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 6

RAB7 silencing impairs mitochondrial membrane potential and mitochondrial function.

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RAB7 silencing impairs mitochondrial membrane potential and mitochondri...
(A) RAB7 siRNA impaired ΔΨm as assessed by TMRE and MitoTracker Green (MitoGreen) flow cytometry. Carbonyl cyanide FCCP was used as a positive control (depolarizes mitochondrial membrane). TMREhi MitoGreenhi cells indicate cells with functional mitochondria, whereas TMRElo MitoGreenhi cells are cells with dysfunctional mitochondria. n = 8 (control) and n = 9 (RAB7). (B) Representative TMRM-stained images show overall reduction and perinuclear accumulation of functional mitochondria in RAB7 siRNA–treated PAECs. Results are representative of 3 experiments. Scale bars: 5 μm. RAB7 knockdown increased mitochondrial motility in peripheral and perinuclear regions. (C) RAB7 knockdown promoted mitochondrial ROS production as indicated by flow cytometry for mitoSOX. n = 6 per group for the representative histogram plots and quantification of MFI. (D) Clustered heatmap shows upregulated glycolysis-related DEGs and downregulated oxidative phosphorylation–related DEGs in bulk RNA-Seq from PAECs plus RAB7 siRNA. Expression is normalized log2-fold. (E) Seahorse high-resolution respirometrics show a reduced OCR with RAB7 siRNA at basal respiration, maximal respiration, and spare respiratory capacity. n = 15 (control) and n = 21 (RAB7). (F) Luminescence assay for lactate: increased lactate production in RAB7 siRNA–treated PAECs. n = 10 (control) and n = 11 (RAB7). (G) ECAR data reveal more rapid acidification (i.e., greater reliance on glycolysis) in the RAB7 siRNA–treated PAECs as shown for glycolysis and the basal and maximum glycolytic rates. n = 12 per group. All graphs show single values and the median ± interquartile range (A, E, and F) or the mean ± SD (C and G) (except B, as bar graphs in B indicate the geometric mean of a log-normal distribution). Data in A–C, E, F, and G are from 2 or more experiments. *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001, by 2-tailed Mann-Whitney U test (A and F), 2-tailed Student’s t test (C), and 2-way ANOVA (E and G) with Holm-Šidák post hoc test and normality testing of residuals (D’Agostino-Pearson).