Suppression of Coronary Atherosclerosis by Helix B Surface Peptide,a Nonerythropoietic,Tissue-Protective Compound Derived from Erythropoietin

Erythropoietin (EPO), a type I cytokine originally identified for its critical role in hematopoiesis, has been shown to have nonhematopoietic, tissue-protective effects, including suppression of atherosclerosis. However, prothrombotic effects of EPO hinder its potential clinical use in nonanemic patients. In the present study, we investigated the antiatherosclerotic effects of helix B surface peptide (HBSP), a nonerythropoietic, tissue-protective compound derived from EPO, by using human umbilical vein endothelial cells (HUVECs) and human monocytic THP-1 cells in vitro and Watanabe heritable hyperlipidemic spontaneous myocardial infarction (WHHLMI) rabbits in vivo. In HUVECs, HBSP inhibited apoptosis (≈70%) induced by C-reactive protein (CRP), a direct mediator of atherosclerosis. By using a small interfering RNA approach, Akt was shown to be a key molecule in HBSP-mediated prevention of apoptosis. HBSP also attenuated CRP-induced production of tumor necrosis factor (TNF)-α and matrix metalloproteinase-9 in THP-1 cells. In the WHHLMI rabbit, HBSP significantly suppressed progression of coronary atherosclerotic lesions as assessed by mean cross-sectional stenosis (HBSP 21.3 ± 2.2% versus control peptide 38.0 ± 2.7%) and inhibited coronary artery endothelial cell apoptosis with increased activation of Akt. Furthermore, TNF-α expression and the number of M1 macrophages and M1/M2 macrophage ratio in coronary atherosclerotic lesions were markedly reduced in HBSP-treated animals. In conclusion, these data demonstrate that HBSP suppresses coronary atherosclerosis, in part by inhibiting endothelial cell apoptosis through activation of Akt and in association with decreased TNF-α production and modified macrophage polarization in coronary atherosclerotic lesions. Because HBSP does not have the prothrombotic effects of EPO, our study may provide a novel therapeutic strategy that prevents progression of coronary artery disease.     

N-Glycosylation and N-Glycan Moieties of CTB Expressed in Rice Seeds

Cholera toxin B subunit (CTB) is widely used as a carrier molecule and mucosal adjuvant and for the expression of fusion proteins of interest. CTB-fusion proteins are also expressed in plants, but the N-glycan structures of CTB have not been clarified. To gain insights into the N-glycosylation and N-glycans of CTB expressed in plants, we expressed CTB in rice seeds with an N-terminal glutelin signal and a C-terminal KDEL sequence and analyzed its N-glycosylation and N-glycan structures. CTB was successfully expressed in rice seeds in two forms: a form with N-glycosylation at Asn32 that included both plant-specific N-glycans and small oligomannosidic N-glycans and a non-N-glycosylated form. N-Glycan analysis of CTB showed that approximately 50 % of the N-glycans had plant-specific M3FX structures and that almost none of the N-glycans was of high-mannose-type N-glycan even though the CTB expressed in rice seeds contains a C-terminal KDEL sequence. These results suggest that the CTB expressed in rice was N-glycosylated through the endoplasmic reticulum (ER) and Golgi N-glycosylation machinery without the ER retrieval.     

The influence of blood glucose on neutrophil function in individuals without diabetes

We assessed the association of neutrophil function with glycated hemoglobin (HbA1c) levels in a Japanese general population. Participants were 809 males and females who were over 20 years old living in the Iwaki region in Aomori Prefecture located in northern Japan. Lifestyle parameters (smoking, alcohol consumption, and exercise habits), HbA1c and neutrophil function such as reactive oxygen species (ROS) production capability and phagocytic activity (PA) were measured. ROS production capability was measured before and after phagocytic stimulus to obtain basal ROS production and stimulated ROS production. Level of HbA1c had a positive correlation with basal ROS production (p=0.053), a negative correlation with stimulated ROS production (p=0.072) and PA (p=0.059) only in post‐menopausal groups, and not in pre‐menopausal groups. However, there were no correlations between levels of HbA1c and neutrophil functions in male. In conclusion, in the present study, despite the presence of diabetes, chronic hyperglycemia was found to cause an increase in daily basal ROS production of neutrophils, and increased susceptibility to infection caused by reduced neutrophilic reaction in females in their menopause. Therefore, from the oxidative point of view, strict glycemic control is necessary to prevent post‐menopausal females from developing diabetic complications in spite of the presence of diabetes.     

Increase of Circulating CD11b+Gr1+ cells and Recruitment into the Synovium in Osteoarthritic Mice with Hyperlipidemia

Although recent studies suggest that hyperlipidemia is a risk factor for osteoarthritis (OA), the link between OA and hyperlipidemia is not fully understood. As the number of activated, circulating myeloid cells is increased during hyperlipidemia, we speculate that myeloid cells contribute to the pathology of OA. Here, we characterized myeloid cells in STR/Ort mice, a murine osteoarthritis model, under hyperlipidemic conditions. Ratios of myeloid cells in bone marrow, the spleen, and peripheral blood were determined by flow cytometry. To examine the influence of the hematopoietic environment, including abnormal stem cells, on the hematopoietic profile of STR/Ort mice, bone marrow transplantations were performed. The relationship between hyperlipidemia and abnormal hematopoiesis was examined by evaluating biochemical parameters and spleen weight of F₂ animals (STR/Ort x C57BL/6J). In STR/Ort mice, the ratio of CD11b⁺Gr1⁺ cells in spleens and peripheral blood was increased, and CD11b⁺Gr1⁺ cells were also present in synovial tissue. Splenomegaly was observed and correlated with the ratio of CD11b⁺Gr1⁺ cells. When bone marrow from GFP-expressing mice was transplanted into STR/Ort mice, no difference in the percentage of CD11b⁺Gr1⁺ cells was observed between transplanted and age-matched STR/Ort mice. Analysis of biochemical parameters in F₂ mice showed that spleen weight correlated with serum total cholesterol. These results suggest that the increase in circulating and splenic CD11b⁺Gr1⁺ cells in STR/Ort mice originates from hypercholesterolemia. Further investigation of the function of CD11b⁺Gr1⁺ cells in synovial tissue may reveal the pathology of OA in STR/Ort mice.     

Vitamin D2 interacts with Human PrPc (90–231) and breaks PrPc oligomerization in vitro

PrP^sc, the pathogenic isoform of PrP^c, can convert PrP^c into PrP^sc through direct interactions. PrP^c oligomerization is a required processing step before PrP^sc formation, and soluble oligomers appear to be the toxic species in amyloid-related disorders. In the current study, direct interactions between vitamin D₂ and human recombinant PrP^c (90–231) were observed by Biacore assay, and 3F4 antibody, specific for amino acid fragment 109–112 of PrP^c, inhibited this interaction. An ELISA study using3F4 antibody showed that PrP^c (101–130), corresponding sequence to human PrP, was affected by vitamin D₂, supporting the results of Biacore studies and suggesting that the PrP^c sequence around the 3F4 epitope was responsible for the interaction with vitamin D₂. Furthermore, the effects of vitamin D₂ on disruption of PrP^c (90–231) oligomerization were elucidated by dot blot analysis and differential protease k susceptibilities. While many chemical compounds have been proposed as potential therapeutic agents for the treatment of scrapie, most of these are toxic. However, given the safety and blood brain barrier permeability of vitamin D₂, we propose that vitamin D₂ may be a suitable agent to target PrP^c in the brain and therefore is a potential therapeutic candidate for prion disease.     

A Decreased Level of Serum Soluble Klotho Is an Independent Biomarker Associated with Arterial Stiffness in Patients with Chronic Kidney Disease

Background

Klotho was originally identified in a mutant mouse strain unable to express the gene that consequently showed shortened life spans. In humans, low serum Klotho levels are related to the prevalence of cardiovascular diseases in community-dwelling adults. However, it is unclear whether the serum Klotho levels are associated with signs of vascular dysfunction such as arterial stiffness, a major determinant of prognosis, in human subjects with chronic kidney disease (CKD).

Methods

We determined the levels of serum soluble Klotho in 114 patients with CKD using ELISA and investigated the relationship between the level of Klotho and markers of CKD-mineral and bone disorder (CKD-MBD) and various types of vascular dysfunction, including flow-mediated dilatation, a marker of endothelial dysfunction, ankle-brachial pulse wave velocity (baPWV), a marker of arterial stiffness, intima-media thickness (IMT), a marker of atherosclerosis, and the aortic calcification index (ACI), a marker of vascular calcification.

Results

The serum Klotho level significantly correlated with the 1,25-dihydroxyvitamin D level and inversely correlated with the parathyroid hormone level and the fractional excretion of phosphate. There were significant decreases in serum Klotho in patients with arterial stiffness defined as baPWV≥1400 cm/sec, atherosclerosis defined as maximum IMT≥1.1 mm and vascular calcification scores of ACI>0%. The serum Klotho level was a significant determinant of arterial stiffness, but not endothelial dysfunction, atherosclerosis or vascular calcification, in the multivariate analysis in either metabolic model, the CKD model or the CKD-MBD model. The adjusted odds ratio of serum Klotho for the baPWV was 0.60 (p = 0.0075).

Conclusions

Decreases in the serum soluble Klotho levels are independently associated with signs of vascular dysfunction such as arterial stiffness in patients with CKD. Further research exploring whether therapeutic approaches to maintain or elevate the Klotho level could improve arterial stiffness in CKD patients is warranted.