Angiogenesis In Cornea Research Articles

Cartilage type IIB procollagen NH<sub>2</sub>-propeptide, PIIBNP, inhibits angiogenesis

<abstract> <p>Cartilage tissue is avascular and resistant to tumor invasion, but the basis for these properties is still unclear. Here we report that the NH<sub>2</sub>-propeptide of type IIB procollagen (PIIBNP), a product of collagen biosynthesis, is capable of inhibiting angiogenesis both <italic>in vitro</italic> and <italic>in vivo</italic>. PIIBNP inhibits tube formation in human umbilical vein cells (HUVEC), inhibits endogenous endothelial cell outgrowth in mouse aortic ring angiogenesis bioassay and is anti-angiogenic in the mouse cornea angiogenesis assay. As α<sub>V</sub>ß<sub>3</sub> and α<sub>V</sub>ß<sub>5</sub> integrins are expressed primarily in endothelial cells, cancer cells and osteoclasts, but not in normal chondrocytes and PIIBNP binds to cell surface integrin α<sub>V</sub>ß<sub>3</sub> and αVß<sub>5</sub>, we propose that natural occurring PIIBNP protects cartilage by targeting endothelial cells during chondrogenesis, thus inhibiting angiogenesis, and rendering the tissue avascular.</p> </abstract>

NDRG1 activates VEGF-A-induced angiogenesis through PLC\u03b31/ERK signaling in mouse vascular endothelial cells

Many diseases, including cancer, have been associated with impaired regulation of angiogenesis, of which vascular endothelial growth factor (VEGF)-A is a key regulator. Here, we test the contribution of N-myc downstream regulated gene 1 (NDRG1) to VEGF-A-induced angiogenesis in vascular endothelial cells (ECs). Ndrg1−/− mice exhibit impaired VEGF-A-induced angiogenesis in corneas. Tumor angiogenesis induced by cancer cells that express high levels of VEGF-A was also reduced in a mouse dorsal air sac assay. Furthermore, NDRG1 deficiency in ECs prevented angiogenic sprouting from the aorta and the activation of phospholipase Cγ1 (PLCγ1) and ERK1/2 by VEGF-A without affecting the expression and function of VEGFR2. Finally, we show that NDRG1 formed a complex with PLCγ1 through its phosphorylation sites, and the inhibition of PLCγ1 dramatically suppressed VEGF-A-induced angiogenesis in the mouse cornea, suggesting an essential role of NDRG1 in VEGF-A-induced angiogenesis through PLCγ1 signaling.

Selective IKK2 inhibitor IMD0354 disrupts NF-\u03baB signaling to suppress corneal inflammation and angiogenesis

Corneal neovascularization is a sight-threatening condition caused by angiogenesis in the normally avascular cornea. Neovascularization of the cornea is often associated with an inflammatory response, thus targeting VEGF-A alone yields only a limited efficacy. The NF-κB signaling pathway plays important roles in inflammation and angiogenesis. Here, we study consequences of the inhibition of NF-κB activation through selective blockade of the IKK complex IκB kinase β (IKK2) using the compound IMD0354, focusing on the effects of inflammation and pathological angiogenesis in the cornea. In vitro, IMD0354 treatment diminished HUVEC migration and tube formation without an increase in cell death and arrested rat aortic ring sprouting. In HUVEC, the IMD0354 treatment caused a dose-dependent reduction in VEGF-A expression, suppressed TNFα-stimulated expression of chemokines CCL2 and CXCL5, and diminished actin filament fibers and cell filopodia formation. In developing zebrafish embryos, IMD0354 treatment reduced expression of Vegf-a and disrupted retinal angiogenesis. In inflammation-induced angiogenesis in the rat cornea, systemic selective IKK2 inhibition decreased inflammatory cell invasion, suppressed CCL2, CXCL5, Cxcr2, and TNF-α expression and exhibited anti-angiogenic effects such as reduced limbal vessel dilation, reduced VEGF-A expression and reduced angiogenic sprouting, without noticeable toxic effect. In summary, targeting NF-κB by selective IKK2 inhibition dampened the inflammatory and angiogenic responses in vivo by modulating the endothelial cell expression profile and motility, thus indicating an important role of NF-κB signaling in the development of pathologic corneal neovascularization.

Genome-wide expression differences in anti-Vegf and dexamethasone treatment of inflammatory angiogenesis in the rat cornea

Angiogenesis as a pathological process in the eye can lead to blindness. In the cornea, suppression of angiogenesis by anti-VEGF treatment is only partially effective while steroids, although effective in treating inflammation and angiogenesis, have broad activity leading to undesirable side effects. In this study, genome-wide expression was investigated in a suture-induced corneal neovascularization model in rats, to investigate factors differentially targeted by dexamethasone and anti-Vegf. Topical treatment with either rat-specific anti-Vegf, dexamethasone, or normal goat IgG (sham) was given to sutured corneas for 48 hours, after which in vivo imaging, tissue processing for RNA microarray, and immunofluorescence were performed. Dexamethasone suppressed limbal vasodilation (P < 0.01) and genes in PI3K-Akt, focal adhesion, and chemokine signaling pathways more effectively than anti-Vegf. The most differentially expressed genes were confirmed by immunofluorescence, qRTPCR and Western blot. Strong suppression of Reg3g and the inflammatory chemokines Ccl2 and Cxcl5 and activation of classical complement pathway factors C1r, C1s, C2, and C3 occurred with dexamethasone treatment, effects absent with anti-Vegf treatment. The genome-wide results obtained in this study provide numerous potential targets for specific blockade of inflammation and angiogenesis in the cornea not addressed by anti-Vegf treatment, as possible alternatives to broad-acting immunosuppressive therapy.

CXCL10 suppression of hem- and lymph-angiogenesis in inflamed corneas through MMP13.

Though not present in the normal adult cornea, both hem- and lymph-angiogenesis can be induced in this tissue after an inflammatory, infectious, or traumatic insult. We previously showed that the chemokine CXCL10 plays a key role in eradicating invading Candida (C.) albicans in C57BL6 mouse corneas. However, even after the clearance of pathogens, infection-induced inflammation and angiogenesis continue to progress in the cornea. The aim of this study is define the role of CXCL10 as a major angiostatic factor in modulating cornea angiogenesis in B6 mouse corneas under pathogenic conditions. We showed that epithelial expression of CXCL10, driven by AAV9 vector, suppressed both infection- and inflammation-induced hem and lymph angiogenesis, whereas the neutralization of CXCL10 as well as its receptor CXCR3 greatly promoted these processes. The inhibitory effect of CXCL10 was unrelated to its antimicrobial activity, but through the suppression of the expression of many angiogenic factors, including VEGFa and c, and MMP-13 in vivo. Inhibition of MMP13 but not TIMPs, attenuated suture-induced neovascularization but had no effects on CXCL10 expression. Strikingly, topical application of CXCL10 post-C. albicans infection effectively blocked both hem- and lymph-angiogenesis and preserved the integrity of sensory nerves in the cornea. Taken together, CXCL10 has strong inhibitory effects on neovascularization, whereas MMP13 is required for neovascularization in C. albicans-infected corneas and the local application of CXCL10 or MMP13 inhibitors, alone or as adjuvant therapy, may target hem- and lymph-angiogenesis in the inflamed corneas.

Triptolide Suppresses Alkali Burn-Induced Corneal Angiogenesis Along with a Downregulation of VEGFA and VEGFC Expression.

Triptolide (TPL) is an active compound extracted from a Chinese herbal medicine tripterygium wilfordii Hook. f. (Celastraceae), which has been used as an anti-inflammatory drug for years. It also inhibits the growth and proliferation of different types of cancer cells. The inhibitory effect of TPL on angiogenesis after chemical-induced corneal inflammation was studied in vivo. The effects of TPL on the proliferation, apoptosis, migration, and tube formation of rat aortic endothelial cells (RAECs) were studied in vitro. Cell proliferation and apoptosis were measured by MTT assay and flow cytometry, respectively. Migration was analyzed using the scratch wound healing assay and transwell assay. Tube formation assay was used to examine angiogenesis. Real-time PCR and Western blot were used to determine the expression of vascular endothelial growth factor A (VEGFA) and VEGFC. To study the in vivo effects of TPL, the mouse model of alkali burn-induced corneal angiogenesis was used. The angiogenesis was analyzed by determining the density of the newly generated blood vessels in corneas. We found that TPL induced apoptosis and inhibited the proliferation of RAECs in a dose-dependent manner. TPL inhibited migration and tube formation of RAECs and decreased the expression of VEGFA and VEGFC in vitro. Furthermore, TPL suppressed alkali burn-induced corneal angiogenesis and inhibited the expression of VEGFA and VEGFC in corneas in vivo. In conclusion, topical TPL as a pharmacological agent has the ability to reduce angiogenesis in cornea and may have clinical indications for the treatment of corneal angiogenesis diseases which have to be further explored. Anat Rec, 300:1348-1355, 2017. © 2017 Wiley Periodicals, Inc.

Impaired differentiation of macrophage lineage cells attenuates bone remodeling and inflammatory angiogenesis in Ndrg1 deficient mice.

N-myc downstream regulated gene 1 (NDRG1) is a responsible gene for a hereditary motor and sensory neuropathy-Lom (Charcot–Marie–Tooth disease type 4D). This is the first study aiming to assess the contribution of NDRG1 to differentiation of macrophage lineage cells, which has important implications for bone remodeling and inflammatory angiogenesis. Ndrg1 knockout (KO) mice exhibited abnormal curvature of the spine, high trabecular bone mass, and reduced number of osteoclasts. We observed that serum levels of macrophage colony-stimulating factor (M-CSF) and macrophage-related cytokines were markedly decreased in KO mice. Differentiation of bone marrow (BM) cells into osteoclasts, M1/M2-type macrophages and dendritic cells was all impaired. Furthermore, KO mice also showed reduced tumor growth and angiogenesis by cancer cells, accompanied by decreased infiltration of tumor-associated macrophages. The transfer of BM-derived macrophages from KO mice into BM-eradicated wild type (WT) mice induced much less tumor angiogenesis than observed in WT mice. Angiogenesis in corneas in response to inflammatory stimuli was also suppressed with decreased infiltration of macrophages. Taken together, these results indicate that NDRG1 deficiency attenuates the differentiation of macrophage lineage cells, suppressing bone remodeling and inflammatory angiogenesis. This study strongly suggests the crucial role of NDRG1 in differentiation process for macrophages.

Protective effects of matrix metalloproteinase-12 following corneal injury

Corneal scarring due to injury is a leading cause of blindness worldwide and results from dysregulated inflammation and angiogenesis during wound healing. Here we demonstrate that the extracellular matrix metalloproteinase MMP12 (macrophage metalloelastase) is an important regulator of these repair processes. Chemical injury resulted in higher expression of the fibrotic markers α-smooth muscle actin and type I collagen, and increased levels of angiogenesis in corneas of Mmp12(-/-) mice compared with corneas of wild-type mice. In vivo, we observed altered immune cell dynamics in Mmp12(-/-) corneas by confocal imaging. We determined that the altered dynamics were the result of an altered inflammatory response, with delayed neutrophil infiltration during the first day and excessive macrophage infiltration 6 days later, mediated by altered expression levels of chemokines CXCL1 and CCL2, respectively. Corneal repair returned to normal upon inhibition of these chemokines. Taken together, these data show that MMP12 has a protective effect on corneal fibrosis during wound repair through regulation of immune cell infiltration and angiogenesis.

Controlled release of imatinib mesylate from PLGA microspheres inhibit craniopharyngioma mediated angiogenesis

Poly(lactic-co-glycolic acid) microspheres loaded with imatinib mesylate has been developed as a new therapeutic strategy to prevent craniopharyngioma recurrence. Microspheres composed of different lactic/glycolic acid ratios, molecular weights and drug compositions were synthesized and loaded with imatinib mesylate by modified double-emulsion/solvent evaporation technique and subsequently characterized by particle-size distribution, scanning electron microscopy, encapsulation efficiency and in vitro drug release. Inhibitory potential of imatinib containing microspheres on tumor neovascularization was investigated on craniopharyngioma tumor samples by rat cornea angiogenesis assay. Results showed that microspheres in different LA:GA ratios [LA:GA 50:50 (G50), 75:25 (G25), 85:15 (G15)] considerably reduced neovascularization induced by recurrent tumor samples in an in vivo angiogenesis assay (P < 0.01). Our data indicate that local delivery of imatinib mesylate to the post-surgical tumoral cavity using biodegradable microspheres may be a promising biologically selective approach to prevent the recurrence of craniopharyngiomas, via inhibition of neovascularization.

Metastatic events of MDA-MB-231 cells induced by angiogenic factors VEGF or MYA1 are inhibited by Tinospora cordifolia hexane fraction (tchf)

ngiogenic growth factors VEGF or MTA1 mediated proliferation, migration and invasion play pivotal role in the process of metastasis. In this paper we have described the preparation of hexane fraction from Tinospora cordifolia (Tchf) and have shown that Tchf has an antiangiogenic activity. HUVEC tube formation and Rat cornea angiogenesis assays were used for validation of antiangiogenic activity of Tchf. Our results on VEGF or MTA1 induced proliferation, migration and invasion of MDA-MB-231 cells show that Tchf and SB202190, a MAP kinase inhibitor effectively inhibits these metastatic processes. Both angiogenic factors VEGF or MTA1 could induce expression of VEGF gene in MDA-MB-231 cells, Tchf repressed VEGF gene expression induced either by VEGF or MTA1. These results clearly indicate for the first time that Tchf is a potent inhibitor of metastatic events which are synergistically trigged by VEGF and MTA1

Identification of novel neutralizing single‐chain antibodies against vascular endothelial growth factor receptor 2

Human vascular endothelial growth factor (VEGF) and its receptor (VEGFR-2/kinase domain receptor [KDR]) play a crucial role in angiogenesis, which makes the VEGFR-2 signaling pathway a major target for therapeutic applications. In this study, a single-chain antibody phage display library was constructed from spleen cells of mice immunized with recombinant human soluble extracellular VEGFR-2/KDR consisting of all seven extracellular domains (sKDR D1-7) to obtain antibodies that block VEGF binding to VEGFR-2. Two specific single-chain antibodies (KDR1.3 and KDR2.6) that recognized human VEGFR-2 were selected; diversity analysis of the clones was performed by BstNI fingerprinting and nucleotide sequencing. The single-chain variable fragments (scFvs) were expressed in soluble form and specificity of interactions between affinity purified scFvs and VEGFR-2 was confirmed by ELISA. Binding of the recombinant antibodies for VEGFR-2 receptors was investigated by surface plasmon resonance spectroscopy. In vitro cell culture assays showed that KDR1.3 and KDR2.6 scFvs significantly suppressed the mitogenic response of human umbilical vein endothelial cells to recombinant human VEGF(165) in a dose-dependent manner, and reduced VEGF-dependent cell proliferation by 60% and 40%, respectively. In vivo analysis of these recombinant antibodies in a rat cornea angiogenesis model revealed that both antibodies suppressed the development of new corneal vessels (p < 0.05). Overall, in vitro and in vivo results disclose strong interactions of KDR1.3 and KDR2.6 scFvs with VEGFR-2. These findings indicate that KDR1.3 and KDR2.6 scFvs are promising antiangiogenic therapeutic agents.

Abstract B48: Inhibition of recurrence of craniopharyngiomas with imatinib mesylate-loaded poly(lactide-co-glycolide) (PLGA) microspheres

Abstract Background: Recurrence is highly seen in craniopharyngiomas. Due to their close proximity to the vital structures, gross-total resection, which is thought to be the major determinant of the recurrence, is not always possible for craniopharyngiomas. So, post-operative long-term effective therapy approaches are needed to prevent the recurrence. Besides, our recent studies showed that there is a positive correlation between the recurrence and angiogenic potential of craniopharyngioma through over activation of PDGF (platelet-derived growth factor) signaling pathway. Objective: In this study, we aimed to investigate the inhibitory potential of local, long-term delivery of imatinib mesylate (PDGFR-B blocker) on recurrence of adult craniopharyngiomas via PLGA-type biodegradable microspheres. Methods: Imatinib mesylate containing PLGA polymers composed of different lactic/glycolic acid concentrations, molecular weights and drug compositions were synthesized by modified double emulsion/solvent evaporation technique. After in vitro characterization of protein holding, surface morphology, entrapment efficiency and drug release kinetics, inhibitory potential of microspheres on neovascularization was tested on craniopharyngioma tumor samples implanted in rat cornea. Results: Imatinib containing PLGA microspheres in different LA:GA ratios (LA:GA, 50:50 (M38); 75:25(M70); 85:15(M71)) considerably reduced the neovascularization induced by recurrent tumor samples in an in vivo rat cornea angiogenesis model, p&amp;lt;0,01. Conclusion: Long-term, local delivery of imatinib mesylate to the post-surgical tumoral cavity using biodegradable microspheres is promising approach to prevent the recurrence of craniopharyngiomas. Citation Information: Cancer Prev Res 2011;4(10 Suppl):B48.

Preclinical anti-angiogenesis and anti-tumor activity of SIM010603, an oral, multi-targets receptor tyrosine kinases inhibitor

SIM010603 is a structurally novel, oral, multi-targeted receptor tyrosine kinase inhibitor. This study investigated the anti-angiogenic and anti-tumor effects of SIM010603. A radiometric protein kinase assay was used for measuring the kinase activity of the 32 protein kinases. Receptor phosphorylation was determined by enzyme-linked immunosorbent assay (ELISA). Cell proliferation was measured by 3-(4, 5-dimethylthiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay. Cell chemotaxis was evaluated by modified Boyden chamber assay. Effect of SIM010603 on angiogenesis was examined by mouse cornea angiogenesis assay. Effect of SIM010603 on xenografts was assessed by tumor growth delay. Effects of SIM010603 on tumor microvascular density (MVD), recruitment of pericytes, and pericyte encapsulation of tumor vessels were analyzed by immunofluorescent staining technique. SIM010603 inhibited stem cell factor receptor (Kit), vascular endothelial growth factor receptor-2 (VEGFR-2), platelet-derived growth factor receptor-β (PDGFR-β), glial cell line-derived neurotrophic factor receptor (Rearranged during Transfection; RET), and Fms-like tyrosine kinase-3 (FLT3) with IC(50) values between 5.0 and 68.1nmol/l. SIM010603 inhibited the phosphorylation of PDGFR-β and VEGFR-2. Moreover, SIM010603 inhibited endothelial cell proliferation, endothelial cells chemotaxis, and corneal angiogenesis. Although SIM010603 exhibited lower activity in regard to proliferation of NCI-H460, MDA-MB-435, and T241-VEGF-A cells (IC(50)>1μmol/l), SIM010603 inhibited tumor growth in these xenograft tumor growth models. SIM010603 reduced tumor MVD in T241-VEGF-A tumor xenograft models and decreased positive signals of CD31, NG2 in MDA-MB-435, and LLC-SW-44 xenograft tumor models. These results support the clinical assessment of SIM010603 as a therapeutic agent for cancer.

Hydrogen andN-Acetyl-l-Cysteine Rescue Oxidative Stress-Induced Angiogenesis in a Mouse CornealAlkali-Burn Model

To investigate the role of reactive oxygen species (ROS) as the prime initiators of the angiogenic response after alkali injury of the cornea and observe the effects of antioxidants in preventing angiogenesis. The corneal epithelia of SOD-1-deficient mice or wild-type (WT) mice were removed after application of 0.15 N NaOH to establish the animal model of alkali burn. ROS production was semiquantitatively measured by dihydroethidium (DHE) fluorescence. Angiogenesis was visualized by CD31 immunohistochemistry. The effects of the specific NF-κB inhibitor DHMEQ, the antioxidant N-acetyl-L-cysteine (NAC), and hydrogen (H2) solution were observed. ROS production in the cornea was enhanced immediately after alkali injury, as shown by increased DHE fluorescence (P<0.01). NF-κB activation and the upregulation of vascular endothelial growth factor (VEGF) and monocyte chemoattractant protein-1 (MCP-1) were significantly enhanced (P<0.01), leading to a significantly larger area of angiogenesis. Angiogenesis in SOD-1-/- mice corneas were significantly higher in WT mice (P<0.01), confirming the role of ROS. Pretreatment with the specific NF-κB inhibitor DHMEQ or the antioxidant NAC significantly reduced corneal angiogenesis by downregulating the NF-κB pathway (P<0.01) in both WT and SOD-1-/- mice. Furthermore, we showed that irrigation of the cornea with hydrogen (H2) solution significantly reduced angiogenesis after alkali-burn injury (P<0.01). Immediate antioxidant therapy with H2-enriched irrigation solution is a new potent treatment of angiogenesis in cornea to prevent blindness caused by alkali burn.

Anti-Angiogenic Activity of a Tetravalent Bispecific Antibody (TAvi6) Targeting VEGF and Angiopoietin-2

AbstractAbstract 4304 Background:VEGF blockade has been validated clinically as a treatment for human cancers. Angiopoietin-2 (Ang-2) expression has been shown to function as a key regulator of blood vessel remodeling and tumor angiogenesis. Several publications described that VEGF and Ang-2 are up-regulated in several hematological tumors like B-CLL, AML or multiple myeloma. In particular, Ang-2 may qualify as a novel prognostic marker in AML and CLL. We have generated TAvi6, a novel bispecific antibody targeting VEGF-A and Ang-2 and tested its anti-tumor and anti-angiogenic activity. TAvi6 is a tetravalent IgG-like bispecific antibody based on bevacizumab and targets Ang-2 with 2 disulfide-stabilized scFvs (LC06) fused to the C-terminus of the heavy chain. Material and Methods:TAvi 6 was profiled in biochemical and cellular (angiogenesis) assays. Antitumoral and anti-angiogenic efficacy was assessed in established s.c. Colo205, s.c. Calu-3 and orthotopic i.m.f.p. KPL-4 xenografts in SCID beige mice. Mice were treated with bevacizumab or <Ang-2> antibody LC06 (10 mg/kg), the respective combination (each 10 mg/kg) and TAvi6 (13.3 mg/kg). In addition, TAvi6 was evaluated for inhibition of metastasis to the lung quantified by Alu-PCR. Tumors were explanted for histological analysis of angiogenesis and inhibition of angiogenesis was assessed in the cornea angiogenesis assay. Results:In biochemical assays (affinity, Tie2-Ang-2 interaction) and cellular assays (Tie2 phosphorylation, HUVEC proliferation, tube formation) TAvi6 shows properties identical to the parental antibodies bevacizumab and LC06. In the orthotopic KPL-4-003 xenograft tumor growth inhibition was 79% for bevacizumab; 39% for LC06; 90% for the combination and 91% for TAvi6. In the s.c. Colo205-009 xenograft TGI was 66% for bevacizumab; 50% for LC06; 78% for the combination and 87% for TAvi6. TAvi6 was able to suppress growth of tumors refractory to 1st-line treatment with bevacizumab (Colo205 xenograft) and suppressed metastasis to the lung significantly (KPL-4 xenograft). In the s.c. Calu-3 xenograft we observed a strong inhibition of angiogenesis by in vivo and ex vivo imaging and an advantage of TAvi6 compared to the antibody combination. Furthermore, TAvi6 potently inhibited VEGF-induced angiogenesis in the mouse corneal angiogenesis assay. Conclusions:We have generated a novel tetravalent IgG-like bispecific antibody targeting VEGF-A and Ang-2 simultaneously. TAvi6 shows identical properties compared to the respective parental antibodies. In particular, TAvi6 blocks angiogenesis in vivo efficacously and mediates strong tumor growth inhibition in various xenograft models with a slight advantage of TAvi6 over the combination of the respective single agents bevacizumab and LC06 in several models. Further studies are foreseen to assess the application of bispecific antibodies targeting Ang-2 and VEGF as novel therapeutic approach for the treatment of hematological cancers. Disclosures:Weidner:Roche: Employment, Equity Ownership. Scheuer:Roche: Employment, Patents & Royalties. Thomas:Roche: Employment, Patents & Royalties. Baehner:Roche: Employment, Patents & Royalties. Seeber:Roche: Employment, Patents & Royalties. Kettenberger:Roche: Employment, Patents & Royalties. Schanzer:Roche: Employment, Patents & Royalties. Brinkmann:Roche: Employment, Patents & Royalties. Regula:Roche: Employment, Patents & Royalties. Klein:Roche: Employment, Equity Ownership, Patents & Royalties.

Endovascular Treatment Increases but Gamma Knife Radiosurgery Decreases Angiogenic Activity of Arteriovenous Malformations

To compare the angiogenic potentials of embolized, gamma knife-treated or untreated cerebral arteriovenous malformations (AVMs), using a rat cornea angiogenesis model. Tissue samples from cerebral AVM patients who were either untreated or had previously been treated with embolization or gamma knife radiosurgery and who had undergone operations for hemorrhage at the Neurosurgery Department or the Neurological Sciences Institute of Marmara University were used. For the macroscopic evaluation of angiogenesis, tissue samples were inoculated in a micropocket created on the rat eye, and the level of angiogenic activity was graded macroscopically for 15 days, with glioblastoma multiforme and normal brain artery tissues serving as positive and negative controls, respectively. For the other part of the experiment, eyes of another set of rats were inoculated with the study samples only using the same cornea angiogenesis model, in which microvessel count and vascular endothelial growth factor assessment was done at days 3, 7, 11, and 15. Based on our macroscopic findings in the cornea angiogenesis model, embolized AVMs exhibited the highest angiogenic activity, followed by untreated AVMs and gamma knife-treated AVMs. Evaluations of vascular endothelial growth factor expression and microvessel counts showed a similar relation among the 3 tissue groups with regard to the level of angiogenic activity, supporting the results of macroscopic examinations. This study, for the first time, provides experimental semiquantitative data to compare the angiogenic potentials of embolized and gamma knife-treated AVM tissues. Embolization may increase angiogenic activity, and gamma knife radiosurgery may decrease it when compared with activity in previously untreated AVMs. These data can be useful to understand why recurrence of AVMs after angiographically demonstrated endovascular occlusion is common but after gamma knife occlusion is rare.

Cytokine secretion by human mesenchymal stem cells cocultured with damaged corneal epithelial cells

We have previously shown that mesenchymal stem cells (MSCs) reduced corneal inflammation and neovascularization in chemically-burned rat corneas in part through paracrine action. In order to identify the molecule(s) involved, we cocultured human MSCs in the following conditions, and examined the alterations in the cytokine secretion profile; (1) human peripheral blood mononuclear cells (hPBMCs), (2) chemically-damaged human corneal epithelial cells (hCECs), (3) hPBMCs/hCECs, (4) hMSCs, (5) hMSCs/hPBMCs, (6) hMSCs/hCECs, (7) hMSCs/hPBMCs/hCECs. We found that the levels of interleukin (IL)-6 and vascular endothelial growth factor (VEGF) by hMSCs markedly increased for hMSC/hCEC cocultures, and this elevation was further remarkable by the addition of hPBMCs. The hMSCs constitutively expressed transforming growth factor (TGF)-β1, matrix metalloproteinase (MMP)-2, and thrombospondin-1. The secretion of MMP-9 by hCECs was significantly suppressed by hMSCs. Tumor necrosis factor (TNF)-α, interferon (IFN)-γ, and IL-10 were not detectable in any cultures. The data presented herein provide the candidate molecules mediating the MSC-mediated modulation of inflammation and angiogenesis in cornea.

Citrullination of CXCL8 by peptidylarginine deiminase alters receptor usage, prevents proteolysis, and dampens tissue inflammation.

Biological functions of proteins are influenced by posttranslational modifications such as on/off switching by phosphorylation and modulation by glycosylation. Proteolytic processing regulates cytokine and chemokine activities. In this study, we report that natural posttranslational citrullination or deimination alters the biological activities of the neutrophil chemoattractant and angiogenic cytokine CXCL8/interleukin-8 (IL-8). Citrullination of arginine in position 5 was discovered on 14% of natural leukocyte-derived CXCL8(1–77), generating CXCL8(1–77)Cit5. Peptidylarginine deiminase (PAD) is known to citrullinate structural proteins, and it may initiate autoimmune diseases. PAD efficiently and site-specifically citrullinated CXCL5, CXCL8, CCL17, CCL26, but not IL-1β. In comparison with CXCL8(1–77), CXCL8(1–77)Cit5 had reduced affinity for glycosaminoglycans and induced less CXCR2-dependent calcium signaling and extracellular signal-regulated kinase 1/2 phosphorylation. In contrast to CXCL8(1–77), CXCL8(1–77)Cit5 was resistant to thrombin- or plasmin-dependent potentiation into CXCL8(6–77). Upon intraperitoneal injection, CXCL8(6–77) was a more potent inducer of neutrophil extravasation compared with CXCL8(1–77). Despite its retained chemotactic activity in vitro, CXCL8(1–77)Cit5 was unable to attract neutrophils to the peritoneum. Finally, in the rabbit cornea angiogenesis assay, the equally potent CXCL8(1–77) and CXCL8(1–77)Cit5 were less efficient angiogenic molecules than CXCL8(6–77). This study shows that PAD citrullinates the chemokine CXCL8, and thus may dampen neutrophil extravasation during acute or chronic inflammation.

Inhibition of Experimental Angiogenesis of Cornea by Various Doses of Doxycycline and Combination of Triamcinolone Acetonide With Low-molecular-weight Heparin and Doxycycline

To evaluate the effect of topically administered doxycycline in various doses; the combination of triamcinolone acetonide and low-molecular-weight heparin (LMWH); and the combination of triamcinolone acetonide and doxycycline on experimental corneal neovascularization in rats. This project is the combination of 2 separate studies. First, the chemical cauterization of corneas in 36 eyes of 36 Long Evans male rats was performed by using silver nitrate/potassium nitrate sticks. Topical instillation of doxycycline at 0.05% (pH = 3.3), 0.1% (pH = 3.1), 1% (pH = 2.3), 2% (pH = 2.1), 2% (pH neutralized to 7.4), and normal saline continued for 7 days. Second, the chemical cauterization of the corneas in 24 eyes of 24 rats was achieved by application of silver nitrate/potassium nitrate sticks. Topical instillation of triamcinolone acetonide (10 microg/mL) and either LMWH (10 mg/mL) or doxycycline (10 mg/mL) was compared with normal saline treatment of 7 days. For both studies, the percent area of the cornea covered by neovascularization and scar in each group was calculated separately by using computer software on digital photographs. All corneas were evaluated histopathologically in study and control groups. The mean percent area of corneal neovascularization determined in the eyes given doxycycline 0.05%, 0.1%, 1%, 2%, and 2% (pH neutralized) study groups and control groups was 69.8% +/- 18.0%, 64.5% +/- 14.0%, 56.4% +/- 20.8%, 54.8% +/- 6.0%, 36.2% +/- 4.3%, and 69.4% +/- 5.7%, respectively. The mean of percent area of neovascularization in the 2% doxycycline (pH neutralized) doxycycline group was significantly less than that of the control group and the <1% doxycycline concentrations (P < 0.05). The percent corneal neovascularization in the 2% (pH neutralized) doxycycline group was not significantly different from that of the 1% and 2% doxycycline groups (P < 0.05). There was no significant difference in percent area of corneal scar between control and study groups (P > 0.05). The mean percent area of corneal neovascularization in triamcinolone acetonide and LMWH, triamcinolone acetonide and doxycycline, and control groups was 2.35% +/- 4.42%, 9.42% +/- 6.8%, and 64.7% +/- 10.0%, respectively. The mean percent area of neovascularization in the triamcinolone acetonide plus LMWH or triamcinolone acetonide plus doxycycline groups was significantly different from that of the control group (P = 0.001 for both). There was no significant difference between study groups with regard to percent area of neovascularization or percent area of corneal scar between the control and study groups. Topically administered combinations of triamcinolone acetonide plus LMWH or triamcinolone acetonide plus doxycycline had effects that contributed to efficient suppression of corneal neovascularization; these drugs were ineffective at similar concentrations used alone. Topically administered 2% (pH neutralized) doxycycline has antiangiogenic effects, which contributed to significant suppression on corneal neovascularization. This drug may be therapeutically beneficial in treatment of corneal neovascularization in clinical trials.

Inhibition of corneal neovascularization with endostatin delivered by adeno-associated viral (AAV) vector in a mouse corneal injury model

The use of a recombinant adeno-associated viral (rAAV) vector carrying endostatin gene as an anti-angiogenesis strategy to treat corneal neovascularization in a mouse model was evaluated. Subconjunctival injection of recombinant endostatin-AAV was used to examine the inhibition of corneal neovascularization induced by silver nitrate cauterization in mice. The results showed that gene expression in corneal tissue was observed as early as 4 days after gene transfer and stably lasted for over 8 months with minimal immune reaction. Subconjunctival injection of a high-titer rAAV-endostatin successfully inhibited neovascularization. Immunohistchemistry staining of CD 31 and endostatin showed that the treatment significantly inhibits angiogenesis in cornea. We concluded that the rAAV was capable of directly delivering genes to the ocular surface epithelium by way of subconjunctival injection and was able to deliver sustained, high levels of gene expression in vivo to inhibit angiogenesis.