Cyclic strain promotes MCP-1 expression in Dahl Salt Sensitive (SS) rat vascular smooth muscle cells (VSMCs) through the TGF-β pathway

Abstract

Prior studies have suggested a role for TGF-β in the generation of hypertension in SS rats. We have shown that production of MCP-1 (CCL-2) and TGF-β1 increase in the vasculature of hypertensive SS rats. This study aims to explore novel mechanisms of hypertension development. We hypothesize that TGF-β1 regulated the production of MCP-1 by VSMCs in the setting of cyclic strain. Primary cultures of SS aortic VSMCs in early (<6) passages were grown in standard fashion in Dulbecco’s Modified Eagle’s Medium supplemented with 10% fetal bovine serum. Cultured cells were identified as smooth muscle cells by their characteristic morphology and positive immunostaining for α-smooth muscle actin. In the first study, VSMCs were treated with recombinant active TGF-β1 (1 ng/mL and 10 ng/mL) for 6 h, then harvested for MCP-1 mRNA quantification by SYBR green qPCR. Medium was also collected to determine MCP-1 levels using ELISA. TGF-β1 increased MCP-1 mRNA expression by 2.0- and 3.1-fold in VSMC incubated in medium containing 1 ng/mL and 10 ng/mL of TGF-β1, respectively. In a second series of studies, VSMCs were seeded onto 6-well BioFlex plates. Some cells were pre-treated with 10 μmol/mL of an ALK4/5 inhibitor. Cyclic strain, an in vitro model of hypertension, was performed using the FlexCell 6000 and a standardized regimen (8 h, 15% elongation @ 1 Hz). Cells and medium were then harvested for mRNA analyses and determination of MCP-1 levels. When compared with expression by VSMCs maintained under static condition, cyclic strain increased (p<0.05) MCP-1 mRNA expression 4.2-fold and TGF-β1 mRNA 1.93-fold. Cyclic strain also increased (p<0.01) MCP-1 protein in the medium (static condition vs cyclic strain, 112.6 ± 7.1 pg/mL vs 222.5 ± 6.5 pg/mL, n=6). Pre-treatment of VSMC with the ALK4/5 inhibitor reduced mRNA expression of MCP-1 mediated by cyclic strain (Cyclic strain vs Cyclic strain + AKL4/5 inhibitor, 4.5 ± 1.06 vs 2.88 ± 0.47, n=6, P < 0.01). Thus, cyclic strain promotes MCP-1 production by VSMCs through the TGF-β pathway. The data support the need for further study of this interaction as well as a potential role in arterial hypertension in this rodent model. Department of Veterans Affairs Basic Sciences R&D (BSRD) Service (1 I01 BX005640) National Institutes of Health (P30 DK079337). This is the full abstract presented at the American Physiology Summit 2024 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.