Apelin Attenuates UVB-Induced Edema and Inflammation by Promoting Vessel Function
2011; Elsevier BV; Volume: 179; Issue: 6 Linguagem: Inglês
10.1016/j.ajpath.2011.08.024
ISSN1525-2191
AutoresMika Sawane, Hiroyasu Kidoya, Fumitaka Muramatsu, Nobuyuki Takakura, Kentaro Kajiya,
Tópico(s)Lipid metabolism and disorders
ResumoApelin, the ligand of the G protein–coupled receptor APJ, is involved in the regulation of cardiovascular functions, fluid homeostasis, and vessel formation. Recent reports indicate that apelin secreted from endothelial cells mediates APJ regulation of blood vessel caliber size; however, the function of apelin in lymphatic vessels is unclear. Here we report that APJ was expressed by human lymphatic endothelial cells and that apelin induced migration and cord formation of lymphatic endothelial cells dose-dependently in vitro. Furthermore, permeability assays demonstrated that apelin stabilizes lymphatic endothelial cells. In vivo, transgenic mice harboring apelin under the control of keratin 14 (K14-apelin) exhibited attenuated UVB-induced edema and a decreased number of CD11b-positive macrophages. Moreover, activation of apelin/APJ signaling inhibited UVB-induced enlargement of lymphatic and blood vessels. Finally, K14-apelin mice blocked the hyperpermeability of lymphatic vessels in inflamed skin. These results indicate that apelin plays a functional role in the stabilization of lymphatic vessels in inflamed tissues and that apelin might be a suitable target for prevention of UVB-induced inflammation. Apelin, the ligand of the G protein–coupled receptor APJ, is involved in the regulation of cardiovascular functions, fluid homeostasis, and vessel formation. Recent reports indicate that apelin secreted from endothelial cells mediates APJ regulation of blood vessel caliber size; however, the function of apelin in lymphatic vessels is unclear. Here we report that APJ was expressed by human lymphatic endothelial cells and that apelin induced migration and cord formation of lymphatic endothelial cells dose-dependently in vitro. Furthermore, permeability assays demonstrated that apelin stabilizes lymphatic endothelial cells. In vivo, transgenic mice harboring apelin under the control of keratin 14 (K14-apelin) exhibited attenuated UVB-induced edema and a decreased number of CD11b-positive macrophages. Moreover, activation of apelin/APJ signaling inhibited UVB-induced enlargement of lymphatic and blood vessels. Finally, K14-apelin mice blocked the hyperpermeability of lymphatic vessels in inflamed skin. These results indicate that apelin plays a functional role in the stabilization of lymphatic vessels in inflamed tissues and that apelin might be a suitable target for prevention of UVB-induced inflammation. The lymphatic vascular system is composed of a dense network of thin-walled capillaries that drain protein-rich lymph from the extracellular space; its function is important for homeostasis of the circulatory and immune systems, maintenance of interstitial fluid composition and volume, and immune cell trafficking in health and in disease.1Cueni L.N. Detmar M. New insights into the molecular control of the lymphatic vascular system and its role in disease.J Invest Dermatol. 2006; 126: 2167-2177Crossref PubMed Scopus (187) Google Scholar, 2Tammela T. Alitalo K. Lymphangiogenesis: molecular mechanisms and future promise.Cell. 2010; 140: 460-476Abstract Full Text Full Text PDF PubMed Scopus (1028) Google Scholar Chronic skin inflammation in mice has been associated with lymphatic endothelial cell (LEC) proliferation, and the skin disease psoriasis exhibited pronounced cutaneous lymphatic hyperplasia,3Kunstfeld R. Hirakawa S. Hong Y.K. Schacht V. Lange-Asschenfeldt B. Velasco P. Lin C. Fiebiger E. Wei X. Wu Y. Hicklin D. Bohlen P. Detmar M. Induction of cutaneous delayed-type hypersensitivity reactions in VEGF-A transgenic mice results in chronic skin inflammation associated with persistent lymphatic hyperplasia.Blood. 2004; 104: 1048-1057Crossref PubMed Scopus (270) Google Scholar indicating that the lymphatic vascular system participates in both acute and chronic inflammation. Acute exposure of skin to UVB irradiation (290 to 320 nm) leads to inflammation associated with epidermal hyperplasia, erythema, vascular hyperpermeability, and edema formation.4Kligman A.M. The treatment of photoaged human skin by topical tretinoin.Drugs. 1989; 38: 1-8Crossref PubMed Scopus (28) Google Scholar, 5Kripke M.L. Ultraviolet radiation and immunology: something new under the sun—presidential address.Cancer Res. 1994; 54: 6102-6105PubMed Google Scholar Previous studies have demonstrated that acute UVB irradiation of both human and mouse skin promotes marked angiogenesis.6Yano K. Kadoya K. Kajiya K. Hong Y.K. Detmar M. Ultraviolet B irradiation of human skin induces an angiogenic switch that is mediated by upregulation of vascular endothelial growth factor and by downregulation of thrombospondin-1.Br J Dermatol. 2005; 152: 115-121Crossref PubMed Scopus (101) Google Scholar, 7Yano K. Kajiya K. Ishiwata M. Hong Y.K. Miyakawa T. Detmar M. Ultraviolet B-induced skin angiogenesis is associated with a switch in the balance of vascular endothelial growth factor and thrombospondin-1 expression.J Invest Dermatol. 2004; 122: 201-208Crossref PubMed Scopus (72) Google Scholar Several angiogenesis factors, including vascular endothelial growth factor-A (VEGF-A), basic fibroblast growth factor, and interleukin-8, were up-regulated in skin after UVB-irradiation.7Yano K. Kajiya K. Ishiwata M. Hong Y.K. Miyakawa T. Detmar M. Ultraviolet B-induced skin angiogenesis is associated with a switch in the balance of vascular endothelial growth factor and thrombospondin-1 expression.J Invest Dermatol. 2004; 122: 201-208Crossref PubMed Scopus (72) Google Scholar, 8Krämer M. Sachsenmaier C. Herrlich P. Rahmsdorf H.J. UV irradiation-induced interleukin-1 and basic fibroblast growth factor synthesis and release mediate part of the UV response.J Biol Chem. 1993; 268: 6734-6741Abstract Full Text PDF PubMed Google Scholar, 9Strickland I. Rhodes L.E. Flanagan B.F. Friedmann P.S. TNF-alpha and IL-8 are upregulated in the epidermis of normal human skin after UVB exposure: correlation with neutrophil accumulation and E-selectin expression.J Invest Dermatol. 1997; 108: 763-768Crossref PubMed Scopus (175) Google Scholar Thrombospondin-1, a potent endogenous angiogenesis inhibitor, was up-regulated.7Yano K. Kajiya K. Ishiwata M. Hong Y.K. Miyakawa T. Detmar M. Ultraviolet B-induced skin angiogenesis is associated with a switch in the balance of vascular endothelial growth factor and thrombospondin-1 expression.J Invest Dermatol. 2004; 122: 201-208Crossref PubMed Scopus (72) Google Scholar Moreover, targeted overexpression of VEGF-A enhanced sensitivity to UVB-induced cutaneous photodamage,10Hirakawa S. Fujii S. Kajiya K. Yano K. Detmar M. Vascular endothelial growth factor promotes sensitivity to ultraviolet B-induced cutaneous photodamage.Blood. 2005; 105: 2392-2399Crossref PubMed Scopus (55) Google Scholar but transgenic overexpression of thrombospondin-1 in the epidermis completely prevented UVB-induced photodamage.11Yano K. Oura H. Detmar M. Targeted overexpression of the angiogenesis inhibitor thrombospondin-1 in the epidermis of transgenic mice prevents ultraviolet-B-induced angiogenesis and cutaneous photo-damage.J Invest Dermatol. 2002; 118: 800-805Crossref PubMed Scopus (83) Google Scholar Taken together, these findings indicate that the cutaneous blood vasculature plays an important role in the mediation of photodamage. A previous study from our research group demonstrated that UVB irradiation caused enlargement of lymphatic vessels with leaky and hyperpermeable function.12Kajiya K. Hirakawa S. Detmar M. Vascular endothelial growth factor-a mediates ultraviolet B-induced impairment of lymphatic vessel function.Am J Pathol. 2006; 169: 1496-1503Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar More recently, we found that activation of the VEGF-C/VEGFR-3 pathway attenuates UVB-induced inflammation by promoting lymphangiogenesis.13Kajiya K. Sawane M. Huggenberger R. Detmar M. Activation of the VEGFR-3 pathway by VEGF-C attenuates UVB-induced edema formation and skin inflammation by promoting lymphangiogenesis.J Invest Dermatol. 2009; 129: 1292-1298Crossref PubMed Scopus (81) Google Scholar These studies point to a crucial role of lymphatic vessels in UVB-induced inflammation. Apelin is an endogenous ligand for the previously orphan G protein–coupled receptor, APJ. The apelin gene (APLN), which is located on the long arm of the human X chromosome, encodes a 77-amino-acid preproprotein that is then cleaved to shorter active peptides.14Hosoya M. Kawamata Y. Fukusumi S. Fujii R. Habata Y. Hinuma S. Kitada C. Honda S. Kurokawa T. Onda H. Nishimura O. Fujino M. Molecular and functional characteristics of APJ Tissue distribution of mRNA and interaction with the endogenous ligand apelin.J Biol Chem. 2000; 275: 21061-21067Crossref PubMed Scopus (426) Google Scholar, 15Tatemoto K. Hosoya M. Habata Y. Fujii R. Kakegawa T. Zou M.X. Kawamata Y. Fukusumi S. Hinuma S. Kitada C. Kurokawa T. Onda H. Fujino M. Isolation and characterization of a novel endogenous peptide ligand for the human APJ receptor.Biochem Biophys Res Commun. 1998; 251: 471-476Crossref PubMed Scopus (1338) Google Scholar The full-length mature peptide, which was originally isolated from bovine stomach extracts, comprises 36 amino acids and is known as apelin-36; the short-length peptide is known as apelin-13. Both peptides activate APJ.16Masri B. Knibiehler B. Audigier Y. Apelin signalling: a promising pathway from cloning to pharmacology.Cell Signal. 2005; 17: 415-426Crossref PubMed Scopus (156) Google Scholar APJ expression has been reported in the cardiovascular system and in the central nervous system.17Devic E. Rizzoti K. Bodin S. Knibiehler B. Audigier Y. Amino acid sequence and embryonic expression of msr/apj, the mouse homolog of Xenopus X-msr and human APJ.Mech Dev. 1999; 84: 199-203Crossref PubMed Scopus (117) Google Scholar, 18O'Dowd B.F. Heiber M. Chan A. Heng H.H. Tsui L.C. Kennedy J.L. Shi X. Petronis A. George S.R. Nguyen T. A human gene that shows identity with the gene encoding the angiotensin receptor is located on chromosome 11.Gene. 1993; 136: 355-360Crossref PubMed Scopus (677) Google Scholar In the brain, the apelin/APJ system plays a role in maintaining body fluid homeostasis and regulating release of vasopressin from the hypothalamus.19De Mota N. Reaux-Le Goazigo A. El Messari S. Chartrel N. Roesch D. Dujardin C. Kordon C. Vaudry H. Moos F. Llorens-Cortes C. Apelin, a potent diuretic neuropeptide counteracting vasopressin actions through inhibition of vasopressin neuron activity and vasopressin release.Proc Natl Acad Sci USA. 2004; 101: 10464-10469Crossref PubMed Scopus (308) Google Scholar In the cardiovascular system, APJ is expressed in endothelial cells, vascular smooth muscle cells, and cardiomyocytes.20Devic E. Paquereau L. Vernier P. Knibiehler B. Audigier Y. Expression of a new G protein-coupled receptor X-msr is associated with an endothelial lineage in Xenopus laevis.Mech Dev. 1996; 59: 129-140Crossref PubMed Scopus (105) Google Scholar, 21Katugampola S.D. Maguire J.J. Matthewson S.R. Davenport A.P. [(125)I]-(Pyr(1))Apelin-13 is a novel radioligand for localizing the APJ orphan receptor in human and rat tissues with evidence for a vasoconstrictor role in man.Br J Pharmacol. 2001; 132: 1255-1260Crossref PubMed Scopus (186) Google Scholar Apelin/APJ in cells of endothelial lineage promotes hypotensive activity22Ishida J. Hashimoto T. Hashimoto Y. Nishiwaki S. Iguchi T. Harada S. Sugaya T. Matsuzaki H. Yamamoto R. Shiota N. Okunishi H. Kihara M. Umemura S. Sugiyama F. Yagami K. Kasuya Y. Mochizuki N. Fukamizu A. Regulatory roles for APJ, a seven-transmembrane receptor related to angiotensin-type 1 receptor in blood pressure in vivo.J Biol Chem. 2004; 279: 26274-26279Crossref PubMed Scopus (359) Google Scholar; the activation of APJ leads to nitric oxide (NO) production by the endothelial cells,23Tatemoto K. Takayama K. Zou M.X. Kumaki I. Zhang W. Kumano K. Fujimiya M. The novel peptide apelin lowers blood pressure via a nitric oxide-dependent mechanism.Regul Pept. 2001; 99: 87-92Crossref PubMed Scopus (561) Google Scholar and this possibly plays a role in the relaxation of smooth muscle cells. Apelin is also essential for blood vessel formation. The apelin/APJ system plays a role in the cardiovascular system of Xenopus laevis24Cox C.M. D'Agostino S.L. Miller M.K. Heimark R.L. Krieg P.A. Apelin, the ligand for the endothelial G-protein-coupled receptor, APJ, is a potent angiogenic factor required for normal vascular development of the frog embryo.Dev Biol. 2006; 296: 177-189Crossref PubMed Scopus (249) Google Scholar and of zebrafish.25Scott I.C. Masri B. D'Amico L.A. Jin S.W. Jungblut B. Wehman A.M. Baier H. Audigier Y. Stainier D.Y. The G protein-coupled receptor Agtrl1b regulates early development of myocardial progenitors.Dev Cell. 2007; 12: 403-413Abstract Full Text Full Text PDF PubMed Scopus (141) Google Scholar Xenopus apelin (Xapelin) is expressed in the region around the presumptive blood vessels during early embryogenesis as Xenopus APJ (Xmsr). Knockdown of Xapelin or Xmsr resulted in a defect of blood vessel formation in the posterior cardinal vein, intersomitic vessels, and vitelline vessels. The regulation of blood vessel formation by apelin in mammals has been described recently. The Apelin/APJ system was shown to be involved in downstream signaling of Ang1/Tie2 in endothelial cells and in regulation of blood vessel diameter during angiogenesis.26Kidoya H. Ueno M. Yamada Y. Mochizuki N. Nakata M. Yano T. Fujii R. Takakura N. Spatial and temporal role of the apelin/APJ system in the caliber size regulation of blood vessels during angiogenesis.EMBO J. 2008; 27: 522-534Crossref PubMed Scopus (196) Google Scholar However, the function of apelin in lymphatic vessels and its role in inflammation is not completely clear. In the present study, we found that the APJ receptor is expressed in lymphatic endothelial cells in vitro and in vivo, and that apelin/APJ signaling promotes stabilization of lymphatic vessels. Moreover, using apelin transgenic mice, we demonstrated that apelin attenuates UVB-induced inflammation by promoting stabilization of lymphatic and blood vessels. These results suggest that apelin might be a suitable target for prevention of UVB-induced skin inflammation and photodamage. Human dermal LECs were isolated from neonatal human foreskins by immunomagnetic purification, as described previously.27Hirakawa S. Hong Y.K. Harvey N. Schacht V. Matsuda K. Libermann T. Detmar M. Identification of vascular lineage-specific genes by transcriptional profiling of isolated blood vascular and lymphatic endothelial cells.Am J Pathol. 2003; 162: 575-586Abstract Full Text Full Text PDF PubMed Scopus (386) Google Scholar The lineage-specific differentiation was confirmed by real-time RT-PCR for the lymphatic vascular markers Prox1, LYVE-1, and podoplanin, as well as by immunostaining for Prox1 and podoplanin, as described previously.28Kajiya K. Hirakawa S. Ma B. Drinnenberg I. Detmar M. Hepatocyte growth factor promotes lymphatic vessel formation and function.EMBO J. 2005; 24: 2885-2895Crossref PubMed Scopus (256) Google Scholar Human umbilical vein endothelial cells (HUVECs) were purchased from PromoCell (Heidelberg, Germany). Cells were cultured in endothelial basal medium (Lonza, Verviers, Belgium) supplemented with supplements provided by the suppliers for up to 11 passages. For Western blot analyses of APJ, Akt, and p-Akt, confluent LECs and HUVECs were homogenized in lysis buffer, and protein concentrations were determined using a DC protein assay kit (Bio-Rad Laboratories, Hercules, CA). Equal amounts of lysates (10 μg protein) were immunoblotted with a rabbit polyclonal antibody against APJ, as described previously.26Kidoya H. Ueno M. Yamada Y. Mochizuki N. Nakata M. Yano T. Fujii R. Takakura N. Spatial and temporal role of the apelin/APJ system in the caliber size regulation of blood vessels during angiogenesis.EMBO J. 2008; 27: 522-534Crossref PubMed Scopus (196) Google Scholar LECs were also cultured with apelin-36 (1000 ng/mL; Peptide Institute, Osaka, Japan) for 2 minutes, followed by homogenization in lysis buffer. Untreated cells were prepared as controls in the same manner. Cell lysates (100 μg total protein each) were immunoprecipitated with antibodies against p-Akt and Akt (Cell Signaling Technology, Danvers, MA). Equal loading was confirmed with an antibody against β-actin (Sigma-Aldrich, St. Louis, MO). The LEC migration assay was performed as described previously,29Hong Y.K. Lange-Asschenfeldt B. Velasco P. Hirakawa S. Kunstfeld R. Brown L.F. Bohlen P. Senger D.R. Detmar M. VEGF-A promotes tissue repair-associated lymphatic vessel formation via VEGFR-2 and the alpha1beta1 and alpha2beta1 integrins.FASEB J. 2004; 18: 1111-1113Crossref PubMed Scopus (256) Google Scholar using 24-well FluoroBlock inserts of 8-μm pore size (Falcon; BD Biosciences, Franklin Lakes, NJ). The bottom sides of the inserts were coated with 10 μg/mL fibronectin (BD Biosciences, Bedford, MA) for 1 hour, followed by incubation with 100 μg/mL of bovine serum albumin. Cells (105 cells in 100 μL) were seeded in serum-free endothelial basal medium into the upper chambers, and were incubated for 5 hours at 37°C in the presence or absence of human recombinant apelin-13 (500 to 1000 ng/mL) or apelin-36 (500 to 1000 ng/mL). Cells on the underside of inserts were stained with Hoechst dye 33342 (Molecular Probes; Invitrogen, Carlsbad, CA). Five different digital images were captured per well, and the number of migrated cells was counted. Cord formation assays were performed as described previously.30Kajiya K. Huggenberger R. Drinnenberg I. Ma B. Detmar M. Nitric oxide mediates lymphatic vessel activation via soluble guanylate cyclase alpha1beta1: impact on inflammation.FASEB J. 2008; 22: 530-537Crossref PubMed Scopus (29) Google Scholar LECs were grown on fibronectin-coated 24-well plates until confluence. In all, 0.5 mL of neutralized isotonic bovine dermal collagen type I (Vitrogen; Celtrix Laboratories, Palo Alto, CA) in the presence or absence of apelin-13 (50 to 1000 ng/mL) or apelin-36 (50 to 1000 ng/mL) was added to the cells. After incubation at 37°C for 24 hours, cells were fixed with 4% paraformaldehyde for 30 minutes at 4°C. Before lymphatic endothelial cells form tubes in collagen gels, endothelial cells connect with each other to make cords in vitro. Representative images were captured, and the total length of cordlike structures per area was measured using IP-LAB software version 4.0. All studies were performed in triplicate. Statistical analyses were performed using the unpaired Student's t-test. LECs were grown into confluence on the fibronectin-coated surface of tissue culture inserts of 0.4-μm pore size (Transwell; Corning, Lowell, MA) and then in serum-free endothelial basal medium for 24 hours. Apelin-13 (500 to 1000 ng/mL) was placed into the upper and lower chambers for 6 hours. Fluorescein isothiocyanate-dextran was added to the upper chambers, and the apparatus was then placed in a CO2 incubator at 37°C. After incubation for 15 minutes, a 100-µL sample was taken from the lower chamber, and the absorbance of fluorescein isothiocyanate-dextran was determined at 492 nm using a spectrophotometer (Fluoroskan Ascent; Thermo Fisher Scientific, Waltham, MA). Transgenic mice harboring apelin under the control of keratin 14 (K14-apelin) were generated on a C57BL/6 background, as described previously.31Kidoya H. Naito H. Takakura N. Apelin induces enlarged and nonleaky blood vessels for functional recovery from ischemia.Blood. 2010; 115: 3166-3174Crossref PubMed Scopus (98) Google Scholar A total of 10 K14-apelin mice and wild-type (WT) mice, 12 weeks old (n = 5/group) were exposed to a single dose of 200 mJ/cm2 of UVB irradiation using 10 Toshiba FL-20 SD fluorescent lamps that deliver energy in the UVB wavelength range (280 to 340 nm) with maximum energy at a wavelength of 305 nm. On day 3 or 4 after UVB irradiation, mouse ears were collected and were processed for histological analysis of frozen sections. Control mice without UVB irradiation were also analyzed. All procedures including UVB irradiation were performed under anesthesia. The study was approved by the ethics committee of Shiseido Research Center in accordance with guidelines of the U.S. National Institutes of Health (7th edition). WT and K14-apelin mice (n = 5/group) were anesthetized with avertin (0.4 g/kg; Sigma-Aldrich), and 1 mL of a 1% solution of Evans Blue dye in 0.9% NaCl was injected intradermally at the inner surface of the rim of the ear, using a 10-mL Hamilton syringe, to visualize the lymphatic vessels. Mouse ears were photographed at 1 and 5 minutes after dye injection. To determine blood vascular permeability, a Miles assay was performed as described previously.31Kidoya H. Naito H. Takakura N. Apelin induces enlarged and nonleaky blood vessels for functional recovery from ischemia.Blood. 2010; 115: 3166-3174Crossref PubMed Scopus (98) Google Scholar Briefly, mice were anesthetized and intravenously injected with 100 µL of a 1% solution of Evans Blue dye in 0.9% NaCl. At 60 minutes after dye injection, ears were photographed and then removed. The dye was eluted from the dissected samples with formamide at 56°C, and the optical density was measured by spectrophotometry (Biotrak II; GE Healthcare, Piscataway, NJ) at 620 nm. Immunofluorescence analysis was performed on cryostat sections (6 μm thick) of mouse ears using antibodies against the macrophage monocyte marker CD11b (Pharmingen; BD Biosciences, San Diego, CA), the blood vessel-specific marker Meca-32 (BD Biosciences), and the lymphatic-specific marker podoplanin (Acris Antibodies, Hiddenhausen, Germany) and using corresponding secondary antibodies labeled with Alexa Fluor 488 or Alexa Fluor 594 (Molecular Probes; Invitrogen). Routine H&E staining was also performed. Sections were examined with an Olympus AX80T microscope (Olympus, Tokyo, Japan), and images were captured with a DP controller digital camera (DP71; Olympus). Morphometric analyses were performed using IP-LAB software version 4.0, as described previously.28Kajiya K. Hirakawa S. Ma B. Drinnenberg I. Detmar M. Hepatocyte growth factor promotes lymphatic vessel formation and function.EMBO J. 2005; 24: 2885-2895Crossref PubMed Scopus (256) Google Scholar Three different fields of each section were examined, and ear thickness and the number of macrophages and average vessel size in the dermis were determined. Statistical analyses were performed using the unpaired Student's t-test. To investigate whether apelin functions in lymphatic endothelial cells, we analyzed the expression of apelin receptor APJ in LECs. Western blot analyses demonstrated that APJ was expressed by both LECs and HUVECs (Figure 1A). Moreover, immunofluorescence analysis of mouse ear skin using antibodies against APJ and the blood vessel marker Meca-32 or the lymphatic marker podoplanin revealed that APJ was expressed by both lymphatic vessels (Figure 1C) and blood vessels (Figure 1B) in vivo. Apelin is known to activate the phosphorylation of Akt in HUVECs.32Masri B. Morin N. Pedebernade L. Knibiehler B. Audigier Y. The apelin receptor is coupled to Gi1 or Gi2 protein and is differentially desensitized by apelin fragments.J Biol Chem. 2006; 281: 18317-18326Crossref PubMed Scopus (122) Google Scholar Treatment of LECs with 1000 ng/mL apelin-36 resulted in the increased phosphorylation of Akt, compared with untreated cells (Figure 1D). Migration assays performed to further characterize the effects of apelin on LEC revealed that both apelin-13 and apelin-36 induced LEC migration in a dose-dependent manner (Figure 1, E and F). To investigate whether apelin stimulation might promote cord formation of lymphatic endothelial cells in vitro, confluent LECs were overlaid with type I collagen. Cord formation of LECs was clearly enhanced in the presence of apelin-13 and apelin-36, compared with control cells (Figure 1, G–I). Morphometric analyses confirmed that both apelin-13 and apelin-36 induced cord formation of LECs dose-dependently (P < 0.01) (Figure 1, J and K). A permeability assay was performed to determine whether apelin contributes to the stabilization of LECs in vitro. LECs were cultured on Transwell culture inserts into confluence, and the concentration of fluorescein isothiocyanate-dextran that permeated across the culture inserts was measured with or without apelin-13. The addition of 500 or 1000 ng/mL of apelin-13 in LECs decreased the fluorescence intensity of permeated fluorescein isothiocyanate-dextran, indicating that apelin promoted the stabilization of LECs (P < 0.01) (Figure 1L). To determine the functional role of apelin in the cutaneous vasculature in vivo, transgenic mice harboring apelin under the control of keratin-14 (K14-apelin) and WT control mice were exposed to 200 mJ cm−2 of UVB irradiation. H&E staining of skin sections at 3 days after UVB irradiation revealed characteristic features of acute photodamage in the ear skin of WT mice, including epidermal hyperplasia and edema formation in the dermis (Figure 2C). Of note, K14-apelin mouse ears irradiated with UVB were closely similar to those of non-UVB-irradiated skin (Figure 2, A, B, and D). In a physiological condition, by contrast, no obvious difference was found between WT and K14-apelin mice. The measurement of skin thickness confirmed that ear swelling was decreased in K14-apelin mouse ears, compared with WT mouse ears, after UVB irradiation (P < 0.05) (Figure 2I). Immunohistochemical staining for a monocyte macrophage marker, CD11b, demonstrated an increased number of infiltrating macrophages in the dermis of WT ears after UVB irradiation, compared with non-UVB-irradiated skin (Figure 2, E–G); however, the ear skin of UVB-irradiated K14-apelin mice exhibited decreased macrophage infiltration in the dermis (Figure 2H). Morphometric analyses confirmed a decreased number of infiltrating macrophages in the dermis of UVB-irradiated K14-apelin mouse ears, compared with WT mice after UVB-irradiation (P < 0.01) (Figure 2J). To investigate how activation of apelin/APJ signaling attenuates edema formation and inflammation induced by UVB irradiation, we analyzed cutaneous lymphatic vessels after UVB irradiation. To visualize lymphatic vessels, Evans Blue dye was injected intradermally into the rim of mouse ears. At 1 and 5 minutes after injection, Evans Blue dye had extravasated from lymphatic vessels in UVB-irradiated WT skin, but such leakage was attenuated in K14-apelin mice (Figure 3, A–D). Next, Miles assay was performed to determine the effects of apelin on blood vessels. UVB exposure induced marked leakage of Evans Blue dye in WT mice (Figure 3E), but such leakage was attenuated in K14-apelin mice (Figure 3F). Quantitative analysis demonstrated that the increase of dye leakage in WT mouse ears was significantly blocked in UVB-irradiated K14-apelin mice (Figure 3G). We performed double immunofluorescence staining for the lymphatic marker podoplanin and the blood vascular marker Meca-32. In a physiological condition, the density of blood vessels was similar between WT and K14-apelin mice, but K14-apelin mice exhibited increased size of blood vessels, as has been demonstrated previously.31Kidoya H. Naito H. Takakura N. Apelin induces enlarged and nonleaky blood vessels for functional recovery from ischemia.Blood. 2010; 115: 3166-3174Crossref PubMed Scopus (98) Google Scholar No great difference in lymphatic vessel formation was immediately evident in K14-apelin mice; however, precise histological examination of skin stained for podoplanin revealed enlarged lymphatic vessels of K14-apelin mice, compared with WT mice (Figure 3, H–M). Enlargement of lymphatic vessels and blood vessels was induced in WT mice after UVB irradiation; surprisingly, however, in K14-apelin mice the UVB-induced enlargement of lymphatic and blood vessels was inhibited (Figure 3, N–S). Morphometric analyses of sections demonstrated that the average size of lymphatic vessels and blood vessels was significantly decreased in skin of UVB-irradiated K14-apelin mice, compared with WT mice after UVB-irradiation (−74%, P < 0.05 for lymphatic vessels; −26%, P < 0.05 for blood vessels; Figure 3, T and U). Apelin has been recently reported to be an important regulator of blood vessel formation. The present study reveals, for the first time, that the apelin receptor APJ is expressed by human lymphatic endothelial cells and that apelin/APJ signaling plays a crucial role in UVB-induced inflammation through stabilization of blood and lymphatic vessels. Acute photodamage of the skin is characterized by epidermal hyperplasia, erythema, and edema formation. Edema is caused by accumulation of extracellular fluid due to excess leakage from hyperpermeable blood vessels33Persson C.G. Role of plasma exudation in asthmatic airways.Lancet. 1986; 2: 1126-1129Abstract PubMed Scopus (166) Google Scholar and by a failure of lymphatic vessels to sufficiently drain the fluid from the interstitium.12Kajiya K. Hirakawa S. Detmar M. Vascular endothelial growth factor-a mediates ultraviolet B-induced impairment of lymphatic vessel function.Am J Pathol. 2006; 169: 1496-1503Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar Moreover, the dysfunction of lymphatic vessels also results in reduced clearance of macrophages from the tissue via lymphatic drainage,34Kataru R.P. Jung K. Jang C. Yang H. Schwendener R.A. Baik J.E. Han S.H. Alitalo K. Koh G.Y. Critical role of CD11b+ macrophages and VEGF in inflammatory lymphangiogenesis, antigen clearance, and inflammation resolution.Blood. 2009; 113: 5650-5659Crossref PubMed Scopus (295) Google Scholar suggesting that the function of lymphatic vessels is profoundly related to the process of UVB-induced inflammation. We have previously reported that skin tissues during days 2 to 4 after UVB irradiation exhibit enlargement of lymphatic vessels and macrophage infiltration.13Kajiya K. Sawane M. Huggenberger R. Detmar M. Activation of the VEGFR-3 pathway by VEGF-C attenuates UVB-induced edema formation and skin inflammation by promoting lymphangiogenesis.J Invest Dermatol. 2009; 129: 1292-1298Crossref PubMed Scopus (81) Google Scholar Surprisingly, K14-apelin mice inhibited the enlargement and hyperpermeability of lymphatic vessels and macrophage infiltration by UVB irradiation, indicating that apelin plays a defensive role in UVB-induced inflammation. How does apelin attenuate skin inflammation? Although the function of the apelin/APJ system in endothelial cells is known to activate endothelial nitric oxide synthase (eNOS), resulting in decreased the blood pressure with vasodilation,22Ishida J. Hashimoto T. Hashimoto Y. Nishiwaki S. Iguchi T. Harada S. Sugaya T. Matsuzaki H. Yamamoto R. Shiota N. Okunishi H. Kihara M. Umemura S. Sugiyama F. Yagami K. Kasuya Y. Mochizuki N. Fukamizu A. Regulatory roles for APJ, a seven-transmembrane receptor related to angiotensin-type 1 receptor in blood pressure in vivo.J Biol Chem. 2004; 279: 26274-26279Crossref PubMed Scopus (359) Google Scholar the present results indicate that apelin attenuates the abnormal enlargement of lymphatic and blood vessels in inflamed skin. Additionally, plasma extravasation was markedly decreased in K14-apelin mice, compared with WT mice after UVB irradiation, indicating a protective role of apelin in blood vessels, as described recently.31Kidoya H. Naito H. Takakura N. Apelin induces enlarged and nonleaky blood vessels for functional recovery from ischemia.Blood. 2010; 115: 3166-3174Crossref PubMed Scopus (98) Google Scholar Moreover, we have previously demonstrated that systemic blockade of lymphatic function by the VEGFR-3 pathway prolongs UVB-induced edema formation and inflammation,35Kajiya K. Detmar M. An important role of lymphatic vessels in the control of UVB-induced edema formation and inflammation.J Invest Dermatol. 2006; 126: 919-921Crossref PubMed Scopus (13) Google Scholar whereas intradermal injection of VEGF-C accelerates the resolution of UVB-induced edema and inflammation by inducing lymphangiogenesis.13Kajiya K. Sawane M. Huggenberger R. Detmar M. Activation of the VEGFR-3 pathway by VEGF-C attenuates UVB-induced edema formation and skin inflammation by promoting lymphangiogenesis.J Invest Dermatol. 2009; 129: 1292-1298Crossref PubMed Scopus (81) Google Scholar In contrast to such VEGF-C treatment, no major differences in the number of lymphatic vessels were observed in skin of K14-apelin mice. It was therefore of considerable interest to see the differential mechanism of attenuating inflammation by apelin. A previous study from our research group demonstrated that acute UVB irradiation increases overextension of lymphatic vessels, which leads to impaired fluid transport and so contributes to prolonged edema formation.12Kajiya K. Hirakawa S. Detmar M. Vascular endothelial growth factor-a mediates ultraviolet B-induced impairment of lymphatic vessel function.Am J Pathol. 2006; 169: 1496-1503Abstract Full Text Full Text PDF PubMed Scopus (81) Google Scholar Of note, the hyperpermeability of lymphatic vessels was blocked in K14-apelin mice after UVB irradiation, compared with that observed in UVB-irradiated WT mice, and the permeability assay in vitro demonstrated that apelin blocked the permeability of human lymphatic endothelial cells. Taken together, these data suggest that inhibiting hyperpermeability by enhancing apelin expression could facilitate transport of tissue fluid, resulting in rapid resolution of edema and the related inflammation induced by UVB. The molecular events that regulate blood vessel formation, especially the caliber size determination of blood vessels by apelin, have been recently suggested. A remarkable study showed that the apelin/APJ system is involved in downstream signaling of Ang1/Tie2 in blood vessel formation.26Kidoya H. Ueno M. Yamada Y. Mochizuki N. Nakata M. Yano T. Fujii R. Takakura N. Spatial and temporal role of the apelin/APJ system in the caliber size regulation of blood vessels during angiogenesis.EMBO J. 2008; 27: 522-534Crossref PubMed Scopus (196) Google Scholar With the present study, we have demonstrated that apelin induces migration and cord formation of LECs and that lymphatic vascular size in K14-apelin mice is greater than in WT mice. Given that apelin induces expression of the junctional proteins claudin-5 and vascular endothelial cadherin (VE-cadherin) in blood vessels, resulting in abundant cell-to-cell contact and regulation of endothelial cell assembly, it is possible that apelin inhibits hyperpermeability of lymphatic vessels and inflammation by UVB-irradiation via the regulation of the junctional protein in lymphatic endothelial cells.26Kidoya H. Ueno M. Yamada Y. Mochizuki N. Nakata M. Yano T. Fujii R. Takakura N. Spatial and temporal role of the apelin/APJ system in the caliber size regulation of blood vessels during angiogenesis.EMBO J. 2008; 27: 522-534Crossref PubMed Scopus (196) Google Scholar Further studies would be needed to clarify a molecular regulation of lymphatic integrity by apelin and to determine whether apelin is involved in downstream Ang1/Tie2 signaling in lymphatic vessels. In summary, the present results indicate that apelin plays a functional role in the stabilization of lymphatic vessels in inflamed tissues. Apelin might be a new suitable target for prevention of UVB-induced skin inflammation. We thank Fumika Miyohashi for her technical assistance.
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