Daniel DiRenzo

A crosstalk between TGF-β/Smad3 and Wnt/β-catenin pathways promotes vascular smooth muscle cell proliferation.

RATIONALEnEndovascular interventions performed for atherosclerotic lesions trigger excessive vascular smooth muscle cell (SMC) proliferation leading to intimal hyperplasia. Our previous studies show that following endovascular injury, elevated TGF-β/Smad3 promotes SMC proliferation and intimal hyperplasia. Furthermore in cultured SMCs, elevated TGF-β/Smad3 increases the expression of several Wnt genes. Here we investigate a crosstalk between TGF-β/Smad3 and Wnt/β-catenin signaling and its role in SMC proliferation.nnnMETHODS AND RESULTSnTo mimic TGF-β/Smad3 up-regulation in vivo, rat aortic SMCs were treated with Smad3-expressing adenovirus (AdSmad3) or AdGFP control followed by stimulation with TGF-β1 (or solvent). AdSmad3/TGF-β treatment up-regulated Wnt2b, Wnt4, Wnt5a, Wnt9a, and Wnt11 (confirmed by qRT-PCR and ELISA), and also increased β-catenin protein as detected by Western blotting. Blocking Wnt signaling using a Frizzled receptor inhibitor (Niclosamide) abolished TGF-β/Smad3-induced β-catenin stabilization. Increasing β-catenin through degradation inhibition (using SKL2001) or by adenoviral expression enhanced SMC proliferation. Furthermore, application of recombinant Wnt2b, Wnt4, Wnt5a, or Wnt9a, but not Wnt11, stabilized β-catenin and stimulated SMC proliferation as well. In addition, increased β-catenin was found in the neointima of injured rat carotid artery where TGF-β and Smad3 are known to be up-regulated.nnnCONCLUSIONSnThese results suggest a novel mechanism whereby elevated TGF-β/Smad3 stimulates the secretion of canonical Wnts which in turn enhances SMC proliferation through β-catenin stabilization. This crosstalk between TGF-β/Smad3 and Wnt/β-catenin canonical pathways provides new insights into the pathophysiology of vascular SMCs linked to intimal hyperplasia.

TGF-β/Smad3 inhibit vascular smooth muscle cell apoptosis through an autocrine signaling mechanism involving VEGF-A

We have previously shown that in the presence of elevated Smad3, transforming growth factor-β (TGF-β) transforms from an inhibitor to a stimulant of vascular smooth muscle cell (SMC) proliferation and intimal hyperplasia (IH). Here we identify a novel mechanism through which TGF-β/Smad3 also exacerbates IH by inhibiting SMC apoptosis. We found that TGF-β treatment led to inhibition of apoptosis in rat SMCs following viral expression of Smad3. Conditioned media from these cells when applied to naive SMCs recapitulated this effect, suggesting an autocrine pathway through a secreted factor. Gene array of TGF-β/Smad3-treated cells revealed enhanced expression of vascular endothelial growth factor (VEGF), a known inhibitor of endothelial cell apoptosis. We then evaluated whether VEGF is the secreted mediator responsible for TGF-β/Smad3 inhibition of SMC apoptosis. In TGF-β/Smad3-treated cells, VEGF mRNA and protein as well as VEGF secretion were increased. Moreover, recombinant VEGF-A inhibited SMC apoptosis and a VEGF-A-neutralizing antibody reversed the inhibitory effect of conditioned media on SMC apoptosis. Stimulation of SMCs with TGF-β led to the formation of a complex of Smad3 and hypoxia-inducible factor-1α (HIF-1α) that in turn activated the VEGF-A promoter and transcription. In rat carotid arteries following arterial injury, Smad3 and VEGF-A expression were upregulated. Moreover, Smad3 gene transfer further enhanced VEGF expression as well as inhibited SMC apoptosis. Finally, blocking either the VEGF receptor or Smad3 signaling in injured carotid arteries abrogated the inhibitory effect of Smad3 on vascular SMC apoptosis. Taken together, our study reveals that following angioplasty, elevation of both TGF-β and Smad3 leads to SMC secretion of VEGF-A that functions as an autocrine inhibitor of SMC apoptosis. This novel pathway provides further insights into the role of TGF-β in the development of IH.

TGF-β/Smad3 Stimulates Stem Cell/Developmental Gene Expression and Vascular Smooth Muscle Cell De-Differentiation

Atherosclerotic-associated diseases are the leading cause of death in the United States. Despite recent progress, interventional treatments for atherosclerosis can be complicated by restenosis resulting from neo-intimal hyperplasia. We have previously demonstrated that TGF-β and its downstream signaling protein Smad3∶1) are up-regulated following vascular injury, 2) together drive smooth muscle cell (SMC) proliferation and migration and 3) enhance the development of intimal hyperplasia. In order to determine a mechanism through which TGF-β/Smad3 promote these effects, Affymetrix gene expression arrays were performed on primary rat SMCs infected with Smad3 and stimulated with TGF-β or infected with GFP alone. More than 200 genes were differentially expressed (>2.0 fold change, p<0.05) in TGF-β/Smad3 stimulated SMCs. We then performed GO term enrichment analysis using the DAVID bioinformatics database and found that TGF-β/Smad3 activated the expression of multiple genes related to either development or cell differentiation, several of which have been shown to be associated with multipotent stem or progenitor cells. Quantitative real-time PCR confirmed up-regulation of several developmental genes including FGF1, NGF, and Wnt11 (by 2.5, 6 and 7 fold, respectively) as well as stem/progenitor cell associated genes CD34 and CXCR4 (by 10 and 45 fold, respectively). In addition, up-regulation of these factors at protein levels were also confirmed by Western blotting, or by immunocytochemistry (performed for CXCR4 and NGF). Finally, TGF-β/Smad3 down regulated transcription of SMC contractile genes as well as protein production of smooth muscle alpha actin, calponin, and smooth muscle myosin heavy chain. These combined results suggest that TGF-β/Smad3 stimulation drives SMCs to a phenotypically altered state of de-differentiation through the up-regulation of developmental related genes.

Halofuginone Stimulates Adaptive Remodeling and Preserves Re-Endothelialization in Balloon-Injured Rat Carotid Arteries

Background—Three major processes, constrictive vessel remodeling, intimal hyperplasia (IH), and retarded re-endothelialization, contribute to restenosis after vascular reconstructions. Clinically used drugs inhibit IH but delay re-endothelialization and also cause constrictive remodeling. Here we have examined halofuginone, an herbal derivative, for its beneficial effects on vessel remodeling and differential inhibition of IH versus re-endothelialization. Methods and Results—Two weeks after perivascular application to balloon-injured rat common carotid arteries, halofuginone versus vehicle (n=6 animals) enlarged luminal area 2.14-fold by increasing vessel size (adaptive remodeling; 123%), reducing IH (74.3%) without inhibiting re-endothelialization. Consistent with its positive effect on vessel expansion, halofuginone reduced collagen type 1 (but not type 3) production in injured arteries as well as that from adventitial fibroblasts in vitro. In support of its differential effects on IH versus re-endothelialization, halofuginone produced greater inhibition of vascular smooth muscle cell versus endothelial cell proliferation at concentrations ≈50 nmol/L. Furthermore, halofuginone at 50 nmol/L effectively blocked Smad3 phosphorylation in smooth muscle cells, which is known to promote smooth muscle cell proliferation, migration, and IH, but halofuginone had no effect on phospho-Smad3 in endothelial cells. Conclusions—Periadventitial delivery of halofuginone dramatically increased lumen patency via adaptive remodeling and selective inhibition of IH without affecting endothelium recovery. Halofuginone is the first reported small molecule that has favorable effects on all 3 major processes involved in restenosis.

Restenosis Inhibition and Re-differentiation of TGFβ/Smad3-activated Smooth Muscle Cells by Resveratrol

To date, there is no periadventitial drug delivery method available in the clinic to prevent restenotic failure of open vascular reconstructions. Resveratrol is a promising anti-restenotic natural drug but subject to low bioavailability when systemically administered. In order to reconcile these two prominent issues, we tested effects of periadventitial delivery of resveratrol on all three major pro-restenotic pathologies including intimal hyperplasia (IH), endothelium impairment, and vessel shrinkage. In a rat carotid injury model, periadventitial delivery of resveratrol either via Pluronic gel (2-week), or polymer sheath (3-month), effectively reduced IH without causing endothelium impairment and vessel shrinkage. In an in vitro model, primary smooth muscle cells (SMCs) were stimulated with elevated transforming growth factor (TGFβ) and its signaling protein Smad3, known contributors to IH. TGFβ/Smad3 up-regulated Kruppel-like factor (KLF5) protein, and SMC de-differentiation which was reversed by KLF5 siRNA. Furthermore, TGFβ/Smad3-stimulated KLF5 production and SMC de-differentiation were blocked by resveratrol via its inhibition of the Akt-mTOR pathway. Concordantly, resveratrol attenuated Akt phosphorylation in injured arteries. Taken together, periadventitial delivery of resveratrol produces durable inhibition of all three pro-restenotic pathologies — a rare feat among existing anti-restenotic methods. Our study suggests a potential anti-restenotic modality of resveratrol application suitable for open surgery.

A Murine Model of Arterial Restenosis: Technical Aspects of Femoral Wire Injury

Cardiovascular disease caused by atherosclerosis is the leading cause of death in the developed world. Narrowing of the vessel lumen, due to atherosclerotic plaque development or the rupturing of established plaques, interrupts normal blood flow leading to various morbidities such as myocardial infarction and stroke. In the clinic endovascular procedures such as angioplasty are commonly performed to reopen the lumen. However, these treatments inevitably damage the vessel wall as well as the vascular endothelium, triggering an excessive healing response and the development of a neointimal plaque that extends into the lumen causing vessel restenosis (re-narrowing). Restenosis remains a major cause of failure of endovascular treatments for atherosclerosis. Thus, preclinical animal models of restenosis are vitally important for investigating the pathophysiological mechanisms as well as translational approaches to vascular interventions. Among several murine experimental models, femoral artery wire injury is widely accepted as the most suitable for studies of post-angioplasty restenosis because it closely resembles the angioplasty procedure that injures both endothelium and vessel wall. However, many researchers have difficulty utilizing this model due to its high degree of technical difficulty. This is primarily because a metal wire needs to be inserted into the femoral artery, which is approximately three times thinner than the wire, to generate sufficient injury to induce prominent neointima. Here, we describe the essential surgical details to effectively overcome the major technical difficulties of this model. By following the presented procedures, performing the mouse femoral artery wire injury becomes easier. Once familiarized, the whole procedure can be completed within 20 min.