Professor University of Wisconsin - Madison, United States
Introduction:: Calcific Aortic Valve Disease (CAVD) is a prevalent valve disease affecting a significant percentage of the population, with the incidence of aortic sclerosis as high as 25% in adults over the age of 65 [1,2]. Although end-stage characteristics of CAVD are well-documented, there still exists a large knowledge gap regarding the onset and progression of disease pathology. This gap has contributed to the lack of pharmaceutical development to tackle this disease with the only intervention being total valve replacement. Biological sex is an important determining factor in not only increased risk for CAVD but also divergence in presentation of the disease pathology. Male sex is a known indicator of higher risk, wherein males exhibit increased calcification as opposed to a prominently fibrotic presentation in females for the same extent of sclerosis. This sex-dependent divergence in disease presentation may be a factor of both inherent cellular sex and sex hormones. We propose that both cellular sex and estrogen (known for its cardioprotective properties) play an important role in mitigating a hallmark of CAVD: fibrosis. To interrogate how fibrosis may be impacted by these factors, we quantified the extent of nascent extracellular matrix (ECM) deposition by valvular cells along with studying specific ECM gene and protein expression in response to a pro-fibrotic pathological stimulus, TGF-b1.
Materials and Methods:: Valvular Interstitial Cells (VICs) were harvested and pooled from naive porcine aortic valves and cultured on TCPS in aVIC media (DMEM media with 10% FBS, 1% penicillin/streptomycin (P/S)) to induce them into a pathological phenotype as activated VICs (aVICs). To evaluate the influence of estrogen (E2), cells were cultured with 5 nM E2 throughout the experiment before being treated with valve-relevant pathological stimulus of 5 ng/mL TGF-b1 over 48h. To ensure there is no interference by E2 in serum, cells were cultured in 10% charcoal stripped (CS) FBS media for 48h prior to being seeded for the experiment at 50,000 cells/cm2. Nascent ECM deposition was visualizsed by culturing cells in nascent ECM media supplemented with and 0.1 mM azidohomoalanine (AHA) and incubated with DBCO-488 as previously described[3]. An Axiovert 200M fluorescent microscope was used to image the cells at 20X magnification. Image J software was used to measure Area fraction of nascent ECM (green) normalizsed against area fraction of nuclei (blue) to quantify amount of nascent ECM deposition. Similarly, collagen I and fibronectin were examined by fluorescently staining for the respective protein and quantified using the technique described to determine protein expression followed by qPCR analysis to understand differences between protein and gene expression. Cell proliferation was also measured using a Click-It EdU Alexa Fluor 488 imaging kit as percentage cells proliferating.
Results, Conclusions, and Discussions:: Photomicrographs of nascent ECM illustrate increased ECM production by cells treated with TGF-b1 across all conditions (Fig 1A). However, this TGF-b1-induced increase in ECM production was noticeably attenuated in both male and female VICs that had received E2, which was confirmed by image intensity quantification (Fig 1B). Sexual dimorphic responses were also observed, as male VICs produced ~2-4-fold more ECM than each of the corresponding female VIC conditions. The highest production of nascent ECM was exhibited by male control aVICs treated with TGF-b1. Other ECM proteins associated with fibrosis, such as fibronectin and collagen I, were also measured, with results indicating that EDA-fibronectin may be downregulated in female VICs. This was supported by qPCR data wherein fibronectin gene expression was 4-fold higher in males. Baseline cell proliferation data showed a trend of decreased cell proliferation with TGF-b1 treatment, particularly in Female E2 cells. Additionally, we also observed higher proliferation in males which may also contribute to the differences in ECM production and fibrosis.
The data suggest that both cellular sex and estrogen have an impact on nascent ECM production with estrogen especially mitigating the effect of the pro-fibrotic stimulus TGF-b1 as there was no significant difference between control and TGF-b1 treated female cells pre-conditioned with E2. Current data establish that there are sex-specific differences in ECM production when conditions simulate fibrosis in valvular cells. However, future studies will be directed towards understanding ECM remodeling involved in fibrosis and the underlying molecular mechanisms driving disease pathology as influenced by both sex and estrogen. This is especially important as the effects of estrogen on valvular fibrosis have not been examined in previous literature.
Acknowledgements (Optional): : This work was supported by NIH R01 HL141181.
References (Optional): : [1] C.M. Otto, B.K. Lind, D.W. Kitzman, B.J. Gersh, D.S. Siscovick, Association of Aortic-Valve Sclerosis with Cardiovascular Mortality and Morbidity in the Elderly, Surv. Anesthesiol. 44 (2000) 23. https://doi.org/10.1097/00132586-200002000-00022.
[2] K.D. O’Brien, Pathogenesis of calcific aortic valve disease: A disease process comes of age (and a good deal more), Arterioscler. Thromb. Vasc. Biol. 26 (2006) 1721–1728. https://doi.org/10.1161/01.ATV.0000227513.13697.ac.
[3] C. Loebel, R.L. Mauck, J.A. Burdick, Local nascent protein deposition and remodelling guide mesenchymal stromal cell mechanosensing and fate in three-dimensional hydrogels, Nat. Mater. 18 (2019) 883–891. https://doi.org/10.1038/s41563-019-0307-6.