Project description:Background: Using proteomics, we strove to reveal novel molecular subtypes of human atherosclerotic lesions, study their associations with histology and imaging and relate them to long-term cardiovascular outcomes. Methods: 219 samples were obtained from 120 patients undergoing carotid endarterectomy. Sequential protein extraction was combined with multiplexed, discovery proteomics. Parallel reaction monitoring for 135 proteins was deployed for targeted validation. A combination of statistical, bioinformatics and machine learning methods was used to perform differential expression, network, pathway enrichment analysis and train and evaluate prognostic models. Results: Our extensive proteomics analysis from the core and periphery of plaques doubled the coverage of the plaque proteome compared to the largest proteomics study on atherosclerosis thus far. Plaque inflammation and calcification signatures were inversely correlated and validated with targeted proteomics. The inflammation signature was enriched with neutrophil-derived proteins, including calprotectin (S100A8/9) and myeloperoxidase. The calcification signature contained fetuin-A, osteopontin, and gamma-carboxylated proteins. Sex differences in the proteome of atherosclerosis were explained by a higher proportion of calcified plaques in women. Single-cell RNA sequencing data attributed the inflammation signature predominantly to neutrophils and macrophages and the calcification signature to smooth muscle cells, except for certain plasma proteins that were not expressed but retained in the plaque, i.e., fetuin-A. Echogenic lesions reflect the collagen content and calcification of plaque but carotid Duplex ultrasound fails to capture the extent of inflammatory protein changes in symptomatic plaques. Applying dimensionality reduction and machine learning on the proteomics data defined 4 distinct plaque phenotypes and revealed key protein signatures linked to smooth muscle cell content, plaque calcification and structural extracellular matrix, which improved the 9-year prognostic AUC by 25% compared to ultrasound and histology. A biosignature of four proteins (CNN1, PROC, SERPH, and CSPG2) independently predicted the progression of atherosclerosis and cardiovascular mortality with an AUC of 75% Conclusion: We combined discovery and targeted proteomics with network reconstruction and clustering techniques to provide molecular insights into protein changes in atherosclerotic plaques. The application of proteomics and machine learning techniques revealed distinct clusters of plaques that inform on disease progression and future adverse cardiovascular events.

Project description:Aims Calcific aortic valve disease (CAVD) is a significant cause of illness and death worldwide. The disease mechanism involves age-associated immuno-osteogenic processes but developmental origins and early disease mechanisms are not fully characterized. Identification of early predictive markers could help optimize therapy decisions and patient follow-up. Methods and Results RNA-sequencing was carried out on human fetal aortic valves at weeks 9, 13, and 22, and adult control, calcified bicuspid (BAV) and tricuspid aortic valves (TAV). K-means clustering was implemented to identify co-expressed gene patterns simultaneously up- and down-regulated over the different conditions. Canonical pathway analysis revealed a predominant immune-metabolic gene signature indicative of ongoing innate and adaptive immune responses, including lymphocyte T-cell metabolic adaptation. Specifically, cytokine and chemokine signalling, cellular migration and proliferation, were all increased in CAVD, while oxidative phosphorylation and protein translation were decreased. Discrete immune-metabolic gene signatures were present at foetal stages and adult controls, indicating that inflammation initiates as soon as the valves are formed and is maintained and amplified during post-natal life. Multiple pathways involved in cellular stress response and neurodegenerative disease were also aberrantly expressed, suggesting that chronic inflammation drives metabolic stress- and age-associated valve pathology in CAVD. Comparing the valve RNA-sequencing dataset with whole blood transcriptomes from asymptomatic individuals with early aortic valve calcification identified a gene signature including the CD28 pathway, highly predictive of patients with CAVD and healthy individuals with moderate levels of aortic valve calcification. Conclusion These data deepen and broaden our understanding of the molecular basis of CAVD, and identify a gene signature for the early detection of aortic valve calcification. Keywords Human foetal valve, CAVD, BAV, inflammation, biomarker, gene signature,PESA

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.

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A Proteomic Atlas of Atherosclerosis: The Contribution of Proteoglycans to Sex Differences, Plaque Phenotypes and Outcomes

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