Granzyme B degrades extracellular matrix and promotes inflammation and choroidal neovascularization.

Age-related macular degeneration (AMD) is a common retinal neurodegenerative disease among the elderly. Neovascular AMD (nAMD), a leading cause of AMD-related blindness, involves choroidal neovascularization (CNV), which can be suppressed by anti-angiogenic treatments. However, current CNV treatments do not work in all nAMD patients. Here we investigate a novel target for AMD. Granzyme B (GzmB) is a serine protease that promotes aging, chronic inflammation and vascular permeability through the degradation of the extracellular matrix (ECM) and tight junctions. Extracellular GzmB is increased in retina pigment epithelium (RPE) and mast cells in the choroid of the healthy aging outer retina. It is further increased in donor eyes exhibiting features of nAMD and CNV. Here, we show in RPE-choroidal explant cultures that exogenous GzmB degrades the RPE-choroid ECM, promotes retinal/choroidal inflammation and angiogenesis while diminishing anti-angiogenic factor, thrombospondin-1 (TSP-1). The pharmacological inhibition of either GzmB or mast-cell degranulation significantly reduces choroidal angiogenesis. In line with our in vitro data, GzmB-deficiency reduces the extent of laser-induced CNV lesions and the age-related deterioration of electroretinogram (ERG) responses in mice. These findings suggest that targeting GzmB, a serine protease with no known endogenous inhibitors, may be a potential novel therapeutic approach to suppress CNV in nAMD.

A complement factor H homolog, heparan sulfation, and syndecan maintain inversin compartment boundaries in C. elegans cilia.

Age-related macular degeneration (AMD) is a leading cause of blindness among the elderly. Canonical disease models suggest that defective interactions between complement factor H (CFH) and cell surface heparan sulfate (HS) result in increased alternative complement pathway activity, cytolytic damage, and tissue inflammation in the retina. Although these factors are thought to contribute to increased disease risk, multiple studies indicate that noncanonical mechanisms that result from defective CFH and HS interaction may contribute to the progression of AMD as well. A total of 60 ciliated sensory neurons in the nematode Caenorhabditis elegans detect chemical, olfactory, mechanical, and thermal cues in the environment. Here, we find that a C. elegans CFH homolog localizes on CEP mechanosensory neuron cilia where it has noncanonical roles in maintaining inversin/NPHP-2 within its namesake proximal compartment and preventing inversin/NPHP-2 accumulation in distal cilia compartments in aging adults. CFH localization and maintenance of inversin/NPHP-2 compartment integrity depend on the HS 3-O sulfotransferase HST-3.1 and the transmembrane proteoglycan syndecan/SDN-1. Defective inversin/NPHP-2 localization in mouse and human photoreceptors with CFH mutations indicates that these functions and interactions may be conserved in vertebrate sensory neurons, suggesting that previously unappreciated defects in cilia structure may contribute to the progressive photoreceptor dysfunction associated with CFH loss-of-function mutations in some AMD patients.

Granzyme B Contributes to Choroidal Neovascularization and Age-Related Macular Degeneration Through Proteolysis of Thrombospondin-1.

Age-related macular degeneration (AMD) is a leading cause of irreversible central vision loss in the elderly. The pathology of neovascular age-related macular degeneration (nAMD), also known as wet AMD, is associated with an abnormal blood vessel growth in the eye and involves an imbalance of proangiogenic and antiangiogenic factors. Thrombospondin (TSP)-1 and TSP-2 are endogenous matricellular proteins that inhibit angiogenesis. TSP-1 is significantly diminished in eyes with AMD, although the mechanisms involved in its reduction are unknown. Granzyme B (GzmB) is a serine protease with an increased extracellular activity in the outer retina and choroid of human eyes with nAMD-related choroidal neovascularization (CNV). This study investigated whether TSP-1 and TSP-2 are GzmB substrates using in silico and cell-free cleavage assays and explored the relationship between GzmB and TSP-1 in human eyes with nAMD-related CNV and the effect of GzmB on TSP-1 in retinal pigment epithelial culture and an explant choroid sprouting assay (CSA). In this study, TSP-1 and TSP-2 were identified as GzmB substrates. Cell-free cleavage assays substantiated the GzmB proteolysis of TSP-1 and TSP-2 by showing dose-dependent and time-dependent cleavage products. TSP-1 and TSP-2 proteolysis were hindered by the inhibition of GzmB. In the retinal pigment epithelium and choroid of human eyes with CNV, we observed a significant inverse correlation between TSP-1 and GzmB, as indicated by lower TSP-1 and higher GzmB immunoreactivity. In CSA, the vascular sprouting area increased significantly with GzmB treatment and reduced significantly with TSP-1 treatment. Western blot showed significantly reduced expression of TSP-1 in GzmB-treated retinal pigment epithelial cell culture and CSA supernatant compared with that in controls. Together, our findings suggest that the proteolysis of antiangiogenic factors such as TSP-1 by extracellular GzmB might represent a mechanism through which GzmB may contribute to nAMD-related CNV. Future studies are needed to investigate whether pharmacologic inhibition of extracellular GzmB can mitigate nAMD-related CNV by preserving intact TSP-1.

Ligand-independent activation of platelet-derived growth factor receptor ß promotes vitreous-induced contraction of retinal pigment epithelial cells.

BACKGROUND: Epiretinal membranes in patients with proliferative vitreoretinopathy (PVR) consist of extracellular matrix and a number of cell types including retinal pigment epithelial (RPE) cells and fibroblasts, whose contraction causes retinal detachment. In RPE cells depletion of platelet-derived growth factor (PDGF) receptor (PDGFR)ß suppresses vitreous-induced Akt activation, whereas in fibroblasts Akt activation through indirect activation of PDGFRα by growth factors outside the PDGF family (non-PDGFs) plays an essential role in experimental PVR. Whether non-PDGFs in the vitreous, however, were also able to activate PDGFRß in RPE cells remained elusive. METHODS: The CRISPR/Cas9 technology was utilized to edit a genomic PDGFRB locus in RPE cells derived from an epiretinal membrane (RPEM) from a patient with PVR, and a retroviral vector was used to express a truncated PDGFRß short of a PDGF-binding domain in the RPEM cells lacking PDGFRß. Western blot was employed to analyze expression of PDGFRß and α-smooth muscle actin, and signaling events (p-PDGFRß and p-Akt). Cellular assays (proliferation, migration and contraction) were also applied in this study. RESULTS: Expression of a truncated PDGFRß lacking a PDGF-binding domain in the RPEM cells whose PDGFRB gene has been silent using the CRISPR/Cas9 technology restores vitreous-induced Akt activation as well as cell proliferation, epithelial-mesenchymal transition, migration and contraction. In addition, we show that scavenging reactive oxygen species (ROS) with N-acetyl-cysteine and inhibiting Src family kinases (SFKs) with their specific inhibitor SU6656 blunt the vitreous-induced activation of the truncated PDGFRß and Akt as well as the cellular events related to the PVR pathogenesis. These discoveries suggest that in RPE cells PDGFRß can be activated indirectly by non-PDGFs in the vitreous via an intracellular pathway of ROS/SFKs to facilitate the development of PVR, thereby providing novel opportunities for PVR therapeutics. CONCLUSION: The data shown here will improve our understanding of the mechanism by which PDGFRß can be activated by non-PDGFs in the vitreous via an intracellular route of ROS/SFKs and provide a conceptual foundation for preventing PVR by inhibiting PDGFRß transactivation (ligand-independent activation).

Inhalable Thioflavin S for the Detection of Amyloid Beta Deposits in the Retina.

We present an integrated delivery technology herein employing the aerosolized method to repurpose thioflavin S for imaging amyloid beta (Abeta) deposits in the retina as a surrogate of Abeta in the brain for early detection of Alzheimer's disease. The data showed that wild type (WT) mice also have Abeta deposits in the retinae, albeit much less than 5XFAD mice. Further, only in 5XFAD mice, significant Abeta deposits were found associated with retinal ganglion cells (RGCs) in whole-mount and cross-section data. Furthermore, the fluorescent signal depicted from thioflavin S corroborates with Abeta immunohistochemistry staining information. Overall, this probe delivery via inhalation method is also applicable to other Abeta-binding molecules, such as Congo red, curcumin, and thioflavin T. The advantage of imaging retinal amyloid deposits compared to the brain counterparts is that the eye is easily accessible by in vivo imaging and it reduces the effort to design a probe that must cross the formidable blood-brain barrier.

Substantially thinner internal granular layer and reduced molecular layer surface in the cerebellar cortex of the Tc1 mouse model of down syndrome - a comprehensive morphometric analysis with active staining contrast-enhanced MRI.

Down Syndrome is a chromosomal disorder that affects the development of cerebellar cortical lobules. Impaired neurogenesis in the cerebellum varies among different types of neuronal cells and neuronal layers. In this study, we developed an imaging analysis framework that utilizes gadolinium-enhanced ex vivo mouse brain MRI. We extracted the middle Purkinje layer of the mouse cerebellar cortex, enabling the estimation of the volume, thickness, and surface area of the entire cerebellar cortex, the internal granular layer, and the molecular layer in the Tc1 mouse model of Down Syndrome. The morphometric analysis of our method revealed that a larger proportion of the cerebellar thinning in this model of Down Syndrome resided in the inner granule cell layer, while a larger proportion of the surface area shrinkage was in the molecular layer.

PDGFRß plays an essential role in patient vitreous-stimulated contraction of retinal pigment epithelial cells from epiretinal membranes.

Platelet-derived growth factor (PDGF) is associated with clinical proliferative vitreoretinopathy (PVR), which is characterized by formation of sub- or epi-retinal membranes that consist of cells including retinal pigment epithelial （RPE） cells and extracellular matrix. RPE cells play an important role in PVR pathogenesis. Previous findings indicated that PDGF receptor (PDGFR)α was essential in experimental PVR induced by fibroblasts. In RPE cells derived from epiretinal membranes from patients with PVR (RPEMs)， Akt was activated by PDGF-B but not PDGF-A, which suggested that PDGFRß was the predominant PDGFR isoform expressed in RPEMs. Indeed, CRISPR/Cas9-mediated depletion of PDGFRß in RPEMs attenuated patient vitreous-induced Akt activation and cellular responses intrinsic to PVR including cell proliferation, migration, and contraction. We conclude that PDGFRß appears to be the PVR relevant PDGFR isoform in RPEMs.

Idelalisib inhibits vitreous-induced Akt activation and proliferation of retinal pigment epithelial cells from epiretinal membranes.

Proliferative vitreoretinopathy (PVR) is a blinding fibrotic eye disease that develops in 8-10% of patients who undergo primary retinal detachment-reparative surgery and in 40-60% of patients with open-globe injury. At present, there is no pharmacological treatment for this devastating disease. Vitreal growth factors activate their respective receptors of cells in the vitreous, trigger their downstream signaling transduction (e.g. phosphoinositide 3 kinases (PI3Ks)/Akt), and drive cellular responses intrinsic to the pathogenesis of PVR. PI3Ks play a central role in experimental PVR. However, which isoform(s) are involved in PVR pathogenesis remain unknown. Herein, we show that p110δ, a catalytic subunit of receptor-regulated PI3K isoform δ, is highly expressed in epiretinal membranes from patients with PVR, and that idelalisib, a specific inhibitor of PI3Kδ, effectively inhibits vitreous-induced Akt activation, proliferation, migration and contraction of retinal pigment epithelial cells derived from an epiretinal membrane of a PVR patient. Small molecules of kinase inhibitors have shown great promise as a class of therapeutics for a variety of human diseases. The data herein suggest that idelalisib is a promising PVR prophylactic.

Blockade of MDM2 with inactive Cas9 prevents epithelial to mesenchymal transition in retinal pigment epithelial cells.

Epithelial to mesenchymal transition (EMT) plays an important role in the pathogenesis of proliferative vitreoretinopathy (PVR). We aimed to demonstrate the role of mouse double minute 2 (MDM2) in transforming growth factor-beta 2 (TGF-ß2)-induced EMT in human retinal pigment epithelial cells (RPEs). Immunofluorescence was used to assess MDM2 expression in epiretinal membranes (ERMs) from patients with PVR. A single guide (sg)RNA targeting the second promoter of MDM2 was cloned into a mutant lentiviral Clustered Regularly Interspaced Short Palindromic Repeats (lentiCRISPR) v2 (D10A and H840A) vector for expressing nuclease dead Cas9 (dCas9)/MDM2-sgRNA in RPEs. In addition, MDM2-sgRNA was also cloned into a pLV-sgRNA-dCas9-Kruppel associated box (KRAB) vector for expressing dCas9 fused with a transcriptional repressor KRAB/MDM2-sgRNA. TGF-ß2-induced expression of MDM2 and EMT biomarkers were assessed by quantitative polymerase chain reaction (q-PCR), western blot, or immunofluorescence. Wound-healing and proliferation assays were used to evaluate the role of MDM2 in TGF-ß2-induced responses in RPEs. As a result, we found that MDM2 was expressed obviously in ERMs, and that TGF-ß2-induced expression of MDM2 and EMT biomarkers Fibronectin, N-cadherin and Vimentin in RPEs. Importantly, we discovered that the dCas9/MDM2-sgRNA blocked TGF-ß2-induced expression of MDM2 and the EMT biomarkers without affecting their basal expression, whereas the dCas9-KRAB/MDM2-sgRNA suppressed basal MDM2 expression in RPEs. These cells could not be maintained continuously because their viability was greatly reduced. Next, we found that Nutlin-3, a small molecule blocking the interaction of MDM2 with p53, inhibited TGF-ß2-induced expression of Fibronectin and N-cadherin but not Vimentin in RPEs, indicating that MDM2 functions in both p53-dependent and -independent pathways. Finally, our experimental data demonstrated that dCas9/MDM2-sgRNA suppressed TGF-ß2-dependent cell proliferation and migration without disturbing the unstimulated basal activity. In conclusion, the CRISPR/dCas9 capability for blocking TGF-ß2-induced expression of MDM2 and EMT biomarkers can be exploited for a therapeutic approach to PVR.

The reduction of XIAP is associated with inflammasome activation in RPE: implications for AMD pathogenesis.

BACKGROUND: Age-related macular degeneration (AMD) is a multifactorial chronic disease of the eye. Several candidate pathways have been hypothesized to play a role in AMD pathogenesis. Our work and those of others suggests inflammasome activity as a mechanism associated with retinal pigment epithelial (RPE) cell demise. X-linked inhibitor of apoptosis protein (XIAP), an anti-apoptosis factor, has recently been shown to regulate inflammasome activity in non-ocular cells. The purpose of this study is to characterize XIAP's regulatory role in RPE. METHODS: Protein lysates of eye tissues from rats (vinpocetine- or aurin tricarboxylic acid complex-treated, ATAC, vs naïve) and mice (wild type vs Caspase-4-/-) were utilized to analyze XIAP protein levels. Immunohistochemistry was used to detect NLRP3 levels in the RPE layer. In vitro inflammasome activation on RPE cells was achieved with L-leucyl-L-leucine methyl ester (Leu-Leu-OMe) stimulation. Levels of XIAP mRNA and 18S RNA were quantified by RT-PCR. Cell culture supernatants were tested directly for secreted IL-1ß by ELISA or concentrated for the detection of secreted IL-18 by western blot. Protein lysates from RPE in cell culture were collected for the measurement of cleaved caspase-1 p20, XIAP, and GAPDH. Data are presented as Mean ± SD. p < 0.05 is considered statistically significant. RESULTS: The XIAP protein level was significantly increased when the inflammasome was inhibited at the "activation" step by ATAC, but not the "priming" step, in vivo. Concomitantly, NLRP3 immunoreactivity was lower in the RPE layer of animals fed with ATAC. In mice where caspase-1 cleavage was impaired by the genetic deficiency in caspase-4, the XIAP protein level increased in eye tissues. In RPE cell culture, Leu-Leu-OMe stimulation led to caspase-1 cleavage, cytokine secretion, and XIAP reduction, which can be abolished by Z-YVAD-FMK. When XIAP siRNA was given as a pre-treatment to RPE in vitro, Leu-Leu-OMe induced IL-1ß/IL-18 secretion was enhanced, whereas overexpressing XIAP reduced IL-1ß secretion under inflammasome activation, both compared to controls cells. CONCLUSIONS: Together, these data suggest XIAP-mediated inhibition of inflammasome activity in RPE may provide insights into the biological consequences of inflammasome activation in RPE and reveals the caspase-1/XIAP/IL-1ß/IL-18 axis as a target for broader applications in AMD biology and treatment design.

Evidence for the activation of pyroptotic and apoptotic pathways in RPE cells associated with NLRP3 inflammasome in the rodent eye.

BACKGROUND: Age-related macular degeneration (AMD) is a devastating eye disease causing irreversible vision loss in the elderly. Retinal pigment epithelium (RPE), the primary cell type that is afflicted in AMD, undergoes programmed cell death in the late stages of the disease. However, the exact mechanisms for RPE degeneration in AMD are still unresolved. The prevailing theories consider that each cell death pathway works independently and without regulation of each other. Building upon our previous work in which we induced a short burst of inflammasome activity in vivo, we now investigate the effects of prolonged inflammasome activity on RPE cell death mechanisms in rats. METHODS: Long-Evans rats received three intravitreal injections of amyloid beta (Aß), once every 4 days, and were sacrificed at day 14. The vitreous samples were collected to assess the levels of secreted cytokines. The inflammasome activity was evaluated by both immunohistochemistry and western blot. The types of RPE cell death mechanisms were determined using specific cell death markers and morphological characterizations. RESULTS: We found robust inflammasome activation evident by enhanced caspase-1 immunoreactivity, augmented NF-κB nuclear translocalization, increased IL-1ß vitreal secretion, and IL-18 protein levels. Moreover, we observed elevated proteolytic cleavage of caspase-3 and gasdermin D, markers for apoptosis and pyroptosis, respectively, in RPE-choroid tissues. There was also a significant reduction in the anti-apoptotic factor, X-linked inhibitor of apoptosis protein, consistent with the overall changes of RPE cells. Morphological analysis showed phenotypic characteristics of pyroptosis including RPE cell swelling. CONCLUSIONS: Our data suggest that two cell death pathways, pyroptosis and apoptosis, were activated in RPE cells after exposure to prolonged inflammasome activation, induced by a drusen component, Aß. The involvement of two distinct cell death pathways in RPE sheds light on the potential interplay between these pathways and provides insights on the future development of therapeutic strategies for AMD.

Understanding AMD by analogy: systematic review of lipid-related common pathogenic mechanisms in AMD, AD, AS and GN.

RATIONALE: Age-related macular degeneration (AMD) is one of the leading causes of blindness among the elderly. Due to its complex etiology, current treatments have been insufficient. Previous studies reveal three systems closely involved in AMD pathogenesis: lipid metabolism, oxidation and inflammation. These systems are also involved in Alzheimer's disease, atherosclerosis and glomerulonephritis. Understanding commonalities of these four diseases may provide insight into AMD etiology. OBJECTIVES: To understand AMD pathogenesis by analogy and suggest ideas for future research, this study summarizes main commonalities in disease pathogenesis of AMD, Alzheimer's disease, atherosclerosis and glomerulonephritis. METHODS: Articles were identified through PubMed, Ovid Medline and Google Scholar. We summarized the common findings and synthesized critical differences. RESULTS: Oxidation, lipid deposition, complement activation, and macrophage recruitment are involved in all four diseases shown by genetic, molecular, animal and human studies. Shared genetic variations further strengthen their connection. Potential areas for future research are suggested throughout the review. CONCLUSIONS: The four diseases share many steps of an overall framework of pathogenesis. Various oxidative sources cause oxidative stress. Oxidized lipids and related molecules accumulate and lead to complement activation, macrophage recruitment and pathology. Investigations that arise under this structure may aid us to better understand AMD pathology.

Structural divergence of essential triad ribbon synapse proteins among placental mammals - Implications for preclinical trials in photoreceptor transplantation therapy.

As photoreceptor transplantation rapidly moves closer to the clinic, verifying graft efficacy in animal models may have unforeseen xenogeneic barriers. Although photoreceptor transplants have most convincingly exhibited functional synaptogenesis in conspecific studies, such evidence (while ruling out false-positives due to: viral graft labeling, fusion/cytosolic transfer, or neuroprotection) has not yet been shown for discordant xenografts. From this, a fundamental question should be raised: is useful xenosynaptogenesis likely between human photoreceptors and mouse retina? The triad ribbon synapse (TRS) that would normally form is unique and contains trans-synaptic proteins essential to its formation and function. Thus, could interspecific structural divergence be present that may inhibit this trans-synaptic bridge in discordant xenografts? In an effort to address this question computationally, we compared eight recently confirmed (including subcellular location) TRS specific (or predominantly expressed at the TRS) proteins among placental mammals (1-to-1 orthologs) using HyPhy selection analysis (a predictive measure of structural divergence) and by using Phyre2 tertiary structural modeling. Here, selection analysis revealed strong positive (diversifying) selection acting on a particularly important TRS protein: pikachurin. This positive selection was localized to its second Laminin-G (LG)-like domain and on its N-terminal domain - a putative region of trans-synaptic interaction. Localization of structural divergence to the N-terminus of each putative post-translational cleavage (PTC) product may suggest neofunctionalization from ancestral uncleaved pikachurin - this would be consistent with a recent counter-paradigm report of pikachurin cleavage predominating at the TRS. From this, we suggest a dual role after cleavage where the N-terminal fragment can still mediate the trans-synaptic bridge, while the C-terminal fragment may act as a diffusible trophic or "homing" factor for bipolar cell dendrite migration. Tertiary structural models mirrored the conformational divergence predicted by selection analysis. With human and mouse pikachurin (as well as other TRS proteins) likely to diverge considerably in structure among placental mammals - alongside known inter-mammalian variation in TRS phenotype and protein repertoire, high levels of diversifying selection acting on genes involving sensation, considerable timespans allowing for genetic drift that can create xenogeneic epistasis, and uncertainty surrounding the extent of xenosynaptogenesis in PPC transplant studies to date - use of distantly related hosts to test human photoreceptor graft therapeutic efficacy should be considered with caution.

LAMELLAR HOLE-ASSOCIATED EPIRETINAL PROLIFERATION: A Clinicopathologic Correlation.

PURPOSE: To correlate clinical and optical coherence tomographic features with histopathological and immunohistochemical findings in an eye undergoing surgical excision of lamellar hole-associated epiretinal proliferation (LHEP). METHODS: An eye with a lamellar macular hole and LHEP without a tractional epiretinal membrane component was identified with spectral-domain optical coherence tomographic imaging and underwent pars plana vitrectomy with LHEP and internal limiting membrane peeling and gas tamponade. The surgically excised LHEP specimen was analyzed with histopathological and immunohistochemical staining using flat-mount preparation techniques. Postsurgical outcomes including visual acuity and optical coherence tomographic imaging were reviewed. RESULTS: With spectral-domain optical coherence tomography, the lamellar macular hole was found to be closed with no residual LHEP after the surgery. Visual acuity improved from 20/200 preoperatively to 20/40 at 6 months after the surgery. Histopathological and immunohistochemical analyses of the LHEP specimen revealed retinal glial cells that reacted positively with anti-glial fibrillary acidic protein and anti-glutamine synthetase, a Müller cell-specific antibody. CONCLUSION: Lamellar macular hole with LHEP may demonstrate closure after pars plana vitrectomy with LHEP and internal limiting membrane peeling and gas tamponade. There was considerable improvement in visual acuity. It is possible that LHEP originates from middle retinal layers of the lamellar hole defect because it contains retinal glial cells, specifically Müller cells.

Age-related increases in amyloid beta and membrane attack complex: evidence of inflammasome activation in the rodent eye.

BACKGROUND: The membrane attack complex (MAC) is a key player in the pathogenesis of age-related macular degeneration (AMD) and is a putative activator of the NLRP3 inflammasome. Amyloid beta (Aß), a component of drusen deposits, has also been implicated in inflammasome activation by our work and those of others. However, the interactions of MAC and Aß are still poorly understood, especially their roles in aging and retinal degenerative pathologies. Since inflammasome activation may represent a key cellular pathway underlying age-related chronic inflammation in the eye, the purpose of this study is to identify the effects associated with MAC and inflammasome activation in the retinal pigment epithelium (RPE)/choroid and to evaluate the therapeutic merits of MAC suppression. METHODS: Adult Long-Evans rats were divided into treatment and control groups. Treatment groups received oral aurin tricarboxylic acid complex (ATAC), a MAC inhibitor, in drinking-water, and control groups received drinking-water alone (No ATAC). Groups were sacrificed at 7.5 or 11.5 months, after approximately 40 days of ATAC treatment. To study age-related changes of Aß and MAC in RPE/choroid, naive animals were sacrificed at 2.5, 7.5, and 11.5 months. Eye tissues underwent immunohistochemistry and western blot analysis for MAC, Aß, NF-κB activation, as well as cleaved caspase-1 and IL-18. Vitreal samples were collected and assessed by multiplex assays for secreted levels of IL-18 and IL-1ß. Statistical analyses were performed, and significance level was set at p ≤ 0.05. RESULTS: In vivo studies demonstrated an age-dependent increase in MAC, Aß, and NF-κB activation in the RPE/choroid. Systemic ATAC resulted in a prominent reduction in MAC formation and a concomitant reduction in inflammasome activation measured by cleaved caspase-1 and secreted levels of IL-18 and IL-1ß, but not in NF-κB activation. In vitro studies demonstrated Aß-induced MAC formation on RPE cells. CONCLUSIONS: Age-dependent increases in Aß and MAC are present in the rodent outer retina. Our results suggest that suppressing MAC formation and subsequent inflammasome activation in the RPE/choroid may reduce chronic low-grade inflammation associated with IL-18 and IL-1ß in the outer retina.

CFH Y402H polymorphism is associated with elevated vitreal GM-CSF and choroidal macrophages in the postmortem human eye.

PURPOSE: Age-related macular degeneration (AMD) is the leading cause of irreversible blindness in people 50 years of age or older in developed countries. The homozygous CC genotype in the complement factor H (CFH) Y402H single nucleotide polymorphism (SNP; rs1061170) is widely recognized as a risk factor for the development of AMD. In this study, we examined vitreal levels of granulocyte macrophage colony-stimulating factor (GM-CSF), a hematopoietic cytokine, and macrophages in the choroid of postmortem human eyes genotyped for the CFH Y402H SNP. METHODS: Twenty-two pairs of postmortem, non-diseased, human donor eyes were obtained. The vitreous and retinal tissues of the left eyes were collected for GM-CSF level measurement and CFH Y402H genotyping, respectively. The right eyes were paraffin-embedded and sectioned for immunohistochemistry using a macrophage and microglia marker, CD68. Cell cultures of RPE cells were stimulated with complement C3a, C5a, 4-hydroxynonenal (HNE), or tumor necrosis factor alpha (TNF-α), and GM-CSF expression was measured with a suspension assay or quantitative PCR. RESULTS: Eyes genotyped with the CC or the CT risk variant of the CFH Y402H SNP showed significantly increased levels of GM-CSF in the vitreous compared to eyes with the protective TT variant (mean ± standard error of mean, 607.54±85.83 pg/ml or 656.32±15.20 pg/ml versus 286.69±81.96 pg/ml, p<0.05). The choroid of eye tissues genotyped with the CC variant showed higher levels of CD68 immunoreactivity than the tissues genotyped with the TT variant (p<0.05). The GM-CSF levels detected in the supernatant of RPE cells in culture treated with HNE or TNF-α were significantly higher compared to the non-treated control (145.88±5.06 pg/ml and 149.32±3.76 pg/ml versus 123.27±4.05 pg/ml, p<0.05). Furthermore, the gene expression of GM-CSF detected in the lysate of RPE cells stimulated with complement C3a or C5a showed significantly increased fold changes compared to the non-treated control (C3a: 2.38±0.31 fold, p<0.05; C5a: 2.84±0.54 fold, p<0.01). CONCLUSIONS: Our data showed a relationship between the CFH Y402H polymorphism and GM-CSF levels in the vitreous and accumulation of choroidal macrophages in the postmortem eye. These data suggest that the at-risk variant of the CFH gene may contribute to the dysregulation of proinflammatory cytokines locally in the eye.

NLRP3 inflammasome: activation and regulation in age-related macular degeneration.

Age-related macular degeneration (AMD) is the leading cause of legal blindness in the elderly in industrialized countries. AMD is a multifactorial disease influenced by both genetic and environmental risk factors. Progression of AMD is characterized by an increase in the number and size of drusen, extracellular deposits, which accumulate between the retinal pigment epithelium (RPE) and Bruch's membrane (BM) in outer retina. The major pathways associated with its pathogenesis include oxidative stress and inflammation in the early stages of AMD. Little is known about the interactions among these mechanisms that drive the transition from early to late stages of AMD, such as geographic atrophy (GA) or choroidal neovascularization (CNV). As part of the innate immune system, inflammasome activation has been identified in RPE cells and proposed to be a causal factor for RPE dysfunction and degeneration. Here, we will first review the classic model of inflammasome activation, then discuss the potentials of AMD-related factors to activate the inflammasome in both nonocular immune cells and RPE cells, and finally introduce several novel mechanisms for regulating the inflammasome activity.

Ranibizumab is a potential prophylaxis for proliferative vitreoretinopathy, a nonangiogenic blinding disease.

Proliferative vitreoretinopathy (PVR) exemplifies a disease that is difficult to predict, lacks effective treatment options, and substantially reduces the quality of life of an individual. Surgery to correct a rhegmatogenous retinal detachment fails primarily because of PVR. Likely mediators of PVR are growth factors in vitreous, which stimulate cells within and behind the retina as an inevitable consequence of a breached retina. Three classes of growth factors [vascular endothelial growth factor A (VEGF-A), platelet-derived growth factors (PDGFs), and non-PDGFs (growth factors outside of the PDGF family)] are relevant to PVR pathogenesis because they act on PDGF receptor α, which is required for experimental PVR and is associated with this disease in humans. We discovered that ranibizumab (a clinically approved agent that neutralizes VEGF-A) reduced the bioactivity of vitreous from patients and experimental animals with PVR, and protected rabbits from developing disease. The apparent mechanism of ranibizumab action involved derepressing PDGFs, which, at the concentrations present in PVR vitreous, inhibited non-PDGF-mediated activation of PDGF receptor α. These preclinical findings suggest that available approaches to neutralize VEGF-A are prophylactic for PVR, and that anti-VEGF-based therapies may be effective for managing more than angiogenesis- and edema-driven pathological conditions.

Vinpocetine inhibits amyloid-beta induced activation of NF-κB, NLRP3 inflammasome and cytokine production in retinal pigment epithelial cells.

Chronic inflammation is a key pathogenic process in age-related macular degeneration (AMD). Amyloid-beta (Aß) is a constituent of AMD drusen and promotes the activation of NLRP3 inflammasome which facilitates the production of cytokines. We investigated the role of transcription factor NF-κB in the activation of inflammasome in the RPE and the effect of vinpocetine, a dietary supplement with inhibitory effect on NF-κΒ. ARPE19/NF-κB-luciferase reporter cells treated with Aß demonstrated enhanced NF-κB activation that was significantly suppressed by vinpocetine. Intraperitoneal injection of vinpocetine (15 mg/kg) inhibited NF-κB nuclear translocation and reduced the expression and activation of NLRP3, caspase-1, IL-1ß, IL-18, and TNF-α in the RPE of adult rats that received intraocular Αß, as measured by retinal immunohistochemistry and Western blot. Cytokine level in the vitreous was assayed using multiplex suspension arrays and revealed significantly lower concentration of MIP-3α, IL-6, IL-1α, IL-1ß, IL-18, and TNF-α in vinpocetine treated animals. These results suggest that the NF-κB pathway is activated by Aß in the RPE and signals the priming of NLRP3 inflammasome and the expression of pro-inflammatory cytokines including the inflammasome substrates IL-1ß and IL-18. NF-κB inhibition may be an effective approach to stem the chronic inflammatory milieu that underlies the development of AMD. Vinpocetine is a potentially useful anti-inflammatory agent that is well-tolerated in long term use.