Effect Of Metformin On Vascular Endothelial Injury Induced By Bevacizumab

Although we are currently making great achievements in cancer treatment and improving the survival rate of cancer patients,cancer is still one of the leading causes of death worldwide.This high mortality rate is not only related to the tumor itself,but also to the complications of tumor combined with vascular disease,chemotherapy resistance,recurrence and metastasis,surgery,radiotherapy and chemotherapy.Cardiovascular disease and stroke are the most harmful among cancer-associated vascular diseases,but some vascular diseases result from treatment with angiogenesis inhibitors.These drugs are increasingly reported to cause endothelial dysfunction,which leads to vascular disease.Bevacizumab is a commonly used angiogenesis inhibitor in clinic.It is a humanized monoclonal antibody of vascular endothelial growth factor VEGF.It is mostly used in the treatment of colorectal cancer and non-small cell lung cancer with remarkable effect.However,many adverse reactions of bevacizumab have been found in clinical application,including thrombosis,hemorrhage,gastrointestinal perforation,hypertension and proteinuria,among which hypertension and proteinuria are the most common,but arterial and venous thrombosis is the most serious.Arterial thromboembolic events include stroke,transient ischemic attack and myocardial infarction.Studies have found that bevacizumab-related vascular complications are mainly related to bevacizumab-induced vascular endothelial dysfunction and endothelial injury.While related inflammatory factors also promote the progress of this process.Regarding the vascular damage caused by bevacizumab,is there a drug that can reduce this damage? After reviewing a large number of documents,it was found that metformin had more evidence for endothelial protection.Previous studies have confirmed that metformin plays an important role in resisting endothelial cell oxidative stress,anti-inflammatory,inhibiting endothelial cell permeability,inhibiting endothelial cell apoptosis,promoting endothelial progenitor cell differentiation and reducing arterial and venous thrombosis.These are all effects of metformin in cancer or other diseases,but there is no evidence that it has a positive effect in cancer patients taking bevacizumab.Therefore,this study explored the effect of bevacizumab on endothelial cells from the clinical,mouse and cellular levels,and demonstrated whether metformin can improve this effect and the corresponding mechanism.This study was divided into three parts to investigate the bevacizumabinduced endothelial injury and the mechanism by which metformin alleviated bevacizumab-induced endothelial injury:The first part: To explore the effects of chemotherapy combined with bevacizumab on endothelial cells injury markers and pro-inflammatory factors in patients with stage IV non-small cell lung cancer and stage IV colorectal cancer who received chemotherapy combined with bevacizumab.The second part: To investigate the effects of bevacizumab on vascular endothelial injury,inflammation and endothelium tissue section microscopic structure in bevacizumab C57 male mice,and to explore whether metformin can alleviate these effects and its mechanism.The third part: To investigate the effect of bevacizumab on human umbilical vein endothelial cell injury,apoptosis and inflammation;to further explore whether metformin can alleviate the above effects and its mechanism.Part One Effects of bevacizumab on endothelial cells injury markers and pro-inflammatory factors in patients with non-small cell lung cancer and colorectal cancerObjective: Collected patients with stage IV non-small cell lung cancer(NSCLC)and stage IV patients who received chemotherapy or chemotherapy combined with bevacizumab in the Department of Immuno-Oncology of our hospital from June 2021 to December 2022.The data of 45 patients with colorectal cancer(CRC)or non-small cell lung cancer(NSCLC)were analyzed to explore the effect of chemotherapy combined with bevacizumab on endothelial cells injury markers and pro-inflammatory factors.Methods: Patients were divided into control group(Control)and bevacizumab group(Bev)according to whether bevacizumab was applied or not.The m-FOLFOX6 chemotherapy regimen was used in CRC patients,and the combination platinum-containing chemotherapy regimen was used in NSCLC patients.Bevacizumab was administered as an intravenous infusion of7.5 mg/kg every 3 weeks.Patient’s age,sex,disease type,platelets,low-density lipoprotein cholesterol(LDL-C),total cholesterol(TC),triglyceride(TG),E-selectin(CD62E),endothelin(ET-1),Thrombin(TM),von Willebrand factor(v WF),tumor necrosis factor alpha(TNF-α),and interleukin-6(IL-6)were collected.Results:1.Comparison of clinical data of two groups of patientsThere were 20 cases in the Control group: 6 with rectal cancer,3 with colon cancer and 11 with lung adenocarcinoma.There were 25 cases in the Bev group: 7 with rectal cancer,6 with colon cancer and 12 with lung adenocarcinoma.There was no significant difference in the general data of each group(P>0.05).2.Effects of bevacizumab on markers of endothelial injury in patients with colorectal cancer or lung adenocarcinomaThe expression of CD62 E,ET-1,TM,and v WF in blood before and after treatment were compared between the two groups,respectively.Before treatment,there was no significant difference in CD62 E,ET-1,TM and v WF between the two groups(P>0.05);but after treatment,in Bev group,the above indexes in the blood were significantly higher than Control group(P<0.001).The expression of CD62 E,ET-1,TM,and v WF in blood before and after treatment were compared in the Bev group and the Control group,respectively.In the Control group,There was no significant difference in the above indexes before and after treatment(P>0.05).In the Bev group,the above indexes were significantly higher after treatment than before treatment(P<0.001).3.Effects of bevacizumab on pro-inflammatory factors in patients with colorectal cancer or lung adenocarcinomaThe levels of IL-6 and TNF-α in the blood before and after treatment were compared between the two groups.There was no significant difference in IL-6and TNF-α between the two groups before treatment(P>0.05).The Bev group was significantly higher than the Control group after treatment(P<0.001).The expression of IL-6 and TNF-α in the blood before and after treatment were compared in the Bev group and the Control group,respectively.Both the control group and the experimental group,the above indexes were all increased after treatment compared with before treatment(P<0.01),but the increase was more significant in Bev group(P<0.001).Conclusions:1.Bevacizumab may cause endothelial cell damage in patients with metastatic colorectal or lung adenocarcinoma.2.Bevacizumab may lead to elevated serum pro-inflammatory cytokines in patients with metastatic colorectal cancer or lung adenocarcinom Part Two Metformin attenuates bevacizumab-induced vascular endothelial injury in mice by upregulating GDF15 and activating the PI3K/AKT/FOXO/PPARγ signaling pathwayObjective: To investigate the effect of bevacizumab on vascular endothelial injury,inflammation and endothelial tissue section microscopic structure in C57 male mice;to further explore whether metformin can alleviate the above-mentioned effects of bevacizumab-induced vascular endothelium and its mechanism.Methods: Grouping: Twenty-four C57 male mice were randomly divided into two groups of eight groups: 1)Control 2)Bev 3)Met 4)Bev+Met5)AAV-GP-1-NC+Bev 6)AAV-GP-1-NC+Bev+Met 7)AAV-GP-1-si GDF15+Bev 8)AAV-GP-1-si GDF15+Bev+Met.Feeding: Met1200mg/kg was given by gavage daily for 7 weeks;Bev 15mg/kg intraperitoneal injection,once every 3 days;AAV-GP-1-si GDF15 or AAV-GP-1-NC tail vein injection(0.2ml/only),2 Repeat the injection after a week.Material collection and detection: Orbital blood was collected for ELISA kit detection of relevant indicators;abdominal aorta was collected and divided into three parts: used for HE staining and observation of tissue sections under microscope,PCR and Wesrern detection.Results:1.Effects of Bev on apoptosis,injury markers and inflammatory factors of mouse abdominal aortic endothelial cellsHE staining showed that Bev increased the apoptosis of mouse abdominal aortic endothelial cells.Bev increased the levels of CD62 E,ET-1,TM and v WF in the venous blood of mice(P<0.001),and increased the expressions of IL-6(P<0.05)and TNF-α(P<0.001).2.Met alleviated the effect of Bev on the endothelium of abdominal aorta in mice.Met treatment reduced Bev-induced apoptosis of mouse abdominal aortic endothelial cells,decreased the expression level of Bev-treated CD62E(P<0.001),v WF and ET-1(P<0.01),TM,IL-6 and TNF-α(P<0.05)in the venous blood of mice.At the same time,Met enhanced the mRNA and protein expressions of GDF15 and PI3 K,PPARγ(P<0.001),and Bcl-2(P<0.05),increased the protein expressions of p-AKT and p-FOXO3 a,and decreased the expression of p-AKT and p-FOXO3 a in Bev-treated mice.Bax mRNA(P<0.01)and protein expression.HE staining of the abdominal aorta of Bev-treated si-NC mice showed that compared with the Bev group,Met combined with Bev reduced the apoptosis of mouse abdominal aortic endothelial cells;In terms of markers and inflammatory factors,Met combined with Bev reduced the levels of CD62 E,TM(P<0.01),ET-1,v WF,IL-6 and TNF-α(P<0.05)in venous blood of mice,and at the same time Increased mRNA and protein expression of GDF15,PI3 K,PPARγ(P<0.001)and Bcl-2(P<0.01),promoted phosphorylation of AKT and FOXO3 protein,and decreased Bax mRNA(P<0.05)and protein expression.3.Inhibition of GDF15 expression can inhibit the expression of PI3K/AKT/FOXO/PPARγ signaling pathwayHE staining of abdominal aorta showed that both si-GDF15 mice and si-NC mice applied with Bev showed apoptosis,but si-GDF15 apoptosis was more significant.On the basis of applying Bev,compared with si-NC mice,the expression of TM(P<0.01),v WF(P<0.001),ET-1,IL-6 and TNF-α(P<0.05)of si GDF15 mice increased;In addition,the mRNA and protein expression of GDF15,PI3 K,PPARγ,Bcl-2 were significantly decreased,but the mRNA and protein expression of Bax were significantly increased(P<0.001).4.Metformin alleviates bevacizumab-induced mouse abdominal aortic endothelial injuryand apoptosis by up-regulating GDF15 and activating PI3K/AKT/FOXO/PPARγ signaling pathwayOn the basis of simultaneous application of Met and Bev,compared with si-NC mice,abdominal aortic endothelial cell apoptosis were significantly increased in si-GDF15 mice,the expression of CD62 E,ET-1,TNF-α(P<0.05),TM,v WF(P<0.01)and IL-6(P<0.01)also increased.In si-GDF15 mice,the mRNA and protein expression of GDF15、PI3K,PPARγ and Bcl-2(P<0.001)were down-regulated,the mRNA and protein expression of Bax were significantly increased(P<0.001).Conclusions:1.Bevacizumab induces inflammation,injury and apoptosis of mouse abdominal aortic endothelial cells.2.Metformin reduces bevacizumab-induced inflammation,injury,and apoptosis in mouse abdominal aortic endothelial cells.3.Metformin may attenuate the effects of bevacizumab-induced mouse abdominal aortic endothelial cells through activation of PI3K/ AKT/ FOXO/PPARγ signaling pathway by GDF15.Part Three Metformin attenuates bevacizumab-induced vascular endothelial injury by upregulating GDF15 and activating PI3K/AKT/FOXO/PPARγ signaling pathway in cellsObjective: To investigate the effect of bevacizumab on vascular endothelial cell inflammation,injury and apoptosis in human umbilical vein endothelial cells;to further explore whether metformin can alleviate the abovementioned effect s of bevacizumab-induced vascular endothelial cells and its mechanism.Methods: Mainly usedRNA interference technology,RT-q PCR,Western-blot,ELISA,Annexin V-FITC technology to detect the expression of CD62 E,ET-1,TM,v WF,TNF-α and IL-6,the expression of GDF15,PI3 K,AKT,FOXO3,PPARγ,BCL-2,BAX in the term of mRNA and protein and cell apoptosis in human umbilical vein endothelial cells in different groups.Results:1.Effects of metformin on bevacizumab-induced apoptosis,markers of vascular endothelial injury and inflammation in HUVECs.Bevacizumab increased apoptosis in HUVECs,whereas metformin treatment decreased apoptosis in bevacizumab-treated HUVECs(P<0.001).Bevacizumab treatment increased CD62 E,ET-1,TM and v WF levels in HUVECs,however,metformin treatment decreased the levels of these markers of endothelial injury except CD62E(P<0.05).Bevacizumab also increased TNF-α and IL-6 expression in HUVECs,but metformin decreased bevacizumab-induced TNF-α and IL-6 expression in HUVECs(P<0.001).Metformin enhanced GDF15 expression and activation of PI3K/ AKT/ FOXO/ PPARγsignaling in bevacizumab-treated HUVECs.2.Effects of GDF15 overexpression on bevacizumab-induced apoptosis,vascular endothelial injury markers and inflammation in HUVECs.After GDF15 plasmid was transfected into HUVECs cells,GDF15 mRNA(P<0.001)and protein expression were up-regulated.After bevacizumab treatment,GDF15 overexpression reduced apoptosis and the expression of TNF-α and IL-6 in HUVECs(P<0.01),and inhibited CD62 E,ET-1,TM and v WF expression(P<0.05),up-regulated PI3 K,PPARγ,Bcl-2 mRNA expression,down-regulated Bax mRNA expression(P<0.01),increased the expression of PI3 K,P-AKT,P-FOXO3,PPARγ and Bcl-2 proteins,and reduced Bax protein expression.3.Effects of GDF15 siRNA on bevacizumab-induced apoptosis,vascular endothelial injury markers and inflammation in HUVECs.After transfection of siRNA,the expression of GDF15 mRNA(P<0.001)and protein expression were down-regulated.GDF15 siRNA attenuated the effects of metformin on bevacizumab-induced apoptosis,TM,v WF,TNF-α and IL-6 expression in HUVECs(P<0.01),but did not reduce the expression of CD62 E or ET-1 in bevacizumab(P>0.05).Attenuated the effects of metformin on PI3 K,PPARγ,Bcl-2 and Bax mRNA expression in bevacizumab-treated HUVECs(P<0.001).At the same time,the expressions of PI3 K,PPARγ,Bax and Bcl-2 proteins showed similar trends as above,the expressions of P-AKT and P-FOXO3 proteins also showed similar trends.4.Effects of LY 294002 on bevacizumab-induced apoptosis,markers of vascular endothelial injury and inflammation in HUVECs.LY 294002 attenuated the effects of metformin on the expression of ET-1,TM,v WF,TNF-α,IL-6 in bevacizumab-induced HUVECs,but had no significant effect on apoptosis or CD62 E expression(P>0.05),down-regulated PI3 K,PPARγ and Bcl-2 mRNA expression,but up-regulated Bax mRNA expression(P<0.01),did not alter GDF15 mRNA expression in bevacizumab and metformin-treated HUVECs(P>0.05).In terms of protein expression,LY294002 decreased the expression of PI3 K,P-AKT,P-FOXO3,PPARγ,Bcl-2 protein,but increased the expression of Bax protein without changing the expression of GDF15 protein.Conclusions:1.Bevacizumab can induce HUVECs inflammation,injury and apoptosis..2.Metformin attenuates bevacizumab-induced inflammation,injury and apoptosis in HUVECs.3.Overexpression of GDF15 attenuates bevacizumab-induced apoptosis,endothelial injury and inflammation in HUVECs.4.Metformin may affect bevacizumab-induced apoptosis,vascular endothelial injury markers and inflammation in HUVECs through GDF15 activation of PI3K/AKT/FOXO/PPARγsignaling pathway.