There is increasing evidence that cyclooxygenase (COX)-2 possess both angiogenic and cardioprotective properties. We examined the effects of hypoxic cardiac myocytes (H9c2 cells) on COX-2 expression in human umbilical vein endothelial cells (HUVECs) to determine the pathway involved in COX-2 regulation. The medium from hypoxic (<1% O2) cardiac myocytes (HMCM) or normoxic cardiac myocytes (21% O2) was added to HUVEC cultures. HMCM induced a transient increase of COX-2 mRNA expression at 1 and 3 h without affecting the COX-1 mRNA level. A similar effect also observed in HMCM from cultured primary cardiac myocytes (rat neonatal cardiac myocytes). The increased COX-2 mRNA was associated with a time-dependent increase in COX-2 protein expression. COX-2 was significantly induced by VEGF (4.86 ± 1.03-fold) and IL-1β (3.93 ± 0.89-fold) and slightly increased by TNF-α but not by FGF2, IGF-1, or PDGFs. Analysis of proteins secreted in HMCM showed increased levels of VEGF but not IL-1β or TNF-α. The HMCM-induced COX-2 expression was inhibited by the addition of an anti-VEGF neutralizing antibody. VEGF induced endothelial cell COX-2 expression by both increasing COX-2 transcription and prolonging the COX-2 mRNA half-life. Furthermore, staurosporine, a nonselective PKC inhibitor, prevented the induction of VEGF by hypoxia. Both a selective PKC-α and -β inhibitor and an inducible nitric oxide synthase (NOS) inhibitor decreased the induction of COX-2 by HMCM and VEGF. Finally, HMCM-induced upregulation of COX-2 was accompanied by upregulation of PGI2 and PGE2. These results suggest that VEGF is one of the principal factors produced by hypoxic myocytes that is responsible for the induction of endothelial cell COX-2 expression. This process likely involves both PKC and NOS pathways. Our findings have important implications regarding the cardiac protection of COX-2 in ischemic heart disease.

Vascular endothelial growth factor (VEGF) is an angiogenic growth factor known to be up-regulated in ischemic heart and hypoxic endothelial cells. However, the transcriptional regulation of VEGF in hypoxia-induced angiogenesis is not fully understood. Transcriptional enhancer factor-1 (TEF-1) is a transcriptional factor family that can regulate many genes expressed in cardiac and skeletal muscle cells by binding to myocytespecific chloramphenicol acetyltransferase heptamer elements in the promoters of these genes. In this study, we demonstrated that related TEF-1 (RTEF-1), a member of the TEF-1 family, is up-regulated in hypoxic endothelial cells. Overexpression of RTEF-1 increases VEGF promoter activity and VEGF expression. Sequential deletion and site-directed mutation analyses of the VEGF promoter demonstrated that a GC-rich region containing four Sp1 response elements, located between ؊114 and ؊50, was essential for RTEF-1 function. This region is beyond the hypoxia-inducible factor-1␣ binding site and does not consist of M-CAT-related elements. By electrophoretic mobility shift assay, RTEF-1 was found to interact with the first Sp1 residue (؊97 to ؊87) of the four consecutive Sp1 elements. Binding activity of RTEF-1 to VEGF promoter is also confirmed by chromatin immunoprecipitation. In addition, induction of VEGF promoter activity by RTEF-1 results in an increase of angiogenic processes including endothelial cells proliferation and vascular structure formation. These results indicate that RTEF-1 acts as a transcriptional stimulator of VEGF by regulating VEGF promoter activity through binding to Sp1 site. In addition, RTEF-1-induced VEGF promoter activity was enhanced in a hypoxic condition, indicating that RTEF-1 may play an important role in the regulation of VEGF under hypoxia.

Increasing evidences have illuminated the fundamental role of inflammation in mediating all stages of atherosclerosis. miR-155, a typical multi-functional miRNA, has recently emerged as a novel component of inflammatory signal transduction in the pathogenesis of atherosclerosis. However, little is known about whether endothelial highly expressed miR-155 can regulate endothelial inflammation-related transcription factors and the predicted role of miR-155 as a negative feedback regulator in endothelial inflammation involved in atherosclerosis. Bioinformatics analysis showed that RELA (nuclear factor-κB p65) is a potential target gene of miR-155 and this was confirmed by a luciferase reporter assay. Our results show that microRNA-155 mediate endothelial inflammation and decrease NFкB p65 and adhesion molecule expression in TNFα-stimulated endothelial cells. Transfection with miR-155 significantly inhibited TNFα-induced monocyte adhesion to endothelium. Inhibition of miR-155 enhanced p65 level and endothelial inflammatory response which was counteracted through the depletion of P65 by Si-P65. On the other hand, knockdown of eNOS, another target of miR-155, while transfecting with miR-155 inhibitor resulted in more significant inflammatory response. miR-155 is highly expressed in TNFα treated HUVECs, deprived of endogenous p65 could reverse TNFα-induced upregulation of miR-155. Thus, TNFα induced miR-155 may serve as a negative feedback regulator in endothelial inflammation involved in atherosclerosis by targeting nuclear transcription factor P65. These results provide a rationale for intervention of intracellular microRNA as possible anti-atherosclerotic targets.

Background-PR39 is a proline-and arginine-rich peptide implicated in wound healing and myocardial ischemia protection. To determine the potential mechanisms of PR39 in ischemia, we examined the role of PR39 in hypoxia-induced apoptosis in vascular endothelial cells. Methods and Results-Hypoxia results in an increase of apoptosis in bovine aortic endothelial cells (BAECs), as determined by terminal deoxynucleotidyl transferase-mediated dUTP biotin nick-end labeling (TUNEL) analysis and caspase-3 activity. Hypoxia induced 66.2Ϯ2.7% TUNEL-positive cells, whereas in the presence of synthesized PR39 peptide, TUNEL-positive cells were reduced to 29.6Ϯ1.9% (PϽ0.05). After 24 hours of hypoxia, the addition of PR39 reduced caspase-3 activity to 3.17Ϯ0.47 pMol/min from 10.52Ϯ0.55 pMol/min in hypoxic BAECs. Moreover, PR39 increased inhibitor of apoptosis protein-2 (IAP-2) gene and protein expression by 3-fold in a time-and dose-dependent manner. The induction of IAP-2 by PR39 conferred an increase in IAP-2 gene transcription and IAP-2 mRNA stability. Furthermore, inhibiting IAP-2 with second mitochondria-derived activator of caspase (Smac) and with small interfering RNA targeting IAP-2 abrogated the ability of PR39 to reduce caspase-3 activity. Conclusions-We provide the first direct evidence for PR39 as an antiapoptotic factor in endothelial cells during hypoxia.These data suggest that PR39 inhibits hypoxia-induced apoptosis and decreases caspase-3 activity in endothelial cells through an increase of IAP-2 expression.

Although many studies have described the roles of microRNAs (miRNAs) in the modulation of the endothelial response to shear stress, the mechanisms remain incompletely understood. Here, we demonstrate that miR-34a expression in endothelial cells was downregulated by atheroprotective physiological high shear stress (HSS), whereas it was upregulated by atheroprone oscillatory shear stress (OSS). Blockade of endogenous miR-34a dramatically decreased basal vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1) protein expression levels. Conversely, miR-34a overexpression increased the protein levels of VCAM-1 and ICAM-1, consequently promoting monocyte adhesion to endothelial cells. Furthermore, miR-34a overexpression attenuated HSS-mediated suppression of VCAM-1 protein expression on endothelial cells, but promoted HSS-induced ICAM-1 expression. In addition, the OSS induction of endothelial cell VCAM-1 and ICAM-1 was suppressed by using an miR-34a inhibitor, which led to a reduction of monocyte adhesion to endothelial cells. Mechanistically, sirtuin 1 overexpression partially prevented miR-34a-induced VCAM-1 and ICAM-1 expression. Subsequent investigation demonstrated that miR-34a increased nuclear factor kB (NF-kB) p65 subunit (also known as RelA) acetylation (on residue Lys310), and silencing NF-kB signaling reduced miR-34a-induced VCAM-1 and ICAM-1 protein expression. These results demonstrate that miR-34a is involved in the flowdependent regulation of endothelial inflammation.

Tamoxifen has shown great success in the treatment of breast cancer; however, long-term treatment can lead to acquired tamoxifen (TOT) resistance and relapse. TOT classically antagonizes estradiol (E2) -dependent breast cancer cell growth, but exerts partial agonist/antagonist behavior on gene expression. Although both E2 and TOT treatment of breast cancer cells results in recruitment of the estrogen receptor (ER) to common and distinct genomic sites, the mechanisms and proteins underlying TOT preferential recruitment of the ER remains poorly defined. To this end, we performed in silico motif-enrichment analyses within the ER-binding peaks in response to E2 or TOT, to identify factors that would specifically recruit ER to genomic binding sites in the presence of TOT as compared to E2. Intriguingly, we found Nkx3-1 and Oct-transcription factor homodimer motifs to be enriched in TOT preferential binding sites and confirmed the critical role of Oct-3/4 (aka Oct-4) in directing ER recruitment to TOT preferential genomic binding sites, by chromatin immunoprecipitation (ChIP) analyses. Further investigation revealed Oct-4 expression to be basally repressed by Nkx3-1 in MCF-7 cells and TOT treatment appeared to elevate Nkx3-1 degradation through a p38MAPK-dependent phosphorylation of the E3 ligase, Skp2 at serine-64 residue, as observed by quantitative mass-spectrometry analyses. Consistently, Oct-4 upon induction by phospho-Ser64-Skp2-mediated proteasomal degradation of Nkx3-1, participated in ER transcriptional complexes along with p38MAPK and Skp2 in a tamoxifen-dependent manner leading to TOT-dependent gene activation and cell proliferation of the TOT-resistant MCF-7-tam breast cancer cells. Notably, Oct-4 levels were highly elevated in MCF-7-tam cells, and appeared critical for their TOT sensitivity in cell proliferation assays. Furthermore, overexpression of Oct-4 enhanced tumor growth in the presence of tamoxifen in mice in vivo. Collectively, our work presents a novel mechanism for tamoxifen-specific gene activation by ER, secondary to its TOT preferential recruitment to genomic sites by specific activation of Oct-4, a phenomenon that appears to underlie tamoxifen resistance in breast cancer cells and in xenograft tumor models, and could be useful in designing therapeutic interventions to improve treatment outcome.

OBJECTIVES: Cystathionine β-synthase is a major enzyme in the metabolism of plasma homocysteine. Hyperhomocysteinemia is positively associated with hypertension and stroke. The present study was performed to examine the possible effects of Cystathionine β-synthase promoter methylation on the development of hypertension and stroke. METHODS: Using quantitative methylation-specific PCR, we determined the Cystathionine β-synthase methylation levels in 218 healthy individuals and 132 and 243 age- and gender-matched stroke and hypertensive patients, respectively. The relative changes in Cystathionine β-synthase promoter methylation were analyzed using the 2 – ΔΔ Ct method. The percent of the methylated reference of Cystathionine β-synthase was used to represent the Cystathionine β-synthase promoter methylation levels. RESULTS: In this study, the Cystathionine β-synthase promoter methylation levels of hypertensive and stroke participants were both higher than that of the healthy individuals (median percentages of the methylated reference were 50.61%, 38.05% and 30.53%, respectively, all p <0.001). Multivariable analysis showed that Cystathionine β-synthase promoter hypermethylation increased the risk of hypertension [odds ratio, OR (95% confidence interval, CI)=1.035 (1.025–1.045)] and stroke [OR (95% CI)=1.015 (1.003–1.028)]. The area under the curve of Cystathionine β-synthase promoter methylation was 0.844 (95% CI: 0.796–0.892) in male patients with hypertension and 0.722 (95% CI: 0.653–0.799) in male patients with stroke. CONCLUSION: Cystathionine β-synthase promoter hypermethylation increases the risk of hypertension and stroke, especially in male patients.

Highlights Standard procedures and appropriate assessment of exercise are proposed for the commonly used animal models related to chronic exercise (e.g., treadmill running, voluntary wheel running, swimming exercise, and resistance exercise) in cardiovascular research. Optimal design of animal exercise studies in cardiovascular research should consider the choice of exercise models, control of exercise protocols, exercise at different stages of disease, and other factors, such as age, sex, and genetic background. An optimal design for studying exercise-induced physiological cardiac growth and its related beneficial effects against cardiovascular diseases is presented.