Introduction: : Aortic Valve Stenosis (AVS) is a sexually dimorphic disease which affects ~20% of adults aged >65 years old and leads to heart failure if left untreated. Male patients with AVS develop increased calcification, whereas female patients experience severe valvular fibrosis prior to calcification. Valvular interstitial cells (VICs) are the most abundant and heterogenous resident fibroblast cell type and transdifferentiate to myofibroblasts and/or osteoblast-like cells which regulate sex-dependent fibro-calcification. However, the cellular mechanisms driving observed sex differences in valve remodeling remain poorly understood. As such, we seek to (1) understand how sex differences in VIC heterogeneity modulate disease progression, (2) identify VIC subtypes near sites of calcification, and (3) identify sex-specific VIC gene signatures that may serve as targets for pharmacologic intervention. Here, we combine single cell RNA sequencing and spatial transcriptomics from sex-separated healthy and diseased patients to probe VIC heterogeneity in diseased aortic valves. We have identified sex-specific gene expression in VICs overlapping with regions of calcification in male valves, while female VICs have increased expression of pro-fibrotic genes.
Materials and
Methods: : Healthy and fibro-calcific human valve tissues were obtained from male (N = 2 healthy, 7 diseased) and female (N=1 healthy, N = 5 diseased) patients and matched by age and disease severity. Alizarin red staining was used to quantify the degree of calcification in sex separated tissues. Valve tissues were bisected and analyzed in parallel with single cell RNA sequencing and spatial transcriptomics pipelines (10X Genomics) to spatially resolve gene expression near sites of calcification. Gene expression was normalized and integrated across patients using the Seurat pipeline. Fibroblast subtypes were identified using unsupervised clustering or Uniform Manifold Approximation and Projection. Differential expression analysis using Wilcoxon rank-sum was run on fibroblast subtypes to identify sex specific gene expression. To identify clustered genes within the tissue, we evaluated Moran’s I test statistic for each gene. Furthermore, gene overlap with calcium regions was evaluated using Fisher’s exact test to determine that observed overlap was significant and not due to random chance (permutation). RNA in situ fluorescence hybridization was used to further spatially resolve transcripts overlapping with calcium in valve tissue.
Results, Conclusions, and Discussions:: We demonstrate VIC heterogeneity and sex-specific fibro-calcification gene signatures that drive calcification in aortic valve disease. Histological analysis of 12 diseased aortic valve leaflets reveals males exhibit increased calcium surface area within male leaflets (Figure A). Our integrated analysis on 65,324 cells reveals fibroblasts are the largest cell population in calcified valves (Figure B). Fibroblast sub-clustering reveals 4 VIC unique subgroups that change in abundance as a function of disease (Figure C). Of note, our gene ontology analysis revealed the VIC 1 population to express genes related to extracellular matrix remodeling. Differential expression analysis shows genes associated with cartilage and bone development COMP, IGFBP7, EGR1 are upregulated in male VIC 1 while females VIC 1 upregulate COL1A1, TNFAIP6, CXCL1 associated with fibroblast activation to myofibroblasts (Figure D). COMP overlaps with regions of calcification specifically in male tissue (Figure E – F). RNA-FISH confirms COMP expression near sites of calcification (Figure G). The enrichment and spatial clustering of pro-calcific genes such as COMP act as potential gene targets for modulating sex specific fibroblast differentiation during AVS progression. Our data support the importance of spatially resolving gene expression in sex-separated aortic valves to understand changes in fibroblast heterogeneity during AVS progression. We have identified a fibroblast subtype expressing sex-dependent genes related to ECM remodeling and pro-calcific genes such as COMP. We also validated COMP localized to regions of calcification in male tissue, implicating COMP as an important modulator of sex-dependent AVS progression. Ongoing efforts will recapitulate valve calcification in a 3D hydrogel model and probe sex specific COMP expression as a function of extra-cellular calcium cues. Together, we have identified sex differences in spatially resolved gene expression, furthering our understanding of how AVS progresses as a function of sex .
