Abstract
Background Vascular smooth muscle cell phenotypic change and consequential intimal hyperplasia (IH) cause arterial stenosis and posttreatment restenosis. Smad3 is a master transcription factor, yet its underlying functional mechanisms in this disease context are not well defined. Methods and Results In cultured smooth muscle cells, Smad3 silencing and overexpression respectively reduced and increased the mRNA and protein of NRP2 (neuropilin 2), a recently reported pro-IH signaling factor. Smad3 silencing attenuated pro-IH smooth muscle cell phenotypes including proliferation, migration, and dedifferentiation (reduced smooth muscle α-actin). While increased Smad3 enhanced these phenotypes, NRP2 silencing abolished this enhancement. Interestingly, the 5' untranslated region but not the promoter of NRP2 was indispensable for Smad3-enhanced transcriptional activity (luciferase assay); both chromatin immunoprecipitation and electrophoretic mobility shift assay showed predominant Smad3 binding in the +51 to +78 bp region of NRP2's 5' untranslated region. In vivo, Smad3 haploinsufficiency reduced NRP2 (immunostaining) and IH (by 47%) in wire-injured mouse femoral arteries. Conclusions Smad3 controls NRP2 expression by occupying its 5' untranslated region in promoting smooth muscle cell phenotypic change in vitro. This and in vivo results shed new light on the long-debated role of Smad3 in IH.