Hideo Kohka Takahashi

Hyperglycaemia provides a suitable environment for infections and the mechanisms of glucose toxicity include the formation of advanced glycation end-products (AGEs), which comprise non-enzymatically glycosylated proteins, lipids, and nucleic acid amino groups. Among AGE-associated phenotypes, glycolaldehyde-derived toxic AGE (AGE-3) is involved in the pathogenesis of diabetic complications. Internalisation of endotoxin by various cell types contributes to innate immune responses against bacterial infection. An endotoxin derived from Gram-negative bacteria, lipopolysaccharide (LPS), was reported to enhance its own uptake by RAW264.7 mouse macrophage-like cells, and an LPS binding protein, CD14, was involved in the LPS uptake. The LPS uptake induced the activation of RAW264.7 leading to the production of chemokine CXC motif ligand (CXCL) 10, which promotes T helper cell type 1 responses. Previously, we reported that AGE-3 was internalised into RAW264.7 cells through scavenger receptor-1 Class A. We hypothesized that AGEs uptake interrupt LPS uptake and impair innate immune response to LPS in RAW264.7 cells. In the present study, we found that AGE-3 attenuated CD14 expression, LPS uptake, and CXCL10 production, which was concentration-dependent, whereas LPS did not affect AGE uptake. AGEs were reported to stimulate the receptor for AGEs and Toll-like receptor 4, which cause inflammatory reactions. We found that inhibitors for RAGE, but not Toll-like receptor 4, restored the AGE-induced suppression of CD14 expression, LPS uptake, and CXCL10 production. These results indicate that the receptor for the AGE-initiated pathway partially impairs the immune response in diabetes patients.

Background: Cerebral small vessel disease (CSVD) is associated with future stroke. Although pathological alteration in small vessels of patients with CSVD can be detected by neuroimaging, diagnosis of CSVD is delayed because it is an asymptomatic disease. The stroke-prone spontaneously hypertensive rat (SHRSP) show similar pathological features to human CSVD and develop stroke-related symptoms with advancing age. Objective: We investigated the time course of haematological parameters in Wistar rats and SHRSP. Material and Methods: Blood cells were analysed using an automated haematological analyser. Results: SHRSP develop stroke-related symptoms including onset of neurological symptoms, decreased body weight and blood brain barrier leakage between 12 and 14 weeks of age. Lymphocyte counts were gradually decreased at 3 weeks before development of stoke-related symptoms and then were further decreased after the development of stroke-related symptoms. The both mean platelet volume and large platelet ratio gradually increased at 3 weeks before the development of stoke-related symptoms. However, although SHRSP showed more microcytic red cells than Wistar rats, the trajectories of change in erythrocyte-related parameters were similar between Wistar rats and SHRSP. Conclusion: Our pilot study suggests that alterations of lymphocyte count and platelet volume predictive indicators for asymptomatic CSVD and symptomatic stroke in SHRSP.

Advanced glycation end-products, especially toxic advanced glycation end-products derived from glyceraldehyde (advanced glycation end-product-2) and glycolaldehyde (advanced glycation end-product-3), are biologically reactive compounds associated with diabetic complications. We previously demonstrated that toxic advanced glycation end-products were internalised into macrophage-like RAW264.7 cells through scavenger receptor-1 class A (CD204). Toxic advanced glycation end-product uptake was inhibited by fucoidan, a sulphated polysaccharide and antagonistic ligand for scavenger receptors, suggesting that sulphated polysaccharides are emerging candidates for treatment of advanced glycation end-product–related diseases. In this study, we compared the effects of six types of sulphated and non-sulphated polysaccharides on toxic advanced glycation end-product uptake in RAW264.7 cells. Fucoidan, carrageenan and dextran sulphate attenuated toxic advanced glycation end-product uptake. Fucoidan and carrageenan inhibited advanced glycation end-product-2–induced upregulation of SR-A, while advanced glycation end-product-3–induced upregulation of scavenger receptor-1 class A was only suppressed by fucoidan. Dextran sulphate did not affect scavenger receptor-1 class A levels in toxic advanced glycation end-product–treated cells. Chondroitin sulphate, heparin and hyaluronic acid failed to attenuate toxic advanced glycation end-product uptake. Heparin and hyaluronic acid had no effect on scavenger receptor-1 class A levels, while chondroitin sulphate inhibited advanced glycation end-product-3–induced upregulation of scavenger receptor-1 class A. Taken together, fucoidan and carrageenan, but not the other sulphated polysaccharides examined, had inhibitory activities on toxic advanced glycation end-product uptake and toxic advanced glycation end-product–induced upregulation of scavenger receptor-1 class A, possibly because of structural differences among sulphated polysaccharides.

Reactive oxygen species (ROS) play important roles in the progression of septic pathogenesis. Recent studies revealed that neutrophil adhesion on vascular wall followed by neutrophil extracellular traps (NETs) release may trigger platelet aggregation and immunothrombus formation in septic organ failure. Additionally, the adherent neutrophils-produced ROS induce the immunothrombus formation and tissue damage. Our previous study indicated that plasma histidine-rich glycoprotein (HRG) levels significantly decreased in cecal ligation and puncture (CLP) septic mice model and administration of HRG dramatically improved the survival rate of CLP mice. However, the role of HRG on neutrophil ROS production and immunothrombosis in septic condition was poorly understood. In this study, we showed that HRG inhibited immunothrombus formation in pulmonary vasculatures by keeping neutrophils quiescent morphologically and functionally using immunohistochemical staining and in vivo imaging methods and suppressed excess extracellular ROS production from neutrophils using isoluminol method. These results suggested that HRG may regulate the uncontrolled activation of circulating neutrophils in septic condition.

Advanced glycation end products (AGEs), produced by non-enzymatic glycation between sugar (or its metabolites) and the amino residues on biomolecules, exert the inflammatory response via the stimulation of some pattern recognition receptors including RAGE. AGEs are known to increase in various age-related diseases, suggesting their involvement in chronic inflammation and tissue remodeling. Therefore, it is likely that the regulation of AGEs signaling will be the potent therapeutic target for prevention and treatment of age-related diseases. Previously, we purified the AGEs-binding factor using affinity gel with AGEs as specific ligand. In this study, we analyzed its properties, and tried to identify the peptides antagonizing the AGEs-RAGE binding. By in vitro AGEs-RAGE binding assay, AGEs-affinity chromatography and MALDI-TOF mass analysis, AGEs-binding factor with molecular mass of 70 kD was isolated. This factor inhibited AGEs-RAGE binding in concentration-dependent manner, and it was revealed that the inhibitory region existed near the N-terminus by the analysis using overlapping-peptides. Additionally, two minimal peptides exhibiting the inhibitory activity were identified. These findings suggested that AGEs-binding factor and its derived inhibitory peptides will have the potential usefulness for regulating chronic inflammation and tissue remodeling.

Hypertensive disorders of pregnancy (HDP) is a pathological condition with hypertension and vascular endothelial cell dysfunction (inflammation) during the gestational period. Recently, it was reported that reduction level of plasma histidine-rich glycoprotein (HRG) in human pregnant patients was correlated with the HDP seriousness. HRG is an anti-inflammatory factor that controls the progression of systemic inflammatory pathology and mainly produced from liver. However, HRG functions in HDP pathology and perinatal physiology have been still unknown. In this study, we examined the involvement of plasma HRG on placental formation and hypertensive event during the gestational period using C57BL/6 mice and HRG gene-deficient (HRG KO) mice. Plasma HRG level was decreased during the gestational period in C57BL/6 mice. In addition, HRG reduction was restored at post-partum and did not depend on the HRG gene expression in liver. Hysterectomy to the gestational C57BL/6 mice also restored the plasma HRG level up to the normal level. When Human plasma-purified HRG was injected to C57BL/6 mice on nulliparity or gestation, biological half-time of the injected HRG was short in the gestational mice compared with the nulliparous mice. Moreover, HRG KO mice showed hypertension by the gestation, whereas rise in blood pressure did not observed in the gestational C57BL/6 mice. The gestational HRG KO mice had fetal growth disorder and placental hyperplasia. These results suggest that HRG may have a physiological function in the gestational period. Pregnant HRG KO mice may be a HDP-like pathological model that causes abnormalities in the placental formation mechanism controlled by HRG.

Stroke is a global health problem and leads to disability. Efficacy of current treatment in both acute and chronic phase of stroke is limited. To accelerate individualized approach to primary prevention for stroke, identification of biological parameter, which is able to predict the risk of stroke development, is an indispensable component. Stroke-prone spontaneously hypertensive rat (SHRSP) is genetic animal model of chronic hypertension that progresses to stroke. Most of SHRSP spontaneously develop stroke including cerebral infarction and cerebral hemorrhage. Hematological tests readily provide health condition information. In this study, we investigated the time course of hematological parameters in Wistar rats and SHRSP. SHRSP develop stroke-related symptoms including onset of neurological symptoms, decreased body weight and blood brain barrier leakage between 12 and 14 weeks of age. Lymphocyte counts were gradually decreased at 3 weeks before development of stoke-related symptoms and then were further decreased after the development of stroke-related symptoms. The platelet volume, as shown by the mean platelet volume and large platelet ratio, gradually increased at 3 weeks before the development of stoke-related symptoms. However, although SHRSP showed more microcytic red cells than Wistar rats, the trajectories of change in erythrocyte-related parameters were similar between Wistar rats and SHRSP. Our findings suggest that alterations of lymphocyte count and platelet volume may be predictive indicators for stroke development in SHRSP.

Advanced glycation end-products (AGEs), which comprise non-enzymatically glycosylated proteins, lipids, and nucleic acid amino groups, play an important role in several diseases and aging processes including angiopathy, renal failure, diabetic complications, and neurodegenerative diseases. Among AGE-associated phenotypes, toxic AGEs, glyceraldehyde-derived AGE-2, and glycolaldehyde-derived AGE-3 are involved in the pathogenesis of diabetic complications. In addition, macrophages are reported to remove extracellular AGEs from tissues via scavenger receptors, leading to the progression of atherosclerosis. In the present study, we found that AGE-2 and AGE-3 enhanced their own endocytic uptake by RAW264.7 mouse macrophage-like cells in a concentration-dependent manner. Furthermore, we demonstrated, for the first time, the morphology of phagocytic macrophages and the endocytosis of AGE particles. The toxic AGEs induced the expression of a scavenger receptor, CD204/scavenger receptors-1 class A (SR-A). Notably, an antibody against CD204 significantly prevented toxic AGE uptake. Moreover, an SR-A antagonistic ligand, fucoidan, also attenuated the AGE-2- and AGE-3-evoked uptake in a concentration-dependent manner. These results indicated that SR-A stimulation, at least in part, plays a role in AGE uptake.