Endothelial cell polarity and extracellular matrix composition require functional ATP6AP2 during developmental and pathological angiogenesis
Nehal R. Patel, Rajan K C, Avery Blanks, Yisu Li, Minolfa C. Prieto, Stryder M. Meadows
Nehal R. Patel, Rajan K C, Avery Blanks, Yisu Li, Minolfa C. Prieto, Stryder M. Meadows
View: Text | PDF
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

×

Figure 2

ATP6AP2 is critical for sprouting angiogenesis.

Options: View larger image (or click on image) Download as PowerPoint
ATP6AP2 is critical for sprouting angiogenesis. (A) Images of IB4+ vesse...
(A) Images of IB4+ vessels from P7 control and Atp6ap2iECKO retinas, with white arrowheads indicating sprouts at the vascular front. Scale bars: 100 µm. (B) Quantification of the number of sprouts in control and Atp6ap2iECKO mice at P7 (n = 4). (C) Images of IB4+ vessels at high magnification showing filopodia in sprouts (white asterisks) at the vascular front . Scale bars: 10 µm. (D) Quantification of the number of filopodia per sprout in control and Atp6ap2iECKO mice at P7 (n = 4). (E) Western blot analysis of ATP6AP2 and β-actin in control-siRNA and Atp6ap2 siRNA–treated TeloHAECs. (F) Densitometric quantification of ATP6AP2 levels in E normalized to β-actin in TeloHAECs following siRNA treatments (n = 3). (G) qPCR analysis of control-siRNA and Atp6ap2 siRNA–treated TeloHAECs for Atp6ap2 mRNA levels normalized to GAPDH transcripts (n = 3; triplicates for each sample). (H) Representative images of TeloHAEC sprouting bead assays embedded in 3D fibrinogen gel at 120 hours following control and Atp6ap2-siRNA treatments. scale bars: 100 µm. Black arrowheads indicate vessel bifurcations. (I) Quantification of the number of sprouts per bead (n = 18). (J) Images of IB4+ vessels at the vascular front of control and Atp6ap2iECKO P7 retinas immunolabeled for Ki67 and the EC-specific nuclear marker ETS transcription factor ERG. Scale bars: 50 µm. (K) Quantification of Ki67+ERG+ proliferative ECs in control and Atp6ap2iECKO mice at P7 (n = 4). (L) Images of ERG+ EC nuclei and IB4+ vessels at the vascular front. Scale bars: 25 µm. (M) Quantification of the number of ERG+ ECs in control and Atp6ap2iECKO P7 mice at the vascular front (n = 4). Data are shown as mean ± SD; 2-tailed unpaired t test. **P < 0.01, ****P < 0.0001.