Considerable evidence indicates that hyperglycemia increases oxidative stress and contributes to the increased incidence of atherosclerosis and cardiovascular complications in diabetic patients. To ...examine the effect of alpha-lipoic acid, a potent natural antioxidant, on atherosclerosis in diabetic mice, 3-month-old apolipoprotein (apo) E-deficient (apoE(-/-)) mice were made diabetic by administering streptozotocin (STZ). At 4 weeks after starting the STZ administration, a high-fat diet with or without alpha-lipoic acid (1.65 g/kg) was given to the mice and to nondiabetic apoE(-/-) controls. At 20 weeks, markers of oxidative stress were significantly lower in both the diabetic apoE(-/-) mice and their nondiabetic apoE(-/-) controls with alpha-lipoic acid supplement than in those without it. Remarkably, alpha-lipoic acid completely prevented the increase in plasma total cholesterol, atherosclerotic lesions, and the general deterioration of health caused by diabetes. These protective effects of alpha-lipoic acid were accompanied by a reduction of plasma glucose and an accelerated recovery of insulin-producing cells in the pancreas, suggesting that part of its effects are attributable to protecting pancreatic beta-cells from damage. Our results suggest that dietary alpha-lipoic acid is a promising protective agent for reducing cardiovascular complications of diabetes.
Glycosylation in type I collagen occurs as O-linked galactosyl- (G-) lesser and glucosylgalactosyl-hydroxylysine (GG-Hyl); however, its biological significance is still not well understood. To ...investigate the function of this modification in bone, we have generated preosteoblast MC3T3-E1 (MC)-derived clones, short hairpin (Sh) clones, in which Glt25d1 gene expression was stably suppressed. In Sh clones, the GLT25D1 protein levels were markedly diminished in comparison to controls (MC and those transfected with the empty vector). In Sh collagen, levels of both G- and GG-Hyl were significantly diminished with a concomitant increase in the level of free-Hyl. In addition, the level of immature divalent cross-links significantly diminished while the level of the mature trivalent cross-link increased. As determined by mass spectrometric analysis, seven glycosylation sites were identified in type I collagen and the most predominant site was at the helical cross-linking site, α1–87. At all of the glycosylation sites, the relative levels of G- and GG-Hyl were markedly diminished, i.e., by ∼50–75%, in Sh collagen, and at five of these sites, the level of Lys hydroxylation was significantly increased. The collagen fibrils in Sh clones were larger, and mineralization was impaired. These results indicate that GLT25D1 catalyzes galactosylation of Hyl throughout the type I collagen molecule and that this modification may regulate maturation of collagen cross-linking, fibrillogenesis, and mineralization.
Nonalcoholic steatohepatitis (NASH) involves liver lipid accumulation (steatosis) combined with hepatic inflammation. The transition towards hepatic inflammation represents a key step in ...pathogenesis, because it will set the stage for further liver damage, culminating in hepatic fibrosis, cirrhosis, and liver cancer. The actual risk factors that drive hepatic inflammation during the progression to NASH remain largely unknown. The role of steatosis and dietary cholesterol in the etiology of diet‐induced NASH was investigated using hyperlipidemic mouse models fed a Western diet. Livers of male and female hyperlipidemic (low‐density lipoprotein receptor–deficient ldlr−/− and apolipoprotein E2 knock‐in APOE2ki) mouse models were compared with livers of normolipidemic wild‐type (WT) C57BL/6J mice after short‐term feeding with a high‐fat diet with cholesterol (HFC) and without cholesterol. Whereas WT mice displayed only steatosis after a short‐term HFC diet, female ldlr−/− and APOE2ki mice showed steatosis with severe inflammation characterized by infiltration of macrophages and increased nuclear factor κB (NF‐κB) signaling. Remarkably, male ldlr−/− and APOE2ki mice developed severe hepatic inflammation in the absence of steatosis after 7 days on an HFC diet compared with WT animals. An HFC diet induced bloated, “foamy” Kupffer cells in male and female ldlr−/− and APOE2ki mice. Hepatic inflammation was found to be linked to increased plasma very low‐density lipoprotein (VLDL) cholesterol levels. Omitting cholesterol from the HFC diet lowered plasma VLDL cholesterol and prevented the development of inflammation and hepatic foam cells. Conclusion: These findings indicate that dietary cholesterol, possibly in the form of modified plasma lipoproteins, is an important risk factor for the progression to hepatic inflammation in diet‐induced NASH. (HEPATOLOGY 2008;48:474–486.)
Macrophage-specific Abca1 knock-out (Abca1–M/–M) mice were generated to determine the role of macrophage ABCA1 expression in plasma lipoprotein concentrations and the innate immune response of ...macrophages. Plasma lipid and lipoprotein concentrations in chow-fed Abca1–M/–M and wild-type (WT) mice were indistinguishable. Compared with WT macrophages, Abca1–M/–M macrophages had a >95% reduction in ABCA1 protein, failed to efflux lipid to apoA-I, and had a significant increase in free cholesterol (FC) and membrane lipid rafts without induction of endoplasmic reticulum stress. Lipopolysaccharide (LPS)-treated Abca1–M/–M macrophages exhibited enhanced expression of pro-inflammatory cytokines and increased activation of the NF-κB and MAPK pathways, which could be diminished by silencing MyD88 or by chemical inhibition of NF-κB or MAPK. In vivo LPS injection also resulted in a higher pro-inflammatory response in Abca1–M/–M mice compared with WT mice. Furthermore, cholesterol depletion of macrophages with methyl-β-cyclodextrin normalized FC content between the two genotypes and their response to LPS; cholesterol repletion of macrophages resulted in increased cellular FC accumulation and enhanced cellular response to LPS. Our results suggest that macrophage ABCA1 expression may protect against atherosclerosis by facilitating the net removal of excess lipid from macrophages and dampening pro-inflammatory MyD88-dependent signaling pathways by reduction of cell membrane FC and lipid raft content.
Stabilin2
(
Stab2
) encodes a large transmembrane protein which is predominantly expressed in the liver sinusoidal endothelial cells (LSECs) and functions as a scavenger receptor for various ...macromolecules including hyaluronans (HA). In DBA/2J mice, plasma HA concentration is ten times higher than in 129S6 or C57BL/6J mice, and this phenotype is genetically linked to the
Stab2
locus.
Stab2
mRNA in the LSECs was significantly lower in DBA/2J than in 129S6, leading to reduced STAB2 proteins in the DBA/2J LSECs. We found a retrovirus-derived transposable element, intracisternal A particle (IAP), in the promoter region of
Stab2
DBA
which likely interferes with normal expression in the LSECs. In contrast, in other tissues of DBA/2J mice, the IAP drives high ectopic
Stab2
DBA
transcription starting within the 5′ long terminal repeat of IAP in a reverse orientation and continuing through the downstream
Stab2
DBA
. Ectopic transcription requires the
Stab2
-IAP element but is dominantly suppressed by the presence of loci on 59.7–73.0 Mb of chromosome (Chr) 13 from C57BL/6J, while the same region in 129S6 requires additional loci for complete suppression. Chr13:59.9–73 Mb contains a large number of genes encoding Krüppel-associated box-domain zinc-finger proteins that target transposable elements-derived sequences and repress their expression. Despite the high amount of ectopic
Stab2
DBA
transcript in tissues other than liver, STAB2 protein was undetectable and unlikely to contribute to the plasma HA levels of DBA/2J mice. Nevertheless, the IAP insertion and its effects on the transcription of the downstream
Stab2
DBA
exemplify that stochastic evolutional events could significantly influence susceptibility to complex but common diseases.
Adult neural stem cells (NSCs) in the mouse subventricular zone (SVZ) serve as a lifelong reservoir for newborn olfactory bulb neurons. Recent studies have identified a slowly dividing subpopulation ...of embryonic neural stem-progenitor cells (NPCs) as the embryonic origin of adult NSCs. Yet, little is known about how these slowly dividing embryonic NPCs are maintained until adulthood while other NPCs are extinguished by the completion of brain development. The extracellular matrix (ECM) is an essential component of stem cell niches and thus a key determinant of stem cell fate. Here we investigated tissue inhibitors of metalloproteinases (TIMPs)-regulators of ECM remodeling-for their potential roles in the establishment of adult NSCs. We found that
,
, and
were expressed at high levels in slowly dividing NPCs compared to rapidly dividing NPCs. Deletion of TIMP3 reduced the number of adult NSCs and neuroblasts in the lateral SVZ. In addition, overexpression of TIMP3 in the embryonic NPCs suppressed neuronal differentiation and upregulated the expression levels of Notch signaling relating genes. These results thus suggest that TIMP3 keeps the undifferentiated state of embryonic NPCs, leading to the establishment and maintenance of adult NSCs.
Adipose tissue (AT) is the body's largest free cholesterol reservoir and abundantly expresses ATP binding cassette transporter A1 (ABCA1), a key cholesterol transporter for high-density lipoprotein ...(HDL) biogenesis. However, the extent to which AT ABCA1 expression contributes to HDL biogenesis in vivo is unknown.
Adipocyte-specific ABCA1 knockout mice (ABCA1(-A/-A)) were generated by crossing ABCA1(floxed) mice with aP2Cre transgenic mice. AT from ABCA1(-A/-A) mice had <10% of wild-type ABCA1 protein expression but normal hepatic and intestinal expression. Deletion of adipocyte ABCA1 resulted in a significant decrease in plasma HDL cholesterol (approximately 15%) and apolipoprotein A-I (approximately 13%) concentrations. AT from ABCA1(-A/-A) mice had a 2-fold increase in free cholesterol content compared with wild-type mice and failed to efflux cholesterol to apolipoprotein A-I. However, cholesterol efflux from AT to plasma HDL was similar for both genotypes of mice. Incubation of wild-type AT explants with apolipoprotein A-I resulted in the formation of multiple discrete-sized nascent HDL particles ranging in diameter from 7.1 to 12 nm; similar incubations with ABCA1(-A/-A) AT explants resulted in nascent HDL <8 nm. Plasma decay and tissue uptake of wild-type (125)I-HDL tracer were similar in both genotypes of recipient mice, suggesting that adipocyte ABCA1 deficiency reduces plasma HDL concentrations solely by reducing nascent HDL particle formation.
We provide in vivo evidence that AT ABCA1-dependent cholesterol efflux and nascent HDL particle formation contribute to systemic HDL biogenesis and that AT ABCA1 expression plays an important role in adipocyte cholesterol homeostasis.
Objective- Dyslipidemia is a component of the metabolic syndrome, an established risk factor for atherosclerotic cardiovascular disease, and is also observed in various autoimmune and chronic ...inflammatory conditions. However, there are limited opportunities to study the impact of acquired dyslipidemia on cardiovascular and immune pathology. Approach and Results- We designed a model system that allows for the conversion to a state of acute hyperlipidemia in adult life, so that the consequences of such a transition could be observed, through conditionally deleting APOE (apolipoprotein E) in the adult mouse. The transition to hypercholesterolemia was accompanied by adaptive immune responses, including the expansion of T lymphocyte helper cell 1, T follicular helper cell, and T regulatory subsets and the formation of germinal centers. Unlike steady-state Apoe
mice, abrupt loss of APOE induced rapid production of antibodies recognizing rheumatoid disease autoantigens. Genetic ablation of the germinal center reduced both autoimmunity and atherosclerosis, indicating that the immune response that follows loss of APOE is independent of atherosclerosis but nevertheless promotes plaque development. Conclusions- Our findings suggest that immune activation in response to hyperlipidemia could contribute to a wide range of inflammatory autoimmune diseases, including atherosclerosis.
Atherosclerosis in different vascular locations leads to distinct clinical consequences, such as ischemic stroke and myocardial infarction. Genome-wide association studies in humans revealed that ...genetic loci responsible for carotid plaque and coronary artery disease were not overlapping, suggesting that distinct genetic pathways might be involved for each location. While elevated plasma cholesterol is a common risk factor, plaque development in different vascular beds is influenced by hemodynamics and intrinsic vascular integrity. Despite the limitation of species differences, mouse models provide platforms for unbiased genetic approaches. Mouse strain differences also indicate that susceptibility to atherosclerosis varies, depending on vascular locations, and that the location specificity is genetically controlled. Quantitative trait loci analyses in mice suggested candidate genes, including
and
, although how each gene affects the location-specific atherosclerosis needs further elucidation. Another unbiased approach of single-cell transcriptome analyses revealed the presence of a small subpopulation of vascular smooth muscle cells (VSMCs), which are "hyper-responsive" to inflammatory stimuli. These cells are likely the previously-reported Sca1
progenitor cells, which can differentiate into multiple lineages in plaques. Further spatiotemporal analyses of the progenitor cells are necessary, since their distribution pattern might be associated with the location-dependent plaque development.
The objective of this study was to examine whether genetically determined differences in the guanylyl cyclase/natriuretic peptide receptor-A gene (Npr1) affect cardiac expression of proinflammatory ...cytokines, hypertrophic markers, nuclear factor-κB (NF-κB), and activating protein-1 (AP-1) in am Npr1 gene-dose–dependent manner. In the present studies, adult male Npr1 gene-disrupted (Npr1−/−), wild-type (Npr1+/+), and gene-duplicated (Npr1++/++) mice were used. The Npr1−/− mice showed 41 mm Hg higher systolic blood pressure and 60% greater heart weight to body weight (HW/BW) ratio; however, Npr1++/++ mice exhibited 15 mm Hg lower systolic blood pressure and 12% reduced HW/BW ratio compared with Npr1+/+ mice. Significant upregulation of gene expression of proinflammatory cytokines and hypertrophic markers along with enhanced NF-κB/AP-1 binding activities were observed in the Npr1−/− mouse hearts. Conversely, hypertrophic markers and proinflammatory cytokines gene expression as well as NF-κB/AP-1 binding activities were markedly decreased in Npr1++/++ mouse hearts compared with wild-type mice. The ventricular guanylyl cyclase activity and cGMP levels were reduced by 96% and 87%, respectively, in Npr1−/− mice; however, these parameters were amplified by 2.8-fold and 3.8-fold, respectively, in Npr1++/++ mice. Echocardiographic analysis revealed significantly increased fractional shortening in Npr1++/++ mice (P < .05) but greatly decreased in Npr1−/− mice (P < .01) hearts compared with Npr1+/+ mice. The present findings suggest that Npr1 represses the expression of cardiac proinflammatory mediators, hypertrophic markers, and NF-κB/AP-1–mediated mechanisms, which seem to be associated in an Npr1 gene-dose–dependent manner.
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