Fig. 4

Pulmonary microvascular endothelial cell injury was mediated by ACE system via Notch 2 receptor. a ACE mRNA level in pulmonary microvascular endothelial cell (PMVEC) in control group, cells stimulated by midkine (20 ng/mL and 100 ng/mL), and normalized to β-actin (upper left). ACE protein level in PMVEC lysates in control group, cells stimulated by midkine (20 ng/mL and 100 ng/mL). Quantitative analysis using densitometry normalized to β-actin was shown (lower left). PMVEC ACE activity determined by FAPGG in control group, cells stimulated by midkine, and cell stimulated by midkine and blocked with inhibitors targeting Notch 2 (DAPT), ALK (LDK378) and EGFR (Erlotinib), respectively. ACE activity was measured at indicated time point (upper right). Angiotensin II in the supernatant was determined after angiotensin I was added to the culture medium, in control group, cells stimulated with midkine, and cells stimulated with midkine blocked by DAPT (lower right). b Notch 2 and ACE protein level in PMVEC in control group, cells stimulated with midkine (100 ng/mL), and cells stimulated by midkine and blocked by DAPT. Quantitative analysis of full length (FL) and intra-cellular domain (NICD) of Notch 2 and ACE, using densitometry, normalized to β-actin. c Cell viability determined by CCK-8 assay of PMVEC in blank, control group, cells stimulated by midkine, cells stimulated by midkine and blocked by DAPT and ACEI respectively. Reactive oxygen species (ROS) production in PMVEC in blank, control group, cells stimulated by midkine, cells stimulated by midkine and blocked by DAPT and ACEI respectively. d The expression of inter-cellular adhesive molecules including VCAM-1 and VE-cadherin were determined by Western blots. The expression of VCAM-1 and VE-cadherin in PMVEC were shown in control group, cells stimulated by midkine, cells stimulated by midkine and blocked by DAPT and ACEI, respectively. e Immunofluorescence staining of VE-Cadherin and ZO-1 in PMVEC in control group, cells stimulated by midkine and cells stimulated by midkine but prohibited by DAPT