Endogenous LXA4 Circuits Are Determinants of Pathological Angiogenesis in Response to Chronic Injury
2009; Elsevier BV; Volume: 176; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2010.090678
ISSN1525-2191
AutoresA. J. Leedom, Aaron B. Sullivan, Baiyan Dong, Denise Lau, Karsten Gronert,
Tópico(s)Biomarkers in Disease Mechanisms
ResumoInflammation and angiogenesis are intimately linked, and their dysregulation leads to pathological angiogenesis in human diseases. 15-lipoxygenase (15-LOX) and lipoxin A4 receptors (ALX) constitute a LXA4 circuit that is a key feature of inflammatory resolution. LXA4 analogs have been shown to regulate vascular endothelial growth factor (VEGF)-A-induced angiogenic response in vitro. 15-LOX and ALX are highly expressed in the avascular and immune-privileged cornea. However, the role of this endogenous LXA4 circuit in pathological neovascularization has not been determined. We report that suture-induced chronic injury in the cornea triggered polymorphonuclear leukocytes (PMN) infiltration, pathological neovascularization, and up-regulation of mediators of inflammatory angiogenesis, namely VEGF-A and the VEGF-3 receptor (FLT4). Up-regulation of the VEGF circuit and neovascularization correlated with selective changes in both 15-LOX (Alox15) and ALX (Fpr-rs2) expression and a temporally defined increase in basal 15-LOX activity. More importantly, genetic deletion of 15-LOX or 5-LOX, key and obligatory enzymes in the formation of LXA4, respectively, led to exacerbated inflammatory neovascularization coincident with increased VEGF-A and FLT4 expression. Direct topical treatment with LXA4, but not its metabolic precursor 15-hydroxyeicosatetraenoic acid, reduced expression of VEGF-A and FLT4 and inflammatory angiogenesis and rescued 15-LOX knockout mice from exacerbated angiogenesis. In summary, our findings and the prominent expression of 15-LOX and ALX in epithelial cells and macrophages place the LXA4 circuit as an endogenous regulator of pathological angiogenesis. Inflammation and angiogenesis are intimately linked, and their dysregulation leads to pathological angiogenesis in human diseases. 15-lipoxygenase (15-LOX) and lipoxin A4 receptors (ALX) constitute a LXA4 circuit that is a key feature of inflammatory resolution. LXA4 analogs have been shown to regulate vascular endothelial growth factor (VEGF)-A-induced angiogenic response in vitro. 15-LOX and ALX are highly expressed in the avascular and immune-privileged cornea. However, the role of this endogenous LXA4 circuit in pathological neovascularization has not been determined. We report that suture-induced chronic injury in the cornea triggered polymorphonuclear leukocytes (PMN) infiltration, pathological neovascularization, and up-regulation of mediators of inflammatory angiogenesis, namely VEGF-A and the VEGF-3 receptor (FLT4). Up-regulation of the VEGF circuit and neovascularization correlated with selective changes in both 15-LOX (Alox15) and ALX (Fpr-rs2) expression and a temporally defined increase in basal 15-LOX activity. More importantly, genetic deletion of 15-LOX or 5-LOX, key and obligatory enzymes in the formation of LXA4, respectively, led to exacerbated inflammatory neovascularization coincident with increased VEGF-A and FLT4 expression. Direct topical treatment with LXA4, but not its metabolic precursor 15-hydroxyeicosatetraenoic acid, reduced expression of VEGF-A and FLT4 and inflammatory angiogenesis and rescued 15-LOX knockout mice from exacerbated angiogenesis. In summary, our findings and the prominent expression of 15-LOX and ALX in epithelial cells and macrophages place the LXA4 circuit as an endogenous regulator of pathological angiogenesis. Formation of a new functional microvasculature, neovascularization, is a fundamental response to ischemia and a salient feature of wound healing. The primary function of newly formed blood vessels is to increase tissue oxygen tension and delivery of essential nutrients and effector cells to restore normal function. However, aberrant neovascularization is a hallmark feature of chronic inflammation and is associated with numerous pathological conditions that include diabetic retinopathy, Crohn's Disease, atherosclerosis, and cancer.1Folkman J Angiogenesis in cancer, vascular, rheumatoid and other disease.Nat Med. 1995; 1: 27-31Crossref PubMed Scopus (7209) Google Scholar, 2Folkman J Angiogenesis: an organizing principle for drug discovery?.Nat Rev Drug Discov. 2007; 6: 273-286Crossref PubMed Scopus (1875) Google Scholar, 3Costa C Incio J Soares R Angiogenesis and chronic inflammation: cause or consequence?.Angiogenesis. 2007; 10: 149-166Crossref PubMed Scopus (346) Google Scholar The growth of microvessels from existing vessels, angiogenesis, is tightly controlled by a range of angiogenic factors and inhibitors; a circuit that is highly evolved in avascular tissues such as the cornea. The vascular endothelial growth factor (VEGF) family of angiogenic factors and their receptors are key mediators of this process, which has led to the recent development and clinical use of anti-VEGF strategies for the treatment of pathological neovascularization in the retina, colon cancer, and lung cancer.2Folkman J Angiogenesis: an organizing principle for drug discovery?.Nat Rev Drug Discov. 2007; 6: 273-286Crossref PubMed Scopus (1875) Google Scholar, 3Costa C Incio J Soares R Angiogenesis and chronic inflammation: cause or consequence?.Angiogenesis. 2007; 10: 149-166Crossref PubMed Scopus (346) Google Scholar, 4Noel A Jost M Lambert V Lecomte J Rakic JM Anti-angiogenic therapy of exudative age-related macular degeneration: current progress and emerging concepts.Trends Mol Med. 2007; 13: 345-352Abstract Full Text Full Text PDF PubMed Scopus (46) Google Scholar, 5Cao Y Langer R A review of Judah Folkman's remarkable achievements in biomedicine.Proc Natl Acad Sci USA. 2008; 105: 13203-13205Crossref PubMed Scopus (48) Google Scholar Many insights into the endogenous role of the VEGF network have been gained by using the cornea,5Cao Y Langer R A review of Judah Folkman's remarkable achievements in biomedicine.Proc Natl Acad Sci USA. 2008; 105: 13203-13205Crossref PubMed Scopus (48) Google Scholar, 6Gimbrone Jr, MA Cotran RS Leapman SB Folkman J Tumor growth and neovascularization: an experimental model using the rabbit cornea.J Natl Cancer Inst. 1974; 52: 413-427Crossref PubMed Scopus (641) Google Scholar which maintains an immune-privileged and avascular state despite expression of VEGF-A and its immediate proximity to the vasculature.7Streilein JW Ocular immune privilege: therapeutic opportunities from an experiment of nature.Nat Rev Immunol. 2003; 3: 879-889Crossref PubMed Scopus (595) Google Scholar Specifically, recent findings have demonstrated that avascularity of the cornea requires expression of a soluble VEGF receptor-1 (sFLT1), which traps VEGF-A.8Ambati BK Nozaki M Singh N Takeda A Jani PD Suthar T Albuquerque RJ Richter E Sakurai E Newcomb MT Kleinman ME Caldwell RB Lin Q Ogura Y Orecchia A Samuelson DA Agnew DW St Leger J Green WR Mahasreshti PJ Curiel DT Kwan D Marsh H Ikeda S Leiper LJ Collinson JM Bogdanovich S Khurana TS Shibuya M Baldwin ME Ferrara N Gerber HP De Falco S Witta J Baffi JZ Raisler BJ Ambati J Corneal avascularity is due to soluble VEGF receptor-1.Nature. 2006; 443: 993-997Crossref PubMed Scopus (562) Google Scholar In addition, the receptor for VEGF-C/VEGF-D, namely VEGF receptor-3 (VEGFR-3, FLT4), is a critical regulator of inflammatory neovascularization.9Chen L Hamrah P Cursiefen C Zhang Q Pytowski B Streilein JW Dana MR Vascular endothelial growth factor receptor-3 mediates induction of corneal alloimmunity. 2004.Ocul Immunol Inflamm. 2007; 15: 275-278Crossref PubMed Scopus (9) Google Scholar, 10Cursiefen C Chen L Saint-Geniez M Hamrah P Jin Y Rashid S Pytowski B Persaud K Wu Y Streilein JW Dana R Nonvascular VEGF receptor 3 expression by corneal epithelium maintains avascularity and vision.Proc Natl Acad Sci USA. 2006; 103: 11405-11410Crossref PubMed Scopus (226) Google Scholar, 11Cursiefen C Ikeda S Nishina PM Smith RS Ikeda A Jackson D Mo JS Chen L Dana MR Pytowski B Kruse FE Streilein JW Spontaneous corneal hem- and lymphangiogenesis in mice with destrin-mutation depend on VEGFR3 signaling.Am J Pathol. 2005; 166: 1367-1377Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar FLT4 is of special of interest because its essential expression during development becomes restricted primarily to lymph vessels and activation of this endothelial receptor is a critical step in initiating lymphangiogenesis. However, FLT4 expression is also up-regulated in microvessels of tumors and wounds, and in macrophages and in addition is constitutively expressed in corneal epithelial cells.3Costa C Incio J Soares R Angiogenesis and chronic inflammation: cause or consequence?.Angiogenesis. 2007; 10: 149-166Crossref PubMed Scopus (346) Google Scholar, 10Cursiefen C Chen L Saint-Geniez M Hamrah P Jin Y Rashid S Pytowski B Persaud K Wu Y Streilein JW Dana R Nonvascular VEGF receptor 3 expression by corneal epithelium maintains avascularity and vision.Proc Natl Acad Sci USA. 2006; 103: 11405-11410Crossref PubMed Scopus (226) Google Scholar, 11Cursiefen C Ikeda S Nishina PM Smith RS Ikeda A Jackson D Mo JS Chen L Dana MR Pytowski B Kruse FE Streilein JW Spontaneous corneal hem- and lymphangiogenesis in mice with destrin-mutation depend on VEGFR3 signaling.Am J Pathol. 2005; 166: 1367-1377Abstract Full Text Full Text PDF PubMed Scopus (51) Google Scholar, 12Tammela T Zarkada G Wallgard E Murtomaki A Suchting S Wirzenius M Waltari M Hellstrom M Schomber T Peltonen R Freitas C Duarte A Isoniemi H Laakkonen P Christofori G Yla-Herttuala S Shibuya M Pytowski B Eichmann A Betsholtz C Alitalo K Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation.Nature. 2008; 454: 656-660Crossref PubMed Scopus (659) Google Scholar, 13Paavonen K Mandelin J Partanen T Jussila L Li TF Ristimaki A Alitalo K Konttinen YT Vascular endothelial growth factors C and D and their VEGFR-2 and 3 receptors in blood and lymphatic vessels in healthy and arthritic synovium.J Rheumatol. 2002; 29: 39-45PubMed Google Scholar A recent report demonstrates that FLT4 is highly expressed in angiogenic sprouts and is a critical regulator of sustained heme-angiogenesis,12Tammela T Zarkada G Wallgard E Murtomaki A Suchting S Wirzenius M Waltari M Hellstrom M Schomber T Peltonen R Freitas C Duarte A Isoniemi H Laakkonen P Christofori G Yla-Herttuala S Shibuya M Pytowski B Eichmann A Betsholtz C Alitalo K Blocking VEGFR-3 suppresses angiogenic sprouting and vascular network formation.Nature. 2008; 454: 656-660Crossref PubMed Scopus (659) Google Scholar which underscores the potential key role of this receptor in pathological neovascularization. Inflammation is intimately associated with neovascularization especially during wound healing and ischemic injury. Lipid autacoids are some of the earliest signals that are released in response to injury or insult. In this regard, the 15-lipoxygenase pathway14Kuhn H O'Donnell VB Inflammation and immune regulation by 12/15-lipoxygenases.Prog Lipid Res. 2006; 45: 334-356Crossref PubMed Scopus (306) Google Scholar, 15Gronert K Lipoxins in the eye and their role in wound healing.Prostaglandins Leukot Essent Fatty Acids. 2005; 73: 221-229Abstract Full Text Full Text PDF PubMed Scopus (50) Google Scholar is of interest as it is one of the most inducible genes in macrophages and highly expressed in mucosal and corneal epithelial cells. Macrophages and epithelial cells are important regulators of angiogenesis, especially in avascular tissue such as the cornea.3Costa C Incio J Soares R Angiogenesis and chronic inflammation: cause or consequence?.Angiogenesis. 2007; 10: 149-166Crossref PubMed Scopus (346) Google Scholar, 10Cursiefen C Chen L Saint-Geniez M Hamrah P Jin Y Rashid S Pytowski B Persaud K Wu Y Streilein JW Dana R Nonvascular VEGF receptor 3 expression by corneal epithelium maintains avascularity and vision.Proc Natl Acad Sci USA. 2006; 103: 11405-11410Crossref PubMed Scopus (226) Google Scholar, 16Cursiefen C Chen L Borges LP Jackson D Cao J Radziejewski C D'Amore PA Dana MR Wiegand SJ Streilein JW VEGF-A stimulates lymphangiogenesis and hemangiogenesis in inflammatory neovascularization via macrophage recruitment.J Clin Invest. 2004; 113: 1040-1050Crossref PubMed Scopus (863) Google Scholar Macrophages have a central and well-documented role in angiogenesis, especially in tumors and inflammatory neovascularization. In the cornea, a well-established model tissue for studying inflammatory neovascularization, VEGF-A recruits macrophages, the major cell type to generate VEGF, which drives inflammatory heme and lymphangiogenesis. Corneal epithelial cells constitutively express the receptor for VEGF-C/VEGF-D (ie, FLT4), which has been proposed as a critical pathway for regulating inflammatory neovascularization.10Cursiefen C Chen L Saint-Geniez M Hamrah P Jin Y Rashid S Pytowski B Persaud K Wu Y Streilein JW Dana R Nonvascular VEGF receptor 3 expression by corneal epithelium maintains avascularity and vision.Proc Natl Acad Sci USA. 2006; 103: 11405-11410Crossref PubMed Scopus (226) Google Scholar Human 15-LOX (ALOX15 and ALOX15B) generate 15S-hydroxyeicosatetraenoic acid (HETE) and mouse 12/15-LOX (Alox15) generates 15S-HETE and 12S-HETE from arachidonic acid. 15-HETE and 12-HETE have been shown to induce proliferation, migration, and tube formation in endothelial cells.17Nie D Tang K Diglio C Honn KV Eicosanoid regulation of angiogenesis: role of endothelial arachidonate 12-lipoxygenase.Hemost Thromb Vasc Biol. 2000; 95: 2304-2311Google Scholar More importantly, 15-HETE is a key intermediate in the formation of the well-studied anti-inflammatory mediator lipoxin A4 (LXA4) that is generated via the rate-limiting enzyme 5-lipoxygenase (5-LOX). A body of work18Bannenberg GL Aliberti J Hong S Sher A Serhan C Exogenous pathogen and plant 15-lipoxygenase initiate endogenous lipoxin a4 biosynthesis.J Exp Med. 2004; 199: 515-523Crossref PubMed Scopus (82) Google Scholar, 19Brezinski ME Serhan CN Selective incorporation of (15S)-hydroxyeicosatetraenoic acid in phosphatidylinositol of human neutrophils: agonist-induced deacylation and transformation of stored hydroxyeicosanoids.Proc Natl Acad Sci USA. 1990; 87: 6248-6252Crossref PubMed Scopus (153) Google Scholar, 20Levy BD Romano M Chapman HA Reilly JJ Drazen J Serhan CN Human alveolar macrophages have 15-lipoxygenase and generate 15(S)-hydroxy-5,8,11-cis-13-trans-eicosatetraenoic acid and lipoxins.J Clin Invest. 1993; 92: 1572-1579Crossref PubMed Scopus (176) Google Scholar, 21Serhan CN Jain A Marleau S Clish C Kantarci A Behbehani B Colgan SP Stahl GL Merched A Petasis NA Chan L Van Dyke TE Reduced inflammation and tissue damage in transgenic rabbits overexpressing 15-lipoxygenase and endogenous anti-inflammatory lipid mediators.J Immunol. 2003; 171: 6856-6865PubMed Google Scholar, 22Levy BD Clish CB Schmidt B Gronert K Serhan CN Lipid mediator class switching during acute inflammation: signals in resolution.Nat Immunol. 2001; 2: 612-619Crossref PubMed Scopus (1098) Google Scholar, 23Nassar GM Morrow JD Roberts II, LJ Lakkis FG Badr KF Induction of 15-lipoxygenase by interleukin-13 in human blood monocytes.J Biol Chem. 1994; 269: 27631-27634Abstract Full Text PDF PubMed Google Scholar, 24Chavis C Godard P Crastes de Paulet A Damon M Formation of lipoxins and leukotrienes by human alveolar macrophages incubated with 15(S)-HETE: a model for cellular cooperation between macrophages and airway epithelial cells.Eicosanoids. 1992; 5: 203-211PubMed Google Scholar has established that the anti-inflammatory actions, which are associated with the up-regulation of 15-LOX and/or 15S-HETE formation are mediated by LXA4 and its G-protein coupled receptor ALX. Recent reports have demonstrated that stable analogs of LXA4 inhibit VEGF induced angiogenic responses in endothelial cells.25Cezar-de-Mello PF Nascimento-Silva V Villela CG Fierro IM Aspirin-triggered Lipoxin A4 inhibition of VEGF-induced endothelial cell migration involves actin polymerization and focal adhesion assembly.Oncogene. 2006; 25: 122-129PubMed Google Scholar, 26Cezar-de-Mello PF Vieira AM Nascimento-Silva V Villela CG Barja-Fidalgo C Fierro IM ATL-1, an analogue of aspirin-triggered lipoxin A4, is a potent inhibitor of several steps in angiogenesis induced by vascular endothelial growth factor.Br J Pharmacol. 2008; 153: 956-965Crossref PubMed Scopus (59) Google Scholar, 27Fierro IM Kutok JL Serhan CN Novel lipid mediator regulators of endothelial cell proliferation and migration: aspirin-triggered-15R-lipoxin A(4) and lipoxin A(4).J Pharmacol Exp Ther. 2002; 300: 385-392Crossref PubMed Scopus (151) Google Scholar These metabolically stable analogs are mimetics of aspirin-triggered LXA4, an endogenous isomer whose synthesis can be triggered by aspirin-acetylated cyclooxygenase-2 rather than 15-LOX. This 15-epi-isomer of LXA4 resists metabolic inactivation and mediates it bioactions, like LXA4, via the ALX receptor. The intimate link between inflammation and angiogenesis and the ability of LXA428Baker N O'Meara SJ Scannell M Maderna P Godson C Lipoxin A4: anti-inflammatory and anti-angiogenic impact on endothelial cells.J Immunol. 2009; 182: 3819-3826Crossref PubMed Scopus (79) Google Scholar and analogs of 15-epi LXA425Cezar-de-Mello PF Nascimento-Silva V Villela CG Fierro IM Aspirin-triggered Lipoxin A4 inhibition of VEGF-induced endothelial cell migration involves actin polymerization and focal adhesion assembly.Oncogene. 2006; 25: 122-129PubMed Google Scholar, 26Cezar-de-Mello PF Vieira AM Nascimento-Silva V Villela CG Barja-Fidalgo C Fierro IM ATL-1, an analogue of aspirin-triggered lipoxin A4, is a potent inhibitor of several steps in angiogenesis induced by vascular endothelial growth factor.Br J Pharmacol. 2008; 153: 956-965Crossref PubMed Scopus (59) Google Scholar, 27Fierro IM Kutok JL Serhan CN Novel lipid mediator regulators of endothelial cell proliferation and migration: aspirin-triggered-15R-lipoxin A(4) and lipoxin A(4).J Pharmacol Exp Ther. 2002; 300: 385-392Crossref PubMed Scopus (151) Google Scholar to inhibit VEGF-A induced angiogenic responses in vitro points toward a potential role of endogenous LXA4 circuits in pathological angiogenesis. However, the endogenous role of 15-LOX in the regulation of angiogenesis remains controversial. Reports have demonstrated that the enzyme or its products promote or inhibit angiogenic responses in in vitro studies.29Mochizuki N Kwon YG 15-lipoxygenase-1 in the vasculature: expanding roles in angiogenesis.Circ Res. 2008; 102: 143-145Crossref PubMed Scopus (19) Google Scholar, 30Bajpai AK Blaskova E Pakala SB Zhao T Glasgow WC Penn JS Johnson DA Rao GN 15(S)-HETE production in human retinal microvascular endothelial cells by hypoxia: novel role for MEK1 in 15(S)-HETE induced angiogenesis.Invest Ophthalmol Vis Sci. 2007; 48: 4930-4938Crossref PubMed Scopus (40) Google Scholar, 31Srivastava K Kundumani-Sridharan V Zhang B Bajpai AK Rao GN 15(S)-hydroxyeicosatetraenoic acid-induced angiogenesis requires STAT3-dependent expression of VEGF.Cancer Res. 2007; 67: 4328-4336Crossref PubMed Scopus (38) Google Scholar, 32Viita H Markkanen J Eriksson E Nurminen M Kinnunen K Babu M Heikura T Turpeinen S Laidinen S Takalo T Yla-Herttuala S 15-lipoxygenase-1 prevents vascular endothelial growth factor A- and placental growth factor-induced angiogenic effects in rabbit skeletal muscles via reduction in growth factor mRNA levels. NO bioactivity, and downregulation of VEGF receptor 2 expression.Circ Res. 2008; 102: 177-184Crossref PubMed Scopus (47) Google Scholar More importantly, the in vivo role of the 15-LOX pathway or the LXA4 circuit in pathological neovascularization remains to be clearly defined. To this end, we assessed the role the 15-LOX pathway and LXA4 circuit in chronic injury-induced inflammatory neovascularization. Here, we report that inflammatory neovascularization and up-regulation of the VEGF circuit correlate with changes in both 15-LOX (Alox15) and LXA4 receptor (ALX) expression and temporally defined 15-LOX activity. More importantly, genetic deletion of 15-LOX or 5-LOX, key enzymes in the formation of LXA4, led to amplified neovascularization and expression of VEGF-A and FLT4 in the avascular cornea during chronic injury. LXA4, but not 15S-HETE, attenuated expression of VEGF-A and FLT4 and the angiogenic response, which provides evidence that selective autacoids from the prominent 15-LOX pathway have an endogenous role in limiting pathological neovascularization. 12/15-lipoxygenase (Alox15) and 5-lipoxygenase (Alox5) deficient mice (6 to 10 weeks, female) were purchased from Jackson Laboratory (Bar Harbor, ME). These Jax Gemm strains have a targeted mutation in 12/15-LOX or in 5-LOX that is in a background C57Bl/6J inbred strain. These mice do not express a functional “leukocyte-type” 12/15-LOX (Alox15) or 5-LOX (Alox5).33Funk CD Prostaglandins and leukotrienes: advances in eicosanoid biology.Science. 2001; 294: 1871-1875Crossref PubMed Scopus (3030) Google Scholar, 34Funk CD Chen XS Johnson EN Zhao L Lipoxygenase genes and their targeted disruption.Prostaglandins Other Lipid Mediat. 2002; 68–69: 303-312Crossref PubMed Scopus (147) Google Scholar Age and gender-matched congenic C57Bl/6J stock 000664 mice (6 to 10 weeks, female) were used as controls. Mice were maintained on a 12-hour day/night cycle and fed ad libitum a standard diet (Rat/Mouse diet LM-485, Harlan Tekland, Madison, WI). All animal studies have been approved by the University of California, Berkeley in accordance with the NIH Guide for the Care and Use of Laboratory Animals and in strict accord with the ARVO Statement for the Use of Animals in Ophthalmic and Vision Research. Mice were anesthetized with ketamine (50 mg/kg) and xylazine (20 mg/kg) intraperitoneally and a drop of tetracaine-HCL 0.5% was applied to the eye to deliver local corneal anesthesia before injury. A single sterile 8.0 silk suture was placed intrastromally extending over the corneal apex, without disrupting the iris. Selected mice were treated topically t.i.d. with LXA4 or 15S-HETE (Cayman Chemical, Ann Arbor, MI), or sterile saline alone (PBS, ph 7.4) for two or seven days. Ethanol from the LXA4 and 15S-HETE solutions were rapidly removed under gentle stream of nitrogen and autacoids immediately resuspended in sterile PBS and applied to the eye (5 μl drop, t.i.d.). Eyes were enucleated at the respective time points under a stereo-microscope and corneas carefully dissected on ice to remove the limbus area and all noncorneal tissue. Isolated corneas were either snap frozen for RNA or lipidomic analyses, or immediately processed for immunohistochemistry. Isolated corneas were rinsed in PBS, fixed in acetone (100%) for 30 minutes, blocked in 2% bovine serum albumin/PBS solution and incubated in PBS containing fluorescein isothiocyanate-conjugated CD31/PECAM-1 overnight (Santa Cruz Biotechnology, Santa Cruz, CA; 1:100). Flatmounts were prepared by sectioning the cornea and fixing them to slides. The images were taken with a Zeiss Axiophot laser scanning confocal microscope and neovascularization was quantified by manually tracing the length of all vessels using Image Pro-Express software (Cyber Media) and was expressed as total pixels. Corneal flatmounts from 5-LOX KO mice and their matched congenic wild-type controls were taken with a Zeiss Axioplan 2 microscope equipped with a Ludl motorized stage and z focus. Mosaic images were taken with a AxioCam MR camera and compiled using MosaiX and Zeiss AxioVision 4.5 software. Myeloperoxidase (MPO) activity, an index of tissue leukocyte infiltration, was measured 48 hours and 7 days post injury.35Biteman B Hassan IR Walker E Leedom AJ Dunn M Seta F Laniado-Schwartzman M Gronert K Interdependence of lipoxin A4 and heme-oxygenase in counter-regulating inflammation during corneal wound healing.FASEB J. 2007; 21: 2257-2266Crossref PubMed Scopus (80) Google Scholar, 36Gronert K Maheshwari N Khan N Hassan IR Dunn M Laniado Schwartzman M A role for the mouse 12/15-lipoxygenase pathway in promoting epithelial wound healing and host defense.J Biol Chem. 2005; 280: 15267-15278Crossref PubMed Scopus (254) Google Scholar In brief, corneas (1 cornea/data point) were homogenized with a hand held tissue grinder in 450 μl of 50 mmol/L potassium phosphate buffer containing 0.5% hexadecyltrimethylammonium bromide (pH 6.0). This was followed by sonication, freeze-thaw three times and a second sonication. The homogenates were then centrifuged and supernatants collected. MPO activity in supernatants was measured by spectrophotometry using o-dianisidine dihydrochloride oxidation as a colorimetric indicator. Calibration curves for MPO activities were established with PMN collected from 12 hours zymosan A-induced peritonitis exudates in mice. RNA from mouse corneas was isolated using RNA Easy Mini Kit (Qiagen Sciences, Maryland). RNA integrity was verified using agarose gel electrophoresis and quantified by spectrophotometry. RNA was reverse transcribed using a High-Capacity cDNA Kit (Applied Biosystems, Foster City, CA). Heme oxygenase (HO)-1 was amplified using 5′-GATAGAGCGCAACAAGCAGAA-3′ and 3′-CAGTGAGGCCCATACCAGAA-5′; VEGFA 5′-TCACCAAAGCCAGCACATAGGAGA-3′ and 3′-TTCGTTTAACTCAAGCTGCCTCGC-5′; Fprl1 5′-CATTTGGTTGGTTCATGTGCAA-3′ and 3′-AATACAGCGGTCCAGTGCAAT-5′; Fpr-rs2 5′-GCCAGG ACTTTCGTGGAGAGAT-3′ and 3′-GATGAACTGGTGCTTGAATCACT-5′; VEGF-R3 (FLT-4) 5′-CTGGCAAATGGTTACTCCATGA-3′ and 3′-ACAACCCGTGTGTCTTCACTG-5′; 12/15 LOX (Alox15) 5′-GCGACGCTGCCCAATCCTAATC-3′ and 3′-ATATGGCCACGCTGTTTTCTACC-5′; and soluble flt1 5′-AGGTGAGCACTGCGGCA-3′ and 3′-ATGAGTCCTTTAATGTTTGAC-5′. Nucleotide primers were selected from the Harvard Primer Bank (pga.mgh.harvard.edu/primerbank/) and verified by the NIH GenBank database or have recently been reported.35Biteman B Hassan IR Walker E Leedom AJ Dunn M Seta F Laniado-Schwartzman M Gronert K Interdependence of lipoxin A4 and heme-oxygenase in counter-regulating inflammation during corneal wound healing.FASEB J. 2007; 21: 2257-2266Crossref PubMed Scopus (80) Google Scholar, 36Gronert K Maheshwari N Khan N Hassan IR Dunn M Laniado Schwartzman M A role for the mouse 12/15-lipoxygenase pathway in promoting epithelial wound healing and host defense.J Biol Chem. 2005; 280: 15267-15278Crossref PubMed Scopus (254) Google Scholar, 37Hassan IR Gronert K Acute changes in dietary omega-3 and omega-6 polyunsaturated fatty acids have a pronounced impact on survival following ischemic renal injury and formation of renoprotective docosahexaenoic acid-derived protectin D1.J Immunol. 2009; 182: 3223-3232Crossref PubMed Scopus (98) Google Scholar, 38Seta F Bellner L Rezzani R Regan RF Dunn MW Abraham NG Gronert K Laniado-Schwartzman M Heme oxygenase-2 is a critical determinant for execution of an acute inflammatory and reparative response.Am J Pathol. 2006; 169: 1612-1623Abstract Full Text Full Text PDF PubMed Scopus (78) Google Scholar, 39Jin Y Arita M Zhang Q Saban DR Chauhan SK Chiang N Serhan CN Dana R Novel anti-inflammatory and pro-resolving lipid mediators block inflammatory angiogenesis.Invest Ophthalmol Vis Sci. 2009; 50: 4743-4752Crossref PubMed Scopus (139) Google Scholar Real-time PCR was performed using Fast SYBR Green Master Mix (Applied Biosystems) with a Step One Plus QPCR system (Applied Biosystems). Amplifications were run in duplicates and efficiencies for each primer pair were established. Comparative quantification of gene expression was performed by Step One software (Applied Biosystems) using the ΔΔCT method. Expression of all genes is referenced to a positive mRNA control that was generated by pooling mRNA from C57Bl/6J mouse spleen and kidney. For endogenous lipid autacoid analysis, corneas were homogenized with a hand-held tissue grinder in 66% methanol (4°C). The methanol contained deuterated internal standards, prostaglandin (PG)E2-d4, 15(S)- HETE-d8, and leukotriene B4 (LTB4)-d4 (400 pg/each), to calculate the recovery of prostanoids or mono-hydroxy- and dihydroxy-containing fatty acids. Lipid autacoids were extracted by solid phase using Accubond ODS-C18 cartridges (Agilent Technologies, Santa Clara, CA)40Gronert K Clish CB Romano M Serhan CN Transcellular regulation of eicosanoid biosynthesis.Methods Mol Biol. 1999; 120: 119-144PubMed Google Scholar and the average extraction recovery of our class specific deuterated internal standard was 81% for PGE2-d4, 60% for LTB4-d4, and 63% for 15-HETE-d8. Eicosanoids were identified and quantified by liquid chromatography (LC)/mass spectrometry (MS)/MS-based lipidomics.37Hassan IR Gronert K Acute changes in dietary omega-3 and omega-6 polyunsaturated fatty acids have a pronounced impact on survival following ischemic renal injury and formation of renoprotective docosahexaenoic acid-derived protectin D1.J Immunol. 2009; 182: 3223-3232Crossref PubMed Scopus (98) Google Scholar, 41Serhan CN Lu Y Hong S Yang R Mediator lipidomics: search algorithms for eicosanoids, resolvins, and protectins.Methods Enzymol. 2007; 432: 275-317Crossref PubMed Scopus (43) Google Scholar, 42Murphy RC Barkley RM Zemski Berry K Hankin J Harrison K Johnson C Krank J McAnoy A Uhlson C Zarini S Electrospray ionization and tandem mass spectrometry of eicosanoids.Anal Biochem. 2005; 346: 1-42Crossref PubMed Scopus (188) Google Scholar, 43Gonzalez-Periz A Horrillo R Ferre N Gronert K Dong B Moran-Salvador E Titos E Martinez-Clemente M Lopez-Parra M Arroyo V Claria J Obesity-induced insulin resistance and hepatic steatosis are alleviated by omega-3 fatty acids: a role for resolvins and protectins.FASEB J. 2009; 23: 1946-1957Crossref PubMed Scopus (469) Google Scholar, 44Chiang N Takano T Clish CB Petasis NA Tai H-H Serhan CN Aspirin-triggered 15-epi-lipoxin A4 (ATL) generation by human leukocytes and murine peritonitis exudates: development of a specific 15-epi-LXA4 ELISA.J Pharmacol Exp Ther. 1998; 287: 779-790PubMed Google Scholar, 45Clish CB Levy BD Chiang N Tai HH Serhan CN Oxidoreductases in lipox
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