Vascular Endothelial Growth Factor Receptor Signaling Research Articles

P1612The ablation of VEGFR-1 signaling promotes angiotensin II induced cardiac dysfunction and sudden death

Abstract Introduction Concentric left ventricular hypertrophy (LVH) and diastolic dysfunction develops as an adaptive response to pressure overload. Eventually this may lead to decompensated hypertrophy characterised by interstitial fibrosis, contractile dysfunction as well as changes in metabolism and electrophysiology, consequentially triggering heart failure. The molecular mechanisms involved in cardiac remodelling are not fully understood but maladaptive angiogenesis could promote the transition from adaptive LVH to decompensated heart failure. Angiogenesis is mediated by vascular endothelial growth factors but their role in LVH has remained unresolved. Purpose In this study, we wanted to investigate whether vascular endothelial growth factor receptor 1 (VEGFR-1) signaling has a role in the progression of LVH and development of heart failure. Methods We used wild type littermate controls and domain specific knock out mouse lacking the intracellular VEGFR-1 tyrosine kinase domain (VEGFR-1 TK−/−) and induced pathological hypertrophy with subcutaneous angiotensin II infusion. We examined the cardiac function with echocardiography and acquired surface ECG signal during the development of LVH. Mice were followed up for 14 days before sacrification and sample collection. Cross-sectional cardiac samples were stained with Masson's trichrome to assess the level of fibrosis and immunostained for lectin to determine capillary area. Additionally, we performed a CD31 whole mount staining to visualise capillary 3D network. To analyse changes in gene expression levels, we performed RT qPCR measurements. Results VEGFR-1 TK deficiency led to increased mortality (33.3%) and lack of adaptive LVH. Whereas wild type mice responded to angiotensin II infusion with a significant increase in ejection fraction (55.5% to 69.9%) within the first 6 days, VEGFR-1 TK−/− mice displayed a 5.2% decrease and without adaptive thickening of the LV anterior wall. The most striking difference was seen in LV volume, where wild type mice displayed a 63.3% reduction but in VEGFR-1 TK−/− mice it remained unaltered after angiotensin II infusion. Histological analyses showed that VEGFR-1 TK−/− mice displayed significant cardiomyocyte hypertrophy combined with ventricular dilatation but without changes in fibrosis or angiogenesis. ECG analysis revealed that VEGFR-1 TK−/− mice exhibited widening of the QRS complex, similar to human LVH, and this was accompanied by increased ANP/BNP levels. Conclusions In this study, we show that the ablation of VEGFR-1 TK signaling has an unexpected role in pressure overload inducing mortality. VEGFR-1 TK−/− mice displayed dilated LVH and a protracted response to angiotensin II infusion, suggesting that VEGFR-1 signaling is required for the adaptive response and concentric hypertrophy of the myocardium.

Regulation of VEGFR2 trafficking and signaling by Rab GTPase-activating proteins

Vascular endothelial growth factor receptor-2 (VEGFR2) and its ligands (VEGFs) are crucial players in vasculogenesis and angiogenesis. General blocking of this signaling system with antibodies or small molecule inhibitors is an established strategy to treat cancer and age-related macular degeneration. Nevertheless, the activated receptor can signal to discrete downstream signaling pathways and the equilibrium between these pathways is modulated by coreceptors and distinct isoforms of VEGF. Here we investigated the influence of Rab GTPase activating proteins (RabGAPs) on VEGFR2 signaling, tube formation, and migration of endothelial cells. We demonstrate that members of the TBC1D10 subfamily of RabGAPs have opposite effects. Whereas TBC1D10A leads to increased Erk1/2 signaling, TBC1D10B lowered Erk1/2 and p38 signaling and reduced tube formation in vitro. TBC1D10A is a RabGAP acting on RAB13 that was shown before to play a role in angiogenesis and we could indeed show colocalization of these two proteins with VEGFR2 in activated cells. In addition, we observed that cells expressing TBC1D10B show lower expression of VEGFR2 and NRP1 on filopodia of activated cells. Taken together, our systematic analysis of influence of RabGAPs on VEGFR2 signaling identifies the TBC1D10 subfamily members as modulators of angiogenesis.

A VEGFR2-MICA bispecific antibody activates tumor-infiltrating lymphocytes and exhibits potent anti-tumor efficacy in mice.

MHC class I-related chain A (MICA) is one of the major ligands for natural killer group 2 member D (NKG2D), which is an activating NK receptor. MICA is expressed on the surface of human epithelial tumor cells, and its shedding from tumor cells leads to immunosuppression. To activate immune response in the tumor microenvironment, we designed an anti-VEGFR2-MICA bispecific antibody (JZC01), consisting of MICA and an anti-VEGFR2 single chain antibody fragment (JZC00) and explored its potential anti-tumor activity. JZC01 targeted vascular endothelial growth factor receptor 2 (VEGFR2) and inhibited tumorigenesis by blocking the VEGFR2 signaling pathway. Additionally, JZC01 promoted NK and CD8+ T cells to release IFN-γ and engaged activated lymphocytes to lysis of VEGFR2-expressing tumor cells. The in vivo anti-tumor activity of JZC01 was investigated by establishing a Lewis lung cancer cell-transplanted mouse model. It effectively reduced the tumor vascular density and increased the infiltration and activation of NK and CD8+ T cells in the tumor microenvironment. Thus, JZC01 functions in anti-tumor angiogenesis and anti-tumor immune activation, and showed improved anti-tumor efficacy combined with docetaxel, which provides a new insight into anti-tumor therapy.

Hydrophobic Interactions Drive Binding between Vascular Endothelial Growth Factor-A (VEGFA) and Polyphenolic Inhibitors.

Some polyphenols have been shown to inhibit, at physiological levels, the VEGF-induced VEGF receptor-2 signaling that causes angiogenesis, allegedly by direct interaction with VEGF and reducing the binding to its receptor VEGFR2. Surface plasmon resonance was used to measure the parameters of binding between VEGF and polyphenols as well as the nature of the interactions by assessing the effect of physico-chemical changes in the solution. CD spectrometry was used to determine any change in the secondary structure of the protein upon binding. The kinetic parameters (ka, kd, and KD) that characterise the binding to VEGF were measured for both inhibitor and non-inhibitor polyphenolic molecules. The effect of changes in the physico-chemical conditions of the solution where the binding occurred indicated that the nature of the interactions between VEGF and EGCG was predominantly of a hydrophobic nature. CD studies suggested that a change in the secondary structure of the protein occurred upon binding. Direct interaction and binding between VEGF and polyphenol molecules acting as inhibitors of the signaling of VEGFR2 has been measured for the first time. The binding between VEGF and EGCG seemed to be based on hydrophobic interactions and caused a change in the secondary structure of the protein.

473-P: Identification of Novel Genes Participating in Diabetic Macrovascular Complications by DNA Methylation Modulation

Diabetic vascular complications contribute to the increased morbidity and mortality of diabetic patients. However, the mechanism of diabetic macrovascular complication remains unclear. Objective: We intend to unleash the role of DNA methylation modification in the pathological process of diabetic macrovascular complication and identify the potential biomarkers that could be beneficial for early intervention. Methods: We screened for differentially methylated genes by methylated DNA immunoprecipitation chip (MeDIP-chip) in diabetic patients. The validation of MeDIP-chip was performed by methylation-specific PCR (MSP). Furthermore, we investigated the expressions of the aberrantly methylated genes and their functions in the model of diabetic mice. Results: Serval aberrantly methylated genes have been explored, including phospholipase C beta 1 (PLCB1), cam kinase I delta (CAMK1D), and dopamine receptor D5 (DRD5) which participated in calcium signaling pathway. Meanwhile, vascular endothelial growth factor B (VEGFB), placental growth factor (PLGF), fatty acid transport protein 3 (FATP3), coagulation factor II thrombin receptor (F2R), and fatty acid transport protein 4 (FATP4) which participated in vascular endothelial growth factor receptor (VEGFR) signaling pathway, were also been found. After MSP and gene expression validation in peripheral blood of patients, PLCB1, PLGF, FATP4, and VEGFB were corroborated. Combining with the detection of fatty acid uptake by radionuclide tracing, DNA methylation regulated VEGFR signaling pathway was demonstrated to mediate the lipid deposition in aortas of diabetic mice. Conclusions: Our study identifies novel biomarkers and therapeutic targets in diabetic macrovascular complications. DNA methylation in VEGFR signaling pathway might play an important role in diabetic macrovascular complications. Disclosure Y. Liao: None. L. Chen: None. T. Zeng: None. Funding National Natural Science Foundation of China

Effect of a Small Physiological Electric Field on Angiogenic Activity in First-Trimester Extravillous Trophoblast Cells.

Electrical stimulation induces significant angiogenesis in vivo. We have shown that electrical stimulation of trophoblast cells has important functions in aspects of angiogenesis. In this study, we investigated the effects of a direct current electrical field on trophoblast angiogenic tube formation. A 6-hour exposure to electric fields ranging from 50 to 150 mV/mm dose dependently increased tube growth and network formation. Additionally, the effect was time dependent, with increased tube formation occurring between 4 and 8 hours, indicating stimulation of trophoblast cell angiogenesis. Electrical fields of small physiological magnitude stimulated vascular endothelial growth factor expression by trophoblast cells in the culture. Electric field treatment also resulted in activation of Akt, while the activity of extracellular-regulated kinase 1/2, p38, and c-Jun NH2-terminal kinase was not significantly changed. Pretreatment with the vascular endothelial growth factor receptor (VEGFR)-2 inhibitor, SU1498, resulted in potent inhibition of tube growth, and the Akt inhibitor, MK-2206 2HCl, significantly reduced electric field-stimulated tubulogenesis. These data suggest the importance of the VEGFR-2 signaling pathway during electric field-induced trophoblastic angiogenesis. This novel evidence indicates that endogenous electrical fields may promote angiogenesis of trophoblast cells by stimulating the VEGFR signaling pathway.

Prenatal inflammation impairs intestinal microvascular development through a TNF-dependent mechanism and predisposes newborn mice to necrotizing enterocolitis.

Prenatal inflammation is a risk factor for necrotizing enterocolitis (NEC), and it increases intestinal injury in a rat NEC model. We previously showed that maldevelopment of the intestinal microvasculature and lack of vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) signaling play a role in experimental NEC. However, whether prenatal inflammation affects the intestinal microvasculature remains unknown. In this study, mouse dams were injected intraperitoneally with lipopolysaccharide (LPS) or saline at embryonic day 17. Neonatal intestinal microvasculature density, endothelial cell proliferation, and intestinal VEGF-A and VEGFR2 proteins were assessed in vivo. Maternal and fetal serum TNF concentrations were measured by ELISA. The impact of TNF on the neonatal intestinal microvasculature was examined in vitro and in vivo, and we determined whether prenatal LPS injection exacerbates experimental NEC via TNF. Here we found that prenatal LPS injection significantly decreased intestinal microvascular density, endothelial cell proliferation, and VEGF and VEGFR2 protein expression in neonatal mice. Prenatal LPS injection increased maternal and fetal serum levels of TNF. TNF decreased VEGFR2 protein in vitro in neonatal endothelial cells. Postnatal TNF administration in vivo decreased intestinal microvasculature density, endothelial cell proliferation, and VEGF and VEGFR2 protein expression and increased the incidence of severe NEC. These effects were ameliorated by stabilizing hypoxia-inducible factor-1α, the master regulator of VEGF. Furthermore, prenatal LPS injection significantly increased the incidence of severe NEC in our model, and the effect was dependent on endogenous TNF. Our study suggests that prenatal inflammation increases the susceptibility to NEC, downregulates intestinal VEGFR2 signaling, and affects perinatal intestinal microvascular development via a TNF mechanism. NEW & NOTEWORTHY This report provides new evidence that maternal inflammation decreases neonatal intestinal VEGF receptor 2 signaling and endothelial cell proliferation, impairs intestinal microvascular development, and predisposes neonatal mouse pups to necrotizing enterocolitis (NEC) through inflammatory cytokines such as TNF. Our data suggest that alteration of intestinal microvascular development may be a key mechanism by which premature infants exposed to prenatal inflammation are at risk for NEC and preserving the VEGF/VEGF receptor 2 signaling pathway may help prevent NEC development.

Analysis of Tumor Angiogenesis and Immune Microenvironment in Non-Functional Pituitary Endocrine Tumors.

Cavernous sinus (CS) invasion is an aggressive behavior exhibited by pituitary neuroendocrine tumors (PitNETs). The cause of CS invasion in PitNETs has not been fully elucidated. The tumor immune microenvironment, known to promote aggressive behavior in various types of tumors, has not been examined for PitNETs. Vascular endothelial growth factor (VEGF)/VEGF receptor (VEGFR) signaling is strongly associated with the tumor immune microenvironment. In the present study, these molecular and histopathological characteristics were examined in invasive non-functional PitNETs (NF-PitNETs). Twenty-seven patients with newly diagnosed NF-PitNETs (with CS invasion: 17, without CS invasion: 10) were analyzed by immunohistochemistry for VEGF-A/VEGFR1 and 2, hypoxia-inducible Factor (HIF), tumor-infiltrating lymphocytes, immunosuppressive cells including regulatory T cells (Tregs) and tumor-associated macrophages (TAMs), and immune checkpoint molecules. Previously validated tumor proliferation markers including mitotic count, Ki-67 index, and p53 were also analyzed for their expressions in NF-PitNETs. VEGF-A and VEGFR1 were expressed on not only vascular endothelial cells, but also on tumor cells. The expressions of VEGF-A and VEGFR1 were significantly higher in NF-PitNETs with CS invasion. The number of TAMs and the expression of PD-L1 were also significantly higher in NF-PitNETs with CS invasion than in NF-PitNETs without CS invasion. The high expression of VEGF-A and VEGFR1 and associated immunosuppressive microenvironment were observed in NF-PitNETs with CS invasion, suggesting that a novel targeted therapy can be applied.

Harmine suppresses bladder tumor growth by suppressing vascular endothelial growth factor receptor 2-mediated angiogenesis.

Angiogenesis is a vital step during the process of oncogenesis of a lot of tumors, with no exception in bladder cancer. One of the useful strategies for the development of new drugs against cancer is targeting angiogenesis. In the present study, we found that a small-molecule natural product, which belonged to the β-carboline alkaloid, named harmine, could strongly inhibit tumor angiogenesis thus exhibiting its ideal treatment efficacy in bladder cancer. In vivo study verified that harmine had the effect of inhibition on human bladder tumor xenograft growth. The inhibitory effect of harmine to bladder cancer growth was coordinated by the effects shown on angiogenesis. To further explore the pharmacological activities of harmine, we tested harmine’s influence on blood vessel formation and found that harmine effectively blocked the microvessel sprouting in rat aortic ring assay when stimulated by vascular endothelial growth factor (VEGF). Furthermore, harmine inhibited human umbilical vein endothelial cell (HUVEC) proliferation as well as chemotactic motility, and when we treated HUVEC cell with harmine, the formation of capillary-like structures was also restrained. Moreover, harmine induced bladder cancer cell apoptosis through triggering the caspase-dependent apoptotic pathway and the downstream vascular endothelial growth factor receptor 2 (VEGFR2) kinase pathway was down-regulated, thus suppressing tumor development signals. Herein, our study demonstrated that natural product harmine might have potential in curing human bladder tumor because of its pharmacological function on tumor angiogenesis, trigged by VEGFR2 signaling pathways.

SFlt‐1 Production in Endothelial Cells is Regulated in Part by VEGF Receptor Signaling

Preeclampsia (PE) is a pregnancy‐specific hypertensive disorder complicating 2–8% of pregnancies worldwide. Although the etiology of PE is still unknown, inadequate perfusion of the placenta and the resulting placental ischemia are believed to be the initiating pathological factors of the disease. The ischemic placenta releases a number of factors that drive the disorder in the mother, including soluble fms‐like tyrosine kinase‐1 (sFlt‐1), a soluble splice variant of vascular endothelial growth factor receptor (VEGFR‐1), containing only the extracellular domain of the receptor. Because sFlt‐1 binds and sequesters vascular endothelial growth factor (VEGF), it acts as an antiangiogenic factor, causing angiogenic imbalance and endothelial dysfunction. While sFlt‐1 in PE is thought to originate primarily in the placenta, another proposed source is the maternal endothelium, although the factors which could cause upregulation of sFlt‐1 in these tissues is unclear. Here we hypothesized that VEGF could directly regulate production of sFlt‐1 in vascular endothelial cells. To test our hypothesis, we treated Human Umbilical Vein Endothelial Cells (HUVECs) with various VEGFR inhibitors to discern whether VEGFRs contribute to sFlt‐1 production. Cells were cultured in a 24 well plate and were treated with the VEGFR1/2 antagonist Vatalanib (Vat), VEGFR2 Kinase Inhibitor (V2KI), the neuropilin inhibitor EG00229, or left untreated as controls. After 24 hours media and cells were collected for ELISA and RNA isolation. sFlt‐1 exists as three main splice variants in human: V2, V3, and V4, with V2 being the predominant subtype. Because of this, expression levels of all three variants, as well as full length Flt‐1 receptor, were measured via qRT‐PCR and normalized to β‐actin. sFlt‐1 V2 expression was reduced by almost 70% (1.25 ± 0.36% vs 0.39 ± 0.04% fold change; p<0.05) in the EG00229 treated group but was not significantly different than controls in Vat and V2KI groups. sFlt‐1 V4 expression levels also showed a downward trend with EG00229 treatment, although this did not reach significance (p=0.065). sFlt‐1 V4 expression levels were not significantly different between Vat and V2KI treatment and the control group. sFlt‐1 V3 expression levels did not significantly change between any of the treated groups and the control group. No effect was observed in any experimental group on full length Flt‐1 expression levels compared to control. Surprisingly, when measured with ELISA, only treatment with Vat significantly decreased media sFlt‐1 levels (6145 ± 411.2pg/ml vs 3264 ± 666.5pg/ml; p<0.01). However, non‐significant trends to decrease media sFlt‐1 in V2KI (6145 ± 411.2pg/ml vs 5220 ± 111.8pg/ml; p=0.1043) and EG00229 (6145 ± 411.2pg/ml vs 5258 ± 378.8pg/ml; p=0.1237) were also observed. While EG00229 treatment changed sFlt‐1 expression levels the most, only pan‐antagonism of VEGFR1 and R2 significantly decreased media sFlt‐1 levels. From this data we concluded that VEGFR1 and R2 signaling could partially sFlt‐1 release through non‐genomic mechanisms. Future studies will look to elucidate the mechanisms through which VEGFR1 and R2 control sFlt‐1 production.Support or Funding InformationThis work was supported in part by NIH grants P01HL51971, P20GM104357, T32HL105324, and R01HL137791This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Two Birds, One Stone: Double Hits on Tumor Growth and Lymphangiogenesis by Targeting Vascular Endothelial Growth Factor Receptor 3.

Vascular endothelial growth factor receptor 3 (VEGFR3) has been known for its involvement in tumor-associated lymphangiogenesis and lymphatic metastasis. The VEGFR3 signaling is stimulated by its main cognate ligand, vascular endothelial growth factor C (VEGF-C), which in turn promotes tumor progression. Activation of VEGF-C/VEGFR3 signaling in lymphatic endothelial cells (LECs) was shown to enhance the proliferation of LECs and the formation of lymphatic vessels, leading to increased lymphatic metastasis of tumor cells. In the past decade, the expression and pathological roles of VEGFR3 in tumor cells have been described. Moreover, the VEGF-C/VEGFR3 axis has been implicated in regulating immune tolerance and suppression. Therefore, the inhibition of the VEGF-C/VEGFR3 axis has emerged as an important therapeutic strategy for the treatment of cancer. In this review, we discuss the current findings related to VEGF-C/VEGFR3 signaling in cancer progression and recent advances in the development of therapeutic drugs targeting VEGF-C/VEGFR3.

HIF prolyl hydroxylase inhibitor FG-4497 enhances mouse hematopoietic stem cell mobilization via VEGFR2/KDR

AbstractIn normoxia, hypoxia-inducible transcription factors (HIFs) are rapidly degraded within the cytoplasm as a consequence of their prolyl hydroxylation by oxygen-dependent prolyl hydroxylase domain (PHD) enzymes. We have previously shown that hematopoietic stem and progenitor cells (HSPCs) require HIF-1 for effective mobilization in response to granulocyte colony-stimulating factor (G-CSF) and CXCR4 antagonist AMD3100/plerixafor. Conversely, HIF PHD inhibitors that stabilize HIF-1 protein in vivo enhance HSPC mobilization in response to G-CSF or AMD3100 in a cell-intrinsic manner. We now show that extrinsic mechanisms involving vascular endothelial growth factor receptor-2 (VEGFR2), via bone marrow (BM) endothelial cells, are also at play. PTK787/vatalanib, a tyrosine kinase inhibitor selective for VEGFR1 and VEGFR2, and neutralizing anti-VEGFR2 monoclonal antibody DC101 blocked enhancement of HSPC mobilization by FG-4497. VEGFR2 was absent on mesenchymal and hematopoietic cells and was detected only in Sca1+ endothelial cells in the BM. We propose that HIF PHD inhibitor FG-4497 enhances HSPC mobilization by stabilizing HIF-1α in HSPCs as previously demonstrated, as well as by activating VEGFR2 signaling in BM endothelial cells, which facilitates HSPC egress from the BM into the circulation.

GEP100/ARF6 regulates VEGFR2 signaling to facilitate high-glucose-induced epithelial-mesenchymal transition and cell permeability in retinal pigment epithelial cells.

Cell permeability and epithelial-mesenchymal transition (EMT) were found to be enhanced in diabetic retinopathy, and the aim of this study was to investigate the underlying mechanism. ARPE-19 cell line or primary retinal pigment epithelial (RPE) cells were cultured under high or normal glucose conditions. Specific shRNAs were employed to knock down ADP-ribosylation factor 6 (ARF6), GEP100, or VEGF receptor 2 (VEGFR2) in ARPE-19 or primary RPE cells. Cell migration ability was measured using Transwell assay. Western blotting was used to measure indicated protein levels. RPE cells treated with high glucose showed increased cell migration, paracellular permeability, EMT, and expression of VEGF. Knockdown of VEGFR2 inhibited the high-glucose-induced effects on RPE cells via inactivation of ARF6 and MAPK pathways. Knockdown ARF6 or GEP100 led to inhibition of high-glucose-induced effects via inactivation of VEGFR2 pathway. Knockdown of ARF6, but not GEP100, decreased high-glucose-induced internalization of VEGFR2. High-glucose enhances EMT and cell permeability of RPE cells through activation of VEGFR2 and ARF6/GEP100 pathways, which form a positive feedback loop to maximize the activation of VEGF/VEGFR2 signaling.

VEGFR-3 Signaling Regulates Triglyceride Retention and Absorption in the Intestine.

The lymphatic system transports dietary lipids absorbed and packaged as chylomicrons by enterocytes, for delivery to the bloodstream. Once considered a passive drainage, chylomicron entry into intestinal lymphatic vessels, or lacteals, is now emerging to be an active process controlled by a dynamic and complex regulation. Vascular endothelial growth factor (VEGF)-C, a major lymphangiogenic factor, regulates lacteal maintenance and function. Little is known about the role of its cognate tyrosine kinase VEGF receptor 3 (VEGFR-3) during lipid absorption. Here we investigated role of VEGFR-3 signaling in triglyceride (TG) absorption and distribution into tissues using the Chy mouse model, which bears an inactivating mutation in the tyrosine kinase domain of VEGFR-3 (heterozygous A3157T mutation resulting in I1053F substitution). Our data show that inactivation of VEGFR-3 tyrosine kinase motif leads to retention of TGs in the enterocytes of the small intestine, decreased postprandial levels of TGs in the plasma and increased excretion of free fatty acids (FFAs) and TGs into their stools. We further show that levels of nitric oxide (NO), required for chylomicron mobilization into the bloodstream, are significantly reduced in the Chy intestine after a fat bolus suggesting a critical role for VEGFR-3 signaling in the generation of NO during lipid absorption. Our data support the hypothesis that VEGFR-3 signaling plays an important role in chylomicron-TG entry into lacteals, possibly affecting TG trafficking to peripheral tissues.

Wound healing complications with lenvatinib identified in a pharmacovigilance database.

The U.S. Food and Drug Administration (FDA) has approved several vascular endothelial growth factor receptor (VEGFR) tyrosine kinase inhibitors, including lenvatinib, for thyroid and renal malignancies. Inhibition of the VEGFR signaling pathway impairs angiogenesis and can disrupt wound healing. The objective of this work was to evaluate wound healing complications as a potential safety risk for patients treated with lenvatinib. We searched the FDA Adverse Event Reporting System (FAERS) database for postmarketing reports of wound healing complications with lenvatinib between 13 February 2015 (FDA approval date) and 15 February 2017. The search identified nine FAERS cases of lenvatinib-associated wound healing complications that were not previously reported in the medical literature. Seven cases involved postoperative wound healing complications, such as impaired healing or wound dehiscence. In our case series, the reported time to identification of delayed wound healing from tissue injury or surgery varied over a wide range (4-58 days). The time of initial lenvatinib exposure relative to the tissue injury was also highly varied in our series, which may have influenced the development and detection of impaired healing. FAERS case-level evidence suggests that lenvatinib may have contributed to wound healing complications based on temporality and biologic plausibility. Healthcare professionals should be aware of this safety risk to facilitate prompt recognition and risk mitigation.

Junctional Adhesion Molecule-a Regulates Vascular Endothelial Growth Factor-a-Dependent Permeability

Introduction: Vascular barrier function is important for maintaining vascular integrity and is regulated by endothelial tight junctions. Breaches in the tight junction result in increased endothelial cell (EC) permeability, which leads to a variety of pathological conditions such as edema, vascular degeneration, diabetic retinopathy, and ischemia reperfusion injury. Junctional adhesion molecule A (JAM-A), is an integral part of the tight junctions of ECs. It has been shown that a lack of Jam-A affects neutrophil extravasation and FGF-2-induced angiogenesis. Role of JAM-A in regulation of vascular permeability is not known.Methods: Jam-A knockout (KO) and age-matched wild-type (WT) control mice of both sexes were used in this study. In vivo vascular permeability was assessed using Miles assay and quantified colorimetrically by extracting Evans blue in formamide at 56oC. The circulating plasma level of vascular endothelial growth factor A (VEGF-A) in WT and Jam-A KO mice was measured by ELISA assay. Surface expression of VEGF receptor 2 (VEGFR2) was analyzed by flow cytometry. Quantitative real- time PCR was used to determine gene transcription. Protein expression was determined by western blotting. VEGF-induced WT and Jam-A KO EC migration was analyzed by both scratch and trans-well assays.Results: Although Jam-A resides at the endothelial tight junctions, genetic ablation of Jam-A does not affect blood vessel structure. However, basal vascular permeability was significantly enhanced (P<0.05) in Jam-A KO mice compared to the WT controls, suggesting that a lack of Jam-A in EC compromises vascular integrity. This was further supported by the fact that there was increased edema in the lungs of the Jam-A KO mice compared to WT controls. Since VEGF is also known as vascular permeability factor, we evaluated the effect of VEGF-A on vascular permeability in Jam-A KO mice. Upon intravenous injection of VEGF-A (100ng/ml), EC permeability was significantly increased in both WT and Jam-A KO mice. In order to determine the cause of this enhanced basal permeability, we evaluated the levels of VEGF-A in WT and Jam-A KO mice. Jam-A deficient mice showed a three-fold (P<0.004) increased level of plasma VEGF-A compared to WT mice. Additionally, VEGF-A protein level was also increased in the ECs from Jam-A KO mice compared to WT controls. Furthermore, mRNA levels of VEGFR2, but not sFlt1, were increased in the Jam-A KO ECs compared to WT ECs. Protein levels as well as surface expression of VEGFR2 were significantly increased (P<0.001) in Jam-A KO ECs compared to WT ECs, suggesting that lack of Jam-A results in increased levels of VEGF-A/VEGFR2. We next evaluated the protein and mRNA levels of both hypoxia-inducible factor-1 α (HIF-1α), a transcription factor known to regulate expression of VEGF-A, and inhibitor of DNA binding 1 (Id1), a transcriptional factor known to regulate expression of VEGFR2. We found that bothHIF-1α and Id1 mRNA as well as protein levels were significantly (P<0.001 and P<0.05 respectively) augmented in Jam-A KO ECs compared to WT ECs. Additionally, overexpression of JAM-A in human umbilical vein endothelial cells (HUVECs) resulted in significantly reduced expression of bothHIF-1α and Id1 proteins. Furthermore, ECs isolated from Jam-A KO mouse showed significantly enhanced (P<0.05) cell migration in response to VEGF-A as compared to WT ECs, suggesting that Jam-A ECs respond favorably to VEGF-A due to increased VEGFR2 on their surface. To confirm that the observed augmented permeability in Jam-A KO mice was primarily due to enhanced VEGFR2 signaling, we inhibited VEGFR2 in mice using DC101 (200μg/kg), a function-blocking antibody. Isotype specific IgG (200μg/kg) was used as control. Inhibition of VEGFR2 completely abrogated basal as well as VEGF-A-induced vascular permeability in both Jam-A KO and WT mice, suggesting that the augmented EC permeability in Jam-A KO mice is not due to structural defect in tight junctions, but due to enhanced VEGF-A/VEGFR2 signaling.Conclusion: Our results suggest that JAM-A positively regulates EC tight junction integrity by suppressing levels of VEGF-A and VEGFR2 through downregulation ofHIF-1α and Id1 transcription factors. DisclosuresNo relevant conflicts of interest to declare.

ACTG1 and TLR3 are biomarkers for alcohol-associated hepatocellular carcinoma.

Alcohol consumption is a risk factor for the development of hepatocellular carcinoma (HCC); however, the association between alcohol and HCC remains unknown. The present study aimed to identify key genes related to alcohol-associated HCC to improve the current understanding of the pathology of this disease. Alcohol-associated and non-alcohol-associated HCC samples in the GSE50579 dataset of the Gene Omnibus Database were analyzed to investigate altered gene expression. Integrated bioinformatics methods were employed to clarify the biological functions of the differentially expressed genes (DEGs), including Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG) and protein-protein interactions (PPIs). The present study reported that candidate biomarker micro (mi)RNAs via TargetScan Human 7.1. DEGs and their associated miRNAs (according to bioinformatics analysis) were validated using reverse transcription-quantitative polymerase chain reaction (RT-qPCR). Additionally, 284 EGs from the GSE50579 dataset were revealed. In GO term analysis, DEGs were closely associated with the ‘regulation of nucleic acid metabolism’. KEGG pathway analysis indicated that the DEGs were tightly engaged in the ‘VEGF and VEGF receptor signaling network’, ‘proteoglycan syndecan-mediated signaling events’, ‘erbB receptor signaling’ and ‘β1 integrin cell surface interactions’. According to the results of PPI and heat map analysis, the main hub genes were centrin 3 (CETN3), Toll-like receptor 3 (TLR3), receptor tyrosine-protein kinase (ERBB4), heat shock protein family member 8, actin γ1 (ACTG1) and α-smooth muscle actin. it was demonstrated that the ACTG1, TLR3, miR-6819-3p and miRΝΑ (miR)-6877-3P had undefined associations. Furthermore, RT-qPCR analysis revealed that miR-6819-3p and miR-6877-3P may enhance the expression levels of ACTG1 and inhibit the expression levels of TLR3 in alcohol-associated HCC tissues. TLR3 and ACTG1 were proposed as potential biomarkers of alcohol-associated HCC. Investigation into the regulatory functions of miR-6819-3p and miR-6877-3P may provide novel insights into the treatment of alcohol-associated HCC.

Vascular endothelial growth factor-C promotes human mesenchymal stem cell migration via an ERK-and FAK-dependent mechanism.

Vascular endothelial cell growth factor-C (VEGF-C) is a member of the VEGF family and plays a role in various biological activities. VEGF-C enhances proliferation and migration of lymphatic endothelial cells and vascular endothelial cells through VEGF receptor 2 (VEGFR2) and/or receptor 3 (VEGFR3), and thereby induces lymphangiogenesis or angiogenesis. However, it remains unclear whether VEGF-C promotes the migration of mesenchymal stem cells (MSCs). Here, we investigated the effects of VEGF-C on the migration of MSCs and evaluated the underlying molecular mechanisms. VEGF-C treatment significantly induced the migration of MSCs, which is accompanied by the promotion of actin cytoskeletal reorganization and focal adhesion assembly. VEGF-C treatment enhanced the phosphorylation of VEGFR2 and VEGFR3 proteins in MSCs, and pretreatment with VEGFR2 and VEGFR3 kinase inhibitors effectively suppressed the VEGF-C-induced MSC migration. In addition, VEGF-C treatment promoted phosphorylation of ERK and FAK proteins in MSCs, and inhibition of VEGFR2 and VEGFR3 signaling pathways abolished the VEGF-C-induced activation of ERK and FAK proteins. Furthermore, treatment with ERK and FAK inhibitors suppressed VEGF-C-induced actin cytoskeletal reorganization and focal adhesion assembly, and then significantly inhibited MSCs migration. These results suggest that VEGF-C-induced MSC migration is mediated via VEGFR2 and VEGFR3, and follows the activation of the ERK and FAK signaling pathway. Thus, VEGF-C may be valuable in tissue regeneration and repair in MSC-based therapy.

Downregulation of VEGFR3 signaling alters cardiac lymphatic vessel organization and leads to a higher mortality after acute myocardial infarction

Heart has a wide lymphatic network but the importance of cardiac lymphatic system in heart diseases has remained unclear. Vascular Endothelial Growth Factor Receptor 3 (VEGFR3) is a key molecule in the development and maintenance of cardiac lymphatic vessels. Here we characterized the role of VEGFR3 in healthy hearts and after myocardial infarction (MI) by using sVEGFR3 transgenic mice expressing a soluble decoy VEGFR3 under K14 promoter and Chy mice which have an inactivating mutation in the VEGFR3 gene. Cardiac lymphatic vessels were significantly dilated in the healthy hearts of sVEGFR3 mice when compared to controls. Lymphatic vessels formed large sheet-like structures in Chy mice. Attenuated VEGFR3 signaling led to a more severe MI predisposing to a significantly higher mortality in sVEGFR3 mice than in control mice. sVEGFR3 mice displayed intramyocardial hemorrhages in the infarcted area indicating hyperpermeability of the vasculature. Furthermore, novel MRI methods TRAFF2 and TRAFF4 and histological analysis revealed a modified structure of the fibrotic infarcted area in sVEGFR3 mice. In conclusion, the downregulation of VEGFR3 signaling modifies the structure of cardiac lymphatic network and causes vascular leakiness and increased mortality after MI.

The Chinese herbal formula Fuzheng Quxie Decoction attenuates cognitive impairment and protects cerebrovascular function in SAMP8 mice.

PurposeThis study was designed to explore the underlying mechanism of action for a Fuzheng Quxie Decoction (FQD) in Alzheimer’s disease (AD), to validate its neuroprotective effects, and to provide experimental support for its predicted mechanism of action.MethodsAn integrative approach to network pharmacology was performed to predict the mechanism of action for treatment of AD with FQD. The predicted mechanism was validated in SAMP8 mice.ResultsWith predicted putative FQD targets and a collection of AD-related genes, 245 possible regulatory targets of FQD were identified for the treatment of AD. Pathway-enrichment analysis for the possible regulatory targets indicated that vascular endothelial growth factor (VEGF) and VEGF-receptor signaling were pivotal in the treatment of AD with FQD. In vivo experiments confirmed the neuroprotective effect and the predicted mechanism of action for treatment of AD with FQD.ConclusionThis study contributes to an understanding of the neuroprotective effect of FQD and its potential mechanism of action for the treatment of AD.