Yuka Ikegame

Comparison of mesenchymal stem cells from adipose tissue and bone marrow for ischemic stroke therapy.

BACKGROUND AIMS Transplantation of mesenchymal stromal cells (MSC) derived from bone marrow (BM) or adipose tissue is expected to become a cell therapy for stroke. The present study compared the therapeutic potential of adipose-derived stem cells (ASC) with that of BM-derived stem cells (BMSC) in a murine stroke model. METHODS ASC and BMSC were isolated from age-matched C57BL/6J mice. These MSC were analyzed for growth kinetics and their capacity to secrete trophic factors and differentiate toward neural and vascular cell lineages in vitro. For in vivo study, ASC or BMSC were administrated intravenously into recipient mice (1 × 10(5) cells/mouse) soon after reperfusion following a 90-min middle cerebral artery occlusion. Neurologic deficits, the degree of infarction, expression of factors in the brain, and the fate of the injected cells were observed. RESULTS ASC showed higher proliferative activity with greater production of vascular endothelial cell growth factor (VEGF) and hepatocyte growth factor (HGF) than BMSC. Furthermore, in vitro conditions allowed ASC to differentiate into neural, glial and vascular endothelial cells. ASC administration showed remarkable attenuation of ischemic damage, although the ASC were not yet fully incorporated into the infarct area. Nonetheless, the expression of HGF and angiopoietin-1 in ischemic brain tissue was significantly increased in ASC-treated mice compared with the BMSC group. CONCLUSIONS Compared with BMSC, ASC have great advantages for cell preparation because of easier and safer access to adipose tissue. Taken together, our findings suggest that ASC would be a more preferable source for cell therapy for brain ischemia than BMSC.

Epidermal Growth Factor Plays a Crucial Role in Mitogenic Regulation of Human Brain Tumor Stem Cells

A cancer stem cell population in malignant brain tumors takes an essential part in brain tumor initiation, growth, and recurrence. Growth factors, such as epidermal growth factor, fibroblast growth factor-2, vascular endothelial growth factor, platelet-derived growth factor, and hepatocyte growth factor, are shown to support the proliferation of neural stem cells and also may play key roles in gliomagenesis. However, the responsible growth factor(s), which controls maintenance of brain tumor stem cells, is not yet uncovered. We have established three cancer stem cell lines from human gliomas. These cells were immunoreactive with the neuronal progenitor markers, nestin and CD133, and established tumors that closely resembled the features of original tumor upon transplantation into mouse brain. Three cell lines retained their self-renewal ability and proliferation only in the presence of epidermal growth factor (>2.5 ng/ml). In sharp contrast, other growth factors, including fibroblast growth factor-2, failed to support maintenance of these cells. The tyrosine kinase inhibitors of epidermal growth factor signaling (AG1478 and gefitinib) suppressed the proliferation and self-renewal of these cells. Gefitinib inhibited phosphorylation of epidermal growth factor receptor as well as Akt kinase and extracellular signal-regulated kinase 1/2. Flow cytometric analysis revealed that epidermal growth factor concentration-dependently increased the population of CD133-positive cells. Gefitinib significantly reduced CD133-positive fractions and also induced their apoptosis. These results indicate that maintenance of human brain tumor stem cells absolutely requires epidermal growth factor and that tyrosine kinase inhibitors of epidermal growth factor signaling potentially inhibit proliferation and induce apoptosis of these cells.

Alzheimer Disease–Associated Peptide, Amyloid β40, Inhibits Vascular Regeneration With Induction of Endothelial Autophagy

Objective—Although the majority of cases of Alzheimer disease (AD) are known to be attributable to the sporadic (nongenetic) form of the disease, the mechanism underlying its cause and progression still remains unclear. Methods and Results—We found that vascular &bgr;-amyloid (A&bgr;), A&bgr;40, inhibited the proliferative activity of human brain vascular endothelial cells (HBECs) without toxic effects on them. This peptide also inhibited tube formation and migration of HBECs. Moreover, A&bgr;40 inhibited ex vivo hippocampal revascularization, reendothelialization, and the differentiation of adult endothelial progenitor cells. Importantly, A&bgr;40 suppressed the proliferative activity of HBECs through the induction of “self-digesting” autophagy. This induction involved the intracellular regulation of class 3 phosphatidylinositol 3-kinase (PI3K) as well as Akt signaling in HBECs. Furthermore, tissue culture of murine brain sections from GFP-LC3 transgenic mice revealed that A&bgr;40 not only reduced the vessel density in hippocampal lesions, but also induced autophagy in neurovascular ECs. Conclusions—Our present findings indicate that the initial progression of AD might be in part driven by A&bgr;40-induced endothelial autophagy and impairment of neurovascular regeneration, suggesting important implications for therapeutic approaches to AD.

The Stent-Eluting Drugs Sirolimus and Paclitaxel Suppress Healing of the Endothelium by Induction of Autophagy

Clinical studies have indicated that the stent-eluting drugs sirolimus and paclitaxel impact restenosis; however, it is still elusive how these drugs affect the vascular endothelium at the molecular and cellular levels. The purpose of this study was to determine whether sirolimus and paclitaxel induce molecular and cellular alterations in the vascular endothelium. Endothelial regrowth was assessed in human aortic endothelial cells and rat aortic endothelium. Molecular and cellular alterations were analyzed in human aortic endothelial cells by Western blot analysis, transmission electron microscopy, and immunofluorescence staining. Green fluorescent protein-LC3 mice were used to analyze autophagic endothelium. Here, we show that sirolimus and paclitaxel differentially induce self-digesting autophagy in vascular endothelial cells with changes in expression of LC3B, p53, and Bcl-2, considerably suppressing re-endothelialization and revascularization. These results suggest that phenotypic alteration in the endothelium by sirolimus or paclitaxel might affect the rates of late stent thrombosis, myocardial infarction, and mortality.

Neutrophil elastase inhibitor prevents ischemic brain damage via reduction of vasogenic edema.

Release of neutrophil elastase is one of the harmful inflammatory reactions in acute cerebral ischemia. Therefore, inhibition of elastase released from neutrophils could be a useful strategy for the treatment of acute stroke. To evaluate this hypothesis, the effect of sivelestat, a selective neutrophil elastase inhibitor was examined in a mouse model of focal ischemia. The results obtained indicate that sivelestat reduced brain edema and vascular permeability, and subsequently improved the neurological deficit in an acute focal ischemia. The architecture of microvessels was analyzed by identifying vascular endothelial cells, which were prelabeled by injecting fluorescein-labeled Griffonia simplicifolia lectin I-isolectin B4 into a tail vein. Most of the microvessels in the infarcted area were structurally destroyed in the control group. In sharp contrast, microvessels in the boundary zone were well maintained in the sivelestat-treated group. Moreover, the expression of angiopoietin-1 was elevated at the ischemic margin in the sivelestat-treated group. Furthermore, the neutrophil elastase inhibitor rescued human brain microvascular endothelial cells in culture from neutrophil elastase-induced damage. These results suggest that neutrophil elastase inhibition could protect blood–brain barrier function in acute cerebral ischemia by augmentation of angiopoietin-1 expression and survival of endothelial cells.

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.

Fate of graft cells: what should be clarified for development of mesenchymal stem cell therapy for ischemic stroke?

Mesenchymal stem cells (MSCs) are believed to be promising for cell administration therapy after ischemic stroke. Because of their advantageous characteristics, such as ability of differentiation into neurovascular lineages, avoidance of immunological problems, and abundance of graft cells in mesodermal tissues, studies regarding MSC therapy have increased recently. However, several controversies are yet to be resolved before a worldwide consensus regarding a standard protocol is obtained. In particular, the neuroprotective effects, the rate of cell migration to the lesion, and differentiation direction differ depending on preclinical observations. Analyses of these differences and application of recent developments in stem cell biology or engineering in imaging modality may contribute to identification of criteria for optimal stem cell therapy in which reliable protocols, which control cell quality and include safe administration procedures, are defined for each recovery phase after cerebral ischemia. In this mini review, we examine controversies regarding the fate of grafts and the prospects for advanced therapy that could be obtained through recent developments in stem cell research as direct conversion to neural cells.

Possible involvement of tumor-producing VEGF-A in the recruitment of lymphatic endothelial progenitor cells from bone marrow

Lymphatic metastasis of human malignant adenocarcinomas is a critical determinant of prognosis. Lymphangiogenesis, the growth of lymphatic vessels, is closely involved in lymphatic metastasis. However, the mechanisms of tumor lymphangiogenesis are not clearly understood. In a previous study, we showed that human gastric cancer MKN45 cells organize neighboring lymphatic vessels via recruitment of bone marrow-derived lymphatic endothelial progenitor cells in a nude mouse xenograft model. The present results also indicated that human colorectal cancer LS174T and breast cancer SK-BR-3 cells promoted lymphangiogenesis as well as the recruitment of lymphatic endothelial progenitor cells from bone marrow. Among growth factors, which are reported to be involved in lymphangiogenesis, only vascular endothelial growth factor (VEGF)-A was extensively secreted by these three types of adenocarcinoma cells in culture. The well-characterized lymphangiogenic factors VEGF-C and VEGF-D in the culture medium of these three types of adenocarcinoma cells were below the detectable levels in ELISA assay. Secretion of epidermal growth factor (EGF) and hepatocyte growth factor (HGF) was not detected. In in vitro culture assay, VEGF-A directly induced the differentiation of bone marrow mononuclear cells into LYVE-1-positive lymphatic endothelial lineage cells. These data collectively suggest the possibility that VEGF-A-rich human adenocarcinomas induce tumor lymphangiogenesis via recruitment of lymphangiogenic endothelial progenitor cells from bone marrow.

Among mesenchymal stem cells: for the best therapy after ischemic stroke

Owing to the several advantages that they have over other cells, mesenchymal stem cells are among the most promising tools in stem cell therapy after ischemic stroke. Mesenchymal stem cells can be obtained from various sources. Therefore, a wide range of careful comparative studies of these cells is required, from the preclinical stage (in vitro and in vivo) to the clinical stage, in order to develop the best therapy with the most appropriate cell type. An in vivo study in this issue of Stem Cell Research & Therapy compares the therapeutic potential of two cell types, obtained from bone marrow and adipose tissue, in a rodent stroke model. This commentary discusses the significance of comparative studies of mesenchymal stem cells, including the related article in translational research.

Differences in Brain Metabolic Impairment between Chronic Mild/Moderate TBI Patients with and without Visible Brain Lesions Based on MRI.

Introduction. Many patients with mild/moderate traumatic brain injury (m/mTBI) in the chronic stage suffer from executive brain function impairment. Analyzing brain metabolism is important for elucidating the pathological mechanisms associated with their symptoms. This study aimed to determine the differences in brain glucose metabolism between m/mTBI patients with and without visible traumatic brain lesions based on MRI. Methods. Ninety patients with chronic m/mTBI due to traffic accidents were enrolled and divided into two groups based on their MRI findings. Group A comprised 50 patients with visible lesions. Group B comprised 40 patients without visible lesions. Patients underwent FDG-PET scans following cognitive tests. FDG-PET images were analyzed using voxel-by-voxel univariate statistical tests. Results. There were no significant differences in the cognitive tests between Group A and Group B. Based on FDG-PET findings, brain metabolism significantly decreased in the orbital gyrus, cingulate gyrus, and medial thalamus but increased in the parietal and occipital convexity in Group A compared with that in the control. Compared with the control, patients in Group B exhibited no significant changes. Conclusions. These results suggest that different pathological mechanisms may underlie cognitive impairment in m/mTBI patients with and without organic brain damage.