Subretinal fibrosis is a result of a wound healing response that follows choroidal neovascularization in neovascular age-related macular degeneration (nAMD). Although anti-vascular endothelial growth ...factor therapy has become a standard treatment that improves visual acuity in many nAMD patients, unsuccessful treatment outcomes have often been attributed to the progression of subretinal fibrosis. In this review, we summarize the cellular and extracellular components of subretinal fibrous membranes and also discuss the possible molecular mechanisms including the functional involvement of growth factors and the inflammatory response in the process. Moreover, we present an murine animal model of subretinal fibrosis that might facilitate greater understanding of the pathophysiology and the development of novel therapeutic strategies for the inhibition of subretinal fibrosis in nAMD.
•Clinical implications of subretinal fibrosis in AMD.•Cellular and extracellular components of subretinal fibrous membranes.•Growth factors and epithelial–mesenchymal transition in subretinal fibrosis.•Immune response related to subretinal fibrosis.•Use of the laser-induced murine model of CNV to study subretinal fibrosis.
Humanin (HN) is a small mitochondrial-encoded peptide with neuroprotective properties. We have recently shown protection of retinal pigmented epithelium (RPE) cells by HN in oxidative stress; ...however, the effect of HN on endoplasmic reticulum (ER) stress has not been evaluated in any cell type. Our aim here was to study the effect of HN on ER stress-induced apoptosis in RPE cells with a specific focus on ER-mitochondrial cross-talk. Dose dependent effects of ER stressors (tunicamycin (TM), brefeldin A, and thapsigargin) were studied after 12 hr of treatment in confluent primary human RPE cells with or without 12 hr of HN pretreatment (1-20 μg/mL). All three ER stressors induced RPE cell apoptosis in a dose dependent manner. HN pretreatment significantly decreased the number of apoptotic cells with all three ER stressors in a dose dependent manner. HN pretreatment similarly protected U-251 glioma cells from TM-induced apoptosis in a dose dependent manner. HN pretreatment significantly attenuated activation of caspase 3 and ER stress-specific caspase 4 induced by TM. TM treatment increased mitochondrial superoxide production, and HN co-treatment resulted in a decrease in mitochondrial superoxide compared to TM treatment alone. We further showed that depleted mitochondrial glutathione (GSH) levels induced by TM were restored with HN co-treatment. No significant changes were found for the expression of several antioxidant enzymes between TM and TM plus HN groups except for the expression of glutamylcysteine ligase catalytic subunit (GCLC), the rate limiting enzyme required for GSH biosynthesis, which is upregulated with TM and TM+HN treatment. These results demonstrate that ER stress promotes mitochondrial alterations in RPE that lead to apoptosis. We further show that HN has a protective effect against ER stress-induced apoptosis by restoring mitochondrial GSH. Thus, HN should be further evaluated for its therapeutic potential in disorders linked to ER stress.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Pericyte loss and deficient vascular platelet-derived growth factor receptor-β (PDGFRβ) signaling are prominent features of the blood–brain barrier breakdown described in Alzheimer’s disease (AD) ...that can predict cognitive decline yet have never been studied in the retina. Recent reports using noninvasive retinal amyloid imaging, optical coherence tomography angiography, and histological examinations support the existence of vascular-structural abnormalities and vascular amyloid β-protein (Aβ) deposits in retinas of AD patients. However, the cellular and molecular mechanisms of such retinal vascular pathology were not previously explored. Here, by modifying a method of enzymatically clearing non-vascular retinal tissue and fluorescent immunolabeling of the isolated blood vessel network, we identified substantial pericyte loss together with significant Aβ deposition in retinal microvasculature and pericytes in AD. Evaluation of postmortem retinas from a cohort of 56 human donors revealed an early and progressive decrease in vascular PDGFRβ in mild cognitive impairment (MCI) and AD compared to cognitively normal controls. Retinal PDGFRβ loss significantly associated with increased retinal vascular Aβ
40
and Aβ
42
burden. Decreased vascular LRP-1 and early apoptosis of pericytes in AD retina were also detected. Mapping of PDGFRβ and Aβ
40
levels in pre-defined retinal subregions indicated that certain geometrical and cellular layers are more susceptible to AD pathology. Further, correlations were identified between retinal vascular abnormalities and cerebral Aβ burden, cerebral amyloid angiopathy (CAA), and clinical status. Overall, the identification of pericyte and PDGFRβ loss accompanying increased vascular amyloidosis in Alzheimer’s retina implies compromised blood–retinal barrier integrity and provides new targets for AD diagnosis and therapy.
To investigate the expression of humanin (HN) in human retinal pigment epithelial (hRPE) cells and its effect on oxidative stress-induced cell death, mitochondrial bioenergetics, and senescence.
...Humanin localization in RPE cells and polarized RPE monolayers was assessed by confocal microscopy. Human RPE cells were treated with 150 μM tert-Butyl hydroperoxide (tBH) in the absence/presence of HN (0.5-10 μg/mL) for 24 hours. Mitochondrial respiration was measured by XF96 analyzer. Retinal pigment epithelial cell death and caspase-3 activation, mitochondrial biogenesis and senescence were analyzed by TUNEL, immunoblot analysis, mitochondrial DNA copy number, SA-β-Gal staining, and p16INK4a expression and HN levels by ELISA. Oxidative stress-induced changes in transepithelial resistance were studied in RPE monolayers with and without HN cotreatment.
A prominent localization of HN was found in the cytoplasmic and mitochondrial compartments of hRPE. Humanin cotreatment inhibited tBH-induced reactive oxygen species formation and significantly restored mitochondrial bioenergetics in hRPE cells. Exogenous HN was taken up by RPE and colocalized with mitochondria. The oxidative stress-induced decrease in mitochondrial bioenergetics was prevented by HN cotreatment. Humanin treatment increased mitochondrial DNA copy number and upregulated mitochondrial transcription factor A, a key biogenesis regulator protein. Humanin protected RPE cells from oxidative stress-induced cell death by STAT3 phosphorylation and inhibiting caspase-3 activation. Humanin treatment inhibited oxidant-induced senescence. Polarized RPE demonstrated elevated cellular HN and increased resistance to cell death.
Humanin protected RPE cells against oxidative stress-induced cell death and restored mitochondrial function. Our data suggest a potential role for HN therapy in the prevention of retinal degeneration, including AMD.
Noninvasive detection of Alzheimer's disease (AD) with high specificity and sensitivity can greatly facilitate identification of at-risk populations for earlier, more effective intervention. AD ...patients exhibit a myriad of retinal pathologies, including hallmark amyloid β-protein (Aβ) deposits.
Burden, distribution, cellular layer, and structure of retinal Aβ plaques were analyzed in flat mounts and cross sections of definite AD patients and controls (n = 37). In a proof-of-concept retinal imaging trial (n = 16), amyloid probe curcumin formulation was determined and protocol was established for retinal amyloid imaging in live patients.
Histological examination uncovered classical and neuritic-like Aβ deposits with increased retinal Aβ42 plaques (4.7-fold; P = 0.0063) and neuronal loss (P = 0.0023) in AD patients versus matched controls. Retinal Aβ plaque mirrored brain pathology, especially in the primary visual cortex (P = 0.0097 to P = 0.0018; Pearson's r = 0.84-0.91). Retinal deposits often associated with blood vessels and occurred in hot spot peripheral regions of the superior quadrant and innermost retinal layers. Transmission electron microscopy revealed retinal Aβ assembled into protofibrils and fibrils. Moreover, the ability to image retinal amyloid deposits with solid-lipid curcumin and a modified scanning laser ophthalmoscope was demonstrated in live patients. A fully automated calculation of the retinal amyloid index (RAI), a quantitative measure of increased curcumin fluorescence, was constructed. Analysis of RAI scores showed a 2.1-fold increase in AD patients versus controls (P = 0.0031).
The geometric distribution and increased burden of retinal amyloid pathology in AD, together with the feasibility to noninvasively detect discrete retinal amyloid deposits in living patients, may lead to a practical approach for large-scale AD diagnosis and monitoring.
National Institute on Aging award (AG044897) and The Saban and The Marciano Family Foundations.
To evaluate cell survival and tumorigenicity of human embryonic stem cell-derived retinal pigment epithelium (hESC-RPE) transplantation in immunocompromised nude rats. Cells were transplanted as a ...cell suspension (CS) or as a polarized monolayer plated on a parylene membrane (PM).
Sixty-nine rats (38 male, 31 female) were surgically implanted with CS (n = 33) or PM (n = 36). Cohort subsets were killed at 1, 6, and 12 months after surgery. Both ocular tissues and systemic organs (brain, liver, kidneys, spleen, heart, and lungs) were fixed in 4% paraformaldehyde, embedded in paraffin, and sectioned. Every fifth section was stained with hematoxylin and eosin and analyzed histologically. Adjacent sections were processed for immunohistochemical analysis (as needed) using the following antibodies: anti-RPE65 (RPE-specific marker), anti-TRA-1-85 (human cell marker), anti-Ki67 (proliferation marker), anti-CD68 (macrophage), and anti-cytokeratin (epithelial marker).
The implanted cells were immunopositive for the RPE65 and TRA-1-85. Cell survival (P = 0.006) and the presence of a monolayer (P < 0.001) of hESC-RPE were significantly higher in eyes that received the PM. Gross morphological and histological analysis of the eye and the systemic organs after the surgery revealed no evidence of tumor or ectopic tissue formation in either group.
hESC-RPE can survive for at least 12 months in an immunocompromised animal model. Polarized monolayers of hESC-RPE show improved survival compared to cell suspensions. The lack of teratoma or any ectopic tissue formation in the implanted rats bodes well for similar results with respect to safety in human subjects.
Age-related macular degeneration (AMD) is the leading cause of blindness among the elderly in developed countries. AMD is classified as either neovascular (NV-AMD) or non-neovascular (NNV-AMD). ...Cumulative damage to the retinal pigment epithelium, Bruch's membrane, and choriocapillaris leads to dysfunction and loss of RPE cells. This causes degeneration of the overlying photoreceptors and consequential vision loss in advanced NNV-AMD (Geographic Atrophy). In NV-AMD, abnormal growth of capillaries under the retina and RPE, which leads to hemorrhage and fluid leakage, is the main cause of photoreceptor damage. Although a number of drugs (e.g., anti-VEGF) are in use for NV-AMD, there is currently no treatment for advanced NNV-AMD. However, replacing dead or dysfunctional RPE with healthy RPE has been shown to rescue dying photoreceptors and improve vision in animal models of retinal degeneration and possibly in AMD patients. Differentiation of RPE from human embryonic stem cells (hESC-RPE) and from induced pluripotent stem cells (iPSC-RPE) has created a potentially unlimited source for replacing dead or dying RPE. Such cells have been shown to incorporate into the degenerating retina and result in anatomic and functional improvement. However, major ethical, regulatory, safety, and technical challenges have yet to be overcome before stem cell-based therapies can be used in standard treatments. This review outlines the current knowledge surrounding the application of hESC-RPE and iPSC-RPE in AMD. Following an introduction on the pathogenesis and available treatments of AMD, methods to generate stem cell-derived RPE, immune reaction against such cells, and approaches to deliver desired cells into the eye will be explored along with broader issues of efficacy and safety. Lastly, strategies to improve these stem cell-based treatments will be discussed.
Age-related macular degeneration (AMD) is a leading cause of blindness in the developed world. The retinal pigment epithelium (RPE) is a critical site of pathology in AMD and αB crystallin expression ...is increased in RPE and associated drusen in AMD. The purpose of this study was to investigate the role of αB crystallin in sodium iodate (NaIO3)-induced retinal degeneration, a model of AMD in which the primary site of pathology is the RPE. Dose dependent effects of intravenous NaIO3 (20-70 mg/kg) on development of retinal degeneration (fundus photography) and RPE and retinal neuronal loss (histology) were determined in wild type and αB crystallin knockout mice. Absence of αB crystallin augmented retinal degeneration in low dose (20 mg/kg) NaIO3-treated mice and increased retinal cell apoptosis which was mainly localized to the RPE layer. Generation of reactive oxygen species (ROS) was observed with NaIO3 in mouse and human RPE which increased further after αB crystallin knockout or siRNA knockdown, respectively. NaIO3 upregulated AKT phosphorylation and peroxisome proliferator-activator receptor-γ (PPARγ) which was suppressed after αB crystallin siRNA knockdown. Further, PPARγ ligand inhibited NaIO3-induced ROS generation. Our data suggest that αB crystallin plays a critical role in protection of NaIO3-induced oxidative stress and retinal degeneration in part through upregulation of AKT phosphorylation and PPARγ expression.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
αB crystallin is a chaperone protein with anti-apoptotic and anti-inflammatory functions and has been identified as a biomarker in age-related macular degeneration. The purpose of this study was to ...determine whether αB crystallin is secreted from retinal pigment epithelial (RPE) cells, the mechanism of this secretory pathway and to determine whether extracellular αB crystallin can be taken up by adjacent retinal cells and provide protection from oxidant stress. We used human RPE cells to establish that αB crystallin is secreted by a non-classical pathway that involves exosomes. Evidence for the release of exosomes by RPE and localization of αB crystallin within the exosomes was achieved by immunoblot, immunofluorescence, and electron microscopic analyses. Inhibition of lipid rafts or exosomes significantly reduced αB crystallin secretion, while inhibitors of classic secretory pathways had no effect. In highly polarized RPE monolayers, αB crystallin was selectively secreted towards the apical, photoreceptor-facing side. In support, confocal microscopy established that αB crystallin was localized predominantly in the apical compartment of RPE monolayers, where it co-localized in part with exosomal marker CD63. Severe oxidative stress resulted in barrier breakdown and release of αB crystallin to the basolateral side. In normal mouse retinal sections, αB crystallin was identified in the interphotoreceptor matrix. An increased uptake of exogenous αB crystallin and protection from apoptosis by inhibition of caspase 3 and PARP activation were observed in stressed RPE cultures. αB Crystallin was taken up by photoreceptors in mouse retinal explants exposed to oxidative stress. These results demonstrate an important role for αB crystallin in maintaining and facilitating a neuroprotective outer retinal environment and may also explain the accumulation of αB crystallin in extracellular sub-RPE deposits in the stressed microenvironment in age-related macular degeneration. Thus evidence from our studies supports a neuroprotective role for αB crystallin in ocular diseases.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
Angiogenesis is regulated by the balance of proangiogenic VEGF
165 and antiangiogenic VEGF
165b splice isoforms. Mutations in
WT1, the Wilms' tumor suppressor gene, suppress VEGF
165b and cause ...abnormal gonadogenesis, renal failure, and Wilms' tumors. In
WT1 mutant cells, reduced VEGF
165b was due to lack of WT1-mediated transcriptional repression of the splicing-factor kinase SRPK1. WT1 bound to the
SRPK1 promoter, and repressed expression through a specific WT1 binding site. In
WT1 mutant cells SRPK1-mediated hyperphosphorylation of the oncogenic RNA binding protein SRSF1 regulated splicing of VEGF and rendered
WT1 mutant cells proangiogenic. Altered VEGF splicing was reversed by wild-type WT1, knockdown of SRSF1, or SRPK1 and inhibition of SRPK1, which prevented in vitro and in vivo angiogenesis and associated tumor growth.
Display omitted
► SR protein kinase 1 is a target for the Wilms Tumor suppressor gene-1 ►
WT1 mutations lead to altered VEGF splicing by hyperphosphorylated SRSF1 ► SRPK1 overexpression regulates VEGF splicing and angiogenesis ► SRPK1 inhibition is antiangiogenic in tumors in vivo