A novel model of subretinal edema induced by DL-alpha aminoadipic acid.

In this study we described a new model of subretinal edema induced by single intraocular injection of DL-alpha-aminoadipic acid (DLAAA) that can be employed to study the mechanism of retinal edema and test the efficacy or potential toxicity of treatments. The progression of subretinal edema was evaluated by fundus photography, fluorescein angiography and optical coherence tomography for up to 4 weeks following DLAAA injection. The VEGF, IL-6, TNF-α, Occludin, ZO-1, AQP4, Kir4.1, GFAP and GS levels were examined in DLAAA models by immunostaining, immumohistochemical staining and Western blot. Additionally, bulk RNA-seq was used to detect the mechanism involved in DLAAA-induced retinal Müller cellular injuries. In vivo and vitro assays were further conducted to confirm the sequencing results. Subretinal edema was successfully induced by DLAAA in New Zealand White rabbits (1.29 mg/eye) and C57BL/6 mice (50 or 100 µg/eye). Our results demonstrated that the disruption of blood-retinal-barrier, including vascular hyperpermeability, inflammation, and Müller cell dysfunction of fluid clearance, was involved in subretinal edema formation in the model. Bulk RNA-seq and in vitro studies indicated the activation of p38 MAPK signaling pathway in DLAAA models. This DLAAA-induced subretinal edema model can be used for mechanistic studies or drug screening.

The Blood-ocular Barriers and their Dysfunction: Anatomy, Physiology, Pathology. / Die Blut-Augen-Schranken und ihre Störungen: Anatomie, Physiologie, Pathologie.

Complex barriers comprise the blood-aqueous (BAB) and the blood-retinal barrier (BRB), and separate anterior and posterior eye chambers, vitreous body, and sensory retina from the circulation. They prevent pathogens and toxins from entering the eye, control movement of fluid, proteins, and metabolites, and contribute to the maintenance of the ocular immune status. Morphological correlates of blood-ocular barriers are tight junctions between neighboring endothelial and epithelial cells, which function as gatekeepers of the paracellular transport of molecules, thereby limiting their uncontrolled access to ocular chambers and tissues. The BAB is composed of tight junctions between endothelial cells of the iris vasculature, endothelial cells of Schlemm's canal inner wall, and cells of the nonpigmented ciliary epithelium. The BRB consists of tight junctions between endothelial cells of the retinal vessels (inner BRB) and epithelial cells of the retinal pigment epithelium (outer BRB). These junctional complexes respond rapidly to pathophysiological changes, thus enabling vascular leakage of blood-derived molecules and inflammatory cells into ocular tissues and chambers. Blood-ocular barrier function, which can be clinically measured by laser flare photometry or fluorophotometry, is compromised in traumatic, inflammatory, or infectious processes, but also frequently contributes to the pathophysiology of chronic diseases of the anterior eye segment and the retina, as exemplified by diabetic retinopathy and age-related macular degeneration.

Microglia increase tight-junction permeability in coordination with Müller cells under hypoxic condition in an in vitro model of inner blood-retinal barrier.

Microglia and Müller cells (MCs) are believed to be critically involved in hypoxia-induced blood-retinal barrier (BRB) disruption, which is a major pathogenic factor of various retinopathies. However, the underlying mechanism remains poorly defined. The inner BRB (iBRB) is primarily formed of microvascular endothelial cells (ECs) with tight junction (TJ), which are surrounded and supported by retinal glial cells. We developed a novel in vitro iBRB model sheet by sandwiching Transwell membrane with layered mouse brain microvascular ECs (bEnd.3) and mouse retinal MCs (QMMuC-1) on each side of the membrane. Using this model, we tested the hypothesis that under hypoxic condition, activated microglia produce inflammatory cytokines such as interleukin (IL)-1ß, which may promote vascular endothelial growth factor (VEGF) production from MCs, leading to TJ disruption. The iBRB model cell sheets were exposed to 1% oxygen for 6 h with or without mouse brain microglia (BV2) or IL-1ß. TJ structure and function were examined by zonula occludens (ZO)-1 immunostaining and fluorescein isothiocyanate permeability assay, respectively. Relative gene expression of IL-1ß in BV2 under normoxic and hypoxic conditions was examined by real-time reverse transcription-polymerase chain reaction. VEGF protein concentration in QMMuC-1 supernatants was measured by enzyme-linked immunosorbent assay. The bEnd.3 cell sheet incubated with BV2 in hypoxic condition or with IL-1ß in normoxic condition showed abnormal localization of ZO-1 and aberrated barrier function. Under normoxic condition, EC-MC iBRB model cell sheet showed lower permeability than bEnd.3 cell sheet. Under hypoxic conditions, the barrier function of EC-MC iBRB model cell sheet was more deteriorated compared to bEnd.3 cell sheet. Under hypoxic condition, incubation of EC-MC iBRB model cell sheet with BV2 cells or IL-1ß significantly increased barrier permeability, and hypoxia-treated BV2 cells expressed significantly higher levels of IL-1ß mRNA. Incubation of QMMuC-1 with IL-1ß increased VEGF production. These results suggest that under hypoxic condition, microglia are activated to release proinflammatory cytokines such as IL-1ß that promote VEGF production from MCs, leading to disruption of iBRB function. Modulating microglia and MCs function may be a novel approach to treat hypoxia-induced retinal BRB dysfunction.

TIM2 modulates retinal iron levels and is involved in blood-retinal barrier breakdown.

Careful control of iron availability in the retina is central to maintenance of iron homeostasis, as its imbalance is associated with oxidative stress and the progression of several retinopathies. Ferritin, known for its role in iron storage and detoxification, has also been proposed as an iron-transporter protein, through its binding to Scara5 and TIM2 membrane receptors. In this study, the presence and iron-related functions of TIM2 in the mouse retina were investigated. Our results revealed for the first time the presence of TIM2 receptors in the mouse retina, mainly in Müller cells. Experimental TIM2 downregulation in the mouse retina promoted, probably due to a compensatory mechanism, Scara5 overexpression that increased retinal ferritin uptake and induced iron overload. Consecutive reactive oxygen species (ROS) overproduction and vascular endothelial growth factor (VEGF) overexpression led to impaired paracellular and transcellular endothelial transport characterized by tight junction degradation and increased caveolae number. In consequence, blood-retinal barrier (BRB) breakdown and retinal edema were observed. Altogether, these results point to TIM2 as a new modulator of retinal iron homeostasis and as a potential target to counteract retinopathy.

Interplay of the Norrin and Wnt7a/Wnt7b signaling systems in blood-brain barrier and blood-retina barrier development and maintenance.

ß-Catenin signaling controls the development and maintenance of the blood-brain barrier (BBB) and the blood-retina barrier (BRB), but the division of labor and degree of redundancy between the two principal ligand-receptor systems-the Norrin and Wnt7a/Wnt7b systems-are incompletely defined. Here, we present a loss-of-function genetic analysis of postnatal BBB and BRB maintenance in mice that shows striking threshold and partial redundancy effects. In particular, the combined loss of Wnt7a and Norrin or Wnt7a and Frizzled4 (Fz4) leads to anatomically localized BBB defects that are far more severe than observed with loss of Wnt7a, Norrin, or Fz4 alone. In the cerebellum, selective loss of Wnt7a in glia combined with ubiquitous loss of Norrin recapitulates the phenotype observed with ubiquitous loss of both Wnt7a and Norrin, implying that glia are the source of Wnt7a in the cerebellum. Tspan12, a coactivator of Norrin signaling in the retina, is also active in BBB maintenance but is less potent than Norrin, consistent with a model in which Tspan12 enhances the amplitude of the Norrin signal in vascular endothelial cells. Finally, in the context of a partially impaired Norrin system, the retina reveals a small contribution to BRB development from the Wnt7a/Wnt7b system. Taken together, these experiments define the extent of CNS region-specific cooperation for several components of the Norrin and Wnt7a/Wnt7b systems, and they reveal substantial regional heterogeneity in the extent to which partially redundant ligands, receptors, and coactivators maintain the BBB and BRB.

Insights into major facilitator superfamily domain-containing protein-2a (Mfsd2a) in physiology and pathophysiology. What do we know so far?

Major facilitator superfamily domain-containing protein-2a (Mfsd2a) which was considered as an orphan transporter has recently gained attention for its regulatory role in the maintenance of proper functioning of the blood-brain barrier. Besides the major role of Mfsd2a in maintaining the barrier function, increasing evidence has emerged with regard to the contributions of Mfsd2a to various biological processes such as transport, cell fusion, cell cycle, inflammation and regeneration, managing tumor growth, functioning of other organs with barrier functions or responses to injury. The purpose of this article is to review the different roles of Mfsd2a and its involvement in the physiological and pathophysiological processes primarily in the central nervous system and throughout the mammalian body under the lights of the current literature.

The role of cldnh during the early retinal development in zebrafish.

Claudin-3, an integral component of tight junction, has recently been shown to be expressed in retinal ganglion cells, retinal pigment cells, and retinal vascular endothelial cells. However, the role of claudin-3 in the development of the neural retina and its vessels remains undefined. This study aimed to investigate the role of zebrafish claudin-h (cldnh), the closest ortholog of mouse and human claudin-3, in the development of the neural retina and its vessels. Cldnh levels in green fluorescent protein transgenic zebrafish were genetically manipulated by cldnh morpholino oligonucleotide (MO) and cldnh mRNA to investigate gene function. The expression of cldnh was analyzed using polymerase chain reaction and immunofluorescence staining. The altered morphological, cellular and molecular events in the cldnh MO-morphant eyes were detected using hematoxylin-eosin staining, fluorescent dye injection, confocal in vivo imaging, BrdU labeling, TUNEL assay, RNA sequencing, and Western blot. We demonstrated that the cldnh protein was expressed in the neural retina and the hyaloid vessel which is the predecessor of the retinal vessel in zebrafish. Cldnh knockdown delayed lamination of the neural retina and reduced its thickness, which might be associated with the downregulation of the retinal development-related genes of atoh7, pcdh17, crx, neurod1, insm1a, sox9b and cdh11, and the upregulation of the cell cycle and apoptosis-associated genes of tp53, cdkn1a and casp8. Cldnh knockdown also reduced the density and interrupted the lumenization of the hyaloid vessels, which might be owing to the downregulation of the vessel formation-related genes of hlx1 and myl7. In conclusion, cldnh was required for the normal development of the neural retina and its vessels in zebrafish, providing a basis for elucidating its role in the pathogenesis of retinal vascular or inflammatory diseases.

Characteristics of Hemichannel-Mediated Substrate Transport in Human Retinal Pigment Epithelial Cells under Deprivation of Extracellular Ca2.

Retinal pigment epithelial (RPE) cells form the outer blood-retinal barrier (BRB) and regulate drug/compound exchange between the neural retina and blood in the fenestrated blood vessels of retinal choroid via membrane transporters. Recent studies have elucidated that RPE cells express hemichannels, which are opened by extracellular Ca2+ depletion and accept several drugs/compounds as a transporting substrate. The objective of this study was to elucidate the hemichannel-mediated compound transport properties of the outer BRB. In human RPE cells, namely ARPE-19 cells, time-dependent uptake of fluorescent hemichannel substrates, such as Lucifer Yellow, sulforhodamine-101 (SR-101), and propidium iodide (PI) was promoted under Ca2+-depleted conditions. The uptake of these substrates under Ca2+-depleted conditions exhibited saturable kinetics with a Michaelis-Menten constant (Km) of 87-109 µM. In addition, SR-101 and PI uptake by ARPE-19 cells was dependent of extracellular Ca2+ concentration, and that under Ca2+-depleted conditions was significantly decreased by typical substrates and/or inhibitors for hemichannels. Moreover, Ca2+-depleted conditions promoted the efflux transport of calcein from ARPE-19 cells, and the promoted calcein efflux transport was significantly inhibited by a typical hemichannel inhibitor. These results suggested that hemichannels at the outer BRB were involved in the influx and efflux transport of drugs/compounds.

RETINAL LEAKAGE INDEX DYNAMICS ON ULTRA-WIDEFIELD FLUORESCEIN ANGIOGRAPHY IN EYES TREATED WITH INTRAVITREAL AFLIBERCEPT FOR PROLIFERATIVE DIABETIC RETINOPATHY IN THE RECOVERY STUDY.

PURPOSE: Characterization of leakage indices on ultra-widefield fluorescein angiography in proliferative diabetic retinopathy treated with intravitreal aflibercept. METHODS: Prospective study enrolling subjects for treatment of proliferative diabetic retinopathy randomized 1:1 to receive 2-mg intravitreal aflibercept every 4 weeks (2q4) or every 12 weeks (2q12). Ultra-widefield fluorescein angiography images obtained at baseline, 24, and 48 weeks were analyzed using a semiautomated leakage segmentation platform. Panretinal and zonal leakage indices were calculated. RESULTS: Forty eyes of 40 subjects were included, and mean age was 48 ± 12.1 years. Mean number of injections was 11 ± 1.7 in the 2q4 arm and 4 ± 0.4 in the 2q12 arm. Median baseline leakage index in the 2q4 and 2q12 groups was 5.1% and 4.3%, respectively (P = 0.28). At 24 and 48 weeks, the 2q4 group significantly improved to 1.1% (-79%, P < 0.0001). At Week 24, the 2q12 group demonstrated nonsignificant improvement (3.4%; -21%, P = 0.47); by Week 48, improvement was significant (1.4%; -68%, P = 0.02). The 2q4 group resulted in lower leakage index compared with the 2q12 group at 24 weeks (1.1% vs. 3.4%, respectively; P = 0.008), but by 48 weeks, leakage index was similar between both groups (1.1% vs. 1.4%, respectively; P = 0.34). CONCLUSION: Proliferative diabetic retinopathy treated with intravitreal aflibercept demonstrated significant leakage index reductions at 1 year. Monthly dosing provided more rapid reduction in leakage index compared with quarterly dosing. TRIAL REGISTRATION: RECOVERY study (NCT02863354); https://clinicaltrials.gov/ct2/show/NCT02863354.

Case Report: Beyond the Blood-retina Barrier: Intravitreal Caspofungin for Fungal Endophthalmitis.

SIGNIFICANCE: Two fungal endophthalmitis cases demonstrate safety and efficiency of intravitreal caspofungin as a new therapy option in fungal endophthalmitis. PURPOSE: The purpose of this study was to evaluate the intravitreal application of caspofungin for the treatment of fungal endophthalmitis because rising resistance to voriconazole and amphotericin B leads to a need for new antifungal therapy options. CASE REPORT: Initially, both patients with fungal endophthalmitis underwent pars plana vitrectomy. Microbiological analysis revealed Aspergillus terreus and Candida dubliniensis, which both possess atypical resistance patterns. Caspofungin has a low bioavailability in the eye when given systemically. It was injected intravitreally into the eyes affected by fungal endophthalmitis. An injection of 100 µg of caspofungin in a volume 0.1 mL was applied repeatedly. Clinical parameters were recorded. Both eyes were stabilized by the treatment. Finally, the intraocular infections with atypical mycotic agents were eliminated. Visual acuity improved to 0.4 logMAR (20/50 Snellen) in the first case and to 1.0 logMAR (20/200 Snellen) in the second case. During the treatment course, we have not seen any toxic effects or damage of intraocular structures related to the intravitreal administration of caspofungin. CONCLUSIONS: In summary, intravitreal caspofungin was effective and well tolerated in both cases. Therefore, caspofungin seems to be a safe and effective intravitreal alternative to voriconazole and amphotericin B in fungal endophthalmitis.

Shear stress modulates inner blood retinal barrier phenotype.

The vascular endothelium responds to the shear stress generated by blood flow and changes function to maintain tissue homeostasis and adapt to injury in pathological conditions. Shear stress in the retinal circulation is altered in patients with retinal vascular diseases, such as diabetic retinopathy. Therefore, we aimed to study the effect of laminar shear stress on barrier properties and on the release of proinflammatory cytokines in human retinal microvascular endothelial cells (HRMEC). HRMEC were cultured in Ibidi flow chambers and exposed to laminar shear stress (0-50 dyn/cm2) for 24-48 h. Tight junction distribution (ZO-1 and claudin-5) and cytokine production were determined by immunofluorescence and ELISA, respectively. The chemotactic effect of conditioned media exposed to shear stress was determined by measuring lymphocyte transmigration in Transwells. We found that cells exposed to moderately low shear stress (1.5 and 5 dyn/cm2) showed enhanced distribution of membrane ZO-1 and claudin-5 and decreased production of the proinflammatory cytokines IL-8, CCL2, and IL-6 compared to static conditions and high shear stress values. Moreover, conditioned media from cells exposed to low shear stress, had the lowest chemotactic effect to recruit lymphocytes compared to conditioned media from cells exposed to static and high shear stress conditions. In conclusion, high shear stress and static flow, associated to impaired retinal circulation, may compromise the inner blood retinal barrier phenotype and barrier function in HRMEC.

Increased serum proteins in non-exudative AMD retinas.

The blood retinal barrier (BRB) closely regulates the retinal microenvironment. Its compromise leads to the accumulation of retinal fluid containing potentially harmful plasma components. While eyes with non-exudative age-related macular degeneration (AMD) were previously felt to have an intact BRB, we propose that the BRB in non-exudative AMD eyes may be subclinically compromised, allowing entry of retina-toxic plasma proteins. We test this hypothesis by measuring retinal levels of abundant plasma proteins that should not cross the intact BRB. Two cohorts of frozen, post mortem neurosensory retinas were studied by Western analysis. One cohort from Alabama had 4 normal controls and 4 eyes with various forms of AMD. Another cohort from Minnesota had 5 intermediate AMD eyes and 5 normals. Both cohorts were age/post mortem interval (PMI) matched. The non-exudative AMD retinas in the Alabama cohort had significantly higher levels of albumin and complement component 9 (C9) than normal controls. The positive control exudative AMD donor retina had higher levels of all but one serum protein. In both macular and peripheral neurosensory retina samples, intermediate AMD retinas in the Minnesota cohort had significantly higher levels of albumin, fibrinogen, IgG, and C9 than controls. Our results suggest that there may be moderate subclinical BRB leakage in non-exudative AMD. Potentially harmful plasma components including complement or iron could enter the neurosensory retina in AMD patients prior to advanced disease. Thus, therapies aiming to stabilize the BRB might have a role in the management of non-exudative AMD.

Protective effects of dexamethasone on hypoxia-induced retinal edema in a mouse model.

Hypoxia-induced retinal edema primarily induced by vascular lesion is seen in various conditions such as diabetic retinopathy (DR) and retinal vein occlusion (RVO). The edematous changes in these conditions occur mainly in intermediate and deep layers of retina as a result of disruption of the inner blood-retinal barrier (iBRB). However, the effect of direct and acute hypoxia on iBRB remains to be elucidated. To investigate direct and acute hypoxia-induced changes in retina, especially in astrocytes/Müller cells that are involved in the maintenance of retinal structure and function, we developed an adult mouse model of hypoxia-induced retinal edema by 24-h exposure in a 6% oxygen environment. Immunohistochemical staining of glial fibrillary acidic protein (GFAP) was enhanced mainly in the superficial layer of the hypoxic retina, corresponding to edematous change. Electron microscopic observation of the hypoxic retina showed vacuole formation in astrocyte/Müller cell foot processes around capillaries in the superficial layer, while no abnormal findings in the perivascular areas were found in intermediate and deep layers. Increase in vascular leakage quantified by Evans blue dye and tight junction breakdown detected by electron-dense tracer were observed in the hypoxia group. In the hypoxic retina, microglia was activated and relative gene expressions of pro-inflammatory cytokines were significantly upregulated. Dexamethasone suppressed these hypoxia-induced pathological reactions. Thus, unlike DR and RVO that induce iBRB breakdown in deeper retinal layers, atmospheric hypoxia induced iBRB disruption with subsequent edematous change mainly in the superficial layer of the retina, and that dexamethasone prevented these pathological changes. In this mouse model, direct and acute hypoxia induces retinal edema in the superficial layer of the retina with morphological changes of astrocytes/Müller cells, and is potentially useful for ophthalmic research in the field related to retinal hypoxia and its treatment.

Inner Blood-Retinal Barrier Regulation in Retinopathies.

The neural retina is protected from the blood circulation by the presence of a highly selective inner blood-retinal barrier (iBRB). The presence of sophisticated tight junctions (TJs) between the endothelial cells (ECs) of the iBRB helps mediate the very low passive permeability of the tissue, permitting entry of nutrients into the retina but excluding harmful toxic material and inflammatory cells. The most highly enriched TJ protein is claudin-5, which is critical in mediating the passive paracellular diffusion barrier properties of the iBRB. In numerous retinal degeneration pathologies, TJ disruption is observed, and a more refined understanding of this disruption could be used for therapeutic benefit.

Tight Junctions of the Outer Blood Retina Barrier.

The outer blood retina barrier (oBRB) formed by the retinal pigment epithelium (RPE) is critical for maintaining retinal homeostasis. Critical to this modified neuro-epithelial barrier is the presence of the tight junction structure that is formed at the apical periphery of contacting cells. This tight junction complex mediates size-selective passive diffusion of solutes to and from the outer segments of the retina. Unlike other epithelial cells, the apical surface of the RPE is in direct contact with neural tissue and it is centrally involved in the daily phagocytosis of the effete tips of photoreceptor cells. While much is known about the intracellular trafficking of material within the RPE, less is known about the role of the tight junction complexes in health and diseased states. Here, we provide a succinct overview of the molecular composition of the RPE tight junction complex in addition to highlighting some of the most common retinopathies that involve a dysregulation of RPE integrity.

Pericytes in Microvessels: From "Mural" Function to Brain and Retina Regeneration.

Pericytes are branched cells located in the wall of capillary blood vessels that are found throughout the body, embedded within the microvascular basement membrane and wrapping endothelial cells, with which they establish a strong physical contact. Pericytes regulate angiogenesis, vessel stabilization, and contribute to the formation of both the blood-brain and blood-retina barriers by Angiopoietin-1/Tie-2, platelet derived growth factor (PDGF) and transforming growth factor (TGF) signaling pathways, regulating pericyte-endothelial cell communication. Human pericytes that have been cultured for a long period give rise to multilineage progenitor cells and exhibit mesenchymal stem cell (MSC) features. We focused our attention on the roles of pericytes in brain and ocular diseases. In particular, pericyte involvement in brain ischemia, brain tumors, diabetic retinopathy, and uveal melanoma is described. Several molecules, such as adenosine and nitric oxide, are responsible for pericyte shrinkage during ischemia-reperfusion. Anti-inflammatory molecules, such as IL-10, TGFß, and MHC-II, which are increased in glioblastoma-activated pericytes, are responsible for tumor growth. As regards the eye, pericytes play a role not only in ocular vessel stabilization, but also as a stem cell niche that contributes to regenerative processes in diabetic retinopathy. Moreover, pericytes participate in melanoma cell extravasation and the genetic ablation of the PDGF receptor reduces the number of pericytes and aberrant tumor microvessel formation with important implications for therapy efficacy. Thanks to their MSC features, pericytes could be considered excellent candidates to promote nervous tissue repair and for regenerative medicine.

Role of lysophosphatidic acid in the retinal pigment epithelium and photoreceptors.

The human retina is a complex structure of organised layers of specialised cells that support the transmission of light signals to the visual cortex. The outermost layer of the retina, the retinal pigment epithelium (RPE), forms part of the blood retina barrier and is implicated in many retinal diseases. Lysophosphatidic acid (LPA) is a bioactive lipid exerting pleiotropic effects in various cell types, during development, normal physiology and disease. Its producing enzyme, AUTOTAXIN (ATX), is highly expressed by the pigmented epithelia of the human eye, including the RPE. Using human pluripotent stem cell (hPSC)-derived retinal cells, we interrogated the role of LPA in the human RPE and photoreceptors. hPSC-derived RPE cells express and synthesize functional ATX, which is predominantly secreted apically of the RPE, suggesting it acts in a paracrine manner to regulate photoreceptor function. In RPE cells, LPA regulates tight junctions, in a receptor-dependent mechanism, with an increase in OCCLUDIN and ZONULA OCCLUDENS (ZO)-1 expression at the cell membrane, accompanied by an increase in the transepithelial resistance of the epithelium. High concentration of LPA decreases phagocytosis of photoreceptor outer segments by the RPE. In hPSC-derived photoreceptors, LPA induces morphological rearrangements by modulating the actin myosin cytoskeleton, as evidenced by Myosin Light Chain l membrane relocation. Collectively, our data suggests an important role of LPA in the integrity and functionality of the healthy retina and blood retina barrier.

Molecular and functional characterization of circulating extracellular vesicles from diabetic patients with and without retinopathy and healthy subjects.

Diabetic retinopathy is a sight-threatening complication of diabetes, characterized by loss of retinal pericytes and abnormal angiogenesis. We previously demonstrated that extracellular vesicles (EVs) derived from mesenchymal stem cells cultured in diabetic-like conditions are able to enter the pericytes, causing their detachment and migration, and stimulating angiogenesis in vitro. The purpose of this work was the molecular and functional characterization of EVs derived from diabetic subjects with or without diabetic retinopathy, compared with healthy controls. Characterization of EVs extracted from serum/plasma of diabetic patients with or without retinopathy, and healthy controls, was performed by FACS and microarray analysis of microRNA (miRNA) content. Relevant miRNA expression was validated through qRT-PCR. EV influence on pericyte detachment, angiogenesis and permeability of the blood-retinal barrier was also investigated. Diabetic subjects had a 2.5 fold higher EV concentration than controls, while expression of surface molecules was unchanged. Microarray analysis revealed 11 differentially expressed miRNAs. Three of them (miR-150-5p, miR-21-3p and miR-30b-5p) were confirmed by qRT-PCR. Plasma EVs from subjects with diabetic retinopathy induced pericyte detachment and pericyte/endothelial cell migration, increased the permeability of pericyte/endothelial cell bilayers and the formation of vessel-like structures, when compared with EVs from controls. In conclusion, circulating EVs show differences between diabetic patients and healthy subjects. EVs extracted from plasma of diabetic retinopathy patients are able to induce features of retinopathy in in vitro models of retinal microvasculature. Our data suggest a role for miR-150-5p, miR-21-3p and miR-30b-5p as potential biomarkers of the onset of diabetic retinopathy.

Retinal pigment epithelium in the pathogenesis of age-related macular degeneration and photobiomodulation as a potential therapy?

The retinal pigment epithelium (RPE) comprises a monolayer of cells located between the neuroretina and the choriocapillaries. The RPE serves several important functions in the eye: formation of the blood-retinal barrier, protection of the retina from oxidative stress, nutrient delivery and waste disposal, ionic homeostasis, phagocytosis of photoreceptor outer segments, synthesis and release of growth factors, reisomerization of all-trans-retinal during the visual cycle, and establishment of ocular immune privilege. Age-related macular degeneration (AMD) is the leading cause of blindness in developed countries. Dysfunction of the RPE has been associated with the pathogenesis of AMD in relation to increased oxidative stress, mitochondrial destabilization and complement dysregulation. Photobiomodulation or near infrared light therapy which refers to non-invasive irradiation of tissue with light in the far-red to near-infrared light spectrum (630-1000 nm), is an intervention that specifically targets key mechanisms of RPE dysfunction that are implicated in AMD pathogenesis. The current evidence for the efficacy of photobiomodulation in AMD is poor but its safety profile and proposed mechanisms of action motivate further research as a novel therapy for AMD.

C-reactive protein isoforms differentially affect outer blood-retinal barrier integrity and function.

The retinal pigment epithelium (RPE) forms the outer blood-retinal barrier (oBRB) and is the prime target of early age-related macular degeneration (AMD). C-reactive protein (CRP), a serum biomarker for chronic inflammation and AMD, presents two different isoforms, monomeric (mCRP) and pentameric (pCRP), that may have a different effect on inflammation and barrier function in the RPE. The results reported in this study suggest that mCRP but not pCRP impairs RPE functionality by increasing paracellular permeability and disrupting the tight junction proteins ZO-1 and occludin in RPE cells. Additionally, we evaluated the effect of drugs commonly used in clinical settings on mCRP-induced barrier dysfunction. We found that a corticosteroid (methylprednisolone) and an anti-VEGF agent (bevacizumab) prevented mCRP-induced ARPE-19 barrier disruption and IL-8 production. Furthermore, bevacizumab was also able to revert mCRP-induced IL-8 increase after mCRP stimulation. In conclusion, the presence of mCRP within retinal tissue may lead to disruption of the oBRB, an effect that may be modified in the presence of corticosteroids or anti-VEGF drugs.