Angiotensin ? Induces Apoptosis Of Cardiac Microvascular Endothelial Cells Via Regulating PTP1B/PI3K/Akt Pathway

The increasing morbidity and mortality of cardiovascular diseases have caused tremendous burden to human society.Clinical and experimental evidence shows that abnormal glucose metabolism,cardiovascular endothelial dysfunction,endoplasmic reticulum stress and insufficient angiogenesis are the important pathogenic factors of cardiovascular diseases.Protein tyrosine phosphatase 1B(PTP1B)is widely expressed in liver,adipose tissue,muscle,pancreas and other metabolic related tissues.By inhibiting the signal transduction of insulin and leptin,it destroys the homeostasis of glucose and lipid metabolism,reduces insulin sensitivity and participates in the regulation of various cellular functions.In recent years,the role of PTP1B in cardiovascular diseases has attracted increasing attention.This study focused on the role and mechanism of PTP1B in Ang II-induced apoptosis of primary myocardial microvascular endothelial cells(CMECs)and provided evidence for elucidating the interaction between endothelial cell apoptosis and RAAS.Objective: To investigate the interaction between endothelial cell apoptosis and activation of the renin-angiotensin-aldosterone system(RAAS).Methods:Male Sprague-Dawley rats of 2 weeks old were selected and cultured.The primary CMECs were isolated and cultured,and the second generation cells were taken for subsequent experiments:1.Screen the optimal concentration for the subsequent experiments: According to the Ang II concentration,the cells were divided into four groups: Control,1nmol,2nmol,4nmol,TUNEL and Caspase-3 activity kits to detect the changes of PTP1B protein levels in different AngII concentrations group.Western blot(WB)assays detect changes in apoptosis-associated proteins.2.To study the effect of AngII on CMECs: The cells were divided into control group,Ang II group,TCS 401(8?M,PTP1B inhibitor)group,Ang II+TCS401 group,four groups.The changes of apoptosis-related proteins were detected by protein immunoblotting(WB).3.To investigate the mechanism of PI3 K attenuating apoptosis of CMECs induced by AngII by regulating PTP1B: cells were divided into control group,Ang II group,Ang II+TCS401 group and Ang II+TCS401+LY294002 group.Apoptosis of cells was detected by Western blotting(WB).Result:1.Ang II induced PTP1B expression in primary rat CMEC cells to increase: TUNEL and Caspase-3 activity assay showed that the expression of PTP1B protein increased with the increase of Ang II concentration,and the highest expression at 4 nmol(p<0.01).2.Ang II significantly increased apoptosis of CMEC in a concentration-dependent manner: Caspase-3 activity assay,TUNEL and WB results showed that compared with control group,Caspase-3 activity in Ang II group increased significantly(p < 0.01),and Bcl-2/Bax ratio decreased(p < 0.05).3.Inhibiting PTP1B reduces apoptosis of CMECs induced by Ang II: TCS 401(8 uM)is used to inhibit PTP1B expression.The results showed that Ang II significantly increased PTP1B protein expression,apoptotic index and caspase-3 activity compared with the control group,and these effects were partly reversed by inhibiting the expression of PTP1B.In addition,Ang II decreased the ratio of anti-apoptotic factor Bcl-2/Bax and increased the ratio of pro-apoptotic factor caspase-3/caspase-3,while TCS 401 treatment significantly reduced these effects.These results suggest that Ang II induces apoptosis of CMEC in a PTP1B-dependent manner.4.Ang II up-regulates PTP1B protein expression and inhibits PI3K/Akt signal transduction: In order to investigate the role of PTP1B in Ang II-induced apoptosis of CMECs,we used WB to detect the changes of PTP1B protein expression.The results showed that PTP1B protein expression was significantly up-regulated in Ang II-treated CMEC in a dose-dependent manner.In addition,the protein expression of p-Akt and PI3 K decreased significantly in a dose-dependent manner after the intervention of Ang II.5.PI3K/Akt pathway was activated by PTP1B inhibitors: after Ang II intervention,the expression of p-Akt and PI3 K was down-regulated,while the expression of PTP1B protein was enhanced.We further explored whether PI3K/Akt pathway was involved in PTP1B-mediated apoptosis of CMEC cells after Ang II stimulation.Western blotting results showed that PI3 K expression and Akt phosphorylation were decreased in Ang II treated cells,and these effects could be partially reversed by co-treatment with PTP1B inhibitors.The data suggest that PI3K/Akt pathway may be downstream signal of PTP1B activated by Ang II.Ang II can induce apoptosis of CMECs by regulating PTP1B/PI3K/Akt pathway.6.LY294002 partially reduces the anti-apoptotic effect of PTP1B inhibitors: In order to further detect the relationship between PTP1B and PI3K/Akt pathway,we used LY294002 inhibitors to inhibit PI3K/Akt pathway.The results showed that compared with Ang II + TCS 401 group,further LY294002 treatment significantly reduced PI3 K expression and Akt phosphorylation,and promoted CMEC cell apoptosis by reducing Bcl-2/Bax ratio and increasing caspase-3/caspase-3 ratio.Conclusion: PI3K/Akt pathway may be downstream signal of activated PTP1B induced by Ang II.Ang II can induce apoptosis of CMECs by regulating PTP1B/PI3K/Akt pathway.