Retinal Leukostasis Research Articles

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, IL1β, 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.

Therapeutic potential of elema-1,3,7(11),8-tetraen-8,12-lactam from Curcuma wenyujin on diabetic retinopathy via anti-inflammatory and anti-angiogenic pathways

Ethnopharmacological relevanceIn traditional Chinese medicine, the causes of diabetic retinopathy (DR) are blood stasis and heat. Curcuma wenyujin Y. H. Chen & C. Ling and its extracts have the effects of promoting blood circulation to remove blood stasis, clearing the heart, and cooling the blood, and have been used in the treatment of DR. Elema-1,3,7 (11),8-tetraen-8,12-lactam (Ele), an N-containing sesquiterpene isolated from this plant. However, the anti-inflammatory and anti-angiogenic effects of Ele and its therapeutic potential in DR are still unknown. Aim of the studyTo evaluate the anti-inflammatory and anti-angiogenic effects of Ele and its therapeutic potential in DR. Materials and methodsIn vitro, anti-inflammatory and anti-angiogenic effects were assessed using TNF-α or VEGF-stimulated HUVECs. Protein expression was analyzed using Western blotting. ICAM-1 and TNF-α mRNA expressions were analyzed using real-time quantitative RT-PCR. The therapeutic potential in DR was assessed using both animal models of STZ-induced diabetes and oxygen-induced retinopathy. The retinal vascular permeability was measured using Evans blue, and the quantitation of retinal leukostasis using FITC-coupled Con A. The retinal neovascular tufts were analyzed using fluorescein angiography and counting pre-retinal vascular lumens. ResultsEle inhibited NF-κB pathway, and ICAM-1, TNF-α mRNA expression in TNF-α- stimulated HUVECs. It also inhibits the multistep process of angiogenesis by inhibiting the phosphorylation of VEGFR2 and its downstream signaling kinases Src, Erk1/2, Akt, and mTOR in VEGF-stimulated HUVECs. Intravitreal injection of Ele can significantly reduce retinal microvascular leakage, leukostasis, and expression of ICAM-1, TNF-α in diabetic rats and inhibits oxygen-induced retinal neovascularization and VEGFR2 phosphorylation in OIR mice. ConclusionsEle has anti-inflammatory and anti-angiogenic effects through inhibiting NF-κB and VEGFR2 signaling pathways, and it may be a potential drug candidate for DR.

Modulation of cGAS-STING signaling by PPARα in a mouse model of ischemia-induced retinopathy

In ischemic retinopathy, overactivated retinal myeloid cells are a crucial driving force of pathological angiogenesis and inflammation. The cyclic GMP-AMP synthase (cGAS) and stimulator of interferon genes (STING) signaling are key regulators of inflammation. This study aims to investigate the association of cGAS-STING signaling with ischemic retinopathy and the regulation of its activation. We found that protein levels of cGAS and STING were markedly up-regulated in retinal myeloid cells isolated from mice with oxygen-induced retinopathy (OIR). Knockout of Sting and pharmacological inhibition of STING both alleviated retinal neovascularization (NV) and reduced retinal vascular leakage in OIR. Further, Sting knockout and STING inhibitor also alleviated leukocyte adhesion to retinal vasculature and infiltration into the retina as well as microglial activation in OIR. These results suggest that cGAS-STING signaling played a pathogenic role in retinal myeloid cell activation and NV in ischemic retinopathy. To identify the regulation of cGAS-STING signaling in OIR, we evaluated the role of transcription factor peroxisome proliferator-activated receptor α (PPARα). The results demonstrated that PPARα was down-regulated in OIR retinas, primarily in myeloid cells. Furthermore, Pparα knockout significantly up-regulated cGAS and STING levels in retinal CD11b+ cells, while PPARα agonist inhibited cGAS-STING signaling and cytosolic mitochondrial DNA (mtDNA) release, a causative feature for cGAS activation. Knockout of Sting ameliorated retinal NV, hyperpermeability, and leukostasis in Pparα-/- mice with OIR. These observations suggest that PPARα regulates cGAS-STING signaling, likely through mtDNA release, and thus, is a potential therapeutic target for ischemic retinopathy.

Deletion of Tgfβ 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‐β (Tgfβ) receptor knock out mouse model and human iPSC‐derived microglia to examine the role of Tgfβ signaling in activated microglia during retinal NV. Using a tamoxifen‐inducible, microglia‐specific Tgfβ receptor type 2 (Tgfβr2) knockout mouse [Tgfβr2 KO (ΔMG)] we show that Tgfβ signaling in microglia actively represses leukostasis in retinal vessels. Furthermore, we show that Tgfβ 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 Tgfβ signaling in activated microglia plays a role in hypoxia‐induced NV where a loss in Tgfβ 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‐β1 (Tgfβ1) in regulating Igf1 expression in hypoxic conditions. Finally, we show that Tgfβ signaling in microglia is essential for microglial homeostasis and that the disruption of Tgfβ signaling in microglia exacerbates retinal NV in OIR by promoting leukostasis and Igf1 expression.

Neutrophil-Derived Proteases Contribute to the Pathogenesis of Early Diabetic Retinopathy.

PurposePrevious studies indicate that leukocytes, notably neutrophils, play a causal role in the capillary degeneration observed in diabetic retinopathy (DR), however, the mechanism by which they cause such degeneration is unknown. Neutrophil elastase (NE) is a protease released by neutrophils which participates in a variety of inflammatory diseases. In the present work, we investigated the potential involvement of NE in the development of early DR.MethodsExperimental diabetes was induced in NE-deficient mice (Elane−/−), in mice treated daily with the NE inhibitor, sivelestat, and in mice overexpressing human alpha-1 antitrypsin (hAAT+). Mice were assessed for diabetes-induced retinal superoxide generation, inflammation, leukostasis, and capillary degeneration.ResultsIn mice diabetic for 2 months, deletion of NE or selective inhibition of NE inhibited diabetes-induced retinal superoxide levels and inflammation, and inhibited leukocyte-mediated cytotoxicity of retinal endothelial cells. In mice diabetic for 8 months, genetic deletion of NE significantly inhibited diabetes-induced retinal capillary degeneration.ConclusionsThese results suggest that a protease released from neutrophils contributes to the development of DR, and that blocking NE activity could be a novel therapy to inhibit DR.

Effect of dual PPAR-α/γ agonist saroglitazar on diabetic retinopathy and oxygen-induced retinopathy

Diabetic retinopathy is a serious complication of diabetes, marked by retinal vascular damage, inflammation, and angiogenesis. This study's objective was to assess the potential benefits of saroglitazar, a peroxisome proliferator-activated receptor-alpha/gamma (PPAR-α/γ) agonist in diabetic retinopathy. Diabetic retinopathy was induced by streptozotocin in Sprague Dawley rats. The effect of saroglitazar was also assessed in the oxygen-induced retinopathy model in newborn rats and VEGF-induced angiogenesis in the chick chorioallantoic membrane (CAM) assay.Treatment of saroglitazar (1 and 4 mg/kg, oral) for 12 weeks significantly ameliorated retinal vascular leakage and leukostasis in the diabetic rats. Saroglitazar decreased oxidative stress, VEGF receptor signalling, NF-κBp65, and ICAM-1 in the retina of diabetic rats. The beneficial effects of saroglitazar (1 and 4 mg/kg, oral) were also observed on the neovascularization in oxygen-induced retinopathy in newborn rats. Saroglitazar also reduced VEGF-induced angiogenesis in CAM assay.This study reveals that saroglitazar has the potential to prevent the progression of retinopathy in diabetic patients.

Therapeutic Potential of Tpl2 (Tumor Progression Locus 2) Inhibition on Diabetic Vasculopathy Through the Blockage of the Inflammasome Complex.

Diabetic retinopathy, one of retinal vasculopathy, is characterized by retinal inflammation, vascular leakage, blood-retinal barrier breakdown, and neovascularization. However, the molecular mechanisms that contribute to diabetic retinopathy progression remain unclear. Approach and Results: Tpl2 (tumor progression locus 2) is a protein kinase implicated in inflammation and pathological vascular angiogenesis. Nε-carboxymethyllysine (CML) and inflammatory cytokines levels in human sera and in several diabetic murine models were detected by ELISA, whereas liquid chromatography-tandem mass spectrometry analysis was used for whole eye tissues. The CML and p-Tpl2 expressions on the human retinal pigment epithelium (RPE) cells were determined by immunofluorescence. Intravitreal injection of pharmacological inhibitor or NA (neutralizing antibody) was used in a diabetic rat model. Retinal leukostasis, optical coherence tomography, and H&E staining were used to observe pathological features. Sera of diabetic retinopathy patients had significantly increased CML levels that positively correlated with diabetic retinopathy severity and foveal thickness. CML and p-Tpl2 expressions also significantly increased in the RPE of both T1DM and T2DM diabetes animal models. Mechanistic studies on RPE revealed that CML-induced Tpl2 activation and NADPH oxidase, and inflammasome complex activation were all effectively attenuated by Tpl2 inhibition. Tpl2 inhibition by NA also effectively reduced inflammatory/angiogenic factors, retinal leukostasis in streptozotocin-induced diabetic rats, and RPE secretion of inflammatory cytokines. The attenuated release of angiogenic factors led to inhibited vascular abnormalities in the diabetic animal model. The inhibition of Tpl2 can block the inflammasome signaling pathway in RPE and has potential clinical and therapeutic implications in diabetes-associated retinal microvascular dysfunction.

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.

Pathogenic role of human C-reactive protein in diabetic retinopathy

Elevated blood levels of C-reactive protein (CRP) are associated with both type 1 and type 2 diabetes and diabetic complications, such as diabetic retinopathy (DR). However, its pathogenic role in DR remains unknown. The present study aims to investigate the potential role of CRP in DR pathogenesis and explore its underlying mechanism. Human CRP transgenic (hCRP-Tg) rats were employed for streptozotocin (STZ)-induced diabetic and oxygen-induced retinopathy (OIR) models. The retina function was monitored by electroretinography (ERG) and retinal thickness was measured by optical coherence tomography (OCT). TUNEL and cell death ELISA were performed to measure the apoptosis. Oxidative stress was detected by the measurement of reactive oxygen species (ROS) in cells and 3-Nitrotyrosine staining in tissue sections. In non-diabetic condition, hCRP-Tg with elevated hCRP levels in the retinas demonstrated declined ERG responses and decreased retinal thickness. In STZ-induced diabetic condition, overexpression of hCRP deteriorated retinal neurodegeneration as shown by ERG and apoptosis assays. hCRP also exacerbated retinal leukostasis and acellular capillary formation induced by diabetes. In the OIR model, overexpression of hCRP exacerbated retinal neovascularization (NV). In retinal cell lines, hCRP treatment induced cell death and over-production of ROS. Furthermore, hCRP-induced overexpression of pro-inflammatory, pro-oxidative, and pro-angiogenic factors was associated with up-regulation of CD32 and the NF-κB signaling in the retinas. Elevated hCRP levels play a pathogenic role in DR. Targeting the hCRP-CD32-NF-κB pathway may represent a novel therapeutic strategy for DR.

Deletion of Thioredoxin-Interacting Protein (TXNIP) Abrogates High Fat Diet-induced Retinal Leukostasis, Barrier Dysfunction and Microvascular Degeneration in a Mouse Obesity Model.

We have shown that a high fat diet (HFD) induces the activation of retinal NOD-like receptor protein (NLRP3)-inflammasome that is associated with enhanced expression and interaction with thioredoxin-interacting protein (TXNIP). Here, the specific contribution of TXNIP and the impact of HFD on retinal leukostasis, barrier dysfunction and microvascular degeneration were investigated. Wild-type (WT) and TXNIP knockout (TKO) mice were fed with normal diet or 60% HFD for 8–18 weeks. TXNIP was overexpressed or silenced in human retinal endothelial cells (REC). At 8 weeks, HFD significantly induced retinal leukostasis and breakdown of the blood–retina barrier in WT mice, but not in TKO mice. In parallel, HFD also induced retinal expression of adhesion molecules and cleaved IL-1β in WT mice, which were also abrogated in TKO mice. In culture, TXNIP overexpression induced NLRP3, IL-1β, and adhesion molecules expression, while TXNIP silencing inhibited them. Blocking the IL-1β receptor significantly suppressed TXNIP-induced expression of NLRP3-inflammasome and adhesion molecules in HREC. Ex-vivo assay showed that leukocytes isolated from WT-HFD, but not from TKO-HFD, induced leukostasis and cell death. At 18 weeks, HFD triggered development of degenerated (acellular) capillaries and decreased branching density in WT but not in TKO mice. Together, HFD-induced obesity triggered early retinal leukostasis and microvascular dysfunction at least in part via TXNIP-NLRP3-inflammasome activation.

Inhibition of acetylcholinesterase attenuated retinal inflammation via suppressing NF-κB activation

Elevated inflammatory cytokines contribute to the pathogenesis of various retinal diseases such as diabetic retinopathy, retinal vasculitis and retinitis. However, the underlying mechanism of retinal inflammation remains largely unknown. Recent studies demonstrated that acetylcholinesterase (ACHE) is an inflammatory indicator in central neural system. This study was aimed to dissect the role of ACHE in retinal inflammation, and its mechanism of action. Retinal inflammation was induced by intravitreal injection of tumor necrosis factor-α (TNF-α) in heterozygous ACHE knockdown mice (ACHE+/-) and wild type mice (ACHE+/+). Donepezil, a well-known ACHE inhibitor, was administrated by daily gavage. Expression of ACHE and intercellular adherent molecule-1 (ICAM-1), infiltration of CD11b+ inflammatory cells, retinal leukostasis and vascular leakage was determined in both ACHE+/- and ACHE+/+ mice. ARPE-19 cells, a human retinal pigment epithelial cell line, were cultured for in vitro assay. Knockdown of ACHE was achieved by lipofectamine-mediated siRNA transfection and pharmaceutical suppression of ACHE was manipulated by donepezil. Cellular expression and distribution of ACHE, ICAM-1, and phosphorylation of NF-κB, IκB and IKKα/β were detected by western-blot analysis or immunocytochemistry. Retinal expression of ACHE was dramatically upregulated, in parallel with increased ICAM-1 expression, enhanced leukostasis and augmented CD11b+ inflammatory cell infiltration as well as vascular hyperpermeability in ACHE+/+ mice injected with TNF-α. However, TNF-α-injected ACHE+/- mice showed lower level of ICAM-1, less leukostasis and fewer infiltrated CD11b+ cells. Moreover, TNF-α-induced retinal vascular leakage was significantly reduced in ACHE+/- mice. Similarly, TNF-α-induced retinal inflammatory response were also attenuated by donepezil intervention. In addition, TNF-α treatment resulted in significant induction of ACHE, upregulation of ICAM-1 and nuclear translocation of NF-κB, phosphorylation of IκB and IKKα/β in ARPE-19 cells. However, inhibition of ACHE reduced TNF-α-induced phosphorylation of NF-κB, IκB and IKKα/β in ARPE-19 cells. The present study reveals a pivotal role of ACHE in retinal inflammation. Inhibition of ACHE attenuates retinal inflammation and retinal leakage likely through suppressing NF-κB signaling activation.

The effect of Shengpuhuang-tang on retinal inflammation in streptozotocin-induced diabetic rats by NF-κB pathway.

Diabetic retinopathy (DR) is a terrible microvascular disorder causing blindness. Retinal inflammation is the early stage in DR, which is believed to play a crucial role in the development of it. Shengpuhuang-tang (ST), a traditional herbal formula, which has effective treatment of fundus bleeding disorder. ST exerts protective effects against DR in rats, but its underlying mechanism of this efficacy remains unknown. Thus, the objective of this study is to examine the mechanism and the efficacy of ST on retinal inflammation in streptozotocin-induced diabetic rats. The administration of ST was initiated at 4 weeks after diabetes induction and continued for 12 weeks. Retinal vessel permeability was evaluated by using FITC-dextran and Evans blue. Retinal leukostasis was evaluated with FITC-coupled concanavalin A lectin (ConA). Moreover, western blotting was performed to detect TNF-α, ICAM-1 and the relative expression levels of IκBα, IKKβ, and p65 in vivo. The results showed that the retinal inflammation in streptozotocin-induced diabetic rats was significantly decreased by ST. ST could decreased the expression levels of TNF-α, ICAM-1 and inhibited the expression of p-IKKβ, p-p65 and IκBα. It could also inhibited the nuclear transfer of p65. In conclusion, these data suggested that ST may have potential treatment strategies against early stage of diabetic retinopathy through NF-κB pathway.

STAT3 activation in circulating myeloid-derived cells contributes to retinal microvascular dysfunction in diabetes

BackgroundLeukostasis is a key patho-physiological event responsible for capillary occlusion in diabetic retinopathy. Circulating monocytes are the main cell type entrapped in retinal vessels in diabetes. In this study, we investigated the role of the signal transducer and activator of transcription 3 (STAT3) pathway in diabetes-induced immune cell activation and its contribution to retinal microvascular degeneration.MethodsForty-one patients with type 1 diabetes (T1D) [mild non-proliferative diabetic retinopathy (mNPDR) (n = 13), active proliferative DR (aPDR) (n = 14), inactive PDR (iPDR) (n = 14)] and 13 age- and gender-matched healthy controls were recruited to the study. C57BL/6 J WT mice, SOCS3fl/fl and LysMCre/+SOCS3fl/fl mice were rendered diabetic by Streptozotocin injection. The expression of the phosphorylated human and mouse STAT3 (pSTAT3), mouse LFA-1, CD62L, CD11b and MHC-II in circulating immune cells was evaluated by flow cytometry. The expression of suppressor of cytokine signalling 3 (SOCS3) was examined by real-time RT-PCR. Mouse plasma levels of cytokines were measured by Cytometric Beads Array assay. Retinal leukostasis was examined following FITC-Concanavalin A perfusion and acellular capillary was examined following Isolectin B4 and Collagen IV staining.ResultsCompared to healthy controls, the expression of pSTAT3 in circulating leukocytes was statistically significantly higher in mNPDR but not aPDR and was negatively correlated with diabetes duration. The expression of pSTAT3 and its inhibitor SOCS3 was also significantly increased in leukocytes from diabetic mice. Diabetic mice had higher plasma levels of IL6 and CCL2 compared with control mice. LysMCre/+SOCS3fl/fl mice and SOCS3fl/fl mice developed comparative levels of diabetes, but leukocyte activation, retinal leukostasis and number of acellular capillaries were statistically significantly increased in LysMCre/+SOCS3fl/fl diabetic mice.ConclusionSTAT3 activation in circulating immune cells appears to contribute to retinal microvascular degeneration and may be involved in DR initiation in T1D.

Fenofibrate-Loaded Biodegradable Nanoparticles for the Treatment of Experimental Diabetic Retinopathy and Neovascular Age-Related Macular Degeneration.

Fenofibrate is a peroxisome proliferator-activated receptor α (PPARα) agonist and has been shown to have therapeutic effects on diabetic retinopathy (DR). However, the effects of fenofibrate through systemic administration are not as potent as desired due to inefficient drug delivery to the retina. The present study aimed to explore the sustained therapeutic effects of fenofibrate-loaded biodegradable nanoparticles (NP) on both DR and neovascular age-related macular degeneration (AMD). Fenofibrate was successfully encapsulated into poly(lactic- co-glycolic acid) (PLGA) NP (Feno-NP), and Feno-NP were optimized by varying polymer composition to achieve high drug loading and prolonged drug release. The Feno-NP made of PLGA 34 kDa demonstrated a drug content of 6% w/w and a sustained drug release up to 60 days in vitro. Feno-NP (PLGA 34 kDa) was selected for following in vivo studies, and one single intravitreal (IVT) injection of Feno-NP into rat eyes with a 30G fine needle maintained sustained fenofibric acid drug level in the eye for more than 60 days. The efficacy of Feno-NP in DR and neovascular AMD was investigated using streptozotocin (STZ)-induced diabetic rats, laser-induced choroidal neovascularization (CNV) rats, and very low-density lipoprotein receptor knockout ( Vldlr -/-) mice. Therapeutic effects of Feno-NP were evaluated by measuring electroretinogram (ERG), retinal vascular leakage, leukostasis, CNV size, and retinal levels of vascular endothelial growth factor (VEGF) and intracellular adhesion molecule-1 (ICAM-1). In diabetic rats, Feno-NP ameliorated retinal dysfunctions, reduced retinal vascular leakage, inhibited retinal leukostasis, and downregulated the overexpression of VEGF and ICAM-1 at 8 weeks after one IVT injection. In addition, Feno-NP reduced retinal vascular leakage and CNV formation in both CNV rats and Vldlr -/- mice. Moreover, no toxicity of Feno-NP or Blank-NP to retinal structure and function was detected. Feno-NP exhibited good physiochemical characteristics and controlled drug release profile, conferring prolonged beneficial effects on DR and neovascular AMD.

Monosodium Urate Contributes to Retinal Inflammation and Progression of Diabetic Retinopathy.

We have investigated the contributing role of monosodium urate (MSU) to the pathological processes associated with the induction of diabetic retinopathy (DR). In human postmortem retinas and vitreous from donors with DR, we have found a significant increase in MSU levels that correlated with the presence of inflammatory markers and enhanced expression of xanthine oxidase. The same elevation in MSU levels was also detected in serum and vitreous of streptozotocin-induced diabetic rats (STZ-rats) analyzed at 8 weeks of hyperglycemia. Furthermore, treatments of STZ-rats with the hypouricemic drugs allopurinol (50 mg/kg) and benzbromarone (10 mg/kg) given every other day resulted in a significant decrease of retinal and plasma levels of inflammatory cytokines and adhesion factors, a marked reduction of hyperglycemia-induced retinal leukostasis, and restoration of retinal blood-barrier function. These results were associated with effects of the hypouricemic drugs on downregulating diabetes-induced levels of oxidative stress markers as well as expression of components of the NOD-like receptor family pyrin domain-containing protein 3 (NLRP3) inflammasome such as NLRP3, Toll-like receptor 4, and interleukin-1β. The outcomes of these studies support a contributing role of MSU in diabetes-induced retinal inflammation and suggest that asymptomatic hyperuricemia should be considered as a risk factor for DR induction and progression.

Fenofibrate for Diabetic Retinopathy.

Fenofibrate is a safe and inexpensive orally administered fibric acid derivative conventionally used to treat dyslipidemia. Two large randomized clinical trials, the Fenofibrate Intervention and Event Lowering in Diabetes (FIELD) and the Action to Control Cardiovascular Risk in Diabetes (ACCORD) studies, demonstrated the benefit of oral fenofibrate in the treatment of patients with type 2 diabetes and diabetic retinopathy (DR), including reduced disease progression and need for laser treatment for diabetic macular edema and proliferative diabetic retinopathy. These findings are supported by results of experimental studies, which have demonstrated beneficial effects of fenofibrate ameliorating retinal vascular leakage and leukostasis, downregulating vascular endothelial growth factor, and reducing endothelial cell and pericyte loss, among others; all of these are characteristic features of DR. In spite of this evidence, fenofibrate is not prescribed routinely for treating patients with diabetes and DR. In FIELD and ACCORD studies, retinopathy was not the primary outcome and this may explain, at least partly, its lack of use for this indication. New trials are now underway to specifically address the effects of fenofibrate in DR; these trials will provide additional and robust data that may support current evidence favoring the use of fenofibrate in this common microvascular complication of diabetes.

Fenofibrate ameliorates diabetic retinopathy by modulating Nrf2 signaling and NLRP3 inflammasome activation.

Oxidative stress and neuroinflammation contribute significantly to the development and progression of diabetic retinopathy. Fenofibrate has received great attention as it benefits diabetic patients by reducing retinal laser requirement. Nuclear factor erythroid-2-related factor 2 (Nrf2) is a master regulator of anti-oxidative defense. Activation of nucleotide binding domain, leucine-rich repeat-containing receptor (NLR), pyrin domain-containing 3 (NLRP3) inflammasome plays a pivotal role in neuroinflammation. The purpose of this study is to determine whether fenofibrate protects retinas from oxidative damage and neuroinflammation via modulating the Nrf2 pathway and blocking NLRP3 inflammasome activation during diabetes. Diabetes is induced by intraperitoneal injection of streptozotocin in mice. Fenofibrate was given to mice in rodent chow. Upregulation of Nrf2 and NLRP3 inflammasome, enhanced ROS formation, and increased leukostasis and vascular leakage were observed in diabetic mouse retinas. Notably, Nrf2 and Caspase-1 were mainly colocalized with glutamine synthetase, one of the Mȕller cell markers. Fenofibrate further increased the expression of Nrf2 and its target gene NQO-1 and HO-1 and reduced ROS formation in diabetic retinas. In addition, retinal expression of NLRP3, Caspase-1 p20, IL-1β p17, and ICAM-1 were dramatically increased in vehicle-treated diabetic mice, which were abolished by fenofibrate intervention. Moreover, fenofibrate treatment also attenuated diabetes-induced retinal leukostasis and vascular leakage in mice. Taken together, fenofibrate attenuates oxidative stress and neuroinflammation in diabetic retinas, which is at least partially through modulating Nrf2 expression and NLRP3 inflammasome activation.

Therapeutic Effects of PPARα Agonist on Ocular Neovascularization in Models Recapitulating Neovascular Age-Related Macular Degeneration.

PurposeThis study was designed to evaluate effects of fenofibric acid (Feno-FA), a peroxisome proliferator–activated receptor-alpha (PPARα) agonist, on ocular neovascularization (NV) in models recapitulating neovascular age-related macular degeneration (AMD), and to explore whether the effects are PPARα dependent.MethodsLaser-induced choroidal NV (CNV) in rats and very low-density lipoprotein receptor knockout (Vldlr−/−) mice received daily intraperitoneal injections of Feno-FA or vehicle. Vascular leakage was examined by fundus fluorescein angiography and permeability assay using Evans blue as tracer. In CNV rats, severity of CNV was evaluated by CNV areas and CNV volume. In Vldlr−/− mice, subretinal NV (SRNV) and intraretinal NV (IRNV) were quantified in choroid flat mount and retina flat mount, respectively. Inflammatory factors were measured using Western blotting and retinal leukostasis assay. Further, Pparα−/− mice and age-matched wild-type (WT) mice were used for laser-induced CNV and treated with Feno-FA to explore the underlying mechanism.ResultsFeno-FA significantly reduced vascular leakage in CNV rats and Vldlr−/− mice, reduced CNV volume in laser-induced CNV rats, and suppressed SRNV and IRNV in Vldlr−/− mice. In addition, Feno-FA downregulated the expression of inflammatory factors, including VEGF, TNF-α, and intercellular cell adhesion molecule-1 (ICAM-1), in the eyecups of CNV rats and decreased adherent retinal leukocytes in Vldlr−/− mice. Furthermore, Pparα−/− mice developed more severe CNV compared with WT mice, and PPARα knockout abolished the beneficial effects of Feno-FA on CNV.ConclusionsFeno-FA has therapeutic effects on ocular NV in models recapitulating neovascular AMD through a PPARα-dependent mechanism.

Targeting of 12/15-Lipoxygenase in retinal endothelial cells, but not in monocytes/macrophages, attenuates high glucose-induced retinal leukostasis

AimsOur previous studies have established a role for 12/15-lipoxygenase (LO) in mediating the inflammatory response in diabetic retinopathy (DR). However, the extent at which the local or systemic induction of 12/15-LO activity involved is unclear. Thus, the current study aimed to characterize the relative contribution of retinal endothelial versus monocytic/macrophagic 12/15-LO to inflammatory responses in DR. Materials & methodsWe first generated a clustered heat map for circulating bioactive lipid metabolites in the plasma of streptozotocin (STZ)-induced diabetic mice using liquid chromatography coupled with mass-spectrometry (LC–MS) to evaluate changes in circulating 12/15-LO activity. This was followed by comparing the in vitro mouse endothelium-leukocytes interaction between leukocytes isolated from 12/15-LO knockout (KO) versus those isolated from wild type (WT) mice using the myeloperoxidase (MPO) assay. Finally, we examined the effects of knocking down or inhibiting endothelial 12/15-LO on diabetes-induced endothelial cell activation and ICAM-1 expression. ResultsAnalysis of plasma bioactive lipids' heat map revealed that the activity of circulating 12/15-LO was not altered by diabetes as evident by no significant changes in the plasma levels of major metabolites derived from 12/15-lipoxygenation of different PUFAs, including linoleic acid (13-HODE), arachidonic acid (12- and 15- HETEs), eicosapentaenoic acid (12- and 15- HEPEs), or docosahexaenoic acid (17-HDoHE). Moreover, leukocytes from 12/15-LO KO mice displayed a similar increase in adhesion to high glucose (HG)-activated endothelial cells as do leukocytes from WT mice. Furthermore, abundant proteins of 12-LO and 15-LO were detected in human retinal endothelial cells (HRECs), while it was undetected (15-LO) or hardly detectable (12-LO) in human monocyte-like U937 cells. Inhibition or knock down of endothelial 12/15-LO in HRECs blocked HG-induced expression of ICAM-1, a well-known identified important molecule for leukocyte adhesion in DR. ConclusionOur data support that endothelial, rather than monocytic/macrophagic, 12/15-LO has a critical role in hyperglycemia-induced ICAM-1 expression, leukocyte adhesion, and subsequent local retinal barrier dysfunction. This may facilitate the development of more precisely targeted treatment strategies for DR.