Fen Gao

Angiotensin-(1-7) treatment ameliorates angiotensin II-induced apoptosis of human umbilical vein endothelial cells.

Angiotensin (Ang)‐(1–7), a metabolite of AngI and AngII, is a counter‐regulatory mediator of AngII. In the present study, we investigated the effects of Ang‐(1–7) on AngII‐induced apoptosis in human umbilical vein endothelial cells (HUVEC). To this end, HUVEC were pretreated with 10−9, 10−8, 10−7 or 10−6 mol/L Ang‐(1–7) at for 30 min before being stimulated with 10−6 mol/L Ang‐II for another 24 h. Acridine orange/ethidium bromide and propidium iodide staining were used to analyse the effects of Ang‐(1–7) on AngII‐induced apoptosis. Alone, 10−6 mol/L Ang‐(1–7) had no effect on the apoptosis of HUVEC following exposure of cells for 30 min, whereas AngII (10−6 mol/L, 24 h) significantly enhanced the number of apoptotic cells (P < 0.01). The AngII‐induced apoptosis of HUVEC was suppressed by 10−9–10−6 mol/L Ang‐(1–7). The anti‐apoptotic effects of Ang‐(1–7) were almost completely abolished by A‐779 (10−6 mol/L, 30 min), a specific Mas receptor antagonist. In addition, Ang‐(1–7) inhibited AngII‐induced accumulation of cleaved caspase 3 and enhanced the expression of the anti‐apoptotic factor Bcl‐2 at both the mRNA and protein levels. Angiotensin II upregulated the expression of lectin‐like oxidized low‐density lipoprotein receptor‐1 (LOX‐1), which is involved in endothelial apoptosis, at both the mRNA and protein levels. This effect was blocked by Ang‐(1–7) in a concentration‐dependent manner, although A‐779 almost completely reversed Ang‐(1–7)‐mediated inhibition of AngII‐induced upregulation of LOX‐1. Silencing of LOX‐1 using short interference RNA enhanced the protective effects of Ang‐(1–7) against AngII‐induced apoptosis in HUVEC. Together, the results suggest that Ang‐(1–7) ameliorates AngII‐induced apoptosis of HUVEC at least in part by suppressing LOX‐1 expression.

Amlodipine Treatment Prevents Angiotensin II-induced Human Umbilical Vein Endothelial Cell Apoptosis

BACKGROUND AND AIMS Amlodipine, a long-acting dihydropyridine calcium channel blocker, is able to improve angiotensin II-mediated vascular endothelial dysfunction. However, the underlying mechanism remains not fully understood. In the present study we attempted to determine whether the protective effect of amlodipine against Ang II-induced endothelial impairment was mediated through blockage of endothelial cell apoptosis. METHODS We pretreated human umbilical venous endothelial cells with increasing doses of amlodipine (10(-8)-10(-6) M) followed by the addition of Ang II. Cell apoptosis was assessed by acridine orange/ethidium bromide staining and by annexin-V/propidium iodide double-labeled cytometry. The involvement of the apoptosis regulators, Bcl-2, Bax, and lectin-like oxidized low-density lipoprotein receptor-1, was determined. RESULTS Pretreatment with amlodipine resulted in a dose-dependent suppression of Ang II-induced HUVEC apoptosis. Moreover, the Bcl-2/Bax ratio was found to be increased in cells pretreated with amlodipine, indicating an enhanced anti-apoptosis potential. Additionally, the induction of LOX-1 by Ang II was remarkably counteracted by the pre-exposure to amlodipine. CONCLUSIONS Our data demonstrate that amlodipine ameliorates Ang II-induced endothelial apoptosis, which is likely associated with the elevation of Bcl-2/Bax ratio and reduction of the LOX-1 expression.

RNA interference targeting the ACE gene reduced blood pressure and improved myocardial remodelling in SHRs

The purpose of the present study was to investigate the effects on blood pressure and myocardial hypertrophy in SHRs (spontaneously hypertensive rats) of RNAi (RNA interference) targeting ACE (angiotensin-converting enzyme). SHRs were treated with normal saline as vehicle controls, with Ad5-EGFP as vector controls, and with recombinant adenoviral vectors Ad5-EGFP-ACE-shRNA, carrying shRNA (small hairpin RNA) for ACE as ACE-RNAi. WKY (Wistar-Kyoto) rats were used as normotensive controls treated with normal saline. The systolic blood pressure of the caudal artery was recorded. Serum levels of ACE and AngII (angiotensin II) were determined using ELISA. ACE mRNA and protein levels were determined in aorta, myocardium, kidney and lung. On day 32 of the experiment, the heart was pathologically examined. The ratios of heart weight/body weight and left ventricular weight/body weight were calculated. The serum concentration of ACE was lower in ACE-RNAi rats (16.37+/-3.90 ng/ml) compared with vehicle controls and vector controls (48.26+/-1.50 ng/ml and 46.67+/-2.82 ng/ml respectively; both P<0.05), but comparable between ACE-RNAi rats and WKY rats (14.88+/-3.15 ng/ml; P>0.05). The serum concentration of AngII was also significantly lower in ACE-RNAi rats (18.24+/-3.69 pg/ml) compared with vehicle controls and vector controls (46.21+/-5.06 pg/ml and 44.93+/-4.12 pg/ml respectively; both P<0.05), but comparable between ACE-RNAi rats and WKY rats (16.06+/-3.11 pg/ml; P>0.05). The expression of ACE mRNA and ACE protein were significantly reduced in the myocardium, aorta, kidney and lung in ACE-RNAi rats compared with that in vehicle controls and in vector controls (all P<0.05). ACE-RNAi treatment resulted in a reduction in systolic blood pressure by 22+/-3 mmHg and the ACE-RNAi-induced reduction lasted for more than 14 days. In contrast, blood pressure was continuously increased in the vehicle controls as well as in the vector controls. The ratios of heart weight/body weight and left ventricular weight/body weight were significantly lower in ACE-RNAi rats (3.12+/-0.23 mg/g and 2.24+/-0.19 mg/g) compared with the vehicle controls (4.29+/-0.24 mg/g and 3.21+/-0.13 mg/g; P<0.05) and the vector controls (4.43+/-0.19 mg/g and 3.13+/-0.12 mg/g; P<0.05). The conclusion of the present study is that ACE-silencing had significant antihypertensive effects and reversed hypertensive-induced cardiac hypertrophy in SHRs, and therefore RNAi might be a new strategy in controlling hypertension.

LOX‑1 is implicated in oxidized low‑density lipoprotein‑induced oxidative stress of macrophages in atherosclerosis.

Induction of oxidative stress has a causal role in atherosclerosis. The aim of the present study was to examine the role of lectin‑like oxidized low‑density lipoprotein receptor‑1 (LOX‑1) in oxidized low‑density lipoprotein (OxLDL)‑induced oxidative stress in atherosclerosis. Small interfering RNA (siRNA) technology was employed to decrease the expression of LOX‑1 in mouse RAW264.7 macrophages and the effects of LOX‑1 silencing on OxLDL‑induced reactive oxygen species (ROS) generation and NADPH oxidase (NOX) expression were investigated. The in vivo effects of reducing LOX‑1 were also examined in a mouse model (ApoE‑/‑) of high‑fat diet‑induced atherosclerosis. Compared with the control cells, OxLDL exposure led to a significant (P<0.05) increase in the intracellular levels of malondialdehyde and ROS and a significant decrease in the activity of superoxide dismutase. Delivery of LOX‑1‑targeting siRNA significantly (P<0.05) reversed the alterations in oxidative stress parameters induced by OxLDL. LOX‑1 silencing downregulated the expression of NOX2, Rac1, p47phox and p22phox and impaired the activation of mitogen‑activated protein kinases in OxLDL‑treated cells. Adenoviral delivery of LOX‑1 siRNA caused a significant increase in the size of the fibrous cap and a decrease in the macrophage content in lesions, compared with the control mice. Western blot analysis demonstrated that the protein expression levels of NOX1, Rac1, p47phox and p22phox in aortic lesions were significantly lower in the LOX‑1 siRNA group than in the control group. LOX‑1 is implicated in OxLDL‑induced oxidative stress of macrophages in atherosclerosis, which in part, involves the regulation of NADPH oxidases.

Correlation between the High Density Lipoprotein and its Subtypes in Coronary Heart Disease.

Background/Aims: To detect the changes of high density lipoprotein (HDL) and its subtypes in serum of patients with coronary heart disease (CHD). Methods: 337 hospitalized patients were selected from our hospital during August, 2014 - January, 2015, and divided into CHD group (n = 190) and control group (n = 127). Lipoprint lipoprotein analyzer was used to classify low density lipoprotein (LDL) particle size and its sub-components, as well as HDL particle size and its sub-components. The changes of the subtypes in patients with CHD were statistically analyzed. The possible mechanism was explored. Results: (1) Compared with the control group, the concentration of HDL in CHD patients reduced, HDLL significantly decreased (P < 0.001), while HDLS increased (P < 0.001); (2) In the patients with HDL less than 1.04 mmol/L among CHD, all HDL subtypes reduced, but HDLL had the most significant decreased; (3) HDL and all HDL subtypes were positively correlated with apolipoprotein A-I (apoA-I), of which, HDLL had the biggest correlation with apoA-I (P < 0.001); (4) HDL subtypes had good correlation with HDL, of which, HDLM had a maximum correlation with HDL (P < 0.001). Conclusion: HDL maturation disorders existed in the serum of CHD patients, HDLL may be protected factor for CHD, whose decrease was closely related wit the risk increase of CHD. The cardiovascular protection function of HDLL may be related with apoA-I content.

Angiotensin-(1-7) stimulates cholesterol efflux from angiotensin II-treated cholesterol-loaded THP-1 macrophages through the suppression of p38 and c-Jun N-terminal kinase signaling.

Angiotensin II (Ang II) and Ang-(1-7) are key effector peptides of the renin-angiotensin system. The present study aimed to investigate the effects of Ang-(1-7) on Ang II-stimulated cholesterol efflux and the associated molecular mechanisms. Differentiated THP-1 macrophages were treated with Ang II (1 µM) and/or Ang-(1-7) (10 and 100 nM) for 24 h and the cholesterol efflux and gene expression levels were assessed. Pharmacological inhibition of peroxisome proliferator-activated receptor (PPAR)γ and mitogen-activated protein kinases (MAPKs) were performed to identify the signaling pathways involved. The results demonstrated that Ang II significantly inhibited the cholesterol efflux from cholesterol-loaded THP-1 macrophages. Treatment with Ang-(1-7) led to a dose-dependent restoration of cholesterol efflux in the Ang II-treated cells. The co-treatment with Ang-(1-7) and Ang II significantly increased the expression levels of adenosine triphosphate (ATP)-binding cassette (ABC)A1 and ABCG1 compared with treatment with Ang II alone. This was coupled with increased expression levels of PPARγ and liver X receptor (LXR)α. The pharmacological inhibition of PPARγ significantly (P<0.05) eliminated the Ang-(1-7)-mediated induction of ABCA1 and ABCG1 mRNA expression. Treatment with Ang-(1-7) caused the inactivation of c-Jun N-terminal kinases (JNK) and p38 MAPK signaling in the Ang II-treated THP-1 macrophages. In addition, the inhibition of JNK or p38 MAPK signaling using specific pharmacological inhibitors mimicked the Ang-(1-7)-induced expression of PPARγ and LXRα. In conclusion, the data demonstrated that treatment with Ang-(1-7) promoted cholesterol efflux in Ang II-treated THP-1 macrophages, partly through inactivation of p38 and JNK signaling and by inducing the expression of PPARγ and LXRα. Ang (1-7) may, therefore, have therapeutic benefits for the treatment of atherosclerosis.

Intermedin attenuates high-glucose exacerbated simulated hypoxia/reoxygenation injury in H9c2 cardiomyocytes via ERK1/2 signaling:

Objective: This study investigated whether and how intermedin (IMD) exerted a protective effect against simulated hypoxia/reoxygenation (H/R) injury in high-glucose-treated H9c2 cells. Methods: Cellular viability was assessed via 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyl-2H-tetrazolium bromide (MTT) assay. Oxidative stress was determined by malondialdehyde and superoxide dismutase content in the culture medium supernatant. Flow cytometry with Annexin V/propidium iodide staining was used to detect the cardiomyocyte apoptosis rate. The protein expression of Bax, Bcl-2, caspase-3, and ERK1/2 was determined by western blot. Results: IMD administration to H9c2 cells during H/R injury decreased oxidative stress product generation and inhibited apoptosis (P < 0.05 or P < 0.01) while these effects were blocked by the ERK1/2 inhibitor (P < 0.05 or P < 0.01). Through the application of a specific ERK1/2 inhibitor, it was demonstrated that IMD mitigates high-glucose-induced oxidative stress and apoptosis via ERK1/2 signaling. Conclusion: Intermedin may be a novel therapeutic agent for mitigating diabetic cardiovascular injury in the clinical setting.