Leukemia cell to endothelial cell communication via exosomal miRNAs

Background and Aim: The miR-17-92 cluster has a potent tumor angiogenesis-regulating activity, and it appears to act as a cluster in which the individual miRNAs show intricate activity. Recently, the existence of extracellular miRNAs enclosed in exosomes has raised the possibility that they play an important role in cell-cell interaction. To elucidate whether or not extracellular miRNAs derived from neoplastic cells transfer into endothelial cells and become functionally active in the recipient cells, we investigated the interaction of a leukemia cell line (K562 cells) and human umbilical vein endothelial cells (HUVECs), because K562 cells release the miR-17-92 cluster, particularly miR-92a, into the extracellular environment. Experimental Procedures: K562 cells and HUVECs were cocultured separately using a Transwell filter (0.4-μm pore, Corning). After coculture for 24 h, the culture medium was collected and extracellular miRNAs were isolated using the mirVana PARIS kit (Ambion). The Pre-miR miRNA precursor (hsa-miR-92a; Ambion) was labeled using the Label IT siRNA Tracker Cy3 kit (Mirus), and K562 cells were transfected with 10 nM of the Cy3-labeled PremiR miRNA precursor. The day following transfection, cells were washed three times with PBS, and the culture medium was replaced with fresh serum-free AIM V medium (Invitrogen). After incubation for 24 h, the culture medium was collected, and the exosomal fraction was isolated using Exoquick (System Biosciences). Results: Transfer of extracellular miR-92a from the donor cells to the endothelial cells. K562 cells transfected with Cy3-labeled Pre-mir-92a were noncontact cocultured with HUVECs. Twenty-four hours after coculture, Cy3-miR-92a signals were detected in the cytoplasm of HUVECs. Moreover, Cy3-miR-92a colocalized with the signals of CD63, an exosomal marker. Exosomal miR-92a directly regulates the target gene. We performed a luciferase reporter assay and examined the expression of integrin α5, a target gene for miR-92a. Luciferase activity was markedly reduced and a significant suppression of integrin α5 expression by extracellular miR-92a was observed in HUVECs. This indicates that exogenous miRNA via exosomal transport can function equally as endogenous miRNA in HUVECs. Exosomal miR-92a enhances endothelial cell migration in HUVECs. We found that HUVECs incorporating exosomal miRNA showed active movement. In addition, wound-healing assay revealed that there were more Cy3-labeled miR-92a signals in migrating HUVECs, but the incorporation of extracellular miR-92a to HUVECs was inhibited by cytochalasin D, a migration inhibitor. This indicates that the incorporation of exosomal miRNA may be closely linked to the movement of HUVECs. Notably, cell migration assay using an 8-μm pore Transwell revealed that an excess of extracellular miR-92a strongly enhanced endothelial cell migration. Conclusions: Our study showed that extracellular miRNAs originating from neoplastic cells are incorporated into endothelial cells via exosomal transport and exert tumor-angiogenic function in endothelial cells. HUVEC migration enhanced the uptake of extracellular miR-92a, and an excess of extracellular miR-92a enhanced HUVEC migration. Our results provide a new insight into the angiogenic function of miR-92a, and revealed that extracellular miRNAs play an important role in neoplasia-to-endothelial cell communication. This abstract is also presented as Poster C25. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the Second AACR International Conference on Frontiers in Basic Cancer Research; 2011 Sep 14-18; San Francisco, CA. Philadelphia (PA): AACR; Cancer Res 2011;71(18 Suppl):Abstract nr PR12.

Transfer of microRNA-486-5p from human endothelial colony forming cell–derived exosomes reduces ischemic kidney injury

Vascular endothelial growth factor (VEGF)-induced endothelial cell migration is a key step in the angiogenic response and is mediated, in part, by an accelerated rate of focal adhesion complex assembly and disassembly. We investigated the signaling pathway by which VEGF regulates focal adhesion complex assembly by examining the signaling proteins involved. VEGF stimulated the tyrosine phosphorylation of the SH2 domain-containing signaling proteins NCK and CRK in human umbilical vein endothelial cells. The signaling pathways that couple the kinase insert domain-containing receptor to NCK and CRK is most likely mediated by another cellular protein, as NCK and CRK were tyrosine-phosphorylated in response to VEGF in cells expressing receptors mutated at each of several candidate SH2 domain-interacting cytosolic tyrosines. In the absence of VEGF treatment, NCK (but not CRK) associated with the p21 GTPase-activated kinase PAK. PAK catalytic activity was augmented after VEGF treatment; an association of PAK with 60- and 90-kDa tyrosine-phosphorylated proteins accompanied this. VEGF stimulated the recruitment of PAK to focal adhesions, and FAK immunoprecipitated with both NCK and PAK in VEGF-treated (but not untreated) human umbilical vein endothelial cells. Inhibition of NCK protein expression using antisense oligonucleotides led to the inhibition of both VEGF-induced focal adhesion assembly and VEGF-induced cell migration, demonstrating a necessary role of NCK in these cellular responses.

Activation and dysfunction of the endothelium underlie many vascular disorders including atherosclerosis, tumor growth, and inflammation. We recently reported that thrombin and vascular endothelial growth factor, but not tumor necrosis factor-alpha, results in dramatic up-regulation of Down syndrome critical region (DSCR)-1 gene in endothelial cells, a negative feedback regulator of calcineurin-NFAT signaling. Constitutive expression of DSCR-1 in activated endothelial cells markedly impaired NFAT nuclear localization, proliferation, tube formation, and tumor growth. The goal of the present study was to elucidate the relative roles of NFAT/DSCR-1 and NF-kappaB/I-kappaB in mediating thrombin-responsive gene expression in endothelial cells. DNA microarrays of thrombin-treated human umbilical vein endothelial cells overexpressing DSCR-1 or constitutive active IkappaBalpha revealed genes that were dependent on NFAT and/or NF-kappaB activity. Vascular cell adhesion molecule-1 was inhibited both by DSCR-1 and I-kappaB at the level of mRNA, protein, promoter activity, and function (monocyte adhesion). Using a combination of transient transfections, electrophoretic mobility shift assays, and chromatin immunoprecipitation, thrombin was shown to induce time-dependent coordinate binding of RelA and NFATc to a tandem NF-kappaB element in the upstream promoter region of vascular cell adhesion molecule-1. Together, these findings suggest that thrombin-mediated activation of endothelial cells involves an interplay between NFAT and NF-kappaB signaling pathways and their negative feedback inhibitors, DSCR-1 and I-kappaB, respectively. As natural brakes in the inflammatory process, DSCR-1 and I-kappaB may lend themselves to therapeutic manipulation in vasculopathic disease states.

The neuropilins-1 and -2 (NRP1 and NRP2) function as receptors for both the semaphorins and vascular endothelial growth factor. In addition to their contribution to the development of the nervous system, NRP1 and NRP2 have been implicated in angiogenesis and tumor progression. Given their importance to cancer and endothelial biology and their potential as therapeutic targets, an important issue that has not been addressed is the impact of metabolic stress conditions characteristic of the tumor microenvironment on their expression and function. Here, we demonstrate that hypoxia and nutrient deprivation stimulate the rapid loss of NRP1 expression in both endothelial and carcinoma cells. NRP2 expression, in contrast, is maintained under these conditions. The lysosomal inhibitors chloroquine and bafilomycin A1 prevented the loss of NRP1 expression, but proteasomal inhibitors had no effect. The hypothesis that NRP1 is degraded by autophagy is supported by the findings that its expression is lost rapidly in response to metabolic stress, prevented with 3-methyladenine and induced by rapamycin. Targeted depletion of NRP2 using small hairpin RNA revealed that NRP2 can function in the absence of NRP1 to mediate endothelial tube formation in hypoxia. Studies aimed at assessing NRP function and targeted therapy in cancer and angiogenesis should consider the impact of metabolic stress.

Recent evidence shows that peroxisome proliferator-activated receptor gamma (PPARgamma) ligands induce the antiangiogenic effect in endothelial cells and tumors. In the present study, we elucidated the involvement of maxi-K channel activation in the antiangiogenic effect of rosiglitazone, a well known PPARgamma ligand in human umbilical vein endothelial cells. We found that the antiangiogenic effects of rosiglitazone were reversed by either bisphenol A diaglycidyl ether, a PPARgamma antagonist, or iberiotoxin, a maxi-K channel blocker. Knockdown of maxi-K channel expression also reversed the antiangiogenic effects. Iberiotoxin reversed the rosiglitazone-induced hyperpolarization while having no effect on the endogenous PPARgamma activation, suggesting that rosiglitazone activates maxi-K channel via PPARgamma. In the rosiglitazone-induced antiangiogenic process, endothelial nitric-oxide synthase-Ser1179 phosphorylation and NO production were significantly elevated, and treatment with the NOS inhibitor N(G)-monomethyl-L-arginine acetate abolished the antiangiogenic and apoptotic effects of rosiglitazone, indicating NO as a key mediator of the rosiglitazone actions. In conclusion, rosiglitazone significantly inhibited VEGF165-induced angiogenesis by a proapoptotic mechanism via PPARgamma-mediated NO production, followed by maxi-K channel opening.

HERG K+ Channels Promote Angiogenesis by Enhancing the Expression Level of VEGF in Leukemia Cells in Vitro.

Endothelial phenotypes are highly regulated in space and time by both transcriptional and post-transcriptional mechanisms. There is increasing evidence that the GATA family of transcription factors function as signal transducers, coupling changes in the extracellular environment to changes in downstream target gene expression. Here we show that human primary endothelial cells derived from large blood vessels express GATA2, -3, and -6. Of these factors, GATA3 was expressed at the highest levels. In DNA microarrays of human umbilical vein endothelial cells (HUVEC), small interfering RNA-mediated knockdown of GATA3 resulted in reduced expression of genes associated with angiogenesis, including Tie2. At a functional level, GATA3 knockdown inhibited angiopoietin (Ang)-1-mediated but not vascular endothelial cell growth factor (VEGF)-mediated AKT signaling, cell migration, survival, and tube formation. In electrophoretic gel mobility shift assays and chromatin immunoprecipitation, GATA3 was shown to bind to regulatory regions within the 5'-untranslated region of the Tie2 gene. In co-immunoprecipitation and co-transfection assays, GATA3 and the Ets transcription factor, ELF1, physically interacted and synergized to transactivate the Tie2 promoter. GATA3 knockdown blocked the ability of Ang-1 to attenuate vascular endothelial cell growth factor stimulation of vascular cell adhesion molecule-1 expression and monocytic cell adhesion. Moreover, exposure of human umbilical vein endothelial cells to tumor necrosis factor-alpha resulted in marked down-regulation of GATA3 expression and reduction in Tie2 expression. Together, these findings suggest that GATA3 is indispensable for Ang-1-Tie2-mediated signaling in large vessel endothelial cells.

Lysophosphatidic Acid Induces Lymphangiogenesis and IL-8 Production in Vitro in Human Lymphatic Endothelial Cells

Background and aim. We have recently shown tumor cell to endothelial cell communication and the induction of endothelial cell migration via exosomes (Umezu et al., Oncogene 2012). The aim of this study was to clarify whether hypoxia promotes angiogenic activity through tumor exosomes. Experimental procedures. We used the human leukemia cell line K562 as exosome-generating cells and human umbilical vein endothelial cells (HUVECs) as exosome target cells. Exosomes derived from K562 cells cultured under normoxic (20%) or hypoxic (1%) condition for 24h were isolated using Exoquick (System Bioscience) and quantitated by nano-particle analysis as well as immunoblotting for the exosomal protein CD63. These exosomes were added to HUVECs cultured on Matrigel for the validation of angiogenic activity, such as tube formation assay. Cellular and exosomal miRNAs responding to hypoxia were analyzed using TaqMan low-density miRNA array (TLDA, Applied Biosystems). Results. The exosomes derived from K562 cells cultured under hypoxic condition enhanced tube formation of HUVECs. Culture under hypoxic condition for 24h affected neither the tumor cell growth nor the amount of exosome secreted into the culture medium, while culture under the same condition induced the up-regulation of HIF-1α expression. The exosomes derived from K562 cells cultured under hypoxic condition for 24h (K562-1%O2-exosomes) markedly enhanced the tube formation of HUVECs compared with the exosomes obtained from K562 cells cultured under normoxic condition (K562-20%O2-exosomes), indicating that K562-1%O2-exosomes potentially included angiogenic factors. Cellular and exosomal miRNA profiling of K562 cells cultured under hypoxic condition. Using TLDA, we profiled the expression of cellular and exosomal miRNAs and observed a fold change of >1.5 under hypoxic condition compared with normoxic condition. We found that a subset of miRNAs, including miR-210, was significantly elevated under hypoxic condition in both cells and exosomes. The exosomal miRNAs derived from K562 cells cultured under hypoxic condition reduced the expression of Ephrin-A3 in HUVECs. We next examined the expression of Ephrin-A3 (EFNA3), an antiangiogenic factor, since miR-210 targets EFNA3. We demonstrated that EFNA3 expression was inhibited when HUVECs were exposed to hypoxic condition for 24h. Similarly, EFNA3 expression was drastically reduced by the addition of K562-1%O2-exosomes into HUVECs. Conclusions. We demonstrated that tumor cells and their exosomes have altered miRNA profiles under hypoxic condition. Although exosomes contain various molecular constituents, such as proteins and mRNAs, altered exosomal compartments under hypoxic condition, including miR-210, affect antiangiogenic factors. Our results suggest the possibility that exosomal miRNA derived from tumor cells cultured under hypoxic condition may partly affect angiogenic activity in endothelial cells. Citation Format: Tomohiro Umezu, Hiroko Tadokoro, Kazuma Ohyashiki, Junko H. Ohyashiki. Hypoxia modulates tumor exosomes, which function as mediators of angiogenesis. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 2305. doi:10.1158/1538-7445.AM2013-2305

Apoptosis and underlying mechanisms were evaluated in human umbilical vein endothelial cells (HUVECs), in target tissues of late diabetic vascular complications [human aortic endothelial cells (HAECs) and human retinal endothelial cells (HRECs)], and in endothelial progenitor cells (EPCs) exposed to FFAs, which are elevated in obesity and diabetes. Saturated stearic acid concentration dependently induced apoptosis that could be mediated via reduced membrane fluidity, because both apoptosis and membrane rigidity are counteracted by eicosapentaenoic acid. PUFAs triggered apoptosis at a concentration of 300 micromol/l in HUVECs, HAECs, and EPCs, but not HRECs, and, in contrast to stearic acid, involved caspase-8 activation. PUFA-induced apoptosis, but not stearic acid-induced apoptosis, strictly correlated (P < 0.01) with protein expression of E2F-1 (r = 0.878) and c-myc (r = 0.966). Lack of c-myc expression and activity owing to quiescence or transfection with dominant negative In373-Myc, respectively, renders HUVECs resistant to PUFA-induced apoptosis. Because c-myc is abundant in growing cells only, apoptosis triggered by PUFAs, but not by saturated stearic acid, obviously depends on the growth/proliferation status of the cells. Finally, this study shows that FFA-induced apoptosis depends on the vascular origin and growth/proliferation status of endothelial cells, and that saturated stearic acid-induced apoptosis and PUFA-induced apoptosis are mediated via different mechanisms.

Angiogenesis is an essential step for many physiological and pathological processes. Tumor necrosis factor (TNF) superfamily cytokines are increasingly recognized as key modulators of angiogenesis. In this study, we tested whether TNF-related activation-induced cytokine (TRANCE), a new member of the TNF superfamily, possesses angiogenic activity in vitro and in vivo. TRANCE stimulated DNA synthesis, chemotactic motility, and capillary-like tube formation in primary cultured human umbilical vein endothelial cells (HUVECs). Both Matrigel plug assay in mice and chick chorioallantoic membrane assay revealed that TRANCE potently induced neovascularization in vivo. TRANCE had no effect on vascular endothelial growth factor (VEGF) expression in HUVECs and TRANCE-induced angiogenic activity was not suppressed by VEGF-neutralizing antibody, implying that TRANCE-induced angiogenesis may be the result of its direct action on endothelial cells. TRANCE evoked a time- and dose-dependent activation of the mitogen-activated protein kinases ERK1/2 and focal adhesion kinase p125(FAK) in HUVECs, which are closely linked to angiogenesis. These signaling events were blocked by the Src inhibitor PP1 or the phospholipase C (PLC) inhibitor. Furthermore, these inhibitors and the Ca(2+) chelator BAPTA-AM suppressed TRANCE-induced HUVEC migration. These results indicate that the angiogenic activity of TRANCE is mediated through the Src-PLC-Ca(2+) signaling cascade upon receptor engagement in endothelial cells, suggesting the role of TRANCE in neovessel formation under physiological and pathological conditions.

Angiogenesis is a highly organized process controlled by a series of molecular events. While much effort has been devoted to identifying angiogenic factors and their reciprocal receptors, far less information is available on the molecular mechanisms underlying directed endothelial cell migration. To search for novel proteins that participate in this process, we used the serial analysis of gene expression (SAGE) transcript profiling approach to identify genes that are selectively expressed in endothelial cells (ECs). Two EC SAGE libraries were constructed from human umbilical vein and artery ECs to enable data-mining against other non-ECs. A novel endothelial protein, Thrombospondin Type I Domain Containing 7A (THSD7A), with preferential expression in placenta vasculature and in human umbilical vein endothelial cells (HUVECs) was identified and targeted for further characterization. Overexpression of a THSD7A carboxyl-terminal fragment in HUVECs inhibited cell migration and disrupted tube formation, while suppression of THSD7A expression enhanced HUVEC migration and tube formation. Immunohistological analysis revealed that THSD7A was expressed at the leading edge of migrating HUVECs, and it co-localized with alpha(V)beta(3) integrin and paxillin. This distribution was dispersed from focal adhesions after disruption of the actin cytoskeleton, suggesting the involvement of THSD7A in cytoskeletal organization. Our results show that THSD7A is a novel placenta endothelial protein that mediates EC migration and tube formation, and they highlight its potential as a new target for anti-angiogenic therapy.

Cigarette smoke exposure impairs VEGF-induced endothelial cell migration: Role of NO and reactive oxygen species

Tumor angiogenesis refers to the sprouting and cooption of proliferating endothelial cells (EC’s) from adjacent pre-existing host vasculature, and is a key target of cancer therapy. Tumor cells exploit their microenvironment by releasing cytokines and growth factors to promote and support angiogenesis. Within this complex tumor microenvironment, we and others have shown that tumors can recruit bone marrow derived endothelial progenitor cells that differentiate into mature bone marrow-derived endothelial cells and incorporate into sprouting tumor neovessels. Under pathological circumstances, such as breast cancer, a clear association between estrogen receptor expression by EC’s, angiogenic activity, and/or tumor invasiveness has been made. Approximately, 80% of breast cancers are hormone-receptor-positive cancers, thus enabling tamoxifen as the mainstay of breast cancer therapy. The roles of the anti-estrogens fulvestrant (ICI) and the dietary supplement 3, 3’-diindolylmethane (DIM) on cell-cell interaction and angiogenesis have not been fully elucidated. This study is designed to evaluate and compare the effect of these antiestrogens on angiogenesis at the cellular and molecular levels using tube formation of human umbilical vein endothelial cells (HUVEC) as an in vitro angiogenesis model. HUVEC cells were treated with serial dilutions of either DIM or ICI in presence and absence of (3nM) estrogen, and subjected to in vitro tube formation, proliferation, migration, and angiogenesis antibody array assays. We report that HUVEC cells are more sensitive to DIM than ICI. At 25 μM concentration, DIM significantly inhibited the crucial steps of angiogenesis including HUVEC cells proliferation, migration, cytokine release, and tube formation in an estrogen independent manner. On the other hand, at 1 μM concentration, ICI significantly exerted an antiangiogenic effects inhibiting HUVEC cells proliferation, migration, and tube formation, but this effect was totally dependent on the presence of estrogen. These results are validated by our observation that HUVEC cells express estrogen receptor beta (ER-β) and not estrogen receptor alpha (ER-α). A correlative effect between the antiangiogenic activity of DIM and ER-β upregulation was noted. We believe that the anti-estrogenic activity of DIM is mediated through the genomic and non-genomic activity of ER-β in endothelial cells predicting a new target for DIM to manifest its antiangiogenic effect. Citation Format: Ghada M. Ben Rahoma, Neha Y. Tuli, Robert B. Bednarczyk, Rachana R. Maniyar, Abraham Mittelman, Jan Geliebter, Raj Tiwari. Dietary supplement 3, 3’-diindolylmethane (DIM) as an antiangiogenic agent in breast cancer. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 3263.