Alteration of developmental and pathological retinal angiogenesis in angptl4-deficient mice

Abstract

Proper vessel maturation, remodeling of endothelial junctions, and recruitment of perivascular cells is crucial for establishing and maintaining vessel functions. In proliferative retinopathies, hypoxia-induced angiogenesis is associated with disruption of the vascular barrier, edema, and vision loss. Therefore, identifying factors that regulate vascular maturation is critical to target pathological angiogenesis. Given the conflicting role of angiopoietin-like-4 (ANGPTL4) reported in the current literature using gain of function systems both in vitro and in vivo, the goal of this study was to characterize angiogenesis, focusing on perinatal retinal vascularization and pathological circumstances in angpl4-deficient mice. We report altered organization of endothelial junctions and pericyte coverage, both leading to impaired angiogenesis and increased vascular leakage that were eventually caught up, suggesting a delay in vessel maturation. In a model of oxygen-induced retinopathy, pathological neovascularization, which results from tissue hypoxia, was also strongly inhibited in angptl4-deficient mice. This study therefore shows that ANGPTL4 tunes endothelial cell junction organization and pericyte coverage and controls vascular permeability and angiogenesis, both during development and in pathological conditions.

FIGURE 1.

Defective developmental angiogenesis in angptl4-deficient pups. A–C, LacZ staining shows expression of angptl4 in endothelial cells at P7 (A), P12 (B), and P17 (C) during developmental angiogenesis of the retina. Scale bar = 500 μm. D–F and G–I, IB4 (red) and GFAP (green) staining allow quantification of the vascular and astrocytic networks, respectively. A, artery; V, vein; VF, vascular front; o, optic nerve. Scale bar = 100 μm. Quantification of filopodia bursts/field of view (J); of artery, capillary and vein diameter (K); of vascular density (% of IB4 positive surface/total field area) (L); of vessel branch points/field of view (M); of astrocyte network density (left panel) and of astrocyte branch points/field of view (right panel) in the avascular (N) and vascular (O) area in angptl4^+/LacZ and angptl4^LacZ/LacZ P6 retinas (n = 8 per group). Shown is quantification by real-time quantitative PCR of vegfa and vegfr2 mRNAs (P) and in angptl4^+/LacZ and angptl4^LacZ/LacZ P7 retinas (n = 3 per group). GFAP, glial fibrillary acidic protein. ns, non significant.

FIGURE 2.

Perturbation of CAV-1 staining in angptl4-deficient pups. A–E, reduced CAV-1 expression in area 1 of angptl4-deficient mice. CAV-1 staining (green) is homogeneous in area 1 of angptl4^+/LacZ vasculature (A and D) but weaker and discontinuous in capillaries from area 1 of angptl4^LacZ/LacZ retinas (B and E). Scale bar = 50 μm. IB4 is shown in red (D and E). C, shown is the ratio of CAV-1-positive vessels to endothelial cell surface (IB4+ vessels). p < 0.01. F–J, reduced CAV-1 expression in areas 2/3 of angptl4-deficient mice. CAV-1 staining (green) is discontinuous in angptl4^+/LacZ vasculature (F and I) but weaker in capillaries from area 2 and rarely present in area 3 (white arrows) of angptl4^LacZ/LacZ retinas (G and J). p < 0.01. IB4 is shown in red. Scale bar = 50 μm. H, shown is the ratio of CAV-1-positive vessels to endothelial cell surface (IB4-positive vessels) (n = 4 per group).

FIGURE 3.

Perturbation of endothelial junction integrity in angptl4-deficient pups. A and B, focal plane by three-dimensional reconstruction of IB4 (blue) staining in angptl4^+/LacZ and angptl4^LacZ/LacZ P7 retinas. ZO-1 (red) (C and D) and VE-CAD (green) (E and F) staining reveals discontinuous endothelial junctions in area 2 of angptl4-deficient vasculature (the arrows point to discontinuous junctions at branch points and the arrowheads indicate tortuous-like junctions) (D and F). Scale bar = 15 μm. G and H, schematic representation of the endothelial surface of IB4 staining (blue) and endothelial junctions of VE-CAD and ZO-1 staining (light blue tracing). Endothelial junctions are poorly developed in angptl4^LacZ/LacZ capillaries (H) compared with mature junctions in control retinas (G).

FIGURE 4.

Defects in pericyte coverage in developmental angptl4^LacZ/LacZretinas. A–E, delay of pericyte coverage in area 2 of P7 angptl4 ^LacZ/LacZ retinas. Representative images of P7 angptl4^+/LacZ (A and C) and angptl4^LacZ/LacZ (B and D) retinas stained with IB4 (red) and NG2 (green) (A–D). The arrows point to abnormal pericytes (B and D) on angptl4 ^LacZ/LacZ capillaries. Scale bar = 25 μm. E, shown is the ratio of pericytes (NG2+ area) to endothelial cell surface (IB4+ vessels), n = 6 per group. p < 0.005.

FIGURE 5.

Increased vessel permeability in angptl4-deficient pups. A, leakage of Evans Blue in P7 angptl4^+/LacZ and angptl4^LacZ/LacZ retinas. Values are expressed as % of the control (angptl4^+/LacZ) value, (angptl4^+/LacZ, n = 4; angptl4^LacZ/LacZ, n = 5). B and G, confocal images of whole mount P7 angptl4^+/LacZ and angptl4^LacZ/LacZ retinas stained with IB4 (blue) and ZO-1 (red). Prior to sacrifice, fluorescent 100 nm microspheres were injected intravenously and flushed from circulation 15 min afterwards. B and C, deconvolution and three-dimensional reconstruction of the boxed region in E. Extravasated microspheres are trapped around tight junctions with a large part outside of the vessel (ZO-1, red). Field dimensions are 200 × 220 × 7 μm. E and G, extravasated microspheres (green) were only observed in angptl4^LacZ/LacZ retinas. Scale bar = 100 μm. n = 6 per group. p < 0.05.

FIGURE 6.

Loss of angptl4 inhibits neovascularization during oxygen-induced retinopathy. A–C, hyperoxia-induced vaso-obliteration is unaffected in angptl4-deficient mice. A and B, images of whole mount P12 angptl4^+/LacZ and angptl4^LacZ/LacZ retinas stained with IB4. The avascular area around the optic nerve (o) is outlined in white. Scale bars = 300 μm. C, values are expressed as % of the control (angptl4^+/LacZ) value (angptl4^+/LacZ, n = 7; angptl4^LacZ/LacZ, n = 11). D–I, neovascularisation at P17 is reduced in angptl4-deficient mice. Images of whole-mount P17 angptl4^+/LacZ and angptl4^LacZ/LacZ retinas stained with IB4 (D–E). The avascular area is outlined in white. Scale bars represent 300 μm. F, values are expressed as % of the control (angptl4^+/LacZ) value, n = 6 per group. G–I, defective pericyte coverage of veins in P17 angptl4^LacZ/LacZ retinas after oxygen-induced retinopathy. Representative images of OIR P17 angptl4^+/LacZ (G) and angptl4^LacZ/LacZ (H) retinas stained with IB4 (red) and NG2 (green) (G–H). Scale bar represents 50 μm. Ratio of NG2-positive vessels to IB4-positive vessels (I), n = 3 per group. p < 0.05.