PAI-1 deficiency drives pulmonary vascular smooth muscle remodeling and pulmonary hypertension

Abstract

Pulmonary arterial hypertension (PAH) is a progressive disease characterized by vasoconstriction and remodeling of small pulmonary arteries (PAs). Central to the remodeling process is a switch of pulmonary vascular cells to a proliferative, apoptosis-resistant phenotype. Plasminogen activator inhibitors-1 and -2 (PAI-1 and PAI-2) are the primary physiological inhibitors of urokinase-type and tissue-type plasminogen activators (uPA and tPA), but their roles in PAH are unsettled. Here, we report that: 1) PAI-1, but not PAI-2, is deficient in remodeled small PAs and in early-passage PA smooth muscle and endothelial cells (PASMCs and PAECs) from subjects with PAH compared with controls; 2) PAI-1^-/- mice spontaneously develop pulmonary vascular remodeling associated with upregulation of mTORC1 signaling, pulmonary hypertension (PH), and right ventricle (RV) hypertrophy; and 3) pharmacological inhibition of uPA in human PAH PASMCs suppresses proproliferative mTORC1 and SMAD3 signaling, restores PAI-1 levels, reduces proliferation, and induces apoptosis in vitro, and prevents the development of SU5416/hypoxia-induced PH and RV hypertrophy in vivo in mice. These data strongly suggest that downregulation of PAI-1 in small PAs promotes vascular remodeling and PH due to unopposed activation of uPA and consequent upregulation of mTOR and transforming growth factor-β (TGF-β) signaling in PASMCs, and call for further studies to determine the potential benefits of targeting the PAI-1/uPA imbalance to attenuate and/or reverse pulmonary vascular remodeling and PH.NEW & NOTEWORTHY This study identifies a novel role for the deficiency of plasminogen activator inhibitor (PAI)-1 and resultant unrestricted uPA activity in PASMC remodeling and PH in vitro and in vivo, provides novel mechanistic link from PAI-1 loss through uPA-induced Akt/mTOR and TGFβ-Smad3 upregulation to pulmonary vascular remodeling in PH, and suggests that inhibition of uPA to rebalance the uPA-PAI-1 tandem might provide a novel approach to complement current therapies used to mitigate this pulmonary vascular disease.

Keywords: plasminogen activator inhibitor (PAI-1); pulmonary hypertension; urokinase PA (uPA).

Graphical abstract

Figure 1.

PAI-1 is deficient in pulmonary vascular cells from small PAH PAs. A: IHC analysis of human lung tissue sections to detect PAI-1 (red). Green—SMA; blue—DAPI; yellow—red and green overlap. Images are representative from three subjects/group. Bar = 50 µm. B–E: immunoblot analysis of PAECs and PASMCs from human control and PAH lungs to detect indicated proteins. Gels for the detection of β-actin were ran separately from the same sample preparations. F: rates of the plasmin generation in control and PAH PASMCs normalized to the cell numbers. G and H: cell proliferation was measured by Ki67 staining (red); data represent the percentile of Ki67-positive cells/total number of cells detected by DAPI (blue). Bar = 50 µm. B–G: data are means ± SD; n = 4–5 human subjects/group. Mann–Whitney U test was used; closed black circles—cells from males; open circles—cells from females. PAs, pulmonary arteries; PAEC, pulmonary artery endothelial cells; PAI-1, plasminogen activator inhibitor-1; PAH, pulmonary arterial hypertension; PASMC, pulmonary artery smooth muscle cells.

Figure 2.

Loss of PAI-1 in mice results in the development of spontaneous PH. A–F: morphological and hemodynamic analysis of 10-mo-old WT C57BL/6J and PAI-1^–/– mice. A and B: representative H&E images (bar = 30 µm) (A) and PA MT (B) from 10 PAs (<150 µm outer diameter) per mouse; C and D: sRVP (C) and RV/(LV+S) (Fulton index) (D). n = 6 mice/WT group (B–D); n = 6 (B and D) and n = 5 (C) mice/PAI-1^–/– group. E and F: IHC analysis of lung tissue sections from PAI-1^–/– and WT mice. Red: phospho-Smad3 (E) or phospho-S6 (F), green—SMA, blue—DAPI. Bar = 20 µm. Representative images from three animals/group. G: human control PASMCs were transfected with siRNA PAI-1 (+) or control scrambled siRNA (–); 48 h posttransfection, immunoblot analysis was performed to detect indicated proteins. Representative images (top) and statistical analysis of n = 5 subjects/group (bottom). Data are means ± SD; Mann–Whitney U test was used; closed black circles—cells from males; open circles—cells from females. PAs, pulmonary arteries; PAI-1, plasminogen activator inhibitor-1; PASMC, pulmonary artery smooth muscle cells; sRVP, systolic RV pressure.

Figure 3.

uPA inhibition reduces proproliferative signaling, restores PAI-1, inhibits proliferation, and induces apoptosis in human PAH PASMCs. Human PAH PASMCs were incubated with 10 µM uPA inhibitors BB2-30F (A) or WX671 (B), or diluent for 48 h followed by immunoblot analysis to detect indicated proteins (A and B), cell proliferation (Ki67) (C), or apoptosis (In Situ Cell Death Detection Kit) (D) assessment. n = 3 subjects/group. Data are means ± SD from three independent experiments, each performed using cells from different human subjects. Bar = 100 µm. Mann–Whitney U test was used; open circles indicate cells from female patients. PAI-1, plasminogen activator inhibitor-1; PAH, pulmonary arterial hypertension; PASMC, pulmonary artery smooth muscle cells; uPA, urokinase-type plasminogen activator.

Figure 4.

Inhibition of uPA prevents the development of SuHx-induced PH in mice. WT (C57BL/6J) male mice were given daily intraperitoneal injections of 10 mg/kg uPA inhibitor BB2-30F or vehicle from days 1–21 of exposure to VEGFR inhibitor SU5416 (20 mg/kg sc once a week) and hypoxia ($\text{F}{\text{I}}_{{\text{O}}_2}$ 10%). Hemodynamic measurements and tissue harvest were made on day 21. Controls (con) were same-age mice kept under normoxia. A: IHC of lung tissue sections. Red—phospho-S6, green—SMA, blue—DAPI. Bar = 50 µm. Images are representative from three animals/group. B and C: representative H&E images (bar = 30 µm) (B) and PA MT (C); n = 6 mice/group, 10 PAs with outer diameter <150 μm per mouse. D–F: sRVP (D), RV/(LV+S) (Fulton index) (E), and PA acceleration time/ejection time (PAAT/PET) ratio (F); C, E, and F: n = 6 mice/group. D: n = 6/4/5 mice/groups Con/SuHx-Veh/SuHx-BB2. C, D, E, and F: one-way ANOVA with Tukey’s multiple comparison test was used; Data are means ± SD. G: schematic representation of the mechanism by which PAI-1 deficiency promotes PA remodeling and PH. PAs, pulmonary arteries; PAI-1, plasminogen activator inhibitor-1; PASMC, pulmonary artery smooth muscle cells; sRVP, systolic RV pressure; uPA, urokinase-type plasminogen activator. [Figure created with BioRender.com.]