Vascular, valvular and kidney calcification manifested in mouse models of adenine-induced chronic kidney disease.

BACKGROUND: Ectopic calcification (EC) involves multiple organ systems in chronic kidney disease (CKD). Previous CKD-animal models primarily focused on a certain histological abnormality but did not show the correlation with calcified development among various tissues. This study compared calcified deposition in various tissues during CKD progression in mice. METHODS: Male 8-week-old C57BL/6J mice were randomly allocated to the seven groups: a basic, adenine, high-phosphorus, or adenine and high-phosphorus diet for 12-16 weeks (Ctl16, A12, P16, or AP16, respectively); an adenine diet for 4-6 weeks; and a high-phosphorus or adenine and high-phosphorus diet for 10-12 weeks (A6 + P10, A4 + P12, or A4 + AP12, respectively). RESULTS: Compared to the Ctl16 mice, the P16 mice only displayed a slight abnormality in serum calcium and phosphorus; the A12 mice had the most serious kidney impairment; the A4 + P12 and A6 + P10 mice had similar conditions of CKD, mineral abnormalities, and mild calcification in the kidney and aortic valves; the A4 + AP12 and AP16 groups had severe kidney impairment, mineral abnormalities and calcification in the kidneys, aortic valves and aortas. Furthermore, calcium-phosphate particles were deposited not only in the tubulointerstitial compartment but in the glomerular and tubular basement membrane. The elemental composition of EC in various tissues matched the calcification of human cardiovascular tissue as determined by energy dispersive spectroscopy. CONCLUSIONS: The severity of CKD was unparalleled with the progression of mineral metabolism disorder and EC. Calcification was closely related in different tissues and observed in the glomerular and tubular basement membranes.

Previous CKD-animal models primarily focused on a certain histological abnormality but lacked investigations of the interplay of EC in various tissues. This study compared calcified deposition in several tissues during CKD progression in mice, which was closely related. The severity of CKD was unparalleled with the development of ectopic calcification. Glomerular and tubular basement membrane calcification was detected in CKD mice, which has been considered extremely rare in clinical.

Scrotal calcinosis in a patient treated with isotretinoin: a rare entity.

Scrotal calcinosis is a rare disorder characterized by multiple papules or nodules of calcification in the scrotal skin. The pathogenesis of this disease is poorly understood. The condition presents as several brown to yellowish asymptomatic nodules on the scrotum. Excision followed by scrotal reconstruction is the treatment of choice. It leaves a good cosmetic result with low chances of recurrence. Newer treatments, such as ablative lasers, have been proposed with very good results. We describe the case of a 28-year-old patient with a history of severe acne treated with oral isotretinoin that presented for scrotal nodules. On laboratory examination, hypercalcemia was found with normal phosphorus, parathyroid hormone, and vitamin D hormone levels. Hypercalcemia was linked to his isotretinoin therapy. Serum calcium concentrations normalized after cessation of isotretinoin and hydration. Because the patient refused surgery, a biopsy of the lesion confirmed the diagnosis of scrotal calcinosis. Then the patient was referred to a cosmetic laser center to treat his condition.

Acute Calcific Band Keratopathy as an Adverse Effect of Recombinant Human Nerve Growth Factor (Cenegermin): A Multicenter Case Series.

PURPOSE: Cenegermin, (OXERVATE) a recently Food and Drug Administration-approved topical formulation of recombinant human nerve growth factor, has been used for the treatment of neurotrophic keratopathy (NK). Corneal deposits have been previously reported as a potential adverse effect; however, the clinical characteristics, visual significance, and treatment options have not been fully described. The purpose of this article is to better characterize corneal deposits occurring during treatment with cenegermin for neurotrophic keratopathy. METHODS: This was a retrospective, multicenter consecutive case series. RESULTS: We identified 5 patients from 3 institutions who developed a white opacity in varying layers of the cornea, consistent with calcium deposition, during treatment with cenegermin. In all cases, the opacity occurred rapidly over the course of a few weeks after initiation of treatment. Histopathologic examination of the cornea from one corneal patient demonstrated extensive calcification of the stroma extending to 90% depth. Before treatment, all patients had stage 2 or 3 NK (Mackie classification). The deposits were visually significant in all patients and did not resolve after cessation of cenegermin. There were no differences in age, sex, etiology of the NK, corneal transplant status, or concurrent medications between the patients who developed a deposit and 15 other patients with stage 2 or 3 NK who did not. One patient was successfully treated with superficial keratectomy with ethylenediaminetetraacetic acid chelation, one patient underwent penetrating keratoplasty, and one patient received a Boston keratoprosthesis. CONCLUSIONS: We report the rapid onset of a corneal opacity after initiation of treatment with cenegermin in patients with stage 2 or 3 NK, consistent with acute calcific band keratopathy. This visually significant adverse finding has not previously been described. We could not identify any risk factors for development. We recommend close monitoring of patients receiving cenegermin therapy because the opacity may be irreversible and may require keratoplasty for visual rehabilitation.

The intermediate-conductance calcium-activated potassium channel KCa3.1 contributes to alkalinization-induced vascular calcification in vitro.

OBJECTIVE: In order to find new strategies for the prevention of vascular calcification in uremic individuals especially treated by dialysis and develop novel therapeutic targets in vascular calcification, we explore the role of KCa3.1 in alkalinization-induced VSMCs calcification in vitro. METHOD: Rat VSMCs calcification model was established by beta-glycerophosphate (ß-GP, 10 mM) induction. The pH of Dulbecco's modified Eagle's medium (DMEM) was adjusted every 24 h with 10 mM HCl or 10 mM NaHCO3 . The mineralization was measured by Alizarin Red staining and O-cresolphthalein complex one method. mRNA and protein expression were detected by RT-PCR and Western blot or immunofluorescence. Ca2+ influx was measured by Elisa. RESULT: The results indicated that alkalization induced an increase in Ca2+ influx to enhance VSMCs calcification. Furthermore, the increase of calcification was associated with the expression of KCa3.1 via advanced expression of osteoblastic differentiation markers alkaline phosphatase (ALP) and Runt-related transcription factor 2 (Runx2). Blocking KCa3.1 with TRAM-34 or shRNA vector can significantly lowered the effects of calcification in the activity of ALP and Runx2 expression. CONCLUSION: Together all, our studies suggested that alkalinization can promote vascular calcification by upregulating KCa3.1 channel and enhancing osteogenic/chondrogenic differentiation by upregulating Runx2. The specific inhibitor TRAM-34 and KCa3.1-shRNA ameliorated VSMCs calcification by downregulating KCa3.1.

Ginkgo Biloba Extract EGB761 Alleviates Warfarin-induced Aortic Valve Calcification Through the BMP2/Smad1/5/Runx2 Signaling Pathway.

ABSTRACT: Calcific aortic valve disease is a common heart disease that contributes to increased cardiovascular morbidity and mortality. There is a lack of effective pharmaceutical therapy because its mechanisms are not yet fully known. Ginkgo biloba extract (EGB761) is reported to alleviate vascular calcification. However, whether EGB761 protects against aortic valve calcification, a disease whose pathogenesis shares many similarities with vascular calcification, and potential molecular mechanisms remain unknown. In this study, porcine aortic valve interstitial cell (pAVIC) calcification was induced by warfarin with or without the presence of EGB761. Immunostaining was performed to establish and characterize the pAVIC phenotype. Calcium deposition and calcium content were examined by Alizarin Red S staining and an intracellular calcium content assay. Alkaline phosphatase activity was detected by the p-nitrophenyl phosphate method. The expression levels of bone morphogenetic protein-2 (BMP2), Runt-related transcription factor 2 (Runx2), homeobox protein MSX-2, and phosphorylated (p)-Smad1/5 were detected by reverse transcription-quantitative polymerase chain reaction (PCR) and Western blot analysis. Consistent with these in vitro data, we also confirmed the suppression of in vivo calcification by EGB761 in the warfarin-induced C57/Bl6 mice. The results indicated that both pAVICs and aortic valves tissue of mice stimulated with warfarin showed increased calcium deposition and expression of osteogenic markers (alkaline phosphatase, BMP2, homeobox protein MSX-2, and Runx2) and promoted p-Smad1/5 translocation from the cytoplasm to the nucleus. The addition of EGB761 significantly inhibited p-Smad1/5 translocation from the cytoplasm to the nucleus, thus suppressing calcification. In conclusion, EGB761 could ameliorate warfarin-induced aortic valve calcification through the inhibition of the BMP2-medicated Smad1/5/Runx2 signaling pathway.

Skin necrosis and calcifications after extravasation of vancomycin: a localised form of calciphylaxis?

Vancomycin is a tricyclic glycopeptide antibiotic produced from Streptococcus orientalis. There is much variation in the literature with regard to the recommended dose, dilution rate and type of infusion. Given the vesicant properties of vancomycin at supratherapeutic doses (>10mg/ml), tissue damage including blistering and necrosis have been reported. We report a rare case of bilateral cutaneous necrosis induced by accidental extravasation of vancomycin when being intravenously administered. The skin surrounding the injection site was marked by the appearance of subcutaneous calcifications. The development of iatrogenic skin calcinosis has not yet been described for the extravasation of vancomycin. The mechanism underlying the calcinosis observed in our case remains unclear, but we hypothesised a form of localised calciphylaxis induced by a local triggering factor. The ulcers progressed to re-epithelialisation following necrosis debridement and local conservative treatments. Given the increased prevalence of meticillin-resistant Staphylococcus aureus, which has prompted clinicians to gradually increase vancomycin dosage, clinicians should be aware of the high risk of skin toxicity in cases of vancomycin high-dose extravasation.

Nanoanalytical analysis of bisphosphonate-driven alterations of microcalcifications using a 3D hydrogel system and in vivo mouse model.

Vascular calcification predicts atherosclerotic plaque rupture and cardiovascular events. Retrospective studies of women taking bisphosphonates (BiPs), a proposed therapy for vascular calcification, showed that BiPs paradoxically increased morbidity in patients with prior acute cardiovascular events but decreased mortality in event-free patients. Calcifying extracellular vesicles (EVs), released by cells within atherosclerotic plaques, aggregate and nucleate calcification. We hypothesized that BiPs block EV aggregation and modify existing mineral growth, potentially altering microcalcification morphology and the risk of plaque rupture. Three-dimensional (3D) collagen hydrogels incubated with calcifying EVs were used to mimic fibrous cap calcification in vitro, while an ApoE-/- mouse was used as a model of atherosclerosis in vivo. EV aggregation and formation of stress-inducing microcalcifications was imaged via scanning electron microscopy (SEM) and atomic force microscopy (AFM). In both models, BiP (ibandronate) treatment resulted in time-dependent changes in microcalcification size and mineral morphology, dependent on whether BiP treatment was initiated before or after the expected onset of microcalcification formation. Following BiP treatment at any time, microcalcifications formed in vitro were predicted to have an associated threefold decrease in fibrous cap tensile stress compared to untreated controls, estimated using finite element analysis (FEA). These findings support our hypothesis that BiPs alter EV-driven calcification. The study also confirmed that our 3D hydrogel is a viable platform to study EV-mediated mineral nucleation and evaluate potential therapies for cardiovascular calcification.

Inorganic-polymerization crosslinked tissue-siloxane hybrid as potential biomaterial for bioprosthetic heart valves.

Bioprosthetic heart valve (BHV) replacement is increasingly used for treating valve-related diseases worldwide but the current commercially used BHVs treated with glutaraldehyde (Glut) often failed within 12-15 years due to degradation, thrombosis, inferior biocompatibility, and calcification. Herein, 3-glycidyloxypropyl trimethoxysilane (GPTMS) was used to crosslink porcine pericardium (PP) at the concentration (vol/vol) of 0.25%, 1%, 2%, and 4% and their performance for potential application in BHVs was evaluated. The crosslinking mechanism mainly involved the ring-opening of epoxide by amine attack and silanol poly-condensation. The stability of collagen in higher concentration (1%, 2%, and 4%) GPTMS crosslinked PPs (GPTMS-PPs) was clearly increased. GPTMS-PPs showed no cytotoxicity and supported the growth of endothelial cells while Glut-PP did not. GPTMS-PPs were less prothrombotic than Glut-PP. GPTMS-PP crosslinked at 1% concentration showed comparable mechanical properties to Glut-PP while had better anti-tearing performance. The subcutaneous implantation in rat for 30 days showed that GPTMS crosslinking was able to effectively inhibit the calcification of BHV.

Extensive Postchemotherapy Calcinosis Cutis in a Non-Hodgkin Lymphoma Patient With Spontaneous Resolution.

We report the case of a 30-year-old woman with stage IV, non-Hodgkin lymphoma. Baseline F-FDG PET/CT scan revealed lymphadenopathy with breasts and skeletal involvement. She received 3 cycles of R-CHOP (rituximab, cyclophosphamide, hydroxydaunorubicin, vincristine, and prednisone) chemotherapy without significant adverse effects. Biochemical examinations before and after chemotherapies were unremarkable. InterimF-FDG PET/CT showed a partial treatment response. However, there was an appearance of FDG-avid coarse calcification in breasts. Cutaneous and subcutaneous regions of elbows, pelvis, and thighs showed similar calcifications. Posttherapy PET/CT showed a significant resolution of calcinosis cutis. This case presents a postchemotherapy idiopathic calcinosis cutis with rapid spontaneous resolution.

Cholesterol (Blood lipid) lowering potential of Rosuvastatin chitosan nanoparticles for atherosclerosis: Preclinical study in rabbit model.

Atherosclerosis is the condition of narrowing of arteries due to plaque buildup on the artery walls. Aortic valve calcification (AVC) is one of the reasons of atherosclerosis which leads to narrowing at the opening of the aortic valve which is commonly referred as Aortic valve stenosis (AS). The Rosuvastatin-chitosan (ROS-chitosan) nanoparticles were prepared using ionotropic gelation method. Nanoparticulate formulation was optimized by 3 factor, 2 level full factorial design to find the effect of independent variables on particle size and percentage encapsulation efficiency. Particle size, encapsulation efficiency, scanning electron microscopy, in vitro drug release of nanoparticles was determined. The adult male rabbit of 4-5 months old were chosen for the study. Hypercholesterolemia was induced in experimental animals by administering diet with Cholesterol and Cholic acid (1.25 % and 0.5% respectively.) Blood lipid profile, interleukin 6 levels and histopathological study was performed. Rosuvastatin was found to be significantly effective in lowering the blood lipid levels. It helps to attenuate atherosclerosis as well as calcification of various valve tissues in experimental animals.

Effect of high-dose vitamin D supplementation on peripheral arterial calcification: secondary analysis of a randomized controlled trial.

Although high-dose vitamin D supplementation is common, effects on arterial calcification remain unexplored. Tibial artery calcification was identified and quantified over 3 years in participants randomized to 400, 4000, or 10,000 IU vitamin D3 daily. High-dose vitamin D supplementation did not affect the development or progression of arterial calcification. INTRODUCTION: To determine whether vitamin D supplementation has a dose-dependent effect on development and progression of arterial calcification. METHODS: This was a secondary analysis of the Calgary Vitamin D Study, a 3-year, double-blind, randomized controlled trial conducted at a single-center in Calgary, Canada. Participants were community-dwelling adults aged 55-70 years with serum 25-hydroxyvitamin D 30-125 nmol/L. Participants were randomized 1:1:1 to receive vitamin D3 400, 4000, or 10,000 IU/day for 3 years. Tibial artery calcification was identified and quantified (in milligrams of hydroxyapatite, mgHA) using high-resolution peripheral quantitative computed tomography (HR-pQCT) at baseline and 6, 12, 24, and 36 months. Changes in calcification over time and treatment group interaction were evaluated using a constrained linear mixed effects model. RESULTS: Of 311 randomized participants, 302 (400: 105, 4000: 96, 10,000: 101) were eligible for analysis of arterial calcification (54% male, mean (SD) age 62 (4) years, mean (SD) 25-hydroxyvitamin D 78.9 (19.9) nmol/L). At baseline, 85 (28%) had tibial artery calcification, and mean (95% CI) calcification quantity was 2.8 mgHA (95% CI 1.7-3.9). In these 85 participants, calcification quantity increased linearly by 0.020 mgHA/month (95% CI 0.012-0.029) throughout the study, with no evidence of a treatment-group effect (p = 0.645 for interaction). No participants developed new arterial calcifications during the study. CONCLUSIONS: In this population of community-dwelling adults who were vitamin D replete at baseline, supplementation with vitamin D 400, 4000, or 10,000 IU/day did not have differential effects on the development or progression of arterial calcification over 3 years. TRIAL REGISTRATION: clinicaltrials.gov (NCT01900860).

The Ryanodine Receptor Contributes to the Lysophosphatidylcholine-Induced Mineralization in Valvular Interstitial Cells.

PURPOSE: Fibrocalcific aortic valve disease (CAVD) is caused by the deposition of calcific nodules in the aortic valve leaflets, resulting in progressive loss of function that ultimately requires surgical intervention. This process is actively mediated by the resident valvular interstitial cells (VICs), which, in response to oxidized lipids, transition from a quiescent to an osteoblast-like state. The purpose of this study was to examine if the ryanodine receptor, an intracellular calcium channel, could be therapeutically targeted to prevent this phenotypic conversion. METHODS: The expression of the ryanodine receptor in porcine aortic VICs was characterized by qRT-PCR and immunofluorescence. Next, the VICs were exposed to lysophosphatidylcholine, an oxidized lipid commonly found in low-density lipoprotein, while the activity of the ryanodine receptor was modulated with ryanodine. The cultures were analyzed for markers of cellular mineralization, alkaline phosphatase activity, proliferation, and apoptosis. RESULTS: Porcine aortic VICs predominantly express isoform 3 of the ryanodine receptors, and this protein mediates the cellular response to LPC. Exposure to LPC caused elevated intracellular calcium concentration in VICs, raised levels of alkaline phosphatase activity, and increased calcific nodule formation, but these changes were reversed when the activity of the ryanodine receptor was blocked. CONCLUSIONS: Our findings suggest blocking the activity of the ryanodine receptor can attenuate the valvular mineralization caused by LPC. We conclude that oxidized lipids, such as LPC, play an important role in the development and progression of CAVD and that the ryanodine receptor is a promising target for pharmacological intervention.

Inhibition of vascular calcification by microRNA-155-5p is accompanied by the inactivation of TGF-ß1/Smad2/3 signaling pathway.

Vascular calcification (VC) is a vital factor for cardiovascular morbidity and mortality. Accumulating data suggest that microRNA (miR) is implicated in the VC. The main purpose of this study is to study the influence of miR-155-5p overexpression on VC development in vitro and in vivo. Immunofluorescence staining, real-time PCR, alizarin red staining, alkaline phosphatase (ALP) activity assay, western blot, luciferase assay, hematoxylin-eosin (HE), Masson's trichrome staining, and calcium content assay were used in this research. The results showed that miR-155-5p was decreased in the rat vascular smooth muscle cells (rVSMCs) undergoing calcification in vitro. MiR-155-5p overexpression reversed the increase of calcification and ALP activity in calcified cells. Further, overexpression of miR-155-5p inhibited the transforming growth factor-ß1 (TGF-ß1)/Smad2/3 signaling pathway, as evidenced by decreased protein expression of TGF-ß1, pSmad-2 and pSmad-3 in rVSMCs. MiR-155-5p was showed to target Smad2 directly. Moreover, miR-155-5p upregulation reduced vascular thickening, fibrosis and calcium content of aorta abdominalis in CaCl2-mediated VC model. Collectively, our results suggest that miR-155-5p overexpression may inhibit VC development through suppressing TGF-ß1/Smad2/3 signaling pathway in vivo and in vitro, indicating that miR-155-5p may act as a potential therapeutic target for VC-related disease.