Abstract 5473: TGFβ Induction of Vascular Smooth Muscle Differentiation Markers is Mediated by Nox4 and p38 Mitogen-activated Protein Kinase
Vascular smooth muscle cells (VSMCs) undergo phenotypic transformation in response to environmental cues. In response to injury, VSMC switch from a “contractile” to “synthetic” phenotype, which is implicated in restenosis and arteriosclerosis. Thus, accelerating VSMC redifferentiation may be one way to limit lesion development. TGFβ induces differentiation in VSMC; however, the mechanism is incompletely understood. Previous work has implicated Nox4 in differentiation. In this study, we hypothesized that TGFβ induces differentiation via Nox4 and p38 mitogen-activated protein kinase (p38) pathways. In VSMCs, treatment with TGFβ (2 ng/mL) for 24 hours increases reactive oxygen species (ROS) production (146±15% vs control) and Nox4 protein expression (150±12% vs control), both of which are reduced by transfection with small interfering RNA against Nox4 (siNox4). At 48 hours, TGFβ maximally induces the expression of the differentiation markers smooth muscle alpha actin (SMA) and calponin (CNN) and increased incorporation of SMA into stress fibers. This induction is blunted by preincubation with the flavin oxidase inhibitor diphenylene iodonium (DPI) (90±8% inhibition) or by siNox4 (100% inhibition). To investigate the role of p38, we first demonstrate p38 is activated by TGFβ at 60 minutes (370±70%) and it is not ROS dependent, because it is not blocked by preincubation with DPI. However, p38 remains activated for up to 24 hours, as measured by continued phosphorylation of its substrates, heat shock protein 27 (650±48%) and the obligatory differentiation transcription factor serum response factor (280±50%). This late activity of p38 is prevented by DPI (68±10% and 57±5% inhibition of HSP27 and SRF). Moreover, the inhibition of p38MAPK partially reverses the upregulation of SMA (60±20% inhibition) and CNN (67%±23 inhibition) by TGFβ, and acts in cooperation with Rho kinase (87±10% and 97±1% inhibition of SMA and CNN). Thus, we conclude that Nox4 and p38 are critical regulators of TGFâ-induced differentiation in VSMC, and that Nox4 maintains the activity of p38. These data raise the possibility that Nox4 and p38 are potential targets in the treatment of cardiovascular diseases in which VSMC dedifferentiation plays a pivotal role.