Caspase-8 modulates physiological and pathological angiogenesis during retina development
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
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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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 7

RIPK3 acts downstream of Casp-8 to regulate angiogenesis.

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RIPK3 acts downstream of Casp-8 to regulate angiogenesis. (A) Western bl...
(A) Western blot showing that knocking down RIPK3 in Casp-8KD ECs rescues the basal hyperphosphorylation of p38. Notice that shCasp-8 HUVECs have increased RIPK3 protein levels. (B) Quantification of Western blots under basal conditions as in A. n = 4. (C) Quantitative PCR (qPCR) of shCtrl or shCasp-8–infected HUVECs showing increased mRNA expression of Ripk3 in the absence of Casp-8. n = 4. (D) Quantification of total tube length of shCtrl or shCasp-8–infected HUVECs that were cotransfected with control siRNA or Ripk3 siRNA and treated with or without VEGF (50 ng/mL) for 4 hours. Ten fields per condition were quantified. n = 3. (E) BrdU+ cells were quantified in control and Casp-8KD ECs transfected with Ripk3 siRNA and with or without VEGF (50 ng/mL) or FGF (50 ng/mL) stimulation for 24 hours. Around 50 cells per condition were quantified. n = 3. (F and G) Representative images of the retinal vasculature stained with IsoB4 in Casp-8WT/RIPK3KO and Casp-8ECKO/RIPK3KO mice. (H and I) Quantitative analysis showing no differences in vessel area (H; n = 11 WT, n = 7 ECKO) and number of branches (I; n = 9 WT, n = 7 ECKO). Data from 4 independent litters. (J) Working model summarizing the role of Casp-8 as a modulator of angiogenesis. Casp-8 inhibits RIPK3 to allow the proper response of ECs to VEGF stimulation (Casp-8WT). If Casp-8 is absent (Casp-8ECKO), increased RIPK3 levels induce p38 hyperphosphorylation, which in turn leads to an impaired response to VEGF stimulation and reduced angiogenesis. For B–D, E, H, and I, data are shown as mean ± SEM. *P < 0.05; ***P <0.001, 1-sample t test (B and C); *P < 0.05; **P < 0.01; ***P < 0.001, 2-way ANOVA with Tukey’s multiple comparisons test (D and E); 2-tailed unpaired Student’s t test (H and I).