Inhibition of Inflammatory Lymphangiogenesis by Integrin α5 Blockade
2007; Elsevier BV; Volume: 171; Issue: 1 Linguagem: Inglês
10.2353/ajpath.2007.060896
ISSN1525-2191
AutoresTina Dietrich-Ntoukas, Jasmine Onderka, Felix Bock, Friedrich E. Kruse, Dörte Vossmeyer, Roland Stragies, Grit Zahn, Claus Cursiefen,
Tópico(s)Corneal Surgery and Treatments
ResumoThe interaction between endothelial cells and extracellular matrix proteins plays an important role in (hem)angiogenesis. Integrins are able to mediate the outgrowth of newly formed blood vessels. In contrast, the role of integrins in lymphangiogenesis, ie, the outgrowth of new from pre-existing lymphatic vessels, has so far been unclear. Here, expression and functional relevance of integrins on lymphatic endothelium in vivo was investigated using the mouse model of combined inflammatory corneal hemangiogenesis and lymphangiogenesis. Immunohistochemistry revealed novel expression of both integrin α5 and αv on both resting and activated lymphatic vessels in vivo. Integrin α5-inhibiting small molecules significantly blocked the outgrowth of new lymphatic vessels into the cornea in a dose-dependent manner. The outgrowth of blood vessels was less significantly affected by this treatment, thus allowing for selective inhibition of lymphangiogenesis at lower dosages. Combined inhibition of integrin α5 and αv using inhibiting molecules did not significantly increase the anti-lymphangiogenic effect in vivo, thus suggesting an important functional role of integrin α5 in lymphangiogenesis. In summary, our findings demonstrate novel expression of specific integrins on growing lymphatic endothelial cells in vivo and reveal their functional role during lymphangiogenesis. This opens new treatment options for selective inhibition of lymphangiogenesis, eg, in oncology and transplant immunology. The interaction between endothelial cells and extracellular matrix proteins plays an important role in (hem)angiogenesis. Integrins are able to mediate the outgrowth of newly formed blood vessels. In contrast, the role of integrins in lymphangiogenesis, ie, the outgrowth of new from pre-existing lymphatic vessels, has so far been unclear. Here, expression and functional relevance of integrins on lymphatic endothelium in vivo was investigated using the mouse model of combined inflammatory corneal hemangiogenesis and lymphangiogenesis. Immunohistochemistry revealed novel expression of both integrin α5 and αv on both resting and activated lymphatic vessels in vivo. Integrin α5-inhibiting small molecules significantly blocked the outgrowth of new lymphatic vessels into the cornea in a dose-dependent manner. The outgrowth of blood vessels was less significantly affected by this treatment, thus allowing for selective inhibition of lymphangiogenesis at lower dosages. Combined inhibition of integrin α5 and αv using inhibiting molecules did not significantly increase the anti-lymphangiogenic effect in vivo, thus suggesting an important functional role of integrin α5 in lymphangiogenesis. In summary, our findings demonstrate novel expression of specific integrins on growing lymphatic endothelial cells in vivo and reveal their functional role during lymphangiogenesis. This opens new treatment options for selective inhibition of lymphangiogenesis, eg, in oncology and transplant immunology. Angiogenesis, ie, the outgrowth of novel blood vessels, plays an important role in tumor growth, metastasis, and other human diseases.1Folkman J Angiogenesis in cancer, vascular, rheumatoid and other disease.Nat Med. 1995; 1: 27-31Crossref PubMed Scopus (7176) Google Scholar Recently, in addition to (hem)angiogenesis, lymphangiogenesis, ie, the outgrowth of novel lymphatic vessels, has gained wide attention for its essential roles in inducing immune responses after organ transplantation as well as mediating tumor growth and metastasis.2Alitalo K Tammela T Petrova TV Lymphangiogenesis in development and human disease.Nature. 2005; 438: 946-953Crossref PubMed Scopus (954) Google Scholar Angiogenesis not only depends on the expression of specific growth factors such as vascular endothelial growth factor (VEGF) and fibroblast growth factor, but also on cell adhesion to the extracellular matrix (ECM). During growth of new blood vessels, adhesion to the ECM, eg, via integrins αvβ3 and αvβ5, regulates proliferation, survival, and motility of endothelial cells.3Varner JA The role of vascular cell integrin αVβ3 and αVβ5 in angiogenesis.EXS. 1997; 79: 361-390PubMed Google Scholar Consequently, integrin antagonists have been shown to be able to block hemangiogenesis in vitro and in vivo.4Friedlander M Brooks PC Shaffer RW Kincaid CM Varner JA Chresh DA Definition of two angiogenic pathways by distinct αV integrins.Science. 1995; 270: 1500-1502Crossref PubMed Scopus (1218) Google Scholar, 5Kim S Bell K Mousa SA Varner JA Regulation of angiogenesis in vivo by ligation of integrin α5β1 with the central cell-binding domain of fibronectin.Am J Pathol. 2000; 156: 1345-1362Abstract Full Text Full Text PDF PubMed Scopus (553) Google ScholarIntegrins, as ubiquitous heterodimeric proteins, are important for cell-cell and cell-ECM connections and are composed of α and β subunits.6Hynes RO Integrins: bidirectional, allosteric signaling machines.Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6764) Google Scholar, 7Stepp MA Corneal integrins and their functions.Exp Eye Res. 2006; 83: 3-15Crossref PubMed Scopus (118) Google Scholar Because integrins serve as transmembrane linkers between their extracellular ligands and the cytoskeleton, they have the capacity to influence cell migration during embryogenesis, angiogenesis, wound healing, immune and nonimmune defense mechanisms, hemostasis, and oncogenic transformation.6Hynes RO Integrins: bidirectional, allosteric signaling machines.Cell. 2002; 110: 673-687Abstract Full Text Full Text PDF PubMed Scopus (6764) Google Scholar, 8Hynes RO A reevaluation of integrins as regulators of angiogenesis.Nat Med. 2002; 8: 918-921Crossref PubMed Scopus (482) Google Scholar During angiogenesis, a significant up-regulation of αvβ3 and α5β1 on activated vascular endothelium has been identified.5Kim S Bell K Mousa SA Varner JA Regulation of angiogenesis in vivo by ligation of integrin α5β1 with the central cell-binding domain of fibronectin.Am J Pathol. 2000; 156: 1345-1362Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar, 9Brooks PC Clark RA Cheresh DA Requirement of vascular integrin αvβ3 for angiogenesis.Science. 1994; 264: 569-571Crossref PubMed Scopus (2717) Google Scholarα5 integrins play a key role during development of the vascular system. Genetic ablation of integrin α5 leads to severe vascular abnormalities.10Francis SE Goh KL Hodivala-Dilke K Bader BL Stark M Davidson D Hynes RO Central roles of α5β1 integrin and fibronectin in vascular development in mouse embryos and embryoid bodies.Arterioscler Thromb Vasc Biol. 2002; 22: 927-933Crossref PubMed Scopus (235) Google Scholar In combination with its extracellular ligand fibronectin, which is able to provide proliferative signals to vascular cells, α5β1 integrin is also up-regulated in tumor blood vessels and plays a role in tumor angiogenesis and growth.5Kim S Bell K Mousa SA Varner JA Regulation of angiogenesis in vivo by ligation of integrin α5β1 with the central cell-binding domain of fibronectin.Am J Pathol. 2000; 156: 1345-1362Abstract Full Text Full Text PDF PubMed Scopus (553) Google Scholar Very recent data have shown that the integrin α5β1 is also involved in the choroidal neovascularization in a laser injury model in mice: α5β1 inhibition caused prevention and regression of choroidal neovascularization.11Umeda N Kachi S Akiyama H Zahn G Vossmeyer D Stragies R Campochiaro PA Suppression and regression of choroidal neovascularization by systemic administration of an alpha5beta1 integrin antagonist.Mol Pharmacol. 2006; 69: 1820-1828Crossref PubMed Scopus (76) Google ScholarIn contrast to α5β1, αv integrins, which are known to also participate in hemangiogenesis by providing survival signals for activated endothelial cells,4Friedlander M Brooks PC Shaffer RW Kincaid CM Varner JA Chresh DA Definition of two angiogenic pathways by distinct αV integrins.Science. 1995; 270: 1500-1502Crossref PubMed Scopus (1218) Google Scholar, 12Drake CJ Cheresh DA Little CD An antagonist of integrin αvβ3 prevents maturation of blood vessels during embryonic neovascularization.J Cell Sci. 1995; 108: 2655-2661Crossref PubMed Google Scholar are not essential for vascular development as genetic ablation models have shown: deletion of αv, β3, and/or β5 fails to block developmental angiogenesis and in some cases may enhance vasculogenesis and angiogenesis.13Bader BL Rayburn H Crowley D Hynes RO Extensive vasculogenesis, angiogenesis, and organogenesis precede lethality in mice lacking all alpha v integrins.Cell. 1998; 95: 507-519Abstract Full Text Full Text PDF PubMed Scopus (541) Google Scholar, 14Hodivala-Dilke KM Reyolds AR Reynolds LE Integrins in angiogenesis: multitalented molecules in a balancing act.Cell Tissue Res. 2003; 314: 131-144Crossref PubMed Scopus (128) Google Scholar Nevertheless, using integrin αvβ3 and αvβ5 antagonists, several groups were able to inhibit hemangiogenesis in vitro and in vivo.4Friedlander M Brooks PC Shaffer RW Kincaid CM Varner JA Chresh DA Definition of two angiogenic pathways by distinct αV integrins.Science. 1995; 270: 1500-1502Crossref PubMed Scopus (1218) Google Scholar, 12Drake CJ Cheresh DA Little CD An antagonist of integrin αvβ3 prevents maturation of blood vessels during embryonic neovascularization.J Cell Sci. 1995; 108: 2655-2661Crossref PubMed Google Scholar, 15Brooks PC Montgomery AM Rosenfeld M Reisfeld RA Hu T Klier G Cheresh DA Integrin αvβ3 antagonists promote tumor regression by inducing apoptosis of angiogenic blood vessels.Cell. 1994; 79: 1157-1164Abstract Full Text PDF PubMed Scopus (2169) Google ScholarIn contrast to the good knowledge on the role of integrins in hemangiogenesis, to date nothing is known about the expression of integrins on lymphatic endothelial cells (LECs) in vivo and their functional relevance in lymphangiogenesis. Preliminary in vitro and in vivo knockout data suggest that lymphatic vessels express integrins such as α1β1 and α2β1,16Hong YK Lange-Aschenfeldt B Velasco P Hirakawa S Kunstfeld R Brown LF Bohlen P Senger DR Detmar M VEGF-A promotes tissue repair-associated lymphatic vessel formation via VEGFR-2 and the α1β1 and α2β1 integrins.FASEB J. 2004; 18: 1111-1113Crossref PubMed Scopus (253) Google Scholar α5, αv, and also α9.17Vlahakis NE Young BA Atakili A Sheppard D The lymphangiogenic endothelial growth factors VEGF-C and -D are ligands for the integrin α9β1.J Biol Chem. 2005; 280: 4544-4552Crossref PubMed Scopus (160) Google Scholar Integrin α9β1 seems to play a critical role during the development of the lymphatic system as shown in integrin α9-deficient mice.18Huang XZ Wu JF Ferrando R Lee JH Wang YL Farese Jr, RV Sheppard D Fatal bilateral chylothorax in mice lacking the integrin α9β1.Mol Cell Biol. 2000; 20: 5208-5215Crossref PubMed Scopus (250) Google Scholar Further knowledge about the role of integrins in lymphangiogenesis may lead to the development of novel specific anti-(lymph)angiogenic therapies, eg, in oncology.Here, we used the murine model of combined inflammatory hemangiogenesis and lymphangiogenesis in the normally avascular cornea to examine expression of integrins α5 and αv and their extracellular ligands fibronectin and vitronectin on lymphatic vascular endothelium in vivo. Because the normal cornea is devoid of both blood and lymphatic vessels but can secondarily be invaded by both vessel types, the cornea is a very useful model system to study to functional role of integrins in lymphangiogenesis in vivo.19Cursiefen 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 (224) Google Scholar Therefore, integrin-blocking small molecules were used to characterize their functional role in lymphangiogenesis and explore novel strategies to inhibit lymphatic vessel outgrowth by specifically targeting integrins.Materials and MethodsAnimalsWe used the mouse model of suture-induced inflammatory corneal neovascularization.20Streilein JW Bradley D Sano Y Sonoda Y Immunosuppressive properties of tissues obtained from eyes with experimentally manipulated corneas.Invest Ophthalmol Vis Sci. 1996; 37: 413-424PubMed Google Scholar Six-week-old BALB/c mice (Charles River, Sulzfeld, Germany) were put under general anesthesia with an intramuscular injection of Ketanest S (8 mg/kg) and Rompun (0.1 ml/kg), and three intrastromal 11-0 nylon sutures (Serag-Wiesner, Naila, Germany) were placed in the cornea in a standardized manner. As described before, this procedure induces combined outgrowth of blood and lymphatic vessels from the limbal arcade (border between physiologically vascularized conjunctiva and normally avascular cornea) into the normally avascular cornea.21Cursiefen C Chen L Dana MR Streilein JW Corneal lymphangiogenesis. Evidence, mechanisms, and implications for corneal transplant immunology.Cornea. 2003; 22: 273-281Crossref PubMed Scopus (200) Google Scholar, 22Cursiefen 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 (851) Google Scholar Animals were treated in accordance with the local animal care committee and the Association for Research in Vision and Ophthalmology Statement for the Use of Animals.AntibodiesFor immunofluorescence, podoplanin served as a specific marker for lymphatic vascular endothelium23Breiteneder-Geleff S Soleiman A Kowalski H Horvat R Amann G Kriehuber E Diem K Weninger W Tschachler E Alitalo K Kerjaschki D Angiosarcomas express mixed endothelial phenotypes of blood and lymphatic capillaries: podoplanin as a specific marker for lymphatic endothelium.Am J Pathol. 1999; 154: 385-394Abstract Full Text Full Text PDF PubMed Scopus (931) Google Scholar: we used the monoclonal Syrian-hamster anti-mouse antibody (Acris Antibodies GmbH, Hiddenhausen, Germany), diluted 1:200. As control staining, we used Syrian-hamster IgG (Santa Cruz Biotechnology, Santa Cruz, CA); all control IgGs were used in a dilution that provided the same quantity of protein as the antibody. Secondary antibody was goat anti-Syrian hamster fluorescein isothiocyanate (FITC) antibody (Dianova, Hamburg, Germany), diluted 1:100. For whole mount preparations, we used LYVE-1 antibody rabbit anti-mouse (a kind gift of D.G. Jackson, Oxford University, Oxford, UK) as specific lymphatic endothelial marker.24Banerji S Ni J Wang SX Clasper S Su J Tammi R Jones M Jackson D LYVE-1, a new homologue of the CD44 glycoprotein, is a lymph-specific receptor for hyaluronan.J Cell Biol. 1999; 144: 789-801Crossref PubMed Scopus (1283) Google Scholar CY3 antibody served as secondary antibody (Dianova). Detection of blood vessels was done using the panendothelial marker CD-31. We used a monoclonal FITC-conjugated rat anti-mouse antibody, diluted 1:50 (Acris Antibodies GmbH). As control staining, we used FITC-conjugated normal rat IgG2A (Santa Cruz Biotechnology). As specific antibody for integrin α5, we used a polyclonal rabbit anti-mouse antibody (Chemicon International, Planegg-München, Germany), diluted 1:50. Integrin αv antibody was polyclonal rabbit anti-mouse antibody (Chemicon International), which was diluted 1:50; control staining was done by normal rabbit IgG (Santa Cruz Biotechnology).A polyclonal rabbit anti-mouse vitronectin antibody (Acris Antibodies GmbH), diluted 1:200, and a polyclonal rabbit anti-mouse fibronectin antibody (Dianova), diluted 1:500, were used. As a control, we used normal rabbit control IgG, diluted 1:100 (Santa Cruz Biotechnology). The secondary antibody for integrin α5/αv and fibronectin/vitronectin was goat anti-rabbit CY3 antibody (Dianova), diluted 1:500.VEGFR-2 antibody, a rabbit anti-mouse antibody, diluted 1:50 (Santa Cruz Biotechnologies), and VEGFR-3 antibody, a goat anti-mouse antibody, diluted 1:100 (R&D Systems, Minneapolis, MN) were used as primary antibodies. For co-localization studies, we used integrin α5β1 rat anti-mouse antibody (diluted 1:25; Chemicon International) as secondary antibody. As a proliferation marker, we used Ki67 antibody (Biozol, Eching, Germany), diluted 1:100. After rinsing, we incubated with the secondary antibodies CY3 (goat anti-rabbit, diluted 1:500; Dianova) and FITC (donkey anti-rat, diluted 1:100; Chemicon International). Normal rabbit IgG and normal goat IgG (Santa Cruz Biotechnologies) were used as controls. For immunofluorescence on LECs in chamber slides, we used integrin α5β1 antibody (mouse anti-human; Chemicon International), VEGFR-3 antibody (rabbit anti-human; Acris Antibodies GmbH), and the proliferation marker Ki67 (Biozol), diluted 1:50.Integrin InhibitorsJSM6427 is a α5β1-specific integrin-inhibiting small molecule further described in Umeda and colleagues.11Umeda N Kachi S Akiyama H Zahn G Vossmeyer D Stragies R Campochiaro PA Suppression and regression of choroidal neovascularization by systemic administration of an alpha5beta1 integrin antagonist.Mol Pharmacol. 2006; 69: 1820-1828Crossref PubMed Scopus (76) Google Scholar JSM6424 is a derivative of JSM6427 recognizing α5β1 as well as αvβ3 integrin. The inhibitory activity of these small molecules was determined using a solid-phase binding assay as described in Umeda and colleagues.11Umeda N Kachi S Akiyama H Zahn G Vossmeyer D Stragies R Campochiaro PA Suppression and regression of choroidal neovascularization by systemic administration of an alpha5beta1 integrin antagonist.Mol Pharmacol. 2006; 69: 1820-1828Crossref PubMed Scopus (76) Google Scholar The integrin inhibitor JSM6427 is an antagonist of binding to integrin α5β1 with an IC50 of 0.55 nmol/L and is at least 1500-fold less potent in the inhibition of binding to αvβ3 and other integrins such as αvβ5 and αIIbβ3. JSM6424 is an antagonist of binding to integrin α5β1 with an IC50 of 0.9 nmol/L and to αvβ3 with an IC50 of 4 nmol/L. For testing of selectivity compared with other nonintegrin receptors, the integrin α5β1 inhibitor was tested in a standard screening assay for activity on 50 nonrelated pharmacological relevant receptors, ion channels, and transporters. The inhibitor showed for all of them no activity or at least 1000-fold less activity than for the target integrin α5β1 and no permeability into cells (Cerep, Paris, France; data not shown). These small molecules are characterized by good tissue penetration because of their low molecular weight and stability in physiological buffers (data not shown). Both small molecules were purchased from Jerini AG, Berlin, Germany.ImmunofluorescenceStaining protocols were standardized for whole mount preparations and cryosections as described previously.22Cursiefen 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 (851) Google Scholar, 25Cursiefen C Schlötzer-Schrehardt U Küchle M Sorokin L Breiteneder-Geleff S Alitalo K Jackson D Lymphatic vessels in vascularized human corneas: immunohistochemical investigation using LYVE-1 and podoplanin.Invest Ophthalmol Vis Sci. 2002; 43: 2127-2135PubMed Google Scholar We used indirect immunofluorescence to localize integrin α5 and αv, fibronectin, and vitronectin on blood and lymphatic vessels in the pathologically vascularized, as well as in normal, nonvascularized mouse corneas and at the limbus. Blood and lymphatic vessels are physiologically present at the limbal border between cornea and conjunctiva. For immunofluorescence, murine eyes were cryopreserved in OCT embedding medium, and 5- to 7-μm cryosections were obtained. Sections were dried (15 minutes, 37°C) and fixed in acetone for 15 minutes on Superfrost slides (Menzel GmbH & Co. KG, Braunschweig, Germany). After rinsing with phosphate-buffered saline (PBS) (3 × 5 minutes on a shaker) and 1 hour incubation in 2% bovine serum albumin (BSA) at room temperature, incubation of primary antibodies followed overnight at 4°C. On the 2nd day, the antibodies were rinsed with PBS (5 × 5 minutes on a shaker) and blocked with 2% BSA (1 hour) and incubated with CD-31 or podoplanin antibody overnight at 4°C in the dark. On the 3rd day, after rinsing with PBS (5 × 5 minutes) and blocking with BSA, secondary antibodies were incubated for 45 minutes at room temperature in the dark. All dilutions were done with 2% BSA in PBS, and all incubations were performed in a humid chamber. After a final rinsing step (5 × 5 minutes PBS), sections were covered with DAKO (Glostrup, Denmark) fluorescent mounting medium and stored at 4°C in the dark. Fluorescence microscopy and photography was done using the Olympus BX51 fluorescence microscope (Olympus Optical Co., Hamburg, Germany) and the F-View II monochrome charge-coupled device camera (Soft Imaging System, Münster, Germany) software provided by analySISB (Soft Imaging Systems).Electron MicroscopyImmunoelectron microscopy was performed on vascularized murine cornea segments as described previously.25Cursiefen C Schlötzer-Schrehardt U Küchle M Sorokin L Breiteneder-Geleff S Alitalo K Jackson D Lymphatic vessels in vascularized human corneas: immunohistochemical investigation using LYVE-1 and podoplanin.Invest Ophthalmol Vis Sci. 2002; 43: 2127-2135PubMed Google Scholar Specimens were embedded in resin (LR-White; London Resin Co., Berkshire, UK) and ultrathin sections were used. Sections were incubated successively in drops of Tris-buffered saline (TBS), 0.05 mol/L glycine in TBS, and TBS-ovalbumin-gelatin. For immunogold labeling, we used a polyclonal rabbit anti-mouse antibody against integrin αv and a polyclonal rabbit anti-mouse antibody against integrin α5 (Chemicon International) diluted in TBS and ovalbumin overnight at 4°C and a 10-nm gold-conjugated goat anti-rabbit secondary antibody (BioCell, Cardiff, UK) diluted in 1:30 TBS-ovalbumin-gelatin for 1 hour at room temperature. After rinsing, sections were stained with uranyl acetate and examined with an electron microscope (LEO 906 E; LEO, Zeiss, Oberkochen, Germany).Systemic Application of Inhibiting Small Molecule SolutionsInhibition of integrin α5β1 function was achieved by a small molecule antagonist, JSM6427 (Jerini AG). We used the following doses of integrin α5β1 inhibitor: 10, 23, and 46 mg/kg/day. For combined inhibition of integrin α5β1 and integrin αvβ3, a combined small molecule antagonist (JSM6424; Jerini AG) was used at a dose of 23 mg/kg/day.Because of rapid elimination of the small molecule inhibitors, systemic application was provided by osmotic pumps (Alzet; Durect Corp., Cupertino, CA). After filling with the inhibiting molecule solution or vehicle solution (control), pumps were incubated in 0.9% NaCl overnight at 37°C to induce pumping function. Osmotic pumps were then placed subcutaneously at the back of the mice after skin incision, simultaneously with the corneal suturing under general anesthesia (see above). The incision was closed by nonresolvable 6-0 silk sutures (Serag-Wiesner). After 1 week, osmotic pumps were changed to new ones that were filled before with solution (inhibiting molecule or vehicle) and again preincubated overnight at 37°C. After a total of 2 weeks of continuous systemic application, mice were sacrificed. The eyes were enucleated, the pumps removed and blood samples taken. Pumps and plasma were analyzed to control sufficient pumping rate and plasma levels. Experiments were repeated three times, with four animals per group. For analysis of macrophage recruitment into the cornea, mice were treated with integrin α5β1 inhibitor JSM6427 (Jerini AG) at a dosage of 46 mg/kg/day (n = 5) or vehicle solution as control (n = 5) for 1 week after corneal suturing as described above.Whole Mount Preparations and ImmunostainingPreparation was done as previously described.22Cursiefen 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 (851) Google Scholar In brief, mice were sacrificed, eyes were enucleated and the cornea dissected from the eye behind the corneal limbus. Corneas were washed 3 × 5 minutes in PBS at room temperature. Fixation was done by acetone for 30 minutes, followed by washing 3 × 5 minutes in PBS. Afterward, we incubated with 2% BSA in PBS for 2 hours at room temperature. As primary antibody, we used LYVE-1 antibody rabbit anti-mouse 1:500 with 2% BSA in PBS, incubated overnight at 4°C. On the 2nd day, after washing 5 × 5 minutes in PBS, the antibody was blocked with 2% BSA in PBS for 2 hours. The secondary antibody CD31-FITC rat anti-mouse (Acris Antibodies GmbH), diluted 1:50 with 2% BSA in PBS, was added for incubation overnight at 4°C in the dark. On day 3, after washing 5 × 5 minutes in PBS, the antibody was blocked with 2% BSA in PBS for 2 hours. The third antibody, CY3 goat anti-rabbit (Dianova), diluted 1:100 with 2% BSA in PBS, was incubated for 45 minutes at room temperature in the dark. For detection of macrophages in the cornea, we used FITC-conjugated rat anti-mouse CD11b antibody (diluted 1:100; Serotec, Oxford, UK) instead of CD31-FITC antibody. As a final step, antibody was washed 5 × 5 minutes in PBS. Corneas were moved to Superfrost slides and covered with DAKO fluorescent mounting medium and stored at 4°C in the dark. Fluorescence microscopy and photography was done using the BX51 camera (Olympus Optical Co., Hamburg, Germany). For analysis, digital photographs were taken as multialignment pictures using ×100 magnification (analySISB; Soft Imaging System).Functional and Statistical AnalysisQuantitative analysis of blood and lymphatic vessels was performed in a standardized procedure using analySISB (Soft Imaging System) software by means of threshold analysis. For measurements, we used rectangles of a standardized size (1.11 mm2), which were aligned along the limbus. The corneal area filled with blood or lymphatic vessels (hemvascularized or lymphvascularized area) was measured in each rectangle. The vascularized areas of the control groups were defined as being 100%. For macrophage analysis, a standardized rectangle in the central cornea was analyzed by threshold analysis as described above. The area filled with macrophages was measured. Subsequent statistical analysis was done using InStat 3 Version 3.06 (GraphPad Software Inc., San Diego, CA). Graphs were drawn using Prism4, version 4.03 (GraphPad Software Inc.).Lymphatic and Blood Endothelial CellsFor in vitro studies, we used human lymphatic microvascular endothelial cells (HMVEC-dLyAd-Der Lym Endo cells; Cambrex Bio Science, Walkersville, MD), which were identified as lymphatic cells via fluorescence-activated cell sorting analysis, revealing more than 95% positivity for podoplanin (as a specific marker for LECs) and for CD31 (as a panendothelial marker) expression. For blood endothelial cells (BECs), we used human umbilical vein endothelial cells (HUVECs; PromoCell, Heidelberg, Germany).Western Blot AnalysisHUVECs (PromoCell) and human lymphatic microvascular endothelial cells (HMVEC-dLyAd-Der Lym Endo cells; Cambrex Bio Science) were cultured in endothelial growth medium (EGM) supplemented with a EGM-2-MV bullet kit (Cambrex Bio Science, Nottingham, UK) in a humidified atmosphere at 37°C, 5% CO2. Cells were grown to 80% confluence. Cells (2 × 106) were lysed in 200 μl of lysis buffer [1% Triton X-100, 150 mmol/L NaCl, 50 mmol/L Tris-HCl, pH 7.5, protease-inhibitor-cocktail (Sigma-Aldrich, Taufkirchen, Germany), 25 mmol/L glycerine phosphate, 50 mmol/L NaF, 10 mmol/L Na4P2O7, and 1 mmol/L orthovanadate] and centrifuged for 5 minutes at 4°C at 12,000 rpm. Cells (7.5 × 104) (corresponding to 18.9 or 9 μg of total protein for HMVECs or HUVECs, respectively) per lane were separated on a 4 to 15% Tris-HCl gel by sodium dodecyl sulfate-polyacrylamide gel electrophoresis under nonreducing conditions and transferred to polyvinylidene difluoride membranes by semidry Western blotting. Twenty ng of human integrin α5 (Chemicon Europe, Chandlers Ford, UK) was run as a positive control. Membranes were blocked with 3% BSA. α5β1 integrin was visualized by rabbit anti-integrin α5 polyclonal antibody (Chemicon Europe) and actin, using a rabbit anti-actin polyclonal antibody (Sigma-Aldrich), respectively, followed by detection with anti-rabbit POD- conjugated antibody (Sigma-Aldrich) and BM chemi-luminescence Western blotting POD substrate (Roche, Penzberg, Germany). The luminescence signal was measured using the Lumi imager (Boehringer-Mannheim GmbH, Mannheim, Germany) and corresponding software.Immunofluorescence on LECs in VitroLECs (see above) were cultivated in chamber slides (Lab-Tek; Nalge Nunc International, Rochester, NY) in 500 μl of EGM medium for 24 hours. After removal of the medium, attached cells were fixed with 500 μl of ice-cold methanol for 10 minutes at room temperature. After three rinsing steps with PBS for 5 minutes, we added heat-inactivated fetal bovine serum for 30 minutes at 37°C. As primary antibody, we used rabbit anti-human Ki67 antibody (diluted 1:50 in 0.2% BSA in PBS; Biozol) overnight at 4°C. CY3 goat anti-rabbit antibody (diluted 1:250, for 1 hour at room temperature in the dark; Dianova) served as secondary antibody. For double staining, podoplanin mouse anti-human antibody (Acris Antibodies GmbH), diluted 1:100, was used, with FITC goat anti-mouse (Acris Antibodies GmbH; diluted 1:100) as secondary antibody. Mou
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