Inhibition Of VEGFR2 Signaling Research Articles

Endothelial Slc35a1 Deficiency Causes Loss of LSEC Identity and Exacerbates Neonatal Lipid Deposition in the Liver in Mice

BACKGROUND & AIMSThe functional maturation of the liver largely occurs after birth. In the early stages of life, the liver of a newborn encounters enormous high-fat metabolic stress caused by the consumption of breast milk. It is unclear how the maturing liver adapts to high lipid metabolism. Liver sinusoidal endothelial cells (LSECs) play a fundamental role in establishing liver vasculature and are decorated with many glycoproteins on their surface. The Slc35a1 gene encodes a CMP-sialic acid transporter responsible for transporting CMP-sialic acids between the cytoplasm and the Golgi apparatus for protein sialylation. This study aimed to determine whether endothelial sialylation plays a role in hepatic vasculogenesis and functional maturation. METHODSEndothelial-specific Slc35a1 knockout mice were generated. Liver tissues were collected for histological analysis, lipidomic profiling, RNA sequencing, confocal immunofluorescence, and immunoblot analyses. RESULTSEndothelial Slc35a1-deficient mice exhibited excessive neonatal hepatic lipid deposition, severe liver damage, and high mortality. Endothelial deletion of Slc35a1 led to sinusoidal capillarization and disrupted hepatic zonation. Mechanistically, VEGFR2 in LSECs was desialylated and VEGFR2 signaling was enhanced in Slc35a1-deficient mice. Inhibition of VEGFR2 signaling by SU5416 alleviated lipid deposition and restored hepatic vasculature in Slc35a1-deficient mice. CONCLUSIONSOur findings suggest that sialylation of LSECs is critical for maintaining hepatic vascular development and lipid homeostasis. Targeting VEGFR2 signaling may be a new strategy to prevent liver disorders associated with abnormal vasculature and lipid deposition.

Abstract 4592: In vivo efficacy evaluation of anti-human VEGFR2 antibodies in humanized B-hVEGFR2 mice

Abstract Angiogenesis, vascular development, vascular permeability, and embryonic hematopoiesis is partly regulated by VEGF binding to VEGFR2, which forms receptor dimers and activates downstream signaling pathways. In addition to developmental cues, VEGFR2 is also associated with disease progression for psoriasis, rheumatoid arthritis, diabetic retinopathy, and more significantly, tumor growth and drug resistance. Given this, inhibition of VEGFR2 signaling is actively being studied, and multiple VEGFR2 inhibitors have entered various phases of clinical studies and have shown promising results. To evaluate in vivo efficacy of anti-human VEGFR2 antibodies, we generated a human VEGFR2 knock-in (B-hVEGFR2) mouse model by replacing the murine Vegfr2 extracellular domain sequence with the corresponding human sequence. We validated human VEGFR2 gene and protein expression in B-hVEGFR2 mice by RT-PCR and flow cytometry, respectively. Furthermore, we observed an anti-tumor effect with anti-human VEGFR2 antibodies in homozygous B-hVEGFR2 mice subcutaneously implanted with MC38 colon cancer cells. Taken together, we established a novel preclinical B-hVEGFR2 mouse model for evaluation of VEGFR2-targeted immunotherapy. Citation Format: Lei Zhao, Chengzhang Shang, Rebecca Soto, Chonghui Liu, Zhaoxue Yu. In vivo efficacy evaluation of anti-human VEGFR2 antibodies in humanized B-hVEGFR2 mice. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4592.

Inhibition of VEGFR-2 by SU5416 increases neonatally glutamate-induced neuronal damage in the cerebral motor cortex and hippocampus.

Vascular endothelial growth factor (VEGF) exerts neuroprotective or proinflammatory effects, depending on what VEGF forms (A-E), receptor types (VEGFR1-3), and intracellular signaling pathways are involved. Neonatal monosodium glutamate (MSG) treatment triggers neuronal death by excitotoxicity, which is commonly involved in different neurological disorders, including neurodegenerative diseases. This study was designed to evaluate the effects of VEGFR-2 inhibition on neuronal damage triggered by excitotoxicity in the cerebral motor cortex (CMC) and hippocampus (Hp) after neonatal MSG treatment. MSG was administered at a dose of 4 g/kg of body weight (b.w.) subcutaneously on postnatal days (PD) 1, 3, 5, and 7, whereas the VEGFR-2 inhibitor SU5416 was administered at a dose of 10 mg/kg b.w. subcutaneously on PD 5 and 7, 30 min before the MSG treatment. Neuronal damage was assessed using hematoxylin and eosin staining, fluoro-Jade staining, and TUNEL assay. Additionally, western blot assays for some proteins of the VEGF-A/VEGFR-2 signaling pathway (VEGF-A, VEGFR-2, PI3K, Akt, and iNOS) were carried out. All assays were performed on PD 6, 8, 10, and 14. Inhibition of VEGFR-2 signaling by SU5416 increases the neuronal damage induced by neonatal MSG treatment in both the CMC and Hp. Moreover, neonatal MSG treatment increased the expression levels of the studied VEGF-A/VEGFR-2 signaling pathway proteins, particularly in the CMC. We conclude that VEGF-A/VEGFR-2 signaling pathway activation could be part of the neuroprotective mechanisms that attempt to compensate for neuronal damage induced by neonatal MSG treatment and possibly also in other conditions involving excitotoxicity.

Perfluorooctane sulfonic acid (PFOS) inhibits vessel formation in a human 3D co-culture angiogenesis model (NCFs/HUVECs)

Perfluorooctane sulfonic acid (PFOS) is a ubiquitous environmental pollutant. In humans, PFOS exposure has been associated with a number of adverse health outcomes, including reduced birth weight. Whether PFOS is capable of affecting angiogenesis and thus possibly fetal development is unknown. Therefore, we investigated 1) the metabolic activity of PFOS-exposed endothelial cells (human umbilical vein endothelial cells, HUVECs), fibroblasts (normal colon fibroblasts, NCFs), and epithelial cells (human colorectal carcinoma cells, HCT116), 2) PFOS-specific inhibition of vascular endothelial growth factor receptor (VEGFR)2 stimulation in KDR/NFAT-RE HEK293 cells, and 3) the antiangiogenic potential of PFOS in a 3D in vitro angiogenesis model of HUVECs and NCFs. In terms of metabolic activity, endothelial cells (HUVECs) were much more sensitive to PFOS than fibroblasts (NCFs) or epithelial cells (HCT116). VEGFR2 signaling in KDR/NFAT-RE HEK293 cells decreased with increasing PFOS concentrations. In co-culture (angiogenesis assay), PFOS treatment resulted in a dose-dependent reduction in tip and branch formation, tip length (μm), and total structural area (μm2) with stable metabolic activity of HUVECs up to high concentrations. We conclude that PFOS possesses antiangiogenic properties. Inhibition of VEGFR2 signaling indicates a possible mechanism of action that can be linked to an existing Adverse Outcome Pathway (AOP43) containing the AO reduced birth weight. Further studies are needed to confirm PFOS-specific adverse effects on angiogenesis, placental perfusion, and fetal growth.

Survivin is critically involved in VEGFR2 signaling-mediated esophageal cancer cell survival

Vascular endothelial growth factor (VEGF) signaling promotes angiogenesis by stimulating the migration and proliferation of endothelial cells. The aim of this study was to investigate the expression of Survivin and VEGF receptor 1/2/3 (VEGFR 1/2/3) in esophageal carcinoma tissues (ECTs), and to explore the therapy effect of the suppression of VEGFR2 signaling. Here, we found that VEGFR2 and Survivin had high expressions and a significant correlation (r = 0.874, P < 0.002) in ECTs. Further, we found that VEGFR2 signaling could activate the AKT1/MDM2/Survivin pathway. The inhibition of VEGFR2 signaling with the XL184 treatment downregulated the phosphorylation of AKT1 and MDM2, and then, increased the activation of Caspase 3/7, resulting in the reduction of cell viability and the apoptosis of HUVECs. Additionally, in the esophageal tumor model, the tumor growth was significantly suppressed by blocking Survivin and the suppression of tumor growth was more effective in the combined treatment by blocking Survivin and Bcl-xl/Bcl-2. Our data thus revealed that Survivin in the signal downstream of VEGFR2 played an important role in esophageal cancer cell survival and might be a potential candidate target for the combined therapy for esophageal cancer.

VEGF signaling regulates the fate of obstructed capillaries in mouse cortex.

Cortical capillaries are prone to obstruction, which over time, could have a major impact on brain angioarchitecture and function. The mechanisms that govern the removal of these obstructions and what long-term fate awaits obstructed capillaries, remains a mystery. We estimate that ~0.12% of mouse cortical capillaries are obstructed each day (lasting >20 min), preferentially in superficial layers and lower order branches. Tracking natural or microsphere-induced obstructions revealed that 75-80% of capillaries recanalized within 24 hr. Remarkably, 30% of all obstructed capillaries were pruned by 21 days, including some that had regained flow. Pruning involved regression of endothelial cells, which was not compensated for by sprouting. Using this information, we predicted capillary loss with aging that closely matched experimental estimates. Genetic knockdown or inhibition of VEGF-R2 signaling was a critical factor in promoting capillary recanalization and minimizing subsequent pruning. Our studies reveal the incidence, mechanism and long-term outcome of capillary obstructions which can also explain age-related capillary rarefaction.

Abstract 3645: Preclinical characterization of a multi-specific DARPin molecule targeting PD1 and VEGF

Abstract Background: Combination therapy of immune-modulating drugs with modalities targeting tumor cells directly or the tumor stroma are increasing in clinical investigation and practice. In particular, the combination of immune checkpoint modulators targeting PD1 and molecules targeting the tumor stroma, like anti-VEGF therapeutics, are being utilized in multiple permutations and have shown promising results. We have developed a new class of protein therapeutics DARPin® molecules for simultaneous targeting of PD1 and VEGF. The multi-specific PD1 and VEGF targeting molecules were tested for inhibition of PD1 and VEGF mediated functions in cellular functional assays and in syngeneic mouse models. Methods: Inhibition of human PD1 functions were tested in cell binding assays, a reporter cell assay (Promega) and in human primary T-cell assays like mixed lymphocyte reaction (MLR). VEGF-A inhibition was tested in functional assays showing inhibition of VEGFR2 signaling. Molecules targeting murine PD1 were analyzed in cell binding assays and splenocyte assays as well as in syngeneic mouse models. The anti-tumoral-effect was monitored by caliper measurement and on T-cell infiltration and angiogenesis by immunohistochemistry. Results: The multi-specific human PD1-VEGF targeting DARPin® molecule inhibits PD1 mediated downregulation of T-cell receptor signaling in a reporter cell assay in the range of the benchmark antibody Nivolumab. Potent picomolar inhibition of VEGF mediated VEGFR2 signaling was also demonstrated for the molecule. Moreover the molecule showed potent inhibition of PD1 in an MLR assay to a similar level as compared to Nivolumab. A murine surrogate molecule was also produced and showed potent PD1 inhibition in vitro. Syngeneic mouse models demonstrate a strong anti-tumoral effect of the surrogate molecule and an increase in the infiltration of T-cells into the tumor was observed upon treatment. The anti-tumoral effect was strongest by combining the anti-PD1 function with the anti-VEGF function. This indicates that the multi-specific DARPin® molecule is functional, shows potency beyond blocking of only PD1 or VEGF and has a strong potential for therapeutic benefit in the clinic. Conclusions: We have generated a multi-specific PD1 and VEGF targeting DARPin® molecule which is potently active in blocking PD1 and VEGF mediated functions in tumor growth and immunity. The potency of the molecule goes beyond targeting PD1 or VEGF alone and has a strong potential for therapeutic benefit in the clinic. * DARPins are small repeat proteins, designed to bind targets with high affinity and specificity, and which can be combined in a modular fashion to produce multi-functional agents. Citation Format: Jennifer Krieg, Schiegg Dieter, Taylor Joanna, Christel Herzog, Laurent Juglair, Nicolo Rigamonti, Mischa Mueller, Ulrike Fiedler, Michael Tobias Stumpp, Dan Snell. Preclinical characterization of a multi-specific DARPin molecule targeting PD1 and VEGF [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 3645. doi:10.1158/1538-7445.AM2017-3645

Endothelial cell regulation of salivary gland epithelial patterning.

Perfusion-independent regulation of epithelial pattern formation by the vasculature during organ development and regeneration is of considerable interest for application in restoring organ function. During murine submandibular salivary gland development, the vasculature co-develops with the epithelium during branching morphogenesis; however, it is not known whether the vasculature has instructive effects on the epithelium. Using pharmacological inhibitors and siRNA knockdown in embryonic organ explants, we determined that VEGFR2-dependent signaling is required for salivary gland epithelial patterning. To test directly for a requirement for endothelial cells in instructive epithelial patterning, we developed a novel ex vivo cell fractionation/reconstitution assay. Immuno-depletion of CD31+ endothelial cells in this assay confirmed a requirement for endothelial cells in epithelial patterning of the gland. Depletion of endothelial cells or inhibition of VEGFR2 signaling in organ explants caused an aberrant increase in cells expressing the ductal proteins K19 and K7, with a reduction in Kit+ progenitor cells in the endbuds of reconstituted glands. Addition of exogenous endothelial cells to reconstituted glands restored epithelial patterning, as did supplementation with the endothelial cell-regulated mesenchymal factors IGFBP2 and IGFBP3. Our results demonstrate that endothelial cells promote expansion of Kit+ progenitor cells and suppress premature ductal differentiation in early developing embryonic submandibular salivary gland buds.

Sry-type HMG box 18 contributes to the differentiation of bone marrow-derived mesenchymal stem cells to endothelial cells

ObjectiveMesenchymal stem cells (MSC) have shown therapeutic potential to engraft and either differentiate into or support differentiation of vascular endothelial cells (EC), smooth muscle cells and cardiomyocytes in animal models of ischemic heart disease. Following intracoronary or transendocardial delivery of MSCs, however, only a small fraction of cells engraft and the majority of those persist as an immature cell phenotype. The goal of the current study was to decipher the molecular pathways and mechanisms that control MSC differentiation into ECs. Vascular endothelial growth factor (VEGF-165) treatment is known to enhance in vitro differentiation of MSCs into ECs. We tested the possible involvement of the Sry-type HMG box (Sox) family of transcription factors in this process. Method and ResultsMSCs were isolated from the bone marrow of Yucatan microswine and underwent a 10 day differentiation protocol. VEGF-165 (50ng/ml) treatment of MSCs in vitro induced a significant increase in the protein expression of VEGFR-2, Sox9 and Sox18, in addition to the EC markers PECAM-1, VE-cadherin and vWF, as determined by Western blot or flow cytometry. siRNA-mediated knockdown of Sox18, as opposed to Sox9, in MSCs prevented VEGF-165-mediated induction of EC markers and capillary tube formation. Inhibition of VEGFR-2 signaling (SC-202850) reduced Sox18 and reduced VEGF-165-induced differentiation of MSCs to ECs. ConclusionHere we demonstrate that VEGF-165 mediates MSC differentiation into ECs via VEGFR-2-dependent induction of Sox18, which ultimately coordinates the transcriptional upregulation of specific markers of the EC phenotype.

O6.10 * GLIOMA CELL VEGFR-2 EXPRESSION IMPAIRS CHEMOTHERAPEUTIC AND ANTIANGIOGENIC TREATMENTS IN PTEN-DEFICIENT GLIOBLASTOMA

BACKGROUND: Given the recent failure of bevacizumab (BEV) in extending overall survival of patients with newly diagnosed glioblastoma, tissue biomarkers predicting response to antiangiogenic treatments are urgently needed. Here, we hypothesized that loss of the tumor suppressor phosphatase and tensin homolog deleted on chromosome 10 (PTEN) is a prerequisite for glioma cell expression of vascular endothelial growth factor receptor (VEGFR)-2 in glioblastoma defining a more aggressive glioblastoma subgroup prone to develop evasive resistance towards antiangiogenic treatments including BEV. METHODS AND RESULTS: Besides its ubiquitous expression on endothelial cells, immunohistochemical analysis of 59 glioblastoma tissues showed glioma cell-bound VEGFR-2 expression was restricted to PTEN-deficient tumor specimens and detectable in 20% of all specimens examined, most pronounced in infiltration zones. In glioma cells, pharmacological and RNAi-mediated inhibition of VEGFR-2 signaling revealed proproliferative, antiinvasive and chemoprotective functions for VEGFR-2 in several experimental paradigms comprising flow cytometry-based cell cycle analyses, in vitro proliferation and invasion assays, organotypic brain slice culture assays, and an MRI-monitored orthotopic mouse model. VEGFR-2-dependent cellular effects were concomitant with activation of three potent resistance factors: 'kappa-light-chain-enhancer' of activated B-cells (NF-κB), protein kinase B (AKT/PKB), and N-myc downstream regulated gene (NDRG)1. Two-photon in vivo microscopy demonstrated that glioma cell expression of VEGFR-2 hampers antiangiogenesis and favors a proinvasive response to treatment with BEV. CONCLUSIONS: Glioma cell expression of VEGFR-2, next to its expression on vessels, indicates a particularly aggressive glioblastoma subgroup prone to develop early resistance to chemotherapy with temozolomide or antiangiogenic treatment with BEV. Loss of PTEN can serve as a readily available surrogate marker to identify those tumors upfront by routine neuropathological methods.

Abstract C147: Itraconazole side-chain analogs reveal a distinct structure-activity relationship for inhibition of hedgehog pathway signaling.

Abstract Itraconazole is an antifungal drug that was recently found to potently inhibit the Hedgehog (Hh) signaling pathway. The mechanism of inhibition of Hh signaling is distinct from that of cyclopamine-competitive inhibitors currently in late-stage clinical development. Itraconazole has also been described as a potent inhibitor of activation of vascular endothelial growth factor receptor-2 (VEGFR-2) and tumor associated angiogenesis. The unexpected activities associated with this FDA-approved agent offer a unique opportunity for expansion of its use to the oncology arena. As such, itraconazole is currently being evaluated in several clinical trials targeting proliferative disease, including basal cell carcinoma and recurrent metastatic non-small cell lung cancer. To date, the target(s) of itraconazole responsible for these activities remain to be determined. In these studies we expand the characterization of the activity profile for itraconazole by examining potency in the context of a Smoothened receptor point-mutation associated with clinical resistance to vismodegib (GDC-0449). Additionally, in the interest of understanding the structure-activity relationship (SAR) guiding Hh pathway targeting and working towards the identification and development of inhibitors demonstrating greater potency and specificity; twenty-five itraconazole side chain analogs were synthesized and assayed for inhibition of Hh pathway activation. Furthermore, the SAR associated with the inhibition of phospho-activatable forms of VEGFR2 is also described. Through these analyses we report that itraconazole maintains potency against the Hh pathway in the context of the D477G Smoothened mutation (mouse homologue to human D473H). We also conclude that SAR trends for targeting of the Hh pathway are divergent from those associated with inhibition of VEGFR2 signaling. Taken together, these results suggest that modification of the sec-butyl side chain of itraconazole can enhance compound activity and differentially affects inhibition of the Hh pathway and VEGFR2 signaling. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the AACR-NCI-EORTC International Conference: Molecular Targets and Cancer Therapeutics; 2011 Nov 12-16; San Francisco, CA. Philadelphia (PA): AACR; Mol Cancer Ther 2011;10(11 Suppl):Abstract nr C147.

Engraftment and Reconstitution of Hematopoiesis Is Dependent on VEGFR2-Mediated Regeneration of Sinusoidal Endothelial Cells

Myelosuppression damages the bone marrow (BM) vascular niche, but it is unclear how regeneration of bone marrow vessels contributes to engraftment of transplanted hematopoietic stem and progenitor cells (HSPCs) and restoration of hematopoiesis. We found that chemotherapy and sublethal irradiation induced minor regression of BM sinusoidal endothelial cells (SECs), while lethal irradiation induced severe regression of SECs and required BM transplantation (BMT) for regeneration. Within the BM, VEGFR2 expression specifically demarcated a continuous network of arterioles and SECs, with arterioles uniquely expressing Sca1 and SECs uniquely expressing VEGFR3. Conditional deletion of VEGFR2 in adult mice blocked regeneration of SECs in sublethally irradiated animals and prevented hematopoietic reconstitution. Similarly, inhibition of VEGFR2 signaling in lethally irradiated wild-type mice rescued with BMT severely impaired SEC reconstruction and prevented engraftment and reconstitution of HSPCs. Therefore, regeneration of SECs via VEGFR2 signaling is essential for engraftment of HSPCs and restoration of hematopoiesis.

Effect of chemical stabilizers of hypoxia-inducible factors on early lung development

Low oxygen stimulates pulmonary vascular development and airway branching and involves hypoxia-inducible factor (HIF). HIF is stable and initiates expression of angiogenic factors under hypoxia, whereas normoxia triggers hydroxylation of the HIF-1alpha subunit by prolyl hydroxylases (PHDs) and subsequent degradation. Herein, we investigated whether chemical stabilization of HIF-1alpha under normoxic (20% O(2)) conditions would stimulate vascular growth and branching morphogenesis in early lung explants. Tie2-LacZ (endothelial LacZ marker) mice were used for visualization of the vasculature. Embryonic day 11.5 (E11.5) lung buds were dissected and cultured in 20% O(2) in the absence or presence of cobalt chloride (CoCl(2), a hypoxia mimetic), dimethyloxalylglycine (DMOG; a nonspecific inhibitor of PHDs), or desferrioxamine (DFO; an iron chelator). Vascularization was assessed by X-gal staining, and terminal buds were counted. The fine vascular network surrounding the developing lung buds seen in control explants disappeared in CoCl(2)- and DFO-treated explants. Also, epithelial branching was reduced in the explants treated with CoCl(2) and DFO. In contrast, DMOG inhibited branching but stimulated vascularization. Both DFO and DMOG increased nuclear HIF-1alpha protein levels, whereas CoCl(2) had no effect. Since HIF-1alpha induces VEGF expression, the effect of SU-5416, a potent VEGF receptor (VEGFR) blocker, on early lung development was also investigated. Inhibition of VEGFR2 signaling in explants maintained under hypoxic (2% O(2)) conditions completely abolished vascularization and slightly decreased epithelial branching. Taken together, the data suggest that DMOG stabilization of HIF-1alpha during early development leads to a hypervascular lung and that airway branching proceeds without the vasculature, albeit at a slower rate.

PE, a new sulfated saponin from sea cucumber, Exhibits anti-angiogenic and anti-tumor activities in vitro and in vivo

Here, we examined the in vitro and in vivo anti-angiogenesis and anti-tumor activities of PE, a new marine-derived compound. Inhibition of angiogenesis was assessed in vitro using proliferation, migration, adhesion, tube-formation and apoptosis assays in PE-treated HMECs and HUVECs. In vivo, CAM assays were used to assess inhibition effect of PE on physiological angiogenesis, and immunofluorescent microscopy was used to examine tumor microvessel density and apoptosis in PE-treated mouse tumor models. Finally, Western blotting analyses were performed to examine the effect of PE on VEGF signaling in HMECs. The results showed that PE inhibited proliferation of HMECs and HUVECs with IC50 values of 2.22°¿0.31 ?M and 1.98°¿0.32 ?M, induced endothelial cell apoptosis at concentrations < 2 ?M, induced dose-dependent suppression of cell migration, cell adhesion and tube formation in HMECs and HUVECs, and showed anti-proliferative activities against several tumor cell lines (IC50 values of ~ 4 ?M). In vivo, PE (5 nM/egg) suppressed spontaneous angiogenesis in our CAM assay, and induced marked growth inhibition in mouse sarcoma 180 and hepatoma 22 models. Specifically, PE treatment reduced mouse sarcoma 180 tumor volume by triggering apoptosis of both tumor and tumor-associated endothelial cells, preferentially targeting on endothelial cells comparable with tumor cells. Finally, PE treatment suppressed the active (phosphorylated) forms of VEGFR2, Akt, ERK, FAK and paxillin, which are involved in endothelial cell survival, proliferation, adhesion and migration. Our results indicate that PE exerts an anti-angiogenic activity associated with inhibition of VEGFR2 signaling, and an anti-tumor activity associated with decreased proliferation of tumor cells and increased apoptosis of both endothelial cells and tumor cells.