Functional interaction of osteogenic transcription factors Runx2 and Vdr in transcriptional regulation of Opn during soft tissue calcification.

Loss of Abcc6 gene expression was identified to be responsible for dystrophic calcification of the heart (DCC) or vessels after acute injury in several strains of laboratory mice. This calcification shares features with osteogenesis and may involve osteogenic factors. Tissue expression of osteopontin (Opn) and 11 osteogenic transcription factors were studied in vivo in mouse models for DCC and in vitro using luciferase reporter gene assays. Compared with DCC-resistant C57BL/6 mice, a significant increase in Opn transcription was demonstrated in necrotic lesions of both DCC-susceptible C3H/He and B6.C3H(Dyscalc1) congenic mice at day 3 after injury. Significant increases in gene expression were also demonstrated for the transcription factors runt domain-containing transcription factor 2 (Runx2), vitamin D receptor (Vdr), SRY (sex-determining region Y)-box 9 protein, and Nfkb1 in C3H/He mice versus C57BL/6 controls. However, only Runx2 remained significantly increased in the B6.C3H(Dyscalc1) congenic mice, which carry only the Dyscalc1 locus with functional Abcc6 deletion on a C57BL/6 genetic background. Luciferase assay use increased Opn promoter activity, which was demonstrated after overexpression of Runx2. A poly-T stretch insertion was identified to stabilize the binding of Runx2, thus significantly enhancing Opn promoter activity. This Runx2-mediated activation was further enhanced by cotransfection with Vdr. Our data suggest a key role of Runx2 in the regulation of Opn in a model of cardiovascular calcification and demonstrate a synergistic cooperation of Runx2 and Vdr.

Ultrafine mapping of Dyscalc1 to an 80-kb chromosomal segment on chromosome 7 in mice susceptible for dystrophic calcification.

In mice, dystrophic cardiovascular calcification (DCC) is controlled by a major locus on proximal mouse chromosome 7 named Dyscalc1. Here we present a strategy that combines in silico analysis, expression analysis, and extensive sequencing for ultrafine mapping of the Dyscalc1 locus. We subjected 15 laboratory mouse strains to freeze-thaw injury of the heart, and association with respective genotypes allowed condensation of the Dyscalc1 locus to 1 Mb. Within this region, 51 known and predicted genes were studied in DCC-susceptible C3H/He and DCC-resistant C57BL/6 mice with respect to mRNA expression in response to injury. Five genes displayed differential expression. Genotyping of seven novel single nucleotide polymorphisms (SNPs) within these genes revealed an 80-Kb region in NZB mice that were found positive for calcification though carrying otherwise alleles from DCC-resistant mice. This microheterogeneity in NZB mice was evolutionary conserved in all DCC-susceptible mouse strains and contains the genes EMP-3, BC013491, and Abcc6 (partially). The flanking SNPs are rs3703247 and NT_039420.5_2757991. mRNA levels of EMP-3 were found to be upregulated in response to injury in both C57BL/6 and C3H/He mice. Sequencing of EMP-3 revealed an SNP leading to an amino acid substitution (p.T153I) that was found in all mouse strains susceptible for DCC but not in resistant strains such as C57BL/6 mice. Thus, the p.T153I changes might affect the biological function of EMP-3 gene product after injury. Using this combined approach, we ultrafine-mapped the Dyscalc1 locus to an 80-Kb region and identified EMP-3 as a new candidate gene for DCC.

Takayasu's arteritis: a case with relapse after urgent coronary revascularization.

BACKGROUND: Vasculitides are commonly unrecognized causes of coronary stenosis and myocardial ischemia. We report on a 24-year old patient with Takayasu's arteritis who underwent urgent percutaneous coronary intervention, suffered from symptomatic restenosis of the left main coronary artery during standard immunosuppressive therapy. CASE PRESENTATION: A 24-year old woman was referred for coronary angiography because of typical progressive angina pectoris. On bicycle ergometry, there were both reproducible symptoms and deep ST segment depressions on precordial leads. Semi-selective angiography of the left coronary artery revealed high-grade ostial stenosis. Because of persistent angina pectoris and electrocardiographic signs of acute myocardial ischemia, immediate percutaneous coronary angioplasty with subsequent implantation of an everolimus-eluting stent was performed. This intervention was performed with excellent angiographic results. Because of several concomitant criteria including hypoechogenicity on postprocedural intravascular ultrasonography, the diagnosis of Takayasu's disease was made. The patient was treated with prednisolone and cyclophosphamide for 5 months. Because of recurrent angina pectoris, another coronary angiography was performed, which revealed high-grade in-stent-restenosis. Immunomodulatory therapy was switched to high-dose prednisolone and the anti-IL-6 receptor antagonist tocilizumab. The high-grade in-stent-restenosis persisted, and aortocoronary bypass graft surgery was performed with two saphenous vein grafts to the left anterior descending and circumflex artery. Since then, the patient has been doing well for 2 years. CONCLUSION: In cases of treatment refractoriness during standard immunosuppressive therapy, more recently developed biological compounds may offer an alternative strategy.

Arterial calcification in mice after freeze-thaw injury.

Vascular calcification is highly correlated with atherosclerosis and cardiovascular disease and is a significant predictor of cardiovascular morbidity and mortality. Studies in mice indicate a genetic contribution to this dystrophic extra osseous calcification. We sought to elaborate a method to induce dystrophic arterial calcification in mice and further examine the pathogenetical mechanisms involved in the phenotype. We established a method of freeze-thaw injury of the infrarenal aorta producing a limited tissue necrosis and histologically investigated the occurrence of dystrophic calcification within the aortic wall 1, 3 and 7 days after injury in C57BL/6 (a mouse strain shown to be resistant to dystrophic cardiac calcification after injury) and C3H/He (susceptible to dystrophic cardiac calcification). C57BL/6 mice exhibited no dystrophic calcification at all within the vessel wall upon injury of the infrarenal aorta (0/5 mice 1 day after injury and 0/10 animals 7 days after injury). By contrast C3H/He mice displayed a remarkable extent of calcification mainly present within the media of the infrarenal aorta which was evident as early as 24 h (three out of five animals 1 day after injury) and reached its maximum extent 7 days after injury (10 out of 10 animals at the seventh postoperative day, p<0.001 compared to C57BL/6 mice). Upon immuno-histological analysis calcification was accompanied by the occurrence of certain bone-matrix associated proteins. Osteopontin and Bone Morphogenetic Protein 2/4 expression was detected co-localized with the calcified lesions. Our results demonstrate that freeze-thaw injury of the infrarenal aorta is a sufficient method to induce dystrophic arterial calcification in mice. We present evidence that the occurrence of arterial calcification in C3H/He mice seems to be actively regulated by certain bone-matrix associated proteins.

Hematopoietic progenitor cells residing in muscle engraft into bone marrow following transplantation.

Hematopoietic and mesenchymal stem cells can potentially be the same cell type or adhere simultaneously in both bone marrow (BM) and muscle. In this study, we asked whether murine BM-derived cells could be tracked in muscle tissue after BM transplantation and whether muscle-derived cells have hematopoietic potential. To answer the first question, we transplanted BM from male BALB/c mice into irradiated female recipients and analyzed for engraftment. We used quantitative polymerase chain reaction (PCR) and fluorescent in situ hybridization techniques for Y chromosome-specific gene probes. A high number of BM-derived cells were located in both the intravascular and extravascular spaces in muscle tissue after BM transplantation. To answer the second question, we analyzed colony-forming potential in vitro with soft-agar assays and the competitive engraftment potential in vivo of muscle-derived cells. Engraftment levels of male cell populations were tested by quantitative PCR. The long-term engraftment potential of muscle-derived cells was low compared with that of BM. We conclude that there is intensive cellular trafficking between BM and muscle tissue. The engraftment potential of muscle-derived stem cells into BM is low and corresponds to the low amounts of hematopoietic colony-forming cells found in muscle tissue.

An alternative splice variant in Abcc6, the gene causing dystrophic calcification, leads to protein deficiency in C3H/He mice.

Dystrophic cardiac calcification (DCC) is an autosomal recessive trait characterized by calcium phosphate deposits in myocardial tissue. The Abcc6 gene locus was recently found to mediate DCC; however, at the molecular level the causative variants remain to be determined. Examining the sequences of Abcc6 cDNA in DCC-resistant C57BL/6 and DCC-susceptible C3H/He mice, we identified a missense mutation (Cys to Thr at codon 619, rs32756904) at the 3'-border of exon 14 that creates an additional donor splice site (GT). Accordingly, an alternative transcript variant was detected, lacking the last 5 bp of exon 14 (-AGG(C/T)GCTgtga-) in DCC-susceptible C3H/He mice that carry the Thr allele. The 5-bp deletion was found to result in premature termination at codon 684, in turn leading to protein deficiency in DCC-susceptible mouse tissue as well as in cells transfected with Abcc6 cDNA lacking the last 5 bp of exon 14. All mouse strains that were found to carry the Thr allele, including C3H/He, DBA/2J, and 129S1/SvJ, were also found to be positive for DCC. In summary, we identified a splice variant leading to a 5-bp deletion in the Abcc6 transcript that gives rise to protein deficiency both in vivo and in vitro. The fact that all mouse strains that carry the deletion also develop dystrophic calcifications further suggests that the underlying splice variant affects the biological function of MRP6 protein and is a cause of DCC in mice.

A locus on chromosome 7 determines dramatic up-regulation of osteopontin in dystrophic cardiac calcification in mice.

Calcification of necrotic tissue is frequently observed in chronic inflammation and atherosclerosis. A similar response of myocardium to injury, referred to as dystrophic cardiac calcinosis (DCC), occurs in certain inbred strains of mice. We now examined a putative inhibitor of calcification, osteopontin, in DCC after transdiaphragmal myocardial freeze-thaw injury. Strong osteopontin expression was found co-localizing with calcification in DCC-susceptible strain C3H/HeNCrlBr, which exhibited low osteopontin plasma concentrations otherwise. Osteopontin mRNA induction was 20-fold higher than in resistant strain C57BL/6NCrlBr, which exhibited fibrous lesions without calcification and little osteopontin expression. Sequence analysis identified several polymorphisms in calcium-binding and phosphorylation sites in osteopontin cDNA. Their potential relevance for DCC was tested in congenic mice, which shared the osteopontin locus with C57BL/6NCrlBr, but retained a chromosomal segment from C3H/HeNCrlBr on proximal chromosome 7. These mice exhibited strong osteopontin expression and DCC comparable to C3H/HeNCrlBr suggesting that a trans-activator of osteopontin transcription residing on chromosome 7 and not the osteopontin gene on chromosome 5 was responsible for the genetic differences in osteopontin expression. A known osteopontin activator encoded by a gene on chromosome 7 is the transforming growth factor-beta1, which was more induced (3.5x) in C3H/HeNCrlBr than in C57BL/6NCrlBr mice.