A Study Of The Role And Mechanisms Of Gsα In Endothelial Barrier And Angiogenesis

Dissertation ⅠGsa modulates endothelial cell permeability through regulation of plasmalemma vesicle-associated protein1.IntroductionEndothelial cells line the inner of all blood vessels,maintain vascular homeostasis,and play a key role not only in regulating smooth muscle cells relaxation,thrombosis,vascular wall remodeling and angiogenesis but also in maintaining barrier function of the vasculature.Intact endothelial barrier function is strictly regulated and is the prerequisite for normal substance exchange between blood and tissues.Some pro-inflammation factors which were released in the pathological conditions could induce abnormal endothelial permeability,accelerated pathological progression,and poor prognosis.Abnormal vascular permeability is the feature of many diseases,such as asthma,chronic colitis,cancer,trauma,and cerebral ischemic stroke.Therefore,it is essential to comprehend the pathogenesis of injured endothelial barrier function.Transendothelial substance exchange is regulated strictly.The vascular barrier,hydrostatic and oncotic forces could influence fluid movement across the endothelium.The transportation of small molecules occurs through receptor-mediated endocytosis or fenestrae,which is the special structure in endothelial cells.Macromolecules are transferred through caveolae,vesicular-vacuolar organelle(VVO)and transendothelial channels.Caveolae,transendothelial channel(TEC)and fenestrae possess thin non-membranous diaphragms,which are built of a scaffold of radial fibrils and demonstrate a central density or knob ultrastructurally as shown in transmission electron microscopy sections.PLVAP protein is the first and only known marker of endothelial stomal diaphragms and fenestral diaphragms.Homozygous stop mutation of PLVAP gene in a newborn caused fetal protein-losing enteropathy(PLE).Homozygous disruption of the PLVAP gene in a mixed background led to growth retardation and decreased survival.The organs with fenestrated capillaries in PLVAP knockout mice showed the disappearance of caveolae,transendothelial channels(TEC)and fenestrate diaphragm,causing hypoproteinemia,hypertriglyceridemia and injured endothelial barrier function.The phenotypes of endothelial PLVAP specifically germline deletion mice were similar to global PLVAP knockout mice,which indicated the important role of PLVAP in endothelial barrier function.The alpha subunit of the stimulatory G protein(Gsα)is widely expressed in many types of cells and organs,which could regulate the nerve system,motor system or endocrine system and is strongly linked to inflammation reaction,cancer,and cardiovascular diseases.As for the cardiovascular system,we have demonstrated its role in smooth muscle cells in angiotensin Ⅱ-induced abdominal aortic aneurysm formation,and in endothelial cells in post-ischemic angiogenesis.There are also researches showing that endothelial Gsα could play roles in hypertension and atherosclerosis.We observed its deficiency could impair the endothelial barrier function,which deserves further investigation.In summary,the following hypothesis is proposed in this study,deficiency of Gsα in endothelial cells could impair endothelial permeability.We would use the endothelial Gsa knocked-out mice to perform a series of experiments in vitro and in vivo and explore the possible signaling pathway that induces injured endothelial barrier function.2.Objectives2.1 To explore the role of endothelial Gsa in vascular homeostasis and endothelial barrier function.2.2 To identify the potential molecular mechanism of endothelial Gsa in regulating the expression of PLVAP.3.Methods3.1 Generation of endothelial-specific Gsα knockout miceGsαflox/flox mice were crossed with Cdh5-CreERT2 mice to get the F1 heterozygotes and the F1 were intercrossed to get the Gsαflox/flox/Cre+mice.The mice were intraperitoneally injected with tamoxifen(lmg/d)for consecutive five days to get the endothelial specifically knocked-out mice.The mice were named GsαECKO mice.The littermates were intraperitoneally injected with the same dose of tamoxifen as CTR.3.2 Growth curve and survival curveThe six-old male CTR and GsαECKO mice were weighed and recorded from the day of accepting Tamoxifen injection to 80 days.The six old male CTR and GsαECKO mice were monitored and recorded survival rates from the day of accepting Tamoxifen injection to 150 days.3.3 Cell experimentHuman umbilical vein endothelial cells(HUVEC)were purchased from Sciencell biotechnology company.The 3rd to 6rd generations were used for experiments.HUVEC was infected with adenovirus-expressing Gsa and lentivirus-expressing CREB1,transfected with Gsa or CREB1 siRNA and treated with the cAMP activator(Forskolin)or the PKA inhibitor(H89).Then Western blot and RT-PCR experiments were performed.3.4 Evans blue dye extravasation assayMale CTR and GsαECKO mice were injected intravenously with 20 mg/kg body weight of sterile EB dye.After 30 min,the mice were anesthetized,and photographs were taken.The whole body was perfused with phosphate buffer saline(PBS),and the lung,heart,kidney,liver,and skin were harvested and dried.The EB dye was extracted from the organs with 1 ml formanide overnight at 55℃ and was determined spectrophotometrically at 600 nm.The amount of EB extracted in formamide was calculated against a standard curve of known EB concentrations.3.5 Blood sampling and biochemical analysisThe blood was isolated from CTR and GsαECKO mice and immediately collected in microcontainer tubes.The serum was used to analyze albumin,alanine aminotransferase(ALT),aspartate aminotransferase(AST),blood urea nitrogen(BIUN),creatinine,urine protein,high-density lipoprotein(HDL),low-density lipoprotein(LDL),triglycerides(TG),and total cholesterol(T-CHO)levels.The whole blood was used for routine blood tests.3.6 Histology and immunofluorescent assayThe mouse organs were harvested after euthanasia,fixed in 4%formalin and embedded in paraffin.5 μm thick sections were cut and performed with hematoxylin/eosin(HE)staining and immunofluorescent staining.3.7 Western blot analysisThe protein levels of Gsα,PLVAP,CREB1,P-CREB1,β-actin,and GAPDH were analyzed.3.8 Real-time PCR(RT-PCR)analysisThe mRNA levels of PLVAP,Gsα and β-actin were analyzed.3.9 Chromatin immunoprecipitation(ChIP)assayChIP assay was performed using HUVEC to validate that CREB1 could bind to the promoter of AGGF1.All the procedures followed the combined protocols supported by the kits.3.10 Luciferase reporter assayThe wild-type Luc construct or mutant construct with deletion of the CRE site in the PLVAP promoter was generated and transfected into HEK-293T cell.After 24 h,cells were treated with forskolin and luciferase activity was analyzed using the dual luciferase assay kit.3.11 Statistical analysisData were expressed as Mean ± SEM and analyzed using GraphPad Prism 9.All data have passed the normality and equal variance test.Statistical comparisons of two groups using an unpaired 2-sided student t-test.For comparisons among more than two groups,the One-Way ANOVA and Bonferroni post-tests were used.*p<0.05 was considered statistically significant.4.Results4.1 Endothelial-specific deletion of Gsα in mice caused edema and impaired postnatal survivalImmunofluorescent assay was performed to detect Gsa expression in the aortic endothelium from CTR and GsaECKO mice,which showed that Gsa decreased significantly in the endothelial cells of GsαECKO mice compared with CTR.At the same time,the level of CAMP and Gsα protein in GsαECKO mice lung tissue was decreased compared with CTR.The average body weight of GsαECKO mice increased significantly at 3 weeks after tamoxifen treatment.GsαECKO mice showed edema,expiratory dyspnea,reduced mobility,decreased blood pressure,and pleural effusion.Strikingly,endothelial Gsa deficiency mice succumbed starting 8 weeks after tamoxifen injection.4.2 GsaECKO mice increased microvascular permeabilityCTR and GsαECKO mice were injected intravenously with EB dye.GsαECKO mice exhibited increased EB-albumin extravasation in the ear,paw,subcutaneous gelatin,and perivascular adipose tissue(PVAT).Compared with CTR mice,EB-albumin extravasation also increased in the lung,heart,kidney and liver of GsαECKO mice.Transmission electron microscopy(TEM)results showed that the caveolae diaphragms of lung capillaries and kidney peritubular capillaries were absent in GsαECKO mice.4.3 GsαECKO mice displayed inflammation infiltration in the lungGross examination revealed numerous petechiae on the pleural surface microvessels of GsαECKO mice lung tissue.The HE staining of GsαECKO mice demonstrated inflammatory cells infiltration and interstitial thickening.The immunofluorescent staining of CD68 in lung tissues indicated that macrophage infiltration increased in GsaECKO mice.4.4 Loss of endothelial Gsa impaired plasma protein homeostasis and blood compositionSince GsαECKO mice showed edema,the biochemical indexes were analyzed to explore the cause of edema.The plasma albumin level in GsαECKO mice was significantly lower than that in CTR.Routine blood test revealed that white blood cells(WBC)including lymphocytes and granulocytes were increased in GsαECKO mice.Compared with CTR,GsαECKO mice also exhibited significantly decreased red blood cells(RBC)and hemoglobin(HGB)level.4.5 Endothelial-specific Gsa knock-out mice displayed decreased adipose tissue and increased plasma lipidSerum lipid levels in GsαECKO mice showed increased plasma triglycerides(TG),total cholesterol(T-CHO),low-density lipoprotein(LDL),and high-density lipoprotein(HDL).White adipose tissue(WAT)deposited in the abdominal wall,retroperitoneum,and gonadal fat pads in GsαECKO mice were much less than that in CTR mice.The histological analysis of subcutaneous adipose tissue(SAT)and visceral adipose tissue(VAT)revealed no significant difference in adipocytes size between CTR and GsαECKO mice.4.6 Gsα deficiency reduced PLVAP expression in endothelial cellsThe protein and mRNA levels of PLVAP were markedly decreased in lung tissue of GsαECKO mice compared with CTR.Then,knocking down Gsa or CREB1 with siRNA decreased the protein and mRNA level of PLVAP in HUVEC.Moreover,H89,as a PKA inhibitor,inhibited PLVAP expression in HUVEC.4.7 Gsα regulates PLVAP expression via CREB1-mediated transcriptionGsa overexpression increased the protein and mRNA levels of PLVAP in HUVEC.HUVEC infected with CREB1 expressing-lentivirus displayed increased PLVAP expression.We also used the cAMP activator-forskolin to treat HUVEC and it enhanced the PLVAP levels.ChIP result demonstrated that CREB1 could bind to the CRE site in the PLVAP promoter.The luciferase reporter assay further demonstrated that the CRE site of the PLVAP promoter is required for Gsα-induced PLVAP gene expression.5.Conclusions5.1 Endothelial Gsα specifically deficiency could induce adult mice to develop phenotypes of edema,hypoproteinemia,hyperlipidemia and decreased survival rate.5.2 Gsa in endothelial cells could maintain the existence of diaphragms and regulate the barrier function of endothelial cells.5.3 Endothelial Gsα could regulate the expression through cAMP/PKA/CREB1 signaling pathway.Dissertation ⅡThe endothelial Gsα is a critical regulator of angiogenesis1.IntroductionAngiogenesis is progress that the new vessels sprout from the pre-existing ones.It can be divided into physiological angiogenesis and pathological angiogenesis,which occurs throughout the life process,from the embryo to the elderly period.Angiogenesis plays an important role in embryonic development,wound healing,tumor growth and metastasis,diabetic retinopathy,myocardial infarction and peripheral vascular lesion.During the last 40 years,scientists had a growing understanding of the exact process of angiogenesis.The treatment and drugs aiming at angiogenesis have widespread adoption in clinical therapy.Increased angiogenesis can effectively treat ischemic heart disease,peripheral vascular disease and wound healing.Otherwise,inhibition of angiogenesis is of great importance in diseases such as cancer,ophthalmic diseases,and rheumatoid arthritis.The angiogenetic process is controlled by a balance of various growth factors and inhibitors.AGGF1(Angiogenic factor with G patch and FHA domains 1)is one of the angiogenic factors.AGGF1 is mainly expressed in endothelial cells and could regulate vascular development by an autocrine or paracrine mode.As a pro-angiogenic factor,AGGF1 promoted angiogenesis through activating PI3K/AKT/GSK3β signaling.Although AGGF1 is well known as a regulator of angiogenesis,how its expression is modulated remains poorly understood.Gsa is the a submit of stimulatory G proteins,activating adenylate cyclase(AC)to produce the second messenger-cyclic adenosine monophosphate(cAMP).cAMP could activate protein kinase A(PKA),then it could phosphorylate CREB1 on Ser133 and transcriptionally activate target genes.Gsa plays different and important roles in various cells and organs.The researchers have shown that endothelial cell-specific deletion of Gsa in mice caused early embryonic lethality with massive hemorrhage and a disorganized vasculature.Deficiency of Gsα in endothelial cells could impair the tube formation ability.But the role of Gsa in angiogenic process of adult mice is still unknown.In summary,the following hypothesis is proposed in this study:loss of Gsa in endothelial cells could impair angiogenesis,which may be due to the downregulation of AGGF1.We would use the inducible endothelial Gsa specifically knocked-out mice,by using the hind limb ischemia model,Matrigel migration in vivo and angiogenesis assay in vitro to investigate the role of endothelial Gsa in angiogenesis.2.Objectives2.1 To explore the specific role and mechanism of endothelial Gsα on angiogenesis.2.2 To investigate the role of endothelial Gsa on the regulation of AGGF1.3.Methods3.1 Construct the endothelial Gsαspecific knocked-out mice.Gsαflox/flox mice were crossed with Cdh5-CreERT2 mice to get the P1 heterozygotes and intercrossed to get the Gsαflox/flox/Cre+ mice.The mice were intraperitoneally injected with tamoxifen(lmg/d)to get the endothelial Gsa specifically knocked-out mice.The mice were named as GsαECKO mice.The littermates which were intraperitoneally injected with the same dose of tamoxifen were named as CTR.3.2 Cell cultureHuman umbilical vein endothelial cell(HUVEC)were purchased from Sciencell biotechnology company.The 3rd to 6rd generations were used for experiments.HUVEC was infected with adenovirus-expressing Gsα,transfected with Gsa or CREB1 siRNA and treated with the cAMP activator(Forskolin)or the PKA inhibitor(H89).Then Western blot and RT-PCR experiments were performed.HUVEC was transfected with AGGF1 siRNA and infected with adenovirus-expressing Gsα,then the cells were performed with wound healing assay,angiogenesis assay in vitro and proliferation assay.3.3 Establish the hindlimb ischemia model.8 weeks aged CTR and GsαECKO mice were performed femoral artery ligation,and hindlimb blood flow monitoring was performed before,day 1,day 3,day 7,and day 14 after surgery respectively.3.4 Tumor and matrigel angiogenesis assayMatrigel with or without LLC(Lewis Lung Carcinoma cells)were injected subcutaneously into dorsal region of CTR and GsαECKO mice.the plugs and tumors were collected after 2 weeks.3.5 Western blot and RT-PCR analysisThe protein levels of Gsα,AGGF1,CREB1,P-CREB1,β-actin,CyclinD1and GAPDH were analyzed.The mRNA level of AGGF1,CREB1 and GAPDH were analyzed.3.6 Immunofluorescent staining of tissue sectionThe mice tissue sections were hydrated and incubated with CD31 or Gsa antibody.The pictures were collected by fluorescence microscopy.3.7 The measurement of cAMP contentWe used the direct cAMP ELISA kit to assay the cAMP level of HUVEC.3.8 Statics analysisAll data were presented as Mean ± SEM.The data distribution was assessed for normality and equal variance test.Statistical comparisons between two groups are involved Student t test and otherwise by one-way analysis of variance and Bonferroni post-tests.*p<0.05 was considered statistically significant.4.Results4.1 The deficiency of Gsα in endothelial cells impaired blood recoveryThe immunofluorescent staining of mice aorta tissue showed Gsa expression significantly decreased in endothelium of GsαECKO mice.The CTR and GsαECKO mice were performed femoral ligation surgery and the blood recovery of ischemic foot was monitored by laser doppler perfusion imaging.The result showed that the blood recovery of ischemic foot in GsαECKO mice was significantly impaired compared with that in CTR mice.Besides,the immunofluorescent staining of CD31 decreased in gastrocnemius of GsαECKO mice compared with CTR mice,which indicated the reduced newly vessels in GsαECKO mice.4.2 Decreased neovascularization of matrigel plug and tumor in GsαECKO miceMatrigel was injected subcutaneously into the CTR and GsαECKO mice and collected after two weeks.The initial angiogenesis activity was lower in GsαECKO mice.The LLC was implanted in the CTR and GsαECKO mice and the tumor size of GsαECKO mice were smaller than that of CTR mice.Worsening neovascularization in GsαECKO mice was also reflected by the decreased CD31 immunofluorescence staining of matrigel or tumor tissue section.4.3 The knockout of Gsa in endothelial cells led to decreased AGGFl expressionWestern blot results showed that reduced AGGF1 protein in the lung tissue of GsαECKO mice compared with CTR.The knockout of Gsa and CREB1 in HUVEC induced the decreasing AGGF1 protein and mRNA levels.HUVEC were treated with H89 and western blot results showed decreased AGGF1 expression.4.4 Endothelial AGGF1 could be regulated by CREB1The overexpression of Gsa in HUVEC could increase the AGGF1 protein and mRNA expression.The cAMP agonist—forskolin,could also increase the AGGF1 protein level.We performed ChIP assay and the result demonstrated that the CREB1 could bind to the CRE site of AGGF1 promoter.The dual luciferase reporter gene experiments demonstrated that CREB1 could transcriptionally activate AGGF1 expression.4.5 Knockout of AGGF1 undermined the proangiogenic effect mediated by endothelial GsαEndothelial Gsα could promote angiogenesis through augmentation of endothelial cells proliferation,migration and sprout formation ability.Otherwise,the knockout of AGGF1 attenuated the proangiogenic ability mediated by Gsα.4.6 Adenoviral overexpression of AGGF1 alleviated the impaired angiogenesis in GsαECKO miceThe CTR and GsαECKO mice were injected with adenovirus-expressing LacZ or AGGF1 via tail vein followed by ligation of femoral artery.The result indicated that AGGF1 could improve the blood perfusion recovery of GsαECKO mice.5.Conclusions5.1 Deficiency of Gsa in endothelial cells could attenuate angiogenesis.5.2 Endothelial Gsa regulates AGGF1 expression through transcription factor—CREB1.5.3 AGGF1 treatment could improve the damaged pro-angiogenic ability caused by knockout of Gsa in endothelial cells.