Mechanisms underlying age-associated exacerbation of pulmonary veno-occlusive disease
Amit Prabhakar, Meetu Wadhwa, Rahul Kumar, Prajakta Ghatpande, Aneta Gandjeva, Rubin M. Tuder, Brian B. Graham, Giorgio Lagna, Akiko Hata
Amit Prabhakar, Meetu Wadhwa, Rahul Kumar, Prajakta Ghatpande, Aneta Gandjeva, Rubin M. Tuder, Brian B. Graham, Giorgio Lagna, Akiko Hata
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Research Article Therapeutics Vascular biology

Mechanisms underlying age-associated exacerbation of pulmonary veno-occlusive disease

Abstract

Pulmonary veno-occlusive disease (PVOD) is a rare but severe form of pulmonary hypertension characterized by the obstruction of pulmonary arteries and veins, causing increased pulmonary artery pressure and leading to right ventricular (RV) heart failure. PVOD is often resistant to conventional pulmonary arterial hypertension (PAH) treatments and has a poor prognosis, with a median survival time of 2–3 years after diagnosis. We previously showed that the administration of a chemotherapy agent mitomycin C (MMC) in rats mediates PVOD through the activation of the eukaryotic initiation factor 2 (eIF2) kinase protein kinase R (PKR) and the integrated stress response (ISR), resulting in the impairment of vascular endothelial junctional structure and barrier function. Here, we demonstrate that aged rats over 1 year exhibit more severe vascular remodeling and RV hypertrophy than young adult rats following MMC treatment. This is attributed to an age-associated elevation of basal ISR activity and depletion of protein phosphatase 1, leading to prolonged eIF2 phosphorylation and sustained ISR activation. Pharmacological blockade of PKR or ISR mitigates PVOD phenotypes in both age groups, suggesting that targeting the PKR/ISR axis could be a potential therapeutic strategy for PVOD.

Authors

Amit Prabhakar, Meetu Wadhwa, Rahul Kumar, Prajakta Ghatpande, Aneta Gandjeva, Rubin M. Tuder, Brian B. Graham, Giorgio Lagna, Akiko Hata

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Figure 2

Administration of C16 or ISRIB 24 days after MMC treatment reverses PVOD phenotypes.

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Administration of C16 or ISRIB 24 days after MMC treatment reverses PVOD...
(A) A scheme of delayed C16 and ISRIB treatment on d24–d32 after the administration of MMC on d0 in aged and young rats (top). The RVSP and the RV/LV+S ratio in vehicle- or MMC-exposed rats with or without C16 or ISRIB treatment are shown as mean ± SEM (bottom). n = 3–7 independent samples. (B) H&E staining of pulmonary vasculature (PAs and PVs) in young and aged rats treated with vehicle or MMC with or without C16 or ISRIB treatment was performed (top). A total number of microvessels was analyzed and distributed in 3 categories based on medial thickening/occlusion of pulmonary vasculature as null (no occlusion), moderate (25%–50% occlusion), and severe (50%–100%) in young (bottom, left) and aged (bottom, right) rats. The data are presented as a faction (%) of vessels in each category relative to the total vessels count and shown as mean ± SEM. Scale bar: 10 μm. n = 3 independent experiments. (C) Trichrome staining of pulmonary vasculature (PAs and PVs) in young and aged rats treated with vehicle or MMC with or without delayed C16 or ISRIB treatment was performed on d32 and shown as mean ± SEM. Scale bar: 10 μm. n = 3 independent experiments. (D) A scheme of MMC treatment in young and aged rats. Lung lysates of young and aged rats 24 days after vehicle or MMC administration were subjected to immunoblot of the indicated proteins. The amounts of indicated proteins, normalized to β-actin, the ratio of p-PKR/t-PKR, and p-eIF2α/t-eIF2α, are shown as mean ± SEM. n = 6 independent samples per condition. (E) Lung sections from young and aged rats administered with vehicle or MMC were subjected to IF staining using anti–p-PKR, anti–t-PKR, anti–p-eIF2α, anti–t-eIF2α, anti-GCN2, and anti-CD31 antibodies, and the images are shown. Cell nuclei were stained with DAPI. The asterisks indicate the location of the vessels. Scale bar: 10 μm. Statistical analysis was performed using 1-way ANOVA with Tukey’s multiple comparisons test (A and D) or 2-way ANOVA with Tukey’s multiple comparisons test (B) with P < 0.05.