Basic Sciences

Tyrosine Kinase Receptor B Protects Against Coronary Artery Disease and Promotes Adult Vasculature Integrity by Regulating Ets1-Mediated VE-Cadherin ExpressionSignificance

Hong Jiang, Shuhong Huang, Xinyun Li, Xian Li, Yun Zhang, Zhe-Yu Chen
https://doi.org/10.1161/ATVBAHA.114.304405
Arteriosclerosis, Thrombosis, and Vascular Biology. 2015;35:580-588
Originally published January 29, 2015

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Abstract

Objective—Tyrosine kinase receptor B (TrkB) is a high-affinity receptor for brain-derived neurotrophic factor. In addition to its nervous system functions, TrkB is also expressed in the cardiovascular system. However, the association of TrkB and coronary artery disease (CAD) remains unknown. We investigated the role of TrkB in the development of CAD and its mechanism.

Approach and Results—We performed a case–control study in 2 independent cohort of Chinese subjects and found –69C>G polymorphisms of TrkB gene significantly associated with CAD. TrkB –69C homozygotes, which corresponded to decreased TrkB expression by luciferase reporter assay, showed increased risk for CAD. Immunofluorescence analysis revealed that TrkB was expressed in the aortic endothelium in atherosclerotic lesions in humans and ApoE–/– mice. TrkB knockdown in the aortic endothelium resulted in vascular leakage in ApoE–/– mice. Mechanistic studies showed that TrkB regulated vascular endothelial cadherin (VE-cadherin) expression through induction and activation of Ets1 transcriptional factor. Importantly, TrkB activation attenuated proatherosclerotic factors induced-endothelial hyperpermeability in human vascular endothelial cells.

Conclusions—Our data demonstrate that TrkB protects endothelial integrity during atherogenesis by promoting Ets1-mediated VE-cadherin expression and plays a previously unknown protective role in the development of CAD.

Introduction

Coronary artery disease (CAD) is the leading cause of death globally. Endothelial 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 atherosclerosis1,2 and inducing atherosclerotic plaque rupture.3

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. The BDNF-TrkB axis has been reported to protect the myocardium against ischemic injury in conditional knockout mice model.4 The BDNF-TrkB axis also 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;5 BDNF deficiency results in endothelial cell apoptosis, intraventricular wall hemorrhage, depressed cardiac contractility, and early postnatal death in mice;6 BDNF-TrkB pathway overexpression in developing mouse hearts resulted in increased cardiac capillary density.6 However, the role of BDNF-TrkB axis in development of CAD remains unknown.

In this study, we first examined the association between CAD and TrkB polymorphism –69C>G or IVS13+40G>A. Then we investigated its mechanism using ApoE–/– mice and human vascular endothelial cells.

Materials and Methods

Materials and Methods are available in the online-only Data Supplement.

Results

TrkB –69C>G Polymorphism Is Associated With CAD and TrkB –69C Homozygotes, Which Corresponded to Decreased TrkB Expression, Showed Increased Risk for CAD

To investigate the association between TrkB and CAD, we compared CAD and nonCAD subjects for the genotype and allele frequency of TrkB polymorphisms –69C>G and IVS13+40G>A, which have been associated with eating disorders by screening the entire TrkB gene,7 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 Shandong group (all P<0.05; Table). This difference was also observed in the Shanxi group (all P<0.05; Table). 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 (P>0.05; Table). All genotype frequencies showed Hardy–Weinberg equilibrium. Statistical powers are all 100% in Shandong and Shanxi group. The clinical characteristics of the 2 cohorts of patients and controls are shown in Table I in the online-only Data Supplement.

View this table:
Table.

Frequency of TrkB –69C/G and IVS13+40G>A Polymorphisms in Patients with CAD and Controls

Polymorphism –69C>G is located in the promoter region of TrkB. It has been reported that approximately one third of promoter variants may alter gene expression to a functionally relevant extent.8 We then constructed luciferase reporter vectors with TrkB promoter segments (–3258 to –11 bp) of contrasting genotypes (–69CC versus –69GG; Figure 1A and 1B) and tested the effect of TrkB –69C>G polymorphism on luciferase reporter gene expression. Regardless of genotype, the promoter significantly promoted luciferase expression (Figure 1C and 1D). Of note, luciferase activity was significantly lower with the –69C construct than with the –69G construct in HeLa cells and human vascular endothelial cells (ECs; Figure 1C and 1D). These data suggested that decreased TrkB expression might be associated with increased risk for CAD.

Figure 1.
Figure 1.

Effects of tyrosine kinase receptor B (TrkB) –69C>G polymorphism on TrkB promoter activity. A, TrkB promoter was cloned into a pGL3 basic vector. Site-directed mutagenesis was used to produce the change of C or G at –69. B, Arrows indicate the change with C or G. C and D, HeLa cells and human vascular endothelial cells (ECs) were transfected with pGL3 basic, pGL3-promoterTrkB-AlleleC, or pGL3-promoterTrkB-AlleleG for 48 h and analyzed for luciferase activity. Data are means±SEM from 3 samples of duplicate determinations in 3 separate experiments. *P<0.05.

TrkB is Expressed in Aortic ECs of Atherosclerotic Lesions in Humans and ApoE–/– Mice

We then investigated the expression of TrkB in aorta. TrkB was prominently expressed in aortic ECs in normal C57BL/6 mice (Figure 2).We next examined TrkB expression in early atherosclerotic lesions of ApoE–/– mice and observed similar results, TrkB was prominently expressed in aortic ECs in early atherosclerotic lesions (Figure 2). Moreover, the expression of TrkB in endothelium is seemed to be reduced in atherosclerotic lesions compared with that in normal endothelium, and its underlining mechanism requires further investigation. We further investigated TrkB expression in the aortas of 10 patients with atherosclerotic lesions. In early atherosclerosis lesions, immunofluorescence staining showed that TrkB expression was prominent in ECs (Figure 2). In consistent with Kraemer et al,9 we found the expression of TrkB in smooth muscle cells in advanced atheroma. In addition to smooth muscle cells, we also found TrkB expression in ECs in advanced atheroma (Figure I in the online-only Data Supplement).

Figure 2.
Figure 2.

Tyrosine kinase receptor B (TrkB) expression on early atherosclerotic lesions in human and ApoE–/– mice aorta. Image of double-immunofluorescence staining for TrkB and CD31 in aorta of normal CA57BL/6 mice (top). Aortic root sections of ApoE–/– mice fed a high-cholesterol diet for 8 wk were performed with immunofluorescence staining for TrkB and CD31 (middle). Image of double-immunofluorescence staining for TrkB and vascular endothelial-cadherin (endothelial cell marker) in early atherosclerotic lesions of human (bottom). Insets are control sections. EC indicates human vascular endothelial cells.

TrkB Prevented EC Barrier Leakage in ApoE–/– Mice

We then investigated the effects of TrkB on the endothelial barrier function in vivo. ApoE–/– mice were systemically infected with adeno-associated virus serotype-9 carrying a Zsgreen reporter gene (AAV9-control), AAV9 carrying small hairpin RNA-TrkB (AAV9-shTrkB), or AAV9-shTrkB plus AAV9 carrying the shRNA-resistant TrkB (AAV9-TrkB) via the tail vein, followed by chow feeding for 8 weeks. After systemic infection of AAV9 virus, which can integrate into genomic DNA and long-term express in the transduced cells, highly efficient expression of reporter gene-Zsgreen was observed in the aortic ECs of ApoE–/– mice (Figure 3A). 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 (Figure 3B–3D). We then assessed the endothelial barrier function using Evans blue assay. In ApoE–/– mice, 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 (Figure 3E). We then examined effects of TrkB knockdown on vascular endothelial cadherin (VE-cadherin) expression. 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 (Figure 3F and 3G).

Figure 3.
Figure 3.

Tyrosine kinase receptor B (TrkB) knockdown in endothelium. ApoE–/– mice were administered with adenoassociated virus serotype-9 (AAV9)-Con, AAV9-shTrkB, or AAV9-shTrkB plus AAV9-TrkB via the tail vein, followed by chow diet feeding for 8 wk. A, The transduction of AAV9 in the aortic wall was determined by immunofluorescence staining for the reporter gene Zsgreen in the aortas of the mice. Bar represents 50 μm. B, Efficacy of small hairpin RNA TrkB (shTrkB) and shRNA-resistant TrkB (srTrkB). A total of 293 cells were cotransfected with the indicated vectors. After 48 h, the cells were collected for testing expression of TrkB using Western blot assay. C, The aortic sections of the ApoE–/– mice with indicated AAV9 were used to test TrkB expression with immunofluorescence analysis. Bar represents 50 μm. D, The total fluorescent intensity of TrkB in the endothelium and the vascular area of aorta were measured with ImagePro-Plus (n=20 sections per sample, ≥3 sites of analysis per slide). The relative fluorescent intensity of TrkB in the endothelium of aorta was calculated by total fluorescent intensity/vascular area ratio from the mice with AAV9-Con (normalized to 1; n=5), AAV9-shTrkB (n=5), or AAV9-shTrkB plus AAV9-TrkB (n=5). E, Effect of TrkB knockdown on endothelial leakage of aorta was determined by Evans blue assay (n=5 per group). *P<0.05. F, Effect of TrkB knockdown on protein expression of VE-cadherin was determined by immunofluorescence staining in aorta of the mice. Bar represents 50 μm. G, Effect of TrkB knockdown on VE-cadherin mRNA level was determined by real-time polymerase chain reaction analysis in aorta of the mice, normalized by GAPDH. *P<0.05. NC indicates no AAV9.

TrkB Protected Endothelial Barrier Integrity in VE-Cadherin–Dependent Manner

Then, we investigated whether TrkB protected endothelial barrier integrity dependent on VE-cadherin in human aortic endothelial cells. The TrkB siRNA efficiently abrogated the expression of the TrkB (Figure II in the online-only Data Supplement). Knockdown of TrkB expression significantly increased fluorescein isothiocyanate-dextran diffusion compared with controls (Figure 4A). VE-cadherin gap was also significantly increased after TrkB knockdown (Figure 4B). Transendothelial migration of leukocytes is a critical event for inflammation, and VE-cadherin gap formation is required for leukocyte transmigration.10 Therefore, we investigated whether TrkB knockdown would enhance the migration of T cells across a monolayer of endothelial cells in a transwell system. Our data showed that TrkB knockdown resulted in the increased transendothelial migration of human T cells (Figure 4C). TrkB knockdown also resulted in significantly decreased mRNA and protein expression of VE-cadherin, and overexpression of TrkB rescued the effects (Figure 4D and 4E). 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 using lentivirus (P<0.05; Figure 4A–4C). We also examined the all effects in human umbilical vein endothelial cells and observed similar results (data not shown). The BDNF-TrkB axis promotes endothelial survival and impairs apoptosis, and then we investigate effects of knockdown or overexpression of TrkB on the proliferation of endothelial cells. We performed the Edu experiments and found that the knockdown or overexpression of TrkB did not significantly affect the endothelial cell proliferation in our study (Figure III in the online-only Data Supplement).

Figure 4.
Figure 4.

Tyrosine kinase receptor B (TrkB) protects endothelial barrier integrity in VE-cadherin–dependent manner in human vascular endothelial cells (ECs). Human aortic endothelial cells were treated with the indicated constructs. A, The permeability of fluorescein isothiocyanate-dextran (40 kDa) in a Transwell system was measured at different times *P<0.05 TrkB siRNA vs control siRNA-treated cells. #P<0.05 TrkB siRNA vs TrkB siRNA plus lentivirus (LV)–VE-cadherin-treated cells. B, Immunofluorescence of VE-cadherin in ECs. A total of 20 random sites per samples were analyzed to determine the gap area. Nuclei were counterstained with DAPI (4’,6-diamidino-2-phenylindole). Yellow stars show gaps. Bar represents 40 μm (C) ECs were incubated with 5×105 T cells (phytohaemagglutinin blasts) per filter. T-cell transendothelial migration was measured by counting cells in the lower chamber after 24 h. D and E, Real-time polymerase chain reaction and Western blot analysis of the mRNA and protein levels, respectively, of VE-cadherin. All experiments were performed in triplicate. *P<0.05.

TrkB Promoted Expression of VE-Cadherin Through Induction and Activation of Ets1 Transcriptional Factor

It has been found that Ets1 transcriptional factor was essential for the expression of VE-cadherin in endothelial cells.11 Moreover, Ets1 is the most abundant Ets factors in ECs.12 We next investigated effects of TrkB signaling on the expression of Ets1 transcriptional factor. TrkB knockdown also led to significantly decreased expression of Ets1 in ECs (Figure 5A), whereas BDNF caused rapid phosphorylation of TrkB (Figure 5B and 5C) and increased expression of Ets1 and VE-cadherin at different time periods after the addition of BDNF (Figure 5D). Next, we examined whether Ets1 would be necessary for the BDNF-mediated upregulation of VE-cadherin by Ets1 knockdown. The Ets1 siRNA efficiently abrogated the expression of the Ets1 (Figure IV in the online-only Data Supplement). The Ets1 siRNA effectively blocked the BDNF-stimulated expression of VE-cadherin (Figure 5E). Therefore, Ets1 is indispensable for the upregulation of VE-cadherin by BDNF. Next, analysis with anti-Ets1 immunofluorescence technique showed that 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 (Figure 5F). Ets1 promoted expression of VE-cadherin by binding to the 2 Ets-binding sites in the VE-cadherin gene promoter in endothelial cells.11 Then, we investigated whether BDNF promoted the Ets1-binding activity. We analyzed nuclear extracts of ECs stimulated with or without BDNF pulled-down using anti-Ets1 antibody. As shown in Figure 5G, BDNF induced a significantly increased level of the immunoprecipitated VE-cadherin promoter.

Figure 5.
Figure 5.

Ets-1 was involved in tyrosine kinase receptor B (TrkB)-regulated expression of VE-cadherin. The human aortic endothelial cells were transfected with the indicated siRNA for 12 h, followed by addition of brain-derived neurotrophic factor (BDNF) (50 ng/mL). A, Western blot analysis of the Ets1 protein levels by TrkB knockdown. B and C, Western blot analysis of the phosphorylated TrkB levels after addition of BDNF with different time or dose. Lysates were immunoprecipitated with TrkB antibody and the immunoprecipitates were then immunoblotted using the anti-Phospho-Tyr antibody. D, Western blot analysis of the Ets1 and VE-cadherin protein levels after addition of BDNF. E, Effects of Ets1 knockdown on BDNF-induced VE-cadherin. F, Immunofluorescence analysis with Ets1 antibody for nuclear translocation of Ets1 after incubation of the cells with BDNF and blocking the pathway by TrkB knockdown. Bar represents 100 μm. G, ChIP assay for Ets1-binding activation. Chromatin was prepared from the cells, and Ets1-DNA complex was pulled-down using anti-Ets1 antibody. Subsequently, the VE-cadherin promoter was amplified by real-time polymerase chain reaction. All experiments were performed in triplicate. *P<0.05.

Proatherosclerotic Factor-Induced Endothelial Hyperpermeability was Attenuated by TrkB Activation

We then investigated whether TrkB signal protected endothelial barrier integrity against endothelial hyperpermeability induced by proatherosclerotic factors, tumor necrosis factor α or oxidized low-density lipoprotein. We found that TrkB activation by BDNF prevented the tumor necrosis factor α-induced increase in fluorescein isothiocyanate-dextran diffusion (Figure 6A), T-cell transendothelial migration (Figure 6B), and gap formation (Figure 6C and 6D). Moreover, TrkB activation blocked the reduced mRNA and protein expression of VE-cadherin induced by tumor necrosis factor α in ECs, and the effects was abrogated by Ets1 siRNA (Figure 6E and 6F). Furthermore, the protective effects of TrkB activation in tumor necrosis factor α-induced endothelial hyperpermeability were all abrogated by VE-cadherin knockdown (Figure 6A–6D). We then examined the effects of TrkB signal on oxidized low-density lipoprotein–induced endothelial hyperpermeability and observed similar results (data not shown).

Figure 6.
Figure 6.

TrkB protects against endothelial hyperpermeability induced by tumor necrosis factor α (TNF-α). The human aortic endothelial cells were transfected with the indicated siRNA for 12 h, followed by addition of brain-derived neurotrophic factor (BDNF) (50 ng/mL) for 12 h, and then the cells were stimulated with TNF-α (50 ng/mL) for 8 h. A and B, Permeability and T-cell transendothelial migration assay in HAECs cultured on collagen-coated transwell filters, and then incubated with fluorescein isothiocyanate-dextran (A) or T cells (B). *P<0.001 vs control cells. &P<0.001 vs cells treated with TNF-α. #P<0.001 vs cells treated with TNF-α plus BDNF. C and D, Gap assay by immunofluorescence staining of VE-cadherin. A total of 20 random sites per samples were analyzed to determine the gap area. Nuclei were counterstained with DAPI. Bar represents 40 μm. E, Real-time polymerase chain reaction and (F) Western blot analysis of the VE-cadherin mRNA and protein level in human vascular endothelial cells treated as indicated. All experiments were performed in triplicate. *P<0.05.

Discussion

CAD is a complex disorder. Although conventional risk factors are important, both rare and common genetic variants account for >50% of susceptibility to CAD. However, identifying the genomic loci associated with increased CAD susceptibility has been a challenge. In this study, our data showed –69C>G polymorphisms of TrkB gene significantly associated with CAD by a case–control study in 2 independent cohort of Chinese subjects, indicating TrkB may be a novel candidate gene for CAD. We also found that –69C>G is a novel functional polymorphism of TrkB. TrkB –69C homozygotes, which corresponded to decreased TrkB expression, showed increased risk for CAD, suggesting that TrkB have a protective role in CAD. Our data demonstrate that TrkB plays a previously unknown protective role in development of CAD. However, there are some limitations in our case–control study. We cannot characterize subjects as controls with stress echo or exercise tolerance test. Because of limited medical resource, the asymptomatic healthy subjects are not suggested to accept the stress echo or exercise tolerance test in China. Therefore, we choose the exclusion criteria as Rossi et al.13 Although a cohort fulfilling these criteria is expected to have a low prevalence of asymptomatic CAD, a small percentage of asymptomatic coronary patients may have been wrongly assigned to controls. Second, there are significant baseline differences in case and control group. To eliminate the effects of confounders, including sex, age, and body mass index, we performed a multiple logistic regression analysis, so the effects of these confounders are eliminated. Considering smoking, hypertension, diabetes mellitus, hypercholesterolemia, and hypertriglyceridemia may account for the observed results, we further investigated the relationship of TrkB –69C/G and the confounders in the patients with CAD and did not find any association (data not shown). Although these baseline differences are seemed not account for the observed results in our case–control study, our results also require further verification in other population. Finally, in this study, we investigated the relationship between TrkB and CAD by examining 2 single nucleotide polymorphisms in 2 independent cohort from different geographic areas. CAD is a complex disorder, several genes in combination and environmental factors can affect atherogenesis. Therefore, the interaction between TrkB and other genes or environmental factors requires further investigation.

Endothelial barrier dysfunction accelerates atherosclerosis and induces atherosclerotic plaque rupture. We found that TrkB is expressed in aortic ECs of atherosclerotic lesions in humans and ApoE–/– mice and has important roles in protecting endothelial barrier’s integrity during atherogenesis. TrkB knockdown in endothelium led to vascular leakage in ApoE–/– mice and TrkB activity protected against endothelial hyperpermeability induced by proatherosclerotic factors in ECs. Our present results showed that 1 major mechanism underlying the protective effect of TrkB on CAD may be through promoting endothelial barrier’s integrity. Similarly, as an angiogenic factor, angiopoietin-114 and angiopoietin-215 have been reported to protect adult vasculature integrity against atherosclerosis. TrkB has also been found in smooth muscle cell of advanced atherosclerotic lesions and to promote smooth muscle cell activity.9 Smooth muscle cell activity appears to have the dual characteristic of promoting atherosclerotic lesions and stabilizing atherosclerotic plaques. Haplodeficient expression of TrkB in ApoE–/– mice led to decreased smooth muscle cell and collagen content, and increased macrophage accumulation in the lesions.9 Therefore, TrkB may stabilize advanced atherosclerotic plaque via simultaneously maintaining endothelial barrier integrity and promoting smooth muscle cell activity. BDNF circulates systemically. It has been reported that the level of plasma BDNF decreased in the aged16 and patients with CAD.17 Consistently, decreased BDNF expression in vascular endothelium was associated with hypertension,18 a common complication of CAD. Reduced TrkB expression in tissues was also found during aging.19,20 Considering that peripheral administration of BDNF activated cardiac TrkB and significantly restored the cardiac dysfunction after myocardial infarction in neuronal BDNF-deficient mice,4 enhancing vascular TrkB activation by increasing plasma BDNF levels may be a useful therapeutic strategy for CAD.

In this study, we identify a new pathway for regulating endothelial permeability via TrkB/Ets1/VE-cadherin. Our data provide the evidence that TrkB is required for regulation of VE-cadherin expression and TrkB signal promoted-synthesis of VE-cadherin is through induction and activation of Ets1 transcriptional factor. VE-cadherin–mediated adhesion junctions are essential to the endothelial barrier function. Many proteases have been found to be induced under atherosclerotic conditions, leading to VE-cadherin disorganization and hyperpermeability in the ECs.21,22 However, normal endothelial cells have the capacity to restore the junctions over the course of several hours because the destruction of the homotypic interactions between the extracellular domains of VE-cadherin induces a rapid resynthesis of VE-cadherin, leading to the restoration of endothelial cell–cell contacts.23 So, in addition to VE-cadherin disorganization, decreased capacity to synthesize VE-cadherin is another important reason of endothelial hyperpermeability during atherogenesis. In this study, we demonstrated that TrkB signaling can promote the synthesis of VE-cadherin and restore the endothelial barrier’s integrity against CAD. However, we cannot exclude the possibility that TrkB signal is also involved in the expression of other transcriptional factors and endothelial cell junction molecules that are also important for vascular permeability and may directly or indirectly contribute to the observed effect.

Systemic gene transfer with virus vectors is an effective tool for investigating gene functions in vivo. However, the aortic wall has been notoriously difficult to transduce with virus vectors. Here, as in Bostick et al,24 we were able to efficiently transduce AAV9 vectors into the aortic ECs of mice. Consistent with Bostick et al,24 limited reporter gene expression was observed in other cell components of the aorta. The poor transduction in other cell components of the aorta might be the result of the intrinsic biological properties of these cells.24 AAVs vectors, which can integrate into mouse genomic DNA and long-term express in the transduced cells, are increasingly being evaluated as part of clinical gene therapy trial.25–27 Recent study showed that AAVs are potentially safe.28 Therefore, systemic infection with AAV9 via the tail vein is an ideal method for investigating gene functions in aortic ECs in mice.

Our data demonstrate that TrkB plays a previously unknown protective role in development of CAD and maintains endothelial integrity during atherogenesis by promoting Ets1-mediated VE-cadherin expression.

Sources of Funding

This study was supported by the National 973 Basic Research Program of China (2011CB503906 and 2012CB518603), the National High-tech Research and Development Program of China (2012AA02A510), the Program of Introducing Talents of Discipline to Universities (B07035), the State Program of National Natural Science Foundation of China for Innovative Research Group (81321061), the National Natural Science Foundation of China (81200209), Shandong Province Natural Science Foundation (ZR2010HQ035), and the Independent Innovation Foundation of Shandong University (2012TS138).

Disclosures

None.

Footnotes

  • The online-only Data Supplement is available with this article at http://atvb.ahajournals.org/lookup/suppl/doi:10.1161/ATVBAHA.114.304405/-/DC1.

  • Nonstandard Abbreviations and Acronyms
    AAV9
    adeno-associated virus serotype-9
    BDNF
    brain-derived neurotrophic factor
    CAD
    coronary artery disease
    ECs
    human vascular endothelial cells
    TrkB
    tyrosine kinase receptor B
    VE-cadherin
    vascular endothelial cadherin

  • Received August 20, 2014.
  • Accepted January 14, 2015.

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Significance

Coronary artery disease (CAD) is the leading cause of death globally. CAD is a complex disorder. Although conventional risk factors are important, genetic factors account for >50% of susceptibility to CAD. The discovery of novel genetic factors could aid in our understanding of pathogenesis of CAD. We found that tyrosine kinase receptor B (TrkB) –69C>G polymorphisms is associated with CAD and TrkB plays a protective role in the development of CAD. Further studies demonstrated that TrkB protected endothelial barrier’s integrity against leakage through promoting Ets1-mediated vascular endothelial cadherin expression, indicating potential mechanisms of TrkB protecting against CAD. Our findings link TrkB to the CAD and extend our knowledge on molecular regulation of endothelial barrier function during atherogenesis. We conclude that enhancing endothelial TrkB activation to protect endothelial barrier integrity may be a useful therapeutic strategy for CAD.

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  • Tyrosine Kinase Receptor B Protects Against Coronary Artery Disease and Promotes Adult Vasculature Integrity by Regulating Ets1-Mediated VE-Cadherin ExpressionSignificance
    Hong Jiang, Shuhong Huang, Xinyun Li, Xian Li, Yun Zhang and Zhe-Yu Chen
    Arteriosclerosis, Thrombosis, and Vascular Biology. 2015;35:580-588, originally published January 29, 2015
    https://doi.org/10.1161/ATVBAHA.114.304405
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