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 5

Impaired endosome-lysosome function in PAH PAECs and RAB7-deficient PAECs.

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Impaired endosome-lysosome function in PAH PAECs and RAB7-deficient PAEC...
(A) Representative optical sections (confocal microscopy) and quantification of pHrodo dextran+ vesicle area and number of vesicles per cell indicate the accumulation of pHrodo dextran after 20 minutes in enlarged vesicles in PAH PAECs (arrows), but not in control PAECs. pHrodo dextran is taken up by endosomes and emits red fluorescence signal when the pH drops during endosomal acidification. n = 9 (control) and n = 17 (PAH). (B) Representative optical sections (confocal microscopy) show an accumulation of pHrodo dextran after 20 minutes in enlarged early endosomes with RAB7 silencing. Early endosomes were identified by transfection of PAECs with baculovirus expressing GFP-labeled RAB5. Quantification of RAB5+ pHrodo dextran+ endosome area and number per cell confirmed that dextran accumulated in enlarged early endosomes following RAB7 silencing. n = 13 per group. (C) Representative optical sections (confocal microscopy) show an accumulation of pHrodo dextran after 20 minutes in enlarged lysosomes in RAB7 siRNA–treated PAECs. Lysosomes were labeled with LysoTracker. Quantification of LysoTracker+ pHrodo dextran+ lysosome area and number per cell confirmed that dextran accumulated in enlarged lysosomes following RAB7 silencing. n = 13 per group. (D) Clustered heatmap shows autophagy-related DEGs that were found to be downregulated in RNA-Seq from PAECs treated with RAB7 siRNA. Expression is normalized log2-fold. (E) Reduced cathepsin B activity also indicates impaired lysosomal autophagy. n = 9 per group. Scale bars: 10 μm (A–C). All graphs show single values and the mean ± SD. Data in A–C are representative of 2 or more experiments. *P < 0.05, **P < 0.01, ****P < 0.0001, by 2-tailed Student’s t test.