The human cardiovascular system is a complex arrangement of specialized structures with distinct functions. The molecular landscape, including the genome, transcriptome and proteome, is pivotal to the biological complexity of both normal and abnormal mammalian processes. Despite our advancing knowledge and understanding of cardiovascular disease (CVD) through the principal use of rodent models, this continues to be an increasing issue in today's world. For instance, as the ageing population increases, so does the incidence of heart valve dysfunction. This may be because of changes in molecular composition and structure of the extracellular matrix, or from the pathological process of vascular calcification in which bone‐formation related factors cause ectopic mineralization. However, significant differences between mice and men exist in terms of cardiovascular anatomy, physiology and pathology. In contrast, large animal models can show considerably greater similarity to humans. Furthermore, precise and efficient genome editing techniques enable the generation of tailored models for translational research. These novel systems provide a huge potential for large animal models to investigate the regulatory factors and molecular pathways that contribute to CVD in vivo. In turn, this will help bridge the gap between basic science and clinical applications by facilitating the refinement of therapies for cardiovascular disease. 2016 The Authors. Published by John Wiley & Sons Ltd.
Patients with end‐stage renal disease (ESRD) have elevated circulating calcium (Ca) and phosphate (Pi), and exhibit accelerated progression of calcific aortic valve disease (CAVD). We hypothesized that matrix vesicles (MVs) initiate the calcification process in CAVD. Ca induced rat valve interstitial cells (VICs) calcification at 4.5 mM (16.4‐fold; p < 0.05) whereas Pi treatment alone had no effect. Ca (2.7 mM) and Pi (2.5 mM) synergistically induced calcium deposition (10.8‐fold; p < 0.001) in VICs. Ca treatment increased the mRNA of the osteogenic markers Msx2, Runx2, and Alpl (p < 0.01). MVs were harvested by ultracentrifugation from VICs cultured with control or calcification media (containing 2.7 mM Ca and 2.5 mM Pi) for 16 hr. Proteomics analysis revealed the marked enrichment of exosomal proteins, including CD9, CD63, LAMP‐1, and LAMP‐2 and a concomitant up‐regulation of the Annexin family of calcium‐binding proteins. Of particular note Annexin VI was shown to be enriched in calcifying VIC‐derived MVs (51.9‐fold; p < 0.05). Through bioinformatic analysis using Ingenuity Pathway Analysis (IPA), the up‐regulation of canonical signaling pathways relevant to cardiovascular function were identified in calcifying VIC‐derived MVs, including aldosterone, Rho kinase, and metal binding. Further studies using human calcified valve tissue revealed the co‐localization of Annexin VI with areas of MVs in the extracellular matrix by transmission electron microscopy (TEM). Together these findings highlight a critical role for VIC‐derived MVs in CAVD. Furthermore, we identify calcium as a key driver of aortic valve calcification, which may directly underpin the increased susceptibility of ESRD patients to accelerated development of CAVD.
Arterial calcification is an important hallmark of cardiovascular disease and shares many similarities with skeletal mineralization. The bone-specific protein osteocalcin (OCN) is an established marker of vascular smooth muscle cell (VSMC) osteochondrogenic transdifferentiation and a known regulator of glucose metabolism. However, the role of OCN in controlling arterial calcification is unclear. We hypothesized that OCN regulates calcification in VSMCs and sought to identify the underpinning signaling pathways. Immunohistochemistry revealed OCN co-localization with VSMC calcification in human calcified carotid artery plaques. Additionally, 3 mM phosphate treatment stimulated OCN mRNA expression in cultured VSMCs (1.72-fold, p < 0.001). Phosphate-induced calcification was blunted in VSMCs derived from OCN null mice (Ocn −/− ) compared with cells derived from wild-type (WT) mice (0.37-fold, p < 0.001). Ocn −/− VSMCs showed reduced mRNA expression of the osteogenic marker Runx2 (0.51-fold, p < 0.01) and the sodium-dependent phosphate transporter, PiT1 (0.70-fold, p < 0.001), with an increase in the calcification inhibitor Mgp (1.42-fold, p < 0.05) compared with WT. Ocn −/− VSMCs also showed reduced mRNA expression of Axin2 (0.13-fold, p < 0.001) and Cyclin D (0.71 fold, p < 0.01), markers of Wnt signaling. CHIR99021 (GSK3β inhibitor) treatment increased calcium deposition in WT and Ocn −/− VSMCs (1 μM, p < 0.001). Ocn −/− VSMCs, however, calcified less than WT cells (1 μM; 0.27-fold, p < 0.001). Ocn −/− VSMCs showed reduced mRNA expression of Glut1 (0.78-fold, p < 0.001), Hex1 (0.77-fold, p < 0.01), and Pdk4 (0.47-fold, p < 0.001). This was accompanied by reduced glucose uptake (0.38-fold, p < 0.05). Subsequent mitochondrial function assessment revealed increased ATP-linked respiration (1.29-fold, p < 0.05), spare respiratory capacity (1.59-fold, p < 0.01), and maximal respiration (1.52-fold, p < 0.001) in Ocn −/− versus WT VSMCs. Together these data suggest that OCN plays a crucial role in arterial calcification mediated by Wnt/β-catenin signaling through reduced maximal respiration. Mitochondrial dynamics may therefore represent a novel therapeutic target for clinical intervention.
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