Genetic Ablation of PDGF-Dependent Signaling Pathways Abolishes Vascular Remodeling and Experimental Pulmonary Hypertension

Abstract

Objective: Despite modern therapies, pulmonary arterial hypertension (PAH) harbors a high mortality. Vascular remodeling is a hallmark of the disease. Recent clinical studies revealed that antiremodeling approaches with tyrosine-kinase inhibitors such as imatinib are effective, but its applicability is limited by significant side effects. Although imatinib has multiple targets, expression analyses support a role for platelet-derived growth factor (PDGF) in the pathobiology of the disease. However, its precise role and downstream signaling events have not been established.

Approach and results: Patients with PAH exhibit enhanced expression and phosphorylation of β PDGF receptor (βPDGFR) in remodeled pulmonary arterioles, particularly at the binding sites for phophatidyl-inositol-3-kinase and PLCγ at tyrosine residues 751 and 1021, respectively. These signaling molecules were identified as critical downstream mediators of βPDGFR-mediated proliferation and migration of pulmonary arterial smooth muscle cells. We, therefore, investigated mice expressing a mutated βPDGFR that is unable to recruit phophatidyl-inositol-3-kinase and PLCγ (βPDGFR(F3/F3)). PDGF-dependent Erk1/2 and Akt phosphorylation, cyclin D1 induction, and proliferation, migration, and protection against apoptosis were abolished in βPDGFR(F3/F3) pulmonary arterial smooth muscle cells. On exposure to chronic hypoxia, vascular remodeling of pulmonary arteries was blunted in βPDGFR(F3/F3) mice compared with wild-type littermates. These alterations led to protection from hypoxia-induced PAH and right ventricular hypertrophy.

Conclusions: By means of a genetic approach, our data provide definite evidence that the activated βPDGFR is a key contributor to pulmonary vascular remodeling and PAH. Selective disruption of PDGF-dependent phophatidyl-inositol-3-kinase and PLCγ activity is sufficient to abolish these pathogenic responses in vivo, identifying these signaling events as valuable targets for antiremodeling strategies in PAH.

Keywords: platelet-derived growth factor; pulmonary hypertension; vascular endothelial growth factor; vascular remodeling.

Figure 1

Expression and site-specific activation of the β platelet-derived growth factor receptor (βPDGFR) in human pulmonary arterial hypertension. Expression of the βPDGFR and phosphorylation of tyrosine residues required for binding of phophatidyl-inositol-3-kinase (Y751) and phospholipase C-γ1 (Y1021) in lung samples of patients with idiopathic pulmonary arterial hypertension (IPAH) and patients without pulmonary hypertension (control). Shown are representative immunostainings (A), representative Western blot analysis (B), and the quantification of Western blot analyses by densitometry (C). Shown is the protein expression/phosphorylation normalized for actin. Data in (C) represent mean values±SEM. From 5 patients in each group. AU indicates arbitrary units. *P<0.05.

Figure 2

Critical role of platelet-derived growth factor receptor (PDGFR)–dependent activation of phophatidyl-inositol-3-kinase (PI3K) and phospholipase C-γ1 (PLCγ) in pulmonary hypertension. A, Expression of βPDGFR and vascular endothelial growth factor receptor (VEGFR) in vascular smooth muscle cells (VSMCs) and endothelial cells (EC), as well as activation (phosphorylation) of these receptors by platelet-derived growth factor (PDGF; 50 ng/mL) or VEGF (50 ng/mL) were assessed by western blot analyses. B and C, PDGF-dependent proliferation (B) and migration (C) of murine pulmonary arterial smooth muscle cells were assessed as BrdU incorporation and by modified Boyden chamber assays, respectively. To assess the role of PI3K and PLCγ, cells were incubated with pharmacological inhibitors: The PI3K inhibitor LY294002 (LY, 10 mmol/L), the PLCγ inhibitor U73122 (U73, 10 mmol/L) or (as a control) the MEK kinase inhibitor PD98059 (PD, 5 mmol/L) 30 minutes before PDGF treatment. Furthermore, cells were treated with both PI3K and PLCγ inhibitors (LY+U73) followed by addition of PDGF. RasGAP indicates GTPase-activating protein of Ras. *P<0.05, **P<0.01.

Figure 3

Characterization of pulmonary arterial smooth muscle cells (PASMCs) isolated from wild-type (WT) and homozygous β platelet-derived growth factor receptor (βPDGFR)^F3/F3 mice. A, Schematic diagram of the intracellular part of the βPDGFR F3 mutant showing lack of the tyrosine phosphorylation sites required for binding of phophatidyl-inositol-3-kinase (PI3K; Tyr-739/750) and phospholipase C-γ1 (PLCγ; Tyr-1020). B, Western blot analysis demonstrating equal protein expression levels of the βPDGFR in PASMC isolated from WT or βPDGFR^F3/F3 mice (GTPase-activating protein of Ras [RasGAP] served as loading control). C, PASMC from WT or βPDGFR^F3/F3 mice was treated with PDGF-BB or vehicle for 5 minutes. Phosphorylation of tyrosines Y1020 and Y750 was assessed by Western blot analysis using phospho- and site-specific antibodies. D, On treatment with PDGF-BB or vehicle, the βPDGFR was immunoprecipitated followed by Western blot analysis to detect total receptor phosphorylation (P-Y) or binding of associated signaling molecules (p85: regulatory subunit of PI3K, PLCγ, RasGAP, and SHP-2). E, To detect PDGF-dependent association of Src with the activated βPDGFR, Src was immunoprecipitated followed by detection of associated βPDGFR, cells treated as in (D). F and G, On stimulation with PDGF-BB or vehicle for the indicated time points, phosphorylation of Akt and Erk 1/2, as well as expression of the cell cycle protein cyclin D1 were assessed. Shown are a representative Western blot analysis (F) and the quantification by densitometry (G). AU indicates arbitrary units. *P<0.05.

Figure 4

Abolished platelet-derived growth factor (PDGF)–dependent proliferation and migration and enhanced susceptibility to apoptosis in F3/F3 pulmonary arterial smooth muscle cells (PASMCs). PDGF-BB–dependent proliferation (A) and PDGF-BB or thrombospondin (TSP-1)–induced migration (B and C) of PASMC from wild-type (WT) or β platelet-derived growth factor receptor (βPDGFR)^F3/F3 mice were assessed by BrdU incorporation or with modified Boyden chamber assays, respectively. C, PDGF-dependent proliferation (BrdU incorporation) of PASMC was analyzed after exposure of cells to hypoxia (1% O₂) or normoxia. D and E, Lung sections of WT and βPDGFR^F3/F3 mice subjected to normoxia (21% O₂) or hypoxia (10% O₂) for 3 weeks stained for PCNA, counterstain with methylgreen. Representative experiment (D) and quantification of 3 independent experiments (E). F and G, Detection of apoptosis by flow cytometric staining of active caspase-3-positive cells. Representative experiment (F) and quantification of 3 independent experiments (G). AU indicates arbitrary units. *P<0.05, **P<0.01.

Figure 5

Hypoxia-induced pulmonary hypertension and right ventricular hypertrophy are abrogated in β platelet-derived growth factor receptor (βPDGFR)^F3/F3 mice. A and B, Right ventricular systolic pressure (RVPsyst) and right ventricular hypertrophy, presented as the ratio of right ventricle (RV) to left ventricle (LV) plus septum (S) weight (RV/LV+S) in mice (genotypes: wild-type, βPDGFR^F3/+, and βPDGFR^F3/F3) subjected to normoxia (21% O₂) or hypoxia (10% O₂) for 3 weeks. C and D, Systolic and diastolic blood pressure (BP) in the various genotypes treated as in (A) and (B) as assessed via cannulation of the carotid artery. E, Heart rate of mice treated as in (A) and (B). n ≥ 6 in each group; *P<0.05, **P<0.01, ***P<0.001.

Figure 6

β platelet-derived growth factor receptor (βPDGFR)^F3/F3 mice are protected from hypoxic pulmonary vascular remodeling. A and B, Mice (genotypes: wild-type (WT), βPDGFR^F3/+, and βPDGFR^F3/F3) were exposed to normoxia (21% O₂) or hypoxia (10% O₂) for 3 weeks. Shown are morphometric analyses of fully muscularized (open bars), partially muscularized (dashed bars), and nonmuscularized (closed bars) small (A: diameter <70 μm) and medium-sized (B: diameter 70–150 μm) pulmonary arteries. C and D, Medial wall thickness in small (C: diameter <50 μm) and medium-sized (D: diameter 50–150 μm) pulmonary arteries of mice treated as in (A) and (B). E and F, Van Gieson staining (E) and double staining for α smooth muscle actin (purple) and the endothelial marker von Willebrand factor (brown; F) in lung sections from mice treated as in (A) and (B). n ≥ 6 in each group; *P<0.05, **P<0.01, ***P<0.001.