Combinatorial inhibitory effects on VSV‐GtsO45GFP trafficking in human endothelial cells due to functional haploinsufficiency of BMPRII, eNOS, STAT5a and STAT5b

Abstract

Lung lesions in idiopathic pulmonary arterial hypertension (PAH) contain enlarged “vacuolated” endothelial and smooth muscle cells with enlarged and fragmented Golgi apparatus and dilated endoplasmic reticulum (ER) cisternae. Familial PAH often involves mutations in or haploinsuffciency of BMPRII but with low penetrance (10–15%) setting up a search for “second‐hit” genes. Recently we observed that acute knockdown of STAT5a or STAT5b or both in HPAECs using siRNAs led to marked Golgi enlargement and fragmentation and cisternal dilatation of the ER. In the present study we used the VSV‐GtsO45GFP ER to Golgi to plasma membrane trafficking assay in EA.hy926 human endothelial cells to quantitatively assess the combinatorial effects of functional haploinsufficiency of BMPRII, eNOS, STAT5a and STAT5b using respective siRNAs. Knockdown of STAT5a, STAT5b, BMPRII, or eNOS one at a time did not significantly affect intracellular trafficking. However, down‐regulation of STAT5a plus STAT5b inhibited VSV‐G trafficking from the ER/Golgi apparatus to the cell surface by 60–70%. STAT5a or STAT5b combined with eNOS siRNA treatments had a similar inhibitory effect. STAT5a or STAT5b combined with BMPRII siRNA also inhibited VSV‐G trafficking. These observations suggest that combinatorial functional haploinsufficiency of BMPRII, eNOS, STAT5a and STAT5b may contribute to intracellular trafficking defects.