in explaining age-associated differences in the response of the vessel wall to injury and its increased susceptibility to developing advanced atherosclerotic lesions remains to be determined but will be guided by studies on intracellular signaling mechanisms. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Atherosclerosis; Vascular injury; Vascular smooth muscle cells; Intima; Neointima; TGF-1; bFGF; PDGF; Calcium; Calmodulin-dependent protein kinases; Integrins
* Corresponding author. Tel.: ⫹011-410-558-8207; fax: ⫹011-410-558-8150. E-mail address: [email protected] (M.T. Crow)
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1. Introduction Cardiovascular disease is the leading cause of death and a major source of disability among the elderly population. Vascular abnormalities, such as atherosclerosis, are a significant factor in the development of many cardiovascular diseases. Epidemiological studies have shown that aging is an independent risk factor for the development of atherosclerosis (Kannel and Gordon, 1980), a vascular abnormality that plays a significant role in the development of many cardiovascular disorders. Animal experiments, in which genetic variations are minimized and exposure to atherosclerosis-inducing stimuli can be precisely controlled, support the notion that aging by itself predisposes the vasculature to advanced atherosclerotic disease. Spagnoli and colleagues (1991) showed that New Zealand white rabbits responded differently to a lipid- and cholesterol-rich diet depending on their age. In particular, younger rabbits developed fewer vascular lesions than their older counterparts, despite the fact that the duration of exposure to the atherogenic stimulus was the same and that changes in blood lipid profiles due to the diet were also indistinguishable between the two age groups. In addition, the majority of lesions in older rabbits were at a significantly more advanced stage of development than in the younger animals. Similar quantitative and qualitative differences in the susceptibility to dietinduced atherosclerosis have been reported in adult and juvenile primates (Weingand et al., 1986). Together, these results indicate that the higher incidence and greater severity of vascular lesions in older animals are the result of biological changes in the organism related to aging.
2. Smooth muscle cells and vascular abnormalities In its more advanced stages, the atherosclerotic lesion is a complex structure of living and dead cells, cellular debris, and macromolecular deposits of lipids, extracellular matrix components, and minerals (reviewed in Bilato and Crow, 1996). Its cellular composition is likely to consist of blood cells recruited from the circulation, vascular smooth muscle cells (VSMCs) already present in the intima (the actual site of lesion development in the vessel-see below), and cells recruited from the underlying medial or adventitial cell layers. A complete understanding of lesion development, however, is seriously hampered by the fact that the exact origins of all the cells that comprise these lesions have not been unambiguously established. A large body of circumstantial evidence, however, suggests important roles for monocytes recruited from the circulation and VSMCs from the medial and intimal cell layers (Schwartz et al., 1995; Bilato and Crow, 1996). The typical arterial vessel wall is composed of three distinct cellular layers. Moving from luminal side inward, the first cellular layer is the intima in which the endothelium resides. In rats, this is the only layer of cells in the intima throughout most of their adult life. In other animals, including humans, other cells are also present in the intima and are located beneath the endothelium. Based on the expression of gene markers, some of these cells may be of smooth muscle cell origin and are accordingly referred to as intimal VSMCs (Schwartz et al., 1995). The size of the intima increases dramatically when the vessel wall is traumatized, such as following mechanical injury inflicted by aggressive inflation of a balloon catheter. The exaggerated intima that develops under these conditions is called the neointima. In rats, the neointima consists almost exclusively of VSMC-like cells, the properties of which are described below. In other animals, including
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humans, the neointima also involves macrophage and leukocyte infiltration (Tanaka et al., 1993). The intima is separated from the medial cell layer by the internal elastic basal lamina. The medial layer consists of variable numbers of elastic laminae and medial VSMCs, each cell being surrounded by an individual basement membrane (Pauly et al., 1992). The medial cell layer is separated from the adventitial cell layer, which contains fibroblast-like cells and blood vessels, by the external elastic lamina. In the intact vessel, medial VSMCs are relatively quiescent with respect to proliferation and are highly specialized as contractile cells to control lumen diameter and thereby regulate blood flow in response to nervous, hormonal, and local influences. When these cells are isolated from the vessel and placed in cell culture, they gradually lose their specialized or differentiated properties and become proliferative and highly motile. In addition, the cells’ gene expression and protein synthesis machinery are redirected from the production of specialized contractile elements to that of supporting cell proliferation and extracellular matrix deposition. This process of switching from the contractile/ differentiated mode to a proliferating/synthetic cell type has been given the name, phenotypic modulation (Campbell and Campbell, 1990). The switch from the contractile to the synthetic phenotype begins as soon as medial VSMCs are isolated from the intact vessel. The process can be somewhat delayed by manipulating the extracellular environment of the isolated VSMCs when they are placed in cell culture. This suggests that the unique features of the extracellular environment of medial VSMCs including their association with an organized elastin matrix and the individual basement membrane surrounding each cell is responsible, in part, for inducing or maintaining the contractile phenotype (Thyberg et al., 1990). Attempts to return synthetic VSMCs to the contractile phenotype by embedding them in reconstituted basement membranes have been only partially successful (Pauly et al., 1992). It is of some interest and possible relevance that medial VSMCs isolated from the vessels of older animals modulate to the synthetic state more rapidly than those isolated from younger animals (Bilato and Crow, 1996). The role that phenotypic modulation plays in vascular abnormalities and the vessel wall’s response to trauma, however, is questionable. Smooth actin-positive cells can be found in atherosclerotic lesions and are abundantly present in human restenotic lesions or the neointima caused by balloon catheter injury in animals (Schwartz et al., 1995), an experimental model for human restenosis. Are these cells synthetic medial VSMCs that have migrated into the lesions from the media? In the case of neointima that forms in response to vessel injury, the answer seems to be no. Although both synthetic medial VSMCs and neointimal VSMCs are non-contractile and proliferate when placed in cell culture, they exhibit strikingly different morphological features and patterns of gene expression (see Schwartz et al., 1995; Pauly et al., 1998). Furthermore, these differences are maintained in culture for many generations, indicating that these phenotypes are not readily interconvertible. Cells with some of the characteristics of neointimal VSMCs can be isolated as a minor subpopulation from the medial cell layer (Bochaton–Piallat et al., 1996), suggesting that these are cells preferentially recruited to the intima upon injury. This be a necessary step for neointimal formation in the balloon injured vessels of young adult rats, since the intima in these animals consists solely of the endothelium and its underlying basement membrane. It is also likely to occur in other animals, including man, since balloon catheter inflation or vessel atherectomy generally removes the complete intima. Interestingly, medial VSMCs isolated from arterial vessels of late embryonic or fetal rats are virtually indistinguishable from neointimal VSMCs (Majesky et al., 1992). Neointimal VSMCs
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may, therefore, arise either from a minor subpopulation of VSMCs in the media that have retained fetal characteristics or from the general population of medial VSMCs that have reverted to the fetal phenotype upon injury of the vessel. Reversion to a fetal phenotype in response to cellular stress has been noted in many different cell types, including cardiomyocytes (reviewed in Crow et al., 1996). What makes neointimal/intimal VSMCs unique in this regard is that the phenotype is stable and persists both in vivo and in cell culture.
3. Age-associated differences in VSMC signaling during the response to vessel injury Experiments published more than 10 years ago established that the response to vessel injury is much greater in vessels of older animals (Hariri et al., 1986). In these studies, two types of vessel injury were compared. In the first, the vessel underwent severe mechanical injury caused by the rapid and repeated overinflation of a balloon catheter. This manipulation resulted in endothelial denudation, platelet binding and activation at the denuded interface, and massive mechanical injury to the underlying medial cell layer resulting in rupture of the elastic laminae and medial cell death. Vessels from either young or old animals mounted a similar response to this type of injury and produced a large neointima at the site of injury. The second type of injury involved gently denuding the vessel of its endothelial cell layer with a fine wire coil. This injury resulted in platelet binding and activation to the denuded vessel wall but produced no significant injury to the underlying medial cell layer. Under these circumstances, the response of younger versus older vessels were markedly different. Vessels in young animals failed to show an increased intima, while vessels in older animals displayed a neointima similar to that seen with the more invasive balloon catheter injury. Subsequent experiments in which vessels from young and old animals were transplanted into different aged hosts established that the age-associated differences in the response to injury were intrinsic to the vessel and unaffected by the age of the donor. One explanation for the differences in responsiveness between young and old vessels has emerged from further analyses of the response to balloon catheter injury in younger animals. These studies established that both platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) are required for the development of the neointima (Lindner et al., 1991). The source of PDGF is activated platelets adhering to the denuded vessel wall, whereas bFGF is released by mechanical stress to VSMCs residing in the media. Infusion of neutralizing antibodies to either PDGF or bFGF blocks neointimal development in response to balloon injury in young animals (Ferns et al., 1991; Lindner et al., 1991). In young vessels, therefore, gentle denudation is not sufficient to evoke neointimal development because there is no bFGF release from the medial cell layer. Subsequently, it has been shown that infusion of bFGF, though itself not enough to elicit neointimal development, enabled young vessels to develop such lesions following gentle denudation (Lindner et al., 1991). Cell culture experiments examining the role of bFGF in PDGF-directed chemotaxis of VSMCs from our laboratory have provided a possible mechanism for this dual growth factor requirement (Bilato et al., 1995). These studies demonstrated that, although bFGF itself was not a chemoattractant, it was required for PDGF to activate the enzyme, calcium/calmodulin-dependent protein kinase II (CamKII). Activation of this enzyme was
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previously shown by the laboratory to be required for PDGF-directed migration of VSMCs isolated from young adult rats and to be an important point of integration for cellular signals and interaction with the environment (Pauly et al., 1995). The signaling requirements for PDGF-directed migration in VSMCs isolated from “older” rats (18 –20 months), however, was quite different than that characterized in the “young” VSMCs and utilized a mechanism that did not require CamKII activation. Consequently, the migration of these VSMCs toward PDGF was unaffected by inhibitors of CamKII and neutralizing antibodies to bFGF (Crow and Bilato, 1996). These results suggested that the difference in the response of young and old rats to gentle arterial endothelium denudation was due, in part, to the fact that the signaling pathway regulating migration of VSMCs from older animals was able to bypass the requirement for CamKII activation and, therefore, the need for bFGF release from the underlying medial VSMCs. How older VSMCs are able to bypass this obligatory integration point is unclear and may involve changes in expression of inhibitors of migration, such as transforming growth factor (TGF)  (see below), or different patterns of integrin expression. Integrins have been shown to be critical for regulating intracellular signaling pathways governing complex biological functions, such as cell migration and proliferation. This last consideration may of particularly importance to emerging therapies for the treatment of vascular disorders based on integrin blockade. One of these involves inhibition of 3 integrin function, which is thought to target not only thrombolytic events via the ␣IIb3 receptor on platelets, but also VSMC function through its ␣v3 receptor. We recently showed that PDGF-migration of VSMCs was inhibited by antagonists of the ␣v3 receptor and involved ␣v3 regulation (via bFGF) of CamKII activation (Bilato et al., 1997). Not surprisingly, migration of VSMCs isolated from old rats was unaffected by these antagonists (Bilato and Crow, 1996). If these results in the rat are applicable to humans, then the effectiveness of anti-␣v3 therapy may significantly reduce vascular disorders in the elderly. Other changes in VSMC may also play a role in the enhanced responsiveness of older vessels to injury. Older VSMCs express more PDGF receptors (Sarzani et al., 1991) and migrate at a significantly greater rate than VSMCs isolated from young adult rats (Li et al., 1997). In addition, although earlier studies concentrated on examining the role for CamKII in the regulation of VSMC function, it is now clear from numerous studies that activation of the mitogen-activated protein kinases (MAPKs), erk1/2, are also important for PDGFstimulated migration (Graf et al., 1997; Lundberg et al., 1998). Our laboratory has shown further that MAPK-mediated cell migration occurs through both CamKII-dependent and -independent mechanisms (Lundberg et al., 1998). About half of all cellular MAPK activity generated by mitogens is microtubule-associated (Reszka et al., 1995), which is thought to phosphorylate microtubule-associated proteins, such as tau (Drewes et al., 1992). This, in turn, may destabilize microtubules and promote cell movement. Ageassociated changes in cytoskeletal proteins occur, and of particular interest, is the upregulation of tubulin, a major component of microtubules (Li et al., 1997). Based on the current data, it is reasonable to hypothesize that age-related increases in tubulin and VSMC migration may allow a shift in the signal transduction pathway toward MAPKmediated microtubule destabilization and away from that regulated by CamKII activation. VSMCs from older animals are also relatively insensitive to the inhibitory influences of TGF-1. These VSMCs exhibit a reduced capacity to bind TGF-1 on the cell surface and an increased ability to actively degrade it (McCaffrey and Falcone, 1993). The mechanism(s) underlying this age-associated defect is unknown, but it has been shown
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that VSMCs from human atherosclerotic and restenotic lesions can acquire resistance to the anti-proliferative effects of TGF1 as a result of the selective loss of functional type II receptors (McCaffrey et al., 1995). Because expression of type I receptors are unaffected and these receptors are preferentially involved in extracellular matrix production, the response of these cells to TGF1 switches from growth arrest to increased fibrosis and uncontrolled growth. Transfection of type II receptors restores the anti-proliferative response in cultured cells, suggesting that intracellular signaling pathways associated with these receptors are intact. More recently, this selective loss in type II TGF- receptors in atherosclerotic lesions has been attributed to microsatellite instability (McCaffrey et al., 1997). Loss of growth control through a genetic mechanism may account for the monoclonal expansion that has been observed in human atherosclerotic lesions (see Schwartz et al., 1995). It is unlikely, however, to account for the age-associated differences seen in response to mechanical vessel injury, unless a significant fraction of cells in the vessel wall have already undergone mutation before the injury is induced.
4. Changes in the vascular wall with aging that may affect VSMC signaling and responsiveness Though intimal thickening can be experimentally induced by trauma, it also occurs during the normal course of vessel aging. In rats, the intima increases up to fivefold compare to young animals. This increase is due predominantly to the accumulation of VSMCs as well as monocytes (Guyton et al., 1983; Li et al., 1999). Both of these cells types are absent from the intima of young rats, which contains only the endothelium and its underlying basement membrane support. A similar thickening of the tunica intima has been reported in humans (Virmani et al., 1991). Thickening of the intima due to VSMC accumulation is likely significant, since regions of intimal thickening are prone to the development of atherosclerosis (reviewed in Schwartz et al., 1995). Changes in the structural architecture of the vessel wall also occur with aging and these changes may be important factors in age-associated increase in the the overall increase in the intima. Extracellular matrix reorganization occurs with aging, such that the volume density of collagen increases with age, whereas that of elastin decreases (Forneri et al., 1992). In addition, collagen fibrils become organized into multi-branched bundles, whereas elastin fibers, which are organized as thick and continuous lamella early in adult life, become disorganized, thinner, and often fragmented with age (Guyton et al., 1983; Forneri et al., 1992). These changes in the relative content and organization of collagen and elastin result in increased fibrosis and contribute to the stiffening of the vascular wall (Lakatta, 1993), an event associated with hypertrophy and the eventual heart failure. The changes in TGF responsiveness described above may be partially responsible for this shift toward increased fibrosis with aging. In addition, increased TGF expression has recently been demonstrated in aging vessels (Li et al., 1999). Changes in elastin biosynthesis and degradation are also likely to play an important role in vascular remodeling and intracellular signaling with aging. Recent genetic studies in which the elastin gene had been deleted from the mouse genome resulted in extensive intimal thickening, indicating that the importance of elastin extends beyond its role in stabilizing arterial structure to regulating VSMCs proliferation and migration during development (Li et al., 1998). Fragmentation and loss of elastin may have similar if not as dramatic effects. Both the accumulation of glycosaminoglycans, which interferes with
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elastin assembly as can the proteolytic degradation of elastin by elastases and metalloproteinases increase with aging (Li et al., 1999). In addition, it has been shown that elastin fragments can act as specific ligands for receptor-mediated signaling in cells (Senior et al., 1984). Paralleling the increase in TFG1 levels in the aging vasculature are marked increases in fibronectin levels (Li et al., 1999). These changes undoubtedly contribute to the shift from an elastic to a fibrotic/stiff vessel. Fibronectin also accelerates the switch from contractile to synthetic phenotype that occurs when medial VSMCs are placed in culture (Thyberg et al., 1990). Interestingly, both fibronectin and TGF- expression are regulated by angiotensin II and chronic administration of angiotensin-converting enzyme (ACE) inhibitors substantially reduce and delay many of the age-associated extracellular matrix and intimal changes described above (Michel et al., 1994). Whether ACE inhibition would affect the response of such “rejuvenated” older vessels to injury is not known but would be valuable in sorting out whether changes in VSMC intracellular signaling pathways with aging are the cause or effect of vessel wall alterations with aging. Finally, another factor that may affect VSMC signaling with advancing age are the effects of advanced glycation endproducts (AGEs). AGEs are the products of nonenzymatic but irreversible glycation and oxidation of proteins and lipids (reviewed in Schmidt et al., 1994). AGEs accumulate on proteins with long half lives, such as collagen and elastin, and occur at an accelerated rate in diabetes. AGEs exert their effects on vascular cells through both direct and indirect mechanisms. Indirectly, they can modify and crosslink extracellular matrix proteins, contributing to increased stiffness of the vessel with age. Directly, they induce a pro-oxidant stress on the cell through a variety of cell surface receptors (Yan et al., 1994). One of these is a member of the immunoglobulin superfamily, referred to as RAGE (receptor for advanced glycation endproducts). Expression of RAGE is elevated in atherosclerotic lesions and in the neointima that forms in response to vessel injury (Crow et al., 1999). AGE-RAGE interactions lead to a proinflammatory pattern of gene expression which is likely to contribute to and exacerbate atherosclerotic lesion development. The development of reagents to block AGE-RAGE interactions and AGE formation will undoubtedly help determine the relative importance of AGEs and RAGE to all vascular abnormalities and especially those whose incidence is markedly elevated in aging vessels.
5. Discussion Currently, the most remarkable differences in intracellular signaling between VSMCs isolated from young and old animals are related to the control of cell migration through the CamKII pathways and the accelerated transition of older VSMCs from the contractile to the synthetic phenotype. These differences may be due to alternative signaling pathways revealed by the inability of older VSMCs to respond to inhibitors, such as TGF-1, or to altered interactions with the extracellular matrix resulting from age-associated shifts in integrin expression. The exact role that these alterations have in explaining ageassociated differences in the response of the vessel wall to injury and its increased susceptibility to developing advanced atherosclerotic lesions remains to be determined but will be guided by such studies on intracellular signaling mechanisms.
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Age-related changes in the signaling and function of vascular smooth muscle cells Martha S. Lundberg, Michael T. Crow* Vascular Biology Unit, Laboratory of Cardiovascular Science, Gerontology Research Center, National Institute on Aging, National Institutes of Health, Baltimore, MD 21224, USA Received 22 March 1999; accepted 29 March 1999
Abstract Aging is an independent risk factor for the development of atheroscelrosis, a vascular abnormality that plays a significant role in the development of many cardiovascular disorders. Animal experiments have demonstrated that aging predisposes the vasculature to advanced atherosclerotic disease and vessel injury and that this predisposition is a function of age-associated changes in the vessel wall itself. Because vascular smooth muscle cells play important roles in the pathogenesis of many vascular disorders, identifying age-associated differences in the way these cells respond to extracellular clues has been an area of active research. Currently, the most remarkable differences in intracellular signaling between vascular smooth muscle cells isolated from young and old animals are related to the control of cell migration through the CamKII pathways and the accelerated transition of older vascular smooth muscle cells from the contractile to the synthetic phenotype. These differences may be due to alternative signaling pathways revealed by the inability of older cells to respond to inhibitors, such as transforming growth factor (TGF)-1, or to altered interactions with the extracellular matrix resulting from ageassociated shifts in integrin expression or changes in the matrix composition of blood vessels. The exact role that these alterations have in explaining age-associated differences in the response of the vessel wall to injury and its increased susceptibility to developing advanced atherosclerotic lesions remains to be determined but will be guided by studies on intracellular signaling mechanisms. © 1999 Elsevier Science Inc. All rights reserved. Keywords: Atherosclerosis; Vascular injury; Vascular smooth muscle cells; Intima; Neointima; TGF-1; bFGF; PDGF; Calcium; Calmodulin-dependent protein kinases; Integrins
* Corresponding author. Tel.: ⫹011-410-558-8207; fax: ⫹011-410-558-8150. E-mail address: [email protected] (M.T. Crow)
0531-5565/99/$ – see front matter © 1999 Elsevier Science Inc. All rights reserved. PII: S 0 5 3 1 - 5 5 6 5 ( 9 9 ) 0 0 0 3 6 - 4
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1. Introduction Cardiovascular disease is the leading cause of death and a major source of disability among the elderly population. Vascular abnormalities, such as atherosclerosis, are a significant factor in the development of many cardiovascular diseases. Epidemiological studies have shown that aging is an independent risk factor for the development of atherosclerosis (Kannel and Gordon, 1980), a vascular abnormality that plays a significant role in the development of many cardiovascular disorders. Animal experiments, in which genetic variations are minimized and exposure to atherosclerosis-inducing stimuli can be precisely controlled, support the notion that aging by itself predisposes the vasculature to advanced atherosclerotic disease. Spagnoli and colleagues (1991) showed that New Zealand white rabbits responded differently to a lipid- and cholesterol-rich diet depending on their age. In particular, younger rabbits developed fewer vascular lesions than their older counterparts, despite the fact that the duration of exposure to the atherogenic stimulus was the same and that changes in blood lipid profiles due to the diet were also indistinguishable between the two age groups. In addition, the majority of lesions in older rabbits were at a significantly more advanced stage of development than in the younger animals. Similar quantitative and qualitative differences in the susceptibility to dietinduced atherosclerosis have been reported in adult and juvenile primates (Weingand et al., 1986). Together, these results indicate that the higher incidence and greater severity of vascular lesions in older animals are the result of biological changes in the organism related to aging.
2. Smooth muscle cells and vascular abnormalities In its more advanced stages, the atherosclerotic lesion is a complex structure of living and dead cells, cellular debris, and macromolecular deposits of lipids, extracellular matrix components, and minerals (reviewed in Bilato and Crow, 1996). Its cellular composition is likely to consist of blood cells recruited from the circulation, vascular smooth muscle cells (VSMCs) already present in the intima (the actual site of lesion development in the vessel-see below), and cells recruited from the underlying medial or adventitial cell layers. A complete understanding of lesion development, however, is seriously hampered by the fact that the exact origins of all the cells that comprise these lesions have not been unambiguously established. A large body of circumstantial evidence, however, suggests important roles for monocytes recruited from the circulation and VSMCs from the medial and intimal cell layers (Schwartz et al., 1995; Bilato and Crow, 1996). The typical arterial vessel wall is composed of three distinct cellular layers. Moving from luminal side inward, the first cellular layer is the intima in which the endothelium resides. In rats, this is the only layer of cells in the intima throughout most of their adult life. In other animals, including humans, other cells are also present in the intima and are located beneath the endothelium. Based on the expression of gene markers, some of these cells may be of smooth muscle cell origin and are accordingly referred to as intimal VSMCs (Schwartz et al., 1995). The size of the intima increases dramatically when the vessel wall is traumatized, such as following mechanical injury inflicted by aggressive inflation of a balloon catheter. The exaggerated intima that develops under these conditions is called the neointima. In rats, the neointima consists almost exclusively of VSMC-like cells, the properties of which are described below. In other animals, including
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humans, the neointima also involves macrophage and leukocyte infiltration (Tanaka et al., 1993). The intima is separated from the medial cell layer by the internal elastic basal lamina. The medial layer consists of variable numbers of elastic laminae and medial VSMCs, each cell being surrounded by an individual basement membrane (Pauly et al., 1992). The medial cell layer is separated from the adventitial cell layer, which contains fibroblast-like cells and blood vessels, by the external elastic lamina. In the intact vessel, medial VSMCs are relatively quiescent with respect to proliferation and are highly specialized as contractile cells to control lumen diameter and thereby regulate blood flow in response to nervous, hormonal, and local influences. When these cells are isolated from the vessel and placed in cell culture, they gradually lose their specialized or differentiated properties and become proliferative and highly motile. In addition, the cells’ gene expression and protein synthesis machinery are redirected from the production of specialized contractile elements to that of supporting cell proliferation and extracellular matrix deposition. This process of switching from the contractile/ differentiated mode to a proliferating/synthetic cell type has been given the name, phenotypic modulation (Campbell and Campbell, 1990). The switch from the contractile to the synthetic phenotype begins as soon as medial VSMCs are isolated from the intact vessel. The process can be somewhat delayed by manipulating the extracellular environment of the isolated VSMCs when they are placed in cell culture. This suggests that the unique features of the extracellular environment of medial VSMCs including their association with an organized elastin matrix and the individual basement membrane surrounding each cell is responsible, in part, for inducing or maintaining the contractile phenotype (Thyberg et al., 1990). Attempts to return synthetic VSMCs to the contractile phenotype by embedding them in reconstituted basement membranes have been only partially successful (Pauly et al., 1992). It is of some interest and possible relevance that medial VSMCs isolated from the vessels of older animals modulate to the synthetic state more rapidly than those isolated from younger animals (Bilato and Crow, 1996). The role that phenotypic modulation plays in vascular abnormalities and the vessel wall’s response to trauma, however, is questionable. Smooth actin-positive cells can be found in atherosclerotic lesions and are abundantly present in human restenotic lesions or the neointima caused by balloon catheter injury in animals (Schwartz et al., 1995), an experimental model for human restenosis. Are these cells synthetic medial VSMCs that have migrated into the lesions from the media? In the case of neointima that forms in response to vessel injury, the answer seems to be no. Although both synthetic medial VSMCs and neointimal VSMCs are non-contractile and proliferate when placed in cell culture, they exhibit strikingly different morphological features and patterns of gene expression (see Schwartz et al., 1995; Pauly et al., 1998). Furthermore, these differences are maintained in culture for many generations, indicating that these phenotypes are not readily interconvertible. Cells with some of the characteristics of neointimal VSMCs can be isolated as a minor subpopulation from the medial cell layer (Bochaton–Piallat et al., 1996), suggesting that these are cells preferentially recruited to the intima upon injury. This be a necessary step for neointimal formation in the balloon injured vessels of young adult rats, since the intima in these animals consists solely of the endothelium and its underlying basement membrane. It is also likely to occur in other animals, including man, since balloon catheter inflation or vessel atherectomy generally removes the complete intima. Interestingly, medial VSMCs isolated from arterial vessels of late embryonic or fetal rats are virtually indistinguishable from neointimal VSMCs (Majesky et al., 1992). Neointimal VSMCs
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may, therefore, arise either from a minor subpopulation of VSMCs in the media that have retained fetal characteristics or from the general population of medial VSMCs that have reverted to the fetal phenotype upon injury of the vessel. Reversion to a fetal phenotype in response to cellular stress has been noted in many different cell types, including cardiomyocytes (reviewed in Crow et al., 1996). What makes neointimal/intimal VSMCs unique in this regard is that the phenotype is stable and persists both in vivo and in cell culture.
3. Age-associated differences in VSMC signaling during the response to vessel injury Experiments published more than 10 years ago established that the response to vessel injury is much greater in vessels of older animals (Hariri et al., 1986). In these studies, two types of vessel injury were compared. In the first, the vessel underwent severe mechanical injury caused by the rapid and repeated overinflation of a balloon catheter. This manipulation resulted in endothelial denudation, platelet binding and activation at the denuded interface, and massive mechanical injury to the underlying medial cell layer resulting in rupture of the elastic laminae and medial cell death. Vessels from either young or old animals mounted a similar response to this type of injury and produced a large neointima at the site of injury. The second type of injury involved gently denuding the vessel of its endothelial cell layer with a fine wire coil. This injury resulted in platelet binding and activation to the denuded vessel wall but produced no significant injury to the underlying medial cell layer. Under these circumstances, the response of younger versus older vessels were markedly different. Vessels in young animals failed to show an increased intima, while vessels in older animals displayed a neointima similar to that seen with the more invasive balloon catheter injury. Subsequent experiments in which vessels from young and old animals were transplanted into different aged hosts established that the age-associated differences in the response to injury were intrinsic to the vessel and unaffected by the age of the donor. One explanation for the differences in responsiveness between young and old vessels has emerged from further analyses of the response to balloon catheter injury in younger animals. These studies established that both platelet-derived growth factor (PDGF) and basic fibroblast growth factor (bFGF) are required for the development of the neointima (Lindner et al., 1991). The source of PDGF is activated platelets adhering to the denuded vessel wall, whereas bFGF is released by mechanical stress to VSMCs residing in the media. Infusion of neutralizing antibodies to either PDGF or bFGF blocks neointimal development in response to balloon injury in young animals (Ferns et al., 1991; Lindner et al., 1991). In young vessels, therefore, gentle denudation is not sufficient to evoke neointimal development because there is no bFGF release from the medial cell layer. Subsequently, it has been shown that infusion of bFGF, though itself not enough to elicit neointimal development, enabled young vessels to develop such lesions following gentle denudation (Lindner et al., 1991). Cell culture experiments examining the role of bFGF in PDGF-directed chemotaxis of VSMCs from our laboratory have provided a possible mechanism for this dual growth factor requirement (Bilato et al., 1995). These studies demonstrated that, although bFGF itself was not a chemoattractant, it was required for PDGF to activate the enzyme, calcium/calmodulin-dependent protein kinase II (CamKII). Activation of this enzyme was
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previously shown by the laboratory to be required for PDGF-directed migration of VSMCs isolated from young adult rats and to be an important point of integration for cellular signals and interaction with the environment (Pauly et al., 1995). The signaling requirements for PDGF-directed migration in VSMCs isolated from “older” rats (18 –20 months), however, was quite different than that characterized in the “young” VSMCs and utilized a mechanism that did not require CamKII activation. Consequently, the migration of these VSMCs toward PDGF was unaffected by inhibitors of CamKII and neutralizing antibodies to bFGF (Crow and Bilato, 1996). These results suggested that the difference in the response of young and old rats to gentle arterial endothelium denudation was due, in part, to the fact that the signaling pathway regulating migration of VSMCs from older animals was able to bypass the requirement for CamKII activation and, therefore, the need for bFGF release from the underlying medial VSMCs. How older VSMCs are able to bypass this obligatory integration point is unclear and may involve changes in expression of inhibitors of migration, such as transforming growth factor (TGF)  (see below), or different patterns of integrin expression. Integrins have been shown to be critical for regulating intracellular signaling pathways governing complex biological functions, such as cell migration and proliferation. This last consideration may of particularly importance to emerging therapies for the treatment of vascular disorders based on integrin blockade. One of these involves inhibition of 3 integrin function, which is thought to target not only thrombolytic events via the ␣IIb3 receptor on platelets, but also VSMC function through its ␣v3 receptor. We recently showed that PDGF-migration of VSMCs was inhibited by antagonists of the ␣v3 receptor and involved ␣v3 regulation (via bFGF) of CamKII activation (Bilato et al., 1997). Not surprisingly, migration of VSMCs isolated from old rats was unaffected by these antagonists (Bilato and Crow, 1996). If these results in the rat are applicable to humans, then the effectiveness of anti-␣v3 therapy may significantly reduce vascular disorders in the elderly. Other changes in VSMC may also play a role in the enhanced responsiveness of older vessels to injury. Older VSMCs express more PDGF receptors (Sarzani et al., 1991) and migrate at a significantly greater rate than VSMCs isolated from young adult rats (Li et al., 1997). In addition, although earlier studies concentrated on examining the role for CamKII in the regulation of VSMC function, it is now clear from numerous studies that activation of the mitogen-activated protein kinases (MAPKs), erk1/2, are also important for PDGFstimulated migration (Graf et al., 1997; Lundberg et al., 1998). Our laboratory has shown further that MAPK-mediated cell migration occurs through both CamKII-dependent and -independent mechanisms (Lundberg et al., 1998). About half of all cellular MAPK activity generated by mitogens is microtubule-associated (Reszka et al., 1995), which is thought to phosphorylate microtubule-associated proteins, such as tau (Drewes et al., 1992). This, in turn, may destabilize microtubules and promote cell movement. Ageassociated changes in cytoskeletal proteins occur, and of particular interest, is the upregulation of tubulin, a major component of microtubules (Li et al., 1997). Based on the current data, it is reasonable to hypothesize that age-related increases in tubulin and VSMC migration may allow a shift in the signal transduction pathway toward MAPKmediated microtubule destabilization and away from that regulated by CamKII activation. VSMCs from older animals are also relatively insensitive to the inhibitory influences of TGF-1. These VSMCs exhibit a reduced capacity to bind TGF-1 on the cell surface and an increased ability to actively degrade it (McCaffrey and Falcone, 1993). The mechanism(s) underlying this age-associated defect is unknown, but it has been shown
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that VSMCs from human atherosclerotic and restenotic lesions can acquire resistance to the anti-proliferative effects of TGF1 as a result of the selective loss of functional type II receptors (McCaffrey et al., 1995). Because expression of type I receptors are unaffected and these receptors are preferentially involved in extracellular matrix production, the response of these cells to TGF1 switches from growth arrest to increased fibrosis and uncontrolled growth. Transfection of type II receptors restores the anti-proliferative response in cultured cells, suggesting that intracellular signaling pathways associated with these receptors are intact. More recently, this selective loss in type II TGF- receptors in atherosclerotic lesions has been attributed to microsatellite instability (McCaffrey et al., 1997). Loss of growth control through a genetic mechanism may account for the monoclonal expansion that has been observed in human atherosclerotic lesions (see Schwartz et al., 1995). It is unlikely, however, to account for the age-associated differences seen in response to mechanical vessel injury, unless a significant fraction of cells in the vessel wall have already undergone mutation before the injury is induced.
4. Changes in the vascular wall with aging that may affect VSMC signaling and responsiveness Though intimal thickening can be experimentally induced by trauma, it also occurs during the normal course of vessel aging. In rats, the intima increases up to fivefold compare to young animals. This increase is due predominantly to the accumulation of VSMCs as well as monocytes (Guyton et al., 1983; Li et al., 1999). Both of these cells types are absent from the intima of young rats, which contains only the endothelium and its underlying basement membrane support. A similar thickening of the tunica intima has been reported in humans (Virmani et al., 1991). Thickening of the intima due to VSMC accumulation is likely significant, since regions of intimal thickening are prone to the development of atherosclerosis (reviewed in Schwartz et al., 1995). Changes in the structural architecture of the vessel wall also occur with aging and these changes may be important factors in age-associated increase in the the overall increase in the intima. Extracellular matrix reorganization occurs with aging, such that the volume density of collagen increases with age, whereas that of elastin decreases (Forneri et al., 1992). In addition, collagen fibrils become organized into multi-branched bundles, whereas elastin fibers, which are organized as thick and continuous lamella early in adult life, become disorganized, thinner, and often fragmented with age (Guyton et al., 1983; Forneri et al., 1992). These changes in the relative content and organization of collagen and elastin result in increased fibrosis and contribute to the stiffening of the vascular wall (Lakatta, 1993), an event associated with hypertrophy and the eventual heart failure. The changes in TGF responsiveness described above may be partially responsible for this shift toward increased fibrosis with aging. In addition, increased TGF expression has recently been demonstrated in aging vessels (Li et al., 1999). Changes in elastin biosynthesis and degradation are also likely to play an important role in vascular remodeling and intracellular signaling with aging. Recent genetic studies in which the elastin gene had been deleted from the mouse genome resulted in extensive intimal thickening, indicating that the importance of elastin extends beyond its role in stabilizing arterial structure to regulating VSMCs proliferation and migration during development (Li et al., 1998). Fragmentation and loss of elastin may have similar if not as dramatic effects. Both the accumulation of glycosaminoglycans, which interferes with
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elastin assembly as can the proteolytic degradation of elastin by elastases and metalloproteinases increase with aging (Li et al., 1999). In addition, it has been shown that elastin fragments can act as specific ligands for receptor-mediated signaling in cells (Senior et al., 1984). Paralleling the increase in TFG1 levels in the aging vasculature are marked increases in fibronectin levels (Li et al., 1999). These changes undoubtedly contribute to the shift from an elastic to a fibrotic/stiff vessel. Fibronectin also accelerates the switch from contractile to synthetic phenotype that occurs when medial VSMCs are placed in culture (Thyberg et al., 1990). Interestingly, both fibronectin and TGF- expression are regulated by angiotensin II and chronic administration of angiotensin-converting enzyme (ACE) inhibitors substantially reduce and delay many of the age-associated extracellular matrix and intimal changes described above (Michel et al., 1994). Whether ACE inhibition would affect the response of such “rejuvenated” older vessels to injury is not known but would be valuable in sorting out whether changes in VSMC intracellular signaling pathways with aging are the cause or effect of vessel wall alterations with aging. Finally, another factor that may affect VSMC signaling with advancing age are the effects of advanced glycation endproducts (AGEs). AGEs are the products of nonenzymatic but irreversible glycation and oxidation of proteins and lipids (reviewed in Schmidt et al., 1994). AGEs accumulate on proteins with long half lives, such as collagen and elastin, and occur at an accelerated rate in diabetes. AGEs exert their effects on vascular cells through both direct and indirect mechanisms. Indirectly, they can modify and crosslink extracellular matrix proteins, contributing to increased stiffness of the vessel with age. Directly, they induce a pro-oxidant stress on the cell through a variety of cell surface receptors (Yan et al., 1994). One of these is a member of the immunoglobulin superfamily, referred to as RAGE (receptor for advanced glycation endproducts). Expression of RAGE is elevated in atherosclerotic lesions and in the neointima that forms in response to vessel injury (Crow et al., 1999). AGE-RAGE interactions lead to a proinflammatory pattern of gene expression which is likely to contribute to and exacerbate atherosclerotic lesion development. The development of reagents to block AGE-RAGE interactions and AGE formation will undoubtedly help determine the relative importance of AGEs and RAGE to all vascular abnormalities and especially those whose incidence is markedly elevated in aging vessels.
5. Discussion Currently, the most remarkable differences in intracellular signaling between VSMCs isolated from young and old animals are related to the control of cell migration through the CamKII pathways and the accelerated transition of older VSMCs from the contractile to the synthetic phenotype. These differences may be due to alternative signaling pathways revealed by the inability of older VSMCs to respond to inhibitors, such as TGF-1, or to altered interactions with the extracellular matrix resulting from age-associated shifts in integrin expression. The exact role that these alterations have in explaining ageassociated differences in the response of the vessel wall to injury and its increased susceptibility to developing advanced atherosclerotic lesions remains to be determined but will be guided by such studies on intracellular signaling mechanisms.
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