Poster T18 - Impact of Hyperglycemia on Vascular Smooth Muscle Cells and Implications for Type 2 Diabetes -Induced Cardiovascular Disease Treatment

Introduction: Type 2 diabetes mellitus (T2DM) is a major public health concern with significant cardiovascular complications (CVD). Despite extensive epidemiological data, the molecular mechanisms relating hyperglycemia to CVD remain incompletely understood. Since this has huge implications in tissue engineering and regenerative medicine approaches, we here investigate the impact of chronic hyperglycemia on SMC phenotype and function using human aortic smooth muscle cells (HASMCs) cultured under varying glucose conditions in vitro, mimicking normal (5 mM/L), pre-diabetic (10 mM/L), and diabetic (20 mM/L) conditions, respectively. Patient-derived T2DM-SMCs were included for comparative analysis.

Materials and

Methods: Primary cryopreserved HASMCs under varying glucose conditions, and T2DM-HASMCs under normal glucose conditions, were cultured for up to 21 days in DMEM containing 10% FBS and 1% Penn/Step. Changes in cell phenotype (morphology, area, perimeter), proliferation, and cytoskeletal organization were determined using staining and imaging techniques. An MFP-3D-BIO atomic force microscope (AFM) was used to characterize the biomechanical changes in cells under these conditions. Finally, the outcomes from metabolomic, proteomic, and RNA-Seq analyses of these cultures were correlated to identify the changes in gene and protein expressions, as well as to identify the inflammatory pathways involved during exposure to hyperglycemia.

Results, Conclusions, and Discussions: Result and Discussion 

Results showed distinct morphological changes with significant increases in cell area, perimeter and F-actin expression in SMCs treated at higher glucose levels. Cell shape index was 0.23 ± 0.01 in SMCs exposed to 10 and 20 mM/L glucose, and 0.48 ± 0.03 for T2DM cells and normal SMCs (5 mM/L) (p < 0.001). AFM analysis showed significant reduction in the Young’s modulus, membrane tether forces, membrane tension, and surface adhesive forces in SMCs with increasing glucose levels. In all these cases, T2DM cells exhibited levels noted in 20 mM/L glucose conditioned cells. A 5 to 6 -fold increase in cell proliferation was observed in SMCs treated with 20 mM/L glucose and in T2DM-SMCs, at day 7, compared to their original seeding density. T2DM-SMCs exhibited elevated levels of pro-inflammatory markers such as IL-6, IL-8, and MCP-1 compared to glucose-treated SMCs. Conversely, growth factors like FGF-2 and TGF-β were higher in SMCs exposed to moderate glucose but lower in T2DM-SMCs. Pathway enrichment analysis showed a significant increase in the expression of inflammatory cytokine-associated pathways, especially involving IL-10, IL-4 and IL-13 signaling in genes that are upregulated at higher glucose levels. Differentially regulated gene (DGE) analysis showed that, compared to SMCs cultured with normal glucose, 513 genes were upregulated, and 590 genes were downregulated in T2DM-SMCs; however, fewer genes (16 – 23) were differentially expressed in SMCs receiving 10 or 20 mM/L glucose. We identified the altered genes involved in ECM organization, elastic fiber synthesis and formation, laminin interactions, and ECM proteoglycans, highlighting the role of hyperglycemia in vascular remodeling and CVD progression.



Conclusions 

Our results suggest that chronic hyperglycemia in SMCs leads to significant morphological, biomechanical, and functional alterations, potentially contributing to the pathogenesis of T2DM-associated CVD. The observed differences in gene expression patterns between in vitro hyperglycemic models and T2DM-SMCs highlight the complexity of T2DM pathophysiology and the need for further exploration of underlying mechanisms.  Nevertheless, these results have significant implications in tissue engineering and regenerative medicine strategies for cardiovascular disease treatment.

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