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

Abstract

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.

STAT3 Protein Regulates Vascular Smooth Muscle Cell Phenotypic Switch by Interaction with Myocardin

“…1, A-D). These observations are consistent with the effects of VEGF on the VSMC phenotypic switch and proliferation (34), suggesting that activated STAT3 signaling pathway is positively correlated with the synthetic VSMC phenotype.…”

“…Correlates with VSMC Synthetic Phenotype-Previous studies have shown that VEGF is a key regulator of physiological and pathological angiogenesis, which inhibits VSMC apoptosis and promotes VSMC proliferation by suppressing the expression of VSMC marker genes (34,35). Our data revealed that although the STAT3 mRNA level in HAVSMCs was not significantly changed after incubating with 50 ng/ml VEGF for different time periods (12,24, and 48 h), the phosphorylated (p)-STAT3 protein expression was significantly elevated to 3-6-fold compared with control levels (Fig.…”

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

“…Indeed, the data showed that Wnt2b, Wnt4, Wnt5a, and Wnt9a all stimulated SMC proliferation compared to the control (Figure 5A). The increase of SMC proliferation unlikely resulted from reduced apoptosis, considering that even basal level of apoptosis (active caspase-3) could not be detected unless apoptosis is induced[11]. Wnt11, which did not stabilize β-catenin, did not enhance SMC proliferation at either of the three concentrations used (Figure 5A) thereby reinforcing the functional specificity of the other 4 Wnts.…”

“…In fact, vascular SMCs are hardy cells. Generally they do not undergo apoptosis until induced with an apoptotic factor such as UV[11]. Taken together, these findings demonstrate that increases in β-catenin can increase SMC proliferation.…”

“…Previous investigators have reported that TGF-β1 inhibits SMC proliferation in vitro [7]. This effect is reversed in the presence of elevated levels of Smad3 and leading to increases in SMC proliferation, migration and de-differentiation while inhibiting apoptosis[8-11]. Upon TGF-β activation, the TGF-β receptor phosphorylates Smad3 which, in a complex with Smad2 and Smad4, translocates to the nucleus to regulate gene expression.…”