Lysophosphatidic Acid Induces Lymphangiogenesis and IL-8 Production in Vitro in Human Lymphatic Endothelial Cells

• DOI: 10.1016/j.ajpath.2012.03.003
• Publication Date: 2012-03-30T00:36:11Z
• Authors: Hong Mu; Tiffany L. Calderone; Michael A. Davies; Victor G. Prieto; Hua Wang; Gordon B. Mills; Menashe Bar-Eli; Jeffrey E. Gershe...
• Keywords: Lymphangiogenesis; Lymphatic Endothelium; Lymphatic vessel

The bioactive phospholipid lysophosphatidic acid (LPA) and its receptors LPA1-3 are aberrantly expressed in many types of human cancer. LPA has been reported to induce tumor cell proliferation, migration, cytokine production. However, whether exerts an effect on lymphatic endothelial cells (LECs) or lymphangiogenesis, a process new vessel formation that is associated with increased metastasis poor prognosis cancer patients, unknown. Here, we show induces survival, tube formation, promotes lymphangiogenesis vitro dermal LECs. In addition, IL-8 expression by enhancing promoter activity via activation the NF-κB pathway Using siRNA neutralizing antibody, revealed plays important role LPA-induced vitro. Moreover, using inhibition, discovered production mediated LPA2 receptor Finally, sentinel afferent explants, demonstrated up-regulates LECs endothelia. 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Mouse anti-human antibody mouse IgG1 control were R&D Systems (Minneapolis, MN). Calcein AM Biotium (Hayward, CA). Human adult PromoCell (Heidelberg, Three lots used, all yielded similar results. cultured medium MV2 (EGM-MV2), comprised basal (EBM-MV2) supplement pack (PromoCell). used between passages 5. made starvation [EBM-MV2 supplemented 1% fetal (FBS), 1 μg/mL ascorbic acid, 0.2 hydrocortisone] 16 hours treatment. concentrations chosen maintain viability. Starvation 5% FBS positive assays. treated 0 μmol/L LPA, proliferation evaluated tetrazolium salt colorimetric assay (MTS), CellTiter 96 AQueous One Solution (Promega, Madison, WI) according manufacturer's instructions. Proliferation examined counting hemocytometer at 72 after treatment LPA. migration measured modified Boyden chamber described previously.24Mu Ohashi Yan Chai Yao Q. Adipokine resistin cells.Cardiovasc 70: 146-157Crossref (179) Inserts (3-μm pores; BD Discovery Labware, Bedford, MA) precoated 1× attachment containing gelatin (Invitrogen Technologies, Carlsbad, CA) use. incubated 24 seeding into upper chambers added lower chambers. Migration assessed staining measuring fluorescence migrated SpectraMax microplate reader (Molecular Devices, Sunnyvale, bottom 485/535 nm excitation/emission wavelengths. ratio LPA-treated compared non-LPA-treated controls. Images captured under Leica DM IL inverted microscope digital camera (Optronics, Goleta, Scratch wounds confluent monolayer subsequent LEC 30 minutes 48 hours. Wound area calculated ImageJ software version 1.43t (NIH, Bethesda, MD), previously.25Mu Wang Li Thymosin beta10 inhibits capillary-like coronary artery Motil Cytoskeleton. 63: 222-230Crossref Tube assays performed total length without they seeded Matrigel (BD Biosciences, San Jose, 6 hours; RNA isolated RiboPure isolation kit (Ambion, Austin, TX) reverse-transcribed MMLV reverse transcriptase (Clontech, Mountain View, Gene quantified quantitative real-time PCR (qPCR) ABI StepOnePlus system TaqMan custom probes (Applied Biosystems; Foster City, Relative determined normalized glyceraldehyde-3-phosphate dehydrogenase (GAPDH) β-actin (ACTB). Probes listed Table 1.Table 1Probes Used qPCR AssaysProbeAssay IDProbe sequenceHuman IL-8Hs00174103_m15′-CAGCTCTGTGTGAAGGTGCAGTTTTGCCAA-3′Human LPA1Hs00173500_m15′-CACAGCCCCAGTTCACAGCCATGAATGAAC-3′Human LPA2Hs00173704_m15′-TGGTCAAGACTGTTGTCATCATCCTGGGGGC-3′Human LPA3Hs00173857_m15′-TGACTGTCTTAGGGGCGTTTGTGGTATGCT-3′Human ACTBHs99999903_m15′-CCTTTGCCGATCCGCCGCCCGTCCACACCC-3′Human GAPDHHs99999905_m15′-TATTGGGCGCCTGGTCACCAGGGCTGCTTT-3′ Open table tab Nontargeting targeted siRNAs (50 nmol/L; Dharmacon, Lafayette, CO) transfected DharmaFECT reagent-1 transfection efficiency GFP generated concomitant GFP-reporter plasmid blocking monitored GAPDH enzyme-linked immunosorbent (ELISA) transfection. effects growth, observed supernatants collected, equal amounts protein (∼3 μg) 96-well plate loaded measurement level ELISA II Biosciences) VEGF-D, (R&D Systems) Two firefly luciferase reporter constructs one driven (−1481 +44 bp) (pGL3-IL-8-Luc) versus (pGL3-Luc)26Mourad-Zeidan A.A. Melnikova V.O. 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Chromatin immunoprecipitation (ChIP) ChIP-IT Express (Active Motif, Briefly, 7 × 106 150-mm culture dishes 60 proteins DNA cross-linked formaldehyde. lysates sonicated generate fragments 1500 bp. Protein magnetic beads μg either anti-phospho-NF-κB p65 (Santa Cruz Biotechnology, Santa Cruz, anti-IgG (negative chromatin-containing overnight 4°C. reversal cross-linking protein/DNA complexes free DNA, primers flanking promoter: 5′-GGGCCATCAGTTGCAAATC-3′ 5′-TTCCTTCCGGTGGTTTCTTC-3′.30Chen L.F. Williams Mu Nakano Duerr J.M. Buckbinder Greene W.C. RelA phosphorylation regulates acetylation.Mol 25: 7966-7975Crossref (368) grown 80% 90% confluency four-well slides serum-free EBM-MV2 being (5 μmol/L). Apoptosis TUNEL 36 DeadEnd fluorometric caspase-3 immunostaining (cleaved) (Biocare Medical, Concord, 30-mm dishes, harvested time points (0, 5, 10, 20, 30, 45, 60, 120 minutes) lysed lysis buffer Triton X-100, 50 HEPES pH 7.4, 150 NaCl, 1.5 MgCl2, EGTA, 100 NaF, NaPPi, 10% glycerol, Na3VO4, complete protease inhibitor cocktail (Roche Diagnostics, Mannheim, concentration mg/mL, denatured SDS 95°C. Reverse-phase array (RPPA) staff MD Anderson Functional Proteomics RPPA Core previously.31Davies Stemke-Hale Tellez Deng Gopal Y.N. Woodman S.E. Calderone Ju Lazar A.J. Prieto V.G. Aldape Gershenwald J.E. Integrated clinical AKT melanoma.Clin Cancer 15: 7538-7546Crossref (192) 32Park E.S. Rabinovsky Carey Hennessy B.T. Agarwal Liu Woo H.G. S.B. Cheong Garraway Weinstein J.N. J.S. Davies Integrative proteomic signatures, mutations, drug responsiveness NCI line set.Mol 9: 257-267Crossref (75) A logarithmic value reflecting relative amount sample analyses. Western blotting previously.33Mu Ohta Kuriyama Shimada Tanihara Yasuda Tanaka Equarin, soluble molecule polarity chick lens equator, involved eye formation.Mech Dev. 120: 143-155Crossref (26) For IHC LECs, (Falcon; Labware); frozen sections fixed cold acetone. Immunostaining Antibodies rabbit phospho-NF-κB (Ser536) (Cell Signaling Technology, Danvers, MA), polyclonal anti-IL-8 Biosource, CA), CXCR1/IL8RA (IL-8 α chain) CXCR2/IL8RB β antibodies Systems), podoplanin (Fitzgerald Industries International, Acton, anti-Prox-1 International), phospho-ERK1/2 Technology), anti-total ERK1/2 Technology). SYTOX Green (Invitrogen-Molecular Probes, visualize nuclei. Confocal images acquired LSM 510 laser-scanning (Carl Zeiss, Thornwood, NY). Small segments (3 mm) anatomically confirmed obtained undergoing intraoperative mapping biopsy procedures part planned surgical Institutional Review Board-approved protocol, previously.34Gershenwald Ross M.I. Sentinel-lymph-node cutaneous melanoma.N Engl 2011; 364: 1738-1745Crossref (104) 3 mL isosulfan blue (Lymphazurin) injected intradermally around immediately mapping; protocol includes intradermal injection 0.5 mCi 99mTc-labeled sulfur colloid surgery. Intraoperative localization aided use handheld gamma counter (Neoprobe Neoprobe Neo Neoprobe, Dublin, OH) visualization dye nodes. Tiny attached harvested. Sentinel placed EGM-MV2 FBS, transported laboratory wet ice, carefully dissected remove perilymphatic tissues dissecting microscope, cut lengthwise expose luminal surface. To ensure adequate experimental controls, segment transected two subsegments. Each pair open subsegments separate wells 20% antibiotics embedded optimal cutting temperature compound (OCT; Sakura Finetek, Torrance, section preparation IHC. four used. Student's t-test evaluate data. Data means ± SEM. P values <0.05 considered statistically significant. Lymphatic lineage immunohistochemical analysis. (passages 5) Prox-1 panendothelial CD31, did not express CD34 (see Supplemental Figure S1 http://ajp.amjpathol.org). As expected, HUVECs both CD31 CD34, Prox-1. dose- time-dependent (Figure 1, B) dose-dependent number 1C). We next tested survival. μmol/L) reduced apoptosis cleaved 1D). Taken together, data demonstrate survival significantly manner, 2, scratch wound 2C). networks Matrigel. promoted form networks, controls 2D). Together behaviors know