Endomucin selectively regulates vascular endothelial growth factor receptor-2 endocytosis through its interaction with AP2.

The endothelial glycocalyx, located at the luminal surface of the endothelium, plays an important role in the regulation of leukocyte adhesion, vascular permeability, and vascular homeostasis. Endomucin (EMCN), a component of the endothelial glycocalyx, is a mucin-like transmembrane glycoprotein selectively expressed by venous and capillary endothelium. We have previously shown that knockdown of EMCN impairs retinal vascular development in vivo and vascular endothelial growth factor 165 isoform (VEGF165)-induced cell migration, proliferation, and tube formation by human retinal endothelial cells in vitro and that EMCN is essential for VEGF165-stimulated clathrin-mediated endocytosis and signaling of VEGF receptor 2 (VEGFR2). Clathrin-mediated endocytosis is an essential step in receptor signaling and is of paramount importance for a number of receptors for growth factors involved in angiogenesis. In this study, we further investigated the molecular mechanism underlying EMCN's involvement in the regulation of VEGF-induced endocytosis. In addition, we examined the specificity of EMCN's role in angiogenesis-related cell surface receptor tyrosine kinase endocytosis and signaling. We identified that EMCN interacts with AP2 complex, which is essential for clathrin-mediated endocytosis. Lack of EMCN did not affect clathrin recruitment to the AP2 complex following VEGF stimulation, but it is necessary for the interaction between VEGFR2 and the AP2 complex during endocytosis. EMCN does not inhibit VEGFR1 and FGFR1 internalization or their downstream activities since EMCN interacts with VEGFR2 but not VEGFR1 or FGFR1. Additionally, EMCN also regulates VEGF121-induced VEGFR2 phosphorylation and internalization.

Computational assessment of pigment epithelium-derived factor as an anti-cancer protein during its interaction with the receptors.

Pigment epithelium-derived factor (PEDF) is a member of the serine proteinase inhibitor (serpin) with antiangiogenic, anti-tumorigenic, antioxidant, anti-atherosclerosis, antithrombotic, anti-inflammatory, and neuroprotective properties. The PEDF can bind to low-density lipoprotein receptor-related protein 6 (LRP6), laminin (LR), vascular endothelial growth factor receptor 1 (VEGFR1), vascular endothelial growth factor receptor 2 (VEGFR2), and ATP synthase ß-subunit receptors. In this study, we aimed to investigate the structural basis of the interaction between PEDF and its receptors using bioinformatics approaches to identify the critical amino acids for designing anticancer peptides. The human ATP synthase ß-subunit was predicted by homology modeling. The molecular docking, molecular dynamics (MD) simulation, and Molecular mechanics/Poisson-Boltzmann surface area (MM/PBSA) were used to study this protein-receptor complex. The molecular docking showed PEDF could bind to the Laminin and VEGFR2 much stronger than ATP synthase ß-subunit, VEGFR1, and LRP6. The PEDF could effectively interact with various receptors during the simulation. The N-terminal of PEDF has an important role in the interaction with the receptors. The MM/PBSA showed the electrostatic (ΔEElec) and van der Waals interactions (ΔEVdW) contributed positively to the binding process of the complexes. The critical amino acids in the binding interaction of PEDF to its receptors in the MD simulation were determined. The interaction mode of 34-mer PEDF to laminin, VEGFR2, and LRP6 were different from VEGFR1, ATP synthase ß-subunit. The 34-mer PEDF has an important role in the interaction with different receptors and these critical amino acids can be used for designing peptides for future therapeutic aims.Communicated by Ramaswamy H. Sarma.

Amorphous selenium nanodots alleviate non-alcoholic fatty liver disease via activating VEGF receptor 1 to further inhibit phosphorylation of JNK/p38 MAPK pathways.

In clinic, there is still no unified standard for the treatment of non-alcoholic fatty liver disease (NAFLD), and the development of effective novel drugs to alleviate NAFLD remains a challenge. This study aimed to explore the effect and mechanism of amorphous selenium nanodots (A SeNDs) in alleviating NAFLD. Model rats with NAFLD were induced by the high-fat diet (HFD). Histomorphology was used to observe liver tissue, automatic biochemical analyzer was used to analyze liver function indicators, and ELISA kit was used to detect the effect of A SeNDs on oxidative stress and inflammatory factors in NAFLD rats. The results exhibited that A SeNDs could reduce hepatocyte steatosis, liver index, blood lipid level, and transaminase level in NAFLD rats. Furthermore, A SeNDs could relieve the oxidative stress and inflammatory reaction and maintain liver tissue structure in NAFLD rats. Mechanistically, A SeNDs (0.3 mg/kg/day) inhibit the phosphorylation of JNK/p38 MAPK pathways after activating vascular endothelial growth factor receptor 1 (VEGFR1) in the liver of rats with NAFLD to allay oxidative stress and inflammatory response and improves hepatic structure and liver function. Therefore, it should be an important method to mitigate NAFLD by supplementing A SeNDs to normalize hepatic structure and liver function.

PlGF Reduction Compromises Angiogenesis in Diabetic Foot Disease Through Macrophages.

Diabetic foot disease (DFD) is a common and serious complication for diabetes and is characterized with impaired angiogenesis. In addition to the well-defined role of vascular endothelial growth factor (VEGF) -A and its defect in the pathogenesis of DFD, another VEGF family member, placental growth factor (PlGF), was also recently found to alter expression pattern in the DFD patients with undetermined mechanisms. This question was thus addressed in the current study. We detected attenuated PlGF upregulation in a mouse DFD model. In addition, the major cell types at the wound to express the unique PlGF receptor, VEGF receptor 1 (VEGFR1), were macrophages and endothelial cells. To assess how PlGF regulates DFD-associated angiogenesis, we injected recombinant PlGF and depleted VEGF1R specifically in macrophages by local injection of an adeno-associated virus (AAV) carrying siRNA for VEGFR1 under a macrophage-specific CD68 promoter. We found that the angiogenesis and recovery of the DFD were both improved by PlGF injection. The PlGF-induced improvement in angiogenesis and the recovery of skin injury were largely attenuated by macrophage-specific depletion of VEGF1R, likely resulting from reduced macrophage number and reduced M2 polarization. Together, our data suggest that reduced PlGF compromises angiogenesis in DFD at least partially through macrophages.

VEGFR1 signaling in retinal angiogenesis and microinflammation.

Five vascular endothelial growth factor receptor (VEGFR) ligands (VEGF-A, -B, -C, -D, and placental growth factor [PlGF]) constitute the VEGF family. VEGF-A binds VEGF receptors 1 and 2 (VEGFR1/2), whereas VEGF-B and PlGF only bind VEGFR1. Although much research has been conducted on VEGFR2 to elucidate its key role in retinal diseases, recent efforts have shown the importance and involvement of VEGFR1 and its family of ligands in angiogenesis, vascular permeability, and microinflammatory cascades within the retina. Expression of VEGFR1 depends on the microenvironment, is differentially regulated under hypoxic and inflammatory conditions, and it has been detected in retinal and choroidal endothelial cells, pericytes, retinal and choroidal mononuclear phagocytes (including microglia), Müller cells, photoreceptor cells, and the retinal pigment epithelium. Whilst the VEGF-A decoy function of VEGFR1 is well established, consequences of its direct signaling are less clear. VEGFR1 activation can affect vascular permeability and induce macrophage and microglia production of proinflammatory and proangiogenic mediators. However the ability of the VEGFR1 ligands (VEGF-A, PlGF, and VEGF-B) to compete against each other for receptor binding and to heterodimerize complicates our understanding of the relative contribution of VEGFR1 signaling alone toward the pathologic processes seen in diabetic retinopathy, retinal vascular occlusions, retinopathy of prematurity, and age-related macular degeneration. Clinically, anti-VEGF drugs have proven transformational in these pathologies and their impact on modulation of VEGFR1 signaling is still an opportunity-rich field for further research.

VEGFR-1 Regulates EGF-R to Promote Proliferation in Colon Cancer Cells.

The relationship between epidermal growth factor (EGF) and vascular endothelial growth factor (VEGF) pathways in tumor growth is well established. EGF induces VEGF production in cancer cells, and the paracrine VEGF activates vascular endothelial cells to promote tumor angiogenesis and thus supports tumor cell growth in an angiogenesis-dependent manner. In this study, we found angiogenesis-independent novel crosstalk between the VEGF and the EGF pathways in the regulation of colon cancer cell proliferation. Stimulation of colon cancer cells with VEGF-A and placental growth factor (PlGF) activated VEGF receptor-1 (VEGFR-1) and increased proliferation activity in an autocrine EGF/EGF receptor (EGF-R)-dependent manner. Mechanistically, VEGFR-1 interacted with and stabilized EGF-R, leading to increased EGF-R protein levels and prolonged its expression on cell surface plasma membrane. In contrast, VEGFR-1 blockade by a neutralizing antibody and an antagonistic peptide of VEGFR-1 suppressed the complex formation of VEGFR-1 and EGF-R and decreased EGF-R expression via a lysosome-dependent pathway, resulting in the suppression of proliferation activity. Our results indicated that VEGFR-1 regulated EGF-R expression to promote proliferation activity in a cell-autonomous-dependent manner.

Effect of inhibitor of differentiation 1 deficiency on ocular neovascularization / 上海交通大学学报（医学版）

Objective · To study the effect of inhibitor of differentiation 1 (ID1) on ocular neovascularization. Methods · The oxygen-induced retinal neovascularization (OIR), laser-induced choroidal neovascularization (CNV) and over-expression of vascular endothelial growth factor (VEGF) (Rho-VEGF) transgenic mice were established. The localization and mRNA level of ID1 in retina of OIR mice and Rho-VEGF transgenic mice were determined by immunofluorescence staining and quantitative real-time PCR. Mice deficient in ID1 (ID1-/-) were used to induce retinal neovascularization in accordance with the above three models, and to compare the changes of ID1 on the number of retinal, subretinal and choroidal neovascularization areas. In order to explore the role ID1 in neovascularization, the numbers and areas of retinal, subretinal and choroidal neovascularization in the mice models with or without ID1 deficiency were compared. Its effect on the related factors, i.e. hypoxia-inducible factor-1α (HIF-1α), VEGF and vascular endothelial growth factor receptor 1/2 (VEGFR1/2) were also observed. Results · Mice deficient in ID1 showed a significant reduction in the area of neovascularization in these three models (P＜0.05). Mice lacking ID1 showed reduced levels of HIF-1α, VEGF and VEGFR 1. Conclusion · ID1 promotes the expression of HIF-1α, VEGF and VEGFR1 in the retina and choroidal neovascularization during hypoxia and oxidative injury.

Effect of inhibitor of differentiation 1 deficiency on ocular neovascularization / 上海交通大学学报（医学版）

Objective • To study the effect of inhibitor of differentiation 1 (ID1) on ocular neovascularization. Methods • The oxygen-induced retinal neovascularization (OIR), laser-induced choroidal neovascularization (CNV) and over-expression of vascular endothelial growth factor (VEGF) (Rho-VEGF) transgenic mice were established. The localization and mRNA level of ID1 in retina of OIR mice and Rho-VEGF transgenic mice were determined by immunofluorescence staining and quantitative real-time PCR. Mice deficient in ID1 (ID1-/-) were used to induce retinal neovascularization in accordance with the above three models, and to compare the changes of ID1 on the number of retinal, subretinal and choroidal neovascularization areas. In order to explore the role ID1 in neovascularization, the numbers and areas of retinal, subretinal and choroidal neovascularization in the mice models with or without ID1 deficiency were compared. Its effect on the related factors, i.e. hypoxia-inducible factor-1α (HIF-1α), VEGF and vascular endothelial growth factor receptor 1/2 (VEGFR1/2) were also observed. Results • Mice deficient in ID1 showed a significant reduction in the area of neovascularization in these three models(P<0.05). Mice lacking ID1 showed reduced levels of HIF-1α, VEGF and VEGFR 1. Conclusion • ID1 promotes the expression of HIF-1α, VEGF and VEGFR1 in the retina and choroidal neovascularization during hypoxia and oxidative injury.

VEGFR1 Signaling Regulates IL-4-Mediated Arginase 1 Expression in Macrophages.

BACKGROUND: Macrophages undergo polarization or activation in response to environmental stimuli, an essential process for proper immune response. Meanwhile, excessive activation of macrophages causes autoimmune diseases. It is therefore crucial to prevent over-activation of macrophage in order to maintain the proper immune response. Arginase 1 (Arg-1) plays a critical role in coordinating the immune response by regulating availability of arginine. OBJECTIVE: To understand the mechanism of Arg-1 regulation. METHODS: Real-time PCR and Western Blot analysis were utilized to examine the Arg-1 levels expressed from the VEGFR1-deleted and VEGFR1-TK-deficient bone marrowderived macrophages (BMDMs). RESULTS: The VEGFR1-mediated signaling suppressed IL-4-induced Arg-1 expression. Deletion of VEGFR1 resulted in elevated Arg-1 expression and the tyrosine kinase domain of VEGFR1 was required for the suppression. Each of three ligands of VEGFR1, VEGF-A, VEGF-B and PIGF, mediated the inhibition to the similar degree. CONCLUSION: Our findings identified a novel function of the VEGFR1 signaling in avoiding over-expression of Arginase 1 potentially to maintain the proper innate immune response.

The imbalance in expression of angiogenic and anti-angiogenic factors as candidate predictive biomarker in preeclampsia.

Preeclampsia is an important pregnancy disorder with serious maternal and fetal complications which its etiology has not been completely understood yet. Early diagnosis and management of disease could reduce its potential side effects. The vascular endothelial growth factor (VEGF) family including VEGF-A is the most potent endothelial growth factor which induces angiogenesis and endothelial cell proliferation and has basic role in vasculogenesis. VEGF and its tyrosine kinase receptors (Flt1 and KDR) are major factors for fetal and placental angiogenic development. Finding mechanisms involved in expression of angiogenic factors may lead to new prognostic and therapeutic points in management of preeclampsia. Recent researches, has shown capability of some anti-angiogenic factors as potential candidate to be used as early predictors for preeclampsia. Soluble fms-like tyrosin kinase-1 (sFlt1) is a truncated splice variant of the membrane-bound VEGF receptor Flt1, that is produced by the placenta and it can bind to angiogenic growth factors and neutraliz, their effects. It is also observed that the ratio of sFlt1 to placental growth factor is valuable as prognostic marker. In this review, VEGF family member's role in angiogenesis is evaluated as biomarkers to be used for prediction of preeclampsia.

Vascular endothelial growth factor receptor 1 expression in nasal polyp tissue.

BACKGROUND: Edema represents a key feature of nasal polyp (NP) disease. Members of the vascular endothelial growth factor (VEGF) family may be involved, but the precise role of VEGF-A, VEGF-B, placental growth factor (PlGF), and their receptors VEGFR1 and VEGFR2 in NP edema formation remains elusive. OBJECTIVE: Exploring the expression of VEGF family members and their receptors and their correlation with clinical, radiological, and edema markers in NP. METHODS: The expression of VEGF-A, VEGF-B, PlGF, VEGFR1, and VEGFR2 was measured in NP (n = 23) and control tissue (n = 22) at mRNA and protein level. Edema was evaluated by measuring albumin levels and wet/dry ratios. Computed tomography (CT) scans were scored using the Lund-Mackay scoring system. IL-5 mRNA expression was determined by real-time RT-PCR. Cell suspensions from NP (n = 10) and control tissue (n = 12) were stimulated in vitro with IL-1ß or TNFα. RESULTS: mRNA expression of VEGFR1 and VEGF-B was significantly higher in NP compared with control tissue. Expression levels of VEGF-B and VEGFR1 significantly correlated with NP albumin content (VEGF-B: P = 0.0208; VEGFR1: P = 0.0293), CT scan scores (VEGF-B: P = 0.0075; VEGFR1: P = 0.0068), and IL-5 mRNA (VEGF-B: P = 0.0027; VEGFR1: P = 0.0001). In vitro stimulation of control and NP tissue cell suspensions with IL-1ß or TNFα significantly reduced the expression of VEGFR2 in control tissue, without altering VEGFR1 and VEGF-B expression. hVEGF-B induced nitric oxide production in NP macrophages (P < 0.05). CONCLUSION: Expression levels of VEGFR1 and VEGF-B correlate with edema and clinical markers of NP disease and therefore represent potential therapeutic targets.