Müller glia (MG) are major retinal supporting cells that participate in retinal metabolism, function, maintenance, and protection. During the pathogenesis of diabetic retinopathy (DR), a ...neurovascular disease and a leading cause of blindness, MG modulate vascular function and neuronal integrity by regulating the production of angiogenic and trophic factors. In this article, I will (1) briefly summarize our work on delineating the role and mechanism of MG-modulated vascular function through the production of vascular endothelial growth factor (VEGF) and on investigating VEGF signaling-mediated MG viability and neural protection in diabetic animal models, (2) explore the relationship among VEGF and neurotrophins in protecting Müller cells in in vitro models of diabetes and hypoxia and its potential implication to neuroprotection in DR and hypoxic retinal diseases, and (3) discuss the relevance of our work to the effectiveness and safety of long-term anti-VEGF therapies, a widely used strategy to combat DR, diabetic macular edema, neovascular age-related macular degeneration, retinopathy of prematurity, and other hypoxic retinal vascular disorders.
The outer blood-retina barrier (BRB) separates the neural retina from the choroidal vasculature, which is responsible for approximately 80% of blood supplies in the eye. To determine the significance ...of outer BRB breakdown in diabetic retinopathy, the outer BRB-specific leakage of macromolecules in diabetic and ischemic rodents was investigated.
Diabetes and ischemia were induced in rodents by streptozotocin and oxygen-induced retinopathy, respectively. Diabetic and ischemic rodents were injected intravenously with fluorescein isothiocyanate (FITC)-dextran. The outer BRB-specific leakage in diabetic and ischemic rodents was visualized by fluorescent microscopy.
A microscopic imaging assay was developed to examine outer BRB breakdown. The outer BRB-specific leakage of fluorescent macromolecules was visualized in diabetic and ischemic rodents. Substantial leakages of macromolecules through the outer BRB in diabetic and ischemic rodents were detected with this assay. The number of severe outer BRB leakage sites is inversely proportional to the size of macromolecules. Significant depletion of occludin in the RPE of ischemic and diabetic rodents was also observed.
For the first time, a microscopic imaging assay for directly visualizing macromolecules leaked through the outer BRB in rodents was developed. Using this assay, the authors demonstrated the significance of outer BRB breakdown in diabetes and ischemia, which will have implications to the understanding, diagnosis, and treatment of diabetic macular edema and other ocular diseases with outer BRB defects. The microscopic imaging assay established in this study will likely be very useful to the development of drugs for macular edema.
Vascular endothelial growth factor (VEGF-A or VEGF) is a major pathogenic factor and therapeutic target for diabetic retinopathy (DR). Since VEGF has been proposed as a survival factor for retinal ...neurons, defining the cellular origin of pathogenic VEGF is necessary for the effectiveness and safety of long-term anti-VEGF therapies for DR. To determine the significance of Müller cell-derived VEGF in DR, we disrupted VEGF in Müller cells with an inducible Cre/lox system and examined diabetes-induced retinal inflammation and vascular leakage in these conditional VEGF knockout (KO) mice.
Leukostasis was determined by counting the number of fluorescently labeled leukocytes inside retinal vasculature. Expression of biomarkers for retinal inflammation was assessed by immunoblotting of TNF-alpha, ICAM-1, and NF-kappaB. Vascular leakage was measured by immunoblotting of retinal albumin and fluorescent microscopic analysis of extravascular albumin. Diabetes-induced vascular alterations were examined by immunoblotting and immunohistochemistry for tight junctions, and by trypsin digestion assays for acellular capillaries. Retinal integrity was analyzed with morphologic and morphometric analyses.
Diabetic conditional VEGF KO mice exhibited significantly reduced leukostasis, expression of inflammatory biomarkers, depletion of tight junction proteins, numbers of acellular capillaries, and vascular leakage compared to diabetic control mice.
Müller cell-derived VEGF plays an essential and causative role in retinal inflammation, vascular lesions, and vascular leakage in DR. Therefore, Müller cells are a primary cellular target for proinflammatory signals that mediates retinal inflammation and vascular leakage in DR.
Autophagy is a conserved feature of lysosome-mediated intracellular degradation. Dysregulated autophagy is implicated as a contributor in neurodegenerative diseases; however, the role of autophagy in ...retinal degeneration remains largely unknown. Here, we report that the photo-activated visual chromophore, all-trans-retinal, modulated autophagosome formation in ARPE19 retinal cells. Increased formation of autophagosomes in these cells was observed when incubated with 2.5 μm all-trans-retinal, a condition that did not cause cell death after 24 h in culture. However, autophagosome formation was decreased at concentrations, which caused cell death. Increased expression of activating transcription factor 4 (Atf4), which indicates the activation of oxidative stress, was recorded in response to light illumination in retinas of Abca4−/−Rdh8−/− mice, which showed delayed clearance of all-trans-retinal after light exposure. Expression of autophagosome marker LC3B-II and mitochondria-specific autophagy, mitophagy, regulator Park2, were significantly increased in the retinas of Abca4−/−Rdh8−/− mice after light exposure, suggesting involvement of autophagy and mitophagy in the pathogenesis of light-induced retinal degeneration. Deletion of essential genes required for autophagy, including Beclin1 systemically or Atg7 in only rod photoreceptors resulted in increased susceptibility to light-induced retinal damage. Increased photoreceptor cell death was observed when retinas lacking the rod photoreceptor-specific Atg7 gene were coincubated with 20 μm all-trans-retinal. Park2−/− mice also displayed light-induced retinal degeneration. Ultra-structural analyses showed mitochondrial and endoplasmic reticulum impairment in retinas of these model animals after light exposure. Taken together, these observations provide novel evidence implicating an important role of autophagy and mitophagy in protecting the retina from all-trans-retinal- and light-induced degeneration.
Background: Autophagy is a conserved process of lysosome-mediated intracellular degradation.
Results: Dysregulation of autophagy is associated with retinal cell death by all-trans-retinal and by light exposure.
Conclusion: Autophagy protects the retina from light-induced retinal degeneration.
Significance: Dynamic autophagy regulation may influence retinal cell survival under stress and disease conditions.
Brain circuits are assembled from a large variety of morphologically and functionally diverse cell types. It is not known how the intermingled cell types of an individual adult brain region differ in ...their expressed genomes. Here we describe an atlas of cell type transcriptomes in one brain region, the mouse retina. We found that each adult cell type expressed a specific set of genes, including a unique set of transcription factors, forming a 'barcode' for cell identity. Cell type transcriptomes carried enough information to categorize cells into morphological classes and types. Several genes that were specifically expressed in particular retinal circuit elements, such as inhibitory neuron types, are associated with eye diseases. The resource described here allows gene expression to be compared across adult retinal cell types, experimenting with specific transcription factors to differentiate stem or somatic cells to retinal cell types, and predicting cellular targets of newly discovered disease-associated genes.
Celotno besedilo
Dostopno za:
DOBA, IZUM, KILJ, NUK, PILJ, PNG, SAZU, UILJ, UKNU, UL, UM, UPUK
Aims/hypothesis
Müller glia (MG) are major sources of retinal cytokines, and their activation is closely linked to retinal inflammation and vascular leakage in diabetic retinopathy. Previously, we ...demonstrated that X-box binding protein 1 (XBP1), a transcription factor activated by endoplasmic reticulum (ER) stress in diabetic retinopathy, is involved in regulation of inflammation in retinal endothelial cells. Now, we have explored the role of
XBP1
and ER stress in the regulation of MG-derived proinflammatory factors, and their influence on vascular permeability in diabetic retinopathy.
Methods
MG-specific conditional
Xbp1
knockout (
Xbp1
Müller−/−
) mice were generated by crossing
Xbp1
flox/flox mice with Müller–Cre transgenic mice. Diabetes was modelled by induction with streptozotocin, and retinal vascular permeability was measured with FITC-conjugated dextran 2 months after induction. Primary Müller cells were isolated from
Xbp1
Müller−/−
and
Xbp1
Müller+/+
mice and exposed to hypoxia and high levels of glucose. Levels of ER-stress and inflammatory factors were examined by real-time PCR, western blotting or immunohistochemistry.
Results
Xbp1
Müller−/−
mice exhibited normal retinal development and retinal function and expressed similar levels of ER-stress and inflammatory genes to
Xbp1
Müller
+/+
littermates. In diabetes-inducing conditions, compared with
Xbp1
Müller+/+
mice,
Xbp1
Müller−/−
mice had higher mRNA levels of retinal
Vegf
(also known as
Vegfa
) and
Tnf-α
(also known as
Tnf
) and ER-stress marker genes
Grp78
(also known as
Hspa5
),
Atf4
,
Chop
(also known as
Ddit3
) and
Atf6
and higher protein levels of vascular endothelial growth factor (VEGF), TNF-α, phospho-c-Jun N-terminal kinase (JNK), 78 kDa glucose-regulated protein (GRP78), phospho-eukaryotic translation initiation factor (eIF)2α and activating transcription factor (ATF)6. Retinal vascular permeability was significantly higher in diabetic
Xbp1
Müller
−/−
mice than in diabetic
Xbp1
Müller
+/+
mice (
p
< 0.01). Results obtained in vitro with primary Müller cells isolated from
Xbp1
Müller
−/−
mice confirmed higher expression levels of inflammatory and ER-stress markers (but not GRP78) than in
cells from Xbp1
Müller
+/+
mice
.
Moreover,
XBP1-deficient Müller cells
were more susceptible to high-glucose- or hypoxia-induced ER stress and inflammation than
cells from Xbp1
Müller
+/+
mice. Inhibition of ER stress with chemical chaperones suppressed hypoxia-induced VEGF and TNF-α production in
XBP1
-deficient
Müller cells
.
Conclusions/interpretation
Our results have revealed an important role of XBP1 and ER stress in MG-driven retinal inflammation, and suggest that targeting ER stress may represent a promising approach for the prevention and treatment of diabetic retinopathy.
Abstract
Azaleas (Ericaceae) comprise one of the most diverse ornamental plants, renowned for their cultural and economic importance. We present a chromosome-scale genome assembly for
Rhododendron ...simsii
, the primary ancestor of azalea cultivars. Genome analyses unveil the remnants of an ancient whole-genome duplication preceding the radiation of most Ericaceae, likely contributing to the genomic architecture of flowering time. Small-scale gene duplications contribute to the expansion of gene families involved in azalea pigment biosynthesis. We reconstruct entire metabolic pathways for anthocyanins and carotenoids and their potential regulatory networks by detailed analysis of time-ordered gene co-expression networks. MYB, bHLH, and WD40 transcription factors may collectively regulate anthocyanin accumulation in
R. simsii
, particularly at the initial stages of flower coloration, and with WRKY transcription factors controlling progressive flower coloring at later stages. This work provides a cornerstone for understanding the underlying genetics governing flower timing and coloration and could accelerate selective breeding in azalea.
To dissect the role of vascular endothelial growth factor receptor-2 (VEGFR2) in Müller cells and its effect on neuroprotection in diabetic retinopathy (DR), we disrupted VEGFR2 in mouse Müller glia ...and determined its effect on Müller cell survival, neuronal integrity, and trophic factor production in diabetic retinas. Diabetes was induced with streptozotocin. Retinal function was measured with electroretinography. Müller cell and neuronal densities were assessed with morphometric and immunohistochemical analyses. Loss of VEGFR2 caused a gradual reduction in Müller glial density, which reached to a significant level 10 months after the onset of diabetes. This observation was accompanied by an age-dependent decrease of scotopic and photopic electroretinography amplitudes and accelerated loss of rod and cone photoreceptors, ganglion cell layer cells, and inner nuclear layer neurons and by a significant reduction of retinal glial cell line-derived neurotrophic factor and brain-derived neurotrophic factor. Our results suggest that VEGFR2-mediated Müller cell survival is required for the viability of retinal neurons in diabetes. The genetically altered mice established in this study can be used as a diabetic animal model of nontoxin-induced Müller cell ablation, which will be useful for exploring the cellular mechanisms of neuronal alteration in DR.
Oxidized lipoproteins stimulate autophagy in advanced atherosclerotic plaques. However, the mechanisms underlying autophagy induction and the role of autophagy in atherogenesis remain to be ...determined. This study was designed to investigate the mechanisms by which 7-ketocholesterol (7-KC), a major component of oxidized lipoproteins, induces autophagy. This study was also designed to determine the effect of autophagy induction on apoptosis, a central event in the development of atherosclerosis. Exposure of human aortic smooth muscle cells to 7-KC increased autophagic flux. Autophagy induction was suppressed by treating the cells with either a reactive oxygen species scavenger or an antioxidant. Administration of 7-KC concomitantly up-regulated Nox4 expression, increased intracellular hydrogen peroxide levels, and inhibited autophagy-related gene 4B activity. Catalase overexpression to remove hydrogen peroxide or Nox4 knockdown with siRNA reduced intracellular hydrogen peroxide levels, restored autophagy-related gene 4B activity, and consequently attenuated 7-KC–induced autophagy. Moreover, inhibition of autophagy aggravated both endoplasmic reticulum (ER) stress and cell death in response to 7-KC. In contrast, up-regulation of autophagic activity by rapamycin had opposite effects. Finally, activation of autophagy by chronic rapamycin treatment attenuated ER stress, apoptosis, and atherosclerosis in apolipoprotein E knockout ( ApoE − / − ) mouse aortas. In conclusion, we demonstrate that up-regulation of autophagy is a cellular protective response that attenuates 7-KC–induced cell death in human aortic smooth muscle cells.
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