Endothelial cell-derived tetrahydrobiopterin prevents aortic valve calcification.

AIMS: Tetrahydrobiopterin (BH4) is a critical determinant of the biological function of endothelial nitric oxide synthase. The present study was to investigate the role of valvular endothelial cell (VEC)-derived BH4 in aortic valve calcification. METHODS AND RESULTS: Plasma and aortic valve BH4 concentrations and the BH4:BH2 ratio were significantly lower in calcific aortic valve disease patients than in controls. There was a significant decrease of the two key enzymes of BH4 biosynthesis, guanosine 5'-triphosphate cyclohydrolase I (GCH1) and dihydrofolate reductase (DHFR), in calcified aortic valves compared with the normal ones. Endothelial cell-specific deficiency of Gch1 in Apoe-/- (Apoe-/-Gch1fl/flTie2Cre) mice showed a marked increase in transvalvular peak jet velocity, calcium deposition, runt-related transcription factor 2 (Runx2), dihydroethidium (DHE), and 3-nitrotyrosine (3-NT) levels in aortic valve leaflets compared with Apoe-/-Gch1fl/fl mice after a 24-week western diet (WD) challenge. Oxidized LDL (ox-LDL) induced osteoblastic differentiation of valvular interstitial cells (VICs) co-cultured with either si-GCH1- or si-DHFR-transfected VECs, while the effects could be abolished by BH4 supplementation. Deficiency of BH4 in VECs caused peroxynitrite formation increase and 3-NT protein increase under ox-LDL stimulation in VICs. SIN-1, the peroxynitrite generator, significantly up-regulated alkaline phosphatase (ALP) and Runx2 expression in VICs via tyrosine nitration of dynamin-related protein 1 (DRP1) at Y628. Finally, folic acid (FA) significantly attenuated aortic valve calcification in WD-fed Apoe-/- mice through increasing DHFR and salvaging BH4 biosynthesis. CONCLUSION: The reduction in endothelial-dependent BH4 levels promoted peroxynitrite formation, which subsequently resulted in DRP1 tyrosine nitration and osteoblastic differentiation of VICs, thereby leading to aortic valve calcification. Supplementation of FA in diet attenuated hypercholesterolaemia-induced aortic valve calcification by salvaging BH4 bioavailability.

Curcumin inhibits calcification of human aortic valve interstitial cells by interfering NF-κB, AKT, and ERK pathways.

The osteogenic differentiation of human aortic valve interstitial cells (hVICs) is the key cellular mechanism of calcified aortic valve disease (CAVD). This study aimed to explore how curcumin (CCM) inhibits the osteogenic differentiation of hVICs and elucidate the molecular mechanisms involved. In this study, CCM inhibited the osteogenic differentiation of hVICs under osteogenic medium (OM) conditions by reversing the OM-induced increase in calcified nodule formation and osteogenesis-specific markers (ALP and Runx2). RNA sequencing identified 475 common differentially expressed genes with Venn diagrams of the different groups. Kyoto Encyclopedia of Genes and Genomes enrichment revealed that the CCM inhibition of hVIC osteogenic differentiation was enriched in the NF-κB, PI3K-AKT, TNF, Jak-STAT, and MAPK signaling pathways. In addition, CCM suppressed the phosphorylation of ERK, IκBα, AKT, and interfered with the translocation of P65 into the cell nucleus in hVICs under OM culture conditions. In conclusion, CCM inhibited the osteogenic differentiation of hVICs via interfering with the activation of NF-κB/AKT/ERK signaling pathways. Our findings provide novel insights into a critical role for CCM in CAVD progression and shed new light on CCM-directed therapeutics for CAVD.

Nobiletin exhibits potent inhibition on tumor necrosis factor alpha-induced calcification of human aortic valve interstitial cells via targeting ABCG2 and AKR1B1.

Inflammation is considered to be one of the initial critical factors in the occurrence of calcific heart valve disease. This study was to prove Nobiletin (NBT) inhibits inflammation-caused calcification of human valve interstitial cells (hVICs) and to elucidate the involved molecular mechanisms. Tumor necrosis factor-alpha (TNF-α)-induced hVICs were treated with or without NBT. Cell growth and calcification of hVICs were assessed. RNA sequencing was utilized to investigate the gene expression changes. Molecular target prediction and docking assay were further performed. NBT interfered with hVIC growth under TNF-α condition in a dose-dependent manner also presented a gradual decrease of positive Alizarin Red S staining, down-regulation of BMP2, and RUNX2 gene expression. Based on the global gene expression cluster, control and TNF-α plus NBT group showed a high similarity versus TNF-α only group. After Venn interaction of differential expression genes (DEGs), 2,236 common DEGs were identified to display different biological functions and signaling pathways. ABCG2 and AKR1B1 were further selected as prediction targets of NBT involved in RELA, TNF, BMP2, RUNX2, etc. interactions in mediating hVIC calcification. The results show that NBT is a natural product to prevent the occurrence of heart valve calcification.

Impact of high-dose atorvastatin therapy and clinical risk factors on incident aortic valve stenosis in patients with cardiovascular disease (from TNT, IDEAL, and SPARCL).

Clinical trials have not provided evidence for a role of statin therapy in reducing aortic valve stenosis (AVS) severity in patients with documented AVS. However, whether statin therapy could prevent the onset of AVS is unknown. Our objectives were (1) to compare the incidence rates of AVS among patients treated with high-dose versus usual-dose statin or placebo and (2) to identify clinical risk factors associated with the development of AVS. We conducted post hoc analyses in 23,508 participants from 3 large-scale multicenter atorvastatin randomized blinded clinical trials: Treating to New Targets, the Incremental Decrease in End Points Through Aggressive Lipid Lowering, and the Stroke Prevention by Aggressive Reduction in Cholesterol Levels. The main outcome measure was the incidence of clinical AVS over a median follow-up of 4.9 years (82 cases). Among patients who developed AVS, 39 (47.6%) were treated with atorvastatin 80 mg and 43 (52.4%) were treated with lower dose statin (atorvastatin 10 mg in Treating to New Targets, simvastatin 20 to 40 mg in Incremental Decrease in End Points Through Aggressive Lipid Lowering, or placebo in Stroke Prevention by Aggressive Reduction in Cholesterol Levels; hazard ratio [HR] 0.91, 95% confidence interval [CI] 0.59 to 1.41, p=0.67). In multivariate analyses forcing treatment, sex, and race into the model, factors that were significantly associated with AVS included age (HR 2.17, 95% CI 1.61 to 2.93, p<0.0001 per 1-SD increment), diabetes (HR 1.67, 95% CI 1.00 to 2.80, p=0.05), vitamin K antagonist use (HR 3.25, 95% CI 2.06 to 5.16, p<0.0001), and previous statin use (HR 2.65, 95% CI 1.54 to 4.60, p=0.0008). In conclusion, random allocation to high-dose versus usual-dose statin therapy or placebo did not impact the incidence of AVS among patients without known AVS. Age, diabetes, vitamin K antagonists, and previous statin use were significant predictors of incident AVS in these high-risk patients.

Bone modifiers and the quest to slow progression of aortic stenosis.

Aortic stenosis (AS) will likely become increasingly frequent with the aging of the American population. The difficulties in treating elderly patients with critical AS emphasize the potential value of a strategy to slow the advancement of aortic valve calcification. Recent prospective trials of statins and angiotensin-converting enzyme inhibitors have been disappointing. New options are needed to achieve a truly effective strategy for retarding the advancement of AS. In this context, the observations of Skolnick et al appearing in this issue of The American Journal of Cardiology are particularly intriguing. In a retrospective review of patients followed for mild or moderate AS, these investigators found that 18 patients receiving treatment for osteoporosis had significantly less decrement in aortic valve area on follow-up echocardiography than 37 not receiving such treatment. The most attractive explanation is an action of drug therapy for osteoporosis, most often bisphosphonates, to retard aortic valve calcification. The mechanism for this action is not clear, although numerous possibilities can be postulated on the basis of the multiple complex processes controlling tissue calcification. In conclusion, the investigators' findings deserve further study to clarify drug impact on aortic valve calcification as well as confirm the clinical findings in a larger and more diverse population. Such investigation should also assess the role of vitamin D and calcium supplementation, common features of treatment for osteoporosis. Currently available results are too preliminary to justify the use of bisphosphonates or other osteoporosis therapies to slow the progression of AS.

[Effects of Xiongshao capsule on blood vessel collagenase gene expression in experimental rabbits with arterial restenosis].

OBJECTIVE: To observe the effects of Xiongshao Capsule (XSC) on blood vessel collagenase gene expression in experimental rabbits with arterial restenosis, and to probe its mechanisms for preventing restenosis. METHODS: Restenosis rabbit model was established by injuring endothelium of abdominal aorta by balloon dilation and feeding with high fatty diet for 6 weeks. Eighty rabbits were randomly allocated into 8 groups, Group A, normal rabbit for control; Group B, rabbit with simple injured arterial endothelium; Group C, model rabbits at different times after modeling (3 days for Group C1, 2 weeks for Group C2, and 6 weeks for Group C3); Group D, model rabbit treated with Probucol for 6 weeks; Group E and F, model rabbit treated with small and large dose of XSC respectively. The effect of XSC on collagenase gene expression during the course of restenosis was observed adopting RT-PCR method and computer image analyzer, and its mechanisms in preventing RS were probed by combined analyzing the change of collagen and patho-morphological examination. RESULTS: Compensatory dilation of lumens appeared at the end of the 2nd week; while 6 weeks after modeling, the diameters of lumens obviously diminished with an apparently increased proliferation index. The cell proliferation inhibiting effect in Group D and F was significant. The total amount of collagen increased and reached the peak at the 2nd week but without conspicuous accumulation on intima, which increased gradually and reached its peak at the 6th week. In Group D-F, especially in Group F, the amount of collagen in vascular wall (intima, media and externa) was lesser than that in Groups C. MMP-1 mRNA showed weak expression in Group A and Group C1-C3; significant difference only existed in comparing Group F with C3 (P < 0.05). CONCLUSION: XSC could markedly increase the MMP-1 mRNA expression in injured portion of vessels, suggesting that its action in preventing RS might be related with the up-regulating of MMP-1 mRNA expression, increasing collagen degradation and reducing collagen deposition in vascular wall.