The Role of Respiratory Microbiota in Lung Cancer

Recently, the impact of microorganisms on tumor growth and metastasis has attracted great attention. The pathogenesis and progression of lung cancer are related to an increase in respiratory bacterial load as well as changes in the bacterial community because the microbiota affects tumors in many ways, including canceration, metastasis, angiogenesis, and treatment. The microbiota may increase tumor susceptibility by altering metabolism and immune responses, promoting inflammation, and increasing toxic effects. The microbiota can regulate tumor metastasis by altering multiple cell signaling pathways and participate in tumor angiogenesis through vascular endothelial growth factors (VEGF), endothelial cells (ECs), inflammatory factors and inflammatory cells. Tumor angiogenesis not only maintains tumor growth at the primary site but also promotes tumor metastasis and invasion. Therefore, angiogenesis is an important mediator of the interaction between microorganisms and tumors. The microbiota also plays a part in antitumor therapy. Alteration of the microbiota caused by antibiotics can regulate tumor growth and metastasis. Moreover, the microbiota also influences the efficacy and toxicity of tumor immunotherapy and chemotherapy. Finally, the effects of air pollution, a risk factor for lung cancer, on microorganisms and the possible role of respiratory microorganisms in the effects of air pollution on lung cancer are discussed.     

蛋白激酶在微血管通透性中的作用

微血管内皮细胞层是一层半选择通透性屏障,可以调节血液中的液体、溶质和血浆蛋白进入组织间隙。在炎症刺激作用下,可通过旁细胞途径和跨细胞途径引起内皮通透性上升。旁细胞通路主要由内皮细胞间的紧密连接、黏附连接和细胞与外基质的黏着斑组成。炎症介质,如脂多糖和肿瘤坏死因子α可激活多种蛋白激酶。活化的蛋白激酶主要包括Rho相关的卷曲蛋白激酶、肌球蛋白轻链激酶、蛋白激酶C、酪氨酸激酶和丝裂原活化蛋白激酶等,参与引发内皮屏障生化和结构改变,旁细胞通路开放,导致通透性上升。该文对上述蛋白激酶在微血管通透性中作用机制的研究进展进行综述。     

Vascular endothelial growth factor gene polymorphisms and colorectal cancer risk: a meta-analysis

Vascular endothelial growth factor (VEGF) is an endothelial cell-specific mitogen involved in a number of pathologic processes, including angiogenesis, tumor growth and metastasis. Polymorphisms of the VEGF gene have been associated with susceptibility to colorectal cancer (CRC). However, the specific association still remains controversial. We made a meta-analysis of the association between VEGF gene polymorphisms and CRC risk. Only eight case-control studies were retrieved, with a total of 2337 CRC patients and 2032 healthy controls. Six VEGF gene polymorphisms were addressed in all studies included, +936C>T (rs3025039), -2578C>A (rs699947), -1154G>A (rs1570360), -634G>C (rs2010963), -460C>T (rs833061), and +405C>G (rs2010963). There was a significant association between -2578C>A polymorphism and susceptibility to CRC in the comparison of C allele carriers (CC + CA) versus AA (odds ratio = 0.77, 95% confidence interval = 0.62-0.96, P = 0.02). No association was found between +936C>T, -1154G>A, -634G>C, -460C>T, and +405C>G with susceptibility to CRC. We conclude that the C allele carrier (CC + CA) of VEGF -2578C>A polymorphism appears to be a protective factor for CRC.     

Estrogen protects renal endothelial barrier function from ischemia-reperfusion in vitro and in vivo

Emerging evidence suggests that renal endothelial function may be altered in ischemia-reperfusion injury. Acute kidney injury is sexually dimorphic, and estrogen protects renal tubular function after experimental ischemic injury. This study tested the hypothesis that during ischemia-reperfusion, estrogen alters glomerular endothelial function to prevent hyperpermeability. Glomerular endothelial cells were exposed to 8-h oxygen-glucose deprivation (OGD) followed by 4- and 8-h reoxygenation-glucose repletion. After 4-h reoxygenation-glucose repletion, transendothelial permeability to Ficoll-70 was reduced, and transendothelial resistance increased, by 17β-estradiol vs. vehicle treatment during OGD (OGD-vehicle: 91.0 ± 11.8%, OGD-estrogen: 102.6 ± 10.8%, P < 0.05). This effect was reversed by coadministration of G protein-coupled receptor 30 (GPR30) antagonist G15 with 17β-estradiol (OGD-estrogen-G15: 89.5 ± 6.9, P < 0.05 compared with 17β-estradiol). To provide preliminary confirmation of this result in vivo, Ficoll-70 was administered to mice 24 h after cardiac arrest and cardiopulmonary resuscitation (CA/CPR). Blood urea nitrogen (BUN) and serum creatinine (SCr) in these mice were elevated within 12 h following CA/CPR and reduced at 24 h by pretreatment with 17β-estradiol (BUN/SCr 17β-estradiol: 34 ± 19/0.2 ± 0.1 vehicle: 92 ± 49/0.5 ± 0.3, n = 8-12, P < 0.05). Glomerular sieving of Ficoll 70 was increased by CA/CPR within 2 h of injury and 17β-estradiol treatment (θ; 17β-estradiol: 0.74 ± 0.26 vs. vehicle: 1.05 ± 0.53, n = 14-15, P < 0.05). These results suggest that estrogen reduces postischemic glomerular endothelial hyperpermeability at least in part through GPR30 and that estrogen may regulate post CA/CPR glomerular permeability in a similar fashion in vivo.     

Biological and anti-vascular activity evaluation of ethoxy-erianin phosphate as a vascular disrupting agent

The effects of ethoxy-erianin phosphate (EBTP) on cell proliferation, mitotic cell arrest, migration, infiltration, and endothelial tubular structures were evaluated in this study. The antiproliferative activity of EBTP and combretastatin A-4P (CA4P) was analyzed on several tumor cells (including MCF-7, HeLa, 2LL, and 2LL-IDO) and on an endothelial cell (human umbilical vein endothelial cells [HUVECs]) as well as a human normal liver cell (L02). The results showed that EBTP possessed antiproliferative activity in the micromole range and was relatively less toxic than CA4P. Treating HUVECs with EBTP caused cell accumulation in the G2/M phase, and wound-healing assays indicated that EBTP inhibited cell migration. Furthermore, EBTP and CA4P destroyed the vasculature in endothelial cells and showed vascular disrupting activity of the chorioallantoic membrane in fertilized chicken eggs. In addition, we found that EBTP suppressed the expression of indoleamine 2,3-dioxygenase (IDO) and significantly inhibited IDO-induced migration and infiltration of 2LL-IDO cells. Administration of EBTP blocked vasculogenic mimicry in 2LL-IDO cells, which was typically observed in tube formation assays of 2LL-IDO cells. Moreover, the results of Lewis lung carcinoma in mice showed a high inhibition rate of EBTP. EBTP is an effective vascular disrupting agent that is superior to CA4P and may prevent and treat malignancy by inhibiting the expression of IDO.     

STRIP2 silencing inhibits vascular smooth muscle cell proliferation and migration via P38–AKT–MMP-2 signaling pathway

STRIP2 (FAM40B) was reported to regulate tumor cell migration. Our study aims to discuss the effect of STRIP2 in mouse aortic smooth muscle cell (MOVAS) proliferation and migration processes, which contributes greatly to atherosclerosis formation. In MOVAS cells, STRIP2 depletion suppressed cell proliferation and migration, which were related to a remarkable decrease in matrix metalloproteinases-2 (MMP-2)/MMP-9 expression. Additionally, P38 mitogen-activated protein kinases and Protein kinase B (AKT) are inactivated while extracellular signal-regulated kinase (ERK1/2) and jun N-terminal kinase (JNK) are activated upon STRIP2 silencing. SB203580 (P38 inhibitor) further reduced AKT phosphorylation (p-AKT) while dehydrocorydaline chloride (Dc; P38 activator) reversed this effect. Furthermore, Dc significantly recovered MMP-2 expression in STRIP2-knockdown cells. As expected, overexpressing STRIP2 exhibited a contrary effect. Dc and AKT activator SC79 reversed the inhibition of cell proliferation and migration induced by STRIP2 silencing. Interestingly, STRIP2 depletion increased vascular endothelial growth factor level significantly. Taken together, STRIP2 contributed to cell proliferation and migration through P38–AKT–MMP-2 signaling in MOVAS cells, indicating the importance of STRIP2 in atherosclerosis.     

Th2 Cytokines Inhibit Lymphangiogenesis

Lymphangiogenesis is the process by which new lymphatic vessels grow in response to pathologic stimuli such as wound healing, inflammation, and tumor metastasis. It is well-recognized that growth factors and cytokines regulate lymphangiogenesis by promoting or inhibiting lymphatic endothelial cell (LEC) proliferation, migration and differentiation. Our group has shown that the expression of T-helper 2 (Th2) cytokines is markedly increased in lymphedema, and that these cytokines inhibit lymphatic function by increasing fibrosis and promoting changes in the extracellular matrix. However, while the evidence supporting a role for T cells and Th2 cytokines as negative regulators of lymphatic function is clear, the direct effects of Th2 cytokines on isolated LECs remains poorly understood. Using in vitro and in vivo studies, we show that physiologic doses of interleukin-4 (IL-4) and interleukin-13 (IL-13) have profound anti-lymphangiogenic effects and potently impair LEC survival, proliferation, migration, and tubule formation. Inhibition of these cytokines with targeted monoclonal antibodies in the cornea suture model specifically increases inflammatory lymphangiogenesis without concomitant changes in angiogenesis. These findings suggest that manipulation of anti-lymphangiogenic pathways may represent a novel and potent means of improving lymphangiogenesis.     

Negative regulation of Gq-mediated pathways in platelets by G(12/13) pathways through Fyn kinase

Platelets contain high levels of Src family kinases (SFKs), but their functional role downstream of G protein pathways has not been completely understood. We found that platelet shape change induced by selective G(12/13) stimulation was potentiated by SFK inhibitors, which was abolished by intracellular calcium chelation. Platelet aggregation, secretion, and intracellular Ca(2+) mobilization mediated by low concentrations of SFLLRN or YFLLRNP were potentiated by SFK inhibitors. However, 2-methylthio-ADP-induced intracellular Ca(2+) mobilization and platelet aggregation were not affected by PP2, suggesting the contribution of SFKs downstream of G(12/13), but not G(q)/G(i), as a negative regulator to platelet activation. Moreover, PP2 potentiated YFLLRNP- and AYPGKF-induced PKC activation, indicating that SFKs downstream of G(12/13) regulate platelet responses through the negative regulation of PKC activation as well as calcium response. SFK inhibitors failed to potentiate platelet responses in the presence of G(q)-selective inhibitor YM254890 or in G(q)-deficient platelets, indicating that SFKs negatively regulate platelet responses through modulation of G(q) pathways. Importantly, AYPGKF-induced platelet aggregation and PKC activation were potentiated in Fyn-deficient but not in Lyn-deficient mice compared with wild-type littermates. We conclude that SFKs, especially Fyn, activated downstream of G(12/13) negatively regulate platelet responses by inhibiting intracellular calcium mobilization and PKC activation through G(q) pathways.     

Mitochondrial DAMPs Increase Endothelial Permeability through Neutrophil Dependent and Independent Pathways

Trauma and sepsis can cause acute lung injury (ALI) and Acute Respiratory Distress Syndrome (ARDS) in part by triggering neutrophil (PMN)-mediated increases in endothelial cell (EC) permeability. We had shown that mitochondrial (mt) damage-associated molecular patterns (DAMPs) appear in the blood after injury or shock and activate human PMN. So we now hypothesized that mitochondrial DAMPs (MTD) like mitochondrial DNA (mtDNA) and peptides might play a role in increased EC permeability during systemic inflammation and proceeded to evaluate the underlying mechanisms. MtDNA induced changes in EC permeability occurred in two phases: a brief, PMN-independent ‘spike’ in permeability was followed by a prolonged PMN-dependent increase in permeability. Fragmented mitochondria (MTD) caused PMN-independent increase in EC permeability that were abolished with protease treatment. Exposure to mtDNA caused PMN-EC adherence by activating expression of adherence molecule expression in both cell types. Cellular activation was manifested as an increase in PMN calcium flux and EC MAPK phosphorylation. Permeability and PMN adherence were attenuated by endosomal TLR inhibitors. EC lacked formyl peptide receptors but were nonetheless activated by mt-proteins, showing that non-formylated mt-protein DAMPs can activate EC. Mitochondrial DAMPs can be released into the circulation by many processes that cause cell injury and lead to pathologic endothelial permeability. We show here that mitochondria contain multiple DAMP motifs that can act on EC and/or PMN via multiple pathways. This can enhance PMN adherence to EC, activate PMN-EC interactions and subsequently increase systemic endothelial permeability. Mitochondrial DAMPs may be important therapeutic targets in conditions where inflammation pathologically increases endothelial permeability.     

Altered gene expression profiles of NIH3T3 cells regulated by human lung cancer associated gene CT120

CT120, a novel membrane-associated gene implicated in lung carcinogenesis, was previously identified from chromosome 17pl 3.3 locus, a hot mutation spot involved in human malignancies. In the present study, we further determined that CT120 ectopic expression could promote cell proliferation activity of NIH3T3 cells using MTS assay, and monitored the downstream effects of CT120 in NIH3T3 cells with Atlas mouse cDNA expression arrays. Among 588 known genes, 133 genes were found to be upregulated or downregulated by CT120. Two major signaling pathways involved in cell proliferation, cell survival and anti-apoptosis were overexpressed and activated in response to CT120:One is the Raf/MEK/Erk signal cascades and the other is the PI3K/Akt signal cascades, suggesting that CT120 might contribute, at least in part, to the constitutively activation of Erk and Akt in human lung caner cells. In addition, some tumor metastasis associated genes cathepsin B, cathepsin D, cathepsin L, MMP-2/TIMP-2 were also upregulated by CT120, upon which CT120 might be involved in tumor invasiveness and metastasis. In addition, CT120 might play an important role in tumor progression through modulating the expression of some candidate “Lung Tumor Progression”genes including B-Raf, Rab-2, BAX, BAG-1, YB-1, and Cdc42.     

Coxsackie- and adenovirus receptor (CAR) is expressed in lymphatic vessels in human skin and affects lymphatic endothelial cell function in vitro

Lymphatic vessels play an important role in tissue fluid homeostasis, intestinal fat absorption and immunosurveillance. Furthermore, they are involved in pathologic conditions, such as tumor cell metastasis and chronic inflammation. In comparison to blood vessels, the molecular phenotype of lymphatic vessels is less well characterized. Performing comparative gene expression analysis we have recently found that coxsackie- and adenovirus receptor (CAR) is significantly more highly expressed in cultured human, skin-derived lymphatic endothelial cells (LECs), as compared to blood vascular endothelial cells. Here, we have confirmed these results at the protein level, using Western blot and FACS analysis. Immunofluorescence performed on human skin confirmed that CAR is expressed at detectable levels in lymphatic vessels, but not in blood vessels. To address the functional significance of CAR expression, we modulated CAR expression levels in cultured LECs in vitro by siRNA- and vector-based transfection approaches. Functional assays performed with the transfected cells revealed that CAR is involved in distinct cellular processes in LECs, such as cell adhesion, migration, tube formation and the control of vascular permeability. In contrast, no effect of CAR on LEC proliferation was observed. Overall, our data suggest that CAR stabilizes LEC-LEC interactions in the skin and may contribute to lymphatic vessel integrity.     

Prospects for the use of peptides and their derivatives,structurally corresponding to the g protein-coupled receptors,in medicine

The regulation of signaling pathways involved in the control of many physiological functions is carried out via the heterotrimeric G protein-coupled receptors (GPCR). The search of effective and selective regulators of GPCR and intracellular signaling cascades coupled with them is one of the important problems of modern fundamental and clinical medicine. Recent data suggest that synthetic peptides and their derivatives, structurally corresponding to the intracellular and transmembrane regions of GPCR, can interact with high efficiency and selectivity with homologous receptors and influence the functional activity of intracellular signaling cascades and fundamental cellular processes controlled by them. GPCR-derived peptides are active in both in vitro and in vivo. They regulate hematopoiesis, angiogenesis and cell proliferation, inhibit tumor growth and metastasis, and prevent the inflammatory diseases and septic shock. These data show great prospects in the development of the new generations of drugs based on GPCR-derived peptides, which can regulate the important functions of the organism.     

Asparaginyl endopeptidase induces endothelial permeability and tumor metastasis via downregulating zonula occludens protein ZO-1

Zona occludens-1 (ZO-1) is a key component of tight junctions that govern the function of the endothelial barrier against tumor metastasis. Factors secreted by tumor cells contribute to the maintenance of tumor vascular networks. How tumor cell-derived protein signals regulate ZO-1 expression is unclear. Here, we explored the effect of tumor cell-secreted asparaginyl endopeptidase (AEP) on the permeability of endothelial cells in the tumor microenvironment. First, we confirmed the existence of AEP in conditioned medium (CM) from AEP-overexpressing MDA-MB-231 and 4T1 cells. Treatment with CM from AEP-overexpressing tumor cells increased the permeability and tumor cell transversal of an endothelial monolayer. Furthermore, CM from AEP-overexpressing tumor cells suppressed endothelial ZO-1 expression, as well as ZO-1-associated nucleic acid binding protein ZONAB. In addition, the level of phosphorylated STAT3 was increased by treatment with AEP-containing CM. A mutation of RGD or blocking integrin αvβ3 with antibody recovered the ZO-1 downregulation induced by AEP. In vivo, a lung metastatic mouse model showed increased endothelial permeability in the AEP-overexpressing group compared with the control group. An orthotopic tumor transplantation model was established using AEP-overexpression and compared with mice receiving control 4T1 cells. Compared with controls, overexpression of AEP increased lung metastatic foci and area, as well as vascular instability in primary tumors or lung metastatic sites. Moreover, endothelial ZO-1 was decreased in the AEP-overexpressing group. Taken together, our data show that tumor cell-derived AEP increases the permeability of endothelial barriers. Interactions between RGD and endothelial integrin αvβ3 mediate this effect by downregulating ZO-1.     

Role of vascular smooth muscle cell in the inflammation of atherosclerosis

Atherosclerosis is a pathologic process occurring within the artery, in which many cell types, including T cell, macrophages, endothelial cells, and smooth muscle cells, interact, and cause chronic inflammation, in response to various inner- or outer-cellular stimuli. Atherosclerosis is characterized by a complex interaction of inflammation, lipid deposition, vascular smooth muscle cell proliferation, endothelial dysfunction, and extracellular matrix remodeling, which will result in the formation of an intimal plaque. Although the regulation and function of vascular smooth muscle cells are important in the progression of atherosclerosis, the roles of smooth muscle cells in regulating vascular inflammation are rarely focused upon, compared to those of endothelial cells or inflammatory cells. Therefore, in this review, we will discuss here how smooth muscle cells contribute or regulate the inflammatory reaction in the progression of atherosclerosis, especially in the context of the activation of various membrane receptors, and how they may regulate vascular inflammation. [BMB Reports 2014; 47(1): 1-7]     

Src-induced Tyrosine Phosphorylation of VE-cadherin Is Not Sufficient to Decrease Barrier Function of Endothelial Monolayers

Activation of Src family kinases (SFK) and the subsequent phosphorylation of VE-cadherin have been proposed as major regulatory steps leading to increases in vascular permeability in response to inflammatory mediators and growth factors. To investigate Src signaling in the absence of parallel signaling pathways initiated by growth factors or inflammatory mediators, we activated Src and SFKs by expression of dominant negative Csk, expression of constitutively active Src, or knockdown of Csk. Activation of SFK by overexpression of dominant negative Csk induced VE-cadherin phosphorylation at tyrosines 658, 685, and 731. However, dominant negative Csk expression was unable to induce changes in the monolayer permeability. In contrast, expression of constitutively active Src decreased barrier function and promoted VE-cadherin phosphorylation on tyrosines 658 and 731, although the increase in VE-cadherin phosphorylation preceded the increase in permeability by 4–6 h. Csk knockdown induced VE-cadherin phosphorylation at sites 658 and 731 but did not induce a loss in barrier function. Co-immunoprecipitation and immunofluorescence studies suggest that phosphorylation of those sites did not impair VE-cadherin ability to bind p120 and β-catenin or the ability of these proteins to localize at the plasma membrane. Taken together, our data show that Src-induced tyrosine phosphorylation of VE-cadherin is not sufficient to promote an increase in endothelial cell monolayer permeability and suggest that signaling leading to changes in vascular permeability in response to inflammatory mediators or growth factors may require VE-cadherin tyrosine phosphorylation concurrently with other signaling pathways to promote loss of barrier function.     

Quantitative proteomics analysis of human endothelial cell membrane rafts: evidence of MARCKS and MRP regulation in the sphingosine 1-phosphate-induced barrier enhancement

Endothelial cell barrier dysfunction results in the increased vascular permeability observed in inflammation, tumor metastasis, angiogenesis, and atherosclerosis. Sphingosine 1-phosphate (S1P), a biologically active phosphorylated lipid growth factor released from activated platelets, enhances the endothelial cell barrier integrity in vitro and in vivo. To begin to identify the molecular mechanisms mediating S1P induced endothelial barrier enhancement, quantitative proteomics analysis (iTRAQ) was performed on membrane rafts isolated from human pulmonary artery endothelial cells in the absence or presence of S1P stimulation. Our results demonstrated that S1P mediates rapid and specific recruitment (1 microM, 5 min) of myristoylated alanine-rich protein kinase C substrate (MARCKS) and MARCKS-related protein (MRP) to membrane rafts. Western blot experiments confirmed these findings with both MARCKS and MRP. Finally, small interfering RNA-mediated silencing of MARCKS or MRP or both attenuates S1P-mediated endothelial cell barrier enhancement. These data suggest the regulation of S1P-mediated endothelial cell barrier enhancement via the cell specific localization of MARCKS and MRP and validate the utility of proteomics approaches in the identification of novel molecular targets.     

Endothelial cell phenotypes in the rheumatoid synovium: activated,angiogenic, apoptotic and leaky

Endothelial cells are active participants in chronic inflammatory diseases. These cells undergo phenotypic changes that can be characterised as activated, angiogenic, apoptotic and leaky. In the present review, these phenotypes are described in the context of human rheumatoid arthritis as the disease example. Endothelial cells become activated in rheumatoid arthritis pathophysiology, expressing adhesion molecules and presenting chemokines, leading to leukocyte migration from the blood into the tissue. Endothelial cell permeability increases, leading to oedema formation and swelling of the joints. These cells proliferate as part of the angiogenic response and there is also a net increase in the turnover of endothelial cells since the number of apoptotic endothelial cells increases. The endothelium expresses various cytokines, cytokine receptors and proteases that are involved in angiogenesis, proliferation and tissue degradation. Associated with these mechanisms is a change in the spectrum of genes expressed, some of which are relatively endothelial specific and others are widely expressed by other cells in the synovium. Better knowledge of molecular and functional changes occurring in endothelial cells during chronic inflammation may lead to the development of endothelium-targeted therapies for rheumatoid arthritis and other chronic inflammatory diseases.     

SC5b-9-Induced Pulmonary Microvascular Endothelial Hyperpermeability Participates in Ventilator-Induced Lung Injury

Mechanical ventilation with large tidal volumes can increase lung alveolar permeability and initiate inflammatory responses, termed ventilator-induced lung injury (VILI). VILI is characterized by an influx of inflammatory cells, increased pulmonary permeability, and endothelial and epithelial cell death. But the underlying molecular mechanisms that regulate VILI remain unclear. The purpose of this study was to investigate the mechanisms that regulate pulmonary endothelial barrier in an animal model of VILI. These data suggest that SC5b-9, as the production of the complement activation, causes increase in rat pulmonary microvascular permeability by inducing activation of RhoA and subsequent phosphorylation of myosin light chain and contraction of endothelial cells, resulting in gap formation. In general, the complement-mediated increase in pulmonary microvascular permeability may participate in VILI.     

GEP100-Arf6-AMAP1-cortactin pathway frequently used in cancer invasion is activated by VEGFR2 to promote angiogenesis

Angiogenesis and cancer invasiveness greatly contribute to cancer malignancy.Arf6 and its effector, AMAP1, are frequently overexpressed in breast cancer, and constitute a central pathway to induce the invasion and metastasis. In this pathway, Arf6 is activated by EGFR via GEP100. Arf6 is highly expressed also in human umbilical vein endothelial cells (HUVECs) and is implicated in angiogenesis. Here, we found that HUVECs also highly express AMAP1, and that vascular endothelial growth factor receptor-2 (VEGFR2) recruits GEP100 to activate Arf6. AMAP1 functions by binding to cortactin in cancer invasion and metastasis. We demonstrate that the same GEP100-Arf6-AMAP1-cortactin pathway is essential for angiogenesis activities, including cell migration and tubular formation, as well as for the enhancement of cell permeability and VE-cadherin endocytosis of VEGF-stimulated HUVECs. Components of this pathway are highly expressed in pathologic angiogenesis, and blocking of this pathway effectively inhibits VEGF- or tumor-induced angiogenesis and choroidal neovascularization. The GEP100-Arf6-AMAP1-cortactin pathway, activated by receptor tyrosine kinases, appears to be common in angiogenesis and cancer invasion and metastasis, and provides their new therapeutic targets.