Hydrogen sulfide prevents arterial medial calcification in rats with diabetic nephropathy

Background Arterial medial calcification (AMC) is associated with a high incidence of cardiovascular risk in patients with type 2 diabetes and chronic kidney disease. Here, we tested whether hydrogen sulfide (H2S) can prevent AMC in rats with diabetic nephropathy (DN). Methods DN was induced by a single injection of streptozotocin and high-fat diet (45% kcal as fat) containing 0.75% adenine in Sprague–Dawley rats for 8 weeks. Results Rats with DN displayed obvious calcification in aorta, and this was significantly alleviated by Sodium Hydrosulfide (NaHS, a H2S donor, 50 μmol/kg/day for 8 weeks) treatment through decreasing calcium and phosphorus content, ALP activity and calcium deposition in aorta. Interestingly, the main endogenous H2S generating enzyme activity and protein expression of cystathionine-γ-lyase (CSE) were largely reduced in the arterial wall of DN rats. Exogenous NaHS treatment restored CSE activity and its expression, inhibited aortic osteogenic transformation by upregulating phenotypic markers of smooth muscle cells SMα-actin and SM22α, and downregulating core binding factor α-1 (Cbfα-1, a key factor for bone formation), protein expressions in rats with DN when compared to the control group. NaHS administration also significantly reduced Stat3 activation, cathepsin S (CAS) activity and TGF-β1 protein level, and improved aortic elastin expression. Conclusions H2S may have a clinical significance for treating AMC in people with DN by reducing Stat3 activation, CAS activity, TGF-β1 level and increasing local elastin level.

Phenotypic characterization of rabbit model for aortic valve stenosis: a novel medial calcification paradigm

Introduction Calcific aortic valve stenosis (CAVS) is the result of subtle, chronic inflammation and osteoblastic differentiation. As we lack human specimens of the early stages, reliable and reproducible animal models are needed to facilitate research. We previously demonstrated the ability of a novel rabbit CAVS vitamin D2 toxicity protocol to produce calcification and valve stenosis (1). We sought to characterize the phenotype of the model at the final stage. Methods Twelve New Zealand Rabbits were randomized 1:1 to control (normal chaw) and experimental group (normal chaw+1% cholesterol+3.500 I.U.s Vitamin D2, in oil in a biscuit) for 7 weeks. Animals were sacrificed and aortic valve cusps were snap frozen or formalin-fixed paraffin embedded. Cusps were then mechanically homogenized in buffer optimized for protein extraction and total protein measured with Bradford method. Part of the extract was subjected to trypsinization, in-gel digestion and untargeted LC-MS/MS. The rest was used to quantitate BMP-2 with total protein-normalized sandwitch competitive ELISA. Thin tissue sections were stained with Masson's trichrome, Von Kossa and H&E. Osteopontin, Bone sialoprotein II (BSPII), tissue non-specific alkaline phosphatase (TNAP) and osteocalcin (OCN) were detected on tissue with immunohistochemistry. Femoral bones from the same animals served as positive controls. Results Aortic valve cusp demonstrate large areas of collagen degradation and calcification in the medial layer, almost sparing the intima. Osteopontin deposits were colocalized with the calcification area in the media, whereas BSPII, TNAP and OCN were not expressed in the lesion, although present in bones. Similarly, BMP-2 levels were not significantly different between groups (experimental = 43.45 vs controls = 62.75 pg/ml, Mann-Whitney U test p=0.496). Proteomic analysis revealed a set of 96 differentially expressed proteins between cases and controls, interestingly including sortilin, osteonectin, beta-crystallin A2, Matrix Gla protein, Na/H exchanger 3, V-type H ATPase subunit D, Y-box binding protein. Conclusion The novel rabbit vitamin D2 toxicity protocol leads to excessive medial calcification of the aortic valve, with overexpression of osteopontin but without other classic markers of CAVS. Proteomics analysis reveals novel pathways with pathophysiological implications for the model and medial calcification. FUNDunding Acknowledgement Type of funding sources: Public grant(s) – National budget only. Main funding source(s): Hellenic Cardiology Society, Hellenic Heart Foundation

Abstract P573: The Interplay Between Mineral and Skeletal Metabolism Biomarkers and Intracranial Internal Carotid Artery Calcifications in Stroke Patients

Background: Intracranial internal carotid artery calcifications (IICAC), considered a phenomenon within the spectrum of atherosclerosis and vascular aging, are frequently encountered in ischemic stroke patients. While intimal IICAC directly contributes to ischemic stroke pathophysiology, the medial counterpart is generally deemed as a risk factor for vascular end-points. Apart from aging and cardiovascular risk factors, the underlying pathophysiology that contributes to development of IICAC is not well understood; herein we studied the interplay between mineral and skeletal metabolism biomarkers, and IICAC presence and pattern. Methods: In a prospective series of 194 ischemic stroke patients (mean±SD age: 69±14 yr), blood samples were collected to determine calcium, phosphorus, magnesium, osteocalcin, parathyroid hormone, and vitamin D levels within 72 hours of symptom onset. IICAC presence and type was determined on admission CT-angiography source images; a medial or intimal type of IICAC category was assigned according to Kockelkoren criteria. Results: A total of 45 (23%) patients had no calcifications, while 95 (49%) had an intimal pattern and 54 (28%) had non-intimal (or medial) pattern. Apart from the well-known factors related with IICAC, such as age, lower glomerular filtration rate (GFR), history of hypertension, diabetes mellitus, coronary artery disease and atrial fibrillation, we identified admission magnesium levels to be associated with IICAC presence and pattern (no calcification: 1.96±0.18 mEq/L, intimal calcification 1.93±0.19 mEq/L, medial calcification: 1.81±0.28 mEq/L; p=0.006). None of the other biomarkers had any significant relationship to IICAC. In multivariate models, a lower magnesium level was significantly associated with medial calcification (each 0.1 mEq/L drop increased the odds by 1.2 (95% CI 1.0-1.4; p=0.046)), in addition to older age, history of diabetes mellitus and lower GFR. Conclusion: Hypomagnesemia is one of the factors well-known to be associated with vascular calcifications in the body. Our findings, extend this relationship to the intracranial vascular bed. No similar association was observed for other biomarkers related to mineral and skeletal metabolism. (Funding: TUBITAK grant 218S753)

Abstract 17021: A Novel Human Relaxin Receptor Agonist, ML290, Attenuates Atherosclerosis- and Chronic Kidney Disease-Induced Vascular Calcification in Mice

Introduction: Vascular calcification is the most significant predictor of cardiovascular morbidity and mortality, but therapeutic options are unavailable. Relaxin has emerged as a vasoprotective molecule, but several drawbacks prevent therapeutic translation. Targeting the relaxin receptor, RXFP1, is safe and well-tolerated in animal models of vascular disease and humans. We identified a biased allosteric agonist of human RXFP1, ML290, and aimed to test the hypothesis that ML290 arrests the progression of vascular calcification in mouse models of atherosclerosis and chronic kidney disease (CKD). Methods and Results: Recurrent treatment with ML290 significantly prevented ( P = 0.0422, n = 8) and reversed ( P = 0.0489, n = 6) atherosclerotic calcification in humanized ( hRXFP1/hRXFP1 ) Apoe -/- mice fed an atherogenic diet. Longitudinal tracing of mineral formation in the aortic arch of these mice revealed the presence of mineral in vehicle- but not ML290-treated mice after 15 weeks of diet. Accelerated mineral growth was observed in vehicle-treated mice after 20 weeks of the diet, which was reduced by ML290 treatment. In humanized mice with CKD, ML290 significantly prevented ( P = 0.0344, n = 9) medial calcification. In vitro , ML290 reduced ( P = 0.0005, n = 3) superoxide production under osteogenic conditions in vascular smooth muscle cells (VSMCs). Osteogenic changes in VSMC phenotype associate with a release of alkaline phosphatase (ALP) in extracellular vesicles (EVs), which promote mineralization. ML290 treatment significantly ( P = 0.0001, n = 3) suppressed the formation of ALP-loaded EVs in vitro . Bone morphogenetic protein-4, an inducer of osteogenic transitions, and caveolin-1, a scaffolding protein required for calcifying EV formation, were significantly ( P = 0.0059, n = 4) down-regulated after 24 h treatment with ML290 compared to vehicle-treated VSMCs under osteogenic conditions. Conclusions: We demonstrate the therapeutic potential for ML290 to mitigate atherosclerosis and CKD-induced vascular calcification in vivo . The actions of ML290 to prevent medial calcification are in part attributed to its ability to limit the release of calcifying EVs as a result of osteogenic differentiation, and to reduce vascular superoxide production.