LRP5 Is Required for Vascular Development in Deeper Layers of the Retina

Background: The low-density lipoprotein receptor-related protein 5 (LRP5) plays an important role in the development of retinal vasculature. LRP5 loss-of-function mutations cause incomplete development of retinal vessel network in humans as well as in mice. To understand the underlying mechanism for how LRP5 mutations lead to retinal vascular abnormalities, we have determined the retinal cell types that express LRP5 and investigated specific molecular and cellular functions that may be regulated by LRP5 signaling in the retina. Methods and Findings: We characterized the development of retinal vasculature in LRP5 mutant mice using specific retinal cell makers and a GFP transgene expressed in retinal endothelial cells. Our data revealed that retinal vascular endothelial cells predominantly formed cell clusters in the inner-plexiform layer of LRP5 mutant retina rather than sprouting out or migrating into deeper layers to form normal vascular network in the retina. The IRES-b-galactosidase (LacZ) report gene under the control of the endogenous LRP5 promoter was highly expressed in Müller cells and was also weakly detected in endothelial cells of the retinal surface vasculature. Moreover, the LRP5 mutant mice had a reduction of a Müller cell-specific glutamine transporter, Slc38a5, and showed a decrease in b-wave amplitude of electroretinogram. Conclusions: LRP5 is not only essential for vascular endothelial cells to sprout, migrate and/or anastomose in the deeper plexus during retinal vasculature development but is also important for the functions of Müller cells and retina

Treatment of two postoperative endophthalmitis cases due to Aspergillus flavus and Scopulariopsis spp. with local and systemic antifungal therapy

<p>Abstract</p> <p>Background</p> <p>Endophthalmitis is the inflammatory response to invasion of the eye with bacteria or fungi. The incidence of endophthalmitis after cataract surgery varies between 0.072–0.13 percent. Treatment of endophthalmitis with fungal etiology is difficult.</p> <p>Case Presentation</p> <p><b>Case 1: </b>A 71-year old male diabetic patient developed postoperative endophthalmitis due to <it>Aspergillus flavus</it>. The patient was treated with topical amphotericin B ophthalmic solution, intravenous (IV) liposomal amphotericin-B and caspofungin following vitrectomy.</p> <p><b>Case 2: </b>A 72-year old male cachectic patient developed postoperative endophthalmitis due to <it>Scopulariopsis </it>spp. The patient was treated with topical and IV voriconazole and caspofungin.</p> <p>Conclusion</p> <p><it>Aspergillus </it>spp. are responsible of postoperative fungal endophthalmitis. Endophthalmitis caused by <it>Scopulariopsis </it>spp. is a very rare condition. The two cases were successfully treated with local and systemic antifungal therapy.</p

Morphological and Functional Changes in the Retina after Chronic Oxygen-Induced Retinopathy

The mouse model of oxygen-induced retinopathy (OIR) has been widely used for studies of retinopathy of prematurity (ROP). This disorder, characterized by abnormal vascularization of the retina, tends to occur in low birth weight neonates after exposure to high supplemental oxygen. Currently, the incidence of ROP is increasing because of increased survival of these infants due to medical progress. However, little is known about changes in the chronic phase after ROP. Therefore, in this study, we examined morphological and functional changes in the retina using a chronic OIR model. Both the a- and b-waves in the OIR model recovered in a time-dependent manner at 4 weeks (w), 6 w, and 8 w, but the oscillatory potential (OP) amplitudes remained depressed following a return to normoxic conditions. Furthermore, decrease in the thicknesses of the inner plexiform layer (IPL) and inner nuclear layer (INL) at postnatal day (P) 17, 4 w, and 8 w and hyperpermeability of blood vessels were observed in conjunction with the decrease in the expression of claudin-5 and occludin at 8 w. The chronic OIR model revealed the following: (1) a decrease in OP amplitudes, (2) morphological abnormalities in the retinal cells (limited to the IPL and INL) and blood vessels, and (3) an increase in retinal vascular permeability via the impairment of the tight junction proteins. These findings suggest that the experimental animal model used in this study is suitable for elucidating the pathogenesis of ROP and may lead to the development of potential therapeutic agents for ROP treatment

Interleukin-10 Promotes Pathological Angiogenesis by Regulating Macrophage Response to Hypoxia during Development

Aberrant angiogenesis in the eye is the most common cause of blindness. The current study examined the role of interleukin-10 (IL-10) in ischemia-induced pathological angiogenesis called neovascularization during postnatal development. IL-10 deficiency resulted in significantly reduced pathological retinal angiogenesis. In contrast to the choroicapillaris where IL-10 interferes with macrophage influx, IL-10 did not prevent anti-angiogenic macrophages from migrating to the retina in response to hypoxia. Instead, IL-10 promoted retinal angiogenesis by altering macrophage angiogenic function, as macrophages from wild-type mice demonstrated increased vascular endothelial growth factor (VEGF) and nitric oxide (NO) compared to IL-10 deficient macrophages. IL-10 appears to directly affect macrophage responsiveness to hypoxia, as macrophages responded to hypoxia with increased levels of IL-10 and STAT3 phosphorylation as opposed to IL-10 deficient macrophages. Also, IL-10 deficient macrophages inhibited the proliferation of vascular endothelial cells in response to hypoxia while wild-type macrophages failed to do so. These findings suggest that hypoxia guides macrophage behavior to a pro-angiogenic phenotype via IL-10 activated pathways

The Angio-Fibrotic Switch of VEGF and CTGF in Proliferative Diabetic Retinopathy

BACKGROUND: In proliferative diabetic retinopathy (PDR), vascular endothelial growth factor (VEGF) and connective tissue growth factor (CTGF) cause blindness by neovascularization and subsequent fibrosis, but their relative contribution to both processes is unknown. We hypothesize that the balance between levels of pro-angiogenic VEGF and pro-fibrotic CTGF regulates angiogenesis, the angio-fibrotic switch, and the resulting fibrosis and scarring. METHODS/PRINCIPAL FINDINGS: VEGF and CTGF were measured by ELISA in 68 vitreous samples of patients with proliferative DR (PDR, N = 32), macular hole (N = 13) or macular pucker (N = 23) and were related to clinical data, including degree of intra-ocular neovascularization and fibrosis. In addition, clinical cases of PDR (n = 4) were studied before and after pan-retinal photocoagulation and intra-vitreal injections with bevacizumab, an antibody against VEGF. Neovascularization and fibrosis in various degrees occurred almost exclusively in PDR patients. In PDR patients, vitreous CTGF levels were significantly associated with degree of fibrosis and with VEGF levels, but not with neovascularization, whereas VEGF levels were associated only with neovascularization. The ratio of CTGF and VEGF was the strongest predictor of degree of fibrosis. As predicted by these findings, patients with PDR demonstrated a temporary increase in intra-ocular fibrosis after anti-VEGF treatment or laser treatment. CONCLUSIONS/SIGNIFICANCE: CTGF is primarily a pro-fibrotic factor in the eye, and a shift in the balance between CTGF and VEGF is associated with the switch from angiogenesis to fibrosis in proliferative retinopathy

Long-term retinal PEDF overexpression prevents neovascularization in a murine adult model of retinopathy

Neovascularization associated with diabetic retinopathy (DR) and other ocular disorders is a leading cause of visual impairment and adult-onset blindness. Currently available treatments are merely palliative and offer temporary solutions. Here, we tested the efficacy of antiangiogenic gene transfer in an animal model that mimics the chronic progression of human DR. Adeno-associated viral (AAV) vectors of serotype 2 coding for antiangiogenic Pigment Epithelium Derived Factor (PEDF) were injected in the vitreous of a 1.5 month-old transgenic model of retinopathy that develops progressive neovascularization. A single intravitreal injection led to long-term production of PEDF and to a striking inhibition of intravitreal neovascularization, normalization of retinal capillary density, and prevention of retinal detachment. This was parallel to a reduction in the intraocular levels of Vascular Endothelial Growth Factor (VEGF). Normalization of VEGF was consistent with a downregulation of downstream effectors of angiogenesis, such as the activity of Matrix Metalloproteinases (MMP) 2 and 9 and the content of Connective Tissue Growth Factor (CTGF). These results demonstrate long-term efficacy of AAV-mediated PEDF overexpression in counteracting retinal neovascularization in a relevant animal model, and provides evidence towards the use of this strategy to treat angiogenesis in DR and other chronic proliferative retinal disorders

Hypoxia-Induced Retinal Angiogenesis in Zebrafish as a Model to Study Retinopathy

Mechanistic understanding and defining novel therapeutic targets of diabetic retinopathy and age-related macular degeneration (AMD) have been hampered by a lack of appropriate adult animal models. Here we describe a simple and highly reproducible adult fli-EGFP transgenic zebrafish model to study retinal angiogenesis. The retinal vasculature in the adult zebrafish is highly organized and hypoxia-induced neovascularization occurs in a predictable area of capillary plexuses. New retinal vessels and vascular sprouts can be accurately measured and quantified. Orally active anti-VEGF agents including sunitinib and ZM323881 effectively block hypoxia-induced retinal neovascularization. Intriguingly, blockage of the Notch signaling pathway by the inhibitor DAPT under hypoxia, results in a high density of arterial sprouting in all optical arteries. The Notch suppression-induced arterial sprouting is dependent on tissue hypoxia. However, in the presence of DAPT substantial endothelial tip cell formation was detected only in optic capillary plexuses under normoxia. These findings suggest that hypoxia shifts the vascular targets of Notch inhibitors. Our findings for the first time show a clinically relevant retinal angiogenesis model in adult zebrafish, which might serve as a platform for studying mechanisms of retinal angiogenesis, for defining novel therapeutic targets, and for screening of novel antiangiogenic drugs

Perturbation of Mouse Retinal Vascular Morphogenesis by Anthrax Lethal Toxin

Lethal factor, the enzymatic moiety of anthrax lethal toxin (LeTx) is a protease that inactivates mitogen activated protein kinase kinases (MEK or MKK). In vitro and in vivo studies demonstrate LeTx targets endothelial cells. However, the effects of LeTx on endothelial cells are incompletely characterized. To gain insight into this process we used a developmental model of vascularization in the murine retina. We hypothesized that application of LeTx would disrupt normal retinal vascularization, specifically during the angiogenic phase of vascular development. By immunoblotting and immunofluorescence microscopy we observed that MAPK activation occurs in a spatially and temporally regulated manner during retinal vascular development. Intravitreal administration of LeTx caused an early delay (4 d post injection) in retinal vascular development that was marked by reduced penetration of vessels into distal regions of the retina as well as failure of sprouting vessels to form the deep and intermediate plexuses within the inner retina. In contrast, later stages (8 d post injection) were characterized by the formation of abnormal vascular tufts that co-stained with phosphorylated MAPK in the outer retinal region. We also observed a significant increase in the levels of secreted VEGF in the vitreous 4 d and 8 d after LeTx injection. In contrast, the levels of over 50 cytokines other cytokines, including bFGF, EGF, MCP-1, and MMP-9, remained unchanged. Finally, co-injection of VEGF-neutralizing antibodies significantly decreased LeTx-induced neovascular growth. Our studies not only reveal that MAPK signaling plays a key role in retinal angiogenesis but also that perturbation of MAPK signaling by LeTx can profoundly alter vascular morphogenesis

Tipping the Balance: Robustness of Tip Cell Selection, Migration and Fusion in Angiogenesis

Vascular abnormalities contribute to many diseases such as cancer and diabetic retinopathy. In angiogenesis new blood vessels, headed by a migrating tip cell, sprout from pre-existing vessels in response to signals, e.g., vascular endothelial growth factor (VEGF). Tip cells meet and fuse (anastomosis) to form blood-flow supporting loops. Tip cell selection is achieved by Dll4-Notch mediated lateral inhibition resulting, under normal conditions, in an interleaved arrangement of tip and non-migrating stalk cells. Previously, we showed that the increased VEGF levels found in many diseases can cause the delayed negative feedback of lateral inhibition to produce abnormal oscillations of tip/stalk cell fates. Here we describe the development and implementation of a novel physics-based hierarchical agent model, tightly coupled to in vivo data, to explore the system dynamics as perpetual lateral inhibition combines with tip cell migration and fusion. We explore the tipping point between normal and abnormal sprouting as VEGF increases. A novel filopodia-adhesion driven migration mechanism is presented and validated against in vivo data. Due to the unique feature of ongoing lateral inhibition, ‘stabilised’ tip/stalk cell patterns show sensitivity to the formation of new cell-cell junctions during fusion: we predict cell fates can reverse. The fusing tip cells become inhibited and neighbouring stalk cells flip fate, recursively providing new tip cells. Junction size emerges as a key factor in establishing a stable tip/stalk pattern. Cell-cell junctions elongate as tip cells migrate, which is shown to provide positive feedback to lateral inhibition, causing it to be more susceptible to pathological oscillations. Importantly, down-regulation of the migratory pathway alone is shown to be sufficient to rescue the sprouting system from oscillation and restore stability. Thus we suggest the use of migration inhibitors as therapeutic agents for vascular normalisation in cancer