Inhibition Of Endothelial Cell Migration Research Articles

Atropisomeric 1-phenylbenzimidazoles affecting microtubule organization: influence of axial chirality.

Benzimidazoles are frequently used in medicinal chemistry. Their anticancer effect is among the most prominent biological activities exhibited by this scaffold. Although numerous benzimidazole derivatives have been synthesized, possible atropisomerism of ortho-substituted 1-phenylbenzimidazoles has been largely overlooked. The aim of this research was to synthesize a small library of novel atropisomeric benzimidazole derivatives and explore their biological activity in various cancer and normal human cell lines. The new unique structural motif provides an interesting 3D architecture with axial chirality, which further contributes to molecular complexity and specificity. Racemates and their separated atropisomers arrested the cell cycle, caused apoptosis, and affected microtubule organization in cancer cells in vitro at different intensities. Moreover, this phenomenon was also verified by the inhibition of endothelial cell migration. These results showed that (+)-atropisomers, especially 5n, exhibit a stronger effect and show promise as agents for cancer therapy.

High-density lipoprotein regulates angiogenesis by affecting autophagy via miRNA-181a-5p.

We previously demonstrated that normal high-density lipoprotein (nHDL) can promote angiogenesis, whereas HDL from patients with coronary artery disease (dHDL) is dysfunctional and impairs angiogenesis. Autophagy plays a critical role in angiogenesis, and HDL regulates autophagy. However, it is unclear whether nHDL and dHDL regulate angiogenesis by affecting autophagy. Endothelial cells (ECs) were treated with nHDL and dHDL with or without an autophagy inhibitor. Autophagy, endothelial nitric oxide synthase (eNOS) expression, miRNA expression, nitric oxide (NO) production, superoxide anion (O2•-) generation, EC migration, and tube formation were evaluated. nHDL suppressed the expression of miR-181a-5p, which promotes autophagy and the expression of eNOS, resulting in NO production and the inhibition of O2•- generation, and ultimately increasing in EC migration and tube formation. dHDL showed opposite effects compared to nHDL and ultimately inhibited EC migration and tube formation. We found that autophagy-related protein 5 (ATG5) was a direct target of miR-181a-5p. ATG5 silencing or miR-181a-5p mimic inhibited nHDL-induced autophagy, eNOS expression, NO production, EC migration, tube formation, and enhanced O2•- generation, whereas overexpression of ATG5 or miR-181a-5p inhibitor reversed the above effects of dHDL. ATG5 expression and angiogenesis were decreased in the ischemic lower limbs of hypercholesterolemic low-density lipoprotein receptor null (LDLr-/-) mice when compared to C57BL/6 mice. ATG5 overexpression improved angiogenesis in ischemic hypercholesterolemic LDLr-/- mice. Taken together, nHDL was able to stimulate autophagy by suppressing miR-181a-5p, subsequently increasing eNOS expression, which generated NO and promoted angiogenesis. In contrast, dHDL inhibited angiogenesis, at least partially, by increasing miR-181a-5p expression, which decreased autophagy and eNOS expression, resulting in a decrease in NO production and an increase in O2•- generation. Our findings reveal a novel mechanism by which HDL affects angiogenesis by regulating autophagy and provide a therapeutic target for dHDL-impaired angiogenesis.

Smooth muscle cell-targeted RNA ligand promotes accelerated reendothelialization in a swine peripheral injury model

The local delivery of antiproliferative agents to inhibit neointimal growth is not specific to vascular smooth muscle cells (VSMC) and delays reendothelialization and vascular healing. This investigation was intended to evaluate the effect of luminal delivery of a VSMC-specific aptamer on endothelial healing. The impact of an RNA aptamer (Apt 14) was first examined on the migration and proliferation of primary cultured porcine aortic endothelial cells (ECs) in response to invitro scratch wound injury. We further evaluated the impact of Apt 14 on reendothelialization when delivered locally in a swine iliofemoral injury model. Although Apt 14 did not affect EC migration and proliferation, invitro results confirmed that paclitaxel significantly inhibited EC migration and proliferation. En face scanning electron microscopy demonstrated confluent endothelium with elongated EC morphology in Apt 14-treated arteries 14 and 28days post-treatment. In contrast, vessels treated with paclitaxel-coated balloons displayed a cobblestone morphology and significant platelet and fibrin attachment at cell junctions. These results provide the first evidence of the efficacy of a cell-targeted RNA aptamer to facilitate endothelial healing in a clinically relevant large animal model.

VEGFa/VEGFR2 autocrine and paracrine signaling promotes cervical carcinogenesis via β-catenin and snail.

VEGF secretion into the tumor microenvironment by cancer cells regulates several oncogenic signaling pathways and cancer-regulated angiogenesis. VEGFR receptors are exclusively present on endothelial cells to maintain their biological homeostasis. The acquisition of unique VEGFR2 receptor and VEGFa in cervical cancer (CC) cells reflects VEGFa/VEGFR2 autocrine machinery. Given the critical role of VEGFR2 in endothelial cell proliferation, migration, and angiogenesis, we explored its function in CC epithelial-mesenchymal transition (EMT) and stemness. Here we report that VEGFR2 regulates cancer-induced angiogenesis and EMT-linked stemness in CC cells via AKT/GSK3β/β-catenin and Snail pathway. Receptor tyrosine kinase inhibitor (RTKi) of VEGFR, Pazopanib (PAZ), shows potential anti-VEGFR2 activity and inhibits VEGFa induced metastatic events such as migration, invasion, and anoikis resistance in CC cells. Similarly, PAZ also attenuates cancer-regulated angiogenesis by inhibiting VE-cadherin internalization in endothelial cells followed by inhibition of endothelial cell migration. Selective depletion of VEGFR2 ligand VEGFa in CC cells also attenuates EMT, metastatic events, and inhibition of cancer-induced angiogenesis. In addition, blocking of VEGFR2 signaling in CC cells via PAZ or shRNA alters the formation of cervical tumorspheres (TS) and their successive generation. Collectively, inhibition of functional VEGFa/VEGFR2 autocrine and paracrine axis ceases the cancer-promoting events in cervical cancer cells. Based on the finding in this study, this oncogenic pathways could be used as a potential therapeutic target in a clinical setting with conventional radio-chemotherapy to achieve synergistic killing of CC cells.

Activation of the cytosolic calcium-independent phospholipase A2 β isoform contributes to TRPC6 externalization via release of arachidonic acid

During vascular interventions, oxidized low-density lipoprotein and lysophosphatidylcholine (lysoPC) accumulate at the site of arterial injury, inhibiting endothelial cell (EC) migration and arterial healing. LysoPC activates canonical transient receptor potential 6 (TRPC6) channels, leading to a prolonged increase in intracellular calcium ion concentration that inhibits EC migration. However, an initial increase in intracellular calcium ion concentration is required to activate TRPC6, and this mechanism remains elusive. We hypothesized that lysoPC activates the lipid-cleaving enzyme phospholipase A2 (PLA2), which releases arachidonic acid (AA) from the cellular membrane to open arachidonate-regulated calcium channels, allowing calcium influx that promotes externalization and activation of TRPC6 channels. The focus of this study was to identify the roles of calcium-dependent and/or calcium-independent PLA2 in lysoPC-induced TRPC6 externalization. We show that lysoPC induced PLA2 enzymatic activity and caused AA release in bovine aortic ECs. To identify the specific subgroup and the isoform(s) of PLA2 involved in lysoPC-induced TRPC6 activation, transient knockdown studies were performed in the human endothelial cell line EA.hy926 using siRNA to inhibit the expression of genes encoding cPLA2α, cPLA2γ, iPLA2β, or iPLA2γ. Downregulation of the β isoform of iPLA2 blocked lysoPC-induced release of AA from EC membranes and TRPC6 externalization, as well as preserved EC migration in the presence of lysoPC. We propose that blocking TRPC6 activation and promoting endothelial healing could improve the outcomes for patients undergoing cardiovascular interventions.

Dihydroartemisinin inhibits endothelial cell migration via the TGF-β1/ALK5/SMAD2 signaling pathway.

Anti-angiogenesis therapy is a novel treatment method for malignant tumors. Endothelial cell (EC) migration is an important part of angiogenesis. Dihydroartemisinin (DHA) exhibits strong anti-angiogenic and anti-EC migration effects; however, the underlying molecular mechanisms are yet to be elucidated. The TGF-β1/activin receptor-like kinase 5 (ALK5)/SMAD2 signaling pathway serves an important role in the regulation of migration. The present study aimed to explore the effects of DHA treatment on EC migration and the TGF-β1/ALK5/SMAD2 signaling pathway. The effects of DHA on human umbilical vein EC migration were assessed using wound healing and Transwell assays. The effects of DHA on the TGF-β1/ALK5/SMAD2 signaling pathway were detected using western blotting. DHA exhibited an inhibitory effect on EC migration in the wound healing and Transwell assays. DHA treatment upregulated the expression levels of ALK5 and increased the phosphorylation of SMAD2 in ECs. SB431542 rescued the inhibitory effect of DHA during EC migration. DHA inhibited EC migration via the TGF-β1/ALK5/SMAD2-dependent signaling pathway, and DHA may be a novel drug for the treatment of patients with malignant tumors.

Abstract 13610: Endothelial Cu Transporter Atp7a Promotes Vegfr2 Signaling and Post-ischemic Neovascularization via Regulating Autophagy

Background: VEGFR2 (KDR/Flk1) signaling in endothelial cells (ECs) plays a central role in angiogenesis. Copper (Cu) is essential micronutrient and has been implicated in angiogenesis. The P-type ATPase transporter ATP7A is key regulator of Cu homeostasis but its role in VEGFR2 signaling in ECs and post-ischemic neovascularization is entirely unknown. Results: Here we show that ATP7A expression was dramatically increased in the angiogenic ECs in mice hindlimb ischemia model. EC-specific ATP7A deficient mice or Cu transporter-dysfunctional ATP7A mut mice showed significant decrease in blood flow recovery and CD31+ capillary density (angiogenesis) in ischemic tissues compared to their control mice. In cultured human ECs, ATP7A knockdown with siRNA significantly inhibited VEGF-induced migration (67%), capillary network formation on Matrigel (46%). Immunofluorescence, co-immunoprecipitation, and proximity ligation assays showed that VEGF stimulated ATP7A translocation from the trans-Golgi network to the plasma membrane where it bound to VEGFR2. Surprisingly, loss of ATP7A promoted VEGF-induced VEGFR2 protein degradation (56%) and inhibited VEGFR2 signaling via enhancing VEGFR2 ubiquitination (2.0-fold) in a Cu-independent manner. This was associated with reduced cell surface VEGFR2 expression, increased VEGFR2 binding to selective autophagic cargo/adaptor p62/SQSTM1 (60%) and LC3-GFP+autophagic puncta (34%) and autolysosome formation (transmission electron microscopy). Inhibition of autophagy by bafilomycin A1 or chloroquine prevented enhanced VEGFR2 degradation in ATP7A-depleted ECs. Furthermore, overexpression of p62, but not p62 lacking ubiquitin binding domain, or autophagy inducer rapamycin treatment increased VEGFR2 degradation (26% and 87%, respectively) and p62 overexpression inhibited ECs migration (29%). Enhanced autophagy flux due to ATP7A dysfunction in vivo was confirmed by employing autophagy reporter CAG-ATP7A mut -RFP-EGFP-LC3 transgenic mice. Conclusion: Our study uncovers the unexpected and novel function of ATP7A to limit autophagic degradation of VEGFR2, thereby promoting VEGFR2 signaling and angiogenesis, which restores neovascularization in ischemic disease.

Carbon monoxide‑releasing molecules protect against blue light exposure and inflammation in retinal pigment epithelial cells.

The most common cause of vision loss among the elderly is age-related macular degeneration (AMD). The aim of the present study was to investigate the potential cytoprotective and anti-inflammatory effects of carbon monoxide-releasing molecules (CORMs), and their ability to activate the expression of nuclear factor erythroid 2-related factor 2 (Nrf2)-related genes in human retinal pigment epithelium (RPE) cells, as well as the inhibition of endothelial cell migration. It was first determined that CORM2 and CORM3 suppressed blue light-induced cell damage. In addition, a decrease in the level of cleaved poly(ADP-ribose) polymerase 1 protein and dissipation of mitochondrial membrane potential were considered to reflect the anti-apoptotic activity of CORMs. Furthermore, CORM2 induced Nrf-2 activation and the expression of the Nrf2-related genes heme oxygenase-1 and glutamate-cysteine ligase. Pretreatment with CORM2 abolished the blue light-induced increase in oxidative stress, suggesting that CORM2-induced antioxidant activity was involved in the cytoprotection against blue light. It was also demonstrated that CORMs markedly suppressed tumor necrosis factor (TNF)α-induced intercellular adhesion molecule-1 expression. Moreover, it was further observed that CORMs exert their inhibitory effects through blocking nuclear factor-κB/p65 nuclear translocation and IκBα degradation in TNFα-treated RPE cells. It was observed that CORM2, but not CORM3, protected against oxidative stress-induced cell damage. CORMs abolished vascular endothelial growth factor-induced migration of endothelial cells. The findings of the present study demonstrated the cytoprotective, antioxidant and anti-inflammatory effects of CORMs on RPE cells and anti-angiogenic effects on endothelial cells, suggesting the potential clinical application of CORMs as anti-AMD agents.

Microvesicles produced by natural killer cells of the NK-92 cell line affect the phenotype and functions of endothelial cells of the EA.Hy926 cell line

Microvesicles (MVs) are small (100-1000 nm) subcellular structures produced by both motionless and activated cells that can transfer molecules to target cells, and regulate physiological and pathological processes. MVs of leukocyte origin, in particular those produced by natural killer cells (NK cells), remain the least studied population of MVs. NK cells can change the functional activity of endothelial cells (ECs) and are involved in regulating angiogenesis. The ability of NK cell-derived MVs to influence the functionality of ECs is understudied currently. We aimed to study the effect of MVs produced by NK cells of the NK-92 cell line on the phenotype, caspase activity, proliferation and migration of ECs of the EA.Hy926 cell line. We cultured ECs in the presence of MVs derived from the NK-92 cell line, and then used flow cytometry to assess changes in EC phenotype, intracellular protein transfer from MVs to ECs, and the relative death of ECs. We used western blot analysis to evaluate the expression of granzyme B in NK cells and in the MVs that they produced, as well as the expression of granzyme B, caspases, extracellular-regulated kinase (ERK) and protein kinase B (AKT) in ECs. We also assessed the proliferation and migration of ECs in the presence of MVs derived from cells of the NK-92 cell line. The results revealed significant differences in the proteomic profiles of cells of the NK-92 cell line and their MV product. Contact between ECs and MVs derived from cells of the NK-92 cell line is accompanied by the following events: a) expression of granzyme B in ECs, b) activation of caspase-9 and caspase-3, with partial EC death, c) appearance of the panleukocyte marker CD45 on ECs, d) decrease in CD105 expression, and increase in CD34 and CD54 expression, and e) inhibition of EC migration. Transfer of ERK (but not AKT) from MVs derived from cells of the NK-92 cell line to ECs, at a concentration 10 times lower than that which causes EC death, leads to an increase in EC proliferation.

Circulating exosomes from patients with peripheral artery disease influence vascular cell migration and contain distinct microRNA cargo

ObjectivePeripheral artery disease (PAD) is a chronic condition characterized by inflammation. Emerging literature suggests that circulating exosomes and their microRNA (miRNA) contents may influence atherosclerosis and vascular remodeling. We hypothesize that circulating exosomes in patients with PAD directly modulate vascular cell phenotype and contain proinflammatory miRNAs. MethodsExosomes (particle size, 30-150 nm) were isolated from plasma of healthy individuals (n = 6), patients with mild PAD (mPAD; median Rutherford class, 2.5; n = 6), and patients with severe PAD (sPAD; median Rutherford class, 4; n = 5). Exosome identity, size, and concentration were determined by Western blot and nanoparticle tracking analysis. Human vascular smooth muscle cell (VSMC) and endothelial cell (EC) migration was assessed by a standard wound closure assay after exposure to exosome preparations. Monocyte-derived macrophages isolated from healthy volunteers were exposed to exosome preparations, and targeted gene expression was analyzed using quantitative polymerase chain reaction. Exosome miRNA cargos were isolated, and a panel of defined, vascular-active miRNAs was assessed by quantitative polymerase chain reaction. ResultsThere was no difference in overall exosome particle concentration or size between the three groups (one-way analysis of variance [ANOVA], P > .05). Compared with exosomes from healthy individuals, exosomes from mPAD and sPAD patients increased VSMC migration (1.0 ± 0.09-fold vs 1.5 ± 0.09-fold vs 2.0 ± 0.12-fold wound closure; ANOVA, P < .0001) and inhibited EC migration (1.8 ± 0.07-fold vs 1.5 ± 0.04-fold vs 1.3 ± 0.02-fold wound closure; ANOVA, P < .01) in a stepwise fashion. Exosomes also induced changes in monocyte-derived macrophage gene expression that did not appear PAD specific. Hierarchical analysis of exosome miRNA revealed distinct clustering of vascular-active miRNAs between the three groups. Several miRNAs that promote inflammatory pathways in vascular cells were expressed at higher levels in exosomes from sPAD patients. ConclusionsCirculating exosomes from individuals with PAD exert in vitro functional effects on VSMCs and ECs that may promote adverse vessel remodeling. Exosomes from healthy individuals, mPAD patients, and sPAD patients contain distinct signatures of immune-regulatory miRNA. Together these data suggest that the proinflammatory cargo of circulating exosomes correlates with atherosclerosis severity in PAD patients and could influence vascular injury and repair.

Revascularization after angiogenesis inhibition favors new sprouting over abandoned vessel reuse

Inhibiting pathologic angiogenesis can halt disease progression, but such inhibition may offer only a temporary benefit, followed by tissue revascularization after treatment stoppage. This revascularization, however, occurs by largely unknown phenotypic changes in pathologic vessels. To investigate the dynamics of vessel reconfiguration during revascularization, we developed a model of reversible murine corneal angiogenesis permitting longitudinal examination of the same vasculature. Following 30 days of angiogenesis inhibition, two types of vascular structure were evident: partially regressed persistent vessels that were degenerate and barely functional, and fully regressed, non-functional empty basement membrane sleeves (ebms). While persistent vessels maintained a limited flow and retained collagen IV+ basement membrane, CD31+ endothelial cells (EC), and α-SMA+ pericytes, ebms were acellular and expressed only collagen IV. Upon terminating angiogenesis inhibition, transmission electron microscopy and live imaging revealed that revascularization ensued by a rapid reversal of EC degeneracy in persistent vessels, facilitating their phenotypic normalization, vasodilation, increased flow, and subsequent new angiogenic sprouting. Conversely, ebms were irreversibly sealed from the circulation by excess collagen IV deposition that inhibited EC migration and prevented their reuse. Fully and partially regressed vessels therefore have opposing roles during revascularization, where fully regressed vessels inhibit new sprouting while partially regressed persistent vessels rapidly reactivate and serve as the source of continued pathologic angiogenesis.

Galloyl Carbohydrates with Antiangiogenic Activity Mediated by Capillary Morphogenesis Gene 2 (CMG2) Protein Binding.

We previously showed that a small molecule of natural origin, 1,2,3,4,6-penta- O-galloyl-β-d-glucopyranose (PGG), binds to capillary morphogenesis gene 2 (CMG2) with a submicromolar IC50 and also has antiangiogenic activity in vitro and in vivo. In this work, we synthetized derivatives of PGG with different sugar cores and phenolic substituents and tested these as angiogenesis inhibitors. In a high-throughput Förster resonant energy transfer-based binding assay, we found that one of our synthetic analogues (1,2,3,4,6-penta- O-galloyl-β-d-mannopyranose (PGM)), with mannose as central core and galloyl substituents, exhibit higher (up to 10×) affinity for CMG2 than the natural glucose prototype PGG and proved to be a potent angiogenesis inhibitor. These findings demonstrate that biochemical CMG2 binding in vitro predicts inhibition of endothelial cell migration ex vivo and antiangiogenic activity in vivo. The molecules herein described, and in particular PGM, might be useful prototypes for the development of novel agents for angiogenesis-dependent diseases, including blinding eye disease and cancer.

Carom, a novel protein induced by Homocysteine suppresses endothelial cell migration through inhibiting Vascular endothelial growth factor receptor 2 (VEGFR2) endocytosis

Hyperhomocysteinemia (HHcy), a syndrome displayed by high concentration of homocysteine (Hcy) in blood has been demonstrated as a significant risk factor for cardiovascular disease (CVD). We have shown that Hcy leaded to endothelial cells (EC) injuries through inhibition of proliferation and migration. Previously we have demonstrated a Hcy specifically induced gene, Carom from cultured EC and shown its inhibition of EC migration by wound healing assay. Here, through further biochemical studies, we explore mechanistic insights behind these regulations. Carom protein belongs to Bin‐Amphiphysin‐Rvs (BAR) superfamily and specifically it is a FBAR protein since it displays Fes/CIP homology (FCH) domain on N‐terminus. Using biotin homologue to label plasma membrane proteins, we find Carom overexpression with adenovirus transduction increases Vascular endothelial growth factor receptor 2 (VEGFR2) internalization in human vein umbilical endothelial cells (HUVEC) after VEGFA stimulation. The same phenomena also exist in Hcy pretreated ECs. This process is mainly dependent on clathrin mediated endocytosis, since both Monensin and dynamin GTPase inhibitor MitMAB eliminate Carom's effects on VEGFR2 endocytosis. The VEGFR2 trafficking is tightly correlated with VEGFR2 protein levels. Carom adenovirus transduction leads to significant reduction of total cellular VEGFR2 levels. Mass spectrometric analysis of Carom interacting proteins in ECs show some partner proteins are involved in subcellular organelle trafficking and cytoskeleton organization, suggesting co‐ordinations between Carom and these partners in regulating receptor endocytosis, sorting and its function. Regulation of VEGFR2 trafficking and the ensuing signaling pathways are one of the major causes accounted for Carom's inhibitory effects on EC migration.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

VEGFA Gene Silencing in CXCR4-Expressing Cells via siRNA Delivery by Means of Targeted Peptide Carrier.

Discovery of small interfering RNA as a tool for specific gene inhibition led to the development of new therapeutic strategy for the treatment of cancers. The efficacious delivery of therapeutic siRNAs into the cells is a crucial step in RNA interference (RNAi) application, but it remains challenging. Non-viral vectors can provide specific cellular uptake, stable siRNA complex formation, and intracellular siRNA release. Recently, we evaluated modular peptide carrier L1 bearing CXCR4 targeting ligand for its ability to condense siRNA and facilitate endosomal escape and VEGFA gene silencing in CXCR4-expressing endothelial and glioblastoma cells. The present chapter showcases the ability of L1 targeted peptide carrier to form complexes with siRNA and provide efficient VEGFA gene knockdown. We showed that siRNA delivery by means of L1 peptide carrier can result in significant decrease of VEGFA gene expression in A172 glioblastoma cells and in EA.hy 926 endothelial cells. Also, delivery of anti-VEGFA siRNA/peptide complexes led to significant inhibition of endothelial cell migration. Our results showed that L1 peptide carrier modified with CXCR4 ligand is a promising tool for targeted siRNA delivery into CXCR4-expressing cancer and endothelial cells.

Autophagy Is a Potential Target for Enhancing the Anti-Angiogenic Effect of Mebendazole in Endothelial Cells.

Mebendazole (MBZ), a microtubule depolymerizing drug commonly used for the treatment of helminthic infections, has recently been noted as a repositioning candidate for angiogenesis inhibition and cancer therapy. However, the definite anti-angiogenic mechanism of MBZ remains unclear. In this study, we explored the inhibitory mechanism of MBZ in endothelial cells (ECs) and developed a novel strategy to improve its anti-angiogenic therapy. Treatment of ECs with MBZ led to inhibition of EC proliferation in a dose-dependent manner in several culture conditions in the presence of vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF) or FBS, without selectivity of growth factors, although MBZ is known to inhibit VEGF receptor 2 kinase. Furthermore, MBZ inhibited EC migration and tube formation induced by either VEGF or bFGF. However, unexpectedly, treatment of MBZ did not affect FAK and ERK1/2 phosphorylation induced by these factors. Treatment with MBZ induced shrinking of ECs and caused G2-M arrest and apoptosis with an increased Sub-G1 fraction. In addition, increased levels of nuclear fragmentation, p53 expression, and active form of caspase 3 were observed. The marked induction of autophagy by MBZ was also noted. Interestingly, inhibition of autophagy through knocking down of Beclin1 or ATG5/7, or treatment with autophagy inhibitors such as 3-methyladenine and chloroquine resulted in marked enhancement of anti-proliferative and pro-apoptotic effects of MBZ in ECs. Consequently, we suggest that MBZ induces autophagy in ECs and that protective autophagy can be a novel target for enhancing the anti-angiogenic efficacy of MBZ in cancer treatment.

Retinal pigment epithelium‐derived transforming growth factor‐β2 inhibits the angiogenic response of endothelial cells by decreasing vascular endothelial growth factor receptor‐2 expression

Transforming growth factor-β (TGF-β) is a multifunctional cytokine that is known to modulate various aspects of endothelial cell (EC) biology. Retinal pigment epithelium (RPE) is important for regulating angiogenesis of choriocapillaris and one of the main cell sources of TGF-β secretion, particularly TGF-β2. However, it is largely unclear whether and how TGF-β2 affects angiogenic responses of ECs. In the current study, we demonstrated that TGF-β2 reduces vascular endothelial growth factor receptor-2 (VEGFR-2) expression in ECs and thereby inhibits vascular endothelial growth factor (VEGF) signaling and VEGF-induced angiogenic responses such as EC migration and tube formation. We also demonstrated that the reduction of VEGFR-2 expression by TGF-β2 is due to the suppression of JNK signaling. In coculture of RPE cells and ECs, RPE cells decreased VEGFR-2 levels in ECs and EC migration. In addition, we showed that TGF-β2 derived from RPE cells is involved in the reduction of VEGFR-2 expression and inhibition of EC migration. These results suggest that TGF-β2 plays an important role in inhibiting the angiogenic responses of ECs during the interaction between RPE cells and ECs and that angiogenic responses of ECs may be amplified by a decrease in TGF-β2 expression in RPE cells under pathologic conditions.

Role of glutamine synthetase in angiogenesis beyond glutamine synthesis.

Glutamine synthetase, encoded by the gene GLUL, is an enzyme that converts glutamate and ammonia to glutamine. It is expressed by endothelial cells, but surprisingly shows negligible glutamine-synthesizing activityin these cells at physiological glutamine levels. Here we show in mice that genetic deletion of Glul in endothelial cells impairs vessel sprouting during vascular development, whereas pharmacological blockade of glutamine synthetase suppresses angiogenesis in ocular and inflammatory skin disease while only minimally affecting healthy adult quiescent endothelial cells. This relies on the inhibition of endothelial cell migration but not proliferation. Mechanistically we show that in human umbilical vein endothelial cells GLUL knockdown reduces membrane localization and activation of the GTPase RHOJ while activating other Rho GTPases and Rho kinase, thereby inducing actin stress fibres and impeding endothelial cell motility. Inhibition of Rho kinase rescues the defect in endothelial cell migration that is induced by GLUL knockdown. Notably, glutamine synthetase palmitoylates itself and interacts with RHOJ to sustain RHOJ palmitoylation, membrane localization and activation. These findings reveal that, in addition to theknown formation of glutamine, the enzyme glutamine synthetase shows unknownactivity in endothelial cell migration during pathological angiogenesisthrough RHOJ palmitoylation.

Abstract 605: Inhibition of the Key Glycolytic Enzyme PFKFB3 with Novel Compounds Suppresses Angiogenesis

Aim: Intraplaque angiogenesis is an important contributor to atherosclerotic plaque growth and instability. Angiogenic signals induce endothelial cells (ECs) to switch their metabolism to being highly glycolytic, enabling their growth and division. Glycolytic modulation by inhibition of theglycolytic activator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) has been shown to reduce angiogenesis. The objective of this study was to identify novel anti-angiogenic compoundswith a potential to efficiently modulate (inhibit) angiogenesis. Methods: Using the human EC line EA.hy926, we studied the effects of PFKFB3 inhibition with 3PO, a weak competitive inhibitor of PFKFB3, and of two potent self-synthesized phenoxindazole analogues (PA-1 and PA-2) on glycolysis, proliferation, migration, matrix metalloproteinase (MMP) activity, and capillary tube formation. Moreover, gene expression of important markers related to angiogenesis were measured at mRNA levelby real-time PCR. Results: PFKFB3 inhibition with all three tested compounds significantly reduced glycolytic activity. While PA-1 and PA-2suppressedcapillary tube formation, 3PO did not have any effect. Accordingly, PA-1 and PA-2 markedly inhibited EC migration, proliferation and wound closing capacity which are essential for neovessel formation. Moreover, these inhibitors downregulated gelatinase gene expression up to 6-fold, as well reduced the activity of proMMP-9 and MMP-2 up to 50% and 30% compared to control, respectively. Gene expression analysis revealed that the PA compounds downregulated PFKFB3 expression whilst 3PO did not. Similarly, markers of migration and angiogenesis, such as CCL5, VCAM-1, VEGFA and VEGFR2, were also markedly reduced (up to 10-fold) by the PA compounds. Conclusions: These findings suggest that PFKFB3 inhibition with PA compounds may interfere with key pro-angiogenic functions, such as endothelial migration, proliferation and capillary-like structure formation and this exerts a multitarget anti-angiogenic activity. Hence, PFKFB3 inhibition with PA compounds is a promising therapeuticapproach to promote plaque stability.

Abstract 435: Inhibition of the Key Glycolytic Enzyme PFKFB3 with Novel Compounds Suppresses Angiogenesis

Aim: Intraplaque angiogenesis is an important contributor to atherosclerotic plaque growth and instability. Angiogenic signals induce endothelial cells (ECs) to switch their metabolism to being highly glycolytic, enabling their growth and division. Glycolytic modulation by inhibition of the glycolytic activator 6-phosphofructo-2-kinase/fructose-2,6-bisphosphatase (PFKFB3) has been shown to reduce angiogenesis. The objective of this study was to identify novel anti-angiogenic compounds with a potential to efficiently modulate (inhibit) angiogenesis. Methods: Using the human EC line EA.hy926, we studied the effects of PFKFB3 inhibition with 3PO, a weak competitive inhibitor of PFKFB3, and of two potent self-synthesized phenoxindazole analogues (PA-1 and PA-2) on glycolysis, proliferation, migration, matrix metalloproteinase (MMP) activity, and capillary tube formation. Moreover, gene expression of important markers related to angiogenesis were measured at mRNA level by real-time PCR. Results: PFKFB3 inhibition with all three tested compounds significantly reduced glycolytic activity. While PA-1 and PA-2 suppressed capillary tube formation, 3PO did not have any effect. Accordingly, PA-1 and PA-2 markedly inhibited EC migration, proliferation and wound closing capacity which are essential for neovessel formation. Moreover, these inhibitors downregulated gelatinase gene expression up to 6-fold, as well reduced the activity of proMMP-9 and MMP-2 up to 50% and 30% compared to control, respectively. Gene expression analysis revealed that the PA compounds downregulated PFKFB3 expression whilst 3PO did not. Similarly, markers of migration and angiogenesis, such as CCL5, VCAM-1, VEGFA and VEGFR2, were also markedly reduced (up to 10-fold) by the PA compounds. Conclusions: These findings suggest that PFKFB3 inhibition with PA compounds may interfere with key pro-angiogenic functions, such as endothelial migration, proliferation and capillary-like structure formation and this exerts a multitarget anti-angiogenic activity. Hence, PFKFB3 inhibition with PA compounds is a promising therapeutic approach to promote plaque stability.

A low-molecular-weight galactofucan from the seaweed, Spatoglossum schröederi, binds fibronectin and inhibits capillary-like tube formation in vitro

A low-molecular-weight (LMW) heterofucan (designated fucan B) was obtained from the brown seaweed, Spatoglossum schröederi, and its activity as an inhibitor of capillary-like tube formation by endothelial cells (ECs) was analyzed. Chemical, infrared and electrophoretic analyses confirmed the identity of fucan B. In contrast to other LMW fucans, fucan B (0.012–0.1 mg/mL) inhibited ECs capillary-like tube formation in a concentration-dependent manner. In addition, fucan B (0.01–0.05 mg/mL) did not affect ECs proliferation. Fucan B also inhibited ECs migration on a fibronectin-coated surface, but not on laminin- or collagen-coated surfaces. Biotinylated fucan B was used as a probe to identify its localization. Confocal microscopy experiments revealed that biotinylated fucan did not bind to the cell surface, but rather only to fibronectin. Our findings suggest that fucan B inhibits ECs capillary-like tube formation and migration by binding directly to fibronectin and blocking fibronectin sites recognized by cell surface ligands. However, further studies are needed to evaluate the in vivo effects of fucan B.