Mouse Model Of Proliferative Retinopathy Research Articles

Triciribine attenuates pathological neovascularization and vascular permeability in a mouse model of proliferative retinopathy

Proliferative retinopathies are the leading cause of irreversible blindness in all ages, and there is a critical need to identify novel therapies. We investigated the impact of triciribine (TCBN), a tricyclic nucleoside analog and a weak Akt inhibitor, on retinal neurovascular injury, vascular permeability, and inflammation in oxygen-induced retinopathy (OIR). Post-natal day 7 (P7) mouse pups were subjected to OIR, and treated (i.p.) with TCBN or vehicle from P14-P16 and compared with age-matched, normoxic, vehicle or TCBN-treated controls. P17 retinas were processed for flat mounts, immunostaining, Western blotting, and qRT-PCR studies. Fluorescein angiography, electroretinography, and spectral domain optical coherence tomography were performed on days P21, P26, and P30, respectively. TCBN treatment significantly reduced pathological neovascularization, vaso-obliteration, and inflammation marked by reduced TNFα, IL6, MCP-1, Iba1, and F4/80 (macrophage/microglia markers) expression compared to the vehicle-treated OIR mouse retinas. Pathological expression of VEGF (vascular endothelial growth factor), and claudin-5 compromised the blood-retinal barrier integrity in the OIR retinas correlating with increased vascular permeability and neovascular tuft formation, which were blunted by TCBN treatment. Of note, there were no changes in the retinal architecture or retinal cell function in response to TCBN in the normoxia or OIR mice. We conclude that TCBN protects against pathological neovascularization, restores blood-retinal barrier homeostasis, and reduces retinal inflammation without adversely affecting the retinal structure and neuronal function in a mouse model of OIR. Our data suggest that TCBN may provide a novel therapeutic option for proliferative retinopathy.

Antibody-Mediated Inhibition of Tspan12 Ameliorates Vasoproliferative Retinopathy Through Suppression of β-Catenin Signaling.

Anti-angiogenic biologicals represent an important concept for the treatment of vasoproliferative diseases. However, the need for continued treatment, the presence of nonresponders, and the risk of long-term side effects limit the success of existing therapeutic agents. Although Tspan12 has been shown to regulate retinal vascular development, nothing is known about its involvement in neovascular disease and its potential as a novel therapeutic target for the treatment of vasoproliferative diseases. Rodent models of retinal neovascular disease, including the mouse model of oxygen-induced retinopathy and the very low density lipoprotein receptor knockout mouse model were analyzed for Tspan/β-catenin regulation. Screening of a phage display of a human combinatorial antibody (Ab) library was used for the development of a high-affinity Ab against Tspan12. Therapeutic effects of the newly developed Ab on vascular endothelial cells were tested in vitro and in vivo in the oxygen-induced retinopathy and very low density lipoprotein receptor knockout mouse model. The newly developed anti-Tspan12 Ab exhibited potent inhibitory effects on endothelial cell migration and tube formation. Mechanistic studies confirmed that the Ab inhibited the interaction between Tspan12 and Frizzled-4 and effectively modulates β-catenin levels and target genes in vascular endothelial cells. Tspan12/β-catenin signaling was activated in response to acute and chronic stress in the oxygen-induced retinopathy and very low density lipoprotein receptor mouse model of proliferative retinopathy. Intravitreal application of the Ab showed significant therapeutic effects in both models without inducing negative side effects on retina function. Moreover, combined intravitreal injection of the Ab with a known vascular endothelial growth factor inhibitor, Aflibercept, resulted in significant enhancement of the therapeutic efficacy of each monotherapy. Combination therapy with the Tspan12 blocking antibody can be used to reduce anti-vascular endothelial growth factor doses, thus decreasing the risk of long-term off-target effects. Tspan12/β-catenin signaling is critical for the progression of vasoproliferative disease. The newly developed anti-Tspan12 antibody has therapeutic effects in vasoproliferative retinopathy and can enhance the potency of existing anti- vascular endothelial growth factor agents.

Effect of Guibi-Tang, a Traditional Herbal Formula, on Retinal Neovascularization in a Mouse Model of Proliferative Retinopathy.

Ocular pathologic angiogenesis is an important causative risk factor of blindness in retinopathy of prematurity, proliferative diabetic retinopathy, and neovascular macular degeneration. Guibi-tang (GBT) is a frequently used oriental herbal formula in East Asian countries, and is also called Qui-pi-tang in Chinese and Kihi-To in Japanese. In the present study, we investigated the preventive effect of GBT on retinal pathogenic neovascularization in a mouse model of oxygen-induced retinopathy (OIR). C57BL/6 mice were exposed to 75% hyperoxia for five days on postnatal day 7 (P7). The mice were then exposed to room air from P12 to P17 to induce ischemic proliferative retinopathy. GBT (50 or 100 mg/kg/day) was intraperitoneally administered daily for five days (from P12 to P16). On P17, Retinal neovascularization was measured on P17, and the expression levels of 55 angiogenesis-related factors were analyzed using protein arrays. GBT significantly decreased retinal pathogenic angiogenesis in OIR mice, and protein arrays revealed that GBT decreased PAI-1 protein expression levels. Quantitative real-time PCR revealed that GBT reduced vascular endothelial growth factor (VEGF), fibroblast growth factor 2 (FGF2), and plasminogen activator inhibitor 1 (PAI-1) mRNA levels in OIR mice. GBT promotes potent inhibitory activity for retinal neovascularization by decreasing VEGF, FGF2, and PAI-1 levels.

Genome-wide association study for sight-threatening diabetic retinopathy reveals association with genetic variation near the GRB2 gene.

Diabetic retinopathy is a serious complication of diabetes mellitus and can lead to blindness. A genetic component, in addition to traditional risk factors, has been well described although strong genetic factors have not yet been identified. Here, we aimed to identify novel genetic risk factors for sight-threatening diabetic retinopathy using a genome-wide association study. Retinopathy was assessed in white Australians with type 2 diabetes mellitus. Genome-wide association analysis was conducted for comparison of cases of sight-threatening diabetic retinopathy (n = 336) with diabetic controls with no retinopathy (n = 508). Top ranking single nucleotide polymorphisms were typed in a type 2 diabetes replication cohort, a type 1 diabetes cohort and an Indian type 2 cohort. A mouse model of proliferative retinopathy was used to assess differential expression of the nearby candidate gene GRB2 by immunohistochemistry and quantitative western blot. The top ranked variant was rs3805931 with p = 2.66 × 10(-7), but no association was found in the replication cohort. Only rs9896052 (p = 6.55 × 10(-5)) was associated with sight-threatening diabetic retinopathy in both the type 2 (p = 0.035) and the type 1 (p = 0.041) replication cohorts, as well as in the Indian cohort (p = 0.016). The study-wide meta-analysis reached genome-wide significance (p = 4.15 × 10(-8)). The GRB2 gene is located downstream of this variant and a mouse model of retinopathy showed increased GRB2 expression in the retina. Genetic variation near GRB2 on chromosome 17q25.1 is associated with sight-threatening diabetic retinopathy. Several genes in this region are promising candidates and in particular GRB2 is upregulated during retinal stress and neovascularisation.

Expressions of survivin and vascular endothelial growth factor in a Murine model of proliferative retinopathy.

To examine the expression of survivin and vascular endothelial growth factor(VEGF) during the development of retinal neovascularization (NV) in a mouse model. A well-characterized murine model of retinal NV was used to study the expression of survivin and VEGF. NV of the retina was induced in mice by exposure to 75% O(2) from postnatal day P7 to P12, followed by return to room air from P12 to P17. Expression of survivin and VEGF protein was analyzed by Immunohistochemistry. In addition, mouse model of proliferative retinopathy was analyzed by retinal fluorescein angiography and quantification analysis. The normal mice had both superfiekal and deep vascular layers that extended from the optic nerve to the periphery. In intraocular pressure(IOP) mice were characterized by represent a typical pattern of pathological retinal NV. There are less or little nuclei of new vessels vascular endothelial cell breaking through the inner retinal than in retinopathy of prematurity (ROP) mice, large clusters of blood vessels were adherent to the internal limiting membrane(ILM) (0.27±0.20 vs 23.38±1.027, t=9.454, P<0.001). During the angiogenic period from P13 to P17, survivin and VEGF protein expression increased in experimental retinas compared with control samples(2.56±0.46 vs 3.34±0.40, t=17.43, P<0.01: 2.18±0.75 vs 4.34±0.25, t=19.61, P<0.01). Protein levels of VEGF and survivn has significantly positive correlation (P<0.05, r=0.411). Correlation was made at the protein levels of survivin expression compared with that of VEGF in a murine model of retinal NV, which suggests a temporal role for survivin and VEGF in new vessel formation in response to hypoxic stimulation.

Abstract 1376: Antiangiogenic properties of the peptide CIGB-300

Abstract CK2 is a highly conserved protein serine/threonine kinase that is ubiquitously distributed in eukaryotes, constitutively active and has been implicated in multiple cellular functions, as well as in tumorigenesis and transformation. Elevated CK2 activity has been associated with the malignant transformation of several tissues and is associated with aggressive tumor behavior. Previously, we have described a peptide CIGB-300 (also named P15-Tat) targeting the acidic phophorylation domain of the CK2 substrates. Different groups around the world have tried to manipulate this biochemical event by targeting the ATP-binding site of CK2 or its gene transcription using antisense oligonucleotides. Otherwise, CIGB-300 peptide was developed following the innovative approach of targeting the phosphoaceptor site on the CK2 substrates rather than the enzyme per se. We demonstrated that CIGB-300 induced apoptosis on tumor cells and tumor regression when injected directly into solid tumors or by systemic administration in mice. In this work we have evaluated the CIGB-300 peptide on the angiogenic process, both in vitro and in vivo. We have focused in the exploration of CIGB-300 properties to inhibit proliferation, tube formation and RNA pattern expression on endothelial cells (HUVEC). Likewise we used the chicken chorioallatoic membrane (CAM) assay as a model in vivo to asses the CIGB-300 activity on angiogenesis. Others authors have studied the role of different CK2 inhibitors on angiogenesis finding that CK2 is involved in endothelial cell proliferation, survival, migration and tube formation. Intraperitoneally administered CK2 inhibitors significantly reduced preretinal neovascularization in a mouse model of proliferative retinopathy. Solid tumors require the growth of new blood vessels (angiogenesis) to grow. Tumor angiogenesis utilizes at least some of the angiogenic signalling pathways that are required during vascular development. Tumor angiogenesis has become an important target for antitumor therapy, with most current therapies aimed at blocking the VEGF pathway. However, not all tumors are responsive to VEGF blockers, and some of them that are responsive initially may become resistant during the course of treatment, thus there is a need to explore other angiogenesis signalling pathways. In this paper we have examined the antiangiogenic properties of CIGB-300 peptide in vitro and in vivo. The data suggests that CK2 is involved in angiogenic processes and CIGB-300 could act as promising antiangiogenic inhibitor. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 101st Annual Meeting of the American Association for Cancer Research; 2010 Apr 17-21; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2010;70(8 Suppl):Abstract nr 1376.

Suppression of Retinal Neovascularization by Erythropoietin siRNA in a Mouse Model of Proliferative Retinopathy

Erythropoietin (EPO), an oxygen-regulated hormone stimulating erythrocyte production, was recently found to be critical for retinal angiogenesis. EPO mRNA expression levels in retina are highly elevated during the hypoxia-induced proliferation phase of retinopathy. The authors investigated the inhibition of retinal EPO mRNA expression with RNA interference as a potential strategy to suppress retinal neovascularization and to prevent proliferative retinopathy. The authors used a mouse model of oxygen-induced retinopathy. Retinal EPO and Epo receptor (EpoR) expression during retinopathy development were quantified with real-time RT-PCR in whole retina and on laser-captured retinal vessels and neuronal layers. Retinal hypoxia was assessed with an oxygen-sensitive hypoxyprobe. A small interference RNA (siRNA) targeting EPO or control negative siRNA was injected intravitreally at postnatal (P) day 12, P14, and P15 during the hypoxic phase, and the effect on neovascularization was evaluated in retinal flatmounts at P17. Retinal EPO mRNA expression in total retina was suppressed during the initial phase of vessel loss in retinopathy and was significantly elevated during the hypoxia-induced proliferative phase in all three neuronal layers in the retina, corresponding to an increased level of retinal hypoxia. EpoR mRNA expression levels also increased during the second neovascular phase, specifically in hypoxia-induced neovascular vessels. Intravitreous injection of EPO siRNA effectively inhibited approximately 60% of retinal EPO mRNA expression and significantly suppressed retinal neovascularization by approximately 40%. Inhibiting EPO mRNA expression with siRNA is effective in suppressing retinal neovascularization, suggesting EPO siRNA is a potentially useful pharmaceutical intervention for treating proliferative retinopathy.

Inhibition of protein kinase CK2 suppresses angiogenesis and hematopoietic stem cell recruitment to retinal neovascularization sites

Ubiquitous protein kinase CK2 participates in a variety of key cellular functions. We have explored CK2 involvement in angiogenesis. As shown previously, CK2 inhibition reduced endothelial cell proliferation, survival and migration, tube formation, and secondary sprouting on Matrigel. Intraperitoneally administered CK2 inhibitors significantly reduced preretinal neovascularization in a mouse model of proliferative retinopathy. In this model, CK2 inhibitors had an additive effect with somatostatin analog, octreotide, resulting in marked dose reduction for the drug to achieve the same effect. CK2 inhibitors may thus emerge as potent future drugs aimed at inhibiting pathological angiogenesis. Immunostaining of the retina revealed predominant CK2 expression in astrocytes. In human diabetic retinas, mRNA levels of all CK2 subunits decreased, consistent with increased apoptosis. Importantly, a specific CK2 inhibitor prevented recruitment of bone marrow-derived hematopoietic stem cells to areas of retinal neovascularization. This may provide a novel mechanism of action of CK2 inhibitors on newly forming vessels.

Increased clonogenic potential of circulating endothelial progenitor cells in patients with type 1 diabetes and proliferative retinopathy

To the Editor: The identification of endothelial progenitor cells (EPCs) along with the description of their essential role in postnatal neovascularisation has been a major achievement for stem cell biology [1]. In particular the finding that the clonogenic potential of EPCs is reduced in patients with high cardiovascular risk [2] represents the first step towards the recognition of the therapeutic effect of EPC transplantation in cardiac and limbic ischaemia [3, 4]. However, two harmful side effects are expected to be associated with EPC transplantation as a consequence of their physiological function: malignant tumour neovascularisation and the induction of proliferative diabetic retinopathy [1]. The contribution of EPCs to tumour neovascularisation has been clearly demonstrated in animal models and confirmed, although to a lesser extent, also in humans [5]. Concerning diabetic retinopathy, recent reports have demonstrated that EPCs play a major role in the retinal neovascularisation of a mouse model of proliferative retinopathy [6], and that the concentration of stromalcell-derived factor 1, the most important chemokine mobilising EPCs, increases with the severity of diabetic retinopathy in vitreous samples of patients affected by type 2 diabetes [7]. These studies are in some way in contrast to two independent reports performed in non-complicated patients showing that type 1 diabetes per se is associated with a reduced number and function of EPCs [8], and that EPCs in type 2 diabetes are impaired in adhesion, proliferation and tubulisation [9]. Finally, a recent study has demonstrated an increased number of CD34+ mononuclear cells in patients affected by type 2 diabetes with both non-proliferative and proliferative retinopathy [10]. Although of interest, these results cannot be easily compared with the ones described above as EPCs represent only one of the different classes of circulating cells staining positively for CD34+ [1]. Taken together, and from a largely speculative point of view, these apparently discrepant results can be explained by a unifying hypothesis. Taking into account the fact that EPCs have a physiological role in the rescue and maintenance of the existing retinal capillary bed [11], it is possible to hypothesise that the reduced number and clonogenic potential of EPCs found in non-complicated diabetic patients [8, 9] might predispose these patients to the later development of diabetic retinopathy. Once the damage is widespread and specific chemokines are produced by the suffering retina, the bone marrow would respond by increasing the production of EPCs, which at this stage, possibly because of the high ambient glucose and/or the badly damaged retina, would give rise to an incompetent neoangiogenesis. To test this hypothesis and verify the possible association between the clonogenic potential of circulating EPCs and proliferative diabetic retinopathy, we isolated EPCs from whole blood obtained from 11 patients with type 1 diabetes and untreated proliferative retinopathy, from 12 patients without retinopathy despite similar age, gender distribution and duration of type 1 diabetes, and from 11 ageand sex-matched non-diabetic controls. Patients affected by type 1 diabetes took no medication other than insulin. Clinical characteristics of subjects included in the study are described in Table 1. Patients were classified with respect to the status of diabetic retinopathy by means of stereoscopic fundus examination and fluorescent angiography. Patients considered for this study either had no signs of diabetic V. Asnaghi . G. Mazzolari . M. R. Pastore . A. Maestroni . D. Ruggieri . L. Luzi . G. Zerbini (*) Renal Pathophysiology of Diabetes Unit, Division of Nutrition–Metabolism, San Raffaele Scientific Institute, Via Olgettina 60, 20132 Milan, Italy e-mail: g.zerbini@hsr.it

Transcription Factor Ets-1 Mediates Ischemia- and Vascular Endothelial Growth Factor-Dependent Retinal Neovascularization

Transcription factor Ets-1 has been reported to regulate angiogenesis in vascular endothelial cells. Here, we investigated a mechanism that may regulate the expression of Ets-1 in vascular endothelial growth factor (VEGF)- and hypoxia-induced retinal neovascularization and that may have potential to inhibit ocular neovascular diseases. VEGF and hypoxia increased Ets-1 expression in cultured bovine retinal endothelial cells. The VEGF-induced mRNA increase of Ets-1 was suppressed by a tyrosine kinase inhibitor (genistein), by inhibitors of MEK (mitogen-activated protein and extracellular signal-regulated kinase kinase) (PD98059 and UO126), and by inhibitors of protein kinase C (GF109203X, staurosporine, and Gö6976). Dominant-negative Ets-1 inhibited VEGF-induced cell proliferation, tube formation, and the expression of neuropilin-1 and angiopoietin-2. In a mouse model of proliferative retinopathy, Ets-1 mRNA was up-regulated. Intravitreal injection of dominant-negative Ets-1 suppressed retinal angiogenesis in a mouse model of proliferative retinopathy. In conclusion, VEGF induces Ets-1 expression in bovine retinal endothelial cells and its expression is protein kinase C/ERK pathway-dependent. Ets-1 up-regulation is involved in the development of retinal neovascularization, and inhibition of Ets-1 may be beneficial in the treatment of ischemic ocular diseases.

Lentivirus-mediated expression of angiostatin efficiently inhibits neovascularization in a murine proliferative retinopathy model.

Ischemic retinal diseases, such as diabetic retinopathy, retinopathy of prematurity, and age-related macular degeneration, are a major cause of blindness worldwide. Angiostatin is an internal peptide fragment of plasminogen that inhibits endothelial proliferation in vitro and tumor growth in vivo. We now demonstrate that HIV vector encoding angiostatin (HIV-angiostatin) can inhibit retinal neovascularization in a mouse model of proliferative retinopathy. Intravitreal injections of HIV-angiostatin led to stable expression of the angiostatin gene in retinal tissue. Retinal neovascularization was histologically quantitated by a masked protocol. Retinal neovascularization in the eye injected with HIV-angiostatin was reduced in 90% (9/10; P=0.025) of animals, compared with the eye injected with phosphate-buffered saline. Reduction of histologically evident neovascular nuclei per 6-microm section averaged 68%, with maximal inhibitory effects of 87%. Neovascularization was not reduced in the eyes injected with HIV vector encoding enhanced green fluorescent protein. This is the first report that HIV-angiostatin can reduce neovascular cell nuclei in a murine proliferative retinopathy model. These data suggest that the anti-angiogenic activity of angiostatin has therapeutic potential for the treatment of retinal neovascularization.

Kinetics of integrin expression in the mouse model of proliferative retinopathy and success of secondary intervention with cyclic RGD peptides.

Vitronectin-receptor-type integrins (alpha(v) beta(3) and alpha(v) beta(5)) are thought to be involved in the selective ablation of tumorigenic and other pathologic angiogenesis. Specifically, it has been shown that ligation inhibition of the alpha(v)-type integrins with cyclic penta-peptid peptide inhibits proliferative retinopathy by almost 80 % in a hypoxia-induced mouse model. On the basis of growth factor and integrin expression dynamics in this model, secondary intervention approaches with cyclic RGDfV peptide were investigated. alpha(v)-integrin expression started immediately after induction of hypoxia (at postnatal day 12, p12) and persisted only during the initial period of neovascularization (until day p14). Vascular endothelial growth factor (VEGF) expression started at high values immediately after return of the mice into room air, and dropped rapidly to low values beyond day 13. In contrast, basic fibroblast growth factor (bFGF) was predominantly expressed during the phase of maximum angiogenesis which was noted between day p17 and 19. Based on these findings, cyclic penta peptide was administered subcutaneously at varying doses (2-20 microg/kg/day) for 5 days beginning either at day p14 (early intervention) or at day p17 (late intervention). Early secondary intervention showed a dose-dependent reduction of new vessels with maximum inhibition of 57 % (control 68.08 +/- 3.21 nuclei/section compared with RGDfV-treated 29.35 +/- 2.39 nuclei/section; p < 0.0001), whereas late secondary intervention had no effect. These data indicate that angiogenesis-related alpha(v)-integrin expression is VEGF- rather than bFGF-dependent, and the efficacy of cyclic penta-peptid (RGDfV)-treatment in proliferative retinopathy is only effective as long as the alpha(v)-integrin target is prominently expressed.

Recombinant angiostatin prevents retinal neovascularization in a murine proliferative retinopathy model.

Retinal neovascularization is central to the pathogenesis of proliferative diabetic retinopathy, the leading cause of blindness among the middle-aged population. Angiostatin, a proteolytic fragment of plasminogen is one of the most promising inhibitors of angiogenesis currently in clinical trials. Here we show that recombinant angiostatin can inhibit retinal neovascularization in a mouse model of proliferative retinopathy. Because proliferative diabetic retinopathy is a recurrent disease, effective therapy will need to be sustained. Recombinant adeno-associated viruses permit long-term expression of transfected genes; however, they can only accommodate a small insert sequence. Thus, we engineered and tested a shortened recombinant angiostatin derivative containing a signal sequence to permit secretion. Recombinant protein was purified from the medium of transfected HEK293 cells and injected subcutaneously into treated animals. The retinal vasculature was analyzed in retinal flat mounts and using immunohistochemically stained sections. Both methods demonstrate that this short, secreted form of angiostatin is effective in reducing the development of blood vessels in a nontumor environment and has therapeutic potential for neovascular retinopathies such as diabetic retinopathy, retinopathy of prematurity, retinal vein occlusion and, possibly, age-related macular degeneration.

Vascular endothelial growth factor/vascular permeability factor expression in a mouse model of retinal neovascularization.

Neovascular diseases of the retina are a major cause of blindness worldwide. Hypoxia is thought to be a common precursor to neovascularization in many retinal diseases, but the factors involved in the hypoxic neovascular response have not been fully identified. To investigate the role of vascular endothelial growth factor/vascular permeability factor (VEGF/VPF) in retinal neovascularization, the expression of VEGF/VPF mRNA and protein were studied in a mouse model of proliferative retinopathy. RNA (Northern) blot analysis revealed that retinal VEGF/VPF mRNA expression increased 3-fold between 6 and 12 hr of relative retinal hypoxia and remained elevated during the development of neovascularization. In situ hybridization localized VEGF/VPF mRNA to cells bodies in the inner nuclear layer of the retina. Immunohistochemical confocal microscopy demonstrated that VEGF/VPF protein levels increase with a time course similar to that of the mRNA. The cells in the inner nuclear layer of the retina that produce VEGF/VPF were identified morphologically as Müller cells. These data suggest that VEGF/VPF expression in the retina plays a central role in the development of retinal ischemia-induced ocular neovascularization.