Inhibition of Sema4D/PlexinB1 signaling alleviates vascular dysfunction in diabetic retinopathy

Article13 January 2020Open Access Source DataTransparent process Inhibition of Sema4D/PlexinB1 signaling alleviates vascular dysfunction in diabetic retinopathy Jie-hong Wu Jie-hong Wu Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Ya-nan Li Ya-nan Li Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author An-qi Chen An-qi Chen Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Can-dong Hong Can-dong Hong Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Chun-lin Zhang Chun-lin Zhang Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Hai-ling Wang Hai-ling Wang Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Yi-fan Zhou Yi-fan Zhou Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Peng-Cheng Li Peng-Cheng Li Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Yong Wang Yong Wang Aier School of Ophthalmology, Wuhan Aier Eye Hospital, Central South University, Wuhan, China Search for more papers by this author Ling Mao Ling Mao Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Yuan-peng Xia Yuan-peng Xia Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Quan-wei He Quan-wei He Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Hui-juan Jin Hui-juan Jin Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Zhen-yu Yue Zhen-yu Yue Department of Neurology and Department of Neuroscience, The Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA Search for more papers by this author Bo Hu Corresponding Author Bo Hu [email protected] orcid.org/0000-0003-1462-8854 Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Jie-hong Wu Jie-hong Wu Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Ya-nan Li Ya-nan Li Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author An-qi Chen An-qi Chen Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Can-dong Hong Can-dong Hong Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Chun-lin Zhang Chun-lin Zhang Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Hai-ling Wang Hai-ling Wang Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Yi-fan Zhou Yi-fan Zhou Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Peng-Cheng Li Peng-Cheng Li Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Yong Wang Yong Wang Aier School of Ophthalmology, Wuhan Aier Eye Hospital, Central South University, Wuhan, China Search for more papers by this author Ling Mao Ling Mao Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Yuan-peng Xia Yuan-peng Xia Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Quan-wei He Quan-wei He Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Hui-juan Jin Hui-juan Jin Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Zhen-yu Yue Zhen-yu Yue Department of Neurology and Department of Neuroscience, The Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA Search for more papers by this author Bo Hu Corresponding Author Bo Hu [email protected] orcid.org/0000-0003-1462-8854 Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China Search for more papers by this author Author Information Jie-hong Wu1,‡, Ya-nan Li1,‡, An-qi Chen1,‡, Can-dong Hong1, Chun-lin Zhang1, Hai-ling Wang1, Yi-fan Zhou1, Peng-Cheng Li2, Yong Wang3, Ling Mao1, Yuan-peng Xia1, Quan-wei He1, Hui-juan Jin1, Zhen-yu Yue4 and Bo Hu *,1 1Department of Neurology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 2Department of Ophthalmology, Union Hospital, Tongji Medical College, Huazhong University of Science and Technology, Wuhan, China 3Aier School of Ophthalmology, Wuhan Aier Eye Hospital, Central South University, Wuhan, China 4Department of Neurology and Department of Neuroscience, The Friedman Brain Institute, Icahn School of Medicine at Mount Sinai, New York, NY, USA ‡These authors contributed equally to this work as first authors *Corresponding author. Tel: +86 27 85726028; Fax: +86 27 85726028; E-mail: [email protected] EMBO Mol Med (2020)12:e10154https://doi.org/10.15252/emmm.201810154 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Diabetic retinopathy (DR) is a common complication of diabetes and leads to blindness. Anti-VEGF is a primary treatment for DR. Its therapeutic effect is limited in non- or poor responders despite frequent injections. By performing a comprehensive analysis of the semaphorins family, we identified the increased expression of Sema4D during oxygen-induced retinopathy (OIR) and streptozotocin (STZ)-induced retinopathy. The levels of soluble Sema4D (sSema4D) were significantly increased in the aqueous fluid of DR patients and correlated negatively with the success of anti-VEGF therapy during clinical follow-up. We found that Sema4D/PlexinB1 induced endothelial cell dysfunction via mDIA1, which was mediated through Src-dependent VE-cadherin dysfunction. Furthermore, genetic disruption of Sema4D/PlexinB1 or intravitreal injection of anti-Sema4D antibody reduced pericyte loss and vascular leakage in STZ model as well as alleviated neovascularization in OIR model. Moreover, anti-Sema4D had a therapeutic advantage over anti-VEGF on pericyte dysfunction. Anti-Sema4D and anti-VEGF also conferred a synergistic therapeutic effect in two DR models. Thus, this study indicates an alternative therapeutic strategy with anti-Sema4D to complement or improve the current treatment of DR. Synopsis Retinal pericyte loss, vascular leakage and neovascularization are the main pathological changes during Diabetic Retinopathy (DR). Here we show that Sema4D/PlexinB1 signaling critically contributes to these processes, and is a therapeutic target in this context. Sema4D was increased in aqueous fluid of DR patients and in retinas of several mouse DR models. Sema4D/PlexinB1 signaling induced both endothelial cell and pericyte dysfunction. Inhibition of Sema4D/PlexinB1 alleviated vascular dysfunction in DR models. Anti-Sema4D and anti-VEGF exhibited a synergistic therapeutic effect. Introduction Diabetic retinopathy (DR) is a common complication of diabetes and a leading cause of blindness in working-aged people (Cheung et al, 2010). The pathology of DR is characterized by early pericyte loss, vascular leakage, the formation of acellular capillaries, and late stage retinal ischemia, leading to an over-compensatory retinal neovascularization (Durham & Herman, 2011). Endothelial cell dysfunction is an important process in DR which leads to retinal vascular leakage and neovascularization. Due to the significant role played by vascular endothelial growth factor A (VEGF) in endothelial cell dysfunction, intravitreal injection (IVI) of its neutralizing antibody has been widely used to treat diabetic macular edema (DME) (Nguyen et al, 2009; Diabetic Retinopathy Clinical Research et al, 2015) and proliferative diabetic retinopathy (PDR) (Sivaprasad et al, 2017) in clinical practice. Although anti-VEGF therapy (such as aflibercept, ranibizumab) has provided some benefits, there are concerns surrounding anti-VEGF treatment. For many of the patients with DME who respond to anti-VEGF therapy, the response may only be temporary. These patients require frequent injections to control edema (Do et al, 2013; Channa et al, 2014; Ashraf et al, 2016). Furthermore, some patients are resistant to anti-VEGF therapy, presenting a challenge with regard to effective disease management (Channa et al, 2014; Ashraf et al, 2016). These patients may experience persistent macular edema over time despite frequent anti-VEGF injections alone (Channa et al, 2014; Ashraf et al, 2016). Moreover, the cost of anti-VEGF agents worldwide is huge, partly due to frequent injections; for example, the total drug expenditures on aflibercept in the United States alone was $2.2 billion in 2016 (Medicare, 2016). Therefore, there is a high demand for additional or alternative treatments to help those non- or poor responders to anti-VEGF and potentially reduce the number of injections. In addition to endothelial cell dysfunction, pericyte loss is another hallmark of DR (Hammes et al, 2002). Pericytes are the vascular cells affected early in DR and pericyte loss leads to secondary changes in endothelial cells (Ejaz et al, 2008), which then initiate or aggravate several pathologic features including abnormal leakage, edema, acellular capillaries and ischemia, subsequently provoke proliferative neovascularization in the retina (Hammes et al, 2002; Ejaz et al, 2008; Durham & Herman, 2011). The development of new therapeutic agents that simultaneously target endothelial cells and pericytes may not only control the disease progression, but could also improve therapeutic outcomes. Semaphorins and their receptors (Plexins) were initially discovered as axon guidance cues in development (Roth et al, 2009; Worzfeld & Offermanns, 2014). They have also been shown to play important roles in a variety of vascular pathophysiological processes (Roth et al, 2009; Worzfeld & Offermanns, 2014). To understand the role of semaphorin molecules in the pathogenesis of DR, we performed a systematic analysis of the expression of semaphorins in oxygen-induced retinopathy (OIR) and streptozotocin (STZ)-induced retinopathy models. We found that Sema4D was significantly increased in both models as well as in the aqueous fluid of DR patients, and the elevated aqueous fluid levels of Sema4D were associated with a poor response to anti-VEGF therapy. We showed that Sema4D induced endothelial cell dysfunction and triggered pericyte loss in DR. Moreover, our study not only revealed the therapeutic advantage of anti-Sema4D over anti-VEGF during multiple injections, but also suggested an improved therapy for DR with a combination of anti-VEGF treatment and the suppression of Sema4D/PlexinB1 signaling. Results Elevated Sema4D expression in mouse models and in the aqueous fluid of patients with diabetic retinopathy To identify specific semaphorins that may contribute to the development of DR, we first performed a screening of the expression of semaphorins in two different models for DR: an STZ-induced mouse DR model, which develops only retinal vascular leakage without neovascularization (Olivares et al, 2017), and oxygen-induced retinopathy (OIR), an animal model of retinopathy of prematurity (ROP) with neovascularization and vascular leakage (Connor et al, 2009). While we found increased mRNA levels in multiple semaphorins, Sema4D was the most obvious semaphorin that increased in both models (Fig 1A and B). Figure 1. Elevated Sema4D expression in mouse models and in the aqueous fluid of patients with DR A. The mRNA levels of semaphorins in age-matched controls in room air or OIR retinas at P17 were analyzed by qPCR (n = 6 per group, *P < 0.05 compared with room air retinas). B. The mRNA levels of semaphorins in retinas 6 months after DM onset were analyzed by qPCR (n = 6 per group, *P < 0.05 compared with vehicle group). C. Optical coherence tomography (OCT) and 3D retinal maps from control patients versus DME patients. The purple and light blue lines in the lower panels indicate the scanning level of OCT on the retinas. D. The levels of sSEMA4D in the aqueous fluid of DME and control individuals were measured at baseline before initial anti-VEGF therapy (P < 0.05 compared with control patients). E, F. Central subfield thickness (CST) and macular volume (MV) were measured by OCT at baseline before initial anti-VEGF therapy and 6 months after initial anti-VEGF therapy. The CST and MV changes were calculated as the baseline value minus the value at 3 months. The correlation curves between the levels of sSema4D in aqueous fluid with changes of CST and MV in response to anti-VEGF therapy are shown. Data information: Data are means ± SD. Statistical test and P-values are reported in Appendix Table S3. Download figure Download PowerPoint Sema4D, a membrane-bound protein, can be shed from the cell surface via proteolytic cleavage to yield a soluble form (sSema4D) during different disease conditions (Maleki et al, 2016). To verify the potential involvement of Sema4D in human DR, we examined the protein levels of sSema4D in aqueous fluid from patients with DR, selected according to their macular thickness as determined by optical coherence tomography (OCT) and 3D retinal maps (Fig 1C). Enzyme-linked immunosorbent assays (ELISA) revealed that sSema4D levels were robustly increased in the aqueous fluid of DR patients compared to that of the control patients (Fig 1D). Moreover, we found that the levels of sSema4D negatively correlated with changes in the central subfield thickness (CST) and macular volume (MV) in response to anti-VEGF therapy (Fig 1E and F). Sema4D is increased in retinas of oxygen-induced retinopathy and streptozotocin-induced diabetes We assessed the retinal levels of Sema4D at different time points in the OIR model. Compared with age-matched room air controls, the mRNA and protein levels of Sema4D increased as a function of time during the neovascularization phase of OIR (Fig 2A–C). In STZ-treated mice, Sema4D protein levels were significantly increased at 3 and 6 months, compared to those in age-matched control mice (Fig 2D–G). Figure 2. Glial Sema4D is increased in DR models A. Sema4D mRNA levels in OIR retinas compared with age-matched controls in room air retinas (n = 6, *P < 0.05 compared with room air retinas). B, C. Western blots (B) and quantification (C) of Sema4D protein levels in OIR retinas compared with age-matched controls in room air retinas (n = 5, *P < 0.05 compared with room air retinas). D–G. Western blots (D, F) and quantification (E, G) of Sema4D protein levels in STZ retinas at three and 6 months of diabetes (n = 6 per group, *P < 0.05 compared with vehicle group). H. Immunofluorescence staining of Sema4D (green) in OIR retinas with GFAP (red), NeuN (blue), and DAPI (gray) (n = 5), and bars indicate 25 μm. I. Fluorescence in situ hybridization with Cy3-labeled RNA probes targeting Sema4D followed by immunofluorescence staining with GFAP (green) in OIR retinas (n = 5), and scale bars indicate 25 μm. J, K. The mRNA levels of Sema4D in cell lysates (J) and secreted protein levels of Sema4D (sSema4D) from conditional medium (K) were increased in primary glial cells after hypoxia (n = 6 per group, *P < 0.05 compared with 0 h group). Data information: Data are means ± SD. Statistical test and P-values are reported in Appendix Table S3. Source data are available online for this figure. Source Data for Figure 2 [emmm201810154-sup-0003-SDataFig2.pdf] Download figure Download PowerPoint We then determined the cell type that expressed Sema4D. Immunofluorescence staining showed that the increased Sema4D signals mainly localized in GFAP+ glial cells (astrocytes and Müller cells) in the OIR model (Fig 2H). Fluorescence in situ hybridization also indicated that Sema4D localized in GFAP+ glial cells in OIR retinas (Fig 2I). Since retinal hypoxia contributes to both DME and PDR (Campochiaro et al, 2016), we used hypoxia stimulation to partly mimic the disease condition. By using isolated primary glial cells, we found that the mRNA levels of Sema4D were evidently increased when the glial cells were exposed to hypoxia (Fig 2J). Meanwhile, the secreted protein levels of Sema4D (sSema4D) from a conditional medium were also increased after hypoxia (Fig 2K). Sema4D is transcriptionally up-regulated by IRF1 To identify the mechanism that regulates the increase of Sema4D, we performed a bioinformatic search for the potential transcription factors that may regulate its expression. By using JASPAR software (http://jaspar.genereg.net), we predicted a transcription factor, IRF1, as a candidate. We first detected the expression of IRF1 in glial cells and found the protein levels of IRF1 increased over time after hypoxia stimulation (Appendix Fig S1A and B). To assess whether IRF1 is required for Sema4D expression, we utilized siRNA to silence IRF1 expression (Appendix Fig S1C). We found that IRF1 siRNA treatment blunted the induction of Sema4D mRNA upon hypoxia stimulation (Appendix Fig S1D). Furthermore, chromatin immunoprecipitation (ChIP) assays demonstrated that hypoxia stimulation induced the recruitment of IRF1 transcription factor to the putative binding site of the Sema4D promoter (Appendix Fig S1E and F). Indeed, IRF1 was increased in retinas during OIR and STZ-induced diabetes, which paralleled the expression pattern change of Sema4D (Appendix Fig S1G–J). ADAM17 induces Sema4D shedding in glial cells Previous studies have reported that Sema4D, a membrane-bound protein, is shed from the cell surface via proteolytic cleavage by certain matrix metalloproteinases (ADAM10, MMP14, ADAMTS4, ADAM17) to yield a soluble form (Maleki et al, 2016). To determine the specific metalloproteinases involved in Sema4D shedding in glial cells, we utilized siRNA to silence individual metalloproteinases (Appendix Fig S2A). We found that silencing ADAM17 only caused a significant decrease of sSema4D in the conditional medium of glial cells (Appendix Fig S2B). Moreover, the treatment of glial cells with TAPI-1, an ADAM17 inhibitor, markedly reduced hypoxia-stimulated sSema4D concentration in the supernatants of the medium of the glial cells in a dose-dependent manner (Appendix Fig S2C). Indeed, ADAM17 protein levels were up-regulated in both OIR and STZ retinas (Appendix Fig S2D–G). Sema4D knockout attenuates pathologic retinal neovascularization and vascular leakage To determine the role of Sema4D in pathologic retinal neovascularization and vascular leakage, we generated a genetic knockout (KO) of Sema4D mice (Appendix Fig S3A–C). By using isolectin B4 staining and Evans blue leakage assays, we found that Sema4D-KO mice showed an obvious reduction of pathologic retinal neovascularization as well as vascular leakage at P17 compared with littermate wild-type (WT) controls in the OIR model (Fig 3A–C). Consistently, the average number of pre-retinal neovascular cells in the retinas of Sema4D-KO mice was significantly decreased compared with littermate WT controls (Fig 3D and E). Figure 3. Sema4D knockout attenuates pathological retinal neovascularization and vascular leakage A–C. Isolectin B4 staining and Evans blue assays were performed to examine pathological retinal neovascularization and vascular leakage in whole-mount retinas at P17 in the OIR model with or without Sema4D. For isolectin B4 staining, multiple overlapping (10–20% overlap) images were obtained with a 4× lens on a fluorescence microscope. The images were merged to visualize the entire retinas. The upper images are representative composite images of entire retinas. The lower images are a cropped enlarged view from the upper entire retina images. The processes were used in all the following isolectin B4 staining figures for the entire retina images (n = 12 for WT in B, n = 13 for Sema4D-KO in B, n = 10 in C, scale bars indicate 200 μm for upper pictures and 100 μm for lower pictures, *P < 0.05 compared with WT group). D, E. Representative HE staining images and quantification of pre-retinal neovascular cells in retinal cross-sections at P17 in the OIR model with or without Sema4D (n = 10. Arrows indicate pre-retinal neovascular cells. Scale bars indicate 20 μm, *P < 0.05 compared with WT group). F, G. Evans blue assays were used to test vascular leakage at 3 months in the STZ model with or without Sema4D. Representative Evans blue fluorescent images are shown. The calculated extracted Evans blue values were used for quantification (n = 12, scale bars indicate 200 μm, *P < 0.05 compared with WT + vehicle group, #P < 0.05 compared with WT + STZ group). H, I. Immunofluorescence staining of isolectin B4 (green) with collagen IV (red) showed that Sema4D knockout attenuated acellular capillary formation at 3 months in the STZ model (n = 6. Arrows indicate acellular capillaries. Scale bars indicate 10 μm, *P < 0.05 compared with WT + vehicle group, #P < 0.05 compared with WT + STZ group). J–L. Immunofluorescence staining of PDGFRβ (green, a pericyte marker) with collagen IV (red) at 3 months in the STZ model (n = 6, scale bars indicate 10 μm, *P < 0.05 compared with WT + vehicle group, #P < 0.05 compared with WT + STZ group). Data information: Data are means ± SD. Statistical test and P-values are reported in Appendix Table S3. Download figure Download PowerPoint To evaluate whether Sema4D KO inhibits the process of DR in an STZ model, we induced diabetes in Sema4D-KO mice and age-matched WT mice. Evans blue leakage assays indicated that diabetic Sema4D-KO mice showed lower vascular leakage than diabetic WT mice (Fig 3F and G). Furthermore, Sema4D KO alleviated diabetes mellitus-induced acellular capillary formation (Figs 3H and I, and EV1A and B). Moreover, by staining pericytes with PDGFRβ and desmin markers, we found that diabetes-induced pericyte loss was markedly blocked by Sema4D KO (Figs 3J–L and EV1C and D). Click here to expand this figure. Figure EV1. Sema4D knockout alleviates acellular capillary formation and pericytes loss in STZ model A, B. Retinal trypsin digestion showed that Sema4D knockout attenuated acellular capillary formation at 3 months in the STZ model (n = 6. Arrows indicate acellular capillaries. Scale bars indicate 20 μm, *P < 0.05 compared with WT + Vehicle group, #P < 0.05 compared with WT + STZ group). C, D. Immunofluorescence staining of desmin (green, a pericyte marker) with collagen IV (red) at 3 months in the STZ model with or without Sema4D (n = 6, scale bars indicate 10 μm, *P < 0.05 compared with WT + Vehicle group, #P < 0.05 compared with WT + STZ group). Data information: Data are means ± SD. Statistical test and P-values are reported in Appendix Table S3. Download figure Download PowerPoint In addition, we evaluated the effect of Sema4D KO on normal retinal vessel development. There was no significant difference between Sema4D-KO mice and littermate WT mice in relation to retinal vascular development at P5, as analyzed by the vascular area, vascular outgrowth, vascular branch points, and sprout number (Appendix Fig S3D–H). Sema4D promotes endothelial cell migration and permeability through the PlexinB1 receptor in vitro We next investigated the function of Sema4D in endothelial cells in vitro. Wound-healing assays indicated that recombinant Sema4D promoted endothelial cell migration in a dose-dependent manner (Fig EV2A and B). To assess the effect of Sema4D on vascular permeability, trans-endothelial electrical resistance (TEER) and permeability to dextran were measured on endothelial monolayers. We found that recombinant Sema4D decreased the TEER value of endothelial monolayers and increased their permeability to dextran (Fig EV2C and D). Click here to expand this figure. Figure EV2. Sema4D regulates endothelial cell function via the PlexinB1 receptor A, B. Wound-healing assays indicated that recombinant Sema4D promoted endothelial cell migration in a dose-dependent manner (n = 5. The vertical red lines indicate the border of the wound. Scale bars indicate 100 μm, *P < 0.05 compared with 0 ng/ml Sema4D group). C, D. Trans-endothelial electrical resistance (TEER) values and dextran permeability assays showed that recombinant Sema4D promoted endothelial monolayer leakage in a dose-dependent manner (n = 6 in C, n = 5 in D, *P < 0.05 compared with 0 ng/ml Sema4D group). E. Knockdown efficiency of PlexinB1 in endothelial cells transfected with lentivirus-mediated CRISPR-plB1 (n = 5, *P < 0.05 compared with CRISPR-wt group). F–I. Endothelial cells transfected with lentivirus-mediated CRISPR-wt or CRISPR-plB1 were treated with or without 1600 ng/ml recombinant Sema4D, and then, wound healing (F and G), TEER value (H), and dextran permeability (I) were measured (n = 5 in G, I. n = 6 in H. The vertical red lines indicate the border of the wound in F. Scale bars indicate 100 μm, *P < 0.05 compared with CRISPR-wt group, #P < 0.05 compared with CRISPR-wt + Sema4D group). Data information: Data are means ± SD. Statistical test and P-values are reported in Appendix Table S3. Source data are available online for this figure. Download figure Download PowerPoint Previous studies have indicated that Sema4D binds with a high affinity to receptor PlexinB1, which mediates the Sema4D signaling afferent (Matsunaga et al, 2016). To evaluate whether PlexinB1 mediates the effects in the above condition, we used lentivirus-mediated CRISPR/Cas9 gene disruption of PlexinB1 (CRISPR-plB1) in endothelial cells (Fig EV2E) and found that the knockdown of PlexinB1 expression significantly blocked the stimulation effect of Sema4D on endothelial cell migration (Fig EV2F and G). Meanwhile, PlexinB1 knockdown also substantially abolished the Sema4D-elicited increased permeability of endothelial monolayers (Fig EV2H and I). Sema4D/PlexinB1 regulates endothelial cell function via mDIA1-Src signaling Through the mining of a large-scale protein–protein network database, we predicted that C210RF59, CENPO, CENPU, mDIA1 (gene name: DIAPH1), HNRNPD, and SGTB were potential binding partners of PlexinB1 (Hein et al, 2015). Among these proteins, only mDIA1 has been reported to participate in angiogenesis and vascular permeability (Gavard et al, 2008; Zhou et al, 2014b, 2018a). In addition, mDIA1 has been shown to form a complex with Src to regulate Src tyrosine kinase signaling (Tominaga et al, 2000; Gavard et al, 2008; Zhou et al, 2018a). Furthermore, Src tyrosine kinase could phosphorylate focal adhesion kinase (Fak) and vascular endothelial cadherin (VE-cadherin), a main component of endothelial adherens junctions, to induce angiogenesis and vascular permeability (Dejana et al, 2008; Zhao & Guan, 2011; Miloudi et al, 2016). Therefore, we next questioned whether PlexinB1 could form a complex with mDIA1/Src upon Sema4D treatment, thereby activating Src tyrosine kinase signaling to regulate endothelial cell function. We performed co-immunoprecipitation to detect complex formation with the indicated antibodies (Fig 4A), and the results showed that the PlexinB1-m DIA1/Src complex formation increased upon stimulation with Sema4D. Meanwhile, we found that Sema4D induced the phosphorylation of Src, VE-cadherin, and Fak (Fig 4B and C) in a time-dependent manner. To determine whether Sema4D works through mDIA1, we knocked down mDIA1 expression using specific siRNA (Fig 4D). The reduced expression of mDIA1 blocked the Sema4D-induced phosphorylation of Src, Fak, and VE-cadherin (Fig 4E and F). Furthermore, sile