VEGFR2 Phosphorylation Research Articles

Propofol attenuated TNF-\u03b1-modulated occludin expression by inhibiting Hif-1\u03b1/ VEGF/ VEGFR-2/ ERK signaling pathway in hCMEC/D3 cells

BackgroundThe levels of tight junction proteins (TJs), especially occludin, correlate with blood-brain barrier (BBB) disruption caused by inflammation in central nervous system (CNS). It has been reported that propofol, the most commonly used anesthetic, could inhibit inflammation response in CNS. In this study, we investigated the effects of tumor necrosis factor-α (TNF-α) and propofol on occludin expression in human cerebral microvascular endothelial cell line, D3 clone (hCMEC/D3 cells), and explored the underlying mechanisms.MethodsThe hCMEC/D3 cells were treated with propofol, followed by TNF-α. The expression and phosphorylation of Hif-1α, VEGF, VEGFR-2, ERK, p38MAPK and occludin were measured by Western blot analysis. The cell viability of hCMEC/D3 cells was measured by cell counting kit-8.ResultsTNF-α (10 ng/ml, 4 h) significantly decreased the expression of occludin, which was attenuated by propofol (25 μM). TNF-α induced Hif-1α/VEGF/VEGFR-2/ERK signaling pathway, while propofol could inhibit it. TNF-α induced the phosphorylation of p38MAPK, while propofol had no effect on it. In addition, the inhibitors of Hif-1α, VEGFR-2, and ERK could reduce the effect of TNF-α on occludin expression.ConclusionTNF-α could decrease the expression of occludin via activating Hif-1α/ VEGF/ VEGFR-2/ ERK signaling pathway, which was attenuated by propofol.

Abstract 4855: AMC303 inhibits tumor growth and metastasis in animal models by targeting CD44v6, a co-receptor of multiple oncogenic receptor tyrosine kinases

Abstract Introduction: CD44v6, a member of the CD44 family, is an essential co-receptor for c-MET, RON and VEGFR-2. The receptor tyrosine kinases (RTKs) c-MET and RON, are key players in tumorigenic pathways, are activated by the ligands HGF and MSP, respectively and homo- or heterodimerize with each other or can be autoactivated due to amplification. The RTK VEGFR-2 is critical for angiogenesis in solid epithelial tumors. The peptidergic inhibitor AMC303, which allosterically and selectively binds to CD44v6, was investigated for its inhibitory effects on ligand-induced and autophosphorylated c-MET activation, and its impact on tumor growth and metastases in vivo. Methods: In vitro, tumor cells were incubated with AMC303 for 30 min prior to induction with an RTK-ligand. Analysis of RTK activation was carried out by Western blotting. For in vivo xenotransplantation human pancreatic tumor cells (L3.6pl) were orthotopically injected. Animals were treated with AMC303 i.p. at 0.1, 1, 10 mg/kg QOD or QWK for 3 weeks to address dose-dependency, the optimal treatment schedule was investigated with one, three or five times weekly administrations. Regression of metastases was investigated at 1 mg/kg QOD. Survival was evaluated with a dose of 1 mg/kg QOD. Immunohistochemical (IHC) analysis was performed for cleaved caspase 6, smooth muscle actin and CD31. Reduction of vessel permeability was assessed in Balb/C mice with the Miles assay and quantification of intra-tumoral FITC dextran after i.v. injection. Results: In vitro, AMC303 inhibited c-MET, VEGFR2 and RON phosphorylation induced by their respective ligands. Notably, AMC303 inhibited c-MET activation in cells with enhanced c-MET signaling caused by the exon 14 skipping mutation, but not c-MET auto-phosphorylation due to gene amplification. In vivo, AMC303 treatment attenuated primary xenograft tumor growth and metastasis and significantly prolonged survival. IHC on treated xenograft-tumors revealed an increase in apoptosis and necrosis along with a reduction in myofibroblast infiltration, angiogenesis and vessel permeability. Pharmacokinetic properties after i.p. administration of AMC303 revealed an terminal half-life of 6h. Administration schedules of AMC303 (1mg/kg) given one, three or five times per week had no significant impact on tumor growth attenuation. AMC303 treatment could be paused for 10 days without incurring significant tumor regrowth. Conclusions: AMC303 inhibits c-MET in vitro in a ligand-dependent fashion and in vivo significantly improved overall survival in an orthotopic pancreatic xenograft mouse tumor model. AMC303 effectively attenuated both primary and metastatic tumor growth by increasing apoptosis, reduction of angiogenesis and vascular normalization. These findings demonstrate the benefit of targeting multiple oncogenic RTK signaling pathways by AMC303. Citation Format: Tina Heumann, Vanessa Al-Rawi, Thorsten Läufer, Martin Augsten, Alexandra Matzke-Ogi, Oliver Coutelle. AMC303 inhibits tumor growth and metastasis in animal models by targeting CD44v6, a co-receptor of multiple oncogenic receptor tyrosine kinases [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 4855.

Abstract 185: Direct cytotoxic effect of a novel anti-angiogenic drug F16 towards U87MG glioblastoma cell line

Abstract Glioblastoma multiforme (GBM) is one of the most aggressive and lethal types of brain cancers with low 5-year survival rate. Therefore, search for better drugs and effective treatment strategies for GBM to improve patient outcomes is continuing. Since GBM is one of the most highly vascularized solid tumors and its growth is angiogenesis-dependent, antagonizing tumor angiogenesis by using angiogenesis inhibitors seems to be one of the promising approaches. In this context, intensive pre-clinical evaluation of our patented anti-angiogenic compound, code-named F16, has exhibited potent anti-angiogenic and anti-tumor activities via selectively antagonizing VEGFR-2 in both in vitro and in vivo models. More importantly, assessment of biochemical parameters that are reflecting the safety of F16 in the in vivo system showed no alterations in the toxicological parameters of heart, liver, kidney, and pancreas, after 30 days of treatment, at the dose of 100 mg/kg body weight. Hence, we tested the direct effects of F16 for inhibiting the growth, angiogenesis and the migratory abilities of the U87MG glioblastoma cells, which are known to express high levels of VEGFR. Our in vitro studies have confirmed potent inhibitory effects of F16 towards the migration and invasion of U87MG cells, and also revealed potent cytotoxic effects (IC50 26 µM) against U87MG cells in comparison to Temozolomide (IC50 430 µM) treatment. In addition, F16 inhibited the phosphorylation of VEGFR-2 through competitive binding and also induced cell cycle arrest and apoptosis by activating p53 pathway in U87MG cells. Furthermore, our in vivo results with ectopically implanted xenograft model confirm the fact that F16 can significantly inhibit tumor growth in the mice implanted with U87MG glioblastoma cell line. Based on our preliminary results, we are proposing that F16 could be a potential candidate for treating GBM, either in monotherapy or in combination with a cytoreductive drug. (This research was supported by the generous funds provided by the Community Foundation of Broward, Florida and also by the Royal Dames of Cancer Research Inc., Ft. Lauderdale, Florida) Citation Format: Mohammad Algahtani, Khalid Alhazzani, Thiagarajan Venkatesan, Ali Alaseem, Sivanesan Dhandayuthapani, Appu Rathinavelu. Direct cytotoxic effect of a novel anti-angiogenic drug F16 towards U87MG glioblastoma cell line [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2019; 2019 Mar 29-Apr 3; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2019;79(13 Suppl):Abstract nr 185.

Design, synthesis, and biological evaluation of tetrahydroisoquinoline-based diaryl urea derivatives for suppressing VEGFR-2 signaling

A novel structural series of tetrahydroisoquinoline-based compounds that incorporate the diaryl urea moiety was designed, synthesized, and biologically evaluated as suppressors of VEFGR-2 signaling. As a consequence, compounds 9k and 9s exhibited comparable or superior cytotoxic activity to that of gefitinib against the tested three cell lines, including A549, MCF-7, and PC-3. Importantly, both of them downregulated the expression of VEGFR-2, and inhibited VEGFR-2 phosphorylation at the concentration of 0.5 or 1.0 μmol/l. Besides, they suppressed human umbilical vein endothelial cell tube formation at the concentration of 4.0 μmol/l. Considering their capability of down-regulating VEGFR-2 expression and inhibiting VEGFR-2 phosphorylation, 9k and 9s may serve as suppressors of angiogenesis for further investigation.

Apatinib preferentially inhibits PC9 gefitinib-resistant cancer cells by inducing cell cycle arrest and inhibiting VEGFR signaling pathway

BackgroundLung cancer is one of the most common and deadly tumors around the world. Targeted therapy for patients with certain mutations, especially by use of tyrosine kinase inhibitors (TKIs) targeting epidermal growth factor receptor (EGFR), has provided significant benefit to patients. However, gradually developed resistance to the therapy becomes a major challenge in clinical practice and an alternative to treat such patients is needed. Herein, we report that apatinib, a novel anti-angiogenic drug, effectively inhibits obtained gefitinib-resistant cancer cells but has no much effect on their parental sensitive cells.MethodsGefitinib-resistant lung cancer cell line (PC9GR) was established from its parental sensitive line (PC9) with a traditional EGFR mutation after long time exposure to gefitinib. Different concentrations of apatinib were used to treat PC9, PC9GR, and other two lung cancer cell lines for its anti-growth effects. RNA sequencing was performed on PC9, PC9GR, and both after apatinib treatment to detect differentially expressed genes and involved pathways. Protein expression of key cycle regulators p57, p27, CDK2, cyclin E2, and pRb was detected using Western blot. Xenograft mouse model was used to assess the anti-tumor activity of apatinib in vivo.ResultsThe established PC9GR cells had over 250-fold increased resistance to gefitinib than its sensitive parental PC9 cells (IC50 5.311 ± 0.455 μM vs. 0.020 ± 0.003 μM). The PC9GR resistance cells obtained the well-known T790M mutation. Apatinib demonstrated much stronger ( ~ fivefold) growth inhibition on PC9GR cells than on PC9 and other two lung cancer cell lines, A549 and H460. This inhibition was mostly achieved through cell cycle arrest of PC9GR cells in G1 phase. RNA-seq revealed multiple changed pathways in PC9GR cells compared to the PC9 cells and after apatinib treatment the most changed pathways were cell cycle and DNA replication where most of gene activities were repressed. Consistently, protein expression of p57, CDK2, cyclin E2, and pRb was significantly impacted by apatinib in PC9GR cells. Oral intake of apatinib in mouse model significantly inhibited establishment and growth of PC9GR implanted tumors compared to PC9 established tumors. VEGFR2 phosphorylation in PC9GR tumors after apatinib treatment was significantly reduced along with micro-vessel formation.ConclusionsApatinib demonstrated strong anti-proliferation and anti-growth effects on gefitinib resistant lung cancer cells but not its parental sensitive cells. The anti-tumor effect was mostly due to apatinib induced cell cycle arrest and VEGFR signaling pathway inhibition. These data suggested that apatinib may provide a benefit to patients with acquired resistance to EGFR-TKI treatment.

Protein Tyrosine Phosphatase, Receptor Type B (PTPRB) Inhibits Brown Adipocyte Differentiation through Regulation of VEGFR2 Phosphorylation.

Brown adipocytes have an important role in the regulation of energy balance through uncoupling protein-1 (UCP-1)-mediated nonshivering thermogenesis. Although brown adipocytes have been highlighted as a new therapeutic target for the treatment of metabolic diseases, such as obesity and type II diabetes in adult humans, the molecular mechanism underlying brown adipogenesis is not fully understood. We recently found that protein tyrosine phosphatase receptor type B (PTPRB) expression dramatically decreased during brown adipogenic differentiation. In this study, we investigated the functional roles of PTPRB and its regulatory mechanism during brown adipocyte differentiation. Ectopic expression of PTPRB led to a reduced brown adipocyte differentiation by suppressing the tyrosine phosphorylation of VEGFR2, whereas a catalytic inactive PTPRB mutant showed no effects on differentiation and phosphorylation. Consistently, the expression of brown adipocyte-related genes, such as UCP-1, PGC-1α, PRDM16, PPAR-γ, and CIDEA, were significantly inhibited by PTPRB overexpression. Overall, these results suggest that PTPRB functions as a negative regulator of brown adipocyte differentiation through its phosphatase activity-dependent mechanism and may be used as a target protein for the regulation of obesity and type II diabetes.

Curcumolide, a unique sesquiterpenoid from Curcuma wenyujin displays anti-angiogenic activity and attenuates ischemia-induced retinal neovascularization

BackgroundTargeting vascular endothelial growth factor is a common treatment strategy for neovascular eye disease, a leading cause of visual impairment and blindness. However, these approaches are limited or carry various complications. Therefore, there is an urgent need for the development of unique therapeutic approaches. PurposeTo investigate the anti-angiogenic effects of curcumolide and its mechanism of action. Methods /Study designsIn this study, we examine the effects of curcumolide on the process of vasculature formation, including cell proliferation, migration, tube formation and apoptosis in vitro using human umbilical vascular endothelial cells (HUVECs). We also assess the anti-angiogenic effects of curcumolide in vivo using a mouse model of oxygen induced retinopathy (OIR). The mechanism of anti-angiogenic effects was investigated by measuring the expression level of various signaling proteins and the molecular docking simulations. ResultsIntravitreal injection of curcumolide reduced the formation of retinal neovascular tufts and VEGFR2 phosphorylation in the murine OIR model at concentrations administered without definite cellular and retinal toxicities. Curcumolide suppressed VEGF-induced HRMECs proliferation, migration and tube formation in a dose-dependent manner. Meanwhile, it promoted caspase-dependent apoptosis. Curcumolide also inhibited VEGF-induced phosphorylation of VEGFR-2 tyrosine kinase, and suppressed downstream protein kinases of VEGFR2, including Src, FAK, ERK, AKT, and mTOR in HRMECs. In silico study revealed that curcumolide bound with ATP-binding sites of the VEGFR2 kinase unit by the formation of a hydrogen bond and hydrophobic interactions. ConclusionCurcumolide has anti-angiogenic activity in HUVECs and in a murine OIR model of ischemia-induced retinal neovascularization, and it might be a potential drug candidate for the treatment of proliferative diabetic retinopathy.

VEGFR-2 Is in a State of Activation in Hair Follicles, Sebaceous Glands, Eccrine Sweat Glands, and Epidermis from Human Scalp: An In Situ Immunohistochemistry Study of Phosphorylated VEGFR-2.

BackgroundRecent research reports that VEGFR-2 is expressed in the whole hair follicle, sebaceous glands, eccrine sweat glands, and epidermis. However, phosphorylated VEGFR-2 was not found, and it could not be ascertained whether the activated form of VEGFR-2 actually participates in the biological control of epidermal appendages. In this study we aimed to determine whether the VEGFR-2 pathway is directly involved in the daily regulation of epidermal appendages biology.Material/MethodsIn this study, we investigated the expression of phosphorylation of VEGFR-2 by immunohistochemical analysis in the epidermis and epidermal appendages in normal human scalp skin.ResultsImmunohistochemical analysis revealed phosphorylation of VEGFR-2 in a whole hair follicle, mainly in the infundibulum basal layer, hair cortex, and medulla in the isthmus, and matrix in the hair bulb. Phosphorylated VEGFR-2 also was found in the sebaceous glands, eccrine sweat glands, and epidermis.ConclusionsTherefore, we suggest that VEGFR-2 activation is involved in routine regulation of human epidermal appendages.

Vascular Endothelial Growth Factor Receptor 3 Regulates Endothelial Function Through β-Arrestin 1.

Receptor signaling is central to vascular endothelial function and is dysregulated in vascular diseases such as atherosclerosis and pulmonary arterial hypertension (PAH). Signaling pathways involved in endothelial function include vascular endothelial growth factor receptors (VEGFRs) and G protein-coupled receptors, which classically activate distinct intracellular signaling pathways and responses. The mechanisms that regulate these signaling pathways have not been fully elucidated and it is unclear what nodes for cross talk exist between these diverse signaling pathways. For example, multifunctional β-arrestin (ARRB) adapter proteins are best known as regulators of G protein-coupled receptor signaling, but their role at other receptors and their physiological importance in the setting of vascular disease are unclear. We used a combination of human samples from PAH, human microvascular endothelial cells from lung, and Arrb knockout mice to determine the role of ARRB1 in endothelial VEGFR3 signaling. In addition, a number of biochemical analyses were performed to determine the interaction between ARRB1 and VEGFR3, signaling mediators downstream of VEGFR3, and the internalization of VEGFR3. Expression of ARRB1 and VEGFR3 was reduced in human PAH, and the deletion of Arrb1 in mice exposed to hypoxia led to worse PAH with a loss of VEGFR3 signaling. Knockdown of ARRB1 inhibited VEGF-C-induced endothelial cell proliferation, migration, and tube formation, along with reduced VEGFR3, Akt, and endothelial nitric oxide synthase phosphorylation. This regulation was mediated by direct ARRB1 binding to the VEGFR3 kinase domain and resulted in decreased VEGFR3 internalization. Our results demonstrate a novel role for ARRB1 in VEGFR regulation and suggest a mechanism for cross talk between G protein-coupled receptors and VEGFRs in PAH. These findings also suggest that strategies to promote ARRB1-mediated VEGFR3 signaling could be useful in the treatment of pulmonary hypertension and other vascular disease.

Recombinant RGD-disintegrin DisBa-01 blocks integrin \u03b1v\u03b23 and impairs VEGF signaling in endothelial cells

BackgroundIntegrins mediate cell adhesion, migration, and survival by connecting the intracellular machinery with the surrounding extracellular matrix. Previous studies demonstrated the interaction between αvβ3 integrin and VEGF type 2 receptor (VEGFR2) in VEGF-induced angiogenesis. DisBa-01, a recombinant His-tag fusion, RGD-disintegrin from Bothrops alternatus snake venom, binds to αvβ3 integrin with nanomolar affinity blocking cell adhesion to the extracellular matrix. Here we present in vitro evidence of a direct interference of DisBa-01 with αvβ3/VEGFR2 cross-talk and its downstream pathways.MethodsHuman umbilical vein (HUVECs) were cultured in plates coated with fibronectin (FN) or vitronectin (VN) and tested for migration, invasion and proliferation assays in the presence of VEGF, DisBa-01 (1000 nM) or VEGF and DisBa-01 simultaneously. Phosphorylation of αvβ3/VEGFR2 receptors and the activation of intracellular signaling pathways were analyzed by western blotting. Morphological alterations were observed and quantified by fluorescence confocal microscopy.ResultsDisBa-01 treatment of endothelial cells inhibited critical steps of VEGF-mediated angiogenesis such as migration, invasion and tubulogenesis. The blockage of αvβ3/VEGFR2 cross-talk by this disintegrin decreases protein expression and phosphorylation of VEGFR2 and β3 integrin subunit, regulates FAK/SrC/Paxillin downstream signals, and inhibits ERK1/2 and PI3K pathways. These events result in actin re-organization and inhibition of HUVEC migration and adhesion. Labelled-DisBa-01 colocalizes with αvβ3 integrin and VEGFR2 in treated cells.ConclusionsDisintegrin inhibition of αvβ3 integrin blocks VEGFR2 signalling, even in the presence of VEGF, which impairs the angiogenic mechanism. These results improve our understanding concerning the mechanisms of pharmacological inhibition of angiogenesis.

Inhibition of VEGFR-2 Phosphorylation and Effects on Downstream Signaling Pathways in Cultivated Human Endothelial Cells by Stilbenes from Vitis Spp.

Stilbenes are phenolic compounds present in different higher plant families that have shown different biological activities, such as antioxidant properties and antitumoral and anti-atherosclerotic effects, among others. Angiogenesis is a key process involved in both cancer and cardiovascular diseases, the vascular endothelial growth factor (VEGF) and its receptor VEGFR-2 being the main triggers. Certain polyphenol compounds, such as flavonoids, have shown a potent capacity to inhibit VEGF and, consequently, angiogenesis. The present work, therefore, aims to evaluate the potential effect of stilbenes on inhibiting VEGF and their subsequent effect on the downstream signaling pathway (PLCγ1, Akt, and eNOS). VEGFR-2 activation was studied through an ELISA assay in the HUVEC line, while the phosphorylation of intracellular downstream proteins PLCγ1, Akt, and eNOS was tested by Western blot. Student's t test was used to determine significant differences between samples. On the one hand, astringin, pallidol, and ω-viniferin showed the lowest IC50 values (2.90 ± 0.27, 4.42 ± 0.67, and 6.10 ± 1.29 μM, respectively) against VEGFR-2 activation. Additionally, VEGF-induced PLCγ1 phosphorylation was significantly inhibited by ε-viniferin, astringin, and ω-viniferin. However, ε-viniferin and pallidol simultaneously enhanced eNOS activation, proving to be via Akt activation in the case of ε-viniferin. For the first time, these data suggest that stilbenes such as astringin, pallidol, ω-viniferin, and ε-viniferin have a potential anti-angiogenic effect and they could be further considered as anti-VEGF ingredients in food and beverages. In addition, ε-viniferin and pallidol significantly allowed eNOS activation and could likely prevent the side effects caused by anti-VEGF hypertension drugs.

Activator of G-protein signaling 8 is involved in VEGF-induced choroidal neovascularization

Choroidal neovascularization (CNV) is associated with age-related macular degeneration (AMD), a major cause of vision loss among elderly people. Vascular endothelial cell growth factor (VEGF) is essential for the development and progression of AMD, and VEGF signaling molecules are effective targets for the treatment of AMD. We recently reported that activator of G-protein signaling 8 (AGS8), a receptor-independent Gβγ regulator, is involved in VEGF-induced angiogenesis in cultured endothelial cells (EC); however, the role of AGS8 in CNV is not yet understood. This study aimed to explore the role of AGS8 in CNV in cultured cells, explanted choroid tissue, and laser-induced CNV in a mouse AMD model. AGS8 knockdown in cultured choroidal EC inhibited VEGF-induced VEGFR-2 phosphorylation, cell proliferation, and migration. AGS8 knockdown also downregulated cell sprouting from mouse choroidal tissue in ex vivo culture. A mouse model of laser-induced CNV, created to analyze the roles of AGS8 in vivo, demonstrated that AGS8 mRNA was significantly upregulated in choroidal lesions and AGS8 was specifically expressed in the neovasculature. Local AGS8 knockdown in intravitreal tissue significantly inhibited laser-induced AGS8 upregulation and suppressed CNV, suggesting that AGS8 knockdown in the choroid has therapeutic potential for AMD. Together, these results demonstrate that AGS8 plays critical roles in VEGF-induced CNV.

Repurposing old carbon monoxide-releasing molecules towards the anti-angiogenic therapy of triple-negative breast cancer.

Triple-negative breast cancer (TNBC) is defined by the lack of expression of the oestrogen and progesterone receptors and HER-2. Recently, carbon monoxide (CO) was found to behave as an important endogenous signalling molecule and to suppress VEGF receptor-2 (VEGFR-2) and protein kinase B phosphorylation. Given that anti-angiogenic drugs exist as one of the few available targeted therapies against TNBC, the aim of this project was to study the effects of CO-releasing molecules (CORMs) on TNBC cell lines and the associated endothelial cells and characterise their anti-angiogenic properties that can be used for the reduction of cancer-driven angiogenesis. Four commercially available CORMs were screened for their cytotoxicity, their effects on cell metabolism, migration, VEGF expression, tube formation and VEGFR-2 activation. The most important result was the reduction in VEGF levels expressed by CORM-treated TNBC cells, along with the inhibition of phosphorylation of VEGFR2 and downstream proteins. The migration and tube formation ability of endothelial cells was also decreased by CORMs, justifying a potential re-purposing of old CORMs towards the anti-angiogenic therapy of TNBC. The additional favourable low cytotoxicity, reduction in the glycolysis levels and downregulation of haem oxygenase-1 in TNBC cells enhance the potential of CORMs against TNBC. In this study, CORM-2 remained the most effective CORM and we propose that CORM-2 may be pursued further as an additional agent in combination with existing anti-angiogenic therapies for a more successful targeting of malignant angiogenesis in TNBC.

Tumor necrosis factor-α regulates angiogenesis of BeWo cells via synergy of PlGF/VEGFR1 and VEGF-A/VEGFR2 axes

ObjectivesTumor necrosis factor-alpha (TNF- α) promotes tumor growth by enhancing tumor angiogenesis; however, the effects on choriocarcinoma remain unknown. We investigated the effects of TNF-α on the production of placental growth factor (PlGF) and vascular endothelial growth factor-A (VEGF-A) in BeWo cells and also examined its significance on the interactions with the endothelial cells by using human umbilical vein endothelial cells (HUVECs). Materials & methodsAfter incubation with TNF-α (10–105 pg/mL), the expression of PlGF and VEGF-A in BeWo cells were assessed by ELISA and RT-PCR. HUVEC tube formation assays were conducted to assess the angiogenic activity of the conditioned medium. The phosphorylation status of VEGFR1 and VEGFR2 in HUVECs under the stimulation of the conditioned medium was assessed by immunoprecipitation and immunoblotting. The same experiments were repeated with recombinant PlGF and VEGF-A to confirm the effects of the growth factors. ResultsLow levels (10–102 pg/mL) of TNF-α enhanced the mRNA and protein levels of PlGF, but the changes in VEGF-A levels were not significant. HUVEC tube formation was promoted by the conditioned medium, and those effects were inhibited by the anti-VEGFR1 antibody and PlGF-siRNA. VEGFR2 was significantly phosphorylated by the conditioned medium, while the effect on VEGFR1 phosphorylation was very weak. HUVEC tube formation was incomplete when recombinant PlGF was used; however, the addition of PlGF promoted the effects of VEGF-A. The addition of PlGF along with VEGF-A also stimulated VEGFR2 phosphorylation. ConclusionsTNF-α promoted PlGF synthesis in BeWo cells and regulated angiogenesis via synergy of the PlGF/VEGFR1 and VEGF-A/VEGFR2 axes.

Beneficial Effects of Deoxyshikonin on Delayed Wound Healing in Diabetic Mice.

Shiunko ointment is composed of five ingredients including Lithospermi Radix (LR), Angelicae Gigantis Radix, sesame seed oil, beeswax, and swine oil. It is externally applied as a treatment for a wide range of skin conditions such as eczema, psoriasis, hair loss, burns, topical wounds, and atopic dermatitis. Deoxyshikonin is the major angiogenic compound extracted from LR. In this study, we investigated the efficacy of LR extract and deoxyshikonin on impaired wound healing in streptozotocin (STZ)-induced diabetic mice. Treatment with LR extract elevated tube formation in human umbilical vein endothelial cells (HUVECs) and exerted antioxidant activity. An open skin wound was produced on the backs of diabetic mice and was then topically treated with deoxyshikonin or vehicle. In addition, deoxyshikonin promoted tube formation in high glucose conditions exposed to HUVECs, and which may be regulated by increased VEGFR2 expression and phosphorylation of Akt and p38. Our results demonstrate that deoxyshikonin application promoted wound repair in STZ-induced diabetic mice. Collectively, these data suggest that deoxyshikonin is an active ingredient of LR, thereby contributing to wound healing in patients with diabetes.

Woodchuck VEGF (wVEGF) characteristics: Model for angiogenesis and human hepatocellular carcinoma directed therapies

Vascular endothelial growth factor (VEGF) stimulates angiogenesis. Human hepatocellular carcinoma (HCC) is a VEGF-driven tumor often associated with chronic hepatitis B or C virus infection. The woodchuck is a well-characterized model of hepatitis B virus related HCC and a valuable tool for translational studies of novel VEGF targeted agents. We cloned the cDNA encoding woodchuck VEGF (wVEGF), transiently expressed it in COS cells and functionally characterized the recombinant protein. The open reading frame of wVEGF contained 645 nucleotides encoding a protein of 214 amino acids. Two protein bands (17 and 25 kDa) were detected in conditioned media of wVEGF expressing COS-1 cells and a single band of 25 kDa was identified in cell lysates. Addition of recombinant wVEGF to COS cells enhanced cell proliferation and stimulated VEGFR2, Akt, ERK1/2, and FAK phosphorylation. Sunitinib, a tyrosine kinase inhibitor, inhibited wVEGF- induced VEGFR2 phosphorylation in a dose-dependent manner. Finally, development of HCC in woodchucks was accompanied by increased laminin and PECAM1 expressing vessels, VEGFR2 expression, increased ligation of VEGF to VEGFR2, and a decrease in collagen IV-positive blood vessels. Our results suggest that woodchuck model can be used further to study angiogenesis and the effect of VEGF directed therapies in human HCC.

Apatinib exerts anti-tumor activity to non-Hodgkin lymphoma by inhibition of the Ras pathway

Apatinib is a tyrosine kinase inhibitor that selectively targets vascular endothelial growth factor receptor-2 (VEGFR-2). Although apatinib has shown promising anti-tumor activity against several types of tumor, its role and underlying mechanism against non-Hodgkin lymphoma (NHL) remain to be explored. Here, we report that apatinib dramatically inhibited in vitro the proliferation of various human NHL cell lines, including Burkitt lymphoma (BL), mantle cell lymphoma (MCL), and diffuse large B-cell lymphoma (DLBCL), in a dose-dependent manner. Moreover, administration of apatinib markedly delayed tumor growth in vivo in a xenograft mouse model derived from human DLBCL OCI-ly3 cells, in association with significantly prolonged survival of tumor-bearing mice. Mechanistically, apatinib suppressed activation of VEGFR2 (manifested by reduced VEGFR2 phosphorylation), accompanied by inhibition of the Ras pathway (reflected by down-regulation Ras, Raf, pMEK1/2, pERK1/2) in OCI-ly1 (GCB subtype of DLBCL) and SU-DHL2 (ABC subtype of DLBCL) cells. Of note, apatinib sharply impaired angiogenesis in vivo in tumor tissues. Together, these results indicate that apatinib displays a marked cytotoxic activity against various types of NHL cells (including BL, MCL, and GCB- or ABC-DLBCL) both in vitro and in vivo. They also suggest that anti-NHL activity of apatinib might be associated with inhibition of tumor cell growth and induction of apoptosis as well as anti-angiogenesis by targeting VEGFR2 and its downstream Ras/Raf/MEK/ERK pathway.

Distinct roles of VE-cadherin for development and maintenance of specific lymph vessel beds.

Endothelial cells line blood and lymphatic vessels and form intercellular junctions, which preserve vessel structure and integrity. The vascular endothelial cadherin, VE-cadherin, mediates endothelial adhesion and is indispensible for blood vessel development and permeability regulation. However, its requirement for lymphatic vessels has not been addressed. During development, VE-cadherin deletion in lymphatic endothelial cells resulted in abortive lymphangiogenesis, edema, and prenatal death. Unexpectedly, inducible postnatal or adult deletion elicited vessel bed-specific responses. Mature dermal lymph vessels resisted VE-cadherin loss and maintained button junctions, which was associated with an upregulation of junctional molecules. Very different, mesenteric lymphatic collectors deteriorated and formed a strongly hyperplastic layer of lymphatic endothelial cells on the mesothelium. This massive hyperproliferation may have been favored by high mesenteric VEGF-C expression and was associated with VEGFR-3 phosphorylation and upregulation of the transcriptional activator TAZ Finally, intestinal lacteals fragmented into cysts or became highly distended possibly as a consequence of the mesenteric defects. Taken together, we demonstrate here the importance of VE-cadherin for lymphatic vessel development and maintenance, which is however remarkably vessel bed-specific.

Novel engineered, membrane-localized variants of vascular endothelial growth factor (VEGF) protect retinal ganglion cells: a proof-of-concept study

Endogenous vascular endothelial growth factor (VEGF-A) can protect retinal ganglion cells (RGC) from stress-induced cell death in ocular hypertensive glaucoma. To exploit the neuroprotective function of VEGF-A for therapeutic application in ocular disorders such as glaucoma while minimizing unwanted vascular side effects, we engineered two novel VEGF variants, eVEGF-38 and eVEGF-53. These variants of the diffusible VEGF-A isoform VEGF121 are expressed as dimeric concatamers and remain tethered to the cell membrane, thus restricting the effects of the engineered VEGF to the cells expressing the protein. For comparison, we tested a Myc-tagged version of VEGF189, an isoform that binds tightly to the extracellular matrix and heparan sulfate proteoglycans at the cell surface, supporting only autocrine and localized juxtacrine signaling. In human retinal endothelial cells (hREC), expression of eVEGF-38, eVEGF-53, or VEGF189 increased VEGFR2 phosphorylation without increasing expression of pro-inflammatory markers, relative to VEGF165 protein and vector controls. AAV2-mediated transduction of eVEGF-38, eVEGF-53, or VEGF189 into primary mouse RGC promoted synaptogenesis and increased the average total length of neurites and axons per RGC by ~ 12-fold, an increase that was mediated by VEGFR2 and PI3K/AKT signaling. Expression of eVEGF-38 in primary RGC enhanced expression of genes associated with neuritogenesis, axon outgrowth, axon guidance, and cell survival. Transduction of primary RGC with any of the membrane-associated VEGF constructs increased survival both under normal culture conditions and in the presence of the cytotoxic chemicals H2O2 (via VEGFR2/PI3K/AKT signaling) and N-methyl-d-aspartate (via reduced Ca2+ influx). Moreover, RGC number was increased in mouse embryonic stem cell-derived retinal organoid cultures transduced with the eVEGF-53 construct. The novel, engineered VEGF variants eVEGF-38 and eVEGF-53 show promise as potential therapeutics for retinal RGC neuroprotection when delivered using a gene therapy approach.

Abstract P277: Ceramide Modulates GPCR And TK Receptor Signaling To Impact Endothelial Functions And Blood Pressure

Ceramides are sphingolipids that modulate a variety of cellular processes, via two major modes of actions: by functioning as second messengers and by regulating the formation of lipid raft, important signaling platform. Receptor-mediated signaling orchestrates multiple pathophysiological processes, hence more than 50% of drugs clinically prescribed target mainly GPCRs. Alterations of ceramide levels are implicated in endothelial dysfunction, a key event in many cardiovascular diseases, including hypertension and atherosclerosis. However, specific molecular mechanisms of how ceramides modulate the structure and the function of GPCRs and TK receptors to impact endothelial functions remains unknown and unexplored. Thus, we generated mice lacking the endothelial sphingolipid de novo biosynthesis, by deleting the serine palmitoyltransferase long subunit 2, of the first enzyme of the pathway, named ECKO-Sptlc2. Systolic blood pressure was markedly increased in ECKO-Sptlc2 vs. control mice (122.1± 1.9 vs. 103.6±0.7 mmHg, n=9). Vasodilation of mesenteric arteries to acetylcholine and histamine (Gq-coupled receptors) and the NO-signaling downstream was preserved suggesting that altered membrane sphingolipid levels did not affect Gq-coupled receptor activation. On the contrary, vasodilation induced by the activation of tyrosine kinase receptors, i.e. VEGFR2 (Emax 32.8±3.5 vs. 55.7±3.6 % vasodilation, n=5), Gi-coupled GPCRs, i.e. S1PR1 (Emax 9.0±11.0 vs. 58.7 ±3.5 % vasodilation, n=5) and flow were markedly reduced. C16-, C24- and C24:1-ceramide are the most abundant ceramides in the endothelium and were markedly reduced by the loss of Sptlc2. Interestingly, the treatment of the mice and endothelial cells in culture with C16-, C24- and C24:1-ceramides, showed that C16-ceramide play a specific role in VEGFR2, IR, S1PR1 and flow mediated vasodilation. Mechanistically, C16-ceramide modulates VEGFR2 phosphorylation and downstream signaling in a concentration-dependent manner. The finding that C16-ceramide affects specific the function of Gi-coupled, TK but not Gq-coupled receptor is of great significance considering that alterations in C16-ceramide strongly correlate with CV conditions, such as CAD and heart failure.