title: Lymphatic endothelial cells efferent to inflamed joints produce iNOS and inhibit lymphatic vessel contraction and drainage in TNF-induced arthritis in mice.
abstract: BACKGROUND
  In this study, we sought to determine the cellular source of inducible nitric oxide synthase (iNOS) induced in lymphatic endothelial cells (LECs) in response to tumor necrosis factor (TNF), the effects of iNOS on lymphatic smooth muscle cell (LSMC) function and on the development of arthritis in TNF-transgenic (TNF-Tg) mice, and whether iNOS inhibitors improve lymphatic function and reduce joint destruction in inflammatory erosive arthritis.
  
  
  METHODS
  We used quantitative polymerase chain reactions, immunohistochemistry, histology, and near-infrared imaging to examine (1) iNOS expression in podoplanin + LECs and lymphatic vessels from wild-type (WT) and TNF-Tg mice, (2) iNOS induction by TNF in WT LECs, (3) the effects of iNOS inhibitors on expression of functional muscle genes in LSMCs, and (4) the effects of iNOS inhibitors on lymphatic vessel contraction and drainage, as well as the severity of arthritis, in TNF-Tg mice.
  
  
  RESULTS
  LECs from TNF-Tg mice had eight fold higher iNOS messenger RNA levels than WT cells, and iNOS expression was confirmed immunohistochemically in podoplanin + LECs in lymphatic vessels from inflamed joints. TNF (0.1 ng/ml) increased iNOS levels 40-fold in LECs. LSMCs cocultured with LECs pretreated with TNF had reduced expression of functional muscle genes. This reduction was prevented by ferulic acid, which blocked nitric oxide production. Local injection of L-N(6)-(1-iminoethyl)lysine 5-tetrazole-amide into inflamed paws of TNF-Tg mice resulted in recovery of lymphatic vessel contractions and drainage. Treatment of TNF-Tg mice with ferulic acid reduced synovial inflammation as well as cartilage and bone erosion, and it also restored lymphatic contraction and drainage.
  
  
  CONCLUSIONS
  iNOS is produced primarily by LECs in lymphatic vessel efferent from inflamed joints of TNF-Tg mice in response to TNF and inhibits LSMC contraction and lymph drainage. Ferulic acid represents a potential new therapy to restore lymphatic function and thus improve inflammatory arthritis by inhibiting local production of nitric oxide by LSMCs.
authors.affiliation: Department of Orthopaedics, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, 725 South Wanping Road, Shanghai, 200032, China.|Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY, 14642, USA.
authors.forename: Qianqian
authors.initials: Q
authors.lastname: Liang
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY, 14642, USA.|Center for Musculoskeletal Research, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY, 14642, USA.
authors.forename: Yawen
authors.initials: Y
authors.lastname: Ju
authors.affiliation: Department of Orthopaedics, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, 725 South Wanping Road, Shanghai, 200032, China.
authors.forename: Yan
authors.initials: Y
authors.lastname: Chen
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY, 14642, USA.
authors.forename: Wensheng
authors.initials: W
authors.lastname: Wang
authors.affiliation: Department of Orthopaedics, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, 725 South Wanping Road, Shanghai, 200032, China.
authors.forename: Jinlong
authors.initials: J
authors.lastname: Li
authors.affiliation: Department of Orthopaedics, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, 725 South Wanping Road, Shanghai, 200032, China.
authors.forename: Li
authors.initials: L
authors.lastname: Zhang
authors.affiliation: Department of Orthopaedics, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, 725 South Wanping Road, Shanghai, 200032, China.|Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY, 14642, USA.
authors.forename: Hao
authors.initials: H
authors.lastname: Xu
authors.affiliation: Departments of Obstetrics and Gynecology, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY, 14642, USA.
authors.forename: Ronald W
authors.initials: RW
authors.lastname: Wood
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY, 14642, USA.|Center for Musculoskeletal Research, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY, 14642, USA.
authors.forename: Edward M
authors.initials: EM
authors.lastname: Schwarz
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY, 14642, USA.|Center for Musculoskeletal Research, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY, 14642, USA.
authors.forename: Brendan F
authors.initials: BF
authors.lastname: Boyce
authors.affiliation: Department of Orthopaedics, Longhua Hospital, Shanghai University of Traditional Chinese Medicine, 725 South Wanping Road, Shanghai, 200032, China. yjwang88@hotmail.com.|Institute of Spine, Shanghai University of Traditional Chinese Medicine, 725 Wan-Ping South Road, Shanghai, 200032, China. yjwang88@hotmail.com.
authors.forename: Yongjun
authors.initials: Y
authors.lastname: Wang
authors.affiliation: Department of Pathology and Laboratory Medicine, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY, 14642, USA. lianping_xing@urmc.rochester.edu.|Center for Musculoskeletal Research, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY, 14642, USA. lianping_xing@urmc.rochester.edu.|Departments of Obstetrics and Gynecology, University of Rochester Medical Center, 601 Elmwood Avenue, Rochester, NY, 14642, USA. lianping_xing@urmc.rochester.edu.
authors.forename: Lianping
authors.initials: L
authors.lastname: Xing
mesh_terms: D000818:Animals; D001169:Arthritis, Experimental / Q000378:metabolism / Q000473:pathology; D001172:Arthritis, Rheumatoid / Q000378:metabolism / Q000473:pathology*; D015153:Blotting, Western; D003373:Coumaric Acids / Q000494:pharmacology; D042783:Endothelial Cells / Q000378:metabolism*; D005434:Flow Cytometry; D016166:Free Radical Scavengers / Q000494:pharmacology; D007150:Immunohistochemistry; D007249:Inflammation / Q000378:metabolism* / Q000473:pathology; D042601:Lymphatic Vessels / Q000378:metabolism*; D051379:Mice; D008810:Mice, Inbred C57BL; D008822:Mice, Transgenic; D009119:Muscle Contraction / Q000502:physiology; D009130:Muscle, Smooth / Q000378:metabolism; D052247:Nitric Oxide Synthase Type II / Q000096:biosynthesis*; D060888:Real-Time Polymerase Chain Reaction; D014409:Tumor Necrosis Factor-alpha
chemical_list: D003373:Coumaric Acids; D016166:Free Radical Scavengers; D014409:Tumor Necrosis Factor-alpha; C004999:ferulic acid; D052247:Nitric Oxide Synthase Type II; C496317:Nos2 protein, mouse
keywords: Ferulic acid; Inducible nitric oxide synthase (iNOS); Lymphatic endothelial cells (LECs); Lymphatic function; Lymphatic smooth muscle cells (LSMC); Rheumatoid arthritis; TNF