Mecanismos fisiopatológicos del remodelado y la calcificación arterial en la cardiopatía isquémica

Zusammenfassung

Abstract Background. Vascular calcification is an independent cardiovascular risk factor and it is frequently associated with clinical conditions such as diabetes, chronic kidney disease, anti-vitamin K drugs, ageing and congenital diseases. Vascular calcification is the main anatomopathological characteristic feature of advanced atherosclerotic lesion type Vb according to Stary's classification. Combination of coronary computerized tomography (CCT) and Agatston score allows quantification of coronary artery calcification (CAC). Mineralization and vascular smooth muscle cell (VSMC) differentiation into osteoblast/chondroblast-like cells are physiopathological characteristics and result from the lack of vascular calcification inhibitors (carboxyated matrix Gla protein, cMGP) and hyperphosphatemia (HPM). Calumenin, an endogenous inhibitor of gamma-carboxylation, as well as both prolil-lysil oxidases and lysil oxidases (e.g.: PLOD and LOX) involved in collagen assembly and cross-linking, may contribute to the development of vascular calcification. Aims. We aimed: i) to assess the role of a single nucleotide polymorphism (SNP) rs1043550 in the 3'-untranslated region (UTR) CALU as well as the role of calumenin during vascular calcification, ii) to explore the role of MEC in a vascular calcification cell model using human and mouse VSMC, and iii) to compare warfarin and acenocumarol effects on VSMC focusing on mineralization and osteoblast differentiation. Methods We recruited prospective steady and stable patients from the Hospital Clínico Universitario Virgen de la Arrixaca (Murcia, Spain) to study the relationship between SNP CALU rs1043550 and CAC assessed by TCC and Agatston score. Arterial tissues were paraffin-embedded for histological assessment of mineralization (Alizarin Red), calumenin and MGP immunohistochemistry, both in association with the underlying CALU allelic variant carriage. TA-cloning was performed to insert 3'UTR CALU in a pGL3 Basic reporter plasmid vector backbone containing LUC gene (p3U-CALUa), and on-site directed mutagenesis was performed to obtain CALU rs1043550 polymorphic variant (p3U-CALUg), thereafter. Luciferase assays were performed in endothelial cells (HUVEC) and VSMC. Human VSMC were used to assess in vitro vascular calcification by using inorganic phosphate HPM conditioning during 2-18 days. VSMC (mVSMC) isolated from wild-type or transgenic mice were used to explore the role of LOX-overexpression on in vitro vascular calcification and chemical inhibitors for evaluating PLOD and LOX/LOXL activities. Sub therapeutic doses of warfarin or acenocumarol were also used for evaluation in calcification involvement. Gene expression was assessed by qPCR. SDS-PAGE and western blot were performed to confirm protein synthesis. Calcium amount from cell monolayers was collected into aqueous acid solutions and quantified by o-cresophtalein method or visualized by Von Kossa staining on cell monolayers. Results. Wild-type allele carriage for SNP CALU rs1043550 was associated with high CAC and lesion severity. Histological analysis in femoral tissues also demonstrated higher residual calcification but lower calumenin and MGP in wild-type homozygous carriers. Moreover, luciferase activity was 30% lowered in p3U-CALUg transfected cells. Accordingly, SNP CALU rs1043550 can impair the stability of mRNA CALU, therefore suggesting that SNP CALU rs1043550 could have functional and protective effects in the vascular calcification scenario. Moreover, calumenin expression and synthesis profile showed an overexpression in early calcification stages of VSMC and it was down-regulated thereafter. Calumenin regulation during calcification was concomitant with progressive increased mineralization, VSMC phenotype repression (SM22- ) and osteoblast markers over-expression (BMP2, RUNX2, SP7, CTNNB1 and BGLAP/osteocalcin). Lowered intracellular calumenin was found concordant with a modest CALU gene repression. Moreover, HPM was associated with the activation of calumenin secretory pathways resulting in calumenin release into the extracellular site and its co-localization with calcium deposits. Moreover, calumenin progressive decrease was associated with a progressive increase in osteocalcin and cMGP levels. Carboxylated osteocalcin is functional and mandatory for mineralized ECM maturation. In addition, calumenin regulation was demonstrated to be partially regulated by miRNA. Potential binding sites in 3'-UTR CALU were bioinformatically predicted for miR-132 and miR-218 and the miR-dependent regulation was confirmed using expression vectors. Both miRNAs were found down-regulated during the early stages of VSMC calcification in association with CALU expression and synthesis. Moreover miRNA mimic assays demonstrated a repression in calumenin at the protein and mRNA level, in BMP2 expression and a lowered mineralization. Moreover, LOX over-expression or its enzymatic activity inhibition (BAPN) were found to increase or decrease mineralization and VSMC differentiation into osteoblast-like cells, respectively. PLOD inhibition (2,2'-dipyridil) was also associated with lower calcium deposition and differentiation. MEC seems to contribute to vascular calcification by regulating cell communication and differentiation processes further than as a mineralization scaffold. Finally, warfarin and acenocumarol leaded to higher calcification than HPM control condition. Our results suggest that acenocumarol is more potent than warfarin, therefore suggesting it may be a good alternative to develop vascular calcification models in VSMC. Interestingly, acenocumarol was associated with an increased expression and synthesis of calumenin in later stages than HPM similarly to hypertrophic chondroblasts.