ApoC-III is a novel inducer of calcification in human aortic valves.

Calcific aortic valve disease (CAVD) occurs when subpopulations of valve cells undergo specific differentiation pathways, promoting tissue fibrosis and calcification. Lipoprotein particles carry oxidized lipids that promote valvular disease, but low-density lipoprotein-lowering therapies have failed in clinical trials, and there are currently no pharmacological interventions available for this disease. Apolipoproteins are known promoters of atherosclerosis, but whether they possess pathogenic properties in CAVD is less clear. To search for a possible link, we assessed 12 apolipoproteins in nonfibrotic/noncalcific and fibrotic/calcific aortic valve tissues by proteomics and immunohistochemistry to understand if they were enriched in calcified areas. Eight apolipoproteins (apoA-I, apoA-II, apoA-IV, apoB, apoC-III, apoD, apoL-I, and apoM) were enriched in the calcific versus nonfibrotic/noncalcific tissues. Apo(a), apoB, apoC-III, apoE, and apoJ localized within the disease-prone fibrosa and colocalized with calcific regions as detected by immunohistochemistry. Circulating apoC-III on lipoprotein(a) is a potential biomarker of aortic stenosis incidence and progression, but whether apoC-III also induces aortic valve calcification is unknown. We found that apoC-III was increased in fibrotic and calcific tissues and observed within the calcification-prone fibrosa layer as well as around calcification. In addition, we showed that apoC-III induced calcification in primary human valvular cell cultures via a mitochondrial dysfunction/inflammation-mediated pathway. This study provides a first assessment of a broad array of apolipoproteins in CAVD tissues, demonstrates that specific apolipoproteins associate with valvular calcification, and implicates apoC-III as an active and modifiable driver of CAVD beyond its potential role as a biomarker.

Tissue formation and host remodeling of an elastomeric biodegradable scaffold in an ovine pulmonary leaflet replacement model.

In pursuit of a suitable scaffold material for cardiac valve tissue engineering applications, an acellular, electrospun, biodegradable polyester carbonate urethane urea (PECUU) scaffold was evaluated as a pulmonary valve leaflet replacement in vivo. In sheep (n = 8), a single pulmonary valve leaflet was replaced with a PECUU leaflet and followed for 1, 6, and 12 weeks. Implanted leaflet function was assessed in vivo by echocardiography. Explanted samples were studied for gross pathology, microscopic changes in the extracellular matrix, host cellular re-population, and immune responses, and for biomechanical properties. PECUU leaflets showed normal leaflet motion at implant, but decreased leaflet motion and dimensions at 6 weeks. The leaflets accumulated α-SMA and CD45 positive cells, with surfaces covered with endothelial cells (CD31+). New collagen formation occurred (Picrosirius Red). Accumulated tissue thickness correlated with the decrease in leaflet motion. The PECUU scaffolds had histologic evidence of scaffold degradation and an accumulation of pro-inflammatory/M1 and anti-inflammatory/M2 macrophages over time in vivo. The extent of inflammatory cell accumulation correlated with tissue formation and polymer degradation but was also associated with leaflet thickening and decreased leaflet motion. Future studies should explore pre-implant seeding of polymer scaffolds, more advanced polymer fabrication methods able to more closely approximate native tissue structure and function, and other techniques to control and balance the degradation of biomaterials and new tissue formation by modulation of the host immune response.

Large-Scale Proteomics Identifies Novel Biomarkers and Circulating Risk Factors for Aortic Stenosis.

BACKGROUND: Limited data exist regarding risk factors for aortic stenosis (AS). The plasma proteome is a promising phenotype for discovery of novel biomarkers and potentially causative mechanisms. OBJECTIVES: The aim of this study was to discover novel biomarkers with potentially causal associations with AS. METHODS: We measured 4,877 plasma proteins (SomaScan aptamer-affinity assay) among ARIC (Atherosclerosis Risk In Communities) study participants in mid-life (visit 3 [V3]; n = 11,430; age 60 ± 6 years) and in late-life (V5; n = 4,899; age 76 ± 5 years). We identified proteins cross-sectionally associated with aortic valve (AV) peak velocity (AVmax) and dimensionless index by echocardiography at V5 and with incident AV-related hospitalization after V3 with the use of multivariable linear and Cox proportional hazard regression. We assessed associations of candidate proteins with changes in AVmax over 6 years and with AV calcification with the use of cardiac computed tomography, replicated analysis in an independent sample, performed Mendelian randomization, and evaluated gene expression in explanted human AV tissue. RESULTS: Fifty-two proteins cross-sectionally were associated with AVmax and dimensionless index at V5 and with risk of incident AV-related hospitalization after V3. Among 3,413 participants in the Cardiovascular Health Study, 6 of those proteins were significantly associated with adjudicated moderate or severe AS, including matrix metalloproteinase 12 (MMP12), complement C1q tumor necrosis factor-related protein 1 (C1QTNF1), and growth differentiation factor-15. MMP12 was also associated with greater increase in AVmax over 6 years, greater degree of AV calcification, and greater expression in calcific compared with normal or fibrotic AV tissue. C1QTNF1 had consistent potential causal effects on both AS and AVmax according to Mendelian randomization analysis. CONCLUSIONS: These findings identify MMP12 as a potential novel circulating biomarker of AS risk and C1QTNF1 as a new putative target to prevent AS progression.

mRNA-miRNA integrative analysis of diabetes-induced cardiomyopathy in rats.

An integrative analysis of miRNA and mRNA expression profiles in left ventricle (LV) of diabetes-induced rats was performed to elucidate the role of miRNAs and their mRNAs target in diabetic cardiomyopathy (DCM). mRNA (GSE4745) and miRNA (GSE44179) datasets were downloaded from Gene Expression Omnibus 2R (GEO2R) and differentially expressed mRNAs and miRNAs were selected. Cardiotoxicity-related mRNAs (n=7) were analyzed by Ingenuity Pathway Analyses 6 (IPA) and regulatory miRNAs (n=639) were identified using TargetScan 7.1. web dataset. The integrative analysis was performed between miRNAs differentially expressed in GSE44179 and regulatory TargetScan-detected miRNAs of mRNAs differentially expressed in GSE4745. Pla2g2a and Hk2 mRNAs were up-and-down regulated, respectively, in GSE4745 on days 3 and 42 after diabetes-induction. The Pla2g2a regulatory miRNAs, rno-miR-877, rno-miR-320 and rno-miR-214, were down-regulated, and Hk2 regulatory miRNAs, rno-miR-17, rno-miR-187, rno-miR-34a, rno-miR-322, rno-miR-188, rno-miR-532 and rno-miR-21, were up-regulated in GSE44179 dataset. These results are suggestive that Pla2g2a and Hk2 mRNAs and their regulatory miRNAs play a role in DCM pathogenesis and they may be potential circulating biomarkers to detect early cardiovascular complications in diabetic patients.

Modulation of miR-26a-5p and miR-15b-5p Exosomal Expression Associated with Clopidogrel-Induced Hepatotoxicity in HepG2 Cells.

Clopidogrel is an essential antiplatelet drug used to prevent thrombosis complications associated with atherosclerosis. However, hepatotoxicity is a potential adverse effect related to clopidogrel therapy. Exosome-derived miRNAs may be useful for improved monitoring of drug response and hepatotoxicity risk. In the present study, the expression of several exosomal miRNAs (miR-26a-5p, miR-145-5p, miR-15b-5p, and miR-4701-3p) and cell-derived mRNA targets (PLOD2, SENP5, EIF4G2, HMGA2, STRADB, and TLK1) were evaluated in HepG2 cells treated with clopidogrel (6.25, 12.5, 25, 50, and 100 µM) for 24 and 48 h. Then, clopidogrel cytotoxicity was evaluated by analyzing DNA fragmentation and the cell cycle profile using flow cytometry. Differential expression of exosome-derived miRNAs and cell-derived mRNAs was analyzed by RT-qPCR. Exposure of HepG2 cells to high concentrations of clopidogrel (50 and 100 µM) for 24 h caused significant DNA fragmentation (17.6 and 44.4%, respectively; p < 0.05) and 48 h (26.8 and 48.9%, respectively; p < 0.05), indicating cellular toxicity. Upregulation of miR-26a-5p and downregulation of miR-15b-5p was observed in cells exposed to 100 µM clopidogrel for 24 and 48 h. The miR-26a-5p target mRNAs HMGA2, EIF4G2, STRADB, and SENP5 were downregulated in HepG2 cells following exposure to cytotoxic concentrations of clopidogrel (50 and 100 µM) for 24 h, and HMGA2 levels remained low after 48 h of treatment. TLK1, a target of miR-15b-5p, was downregulated by 50 and 100 µM clopidogrel at 24 h. In conclusion, our results suggest that exposure to high concentrations of clopidogrel modulates the expression of exosomal miR-26a-5p and miR-15b-5p and their target mRNAs in HepG2 cells. Dysregulation of these miRNAs maybe modulate the regulatory pathways involved in clopidogrel-induced liver injury.