Effect of OPG knockout on left ventricular systolic function of mouse
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Objective To investigate the relationships between serum levels of receptor activator of nuclear factor-kappa B ligand(RANKL),estrogen and heart function in osteoprotegerin(OPG) deficience-induced mouse osteoporosis. MethodsOPG knockout mouse model and control group were established.Serum OPG and RANKL levels were measured by ELISA.Serum estrogen was detected by radioimmunology,and cardiac ultrasonography was performed to assess the heart function.Results The heart weight,the ratio of heart weight to body weight and transverse area of myocardial cells of left ventricle were significantly increased in OPG knockout mice compared to the control group(P0.01).Left ventricular systolic diameter and left ventricular end-systolic volume in OPG knockout mice were significantly higher than those in the control group(P0.05).The serum RANKL level in OPG knockout mice was higher than that of the control group(P0.001),while there was no significant change in the serum estrogen level(P0.05). Conclusion Deficience of OPG induces up-regulation of serum RANKL level and myocardial hypertrophy,but has no effect on serum estrogen level.Keywords:
Knockout mouse
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The receptor activator of the nuclear factor kappa-B (NF-κB) ligand (RANKL), its membrane receptor RANK and its decoy receptor osteoprotegerin (OPG) are all members of the tumour necrosis factor family involved in bone metabolism and immune response. We evaluated the activation of the OPG/RANKL/RANK pathway in patients undergoing cardiac surgery with and without cardiopulmonary bypass (CPB). Twenty consecutive patients undergoing elective coronary artery surgery were enrolled in the study and assigned either to the on-pump or to the off-pump group. Pre- and postoperative serum levels of OPG and RANKL were evaluated by enzyme-linked immunosorbent assay; gene expression of OPG, RANKL, RANK and NF-κB p50 subunits were determined by real-time polymerase chain reaction in peripheral blood T-cells and monocytes. Serum levels of OPG significantly increased after surgery in both groups, whereas serum levels of RANKL did not differ over time. T-cells from the on-pump group showed increased gene expression of OPG, RANKL and RANK after the intervention, whereas no mRNA variation for these genes was detected in T-cells from off-pump patients. Gene expression of p50 subunit increased in T-cells and monocytes from both groups. Cardiac surgery induces the activation of the OPG/RANKL/RANK pathway; both on- and off-pump procedures are associated with increased postoperative OPG serum levels and up-regulation of the NF-κB p50 subunit.
RANK Ligand
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Objective:To study the change and meaning of osteoprotegerin(OPG) receptor activator of NF-κB ligand(RANKL) during the process of the rat arterial calcification.Method:The 60 SD rats were divided randomly into control group,atorvastatin group and calcified group.Each group was also divided into the early sub-group and late sub-group.Next,the calcified group was taken the intragastric administration with Warfarin;the atorvastatin group was taken the intragastric administration with both Warfarin and atorvastatin.The early and late sub-groups were detected at 17 days and 34 days,respectively.Von Kossa staining,ALP activity assay and osteocalcin assayed with Western Blot were used to detect the calcification level;Real Time PCR was used to detect the expression of OPG and RANKL.Result:There was great expression of OPG but no expression of RANKL in control group neither early nor late sub-group.The expression of OPG was increased in the early sub-group and decreased in the late sub-group in atorvastatin group and calcified group.However,the expression of RANKL in these two groups was increased all the time during the calcification.Among the three groups,the ratio of OPG/RANKL was decreased along with the increase of calcification level.And compared with the early sub-groups,the ratio of OPG/RANKL in the late sub-groups was also decreased along with the increase of calcification level in each group.Conclusion:The ratio of OPG/RANKL has a negative correlation with the level of the arterial calcification,and atorvastatin could significantly inhibit the arterial calcification.
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The present study aims to study the role of receptor activator of nuclear factor kappa B ligand/receptor activator of nuclear factor kappa B/osteoprotegerin (RANKL/RANK/OPG) system in cardiac hypertrophy in a spontaneous hypertension rat (SHR) model and the effects of amlodipine and atorvastatin intervention. Thirty-six-week-old male SHRs were randomly divided into four groups: 1) SHR control group; 2) amlodipine alone (10 mg/kg/d) group, 3) atorvastatin alone (10 mg/kg/d) group, 4) combination of amlodinpine and atorvastatin (10 mg/kg/d for each) group. Same gender, weight, and age of Wistar-Kyoto (WKY) rats with normal blood pressure were used as normal control. Drugs were administered by oral gavage over 12 weeks. The thicknesses of left ventricle walls, left ventricle weight, and cardiac function were measured by transthoracic echocardiography. Left ventricular pressure and function were assessed by hemodynamic examination. Cardiomyocyte hypertrophy and collagen accumulation in cardiac tissue were measured by hematoxylin and eosin (HE) and Masson staining, respectively. The hydroxyproline content of cardiac tissue was examined by biochemistry technique. RANKL, RANK and OPG mRNA, protein expression and tissue localization were studied by RT-PCR, Immunohistochemistry and Western blot. Treatment with amlodipine or atorvastatin alone significantly decreased left ventricular mass index, cardiomyocyte cross-sectional area and interstitial fibrosis in SHR (each P < 0.05). Moreover, combined amlodipine and atorvastatin treatment induced significant reversal of left ventricular hypertrophy and decreased cardiomyocyte cross-sectional area and interstitial fibrosis in SHR to a greater extent than each agent alone (P < 0.05). Compared with WKY rats, the myocardial expression of RANKL, RANK, and OPG was increased. Both amlodipine and atorvastatin reduced RANKL, RANK, and OPG expression, with the best effects seen with the combination. Based on our results, activation of the RANKL/RANK/OPG system may be an important factor leading to ventricular remodeling in SHR rats. Amlodipine and atorvastatin could improve ventricular remodeling in SHR rats through intervention with the RANKL/RANK/OPG system.
Amlodipine
Cardiac Fibrosis
Myocardial fibrosis
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Myocardial fibrosis
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Background. Osteoprotegerin (OPG) is a soluble glycoprotein of the tumor necrosis factor (TNF) receptor superfamily, which was initially identified as a key regulator in bone turnover. It acts as a decoy receptor for the receptor activator of nuclear factor kB ligand (RANKL) and for the TNF-related apoptosis-inducing ligand (TRAIL), counterbalancing their biological effects. OPG is produced by a wide range of tissues, including the cardiovascular system, and its levels are particularly high in aortic and renal arteries. Several studies have clearly demonstrated that the serum levels of OPG are elevated in diabetic and nondiabetic patients affected by cardiovascular diseases, and increased levels of OPG represent a risk factor for cardiovascular mortality, especially in diabetic patients. However, in spite of the reported findings, the physiopathological role of elevated serum levels of OPG in vascular biology and in pancreatic islet function are not well understood.
Aim of the study. The aims of our studies were:
Study 1. Evaluate the potential role of OPG in the pathogenesis of diabetes associated atherosclerosis.
Study 2. Investigate OPG effects on pancreatic islet function and its interaction with local pancreatic renin-angiotensin system (RAS).
Materials and Methods. Study 1.A. In vivo study: 80 apoE knockout male mice were further randomized into 4 groups (n=20) and followed for 3 months. One group of non diabetic animals received an intraperitoneal (i.p.) injection of vehicle and served as a control; another group of non-diabetic animals received every 3 weeks an i.p. injection of human recombinant OPG (OPG). The other two groups, rendered diabetic by 5 daily i.p. injections of streptozotocin (55mg/Kg/die), received injections of OPG or an equivalent volume of vehicle. At the end of the study, animals were culled, the blood was collected for biochemical analysis, and the entire aorta was excised out to study the total plaques extent and to evaluate the lesion composition and complexity of the aortic plaques. B. In vitro study: Murine vascular smooth muscle cells (VMSC) were treated with different concentrations of OPG, TGFβ and SB431542 (TGFβ- type 1 receptor inhibitor). Subsequently, cellular proliferation and pro-fibrotic markers gene expression were evaluated at different time points. OPG protein release was measured in growth media (ELISA technique).
Study 2. 40 male mice C57Bl/6J, aged 10 weeks, were randomized into 4 groups (n=10) and studied for 3 months. Group 1 received every 3 weeks an i.p. injection of vehicle and served
as a control. Group 2 received every 3 weeks an i.p. injection of OPG. Group 3 received the ACE inhibitor ramipril at the dose of 10mg/Kg/die in drinking water in co-treatment with i.p. injections of vehicle. Group 4 received ramipril in co-treatment with i.p. injections of OPG. At the end of the study, animals were culled, the blood was collected for biochemical analysis, and the pancreas was dissected out for subsequent quantitative RT-PCR measurements and immunohistochemical analysis.
Results. Study 1.A. At the end of the study, diabetic animals injected with OPG presented a significant increase in total plaques extent, with an increase of smooth muscle cells content in aortic plaques. Moreover OPG treated animals showed an increase in the collagen content in aortic media in respect to control mice. B. OPG promoted VSMC proliferation and pro-fibrotic markers gene expression. TGFβ treatment of VSMC induced a dose-dependent increase of OPG gene and protein expression, that was completed prevented by pre-treatment with the SB431542 inhibitor.
Study 2. OPG-treated animals showed increased islet monocyte-macrophage infiltration, fibrosis and apoptosis with reduction of islet function. The remodeling of islet architecture was associated with increased pancreatic expression of components of the RAS, growth factor genes (TGFβ and CTGF) and inflammatory molecules (MCP-1 and VCAM-1). Prevention of these changes with improvement of insulin secretion was observed in ramipril treated animals.
Conclusion. Study 1.A-B OPG seems to play an important pathogenetic role in the development and progression of diabetic atherosclerosis.
Study 2. Our data suggest that OPG might play an important role in promoting beta cell dysfunction and the upregulation of the local RAS represents one possible mechanism responsible for the OPG-induced beta cell dysfunction.
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Bone remodeling
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S-amlodipine has been broadly used to treat hypertension, but its protective effects and underlying mechanism remain controversial. The purpose of our study was to investigate the mechanism by which S-amlodipine improves endothelial dysfunction. Specifically, we investigated if S-amlodipine regulates RANK/RANKL/OPG and micro-RNA 155 (miR-155) levels. Spontaneous hypertensive rats (SHR) were randomly divided into two groups: SHR (n = 12) and S-amlodipine (n = 12). We found that left ventricular ejection fraction (LVEF) increased significantly in the S-amlodipine group compared to the SHR group. After 10 weeks of S-amlodipine treatment, tumor necrosis factor-α (TNF-α) and interleukin-6 (IL-6) levels were significantly lower and eNOS and NO production was significantly higher in the S-amlodipine group compared to the SHR group. In human umbilical vein endothelial cells (HUVECs), miR-155, RANK, and RANKL levels were significantly decreased, while OPG mRNA levels were significantly increased in the S-amlodipine group. HUVECs were transfected with miR-155 mimics or an inhibitor to determine the relationship between miR-155 and RANK/RANKL/OPG and NF-κB signaling. OPG mRNA levels following miR-155 inhibition were significantly higher compared to levels following treatment with miR-155 mimics. S-amlodipine significantly inhibited RANKL expression and NF-κB phosphorylation, and there were no significant differences in response to the NF-κB inhibitor (Bay110785). RANKL expression and NF-κB phosphorylation significantly decreased in the miR-155 inhibitor group. Furthermore, OPG protein expression significantly increased in response to miR-155 inhibition and S-amlodipine treatment (all p < 0.05). Our results indicate that S-amlodipine inhibits inflammation and protects against endothelial dysfunction, likely via regulating the RANK/RANKL/OPG pathway, which appears to be downstream of miR-155.
Amlodipine
Endothelial Dysfunction
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