Calcification Of Valve Interstitial Cells Research Articles

The m6A demethylase ALKBH5 is a novel epigenetic regulator of aortic valve calcification.

Calcific aortic valve disease (CAVD) is a common heart valve disease with significant clinical consequences. The mechanisms that drive the pathogenesis of CAVD remain to be fully elucidated. N6-methyladenosine (m6A), the most prevalent RNA epigenetic regulator, has recently been implicated in cardiovascular disease, but its role in CAVD has yet to be investigated. In this study, we investigated the potential function of m6A modification in CAVD. Using clinical samples from CAVD patients in combination with human valve interstitial cell (hVIC) calcification model, we screened the expression of m6A modulators and discovered that ALKBH5 alkB homolog 5, RNA demethylase (ALKBH5), a key m6A demethylase, was significantly down-regulated in calcified hVICs and human aortic valves. Consistently, increased m6A levels were seen in calcified hVICs, and treated with 3-deazaadenosine (DAA), an inhibitor of m6A modification, significantly reduced hVIC osteogenic differentiation and calcification. In addition, we showed that silencing of ALKBH5 expression increased global m6A levels, and accelerated hVIC osteogenic differentiation and calcification, whereas overexpression of ALKBH5 resulted in the opposite effect. We demonstrated that ALKBH5 directly modulate m6A levels of TGFBR2 and its mRNA stability, leading to altered TGFBR2 expression and SMAD2 signaling in hVICs. We further showed that inhibition of TGFBR2 or knockdown of SMAD2 attenuated ALKBH5 knockdown-induced hVIC osteogenic differentiation and calcification. The expression of the m6A reader protein YTH N6-methyladenosine RNA binding protein F1 (YTHDF1) was upregulated during the process of hVIC calcification. Intriguingly, we revealed that the ALKBH5 silencing-induced increased hVIC osteogenic differentiation and calcification were abolished after knockdown of YTHDF1. These data suggest a potential role YTHDF1 in aortic valve calcification. This study showed that ALKBH5 attenuated aortic valve calcification through the TGFBR2/SMAD2 signaling pathway via direct m6A modification of TGFBR2. .

Effect of Glucagon-like Peptide-1 Receptor Agonism on Aortic Valve Stenosis Risk: A Mendelian Randomization Analysis.

Background: Aortic valve repair is currently the only effective treatment for calcific aortic valve stenosis (CAVS), as no pharmacological therapies exist to prevent or slow its progression. Recent promising results showed that glucagon-like peptide-1 (GLP-1) attenuates the calcification of aortic valve interstitial cells. Therefore, we conducted a two-sample Mendelian randomization analysis to investigate the effect of GLP-1 receptor agonism (GLP-1Ra) on the risk of CAVS. Methods: The inverse variance weighted (IVW) method was used to obtain the primary causal inference, and several sensitivity analyses, including MR-Egger, were performed to assess the robustness of the results. Results: Based on the IVW estimates, the GLP-1Ra showed a neutral effect on the risk of CAVS (odds ratio [OR] per 1 mmol/mol decrease in glycated hemoglobin = 0.87, 95% CI = [0.69, 1.11], p = 0.259; I2 = 4.5%, Cohran's Q = 2.09, heterogeneity p = 0.35; F statistic = 16.8). A non-significant effect was also derived by the sensitivity analyses. No evidence of horizontal pleiotropy was identified. Conclusions: GLP-1Ra was not significantly associated with the risk of CAVS. Furthermore, pragmatically designed studies are required to evaluate the effect of GLP-1Ra on the clinical course of CAVS in different patient subgroups.

Role of CD34 in calcification of human aortic valve interstitial cells from patients with aortic valve stenosis

Various osteogenic factors are involved in ectopic human aortic valve calcification; however, the key cell species involved in calcification remains unclear. In a previous study, we reported that mesenchymal stem (CD73, 90, 105) and endothelial (VEGFR2) cell markers are positive in almost all human aortic valve interstitial cells (HAVICs) obtained from a patient with calcified aortic valve stenosis (CAVS). Further, CD34-negative HAVICs are highly sensitive to calcification stimulations. Here, we aimed to pathophysiologically clarify the role of CD34 in HAVICs obtained from individual patients with severe CAVS. A DNA microarray between CD34-positive and CD34-negative HAVICs, separated by fluorescence-activated cell sorting, indicated that tenascin X (TNX) mRNA expression significantly decreased in CD34-negative cells. Furthermore, the inflammatory cytokines, tumor necrosis factor (TNF)-α and interleukin (IL)-1β significantly downregulated CD34 expression in HAVICs. TGF-β, a key cytokine of endothelial-mesenchymal transition, did not affect HAVIC calcification. CD34 overexpression strongly inhibited TNF-α- and IL-1β-induced calcification and maintained TNX mRNA expression. These results suggest one possibility that CD34 is an inhibitory regulator of valve calcification. Furthermore, TNF-α- and IL-1β-induced CD34 downregulation in HAVICs contributes to HAVIC calcification by downregulating TNX protein expression.

Hyperglycemia-simulating environment attenuated experimentally induced calcification in cultured human aortic valve interstitial cells

Objectives: The role of diabetes mellitus as a risk factor for the development of calcific aortic valve disease has not been fully clarified. Aortic valve interstitial cells (VICs) have been suggested to be crucial for calcification of the valve. Induced calcification in cultured VICs is a good in vitro model for aortic valve calcification. The purpose of this study was to investigate whether increased glucose levels increase experimentally induced calcification in cultured human VICs. Design: VICs were isolated from explanted calcified aortic valves after valve replacement. Osteogenic medium induced calcification of cultured VICs at different glucose levels (5, 15, and 25 mM). Calcium deposits were visualized using Alizarin Red staining and measured spectrophotometrically. Results: The higher the glucose concentration, the lower the level of calcification. High glucose (25 mM) reduced calcification by 52% compared with calcification at a physiological (5 mM) glucose concentration (correlation and regression analysis: r = −0.55, p = .025 with increased concentration of glucose). Conclusions: In vitro hyperglycemia-like conditions attenuated calcification in VICs. High glucose levels may trigger a series of events that secondarily stimulate calcification of VICs in vivo.

Atorvastatin reduces calcification in valve interstitial cells via the NF-κB signalling pathway by promoting Atg5-mediated autophagy.

Aortic valve calcification (AVC) is a common cardiovascular disease and a risk factor for sudden death. However, the potential mechanisms and effective therapeutic drugs need to be explored. Atorvastatin is a statin that can effectively prevent cardiovascular events by lowering cholesterol levels. However, whether atorvastatin can inhibit AVC by reducing low-density lipoprotein (LDL) and its possible mechanism of action require further exploration. In the current study, we constructed an in vitro AVC model by inducing calcification of the valve interstitial cells. We found that atorvastatin significantly inhibited osteogenic differentiation, reduced the deposition of calcium nodules in valve interstitial cells, and enhanced autophagy in calcified valve interstitial cells, manifested by increased expression levels of the autophagy proteins Atg5 and LC3B-II/I and the formation of smooth autophagic flow. Atorvastatin inhibited the NF-κB signalling pathway and the expression of inflammatory factors mediated by NF-κB in calcified valve interstitial cells. The activation of the NF-κB signalling pathway led to the reversal of atorvastatin's effect on enhancing autophagy and alleviating valve interstitial cell calcification. In conclusion, atorvastatin inhibited the NF-κB signalling pathway by upregulating autophagy, thereby alleviating valve interstitial cell calcification, which was conducive to improving AVC.

Effects of LP533401 on vascular and bone calcification in hyperlipidemic mice

Peripheral serotonin levels are associated with cardiovascular disease risk. We previously found that serum serotonin levels are higher in hyperlipidemic mice than wild-type mice. Evidence also suggests that serotonin regulates biomineralization, in that serotonin treatment augments TNF-a-induced matrix calcification of aortic valve interstitial cells and that a selective inhibitor of peripheral serotonin, LP533401, rescues bone loss induced by ovariectomy in mice. Thus, in the present study, we examined the effects of LP533401 on both skeletal bone mineral density (BMD) and aortic calcification in both young and older hyperlipidemic mice susceptible to calcific atherosclerosis and bone loss. By serial in vivo microCT imaging, we assessed BMD and aortic calcification of Apoe−/− mice fed an atherogenic (high cholesterol) diet alone or mixed with LP533401. Results show that in the young mice, LP533401 blunted skeletal bone loss in lumbar vertebrae but not in femurs. LP533401 also blunted the initial development of aortic calcification but not its progression. Echocardiographic analysis showed that LP533401 blunted both hyperlipidemia-induced cardiac hypertrophy and left ventricular dysfunction. In the older mice, LP533401 increased the BMD of lumbar vertebrae but not of femurs. The aortic calcification progressed in both controls and LP533401-treated mice, but, at post-treatment, LP533401-treated mice had significantly less aortic calcification than the controls. These findings suggest that LP533401 mitigates adverse effects of hyperlipidemia on skeletal and vascular tissues in site- and stage-dependent manners.

Metformin ameliorates valve interstitial cell calcification by promoting autophagic flux

Calcific aortic valve disease (CAVD) is the most common heart disease of the developed world. It has previously been established that metformin administration reduces arterial calcification via autophagy; however, whether metformin directly regulates CAVD has yet to be elucidated. In the present study we investigated whether metformin alleviates valvular calcification through the autophagy-mediated recycling of Runx2. Calcification was reduced in rat valve interstitial cells (RVICs) by metformin treatment (0.5–1.5 mM) (P < 0.01), with a marked decrease in Runx2 protein expression compared to control cells (P < 0.05). Additionally, upregulated expression of Atg3 and Atg7 (key proteins required for autophagosome formation), was observed following metformin treatment (1 mM). Blocking autophagic flux using Bafilomycin-A1 revealed colocalisation of Runx2 with LC3 puncta in metformin treated RVICs (P < 0.001). Comparable Runx2 accumulation was seen in LC3 positive autolysosomes present within cells that had been treated with both metformin and hydroxychloroquine in combination (P < 0.001). Mechanistic studies employing three-way co-immunoprecipitation with Runx2, p62 and LC3 suggested that Runx2 binds to LC3-II upon metformin treatment in VICs. Together these studies suggest that the utilisation of metformin may represent a novel strategy for the treatment of CAVD.

Protective effect of niclosamide on calcification of aortic valve interstitial cells and its underlying molecular mechanism

Protective effect of niclosamide on calcification of aortic valve interstitial cells and its underlying molecular mechanism

Decreased expression of RPL15 and RPL18 exacerbated the calcification of valve interstitial cells during aortic valve calcification.

Calcific aortic valve disease (CAVD)is the most common valvular heart disease, with an increasing prevalence due to an aging population. The pathobiology of CAVD is a multifaceted and actively regulated process, but the detailed mechanisms have not been elucidated. The present study aims to identify the differentially expressed genes (DEGs)in calcified aortic valve tissues, and to analyze the correlation between DEGs and clinical features in CAVD patients. The DEGs were screened by microarray in normal and CAVD groups (n = 2 for each group), and confirmed by quantitative real-time polymerase chain reaction in normal (n = 12) and calcified aortic valve tissues (n = 34). A total of 1048 DEGs were identified in calcified aortic valve tissues, including 227 upregulated mRNAs and 821 downregulated mRNAs. Based on multiple bioinformatic analyses, three 60S ribosomal subunit components (RPL15,RPL18, and RPL18A), and two 40S ribosomal subunit components (RPS15and RPS21) were identified as the top 5 hub genes in the protein-protein interaction network of DEGs. The expression of RPL15 and RPL18 was also found significantly decreased in calcified aortic valve tissues (both p < .01), and negatively correlated with the osteogenic differentiation marker OPN in CAVD patients (both p < .01). Moreover, inhibition of RPL15 or RPL18 exacerbated the calcification of valve interstitial cells under osteogenic induction conditions. The present study proved that decreased expression of RPL15 and RPL18 was closely associated with aortic valve calcification, which provided valuable clues to find therapeutic targets for CAVD.

Niclosamide Inhibits Aortic Valve Interstitial Cell Calcification by Interfering with the GSK-3β/β-Catenin Signaling Pathway.

The most common heart valve disorder is calcific aortic valve stenosis (CAVS), which is characterized by a narrowing of the aortic valve. Treatment with the drug molecule, in addition to surgical and transcatheter valve replacement, is the primary focus of researchers in this field. The purpose of this study is to determine whether niclosamide can reduce calcification in aortic valve interstitial cells (VICs). To induce calcification, cells were treated with a pro-calcifying medium (PCM). Different concentrations of niclosamide were added to the PCM-treated cells, and the level of calcification, mRNA, and protein expression of calcification markers was measured. Niclosamide inhibited aortic valve calcification as observed from reduced alizarin red s staining in niclosamide treated VICs and also decreased the mRNA and protein expressions of calcification-specific markers: runt-related transcription factor 2 and osteopontin. Niclosamide also reduced the formation of reactive oxygen species, NADPH oxidase activity and the expression of Nox2 and p22phox. Furthermore, in calcified VICs, niclosamide inhibited the expression of β-catenin and phosphorylated glycogen synthase kinase (GSK-3β), as well as the phosphorylation of AKT and ERK. Taken together, our findings suggest that niclosamide may alleviate PCM-induced calcification, at least in part, by targeting oxidative stress mediated GSK-3β/β-catenin signaling pathway via inhibiting activation of AKT and ERK, and may be a potential treatment for CAVS.

Hypoxia-inducible factor activation promotes osteogenic transition of valve interstitial cells and accelerates aortic valve calcification in a mice model of chronic kidney disease.

Valve calcification (VC) is a widespread complication in chronic kidney disease (CKD) patients. VC is an active process with the involvement of in situ osteogenic transition of valve interstitial cells (VICs). VC is accompanied by the activation of hypoxia inducible factor (HIF) pathway, but the role of HIF activation in the calcification process remains undiscovered. Using in vitro and in vivo approaches we addressed the role of HIF activation in osteogenic transition of VICs and CKD-associated VC. Elevation of osteogenic (Runx2, Sox9) and HIF activation markers (HIF-1α and HIF-2α) and VC occurred in adenine-induced CKD mice. High phosphate (Pi) induced upregulation of osteogenic (Runx2, alkaline-phosphatase, Sox9, osteocalcin) and hypoxia markers (HIF-1α, HIF-2α, Glut-1), and calcification in VICs. Down-regulation of HIF-1α and HIF-2α inhibited, whereas further activation of HIF pathway by hypoxic exposure (1% O2) or hypoxia mimetics [desferrioxamine, CoCl2, Daprodustat (DPD)] promoted Pi-induced calcification of VICs. Pi augmented the formation of reactive oxygen species (ROS) and decreased viability of VICs, whose effects were further exacerbated by hypoxia. N-acetyl cysteine inhibited Pi-induced ROS production, cell death and calcification under both normoxic and hypoxic conditions. DPD treatment corrected anemia but promoted aortic VC in the CKD mice model. HIF activation plays a fundamental role in Pi-induced osteogenic transition of VICs and CKD-induced VC. The cellular mechanism involves stabilization of HIF-1α and HIF-2α, increased ROS production and cell death. Targeting the HIF pathways may thus be investigated as a therapeutic approach to attenuate aortic VC.

Identification of TNFα-mediated inflammation as potential pathological marker and therapeutic target for calcification progress of congenital bicuspid aortic valve

BackgroudCongenital bicuspid aortic valve (cBAV) develops calcification and stenotic obstruction early compared with degenerative tricuspid aortic valve (dTAV), which requires surgical intervention. Here we report a comparative study of patients with cBAV or dTAV to identify risk factors associated with the rapid development of calcified bicuspid valves. MethodsA total of 69 aortic valves (24 dTAV and 45 cBAV) were collected at the time of surgical aortic valve replacement for comparative clinical characteristics. Ten samples were randomly selected from each group for histology, pathology, and inflammatory factors expression and comparison analyses. OM-induced calcification in porcine aortic valve interstitial cell cultures were prepared for illustrating the underlying molecular mechanisms about calcification progress of cBAV and dTAV. ResultsWe found that cBAV patients have increased cases of aortic valve stenosis compared with dTAV patients. Histopathological examinations revealed increased collagens deposition, neovascularization and infiltrations by inflammatory cells, especially T-lymphocytes and macrophages. We identified that tumor necrosis factor α (TNFα) and its regulated inflammatory cytokines are upregulated in cBAV. Further in vitro study indicated that TNFα-NFκB and TNFα-GSK3β pathway accelerate aortic valve interstitial cells calcification, while inhibition of TNFα significantly delays this process. ConclusionThe finding of intensified TNFα-mediated inflammation in the pathological cBAV advocates the inhibition of TNFα as a potential treatment for patients with cBAV by alleviating the progress of inflammation-induced valve damage and calcification.

Capsaicin inhibits aortic valvular interstitial cell calcification via the redox-sensitive NFκB/AKT/ERK1/2 pathway

Calcific aortic valve stenosis (CAVS), the third most prevalent cardiovascular disorder is known to impose a huge social and economic burden on patients. However, no pharmacotherapy has yet been established. Aortic valve replacement is the only treatment option, although its lifelong efficacy is not guaranteed and involves inevitable complications. So, there is a crucial need to find novel pharmacological targets to delay or prevent CAVS progression. Capsaicin is well known for its anti-inflammatory and antioxidant properties and has recently been revealed to inhibit arterial calcification. We thus investigated the effect of capsaicin in attenuating aortic valve interstitial cells (VICs) calcification induced by pro-calcifying medium (PCM). Capsaicin reduced the level of calcium deposition in calcified VICs, along with reductions in gene and protein expression of the calcification markers Runx2, osteopontin, and BMP2. Based on Gene Ontology biological process and Kyoto Encyclopedia of Genes and Genomes pathway analysis oxidative stress, AKT and AGE–RAGE signaling pathways were selected. The AGE–RAGE signaling pathway activates oxidative stress and inflammation-mediated pathways including ERK and NFκB signaling pathways. Capsaicin successfully inhibited oxidative stress- and reactive oxygen species-related markers NOX2 and p22phox. The markers of the AKT, ERK1/2, and NFκB signaling pathways, namely, phosphorylated AKT, ERK1/2, NFκB, and IκBα were upregulated in calcified cells, while being significantly downregulated upon capsaicin treatment. Capsaicin attenuates VICs calcification in vitro by inhibition of redox-sensitive NFκB/AKT/ERK1/2 signaling pathway, indicating its potential as a candidate to alleviate CAVS.

Inhibition of miR-101-3p prevents human aortic valve interstitial cell calcification through regulation of CDH11/SOX9 expression

BackgroundCalcific aortic valve disease (CAVD) is the second leading cause of adult heart diseases. The purpose of this study is to investigate whether miR-101-3p plays a role in the human aortic valve interstitial cells (HAVICs) calcification and the underlying mechanisms.MethodsSmall RNA deep sequencing and qPCR analysis were used to determine changes in microRNA expression in calcified human aortic valves.ResultsThe data showed that miR-101-3p levels were increased in the calcified human aortic valves. Using cultured primary HAVICs, we demonstrated that the miR-101-3p mimic promoted calcification and upregulated the osteogenesis pathway, while anti-miR-101-3p inhibited osteogenic differentiation and prevented calcification in HAVICs treated with the osteogenic conditioned medium. Mechanistically, miR-101-3p directly targeted cadherin-11 (CDH11) and Sry-related high-mobility-group box 9 (SOX9), key factors in the regulation of chondrogenesis and osteogenesis. Both CDH11 and SOX9 expressions were downregulated in the calcified human HAVICs. Inhibition of miR-101-3p restored expression of CDH11, SOX9 and ASPN and prevented osteogenesis in HAVICs under the calcific condition.ConclusionmiR-101-3p plays an important role in HAVIC calcification through regulation of CDH11/SOX9 expression. The finding is important as it reveals that miR-1013p may be a potential therapeutic target for calcific aortic valve disease.

Disturbing effect of cepharanthine on valve interstitial cells calcification via regulating glycolytic metabolism pathways.

Osteogenic differentiation of valve interstitial cells (VICs) directly leads to aortic valve calcification, which is a common cardiovascular disease caused by inflammation and metabolic disorder. There is still no ideal drug for its treatment and prevention. The purpose of this study was to explore the effect and molecular mechanism of cepharanthine (CEP), a natural product, on inhibiting the osteogenic differentiation of VICs. First, CCK8 assay was used to evaluate cell viability of CEP on VICs. CEP concentration of 10μM was the effective dose with slight cytotoxicity, which was used for further study. The alizarin red staining analysis showed that CEP significantly inhibited calcium deposition caused by osteogenic medium related calcification induction. In order to explore the anti-calcification molecular mechanism of CEP, transcriptome and metabolome were synchronously used to discover the possible molecular mechanism and target of CEP. The results showed that CEP inhibited valve calcification by regulating the glycolytic pathway. The molecular docking of CEP and selected key factors in glycolysis showed significant binding energies for GLUT1 (-11.3kcal/mol), ENO1 (-10.6kcal/mol), PKM (-9.8kcal/mol), HK2 (-9.2kcal/mol), PFKM (-9.0kcal/mol), and PFKP (-8.9kcal/mol). The correlation analysis of RUNX2 expression and cellular lactate content showed R2 of 0.7 (p < 0.001). In conclusion, this study demonstrated that CEP inhibited osteoblastic differentiation of VICs by interfering with glycolytic metabolisms via downregulation of the production of lactate and glycolysis-associated metabolites.

Multi-omics of in vitro aortic valve calcification.

Heart valve calcification is an active cellular and molecular process that partly remains unknown. Osteogenic differentiation of valve interstitial cells (VIC) is a central mechanism in calcific aortic valve disease (CAVD). Studying mechanisms in CAVD progression is clearly needed. In this study, we compared molecular mechanisms of osteogenic differentiation of human VIC isolated from healthy donors or patients with CAVD by RNA-seq transcriptomics in early timepoint (48 h) and by shotgun proteomics at later timepoint (10th day). Bioinformatic analysis revealed genes and pathways involved in the regulation of VIC osteogenic differentiation. We found a high amount of stage-specific differentially expressed genes and good accordance between transcriptomic and proteomic data. Functional annotation of differentially expressed proteins revealed that osteogenic differentiation of VIC involved many signaling cascades such as: PI3K-Akt, MAPK, Ras, TNF signaling pathways. Wnt, FoxO, and HIF-1 signaling pathways were modulated only at the early timepoint and thus probably involved in the commitment of VIC to osteogenic differentiation. We also observed a significant shift of some metabolic pathways in the early stage of VIC osteogenic differentiation. Lentiviral overexpression of one of the most upregulated genes (ZBTB16, PLZF) increased calcification of VIC after osteogenic stimulation. Analysis with qPCR and shotgun proteomics suggested a proosteogenic role of ZBTB16 in the early stages of osteogenic differentiation.

Identification of MMP9 as a Novel Biomarker to Mitochondrial Metabolism Disorder and Oxidative Stress in Calcific Aortic Valve Stenosis.

Calcific aortic valve stenosis (CAVS) is the most common heart valve disorder among humans. To date, no effective method has been identified to prevent this disease. Herein, we aimed to identify novel diagnostic and mitochondria-related biomarkers of CAVS, based on two machine learning algorithms. We further explored their association with infiltrating immune cells and studied their potential function in CAVS. The GSE12644, GSE51472, and GSE83453 expression profiles were downloaded from the Gene Expression Omnibus (GEO) repository. The GSE12644 and GSE51472 datasets were integrated to identify differentially expressed genes (DEGs). GSE12644 contains 10 normal and 10 CAVS samples, whereas GSE51472 contains 5 normal and 10 CAVS samples. GO and KEGG assays of DEGs were conducted, and the correlation between matrix metalloproteinase 9 (MMP9) expression and immune cell infiltration was explored, using CIBERSORT. The LASSO regression model and SVM-RFE analysis were used to identify diagnostic genes. The expression of MMP9 in CAVS and non-CAVS samples was measured using RT-PCR, western blotting and immunohistochemistry. A series of functional experiments were performed to explore the potential role of MMP9 in mitochondrial metabolism and oxidative stress during CAVS progression. Twenty-two DEGs were identified, of which six genes (SCG2, PPBP, TREM1, CCL19, WIF1, and MMP9) were ultimately distinguished as diagnostic genes in CAVS. Of these, MMP9 was indicated as a mitochondria-related gene, the expression and diagnostic value of which were further confirmed in the GSE83453 dataset. Correlation analysis revealed a positive correlation between MMP9 and infiltrating immune cells. In our cohort, MMP9 expression was distinctly increased in CAVS samples, and its inhibition attenuated the calcification of valve interstitial cells (VICs) by suppressing mitochondrial damage and oxidative stress. Taken together, our findings suggest MMP9 as a novel mitochondrial dysfunction biomarker and therapeutic target for CAVS.

The Natural Product Andrographolide Ameliorates Calcific Aortic Valve Disease by Regulating the Proliferation of Valve Interstitial Cells via the MAPK-ERK Pathway.

Calcific aortic valve disease (CAVD) is an active pathobiological process that involves fibrosis and calcification of aortic valve leaflets, thereby causing cardiac hemodynamic changes and eventually heart failure. Cell proliferation changes at the initial stage of CAVD are an important target for pharmaceutical intervention. This study aimed to investigate whether andrographolide (AGP) could inhibit the proliferation of valve interstitial cells (VICs) in vitro and in vivo to delay the process of CAVD. Cell proliferative factors were tested in both healthy and CAVD aortic valve samples. Cell cycle, cell growth, and calcification of VICs were assessed using flow cytometry, CCK8 assay, EdU staining, and Alizarin Red S staining. The expression of cell proliferative factors and osteogenic factors were quantified by qRT-PCR or immunofluorescence staining. The interaction between AGP and ERK (extracellular regulated protein kinases) was detected by molecular docking. In addition, a high-fat diet-fed animal model was used to verify the effect of AGP on CAVD in vivo. In conclusion, we found that AGP ameliorates aortic valve incrassation by inhibiting cell proliferation via the MAPK-ERK signaling pathway. Therefore, AGP is a promising drug that prevents the occurrence of CAVD via regulating cell proliferation.

Galectin-3 promotes calcification of human aortic valve interstitial cells via the NF-kappa B signaling pathway.

Calcific aortic valve disease (CAVD) is an active pathobiological process that takes place at the cellular and molecular levels. It involves fibrosis and calcification of aortic valve leaflets, which eventually contributes to heart failure. Galectin-3 (Gal-3), a β-galactoside-binding lectin, is involved in myocardial fibrosis and remodeling. Our study aimed to explore how Gal-3 promoted the osteogenic differentiation of human aortic valve interstitial cells (hVICs) along with elucidating the underlying molecular mechanisms. To determine the Gal-3 expression in this study, we included the blood samples and aortic valves (AVs) from patients with CAVD (n=20) and normal controls (n=20). The hVICs were stimulated by Osteogenic medium (OM) and were treated with or without recombinant human Gal-3. Calcified transformation of hVICs was assessed by Alizarin Red S staining and osteogenic gene/protein expression. RNA-sequencing was performed for all different treatments to investigate differentially expressed genes (DEGs) along with exploring the enriched pathways for potential molecular targets of Gal-3. The targets were further detected using Western blotting and immunofluorescence staining. Gal-3 levels were found to be significantly increased in CAVD patients. Treatment of valve interstitial cells (VICs) with Gal-3 led to a marked increase in Runx2 and ALP-mRNA/protein expression levels as well as calcification. Gene expression profiles of hVICs cultured with or without Gal-3 revealed 79 upregulated genes and 82 down-regulated genes, which were highly enriched in TNF and NF-κB signaling pathways. Furthermore, Gal-3 could activate the phosphorylation of IκBα and interfere with the translocation of p65 into the cell nucleus of hVICs. However, inhibition of this pathway can suppress the osteogenic differentiation by Gal-3. Gal-3 acts as a positive regulator of osteogenic differentiation by activating the NF-κB signaling pathway in hVICs. Our findings provide novel mechanistic insights into the critical role of Gal-3 in the CAVD progression.

Mechanism of Endoplasmic Reticulum Stress Pathway in the Osteogenic Phenotypic Transformation of Aortic Valve Interstitial Cells

Background and PurposeCalcific Aortic Valve Disease (CAVD) is a crucial component of degenerative valvular disease in old age and with the increasing prevalence of the aging population. we hope that by modeling valvular osteogenesis and intervening with endoplasmic reticulum stress inhibitor TUDCA to observe the effect of endoplasmic reticulum stress on valve osteogenesisMethodsIn this study, rabbit heart valvular interstitial cells (VICs) were isolated and cultured. They treated with ox-LDL (Oxidized Low Density Lipoprotein) stimulation to establish a model of valvular osteogenic transformation. BMP2 (Bone Morphogenetic Protein 2), PERK (Protein kinase R-like endoplasmic reticulum kinase), CHOP (CCAAT/enhancer-binding protein homologous protein) and transcriptional regulatory factor ATF4 (Activating Transcription Factor 4 )were recorded after intervention with ER stress inhibitor TUDCA. The effects of er stress on valvular osteogenic transformation were analyzed.ResultAfter stimulation of VICs with ox-LDL, the expression levels of BMP2, PERK, CHOP, and ATF4 increased. However, TUDCA treatment can alleviate the increased expression levels of BMP2, PERK ATF4, and CHOP under ox-LDL stimulation to a certain extent.ConclusionThe endoplasmic reticulum stress signaling pathway is involved in ox-LDL-induced calcification of rabbit valve interstitial cells. Inhibition of endoplasmic reticulum stress using TUDCA can improve the progression of rabbit aortic valve calcification.