平滑肌與內皮細胞之增生在血管疾病的病程中扮演著重要的角色。平滑肌細胞增生可以被細胞外微環境所產生之物理與化學變化所驅動。這些變化包括了接觸面基質的組成、細胞激素及生長因子的刺激，而這些刺激所造成之平滑肌細胞增生會惡化成動脈硬化之主要特徵，血管壁內膜增生。當動脈壁因血管疾病而產生硬化時，內皮細胞也會因應此細胞外機械力的變化而促其產生增生之現象，伴隨著發炎反應及加劇血管內皮功能障礙。本篇論文將探討平滑肌與內皮細胞遭受到不同胞外刺激時所引起之細胞增生分子機制。
為了要釐清物理性單層型對照於纖維型膠原蛋白與化學性PDGF-BB/IL-1對照於溶劑組刺激對調控平滑肌細胞增生之分子機制，實驗為將平滑肌細胞培養於單層型或纖維型之第一型膠原蛋白上；同時，也將培養於纖維型之膠原蛋白上之平滑肌細胞同時給予PDGF-BB/IL-1之刺激。結果發現這些物理性及化學性的刺激能引發相同之細胞週期訊息，其中包括了增加CDK-4/6與cyclins-A/D1之蛋白表現，以及增加Rb之磷酸化，使E2F2/3得以與Rb分離。這個細胞週期可以被PI3K所引起之Akt與p38 MAPK，在上游進行正負向的調控。而纖維型膠原蛋白能降解平滑肌細胞之p66Shc，此蛋白上之serine-36磷酸化能夠調控單層型膠原蛋白及PDGF-BB/IL-1之刺激所造成之細胞周期及增生。單層型膠原蛋白能透過1 integrin，而PDGF-BB/IL-1之刺激則是透過PDGF受體型，都能造成p66Shc表現增加，進而驅動下游平滑肌之增生訊息。這些結果說明了物理性及化學性之刺激都能夠藉由p66Shc，進而活化下游之PI3K，而其所引起之Akt與p38 MAPK都能夠正負調控平滑肌細胞之細胞週期及增生。
為了要了解胞外基質之機械特性，對內皮細胞增生之分子機制之影響，實驗以高硬度HSG，21.5 kPa與低硬度LSG，1.72 kPa之水凝膠做為內皮細胞貼附之細胞外基質，用以研究內皮細胞之增生機制。研究結果發現，相較於低硬度膠，內皮細胞生長於高硬度膠上表現出高生長率、明顯之stress fibers與較高之RhoA活性。而抑制RhoA活性則能夠減弱高硬度膠所引起之stress fibers及細胞增生。Src及Vav2RhoA上游正向調控蛋白之磷酸化參與在高硬度膠所引起之內皮細胞增生。當培養於低硬度膠時，內皮細胞則會高度表現SEPT9，其為RhoA上游之負向調控蛋白。抑制SEPT9能增加內皮細胞在低硬度膠之RhoA活性、Src/Vav2之磷酸化及促進細胞增生。進一步的研究更發現，內皮細胞在低硬度的膠上會有較多不活化態的v3 integrin，其能夠造成SEPT9在低硬度膠上表現量增加、進而減弱Src/Vav2之磷酸化、抑制RhoA活性及其所主導之內皮細胞增生。這些結果說明了SEPT9/Src/Vav2/RhoA之訊息傳導路徑，對機械力所調控之內皮細胞增生扮演著重要的分子機制。
藉由此篇論文的研究，我們1定義出p66Shc，能夠同時調控著物理及化學性刺激所造成之平滑肌細胞之細胞週期及增生，2也定義了SEPT9所主導之新訊息路徑，能夠調控細胞外基質之機械特性所造成之內皮細胞增生。由於平滑肌與內皮細胞之增生在血管疾病的病程中扮演著重要的角色，此篇論文之研究結果可以幫助了解這些細胞在病生理過程中之分子層面、並提供未來醫療用途之藥物標靶之基礎。

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.

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