Targeting Smad-Mediated TGFß Pathway in Coronary Artery Bypass Graft

Abstract

Revascularization surgeries such as coronary artery bypass grafting (CABG) are sometimes necessary to manage coronary heart disease (CHD). However, more than half of these surgeries fail within 10 years due to the development of intimal hyperplasia (IH) among others. The cytokine transforming growth factor-beta (TGFß) and its signaling components have been found to be upregulated in diseased or injured vessels, and to promote IH after grafting. Interventions that globally inhibit TGFß in CABG have yielded contrasting outcomes in in vitro and in vivo studies including clinical trials. With advances in molecular biology, it becomes clear that TGFß exhibits both protective and damaging roles, and only specific components such as some Smad-dependent TGFß signaling mediate vascular IH. The activin receptor-like kinase (ALK)-mediated Smad-dependent TGFß signaling pathways have been found to be activated in human vascular smooth muscle cells (VSMCs) following injury and in hyperplastic preimplantation vein grafts. It appears that focused targeting of TGFß pathway constitutes a promising therapeutic target to improve the outcome of CABG. This study dissects the role of TGFß pathway in CABG failure, with particular emphasis on the therapeutic potentials of specific targeting of Smad-dependent and ALK-mediated signaling.

Introduction

Cardiovascular diseases rate as the leading cause of mortality globally with more than 17 million deaths annually, and projected to reach 23.4 million in 2030, about half of which will be attributable to coronary heart disease (CHD). ^1,2 CHD results when the coronary arteries become narrowed or clogged leading to diminished blood supply to the heart. Chronic CHD was initially regarded as a cholesterol storage disease, but is currently considered as a complex cellular and molecular interaction involving vascular and blood cells and their products culminating into the formation of atherosclerotic plaque and vessel narrowing. ^{3 -5} Several risk factors play critical role in the development and progression of the disease including genetics, other diseases and lifestyle. ^{6 -8} Acute Coronary Syndrome (ACS) is thought to result from sudden physical disruption of a previously formed atherosclerotic plaque such as rupture of atheromatous plaque, superficial erosion, intraplaque hemorrhage, and the erosion of a calcified nodule leading to a lethal coronary thrombosis. ^3,9,10

CHD Management and Revascularization Surgeries

Patients diagnosed with CHD are at increased risk of other cardiovascular events such as myocardial infarction, stroke and death, and rigorous control of modifiable risk factors reduces incidence of these endpoints including the need for revascularization surgeries. ^3,6,11 Drug therapy in CHD is aimed at reducing oxygen requirements of the heart and/or enhancement of its blood supply. ¹¹ However, in certain instances revascularization surgeries are necessary in order to avoid fatal endpoints and to restore forward coronary artery blood flow. ¹² Commonly employed revascularization procedures in patients with multivessel CHD are coronary artery bypass graft (CABG) and percutaneous coronary intervention (PCI). ^12,13 Hlatky et al have shown that long-term mortality is the same following CABG and PCI when 10 randomized trials were pooled, and recommended that decision be subject to patients’ choice for other endpoints. ¹⁴ However, the SYNTAX trial has shown the superiority of CABG over PCI surgery with significantly lower cardiac related mortality rates compared to PCI, ¹⁵ particularly in high-risk patients, such as diabetics and patients aged 65 years and above with multi-vessel disease. ^{16 -18} CABG is currently the preferred method of revascularization in patients with severe, multi-vessel coronary artery disease and about 300,000 grafts are carried out annually worldwide. ¹⁹ Several venous and arterial conduits have been trialed over the years as grafts for CAB, with different rates of graft failure. Arterial grafts (e.g. internal mammary, gastroepiploic and radial arteries) generally have higher patency rates, but harvesting enough graft tissue can prove difficult, thus requiring multiple and often complicated operations. ^20,21 Since its first use in 1969, the long saphenous vein has been the preferred vessel for use in CABG. ^22,23 Although more plentiful and easier to harvest, saphenous vein bypass grafts are generally associated with poorer outcomes compared to arterial grafts, with about 50% failing within 10 years of implantation. ^19,24 A meta-analysis of 5 CABG clinical trials including 1036 patients revealed superiority of the radial artery grafts over saphenous vein grafts in terms of adverse cardiac events and the risk of restenosis. ²⁵ Emerging revascularization modifications include stimulation of arteriogenesis by gene, protein, or cell therapy and it is hoped that these will decrease failure of CABG and PCI surgeries significantly. ^{26 -28}

Vascular Remodeling, Neointima Formation and Vein Graft Failure (VGF)

Vascular remodeling of CABG is caused by acute thrombosis, neointima formation and atherosclerosis arising from dynamic and complex interactions between cells of different lineages, including endothelial cells, vascular smooth muscle cells, activated thrombocytes, and migrating inflammatory cells. ^{26,29 -32} Endothelial to mesenchymal transition (EndMT) is a key feature of vein graft remodeling, where endothelium loses its properties and assumes the phenotype of immature smooth muscle cells. ³¹ Vascular remodeling and intimal hyperplasia (IH) are physiological responses required for proper adaptation of the vein graft in the new arterial environment, however, in some patients aberrant and excessive vascular remodeling leads to progressive narrowing and occlusion of the graft conduit. ²⁴ Vascular injury associated with the process of vessel harvest, quality of the vessel itself as well as handling of the vessel graft when making anastomosis, and other factors related to the graft surgery influence the development of VGF. ^{33 -35} New strategies are being introduced to enhance patency rates and reduce graft failure ranging from use of adjuvant drugs and local gene therapy to the improvement of vein harvesting and grafting techniques. ^26,28 Early VGF results from thrombotic occlusions which occur during the acute postoperative period (∼ month). ³² Midterm VGF, which accounts for 15-30% of VGF, occurs within a year of surgery and is mainly caused by fibrointimal hyperplasia leading to occlusive stenosis, whereas late VGF is mediated primarily by atherosclerosis involving atheromatous changes and fibrotic deposition within the next 10 years. ^24,36 Early thrombotic occlusions are mainly attributed to technical errors at time of vessel harvesting or grafting and are caused by imbalances related to clotting mechanism such as reduced tissue plasminogen activator, heparin and thrombomodulin. ³² Although the precise stimulus for intimal hyperplasia is yet to be fully elucidated, it is thought to be a response of the vascular smooth muscle cells (VSMCs) to physical, cellular and humoral factors combined with endothelial dysfunction. ^24,37,38

IH results from migration and proliferation of VSMCs into the vascular tunica intima stimulated by growth factors, cytokines and other biochemical mediators released by the injured endothelium, platelets and activated macrophages ^30,38,39 Shear stress on endothelium, a change in flow pattern from the previously adapted lower venous to a new higher arterial pulsatile pressure, promotes SMC proliferation in the vein graft as well as increases levels of adhesion molecules and chemotactic proteins thereby facilitating leukocyte infiltration. ^{40 -42} The presence of macrophages in the intima and T-lymphocytes in the adventitia suggests that immune cells play a role in neointimal formation.

The mature neointima is principally composed of vascular smooth muscle cells and extracellular matrix proteins, and structurally resembles tunica intima and media of normal artery. ⁴³ Late graft stenosis is mainly due to atherosclerosis which culminates into fully formed plaque. At this stage, there is intimal fibrosis and reduction in cellularity with concomitant apoptosis of migratory SMCs. ³⁸ Monocytes infiltrating the neointima turn to macrophages subsequently forming foam cells upon uptake of lipids, serving as the foundation for development of superimposed atherosclerosis (Figure 1). Leukocyte and platelet adhesion ultimately ensues and plaque formation progresses slowly leading to intraluminal stenosis. ^38,44 Highly diffuse and concentric atheromas may eventually result within the graft and can be calcified, develop fibrous caps or rupture causing thrombotic occlusion of the graft atheromas. ^{45 -47} Several cytokines and growth factors such as platelet derived growth factor (PDGF) and transforming growth factor-beta (TGFβ) and other biochemical mediators originating from injured endothelium, platelets and activated macrophages are found to mediate neointima formation and CABG failure (Figure 1). ^31,39 TGFβ is an essential factor in neointima formation and atherosclerosis with several studies showing it to be upregulated at sites of vascular injury and elevated levels detected following arterial balloon injury. ^{48 -52}

Figure 1.

Development of neointima. (A) healthy vessel. (B) Physical injury of the endothelium mediated by factors including shear stress predisposes the endothelial cells to release cytokines such as TGFβ and other chemo-attractants, inducing migration and proliferation of smooth muscle cells (SMCs) into the tunica intima. (C) The migratory SMCs together with infiltrating leukocytes, platelets and fibroblasts form the neointima which eventually leads to vascular graft restenosis. Parts of the figure were drawn by using images from Servier Medical Art by Servier, licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).

TGFβ; Signaling Pathways and the Role of Smad Proteins

TGFβ1 is the prototype of the TGFβ superfamily of pleiotropic cytokines, which also comprises the growth differentiation factors (GDFs), activins, inhibins, bone morphogenetic proteins (BMPs), Müllerian-inhibiting substance (MIS) and other structurally related cytokines. ^53,54 The TGFβ proteins share a basic cysteine knot structure with widely distributed expression in almost every cell in the human body with varied roles in embryonic development and regulation of cellular homeostasis including proliferation, differentiation, apoptosis, and extracellular matrix remodeling in a cell and context specific manner. ^31,54,55

Cells secrete TGFβ as a large latent complex consisting of a mature dimeric molecule known as latency-associated propeptite (LAP), and a latent TGFβ binding protein (LTBP). ^56,57 It is essential for TGFβ to be liberated from this latent complex and activated before it can bind to the signaling receptors and exert its effect. ⁵⁸ TGFβ binds to two tetrameric transmembrane receptor kinases, the signal propagating TGFβ type 1 and the activator TGFβ type 2 receptor (ALK1 or ALK5). ⁵⁷ Specifically, TGFβ binds exclusively to ALK5, whereas BMP9 and BMP10 bind to ALK1 with high affinity. ⁵⁹ The TGFβ receptors (TGFβR1 and TGFβR2) are structurally single transmembrane-spanning proteins with an extracellular cysteine-rich ligand-binding domain and an intracellular serine-threonine kinase domain. ⁶⁰ Interactions of TGFβ with its receptors tend to be complex and selective involving the receptors’ four subunits working with each other in an interdependent manner leading to activation of both Smad family-dependent signal transduction (the canonical TGFβ signaling pathway) and other, non-canonical or Smad-independent pathways. ^59,61

Eight types of Smad proteins (Smad1 to Smad8) exist in vertebrates activated either through phosphorylation by the TGFβ and Activin receptors (ActR); TGFβR2/ALK-5 and ActR1B in the case of Smad2 and Smad3 through ALK1, ALK2, BMP receptor 1-A (BMPR1A)/ALK-3 and BMPR1B/ALK-6 in the case of Smad1, Smad5 and Smad8. ^62,63 Functionally, Smad proteins are categorized into three groups; receptor-associated Smads (R-Smads) including Smad1, Smad2, Smad3, Smad5 and Smad8; co-operating Smads (Co-Smads) group consisting of only Smad4; and inhibitory Smads (I-Smads) formed by Smad6 and Smad7. ^59,61 Ligand binding and hetero-tetrameric receptor complex formation leads to recruitment and subsequent phosphorylation of R-Smads by the receptor’s cytoplasmic Serine/Threonine kinase domain. ⁵⁹ Typically, in Smad-mediated signaling, Smad2 and Smad3 are activated by the TGFβR2/ALK-5 complex while Smad1, Smad5 and Smad8 are activated by TGFβR2/ALK-1 complex (Figure 2). ⁶⁴ TGFβ binding to the TGFβR2 results into phosphorylation of the specific serine and threonine residues in the glycine-serine enriched (GS)-domain of the TGFβR1/ALK-5 by the TGFβR2 kinase. ^{65 -67} Activated TGFβR phosphorylates Smad2 and Smad3 at their carboxyl termini and the phosphorylated Smad2 and Smad3 form a complex with Smad4. ^68,69 On the other hand, binding of TGFβ to the TGFβR2-ALK-1 complex or the binding of the BMPs to the type 1 receptors ALK-1, -2, -3, or -6, and the type 2 receptors BMPRII, ActRIIA, or ActRIIB result into the phosphorylation of Smad1, Smad5 and Smad8. ⁶⁰ Activated R-Smads form heteromeric complexes with the co-Smads followed by translocation of the complex into the nucleus. ⁶¹ Smads shuttle between cytoplasm and nucleus through nuclear pore with the aid of nucleoporins and specialized nuclear factors at basal and activated oligomeric state respectively. ⁷⁰ Smad complexes in the nucleus bind to DNA at specific Smad-binding elements through high affinity and selectivity of their target promoters with the complimentary binding elements. ⁷¹ The type of genes to be targeted by the R-Smad complexes and whether the target genes will be upregulated or downregulated are largely determined by the particular transcription factors, chromatin remodelers and histone readers and modifiers binding to the R-Smad complexes. ⁵⁹ I-Smads downregulate TGFβ receptors and the interaction of R-Smads and Co-Smad, with Smad6 specifically inhibiting BMP-induced signaling, whereas Smad7 inhibits signaling initiated by both TGFβ and BMP. ⁷² Smad ubiquitin regulatory factors (Smurfs), type E3 ligases homologous to the E6-accessory protein (HECT), interact with I-Smads to facilitate the ubiquitylation and proteasomal degradation of TGFβ receptors. ^72,73 TGFβ binding to receptors also activates other non-Smad pathways such as PI3K-Akt, Jun N-terminal kinase (JNK), Erk, p38MAPK and GTPases (rhoA and Cdc42) capable of regulating Smad signaling as well as eliciting Smad-independent TGFβ responses ^63,74 TGFβ-mediated transcriptional response is regulated by factors surrounding ligand-receptor interaction, transcription factors binding to activated Smad proteins which dictate genes to be targeted and the epigenetic status of the cell itself (Figure 2). ⁵⁹

Figure 2.

Smad-dependent TGFβ signaling pathways. Mature TGFβ dimers binds to two receptors (ALK1 or ALK5) to activate Smad-dependent or Smad-independent pathways. ALK1 and ALK5 ligand activation leads to phosphorylation of pSmad1/5/8 and pSmad2/3 respectively. The receptor-activated Smads (R-Smads) interact with Co-Smads and the complex translocates to nucleus where they bind to DNA with the aid of specific transcription factors leading to cellular responses through effects on target gene expression. Parts of the figure were drawn by images pictures from Servier Medical Art by Servier, licensed under a Creative Commons Attribution 3.0 Unported License (https://creativecommons.org/licenses/by/3.0/).

In human saphenous vein, the widely used CABG source, it has been shown that TGFβ can bind to both ALK1 and ALK5, to separately activate both Smad1/5- and Smad2-mediated pathways. ⁷⁵ Several studies have indicated the role of Smad-mediated components in vascular function and pathology through IH including in CABG failure. Moreover, TGFβ protects from atherosclerosis and it was shown to inhibit the uptake of modified low-density lipoprotein in macrophages through Smad 2 and Smad3. ⁵² Disruption of TGFβ signaling in ApoE-knockout mice resulted in increased aortic lesion surface area, size and expression of IFN-γ mRNA compared to the control mice. ⁷⁶ It also appears that the 2 distinct Smad-dependent TGFβ pathways mediate different roles in blood vessels. ⁷⁵

TGFβ Pathway in CABG Failure

The role of TGFβ-mediated cellular proliferation, differentiation and apoptosis in vascular physiology and pathologies including CABG failure has been described in several studies. The presence of TGFβ1 in the neointima of rat carotid arteries was first shown by Madri et al. and the authors suggested a possible role of TGFβ in extracellular matrix (ECM) formation. ⁴⁸ These findings were supported by Majesky et al., who demonstrated that TGFβ1 was upregulated in SMCs in rat carotid arteries 6 hours after injury and remained significantly elevated for 2 weeks, during which an active neointimal thickening process was in place. ⁷⁷ Subsequently, many studies reported upregulation of TGFβ in human and animal vascular injury and restenotic lesions. ^{49,78 -80} For example, there was higher expression of TGFβl and PDGF-A mRNA in vein grafts prior to formation of neointima suggesting a role for these growth factors in the development of vein graft intimal hyperplasia. ⁸¹ Similarly, significantly elevated levels of active TGFβ1 were observed from 2 hours to 7 days post-angioplasty in porcine coronary angioplasty model. ⁷⁹ In addition, TGFβ1 was shown to be localized to the SMCs and endothelial cells (ECs) by immunohistochemical staining. ^49,79 In another study, the expression level of BMP2, was significantly higher in calcium chloride-induced calcifying rat carotid artery. Interestingly, there was also increased neointima formation in calcifying vessels, which might have resulted from BMP-2 overexpression. ⁸²

Experimental studies have shown that upregulation of TGFβ or addition of exogenous TGFβ resulted in increased intimal thickening or neointima formation. Treatment with TGFβ1 was shown to result in hypertrophy of VSMC in time and concentration dependent manner. ⁸³ Expression of TGFβ1 in uninjured porcine arteries resulted in increased ECM production and cellular proliferation. ⁵⁰ In another study, significantly higher intimal thickening was observed in injured rabbit carotid arteries treated with 10 µg/kg TGFβ and 10 mg/kg aspirin, compared to vessels treated with 10 mg/kg aspirin only. ⁸⁴ Likewise, treatment of rat vein grafts with adenovirus expressing TGFβ mRNA resulted in a larger collagen-rich neointima, compared with control. ⁸⁵ Furthermore, even in uninjured rat carotid arteries, adenoviral overexpression of TGFβ1 in the endothelium lead to considerable intimal thickening with cellular proliferation. ⁸⁶ Exogenous BMP-2 was shown to significantly increase proliferation of VSMCs under calcifying conditions, indicating a link between calcification, IH and the role this might play in CABG failure. ⁸²

In contrast, overexpressing TGFβ3 inhibited vascular remodeling and stenosis after coronary angioplasty in pigs, ⁸⁷ and reduced vessel wall thickness by 30% in goat carotid arteries after anastomosis. ⁸⁸ Other studies have also shown inhibitory effects of TGFβ on SMC proliferation and migration. ^{83,89 -91} This suggests differences in the response of SMCs depending on specific TGFβ isoforms, heterogeneity of SMCs and the complexity of the TGFβ receptors, presence of other growth factors and intracellular signaling. ^83,87,89,92 Nevertheless, most evidence from animal studies strongly indicate that TGFβ mediates vascular remodeling and promotes the proliferation and migration of VSMCs. In addition, several in vivo interventional studies have demonstrated that inhibition of TGFβ1 reduced intimal thickening, adventitial myofibroblast formation, collagen deposition in rat and rabbit carotid artery balloon injury. ^{51,85,93 -95}

Smad Proteins in Intimal Hyperplasia

Smad proteins display diverse functions in vascular physiology and pathology. For example, Smad3 primarily mediates TGFβ1-induced SM22α expression, whereas Smad4 is essential in vascular development and SMC function. ^96,97 On the other hand, Smad6 and Smad7 collectively known as I-Smads repress SM22α activation. The role of the canonical Smad2/3 pathway in mediating the intracellular signaling TGFβ pathway in IH has been indicated. Several in vivo and in vitro studies have demonstrated the upregulation of Smad proteins in vascular injury. Interventional overexpression of Smad proteins results into IH, whereas inhibition of some Smads leads to reduced IH (Table 1). The activated Smad1, Smad2, Smad3, and Smad5 were observed to be upregulated in rabbit femoral and iliac arteries following balloon injury. ⁹⁸ Using mouse carotid ligation model, LeClair et al. have shown an increased expression of activated Smad2 and 3 in adventitital fibroblasts and neointimal SMCs during remodelling. ⁹⁹ Subsequently, this was supported by other studies in rat and rabbit models of vascular injury. ^80,100,101 Substantiating this, Edlin et al. demonstrated higher levels of alpha-actin+ and expression of Smad3 in human femoral artery restenotic lesions compared with primary atherosclerotic lesions. ¹⁰² Medial Smad3 overexpression via gene transfer resulted in adventitial changes including increased collagen production in injured arteries. ¹⁰⁰ Similar infection of injured rat carotid artery with adenovirus-expressing Smad3 (AdSmad3) lead to increased IH and VSMC proliferation. In addition, it was shown that this might be mediated by p27, a cyclin-dependent kinase inhibitor. ⁸⁰

Table 1.

The Role of Components of Smad-Mediated TGFβ Signaling Pathway in Vascular Injury and Hyperplasia.

Vascular injury upregulates of Smad-associated proteins
Experimental model of injury	Smad-associated protein upregulated		Reference
Ballon injury in rabbit femoral and iliac arteries	Smad1, 2, 3, and 5		Sluijter et al. 98
Injured mouse carotid ligation model	Smad2 and 3		LeClair et al. 99
Rabbit abdominal aortic balloon angioplasty model	Smad2		Zeng et al. 101
Rat carotid injury	Smad3		Kundi et al. 100
Human restenotic lesions	Smad3		Edlin et al. 102
Balloon injury in the rat carotid artery.	Smad3		Tsai et al. 80
Overexpression of Smad pathway components mediates IH
Experimental model	Intervention	Findings	Reference
Injured artery	Smad3 overexpression	Increased intima-to-media ratio, PCNA expression and adventitial collagen	Kundi et al. 100
Injured rat carotid artery	Smad3 overexpression	Increased intima-to-media ratio and PCNA expression	Tsai et al. 80
Inhibition of Smad pathway components
Experimental model	Intervention	Outcome	Reference
Rat carotid balloon angioplasty	Adenoviral overexpression of Smad7	Decreased IH, reduced medial and intimal cell proliferation	Tsai et al. 80
Rat carotid balloon injury	Adenoviral overexpression of Smad7	Reduced neointima formation and collagen deposition	Mallawaarachichi et al. 103
Rat carotid balloon injury	SM16, ALK5/ALK4 inhibitor	Reduced intimal collagen production	Fu et al. 104
Smad3-null mice	Femoral artery injury	Larger IH compared to wild type	Kobayashi et al. 105
VSMC serum stimulation	Smad2 and Smad3 knockout; SB431542	Reduced VSMC proliferation and migration	Mao et al. 96
Rat carotid artery balloon injury	SB431542, ALK4/5/7 inhibitor	Reduced IH, and SMC proliferation, reduced recruitment of Mesenchymal Stem Cells	Zhao et al. 95
Mouse acute carotid artery injury	LDN193189, Smad1/5 inhibitor	Reduced neointima formation	Low et al. 106

Given the strong evidence indicating functions of Smad pathway components in IH, many studies investigated the effect of inhibition of the pathway proteins (Table 1). Mallawaarachichi et al. perivascularly treated rat balloon-catheter injured carotid arteries with adenovirus expressing Smad7 (AdSmad7), a known inhibitor of the TGFβ/Smad3 pathway, and reported reduced neointima formation and collagen deposition, which was linked to decreased Smad2 phosphorylation. ¹⁰³ This was corroborated by Tsai et al., who also showed that AdSmad7 attenuated IH in rat carotid arteries, evident as lowered intima-to-media ratio as well as reduced medial and intimal cell proliferation. ⁸⁰ In addition, Smad2 and Smad3 shRNAi mediated silencing significantly reduced VSMC proliferation and migration. ⁹⁶ In a recent study, Low et al. treated acute vascular injury in mice with LDN193189 (Figure 3), an inhibitor of Smad1/5 phosphorylation downstream of ALK1/ALK2. A substantial reduction in neointima was observed in LDN193189-treated mice compared to control. ¹⁰⁶ However, wire injured femoral arteries of Smad3-null mice displayed large neointimal hyperplasia compared with wild-type mice, suggesting that that Smad3 is protective, ¹⁰⁵ contrary to the previous hypothesis that it mediates restenosis. ¹⁰⁷ However, overexpression of Smad7 inhibited angiotensin II and TGFβ-induced expression of CTGF and procollagen, as well as the production of fibronectin in VSMCs. ¹⁰⁸

Figure 3.

Chemical structures of some inhibitors of Smad and ALK.

The Emerging Role of ALK Components of Smad-Mediated Pathway

Although it was initially assumed that ALK1 was an endothelial cell-specific receptor, recent studies have shown that ALK1 is expressed in several cells in different body parts including SMCs, myofibroblast, monocytes, myoblasts, hepatocytes, chondrocytes, macrophages, and fibroblasts. ALK1 appears to play vital role in vascular physiology and angiogenesis and has been associated with pathogenesis of several cardiovascular diseases, but its specific role is still unfolding. ^60,109 Although ALK1 is abundant in the healthy endothelium, it has been found to be upregulated in SMCs of injured or atherogenic vessels. ^110,111 BMP 9 and BMP10 are high affinity ligands of ALK1 in the endothelium, and activate the Smad1/5/8 signaling. ¹¹² On the other hand, ALK5 is the principal type I TGFβ receptor with ubiquitous distribution and it mediates most cellular responses to TGFβ. In healthy adult animals, ALK5 is chiefly localized to the medial vascular SMCs. ¹¹⁰

The deficiency of ALK1 is linked to arteriovenous malformations (AVMs), suggesting that this kinase is protective to vessels. Krüppel-like factor 6 (KLF6) was shown to mediate upregulation of ALK1 in ECs during vascular injury through a synergy with Sp1 by binding to ALK1 promoter, and via EC-VSMC paracrine interaction in VSMCs during vascular remodeling. ¹¹¹ ALK1 and its signaling components were also found to be highly expressed in preimplantation saphenous veins showing substantial pre-existing neointima formation, indicating its role in graft restenosis. ¹⁰⁶ ALK1’s role in regulating cell proliferation and migration and the modulation of extracellular matrix (ECM) protein expression strongly indicates an important function in cardiovascular remodeling and suggests that the ALK1/smad1/5/8 pathway is potentially a powerful therapeutic target. ^71,109,113 Induction of the phosphorylation of Smad1/5 and Smad2 by the binding of TGFβ to ALK1 and ALK5 respectively has been demonstrated in human saphenous vein SMCs using ALK inhibitors and siRNA approaches.

Fu et al. treated rat carotid balloon injury model with SM16 (Figure 3), a small molecule inhibitor of ALK5/ALK4 kinase. This inhibited Smad2/3 phosphorylation resulting in reduced intimal collagen production, but not intimal SMC proliferation, compared to control. ¹⁰⁴ Similarly, inhibition of ALK5 using SB431542 significantly attenuated serum stimulation induced SMC migration. ⁹⁶ In addition, treatment of a model of balloon-induced injury with SB431542, an ALK4/5/7 inhibitor, was shown to reduce IH, and the expression of TGFβ1, TGFβ1RI, and Smad2/3, although higher level of phosphorylated Smad2/3 was observed. ⁹⁵ More recently, dsiRNA-mediated knockdown of ALK1 resulted in reduced expression of Smad1/5, while knockdown of ALK5 downregulated Smad2. Similarly, treatment with ALK1 kinase inhibitor KO2288/KO and the ALK5 kinase inhibitor SB525334/SB (Figure 3) downregulated Smad1/5 and Smad2 respectively. ¹⁰⁶ Specifically, while ALK5 signaling was activated in both intact and injured vessels, ALK1 signaling was only found to be active in injured vessels. Thus, it appears that ALK-1 and Smad1/5 activation are the pathological hallmark of the SMC response to acute vascular injury, promoting neointima formation and inward remodeling. ¹⁰⁶

Targeting the Smad-Mediated TGFB Pathway in CABG

TGFβ inhibitor, pirfenidone, has been approved for the treatment of idiopathic pulmonary fibrosis by FDA in 2014. ¹¹⁴ Galunisertib (LY2157299 monohydrate), a small molecule inhibitor of the ALK5 kinase, is currently in phase II/III clinical trials for management of various types of cancer either as single or combination therapy, with preliminary data showing good promise. ^{115 -118} Given the success of TGFβ inhibitors in clinical trials for cancer and fibrosis therapy, and the encouraging evidence available from in vitro and in vivo studies, targeting TGFβ appears promising in restenotic disease. ¹¹⁹ Oral administration of tranilast, a non-specific inhibitor of TGFβ biosynthesis demonstrated reduced risk of restenosis compared with placebo (17.6% vs. 39.4% at 3 months) in an early small scale clinical trials. ^120,121 However, in the large-scale randomized double-blind clinical trial PRESTO (Prevention of REStenosis with Tranilast and its Outcomes) involving 11,484 patients after PCI, tranilast did not improve measures of restenosis. In addition, tranilast treatment was associated with liver adverse effects, that were reversed upon cessation of treatment. ¹²²

With better understanding of the complexity of TGFβ signaling pathway in different patients, it now becomes clear that global inhibition of TGFβ might not be beneficial in preventing CABG failure and other vascular pathologies. Thus, TGFβ-based therapies against restenosis need to be focused to a particular pathway component to avoid undesired side effects. ⁷⁵ Smad proteins and pathway components such as ALK1 and ALK5 represent promising targets, that should be thoroughly explored in the prevention of CABG failure. TGFβ signaling pathway mediates several functions in other organ systems, making systemic deliveries risky. Thus, TGFβ targeted therapies in restenosis should be specific and can utilize the many delivery system becoming available such as catheter- or stent-based drug delivery devices, intraluminal delivery, gene therapy, treatment of pre-implantation vessels during surgery as well as the emerging smart drug design. Great emphasis should also be placed on personalized medicine before and after CABG.

Conclusion

Restenosis remains a crucial challenge to revascularization surgeries such as CABG, resulting in deaths and disabilities worldwide. IH, a major process that leads to CABG failure, is mediated by specific components of TGFβ signaling pathway. Specifically, the role Smad proteins, ALK-1, and ALK-5 play in the pathogenesis of restenosis is becoming clearer. This indicates that these kinases represent promising targets for the improvement of clinical outcomes of CHD patients undergoing revascularization surgeries.