Zinc ameliorates human aortic valve calcification through GPR39 mediated ERK1/2 signalling pathway.

AIMS: Calcific aortic valve disease (CAVD) is the most common heart valve disease in the Western world. It has been reported that zinc is accumulated in calcified human aortic valves. However, whether zinc directly regulates CAVD is yet to be elucidated. The present study sought to determine the potential role of zinc in the pathogenesis of CAVD. METHODS AND RESULTS: Using a combination of a human valve interstitial cell (hVIC) calcification model, human aortic valve tissues, and blood samples, we report that 20 µM zinc supplementation attenuates hVIC in vitro calcification, and that this is mediated through inhibition of apoptosis and osteogenic differentiation via the zinc-sensing receptor GPR39-dependent ERK1/2 signalling pathway. Furthermore, we report that GPR39 protein expression is dramatically reduced in calcified human aortic valves, and there is a significant reduction in zinc serum levels in patients with CAVD. Moreover, we reveal that 20 µM zinc treatment prevents the reduction of GPR39 observed in calcified hVICs. We also show that the zinc transporter ZIP13 and ZIP14 are significantly increased in hVICs in response to zinc treatment. Knockdown of ZIP13 or ZIP14 significantly inhibited hVIC in vitro calcification and osteogenic differentiation. CONCLUSIONS: Together, these findings suggest that zinc is a novel inhibitor of CAVD, and report that zinc transporter ZIP13 and ZIP14 are important regulators of hVIC in vitro calcification and osteogenic differentiation. Zinc supplementation may offer a potential therapeutic strategy for CAVD.

Lack of Endothelial Nitric Oxide Synthase Accelerates Ectopic Calcification in Uremic Mice Fed an Adenine and High Phosphorus Diet.

Ectopic calcification is a risk of cardiovascular disease in chronic kidney disease (CKD) patients, and impaired endothelial nitric oxide synthase (eNOS) is involved in the CKD complications. However, whether eNOS dysfunction is a cause of ectopic calcification in CKD remains to be elucidated. To address this issue, we investigated the role of eNOS in ectopic calcification in mice with renal injury caused by an adenine and high-phosphorus (Ade + HP) diet. DBA/2J mice, a calcification-sensitive strain, were fed Ade + HP for 3 weeks. Expression levels of eNOS-related genes were reduced significantly in their calcified aorta. C57BL/6J is a calcification-resistant strain, and wild-type mice showed mild calcified lesions in the aorta and kidney when given an Ade + HP diet for 4 weeks. In contrast, a lack of eNOS led to the development of severe aortic calcification accompanied by an increase in runt-related transcription factor 2, an osteochondrogenic marker. Increased renal calcium deposition and the tubular injury score were remarkable in mice lacking eNOS-fed Ade + HP. Exacerbation of ectopic calcification by a lack of eNOS is associated with increased oxidative stress markers such as nicotinamide adenine dinucleotide phosphate oxidases. In conclusion, eNOS is critically important in preventing ectopic calcification. Therefore, the maintenance of eNOS is useful to reduce cardiovascular disease events and to improve prognosis in CKD patients.

Novel PHEX nonsense mutation in a patient with X-linked hypophosphatemic rickets and review of current therapeutic regimens.

INTRODUCTION: The most common form of familial hypophosphatemic rickets is X-linked. PHEX has been identified as the gene defective in this phosphate wasting disorder leading to decreased renal phosphate reabsorption, hypophosphatemia and inappropriate concentrations of 1,25-dihydroxyvitamin D in regard to hypophosphatemia. Clinical manifestation are skeletal deformities, short stature, osteomalacia, dental abscesses, bone pain, and loss of hearing. SUBJECTS AND METHODS: We report 3 cases of hypophosphatemic rickets with genetic mutational analysis of the PHEX gene. In 1 male patient an unknown nonsense mutation was found in exon 7, codon 245 (c.735T>G, Tyr245Term, Y245X). In both female patients known mutations were found: c.682delTC (exon 6, codon 228) and c.1952G>C (exon 19, codon 651, R651P). Age at diagnosis ranged from early childhood to the age of 35 years. Clinical complications were hip replacement in 1 patient, mild nephrocalcinosis in 2 patients and loss of hearing in 1 patient. All 3 patients have been treated with phosphate supplements and receive 1,25-dihydroxyvitamin D. Under this regimen all patients show stable biochemical markers with slight hyperparathyreoidism. In all patients at least one family member is affected by rickets, as well. CONCLUSIONS: We report a novel nonsense mutation of PHEX that has not been identified so far. The recent discovery of FGF23 and MEPE has changed our understanding of the kidney-bone metabolism, but also raises concerns about the efficacy of current therapeutic regimens that are reviewed in this context.

Calcification of human valve interstitial cells is dependent on alkaline phosphatase activity.

BACKGROUND AND AIM OF THE STUDY: The calcification of heart valves is associated with valve degeneration and failure, but the mechanisms involved are poorly understood. The presence of lamellar bone has been demonstrated in calcified aortic valves. Since osseous calcification is closely associated with alkaline phosphatase (ALP) activity, it was hypothesized that ALP activity might be implicated in the calcification of isolated leaflet interstitial cells (ICs). METHODS: Human valve leaflet ICs were isolated from transplant-explanted hearts at the time of transplantation (n = 12). RESULTS: Isolated leaflet ICs expressed the fibroblast-specific antigen (100% of cells) and smooth muscle (SM) alpha-actin (70-80% of cells), but osteoblastic markers were not expressed. Cultured ICs did not calcify spontaneously, however when the growth medium was supplemented with beta-glycerophosphate (an organic phosphate) it induced the formation of calcified nodules that expressed osteonectin and ALP, but not SM alpha-actin. Beta-glycerophosphate-induced calcification of ICs showed a time-dependent effect on the calcium content of treated cells over a 14-day period. ALP activity was considerably increased in beta-glycerophosphate-treated ICs, and this correlated with the calcium content (r = 0.5: p = 0.01). Levamisol (an ALP inhibitor) inhibited the beta-glycerophosphate-induced calcification process, as well as the expression of osteoblastic differentiation markers. CONCLUSION: Isolated and cultured leaflet ICs did not calcify spontaneously, though organic phosphate induced the formation of calcified nodules that expressed osteoblastic markers. The calcification of isolated ICs was seen to be dependent on ALP activity.

Glycosaminoglycan-degrading enzymes in porcine aortic heart valves: implications for bioprosthetic heart valve degeneration.

BACKGROUND AND AIM OF THE STUDY: Glutaraldehyde (GA)-fixed aortic valves used in heart valve replacement surgery have limited durability due to tissue degeneration and calcification. Despite their structural and functional importance, very little is known about the fate of glycosaminoglycans (GAGs) within the extracellular matrix of bioprosthetic heart valves. The study aim was to investigate the stability of GAGs in GA-fixed tissues and to identify enzymatic mechanisms that may be responsible for GAG degeneration. METHODS: Porcine aortic valve cusps were fixed with GA and implanted subdermally in rats for 21 days. Fresh, fixed and explanted cusps were analyzed for GAG content by hexosamine determination, and GAG-degrading enzyme activity was evaluated using zymography. GAG classes in fresh cusps were also assessed by flurorophore-assisted carbohydrate electrophoresis. Fresh and GA-fixed cusps were also exposed in vitro to hyaluronidase and chondroitinase in order to test the susceptibility of cusp GAGs towards enzymatic degradation. RESULTS: Native aortic cusps contained -3.5% GAGs by dry weight, consisting of hyaluronic acid, chondroitin sulfate and dermatan sulfate. Significantly lower GAG levels were found in aortic cusps after fixation with GA, and even lower levels were found after subdermal implantation in rats. GAG levels in GA-fixed cusps were also significantly reduced by in-vitro incubation with hyaluronidase and chondroitinase. Novel GAG-degrading enzymes were detected in considerable levels in native cusps, in lower levels in GA-fixed cusps and significantly increased levels after subdermal implantation of GA-fixed cusps. CONCLUSION: The combined action of active GAG-degrading enzymes and the failure of GA to stabilize GAGs towards enzymatic digestion may contribute significantly to bioprosthetic heart valve degeneration and subsequent structural failure.

Hypothalamic digoxin related membrane Na+-K+ ATPase inhibition and familial basal ganglia calcification.

The isoprenoid pathway produces three key metabolites-digoxin (membrane sodium-potassium ATPase inhibitor and regulator of intracellular calcium-magnesium ratios), dolichol (regulator of N-glycosylation of proteins) and ubiquinone (free radical scavenger). The pathway was assessed in a rare and specific type of familial basal ganglia calcification described. The family had a coexistence of basal ganglia calcification (six out of 10 cases), schizophrenia, Parkinson's disease, Alzheimer's disease, rheumatoid arthritis, systemic tumours and syndrome X and were all right hemispheric dominant. The isoprenoid pathway was also studied for comparison in right hemispheric dominant, bihemispheric dominant and left hemispheric dominant individuals. The isoprenoid pathway was upregulated with increased digoxin synthesis in familial basal ganglia calcification. Membrane sodium-potassium ATPase inhibition can lead on to increase in intracellular calcium and calcification of the basal ganglia. There was increase in tryptophan catabolites and a reduction in tyrosine catabolites. There was also an increase in dolichol and glycoconjugate levels with reduced lysosomal stability in these patients. The ubiquinone levels were low and free radical levels increased. The cholesterol-phospholipid ratio was increased and glycoconjugate level of the RBC membrane reduced in these group of patients. No significance difference was noted in family members with and without basal ganglia calcification. This findings were correlated with the pathogenesis of syndrome X, immune mediated diseases, degenerations, tumours and psychiatric disorders noted in the familial basal ganglia calcification described. The biochemical patterns obtained in familial basal ganglia calcification correlated with those in right hemispheric dominance.