Essential role of protein kinase R in the pathogenesis of pulmonary veno-occlusive disease
Amit Prabhakar, Rahul Kumar, Meetu Wadhwa, Abhilash Barpanda, Joseph Lyons, Asavari Gowda, Simren Gupta, Ananyaa Arvind, Prajakta Ghatpande, Arun P. Wiita, Brian B. Graham, Giorgio Lagna, Akiko Hata
Amit Prabhakar, Rahul Kumar, Meetu Wadhwa, Abhilash Barpanda, Joseph Lyons, Asavari Gowda, Simren Gupta, Ananyaa Arvind, Prajakta Ghatpande, Arun P. Wiita, Brian B. Graham, Giorgio Lagna, Akiko Hata
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Research Article Cell biology Vascular biology

Essential role of protein kinase R in the pathogenesis of pulmonary veno-occlusive disease

Abstract

Pulmonary veno-occlusive disease (PVOD) is a rare and severe subtype of pulmonary arterial hypertension, characterized by progressive remodeling of small pulmonary arteries and veins with no therapies. Using a mitomycin C–induced (MMC-induced) rat model, we previously demonstrated that protein kinase R–mediated (PKR-mediated) integrated stress response (ISR) drives endothelial dysfunction and vascular remodeling. To determine whether PKR is the primary mediator of ISR and the pathogenesis, we treated control (Ctrl) and PKR-knockout (KO) mice with the same dose of MMC. Consistent with rat data, Ctrl mice displayed ISR activation, vascular remodeling, and pulmonary hypertension after MMC treatment, while KO mice showed none of these phenotypes. Proteomic analysis revealed that MMC-mediated ISR activation attenuated protein synthesis in Ctrl but not in KO mice. These findings underscore the critical role of PKR-dependent ISR activation and subsequent perturbation of proteostasis as central mechanisms driving PVOD pathogenesis and identify PKR as a promising therapeutic target.

Authors

Amit Prabhakar, Rahul Kumar, Meetu Wadhwa, Abhilash Barpanda, Joseph Lyons, Asavari Gowda, Simren Gupta, Ananyaa Arvind, Prajakta Ghatpande, Arun P. Wiita, Brian B. Graham, Giorgio Lagna, Akiko Hata

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Figure 2

PKR-deficient mice do not develop PVOD phenotypes.

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PKR-deficient mice do not develop PVOD phenotypes. (A) RVSP (mmHg) and R...
(A) RVSP (mmHg) and RV/LV+S ratio in vehicle- (Veh) or MMC-administered control (Ctrl) and KO mice. Male and female animals are indicated as black circles and blue triangles, respectively. n = 8–9 independent samples per group. (B) RV wall thickness (mm) of Veh- or MMC-treated Ctrl and KO mice was measured and shown as mean ± SEM (left). The wall thickness was measured at 5 locations, and the mean value was calculated per sample. n = 6 independent samples per group. (C) Microfil casting of the lung vasculature in Ctrl and KO mice treated with either Veh or MMC on day 5 (d5). Holistic images of the entire lung are displayed (left). Scale bars: 0.5 cm. The number of junctions and branches per cm² of distal pulmonary vessels was quantified, with the data presented as mean ± SEM (right). n = 4 independent samples per group. (D) H&E staining of pulmonary vessels (PA and PV; arrows) in Ctrl and KO mice administered Veh or MMC (left). The third column is a magnified image of the black rectangle area in the second column (left). The fraction (%) of moderately (25%–40% occlusion) and severely (>40% occlusion) occluded vessels were counted and shown as mean ± SEM (right). Scale bars: 10 μm. n = 5 independent samples. (E) MSB staining visualizing collagens (blue) and smooth muscle cells (pink) in the lungs of Ctrl (on d5) and KO mice (on d5 and d10) following Veh or MMC administration. Scale bars: 10 μm. (F) PAs and PVs from Ctrl and KO mice on d5 or d10 after vehicle or MMC administration were stained with an anti-αSMA (red) and anti–VE-Cad (green) antibody for smooth muscle cells and endothelial cells, respectively. The merged images of αSMA and VE-Cad staining are shown. An asterisk indicates the location of the vessel (top). Scale bars: 50 μm. The signal intensities of αSMA and VE-Cad are quantified and are presented as mean ± SEM (bottom). (G) The permeability of pulmonary vasculature was assessed by injecting EB dye in Ctrl and KO mice administered with vehicle or MMC. The lung was harvested on d5 or d10, and the image was taken when the lung became translucent (left). Representative images of the whole lung and the largest lobe are presented. Scale bar: 0.5 cm. The relative intensities of EB staining were quantified and are presented as mean ± SEM (right). Statistical analysis was performed using 1-way ANOVA with Tukey’s multiple-comparison test (A) or 2-way ANOVA with Tukey’s multiple-comparison test (B–D, F, and G).