The Role And Mechanism Of Tyrosine Kinase Receptor B In Coronary Artery Disease

BackgroundCoronary artery disease (CAD) is the leading cause of death globally. Endothelial barrier’s dysfunction has been recognized as an initiating factor for the formation of atherosclerotic lesions and associated with all stages of atherosclerosis. Vascular endothelium covers the luminal surface of the blood vasculature and provides a physical barrier that controls the traffic of plasma proteins and circulating cells across the blood vessel. Endothelial barrier dysfunction leads to lipoprotein leakage and monocyte extravasation into the vessel walls, thereby accelerating atherosclerosis and inducing atherosclerotic plaque rupture.The tyrosine kinase receptor B (TrkB) is a high-affinity receptor for brain-derived neurotrophic factor (BDNF). The BDNF-TrkB pathway plays critical roles in the survival, growth, maintenance, and death of central and peripheral neurons. In addition to its nervous system functions, TrkB is also expressed in the cardiovascular system. It has been reported that TrkB is involved in cell adhesion. TrkB-expressing tool cells displayed varying degrees of aggregation depending on the mount of TrkB protein expressed. The observed adhesion was Ca21-, Mg21-, and temperature-dependent, characteristics shared by the cadherin family of adhesion molecules. Cell adhesions also play important roles in cardiovascular development. The BDNF-TrkB axis plays critical roles in cardiovascular development through promoting endothelial survival. Mice with a disrupted TrkB gene lack a significant proportion of intramyocardial blood vessels and showed early postnatal death; BDNF deficiency results in endothelial cell apoptosis, intraventricular wall hemorrhage, depressed cardiac contractility, and early postnatal death in mice; BDNF-TrkB pathway overexpression in developing mouse hearts resulted in increased cardiac capillary density. However, the role of BDNF-TrkB axis in development of CAD remains unknown. The present study investigates the association of TrkB and CAD, the roles of TrkB in aortas and its mechanism. We aimed to find a novel pathological mechanism of CAD and provide novel ideal and target for diagnosis and treatment of CAD.Objectives:1. To investigate the association of TrkB polymophisms and CAD. Aim to find whether TrkB is a novel susceptibility gene of CAD.2. To investigate the effects of TrkB on endothelial permeability. Aim to find the roles of TrkB in regulating endothelial permeability and its mechanism.3. To investigate whether TrkB signal protected endothelial barrier integrity against endothelial hyperpermeability induced by proatherosclerotic factors. Aim to find a novel target of protecting endothelium against endothelial injury.Methods1. Patients and Controls We recruited two independent cohorts including patients with CAD and controls from different geographic areas. We collected the clinical data and blood of all subjects.2. Genotype AnalysisGenomic DNA was isolated from whole blood using standard procedures. All of the genotyping involved real-time PCR with Taqman probes.3. Reporter Gene AssaysWe constructed pGL3-TrkB-69C and pGL3-TrkB-69G vectors using gene cloning and site-directed mutagenesis techniques. The reporter constructs were transfected in HeLa cells and ECs with the Lipofectamine 2000. The cells were harvested and analyzed on a fluorescence plate reader.4. Analysis of TrkB expression in human and mouse atherosclerotic plaquesFrozen sections of human and mouse aortas with atherosclerotic plaques were prepared. The protein expression of TrkB in the aorta was detected using conventional immunofluorescence with anti-TrkB antibody.5. Adeno-associated virus serotype-9 (AAV9) constructionThree pairs of chemically synthesized small interfering RNA were introduced into endothelial cells, respectively. Then screen out the most efficient sequences, which will use for preparation of TrkB knockdown virus. Using conventional cloning techniques to prepare TrkB-overexpression plasmid, then TrkB-overexpression vector was modified by site-directed mutagenesis to resistant the role of the shRNA. Finally, all AAV9 were generated at Shenzhen Baienwei Company, China.6. In vivo endothelial barrier assaysIn vivo endothelial barrier function was measured using Evans blue assay. The mice were injected via the tail vein with Evans blue. After 45 minutes, the mice were perfused through the left ventricle with PBS, and the aorta was excised for analysis of the vessel wall leakage of dye.7. Cell CultureHuman aortic endothelial cells were cultured in endothelial cell medium. HeLa cells and 293T cells were grown in DMEM supplemented with 10% fetal bovine serum. All of the cells were cultured at 37℃ with 5% CO2. All assays were performed in triplicate.8. Paracellular Permeability in vitroUsing transwell chamber to detecte how many FITC-labeled dextran were entered through endothelial monolayer into the lower chamber. The fluorescent content of the samples was measured with a fluorescence plate reader at an excitation of 485 nm and an emission of 530 nm.9. Transendothelial migrationUsing transwell chamber to detecte how many T cells were entered through endothelial monolayer into the lower chamber. The results are expressed as the percentage of transmigrated cells.10. Lentiviral constructionUsing conventional cloning techniques we cloned the target genes into a pCCL.PGK.TetLinker. WPRE vector. The plasmid was mixed with ENV plasmid (VSV-G), packaging plasmid (pMDLg/pRRE), and pRSV-REV and transfected into 293T cells with the Lipofectamine 2000.11. Real-Time PCRSYBR green real-time PCR and quantitative assays used the Light Cycler 2.0 (Roche Diagnostics, Mannheim, Germany). The relative expression of genes was obtained by 2-△△Ct calculation.12. Western Blot AnalysisThe proteins were separated using 10% SDS-PAGE, transferred to polyvinylidene difluoride membranes, and then incubated with antibodies for anti-TrkB, anti-VE-cadherin, or anti-tubulin, followed by secondary antibody incubation. The bands were detected with AlphaChem 9900, and quantified with AlphaChem software.13. ChIP assayChromatin was purified from the ECs, and then immunoprecipitated using anti-Etsl antibody and IgG was used as control. VE-cadherin promoter was amplified by real-time PCR, and its level was plotted relative to the input, and GAPDH was used as negative control. These experiments were performed in triplicate.14. ImmunofluorescenceCryosections were fixed in 4% paraformaldehyde for 10 minute. After incubation with the appropriate antibodies antibodies and DAPI, the sections were observed using a fluorescence microscope.15. Statistical analysisAll of the statistical tests were performed with SPSS 15.0 software (SPSS, Chicago, IL).Results1. TrkB-69C＞G Polymorphism Is Associated With CAD.We compared CAD and nonCAD subjects for the genotype and allele frequency of TrkB polymorphisms -69C>G and IVS13+40G>A in 2 independent cohort from different geographic areas. The frequency of the TrkB -69C>G genotype and allele significantly differed between patients with CAD and controls in the 2 independent cohort.Multiple logistic regression analysis revealed homozygous TrkB -69C, rather than TrkB -69G carriers, were associated with an increased risk of CAD in the Shandong group (odds ratio,2.1; 95% confidence interval, 1.68-2.62; P＜0.01) and in the Shanxi group (odds ratio,2.0; 95% confidence interval,1.69-2.67; P＜0.01), after adjusting sex, age, and body mass index.There was no association between the TrkB IVS13+40G>A polymorphism and CAD.2. TrkB-69C Homozygotes, Which Corresponded to Decreased TrkB Expression, Showed Increased Risk for CAD.We tested the effect of TrkB-69C>G polymorphism on luciferase reporter gene expression. Regardless of genotype, the promoter significantly promoted luciferase expression.Importantly, luciferase activity was significantly lower with the -69C construct than with the -69G construct in HeLa cells and human vascular endothelial cells. These data suggested that decreased TrkB expression might be associated with increased risk for CAD.3. TrkB is Expressed in Aortic ECs of Atherosclerotic Lesions in Humans and ApoE-/-Mice.Immunofluorescence assy showed that TrkB was prominently expressed in aortic ECs in normal C57BL/6 mice.In ApoE-/-mice TrkB was also prominently expressed in aortic ECs in early atherosclerotic lesions.In early atherosclerosis lesions of human, immunofluorescence staining showed that TrkB expression was prominent in ECs.4. TrkB Prevented EC Barrier Leakage in ApoE-/-Mice.ApoE-/-mice were systemically infected with adeno-associated virus serotype-9 via the tail vein, and then the highly efficient expression of reporter gene-Zsgreen was observed in the aortic ECs of ApoE-/- mice.The introduction of AAV9-shTrkB to ApoE-/-mice led to a 93% decrease in TrkB expression in endothelium, which was blocked by overexpressing AAV9-TrkB.Evans blue assay demonstrated that the knockdown of TrkB expression in aortic ECs resulted in a 30% increase in Evans blue deposition in the aorta, and these effects were rescued by AAV9-TrkB in ApoE-/-mice.Immunofluorescence staining and real-time polymerase chain reaction revealed that VE-cadherin protein and mRNA expression were decreased in aortic endothelium in response to AAV9-shTrkB, and the effects were rescued by AAV9-TrkB.5. TrkB Protected Endothelial Barrier Integrity in VE-Cadherin-Dependent Manner.TrkB knockdown significantly increased fluorescein isothiocyanate-dextran diffusion, VE-cadherin gap formation, and migration of T cells across a monolayer of endothelial cells compared with controls.TrkB knockdown also resulted in significantly decreased mRNA and protein expression of VE-cadherin, and overexpression of TrkB rescued the effects.Importantly, the increases in fluorescein isothiocyanate-dextran diffusion, gaps and migration of T cells induced by depleting TrkB were all prevented by overexpressing VE-cadherin.6. TrkB Promoted Expression of VECadherin through Induction and Activation of Etsl Transcriptional Factor.We investigated effects of TrkB signaling on the expression of Ets1 transcriptional factor. TrkB knockdown led to significantly decreased expression of Ets1 in ECs, whereas BDNF caused rapid phosphorylation of TrkB and increased expression of Ets1.Next, we examined whether Ets1 would be necessary for the BDNF-mediated upregulation of VE-cadherin. The Ets1 siRNA effectively blocked the BDNF-stimulated expression of VE-cadherin.Next, BDNF treatment not only increased Ets1 expression but also stimulated the nuclear localization of Ets1 proteins, whereas TrkB knockdown led to decreased Ets1 expression and nuclear localization.Etsl promoted expression of VE-cadherin by binding to the 2 Ets-binding sites in the VE-cadherin gene promoter in endothelial cells.Our data showed that BDNF promoted the Ets1-binding activity.7. Proatherosclerotic Factor-Induced Endothelial Hyperpermeability Was Attenuated by TrkB Activation.We found that TrkB activation by BDNF prevented the tumor necrosis factor α-induced increase in fluorescein isothiocyanate-dextran diffusion, T-cell transendothelial migration, and gap formation.Moreover, TrkB activation blocked the reduced mRNA and protein expression of VE-cadherin induced by tumor necrosis factor a in ECs, and the effects was abrogated by Ets1 siRNA.The protective effects of TrkB activation in tumor necrosis factor α-induced endothelial hyperpermeability were all abrogated by VE-cadherin or Ets1 knockdown.Conclusions1. TrkB-69C>G polymorphism is associated with CAD. TrkB-69C homozygotes, which corresponded to decreased TrkB expression, showed increased risk for CAD. So TrkB may be a novel susceptibility gene of CAD and play a protective role in the pathogenesis of CAD.2. TrkB is prominently expressed in the aortic ECs in early atherosclerotic lesions.3. TrkB protects endothelial barrier integrity by promoting Etsl-mediated synthesis of VE-cadherin.4. TrkB activation protects against proatherosclerotic factor-induced endothelial hyperpermeability.BackgroundAtherosclerosis is the pathological basis of cardiovascular diseases, such as coronary heart disease (CAD). Endothelial barrier dysfunction has been recognized as an initiating factor for the formation of atherosclerotic lesions. Endothelial barrier dysfunction leads to lipoprotein leakage and monocyte extravasation into the vessel walls, thereby accelerating atherosclerosis.Tyrosine kinase receptor B (TrkB) is a high-affinity receptor for brain-derived neurotrophic factor (BDNF). The BDNF-TrkB pathway plays critical roles in the survival, growth, and maintenance of central and peripheral neurons. In addition to its nervous system functions, TrkB also functions in the cardiovascular system. The BDNF-TrkB pathway has been reported to protect the myocardium against ischemic injury. The pathway also plays important roles in the survival and maintenance of vascular endothelial cells. BDNF deficiency results in endothelial cell apoptosis, intraventricular wall hemorrhage, depressed cardiac contractility and early postnatal death in mice. Mice with a disrupted TrkB gene lack a significant proportion of intramyocardial blood vessels and showed early postnatal death. We recently found that TrkB was prominently expressed in the endothelium of atherosclerotic lesions and maintained endothelial barrier integrity by promoting VE-cadherin synthesis in ApoE-/-mice. In agreement with our results, Matsuda et al also found that the single addition of BDNF into a culture of human microvascular endothelial cells increased the expression of VE-cadherin. VE-cadherin performs an important role in protecting endothelial barrier integrity against atherosclerosis. However, the association between endothelial TrkB and atherosclerosis is not completely clear.This study investigated whether endothelial damage affected the expression of endothelial TrkB, leading to accelerate the development of atherosclerosis. We aimed to demonstrate the roles of TrkB in the pathological process of AS, provide a novel idea for the prevetion and therapy of atherosclerosis.Objectives:1. To investigate the expression of endothelial TrkB in the atheroma of ApoE-/-mice compared with the atheroma-free aorta of WT mice.2. To investigate the effects of TrkB knockdown on development of atherosclerosis in ApoE-/-mice.3. To investigate the effects of TrkB on the cleavage of VE-cadherin and its mechanism.Methods1. Animal study protocolApoE-/-mice received a single systemic administration of adeno-associated virus serotype-9 (AAV9) with or without shRNA specifically targeting TrkB via the tail vein. To induce early atherosclerotic lesion development, the ApoE-/- mice were fed a high-cholesterol diet for 8 weeks. Blood and aortas were collected for next analyses.2. Oil Red O stainingThe aortic trees and cross-sections of the aortic sinuses were fixed with 4% paraformaldehyde. After washing with PBS, the samples were stained with Oil Red O.3. Immunofluorescence analysisThe cryosections of the aortic sinus were fixed with 4% paraformaldehyde. After washing with PBS and incubating with primary and secondary antibodies and DAPI, the sections were observed using fluorescence microscopy.4. WB analysisThe preparation of the cell lysates, measurement of the protein concentrations, sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), electrophoretic transfer, sequential incubation with primary and second antibodies and film development were performed by conventional methods.5. Real-Time PCRTotal RNA was isolated from the aortas of the mice. First cDNA was synthesized using AMV reverse transcriptase. SYBR green real-time PCR and quantitative assays used the Light Cycler 2.0 (Roche Diagnostics, Mannheim, Germany). The relative expression of the genes was obtained through the 2-AACt calculation.6. Cell CultureHuman aortic endothelial cells were cultured in endothelial cell medium. All cells were cultured at 37℃ with 5% CO2.7. Statistical analysisAll statistical tests were 2-tailed with P＜0.05 set as the significance level, and they were performed using the SPSS 15.0 software (SPSS, Chicago, IL).Results1. TrkB expression in the endothelium is down regulated in atherosclerotic lesions of ApoE-/-mice compared with the atheroma-free aorta of WT mice.The relative fluorescent intensity analysis demonstrated that the TrkB expression observed in the endothelium was decreased by 35% in the atheroma of ApoE-/- mice compared with the atheroma-free aorta of WT mice.Western blot analysis demonstrated that TrkB expressions in HAECs were significantly inhibited after TNF-a or ox-LDL administration.2. TrkB confers atheroprotection in apoE-/- miceApoE-/- mice were systemically infected with Adeno-associated virus serotype-9 (AAV9) carrying a Zsgreen reporter gene (AAV9-control) or AAV9 carrying small hairpin RNA-TrkB (AAV9-shTrkB) via the tail vein and fed with an atherogenic diet for 8 weeks. After systemic infection, highly efficient expression of the reporter gene Zsgreen was observed in the endothelial layer of atherosclerotic lesions in ApoE-/- mice.Western blot analysis revealed a significant 91% reduction of TrkB expression in the aorta of the ApoE-/- mice infected with AAV9-shTrkB compared with the control mice with AAV9-Control infection.The introduction of AAV9-shTrkB into apoE-/- mice increased the lesion area in aortic trees by 40% compared with the introduction of AAV9-control. Similar results were also found in the intimal area of aortic sinus cross-sections from the mice.3. TrkB knockdown leads to increased lipid deposition, macrophage infiltration and inflammatory responses in the atherosclerotic lesions of ApoE-/- miceLipid deposition in the lesion, as demonstrated by the Oil Red O-positive region, was significantly increased by 35% in ApoE-/- mice infected with AAV9-shTrkB.The infiltration of macrophages into the vascular wall, as assessed by immunofluorescence with MOMA-2 antibody, was significantly increased by 40% with the AAV9-shTrkB.Real-time PCR showed that the mRNA levels of the proinflammatory markers, including nuclear factor-KB, intercellular adhesion molecule-1, vascular cell adhesion molecule-1, E-selectin, tumor necrosis factor-a, and interleukin-6, in the aortas of ApoE-/- mice were significantly increased in the TrkB knockdown group compared with those in control group.4. BDNF prevented TNF-a-induced-shedding of VE-cadherin by reducing its tyrosine phosphorylation and enhancing the binding of VE-cadherin and VE-PTP in HAECsBy comparing the mRNA and protein levels of VE-cadherin after TrkB knockdown, we found that VE-cadherin protein was decreased by 79%, whereas the VE-cadherin mRNA was only decreased by 43%. These data suggested that TrkB might be involved in both the VE-cadherin synthesis and cleavage. Our data revealed that the shedding fragments of VE-cadherin were significantly reduced in the medium of TNF-a-stimulated HAECs by preincubation with BDNF, and the effects were blocked by TrkB siRNA.BDNF reduced the tyrosine phosphorylation levels of VE-cadherin induced by TNF-a, and the effects were blocked by TrkB siRNA.Then, we evaluated whether BDNF promoted the binding of VE-PTP and VE-cadherin. Our data revealed that the levels of the immunoprecipitated VE-PTP were significantly higher in HAECs treated with BDNF, and the effects were blocked by TrkB siRNA.The aortas of ApoE-/- mice with TrkB knockdown also displayed increased tyrosine phosphorylation of VE-cadherin and reduced binding of VE-PTP and VE-cadherin compared with control mice.Conclusions1. TrkB is an endothelial injury-response molecule during atherogenesis, its expression in the endothelium to be downregulated in atherosclerotic lesions of ApoE-/- mice compared with the atheroma-free aorta of WT mice.2. Endothelial TrkB affects both the synthesis and shedding of VE-cadherin and protects against early atherosclerotic lesion development in ApoE-/-mice.3. We further elucidate the mechanism by which TrkB preventing VE-cadherin shedding. Our data for the first time demonstrated that BDNF/TrkB signaling involved in the tyrosine phosphorylation of VE-cadherin and the interaction between VE-PTP and VE-cadherin.