VEGF receptor signaling links inflammation and tumorigenesis in colitis-associated cancer

Whereas the inhibition of vascular endothelial growth factor (VEGF) has shown promising results in sporadic colon cancer, the role of VEGF signaling in colitis-associated cancer (CAC) has not been addressed. We found that, unlike sporadic colorectal cancer and control patients, patients with CAC show activated VEGFR2 on intestinal epithelial cells (IECs). We then explored the function of VEGFR2 in a murine model of colitis-associated colon cancer characterized by increased VEGFR2 expression. Epithelial cells in tumor tissue expressed VEGFR2 and responded to VEGF stimulation with augmented VEGFR2-mediated proliferation. Blockade of VEGF function via soluble decoy receptors suppressed tumor development, inhibited tumor angiogenesis, and blocked tumor cell proliferation. Functional studies revealed that chronic inflammation leads to an up-regulation of VEGFR2 on IECs. Studies in conditional STAT3 mutant mice showed that VEGFR signaling requires STAT3 to promote epithelial cell proliferation and tumor growth in vivo. Thus, VEGFR-signaling acts as a direct growth factor for tumor cells in CAC, providing a molecular link between inflammation and the development of colon cancer.     

Taspine downregulates VEGF expression and inhibits proliferation of vascular endothelial cells through PI3 kinase and MAP kinase signaling pathways

Taspine is an active component isolated from Radix et Rhizoma Leonticis with inhibiting tumor angiogenic properties. The molecular mechanism(s) of taspine on tumor angiogenic inhibition have not been well documented. The aim of this study was to elucidate in detail the effects of taspine on genetic expressions of VEGF in human umbilical vein endothelial cells, and on VEGFR2-mediated intracellular signaling of human umbilical vein endothelial cells. The genetic expression of vascular endothelial growth factor (VEGF) in the human umbilical vein endothelial cells (HUVECs) treated with taspine in vitro was measured by the ELISA and RT-PCR methods. The effects of taspine on cell proliferation of HUVECs and HUVECs induced by VEGF165 were considered by using MTT assay. And also, a western blot was used to detect Akt and Erk1/2 expressions and their phosphorylation levels in HUVECs treated with taspine. Our results show that VEGF protein and mRNA expressions in the cells treated with taspine were significantly decreased. Taspine also significantly inhibited cell proliferation of HUVECs induced by VEGF165. HUVECs treated with taspine showed decreased Akt and Erk1/2 activities.     

Fasudil-induced hypoxia-inducible factor-1alpha degradation disrupts a hypoxia-driven vascular endothelial growth factor autocrine mechanism in endothelial cells

Hypoxic response of endothelial cells (EC) is an important component of tumor angiogenesis. Especially, hypoxia-inducible factor-1 (HIF-1)-dependent EC-specific mechanism is an essential component of tumor angiogenesis. Recently, the Rho/Rho-associated kinase (ROCK) signaling has been shown to play a key role in HIF-1alpha induction in renal cell carcinoma and trophoblast. The present study was designed to investigate whether low oxygen conditions might modulate HIF-1alpha expression through the Rho/ROCK signaling in human umbilical vascular ECs (HUVEC). Pull-down assay showed that hypoxia stimulated RhoA activity. Under hypoxic conditions, HUVECs transfected with small interfering RNA of RhoA and ROCK2 exhibited decreased levels of HIF-1alpha protein compared with nontargeted small interfering RNA transfectants, whereas HIF-1alpha mRNA levels were not altered. One of ROCK inhibitors, fasudil, inhibited hypoxia-induced HIF-1alpha expression without altering HIF-1alpha mRNA expression. Furthermore, proteasome inhibitor prevented the effect of fasudil on HIF-1alpha expression, and polyubiquitination was enhanced by fasudil. These results suggested that hypoxia-induced HIF-1alpha expression is through preventing HIF-1alpha degradation by activating the Rho/ROCK signaling in ECs. Furthermore, hypoxia induced both vascular endothelial growth factor (VEGF) and VEGF receptor-2 expression through the Rho/ROCK/HIF-1alpha signaling in HUVECs. Thus, augmented VEGF/VEGF receptor-2 autocrine mechanism stimulated HUVEC migration under hypoxic conditions. In summary, the Rho/ROCK/HIF-1alpha signaling is an essential mechanism for hypoxia-driven, VEGF-mediated autocrine loop in ECs. Therefore, fasudil might have the antimigratory effect against ECs in tumor angiogenesis.     

AIP1 functions as an endogenous inhibitor of VEGFR2-mediated signaling and inflammatory angiogenesis in mice

ASK1-interacting protein-1 (AIP1), a recently identified member of the Ras GTPase-activating protein family, is highly expressed in vascular ECs and regulates EC apoptosis in vitro. However, its function in vivo has not been established. To study this, we generated AIP1-deficient mice (KO mice). Although these mice showed no obvious defects in vascular development, they exhibited dramatically enhanced angiogenesis in 2 models of inflammatory angiogenesis. In one of these models, the enhanced angiogenesis observed in the KO mice was associated with increased VEGF-VEGFR2 signaling. Consistent with this, VEGF-induced ear, cornea, and retina neovascularization were greatly augmented in KO mice and the enhanced retinal angiogenesis was markedly diminished by overexpression of AIP1. In vitro, VEGF-induced EC migration was inhibited by AIP1 overexpression, whereas it was augmented by both AIP1 knockout and knockdown, with the enhanced EC migration caused by AIP1 knockdown being associated with increased VEGFR2 signaling. We present mechanistic data that suggest AIP1 is recruited to the VEGFR2-PI3K complex, binding to both VEGFR2 and PI3K p85, at a late phase of the VEGF response, and that this leads to inhibition of VEGFR2 signaling. Taken together, our data demonstrate that AIP1 functions as an endogenous inhibitor in VEGFR2-mediated adaptive angiogenesis in mice.     

Targeting prion-like protein doppel selectively suppresses tumor angiogenesis

Controlled and site-specific regulation of growth factor signaling remains a major challenge for current antiangiogenic therapies, as these antiangiogenic agents target normal vasculature as well tumor vasculature. In this article, we identified the prion-like protein doppel as a potential therapeutic target for tumor angiogenesis. We investigated the interactions between doppel and VEGFR2 and evaluated whether blocking the doppel/VEGFR2 axis suppresses the process of angiogenesis. We discovered that tumor endothelial cells (TECs), but not normal ECs, express doppel; tumors from patients and mouse xenografts expressed doppel in their vasculatures. Induced doppel overexpression in ECs enhanced vascularization, whereas doppel constitutively colocalized and complexed with VEGFR2 in TECs. Doppel inhibition depleted VEGFR2 from the cell membrane, subsequently inducing the internalization and degradation of VEGFR2 and thereby attenuating VEGFR2 signaling. We also synthesized an orally active glycosaminoglycan (LHbisD4) that specifically binds with doppel. We determined that LHbisD4 concentrates over the tumor site and that genetic loss of doppel in TECs decreases LHbisD4 binding and targeting both in vitro and in vivo. Moreover, LHbisD4 eliminated VEGFR2 from the cell membrane, prevented VEGF binding in TECs, and suppressed tumor growth. Together, our results demonstrate that blocking doppel can control VEGF signaling in TECs and selectively inhibit tumor angiogenesis.     

Targeting FGFR/PDGFR/VEGFR impairs tumor growth, angiogenesis, and metastasis by effects on tumor cells, endothelial cells, and pericytes in pancreatic cancer

Activation of receptor tyrosine kinases, such as fibroblast growth factor receptor (FGFR), platelet-derived growth factor receptor (PDGFR), and VEGF receptor (VEGFR), has been implicated in tumor progression and metastasis in human pancreatic cancer. In this study, we investigated the effects of TKI258, a tyrosine kinase inhibitor to FGFR, PDGFR, and VEGFR on pancreatic cancer cell lines (HPAF-II, BxPC-3, MiaPaCa2, and L3.6pl), endothelial cells, and vascular smooth muscle cells (VSMC). Results showed that treatment with TKI258 impaired activation of signaling intermediates in pancreatic cancer cells, endothelial cells, and VSMCs, even upon stimulation with FGF-1, FGF-2, VEGF-A, and PDGF-B. Furthermore, blockade of FGFR/PDGFR/VEGFR reduced survivin expression and improved activity of gemcitabine in MiaPaCa2 pancreatic cancer cells. In addition, motility of cancer cells, endothelial cells, and VSMCs was reduced upon treatment with TKI258. In vivo, therapy with TKI258 led to dose-dependent inhibition of subcutaneous (HPAF-II) and orthotopic (L3.6pl) tumor growth. Immunohistochemical analysis revealed effects on tumor cell proliferation [bromodeoxyuridine (BrdUrd)] and tumor vascularization (CD31). Moreover, lymph node metastases were significantly reduced in the orthotopic tumor model when treatment was initiated early with TKI258 (30 mg/kg/d). In established tumors, TKI258 (30 mg/kg/d) led to significant growth delay and improved survival in subcutaneous and orthotopic models, respectively. These data provide evidence that targeting FGFR/PDFGR/VEGFR with TKI258 may be effective in human pancreatic cancer and warrants further clinical evaluation.     

Vascular endothelial growth factor and angiopoietin-1 stimulate postnatal hematopoiesis by recruitment of vasculogenic and hematopoietic stem cells

Tyrosine kinase receptors for angiogenic factors vascular endothelial growth factor (VEGF) and angiopoietin-1 (Ang-1) are expressed not only by endothelial cells but also by subsets of hematopoietic stem cells (HSCs). To further define their role in the regulation of postnatal hematopoiesis and vasculogenesis, VEGF and Ang-1 plasma levels were elevated by injecting recombinant protein or adenoviral vectors expressing soluble VEGF(165), matrix-bound VEGF(189), or Ang-1 into mice. VEGF(165), but not VEGF(189), induced a rapid mobilization of HSCs and VEGF receptor (VEGFR)2(+) circulating endothelial precursor cells (CEPs). In contrast, Ang-1 induced delayed mobilization of CEPs and HSCs. Combined sustained elevation of Ang-1 and VEGF(165) was associated with an induction of hematopoiesis and increased marrow cellularity followed by proliferation of capillaries and expansion of sinusoidal space. Concomitant to this vascular remodeling, there was a transient depletion of hematopoietic activity in the marrow, which was compensated by an increase in mobilization and recruitment of HSCs and CEPs to the spleen resulting in splenomegaly. Neutralizing monoclonal antibody to VEGFR2 completely inhibited VEGF(165), but not Ang-1-induced mobilization and splenomegaly. These data suggest that temporal and regional activation of VEGF/VEGFR2 and Ang-1/Tie-2 signaling pathways are critical for mobilization and recruitment of HSCs and CEPs and may play a role in the physiology of postnatal angiogenesis and hematopoiesis.     

VEGFR2 induces c-Src signaling and vascular permeability in vivo via the adaptor protein TSAd

Regulation of vascular endothelial (VE) growth factor (VEGF)-induced permeability is critical in physiological and pathological processes. We show that tyrosine phosphorylation of VEGF receptor 2 (VEGFR2) at Y951 facilitates binding of VEGFR2 to the Rous sarcoma (Src) homology 2-domain of T cell-specific adaptor (TSAd), which in turn regulates VEGF-induced activation of the c-Src tyrosine kinase and vascular permeability. c-Src was activated in vivo and in vitro in a VEGF/TSAd-dependent manner, and was regulated via increased phosphorylation at pY418 and reduced phosphorylation at pY527. Tsad silencing blocked VEGF-induced c-Src activation, but did not affect pathways involving phospholipase Cγ, extracellular regulated kinase, and endothelial nitric oxide. VEGF-induced rearrangement of VE-cadherin-positive junctions in endothelial cells isolated from mouse lungs, or in mouse cremaster vessels, was dependent on TSAd expression, and TSAd formed a complex with VE-cadherin, VEGFR2, and c-Src at endothelial junctions. Vessels in tsad(-/-) mice showed undisturbed flow and pressure, but impaired VEGF-induced permeability, as measured by extravasation of Evans blue, dextran, and microspheres in the skin and the trachea. Histamine-induced extravasation was not affected by TSAd deficiency. We conclude that TSAd is required for VEGF-induced, c-Src-mediated regulation of endothelial cell junctions and for vascular permeability.     

Autocrine VEGF-VEGFR2-Neuropilin-1 signaling promotes glioma stem-like cell viability and tumor growth

Although vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) is traditionally regarded as an endothelial cell protein, evidence suggests that VEGFRs may be expressed by cancer cells. Glioblastoma multiforme (GBM) is a lethal cancer characterized by florid vascularization and aberrantly elevated VEGF. Antiangiogenic therapy with the humanized VEGF antibody bevacizumab reduces GBM tumor growth; however, the clinical benefits are transient and invariably followed by tumor recurrence. In this study, we show that VEGFR2 is preferentially expressed on the cell surface of the CD133(+) human glioma stem-like cells (GSCs), whose viability, self-renewal, and tumorigenicity rely, at least in part, on signaling through the VEGF-VEGFR2-Neuropilin-1 (NRP1) axis. We find that the limited impact of bevacizumab-mediated VEGF blockage may reflect ongoing autocrine signaling through VEGF-VEGFR2-NRP1, which is associated with VEGFR2-NRP1 recycling and a pool of active VEGFR2 within a cytosolic compartment of a subset of human GBM cells. Whereas bevacizumab failed to inhibit prosurvival effects of VEGFR2-mediated signaling, GSC viability under unperturbed or radiation-evoked stress conditions was attenuated by direct inhibition of VEGFR2 tyrosine kinase activity and/or shRNA-mediated knockdown of VEGFR2 or NRP1. We propose that direct inhibition of VEGFR2 kinase may block the highly dynamic VEGF-VEGFR2-NRP1 pathway and inspire a GBM treatment strategy to complement the currently prevalent ligand neutralization approach.     

Fibroblast growth factors: the keepers of endothelial normalcy

Endothelial cells (ECs) under physiologic and pathologic conditions are capable of substantial plasticity that includes the endothelial-mesenchymal transition (EndMT). Notably, in the hypoxic pulmonary circulation EndMT likely drives increases in the pulmonary arterial blood pressure, leading to pulmonary arterial hypertension (PAH). However, it is unclear whether suppressing EndMT can prevent PAH development or mitigate established disease. In this issue of the JCI, Woo et al. generated mice with EC-specific deletion of FGFR1 and -2 and mice with EC-specific expression of a constitutively active FGFR1 to determine the role of FGF signaling in PAH. Mice with FGFR1/2 deletion in ECs that were exposed to hypoxic conditions developed extensive EndMT and more severe PAH than control mice. Animals with the constitutively active endothelial FGFR were protected from hypoxia-induced EndMT and PAH development. These findings suggest that FGF signaling may promote vascular resilience and prevent hypoxia-induced development of EndMT and PAH.     

Tumor VEGF:VEGFR2 autocrine feed-forward loop triggers angiogenesis in lung cancer

The molecular mechanisms that control the balance between antiangiogenic and proangiogenic factors and initiate the angiogenic switch in tumors remain poorly defined. By combining chemical genetics with multimodal imaging, we have identified an autocrine feed-forward loop in tumor cells in which tumor-derived VEGF stimulates VEGF production via VEGFR2-dependent activation of mTOR, substantially amplifying the initial proangiogenic signal. Disruption of this feed-forward loop by chemical perturbation or knockdown of VEGFR2 in tumor cells dramatically inhibited production of VEGF in vitro and in vivo. This disruption was sufficient to prevent tumor growth in vivo. In patients with lung cancer, we found that this VEGF:VEGFR2 feed-forward loop was active, as the level of VEGF/VEGFR2 binding in tumor cells was highly correlated to tumor angiogenesis. We further demonstrated that inhibition of tumor cell VEGFR2 induces feedback activation of the IRS/MAPK signaling cascade. Most strikingly, combined pharmacological inhibition of VEGFR2 (ZD6474) and MEK (PD0325901) in tumor cells resulted in dramatic tumor shrinkage, whereas monotherapy only modestly slowed tumor growth. Thus, a tumor cell-autonomous VEGF:VEGFR2 feed-forward loop provides signal amplification required for the establishment of fully angiogenic tumors in lung cancer. Interrupting this feed-forward loop switches tumor cells from an angiogenic to a proliferative phenotype that sensitizes tumor cells to MAPK inhibition.     

Protein kinase C-{alpha} mediates epidermal growth factor receptor transactivation in human prostate cancer cells

Progression of human prostate cancer to a malignancy that is refractory to androgen-ablation therapy renders the disease resistant to available treatment options and accounts for the high prostate cancer mortality rate. Epidermal growth factor receptor (EGFR) expression in human prostate cancer specimens increases with disease progression to androgen-refractory prostate cancer, and experimental models implicate EGFR-dependent signaling to Erk1/2 activation in the androgen-refractory prostate cancer phenotype. 12-O-Tetradecanoylphorbol-13-acetate (TPA)-induced Erk1/2 activation in human prostate cancer PC-3 cells is a paradigm of diacylglycerol-induced EGFR transactivation in androgen-independent prostate cancer. In this report, we establish an obligatory role for TPA-induced protein kinase C (PKC)-alpha activation in EGFR transactivation and signaling to Erk1/2 activation in PC-3 cells. TPA-regulated molecules include PKCs, PKDs, and Ras guanyl nucleotide-releasing proteins. The PKC-selective inhibitors GF109203X and Go6983 each blocked TPA-induced EGFR transactivation, indicating a requirement for PKC. PC-3 cells express four PKC isozymes. Prolonged bryostatin 1 treatment abrogated PKCalpha expression without altering expression levels of the other PKC isozymes. Pharmacologic PKCalpha "knockdown" abrogated TPA-induced Erk1/2 activation without affecting the EGF/EGFR-induced response, indicating that PKCalpha was required for EGFR transactivation but dispensable for signaling of ligand-activated EGFR to Erk1/2 activation. We corroborated this by showing that Go6976, which is a PKCalpha-selective inhibitor in PC-3 cells, likewise abolished TPA-induced Erk1/2 activation and did not inhibit EGF/EGFR-induced Erk1/2 activation. Go6976 had similar effects in DU145 cells, providing evidence for a common PKCalpha-dependent Erk1/2 activation mechanism in androgen-independent human prostate cancer cells of distinct genetic origin. These results constitute a rational basis for selective PKCalpha inhibition as a modality of prostate cancer therapy.     

A Compartment Model of VEGF Distribution in Humans in the Presence of Soluble VEGF Receptor-1 Acting as a Ligand Trap

Vascular endothelial growth factor (VEGF), through its activation of cell surface receptor tyrosine kinases including VEGFR1 and VEGFR2, is a vital regulator of stimulatory and inhibitory processes that keep angiogenesis – new capillary growth from existing microvasculature – at a dynamic balance in normal physiology. Soluble VEGF receptor-1 (sVEGFR1) – a naturally-occurring truncated version of VEGFR1 lacking the transmembrane and intracellular signaling domains – has been postulated to exert inhibitory effects on angiogenic signaling via two mechanisms: direct sequestration of angiogenic ligands such as VEGF; or dominant-negative heterodimerization with surface VEGFRs. In pre-clinical studies, sVEGFR1 gene and protein therapy have demonstrated efficacy in inhibiting tumor angiogenesis; while in clinical studies, sVEGFR1 has shown utility as a diagnostic or prognostic marker in a widening array of angiogenesis–dependent diseases. Here we developed a novel computational multi-tissue model for recapitulating the dynamic systemic distributions of VEGF and sVEGFR1. Model features included: physiologically-based multi-scale compartmentalization of the human body; inter-compartmental macromolecular biotransport processes (vascular permeability, lymphatic drainage); and molecularly-detailed binding interactions between the ligand isoforms VEGF₁₂₁ and VEGF₁₆₅, signaling receptors VEGFR1 and VEGFR2, non-signaling co-receptor neuropilin-1 (NRP1), as well as sVEGFR1. The model was parameterized to represent a healthy human subject, whereupon we investigated the effects of sVEGFR1 on the distribution and activation of VEGF ligands and receptors. We assessed the healthy baseline stability of circulating VEGF and sVEGFR1 levels in plasma, as well as their reliability in indicating tissue-level angiogenic signaling potential. Unexpectedly, simulated results showed that sVEGFR1 – acting as a diffusible VEGF sink alone, i.e., without sVEGFR1-VEGFR heterodimerization – did not significantly lower interstitial VEGF, nor inhibit signaling potential in tissues. Additionally, the sensitivity of plasma VEGF and sVEGFR1 to physiological fluctuations in transport rates may partially account for the heterogeneity in clinical measurements of these circulating angiogenic markers, potentially hindering their diagnostic reliability for diseases.     

VEGF released from a fibrin biomatrix increases VEGFR‐2 expression and improves early outcome after ischaemia–reperfusion injury

Skeletal ischaemia–reperfusion (I–R) injury may influence patient outcome after severe vascular trauma or clamping of major vessels. The aim of this study was to observe whether locally applied vascular endothelial growth factor (VEGF) in fibrin could induce the expression of VEGF‐receptor‐2 (VEGFR‐2) and improve the outcome after I–R injury. Transgenic mice expressing VEGFR‐2 promoter‐controlled luciferase were used for the assessment of VEGFR‐2 expression. Ischaemia was induced for 2 h by a tension‐controlled tourniquet to the hind limb, followed by 24 h of reperfusion. The animals were locally injected subcutaneously with fibrin sealant containing 20 or 200 ng VEGF; control animals received no treatment or fibrin sealant application. In vivo VEGFR‐2 expression was quantified upon administration of luciferin at several observation times. For oedema and inflammation quantification, wet:dry ratio measurements and a myeloperoxidase assay of the muscle tissue were performed. Laser Doppler imaging showed that ischaemia was present and that the blood flow had returned to baseline levels after 24 h of reperfusion. VEGFR‐2 expression levels in the fibrin + 200 ng VEGF were significantly higher than in all other groups. Granulocyte infiltration was reduced in both treatment groups, as well as reduced oedema formation. These results showed that VEGF released from fibrin had a positive effect on early I–R outcome in a mouse model, possibly via VEGFR‐2. Copyright © 2016 John Wiley & Sons, Ltd.     

P2Y12 receptor-mediated potentiation of thrombin-induced thromboxane A2 generation in platelets occurs through regulation of Erk1/2 activation

BACKGROUND: Thromboxane A2 (TXA2) is a positive feedback lipid mediator that is generated upon stimulation of platelets with various agonists. Aspirin works as an antithrombotic drug by blocking the generation of TXA2. The aim of this study was to evaluate the role of the purinergic P2Y receptors in thrombin-induced TXA2 generation. RESULTS: PAR1-activating peptide (SFLLRN), PAR4-activating peptide (AYPGKF), and thrombin, induced the activation of cytosolic phospholipase A2 (cPLA2), release of arachidonic acid (AA) from membrane-bound phospholipids, and subsequent TXA2 generation in human platelets. The actions of these agonists were significantly inhibited in the presence of the P2Y12 receptor antagonist, AR-C69931MX, but not the P2Y1 receptor antagonist, MRS2179. In addition, AYPGKF- and thrombin-induced TXA2 generation was significantly reduced in platelets from mice dosed with clopidogrel, confirming the results obtained with the human platelets. Also, Pearl mouse platelets that lack releasable nucleotides generated significantly less TXA2 when compared with the wild-type littermates in response to PAR stimulation. Inhibition of extracellular signal-regulated protein kinase 1/2 (Erk 1/2) activation using U0126, an inhibitor of MAP kinase kinase (MEK), suppressed PAR-mediated cPLA2 phosphorylation and TXA2 generation. Further, platelets that were pretreated with AR-C69931MX, as well as Pearl mouse platelets, displayed the reduced levels of Erk1/2 phosphorylation upon stimulation with the PAR agonists. CONCLUSIONS: Based on these findings, we conclude that thrombin-induced Erk1/2 activation is essential for PAR-mediated TXA2 generation, which is potentiated by the P2Y12 receptor-mediated signaling pathway but not the P2Y1 receptor-mediated signaling pathway. Finally, using selective inhibitors of Src kinases, we show that PAR-mediated Src activation precedes Erk1/2 activation.     

Precise Scheduling of Chemotherapy Primes VEGF-producing Tumors for Successful Systemic Oncolytic Virotherapy

We have previously reported that a burst of vascular endothelial growth factor (VEGF) signaling to tumor-associated endothelium induces a proviral state, during which systemically delivered oncolytic reovirus can replicate in endothelium, thereby inducing immune-mediated vascular collapse and significant antitumor therapy. Using chimeric receptors, we show here that induction of the proviral state proceeds through VEGFR2, but not VEGFR1, signaling in endothelial cells. In contrast, innate immune activation by reovirus-exposed endothelial cells was predominantly through VEGFR1. By screening conventional chemotherapies for their ability to induce similar effects in combination with reovirus both in vitro and in vivo, we observed that the proviral state could also be induced in endothelial cells exposed to VEGF during rebound from paclitaxel-mediated inhibition of VEGF signaling. We translated these in vitro findings in vivo by careful scheduling of paclitaxel chemotherapy with systemic virotherapy, neither of which alone had therapeutic effects against B16 tumors. Systemic availability of reovirus during endothelial cell recovery from paclitaxel treatment allowed for endothelial replication of the virus, immune-mediated therapy, and tumor cures. Therefore, careful scheduling of combination viro- and chemotherapies, which preclinical testing suggests are individually ineffective against tumor cells, can lead to rational new clinical protocols for systemic treatments with oncolytic viruses.     

Endothelial epsin deficiency decreases tumor growth by enhancing VEGF signaling

Epsins are a family of ubiquitin-binding, endocytic clathrin adaptors. Mice lacking both epsins 1 and 2 (Epn1/2) die at embryonic day 10 and exhibit an abnormal vascular phenotype. To examine the angiogenic role of endothelial epsins, we generated mice with constitutive or inducible deletion of Epn1/2 in vascular endothelium. These mice exhibited no abnormal phenotypes under normal conditions, suggesting that lack of endothelial epsins 1 and 2 did not affect normal blood vessels. In tumors, however, loss of epsins 1 and 2 resulted in disorganized vasculature, significantly increased vascular permeability, and markedly retarded tumor growth. Mechanistically, we show that VEGF promoted binding of epsin to ubiquitinated VEGFR2. Loss of epsins 1 and 2 specifically impaired endocytosis and degradation of VEGFR2, which resulted in excessive VEGF signaling that compromised tumor vascular function by exacerbating nonproductive leaky angiogenesis. This suggests that tumor vasculature requires a balance in VEGF signaling to provide sufficient productive angiogenesis for tumor development and that endothelial epsins 1 and 2 negatively regulate the output of VEGF signaling. Promotion of excessive VEGF signaling within tumors via a block of epsin 1 and 2 function may represent a strategy to prevent normal angiogenesis in cancer patients who are resistant to anti-VEGF therapies.     

运动对高脂饲养大鼠白色脂肪组织血管内皮生成相关因子的影响

目的:观察8周中等强度有氧运动干预以及高脂饮食对雄性大鼠血管内皮生成相关因子的影响.方法:40只3周龄SPF级雄性SD大鼠适应性喂养3天后,随机分为标准安静组(CS)、标准运动组(CE)、高脂安静组(HS)、高脂运动组(HE).标准组大鼠喂饲普通饲料(D12450B),高脂组大鼠喂饲高脂饲料(D12451),所有大鼠均...     

Imatinib inhibits VEGF-independent angiogenesis by targeting neuropilin 1–dependent ABL1 activation in endothelial cells

To enable new blood vessel growth, endothelial cells (ECs) express neuropilin 1 (NRP1), and NRP1 associates with the receptor tyrosine kinase VEGFR2 after binding the vascular endothelial growth factor A (VEGF) to enhance arteriogenesis. We report that NRP1 contributes to angiogenesis through a novel mechanism. In human and mouse ECs, the integrin ligand fibronectin (FN) stimulated actin remodeling and phosphorylation of the focal adhesion component paxillin (PXN) in a VEGF/VEGFR2-independent but NRP1-dependent manner. NRP1 formed a complex with ABL1 that was responsible for FN-dependent PXN activation and actin remodeling. This complex promoted EC motility in vitro and during angiogenesis on FN substrates in vivo. Accordingly, both physiological and pathological angiogenesis in the retina were inhibited by treatment with Imatinib, a small molecule inhibitor of ABL1 which is widely used to prevent the proliferation of tumor cells that express BCR-ABL fusion proteins. The finding that NRP1 regulates angiogenesis in a VEGF- and VEGFR2-independent fashion via ABL1 suggests that ABL1 inhibition provides a novel opportunity for anti-angiogenic therapy to complement VEGF or VEGFR2 blockade in eye disease or solid tumor growth.New blood vessels arise from preexisting ones in the process of physiological angiogenesis, both during embryonic development and in the female reproductive cycle. In addition, angiogenic vessel growth is closely associated with the progression of various cancers and eye diseases (Welti et al., 2013). Thus, angiogenesis promotes the growth of solid tumors and their metastasis, whereas abnormal blood vessel growth in the eye impairs visual function. For example, abnormal choroidal angiogenesis is a pathological feature of the “wet” form of age-related macular degeneration (AMD), whereas excessive retinal angiogenesis leads to vascular malformations that protrude into the vitreous in patients with proliferative diabetic retinopathy (PDR) or retinopathy of prematurity (ROP; Campochiaro, 2013).The vascular endothelial growth factor A (VEGF) is a key mediator of both physiological and pathological angiogenesis and a validated target for anti-angiogenesis therapy in the clinic (Kim and D’Amore, 2012; Welti et al., 2013). For example, anti-VEGF therapy stabilized sight in >90% and significantly improved vision in ∼30% of patients with wet AMD over a 2-yr treatment period (Rosenfeld et al., 2006). However, the efficacy of anti-VEGF in AMD has been mainly attributed to reduced vascular leak rather than to an effect on neoangiogenesis (Campochiaro, 2013). The incomplete sensitivity of pathological eye vessels to anti-VEGF therapy may suggest that VEGF-independent pathways also contribute to ocular angiogenesis. Moreover, long-term anti-VEGF treatment has been proposed to pose likely risks, as preclinical studies for several different eye diseases revealed excessive neuronal cell death in the retina after VEGF blockade (Nishijima et al., 2007; Saint-Geniez et al., 2008; Foxton et al., 2013). Yet clinical data from long-term studies of patients with continuous anti-VEGF treatment are not available. These considerations, combined with the observation that tumor vessels can develop resistance to anti-VEGF therapy (Casanovas et al., 2005; Shojaei et al., 2007), highlight the need to identify effective anti-angiogenesis therapies that are based on VEGF-independent targets and can be used in combination with or independently of anti-VEGF therapy to improve outcome for patients.Neuropilin 1 (NRP1) is a non-catalytic receptor for the VEGF165 isoform of VEGF that complexes with VEGFR2 to potentiate signal transduction in endothelial cells (ECs; e.g., Mamluk et al., 2002; Koch et al., 2011). Thus, the NRP1 cytoplasmic tail recruits a trafficking complex that directs VEGFR2 along an endocytic pathway that prevents receptor dephosphorylation to augment MAPK signaling via ERK1 and ERK2 (Salikhova et al., 2008; Ballmer-Hofer et al., 2011; Lanahan et al., 2013). This NRP1 function is essential for arteriogenesis, which depends on luminal vessel growth, but is dispensable for angiogenesis, driven by vessel sprouting, branching, and fusion (Fantin et al., 2011; Lanahan et al., 2013). Additionally, NRP1 is able to interact with extracellular matrix (ECM) receptors of the integrin family independently of VEGFR2 (Murga et al., 2005; Fukasawa et al., 2007; Valdembri et al., 2009). However, the relative significance of NRP1 for VEGF/VEGFR2-dependent versus integrin ligand-stimulated, but VEGFR2-independent processes for angiogenesis in vivo has not previously been determined. Moreover, the intracellular pathways that may be regulated by NRP1 in a VEGF/VEGFR2-independent fashion have, surprisingly, not yet been defined.Here, we demonstrate that NRP1 promotes the phosphorylation of integrin targets such as paxillin (PXN) and concomitant actin remodeling in fibronectin (FN)-stimulated human ECs. Rather than using VEGFR2, NRP1-dependent PXN activation was found to rely on NRP1 association with ABL1, a nonreceptor tyrosine kinase with an actin-binding/bundling domain that links phosphoregulation to actin remodeling in a diverse range of cell types (Colicelli, 2010). Accordingly, knockdown of NRP1 or ABL1 by siRNA technology or with genetic tools inhibited actin remodeling and PXN phosphorylation, and consequently the migration of human and mouse ECs on FN in vitro. Inhibition of ABL1 kinase activity with Imatinib, an FDA-approved drug used to treat cell proliferation in leukemia caused by ABL1-BCR fusion protein, demonstrated similar effects on ECs in vitro and reduced vascular sprouting and branching on FN-rich templates during angiogenesis in vivo. Moreover, Imatinib treatment curbed pathological blood vessel growth in a mouse model of neovascular eye disease similarly to targeting NRP1 in ECs. ABL1 therefore presents a novel therapeutic opportunity for anti-angiogenic therapy.