| Background:Cardiovascular disease and its complications are common and frequently-occurring diseases.Although prevention,diagnosis and treatment interventions have made significant progress in strategies to prevent cardiovascular disease,coronary artery disease remains the leading cause of global disease mortality and morbidity.Since not everyone has the same risk of future cardiovascular event(CVE),an important challenge for cardiovascular medicine is how to accurately predict who will develop atherosclerosis-related complications,such as acute coronary syndrome.Atherosclerosis is the pathological basis of the onset of cardiovascular disease.At present,it is a hot topic of cardiovascular and clinical research to study the mechanism of atherosclerosis and to find effective intervention targets.Domestic and international studies have found that the mainstream mechanisms of atherosclerosis include lipid infiltration,chronic inflammation,endothelial damage,oxidative stress injury and blood flow shear.Recent studies have found that angiogenesis is also an important mechanism affecting the development of atherosclerosis.Based on the role of angiogenesis in atherosclerosis,research on angiogenesis has also become a special area of research.To explore the mechanism of angiogenesis during the development of atherosclerotic plaque,to find and intervene new targets affecting angiogenesis,so as to prevent or even avoid the occurrence of atherosclerotic disease,is one of the research hotspots in the field of cardiovascular research at present.Endothelial cells play a natural barrier function in the blood vessel wall,and play an important role in inflammation and angiogenesis by balancing the release of cells and molecular components at the paracrine and autocrine levels,preventing pathogen invasion and maintaining vascular integrity.The integrity of the vascular endothelium is very important for the balance of the circulating vascular system.Vascular dysfunction is the pathophysiological basis of atherosclerotic disease,and endothelial dysfunction can be considered as the initial stage of atherosclerosis.Plaque neovascularization consists of a network of capillaries that nourish blood vessels from the adventitia and extend to the intimal layer of atherosclerotic lesions and other types of vascular damage.The function of these plaque capillaries is considered to be an important regulator of plaque growth and lesion instability.Some molecular pathways have similar roles in the pathogenesis and progression of atherosclerosis and cancer,and the common mechanisms associated with these two diseases include oxidative stress and the resulting cellular damage.The role of angiogenesis in atherosclerosis and other cardiovascular diseases is still an unresolved question.At present,many studies have shown that the formation of new blood vessels contributes to the growth of atherosclerotic lesions,and is a key factor leading to plaque rupture and instability.Prevention of angiogenesis in atherosclerotic plaques may play an important role in stabilizing vulnerable plaques.ErbB2(Erythroblastic leukemia viral oncogene homolog 2),also known as Her2 in humans,is one of the important members of the epidermal growth factor receptor(EGFR).Erbin(ErbB2 interacting protein),which contains 16 leucine-rich repeats(LRR)at the amino terminus and a PDZ(PSD95/Discslarge/ZO-1)domain terminus at the carboxy terminus,belonging to the new LAP protein Family members,by PDZ domain tightly bound to ErbB2,first reported specific interaction with ErbB2[1].It is reported that Erbin is expressed in most human tissues,especially in the brain,heart,kidneys,muscles and stomach.Erbin is an interacting protein of ErbB2/Her2,which directly and specifically interacts with ErbB2/Her2 and localizes ErbB2/Her2 to the basolateral membrane of epithelial cells.ErbB2 has been shown to play an important role in vascular development because ErbB2 increases the synthesis of vascular endothelial growth factor(VEGF),resulting in increased angiogenesis.However,the mechanism by which ErbB2 works is not clear enough.ErbB2/Her2 plays an extremely important role in cell proliferation and differentiation,especially in angiogenesis.ErbB2 not only promotes the expression of VEGF,but also increases the invasive ability of endothelial cells and further promotes angiogenesis.ErbB2 is expressed in the coronary vascular endothelium and plays an irreplaceable role in the development of coronary vessels.At present,the following problems still need to be explored and solved:1 It is not clear whether Erbin is expressed in blood vessels;2 If Erbin is expressed in blood vessels,how to determine its specific cell localization;3Erbin affects the biological function of endothelial cells;4Erbin affects cells Specific signal transduction pathways for biological functions.Based on previous studies,we designed current experiments to verify the relationship between Erbin and vascular and endothelial cells,and the effects of Erbin on endothelial cell function,and further explore its molecular biological mechanisms or signaling pathways that affect cell function.Objective:Human umbilical vein endothelial cells were used as a cell model to interfere with the expression of endogenous Erbin by transfecting Erbin with lentivirus,and then observe the changes of proliferation,apoptosis,migration and tube formation of human umbilical vein endothelial cells.In vitro experiments were carried out to clarify the related signaling pathways of Erbin on human umbilical vein endothelial cells,and further in vivo experiments were conducted to explore the mechanism of Erbin affecting angiogenesis,and to find new targets affecting angiogenesis and atherosclerosis.Part Ⅰ:To explore the effects of Erbin on the proliferation,apoptosis,migration and tube formation of human venous endothelial cells.1.Tissue immunofluorescence technique to detect the expression and localization of Erbin in blood vessels.Human umbilical cord was taken for histological fixation,paraffin-embedded and sectioned,and tissue immunofluorescence was performed to observe the expression and localization of Erbin in blood vessels.2.Construction and screening of lentiviruses that interfere with endogenous ErbinWestern blot was used to determine the most effective interference sequence to inhibit endogenous Erbin expression after three different lentiviruses were constructed by encoding Erbin siRNA or disorderly sequence and infected with human umbilical vein endothelial cells.3.Explore the effect of Erbin on proliferation and apoptosis of human umbilical vein endothelial cells3.1 To detect the effect of Erbin on the proliferation of endothelial cells by CCK-8 assayHuman umbilical vein endothelial cells were transfected with the lentivirus of Erbin,and the proliferation of endothelial cells was detected at different time points using CCK-8 proliferation kit.3.2 To detect the effect of Erbin on apoptosis by flow cytometryHuman umbilical vein endothelial cells were transfected with the lentivirus of Erbin.The Annexin V-FITC/PI cell apoptosis assay kit was used for double staining,and flow cytometry was used to detect the changes of endothelial cell apoptosis.4.Explore the effect of Erbin on the migration and tube formation of human umbilical vein endothelial cells4.1 To detect the effect of Erbin on cell migration ability by cell scratch test and Transwell migration test.HUVECs were transfected with the lentivirus of Erbin,and the changes of endothelial cell migration ability were detected by cell scratch test and Transwell migration test.4.2 To detect the effect of Erbin on the angiogenic ability of human umbilical vein endothelial cells by endothelial cell tube formation assayThe lentivirus of Erbin infects HUVECs,and tube formation assay were performed to detect the ability of endothelial cells to form tubes.5.Statistical analysisData in the study were evaluated with predictive analytics software statistics 18.0(SPSS Inc,Chicago,IL).One-way analysis of variance(ANOVA)was used to compare data on normal distribution between different test groups,while the nonparametric variables were analyzed by the Mann-Whitney U test.Statistical significance was confirmed as P<0.05.Each assay was performed in triplicate.Part Ⅱ:To explore the the mechanism of Erbin affecting the migration and tube formation of human umbilical vein endothelial cells.1.To explore the effect of Erbin on ERK signaling pathway in human umbilical vein endothelial cells by Western blotAfter human umbilical vein endothelial cells were transfected with the lentivirus containing Erbin RNAi or the negative control virus,the correspondingly treated cells were collected,and the cell protein was extracted.Western blot was used to detect the expression of Erbin with GAPDH as an internal reference.The expression of t-ERK and p-ERK was detected by using the corresponding non-phosphorylation state as an internal reference.2.To explore the effect of Erbin on Smad signal transduction pathway in human umbilical vein endothelial cells2.1 Western blot analysis of the effect of Erbin on Smad signaling pathway in human umbilical vein endothelial cellsAfter human umbilical vein endothelial cells were transfected with the lentivirus containing Erbin RNAi or the negative control virus,the correspondingly treated cells were collected,and the cell protein was extracted.The expression of Erbin was detected by Western blot with GAPDH as an internal reference.As well as the expression of t-Smad2,p-Smad2,t-Smad3,p-Smad3,t-Smad1 p-Smad1/5,and t-Smad5 were detected with the corresponding non-phosphorylation state as the internal reference.2.2 Cellular immunofluorescence staining for phosphorylation and nuclear translocation of Smad1/5After transfected with the lentivirus containing Erbin RNAi or the negative control virus,we used cell immunofluorescence technology to detect the changes in the expression levels of t-Smad1,p-Smad1/5 and t-Smad5 outside the core.2.3 Changes of umbilical vein endothelial cell migration and tube formation after Smad siRNA blocking Smadl/5 pathwayTo observe the effect of Smad pathway block on umbilical vein endothelial cell migration and tubular structure formation after the human umbilical vein endothelial cells which were transfected with the lentivirus containing Erbin RNAi or the negative control virus pretreated with Smad siRNA,and the related Smad1/5 signaling pathway was inhibited.3.Statistical analysisThe data in the study was evaluated using predictive analysis software PASW Statistics 18.0(SPSS Inc,Chicago,IL).Normal distribution data were analyzed by one-way ANOVA and non-parametric variables are analyzed using the Mann-Whitney U test.Statistical significance was confirmed as P<0.05.ResultsPart Ⅰ:Erbin is positively expressed in vascular endothelial cells and can promote the migration and tube formation of human umbilical vein endothelial cells.1.Immunofluorescence technique shows that Erbin is mainly expressed in human umbilical vein endothelial cells.After obtaining the fresh umbilical vein tissue of the human body,the expression of Erbin in human umbilical vein was found by immunofluorescence technique,and the colocalization of Erbin and umbilical vein endothelial cells in the blood vessels was also observed.2.The Erbin interference lentiviral sequence were constructed successfully.After infection of human umbilical vein endothelial cells with three different lentiviruses encoding Erbin siRNA or scrambled sequences,Western blotting was used to determine the most effective interference sequence for inhibition of endogenous Erbin expression.3.Erbin has no effect on proliferation and apoptosis of human umbilical vein endothelial cellsCell viability was measured by CCK-8 proliferation kit at different time points after transfection of human umbilical vein endothelial cells with lentivirus.There was no difference between LV control group and LV-Erbin experimental group,confirming that Erbin has no effect on proliferation of human umbilical vein endothelial cells.Endothelial cells of human umbilical vein were transfected with lentivirus for 48 hours,then stimulated with H202(300 μM)for 24 hours to induce apoptosis.After anexcinV/PI double staining,flow cytometry was used to detect endothelial cell apoptosis.There was no significant difference between the LV control group and the LV-Erbin experimental group,confirming that Erbin had no significant effect on the apoptosis of human umbilical vein endothelial cells.4.Erbin promotes migration and tube formation of human umbilical vein endothelial cellsAfter Erbin interference lentivirus was transfected into human umbilical vein endothelial cells to inhibit the formation of endogenous Erbin,cell scratch test and Transwell migration assay were used to detect that the migration ability and tube formation ability of human umbilical vein endothelial cells decreased.Part Ⅱ:Mechanism of Erbin Promoting Migration and Tube Formation of Human Umbilical Vein Endothelial Cells1.Erbin has no significant effect on the phosphorylation level of ERK proteinAfter human umbilical vein endothelial cells were transfected with with lentivirus of Erbin,the corresponding cells were collected,and the cell protein was extracted.Western blot was used to detect the expression of Erbin with GAPDH as an internal reference.The expression of t-ERK and p-ERK was detected by the corresponding non-phosphorylation state as an internal reference,and there was no significant difference between the LV control group and the LV-Erbin group.2.Erbin affects the migration and formation of human umbilical vein endothelial cells mainly by inhibiting Smad1/5 phosphorylation2.1 Erbin has no significant effect on the phosphorylation level of Smad2/3 proteinAfter the endogenous Erbin was was knocked down in human umbilical vein endothelial cells by the lentivirus of Erbin,Western blot was used to detect the protein of t-Smad2,p-Smad2,t-Smad3 and p-Smad3 with corresponding non-phosphorylation status as internal reference.There was no significant change in the expression level.2.2 Erbin inhibits Smad1/5 phosphorylation and nuclear translocation.After using interfere with lentiviral of Erbin knockdown of endogenous Erbin in human umbilical vein endothelial cells,t-Smadl,p-Smad1/5 and t-Smad5 were detected by Western blot with corresponding non-phosphorylation status as internal reference,and it was found that low Erbin knockdown not only promoted the phosphorylation of Smad1/5,but also promoted the nuclear translocation of phosphorylation of Smad1/5.2.3 Cellular immunofluorescence staining found that Erbin inhibits Smad1/5 phosphorylation and nuclear translocation in human umbilical vein endothelial cellsIn Human umbilical vein endothelial cells,the inhibition of endogenous Erbin not only promotes phosphorylation of Smad1/5,but also promotes nuclear translocation of Smad1/5 after transfected with the lentiviral of Erbin.2.4 The inhibition of Smad1/5 pathway eliminates the ability of Erbin knockdown to inhibit migration and tube formation of human umbilical vein endothelial cellsSmadl siRNA,Smad5 siRNA,and both Smad1/5 siRNA were applied to pretreat the cells for 2 hours,and the results demonstrated that simultaneously applied Smadl and Smad5 siRNAs had a more efficient effect on the inhibition of the Smad1/5 signaling pathway.Moreover,the blocking of Smad1/5 pathway significantly eliminated the inhibitory effect of Erbin knockdown on umbilical vein endothelial cell migration and tubular structure formation,which strongly suggested that Erbin mainly promoted endothelial cell migration and tubular structure formation through Smadl/5 pathway.Conclusions:1.Erbin is expressed in human umbilical vein endothelial cells.2.Erbin has no effect on the proliferation and apoptosis of human umbilical vein endothelial cells.3.Erbin promotes the migration and tube formation of human umbilical vein endothelial cells.4.Erbin has no effect on the phosphorylation of ERK and Smad2/3 proteins,and may promote the migration and formation of human umbilical vein endothelial cells by inhibiting Smadl/5 phosphorylation and phosphorylation of Smad1/5 nuclear translocation. |
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