Neuroblastoma suppressor of tumorigenicity 1 is associated with the severity of interstitial fibrosis and kidney function decline in IgA nephropathy.

INTRODUCTION: Recently, circulating neuroblastoma suppressor of tumorigenicity 1 (NBL1) was shown to be strongly associated with kidney disease progression and histological lesions in patients with diabetic kidney disease. This study aimed to examine whether serum NBL1 level was also associated with kidney function and renal histological findings in patients with IgA nephropathy. METHODS: We evaluated the levels of NBL1 in 109 patients with newly diagnosed biopsy-proven primary IgAN, between 2009 and 2018, at the Nihon University School of Medicine Itabashi Hospital, Tokyo, Japan, using serum obtained immediately before the renal biopsy, and examined the relationship between serum NBL1, renal function and renal histological findings assessed using the Oxford Classification (MEST score). Furthermore, we analyzed the association of serum NBL1 with kidney function decline over time in patients with IgA nephropathy who had follow-up data on the estimated glomerular filtration rate (n = 76). RESULTS: Serum NBL1 levels in patients with newly diagnosed IgA nephropathy were elevated, as compared to those in healthy individuals (n = 93). Logistic regression analysis demonstrated that the serum NBL1 level was independently and significantly associated with tubular atrophy/interstitial fibrosis. Immunohistochemical staining revealed that NBL1 was highly expressed in the tubulointerstitium. Furthermore, Spearman's rank correlation identified a significant correlation between serum NBL1 level and estimated glomerular filtration rate slope. CONCLUSIONS: The serum NBL1 level was significantly associated with the severity of renal interstitial fibrosis and kidney disease progression in patients with newly diagnosed IgA nephropathy. Thus, circulating NBL1 may serve as a good biomarker for evaluating renal interstitial fibrosis and the risk of kidney disease progression.

Gut-Microbiota Dysbiosis in Stroke-Prone Spontaneously Hypertensive Rats with Diet-Induced Steatohepatitis.

Metabolic-dysfunction-associated fatty-liver disease (MAFLD) is the principal worldwide cause of liver disease. Individuals with nonalcoholic steatohepatitis (NASH) have a higher prevalence of small-intestinal bacterial overgrowth (SIBO). We examined gut-microbiota isolated from 12-week-old stroke-prone spontaneously hypertensive-5 rats (SHRSP5) fed on a normal diet (ND) or a high-fat- and high-cholesterol-containing diet (HFCD) and clarified the differences between their gut-microbiota. We observed that the Firmicute/Bacteroidetes (F/B) ratio in both the small intestines and the feces of the SHRSP5 rats fed HFCD increased compared to that of the SHRSP5 rats fed ND. Notably, the quantities of the 16S rRNA genes in small intestines of the SHRSP5 rats fed HFCD were significantly lower than those of the SHRSP5 rats fed ND. As in SIBO syndrome, the SHRSP5 rats fed HFCD presented with diarrhea and body-weight loss with abnormal types of bacteria in the small intestine, although the number of bacteria in the small intestine did not increase. The microbiota of the feces in the SHRSP5 rats fed HFCD was different from those in the SHRP5 rats fed ND. In conclusion, there is an association between MAFLD and gut-microbiota alteration. Gut-microbiota alteration may be a therapeutic target for MAFLD.

Development of gene silencer pyrrole-imidazole polyamides targeting GSK3ß for treatment of polycystic kidney diseases.

The cyclic adenosine monophosphate (cAMP)-response element binding protein (CREB)-glycogen synthase kinase 3ß (GSK3ß) signaling pathway was reported to be involved in the progression of autosomal dominant polycystic kidney diseases (ADPKD). We designed and synthesized pyrrole-imidazole (PI) polyamides as novel gene-silencers to prevent binding of CREB on the GSK3ß gene promoter and examined the effects of the PI polyamides on proliferation and cyst formation of mouse collecting duct M1 cells. The GSK3ß PI polyamides significantly inhibited expression of GSK3ß mRNA in M1 cells with forskolin. To obtain cells as collecting ducts from ADPKD, the PKD1 gene was knocked down by shRNA. Lower concentrations of forskolin significantly stimulated proliferation of PKD1 knock-down M1 cells, whereas GSK3ß PI polyamide significantly inhibited proliferation of PKD1 knock-down M1 cells with forskolin. Stimulation with forskolin for 5 days induced enlargement of cysts from PKD1 knock-down M1 cells. GSK3ß PI polyamides significantly suppressed the enlargement of cysts with forskolin stimulation in PKD1 knock-down M1 cells. Thus, the present study showed that transcriptional suppression of the GSK3ß gene by PI polyamides targeting the binding of CREB inhibited the proliferation and cyst formation of PKD1 knock-down M1 cells. The GSK3ß PI polyamides may potentially be novel medicines for ADPKD.

Twist-related protein 1 induces epithelial-mesenchymal transition and renal fibrosis through the upregulation of complement 3.

We have demonstrated that complement 3 (C3) is upregulated and induces epithelial-mesenchymal transition (EMT) phenomenon and renal fibrosis in unilateral ureteral obstruction (UUO) kidney. We investigated roles of twist-related protein 1 (TWIST1) in EMT phenomenon and renal fibrosis through C3 upregulation in a mouse UUO model with gene silencer pyrrole-imidazole (PI) polyamides targeting TWIST1. We designed and synthesized PI polyamides targeting TWIST1 binding site on mouse pre-pro C3 promoter. Increased expression C3 mRNA with interferon-γ was significantly inhibited with PI polyamide in nephrotubular epithelial cells. Immunofluorescence showed suppression of E-cadherin and enhancement of α-smooth muscle actin (α-SMA) stainings as EMT phenomena in UUO kidney. TWIST1 and C3 expression was significantly increased in UUO kidney versus contralateral unobstructed kidney (CUK). Expression of transforming growth factor-ß1 (TGF-ß1), α-SMA and renin mRNAs was increased in UUO kidney versus CUK. Systemic administration of TWIST1 PI polyamide significantly suppressed increased C3 expression in UUO kidney versus CUK. PI polyamide administration also suppressed the increased expression of TGF-ß1, α-SMA and renin mRNAs and histologically improved renal fibrosis in UUO kidney. These findings indicate that TWIST1 induces EMT phenomenon and renal fibrosis by TGF-ß1 upregulation of C3 in mouse UUO model and that TWIST1 PI polyamide may be a novel medicine for renal fibrosis.

Transcriptional Suppression of Diabetic Nephropathy with Novel Gene Silencer Pyrrole-Imidazole Polyamides Preventing USF1 Binding to the TGF-ß1 Promoter.

Upstream stimulatory factor 1 (USF1) is a transcription factor that is increased in high-glucose conditions and activates the transforming growth factor (TGF)-ß1 promoter. We examined the effects of synthetic pyrrole-imidazole (PI) polyamides in preventing USF1 binding on the TGF-ß1 promoter in Wistar rats in which diabetic nephropathy was established by intravenous administration of streptozotocin (STZ). High glucose induced nuclear localization of USF1 in cultured mesangial cells (MCs). In MCs with high glucose, USF1 PI polyamide significantly inhibited increases in promoter activity of TGF-ß1 and expression of TGF-ß1 mRNA and protein, whereas it significantly decreased the expression of osteopontin and increased that of h-caldesmon mRNA. We also examined the effects of USF1 PI polyamide on diabetic nephropathy. Intraperitoneal injection of USF1 PI polyamide significantly suppressed urinary albumin excretion and decreased serum urea nitrogen in the STZ-diabetic rats. USF1 PI polyamide significantly decreased the glomerular injury score and tubular injury score in the STZ-diabetic rats. It also suppressed the immunostaining of TGF-ß1 in the glomerulus and proximal tubules and significantly decreased the expression of TGF-ß1 protein from kidney in these rats. These findings indicate that synthetic USF1 PI polyamide could potentially be a practical medicine for diabetic nephropathy.

Contribution of TGF-ß1 and Effects of Gene Silencer Pyrrole-Imidazole Polyamides Targeting TGF-ß1 in Diabetic Nephropathy.

TGF-ß1 has been known to induce diabetic nephropathy with renal fibrosis and glomerulosclerosis. DNA-recognized peptide compound pyrrole-imidazole (PI) polyamides as novel biomedicines can strongly bind promoter lesions of target genes to inhibit its transcription. We have developed PI polyamide targeting TGF-ß1 for progressive renal diseases. In the present study, we evaluated the contribution of TGF-ß1 in the pathogenesis of diabetic nephropathy, and examined the effects of PI polyamide targeting TGF-ß1 on the progression of diabetic nephropathy in rats. For in vitro experiments, rat renal mesangial cells were incubated with a high (25 mM) glucose concentration. Diabetic nephropathy was established in vivo in eight-week-old Wistar rats by intravenously administering 60 mg/kg streptozotocin (STZ). We examined the effects of PI polyamide targeting TGF-ß1 on phenotype and the growth of mesangial cells, in vitro, and the pathogenesis of diabetic nephropathy in vivo. High glucose significantly increased expression of TGF-ß1 mRNA, changed the phenotype to synthetic, and increased growth of mesangial cells. STZ diabetic rats showed increases in urinary excretions of protein and albumin, glomerular and interstitial degenerations, and podocyte injury. Treatment with PI polyamide targeting TGF-ß1 twice weekly for three months improved the glomerular and interstitial degenerations by histological evaluation. Treatment with PI polyamide improved podocyte injury by electron microscopy evaluation. These findings suggest that TGF-ß1 may be a pivotal factor in the progression of diabetic nephropathy, and PI polyamide targeting TGF-ß1 as a practical medicine may improve nephropathy.

Lipoprotein Lipase Deficiency Arising in Type V Dyslipidemia.

A 40-year-old Japanese man presented with child-onset hypertriglyceridemia recently complicated by diabetes mellitus. The patient's diabetes mellitus was maintained, but he had persistent insulin resistance. The patient also had persistent severe hypertriglyceridemia (1,224-4,104 mg/dL), despite the administration of bezafibrate and ezetimibe. Type V dyslipidemia was revealed by agarose gel electrophoresis and the refrigerator test, and a significantly reduced post-heparin lipoprotein lipase mass of 26 ng/mL was confirmed. Genetic testing confirmed two heterozygous LPL variants, p.Tyr88X and p.Gly215Glu in trans; thus, the patient was diagnosed with lipoprotein lipase deficiency. Lipoprotein lipase deficiency typically arises in type I dyslipidemia, but is latent in type V dyslipidemia.

The adrenal gland circadian clock exhibits a distinct phase advance in spontaneously hypertensive rats.

The circadian clock influences a multitude of cellular and biological processes, including blood pressure control. Spontaneously hypertensive rats (SHR) exhibit aberrant circadian rhythms affecting cardiovascular parameters, and they also have abnormal clock gene expression profiles in several organs. Given the important role of the adrenal gland in orchestrating circadian oscillations, we investigated the adrenal gland circadian clock in SHR and control Wistar-Kyoto rats maintained under a 12-hour light-dark cycle. Adrenal glands, livers, and serum samples were collected every 4 h and mRNA was extracted for analysis of clock gene expression. Serum levels of corticosterone and aldosterone were also analyzed. Overall, the circadian profiles of Bmal1, Per2, Per3, Cry1, RevErba, Revervb, and Dbp gene expression in SHR adrenal glands were phase-advanced relative to controls. The expression profile of StAR (a representative gene under circadian control in the adrenal gland), as well as the circadian rhythms of serum concentrations of corticosteroid and aldosterone were also phase advanced. E4bp4 gene expression was significantly higher during the dark period, yet the expression of its transcriptional activator, Rora, was significantly lower throughout the 24 h period in SHR adrenal glands than in controls. This paradoxical high E4bp4 gene expression was, however, not observed in the liver. In addition, Per1, Per2, Per3, Reverba, and Reverbb mRNA tended to be lower in SHR adrenal glands than in controls. Thus, we conclude that SHR possess an abnormal adrenal circadian clock, which may affect the transcriptional regulation of clock-controlled genes, and steroid hormone secretion by the adrenal gland.

Association of mRNA expression of iron metabolism-associated genes and progression of non-alcoholic steatohepatitis in rats.

BACKGROUND: Excess iron is associated with non-alcoholic steatohepatitis (NASH). RESULTS: mRNA expression of duodenal cytochrome b, divalent metal transporter 1, ferroportin 1, hepcidin, hephaestin and transferrin receptor 1 in liver were higher in high fat, high cholesterol-containing diet (HFCD) group than in normal diet (ND) group. mRNA levels of divalent metal transporter 1 and transferrin receptor 1, which stimulate iron absorption and excretion, were enhanced in small intestine. Epithelial mucosa of small intestine in HFCD group was characterized by plasma cell and eosinophil infiltration and increased vacuoles. Iron absorption was enhanced in this NASH model in the context of chronic inflammation of small intestinal epithelial cells, consequences of intestinal epithelial cell impairment caused by HFCD. Iron is transported to hepatocytes via portal blood, and abnormalities in iron absorption and excretion occur in small intestine from changes in iron transporter expression, which also occurs in NASH liver. Knockdown of hepcidin antimicrobial peptide led to enhanced heavy chain of ferritin expression in human hepatocytes, indicating association between hepcidin production and iron storage in hepatocytes. CONCLUSIONS: Iron-related transporters in liver and lower/upper portions of small intestine play critical roles in NASH development. METHODS: Expression of iron metabolism-related genes in liver and small intestine was analyzed in stroke-prone spontaneously hypertensive rats (SHR-SP), which develop NASH. Five-week-old SHR-SP fed ND or HFCD were examined. mRNA and protein levels of iron metabolism-related genes in liver and small intestine from 12- and 19-week-old rats were evaluated by real-time RT-PCR and immunohistochemistry or Western blot.

Taurine and magnesium supplementation enhances the function of endothelial progenitor cells through antioxidation in healthy men and spontaneously hypertensive rats.

Endothelial damage is repaired by endothelial progenitor cells (EPCs), which are pivotal in preventing cardiovascular diseases and prolonging lifespan. The WHO Cardiovascular Diseases and Alimentary Comparison Study demonstrated that dietary taurine and magnesium (Mg) intake suppresses cardiovascular diseases. We herein evaluate the effects of taurine and Mg supplementation on EPC function and oxidative stress in healthy men and spontaneously hypertensive rats (SHRs). Healthy men received taurine (3 g per day) or Mg (340 mg per day) for 2 weeks. SHRs and Wistar-Kyoto (WKY) rats were housed with high-salt drinking water (1% NaCl). The SHRs received 3% taurine solution and/or a high-Mg (600 mg per 100 g) diet for 4 weeks. Their peripheral blood mononuclear cells were separated to quantify EPC colony formation. Oxidative stress markers in their peripheral blood were evaluated using a free radical analytical system and a thiobarbituric acid reactive substance (TBARS) assay. Taurine and Mg supplementation significantly increased EPC colony numbers and significantly decreased free radical levels and TBARS scores in healthy men. Taurine and Mg supplementation significantly increased EPC colony numbers and significantly decreased TBARS scores and free radical levels in SHRs. Nicotinamide adenine dinucleotide phosphate oxidase component mRNA expression was significantly higher in the renal cortex of salt-loaded SHRs than in WKY rats, in which it was suppressed by taurine and Mg supplementation. Taurine and Mg supplementation increased EPC colony formation in healthy men and improved impaired EPC function in SHRs through antioxidation, indicating that the dietary intake of taurine and Mg may prolong lifespan by preventing the progression of cardiovascular diseases.

Reduction of Dimethylnitrosamine-Induced Liver Fibrosis by the Novel Gene Regulator PI Polyamide Targeting Transforming Growth Factor ß1 Gene.

Pyrrole-imidazole (PI) polyamide is a novel gene regulating agent that competitively inhibits transcription factor binding to the promoter of the specific target gene. Liver fibrosis is an integral stage in the development of chronic liver disease, and transforming growth factor ß (TGFß) is known to play a central role in the progression of this entity. The aim of this study was to evaluate the effect of PI polyamide targeting TGFß1 on rat liver fibrosis. PI polyamide was designed to inhibit activator protein 1 (AP-1) transcription factor binding to the TGFß1 gene promoter. The effect of PI polyamide on hepatic stellate cells was evaluated by real time polymerase chain reaction (PCR) in RI-T cells. To determine the effect of PI polyamide in vivo, PI polyamide was intravenously administered at a dose of 3 mg/kg/week in dimethylnitrosamine (DMN)-induced rat model of liver fibrosis. Treatment of RI-T cells with 1.0 µM PI polyamide targeting TGFß1 significantly inhibited TGFß1 mRNA expression. Azan staining showed that DMN treatment significantly increased areas of fibrous materials compared with controls. PI polyamide targeting TGFß1 significantly decreased the fibrous area compared with DMN group. mRNA expression levels of α-smooth muscle actin and matrix metalloproteinase-2 were significantly increased in DMN-treated group compared with control. Treatment with TGFß1 PI polyamide significantly decreased mRNA expression of these genes compared with DMN group. The novel gene regulator PI polyamide targeting TGFß1 may be a feasible therapeutic agent for the treatment of chronic liver disease.

A novel gene regulator, pyrrole-imidazole polyamide targeting ABCA1 gene increases cholesterol efflux from macrophages and plasma HDL concentration.

UNLABELLED: Pyrrole-imidazole (PI) polyamides are nuclease-resistant novel compounds that inhibit transcription factors by binding to the minor groove of DNA. A PI polyamide that targets mouse ABCA1 and increases ABCA1 gene expression was designed and evaluated as an agent to increase plasma HDL concentration. A PI polyamide was designed to bind the activator protein-2 binding site of the mouse ABCA1 promoter. The effect of this PI polyamide on ABCA1 expression was evaluated by real-time RT-PCR and Western blotting using RAW264 cells. In vivo effects of this polyamide on ABCA1 gene expression and plasma HDL level were examined in C57B6 mice. One milligram per kilogram of body weight of PI polyamide was injected via the tail veins every 2 days for 1 week, and plasma lipid profiles were evaluated. PI polyamide showed a specific binding to the target DNA in gel mobility shift assay. Treatment of RAW264 cells with 1.0 µM PI polyamide significantly increased ABCA1 mRNA expression. PI polyamide also significantly increased apolipoprotein AI-mediated HDL biogenesis in RAW264 cells. Cellular cholesterol efflux mediated by apolipoprotein AI was significantly increased by the PI polyamide treatment. PI polyamide significantly increased expression of ABCA1 mRNA in the liver of C57B6 mice. Plasma HDL concentration was increased by PI polyamide administration. All of the HDL sub-fractions showed a tendency to increase after PI polyamide administration. The designed PI polyamide that targeted ABCA1 successfully increased ABCA1 expression and HDL biogenesis. This novel gene-regulating agent is promising as a useful compound to increase plasma HDL concentration. KEY MESSAGES: A novel pyrrole-imidazole (PI) polyamide binds to ABCA1. PI polyamide interfered with binding of AP-2ɑ protein to the ABCA1 gene promoter. PI polyamide inhibited the AP-2ɑ-mediated reduction of ABCA1 gene and protein expression. PI polyamide increased ABCA1 protein and apolipoprotein AI mediated HDL biogenesis. PI polyamide is a new gene regulator for the prevention of atherosclerotic diseases.

Effects of an angiotensin II receptor blocker on the impaired function of endothelial progenitor cells in patients with essential hypertension.

BACKGROUND: Endothelial progenitor cells (EPCs) induce neovascularization and repair vascular damage. We have demonstrated that EPC function is impaired in hypertensive rats with increases in oxidative stress and that angiotensin II receptor blockers improved the impaired function of EPCs. In this study, we investigated basal EPC functions in normotensive control subjects and patients with essential hypertension and the effect of losartan on EPC function in hypertensive patients. METHODS: Eighteen normotensive control subjects and 36 patients with essential hypertension who were undergoing treatment participated in the study. Hypertensive patients were randomly selected to receive 50mg of losartan or 4 mg of trichlormethiazide daily for 4 weeks. Peripheral blood mononuclear cells were isolated and cultured to assay EPC colony formation. Blood pressure, biological examination, and oxidative stress were evaluated in all subjects. RESULTS: The number of EPC colonies was significantly lower in patients with essential hypertension than in normotensive control subjects. EPC colony number was significantly and inversely correlated with systolic and diastolic blood pressure in all subjects. EPC colony number was significantly increased by treatment with losartan in patients with essential hypertension but not affected by treatment with trichlormethiazide. CONCLUSIONS: EPC function was inversely correlated with blood pressure and was impaired in essential hypertension. Losartan significantly improved the impaired EPC function in hypertensive patients. Impaired EPC function may determine the cardiovascular complications in essential hypertension. The improvement of EPC function with the administration of angiotensin II receptor blockers is considered to be one of the cardiovascular protective effects.

Development of pyrrole-imidazole polyamide targeting fc receptor common gamma chain for the treatment of immune-complex related renal disease.

Fcγ receptors I and III are thought to be involved in the development of lupus nephritis. Expression of Fc receptor common gamma chain (FcRγ) is necessary for the stable expression of Fcγ receptors I and III. The aim of this study was to develop a novel agent for the treatment of immune complex related renal disease using a gene regulator, pyrrole(Py)-imidazole(Im) (PI) polyamide, targeting the mouse FcRγ gene promoter. Two PI polyamides targeting FcRγ promoters were designed and synthesized. The effect of the PI polyamides on FcRγ mRNA expression was evaluated in J774.A cells by real-time polymerase chain reaction (PCR), and CD16/32 protein expression was determined by immunocytochemical analysis and flow cytometry. The effects of these polyamides on FcRγ gene expression and CD16/32 protein expression were evaluated in mouse peripheral blood mononuclear cells (PBMCs). One milligram per kilogram body weight of PI polyamide was injected via the tail vein every 2 d for 1 week and PBMCs were collected and analyzed. PI polyamide showed a specific binding to the target DNA in a gel mobility shift assay. Treatment of J774.A cells with 1.0 µM PI polyamide 1 significantly reduced FcRγ mRNA expression and CD16/32 surface protein expression in J774.A cells. Similarly, PI polyamide significantly decreased expression of FcRγ mRNA and CD16/32 in the PBMCs of C57B6 mice. PI polyamide designed to bind the FcRγ promoter decreased FcRγ gene and CD16/32 protein expression. PI polyamide targeting the FcRγ gene may be a novel gene regulator for the prevention of lupus nephritis or other immune complex-related disease.

Obesity alters the expression profile of clock genes in peripheral blood mononuclear cells.

INTRODUCTION: The aim of this study was to investigate the association between the variation in expression profile of clock genes and obesity using peripheral blood mononuclear (PMN) cells. MATERIAL AND METHODS: The subjects comprised 10 obese patients and 10 healthy volunteers. Blood was collected at different time-points during the day and levels of blood sugar, IRI, adiponectin and leptin were determined. Peripheral blood mononuclear cells were sampled, and expression levels of brain and muscle Arnt-like protein-1 (BMAL1), Period (PER)1, PER2, Cryptochrome (CRY)1, CRY2, and REV-ERBα mRNA were quantified. RESULTS: During the day, the expression levels of BMAL1, CRY1, CRY2 and PER2 genes in PMN cells of the obese group were all significantly higher compared to those in the non-obese group. In addition, expression of BMAL1, CRY1, CRY2 and PER2 genes in PMN cells increased between 12:00 and 21:00 in the obese group. In PMN cells of both groups, PER1 gene expression showed a bimodal pattern, with high expression at 9:00 and 18:00. CONCLUSIONS: Differences were observed in the expression profile variation of clock genes between the obese and non-obese groups. This study reveals the differences in clock gene expression profiles between obese and non-obese subjects, with evidence for two distinct chronotypes, and suggests a contribution of these chronotypes to fat accumulation in humans.

Moderate low temperature preserves the stemness of neural stem cells and suppresses apoptosis of the cells via activation of the cold-inducible RNA binding protein.

We hypothesized that one of the mechanisms underlying the protection of brain injury by therapeutic hypothermia is associated with preservation of neural stem cells. We investigated effects of moderate low temperature and the contribution of a cold-inducible molecule for the stemness of neural stem cells. The MEB5 mouse neural stem cell line was cultured in the presence or absence of EGF, and apoptosis, mRNA expression, and immunocytochemistry of the differentiation markers nestin and GFAP were evaluated at 37 or 32°C. We investigated the contribution of the cold-inducible RNA binding protein (CIRP) on apoptosis and differentiation of MEB5 cells at 32°C. EGF deprivation increased the number of apoptotic cells, decreased expression of nestin, and increased expression of GFAP. The moderate low temperature prevented apoptosis and decreases in expression of GFAP in MEB5 by EGF deprivation. The moderate low temperature significantly increased expression of CIRP. siRNA against CIRP significantly increased the apoptotic cell population of MEB5 cells via EGF deprivation at 32°C. These findings suggest that moderate low temperature preserved stemness of neural stem cells and prevented cell apoptosis via the stimulation of CIRP, and one of the mechanisms of rescue of brain injury by the moderate hypothermia is associated with preservation of neural stem cells.

Appropriate doses of granulocyte-colony stimulating factor reduced atherosclerotic plaque formation and increased plaque stability in cholesterol-fed rabbits.

AIM: To evaluate the dose-response effects of granulocyte-colony stimulating factor (G-CSF) on atherosclerosis, we examined how G-CSF treatment at different concentrations affects atherosclerotic plaque formation in the aorta of cholesterol-fed rabbits. METHODS: Japanese White rabbits (n=8 each) fed on a 1.5% cholesterol diet were subcutaneously injected with G-CSF at 50 (GL), 100 (GM), or 300 microg/kg/day (GH) for five days, or with 3 cycles of G-CSF at 100 microg/kg/day at 3-week intervals (GM3), or human serum albumin (Control). The extent and composition of atherosclerosis was evaluated 14 weeks after cholesterol feeding. RESULTS: Although G-CSF treatment did not affect plasma lipid levels, the percentage of aortic surface involvement in the GM3 group was significantly decreased (p<0.05) compared with the Control group. Histological analysis revealed that the intima media ratio was also diminished in GM and GM3 groups. The extent of intimal smooth muscle cell accumulation was higher in GL and GM3 groups than in the Control group. TIMP-2 mRNA expression in the aortic tissue was increased by G-CSF treatment. CONCLUSIONS: Our results suggest that appropriate doses of G-CSF reduced atherosclerotic plaque formation and increased plaque stability in cholesterol-fed rabbits.

Influence of genetic polymorphisms in oxidative stress related genes and smoking on plasma MDA-LDL, soluble CD40 ligand, E-selectin and soluble ICAM1 levels in patients with coronary artery disease.

BACKGROUND: Previous studies have shown that oxidative stress plays an important role in coronary heart disease. Polymorphisms in key enzymes that regulate oxidative stress may play a role in atherogenicity and were investigated in this study. MATERIAL/METHODS: One hundred and forty-three patients with angiographically proven coronary artery disease were studied. The effect of the C242T polymorphism of the p22phox gene, an essential component of the NADH/NADPH oxidase, and glutathione-S-transferase T1, M1 and P1 polymorphisms on plasma MDA-LDL, soluble CD40 ligand, E-selectin and soluble ICAM1 levels was determined. Genotyping of the p22 phox C242T polymorphism was performed by RFLP analysis, and GSTT1, GSTM1 and GSTP1 genotypes were determined using a multiplex PCR assay. The MDA-LDL, sCD40L, E-selectin and sICAM1 levels were determined using ELISA. RESULTS: Patients with the TT or TC genotype of the p22 phox C242T polymorphism had significantly higher plasma MDA-LDL levels compared to those of the CC genotype. Plasma E-selectin and soluble ICAM1 levels were significantly higher in the TT or TC genotype compared to that of the CC genotype. In GSTT1+ patients, plasma MDA-LDL levels were significantly higher than those of GSTT1- patients. CONCLUSIONS: Genetic polymorphism of the p22 phox gene had a significant effect on plasma lipid peroxidation and endothelial function through oxidative stress. The results of this study confirm the effect of NADH/NADPH oxidase on atherogenecity.

A novel gene silencer, pyrrole-imidazole polyamide targeting human lectin-like oxidized low-density lipoprotein receptor-1 gene improves endothelial cell function.

Pyrrole-imidazole polyamide can be combined in antiparallel side-by-side dimeric complexes along the minor groove of DNA in a sequence-specific manner. Pyrrole-imidazole polyamides are effective inhibitors of transcription factors as well as viral repressors and transactivators. Recently, lectin-like oxidized low-density lipoprotein receptor-1 (LOX-1) was reported to be a major factor contributing to the pathogenesis of coronary atherosclerosis. In this study, we designed a pyrrole-imidazole polyamide specific for the LOX-1 gene and evaluated its effect on LOX-1 gene transcription. A pyrrole-imidazole polyamide was designed to target the AP-1 binding site of the LOX-1 gene and synthesized by solid phase methods. This pyrrole-imidazole polyamide significantly inhibited LOX-1 promoter activity in HEK293 cells, determined by the luciferase assay. LOX-1 mRNA expression was also inhibited by the pyrrole-imidazole polyamide at a concentration of 10-9 mol/l in human umbilical vein endothelial cells (HUVEC), determined by the real-time PCR method. HUVEC were treated by pyrrole-imidazole polyamide targeting the LOX-1 gene, and apoptosis was assessed using Hoechst stain, terminal deoxy nucleotidyl transferase-mediated UTP end labeling method, and dye-uptake bioassay. Treatment of HUVEC for 72 h with LOX-1 targeted pyrrole-imidazole polyamide decreased apoptosis induced by angiotensin II and oxidized low-density lipoprotein (ox-LDL) loading in all assays. This novel therapeutic agent, pyrrole-imidazole polyamide, could specifically inhibit LOX-1 gene expression by reducing the promoter activity of the gene. Pyrrole-imidazole polyamide seems to be a powerful promising new agent that can be used to explore therapies based on inhibition of transcription. Molecular recognition of DNA by small molecules could provide insight into the development of new human medicines.

Atherogenic dyslipidemia and altered hepatic gene expression in SHRSP.Z-Leprfa/IzmDmcr rats.

We investigated lipid and lipoprotein abnormalities in SHRSP fatty rats as a new animal model of metabolic syndrome. We examined differentially expressed genes in the liver, one of the major tissues contributing to lipid metabolism. Using gel filtration high performance liquid chromatography, increased cholesterol concentrations of small particle size low-density lipoprotein (LDL) fractions were observed in SHRSP fatty rats, whereas the Zucker Fatty strain did not show a similar elevation of cholesterol content. Existence of apolipoprotein B in these fractions was confirmed by Western blotting. The small particle size of the LDL fractions was significantly decreased by a 4-week fenofibrate treatment. Microarray analysis identified seventeen genes that were significantly upregulated and ten that were significantly decreased in liver tissues of SHRSP fatty rats compared with levels in SHRSP rats. Stearoyl-coenzyme A desaturase 1, fatty acid synthase, ATP citrate lyase, and sterol regulatory element binding factor 1 genes were among the upregulated genes. These findings suggest that SHRSP fatty rats carry small dense LDL like particles which is a common lipid abnormality in the metabolic syndrome. Three of ten genes upregulated in liver tissues of SHRSP fatty rats play a role in this metabolic abnormality and are a therapeutic target of this metabolic syndrome.