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AimsCalcific aortic valve disease (CAVD) is the most common valve disease, which consists of a chronic interplay of inflammation, fibrosis, and calcification. In this study, sortilin (SORT1) was identified as a novel key player in the pathophysiology of CAVD, and its role in the transformation of valvular interstitial cells (VICs) into pathological phenotypes is explored.Methods and resultsAn aortic valve (AV) wire injury (AVWI) mouse model with sortilin deficiency was used to determine the effects of sortilin on AV stenosis, fibrosis, and calcification. In vitro experiments employed human primary VICs cultured in osteogenic conditions for 7, 14, and 21 days; and processed for imaging, proteomics, and transcriptomics including single-cell RNA-sequencing (scRNA-seq). The AVWI mouse model showed reduced AV fibrosis, calcification, and stenosis in sortilin-deficient mice vs. littermate controls. Protein studies identified the transition of human VICs into a myofibroblast-like phenotype mediated by sortilin. Sortilin loss-of-function decreased in vitro VIC calcification. ScRNA-seq identified 12 differentially expressed cell clusters in human VIC samples, where a novel combined inflammatory myofibroblastic-osteogenic VIC (IMO-VIC) phenotype was detected with increased expression of SORT1, COL1A1, WNT5A, IL-6, and serum amyloid A1. VICs sequenced with sortilin deficiency showed decreased IMO-VIC phenotype.ConclusionSortilin promotes CAVD by mediating valvular fibrosis and calcification, and a newly identified phenotype (IMO-VIC). This is the first study to examine the role of sortilin in valvular calcification and it may render it a therapeutic target to inhibit IMO-VIC emergence by simultaneously reducing inflammation, fibrosis, and calcification, the three key pathological processes underlying CAVD.

Background: Rheumatic heart valve disease (RHVD) is a leading cause of cardiovascular death in low- and middle-income countries and affects predominantly women. The underlying mechanisms of chronic valvular damage remain unexplored and regulators of sex predisposition are unknown. Methods: Proteomics analysis of human heart valves (nondiseased aortic valves, nondiseased mitral valves [NDMVs], valves from patients with rheumatic aortic valve disease, and valves from patients with rheumatic mitral valve disease; n=30) followed by system biology analysis identified ProTα (prothymosin alpha) as a protein associated with RHVD. Histology, multiparameter flow cytometry, and enzyme-linked immunosorbent assay confirmed the expression of ProTα. In vitro experiments using peripheral mononuclear cells and valvular interstitial cells were performed using multiparameter flow cytometry and quantitative polymerase chain reaction. In silico analysis of the RHVD and Streptococcus pyogenes proteomes were used to identify mimic epitopes. Results: A comparison of NDMV and nondiseased aortic valve proteomes established the baseline differences between nondiseased aortic and mitral valves. Thirteen unique proteins were enriched in NDMVs. Comparison of NDMVs versus valves from patients with rheumatic mitral valve disease and nondiseased aortic valves versus valves from patients with rheumatic aortic valve disease identified 213 proteins enriched in rheumatic valves. The expression of the 13 NDMV-enriched proteins was evaluated across the 213 proteins enriched in diseased valves, resulting in the discovery of ProTα common to valves from patients with rheumatic mitral valve disease and valves from patients with rheumatic aortic valve disease. ProTα plasma levels were significantly higher in patients with RHVD than in healthy individuals. Immunoreactive ProTα colocalized with CD8 + T cells in RHVD. Expression of ProTα and estrogen receptor alpha correlated strongly in circulating CD8 + T cells from patients with RHVD. Recombinant ProTα induced expression of the lytic proteins perforin and granzyme B by CD8 + T cells as well as higher estrogen receptor alpha expression. In addition, recombinant ProTα increased human leukocyte antigen class I levels in valvular interstitial cells. Treatment of CD8 + T cells with specific estrogen receptor alpha antagonist reduced the cytotoxic potential promoted by ProTα. In silico analysis of RHVD and S pyogenes proteomes revealed molecular mimicry between human type 1 collagen epitope and bacterial collagen-like protein, which induced CD8 + T-cell activation in vitro. Conclusions: ProTα-dependent CD8 + T-cell cytotoxicity was associated with estrogen receptor alpha activity, implicating ProTα as a potential regulator of sex predisposition in RHVD. ProTα facilitated recognition of type 1 collagen mimic epitopes by CD8 + T cells, suggesting mechanisms provoking autoimmunity.

BACKGROUND: Rheumatic heart valve disease (RHVD) is a leading cause of cardiovascular death in low-middle-income countries and affects predominantly women. However, the underlying mechanisms of valvular damage remain scant, and regulators of sex predisposition are unknown. METHODS: Proteomics analysis of human heart valves (Non-Diseased Aortic Valves/NDAV; Non-Diseased Mitral Valves/NDMV; Rheumatic Aortic Valve Disease/RAVD; Rheumatic Mitral Valve Disease/RMVD; n=30), followed by system biology analysis, identified prothymosin alpha (ProTα) as a protein associated with RHVD. Histology, multiparameter flow cytometry and ELISA confirmed the expression of ProTα. In vitro experiments using peripheral mononuclear cells and valvular interstitial cells (VICs) were performed using multiparameter flow cytometry and qPCR. In silico analysis of the RHVD and S. pyogenes proteomes were employed to identify mimic epitopes. RESULTS: A comparison of NDMV and NDAV proteomes established the baseline differences between non-diseased aortic and mitral valves. Thirteen unique proteins were enriched in NDMV. Comparison of NDMV vs. RMVD and NDAV vs. RAVD identified 213 proteins enriched in rheumatic valves. The expression of the 13 NDMV-enriched proteins was evaluated across the 213 proteins enriched in diseased valves, resulting in the discovery of ProTα common to RMVD and RAVD. ProTα plasma levels were significantly higher in RHVD than healthy individuals. Immunoreactive ProTα colocalized with CD8(+) T-cells in RHVD. Expression of ProTα and estrogen receptor alpha (ERα) correlated strongly in circulating CD8(+) T-cells from RHVD patients. Recombinant ProTα induced expression of lytic proteins: perforin and granzyme-B, by CD8(+) T-cells as well as higher ERα expression. In addition, recombinant ProTα increased levels of HLA-I in VICs. Treatment of CD8(+) T-cells with specific ERα antagonist reduced cytotoxic potential promoted by ProTα. In silico analysis of RHVD and S. pyogenes proteomes revealed cross-reactivity between human type 1 collagen epitope and bacterial collagen-like protein, which induced CD8+ T-cells activation in vitro. CONCLUSIONS: ProTα-dependent CD8(+) T-cell cytotoxicity was associated with ERα activity, implicating ProTα as a potential regulator of sex predisposition in RHVD. Moreover, ProTα facilitated recognition of type 1 collagen cross-reactive epitopes by CD8 T-cells, suggesting mechanisms provoking autoimmunity.