RESUMEN

Based on high-throughput transcriptomic sequencing, SNHG3 was among the most highly expressed long noncoding RNAs in calcific aortic valve disease. SNHG3 upregulation was verified in human and mouse calcified aortic valves. Moreover, in vivo and in vitro studies showed SNHG3 silencing markedly ameliorated aortic valve calcification. In-depth functional assays showed SNHG3 physically interacted with polycomb repressive complex 2 to suppress the H3K27 trimethylation BMP2 locus, which in turn activated BMP2 expression and signaling pathways. Taken together, SNHG3 promoted aortic valve calcification by upregulating BMP2, which might be a novel therapeutic target in human calcific aortic valve disease.

RESUMEN

Aims: Calcific aortic valve disease (CAVD) is a progressive heart disease that is particularly prevalent in elderly patients. The current treatment of CAVD is surgical valve replacement, but this is not a permanent solution, and it is very challenging for elderly patients. Thus, a pharmacological intervention for CAVD may be beneficial. In this study, we intended to rescue aortic valve (AV) calcification through inhibition of TGFß1 and SMAD3 signaling pathways. Methods and Results: The klotho gene, which was discovered as an aging-suppressor gene, has been observed to play a crucial role in AV calcification. The klotho knockout (Kl -/-) mice have shorter life span (8-12 weeks) and develop severe AV calcification. Here, we showed that increased TGFß1 and TGFß-dependent SMAD3 signaling were associated with AV calcification in Kl -/- mice. Next, we generated Tgfb1- and Smad3-haploinsufficient Kl -/- mice to determine the contribution of TGFß1 and SMAD3 to the AV calcification in Kl -/- mice. The histological and morphometric evaluation suggested a significant reduction of AV calcification in Kl -/-; Tgfb1 ± mice compared to Kl -/- mice. Smad3 heterozygous deletion was observed to be more potent in reducing AV calcification in Kl -/- mice compared to the Kl -/-; Tgfb1 ± mice. We observed significant inhibition of Tgfb1, Pai1, Bmp2, Alk2, Spp1, and Runx2 mRNA expression in Kl -/-; Tgfb1 ± and Kl -/-; Smad3 ± mice compared to Kl -/- mice. Western blot analysis confirmed that the inhibition of TGFß canonical and non-canonical signaling pathways were associated with the rescue of AV calcification of both Kl -/-; Tgfb1 ± and Kl -/-; Smad3 ± mice. Conclusion: Overall, inhibition of the TGFß1-dependent SMAD3 signaling pathway significantly blocks the development of AV calcification in Kl -/- mice. This information is useful in understanding the signaling mechanisms involved in CAVD.

RESUMEN

There are currently no pharmacological therapies for calcific aortic valve disease (CAVD). Here, we evaluated the role of protein tyrosine phosphatase 1B (PTP1B) inhibition in CAVD. Up-regulation of PTP1B was critically involved in calcified human aortic valve, and PTP1B inhibition had beneficial effects in preventing fibrocalcific response in valvular interstitial cells and LDLR-/- mice. In addition, we reported a novel function of PTP1B in regulating mitochondrial homeostasis by interacting with the OPA1 isoform transition in valvular interstitial cell osteogenesis. Thus, these findings have identified PTP1B as a potential target for preventing aortic valve calcification in patients with CAVD.

RESUMEN

Expression of a neuropilin-like protein, DCBLD2, is reduced in human calcific aortic valve disease (CAVD). DCBLD2-deficient mice develop bicuspid aortic valve (BAV) and CAVD, which is more severe in BAV mice compared with tricuspid littermates. In vivo and in vitro studies link this observation to up-regulated bone morphogenic protein (BMP)2 expression in the presence of DCBLD2 down-regulation, and enhanced BMP2 signaling in BAV, indicating that a combination of genetics and BAV promotes aortic valve calcification and stenosis. This pathway may be a therapeutic target to prevent CAVD progression in BAV.

RESUMEN

INTRODUCTION: Hydrogen sulfide (H2S) was revealed to inhibit aortic valve calcification and inflammation was implicated in the pathogenesis of calcific aortic valve disease (CAVD). OBJECTIVES: We investigate whether H2S inhibits mineralization via abolishing inflammation. METHODS AND RESULTS: Expression of pro-inflammatory cytokines, interleukin-1ß (IL-1ß) and tumor necrosis factor α (TNF-α) were increased in patients with CAVD and in calcified aortic valve of ApoE-/- mice. Administration of H2 2S releasing donor (4-methoxyphenyl piperidinylphosphinodithioc acid (AP72)) exhibited inhibition on both calcification and inflammation in aortic valve of apolipoprotein E knockout mice (ApoE-/-) mice is reflected by lowering IL-1ß and TNF-α levels. Accordingly, AP72 prevented the accumulation of extracellular calcium deposition and decreased nuclear translocation of nuclear factor-κB (NF-κB) in human valvular interstitial cells (VIC). This was also accompanied by reduced cytokine response. Double-silencing of endogenous H2S producing enzymes, Cystathionine gamma-lyase (CSE) and Cystathionine beta-synthase (CBS) in VIC exerted enhanced mineralization and higher levels of IL-1ß and TNF-α. Importantly, silencing NF-κB gene or its pharmacological inhibition prevented nuclear translocation of runt-related transcription factor 2 (Runx2) and subsequently the calcification of human VIC. Increased levels of NF-κB and Runx2 and their nuclear accumulation occurred in ApoE-/- mice with a high-fat diet. Administration of AP72 decreased the expression of NF-κB and prevented its nuclear translocation in VIC of ApoE-/- mice on a high-fat diet, and that was accompanied by a lowered pro-inflammatory cytokine level. Similarly, activation of Runx2 did not occur in VIC of ApoE-/- mice treated with H2S donor. Employing Stimulated Emission Depletion (STED) nanoscopy, a strong colocalization of NF-κB and Runx2 was detected during the progression of valvular calcification. CONCLUSIONS: Hydrogen sulfide inhibits inflammation and calcification of aortic valve. Our study suggests that the regulation of Runx2 by hydrogen sulfide (CSE/CBS) occurs via NF-κB establishing a link between inflammation and mineralization in vascular calcification.

RESUMEN

Calcific aortic valve disease (CAVD) is the result of maladaptive fibrocalcific processes leading to a progressive thickening and stiffening of aortic valve (AV) leaflets. CAVD is the most common cause of aortic stenosis (AS). At present, there is no effective pharmacotherapy in reducing CAVD progression; when CAVD becomes symptomatic it can only be treated with valve replacement. Inflammation has a key role in AV pathological remodeling; hence, anti-inflammatory therapy has been proposed as a strategy to prevent CAVD. Cyclooxygenase 2 (COX-2) is a key mediator of the inflammation and it is the target of widely used anti-inflammatory drugs. COX-2-inhibitor celecoxib was initially shown to reduce AV calcification in a murine model. However, in contrast to these findings, a recent retrospective clinical analysis found an association between AS and celecoxib use. In the present study, we investigated whether variations in COX-2 expression levels in human AVs may be linked to CAVD. We extracted total RNA from surgically explanted AVs from patients without CAVD or with CAVD. We found that COX-2 mRNA was higher in non-calcific AVs compared to calcific AVs (0.013 ± 0.002 vs. 0.006 ± 0.0004; p < 0.0001). Moreover, we isolated human aortic valve interstitial cells (AVICs) from AVs and found that COX-2 expression is decreased in AVICs from calcific valves compared to AVICs from non-calcific AVs. Furthermore, we observed that COX-2 inhibition with celecoxib induces AVICs trans-differentiation towards a myofibroblast phenotype, and increases the levels of TGF-ß-induced apoptosis, both processes able to promote the formation of calcific nodules. We conclude that reduced COX-2 expression is a characteristic of human AVICs prone to calcification and that COX-2 inhibition may promote aortic valve calcification. Our findings support the notion that celecoxib may facilitate CAVD progression.

Asunto(s)

Estenosis de la Válvula Aórtica/tratamiento farmacológico , Válvula Aórtica/patología , Calcinosis/tratamiento farmacológico , Ciclooxigenasa 2/genética , Inflamación/tratamiento farmacológico , Factor de Crecimiento Transformador beta/genética , Anciano , Anciano de 80 o más Años , Animales , Válvula Aórtica/efectos de los fármacos , Estenosis de la Válvula Aórtica/genética , Estenosis de la Válvula Aórtica/patología , Apoptosis/efectos de los fármacos , Calcinosis/genética , Calcinosis/patología , Celecoxib/administración & dosificación , Femenino , Regulación de la Expresión Génica/efectos de los fármacos , Humanos , Inflamación/genética , Inflamación/patología , Masculino , Ratones , Persona de Mediana Edad , ARN Mensajero/genética

RESUMEN

This study sought to investigate whether reactive oxygen species (ROS)-generating reduced nicotinamide adenine dinucleotide phosphate oxidase 2 (Nox2) contributes to calcific aortic valve disease (CAVD) or whether celastrol, a natural Nox2 inhibitor, may provide potential therapeutic target for CAVD. CAVD is an active and cellular-driven fibrocalcific process characterized by differentiation of aortic valvular interstitial cells (AVICs) toward an osteogenic-like phenotype. ROS levels increase in calcified aortic valves, while the sources of ROS and their roles in the pathogenesis of CAVD are elusive. The roles of Nox2 and the effects of celastrol were studied using cultured porcine AVICs in vitro and a rabbit CAVD model in vivo. Nox2 proteins were significantly upregulated in human aortic valves with CAVD. In vitro, Nox2 was markedly induced upon stimulation of AVICs with osteogenic medium, along with the increases in ROS production and calcium nodule formation. Celastrol significantly decreased calcium deposition of AVICs by 35%, with a reduction of ROS generation. Knockdown of endogenous Nox2 substantially suppressed AVIC calcification by 39%, the inhibitory effect being similar to celastrol treatment. Mechanistically, either celastrol treatment or knockdown of Nox2 significantly inhibited glycogen synthase kinase 3 beta/ß-catenin signaling, leading to attenuation of fibrogenic and osteogenic responses of AVICs. In a rabbit CAVD model, administration of celastrol significantly reduced aortic valve ROS production, fibrosis, calcification, and severity of aortic stenosis, with less left ventricular dilatation and better preserved contractile function. Upregulation of Nox2 is critically involved in CAVD. Celastrol is effective to alleviate CAVD, likely through the inhibition of Nox2-mediated glycogen synthase kinase 3 beta/ß-catenin pathway in AVICs.

RESUMEN

Calcific aortic valve disease is a progressive fibrocalcific process that can only be treated with valve replacement. Cadherin-11 has recently been identified as a potential therapeutic target for calcific aortic valve disease. The already approved drug celecoxib, a cyclooxygenase-2 inhibitor, binds cadherin-11, and was investigated as a therapeutic against calcific aortic valve disease. Unexpectedly, celecoxib treatment led to hallmarks of myofibroblast activation and calcific nodule formation in vitro. Retrospective electronic medical record analysis of celecoxib, ibuprofen, and naproxen revealed a unique association of celecoxib use and aortic stenosis.