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Deficiency in the anti-aging gene Klotho promotes aortic valve fibrosis through AMPK?-mediated activation of RUNX2.

ABSTRACT: Fibrotic aortic valve disease (FAVD) is an important cause of aortic stenosis, yet currently there is no effective treatment for FAVD due to its unknown etiology. The purpose of this study was to investigate whether deficiency in the anti-aging Klotho gene (KL) promotes high-fat-diet-induced FAVD and to explore the underlying molecular mechanism. Heterozygous Klotho-deficient (KL(+/-) ) mice and WT littermates were fed with a high-fat diet (HFD) or normal diet for 13 weeks, followed by treatment with the AMPK? activator (AICAR) for an additional 2 weeks. A HFD caused a greater increase in collagen levels in the aortic valves of KL(+/-) mice than of WT mice, indicating that Klotho deficiency promotes HFD-induced aortic valve fibrosis (AVF). AMPK? activity (pAMPK?) was decreased, while protein expression of collagen I and RUNX2 was increased in the aortic valves of KL(+/-) mice fed with a HFD. Treatment with AICAR markedly attenuated HFD-induced AVF in KL(+/-) mice. AICAR not only abolished the downregulation of pAMPK? but also eliminated the upregulation of collagen I and RUNX2 in the aortic valves of KL(+/-) mice fed with HFD. In cultured porcine aortic valve interstitial cells, Klotho-deficient serum plus cholesterol increased RUNX2 and collagen I protein expression, which were attenuated by activation of AMPK? by AICAR. Interestingly, silencing of RUNX2 abolished the stimulatory effect of Klotho deficiency on cholesterol-induced upregulation of matrix proteins, including collagen I and osteocalcin. In conclusion, Klotho gene deficiency promotes HFD-induced fibrosis in aortic valves, likely through the AMPK?-RUNX2 pathway.

SUBMITTER: Chen J

PROVIDER: S-EPMC5013020 | biostudies-literature | 2016 Oct

REPOSITORIES: biostudies-literature

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Deficiency in the anti-aging gene Klotho promotes aortic valve fibrosis through AMPKα-mediated activation of RUNX2.

Chen Jianglei J Lin Yi Y Sun Zhongjie Z

Aging cell 20160531 5

Fibrotic aortic valve disease (FAVD) is an important cause of aortic stenosis, yet currently there is no effective treatment for FAVD due to its unknown etiology. The purpose of this study was to investigate whether deficiency in the anti-aging Klotho gene (KL) promotes high-fat-diet-induced FAVD and to explore the underlying molecular mechanism. Heterozygous Klotho-deficient (KL(+/-) ) mice and WT littermates were fed with a high-fat diet (HFD) or normal diet for 13 weeks, followed by treatment ...[more]

PMID: 27242197

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Project description:Background: Calcific aortic valve disease is common in the aging population and is characterized by the histological changes of the aortic valves including extracellular matrix remodeling, osteochondrogenic differentiation, and calcification. Combined, these changes lead to aortic sclerosis, aortic stenosis (AS), and eventually to heart failure. Runt-related transcription factor 2 (Runx2) is a transcription factor highly expressed in the calcified aortic valves. However, its definitive role in the progression of calcific aortic valve disease (CAVD) has not been determined. In this study, we utilized constitutive and transient conditional knockout mouse models to assess the molecular, histological, and functional changes in the aortic valve due to Runx2 depletion. Methods: Lineage tracing studies were performed to determine the provenance of the cells giving rise to Runx2+ osteochondrogenic cells in the aortic valves of LDLr-/- mice. Hyperlipidemic mice with a constitutive or temporal depletion of Runx2 in the activated valvular interstitial cells (aVICs) and sinus wall cells were further investigated. Following feeding with a diabetogenic diet, the mice were examined for changes in gene expression, blood flow dynamics, calcification, and histology. Results: The aVICs and sinus wall cells gave rise to Runx2+ osteochondrogenic cells in diseased mouse aortic valves. The conditional depletion of Runx2 in the SM22α+ aVICs and sinus wall cells led to the decreased osteochondrogenic gene expression in diabetic LDLr-/- mice. The transient conditional depletion of Runx2 in the aVICs and sinus wall cells of LDLr-/-ApoB100 CAVD mice early in disease led to a significant reduction in the aortic peak velocity, mean velocity, and mean gradient, suggesting the causal role of Runx2 on the progression of AS. Finally, the leaflet hinge and sinus wall calcification were significantly decreased in the aortic valve following the conditional and temporal Runx2 depletion, but no significant effect on the valve cusp calcification or thickness was observed. Conclusions: In the aortic valve disease, Runx2 was expressed early and was required for the osteochondrogenic differentiation of the aVICs and sinus wall cells. The transient depletion of Runx2 in the aVICs and sinus wall cells in a mouse model of CAVD with a high prevalence of hemodynamic valve dysfunction led to an improved aortic valve function. Our studies also suggest that leaflet hinge and sinus wall calcification, even in the absence of significant leaflet cusp calcification, may be sufficient to cause significant valve dysfunctions in mice.

Project description:AimsThere is evidence to suggest that the subtype of aortic stenosis (AS), the degree of myocardial fibrosis (MF), and level of aortic valve calcification (AVC) are associated with adverse cardiac outcome in AS. Because little is known about their respective contribution, we sought to investigate their relative importance and interplay as well as their association with adverse cardiac events following transcatheter aortic valve replacement (TAVR).Methods and resultsOne hundred consecutive patients with severe AS and indication for TAVR were prospectively enrolled between January 2017 and October 2018. Patients underwent transthoracic echocardiography, multidetector computed tomography, and left ventricular endomyocardial biopsies at the time of TAVR. The final study cohort consisted of 92 patients with a completed study protocol, 39 (42.4%) of whom showed a normal ejection fraction (EF) high-gradient (NEFHG) AS, 13 (14.1%) a low EF high-gradient (LEFHG) AS, 25 (27.2%) a low EF low-gradient (LEFLG) AS, and 15 (16.3%) a paradoxical low-flow, low-gradient (PLFLG) AS. The high-gradient phenotypes (NEFHG and LEFHG) showed the largest amount of AVC (807 ± 421 and 813 ± 281 mm3 , respectively) as compared with the low-gradient phenotypes (LEFLG and PLFLG; 503 ± 326 and 555 ± 594 mm3 , respectively, P < 0.05). Conversely, MF was most prevalent in low-output phenotypes (LEFLG > LEFHG > PLFLG > NEFHG, P < 0.05). This was paralleled by a greater cardiovascular (CV) mortality within 600 days after TAVR (LEFLG 28% > PLFLG 26.7% > LEFHG 15.4% > NEFHG 2.5%; P = 0.023). In patients with a high MF burden, a higher AVC was associated with a lower mortality following TAVR (P = 0.045, hazard ratio 0.261, 95% confidence interval 0.07-0.97).ConclusionsMF is associated with adverse CV outcome following TAVR, which is most prevalent in low EF situations. In the presence of large MF burden, patients with large AVC have better outcome following TAVR. Conversely, worse outcome in large MF and relatively little AVC may be explained by a relative prominence of an underlying cardiomyopathy. The better survival rates in large AVC patients following TAVR indicate TAVR induced relief of severe AS-associated pressure overload with subsequently improved outcome.

Project description:BackgroundCalcific aortic valve disease (CAVD) is a common cardiovascular disease with high morbidity and mortality, and no effective prevention or treatment is available. In recent years, increasing evidence has shown that noncoding RNAs (ncRNAs) play an important role in the pathogenesis and prognosis of CAVD. Several associated circular RNAs (circRNAs) have been reported to be involved in CAVD, such as circRIC3 and TGFBR2. However, the limited number of circRNAs identified in CAVD warrants further in-depth investigation, and the comprehensive elucidation of their role in the key mechanisms of this disease is needed.MethodsThe expression of circRNAs and microRNAs (miRNAs) were analyzed by RNA sequencing. Quantitative real-time polymerase chain reaction (qRT-PCR) was conducted to analyze the expression of circRNA ARHGAP10 (circARHGAP10), miR-335-3p, and RUNX2. Luciferase reporter assay, pull-down assay, and RNA binding protein immunoprecipitation (RIP) assay were performed to evaluate the binding of miR-335-3p to circARHGAP10 or RUNX2. Alizarin red S staining showed the formation of calcified nodules in valve interstitial cells (VICs). The expression of circARHGAP10 and miR-335-3p was altered through lentivirus infection. Alkaline phosphatase (ALP) activity was used to verify the correlation between circARHGAP10 and miR-335-3p. The expression of proteins was assessed via Western blot. RNA fluorescence in situ hybridization (FISH) was used to confirm the localization of circARHGAP10 in the cytoplasm of VICs. Immunofluorescence was used to detect the expression level of RUNX2. ApoE-/- mice were used to construct a CAVD model, circARHGAP10 short hairpin RNA (shRNA) and miR-335-3p inhibitor lentivirus were intraperitoneally injected, and scramble and inhibitor normal control (NC) lentivirus were injected as controls, followed by hematoxylin and eosin (HE) staining.ResultsThrough RNA sequencing, we found that circARHGAP10 (hsa_circ_0008975) was highly expressed in calcific aortic valves. CircARHGAP10 knockdown effectively inhibited the extent of osteogenic differentiation of VICs. We then found that circARHGAP10 was a competing endogenous RNA (ceRNA) of miR-355-3p and that miR-355-3p targeted RUNX2. In vitro experiments confirmed that circARHGAP10 regulated the osteogenic differentiation of VICs through the miR-355-3p/RUNX2 pathway, and this was validated in vivo using an ApoE-/- mouse model.ConclusionsThese findings provide a foundation for circRNA-directed diagnostics and therapeutics for CAVD.

Dataset Information

Deficiency in the anti-aging gene Klotho promotes aortic valve fibrosis through AMPK?-mediated activation of RUNX2.

Publications

Deficiency in the anti-aging gene Klotho promotes aortic valve fibrosis through AMPKα-mediated activation of RUNX2.

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