Sortilin enhances fibrosis and calcification in aortic valve disease by inducing interstitial cell heterogeneity

Abstract

Aims: Calcific 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 results: An 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.

Conclusion: Sortilin 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.

Keywords: Aortic stenosis; Calcification; Fibrosis; Inflammation; Single-cell RNA-sequencing; Sortilin.

Structured Graphical Abstract

Multi-omic approach to identify the role of sortilin in mediating fibrosis and calcification in calcific aortic valve disease (CAVD). Aortic valve (AV) wire injury in sortilin wild-type (Sort1^+/+) and deficient mice (Sort1^−/−) showed decreased collagen deposition and calcification in mouse AVs. Valvular interstitial cells (VICs) were collected from human CAVD tissue and cultured in osteogenic conditions. VICs collected at varying time points of culture (Days 7, 14, and 21) were processed for flow cytometry, proteomics, and single-cell RNA-sequencing (scRNA-seq). Multiomics data identified increased sortilin expression following the osteogenic culture of VICs. Protein analysis and scRNA-seq identified increased expression of WNT5a, MAPK, YAP, and IL-6 regulated by the expression of sortilin. ScRNA-seq identified a transitionary VIC subpopulation with an activated myofibroblast phenotype that later transitioned into a combined myofibroblast and osteogenic phenotype. ScRNA-seq data identified an inflammatory myofibroblastic-osteogenic VIC (IMO-VIC) subpopulation that may be a key player in the pathogenesis of CAVD under the regulation of sortilin.

Figure 1

Sortilin-mediated effects on functional and morphometric properties of murine aortic valves. (A) Associations with aortic stenosis of 555 variants within 50 kb of SORT1 in the meta-analysis of the GERA and UK Biobank cohorts (302,597 participants; 6912 cases). GERA, Genetic Epidemiology Research on Adult Health and Aging. (B) In vivo study design: high-fat, high-cholesterol diet initiation and aortic valve wire injury (AVWI), sham procedure or no procedure at 10 weeks of age, monthly serial echocardiographic exams. (C) Left panel: representative parasternal long axis views for aortic valve opening diameter; middle panel: colour flow Doppler of the aortic valve; right panel, flow velocity interrogation of the aortic valve (n = 10–12). (D) Aortic valve opening diameter and (E) peak systolic aortic valve flow velocity for the study groups over 16 weeks, blue asterisk represents AVWI Ldlr^−/−/Sort1^+/+ and black asterisks represent control groups. Aortic valve stenosis and increases in peak systolic aortic valve flow for AVWI Ldlr^−/−/Sort1^+/+ vs. AVWI Ldlr^−/−/Sort1^−/− (blue asterisk, P < 0.05) and for Ldlr^−/−/Sort1^+/+ and Ldlr^−/−/Sort1^−/− vs. all other groups (black asterisk, P < 0.01) over the time of echocardiographic measurements. (F) Representative collagen staining of aortic valve leaflets using picrosirius staining. Higher collagen signal and fibrosis detected inAVWI Ldlr^−/−/Sort1^+/+ vs. AVWI Ldlr^−/−/Sort1^−/− (P < 0.05) and greater collagen signal detected in AVWI vs. sham mice Ldlr^−/−/Sort1^+/+ and Ldlr^−/−/Sort1^−/− (P < 0.001 and P < 0.0001, respectively). Representative immunofluorescence imaging of myofibroblast activation (α-smooth muscle actin) in aortic valve leaflets. (G) Calcification molecular imaging of all three aortic valve leaflets by multiphoton and confocal microscopy. Calcification intensity per aortic valve: increased calcification detected in AVWI Ldlr^−/−/Sort1^+/+ vs. AVWI Ldlr^−/−/Sort1^−/− (P < 0.05) and aortic valve wire injury mice vs. sham Ldlr^−/−/Sort1^+/+ and Ldlr^−/−/Sort1^−/− (P < 0.001 and P < 0.05, respectively). Representative immunofluorescence imaging of alkaline phosphatase in aortic valve leaflets. One-way ANOVA performed for statistical analysis (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). Scale bars for (F and G) (picrosirius and calcification staining): 500 μm. Scale bars for (F and G) (α-smooth muscle actin and alkaline phosphatase staining):100 μm.

Figure 2

Sortilin is expressed by valvular interstitial cells identified in calcific aortic valve disease. (A) Schematic highlighting the use of freshly obtained aortic valve samples from patients with calcific aortic valve disease. Valvular interstitial cells were expanded in growth medium and then stimulated in control or osteogenic media (control media, osteogenic media, respectively). (B) Representative imaging of fibrotic (picrosirius) and calcified regions of the aortic valve. Sortilin expressed in fibrotic collagen-rich (top) and calcified aortic valve regions (bottom) (n = 3). (C) Representative immunofluorescence imaging identifying cells in calcific aortic valve disease samples that co-express sortilin, alkaline phosphatase activity, SM22α, near areas of calcification (n = 3). (D) Single-cell RNA-sequencing analysis and uniform manifold approximation and projection of valvular interstitial cells expanded in growth media. Uniform manifold approximation and projection highlights overall valvular interstitial cell heterogeneity and heterogeneity in the expression of sortilin (n = 3). (E) Representative immunofluorescence imaging of valvular interstitial cells cultured in osteogenic media with small interfering RNA sicontrol and siSORT1 for 14 days. Valvular interstitial cells increase the expression of sortilin in osteogenic media conditions. In the bottom panel, valvular interstitial cells co-express sortilin and α-smooth muscle actin (n = 3). (F) Quantitative polymerase chain reaction (n = 6) and (G) western blot analysis to identify protein and mRNA transcripts levels of sortilin by valvular interstitial cells over 21 days of culture in osteogenic media sicontrol and siSORT1 conditions (n = 5). A significant increase in sortilin detected at day 21 in osteogenic media conditions compared with control media (P < 0.05). Two-way ANOVA performed for statistical analysis (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). Scale bars for (B) (collagen/calcification staining) 500 μm; (C) high magnification 100 μm, low magnification 50 μm. (E) 2 μm.

Figure 3

Flow cytometry identifies a subset of valvular interstitial cells that are larger in size and co-express sortilin with myofibroblastic-osteogenic markers. (A) Flow cytometry gating strategy to identify mean fluorescence intensities of proteins expressed by valvular interstitial cells cultured in control media and osteogenic media conditions for 21 days. (B) Overlapping histograms of mean fluorescence intensities for compensation control, control media and osteogenic media groups. Gates are determined based on compensation controls. Greater mean fluorescence intensities identified by valvular interstitial cells cultured in osteogenic media conditions for myofibroblast activation marker (α-smooth muscle actin) and osteogenic markers (osteopontin, osteocalcin, and alkaline phosphatase) (n = 5). (C) Gating strategy for three valvular interstitial cell sub-populations based on valvular interstitial cell complexity (side scatter (SSC)) and size (forward scatter (FSC)), where larger-sized valvular interstitial cells are identified in P2 gate, supported by representative bright field images (n = 6). (D) Quantification of mean fluorescence intensities for myofibroblast and osteogenic proteins expressed by valvular interstitial cells in P1, P2, and P3 gates. Sub-gate P2 includes valvular interstitial cells that increase the expression of sortilin (P < 0.001), α-smooth muscle actin (P < 0.001), osteocalcin (P < 0.001) and alkaline phosphatase (P < 0.05) in osteogenic media conditions compared with control media (n = 6). Black asterisks identify differences within the P2 gate, whereas blue asterisks represent significant differences between P2 and other respective P-gates with blue asterisks. (E) Quantification of myofibroblast and osteogenic mRNA transcript levels by quantitative polymerase chain reaction at days 7, 14, and 21 of osteogenic media culture (n = 6). Valvular interstitial cells increase the expression of sortilin in conjunction with increases in COL1A1, ACTA2, and ALPL over 21 days of culture. Two-way ANOVA performed for statistical analysis (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). Scale bar for (C), 25 μm.

Figure 4

Silencing sortilin decreases valvular interstitial cell myofibroblastic-osteogenic phenotypes and decreases in vitro calcification. (A) Representative immunofluorescence imaging of valvular interstitial cells cultured in osteogenic media conditions with small interfering RNA SORT1 silencing. Valvular interstitial cells with decreased expression of sortilin also decreased the expression of myofibroblast activation proteins α-smooth muscle actin, collagen 1A1 compared with control media (CM)sicontrol and osteogenic media (OM) sicontrol groups (n = 3). (B) Quantification of myofibroblast and osteogenic mRNA transcript levels by quantitative polymerase chain reaction at days 7, 14, and 21 of culture. Plus indicates siSORT1 and minus indicates sicontrol. Valvular interstitial cells decrease the expression of myofibroblastic-osteogenic transcripts when sortilin is silenced in osteogenic media conditions over 21 days of culture (P < 0.001) (n = 5). (C) Representative immunofluorescence imaging identifying sortilin and alkaline phosphatase by valvular interstitial cells cultured in osteogenic conditions (n = 3). (D) Quantification of alkaline phosphatase activity shows decreased activity following siSORT1 (P < 0.05) (n = 4, Day 14). (E) Alizarin staining to identity in vitro calcification. SORT1 silencing significantly reduces valvular interstitial cell calcification (P < 0.05) (n = 3). (F) Label-free proteomics by two group comparisons via t-test, false discovery rate q < 0.05. Heat map representing statistically filtered protein abundances for each valvular interstitial cell sample further underscore the contrast between the culture conditions, control media and osteogenic media, including 580 differentiating protein variables (n = 5). Right panel: schematic pathway network depicting Gene Ontology Biological Process terms enriched in differentially expressed proteins (see Supplementary figures for labeled pathways and figure details). (G) Heat map representing statistically filtered protein abundances for valvular interstitial cell sample cultured in control media and osteogenic media and treated with sicontrol or with siSORT1 treatment (including timepoints days 7, 14, and 21, two group comparison between by t-test sicontrol vs. siSORT1, q < 0.05). Heat map underscores the contrast in valvular interstitial cell protein profiles after sortilin silencing, including 947 differentiating protein variables (n = 5). Proteins decreased in valvular interstitial cells are represented in blue and increased proteins are represented in yellow. Right panel: schematic pathway network depicting Gene Ontology Biological Process terms enriched in Cluster 1 (i.e. enriched in sicontrol; see Supplementary figures for labeled pathways and figure details). Two-way ANOVA performed for statistical analysis (*P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001). Scale bar for (A and C): 25 μm.

Figure 5

Single-cell RNA-sequencing identifies heterogeneity in valvular interstitial cell cultures and novel clusters in osteogenic conditions. (A) Uniform manifold approximation and projection projections of valvular interstitial cells sequenced at days 0, 14, and 21 in growth media (GM), control media (CM) and osteogenic media (OM). Common cluster to all conditions highlighted by the box. Deconstructed uniform manifold approximation and projection based on culture conditions. (B) Deconstructed uniform manifold approximation and projection highlighting emergence of valvular interstitial cell clusters based on the day of culture (Day 0, Day 14 , and Day 21). Novel valvular interstitial cell population at Day 21 in osteogenic media highlighted by box. (C) Uniform manifold approximation and projection projections highlighting heterogeneity in valvular interstitial cell cultures by identifying 12 unique clusters over 21 days of culture, in addition to the emergence novel Cluster 10 at day 21 of culture only in osteogenic media conditions. (D) Violin plots identifying the expression of SORT1 in 12 valvular interstitial cell clusters based on culture conditions (growth media, control media, and osteogenic media). SORT1 increases in osteogenic media conditions in all clusters, whereas SORT1 is exclusively identified in osteogenic media conditions for Cluster 11. (E) Uniform manifold approximation and projection projections of SORT1 and myofibroblast activation genes (COL1A1, ACTA2, TAGLN, and MAPK11) and osteogenic gene (alkaline phosphatase) in valvular interstitial cell populations. Enrichment of SORT1 identified in clusters (arrows) detected only in osteogenic media conditions; based on intensity scale. (F) Heat map highlighting the enrichment of top 50 genes per cluster. (G) Uniform manifold approximation and projection projections of top enriched genes identified mainly in osteogenic media conditions. n = 3 for each culture condition and time point. Uniform manifold approximation and projection and heat map display normalized gene expression.

Figure 6

A distinct cluster emerges in osteogenic conditions that represents an inflammatory myofibroblastic-osteogenic phenotype-valvular interstitial cell. (A) Uniform manifold approximation and projection highlighting (underline) the clusters in osteogenic media conditions that are further analysed. Pseudotime analysis (slingshot) identifies baseline clusters (3 and 4) and terminal clusters (10 and 11). (B) Heat map displaying enriched genes in Clusters 1, 6, 10, and 11, identified only in osteogenic media conditions. (C) Violin plots identifying the expression levels of genes enriched in Clusters 1, 6, 10, 11. Cluster 10 exhibits both myofibroblastic-osteogenic and inflammatory phenotypes. (D) Single-cell RNA-sequencing analysis and uniform manifold approximation and projection projections of valvular interstitial cells transfected with sicontrol and siSORT1 for 21 days in osteogenic media conditions. Clusters are identified based on culture media and small interfering RNA treatment. (E) Uniform manifold approximation and projection projections highlighting the 11 clusters identified in transfected sicontrol and siSORT1 valvular interstitial cells. Pseudotime analysis identifies various cell trajectories from Clusters 0, 3, 5, 6 and 10 (sicontrol) to Clusters 1 and 4 (siSORT1). (F) Uniform manifold approximation and projection projections highlighting enriched genes from (B). Decreases in the expression of SORT1, ACTA2, COL1A1, WNT5A, IL6, and serum amyloid A1 identified in Clusters 1 and 4, following siSORT1 at 21 days of culture. (G) Representative immunofluorescence imaging of human aortic valves that identify the co-expression of sortilin, SM22α and IL-6 and sortilin with WNT5a (n = 3). Single-cell RNA-sequencing: n = 3 for control media sicontrol and CMsiSORT1 groups and n = 5 for osteogenic media sicontrol and siSORT1 groups). Uniform manifold approximation and projection and heat map display normalized gene expression. Scale bar for (G): 100 μm.