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Fig. 1 | Arthritis Research & Therapy

Fig. 1

From: Lymphatic endothelial cells efferent to inflamed joints produce iNOS and inhibit lymphatic vessel contraction and drainage in TNF-induced arthritis in mice

Fig. 1

Lymphatic endothelial cells (LECs) in efferent vessels from arthritic paws of tumor necrosis factor–transgenic (TNF-Tg) mice express high levels of inducible nitric oxide synthase (iNOS). a Podoplanin-positive (PDPN+) LECs were isolated from joints of 7-month-old TNF-Tg mice and their wild-type (WT) littermates using phycoerythrin (PE) anti-PDPN antibody/anti-PE microbeads passed through an LS column. Cells were subjected to flow cytometry to assess their surface expression of LEC markers. b The expression levels of TNF, endothelial nitric oxide synthase (eNOS), and iNOS in LECs were determined by quantitative polymerase chain reaction (qPCR). The fold changes normalized to actin were calculated using a WT sample value as 1. Values are mean ± SD of five mice. c A murine LEC line was treated with different concentrations of TNF for 24 h. The expression levels of TNF, eNOS, and iNOS were determined by qPCR. The fold changes were calculated using phosphate-buffered saline (PBS)-treated sample values as 1. Values are mean ± SD of three samples. *p < 0.05 vs. PBS-treated samples. The expression levels of iNOS protein were determined by Western blot analysis. d Collecting lymphatic vessels and surrounding tissues were harvested from TNF-Tg mice older than 5 months of age. Frozen sections were stained with hematoxylin and eosin (H&E) or double–immunofluorescence-stained with anti-PDPN for LECs and anti-iNOS antibodies. H&E-stained section (a) shows a lymphatic vessel with an open lumen (indicated by blue arrows). Immunohistochemically stained sections show (b) PDPN+ lymphatic vessel endothelium (red), (c) iNOS+ vessels (green), and (d) merged PDPN+/iNOS+ LECs (yellow). Bar represents 200 μm. mRNA messenger RNA, LYVE-1 lymphatic vessel endothelial hyaluronan receptor 1, VEGFR3 vascular endothelial growth factor receptor 3

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