Fig. 2

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