Fig 1: Nitric oxide (NO) from tumor necrosis factor (TNF)-treated lymphatic endothelial cells (LECs) reduces the expression levels of functional muscle genes in lymphatic smooth muscle cells (LSMCs), which is prevented by a selective inducible nitric oxide synthase (iNOS) inhibitor. a A schematic cartoon of the coculture model used to assess paracrine effects of NO induced by TNF in LECs on LSMC gene expression. b The effect of 1 ng/ml TNF on expression levels of functional muscle genes in LSMCs was determined by quantitative polymerase chain reaction (qPCR) and Western blot analysis. The signal intensity of bands on the Western blot was quantified by densitometry. c LECs were treated with TNF as in (a) with or without the selective NOS inhibitor aminoguanidine hemisulfate salt (Ami) or Ami alone and then cocultured with LSMCs for another 24 h. The expression levels of functional muscle genes were assessed by qPCR. The fold changes were calculated using phosphate-buffered saline (PBS)-treated sample values as 1. Values are mean ± standard deviation (SD) of three samples. *p < 0.05 vs. PBS-treated samples. d NO levels in the conditioned medium of TNF-treated LECs with or without different amounts of Ami or Ami alone were measured by using a Griess method. Values are mean ± SD of three samples. The experiments were repeated twice with similar results. *p < 0.05 vs. samples without Ami and TNF-treated; # p < 0.05 vs. TNF-treated samples. NF-κB2 nuclear factor κB2, sMYH smooth muscle myosin heavy chain, SMα smooth muscle actin
Fig 2: Decreased lymphatic clearance in tumor necrosis factor–transgenic (TNF-Tg) mice is associated with the progression of arthritis. Indocyanine green ([ICG] 0.1 mg/ml in 6 μl) was injected intradermally into the footpads of 1-, 2.5-, and 5-month-old TNF-Tg mice and their wild-type (WT) littermates. Lymphatic vessels of the entire leg were examined using near-infrared ICG imaging immediately and at 6 and 24 h post-ICG administration. The ICG fluorescence signal intensity at the footpads (outlined by the red circles) was recorded. The ICG clearance was calculated. Shown are a representative ICG signal (a) and the percentage of ICG clearance (b). Values are mean ± standard deviation of four or five mice. *p < 0.05 vs. WT mice
Fig 3: Ferulic acid (FLA) reduces joint tissue damage in tumor necrosis factor–transgenic (TNF-Tg) mice. Three-month-old TNF-Tg and wild-type (WT) mice were treated with FLA or saline, as described in Fig. 5. a Representative Alcian Blue/Orange G (ABOG)- and tartrate-resistant acid phosphatase (TRAP)-stained sections show decreased joint tissue damage, including decreased cartilage (blue arrow) and bone erosion (black arrow) and TRAP+ osteoclasts in an FLA-treated mouse which has relatively normal joint morphology. Quantitation of inflammatory area (b), bone area (c), cartilage area (d), and TRAP+ area (e). Values are the mean ± standard deviation of six to eight legs per group. *p < 0.05 vs. WT mice; # p < 0.05 vs. saline-treated TNF-Tg mice
Fig 4: Differential effects of selective inducible nitric oxide synthase (iNOS) inhibition vs. total nitric oxide synthase (NOS) inhibition on lymphatic vessel contractions and lymph drainage in tumor necrosis factor–transgenic (TNF-Tg) mice with flaring arthritis. TNF-Tg mice more than 5 months old with severe ankle arthritis were used. a TNF-Tg mice (n = 5) were subjected to real-time near-infrared indocyanine green (NIR-ICG) imaging to quantify the afferent lymphatic pulse before, during, and following a direct injection of saline (a, b) or L-N6-(1-iminoethyl)lysine 5-tetrazole-amide (L-NIL) (c, d) into the footpad. Representative NIR-ICG images illustrate the lack of ICG lymphatic drainage from the injection site in the footpad to the region of the popliteal lymph node at 1 h postinjection (a, c). No remarkable changes were observed 9 minutes after saline injection (b). However, ICG was detected in the primary lymphatic vessel (LV; arrow) and the popliteal lymph node (PLN; arrowhead) within 4 minutes after L-NIL injection (d). b The lymphatic vessel contraction frequency was determined by assessing pulses in the NIR-ICG signal intensity in the region of interest (red circles), which was graphed over time during the imaging session. Note the recovery of lymphatic pulses (red arrows) after the iNOS inhibitor L-NIL (green line) injection, while saline did not affect lymphatic contraction frequency (orange line). c TNF-Tg mice and their wild-type (WT) littermates (n = 4) underwent pre- and posttreatment NIR-ICG imaging to assess the effects of 6 weeks of saline, L-NIL, and N ω-nitro-L-arginine methyl ester (l-NAME) on lymphatic vessel contractions (c) and percentage ICG clearance (d). Figure 4c, *P<0.05 between groups. Figure 4d, *P<0.05 vs WT. #P<0.05 vs. TNF-Tg
Fig 5: 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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