Hideaki Wakita

A Novel Stem Cell Source for Vasculogenesis in Ischemia: Subfraction of Side Population Cells from Dental Pulp

Cell therapy with stem cells and endothelial progenitor cells (EPCs) to stimulate vasculogenesis as a potential treatment for ischemic disease is an exciting area of research in regenerative medicine. EPCs are present in bone marrow, peripheral blood, and adipose tissue. Autologous EPCs, however, are obtained by invasive biopsy, a potentially painful procedure. An alternative approach is proposed in this investigation. Permanent and deciduous pulp tissue is easily available from teeth after extraction without ethical issues and has potential for clinical use. We isolated a highly vasculogenic subfraction of side population (SP) cells based on CD31 and CD146, from dental pulp. The CD31−;CD146− SP cells, demonstrating CD34+ and vascular endothelial growth factor‐2 (VEGFR2)/Flk1+, were similar to EPCs. These cells were distinct from the hematopoietic lineage as CD11b, CD14, and CD45 mRNA were not expressed. They showed high proliferation and migration activities and multilineage differentiation potential including vasculogenic potential. In models of mouse hind limb ischemia, local transplantation of this subfraction of SP cells resulted in successful engraftment and an increase in the blood flow including high density of capillary formation. The transplanted cells were in proximity of the newly formed vasculature and expressed several proangiogenic factors, such as VEGF‐A, G‐CSF, GM‐CSF, and MMP3. Conditioned medium from this subfraction showed the mitogenic and antiapoptotic activity on human umbilical vein endothelial cells. In conclusion, subfraction of SP cells from dental pulp is a new stem cell source for cell‐based therapy to stimulate angiogenesis/vasculogenesis during tissue regeneration.

Glial activation and white matter changes in the rat brain induced by chronic cerebral hypoperfusion: an immunohistochemical study.

Activation of glial cells and white matter changes (rarefaction of the white matter) induced in the rat brain by permanent bilateral occlusion of the commom carotid arteries were immunohistochemically investigated up to 90 days. One day after ligation of the arteries, expression of the major histocompatibility complex (MHC) class I antigen in microglia increased in the white matter including the optic nerve, optic tract, corpus callosum, internal capsule, anterior commissure and traversing fiber bundles of the caudoputamen. After 3 days of occlusion, MHC class I antigen was still elevated and in addition MHC class II antigen and leukocyte common antigen were up-regulated in the microglia in these same regions. Astroglia, labeled with glial fibrillary acidic protein, increased in number in these regions after 7 days of occlusion. A few lymphocytes, labeled with CD4 or CD8 antibodies, were scattered in the neural parenchyma 1 h after occlusion. Activation of glial cells and infiltration of lymphocytes persisted after 90 days of occlusion in the white matter and the retinofugal pathway. However, cellular activation and infiltration in microinfarcts of the gray matter was less extensive and was substantially diminished 30 days after occlusion. The white matter changes were most intense in the optic nerve and optic tract, moderate in the medial part of the corpus callosum, internal capsule and anterior commissure, and slight in the fiber bundles of the caudoputamen. These results indicated that chronic cerebral hypoperfusion induced glial activation preferentially in the white matter. This activation seemed to be an early indicator of the subsequent changes in the white matter.

Alterations of the blood-brain barrier and glial cells in white-matter lesions in cerebrovascular and Alzheimer's disease patients

BACKGROUND AND PURPOSE The underlying cause of white-matter lesions, which are frequent findings in cerebrovascular disease (CVD) and Alzheimers disease (AD), remains uncertain. We performed immunohistochemical analysis of serum protein extravasation to investigate the function of the blood-brain barrier in white-matter lesions. METHODS White-matter lesions were estimated by use of Kluver-Barrera staining in patients diagnosed clinicopathologically as having ischemic CVD (n = 14) and AD (n = 12) and from nonneurological control subjects (n = 6). Axonal damages were investigated by use of immunohistochemistry for amyloid protein precursor. Alteration of the blood-brain barrier was examined with fibrinogen and immunoglobulins used as markers. The numbers of HLA-DR-positive microglia and glial fibrillary acidic protein-positive astroglia were examined comparatively. RESULTS White-matter lesions were graded as normal (grade 0) in 14 of the 32 cases (44%), slight (grade I) in 10 cases (31%), moderate (grade II) in 6 cases (19%), and severe (grade III) in 2 cases (6%). Amyloid precursor protein was accumulated most frequently in grade II white-matter lesions. Immunohistochemistry for serum proteins labeled astroglial cell bodies and their processes, which seemed to have sequestered extravasated proteins. The groups with detectable white-matter lesions had significantly higher grading scores for fibrinogen and immunoglobulins than the control group (P < .05). Although the higher scores for serum protein extravasation were statistically significant in ischemic CVD cases (P < .05), there was no significant increase in AD cases. Activated microglia and astroglia were more numerous in the groups with white-matter lesions in both ischemic CVD and AD cases, although this increase in the number of astroglia was not evident in regions with clasmatodendrosis. CONCLUSIONS Dysfunction of the blood-brain barrier is more prominent in white-matter lesions seen in ischemic CVD than in AD and may have a role in the pathogenesis of cerebrovascular white-matter lesions.

Protein SUMOylation is massively increased in hibernation torpor and is critical for the cytoprotection provided by ischemic preconditioning and hypothermia in SHSY5Y cells

Hibernation torpor provides an excellent natural model of tolerance to profound reductions in blood flow to the brain and other organs. Here, we report that during torpor of 13-lined ground squirrels, massive SUMOylation occurs in the brain, liver, and kidney. The level of small ubiquitin-related modifier (SUMO) conjugation coincides with the expression level of Ubc9, the SUMO specific E2-conjugating enzyme. Hypothermia alone also increased SUMO conjugation, but not as markedly as hibernation torpor. Increased SUMO conjugation (induced by Ubc9 overexpression, ischemic preconditioning (PC) ± hypothermia) was necessary and sufficient for tolerance of SHSY5Y neuroblastoma cells to oxygen/glucose deprivation (OGD) (‘in vitro ischemia‘); decreased SUMO conjugation (induced by a dominant-negative Ubc9) severely reduced tolerance to OGD in these cells. These data indicate that post-translational modification of proteins by SUMOylation is a prominent feature of hibernation torpor and is critical for cytoprotection by ischemic PC ± hypothermia in SHSY5Y cells subjected to OGD.

Axonal damage and demyelination in the white matter after chronic cerebral hypoperfusion in the rat.

Cerebral white matter (WM) lesions are observed frequently in human ischemic cerebrovascular disease and have been thought to contribute to cognitive impairment. This type of lesion can be experimentally induced in rat brains under chronic cerebral hypoperfusion by the permanent occlusion of both common carotid arteries. However, it remains uncertain whether chronic ischemia can damage both the gray and white matter, and whether it can induce demyelination with or without axonal damage. Therefore, we examined axonal damage using immunohistochemistry for the amyloid beta/A4 precursor protein (APP), chromogranin A (CgA) and demyelination using immunohistochemistry for the encephalitogenic peptide (EP) in this model. Severe WM lesions such as vacuolation and the loss of nerve fibers appeared in the optic nerve and optic tract after 3 days of ligation, and less intense changes were observed in the corpus callosum, internal capsule, and fiber bundles of the caudoputamen after 7 days with Klüver-Barrera and Bielschowsky staining. These WM lesions persisted even after 30 days. The APP, CgA, and EP-immunopositive fibers increased in number from 1 to 30 days after the ligation in the following WM regions: the optic nerve, optic tract, corpus callosum, internal capsule, and fiber bundles of the caudoputamen. In contrast, only a few APP, CgA, or EP-immunopositive fibers were detected in the gray matter regions, including the cerebral cortex and hippocampus. These results indicate that the WM is more susceptible to chronic cerebral hypoperfusion than the gray matter, with an involvement of both axonal and myelin components. Furthermore, immunohistochemistry for APP, CgA, and EP is far superior to routine histological staining in sensitivity and may become a useful tool to investigate WM lesions caused by various pathoetiologies.

Cyclooxygenase-2 is induced in microglia during chronic cerebral ischemia in humans

Abstract Cyclooxygenase-2 (COX-2) is known to be up-regulated in ischemic rodent brains, but only little information is available for the human brain. Using immunohistochemistry for COX-2, we investigated brains from control subjects and from patients with cerebrovascular diseases. COX-2 was markedly up-regulated in the neurons and endothelial cells in acute cerebral infarction, but was detected sparsely at chronic stages in these cellular compartments. In contrast, COX-2 immunoreactivity in glial cells was localized to the perinuclear region even in control brains. This immunolabeling was more intense and occurred also in the glial cytoplasm in the brains with chronic cerebral ischemia such as Binswanger’s disease. Double-labeling immunohistochemistry confirmed that COX-2-immunoreactive glia were mostly microglia. These results indicate that prostanoid synthesis is up-regulated in microglia during chronic cerebral ischemia, and that these cells may be involved in tissue repair or inflammation-mediated cell responses.

Chronic cerebral hypoperfusion induced by right unilateral common carotid artery occlusion causes delayed white matter lesions and cognitive impairment in adult mice

Some lines of evidence have suggested that subcortical ischemic vascular dementia (SIVD) is a common form of vascular dementia (VaD), and that its pathological changes are the development of ischemic white matter (WM) lesions under chronic hypoperfusion and lacunes. Here, we have developed a novel mouse model of VaD with WM lesions, which was induced by right unilateral common carotid artery occlusion (rUCCAO). The mice subjected to rUCCAO exhibited chronic cerebral hypoperfusion in the cerebral hemisphere ipsilateral to rUCCAO monitored using a laser-Doppler flow meter (p<0.01), and significant WM damage in the corpus callosum (p<0.05) and deficits in object recognition test correlated with the damage of frontal-subcortical circuits (p<0.01). However, no differences in spontaneous alternation or spontaneous motor activity were observed. Furthermore, the levels of pro-inflammatory cytokines, such as interleukin-1beta (IL-1beta) and interleukin-6 (IL-6), significantly increased (p<0.01), and those of anti-inflammatory cytokines, such as interleukin-4 (IL-4) and interleukin-10 (IL-10), significantly decreased in the ischemic brain (p<0.05). These results suggest that this model is a useful tool for investigating the associations among inflammatory reactions, cognitive impairment, and WM damage, which may help elucidating the pathomechanism of VaD, particularly SIVD.

Blood-brain barrier disruption in white matter lesions in a rat model of chronic cerebral hypoperfusion

Blood–brain barrier damage has been implicated in the pathogenesis of cerebrovascular white matter lesions. This type of lesion is responsible for cognitive impairment in the elderly and can be induced by permanent ligation of the bilateral common carotid arteries in the rat. Because it is unclear whether the blood–brain barrier is impaired, we examined whether vascular permeability to horseradish peroxidase is altered using this model. According to light microscopic results, the reaction product of horseradish peroxidase was most intensely localized to the paramedian part of the corpus callosum in the brain, occurring to a small degree at 3 hours, day 1, markedly on day 3, but reduced on days 7 and 14. By electron microscopic study of the same area, the reaction product of horseradish peroxidase was localized to the plasmalemmal vesicles in the endothelial cells 3 hours after ligation, but appeared in the cytoplasm on days 1 and 3, suggesting a diffuse leakage of horseradish peroxidase. In addition, the reaction product was dispersed into the cytoplasm of glial cells in the perivascular regions on day 3. The luminal surface of the endothelial cell cytoplasm appeared irregular on day 7, suggesting a conformational change of the endothelial cells. Collagen fibrils proliferated in the thickened basal lamina and mitochondria degenerated in the pericyte on days 7 and 14. Perivascular glial endfeet were swollen throughout the survival period. In sham-operated rats, the reaction product of horseradish peroxidase was not observed at any time interval, except in vesicular structures. These findings indicate that chronic cerebral hypoperfusion induces blood–brain barrier damage with subsequent morphologic changes of the vascular structures in the corpus callosum. An extravasation of macromolecules, such as proteases and immunoglobulins, may contribute to the pathogenesis of white matter lesions.

Protective Effect of Cyclosporin A on White Matter Changes in the Rat Brain After Chronic Cerebral Hypoperfusion

BACKGROUND AND PURPOSE Activation of glial cells and rarefaction of the white matter have been reported in rat brain after bilateral permanent occlusion of the common carotid arteries. Using this model, we investigated the effects of the immunosuppressant cyclosporin A on the activation of glial cells and the white matter rarefaction. METHODS Both common carotid arteries were ligated bilaterally in 40 male Wistar rats. Twenty-two of these rats received an intraperitoneal injection of cyclosporin A, and the remaining 18 received a vehicle-solution injection. Microglia/macrophages were investigated with immunohistochemistry for the major histocompatibility complex class I and II antigens as well as for leukocyte common antigen. Astroglia were examined with glial fibrillary acidic protein as a marker. Activation of glial cells and white matter rarefaction were then investigated from 7 to 30 days after the ligation. RESULTS In vehicle-treated animals, there was a persistent and extensive activation of both microglia/macrophages and astroglia in the white matter, including the optic nerve, optic tract, corpus callosum, internal capsule, and traversing fiber bundles of the caudoputamen. In cyclosporin A-treated rats, the number of activated microglia/macrophages was significantly reduced (P < .01) to approximately one fifth of that in vehicle-treated animals. Similarly, rarefaction of the white matter was much less intense in cyclosporin A-treated rats (P < .01). CONCLUSIONS Cyclosporin A suppressed both glial activation and white matter changes after chronic cerebral hypoperfusion. These results suggest that immunologic reaction may play a role in the pathogenesis of the white matter changes and that the present model may be useful in investigating the pathophysiology of white matter changes induced by chronic cerebral hypoperfusion.

Chronic cerebral hypoperfusion induces MMP-2 but not MMP-9 expression in the microglia and vascular endothelium of white matter

White matter lesions are closely associated with cognitive impairment and motor dysfunction in the aged. To explore the pathophysiology of these lesions, the authors examined the expression of matrix metalloproteinase-2 (MMP-2) and MMP-9 in the white matter in a rat model of chronic cerebral hypoperfusion. After bilateral clipping of the common carotid arteries, myelin staining revealed demyelinating changes in the optic tract and the corpus callosum on day 7. Zymographic analyses indicated an increase in the level of MMP-2, but not MMP-9, after the hypoperfusion. Immunohistochemical analyses revealed the presence (most abundantly on day 3) of MMP-2–expressing activated microglia in the optic tract and corpus callosum. In contrast, the capillary endothelial cells expressed MMP-2 later. IgM-immunoreactive glial cells were absent in the sham-operated animals, but were present in the hypoperfused animals by day 3, reflecting the disrupted blood–brain barrier. These findings suggest that the main sources of the elevated MMP-2 were the microglia and the endothelium, and that these cells may contribute to the remodeling of the white matter myelin and microvascular beds in chronic cerebral hypoperfusion.