Activated monocytes resist elimination by retinal pigment epithelium and downregulate their OTX2 expression via TNF‐α

Orthodenticle homeobox 2 (OTX2) controls essential, homeostatic retinal pigment epithelial (RPE) genes in the adult. Using cocultures of human CD14⁺ blood monocytes (Mos) and primary porcine RPE cells and a fully humanized system using human‐induced pluripotent stem cell‐derived RPE cells, we show that activated Mos markedly inhibit RPEOTX2 expression and resist elimination in contact with the immunosuppressive RPE. Mechanistically, we demonstrate that TNF‐α, secreted from activated Mos, mediates the downregulation of OTX2 and essential RPE genes of the visual cycle among others. Our data show how subretinal, chronic inflammation and in particular TNF‐α can affect RPE function, which might contribute to the visual dysfunctions in diseases such as age‐related macular degeneration (AMD) where subretinal macrophages are observed. Our findings provide important mechanistic insights into the regulation of OTX2 under inflammatory conditions. Therapeutic restoration of OTX2 expression might help revive RPE and visual function in retinal diseases such as AMD.     

Glycyrrhizin protects against sodium iodate‐induced RPE and retinal injury though activation of AKT and Nrf2/HO‐1 pathway

Glycyrrhizin is a bioactive triterpenoid saponin extracted from a traditional Chinese medicinal herb, glycyrrhiza, and has been reported to protect the organs such as liver and heart from injuries. However, there is no report about the effects of glycyrrhizin on atrophic age‐related macular degeneration (AMD). This study investigated the effects of glycyrrhizin on retinal pigment epithelium (RPE) in vitro and retina of mice in vivo treated with sodium iodate (SI). Glycyrrhizin significantly inhibited SI‐induced reactive oxygen species (ROS), and decreased apoptosis of RPE in vitro. The underlying mechanisms included increased phosphorylation of Akt, and increased expression of nuclear factor erythroid 2‐related factor2 (Nrf‐2) and HO‐1, thereby protecting RPE from SI‐induced ROS and apoptosis. Furthermore, glycyrrhizin significantly decreased the apoptosis of retinal cells in vivo, resulting in the inhibition of thinning of retina, decreasing the number of drusen and improving the function of retina. These findings suggested that glycyrrhizin may be a potential candidate for the treatment of atrophic AMD in clinical practice.     

Expression of VEGFA‐regulating miRNAs and mortality in wet AMD

MicroRNAs (miRNAs) regulate gene expression; many of them act in the retinal pigment epithelium (RPE), and RPE degeneration is known to be a critical factor in age‐related macular degeneration (AMD). Repeated injections with anti‐VEGFA (vascular endothelial growth factor A) are the only effective therapy in wet AMD. We investigated the correlation between the expression of 18 miRNAs involved in the regulation of the VEGFA gene in serum of 76 wet AMD patients and 70 controls. Efficacy of anti‐VEGFA treatment was evaluated by counting the number of injections delivered up to 12 years. In addition, we compared the relative numbers of deaths in patient with AMD and control groups. We observed a decreased expression of miR‐34‐5p, miR‐126‐3p, miR‐145‐5p and miR‐205‐5p in wet AMD patients as compared with controls. These miRNAs are involved in the regulation of angiogenesis, cytoprotection and protein clearance. No miRNA was significantly correlated with the treatment outcome. Wet AMD patients had greater mortality than controls, and their survival was inversely associated with the number of anti‐VEGFA injections per year. No association was observed between miRNA expression and mortality. Our study emphasizes the need to clarify the role of miRNA regulation in AMD pathogenesis.     

Lysosomal-mediated waste clearance in retinal pigment epithelial cells is regulated by CRYBA1/βA3/A1-crystallin via V-ATPase-MTORC1 signaling

In phagocytic cells, including the retinal pigment epithelium (RPE), acidic compartments of the endolysosomal system are regulators of both phagocytosis and autophagy, thereby helping to maintain cellular homeostasis. The acidification of the endolysosomal system is modulated by a proton pump, the V-ATPase, but the mechanisms that direct the activity of the V-ATPase remain elusive. We found that in RPE cells, CRYBA1/βA3/A1-crystallin, a lens protein also expressed in RPE, is localized to lysosomes, where it regulates endolysosomal acidification by modulating the V-ATPase, thereby controlling both phagocytosis and autophagy. We demonstrated that CRYBA1 coimmunoprecipitates with the ATP6V0A1/V₀-ATPase a1 subunit. Interestingly, in mice when Cryba1 (the gene encoding both the βA3- and βA1-crystallin forms) is knocked out specifically in RPE, V-ATPase activity is decreased and lysosomal pH is elevated, while cathepsin D (CTSD) activity is decreased. Fundus photographs of these Cryba1 conditional knockout (cKO) mice showed scattered lesions by 4 months of age that increased in older mice, with accumulation of lipid-droplets as determined by immunohistochemistry. Transmission electron microscopy (TEM) of cryba1 cKO mice revealed vacuole-like structures with partially degraded cellular organelles, undigested photoreceptor outer segments and accumulation of autophagosomes. Further, following autophagy induction both in vivo and in vitro, phospho-AKT and phospho-RPTOR/Raptor decrease, while pMTOR increases in RPE cells, inhibiting autophagy and AKT-MTORC1 signaling. Impaired lysosomal clearance in the RPE of the cryba1 cKO mice also resulted in abnormalities in retinal function that increased with age, as demonstrated by electroretinography. Our findings suggest that loss of CRYBA1 causes lysosomal dysregulation leading to the impairment of both autophagy and phagocytosis.     

Serine racemase deficiency attenuates choroidal neovascularization and reduces nitric oxide and VEGF levels by retinal pigment epithelial cells

Choroidal neovascularization (CNV) is a leading cause of blindness in age‐related macular degeneration. Production of vascular endothelial growth factor (VEGF) and macrophage recruitment by retinal pigment epithelial cells (RPE) significantly contributes to the process of CNV in an experimental CNV model. Serine racemase (SR) is expressed in retinal neurons and glial cells, and its product, d ‐serine, is an endogenous co‐agonist of N‐methyl‐d ‐aspartate receptor. Activation of the receptor results in production of nitric oxide (^.NO), a molecule that promotes retinal and choroidal neovascularization. These observations suggest possible roles of SR in CNV. With laser‐injured CNV mice, we found that inactivation of SR‐coding gene (Srr_null) significantly reduced CNV volume, neovascular density, and invading macrophages. We exploited the underlying mechanism in vivo and ex vivo. RPE from wild‐type (WT) mice expressed SR. To explore the possible downstream target of SR inactivation, we showed that choroid/RPE homogenates extracted from laser‐injured Srr_null mice contained less inducible nitric oxide synthase and decreased phospho‐VEGFR2 compared to amounts in WT mice. In vitro, inflammation‐primed WT RPEs expressed more inducible NOS, produced more^.NO and VEGF than did inflammation‐primed Srr_null RPEs. When co‐cultured with inflammation‐primed Srr_null RPE, significantly fewer RF/6A‐a cell line of choroidal endothelial cell, migrated to the opposite side of the insert membrane than did cells co‐cultured with pre‐treated WT RPE. Altogether, SR deficiency reduces RPE response to laser‐induced inflammatory stimuli, resulting in decreased production of a cascade of pro‐angiogenic cytokines, including^.NO and VEGF, and reduced macrophage recruitment, which contribute synergistically to attenuated angiogenesis.

     

Advanced glycation end products‐related modulation of cathepsin L and NF‐κB signalling effectors in retinal pigment epithelium lead to augmented response to TNFα

The retinal pigment epithelium (RPE) plays a central role in neuroretinal homoeostasis throughout life. Altered proteolysis and inflammatory processes involving RPE contribute to the pathophysiology of age‐related macular degeneration (AMD), but the link between these remains elusive. We report for the first time the effect of advanced glycation end products (AGE)—known to accumulate on the ageing RPE's underlying Bruch's membrane in situ—on both key lysosomal cathepsins and NF‐κB signalling in RPE. Cathepsin L activity and NF‐κB effector levels decreased significantly following 2‐week AGE exposure. Chemical cathepsin L inhibition also decreased total p65 protein levels, indicating that AGE‐related change of NF‐κB effectors in RPE cells may be modulated by cathepsin L. However, upon TNFα stimulation, AGE‐exposed cells had significantly higher ratio of phospho‐p65(Ser536)/total p65 compared to non‐AGEd controls, with an even higher fold increase than in the presence of cathepsin L inhibition alone. Increased proportion of active p65 indicates an AGE‐related activation of NF‐κB signalling in a higher proportion of cells and/or an enhanced response to TNFα. Thus, NF‐κB signalling modulation in the AGEd environment, partially regulated via cathepsin L, is employed by RPE cells as a protective (para‐inflammatory) mechanism but renders them more responsive to pro‐inflammatory stimuli.     

Amyloid β induces NLRP3 inflammasome activation in retinal pigment epithelial cells via NADPH oxidase‐ and mitochondria‐dependent ROS production

Amyloid β (Aβ)‐induced chronic inflammation is believed to be a key pathogenic process in early‐stage age‐related macular degeneration (AMD). Nucleotide oligomerization domain (NOD)‐like receptor family, pyrin domain containing 3 (NLRP3) inflammasome activation triggered by Aβ is responsible for retinal pigment epithelium (RPE) dysfunction in the onset of AMD; however, the detailed molecular mechanism remains unclear. In this study, we investigated the involvement of NADPH oxidase‐ and mitochondria‐derived reactive oxygen species (ROS) in the process of Aβ_1–40‐induced NLRP3 inflammasome activation in LPS‐primed ARPE‐19 cells. The results showed that Aβ_1–40 could induce excessive ROS generation, MAPK/NF‐κB signaling activation and subsequently NLRP3 inflammasome activation in LPS‐primed ARPE‐19 cells. Furthermore, the inductive effect of Aβ_1–40 on NLRP3 inflammasome activation was mediated in a manner dependent on NADPH oxidase‐ and mitochondria‐derived ROS. Our findings may provide a novel insight into the molecular mechanism by which Aβ contributes to the early‐stage AMD.     

Alternative Complement Pathway Deficiency Ameliorates Chronic Smoke-Induced Functional and Morphological Ocular Injury

Background

Age-related macular degeneration (AMD), a complex disease involving genetic variants and environmental insults, is among the leading causes of blindness in Western populations. Genetic and histologic evidence implicate the complement system in AMD pathogenesis; and smoking is the major environmental risk factor associated with increased disease risk. Although previous studies have demonstrated that cigarette smoke exposure (CE) causes retinal pigment epithelium (RPE) defects in mice, and smoking leads to complement activation in patients, it is unknown whether complement activation is causative in the development of CE pathology; and if so, which complement pathway is required.

Methods

Mice were exposed to cigarette smoke or clean, filtered air for 6 months. The effects of CE were analyzed in wildtype (WT) mice or mice without a functional complement alternative pathway (AP; CFB^−/−) using molecular, histological, electrophysiological, and behavioral outcomes.

Results

CE in WT mice exhibited a significant reduction in function of both rods and cones as determined by electroretinography and contrast sensitivity measurements, concomitant with a thinning of the nuclear layers as measured by SD-OCT imaging and histology. Gene expression analyses suggested that alterations in both photoreceptors and RPE/choroid might contribute to the observed loss of function, and visualization of complement C3d deposition implies the RPE/Bruch''s membrane (BrM) complex as the target of AP activity. RPE/BrM alterations include an increase in mitochondrial size concomitant with an apical shift in mitochondrial distribution within the RPE and a thickening of BrM. CFB^−/− mice were protected from developing these CE-mediated alterations.

Conclusions

Taken together, these findings provide clear evidence that ocular pathology generated in CE mice is dependent on complement activation and requires the AP. Identifying animal models with RPE/BrM damage and verifying which aspects of pathology are dependent upon complement activation is essential for developing novel complement-based treatment approaches for the treatment of AMD.     

Cigarette smoke-related hydroquinone dysregulates MCP-1, VEGF and PEDF expression in retinal pigment epithelium in vitro and in vivo

Background

Age-related macular degeneration (AMD) is the leading cause of legal blindness in the elderly population. Debris (termed drusen) below the retinal pigment epithelium (RPE) have been recognized as a risk factor for dry AMD and its progression to wet AMD, which is characterized by choroidal neovascularization (CNV). The underlying mechanism of how drusen might elicit CNV remains undefined. Cigarette smoking, oxidative damage to the RPE and inflammation are postulated to be involved in the pathophysiology of the disease. To better understand the cellular mechanism(s) linking oxidative stress and inflammation to AMD, we examined the expression of pro-inflammatory monocyte chemoattractant protein-1 (MCP-1), pro-angiogenic vascular endothelial growth factor (VEGF) and anti-angiogenic pigment epithelial derived factor (PEDF) in RPE from smoker patients with AMD. We also evaluated the effects of hydroquinone (HQ), a major pro-oxidant in cigarette smoke on MCP-1, VEGF and PEDF expression in cultured ARPE-19 cells and RPE/choroids from C57BL/6 mice.

Principal Findings

MCP-1, VEGF and PEDF expression was examined by real-time PCR, Western blot, and ELISA. Low levels of MCP-1 protein were detected in RPE from AMD smoker patients relative to controls. Both MCP-1 mRNA and protein were downregulated in ARPE-19 cells and RPE/choroids from C57BL/6 mice after 5 days and 3 weeks of exposure to HQ-induced oxidative injury. VEGF protein expression was increased and PEDF protein expression was decreased in RPE from smoker patients with AMD versus controls resulting in increased VEGF/PEDF ratio. Treatment with HQ for 5 days and 3 weeks increased the VEGF/PEDF ratio in vitro and in vivo.

Conclusion

We propose that impaired RPE-derived MCP-1-mediated scavenging macrophages recruitment and phagocytosis might lead to incomplete clearance of proinflammatory debris and infiltration of proangiogenic macrophages which along with increased VEGF/PEDF ratio favoring angiogenesis might promote drusen accumulation and progression to CNV in smoker patients with dry AMD.     

Lipocalin‐2 released in response to cerebral ischaemia mediates reperfusion injury in mice

Thrombolysis remains the only effective therapy to reverse acute ischaemic stroke. However, delayed treatment may cause serious complications including hemorrhagic transformation and reperfusion injury. The level of lipocalin‐2 (LCN2) is elevated in the plasma of ischaemic stroke patients, but its role in stroke is unknown. Here, we show that LCN2 was acutely induced in mice after ischaemic stroke and is an important mediator of reperfusion injury. Increased levels of LCN2 were observed in mouse serum as early as 1 hr after transient middle cerebral artery occlusion (tMCAO), reaching peak levels at 23 hrs. LCN2 was also detected in neutrophils infiltrating into the ipsilateral hemisphere, as well as a subset of astrocytes after tMCAO, but not in neurons and microglia. Stroke injury, neurological deficits and infiltration of immune cells were markedly diminished in LCN2 null mice after tMCAO, but not after permanent MCAO (pMCAO). In vitro, recombinant LCN2 protein induced apoptosis in primary cultured neurons in a dose‐dependent manner. Our results demonstrate that LCN2 is a neurotoxic factor secreted rapidly in response to cerebral ischaemia, suggesting its potential usage as an early stroke biomarker and a novel therapeutic target to reduce stroke‐reperfusion injury.     

Aged regulatory T cells protect from autoimmune inflammation despite reduced STAT3 activation and decreased constraint of IL-17 producing T cells

Regulatory T cells (Tregs) are specialized CD4⁺ T lymphocytes helping defend against autoimmunity and inflammation. Although age is associated with increased inflammation and autoimmunity, few reports address age effects of immune regulation or auto‐aggressive T cells. We show here that young and aged naïve CD4⁺ T cells are equivalently auto‐aggressive in vivo in T cell‐driven autoimmune colitis. Young and aged CD4⁺ Tregs equally suppressed age‐matched T cell proliferation in vitro and controlled clinical and pathologic T cell‐driven autoimmune colitis, suggesting equivalent regulatory function. However, whereas young and aged CD4⁺ Tregs suppressed interferon (IFN)‐γ⁺ T cells equivalently in this model, aged CD4⁺ Tregs unexpectedly failed to restrain interleukin (IL)‐17⁺ T cells. Nonetheless, young and aged CD4⁺ Tregs equally restrained IL‐17⁺ T cells in vivo during acute inflammation, suggesting a chronic inflammation‐related defect in aged CD4⁺ Tregs. In support, aged Tregs expressed reduced STAT3 activation, a defect associated with poor IL‐17‐producing T cell restraint. Aged naïve mice had markedly increased programmed death (PD)‐1⁺ T cells, but these exhibited no significant auto‐aggressive or regulatory functions in T cell‐driven colitis. Young CD8⁺ CD122^? T cells induce autoimmune bone marrow failure, but we show that aged CD8⁺ CD122^? T cells do not. These data demonstrate no apparent age‐related increase in auto‐aggressive T cell behavior, but disclose previously unrecognized functional defects in aged CD4⁺ Tregs during chronic inflammation. IL‐17 can be inflammatory and contributes to certain autoimmune disorders. Reduced aged Treg function during chronic inflammation and reduced IL‐17 restraint could contribute to age‐related inflammation or autoimmunity.     

Retinal pigment epithelial cell multinucleation in the aging eye – a mechanism to repair damage and maintain homoeostasis

Retinal pigment epithelial (RPE) cells are central to retinal health and homoeostasis. Dysfunction or death of RPE cells underlies many age‐related retinal degenerative disorders particularly age‐related macular degeneration. During aging RPE cells decline in number, suggesting an age‐dependent cell loss. RPE cells are considered to be postmitotic, and how they repair damage during aging remains poorly defined. We show that RPE cells increase in size and become multinucleate during aging in C57BL/6J mice. Multinucleation appeared not to be due to cell fusion, but to incomplete cell division, that is failure of cytokinesis. Interestingly, the phagocytic activity of multinucleate RPE cells was not different from that of mononuclear RPE cells. Furthermore, exposure of RPE cells in vitro to photoreceptor outer segment (POS), particularly oxidized POS, dose‐dependently promoted multinucleation and suppressed cell proliferation. Both failure of cytokinesis and suppression of proliferation required contact with POS. Exposure to POS also induced reactive oxygen species and DNA oxidation in RPE cells. We propose that RPE cells have the potential to proliferate in vivo and to repair defects in the monolayer. We further propose that the conventionally accepted ‘postmitotic’ status of RPE cells is due to a modified form of contact inhibition mediated by POS and that RPE cells are released from this state when contact with POS is lost. This is seen in long‐standing rhegmatogenous retinal detachment as overtly proliferating RPE cells (proliferative vitreoretinopathy) and more subtly as multinucleation during normal aging. Age‐related oxidative stress may promote failure of cytokinesis and multinucleation in RPE cells.     

Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD

Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.     

An animal model of age-related macular degeneration in senescent Ccl-2- or Ccr-2-deficient mice

The study and treatment of age-related macular degeneration (AMD), a leading cause of blindness, has been hampered by a lack of animal models. Here we report that mice deficient either in monocyte chemoattractant protein-1 (Ccl-2; also known as MCP-1) or its cognate C-C chemokine receptor-2 (Ccr-2) develop cardinal features of AMD, including accumulation of lipofuscin in and drusen beneath the retinal pigmented epithelium (RPE), photoreceptor atrophy and choroidal neovascularization (CNV). Complement and IgG deposition in RPE and choroid accompanies senescence in this model, as in human AMD. RPE or choroidal endothelial production of Ccl-2 induced by complement C5a and IgG may mediate choroidal macrophage infiltration into aged wild-type choroids. Wild-type choroidal macrophages degrade C5 and IgG in eye sections of Ccl2(-/-) or Ccr2(-/-) mice. Impaired macrophage recruitment may allow accumulation of C5a and IgG, which induces vascular endothelial growth factor (VEGF) production by RPE, possibly mediating development of CNV. These models implicate macrophage dysfunction in AMD pathogenesis and may be useful as a platform for validating therapies.     

Directional protein secretion by the retinal pigment epithelium: roles in retinal health and the development of age‐related macular degeneration

The structural and functional integrity of the retinal pigment epithelium (RPE) is fundamental for maintaining the function of the neuroretina. These specialized cells form a polarized monolayer that acts as the retinal–blood barrier, separating two distinct environments with highly specialized functions: photoreceptors of the neuroretina at the apical side and Bruch's membrane/highly vascularized choriocapillaris at the basal side. The polarized nature of the RPE is essential for the health of these two regions, not only in nutrient and waste transport but also in the synthesis and directional secretion of proteins required in maintaining retinal homoeostasis and function. Although multiple malfunctions within the RPE cells have been associated with development of age‐related macular degeneration (AMD), the leading cause of legal blindness, clear causative processes have not yet been conclusively characterized at the molecular and cellular level. This article focuses on the involvement of directionally secreted RPE proteins in normal functioning of the retina and on the potential association of incorrect RPE protein secretion with development of AMD. Understanding the importance of RPE polarity and the correct secretion of essential structural and regulatory components emerge as critical factors for the development of novel therapeutic strategies targeting AMD.     

Dysregulated autophagy in the RPE is associated with increased susceptibility to oxidative stress and AMD

Autophagic dysregulation has been suggested in a broad range of neurodegenerative diseases including age-related macular degeneration (AMD). To test whether the autophagy pathway plays a critical role to protect retinal pigmented epithelial (RPE) cells against oxidative stress, we exposed ARPE-19 and primary cultured human RPE cells to both acute (3 and 24 h) and chronic (14 d) oxidative stress and monitored autophagy by western blot, PCR, and autophagosome counts in the presence or absence of autophagy modulators. Acute oxidative stress led to a marked increase in autophagy in the RPE, whereas autophagy was reduced under chronic oxidative stress. Upregulation of autophagy by rapamycin decreased oxidative stress-induced generation of reactive oxygen species (ROS), whereas inhibition of autophagy by 3-methyladenine (3-MA) or by knockdown of ATG7 or BECN1 increased ROS generation, exacerbated oxidative stress-induced reduction of mitochondrial activity, reduced cell viability, and increased lipofuscin. Examination of control human donor specimens and mice demonstrated an age-related increase in autophagosome numbers and expression of autophagy proteins. However, autophagy proteins, autophagosomes, and autophagy flux were significantly reduced in tissue from human donor AMD eyes and 2 animal models of AMD. In conclusion, our data confirm that autophagy plays an important role in protection of the RPE against oxidative stress and lipofuscin accumulation and that impairment of autophagy is likely to exacerbate oxidative stress and contribute to the pathogenesis of AMD.     

Autophagy deficiency in myeloid cells increases susceptibility to obesity-induced diabetes and experimental colitis

Autophagy, which is critical for the proper turnover of organelles such as endoplasmic reticulum and mitochondria, affects diverse aspects of metabolism, and its dysregulation has been incriminated in various metabolic disorders. However, the role of autophagy of myeloid cells in adipose tissue inflammation and type 2 diabetes has not been addressed. We produced mice with myeloid cell-specific deletion of Atg7 (autophagy-related 7), an essential autophagy gene (Atg7 conditional knockout [cKO] mice). While Atg7 cKO mice were metabolically indistinguishable from control mice, they developed diabetes when bred to ob/w mice (Atg7 cKO-ob/ob mice), accompanied by increases in the crown-like structure, inflammatory cytokine expression and inflammasome activation in adipose tissue. Mφs (macrophages) from Atg7 cKO mice showed significantly higher interleukin 1 β release and inflammasome activation in response to a palmitic acid plus lipopolysaccharide combination. Moreover, a decrease in the NAD⁺:NADH ratio and increase in intracellular ROS content after treatment with palmitic acid in combination with lipopolysaccharide were more pronounced in Mφs from Atg7 cKO mice, suggesting that mitochondrial dysfunction in autophagy-deficient Mφs leads to an increase in lipid-induced inflammasome and metabolic deterioration in Atg7 cKO-ob/ob mice. Atg7 cKO mice were more susceptible to experimental colitis, accompanied by increased colonic cytokine expression, T helper 1 skewing and systemic bacterial invasion. These results suggest that autophagy of Mφs is important for the control of inflammasome activation in response to metabolic or extrinsic stress, and autophagy deficiency in Mφs may contribute to the progression of metabolic syndrome associated with lipid injury and colitis.     

Neuron-specific TGF-beta signaling deficiency results in retinal detachment and cataracts in mice

We generated a mouse model (cKO) with a conditional deletion of TGF-beta signaling in the retinal neurons by crossing TGF-beta receptor I (TGF-beta RI) floxed mice with nestin-Cre mice. Almost all of the newborn cKO mice had retinal detachment at the retinal pigment epithelium (RPE)/photoreceptor layer junction of the neurosensory retina (NSR). The immunostaining for chondroitin-6-sulfate showed a very weak reaction in cKO mice in contrast to intense staining in the photoreceptor layer in wild-type mice. Macroscopic cataracts, in one or both eyes, were observed in 50% of the mice by 6 months of age, starting as early as the first month after birth. The cKO mouse model demonstrates that the TGF-beta signaling deficiency in retinal cells leads to decreased levels of chondroitin sulfate proteoglycan in the retinal interphotoreceptor matrix. This in turn causes retinal detachment due to the loss of adhesion of the NSR to RPE.     

HTRA1, an age‐related macular degeneration protease,processes extracellular matrix proteins EFEMP1 and TSP1

High‐temperature requirement protein A1 (HTRA1) is a serine protease secreted by a number of tissues including retinal pigment epithelium (RPE). A promoter variant of the gene encoding HTRA1 is part of a mutant allele that causes increased HTRA1 expression and contributed to age‐related macular degeneration (AMD) in genomewide association studies. AMD is characterized by pathological development of drusen, extracellular deposits of proteins and lipids on the basal side of RPE. The molecular pathogenesis of AMD is not well understood, and understanding dysregulation of the extracellular matrix may be key. We assess the high‐risk genotype at 10q26 by proteomic comparison of protein levels of RPE cells with and without the mutation. We show HTRA1 protein level is increased in high‐risk RPE cells along with several extracellular matrix proteins, including known HTRA1 cleavage targets LTBP‐1 and clusterin. In addition, two novel targets of HTRA1 have been identified: EFEMP1, an extracellular matrix protein mutated in Doyne honeycomb retinal dystrophy, a genetic eye disease similar to AMD, and thrombospondin 1 (TSP1), an inhibitor of angiogenesis. Our data support the role of RPE extracellular deposition with potential effects in compromised barrier to neovascularization in exudative AMD.