Brain Pericytes Acquire Stemness via the Nrf2-Dependent Antioxidant System.

Pericytes (PCs) are a mural support cell population elongated at intervals along the walls of capillaries. Recent studies reported that PCs are multipotent cells that are activated in response to tissue injury and contribute to the regenerative process. Using a C.B-17 mouse model of ischemic stroke, it has been proposed that normal brain pericytes (nPCs) are converted to ischemic pericytes (iPCs), some of which function as multipotent stem cells. Furthermore, oxygen-glucose deprivation (OGD) promoted mesenchymal-epithelial transition in nPCs; however, nestin was not induced under OGD conditions. Therefore, further studies are needed to elucidate the PC reprogramming phenomenon. We herein isolated nPCs from the cortex of C.B-17 mice, and compared the traits of iPCs and nPCs. The results obtained showed that nPCs and iPCs shared common pericytic markers. Furthermore, intercellular levels of reactive oxygen species and the nuclear accumulation of nuclear factor erythroid-2-related factor 2 (Nrf2), a key player in antioxidant defenses, were higher in iPCs than in nPCs. OGD/reoxygenation and a treatment with tBHQ, an Nrf2 inducer, increased nestin levels in nPCs. Moreover, epithelial marker levels, including nestin, Sox2, and CDH1 (E-cadherin) mRNAs, were elevated in Nrf2-overexpressing PCs, which formed neurosphere-like cell clusters that differentiated into Tuj1-positive neurons. The present results demonstrate that oxidative stress and Nrf2 are required for the generation of stem cells after stroke and will contribute to the development of novel therapeutic strategies for ischemic stroke.

Chlorogenic Acid Activates Nrf2/SKN-1 and Prolongs the Lifespan of Caenorhabditis elegans via the Akt-FOXO3/DAF16a-DDB1 Pathway and Activation of DAF16f.

Chlorogenic acid (CGA) is the most abundant polyphenol in coffee. It has been widely reported to exhibit antioxidant activity by activating nuclear factor erythroid 2-related factor 2 (Nrf2) potentially via the canonical Kelch-like-ECH-associated protein 1 (Keap1)-Nrf2 pathway. We herein demonstrated that the knockdown of WD40 repeat protein 23 (WDR23), but not Keap1, abolished the effects of CGA on the activation of Nrf2. CGA decreased the expression of DDB1, an adaptor for WDR23-Cullin 4A-RING ligase (CRL4AWDR23). FOXO3, a major target for inactivation by the PI3K/Akt pathway, was identified as the transcription factor responsible for the basal and CGA-inhibited expression of the DDB1 gene. CGA blocked FOXO3 binding to importin-7 (IPO7), thereby inhibiting the nuclear accumulation of FOXO3, down-regulating the expression of DDB1, inhibiting the activity of CRL4WDR23, and ultimately increasing that of Nrf2. This pathway was conserved in Caenorhabditis elegans, and CGA extended the lifespan partly through this pathway. We found that in C. elegans, the isoform DAF-16a, but not DAF-16f, regulated the expression levels of ddb-1 mRNA and SKN-1 protein. CGA prolonged the mean lifespan of DAF-16a- and DAF-16f-rescued worms by 24% and 9%, respectively, suggesting that both isoforms involve in lifespan-extending effects of CGA, with DAF-16a being more important than DAF-16f. Based on these results, we established a novel Akt-FOXO3/DAF16a-DDB1 axis that links nutrient sensing and oxidative stress response pathways. Our results also provide a novel molecular mechanism for Nrf2/SKN-1 activation by CGA and the increased lifespan of C. elegans by CGA via this pathway.

Yeast ß-glucan Increases Etoposide Sensitivity in Lung Cancer Cell Line A549 by Suppressing Nuclear Factor Erythroid 2-Related Factor 2 via the Noncanonical Nuclear Factor Kappa B Pathway.

Etoposide is regarded as one of the main standard cytotoxic drugs for lung cancer. However, mutations in Kelch-like ECH-associated protein 1 (Keap1), the main regulator of nuclear factor erythroid 2-related factor 2 (Nrf2), are often detected in lung cancer and lead to chemoresistance. Since the aberrant activation of Nrf2 enhances drug resistance, the suppression of the Nrf2 pathway is a promising therapeutic strategy for lung cancer. We herein used the human lung adenocarcinoma cell line A549 because it harbors a Keap1 loss-of-function mutation. A treatment with ß-glucan, a major component of the fungal cell wall, reduced Nrf2 protein levels; downregulated the expression of cytochrome P450 3A5, UDP glucuronosyltransferase 1A1, and multidrug resistance protein 1; and increased etoposide sensitivity in A549 cells. Furthermore, the ephrin type-A receptor 2 (EphA2) receptor was important for the recognition and biologic activity of ß-glucan in A549 cells. EphA2 signaling includes nuclear factor kappa B (NF-κB), signal transducer and activator of transcription 3 (STAT3), and p38 mitogen-activated protein kinase (MAPK). However, treatment of cells with stattic (STAT3 inhibitor) or SB203580 (p38 MAPK inhibitor) did not diminish the effects of ß-glucan. In contrast, knockdown of v-rel reticuloendotheliosis viral oncogene homolog B (RelB) abolished the effects of ß-glucan, suggesting the involvement of the noncanonical NF-κB pathway. The ß-glucan effects were also attenuated by the knockdown of WD40 Repeat protein 23 (WDR23). The ß-glucan treatment and RelB overexpression induced the expression of Cullin-4A (CUL4A), which increased WDR23 ligase activity and promoted the subsequent depletion of Nrf2. These results revealed a novel property of ß-glucan as a resistance-modifying agent in addition to its widely reported immunomodulatory effects for lung cancer therapy via the EphA2-RelB-CUL4A-Nrf2 axis. SIGNIFICANCE STATEMENT: Chemotherapeutic resistance remains a major obstacle in cancer therapy despite extensive efforts to elucidate the underlying molecular mechanisms and overcome multidrug resistance. The present study revealed a novel resistance-modifying property of ß-glucan, thereby expanding our knowledge on the beneficial roles of ß-glucan and providing an alternative strategy to prevent drug resistance by cancer. The present results provide evidence for the involvement of a novel mode of NF-κB and Nrf2 crosstalk in the drug resistance phenotype.

Bisphenol A stabilizes Nrf2 via Ca2+ influx by direct activation of the IP3 receptor.

Bisphenol A (BPA) is an endocrine-disrupting chemical used in polycarbonate and epoxy resins. Previously, we found that BPA stabilized the protein levels of nuclear factor erythroid 2-related factor 2 (Nrf2) by inducing Ca2+ efflux into the cytosol, followed by nitric oxide synthase activation, resulting in the enhanced nitrosylation of Keap1, which is a negative regulator of Nrf2. However, the mechanisms behind the stimulation of Ca2+ efflux by BPA remain unknown. In the present study, we found that BPA stimulated Ca2+ efflux into the cytosol from the ER, but not from outside of cells through the plasma membrane in Hep3B cells. Ca2+ efflux and Nrf2 stabilization by BPA were inhibited by an inhibitor of the inositol 1,4,5-trisphosphate (IP3) receptor, 2-aminoethoxydiphenylborane, in the endoplasmic reticulum. IP3 is produced by activation of phospholipase C (PLC) from a membrane lipid, phosphatidylinositol 4,5-bisphosphate (PIP2). The induction of Nrf2 by BPA was not inhibited by a PLC inhibitor, U-73122, suggesting that BPA does not induce the production of IP3 via PLC activation. We found that BPA bound directly to the IP3 binding core domain of the IP3 receptor, and BPA competed with IP3 on this site. In addition, overexpression of this domain of the IP3 receptor in Hep3B cells inhibited the stabilization of Nrf2 by BPA. These results clarified that the IP3 receptor is a new target of BPA, and that BPA induces Ca2+ efflux from the endoplasmic reticulum via activation of the IP3 receptor.

Utility of an adverse drug event database based on the narrative accounts of patients with breast cancer.

Patient narratives of adverse drug events (ADEs) often differ from the symptoms listed on the package inserts of pharmaceutical products using common ADE terminology and could be a source of great comfort to patients with the same disease. To explore this idea, we analyzed written narratives obtained from 48 patients with breast cancer using the NPO Corporation Database of Individual Patients' Experiences, Japan (DIPEx-Japan). Our analysis aimed to determine the utility of an "Adverse Drug Event Database" for use in clinical settings as a novel source of disease information in patients' own words. An analysis of transcripts from 29 patients, in which they recounted their treatment drugs and the time of onset and duration of ADEs in great detail, revealed several discrepancies between the language they used to describe various side effects and the standard ADE terminology on package inserts. We conclude that the language used to describe ADEs on package inserts is insufficient for helping patients as they struggle to recognize, internalize, and overcome ADEs, and argue the need for available, detailed information in the words of real patients about the nature of the ADEs predicted, as well as their clinical course and duration. Such information would be invaluable in supplementing the standardized language used on package inserts. Databases of patients' narrative accounts of ADEs are needed as information sources that can be reliably disseminated among patients.

Isolation and Characterization of Cerebellum-Derived Stem Cells in Poststroke Human Brain.

There is compelling evidence that the mature central nervous system (CNS) harbors stem cell populations outside conventional neurogenic regions. We previously demonstrated that brain pericytes (PCs) in both mouse and human exhibit multipotency to differentiate into various neural lineages following cerebral ischemia. PCs are found throughout the CNS, including cerebellum, but it remains unclear whether cerebellar PCs also form ischemia-induced multipotent stem cells (iSCs). In this study, we demonstrate that putative iSCs can be isolated from poststroke human cerebellum (cerebellar iSCs [cl-iSCs]). These cl-iSCs exhibited multipotency and differentiated into electrophysiologically active neurons. Neurogenic potential was also confirmed in single-cell suspensions. DNA microarray analysis revealed highly similar gene expression patterns between PCs and cl-iSCs, suggesting PC origin. Global gene expression comparison with cerebral iSCs revealed general similarity, but cl-iSCs differentially expressed certain cerebellum-specific genes. Thus, putative iSCs are present in poststroke cerebellum and possess region-specific traits, suggesting potential capacity to regenerate functional cerebellar neurons following ischemic stroke.

Comparative Characterization of Ischemia-Induced Brain Multipotent Stem Cells with Mesenchymal Stem Cells: Similarities and Differences.

Mesenchymal stem cells (MSCs) are multipotent stem cells localized to the perivascular regions of various organs, including bone marrow (BM). While MSC transplantation represents a promising stem cell-based therapy for ischemic stroke, increasing evidence indicates that exogenously administered MSCs rarely accumulate in the injured central nervous system (CNS). Therefore, compared with MSCs, regionally derived brain multipotent stem cells may be a superior source to elicit regeneration of the CNS following ischemic injury. We previously identified ischemia-induced multipotent stem cells (iSCs) as likely originating from brain pericytes/perivascular cells (PCs) within poststroke regions. However, detailed characteristics of iSCs and their comparison with MSCs remains to be investigated. In the present study, we compared iSCs with BM-derived MSCs, with a focus on the stemness and neuron-generating activity of each cell type. From our results, stem and undifferentiated cell markers, including c-myc and Klf4, were found to be expressed in iSCs and BM-MSCs. In addition, both cell types exhibited the ability to differentiate into mesoderm lineages, including as osteoblasts, adipocytes, and chondrocytes. However, compared with BM-MSCs, high expression of neural stem cell markers, including nestin and Sox2, were found in iSCs. In addition, iSCs, but not BM-MSCs, formed neurosphere-like cell clusters that differentiated into functional neurons. These results demonstrate that iSCs are likely multipotent stem cells with the ability to differentiate into not only mesoderm, but also neural, lineages. Collectively, our novel findings suggest that locally induced iSCs may contribute to CNS repair by producing neuronal cells following ischemic stroke.

Ischemic stroke activates the VE-cadherin promoter and increases VE-cadherin expression in adult mice.

Endothelial cells (ECs) are a key component of the blood-brain barrier (BBB). Healthy ECs in the BBB form inter-endothelial junctions, including adherens junctions (AJs). Under pathological conditions, such as after ischemic stroke, the BBB may be functionally compromised. However, gene and protein expression patterns involving endothelial AJs have not been well studied. Because expression levels of endothelial AJs are considered to be related to BBB functionality, we investigated the expression pattern of a representative endothelial AJ marker, VE-cadherin, in healthy and diseased mice. We first examined the expression of VE-cadherin in developing mouse brains. In addition, to a mouse model of cerebral infarction, we investigated the expression pattern of VE-cadherin in pathologic brains. Furthermore, using the Cre-LoxP system, we established a strain of mice expressing yellow fluorescent protein (YFP) under the control of the VE-cadherin promoter and investigated the expression pattern of YFP-expressing ECs in developing and pathologic murine brains. VE-cadherin protein and YFP expression driven by the VE-cadherin promoter both showed that VE-cadherin expression was weak during embryonic stages, followed by a steady increase postnatally, which then decreased during adulthood. However, following ischemic stroke, imunohistochemistry of VE-cadherin demonstrated an upregulation in ECs within ischemic regions, concomitant with YFP upregulation. These findings reveal that ischemic stroke activates the VE-cadherin promoter and increases VE-cadherin protein expression, which suggests that endothelial VE-cadherin is involved in the reconstruction of the BBB following ischemic stroke.

Novel Regenerative Therapies Based on Regionally Induced Multipotent Stem Cells in Post-Stroke Brains: Their Origin, Characterization, and Perspective.

Brain injuries such as ischemic stroke cause severe neural loss. Until recently, it was believed that post-ischemic areas mainly contain necrotic tissue and inflammatory cells. However, using a mouse model of cerebral infarction, we demonstrated that stem cells develop within ischemic areas. Ischemia-induced stem cells can function as neural progenitors; thus, we initially named them injury/ischemia-induced neural stem/progenitor cells (iNSPCs). However, because they differentiate into more than neural lineages, we now refer to them as ischemia-induced multipotent stem cells (iSCs). Very recently, we showed that putative iNSPCs/iSCs are present within post-stroke areas in human brains. Because iNSPCs/iSCs isolated from mouse and human ischemic tissues can differentiate into neuronal lineages in vitro, it is possible that a clearer understanding of iNSPC/iSC profiles and the molecules that regulate iNSPC/iSC fate (e.g., proliferation, differentiation, and survival) would make it possible to perform neural regeneration/repair in patients following stroke. In this article, we introduce the origin and traits of iNSPCs/iSCs based on our reports and recent viewpoints. We also discuss their possible contribution to neurogenesis through endogenous and exogenous iNSPC/iSC therapies following ischemic stroke.

Identification of Multipotent Stem Cells in Human Brain Tissue Following Stroke.

Perivascular regions of the brain harbor multipotent stem cells. We previously demonstrated that brain pericytes near blood vessels also develop multipotency following experimental ischemia in mice and these ischemia-induced multipotent stem cells (iSCs) can contribute to neurogenesis. However, it is essential to understand the traits of iSCs in the poststroke human brain for possible applications in stem cell-based therapies for stroke patients. In this study, we report for the first time that iSCs can be isolated from the poststroke human brain. Putative iSCs were derived from poststroke brain tissue obtained from elderly stroke patients requiring decompressive craniectomy and partial lobectomy for diffuse cerebral infarction. Immunohistochemistry showed that these iSCs were localized near blood vessels within poststroke areas containing apoptotic/necrotic neurons and expressed both the stem cell marker nestin and several pericytic markers. Isolated iSCs expressed these same markers and demonstrated high proliferative potential without loss of stemness. Furthermore, isolated iSCs expressed other stem cell markers, such as Sox2, c-myc, and Klf4, and differentiated into multiple cells in vitro, including neurons. These results show that iSCs, which are likely brain pericyte derivatives, are present within the poststroke human brain. This study suggests that iSCs can contribute to neural repair in patients with stroke.

Brain pericytes serve as microglia-generating multipotent vascular stem cells following ischemic stroke.

BACKGROUND: Microglia are the resident macrophage population of the central nervous system (CNS) and play essential roles, particularly in inflammation-mediated pathological conditions such as ischemic stroke. Increasing evidence shows that the population of vascular cells located around the blood vessels, rather than circulating cells, harbor stem cells and that these resident vascular stem cells (VSCs) are the likely source of some microglia. However, the precise traits and origins of these cells under pathological CNS conditions remain unclear. METHODS: In this study, we used a mouse model of cerebral infarction to investigate whether reactive pericytes (PCs) acquire microglia-producing VSC activity following ischemia. RESULTS: We demonstrated the localization of ionized calcium-binding adaptor molecule 1 (Iba1)-expressing microglia to perivascular regions within ischemic areas. These cells expressed platelet-derived growth factor receptor-ß (PDGFRß), a hallmark of vascular PCs. PDGFRß(+) PCs isolated from ischemic, but not non-ischemic, areas expressed stem/undifferentiated cell markers and subsequently differentiated into various cell types, including microglia-like cells with phagocytic capacity. CONCLUSIONS: The study results suggest that vascular PCs acquire multipotent VSC activity under pathological conditions and may thus be a novel source of microglia.

Brain vascular pericytes following ischemia have multipotential stem cell activity to differentiate into neural and vascular lineage cells.

Brain vascular pericytes (PCs) are a key component of the blood-brain barrier (BBB)/neurovascular unit, along with neural and endothelial cells. Besides their crucial role in maintaining the BBB, increasing evidence shows that PCs have multipotential stem cell activity. However, their multipotency has not been considered in the pathological brain, such as after an ischemic stroke. Here, we examined whether brain vascular PCs following ischemia (iPCs) have multipotential stem cell activity and differentiate into neural and vascular lineage cells to reconstruct the BBB/neurovascular unit. Using PCs extracted from ischemic regions (iPCs) from mouse brains and human brain PCs cultured under oxygen/glucose deprivation, we show that PCs developed stemness presumably through reprogramming. The iPCs revealed a complex phenotype of angioblasts, in addition to their original mesenchymal properties, and multidifferentiated into cells from both a neural and vascular lineage. These data indicate that under ischemic/hypoxic conditions, PCs can acquire multipotential stem cell activity and can differentiate into major components of the BBB/neurovascular unit. Thus, these findings support the novel concept that iPCs can contribute to both neurogenesis and vasculogenesis at the site of brain injuries.

Alkaloid constituents from flower buds and leaves of sacred lotus (Nelumbo nucifera, Nymphaeaceae) with melanogenesis inhibitory activity in B16 melanoma cells.

Methanolic extracts from the flower buds and leaves of sacred lotus (Nelumbo nucifera, Nymphaeaceae) were found to show inhibitory effects on melanogenesis in theophylline-stimulated murine B16 melanoma 4A5 cells. From the methanolic extracts, a new alkaloid, N-methylasimilobine N-oxide, was isolated together with eleven benzylisoquinoline alkaloids. The absolute stereostructure of the new alkaloid was determined from chemical and physicochemical evidence. Among the constituents isolated, nuciferine, N-methylasimilobine, (-)-lirinidine, and 2-hydroxy-1-methoxy-6a,7-dehydroaporphine showed potent inhibition of melanogenesis. Comparison of the inhibitory activities of synthetic related alkaloids facilitated characterization of the structure-activity relationships of aporphine- and benzylisoquinoline-type alkaloids. In addition, 3-30 µM nuciferine and N-methylasimilobine inhibited the expression of tyrosinase mRNA, 3-30 µM N-methylasimilobine inhibited the expression of TRP-1 mRNA, and 10-30 µM nuciferine inhibited the expression of TRP-2 mRNA.