Activation of HIF-1α/VEGF-A pathway by deferoxamine ameliorates retinal hypoxia in a rat subarachnoid hemorrhage model.

BACKGROUND AND PURPOSE: Subarachnoid hemorrhage (SAH) is associated with sustained vasoconstriction in retinal vessels and vasoconstriction leads to retinal ischemia and hypoxia. Our previous finding also revealed the changes in hypoxia-related elements in the retina after SAH, further lending weight to the hypothesis that retinal vasospasm and hypoxia after SAH. Deferoxamine is a high-affinity iron chelator with reported neuroprotective effects against stroke. Here, we aimed to explore the effects of deferoxamine on retinal hypoxia after SAH. METHODS: SAH was established and deferoxamine was injected intraperitoneally for 3 days in the treatment group. To detect retinal new vessels, platelet endothelial cell adhesion molecule (CD31) was labeled by immunofluorescence and immunohistochemistry. Furthermore, the effects of deferoxamine on the expression of vascular endothelial growth factor A (VEGF-A) and hypoxia-inducible factor-1α (HIF-1α) were revealed by western blot analysis. RESULTS: The immunofluorescence and immunohistochemical staining of CD31 revealed a marked increase in new vessels in the retinal ganglion cell layer after deferoxamine treatment. By western blot analysis, HIF-1α and VEGF-A increased gradually in the first day and then rebounded to a new level on day 7. A deferoxamine-induced increase in HIF-1α/VEGF-A expression was also confirmed by western blot. CONCLUSIONS: Our findings suggest that modulating the application of deferoxamine may offer therapeutic approaches to alleviate retinal complications after SAH.

A20 Establishes Negative Feedback With TRAF6/NF-κB and Attenuates Early Brain Injury After Experimental Subarachnoid Hemorrhage.

Nuclear factor (NF)-κB-ty -50mediated neuroinflammation plays a crucial role in early brain injury (EBI) after subarachnoid hemorrhage (SAH). As an important negative feedback regulator of NF-κB, A20 is essential for inflammatory homeostasis. Herein, we tested the hypothesis that A20 attenuates EBI by establishing NF-κB-associated negative feedback after experimental SAH. In vivo and in vitro models of SAH were established. TPCA-1 and lentivirus were used for NF-κB inhibition and A20 silencing/overexpression, respectively. Cellular localization of A20 in the brain was determined via immunofluorescence. Western blotting and enzyme-linked immunosorbent assays were applied to observe the expression of members of the A20/tumor necrosis factor receptor-associated factor 6 (TRAF6)/NF-κB pathway and inflammatory cytokines (IL-6, IL-1ß, TNF-α). Evans blue staining, TUNEL staining, Nissl staining, brain water content, and modified Garcia score were performed to evaluate the neuroprotective effect of A20. A20 expression by astrocytes, microglia, and neurons was increased at 24 h after SAH. A20 and inflammatory cytokine levels were decreased while TRAF6 expression was elevated after NF-κB inhibition. TRAF6, NF-κB, and inflammatory cytokine levels were increased after A20 silencing but suppressed with A20 overexpression. Also, Bcl-2, Bax, MMP-9, ZO-1 protein levels; Evans blue, TUNEL, and Nissl staining; brain water content; and modified Garcia score showed that A20 exerted a neuroprotective effect after SAH. A20 expression was regulated by NF-κB. In turn, increased A20 expression inhibited TRAF6 and NF-κB to reduce the subsequent inflammatory response. Our data also suggest that negative feedback regulation mechanism of the A20/TRAF6/NF-κB pathway and the neuroprotective role of A20 to attenuate EBI after SAH.

Deferoxamine reduces amyloid-beta peptides genesis and alleviates neural apoptosis after traumatic brain injury.

Traumatic brain injury (TBI) is recognized as the most influential risk factor for neurodegenerative diseases later in life, including Alzheimer's disease. The aberrant genesis of amyloid-ß peptides, which is triggered by TBI, is associated with the development of Alzheimer's disease. Evidence suggests that iron plays a role in both the production of amyloid-ß and its neurotoxicity, and iron overload has been noted in the brain after TBI. We therefore investigated the effects of an iron-chelating treatment on amyloid-ß genesis in a weight-drop model of TBI in mice. Human brain samples were obtained from patients undergoing surgery for severe brain trauma. The Institute of Cancer Research mice were treated with deferoxamine by intraperitoneal injection after TBI induction. Changes in amyloid-ß(1-42) were assessed using western blot and immunohistochemical staining. Ferritin was also detected using western blot to investigate iron deposition in the mice brain. Immunofluorescent terminal deoxynucleotidyl transferase-mediated dUTP nick end labeling was also performed to evaluate neural apoptosis. The amyloid-ß(1-42) was markedly elevated after TBI in both humans and mice. Deferoxamine treatment in mice significantly decreased the levels of both amyloid-ß(1-42) and ferritin in the brain, and reduced TBI-induced neural cell apoptosis. The iron chelator deferoxamine can alleviate the increase of amyloid-ß(1-42) in the brain after TBI, and may therefore be a potential therapeutic strategy to prevent TBI patients from undergoing neurodegenerative processes.

Retinal hypoxia after experimental subarachnoid hemorrhage.

BACKGROUND AND PURPOSE: The patients who survive subarachnoid hemorrhage (SAH) often have long-term neurological complications. There are no reports about the pathological change of retina after SAH. METHODS: An experimental model of SAH was established by injecting autologous blood into the prechiasmatic cistern of Sprague-Dawley rats. Hematoxylin and eosin (HE) staining was performed to show the alternation of morphology in retina after SAH. To detect the retinal new vessels (NVs), CD31 was labelled by immunofluorescence and immunohistochemistry. The time-course expressions of vascular endothelial growth factor (VEGF)-A and hypoxia-inducible factor-1α (HIF-1 α) was also revealed by Western blot analysis. RESULTS: A clear reduction of retinal ganglion cells (RGCs) was noticed after SAH. The immunofluorescence and immunohistochemical staining of CD31 reveals a large number of NVs in RGC layer after SAH compared with the normal controls. The level of VEGF-A in the retina after SAH was increased and peaked at 12h and 14 d. The expression of HIF-1α in the retina increased as early as 3 h after SAH, reached a peak at 12 h after SAH. CONCLUSIONS: The results showed that SAH induced the retina hypoxia resulting in the reduction of RGCs, increase of NVs and activation of NVs related HIF-1α/VEGF-A pathway.

Neuroprotective role of glutathione peroxidase 4 in experimental subarachnoid hemorrhage models.

BACKGROUND AND PURPOSE: Early brain injury is an essential pathological process after subarachnoid hemorrhage (SAH), with many cell death modalities. Ferroptosis is a newly discovered regulated cell death caused by the iron-dependent accumulation of lipid peroxidation, which can be prevented by glutathione peroxidase 4 (GPX4). Our study aimed to investigate the role of GPX4 in neuronal cell death after experimental SAH. METHODS: In vivo experimental SAH was induced by injecting autologous arterial blood into the prechiasmatic cistern in male Sprague-Dawley rats. Meanwhile, the in vitro SAH model was performed with primary rat cortical neurons cultured in medium containing hemoglobin (Hb). Adenovirus was used to overexpress GPX4 before experimental SAH. GPX4 expression was detected by western blot and immunofluorescence experiments. Malondialdehyde (MDA) was measured to evaluate the level of lipid peroxidation. Nissl staining was employed to assess cell death in vivo, whereas lactate dehydrogenase (LDH) release was used to evaluate cell damage in vitro. The brain water content and neurological deficits were evaluated to determine brain injury. RESULTS: Endogenous GPX4 was mainly expressed in neurons, and its expression decreased at 24 h after experimental SAH. Overexpression of GPX4 significantly reduced lipid peroxidation and cell death in the experimental SAH models both in vivo and in vitro. Moreover, overexpression of GPX4 ameliorated brain edema and neurological deficits at 24 h after SAH. CONCLUSIONS: The decrease of GPX4 expression potentially plays an important role in ferroptosis during early brain injury after SAH. Overexpression of GPX4 has a neuroprotective effect after SAH.

Cross-talk between microRNA-let7c and transforming growth factor-ß2 during epithelial-to-mesenchymal transition of retinal pigment epithelial cells.

AIM: To explore the roles of microRNA-let7c (miR-let7c) and transforming growth factor-ß2 (TGF-ß2) and cellular signaling during epithelial-to-mesenchymal transition (EMT) of retinal pigment epithelial cells. METHODS: Retinal pigment epithelial (ARPE-19) cells were cultured with no serum for 12h, and then with recombinant human TGF-ß2 for different lengths of time. ARPE-19 cells were transfected with 1×106 TU/mL miR-let7c mimcs (miR-let7cM), miR-let7c mimcs negative control (miR-let7cMNC) and miR-let7c inhibitor (miR-let7cI) using the transfection reagent. The expression of keratin-18, vimentin, N-cadherin, IKB alpha, p65 were detected by Western blot, quantitative polymerase chain reaction and immunofluorescence. RESULTS: The expression of miR-let7c was dramatically reduced and the nuclear factor-kappa B (NF-κB) signaling pathway was activated after induction by TGF-ß2 (P<0.05). In turn, overexpressed miR-let7c significantly inhibited TGF-ß2-induced EMT (P<0.05). However, miR-let7c was unable to inhibit TGF-ß2-induced EMT when the NF-κB signaling pathway was inhibited by BAY11-7082 (P<0.01). CONCLUSION: The miR-let7c regulates TGF-ß2-induced EMT through the NF-κB signaling pathway in ARPE-19 cells.

Dihydroartemisinin initiates ferroptosis in glioblastoma through GPX4 inhibition.

It has been demonstrated from previous studies about the killing effect of dihydroartemisinin (DHA) on glioblastoma, which involves multiple aspects: cytotoxicity, cell cycle arrest and invasion inhibition. DHA has the advantages of low cytotoxicity to normal cells, selective killing effect and low drug resistance, making it one of the popular anti-tumor research directions. Ferroptosis is a newly discovered form of cell death characterized by iron dependence and lipid reactive oxygen species (ROS) accumulation. In the present study, we found differences in the expression of transferrin receptors in normal human astrocytes (NHA) and glioblastoma cells (U87 and A172), which may be one of the mechanisms of DHA selective killing effect. Through the determination of ferroptosis-related protein expression, we found that the significant decrease of GPX4, accompanied by the constant expression of xCT and ACSL4, suggesting GPX4 was a pivotal target for DHA-activated ferroptosis in glioblastoma. Total and lipid ROS levels were increased and all these results could be reversed by the ferroptosis inhibitor, ferrostatin-1. These findings demonstrated ferroptosis would be a critical component of cell death caused by DHA and GPX4 was the main target. All these results provide a novel treatment direction to glioblastoma. The association between ferroptosis and polyamines is also discussed, which will provide new research directions for ferroptosis caused by DHA in glioblastoma.

FOXO4 expression associates with glioblastoma development and FOXO4 expression inhibits cell malignant phenotypes in vitro and in vivo.

BACKGROUND AND AIM: Forkhead box protein O4 (FOXO4) is a transcription factor, and aberrant FOXO4 expression is associated with development of various human cancers. This study explored the role of FOXO4 in glioma in vitro and in vivo. METHODS: FOXO4 expression was first assessed in normal brain tissues, low-grade glioma, glioblastoma multiforme (GBM), normal human astrocytes (HA), and GBM cell lines, while manipulation of FOXO4 expression in glioma cell lines was assessed using qRT-PCR, Western blot, and cell viability CCK-8, Transwell, and a nude mouse subcutaneous xenograft assays. KEY FINDINGS: The data showed downregulated FOXO4 expression in GBM tissues and cell lines. FOXO4 overexpression induced by transfection with FOXO4 cDNA significantly inhibited GBM cell proliferation, migration, and invasion, but increased tumor cells to undergo apoptosis in vitro, while suppressed growth of GBM cell subcutaneous xenografts in nude mice. In conclusion, FOXO4 possesses an anti-cancer glioma activity, which could be a novel target for future control of GBM.

The role of mechanical stretch and TGF-ß2 in epithelial-mesenchymal transition of retinal pigment epithelial cells.

AIM: To explore the effects and mechanisms of mechanical stress and transforming growth factor-beta2 (TGF-ß2) on epithelial-mesenchymal transition (EMT) in cultured human retinal pigment epithelial (RPE) cells. METHODS: Human RPE cells were inoculated on BioFex 6-well plates and RPE cells received 0, 1, 2, 3, or 4 mild stretch injuries delivered 3h apart after 24h of culture. The device of mechanical stress parameters were set to sine wave, frequency 1 Hz, stretch strength 20%. For treatment with TGF-ß2, when the inoculated RPE cells in 6-well plates were around 60% confluent, serum was reduced to 0 for 12h and recombinant human TGF-ß2 (0, 1, 5, 10 ng/mL) was added for 48h. α-SMA, Vimentin and N-Cadherin, fibronectin proteins expressions were detected by Western blotting, confocal cell immunofluorescence and quantitative real-time polymerase chain reaction (qRT-PCR). Then we detected the change of miRNA-29b and ascertained the changes of phosphatidylinositol 3-kinase-serine threonine protein kinase (PI3K/Akt) pathway after RPE cells were stretched by the device of mechanical stress and induced by TGF-ß2 by Western blotting, confocal cell immunofluorescence and qRT-PCR. RESULTS: Mechanical stress induce EMT and activate the PI3K/Akt pathway in ways that lead to the EMT process. TGF-ß2 induce RPE cells EMT and in a certain range and TGF-ß2 decrease the miRNA-29b expression in RPE cells, and the inhibitory effect is more obvious with the increase of TGF-ß2 concentration. CONCLUSION: Our findings are crucial steps in determining the critical roles of the PI3K/Akt signaling pathway and miRNA-29b in pathogenesis of proliferative vitreoretinopathy (PVR) which may be a potential target for preventing or treating PVR.