Professor University of Wisconsin - Madison, United States
Introduction:: With over 10 million aging individuals in the US affected, CAVD is the most common valvular disease worldwide [1]. A gap in the understating of mechanisms underlying disease pathology prevents the development of effective treatments, leaving total valve replacement as the only treatment option [3]. CAVD is a progressive condition defined by ongoing alterations to the valve extracellular matrix, which leads to valve sclerosis in the early stages and valve stenosis in the late stages. CAVD also exhibits sexual dimorphism, wherein the pathological features differ in males and females [2]. In order to study what aspects of ECM remodeling contribute to sex dimorphism in CAVD, we designed stage-specific, disease-inspired tissue-engineered valve models that mimic collagen enrichment as observed during disease progression. Specifically, we intended to decouple which aspect of collagen enrichment drives fibrosis in CAVD: total protein enrichment or increased fiber density. Understanding which of these factors leads to CAVD fibrosis will help us understand the underlying processes leading to the divergence in the sex-dependent presentation of CAVD.
Materials and Methods:: 3-D disease-inspired scaffolds were developed to mimic collagen enrichment as a hallmark of CAVD. All models were created using a GelMA (Gelatin Methacrylate) based hydrogel (4% w/v) with an interpenetrating network of collagen fibers [4]. Scaffolds mimicking early-stage disease contained 0.75 mg/mL (1X) collagen I (Col1), while late-stage disease scaffolds contained 1.5 mg/mL (2X) Col1. In order to distinguish whether VIC (Valvular Interstitial Cells) fibrotic responses result as a consequence of total protein enrichment or increased presence of fibers, a third condition was introduced. A new late-stage model was created by combining 0.75 mg/mL (1X) Col1 + 0.75 mg/mL (1X) GelMa to maintain a 2X concentration of total collagenous protein without altering the fiber density in the system compared to early-stage. Female and male VICS were cultured independently as a mixture of qVICs (quiescent valvular interstitial cells) and aVICs (activated valvular interstitial cells) at a 50% ratio for a total concentration of 4 x 10^6 cells/mL was embedded and cultured in these systems for a period of 6 days. Samples were then collected and assayed for expression of fibrotic markers of ECM remodeling.
Results, Conclusions, and Discussions:: When compared culture in early-stage disease models, preliminary results from female and male VICs revealed enhanced expression of FN, Col1A1, CHSY1, and ACTA2 fibrotic markers at the late-stage disease-inspired scaffold where both protein and fiber enrichment was present. Sex-dimorphic features were observed between females and males in the early-stage model, as indicated by increased expression in males versus females for TGFB-1, ACTA2, FN, and CHSY1 and, at the late-stage model for ACTA2, FN, and Col1A1 (Figure 1). No statistically significant differences were observed between the early-stage disease model and our modified late-stage model (increased protein enrichment but unchanged fiber content) in terms of stage transition or sexual dimorphism. These findings imply that collagen enrichment during the late stages of disease progression increases fibrosis in female and male VICs and that the fibrillar structure of collagen is key in driving this process. The development and application of stage- and sex-specific engineered disease models provide a chance to unravel the reasons and consequences of sexual dimorphic behaviors in CAVD progression, thereby increasing our understanding of the disease and advancing research into improved treatments.
Acknowledgements (Optional): : This investigation was supported by NIH R01 HL141181 and the National Institutes of Health, under Ruth L. Kirschstein National Research Service Award T32 HL 007936 from the National Heart Lung and Blood Institute to the University of Wisconsin-Madison Cardiovascular Research Center.
References (Optional): :
Grande-Allen, K. J., (2023) Frontiers in cardiovascular medicine
