Reversal of experimental pulmonary hypertension by PDGF inhibition

Abstract

Progression of pulmonary hypertension is associated with increased proliferation and migration of pulmonary vascular smooth muscle cells. PDGF is a potent mitogen and involved in this process. We now report that the PDGF receptor antagonist STI571 (imatinib) reversed advanced pulmonary vascular disease in 2 animal models of pulmonary hypertension. In rats with monocrotaline-induced pulmonary hypertension, therapy with daily administration of STI571 was started 28 days after induction of the disease. A 2-week treatment resulted in 100% survival, compared with only 50% in sham-treated rats. The changes in RV pressure, measured continuously by telemetry, and right heart hypertrophy were reversed to near-normal levels. STI571 prevented phosphorylation of the PDGF receptor and suppressed activation of downstream signaling pathways. Similar results were obtained in chronically hypoxic mice, which were treated with STI571 after full establishment of pulmonary hypertension. Moreover, expression of the PDGF receptor was found to be significantly increased in lung tissue from pulmonary arterial hypertension patients compared with healthy donor lung tissue. We conclude that STI571 reverses vascular remodeling and cor pulmonale in severe experimental pulmonary hypertension regardless of the initiating stimulus. This regimen offers a unique novel approach for antire-modeling therapy in progressed pulmonary hypertension.

Figure 1

Impact of STI571 treatment on hemodynamics and gas exchange in MCT- and hypoxia-induced pulmonary hypertension. (A) RVSP (in mmHg) in the different treatment groups is shown. (B) Effect of STI571 on the course of RVSP in MCT-induced pulmonary hypertension measured by telemetry. MCT (s.c.) was applied at day 0 after animals had recovered from surgery for catheter implantation. Pulmonary hypertension developed progressively until day 28. STI571 was applied by daily i.p. injections at a dose of 50 mg/kg/d from day 28 to 42. In addition, systemic arterial pressure (SAP; in mmHg) (C), cardiac index (CI; in ml/min per 100 g body weight) (D), and oxygenation index (PaO₂/FiO₂) (E) are given for the different experimental groups. STI571 was applied at doses of 1, 10, and 50 mg/kg/d. (F) RVSP (in mmHg) in the different treatment groups of chronically hypoxic mice. STI571 was applied at doses of 50 and 100 mg/kg/d by gavage from day 21 to 35. *P < 0.05 versus control; ^†P < 0.05 versus MCT at day 28 or hypoxia at day 21; ^‡P < 0.05 versus MCT at day 42 or hypoxia at day 35.

Figure 2

Effects of STI571 on right heart hypertrophy and survival. Ratio of RV to LV plus septum weight (RV/LV+S) (A) and survival rates of STI571-treated versus sham-treated (MCT control; filled circles) animals (B) are shown. Treatment at doses of 1, 10, and 50 mg/kg/d was started at day 28 after MCT injection; the different doses are indicated. (C) RV/LV+S values of chronically hypoxic mice. STI571 was applied at doses of 50 and 100 mg/kg/d by gavage from day 21–35. *P < 0.05 versus control; ^†P < 0.05 versus MCT at day 28 or hypoxia at day 21; ^‡P < 0.05 versus MCT at day 42 or hypoxia at day 35.

Figure 3

Effects of STI571 on the degree of muscularization (A–C), medial wall thickness of small pulmonary arteries (D–F), and PDGF-B expression (G–I). A, D, and G: Day 0. B, E, and H: Day 42. C, F, and I: Day 42 treated with 50 mg/kg/d STI571. (A–C) The degree of muscularization is demonstrated by von Willebrand (brown) and α–smooth muscle actin (purple) staining for identifying endothelium and vascular SMCs, respectively. (D–F) Changes in medial wall thickness in pulmonary arteries are demonstrated. (G–I) PDGF-B staining is demonstrated in the medial layer of small pulmonary arteries. Scale bars: 20 μm. The arrows indicate pulmonary arteries.

Figure 4

Effects of STI571 on the degree of muscularization; medial wall thickness of pulmonary arteries sized 25–50 μm, 51–100 μm, and greater than 100 μm; and internal lumen area of pulmonary arteries sized 25–50 μm. (A) Proportion of non- (N), partially (P), or fully (M) muscularized pulmonary arteries, as percentage of total pulmonary artery cross section (sized 25–50 μm). A total of 60–80 intraacinar vessels was analyzed in each lung. (B–D) Medial wall thickness of pulmonary arteries sized 25–50 μm (B); 51–100 μm (C); and greater than 100 μm (D). (E) The internal lumen area of vessels between 25 and 50 μm. Results from rats exposed to MCT for 28 and 42 days and STI571-treated rats (treatment from day 28 to 42 with 50 mg/kg/d) are presented. (F) Proportion of non-, partially, or fully muscularized pulmonary arteries from chronically hypoxic mice treated with STI571 (sized 20–70 μm). STI571 was applied at a dose of 100 mg/kg/d by gavage from day 21 to 35. *P < 0.05 versus control; ^†P < 0.05 versus MCT at day 28 or hypoxia at day 21; ^‡P < 0.05 versus MCT at day 42 or hypoxia at day 35.

Figure 5

MMP expression (A) and densitometric quantification (B) in pulmonary arteries. Homogenates of lung tissue were examined from MCT-challenged animals receiving STI571 at 50 mg/kg/d, as compared with controls and nontreated MCT animals. Western blots for the pro and active forms of MMP-2 and MMP-9 (A) and densitometric quantification normalized to GAPDH (B) are shown. *P < 0.05 versus control; ^†P < 0.05 versus MCT at day 42.

Figure 6

Increased PDGFRβ expression/phosphorylation in MCT-induced pulmonary hypertension. Western blot analysis was used to assess expression of PDGFRβ and PDGFRβ phosphorylation (P-PDGFRβ) in rat lungs from controls and animals treated with MCT (14 days), MCT (28 days), MCT (42 days), and MCT (42 days)/STI571. Immunoblots are representative of 4 individual lungs from each group, showing identical results. Quantification of PDGFRβ and PDGFRβ phosphorylation is shown in the bar graphs. PDGFRβ and PDGFRβ phosphorylation are normalized to GAPDH. *P < 0.05 versus control; ^†P < 0.05 versus MCT at day 28; ^‡P < 0.05 versus MCT at day 42.

Figure 7

Increased PDGFRβ expression/phosphorylation in hypoxia-induced pulmonary hypertension. Western blot analysis was used to assess expression of PDGFRβ and PDGFRβ phosphorylation in mouse lungs from controls and animals treated with hypoxia (35 days) and hypoxia (35 days)/STI571. Immunoblots are representative of 4 individual lungs from each group, showing identical results. Quantification of PDGFRβ and PDGFRβ phosphorylation is shown in the bar graphs. PDGFRβ and PDGFRβ phosphorylation are normalized to GAPDH.

Figure 8

Increased PDGFRβ signaling (ERK1/2 phosphorylation) in MCT-induced pulmonary hypertension. Western blot analysis was used to assess expression of ERK1/2 phosphorylation (P-ERK1/2) in rat lungs from controls and animals treated with MCT (14 days), MCT (28 days), MCT (42 days), and MCT (42 days)/STI571. Immunoblots are representative of 4 individual lungs from each group, showing identical results. Quantification of ERK1/2 phosphorylation is shown in the bar graph. ERK1/2 phosphorylation is normalized to ERK1.

Figure 9

Change in expression of PDGFRβ in PAH lungs. Expression of PDGFRβ in lung homogenates from patients with PAH (n = 4) and healthy donors (n = 4) and densitometric quantification of the signal intensity. Western blot analysis was performed with anti-PDGFRβ antibody. The specific antibody recognizes protein at a molecular weight of ∼185 kDa. Quantification of PDGFRβ is shown in the bar graph.

Figure 10

Effect of STI571 on serum-induced proliferation ([³H]thymidine incorporation) (A), apoptosis (B) and PDGF-AA–, PDGF-BB–, IGF-1–, EGF-, or bFGF-induced proliferation (C) in pulmonary rat SMCs. STI571 dose-dependently inhibited proliferation of pulmonary artery rat SMCs stimulated with 10% FBS/DMEM. For comparison, values for control cells maintained in 0.1% FBS are given. Values are expressed as counts per minute/milligram protein or percent per field derived from 6–8 separate isolates. *P < 0.05 versus control; ^†P < 0.05 versus serum (10%) or the different growth factors.

Figure 11

Effect of STI571 on rat PA-SMC proliferation (A) and apoptosis (B) in MCT-induced pulmonary hypertension. Medial hypertrophy of pulmonary resistance vessels was associated with an increased number of proliferating vascular cells in MCT-induced pulmonary hypertension. Immunohistochemistry for PCNA (red nuclei are PCNA-positive cells; A) revealed increased proliferation in MCT-challenged animals (as compared with controls). Regression of medial hypertrophy induced by STI571 (50 mg/kg/d) was attributed to reduced SMC proliferation. Apoptosis (assessed by in situ TUNEL assay) was virtually absent in control animals and very low in pulmonary arteries from MCT animals at different time points. Apoptosis (green cells are TUNEL-positive cells; B) was increased in animals treated with STI571. Scale bar: 100 μm. Arrows indicate PCNA- and TUNEL- positive cells in A and B, respectively.

Figure 12

Schematic illustration of the proposed signaling events in MCT- or hypoxia-induced pulmonary hypertension and the impact of STI571. This scheme depicts the proposed mechanism of disease induction by MCT or hypoxia and the possible interaction of STI571 with the involved signaling events. Induction of pathways and/or cellular events is symbolized by black arrows (solid lines indicate proven evidence; dashed lines indicate postulated mechanisms). Green lines and arrows indicate the actions of STI571 in this model.