Graduate Student University of Florida Gainesville, Florida, United States
Introduction:: Calcified Aortic Valve Disease (CAVD) is the most common among all valvular diseases affecting up to 13% of the population in the U.S. Recent studies have demonstrated that changes in diet impact the gut microbiome leading to the production of metabolites that can affect cardiovascular health. TMAO (Trimethylamine N oxide) is a gut microbiome-derived metabolite generated from micronutrients such as choline and carnitine. It has been proposed as a driver and circulating biomarker for atherosclerosis. Because atherosclerosis and CAVD share several risk factors and similar molecular triggers, we hypothesize that TMAO could also promote the onset of CAVD by stimulating valve interstitial cell (VIC) activation.
Materials and Methods:: We isolated VICs from female porcine aortic valves. Traditional culture on tissue culture polystyrene leads to spontaneous VIC activation. Because VICs in a healthy aortic valve exhibit a quiescent phenotype, we generated quiescent VICs (qVICs) for all experiments using a protocol previously developed by our lab. Briefly, we cultured the VICs on collagen-coated plates and supplemented them with insulin and BFGF in DMEM containing 2% FBS to induce quiescence for 10 days. The cells were then seeded at a density of 3200 per square centimeter and treated with different concentrations of TMAO in media containing 2% serum. Controls included untreated qVICs, activated VICs (aVICS), and qVICs treated with transforming growth factor-β (a promyofibroblastic growth factor). After 3 days of treatment, we analyzed VIC phenotype to assess their activation towards a myofibroblastic phenotype. More specifically, we examined the effects of TMAO on the expression of alpha smooth muscle actin (αSMA), proliferation, apoptosis, and extracellular matrix deposition.
Results, Conclusions, and Discussions:: Results and Discussion. qVICs treated with TMAO exhibited increased proliferation compared to the control group (untreated qVICs) reaching levels similar to those of activated VICs and cells treated with transforming growth factor (Figure 1A). Additionally, the expression of α-SMA increased in the qVICs treated with TMAO compared to control group (Figure 1B). Similarly, cells treated with TMAO exhibited a morphology similar to that of aVICs and the transforming growth factor-treated group. (Figure 1B). Finally, the apoptosis data shows no significant variation between experimental and control groups. Despite treatment with different concentrations of TMAO, there were no notable differences observed between doses for all assays. These results indicate the need to optimize the TMAO concentration for future experiments. Based on these results, we also expect to see differences in ECM deposition after TMAO treatment.
Conclusion. These results demonstrate that TMAO, a gut microbiome-derived metabolite, promotes the activation of quiescent VICs towards myofibroblastic phenotype in vitro. Because sexual dimorphism plays a critical role in CAVD, we plan to expand the project to study the effect of TMAO on qVICS of both sexes. Overall, these findings suggest that TMAO could play a role in the development and progression of aortic valve disease and point to the importance of considering host-microbe interactions in the progression of cardiovascular disease.
