Voltar
Artigo Acesso aberto Revisado por pares
BIBTEX RIS

Retinal Neurons Curb Inflammation and Enhance Revascularization in Ischemic Retinopathies via Proteinase-Activated Receptor-2

2014; Elsevier BV; Volume: 185; Issue: 2 Linguagem: Inglês

10.1016/j.ajpath.2014.10.020

ISSN

1525-2191

Autores

Nicholas Sitaras, José Carlos Rivera, Baraa Noueihed, Milsa Bien-Aimé, Karine Zaniolo, Samy Omri, David Hamel, Tang Zhu, Pierre Hardy, Przemysław Sapieha, Jean‐Sébastien Joyal, Sylvain Chemtob,

Tópico(s)

Retinal and Optic Conditions

Resumo

Ischemic retinopathies are characterized by sequential vaso-obliteration followed by abnormal intravitreal neovascularization predisposing patients to retinal detachment and blindness. Ischemic retinopathies are associated with robust inflammation that leads to generation of IL-1β, which causes vascular degeneration and impairs retinal revascularization in part through the liberation of repulsive guidance cue semaphorin 3A (Sema3A). However, retinal revascularization begins as inflammation culminates in ischemic retinopathies. Because inflammation leads to activation of proteases involved in the formation of vasculature, we hypothesized that proteinase-activated receptor (Par)-2 (official name F2rl1) may modulate deleterious effects of IL-1β. Par2, detected mostly in retinal ganglion cells, was up-regulated in oxygen-induced retinopathy. Surprisingly, oxygen-induced retinopathy–induced vaso-obliteration and neovascularization were unaltered in Par2 knockout mice, suggesting compensatory mechanisms. We therefore conditionally knocked down retinal Par2 with shRNA-Par2–encoded lentivirus. Par2 knockdown interfered with normal revascularization, resulting in pronounced intravitreal neovascularization; conversely, the Par2 agonist peptide (SLIGRL) accelerated normal revascularization. In vitro and in vivo exploration of mechanisms revealed that IL-1β induced Par2 expression, which in turn down-regulated sequentially IL-1 receptor type I and Sema3A expression through Erk/Jnk-dependent processes. Collectively, our findings unveil an important mechanism by which IL-1β regulates its own endothelial cytotoxic actions by augmenting neuronal Par2 expression to repress sequentially IL-1 receptor type I and Sema3A expression. Timely activation of Par2 may be a promising therapeutic avenue in ischemic retinopathies. Ischemic retinopathies are characterized by sequential vaso-obliteration followed by abnormal intravitreal neovascularization predisposing patients to retinal detachment and blindness. Ischemic retinopathies are associated with robust inflammation that leads to generation of IL-1β, which causes vascular degeneration and impairs retinal revascularization in part through the liberation of repulsive guidance cue semaphorin 3A (Sema3A). However, retinal revascularization begins as inflammation culminates in ischemic retinopathies. Because inflammation leads to activation of proteases involved in the formation of vasculature, we hypothesized that proteinase-activated receptor (Par)-2 (official name F2rl1) may modulate deleterious effects of IL-1β. Par2, detected mostly in retinal ganglion cells, was up-regulated in oxygen-induced retinopathy. Surprisingly, oxygen-induced retinopathy–induced vaso-obliteration and neovascularization were unaltered in Par2 knockout mice, suggesting compensatory mechanisms. We therefore conditionally knocked down retinal Par2 with shRNA-Par2–encoded lentivirus. Par2 knockdown interfered with normal revascularization, resulting in pronounced intravitreal neovascularization; conversely, the Par2 agonist peptide (SLIGRL) accelerated normal revascularization. In vitro and in vivo exploration of mechanisms revealed that IL-1β induced Par2 expression, which in turn down-regulated sequentially IL-1 receptor type I and Sema3A expression through Erk/Jnk-dependent processes. Collectively, our findings unveil an important mechanism by which IL-1β regulates its own endothelial cytotoxic actions by augmenting neuronal Par2 expression to repress sequentially IL-1 receptor type I and Sema3A expression. Timely activation of Par2 may be a promising therapeutic avenue in ischemic retinopathies. Ischemic retinopathies, such as retinopathy of prematurity and in some circumstances diabetic retinopathy, are the main causes of severe visual impairment in children and working class populations in industrialized countries.1Yau J.W.Y. Rogers S.L. Kawasaki R. Lamoureux E.L. Kowalski J.W. Bek T. et al.The Meta-Analysis for Eye Disease (Meta-eye) Study GroupGlobal prevalence and major risk factors of diabetic retinopathy.Diabetes Care. 2012; 35: 556-564Crossref PubMed Scopus (2742) Google Scholar, 2Gilbert C. Rahi J. Eckstein M. O'Sullivan J. Foster A. Retinopathy of prematurity in middle-income countries.Lancet. 1997; 350: 12-14Abstract Full Text Full Text PDF PubMed Scopus (357) Google Scholar Ischemic retinopathies are biphasic diseases characterized by a degeneration of the retinal vascular bed, resulting in tissue hypoxia that triggers a compensatory albeit anarchic and deregulated vessel proliferation into the vitreous, which in severe instances can result in retinal detachment and blindness.3Sapieha P. Hamel D. Shao Z. Rivera J.C. Zaniolo K. Joyal J.S. Chemtob S. Proliferative retinopathies: angiogenesis that blinds.Int J Biochem Cell Biol. 2010; 42: 5-12Crossref PubMed Scopus (110) Google Scholar Importantly, retinal ischemia from vascular decay also causes neuronal dysfunction and demise.4Fulton A. Hansen R. Moskowitz A. Akula J. The neurovascular retina in retinopathy of prematurity.Prog Retin Eye Res. 2009; 28: 452-482Crossref PubMed Scopus (112) Google Scholar Therefore, therapeutic interventions that help alleviate ischemic stress of the neural retina would be desirable to prevent both vascular and neuronal adverse consequences in patients with ischemic retinopathies. In ischemic retinopathies, oxidative stress elicits microglia and resident macrophages to release inflammatory cytokines,5Demircan N. Safran B.G. Soylu M. Ozcan A.A. Sizmaz S. Determination of vitreous interleukin-1 (il-1) and tumour necrosis factor (tnf) levels in proliferative diabetic retinopathy.Eye (Lond). 2006; 20: 1366-1369Crossref PubMed Scopus (313) Google Scholar, 6Mocan M.C. Kadayifcilar S. Eldem B. Elevated intravitreal interleukin-6 levels in patients with proliferative diabetic retinopathy.Can J Ophthalmol. 2011; 41: 747-752Abstract Full Text PDF Google Scholar, 7Krady J.K. Basu A. Allen C.M. Xu Y. LaNoue K.F. Gardner T.W. Levison S.W. Minocycline reduces proinflammatory cytokine expression, microglial activation, and caspase-3 activation in a rodent model of diabetic retinopathy.Diabetes. 2005; 54: 1559-1565Crossref PubMed Scopus (439) Google Scholar, 8Rivera J.C. Sitaras N. Noueihed B. Hamel D. Madaan A. Zhou T. Honore J. Quiniou C. Joyal J. Hardy P. Sennlaub F. Lubell W. Chemtob S. Microglia and IL-1β in ischemic retinopathy elicit microvascular degeneration through neuronal Semaphorin3A.Arterioscler Thromb Vasc Biol. 2013; 133: 1881-1891Crossref Scopus (106) Google Scholar notably IL-1β, which further recruits and activates inflammatory cells thereby exacerbating retinal inflammation.8Rivera J.C. Sitaras N. Noueihed B. Hamel D. Madaan A. Zhou T. Honore J. Quiniou C. Joyal J. Hardy P. Sennlaub F. Lubell W. Chemtob S. Microglia and IL-1β in ischemic retinopathy elicit microvascular degeneration through neuronal Semaphorin3A.Arterioscler Thromb Vasc Biol. 2013; 133: 1881-1891Crossref Scopus (106) Google Scholar, 9dell'Omo R. Semeraro F. Bamonte G. Cifariello F. Romano M.R. Costagliola C. Vitreous mediators in retinal hypoxic diseases.Mediators Inflamm. 2013; : 1-16Crossref Scopus (41) Google Scholar, 10Ishida S. Yamashiro K. Usui T. Kaji Y. Ogura Y. Hia T. Honda Y. Oguchi Y. Adamis A. Leukocytes mediate retinal vascular remodeling during dvelopment and vaso-obliteration in disease.Nat Med. 2003; 9: 781-789Crossref PubMed Scopus (197) Google Scholar We found in a murine model of oxygen-induced retinopathy (OIR) that hyperoxia-activated microglia secretes IL-1β through an autocrine feedback loop.8Rivera J.C. Sitaras N. Noueihed B. Hamel D. Madaan A. Zhou T. Honore J. Quiniou C. Joyal J. Hardy P. Sennlaub F. Lubell W. Chemtob S. Microglia and IL-1β in ischemic retinopathy elicit microvascular degeneration through neuronal Semaphorin3A.Arterioscler Thromb Vasc Biol. 2013; 133: 1881-1891Crossref Scopus (106) Google Scholar IL-1β, in turn, induces the release of repulsive cue semaphorin 3A (Sema3A) from neighboring retinal ganglion cells (RGCs), which contributes to vaso-obliteration8Rivera J.C. Sitaras N. Noueihed B. Hamel D. Madaan A. Zhou T. Honore J. Quiniou C. Joyal J. Hardy P. Sennlaub F. Lubell W. Chemtob S. Microglia and IL-1β in ischemic retinopathy elicit microvascular degeneration through neuronal Semaphorin3A.Arterioscler Thromb Vasc Biol. 2013; 133: 1881-1891Crossref Scopus (106) Google Scholar and hinders normal revascularization.11Joyal J.S. Sitaras N. Binet F. Rivera J.C. Stahl A. Zaniolo K. Shao Z. Polosa A. Zhu T. Hamel D. Djavari M. Kunik D. Honoré J.C. Picard E. Zabeida A. Varma D.R. Hickson G. Mancini J. Klagsbrun M. Costantino S. Beauséjour C. Lachapelle P. Smith L.E. Chemtob S. Sapieha P. Ischemic neurons prevent vascular regeneration of neural tissue by secreting semaphorin 3A.Blood. 2011; 117: 6024-6035Crossref PubMed Scopus (148) Google Scholar This self-sustained and potentially unrelenting destructive inflammatory cascade, if kept unchecked, could result in severe obliteration of the microvascular network of the retina; however, this is not the case because revascularization eventually occurs, albeit slowly (during several days).12Dorfman A. Dembinska O. Chemtob S. Lachapelle P. Early manifestations of postnatal hyperoxia on the retinal structure and function of the neonatal rat.Invest Ophthalmol Vis Sci. 2008; 49: 458-466Crossref PubMed Scopus (40) Google Scholar Classic homologous down-regulation of IL receptor type I (Il-1RI) by its own natural ligand has not been observed; in fact, the contrary generally occurs.13Teshima S. Nakanishi H. Nishizawa M. Kitagawa K. Kaibori M. Yamada M. Habara K. Kwon A. Kamiyama Y. Ito S. Okumura T. Up-regulation of IL-1 receptor through PI3K/Akt is essential for the induction of iNOS gene expression in hepatocytes.J Hepatol. 2004; 40: 616-623Abstract Full Text Full Text PDF PubMed Scopus (66) Google Scholar, 14Skundric D.S. Bealmear B. Lisak R.P. Induced upregulation of IL-1, IL-1RA and IL-1R type I gene expression by Schwann cells.J Neuroimmunol. 1997; 74: 9-18Abstract Full Text Full Text PDF PubMed Scopus (39) Google Scholar Thus, it is reasonable to postulate the existence of an alternate intrinsic regulatory mechanism that counteracts the self-sustained and potentially harmful IL-1β–induced inflammatory cascade. Inflammation is associated with the release and activation of various proteases involved in ensued vascularization.15Kowluru R. Zhong Q. Santos J. Matrix metalloproteinases in diabetic retinopathy: potential role of MMP-9.Expert Opin Investig Drugs. 2012; 21: 797-805Crossref PubMed Scopus (123) Google Scholar Proteinase-activated receptors (Pars) are G protein–coupled receptors activated by circulating proteases via cleavage of their N-terminus, thus revealing a tethered ligand, which subsequently binds to their second extracellular loop to elicit intracellular signaling.16Soh U.J.K. Dores M.R. Chen B. Trejo J. Signal transduction by protease-activated receptors.Br J Pharmacol. 2010; 160: 191-203Crossref PubMed Scopus (232) Google Scholar Of the four members of the proteinase-activated receptor (Par) family (Par1 through Par4),16Soh U.J.K. Dores M.R. Chen B. Trejo J. Signal transduction by protease-activated receptors.Br J Pharmacol. 2010; 160: 191-203Crossref PubMed Scopus (232) Google Scholar Par2 (official name F2rl1) is recognized for its marked proangiogenic properties in the retina.17Uusitalo-Jarvinen H. Kurokawa T. Mueller B.M. Andrade-Gordon P. Friedlander M. Ruf W. Role of protease activated receptor 1 and 2 signaling in hypoxia-induced angiogenesis.Arterioscler Thromb Vasc Biol. 2007; 27: 1456-1462Crossref PubMed Scopus (98) Google Scholar, 18Belting M. Dorrell M.I. Sandgren S. Aguilar E. Ahamed J. Dorfleutner A. Carmeliet P. Mueller B.M. Friedlander M. Ruf W. Regulation of angiogenesis by tissue factor cytoplasmic domain signaling.Nat Med. 2004; 10: 502-509Crossref PubMed Scopus (312) Google Scholar Interestingly, Par2 is robustly activated by IL-1β in different cell types, including neurons and endothelial cells19Tsai S.H. Sheu M.T. Liang Y.C. Cheng H.T. Fang S.S. Chen C. TGF-β inhibits IL-1β-activated PAR-2 expression through multiple pathways in human primary synovial cells.J Biomed Sci. 2009; 16: 97-111Crossref PubMed Scopus (17) Google Scholar, 20Ritchie E. Saka M. MacKenzie C. Drummond R. Wheeler-Jones C. Kanke T. Plevin R. Cytokine upregulation of proteinase-activated-receptors 2 and 4 expression mediated by p38 MAP kinase and inhibitory kappa B kinase β in human endothelial cells.Br J Pharmacol. 2007; 150: 1044-1054Crossref PubMed Scopus (65) Google Scholar, 21Boileau C. Amiable N. Martel-Pelletier J. Fahmi H. Duval N. Pelletier J. Activation of proteinase-activated receptor 2 in human osteoarthritic cartilage upregulates catabolic and proinflammatory pathways capable of inducing cartilage degradation: a basic science study.Arthritis Res Ther. 2007; 9: R121Crossref PubMed Scopus (56) Google Scholar, 22Noorbakhsh F. Vergnolle N. McArthur J.C. Silva C. Andrade-Gordon P. Vodjgani M. Hollenberg M. Power C. Protease-activated receptor-2 induction by neuroinflammation prevents neuronal death during HIV infection.J Immunol. 2005; 174: 7320-7329Crossref PubMed Scopus (82) Google Scholar; IL-1β–mediated inflammation induces the expression of various proteases, which can cleave and activate Par2.23Suharti C. van Gorp E.C. Setiati T.E. Dolmans W.M. Djokomoeljanto R.J. Hack C.E. Hugo ten C. van der Meer J.W. The role of cytokines in activation of coagulation and fibrinolysis in dengue shock syndrome.Thromb Haemost. 2002; 87: 42-46PubMed Google Scholar This may appear rather counterintuitive because amplified IL-1β increases endothelial cell death in OIR,8Rivera J.C. Sitaras N. Noueihed B. Hamel D. Madaan A. Zhou T. Honore J. Quiniou C. Joyal J. Hardy P. Sennlaub F. Lubell W. Chemtob S. Microglia and IL-1β in ischemic retinopathy elicit microvascular degeneration through neuronal Semaphorin3A.Arterioscler Thromb Vasc Biol. 2013; 133: 1881-1891Crossref Scopus (106) Google Scholar, 11Joyal J.S. Sitaras N. Binet F. Rivera J.C. Stahl A. Zaniolo K. Shao Z. Polosa A. Zhu T. Hamel D. Djavari M. Kunik D. Honoré J.C. Picard E. Zabeida A. Varma D.R. Hickson G. Mancini J. Klagsbrun M. Costantino S. Beauséjour C. Lachapelle P. Smith L.E. Chemtob S. Sapieha P. Ischemic neurons prevent vascular regeneration of neural tissue by secreting semaphorin 3A.Blood. 2011; 117: 6024-6035Crossref PubMed Scopus (148) Google Scholar opposing the angiogenic effects of Par2. We therefore hypothesize that in OIR Par2 exerts its angiogenic properties by countering the cytotoxic actions of IL-1β. Using a mouse model of OIR,24Smith L.E. Wesoloiuski E. McLellan A. Kostyk S.K. D'Amato R. Sullivan R. D'Amore P.A. Oxygen-induced retinopathy in the mouse.Invest Ophthalmol Vis Sci. 1994; 35: 101-111PubMed Google Scholar which mimics the cardinal features of ischemic retinopathies, we have uncovered a new mechanism of action for Par2 in retinal revascularization. By conditionally knocking down Par2 [using a lentiviral (Lv)–encoded short hairpin (sh) RNA], we found that IL-1β regulates neuronal Par2 expression, which in turn reduces oxygen-induced vaso-obliteration and enhances desirable revascularization of the retina. We found that Par2 down-regulates IL-1RI specifically in RGCs, which in turn curtails the secretion of Sema3A, thereby facilitating retinal revascularization. Our findings unveil a novel property for Par2 in modulating inflammation, which in the context of ischemic retinopathies limits the vaso-obliterative effects of amplified IL-1β, allowing desirable revascularization and consequently reducing pathologic intravitreal neovascularization. Adult C57BL6/J mice [wild type (WT)] and Par2 knockout mice (B6.Cg-F2rl1tm1Mslb or Par2−/−) were purchased from Jackson Laboratories (Bar Harbor, ME). Par2−/− mice were genotyped as described by Jackson Laboratories. Adoptive CD-1 lactating females were purchased from Charles Rivers Laboratories (Sainte-Hyacinthe, Canada) to tend to hyperoxia-exposed mice pups. All experiments were conducted in accordance with the Association for Research in Vision and Ophthalmology statement regarding use of animals in ophthalmic and vision research and were approved by Maisonneuve-Rosemont and Sainte-Justine Research Center animal care committees. Mice pups were exposed from postnatal day 7 to postnatal day 12 to 75% oxygen using a BioSpherix oxycycler (BioSpherix, Lacona, NY). Vaso-obliteration and neovascularization were assessed in hyperoxia-exposed mice pups at postnatal days 12 and 17, respectively, as described previously.24Smith L.E. Wesoloiuski E. McLellan A. Kostyk S.K. D'Amato R. Sullivan R. D'Amore P.A. Oxygen-induced retinopathy in the mouse.Invest Ophthalmol Vis Sci. 1994; 35: 101-111PubMed Google Scholar, 25Stahl A. Connor K.M. Sapieha P. Chen J. Dennison R.J. Krah N.M. Seaward M.R. Willett K.L. Aderman C.M. Guerin K.I. Hua J. Lofqvist C. Hellstrom A. Smith L.E. The mouse retina as an angiogenesis model.Invest Ophthalmol Vis Sci. 2010; 51: 2813-2826Crossref PubMed Scopus (439) Google Scholar Concisely, mice pups were fully anesthetized in 3% isoflurane in oxygen and decapitated using a guillotine. Eyes were enucleated and fixed in 4% paraformaldehyde solution for 1 hour at room temperature. Retinas were dissected and stained overnight at 4°C with fluorescein-labeled Griffonia (Bandeiraea) simplicifolia lectin 1, isolectin B4 (Vector Labs, Burlingame, CA; 1:100). Lectin-stained retinas were whole mounted onto Superfrost/Plus microscope slides (Thermo Fisher Scientific, Waltham, MA) with the photoreceptor side down and imbedded in Fluoro-gel (Electron Microscopy Sciences, Hatfield, PA) and imaged at 10× using a Zeiss AxioObserver.Z1 (Zeiss, San Diego, CA). Images were merged into a single file using the MosiaX option in the AxioVision software version 4.6.5 (Zeiss). Quantification of vaso-obliteration and neovascularization was assessed using the SWIFT_NV methods as previously described.26Stahl A. Connor K.M. Sapieha P. Willett K.L. Krah N.M. Dennison R.J. Chen J. Guerin K.I. Smith L.E. Computer-aided quantification of retinal neovascularization.Angiogenesis. 2009; 12: 297-301Crossref PubMed Scopus (116) Google Scholar The commercial IL-1 receptor antagonist (IL-1Ra) anakinra (Kineret; Swedish Orphan Biovitrum AB, Stockholm, Sweden) was administered intraperitoneally from postoperative day 7 (P7) to P8 at 20 mg/kg/d. Third-generation Lv (HIV-1) was prepared as previously described.27Dull T. Zufferey R. Kelly M. Mandel R.J. Nguyen M. Trono D. Naldini L. A third-generation lentivirus vector with a conditional packaging system.J Virol. 1998; 72: 8463Crossref PubMed Google Scholar A p24 enzyme-linked immunosorbent assay kit (Clontech, Mountain View, CA) was used to quantify Lv titers for Lv shRNA against green fluorescent protein (GFP) (8.5 ng/μL), Lv shPar2 (9.6 ng/μL), and Lv GFP (15.0 ng/μL). Animals were anesthetized in 3% isolfurane in oxygen and injected intravitreally either at P3 with 1.0 μL of Lv particles (described above) or at P7 with 10 μmol of anakinra or P7 and P9 or P12 and P14 with 100 μmol of NH2-SLIGRL (Elim Biopharm, Hayward, CA) using a Hamilton syringe equipped with 50-gauge glass capillary. Eyes were enucleated from mice pups and fixed in 4% paraformaldehyde at room temperature for 2 hours and saturated overnight at 4°C in a 30% sucrose solution before embedding in optimal cutting temperature compound (Tissue-Tek, Sakura, Torrance, CA). Coronal sections of 10 μm were sectioned using a Cryostat CM3050S (Leica Microsystems, Concord, Canada). Sections were subsequently washed with phosphate-buffered saline, blocked, and permeabilized for 1 hour at room temperature and subsequently incubated with fluorescein-labeled Griffonia (Bandeiraea) simplicifolia lectin 1, isolectin B4 (Vector Labs; 1:100) for retinal vasculature. Antibodies to rabbit βIII-tubulin (ECM Biosciences, Versailles, KY; 1:1000), mouse βIII-tubulin (Sigma-Aldrich, St Louis, MO; 1:1500), mouse Par2 (SAM11, Invitrogen, Carlsbad, CA; 1:500), rat CD31 (BD Biosciences, Franklin Lakes, NJ; 1:100), rabbit IL-1RI (Santa Cruz Biotechnology, Santa Cruz, CA; 1:400), rabbit Sema3A (Abcam, Cambridge, MA, 1:500), or rabbit vascular endothelial growth factor (VEGF) (Santa Cruz Biotechnology; 1:200), whereas fluoresceinated secondary antibodies (goat anti-mouse IgG Alexa Fluor 488, 594, and/or 647 and goat anti-rabbit IgG Alexa Fluor 488, 594, and/or 647; Invitrogen) were used for localization studies according to the manufacturer's recommendations. Samples were visualized using 30× or 60× objectives with an IX81 confocal microscope (Olympus, Richmond Hill, Canada), and images were obtained with Fluoview software version 3.1 (Olympus). The specificity of Par2 SAM11 monoclonal antibody was confirmed on sagittal sections from Par2−/− mice, revealing absence of immunoreactivity. Eyes were enucleated and immediately embedded in optimal cutting temperature compound and snap frozen in liquid nitrogen and subsequently cut into 16-μm coronal sections onto MembraneSlide 1.0 PEN nuclease free slides (Zeiss). To visualize vessels, sections were prepared as previously described.11Joyal J.S. Sitaras N. Binet F. Rivera J.C. Stahl A. Zaniolo K. Shao Z. Polosa A. Zhu T. Hamel D. Djavari M. Kunik D. Honoré J.C. Picard E. Zabeida A. Varma D.R. Hickson G. Mancini J. Klagsbrun M. Costantino S. Beauséjour C. Lachapelle P. Smith L.E. Chemtob S. Sapieha P. Ischemic neurons prevent vascular regeneration of neural tissue by secreting semaphorin 3A.Blood. 2011; 117: 6024-6035Crossref PubMed Scopus (148) Google Scholar Retinal sections were laser microdissected with the Zeiss (Observer.Z1) Palm Microbeam laser microscope system. Isolated retinal RNA was transcribed into cDNA for quantitative real-time PCR analysis (see RT-PCR and Quantitative Real-Time PCR). Eyes were enucleated and retinas dissected and placed into commercial radioimmunoprecipitation assay buffer (Cell Signaling Technology, Danvers, MA) and homogenized using Precellys 24 homogenizer (Bertin Technologies, Montigny-le-Bretonneux, France). Samples were centrifuged, and 30 μg of pooled retinal lysate from two different animals was loaded on an SDS-PAGE gel and subsequently electroblotted onto either polyvinylidene difluoride or nitrocellulose membrane (BioRad, Hercules, CA). After blocking, the membranes were blotted with mouse antibody to Par2 (1:400, SAM11, Invitrogen), mouse antibody to β-actin (1:1000, Santa Cruz Biotechnology), rabbit antibody to VEGF (1:200, Santa Cruz Biotechnology), rabbit antibody to IL-1RI (1:400, Santa Cruz Biotechnology), goat antibody to IL-1β (1:400, R&D Systems, Minneapolis, MN), rabbit antibody to Sema3A (1:1000, Abcam), rabbit antibody to total (1:500, Cell Signaling) or phosphorylated Irak1 (1:500, Sigma-Aldrich), rabbit or mouse antibody to total and phosphorylated Erk1/2 (1:1000, Cell Signaling), rabbit antibody to total and phosphorylated p38 (1:1000, Cell Signaling), or rabbit or mouse antibody to total and phosphorylated Jnk (1:1000, Cell Signaling). After washing, membranes were incubated with 1:5000 horseradish peroxidase–conjugated anti-mouse or 1:2000 horseradish peroxidase anti-goat or anti-rabbit secondary antibodies (Millipore, Billerica, MA). Membranes were imaged with LAS-3000 imager, and bands were assessed using densitometry plugins in Multi Gauge software version 4.0 (FujiFilm, Tokyo, Japan). Specificity of SAM11 antibody against Par2 was tested in lysates from Par2−/− mice retina using WT mice retina as control; immunoblots using Par2 SAM11 antibody on cell lysates from various cell lines expressing Par2 or not have also been provided. Freshly dissected whole retina or laser-capture microdissected samples were processed using RiboZol RNA Extraction Reagent (AMRESCO, Solon, Ohio) as indicated in the manufacturer's instructions. Genomic DNA was removed using DNase I (Invitrogen). Approximately 1 μg of total RNA was reverse transcribed into cDNA using iScript RT Supermix (BioRad) as indicated in the manufacturer's instructions. cDNA was analyzed by quantitative real-time PCR using iQ SYBR Green Supermix (BioRad) with primers targeting mouse Par2 (forward 5′-TGACCACGGTCTTTCTTCCG-3′ and reverse 5′-TCAGGGGGAACCAGATGACA-3′), rat Par2 (forward 5′-TGGGAGGTATCACCCTTCTG-3′ and reverse 5′-GGGGAACCAGATGACAGAGA-3′), mouse Sema3A (forward 5′-GCTCCTGCTCCGTAGCCTGC-3′ and reverse 5′-TCGGCGTTGCTTTCGGTCCC-3′), mouse VEGF-A (forward 5′-GCCCTGAGTCAAGAGGACAG-3′ and reverse 5′-CTCCTAGGCCCCTCAGAAGT-3′), mouse and rat IL-1RI (forward 5′-TGAATGTGGCTGAAGAGCAC-3′ and reverse 5′-CGTGACGTTGCAGACAGTT-3′), and mouse IL-1β (forward 5′-CTGGTACATCAGCACCTCACA-3′ and reverse 5′-GAGCTCCTTAACATGCCCTG-3′) [designed using Primer3 (National Center for Biotechnology Information)]. Quantitative gene expression analysis was assessed using the ABI 7500 Real-Time PCR system (Applied Biosystems, Foster City, CA) and compared with control genes cyclophilin A (forward 5′-CAGACGCCACTGTCGCTTT-3′ and reverse 5′-TGTCTTTGGAACTTTGTCTGCAA-3′) or 18S primers (Ambion, Austin, TX) using the ΔΔCT quantification. The RGC-5 cell line was kindly provided by Neeraj Agarwal (National Eye Institute, Bethesda, MD), which was prepared as previously described.28Sapieha P. Sirinyan M. Hamel D. Zaniolo K. Joyal J.S. Cho J.H. Honoré J.C. Kermorvant-Duchemin E. Varma D.R. Tremblay S. Leduc M. Rihakova L. Hardy P. Klein W.H. Mu X. Mamer O. Lachapelle P. Di Polo A. Beauséjour C. Andelfinger G. Mitchell G. Sennlaub F. Chemtob S. The succinate receptor GPR91 in neurons has a major role in retinal angiogenesis.Nat Med. 2008; 14: 1067-1076Crossref PubMed Scopus (282) Google Scholar Undifferentiated RGC-5 samples were incubated overnight with Lv particles that contained shRNA. The next day, media was changed and incubated for 48 hours before selection with 5 μg/mL of puromycin (Sigma-Aldrich) for 7 days. Cells were differentiated thereafter with 1 μmol/L staurosporine for 1 hour (Sigma-Aldrich). Rat brain microvascular endothelial cells (RBMVECs) were obtained from (Cell Applications Inc, San Diego, CA; catalog number R840-05a) and used between passages 2 and 7 (brain and retina microvascular endothelial cells share numerous common properties29Chang-Ling T. Neill A.L. Hunt N.H. Early microvascular changes in murine cerebral malaria detected in retinal wholemounts.Am J Pathol. 1992; 140: 1121-1130PubMed Google Scholar). RGC-5 samples were cultured in Dulbecco's modified Eagle's medium (Invitrogen) supplemented with 10% fetal bovine serum (Cell Applications) and 1% penicillin/streptomycin (Cell Applications) at 37°C and 5% CO2 whereas RBMVECs were cultured as indicated in the manufacturer's instructions. Cells were starved 4 hours before treatment with 1.0 or 10.0 ng/mL of recombinant murine IL-1β (PeproTech, Rocky Hill, NJ) or 0, 40, or 100 μmol/L SLIGRL-NH2. Commercial inhibitors for Erk1/2 (U0126), Jnk (SP600125), and p38 (SB203580) were purchased from Sigma-Aldrich and used at 10 μmol/L approximately 60 minutes before SLIGRL-NH2 treatment. Terminally differentiated RGC-5 conditioned media were seeded (106 cells) and starved 4 hours before treatment with 0 or 100 μmol/L NH2-SLIGRL and exposed to 5.0 ng/mL of recombinant murine IL-1β (PeproTech). Supernatants were collected 24 hours later, centrifuged briefly, and filtered through 0.22-μm filters (Millipore) and distributed for proliferation assays (see below). Approximately 104 RBMVECs per well were seeded in 24-well plates and starved 4 hours before exposure to RGC-5 conditioned media. Neutralizing antibody to Sema3A was used at 2 μg/mL (Abcam). After 24 hours, 50 μL of a 5 μg/mL solution of thiazolyl blue tetrazolium bromide (Sigma-Aldrich) was added and cells incubated for 2 to 3 hours. Supernatants were aspirated and cells were lyzed and resuspended in acidified isopropanol. Duplicate absorbance readings were taken at 565 nm using an Infinite M1000 Pro plate reader (Tecan, San Jose, CA). Aortae were isolated from adult Par2−/− mice, sectioned into 1-mm rings, and placed into growth factor–reduced Matrigel (BD Biosciences) in 24-well plates. Rings were cultured in RBMVEC supplemented endothelial basal medium (Cell Applications Inc, San Diego, CA) 4 to 5 days before a 48 hours exposure to RGC-5 conditioned media. Treated rings were photomicrographed using AxioObserver (Zeiss) and microvascular growth assessed using Image Pro version 4.5 (Media Cybernetics, Rockville, MD). Neutralizing antibody to mouse VEGF164 was used at concentrations of 20 μg/mL (R&D Systems). Results are presented as means ± SEM. for all studies. One-way or two-way analysis of variance with significance α = 0.05 was used for processing data. Bonferroni post hoc analysis was used for calculating significance between groups. Two-tailed Student's t-tests were used to test for significance between two means. Retinas from WT mice subjected to 75% oxygen for 5 days (P7 to P12)24Smith L.E. Wesoloiuski E. McLellan A. Kostyk S.K. D'Amato R. Sullivan R. D'Amore P.A. Oxygen-induced retinopathy in the mouse.Invest Ophthalmol Vis Sci. 1994; 35: 101-111PubMed Google Scholar were analyzed at different time points during OIR. During the early phases of vaso-obliteration (at P8), Par2 protein levels surged approximately threefold compared with normoxic controls (normalized versus P5) (Figure 1, A and B). Par2 expression in retina remained high during the hyperoxic phase at P12 (Figure 1, A and B); by P17, Par2 levels were comparable to normoxic controls. Par2−/− mice had no immunoreactivity to SAM11 Par2 monoclonal antibody (Supplemental Figure S1). Immunofluorescence analysis on sagittal sections from P8 and P12 normoxic retinas had preferential distribution of Par2 in βIII-tubulin positive RGCs (Figure 1C and Supplemental Figure S2A), which robustly increa

Referência(s)