Poster BB3 - Effect of High Wall Shear Stress and Nitric Oxide Deficiency on Vascular Smooth Muscle Cell Phenotype

Introduction: Nitric Oxide (NO) produced by vascular endothelial cells (ECs) plays an important role in maintaining vascular homeostasis (Harrison et al., 2006) (Cai and Harrison, 2000). Production of NO is regulated by ECs in response to hemodynamic shear stress (wall shear stress; WSS), and endothelial dysfunction, particularly defects of NO production, accompanies a number of cardiovascular diseases (Harrison et al., 2006) (Cai and Harrison, 2000). Recently, extremely higher WSS (~20 Pa) has been pointed out as a factor in the formation and development of aortic aneurysm and dissection (Kimura et al., 2017) (Rooprai, et al. 2019); however, the detailed mechanism remains unclear. Phenotypic switching of vascular smooth muscle cells (SMCs) from contractile to synthetic states is involved in the pathogenesis of aortic diseases, and their phenotypic modulation can be affected by EC-derived NO (Harrison et al., 2006) (van de Pol et al., 2017). Here we investigated the role of EC-derived NO in the phenotypic switching of SMCs under higher WSS to understand the pathogenesis of aortic diseases.

Materials and

Methods: A centrifugally compressed cell–collagen combined construct (C6) (Hiroshima et al., 2022) (Ye et al., 2023) was constructed with primary human aortic SMCs. Type I collagen gel containing the SMCs was compressed by a 1,000 g centrifugal force with a plate centrifuge. The compressed gel (C6) was cultured for phenotype control in serum-free D-MEM / Ham’s F12 supplemented 1 % Insulin-Transferrin-Selenium-Ethanolamine Supplement for 7 days (Han et al., 2017). Human aortic ECs were subsequently seeded on the C6 and cocultured with phenotype-controlled SMCs for 1-2 days. The coculture model was then exposed to 20 Pa WSS for 24 h in the presence or absence of 1mM L-NAME, an inhibitor of endothelial nitric oxide synthase (eNOS). The exposure to 2 Pa WSS as a physiological condition was also performed for comparison. After the exposure to WSS, expression of contractile phenotype protein markers, αSMA and Calponin1, of SMCs in the C6 was assessed with Western blotting.

Results, Conclusions, and Discussions: In the absence of the eNOS inhibitor, increased Calponin1 expression in the SMCs was detected under the 20 Pa WSS compared to the 2 Pa WSS condition, whereas expression of αSMA for 20 Pa was similar to that for 2 Pa. On the other hand, under the L-NAME-treated condition, the expression of both αSMA and Calponin1 was decreased by approximately 30% by exposure to 20 Pa WSS condition compared to 2 Pa, indicating a phenotypic shift of SMCs to synthetic states.
SMCs in the healthy artery wall show contractile states expressing a specific set of contractile proteins, and the suppression of the contractile proteins is frequently associated with the progression of aortic diseases (van de Pol et al., 2017). Although it was anticipated that higher WSS would lead to decreased contractile protein expression in SMCs, our results suggest that EC-derived NO could maintain the contractile phenotypes of SMCs. Deficiency in NO due to abnormal expression of eNOS disrupts vascular homeostasis and relates to numerous vascular diseases. In addition, decreased expression of eNOS in aneurysm tissue, which is supposed to be arising from pathologically higher WSS, has been previously reported (Mohamed et al., 2012). Our results demonstrate that endothelial dysfunction may be involved in the phenotypic switching under higher WSS conditions.
In this study, we assessed the combined effect of higher WSS and EC-derived NO on the expression of the contractile SMC protein markers. Our results indicate that defects in NO production may induce phenotypic switching to synthetic SMCs under pathologically higher WSS conditions.

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