Apelin-13 upregulates Egr-1 expression in rat vascular smooth muscle cells through the PI3K/Akt and PKC signaling pathways.

Previous studies have shown that Apelin-13 upregulates early growth response factor-1 (Egr-1) via the extracellular signal-regulated protein kinase (ERK) signaling pathway. Apelin-13 induces proliferation and migration of vascular smooth muscle cells (VSMCs) as well as the upregulation of osteopontin (OPN) via the upregulation of Egr-1. This study was designed to further explore the activity of Apelin-13 in VSMCs by investigating members of the mitogen-activated protein kinase (MAPK) family, in particular Jun kinase (JNK) and p38 mitogen-activated protein kinase (P38). We also examined whether the phosphatidylinositol 3 kinase (PI3K)/protein kinase B (Akt) and protein kinase C (PKC) signaling pathways were involved in the regulation of Egr-1 by Apelin-13. We treated rat aortic VSMCs with Apelin-13 and examined the expression of JNK, p-JNK, P38, and p-P38 to investigate whether Apelin-13-mediated increases in Egr-1 occurred through the JNK and P38 signaling pathways. We then pretreated VSMCs with the Gi protein inhibitor pertussis toxin (PTX) and the Gq inhibitor YM254890, added Apelin-13 and looked for changes in Egr-1 expression. Finally, we pretreated with the PI3K inhibitor LY294002 and the PKC inhibitor GF109203X, and treated with Apelin-13. Our results showed that JNK and P38 did not participate in Apelin-13-mediated increase in Egr-1. Instead, Apelin-13 upregulation of Egr-1 was mediated by a PTX-sensitive Gi protein. Apelin-13 did increase ERK phosphorylation through the PI3K/Akt and PKC signaling pathways, resulting in changes in Egr-1 expression. These data provide important targets for future studies to modulate vascular remodeling.

Short-term rosuvastatin treatment for the prevention of contrast-induced acute kidney injury in patients receiving moderate or high volumes of contrast media: a sub-analysis of the TRACK-D study.

BACKGROUND: Current randomized trials have demonstrated the effects of short-term rosuvastatin therapy in preventing contrast-induced acute kidney injury (CIAKI). However, the consistency of these effects on patients administered different volumes of contrast media is unknown. METHODS: In the TRACK-D trial, 2998 patients with type 2 diabetes and concomitant chronic kidney disease (CKD) who underwent coronary/peripheral arterial angiography with or without percutaneous intervention were randomized to short-term (2 days before and 3 days after procedure) rosuvastatin therapy or standard-of-care. This prespecified analysis compared the effects of rosuvastatin versus standard therapy in patients exposed to (moderate contrast volume [MCV], 200-300 ml, n = 712) or (high contrast volume [HCV], ≥ 300 ml, n = 220). The primary outcome was the incidence of CIAKI. The secondary outcome was a composite of death, dialysis/hemofiltration or worsened heart failure at 30 days. RESULTS: Rosuvastatin treatment was associated with a significant reduction in CIAKI compared with the controls (2.1% vs. 4.4%, P = 0.050) in the overall cohort and in patients with MCV (1.7% vs. 4.5%, P = 0.029), whereas no benefit was observed in patients with HCV (3.4% vs. 3.9%, P = 0.834). The incidence of secondary outcomes was significantly lower in the rosuvastatin group compared with control group (2.7% vs. 5.3%, P = 0.049) in the overall cohort, but it was similar between the patients with MCV (2.0% vs. 4.2%, P = 0.081) or HCV (5.1% vs. 8.8%, P = 0.273). CONCLUSIONS: Periprocedural short-term rosuvastatin treatment is effective in reducing CIAKI and adverse clinical events for patients with diabetes and CKD after their exposure to a moderate volume of contrast medium.

Apelin-13-induced proliferation and migration induced of rat vascular smooth muscle cells is mediated by the upregulation of Egr-1.

Apelin-13 plays an important role in the migration and proliferation of vascular smooth muscle cells (VSMCs); however, the underlying mechanisms are still unclear. Egr-1 is a nuclear transcription factor, which is considered to be the critical initiating factor of the processes of VSMC proliferation and migration. Egr-1 is known to regulate the expression of osteopontin (OPN), which is a marker of the phenotypic modulation that is a necessary condition of VSMC proliferation and migration. We hypothesized that the role of Apelin-13 is mediated via upregulation of Egr-1. To test this hypothesis, we analyzed the effects of Apelin-13 treatment on Egr-1 mRNA and protein expression in A10 rat aortic VSMCs by RT-PCR and Western blotting, respectively. Results showed that, Apelin-13 upregulated the expression of Egr-1. Furthermore, treatment with the extracellular-regulated protein kinase (ERK) inhibitor, PD98059, inhibited the upregulation of Egr-1 by Apelin-13. In addition, this upregulation was inhibited by treatment of VSMCs with the Egr-1 specific deoxyribozyme ED5 (DNAenzyme/10-23 DRz). Furthermore, ED5 treatment was found to significantly inhibit Apelin-13-induced migration and proliferation of VSMCs using transwell and MTT assays, respectively. The evaluation of OPN mRNA and protein expression levels by RT-PCR and Western blot analyses revealed that ED5 treatment also inhibited Apelin-13-induced OPN upregulation. The results of this study indicated that Apelin-13 upregulates Egr-1 via ERK. Furthermore, Apelin-13 induced the proliferation and migration of VSMCs as well as the upregulation of OPN via the upregulation of Egr-1. These results will provide an important theoretical and experimental basis for the control of inappropriate remodeling of vessel walls, and will hopefully lead to the prevention and treatment of vascular remodeling diseases.

Effects of tirofiban on the reperfusion-related no-reflow in rats with acute myocardial infarction.

OBJECTIVE: To investigate the effects of tirofiban on the no-reflow phenomenon of acute myocardial infarction (AMI) rats received reperfusion, as well as the underlying mechanisms. METHODS: Fifty-six male Sprague-Dawley rats were randomly divided into four groups: Sham operation group (Sham), AMI/reperfusion group (AMI/R), Tirofiban group (Tiro) and Tiro+N-nitro-L-arginine group (L-NNA; an endothelial nitric oxide synthase inhibitor). To generate the animal model mimicking the no-reflow phenomenon, the rats first received occlusion of the left anterior descending coronary artery for 60 min and then followed by reperfusion for 120 min. Area of no-reflow, area at risk and area of necrosis were measured by thioflavine S, Evans blue and triphenyl tetrazolium chloride staining, respectively. Haemodynamic function was measured at the end. In the meantime, nitric oxide synthase (NOS) activity was determined by a NOS assay kit. The expression of myocardial endothelial nitric oxide synthase (eNOS) was determined by an enzyme-linked immunosorbent assay (ELISA). The expression of phosphorylated eNOS at Ser(1177) (p-eNOS Ser(1177)) and vascular endothelial-cadherin (VE-cadherin) were determined by western blot. RESULTS: Compared with AMI/R group, tirofiban significantly reduced the no-reflow area and infarct size (all P < 0.05). Tirofiban elevated eNOS activity, lessen inducible nitric oxide synthase (iNOS) activity and increased the expression of Ser(1177) phosphorylated eNOS and VE-cadherin in the ischemic myocardium (all P < 0.05). No statistical differences were found in the expression of eNOS among the four groups. Also, tirofiban improved cardiac function with significantly higher levels of left ventricular end systolic pressure, maximum change rate of left ventricular pressure rise and fall, heart rate, and lower level of left ventricular end diastolic pressure than those of the AMI/R group (all P < 0.05). Whereas, these effects of tirofiban were partially abolished by L-NNA. CONCLUSIONS: Tirofiban could reduce the size of no-reflow and infarct. A possible mechanism underlying this effect is that tirofiban could protect the structural and functional integrity of microvascular endothelium which is partially regulated by eNOS activity.

Apelin protects against cardiomyocyte apoptosis induced by glucose deprivation.

BACKGROUND: Apoptosis is a major cause of ischemic heart dysfunction. Apelin, the endogenous ligand for the G-protein-coupled APJ receptor, has been reported to exert cardioprotective effects during myocardial injury. The aim of this study was to investigate the effects of apelin on apoptosis of rat cardiomyocytes induced by glucose deprivation (GD) and study the related signaling pathway. METHODS: Apelin and APJ mRNA expression were determined by RT-PCR in neonatal rat cardiomyocytes during different durations of GD. Cardiomyocyte apoptosis was detected by annexin V-FITC/propidium iodide (PI) staining after GD for 12 hours with or without apelin-13 (10 and 100 nmol/L) pretreatment. Protein levels of Akt and the mammalian target of rapamycin (mTOR) as well as cell apoptosis were detected in the presence or absence of LY294002 (a phosphatidylinositol 3-kinases (PI3K) inhibitor) or rapamycin (a mTOR inhibitor). RESULTS: Apelin mRNA expression was up-regulated when cardiomyocytes were exposed to GD for 6, 12, 18, and 24 hours compared with the base level (P > 0.05, P < 0.01, P < 0.01, P < 0.01). However, when cardiomyocytes were exposed to GD for up to 36 hours, apelin mRNA expression was 17% lower than the base level (P < 0.05). APJ mRNA expression paralleled that of apelin. Apelin-13 pretreatment at 100 nmol/L significantly inhibited GD-induced cardiomyocyte apoptosis (P < 0.05) and increased Akt and mTOR phosphorylation (P < 0.01, P < 0.01). At the same time apelin-13 (100 nmol/L) up-regulated Bcl-2 protein expression and down-regulated Bax and cleaved caspase-3 expression (P < 0.01, P < 0.05, P < 0.05). The anti-apoptotic effect of apelin-13 was blocked by LY294002 (P < 0.01) but not by rapamycin. CONCLUSIONS: The endogenous apelin-APJ system is compensatorily up-regulated and ultimately down-regulated following sustained myocardial ischemia. Apelin protects against ischemic cardiomyocyte apoptosis via activation of the PI3K/Akt pathway.