Caspase-8 modulates physiological and pathological angiogenesis during retina development
Nathalie Tisch, … , Hellmut G. Augustin, Carmen Ruiz de Almodovar
Nathalie Tisch, … , Hellmut G. Augustin, Carmen Ruiz de Almodovar
Published December 2, 2019; First published August 27, 2019
Citation Information: J Clin Invest. 2019;129(12):5092-5107. https://doi.org/10.1172/JCI122767.
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Categories: Research Article Angiogenesis

Caspase-8 modulates physiological and pathological angiogenesis during retina development

Abstract

During developmental angiogenesis, blood vessels grow and remodel to ultimately build a hierarchical vascular network. Whether, how, cell death signaling molecules contribute to blood vessel formation is still not well understood. Caspase-8 (Casp-8), a key protease in the extrinsic cell death–signaling pathway, regulates cell death via both apoptosis and necroptosis. Here, we show that expression of Casp-8 in endothelial cells (ECs) is required for proper postnatal retina angiogenesis. EC-specific Casp-8–KO pups (Casp-8ECKO) showed reduced retina angiogenesis, as the loss of Casp-8 reduced EC proliferation, sprouting, and migration independently of its cell death function. Instead, the loss of Casp-8 caused hyperactivation of p38 MAPK downstream of receptor-interacting serine/threonine protein kinase 3 (RIPK3) and destabilization of vascular endothelial cadherin (VE-cadherin) at EC junctions. In a mouse model of oxygen-induced retinopathy (OIR) resembling retinopathy of prematurity (ROP), loss of Casp-8 in ECs was beneficial, as pathological neovascularization was reduced in Casp-8ECKO pups. Taking these data together, we show that Casp-8 acts in a cell death–independent manner in ECs to regulate the formation of the retina vasculature and that Casp-8 in ECs is mechanistically involved in the pathophysiology of ROP.

Authors

Nathalie Tisch, Aida Freire-Valls, Rosario Yerbes, Isidora Paredes, Silvia La Porta, Xiaohong Wang, Rosa Martín-Pérez, Laura Castro, Wendy Wei-Lynn Wong, Leigh Coultas, Boris Strilic, Hermann-Josef Gröne, Thomas Hielscher, Carolin Mogler, Ralf H. Adams, Peter Heiduschka, Lena Claesson-Welsh, Massimiliano Mazzone, Abelardo López-Rivas, Thomas Schmidt, Hellmut G. Augustin, Carmen Ruiz de Almodovar

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Figure 3

Loss of Casp-8 in ECs impairs sprouting, proliferation, and migration.

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Loss of Casp-8 in ECs impairs sprouting, proliferation, and migration. (...
(A) Representative images of the bead-sprouting assay using HUVECs transfected with control siRNA (siCtrl) or Casp-8 siRNA (siCasp-8) and treated with VEGF (50 ng/ml) for 24 hours. (B) Quantitative analysis of total sprout length showing that VEGF is not able to induce vessel sprouting in the absence of Casp-8. Approximately 20 beads per condition were quantified. n = 4. (C) Representative images of the retinal vasculature costained with IsoB4, EdU (labels proliferating cells), and ERG (labels EC nuclei) in Casp-8WT and Casp-8ECKO mice. IsoB4 single channel was transformed to gray colors and inverted with ImageJ for better visualization. (D and E) Masks obtained by ImageJ of ERG+ cells (D) and of ERG+ EdU+ proliferating ECs (E). (F and G) Quantification of total number of ECs per retina area (F; n = 7 WT, n = 12 ECKO) and proliferating ECs per vessel area (G; n = 4 WT, n = 7 ECKO), revealing lower absolute EC numbers and fewer proliferating ECs in Casp-8ECKO retinas compared with Casp-8WT littermates. Data from 2 independent litters. (H) Single-cell motility tracks of HUVECs infected with a control (shCtrl) or Casp-8 shRNA lentivirus (shCasp-8) and treated with VEGF (50 ng/ml) for 12 hours. Migration origin of each cell was overlaid at the zero-crossing point. (I) Quantification of the total migration distance of HUVECs in H showing that VEGF-induced migration was impaired in Casp-8KD ECs. At least 30 cells per condition were quantified. n = 3. For B, F, G, and I, data are shown as mean ± SEM. *P < 0.05; ***P < 0.001, 2-way ANOVA with Bonferroni’s multiple comparisons test (B and I); *P < 0.05, 2-tailed unpaired Student’s t test (F and G). Scale bars: 200 μm (A); 50 μm (C).