Naoyuki Iso-O

Facilitated angiogenesis induced by heme oxygenase-1 gene transfer in a rat model of hindlimb ischemia.

Heme oxygenase-1 (HO-1) is an inducible form of heme oxygenase that catabolizes heme to carbon monoxide, biliverdin, and ferrous iron. We have investigated whether HO-1 can induce angiogenic effects in vivo. Rats were subjected to a bolus injection of either wild type adenovirus (ad-wt) or adenovirus encoding HO-1 (ad-HO-1) through the right femoral artery, which was then removed immediately. HO-1 gene transfer resulted in about a sixfold increase in HO-1 protein levels as compared to the non-treated animals. The increase in both blood flow and capillary density was significantly greater in the ischemic hindlimbs that had been injected with ad-HO-1 than in those injected with ad-wt. These angiogenic effects of ad-HO-1 infection could be completely abolished by treating the animals with the HO inhibitor, zinc protoporphyrin, indicating that they were specifically due to the expression of HO-1. Thus, HO-1 gene transfer improves the blood flow in ischemic hindlimb, at least in part, via angiogenesis facilitated by the induction of this molecule.

Thiazolidinediones Enhance Sodium-Coupled Bicarbonate Absorption from Renal Proximal Tubules via PPARγ-Dependent Nongenomic Signaling

Thiazolidinediones (TZDs) improve insulin resistance by activating a nuclear hormone receptor, peroxisome proliferator-activated receptor γ (PPARγ). However, the use of TZDs is associated with plasma volume expansion through a mechanism that remains to be clarified. Here we showed that TZDs rapidly stimulate sodium-coupled bicarbonate absorption from the renal proximal tubule in vitro and in vivo. TZD-induced transport stimulation is dependent on PPARγ-Src-EGFR-ERK and observed in rat, rabbit and human, but not in mouse proximal tubules where Src-EGFR is constitutively activated. The existence of PPARγ-Src-dependent nongenomic signaling, which requires the ligand-binding ability, but not the transcriptional activity of PPARγ, is confirmed in mouse embryonic fibroblast cells. The enhancement of the association between PPARγ and Src by TZDs supports an indispensable role of Src in this signaling. These results suggest that the PPARγ-dependent nongenomic stimulation of renal proximal transport is also involved in TZD-induced volume expansion.

Isoform-Dependent Cholesterol Efflux From Macrophages by Apolipoprotein E Is Modulated by Cell Surface Proteoglycans

Objective—Apolipoprotein E (apoE) mediates cellular cholesterol efflux and plays a crucial role in the inhibition of atherogenesis. We investigated whether there is an isoform-specific difference in its function for cholesterol efflux from cholesterol-loaded RAW264.7 cells, a murine macrophage cell line that lacks endogenous apoE expression. Methods and Results—When human apoE was expressed in RAW264.7 cells, apoE2 reduced cellular total cholesterol (TC) and esterified cholesterol (EC) levels significantly, whereas apoE3 and apoE4 had no effect. However, treatment of cells with 4-methylumbelliferyl-7-&bgr;-d-xyloside (&bgr;-DX) resulted in all 3 isoforms’ reducing cellular TC and EC contents significantly. We also investigated the effect of exogenously derived apoE on cholesterol efflux by utilizing the medium harvested from HeLa cells expressing apoE. ApoE2 and E3 reduced both cellular TC and EC contents significantly, whereas apoE4 did not. However, treatment of the cells with &bgr;-DX resulted in all 3 exogenously derived apoE isoforms’ reducing TC and EC contents significantly. The binding ability of apoE to heparan sulfate proteoglycans examined by heparinase I treatment revealed less binding ability of apoE2 compared with that of apoE3 or apoE4. Conclusions—The present study clarified the differential cellular cholesterol-modulating effect of apoE isoforms in macrophages, which would be due to the difference in their binding to proteoglycans.

Increased cholesterol biosynthesis and hypercholesterolemia in mice overexpressing squalene synthase in the liver

Squalene synthase (SS) is the first committed enzyme for cholesterol biosynthesis, located at a branch point in the mevalonate pathway. To examine the role of SS in the overall cholesterol metabolism, we transiently overexpressed mouse SS in the livers of mice using adenovirus-mediated gene transfer. Overexpression of SS increased de novo cholesterol biosynthesis with increased 3-hydroxy-3-methyglutaryl-CoA (HMG-CoA) reductase activity, in spite of the downregulation of its own mRNA expression. Furthermore, overexpression of SS increased plasma concentrations of LDL, irrespective of the presence of functional LDL receptor (LDLR). Thus, the hypercholesterolemia is primarily caused by increased hepatic production of cholesterol-rich VLDL, as demonstrated by the increases in plasma cholesterol levels after intravenous injection of Triton WR1339. mRNA expression of LDLR was decreased, suggesting that defective LDL clearance contributed to the development of hypercholesterolemia. Curiously, the liver was enlarged, with a larger number of Ki-67-positive cells. These results demonstrate that transient upregulation of SS stimulates cholesterol biosynthesis as well as lipoprotein production, providing the first in vivo evidence that SS plays a regulatory role in cholesterol metabolism through modulation of HMG-CoA reductase activity and cholesterol biosynthesis.

Modulation of lipid metabolism with the overexpression of NPC1L1 in mouse liver

Niemann-Pick C1-like 1 protein (NPC1L1), a transporter crucial in intestinal cholesterol absorption, is expressed in human liver but not in murine liver. To elucidate the role of hepatic NPC1L1 on lipid metabolism, we overexpressed NPC1L1 in murine liver utilizing adenovirus-mediated gene transfer. C57BL/6 mice, fed on normal chow with or without ezetimibe, were injected with NPC1L1 adenovirus (L1-mice) or control virus (Null-mice), and lipid analyses were performed five days after the injection. The plasma cholesterol levels increased in L1-mice, and FPLC analyses revealed increased cholesterol contents in large HDL lipoprotein fractions. These fractions, which showed α-mobility on agarose electrophoresis, were rich in apoE and free cholesterol. These lipoprotein changes were partially inhibited by ezetimibe treatment and were not observed in apoE-deficient mice. In addition, plasma and VLDL triglyceride (TG) levels decreased in L1-mice. The expression of microsomal triglyceride transfer protein (MTP) was markedly decreased in L1-mice, accompanied by the reduced protein levels of forkhead box protein O1 (FoxO1). These changes were not observed in mice with increased hepatic de novo cholesterol synthesis. These data demonstrate that cholesterol absorbed through NPC1L1 plays a distinct role in cellular and plasma lipid metabolism, such as the appearance of apoE-rich lipoproteins and the diminished VLDL-TG secretion.

LXR agonist increases apoE secretion from HepG2 spheroid, together with an increased production of VLDL and apoE-rich large HDL

BackgroundThe physiological regulation of hepatic apoE gene has not been clarified, although the expression of apoE in adipocytes and macrophages has been known to be regulated by LXR.Methods and ResultsWe investigated the effect of TO901317, a LXR agonist, on hepatic apoE production utilizing HepG2 cells cultured in spheroid form, known to be more differentiated than HepG2 cells in monolayer culture. Spheroid HepG2 cells were prepared in alginate-beads. The secretions of albumin, apoE and apoA-I from spheroid HepG2 cells were significantly increased compared to those from monolayer HepG2 cells, and these increases were accompanied by increased mRNA levels of apoE and apoA-I. Several nuclear receptors including LXRα also became abundant in nuclear fractions in spheroid HepG2 cells. Treatment with TO901317 significantly increased apoE protein secretion from spheroid HepG2 cells, which was also associated with the increased expression of apoE mRNA. Separation of the media with FPLC revealed that the production of apoE-rich large HDL particles were enhanced even at low concentration of TO901317, and at higher concentration of TO901317, production of VLDL particles increased as well.ConclusionsLXR activation enhanced the expression of hepatic apoE, together with the alteration of lipoprotein particles produced from the differentiated hepatocyte-derived cells. HepG2 spheroids might serve as a good model of well-differentiated human hepatocytes for future investigations of hepatic lipid metabolism.

Case Report: Cytokine-Induced Hypoalbuminemia in a Patient with Hemophagocytic Syndrome (Direct in Vitro Evidence for the Role of Tumor Necrosis Factor-α)

Hemophagocytic syndrome (HPS) is a rare, potentially lethal hematological disorde r, characterized by in® ltration of hemophagocytic histiocyte s into various tissues and organs. It can take a varie ty of clinical forms such as familial erythrophagocytic lymphohistiocytosis (FEL), virus-assoc iated hemophagocytic syndrome (VAHS), histiocytic medullary reticulosis, malignant histiocytosis, or malignancy-assoc iated hemophagocyti c syndrome (1± 6). Common clinical manife stations include high fever, weight loss, pancytopenia, hepatosplenomegaly, jaundice , and disseminated intravascular coagulopathy (1± 6). Hypoalbuminemia has also been observed frequently in HPS, although its pathogenesis has not been fully elucidated (5). Cytokine s are multifunctional local peptide mediators released from reticuloendothe lial cells including the macrophage /monocyte /histiocyte system (7). In the live r, in ̄ ammatory cytokine s, ie , tumor necrosis factora (TNFa ), interleukins (IL-1 and IL-6), together with interferong (IFNg ), have been shown in both in vitro and in vivo studie s to enhance the synthesis of acute -phase proteins, but to suppress that of albumin (8 ± 10) . Clinically, elevated plasma concentrations of these cytokine s have been detected in patients with septic shock, cirrhosis, or alcoholic live r disease, disorders that are frequently accompanie d by hypoalbum inemia (11± 15) . It has therefore been postulated that these cytokine s may be involve d in the pathoge nesis of hypoalbumine mia unde r such conditions (11± 15) . In HPS, an increase in the serum leve ls of these cytokine s has also been reported, and this is known to be corre lated positive ly with disease activity and negative ly with prognosis (2, 3). It is therefore reasonable to speculate that these cytokine s play a role in the pathoge nesis of hypoalbum inemia in patients with HPS, although the relationships between overproduction of cytokine s and hypoalbum inemia in HPS have not been reported. In this communication, we report a case of HPS characterized by marked hypoalbumine mia and ele vated plasma levels of TNFa and IFNg and evidence obtained from an in vitro experiment supporting the crucial pathogenetic role of TNFa in severe hypoalbum inemia in this case .

Identification of a novel mutation for phytosterolemia. Genetic analyses of 2 cases.

BACKGROUND Phytosterolemia is one of the genetic disorders causing hypercholesterolemia and atherosclerosis together with the accumulation of plant sterol in plasma and tissues. The mutations in ABCG5 and ABCG8 genes, encoding sterolin-1 and -2, respectively, are responsible for phytosterolemia. METHODS We performed genetic analyses on 2 Japanese phytosterolemia patients. RESULTS We identified 2 mutations in the ABCG5 gene in these patients. The first patient was homozygous for a novel mutation, which was a 19-base pair tandem repeat insertion in exon 7, leading to a premature termination at codon 288. The second patient was a compound heterozygote; one of the mutations was the same as that found in the first patient, while the other mutation was a C to T substitution in exon 10, resulting in a premature termination at codon 446 (R446X). No other mutation was found in the ABCG5 and ABCG8 genes. CONCLUSIONS This result was concordant with previous observations that found most Asian phytosterolemia patients possessed mutations in the ABCG5 gene, and the site of the novel mutation was completely different from these previous reports, necessitating the extensive analyses for phytosterolemia.

Genetic analysis of phytosterolaemia.

Two women with multiple xanthomas, intermittent arthritis and thrombocytopenia were diagnosed as phytosterolaemia, an autosomal-recessive lipid storage disease, based on their increased serum concentrations of β-sitosterol, campesterol and sitostanol. The gene responsible for this disease is located within a distance of 18 cM between microsatellite markers of D2S1788 and D2S1352 at chromosome 2p21. We genotyped the patients and their family members with 16 microsatellite markers around this locus. The results from the homozygosity mapping of one family suggested that the gene was located within the distance of 12.6 cM between D2S2328 and D2S1352. We have shortened the genetic distance by 5.4 cM.

Association between Deletion Polymorphism of Angiotensin Converting Enzyme Gene and Proteinuria in Japanese Overweight Men

Association between Deletion Polymorphism of Angiotensin Converting Enzyme Gene and Proteinuria in Japanese Overweight Men: Yoshiaki Hashimoto, et al. Departments of Clinical Laboratory Medicine, The University of Tokyo Hospital—A cross‐sectional study was performed in 237 overweight men (BMI: 25.0‐34.8 kg/m2) to investigate the relationship between an insertion (I)/ deletion (D) polymorphism of intron 16 of the angiotensin converting enzyme (ACE) gene and proteinuria. Proteinuria (± or greater) was determined with a reagent strip. The prevalences of proteinuria were 9.2,13.3 and 24.3% in the subjects with the ACE II, ID and DD genotype, respectively. There was a significant difference between the II and the DD genotypes in the prevalence of proteinuria. No difference among the three genotypes was observed in blood pressure, total cholesterol, HDL‐cholesterol, triglycerides and HbA1c. When the subjects were divided into two groups according to the result of urinalysis, the prevalence of the DD genotype was significantly higher in the group with proteinuria (28.1 %) than in the group without it (13.7%). Multiple logistic regression analysis showed that the ACE DD genotype was independently related to proteinuria. The results suggest that the ACE DD genotype may be an independent risk factor of proteinuria in Japanese overweight men. It is specially important for subjects with the ACE DD genotype to prevent the other risk factors of proteinuria such as diabetes, hypertension and obesity by keeping healthy life‐style habits.