LYVE-1 expression is regulated by BMP9 both in vivo in lymphatic collecting vessels of the mesentery and in vitro in cultured LECs. (A) P0 lymphatic mesenteric collecting vessels were stained for LYVE-1 (red) and Prox1 (green). The dashed lines delineate the lymphatic vessels. Arrow indicates a valve. A nonlymphatic LYVE-1 immunoreactivity is also observed in isolated cells outside vessels, which probably correspond to macrophages. (B) Time-dependent inhibition of LYVE-1 mRNA expression in LECs treated with BMP9 (10 ng/mL); the results are presented as mRNA fold changes measured in BMP9-treated cells vs nontreated cells at each time point. Data are the mean ± SE from 6 independent experiments performed in duplicate. *P ≤ .05, significantly different from cytotoxic T lymphocyte (CTL) by Mann-Whitney U test. (C,E) Flow cytometry detection of LYVE-1 protein expression in LECs transfected for 24 hours with scramble siRNA or siRNA targeting Alk1 and then stimulated with (pink) or without (green) 10 ng/mL BMP9 for 48 hours. NS corresponds to fluorescence-activated cell sorter analysis in absence of antibodies (black). (D) LYVE-1 mRNA expression in LECs transfected for 24 hours with scrambled siRNA (siScr) or siRNA targeting Alk1 and then stimulated with or without 10 ng/mL BMP9 for 24 hours; data represent mean ± SE (n = 6). **P ≤ .01, significantly different from CTL by Mann-Whitney U test. Inset shows Alk1 mRNA downregulation by siAlk1 (mean ± SE, n = 4). *P ≤ .05, significantly different from siCTL by Mann-Whitney U test.