Sou Sugitani

The growth factor progranulin attenuates neuronal injury induced by cerebral ischemia-reperfusion through the suppression of neutrophil recruitment.

BackgroundTo improve the clinical outcome of patients who suffered ischemic stroke, cerebral ischemia-reperfusion (I/R) injury is one of the major concerns that should be conquered. Inflammatory reactions are considered a major contributor to brain injury following cerebral ischemia, and I/R exacerbates these reactions. The aim of this study was to investigate the possible ameliorative effects of progranulin (PGRN) against I/R injury in mice.MethodsIn vivo I/R was induced in four-week-old male ddY mice by 2 h of MCAO (middle cerebral artery occlusion) followed by 22 h of reperfusion. We evaluate expression of PGRN in I/R brain, efficacy of recombinant-PGRN (r-PGRN) treatment and its therapeutic time-window on I/R injury. Two hours after MCAO, 1.0 ng of r-PRGN or PBS was administered via intracerebroventricular. We assess neutrophil infiltration, expression of tumor necrosis factor (TNF)-α, matrix metalloproteinase-9 (MMP-9) and phosphorylation of nuclear factor-κB (NF-κB) by immunofluorescense staining and Western blotting. We also investigate neutrophil chemotaxis and intercellular adhesion molecule-1 (ICAM-1) expression in vitro inflammation models using isolated neutrophils and endothelial cells.ResultsWe found that expression of PGRN was decreased in the I/R mouse brain. r-PGRN treatment at 2 h after MCAO resulted in a reduction in the infarct volume and decreased brain swelling; this led to an improvement in neurological scores and to a reduction of mortality rate at 24 h and 7 d after MCAO, respectively. Immunohistochemistry, Western blotting, and gelatin zymography also confirmed that r-PGRN treatment suppressed neutrophil recruitment into the I/R brain, and this led to a reduction of NF-κB and MMP-9 activation. In the in vitro inflammation models, PGRN suppressed both the neutrophil chemotaxis and ICAM-1 expression caused by TNF-α in endothelial cells.ConclusionsPGRN exerted ameliorative effects against I/R-induced inflammation, and these effects may be due to the inhibition of neutrophil recruitment into the I/R brain.

The conditioned medium of murine and human adipose-derived stem cells exerts neuroprotective effects against experimental stroke model

This study investigated the possible ameliorative effects of adipose-derived stem cells-conditioned medium (ASC-CM) on experimental ischemic stroke. In vivo ischemic stroke was induced in mice after 2h of middle cerebral artery occlusion (MCAO) followed by 22 h reperfusion. Culture of SH-SY5Y human neuroblastoma cells with 100 μM glutamate for 24h was used as an in vitro neuronal apoptosis model. Intracerebroventricular (i.c.v.) administration of 30- and 100-fold concentrated murine ASC-CM 1h prior to MCAO resulted in a dose-dependent reduction in the infarct volume and the brain swelling. The administration of murine ASC-CM immediately after MCAO was also effective, but administration 2h after MCAO was not. Neuroprotective effects of murine ASC-CM were also confirmed in an in vitro model. Pretreatment with 100-fold concentrated murine ASC-CM at 10% of the total culture volume significantly reduced glutamate-induced excitotoxicity in the SH-SY5Y cells. Similar reduction in the MCAO-induced infarction volume was seen following i.c.v. administration of 100-fold concentrated human ASC-CM or murine ASC-CM. In conclusion, ASC-CM appears to exert ameliorative effects on experimental ischemic stroke i\n both in vivo and in vitro models. These findings suggest the feasibility of ASC-CM administration as a therapy for acute stage stroke.

Role of endoplasmic reticulum stress in light‐induced photoreceptor degeneration in mice

Exposure to excessive levels of light induces photoreceptor apoptosis and can be a causative factor in age‐related macular degeneration (AMD). However, the cellular events that mediate this apoptotic response are poorly understood. Here, we investigated the roles of endoplasmic reticulum (ER) stress in light‐induced cell death in the murine retina and murine photoreceptor cells (661W). Excessive light exposure induced retinal dysfunction, photoreceptor degeneration, and apoptosis. Furthermore, the accumulation of polyubiquitinated proteins and the transcriptional expression of ER stress‐related factors, including 78‐kDa glucose‐regulated protein (GRP78)/immunoglobulin‐binding protein (BiP) and C/EBP‐homologous protein (CHOP), were increased in light‐exposed retinas. Light exposure also induced both cell death and up‐regulation of polyubiquitinated proteins, S‐opsin aggregation, bip and chop mRNAs in 661W cells in vitro. Knock‐down of chop mRNA inhibited photoreceptor cell death induced by light exposure. Furthermore, treatment with BiP inducer X (BIX), an ER stress inhibitor, induced bip mRNA and reduced both chop expression and light‐induced photoreceptor cell death. These data indicate that excessive ER stress may induce photoreceptor cell death in light‐exposed retinas via activation of the CHOP‐dependent apoptotic pathway, suggesting that the ER stress may play a pivotal role in light exposure‐induced retinal damage.

Mild Endoplasmic Reticulum Stress Promotes Retinal Neovascularization via Induction of BiP/GRP78

Endoplasmic reticulum (ER) stress occurs as a result of accumulation of unfolded or misfolded proteins in the ER and is involved in the mechanisms of various diseases, such as cancer and neurodegeneration. The goal of the present study was to clarify the relationship between ER stress and pathological neovascularization in the retina. Proliferation and migration of human retinal microvascular endothelial cells (HRMEC) were assessed in the presence of ER stress inducers, such as tunicamycin and thapsigargin. The expression of ER chaperone immunoglobulin heavy-chain binding protein (BiP), known as Grp78, was evaluated by real time RT-PCR, immunostaining, and Western blotting. Tunicamycin or thapsigargin was injected into the intravitreal body of oxygen-induced retinopathy (OIR) model mice at postnatal day 14 (P14) and retinal neovascularization was quantified at P17. The expression and localization of BiP in the retina was also evaluated in the OIR model. Exposure to tunicamycin and thapsigargin increased the proliferation and migration of HRMEC. Tunicamycin enhanced the expression of BiP in HRMEC at both the mRNA level and at the protein level on the cell surface, and increased the formation of a BiP/T-cadherin immunocomplex. In OIR model mice, retinal neovascularization was accelerated by treatments with ER stress inducers. BiP was particularly observed in the pathological vasculature and retinal microvascular endothelial cells, and the increase of BiP expression was correlated with retinal neovascularization. In conclusion, ER stress may contribute to the formation of abnormal vasculature in the retina via BiP complexation with T-cadherin, which then promotes endothelial cell proliferation and migration.

Progranulin, a Major Secreted Protein of Mouse Adipose-Derived Stem Cells, Inhibits Light-Induced Retinal Degeneration

Adipose tissue stromal vascular fraction contains mesenchymal stem cells, which show protective effects when administered to damaged tissues, mainly through secreted trophic factors. We examined the protective effects of adipose‐derived stem cells (ASCs) and ASC‐conditioned medium (ASC‐CM) against retinal damage and identified the neuroprotective factors in ASC‐CM. ASCs and mature adipocytes were isolated from mouse subcutaneous tissue. ASCs were injected intravitreally in a mouse model of light‐induced retinal damage, and ASC injection recovered retinal function as measured by electroretinogram and inhibited outer nuclear layer, thinning, without engraftment of ASCs. ASC‐CM and mature adipocyte‐conditioned medium were collected after 72 hours of culture. In vitro, H2O2‐ and light‐induced cell death was reduced in a photoreceptor cell line with ASC‐CM but not with mature adipocyte‐conditioned medium. In vivo, light‐induced photoreceptor damage was evaluated by measurement of outer nuclear layer thickness at 5 days after light exposure and by electroretinogram recording. ASC‐CM significantly inhibited photoreceptor degeneration and retinal dysfunction after light exposure. Progranulin was identified as a major secreted protein of ASCs that showed protective effects against retinal damage in vitro and in vivo. Furthermore, progranulin phosphorylated extracellular signal‐regulated kinase, cAMP response element binding protein, and hepatocyte growth factor receptor, and protein kinase C signaling pathways were involved in the protective effects of progranulin. These findings suggest that ASC‐CM and progranulin have neuroprotective effects in the light‐induced retinal‐damage model. Progranulin may be a potential target for the treatment of the degenerative diseases of the retina.

Diabetes mellitus aggravates hemorrhagic transformation after ischemic stroke via mitochondrial defects leading to endothelial apoptosis.

Diabetes is a crucial risk factor for stroke and is associated with increased frequency and poor prognosis. Although endothelial dysfunction is a known contributor of stroke, the underlying mechanisms have not been elucidated. The aim of this study was to elucidate the mechanism by which chronic hyperglycemia may contribute to the worsened prognosis following stroke, especially focusing on mitochondrial alterations. We examined the effect of hyperglycemia on hemorrhagic transformation at 24 hours after middle cerebral artery occlusion (MCAO) in streptozotocin (STZ) -induced diabetic mice. We also examined the effects of high-glucose exposure for 6 days on cell death, mitochondrial functions and morphology in human brain microvascular endothelial cells (HBMVECs) or human endothelial cells derived from induced pluripotent stem cells (iCell endothelial cells). Hyperglycemia aggravated hemorrhagic transformation, but not infarction following stroke. High-glucose exposure increased apoptosis, capase-3 activity, and release of apoptosis inducing factor (AIF) and cytochrome c in HBMVECs as well as affected mitochondrial functions (decreased cell proliferation, ATP contents, mitochondrial membrane potential, and increased matrix metalloproteinase (MMP)-9 activity, but not reactive oxygen species production). Furthermore, morphological aberration of mitochondria was observed in diabetic cells (a great deal of fragmentation, vacuolation, and cristae disruption). A similar phenomena were seen also in iCell endothelial cells. In conclusion, chronic hyperglycemia aggravated hemorrhagic transformation after stroke through mitochondrial dysfunction and morphological alteration, partially via MMP-9 activation, leading to caspase-dependent apoptosis of endothelial cells of diabetic mice. Mitochondria-targeting therapy may be a clinically innovative therapeutic strategy for diabetic complications in the future.

Annatto prevents retinal degeneration induced by endoplasmic reticulum stress in vitro and in vivo

SCOPE Annatto (Bixa orellana) seeds have been used as a colorant in butter and in a variety of other foods. In this study, we investigated the amelioration of retinal damage by an acetone extract of annatto (A-ext.), bixin (a main component of annatto), and four bixin derivatives (Bx-1, Bx-2, Bx-3, and Bx-4) that we have synthesized. METHODS AND RESULTS We used cultured retinal ganglion cells (RGC-5) to examine in vitro effects of A-ext. on stress pathways, focusing on intracellular oxidation induced by reactive oxygen species, expression of endoplasmic reticulum (ER) stress-related proteins, caspase-3 activation, and cell membrane damage. In vivo retinal damage in mice following intravitreous injection of tunicamycin was evaluated by counting the cell numbers in the ganglion cell layer (GCL) and measuring the thickness of outer nuclear layer (ONL). A-ext., bixin, and Bx-1 treatment inhibited both tunicamycin- and H₂O₂-induced cell death. Bixin derivatives also inhibited tunicamycin-induced cell death. Treatment with A-ext., bixin, and Bx-1 reduced tunicamycin-induced caspase-3 activity and inhibited the inversion of phosphatidylserine, an early apoptotic event without antioxidant effect or reduction of ER stress itself. A-ext., bixin, and Bx-1 significantly inhibited the tunicamycin-induced loss of cells from the GCL, and these materials also suppressed the tunicamycin-induced thinning of ONL. CONCLUSION A-ext., its main component bixin, and bixin derivatives may therefore be useful for preventive and therapeutic treatment of retinal-related diseases.

Protective effects of placental growth factor on retinal neuronal cell damage

Placental growth factor (PlGF) is a member of the vascular endothelial growth factor family. Although it has been reported that PlGF protects against neuronal damage in the brain, little is known about the effects of PlGF in the retina. Therefore, we investigated the effects of PlGF on retinal neuronal cells. To evaluate the effects of PlGF against L‐buthionine‐(S,R)‐sulfoximine (BSO)/glutamate cell death, oxygen–glucose deprivation (OGD)‐induced cell death, and light‐induced cell death, RGC‐5 and 661W cells were used. We evaluated the mechanism responsible for the protective effects of PlGF against retinal neuronal cell death by performing the examinations with U1026, which is a mitogen‐activated protein kinase (MEK) inhibitor, and LY294002, which is a phosphoinositide 3‐kinase (PI3K) inhibitor. In addition, we measured caspase‐3/7 activity in RGC‐5 cells and 661W cells. PlGF protected against RGC‐5 cell death induced by BSO/glutamate and OGD and against 661W cell death induced by light irradiation. Moreover, an anti‐PlGF antibody negated these protective effects. The protective effects of PlGF against OGD‐induced RGC‐5 cell death and light‐induced 661W cell death were suppressed by using an anti‐PlGF antibody, U1026, and LY294002. Treatment with PlGF suppressed caspase‐3/7 activity in both cell lines. We demonstrated for the first time that PlGF exerts a protective effect by inhibiting the activation of caspase‐3/7 through the MEK and PI3K pathway in retinal neuronal cells. These data suggest that PlGF may be an important protective factor in the retina.

Progranulin promotes the retinal precursor cell proliferation and the photoreceptor differentiation in the mouse retina

Progranulin (PGRN) is a secreted growth factor associated with embryo development, tissue repair, and inflammation. In a previous study, we showed that adipose-derived stem cell-conditioned medium (ASC-CM) is rich in PGRN. In the present study, we investigated whether PGRN is associated with retinal regeneration in the mammalian retina. We evaluated the effect of ASC-CM using the N-methyl-N-nitrosourea-induced retinal damage model in mice. ASC-CM promoted the differentiation of photoreceptor cells following retinal damage. PGRN increased the number of BrdU+ cells in the outer nuclear layer following retinal damage some of which were Rx (retinal precursor cell marker) positive. PGRN also increased the number of rhodopsin+ photoreceptor cells in primary retinal cell cultures. SU11274, a hepatocyte growth factor (HGF) receptor inhibitor, attenuated the increase. These findings suggest that PGRN may affect the differentiation of retinal precursor cells to photoreceptor cells through the HGF receptor signaling pathway.