Yun-Zheng Le

Müller cell-derived VEGF is essential for diabetes-induced retinal inflammation and vascular leakage

OBJECTIVE Vascular endothelial growth factor (VEGF-A or VEGF) is a major pathogenic factor and therapeutic target for diabetic retinopathy (DR). Since VEGF has been proposed as a survival factor for retinal neurons, defining the cellular origin of pathogenic VEGF is necessary for the effectiveness and safety of long-term anti-VEGF therapies for DR. To determine the significance of Müller cell-derived VEGF in DR, we disrupted VEGF in Müller cells with an inducible Cre/lox system and examined diabetes-induced retinal inflammation and vascular leakage in these conditional VEGF knockout (KO) mice. RESEARCH DESIGN AND METHODS Leukostasis was determined by counting the number of fluorescently labeled leukocytes inside retinal vasculature. Expression of biomarkers for retinal inflammation was assessed by immunoblotting of TNF-α, ICAM-1, and NF-κB. Vascular leakage was measured by immunoblotting of retinal albumin and fluorescent microscopic analysis of extravascular albumin. Diabetes-induced vascular alterations were examined by immunoblotting and immunohistochemistry for tight junctions, and by trypsin digestion assays for acellular capillaries. Retinal integrity was analyzed with morphologic and morphometric analyses. RESULTS Diabetic conditional VEGF KO mice exhibited significantly reduced leukostasis, expression of inflammatory biomarkers, depletion of tight junction proteins, numbers of acellular capillaries, and vascular leakage compared to diabetic control mice. CONCLUSIONS Müller cell-derived VEGF plays an essential and causative role in retinal inflammation, vascular lesions, and vascular leakage in DR. Therefore, Müller cells are a primary cellular target for proinflammatory signals that mediates retinal inflammation and vascular leakage in DR.

Conditional gene knockout using Cre recombinase.

Cre recombinase has become an important instrument for achieving precise genetic manipulation in mice. Many of these desired genetic manipulations rely on Cres ability to direct spatially and temporally specified excision of a predesignated DNA sequence that has been flanked by directly repeated copies of the loxP recombination site. Success in achieving such conditional mutagenesis in mice depends both on the careful design of conditional alleles and on reliable detection of cre gene expression. These procedures include PCR, immunohistochemistry and the use of a recombination-proficient GFP-tagged Cre protein.

Müller cell-derived VEGF is a significant contributor to retinal neovascularization.

Vascular endothelial growth factor (VEGF‐A) is a major pathogenic factor and a therapeutic target for age‐related macular degeneration, diabetic retinopathy, and retinopathy of prematurity. Despite intensive effort in the field, the cellular mechanisms of VEGF action remain virtually uninvestigated. This situation makes it difficult to design cellular target‐based therapeutics for these diseases. In light of the recent finding that VEGF is a potential neurotrophic factor, revealing the cellular mechanisms of VEGF action becomes necessary to preserve its beneficial effect and inhibit its pathological function in long‐term anti‐VEGF therapeutics for ocular vascular diseases. We therefore generated conditional VEGF knockout mice with an inducible Cre/lox system and determined the significance of Müller cell‐derived VEGF in retinal development and maintenance and ischaemia‐induced neovascularizartion and vascular leakage. Retinal development in the conditional VEGF knockout mice was analysed by examining retinal and choroidal vasculatures and retinal morphology and function. Ischaemia‐induced retinal neovascularization and vascular leakage in the conditional VEGF knockout mice were analysed with fluorescein angiography, quantification of proliferative neovascular cells, immunohistochemistry, and immunoblotting using an oxygen‐induced retinopathy model. Our results demonstrated that disruption of Müller cell‐derived VEGF resulted in no apparent defects in retinal and choroidal vasculatures and retinal morphology and function, significant inhibition of the ischaemia‐induced retinal neovascularization and vascular leakage, and attenuation of the ischaemia‐induced breakdown of the blood‐retina barrier. These results suggest that the retinal Müller cell‐derived VEGF is a major contributor to ischaemia‐induced retinal vascular leakage and pre‐retinal and intra‐retinal neovascularization. The observation that a significant, but not complete, reduction of VEGF in the retina does not cause detectable retinal degeneration suggests that appropriate doses of anti‐VEGF agents may be important to the safe treatment of retinal vascular diseases. Copyright

IFT20 is required for opsin trafficking and photoreceptor outer segment development

Intraflagellar transport (IFT) 20 is required for the formation of photoreceptor outer segments, and deletion of this gene in mature cells leads to opsin accumulation in the cell body. IFT20 interacts with opsin photopigments both in the context of the IFT particle and independent of the particle, suggesting that opsin receptors are cargo for the IFT system.

7-Ketocholesterol Induces Autophagy in Vascular Smooth Muscle Cells through Nox4 and Atg4B

Oxidized lipoproteins stimulate autophagy in advanced atherosclerotic plaques. However, the mechanisms underlying autophagy induction and the role of autophagy in atherogenesis remain to be determined. This study was designed to investigate the mechanisms by which 7-ketocholesterol (7-KC), a major component of oxidized lipoproteins, induces autophagy. This study was also designed to determine the effect of autophagy induction on apoptosis, a central event in the development of atherosclerosis. Exposure of human aortic smooth muscle cells to 7-KC increased autophagic flux. Autophagy induction was suppressed by treating the cells with either a reactive oxygen species scavenger or an antioxidant. Administration of 7-KC concomitantly up-regulated Nox4 expression, increased intracellular hydrogen peroxide levels, and inhibited autophagy-related gene 4B activity. Catalase overexpression to remove hydrogen peroxide or Nox4 knockdown with siRNA reduced intracellular hydrogen peroxide levels, restored autophagy-related gene 4B activity, and consequently attenuated 7-KC-induced autophagy. Moreover, inhibition of autophagy aggravated both endoplasmic reticulum (ER) stress and cell death in response to 7-KC. In contrast, up-regulation of autophagic activity by rapamycin had opposite effects. Finally, activation of autophagy by chronic rapamycin treatment attenuated ER stress, apoptosis, and atherosclerosis in apolipoprotein E knockout (ApoE(-/-)) mouse aortas. In conclusion, we demonstrate that up-regulation of autophagy is a cellular protective response that attenuates 7-KC-induced cell death in human aortic smooth muscle cells.

Autophagy Protects the Retina from Light-induced Degeneration

Background: Autophagy is a conserved process of lysosome-mediated intracellular degradation. Results: Dysregulation of autophagy is associated with retinal cell death by all-trans-retinal and by light exposure. Conclusion: Autophagy protects the retina from light-induced retinal degeneration. Significance: Dynamic autophagy regulation may influence retinal cell survival under stress and disease conditions. Autophagy is a conserved feature of lysosome-mediated intracellular degradation. Dysregulated autophagy is implicated as a contributor in neurodegenerative diseases; however, the role of autophagy in retinal degeneration remains largely unknown. Here, we report that the photo-activated visual chromophore, all-trans-retinal, modulated autophagosome formation in ARPE19 retinal cells. Increased formation of autophagosomes in these cells was observed when incubated with 2.5 μm all-trans-retinal, a condition that did not cause cell death after 24 h in culture. However, autophagosome formation was decreased at concentrations, which caused cell death. Increased expression of activating transcription factor 4 (Atf4), which indicates the activation of oxidative stress, was recorded in response to light illumination in retinas of Abca4−/−Rdh8−/− mice, which showed delayed clearance of all-trans-retinal after light exposure. Expression of autophagosome marker LC3B-II and mitochondria-specific autophagy, mitophagy, regulator Park2, were significantly increased in the retinas of Abca4−/−Rdh8−/− mice after light exposure, suggesting involvement of autophagy and mitophagy in the pathogenesis of light-induced retinal degeneration. Deletion of essential genes required for autophagy, including Beclin1 systemically or Atg7 in only rod photoreceptors resulted in increased susceptibility to light-induced retinal damage. Increased photoreceptor cell death was observed when retinas lacking the rod photoreceptor-specific Atg7 gene were coincubated with 20 μm all-trans-retinal. Park2−/− mice also displayed light-induced retinal degeneration. Ultra-structural analyses showed mitochondrial and endoplasmic reticulum impairment in retinas of these model animals after light exposure. Taken together, these observations provide novel evidence implicating an important role of autophagy and mitophagy in protecting the retina from all-trans-retinal- and light-induced degeneration.

Ischaemia-induced retinal neovascularisation and diabetic retinopathy in mice with conditional knockout of hypoxia-inducible factor-1 in retinal Müller cells

Aims/hypothesisRetinal Müller cells are known to produce inflammatory and angiogenic cytokines, which play important roles in diabetic retinopathy. Hypoxia-inducible factor (HIF)-1 has been shown to play a crucial role in retinal inflammation and neovascularisation. We sought to determine the role of Müller cell-derived HIF-1 in oxygen-induced retinopathy (OIR) and diabetic retinopathy using conditional Hif-1α (also known as Hif1a) knockout (KO) mice.MethodsConditional Hif-1α KO mice were generated by crossing mice expressing cyclisation recombinase (cre, also known as P1_gp003) in Müller cells with floxed Hif-1α mice and used for OIR and streptozotocin-induced diabetes to induce retinal neovascularisation and inflammation, respectively. Abundance of HIF-1α and pro-angiogenic and pro-inflammatory factors was measured by immunoblotting and immunohistochemistry. Retinal neovascularisation was visualised by angiography and quantified by counting pre-retinal nuclei. Retinal inflammation was evaluated by leucostasis and vascular leakage.ResultsWhile the Hif-1α KO mice showed significantly decreased HIF-1α levels in the retina, they exhibited no apparent histological or visual functional abnormalities under normal conditions. Compared with wild-type counterparts, Hif-1α KO mice with OIR demonstrated attenuated overproduction of vascular endothelial growth factor (VEGF) and intercellular adhesion molecule (ICAM)-1, reduced vascular leakage and alleviated neovascularisation in the retina. Under diabetes conditions, disruption of Hif-1α in Müller cells attenuated the increases of retinal vascular leakage and adherent leucocytes, as well as the overproduction of VEGF and ICAM-1.Conclusions/interpretationMüller cell-derived HIF-1α is a key mediator of retinal neovascularisation, vascular leakage and inflammation, the major pathological changes in diabetic retinopathy. Müller cell-derived HIF-1α is therefore a promising therapeutic target for diabetic retinopathy.

Inducible Expression of Cre Recombinase in the Retinal Pigmented Epithelium

PURPOSE The retinal pigmented epithelium (RPE) expresses many genes that play important roles in the support and maintenance of photoreceptors. The present study was conducted to develop a system amenable to the dissection of the temporal function of these genes, specifically within RPE cells. Transgenic mice were generated and characterized in which the expression of Cre recombinase could be specifically induced within the RPE. METHODS Transgenic mice carrying the human vitelliform macular dystrophy-2 (VMD2) promoter (P(VMD2))-directed reverse tetracycline-dependent transactivator (rtTA) and the tetracycline-responsive element (TRE)-directed cre were generated. Inducible Cre expression was achieved by feeding doxycycline to these mice and was characterized by using a Cre-activatable lacZ reporter mouse strain (R26R). RESULTS A beta-galactosidase assay of rtTA/Cre-R26R mice demonstrated that the basal level of Cre expression without doxycycline induction was negligible. Addition of doxycycline led to induction of RPE-specific Cre expression/function at least from embryonic day 9 to postnatal day 60. The highest induction occurred at approximately postnatal day 4. As measured by ERG and histology, retinal function and morphology were normal in 10-month-old rtTA/Cre mice that were treated with doxycycline at weaning age. CONCLUSIONS Transgenic mice were generated that express Cre recombinase in the RPE in an inducible fashion. These mice will be useful for studies of the RPE-specific role of genes that are expressed in the RPE as well as other cells, particularly for avoiding embryonic lethality and dissecting the function of genes that play dual roles in development and adulthood.

Photoreceptor avascular privilege is shielded by soluble VEGF receptor-1

Optimal phototransduction requires separation of the avascular photoreceptor layer from the adjacent vascularized inner retina and choroid. Breakdown of peri-photoreceptor vascular demarcation leads to retinal angiomatous proliferation or choroidal neovascularization, two variants of vascular invasion of the photoreceptor layer in age-related macular degeneration (AMD), the leading cause of irreversible blindness in industrialized nations. Here we show that sFLT-1, an endogenous inhibitor of vascular endothelial growth factor A (VEGF-A), is synthesized by photoreceptors and retinal pigment epithelium (RPE), and is decreased in human AMD. Suppression of sFLT-1 by antibodies, adeno-associated virus-mediated RNA interference, or Cre/lox-mediated gene ablation either in the photoreceptor layer or RPE frees VEGF-A and abolishes photoreceptor avascularity. These findings help explain the vascular zoning of the retina, which is critical for vision, and advance two transgenic murine models of AMD with spontaneous vascular invasion early in life. DOI: http://dx.doi.org/10.7554/eLife.00324.001

X-Box Binding Protein 1 Is Essential for the Anti-Oxidant Defense and Cell Survival in the Retinal Pigment Epithelium

Damage to the retinal pigment epithelium (RPE) is an early event in the pathogenesis of age-related macular degeneration (AMD). X-box binding protein 1 (XBP1) is a key transcription factor that regulates endoplasmic reticulum (ER) homeostasis and cell survival. This study aimed to delineate the role of endogenous XBP1 in the RPE. Our results show that in a rat model of light-induced retinal degeneration, XBP1 activation was suppressed in the RPE/choroid complex, accompanied by decreased anti-oxidant genes and increased oxidative stress. Knockdown of XBP1 by siRNA resulted in reduced expression of SOD1, SOD2, catalase, and glutathione synthase and sensitized RPE cells to oxidative damage. Using Cre/LoxP system, we generated a mouse line that lacks XBP1 only in RPE cells. Compared to wildtype littermates, RPE-XBP1 KO mice expressed less SOD1, SOD2, and catalase in the RPE, and had increased oxidative stress. At age 3 months and older, these mice exhibited apoptosis of RPE cells, decreased number of cone photoreceptors, shortened photoreceptor outer segment, reduced ONL thickness, and deficit in retinal function. Electron microscopy showed abnormal ultrastructure, Bruchs membrane thickening, and disrupted basal membrane infolding in XBP1-deficient RPE. These results indicate that XBP1 is an important gene involved in regulation of the anti-oxidant defense in the RPE, and that impaired activation of XBP1 may contribute to RPE dysfunction and cell death during retinal degeneration and AMD.