The effect of the polymerization state of type I collagen on the phenotype of vascular smooth muscle cells

• Main Authors: Bor-yow Lin, 林伯祐
• Other Authors: Meei-jyh Jiang
• Format: Others
• Language: en_US
• Published: 2008
• Online Access: http://ndltd.ncl.edu.tw/handle/75167555522909514217

碩士 === 國立成功大學 === 細胞生物及解剖學研究所 === 96 === Most cells in multicellular organisms are surrounded by complex structural molecules that make up the extracellular matrix (ECM). Collagens are major structural components of the extracellular matrix of the artery wall. During smooth muscle cells (SMCs) differentiation, immature SMCs develop from synthetic phenotype to contractile phenotype characterized by the expression of various differentiation markers such as smooth muscle ��-actin (SM-α-actin), SM22, calponin, h-caldesmon (h-CaD) and smooth muscle myosin heavy chain (SM-MHC). SMCs cultured on polymerized collagen retain their contractile phenotype and mimic many of the characteristics of medial SMCs in vivo whereas SMCs cultured on monomeric collagen are proliferative. This study examined the effect of type I collagen polymerization and stiffness on the behavior of cultured SMCs. Rat aortic SMCs (RASMCs) were cultured on monomeric collagen-coated (mC), polymeric collagen-coated (pC) or polymeric collagen gel (pG) with uncoated dish as control. The distribution of actin filament, microtubules, and SM-α-actin was examined. RASMCs appeared more extended on mC but smaller and with many filopodia on polymeric collagen. Actin filaments detected by phalloidin mainly appeared as stress fibers traversing the cell in RASMCs of mC and control. In contrast, actin filaments formed a meshwork in more than 80% of RASMCs cultured on polymeric collagen, exhibiting prominent structures analogous to focal adhesions. Similar results were observed in SM-α-actin distribution. No difference was detected in microtubule distribution among different conditions. To investigate the signaling pathways from type I collagen, FAK phosphorylation and expression and the distribution of focal adhesion-associated protein, vinculin, were examined. FAK was phosphorylated in RASMCs cultured on both monomeric and polymeric collagen, but the expression of FAK was decreased in RASMCs cultured on pG. Vinculin was co-localized with actin filament along stress fibers but not at actin filament meshwork. To examine the role of integrin in actin filament organization in RASMCs cultured on different forms of collagen, the actin filament organization was examined by phalloidin staining following functional blocking of β1, α1 or α2 integrin with specific antibodies. Using immunofluorescence, β1 integrin was detected at focal adhesions, exhibiting partial co-localization with actin stress fibers and actin meshwork. Functional blocking of β1, α1 or α2 integrin with specific antibody showed that β1 integrin and at least in part, α1 integrin were required for RASMCs spreading, filopodia and actin stress fiber formation in RASMCs cultured on different forms of collagen. The expression of VSMC differentiation markers was examined by Western blot. The expression of differentiation markers, SM-MHC and h-CaD, but not SM-α-actin, decreased in RASMCs cultured on polymeric collagen. These results suggest that the polymerization state of type I collagen substratum modulates cell morphology, phenotypic change and actin filament organization of VSMC in β1 integrin-dependent manner.