TNFAIP8 overexpression aggravates retinal pathophysiological features of diabetic retinopathy.

Our previous research shown that tumor necrosis factor-alpha-induced protein 8 (TNFAIP8) is elevated in the plasma extracellular vesicles and vitreous humor in diabetic retinopathy (DR). TNFAIP8 also significantly increases the viability of human retinal microvascular endothelial cells (HRMECs) and promotes cell migration and tube formation in vitro. To comprehensively explore its role in DR, we investigated the effect of TNFAIP8 on DR development using an animal model in this study. A TNFAIP8-overexpressing adeno-associated virus (AAV) vector and streptozotocin-induced mouse model was used. The AAV-TNFAIP8 vector was injected into the mice intravitreally, and the effect was evaluated. The evaluation included analysis of retinal structure and function using electroretinography, optical coherence tomography, and histological assessment. The influence of TNFAIP8 on the avascular area, retinal leukostasis, and the expression levels of inflammatory factors was also determined. TNFAIP8 significantly decreased a/b-wave amplitude and retinal thickness in diabetic mice. Histological assessment showed that TNFAIP8 aggravated pathological abnormalities with distorted organization of the retina. TNFAIP8 also significantly increased the avascular area, leukostasis, and the expression of inflammatory factors, such as TNFα, IL1ß, ICAM1, and GFAP, in the retina. The results of this study support the role of TNFAIP8 in DR pathogenesis. A mechanistic understanding of TNFAIP8 may offer novel therapeutic strategies.

Deletion of Tgfß signal in activated microglia prolongs hypoxia-induced retinal neovascularization enhancing Igf1 expression and retinal leukostasis.

Retinal neovascularization (NV) is the major cause of severe visual impairment in patients with ischemic eye diseases. While it is known that retinal microglia contribute to both physiological and pathological angiogenesis, the molecular mechanisms by which these glia regulate pathological NV have not been fully elucidated. In this study, we utilized a retinal microglia-specific Transforming Growth Factor-ß (Tgfß) receptor knock out mouse model and human iPSC-derived microglia to examine the role of Tgfß signaling in activated microglia during retinal NV. Using a tamoxifen-inducible, microglia-specific Tgfß receptor type 2 (Tgfßr2) knockout mouse [Tgfßr2 KO (ΔMG)] we show that Tgfß signaling in microglia actively represses leukostasis in retinal vessels. Furthermore, we show that Tgfß signaling represses expression of the pro-angiogenic factor, Insulin-like growth factor 1 (Igf1), independent of Vegf regulation. Using the mouse model of oxygen-induced retinopathy (OIR) we show that Tgfß signaling in activated microglia plays a role in hypoxia-induced NV where a loss in Tgfß signaling microglia exacerbates and prolongs retinal NV in OIR. Using human iPSC-derived microglia cells in an in vitro assay, we validate the role of Transforming Growth Factor-ß1 (Tgfß1) in regulating Igf1 expression in hypoxic conditions. Finally, we show that Tgfß signaling in microglia is essential for microglial homeostasis and that the disruption of Tgfß signaling in microglia exacerbates retinal NV in OIR by promoting leukostasis and Igf1 expression.

Effects of Diabetes on Microcirculation and Leukostasis in Retinal and Non-Ocular Tissues: Implications for Diabetic Retinopathy.

Changes in retinal microcirculation are associated with the development of diabetic retinopathy (DR). However, it is unclear whether such changes also develop in capillary beds of other non-retinal tissues. Here, we investigated microcirculatory changes involving velocity of rolling neutrophils, adherence of neutrophils, and leukostasis during development of retinal vascular lesions in diabetes in other non-retinal tissues. Intravital microscopy was performed on post-capillary venules of cremaster muscle and ear lobe of mice with severe or moderate diabetes and compared to those of non-diabetic mice. Additionally, number and velocity of rolling leukocytes, number of adherent leukocytes, and areas of leukostasis were quantified, and retinal capillary networks were examined for acellular capillaries (AC) and pericyte loss (PL), two prominent vascular lesions characteristic of DR. The number of adherent neutrophils and areas of leukostasis in the cremaster and ear lobe post-capillary venules of diabetic mice was increased compared to those of non-diabetic mice. Similarly, a significant increase in the number of rolling neutrophils and decrease in their rolling velocities compared to those of non-diabetic control mice were observed and severity of diabetes exacerbated these changes. Understanding diabetes-induced microcirculatory changes in cremaster and ear lobe may provide insight into retinal vascular lesion development in DR.

Systemic alterations in leukocyte subsets and the protective role of NKT cells in the mouse model of diabetic retinopathy.

The involvement of leukocytes in the pathophysiology of DR has mostly examined the role of monocytes and neutrophils with little emphasis on other immune cell types. In this study, we determined the systemic alterations in T cell subsets, myeloid cell types, NK cells, and NKT cells in the streptozotocin (STZ) mouse model of diabetic retinopathy (DR), and the role of NKT cells on retinal leukostasis and permeability changes. C57BL/6 J mice were made diabetic with 60 mg/kg dose of STZ given for 5-days. Flow cytometry assay measured the frequency of leukocyte subsets in the peripheral blood, spleen, and bone marrow of STZ- and vehicle-treated C57BL/6 J mice. Our results showed an increased proportion of memory CD8 T cells and interferon-gamma (IFN-γ) secreting CD8 T cells in the bone marrow of STZ-treated compared to control mice. Subsequently, increased production of inflammatory monocytes in the bone marrow and an enhanced frequency of CD11b + cells in the diabetic retina were seen in STZ-treated compared to control mice. The diabetic mice also exhibited a decrease in total NKT and CD4+NKT cells. A monoclonal antibody-based approach depleted NKT cells from STZ-treated mice, followed by measurements of retinal vascular permeability and leukostasis. The depletion of NKT cells in STZ-treated mice resulted in a significant increase in vascular permeability in the retinal tissue. Together, our results strongly imply the involvement of NKT cells in regulating the pathophysiology of the diabetic retina.

Inactivation of Endothelial ADAM17 Reduces Retinal Ischemia-Reperfusion Induced Neuronal and Vascular Damage.

Retinal ischemia contributes to visual impairment in ischemic retinopathies. A disintegrin and metalloproteinase ADAM17 is implicated in multiple vascular pathologies through its ability to regulate inflammatory signaling via ectodomain shedding. We investigated the role of endothelial ADAM17 in neuronal and vascular degeneration associated with retinal ischemia reperfusion (IR) injury using mice with conditional inactivation of ADAM17 in vascular endothelium. ADAM17Cre-flox and control ADAM17flox mice were subjected to 40 min of pressure-induced retinal ischemia, with the contralateral eye serving as control. Albumin extravasation and retinal leukostasis were evaluated 48 h after reperfusion. Retinal morphometric analysis was conducted 7 days after reperfusion. Degenerate capillaries were assessed by elastase digest and visual function was evaluated by optokinetic test 14 and 7 days following ischemia, respectively. Lack of ADAM17 decreased vascular leakage and reduced retinal thinning and ganglion cell loss in ADAM17Cre-flox mice. Further, ADAM17Cre-flox mice exhibited a remarkable reduction in capillary degeneration following IR. Decrease in neurovascular degeneration in ADAM17Cre-flox mice correlated with decreased activation of caspase-3 and was associated with reduction in oxidative stress and retinal leukostasis. In addition, knockdown of ADAM17 resulted in decreased cleavage of p75NTR, the process known to be associated with retinal cell apoptosis. A decline in visual acuity evidenced by decrease in spatial frequency threshold observed in ADAM17flox mice was partially restored in ADAM17-endothelial deficient mice. The obtained results provide evidence that endothelial ADAM17 is an important contributor to IR-induced neurovascular damage in the retina and suggest that interventions directed at regulating ADAM17 activity can be beneficial for alleviating the consequences of retinal ischemia.

Suppression of Ocular Vascular Inflammation through Peptide-Mediated Activation of Angiopoietin-Tie2 Signaling.

Persistent inflammation is a complication associated with many ocular diseases. Changes in ocular vessels can amplify disease responses and contribute to vision loss by influencing the delivery of leukocytes to the eye, vascular leakage, and perfusion. Here, we report the anti-inflammatory activity for AXT107, a non-RGD, 20-mer αvß3 and α5ß1 integrin-binding peptide that blocks vascular endothelial growth factor (VEGF)-signaling and activates tyrosine kinase with immunoglobulin and EGF-like domains 2 (Tie2) using the normally inhibitory ligand angiopoietin 2 (Ang2). Tumor necrosis factor α (TNFα), a central inflammation mediator, induces Ang2 release from endothelial cells to enhance its stimulation of inflammation and vascular leakage. AXT107 resolves TNFα-induced vascular inflammation in endothelial cells by converting the endogenously released Ang2 into an agonist of Tie2 signaling, thereby disrupting both the synergism between TNFα and Ang2 while also preventing inhibitor of nuclear factor-κB α (IκBα) degradation directly through Tie2 signaling. This recovery of IκBα prevents nuclear factor kappa-light-chain-enhancer of activated B cells (NF-κB) nuclear localization, thereby blocking NF-κB-induced inflammatory responses, including the production of VCAM-1 and ICAM-1, leukostasis, and vascular leakage in cell and mouse models. AXT107 also decreased the levels of pro-inflammatory TNF receptor 1 (TNFR1) without affecting levels of the more protective TNFR2. These data suggest that AXT107 may provide multiple benefits in the treatment of retinal/choroidal and other vascular diseases by suppressing inflammation and promoting vascular stabilization.

The peroxisome proliferator-activated receptor-ß/δ antagonist GSK0660 mitigates retinal cell inflammation and leukostasis.

Diabetic retinopathy (DR) is triggered by retinal cell damage stimulated by the diabetic milieu, including increased levels of intraocular free fatty acids. Free fatty acids may serve as an initiator of inflammatory cytokine release from Müller cells, and the resulting cytokines are potent stimulators of retinal endothelial pathology, such as leukostasis, vascular permeability, and basement membrane thickening. Our previous studies have elucidated a role for peroxisome proliferator-activated receptor-ß/δ (PPARß/δ) in promoting several steps in the pathologic cascade in DR, including angiogenesis and expression of inflammatory mediators. Furthermore, PPARß/δ is a known target of lipid signaling, suggesting a potential role for this transcription factor in fatty acid-induced retinal inflammation. Therefore, we hypothesized that PPARß/δ stimulates both the induction of inflammatory mediators by Müller cells as well the paracrine induction of leukostasis in endothelial cells (EC) by Müller cell inflammatory products. To test this, we used the PPARß/δ inhibitor, GSK0660, in primary human Müller cells (HMC), human retinal microvascular endothelial cells (HRMEC) and mouse retina. We found that palmitic acid (PA) activation of PPARß/δ in HMC leads to the production of pro-angiogenic and/or inflammatory cytokines that may constitute DR-relevant upstream paracrine inflammatory signals to EC and other retinal cells. Downstream, EC transduce these signals and increase their synthesis and release of chemokines such as CCL8 and CXCL10 that regulate leukostasis and other cellular events related to vascular inflammation in DR. Our results indicate that PPARß/δ inhibition mitigates these upstream (MC) as well as downstream (EC) inflammatory signaling events elicited by metabolic stimuli and inflammatory cytokines. Therefore, our data suggest that PPARß/δ inhibition is a potential therapeutic strategy against early DR pathology.

miR-15a/16 reduces retinal leukostasis through decreased pro-inflammatory signaling.

BACKGROUND: Hyperglycemia is a significant risk factor for diabetic retinopathy and induces increased inflammatory responses and retinal leukostasis, as well as vascular damage. Although there is an increasing amount of evidence that miRNA may be involved in the regulation in the pathology of diabetic retinopathy, the mechanisms by which miRNA mediate cellular responses to control onset and progression of diabetic retinopathy are still unclear. The purpose of our study was to investigate the hypothesis that miR-15a/16 inhibit pro-inflammatory signaling to reduce retinal leukostasis. METHODS: We generated conditional knockout mice in which miR-15a/16 are eliminated in vascular endothelial cells. For the in vitro work, human retinal endothelial cells (REC) were cultured in normal (5 mM) glucose or transferred to high glucose medium (25 mM) for 3 days. Transfection was performed on REC in high glucose with miRNA mimic (hsa-miR-15a-5p, hsa-miR-16-5p). Statistical analyses were done using unpaired Student t test with two-tailed p value. p < 0.05 was considered significant. Data are presented as mean ± SEM. RESULTS: We demonstrated that high glucose conditions decreased expression of miR-15a/16 in cultured REC. Overexpression of miR-15a/16 with the mimic significantly decreased pro-inflammatory signaling of IL-1ß, TNFα, and NF-κB in REC. In vivo data demonstrated that the loss of miR-15a/16 in vascular cells led to increased retinal leukostasis and CD45 levels, together with upregulated levels of IL-1ß, TNFα, and NF-κB. CONCLUSIONS: The data indicate that miR-15a/16 play significant roles in reducing retinal leukostasis, potentially through inhibition of inflammatory cellular signaling. Therefore, we suggest that miR-15a/16 offer a novel potential target for the inhibition of inflammatory mediators in diabetic retinopathy.

NFAT isoforms play distinct roles in TNFα-induced retinal leukostasis.

The objective of this study was to determine the role of individual NFAT isoforms in TNFα-induced retinal leukostasis. To this end, human retinal microvascular endothelial cells (HRMEC) transfected with siRNA targeting individual NFAT isoforms were treated with TNFα, and qRT-PCR was used to examine the contribution of each isoform to the TNFα-induced upregulation of leukocyte adhesion proteins. This showed that NFATc1 siRNA increased ICAM1 expression, NFATc2 siRNA reduced CX3CL1, VCAM1, SELE, and ICAM1 expression, NFATc3 siRNA increased CX3CL1 and SELE expression, and NFATc4 siRNA reduced SELE expression. Transfected HRMEC monolayers were also treated with TNFα and assayed using a parallel plate flow chamber, and both NFATc2 and NFATc4 knockdown reduced TNFα-induced cell adhesion. The effect of isoform-specific knockdown on TNFα-induced cytokine production was also measured using protein ELISAs and conditioned cell culture medium, and showed that NFATc4 siRNA reduced CXCL10, CXCL11, and MCP-1 protein levels. Lastly, the CN/NFAT-signaling inhibitor INCA-6 was shown to reduce TNFα-induced retinal leukostasis in vivo. Together, these studies show a clear role for NFAT-signaling in TNFα-induced retinal leukostasis, and identify NFATc2 and NFATc4 as potentially valuable therapeutic targets for treating retinopathies in which TNFα plays a pathogenic role.

FT011, a Novel Cardiorenal Protective Drug, Reduces Inflammation, Gliosis and Vascular Injury in Rats with Diabetic Retinopathy.

Diabetic retinopathy features inflammation as well as injury to glial cells and the microvasculature, which are influenced by hypertension and overactivity of the renin-angiotensin system. FT011 is an anti-inflammatory and anti-fibrotic agent that has been reported to attenuate organ damage in diabetic rats with cardiomyopathy and nephropathy. However, the potential therapeutic utility of FT011 for diabetic retinopathy has not been evaluated. We hypothesized that FT011 would attenuate retinopathy in diabetic Ren-2 rats, which exhibit hypertension due to an overactive extra-renal renin-angiotensin system. Diabetic rats were studied for 8 and 32 weeks and received intravitreal injections of FT011 (50 µM) or vehicle (0.9% NaCl). Comparisons were to age-matched controls. In the 8-week study, retinal inflammation was examined by quantitating vascular leukocyte adherence, microglial/macrophage density and the expression of inflammatory mediators. Macroglial Müller cells, which exhibit a pro-inflammatory and pro-angiogenic phenotype in diabetes, were evaluated in the 8-week study as well as in culture following exposure to hyperglycaemia and FT011 (10, 30, 100 µM) for 72 hours. In the 32-week study, severe retinal vasculopathy was examined by quantitating acellular capillaries and extracellular matrix proteins. In diabetic rats, FT011 reduced retinal leukostasis, microglial density and mRNA levels of intercellular adhesion molecule-1 (ICAM-1). In Müller cells, FT011 reduced diabetes-induced gliosis and vascular endothelial growth factor (VEGF) immunolabeling and the hyperglycaemic-induced increase in ICAM-1, monocyte chemoattractant protein-1, CCL20, cytokine-induced neutrophil chemoattractant-1, VEGF and IL-6. Late intervention with FT011 reduced acellular capillaries and the elevated mRNA levels of collagen IV and fibronectin in diabetic rats. In conclusion, the protective effects of FT011 in cardiorenal disease extend to key elements of diabetic retinopathy and highlight its potential as a treatment approach.

Role of the receptor for advanced glycation endproducts (RAGE) in retinal vasodegenerative pathology during diabetes in mice.

AIMS/HYPOTHESIS: The receptor for AGEs (RAGE) is linked to proinflammatory pathology in a range of tissues. The objective of this study was to assess the potential modulatory role of RAGE in diabetic retinopathy. METHODS: Diabetes was induced in wild-type (WT) and Rage (-/-) mice (also known as Ager (-/-) mice) using streptozotocin while non-diabetic control mice received saline. For all groups, blood glucose, HbA1c and retinal levels of methylglyoxal (MG) were evaluated up to 24 weeks post diabetes induction. After mice were killed, retinal glia and microglial activation, vasopermeability, leucostasis and degenerative microvasculature changes were determined. RESULTS: Retinal expression of RAGE in WT diabetic mice was increased after 12 weeks (p < 0.01) but not after 24 weeks. Rage (-/-) mice showed comparable diabetes but accumulated less MG and this corresponded to enhanced activity of the MG-detoxifying enzyme glyoxalase I in their retina when compared with WT mice. Diabetic Rage (-/-) mice showed significantly less vasopermeability, leucostasis and microglial activation (p < 0.05-0.001). Rage (-/-) mice were also protected against diabetes-related retinal acellular capillary formation (p < 0.001) but not against pericyte loss. CONCLUSIONS/INTERPRETATION: Rage (-/-) in diabetic mice is protective against many retinopathic lesions, especially those related to innate immune responses. Inhibition of RAGE could be a therapeutic option to prevent diabetic retinopathy.

Silybin reduces obliterated retinal capillaries in experimental diabetic retinopathy in rats.

Silybin has been previously reported to possess anti-inflammatory properties, raising the possibility that it may reduce vascular damage in diabetic retinopathy. Present study was designed to investigate this potential effect of silybin and its underlying mechanisms in experimental diabetic retinopathy. Diabetes was induced with streptozotocin (STZ) plus high-fat diet in Sprague-Dawley rats, and silybin was administrated for 22 weeks after the induction of diabetes. Histochemical and immunofluorescence techniques were used to assess the obliterated retinal capillaries, leukostasis, and level of retinal intercellular adhesion molecule-1 (ICAM-1). Western blot was performed to quantitate the expression of retinal ICAM-1. Results showed that silybin treatment significantly prevented the development of obliterated retinal capillaries in diabetes, compared with vehicle treatment. In addition, leukostasis and level of the retinal ICAM-1 were found to decrease considerably in silybin-treated diabetic groups. In conclusion, these results indicate that silybin reduces obliterated retinal capillaries in experimental diabetes, and the recovered retinal vascular leukostasis and level of ICAM-1 at least partly contributes to the preventive effect of silybin.

Negatively regulating TLR4/NF-κB signaling via PPARα in endotoxin-induced uveitis.

Toll-like receptor (TLR) signaling plays a fundamental role in the induction and progression of autoimmune disease. In the present study, we showed that lipopolysaccharide (LPS), a TLR4 ligand, functions as an antagonist of peroxisome proliferator-activated receptor alpha (PPARα), a nuclear transcription factor. Using endotoxin induced uveitis (EIU) as a model, we found that TLR was negatively regulated by PPARα. Our data revealed that treatment with the PPARα agonist fenofibrate dramatically prevented LPS-induced uveitis and inhibited TLR/ Nuclear factor-kappaB (NF-κB) signaling during inflammation. Evaluation of the severity of anterior uveitis further showed that PPARα agonist treatment significantly decreased inflammatory cell infiltration, total protein concentration, vessel density, inflammatory cytokine production, and clinical scores in the anterior section of the eye during EIU. Moreover, fenofibrate administration recovered retinal function and decreased the production of inflammatory cytokines, retinal vascular leukostasis, and inflammatory cell infiltration into the posterior section of the eyes during EIU. In vitro studies further showed that down-regulation or deletion of PPARα led to increased TLR4 levels and the activation of NF-κB signaling in RPE cells and also blocked the anti-inflammatory effects of fenofibrate. Furthermore, activation or up-regulation of PPARα decreased TLR4 levels and inhibited the NF-κB signaling pathway induced by LPS in RPE cells. In TLR4-expressing reporter cells, activation or up-regulation of PPARα partially inhibited the activation of NF-κB and also decreased TLR4 transcriptional activity. In conclusion, the activation of PPARα represents a novel therapeutic strategy for human uveitis, as PPARα negatively regulates TLR4 activity and therefore exerts anti-inflammatory actions.

Antagonism of CD11b with neutrophil inhibitory factor (NIF) inhibits vascular lesions in diabetic retinopathy.

Leukocytes and proteins that govern leukocyte adhesion to endothelial cells play a causal role in retinal abnormalities characteristic of the early stages of diabetic retinopathy, including diabetes-induced degeneration of retinal capillaries. Leukocyte integrin αmß2 (CD11b/CD18, MAC1), a protein mediating adhesion, has been shown to mediate damage to endothelial cells by activated leukocytes in vitro. We hypothesized that Neutrophil Inhibitory Factor (NIF), a selective antagonist of integrin αmß2, would inhibit the diabetes-induced degeneration of retinal capillaries by inhibiting the excessive interaction between leukocytes and retinal endothelial cells in diabetes. Wild type animals and transgenic animals expressing NIF were made diabetic with streptozotocin and assessed for diabetes-induced retinal vascular abnormalities and leukocyte activation. To assess if the leukocyte blocking therapy compromised the immune system, animals were challenged with bacteria. Retinal superoxide production, leukostasis and leukocyte superoxide production were increased in wild type mice diabetic for 10 weeks, as was the ability of leukocytes isolated from diabetic animals to kill retinal endothelial cells in vitro. Retinal capillary degeneration was significantly increased in wild type mice diabetic 40 weeks. In contrast, mice expressing NIF did not develop any of these abnormalities, with the exception that non-diabetic and diabetic mice expressing NIF generated greater amounts of superoxide than did similar mice not expressing NIF. Importantly, NIF did not significantly impair the ability of mice to clear an opportunistic bacterial challenge, suggesting that NIF did not compromise immune surveillance. We conclude that antagonism of CD11b (integrin αmß2) by NIF is sufficient to inhibit early stages of diabetic retinopathy, while not compromising the basic immune response.

MyD88-dependent pathways in leukocytes affect the retina in diabetes.

BACKGROUND: Previous studies by us and other have provided evidence that leukocytes play a critical role in the development of diabetic retinopathy, suggesting a possible role of the innate immune system in development of the retinopathy. Since MyD88 is a convergence point for signaling pathways of the innate immune system (including Toll-Like Receptors (TLRs) and interleukin-1ß (IL-1ß)), the purpose of this study was to assess the role of MyD88 and its dependent pathways on abnormalities that develop in retina and white blood cells related to diabetic retinopathy. METHODS: C57BL/6J mice were made diabetic with streptozotocin. Chimeric mice were generated in which MyD88-dependent pathways were deleted from bone marrow-derived only. Mice were sacrificed at 2 mos of diabetes for assessment of, leukostasis, albumin accumulation in neural retina, leukocyte-mediated killing of retinal endothelial cells, and cytokine/chemokine generation by retinas of diabetic mice in response to TLR agonists. RESULTS: IL-6 and CXCL1 were generated in retinas from diabetic (but not nondiabetic mice) following incubation with Pam3CysK/TLR2, but incubation with other TLR ligands or IL-1ß did not induce cytokine production in retinas from nondiabetic or diabetic mice. Diabetes-induced abnormalities (leukostasis, ICAM-1 expression on the luminal surface of the vascular endothelium, retinal superoxide generation) were significantly inhibited by removing either MyD88 or the signaling pathways regulated by it (TLRs 2 and 4, and IL-1ß) from bone marrow-derived cells only. Leukocyte-mediated killing of endothelial cells tended to be decreased in the marrow-derived cells lacking TLR2/4, but the killing was significantly exacerbated if the marrow cells lacked MyD88 or the receptor for IL-1ß (IL-1ßr). CONCLUSIONS: MyD88-dependent pathways play an important role in the development of diabetes-induced inflammation in the retina, and inhibition of MyD88 might be a novel target to inhibit early abnormalities of diabetic retinopathy and other complications of diabetes.

Aquaporin 4 knockdown exacerbates streptozotocin-induced diabetic retinopathy through aggravating inflammatory response.

Diabetic retinopathy is a leading cause of reduced visual acuity and acquired blindness. Diabetes is known to alter the amount of retinal expression of the water-selective channels aquaporin 4 (AQP4). However, the function and impact of AQP4 in diabetic retinopathy is not well understood. In the present work, diabetes was induced by intraperitoneal injection of streptozotocin in Sprague-Dawley rats. Two weeks later, AQP4 shRNA (r) lentiviral particles or negative lentiviral particles were delivered by intravitreal injection to the eyes. Gene delivery was confirmed by quantitative reverse-transcriptase polymerase chain reaction (qRT-PCR) and Western blotting analysis. Eight weeks later, BRB breakdown was measured using Evans blue dye. Images of retinal sections were obtained and the thicknesses of the retinas were determined. Retinal leukostasis measurement was performed using acridine orange leukocyte fluorography. The mRNA levels of IL-1ß, IL-6, intercellular adhesion molecule 1 (ICAM-1), glial fibrillary acidic protein (GFAP) and vascular endothelial growth factor (VEGF) were determined using qRT-PCR method. AQP4 shRNA (r) lentiviral particles or negative lentiviral particles were transfected into rMC-1 cells to investigate its effect on inflammation induced by high glucose. Incubation with IL-1ß or IL-6 was performed to test their effect on AQP4 expression in rMC-1 cells. In the current work, it was found that AQP4 expression was enhanced in the retina of diabetic rats. AQP4 knockdown led to exacerbation of retinopathy including enhancing retinal vascular permeability, retinal thickness, pro-inflammatory factors expression, and VEGF and GFAP expression in retinas of diabetic rats. AQP4 knockdown enhanced the expression of pro-inflammatory cytokines induced by high glucose in rMC-1 cells. In addition, AQP4 knockdown enhanced the release of IL-6 and VEGF from rMC-1 cells into the medium. Moreover, it was found that incubation with IL-1ß or IL-6 suppressed AQP4 expression in rMC-1 cells. These results suggested that streptozotocin injection induced diabetes resulted in compensatory increases of AQP4 expression, and downregulation of AQP4 exacerbated diabetic retinopathy through aggravating inflammatory response, at last in part. Therefore, regulation of retinal function by AQP4 may attenuate diabetic retinopathy, offering a promising therapeutic strategy for diabetic retinopathy.

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

AIMS/HYPOTHESIS: Retinal 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. METHODS: Conditional 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. RESULTS: While 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/INTERPRETATION: Müller 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.

Role of alpha 4 integrin (CD49d) in the pathogenesis of diabetic retinopathy.

PURPOSE: The pathophysiology of diabetic retinopathy is mediated by leukocyte adhesion to the vascular endothelium of the diabetic retina, which results in endothelial injury, blood-retina barrier breakdown, and capillary nonperfusion. Leukocyte adhesion is triggered by the interaction of vascular endothelium adhesion molecules, such as ICAM-1, with leukocyte integrins, such as CD18. Inhibition of ICAM-1/CD18 signaling suppresses but does not completely abolish the cardinal manifestations of diabetic retinopathy, suggesting a role for additional adhesion molecules. Integrin alpha 4 (CD49d), in complex with integrin beta1, forms very late antigen-4 (VLA-4), which interacts with vascular cell adhesion molecule-1. The authors have now studied the role of integrin alpha 4/CD49d in the pathogenesis of diabetic retinopathy. METHODS: Diabetes mellitus was induced in Long Evans rats with streptozotocin, and an anti-alpha 4 integrin/CD49d neutralizing antibody was injected 5 and 10 days later. Two weeks after streptozotocin administration, vascular leakage was quantified with the Evans Blue technique. Leukostasis was measured with a static adhesion assay ex vivo and the FITC-lectin perfusion method in vivo. Retinal VEGF and TNF-alpha levels and NF-kappaB activity were measured by ELISA. RESULTS: Blockade of alpha 4 integrin/CD49d attenuated the diabetes-induced upregulation of NF-kappaB activation, VEGF, and TNF-alpha protein levels and reduced significantly diabetes-induced leukocyte adhesion and vascular leakage. CONCLUSIONS: These data identify alpha 4 integrin/CD49d as a mediator of leukocyte adhesion and the resultant early signature abnormalities of diabetic retinopathy. Inhibition of this signaling pathway may hold promise for clinical activity in patients with diabetes.