Transforming Growth Factor- Beta and its Signaling Mediator Smad3 Enhance Cell Survival After Vascular Injury

Abstract

Introduction: It is well established that Transforming Growth Factor-beta (TGF-β) promotes intimal hyperplasia (IH); levels of this cytokine are elevated after vascular injury and blocking TGF-β results in inhibition of the hyperplastic response. IH is usually accompanied by inhibition of apoptosis/increased cell survival, which contributes to the increased cellularity that accompanies hyperplastic plaque. Paradoxically, TGF-β traditionally is thought to be pro-apoptotic. We have previously shown injured smooth muscle cells (SMCs) contain elevated levels of Smad3 and may respond to TGF-β differently compared to normal SMCs. in the current study, we tested a hypothesis that TGF-β in the presence of elevated Smad3 may be anti-apoptotic. Methods: Cultured aortic SMCs were infected with adenovirus expressing Smad3 or GFP and then stimulated with TGF-β for 24 hours. Apoptosis was induced with 5 minutes of ultraviolet light. Cleaved Caspase-3 immunofluorescence and Terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) were used to detect apoptosis. Male Sprague-Dawley rats underwent carotid balloon injury followed by intraluminal infection with adenovirus expressing Smad3; or GFP control and arteries harvested at 3 days were subject to TUNEL staining and immunohistochemistry for cleaved Caspase-3. Akt activation was measured with phospho-Ser473 Akt (p-Akt). SIS3 was used to inhibit Smad3 activation. Results: in vitro, stimulation of SMCs withTGF-β reduced the number of TUNEL positive cells (Control = 35.0 vs TGF-β = 22.6%, p < 0.05) and diminished activation of Caspase-3 (31.4 vs 21.8%, p < 0.01) suggesting that TGF-β has an anti-apoptotic or protective effect on vascular SMCs. These protective effects were reversed by knockdown (siRNA) of TGF-β’s primary signaling protein, Smad3 (cleaved Caspase-3, p < 0.05). Moreover, overexpression of Smad3 in SMCs further enhanced TGF-β’s antiapototic effect (cleaved Caspase-3, p < 0.05). TGF-β phosphorylation of Akt was time-dependent and significantly enhanced when Smad3 was overexpressed. Both chemical inhibition and knockdown of Smad3 prevented phosphorylation of Akt (n = 3, p < 0.01). in injured carotid arteries, overexpression of Smad3 resulted in a dramatic inhibition of apoptosis but increased phosphorylation of Akt in the medial layer of the arterial wall: cleaved Caspase-3 (GFP = 35.2 vs Smad3 = 4.9, p < 0.01), TUNEL (32.1 vs 8.5, p < 0.01) and p-Akt (6.2 vs 22.4 p < 0.05). Conclusions: Our data show that TGF-β promotes SMC survival both in vitro and in vivo. This anti-apoptotic effect may well be one of the primary mechanisms through which TGF-β enhances intimal hyperplasia. Knockout and overexpression studies suggest that TGF-β’s inhibitory effect on apoptosis is mediated through the signaling protein Smad3. the correlation with Akt activation suggests that this may be mediated by the Akt-cell survival pathway.