PDF(Pubmed)
Abstract：
BACKGROUND: The contractile phenotype of vascular smooth muscle cells (VSMCs) results in good diastolic and contractile capacities, and its altered function is the main pathophysiological basis for diseases such as hypertension. VSMCs exist as a synthetic phenotype in vitro, making it challenging to maintain a contractile phenotype for research. It is widely recognized that the common medium in vitro is significantly less crowded than in the in vivo environment. Additionally, VSMCs have a heightened sense for detecting changes in medium crowding. However, it is unclear whether macromolecular crowding (MMC) helps maintain the VSMCs contractile phenotype.
OBJECTIVE: This study aimed to explore the phenotypic, behavioral and gene expression changes of VSMCs after increasing the crowding degree by adding carrageenan (CR).
METHODS: The degree of medium crowding was examined by a dynamic light scattering assay; VSMCs survival and activity were examined by calcein/PI cell activity and toxicity and CCK-8 assays; VSMCs phenotypes and migration were examined by WB and wound healing assays; and gene expression was examined by transcriptomic analysis and RT-qPCR.
RESULTS: Notably, 225 μg/mL CR significantly increased the crowding degree of the medium and did not affect cell survival. Simultaneously, CR significantly promoted the contraction phenotypic marker expression in VSMCs, shortened cell length, decreased cell proliferation, and inhibited cell migration. CR significantly altered gene expression in VSMCs. Specifically, 856 genes were upregulated and 1207 genes were downregulated. These alterations primarily affect the cellular ion channel transport, microtubule movement, respiratory metabolism, amino acid transport, and extracellular matrix synthesis. The upregulated genes were primarily involved in the cytoskeleton and contraction processes of VSMCs, whereas the downregulated genes were mainly involved in extracellular matrix synthesis.
CONCLUSIONS: The in vitro study showed that VSMCs can maintain the contractile phenotype by sensing changes in the crowding of the culture environment, which can be maintained by adding CR.
OBJECTIVE: This study aimed to explore the phenotypic, behavioral and gene expression changes of VSMCs after increasing the crowding degree by adding carrageenan (CR).
METHODS: The degree of medium crowding was examined by a dynamic light scattering assay; VSMCs survival and activity were examined by calcein/PI cell activity and toxicity and CCK-8 assays; VSMCs phenotypes and migration were examined by WB and wound healing assays; and gene expression was examined by transcriptomic analysis and RT-qPCR.
RESULTS: Notably, 225 μg/mL CR significantly increased the crowding degree of the medium and did not affect cell survival. Simultaneously, CR significantly promoted the contraction phenotypic marker expression in VSMCs, shortened cell length, decreased cell proliferation, and inhibited cell migration. CR significantly altered gene expression in VSMCs. Specifically, 856 genes were upregulated and 1207 genes were downregulated. These alterations primarily affect the cellular ion channel transport, microtubule movement, respiratory metabolism, amino acid transport, and extracellular matrix synthesis. The upregulated genes were primarily involved in the cytoskeleton and contraction processes of VSMCs, whereas the downregulated genes were mainly involved in extracellular matrix synthesis.
CONCLUSIONS: The in vitro study showed that VSMCs can maintain the contractile phenotype by sensing changes in the crowding of the culture environment, which can be maintained by adding CR.
摘要：
背景：血管平滑肌细胞（VSMC）的收缩表型导致良好的舒张和收缩能力，其功能改变是高血压等疾病的主要病理生理基础。VSMC在体外作为合成表型存在，使其具有挑战性，以保持收缩表型的研究。广泛认识到，体外普通培养基比体内环境明显不拥挤。此外,VSMC对检测中等拥挤的变化具有增强的感觉。然而,目前尚不清楚大分子拥挤(MMC)是否有助于维持VSMC收缩表型.
目的：本研究旨在探讨表型，添加角叉菜胶（CR）增加拥挤度后，VSMCs的行为和基因表达变化。
方法：通过动态光散射测定法检查培养基拥挤程度；通过钙黄绿素/PI细胞活性和毒性以及CCK-8测定法检查VSMC的存活和活性；通过WB和伤口愈合测定法检查VSMC的表型和迁移；并通过转录组学分析和RT-qPCR检查基因表达。
结果：值得注意的是，225μg/mLCR显著增加培养基的拥挤程度，不影响细胞存活。同时,CR显著促进VSMCs中收缩表型标记的表达,缩短细胞长度，细胞增殖减少，并抑制细胞迁移。CR显著改变VSMC中的基因表达。具体来说,856个基因上调，1207个基因下调。这些改变主要影响细胞离子通道的运输，微管运动，呼吸代谢,氨基酸运输，和细胞外基质合成。上调的基因主要参与VSMC的细胞骨架和收缩过程，而下调的基因主要参与细胞外基质的合成。
结论：体外研究表明，VSMCs可以通过感知培养环境拥挤的变化来维持收缩表型，可以通过添加CR来维护。
目的：本研究旨在探讨表型，添加角叉菜胶（CR）增加拥挤度后，VSMCs的行为和基因表达变化。
方法：通过动态光散射测定法检查培养基拥挤程度；通过钙黄绿素/PI细胞活性和毒性以及CCK-8测定法检查VSMC的存活和活性；通过WB和伤口愈合测定法检查VSMC的表型和迁移；并通过转录组学分析和RT-qPCR检查基因表达。
结果：值得注意的是，225μg/mLCR显著增加培养基的拥挤程度，不影响细胞存活。同时,CR显著促进VSMCs中收缩表型标记的表达,缩短细胞长度，细胞增殖减少，并抑制细胞迁移。CR显著改变VSMC中的基因表达。具体来说,856个基因上调，1207个基因下调。这些改变主要影响细胞离子通道的运输，微管运动，呼吸代谢,氨基酸运输，和细胞外基质合成。上调的基因主要参与VSMC的细胞骨架和收缩过程，而下调的基因主要参与细胞外基质的合成。
结论：体外研究表明，VSMCs可以通过感知培养环境拥挤的变化来维持收缩表型，可以通过添加CR来维护。
