Vascular Endothelial Growth Factor Receptor Signaling Research Articles

Microbial Metabolite Deoxycholic Acid Promotes Vasculogenic Mimicry Formation in Intestinal Carcinogenesis

Background: High-fat diet (HFD) leads to exposure to gut microbial metabolite secondary bile acids such as deoxycholic acid (DCA) in the intestine, which is closely linked to colorectal cancer (CRC). Vasculogenic mimicry (VM) is a critical event for cancer malignant transformation. We aimed to investigate the crucial roles of DCA on VM and the intestinal carcinogenesis. Methods: The effects of HFD on VM formation and epithelial-mesenchymal transition (EMT) in human CRC tissues were investigated. The fecal DCA level was detected in the HFD treated- Apc min/+ mice. The four-week Apc min/+ mice were treated with 0·2% DCA for 12 weeks. Intestinal tumor tissues were collected for detection of VM, EMT, and vascular endothelial growth factor receptor 2 (VEGFR2) signaling. The effects of DCA on tube formation, EMT, and VEGFR2 signaling of HCT-116 cells were evaluated. Findings: Compared with the normal diet, HFD exacerbated VM formation and EMT in CRC patients. HFD could significantly increase fecal DCA level in Apc min/+ mice. More importantly, DCA promoted tumor cell proliferation and EMT, and increased VM formation accompanied by VEGFR2 activation, which led to intestinal carcinogenesis. DCA enhanced proliferation and migration of HCT-116 cells, and induced EMT and vitro tube formation. Furthermore, the silence of VEGFR2 blunted DCA-induced EMT, VM formation, and migration. Interpretation: Microbial metabolite DCA promoted VM formation and EMT through VEGFR2 activation, which further exacerbated intestinal carcinogenesis. Funding: This study was supported by the National Natural Science Foundation of China and the Key Project of Science and Technology Pillar Program of Tianjin. Declaration of Interest: The authors declare no conflicts of interest. Ethical Approval: This study was approved by the Ethics Committee of General Hospital, Tianjin Medical University, China.

Paladin is a phosphoinositide phosphatase regulating endosomal VEGFR2 signalling and angiogenesis.

Cell signalling governs cellular behaviour and is therefore subject to tight spatiotemporal regulation. Signalling output is modulated by specialized cell membranes and vesicles which contain unique combinations of lipids and proteins. The phosphatidylinositol 4,5‐bisphosphate (PI(4,5)P2), an important component of the plasma membrane as well as other subcellular membranes, is involved in multiple processes, including signalling. However, which enzymes control the turnover of non‐plasma membrane PI(4,5)P2, and their impact on cell signalling and function at the organismal level are unknown. Here, we identify Paladin as a vascular PI(4,5)P2 phosphatase regulating VEGFR2 endosomal signalling and angiogenesis. Paladin is localized to endosomal and Golgi compartments and interacts with vascular endothelial growth factor receptor 2 (VEGFR2) in vitro and in vivo. Loss of Paladin results in increased internalization of VEGFR2, over‐activation of extracellular regulated kinase 1/2, and hypersprouting of endothelial cells in the developing retina of mice. These findings suggest that inhibition of Paladin, or other endosomal PI(4,5)P2 phosphatases, could be exploited to modulate VEGFR2 signalling and angiogenesis, when direct and full inhibition of the receptor is undesirable.

The antihypertension effect of hydrogen sulfide (H2S) is induced by activating VEGFR2 signaling pathway

AimsPrevious studies demonstrated that H2S has an antihypertension effect on hypertension, but the mechanism involved is unclear until now. The aim of the study is to elucidate the effect of H2S on PH and the mechanism involved. Main methodsIn this study, GYY4137 (a H2S donor) were administered to spontaneously hypertensive rats (SHR) and normotensive Wistar-Kyoto rats (WKY) by intraperitoneally injection daily for consecutive 14 days. Systolic blood pressure (SBP), endothelial-dependent relaxation (EDR), plasma malondialdehyde (MDA), superoxide dismutase (SOD), and H2S levels were measured. Human umbilical vein endothelial cells (HUVECs) were also used to elucidate the mechanism involved in the protect effect of H2S on the injured vessels. Key findingsOur results showed that GYY4137 normalized the SBP (P < 0.0001), increased EDR (P < 0.01), reduced oxidative stress (increased the content of SOD and reduced the content of MDA) of SHR. Meanwhile, GYY4137 could promote the proliferation (P < 0.01) and migration (P < 0.01) of HUVECs, increase the expression of endothelial NO synthase (eNOS) and Vascular Endothelial Growth Factor Receptor 2 (VEGFR2) both in SHR and HUVECs treated with GYY4137. In addition to the above results, the PIP3/Akt signaling pathway was activated and the expression of caspase 3 was increased by GYY4137. However, all the above effects of GYY4137 were blocked by ZD6474 (a VEGFR2 inhibitor). SignificanceGYY4137 had a hypotensive and vascular protect effect on PH. This effect might be mediated through upregulating the expression of VEGFR2, which subsequently alleviating oxidant-provoked vascular endothelial dysfunction, and promoting the proliferation and migration of endothelial cells in SHR.

MiR-25-3p promotes endothelial cell angiogenesis in aging mice via TULA-2/SYK/VEGFR-2 downregulation.

In aging, the regulation of angiogenesis is a dynamic and complex process. We aimed to identify and characterize microRNAs that regulate angiogenesis during aging. We showed that, in response to vascular endothelial senescence, microRNA-25-3p (miR-25-3p) plays the role of an angiogenic microRNA by targeting TULA-2 (T-cell ubiquitin ligand-2)/SYK (spleen tyrosine kinase)/VEGFR-2 (vascular endothelial growth factor receptor 2) signaling in vitro and in vivo. Mechanistic studies demonstrated that miR-25-3p inhibits a TULA-2/SYK/VEGFR-2 signaling pathway in endothelial cells. In old endothelial cells (OECs), upregulation of miR-25-3p inhibited the expression of TULA-2, which caused downregulation of the interaction between TULA-2 and SYK and increased phosphorylation of SYK Y323. The increased SYK Y323 phosphorylation level upregulated the phosphorylation of VEGFR-2 Y1175, which plays a vital role in angiogenesis, while miR-25-3p downregulation in YECs showed opposite effects. Finally, a salvage study showed that miR-25-3p upregulation promoted capillary regeneration and hindlimb blood flow recovery in aging mice with hindlimb ischemia. These findings suggest that miR-25-3p acts as an agonist of TULA-2/SYK/VEGFR-2 and mediates the endothelial cell angiogenesis response, which shows that the miR-25-3p/TULA-2 pathway may be potential therapeutic targets for angiogenesis during aging.

BMP6/TAZ-Hippo signaling modulates angiogenesis and endothelial cell response to VEGF

The BMP/TGFβ-Smad, Notch and VEGF signaling guides formation of endothelial tip and stalk cells. However, the crosstalk of bone morphogenetic proteins (BMPs) and vascular endothelial growth factor receptor 2 (VEGFR2) signaling has remained largely unknown. We demonstrate that BMP family members regulate VEGFR2 and Notch signaling, and act via TAZ-Hippo signaling pathway. BMPs were found to be regulated after VEGF gene transfer in C57/Bl6 mice and in a porcine myocardial ischemia model. BMPs 2/4/6 were identified as endothelium-specific targets of VEGF. BMP2 modulated VEGF-mediated endothelial sprouting via Delta like Canonical Notch Ligand 4 (DLL4). BMP6 modulated VEGF signaling by regulating VEGFR2 expression and acted via Hippo signaling effector TAZ, known to regulate cell survival/proliferation, and to be dysregulated in cancer. In a matrigel plug assay in nude mice BMP6 was further demonstrated to induce angiogenesis. BMP6 is the first member of BMP family found to directly regulate both Hippo signaling and neovessel formation. It may thus serve as a target in pro/anti-angiogenic therapies.

Discovery of novel indolyl-1,2,4-triazole hybrids as potent vascular endothelial growth factor receptor-2 (VEGFR-2) inhibitors with potential anti-renal cancer activity

Targeting VEGFR-2 signaling pathway is well-established as an important approach for the treatment of solid tumors, particularly renal cancer. Herein, novel indolyl-1,2,4-triazole hybrids were designed and synthesized as VEGFR-2 kinase inhibitors with potential anti-renal cancer activity. The structures of the newly synthesized compounds were confirmed based on their spectral and elemental analyses. The results of in vitro kinase assay indicated that all target compounds revealed submicromolar inhibition of VEGFR-2 kinase enzyme. Analogs 5c, 5d and 9b emerged as the most active compounds (IC50 = 0.034–0.064 µM), showing VEGFR-2 inhibitory activity much superior to that of sunitinib reference drug (IC50 = 0.075 µM). Moreover, compounds 5a, 8c, 9d, 12c were equipotent to sunitinib against VEGFR-2 kinase. Additionally, the most potent compounds were further examined for their anticancer activity against two human renal cancer cell lines. All screened compounds effectively inhibited the growth of the two tested cell lines with IC50 values spanning from sub-micromolar to low micromolar levels. Compounds 5b, 5d, 11c and 12c were three to five-fold more potent than sunitinib against CAKI-1 cell line. Analogue 8c was superior/comparable to sunitinib against CAKI-1/A498 cell lines. Moreover, compound 9d showed double potency of sunitinib against A498 cell line. Besides, compounds 8c and 12c demonstrated a safety profile much better than that of sunitinib against non-cancer human renal cells. As well, the docked models of title compounds revealed strong interactions with key residues within the active site of VEGFR-2 kinase.

PR1P Stabilizes VEGF and Upregulates Its Signaling to Reduce Elastase-induced Murine Emphysema.

Emphysema is a progressive and fatal lung disease with no cure that is characterized by thinning, enlargement, and destruction of alveoli, leading to impaired gas exchange. Disease progression is due in part to dysregulation of VEGF (vascular endothelial growth factor) signaling in the lungs and increased lung-cell apoptosis. Here we asked whether PR1P (Prominin-1-derived peptide), a novel short peptide we designed that increases VEGF binding to endothelial cells, could be used to improve outcome in in vitro and in vivo models of emphysema. We used computer simulation and in vitro and in vivo studies to show that PR1P upregulated endogenous VEGF receptor-2 signaling by binding VEGF and preventing its proteolytic degradation. In so doing, PR1P mitigated toxin-induced lung-cell apoptosis, including from cigarette-smoke extract in vitro and from LPS in vivo in mice. Remarkably, inhaled PR1P led to significantly increased VEGF concentrations in murine lungs within 30 minutes that remained greater than twofold above that of control animals 24 hours later. Finally, inhaled PR1P reduced acute lung injury in 4- and 21-day elastase-induced murine emphysema models. Taken together, these results highlight the potential of PR1P as a novel therapeutic agent for the treatment of emphysema or other lung diseases characterized by VEGF signaling dysregulation.

Small extracellular vesicle-bound vascular endothelial growth factor secreted by carcinoma-associated fibroblasts promotes angiogenesis in a bevacizumab-resistant manner.

The blood vessel growth inhibitor bevacizumab targets vascular endothelial growth factor (VEGF), a crucial regulator of angiogenesis. Recently, small extracellular vesicles (sEVs) have been demonstrated to be important vehicles in the transport of growth factors to target cells. In this study, we isolated primary carcinoma-associated fibroblasts (CAFs) from four human oral squamous cell carcinoma (OSCC) specimens. Compared with other non-extracellular vesicle components, CAF-derived sEVs were found to be the main regulators of angiogenesis. The ability of CAF sEVs to activate VEGF receptor 2 (VEGFR2) signaling in human umbilical vein endothelial cells (HUVEC) was dependent on the association between sEVs and VEGF. In addition, sEV-bound VEGF secreted by CAFs further activated VEGFR2 signaling in HUVEC in a bevacizumab-resistant manner. VEGF was found to interact with heparan sulfate proteoglycans on the CAF sEV surface and could be released by heparinase I/III. The bioactivity of the dissociated VEGF was retained in vitro and in vivo and could be neutralized by bevacizumab. These findings suggest that the combined use of heparinase and bevacizumab might inhibit angiogenesis in patients with high levels of sEV-bound VEGF.

Piceatannol, a Natural Analog of Resveratrol, Exerts Anti-angiogenic Efficiencies by Blockage of Vascular Endothelial Growth Factor Binding to Its Receptor.

Piceatannol is also named as trans-3,4,3′,5′-tetrahydroxy-stilbene, which is a natural analog of resveratrol and a polyphenol existing in red wine, grape and sugar cane. Piceatannol has been proved to possess activities of immunomodulatory, anti-inflammatory, antiproliferative and anticancer. However, the effect of piceatannol on VEGF-mediated angiogenesis is not known. Here, the inhibitory effects of piceatannol on VEGF-induced angiogenesis were tested both in vitro and in vivo models of angiogenesis. In human umbilical vein endothelial cells (HUVECs), piceatannol markedly reduced the VEGF-induced cell proliferation, migration, invasion, as well as tube formation without affecting cell viability. Furthermore, piceatannol significantly inhibited the formation of subintestinal vessel in zebrafish embryos in vivo. In addition, we identified the underlying mechanism of piceatannol in triggering the anti-angiogenic functions. Piceatannol was proposed to bind with VEGF, thus attenuating VEGF in activating VEGF receptor and blocking VEGF-mediated downstream signaling, including expressions of phosphorylated eNOS, Erk and Akt. Furthermore, piceatannol visibly suppressed ROS formation, as triggered by VEGF. Moreover, we further determined the outcome of piceatannol binding to VEGF in cancer cells: piceatannol significantly suppressed VEGF-induced colon cancer proliferation and migration. Thus, these lines of evidence supported the conclusion that piceatannol could down regulate the VEGF-mediated angiogenic functions with no cytotoxicity via decreasing the amount of VEGF binding to its receptors, thus affecting the related downstream signaling. Piceatannol may be developed into therapeutic agents or health products to reduce the high incidence of angiogenesis-related diseases.

Rac Signaling Drives Clear Cell Renal Carcinoma Tumor Growth by Priming the Tumor Microenvironment for an Angiogenic Switch.

Clear cell renal cell carcinoma (ccRCC) remains a common cause of cancer mortality. Better understanding of ccRCC molecular drivers resulted in the development of antiangiogenic therapies that block the blood vessels that supply tumors with nutrients for growth and metastasis. Unfortunately, most ccRCC patients eventually become resistant to initial treatments, creating a need for alternative treatment options. We investigated the role of the small GTPase Rac1 in ccRCC. Analysis of ccRCC clinical samples indicates that Rac signaling drives disease progression and predicts patients with poorer outcomes. Investigation of Rac1 identifies multiple roles for Rac1 in the pathogenesis of ccRCC. Rac1 is overexpressed in RCC cell lines and drives proliferation and migratory/metastatic potential. Rac1 is also critical for endothelial cells to grow and form endothelial tubular networks potentiated by angiogenic factors. Importantly, Rac1 controls paracrine signaling of angiogenic factors including VEGF from renal carcinoma cells to surrounding blood vessels. A novel Rac1 inhibitor impaired the growth and migratory potential of both renal carcinoma cells and endothelial cells and reduced VEGF production by RCC cells, thereby limiting paracrine signaling both in vitro and in vivo Lastly, Rac1 was shown to be downstream of VEGF receptor (VEGFR) signaling and required for activation of MAPK signaling. In combination with VEGFR2 inhibitors, Rac inhibition provides enhanced suppression of angiogenesis. Therefore, targeting Rac in ccRCC has the potential to block the growth of tumor cells, endothelial cell recruitment, and paracrine signaling from tumor cells to other cells in the tumor microenvironment.

Revision to psychopharmacology mRNA and microRNA profiles are associated with stress susceptibility and resilience induced by psychological stress in the prefrontal cortex

The prefrontal cortex is associated with many mental neurological diseases. The mRNA and microRNA profiles of stress susceptibility and resilience induced by psychological stress in the prefrontal cortex remain to be elucidated. The C57 observer was placed in the cage next to the CD1 mouse and suffered psychological stress by watching the CD1 attacking another C57 mouse. After 5days of psychological stress, the degree of fear memory and anxiety of mice were measured by social interaction test and elevated plus maze (EPM). The prefrontal cortex was extracted and mRNA and microRNA profiles were analyzed by high-throughput sequencing. In susceptible mice versus resilient mice, the downregulation of genes involved in serotonergic synapse may be related to the susceptibility to psychological stress. The imbalanced regulation of genes involved in VEGF, p53, chemokine, Ras, sphingolipid, GnRH, MAPK, and NOD-like receptor signaling pathways may be related to the susceptibility to psychological stress. Compared with control mice, susceptible mice and resilient mice have changed genes involved in serotonergic synapse, neuroactive ligand-receptor interaction, axon guidance, calcium, cAMP, GnRH, estrogen, PI3K-Akt, MAPK, Rap1, and Ras signaling pathways, these changes may be related to psychological stress processing. The sequencing results of mRNAs and microRNAs were verified by qRT-PCR and dual-luciferase reporter assay. The downregulation of genes involved in serotonergic synapse and imbalance of signaling pathways in the prefrontal cortex may be related to susceptibility to psychological stress.

Generating a Novel Bispecific Nanobody to Enhance Antitumor Activity

AbstractTumor cells express high levels of human epidermal growth factor receptor 2 (HER2) and vascular endothelial growth factor receptor 2 (VEGFR2), which are closely related to their proliferation and survival. Cancer treatments that target a single signaling pathway may result in immune pathway escape or drug resistance. Based on the correlation between the HER2 and VEGFR2 signaling pathways, we speculated that targeting the two pathways simultaneously may produce a synergistic effect and avoid occurrence of drug resistance, resulting in improved efficacy. Anti-VEGFR2 nanobody 3VGR19–3 and anti-HER2 nanobody 2D3 were combined to construct a bispecific nanobody (Bi-Nb). They can recognize both HER2 and VEGFR2 (both highly expressed in HT-29 cells) to simultaneously block the two signaling pathways. We verified the affinity of the Bi-Nb to its targets using the surface plasmon resonance technology, and test its effects to inhibit tumor cell growth and promote cell apoptosis in vitro by the Cell Counting Kit-8 assay and apoptosis assay. In summary, we have successfully constructed a Bi-Nb, and verified its tumor-suppressing effects in vitro. Compared with a single monospecific nanobody, our Bi-Nb showed superior antitumor effect, which provides a new perspective for treatment of tumors with high HER2 and VEGFR2 expression.

Cyperenoic acid, a sesquiterpene derivative from Croton crassifolius, inhibits tumor growth through anti-angiogenesis by attenuating VEGFR2 signal pathway in breast cancer

BackgroundCyperenoic acid, one of the main chemical constituents of the root of Croton crassifolius, exhibited potent anti-angiogenic property on the zebrafish embryo model with little cytotoxicity. Nevertheless, its anti-angiogenic mechanism and anti-tumor effect have not been investigated. PurposeTo investigate the anti-angiogenic mechanisms of cyperenoic acid and evaluate it whether could exert anti-tumor effect by inhibiting angiogenesis. Study designTargeting vascular endothelial growth factor receptor-2 (VEGFR2) pathway to inhibit tumor angiogenesis is a significant strategy for cancer treatment. Initially, the anti-angiogenic effect of cyperenoic acid as well as the mechanisms of the action was studied using both in-vitro and in-vivo methodologies. Then, its anti-tumor effect through anti-angiogenesis by attenuating VEGFR2 signaling pathway was evaluated. MethodsThe in-vitro inhibitory effect of cyperenoic acid on the vascular endothelial growth factor (VEGF)-induced angiogenesis was evaluated using human umbilical vein endothelial cells (HUVECs) model. Moreover, its ex-vivo and in-vivo effects were evaluated using the aortic ring assay and the matrigel plug assay. The influence of the cyperenoic acid on tyrosine phosphorylation of VEGFR2 was studied by western blotting assay and the influence on downstream signaling pathway of VEGFR2 also be detected. Computer-docking simulations were carried out to study the interaction between cyperenoic acid and VEGFR2. Finally, its inhibitory effect on tumor growth was studied using breast cancer xenograft model. ResultsCyperenoic acid possessed little toxicity to HUVECs, but it significantly inhibited VEGF-induced proliferation, invasion, migration and tube formation of HUVECs. Moreover, it inhibited VEGF-induced sprout formation ex vivo and vessel formation in vivo. Further mechanistic study showed that cyperenoic acid could suppress VEGFR2 tyrosine kinase activity and alter its downstream signaling pathways in VEGF-induced HUVECs. In addition, it could form two hydrogen bonds with the ATP binding pocket of the VEGFR2 kinase domain by docking. For breast cancer xenograft model, cyperenoic acid suppressed tumor growth, but no obvious toxic pathologic changes were observed in mice. Besides, it suppressed the phosphorylation of VEGFR2 in tumor, demonstrating its anti-angiogenic ability in vivo partly targeting the VEGFR2. ConlusionCyperenoic acid could exert anti-tumor effect in breast cancer by inhibiting angiogenesis via VEGFR2 signaling pathway.

The CCL2/CCR2 axis is critical to recruiting macrophages into acellular nerve allograft bridging a nerve gap to promote angiogenesis and regeneration

Acellular nerve allografts (ANAs) are increasingly used to repair nerve gaps following injuries. However, these nerve scaffolds have yet to surpass the regenerative capabilities of cellular nerve autografts; improved understanding of their regenerative mechanisms could improve design. Due to their acellular nature, both angiogenesis and diverse cell recruitment is necessary to repopulate these scaffolds to promote functional regeneration. We determined the contribution of angiogenesis to initial cellular repopulation of ANAs used to repair nerve gaps, as well as the signaling that drives a significant portion of this angiogenesis. Wild-type (WT) mice with nerve gaps repaired using ANAs that were treated with an inhibitor of VEGF receptor signaling severely impaired angiogenesis within ANAs, as well as hampered cell repopulation and axon extension into ANAs. Similarly, systemic depletion of hematogenous-derived macrophages, but not neutrophils, in these mice models severely impeded angiogenesis and subsequent nerve regeneration across ANAs suggesting hematogenous-derived macrophages were major contributors to angiogenesis within ANAs. This finding was reinforced using CCR2 knockout (KO) models. As macrophages represented the majority of CCR2 expressing cells, a CCR2 deficiency impaired angiogenesis and subsequent nerve regeneration across ANAs. Furthermore, an essential role for CCL2 during nerve regeneration across ANAs was identified, as nerves repaired using ANAs had reduced angiogenesis and subsequent nerve regeneration in CCL2 KO vs WT mice. Our data demonstrate the CCL2/CCR2 axis is important for macrophage recruitment, which promotes angiogenesis, cell repopulation, and subsequent nerve regeneration and recovery across ANAs used to repair nerve gaps.

Lymphangiogenesis induced by vascular endothelial growth factor receptor 1 signaling contributes to the progression of endometriosis in mice

Lymphangiogenesis is related to the growth of endometriosis. Here, we examined whether vascular endothelial growth factor (VEGF) receptor 1 (VEGFR1) signaling plays a role in lymphangiogenesis during endometriosis. Endometrial fragments from wild-type (WT) mice transplanted into the peritoneal wall of host WT mice (WT→WT) developed well and displayed enhanced lymphangiogenesis associated with increases in mRNA levels of VEGF-C and VEGF-D. Compared with WT mice, the implant size and lymphangiogenesis were reduced, when endometrial fragments from mice lacking the VEGFR1 tyrosine kinase (TK) domain (TK-/-) were transplanted into host TK-/- mice (TK-/-→TK-/-). Treatment of WT→WT mice with the VEGFR3 kinase inhibitor suppressed the size of implants and lymphangiogenesis. Immunofluorescence analyses demonstrated that VEGF-C and VEGF-D were expressed in both CD11b+ and S100A4+ cells. TK-/-→TK-/- mice had lower numbers of CD11b+ and S100A4+ cells than WT→WT mice. When isolated bone marrow (BM)-derived macrophages or culture murine fibroblasts were stimulated with placental growth factor (PlGF), a specific agonist of VEGFR1, the levels of VEGF-C and VEGF-D were increased in a VEGFR1-dependent manner. These results suggest that VEGFR1 signaling in macrophages and fibroblasts contributes to the growth of endometrial implants and lymphangiogenesis.

CM082, a novel angiogenesis inhibitor, enhances the antitumor activity of gefitinib on epidermal growth factor receptor mutant non-small cell lung cancer in vitro and in vivo.

BackgroundCM082 is a novel angiogenesis inhibitor targeting vascular endothelial growth factor receptor (VEGFR) and platelet‐derived growth factor receptor (PDGFR). The purpose of this research was to evaluate the antitumor activity of CM082 combined with gefitinib on epidermal growth factor receptor (EGFR) mutant non‐small cell lung cancer (NSCLC) in vitro and in vivo.MethodsThe effect of CM082 on human umbilical vein endothelial cells (HUVECs) was assessed. In vitro and in vivo efficacy of CM082 combined with gefitinib on EGFR NSCLC cell lines (HCC827 harboring E746_A750 deletion and H3255 harboring L858R) and a xenograft model was evaluated.ResultsCM082 inhibited VEGF‐induced cell growth, phosphorylation of VEGFR and downstream signaling molecules, tube formation, and cell migration of HUVECs. Furthermore, CM082 combined with gefitinib was more effective in inhibiting growth and colony formation and inducing apoptosis of H3255 and HCC827 cells in vitro than monotherapy. Moreover, tumor growth inhibition by the combination in a H3255 xenograft model was 107.7% more than that by gefitinib (93.6%) (P < 0.0001) and CM082 (51.4%) (P < 0.0001) alone. In addition, coadministration had a better effect on inhibiting cell proliferation, inducing apoptosis, and inhibiting the expression of CD31 and VEGF‐A. The combination therapy had a stronger inhibition effect on STAT3 phosphorylation than monotherapy.ConclusionsCM082, a novel angiogenesis inhibitor, enhances the antitumor activity of gefitinib on EGFR mutant NSCLC by inhibiting proliferation and angiogenesis and promoting apoptosis of tumor cells.Key points Significant findings of the study CM082, a novel angiogenesis inhibitor, enhances the antitumor activity of gefitinib on EGFR mutant NSCLC by inhibiting proliferation and angiogenesis and promoting apoptosis of tumor cells. What this study adds These findings justify evaluation of the efficacy of CM082 combined with gefitinib in patients with EGFR mutant advanced NSCLC in clinical trials.

Abnormal Vascular Phenotypes Associated with the Timing of Interruption of Retinal Vascular Development in Rats.

Pathological angiogenesis is a leading cause of blindness in several retinal diseases. The key driving factor inducing pathological angiogenesis is the pronounced hypoxia leading to a marked, increased production of vascular endothelial growth factor (VEGF). The aim of this study was to determine whether the abnormal vascular growth occurs in a manner dependent on the degree of the vascular defects. Vascular defects of two different degrees were created in the retina by subcutaneously treating neonatal rats with the VEGF receptor (VEGFR) tyrosine kinase inhibitor KRN633 on postnatal day (P) 4 and P5 (P4/5) or P7 and P8 (P7/8). The structure of the retinal vasculature changes was examined immunohistochemically. Prevention of vascular growth and regression of some preformed capillaries were observed on the next day, after completion of each treatment (i.e., P6 and P9). The vascular regrowth occurred as a result of eliminating the inhibitory effect on the VEGFR signaling pathway. KRN633 (P4/5)-treated rats exhibited a retinal vasculature with aggressive intravitreal neovascularization on P21. On the other hand, the appearance of tortuous arteries is a representative vascular pathological feature in retinas of KRN633 (P7/8)-treated groups. These results suggest that an interruption of the retinal vascular development at different time points induces different vascular pathological features in the retina. Pharmacological agents targeting the VEGF signaling pathway are useful for creating an abnormal retinal vasculature with various pathological features in order to evaluate the efficacy of anti-angiogenic compounds.

Lymphangiogenesis and accumulation of reparative macrophages contribute to liver repair after hepatic ischemia-reperfusion injury.

Hepatic tissue repair plays a critical role in determining the outcome of hepatic ischemia-reperfusion (I/R) injury. Hepatic lymphatics participate in the clearance of dead tissues and contribute to the reparative process after acute hepatic injury; however, it remains unknown whether lymphangiogenesis in response to hepatic inflammation is involved in liver repair. Herein, we determined if hepatic lymphangiogenesis improves liver repair after hepatic I/R injury. Using a mouse model of hepatic I/R injury, we investigated hepatic lymphatic structure, growth, and function in injured murine livers. Hepatic I/R injury enhanced lymphangiogenesis around the portal tract and this was associated with increased expression of pro-lymphangiogenic growth factors including vascular endothelial growth factor (VEGF)-C and VEGF-D. Recombinant VEGF-D treatment facilitated liver repair in association with the expansion of lymphatic vessels and increased expression of genes related to the reparative macrophage phenotype. Treatment with a VEGF receptor 3 (VEGFR3) inhibitor suppressed liver repair, lymphangiogenesis, drainage function, and accumulation of VEGFR3-expressing reparative macrophages. VEGF-C and VEGF-D upregulated expression of genes related to lymphangiogenic factors and the reparative macrophage phenotype in cultured macrophages. These results suggest that activation of VEGFR3 signaling increases lymphangiogenesis and the number of reparative macrophages, both of which play roles in liver repair. Expanded lymphatics and induction of reparative macrophage accumulation may be therapeutic targets to enhance liver repair after hepatic injury.

EXPRESS: Gremlin1 blocks vascular endothelial growth factor signalling in the pulmonary microvascular endothelium.

The bone morphogenetic protein (BMP) antagonist gremlin 1 plays a central role in the pathogenesis of hypoxic pulmonary hypertension (HPH). Recently, non‐canonical functions of gremlin 1 have been identified, including specific binding to the vascular endothelial growth factor receptor‐2 (VEGFR2). We tested the hypothesis that gremlin 1 modulates VEGFR2 signaling in the pulmonary microvascular endothelium.We examined the effect of gremlin 1 haploinsufficiency on the expression of VEGF responsive genes and proteins in the hypoxic (10% O2) murine lung in vivo. Using human microvascular endothelial cells in vitro we examined the effect of gremlin 1 on VEGF signaling.Gremlin 1 haploinsufficiency (Grem1+/–) attenuated the hypoxia‐induced increase in gremlin 1 observed in the wild‐type mouse lung. Reduced gremlin 1 expression in hypoxic Grem1+/– mice restored VEGFR2 expression and endothelial nitric oxide synthase (eNOS) expression and activity to normoxic values. Recombinant monomeric gremlin 1 inhibited VEGFA‐induced VEGFR2 activation, downstream signaling, and VEGF‐induced increases in Bcl‐2, cell number, and the anti‐apoptotic effect of VEGFA in vitro.These results show that the monomeric form of gremlin 1 acts as an antagonist of VEGFR2 activation in the pulmonary microvascular endothelium. Given the previous demonstration that inhibition of VEGFR2 causes marked worsening of HPH, our results suggest that increased gremlin 1 in the hypoxic lung, in addition to blocking BMP receptor type‐2 (BMPR2) signaling, contributes importantly to the development of PH by a non‐canonical VEGFR2 blocking activity.

Synchronous inhibition of mTOR and VEGF/NRP1 axis impedes tumor growth and metastasis in renal cancer

Clear cell renal cell carcinoma (ccRCC) is known for its highly vascular phenotype which is associated with elevated expression of vascular endothelial growth factor A (VEGF), also known as vascular permeability factor (VPF). Accordingly, VEGF has been an attractive target for antiangiogenic therapies in ccRCC. Two major strategies have hitherto been utilized for VEGF-targeted antiangiogenic therapies: targeting VEGF by antibodies, ligand traps or aptamers, and targeting the VEGF receptor signaling via antibodies or small-molecule tyrosine-kinase inhibitors (TKIs). In the present article we utilized two entirely different approaches: targeting mammalian target of rapamycin (mTOR) pathway that is known to be involved in VEGF synthesis, and disruption of VEGF/Neuroplin-1 (NRP1) axis that is known to activate proangiogenic and pro-tumorigenic signaling in endothelial and tumor cells, respectively. Everolimus (E) and a small-molecule inhibitor EG00229 (G) were used for the inhibition of mTOR and the disruption of VEGF/NRP1 axis, respectively. We also exploited a liposomal formulation decorated with a proprietary tumor-targeting-peptide (TTP) to simultaneously deliver these two agents in a tumor-targeted manner. The TTP-liposomes encapsulating both Everolimus and EG00229 (EG-L) demonstrated higher in vitro and in vivo growth retardation than the single drug-loaded liposomes (E-L and G-L) in two different ccRCC models and led to a noticeable reduction in lung metastasis in vivo. In addition, EG-L displayed remarkable inhibition of tumor growth in a highly aggressive syngeneic immune-competent mouse model of ccRCC developed in Balb/c mice. Taken together, this study demonstrates an effective approach to achieve improved therapeutic outcome in ccRCC.