Hypolipidemic and Anti-Inflammatory Effects of Curcuma longa-Derived Bisacurone in High-Fat Diet-Fed Mice.

Turmeric (Curcuma longa) contains various compounds that potentially improve health. Bisacurone is a turmeric-derived compound but has been less studied compared to other compounds, such as curcumin. In this study, we aimed to evaluate the anti-inflammatory and lipid-lowering effects of bisacurone in high-fat diet (HFD)-fed mice. Mice were fed HFD to induce lipidemia and orally administered bisacurone daily for two weeks. Bisacurone reduced liver weight, serum cholesterol and triglyceride levels, and blood viscosity in mice. Splenocytes from bisacurone-treated mice produced lower levels of the pro-inflammatory cytokines IL-6 and TNF-α upon stimulation with a toll-like receptor (TLR) 4 ligand, lipopolysaccharide (LPS), and TLR1/2 ligand, Pam3CSK4, than those from untreated mice. Bisacurone also inhibited LPS-induced IL-6 and TNF-α production in the murine macrophage cell line, RAW264.7. Western blot analysis revealed that bisacurone inhibited the phosphorylation of IKKα/ß and NF-κB p65 subunit, but not of the mitogen-activated protein kinases, p38 kinase and p42/44 kinases, and c-Jun N-terminal kinase in the cells. Collectively, these results suggest that bisacurone has the potential to reduce serum lipid levels and blood viscosity in mice with high-fat diet-induced lipidemia and modulate inflammation via inhibition of NF-κB-mediated pathways.

Synthesis and evaluation of a deltic guanidinium analogue of a cyclic RGD peptide.

A guanidine group is abundantly found in natural products and drugs. Guanidine has the highest basicity among many common functional groups in nature. Because of its high basicity, it generally exists as a protonated guanidinium and functions as a cationic hydrogen bond donor. Finding an appropriate bioisostere of guanidinium is challenging because of its high basicity and unique trigonal planar shape. In this study, we explored the possibility of "deltic guanidinium" as a bioisostere of guanidinium using a cyclic arginine-glycine-aspartic acid (RGD) peptide as a parent compound. We synthesized c(deltic RGDyK), in which a guanidinium group of an arginine residue in c(RGDyK) is replaced with deltic guanidinium. A target binding assay, biodistribution study, and metabolic stability assay were conducted with c(deltic RGDyK) and its radioiodinated variant. The deltic guanidinium analog peptides exhibited similar biological properties to the parent peptides and improved in vivo stability, indicating that deltic guanidinium could work as a unique bioisostere of guanidinium.

[Development of Photocatalysts-Coated Polyethylene Terephthalate Film for Degradation and Elimination of Chemotherapy Drugs].

Prolonged exposure to anticancer drugs can lead to health damage in medical professionals as well as in patients and their families, and can be a serious issue. The risk of occupational acute exposure to anticancer drugs in medical professionals has largely been reduced following the establishment of guidelines for safe handling of hazardous drugs. However, the problem of pollution associated with residual anticancer drugs attached to the surface of floors and walls in the prescription laboratory, treatment room and the lavatory remains unsolved. We have recently developed a polyethylene terephthalate(PET) film coated with photocatalysts such as titanium dioxide and tungsten oxide. In the present study, the ability of the PET film to degrade anticancer drugs was tested by dripping anticancer drug solution on to the surface of the film followed by irradiation with LED light. The contents of several anticancer drugs, including 5-fluorouracil, cyclophosphamide, and cisplatin, were greatly reduced after 24 hr irradiation. Therefore, the present photocatalysts-coated PET film may become a useful tool to reduce the risk for exposure to hazardous agents including anticancer drugs.

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.

Heparin-induced thrombocytopenia associated with polycythemia vera during the treatment of acute coronary syndrome.

Here, we report a case of heparin-induced thrombocytopenia (HIT) associated with polycythemia vera (PV) during the treatment of acute coronary syndrome. An 84-year-old woman with pre-existing PV had an acute myocardial infarction and developed HIT after using heparin. An additional myocardial infarction was caused by HIT, and caused marked damage to her cardiac function. However, she was successfully treated with argatroban infusion and intensive care. In this case, we suspected HIT at an extremely early stage, when the decline in platelet count remained at 16%, which might have prevented further thrombosis. Subsequently, the nadir in the platelet count remained at 32%, which resulted in "intermediate possibility of HIT" according to the 4Ts score; thus, further detailed serological examination may be required for accurate diagnosis of HIT. .

Pyrrole-imidazole polyamide-mediated silencing of KCNQ1OT1 expression induces cell death in Wilms' tumor cells.

KvDMR (an intronic CpG island within the KCNQ1 gene) is one of the imprinting control regions on human chromosome 11p15.5. Since KvDMR exists within the promoter region of KCNQ1OT1 (antisense transcript of KCNQ1), it is likely that genomic alterations of this region including deletion, paternal uniparental disomy and de-methylation in maternal allele lead to aberrant overexpression of KCNQ1OT1. Indeed, de-methylation of KvDMR accompanied by uncontrolled overexpression of KCNQ1OT1 occurs frequently in Beckwith-Wiedemann syndrome (BWS), and around 10% of BWS patients developed embryonal tumors (Wilms' tumor or hepatoblastoma). These observations strongly suggest that silencing of KCNQ1OT1 expression might suppress its oncogenic potential. In the present study, we designed two pyrrole-imidazole (PI) polyamides, termed PI-a and PI-b, which might have the ability to bind to CCAAT boxes of the KCNQ1OT1 promoter region, and investigated their possible antitumor effect on Wilms' tumor-derived G401 cells. Gel retardation assay demonstrated that PI-a and PI-b specifically bind to their target sequences. Microscopic observations showed the efficient nuclear access of these PI polyamides. Quantitative real-time PCR analysis revealed that the expression level of KCNQ1OT1 was significantly decreased when treated with PI-a and PI-b simultaneously but not with either PI-a or PI-b single treatment. Consistent with these results, the combination of PI-a and PI-b resulted in a significant reduction in viability of G401 cells in a dose-dependent manner. Furthermore, FACS analysis demonstrated that combinatory treatment with PI-a and PI-b induces cell death as compared with control cells. Taken together, our present observations strongly suggest that the combinatory treatment with PI polyamides targeting KCNQ1OT1 might be a novel therapeutic strategy to cure patients with tumors over-expressing KCNQ1OT1.

Systematic implantation of dedifferentiated fat cells ameliorated monoclonal antibody 1-22-3-induced glomerulonephritis by immunosuppression with increases in TNF-stimulated gene 6.

INTRODUCTION: Implantation of mesenchymal stem cells (MSCs) has recently been reported to repair tissue injuries through anti-inflammatory and immunosuppressive effects. We established dedifferentiated fat (DFAT) cells that show identical characteristics to MSCs. METHODS: We examined the effects of 10(6) of DFAT cells infused through renal artery or tail vein on monoclonal antibody (mAb) 1-22-3-induced glomerulonephritis (as an immunological type of renal injury) and adriamycin-induced nephropathy (as a non-immunological type of renal injury) in rats. The mAb 1-22-3-injected rats were also implanted with 10(6) of DFAT cells transfected with TSG-6 siRNA through tail vein. RESULTS: Although DFAT cells transfused into blood circulation through the tail vein were trapped mainly in lungs without reaching the kidneys, implantation of DFAT cells reduced proteinuria and improved glomerulosclerosis and interstitial fibrosis. Implantation of DFAT cells through the tail vein significantly decreased expression of kidney injury molecule-1, collagen IV and fibronectin mRNAs, whereas nephrin mRNA expression was increased. Implantation of DFAT cells did not improve adriamycin-induced nephropathy, but significantly decreased the glomerular influx of macrophages, common leukocytes and pan T cells. However, the glomerular influx of helper T cells, was increased. Implantation of DFAT cells decreased expression of interleukin (IL)-6 and IL-12ß mRNAs and increased expression of TNF-stimulated gene (TSG)-6 mRNA in renal cortex from mAb 1-22-3-injected rats. The basal level of TSG-6 protein was significantly higher in DFAT cells than in fibroblasts. Expression of TSG-6 mRNA in MCs cocultured with DFAT cells was significantly higher than in mesangial cells or DFAT cells alone. Systematic implantation of DFAT cells with TSG-6 siRNA through tail vein did not improve proteinuria, renal dysfunction and renal degeneration in the mAb 1-22-3-injected rats. CONCLUSION: Systematic implantation of DFAT cells effectively ameliorated mAb 1-22-3-induced glomerulonephritis through immunosuppressive effects accompanied by the suppression of macrophage infiltration and expression of IL-6, IL-10 and IL-12ß, and increased production of serum and renal TSG-6 that improved the mAb 1-22-3-induced renal degeneration by the immunosuppressive effects of TSG-6. Thus DFAT cells will be suitable cell source for the treatment of immunological progressive renal diseases.

Inhibition of human osteosarcoma cell migration and invasion by a gene silencer, pyrrole-imidazole polyamide, targeted at the human MMP9 NF-κB binding site.

Osteosarcoma is one of the most prevalent bone tumors, occurring mostly in adolescence. However, no noticeable progress has been achieved in developing new therapeutic agents for this disease. Matrix metalloproteinase 9 (MMP9), a type IV collagenase, is a known anticancer target and is overexpressed in osteosarcomas. MMPs can degrade components of the extracellular matrix and are known to be involved in tumor invasion and metastasis. In the present study, we designed and synthesized a pyrrole-imidazole polyamide (HN.49), a gene-silencing agent that specifically targets the nuclear factor-kappa B (NF-κB) binding site of the human MMP9 promoter. We then examined the effect of HN.49 on the enzyme activity of MMP9 and the migration activity of osteosarcoma cells in vitro. It was clearly shown that HN.49 polyamide reduced the expression level of MMP9 mRNA and the enzymatic activity of MMP-9 in SaOS-2 cells. Moreover, HN.49 polyamide inhibited migration and invasion by SaOS-2 cells in in vitro wound-closure and matrigel-invasion assays. These results indicate that HN.49 may be a potential therapeutic agent for inhibiting the invasion and metastasis of osteosarcoma.

Inhibition of malignant phenotypes of human osteosarcoma cells by a gene silencer, a pyrrole-imidazole polyamide, which targets an E-box motif.

Gene amplification and/or overexpression of the transcription factor c-MYC, which binds to the E-box sequence (5'-CACGTG-3'), has been observed in many human tumors. In this study, we have designed 5 pyrrole-imidazole (PI) polyamides recognizing E-box, and found that, among them, Myc-6 significantly suppresses malignant phenotypes of human osteosarcoma MG63 cells both in vitro and in vivo. Intriguingly, knockdown of the putative Myc-6 target MALAT1 encoding long noncoding RNA remarkably impaired cell growth of MG63 cells. Collectively, our present findings strongly suggest that Myc-6 exerts its tumor-suppressive ability at least in part through the specific down-regulation of MALAT1.

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.

Nutrition education in Japanese medical schools: a follow-up survey.

A questionnaire survey was used to determine the status of nutrition education in Japanese medical schools in 2009. A similar survey was conducted in 2004, at which time nutritional education was determined to be inadequate in Japanese medical schools. The current questionnaire was sent to the directors of Centers for Medical Education of 80 medical schools, who represented all medical schools in Japan. Sixty-seven medical schools (83.8%) responded, of which 25 schools (37.3%) offered dedicated nutrition courses and 36 schools (53.7%) did not offer dedicated nutrition courses but offered something related to nutrition in other courses; six schools (9.0%) did not offer any nutrition education. Overall, 61 schools (91.0%) offered at least some nutritional topics in their undergraduate education. Nevertheless, only 11 schools (16.4%) seem to dedicate more than 5 hours to substantial nutrition education as judged by their syllabi. Although the mean length of the course was 11 hours, substantial nutrition education accounted for only 4.2 hours. Of the 25 medical schools that offered dedicated nutrition courses, seven schools offered the nutrition course as a stand-alone course and 18 schools offered it as an integrated course. In conclusion, the status of nutrition education in Japan has improved slightly but is still inadequate.

Effects of the herbal medicine composition "Saiko-ka-ryukotsu-borei-To" on the function of endothelial progenitor cells in hypertensive rats.

Endothelial progenitor cells (EPCs) are known to repair vascular injuries. Recent studies suggest that Saiko-ka-ryukotsu-borei-To (SKRBT), a traditional herbal medicine that has been used to treat stress-related neuropsychiatric disorders, has protective effects on cardiovascular diseases such as hypertension and arteriosclerosis. Spontaneously hypertensive rats (SHRs) were fed diets containing lyophilized SKRBT extract for 6 weeks. Peripheral blood mononuclear cells (MNCs) were isolated and cultured to assay EPC colony formation. Oxidative stress in MNCs was evaluated by thiobarbituric acid reactive substance (TBARS) assay and flowcytometric analyses. Treatment with SKRBT increased EPC colony numbers significantly (p<0.05) with decrease in oxidative stress and without affecting blood pressure in SHRs. Treatment with SKRBT did not reduce the expression of nicotinamide adenine dinucleotide phosphate (NADPH) oxidase subunits in cardiovascular organs. Serum IL-6 level was significantly reduced. SKRBT is a feasible herbal medicine that protects against cardiovascular diseases through an increase in EPC function along with anti-oxidative effects, and may affect the link between chronic inflammation and cardiovascular disease.

2-Nitroimidazole-tricarbocyanine conjugate as a near-infrared fluorescent probe for in vivo imaging of tumor hypoxia.

We developed a novel near-infrared (NIR) fluorescent probe, GPU-167, for in vivo imaging of tumor hypoxia. GPU-167 comprises a tricarbocyanine dye as an NIR fluorophore and two 2-nitroimidazole moieties as exogenous hypoxia markers that undergo bioreductive activation and then selective entrapment in hypoxic cells. After treatment with GPU-167, tumor cells contained significantly higher levels of fluorescence in hypoxia than in normoxia. In vivo fluorescence imaging specifically detected GPU-167 in tumors 24 h after administration. Ex vivo analysis revealed that fluorescence showed a strong correlation with hypoxia inducible factor (HIF)-1 active hypoxic regions. These data suggest that GPU-167 is a promising in vivo optical imaging probe for tumor hypoxia.

Anderson's disease/chylomicron retention disease in a Japanese patient with uniparental disomy 7 and a normal SAR1B gene protein coding sequence.

BACKGROUND: Anderson's Disease (AD)/Chylomicron Retention Disease (CMRD) is a rare hereditary hypocholesterolemic disorder characterized by a malabsorption syndrome with steatorrhea, failure to thrive and the absence of chylomicrons and apolipoprotein B48 post-prandially. All patients studied to date exhibit a mutation in the SAR1B gene, which codes for an essential component of the vesicular coat protein complex II (COPII) necessary for endoplasmic reticulum to Golgi transport. We describe here a patient with AD/CMRD, a normal SAR1B gene protein coding sequence and maternal uniparental disomy of chromosome 7 (matUPD7). METHODS AND RESULTS: The patient, one of two siblings of a Japanese family, had diarrhea and steatorrhea beginning at five months of age. There was a white duodenal mucosa upon endoscopy. Light and electron microscopy showed that the intestinal villi were normal but that they had lipid laden enterocytes containing accumulations of lipid droplets in the cytoplasm and lipoprotein-size particles in membrane bound structures. Although there were decreased amounts in plasma of total- and low-density lipoprotein cholesterol, apolipoproteins AI and B and vitamin E levels, the triglycerides were normal, typical of AD/CMRD. The presence of low density lipoproteins and apolipoprotein B in the plasma, although in decreased amounts, ruled out abetalipoproteinemia. The parents were asymptomatic with normal plasma cholesterol levels suggesting a recessive disorder and ruling out familial hypobetalipoproteinemia. Sequencing of genomic DNA showed that the 8 exons of the SAR1B gene were normal. Whole genome SNP analysis and karyotyping revealed matUPD7 with a normal karyotype. In contrast to other cases of AD/CMRD which have shown catch-up growth following vitamin supplementation and a fat restricted diet, our patient exhibits continued growth delay and other aspects of the matUPD7 and Silver-Russell Syndrome phenotypes. CONCLUSIONS: This patient with AD/CMRD has a normal SAR1B gene protein coding sequence which suggests that factors other than the SAR1B protein may be crucial for chylomicron secretion. Further, this patient exhibits matUPD7 with regions of homozygosity which might be useful for elucidating the molecular basis of the defect(s) in this individual. The results provide novel insights into the relation between phenotype and genotype in these diseases and for the mechanisms of secretion in the intestine.

Development of a novel gene silencer pyrrole-imidazole polyamide targeting human connective tissue growth factor.

Pyrrole-imidazole (PI) polyamide can bind to specific sequences in the minor groove of double-helical DNA and inhibit transcription of the genes. We designed and synthesized a PI polyamide to target the human connective tissue growth factor (hCTGF) promoter region adjacent to the Smads binding site. Among coupling activators that yield PI polyamides, 1-[bis(dimethylamino)methylene]-5-chloro-1H-benzotriazolium 3-oxide hexafluorophosphate (HCTU) was most effective in total yields of PI polyamides. A gel shift assay showed that a PI polyamide designed specifically for hCTGF (PI polyamide to hCTGF) bound the appropriate double-stranded oligonucleotide. A fluorescein isothiocyanate (FITC)-conjugated PI polyamide to CTGF permeated cell membranes and accumulated in the nuclei of cultured human mesangial cells (HMCs) and remained there for 48 h. The PI polyamide to hCTGF significantly decreased phorbol 12-myristate acetate (PMA)- or transforming growth factor-ß1 (TGF-ß1)-stimulated luciferase activity of the hCTGF promoter in cultured HMCs. The PI polyamide to hCTGF significantly decreased PMA- or TGF-ß1-stimulated expression of hCTGF mRNA in a dose-dependent manner. The PI polyamide to hCTGF significantly decreased PMA- or TGF-ß1-stimulated levels of hCTGF protein in HMCs. These results indicate that the developed synthetic PI polyamide to hCTGF could be a novel gene silencer for fibrotic diseases.

Treatment with valsartan stimulates endothelial progenitor cells and renal label-retaining cells in hypertensive rats.

OBJECTIVE: The pathogenesis of hypertension is dependent on tissue angiotensin (Ang) II, which induces cardiovascular and renal remodeling. The presence of label-retaining cells (LRCs) as renal stem cells has been reported in nephrotubulus. We examined effects of treatment with valsartan on endothelial progenitor cells (EPCs) and renal LRCs in stroke-prone spontaneously hypertensive rats (SHR-SP). METHODS: SHR-SP were salt-loaded and treated with hydralazine or valsartan. Peripheral blood mononuclear cells (MNCs) were cultured to assess EPC colony formation and migration. LRCs were labeled for 1 week with bromodeoxyuridine (BrdU) and were detected after a 2-week chase period. We measured expression of c-kit and Pax-2 mRNAs in renal medulla. RESULTS: Colony formation and migration of EPCs were suppressed in salt-loaded SHR-SP. Treatment with valsartan markedly stimulated these EPC functions. There was no difference in the number of renal LRCs in normotensive Wistar-Kyoto rats and SHR-SP. Treatment with valsartan significantly improved renal tubular degeneration and increased the number of LRCs in renal medulla from salt-loaded SHR-SP. Treatment with valsartan significantly increased expression of c-kit and Pax-2 mRNAs in renal medulla from salt-loaded SHR-SP. CONCLUSION: These findings suggest that ARBs have cardiovascular and renal protective effects through an antioxidative action that stimulates ECP function and increases the number of the self-repairing renal LRCs.

Protective effects of statin on cardiac fibrosis and apoptosis in adrenomedullin-knockout mice treated with angiotensin II and high salt loading.

Statins exert pleiotropic effects, including antioxidative and cellular protective effects. Endogenous adrenomedullin (AM) induces anti-inflammatory, anti-fibrotic and proangiogenic effects. We examined the effects of simvastatin on cardiac fibrosis and apoptosis in AM heterozygous knockout (AM(+/-)) mice treated with angiotensin (Ang) II and high salt loading. Seven-week-old AM(+/-) mice were infused with Ang II while on a high-salt diet with or without simvastatin for 2 weeks. Hearts were stained by hematoxylin-eosin or Masson's trichrome, and were immunostained with isolectin B(4) and α-smooth muscle actin antibodies. Expression of c-Kit and Sca-1 messenger RNA (mRNA) was evaluated by real-time PCR analysis. Apoptotic cells in hearts were identified by terminal deoxynucleotidyl transferase-mediated UTP end labeling (TUNEL) staining. Hearts from Ang II/salt loading AM(+/-) mice showed marked perivascular fibrosis around coronary arteries. Treatment with simvastatin significantly inhibited the fibrosis around coronary arteries in Ang II/salt-loading AM(+/-) mice. Expression of c-Kit and Sca-1 mRNAs in hearts from Ang II/salt-loading AM(+/-) mice was significantly lower than in hearts from wild-type mice. Treatment with simvastatin significantly increased the suppressed expression of c-Kit and Sca-1 mRNAs. In addition, treatment with simvastatin significantly increased the number of isolectin B(4)-positive capillary arteries in hearts from Ang II/salt-loading AM(+/-) mice. Ang II/high salt significantly increased apoptotic cells in hearts from AM(+/-) mice; this trend was reversed by treatment with simvastatin. Thus, statins have potent cardioprotective effects that may be associated with anti-fibrotic, proangiogenic and anti-apoptotic effects in Ang II/salt-loading AM(+/-) mice.

Zinc-finger transcriptional factor Sall1 induces angiogenesis by activation of the gene for VEGF-A.

Zinc-finger transcriptional factor Sall1 modulates gene expression and regulates organogenesis, including kidney development. Angiogenesis induced by vascular endothelial growth factor (VEGF) is also required for organogenesis. We investigated whether Sall1 induces angiogenesis through VEGF gene activation. Sall1 gene transfer induced marked neovascularization in rat cornea and in mouse embryoid bodies (EBs). The neovascularization in EBs was abolished by co-administration of anti-VEGF antibody. Sall1 gene transfer in Swiss 3T3 cells significantly increased the expression of VEGF-A mRNA but did not markedly increase the expression of fibroblast growth factor-2, epidermal growth factor, hepatocyte growth factor and ETS-1 mRNA. Sall1 gene transfer significantly increased VEGF-A protein levels in conditioned medium from cultured fibroblasts. Sall1 gene transfer significantly increased VEGF-A promoter activity in HEK293T cells as compared with cells transfected with mock vector or truncated Sall1. These results suggest that Sall1 induces angiogenesis by stimulating VEGF-A promoter activity.

Ubiquitin-positive foam cells are identified in the aortic and mitral valves with atherosclerotic involvement.

AIM: Our aim was to determine the roles of the ubiquitin (Ub)-proteasome system (UPS) in valvular diseases by immunohistochemically identifying Ub-positive cells in aortic and mitral valves and determining if Ub+cells were associated with the severity of valvular diseases. METHODS: We evaluated surgically removed aortic and mitral valves from 60 patients (mean age, 64.5 years) for thickening, fibrosis, foam cell infiltration, thrombus, and atheromatous plaques by using grading scores. U+cells were detected immunohistochemically. RESULTS: We found Ub+cells in 16 (26.7%) of the 60 patients. Eleven (28.2%) of the 39 aortic valves and 5 (23.8%) of the 21 mitral valves were Ub-positive. Ub was found with granular depositions in the cytoplasm of monocyte-derived foam cells that were CD68+. The aortic valvular thickness of the Ub+group was significantly greater than that of the Ub- group (3.9+/-1.6mm vs. 3.2+/-1.6mm, p<0.05). Foam cells and fibrosis were greater in the Ub+group (p<0.05), and calcifications were prominent in aortic valves. There was no difference in the number of apoptotic cells in Ub+ and Ub- groups. Ub+cells were present in the affected valves and ubiquitinated proteins were accumulated in macrophage-derived foam cells. CONCLUSIONS: Ub+ foam cells are present in valves that are vulnerable to valvular disease, and UPS may contribute to the development of atherosclerosis through the inflammatory process.

Effects of atorvastatin on angiogenesis in hindlimb ischemia and endothelial progenitor cell formation in rats.

AIM: To investigate the mechanisms underlying the pro-angiogenic effects of statin, the effects of atorvastatin were investigated on the expression of angiogenic factors in ischemic hindlimbs of rats. The function and number of endothelial progenitor cells (EPCs) were investigated in hypertensive rats. METHODS: Hindlimb ischemia rats were administered 10 or 30 mg/kg/day atorvastatin orally for 2 weeks. Angiogenesis was evaluated by a laser Doppler and by Isolectin-B4 immunostaining. The expressions of VEGF, IL-8, angiopoietin (Ang)-1, Ang-2, eNOS, and hemoxidase (HO)-1 were evaluated by Western blotting and immunohistochemistry. Spontaneously hypertensive rats (SHR) were administered 10 mg/kg/day atorvastatin. EPC function was evaluated by colony formation and migration. The EPC number was evaluated by CD34-positive cells. RESULTS: A lowdose of atorvastatin, but not a highdose, significantly increased regional blood flow. Atorvastatin significantly increased the expressions of VEGF, IL-8, Ang-1, Ang-2, eNOS, and HO-1 proteins in ischemic hindlimbs. Atorvastatin significantly increased the number and colony formation of EPCs and decreased oxidation in mononuclear cells from SHR. CONCLUSION: Atorvastatin strongly induced angiogenesis with increases in angiogenic cytokines, HO-1 and EPC numbers. Statins are thus considered potertial agents for therapeutic angiogenesis.