Jian-Xiong Chen

Hypoxia increases Hsp90 binding to eNOS via PI3K-Akt in porcine coronary artery endothelium

This study examines the molecular mechanisms by which hypoxia regulates phosphorylated endothelial nitric oxide synthase (eNOS) activity and NO production in porcine coronary artery endothelial cells (PCAEC). Exposure to hypoxia (pO2=10 mmHg) for periods up to 3 h resulted in a time-dependent increase in eNOS protein expression and an early (15 min) and sustained increase in eNOS phosphorylation at Ser-1177. Exposure to hypoxia for 30 min led to a doubling in eNOS activity (control=6.2±4.4 vs hypoxia=14.1±5.0 fmol cGMP/μg protein, P<0.05) and NO release (control=5.9±0.8 vs hypoxia=11.8±1.2 nM/μg protein, P<0.05). Hypoxia also led to a significant increase in Akt phosphorylation and upregulation of Hsp90 binding to eNOS. Pretreatment of cells with either 1 μg/ml geldanamycin (a specific inhibitor of Hsp90) or 500 nM wortmannin (a specific PI3 kinase inhibitor) suppressed hypoxia-stimulated Akt and eNOS phosphorylation and significantly attenuated hypoxia-stimulated Hsp90 binding to eNOS. Both eNOS activity and NO production were inhibited by geldanamycin and wortmannin. Although hypoxia led to early activation of p42/44 mitogen-activated protein kinases (MAPK), inhibition of their pathway by PD98059 did not suppress hypoxia-stimulated eNOS phosphorylation and eNOS activity. These data demonstrate that hypoxia leads to increased eNOS phosphorylation via stimulated Hsp90 binding to eNOS and activation of the PI3–Akt pathway. We conclude that a coordinated interaction between Hsp90 and PI3–Akt may be an important mechanism by which eNOS activity and NO production is upregulated in hypoxic heart.

Critical role of angiopoietins/Tie-2 in hyperglycemic exacerbation of myocardial infarction and impaired angiogenesis

Angiopoietin-1 (Ang-1) and angiopoietin-2 (Ang-2) are the two ligands of the Tie-2 receptor, a receptor tyrosine kinase that is expressed on the endothelium. A balanced angiopoietin/Tie-2 system is critical for the maintenance of vascular integrity. We investigated the potential role of a disrupted angiopoietin/Tie-2 system on hyperglycemic exacerbation of myocardial infarction and impaired angiogenesis. Using streptozotocin (STZ) mice subjected to myocardial ischemia, we examined the effects of shifting the Ang-2-to-Ang-1 ratio on myocardial infarction size, apoptosis, bone marrow (BM) cell-endothelial progenitor cell (EPC) differentiation, and angiogenesis. In control mice, myocardial ischemia increased expression of both Ang-2 and Tie-2. In STZ mice, Ang-2 expression was elevated, whereas Tie-2 expression was reduced, and neither was significantly altered by ischemia. Myocardial infarct size and apoptosis were increased in STZ compared with control mice. Using in vivo administration of an adenovirus containing Ang-1 or Ang-2, we found that shifting the Ang-2-to-Ang-1 ratio to favor Ang-1 reduced myocardial apoptosis and infarct size in STZ mice, while shifting the Ang-2-to-Ang-1 ratio to favor Ang-2 resulted in a significant increase in myocardial infarct size and apoptosis in control mice. Myocardial ischemia-stimulated BM cell-EPC differentiation was inhibited and myocardial angiogenesis was reduced in STZ mice. Systemic administration of Ad-Ang-1 restored BM cell-EPC differentiation and increased myocardial VEGF expression and angiogenesis in STZ mice. Our data demonstrate that disturbed angiopoietin/Tie-2 signaling contributes to the hyperglycemic exacerbation of myocardial infarction and impaired angiogenesis. Restoration of the Ang-2-to-Ang-1 ratio may be a novel therapeutic strategy for the treatment of diabetic myocardial ischemic diseases.

Nitric oxide donors regulate nitric oxide synthase in bovine pulmonary artery endothelium

This study examined the notion that exogenous generation of nitric oxide (NO) modulates NOS gene expression and activity. Bovine pulmonary artery endothelial cells (BPAEC) were treated with the NO donors, 1 mM SNAP (S‐nitroso‐N‐acetylpenicillamine), 0.5 mM SNP (sodium nitroprusside) or 0.2 μM NONOate (spermine NONOate) in medium 199 containing 2% FBS. Controls included untreated cells and cells exposed to 1 mM NAP (N‐acetyl‐D‐penicillamine). NOS activity was assessed using a fibroblast‐reporter cell assay; intracellular Ca2+ concentrations were assessed by Fura‐2 microfluorometry; and NO release was measured by chemiluminescence. Constitutive endothelial (e) and inducible (i) NOS gene and protein expression were examined by northern and western blot analysis, respectively. Two hours exposure to either SNAP or NONOate caused a significant elevation in NO release from the endothelial cells (SNAP = 51.4 ± 5.9; NONOate = 23.8 ± 4.2; control = 14.5 ± 2.8 μM); but A23187 (3 μM)‐stimulated NO release was attenuated when compared to controls. Treatment with either SNAP or NONOate for 2 h also resulted in a significant increase in NOS activity in endothelial homogenates (SNAP = 23.6 ± 2.5; NONOate = 29.8 ± 7.7; control = 14.5 ± 2.5 fmol cGMP/μg per 106 cells). Exposure to SNAP and SNP, but not NONOate, for 1 h caused an increase in intracellular calcium. Between 4 and 8 h, SNAP and NONOate caused a 2‐ to 3‐fold increase in eNOS, but not iNOS, gene (P < 0.05) and protein expression. NAP had little effect on either eNOS gene expression, activity or NO production. Our data indicate that exogenous generation of NO leads to a biphasic response in BPAEC, an early increase in intracellular Ca2+, and increases in NOS activity and NO release followed by increased expression of the eNOS gene, but not the iNOS gene. We conclude that eNOS gene expression and activity are regulated by a positive‐feedback regulatory action of exogenous NO. J. Cell. Physiol. 186:116–123, 2001.

Glutathione mediates LPS-stimulated COX-2 expression via early transient p42/44 MAPK activation.

This study examines whether endotoxin (LPS)‐stimulated COX‐2 is modulated by an interaction between mitogen activated protein kinases (MAPK) and intracellular glutathione. Bovine pulmonary artery endothelial cells (BPAEC) were pretreated for 30 min with the following prior to addition of 0.1 μg/ml endotoxin in 2% FBS in medium 199: 5 mM N‐acetylcysteine (NAC) or 5 mM glutathione ethyl ester (GSE) (modulators of intracellular glutathione); 10 μM SB203580 or 25 μM PD98059 (inhibitors of p38 and p42/44 MAPKs, respectively). End‐points included assessment of COX‐1 and COX‐2 gene expression by reverse transcription polymerase chain reaction (RT‐PCR); COX‐1, COX‐2, p38, and p42/44 protein by Western analysis; and measurement of PGE2 and 6‐keto‐PGF1α releases by GC/MS. Both GSE and NAC resulted in significant exacerbation of the LPS‐stimulated increase in COX‐2 gene and protein expression and prostaglandin release, and suppressed the LPS‐induced decrease in COX‐1. LPS caused a biphasic activation of p42/44 MAPKs, an early increase peaking at 30 min and a second sustained phase, lasting up to 24 h; LPS also caused an early and sustained increase p38 MAPK activity. Pretreatment of cells with either GSE or NAC increased the early LPS‐stimulated activation of p42/44 but had little effect on the sustained phase. Inhibition of either p38 or p42/44 MAPKs suppressed LPS‐stimulated COX‐2 gene and protein expression, and prostaglandin release (P < 0.05) but had little effect on COX‐1. We conclude that intracellular glutathione modulates LPS‐stimulated COX‐2 gene expression and prostaglandin synthesis in BPAEC via early activation of p42/44 MAPKs. J. Cell. Physiol. 197: 86–93, 2003© 2003 Wiley‐Liss, Inc.

LPS causes pericyte loss and microvascular dysfunction via disruption of Sirt3/angiopoietins/Tie-2 and HIF-2α/Notch3 pathways.

Recent studies reveal a crucial role of pericyte loss in sepsis-associated microvascular dysfunction. Sirtuin 3 (SIRT3) mediates histone protein post-translational modification related to aging and ischemic disease. This study investigated the involvement of SIRT3 in LPS-induced pericyte loss and microvascular dysfunction. Mice were exposed to LPS, expression of Sirt3, HIF-2α, Notch3 and angiopoietins/Tie-2, pericyte/endothelial (EC) coverage and vascular permeability were assessed. Mice treated with LPS significantly reduced the expression of SIRT3, HIF-2α and Notch3 in the lung. Furthermore, exposure to LPS increased Ang-2 while inhibited Ang-1/Tie-2 expression with a reduced pericyte/EC coverage. Intriguingly, knockout of Sirt3 upregulated Ang-2, but downregulated Tie-2 and HIF-2α/Notch3 expression which resulted in a dramatic reduction of pericyte/EC coverage and exacerbation of LPS-induced vascular leakage. Conversely, overexpression of Sirt3 reduced Ang-2 expression and increased Ang-1/Tie-2 and HIF-2α/Notch3 expression in the LPS treated mice. Overexpression of Sirt3 further prevented LPS-induced pericyte loss and vascular leakage. This was accompanied by a significant reduction of the mortality rate. Specific knockout of prolyl hydroxylase-2 (PHD2) increased HIF-2α/Notch3 expression, improved pericyte/EC coverage and reduced the mortality rate in the LPS-treated mice. Our study demonstrates the importance of SIRT3 in preserving vascular integrity by targeting pericytes in the setting of LPS-induced sepsis.

Angiopoietin-1 protects myocardial endothelial cell function blunted by angiopoietin-2 and high glucose condition

Aim:To evaluate the effects of angiopoietin-1 (Ang-1) on myocardial endothelial cell function under high glucose (HG) condition.Methods:Mouse heart myocardial endothelial cells (MHMECs) were cultured and incubated under HG (25 mmol/L) or normal glucose (NG, 5 mmol/L) conditions for 72 h. MTT was used to determine cellular viability, and TUNEL assay and caspase-3 enzyme linked immunosorbent assays were used to assay endothelial apoptosis induced by serum starvation. Immunoprecipitation and Western blot analysis were used to analyze protein phosphorylation and expression. Endothelial tube formation was used as an in vitro assay for angiogenesis.Results:Exposure of MHMECs to HG resulted in dramatic decreases in phosphorylation of the Tie-2 receptor and its downstream signaling partners, Akt/eNOS, compared to that under NG conditions. Ang-1 (250 ng/mL) increased Tie-2 activation, inhibited cell apoptosis, and promoted angiogenesis. Ang-1-mediated protection of endothelial function was blunted by Ang-2 (25 ng/mL).Conclusion:Ang-1 activates the Tie-2 pathway and restores hyperglycemia-induced myocardial microvascular endothelial dysfunction. This suggests a protective role of Ang-1 in the ischemic myocardium, particularly in hearts affected by hyperglycemia or diabetes.

Apelin Gene Therapy Increases Autophagy Via Activation of Sirtuin 3 in Diabetic Heart

Heart failure is the leading cause of death in diabetic patients. Recently we showed that apelin gene therapy attenuates heart failure following myocardial infarction. This study further explored the potential mechanisms by which apelin may reduce cardiac injury in Postmyocardial infarction (MI)) model of diabetes. Wild type and Sirt3 knockout (Sirt3 KO) mice were induced into diabetes by intra-peritoneal (i.p.) Streptozotocin (STZ). STZ mice were then subjected to MI followed by immediate intramyocardial injection with Adenovirus-apelin (Ad-apelin). Ad-apelin treatment resulted in over expression of apelin in the ischemic hearts of STZ mice. Apelin over expression led to a significant increase in Sirt3 expression. Apelin over expression significantly reduced gp91phox expression. This was accompanied by a significant reduction of reactive oxygen species formation. Ad-apelin treatment also dramatically reduced NF-κb-p65 expression in WT-STZ mice. Over expression of apelin further enhanced autophagy markers (LC3-II and beclin-1) expression in post-MI heart. Most intriguingly, knockout of Sirt3 in STZ mice abolished these beneficial effects of apelin treatment. In vitro, knockout of Sirt3 in EPCs significantly enhanced high glucose-induced ROS formation. Conversely, treatment of Sirt3 KO-EPCs with NADPH oxidase inhibitor led to two fold increase in LC3-II levels. Our studies demonstrate that apelin increases autophagy via up regulation of Sirt3 and suppression of ROS-NF-κb pathway in diabetic heart.

Emerging role of SIRT3 in endothelial metabolism, angiogenesis, and cardiovascular disease: HE et al.

Sirtuin 3 (SIRT3) a mitochondrial enzyme that plays an important role in energy homeostasis, cardiac remodeling, and heart failure (HF). The expression of SIRT3 declines with advanced age, cardiovascular, and metabolic diseases. Accumulating evidence suggests that SIRT3 plays a critical role in protecting the heart from cardiac hypertrophy, cardiac dysfunction associated with HF, and in the protection of cardiac cells from stress‐mediated cell death. Clinical studies have demonstrated that HF with preserved ejection fraction (HFpEF) in patients present with abnormalities in coronary microcirculation related to endothelial dysfunction and coronary microvascular rarefaction. Although SIRT3‐mediated regulation of mitochondrial homeostasis and heart function has been intensively investigated, the effect of SIRT3 on endothelial cell (EC) glycolytic metabolism and microvascular function has not been well studied. ECs utilize glycolysis for generating ATP rather than oxidative phosphorylation to maintain their normal functions and promote angiogenesis and EC–cardiomyocyte interactions. Emerging evidence indicates that SIRT3 is involved in the regulation of endothelial metabolism and angiogenesis and thus affects the development of cardiovascular diseases associated with aging. This review will discuss the current knowledge of SIRT3 and its functional role on endothelial metabolism, cardiac function, and cardiovascular diseases.

Curcumin enhances vascular contractility via induction of myocardin in mouse smooth muscle cells

A variety of cardiovascular diseases is accompanied by the loss of vascular contractility. This study sought to investigate the effects of curcumin, a natural polyphenolic compound present in turmeric, on mouse vascular contractility and the underlying mechanisms. After mice were administered curcumin (100 mg·kg-1·d-1, ig) for 6 weeks, the contractile responses of the thoracic aorta to KCl and phenylephrine were significantly enhanced compared with the control group. Furthermore, the contractility of vascular smooth muscle (SM) was significantly enhanced after incubation in curcumin (25 μmol/L) for 4 days, which was accompanied by upregulated expression of SM marker contractile proteins SM22α and SM α-actin. In cultured vascular smooth muscle cells (VSMCs), curcumin (10, 25, 50 μmol/L) significantly increased the expression of myocardin, a “master regulator” of SM gene expression. Curcumin treatment also significantly increased the levels of caveolin-1 in VSMCs. We found that as a result of the upregulation of caveolin-1, curcumin blocked the activation of notch1 and thereby abolished Notch1-inhibited myocardin expression. Knockdown of caveolin-1 or activation of Notch1 signaling with Jagged1 (2 μg/mL) diminished these effects of curcumin in VSMCs. These findings suggest that curcumin induces the expression of myocardin in mouse smooth muscle cells via a variety of mechanisms, including caveolin-1-mediated inhibition of notch1 activation and Notch1-mediated repression of myocardin expression. This may represent a novel pathway, through which curcumin protects blood vessels via the beneficial regulation of SM contractility.