Elucidation of Long non-coding RNA in epigenetic regulation in aortic disease and their potential therapeutic role

Abstract: Cardiovascular diseases (CVDs) remain the leading cause of mortality worldwide, with aortic valve stenosis (AVS) being a significant contributor, particularly in elderly populations. Calcific aortic valve disease (CAVD), the primary cause of AVS, involves active pathophysiological processes such as inflammation, fibrosis, and calcification within the valve. Recent studies have indicated that long non-coding RNA may be involved in pathogenesis of AVS. However, a systemic analysis is required to understand causal relationship of lncRNA in AVS pathology and examine potential therapeutic benefits. Here in, we aim to investigate the role of the long non-coding RNAs MALAT-1 and TUG-1 in AVS progression and explore their potential role as therapeutic targets.

Methods and Results: Our recent deep sequencing from human aortic valve tissue explants revealed that MALAT1 and TUG1 is dysregulated in patients compare to control. To illustrate respective causal role in valve cells, valvular endothelial cells (VECs), and valve interstitial cells (VICs) were transfected with siRNAs targeting MALAT-1 and TUG-1 to assess impacts on cellular viability, migration, angiogenesis, and pro-calcific markers. Preliminary data show that siRNA-mediated knockdowns of MALAT-1 and TUG-1 in valve cells dysregulated lncRNA significantly (P<0.05). However, loss of function experiments indicates lncRNAs do not affect functional properties evaluated by scratch-wound healing and network formation assays. In addition, cell viability and cell toxicity were evaluated post-knockdown by reduction of MTT and LDH (lactate dehydrogenase) release upon induction of oxidative stress with H₂O₂ (100uM). These results show a significant increase in cell viability and a significant reduction in LDH release in ECs (P<0.05) suggesting that lncRNAs may be involved cell viability and survival. While analyses of pro-inflammatory genes (IL-1β, IL-6, NPLR3, ICAM-1, and CASP-1) revealed that these lncRNAs may interplay with immunogenic activity in these cell-types.

Perspectives and Future directions: Our preliminary data hinting towards an involvement of these lncRNAs on inflammatory and calcification pathways-associated with AVS, providing insight into their therapeutic potential. To translate our in vitro data, we will investigate the role of MALAT-1 and TUG-1 in modulating cellular crosstalk and communication between VICs and VECs using in vitro co-culture experiments by using patient-derived cells. In addition, we will employ an experimental murine model of AVS with LDLR^-/- mice which will provide us more knowledge on these lncRNA regulation in AVS pathologies and unveil potential therapeutic potential in future.