Canagliflozin inhibits PASMCs proliferation via regulating SGLT1/AMPK signaling and attenuates artery remodeling in MCT-induced pulmonary arterial hypertension.

Pulmonary arterial hypertension (PAH) was a devastating disease characterized by artery remodeling, ultimately resulting in right heart failure. The aim of this study was to investigate the effects of canagliflozin (CANA), a sodium-glucose cotransporter 2 inhibitor (SGLT2i) with mild SGLT1 inhibitory effects, on rats with PAH, as well as its direct impact on pulmonary arterial smooth muscle cells (PASMCs). PAH rats were induced by injection of monocrotaline (MCT) (40 mg/kg), followed by four weeks of treatment with CANA (30 mg/kg/day) or saline alone. Pulmonary artery and right ventricular (RV) remodeling and dysfunction in PAH were alleviated with CANA, as assessed by echocardiography. Hemodynamic parameters and structural of pulmonary arteriole, including vascular wall thickness and wall area, were reduced by CANA. RV hypertrophy index, cardiomyocyte hypertrophy, and fibrosis were decreased with CANA treatment. PASMCs proliferation was inhibited by CANA under stimulation by platelet-derived growth factor (PDGF)-BB or hypoxia. Activation of AMP kinase (AMPK) was induced by CANA treatment in cultured PASMCs in a time- and concentration-dependent manner. These effects of CANA were attenuated when treatment with compound C, an AMPK inhibitor. Abundant expression of SGLT1 was observed in PASMCs and pulmonary arteries, while SGLT2 expression was undetectable. SGLT1 increased in response to PDGF-BB or hypoxia stimulation, while PASMCs proliferation was inhibited and beneficial effects of CANA were counteracted by knockdown of SGLT1. Our research demonstrated for the first time that CANA inhibited the proliferation of PASMCs by regulating SGLT1/AMPK signaling and thus exerted an anti-proliferative effect on MCT-induced PAH.

Exploring potential therapeutic agents for lipopolysaccharide-induced septic cardiomyopathy based on transcriptomics using bioinformatics.

Septic cardiomyopathy (SCM) is a common and severe complication of sepsis, characterized by left ventricular dilation and reduced ejection fraction leading to heart failure. The pathogenesis of SCM remains unclear. Understanding the SCM pathogenesis is essential in the search for effective therapeutic agents for SCM. This study was to investigate the pathophysiology of SCM and explore new therapeutic drugs by bioinformatics. An SCM rat model was established by injection of 10 mg/kg lipopolysaccharide (LPS) for 24 h, and the myocardial tissues were collected for RNA sequencing. The differentially expressed genes (DEGs) between LPS rats and control (Ctrl) with the thresholds of |log2fold change|≥ 1 and P < 0.05. A protein-protein interaction (PPI) network was constructed based on the DEGs. The hub genes were identified using five algorithms of Cytoscape in the PPI networks and validated in the GSE185754 dataset and by RT-qPCR. The hub genes were analyzed by Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG), as well as Gene set enrichment analyses (GSEA). In addition, the miRNAs of hub genes were predicted through miRWalk, and the candidate therapeutic drugs were identified using the Connectivity Map (CMAP) database. This study revealed the identified hub genes (Itgb1, Il1b, Rac2, Vegfa) and key miRNAs (rno-miR-541-5p, rno-miR-487b-3p, rno-miR-1224, rno-miR-378a-5p, rno-miR-6334, and rno-miR-466b-5p), which were potential biological targets and biomarkers of SCM. Anomalies in cytokine-cytokine receptor interactions, complement and coagulation cascades, chemokine signaling pathways, and MAPK signaling pathways also played vital roles in SCM pathogenesis. Two high-confidence candidate compounds (KU-0063794 and dasatinib) were identified from the CMAP database as new therapeutic drugs for SCM. In summary, these four identified hub genes and enrichment pathways may hold promise for diagnosing and treating SCM.

Comprehensive analyses of m6A RNA methylation patterns and related immune microenvironment in idiopathic pulmonary arterial hypertension.

Idiopathic pulmonary arterial hypertension (IPAH) is a life-threatening disease with a poor prognosis and high heritability, characterized by elevated pulmonary vascular resistance (PVR) and pulmonary artery pressure. N6-methyladenosine (m6A) RNA modification influences many RNA metabolism pathways. However, the position of m6A methylation regulators in IPAH remains unknown. Therefore, the study aims to disclose the function m6A regulators exert in the pathological mechanisms of IPAH and the immune microenvironment involved. The GSE117261 dataset was downloaded from the Gene Expression Omnibus (GEO) to screen the differentially expressed genes (DEGs) between normal and IPAH samples. Functional and pathway enrichment analyses of DEGs were then conducted by Gene ontology (GO) analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG). We also identified the differentially-expressed m6A (DEm6A) regulators between normal and IPAH samples. Key m6A regulators related to the prediction of IPAH were selected using the random forest model. The results showed that FMR1, RBM15, HNRNPA2B1 and IGFBP3 were upregulated in IPAH. In contrast, LRPPRC was downregulated. The single sample gene set enrichment analysis (ssGSEA) method was then adopted to estimate the immune microenvironment in distinct m6A clusters and m6A phenotype-related genes (PRGs) clusters, respectively. Furthermore, we calculated the m6A score via principal component analysis (PCA), and the Sankey diagram was selected to present the correlation among the m6A clusters, m6A PRGs clusters and m6A score. Finally, quantitative RT-PCR and Western blotting were used to validate the key genes in human pulmonary artery smooth muscle cells (HPASMCs) treated by human platelet-derived growth factor-BB (PDGF-BB). The relative mRNA and protein expression levels of FMR1 were significantly elevated, however, the relative mRNA and protein expression levels of LRPPRC were downregulated. Besides, the relative mRNA level of HNRNPA2B1 was increased. Generally, this bioinformatics analysis might provoke more insights into diagnosing and treating IPAH.

Bioinformatics analysis of the immune cell infiltration characteristics and correlation with crucial diagnostic markers in pulmonary arterial hypertension.

BACKGROUND: Pulmonary arterial hypertension (PAH) is a pathophysiological syndrome, characterized by pulmonary vascular remodeling. Immunity and inflammation are progressively recognized properties of PAH, which are crucial for the initiation and maintenance of pulmonary vascular remodeling. This study explored immune cell infiltration characteristics and potential biomarkers of PAH using comprehensive bioinformatics analysis. METHODS: Microarray data of GSE117261, GSE113439 and GSE53408 datasets were downloaded from Gene Expression Omnibus database. The differentially expressed genes (DEGs) were identified in GSE117261 dataset. The proportions of infiltrated immune cells were evaluated by CIBERSORT algorithm. Feature genes of PAH were selected by least absolute shrinkage and selection operator (LASSO) regression analysis and validated by fivefold cross-validation, random forest and logistic regression. The GSE113439 and GSE53408 datasets were used as validation sets and logistic regression and receiver operating characteristic (ROC) curve analysis were performed to evaluate the prediction value of PAH. The PAH-associated module was identified by weighted gene association network analysis (WGCNA). The intersection of genes in the modules screened and DEGs was used to construct protein-protein interaction (PPI) network and the core genes were selected. After the intersection of feature genes and core genes, the hub genes were identified. The correlation between hub genes and immune cell infiltration was analyzed by Pearson correlation analysis. The expression level of LTBP1 in the lungs of monocrotaline-induced PAH rats was determined by Western blotting. The localization of LTBP1 and CD4 in lungs of PAH was assayed by immunofluorescence. RESULTS: A total of 419 DEGs were identified, including 223 upregulated genes and 196 downregulated genes. Functional enrichment analysis revealed that a significant enrichment in inflammation, immune response, and transforming growth factor ß (TGFß) signaling pathway. CIBERSORT analysis showed that ten significantly different types of immune cells were identified between PAH and control. Resting memory CD4+ T cells, CD8+ T cells, γδ T cells, M1 macrophages, and resting mast cells in the lungs of PAH patients were significantly higher than control. Seventeen feature genes were identified by LASSO regression for PAH prediction. WGCNA identified 15 co-expression modules. PPI network was constructed and 100 core genes were obtained. Complement C3b/C4b receptor 1 (CR1), thioredoxin reductase 1 (TXNRD1), latent TGFß binding protein 1 (LTBP1), and toll-like receptor 1 (TLR1) were identified as hub genes and LTBP1 has the highest diagnostic efficacy for PAH (AUC = 0.968). Pearson correlation analysis showed that LTBP1 was positively correlated with resting memory CD4+ T cells, but negatively correlated with monocytes and neutrophils. Western blotting showed that the protein level of LTBP1 was increased in the lungs of monocrotaline-induced PAH rats. Immunofluorescence of lung tissues from rats with PAH showed increased expression of LTBP1 in pulmonary arteries as compared to control and LTBP1 was partly colocalized with CD4+ cells in the lungs. CONCLUSION: LTBP1 was correlated with immune cell infiltration and identified as the critical diagnostic maker for PAH.

Zinc promotes cell proliferation via regulating metal-regulatory transcription factor 1 expression and transcriptional activity in pulmonary arterial hypertension.

Metal responsive transcription factor 1 (MTF-1) is a zinc-dependent transcription factor involved in the development of pulmonary arterial hypertension (PAH), which is a life-threatening disease characterized by elevated pulmonary artery pressure and pulmonary vascular remodeling. However, little is known about the role and regulatory signaling of MTF-1 in PAH. This study aimed to investigate the effect and mechanism of MTF-1 on the proliferation of pulmonary arterial smooth muscle cells (PASMCs). Several techniques including intracellular-free zinc detected by fluorescent indicator-fluozinc-3-AM, western blot, luciferase reporter, and cell proliferation assay were conducted to perform a comprehensive analysis of MTF-1 in proliferation of PASMCs in PAH. Increased cytosolic zinc was shown in monocrotaline (MCT)-PASMCs and ZnSO4-treated PASMCs, which led to overexpression and overactivation of MTF-1, followed by the up-regulation of placental growth factor (PlGF). Elevated MTF-1 and PlGF were observed in western blot, and high transcriptional activity of MTF-1 was confirmed by luciferase reporter in ZnSO4-treated cells. Further investigation of cell proliferation revealed a favorable impact of zinc ions on PASMCs proliferation, with the deletion of Mtf-1/Plgf attenuating ZnSO4-induced proliferation. Flow cytometry analysis showed that blockade of PKC signaling inhibited the cell cycle of MCT-PASMCs and ZnSO4-treated PASMCs. The Zinc/PKC/MTF-1/PlGF pathway is involved in the up-regulatory effect on the PASMCs proliferation in the process of PAH. This study provided novel insight into zinc homeostasis in the pathogenesis of PAHs, and the regulation of MTF-1 might be a potential target for therapeutic intervention in PAH.

TNF-α contributes to sarcopenia through caspase-8/caspase-3/GSDME-mediated pyroptosis.

Sarcopenia has become a leading cause of disability and mortality in the elderly. It has been reported that programmed cell death (PCD) is associated with the development of sarcopenia that is characterized by reduction of muscle fiber size and number. TNF-α is also validated to play a prominent role in sarcopenia through its complex signaling pathways including cell death signaling. However, it is still unclear whether TNF-α contributes to sarcopenia by mediating pyroptosis, one type of PCD. Here, we first established naturally aged mice with sarcopenia model and confirmed an inflammatory state represented by TNF-α in aged mice. Evidence of GSDME-mediated pyroptosis and activation of apoptotic caspase-8/-3 were also found in skeletal muscle cells of aged mice with sarcopenia. We demonstrated that TNF-α triggered GSDME-mediated pyroptosis in myotubes through activating caspase-8 and caspase-3 by using caspase-8 and caspase-3 inhibitors. Comparing the activation of caspase-8 and GSDME expression between TNF Complex IIa and TNF Complex IIb, TNF-α was found to be more inclined to assemble TNF Complex IIb in activating caspase-8 and triggering pyroptosis. Moreover, pyroptotic myotubes were validated to result in decreased expression of MHC1 and finally loss of myotubes by knockdown of GSDME. Our work reveals a novel mechanism that TNF-ɑ/caspase-8/caspase-3/GSDME signaling-mediated pyroptosis contributes to the development of sarcopenia. Caspase-3/GSDME signaling-mediated pyroptosis may be a promising therapeutic target for sarcopenia.

The identification and verification of hub genes associated with pulmonary arterial hypertension using weighted gene co-expression network analysis.

BACKGROUND: Pulmonary arterial hypertension (PAH) is characterized by a progressive increase in pulmonary vascular resistance and pulmonary arterial pressure, with complex etiology, difficult treatment and poor prognosis. The objective of this study was to investigate the potential biomarkers for PAH based on bioinformatics analysis. METHODS: The GSE117261 datasets were downloaded from the Gene Expression Omnibus database. Differentially expressed genes (DEGs) were identified by screening PAH patients and controls. Then the DEGs were analyzed using a Weighted Gene Co-expression Network Analysis (WGCNA) and the key modules were determined, and to further explore their potential biological functions via Gene Ontology analysis (GO), Kyoto Encyclopedia of Genes and Genomes Pathway analysis (KEGG), and Gene Set Enrichment Analysis (GSEA). Moreover, Protein-protein interaction (PPI) networks were constructed to identify hub gene candidates in the key modules. Finally, real-time quantitative polymerase chain reaction was supplied to detect the expressions of hub genes in human pulmonary arterial smooth cells treated with cobalt chloride (COCl2) which was used to mimic hypoxia. RESULTS: There were 2299 DEGs identified. WGCNA indicated that yellow module was the key one correlated with PAH. GO and KEGG analysis demonstrated that genes in the yellow module were mainly enriched in 'Pathways in cancer'. GSEA revealed that 'HALLMARK_MYC_TARGETS_V1' was remarkably enriched in PAH. Based on the PPI network, vascular endothelial growth factor A, proto-oncogene receptor tyrosine kinase (KIT), PNN interacting serine and arginine rich protein (PNISR) and heterogeneous nuclear ribonucleoprotein H1 (HNRNPH1) were identified as the hub genes. Additionally, the PCR indicated that the elevated expressions of PNISR and HNRNPH1 were in line with the bioinformatics analysis. ROC analysis determined that PNISR and HNRNPH1 may be potential biomarkers to provide better diagnosis of PAH. CONCLUSION: PNISR and HNRNPH1 were potential biomarkers to diagnosis PAH. In summary, the identified DEGs, modules, pathways, and hub genes provide clues and shed light on the potential molecular mechanisms of PAH.

Reduced Neutrophil Extracellular Trap Formation During Ischemia Reperfusion Injury in C3 KO Mice: C3 Requirement for NETs Release.

Complement C3 plays a prominent role in inflammatory processes, and its increase exacerbates ischemia reperfusion injury (IRI)-induced acute kidney injury (AKI). Infiltrated neutrophils can be stimulated to form neutrophil extracellular traps (NETs), leading to renal injury. However, the relationship between the increase of C3 and the release of NETs in AKI was not clear. Here we found that IRI in the mouse kidney leads to increased neutrophils infiltration and NET formation. Furthermore, neutrophils depletion by anti-Ly6G IgG (1A8) did not reduce C3 activation but reduced kidney injury and inflammation, indicating a link between neutrophils infiltration and renal tissue damage. Pretreatment with 1A8 suppressed ischemia-induced NET formation, proving that extracellular traps (ETs) in renal tissue were mainly derived from neutrophils. Renal ischemia injury also leads to increased expression of C3. Moreover, C3 KO mice (C3 KO) with IRI exhibited attenuated kidney damage and decreased neutrophils and NETs. In vitro, C3a primed neutrophils to form NETs, reflected by amorphous extracellular DNA structures that colocalized with CitH3 and MPO. These data reveal that C3 deficiency can ameliorate AKI by reducing the infiltration of neutrophils and the formation of NETs. Targeting C3 activation may be a new therapeutic strategy for alleviating the necroinflammation of NETs in AKI.

The zinc transporter ZIP12 regulates monocrotaline-induced proliferation and migration of pulmonary arterial smooth muscle cells via the AKT/ERK signaling pathways.

BACKGROUND: The zinc transporter ZIP12 is a membrane-spanning protein that transports zinc ions into the cytoplasm from the extracellular space. Recent studies demonstrated that upregulation of ZIP12 is involved in elevation of cytosolic free zinc and excessive proliferation of pulmonary arterial smooth muscle cells (PASMCs) induced by hypoxia. However, the expression of ZIP12 and its role in pulmonary arterial hypertension (PAH) induced by monocrotaline (MCT) in rats have not been evaluated previously. The aim of this study was to investigate the effect of ZIP12 on the proliferation and migration of PASMCs and its underlying mechanisms in MCT-induced PAH. METHODS: A PAH rat model was generated by intraperitoneal injection of 20 mg/kg MCT twice at one-week intervals. PASMCs were isolated from the pulmonary arteries of rats with MCT-induced PAH or control rats. The expression of ZIP12 and related molecules was detected in the lung tissues and cells. A ZIP12 knockdown lentivirus and an overexpressing lentivirus were constructed and transfected into PASMCs derived from PAH and control rats, respectively. EdU assays, wound healing assays and Western blotting were carried out to explore the function of ZIP12 in PASMCs. RESULTS: Increased ZIP12 expression was observed in PASMCs derived from MCT-induced PAH rats. The proliferation and migration of PASMCs from PAH rats were significantly increased compared with those from control rats. These results were corroborated by Western blot analysis of PCNA and cyclin D1. All these effects were significantly reversed by silencing ZIP12. Comparatively, ZIP12 overexpression resulted in the opposite effects as shown in PASMCs from control rats. Furthermore, selective inhibition of AKT phosphorylation by LY294002 abolished the effect of ZIP12 overexpression on enhancing cell proliferation and migration and partially suppressed the increase in ERK1/2 phosphorylation induced by ZIP12 overexpression. However, inhibition of ERK activity by U0126 resulted in partial reversal of this effect and did not influence an increase in AKT phosphorylation induced by ZIP12 overexpression. CONCLUSIONS: ZIP12 is involved in MCT-induced pulmonary vascular remodeling and enhances the proliferation and migration of PASMCs. The mechanism of these effects was partially mediated by enhancing the AKT/ERK signaling pathways.

Zinc-mediated activation of CREB pathway in proliferation of pulmonary artery smooth muscle cells in pulmonary hypertension.

BACKGROUND: Transcription factor CREB is involved in the development of pulmonary hypertension (PH). However, little is known about the role and regulatory signaling of CREB in PH. METHODS: A series of techniques, including bioinformatics methods, western blot, cell proliferation and luciferase reporter assay were used to perform a comprehensive analysis of the role and regulation of CREB in proliferation of pulmonary artery smooth muscle cells (PASMCs) in PH. RESULTS: Using bioinformatic analysis of the differentially expressed genes (DEGs) identified in the development of monocrotaline (MCT)- and hypoxia-induced PH, we found the overrepresentation of CRE-containing DEGs. Western blot analysis revealed a sustained increase in total- and phosphorylated-CREB in PASMCs isolated from rats treated with MCT. Similarly, an enhanced and prolonged serum-induced CREB phosphorylation was observed in hypoxia-pretreated PASMCs. The sustained CREB phosphorylation in PASMCs may be associated with multiple protein kinases phosphorylated CREB. Additionally, hierarchical clustering analysis showed reduced expression of the majority of CREB phosphatases in PH, including regulatory subunits of PP2A, Ppp2r2c and Ppp2r3a. Cell proliferation analysis showed increased PASMCs proliferation in MCT-induced PH, an effect relied on CREB-mediated transcriptional activity. Further analysis revealed the raised intracellular labile zinc possibly from ZIP12 was associated with reduced phosphatases, increased CREB-mediated transcriptional activity and PASMCs proliferation. CONCLUSIONS: CREB pathway was overactivated in the development of PH and contributed to PASMCs proliferation, which was associated with multiple protein kinases and/or reduced CREB phosphatases and raised intracellular zinc. Thus, this study may provide a novel insight into the CREB pathway in the pathogenesis of PH. Video abstract.

Transcription factor CREB plays an important role in the development of pulmonary hypertension (PH). However, paradoxical roles have been reported in the pathogenesis of PH, and the regulatory mechanisms of CREB activation in pulmonary artery smooth muscle cells (PASMCs) proliferation remained unknown. In this study, we showed that CRE-containing genes were overrepresented among the differentially expressed genes in experimental PH, which resulted from the sustained activation of CREB pathway. The sustained activation of CREB pathway may be associated with the activation of multiple protein kinases that positively regulate CREB and down-regulation of numerous phosphatases involved in CREB dephosphorylation. Additionally, we found that the proliferation of PAMSCs was dependent on the CREB-mediated transcriptional activity in experimental PH. Moreover, the raised intracellular labile zinc possibly from ZIP12 may be associated with reduced protein phosphatases, increased CREB-mediated transcriptional activity and PASMCs proliferation. Collectively, we found CREB-mediated transcriptional activity in the proliferation of PASMCs in PH, which may be associated with multiple protein kinases and/or reduced phosphatases and elevated intracellular zinc. This study may reveal a critical role of zinc-mediated activation of CREB pathway in the proliferation of PASMCs, thus providing a more comprehensive understanding of CREB pathway in the pathogenesis of PH.

[Corrigendum] Combination treatment of adipose­derived stem cells and adiponectin attenuates pulmonary arterial hypertension in rats by inhibiting pulmonary arterial smooth muscle cell proliferation and regulating the AMPK/BMP/Smad pathway.

Following the publication of the above article, an interested reader drew the authors' attention that the data featured in Fig. 1B (for adipogenic differentiation of adipose­derived stem cells) and Fig. 1F (for expression of green fluorescent protein of adipose­derived stem cells) of the above article appeared to have already been published as Fig. 1A (for adipogenic differentiation of adipose­derived stem cells) and Fig. 2D (for expression of green fluorescent protein of adipose­derived stem cells) in the following article: Luo L, Lin T, Zheng S, Xie Z, Chen M, Lian G, Xu C, Wang H and Xie L: Adipose­derived stem cells attenuate pulmonary arterial hypertension and ameliorate pulmonary arterial remodeling in monocrotaline­induced pulmonary hypertensive rats. Clin Exp Hypertens 37: 241­248, 2015. The authors consulted their original data and were able to determine that the duplication of these figure parts had arisen inadvertently during the process of compiling the figure. The revised version of Fig. 1, featuring the corrected data panels for the above­mentioned experiments in Fig. 1B and F, is shown on the next page. The authors confirm that the errors associated with this figure did not have any significant impact on either the results or the conclusions reported in this study, and are grateful to the Editor of International Journal of Molecular Medicine for allowing them the opportunity to publish this Corrigendum. Furthermore, they apologize to the readership of the Journal for any inconvenience caused. [the original article was published in International Journal of Molecular Medicine 41: 51­60, 2018; DOI: 10.3892/ijmm.2017.3226].

Influence of atorvastatin on metabolic pattern of rats with pulmonary hypertension.

BACKGROUND: Metabonomics has been widely used to analyze the initiation, progress, and development of diseases. However, application of metabonomics to explore the mechanism of pulmonary arterial hypertension (PAH) are poorly reported. This study aimed to investigate the influence of atorvastatin (Ato) on metabolic pattern of rats with pulmonary hypertension. METHODS: PAH animal model was established using monocrotaline (MCT). The mean pulmonary artery pressure (mPAP) and right ventricular hypertrophy index (RVHI) were measured. The microstructure of pulmonary arterioles was observed by HE staining. Nuclear magnetic resonance was used to detect and analyze the serum metabolites. The levels of glycogen synthase kinase-3ß (GSK-3ß), hexokinase 2 (HK-2), sterol regulatory element-binding protein 1c (SREBP-1c), and carnitine palmitoyltransferase I (CPT-1) in the lung tissues were measured. RESULTS: Ato significantly improved lung function by decreasing mPAP, RVHI, wall thickness, and wall area. Differences in metabolic patterns were observed among normal, PAH, and Ato group. The levels of GSK-3ß and SREBP-1c were decreased, but HK-2 and CPT-1 were increased in the group PAH. Ato treatment markedly reversed the influence of MCT. CONCLUSION: Ato significantly improved the pulmonary vascular remodeling and pulmonary hypertension of PAH rats due to its inhibition on Warburg effect and fatty acid ß oxidation.

Development of a nomogram for screening the risk of left ventricular hypertrophy in Chinese hypertensive patients.

Left ventricular hypertrophy (LVH) is an important risk factor for cardiovascular morbidity and mortality in hypertensives. Therefore, early identification of at-risk patients is necessary. The objective of this study was to estimate the risk of LVH among Chinese hypertensives by designing a nomogram. 832 hypertensives were divided into two groups based on the presence of LVH. The least absolute shrinkage and selection operator (LASSO) regression and multivariable logistic regression were successively applied for optimal variable selection and nomogram construction. Discrimination power, calibration, and clinical usefulness were evaluated using the receiver operating characteristic (ROC) curve, calibration curve, and decision curve analysis. Internal validation was performed using the bootstrap method. The nomogram included five predictors, namely gender, duration of hypertension, age, body mass index (BMI), and systolic blood pressure. The area under the ROC curve (AUC) was 0.724 (95% CI: 0.687-0.761), indicating moderate discrimination. The calibration curve showed an excellent agreement between the predicted LVH and the actual LVH probability. The risk threshold between 5% and 72% according to the decision curve analysis, and the nomogram is clinically beneficial. Internal validation by bootstrapping with 1000 samples showed a good C-index of 0.715, which suggested that the predictive abilities for the training set and testing set were in consistency. Our study proposed a nomogram that can be utilized to assess the LVH risk rapidly for Chinese hypertensives. This tool could be useful in identifying patients at high risk for LVH. Further studies are required to ascertain the stability and applicability of this nomogram.

Relationship between high-normal albuminuria and arterial stiffness in Chinese population.

High-normal albuminuria is related to the morbidity and mortality of cardiovascular disease. Arterial stiffness has been regarded as a predictor of cardiovascular disease. However, the relationship between high-normal albuminuria and arterial stiffness is uncertain in Chinese population. A total of 1343 Chinese participants (aged 58.9 ± 12.1 years, 63.53% male) were included in this study. High-normal albuminuria was defined as urinary albumin-to-creatinine ratio (UACR) above the median within normal albuminuria. Based on the level of UACR, all participants were divided into low-normal albuminuria group (UACR < 6.36 mg/g, n = 580), high-normal albuminuria group (6.36 mg/g ≤ UACR < 30 mg/g, n = 581), microalbuminuria (30 mg/g ≤ UACR < 300 mg/g, n = 162), and macroalbuminuria (UACR ≥ 300 mg/g, n = 20). Arterial stiffness was assessed by measuring carotid-femoral pulse wave velocity (cfPWV). With the increment of UACR, the level of cfPWV was increased gradually (P < .001). Stepwise multiple regression analysis showed that systolic blood pressure, age, serum creatinine, heart rate, logarithmic (LG)-transformed UACR, and fasting plasma glucose were independently associated with cfPWV in all subjects (P < .001). LG-UACR was found to be related to cfPWV in high-normal albuminuria and macroalbuminuria subjects. After further stratification in the high-normal albuminuria subjects, their relation remained in male, elderly over 65 years old, or normotensives. In summary, UACR is associated with arterial stiffness in subjects with proteinuria excretion in high normal level. High-normal albuminuria might be an early indicator of arterial stiffness, especially in male, elderly, or normotensives in Chinese population. Furthermore, age and blood pressure are still observed to be the most important risk factor of arterial stiffness.

Role of 20-hydroxyeicosatetraenoic acid in pulmonary hypertension and proliferation of pulmonary arterial smooth muscle cells.

OBJECTIVE: To investigate the level of 20-Hydroxyeicosatetraenoic acid (20-HETE) in model of pulmonary hypertension (PH) and its effect on the proliferation of pulmonary arterial smooth muscle cells (PASMCs). METHODS: Twenty male Sprague-Dawley rats were randomly divided into two groups, including control group and PH group. PH was induced by intra-peritoneal injection of 20 mg/kg monocrotaline (MCT) twice in a week in 10 rats, and control rats were given equal amount of saline. Mean pulmonary arterial pressure (mPAP), right ventricular hypertrophy index (RVHI) and pulmonary vascular remodeling index (WA%, WT%) were assessed at the week 4. The levels of 20-HETE were analysed by liquid chromatography tandem-mass spectrometry (LC-MS/MS). EdU test was used to determine the proliferation of PASMCs. Intracellular levels of reactive oxygen species (ROS) were detected using DCFH-DA dye. RESULTS: (1) Prominent right ventricular hypertrophy and pulmonary vascular remodeling were verified in PH rats; (2) 20-HETE levels in lung tissue and serum were significantly lifted in PH rats; (3) Increased 20-HETE levels in cell culture supernatants were significantly noted in hypoxia condition; (4) Proliferation of PASMCs was induced by 20-HETE and hypoxia, and was inhibited by HET0016; (5) Production of ROS was elevated by 20-HETE and hypoxia, and was reduced by HET0016; CONCLUSION: Vascular remodeling was demonstrated in PH rats. 20-HETE levels were significantly increased in PH rats and under hypoxia condition. PASMCs proliferation and ROS production were elevated by 20-HETE and could be inhibited by HET0016, a specific inhibitor of 20-HETE. Taken together, changes in the level of 20-HETE may be related to the excessive proliferation of PASMCs in PH rats.

Transcriptomic analysis identifies Toll-like and Nod-like pathways and necroptosis in pulmonary arterial hypertension.

Inflammation and immunity play a causal role in the pathogenesis of pulmonary vascular remodelling and pulmonary arterial hypertension (PAH). However, the pathways and mechanisms by which inflammation and immunity contribute to pulmonary vascular remodelling remain unknown. RNA sequencing was used to analyse the transcriptome in control and rats injected with monocrotaline (MCT) for various weeks. Using the transcriptional profiling of MCT-induced PAH coupled with bioinformatics analysis, we clustered the differentially expressed genes (DEGs) and chose the increased expression patterns associated with inflammatory and immune response. We found the enrichment of Toll-like receptor (TLR) and Nod-like receptor (NLR) pathways and identified NF-κB-mediated inflammatory and immune profiling in MCT-induced PAH. Pathway-based data integration and visualization showed the dysregulated TLR and NLR pathways, including increased expression of TLR2 and NLRP3, and their downstream molecules. Further analysis revealed that the activation of TLR and NLR pathways was associated with up-regulation of damage-associated molecular patterns (DAMPs) and RIPK3-mediated necroptosis was involved in the generation of DAMPs in MCT-induced PAH. Collectively, we identify RIPK3-mediated necroptosis and its triggered TLR and NLR pathways in the progression of pulmonary vascular remodelling, thus providing novel insights into the mechanisms underlying inflammation and immunity in the pathogenesis of PAH.

miR-125a-5p inhibits glycolysis by targeting hexokinase-II to improve pulmonary arterial hypertension.

PURPOSE: The aim of this study was to investigate the effect of microRNAs on the proliferation of pulmonary arterial smooth muscle cells (PASMCs) as a result of targeting hexokinase-II (HK-II) and its mechanism of action. RESULTS: Differences in metabolic patterns were found between the normal group and monocrotaline-induced pulmonary arterial hypertension (MCT-PH) group. miR-125a-5p decreased glycolysis levels of monocrotaline (MCT)-induced PASMCs by targeting HK-II and inhibiting its proliferation. In vivo experiments found that miR-125a-5p agomir upregulated HK-II expression in the MCT-PH. Right ventricular hypertrophy was reversed and cardiac function improved as a result of decreased mean pulmonary artery pressure (mPAP). CONCLUSION: In vitro and in vivo experiments both confirmed that miR-125a-5p could inhibit cell glycolysis and PASMC proliferation to improve PAH by targeting HK-II. METHODS: HK-II overexpression was constructed, and differentially expressed microRNAs were screened for using microarrays. Serum metabolites were detected using Nuclear Magnetic Resonance (NMR). Through screening for characteristic metabolites in rat body fluids and by analyzing biological functions, disordered metabolic pathways were identified. Activity of the miR-125a-5p target HK-II was measured using a luciferase reporter assay. Expression of downstream molecules was measured by RT-qPCR and/or western blot. Glucose consumption and lactic acid production were analyzed and used as a reflection of glycolysis.

RNA sequencing analysis of monocrotaline-induced PAH reveals dysregulated chemokine and neuroactive ligand receptor pathways.

Pulmonary arterial hypertension (PAH) is a serious disease characterized by elevated pulmonary artery pressure, inflammatory cell infiltration and pulmonary vascular remodeling. However, little is known about the pathogenic mechanisms underlying the disease onset and progression. RNA sequencing (RNA-seq) was used to identify the transcriptional profiling in control and rats injected with monocrotaline (MCT) for 1, 2, 3 and 4 weeks. A total of 23200 transcripts and 280, 1342, 908 and 3155 differentially expressed genes (DEGs) were identified at the end of week 1, 2, 3 and 4, of which Svop was the common top 10 DEGs over the course of PAH progression. Functional enrichment analysis of DEGs showed inflammatory/immune response occurred in the early stage of PAH development. KEGG pathway enrichment analysis of DEGs showed that cytokine-cytokine receptor interaction and neuroactive ligand-receptor interaction were in the initiation and progression of PAH. Further analysis revealed impaired expression of cholinergic receptors, adrenergic receptors including alpha1, beta1 and beta2 receptor, and dysregulated expression of γ-aminobutyric acid receptors. In summary, the dysregulated inflammation/immunity and neuroactive ligand receptor signaling pathways may be involved in the onset and progression of PAH.

Electrochemiluminescence biosensor for miRNA-21 based on toehold-mediated strand displacement amplification with Ru(phen)32+ loaded DNA nanoclews as signal tags.

A novel electrochemiluminescence (ECL) biosensor was developed for high sensitive and selective detection of miRNA-21 based on the efficient and specific toehold-mediated strand displacement (TMSD) amplification with Ru(phen)32+ loaded DNA nanoclews (NCs-Ru(phen)32+) as signal tags. The stable DNA nanoclews, synthesized by a simple rolling circle amplification reaction, were employed to load with Ru(phen)32+ efficiently as ECL signal tags to amplify the signals. As for TMSD, the substrate strand (Sub) was initially hybridized with P1 and P2 to form DNA duplex structures with a toehold 1. miRNA-21 could hybridize with the toehold 1 and trigger the TMSD amplification with the help of assist strand, releasing lots of P1 stands from DNA duplex structures. The TMSD technique realized the converting and amplification of the single miRNA-21 input to lots of output DNA (namely P1) with good selectivity, simultaneously. Output P1 were designed to expand the stem-locked region of HP, which were immobilized on the Au electrodes firstly. Subsequently, the opened HP could hybridize with the Ru(phen)32+, capturing the ECL signal tags closed to the sensing surface. The ECL intensity of the system had a linear relationship with the logarithm of the miRNA-21 concentration in the range of 1.0 fM to 100 pM with a limit of detection of 0.65 fM. The strategy was further applied to detect miRNA-21 in complex samples, and the result was consistent with the qRT-PCR.

Low-Grade Albuminuria Is Associated with Left Ventricular Hypertrophy and Diastolic Dysfunction in Patients with Hypertension.

INTRODUCTION: Microalbuminuria is a risk factor for cardiovascular morbidity and mortality in hypertensive patients. However, the relationship between low-grade albuminuria, a higher level of albuminuria below microalbuminuria threshold, and hypertension-related organ damage is unclear. Left ventricular (LV) hypertrophy (LVH) is well recognized to be a subclinical organ damage of hypertension, and LV diastolic dysfunction is also reported to be an early functional cardiac change of hypertension that predicts heart failure. The present study aimed to investigate the association of low-grade albuminuria with LVH and LV diastolic dysfunction in hypertensive patients. METHODS: This cross-sectional observational clinical study was retrospectively performed in 870 hypertensive patients admitted to our hospital. Urinary albumin to creatinine ratio (UACR) was calculated to assess the levels of albuminuria: macroalbuminuria (≥300 mg/g), microalbuminuria (≥30 mg/g, but <300 mg/g), and normal albuminuria (<30 mg/g). Low-grade albuminuria was defined as sex-specific highest tertile within normal albuminuria (8.1-29.6 mg/g in males and 11.8-28.9 mg/g in females). LVH and LV diastolic dysfunction were identified as recommended by American Society of Echocardiography. RESULTS: Of the 870 patients, 765 (87.9%) had normal albuminuria, 77 (8.9%) had microalbuminuria, and 28 (3.2%) had macroalbuminuria. Percentage of LVH and LV diastolic dysfunction was increased with ascending UACR. UACR was independently associated with LVH and LV diastolic dysfunction, even in patients with normal albuminuria. Multivariable logistic regression showed that the patients with the highest tertile within normal albuminuria had nearly 80% increase in LVH and nearly 60% increase in LV diastolic dysfunction (adjusted OR for LVH 1.788, 95% CI 1.181-2.708, p = 0.006; adjusted OR for LV diastolic dysfunction 1.567, 95% CI 1.036-2.397, p = 0.034). After further stratification analyses in patients with normal albuminuria, it was shown that this independent association persisted in female patients, those who were younger than 70 years old, and those with duration of hypertension <15 years. CONCLUSION: Low-grade albuminuria was associated with LVH and LV diastolic dysfunction in hypertensive patients, especially in patients younger than 70 years old, and those with duration of hypertension <15 years.