Growth Factor Midkine Aggravates Pulmonary Arterial Hypertension via Surface Nucleolin

Figures

• 

  Figure 1. Midkine (MK) expression levels in patients with pulmonary arterial hypertension (PAH) and hypoxia-induced PAH mice. (a) Serum MK levels in patients who underwent right-heart catheterization to determine pulmonary vascular resistance (PVR). (b) Relative MK mRNA expression in mouse lungs 4 weeks after the exposure to normoxia or hypoxia. (c) MK protein expression in mouse lungs during the exposure to hypoxic conditions. Expression levels were normalized to those of β-tubulin. Full-length blots are presented in Supplementary Figure 1 (d) Mouse lungs 4 weeks after the exposure to normoxia or hypoxia were stained with antibodies against MK (red), nuclear marker (DAPI, blue), and cell type markers (green) for vascular smooth muscle cell (α-smooth muscle actin (SMA)), stromal cell (vimentin), and endothelial cell (platelet endothelial cell adhesion molecule (PECAM)). Arrowheads in each image show both MK and the cell type marker positive cells. (e) Fluorescent intensity was quantified and normalized to the background intensity by using ImageJ software (version 1.42; https://imagej.nih.gov/ij/). (f) Serum MK concentrations in mice exposed to normoxia or hypoxia were evaluated. Data are…

• 

  Figure 2. Systemic midkine (MK) knockdown effects on pulmonary arterial remodeling in chronic hypoxia-induced pulmonary arterial hypertension (PAH) mice. (a) Representative Elastica-Masson staining images of mouse lung transverse sections at 4 weeks after the exposure to normoxia or hypoxia. Scale bars, 50 µm. (b) Degree of muscularization of pulmonary arterial vessels (outer diameter 20–100) in mice exposed to normoxia or hypoxia (n = 5–9 each). WT, wild-type mice; MK-KO, midkine knockdown mice. (c) Pulmonary arterial remodeling evaluated by the percentage of medial wall thickness (n = 5–9 each). (d) Right hypertrophy [ratio of right ventricle (RV)/left ventricle plus septum (LV + S)] (n = 5–9 each). (e) Pulmonary artery acceleration time (PAAT)/ pulmonary artery ejection time (PAET), determined by echocardiography (n = 5–15 each). (f) Western blots analysis of EGFR and ERK1/2 phosphorylation rates in mouse lungs (n = 5). Full-length blots are presented in Supplementary Figure 2. (g) Western blots analysis for Ccnb1, PCNA, and β-tubulin levels in mouse lungs (n = 5). Full-length blots are presented in Supplementary Figure 3. *ANOVA post-hoc Tukey’s honest significant difference, P < 0.05,…

• 

  Figure 3. Midkine (MK) effects on pulmonary arterial smooth muscle cell (PASMC) migration and proliferation mediated through nucleolin/EGFR pathway. (a) Representative scratch assay images of PASMCs treated with vehicle or MK under normoxic or hypoxic conditions. (b) Quantification of the scratch area coverage by the migrating cells using ImageJ software (version 1.42; https://imagej.nih.gov/ij/). Migration levels were normalized to that of the control (n = 18). (c) PASMC proliferation assay, after the stimulation with or without MK (n = 18). (d) Representative ERK1/2, EGFR and proliferating cell nuclear antigen (PCNA) expression results in PASMCs exposed to hypoxia or normoxia and/or MK treatment. Full-length blots are presented in Supplementary Figure 4. (e) Representative images of EGFR, ERK1/2, PCNA, nucleolin (NCL), and β-tubulin expression in PASMCs, and their quantification, following the treatment of PASMCs with MK in cells where nucleolin expression was inhibited (n = 6). Full-length blots are presented in Supplementary Figure 5. (f) Representative image of western blots of ERK1/2, PCNA, β-tubulin, and EGFR. PASMC was treated with midkine under downregulation of EGFR using siRNA.…

• 

  Figure 4. Hypoxia induces cell surface translocation of nucleolin under hypoxic conditions. (a) PASMCs were lysed following the exposure to normoxia or hypoxia (FiO2 0.03) for 1 h. Nucleolin (NCL), low density lipoprotein related protein 1 (LRP1), integrin β1, epidermal growth factor receptor (EGFR), and β-tubulin expression levels were evaluated. Representative images of western blot, and their quantification (n = 4). Full-length blots are presented in Supplementary Figure 7. (b) Nucleolin (NCL), LRP1, integrin β1, lamin B, and Na/K-ATPase expression in nuclear or membrane fractions of pulmonary arterial smooth muscle cells (PASMCs) after their exposure to hypoxia or normoxia, and their quantification (n = 6). Full-length blots are presented in Supplementary Figure 8. (c) The distribution of green fluorescent protein (GFP)-tagged nucleolin was observed under hypoxia, using time-lapse microscopy. (d) Confocal laser microscopy image of GFP-tagged nucleolin and Na/K-ATPase, under hypoxic conditions. (e) PASMCs were treated with biotinylated midkine (MK) or vehicle, and exposed to normoxia or hypoxia, and midkine-nucleolin binding was analyzed. Full-length blots are presented in Supplementary…

• 

  Figure 5. AS1411 suppresses the development of pulmonary arterial hypertension (PAH) by inhibiting midkine (MK)-nucleolin (NCL)-EGFR axis. (a) Pulmonary arterial smooth muscle cells (PASMCs) were treated with vehicle or AS1411, which was followed with the treatment with biotinylated MK. Full-length blots are presented in Supplementary Figure 11. (b) Phosphorylated ERK1/2 and EGFR (p-ERK1/2 and p-EGFR) and total ERK1/2 and EGFR (t-ERK1/2 and t-EGFR) expression levels in PASMCs treated with MK after AS1411 pretreatment. Full-length blots are presented in Supplementary Figure 12. (c) Representative images of MK-induced migration of PASMCs after the pretreatment of cells with placebo and AS1411, and its quantification using ImageJ software (version 1.42; https://imagej.nih.gov/ij/). (d) MK-induced proliferation of PASMCs under hypoxic conditions. (e) Study design. Rats were divided into three groups, and the rats were treated with AS1411 (10 mg/kg/day) or the vehicle for consecutive 5 days after the establishment of PAH. Normoxic control was treated with vehicle and exposed to hypoxic conditions for 3 weeks, which was followed by the re-exposure to normoxia. (f) Western blot analysis midkine…

• 

  Figure 6. Proposed mechanism underlying midkine (MK) effects on proliferation and migration of pulmonary arterial smooth muscle cells (PASMCs). MK contributes to the development of pulmonary arterial remodeling via cell surface nucleolin translocation under hypoxic conditions, and promotes the activation of epidermal growth factor receptor (EGFR) by inducing the interactions between nucleolin and EGFR. The inhibition of the MK binding to nucleolin by AS1411 suppresses the proliferation of PASMCs and attenuates the development of pulmonary arterial hypertension (PAH).