Muhidien Soufi

Risk of Coronary Artery Disease Associated With Polymorphism of the Cytochrome P450 Epoxygenase CYP2J2

Background—Cytochrome P450 (CYP) 2J2 is expressed in the vascular endothelium and metabolizes arachidonic acid to biologically active epoxyeicosatrienoic acids (EETs). The EETs are potent endogenous vasodilators and inhibitors of vascular inflammation. However, it is not known whether genetic polymorphisms of CYP2J2 are associated with increased cardiovascular risks. Methods and Results—All 9 exons of the CYP2J2 gene and its proximal promoter were sequenced in 132 patients to identify potential variants. Functional consequence of a single nucleotide polymorphism (SNP) in the promoter of CYP2J2 was further evaluated by use of transcription factor-binding and reporter assays. A total of 17 polymorphisms were identified. One of the most relevant polymorphisms in terms of frequency and functional importance is located at −50 (G-50T) in the proximal promoter of CYP2J2. Screening of 289 patients with coronary artery disease and 255 control subjects revealed 77 individuals with the G-50T SNP (17.3% of coronary artery disease patients, 10.6% of control subjects; P=0.026). The association of the G-50T polymorphism remained significant after adjustment for age, gender, and conventional cardiovascular risk factors (OR, 2.23; 95% CI, 1.04 to 4.79). The G-50T mutation resulted in the loss of binding of the Sp1 transcription factor to the CYP2J2 promoter and resulted in a 48.1±2.4% decrease in CYP2J2 promoter activity (P<0.01). Plasma concentrations of stable EET metabolites were significantly lower in individuals with the G-50T SNP. Conclusions—A functionally relevant polymorphism of the CYP2J2 gene is independently associated with an increased risk of coronary artery disease.

In vivo stable-isotope kinetic study suggests intracellular assembly of lipoprotein(a)

OBJECTIVE Lipoprotein(a) [Lp(a)] consists of apolipoprotein B-100 (apoB-100) as part of an LDL-like particle and the covalently linked glycoprotein apolipoprotein(a) [apo(a)]. Detailed mechanisms of its biosynthesis, assembly, secretion and catabolism are still poorly understood. To address the Lp(a) assembly mechanism, we studied the in vivo kinetics of apo(a) and apoB-100 from Lp(a) and LDL apoB-100 in nine healthy probands using stable-isotope methodology. METHODS The level of isotope enrichment was used to calculate the fractional synthesis rate (FSR), production rate (PR) and retention time (RT) using SAAMII software and multicompartmental modeling. RESULTS We observed a similar mean PR for apo(a) (1.15 nmol/kg/d) and apoB-100 (1.31 nmol/kg/d) from Lp(a), which differed significantly from the PR for apoB-100 from LDL (32.6 nmol/kg/d). Accordingly, mean FSR and RT values for Lp(a)-apo(a) were similar to those of Lp(a)-apoB and different from those for LDL-apoB. CONCLUSION Two different kinetic apoB pools within Lp(a) and LDL suggest intracellular Lp(a) assembly from apo(a) and newly synthesized LDL.

Molecular Mechanisms Involved in Atherosclerosis

Background: Research in atherosclerosis is a good example how helpful different disciplines such as clinicians, epidemiologists and basic science can collaborate. In recent years our knowledge on cellular and subcellular mechanisms involved in initiation and progress of atherosclerosis has expanded due to the shared knowledge of different disciplines and thanks to new technologies in molecular biology. Pathophysiology of LDL and HDL Metabolism: The understanding of the molecular basis of inborn errors of LDL metabolism – such as familial hypercholesterolemia due to a defect of the LDL receptor – provided us new insights in physiology and pathophysiology of LDL metabolism. Most recently we have learned much about the vasoprotective HDL cholesterol. HDL is the major player in reverse cholesterol transport and some of its receptors such as ABCA1 and SR-BI were identified. This knowledge gives us a deeper understanding of the complex system which performs reverse cholesterol transport from peripheral tissue and the vessel wall back to the liver. Plaque Formation: Furthermore the process of formation and progression of the atherosclerotic plaque has been the focus of recent research. The stability or instability of plaques is depending on the complex interaction of adhesion molecules, monocytes, macrophages, endothelial cells, cytokines, transmitters and proteinases. Since we are unable to prevent plaque formation completely, the stabilization of plaques is a major goal for the coming years. Despite some success (such as the use of statines and ACE inhibitors) there is still a long way to go.Hintergrund: Durch die Nutzung neuer molekularbiologischer Techniken hat sich in den letztenJahren unser Wissen über molekulare und zelluläre Mechanismen der Pathogenese der Atherosklerose deutlich erweitert. Der Kampf gegen den Herzinfarkt ist ein beeindruckendes Beispiel, wie sehr Kliniker, Epidemiologen und Grundlagenforscher voneinander bei ihrer Arbeit profitieren können. Waren es ursprünglich rein klinische und epidemiologische Beobachtungen, die uns die Bedeutung erhöhter Cholesterinwerte für die Atherosklerose nahe legten, so kennen wir heute eine Vielzahl (wenngleich auch noch lange nicht alle) der hierfür verantwortlichen molekularen Mechanismen. Pathophysiologie des LDL- und HDL-Metabolismus: So wissen wir, dass bei der familiären Hypercholesterinämie der primäre Defekti bei einem mutierten LDL-Rezeptor zu suchen ist, der zu einer Erhöhung der atherogenen Lipoproteine, dem LDL-Cholesterin, führt. Die vasoprotektiven Lipoproteine, das HDL-Cholesterin, werden derzeit mit Hochdruck erforscht. Dabei gelang erst vor kurzem die Beschreibung einiger wesentlicher Rezeptoren wie ABCA1 und SR-BI, die uns helfen, das komplexe System des Cholesterinrücktransports vom peripheren Gewebe und der Gefäßwand zurück zur “Entgiftungszentrale” der Leber zu verstehen. Plaqueformation: Wir sehen mittlerweile den atheromatösen Plaque als ein dynamisches, überaus vulnerables Gebilde. Die Stabilität oder Instabilität eines solchen Plaques entscheidet über Leben oder Tod der Betroffenen und ist das Ergebnis aus dem Wechselspiel einer Vielzahl von Einzelspielern: Adhäsionsmolekülen, Monozyten, Makrophagen, Endothelzellen, Zytokinen, zellulären Transmittern und Proteinasen. Solange wird nicht in der Lage sind, die Plaqueentstehung gänzlich zu verhindern, werden wir versuchen müssen, die Stabilität der Plaques zu erhöhen. Mit einigen therapeutischen Ansätzen ist uns dies bereits gelungen (CSE-Hemmer-Therapie, ACE-Hemmer-Therapie), wenngleich noch ein langer Weg vor uns liegt.

The impact of severe LDL receptor mutations on SREBP-pathway regulation in homozygous familial hypercholesterolemia (FH)

Familial hypercholesterolemia (FH), Niemann-Pick disease type C (NPC) and Tangier disease (TD) are genetic inherited disorders with impaired processing of cholesterol, caused by mutations in genes that regulate cellular uptake, intracellular movement and transport of cholesterol. Various studies have shown a crucial regulatory role of the SREBP-pathway for cellular cholesterol homeostasis in these diseases. Since cholesterol is an essential structural component of cells, we assessed the impact of a severe FH causing LDLR mutation (FH p.W556R) on the SREBP pathway in primary FH fibroblasts. Primary FH fibroblasts derived from patients with the LDL receptor mutation p.W556R were used for gene expression experiments. Gene expression studies revealed increased expressions of SREBP regulated genes HMGCR, LDLR, SREBP-2, SREBP-1, SR-BI, INSIG-1, but interestingly not SCAP. In contrast expression of ABCA1, was strongly decreased in homozygous, but not in heterozygous p.W556R fibroblasts. Gene expression experiments with LDL receptor lacking primary FH fibroblasts, revealed that SR-BI and ABCA1 are important regulators for cholesterol acquisition in FH cells, consistent with findings in cells from NPC and TD patients.

Molecular basis of obesity and the risk for cardiovascular disease.

Atherosclerosis and cardiovascular disease (CVD) are the main causes of death in the Western world, for both men and women. The onset and development of diseases of the cardiovascular and cerebrovascular system are strongly dependent on multiple risk factors that promote pathologic conditions like atherosclerosis, hypertension and thrombosis. Besides genetic factors also environmental influences such as diet composition are known to be closely related to CVD. In this context obesity has been postulated as an independent cardiovascular risk factor. Data from the Framingham Heart Study have consistently shown that increasing degrees of obesity are accompanied by greater rates of CVD. At present, obesity affects 10–35% of the European and US population and increases steadily. As obesity is a serious health problem which promotes metabolic abnormalities (insulin resistance, hyperinsulinemia and dyslipidemia) and dramatically increases the risk for CVD, this review will focus on the epidemiologic and genetic background of obesity. Furthermore, the molecular mechanisms involved in obesity development and their contribution to CVD will be discussed.ZusammenfassungAtherosklerose und die damit vergesellschafteten kardiovaskulären Erkrankungen sind nach wie vor die Haupttodesursache in der westlichen Welt, sowohl bei Männern als auch bei Frauen. Entstehung und Fortschreiten dieser Erkrankung werden durch eine Reihe von Risikofaktoren getrieben, die letztendlich zur Atherosklerose, aber auch zu Bluthochdruck sowie erhöhter Thromboseneigung führen können. Neben genetischen Faktoren spielen Umwelt- sowie Ernährungsfaktoren eine große Rolle. In diesem Zusammenhang wurde auch das Vorhandensein von Übergewicht als kardiovaskulärer Risikofaktor beschrieben. Die Daten der Framingham Heart Study zeigten, dass es mit zunehmendem Übergewicht zum vermehrten Auftreten koronarer Herzerkrankungen (KHK) kommt. Derzeit sind zwischen 10% und 35% aller Europäer und US-Amerikaner übergewichtig—mit rasch steigender Tendenz. Dadurch wird Übergewicht zu einem ernsthaften Gesundheitsproblem. In der Folge führt krankhaftes Übergewicht zu einer ganze Reihe von atheroskleroserelevanten Stoffwechselstörungen, wie Insulinresistenz, Hyperinsulinismus und Dyslipidämien, und in der Folge zu einer dramatischen Zunahme kardiovaskulärer Erkrankungen. Diese Arbeit wird sich mit den epidemiologischen und genetischen Ursachen von Übergewicht auseinander setzen. Darüber hinaus werden die molekularen Mechanismen, die zur Adipositas und in der Folge zur KHK führen, sowie sich hieraus entwickelnde neue Therapieansätze diskutiert.

Increased Production of HDL ApoA-I in Homozygous Familial Defective ApoB-100

Familial defective apolipoprotein (apo) B-100 (FDB) is a frequent cause of hypercholesterolemia. Hypercholesterolemia in homozygous FDB is less severe than in homozygotes for familial hypercholesterolemia. Recently, we showed decreased low density lipoprotein (LDL) apoB-100 fractional catabolism and decreased production of LDL due to an enhanced removal of apoE-containing precursors in a patient with homozygous FDB. The effects of defective apoB-100 on high density lipoprotein (HDL) metabolism are unknown. We studied HDL apoA-I metabolism in this FDB patient and in 6 control subjects by using (2)H(3)-L-leucine as a tracer. ApoA-I levels were normal in all study subjects. However, the fractional catabolic rate and the production rate of apoA-I were increased, by 79% and 70%, respectively, in FDB; the fractional catabolic rate of apoA-I in FDB was 0.34 day(-1) compared with 0.19+/-0.03 day(-1) in normal controls. The production rate of apoA-I in FDB was 18.4 mg. kg(-1). d(-1) compared with 10.8+/-2.3 mg. kg(-1). d(-1) in controls. Thus, we have shown for the first time that defective apoB-100 may influence HDL kinetics. The increase in total HDL turnover might enhance reverse cholesterol transport and could contribute to the seemingly benign clinical course of FDB compared with that of familial hypercholesterolemia.

A combined LDL receptor/LDL receptor adaptor protein 1 mutation as the cause for severe familial hypercholesterolemia.

Familial hypercholesterolemia (FH) results from impaired catabolism of plasma low density lipoproteins (LDL), thus leading to high cholesterol, atherosclerosis, and a high risk of premature myocardial infarction. FH is commonly caused by defects of the LDL receptor or its main ligand apoB, together mediating cellular uptake and clearance of plasma LDL. In some cases FH is inherited by mutations in the genes of PCSK9 and LDLRAP1 (ARH) in a dominant or recessive trait. The encoded proteins are required for LDL receptor stability and internalization within the LDLR pathway. To detect the underlying genetic defect in a family of Turkish descent showing unregular inheritance of severe FH, we screened the four candidate genes by denaturing gradient gel electrophoresis (DGGE) mutation analysis. We identified different combinatory mixtures of LDLR- and LDLRAP1-gene defects as the cause for severe familial hypercholesterolemia in this family. We also show for the first time that a heterozygous LDLR mutation combined with a homozygous LDLRAP1 mutation produces a more severe hypercholesterolemia phenotype in the same family than a homozygous LDLR mutation alone.

Genetics and kinetics of familial hypercholesterolemia, with the special focus on FH- Marburg p.W556R

OBJECTIVE Familial hypercholesterolemia (FH) is an autosomal dominant inherited disorder, caused by mutations in the low density lipoprotein receptor (LDLR) gene. FH is characterized by elevated plasma LDL cholesterol, premature atherosclerosis and high risk of premature myocardial infarction. Extended work has been done to understand both, the primary genetic defect as well as the in vivo kinetic consequences of this disease. Both approaches, genetics and kinetics, are challenging but also fruitful approaches for a better understanding of this devastating disease. For this we reviewed the recent literature and used our in vitro and in vivo data on one of the most frequently occurring types of FH, the FH(Marburg) p.W556R. METHODS To identify the primary genetic defect of the FH(Marburg) we used denaturing gradient gel electrophoresis (DGGE) mutation analysis. In vivo kinetic studies were performed in a heterozygote FH(Marburg) subject and in 5 healthy control subjects utilizing a stable isotope tracer kinetic approach with 3D-leucine. RESULTS DGGE screening of the LDLR gene identified a tryptophan (W) to arginine (R) substitution at residue 556 (p.W556R) in the fifth conserved YWTD repeat of the LDLR-beta-propeller in FH(Marburg). In vivo kinetic studies in a heterozygote FH subject for FH(Marburg) and in 5 healthy control subjects demonstrated a severe decrease in LDL FCR and a mild increase of LDL PR in FH compared to healthy controls. CONCLUSIONS The LDLR mutation p.W556R is a frequent and severe defect for FH. This defect has a major influence on the in vivo lipoprotein kinetics and lipid levels. In a heterozygote FH patient we found a dual defect for the increase in LDL cholesterol, namely a decrease in the fractional catabolic rate (FCR) of LDL but also an increase in LDL production rate (PR). By this a well defined, single genetic defect may have a series of different in vivo metabolic consequences which could be used for potential therapeutic approaches to this disease.

Pharmacogenetic aspects in familial hypercholesterolemia with the special focus on FHMarburg (FH p.W556R)

Objective Familial hypercholesterolemia (FH) is an autosomal dominant inherited disorder caused by mutations in the low density lipoprotein receptor (LDLR) gene. FH is characterized by elevated plasma LDL cholesterol, premature atherosclerosis, and a high risk of premature myocardial infarction. In general, mutations within LDLR gene can cause five different classes of defects, namely: class I defect: no LDLR synthesis; class II defect: no LDLR transport; class III defect: no low density lipoprotein (LDL) to LDLR binding; class IV defect: no LDLR/LDL internalization; and class V defect: no LDLR recycling. One might expect that both the class of LDLR defect as well as the precise mutation influences the severity of hypercholesterolemia on one hand and the response on drug treatment on the other. To clarify this question we studied the effect of the LDLR mutation p.W556R in two heterozygote subjects. Results We found that two heterozygote FH patients with the LDLR mutation p.W556R causing a class II LDLR defect (transport defective LDLR) respond exceedingly well to the treatment with simvastatin 40 mg/ezetimibe 10 mg. There was a LDL cholesterol decrease of 55 and 64%, respectively. In contrast, two affected homozygote p.W556R FH patients, in the mean time undergoing LDL apheresis, had no response to statin but a 15% LDL cholesterol decrease on ezetimibe monotherapy. Conclusions The LDLR mutation p.W556R is a frequent and severe class II defect for FH. The affected homozygote FH patients have a total loss of the functional LDLR and—as expected—do not respond on statin therapy and require LDL apheresis. In contrast, heterozygote FH patients with the same LDLR defect respond exceedingly well to standard lipid-lowering therapy, illustrating that the knowledge of the primary LDLR defect enables us to foresee the expected drug effects.

Lipids and lipoproteins in women.

AbstractAtherosclerosis and coronary artery disease (CAD) are the main causes of death in the Western world, for both men and women. However, in premenopausal women CAD is less frequent than in men, but in elderly women (e.g., > 75 years) myocardial infarction (MI) occurs even more often than in men. In summary, women suffer from CAD and MI but at a later age than men. Therefore it is important to observe and compare the cardiovascular risk factors in women and men. The typical CAD risk factors such as hyperlipidemia, smoking, arterial hypertension, diabetes mellitus, obesity, physical inactivity, and unhealthy nutrition are increasingly important for both genders. Many of these factors are comparable between men and women, but due to hormonal influences especially the lipoprotein metabolism shows some striking differences between men and women, but interestingly enough also between pre- and postmenopausal women. Therefore this paper will focus especially on the gender-specific differences in lipid metabolism as a potential target which might explain both the gender-specific and also pre- and postmenopausal differences in the occurrence of CAD.ZusammenfassungAtherosklerose und koronare Herzerkrankung (KHK) sind die häufigsten Todesursachen in den westlichen Industrienationen, und dies gilt sowohl für Männer als auch für Frauen. Allerdings ist die KHK bei jüngeren, prämenopausalen Frauen wesentlich seltener als bei Männern. Bei älteren Frauen (besonders bei > 75-jährigen) kommt es hingegen häufiger zum Herzinfarkt als bei älteren Männern. Insofern erkranken Frauen sehr wohl an KHK und Herzinfarkt, allerdings in einem späteren Alter. Daher erscheint es lohnenswert, kardiovaskuläre Risikofaktoren in ihrer unterschiedlichen Ausprägung bei Männern und Frauen zu erforschen. Die typischen Risikofaktoren der KHK wie Hyperlipidämie, Rauchen, arterielle Hypertonie, Diabetes mellitus, Übergewicht, körperliche Inaktivität und ungesunde Ernährung spielen in zunehmendem Maße für beide Geschlechter eine große Rolle. Viele dieser Risikofaktoren sind bei Männern wie bei Frauen mit vergleichbarer Bedeutung zu finden. Aufgrund der unterschiedlichen Hormonsituation finden sich aber beim Lipidstoffwechsel deutliche Unterschiede zwischen Mann und Frau sowie interessanterweise auch zwischen prä- und postmenopausalen Frauen. Daher wird sich dieser Beitrag mit den geschlechtsspezifischen Unterschieden des Lipidstoffwechsels beschäftigen. Hier liegt ein möglicher Ansatz zur Erklärung der unterschiedlichen kardiovaskulären Erkrankungsraten, sowohl im Vergleich zwischen Männern und Frauen als auch im Vergleich zwischen prä- und postmenopausalen Frauen.