Genetic Evidence for Causal Relationships between Plasma Eicosanoid Levels and Cardiovascular Disease.

Cardiovascular diseases are the most common causes of mortality and disability worldwide. Eicosanoids are a group of bioactive metabolites that are mainly oxidized by arachidonic acid. Eicosanoids play a diverse role in cardiovascular diseases, with some exerting beneficial effects while others have detrimental consequences. However, a causal relationship between eicosanoid levels and cardiovascular disease remains unclear. Six single nucleotide polymorphisms (SNPs) with strong associations with plasma eicosanoid levels were selected. Summary-level data for cardiovascular disease were obtained from publicly available genome-wide association studies. A two-sample MR analysis identified that plasma eicosanoid levels were inversely correlated with unstable angina pectoris (OR 1.06; 95% CI 1-1.12; p = 0.04), myocardial infarction (OR 1.05; 95% CI 1.02-1.09; p = 0.005), ischemia stroke (OR 1.05; 95% CI 1-1.11; p = 0.047), transient ischemic attack (OR 1.03; 95% CI 1-1.07; p = 0.042), heart failure (OR 1.03; 95% CI 1.01-1.05; p = 0.011), and pulmonary embolism (OR 1.08; 95% CI 1.02-1.14; p = 1.69 × 10-6). In conclusion, our data strongly suggest a genetic causal link between high plasma eicosanoid levels and an increased cardiovascular disease risk. This study provides genetic evidence for treating cardiovascular diseases.

Integrated Control of Fatty Acid Metabolism in Heart Failure.

Disrupted fatty acid metabolism is one of the most important metabolic features in heart failure. The heart obtains energy from fatty acids via oxidation. However, heart failure results in markedly decreased fatty acid oxidation and is accompanied by the accumulation of excess lipid moieties that lead to cardiac lipotoxicity. Herein, we summarized and discussed the current understanding of the integrated regulation of fatty acid metabolism (including fatty acid uptake, lipogenesis, lipolysis, and fatty acid oxidation) in the pathogenesis of heart failure. The functions of many enzymes and regulatory factors in fatty acid homeostasis were characterized. We reviewed their contributions to the development of heart failure and highlighted potential targets that may serve as promising new therapeutic strategies.

Dendritic cell-mediated chronic low-grade inflammation is regulated by the RAGE-TLR4-PKCß1 signaling pathway in diabetic atherosclerosis.

BACKGROUND: The unique mechanism of diabetic atherosclerosis has been a central research focus. Previous literature has reported that the inflammatory response mediated by dendritic cells (DCs) plays a vital role in the progression of atherosclerosis. The objective of the study was to explore the role of DCs in diabetes mellitus complicated by atherosclerosis. METHODS: ApoE-/- mice and bone marrow-derived DCs were used for in vivo and in vitro experiments, respectively. Masson's staining and Oil-red-O staining were performed for atherosclerotic lesion assessment. The content of macrophages and DCs in plaque was visualized by immunohistochemistry. The expression of CD83 and CD86 were detected by flow cytometry. The fluctuations in the RNA levels of cytokines, chemokines, chemokine receptors and adhesions were analyzed by quantitative RT-PCR. The concentrations of IFN-γ and TNF-α were calculated using ELISA kits and the proteins were detected using western blot. Coimmunoprecipitation was used to detect protein-protein interactions. RESULTS: Compared with the ApoE-/- group, the volume of atherosclerotic plaques in the aortic root of diabetic ApoE-/- mice was significantly increased, numbers of macrophages and DCs were increased, and the collagen content in plaques decreased. The expression of CD83 and CD86 were significantly upregulated in splenic CD11c+ DCs derived from mice with hyperglycemia. Increased secretion of cytokines, chemokines, chemokine receptors, intercellular cell adhesion molecule (ICAM), and vascular cell adhesion molecule (VCAM) also were observed. The stimulation of advanced glycation end products plus oxidized low-density lipoprotein, in cultured BMDCs, further activated toll-like receptor 4, protein kinase C and receptor of AGEs, and induced immune maturation of DCs through the RAGE-TLR4-PKCß1 signaling pathway that was bound together by intrinsic structures on the cell membrane. Administering LY333531 significantly increased the body weight of diabetic ApoE-/- mice, inhibited the immune maturation of spleen DCs, and reduced atherosclerotic plaques in diabetic ApoE-/- mice. Furthermore, the number of DCs and macrophages in atherosclerotic plaques was significantly reduced in the LY333531 group, and the collagen content was increased. CONCLUSIONS: Diabetes mellitus aggravates chronic inflammation, and promotes atherosclerotic plaques in conjunction with hyperlipidemia, which at least in part through inducing the immune maturation of DCs, and its possible mechanism of action is through the RAGE-TLR4-pPKCß1 signaling pathway.

iPLA2ß Contributes to ER Stress-Induced Apoptosis during Myocardial Ischemia/Reperfusion Injury.

Both calcium-independent phospholipase A2 beta (iPLA2ß) and endoplasmic reticulum (ER) stress regulate important pathophysiological processes including inflammation, calcium homeostasis and apoptosis. However, their roles in ischemic heart disease are poorly understood. Here, we show that the expression of iPLA2ß is increased during myocardial ischemia/reperfusion (I/R) injury, concomitant with the induction of ER stress and the upregulation of cell death. We further show that the levels of iPLA2ß in serum collected from acute myocardial infarction (AMI) patients and in samples collected from both in vivo and in vitro I/R injury models are significantly elevated. Further, iPLA2ß knockout mice and siRNA mediated iPLA2ß knockdown are employed to evaluate the ER stress and cell apoptosis during I/R injury. Additionally, cell surface protein biotinylation and immunofluorescence assays are used to trace and locate iPLA2ß. Our data demonstrate the increase of iPLA2ß augments ER stress and enhances cardiomyocyte apoptosis during I/R injury in vitro and in vivo. Inhibition of iPLA2ß ameliorates ER stress and decreases cell death. Mechanistically, iPLA2ß promotes ER stress and apoptosis by translocating to ER upon myocardial I/R injury. Together, our study suggests iPLA2ß contributes to ER stress-induced apoptosis during myocardial I/R injury, which may serve as a potential therapeutic target against ischemic heart disease.

Identification of differentially expressed genes in the endothelial precursor cells of patients with type 2 diabetes mellitus by bioinformatics analysis.

Type 2 diabetes mellitus (DM) is a metabolic disease with worldwide prevalence that is associated with a decrease in the number and function of endothelial progenitor cells (EPCs). The aim of the present study was to explore the potential hub genes of EPCs in patients with type 2 DM. Differentially expressed genes (DEGs) were screened from a public microarray dataset (accession no. GSE43950). Pathway and functional enrichment analyses were performed using the Database for Annotation, Visualization and Integrated Discovery. The protein-protein interaction (PPI) network was visualized. The most significantly clustered modules and hub genes were identified using Cytoscape. Furthermore, hub genes were validated by quantitative PCR analysis of EPCs isolated from diabetic and normal subjects. Subsequently, weighted gene co-expression network analysis (WGCNA) was performed to identify the modules incorporating the genes exhibiting the most significant variance. A total of 970 DEGs were obtained and they were mainly accumulated in inflammation-associated pathways. A total of 9 hub genes were extracted from the PPI network and the highest differential expression was determined for the interleukin 8 (IL8) and CXC chemokine ligand 1 (CXCL1) genes. In the WGCNA performed to determine the modules associated with type 2 DM, one module incorporated IL8 and CXCL1. Finally, pathway enrichment of 10% genes in the pink module ordered by intramodular connectivity (IC) was associated with the IL17 and the chemokine signaling pathways. The present results revealed that the expression of IL8 and CXCL1 may serve important roles in the pathophysiology of EPCs during type 2 DM and inflammatory response may be critical for the reduced number and hypofunction of EPCs isolated from patients with diabetes.

GRP78 (Glucose-Regulated Protein of 78 kDa) Promotes Cardiomyocyte Growth Through Activation of GATA4 (GATA-Binding Protein 4).

The heart manifests hypertrophic growth in response to elevation of afterload pressure. Cardiac myocyte growth involves new protein synthesis and membrane expansion, of which a number of cellular quality control machineries are stimulated to maintain function and homeostasis. The unfolded protein response is potently induced during cardiac hypertrophy to enhance protein-folding capacity and eliminate terminally misfolded proteins. However, whether the unfolded protein response directly regulates cardiac myocyte growth remains to be fully determined. Here, we show that GRP78 (glucose-regulated protein of 78 kDa)-an endoplasmic reticulum-resident chaperone and a critical unfolded protein response regulator-is induced by cardiac hypertrophy. Importantly, overexpression of GRP78 in cardiomyocytes is sufficient to potentiate hypertrophic stimulus-triggered growth. At the in vivo level, TG (transgenic) hearts overexpressing GRP78 mount elevated hypertrophic growth in response to pressure overload. We went further to show that GRP78 increases GATA4 (GATA-binding protein 4) level, which may stimulate Anf (atrial natriuretic factor) expression and promote cardiac hypertrophic growth. Silencing of GATA4 in cultured neonatal rat ventricular myocytes significantly diminishes GRP78-mediated growth response. Our results, therefore, reveal that protein-folding chaperone GRP78 may directly enhance cardiomyocyte growth by stimulating cardiac-specific transcriptional factor GATA4.

Green Tea Polyphenol Epigallocatechin-3-Gallate Promotes Reendothelialization in Carotid Artery of Diabetic Rabbits by Reactivating Akt/eNOS Pathway.

Background: Epigallocatechin gallate (EGCG) is the most abundant catechin in green tea and has proven benefits on endothelial cells in diabetes. However, it remains unclear whether EGCG could improve function of late endothelial progenitor cells (L-EPCs) in diabetes. Methods: Thirty-six rabbits were randomized into six groups. Thirty diabetic rabbits were induced by a single dose of alloxan (100 mg/kg injection intraperitoneally). All of them were given intragastrically EGCG (50 mg/kg/day) or saline for 7 days after carotid injury. In autotransfusion experiment, L-EPCs were cultured with pre-treated EGCG (40 µM for 72 h) and then were injected into the site of injured vascular. Proliferation and migration of EGCG pre-treated L-EPCs in high glucose condition were assessed by EDU incorporation assay and modified Boyden chamber assay, respectively. The mRNA and protein expression of Akt-eNOS pathway were detected by real-time PCR and western blot. Results: Reendothelialization rate in injured carotid artery of diabetic rabbits was augmented in the EGCG group (50 mg/kg/d for 7 days) compared with the non-EGCG group (74.2 ± 4.6% vs. 25.6 ± 5.9%, P < 0.001). EGCG pre-treated L-EPCs autologous transfusion also accelerated the diabetic rabbits' carotid reendothelialization compared with the diabetic sham-operated group (65.6 ± 8.5% vs. 32.9 ± 5.0%, P = 0.011). In vitro studies showed, 40 µM EGCG treatment for 72 h recovered L-EPCs' proliferation and migration, as well as restored the phosphorylation level of Akt and eNOS blocked by high glucose condition. Conclusion: EGCG accelerated reendothelialization in diabetic rabbits after carotid injury in part by reactivating the Akt/eNOS pathway, which might contribute to recovering proliferation and migration of L-EPCs impaired by high glucose.

Thymosin ß4 promotes glucose-impaired endothelial progenitor cell function via Akt/endothelial nitric oxide synthesis signaling pathway.

Circulating endothelial progenitor cells (EPCs) are a subtype of hematopoietic stem cells, which can differentiate into endothelial cells and restore endothelial function. However, high glucose decreases the number and impairs the function of EPCs. A previous study showed that thymosin ß4 (Tß4), a pleiotropic peptide beneficial for multiple functions of various types of cells, could promote EPC migration and dose-dependently upregulate the phosphorylation of Akt and endothelial nitric oxide synthesis signaling (eNOS). In present study, the hypothesis that Tß4 can improve glucose-suppressed EPC functions via the Akt/eNOS signaling pathway and restores the production of nitric oxide (NO) is investigated. EPCs were isolated from the peripheral blood of healthy volunteers and formed a cobblestone shape after 3-4 weeks of cultivation. Then, EPCs were treated with high concentrations of glucose (25 mM) for 4 days and administrated with Tß4 for further study. Transwell migration and tube formation assays were performed to access the migratory and angiogenic ability of EPCs. In addition, the quantity of Akt, eNOS and the concentration of nitric oxide (NO) was investigated. Functional studies showed that high concentrations of glucose significantly suppressed EPC function, while this adverse effect was reversed by the administration of Tß4. In addition, Akt small interfering (si)RNA and eNOS siRNA were demonstrated to reduce the protective effect of Tß4 against glucose-impaired EPC functions. These findings suggest that Tß4 improves glucose-impaired EPC functions via the Akt/eNOS signaling pathway.

Thymosin ß4 promotes endothelial progenitor cell angiogenesis via a vascular endothelial growth factor­dependent mechanism.

Endothelial progenitor cells (EPCs) are a promising cell source for tissue repair and regeneration, predominantly through angiogenesis promotion. Paracrine functions serve a pivotal role in EPC­mediated angiogenesis, which may be impaired by various cardiovascular risk factors. Therefore, it is important to identify a solution that optimizes the paracrine function of EPCs. Thymosin ß4 (Tß4) is a peptide with the potential to promote tissue regeneration and wound healing. A previous study demonstrated that Tß4 enhances the EPC­mediated angiogenesis of the ischemic myocardium. In the present study, whether Tß4 improved angiogenesis by enhancing the paracrine effects of EPCs was investigated. A tube formation assay was used to assess the effect of angiogenesis, and the paracrine effects were measured using an ELISA kit. The results indicated that Tß4 improved the paracrine effects of EPCs, evidenced by an increase in the expression of vascular endothelial growth factor (VEGF). EPC­conditioned medium (EPC­CM) significantly promoted human umbilical vein endothelial cell angiogenesis in vitro, which was further enhanced by pretreatment with Tß4. The effect of Tß4 pretreated EPC­CM on angiogenesis was abolished by VEGF neutralizing antibody in vitro, indicating that increased VEGF secretion had a pivotal role in Tß4­mediated EPC angiogenesis. Furthermore, transplantation of EPCs pretreated with Tß4 into infarcted rat hearts resulted in significantly higher VEGF expression in the border zone, compared with EPC transplantation alone. To further investigate whether the Akt/eNOS pathway was involved in Tß4­induced VEGF secretion in EPCs, the expression levels of VEGF in EPC­CM were significantly decreased following knockdown of Akt or eNOS by small interfering RNA transfection. In conclusion, Tß4 significantly increased angiogenesis by enhancing the paracrine effects of EPCs, evidenced by the increased expression of VEGF. The RAC­α serine/threonine­protein kinase/endothelial nitric oxide synthase signal transduction pathway was involved in the regulation of Tß4­induced VEGF secretion in EPCs. Further studies are required to investigate the long­term prognosis of patients with coronary heart disease following Tß4­pretreated EPC transplantation.

Glucose-regulated protein 78 is essential for cardiac myocyte survival.

Secretory and transmembrane proteins rely on proper function of the secretory pathway for folding, posttranslational modification, assembly, and secretion. Accumulation of misfolded proteins in the endoplasmic reticulum (ER) stimulates the unfolded protein response (UPR), which communicates between the ER and other organelles to enhance ER-folding capacity and restore cellular homeostasis. Glucose-regulated protein of 78 kDa (GRP78), an ER-resident protein chaperone, is a master regulator of all UPR signaling branches. Accumulating studies have established a fundamental role of GRP78 in protein folding, ER stress response, and cell survival. However, role of GRP78 in the heart remains incompletely characterized. Here we showed that embryos lacking GRP78 specifically in cardiac myocytes manifest cardiovascular malformations and die in utero at late gestation. We went further to show that inducible knockout of GRP78 in adult cardiac myocytes causes early mortality due to cardiac cell death and severe decline in heart performance. At the cellular level, we found that loss of GRP78 increases apoptotic cell death, which is accompanied by reduction in AKT signaling and augmentation of production for reactive oxygen species. Importantly, enhancing AKT phosphorylation and activity leads to decreases in oxidative stress and increases in cardiac myocyte survival. Collectively, our results demonstrate an essential role of GRP78 in ensuring normal cardiogenesis and maintaining cardiac contractility and function.

Endoplasmic Reticulum Chaperone GRP78 Protects Heart From Ischemia/Reperfusion Injury Through Akt Activation.

RATIONALE: Restoration of coronary artery blood flow is the most effective means of ameliorating myocardial damage triggered by ischemic heart disease. However, coronary reperfusion elicits an increment of additional injury to the myocardium. Accumulating evidence indicates that the unfolded protein response (UPR) in cardiomyocytes is activated by ischemia/reperfusion (I/R) injury. Xbp1s (spliced X-box binding protein 1), the most highly conserved branch of the unfolded protein response, is protective in response to cardiac I/R injury. GRP78 (78 kDa glucose-regulated protein), a master regulator of the UPR and an Xbp1s target, is upregulated after I/R. However, its role in the protective response of Xbp1s during I/R remains largely undefined. OBJECTIVE: To elucidate the role of GRP78 in the cardiomyocyte response to I/R using both in vitro and in vivo approaches. METHODS AND RESULTS: Simulated I/R injury to cultured neonatal rat ventricular myocytes induced apoptotic cell death and strong activation of the UPR and GRP78. Overexpression of GRP78 in neonatal rat ventricular myocytes significantly protected myocytes from I/R-induced cell death. Furthermore, cardiomyocyte-specific overexpression of GRP78 ameliorated I/R damage to the heart in vivo. Exploration of underlying mechanisms revealed that GRP78 mitigates cellular damage by suppressing the accumulation of reactive oxygen species. We go on to show that the GRP78-mediated cytoprotective response involves plasma membrane translocation of GRP78 and interaction with PI3 kinase, culminating in stimulation of Akt. This response is required as inhibition of the Akt pathway significantly blunted the antioxidant activity and cardioprotective effects of GRP78. CONCLUSIONS: I/R induction of GRP78 in cardiomyocytes stimulates Akt signaling and protects against oxidative stress, which together protect cells from I/R damage.

Apelin Ameliorates High Glucose-Induced Downregulation of Connexin 43 via AMPK-Dependent Pathway in Neonatal Rat Cardiomyocytes.

Diabetes Mellitus is a common disorder, with increasing risk of cardiac arrhythmias. Studies have shown that altered connexin expression and gap junction remodeling under hyperglycemia contribute to the high prevalence of cardiac arrhythmias and even sudden death. Connexin 43 (Cx43), a major protein that assembles to form cardiac gap junctions, has been found to be downregulated under high glucose conditions, along with inhibition of gap junctional intercellular communication (GJIC). While, apelin, a beneficial adipokine, increases Cx43 protein expression in mouse and human embryonic stem cells during cardiac differentiation. However, it remains unknown whether apelin influences GJIC capacity in cardiomyocytes. Here, using Western blotting and dye transfer assays, we found that Cx43 protein expression was reduced and GJIC was impaired after treatment with high glucose, which, however, could be abrogated after apelin treatment for 48 h. We also found that apelin increased Cx43 expression under normal glucose. Real-time PCR showed that the Cx43 mRNA was not significantly affected under high glucose conditions in the presence of apelin or high glucose and apelin. High glucose decreased the phosphorylation of AMPKα; however, apelin activated AMPKα. Interestingly, we found that Cx43 expression was increased after treatment with AICAR, an activator of AMPK signaling. AMPKα inhibition mediated with transfection of siRNA-AMPKα1 and siRNA-AMPKα2 abolished the protective effect of apelin on Cx43 expression. Our data suggest that apelin attenuates high glucose-induced Cx43 downregulation and improves the loss of functional gap junctions partly through the AMPK pathway.

The Rho kinase inhibitor, fasudil, ameliorates diabetes-induced cardiac dysfunction by improving calcium clearance and actin remodeling.

Previous study showed inhibition of RhoA and Rho kinase (ROCK) activity with fasudil could alleviate diabetes-induced cardiac dysfunction partially due to improvement of myocardial fibrosis. However, the effect of fasudil on intracellular calcium cycling and actin remodeling, both of which are important to regulate excitation-contract coupling, is still not fully elucidated. In this study, a diabetic cardiomyopathy model was induced by a single intraperitoneal injection of streptozotocin (STZ) in male Sprague Dawley rats. Diabetic rats were treated with fasudil or placebo for 8 weeks. We found that long-term administration of fasudil, a specific Rho kinase inhibitor, significantly ameliorated diabetes-induced contractile dysfunction both at cellular and whole organ levels. Fasudil-treated rats displayed improved diastolic intracellular calcium ([Ca2+]i) removal and rescued expression of protein responsible for [Ca2+]i clearance. Furthermore, our study indicated that fasudil treatment normalized the phosphorylation of the PKCß2/Akt pathway in the diabetic heart, which might be the underlying mechanism accounting for the protective effect of fasudil on [Ca2+]i clearance. In addition, compared to the diabetes group, fasudil also normalized the G/F-actin ratio by preventing cofilin phosphorylation and promoted F-actin organization, suggesting a beneficial effect on actin remodeling. These findings indicate the protective effect of fasudil against diabetes-induced cardiac dysfunction via modulation of Ca2+ handling and actin remodeling. Overactivation of RhoA/ROCK plays a key role in the development of DCM. Inhibition of ROCK activity with fasudil improved [Ca2+]i removal in diabetic cardiomyocytes. Fasudil normalized the G/F-actin ratio and promoted F-actin organization. ROCK may be an excellent therapeutic target for the treatment of DCM. KEY MESSAGE: Overactivation of RhoA/ROCK plays a key role in the development of DCM. Inhibition of ROCK activity with fasudil improved [Ca2+]i removal in diabetic cardiomyocytes. Fasudil normalized the G/F-actin ratio and promoted F-actin organization. ROCK may be an excellent therapeutic target for the treatment of DCM.

Resveratrol prevents endothelial progenitor cells from senescence and reduces the oxidative reaction via PPAR­Î³/HO­1 pathways.

Increasing evidence suggests endothelial progenitor cells (EPCs) improve neovascularization and endothelium regeneration. Resveratrol (RSV) is a natural polyphenolic compound, which has been demonstrated to exert multiple protective effects on the cardiovascular system, including inhibition of platelet adhesion and aggregation, reduction of myocardial ischemia­reperfusion injury, and suppression of neointimal hyperplasia of injured vascular tissue. The present study investigated the role of RSV on levels of oxidative stress and senescence of EPCs, and the effects of RSV on vascular­promoting and/or vascular­healing capacity of EPCs. It was demonstrated that EPCs could promote the repair of endothelium of the injured artery. RSV reduced the oxidative reaction of EPCs and inhibited EPC senescence, and these effects may occur via the peroxisome proliferator­activated receptor­Î³/heme oxygenase­1 signaling pathways.

The Impact of Rosuvastatin on the Density Score of Coronary Artery Calcification in Coronary Artery Disease Patients with Type 2 Diabetes Mellitus: Rationale and Design of RosCal Study.

INTRODUCTION: More and more evidence suggests that the density of calcification plays an important role in the plaque stability. However, few studies have investigated the statin treatment on the density of plaque calcification in patients with both coronary artery disease (CAD) and type 2 diabetes mellitus. METHODS: One hundred and twenty-two CAD patients with type 2 diabetes with confirmed coronary artery calcification (CAC) will be recruited consecutively in a 12-month period. These patients will receive rosuvastatin (20 mg/day) therapy in the next 24 months. Blood tests and adverse events will be collected at routine follow-up of 1, 3, 6, 12, 18 and 24 months. The primary endpoint will be the change of CAC density score measured by coronary CT angiography after 24 months' treatment of rosuvastatin (20 mg/day) compared with baseline. The secondary endpoints will be the change of serum sclerostin and the effect on the volume score of CAC in those patients. RESULTS: We expect that rosuvastatin could both increase the density of CAC to improve plaque stability and up-regulate serum sclerostin, which would suggest the underlying mechanism of the plaque stabilization by a statin. CONCLUSION: This study would be the first to demonstrate the impact of rosuvastatin on the density score of coronary artery calcification in CAD patients with type 2 diabetes. This study has been registered in ClinicalTrials.gov (NCT02418884).

Activation of liver X receptor attenuates lysophosphatidylcholine-induced IL-8 expression in endothelial cells via the NF-κB pathway and SUMOylation.

The liver X receptor (LXR) is a cholesterol-sensing nuclear receptor that has an established function in lipid metabolism; however, its role in inflammation is elusive. In this study, we showed that the LXR agonist GW3965 exhibited potent anti-inflammatory activity by suppressing the firm adhesion of monocytes to endothelial cells. To further address the mechanisms underlying the inhibition of inflammatory cell infiltration, we evaluated the effects of LXR agonist on interleukin-8 (IL-8) secretion and nuclear factor-kappa B (NF-κB) activation in human umbilical vein endothelial cells (HUVECs). The LXR agonist significantly inhibited lysophosphatidylcholine (LPC)-induced IL-8 production in a dose-dependent manner without appreciable cytotoxicity. Western blotting and the NF-κB transcription activity assay showed that the LXR agonist inhibited p65 binding to the IL-8 promoter in LPC-stimulated HUVECs. Interestingly, knockdown of the indispensable small ubiquitin-like modifier (SUMO) ligases Ubc9 and Histone deacetylase 4 (HDAC4) reversed the increase in IL-8 induced by LPC. Furthermore, the LPC-induced degradation of inhibitory κBα was delayed under the conditions of deficient SUMOylation or the treatment of LXR agonist. After enhancing SUMOylation by knockdown SUMO-specific protease Sentrin-specific protease 1 (SENP1), the inhibition of GW3965 was rescued on LPC-mediated IL-8 expression. These findings indicate that LXR-mediated inflammatory gene repression correlates to the suppression of NF-κB pathway and SUMOylation. Our results suggest that LXR agonist exerts the anti-atherosclerotic role by attenuation of the NF-κB pathway in endothelial cells.

NADPH oxidase activation played a critical role in the oxidative stress process in stable coronary artery disease.

OBJECTIVES: The study was designed to investigate the oxidative stress levels of endothelial progenitor cells (EPCs) in stable coronary artery disease (CAD) and to explore the underlying mechanisms of NADPH oxidase activation and subsequent EPCs dysfunction. METHODS: EPCs were isolated from patients with stable CAD (n=50) and matched healthy volunteers (n=50). NADPH oxidase activation was detected by measuring the expression of each subunit using western blotting and qPCR analyses and the membrane translocation of p47phox using immunofluorescence. The in vivo angiogenesis capacity was evaluated using immunofluorescence by transplanting EPCs into a rat hind limb ischemia model. The PKC inhibitor GÖ-6983 was used to determine the role of PKC in NADPH oxidase activation. RESULTS: Oxidative stress level was increased and the in vivo angiogenesis capacity was impaired in EPCs obtained from CAD subjects with the activation of NADPH oxidase. P47phox membrane translocation increased in CAD group vs controls. These effects were resolved by NADPH oxidase inhibition. Up-regulation of PKCα/ß2 was found in EPCs from CAD subjects, PKC inhibition GÖ-6983 could reduce the expression and activity of NADPH oxidation. CONCLUSIONS: NADPH oxidase activation via p47phox membrane translocation played a critical role in the initiation and progression of CAD, and the PKCα/ß2 signaling pathway might be involved.

Stromal cell-derived factor-1α prevents endothelial progenitor cells senescence and enhances re-endothelialization of injured arteries via human telomerase reverse transcriptase.

Recent studies have suggested that endothelial progenitor subpopulation (EPCs) number and activity were associated with EPCs senescence. Our previous study had shown that stromal cell-derived factor-1alpha (SDF-1α) could prevent EPCs senescence, which may be via telomerase. In this study, we further investigated the role of human telomerase reverse transcriptase (h-TERT) on the protective effect of SDF-1α against senescence. Knockdown h-TERT abrogated the protective effect of SDF-1α and abolished the effects of SDF-1α on migration and proliferation. Moreover, it inhibited EPCs recruitment. In conclusion, h-TERT served a critical role in the progress that SDF-1α prevented EPCs senescence and enhanced re-endothelialization of the injured arteries.