scholarly journals The Protective Effects of Endogenous PACAP in Oxygen-Induced Retinopathy

2021 ◽  
Author(s):  
Timea Kvarik ◽  
Dora Reglodi ◽  
Dora Werling ◽  
Alexandra Vaczy ◽  
Petra Kovari ◽  
...  
Keyword(s):  
Western Blot ◽  
Protective Effects ◽  
Wild Type ◽  
Avascular Area ◽  
Akt Phosphorylation ◽  
Oxygen Induced Retinopathy ◽  
Apoptotic Protein ◽  
Angiogenic Proteins ◽  
Pituitary Adenylate Cyclase ◽  
Retinal Structure

AbstractPituitary adenylate cyclase–activating polypeptide (PACAP) is a neuropeptide having trophic and protective functions in neural tissues, including the retina. Previously, we have shown that intravitreal PACAP administration can maintain retinal structure in the animal model of retinopathy of prematurity (ROP). The purpose of this study is to examine the development of ROP in PACAP-deficient and wild-type mice to reveal the function of endogenous PACAP. Wild-type and PACAP-knockout (KO) mouse pups at postnatal day (PD) 7 were maintained at 75% oxygen for 5 consecutive days then returned to room air on PD12 to develop oxygen-induced retinopathy (OIR). On PD15, animals underwent electroretinography (ERG) to assess visual function. On PD16, eyes were harvested for either immunohistochemistry to determine the percentage of the central avascular retinal area or molecular analysis to assess angiogenesis proteins by array kit and anti-apoptotic protein kinase B (Akt) change by western blot. Retinas of PACAP-deficient OIR mice showed a greater central avascular area than that of the wild types. ERG revealed significantly decreased b-wave amplitude in PACAP KO compared to their controls. Several angiogenic proteins were upregulated due to OIR, and 11 different proteins markedly increased in PACAP-deficient mice, whereas western blot analysis revealed a reduction in Akt phosphorylation, suggesting an advanced cell death in the lack of PACAP. This is the first study to examine the endogenous effect of PACAP in the OIR model. Previously, we have shown the beneficial effect of exogenous local PACAP treatment in the rat OIR model. Together with the present findings, we suggest that PACAP could be a novel retinoprotective agent in ROP.

scholarly journals The Protective Effects of Endogenous PACAP in Oxygen-induced Retinopathy 

2021 ◽  
Author(s):  
Timea Kvarik ◽  
Dora Werling ◽  
Alexandra Vaczy ◽  
Petra Kovari ◽  
Edina Szabo ◽  
...  
Keyword(s):  
Protein Kinase B ◽  
Protective Effects ◽  
Wild Type ◽  
Avascular Area ◽  
Oxygen Induced Retinopathy ◽  
Angiogenic Proteins ◽  
Pituitary Adenylate Cyclase ◽  
Neural Tissues ◽  
Retinal Structure ◽  
Deficient Mice

Abstract Purpose: Pituitary adenylate cyclase-activating polypeptide (PACAP) is a neuropeptide having trophic and protective functions in neural tissues including the retina. Previously we have shown that intravitreal PACAP administration can maintain retinal structure in the animal model of retinopathy of prematurity (ROP). The purpose of this study is to examine the development of ROP in PACAP-deficient and wild-type mice to reveal the function of endogenous PACAP. Methods: Wild-type and PACAP-KO mouse pups at postnatal day (PD) 7 were maintained at 80% oxygen for 5 consecutive days then returned to room air on PD12 to develop oxygen-induced retinopathy (OIR). On PD15 animals underwent electroretinography (ERG) to assess visual function. On PD16 eyes were harvested for either immunohistochemistry to determine the percentage of central avascular retinal area and neovascular tuft formation or molecular analysis to assess angiogenesis proteins by array kit and antiapoptotic protein kinase B (Akt) change by western blot. Results: The retina of PACAP deficient OIR mice showed greater central avascular area than that of the wild types. ERG revealed significantly decreased b-wave amplitude in PACAP KO compared to their controls. Several angiogenic proteins were upregulated as a results of OIR and 11 further proteins markedly increased in PACAP deficient mice. Conclusion: This is the first study to examine the endogenous effect of PACAP in OIR model. Previously we have shown the beneficial effect of exogenous local PACAP treatment in the rat OIR model. Together with the present findings we suppose that PACAP could be a novel retinoprotective agent in ROP.

scholarly journals Protective Role of Endogenous PACAP in Inflammation-induced Retinal Degeneration

2018 ◽  
Vol 24 (30) ◽  
pp. 3534-3542 ◽  
Author(s):  
Alexandra Vaczy ◽  
Petra Kovari ◽  
Krisztina Kovacs ◽  
Kinga Farkas ◽  
Edina Szabo ◽  
...  
Keyword(s):  
Protective Role ◽  
Protective Effects ◽  
Wild Type ◽  
Retinal Degenerations ◽  
Enhanced Sensitivity ◽  
Functional Differences ◽  
Pituitary Adenylate Cyclase ◽  
Retinal Inflammation ◽  
Lps Treatment

Purpose: Pituitary adenylate Cyclase-Activating Polypeptide (PACAP) is a neuroprotective peptide that has been shown to exert protective effects in different models of neurodegenerative diseases, including retinal degenerations. Data obtained from PACAP-deficient (PACAP KO) mice provide evidence that endogenous PACAP has a neuroprotective role in different pathologies. PACAP KO mice show enhanced sensitivity to different insults, such as oxidative stress, hypoxia and inflammation. The aim of the present study was to investigate the protective effects of endogenous PACAP in retinal inflammation. Methods: Endotoxin-induced eye inflammation was induced by intraperitoneal injection of lipopolysaccharide (LPS) in PACAP KO and wild-type (Wt) mice. After LPS treatment, retinas were processed for histological examination. To detect the alterations of different proteins and cytokines, immunohistochemical, western blot and cytokine array were used. We also performed dark-adapted electroretinography (ERG) to detect the functional differences. Results: The thickness of nearly all layers was significantly less in LPS-injected PACAP KO mice compared to Wt animals. Increased expression of Glial Fibrillary Acidic Protein (GFAP) was induced in Müller glial cells after LPS treatment, which was more intense in PACAP KO mice. The levels of pAkt and pGSK were decreased in PACAP KO group during inflammation. LPS treatment significantly increased cytokines (sICAM-1, JE, TIMP-1) in both treated groups, but it was more expressed in PACAP KO animals. Furthermore, ERG responses were disturbed after LPS injection in PACAP KO mice. Conclusion: Our results showed that endogenous PACAP has a protective role in LPS-caused retinal inflammation.

Rab7b suppresses autophagy and induces apoptosis of K562 leukemic cells treated with homoharringtonine

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 4884-4884 ◽  
Author(s):  
Peilin Lu ◽  
Donghua He ◽  
Yang Yang ◽  
Pengfei Hu ◽  
Yi Zhao ◽  
...  
Keyword(s):  
Western Blot ◽  
Blot Analysis ◽  
Western Blot Analysis ◽  
Annexin V ◽  
K562 Cells ◽  
Leukemia Cell Line ◽  
Leukemia Cells ◽  
Rab Proteins ◽  
Wild Type ◽  
Akt Phosphorylation

Abstract Abstract 4884 Homoharringtonine is an effective anti-leukemia medicine developed by Chinese. It has been found to induce differentiation and apoptosis of leukemia cells. However, the mechanism of its anti-leukemia function has not been fully understood. Rab is a kind of G protein of Ras superfamily and participates in endocytosis and exocytosis of protein transport process. In recent studies, some Rab proteins such as Rab7 are found to be associated with cellular autophagy and apoptosis. We previously identified a new small GTPase homologous to Rab7, named Rab7b, which is selectively expressed in promyeloid and monocytic cells and is localized to lysosome-associated compartments. To investigate the roles of Rab7b in acute myeloid leukemia, we used leukemia cell line K562 as a model in the present study. After treatment of K562 cells with various doses of HHT, cell viability and apoptosis were measured by MTT assay and Annexin V/PI staining respectively. Protein expression of LC3, a marker of autophagy, and caspase3, 9, ERK1/2,Akt were determined by Western blot analysis. Using stable gene transfection, several Rab7b variants, including Rab7b wild-type, active mutant Rab7b-Q67L and localization-deficient mutant Rab7b-ΔCC as well as Rab7b RNAi were transfected in to K562 cells and their roles in regulation of apoptosis in K562 leukemia cells induced by HHT were further evaluated. Our data showed that the viability of the K562 cells was greatly reduced by HHT treatment in a dose- and time- dependant manner. Treatment of the K562 with HHT significantly increases apoptosis in the cells as measured by Annexin V/PI staining. Using Western blot analysis, we further determined that the expression of caspase3, 9 was increased, and ERK1/2 augmented with Akt was suppressed in the cells treated with HHT. After suppressing autophagy with 3-MA, apoptosis was enhanced in the K562 cells treated with HHT. (p<0.05). By constitutively expression of Rab7B and variants in K562 cells, we found that the rate of apoptotic cells are much higher in the K562 cells transfected with Rab7b wild-type and Rab7b-Q67L variants, along with increased expression of caspase3, 9, ERK1/2 and decreased expression of Akt in the transfectants with Rab7b wild-type and active mutant Rab7b-Q67L. Our study suggests that HHT is able to suppress autophagy and enhance apoptosis in K562 leukemia cells in a caspase-dependent way, which is associated with suppression of Akt phosphorylation and upregulation of ERK1/2. Over-expression of Rab7b can enhance HHT induced apoptosis in K562 cells, which may also be associated with suppression of Akt phosphorylation and upregulation of ERK1/2. Taken together, our study elucidates a new recognition for the mechanism of HHT in anti-leukemia therapy and provides a new insight into understanding the relationship between autophagy and apoptosis in leukemia cells induced chemotherapy. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Reduced Levels of Drp1 Protect against Development of Retinal Vascular Lesions in Diabetic Retinopathy

Cells ◽  
2021 ◽  
Vol 10 (6) ◽  
pp. 1379
Author(s):  
Dongjoon Kim ◽  
Hiromi Sesaki ◽  
Sayon Roy
Keyword(s):  
Diabetic Retinopathy ◽  
Cell Loss ◽  
Vascular Lesions ◽  
Protective Effects ◽  
Diabetic Mice ◽  
Wild Type ◽  
Apoptotic Genes ◽  
Retinal Vascular Lesions ◽  
Acellular Capillaries ◽  
Pericyte Loss

High glucose (HG)-induced Drp1 overexpression contributes to mitochondrial dysfunction and promotes apoptosis in retinal endothelial cells. However, it is unknown whether inhibiting Drp1 overexpression protects against the development of retinal vascular cell loss in diabetes. To investigate whether reduced Drp1 level is protective against diabetes-induced retinal vascular lesions, four groups of mice: wild type (WT) control mice, streptozotocin (STZ)-induced diabetic mice, Drp1+/− mice, and STZ-induced diabetic Drp1+/− mice were examined after 16 weeks of diabetes. Western Blot analysis indicated a significant increase in Drp1 expression in the diabetic retinas compared to those of WT mice; retinas of diabetic Drp1+/− mice showed reduced Drp1 level compared to those of diabetic mice. A significant increase in the number of acellular capillaries (AC) and pericyte loss (PL) was observed in the retinas of diabetic mice compared to those of the WT control mice. Importantly, a significant decrease in the number of AC and PL was observed in retinas of diabetic Drp1+/− mice compared to those of diabetic mice concomitant with increased expression of pro-apoptotic genes, Bax, cleaved PARP, and increased cleaved caspase-3 activity. Preventing diabetes-induced Drp1 overexpression may have protective effects against the development of vascular lesions, characteristic of diabetic retinopathy.

scholarly journals HGG-26. H3G34V MUTATION AFFECTS GENOMIC H3K36 METHYLATION IN PEDIATRIC GLIOMA

2020 ◽  
Vol 22 (Supplement_3) ◽  
pp. iii348-iii348
Author(s):  
Tina Huang ◽  
Andrea Piunti ◽  
Elizabeth Bartom ◽  
Jin Qi ◽  
Rintaro Hashizume ◽  
...  
Keyword(s):  
Western Blot ◽  
Allelic Frequency ◽  
Glioma Cell Line ◽  
Fluorescent Imaging ◽  
Wild Type ◽  
Gene Expressions ◽  
Pediatric Glioma ◽  
Genes Encoding ◽  
Normal Human

Abstract BACKGROUND Histone H3.3 mutation (H3F3A) occurs in 50% of cortical pediatric high-grade gliomas. This mutation replaces glycine 34 with arginine or valine (G34R/V), impairing SETD2 activity (H3K36-specific trimethyltransferase), resulting in reduced H3K36me on H3G34V nucleosomes relative to wild-type. This contributes to genomic instability and drives distinct gene expressions associated with tumorigenesis. However, it is not known if this differential H3K36me3 enrichment is due to H3G34V mutant protein alone. Therefore, we set to elucidate the effect of H3G34V on genomic H3K36me3 enrichment in vitro. METHODS Doxycycline-inducible short hairpin RNA (shRNA) against H3F3A was delivered via lentivirus to established H3G34V mutant pediatric glioma cell line KNS42, and H3G34V introduced into H3.3 wild type normal human astrocytes (NHA). Transfections were confirmed by western blot, fluorescent imaging, and flow cytometry, with resulting H3.3WT and H3K36me3 expression determined by western blot. H3.3WT, H3K36me3, and H3G34V ChIP-Seq was performed to evaluate genomic enrichment. RESULTS Complete knockdown of H3G34V was achieved with DOX-induced shRNA, with no change in total H3.3, suggesting disproportionate allelic frequency of genes encoding H3.3 (H3F3A and H3F3B). Modest increase in H3K36me3 occurred after H3F3A-knockdown from KNS42, suggesting H3G34V alone impacts observed H3K36me3 levels. Distinct H3K36me3 genomic enrichment was observed with H3G34V knock-in. CONCLUSIONS We demonstrate that DOX-inducible knockdown of H3F3A in an H3G34V mutant pediatric glioma cells and H3G34V mutation transduction in wild-type astrocytes affects H3K36me3 expression. Further evaluation by ChIP-Seq analysis for restoration of wild-type genomic H3K36me3 enrichment patterns with H3G34V knockdown, and mutant H3K36me3 patterns with H3G34V transduction, is currently underway.

scholarly journals Adiponectin inhibits leptin signalling via multiple mechanisms to exert protective effects against hepatic fibrosis

2011 ◽  
Vol 440 (3) ◽  
pp. 385-395 ◽  
Author(s):  
Jeffrey A. Handy ◽  
Ping P. Fu ◽  
Pradeep Kumar ◽  
Jamie E. Mells ◽  
Shvetank Sharma ◽  
...  
Keyword(s):  
Hepatic Fibrosis ◽  
Leptin Receptor ◽  
Tyrosine Phosphatase ◽  
Janus Kinase ◽  
Protective Effects ◽  
Wild Type ◽  
Matrix Deposition ◽  
Matrix Metalloproteinase 1 ◽  
Expression And Activity

Adiponectin is protective against hepatic fibrosis, whereas leptin promotes fibrosis. In HSCs (hepatic stellate cells), leptin signals via a JAK2 (Janus kinase 2)/STAT3 (signal transducer and activator of transcription 3) pathway, producing effects that enhance ECM (extracellular matrix) deposition. SOCS-3 (suppressor of cytokine signalling-3) and PTP1B (protein tyrosine phosphatase 1B) are both negative regulators of JAK/STAT signalling, and recent studies have demonstrated a role for adiponectin in regulating SOCS-3 expression. In the present study we investigate mechanisms whereby adiponectin dampens leptin signalling and prevents excess ECM production. We treated culture-activated rat HSCs with recombinant adiponectin, leptin, both or neither, and also treated adiponectin knockout (Ad−/−) and wild-type mice with leptin and/or carbon tetrachloride (CCl4) or saline. We analyse JAK2 and Ob-Rb (long form of the leptin receptor) phosphorylation, and PTP1B expression and activity. We also explore potential mechanisms through which adiponectin regulates SOCS-3–Ob-Rb association. Adiponectin inhibits leptin-stimulated JAK2 activation and Ob-Rb phosphorylation in HSCs, whereas both were increased in Ad−/− mice. Adiponectin stimulates PTP1B expression and activity in vitro, whereas PTP1B expression was lower in Ad−/−mice than in wild-type mice. Adiponectin also promotes SOCS-3–Ob-R association and blocks leptin-stimulated formation of extracellular TIMP-1 (tissue inhibitor of metalloproteinases-1)–MMP-1 (matrix metalloproteinase-1) complexes in vitro. These results suggest two novel mechanisms whereby adiponectin inhibits hepatic fibrosis: (i) by promoting binding of SOCS-3 to Ob-Rb, and (ii) by stimulating PTP1B expression and activity, thus inhibiting JAK2/STAT3 signalling at multiple points.

Protective effects of pituitary adenylate cyclase activating polypeptide against neurotoxic agents

2018 ◽  
Vol 66 ◽  
pp. 185-194 ◽  
Author(s):  
D. Reglodi ◽  
A. Tamas ◽  
A. Jungling ◽  
A. Vaczy ◽  
A. Rivnyak ◽  
...  
Keyword(s):  
Adenylate Cyclase ◽  
Protective Effects ◽  
Pituitary Adenylate Cyclase

scholarly journals N-Acetylcysteine Reduces the Pro-Oxidant and Inflammatory Responses during Pancreatitis and Pancreas Tumorigenesis

Antioxidants ◽  
2021 ◽  
Vol 10 (7) ◽  
pp. 1107
Author(s):  
Marie-Albane Minati ◽  
Maxime Libert ◽  
Hajar Dahou ◽  
Patrick Jacquemin ◽  
Mohamad Assi
Keyword(s):  
Nuclear Translocation ◽  
Inflammatory Responses ◽  
Acinar Cells ◽  
Pancreatic Acinar Cells ◽  
Glutathione Metabolism ◽  
Protective Effects ◽  
Wild Type ◽  
Kras Mutations ◽  
Oxidative Damages ◽  
Factor Α

Pancreatitis, an inflammation of the pancreas, appears to be a main driver of pancreatic cancer when combined with Kras mutations. In this context, the exact redox mechanisms are not clearly elucidated. Herein, we treated mice expressing a KrasG12D mutation in pancreatic acinar cells with cerulein to induce acute pancreatitis. In the presence of KrasG12D, pancreatitis triggered significantly greater redox unbalance and oxidative damages compared to control mice expressing wild-type Kras alleles. Further analyses identified the disruption in glutathione metabolism as the main redox event occurring during pancreatitis. Compared to the wild-type background, KrasG12D-bearing mice showed a greater responsiveness to treatment with a thiol-containing compound, N-acetylcysteine (NAC). Notably, NAC treatment increased the pancreatic glutathione pool, reduced systemic markers related to pancreatic and liver damages, limited the extent of pancreatic edema and fibrosis as well as reduced systemic and pancreatic oxidative damages. The protective effects of NAC were, at least, partly due to a decrease in the production of tumor necrosis factor-α (TNF-α) by acinar cells, which was concomitant with the inhibition of NF-κB(p65) nuclear translocation. Our data provide a rationale to use thiol-containing compounds as an adjuvant therapy to alleviate the severity of inflammation during pancreatitis and pancreatic tumorigenesis.

scholarly journals Increased Association of Deamidated αA-N101D with Lens Membrane of Transgenic αAN101D vs. Wild Type αA Mice: Potential Effects on Intracellular Ionic Imbalance and Membrane Disorganization

2020 ◽  
Author(s):  
Om Srivast ◽  
Kiran Srivast ◽  
Roy Joseph ◽  
Landon Wilson
Keyword(s):  
Epithelial Cells ◽  
Western Blot ◽  
Immunogold Labeling ◽  
Human Lens ◽  
Membrane Association ◽  
Related Protein ◽  
Wild Type ◽  
Age Related ◽  
Ionic Imbalance

Abstract We have generated two mouse models, in one by inserting the human lens αAN101D transgene in CRYαAN101D mice, and in the other by inserting human wild-type αA-transgene in CRYαAWT mice. The CRYαAN101D mice developed cortical cataract at about 7-months of age relative to CRYαAWT mice. The objective of the study was to determine the following relative changes in the lenses of CRYαAN101D- vs. CRYαAWT mice: age-related changes with specific emphasis on protein insolubilization, relative membrane-association of αAN101D vs. WTαA proteins, and changes in intracellular ionic imbalance and membrane organization. Methods: Lenses of varying ages from CRYαAWT and CRYαAN101D mice were compared for an age-related protein insolubilization. The relative lens membrane-association of the αAN101D- and WTαA proteins in the two types of mice was determined by immunohistochemical-, immunogold-labeling-, and western blot analyses. The relative levels of membrane-binding of recombinant αAN101D- and WTαA proteins was determined by an in vitro assay, and the levels of intracellular Ca2+ uptake and Na, K-ATPase mRNA were determined in the cultured epithelial cells from lenses of the two types of mice.Results: Compared to the lenses of CRYαAWT, the lenses of CRYαAN101D mice exhibited: (A) An increase in age-related protein insolubilization beginning at about 4-months of age. (B) A greater lens membrane-association of αAN101D- relative to WTαA protein during immunogold-labeling- and western blot analyses, including relatively a greater membrane swelling in the CRYαAN101D lenses. (C) During in vitro assay, the greater levels of binding αAN101D- relative to WTαA protein to membranes was observed. (D) The 75% lower level of Na, K-ATPase mRNA but 1.5X greater Ca2+ uptake were observed in cultured lens epithelial cells of CRYαAN101D- than those of CRYαAWT mice. Conclusions: The results show that an increased lens membrane association of αAN101D--relative WTαA protein in CRYαAN101D mice than CRYαAWT mice occurs, which causes intracellular ionic imbalance, and in turn, membrane swelling that potentially leads to cortical opacity.

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