Combined inhibition of MET and VEGF enhances therapeutic efficacy of EGFR TKIs in EGFR-mutant non-small cell lung cancer with concomitant aberrant MET activation.

BACKGROUND: Aberrant activation of mesenchymal epithelial transition (MET) has been considered to mediate primary and acquired resistance to epidermal growth factor receptor (EGFR) tyrosine kinase inhibitors (TKIs) in EGFR-mutant non-small cell lung cancer (NSCLC). However, mechanisms underlying this process are not wholly clear and the effective therapeutic strategy remains to be determined. METHODS: The gefitinib-resistant NSCLC cell lines were induced by concentration increase method in vitro. Western blot and qPCR were used to investigate the relationship between MET and vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) signaling pathway. Double luciferase reporter gene and co-immunoprecipitation were used to further reveal the regulation mechanism between MET and VEGF/VEGFR2. The effect of combined inhibition of MET and VEGF/VEGFR2 signaling pathway on the therapeutic sensitivity of EGFR-TKI in gefitinib resistant cell lines with MET aberration was verified ex vivo and in vivo. RESULTS: We successfully obtained two gefitinib-resistant NSCLC cell lines with EGFR mutation and abnormal activation of MET. We observed that MET formed a positive feedback loop with the VEGF/VEGFR2 signaling, leading to persistent downstream signaling activation. Specifically, MET up-regulated VEGFR2 expression in a MAPK/ERK/ETS1-dependent manner, while VEGF promoted physical interaction between VEGFR2 and MET, thereby facilitating MET phosphorylation. A MET inhibitor, crizotinib, combined with an anti-VEGF antibody, bevacizumab, enhanced the sensitivity of NSCLC cells to gefitinib and synergistically inhibited the activation of downstream signaling in vitro. Dual inhibition of MET and VEGF combined with EGFR TKIs markedly restrained tumor growth in both human NSCLC xenograft models and in an EGFR/MET co-altered case. CONCLUSIONS: Our work reveals a positive feedback loop between MET and VEGF/VEGFR2, resulting in continuous downstream signal activation. Combined inhibition of MET and VEGF/VEGFR2 signaling pathway may be beneficial for reversing EGFR TKIs resistance.

Polyene phosphatidylcholine promotes tibial fracture healing in rats by stimulating angiogenesis dominated by the VEGFA/VEGFR2 signaling pathway.

One of the factors that predispose to fractures is liver damage. Interestingly, fractures are sometimes accompanied by abnormal liver function. Polyene phosphatidylcholine (PPC) is an important liver repair drug. We wondered if PPC had a role in promoting fracture healing. A rat model of tibial fracture was developed using the modified Einhorn model method. X-rays were used to detect the progression of fracture healing. Progress of ossification and angiogenesis at the fracture site were analyzed by Safranin O/fast green staining and CD31 immunohistochemistry. To investigate whether PPC has a direct angiogenesis effect, HUVECs were used. We performed MTT, wound healing, Transwell migration, and tube formation assays. Finally, RT-qPCR and Western blot analysis were used to study the underlying mechanism. The results showed that PPC significantly shortened the apparent recovery time of mobility in rats. PPC treatment significantly promoted the formation of cartilage callus, endochondral ossification, and angiogenesis at the fracture site. In vitro, PPC promoted the proliferative viability of HUVECs, their ability to heal wounds, and their ability to penetrate membranes in the Transwell apparatus and increased the tube formation of cells. The transcription of VEGFA, VEGFR2, PLCγ, RAS, ERK1/2 and MEK1/2 was significantly up regulated by PPC. Further, the protein level results demonstrated a significant increase in the expression of VEGFA, VEGFR2, MEK1/2, and ERK1/2 proteins. In conclusion, our findings suggest that PPC promotes angiogenesis by activating the VEGFA/VEGFR2 and downstream signaling pathway, thereby accelerating fracture healing.

Cediranib ameliorates portal hypertensive syndrome via inhibition of VEGFR-2 signaling in cirrhotic rats.

Portal hypertension (PHT) is a syndrome caused by systemic and portal hemodynamic disturbances with the progression of cirrhosis. However, the exact mechanisms regulating angiogenesis-related responses in PHT remain unclear. Cediranib is a potent inhibitor of vascular endothelial growth factor receptor (VEGFR) tyrosine kinases, exhibiting a greater affinity for VEGFR-2. Liver cirrhosis was induced by common bile duct ligation (BDL) in Sprague-Dawley rats. Sham-operated rats were controls. BDL and sham rats were randomly allocated to receive Cediranib or vehicle after BDL. On the 28th day, portal hypertension related parameters were surveyed. Cediranib treatment could significantly reduce the portal pressure (PP) in BDL rats, while it did not affect the mean arterial pressure (MAP) in sham groups and BDL groups. Cediranib treatment could significantly affect the stroke volume (SV), cardiac output (CO), cardiac index (CI), systemic vascular resistance (SVR), superior mesenteric artery (SMA) flow and SMA resistance in BDL groups and BDL with Cediranib groups. Cediranib treatment could improve the mesenteric vascular remodeling and contractility. Cediranib treatment significantly reduced mesenteric vascular density. And phospho-VEGFR-2 was significantly downregulated by Cediranib. On the other hand, phospho-endothelial Nitric Oxide Synthases (phospho-eNOS) expressions were upregulated. Cediranib not only improved splanchnic hemodynamics, extrahepatic vascular remodeling and vasodilation, but also alleviated intrahepatic fibrosis and collagen deposition significantly. Cediranib treatment could reduce intrahepatic angiogenesis between BDL-vehicle and BDL-Cediranib rats. In conclusion, Cediranib could improve extrahepatic hyperdynamic circulation by inhibiting extrahepatic angiogenesis through inhibition of the VEGFR-2 signaling pathway, portal collateral circulation formation, as well as eNOS-mediated vasodilatation and vascular remodeling, and at the same time, Cediranib improved intrahepatic fibrogenesis and angiogenesis, which together alleviate cirrhotic PHT syndrome.

Saponin of Aralia taibaiensis promotes angiogenesis through VEGF/VEGFR2 signaling pathway in cerebral ischemic mice.

ETHNOPHARMACOLOGICAL RELEVANCE: Aralia taibaiensis is known for its ability to promote blood circulation and dispel blood stasis, activate meridians and remove arthralgia. The saponins of Aralia taibaiensis (sAT) are the main active components that are often used to treat cardiovascular and cerebrovascular diseases. However, it has not been reported whether sAT can improve ischemic stroke (IS) by promoting angiogenesis. AIM OF THE STUDY: In this study, we investigated the potential of sAT to promote post-ischemic angiogenesis in mice and determined the underlying mechanism through in vitro experiments. METHODS: To establish the middle cerebral artery occlusion (MCAO) mice model in vivo. First of all, we examined the neurological function, brain infarct volume, and degree of brain swelling in MCAO mice. We also observed pathological changes in brain tissue, ultrastructural changes in blood vessels and neurons, and the degree of vascular neovascularization. Additionally, we established the oxygen-glucose deprivation/reoxygenation (OGD/R) -human umbilical vein endothelial cells (HUVECs) model in vitro to detect the survival, proliferation, migration and tube formation of OGD/R HUVECs. Finally, we verified the regulatory mechanism of Src and PLCγ1 siRNA on sAT promoting angiogenesis by cell transfection technique. RESULTS: In the cerebral ischemia-reperfusion mice, sAT distinctly improved the cerebral infarct volume, brain swelling degree, neurological dysfunction, and brain histopathological morphology due to cerebral ischemia/reperfusion injury. It also increased the double positive expression of BrdU and CD31 in brain tissue, promoted the release of VEGF and NO and decreased the release of NSE and LDH. In the OGD/R HUVECs, sAT significantly improved cell survival, proliferation, migration and tube formation, promoted the release of VEGF and NO, and increased the expression of VEGF, VEGFR2, PLCγ1, ERK1/2, Src and eNOS. Surprisingly, the effect of sAT on angiogenesis was inhibited by Src siRNA and PLCγ1 siRNA in OGD/R HUVECs. CONCLUSION: The results proved that sAT promotes angiogenesis in cerebral ischemia-reperfusion mice and its mechanism is to regulate VEGF/VEGFR2 and then regulate Src/eNOS and PLCγ1/ERK1/2.

Filamin A pre-mRNA editing modulates vascularization and tumor growth.

Adenosine to inosine (A to I) editing is mediated by adenosine deaminases acting on RNA (ADAR) enzymes. Inosines are interpreted as guanosines by the translational machinery. Consequently, A to I editing in mRNAs can lead to their recoding and the formation of proteins not encoded in the genome. Filamin A is an actin-crosslinking protein. A to I editing in the filamin pre-mRNA leads to the exchange of a glutamine to an arginine in a highly interactive domain of the protein. However, the consequences of this editing event are still poorly understood. Here we show, using transgenic mice expressing either constitutively edited or constitutively uneditable filamin A that filamin A editing critically controls angiogenesis in tumors but also in a mouse ischemia model. Hyper-editing reduces angiogenesis, while hypoediting leads to increased angiogenesis, possibly by altering vascular endothelial growth factor receptor 2 (VEGFR2) turnover. Further, FLNA editing of the tumor itself seemingly affects its metastatic potential by changing its interaction with the extracellular matrix. We therefore identify filamin A editing as a critical component for angiogenesis, tumor growth, and metastasis formation.

Morphine disrupts macrophage functions even during HIV infection.

HIV-associated neurocognitive impairment (HIV-NCI) is a debilitating comorbidity that reduces quality of life in 15-40% of people with HIV (PWH) taking antiretroviral therapy (ART). Opioid use has been shown to increase neurocognitive deficits in PWH. Monocyte-derived macrophages (MDMs) harbor HIV in the CNS even in PWH on ART. We hypothesized that morphine (MOR), a metabolite of heroin, further dysregulates functional processes in MDMs to increase neuropathogenesis. We found that, in uninfected and HIV-infected primary human MDMs, MOR activates these cells by increasing phagocytosis and up-regulating reactive oxygen species. Effects of MOR on phagocytosis were dependent on µ-opioid receptor activity and were mediated, in part, by inhibited lysosomal degradation of phagocytized substrates. All results persisted when cells were treated with both MOR and a commonly prescribed ART cocktail, suggesting minimal impact of ART during opioid exposure. We then performed mass spectrometry in HIV-infected MDMs treated with or without MOR to determine proteomic changes that suggest additional mechanisms by which opioids affect macrophage homeostasis. Using downstream pathway analyses, we found that MOR dysregulates ER quality control and extracellular matrix invasion. Our data indicate that MOR enhances inflammatory functions and impacts additional cellular processes in HIV-infected MDMs to potentially increases neuropathogenesis in PWH using opioids.

Novel artemisinin derivative FO8643 with anti-angiogenic activity inhibits growth and migration of cancer cells via VEGFR2 signaling.

The vascular endothelial growth factor receptor 2 (VEGFR2) is widely recognized as a key effector in angiogenesis and cancer progression and has been considered a critical target for the development of anti-cancer drugs. Artemisinin (ARS) and its derivatives exert profound efficacy in treating not only malaria but also cancer. As a novel ARS-type compound, FO8643 caused significant suppression of the growth of a panel of cancer cells, including both solid and hematologic malignancies. In CCRF-CEM leukemia cells, FO8643 dramatically inhibited cell proliferation coupled with increased apoptosis and cell cycle arrest. Additionally, FO8643 restrained cell migration in the 2D wound healing assay as well as in a 3D spheroid model of human hepatocellular carcinoma HUH-7 cells. Importantly, SwissTargetPrediction predicted VEGFR2 as an underlying target for FO8643. Molecular docking simulation further indicated that FO8643 formed hydrogen bonds and hydrophobic interactions within the VEGFR2 kinase domain. Moreover, FO8643 directly inhibited VEGFR2 kinase activity and its downstream action including MAPK and PI3K/Akt signaling pathways in HUH-7 cells. Encouragingly, FO8643 decreased angiogenesis in the chorioallantoic membrane assay in vivo. Collectively, FO8643 is a novel ARS-type compound exerting potential VEGFR2 inhibition. FO8643 may be a viable drug candidate in cancer therapy.

SH2-Domain Protein Isolation Using Synthetic Phosphorylated Peptides to Study VEGFR2 Signaling.

Vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) signaling pathways are tightly regulated multistep chain reactions that involve a wide range of molecular interactions and enzymatic activities. The first signal induced by VEGF binding to VEGFR2, is the activation of the receptor tyrosine kinase and autophosphorylation of intracellular tyrosine residues of the receptor. In endothelial cells, five tyrosine residues in the VEGFR2 intracellular domain are essential in signal transmission and in the respective regulation of cellular processes. Because of their number and their localization on the receptor, it is challenging to locate the proteins with which these tyrosine residues interact that result in further downstream signaling cascades. In this chapter, we describe a method to precipitate phosphotyrosine binding proteins using phosphotyrosine-containing synthetic peptides immobilized to magnetic beads. The identity of the precipitated proteins is determined by mass spectrometry and the findings validated by Western blot. Using this method, we identified and verified two proteins, growth factor receptor binding-2 (GRB2) and phosphoinositide 3'-kinase (PI3Kp85), binding to the tyrosine 1214 of VEGFR2. Thereby, we can predict the signaling pathways downstream of pY1214.

Inhibition of Metastatic Hepatocarcinoma by Combined Chemotherapy with Silencing VEGF/VEGFR2 Genes through a GalNAc-Modified Integrated Therapeutic System.

Hepatocellular carcinoma (HCC) is a highly malignant tumor related to high mortality and is still lacking a satisfactory cure. Tumor metastasis is currently a major challenge of cancer treatment, which is highly related to angiogenesis. The vascular endothelial growth factor (VEGF)/VEGFR signaling pathway is thus becoming an attractive therapeutic target. Moreover, chemotherapy combined with gene therapy shows great synergistic potential in cancer treatment with the promise of nanomaterials. In this work, a formulation containing 5-FU and siRNA against the VEGF/VEGFR signaling pathway into N-acetyl-galactosamine (GalNAc)-modified nanocarriers is established. The targeting ability, biocompatibility and pH-responsive degradation capacity ensure the efficient transport of therapeutics by the formulation of 5-FU/siRNA@GalNAc-pDMA to HCC cells. The nano-construct integrated with gene/chemotherapy exhibits significant anti-metastatic HCC activity against C5WN1 liver cancer cells with tumorigenicity and pulmonary metastasis in the C5WN1-induced tumor-bearing mouse model with a tumor inhibition rate of 96%, which is promising for future metastatic HCC treatment.

Murine Beta-Amyloid (1-42) Oligomers Disrupt Endothelial Barrier Integrity and VEGFR Signaling via Activating Astrocytes to Release Deleterious Soluble Factors.

Transgenic mouse models of Alzheimer's disease (AD) overexpress mutations of the human amyloid protein precursor (APP) and presenilin-1 (PSEN1) genes, which are known causes of amyloid pathology in familial AD. However, animal models for studying AD in the context of aging and age-related co-morbidities, such as blood-brain barrier (BBB) disruptions, are lacking. More recently, aged and progeroid mouse models have been proposed as alternatives to study aging-related AD, but the toxicity of murine amyloid-beta protein (Aß) is not well defined. In this study, we aimed to study the potential toxicity of murine Aß on brain endothelial cells and astrocytes, which are important components of the BBB, using mouse brain endothelial cells (bEnd.3) and astrocytes (C8-D1A). Murine-soluble Aß (1-42) oligomers (sAßO42) (10 µM) induced negligible injuries in an endothelial monolayer but induced significant barrier disruptions in a bEnd.3 and C8-D1A co-culture. Similar results of endothelial perturbation were observed in a bEnd.3 monolayer treated with astrocyte-conditioned medium (ACM) generated by astrocytes exposed to sAßO42 (ACM-sAßO42), while additional exogenous sAßO42 did not cause further damage. Western blot analysis showed that ACM-sAßO42 altered the basal activities of vascular endothelial growth factor receptor 2 (VEGFR2), eNOS, and the signaling of the MEK/ERK and Akt pathways in bEnd.3. Our results showed that murine sAßO42 was moderately toxic to an endothelial and astrocyte co-culture. These damaging effects on the endothelial barrier were induced by deleterious soluble factors released from astrocytes, which disrupted endothelial VEGFR2 signaling and perturbed cell survival and barrier stabilization.

LncRNA NKX2-1-AS1 promotes tumor progression and angiogenesis via upregulation of SERPINE1 expression and activation of the VEGFR-2 signaling pathway in gastric cancer.

Long noncoding RNAs (lncRNAs) can compete with endogenous RNAs to modulate the gene expression and contribute to oncogenesis and tumor metastasis. lncRNA NKX2-1-AS1 (NKX2-1 antisense RNA 1) plays a pivotal role in cancer progression and metastasis; however, the contribution of aberrant expression of NKX2-1-AS1 and the mechanism by which it functions as a competing endogenous RNA (ceRNA) in gastric cancer (GC) remains elusive. NKX2-1-AS1 expression was detected in paired tumor and nontumor tissues of 178 GC patients by quantitative reverse transcription PCR (qRT-PCR). Using loss-of-function and gain-of-function experiments, the biological functions of NKX2-1-AS1 were evaluated both in vitro and in vivo. Further, to assess that NKX2-1-AS1 regulates angiogenic processes, tube formation and co-culture assays were performed. RNA binding protein immunoprecipitation (RIP) assay, a dual-luciferase reporter assay, quantitative PCR, Western blot, and fluorescence in situ hybridization (FISH) assays were performed to determine the potential molecular mechanism underlying this ceRNA. The results indicated that NKX2-1-AS1 expression was upregulated in GC cell lines and tumor tissues. Overexpression of NKX2-1-AS1 was significantly associated with tumor progression and enhanced angiogenesis. Functionally, NKX2-1-AS1 overexpression promoted GC cell proliferation, metastasis, invasion, and angiogenesis, while NKX2-1-AS1 knockdown restored these effects, both in vitro and in vivo. RIP and dual-luciferase assays revealed that the microRNA miR-145-5p is a direct target of NKX2-1-AS1 and that NKX2-1-AS1 serves as a ceRNA to sponge miRNA and regulate angiogenesis in GC. Moreover, serpin family E member 1 (SERPINE1) is an explicit target for miR-145-5p; besides, the NKX2-1-AS1/miR-145-5p axis induces the translation of SERPINE1, thus activating the VEGFR-2 signaling pathway to promote tumor progression and angiogenesis. NKX2-1-AS1 overexpression is associated with enhanced tumor cell proliferation, angiogenesis, and poor prognosis in GC. Collectively, NKX2-1-AS1 functions as a ceRNA to miR-145-5p and promotes tumor progression and angiogenesis by activating the VEGFR-2 signaling pathway via SERPINE1.

Lymphatic Endothelial Cell Junctions: Molecular Regulation in Physiology and Diseases.

Lymphatic endothelial cells (LECs) lining lymphatic vessels develop specialized cell-cell junctions that are crucial for the maintenance of vessel integrity and proper lymphatic vascular functions. Successful lymphatic drainage requires a division of labor between lymphatic capillaries that take up lymph via open "button-like" junctions, and collectors that transport lymph to veins, which have tight "zipper-like" junctions that prevent lymph leakage. In recent years, progress has been made in the understanding of these specialized junctions, as a result of the application of state-of-the-art imaging tools and novel transgenic animal models. In this review, we discuss lymphatic development and mechanisms governing junction remodeling between button and zipper-like states in LECs. Understanding lymphatic junction remodeling is important in order to unravel lymphatic drainage regulation in obesity and inflammatory diseases and may pave the way towards future novel therapeutic interventions.

RTK signaling requires C3ar1/C5ar1 and IL-6R joint signaling to repress dominant PTEN, SOCS1/3 and PHLPP restraint.

How receptor tyrosine kinase (RTK) growth signaling is controlled physiologically is incompletely understood. We have previously provided evidence that the survival and mitotic activities of vascular endothelial cell growth factor receptor-2 (VEGFR2) signaling are dependent on C3a/C5a receptor (C3ar1/C5ar1) and IL-6 receptor (IL-6R)-gp130 joint signaling in a physically interactive platform. Herein, we document that the platelet derived and epidermal growth factor receptors (PDGFR and EGFR) are regulated by the same interconnection and clarify the mechanism underlying the dependence. We show that the joint signaling is required to overcome dominant restraint on RTK function by the combined repression of tonically activated PHLPP, SOCS1/SOCS3, and CK2/Fyn dependent PTEN. Signaling studies showed that augmented PI-3KÉ£ activation is the process that overcomes the multilevel growth restraint. Live-cell flow cytometry and single-particle tracking indicated that blockade of C3ar1/C5ar1 or IL-6R signaling suppresses RTK growth factor binding and RTK complex formation. C3ar1/C5ar1 blockade abrogated growth signaling of four additional RTKs. Active relief of dominant growth repression via joint C3ar1/C5ar1 and IL-6R joint signaling thus enables RTK mitotic/survival signaling.

Anti-VEGF/VEGFR2 Monoclonal Antibodies and their Combinations with PD-1/PD-L1 Inhibitors in Clinic.

The vascular endothelial growth factor (VEGF)/VEGF receptor 2 (VEGFR2) signaling pathway is one of the most important pathways responsible for tumor angiogenesis. Currently, two monoclonal antibodies, anti-VEGF-A antibody Bevacizumab and anti-VEGFR2 antibody Ramucizumab, have been approved for the treatment of solid tumors. At the same time, VEGF/VEGFR2 signaling is involved in the regulation of immune responses. It is reported that the inhibition of this pathway has the capability to promote vascular normalization, increase the intra-tumor infiltration of lymphocytes, and decrease the number and function of inhibitory immune cell phenotypes, including Myeloid-derived suppressor cells (MDSCs), regulatory T cells (Tregs) and M2 macrophages. On this basis, a number of clinical studies have been performed to investigate the therapeutic potential of VEGF/VEGFR2-targeting antibodies plus programmed cell death protein 1 (PD-1)/ programmed cell death ligand 1 (PD-L1) inhibitors in various solid tumor types. In this context, VEGF/VEGFR2- targeting antibodies, Bevacizumab and Ramucizumab are briefly introduced, with a description of the differences between them, and the clinical studies involved in the combination of Bevacizumab/ Ramucizumab and PD-1/PD-L1 inhibitors are summarized. We hope this review article will provide some valuable clues for further clinical studies and usages.

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

BACKGROUND: Targeting 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. PURPOSE: To investigate the anti-angiogenic effects of curcumolide and its mechanism of action. METHODS /STUDY DESIGNS: In 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. RESULTS: Intravitreal 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. CONCLUSION: Curcumolide 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.

Tussilagone Suppresses Angiogenesis by Inhibiting the VEGFR2 Signaling Pathway.

Tussilagone (TSL) is a sesquiterpenoid isolated from Tussilago farfara, which has been used as a traditional medicine for the treatment of asthma and bronchitis. It also takes part in the anti-inflammatory and antioxidant effects, but its role in angiogenesis is unknown. Angiogenesis is a cancer feature that is essential for supplying oxygen and nutrients to all proliferating tumor cells. Here, we demonstrated that TSL significantly inhibited the proliferation, migration, invasion, and tube formation of primary human umbilical vascular endothelial cell (HUVEC) in vitro. Also, TSL inhibited vascular endothelial growth factor (VEGF)-induced angiogenesis revealed by Matrigel plug assay in vivo. At present, we observed that TSL inhibited the activity of VEGFR2 signal pathway induced by VEGF. These findings suggested that TSL may serve as a potential therapeutic target in the angiogenesis.

Tumor and endothelial cells collaborate via transcellular receptor complexes.

Many cellular signaling pathways are initiated by cell-surface ligand-sensing complexes that incorporate not just one but multiple receptors. Most studies focus on receptors coexpressed on a single cell (cis interactions), but complexes containing receptors on adjacent cells (trans interactions) are also possible. Recent work by Morin et al published in this journal provides critical evidence for such trans interactions between Neuropilin-1 (NRP1) expressed on human tumor cells and vascular endothelial growth factor receptor 2 (VEGFR2) expressed on adjacent endothelial cells, with the ligand VEGFA binding and bridging the two receptors. They show that the formation of these complexes is correlated with reduced tumor proliferation and increased patient survival. They also observe trans NRP1-VEGFA-VEGFR2 repressing angiogenesis and cis NRP1-VEGFA-VEGFR2 increasing angiogenesis in selected cancers. The distinct molecular signature of each tumor and each patient will determine which type of complexes dominate and will influence prognosis and treatment. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Therapeutic efficacy of a synthetic epsin mimetic peptide in glioma tumor model: uncovering multiple mechanisms beyond the VEGF-associated tumor angiogenesis.

Binding of epsin ubiquitin-interacting motif (UIM) with ubiquitylated VEGFR2 is a critical mechanism for epsin-dependent VEGFR2 endocytosis and physiological angiogenesis. Deletion of epsins in vessel endothelium produces uncontrolled tumor angiogenesis and retards tumor growth in animal models. The aim of this study is to test the therapeutic efficacy and targeting specificity of a chemically-synthesized peptide, UPI, which compete for epsin binding sites in VEGFR2 and potentially inhibits Epsin-VEGFR2 interaction in vivo, in an attempt to reproduce an epsin-deficient phenotype in tumor angiogenesis. Our data show that UPI treatment significantly inhibits and shrinks tumor growth in GL261 glioma tumor model. UPI peptide specifically targets VEGFR2 signaling pathway revealed by genetic and biochemical approaches. Furthermore, we demonstrated that UPI peptide treatment caused serious thrombosis in tumor vessels and damages tumor cells after a long-term UPI peptide administration. Besides, we revealed that UPI peptides were unexpectedly targeted cancer cells and induced apoptosis. We conclude that UPI peptide is a potent inhibitor to glioma tumor growth through specific targeting of VEGFR2 signaling in the tumor vasculature and cancer cells, which may offer a potentially novel treatment for cancer patients who are resistant to current anti-VEGF therapies.

I131 reinforces antitumor activity of metuximab by reversing epithelial-mesenchymal transition via VEGFR-2 signaling in hepatocellular carcinoma.

CD147 is highly expressed in hepatocellular carcinoma (HCC) and associated with the invasion and metastasis of HCC. The efficacy of I131 -metuximab (I131 -mab), a newly developed agent that targets CD147, as a radio-immunotherapy for local HCC, has been validated in clinical practice. However, the synergistic anticancer activity and molecular mechanism of different conjugated components within I131 -mab remain unclear. In this study, the cytological experiments proved that I131 -mab inhibited the proliferation and invasion of HCC cells. Mechanically, this inhibition effect was mainly mediated by the antibody component part of I131 -mab, which could reverse the epithelial-mesenchymal transition of HCC cells partially by suppressing the phosphorylation of VEGFR-2. The inhibitory effect of I131 on HCC cell proliferation and invasion is limited, whereas, when combined with metuximab, I131 significantly enhanced the sensitivity of HCC cells to CD147-mab and consequently reinforced the anticancer effects of CD147-mab, suggesting that the two components of I131 -mab exerted synergistic anti-HCC capability. Furthermore, the experiments using SMMC-7721 human HCC xenografts in athymic nude mice showed that I131 -mab and CD147-mab significantly inhibited the growth of xenograft tumors and that I131 -mab was more effective than CD147-mab. In conclusion, our results elucidated the mechanism underlying the anti-HCC effects of I131 -mab and provided a theoretical foundation for the clinical application of I131 -mab.

Circular RNA MYLK as a competing endogenous RNA promotes bladder cancer progression through modulating VEGFA/VEGFR2 signaling pathway.

Accumulating evidences indicate that circular RNAs (circRNAs) play a vital role in modulating gene expression. However, the mechanisms underlying circRNAs remain largely elusive. Here, we screened circRNA and mRNA expression profiles of bladder carcinoma (BC) using microarray analysis. We found that circRNA-MYLK and VEGFA were significantly up-regulated and co-expressed in BC. Importantly, circRNA-MYLK levels were related to the progression of stage and grade of BC. Mechanistically, we demonstrated that circRNA-MYLK could directly bind to miR-29a and relieve suppression for target VEGFA, which activated VEGFA/VEGFR2 signaling pathway. Functionally, we found that ectopically expressing circRNA-MYLK accelerated cell proliferation, migration, tube formation of HUVEC and rearranged cytoskeleton. Moreover, up-regulating circRNA-MYLK promoted epithelial-mesenchymal transition (EMT). Whereas circRNA-MYLK knockdown decreased cell proliferation, motility, and induced apoptosis. Finally, up-regulating circRNA-MYLK promoted the growth, angiogenesis and metastasis of BC xenografts. Taken together, this study demonstrated for the first time that circRNA-MYLK might function as competing endogenous RNA (ceRNA) for miR-29a, which could contribute to EMT and the development of BC through activating VEGFA/VEGFR2 and downstream Ras/ERK signaling pathway. Our data suggest that circRNA-MYLK would be a promising target for BC diagnosis and therapy.