Hyperoxia Therapy of Pre-Proliferative Ischemic Retinopathy in a Mouse Model
ABSTRACT To investigate the therapeutic use and mechanisms of action of normobaric hyperoxia to promote revascularization and to prevent neovascularization in a mouse model of oxygen-induced ischemic retinopathy.
Hyperoxia treatment (HT, 40%-75% oxygen) was initiated on postnatal day (P) 14 during the pre-proliferative phase of ischemic retinopathy. Immunohistochemistry, ELISA, and quantitative PCR were used to assess effects on retinal vascular repair and pathologic angiogenesis in relation to glial cell injury, VEGF protein, and mRNA levels of key mediators of pathologic angiogenesis. Effects of intravitreal injections of VEGF and the VEGF inhibitor VEGFR1/Fc fusion protein were also studied.
Administration of HT during the ischemic pre-proliferative phase of retinopathy effectively accelerated the process of revascularization while preventing the development of vitreous neovascularization. HT enhanced the formation of specialized endothelial tip cells at the edges of the repairing capillary networks and blocked the overexpression of several molecular mediators of angiogenesis, inflammation, and extracellular proteolysis. HT markedly reduced the reactive expression of GFAP in Müller cells and improved the morphology of astrocytes in the avascular region of the retina. Exogenous VEGF administered into the vitreous on P14 was not sufficient to cause vitreous neovascularization in the HT mice. Injection of the VEGF antagonist VEGFR1/Fc blocked both pathologic and physiological angiogenesis and did not rescue astrocytes.
HT may be clinically useful to facilitate vascular repair while blocking neovascularization in the pre-proliferative stage of ischemic retinopathy by correcting a broad range of biochemical and cellular abnormalities.
[Show abstract] [Hide abstract]
ABSTRACT: The mouse model of oxygen-induced retinopathy (OIR) is commonly used to investigate various aspects of the pathogenesis of the retinopathy of prematurity (ROP) as well as angiogenesis in general. Retinal astrocytes were suggested to be involved in retinal angiogenesis. This study aimed to describe their localization and cell density during the course of physiological vascularization and pathological revascularization. Mice expressing H2B-GFP (green fluorescent protein fused to histone 2B) from the endogenous Pdgfra promoter were kept in 75% oxygen from P7 (post natal day 7) to P12 (mouse model of OIR). Retinal flatmounts or cryosections were immunostained for glial fibrillary acidic protein (Gfap), glutamine synthetase (Glul), collagen IV (Col IV), desmin (Des), caspase 3 (Casp3), paired box 2 (Pax2), or Ki67. Astrocytic nuclei were counted with the ImageJ macro AuTOCellQuant. The hypoxic state of the retina was investigated by Hypoxyprobe. The GFP signal of the Pdgfra reporter mice co-localized with Pax2, a nuclear marker for retinal astrocytes. This bright label was much easier to quantify than Gfap or Pax2 staining. Quantification of the cell density of astrocytes during physiological development specified the spreading of astrocytes in a concentrical wave from the optic nerve head towards the periphery. Astrocyte density was reduced during the remodelling of the primary vascular plexus into a hierarchical vascular tree (maximal astrocyte density at P1: 2800 astrocytes / mm2, final astrocyte density: 800 astrocytes / mm2). In the OIR model, cell density of astrocytes was elevated in the peripheral vascularized zone. In contrast, astrocyte density dropped to a half (400 astrocytes / mm2) of the normal value in the central avascular zone during the hyperoxic phase between P8 and P10 by apoptosis and rose only after P17 as the retinal network normalized. An additional drop of astrocyte density was observed within the angles between the large vessels of the central avascular zone during hypoxia between P12 and P17. Astrocyte density was not altered at vascular tufts. The hyperoxia effect on astrocytes including the reduced astrocyte density is not the reason for vascular tuft formation. Hypoxia-affected astrocytes in combination with a reduced astrocytic network in the central avascular zone during the hypoxic phase are important determinants in the formation of pathological features during retinal revascularization.Molecular and Cellular Neuroscience 06/2013; 56. DOI:10.1016/j.mcn.2013.06.001 · 3.73 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Purpose: Retinal neovascularization is a major cause of vision loss in ischemia-induced retinopathy. Tumor necrosis factor (TNF)-like weak inducer of apoptosis (TWEAK) and its receptor, Fn14, have been implicated in angiogenesis but their role in retinal diseases is unknown. The goal of this study was to investigate the role of TWEAK/Fn14 pathway in retinal neovascularization. Methods: Studies were performed in a mouse model of oxygen-induced retinopathy (OIR) and in primary human retinal microvascular endothelial cells (HRMECs). Hyperoxia treatment was initiated on postnatal day (P)14. Immunohistochemistry and quantitative PCR were used to assess retinal vascular changes in relation to expression of Fn14 and TWEAK. Results: Fn14 mRNA was prominently increased from P13 to P17 in OIR retinas, whereas TWEAK level was slightly decreased. These alterations were normalized by hyperoxia treatment and were more striking in isolated retinal vessels. There was a discernible shift in the immunoreactivity of Fn14 and TWEAK from the neuronal layers in the normal retina to the neovascular tufts in that of OIR. Blockade of TWEAK/Fn14 significantly prevented retinal neovascularization while slightly accelerated revascularization. In contrast, activation of Fn14 positively regulated survival pathways in the Bcl2 family and robustly enhanced HRMEC survival. Furthermore, gene analysis revealed the regulatory region of Fn14 gene contains several conserved hypoxia inducible factor (HIF)-1α binding sites. Overexpression of HIF-1α prominently induced Fn14 expression in HRMECs. Conclusion: The TWEAK/Fn14 pathway is involved in the development of pathological retinal neovascularization during retinal ischemia. HIF-1α is likely implicated in the upregulation of Fn14.Investigative ophthalmology & visual science 01/2014; 55(2). DOI:10.1167/iovs.13-12812 · 3.66 Impact Factor
[Show abstract] [Hide abstract]
ABSTRACT: Oxygen-induced retinopathy (OIR) in mice is a popular model system to study pathological angiogenesis in the retinal vasculature. The system is based on vessel depletion by exposure to hyperoxia, which results in acute retinal hypoxia upon return to room air. This hypoxia then triggers neovascularization in the remaining vessels after 5 days. Here we aimed to establish an additional and earlier experimental readout of the vascular response to hypoxia by quantifying the tortuosity of retinal arteries after 2 days. Mouse pups from three different mouse strains were exposed to hyperoxia from postnatal day (P) 7 to P12 and retinas were analysed at P12, P14 and P17. Hypoxia was assessed by staining with the hypoxia marker EF5 and by measuring Vegf mRNA by qPCR. The retinal vasculature was stained in whole mount retinas and tortuosity of radial arterioles was quantified. C57BL/6J mice were used because the vascular response at P17 is well characterised in this strain. We also used C3H/HeJ mice, which contain the retinal degeneration 1 (Rd1) mutation (Pde6b(Rd1)) and have abnormally thin retinas. These thinner, C3H/HeJ retinas do not become ischemic during the OIR model and do not develop neovascularization. They can therefore be used as a control. In addition, we included C3H/HeJ mice that lack the Rd1 mutation (C3H/He(Rd1-)), with normal thickness retinas, to control for strain differences between C57BL/6J and C3H/HeJ. Quantification of vessel tortuosity at P14 showed tortuous arteries in normal thickness retinas (C57BL/6J and C3H/He(Rd1-)) and straight arteries in the thin C3H/HeJ retinas. This correlated with hypoxia, which was severe in normal thickness retinas and mild in the thin C3H/HeJ retinas. Furthermore, at P17 the normal thickness retinas showed strong neovascularisation whereas in the thin C3H/HeJ retinas the retinal vasculature regenerated normally. In conclusion we have demonstrated that arterial tortuosity can act as an early readout for hypoxia in the OIR model before neovascularisation develops.Experimental Eye Research 01/2014; 120. DOI:10.1016/j.exer.2013.12.020 · 3.02 Impact Factor