Project description:Calcific aortic valve disease (CAVD) is a leading cause of cardiovascular morbidity and mortality with no effective medical therapy. We previously identified carnitine O-octanoyltransferase (CROT) as a mediator of mitochondrial dysfunction and vascular calcification. However, its role in CAVD remains unexplored. Here, we investigate whether CROT contributes to CAVD progression by promoting the release of pro-calcific mitochondria-derived extracellular vesicles (mitoEVs). Methods and Results: Human valvular interstitial cells (VICs) were isolated from aortic valves obtained from patients with aortic stenosis (AS; n=34 donors). VICs were cultured in osteogenic medium (OM) to induce calcification, which was evaluated by Alizarin Red staining. Proteomic analysis was performed to identify differentially abundant proteins between VICs cultured in normal medium (NM) and OM, with the goal of elucidating mechanisms linking CROT to VIC calcification. Mitochondrial function was assessed using the Seahorse XF Analyzer to measure oxygen consumption rate (OCR), and mitochondrial morphology was examined by MitoTracker staining. Extracellular vesicles (EVs) were isolated from VIC-conditioned media by ultracentrifugation and characterized for particle size and concentration using NanoSight Pro. In vivo, AS was induced in C57BL/6J, Ldlr⁻/⁻ Crot (Sham, n=12), Ldlr-/- Crot+/+ (Aortic valve wire injury; AVWI, n=19) and Ldlr-/- Crot-/- (AVWI, n=23). Disease progression was monitored by echocardiography, and aortic valve calcification was visualized using OsteoSense 680EX near-infrared fluorescence-based molecular imaging. OM–induced calcification in VICs was reduced by siRNA-mediated CROT silencing (siCROT; p<0.05). Proteomic analysis revealed that mitochondrial-associated proteins were markedly altered following siCROT silencing. Examination of mitochondrial morphology showed that OM culture induced marked mitochondrial fragmentation, which was restored by siCROT treatment. Western blotting and flow cytometry analyses indicated that OM-induced mitochondrial fragmentation promotes the release of mitoEVs from VICs, which in turn contributes to calcification. Immunofluorescence revealed mitoEVs within the extracellular space, colocalizing with calcification. In vivo, CROT deficiency attenuated the progression of AS in AVWI mouse model by reducing valvular calcification. Conclusion: Osteogenic stimulation disrupts mitochondrial dynamics in VICs, promoting the release of pro-calcific mitoEVs. This study identifies CROT as a key regulator of this process, where CROT inhibition restores mitochondrial homeostasis and suppresses VIC calcification. These findings highlight CROT as a promising therapeutic target for the treatment of CAVD.

Project description:Introduction: Renal failure is associated with aortic valve calcification. Using our rat model of uraemia-induced reversible aortic valve calcification, we assessed the role of apoptosis and survival pathways in aortic valve calcification. We also explored the effects of raloxifene - an estrogen receptor modulator on valvular calcification. Methods: Gene array analysis was performed in aortic valves obtained from 3 groups of rats (n=7 each): calcified valves from rats fed with uremic diet -high-adenine (0.75%), high-phosphate diet (1.5%), valves after calcification resolution following diet cessation (reversibility) and control. In addition, four groups of rats (n=10 each) were used in order to evaluate the effect of raloxifene in aortic valve calcification: three groups as mentioned above and a fourth group fed with the uremic diet which also received daily raloxifene. Evaluation of these groups included imaging, histology and antigen expression analysis. Results: Gene array results showed that the majority of the expressed genes that were altered were from the diet group valves. Most apoptosis-related genes were changed in a pro-apoptotic direction in calcified valves. Apoptosis and decrease in several survival pathways were confirmed in calcified valves. Resolution of aortic valve calcification was accompanied by decreased apoptosis and upregulation of these ant-apoptotic pathways. Imaging and histology demonstrated that raloxifene significantly decreased aortic valve calcification. Conclusion: Downregulation of several survival pathways and apoptosis are involved in the pathogenesis of aortic valve calcification. The beneficial effect of raloxifene in valve calcification is related to apoptosis modulation. This novel observation is important for developing remedies for aortic valve calcification in patients with renal failure. Introduction: Renal failure is associated with aortic valve calcification. Using our rat model of uraemia-induced reversible aortic valve calcification, we assessed the role of apoptosis and survival pathways in aortic valve calcification. We also explored the effects of raloxifene - an estrogen receptor modulator on valvular calcification. Methods: Gene array analysis was performed in aortic valves obtained from 3 groups of rats (n=7 each): calcified valves from rats fed with uremic diet -high-adenine (0.75%), high-phosphate diet (1.5%), valves after calcification resolution following diet cessation (reversibility) and control. In addition, four groups of rats (n=10 each) were used in order to evaluate the effect of raloxifene in aortic valve calcification: three groups as mentioned above and a fourth group fed with the uremic diet which also received daily raloxifene. Evaluation of these groups included imaging, histology and antigen expression analysis. Results: Gene array results showed that the majority of the expressed genes that were altered were from the diet group valves. Most apoptosis-related genes were changed in a pro-apoptotic direction in calcified valves. Apoptosis and decrease in several survival pathways were confirmed in calcified valves. Resolution of aortic valve calcification was accompanied by decreased apoptosis and upregulation of these ant-apoptotic pathways. Imaging and histology demonstrated that raloxifene significantly decreased aortic valve calcification. Conclusion: Downregulation of several survival pathways and apoptosis are involved in the pathogenesis of aortic valve calcification. The beneficial effect of raloxifene in valve calcification is related to apoptosis modulation. This novel observation is important for developing remedies for aortic valve calcification in patients with renal failure.

Project description:Valve remodeling is a complex process involving extracellular matrix organization, development of trilaminar structures, and physical elongation of valve leaflets. However, the cellular and molecular mechanisms regulating valve remodeling and their roles in congenital valve disorders remain poorly understood. Semilunar valves and atrioventricular valves from healthy and age-matched human fetal hearts with pulmonary stenosis (PS) were collected. Single-Cell RNA-sequencing (scRNA-seq) was performed to determine the transcriptomic landscape of multiple valvular cell subtypes in valve remodeling and disease. Spatial localization of newly-identified cell subtypes was determined via immunofluorescence and RNA in situ hybridization. The molecular mechanisms mediating valve development was investigated utilizing primary human fetal valve interstitial cells (VICs) and endothelial cells (VECs). scRNA-seq analysis of healthy human fetal valves identified a novel APOE+ elastin-producing VIC subtype (Elastin-VIC) spatially located underneath VECs sensing the unidirectional flow. Knockdown of APOE in fetal VICs resulted in significant elastogenesis defects. In pulmonary valve with PS, we observed decreased expression of APOE and other genes regulating elastogenesis such as EMILIN1 and LOXL1, as well as elastin fragmentation. These findings suggested the crucial role of APOE in regulating elastogenesis during valve remodeling. Furthermore, cell-cell interaction analysis revealed that JAG1 from unidirectional VECs activates NOTCH signaling in Elastin-VICs through NOTCH3. In vitro Jag1 treatment in VICs increased the expression of elastogenesis-related genes and enhanced contractile functions with related gene expression. This was accompanied by activation of NOTCH signaling and elastogenesis observed in VICs co-cultured with VECs in the presence of unidirectional flow. Notably, we found that the JAG1-NOTCH3 signaling pair was drastically reduced in the PS valves. Lastly, we demonstrated that APOE is indispensable for JAG1-induced NOTCH activation in VICs, reinforcing the presence of a synergistic intrinsic and external regulatory network involving APOE and NOTCH signaling that is responsible for regulating elastogenesis during human valve remodeling. scRNA-seq analysis of human fetal valves identified a novel Elastin-VIC subpopulation, and revealed mechanism of intrinsic APOE and external NOTCH signaling from VECs sensing unidirectional flow in regulating elastogenesis during valve remodeling. These mechanisms may contribute to the pathogenesis of elastic malformation in congenital valve disease.

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Aortic valve calcification is induced by the loss of ALDH1A1 and can be prevented by agonists of retinoic acid receptor alpha: preclinical evidence for drug repositioning, 2

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