Gene expression in atherosclerotic lesion of ApoE deficient mice

BACKGROUND: Atherosclerosis, the major cause of mortality and invalidity in industrialized countries, is a multifactorial disease associated with high plasma cholesterol levels and inflammation in the vessel wall. Many different genes have previously been demonstrated in atherosclerosis, although limited numbers of genes are dealt with in each study. In general, data on dynamic gene expression during disease progress is limited and large-scale evaluation of gene expression patterns during atherogenesis could lead to a better understanding of the key events in the pathogenesis of atherosclerosis. We have therefore applied a mouse gene filter array to analyze gene expression in atherosclerotic ApoE-deficient mice. MATERIALS AND METHODS: ApoE-deficient mice were fed atherogenic western diet for 10 or 20 weeks and aortas isolated. C57BL/6 mice on normal chow were used as controls. The mRNAs of 15 animals were pooled and hybridized onto commercially available Clontech mouse gene array filters. RESULTS: The overall gene expression in the ApoE-deficient and control mice correlated well at both time points. Gene expression profiling showed varying patterns including genes up-regulated at 10 or 20 weeks only. At 20 weeks of diet, an increasing number of up-regulated genes were found in ApoE-deficient mice. CONCLUSIONS: The gene expression in atherogenesis is not a linear process with a maximal expression at advanced lesion stage. Instead, several genes demonstrate a dynamic expression pattern with peaks at the intermediate lesions stage. Thus, detailed evaluation of gene expression at several time points should help understanding the development of atherosclerosis and establishment of preventive intervention.     

Targeted expression of human vitamin D receptor in adipocytes decreases energy expenditure and induces obesity in mice

Our previous studies demonstrated a high fat diet-resistant lean phenotype of vitamin D receptor (VDR)-null mutant mice mainly due to increased energy expenditure, suggesting an involvement of the VDR in energy metabolism. Here, we took a transgenic approach to further define the role of VDR in adipocyte biology. We used the aP2 gene promoter to target the expression of the human (h) VDR in adipocytes in mice. In contrast to the VDR-null mice, the aP2-hVDR Tg mice developed obesity compared with the wild-type counterparts without changes in food intake. The increase in fat mass was mainly due to markedly reduced energy expenditure, which was correlated with decreased locomotive activity and reduced fatty acid β-oxidation and lipolysis in the adipose tissue in the transgenic mice. Consistently, the expression of genes involved in the regulation of fatty acid transport, thermogenesis, and lipolysis were suppressed in the transgenic mice. Taken together, these data confirm an important role of the VDR in the regulation of energy metabolism.     

Glucose induces and leptin decreases expression of uncoupling protein-2 mRNA in human islets

Elevated islet uncoupling protein-2 (UCP-2) impairs beta-cell function and UCP-2 may be increased in clinical obesity and diabetes. We investigated the effects of glucose and leptin on UCP-2 expression in isolated human islets. Human islets were incubated for 24 h with glucose (5.5-22 mmol/l)+/-leptin (0-10 nmol/l). Some islet batches were incubated at high (22 mmol/l), and subsequently lower (5.5 mmol/l), glucose to assess reversibility of effects. Leptin effects on insulin release were also measured. Glucose dose-dependently increased UCP-2 expression in all islet batches, maximally by three-fold. This was not fully reversed by subsequently reduced glucose levels. Leptin decreased UCP-2 expression by up to 75%, and maximally inhibited insulin release by 47%, at 22 mmol/l glucose. This is the first report of UCP-2 expression in human islets and provides novel evidence of its role in the loss of beta-cell function in diabetes.     

Effect of paricalcitol and calcitriol on aortic wall remodeling in uninephrectomized ApoE knockout mice

Despite an only minor reduction in the glomerular filtration rate, uninephrectomy (UNX) markedly accelerates the rate of growth of atherosclerotic plaques in ApoE-/- mice. It has been suggested that vitamin D receptor (VDR) activation exerts an antiproliferative effect on vascular smooth muscle cells, but the side effects may limit its use. To assess a potentially different spectrum of actions, we compared the effects of paricalcitol and calcitriol on remodeling and calcification of the aortic wall in sham-operated and UNX ApoE-/- mice on a diet with normal cholesterol content. Sham-operated and UNX mice were randomly allotted to treatment with solvent, calcitriol (0.03 μg/kg) or paricalcitol (0.1 μg/kg) 5 times/wk intraperitoneally for 10 wk. Semithin (0.6 μm) sections of the aorta were analyzed by 1) morphometry, 2) immunohistochemistry, and 3) Western blotting of key proteins involved in vascular calcification and growth. Compared with sham-operated animals (5.6 ± 0.24), the wall-to-lumen ratio (x100) of the aorta was significantly higher in solvent- and calcitriol-treated UNX animals (6.64 ± 0.27 and 7.17 ± 0.81, respectively, P < 0.05), but not in paricalcitol-treated UNX (6.1 5 ± 0.32). Similar differences were seen with respect to maximal plaque height. Expression of transforming growth factor (TGF)-β1 in aortic intima/plaque was also significantly higher in UNX solvent and UNX calcitriol compared with sham-operated and UNX paricalcitol animals. Treatment with both paricalcitol and calcitriol caused significant elevation of VDR expression in the aorta. While at the dose employed paricalcitol significantly reduced TGF-β expression in plaques, calcitriol in contrast caused significant vascular calcification and elevated expression of related proteins (BMP2, RANKL, and Runx2).     

Inhibition of Cdc42 activity extends lifespan and decreases circulating inflammatory cytokines in aged female C57BL/6 mice

Cdc42 is a small RhoGTPase regulating multiple functions in eukaryotic cells. The activity of Cdc42 is significantly elevated in several tissues of aged mice, while the Cdc42 gain‐of‐activity mouse model presents with a premature aging‐like phenotype and with decreased lifespan. These data suggest a causal connection between elevated activity of Cdc42, aging, and reduced lifespan. Here, we demonstrate that systemic treatment of aged (75‐week‐old) female C57BL/6 mice with a Cdc42 activity‐specific inhibitor (CASIN) for 4 consecutive days significantly extends average and maximum lifespan. Moreover, aged CASIN‐treated animals displayed a youthful level of the aging‐associated cytokines IL‐1β, IL‐1α, and INFγ in serum and a significantly younger epigenetic clock as based on DNA methylation levels in blood cells. Overall, our data show that systemic administration of CASIN to reduce Cdc42 activity in aged mice extends murine lifespan.     

Reduction in Tcf7l2 expression decreases diabetic susceptibility in mice

Objective: The WNT signaling pathway effector gene TCF7L2 has been associated with an increased risk of type 2 diabetes. However, it remains unclear how this gene affects diabetic pathogenesis. The goal of this study was to investigate the effects of Tcf7l2 haploinsufficiency on metabolic phenotypes in mice.Experimental Design: Tcf7l2 knockout (Tcf7l^-/-) mice were generated. Because of the early mortality of Tcf7l2^-/- mice, we characterized the metabolic phenotypes of heterozygous Tcf7l2^+/- mice in comparison to the wild-type controls. The mice were fed a normal chow diet or a high fat diet (HFD) for 9 weeks.Results: The Tcf7l2^+/- mice showed significant differences from the wild-type mice with regards to body weight, fasting glucose and insulin levels. Tcf7l2^+/- mice displayed improved glucose tolerance. In the liver of Tcf7l2^+/- mice fed on the HFD, reduced lipogenesis and hepatic triglyceride levels were observed when compared with those of wild-type mice. Furthermore, the Tcf7l2^+/- mice fed on the HFD exhibited decreased peripheral fat deposition. Immunohistochemistry in mouse pancreatic islets showed that endogenous expression of Tcf7l2 was upregulated in the wild-type mice, but not in the Tcf7l2^+/- mice, after feeding with the HFD. However, the haploinsufficiency of Tcf7l2 in mouse pancreatic islets resulted in little changes in glucose-stimulated insulin secretion.Conclusion: These results suggest that decreased expression of Tcf7l2 confers reduction of diabetic susceptibility in mice via regulation on the metabolism of glucose and lipid.     

Lack of ABCA1 considerably decreases brain ApoE level and increases amyloid deposition in APP23 mice

ABCA1 (ATP-binding cassette transporter A1) is a major regulator of cholesterol efflux and high density lipoprotein (HDL) metabolism. Mutations in human ABCA1 cause severe HDL deficiencies characterized by the virtual absence of apoA-I and HDL and prevalent atherosclerosis. Recently, it has been reported that the lack of ABCA1 causes a significant reduction of apoE protein level in the brain of ABCA1 knock-out (ABCA1-/-) mice. ApoE isoforms strongly affect Alzheimer disease (AD) pathology and risk. To determine further the effect of ABCA1 on amyloid deposition, we used APP23 transgenic mice in which the human familial Swedish AD mutant is expressed only in neurons. We demonstrated that the targeted disruption of ABCA1 increases amyloid deposition in APP23 mice, and the effect is manifested by an increased level of Abeta immunoreactivity, as well as thioflavine S-positive plaques in brain parenchyma. We found that the lack of ABCA1 also considerably increased the level of cerebral amyloid angiopathy and exacerbated cerebral amyloid angiopathy-related microhemorrhage in APP23/ABCA1-/- mice. Remarkably, the elevation in parenchymal and vascular amyloid in APP23/ABCA1-/- mice was accompanied by a dramatic decrease in the level of soluble brain apoE, although insoluble apoE was not changed. The elevation of insoluble Abeta fraction in old APP23/ABCA1-/- mice, accompanied by a lack of changes in APP processing and soluble beta-amyloid in young APP23/ABCA1-/- animals, supports the conclusion that the ABCA1 deficiency increases amyloid deposition. These results suggest that ABCA1 plays a role in the pathogenesis of parenchymal and cerebrovascular amyloid pathology and thus may be considered a therapeutic target in AD.     

RhoA induces expression of inflammatory cytokine in adipocytes

Rho GTPase regulates actin cytoskeleton organization and assembly in many cell types, however, its significance in adipose tissue is not well characterized. Here, we demonstrate high RhoA activity in adipose tissues of C57BL/6J mice. To determine the effect of RhoA activation on 3T3-L1 cells, stable cell lines overexpressing G14VRhoA fused to destabilizing domain of FKBP12 (DD-G14VRhoA-L1) were generated. Treatment of DD-G14VRhoA-L1 cells with Shield1 following their differentiation into adipocytes, resulted in the appearance of thick cortical actin filaments, and increased the mRNA expression levels of plasminogen activator inhibitor type-1 (PAI-1) and monocyte chemoattractant protein-1 (MCP-1). The induction of PAI-1 and MCP-1 was inhibited by treatment with a Rho-associated kinase (ROCK) inhibitor, Y-27632. In 3T3-L1 adipocytes, tumor necrosis factor-α activated RhoA and increased mRNA expression of PAI-1 and MCP-1, and their treatment with Y-27632 partially inhibited these changes. The results indicate that RhoA-ROCK pathway induces inflammatory cytokine expression in adipocytes.     

Reactive oxygen species-selective regulation of aortic inflammatory gene expression in Type 2 diabetes

Vascular diseases are a major complication of diabetes mellitus (DM), although their etiology is poorly understood. NADPH oxidase-derived reactive oxygen species (ROS) production and inflammation are potential mediators of DM-associated vascular diseases. Using db/db mice as a Type 2 diabetes model, we examined the relationship between NADPH oxidase-derived ROS and vascular inflammation. When compared with control m+/+ mice, aortas from 4- and 12-wk-old db/db mice had higher NADPH oxidase activity and increased superoxide levels, leading to NADPH oxidase-dependent impaired vasodilation at 12 wk. Diabetes progression from 4 to 12 wk led to increased Nox1, Nox4, and p22(phox) subunit mRNAs and induced the expression of a group of matrix remodeling-related cytokines: connective tissue growth factor (CTGF), bone morphogenetic protein 4 (BMP-4), and osteopontin (OPN). After 8 wk of treatment with the superoxide scavenger Tempol, 12-wk-old db/db mice had lower superoxide production, reduced plasma glucose and lipids, and lower BMP-4 and OPN protein expression when compared with nontreated mice. No changes were observed with Tempol in CTGF or m+/+ mice. The ability of Tempol to reverse ROS production as well as OPN and BMP-4, but not CTGF, induction suggests that DM-induced vascular inflammation involves both ROS-sensitive and -insensitive pathways.     

Non-imprinted Igf2r expression decreases growth and rescues the Tme mutation in mice

In the mouse the insulin-like growth factor receptor type 2 gene (Igf2r) is imprinted and maternally expressed. Igf2r encodes a trans-membrane receptor that transports mannose-6-phosphate tagged proteins and insulin-like growth factor 2 to lysosomes. During development the receptor reduces the amount of insulin-like growth factors and thereby decreases embryonic growth. The dosage of the gene is tightly regulated by genomic imprinting, leaving only the maternal copy of the gene active. Although the function of Igf2r in development is well established, the function of imprinting the gene remains elusive. Gene targeting experiments in mouse have demonstrated that the majority of genes are not sensitive to gene dosage, and mice heterozygous for mutations generally lack phenotypic alterations. To investigate whether reduction of Igf2r gene dosage by genomic imprinting has functional consequences for development we generated a non-imprinted allele (R2Delta). We restored biallelic expression to Igf2r by deleting a critical element for repression of the paternal allele (region 2) in mouse embryonic stem cells. Maternal inheritance of the R2Delta allele has no phenotype; however, paternal inheritance results in biallelic expression of Igf2r, which causes a 20% reduction in weight late in embryonic development that persists into adulthood. Paternal inheritance of the R2Delta allele rescues the lethality of a maternally inherited Igf2r null allele and a maternally inherited Tme (T-associated maternal effect) mutation. These data show that the biological function of imprinting Igf2r is to increase birth weight and they also establish Igf2r as the Tme gene.     

Lentiviral-mediated over-expression of hyaluronan synthase-1 (HAS-1) decreases the cellular inflammatory response and results in regenerative wound repair

Fetal wounds have been found to have increased levels of high-molecular-weight hyaluronan (HMW-HA) compared with those of adults. The primary enzyme responsible for producing HMW-HA is hyaluronic acid synthase-1 (HAS-1). We hypothesized that over-expression of HAS-1 in adult dermal wounds would decrease inflammation and promote regenerative healing. To test this hypothesis, the flanks of adult C57Bl/6 mice were treated with a lentiviral construct containing either HAS-1-GFP or GFP transgenes. After 48 h, a 4-mm excisional wound was made at the site of treatment. Wounds were harvested at days 3, 7, or 28 after wounding. Wound phenotype was assessed by histology to examine tissue architecture and immunohistochemistry for CD45. At 7 and 28 days, lenti-HAS-1-treated wounds demonstrated the restoration of the normal dermal elements and organized collagen fiber orientation. In contrast, the lenti-GFP-treated wounds lacked normal dermal architecture and demonstrated a disorganized collagen scar. At 3 and 7 days, wounds treated with lenti-HAS-1 exhibited a significant decrease in the number of inflammatory cells when compared with wounds treated with lenti-GFP. Thus, HAS-1 over-expression promotes dermal regeneration, in part by decreasing the inflammatory response and by recapitulation of fetal extracellular matrix HMW-HA content.     

Double-label expression studies of prostacyclin synthase, thromboxane synthase and COX isoforms in normal aortic endothelium

We have performed double-label immunofluorescence microscopy studies to evaluate the extent of co-localization of prostacyclin synthase (PGIS) and thromboxane synthase (TXS) with cyclooxygenase (COX)-1 and COX-2 in normal aortic endothelium. In dogs, COX-2 expression was found to be restricted to small foci of endothelial cells while COX-1, PGIS and TXS were widely distributed throughout the endothelium. Quantification of the total cross-sectioned aortic endothelium revealed a 6- to 7-fold greater expression of COX-1 relative to COX-2 (55 vs. 8%) and greater co-distribution of PGIS with COX-1 compared to COX-2 (19 vs. 3%). These results are in contrast to the extensive co-localization of PGIS and COX-2 in bronchiolar epithelium. In rat and human aortas, immunofluorescence studies also showed significant COX-1 and PGIS co-localization in the endothelium. Only minor focal COX-2 expression was detected in rat endothelium, similar to the dog, while COX-2 was not detected in human specimens. Inhibition studies in rats showed that selective COX-1 inhibition caused a marked reduction of 6-keto-PGF(1alpha) and TXB(2) aortic tissue levels, while COX-2 inhibition had no significant effect, providing further evidence for a functionally larger contribution of COX-1 to the synthesis of prostacyclin and thromboxane in aortic tissue. The data suggest a major role for COX-1 in the production of both prostacyclin and thromboxane in normal aortic tissue. The extensive co-localization of PGIS and COX-2 in the lung also indicates significant tissue differences in the co-expression patterns of these two enzymes.     

Involvement of various combinations of endogenous inflammatory cytokines in Listeria monocytogenes-induced expression of inducible nitric oxide synthase in mice

Abstract Using an in vitro infection of spleen cells with Listeria monocytogenes , the relationship between endogenous cytokines and the expression of inducible nitric oxide synthase (iNOS) was examined. When all interferon (IFN)-γ, tumor necrosis factor (TNF)-α and interleukin (IL)-1 α, or the combination of IFN-γ with either TNF-α or IL-1 α were neutralized by antibodies, there was a significant reduction of iNOS expression and nitrite production in culture. However, there was no reduction of iNOS expression and nitrite production when these cytokines were individually neutralized. After the depletion of natural killer cells, there was no change in the expression of Listeria -induced iNOS and nitrite production although the IFN-γ production was abrogated. Neutralization of TNF-α and IL-1 α in natural killer cell-depleted culture resulted in the reduction of iNOS expression. Thus, various combinations of cytokines appeared to play an important role in iNOS induction by L. monocytogenes .     

TRAIL induces apoptosis and inflammatory gene expression in human endothelial cells

Human TRAIL can efficiently kill tumor cells in vitro and kill human tumor xenografts in mice with little effect on normal mouse cells or tissues. The effects of TRAIL on normal human tissues have not been described. In this study, we report that endothelial cells (EC), isolated from human umbilical veins or human dermal microvessels, express death domain-containing TRAIL-R1 and -R2. Incubation with TRAIL for 15 h causes approximately 30% of cultured EC to die, as assessed by propidium iodide uptake. Death is apoptotic, as assessed by Annexin V staining, 4',6'-diamidino-2-phenylindole staining, and DNA fragment ELISA. EC death is increased by cotreatment with cycloheximide but significantly reduced by caspase inhibitors or transduced dominant-negative Fas-associated death domain protein. In surviving cells, TRAIL activates NF-kappaB, induces expression of E-selectin, ICAM-1, and IL-8, and promotes adhesion of leukocytes. Injection of TRAIL into human skin xenografts promotes focal EC injury accompanied by limited neutrophil infiltration. These data suggest that TRAIL is an inducer of tissue injury in humans, an outcome that may influence antitumor therapy with TRAIL.     

The cloning and over-expression of PABA synthase in E. coli

Both the genes encoding E. coli p-aminobenzoic acid synthase have been cloned and an overproducing strain has been obtained. The partial purification of the large subunit is described. The kinetic properties of the cloned enzyme, while similar to those reported for the B. subtilis enzyme, show some differences to those reported for the S. griseus enzyme.