ABSTRACT
Macrophages play a pivotal role in atherosclerosis through a variety of events related to cellular oxidative stress. This process is mainly due to an excessive production of reactive oxygen species whose elimination occurs through antioxidant systems including the thioredoxin (Trx) system. In this paper, we investigated whether the Trx system would exhibit circadian rhythmicity in dexamethasone synchronized cultured macrophages and monitored the impact of the rhythmicity of Trx-1 on markers of atherosclerosis. We found that the clock-related genes BMAL-1, PER-2, CRY-1 and REV ERB α exhibited a robust circadian expression. However, the Trx genes family (Trx-1, Trx-2, TrxR1 and TXNIP) did not exhibit a circadian expression at the mRNA level in spite of the presence of E-box elements within the promoter regions of TrxR1 and TXNIP genes. Nevertheless, both Trx-1 and TXNIP exhibited a circadian expression at the protein level and proteasome inhibition abolished the rhythmicity of Trx-1. Moreover, we found a link between low Trx-1 level and elevated atherogenic markers such as 4-HNE, TNF-α and cholesterol accumulation in macrophages. Our results indicate that the Trx gene family does not exhibit the same circadian regulation and that the presence of E-box elements in the TXNIP promoter is not sufficient to ensure a circadian rhythmicity at the transcriptional level. In addition, since a link was found between a low level of Trx-1 protein during circadian rhythm and high levels of atherogenic markers, administration of Trx-1 at certain time points could be an interesting approach to protect against atherosclerosis development.
Subject(s)
Atherosclerosis , Macrophages, Peritoneal , Animals , Circadian Rhythm , Macrophages, Peritoneal/metabolism , Mice , Oxidative Stress , Thioredoxins/genetics , Thioredoxins/metabolismABSTRACT
AIM: To explore the isokinetic concentric strength of the knee muscle groups, and the relationship between the isokinetic knee extensors strength and the vertical jump performance in young elite female basketball players. METHODS: Eighteen elite female basketball players performed a countermovement jump, and an isokinetic knee test using a Biodex dynamometer. The maximal isokinetic peak torque of the knee extensor and flexor muscles was recorded at four angular velocities (90°/s, 180°/s, 240°/s and 300°/s) for the dominant and non-dominant legs. The conventional hamstring/quadriceps ratio (H/Q) was assessed at each angular velocity for both legs. RESULTS: There was no significant difference between dominant and non-dominant leg whatever the angular velocity (all P>0.05). However, the H/Q ratio enhanced as the velocity increased from 180°/s to 300°/s (P<0.05). Furthermore, low to high significant positive correlations were detected between the isokinetic measures of the knee extensors and the vertical jump height. The highest one was found for the knee extensors peak torque at a velocity of 240°/s (r=0.88, P<0.001). CONCLUSION: The results accounted for an optimal velocity at which a strong relationship could be obtained between isokinetic knee extensors strength and vertical jump height. Interestingly, the H/Q ratio of the young elite female basketball players in the present study was unusual as it was close to that generally observed in regular sportsmen.
Subject(s)
Athletes , Basketball , Knee Injuries/prevention & control , Knee Joint/physiology , Knee/physiology , Muscle Contraction/physiology , Muscle Strength/physiology , Muscle, Skeletal/physiology , Adolescent , Analysis of Variance , Basketball/physiology , Biomechanical Phenomena , Exercise/physiology , Female , Humans , Knee Joint/metabolism , Leg/physiology , Muscle, Skeletal/metabolism , Oxygen Consumption/physiology , Thigh/physiology , TorqueABSTRACT
The hypothesis that ethnicity and sport practice influence the relationship between maximal power in cycling (P(max)) and countermovement jump (CMJ) has been studied by relating CMJ and P(max) in two groups (volleyball players, VB, and physical education students, PES) including subjects with Caucasian (67 C) or West African (39 WA) origins. Maximal power of the arms (P(max) Arms) was also measured. A two-way analysis of variance (groups × ethnicity) showed significant effects of both factors upon CMJ, which was higher in WA and VB, P = 0.002 and P < 0.001, respectively. Within WA, CMJ was significantly higher in VB (0.732 ± 0.057 m) than in PES (0.661 ± 0.082 m), although there was no difference in P(max) (14.7 ± 1.7 vs 14.7 ± 1.9 W/kg). CMJ was significantly higher in WA (0.69 ± 0.08 vs 0.65 ± 0.09 m in C, P = 0.002) without significant interethnic difference in P(max) (14.7 ± 1.8 in WA, and 14.8 ± 1.9 W/kg in C). The CMJ-P(max) relationships were different in C and WA (P = 0.003). Therefore, CMJ predicted from P(max) would be underestimated in WA. The same difference was observed for the relationship between CMJ and P(max) Arms. These results were confirmed by the comparison with previous P(max) -CMJ relationship in the literature, collected in Caucasian and African subjects with the same protocols.
Subject(s)
Arm/physiology , Black People , Leg/physiology , Sports/physiology , White People , Adult , Athletic Performance/physiology , Bicycling/physiology , Biomechanical Phenomena , Exercise Test , Female , Humans , Male , Muscle Strength/physiology , VolleyballABSTRACT
Peroxisome proliferator-activated receptor delta (PPAR-delta) is a transcription factor implicated in metabolism and inflammation. The +294T/C polymorphism in the PPAR-delta gene is associated with risk of coronary artery disease (CAD) in dyslipidemic women and hypercholesterolemic men. Whether this polymorphism influences the risk of CAD in the absence of dyslipidemia was not known, so we investigated a possible association of this polymorphism with plasma lipid and lipoprotein levels and with risk and outcome of CAD in a normolipidemic Tunisian population. Genotyping was performed by PCR-RFLP in 112 CAD patients and 113 healthy volunteers. The C-allele was significantly more frequent in patients than in controls (0.320 vs 0.189, P = 0.001). This association remained significant after adjustment for age, gender, body mass index, smoking, hypertension, and high-density lipoprotein cholesterol. Subjects carrying either one or two copies of the C-allele had a 2.7-fold higher risk of CAD than subjects homozygous for the T-allele. PPAR-delta genotypes were not associated with lipoprotein concentrations or outcome of CAD. We conclude that PPAR-delta +294T/C polymorphism is an independent risk factor of CAD in normolipidemic Tunisian subjects. The lack of association with lipoprotein concentrations suggests that the effect of the polymorphism on CAD is not mediated through lipoprotein levels in this population and that it may influence the atherosclerotic process through mechanisms involving inflammation.
Subject(s)
Coronary Artery Disease/genetics , Genetic Predisposition to Disease , Lipids/blood , PPAR gamma/genetics , Polymorphism, Single Nucleotide/genetics , Body Mass Index , Case-Control Studies , Coronary Artery Disease/blood , Female , Humans , Lipoproteins/blood , Male , Middle Aged , Treatment Outcome , TunisiaABSTRACT
Familial hypercholesterolemia (FH) is an autosomal dominant disease caused by mutations in the low-density lipoprotein receptor (LDLR), apolipoprotein B (APOB), and proprotein convertase subtilisin/kexin type 9 (PCSK9) genes. In previous studies, we have identified novel mutations in Tunisian FH families. In this study, we have extended our investigation to additional families. Five unrelated probands were screened for mutations in the LDLR and APOB genes, using direct sequencing and enzymatic restriction. We identified two novel LDLR mutations: a missense mutation in exon 7: p.Gly343Cys (c.1027G>T), and a nonsense mutation in exon 17: p.Lys816X (c.2446A>T). Using the PolyPhen and SIFT prediction computer programs the p.Gly343Cys is predicted to have a deleterious effect on LDL receptor activity. The missense mutation we found in exon 3, p.Cys89Trp (c.267C>G), has previously been identified in patients from United Kingdom and Spain, and is reported here for the first time in the Tunisian population. Finally, the framshift mutation in exon 10, p.Ser493ArgfsX44, is reported here for the fourth and fifth time in Tunisian families. The latter is the most frequent FH-causing mutation in Tunisia. These LDLR gene mutations enrich the spectrum of mutations causing FH in the Tunisian population. The framshift mutation, p.Ser493ArgfsX44, seems to be a founder mutation in this population.
Subject(s)
DNA Mutational Analysis , Hyperlipoproteinemia Type II/diagnosis , Hyperlipoproteinemia Type II/genetics , Mutation , Receptors, LDL/genetics , Adolescent , Adult , Aged , Exons , Family Health , Female , Heterozygote , Humans , Hyperlipoproteinemia Type II/epidemiology , Male , Pedigree , Polymorphism, Genetic , TunisiaABSTRACT
Ochronosis or alkaptonuria is a rare inherited disease. It is characterized by the deposition of dark pigments in collagen-rich tissues, which leads to clinical manifestations such as arthropathy. The ochronotic pigment can be found in the sclera, the conjunctiva, and the limbic cornea. Vision is usually not affected. We report the case of 47-year-old patient who complained of lower back pain. Ophthalmologic examination showed dark pigments in the conjunctiva. The increased levels of homogentisic acid in urine confirmed the diagnosis of ochronosis.
Subject(s)
Conjunctiva/pathology , Homogentisic Acid/analysis , Lumbar Vertebrae/pathology , Ochronosis/pathology , Spinal Osteophytosis/etiology , Biopsy , Calcinosis/diagnostic imaging , Calcinosis/etiology , Homogentisic Acid/urine , Humans , Intervertebral Disc/diagnostic imaging , Intervertebral Disc/pathology , Low Back Pain/diagnostic imaging , Low Back Pain/etiology , Lumbar Vertebrae/diagnostic imaging , Male , Middle Aged , Ochronosis/complications , Ochronosis/diagnosis , Radiography , Sclera/pathology , Skin Pigmentation , Spinal Osteophytosis/diagnostic imagingABSTRACT
BACKGROUND: Familial hypercholesterolemia (FH) is an autosomal dominant inherited disease caused by mutations in either the low-density lipoprotein receptor, the apolipoprotein B or the proprotein convertase subtilisin/kexin type 9 genes. It is characterized by a high concentration of low-density lipoprotein (LDL), which frequently gives rise to premature coronary disease. In this study, we report a novel splice site mutation of the LDL receptor gene in a Tunisian family. METHODS: Seven patients from the family were screened for mutations in the LDLR gene and the apoB gene, using direct sequencing. RT-PCR and study on cultured skin fibroblast were realised to characterize the effect of novel mutation. RESULTS: Direct sequencing of the promoter and 18 exons reveals a G>A substitution in the splice site junction of intron 8 (c.1186+1 G>A). Study on cultured skin fibroblasts showed a residual activity of 10% of the LDL receptor. Reverse transcription, amplification and direct sequencing of RNA from patient's lymphocytes reveal a deletion of the final 51 bp of exon 8 preserving the reading frame. CONCLUSIONS: The study identified a novel splice mutation c.1186+1 G>A in the LDL receptor gene. It causes the utilization of a new cryptic donor splice site 51 bp downstream from the normal site.
Subject(s)
Apolipoproteins B/genetics , Fibroblasts/metabolism , Hypercholesterolemia/genetics , Mutation , RNA Splice Sites , Receptors, LDL/genetics , Adult , Aged , Apolipoproteins B/metabolism , Cells, Cultured , Family , Female , Humans , Hypercholesterolemia/blood , Introns , Male , Middle Aged , Receptors, LDL/metabolism , TunisiaABSTRACT
The fate and phenotype of lesion macrophages is regulated by cellular oxidative stress. Thioredoxin-1 (Trx-1) plays a major role in the regulation of cellular redox balance, with resultant effects on gene expression and cellular responses including cell growth and death. Trx-1 activity is inhibited by interaction with vitamin D-upregulated protein-1 (VDUP-1). Peroxisome proliferator-activated receptor gamma (PPARgamma) is expressed by human monocyte-derived macrophages (HMDM) and PPARgamma agonism has been reported to decrease expression of inflammatory genes and to promote apoptosis of these cells. To determine whether VDUP-1 may be involved in regulating the effects of PPARgamma agonists in macrophages, we investigated the effect of a synthetic PPARgamma agonist (GW929) on the expression of VDUP-1 in HMDM. GW929 concentration-dependently increased HMDM expression of VDUP-1 (mRNA and protein). Transfection of different fragments of the VDUP-1 promoter as well as gel shift analysis revealed the presence of functional PPARgamma response elements (PPRE) in the promoter. Under conditions in which PPAR agonism altered levels of VDUP-1, caspase-3 activity, and macrophage apoptosis were also elevated. The results suggest that PPARgamma activation stimulates apoptosis in human macrophages by altering the cellular redox balance via regulation of VDUP-1.
Subject(s)
Apoptosis/drug effects , Carrier Proteins/metabolism , Gene Expression Regulation/drug effects , Macrophages/drug effects , PPAR gamma/agonists , Aorta/cytology , Carrier Proteins/genetics , Caspase 3/analysis , Caspase 3/metabolism , Cell Culture Techniques , Cell Differentiation , Cells, Cultured , Dose-Response Relationship, Drug , Electrophoretic Mobility Shift Assay , Genes, Reporter , Humans , In Situ Nick-End Labeling , Luciferases/metabolism , Macrophages/metabolism , Monocytes/cytology , Monocytes/metabolism , Muscle, Smooth, Vascular/cytology , Plasmids , Promoter Regions, Genetic , RNA, Messenger/metabolism , RNA, Small Interfering/metabolism , Response Elements , Transfection , U937 CellsABSTRACT
Following the arrival of blue-tongue in Tunisia, the authors report the results of the first survey made in Monastir. They show the existence of nine species of Culicoides, three of which are new to the country IC. paolae, C. imicola, C. newsteadi), that now brings to 22 the number of the known species.
Subject(s)
Ceratopogonidae/classification , Ceratopogonidae/parasitology , Animals , Female , Male , Phylogeny , TunisiaABSTRACT
OBJECTIVE: Cholesterol accumulation in macrophages is known to alter macrophage biology. In this article we studied the impact of macrophage cholesterol loading on gene expression and identified a novel gene implicated in cell death. METHODS AND RESULTS: The regulated in development and DNA damage response 2 (REDD2) gene was strongly upregulated as THP-1 macrophages are converted to foam cells. These results were confirmed by Northern blot of RNA from human monocyte-derived macrophages (HMDM) treated with oxidized LDL (oxLDL). Human REDD2 shares 86% amino acid sequence identity with murine RTP801-like protein, which is 33% identical to RTP801, a hypoxia-inducible factor 1-responsive gene involved in apoptosis. Treatment of HMDM with desferrioxamine, a molecule that mimics the effect of hypoxia, increased expression of REDD2 in a concentration-dependent fashion. Transfection of U-937 and HMEC cells with a REDD2 expression vector increased the sensitivity of the cells for oxLDL-induced cytotoxicity, by inducing a shift from apoptosis toward necrosis. In contrast, suppression of mRNA expression using siRNA approach resulted in increased resistance to oxLDL treatment. CONCLUSIONS: We showed that stimulation of REDD2 expression in macrophages increases oxLDL-induced cell death, suggesting that REDD2 gene might play an important role in arterial pathology.
Subject(s)
Cell Death/physiology , Hypoxia/pathology , Lipoproteins, LDL/pharmacology , Macrophages/drug effects , Macrophages/physiology , Proteins/physiology , Up-Regulation/drug effects , Up-Regulation/physiology , Adaptor Proteins, Signal Transducing , Arteriosclerosis/genetics , Cell Line , Cell Line, Tumor , Cells, Cultured , DNA/genetics , Deferoxamine/pharmacology , Dose-Response Relationship, Drug , Endothelium, Vascular/chemistry , Endothelium, Vascular/metabolism , Foam Cells/physiology , Humans , Monocytes/physiology , RNA, Messenger/biosynthesis , RNA, Messenger/genetics , Reverse Transcriptase Polymerase Chain Reaction/methods , Transfection/methods , U937 Cells/chemistry , U937 Cells/metabolismABSTRACT
Uptake of modified low-density lipoproteins (LDLs) by macrophages in the arterial wall is an important event in atherogenesis. Indeed, oxidatively modified LDLs (oxLDLs) are known to affect various cellular processes by modulating oxidation-sensitive signaling pathways. Here we found that the ubiquitous 55 kDa selenoprotein thioredoxin reductase 1 (TrxR1), which is a key enzyme for cellular redox control and antioxidant defense, was upregulated in human atherosclerotic plaques and expressed in foam cells. Using reverse transcription polymerase chain reaction analysis, we also found that oxLDLs, but not native LDLs (nLDLs), dose-dependently increased TrxR1 mRNA in human monocyte-derived macrophages (HMDMs). This stimulating effect was specific for oxLDLs, as pro-inflammatory factors, such as lipopolysaccharides (LPSs), interleukin-1beta (IL-1beta), interleukin-6 (Il-6), and tumor necrosis factor alpha (TNFalpha), under the same conditions, failed to induce TrxR1 mRNA levels to the same extent. Moreover, phorbol ester-differentiated THP-1 cells or HMDMs transiently transfected with TrxR1 promoter fragments linked to a luciferase reporter gene allowed identification of a defined promoter region as specifically responding to the phospholipid component of oxLDLs (p <.05 vs. phospholipid component of nLDLs). Gel mobility shift analyses identified a short 40-nucleotide stretch of the promoter carrying AP-1 and HoxA5 consensus motifs that responded with an altered shift pattern in THP-1 cells treated with oxLDLs, however, without evident involvement of either the Fos, Jun, Nrf2 or HoxA5 transcription factors.
Subject(s)
Carotid Artery Diseases/enzymology , Gene Expression Regulation, Enzymologic , Lipoproteins, LDL/pharmacology , Macrophages/enzymology , Promoter Regions, Genetic/genetics , Thioredoxin-Disulfide Reductase/genetics , Base Sequence , Carotid Artery Diseases/surgery , Cell Line, Tumor , Endarterectomy, Carotid , Humans , Molecular Sequence Data , Monocytes/physiology , RNA, Messenger/genetics , Thioredoxin Reductase 1 , TransfectionABSTRACT
Metalloproteinases (MMP)-2 and MMP-9 play a role in smooth muscle cell (SMC) migration from the media to the intima following arterial injury. Intravenous administration of adenovirus encoding tissue inhibitor of metalloproteinase-1 (TIMP-1) into balloon-injured rat arteries (3 x 10(11) viral particles/rat; n=7) resulted in a transient expression of TIMP-1 and a significant inhibition of neointima thickening within 16 days ( approximately 40% vs. control; P=0.012). Three days after injury, the number of intimal SMCs was decreased by approximately 98% in TIMP-1-treated rats. However, no alteration was seen in intimal SMC proliferation after 13 days of injury. Therefore, our results show that systemic gene transfer of TIMP-1 is a promising approach in early restenosis treatment.
Subject(s)
Angioplasty, Balloon/adverse effects , Carotid Arteries/pathology , Carotid Stenosis/therapy , Genetic Therapy , Tissue Inhibitor of Metalloproteinase-1/genetics , Adenoviridae/genetics , Animals , Carotid Stenosis/etiology , Carotid Stenosis/pathology , Cell Movement , Genetic Vectors/administration & dosage , Humans , Hyperplasia , Injections, Intravenous , Kinetics , Male , Matrix Metalloproteinases/metabolism , Muscle, Smooth, Vascular/physiopathology , Rats , Rats, Sprague-Dawley , Tissue Inhibitor of Metalloproteinase-1/bloodABSTRACT
We have investigated the lipoprotein lipase (LPL) gene of a 2-year-old patient presenting classical features of the familial LPL deficiency including undetectable LPL activity. DNA sequence analysis of exon 5 identified the patient as a homozygote for the Gly188Glu mutation, frequently involved in this disease. A review of cases of LPL deficiency with molecular study of the LPL gene showed a total number of 221 reported mutations involved in this disease. Gly188Glu was involved in 23.5 % of cases and 74.6 % of mutations were clustered in exons 5 and 6. Based on these observations, we propose a method of screening for mutations in this gene.
Subject(s)
Hyperlipoproteinemia Type I/genetics , Lipoprotein Lipase/deficiency , Lipoprotein Lipase/genetics , Mutation, Missense , Amino Acid Substitution , Consanguinity , Exons , Female , Homozygote , Humans , Infant , Lipolysis , Lipoprotein Lipase/chemistry , Male , Sequence DeletionABSTRACT
A new polymorphism located at position -629 (CETP/-629A/C) in the promoter of the cholesteryl ester transfer protein (CETP) gene is described. The -629A allele was associated with lower CETP mass (P<0. 0001) and higher high density lipoprotein cholesterol (P<0.001) than the C allele in a sample of 536 control subjects from the ECTIM study. Transfection studies in HepG2 cells with a luciferase expression vector incorporating a 777-bp fragment of the CETP promoter and containing either A or C at position -629 showed significantly lower luciferase activity with the promoter fragment of the A allele (-25%, P<0.05). By gel-shift assay, DNA-protein interactions were evaluated in nuclear extracts of HepG2 cells with the use of 2 probes (A or C probe) composed of 20 bp of the promoter sequence surrounding the polymorphic site. Two specific complexes of distinct migration rate were identified with the A and the C probe. Competition with an excess of oligonucleotide containing the Sp1 consensus binding site showed that a protein(s) of the Sp transcription factor family was implicated in complex formation with the A probe but not with the C probe. Incubation with specific antibodies indicated that Sp1 and Sp3 bound specifically to the A probe. We introduced mutations in the -629-Sp1 binding site to test its functionality and to define the characteristics of transcription factor binding. We showed, by gel-shift assay, that no nuclear proteins bound to the mutated sequence. Transient transfection of HepG2 cells revealed that the expression of the mutated fragment was significantly increased compared with that of the A promoter fragment (25%, P<0.05). The mutated fragment displayed the same activity as that of the C promoter. These results indicate that Sp1 and/or Sp3 repress CETP promoter activity, whereas nuclear factors binding the C allele are without effect on promoter expression.
Subject(s)
Carrier Proteins/genetics , Cholesterol, HDL/blood , Glycoproteins , Polymorphism, Genetic/physiology , Promoter Regions, Genetic/genetics , Adult , Binding Sites/physiology , Carrier Proteins/blood , Carrier Proteins/metabolism , Cholesterol Ester Transfer Proteins , DNA-Binding Proteins/genetics , Electrophoresis, Gel, Two-Dimensional , Gene Frequency , Humans , Middle Aged , Peptide Fragments/metabolism , Sp1 Transcription Factor/genetics , Sp1 Transcription Factor/metabolism , Sp2 Transcription Factor , Transcription Factors/genetics , Transcription, GeneticABSTRACT
Tissue factor pathway inhibitor is the major potent physiologic inhibitor of tissue factor-induced coagulation. Several potential binding sites for transcription factors have been described in the 750 bp of the 5' flanking region of the human tissue factor pathway inhibitor gene reported earlier. To identify elements that regulate the expression of tissue factor pathway inhibitor in endothelial, hepatocyte, and monocyte cells, the sequence of an additional 770 bp of tissue factor pathway inhibitor was determined. Comparison of this new sequence as well as that reported earlier with consensus sequences for transcription factor binding sites provided matches for GATA-2, SP1, and c-Myc sequences. Moreover, plasmids containing deletion mutants of the 5' tissue factor pathway inhibitor promoter region and the luciferase reporter gene were transfected into HepG2, ECV304, and THP1 cells. Three negative regulatory elements were localized between -548 to -390, - 390 to -75, and -1158 to -796 relative to the transcriptional start, respectively, in HepG2, ECV304 and THP-1 cells.
Subject(s)
Lipoproteins/genetics , Promoter Regions, Genetic/genetics , Base Sequence , Cell Line , Gene Expression Regulation , Humans , Molecular Sequence Data , Sequence Analysis, DNAABSTRACT
BACKGROUND: To define the role of metalloproteinases (MMPs) in the development of lipid-rich atherosclerotic lesions in relation to the balance between proteolytic and antiproteolytic activities, we investigated the impact of adenovirus-mediated elevation in the circulating levels of human tissue inhibitor of MMP (TIMP-1) in atherosclerosis-susceptible apolipoprotein E-deficient (apoE(-/-)) mice. METHODS AND RESULTS: Infusion of apoE(-/-) mice fed a lipid-rich diet with rAd.RSV.TIMP-1 (1x10(11) viral particles) resulted in high hepatic expression of TIMP-1. At 2 weeks after injection, plasma TIMP-1 levels ranged from 7 to 24 micrograms/mL (mean 14.8+/-6.8). Marked overexpression of TIMP-1 was transient, with levels of TIMP-1 decreasing to 2.5 to 8 micrograms/mL (mean 4.3+/-2.1) at 4 weeks. Plasma lipid and lipoprotein levels in mice treated with rAd.RSV.TIMP-1 were similar to those treated with rAd.RSV.betaGal. However, rAd.RSV.TIMP-1-infused mice displayed a marked reduction (approximately 32%; P<0.05) in mean lesion area per section (512+/-121 micrometers(2)x10(3); n=12 sections from 4 animals) as compared with rAd.RSV.betaGal-infused mice (750+/-182 micrometers(2)x10(3); n=12 sections from 4 animals). Similarly, marked reduction in macrophage deposition as well as MMP-2, MMP-3, and MMP-13 antigens was observed. CONCLUSIONS: Histological and immunohistologic analyses of atherosclerotic lesions revealed increases in collagen, elastin, and smooth muscle alpha-actin content in mice treated with rAd.RSV.TIMP-1. These qualitative and quantitative features were the consequence of TIMP-1 infiltration from plasma to arterial intima, as immunohistochemical analyses revealed an abundance of TIMP-1 specifically in lesions of rAd.RSV. TIMP-1-treated mice.
Subject(s)
Adenoviridae , Apolipoproteins E/deficiency , Arteriosclerosis/pathology , Arteriosclerosis/prevention & control , Gene Transfer Techniques , Genetic Vectors , Tissue Inhibitor of Metalloproteinase-1/blood , Animals , Arteriosclerosis/etiology , Dietary Fats/administration & dosage , Dietary Fats/adverse effects , Female , Immunohistochemistry , Mice , Mice, Inbred C57BL , Up-RegulationABSTRACT
BACKGROUND: The accumulation of macrophage-derived foam cells in atherosclerotic lesions correlates with increased local release of matrix-degrading metalloproteinases (MMPs) and a thin fibrous cap. The activity of these enzymes is controlled by specific tissue inhibitors of metalloproteinases (TIMPs). METHODS AND RESULTS: Because oxidized low-density lipoprotein (OxLDL) modulates gene expression, we investigated the effect of these particles on the levels of MMP-1, MMP-3, MMP-9, TIMP-1, and TIMP-2 in the culture media of human monocyte-derived macrophages. OxLDL but not native LDL or high-density lipoprotein reduced the level of TIMP-1 in a dose-dependent manner with maximal effect (60% of control) at approximately 100 microg protein/mL. In addition, Northern blotting revealed marked reduction in the abundance of TIMP-1 mRNA in OxLDL-treated cells. Evaluation of the effect of oxysterol components of OxLDL on TIMP-1 production revealed that 25-hydroxycholesterol (1 microg/mL) was the most potent inhibitor ( approximately 30% of control). Such inhibition was partially mediated by interleukin (IL)-8. Indeed, IL-8 (2.5 ng/mL) induced maximal inhibition of TIMP-1 accumulation (30% of control) in 4 of 6 cell preparations. In addition, the inhibitory effect of OxLDL-treated cells in the presence of an anti-IL-8 neutralizing antibody was partially reversed. CONCLUSIONS: Immunohistochemical analyses of human atherosclerotic plaques revealed the expression of TIMP-1 in some but not all macrophage-rich and IL-8-rich areas. Therefore, IL-8 may play a potential atherogenic role by inhibiting local TIMP-1 expression, thereby leading to an imbalance between MMPs and TIMPs at focal sites in the atherosclerotic plaque.
Subject(s)
Arteriosclerosis/metabolism , Collagenases/metabolism , Interleukin-8/metabolism , Macrophages/enzymology , Tissue Inhibitor of Metalloproteinase-1/genetics , Antibodies, Monoclonal , Antigens, CD/analysis , Antigens, CD/immunology , Antigens, Differentiation, Myelomonocytic/analysis , Antigens, Differentiation, Myelomonocytic/immunology , Arteriosclerosis/pathology , Carotid Arteries/chemistry , Carotid Arteries/enzymology , Carotid Arteries/pathology , Cells, Cultured , Cholesterol, LDL/metabolism , Cholesterol, LDL/pharmacology , Collagenases/analysis , Collagenases/immunology , Gene Expression Regulation, Enzymologic , Humans , Interleukin-8/analysis , Interleukin-8/immunology , Macrophages/chemistry , Macrophages/drug effects , Matrix Metalloproteinase 1 , Matrix Metalloproteinase 3/analysis , Matrix Metalloproteinase 3/immunology , Matrix Metalloproteinase 3/metabolism , Matrix Metalloproteinase 9 , Monocytes/chemistry , Monocytes/drug effects , Monocytes/enzymology , Oxidation-Reduction , Tissue Inhibitor of Metalloproteinase-1/immunology , Tissue Inhibitor of Metalloproteinase-2/analysis , Tissue Inhibitor of Metalloproteinase-2/genetics , Tissue Inhibitor of Metalloproteinase-2/immunologyABSTRACT
We have studied the underlying molecular defect in a patient presenting with recurrent pancreatitis, hypertriglyceridemia, and virtually undetectable postheparin plasma lipoprotein lipase (LPL) mass and activity, who normalized her triglycerides 3 to 6 months after initiation of either medium-chain triglyceride (MCT) oil or omega-3 fatty acid (omega-3-FA) therapy. After treatment, postheparin plasma LPL activity and mass ranged from 24% to 39% of normal and LPL specific activity was normal (1.0 nmol.ng-1.min-1). On discontinuation of MCT oil or omega-3-FA, plasma triglyceride increased to > 2000 mg/dL. Northern blotting revealed both normal size and abundance of LPL mRNA isolated from adipocytes as well as macrophages. Sequence analysis of the LPL gene, which included all 10 exons, intron-exon splice junctions, and 1.7 kb of the 5'-flanking region, and of LPL cDNA failed to identify any mutations. ApoC-II activity and mass assays revealed the presence of normal levels of a fully functional cofactor as well as the absence of circulating plasma inhibitors of lipase function. In summary, we describe a unique patient presenting with classical features of the familial chylomicronemia syndrome who manifests an unusually beneficial therapeutic response to MCT oil and omega-3-FA therapy. Unlike that in most patients with LPL deficiency, the chylomicronemia in this patient is not caused by a mutation in the structural LPL gene but possibly by a posttranscriptional defect. Thus, a subset of LPL-deficient patients with unique genetic defects respond to therapy by normalizing fasting plasma triglycerides; a therapeutic trial with MCT oil should be considered in all patients presenting with the familial chylomicronemia syndrome.
Subject(s)
Chylomicrons/blood , Fatty Acids, Omega-3/therapeutic use , Lipase/blood , Lipid Metabolism, Inborn Errors/diet therapy , Triglycerides/therapeutic use , Adipose Tissue/enzymology , Child , Female , Gene Expression Regulation, Enzymologic , Humans , Lipoprotein Lipase/blood , Lipoprotein Lipase/genetics , Liver/enzymology , Macrophages/enzymology , Male , Pedigree , RNA, Messenger/metabolismABSTRACT
Lecithin:cholesterol acyltransferase (LCAT) is responsible for the formation of the majority of plasma cholesteryl esters. Familial LCAT deficiency is associated with corneal opacity, anemia and proteinurea and typically results in renal failure in the 4-5th decade; this syndrome is equally characterized by the quasi-absence of plasma LCAT activity with variable enzyme mass and very low levels of plasma cholesteryl esters. In this study, we report detailed analyses of plasma lipids and lipoprotein profile in two sisters (CM and ML) presenting classical homozygous LCAT-deficiency; the younger sibling (CM) had proteinurea from an early age whereas the older sister (ML) has never exhibited renal dysfunction. We investigated the molecular defect in the 45 year-old woman (proband CM) exhibiting all clinical and biochemical features of familial LCAT deficiency: a plasma cholesterol level of 105 mg/dl, of which 95% was unesterified, an HDL-cholesterol of 6.5 mg/dl and an apo A-I level of 52 mg/dl. The proband (CM) displayed a plasma cholesterol esterification rate which corresponded to 2% of normal LCAT activity; plasma LCAT protein concentration was 0.56 microg/ml and equivalent to approximately 10% of normal LCAT mass. Analysis by single strand conformation polymorphism (SSCP) of the PCR products corresponding to exons 4 and 5 of the LCAT gene revealed a visible band shift. Sequence analyses of exons 4 + 5 revealed two separate single point mutations: a C --> T transition replacing Arg147 by Trp and a T --> G transition converting Tyr171 to a stop codon. The presence of these two point mutations was confirmed by restriction enzyme analyses: the C --> T transition abolished a MwoI site whereas the T --> G transition created an AvrII site. The Arg147 mutation was associated with a non-secreted protein. The Tyr171 mutation resulted in formation of a truncated protein lacking the catalytic site. In summary, we have identified an LCAT deficient patient corresponding to a compound heterozygote for the Arg147 --> Trp mutation and a new molecular defect involving a Tyr171 --> Stop mutation in the LCAT gene.
Subject(s)
Lecithin Cholesterol Acyltransferase Deficiency/genetics , Phosphatidylcholine-Sterol O-Acyltransferase/genetics , Point Mutation , Apolipoprotein A-I/metabolism , Arginine , Cholesterol/blood , Cholesterol Esters/blood , Cholesterol, HDL/blood , DNA/chemistry , Female , Heterozygote , Humans , Lecithin Cholesterol Acyltransferase Deficiency/blood , Middle Aged , Pedigree , Phosphatidylcholine-Sterol O-Acyltransferase/blood , Polymerase Chain Reaction , Polymorphism, Single-Stranded Conformational , Sequence Analysis, DNA , TyrosineABSTRACT
We present a review of the literature and results of a survey involving 50 closed scrotal traumas. Based on this analysis, we propose an anatomoclinical classification of scrotal contusions based on what we consider to be the most appropriate therapeutic management.