Adiponectin Ameliorates Angiotensin Ⅱ-induced Vascular Endothelial Damage

Adiponectinis an adipocyte-specific adipocytokine that possesses anti-atherogenic and anti-diabetic properties.It has been shown to have a beneficial effect on the cardiovascular system,but it remains to be elucidated whether adiponectin has a therapeutic effect on vascular damage induced by the potential vasoactive substance angiotensin II（Ang II）.In this study,the effects of adiponectin on Ang II-induced vascular endothelial damage and the underline mechanisms were investigated.Objective:1.To explore whether adiponectin ameliorates Ang II-induced vascular endothelial damage;2.To investigate the involved mechanisms that adiponectin prevent vascular endothelial cels from damage and apoptosis with the support of RNAi technique.Method:1.Adiponectin ameliorates Ang II-induced vascular endothelial damage:Humanumbilical vein endothelial cells（HUVECs）from Lonza（Walkersville,USA）were cultured in EBM-2 media with supplemental growth factors according to manufacturer’s instructions.Cells were pretreated with adiponectin at 1,5,or 10μg/ml for 4h before adding 1-μM Ang II.Levels of intracellular ROS were determined by fluorescence dye 2’,7’-dichlorofluorescein-diacetate（DCFH-DA）. Average fluorescence intensity was used to represent levels of intracellular ROS. Levels of 4-hydroxy-2-nonenal（4-HNE）found in HUVECs were used to index lipid peroxidation.Cell viability of HUVECs was determined by MTT reduction assay.Cell viability was indexed by OD values measured at 570 nm using a microplate reader.Cell membrane integrity was indexed by the release of lactate dehydrogenase（LDH）into the medium.Absorbance was read at 490 nm and calculated as the percentage of cell death.Apoptosis was determined by terminal deoxynucleotidyl transferase dUTP nick end labeling（TUNEL）assay.Levels of activated caspase-3 and poly（ADP-ribose）polymerase（PARP）were also evaluated by Western Blot analysis.2.The involved mechanisms that adiponectin prevent vascular endothelial cells damage and apoptosis from influence of Ang II with the support of RNAi technique:siRNA of adiponectin receptors（AdipoR）type 1 and 2 were synthesized according to Human AdipoR DNA sequence provided in GeneBank. Levels of AdipoR were evaluated again by Western Blot analysis to validate the effect of RNAi.After successful RNAi,levels of LDH and the expression oflectin-like,oxidized low-density lipoprotein receptor-1（LOX-1）were evaluated toexplore which AdipoR was involved in the progress of apoptosis prevention. SiRNA of adaptor protein with phosphotyrosine binding,pleckstrin homology domainsandaleucinezippermotif（APPL1）andadenosine monophosphateactivated protein kinase（AMPK）were synthesized respectively according to Human APPL1 and AMPK DNA sequence provided in GeneBank. Levels of APPL1 and AMPK were evaluated again by Western Blot analysis to validate the effect of RNAi.After successful RNAi,the expression of LOX-1 was evaluated to explore whether APPL1-AMPK pathway was involved in the progress of apoptosis prevention.Levels of multiple anti-apoptotic proteins including Bcl-2 and inhibitory apoptotic protein-1（cIAP-1）were also evaluated by Western Blot analysis.Result:1.Adiponectin ameliorates Ang II-induced vascular endothelial damage:a)ROS levels in Ang II-treated cells were significantly higher than in controls and that this could be attenuated by adiponectin in a dose-dependent manner from 1 to 10μg/ml（5.89×103±5.30×102 vs2.83×103±2.88×102,p<0.001）.Consistently,Ang II treatment led to increased levels of 4-HNE,but was prevented by adiponectin（1.22×103±1.06×102 vs 5.53×102±55.9,p<0.001）.In addition,treatment with adiponectin reduced the basal levels of ROS and 4-HNE.b)Both MTT and LDH assays displayed that 24-h treatment with Ang II led to impaired cell viability.Notably,Ang II-induced cell death was effectively prevented by adiponectin treatment in a dose-dependent manner.c)TUNEL staining was used to detect apoptosis following Ang II and adiponectin treatment.In the result,Ang II significantly promoted apoptosis in cells.However,adiponectin treatment ameliorated these patterns of apoptosis.The apoptotic rate of HUVECs was significantly decreased（61.00±8.67%vs 22.00±2.19%,p<0.001）.our results demonstrate that caspase-3 is activated in HUVECs after Ang II treatment and that the activation of caspase-3 is significantly inhibited by adiponectin treatment（2.65×10-1±3.46×10-2 vs 1.11×10-1±1.54×10-2,p<0.001）.Our data also indicated that Ang II treatment increased the levels of cleaved PARP,which was prevented by adiponectin（5.14×10-1±7.66×10-2 vs 1.47×10-1±2.68×10-2,p<0.001）.Our results indicate that Ang II treatment led to the up-regulation of LOX-1expression.However,up-regulation of LOX-1 was significantly inhibited by adiponectin treatment（3.65×10-1±3.81×10-2 vs 1.23×10-1±1.39×10-2,p<0.01）.2.adiponectin prevent vascular endothelial cells damage and apoptosis from effect of Ang II through AdipoR1-APPL1-AMPK-LOX1 pathwaya)AdipoR1 is the specific receptor for adiponectin involved in the HUVECs protection Successful knockdown was confirmed by Western blot analysis（1.38±0.15 vs 0.45±0.09,p<0.01）.Results show that the protective effects of adiponectin against Ang II-induced cytotoxicity were lost in HUVECs with knockdown of AdipoR1 but not AdipoR2.b)Successful knockdown of APPL1 promotes HUVECs apoptosis Successful knockdown was confirmed by Western blot analysis（7.09×10-1±1.21×10-2 vs 1.83×10-1±3.82×10-2,p<0.01）.We found that the protective effects of adiponectin against cytotoxicity under Ang II treatment were lost in cells with knockdown of APPL1 expression.Notably,it was found that the protective effects of adiponectin against Ang II-induced up-regulation of LOX-1 were lost in HUVECs with knock down of APPL1.c)AMPK activation is involved in HUVECs protection. Successful knockdown was confirmed by Western blot analysis（7.98×10-1±1.12×10-2 vs 2.06×10-1±6.82×10-2,p<0.01）.The results show that the protective effects of adiponectin against Ang II cytotoxicity were lost in cells with knockdown of AMPK expression.Importantly,our results also demonstrated that the protective effects of adiponectin against Ang II-induced up-regulation of LOX-1 were lost in HUVECs with knockdown of AMPK.d)Expression of cIAP-1 and Bcl-2/Bax ratio indicates HUVECs apoptosis Findings show that cIAP-1 was significantly decreased in HUVECs after Ang II treatment,an effect that was markedly prevented by adiponectin treatment(5.93×10^-1±1.11×10^-1 vs 1.26±1.52×10^-1,p<0.01).The Bcl-2/Bax ratio was significantly reduced in cells treated with Ang II.In contrast,adiponectin treatment prevented Bcl-2/Bax reduction,indicating potential for enhanced anti-apoptotic activity（6.73×10-1±8.20×10-1 vs 2.59×10-1±3.16×10-2,p<0.01）.Conclusion:1.The present study demonstrates that adiponectin works to protect HUVECs against Ang II-induced cytotoxicity by attenuating oxidative stress,increasing cell viability,and preventing apoptosis.2.In addition,the protective effects of adiponectin depend on its ability to bind to its associated receptors,AdipoR1 and AdipoR2.Importantly,our results demonstrate that adiponectin treatment modulates the apoptotic pathway by reducing the expression of LOX-1 and upregulating both cIAP-1 expression and the Bcl-2/Baxratio.