Keisuke Kuwabara

Hypoxic induction of interleukin-8 gene expression in human endothelial cells.

Because leukocyte-mediated tissue damage is an important component of the pathologic picture in ischemia/reperfusion, we have sought mechanisms by which PMNs are directed into hypoxic tissue. Incubation of human endothelial cells (ECs) in hypoxia, PO2 approximately 14-18 Torr, led to time-dependent release of IL-8 antigen into the conditioned medium; this was accompanied by increased chemotactic activity for PMNs, blocked by antibody to IL-8. Production of IL-8 by hypoxic ECs occurred concomitantly with both increased levels of IL-8 mRNA, based on polymerase chain reaction analysis, and increased IL-8 transcription, based on nuclear run-on assays. Northern analysis of mRNA from hypoxic ECs also demonstrated increased levels of mRNA for macrophage chemotactic protein-1, another member of the chemokine superfamily of proinflammatory cytokines. IL-8 gene induction was associated with the presence of increased binding activity in nuclear extracts from hypoxic ECs for the NF-kB site. Studies with human umbilical vein segments exposed to hypoxia also demonstrated increased elaboration of IL-8 antigen compared with normoxic controls. In mice exposed to hypoxia (PO2 approximately 30-40 Torr), there was increased pulmonary leukostasis, as evidenced by increased myeloperoxidase activity in tissue homogenates. In parallel, increased levels of transcripts for IP-10, a murine homologue in the chemokine family related to IL-8, were observed in hypoxic lung tissue. Taken together, these data suggest that hypoxia constitutes a stimulus for leukocyte chemotaxis and tissue leukostasis.

Rage: A Novel Cellular Receptor for Advanced Glycation End Products

Exposure of proteins to reducing sugars results in nonenzymatic glycation with the ultimate formation of advanced glycation end products (AGEs). One means through which AGEs modulate cellular functions is through binding to specific cell surface acceptor molecules. The receptor for AGEs (RAGE) is such a receptor and is a newly identified member of the immunoglobulin superfamily expressed on endothelial cells (ECs), mononuclear phagocytes (MPs), and vascular smooth muscle cells (SMCs) in both vivo and in vitro. Binding of AGEs to RAGE results in induction of cellular oxidant stress, as exemplified by the generation of thiobarbituric acid-reactive substances, expression of heme oxygenase type I, and activation of the transcription factor NF-kB, with consequences for a range of cellular functions. AGEs on the surface of diabetic red cells enhance binding to endothelial RAGE and result in enhanced oxidant stress in the vessel wall. By using reagents to selectively block access to RAGE, the role of this receptor in AGE-mediated perturbation of cellular properties can be dissected in detail.

Contribution of Microglia/Macrophages to Expansion of Infarction and Response of Oligodendrocytes After Focal Cerebral Ischemia in Rats

BACKGROUND AND PURPOSE The purpose of this study was (1) to examine the contribution of microglia and macrophages with their interleukin-1beta production and (2) to assess the vulnerability and response of oligodendrocytes in cerebral infarction. METHODS Male Wistar rats were subjected to permanent occlusion of the left middle cerebral artery. Expansion of ischemic infarction and response of oligodendrocytes were investigated together with accumulation of inflammatory cells, production of interleukin-1beta, and disruption of the blood-brain barrier. Apoptotic cell death was inferred from fragmented DNA and the expression of proapoptotic Bax protein. RESULTS During expansion of infarction, amoeboid microglia and extravasation of serum albumin were observed not only in the infarcted area but also in the adjacent surviving area, whereas macrophages accumulated along the boundary and granulocytes migrated into the center of the infarction. Both amoeboid microglia and macrophages produced interleukin-1beta, an inflammatory cytokine, during an early ischemic period. Furthermore, macrophages within the infarcted tissue expressed Bax protein and subsequently showed fragmented nuclear DNA. Oligodendrocytes were detected in the infarcted area even after 24 hours following middle cerebral artery occlusion, but they subsequently developed fragmented DNA. A week after onset of ischemia, oligodendrocytes were found to be accumulated in the intact area bordered with the infarct together with reactive astrocytes. CONCLUSIONS Our results suggest the importance of amoeboid microglia, macrophages, and their interleukin-1beta production in gradual expansion of cerebral infarction. Resident oligodendrocytes may be resistant to ischemic insults, and oligodendrocytes accumulated at the border of the infarction may participate in tissue repair after cerebral infarction.

Influence of oxidative stress on induced tolerance to ischemia in gerbil hippocampal neurons

We investigated whether reversible oxidative stress induced by the administration of the superoxide dismutase inhibitor, diethyldithiocarbamate, could induce tolerance to subsequent cerebral ischemia in gerbil hippocampal neurons. Mature male gerbils received intraperitoneal injections of diethyldithiocarbamate (1.0 g/kg), which led to reduced superoxide dismutase activity and increases in thiobarbituric acid-reactive substance in the brain. Cerebral ischemia was produced by occluding the bilateral common carotid arteries for 5 min, either 2 or 4 days after diethyldithiocarbamate injection. One week after ischemia, samples from each brain were stained with hematoxylin-eosin to evaluate ischemic neuronal damage in the hippocampal CA1 sector. Diethyldithiocarbamate treatment 4 days before ischemia had significant protective effects against cerebral ischemia, while diethyldithiocarbamate 2-day pretreatment and vehicle treatment failed to show neuroprotection. Biochemical examinations showed a clear induction of heat shock protein 72 and a significant increase in manganese-containing superoxide dismutase in the hippocampus in animals treated with diethyldithiocarbamate 4 days prior to ischemia. These results suggested that the oxidative stress caused by diethyldithiocarbamate could induced tolerance to ischemia in the gerbil brain, and that the increase in the biosynthesis of manganese-containing superoxide dismutase and heat shock protein 72 could provide a biochemical explanation of the tolerance induced under these conditions.

Hyperthermia-Induced Neuronal Protection against Ischemic Injury in Gerbils

We investigated the effect of hyperthermic pretreatment before induction of ischemia using a gerbil model of 5-min forebrain ischemia. A single hyperthermic treatment 18 h before ischemia exhibited a partial protective effect, and repetitive hyperthermic pretreatments at 18-h intervals before ischemia showed clear protection against neuronal death in the CA1 area of the hippocampus, whereas single hyperthermic treatment 3, 6, 24, or 50 h before ischemia exhibited little protective effect. This transient and cumulative neuroprotective effect of hyperthermic pretreatment strongly suggested the involvement of stress reactions after hyperthermia in the protective mechanism against ischemic neuronal death.

150-kDa Oxygen-regulated Protein (ORP150) Suppresses Hypoxia-induced Apoptotic Cell Death

To determine the contribution of 150-kDa oxygen-regulated protein (ORP150) to cellular processes underlying adaptation to hypoxia, a cell line stably transfected to overexpress ORP150 antisense RNA was created. In human embryonic kidney (HEK) cells stably overexpressing ORP150 antisense RNA, ORP150 antigen and transcripts were suppressed to low levels in normoxia and hypoxia, whereas wild-type cells showed induction of ORP150 with oxygen deprivation. Inhibition of ORP150 in antisense transfectants was selective, as hypoxia-mediated enhancement of glucose-regulated protein (GRP) 78 and GRP94 was maintained. However, antisense ORP150 transfectants displayed reduced viability when subjected to hypoxia, compared with wild-type and sense-transfected HEK cells. In contrast, diminished levels of ORP150 had no effect on cytotoxicity induced by other stimuli, including oxygen-free radicals and sodium arsenate. Although cellular ATP content was similar in hypoxia, compared with ORP150 antisense transfectants and wild-type HEK cells, suppression of ORP150 expression was associated with accelerated apoptosis. Hypoxia-mediated cell death in antisense HEK transfectants did not cause an increase in caspase activity or in cytoplasmic cytochromec antigen. A well recognized inducer of apoptosis in HEK cells, staurosporine, caused increased caspase activity and cytoplasmic cytochrome c levels in both wild-type and antisense cells. These data indicate that ORP150 has an important cytoprotective role in hypoxia-induced cellular perturbation and that ORP150-associated inhibition of apoptosis may involve mechanisms distinct from those triggered by other apoptotic stimuli.

Amelioration of Hippocampal Neuronal Damage After Global Ischemia by Neuronal Overexpression of BCL-2 in Transgenic Mice

BACKGROUND AND PURPOSE Reports suggesting the involvement of apoptosis in ischemic neuronal damage have been accumulating, and protection against apoptotic death by BCL-2 has been shown in many types of cells. Overexpression of BCL-2 has been shown to reduce infarct size after focal ischemia. The purpose of the present study was to assess whether BCL-2 exerted its effect on selective neuronal vulnerability after transient global ischemia. METHODS Transgenic mice overexpressing BCL-2 in neurons and their littermates were subjected to transient forebrain ischemia for 12 minutes, and the hippocampus was examined 7 days later with conventional histology, immunohistochemistry, and in situ terminal deoxynucleotidyl transferase-mediated dUTP-biotin nick end-labeling of fragmented DNA. RESULTS Although both types of mice showed a similar degree of ischemic insult, transgenic mice showed a lesser degree of neuronal death together with DNA fragmentation in the hippocampus than their littermates. CONCLUSIONS Overexpression of BCL-2 in neurons mitigates selective neuronal vulnerability in the hippocampus of transgenic mice after transient global ischemia.

Exposure of Astrocytes to Hypoxia/Reoxygenation Enhances Expression of Glucose-Regulated Protein 78 Facilitating Astrocyte Release of the Neuroprotective Cytokine Interleukin 6

Abstract: Astrocytes exposed to hypoxia (H) or hypoxia/reoxygenation (H/R) maintain cell viability and display changes in protein biosynthesis. Sodium dodecyl sulfate‐polyacrylamide gel electrophoresis of metabolically labeled astrocytes exposed to H showed induction of an ≈78‐kDa polypeptide that demonstrated sequence identity with glucose‐regulated protein (GRP) 78. Cell lysates from H/R astrocytes displayed induction of neuroprotective interleukin (IL) 6, which was present in a high‐molecular‐weight complex also containing GRP78, suggesting that GRP78 might be functioning as a chaperone during cellular stress consequent on H/R. Introduction of anti‐sense oligonucleotide to GRP78 into astrocytes prevented expression of the protein and suppressed H/R‐induced astrocyte release of IL‐6 by ≈50%. These data indicate that modulation of astrocyte properties during oxygen deprivation results, in part, from intracellular glucose depletion and subsequent expression of GRP78, which sustains generation of neuroprotective IL‐6 under the stress of H/R.

Involvement of ICAM-1 in the progression of atherosclerosis in APOE-knockout mice

Recent clinical evidence has indicated that the level of soluble ICAM-1 (sICAM-1) is correlated with the severity of atherosclerosis and can predict future cardiovascular events. Here, using apolipoprotein E (APOE)-deficient mice, we investigated the level of sICAM-1 in parallel with endothelial ICAM-1 expression and aortic atherosclerosis. We also examined the effect of ICAM-1 deficiency during the progression of atherosclerosis using double knockout mice. The level of sICAM-1 increased significantly in parallel with the progression of atherosclerosis in APOE-deficient mice, while the sICAM-1 level remained constant in wild-type mice from 3 to 10 months of age. ICAM-1 staining was detected in virtually all endothelial cells, however, ICAM-1 was expressed strongly in the endothelium overlying atheromatous palque in APOE-deficient mice. Deficiency of ICAM-1 in APOE-deficient mice significantly reduced lesions after 5 and 10 months. The present study supported the notion that the level of sICAM-1 is closely related with the severity of atherosclerosis and cardiovascular events, and also suggested that inhibition of ICAM-1 can delay the progression of atherosclerosis.

Higher Levels of Interleukin-6 Are Associated With Lower Echogenicity of Carotid Artery Plaques

Background and Purpose— Echo-lucent carotid plaques can be fragile and vulnerable to rupture, representing a risk factor for ischemic stroke. Given the studies showing that elevated levels of circulating inflammatory markers are predictive of cardiovascular events, we sought to determine whether higher levels of serum interleukin-6 (IL-6) and high-sensitivity C-reactive protein (hsCRP) are associated with lower echogenicity of carotid plaques. Methods— The study comprised 246 patients who had carotid atherosclerotic plaques as evidenced by ultrasound. Using acoustic densitometry, we quantified the echogenicity of the largest plaque in each patient by integrated backscatter analysis. Serum IL-6 and hsCRP levels were determined in all patients. Results— Both log-transformed IL-6 and hsCRP concentrations were negatively correlated with carotid plaque echogenicity (r =−0.28, P <0.001, and r =−0.14, P <0.05, respectively). When traditional atherosclerotic risk factors, plaque thickness, and medication use were controlled for, IL-6 levels were inversely associated with plaque echogenicity (&bgr;=−0.21, P <0.01), whereas such an association was of borderline significance for hsCRP (&bgr;=−0.12, P =0.06). Conclusions— Higher IL-6 levels, in addition to hsCRP levels, appear to be associated with lower echogenicity of carotid plaques, suggesting a link between inflammation and potential risk of plaques.