Carrageenan maintains the contractile phenotype of vascular smooth muscle cells by increasing Macromolecular crowding in vitro

Abstract

Background The contractile phenotype of vascular smooth muscle cells (VSMCs) has a good diastolic and contractile capacity, and their altered function is the main pathophysiological basis for diseases such as hypertension. VSMCs always exist in a synthetic phenotype in vitro, making it challenging to maintain the contractile phenotype for research. It is widely recognized that common medium in vitro is significantly less crowded than the in vivo environment. Additionally, VSMCs have a heightened sense of touch to detect medium crowding changes. However, it is unclear whether macromolecular crowding (MMC) can help maintain the contractile phenotype of VSMCs. Purpose To study and obtain the phenotypic, behavioral and gene expression changes of VSMCs after increasing the crowding degree by adding carrageenan (CR). Methods The medium crowding degree was examined by the dynamic light scattering assay;VSMCs survival and activity were examined by the Calcein/PI cell activity and toxicity assay and CCK-8 assays༛VSMCs phenotype and migration were examined by WB and wound healing assay༛Gene expression was examined by transcriptomic analysis and RT-qPCR. Results 225 µg/mL CR significantly increased the crowding degree of the medium and did not affect cell survival. Simultaneously, CR significantly promoted contraction phenotypic markers expression in VSMCs, cell length was shortened, cell proliferation was decreased, and cell migration was inhibited. CR significantly altered gene expression in VSMCs. Specifically, 856 genes were up-regulated and 1207 genes were down-regulated. These alterations primarily affected cellular ion channel transport, microtubule movement, cellular respiratory metabolism, amino acid transport, and extracellular matrix synthesis. The up-regulated genes were primarily involved in the cytoskeleton and the contraction process of VSMCs, while the down-regulated genes were mainly involved in extracellular matrix synthesis. Conclusion The study showed that VSMCs in vitro can maintain the contractile phenotype by sensing changes in the culture environment's crowding, which can be maintained by adding a concentration of CR.