Blood-retinal Barrier Research Articles

Retinal Cell Transplantation, Biomaterials, and In Vitro Models for Developing Next-generation Therapies of Age-related Macular Degeneration.

Retinal pigment epithelium (RPE) cells grown on a scaffold, an RPE patch, have potential to ameliorate visual impairment in a limited number of retinal degenerative conditions. This tissue-replacement therapy is suited for age-related macular degeneration (AMD), and related diseases. RPE cells must be transplanted before the disease reaches a point of no return, represented by the loss of photoreceptors. Photoreceptors are specialized, terminally differentiated neurosensory cells that must interact with RPE’s apical processes to be functional. Human photoreceptors are not known to regenerate. On the RPE’s basal side, the RPE transplant must induce the reformation of the choriocapillaris, thereby re-establishing the outer blood-retinal barrier. Because the scaffold is positioned between the RPE and choriocapillaris, it should ideally degrade and be replaced by the natural extracellular matrix that separates these tissues. Besides biodegradable, the scaffolds need to be nontoxic, thin enough to not affect the focal length of the eye, strong enough to survive the transplant procedure, yet flexible enough to conform to the curvature of the retina. The challenge is patients with progressing AMD treasure their remaining vision and fear that a risky surgical procedure will further degrade their vision. Accordingly, clinical trials only treat eyes with severe impairment that have few photoreceptors to interact with the transplanted patch. Although safety has been demonstrated, the cell-replacement mechanism and efficacy remain difficult to validate. This review covers the structure of the retina, the pathology of AMD, the limitations of cell therapy approaches, and the recent progress in developing retinal therapies using biomaterials.

Translational and clinical advancements in management of proliferative vitreoretinopathy.

Despite advancement in the surgical instrumentation and techniques, proliferative vitreoretinopathy (PVR) remains the most common cause for failure of rhegmatogenous retinal detachment (RRD) repair. This review discusses ongoing translational and clinical advancements in PVR. PVR represents an exaggerated and protracted scarring process that can occur after RRD. The primary cell types involved are retinal pigment epithelium, glial, and inflammatory cells. They interact with growth factors and cytokines derived from the breakdown of the blood-retinal barrier that trigger a cascade of cellular processes, such as epithelial-mesenchymal transition, cell migration, chemotaxis, proliferation, elaboration of basement membrane and collagen and cellular contraction, leading to overt retinal pathology. Although there are currently no medical therapies proven to be effective against PVR in humans, increased understanding of the risks factors and pathophysiology have helped guide investigations for molecular targets of PVR. The leading therapeutic candidates are drugs that mitigate growth factors, inflammation, and proliferation are the leading therapeutic candidates. Although multiple molecular targets have been investigated to prevent and treat PVR, none have yet demonstrated substantial evidence of clinical benefit in humans though some show promise. Advancements in our understanding of the pathophysiology of PVR may help develop a multipronged approach for this condition.

C1q/TNF-Related Protein 3 Prevents Diabetic Retinopathy via AMPK-Dependent Stabilization of Blood-Retinal Barrier Tight Junctions.

Background The impairment of the inner blood–retinal barrier (iBRB) increases the pathological development of diabetic retinopathy (DR), a severe complication in diabetic patients. Identifying approaches to preserving iBRB integrity and function is a significant challenge in DR. C1q/tumor necrosis factor-related protein-3 (CTRP3) is a newly discovered adipokine and a vital biomarker, predicting DR severity. We sought to determine whether and how CTRP3 affects the pathological development of non-proliferative diabetic retinopathy (NPDR). Methods To clarify the pathophysiologic progress of the blood–retinal barrier in NPDR and explore its potential mechanism, a mouse Type 2 diabetic model of diabetic retinopathy was used. The capillary leakage was assessed by confocal microscope with fluorescent-labeled protein in vivo. Furthermore, the effect of CTRP3 on the inner blood–retinal barrier (iBRB) and its molecular mechanism was clarified. Results The results demonstrated that CTRP3 protects iBRB integrity and resists the vascular permeability induced by DR. Mechanistically, the administration of CTRP3 activates the AMPK signaling pathway and enhances the expression of Occludin and Claudin-5 (tight junction protein) in vivo and in vitro. Meanwhile, CTRP3 improves the injury of human retinal endothelial cells (HRMECs) induced by high glucose/high lipids (HG/HL), and its protective effects are AMPK-dependent. Conclusions In summary, we report, for the first time, that CTRP3 prevents diabetes-induced retinal vascular permeability via stabilizing the tight junctions of the iBRB and through the AMPK-dependent Occludin/Claudin-5 signaling pathway, thus critically affecting the development of NPDR.

Permeability, anti-inflammatory and anti-VEGF profiles of steroidal-loaded cationic nanoemulsions in retinal pigment epithelial cells under oxidative stress

Age-related macular degeneration (AMD) is defined as a degenerative, progressive and multifactorial disorder that affects the macula with a complex etiology. The retinal pigment epithelium is a monolayer of cells that has the function to separate the surface of the choroid from the neural retina that is involved in the signal transduction leading to vision. The blood-aqueous barrier and the blood retinal barrier limit the permeation of drugs into the retina and thereby reducing their efficacy. Triamcinolone acetonide (TA) is widely used as anti-inflammatory and immunomodulatory drug that promotes the inhibition of the inflammatory processes. The factors that stimulate or inhibit angiogenesis in AMD create a local balance that is responsible for the growth of sub-retinal neovascularization. In AMD, the main angiogenic stimulus is the vascular endothelial growth factor (VEGF). In this work, nanoemulsions with cationic surfactants (mono- and dicationic DABCO and quinuclidine) were produced to deliver TA, and were found to reduce the production of tumor necrosis factor alpha (TNF-α), which stimulates the choroidal neovascularization development by upregulating the VEGF production, and consequently decreased the VEGF levels. Our results support the potential use of mono- and dicationic DABCO and quinuclidine-based cationic nanoemulsions for the delivery of TA in the treatment of AMD.

Yiqi Tongluo Fang could preventive and delayed development and formation of diabetic retinopathy through antioxidant and anti-inflammatory effects

BackgroundYiqi Tongluo Fang (YQTLF) is an effective prescription for the treatment of diabetic retinopathy (DR), but its mechanism of action remains unclear. MethodThe content of YQTLF was determined using liquid and gas chromatography-mass spectrometry (LC-MS and GC-MS, respectively). Twenty-five Sprague Dawley (SD) rats were randomly selected as the normal control group. One hundred SD streptozotocin-induced diabetes (type 1) rats were randomly divided into diabetic control, diabetic+insulin+ calcium dobesilate (CaD), and diabetic+insulin+ YQTLF groups, with 25 rats in each group. Bodyweight level was measured every 2 weeks. After 12 weeks of gavage, the glucose levels, lipids, oxidative stress, inflammation, retinal histopathology, and the blood-retinal barrier were assessed in each group. The p38 MAPK pathway was changed to explore its internal mechanism. The measurement data were expressed as mean ± standard deviation, and different statistical methods were used according to a normal distribution, square error, or not. ResultsA total of 1024 valid peaks were identified in YQTLF using GC-MS. YQTLF significantly lowered the fasting blood glucose levels in diabetic rats. YQTLF early inhibited changes in retinal histology, capillaries, cells, and tight junction proteins (such as ZO-1, occludin, claudin-5, and VE-cadherin) before the formation and development of DR. These findings correlated with the alleviation of glucolipid metabolism, inflammation, and oxidative stress. The lncRNA MALAT1 and the PRC 2/p38 MAPK-related pathway, such as the expression of EZH2, SUZ12, EED, p38 MAPK, MMP-9, and VEGFR, were also correlated. ConclusionWe have demonstrated the molecular and cellular mechanisms underlying the preventive and delayed development and formation of DR. YQTLF prevents changes in dyslipidemia, retinal histology, capillaries, cells, and tight junction proteins. These protective effects appear to be linked to its antioxidant and anti-inflammatory effects, which prevent the activation of intracellular signaling pathways, such as the lncRNA MALAT1 and PRC 2/p38 MAPK-related pathway.

A Splicing Mutation in Slc4a5 Results in Retinal Detachment and Retinal Pigment Epithelium Dysfunction.

Fluid and solute transporters of the retinal pigment epithelium (RPE) are core components of the outer blood–retinal barrier. Characterizing these transporters and their role in retinal homeostasis may provide insights into ocular function and disease. Here, we describe RPE defects in tvrm77 mice, which exhibit hypopigmented patches in the central retina. Mapping and nucleotide sequencing of tvrm77 mice revealed a disrupted 5’ splice donor sequence in Slc4a5, a sodium bicarbonate cotransporter gene. Slc4a5 expression was reduced 19.7-fold in tvrm77 RPE relative to controls, and alternative splice variants were detected. SLC4A5 was localized to the Golgi apparatus of cultured human RPE cells and in apical and basal membranes. Fundus imaging, optical coherence tomography, microscopy, and electroretinography (ERG) of tvrm77 mice revealed retinal detachment, hypopigmented patches corresponding to neovascular lesions, and retinal folds. Detachment worsened and outer nuclear layer thickness decreased with age. ERG a- and b-wave response amplitudes were initially normal but declined in older mice. The direct current ERG fast oscillation and light peak were reduced in amplitude at all ages, whereas other RPE-associated responses were unaffected. These results link a new Slc4a5 mutation to subretinal fluid accumulation and altered light-evoked RPE electrophysiological responses, suggesting that SLC4A5 functions at the outer blood–retinal barrier.

Understanding Neurodegeneration from a Clinical and Therapeutic Perspective in Early Diabetic Retinopathy.

Recent evidence indicates that neurodegeneration is a critical element of diabetic retinopathy (DR) pathogenesis. The neuronal cells’ apoptosis contributes to microvascular impairment and blood–retinal barrier breakdown. Therefore, neurodegeneration represents an early intervention target to slow and prevent the development of microvascular alterations visible on clinical examination. Multimodal imaging features and functional assessment can permit the identification of neuronal damage in a subclinical stage before the recognition of DR signs. Clinical features of neurodegeneration are crucial in identifying patients at high risk of developing a vascular impairment and, thus, serve as outcome measures to understand the efficacy of supplementation. The optimal approach for targeting neurodegeneration contemplates the use of topical compounds that possibly act on different elements of the pathogenic cascade. To date, clinical trials available on humans tested three different topical agents, including brimonidine, somatostatin, and citicoline, with promising results.

Ex Vivo and In Vitro Analysis Identify a Detrimental Impact of Neutrophil Extracellular Traps on Eye Structures in Equine Recurrent Uveitis.

Equine recurrent uveitis (ERU) is a common ocular disease of horses and described as a model for human autoimmune uveitis. This immune-mediated, inflammatory condition progressively destroys the eye, ultimately leading to blindness. Genetic and autoimmune factors, next to infections with Leptospira, are discussed as key factors in the pathogenesis. Furthermore, a release of neutrophil extracellular traps (NETs) by activated neutrophils is involved. NETs are composed of decondensed chromatin and proteins that can immobilize invading pathogens. However, if NETs accumulate, they can contribute to detrimental autoimmune processes. Thus, we aimed to investigate the impact of NETs in ERU patients. Therefore, we quantified several NET-markers (cell-free DNA, nucleosomes, citrullinated histone H3, histone-myeloperoxidase complexes, interleukin-17, equine cathelicidin 1 and DNase I activity) and NET-autoantibodies in sera and vitreous body fluids (VBF) of ERU-diseased horses and correlated the data with the disease status (signalment, ERU scores and Leptospira infection status). NET markers were detected to varying degrees in VBF of diseased horses, and partially correlated to disease severity and the presence of Leptospira spp. Cell-free DNA and nucleosomes as NET markers correlate with ERU severity in total and VBF scores, despite the presence of active DNases. Additionally, a significant correlation between fundus affection in the eye and NET autoantibodies was detectable. Therefore, we further investigated the influence of VBF samples from equine patients and isolated NETs on the blood-retina barrier in a cell culture model. VBF of diseased horses significantly induced cytotoxicity in retinal pigment epithelial cells. Moreover, partially digested NETs also resulted in cytotoxic effects. In the presence of lipopolysaccharide (LPS), the main component of the leptospiral surface, both undigested and completely digested NETs were cytotoxic. Correlations between the ERU-scores and Leptospira were also calculated. Detection of leptospiral DNA, and antibody titers of the serovar Grippotyphosa correlated with disease severity. In addition, a correlation between Leptospira and several NET markers was observed in VBF. Altogether, our findings suggest a positive correlation between NET markers with disease severity and involvement of Leptospira in the VBF of ERU-diseased horses, as well as a cytotoxic effect of NETs in eyes.

Chlorogenic acid improves diabetic retinopathy by alleviating blood-retinal-barrier dysfunction via inducing Nrf2 activation.

As one of the major diabetic microvascular complications, diabetic retinopathy (DR) is mainly initiated by the blood-retinal barrier (BRB) dysfunction. Chlorogenic acid (CGA) is a natural polyphenolic compound in Lonicerae Japonicae Flos, which traditionally has the beneficial function for eyes and is commonly included in many anti-diabetic formulas. In this study, the potential protective mechanism of CGA against DR was investigated. Streptozotocin (STZ) was used to induce diabetes in mice. CGA attenuated BRB dysfunction and reversed endothelial-mesenchymal transition (EndoMT) and epithelial-mesenchymal transition (EMT) in retinas in vivo. CGA inhibited microglia activation and reduced tumor necrosis factor (TNF)α release both in vivo and in vitro. CGA promoted nuclear factor erythroid 2-related factor 2 (Nrf2) activation and prevented EndoMT/EMT in TNFα-treated human retinal endothelial cells (HRECs) or retinal pigment epithelial APRE19 cells. CGA alleviated endothelial/epithelial barrier oxidative injury in HRECs or APRE19 cells stimulated with TNFα, but this effect was disappeared in cells co-incubated with Nrf2 inhibitor. Additionally, the CGA-supplied alleviation on BRB damage and EndoMT/EMT was markedly weakened in retinas from STZ-treated Nrf2 knock-out mice. All results suggest that CGA improves DR through attenuating BRB injury by reducing microglia-initiated inflammation and preventing TNFα-induced EndoMT/EMT and oxidative injury via inducing Nrf2 activation.

Structure and function of the retina of low-density lipoprotein receptor-related protein 5 (Lrp5)-deficient rats

Loss-of-function mutations in the Wnt co-receptor, low-density lipoprotein receptor-related protein 5 (LRP5), result in familial exudative vitreoretinopathy (FEVR), osteoporosis-pseudoglioma syndrome (OPPG), and Norrie disease. CRISPR/Cas9 gene editing was used to produce rat strains deficient in Lrp5. The purpose of this study was to validate this rat model for studies of hypovascular, exudative retinopathies. The retinal vasculature of wildtype and Lrp5 knockout rats was stained with Giffonia simplifolia isolectin B4 and imaged by fluorescence microscopy. Effects on retinal structure were investigated by histology. The integrity of the blood-retina barrier was analyzed by measurement of permeability to Evans blue dye and staining for claudin-5. Retinas were imaged by fundus photography and SD-OCT, and electroretinograms were recorded. Lrp5 gene deletion led to sparse superficial retinal capillaries and loss of the deep and intermediate plexuses. Autofluorescent exudates were observed and are correlated with increased Evans blue permeability and absence of claudin-5 expression in superficial vessels. OCT images show pathology similar to OCT of humans with FEVR, and retinal thickness is reduced by 50% compared to wild-type rats. Histology and OCT reveal that photoreceptor and outer plexiform layers are absent. The retina failed to demonstrate an ERG response. CRISPR/Cas9 gene-editing produced a predictable rat Lrp5 knockout model with extensive defects in the retinal vascular and neural structure and function. This rat model should be useful for studies of exudative retinal vascular diseases involving the Wnt and norrin pathways.

Effects of Excess Iron on the Retina: Insights From Clinical Cases and Animal Models of Iron Disorders.

Iron plays an important role in a wide range of metabolic pathways that are important for neuronal health. Excessive levels of iron, however, can promote toxicity and cell death. An example of an iron overload disorder is hemochromatosis (HH) which is a genetic disorder of iron metabolism in which the body’s ability to regulate iron absorption is altered, resulting in iron build-up and injury in several organs. The retina was traditionally assumed to be protected from high levels of systemic iron overload by the blood-retina barrier. However, recent data shows that expression of genes that are associated with HH can disrupt retinal iron metabolism. Thus, the effects of iron overload on the retina have become an area of research interest, as excessively high levels of iron are implicated in several retinal disorders, most notably age–related macular degeneration. This review is an effort to highlight risk factors for excessive levels of systemic iron build-up in the retina and its potential impact on the eye health. Information is integrated across clinical and preclinical animal studies to provide insights into the effects of systemic iron loading on the retina.

Na+-coupled lipid release by MFSD2A transporter

Major facilitator superfamily domain containing 2A (MFSD2A) is a membrane protein that is highly enriched within endothelial cells of the blood-brain and blood-retinal barriers where it mediates Na+-dependent uptake of the ω-3 fatty acid docosahexaenoic acid (DHA) in the form of lysophosphatidylcholine (LPC-DHA) into the brain and eyes, respectively. MFSD2A shares the 12 transmembrane domain architecture with other MFS family members. However, the molecular details of the transport process remain unknown, because unlike most MFS proteins, MFSD2A transports lysolipids rather than water-soluble molecules, suggesting that molecular mechanism of MFSD2A-mediated transport can be different and more akin to that of flippases.

Features of Local Expression of Genes of Immune Response Cytokines and Trophic and Vasoregulatory Factors in Modeling of Retinal Pigment Epithelium Atrophy.

Local expression of genes encoding IL-1β, IL-18, MCP-1/CCL2, PEDF, VEGF-A, and ZO-1 in the retina-retinal pigment epithelium-chorioidea tissue complex was studied in healthy rabbits and animals with simulated retinal pigment epithelium atrophy. Retinal pigment epithelium atrophy was modeled by single subretinal injection of 0.01 ml 0.9% NaCl (group 1; n=17) or 0.01 ml solution containing angiogenesis inhibitor bevacizumab in a dose of 0.025 mg (group 2; n=18). The gene expression was evaluated by reverse transcription PCR. In 27.7% cases, atrophic changes in the fundus were accompanied by a significant increase of IL-1β gene expression and in more than 50% cases by an increase in VEGF-A and MCP-1/CCL2 mRNA levels. These factors contribute to an increase in the permeability of the blood-retina barrier and abolition of the immune privilege of the posterior eye segment, which should be taken into account when testing invasive approaches, in particular, for approbation of various options of replacement therapy with retinal pigment epithelium stem cells and development and use of neuroprotectors and drugs of targeted action.

Freshly isolated retinal capillaries to determine efflux transporter function at the inner BRB

Since it has been known that in vitro cell lines for analyzing drug transport at the inner blood-retinal barrier (BRB) do not completely retain several in vivo functions, new ex vivo/in vitro methods to evaluate drug transport across the inner BRB help us understand the role of this barrier in maintaining the homeostasis of vision and regulating drug distribution to the retina. To expand the limitations of existing in vitro approaches, we established a protocol to isolate fresh rat retinal capillaries as ex vivo model of the inner BRB. Fresh retinal capillaries were prepared by applying serial filtration steps and using density gradient centrifugation. We performed mRNA and protein analyses by reverse transcription-polymerase chain reaction and immunostaining that indicated expression of marker proteins such as facilitative glucose transporter 1 and claudin-5 in freshly isolated rat retinal capillaries. We also used fluorescent transporter substrates to characterize functional activity of organic anion transporter (Oat) 3, P-glycoprotein (P-gp), breast cancer resistance protein (Bcrp), and multidrug resistance-associated protein (Mrp) 4 in isolated retinal capillaries. Capillary luminal accumulation of fluorescent substrates of P-glycoprotein and Bcrp was decreased in the presence of transporter inhibitors. Moreover, luminal accumulation of the Oat3 and Mrp4 substrate, 8-(2-[fluoresceinyl]aminoethylthio) adenosine-3′,5′-cyclic monophosphate (8-[fluo]-cAMP), was reduced by substrates/inhibitors of Oat3 and Mrp4. In conclusion, our study shows that freshly isolated retinal capillaries retain marker protein expression and transporter functional activity. It is suggested that isolated retinal capillaries are a useful tool to study transport across the inner BRB. Using freshly isolated retinal capillaries, we anticipate applying this approach to determine the role of transporters at the inner BRB during pathophysiological states of the eye and evaluate the drug delivery to the retina.

Drug Delivery Challenges and Current Progress in Nanocarrier-Based Ocular Therapeutic System.

Drug instillation via a topical route is preferred since it is desirable and convenient due to the noninvasive and easy drug access to different segments of the eye for the treatment of ocular ailments. The low dose, rapid onset of action, low or no toxicity to the local tissues, and constrained systemic outreach are more prevalent in this route. The majority of ophthalmic preparations in the market are available as conventional eye drops, which rendered <5% of a drug instilled in the eye. The poor drug availability in ocular tissue may be attributed to the physiological barriers associated with the cornea, conjunctiva, lachrymal drainage, tear turnover, blood–retinal barrier, enzymatic drug degradation, and reflex action, thus impeding deeper drug penetration in the ocular cavity, including the posterior segment. The static barriers in the eye are composed of the sclera, cornea, retina, and blood–retinal barrier, whereas the dynamic barriers, referred to as the conjunctival and choroidal blood flow, tear dilution, and lymphatic clearance, critically impact the bioavailability of drugs. To circumvent such barriers, the rational design of the ocular therapeutic system indeed required enriching the drug holding time and the deeper permeation of the drug, which overall improve the bioavailability of the drug in the ocular tissue. This review provides a brief insight into the structural components of the eye as well as the therapeutic challenges and current developments in the arena of the ocular therapeutic system, based on novel drug delivery systems such as nanomicelles, nanoparticles (NPs), nanosuspensions, liposomes, in situ gel, dendrimers, contact lenses, implants, and microneedles. These nanotechnology platforms generously evolved to overwhelm the troubles associated with the physiological barriers in the ocular route. The controlled-drug-formulation-based strategic approach has considerable potential to enrich drug concentration in a specific area of the eye.

Microphysiological Neurovascular Barriers to Model the Inner Retinal Microvasculature.

Blood-neural barriers regulate nutrient supply to neuronal tissues and prevent neurotoxicity. In particular, the inner blood-retinal barrier (iBRB) and blood–brain barrier (BBB) share common origins in development, and similar morphology and function in adult tissue, while barrier breakdown and leakage of neurotoxic molecules can be accompanied by neurodegeneration. Therefore, pre-clinical research requires human in vitro models that elucidate pathophysiological mechanisms and support drug discovery, to add to animal in vivo modeling that poorly predict patient responses. Advanced cellular models such as microphysiological systems (MPS) recapitulate tissue organization and function in many organ-specific contexts, providing physiological relevance, potential for customization to different population groups, and scalability for drug screening purposes. While human-based MPS have been developed for tissues such as lung, gut, brain and tumors, few comprehensive models exist for ocular tissues and iBRB modeling. Recent BBB in vitro models using human cells of the neurovascular unit (NVU) showed physiological morphology and permeability values, and reproduced brain neurological disorder phenotypes that could be applicable to modeling the iBRB. Here, we describe similarities between iBRB and BBB properties, compare existing neurovascular barrier models, propose leverage of MPS-based strategies to develop new iBRB models, and explore potentials to personalize cellular inputs and improve pre-clinical testing.

Astrocytic p75NTR expression provoked by ischemic stroke exacerbates the blood-brain barrier disruption.

The disruption of the blood-brain barrier (BBB) plays a critical role in the pathology of ischemic stroke. p75 neurotrophin receptor (p75NTR ) contributes to the disruption of the blood-retinal barrier in retinal ischemia. However, whether p75NTR influences the BBB permeability after acute cerebral ischemia remains unknown. The present study investigated the role and underlying mechanism of p75NTR on BBB integrity in an ischemic stroke mouse model, middle cerebral artery occlusion (MCAO). After 24 h of MCAO, astrocytes and endothelial cells in the infarct-affected brain area up-regulated p75NTR . Genetic p75NTR knockdown (p75NTR+/- ) or pharmacological inhibition of p75NTR using LM11A-31, a selective inhibitor of p75NTR , both attenuated brain damage and BBB leakage in MCAO mice. Astrocyte-specific conditional knockdown of p75NTR mediated with an adeno-associated virus significantly ameliorated BBB disruption and brain tissue damage, as well as the neurological functions after stroke. Further molecular biological examinations indicated that astrocytic p75NTR activated NF-κB and HIF-1α signals, which upregulated the expression of MMP-9 and vascular endothelial growth factor (VEGF), subsequently leading to tight junction degradation after ischemia. As a result, increased leukocyte infiltration and microglia activation exacerbated brain injury after stroke. Overall, our results provide novel insight into the role of astrocytic p75NTR in BBB disruption after acute cerebral ischemia. The p75NTR may therefore be a potential therapeutic target for the treatment of ischemic stroke.

Endostatin Inhibits Blood-Retinal Barrier Breakdown in Diabetic Rats by Increasing the Expression of ICAM-1 and VCAM-1 and Decreasing the Expression of VEGF.

Endostatin has become the strongest endogenous angiogenesis inhibitor due to suppressing VEGF expression. The purpose of this study was to assess the impact of endostatin on the blood-retinal barrier (BRB) in diabetic rats. SD rats were induced to develop diabetes by streptozotocin, and endostatin was administrated by intravitreal injection. The body weight, the level of blood glucose, the expressions of C-reactive protein (CRP), adhesion molecules intercellular cell adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), junction proteins (occludin, claudin-5, and zonula occluden-1), and VEGF were measured in rats' retinas of diabetes. The BRB breakdown was evaluated using Evans blue. The level of CRP and adhesion molecules (ICAM-1 and VCAM-1) was increased in retinas of diabetic rats, while endostatin significantly inhibited the upregulation of these. Diabetes increased the BRB permeability and retinal thickness. Diabetes also decreased the levels of occludin, claudin-5, and ZO-1 in retinals. These changes were inhibited by endostatin treatment. Upregulation of vascular endothelial growth factor (VEGF), transforming growth factor-β (TGF-β), and protein kinase C- (PKC-) β2 was also reversed by endostatin in retinas of diabetic rats. Endostatin provides protection against diabetic retinopathy, which may involve its barrier-enhancing effects.

Tissue Inhibitor of Metalloproteinase-3 Ameliorates Diabetes-Induced Retinal Inflammation.

Purpose: Endogenous tissue inhibitor of matrix metalloproteinase-3 (TIMP-3) has powerful regulatory effects on inflammation and angiogenesis. In this study, we investigated the role of TIMP-3 in regulating inflammation in the diabetic retina.Methods: Vitreous samples from patients with proliferative diabetic retinopathy (PDR) and non-diabetic patients were subjected to Western blot analysis. Streptozotocin-treated rats were used as a preclinical diabetic retinopathy (DR) model. Blood-retinal barrier (BRB) breakdown was assessed with fluorescein isothiocyanate (FITC)-conjugated dextran. Rat retinas, human retinal microvascular endothelial cells (HRMECs) and human retinal Müller glial cells were studied by Western blot analysis and ELISA. Adherence of human monocytes to HRMECs was assessed and in vitro angiogenesis assays were performed.Results: Tissue inhibitor of matrix metalloproteinase-3 in vitreous samples was largely glycosylated. Intravitreal injection of TIMP-3 attenuated diabetes-induced BRB breakdown. This effect was associated with downregulation of diabetes-induced upregulation of the p65 subunit of NF-κB, intercellular adhesion molecule-1 (ICAM-1), and vascular endothelial growth factor (VEGF), whereas phospho-ERK1/2 levels were not altered. In Müller cell cultures, TIMP-3 significantly attenuated VEGF upregulation induced by high-glucose (HG), the hypoxia mimetic agent cobalt chloride (CoCl2) and TNF-α and attenuated MCP-1 upregulation induced by CoCl2 and TNF-α, but not by HG. TIMP-3 attenuated HG-induced upregulation of phospho-ERK1/2, caspase-3 and the mature form of ADAM17, but not the levels of the p65 subunit of NF-κB and the proform of ADAM17 in Müller cells. TIMP-3 significantly downregulated TNF-α-induced upregulation of ICAM-1 and VCAM-1 in HRMECs. Accordingly, TIMP-3 significantly decreased spontaneous and TNF-α- and VEGF-induced adherence of monocytes to HRMECs. Finally, TIMP-3 significantly attenuated VEGF-induced migration, chemotaxis and proliferation of HRMECs.Conclusion: In vitro and in vivo data point to anti-inflammatory and anti-angiogenic effects of TIMP-3 and support further studies for its applications in the treatment of DR.

The Long Pentraxin PTX3 as a New Biomarker and Pharmacological Target in Age-Related Macular Degeneration and Diabetic Retinopathy.

Age related macular degeneration (AMD) and diabetic retinopathy (DR) are multifactorial, neurodegenerative and inflammatory diseases of the eye primarily involving cellular and molecular components of the outer and inner blood-retina barriers (BRB), respectively. Largely contributed by genetic factors, particularly polymorphisms in complement genes, AMD is a paradigm of retinal immune dysregulation. DR, a major complication of diabetes mellitus, typically presents with increased vascular permeability and occlusion of the retinal vasculature that leads, in the proliferative form of the disease, to neovascularization, a pathogenic trait shared with advanced AMD. In spite of distinct etiology and clinical manifestations, both pathologies share common drivers, such as chronic inflammation, either of immune (in AMD) or metabolic (in DR) origin, which initiates and propagates degeneration of the neural retina, yet the underlying mechanisms are still unclear. As a soluble pattern recognition molecule with complement regulatory functions and a marker of vascular damage, long pentraxin 3 (PTX3) is emerging as a novel player in ocular homeostasis and a potential pharmacological target in neurodegenerative disorders of the retina. Physiologically present in the human eye and induced in inflammatory conditions, this protein is strategically positioned at the BRB interface, where it acts as a “molecular trap” for complement, and modulates inflammation both in homeostatic and pathological conditions. Here, we discuss current viewpoints on PTX3 and retinal diseases, with a focus on AMD and DR, the roles therein proposed for this pentraxin, and their implications for the development of new therapeutic strategies.