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 4

Effects of MMC on the lung proteomic landscape in Ctrl and KO mice.

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Effects of MMC on the lung proteomic landscape in Ctrl and KO mice. (A) ...
(A) Lungs harvested on day 5 from Veh- or MMC-treated Ctrl and KO mice were subjected to MS analysis. The principal component analysis of the MS data for lungs from Veh- or MMC-treated Ctrl and KO mice, conducted in triplicate, is shown. (B) A hierarchically clustered heatmap of differentially expressed proteins (DEPs) that are statistically significant (P < 0.05 by 2-tailed Student’s t test) in the lungs of Veh- or MMC-treated Ctrl and KO mice. n = 3 per group. (C) Volcano plots compare the proteome of MMC-treated versus Veh-treated Ctrl and KO mouse lungs (top). A larger circle size represents a lower P value, while the color gradient from blue to red corresponds to increasing log2(FC) values, with blue indicating lower values and magenta indicating higher values. Pie charts demonstrate the fraction (%) of DEPs upregulated (magenta) or downregulated (blue), with a threshold of log2(FC) greater than 0.8 or less than –0.8 in Ctrl and KO mice (bottom). The gray part represents the fraction of DEPs with –0.8 ≤ log2(FC) ≤ 0.8. n = 3 per group. (D) Top 15 pathways most enriched in upregulated (magenta) and downregulated (blue) DEPs in the lungs of Ctrl (top) and KO mice (bottom) following MMC treatment. The green, red, and orange asterisks indicate pathways related to protein synthesis/modifications, RNA metabolism, and mitochondrial ATP synthesis, respectively. (E) The bar graphs indicate the fold change in ribosomal proteins (left panel) and components of the mitochondrial electron transport chain (ETC) (right panel) between vehicle and MMC treatment in Ctrl and KO mice. Magenta and blue colors indicate upregulated and downregulated proteins, respectively. Darker colored bars represent P < 0.05. Differential expression of proteins was assessed using Welch’s t test.