Multiomics of Tissue Extracellular Vesicles Identifies Unique Modulators of Atherosclerosis and Calcific Aortic Valve Stenosis.

BACKGROUND: Fewer than 50% of patients who develop aortic valve calcification have concomitant atherosclerosis, implying differential pathogenesis. Although circulating extracellular vesicles (EVs) act as biomarkers of cardiovascular diseases, tissue-entrapped EVs are associated with early mineralization, but their cargoes, functions, and contributions to disease remain unknown. METHODS: Disease stage-specific proteomics was performed on human carotid endarterectomy specimens (n=16) and stenotic aortic valves (n=18). Tissue EVs were isolated from human carotid arteries (normal, n=6; diseased, n=4) and aortic valves (normal, n=6; diseased, n=4) by enzymatic digestion, (ultra)centrifugation, and a 15-fraction density gradient validated by proteomics, CD63-immunogold electron microscopy, and nanoparticle tracking analysis. Vesiculomics, comprising vesicular proteomics and small RNA-sequencing, was conducted on tissue EVs. TargetScan identified microRNA targets. Pathway network analyses prioritized genes for validation in primary human carotid artery smooth muscle cells and aortic valvular interstitial cells. RESULTS: Disease progression drove significant convergence (P<0.0001) of carotid artery plaque and calcified aortic valve proteomes (2318 proteins). Each tissue also retained a unique subset of differentially enriched proteins (381 in plaques; 226 in valves; q<0.05). Vesicular gene ontology terms increased 2.9-fold (P<0.0001) among proteins modulated by disease in both tissues. Proteomics identified 22 EV markers in tissue digest fractions. Networks of proteins and microRNA targets changed by disease progression in both artery and valve EVs revealed shared involvement in intracellular signaling and cell cycle regulation. Vesiculomics identified 773 proteins and 80 microRNAs differentially enriched by disease exclusively in artery or valve EVs (q<0.05); multiomics integration found tissue-specific EV cargoes associated with procalcific Notch and Wnt signaling in carotid arteries and aortic valves, respectively. Knockdown of tissue-specific EV-derived molecules FGFR2, PPP2CA, and ADAM17 in human carotid artery smooth muscle cells and WNT5A, APP, and APC in human aortic valvular interstitial cells significantly modulated calcification. CONCLUSIONS: The first comparative proteomics study of human carotid artery plaques and calcified aortic valves identifies unique drivers of atherosclerosis versus aortic valve stenosis and implicates EVs in advanced cardiovascular calcification. We delineate a vesiculomics strategy to isolate, purify, and study protein and RNA cargoes from EVs entrapped in fibrocalcific tissues. Integration of vesicular proteomics and transcriptomics by network approaches revealed novel roles for tissue EVs in modulating cardiovascular disease.

Induced pluripotent stem cell-derived smooth muscle cells to study cardiovascular calcification.

Cardiovascular calcification is the lead predictor of cardiovascular events and the top cause of morbidity and mortality worldwide. To date, only invasive surgical options are available to treat cardiovascular calcification despite the growing understanding of underlying pathological mechanisms. Key players in vascular calcification are vascular smooth muscle cells (SMCs), which transform into calcifying SMCs and secrete mineralizing extracellular vesicles that form microcalcifications, subsequently increasing plaque instability and consequential plaque rupture. There is an increasing, practical need for a large scale and inexhaustible source of functional SMCs. Here we describe an induced pluripotent stem cell (iPSC)-derived model of SMCs by differentiating iPSCs toward SMCs to study the pathogenesis of vascular calcification. Specifically, we characterize the proteome during iPSC differentiation to better understand the cellular dynamics during this process. First, we differentiated human iPSCs toward an induced-SMC (iSMC) phenotype in a 10-day protocol. The success of iSMC differentiation was demonstrated through morphological analysis, immunofluorescent staining, flow cytometry, and proteomics characterization. Proteomics was performed throughout the entire differentiation time course to provide a robust, well-defined starting and ending cell population. Proteomics data verified iPSC differentiation to iSMCs, and functional enrichment of proteins on different days showed the key pathways changing during iSMC development. Proteomics comparison with primary human SMCs showed a high correlation with iSMCs. After iSMC differentiation, we initiated calcification in the iSMCs by culturing the cells in osteogenic media for 17 days. Calcification was verified using Alizarin Red S staining and proteomics data analysis. This study presents an inexhaustible source of functional vascular SMCs and calcifying vascular SMCs to create an in vitro model of vascular calcification in osteogenic conditions, with high potential for future applications in cardiovascular calcification research.

Lipoprotein(a) Induces Vesicular Cardiovascular Calcification Revealed With Single-Extracellular Vesicle Analysis.

Lipoprotein(a) (Lp[a]) blood levels >50 mg/dL is a major cardiovascular disease risk factor in humans. Lp(a) associates with increased cardiovascular calcification, a critical pathology with no clinically available drug therapies. The mechanisms through which Lp(a) increases cardiovascular calcification risk remain undefined. We hypothesized that Lp(a) promotes the release of calcifying extracellular vesicles (EVs) that contribute to formation of microcalcification in cardiovascular tissues. Here, we show Lp(a) increased calcification in both primary human smooth muscle cells (SMCs) and valvular interstitial cells (VICs), potentially through inflammation-related mechanisms that were suppressed with E06 antibody that neutralizes pro-inflammatory oxidized phospholipids. Incubating human SMCs and VICs with Lp(a) altered the composition of EVs, increasing CD29+/tetraspanin- microvesicle release, demonstrated with a tailored single-EV microarray assay that can distinguish multivesicular body-derived exosomes and plasma membrane budded microvesicles at a single-vesicle level. Lp(a) stimulation led to release of SMC and VIC EVs that readily calcified in acellular 3D-collagen hydrogels mimicking formation of ectopic microcalcification occurring in extracellular matrix of human atherosclerotic arteries and stenotic aortic valves. Our study mechanistically demonstrates that Lp(a) partially mediates cardiovascular calcification formation via inducing the release of calcifying EVs. Additionally, we provide a customized method to assess calcifying EVs at a single-vesicle level that can be more broadly applied to assist in quantitatively differentiating exosome and microvesicle EV subpopulations.

CROT (Carnitine O-Octanoyltransferase) Is a Novel Contributing Factor in Vascular Calcification via Promoting Fatty Acid Metabolism and Mitochondrial Dysfunction.

OBJECTIVE: Vascular calcification is a critical pathology associated with increased cardiovascular event risk, but there are no Food and Drug Administration-approved anticalcific therapies. We hypothesized and validated that an unbiased screening approach would identify novel mediators of human vascular calcification. Approach and Results: We performed an unbiased quantitative proteomics and pathway network analysis that identified increased CROT (carnitine O-octanoyltransferase) in calcifying primary human coronary artery smooth muscle cells (SMCs). Additionally, human carotid artery atherosclerotic plaques contained increased immunoreactive CROT near calcified regions. CROT siRNA reduced fibrocalcific response in calcifying SMCs. In agreement, histidine 327 to alanine point mutation inactivated human CROT fatty acid metabolism enzymatic activity and suppressed SMC calcification. CROT siRNA suppressed type 1 collagen secretion, and restored mitochondrial proteome alterations, and suppressed mitochondrial fragmentation in calcifying SMCs. Lipidomics analysis of SMCs incubated with CROT siRNA revealed increased eicosapentaenoic acid, a vascular calcification inhibitor. CRISPR/Cas9-mediated Crot deficiency in LDL (low-density lipoprotein) receptor-deficient mice reduced aortic and carotid artery calcification without altering bone density or liver and plasma cholesterol and triglyceride concentrations. CONCLUSIONS: CROT is a novel contributing factor in vascular calcification via promoting fatty acid metabolism and mitochondrial dysfunction, as such CROT inhibition has strong potential as an antifibrocalcific therapy.

Annexin A1-dependent tethering promotes extracellular vesicle aggregation revealed with single-extracellular vesicle analysis.

Extracellular vesicles (EVs) including plasma membrane-derived microvesicles and endosomal-derived exosomes aggregate by unknown mechanisms, forming microcalcifications that promote cardiovascular disease, the leading cause of death worldwide. Here, we show a framework for assessing cell-independent EV mechanisms in disease by suggesting that annexin A1 (ANXA1)-dependent tethering induces EV aggregation and microcalcification. We present single-EV microarray, a method to distinguish microvesicles from exosomes and assess heterogeneity at a single-EV level. Single-EV microarray and proteomics revealed increased ANXA1 primarily on aggregating and calcifying microvesicles. ANXA1 vesicle aggregation was suppressed by calcium chelation, altering pH, or ANXA1 neutralizing antibody. ANXA1 knockdown attenuated EV aggregation and microcalcification formation in human cardiovascular cells and acellular three-dimensional collagen hydrogels. Our findings explain why microcalcifications are more prone to form in vulnerable regions of plaque, regulating critical cardiovascular pathology, and likely extend to other EV-associated diseases, including autoimmune and neurodegenerative diseases and cancer.

Simulations of morphotype-dependent hemodynamics in non-dilated bicuspid aortic valve aortas.

Bicuspid aortic valves (BAVs) generate flow abnormalities that may promote aortopathy. While positive helix fraction (PHF) index, flow angle (θ), flow displacement (d) and wall shear stress (WSS) exhibit abnormalities in dilated BAV aortas, it is unclear whether those anomalies stem from the abnormal valve anatomy or the dilated aorta. Therefore, the objective of this study was to quantify the early impact of different BAV morphotypes on aorta hemodynamics prior to dilation. Fluid-structure interaction models were designed to quantify standard peak-systolic flow metrics and temporal WSS characteristics in a realistic non-dilated aorta connected to functional tricuspid aortic valve (TAV) and type-I BAVs. While BAVs generated increased helicity (PHF>0.68) in the middle ascending aorta (AA), larger systolic flow skewness (θ>11.2°) and displacement (d>6.8mm) relative to the TAV (PHF=0.51; θ<5.5°; d<3.3mm), no distinct pattern was observed between morphotypes. In contrast, WSS magnitude and directionality abnormalities were BAV morphotype- and site-dependent. Type-I BAVs subjected the AA convexity to peak-systolic WSS overloads (up to 1014% difference vs. TAV). While all BAVs increased WSS unidirectionality on the proximal AA relative to the TAV, the most significant abnormality was achieved by the BAV with left-right-coronary cusp fusion on the wall convexity (up to 0.26 decrease in oscillatory shear index vs. TAV). The results indicate the existence of strong hemodynamic abnormalities in non-dilated type-I BAV AAs, their colocalization with sites vulnerable to dilation and the superior specificity of WSS metrics over global hemodynamic metrics to the valve anatomy.

Bicuspid aortic valve hemodynamics does not promote remodeling in porcine aortic wall concavity.

AIM: To investigate the role of type-I left-right bicuspid aortic valve (LR-BAV) hemodynamic stresses in the remodeling of the thoracic ascending aorta (AA) concavity, in the absence of underlying genetic or structural defects. METHODS: Transient wall shear stress (WSS) profiles in the concavity of tricuspid aortic valve (TAV) and LR-BAV AAs were obtained computationally. Tissue specimens excised from the concavity of normal (non-dilated) porcine AAs were subjected for 48 h to those stress environments using a shear stress bioreactor. Tissue remodeling was characterized in terms of matrix metalloproteinase (MMP) expression and activity via immunostaining and gelatin zymography. RESULTS: Immunostaining semi-quantification results indicated no significant difference in MMP-2 and MMP-9 expression between the tissue groups exposed to TAV and LR-BAV AA WSS (P = 0.80 and P = 0.19, respectively). Zymography densitometry revealed no difference in MMP-2 activity (total activity, active form and latent form) between the groups subjected to TAV AA and LR-BAV AA WSS (P = 0.08, P = 0.15 and P = 0.59, respectively). CONCLUSION: The hemodynamic stress environment present in the concavity of type-I LR-BAV AA does not cause any significant change in proteolytic enzyme expression and activity as compared to that present in the TAV AA.

Etiology of bicuspid aortic valve disease: Focus on hemodynamics.

The bicuspid aortic valve (BAV) is the most common form of inheritable cardiac defect. Although this abnormality may still achieve normal valvular function, it is often associated with secondary valvular and aortic complications such as calcific aortic valve disease and aortic dilation. The clinical significance and economic burden of BAV disease justify the need for improved clinical guidelines and more robust therapeutic modalities, which address the root-cause of those pathologies. Unfortunately, the etiology of BAV valvulopathy and aortopathy is still a debated issue. While the BAV anatomy and its secondary complications have been linked historically to a common genetic root, recent advances in medical imaging have demonstrated the existence of altered hemodynamics near BAV leaflets prone to calcification and BAV aortic regions vulnerable to dilation. The abnormal mechanical stresses imposed by the BAV on its leaflets and on the aortic wall could be transduced into cell-mediated processes, leading ultimately to valvular calcification and aortic medial degeneration. Despite increasing evidence for this hemodynamic etiology, the demonstration of the involvement of mechanical abnormalities in the pathogenesis of BAV disease requires the investigation of causality between the blood flow environment imposed on the leaflets and the aortic wall and the local biology, which has been lacking to date. This editorial discusses the different hypothetical etiologies of BAV disease with a particular focus on the most recent advances in cardiovascular imaging, flow characterization techniques and tissue culture methodologies that have provided new evidence in support of the hemodynamic theory.

Bicuspid aortic valve hemodynamics induces abnormal medial remodeling in the convexity of porcine ascending aortas.

The type-I bicuspid aortic valve (BAV), which differs from the normal tricuspid aortic valve (TAV) most commonly by left-right coronary cusp fusion, is frequently associated with secondary aortopathies. While BAV aortic dilation has been linked to a genetic predisposition, hemodynamics has emerged as a potential alternate etiology. However, the link between BAV hemodynamics and aortic medial degeneration has not been established. The objective of this study was to compare the regional wall shear stresses (WSS) in a TAV and BAV ascending aorta (AA) and to isolate ex vivo their respective impact on aortic wall remodeling. The WSS environments generated in the convex region of a TAV and BAV AA were predicted through fluid-structure interaction (FSI) simulations in an aorta model subjected to both valvular flows. Remodeling of porcine aortic tissue exposed to TAV and BAV AA WSS for 48 h in a cone-and-plate bioreactor was investigated via immunostaining, immunoblotting and zymography. FSI simulations revealed the existence of larger and more unidirectional WSS in the BAV than in the TAV AA convexity. Exposure of normal aortic tissue to BAV AA WSS resulted in increased MMP-2 and MMP-9 expressions and MMP-2 activity but similar fibrillin-1 content and microfibril organization relative to the TAV AA WSS treatment. This study confirms the sensitivity of aortic tissue to WSS abnormalities and demonstrates the susceptibility of BAV hemodynamic stresses to focally mediate aortic medial degradation. The results provide compelling support to the important role of hemodynamics in BAV secondary aortopathy.