scholarly journals MitoROS due to loss of Slc4a11 in corneal endothelial cells induces ER stress, lysosomal dysfunction and impairs autophagy

2020 ◽  
Author(s):  
Rajalekshmy Shyam ◽  
Diego G. Ogando ◽  
Moonjung Choi ◽  
Joseph A. Bonanno
Keyword(s):  
Endothelial Cells ◽  
Er Stress ◽  
Corneal Edema ◽  
Corneal Endothelial Cells ◽  
Autophagy Flux ◽  
Unfolded Protein ◽  
Stress Markers ◽  
Lysosomal Dysfunction ◽  
Mitochondrial Ros ◽  
Elevated Expression

AbstractRecent studies from Slc4a11 KO mice have identified mitochondrial dysfunction as a major contributor toward oxidative stress and cell death in Congenital Hereditary Endothelial Dystrophy. Here we asked if this stress activated autophagy in the Slc4a11 KO cell line and in KO mouse endothelial tissue. Early indicators of autophagy, phospho-mTOR and LC3-II indicated activation, however P62 was elevated suggesting an impairment of autophagy flux. The activity and the number of lysosomes, the organelle responsible for the final degradation of autophagy substrates, were found to be reduced in the KO. In addition, the expression of the master regulator of lysosomal function and biogenesis, TFEB, was significantly reduced in the KO corneal endothelia. Also, we observed increased Unfolded Protein Response, as well as elevated expression of ER stress markers, BIP and CHOP. To test if lysosomal and ER stress stems from elevated mitochondrial ROS, we treated Slc4a11 KO corneal endothelial cells with the mitochondrial ROS quencher, MitoQ. MitoQ restored lysosomal enzymes as well as TFEB, reduced ER stress, and increased autophagy flux. MitoQ injections of Slc4a11 KO mice decreased corneal edema, the major phenotype associated with CHED. We conclude that mitochondrial ROS causes ER stress and lysosomal dysfunction with impairment of autophagy in Slc4a11 KO corneal endothelium. Our study is the first to identify the presence as well as cause of lysosomal dysfunction and ER stress in an animal model of CHED, and to characterize inter-organelle relationship in a corneal cell type.

scholarly journals Utilização do flap de Gundersen para tratamento de disfunção endotelial em equino – relato de caso

2021 ◽  
Vol 4 (5) ◽  
pp. 32-38
Author(s):  
Cleydianne Rodrigues de Almeida ◽  
◽  
Cleyber José da Trindade de Fátima ◽  
Rômulo Vitelli Rocha Peixoto ◽  
Anderson Farias ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Endothelial Dysfunction ◽  
Corneal Ulcer ◽  
Corneal Edema ◽  
Corneal Endothelial Cells ◽  
Eye Care ◽  
Visual Axis ◽  
Lens Dislocation ◽  
Uveitis Anterior ◽  
Ulcerative Keratitis

Endothelial dysfunction in horses is associated with dystrophy or degeneration of corneal endothelial cells, clinically presented as a diffuse corneal edema unresponsive to conventional treatments. The main causes of such injury are trauma, ulcerative keratitis, recurrent uveitis, anterior lens dislocation and glaucoma. This paper aims to report a case of endothelial dysfunction in a mare, diagnosed with endothelial dysfunction after uveitis, glaucoma and indolent corneal ulcer. For correction, a superficial lamellar keratectomy followed by permanent conjunctival graft, described as a Gundersen flap, was performed. After intensive eye care, he returned to his athletic functions, maintained corneal and visual axis transparency and ocular reflexes.

scholarly journals Leukemia-Associated HSC Vascular Niche Is Negatively Regulated By PERK of Unfolded Protein Response (UPR)

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 2486-2486
Author(s):  
Lan Zhou ◽  
Cui Liu ◽  
Stanley A Adoro ◽  
Lechuang Chen ◽  
Diana Ramirez ◽  
...  
Keyword(s):  
Bone Marrow ◽  
Endothelial Cells ◽  
Er Stress ◽  
Leukemia Cell ◽  
Unfolded Protein ◽  
Vascular Niche ◽  
Activating Transcription Factor ◽  
Protein Response

T cell acute lymphoblastic leukemia (T-ALL) is an aggressive hematological malignancy derived from early T cell progenitors. Diffuse infiltration of the bone marrow by T-ALL is associated with worse prognosis. We previously reported that actively proliferating leukemia cells inhibit normal hematopoietic stem and progenitor cell (HSPC) proliferation and homing to the perivascular region. We found that aberrant Notch activation in the stroma plays an important role in negatively regulating the expression of CXLC12 on osteoblasts and their differentiation. However, the underlying molecular mechanism that leads to the suppression of hematopoiesis and decreased HSPC in the vascular niche is unclear. It has been demonstrated that rapid cellular proliferation associated with oncogenic activity such as MYC in T-ALL leads to a global increase in protein synthesis and an increase in misfolded/unfolded polypeptides in the endoplasmic reticulum (ER), referred to as unfolded protein response (UPR) or ER stress. Elevated ER stress leads to activation of at least three types of ER stress transducers through the release of inhibitory binding by glucose-regulated chaperone protein (GRP78/BIP): the protein kinase RNA-like ER kinase (PERK), the inositol-requiring enzyme 1 (IRE1), and the activating transcription factor 6 (ATF6). Activation of PERK phosphorylates eIF2 to repress global translation with the exception of a small number of proteins including ATF4 (activating transcription factor-4). ATF4 regulates genes involved in restoring ER homeostasis and genes in apoptosis. Here, we studied the role of UPR in the regulation of HSC niche function in the setting of T-ALL progression. Using in vitro assays in which T-ALL leukemia cells driven by activated Notch1 (ICN1) were co-cultured with endothelial cells (MILE SVEN 1, MS1), and in vivo ICN1-driven T-ALL model, we found that PERK-eIF2a-ATF4 pathway was activated in both MS1 cells and BM endothelial cells isolated from T-ALL mice, while IRE1 and ATF6 pathways were only mildly altered. The activation of PERK was accompanied with the increased expression of Jagged1 and suppressed expression of CXCL12 in both cultured endothelial cells and bone marrow endothelial cells from leukemia mice. PERK inhibitor (GSK2606414) treatment of co-cultured cells largely restored CXCL12 expression, which was also negatively regulated by Jagged1, and accelerated the leukemia cell apoptosis as indicated by the enhanced annexin staining. These findings suggest that PERK is the upstream regulator of Jagged1 and CXCL12 in the endothelial cells; however, the function of cell-autonomous PERK on leukemia cell survival needs to be further clarified. To understand the role of PERK in bone marrow endothelium during leukemia development in vivo, we examined T-ALL leukemia progression and its effect on vascular niche function in VE-CadherinERT2/PERKF/F mice in which Perk was specifically deleted in endothelial cells. Consistent with in vitro findings, T-ALL development induced endothelial PERK-eIF2a-ATF4 activation, while up-regulated Jagged1 and down-regulated CXCL12 were also identified in isolated BM endothelial cells. Compared to the wild type mice, VE-CadherinERT2/PERKF/F mice showed attenuated leukemia progression, increased HSPC (Lin-Sca-1+c-kit+) frequency, and improved survival. Taken together, our findings suggest that PERK activation in BM endothelial cells is a key regulator of the leukemia vascular niche to promote leukemia progression and to suppress normal hematopoiesis. Therefore, targeting PERK may offer an effective strategy in restoring normal HSPC homeostasis and limiting leukemia progression. Disclosures No relevant conflicts of interest to declare.

scholarly journals NAD+ precursors protect corneal endothelial cells from UVB-induced apoptosis

2020 ◽  
Vol 318 (4) ◽  
pp. C796-C805 ◽  
Author(s):  
Can Zhao ◽  
Wenjing Li ◽  
Haoyun Duan ◽  
Zongyi Li ◽  
Yanni Jia ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Cell Apoptosis ◽  
Corneal Endothelium ◽  
Corneal Edema ◽  
Ultraviolet B ◽  
Subconjunctival Injection ◽  
Corneal Endothelial Cells ◽  
Nicotinamide Phosphoribosyltransferase ◽  
Mesenchymal Transition ◽  
Induced Apoptosis

Excessive exposure of the eye to ultraviolet B light (UVB) leads to corneal edema and opacification because of the apoptosis of the corneal endothelium. Our previous study found that nicotinamide (NIC), the precursor of nicotinamide adenine dinucleotide (NAD), could inhibit the endothelial-mesenchymal transition and accelerate healing the wound to the corneal endothelium in the rabbit. Here we hypothesize that NIC may possess the capacity to protect the cornea from UVB-induced endothelial apoptosis. Therefore, a mouse model and a cultured cell model were used to examine the effect of NAD+ precursors, including NIC, nicotinamide mononucleotide (NMN), and NAD, on the UVB-induced apoptosis of corneal endothelial cells (CECs). The results showed that UVB irradiation caused apparent corneal edema and cell apoptosis in mice, accompanied by reduced levels of NAD+ and its key biosynthesis enzyme, nicotinamide phosphoribosyltransferase (NAMPT), in the corneal endothelium. However, the subconjunctival injection of NIC, NMN, or NAD+ effectively prevented UVB-induced tissue damage and endothelial cell apoptosis in the mouse cornea. Moreover, pretreatment using NIC, NMN, and NAD+ increased the survival rate and inhibited the apoptosis of cultured human CECs irradiated by UVB. Mechanistically, pretreatment using nicotinamide (NIC) recovered the AKT activation level and decreased the BAX/BCL-2 ratio. In addition, the capacity of NIC to protect CECs was fully reversed in the presence of the AKT inhibitor LY294002. Therefore, we conclude that NAD+ precursors can effectively prevent the apoptosis of the corneal endothelium through reactivating AKT signaling; this represents a potential therapeutic approach for preventing UVB-induced corneal damage.

Abstract 1313: Pro-atherogenic Effects Of Lipid Peroxidation Products: Role Of The Unfolded Protein Response

Circulation ◽  
2007 ◽  
Vol 116 (suppl_16) ◽  
Author(s):  
Elena Vladykoskaya ◽  
Petra Haberzettl ◽  
Yonis Ahmed ◽  
Bradford G Hill ◽  
Srinivas D Sithu ◽  
...  
Keyword(s):  
Endothelial Cells ◽  
Er Stress ◽  
Unfolded Protein Response ◽  
Initiation Factor ◽  
Chemical Chaperone ◽  
Unfolded Protein ◽  
The Unfolded Protein Response ◽  
Protein Response ◽  
Jnk Activation

Endoplasmic reticulum (ER) stress and the unfolded protein response (UPR) are associated with atherosclerosis. Expression of UPR target genes such as activating transcription factor 3 (ATF3) and ATF4 is markedly increased in human atherosclerotic lesions. Staining for these proteins co-localizes with the staining with antibodies that recognize the aldehydic epitopes of oxidized LDL, suggesting that lipid-derived aldehydes could be involved in mediating ER stress and UPR. We examined the role of phospholipid aldehyde, 1-palmitoyl-2-(5-oxovaleroyl)- sn -glycero-3-phosphocholine (POVPC), unsaturated lipid-derived aldehydes- 4-hydroxy, trans -2-nonenal (HNE) and acrolein in the induction of ER-stress and UPR in human aortic endothelial cells (HAEC) and human umbical vein endothelial cells (HUVEC). POVPC, HNE and acrolein (10 –25 μM) increased the phosphorylation of eIF2α (eukaryotic initiation factor-2α) by 1.5–5 fold (P<0.001) and induced its downstream effector proteins - ATF4 (1.5–3.5 fold; P<0.001) and ATF3 (4–10 fold; P<0.0001). Incubation of HAEC with these aldehydes also increased the adhesion of THP-1 cells (monocyte) to HAEC by 1.4–1.6 fold (P<0.01). Moreover, incubation of endothelial cells with POVPC increased the mRNA level of the pro-inflammatory cytokine IL-8 by >25 fold (P<0.0001). Chemical chaperone, phenyl butyric acid (PBA), diminished aldehydes-induced expression of ATF3 and ATF4 proteins, endothelial cell-monocyte adhesion and IL-8 formation by 80–95% (P<0.001). POVPC (10–25 μM) also activated JNK by (3–6 fold) in HAEC. Reduction of POVPC to its corresponding alcohol, 1-palmitoyl-2-(5-hydroxyvaleroyl)- sn -glycero-3-phosphocholine (PHVPC) inhibited JNK activation by 74 ± 14 % (P<0.001). Pharmacological inhibition of JNK, inhibited the aldehyde-induced induction of ATF3 and ATF4 proteins by 70–90 % (P<0.001) but not the phosphorylation of eIF2α, and PBA inhibited the POVPC-induced JNK activation by 85 ± 11 % (P<0.001). These data suggest that lipoprotein oxidation products activate endothelial cells in part by inducing ER-stress and their inflammatory signaling could be attenuated by chemical chaperones of protein folding.

scholarly journals O-GlcNAc signaling attenuates ER stress-induced cardiomyocyte death

2009 ◽  
Vol 297 (5) ◽  
pp. H1711-H1719 ◽  
Author(s):  
Gladys A. Ngoh ◽  
Tariq Hamid ◽  
Sumanth D. Prabhu ◽  
Steven P. Jones
Keyword(s):  
Cell Death ◽  
Er Stress ◽  
Cardiac Myocyte ◽  
Brefeldin A ◽  
Unfolded Protein ◽  
Homologous Protein ◽  
Stress Markers ◽  
The Unfolded Protein Response ◽  
Glcnac Transferase ◽  
Protein Response

We previously demonstrated that the O-linked β- N-acetylglucosamine ( O-GlcNAc) posttranslational modification confers cardioprotection at least partially through mitochondrial-dependent mechanisms, but it remained unclear if O-GlcNAc signaling interfered with other mechanisms of cell death. Because ischemia/hypoxia causes endoplasmic reticulum (ER) stress, we ascertained whether O-GlcNAc signaling could attenuate ER stress-induced cell death per se. Before induction of ER stress (with tunicamycin or brefeldin A), we adenovirally overexpressed O-GlcNAc transferase (AdOGT) or pharmacologically inhibited O-GlcNAcase [via O-(2-acetamido-2-deoxy-d-glucopyranosylidene) amino- N-phenylcarbamate] to augment O-GlcNAc levels or adenovirally overexpressed O-GlcNAcase to reduce O-GlcNAc levels. AdOGT significantly ( P < 0.05) attenuated the activation of the maladaptive arm of the unfolded protein response [according to C/EBP homologous protein (CHOP) activation] and cardiomyocyte death (reflected by percent propidium iodide positivity). Moreover, pharmacological inhibition of O-GlcNAcase significantly ( P < 0.05) mitigated ER stress-induced CHOP activation and cardiac myocyte death. Interestingly, overexpression of GCA did not alter ER stress markers but exacerbated brefeldin A-induced cardiomyocyte death. We conclude that enhanced O-GlcNAc signaling represents a partially proadaptive response to reduce ER stress-induced cell death. These results provide new insights into a possible interaction between O-GlcNAc signaling and ER stress and may partially explain a mechanism of O-GlcNAc-mediated cardioprotection.

scholarly journals Autophagy, Apoptosis, the Unfolded Protein Response, and Lung Function in Idiopathic Pulmonary Fibrosis

Cells ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1642
Author(s):  
Pawan Sharma ◽  
Javad Alizadeh ◽  
Maya Juarez ◽  
Afshin Samali ◽  
Andrew J. Halayko ◽  
...  
Keyword(s):  
Lung Function ◽  
Idiopathic Pulmonary Fibrosis ◽  
Caspase 3 ◽  
Tissue Expression ◽  
Cell Stress ◽  
Autophagy Flux ◽  
Unfolded Protein ◽  
Stress Markers ◽  
The Unfolded Protein Response ◽  
Protein Response

Autophagy, apoptosis, and the unfolded protein response (UPR) are fundamental biological processes essential for manifold cellular functions in health and disease. Idiopathic pulmonary fibrosis (IPF) is a progressive and lethal pulmonary disorder associated with aging that has limited therapies, reflecting our incomplete understanding. We conducted an observational study linking molecular markers of cell stress response pathways (UPR: BiP, XBP1; apoptosis: cleaved caspase-3; autophagy: LC3β) in lung tissues from IPF patients and correlated the expression of these protein markers to each subject’s lung function measures. We hypothesized that changes in lung tissue expression of apoptosis, autophagy, and UPR markers correlate with lung function deficits in IPF. The cell stress markers BiP, XBP1, LC3β puncta, and cleaved caspase-3 were found to be elevated in IPF lungs compared to non-IPF lungs, and, further, BiP and cleaved caspase-3 co-localized in IPF lungs. Considering lung function independently, we observed that increased XBP1, BiP, and cleaved caspase-3 were each associated with reduced lung function (FEV1, FVC, TLC, RV). However, increased lung tissue expression of LC3β puncta was significantly associated with increased diffusion capacity (DLCO), an indicator of alveolar–capillary membrane function. Similarly, the co-localization of UPR (XBP1, BiP) and autophagy (LC3β puncta) markers was positively correlated with increased lung function (FEV1, FVC, TLC, DLCO). However, the presence of LC3β puncta can indicate either autophagy flux inhibition or activation. While the nature of our observational cross-sectional study design does not allow conclusions regarding causal links between increased expression of these cell stress markers, lung fibrosis, and lung function decline, it does provide some insights that are hypothesis-generating and suggests that within the milieu of active UPR, changes in autophagy flux may play an important role in determining lung function. Further research is necessary to investigate the mechanisms linking UPR and autophagy in IPF and how an imbalance in these cell stress pathways can lead to progressive fibrosis and loss of lung function. We conclude by presenting five testable hypotheses that build on the research presented here. Such an understanding could eventually lead to the development of much-needed therapies for IPF.

scholarly journals Persimmon Leaves (Diospyros kaki) Extract Enhances the Viability of Human Corneal Endothelial Cells by Improving Na+-K+-ATPase Activity

2022 ◽  
Vol 15 (1) ◽  
pp. 72
Author(s):  
Ramsha Afzal ◽  
Hyung Bin Hwang
Keyword(s):  
Endothelial Cells ◽  
Atpase Activity ◽  
Basolateral Membrane ◽  
Corneal Edema ◽  
Ethanol Extract ◽  
Diospyros Kaki ◽  
Corneal Endothelial Cells ◽  
Human Corneal Endothelial Cells ◽  
Corneal Decompensation

The Na+/K+-ATPase, present in the basolateral membrane of human corneal endothelial cells (HCECs), is known to play an important role for corneal transparency. Na+/K+-ATPase dysfunction is one of the major causes of corneal decompensation. The ethanol extract of Diospyros kaki (EEDK) has been reported to increase corneal cell viability. Thus, we treated HCECs with EEDK and studied its effects on HCECs survival and Na+/K+-ATPase against cytotoxic drugs like staurosporine (ST) and ouabain (OU). Firstly, survival assays, (MTT assay and live dead-imaging) showed that decreased HCECs viability by ST and OU was significantly recovered by EEDK co-treatment. Secondly, Na+/K+-ATPase activity assays revealed that EEDK enhanced Na+/K+-ATPase enzymatic activity (* p < 0.01) with/without ST and OU. Finally, Na+/K+-ATPase expression analysis (Western Blot and confocal microscopy) demonstrated that EEDK treatment with/without ST and OU facilitates Na+/K+-ATPase expression in HCECs. Taken together, our findings led us to the conclusion that EEDK might aid HCECs survival in vitro by increasing the activity and expression of Na+/K+-ATPase enzyme. Since Na+/K+-ATPase activity is important to maintain cellular function of HCECs, we suggest that EEDK can be a potential effective agent against corneal edema and related corneal disorders.

scholarly journals FoxO suppresses endoplasmic reticulum stress to inhibit growth of Tsc1-deficient tissues under nutrient restriction

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Avantika Gupta ◽  
Hugo Stocker
Keyword(s):  
Endoplasmic Reticulum ◽  
Er Stress ◽  
Target Genes ◽  
Degradation Pathway ◽  
Nutrient Restriction ◽  
Loss Of Function ◽  
Er Stress Response ◽  
Stress Markers ◽  
Stress Response Pathway ◽  
Elevated Expression

The transcription factor FoxO has been shown to block proliferation and progression in mTORC1-driven tumorigenesis but the picture of the relevant FoxO target genes remains incomplete. Here, we employed RNA-seq profiling on single clones isolated using laser capture microdissection from Drosophila larval eye imaginal discs to identify FoxO targets that restrict the proliferation of Tsc1-deficient cells under nutrient restriction (NR). Transcriptomics analysis revealed downregulation of endoplasmic reticulum-associated protein degradation pathway components upon foxo knockdown. Induction of ER stress pharmacologically or by suppression of other ER stress response pathway components led to an enhanced overgrowth of Tsc1 knockdown tissue. Increase of ER stress in Tsc1 loss-of-function cells upon foxo knockdown was also confirmed by elevated expression levels of known ER stress markers. These results highlight the role of FoxO in limiting ER stress to regulate Tsc1 mutant overgrowth.

scholarly journals Human CPA1 mutation causes digestive enzyme misfolding and chronic pancreatitis in mice

Gut ◽  
2018 ◽  
Vol 68 (2) ◽  
pp. 301-312 ◽  
Author(s):  
Eszter Hegyi ◽  
Miklós Sahin-Tóth
Keyword(s):  
Chronic Pancreatitis ◽  
Er Stress ◽  
Digestive Enzyme ◽  
Pancreatic Disease ◽  
Mutant Mice ◽  
Inflammatory Disorder ◽  
Loss Of Function ◽  
Stress Markers ◽  
Elevated Expression

ObjectiveChronic pancreatitis is a progressive, relapsing inflammatory disorder of the pancreas, which often develops in the background of genetic susceptibility. Recently, loss-of-function mutations in CPA1, which encodes the digestive enzyme carboxypeptidase A1, were described in sporadic early onset cases and in hereditary pancreatitis. Mutation-induced misfolding of CPA1 and associated endoplasmic reticulum (ER) stress was suggested as potential disease mechanism; however, in vivo evidence has been lacking. The objective of the present study was to create a mouse model that recapitulates features of CPA1-associated chronic pancreatitis.DesignWe knocked-in the most frequently occurring p.N256K human CPA1 mutation to the mouse Cpa1 locus. Mutant mice were characterised with respect to pancreas pathology and ER stress and compared with C57BL/6N and CPA1 null control mice.ResultsIn the CPA1 N256K mutant mice, we observed hallmarks of chronic pancreatitis that included progressive acinar cell atrophy, inflammatory cell infiltration, fibrosis and acinar-ductal metaplasia. In contrast, similarly to the C57BL/6N mice, the CPA1 null control strain exhibited no signs of pancreatic disease. Mutation p.N256K induced misfolding of mouse CPA1 and resulted in elevated expression of ER stress markers Hspa5 (BiP) and Ddit3 (CHOP) both in cell culture and mutant mice.ConclusionThe results offer categorical evidence that CPA1 mutations elicit enzyme misfolding and cause chronic pancreatitis via an ER stress-related mechanism.

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