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.

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.