Mechanical Stretch Can Modulate Calcifying Potential of Caveolin-1-dependent Extracellular Vesicles from Vascular Smooth Muscle Cells

Introduction:: Hypertension-induced mechanical stress is a known risk factor for vascular remodeling, including vascular calcification, the most significant predictor of cardiovascular morbidity. Caveolin-1 (CAV1), an integral structural component of plasma membrane invaginations, is a mechanosensitive protein that is required for the formation of calcifying extracellular vesicles (EVs) from vascular smooth muscle cells (VSMCs). Calcifying EVs provide the necessary components for mineral formation in vascular calcification. Recent studies have suggested that different stretch profiles can alter VSMC phenotype; however, the role of mechanics in CAV1-induced EV formation from VSMCs has not been reported.


Materials and Methods:: Mouse VSMCs were exposed to three different mechanical stretch profiles, uniaxial (Uni), variable equibiaxial (VS) and monotonic equibiaxial (MS) stretch (10% mean amplitude, 0.5Hz, 72h). Non-stretched (NS) VSMCs were used as baseline for comparisons. Following mechanical stimulation, CAV1 in VSMCs and EVs that were collected from culture media were analyzed via western blotting. Phalloidin staining was performed to assess changes in actin filament orientation within SMCs. Mineralization potential of EVs was measured by incubating EVs in phosphate solution and measuring mineral-induced light scatter at 340nm. Annexin V, a calcium-binding protein involved in EV mineral formation, was also assessed.


Results, Conclusions, and Discussions:: Immunofluorescence showed that stress fibers appear oriented in a uniform direction in VSMCs that were exposed to 10% mechanical stretch compared to non-stretched cells (Fig 1A). Our western blot results showed no significant differences in CAV1 content within the cells (Fig 1B). However, CAV1 content in the released EVs from VSMCs exposed to MS was significantly increased by 5.3-fold, 2.5-fold and 2.1-fold compared to NS, Uni and VS, respectively (Fig 1C). The mineralization assay revealed a significant increase of 20.9±1%, 12.4 ±1% and 18.3±1% in calcifying potential of released EVs from VSMCs exposed to MS, compared to NS, Uni and VS VSMCs, respectively (Fig 1D). Annexin V content in EVs isolated from VSMCs exposed to MS was 10.2-fold, 2.5-fold and 2.7-fold higher compared to NS, Uni and VS, respectively (Fig 1E). These data increased liberation of CAV1-positive pro-calcific EVs from VSMCs exposed to the monotonic equiaxial stretch compared to other stretch profiles. Future studies will seek to determine the mechanotransductive mechanisms responsible for differences in EV release and the implications for the role of mechanics in pathological vascular remodeling.

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