Endothelial cell polarity and extracellular matrix composition require functional ATP6AP2 during developmental and pathological angiogenesis
Nehal R. Patel, … , Minolfa C. Prieto, Stryder M. Meadows
Nehal R. Patel, … , Minolfa C. Prieto, Stryder M. Meadows
Published August 23, 2022
Citation Information: JCI Insight. 2022;7(19):e154379. https://doi.org/10.1172/jci.insight.154379.
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Research Article Angiogenesis Vascular biology

Endothelial cell polarity and extracellular matrix composition require functional ATP6AP2 during developmental and pathological angiogenesis

Abstract

The (Pro)renin receptor ([P]RR), also known as ATP6AP2, is a single-transmembrane protein that is implicated in a multitude of biological processes. However, the exact role of ATP6AP2 during blood vessel development remains largely undefined. Here, we use an inducible endothelial cell–specific (EC-specific) Atp6ap2-KO mouse model to investigate the role of ATP6AP2 during both physiological and pathological angiogenesis in vivo. We observed that postnatal deletion of Atp6ap2 in ECs results in cell migration defects, loss of tip cell polarity, and subsequent impairment of retinal angiogenesis. In vitro, Atp6ap2-deficient ECs similarly displayed reduced cell migration, impaired sprouting, and defective cell polarity. Transcriptional profiling of ECs isolated from Atp6ap2 mutant mice further indicated regulatory roles in angiogenesis, cell migration, and extracellular matrix composition. Mechanistically, we provided evidence that expression of various extracellular matrix components is controlled by ATP6AP2 via the ERK pathway. Furthermore, Atp6ap2-deficient retinas exhibited reduced revascularization in an oxygen-induced retinopathy model. Collectively, our results demonstrate a critical role of ATP6AP2 as a regulator of developmental and pathological angiogenesis.

Authors

Nehal R. Patel, Rajan K C, Avery Blanks, Yisu Li, Minolfa C. Prieto, Stryder M. Meadows

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Figure 3

ATP6AP2 regulates endothelial cell migration and polarity in vivo and in vitro.

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ATP6AP2 regulates endothelial cell migration and polarity in vivo and in...
(A) Representative images of ERG+ nuclei of ECs at the vascular front in P7 control and Atp6ap2iECKO retinas. Scale bars: 25 µm. (B) Quantification of nuclear ellipticity in control and Atp6ap2iECKO mice at P7 (n = 4). (C) Scratch wound assays performed on TeloHAEC monolayers following control and Atp6ap2 siRNA treatments. Images at 0 and 40 hours following the scratch. Scale bars: 75 µm. (D) Quantification of the wound closure and rate of migration in control and Atp6ap2 siRNA–treated TeloHAECs at 40 hours (n = 12). (E) Images of fluorescently labeled IB4+ vessels, ERG+ nuclei in ECs, and Golgi Matrix Protein GM130+ Golgi apparatuses at the vascular front with their respective insets (white dashed-line boxed) highlighting tip cells in control and Atp6ap2iECKO mice at P7. Scale bars: 50 µm and 15 μm (insets). White arrowheads denote the Golgi apparatus position in respect to the nucleus. White arrows indicate direction of the retinal vasculature outgrowth. (F) Images of DAPI+ nuclei, GM130+ Golgi apparatuses, and F-actin+ cytoskeleton of indicated TeloHAECs at 12 hours after initiating cell migration in a scratch assay . Scale bars: 50 µm. White arrows indicate the direction of migration. (G) Rose plots showing the Golgi apparatus polarization in respect to the nucleus in both control and Atp6ap2 siRNA–treated TeloHAECs at 12 hours after scratch (n = 52). (H) Western blot analysis of PAR3, PARVA, and β-actin from control and Atp6ap2iECKO iLECs at P7 (n = 3). (I) Densitometric quantifications of PAR3 and PARVA levels in control and Atp6ap2iECKO iLECs from H. Data are shown as mean ± SD; 2-tailed unpaired t test. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.