Lymphatic endothelial cells express ALK1. (A) RT-PCR analysis of gene expression in HUVECs and human microvascular dermal neonatal lymphatic endothelial cells. RNA from a mixture of human cell lines with (+) and without (−) reverse transcriptase enzyme was used as a positive and negative control. Sizes of PCR products are indicated. (B) Expression level of Smad6 in HUVECs and lymphatic endothelial cells stimulated with 50 pg/mL rhBMP9 or 500 pg/mL rhBMP10 for 3 hours. Inhibition of rhBMP9 and rhBMP10 induced up-regulation of Smad6 expression by pretreatment with 10 μg/mL ALK1Fc. Quantitative RT-PCR results are normalized to glyceraldehyde 3-phosphate dehydrogenase and then to the untreated (UT) sample. Gray squares represent individual data points (n = 3). (C) Expression level of Smad6 in lymphatic endothelial cells stimulated with rhBMP9 150 pg/mL and transfected with siRNA targeting the Alk1-7, Smad1, 4, 5, or endoglin (Eng). Gray squares represent individual data points (n = 3).