C1q/Tumor Necrosis Factor-Related Protein-9 Is a Novel Vasculoprotective Cytokine That Restores High Glucose-Suppressed Endothelial Progenitor Cell Functions by Activating the Endothelial Nitric Oxide Synthase.

BACKGROUND: This study investigated whether gCTRP9 (globular C1q/tumor necrosis factor-related protein-9) could restore high-glucose (HG)-suppressed endothelial progenitor cell (EPC) functions by activating the endothelial nitric oxide synthase (eNOS). METHODS AND RESULTS: EPCs were treated with HG (25 mmol/L) and gCTRP9. Migration, adhesion, and tube formation assays were performed. Adiponectin receptor 1, adiponectin receptor 2, and N-cadherin expression and AMP-activated protein kinase, protein kinase B, and eNOS phosphorylation were measured by Western blotting. eNOS activity was determined using nitrite production measurement. In vivo reendothelialization and EPC homing assays were performed using Evans blue and immunofluorescence in mice. Treatment with gCTRP9 at physiological levels enhanced migration, adhesion, and tube formation of EPCs. gCTRP9 upregulated the phosphorylation of AMP-activated protein kinase, protein kinase B, and eNOS and increased nitrite production in a concentration-dependent manner. Exposure of EPCs to HG-attenuated EPC functions induced cellular senescence and decreased eNOS activity and nitric oxide synthesis; the effects of HG were reversed by gCTRP9. Protein kinase B knockdown inhibited eNOS phosphorylation but did not affect gCTRP9-induced AMP-activated protein kinase phosphorylation. HG impaired N-cadherin expression, but treatment with gCTRP9 restored N-cadherin expression after HG stimulation. gCTRP9 restored HG-impaired EPC functions through both adiponectin receptor 1 and N-cadherin-mediated AMP-activated protein kinase /protein kinase B/eNOS signaling. Nude mice that received EPCs treated with gCTRP9 under HG medium showed a significant enhancement of the reendothelialization capacity compared with those with EPCs incubated under HG conditions. CONCLUSIONS: CTRP9 promotes EPC migration, adhesion, and tube formation and restores these functions under HG conditions through eNOS-mediated signaling mechanisms. Therefore, CTRP9 modulation could eventually be used for vascular healing after injury.

Fluid Shear Stress Ameliorates Prehypertension-Associated Decline in Endothelium-Reparative Potential of Early Endothelial Progenitor Cells.

This study investigated the effects of prehypertension and shear stress on the reendothelialization potential of human early EPCs and explored its potential mechanisms. Early EPCs from the prehypertensive patients showed reduced migration and adhesion in vitro and demonstrated a significantly impaired in vivo reendothelialization capacity. Shear stress pretreatment markedly promoted the in vivo reendothelialization capacity of EPCs. Although basal CXCR4 expression in early EPCs from prehypertensive donors was similar to that from healthy control, SDF-1-induced phosphorylation of CXCR4 was lower in prehypertensive EPCs. Shear stress up-regulated CXCR4 expression and increased CXCR4 phosphorylation, and restored the SDF-1/CXCR4-dependent JAK-2 phosphorylation in prehypertensive EPCs. CXCR4 knockdown or JAK-2 inhibitor treatment prevents against shear stress-induced increase in the migration, adhesion and reendothelialization capacity of the prehypertensive EPCs. Collectively, CXCR4 receptor profoundly modulates the reendothelialization potential of early EPCs. The abnormal CXCR4-mediated JAK-2 signaling may contribute to impaired functions of EPCs from patients with prehypertension.

5mC modification patterns provide novel direction for early acute myocardial infarction detection and personalized therapy.

Background: Most deaths from coronary artery disease (CAD) are due to acute myocardial infarction (AMI). There is an urgent need for early AMI detection, particularly in patients with stable CAD. 5-methylcytosine (5mC) regulatory genes have been demonstrated to involve in the progression and prognosis of cardiovascular diseases, while little research examined 5mC regulators in CAD to AMI progression. Method: Two datasets (GSE59867 and GSE62646) were downloaded from Gene Expression Omnibus (GEO) database, and 21 m5C regulators were extracted from previous literature. Dysregulated 5mC regulators were screened out by "limma." The least absolute shrinkage and selection operator (LASSO) and support vector machine recursive feature elimination (SVM-RFE) algorithm were employed to identify hub 5mC regulators in CAD to AMI progression, and 43 clinical samples (Quantitative real-time PCR) were performed for expression validation. Then a logistic model was built to construct 5mC regulator signatures, and a series of bioinformatics algorithms were performed for model validation. Besides, 5mC-associated molecular clusters were studied via unsupervised clustering analysis, and correlation analysis between immunocyte and 5mC regulators in each cluster was conducted. Results: Nine hub 5mC regulators were identified. A robust model was constructed, and its prominent classification accuracy was verified via ROC curve analysis (area under the curve [AUC] = 0.936 in the training cohort and AUC = 0.888 in the external validation cohort). Besides, the clinical effect of the model was validated by decision curve analysis. Then, 5mC modification clusters in AMI patients were identified, along with the immunocyte infiltration levels of each cluster. The correlation analysis found the strongest correlations were TET3-Mast cell in cluster-1 and TET3-MDSC in cluster-2. Conclusion: Nine hub 5mC regulators (DNMT3B, MBD3, UHRF1, UHRF2, NTHL1, SMUG1, ZBTB33, TET1, and TET3) formed a diagnostic model, and concomitant results unraveled the critical impact of 5mC regulators, providing interesting epigenetics findings in AMI population vs. stable CAD.

Importance of ß2AR elevation for re-endothelialization capacity mediated by late endothelial progenitor cells in hypertensive patients.

Dysfunction of late endothelial progenitor cells (EPCs) has been suggested to be associated with hypertension. ß2-Adrenergic receptor (ß2AR) is a novel and key target for EPC homing. Here, we proposed that attenuated ß2AR signaling contributes to EPCs dysfunction, whereas enhanced ß2AR signaling restores EPCs' functions in hypertension. EPCs derived from hypertensive patients exhibited reduced cell number, impaired in vitro migratory and adhesion abilities, and impaired re-endothelialization after transplantation in nude mice with carotid artery injury. ß2AR expression of EPCs from hypertensive patients was markedly downregulated, whereas the phosphorylation of the p38 mitogen-activated protein kinase (p38-MAPK) was elevated. The cleaved caspase-3 levels were elevated in EPCs. The overexpression of ß2AR in EPCs from hypertensive patients inhibited p38-MAPK signaling, whereas it enhanced in vitro EPC proliferation, migration, and adhesion and in vivo re-endothelialization. The ß2AR-mediated effects were attenuated by treating the EPCs with a neutralizing monoclonal antibody against ß2AR, which could be partially antagonized by the p38-MAPK inhibitor SB203580. Moreover, shear stress stimulation, a classic nonpharmacological intervention, increased the phosphorylation levels of ß2AR and enhanced the in vitro and in vivo functions of EPCs from hypertensive patients. Collectively, the current investigation demonstrated that impaired ß2AR/p38-MAPK/caspase-3 signaling at least partially reduced the re-endothelialization capacity of EPCs from hypertensive patients. Restoration of ß2AR expression and shear stress treatment could improve their endothelial repair capacity by regulating the p38-MAPK/caspase-3 signaling pathway. The clinical significance of ß2AR in endothelium repair still requires further investigation.NEW & NOTEWORTHY Impaired ß2-adrenergic receptor (ß2AR) expression with an elevation of p38-MAPK/caspase-3 signaling at least partially contributes to the decline of re-endothelialization capacity of late endothelial progenitor cells (EPCs) from hypertensive patients. ß2AR gene transfer and shear stress treatment improve the late EPC-mediated enhancement of the re-endothelialization capacity in hypertensive patients through activating ß2AR/p38-MAPK/caspase-3 signaling. The present study is the first to reveal the potential molecular mechanism of the impaired endothelium-reparative capacity of late EPCs in hypertension after vascular injury and strongly suggests that ß2AR is a novel and crucial therapeutic target for increasing EPC-mediated re-endothelialization capacity in hypertension.

The effect of fluid shear stress in hydrogen sulphide production and cystathionine γ-lyase expression in human early endothelial progenitor cells.

BACKGROUND: Physiological fluid shear stress has been shown to have a beneficial impact on vascular homeostasis. Endothelial progenitor cells (EPCs) make a significant contribution to maintaining endothelial integrity. Therefore, we hypothesised that shear stress-induced endothelium protection plays a role in hydrogen sulphide (H2S) production and up-regulation of cystathionine γ-lyase (CSE) expression in EPCs. METHODS: Human EPC-derived CSE activity was detected by colorimetric assay, and H2S production was evaluated by membrane adsorption method. Cell proliferation, migration, and adhesion were assessed by MTT, Transwell, and endothelial cell-mediated adhesion assays, respectively. Real-time polymerase chain reaction (RT-PCR) was carried out to analyse gene expression. Protein expression was analysed by western blot. RESULTS: Human EPCs were treated with shear stress levels of 5-25 dyn/cm2 for up to 3 h, and 25 dyn/cm2 for up to 24 h. H2S production and CSE mRNA expression in the EPCs were increased by shear stress in a dose-dependent manner in vitro. Likewise, time-dependent shear stress also significantly enhanced CSE protein expression. Compared to static condition, shear stress improved EPCs proliferation, migration and adhesion capacity. Knockdown of CSE expression by small interfering RNA substantially eliminated the shear stress-induced above functions of human EPCs in vitro. CONCLUSIONS: This study gives new insight into the regulatory effect of physiological shear stress on the CSE/H2S system in human EPCs. Our findings may contribute to the development of vascular protective research, although the relevant evidence is admittedly indirect.

Exogenous Hydrogen Sulfide Attenuates High Glucose-Induced Cardiotoxicity by Inhibiting NLRP3 Inflammasome Activation by Suppressing TLR4/NF-κB Pathway in H9c2 Cells.

BACKGROUND/AIMS: This study aimed to investigate whether exogenous hydrogen sulfide (H2S) confered cardiac protection against high glucose (HG)-induced injury by inhibiting NLRP3 inflammasome activation via a specific TLR4/NF-κB pathway. METHODS: H9c2 cardiac cells were exposed to 33 mM glucose for 24 h to induce HG-induced cytotoxicity. The cells were pretreated with NaHS (a donor of H2S) before exposure to HG. Cell viability, cell apoptosis, intracellular reactive oxygen species (ROS), mitochondrial membrane potential (MMP), and TLR4, NF-κB, NLRP3 inflammasome, IL-1ß, IL-18 and caspase-3 expression were measured by standard methods. RESULTS: H2S attenuated HG-induced cell apoptosis, ROS expression and loss of MMP and reduced the expression of NLRP3, ASC, pro-caspase-1, caspase-1, IL-1ß, IL-18 and caspase-3. In addition, H2S inhibited the HG-induced activation of TLR4 and NF-κB. Furthermore, NLRP3 inflammasome activation was regulated by the TLR4 and NF-κB pathway. CONCLUSION: The present study demonstrated for the first time that H2S appears to suppress HG-induced cardiomyocyte inflammation and apoptosis by inhibiting the TLR4/NF-κB pathway and its downstream NLRP3 inflammasome activation. Thus H2S might possess potential in the treatment of diabetic cardiomyopathy.

Exogenous hydrogen sulfide protects against high glucose­induced inflammation and cytotoxicity in H9c2 cardiac cells.

Hyperglycemia serves an important role in the pathogenesis of diabetic cardiomyopathy. The aim of the present study was to investigate whether exogenous hydrogen sulfide (H2S) protects against high glucose­induced inflammation and cytotoxicity in cardiac cells by inhibiting the p38 mitogen­activated protein kinase (MAPK)/nuclear factor­κB (NF­κB), cyclooxygenase­2 (COX­2) and inducible nitric oxide synthase (iNOS) signaling pathways. Rat H9c2 myocardium cells were exposed to 33 mM glucose (high glucose, HG) for 24 h to stimulate HG­induced cytotoxicity. One group of cells was pretreated with NaHS (a donor of H2S) prior to HG exposure, and cell viability was determined using the Cell Counting Kit­8 assay. The protein expression levels of p38MAPK, the phosphorylated p65 subunit of NF­κB, iNOS, COX­2 and caspase­3 were analyzed by western blotting, and the protein expression levels of interleukin (IL)­1ß and IL­6 were detected by enzyme­linked immunosorbent assay (ELISA). Pretreatment of H9c2 cells with NaHS for 30 min prior to exposure to HG significantly ameliorated the expression of p38MAPK and NF­κB. In addition, pretreatment with NaHS markedly attenuated p38MAPK/NF­κB­mediated cytotoxicity and inflammation, as evidenced by the significant increase in cell viability and decrease in iNOS, COX­2, IL­1ß and IL­6 expression levels. Furthermore, treatment of cells with NaHS significantly decreased the expression of caspase­3, which suggested that NaHS attenuated HG­induced apoptosis. In conclusion, the results of the present study provided evidence to suggest that exogenous H2S protects against HG­induced cytotoxicity and inflammation in H9c2 cardiac cells. H2S may exert these cytoprotective effects via inhibition of the p38MAPK/NF­κB, COX­2 and iNOS signaling pathways.

Design, synthesis, and antihypertensive activity of curcumin-inspired compounds via ACE inhibition and vasodilation, along with a bioavailability study for possible benefit in cardiovascular diseases.

This study describes the synthesis of a novel series of curcumin-inspired compounds via a facile synthetic route. The structures of these derivatives were ascertained using various spectroscopic and analytic techniques. The pharmacological effects of the target analogs were assessed by assaying their inhibition of angiotensin-converting enzyme (ACE). All of the synthesized derivatives exhibited considerable inhibition of ACE, with half-maximal inhibitory concentrations ranging from 1.23 to 120.32 µM. In a docking analysis with testicular ACE (tACE), the most promising inhibitor (4j) was efficiently accommodated in the deep cleft of the protein cavity, making close interatomic contacts with Glu162, His353, and Ala356, comparable with lisinopril. Compounds 4i, 4j, 4k, and 4l were further selected for determination of their vasodilator activity (cardiac output and stroke volume) on isolated rat hearts using the Langendorff technique. The bioavailability of compound 4j was determined in experimental mice.

Calcitriol prevents peripheral RSC96 Schwann neural cells from high glucose & methylglyoxal-induced injury through restoration of CBS/H2S expression.

A meta-analysis has suggested that vitamin D deficiency is involved in diabetic peripheral neuropathy (DPN) and the levels of hydrogen sulfide (H2S) are also decreased in type 2 diabetes. The injection of vitamin D induces cystathionine-ß-synthase (CBS) expression and H2S generation. However, it remains unclear whether the supplementation of vitamin D prevents DPN through improvement of CBS/H2S expression. In the present study, RSC96 cells, a rat Schwann cell line, were exposed to high glucose and methylglyoxal (HG&MG) to simulate diabetic peripheral nerve injury in vivo. Before the exposure to HG&MG, the cells were preconditioned with calcitriol (CCT), an active form of vitamin D, and then CCT-mediated neuroprotection was investigated in respect of cellular viability, superoxide anion (O2(-)) generation, inducible nitric oxide (NO) synthase (iNOS)/NO expression, mitochondrial membrane potential (MMP), as well as CBS expression and activity. It was found that both high glucose and MGO decreased cell viability and co-treatment with the two induced a more serious injury in RSC96 cells. Therefore, the exposure to HG&MG was used in the present study. The exposure to HG&MG markedly induced iNOS expression, NO and O2(-) generation, as well as MMP loss. In addition, the exposure to HG&MG depressed CBS expression and activity in RSC96 cells. However, the preconditioning with CCT significantly antagonized HG&MG-induced cell injury including the decreased viability, iNOS overexpression, NO and O2(-) accumulation, as well as MMP loss. CCT also partially restored the decreased CBS expression and activity triggered by HG&MG, while the inhibition of CBS with hydroxylamine attenuated CCT-mediated neuroprotection. Moreover, the exogenous donation of H2S produced similar cellular protective effects to CCT. The data indicate that the supplementation of vitamin D prevents HG&MG-induced peripheral nerve injury involving the restoration of endogenous H2S system, which may provide a basal support for the treatment of DPN with vitamin D clinically.

Exogenous hydrogen sulfide alleviates high glucose-induced cardiotoxicity via inhibition of leptin signaling in H9c2 cells.

Hydrogen sulfide (H2S) protects cardiomyoblasts against high glucose (HG)-induced injury by inhibiting the activation of p38 mitogen-activated protein kinase (MAPK). This study aims to determine whether the leptin-p38 MAPK pathway is involved in HG-induced injury and whether exogenous H2S prevents the HG-induced insult through inhibition of the leptin-p38 MAPK pathway in H9c2 cells. H9c2 cells were treated with 35 mM glucose (HG) for 24 h to establish a HG-induced cardiomyocyte injury model. Cell viability; mitochondrial membrane potential (ΔΨ m); apoptosis; reactive oxygen species (ROS) level; and leptin, leptin receptor, and p38 MAPK expression level were measured by the methods indicated. The results showed pretreatment of H9c2 cells with NaHS before exposure to HG led to an increase in cell viability, decrease in apoptotic cells, ROS generation, and a loss of ΔΨ m. Exposure of H9c2 cells to 35 mM glucose for 24 h significantly upregulated the expression levels of leptin and leptin receptors. The increased expression levels of leptin and leptin receptors were markedly attenuated by pretreatment with 400 µM NaHS. In addition, the HG-induced increase in phosphorylated (p) p38 MAPK expression was ameliorated by pretreatment with 50 ng/ml leptin antagonist. In conclusion, the present study has demonstrated for the first time that the leptin-p38 MAPK pathway contributes to the HG-induced injury in H9c2 cells and that exogenous H2S protects H9c2 cells against HG-induced injury at least in part by inhibiting the activation of leptin-p38 MAPK pathway.

Inhibition of ROS-activated ERK1/2 pathway contributes to the protection of H2S against chemical hypoxia-induced injury in H9c2 cells.

Hydrogen sulfide (H(2)S) has been shown to exert cardioprotective effects. However, the roles of extracellular signal-regulated protein kinases 1/2 (ERK1/2) in H(2)S-induced cardioprotection have not been completely elucidated. In this study, cobalt chloride (CoCl(2)), a chemical hypoxia mimetic agent, was applied to treat H9c2 cells to establish a chemical hypoxia-induced cardiomyocyte injury model. The results showed that pretreatment with NaHS (a donor of H(2)S) before exposure to CoCl(2) attenuated the decreased cell viability, the increased apoptosis rate, the loss of mitochondrial membrane potential (ΔΨm), and the intracellular accumulation of reactive oxygen species (ROS) in H9c2 cells. Exposure of H9c2 cells to CoCl(2) or hydrogen peroxide (H(2)O(2)) upregulated expression of phosphorylated (p) ERK1/2, which was reduced by pretreatment with NaHS or N-acetyl-L-cysteine, a ROS scavenger. More importantly, U0126, a selective inhibitor of ERK1/2, mimicked the above cytoprotection of H(2)S against CoCl(2)-induced injury in H9c2 cells. In conclusion, these results indicate that H(2)S protects H9c2 cells against chemical hypoxia-induced injury partially by inhibiting ROS-mediated activation of ERK1/2.

Cyclooxygenase mediates cardioprotection of angiotensin-(1-7) against ischemia/reperfusion-induced injury through the inhibition of oxidative stress.

Angiotensin (Ang)-(1-7) exhibits cardioprotective effects in myocardial ischemia reperfusion (I/|R)-induced injury. However, the roles of oxidation and cyclooxygenase (COX) in the cardioprotection of Ang-(1-7) remain unclear. This study was conducted to investigate whether oxidation and COX were involved in the cardioprotection of Ang-(1-7) against I/|R-induced injury in isolated rat hearts. The hearts were subjected to 15 min regional ischemia followed by 30 min reperfusion. Myocardial I/|R treatment induced significant cardiac dysfunction, including ventricular arrhythmia (VA) and a reduction of left ventricular systolic pressure (LVSP), cardiomyocyte apoptosis and oxidative stress, manifesting as an increase in malondialdehyde (MDA) production and a decrease in superoxide dismutase (SOD) activity. Pretreatment of the hearts with 1.0 nmol/|l Ang-(1-7) for 30 min prior to ischemia considerably attenuated I/|R-induced VA, apoptosis and MDA production, and enhanced LVSP and SOD activity. These cardioprotective effects of Ang-(1-7) were antagonized by the intraperitoneal injection of 5 mg/|kg body weight indomethacin (IDM, a COX inhibitor), presenting as an enhancement of VA, apoptosis and MDA production as well as a reduction of LVSP and SOD activity. In conclusion, COX mediated Ang-(1-7)-induced cardioprotection via its antioxidative mechanism.

[ERK1/2 mediates edaravone-triggered protection against myocardial damage induced by isoprenaline in H9c2 cells].

OBJECTIVE: To explore the effect of extracellular signal regulated kinase 1/2 (ERK1/2) on edaravone (EDA)-triggered protection against myocardial toxicity induced by isoprenaline (ISO) in H9c2 myocardial cells (H9c2 cells). METHODS: H9c2 cells were exposed to ISO at different concentrations to establish a cardiac toxicity model induced by persistent excitation of ß1 receptor. EDA was added before ISO as a pretreatment. PD-98059, an ERK1/2 inhibitor, was administered 1 h prior to EDA to inhibit the phosphorylation of ERK1/2. Cell viability was measured using cell counter kit (CCK-8). The expressions of p-ERK1/2 and t-ERK1/2 were tested by Western blotting. Mitochondrial membrane potential (MMP) was detected by Rhodamine123 (Rh123) staining and photofluorography. RESULTS: Exposure of H9c2 cells to 80 µmol/L ISO for 24 h down-regulated ERK1/2 phosphorylation and repressed MMP. Pretreatment with 10-40 µmol/L EDA for 1 h inhibited ISO-induced myocardial toxicity and pretreatment of 40 µmol/L EDA partially rescued ERK1/2 phosphorylation and MMP level. PD-98059 abolished cardiac protection of EDA, leading to myocardial toxicity and MMP loss. CONCLUSION: EDA can protect H9c2 cells against myocardial injury induced by ISO by suppressing ISO-triggered inhibition of ERK1/2 activation.