Trimethylamine Oxide (TMAO), a gut metabolite, activates quiescent valvular interstitial cells through the PERK pathway

Introduction: Over the past three decades, the global incidence of Calcific Aortic Valve Disease (CAVD) has tripled, a trend that has coincided with notable shifts in dietary patterns. Gut metabolites act as crucial mediators in this complex interplay between dietary habits and CAVD development. Recent studies suggest that trimethylamine oxide (TMAO), a gut metabolite derived from micronutrients like choline and carnitine, may be a biomarker for atherosclerosis. TMAO serum levels have also been correlated with clinical CAVD severity; however, it is not clear whether this metabolite is a causative factor in the progression of CAVD. Given the shared risk factors and molecular triggers between atherosclerosis and CAVD, we hypothesize that TMAO could contribute to CAVD by activating valve interstitial cells (VIC), the primary cell type found in the aortic valve, towards a diseased phenotype.

Materials and

Methods: Physiologically, VICs typically exhibit a quiescent phenotype. However, during CAVD development, these cells undergo a transition, adopting an activated fibroblast phenotype and expressing αSMA. Testing our hypothesis using traditional in vitro techniques is challenging because culture on tissue culture polystyrene leads to spontaneous VIC activation. To address this problem, we generated quiescent VICs (qVICs) for all experiments using a protocol previously developed by our lab. Briefly, we cultured VICs isolated from porcine aortic valves on collagen-coated plates and supplemented them with insulin and basic fibroblast growth factor and insulin in DMEM containing 2% FBS for 10 days. The cells were then seeded at a density of 10,000 cells per cm2 and treated with TMAO (25μM to 150μM) in media containing 2% serum. Controls included untreated qVICs, activated VICs (aVICS), and qVICs treated with transforming growth factor-β (TGF- β), a pro myofibroblastic growth factor. After 3 days of treatment, we analyzed the VIC phenotype to assess their activation towards a myofibroblastic phenotype. More specifically, we examined the expression of alpha-smooth muscle actin (αSMA), proliferation, apoptosis, and extracellular matrix deposition. To evaluate the pathway in which TMAO impacts the qVICs phenotype, qVICs were seeded at a density of 10,000 cells/cm2 and treated after 24 hours with GSK2656157, Protein Kinase R-like ER kinase (PERK) inhibitor at a concentration of 1μM for 1 hour. Following this initial treatment, the cells were cultured in 150 µM TMAO. This feeding cycle was repeated every other day for a total duration of 3 days.

Results, Conclusions, and Discussions: TMAO treatment had no significant effect on proliferation or α-SMA expression in aVICs (i.e. VICs plated directly on plastic following standard culture practices in the field). In contrast, qVICs treated with TMAO exhibited increased expression of α-SMA compared to the control group (untreated qVICs), reaching levels similar to those of activated VICs and cells treated with TGF- β (Figure 1A-B). In conjunction with an increase in αSMA expression, higher rates of proliferation were noted in qVICs treated with TMAO (Figure 1C), another marker of VIC activation. Because CAVD has different presentations in males and females, we also examined the effect of TMAO on qVICs isolated from porcine hearts of both sexes. No sex-related differences were observed in the response to this metabolite.

We then studied the impact of TMAO treatment beyond VIC activation. . TMAO treatment significantly increased qVIC deposition of fibronectin (Figure 1D) and vascular endothelial growth factor after 5 days of treatment. Because both fibrosis and angiogenesis are hallmarks of CAVD progression, these results suggest an active role for TMAO in the pathogenesis of this disease.

Finally, we sought to identify the mechanism mediating the activation of qVICs in response to TMAO treatment. Based on the atherosclerosis literature, we explored the role of the PERK pathway. Inhibition of the PERK pathway successfully blocked the effects of TMAO with no increases in αSMA (Figure 1E), proliferation, or fibronectin (Figure 1F) observed after treatment with both TMAO and the inhibitor. PERK inhibition had no impact on TGF-β-induced activation. Thus, we successfully identified the pathway employed by this gut metabolite to initiate VIC activation.

These results demonstrate that TMAO, a gut microbiome-derived metabolite, promotes the activation of quiescent VICs towards a myofibroblastic phenotype in vitro, a key initial step in the development of CAVD. This effect was only observed on VICs pre-treated to achieve a quiescent phenotype, demonstrating the need for engineering approaches that preserve yield physiologically relevant cell phenotypes. Overall, these findings suggest that TMAO could play a role in early aortic valve disease, and point to the importance of considering host-microbe interactions in the progression of cardiovascular disease.

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