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• W2537300122 abstract "The vasculature influences the progression and resolution of tissue inflammation. Capillaries express vascular endothelial growth factor (VEGF) receptors, including neuropilins (NRPs), which regulate interstitial fluid flow. NRP2, a receptor of VEGFA and semaphorin (SEMA) 3F ligands, is expressed in the vascular and lymphatic endothelia. Previous studies have demonstrated that blocking VEGF receptor 2 attenuates VEGFA-induced vascular permeability. The inhibition of NRP2 was hypothesized to decrease vascular permeability as well. Unexpectedly, massive tissue swelling and edema were observed in Nrp2−/− mice compared with wild-type littermates after delayed-type hypersensitivity reactions. Vascular permeability was twofold greater in inflamed blood vessels in Nrp2-deficient mice compared to those in Nrp2-intact littermates. The addition of exogenous SEMA3F protein inhibited vascular permeability in Balb/cJ mice, suggesting that the loss of endogenous Sema3F activity in the Nrp2-deficient mice was responsible for the enhanced vessel leakage. Functional lymphatic capillaries are necessary for draining excess fluid after inflammation; however, Nrp2-mutant mice lacked superficial lymphatic capillaries, leading to 2.5-fold greater fluid retention and severe lymphedema after inflammation. In conclusion, Nrp2 deficiency increased blood vessel permeability and decreased lymphatic vessel drainage during inflammation, highlighting the importance of the NRP2/SEMA3F pathway in the modulation of tissue swelling and resolution of postinflammatory edema. The vasculature influences the progression and resolution of tissue inflammation. Capillaries express vascular endothelial growth factor (VEGF) receptors, including neuropilins (NRPs), which regulate interstitial fluid flow. NRP2, a receptor of VEGFA and semaphorin (SEMA) 3F ligands, is expressed in the vascular and lymphatic endothelia. Previous studies have demonstrated that blocking VEGF receptor 2 attenuates VEGFA-induced vascular permeability. The inhibition of NRP2 was hypothesized to decrease vascular permeability as well. Unexpectedly, massive tissue swelling and edema were observed in Nrp2−/− mice compared with wild-type littermates after delayed-type hypersensitivity reactions. Vascular permeability was twofold greater in inflamed blood vessels in Nrp2-deficient mice compared to those in Nrp2-intact littermates. The addition of exogenous SEMA3F protein inhibited vascular permeability in Balb/cJ mice, suggesting that the loss of endogenous Sema3F activity in the Nrp2-deficient mice was responsible for the enhanced vessel leakage. Functional lymphatic capillaries are necessary for draining excess fluid after inflammation; however, Nrp2-mutant mice lacked superficial lymphatic capillaries, leading to 2.5-fold greater fluid retention and severe lymphedema after inflammation. In conclusion, Nrp2 deficiency increased blood vessel permeability and decreased lymphatic vessel drainage during inflammation, highlighting the importance of the NRP2/SEMA3F pathway in the modulation of tissue swelling and resolution of postinflammatory edema. The endothelium plays a crucial role in inflammatory reactions within tissues, and chronic inflammatory diseases (eg, psoriasis, eczema) are associated with neovascularization. The vasculature mediates two phases of acute inflammation: the leakage phase, modulated by endothelial cells (ECs), which results in edema and swelling, and the drainage phase, controlled by lymphatic ECs (LECs), which clears interstitial fluid and results in resolution and return to homeostasis.1Halin C. Detmar M. Chapter 1. Inflammation, angiogenesis, and lymphangiogenesis.Methods Enzymol. 2008; 445: 1-25Crossref PubMed Scopus (51) Google Scholar The cutaneous delayed-type hypersensitivity (DTH) assay (also called the contact hypersensitivity assay) has been routinely used for studying the inflammatory endothelium. Inflamed vessels undergo remodeling characterized by increased permeability, increased flow, and an influx of immune cells.2Huggenberger R. Detmar M. The cutaneous vascular system in chronic skin inflammation.J Investig Dermatol Symp Proc. 2011; 15: 24-32Abstract Full Text Full Text PDF PubMed Scopus (107) Google Scholar The vascular endothelial growth factors (VEGFs) A and C pathways are known to be pivotal in regulating vascular permeability and lymphatic drainage, respectively, during inflammation.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, 4Kajiya K. Detmar M. An important role of lymphatic vessels in the control of UVB-induced edema formation and inflammation.J Invest Dermatol. 2006; 126: 919-921Abstract Full Text Full Text PDF PubMed Scopus (13) Google Scholar, 5Huggenberger R. Ullmann S. Proulx S.T. Pytowski B. Alitalo K. Detmar M. Stimulation of lymphangiogenesis via VEGFR-3 inhibits chronic skin inflammation.J Exp Med. 2010; 207: 2255-2269Crossref PubMed Scopus (186) Google Scholar, 6Huggenberger R. Siddiqui S.S. Brander D. Ullmann S. Zimmermann K. Antsiferova M. Werner S. Alitalo K. Detmar M. An important role of lymphatic vessel activation in limiting acute inflammation.Blood. 2011; 117: 4667-4678Crossref PubMed Scopus (185) Google Scholar For example, inflammation increases VEGFA, also called vascular permeability factor, which binds VEGF receptor (VEGFR)-2 in ECs to cause fluid leakage, resulting in edema.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, 7Brown L.F. Harrist T.J. Yeo K.T. Stahle-Backdahl M. Jackman R.W. Berse B. Tognazzi K. Dvorak H.F. Detmar M. Increased expression of vascular permeability factor (vascular endothelial growth factor) in bullous pemphigoid, dermatitis herpetiformis, and erythema multiforme.J Invest Dermatol. 1995; 104: 744-749Abstract Full Text PDF PubMed Scopus (137) Google Scholar Keratin 14–VEGFA transgenic mice display excessive edema and psoriasis-like symptoms,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, 8Xia Y.P. Li B. Hylton D. Detmar M. Yancopoulos G.D. Rudge J.S. Transgenic delivery of VEGF to mouse skin leads to an inflammatory condition resembling human psoriasis.Blood. 2003; 102: 161-168Crossref PubMed Scopus (312) Google Scholar and the inhibition of VEGFR2 with blocking antibodies during inflammation in hemizygous keratin 14–VEGFA mice inhibits the extent of edema.5Huggenberger R. Ullmann S. Proulx S.T. Pytowski B. Alitalo K. Detmar M. Stimulation of lymphangiogenesis via VEGFR-3 inhibits chronic skin inflammation.J Exp Med. 2010; 207: 2255-2269Crossref PubMed Scopus (186) Google Scholar During resolution, fluid that leaks from blood vessels into the interstitial space is drained by lymphatic capillaries through lymphatic ducts to lymph nodes. Lymphatic capillary dysfunction can result in lymphedema.9Witte C.L. Witte M.H. Disorders of lymph flow.Acad Radiol. 1995; 2: 324-334Abstract Full Text PDF PubMed Scopus (38) Google Scholar Keratin 14–VEGFC transgenic mice stimulate lymphangiogenesis, providing a conduit for drainage and exhibit reduced lymphedema after inflammation,6Huggenberger R. Siddiqui S.S. Brander D. Ullmann S. Zimmermann K. Antsiferova M. Werner S. Alitalo K. Detmar M. An important role of lymphatic vessel activation in limiting acute inflammation.Blood. 2011; 117: 4667-4678Crossref PubMed Scopus (185) Google Scholar while antibodies used for blocking VEGFR3 during inflammation promote lymphedema.5Huggenberger R. Ullmann S. Proulx S.T. Pytowski B. Alitalo K. Detmar M. Stimulation of lymphangiogenesis via VEGFR-3 inhibits chronic skin inflammation.J Exp Med. 2010; 207: 2255-2269Crossref PubMed Scopus (186) Google Scholar Neuropilins (NRPs) are transmembrane coreceptors that mediate both stimulatory signals from VEGF family proteins and inhibitory signals from class 3 semaphorin (SEMA3) ligands.10Fujisawa H. Kitsukawa T. Receptors for collapsin/semaphorins.Curr Opin Neurobiol. 1998; 8: 587-592Crossref PubMed Scopus (116) Google Scholar, 11Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor.Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2072) Google Scholar, 12Favier B. Alam A. Barron P. Bonnin J. Laboudie P. Fons P. Mandron M. Herault J.P. Neufeld G. Savi P. Herbert J.M. Bono F. Neuropilin-2 interacts with VEGFR-2 and VEGFR-3 and promotes human endothelial cell survival and migration.Blood. 2006; 108: 1243-1250Crossref PubMed Scopus (226) Google Scholar ECs express two NRP receptors, NRP1 and NRP2, during development.11Soker S. Takashima S. Miao H.Q. Neufeld G. Klagsbrun M. Neuropilin-1 is expressed by endothelial and tumor cells as an isoform-specific receptor for vascular endothelial growth factor.Cell. 1998; 92: 735-745Abstract Full Text Full Text PDF PubMed Scopus (2072) Google Scholar, 13Herzog Y. Kalcheim C. Kahane N. Reshef R. Neufeld G. Differential expression of neuropilin-1 and neuropilin-2 in arteries and veins.Mech Dev. 2001; 109: 115-119Crossref PubMed Scopus (215) Google Scholar NRP1 is essential for angiogenesis and cardiovascular development, and Nrp1-knockout (KO) mice die in utero from vascular defects.13Herzog Y. Kalcheim C. Kahane N. Reshef R. Neufeld G. Differential expression of neuropilin-1 and neuropilin-2 in arteries and veins.Mech Dev. 2001; 109: 115-119Crossref PubMed Scopus (215) Google Scholar, 14Kawasaki T. Kitsukawa T. Bekku Y. Matsuda Y. Sanbo M. Yagi T. Fujisawa H. A requirement for neuropilin-1 in embryonic vessel formation.Development. 1999; 126: 4895-4902Crossref PubMed Google Scholar NRP2 is expressed in capillary (and venous) ECs of the blood and lymphatic system and is not required for developmental angiogenesis.13Herzog Y. Kalcheim C. Kahane N. Reshef R. Neufeld G. Differential expression of neuropilin-1 and neuropilin-2 in arteries and veins.Mech Dev. 2001; 109: 115-119Crossref PubMed Scopus (215) Google Scholar, 15Yuan L. Moyon D. Pardanaud L. Breant C. Karkkainen M.J. Alitalo K. Eichmann A. Abnormal lymphatic vessel development in neuropilin 2 mutant mice.Development. 2002; 129: 4797-4806Crossref PubMed Google Scholar Nrp2-deficient mice survive to adulthood and are fertile but smaller in size than are wild-type (WT) littermates.16Giger R.J. Cloutier J.F. Sahay A. Prinjha R.K. Levengood D.V. Moore S.E. Pickering S. Simmons D. Rastan S. Walsh F.S. Kolodkin A.L. Ginty D.D. Geppert M. Neuropilin-2 is required in vivo for selective axon guidance responses to secreted semaphorins.Neuron. 2000; 25: 29-41Abstract Full Text Full Text PDF PubMed Scopus (367) Google Scholar, 17Walz A. Rodriguez I. Mombaerts P. Aberrant sensory innervation of the olfactory bulb in neuropilin-2 mutant mice.J Neurosci. 2002; 22: 4025-4035Crossref PubMed Google Scholar Nrp2−/− embryos have fewer lymphatic vessels than do Nrp2+/+ embryos but do not show overt signs of edema.15Yuan L. Moyon D. Pardanaud L. Breant C. Karkkainen M.J. Alitalo K. Eichmann A. Abnormal lymphatic vessel development in neuropilin 2 mutant mice.Development. 2002; 129: 4797-4806Crossref PubMed Google Scholar, 18Xu Y. Yuan L. Mak J. Pardanaud L. Caunt M. Kasman I. Larrivee B. Del Toro R. Suchting S. Medvinsky A. Silva J. Yang J. Thomas J.L. Koch A.W. Alitalo K. Eichmann A. Bagri A. Neuropilin-2 mediates VEGF-C-induced lymphatic sprouting together with VEGFR3.J Cell Biol. 2010; 188: 115-130Crossref PubMed Scopus (253) Google Scholar Few studies have analyzed the physiologic function of the NRP2 receptor in ECs or LECs beyond developmental stages; however, mutations in the human NRP2 gene have been found in families with primary lymphedema.19Ferrell R.E. Kimak M.A. Lawrence E.C. Finegold D.N. Candidate gene analysis in primary lymphedema.Lymphatic Res Biol. 2008; 6: 69-76Crossref PubMed Scopus (50) Google Scholar Normally, NRP2 is down-regulated or absent in adult quiescent capillaries, but NRP2 can be up-regulated during ischemia.20Klagsbrun M. Takashima S. Mamluk R. The role of neuropilin in vascular and tumor biology.Adv Exp Med Biol. 2002; 515: 33-48Crossref PubMed Scopus (175) Google Scholar, 21Caunt M. Mak J. Liang W.C. Stawicki S. Pan Q. Tong R.K. Kowalski J. Ho C. Reslan H.B. Ross J. Berry L. Kasman I. Zlot C. Cheng Z. Le Couter J. Filvaroff E.H. Plowman G. Peale F. French D. Carano R. Koch A.W. Wu Y. Watts R.J. Tessier-Lavigne M. Bagri A. Blocking neuropilin-2 function inhibits tumor cell metastasis.Cancer Cell. 2008; 13: 331-342Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar Since NRP2 is a mediator of the VEGFA and VEGFC pathways12Favier B. Alam A. Barron P. Bonnin J. Laboudie P. Fons P. Mandron M. Herault J.P. Neufeld G. Savi P. Herbert J.M. Bono F. Neuropilin-2 interacts with VEGFR-2 and VEGFR-3 and promotes human endothelial cell survival and migration.Blood. 2006; 108: 1243-1250Crossref PubMed Scopus (226) Google Scholar, 22Coma S. Allard-Ratick M. Akino T. van Meeteren L.A. Mammoto A. Klagsbrun M. GATA2 and Lmo2 control angiogenesis and lymphangiogenesis via direct transcriptional regulation of neuropilin-2.Angiogenesis. 2013; 16: 939-952Crossref PubMed Scopus (36) Google Scholar, 23Karpanen T. Heckman C.A. Keskitalo S. Jeltsch M. Ollila H. Neufeld G. Tamagnone L. Alitalo K. Functional interaction of VEGF-C and VEGF-D with neuropilin receptors.FASEB J. 2006; 20: 1462-1472Crossref PubMed Scopus (240) Google Scholar and is expressed in both ECs and LECs, we hypothesized that NRP2 may play a role in the regulation of fluid dynamics after inflammation during both the leakage and drainage phases. We predicted that inhibiting NRP2 would have effects similar to inhibiting VEGFR2 since both are coreceptors for VEGFA. However, our results show the opposite effect—Nrp2-deficient mice are hyperpermeable. Additionally, we demonstrate that adult Nrp2-mutant mice show extensive lymphedema under inflammatory conditions. These phenotypes are due to the promiscuous nature of the Nrp2 receptor, which also binds to Sema3F, an endogenous angiogenesis and lymphangiogenesis inhibitor.24Wu F. Zhou Q. Yang J. Duan G.J. Ou J.J. Zhang R. Pan F. Peng Q.P. Tan H. Ping Y.F. Cui Y.H. Qian C. Yan X.C. Bian X.W. Endogenous axon guiding chemorepulsant semaphorin-3F inhibits the growth and metastasis of colorectal carcinoma.Clin Cancer Res. 2011; 17: 2702-2711Crossref PubMed Scopus (38) Google Scholar, 25Doci C.L. Mikelis C.M. Lionakis M.S. Molinolo A.A. Gutkind J.S. Genetic identification of SEMA3F as an antilymphangiogenic metastasis suppressor gene in head and neck squamous carcinoma.Cancer Res. 2015; 75: 2937-2948Crossref PubMed Scopus (30) Google Scholar All mice were maintained under specific pathogen-free conditions in a facility accredited by the Association for Assessment and Accreditation of Laboratory Animal Care. The care and experimental procedures were conducted in compliance with the NIH's Guide for the Care and Use of Laboratory Animals26Committee for the Update of the Guide for the Care and Use of Laboratory Animals National Research CouncilGuide for the Care and Use of Laboratory Animals.Eighth Edition. National Academies Press, Washington, DC2011Crossref Google Scholar and were approved by the Institutional Animal Care and Use Committee at Boston Children's Hospital (Boston, MA). Nrp2+/LacZ mice were a gift from Dr. Seiji Takashima (Osaka University Graduate School of Medicine, Osaka, Japan).27Takashima S. Kitakaze M. Asakura M. Asanuma H. Sanada S. Tashiro F. Niwa H. Miyazaki Ji J. Hirota S. Kitamura Y. Kitsukawa T. Fujisawa H. Klagsbrun M. Hori M. Targeting of both mouse neuropilin-1 and neuropilin-2 genes severely impairs developmental yolk sac and embryonic angiogenesis.Proc Natl Acad Sci U S A. 2002; 99: 3657-3662Crossref PubMed Scopus (322) Google Scholar These mice were generated by replacing the first coding exon of Nrp2 with a promoterless Escherichia coli β-galactosidase gene. These mice were backcrossed to the C57BL/6J strain for >10 generations. Nrp2Lacz/LacZ pups die soon after birth. Nrp2+/LacZ mice are of normal size and weight and are indistinguishable from Nrp2+/+ littermates. Nrp2+/gfp mice (also known as Nrp2tm1.2Mom/MomJ; stock number 006700) were purchased from The Jackson Laboratory (Bar Harbor, ME) and maintained in the C57BL/6J background. Nrp2gfp/gfp mice (functionally referred to as Nrp2−/− mice) are viable, fertile, and nonedematous at baseline. C57BL/6J and Balb/cJ mice were purchased from The Jackson Laboratory. DTH reactions were induced in the ears of female mice (n = 5 mice per group) at 8 to 12 weeks of age, as previously described.28Mamluk R. Klagsbrun M. Detmar M. Bielenberg D.R. Soluble neuropilin targeted to the skin inhibits vascular permeability.Angiogenesis. 2005; 8: 217-227Crossref PubMed Scopus (34) Google Scholar DTH was performed on several strains, including Balb/cJ; C57BL/6J; Nrp2+/LacZ and the resultant offspring from Nrp2+/gfp × Nrp2+/gfp matings, including Nrp2+/+ (WT), Nrp2+/gfp (heterozygous, called Nrp2+/−), and Nrp2gfp/gfp (KO, called Nrp2−/−). Mice were sensitized by the topical application of a 2% oxazolone (4-ethoxymethylene-2 phenyl-2-oxazoline-5-one; Sigma-Aldrich, St Louis, MO) solution in acetone/olive oil (4:1 vol/vol) to the shaved abdomen (50 μL) and to each paw (5 μL). Mice were challenged on the right ear 5 days after sensitization (day 0) by topical application of a 1% oxazolone solution (20 μL total; 10 μL on each side of the right ear), and left ears were treated with vehicle alone. The thicknesses of vehicle- and oxazolone-treated ears were measured using a Mitutoyo gauge, daily for up to 10 days, as described for the mouse ear-swelling test.29Gad S.C. Dunn B.J. Dobbs D.W. Reilly C. Walsh R.D. Development and validation of an alternative dermal sensitization test: the mouse ear swelling test (MEST).Toxicol Appl Pharmacol. 1986; 84: 93-114Crossref PubMed Scopus (283) Google Scholar The increase in ear thickness over the baseline thickness (measured in microns) was used as a measurement of the extent of inflammation and plotted versus time. The experiment comparing WT, heterozygous, and KO mice was repeated three times. Miles assays were performed on female Balb/cJ mice (n = 4 mice per group) at 8 weeks of age. Mice were shaved 1 day before the experiment. Mice were anesthetized with tribromoethanol delivered i.p. Evans Blue dye (100 μL of a 1% solution in normal saline) was injected via the tail vein. After 10 minutes, proteins (50 μL) or vehicle were injected s.c. to induce permeability, including phosphate-buffered saline (PBS), recombinant human VEGFA165 (200 ng/mL = 4.76 nmol/L) (R&D Systems, Minneapolis, MN), recombinant human VEGFA165 (4.76 nmol/L) plus SEMA3F (95 nmol/L; 20-fold excess), recombinant human VEGFA165 (4.76 nmol/L) plus SEMA3F (950 nmol/L; 200-fold excess), and recombinant human VEGFA165 (4.76 nmol/L) plus bevacizumab (950 nmol/L; 200-fold excess) (Genentech, South San Francisco, CA). SEMA3F protein was purified as previously described.30Bielenberg D.R. Shimizu A. Klagsbrun M. Semaphorin-induced cytoskeletal collapse and repulsion of endothelial cells.Methods Enzymol. 2008; 443: 299-314Crossref PubMed Scopus (20) Google Scholar After 20 minutes, the mice were euthanized in a CO2 chamber, and the area of skin that included the extravasated dye was excised and imaged. Evans Blue dye was extracted from the skin pieces by incubation in formamide at room temperature for 5 days. The absorbance at 620 nm was measured using a spectrophotometer. This experiment was repeated three independent times with 4 mice per group in the first two experiments and 3 mice per group in the third experiment, for a total of 11 mice per group. The means ± SEM of each experiment were plotted and compared. Modified Miles assays were performed on the female, adult (8- to 12-week-old) resultant offspring from Nrp2+/gfp × Nrp2+/gfp matings, including Nrp2+/+ (WT), Nrp2+/− (heterozygous), and Nrp2−/− (KO), on day 2 after DTH (n = 3 mice per group). Evans Blue dye (100 μL of a 1% solution in normal saline) was injected via the tail vein. Dye leaked from blood vessels into the interstitial space in the challenged ears; vehicle-treated ears were used as controls. WT mice not undergoing DTH reactions were injected in the ear with recombinant human VEGFA165 as a positive control. Extravasated dye was quantified in a spectrophotometer (620 nm). Evans Blue dye (10 μL of a 1% solution in normal saline) was injected into the tip of vehicle-treated or oxazolone-challenged ears on the 3rd day after DTH in isoflurane-anesthetized Balb/cJ mice (n = 5 mice). Mice were imaged within 5 minutes for the visualization of lymphatic vessels. Dye-retention experiments were performed as described.6Huggenberger R. Siddiqui S.S. Brander D. Ullmann S. Zimmermann K. Antsiferova M. Werner S. Alitalo K. Detmar M. An important role of lymphatic vessel activation in limiting acute inflammation.Blood. 2011; 117: 4667-4678Crossref PubMed Scopus (185) Google Scholar Evans Blue dye (5 μL of a 1% solution in normal saline) was injected into the tip of oxazolone-challenged ears on the 4th day after DTH in isoflurane-anesthetized Nrp2+/+ or Nrp2−/− mice (n = 5 mice per group). After 16 hours, the mice were euthanized, and ear pieces of equal area were incubated in formamide for the extraction of the dye. After 48 hours, the absorbance was measured in a spectrophotometer at 620 nm. Tissues were embedded in OCT compound and frozen in liquid nitrogen. Cryosections (8 μm) were fixed in cold acetone for 5 minutes and washed with PBS. Endogenous peroxidases were blocked in a 3% H2O2 solution in methanol for 12 minutes and washed with PBS. Endogenous proteins were blocked with Tris-HCl, NaCl blocking buffer (PerkinElmer, Waltham, MA) for 20 minutes at room temperature. Sections were incubated overnight at 4°C in rabbit anti-mouse lymphatic vessel endothelial hyaluronic acid receptor 1 antibody (catalog number 102-PA50; ReliaTech GmbH, Wolfenbuttel, Germany) in Tris-HCl, NaCl blocking buffer. The next day, tissues were washed in PBS and incubated in biotinylated goat anti-rabbit IgG (Vector Laboratories, Burlingame, CA) for 1 hour. Sections were washed in PBS and incubated with alkaline phosphatase–conjugated avidin (Vectastain ABC-AP kit; Vector Laboratories) for 30 minutes. Sections were washed in PBS, visualized using Vector Red Substrate (Vector Laboratories), and counterstained with hematoxylin (Sigma-Aldrich). Nrp2+/LacZ mice cryosections were fixed in cold methanol for 5 minutes. β-Galactosidase activity was detected by incubation in X-gal reagent [1 mg/mL 5-bromo-4-chloro-3-indolyl-β-d-galactopyranoside (Gold Biotechnology, Olivette, MO) in dimethyl sulfoxide, 5 mmol/L K3Fe(CN)6, 5 mmol/L K4Fe(CN)6, 2 mmol/L MgCl2 in PBS (pH 6.5)] overnight at 37°C. Sections were washed in PBS and counterstained in eosin (Sigma-Aldrich). Paraffin sections (4 μm) were dewaxed in xylene and rehydrated through a graded series of ethanol (100%, 95%, 70%, 50%) to water. Endogenous peroxidases and proteins were blocked as described in the previous paragraph. Sections were incubated overnight at 4°C in rabbit anti-SEMA3F antibody,31Coma S. Amin D.N. Shimizu A. Lasorella A. Iavarone A. Klagsbrun M. Id2 promotes tumor cell migration and invasion through transcriptional repression of semaphorin 3F.Cancer Res. 2010; 70: 3823-3832Crossref PubMed Scopus (72) Google Scholar rabbit monoclonal anti-mouse Nrp2 antibody (catalog number 3366; Cell Signaling, Beverly, MA), or Syrian hamster anti-mouse podoplanin (ReliaTech) in Tris-HCl, NaCl blocking buffer. The next day, tissues were washed in PBS and incubated in biotinylated goat anti-rabbit IgG or anti-hamster IgG (Vector Laboratories) for 1 hour. Sections were washed in PBS and incubated with either horseradish peroxidase–conjugated avidin (Vectastain Elite ABC kit; Vector Laboratories) or alkaline phosphatase–conjugated avidin for 30 minutes. Sections were washed in PBS, visualized using the DAB (3,3-diaminobenzidine) Substrate Kit (Vector Laboratories) or Ferangi Blue Chromogen Kit (Biocare Medical, Concord, CA), and counterstained with hematoxylin (Sigma-Aldrich). Protein lysates were separated with 7.5% SDS-PAGE, transferred to nitrocellulose membranes, blocked in skim milk for 30 minutes at room temperature, and incubated overnight at 4°C in rabbit monoclonal anti-mouse Nrp2 (catalog number 3366; Cell Signaling) or in rabbit anti-SEMA3F antibody.31Coma S. Amin D.N. Shimizu A. Lasorella A. Iavarone A. Klagsbrun M. Id2 promotes tumor cell migration and invasion through transcriptional repression of semaphorin 3F.Cancer Res. 2010; 70: 3823-3832Crossref PubMed Scopus (72) Google Scholar Membranes were washed in Tris-buffered saline with Tween 20 and incubated in horseradish peroxidase–linked donkey anti-rabbit (catalog number NA934V; GE Healthcare Bio-Sciences, Pittsburgh, PA) for 1 hour at room temperature. Blots were washed in Tris-buffered saline with Tween 20 and exposed to Western Lightning Plus ECL (enhanced chemiluminescence) (PerkinElmer). Blots were stripped (Reblot Plus; EMD Millipore, Billerica, MA) and reprobed with anti-integrin α5 (catalog number 4705; Cell Signaling) to normalize for protein loading. Significance was measured with an unpaired t-test. Procedures were performed in compliance with NIH, Association for Assessment and Accreditation of Laboratory Animal Care, and Institutional Animal Care and Use Committee regulations at Boston Children's Hospital. DTH reactions in C57BL/6J mice resulted in tissue swelling that reached maximal levels by 1 day after challenge and caused hyperplasia and remodeling by day 3 (Figure 1, A and B). Inflammation-induced vascular permeability was evident by increased redness and swelling in the challenged ears (Supplemental Figure S1A). Lymphangiography showed pooled dye and retrograde lymph flow in inflamed ears compared to unchallenged ears (Supplemental Figure S1B).32Migliozzi M.T. Mucka P. Bielenberg D.R. Lymphangiogenesis and metastasis-A closer look at the neuropilin/semaphorin3 axis.Microvasc Res. 2014; 96C: 68-76Crossref Scopus (11) Google Scholar Immunoblot analysis of C57BL/6J mice ear proteins after DTH showed nearly twofold increased expression of Nrp2 by day 1 and nearly threefold by days 5 to 7 (Figure 1C). Moreover, Nrp2 expression (β-galactosidase activity) was up-regulated in inflamed endothelium in Nrp2+/LacZ mice (Figure 1D). Nrp2 is expressed in both vascular ECs and LECs (Supplemental Figure S1C). Nrp2 was found in inflamed blood capillaries (Figure 1E) and colocalized with podoplanin staining on lymphatic vessels after inflammation (Figure 1F). Noninflamed adult mouse ears expressed Nrp2 only in melanocytes of hair follicles (Figure 1D), not in capillaries, consistent with findings from previous reports.20Klagsbrun M. Takashima S. Mamluk R. The role of neuropilin in vascular and tumor biology.Adv Exp Med Biol. 2002; 515: 33-48Crossref PubMed Scopus (175) Google Scholar, 21Caunt M. Mak J. Liang W.C. Stawicki S. Pan Q. Tong R.K. Kowalski J. Ho C. Reslan H.B. Ross J. Berry L. Kasman I. Zlot C. Cheng Z. Le Couter J. Filvaroff E.H. Plowman G. Peale F. French D. Carano R. Koch A.W. Wu Y. Watts R.J. Tessier-Lavigne M. Bagri A. Blocking neuropilin-2 function inhibits tumor cell metastasis.Cancer Cell. 2008; 13: 331-342Abstract Full Text Full Text PDF PubMed Scopus (280) Google Scholar, 33Bielenberg D.R. Pettaway C.A. Takashima S. Klagsbrun M. Neuropilins in neoplasms: expression, regulation, and function.Exp Cell Res. 2006; 312: 584-593Crossref PubMed Scopus (227) Google Scholar Nrp2 expression was absent in quiescent adult vasculature (Figure 1D) and in Nrp2−/− mouse ear sections (Figure 1G). Adult female Nrp2 KO and WT mice have ears of equal thickness (data not shown), suggesting that Nrp2-deficient mice are not normally edematous. In unchallenged ears, vascular permeability was slightly (but not significantly) greater in Nrp2−/− vessels than in Nrp2+/+ vessels (Figure 2, A and B). However, on day 2 after inflammation, vascular leakage in inflamed ears, as measured by a modified Miles assay, was double in Nrp2 KO mice compared to WT mice (Figure 2, A and B). Thus, Nrp2 deficiency increased edema due to increased vascular permeability. NRP2 binds inhibitory SEMA3F proteins as well as VEGF proteins.34Bielenberg D.R. Hida Y. Shimizu A. Kaipainen A. Kreuter M. Kim C.C. Klagsbrun M. Semaphorin 3F, a chemorepulsant for endothelial cells, induces a poorly vascularized, encapsulated, nonmetastatic tumor phenotype.J Clin Invest. 2004; 114: 1260-1271Crossref PubMed Scopus (241) Google Scholar, 35Geretti E. Shimizu A. Kurschat P. Klagsbrun M. Site-directed mutagenesis in the B-neuropilin-2 domain selectively enhances its affinity to VEGF165, but not to semaphorin 3F.J Biol Chem. 2007; 282: 25698-25707Crossref PubMed Scopus (37) Google Scholar We hypothesized that the edema seen in inflamed Nrp2−/− mice may ha" @default.
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• W2537300122 title "Inflammation and Lymphedema Are Exacerbated and Prolonged by Neuropilin 2 Deficiency" @default.
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