[Frontiers in Bioscience S1, 236-245, June 1, 2009]
TGF-beta signaling in atherosclerosis and restenosis
The George Washington University Medical Center, Department of Biochemistry and Molecular Biology, The Catherine Birch McCormick Genomics Center, and The Richard B. and Lynne V. Cheney Cardiovascular Institute, Washington, D.C. USA
TABLE OF CONTENTS
1. ABSTRACT
Current theories suggest that atherosclerotic and restenotic lesions result from imbalances between systems that are proinflammatory/fibroproliferative versus the endogenous inhibitory systems that normally limit inflammation and vascular wound repair. Abnormalities in one of the major regulatory pathways, the transforming growth factor-beta (TGF-beta) system, has been characterized in both animal models and in human lesions and lesion-derived cells. TGF-beta signaling is capable of regulating many of the key aspects of atherosclerosis and restenosis: inflammation, chemotaxis, fibrosis, proliferation, and apoptosis. There are significant decreases in TGF-beta activity in patients with atherosclerosis, and equally important changes in the way cells respond to TGF-beta during atherogenesis. Evidence from multiple sources indicates that experimental modulation of TGF-beta activity, or TGF-beta responses, changes the course of atherosclerosis and intimal hyperplasia. Cells derived from human lesions produce adequate TGF-beta levels, but are resistant to the antiproliferative and apoptotic effects of TGF-beta. An evolving theory describes TGF-beta as a major orchestrator of the vascular repair process, with observable defects in its production, activation, and cellular responses during the atherosclerotic and restenotic processes.
2. INTRODUCTION
2.1 Atherosclerosis
Cardiovascular diseases remain the largest cause of death and morbidity in the Western hemisphere. Despite major advances is diagnosis, risk management, and treatment, roughly 40% of all deaths are attributable to heart disease, stroke, and atherosclerosis (1). Americans have about a 2-fold greater risk of dying of atherosclerotic diseases than cancer. The major pathophysiological causes of vascular disease are raised 'fibro-fatty' lesions which occlude blood flow and trigger thrombosis, as shown histologically in Figure 1. Atherosclerotic vascular changes are a major contributing factor to life-threatening events such as myocardial infarction, stroke, aneurysm, and pulmonary embolism. The incidence of coronary and aortic atherosclerosis increases strongly with advancing age, such that by the age of 65 as much as 50% of the coronary artery surface can be covered by raised atherosclerotic lesions (2).
2.2 Angioplasty/Restenosis
Patients presenting with clinical and angiographic evidence of occlusive coronary atherosclerosis are usually offered either coronary bypass surgery or angioplasty. Balloon angioplasty, typically with endovascular stent placement, is performed on more than 450,000 patients per year in the U.S., and achieves initial reperfusion in more than 95% of cases. Given the high success rate, short hospitalization, and minimally invasive nature, angioplasty is likely to remain a treatment of choice for coronary and peripheral atherosclerosis. The initially high success rate of angioplasty is offset by progressive fibroproliferative reclosure of the artery, termed restenosis, over the 3 to 6 month period post-angioplasty, though this complication is significantly reduced by newer drug-eluting stents.
2.3 Pathophysiology of atherosclerosis and restenosis
Atherosclerosis, derived from the Greek 'athero' for gruel, and 'skleros' for hard, is thought to result from chronic or repetitive insults to the vessel wall which trigger a hyperplastic response with prominent inflammatory components. Many of the damaging agents are known: elevated lipids, especially in their oxidized/modified forms, hydrocarbons from smoking, elevated glucose from diabetes, elevated blood pressure and shear forces due to hypertension, and potentially, autoimmune or infectious damage. While these vascular insults are tolerated and repaired efficiently in younger people, advancing age is associated with imbalances in the repair processes leading to accumulation of repair cells, excess extracellular matrix, inefficient removal of cholesterol-rich debris, and persistent inflammation. As shown in Figure 1, these lesions become lethal when the plaque ruptures, exposing the cholesterol and procoagulant-rich debris to blood, precipitating thrombosis and occluding blood flow to the heart, brain, or other vital organs.
The restenotic process may be an interesting microcosm of the atherosclerotic process because it is a highly-defined, acute vascular insult. Balloon angioplasty causes severe arterial injury, creating intimal "flaps" of partially dissected lesion (Figure 2), and fissures in the lesion, with accompanying mural thrombus. Histologically, these flaps and fissures are consumed with a fibroproliferative lesion over a period of several months. The course of injury-induced intimal hyperplasia probably involves: 1) endothelial injury and connective layer dissections, 2) mural thrombosis, 3) migration of smooth muscle cells (SMC) and myofibroblasts, and infiltration of circulating monocytes/lymphocytes, 4) increased cell proliferation within the neointima, 5) accumulation of extracellular matrix, 6) reendothelialization, 7) partial or complete resolution via apoptosis (3) and 8) tissue remodeling involving wound contraction. As shown in Figure 2, these lesion are principally fibrotic and can largely reclose an artery in the absence of drug-eluting stents.
2.4 Regression
Rats and rabbits both develop vascular lesions after balloon injury, though these lesions will spontaneously regress over a period of weeks after injury. However, injury to old rats leads to a more aggressive proliferative wave, and a failure to resolve the lesion after reendothelialization. This excessive response in the aged artery is due to intrinsic changes in the artery wall (4-6). Likewise, evidence from the PDAY study, and others, strongly indicates that atherosclerotic lesions occur reversibly in young adults, but lesions become more persistent with advancing age (7). Serial angiographic analysis in humans indicates that restenosis occurs in most patients after angioplasty, but the majority of patients show spontaneous regression of the lesion (8). Thus, one of the major questions is why some patients heal normally, without persistent hyperplasia, while others develop an occlusive vascular hyperplasia in response to the same injury. A reasonable theory is that atherosclerosis and restenosis reflect an underlying failure in the regression of wound repair due to a defect in the apoptotic process (9).
3. Production and localization of TGF-beta1 during the repair of vascular damage
Essentially all cells present in the arterial wall during vascular damage and repair are capable of producing transforming growth factor-beta (TGF-beta). TGF-beta is a small family of TGF-beta1, beta2, and beta3 in mammals, with an extended family of TGF-beta-like proteins including the bone morphogenic proteins (BMPs), inhibins, activins, and growth/differentiation factors (GDFs). Immunostaining reveals high levels of active TGF-beta1 within fibroproliferative regions of the human lesion (Figure 3). TGF-beta1 is produced as a 26 kD dimer which then reassociates with a precursor sequence to form a latent complex (10). Latent TGF-beta can be activated by acidic conditions (11), plasmin (12), transglutaminase (13), and thrombospondin (14). TGF-beta and its latent complex binds to several key extracellular matrix proteins that include fibronectin (15), thrombospondin (16), and type IV collagen (17), which localize TGF-beta in the extracellular matrix in its latent form (18). Thus, latent TGF-beta is a 'cryptocrine' factor, which resides in a cryptic, latent form that can be activated by local proteolytic activity (19).
TGF-beta1 is a heparin-binding protein (20, 21), and its association with heparin prevents the binding of TGF-beta1 to the activated form of alpha2-macroglobulin, the principal soluble inactivating pathway for TGF-beta (21). This action of heparin on TGF-beta probably contributes to the antiproliferative effect of heparin on SMC (21, 22). At sites of vascular injury, TGF-beta1 would be released from degranulating platelets in the mural thrombus, and equally importantly, TGF-beta1 production is increased in restenotic lesions (23) and in the balloon injured rat carotid artery (24, 25).
4. Effects of TGF-betas on vascular cells
TGF-beta exerts potent and diverse actions on each of the cell types involved in vascular disease (26). TGF-beta frequently exerts bifunctional effects that are dependent upon the context in which the particular cell type encounters the TGF-beta signal. Biologically, this context-dependent effect would allow TGF-beta to orchestrate an entire repair process that might involve opposite effects at different times. For additional perspectives on the relationship of TGF-beta to vascular disease, other excellent recent reviews are highly recommended (27).
4.1. Endothelial cells
Endothelial cells tend to be strongly inhibited by TGF-beta both with respect to their proliferation and migration (28, 29). This inhibition is associated with increases in extracellular matrix and proteoglycan synthesis (30). The in vitro antiproliferative effect of TGF-beta may explain in vivo studies of TGF-beta treatment which have observed delays in reendothelialization of the denuded artery (31). It seems likely that the anti-migratory and anti-proliferative effects of TGF-beta are most relevant to the anti-angiogenic effect of TGF-beta. Importantly, TGF-beta can have a dose-dependent bifunctional effect on angiogenesis induced by VEGF and FGF (32) which parallels its ability to stimulate endothelial migration and proliferation at low concentrations, but inhibit both at higher concentrations (33). Conceptually, this in vitro dose-dependent effect probably mimics an in vivo concentration gradient of TGF-beta designed to attract cells from a distance, but then retain them at the site of injury for repair.
4.2. Smooth muscle cells (SMC)
Smooth muscle cells (SMC), and closely related myofibroblasts, which compose much of the atherosclerotic lesion, have been extensively studied for their response to TGF-beta. Typically, TGF-beta is a potent inhibitor of migration and proliferation of SMC (34, 35), though in rat aortic SMC, TGF-beta can stimulate the production and release of mitogens, such as PDGF, which can have mitogenic effects on SMC (36), particularly when the SMC are quiescent or highly confluent. SMC derived from the chick embryonic cardiac neural crest are of ectodermal origin and are thought to compose the proximal portions of the coronary arteries. These ectodermal cells are growth-stimulated by TGF-beta, while cells derived from the mesoderm are growth inhibited (37). TGF-beta is a particularly potent modulator of the human SMC phenotype, as it regulates cytoskeletal actin reorganization and cell spreading (35).
The effects of TGF-beta on apoptosis are cell-type dependent (38) and context-dependent. Typically, TGF-beta will induce apoptosis of both vascular SMC and endothelial cells. However, in circumstances where apoptosis is initiated by other factors, TGF-beta can exert a protective effect. Cells derived from human vascular lesions appear prone to spontaneous apoptosis (39), though once the culture is established the lesion-derived cells are typically completely resistant to TGF-beta-induced apoptosis (40). Physiologically, TGF-beta-induced apoptosis may be a critical feature of wound or lesion regression after repair is completed.
4.3. Macrophages
Macrophages exhibit a complex interaction with TGF-beta because the effect is dependent upon their state of activation. TGF-beta tends to suppress the activation of monocytes to macrophages (41), a key step in atherogenesis. TGF-beta suppresses both the proliferation and migration of cells in the monocyte/macrophage lineage. However, once activated, TGF-beta stimulates macrophages to produce urokinase, leading to plasmin generation (42). Within the lesion, macrophages commonly colocalize to regions containing active TGF-beta (40, 43).
4.4. Lymphocytes
Lymphocytes are potently suppressed by TGF-beta under most conditions tested. TGF-beta induces apoptosis in B and T lymphocytes in culture at very low concentrations (44). Within the vascular lesion, lymphocytes express the Type I but not the Type II receptor, suggesting that limited resistance to TGF-beta may be required for their function in the plaque (40).
5. Effects of TGF-beta and TGF-beta neutralization on vascular disease
Overexpression of TGF-beta, via direct cDNA transfection into the artery wall at the time of vascular injury, markedly increases intimal and medial thickness, principally by increasing the extracellular matrix component (45). Likewise, infusion of TGF-beta at the time of balloon catheter injury in the rat also causes a two-fold increase in the thickness of the neointima and increases the content of matrix in the lesion (24). Rabbit arteries showed a similar fibrotic response to vascular injury with infusion of TGF-beta (46).
Likewise, infusions of a neutralizing antibody to TGF-beta at the time of balloon catheter injury to the rat aorta markedly reduced intimal hyperplasia and fibrosis (25). Experimentally defeating the TGF-beta response in T cells by expression of dominant negative Type II receptor expression in ApoE (47) and LDL receptor (48) knockout mice, or by systemic treatment with a soluble TGF-beta Type II receptor (49), all cause exaggerated atherosclerosis. However, systemic treatment by injection of a soluble Type II receptor into ApoE -/- mice reduced lesion size and switched it from a fibrotic to an inflammatory lesion (50).
5.1. Expression of TGF-betas and receptors during vascular repair
5.1.1. Animal models
Circulating levels of TGF-beta may be an important factor that modulates the vessel wall response to injury. Apo(a), which interferes with the normal activation of TGF-beta1 in the artery wall, contributes to excessive cell proliferation in animal models of lipid-induced injury (51). After vascular injury to the rat carotid, expression of TGF-beta1 mRNA in the vessel wall is elevated within 6 hours, peaks at 24 hours, with sustained elevations for up to 2 weeks, coinciding with the development of the fibroproliferative neointima (24). TGF-beta1 production is increased in arteries after mechanical injury (24, 25), hypercholesterolemia (43), and DOC/salt hypertension (52). Both Type I and Type II receptors for TGF-beta1 are increased after balloon injury to the rabbit (46).
5.1.2. Human atherosclerosis
Immunohistochemical analysis of early human lesions indicates that TGF-beta1 and TGF-beta3 isoforms are present in the lesion, in association with SMC, macrophages, and foam cells. Both the Type I Alk5 receptor and the Type II receptor were detected at elevated levels in the early fibrofatty streak (53). However, as shown in Figure 3, advanced atherosclerotic lesions show only isolated areas that express either active TGF-beta1 or the receptors (40, 53). The majority of the advanced fibrous lesion expresses low and variable levels of the Type I receptor, but generally much lower levels of the Type II receptor.
5.2. Circulating/soluble levels of TGF-beta in atherosclerosis
TGF-beta has important autocrine, paracrine, and endocrine effects, and thus, it is important to consider soluble and circulating levels. Accumulating evidence suggests that TGF-beta levels in plasma are reduced in patients with atherosclerosis (54-56). Vessel wall levels, much of which would be interstitial and matrix-associated, are also reduced in atherosclerotic regions, further suggesting a reduced bioactive TGF-beta signal during atherogenesis (27). The combination of reduced levels, and reduced responsiveness would greatly reduce the overall TGF-beta signal in the atherosclerotic environment. On the basis of this and other evidence, a "protective cytokine" theory of atherosclerosis has been forwarded, which highlights the potential importance of reduced TGF-beta bioactivity in atherosclerosis (57).
5.3. Resistance to the antiproliferative effect of TGF-beta1
The sharp increase in atherosclerotic disease with age implies that underlying age-related changes in the vascular wall may exacerbate vascular injury (4, 5), partially due to age-related resistance to inhibitors such as TGF-beta (6, 58, 59). SMC derived from old animals produce normal amounts of both active and latent TGF-beta1 (60). However, the old SMC showed no inhibition of DNA synthesis in response to TGF-beta1 over the range 0-5 ng/ml, while young SMC were inhibited 50% at 50 pg/ml (60). A similar age-dependent response to TGF-beta has been observed in SMC derived from the spontaneously hypertensive rat (61). Interestingly, elevated cholesterol levels reduce the responsiveness to TGF-beta in vascular cells by modulating Type II/Type receptor binding profiles (62, 63).
In both rat and human cells, this acquired resistance is associated with a preferential down-regulation of the Type II receptors (40, 60), although additional changes in negative regulators of signaling, such as Smurf2, have been observed (64). Transfection of Type II receptors partially corrects the response of human LDC and old rat SMC (65), suggesting that the intracellular signaling system remains at least partially functional. The remaining resistance could be due to changes in the key SMAD signaling pathways that have been observed in cells of the fibrofatty lesion (66). A small subset of patients possess lesion cells with acquired mutations in a microsatellite region of the Type II receptor (67, 68). However, it is likely that regulation of Type II receptor levels, and changes in the intracellular signaling pathways are the major modes of resistance. For instance, changes in the E3 ubiquitin ligases, Smurf1 and Smurf2, could account for reduced TGF-beta signaling via SMAD interactions (69, 70). Recently, a number of other potentially important TGF-beta signaling modulators have been described, including Arkadia (71), Nedd4-2 (72), and WWP1 (73). Also, TGF-beta is capable signaling both through SMAD-dependent, and SMAD-independent pathways to modulate vessel wall functions (74).
6. SUMMARY AND CONCLUSIONS
The evidence is compelling that TGF-beta activity and responses are key modulators of the normal, and abnormal vascular repair processes. As diagrammed schematically in Figure 4, the TGF-beta system involves tight regulation of this signal at multiple levels. Reduced TGF-beta activity is a feature of atherosclerosis, both in the vessel wall and in circulating levels, thereby favoring a pro-inflammatory environment. Concurrent with the decreased TGF-beta activity, there is a reduced and altered response to TGF-beta by subsets of cells in the atherosclerotic lesion, which may be symptomatic of broader changes because lesion cells are also resistant to the effects of glucocorticoids (75). TGF-beta resistance, on one hand, may allow vascular repair cells to tolerate hostile environments and repair the damage, but if it persists, lesion cells would excessively repair the artery wall. Thus, determining the molecular basis of the imbalances in this key system may be critical in understanding occlusive vascular diseases. While it is tempting to categorize TGF-beta as either a 'protective' or 'pathologic' factor, it is more likely that TGF-beta is instrumental in both normal physiologic vascular repair, and a component of dysregulated vascular disease.
7. ACKNOWLEDGEMENTS
The author is grateful for support from the NIH/National Institutes on Aging MERIT Award AG12712 and the generous support of the St. Laurent Institute, The Catherine Birch McCormick Genomics Center, and The Richard B. and Lynne V. Cheney Cardiovascular Institute. The author greatly appreciates the expert editorial support of Neil Hammell (GW).
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Key Words: TGF-beta, Atherosclerosis, Restenosis, Review
Send correspondence to: Tim McCaffrey, The George Washington Medical Center, Department of Biochemistry and Molecular Biology, 2300 I Street NW, Ross Hall 541, Washington, D.C. 20037, Tel: 202-994-8919, Fax: 202-994-8924, E-mail:mcc@gwu.edu