Multi-Omics Profiling of Human Endothelial Cells from the Coronary Artery and Internal Thoracic Artery Reveals Molecular but Not Functional Heterogeneity.

Major adverse cardiovascular events occurring upon coronary artery bypass graft surgery are typically accompanied by endothelial dysfunction. Total arterial revascularisation, which employs both left and right internal thoracic arteries instead of the saphenous vein to create a bypass, is associated with better mid- and long-term outcomes. We suggested that molecular profiles of human coronary artery endothelial cells (HCAECs) and human internal mammary artery endothelial cells (HITAECs) are coherent in terms of transcriptomic and proteomic signatures, which were then investigated by RNA sequencing and ultra-high performance liquid chromatography-mass spectrometry, respectively. Both HCAECs and HITAECs overexpressed molecules responsible for the synthesis of extracellular matrix (ECM) components, basement membrane assembly, cell-ECM adhesion, organisation of intercellular junctions, and secretion of extracellular vesicles. HCAECs were characterised by higher enrichment with molecular signatures of basement membrane construction, collagen biosynthesis and folding, and formation of intercellular junctions, whilst HITAECs were notable for augmented pro-inflammatory signaling, intensive synthesis of proteins and nitrogen compounds, and enhanced ribosome biogenesis. Despite HCAECs and HITAECs showing a certain degree of molecular heterogeneity, no specific markers at the protein level have been identified. Coherence of differentially expressed molecular categories in HCAECs and HITAECs suggests synergistic interactions between these ECs in a bypass surgery scenario.

Extracellular vesicles stimulate smooth muscle cell migration by presenting collagen VI.

The extracellular matrix (ECM) supports blood vessel architecture and functionality and undergoes active remodelling during vascular repair and atherogenesis. Vascular smooth muscle cells (VSMCs) are essential for vessel repair and, via their secretome, are able to invade from the vessel media into the intima to mediate ECM remodelling. Accumulation of fibronectin (FN) is a hallmark of early vascular repair and atherosclerosis and here we show that FN stimulates VSMCs to secrete small extracellular vesicles (sEVs) by activating the ß1 integrin/FAK/Src pathway as well as Arp2/3-dependent branching of the actin cytoskeleton. Spatially, sEV were secreted via filopodia-like cellular protrusions at the leading edge of migrating cells. We found that sEVs are trapped by the ECM in vitro and colocalise with FN in symptomatic atherosclerotic plaques in vivo. Functionally, ECM-trapped sEVs induced the formation of focal adhesions (FA) with enhanced pulling forces at the cellular periphery. Proteomic and GO pathway analysis revealed that VSMC-derived sEVs display a cell adhesion signature and are specifically enriched with collagen VI. In vitro assays identified collagen VI as playing the key role in cell adhesion and invasion. Taken together our data suggests that the accumulation of FN is a key early event in vessel repair acting to promote secretion of collage VI enriched sEVs by VSMCs. These sEVs stimulate migration and invasion by triggering peripheral focal adhesion formation and actomyosin contraction to exert sufficient traction forces to enable VSMC movement within the complex vascular ECM network.

Proteolytic Degradation Is a Major Contributor to Bioprosthetic Heart Valve Failure.

Background Whereas the risk factors for structural valve degeneration (SVD) of glutaraldehyde-treated bioprosthetic heart valves (BHVs) are well studied, those responsible for the failure of BHVs fixed with alternative next-generation chemicals remain largely unknown. This study aimed to investigate the reasons behind the development of SVD in ethylene glycol diglycidyl ether-treated BHVs. Methods and Results Ten ethylene glycol diglycidyl ether-treated BHVs excised because of SVD, and 5 calcified aortic valves (AVs) replaced with BHVs because of calcific AV disease were collected and their proteomic profile was deciphered. Then, BHVs and AVs were interrogated for immune cell infiltration, microbial contamination, distribution of matrix-degrading enzymes and their tissue inhibitors, lipid deposition, and calcification. In contrast with dysfunctional AVs, failing BHVs suffered from complement-driven neutrophil invasion, excessive proteolysis, unwanted coagulation, and lipid deposition. Neutrophil infiltration was triggered by an asymptomatic bacterial colonization of the prosthetic tissue. Neutrophil elastase, myeloblastin/proteinase 3, cathepsin G, and matrix metalloproteinases (MMPs; neutrophil-derived MMP-8 and plasma-derived MMP-9), were significantly overexpressed, while tissue inhibitors of metalloproteinases 1/2 were downregulated in the BHVs as compared with AVs, together indicative of unbalanced proteolysis in the failing BHVs. As opposed to other proteases, MMP-9 was mostly expressed in the disorganized prosthetic extracellular matrix, suggesting plasma-derived proteases as the primary culprit of SVD in ethylene glycol diglycidyl ether-treated BHVs. Hence, hemodynamic stress and progressive accumulation of proteases led to the extracellular matrix degeneration and dystrophic calcification, ultimately resulting in SVD. Conclusions Neutrophil- and plasma-derived proteases are responsible for the loss of BHV mechanical competence and need to be thwarted to prevent SVD.

Calciprotein Particles Cause Physiologically Significant Pro-Inflammatory Response in Endothelial Cells and Systemic Circulation.

Calciprotein particles (CPPs) represent an inherent mineral buffering system responsible for the scavenging of excessive Ca2+ and PO43- ions in order to prevent extraskeletal calcification, although contributing to the development of endothelial dysfunction during the circulation in the bloodstream. Here, we performed label-free proteomic profiling to identify the functional consequences of CPP internalisation by endothelial cells (ECs) and found molecular signatures of significant disturbances in mitochondrial and lysosomal physiology, including oxidative stress, vacuolar acidification, accelerated proteolysis, Ca2+ cytosolic elevation, and mitochondrial outer membrane permeabilisation. Incubation of intact ECs with conditioned medium from CPP-treated ECs caused their pro-inflammatory activation manifested by vascular cell adhesion molecule 1 (VCAM1) and intercellular adhesion molecule 1 (ICAM1) upregulation and elevated release of interleukin (IL)-6, IL-8, and monocyte chemoattractant protein-1/ C-C motif ligand 2 (MCP-1/CCL2). Among the blood cells, monocytes were exclusively responsible for CPP internalisation. As compared to the co-incubation of donor blood with CPPs in the flow culture system, intravenous administration of CPPs to Wistar rats caused a considerably higher production of chemokines, indicating the major role of monocytes in CPP-triggered inflammation. Upregulation of sICAM-1 and IL-8 also suggested a notable contribution of endothelial dysfunction to systemic inflammatory response after CPP injections. Collectively, our results demonstrate the pathophysiological significance of CPPs and highlight the need for the development of anti-CPP therapies.

Multi-omics of in vitro aortic valve calcification.

Heart valve calcification is an active cellular and molecular process that partly remains unknown. Osteogenic differentiation of valve interstitial cells (VIC) is a central mechanism in calcific aortic valve disease (CAVD). Studying mechanisms in CAVD progression is clearly needed. In this study, we compared molecular mechanisms of osteogenic differentiation of human VIC isolated from healthy donors or patients with CAVD by RNA-seq transcriptomics in early timepoint (48 h) and by shotgun proteomics at later timepoint (10th day). Bioinformatic analysis revealed genes and pathways involved in the regulation of VIC osteogenic differentiation. We found a high amount of stage-specific differentially expressed genes and good accordance between transcriptomic and proteomic data. Functional annotation of differentially expressed proteins revealed that osteogenic differentiation of VIC involved many signaling cascades such as: PI3K-Akt, MAPK, Ras, TNF signaling pathways. Wnt, FoxO, and HIF-1 signaling pathways were modulated only at the early timepoint and thus probably involved in the commitment of VIC to osteogenic differentiation. We also observed a significant shift of some metabolic pathways in the early stage of VIC osteogenic differentiation. Lentiviral overexpression of one of the most upregulated genes (ZBTB16, PLZF) increased calcification of VIC after osteogenic stimulation. Analysis with qPCR and shotgun proteomics suggested a proosteogenic role of ZBTB16 in the early stages of osteogenic differentiation.

Crenigacestat (LY3039478) inhibits osteogenic differentiation of human valve interstitial cells from patients with aortic valve calcification in vitro.

Calcific aortic valve disease (CAVD) is one of the dangerous forms of vascular calcification. CAVD leads to calcification of the aortic valve and disturbance of blood flow. Despite high mortality, there is no targeted therapy against CAVD or vascular calcification. Osteogenic differentiation of valve interstitial cells (VICs) is one of the key factors of CAVD progression and inhibition of this process seems a fruitful target for potential therapy. By our previous study we assumed that inhibitors of Notch pathway might be effective to suppress aortic valve leaflet calcification. We tested CB-103 and crenigacestat (LY3039478), two selective inhibitors of Notch-signaling, for suppression of osteogenic differentiation of VICs isolated from patients with CAVD in vitro. Effect of inhibitors were assessed by the measurement of extracellular matrix calcification and osteogenic gene expression. For effective inhibitor (crenigacestat) we also performed MTT and proteomics study for better understanding of its effect on VICs in vitro. CB-103 did not affect osteogenic differentiation. Crenigacestat completely inhibited osteogenic differentiation (both matrix mineralization and Runx2 expression) in the dosages that had no obvious cytotoxicity. Using proteomics analysis, we found several osteogenic differentiation-related proteins associated with the effect of crenigacestat on VICs differentiation. Taking into account that crenigacestat is FDA approved for clinical trials for anti-tumor therapy, we argue that this drug could be considered as a potential inhibitor of cardiovascular calcification.