Angiostatic Activity Research Articles

Peptidylarginine Deiminases Inhibitors Decrease Endothelial Cells Angiogenic Potential by Affecting Akt Signaling and the Expression and Secretion of Angiogenic Factors.

Endothelial cells (ECs) play a crucial role in various physiological processes, particularly those related to the cardiovascular system, but also those affecting the entire organism. The biology of ECs is regulated by multiple biochemical stimuli and epigenetic drivers that govern gene expression. We investigated the angiogenic potential of ECs from a protein citrullination perspective, regulated by peptidyl-arginine deiminases (PADs) that modify histone and non-histone proteins. Although the involvement of PADs has been demonstrated in several physiological processes, inflammation-related disorders and cancer, their role in angiogenesis remains unclear. To elucidate the role of PADs in endothelial angiogenesis, we used two human EC models: primary vein (HUVECs) and microvascular endothelial cells (HMEC-1). PADs activity was inhibited using irreversible inhibitors: BB-Cl-amidine, Cl-amidine and F-amidine. We analyzed all three steps of angiogenesis in vitro : proliferation, migration, and capillary-like tube formation, as well as secretory activities, gene expression and signaling in ECs. All used PAD inhibitors reduced the histone H3 citrullination (H3cit) mark, inhibited endothelial cell migration and capillary-like tube formation, and favored an angiostatic activity in HMEC-1 cells, by increasing PEDF (pigment epithelium-derived factor) and reducing VEGF (vascular endothelial growth factor) mRNA expression and protein secretion. Additionally, BB-Cl-amidine reduced the total activity of MMPs (Matrix metalloproteinases). The observed effects were underlined by the inhibition of Akt phosphorylation.>. Our findings suggest that pharmacological inhibitors of citrullination are promising therapeutic agents to target angiogenesis.

Natural carboxyterminal truncation of human CXCL10 attenuates glycosaminoglycan binding, CXCR3A signaling and lymphocyte chemotaxis, while retaining angiostatic activity.

Interferon-γ-inducible protein of 10kDa (IP-10/CXCL10) is a dual-function CXC chemokine that coordinates chemotaxis of activated T cells and natural killer (NK) cells via interaction with its G protein-coupled receptor (GPCR), CXC chemokine receptor 3 (CXCR3). As a consequence of natural posttranslational modifications, human CXCL10 exhibits a high degree of structural and functional heterogeneity. However, the biological effect of natural posttranslational processing of CXCL10 at the carboxy (C)-terminus has remained partially elusive. We studied CXCL10(1-73), lacking the four endmost C-terminal amino acids, which was previously identified in supernatant of cultured human fibroblasts and keratinocytes. Relative levels of CXCL10(1-73) and intact CXCL10(1-77) were determined in synovial fluids of patients with rheumatoid arthritis (RA) through tandem mass spectrometry. The production of CXCL10(1-73) was optimized through Fmoc-based solid phase peptide synthesis (SPPS) and a strategy to efficiently generate human CXCL10 proteoforms was introduced. CXCL10(1-73) was compared to intact CXCL10(1-77) using surface plasmon resonance for glycosaminoglycan (GAG) binding affinity, assays for cellmigration, second messenger signaling downstream of CXCR3, and flow cytometry of CHO cells and primary human T lymphocytes and endothelial cells. Leukocyte recruitment in vivo upon intraperitoneal injection of CXCL10(1-73) was also evaluated. Natural CXCL10(1-73) was more abundantly present compared to intact CXCL10(1-77) in synovial fluids of patients with RA. CXCL10(1-73) had diminished affinity for GAG including heparin, heparan sulfate and chondroitin sulfate A.Moreover, CXCL10(1-73) exhibited an attenuated capacity to induce CXCR3A-mediated signaling, as evidenced in calcium mobilization assays and through quantification of phosphorylated extracellular signal-regulated kinase-1/2 (ERK1/2) and protein kinase B/Akt. Furthermore, CXCL10(1-73) incited significantly less primary human T lymphocyte chemotaxis in vitro and peritoneal ingress of CXCR3+ T lymphocytes in mice. In contrast, loss of the four endmost C-terminal residues did not affect the inhibitory properties of CXCL10 on migration, proliferation, wound closure, phosphorylation of ERK1/2, and sprouting of human microvascular endothelial cells. Our study shows that the C-terminal residues Lys74-Pro77 of CXCL10 are important for GAG binding, signaling through CXCR3A, T lymphocyte chemotaxis, but dispensable for angiostasis.

Proteoglycans of basement membranes: Crucial controllers of angiogenesis, neurogenesis, and autophagy.

Anti-angiogenic therapy is an established method for the treatment of several cancers and vascular-related diseases. Most of the agents employed target the vascular endothelial growth factor A, the major cytokine stimulating angiogenesis. However, the efficacy of these treatments is limited by the onset of drug resistance. Therefore, it is of fundamental importance to better understand the mechanisms that regulate angiogenesis and the microenvironmental cues that play significant role and influence patient treatment and outcome. In this context, here we review the importance of the three basement membrane heparan sulfate proteoglycans (HSPGs), namely perlecan, agrin and collagen XVIII. These HSPGs are abundantly expressed in the vasculature and, due to their complex molecular architecture, they interact with multiple endothelial cell receptors, deeply affecting their function. Under normal conditions, these proteoglycans exert pro-angiogenic functions. However, in pathological conditions such as cancer and inflammation, extracellular matrix remodeling leads to the degradation of these large precursor molecules and the liberation of bioactive processed fragments displaying potent angiostatic activity. These unexpected functions have been demonstrated for the C-terminal fragments of perlecan and collagen XVIII, endorepellin and endostatin. These bioactive fragments can also induce autophagy in vascular endothelial cells which contributes to angiostasis. Overall, basement membrane proteoglycans deeply affect angiogenesis counterbalancing pro-angiogenic signals during tumor progression, and represent possible means to develop new prognostic biomarkers and novel therapeutic approaches for the treatment of solid tumors.

Melanocytes as emerging key players in niche regulation of limbal epithelial stem cells

PurposeLimbal melanocytes (LMel) represent essential components of the corneal epithelial stem cell niche and are known to protect limbal epithelial stem/progenitor cells (LEPCs) from UV damage by transfer of melanosomes. Here, we explored additional functional roles for LMel in niche homeostasis, immune regulation and angiostasis. MethodsHuman corneoscleral tissues were morphologically analyzed in normal, inflammatory and wound healing conditions. The effects of LMel on LEPCs were analyzed in direct and indirect co-culture models using electron microscopy, immunocytochemistry, qRT-PCR, Western blotting and functional assays; limbal mesenchymal stromal cells and murine embryonic 3T3 fibroblasts served as controls. The immunophenotype of LMel was assessed by flow cytometry before and after interferon-γ stimulation, and their immunomodulatory properties were analyzed by mixed lymphocytes reaction, monocyte adhesion assays and cytometric bead arrays. Their angiostatic effects on human umbilical cord endothelial cells (HUVECs) were evaluated by proliferation, migration, and tube formation assays. ResultsLMel and LEPCs formed structural units in the human limbal stem cell niche in situ, which could be functionally replicated, including melanosome transfer, by co-cultivation in vitro. LMel supported LEPCs during clonal expansion and during epithelial wound healing by stimulating proliferation and migration, and suppressed their differentiation through direct contact and paracrine effects. Under inflammatory conditions, LMel were increased in numbers and upregulated expression of ICAM-1 and MHC II molecules (HLA-DR), but lacked expression of HLA-G, -DP, -DQ and costimulatory molecules CD80 and CD86. They were also found to be potent suppressors of alloreactive T- cell proliferation and cytokine secretion, which largely depended on direct cell-cell interaction. Moreover, the LMel secretome exerted angiostatic activity by inhibiting vascular endothelial cell proliferation and capillary network formation. ConclusionThese findings suggest that LMel are not only professional melanin-producing cells, but exert various non-canonical functions in limbal niche homeostasis by regulating LEPC maintenance, immune responses, and angiostasis. Their potent regulatory, immunomodulatory and anti-angiogenic properties may have important implications for future regenerative cell therapies.

The effects of glucose and fatty acids on CXCL10 expression in skeletal muscle cells.

Skeletal muscles produce secretory factors termed as myokines, which alter physiological functions of target tissues. We recently identified C-X-C chemokine ligand 10 (CXCL10) as a novel myokine, which is downregulated in response to exercise. In the present study, we investigated whether the nutritional changes affect CXCL10 expression in mouse skeletal muscle. Expression of CXCL10 was evaluated in mice fed a normal diet or a high fat diet for 10weeks. In animals fed on HFD, Cxcl10 expression was significantly induced in fast-twitched muscles, and was accompanied by increased blood glucose and free fatty acid levels. In vitro experiments using C2C12 myotubes suggested that the increased levels of glucose and palmitic acids directly enhanced CXCL10 expression. Interestingly, the effect of palmitic acids was attenuated by palmitoleic acids. Considering its potent angiostatic activity, induction of CXCL10 by nutritional changes may contribute to the impairment of microvascular networks in skeletal muscles.

Anti-angiogenic effects of crenolanib are mediated by mitotic modulation independently of PDGFR expression

BackgroundCrenolanib is a tyrosine kinase inhibitor targeting PDGFR-α, PDGFR-β and Fms related tyrosine kinase-3 (FLT3) that is currently evaluated in several clinical trials. Although platelet-derived growth factor receptor (PDGFR) signalling pathway is believed to play an important role in angiogenesis and maintenance of functional vasculature, we here demonstrate a direct angiostatic activity of crenolanib independently of PDGFR signalling.MethodsThe activity of crenolanib on cell viability, migration, sprouting, apoptosis and mitosis was assessed in endothelial cells, tumour cells and fibroblasts. Alterations in cell morphology were determined by immunofluorescence experiments. Flow-cytometry analysis and mRNA expression profiles were used to investigate cell differentiation. In vivo efficacy was investigated in human ovarian carcinoma implanted on the chicken chorioallantoic membrane (CAM).ResultsCrenolanib was found to inhibit endothelial cell viability, migration and sprout length, and induced apoptosis independently of PDGFR expression. Treated cells showed altered actin arrangement and nuclear aberrations. Mitosis was affected at several levels including mitosis entry and centrosome clustering. Crenolanib suppressed human ovarian carcinoma tumour growth and angiogenesis in the CAM model.ConclusionsThe PDGFR/FLT3 inhibitor crenolanib targets angiogenesis and inhibits tumour growth in vivo unrelated to PDGFR expression. Based on our findings, we suggest a broad mechanism of action of crenolanib.

Abstract 2048: Interferon-γ triggers an anti-tumorigenic chain reaction in the tumor vessels of colorectal carcinoma

Abstract In colorectal carcinoma (CRC), a Th1-tumor microenvironment (TME) is associated with improved prognosis of the patients. The lead cytokine of the Th1-response is interferon (IFN)-γ. IFN-γ is predominantly regarded as an immunomodulatory cytokine. At present, its putative tumor blood vessel-directed anti-tumorigenic effects have not been investigated comprehensively. We demonstrate that the vascular effects of IFN-γ in CRC trigger a two-step anti-tumorigenic chain reaction. First, IFN-γ exerts direct angiostatic activity on endothelial cells. This was detected in vitro using primary endothelial cell cultures and in vivo in Th1-dominated inflammatory reactions of the colon using mouse models with specific deletion of the IFN-γ receptor-2 in endothelial cells and treatment with neutralizing anti-IFN-γ antibodies. Angiostatic IFN-γ effects could be validated in human CRC tissues using the cellular IFN-γ activation marker guanylate binding protein-1 which confirmed reduced angiogenic activity of vessels exposed to IFN-γ at that single cell level. IFN-γ-induced angiostasis was associated with a highly significantly improved cancer-related 5-year survival of the CRC patients (n=388). In a second step, angiostatic activity of IFN-γ resulted in the maintenance of mature tumor vessels in CRC tissues which expressed and secreted the anti-tumorigenic protein SPARCL1 in human CRC tissues (n = 42). This was significantly repressed in CRC tissues lacking a Th1 response. Functional analyses showed that SPARCL1 inhibited angiogenic activity of cultivated endothelial cells in different in vitro tests of angiogenesis (endothelial cell proliferation, migration, spreading, 3D-sprouting and capillary formation in matrigel), both after retrovirally triggered recombinant expression in endothelial cells or after adding the recombinantly purified human SPARCL1 protein. Interestingly, secreted SPARCL1 has also been shown to inhibit proliferation and migration of human CRC tumor cell lines. We could confirm the anti-tumor cell activity of soluble purified SPARCL1 using different tumor cell lines derived from mouse colon tumors (MC38) or mouse melanomas (B16F10), respectively. SPARCL1 inhibited proliferation and migration of both cell lines significantly. Using a mouse model system with a general knock out of the SPARCL1 gene, we obtained first evidence that the growth of metastatically spread MC38 cells in the lungs is repressed by SPARCL1 in vivo. Altogether our study demonstrates that the tumor vessel-directed effects of an IFN-γ-dominated tumor microenvironment in CRC trigger a two-step anti-tumorigenic chain reaction. This provides further insight into the potent anti-tumorigenic activity of a Th1-TME in CRC that may be of relevance for selection of patients' for anti-angiogenic therapy regiments. Moreover, our findings indicate novel pathways which may be prone to tumor immune evasion. Citation Format: Michael Stürzl, Victoria Langer, Daniela Regensburger, Clara Tenkerian, Annika Klingler, Maximilian Waldner, Christoph Becker, Valerie Meniel, Robert Grützmann, Carol-Immanuel Geppert, Nathalie Britzen-Laurent, Elisabeth Naschberger. Interferon-γ triggers an anti-tumorigenic chain reaction in the tumor vessels of colorectal carcinoma [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2018; 2018 Apr 14-18; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2018;78(13 Suppl):Abstract nr 2048.

Dual regulation of Stat1 and Stat3 by the tumor suppressor protein PML contributes to interferon α-mediated inhibition of angiogenesis

IFNs are effective in inhibiting angiogenesis in preclinical models and in treating several angioproliferative disorders. However, the detailed mechanisms of IFNα-mediated anti-angiogenesis are not completely understood. Stat1/2/3 and PML are IFNα downstream effectors and are pivotal regulators of angiogenesis. Here, we investigated PML's role in the regulation of Stat1/2/3 activity. In Pml knock-out (KO) mice, ablation of Pml largely reduces IFNα angiostatic ability in Matrigel plug assays. This suggested an essential role for PML in IFNα's anti-angiogenic function. We also demonstrated that PML shared a large cohort of regulatory genes with Stat1 and Stat3, indicating an important role of PML in regulating Stat1 and Stat3 activity. Using molecular tools and primary endothelial cells, we demonstrated that PML positively regulates Stat1 and Stat2 isgylation, a ubiquitination-like protein modification. Accordingly, manipulation of the isgylation system by knocking down USP18 altered IFNα-PML axis-mediated inhibition of endothelial cell migration and network formation. Furthermore, PML promotes turnover of nuclear Stat3, and knockdown of PML mitigates the effect of LLL12, a selective Stat3 inhibitor, on IFNα-mediated anti-angiogenic activity. Taken together, we elucidated an unappreciated mechanism in which PML, an IFNα-inducible effector, possess potent angiostatic activity, doing so in part by forming a positive feedforward loop with Stat1/2 and a negative feedback loop with Stat3. The interplay between PML, Stat1/Stat2, and Stat3 contributes to IFNα-mediated inhibition of angiogenesis, and disruption of this network results in aberrant IFNα signaling and altered angiostatic activity.

A recurrent WARS mutation is a novel cause of autosomal dominant distal hereditary motor neuropathy.

Distal hereditary motor neuropathy is a heterogeneous group of inherited neuropathies characterized by distal limb muscle weakness and atrophy. Although at least 15 genes have been implicated in distal hereditary motor neuropathy, the genetic causes remain elusive in many families. To identify an additional causal gene for distal hereditary motor neuropathy, we performed exome sequencing for two affected individuals and two unaffected members in a Taiwanese family with an autosomal dominant distal hereditary motor neuropathy in which mutations in common distal hereditary motor neuropathy-implicated genes had been excluded. The exome sequencing revealed a heterozygous mutation, c.770A > G (p.His257Arg), in the cytoplasmic tryptophanyl-tRNA synthetase (TrpRS) gene (WARS) that co-segregates with the neuropathy in the family. Further analyses of WARS in an additional 79 Taiwanese pedigrees with inherited neuropathies and 163 index cases from Australian, European, and Korean distal hereditary motor neuropathy families identified the same mutation in another Taiwanese distal hereditary motor neuropathy pedigree with different ancestries and one additional Belgian distal hereditary motor neuropathy family of Caucasian origin. Cell transfection studies demonstrated a dominant-negative effect of the p.His257Arg mutation on aminoacylation activity of TrpRS, which subsequently compromised protein synthesis and reduced cell viability. His257Arg TrpRS also inhibited neurite outgrowth and led to neurite degeneration in the neuronal cell lines and rat motor neurons. Further in vitro analyses showed that the WARS mutation could potentiate the angiostatic activities of TrpRS by enhancing its interaction with vascular endothelial-cadherin. Taken together, these findings establish WARS as a gene whose mutations may cause distal hereditary motor neuropathy and alter canonical and non-canonical functions of TrpRS.

Combination of ruthenium(II)-arene complex [Ru(\u03b76-p-cymene)Cl2(pta)] (RAPTA-C) and the epidermal growth factor receptor inhibitor erlotinib results in efficient angiostatic and antitumor activity

Ruthenium-based compounds show strong potential as anti-cancer drugs and are being investigated as alternatives to other well-established metal-based chemotherapeutics. The organometallic compound [Ru(η6-p-cymene)Cl2(pta)], where pta = 1,3,5-triaza-7-phosphaadamantane (RAPTA-C) exhibits broad acting anti-tumor efficacy with intrinsic angiostatic activity. In the search for an optimal anti-angiogenesis drug combination, we identified synergistic potential between RAPTA-C and the epidermal growth factor receptor (EGFR) inhibitor, erlotinib. This drug combination results in strong synergistic inhibition of cell viability in human endothelial (ECRF24 and HUVEC) and human ovarian carcinoma (A2780 and A2780cisR) cells. Additionally, erlotinib significantly enhances the cellular uptake of RAPTA-C relative to treatment with RAPTA-C alone in human ovarian carcinoma cells, but not endothelial cells. Drug combinations induce the formation of chromosome bridges that persist after mitotic exit and delay abscission in A2780 and A2780cisR, therefore suggesting initiation of cellular senescence. The therapeutic potential of these compounds and their combination is further validated in vivo on A2780 tumors grown on the chicken chorioallantoic membrane (CAM) model, and in a preclinical model in nude mice. Immunohistochemical analysis confirms effective anti-angiogenic and anti-proliferative activity in vivo, based on a significant reduction of microvascular density and a decrease in proliferating cells.

Non-receptor type, proline-rich protein tyrosine kinase 2 (Pyk2) is a possible therapeutic target for Kawasaki disease

Kawasaki disease (KD) is a paediatric vasculitis whose pathogenesis remains unclear. Based on experimental studies using a mouse model for KD, we report here that proline-rich protein tyrosine kinase 2 (Pyk2) plays a critical role in the onset of KD-like murine vasculitis.The mouse model for KD was prepared by administrating a Candida albicans water-soluble fraction (CAWS). Unlike CAWS-treated WT mice, CAWS-treated Pyk2-Knockout (Pyk2-KO) mice did not develop apparent vasculitis. A sustained increase in MIG/CXCL9 and IP-10/CXCL10, both of which have potent angiostatic activity, was observed in CAWS-treated Pyk2-KO mice. CAWS-induced activation of STAT3, which negatively regulates the expression of these chemokines, was also attenuated in macrophages derived from Pyk2-KO mice.The present study suggests that defects in Pyk2 suppress KD-like experimental vasculitis, presumably through CXCL9- and CXCL10-dependent interference with neo-angiogenesis. Since Pyk2-KO mice show no life-threatening phenotype, Pyk2 may be a promising therapeutic molecular target for KD.

Recombinant Parvoviruses Armed to Deliver CXCL4L1 and CXCL10 Are Impaired in Their Antiangiogenic and Antitumoral Effects in a Kaposi Sarcoma Tumor Model Due To the Chemokines' Interference with the Virus Cycle.

Application of oncolytic viruses is a valuable option to broaden the armament of anticancer therapies, as these combine specific cytotoxic effects and immune-stimulating properties. The self-replicating H-1 parvovirus (H-1PV) is a prototypical oncolytic virus that, besides targeting tumor cells, also infects endothelial cells, thus combining oncolytic and angiostatic traits. To increase its therapeutic value, H-1PV can be armed with cytokines or chemokines to enhance the immunological response. Some chemokines-more specifically, the CXCR3 ligands CXCL4L1 and CXCL10-combine immune-stimulating properties with angiostatic activity. This study explores the therapeutic value of recombinant parvoviruses carrying CXCL4L1 or CXCL10 transgenes (Chi-H1/CXCL4L1 or Chi-H1/CXCL10, respectively) to inhibit the growth of the human Kaposi sarcoma cell line KS-IMM. KS-IMM cells infected by Chi-H1/CXCL4L1 or Chi-H1/CXCL10 released the corresponding chemokine and showed reduced migratory capacity. Therefore, the antitumoral capacity of Chi-H1/CXCL4L1 or Chi-H1/CXCL10 was tested in mice. Either in vitro infected KS-IMM cells were injected or subcutaneously growing KS-IMM xenografts were treated by peritumoral injections of the different viruses. Surprisingly, the transgenes did not increase the antitumoral effect of natural H-1PV. Further experiments indicated that CXCL4L1 and CXCL10 interfered with the expression of the viral NS1 protein in KS-IMM cells. These results indicate that the outcome of parvovirus-based delivery of CXCR3 ligands might be tumor cell type dependent, and hence its application must be considered carefully.

Degradomic and yeast 2-hybrid inactive catalytic domain substrate trapping identifies new membrane-type 1 matrix metalloproteinase (MMP14) substrates: CCN3 (Nov) and CCN5 (WISP2)

Members of the CCN family of matricellular proteins are cytokines linking cells to the extracellular matrix. We report that CCN3 (Nov) and CCN5 (WISP2) are novel substrates of MMP14 (membrane-type 1-matrix metalloproteinase, MT1-MMP) that we identified using MMP14 “inactive catalytic domain capture” (ICDC) as a yeast two-hybrid protease substrate trapping platform in parallel with degradomics mass spectrometry screens for MMP14 substrates. CCN3 and CCN5, previously unknown substrates of MMPs, were biochemically validated as substrates of MMP14 and other MMPs in vitro—CCN5 was processed in the variable region by MMP14 and MMP2, as well as by MMP1, 3, 7, 8, 9 and 15. CCN1, 2 and 3 are proangiogenic factors yet we found novel opposing activity of CCN5 that was potently antiangiogenic in an aortic ring vessel outgrowth model. MMP14, a known regulator of angiogenesis, cleaved CCN5 and abrogated the angiostatic activity. CCN3 was also processed in the variable region by MMP14 and MMP2, and by MMP1, 8 and 9. In addition to the previously reported cleavages of CCN1 and CCN2 by several MMPs we found that MMPs 8, 9, and 1 process CCN1, and MMP8 and MMP9 also process CCN2. Thus, our study reveals additional and pervasive family-wide processing of CCN matricellular proteins/cytokines by MMPs. Furthermore, CCN5 cleavage by proangiogenic MMPs results in removal of an angiogenic brake held by CCN5. This highlights the importance of thorough dissection of MMP substrates that is needed to reveal higher-level control mechanisms beyond type IV collagen and other extracellular matrix protein remodelling in angiogenesis. SummaryWe find CCN family member cleavage by MMPs is more pervasive than previously reported and includes CCN3 (Nov) and CCN5 (WISP2). CCN5 is a novel antiangiogenic factor, whose function is abrogated by proangiogenic MMP cleavage. By processing CCN proteins, MMPs regulate cell responses angiogenesis in connective tissues.

Identification of a residue crucial for the angiostatic activity of human mini tryptophanyl-tRNA synthetase by focusing on its molecular evolution.

Human tryptophanyl-tRNA synthetase (TrpRS) exists in two forms: a full-length TrpRS and a mini TrpRS. We previously found that human mini, but not full-length, TrpRS is an angiostatic factor. Moreover, it was shown that the interaction between mini TrpRS and the extracellular domain of vascular endothelial (VE)-cadherin is crucial for its angiostatic activity. However, the molecular mechanism of the angiostatic activity of human mini TrpRS is only partly understood. In the present study, we investigated the effects of truncated (mini) form of TrpRS proteins from human, bovine, or zebrafish on vascular endothelial growth factor (VEGF)-stimulated chemotaxis of human umbilical vein endothelial cells (HUVECs). We show that both human and bovine mini TrpRSs inhibited VEGF-induced endothelial migration, whereas zebrafish mini TrpRS did not. Next, to identify residues crucial for the angiostatic activity of human mini TrpRS, we prepared several site-directed mutants based on amino acid sequence alignments among TrpRSs from various species and demonstrated that a human mini K153Q TrpRS mutant cannot inhibit VEGF-stimulated HUVEC migration and cannot bind to the extracellular domain of VE-cadherin. Taken together, we conclude that the Lys153 residue of human mini TrpRS is a VE-cadherin binding site and is therefore crucial for its angiostatic activity.

Endothelial cell dysfunction in globoid cell leukodystrophy.

Angiogenesis plays a pivotal role in the physiology and pathology of the brain. Microvascular alterations have been observed in various neurodegenerative disorders, including genetic leukodystrophies. Globoid cell leukodystrophy (GLD) is a lysosomal storage disease caused by β-galactosylceramidase (GALC) deficiency and characterized by the accumulation of the neurotoxic metabolite psychosine in the central nervous system and peripheral tissues. Structural and functional alterations occur in the microvascular endothelium of the brain of GLD patients and twitcher mice, a murine model of the disease. In addition, increased vessel permeability and a reduced capacity to respond to proangiogenic stimuli characterize the endothelium of twitcher animals. On the one hand, these alterations may depend, at least in part, on the local and systemic angiostatic activity exerted by psychosine on endothelial cells. On the other hand, studies performed in vivo on zebrafish embryos and in vitro on human endothelial cells suggest that GALC downregulation may also lead to psychosine-independent neuronal and vascular defects. Together, experimental observations indicate that endothelial cell dysfunctions may represent a novel pathogenic mechanism in human leukodystrophies, including GLD. A better understanding of the molecular mechanisms responsible for these microvascular alterations may provide new insights for the therapy of GLD. © 2016 Wiley Periodicals, Inc.

Hypoxia paradoxically inhibits the angiogenic response of isolated vessel explants while inducing overexpression of vascular endothelial growth factor.

This study was designed to investigate how changes in O2 levels affected angiogenesis in vascular organ culture. Although hypoxia is a potent inducer of angiogenesis, aortic rings cultured in collagen paradoxically failed to produce an angiogenic response in 1-4% O2. Additionally, aortic neovessels preformed in atmospheric O2 lost pericytes and regressed at a faster rate than control when exposed to hypoxia. Aortic explants remained viable in hypoxia and produced an angiogenic response when returned to atmospheric O2. Hypoxic aortic rings were unresponsive to VEGF, while increased oxygenation of the system dose-dependently enhanced VEGF-induced angiogenesis. Hypoxia-induced refractoriness to angiogenic stimulation was not restricted to the aorta because similar results were obtained with vena cava explants or isolated endothelial cells. Unlike endothelial cells, aorta-derived mural cells were unaffected by hypoxia. Hypoxia downregulated expression in aortic explants of key signaling molecules including VEGFR2, NRP1 and Prkc-beta while upregulating expression of VEGFR1. Medium conditioned by hypoxic cultures exhibited angiostatic and anti-VEGF activities likely mediated by sVEGFr1. Hypoxia reduced expression of VEGFR1 and VEGFR2 in endothelial cells while upregulating VEGFR1 in macrophages and VEGF in both macrophages and mural cells. Thus, changes in O2 levels profoundly affect the endothelial response to angiogenic stimuli. These results suggest that hypoxia-induced angiogenesis is fine-tuned by complex regulatory mechanisms involving not only production of angiogenic factors including VEGF but also differential regulation of VEGFR expression in different cell types and production of inhibitors of VEGF function such as sVEGFR1.

Abstract 5223: A novel pro-angiogenic role for IDO1 in inflammatory tumor promotion

Abstract Chronic inflammation is a major contributing factor in cancer, but, due to the complex multifactorial nature of inflammation, there remains limited understanding of specific pathogenic determinants that might be targeted therapeutically. The tryptophan-catabolizing enzyme IDO1 (indoleamine 2,3-dioxygenase) has emerged as an intriguing, pro-tumorigenic regulator of immune function in this regard. Because IDO1 can be elevated in chronic inflammatory states even prior to the initiation of cancer, it may represent one of the earliest determinants directing the immune response towards supporting rather than eliminating tumors. Genetic studies in mice have clearly established the tumor-promoting role of IDO1, but what this actually entails remains uncertain. Here we present evidence to support the novel hypothesis that a principle means by which IDO1 facilitates tumorigenesis is by mitigating immune-based angiostasis. Tumor angiogenesis is characterized by excessive and disorganized blood vessel growth much like that induced by ischemia where immune cells have been shown to be important for limiting neovascularization. Likewise, an anti-angiogenic response may be a factor in tumor immunity. In particular, IFNγ, an inflammatory cytokine, long recognized as a major inducer of IDO1, has been shown to exert angiostatic activity against developing tumors, which was implicated in these studies as the primary mechanism for both CD4 and CD8 T cell dependent tumor rejection. Our recently reported finding that the loss of IDO1 resulted in diminished pulmonary vascularization (Smith, Cancer Discovery 2012) suggested the possibility that IDO1 might be working at cross purposes to limit IFNγ-mediated angiostasis. In this same study IDO1 loss was also associated with the attenuated induction of the pro-angiogenic inflammatory cytokine IL6. To directly investigate the role of IDO1 in pathologic angiogenesis, we have utilized an oxygen-induced retinopathy (OIR) model. As predicted, neovascularization in the OIR model was significantly reduced in Ido1-/- mice. Consistent with the hypothesis that IDO1 supports neovascularization primarily by counteracting the angiostatic activity of IFNγ, neovascularization in double knockout Ifng-/- Ido1-/- mice reverted back to wild type levels. Il6-/- mice, on the other hand, exhibited reduced neovascularization which was likewise reversed by the concurrent elimination of IFNγ. In conjunction with these angiogenesis studies, we have also examined pulmonary metastasis development by 4T1 breast carcinoma isografts. Loss of either IDO1 or IL6 resulted in resistance to pulmonary metastases that, in both cases, was abrogated by the concurrent loss of IFNγ. Taken together, these findings have led us to propose a conceptually novel working hypothesis that, in the context of an inflammatory cytokine milieu, IDO1 plays a key role in supporting tumor angiogenesis. Citation Format: Arpita Mondal, James B. DuHadaway, Erika Sutanto-Ward, Courtney Smith, George C. Prendergast, Arturo Bravo-Nuevo, Alexander J. Muller. A novel pro-angiogenic role for IDO1 in inflammatory tumor promotion. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5223. doi:10.1158/1538-7445.AM2015-5223

Abstract 5552: Medicinal plants show anticancer splicing activity of apoptotic genes

Abstract Oncogenesis is associated with splicing switches and a disruption of splicing regulation being specific for cancer formation and tumour progression. An association between differential expression of splicing isoforms and tumour progression has been shown for several proteins, such as MDM2 and survivin. Currently the analysis of cancer-specific alternative splicing is a promising step forward in basic and translational molecular biology. Traditional medicine has a long history and is still the major source of medicine in developing countries. Approximately 70% of the South African population consults traditional healers, perpetuating the need for scientific appraisal of traditional medicine as a means to establish its efficiency and safety Also, pharmacological and phytochemical insights into several plants have led to the discovery of novel chemicals and therefore novel drugs. Alternatively, such novel chemical structures can serve as lead compounds/templates for the design of new drugs. The aim was to ascertain if the South African medicinal plants have anticancer splicing activity. 10c cells were treated with Tulbhaghia violacea and Cotyledon orbiculata, followed by mRNA extraction and RT-PCR. The results showed that Tulbhaghia violacea and Cotyledon orbiculata extracts have anti-cancer splicing activity on the BCLX and the AXL apoptosis genes. Additionally Cotyledon orbiculata extract has an anticancer splicing activity of the angiogenesis gene VEGF165. VEGF Elisa also confirmed the VEGF165 VEGF165b splicing switch. We have shown that South African medicinal plants have anti-cancer splicing activity. The anti-cancer splicing activity of the VEGF gene by Cotyledon orbiculata could mean that this extract has both angiostatic and apoptotic anti-cancer activities. We are continuing to screen more medicinal plants and will select those extracts with anti-cancer splicing activity for further studies. These further studies should identify numerous splicing pathways and completely elucidate the splicing target compounds that may serve as novel anti-cancer drugs or lead compounds. Note: This abstract was not presented at the meeting. Citation Format: Zodwa Dlamini, David Bates. Medicinal plants show anticancer splicing activity of apoptotic genes. [abstract]. In: Proceedings of the 106th Annual Meeting of the American Association for Cancer Research; 2015 Apr 18-22; Philadelphia, PA. Philadelphia (PA): AACR; Cancer Res 2015;75(15 Suppl):Abstract nr 5552. doi:10.1158/1538-7445.AM2015-5552

Antiangiogenic and Anticancer Properties of Bifunctional Ruthenium(II)-p-Cymene Complexes: Influence of Pendant Perfluorous Chains.

Two bifunctional ruthenium(II)-p-cymene complexes with perfluorinated side chains, attached via pyridine ligands, have been evaluated in a series of in vitro and in vivo assays. Their effects on human endothelial (ECRF24 and HUVEC) cells, noncancerous human embryonic kidney (HEK-293) cells, and various human tumor cells were investigated. The complex with the shorter chain, 1, inhibits the proliferation of the tumor cell lines and ECRF24, whereas 2 selectively inhibits ECRF24 and HUVEC proliferation. Neither inhibits the migration of ECRF24 cells whereas both compounds inhibit sprout formation in HUVEC cells. Using three preclinical models, i.e., vasculature formation in the chorioallantoic membrane (CAM) of the chicken embryo, human A2780 ovarian carcinoma tumors xenografted on the CAM, and human LS174T colorectal adenocarcinoma tumors grown in athymic mice, the angiostatic and anticancer activities of these two complexes were studied. Overall, 1 inhibited tumor growth predominantly through an anticancer effect whereas 2 inhibited tumor growth predominately via an antiangiogenic mechanism.

Interferon Gamma Counteracts the Angiogenic Switch and Induces Vascular Permeability in Dextran Sulfate Sodium Colitis in Mice.

Interferon (IFN)-γ is a central pathogenesis factor in inflammatory bowel disease (IBD) with pleiotropic effects on many different cell types. However, as yet, the immune modulatory functions of IFN-γ in IBD have been predominantly investigated. Based on previous studies showing that IFN-γ acts antiangiogenic in colorectal carcinoma, we investigated the effects of IFN-γ on the vascular system in IBD. Colon tissues of patients with IBD and dextran sulfate sodium-induced colitis in mice were subjected to immunohistochemistry, quantitative real-time polymerase chain reactions, and in situ hybridization to quantify cell activation, angiogenesis, and immune responses. Vascular structure and permeability in mice were analyzed by ultramicroscopy and in vivo confocal laser endomicroscopy. We showed a significantly increased blood vessel density in IBD and dextran sulfate sodium colitis. In mice, this was associated with a disorganized blood vessel structure and profound vascular leakage. As compared with genes associated with angiogenesis, genes associated with inflammatory cell activation including IFN-γ were more strongly upregulated in colitis tissues. IFN-γ exerted direct effects on endothelial cells in IBD tissues in vivo, as indicated by the expression of IFN-γ-induced guanylate binding protein 1 (GBP-1). Neutralization of IFN-γ in the acute dextran sulfate sodium colitis model demonstrated that this cytokine exerts endogenous angiostatic activity in IBD and contributes to increased vascular permeability. The dissection of the pleiotropic activities of IFN-γ in IBD provides new insights to the pathological functions of this cytokine and may be of high relevance for the optimization of combination therapy approaches.