Although the nature and consequences of oxidative changes in the chemical constituents of low density lipoproteins (LDLs) have been extensively examined, the physical dynamics of LDL oxidation and ...the influence of physical organization on the biological effects of oxidized LDLs have remained relatively unexplored. To address these issues, in the present studies we monitored surface- and core-specific peroxidative stress relative to temporal changes in conjugated dienes (CDs), particle charge (an index of oxidative protein modification), and LDL-macrophage interactions. Peroxidative stress in LDL surface and core compartments was evaluated with the site-specific, oxidation-labile fluorescent probes parinaric acid (PnA) and PnA cholesteryl ester (PnCE), respectively. When oxidation was initiated by Copper+, oxidative loss of the core probe (PnCE) closely followed that of the surface probe (PnA), as indicated by the time to 50% probe depletion (t1/2; 15.5 +/- 7.8 and 30.4 +/- 12 minutes for PnA and PnCE, respectively). Both probes were more resistant in LDL exposed to Iron+ (t1/2, 53.2 +/- 8.1 and 346.7 +/- 155.4 minutes), although core probe resistance was much greater with this oxidant (PnCE t1/2 /PnA t1/2, 5.8 vs 2.0 for Copper+). Despite differences in the rate and extent of oxidative changes in Copper sup 2+ - versus Iron+ -exposed LDLs, PnCE loss occurred in close correspondence with CD formation and appeared to precede changes in particle charge under both conditions. Exposure of LDLs to hemin, a lipophilic Iron+ -containing porphyrin that becomes incorporated into the LDL particle, resulted in rapid loss of PnCE and simultaneous changes in particle charge, even at concentrations that yielded increases in CDs and thiobarbituric acid-reactive substances similar to those obtained with free Iron+. These results suggest that oxidation of the LDL hydrophobic core occurs in conjunction with accelerated formation of CDs and may be essential for LDL protein modification. In accordance with the known effects of oxidative protein modifications on LDL receptor recognition, exposure of LDLs to Copper+ and hemin but not Iron+ produced particles that were readily processed by macrophages. Thus, the physical site of oxidative injury appears to be a critical determinant of the chemical and biological properties of LDLs, particularly when oxidized by Iron+. (Arterioscler Thromb Vasc Biol. 1996;16:1580-1587.)
Sequence polymorphisms in a 58kb interval on chromosome 9p21 confer a markedly increased risk for coronary artery disease (CAD), the leading cause of death worldwide
1
,
2
. The variants have a ...substantial impact on the epidemiology of CAD and other life-threatening vascular conditions since nearly a quarter of Caucasians are homozygous for risk alleles. However, the risk interval is devoid of protein-coding genes and the mechanism linking the region to CAD risk has remained enigmatic. Here we show that deletion of the orthologous 70kb noncoding interval on mouse chromosome4 affects cardiac expression of neighboring genes, as well as proliferation properties of vascular cells. Chr4
Δ70kb/Δ70kb
mice are viable, but show increased mortality both during development and as adults. Cardiac expression of two genes near the noncoding interval,
Cdkn2a
and
Cdkn2b
, is severely reduced in chr4
Δ70kb/Δ70kb
mice, indicating that distant-acting gene regulatory functions are located in the noncoding CAD risk interval. Allele-specific expression of
Cdkn2b
transcripts in heterozygous mice revealed that the deletion affects expression through a
cis
-acting mechanism. Primary cultures of chr4
Δ70kb/Δ70kb
aortic smooth muscle cells exhibited excessive proliferation and diminished senescence, a cellular phenotype consistent with accelerated CAD pathogenesis. Taken together, our results provide direct evidence that the CAD risk interval plays a pivotal role in regulation of cardiac
Cdkn2a/b
expression and suggest that this region affects CAD progression by altering the dynamics of vascular cell proliferation.
Celotno besedilo
Dostopno za:
DOBA, IJS, IZUM, KILJ, NUK, PILJ, PNG, SAZU, SIK, UILJ, UKNU, UL, UM, UPUK
The in vivo analysis of lipoprotein(a) (Lp(a)), an independent atherosclerosis risk factor in humans, has been limited in part by its restricted distribution among mammals. Although transgenic mice ...have been created containing Lp(a), the relatively small size of the mouse has precluded some studies. To examine the properties of this molecule in a significantly larger mammal, we have used a 270-kilobase yeast artificial chromosome clone containing the human apolipoprotein(a) (apo(a)) gene and a 90-kilobase P1 phagemid clone containing the human apolipoprotein B (apoB) gene to create transgenic rabbits that express either or both transgenes. Expression of both transgenes was tissue specific and localized predominantly to the liver. Average apolipoprotein plasma levels in the rabbits were 2.5 mg/dl for apo(a) and 17.6 mg/dl for human apoB. In contrast to observations in apo(a) transgenic mice, we found that apo(a) plasma levels in the rabbits were stable throughout sexual maturity. Also, apo(a) formed a covalent association with the endogenous rabbit apoB albeit with a lower efficiency than its association with human apoB. The analysis of Lp(a) transgenic rabbits has provided new insights into apo(a) expression and Lp(a) assembly. In addition, these transgenic rabbits potentially will provide an improved experimental model for the in vivo analysis of Lp(a) and its role in promoting atherosclerosis and restenosis.
The role of apolipoprotein A-II (apoA-II) in high density lipoprotein (HDL) structure and metabolism has been studied previously
in transgenic mice overexpressing either human or murine apoA-II. ...These studies have shown differences between these two groups
of transgenic animals in the levels of very low density, low density, and high density lipoproteins, in the HDL particle size
distribution, and in the relationship between apoA-II levels and lipoprotein levels. To determine whether these differences
are due to the fact that human apoA-II is dimeric and murine apoA-II monomeric, we have examined the effects of monomeric
human apoA-II (hA-II ) in transgenic mice. Site-directed mutagenesis (Cys 6 Ser) was used to generate 15 transgenic founder lines of hA-II mice, that contained plasma hA-II concentrations over a 10-fold range (11 mg/dl to 185 mg/dl). The hA-II floated in the d ⤠1.21 g/ml fraction and migrated as an apoA-II monomer by nonreducing SDS-polyacrylamide gel electrophoresis. HDL levels
were not correlated with hA-II levels ( r = â0.26); HDL particle size and size distribution, as well as very low density and low density lipoprotein levels and sizes,
were unchanged compared to nontransgenic control mice. These results suggest that differences between mice overexpressing
human dimeric apoA-II and those overexpressing murine apoA-II are the result of sequence differences between these two apoA-II
molecules and are not solely due to the fact that human apoA-II exists as a dimer.
No evidence of premature vascular disease is found in apolipoprotein A-I(Milano) (apoA-I(M)) human carriers, despite very low high density lipoprotein (HDL) cholesterol levels. Whether apoA-I(M) may ...impart a "gain of function" in atherosclerosis protection compared to wild-type apoA-I is hotly debated. To address this question, knock-in mice expressing human apoA-I or apoA-I(M) were crossed with atherosclerosis-susceptible mice expressing the human apoB/A-II transgene (h-B/A-II/A-I(Hu/Hu) and h-B/A-II/A-I(M)(Hu/Hu)). On a chow diet, h-B/A-II/A-I(M)(Hu/Hu) mice were characterized by low HDL cholesterol levels compared to h-B/A-II/A-I(Hu/Hu) mice (35.65+/-8.00 mg/dl versus 58.09+/-13.50mg/dl, respectively; p<0.005). Gender differences in response to high fat diet were observed in both h-B/A-II/A-I(M)(Hu/Hu) and h-B/A-II/A-I(Hu/Hu) lines. h-B/A-II/A-I(M)(Hu/Hu) females had higher total cholesterol levels compared to h-B/A-II/A-I(Hu/Hu) females (895.08+/-183.07 mg/dl versus 544.43+/-116.42 mg/dl; p<0.05) and developed larger atherosclerotic lesions (148,260+/-78,924 microm(2) versus 54,132+/-43,204 microm(2), respectively; p<0.05). On the contrary, no difference in mean lesion area was found between h-B/A-II/A-I(M)(Hu/Hu) and h-B/A-II/A-I(Hu/Hu) males (19,779+/-6,098 microm(2) versus 15,706+/-13,095 microm(2); p=0.685). Our data suggest that, in the atherosclerosis-susceptible human apoB/A-II mouse model, expression of the human apoA-I(M) gene does not have protective advantage over that of the apoA-I gene.
Objective— The apolipoprotein(a) apo(a) gene locus is the major determinant of the circulating concentration of the atherothrombogenic lipoprotein Lp(a). In vitro analysis of the intergenic region ...between the apo(a) and plasminogen genes revealed the presence of a putative apo(a) transcription control region (ACR) approximately 20 kb upstream of the apo(a) gene that significantly increases the minimal promoter activity of the human apo(a) gene. Methods and Results— To examine the function of the ACR in its natural genomic context, we used the Cre- lox P recombination system to generate 2 nearly identical apo(a)–yeast artificial chromosome transgenic mouse lines that possess a single integration site for the human apo(a) transgene in the mouse genome but differ by the presence or absence of the ACR enhancer. Analysis of the 2 groups of animals revealed that the deletion of the ACR was associated with 30% reduction in plasma and mRNA apo(a) levels. Apo(a)–yeast artificial chromosome transgenic mice with and without the ACR sequence were similar in all other aspects of apo(a) regulation, including liver-specific apo(a) expression and alteration in expression levels in response to sexual maturation and a high-fat diet. Conclusions— This study provides the first experimental in vivo evidence for a functional role of the ACR enhancer in determining levels of apo(a) expression.
Gradient gel electrophoresis in conjunction with automated densitometry was applied to the identification and estimation of subpopulations of high-density lipoproteins (HDL) in the ultracentrifugal d ...less than or equal to 1.200 fraction from human plasma. The frequency distribution of relative migration distances (RF values) of subpopulation peaks in HDL patterns of a group (n = 194) of human subjects showed five apparent maxima: two in the RF range associated with the HDL2 subclass, and three in the RF range of the HDL3 subclass. HDL within RF intervals bounding these maxima were designated (HDL2b)gge, (HDL2a)gge, (HDL3a)gge, (HDL3b)gge and (HDL3c)gge and were shown to correspond approximately to material determined by analytic ultracentrifugation within the HDL2b, HDL2a and HDL3 components. Material represented by the HDL2a component, as resolved by three-component analysis of the ultracentrifugal Schlieren pattern, was found by gradient gel electrophoresis to be polydisperse in particle size. Mean hydrated densities and particle sizes of HDL corresponding to those with RF values of the frequency maxima were: 1.085 g/ml and 10.57 nm in the (HDL2b)gge; 1.115 g/ml and 9.16 nm in the (HDL2a)gge; 1.136 g/ml and 8.44 nm in the (HDL3a)gge; 1.154 g/ml and 7.97 nm in the (HDL3b)gge; and 1.171 g/ml and 7.62 nm in the (HDL3c)gge. The mean hydrated density values of the subpopulations within the (HDL3a)gge and (HDL3b)gge were comparable to those of the HDL3L and HDL3D components recently characterized by zonal ultracentrifugation. High order and statistically significant correlations between densitometric scans of the (HDL2b)gge, (HDL2a)gge and (HDL3)gge material, as obtained from gradient gels, and plasma concentrations of the HDL2b, HDL2a and HDL3 components, as obtained from analytic ultracentrifugation, were demonstrated.
Apolipoproteins A-I and A-II comprise approximately 70 and 20%, respectively, of the total protein content of HDL. Evidence suggests that apoA-I plays a central role in determining the structure and ...plasma concentration of HDL, while the role of apoA-II is uncertain. To help define the function of apoA-II and determine what effect increasing its plasma concentration has on HDL, transgenic mice expressing human apoA-II and both human apoA-I and human apoA-II were produced. Human apoA-II mRNA is expressed exclusively in the livers of transgenic animals, and the protein exists as a dimer as it does in humans. High level expression of human apoA-II did not increase HDL concentrations or decrease plasma concentrations of murine apoA-I and apoA-II in contrast to what was observed in mice overexpressing human apoA-I. The primary effect of overexpressing human apoA-II was the appearance of small HDL particles composed exclusively of human apoA-II. HDL from mice transgenic for both human apoA-I and human apoA-II displayed a unique size distribution when compared with either apoA-I or apoA-II transgenic mice and contain particles with both these human apolipoproteins. These results in mice, indicating that human apoA-II participates in determining HDL size, parallel results from human studies.
The in vivo analysis of lipoprotein(a) (Lp(a)), an independent atherosclerosis risk factor in humans, has been limited in part by its restricted distribution among mammals. Although transgenic mice ...have been created containing Lp(a), the relatively small size of the mouse has precluded some studies. To examine the properties of this molecule in a significantly larger mammal, we have used a 270-kilobase yeast artificial chromosome clone containing the human apolipoprotein(a) (apo(a)) gene and a 90-kilobase P1 phagemid clone containing the human apolipoprotein B (apoB) gene to create transgenic rabbits that express either or both transgenes. Expression of both transgenes was tissue specific and localized predominantly to the liver. Average apolipoprotein plasma levels in the rabbits were 2.5 mg/dl for apo(a) and 17.6 mg/dl for human apoB. In contrast to observations in apo(a) transgenic mice, we found that apo(a) plasma levels in the rabbits were stable throughout sexual maturity. Also, apo(a) formed a covalent association with the endogenous rabbit apoB albeit with a lower efficiency than its association with human apoB. The analysis of Lp(a) transgenic rabbits has provided new insights into apo(a) expression and Lp(a) assembly. In addition, these transgenic rabbits potentially will provide an improved experimental model for the in vivo analysis of Lp(a) and its role in promoting atherosclerosis and restenosis.
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