TGF-β1-directed Smad3 stimulates proliferation and alters morphology of rat primary vascular smooth muscle cells

Abstract

With cardiovascular disease (CVD) being the leading cause of morbidity and death in the United States and worldwide (2, 28), studying the mechanisms of these pathologies is imperative. Recent studies have shown that a correlation exists between the activation of synthetic and growth-promoting transforming growth factor-β1 (TGF-β1) and the pathogenesis of CVD (13). Cell-to-cell adhesion through components of the extracellular matrix (ECM) is required for normal growth conditions; however, these adhesive interactions have also been linked to CVD pathogenesis. TGF-β1 is thought to synthesize ECM elements through a Smad3-dependent pathway. Considering the correlation between TGF-β1 and CVD, studying its mechanistic effects on cell proliferation and migration could prove beneficial in combatting CVD pathologies. Past studies involving TGF-β1 have generally focused on its intracellular Smad3 signals, and results have shown conflicting effects of Smad3 and even it switching between pro-growth and anti-growth phenotypes depending on concentration and cell density (9, 21). In the current study in order to study the impact of Smad3 on CVD pathogenesis, rat primary vascular smooth muscle cells (VSMCs) from the thoracic aorta were harvested, grown and maintained, and three treatment groups were used: naïve (no virus) serving as control, and adenovirus infection with either green fluorescent protein (AdGFP) or Smad3 (AdSmad3) to overexpress Smad3. Proliferation was measured using flow cytometry and automatic cell proliferation and viability analyses to assess differences in cell number (proliferation), cell viability, and cell morphology between groups. Crystal violet attachment was performed as a function of studying differences in the ECM composition between the groups. Through these efforts in studying TGF-β1-directed Smad3 and its potential role in mediating VSMC growth, we hope these results will facilitate our understanding of the mechanisms associated with the pathogenesis of CVD and ultimately will discover a new potential therapeutic target in TGF-β1-directed Smad3.