Laboratory of Cell Biology and Functional Anatomy, Department of Anatomy, Institute of Biomedical Sciences, University of São Paulo, Cidade Universitária, São Paulo, Brazil.
Mol Cell Biochem. 2010 Aug;341(1-2):279-89. doi: 10.1007/s11010-010-0459-9. Epub 2010 Apr 18.
Primary cultures of vascular smooth muscle cells (VSMCs) from rats offer a good model system to examine the molecular basis of mechanism of vascular contraction-relaxation. However, during pathological conditions such as atherosclerosis and hypertension, VSMCs characteristically exhibit phenotypic modulation, change from a quiescent contractile to a proliferative synthetic phenotype, which impairs this mechanism of vascular contraction-relaxation. Taking in account that Myosin light chain (MLC) and ERK1/2 directly participate in the process of vascular contraction, the aim of the current study was to analyze the involvement of MLC and ERK1/2 signaling during the process of VSMCs phenotypic modulation. Primary cultures of VSMCs from rat thoracic aortas were isolated and submitted to different number of passages or to freezing condition. Semi-quantitative RT-PCR was used to evaluate the mRNA levels of VSMCs differentiation markers, and western blot assays were used to determine the MLC and ERK1/2 phosphorylation levels during VSMCs phenotypic modulation. Also, immunocytochemical experiments were performed to evaluate morphological alterations occurred during the phenotypic modulation. Elevated number of passages (up to 4) as well as the freezing/thawing process induced a significant phenotypic modulation in VSMCs, which was accompanied by diminished MLC and ERK1/2 phosphorylation levels. Phosphorylation of MLC was suppressed completely by the treatment with a synthetic inhibitor of MEK-1, a direct upstream of ERK1/2, PD98059. These findings provide that ERK1/2-promoted MLC phosphorylation is impaired during VSMCs phenotypic modulation, suggesting that ERK1/2 signaling pathway may represent a potential target for understanding the pathogenesis of several vascular disease processes frequently associated to this condition.
大鼠血管平滑肌细胞（VSMCs）的原代培养为研究血管收缩-舒张机制的分子基础提供了良好的模型系统。然而，在动脉粥样硬化和高血压等病理条件下，VSMCs表现出典型的表型调节，从静止的收缩型转变为增殖的合成型，这会损害血管收缩-舒张机制。考虑到肌球蛋白轻链（MLC）和 ERK1/2 直接参与血管收缩过程，本研究旨在分析 MLC 和 ERK1/2 信号通路在 VSMCs 表型调节过程中的作用。从大鼠胸主动脉中分离并培养 VSMCs，进行不同数量的传代或冷冻处理。半定量 RT-PCR 用于评估 VSMCs 分化标志物的 mRNA 水平，Western blot 检测用于评估 VSMCs 表型调节过程中 MLC 和 ERK1/2 的磷酸化水平。同时，进行免疫细胞化学实验以评估表型调节过程中发生的形态学改变。传代次数的增加（高达 4 次）以及冷冻/解冻过程诱导了 VSMCs 的显著表型调节，伴随着 MLC 和 ERK1/2 磷酸化水平的降低。通过使用 MEK-1 的合成抑制剂 PD98059 处理，完全抑制了 MLC 的磷酸化。这些发现表明，在 VSMCs 表型调节过程中，ERK1/2 促进的 MLC 磷酸化受损，提示 ERK1/2 信号通路可能是理解与该情况相关的几种血管疾病发病机制的潜在靶点。
