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Cardiovascular Research 2007 74(2):223-234; doi:10.1016/j.cardiores.2007.02.012
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Copyright © 2007, European Society of Cardiology

Understanding the role of transforming growth factor-β1 in intimal thickening after vascular injury

Razi Khana,*, Alex Agrotisb and Alex Bobikb

aDepartment of Internal Medicine, University of Western Ontario, Suite 1604, 1265 Richmond St. North, London, Ontario, Canada N6A 3M1
bCell Biology Laboratory, Baker Heart Research Institute, 75 Commercial Road, Melbourne, Victoria 3004, Australia

* Corresponding author. Tel.: +1 519 858 4971. Email address: razi.khan{at}gmail.com

Received 22 December 2006; revised 4 February 2007; accepted 8 February 2007


   Abstract
Top
 Abstract
1. Introduction
 2. Pathology of intimal...
 3. Biology of TGF-β1
 4. Involvement of TGF-β1...
 5. TGF-β1 modulation after...
 6. Direct inhibition of...
 7. Inhibition of TGF-β1...
 8. Inhibition of TGF-β1...
 9. Conclusion and future...
 References
 
Intimal thickening is the most important cause of in-stent restenosis. The pathology of intimal thickening is attributable to a local inflammatory response after vascular injury which results in the production of cytokines. Transforming growth factor-β1 (TGF-β1) is a profibrotic cytokine that is involved in the induction of intimal thickening. Up-regulation of TGF-β1 after arterial injury results in the activation of various downstream pathways which stimulate the proliferation and migration of vascular smooth muscle cells, as well as the production of local extracellular matrix proteins. Recent evidence suggests that antagonizing TGF-β1 activity with direct or indirect inhibitors may attenuate or prevent intimal thickening. Additionally, TGF-β1 synthesis, activation and downstream regulation may also serve as significant sources of treatment. This review attempts to show the role of TGF-β1 in the pathology of intimal thickening and underlines the importance of TGF-β1 as a target for therapy.

KEYWORDS Transforming growth factor-β1; Intimal thickening; Percutaneous coronary intervention; Vascular smooth muscle cell; Extracellular matrix; Transforming growth factor-β3; Tranilast


   1. Introduction
Top
 Abstract
 1. Introduction
2. Pathology of intimal...
 3. Biology of TGF-β1
 4. Involvement of TGF-β1...
 5. TGF-β1 modulation after...
 6. Direct inhibition of...
 7. Inhibition of TGF-β1...
 8. Inhibition of TGF-β1...
 9. Conclusion and future...
 References
 
Every year, over 500000 patients in the United States undergo percutaneous coronary intervention (PCI) [1]. Currently, the average rates of restenosis for drug-eluting and bare metal stents are 8.9% and 29% respectively at 6 months after PCI [2]. Intimal thickening (also referred to as neointimal or intimal hyperplasia) is the most important cause of in-stent restenosis [3].

Transforming growth factor-β1 (TGF-β1) is a profibrotic cytokine implicated in the development of diabetic nephropathy, hepatic fibrosis, ischemic and dilated cardiomyopathies, arrhythmia and valvular disease [4–6]. Recently, both in vitro and in vivo studies have shown that TGF-β1 plays an integral role in promoting intimal thickening [7–9]. After arterial injury, TGF-β1 has been shown to stimulate extracellular matrix (ECM) protein expression and vascular smooth muscle cell (VSMC) proliferation and migration, leading to eventual luminal narrowing [10,11]. Concordantly, attenuating TGF-β1 activity with tranilast, TGF-β3, or direct TGF-β1 inhibitors has been efficacious in diminishing intimal thickening and in-stent restenosis [12–15].

This review outlines the evidence correlating TGF-β1 with intimal thickening after arterial injury, as well as describing the molecular mechanisms of TGF-β1 action within the vessel wall. It also aims to show that TGF-β1 is an appropriate target for treatment and prevention of in-stent restenosis.


   2. Pathology of intimal thickening
Top
 Abstract
 1. Introduction
 2. Pathology of intimal...
3. Biology of TGF-β1
 4. Involvement of TGF-β1...
 5. TGF-β1 modulation after...
 6. Direct inhibition of...
 7. Inhibition of TGF-β1...
 8. Inhibition of TGF-β1...
 9. Conclusion and future...
 References
 
Intimal thickening has a physiological role within the body, most notably in the closing of the ductus arteriosus at birth. Unfortunately, it is also involved in a number of organ pathologies such as pulmonary hypertension and coronary artery vasculopathy after cardiac transplant [16,17]. Intimal thickening is an important cause of luminal restenosis, particularly after PCI and coronary artery bypass surgery. Both balloon angioplasty and stenting of the arterial wall result in trauma and denudation of the endothelial layer of the vessel, removing the non-permeable barrier protecting both underlying subintimal and smooth muscle layers. At the trauma site, the exposure of subintimal layers results in the aggregation and accumulation of leukocytes and platelets, which, in turn, interact via adhesion molecules, intensifying the local inflammatory response [18]. Release of cytokines and growth factors, such as TGF-β1 and platelet-derived growth factor (PDGF), from both platelets and leukocytes, between days 3 and 14 after intervention, results in phenotypic modulation, proliferation and migration of VSMCs [19]. However, after two months, VSMC proliferation is no longer a major factor contributing to in-stent restenosis, with areas of intimal thickening from human artery specimens often being cell-depleted [7]. Instead, areas of intimal thickening are associated with increased collagen expression, indicating that enhanced ECM expression may be the predominant cause of late in-stent restenosis [7]. Concordantly, Fattori and Piva noted that although VSMC proliferation may be paramount to intimal thickening, the majority of in-stent restenosis lesions consist of ECM and collagen, with cells accounting for only 11% of the growth [20]. Similarly, Pickering et al. noted the collagen area fraction of restenotic lesions after balloon angioplasty was approximately 80% [21]. Thus, in-stent restenosis appears to be dependent upon both VSMC proliferation and increased ECM protein expression. Additionally, the adventitial layer may also be a major contributor to the process of arterial remodeling and restenosis subsequent to vascular injury, via mechanisms involving matrix accumulation, angiogenesis, and luminal migration of adventitial fibroblasts [22,23].


   3. Biology of TGF-β1
Top
 Abstract
 1. Introduction
 2. Pathology of intimal...
 3. Biology of TGF-β1
4. Involvement of TGF-β1...
 5. TGF-β1 modulation after...
 6. Direct inhibition of...
 7. Inhibition of TGF-β1...
 8. Inhibition of TGF-β1...
 9. Conclusion and future...
 References
 
TGF-β1 is part of a superfamily of proteins serving critical roles in ECM production, regulation of cell growth, differentiation, migration and apoptosis in different organ systems [24]. The three mammalian TGF-β isoforms, TGF-β1, -β2, and -β3, are encoded by distinct genes. TGF-β1 is expressed in endothelial cells, VSMCs, myofibroblasts and hematopoeitc cells [25]. TGF-β2 is expressed in epithelial cells and neurons, while TGF-β3 is mesenchymally expressed.

TGF-β1 is initially produced as a large precursor molecule (44 kDa) containing an N-terminal latency-associated pro-peptide (LAP), which is cleaved intracellularly [26]. After cleavage, a small latent complex (100 kDa) is formed when mature TGF-β1 (25 kDa) binds non-covalently to LAP [27]. The small latent complex then covalently binds to latent TGF-β1 binding proteins, forming a large latent secreted complex (290 kDa), biologically inactive and unable to bind TGF-β1 receptors [28]. Vascular TGF-β1 activation requires interaction between furin-like proprotein convertases, plasmin, thrombospondin, urokinase-type plasminogen activator and mannose-6-phosphate/insulin-like growth factor II [29]. In particular, furin-like proprotein convertases are found to be involved in the activation of TGF-β1 after arterial injury [30]. There is also evidence that after arterial injury, local increases in both MMP-2 and MMP-9 (both of which are increased in expression by TGF-β1 in aortic endothelial cells and VSMCs), may act to augment the bioavailability of TGF-β1, creating a feedback loop promoting intimal thickening [31–33].

Active TGF-β1 initiates cell signaling by binding to TGF-β1 receptor type II (TβR-II), which then recruits and dimerizes with TGF-β1 receptor type I (TβR-I/ALK-5 or ALK-1) [34]. The formation of a heterotrimeric complex (TGF-β1, TβR-II and TβR-I) results in the activation of TβR-II and TβR-I intracellular serine–threonine kinases and stimulates signaling via the Smad pathway [35]. Activated TβR-I phosphorylates Smad2, which complexes with Smad3 and Smad4 [36]. This Smad complex then translocates into the nucleus and regulates gene expression by interacting with transcription factors or directly with DNA. Additionally, TGF-β1 has been noted to exert biological effects through Smad-independent downstream pathways, involving p38 MAPK, JNK and protein kinase-C ([35], and Fig. 1). Within the vasculature, endothelial cells, VSMCs, mesenchymal cells, macrophages, and lymphocytes all express TβR-II and TβR-I (either ALK-5 and/or ALK-1) and are responsive to TGF-β1 via these Smad-dependent or Smad-independent signaling pathways [29].


Figure 1
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  		Fig. 1 Schematic depiction of TGF-β1 secretion, activation, and signaling via Smad-dependent and Smad-independent pathways. The TGF-β1 dimer, secreted as part of a larger complex with the latency-associated peptide (LAP) and the latent TGF-β binding protein (LTBP) dissociates and becomes active, most notably via proteolytic cleavage by furin-like protein convertases. Subsequent to binding to the TβR-II:TβR-I (either ALK-5 or ALK-1) receptor complex, Smads2 and 3 are phosphorylated and translocate to the nucleus as a trimeric complex with Smad4; this trimeric Smad complex, in combination with other transcriptional co-activators (CBP/P300) or co-repressors (Sno), then acts to either stimulate (upward arrow) or inhibit (downward arrow) the expression of specific genes. In addition, Smad-independent signaling can also occur via pathways that include TAK-1, p38 MAPK, ERK-1/ERK-2, and JNK. The sites of action of various inhibitors of TGF-β1 activity (tranilast, Smad7, and sirolimus) are shown by dotted-line arrows.

 

   4. Involvement of TGF-β1 in intimal thickening
Top
 Abstract
 1. Introduction
 2. Pathology of intimal...
 3. Biology of TGF-β1
 4. Involvement of TGF-β1...
5. TGF-β1 modulation after...
 6. Direct inhibition of...
 7. Inhibition of TGF-β1...
 8. Inhibition of TGF-β1...
 9. Conclusion and future...
 References
 
4.1 Correlating TGF-β1 with intimal thickening
The use of balloon catheter denudation in rats and coronary angioplasty or stenting in porcine models has helped to establish the association of TGF-β1 with restenosis after intervention. Madri et al. first noted increased immunostaining for TGF-β1 in the intimal regions of rat carotid arteries 10 weeks after balloon catheter injury [37]. Majesky et al. found that VSMCs had significantly enhanced TGF-β1 levels 6 h after carotid injury in rats, being 5–7 fold above baseline within 24 h, and remaining elevated for 2 weeks as intimal thickening occurred [38]. In porcine coronary arteries, balloon angioplasty injury resulted in significantly elevated active TGF-β1 levels between 2 h and 7 days after intervention, returning to baseline 28 days later [39]. In this study, TGF-β1 was highly expressed in the intima and adventitia.

Treatment with or enhanced local expression of TGF-β1 in animal models has been noted to hasten arterial changes after injury. Kanzaki et al. found that administration of TGF-β1 before carotid balloon catheterization in rabbits resulted in greater intimal thickening after the procedure, with the first signs of thickening observed on day 7 and progressively increasing until day 21 [40]. Transduction of a constitutively active TGF-β1 gene into rat carotid arteries was similarly associated with significantly increased intimal thickening after vascular injury, which then regressed in the absence of TGF-β1 [8]. Finally, direct transfer of the TGF-β1 gene into porcine coronary arteries resulted in elevated local ECM production, again leading to substantial intimal thickening [9].

In human specimens, Nikol et al. noted elevated TGF-β1 levels in VSMCs of coronary and peripheral arteries, and that restenotic lesions after PCI had higher TGF-β1 levels compared with primary lesions [41]. Chung et al., examining human restenotic lesions after stenting, found that 16 of 20 vessels retrieved via atherectomy were positively stained for TGF-β1 [7]. Similarly, Yutani et al., looking at atherectomized coronary vessels after stent restenosis, noted that the intima in 80% of arteries were TGF-β1-positive [42].

4.2 Effect of TGF-β1 on VSMC proliferation
TGF-β1 has been shown to have a predominantly anti-mitogenic effect in most cell lines. However, in VSMCs, TGF-β1 has been noted to both induce and inhibit proliferation. The effects of TGF-β1 on VSMC proliferation appear to depend on cellular density, the local concentration of TGF-β1 and the presence of other growth factors. In VSMC cultures, Majack found that TGF-β1 stimulated proliferation at monolayer densities, while inhibiting proliferation at subconfluent densities [10]. Hwang et al. found that treatment of porcine coronary artery VSMCs with TGF-β1, at a concentration of 0.025 ng/ml, stimulated proliferation, but inhibited growth at concentrations of greater than 0.1 ng/ml [43].

The presence of other growth factors also appears to influence the effects of TGF-β1 on VSMCs. TGF-β1 has been shown to promote rat aortic VSMC mitogenesis indirectly by inducing PDGF-AA and thrombospondin [44,45]. Additionally, PDGF-BB and TGF-β1 have been shown to synergistically enhance VSMC proliferation when compared to TGF-β1 alone [46]. TGF-β1 has also been shown to enhance DNA synthesis in VSMCs when combined with potent mitogens such as fibroblastic growth factor (FGF-2) and epidermal growth factor (EGF) [47].

In vivo, Majesky et al. noted that infusion of recombinant TGF-β1 into rats after arterial injury resulted in increased intimal VSMC proliferation, while no significant changes were found in the tunica media of the vessel, suggesting fundamental differences between intimal and medial VSMCs [38]. Similarly, Schulick et al. noted that localized over-expression of TGF-β1 in the endothelium of uninjured rats resulted in intimal thickening, with marked cellular proliferation when compared to controls [8].

4.3 TGF-β1-mediated up-regulation of integrins in VSMC migration
Along with proliferation, VSMC migration is also integral to intimal thickening. Vitronectin and osteopontin, both extracellular cell adhesion molecules, enhance VSMC migration after arterial injury by binding to integrin transmembrane cellular receptors. {alpha}Vβ3 integrins have been demonstrated to be integral to VSMC migration from the tunica media to the intima [48]. In porcine models, injury secondary to PCI has been shown to induce {alpha}Vβ3 integrins within the arterial neointima at 14 days, reaching peak levels at 28 days [49]. In the same study, the use of {alpha}Vβ3 integrins inhibitors was noted to significantly reduce intimal thickening 28 days after PCI. Within neointimal lesions, expression of integrins has been noted to be largely regulated by cytokines [50]. In particular, TGF-β1 potently up-regulates {alpha}Vβ3 integrins expression in VSMCs [51]. Concordantly, pre-treatment of VSMCs with TGF-β1 was associated with enhanced vitronectin-driven migration [52]. Finally, inhibition of TGF-β1 action has been noted to prevent {alpha}Vβ3 integrin up-regulation after carotid injury in rats [53].

4.4 TGF-β1-mediated up-regulation of arterial ECM proteins
ECM proteins have been shown to constitute up to 80% of intimal mass in restenotic lesions [54]. TGF-β1 has been documented to be one of the most potent up-regulators of collagen synthesis in VSMC cultures [55]. Amento et al. noted that TGF-β1 elevated the synthesis of collagen types I and III in human VSMC cultures [11]. TGF-β1 has been shown to up-regulate collagen type I, usually the most abundant ECM protein in intimal thickening, by acting on the proximal COL1A2 gene promoter [56]. Furthermore, Rasmussen et al. noted that VSMCs from injured rat aortas produced more fibronectin and proteoglycans and contained greater amounts of TGF-β1 compared to non-injured VSMCs [57]. Administration of TGF-β1-directed antibody prevented ECM matrix protein expression from injured VSMC lines. In human specimens, Majesky et al. found the vast majority of intimal VSMCs stained TGF-β1-positive after balloon angioplasty, and noted this staining was associated with increased intimal levels of fibronectin, and collagen types I and III [38].

Therefore, evidence from both human and animal studies demonstrates that TGF-β1 up-regulation following arterial injury appears to increase intimal thickening (Fig. 2), thereby implicating TGF-β1 in contributing to restenosis after coronary intervention.


Figure 2
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  		Fig. 2 Mechanisms of TGF-β1-induced intimal thickening. Subsequent to vascular injury, activated TGF-β1 derived from either the vessel itself or from platelets induces VSMCs to de-differentiate and adventitial fibroblasts to undergo transition to a myofibroblast state. Subsequently, these myofibroblasts migrate towards the vessel lumen and produce collagen, as do the migrating and proliferating VSMCs, which produce extracellular matrix that includes collagen, fibronectin, and various proteoglycan species. Cell migration is facilitated by TGF-β1-induced increases in expression of {alpha}vβ3 integrin, MMP-2/-9, and decreases in expression of TIMP. VSMC proliferation and migration is enhanced by TGF-β1-mediated expression of factors which include PDGF-A and PDGF-B, EGF, and FGF-2, and the survival of these VSMCs is augmented by TGF-β1. Increased perivascular collagen content may also be seen. Intimal thickening occurs by these cellular/fibrotic processes, and in concert with an increased constrictive remodeling, and a loss of lumen area, can ultimately lead to restenosis.

 

   5. TGF-β1 modulation after vascular injury
Top
 Abstract
 1. Introduction
 2. Pathology of intimal...
 3. Biology of TGF-β1
 4. Involvement of TGF-β1...
 5. TGF-β1 modulation after...
6. Direct inhibition of...
 7. Inhibition of TGF-β1...
 8. Inhibition of TGF-β1...
 9. Conclusion and future...
 References
 
5.1 Up-regulation of TGF-β1
In experimental models, balloon- and stent-induced vascular injuries have been noted to stimulate the expression of transcription factors such as early growth response factor-1 (egr-1) [58]. Elevated levels of egr-1 have been localized to atheromatous VSMCs in both mice and humans [59]. Administration of DNA enzymes cleaving egr-1 inhibited VSMC proliferation in culture, and also attenuated intimal thickening after carotid balloon injury in rats [60]. Similarly, in porcine models, the intraluminal administration of DNA enzymes after coronary artery stenting inhibited VSMC proliferation and intimal thickening by up to 30% when compared to controls [61]. Finally, synthetic DNA, serving as a local egr-1 decoy, was shown to reduce TGF-β1 gene expression after carotid balloon injury, while significantly decreasing intimal thickening in hypercholesterolemic rabbits [62]. Binding sites for egr-1 have been identified within the proximal TGF-β1 gene promoter [63]. In murine lung models, egr-1 has also been shown to be essential in TGF-β1-mediated remodeling and fibrosis after injury [64].

5.2 Downstream TGF-β1 signal cascade
Enhanced vascular expression of TGF-β1 has been correlated with increased Smad activity. Adenoviral TGF-β1 transfection, producing greater local expression of the cytokine, resulted in a time- and concentration-dependent increase in aortic VSMC Smad2 phosphorylation [65]. Additionally, increased TGF-β1 expression in mouse aortic cells was associated with greater Smad2 phosphorylation and increased collagen type I expression [66]. More recently, adenoviral over-expression of Smad7, a Smad complex antagonist, within the adventitia of balloon-injured rat carotid arteries resulted in antagonism of TGF-β1 signaling for up to 14 days, as demonstrated by the diminished Smad2 phosphorylation [67], and was accompanied by a decreased adventitial expression of {alpha}-smooth muscle actin, a reduction in adventitial cell migration to the neointima, and an attenuation of both the loss of lumen area and the increased perivascular collagen content subsequent to injury [67].

Along with the Smad pathway, other TGF-β1 downstream mediators may also be involved in intimal thickening. Immunohistochemical analysis of medial cells after vascular injury indicated elevated levels of p38 MAPK, and administration of p38 MAPK inhibitors was associated with reduced VSMC proliferation, ECM protein production and intimal-to-media ratios [68]. Concordantly, adenoviral transfection of VSMCs with p38 MAPK dominant negative mutants attenuated intimal thickening, and was associated with reduced VSMC migration and proliferation [69]. Ju et al. also noted that p38 MAPK inhibitors produced a concentration-dependent inhibition of TGF-β1-induced fibronectin up-regulation following balloon injury in rabbits [70].

5.3 TGF-β1-induction of proteins associated with vascular injury
Although TGF-β1 acts directly to up-regulate ECM expression, it also stimulates the synthesis of modulator proteins that amplify its pathogenic effects, such as plasminogen activator inhibitor-1 (PAI-1) in endothelial cells in vivo [71] via phosphorylated Smad proteins enhancing PAI-1 gene promoter activity [72]. Additionally, inhibition of the TGF-β1 downstream regulators, MEK and p38 MAPKs, can also prevent TGF-β1-induced PAI-1 expression [73]. In examining the relevance of the TGF-β1-PAI-1 cascade in vascular injury, Otsuka et al., using mice models, found that localized adenovirus-mediated TGF-β1 over-expression resulted in ECM-rich intimal thickening partially mediated through the induction of PAI-1 [74]. In this study, over-expression of arterial TGF-β1 by 6–10 fold in PAI-1 deficient mice was unable to cause intimal thickening, consistent with previous studies showing over-expression of PAI-1 was associated with enhanced intimal thickening, whereas PAI-1 deficiencies were correlated with corresponding reductions in intimal thickening [75,76].

Connective tissue growth factor (CTGF), regulated by TGF-β1, is thought to be an important up-regulator of ECM proteins after PCI, and CTGF expression has been shown to parallel elevations in local TGF-β1 levels after balloon injury in rats [77]. After inducing graft injury in monkey aortas, Geary et al. noted that intimal VSMCs expressed greater than 3-fold higher levels of CTGF when compared to non-injured VSMCs [78]. In the vasculature, TGF-β1 likely up-regulates CTGF via the Smad pathway. In human aortic VSMC cultures, Fu et al. found that peroxismal proliferator-activated receptors attenuated the up-regulation of CTGF by TGF-β1 through inhibition of Smad3 [79].

5.4 Synergism of TGF-β1 with other growth factors
TGF-β1 may also act synergistically with other growth factors such as PDGF to promote intimal thickening. Endogenous PDGF has been shown to stimulate VSMC proliferation, contributing to restenosis after PCI [80]. TGF-β1 has been noted to enhance expression of PDGF-AA from VSMC and vascular endothelial cell cultures [44,81,82]. In mitogenic assays, TGF-β1 was shown to potentiate the proliferative effects of PDGF-AA on VSMCs [44]. Nishio and Watanabe found that co-administration of TGF-β1 and PDGF-BB led to a 15-fold increase in aortic VSMC proliferation when compared to controls [83]. Additionally, PDGF-specific antibodies have been shown to inhibit TGF-β1-mediated collagen up-regulation in human aortic VSMC cultures [81]. In vivo, TGF-β1 stimulation of DNA synthesis in human VSMCs was significantly attenuated by PDGF-A neutralizing ribozymes [84].


   6. Direct inhibition of TGF-β1
Top
 Abstract
 1. Introduction
 2. Pathology of intimal...
 3. Biology of TGF-β1
 4. Involvement of TGF-β1...
 5. TGF-β1 modulation after...
 6. Direct inhibition of...
7. Inhibition of TGF-β1...
 8. Inhibition of TGF-β1...
 9. Conclusion and future...
 References
 
The importance of TGF-β1 in post-interventional restenosis suggests the need for treatments targeting its effects after vascular injury. Several studies have shown that local, direct inhibition of TGF-β1 may be efficacious in preventing luminal stenosis. In animal models, different techniques have been employed for direct inhibition of TGF-β1. The first, recombinant soluble TβR-II, inhibits the actions of TGF-β1 by competing with cell-surface receptors for binding of active TGF-β1. Smith et al., using the balloon catheter carotid denudation model in rats, found that the administration of soluble TβR-II was able to markedly attenuate adventitial myofibroblast transition and adventitial fibrosis, and reduced intimal thickening by up to 65% when compared to controls, resulting in an 88% increase in luminal area in comparison to non-treated rats [12]. Along with increasing luminal size, Ryan et al. found that administration of soluble TβR-II was associated with significantly reduced collagen type I expression within the intima [85]. Similar results were obtained with in vivo analysis after angioplasty in porcine coronary arteries, in which treatment with soluble TβR-II increased the luminal size of injured arteries by 40% compared to controls, primarily because of the inhibitory effects of TβR-II on constrictive remodeling. However, intimal-to-medial ratios were also decreased significantly in animals receiving soluble TβR-II [86].

Ribozymes are RNA effector molecules that catalyze the cleavage of target mRNA and inhibit gene expression. Transfection of chimeric DNA–RNA ribozymes inhibiting TGF-β1 mRNA expression in VSMCs from spontaneously hypertensive rats also inhibited cellular proliferation [87]. In vivo, the transfection of ribozymes targeting TGF-β1 into the carotid arteries of rats immediately after balloon injury prevented characteristic intima formation [88]. Such inhibition was associated with attenuated collagen synthesis, with significant reductions in collagen type I and type III mRNA expression two weeks after injury when compared to controls.

Antisense DNA binds to the pre-mRNA or mRNA of target molecules, inhibiting their translation. Transfection of TGF-β1 antisense oligonucleotides into the carotid arteries of rabbits after balloon injury was associated with a 40% reduction in intimal thickening 23 days after injury when compared to controls [89]. Rabbits treated with TGF-β1 antisense oligonucleotides were also noted to have reduced proteoglycan synthesis in the media and subendothelium after injury.

The use of local, direct TGF-β1 inhibitors have proven efficacious in animal models. They offer the promise of preventing in-stent restenosis while simultaneously limiting systemic side effects. However, further human studies are required in order to determine the clinical utility of direct TGF-β1 inhibition.


   7. Inhibition of TGF-β1 with tranilast
Top
 Abstract
 1. Introduction
 2. Pathology of intimal...
 3. Biology of TGF-β1
 4. Involvement of TGF-β1...
 5. TGF-β1 modulation after...
 6. Direct inhibition of...
 7. Inhibition of TGF-β1...
8. Inhibition of TGF-β1...
 9. Conclusion and future...
 References
 
Tranilast (N-(3,4-dimethoxycinnamoyl) anthranilic acid) was first shown to inhibit the actions of TGF-β1 on fibroblasts, attenuating collagen synthesis and phenotypic modulation [90]. Subsequently, in vitro studies indicated that tranilast could inhibit TGF-β1-mediated VSMC proliferation and collagen expression [91]. Tranilast has also been shown to prevent PDGF and TGF-β1-mediated up-regulation of TβR-I and TβR-II in VSMC cultures [92]. In vivo, Ward et al. first noted that tranilast significantly inhibited the up-regulation of local arterial TGF-β1, TβR-I and TβR-II expression which normally followed carotid balloon injury in rat models [92]. In the same study, tranilast also decreased TGF-β1-induced up-regulation of {alpha}vβ3 integrins and was associated with concordantly lower VSMC migration after injury when compared to controls.

Oral administration of tranilast was noted to reduce intimal area by 23% after balloon angioplasty in the coronary arteries of pigs, as well as significantly lowering intimal area normalized to injury score after stenting [93]. After endoluminal stenting, Ward et al. noted that tranilast attenuated local elevation of TGF-β1 and its receptors by 65% and 80% respectively ([13]; also see Fig. 3). Treatment was simultaneously associated with a 43% reduction in mean intimal thickening when compared to controls 28 days after stenting.


Figure 3
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  		Fig. 3 Examining the effects of tranilast on the intimal expression of TGF-β1, TβR-I (ALK-5) and TβR-II in areas surrounding stent struts 4 weeks after coronary artery stenting. (The dark grey colour represents immunoreactive peptides, with sections counterstained with hematoxylin). Expression of all three proteins is markedly attenuated by tranilast. Adapted, with permission, from Ward et al. [13].

 
Four clinical trials have examined the efficacy of oral tranilast therapy for prevention of restenosis. In the first study (n=205), the restenosis rate for patients receiving 600 mg/day of tranilast was 12.7%, compared to 38% seen with the control group 3 months after PCI [94]. The Tranilast Restenosis Following Angioplasty Trial (TREAT, n=255) noted that tranilast treatment of 600 mg/day, but not 300 mg/day, was associated with a relative risk reduction in restenosis of greater than 20% at 3 months after intervention [14]. The TREAT 2 trial (n=112) similarly showed that the rate of restenosis in patients treated with 600 mg/day of tranilast was 18.8% as opposed to the 44.1% documented in controls (p<0.00005) 3 months after PCI [95].

In North America, the large-scale Prevention of REStenosis with Tranilast and its Outcomes (PRESTO, n=11484) trial examined the effects of bi-daily 300 mg (600 mg/day) and 450 mg (900 mg/day) treatment of tranilast on restenosis after PCI [96]. Examination at 1 and 3 months after intervention revealed that neither tranilast treatment significantly reduced restenosis when compared with placebo. The size and design (randomized, double blind) of this trial suggest that its results are definitive. Therefore tranilast is no longer targeted as a potential oral therapy for attenuation of in-stent restenosis.

The negative results from the PRESTO trial may also emphasize the need for local delivery in order to achieve and sustain optimal arterial levels of TGF-β1 inhibitors. A number of clinical trials have shown that oral administration of sirolimus, used to coat the drug-eluting stents that significantly reduce restenosis after intervention, failed to suppress intimal thickening after arterial injury, indicating that efficacy of oral and local therapy for drugs must be gauged separately [97,98].


   8. Inhibition of TGF-β1 with TGF-β3
Top
 Abstract
 1. Introduction
 2. Pathology of intimal...
 3. Biology of TGF-β1
 4. Involvement of TGF-β1...
 5. TGF-β1 modulation after...
 6. Direct inhibition of...
 7. Inhibition of TGF-β1...
 8. Inhibition of TGF-β1...
9. Conclusion and future...
 References
 
TGF-β3 has been shown to antagonize the actions of TGF-β1, with administration of TGF-β3 to human dermal fibroblasts stimulated with TGF-β1 causing down-regulation of collagen and TGF-β1 mRNA levels [99]. Exogenous administration of TGF-β3 to rodent cutaneous tissue was shown to attenuate scarring, primarily through reduction in inflammatory cell accumulation, as well as decreased fibronectin and collagen deposition [100].

Adenoviral transfection of TGF-β3 into porcine arteries after angioplasty resulted in significant reduction of luminal loss 28 days after intervention when compared to controls, primarily through the inhibition of constrictive remodeling [101]. Ghosh et al. examined the effects of transection and reanastomosis of the common carotid arteries in goat models, noting significant intimal thickening after the procedure [15]. Localized administration of TGF-β3 attenuated vessel thickness by 30%, and reduced cellular content by 20% after reanastamosis. There was also a significantly decreased level of collagen type VIII, a potent stimulator of VSMC proliferation and migration, and elastin fibres found in the TGF-β3-treated goats.

A summary of the in vivo animal studies employing different approaches to target TGF-β1 activity after vascular injury is presented in tabular form (see Table 1).


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  		Table 1 A summary of the in vivo animal studies employing different approaches to target TGF-β1 activity after vascular injury

 

   9. Conclusion and future directions
Top
 Abstract
 1. Introduction
 2. Pathology of intimal...
 3. Biology of TGF-β1
 4. Involvement of TGF-β1...
 5. TGF-β1 modulation after...
 6. Direct inhibition of...
 7. Inhibition of TGF-β1...
 8. Inhibition of TGF-β1...
 9. Conclusion and future...
References
 
A myriad of studies have now implicated TGF-β1 as a key molecule in the pathological response to vascular injury. Whilst most of this information has been obtained from in vitro studies and injured "normal" vessels in animal models, rather than from atheromatous human arteries, the mechanistic data gathered nonetheless has highlighted various approaches to target TGF-β1 activity subsequent to vascular injury. These include neutralizing antibodies [57], soluble TβR-II [12], DNA enzymes [61], chimeric ribozymes [88], antisense DNA [89], Smad7 over-expression [67], or TGF-β3 over-expression [101]. Newly developed selective TβR-I/ALK-5 kinase inhibitors are also showing some promise as potential therapy, being capable of inhibiting TGF-β1 activity in vitro [102,103] and TGF-β1-mediated fibrosis in vivo [104,105]. Additionally, targeting of specific TGF-β1 signaling pathways downstream of the receptors, such as JNK [45], p38 MAPK, or ERK [106–108] may also be of utility. Finally, regulating the vascular bioavailability of mature TGF-β1, via the selective binding of the glycoprotein emilin-1 to the TGF-β1 pro-peptide [109,110], may also be a future therapeutic approach towards the treatment of intimal thickening. It is also conceivable that a combination of these various inhibitors may represent an attractive approach towards targeting TGF-β1 activity subsequent to vascular injury.

Concordantly, the modes of drug/treatment delivery employed to inhibit TGF-β1 activity may greatly affect efficacy of therapies directed towards intimal thickening. Orally delivered medications have not been able to limit restenosis because of an inability to attain sufficient local concentrations at injury sites, and have also been associated with systemic side effects [96]. Thus far, the use of drug-eluting stents has allowed for localized high drug concentrations at the site of vessel injury, and they also permit sustained delivery of a drug over an extended period of time. Unfortunately, stent based drug delivery is not without risks, such as thrombotic vessel occlusions after PCI and inflammation at the site of injury [111]. Previous animal studies have also shown that stents coated with a high concentration of taxanes (e.g. paclitaxel) had incomplete arterial healing at the injury site [112]. In this regard, it would seem that the emergence of nanoparticle technology is likely to play a key role in future therapeutic targeting of the TGF-β1 system in the vasculature and the prevention of intimal thickening, allowing for a more precise release of TGF-β1 antagonists subsequent to vascular injury. The potential for nanoparticles, carrying the therapeutic agents either as DNA, peptides, or low molecular weight compounds [113], to be beneficial towards the targeting of TGF-β1-induced intimal thickening and restenosis has been demonstrated from studies showing that various nanoparticle formulations have reduced intimal thickening in both rat [114] and porcine injured arteries [115]. Additionally, there is also evidence to indicate that local delivery of autologous endothelial progenitor cells subsequent to vascular injury can markedly attenuate intimal thickening [116]. It is therefore conceivable that a component of future therapy to target intimal thickening could consist of such "ex-vivo modified" cells, genetically manipulated to over-express inhibitors of TGF-β1 activity, being delivered to the site of injury by either catheter based methodology [117] or via coated stents seeded with such cells [118].

In conclusion, whilst a number of diverse cytokines are involved in the development of intimal thickening and restenosis after PCI, evidence suggests that TGF-β1 plays an integral role in the development of this pathology. Concordantly, whilst the complexity of cytokines and mechanisms involved in the restenotic process presents a significant challenge in terms of designing effective long-term clinical therapy to target this pathology after PCI, inhibiting the actions of TGF-β1 may represent a key component of such an approach.


   Notes
 
Time for primary review 18 days


   References
Top
 Abstract
 1. Introduction
 2. Pathology of intimal...
 3. Biology of TGF-β1
 4. Involvement of TGF-β1...
 5. TGF-β1 modulation after...
 6. Direct inhibition of...
 7. Inhibition of TGF-β1...
 8. Inhibition of TGF-β1...
 9. Conclusion and future...
 References
 

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