Abstract
Cell proliferation of smooth muscle cells (SMCs) and endothelial cells (ECs) play important roles in the pathologenesis of vascular diseases. SMC proliferation can be triggered by mechanical and chemical changes in the extracellular microenvironment. This includes surface composition, cytokines and growth factors, which contribute to the neointima formation, a prominent feature in athersclerosis. ECs also respond to arterial stiffening during the progression of vascular diseases and when proliferation increases, inflammation and cell dysfunction advance. Here, we study the molecular mechanisms in the regulation of SMC and EC proliferation. To elucidate the mechanisms by which physical (monomeric vs. fibrillar collagens) and chemical (platelet-derived growth factor (PDGF)-BB/interleukin (IL)-1 vs. vehicle controls) stimuli modulate the cell cycle and proliferation, SMCs were cultured on monomeric or fibrillar type I collagens. In parallel experiments, SMCs on fibrillar collagen were co-stimulated with PDGF-BB/IL-1. These physical and chemical factors induced common cell cycle signaling events, including upregulation of cyclin-dependent kinase-4/6 and cyclins A/D1, phosphorylation of retinoblastoma (Rb) and its dissociation with E2F2/3. The physical and chemical inductions of SMC cycle signaling and progression were oppositely regulated by phosphatidylinositol 3-kinase (PI3K)-mediated Akt and p38 mitogen-activated protein kinase (MAPK). Fibrillar collagen degraded p66Shc, whose induction and Ser36-phosphorylation regulated monomeric collagen- and PDGF-BB/IL-1-induced SMC cycle signaling and progression. These physical and chemical modulations in p66Shc were mediated by 1 integrin and PDGF receptor-, respectively. These results demonstrate that fibrillar collagen-regulated p66Shc converge the physical and chemical stimuli to modulate SMC cycle and proliferation through PI3K-mediated Akt and p38 MAPK, and they oppositely regulate common downstream cell cycle signaling cascades. To demonstrate the mechanism of extracellular matrix (ECM) mechanics on EC proliferation, hydrogels with high stiffness (HSG, 21.5 kPa) in comparison to those with low stiffness (LSG, 1.72 kPa) were used to study EC proliferation. ECs cultured on HSG showed a higher proliferative rate, more prominent stress fibers and higher RhoA activity when compared with ECs on LSG. Blocking RhoA attenuated stress fiber formation and proliferation of ECs on HSG but had little effect on ECs on LSG. Phosphorylations of Src and Vav2, positive RhoA upstream effectors, were involved in HSG-mediated RhoA activation and EC proliferation but exhibited nominal effects on ECs grown on LSG. Septin 9 (SEPT9), the negative upstream effector for RhoA, was significantly higher in ECs on LSG. SEPT9 inhibition increased RhoA activation, Src/Vav2 phosphorylation levels, and EC proliferation on LSG, but ECs on HSG showed only minor responses. Furthermore, ECs on LSG had an inactivation of αvβ3 integrin which caused an increase of SEPT9 expression that attenuated Src/Vav2 phosphorylation levels and inhibited RhoA-dependent EC proliferation. These results demonstrate that the SEPT9/Src/Vav2/RhoA pathway constitutes an important molecular mechanism for the mechanical regulation of EC proliferation. In summary, our results 1 identified p66Shc is the convergent molecule that responds to physical and chemical stimuli resulting in SMC cell cycle regulation and proliferation, and 2 generated novel insight into the SEPT9-mediated pathway in ECM mechanics-regulated EC proliferation. Since both SMC and EC proliferation play important roles in the progression of vascular diseases, our results may enhance the molecular understanding of the pathophysiological process and may provide foundations for molecular target therapeutic applications in the future.