Lumican promotes calcific aortic valve disease through H3 histone lactylation.

BACKGROUND AND AIMS: Valve interstitial cells (VICs) undergo a transition to intermediate state cells before ultimately transforming into the osteogenic cell population, which is a pivotal cellular process in calcific aortic valve disease (CAVD). Herein, this study successfully delineated the stages of VIC osteogenic transformation and elucidated a novel key regulatory role of lumican (LUM) in this process. METHODS: Single-cell RNA-sequencing (scRNA-seq) from nine human aortic valves was used to characterize the pathological switch process and identify key regulatory factors. The in vitro, ex vivo, in vivo, and double knockout mice were constructed to further unravel the calcification-promoting effect of LUM. Moreover, the multi-omic approaches were employed to analyse the molecular mechanism of LUM in CAVD. RESULTS: ScRNA-seq successfully delineated the process of VIC pathological transformation and highlighted the significance of LUM as a novel molecule in this process. The pro-calcification role of LUM is confirmed on the in vitro, ex vivo, in vivo level, and ApoE-/-//LUM-/- double knockout mice. The LUM induces osteogenesis in VICs via activation of inflammatory pathways and augmentation of cellular glycolysis, resulting in the accumulation of lactate. Subsequent investigation has unveiled a novel LUM driving histone modification, lactylation, which plays a role in facilitating valve calcification. More importantly, this study has identified two specific sites of histone lactylation, namely, H3K14la and H3K9la, which have been found to facilitate the process of calcification. The confirmation of these modification sites' association with the expression of calcific genes Runx2 and BMP2 has been achieved through ChIP-PCR analysis. CONCLUSIONS: The study presents novel findings, being the first to establish the involvement of lumican in mediating H3 histone lactylation, thus facilitating the development of aortic valve calcification. Consequently, lumican would be a promising therapeutic target for intervention in the treatment of CAVD.

Modulation of anti-cardiac fibrosis immune responses by changing M2 macrophages into M1 macrophages.

BACKGROUND: Macrophages play a crucial role in the development of cardiac fibrosis (CF). Although our previous studies have shown that glycogen metabolism plays an important role in macrophage inflammatory phenotype, the role and mechanism of modifying macrophage phenotype by regulating glycogen metabolism and thereby improving CF have not been reported. METHODS: Here, we took glycogen synthetase kinase 3ß (GSK3ß) as the target and used its inhibitor NaW to enhance macrophage glycogen metabolism, transform M2 phenotype into anti-fibrotic M1 phenotype, inhibit fibroblast activation into myofibroblasts, and ultimately achieve the purpose of CF treatment. RESULTS: NaW increases the pH of macrophage lysosome through transmembrane protein 175 (TMEM175) and caused the release of Ca2+ through the lysosomal Ca2+ channel mucolipin-2 (Mcoln2). At the same time, the released Ca2+ activates TFEB, which promotes glucose uptake by M2 and further enhances glycogen metabolism. NaW transforms the M2 phenotype into the anti-fibrotic M1 phenotype, inhibits fibroblasts from activating myofibroblasts, and ultimately achieves the purpose of treating CF. CONCLUSION: Our data indicate the possibility of modifying macrophage phenotype by regulating macrophage glycogen metabolism, suggesting a potential macrophage-based immunotherapy against CF.

FOXO1 regulates RUNX2 ubiquitination through SMURF2 in calcific aortic valve disease.

The prevalence of calcific aortic valve disease (CAVD) remains substantial while there is currently no medical therapy available. Forkhead box O1 (FOXO1) is known to be involved in the pathogenesis of cardiovascular diseases, including vascular calcification and atherosclerosis; however, its specific role in calcific aortic valve disease remains to be elucidated. In this study, we identified FOXO1 significantly down-regulated in the aortic valve interstitial cells (VICs) of calcified aortic valves by investigating clinical specimens and GEO database analysis. FOXO1 silencing or inhibition promoted VICs osteogenic differentiation in vitro and aortic valve calcification in Apoe-/- mice, respectively. We identified that FOXO1 facilitated the ubiquitination and degradation of RUNX2, which process was mainly mediated by SMAD-specific E3 ubiquitin ligase 2 (SMURF2). Our discoveries unveil a heretofore unacknowledged mechanism involving the FOXO1/SMURF2/RUNX2 axis in CAVD, thereby proposing the potential therapeutic utility of FOXO1 or SMURF2 as viable strategies to impede the progression of CAVD.

AVCAPIR: A Novel Procalcific PIWI-Interacting RNA in Calcific Aortic Valve Disease.

BACKGROUND: Calcification of the aortic valve leads to increased leaflet stiffness and consequently results in the development of calcific aortic valve disease (CAVD). However, the underlying molecular and cellular mechanisms of calcification remain unclear. Here, we identified a novel aortic valve calcification-associated PIWI-interacting RNA (piRNA; AVCAPIR) that increases valvular calcification and promotes CAVD progression. METHODS: Using piRNA sequencing, we identified piRNAs contributing to the pathogenesis of CAVD that we termed AVCAPIRs. High-cholesterol diet-fed ApoE-/- mice with AVCAPIR knockout were used to examine the role of AVCAPIR in aortic valve calcification (AVC). Gain- and loss-of-function assays were conducted to determine the role of AVCAPIR in the induced osteogenic differentiation of human valvular interstitial cells. To dissect the mechanisms underlying AVCAPIR-elicited procalcific effects, we performed various analyses, including an RNA pulldown assay followed by liquid chromatography-tandem mass spectrometry, methylated RNA immunoprecipitation sequencing, and RNA sequencing. RNA pulldown and RNA immunoprecipitation assays were used to study piRNA interactions with proteins. RESULTS: We found that AVCAPIR was significantly upregulated during AVC and exhibited potential diagnostic value for CAVD. AVCAPIR deletion markedly ameliorated AVC in high-cholesterol diet-fed ApoE-/- mice, as shown by reduced thickness and calcium deposition in the aortic valve leaflets, improved echocardiographic parameters (decreased peak transvalvular jet velocity and mean transvalvular pressure gradient, as well as increased aortic valve area), and diminished levels of osteogenic markers (Runx2 and Osterix) in aortic valves. These results were confirmed in osteogenic medium-induced human valvular interstitial cells. Using unbiased protein-RNA screening and molecular validation, we found that AVCAPIR directly interacts with FTO (fat mass and obesity-associated protein), subsequently blocking its N6-methyladenosine demethylase activity. Further transcriptomic and N6-methyladenosine modification epitranscriptomic screening followed by molecular validation confirmed that AVCAPIR hindered FTO-mediated demethylation of CD36 mRNA transcripts, thus enhancing CD36 mRNA stability through the N6-methyladenosine reader IGF2BP1 (insulin-like growth factor 2 mRNA binding protein 1). In turn, the AVCAPIR-dependent increase in CD36 stabilizes its binding partner PCSK9 (proprotein convertase subtilisin/kexin type 9), a procalcific gene, at the protein level, which accelerates the progression of AVC. CONCLUSIONS: We identified a novel piRNA that induced AVC through an RNA epigenetic mechanism and provide novel insights into piRNA-directed theranostics in CAVD.

A gadoxetic acid-enhanced MRI-based model using LI-RADS v2018 features for preoperatively predicting Ki-67 expression in hepatocellular carcinoma.

PURPOSE: To construct a gadoxetic acid-enhanced MRI (EOB-MRI) -based multivariable model to predict Ki-67 expression levels in hepatocellular carcinoma (HCC) using LI-RADS v2018 imaging features. METHODS: A total of 121 patients with HCC who underwent EOB-MRI were enrolled in this study. The patients were divided into three groups according to Ki-67 cut-offs: Ki-67 ≥ 20% (n = 86) vs. Ki-67 < 20% (n = 35); Ki-67 ≥ 30% (n = 73) vs. Ki-67 < 30% (n = 48); Ki-67 ≥ 50% (n = 45) vs. Ki-67 < 50% (n = 76). MRI features were analyzed to be associated with high Ki-67 expression using logistic regression to construct multivariable models. The performance characteristic of the models for the prediction of high Ki-67 expression was assessed using receiver operating characteristic curves. RESULTS: The presence of mosaic architecture (p = 0.045), the presence of infiltrative appearance (p = 0.039), and the absence of targetoid hepatobiliary phase (HBP, p = 0.035) were independent differential factors for the prediction of high Ki-67 status (≥ 50% vs. < 50%) in HCC patients, while no features could predict high Ki-67 status with thresholds of 20% (≥ 20% vs. < 20%) and 30% (≥ 30% vs. < 30%) (p > 0.05). Four models were constructed including model A (mosaic architecture and infiltrated appearance), model B (mosaic architecture and targetoid HBP), model C (infiltrated appearance and targetoid HBP), and model D (mosaic architecture, infiltrated appearance and targetoid HBP). The model D yielded better diagnostic performance than the model C (0.776 vs. 0.669, p = 0.002), but a comparable AUC than model A (0.776 vs. 0.781, p = 0.855) and model B (0.776 vs. 0.746, p = 0.076). CONCLUSIONS: Mosaic architecture, infiltrated appearance and targetoid HBP were sensitive imaging features for predicting Ki-67 index ≥ 50% and EOB-MRI model based on LI-RADS v2018 features may be an effective imaging approach for the risk stratification of patients with HCC before surgery.

Gli1 promotes the phenotypic transformation of valve interstitial cells through Hedgehog pathway activation exacerbating calcific aortic valve disease.

Calcific aortic valve disease (CAVD) is the most prevalent human valve disease worldwide. Multiple factors induce "irreversible" pathological changes in the aortic valve leaflets, resulting in changes in cardiac hemodynamics, eventually leading to heart failure. However, no effective pharmaceutical interventions have been found and prosthetic valve replacement is the only curative approach. Glioma-associated oncogene 1 (Gli1) exerts a regulatory role on cardiovascular diseases, and it is already a therapeutic target to combat tumors. Our research aimed to explore the role and basic mechanism of Gli1 in CAVD, to pave the way for the discovery of effective drugs in the treatment of CAVD. Human aortic valve tissues were obtained to evaluate Gli1 expression and primary valve interstitial cells (VICs) were used to perform related experiments. The results showed that Gli1 promoted cell proliferation and significantly accelerated cell osteogenic transformation through the up-regulation of the osteogenic factors Runx2 and Alp, in turn through the AKT signaling pathway by targeting P130cas expression. Furthermore, Gli1 was activated by TGF-ß and sonic hedgehog through the canonical and non-canonical Hedgehog signaling pathways in VICs. Our results indicated that Gli1 promoted cell proliferation and accelerated cell osteogenic transformation in VICs, providing a new strategy for the therapy of CAVD by targeting Gli1.

RNA-sequencing of human aortic valves identifies that miR-629-3p and TAGLN miRNA-mRNA pair involving in calcified aortic valve disease

This study aimed to uncover the microRNA and messenger RNA (miRNA/mRNA) interactions in the pathophysiological process of calcified aortic valve disease (CAVD) of the human aortic valve. RNA sequencing of six selected samples (3 healthy control samples vs. 3 CAVD samples) was performed to obtain mRNA and miRNA sequences, and differential expression (DE) analysis of miRNA and mRNAs was performed. To build a CAVD-specific miRNA-mRNA interactome, the upregulated mRNAs and downregulated miRNAs were selected, followed by the establishment of inverse DE of mRNA-miRNA co-expression network based on Pearson’s correlation coefficient using miRanda in the R language software. Subsequently, pathway enrichment analysis was performed to elucidate CAVD-related pathways that were likely mediated by miRNA regulatory mechanisms. In addition, miRNAs with an mRNA correlation greater than 0.9 in the co-expression network were selected for anti-calcification verification in a CAVD cellular model. We identified 216 mRNAs (99 downregulated and 117 upregulated) and 602 miRNAs (371 downregulated and 231 upregulated) that were differentially expressed between CAVD and healthy aortic valves. After applying Pearson’s correlation toward miRNA-mRNA targets, a regulatory network of 67 miRNAs targeting 76 mRNAs was created. The subsequent pathway enrichment analysis of these targeted mRNAs elucidated that genes within the focal adhesion pathway are likely mediated by miRNA regulatory mechanisms. The selected hsa-miR-629-3p and TAGLN pair exhibited anti-calcification effects on osteogenic differentiation-induced human aortic valve interstitial cells (hVICs). On integrating the miRNA and mRNA sequencing data for healthy aortic valves and those with CAVD, the CAVD-associated miRNA-mRNA interactome and related pathways were elucidated. (AU)

Dihydromyricetin ameliorates osteogenic differentiation of human aortic valve interstitial cells by targeting c-KIT/interleukin-6 signaling pathway.

Aims: Calcific aortic valve disease (CAVD) is a chronic cardiovascular disease with high morbidity that lacks effective pharmacotherapeutics. As a natural flavonoid extracted from Ampelopsis grossedentata, dihydromyricetin (DHM) has been shown to be effective in protecting against atherosclerosis; yet, the therapeutic role of DHM in CAVD remains poorly understood. Herein, we aimed to clarify the therapeutic implications of DHM in CAVD and the underlying molecular mechanisms in human valvular interstitial cells (hVICs). Methods and Results: The protein levels of two known osteogenesis-specific genes (alkaline phosphatase, ALP; runt-related transcription factor 2, Runx2) and calcified nodule formation in hVICs were detected by Western blot and Alizarin Red staining, respectively. The results showed that DHM markedly ameliorated osteogenic induction medium (OM)-induced osteogenic differentiation of hVICs, as evidenced by downregulation of ALP and Runx2 expression and decreased calcium deposition. The SwissTargetPrediction database was used to identify the potential AVC-associated direct protein target of DHM. Protein-protein interaction (PPI) analysis revealed that c-KIT, a tyrosine-protein kinase, can act as a credible protein target of DHM, as evidenced by molecular docking. Mechanistically, DHM-mediated inhibition of c-KIT phosphorylation drove interleukin-6 (IL-6) downregulation in CAVD, thereby ameliorating OM-induced osteogenic differentiation of hVICs and aortic valve calcification progression. Conclusion: DHM ameliorates osteogenic differentiation of hVICs by blocking the phosphorylation of c-KIT, thus reducing IL-6 expression in CAVD. DHM could be a viable therapeutic supplement to impede CAVD.

RNA-sequencing of human aortic valves identifies that miR-629-3p and TAGLN miRNA-mRNA pair involving in calcified aortic valve disease.

This study aimed to uncover the microRNA and messenger RNA (miRNA/mRNA) interactions in the pathophysiological process of calcified aortic valve disease (CAVD) of the human aortic valve. RNA sequencing of six selected samples (3 healthy control samples vs. 3 CAVD samples) was performed to obtain mRNA and miRNA sequences, and differential expression (DE) analysis of miRNA and mRNAs was performed. To build a CAVD-specific miRNA-mRNA interactome, the upregulated mRNAs and downregulated miRNAs were selected, followed by the establishment of inverse DE of mRNA-miRNA co-expression network based on Pearson's correlation coefficient using miRanda in the R language software. Subsequently, pathway enrichment analysis was performed to elucidate CAVD-related pathways that were likely mediated by miRNA regulatory mechanisms. In addition, miRNAs with an mRNA correlation greater than 0.9 in the co-expression network were selected for anti-calcification verification in a CAVD cellular model. We identified 216 mRNAs (99 downregulated and 117 upregulated) and 602 miRNAs (371 downregulated and 231 upregulated) that were differentially expressed between CAVD and healthy aortic valves. After applying Pearson's correlation toward miRNA-mRNA targets, a regulatory network of 67 miRNAs targeting 76 mRNAs was created. The subsequent pathway enrichment analysis of these targeted mRNAs elucidated that genes within the focal adhesion pathway are likely mediated by miRNA regulatory mechanisms. The selected hsa-miR-629-3p and TAGLN pair exhibited anti-calcification effects on osteogenic differentiation-induced human aortic valve interstitial cells (hVICs). On integrating the miRNA and mRNA sequencing data for healthy aortic valves and those with CAVD, the CAVD-associated miRNA-mRNA interactome and related pathways were elucidated. Additional cell function data demonstrated anti-calcification effects of the selected hsa-miR-629-3p targeting TAGLN, validating that it is a potential therapeutic target for inhibiting CAVD.

CEND1 deficiency induces mitochondrial dysfunction and cognitive impairment in Alzheimer's disease.

Alzheimer's disease (AD) is the most common form of neurodegenerative disease featured with memory loss and cognitive function impairments. Chronic mitochondrial stress is a vital pathogenic factor for AD and finally leads to massive neuronal death. However, the underlying mechanism is unclear. By proteomic analysis, we identified a new mitochondrial protein, cell-cycle exit and neuronal differentiation 1 (CEND1), which was decreased significantly in the brain of 5xFAD mice. CEND1 is a neuronal specific protein and locates in the presynaptic mitochondria. Depletion of CEND1 leads to increased mitochondrial fission mediated by upregulation of dynamin related protein 1 (Drp1), resulting in abnormal mitochondrial functions. CEND1 deficiency leads to cognitive impairments in mice. Overexpression of CEND1 in the hippocampus of 5xFAD mice rescued cognitive deficits. Moreover, we identified that CDK5/p25 interacted with and phosphorylated CEND1 which promoted its degradation. Our study provides new mechanistic insights in mitochondrial function regulations by CEND1 in Alzheimer's disease.

Prosthesis Selection for Aortic Valve Replacement With Concomitant Coronary Artery Bypass Grafting.

BACKGROUND: The optimal prosthesis for aortic valve replacement (AVR) with concomitant coronary artery bypass grafting (CABG) is controversial. We investigated postoperative outcomes in these patients with a biological prosthesis or mechanical prosthesis. METHODS: A retrospective cohort analysis was performed of 2485 patients aged 50 to 69 years who underwent AVR+CABG in Hubei province hospitals from 2002 to 2018. The median follow-up duration was 6.5 years (interquartile range, 0-15.8 years). Propensity score matching for 18 baseline characteristics yielded 346 patient pairs between bioprosthetic and mechanical prosthetic groups. End points were death, stroke, major bleeding event, and reoperation. RESULTS: No differences in survival, stroke, or overall reoperation rates were observed between the bioprosthetic and mechanical valve groups. The 15-year cumulative incidence of reoperation due to prosthesis failure/dysfunction was higher in the bioprosthetic group (hazard ratio [HR], 2.72; 95% confidence interval [CI], 1.26-5.88; P = .011), whereas the 15-year cumulative incidence of reoperation due to coronary artery disease progression/bypass failure was similar between 2 groups (HR, 0.76; 95% CI, 0.37-1.57; P = .459). Mechanical valves were associated with a higher 15-year cumulative incidence of major bleeding events compared with bioprosthetic valves (HR, 1.92; 95% CI, 1.16-3.19; P = .012). CONCLUSIONS: Long-term survival, overall reoperation, or stroke incidence was comparable among the 2 groups, while patients with a mechanical valve showed a greater likelihood of major bleeding events. Regarding the limited durability of bioprosthetic valves, a larger sample size monitored for 15 or more years will be necessary to determine the optimal aortic valve prosthesis for patients aged 50 to 69 years undergoing concurrent AVR and CABG.

Ciliary protein Kif7 regulates Gli and Ezh2 for initiating the neuronal differentiation of enteric neural crest cells during development.

Gastrointestinal motility disorders occur frequently in patients with ciliopathy, but the underlying genetic link is unclear. The ciliary protein Kif7 can positively or negatively regulate Hedgehog signaling in different cellular contexts. Mice with neural crest cell (NCC)­specific Kif7 deficiency show a marked reduction of enteric NOS+ inhibitory neurons. Malformation of enteric nervous system (ENS) causes growth retardation and gut motility defect in mice. Mechanistically, Kif7 inhibits Gli2 in enteric NCCs (ENCCs), where Gli2 positively regulates the expression of Ezh2 by inhibiting the miR124-mediated suppression. In developing ENCCs, Ezh2 is a master regulator of 102 core genes underlying ENCC differentiation. Deletion of Gli2 or inhibition of Ezh2 favors the neurogenic lineage differentiation of mouse and human ENCCs and rescues the ENS defects of Kif7 mutants. In summary, Hedgehog signal, via Kif7-Gli-Ezh2, controls the timely expressions of the core genes to mediate the differentiation of ENCCs.

DUSP26 induces aortic valve calcification by antagonizing MDM2-mediated ubiquitination of DPP4 in human valvular interstitial cells.

AIMS: The morbidity and mortality rates of calcific aortic valve disease (CAVD) remain high while treatment options are limited. Here, we evaluated the role and therapeutic value of dual-specificity phosphatase 26 (DUSP26) in CAVD. METHODS AND RESULTS: Microarray profiling of human calcific aortic valves and normal controls demonstrated that DUSP26 was significantly up-regulated in calcific aortic valves. ApoE-/- mice fed a normal diet or a high cholesterol diet (HCD) were infected with adeno-associated virus serotype 2 carrying DUSP26 short-hairpin RNA to examine the effects of DUSP26 silencing on aortic valve calcification. DUSP26 silencing ameliorated aortic valve calcification in HCD-treated ApoE-/- mice, as evidenced by reduced thickness and calcium deposition in the aortic valve leaflets, improved echocardiographic parameters (decreased peak transvalvular jet velocity and mean transvalvular pressure gradient, as well as increased aortic valve area), and decreased levels of osteogenic markers (Runx2, osterix, and osteocalcin) in the aortic valves. These results were confirmed in osteogenic medium-induced human valvular interstitial cells. Immunoprecipitation, liquid chromatography-tandem mass spectrometry, and functional assays revealed that dipeptidyl peptidase-4 (DPP4) interacted with DUSP26 to mediate the procalcific effects of DUSP26. High N6-methyladenosine levels up-regulated DUSP26 in CAVD; in turn, DUSP26 activated DPP4 by antagonizing mouse double minute 2-mediated ubiquitination and degradation of DPP4, thereby promoting CAVD progression. CONCLUSION: DUSP26 promotes aortic valve calcification by inhibiting DPP4 degradation. Our findings identify a previously unrecognized mechanism of DPP4 up-regulation in CAVD, suggesting that DUSP26 silencing or inhibition is a viable therapeutic strategy to impede CAVD progression.

Factors influencing osteogenic differentiation of human aortic valve interstitial cells.

OBJECTIVE: Human aortic valve interstitial cells redifferentiate into an osteoblast-like phenotype, which is the key cellular mechanism of aortic valve calcification. Methyltransferase-like 3, the N6-methyladenosine methylation writer, has emerged as a new layer of epigenetic regulation for osteogenic differentiation of bone mesenchymal stem cells. The current study sought to determine whether methyltransferase-like 3 also plays a role in the osteogenic differentiation of human aortic valve interstitial cells. METHODS: Aortic valves from patients with aortic stenosis (n = 50) and normal controls (n = 50) were subjected to determination of methyltransferase-like 3 expression. Mineralized bone matrix formation was assessed by Alizarin Red staining. The interaction between methyltransferase-like 3 and twist-related protein 1 was confirmed via luciferase reporter and N6-methyladenosine methylated RNA immunoprecipitation quantitative reverse-transcription polymerase chain reaction. RESULTS: Methyltransferase-like 3 was highly expressed in human calcified aortic valves (1.61 ± 0.50) versus normal valves (3.07 ± 0.62; P < .0001). Osteogenic stimulation for 7 days resulted in a 2.15 ± 0.16-fold increase (P < .0001) in methyltransferase-like 3 protein level compared with the control group in human aortic valve interstitial cells. Functionally, methyltransferase-like 3 acted as a positive regulator of osteogenic differentiation of human aortic valve interstitial cells. Mechanistically, methylated RNA immunoprecipitation quantitative reverse-transcription polymerase chain reaction identified twist-related protein 1 as a target of methyltransferase-like 3-mediated m6A modification. Moreover, N6-methyladenosine-mediated twist-related protein 1 mRNA inhibition relied on the m6A binding protein YTH-domain family member 2-dependent pathway. CONCLUSIONS: Methyltransferase-like 3 promotes osteogenic differentiation of human aortic valve interstitial cells by inhibiting twist-related protein 1 through an N6-methyladenosine YTH-domain family member 2-dependent pathway. Our findings provide novel mechanistic insights into a critical role of methyltransferase-like 3 in the aortic valve calcification progression and shed new light on N6-methyladenosine-directed diagnostics and therapeutics in aortic valve calcification.

Cell-Type Transcriptome Atlas of Human Aortic Valves Reveal Cell Heterogeneity and Endothelial to Mesenchymal Transition Involved in Calcific Aortic Valve Disease.

OBJECTIVE: Leaflet thickening, fibrosis, and hardening are early pathological features of calcific aortic valve disease (CAVD). An inadequate understanding of the resident aortic valve cells involved in the pathological process may compromise the development of therapeutic strategies. We aim to construct a pattern of the human aortic valve cell atlas in healthy and CAVD clinical specimens, providing insight into the cellular origins of CAVD and the complex cytopathological differentiation process. Approach and Results: We used unbiased single-cell RNA sequencing for the high-throughput evaluation of cell heterogeneity in 34 632 cells isolated from 6 different human aortic valve leaflets. Cellular experiments, in situ localization, and bulk sequencing were performed to verify the differences between normal, healthy valves and those with CAVD. By comparing healthy and CAVD specimens, we identified 14 cell subtypes, including 3 heterogeneous subpopulations of resident valve interstitial cells, 3 types of immune-derived cells, 2 types of valve endothelial cells, and 6 novel valve-derived stromal cells found particularly in CAVD leaflets. Combining additional verification experiments with single-cell transcriptome profiling provided evidence of endothelial to mesenchymal transition involved in lesion thickening of the aortic valve leaflet. CONCLUSIONS: Our findings deconstructed the aortic valve cell atlas and suggested novel functional interactions among resident cell subpopulations. Our findings may provide insight into future targeted therapies to prevent CAVD.

Caffeic Acid Phenethyl Ester Ameliorates Calcification by Inhibiting Activation of the AKT/NF-κB/NLRP3 Inflammasome Pathway in Human Aortic Valve Interstitial Cells.

Calcific aortic valve disease (CAVD) occurs via a pathophysiological process that includes inflammation-induced osteoblastic differentiation of aortic valvular interstitial cells (AVICs). Here, we investigated the role of the anti-inflammatory compound caffeic acid phenethyl ester (CAPE) in inhibiting CAVD. Human AVICs were isolated and cultured in osteogenic induction medium (OM) with or without 10 µM CAPE. Cell viability was assessed using CCK8 assays and calcified transformation of AVICs was evaluated by Alizarin Red staining and osteogenic gene/protein expression. RNA-sequencing was conducted to identify differentially expressed genes (DEGs) and enrichment in associated pathways, as potential molecular targets through which CAPE inhibits osteogenic induction. The regulatory effects of CAPE on activation of the AKT/NF-κB and NLRP3 inflammasome were evaluated by Western blot analysis and immunofluorescent staining. CAPE slowed the growth of AVICs cultured in OM but did not show significant cytotoxicity. In addition, CAPE markedly suppressed calcified nodule formation and decreased gene/protein expression of RUNX2 and ALP in AVICs. Gene expression profiles of OM-induced AVICs cultured with or without CAPE revealed 518 common DEGs, which were highly enriched in the NOD-like receptor, PI3K-AKT, and NF-κB signaling pathways. Furthermore, CAPE inhibited phosphorylation of AKT, ERK1/2, and NF-κB, and suppressed NLRP3 inflammasome activation in AVICs cultured in OM. Thus, CAPE is implicated as a potent natural product for the prevention of CAVD by inhibiting activation of the AKT/NF-κB pathway and NLRP3 inflammasome.

The Tumor-Suppressive Human Circular RNA CircITCH Sponges miR-330-5p to Ameliorate Doxorubicin-Induced Cardiotoxicity Through Upregulating SIRT6, Survivin, and SERCA2a.

RATIONALE: Doxorubicin is one of the most potent antitumor agents available; however, its clinical use is restricted because it poses a risk of severe cardiotoxicity. Previous work has established that CircITCH (circular RNA ITCH [E3 ubiquitin-protein ligase]) is a broad-spectrum tumor-suppressive circular RNA and that its host gene, ITCH (E3 ubiquitin protein ligase), is involved in doxorubicin-induced cardiotoxicity (DOXIC). Whether CircITCH plays a role in DOXIC remains unknown. OBJECTIVE: We aimed to dissect the role of CircITCH in DOXIC and further decipher its potential mechanisms. METHODS AND RESULTS: Circular RNA sequencing was performed to screen the potentially involved circRNAs in DOXI pathogenesis. Quantitative polymerase chain reaction and RNA in situ hybridization revealed that CircITCH was downregulated in doxorubicin-treated human-induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) as well as in the autopsy specimens from cancer patients who suffered from doxorubicin-induced cardiomyopathy. Cell death/viability assays, detection of cardiomyocyte necrosis markers, microelectrode array, and cardiomyocyte functional assays revealed that CircITCH ameliorated doxorubicin-induced cardiomyocyte injury and dysfunction. Detection of cellular/mitochondrial oxidative stress and DNA damage markers verified that CircITCH alleviated cellular/mitochondrial oxidative stress and DNA damage induced by doxorubicin. RNA pull-down assays, Ago2 immunoprecipitation and double fluorescent in situ hybridization identified miR-330-5p as a direct target of CircITCH. Moreover, CircITCH was found to function by acting as an endogenous sponge that sequestered miR-330-5p. Bioinformatic analysis, luciferase reporter assays, and quantitative polymerase chain reaction showed that SIRT6 (sirtuin 6), BIRC5 (baculoviral IAP repeat containing 5, Survivin), and ATP2A2 (ATPase sarcoplasmic/endoplasmic reticulum Ca2+ transporting 2, SERCA2a [SR Ca2+-ATPase 2]) were direct targets of miR-330-5p and that they were regulated by the CircITCH/miR-330-5p axis in DOXIC. Further experiments demonstrated that CircITCH-mediated alleviation of DOXIC was dependent on the interactions between miR-330-5p and the 3'-UTRs of SIRT6, BIRC5, and ATP2A2 mRNA. Finally, AAV9 (adeno-associated virus serotype 9) vector-based overexpression of the well-conserved CircITCH partly prevented DOXIC in mice. CONCLUSIONS: CircITCH represents a novel therapeutic target for DOXIC because it acts as a natural sponge of miR-330-5p, thereby upregulating SIRT6, Survivin and SERCA2a to alleviate doxorubicin-induced cardiomyocyte injury and dysfunction.

Melatonin ameliorates aortic valve calcification via the regulation of circular RNA CircRIC3/miR-204-5p/DPP4 signaling in valvular interstitial cells.

Calcific aortic valve disease (CAVD) is highly prevalent with marked morbidity and mortality rates and a lack of pharmaceutical treatment options because its mechanisms are unknown. Melatonin is reported to exert atheroprotective effects. However, whether melatonin protects against aortic valve calcification, a disease whose pathogenesis shares many similarities to that of atherosclerosis, and the underlying molecular mechanisms remain unknown. In this study, we found that the intragastric administration of melatonin for 24 weeks markedly ameliorated aortic valve calcification in high cholesterol diet (HCD)-treated ApoE-/- mice, as evidenced by reduced thickness and calcium deposition in the aortic valve leaflets, improved echocardiographic parameters (decreased transvalvular peak jet velocity and increased aortic valve area), and decreased osteogenic differentiation marker (Runx2, osteocalcin, and osterix) expression in the aortic valves. Consistent with these in vivo data, we also confirmed the suppression of in vitro calcification by melatonin in hVICs. Mechanistically, melatonin reduced the level of CircRIC3, a procalcification circular RNA, which functions by acting as a miR-204-5p sponge to positively regulate the expression of the procalcification gene dipeptidyl peptidase-4 (DPP4). Furthermore, CircRIC3 overexpression abolished the inhibitory effects of melatonin on hVIC osteogenic differentiation. Taken together, our results suggest that melatonin ameliorates aortic valve calcification via the regulation of CircRIC3/miR-204-5p/DPP4 signaling in hVICs; therefore, melatonin medication might be considered a novel pharmaceutical strategy for CAVD treatment.

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.

The natural compound andrographolide inhibits human aortic valve interstitial cell calcification via the NF-kappa B/Akt/ERK pathway.

Calcific aortic valve disease (CAVD) is caused by valve interstitial cells (VICs) initiating the thickening and calcification of valve leaflets. The present study aimed to investigate whether andrographolide (AGP) could attenuate the calcification of human valve interstitial cells (hVICs). hVICs stimulated by osteoblastic medium (OM) were treated with or without AGP. RNA sequencing was utilized to investigate changes in gene expression. Cell growth and calcification of hVICs were assessed using a CCK8 assay and Alizarin Red S staining, respectively. The expression of the two calcification-related markers, RUNX2 and ALP, were quantified by qRT-PCR, Western blotting, and immunofluorescent staining. The results indicate that hVICs treated with OM plus AGP exhibited decreased Alizarin Red S staining compared with cells treated with OM only in addition to down-regulation of ALP and RUNX2. Mappings of differentially expressed genes (DEGs) in different groups using Venn diagrams during analysis of gene expression profiles, 653 common DEGs were identified that displayed different biological functions and signaling pathways after treatment with AGP. RELA, a core factor of the NF-κB pathway was inhibited by AGP in addition to phosphorylation of AKT and ERK1/2. Thus, AGP attenuated calcification of hVICs. These results demonstrate that AGP, a promising natural product, can attenuate the process of CAVD.