Ucma/GRP inhibits phosphate-induced vascular smooth muscle cell calcification via SMAD-dependent BMP signalling

Vascular calcification (VC) is the process of deposition of calcium phosphate crystals in the blood vessel wall, with a central role for vascular smooth muscle cells (VSMCs). VC is highly prevalent in chronic kidney disease (CKD) patients and thought, in part, to be induced by phosphate imbalance. The molecular mechanisms that regulate VC are not fully known. Here we propose a novel role for the mineralisation regulator Ucma/GRP (Upper zone of growth plate and Cartilage Matrix Associated protein/Gla Rich Protein) in phosphate-induced VSMC calcification. We show that Ucma/GRP is present in calcified atherosclerotic plaques and highly expressed in calcifying VSMCs in vitro. VSMCs from Ucma/GRP(-/-) mice showed increased mineralisation and expression of osteo/chondrogenic markers (BMP-2, Runx2, beta-catenin, p-SMAD1/5/8, ALP, OCN), and decreased expression of mineralisation inhibitor MGP, suggesting that Ucma/GRP is an inhibitor of mineralisation. Using BMP signalling inhibitor noggin and SMAD1/5/8 signalling inhibitor dorsomorphin we showed that Ucma/GRP is involved in inhibiting the BMP-2-SMAD1/5/8 osteo/chondrogenic signalling pathway in VSMCs treated with elevated phosphate concentrations. Additionally, we showed for the first time evidence of a direct interaction between Ucma/GRP and BMP-2. These results demonstrate an important role of Ucma/GRP in regulating osteo/chondrogenic differentiation and phosphate-induced mineralisation of VSMCs.NWO ZonMw [MKMD 40-42600-98-13007]; FCT [SFRH/BPD/70277/2010]info:eu-repo/semantics/publishedVersio

Vascular calcifications, vertebral fractures and mortality in haemodialysis patients

Background. Vascular calcifications and the bone fractures caused by abnormal bone fragility, also called osteoporotic fractures, are frequent complications associated with chronic kidney diseases (CKD). The aim of this study was to investigate the association between vascular calcifications, osteoporotic bone fractures and survival in haemodialysis (HD) patients

Microcalcifications in breast cancer: novel insights into the molecular mechanism and functional consequence of mammary mineralisation.

BACKGROUND: Mammographic microcalcifications represent one of the most reliable features of nonpalpable breast cancer yet remain largely unexplored and poorly understood. METHODS: We report a novel model to investigate the in vitro mineralisation potential of a panel of mammary cell lines. Primary mammary tumours were produced by implanting tumourigenic cells into the mammary fat pads of female BALB/c mice. RESULTS: Hydroxyapatite (HA) was deposited only by the tumourigenic cell lines, indicating mineralisation potential may be associated with cell phenotype in this in vitro model. We propose a mechanism for mammary mineralisation, which suggests that the balance between enhancers and inhibitors of physiological mineralisation are disrupted. Inhibition of alkaline phosphatase and phosphate transport prevented mineralisation, demonstrating that mineralisation is an active cell-mediated process. Hydroxyapatite was found to enhance in vitro tumour cell migration, while calcium oxalate had no effect, highlighting potential consequences of calcium deposition. In addition, HA was also deposited in primary mammary tumours produced by implanting the tumourigenic cells into the mammary fat pads of female BALB/c mice. CONCLUSION: This work indicates that formation of mammary HA is a cell-specific regulated process, which creates an osteomimetic niche potentially enhancing breast tumour progression. Our findings point to the cells mineralisation potential and the microenvironment regulating it, as a significant feature of breast tumour development

Abdominal aortic calcification quantified by the Morphological Atherosclerotic Calcification Distribution (MACD) index is associated with features of the metabolic syndrome

<p>Abstract</p> <p>Background</p> <p>Abdominal aortic calcifications (AAC) predict cardiovascular mortality. A new scoring model for AAC, the Morphological Atherosclerotic Calcification Distribution (MACD) index may contribute with additional information to the commonly used Aortic Calcification Severity (AC24) score, when predicting death from cardiovascular disease (CVD). In this study we investigated associations of MACD and AC24 with traditional metabolic-syndrome associated risk factors at baseline and after 8.3 years follow-up, to identify biological parameters that may account for the differential performance of these indices.</p> <p>Methods</p> <p>Three hundred and eight healthy women aged 48 to 76 years, were followed for 8.3 ± 0.3 years. AAC was quantified using lumbar radiographs. Baseline data included age, weight, blood pressure, blood lipids, and glucose levels. Pearson correlation coefficients were used to test for relationships.</p> <p>Results</p> <p>At baseline and across all patients, MACD correlated with blood glucose (r²= 0.1, P< 0.001) and to a lesser, but significant extent with traditional risk factors (p < 0.01) of CVD. In the longitudinal analysis of correlations between baseline biological parameters and the follow-up calcification assessment using radiographs we found LDL-cholesterol, HDL/LDL, and the ApoB/ApoA ratio significantly associated with the MACD (P< 0.01). In a subset of patients presenting with calcification at both baseline and at follow-up, all cholesterol levels were significantly associated with the MACD (P< 0.01) index. AC24 index was not correlated with blood parameters.</p> <p>Conclusion</p> <p>Patterns of calcification identified by the MACD, but not the AC24 index, appear to contain useful biological information perhaps explaining part of the improved identification of risk of cardiovascular death of the MACD index. Correlations of MACD but not the AC24 with glucose levels at baseline suggest that hyperglycemia may contribute to unique patterns of calcification indicated by the MACD.</p

Prognostic significance of osteopontin expression in early-stage non-small-cell lung cancer

Osteopontin (OPN) is a multifunctional protein, which has recently been shown to be linked to tumorigenesis, progression and metastasis in different malignancies. Since non-small-cell lung cancer (NSCLC)'s prognosis remains bad, with few predictors of outcome, the purpose of this study was to evaluate if OPN might be involved in NSCLC's biology and therefore represent a prognostic marker and a target for new therapeutic trials. Immunohistochemistry was used to detect OPN expression, evaluated as percentage of neoplastic cells with cytoplasmic immunoreactivity, in a wide cohort of patients with stage I NSCLC (136 cases). The median value of this series (20% of positive cells) was used as the cutoff value to distinguish tumours with low (<20%) from tumours with high (⩾20%) OPN expression. A statistically significant correlation between high levels of OPN and shorter overall (P=0.034) and disease-free (P=0.011) survival in our patients was shown. Our results support the hypothesis that high OPN expression is a significantly unfavourable prognostic factor for the survival of patients with stage I NSCLC. This conclusion has notable importance in terms of the biological characterization of early-stage tumours and therapeutic opportunities

Combination of Sleeping Beauty transposition and chemically induced dimerization selection for robust production of engineered cells

The main methods for producing genetically engineered cells use viral vectors for which safety issues and manufacturing costs remain a concern. In addition, selection of desired cells typically relies on the use of cytotoxic drugs with long culture times. Here, we introduce an efficient non-viral approach combining the Sleeping Beauty (SB) Transposon System with selective proliferation of engineered cells by chemically induced dimerization (CID) of growth factor receptors. Minicircles carrying a SB transposon cassette containing a reporter transgene and a gene for the F36VFGFR1 fusion protein were delivered to the hematopoietic cell line Ba/F3. Stably-transduced Ba/F3 cell populations with >98% purity were obtained within 1 week using this positive selection strategy. Copy number analysis by quantitative PCR (qPCR) revealed that CID-selected cells contain on average higher copy numbers of transgenes than flow cytometry-selected cells, demonstrating selective advantage for cells with multiple transposon insertions. A diverse population of cells is present both before and after culture in CID media, although site-specific qPCR of transposon junctions show that population diversity is significantly reduced after selection due to preferential expansion of clones with multiple integration events. This non-viral, positive selection approach is an attractive alternative for producing engineered cells

Association of Osteocalcin and Abdominal Aortic Calcification in Older Women: The Study of Osteoporotic Fractures

Osteocalcin (OC) is produced by osteoblasts and vascular smooth muscle cells. In animal models, serum OC levels are strongly correlated with vascular calcium content, however, the association of OC with vascular calcification in humans is uncertain. The Study of Osteoporotic Fractures (SOF) enrolled community-living women, age ≥65 years. The present study included a subsample of 363 randomly selected SOF participants. Serum total OC was measured by ELISA, and abdominal aortic calcification (AAC) was evaluated on lateral lumbar radiographs. We examined the cross-sectional association between serum OC and AAC. The mean serum OC level was 24 ± 11 ng/ml and AAC was present in 188 subjects (52%). We observed no association of OC and AAC in either unadjusted or adjusted analyses. For example, each standard deviation higher OC level was associated with an odds ratio (OR) for AAC prevalence (AAC score >0) near unity (OR = 1.06; 95% CI, 0.82–1.36) in models adjusted for CVD risk factors. Further adjustment for intact parathyroid hormone, bone-specific alkaline phosphatase, 25-hydroxyvitamin D, and hip and spine bone mineral density did not materially change the results (OR = 1.22; 95% CI, 0.86–1.75). Similarly, higher OC levels were not associated with severity of AAC (P = 0.87). In conclusion, among community-living older women, serum OC is not associated with AAC. These findings suggest that serum OC levels may more closely reflect bone formation than vascular calcification in humans

Short-term effects of amelogenin gene splice products A+4 and A-4 implanted in the exposed rat molar pulp

In order to study the short-time effects of two bioactive low-molecular amelogenins A+4 and A-4, half-moon cavities were prepared in the mesial aspect of the first maxillary molars, and after pulp exposure, agarose beads alone (controls) or beads soaked in A+4 or A-4 (experimental) were implanted into the pulp. After 1, 3 or 7 days, the rats were killed and the teeth studied by immunohistochemistry. Cell proliferation was studied by PCNA labeling, positive at 3 days, but decreasing at day 7 for A+4, whilst constantly high between 3 and 7 days for A-4. The differentiation toward the osteo/odontoblast lineage shown by RP59 labeling was more apparent for A-4 compared with A+4. Osteopontin-positive cells were alike at days 3 and 7 for A-4. In contrast, for A+4, the weak labeling detected at day 3 became stronger at day 7. Dentin sialoprotein (DSP), an in vivo odontoblast marker, was not detectable until day 7 where a few cells became DSP positive after A-4 stimulation, but not for A+4. These results suggest that A +/- 4 promote the proliferation of some pulp cells. Some of them further differentiate into osteoblast-like progenitors, the effects being more precocious for A-4 (day 3) compared with A+4 (day 7). The present data suggest that A +/- 4 promote early recruitment of osteogenic progenitors, and evidence functional differences between A+4 and A-4

Risk factors of one year increment of coronary calcifications and survival in hemodialysis patients

<p>Abstract</p> <p>Background</p> <p>Heart and coronary calcifications in hemodialysis patients are of very common occurrence and linked to cardiovascular events and mortality. Several studies have been published with similar results. Most of them were mainly cross-sectional and some of the prospective protocols were aimed to evaluate the results of the control of altered biochemical parameters of mineral disturbances with special regard to serum calcium, phosphate and CaxP with the use of calcium containing and calcium free phosphate chelating agents. The aim of the present study was to evaluate in hemodialysis patients classic and some non classic risk factors as predictors of calcification changes after one year and to evaluate the impact of progression on survival.</p> <p>Methods</p> <p>81 patients on hemodialysis were studied, with a wide age range and HD vintage. Several classic parameters and some less classic risk factors were studied like fetuin-A, CRP, 25-OHD and leptin. Calcifications, as Agatston scores, were evaluated with Multislice CT basally and after 12-18 months.</p> <p>Results</p> <p>Coronary artery calcifications were observed in 71 of 81 patients. Non parametric correlations between Agatston scores and Age, HD Age, PTH and CRP were significant. Delta increments of Agatston scores correlated also with serum calcium, CaxP, Fetuin-A, triglycerides and serum albumin. Logistic regression analysis showed Age, PTH and serum calcium as important predictors of Delta Agatston scores. LN transformation of the not normally distributed variables restricted the significant correlations to Age, BMI and CRP. Considering the Delta Agatston scores as dependent, significant predictors were Age, PTH and HDL. A strong association was found between basal calcification scores and Delta increment at one year. By logistic analysis, the one year increments in Agatston scores were found to be predictors of mortality. Diabetic and hypertensive patients have significantly higher Delta scores.</p> <p>Conclusions</p> <p>Progression of calcification is of common occurrence, with special regard to elevated basal scores, and is predictive of survival. Higher predictive value of survival is linked to the one year increment of calcification scores. Some classic and non classic risk factors play an important role in progression. Some of them could be controlled with appropriate management with possible improvement of mortality.</p

Modulation of calcification of vascular smooth muscle cells in culture by calcium antagonists, statins, and their combination

Background Vascular calcification is an organized process in which vascular smooth muscle cells (VSMCs) are implicated primarily. The purpose of the present study was to assess the effects of calcium antagonists and statins on VSMC calcification in vitro. Methods VSMC calcification was stimulated by incubation in growth medium supplemented with 10 mmol/l β-glycerophosphate, 8 mmol/l CaCl2, 10 mmol/l sodium pyruvate, 1 μmol/l insulin, 50 μg/ml ascorbic acid, and 100 nmol/l dexamethasone (calcification medium). Calcification, proliferation, and apoptosis of VSMCs were quantified. Results Calcium deposition was stimulated dose-dependently by β-glycerophosphate, CaCl2, and ascorbic acid (all P < 0.01). Addition of amlodipine (0.01–1 μmol/l) to the calcification medium did not affect VSMC calcification. However, atorvastatin (2–50 μmol/l) stimulated calcium deposition dose-dependently. Combining treatments stimulated calcification to a degree similar to that observed with atorvastatin alone. Both atorvastatin and amlodipine inhibited VSMC proliferation at the highest concentration used. Only atorvastatin (50 μmol/l) induced considerable apoptosis of VSMCs. Conclusion In vitro calcification of VSMCs is not affected by amlodipine, but is stimulated by atorvastatin at concentrations ≥10 μmol/l, which could contribute to the plaque-stabilizing effect reported for statins