(D-141) Uncovering the Protective Axis of the Renin-Angiotensin System in a 3D Valve-On-Chip Model

Uncovering the Protective Axis of the Renin-Angiotensin System in a 3D Valve-On-Chip Model

Gustavo Vaca-Diez, Ishita Tandon, Kartik Balachandran

Department of Biomedical Engineering, University of Arkansas, Fayetteville, Arkansas

Introduction: The Renin-angiotensin system (RAS) is a fundamental component in cardiovascular physiology. Current research is now focused on the counter-regulatory axis of the RAS, facilitated by its key player: the angiotensin-converting enzyme 2 (ACE2). Current understanding suggests that its products possess vasorelaxant properties and play a protective role that counterbalances the effects of the classical RAS axis. RAS has been implicated in the development of fibrosis in early aortic valve pathology. A better understanding of the counter-regulatory RAS in valve pathophysiology could lead to preventive treatments for calcific aortic valve disease (CAVD) and aortic stenosis (AS). The utilization of in vitro valve mimetic models can provide insights into the expression of ACE2 and its potential in counterbalancing effects on vasoconstriction in response to angiotensin II. The study consists of two objectives: 1. Assessing ACE2 expression in porcine valve interstitial cells (pVICs) using a simple 2D model and 2. Further assessing expression of ACE2 and its role in a 3D model containing extracellular matrix components.

Materials and Methods: pVICs were cultured in standard 2D model using quiescence inducing media (2% FBS, insulin, FGF2) and exposed to angiotensin II (Ang II) for 5 days. We evaluated the pathologic effects by measuring alpha smooth muscle actin (αSMA) using immunocytochemistry. We will evaluate ACE2 mRNA expression with RT-qPCR. For objective 2, we will construct a 3D culture that mimics the fibrosa and spongiosa layers of the aortic valve using collagen and glycosaminoglycans (GAGs) based hydrogels assembled in a bilayer with embedded pVICs (Figure 1D). These 3D constructs will be cultured in quiescent media (2% FBS, insulin, FGF2) as well. Exposure to Ang II for 5 days and measurements will be replicated as in 2D culture.

Results and Discussion: Our results have shown increased expression of ACE2 in the Ang II exposed group (Figure 1B). A similar trend is present in the expression of αSMA (Figure 1C). Previous studies have shown that activation of the Angiotensin II Type 1 Receptor (AT1R) by Ang II induces the expression of ACE2. However, excess Ang II also activates AT2R, which counteracts the effects of AT1R, hypothetically leading to the reduction of ACE2 expression. Ongoing studies will obtain levels of mRNA expression of ACE2 and investigate these counterbalancing effects in 3D valve models.

Conclusion: Our study aimed to investigate the impact of angiotensin II on ACE2 expression and profibrotic RAS processes in the aortic valve. Our work aims to lay a foundation in filling a significant gap in research on the counter-regulatory axis of the RAS in aortic valve pathophysiology, which may inform the development of new therapeutic strategies for valvular heart disease.