[New AHA and ACC guidelines on the treatment of blood cholesterol to reduce atherosclerotic cardiovascular risk : Statement of the D•A•CH Society for Prevention of Cardiovascular Diseases, the Austrian Atherosclerosis Society and the Working Group on Lipids and Atherosclerosis (AGLA) of the Swiss Society for Cardiology]. / Neue AHA- und ACC-Leitlinie zur Risikoreduktion von Herz-Kreislauf-Erkrankungen durch Cholesterinsenkung : Stellungnahme der D•A•CH-Gesellschaft Prävention von Herz-Kreislauf-Erkrankungen e. V., der Österreichischen Atherosklerose Gesellschaft und der Arbeitsgruppe Lipide und Atherosklerose (AGLA) der Schweizer Gesellschaft für Kardiologie.

Guidelines for the reduction of cholesterol to prevent atherosclerotic vascular events were recently released by the American Heart Association and the American College of Cardiology. The authors claim to refer entirely to evidence from randomized controlled trials, thereby confining their guidelines to statins as the primary therapeutic option. The guidelines derived from these trials do not specify treatment goals, but refer to the percentage of cholesterol reduction by statin medication with low, moderate, and high intensity. However, these targets are just as little tested in randomized trials as are the cholesterol goals derived from clinical experience. The same applies to the guidelines of the four patient groups which are defined by vascular risk. No major statin trial has included patients on the basis of their global risk; thus the allocation criteria are also arbitrarily chosen. These would actually lead to a significant increase in the number of patients to be treated with high or maximum dosages of statins. Also, adhering to dosage regulations instead of cholesterol goals contradicts the principles of individualized patient care. The option of the new risk score to calculate lifetime risk up to the age of 80 years in addition to the 10-year risk can be appreciated. Unfortunately it is not considered in the therapeutic recommendations provided, despite evidence from population and genetic studies showing that even a moderate lifetime reduction of low-density lipoprotein (LDL) cholesterol or non-HDL cholesterol has a much stronger effect than an aggressive treatment at an advanced age. In respect to secondary prevention, the new American guidelines broadly match the European guidelines. Thus, the involved societies from Germany, Austria and Switzerland recommend continuing according to established standards, such as the EAS/ESC guidelines.

Expression of fat mobilizing genes in human epicardial adipose tissue.

BACKGROUND: Epicardial adipose tissue (EAT) mass correlates with metabolic syndrome and coronary artery disease (CAD). However, little is known about the expression of genes involved in triglyceride (TG) storage and mobilization in EAT. We therefore analyzed the expression of genes involved in fat mobilization in EAT in comparison to subcutaneous abdominal adipose tissue (AAT) in CAD patients and in controls. METHODS: EAT and AAT were obtained during coronary artery bypass graft (CABG) surgery from 16 CAD patients and from 14 non-CAD patients presenting for valve surgery. The state of atherosclerosis was assessed by angiography. RNA from tissues were extracted, reversibly transcribed and quantified by real time polymerase chain reaction (RT-PCR). The following genes were analyzed: perilipin-1 and -5 (PLIN1, PLIN5), lipoprotein lipase (LPL), hormone sensitive lipase (HSL), adipose triglyceride lipase (ATGL), comparative gene identification-58 (CIG-58), angiopoietin like protein 4 (ANGPTL4), in addition to interleukine-6 (IL-6), leptin (LEP) and adiponectin (ADPN). RESULTS: A significant expression of all listed genes could be observed in EAT. The relative expression pattern of the 10 genes in EAT was comparable to the expression in AAT, yet there was a significantly higher overall expression in AAT. The expression of the listed genes was not different between CAD patients and controls. CONCLUSION: It is suggested that the postulated difference in EAT volume between CAD patients and non-CAD patients is not caused by a differential mRNA expression of fat mobilizing genes. Further work on protein levels and enzyme activities will be necessary to get a complete picture.

Undifferentiated human mesenchymal stem cells (hMSCs) are highly sensitive to mechanical strain: transcriptionally controlled early osteo-chondrogenic response in vitro.

OBJECTIVE: Physical cues play a crucial role in skeletogenesis and osteochondral regeneration. Although human mesenchymal stem cells (hMSCs) offer considerable therapeutic potential, little is known about the molecular mechanisms that control their differentiation. We hypothesized that mechanical strain might be an inherent stimulus for chondrogenic and/or osteogenic differentiation in undifferentiated hMSCs, where c-Fos (FOS) might play a major role in mechanotransduction. METHOD: hMSCs from 10 donors were intermittently stimulated by cyclic tensile strain (CTS) at 3000 mustrain for a period of 3 days. Differential gene expression of strained and unstrained hMSCs was analysed by real-time RT-PCR for several marker genes, including the transcription factors FOS, RUNX2, SOX9, and others. Additionally, alkaline phosphatase activity (ALP) was determined kinetically. RESULTS: The application of CTS significantly stimulated the expression levels of the early chondrogenic and osteogenic marker genes (SOX9, LUM, DCN; RUNX2, SPARC, SPP1, ALPL); this was accompanied by stimulation of ALP activity (+38%+/-12 standard error of mean, P<0.05). Matrix analysis revealed that the osteo-chondrogenic response followed a coordinated expression pattern, in which FOS was attributed to early osteogenic but not chondrogenic differentiation. CONCLUSION: Undifferentiated hMSCs are highly sensitive to mechanical strain with a transcriptionally controlled osteo-chondrogenic differentiation response in vitro.

Diagnosis of families with familial hypercholesterolaemia and/or Apo B-100 defect by means of DNA analysis of LDL-receptor gene mutations.

BACKGROUND: One major problem of using hypercholesterolaemia alone as a primary criterion for diagnosing familial hypercholesterolaemia (FH) is that 15-40% of relatives may be misdiagnosed because plasma lipid levels in FH heterozygotes overlap with those in the general population. SETTING: General Hospital/University of Vienna, Department of Pediatrics, Outpatient lipid clinic. METHODS: As a part of the MED-PED (make early diagnosis-prevent early death) project we are currently investigating children, adolescents and their relatives who are suspected to be affected with FH in our out-patient clinic for metabolic diseases using MED-PED inclusion criteria and confirming the diagnosis by means of DNA analysis. PATIENTS: 263 patients with premature atherosclerosis and/or hypercholesterolaemia: 116 children (mean age 11.6 +/- 4.1 years; 57 girls and 59 boys) and 147 adults (64 women, mean age 41.5 +/- 13.7 years; 83 men, mean age 42.8 +/- 10.8 years). RESULTS: 119 patients with mutations have been detected; 56 children with either low density lipoprotein receptor (LDLR) and/or ApoB mutations (27 girls and 29 boys; mean total cholesterol (TC) 275 +/- 71 mg/dl, triglycerides (TG) 101 +/- 57 mg/dl, high-density lipoprotein cholesterol (HDL-C) 49 +/- 12 mg/dl, low-density lipoprotein cholesterol (LDL-C) 198 +/- 67 mg/dl) and one boy with a homozygous. LDLR mutation. A further 62 adults with LDLR and/or ApoB mutations were documented; 33 women (mean age 36.9 +/- 11.1 years; mean TC 283 +/- 76 mg/dl, TG 137 +/- 78 mg/dl, HDL-C 55 +/- 17 mg/dl, LDL-C 210 +/- 67 mg/dl) and 29 men (mean age 45.0 +/- 10.6 years; mean TC 301 +/- 87 mg/dl, TG 163 +/- 112 mg/dl, HDL-C 42 +/- 12 mg/dl, LDL-C 233 +/- 83 mg/dl). In 32 of these subjects (11 children (21%), 21 adults (42%)), serum lipid levels were lower than the diagnostic MED-PED limits adopted, so that they might have been misclassified without an additional DNA analysis. CONCLUSION: In our study, diagnosis of FH and related disorders (ApoB-100 defect) by means of conventional laboratory methods missed at least 21% in children and 42% in adults affected with LDLR and/or ApoB gene mutations. Genetic FH diagnosis provides a tool for specific diagnosis of mutation carrier status.

Therapy of hyper-Lp(a).

Lipoprotein (a) [Lp(a)] appears to be one of the most atherogenic lipoproteins. It consists of a low-density lipoprotein (LDL) core in addition to a covalently bound glycoprotein, apolipoprotein (a) [apo(a)]. Apo(a) exists in numerous polymorphic forms. The size polymorphism is mediated by the variable number of kringle-4 Type-II repeats found in apo(a). Plasma Lp(a) levels are determined to more than 90% by genetic factors. Plasma Lp(a) levels in healthy individuals correlate significantly high with apo(a) biosynthesis and not with its catabolism. There are several hormones known to have a strong impact on Lp(a) metabolism. In certain diseases, such as kidney disease, Lp(a) catabolism is impaired leading to up to fivefold elevations. Lp(a) levels rise with age but are otherwise influenced only little by diet and lifestyle. There is no safe and efficient way of treating individuals with elevated plasma Lp(a) concentrations. Most of the lipid-lowering drugs have either no significant influence on Lp(a) or exhibit a variable effect in patients with different forms of primary and secondary hyperlipoproteinemia. There is without doubt a strong need to concentrate on the development of specific medications to selectively target Lp(a) biosynthesis, Lp(a) assembly and Lp(a) catabolism. So far only anabolic steroids were found to drastically reduce Lp(a) plasma levels. This class of substance cannot, of course, be used for treatment of patients with hyper-Lp(a). We recommend that the mechanism of action of these drugs be studied in more detail and that the possibility of synthesizing derivatives which may have a more specific effect on Lp(a) without having any side effects be pursued. Other strategies that may be of use in the development of drugs for treatment of patients with hyper-Lp(a) are discussed in this review.

Human paraoxonase 1 gene polymorphisms and the risk of coronary heart disease: a community-based study.

Published data on the association between paraoxonase1 (PON1) polymorphisms and coronary heart disease (CHD) have yielded controversial results. The objective of this study was to determine the possible relationship between the two human PON1 amino acid variants, the Leu55Met and the Gln192Arg polymorphism, and the risk of CHD in a community-dwelling cohort of European ancestry. PON1 genotypes of 152 women and 151 men out of 1,998 randomly selected individuals aged 44-75 years were determined by polymerase chain reaction-based restriction enzyme digestion. Study participants underwent cardiological examination including a structured clinical interview, resting ECG, exercise testing and echocardiography. The diagnosis of CHD was based on history and/or appropriate findings during cardiac examination. Evidence for CHD was found in 43 (14.2%) study participants. The Leu/Leu (LL), Leu/Met (LM) and Met/Met (MM) genotypes at position 55 were noted in 131 (43.2%), 128 (42.2%) and 44 (14.5%) subjects; the Gln/Gln (QQ), Gln/Arg (QR) and Arg/Arg (RR) genotypes at codon 192 occurred in 167 (55.1%), 118 (38.9%) and 18 (5.9%) individuals, respectively. Homozygosity for the 55L-allele was significantly associated with CHD (p = 0.02), while the Gln192Arg polymorphism had no effect (p = 0.16). Logistic regression analysis demonstrated age (odds ratio 1.06/year), smoking (odds ratio 2.86), HDL cholesterol (odds ratio 0.94/mg/dl) and the paraoxonase LL genotype (odds ratio 2.25) to be significant predictors of CHD. These data suggest that the paraoxonase LL genotype at position 55 may present a risk factor for CHD.

Lipoprotein (a) binds and inactivates tissue factor pathway inhibitor: a novel link between lipoproteins and thrombosis.

Lipoprotein (a) [Lp(a)] has been associated with both anti-fibrinolytic and atherogenic effects. However, no direct link currently exists between this atherogenic lipoprotein and intravascular coagulation. The current study examined the binding and functional effects of Lp(a), its lipoprotein constituents, apoliprotein (a) [apo(a)] and low-density lipoprotein (LDL), and lysine-plasminogen (L-PLG), which shares significant homology with apo(a), on tissue factor pathway inhibitor (TFPI), a major regulator of tissue factor-mediated coagulation. Results indicate that Lp(a), apo(a), and PLG but not LDL bound recombinant TFPI (rTFPI) in vitro and that apo(a) bound to a region spanning the last 37 amino acid residues of the c-terminus of TFPI. The apparent binding affinity for TFPI was much higher for Lp(a) (KD approximately 150 nM) compared to PLG (KD approximately 800 nM) and nanomolar concentrations of apo(a) (500 nM) inhibited PLG binding to TFPI. Lp(a) also inhibited in a concentration-dependent manner rTFPI activity and endothelial cell surface TFPI activity in vitro, whereas PLG had no such effect. Moreover physiologic concentrations of PLG (2 microM) had no effect on the concentration-dependent inhibition of TFPI activity induced by Lp(a). In human atherosclerotic plaque, apo(a) and TFPI immunostaining were shown to coexist in smooth muscle cell-rich areas of the intima. These data suggest a novel mechanism whereby Lp(a) through its apo(a) moiety may promote thrombosis by binding and inactivating TFPI.

Mechanism of the interaction of beta(2)-glycoprotein I with negatively charged phospholipid membranes.

In an attempt to understand the multifunctional involvement of beta(2)-glycoprotein I (beta(2)GPI) in autoimmune diseases, thrombosis, atherosclerosis, and inflammatory processes, substantial interest is focused on the interaction of beta(2)GPI with negatively charged ligands, in particular, with acidic phospholipids. In this study, unilamellar vesicles composed of cardiolipin were used as in vitro membrane system to test and further refine a model of interaction based on the crystal structure of beta(2)GPI. The data suggest that beta(2)GPI anchors to the membrane surface with its hydrophobic loop adjacent to the positively charged lysine rich region in domain V. Subsequently, beta(2)GPI penetrates the membrane interfacial headgroup region as indicated by a restriction of the lipid side chain mobility, but without formation of a nonbilayer lipid phase. A structural rearrangement of beta(2)GPI upon lipid binding was detected by microcalorimetry and may result in the exposure of cryptic epitopes located in the complement control protein domains. This lipid-dependent conformational change may induce oligomerization of beta(2)GPI and promote intermolecular associations. Thus, the aggregation tendency of beta(2)GPI may serve as the basis for the formation of a molecular link between cells but may also be an essential feature for binding of autoantibodies and hence determine the role of beta(2)GPI in autoimmune diseases.

Adenovirus-mediated rescue of lipoprotein lipase-deficient mice. Lipolysis of triglyceride-rich lipoproteins is essential for high density lipoprotein maturation in mice.

Lipoprotein lipase (LPL) is the rate-limiting enzyme for the hydrolysis of triglycerides and the subsequent uptake of free fatty acids in extrahepatic tissues. Deficiency of LPL in humans (Type I hyperlipoproteinemia) is associated with massive chylomicronemia, low high density lipoprotein (HDL) cholesterol levels, and recurrent attacks of pancreatitis when not controlled by a strict diet. In contrast to humans, homozygous LPL knock-out mice (L0) do not survive suckling and die between 18 and 24 h after birth. In this study, an adenovirus-based protocol was utilized for the transient expression of LPL during the suckling period in an effort to rescue L0 mice. After a single intraperitoneal injection of 5x10(9) plaque-forming units of LPL-expressing virus immediately after birth, more than 90% of L0 mice survived the first days of life. 3% of L0 mice survived the entire suckling period and lived for up to 20 months, although LPL activity in mouse tissues and postheparin plasma was undetectable in all animals after 6 weeks of age. Adult LPL-deficient mice were smaller than their littermates until 2-3 months of age and exhibited very high triglyceride levels in the fed (4997 +/- 1102 versus 113.4 +/- 18.7 mg/dl) and fasted state (2007 +/- 375 versus 65.5 +/- 7.4 mg/dl). Plasma total cholesterol levels, free fatty acids, and ketone bodies were elevated in L0 mice, whereas plasma glucose was normal. Most strikingly, L0 mice lacked apoA-I-containing prebeta-HDL particles as well as mature HDL resulting in undetectable HDL cholesterol and HDL-apoA-I levels. HDL deficiency in plasma was evident despite normal apoA-I mRNA levels in the liver and normal apoA-I protein levels in plasma, which were predominantly found in the chylomicron fraction. The absence of prebeta-HDL and mature HDL particles supports the concept that the lipolysis of triglyceride-rich lipoproteins is an essential step for HDL maturation.

Angiotensinogen polymorphism M235T, carotid atherosclerosis, and small-vessel disease-related cerebral abnormalities.

The angiotensinogen M235T polymorphism has been linked to hypertension and cardiovascular disease. We studied the role of this polymorphism as a risk factor for carotid atherosclerosis and small-vessel disease-related brain abnormalities. A total of 431 randomly selected community-dwelling subjects without clinical evidence for strokes underwent angiotensinogen genotyping and carotid Duplex scanning; 1.5-T brain magnetic resonance imaging (MRI) was done in 396 individuals. At 3-year follow-up, we reexamined 343 and 267 study participants by ultrasound and brain MRI, respectively. Carotid atherosclerosis was graded on a 5-point scale. Small-vessel disease-related brain abnormalities were deep or subcortical white matter lesions or lacunes. Progression of carotid atherosclerosis and MRI findings was rated by direct imaging comparison by 3 independent raters. The M/M, M/T, and T/T genotypes were seen in 20.9%, 52.9%, and 18.1% of subjects, respectively. The M235T polymorphism was neither associated with baseline carotid findings nor with progression of carotid atherosclerosis. There was a trend toward more frequent small-vessel disease-related MRI abnormalities in the T/T than in the other genotypes at the baseline examination. Progression of brain lesions occurred significantly more commonly in T/T than in M/M and M/T carriers (P<0.001). Logistic regression analysis identified the T/T genotype (odds ratio, 3.19; P=0.002) and arterial hypertension (odds ratio, 3.06; P=0.03) as significant independent predictors of lesion progression. These data suggest that the angiotensinogen T/T genotype at position 235 is a genetic marker for brain lesions from and progression of small vessel disease but not for extracranial carotid atherosclerosis.

Role of various tissues in apo(a) fragmentation and excretion of fragments by the kidney.

BACKGROUND: Lipoprotein(a) [Lp(a)] is an atherothrombotic plasma lipoprotein with unknown function. Little is known about the catabolism of this lipoprotein, in particular the steps related to apolipoprotein(a) [apo(a)] fragmentation and excretion by the kidney. MATERIAL AND METHODS: High plasma levels (up to 9 mg dL(-1)) of the N-terminal fragment of apo(a) were expressed in mice by adenovirus mediated gene transfer. Plasma of such N-apo(a) mice was injected into acceptor mice and the fragmentation and urinary secretion of N-apo(a) were followed by immunochemical techniques. RESULTS: Mice transduced with N-Ad expressed apo(a)-fragments with 3-11 kringle-IV (KIV) repeats. Injection of N-apo(a)-plasma from donor mice into acceptor mice resulted in fragmentation of N-apo(a)s with 3-11 KIVs yielding smaller peptides down to 2 KIVs. Secretion of N-apo(a)-fragments with 2 to maximally 6 KIVs into urine occurred as early as 2 min after injection. Immunohistochemical studies of kidney suggested filtration as a mechanism of apo(a)-fragment excretion. When N-apo(a) was incubated in vitro with various tissues from perfused mice, skeletal muscle and kidney followed by liver and spleen contributed to fragmentation. Tissues from unperfused organs, or the addition of normal mouse plasma, caused marked reduction in N-apo(a) fragmentation. EDTA, and not aprotinin or leupeptin, prevented apo(a) cleavage. CONCLUSION: Here we provide evidence that apo(a) is cleaved by metalloproteinases located on skeletal muscle, kidney and other organs. Small apo(a)-fragments up to a size of 6 KIVs are excreted into urine, yet a major portion of apo(a) fragments is removed from circulation extrarenally.

Lipoprotein lipase mediates the uptake of glycated LDL in fibroblasts, endothelial cells, and macrophages.

The nonenzymatic glycation of LDL is a naturally occurring chemical modification of apolipoprotein (apo)-B lysine residues by glucose. Once glycated, LDL is only poorly recognized by lipoprotein receptors including the LDL receptor (LDL-R), the LDL-R-related protein (LRP), and scavenger receptors. Glycated LDL (gLDL) is a preferred target for oxidative modifications. Additionally, its presence initiates different processes that can be considered "proatherogenic." Thus, LDL glycation might contribute to the increased atherosclerotic risk of patients with diabetes and familial hypercholesterolemia. Here we investigate whether lipoprotein lipase (LPL) can mediate the cellular uptake of gLDL. The addition of exogenous LPL to the culture medium of human skin fibroblasts, porcine aortic endothelial cells, and mouse peritoneal macrophages enhanced the binding, uptake, and degradation of gLDL markedly, and the relative effect of LPL on lipoprotein uptake increased with the degree of apoB glycation. The efficient uptake of gLDL by LDL-R-deficient fibroblasts and LRP-deficient Chinese hamster ovary cells in the presence of LPL suggested a mechanism that was independent of the LDL-R and LRP. In macrophages, the uptake of gLDL was also correlated with their ability to produce LPL endogenously. Mouse peritoneal macrophages from genetically modified mice, which lacked LPL, exhibited a 75% reduction of gLDL uptake compared with normal macrophages. The LPL-mediated effect required the association of the enzyme with cell surface glycosaminoglycans but was independent of its enzymatic activity. The uptake of gLDL in different cell types by an LPL-mediated process might have important implications for the cellular response after gLDL exposure as well as the removal of gLDL from the circulation.

Adenovirus-mediated apo(a)-antisense-RNA expression efficiently inhibits apo(a) synthesis in vitro and in vivo.

Apo(a) is a very atherogenic plasma protein without apparent function, which is highly expressed in humans. The variation in plasma Lp(a) concentration among individuals is considerable. Approximately 10-15% of the white population exhibit plasma Lp(a) concentrations above the atherogenic cut-off value of approximately 30 mg/dl. Since there is currently no safe way of treating those patients with drugs, we have tested the possibility of interfering with apo(a) biosynthesis by adenovirus-mediated expression of antisense apo(a) mRNA comprising the 5' UTR, the signal sequence and the first three kringles of native apo(a). Transduction of rat hepatoma McA RH 7777 cells which stably expressed apo(a) with 18 kringle IV (KIV) domains with apo(a)-antisense adenovirus (AS-Ad) at multiplicity of infection (MOI) of 30 reduced apo(a) synthesis to 23% as compared with control cells. As apo(a) is not synthesized in laboratory animals, we induced biosynthesis of the N-terminal fragments of apo(a) in mice by adenovirus-mediated gene transfer. Cotransduction of these mice with AS-Ad, which expressed up to eight times higher amounts of apo(a) than stable transgenic apo(a) mice, led to an almost complete disappearance of apo(a) from plasma. We conclude that the proposed AS-construct is very efficient in interfering with apo(a) biosynthesis in vivo. The strategy of inducing the synthesis of a nonexpressed protein followed by knocking it out by AS technology may also be applicable to other systems.

Impact of apolipoprotein(a) on in vitro angiogenesis.

Angiostatin, which consists of the kringle I-IV domains of plasminogen and which is secreted into urine, is an efficient inhibitor of angiogenesis and tumor growth. Because N-terminal apolipoprotein(a) [apo(a)] fragments, which also contain several types of kringle IV domains, are found in urine as well, we evaluated the potential angiostatic properties of these urinary apo(a) fragments and of a recombinant form of apo(a) [r-apo(a)]. We used human microvascular endothelial cell (hMVEC)-based in vitro assays of tube formation in 3-dimensional fibrin matrixes. Purified urinary apo(a) fragments or r-apo(a) inhibited the basic fibroblast growth factor/tumor necrosis factor-alpha-induced formation of capillary-like structures. At concentrations varying from 0.2 to 10 microgram/mL, urinary apo(a) fragments inhibited tube formation by as much as 70%, whereas there was complete inhibition by r-apo(a). The highest concentrations of both inhibitors also reduced urokinase plasminogen activator production of basic fibroblast growth factor-induced hMVEC proliferation. The inhibitors had no effect on plasminogen activator inhibitor-1 expression. If our in vitro model for angiogenesis is valid for the in vivo situation as well, our data point toward the possibility that apo(a) may also be physiologically operative in modulating angiogenesis, as the concentration of free apo(a) found in humans exceeds that tested herein.

Urinary excretion of apolipoprotein(a): relation to other plasma proteins.

The atherogenic lipoprotein Lp(a) consists of an LDL-like core and apo(a), linked to apoB via a thiol bridge. Apo(a) fragments ranging in size from 60 to 220 kDa are excreted into urine and the excretion rate correlates significantly with the plasma levels of Lp(a). In order to study the interrelationship of apo(a) secretion with that of other plasma proteins, urinary apo(a) and protein secretion of five probands were followed for 24 h at different urinary densities. The excretion rate of apo(a) fragments, despite their high molecular weight, was highest, followed by apoD, orosomucoid, albumin and beta(2)-glycoprotein-I (beta2-GI) and plasminogen (1.58, 0.87, 0.095, 0.027, 0.013 and <0.001%/day, respectively). There was a highly significant correlation between apo(a), apoD and beta2-GI concentrations but not with albumin and orosomucoid concentrations in urine. The only protein that was fragmented in urine was apo(a) while the other proteins had molecular weights comparable to those in plasma. We conclude that a previously suggested fragmentation of apo(a) by the kidney is not a rate-limiting step in its excretion. Since plasminogen, another kringle-IV-containing plasma compound, and fragments thereof, are undetectable in urine under identical experimental conditions, it is very unlikely that the characteristic kringle structure is responsible for the high excretion rate of apo(a).

Lipoprotein (a) up-regulates the expression of the plasminogen activator inhibitor 2 in human blood monocytes.

Elevated plasma lipoprotein (a) (Lp[a]) and cardiac events show a modest but significant association in various clinical studies. However, the influence of high Lp(a) on the gene expression in blood monocytes as a major cell involved in atherogenesis is poorly described. To identify genes influenced by elevated serum Lp(a), the gene expression was analyzed on a complementary DNA microarray comparing monocytes from a patient with isolated Lp(a) hyperlipidemia and coronary heart disease with monocytes from a healthy blood donor with low Lp(a). By using this approach, numerous genes were found differentially expressed in patient-versus-control monocytes. Verification of these candidates by Northern blot analysis or semiquantitative polymerase chain reaction in monocytes from additional patients with Lp(a) hyperlipidemia and healthy blood donors with elevated Lp(a) confirmed a significant induction of plasminogen activator inhibitor type 2 (PAI-2) messenger RNA (mRNA) in monocytes from male, but not from female, individuals with high Lp(a), indicating that this observation is gender specific. This led also to increased intracellular and secreted PAI-2 protein in monocytes from male probands with Lp(a) hyperlipidemia. Plasminogen activator inhibitor type 1 (PAI-1) mRNA was found suppressed only in the patients' monocytes and not in healthy probands with high Lp(a) levels. Purified Lp(a) induced PAI-2 mRNA and protein and reduced PAI-1 expression in monocytes isolated from various controls. The finding that PAI-2 is elevated in monocytes from male patients with isolated Lp(a) hyperlipidemia and male healthy probands with high Lp(a) and that purified Lp(a) up-regulates PAI-2 in control monocytes in vitro indicate a direct, but gender-specific, effect of Lp(a) for the induction of PAI-2 expression.

Angiotensinogen gene promoter haplotype and microangiopathy-related cerebral damage: results of the Austrian Stroke Prevention Study.

BACKGROUND AND PURPOSE: Microangiopathy-related cerebral damage (MARCD) is a common finding in the elderly. It may lead to cognitive impairment and gait disturbances. Arterial hypertension and age are the most important risk factors. We assessed the association between MARCD and sequence alterations in the promoter region of the angiotensinogen (AGT) gene. METHODS: We studied 410 randomly selected community-dwelling individuals aged 50 to 75 years. MARCD was defined as early confluent or confluent white matter hyperintensities or lacunes on a 1.5-T MRI. The AGT promoter was analyzed by temporal temperature gradient gel electrophoresis and automated sequencing. RESULTS: We detected 4 polymorphic sites, at positions -6, -20, -153, and -218. They created 5 haplotypes, which we coded as A (-6:g, -20:a, -153:g, -218g), B (-6:a, -20:c, -153:g, -218:g), C (-6:a, -20:c, -153:a, -218:g), D (-6:a, -20:a, -153:g, -218:g), and E (-6:a, -20:a, -153:g, -218:a). MARCD was seen in 7 subjects (63.6%) carrying 2 copies of the B haplotype (B/B), in 12 subjects (38.7%) carrying 1 copy of the B haplotype in the absence of the A haplotype (B+/A-), but in only 70 subjects (19.0%) in the remaining cohort (P:<0.001). The odds ratios for the B/B and the B+/A- genotypes were 8.0 (95% CI, 2.1 to 31.1; P:=0.003) and 1.8 (95% CI, 0.8 to 4.2; P:=0.14) after adjustment for possible confounders. CONCLUSIONS: The B haplotype of the AGT promoter in the absence of the wild-type A haplotype might represent a genetic susceptibility factor for MARCD.

Silent mutations in secondary Shine-Dalgarno sequences in the cDNA of human serum amyloid A4 promotes expression of recombinant protein in Escherichia coli.

The serum amyloid A (SAA) superfamily comprises a number of differentially expressed genes with a high degree of homology in mammalian species. SAA4, an apolipoprotein constitutively expressed only in humans and mice, is associated almost entirely with lipoproteins of the high-density range. The presence of SAA4 mRNA and protein in macrophage-derived foam cells of coronary and carotid arteries suggested a specific role of human SAA4 during inflammation including atherosclerosis. Here we underline the importance of ribosome binding site (rbs)-like sequences (also known as Shine-Dalgarno sequences) in the SAA4 cDNA for expression of recombinant SAA4 protein in Escherichia coli. In contrast to rbs sequences coded by the expression vectors, rbs-like sequences in the cDNA of target protein(s) are known to interfere with protein translation via binding to the small 16S ribosome subunit, yielding low or even no expression. Here we show that PCR mutations of two rbs-like sequences in the human SAA4 cDNA promote expression of considerable amounts of recombinant SAA4 in E.coli.

Apo(a)-kringle IV-type 6: expression in Escherichia coli, purification and in vitro refolding.

Lipoprotein (a) [Lp(a)] belongs to the class of highly thrombo-atherogenic lipoproteins. The assembly of Lp(a) from LDL and the specific apo(a) glycoprotein takes place extracellularly in a two-step process. First, an unstable complex is formed between LDL and apo(a) due to the interaction of the unique kringle (K) IV-type 6 (T6) in apo(a) with amino groups on LDL, and in the second step this complex is stabilized by a disulfide bond between apo(a) KIV-T9 and apoB(100). In order to understand this process better, we overexpressed and purified apo(a) KIV-T6 in Escherichia coli. Recombinant KIV-T6 was expressed as a His-tag fusion protein under control of the T7 promoter in BL21 (DE3) strain. After one-step purification by affinity chromatography the yield was 7 mg/l of bacterial suspension. Expressed fusion apo(a) KIV-T6 was insoluble in physiological buffers and it also lacked the characteristic kringle structure. After refolding using a specific procedure, high-resolution (1)H-NMR spectroscopy revealed kringle structure-specific signals. Refolded KIV-T6 bound to Lys-Sepharose with a significantly lower affinity than recombinant apo(a) (EC(50) with epsilon-ACA 0.47 mM versus 2-11 mM). In competition experiments a 1000-fold molar excess of KIV-T6 was needed to reach 60% inhibition of Lp(a) assembly.