Inhibition of retinal angiogenesis by peptides derived from thrombospondin-1. (25/515)

PURPOSE: Thrombospondin (TSP)1 is a tumor suppressor with activity that is associated with its ability to inhibit neovascularization. Previous studies have mapped this antiangiogenic activity to the type 1 repeats and the amino-terminal portion of the molecule within the procollagen-like domain. The present study was performed to investigate the ability of TSP-1 and peptides derived from the type 1 repeats to inhibit retinal angiogenesis. METHODS: TSP-1 and peptides with tryptophan-rich, heparin-binding sequences and transforming growth factor (TGF)-beta1 activation sequences were evaluated in two models of retinal angiogenesis: a retinal explant assay and a rat model of retinopathy of prematurity (ROP). RESULTS: Platelet-derived TSP-1 inhibited angiogenesis in both experimental models. Peptides from the native TSP-1 sequence, which contained both the tryptophan-rich repeat and the TGF-beta1 activation sequence, were the most potent inhibitors of endothelial cell outgrowth in the retinal explant assay. In contrast, a peptide containing only the tryptophan-rich, heparin-binding sequence was most active in inhibiting neovascular disease in the rat ROP model. CONCLUSIONS: These results indicate that the type 1 repeats of TSP-1 contain two subdomains that may independently influence the process of neovascularization, and that peptides derived from these type 1 repeats may be promising pharmacologic agents for treatment of retinal angiogenesis.  (+info)

Pentoxifylline inhibition of vasculogenesis in the neonatal rat retina. (26/515)

PURPOSE: The zeta isozyme of protein kinase C (PKC) is essential for activation of the transcription factor nuclear factor (NF)kappaB and transcription of vascular endothelial growth factor (VEGF). This study examined the antiangiogenic potential of an existing drug, pentoxifylline (PTX), which inhibits PKC-dependent activation of NFkappaB and is reported to prevent hypoxia-induced expression of VEGF. METHODS: Neovascularization was induced by maintaining neonatal rats for 10 full days in 80% oxygen, interrupted daily by 30 minutes in room air followed by a progressive return to 80% oxygen. On experimental day 11, they were placed in room air until they were killed on day 17. Daily intraperitoneal injections of PTX in saline (25 or 75 mg/kg per day), or saline alone, were administered from day 6 through day 16. Retinal neovascularization was scored, and avascular areas (AVAs) were measured in ADPase stained retinas. RESULTS: PTX inhibited radial extension of retinal vessels, causing increases in AVA of 65% (P < 0.01) and 33% (P < 0.15) at the lower and upper doses, respectively. A significant increase in mean neovascular score was seen at the lower dose (P < 0.0001), but analysis of variance indicated that neovascularization was strongly and positively influenced by the AVA (P < 0.0001) and only weakly stimulated by PTX (P < 0.05). CONCLUSIONS: Systemic PTX significantly inhibited VEGF-mediated retinal vasculogenesis, but was not effective in reducing neovascularization in the oxygen-exposed neonatal rat.  (+info)

Dexamethasone and critical effect of timing on retinopathy. (27/515)

PURPOSE: Administration of corticosteroids soon after birth has been reported to have deleterious, protective, and no effect on retinopathy of prematurity. Conflicting results may be due to timing of corticosteroid administration. The goal of this study was to determine effects of pretreatment and late dexamethasone on retinopathy in a mouse model. METHODS: The C57BL6 mouse model of oxygen-induced retinopathy (by placing animals in 75% oxygen from postnatal days 7 through 12) was used to create retinal neovascularization. Dexamethasone at 0.5 mg/kg per day was administered from day 1 through day 5 in the pretreatment group. The late-treatment group received 5 days of dexamethasone at the same dose beginning on day 12. Mice were killed at days 17 through 20, and retinal vasculature was assessed by a retinal scoring system of wholemount preparation after high-molecular-weight fluorescein-labeled dextran perfusion. In addition, retinal neovascularization was assessed by quantification of extraretinal neovascular nuclei in retinal sections. Statistical significance was defined as P: < 0.05 and was determined by the Kruskal-Wallis test, Mann-Whitney test, and Student's t-test. RESULTS: Oxygen-exposed animals that received treatment with dexamethasone before oxygen exposure had an improvement in retinopathy, with a median score of 6 (5,7; 25th,75th quartiles) compared with 10 (8,11) in the untreated oxygen-exposed (P: < 0.05). The group treated late (after oxygen exposure) with dexamethasone had a median score of 10 (9,11). Pretreatment reduced extraretinal vascularization, when assessed by quantification of neovascular nuclei, to a mean +/- SEM of 19 +/- 9, significantly less than in the untreated oxygen-exposed group (55 +/- 12; P: < 0.05). No difference was observed in the late-treatment group when compared with the untreated oxygen-exposed group. Significant growth retardation, indicated by body weight, was observed in the pretreatment (P: < 0.01) and late-treatment (P: < 0. 05) groups when compared with the control group. CONCLUSIONS: Timing of dexamethasone administration was critical to the inhibition of development of retinopathy in the mouse model. Degree of growth retardation, measured by body weight, also appeared to be time dependent. These data may explain the different results of clinical observations with respect to corticosteroid treatment, timing, and development of retinopathy.  (+info)

Retinal VEGF mRNA measured by SYBR green I fluorescence: A versatile approach to quantitative PCR. (28/515)

PURPOSE: To determine whether continuous monitoring of SYBR Green I fluorescence provides a reliable and flexible method of quantitative RT-PCR. Our aims were (i) to test whether SYBR Green I analysis could quantify a wide range of known VEGF template concentrations, (ii) to apply this method in an experimental model, and (iii) to determine whether 20 existing primer pairs could be used to quantify their cognate mRNAs. METHODS: Real-time quantitative PCR was performed using a LightCycler rapid thermal cycler (Roche). Retinal VEGF mRNA levels were measured in a murine model of oxygen-induced retinopathy during vaso-obliterative and hypoxic phases. RESULTS: This technique was able to detect as few as 10 control template copies, with quantitative data available routinely for 1000 or more copies. The levels of retinal VEGF mRNA expression followed the hypoxia-induced pattern determined previously by conventional methods. All gene-specific primer pairs which amplify a specific product by conventional PCR were successfully converted to SYBR Green analysis, including those for housekeeping genes glyceraldehyde phosphate dehydrogenase (GAPDH), cyclophilin, and acidic ribosomal phosphoprotein PO (ARP/36B4) and for 28S rRNA. In each case melting curve analysis and agarose gel electrophoresis confirmed the specificity of the amplification product. CONCLUSIONS: The sequence-independent detection of DNA with SYBR Green I means that it can be used to quantify the amplification of any cDNA using gene-specific primers. This rapid and flexible method is ideally suited for researchers in vision science wishing to quantify mRNAs from many different genes because it does not require investment in gene-specific hybridization probes.  (+info)

Retinal neovascularization is prevented by blockade of the renin-angiotensin system. (29/515)

Both angiotensin II and vascular endothelial growth factor are angiogenic agents that have recently been implicated in the pathogenesis of proliferative diabetic retinopathy. In this study, retinal neovascularization was examined in a model of retinopathy of prematurity with the use of neonatal transgenic (mRen-2)27 rats, which overexpress renin in tissues, and Sprague-Dawley rats. Blockers of the renin-angiotensin system were administered during the neovascularization period. The ACE inhibitor lisinopril and the angiotensin type 1 receptor antagonist losartan both increased retinal renin levels and prevented inner retinal blood vessel growth. Quantitative in situ hybridization revealed that the expression of vascular endothelial growth factor and its type 2 receptor in the inner retina and proliferating blood vessels were increased in rats with retinopathy of prematurity. Lisinopril reduced both retinal vascular endothelial growth factor and its type 2 receptor mRNA in retinopathy of prematurity rats, whereas losartan had no effect. It is predicted that agents that interrupt the renin-angiotensin system may play an important role as retinoprotective agents in various forms of proliferative retinopathy.  (+info)

The balance between proteinases and inhibitors in a murine model of proliferative retinopathy. (30/515)

PURPOSE: To examine the expression of matrix metalloproteinases (MMPs) and their inhibitors during the development of retinal neovascularization (NV) in a mouse model. METHODS: A well-characterized murine model of retinal NV was used to study the expression of specific MMPs (MMP-2, MMP-9, and MT1-MMP) and tissue inhibitor of metalloproteinases (TIMPs types 1, 2, and 3). NV of the retina was induced in mice by exposure to 75% O(2) from postnatal day (P)7 to P12, followed by return to room air from P12 to P17. Expression of MMP mRNA was analyzed by reverse transcription-polymerase chain reaction (RT-PCR). In addition, retinal tissue removed from control (without NV) and experimental animals (with NV) was analyzed for the expression of TIMP-1, TIMP-2, and TIMP-3 mRNA and protein using RT-PCR and Western blot analysis. RESULTS: During the angiogenic period from P13 to P17, MMP-2 and -9, and MT1-MMP message expression increased in experimental retinas compared with control samples. The TIMP-2 message and protein levels increased steadily in the retina of control animals until P17. This was in contrast to that seen in the retinas of the experimental animals in which TIMP-2 message and protein remained low and significantly less than in control samples. There were no significant changes in TIMP-3 message levels in retinal tissues, and TIMP-1 message and protein were undetectable. CONCLUSIONS: Correlation was made at the mRNA and protein levels of TIMP expression compared with that of MMPs in a murine model of retinal NV, which suggests a temporal role for MMP-2 and -9, MT1-MMP, and TIMP-2 in new vessel formation in response to hypoxic stimulation.  (+info)

Reduced severity of oxygen-induced retinopathy in eNOS-deficient mice. (31/515)

PURPOSE: Exposure of premature human infants to hyperoxia results in the obliteration of developing retina capillaries, leading to a vision-threatening retinopathy termed retinopathy of prematurity (ROP). The authors hypothesized that this process may be mediated in part by endothelial nitric oxide (NO)-derived oxidants such as peroxynitrite and tested this hypothesis in a mouse model of ROP. METHODS: Normal mice, mice treated with the nitric oxide synthase (NOS) inhibitor N:(G)-nitro-L-arginine (L-NNA), and knockout mice carrying a homozygous targeted disruption of the gene for endothelial NOS (eNOS) were studied in an experimental model of ROP. Retinas were compared for extent of capillary obliteration in hyperoxia, vascular endothelial growth factor (VEGF) expression, nitrotyrosine formation, and vitreous neovascularization. RESULTS: Oxygen-induced retinal vaso-obliteration was significantly reduced by L-NNA treatment (43% decrease from controls). The eNOS-deficient mice showed a similar reduction in vaso-obliteration (46% decrease from controls), and vitreous neovascularization was also substantially reduced (threefold decrease). Retinal nitrotyrosine formation, a measure of in situ peroxynitrite modification of proteins, was significantly elevated in normal mice during hyperoxia, in a spatial and temporal pattern consistent with a role in oxygen-induced vaso-obliteration. This was not seen in eNOS-deficient mice. VEGF expression was similar in both groups of mice, although suppression in hyperoxia was slightly blunted in eNOS-deficient mice. CONCLUSIONS: These data suggest a role for NO and peroxynitrite in the pathogenesis of ROP. Therapies aimed at modulation of eNOS activity may have therapeutic potential for preventing ROP.  (+info)

The effect of an angiostatic steroid on neovascularization in a rat model of retinopathy of prematurity. (32/515)

PURPOSE: The inhibition of angiogenesis by angiostatic steroids has been demonstrated in a variety of systems, including rabbit and rat cornea. There is considerable interest in the therapeutic potential of this class of compounds for angiogenic ocular conditions such as diabetic retinopathy, macular degeneration, and retinopathy of prematurity (ROP). This study was designed to test the capacity of an angiostatic steroid, anecortave acetate, to inhibit retinal neovascularization using a rat model of ROP and to investigate the mechanism of the effect. METHODS: At birth, rats were placed in an atmosphere of varying oxygen that produces retinal neovascular changes that approximate human ROP. The rats then received intravitreal injections of either anecortave acetate or vehicle at varying times, and all were subsequently placed in room air. Retinas were assessed for plasminogen activator inhibitor (PAI)-1 mRNA level by RNase protection assay at 1, 2, and 3 days after injection and for normal and abnormal blood vessel growth 3 days later. RESULTS: A significant reduction in the severity of abnormal retinal neovascularization was observed in the steroid-treated eyes compared with vehicle-injected eyes in ROP rats, yet the extent of normal total retinal vascular area was not significantly different. The drug had no effect on either retinal vascular area or neovascularization when tested in room air-raised control rats. Drug-injected eyes demonstrated a six- to ninefold increase in PAI-1 mRNA at 1 to 3 days after injection. CONCLUSIONS: This study represents the first therapeutic effect of an angiostatic steroid in an animal model of neovascular retinopathy. Additionally, the induction of PAI-1 indicates a mechanism of action for this class of compounds, and this is a novel finding in vivo. Because anecortave acetate significantly inhibited pathologic retinal angiogenesis in this model, while not significantly affecting normal intraretinal vessels, it holds therapeutic potential for a number of human ocular conditions in which angiogenesis plays a critical pathologic role.  (+info)