RAB7 deficiency impairs pulmonary artery endothelial function and promotes pulmonary hypertension
Bryce Piper, … , David M. Eckmann, Laszlo Farkas
Bryce Piper, … , David M. Eckmann, Laszlo Farkas
Published November 28, 2023
Citation Information: J Clin Invest. 2024;134(3):e169441. https://doi.org/10.1172/JCI169441.
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Research Article Pulmonology

RAB7 deficiency impairs pulmonary artery endothelial function and promotes pulmonary hypertension

Abstract

Pulmonary arterial hypertension (PAH) is a devastating and progressive disease with limited treatment options. Endothelial dysfunction plays a central role in the development and progression of PAH, yet the underlying mechanisms are incompletely understood. The endosome-lysosome system is important to maintain cellular health, and the small GTPase RAB7 regulates many functions of this system. Here, we explored the role of RAB7 in endothelial cell (EC) function and lung vascular homeostasis. We found reduced expression of RAB7 in ECs from patients with PAH. Endothelial haploinsufficiency of RAB7 caused spontaneous pulmonary hypertension (PH) in mice. Silencing of RAB7 in ECs induced broad changes in gene expression revealed via RNA-Seq, and RAB7-silenced ECs showed impaired angiogenesis and expansion of a senescent cell fraction, combined with impaired endolysosomal trafficking and degradation, suggesting inhibition of autophagy at the predegradation level. Furthermore, mitochondrial membrane potential and oxidative phosphorylation were decreased, and glycolysis was enhanced. Treatment with the RAB7 activator ML-098 reduced established PH in rats with chronic hypoxia/SU5416. In conclusion, we demonstrate for the first time to our knowledge the fundamental impairment of EC function by loss of RAB7, causing PH, and show RAB7 activation to be a potential therapeutic strategy in a preclinical model of PH.

Authors

Bryce Piper, Srimathi Bogamuwa, Tanvir Hossain, Daniela Farkas, Lorena Rosas, Adam C. Green, Geoffrey Newcomb, Nuo Sun, Jose A. Ovando-Ricardez, Jeffrey C. Horowitz, Aneel R. Bhagwani, Hu Yang, Tatiana V. Kudryashova, Mauricio Rojas, Ana L. Mora, Pearlly Yan, Rama K. Mallampalli, Elena A. Goncharova, David M. Eckmann, Laszlo Farkas

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Figure 4

Loss of RAB7 induces endothelial dysfunction and senescence in PAECs.

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Loss of RAB7 induces endothelial dysfunction and senescence in PAECs. (A...
(A) The clustered heatmap shows increased expression of antiangiogenic genes and reduced expression of angiogenic and EC barrier function genes in RNA-Seq of PAECs after RAB7 siRNA treatment versus treatment with control siRNA. n = 3 per group. (B) Representative phase-contrast images after 24 hours and quantification of total network length in RAB7 siRNA–treated PAECs. n = 6 per group. (C) Representative phase-contrast images after 16 hours of a gap closure assay and quantification of the percentage of gap closure in RAB7 siRNA–transfected PAECs versus treatment with control siRNA. n = 7 per group. (D) Clustered heatmap of DEGs of senescence-associated gene expression pattern (Ingenuity Pathway Analysis) in RNA-Seq of PAECs treated with RAB7 siRNA. n = 3 per group. (E) Representative immunoblots and densitometric quantification for RAB7 and p16 in PAECs treated with RAB7 siRNA. n = 3 per group. (F) Representative images and quantification of the fraction of SA–β-gal+ PAECs after RAB7 siRNA treatment versus treatment with control siRNA. n = 6 per group. Data are shown as single values and the median ± IQR (B and C) or the mean ± SD (E and F). *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001, by 2-tailed Mann-Whitney U test (B and C) or 2-tailed Student’s t test (E and F). Heatmap data are normalized log2 fold expression. Data in B, C, E, and F are representative of 2 or more experiments. Scale bars: 250 μm (B and C) and 200 μm (F).