High throughput echocardiography in conscious mice: training and primary screens.

PURPOSE: Genetic engineering techniques led to an exponential increase in the number of transgenic and knock-out mouse models. For many genetically modified mice, high throughput echocardiography is an essential part of a systematic screening workflow. Many researchers perform mouse echocardiography in conscious animals to avoid anesthesia-induced impairment of cardiac function. However, it has been controversially discussed whether mice need to be habituated to handling before their cardiac function can be assessed. The aim of this study was to test the influence of training on parameters assessed during conscious mouse echocardiography. In addition, we tested whether a simple and fast echocardiography protocol has sufficient sensitivity and specificity for primary screening. MATERIALS AND METHODS: Examined parameters include fractional shortening, heart rate and respiratory rate. A total of 139 mice were examined in this study with a total of 587 echocardiograms. 103 mice were examined on five consecutive days (with examinations on day 1 - 4 regarded as training), 36 mice were only examined on day 1 and 5. RESULTS: Fractional shortening, heart rate and respiratory rate did not show any statistically significant difference between day 1 and day 5 in both groups. The sensitivity and specificity of fractional shortening assessment for predicting a homozygote knock out genotype were 86 % and 97 %, respectively. CONCLUSION: We conclude that conscious mouse echocardiography can be performed in untrained mice. Fractional shortening measurements may suffice for correct phenotyping in a high throughput setting.

[New approaches and indications for the analysis of platelet function in cardiology]. / Neue Ansätze und Indikationen zur Plättchenfunktionsdiagnostik in der Kardiologie.

Inhibiting platelet function is a key therapeutic principle in cardiology because platelets play a pivotal role in triggering cardiovascular events. In addition to acetylsalicylic acid, a cyclooxygenase inhibitor, ADP-receptor blockers are frequently used for anti-platelet therapy. This therapy does not abolish platelet activation and aggregation. Platelets may still be activated by alternative routes such as the thrombin receptor-mediated pathway. New, more potent inhibitors of platelet function continue to lower the risk of ischaemic events but several trials and clinical registries have also shown that this advantage was frequently offset by an increased risk of bleeding complications. As a consequence, the individual risk of ischaemia and bleeding of a patient must be taken into consideration to select the platelet inhibitor offering the best benefit-risk ratio. Modern laboratory diagnostics may help to achieve this goal by complementing functional platelet tests with pharmacogenomic analyses consistent with the idea of "personalized medicine".

Complex genetic control of HDL levels in mice in response to an atherogenic diet. Coordinate regulation of HDL levels and bile acid metabolism.

Inbred strains of mice differ in susceptibility to atherogenesis when challenged with a high fat, high cholesterol diet containing 0.5% cholic acid. Studies of recombinant inbred (RI) strains derived from the susceptible strain C57BL/6J (B6) and the resistant strains C3H/HeJ (C3H) and BALB/cJ have revealed an association between fatty streak lesion size and a decrease in high density lipoprotein (HDL) levels on the diet. To better understand the genetic factors contributing to HDL metabolism and atherogenesis in response to the diet, we studied mice derived from an intercross between B6 and C3H using a complete linkage map approach. A total of 185 female progeny were typed for 134 genetic markers spanning the mouse genome, resulting in an average interval of about 10 cM between markers. A locus on distal chromosome 1 containing the apolipoprotein AII gene was linked to HDL-cholesterol levels on both the chow and the atherogenic diets, but this locus did not contribute to the decrease in HDL-cholesterol in response to the diet. At least three distinct genetic loci, on chromosomes 3, 5, and 11, exhibited evidence of linkage to a decrease in HDL-cholesterol after a dietary challenge. Since a bile acid (cholic acid) is required for the diet induced changes in HDL levels and for atherogenesis in these strains, we examined cholesterol-7-alpha hydroxylase (C7AH) expression. Whereas B6 mice exhibited a large decrease in C7AH mRNA levels in response to the diet, C3H showed an increase. Among the intercross mice, multiple loci contributed to the regulation of C7AH mRNA levels in response to the diet, the most notable of which coincided with the loci on chromosomes 3, 5, and 11 controlling HDL levels in response to the diet. None of these loci were linked to the C7AH structural gene which we mapped to proximal chromosome 4. These studies reveal coordinate regulation of C7AH expression and HDL levels, and they indicate that the genetic factors controlling HDL levels are more complex than previously suggested by studies of RI strains. Furthermore, we observed that two of the loci for C7AH expression contributed to differences in gallstone formation between these strains.

[Platelet inhibitors from a cardiological viewpoint: evidence based therapy and new concepts for the acute coronary syndrome without ST-segment elevation]. / Plättchenhemmer aus kardiologischer Sicht. Evidenzbasierte Therapie und neue Ansätze beim akuten Koronarsyndrom ohne ST-Streckenhebung.

The pathophysiology of the acute coronary syndrome is related to a vulnerable, inflammatory plaque promoting activation and aggregation of platelets. Therefore, the inhibition of platelet function is a primary goal of the treatment of the acute coronary syndrome. In recent years this field has made substantial progress, which is reflected by frequently updated treatment guidelines. New dose regimens and therapeutic strategies, but also innovative pharmacological principles evolved recently. Some of these novel drugs are currently tested in clinical trials. Increasing experience with the currently used platelet inhibitors also revealed potential shortcomings of these substances, such as drug resistance. This review will give a cardiological perspective of some important clinical aspects and new developments in the field of platelet inhibitors.

New Dyscalc loci for myocardial cell necrosis and calcification (dystrophic cardiac calcinosis) in mice.

Dystrophic cardiac calcinosis (DCC) occurs among certain inbred strains of mice and involves necrosis and subsequent calcification as response of myocardial tissue to injury. Using a complete linkage map approach, we investigated the genetics of DCC in an F(2) intercross of resistant C57BL/6J and susceptible C3H/HeJ inbred strains and identified previously a major predisposing quantitative trait locus (QTL), Dyscalc1, on proximal chromosome 7. Analysis of inheritance suggested, however, that DCC is influenced by additional modifier QTL, which have as yet not been mapped. Here, we report the identification by composite interval mapping of the DCC loci Dyscalc2, Dyscalc3, and Dyscalc4 on chromosomes 4, 12 and 14, respectively. Together, the four Dyscalc loci explained 47% of the phenotypic variance of DCC, which was induced by a high-fat diet. Additive epistasis between Dyscalc1 and Dyscalc2 enhanced DCC. Examining recombinant inbred strains, we propose a 10-cM interval containing Dyscalc1 and discuss potential candidate genes.

Vitamins C, E and A and heme oxygenase in rats fed methyl/folate-deficient diets.

There is evidence that the development of hepatocarcinoma in rats fed a methyl-deficient diet is associated with oxidative stress. We investigated, therefore, whether the tissue concentrations of the antioxidant vitamins ascorbic acid (AA) and alpha- and gamma-tocopherol (T) are altered in methyl/folate deficiency. We also measured retinol concentrations in tissues and hepatic mRNA expression of heme oxygenase (HO1). A 6% gelatin, 6% casein diet, devoid of choline and folate (CFD) was selected based on the high rate of tumor development in rats fed this diet. Spectrophotometric measurement of AA and HPLC determination of tissue T and retinol showed decreased concentrations of AA in blood; alpha- and gamma-T in lung, heart and plasma, alpha-T and retinol in liver; retinol in lung; and increased expression of hepatic HO1 mRNA. Similar alterations in tissue vitamin concentrations were found when the CFD diet devoid of niacin (CFND) was fed. Reducing alpha-T in the CFND diet (CFNED) further decreased hepatic alpha-T concentrations. These results show that chronic methyl/folate deficiency is associated with a compromised antioxidant defense system.

Study of causes underlying the low atherosclerotic response to dietary hypercholesterolemia in a selected strain of rabbits.

We have recently characterized a strain of rabbits that shows a low atherosclerotic response (LAR) to dietary hypercholesterolemia in contrast to the usual high atherosclerotic response (HAR) of rabbits [1]. Presently, we have focused on three well established and important stages of atherogenesis, i.e., monocyte adhesion to endothelium, cell mediated peroxidative modification of lipoproteins and induction of a receptor that recognizes modified low density lipoprotein (LDL). The results obtained show that (1) beta-very low density lipoprotein (beta-VLDL) from LAR and HAR rabbits enhanced monocyte adhesion to endothelial cells to the same extent; (2) Cell mediated peroxidation of LDL and beta-VLDL, tested by loss of alpha-tocopherol and formation of thiobarbituric acid reacting substances (TBARS), was compared using macrophages, fibroblasts and smooth muscle cells (SMC) of LAR and HAR rabbits and no significant differences were found; (3) Induction of scavenger receptor by phorbol ester (phorbol 12-myristate 13-acetate (PMA)) and platelet-derived growth factor-BB (PDGF-BB) was determined in SMC or fibroblasts from LAR and HAR rabbits using 1,1'-dioctadecyl-3,3,3',3'-tetramethylindocarbocyanine perchlorate-acetylated LDL (DiL-acLDL). We found a significantly higher uptake of DiI-acLDL in SMC and fibroblasts derived from HAR rabbits as compared with cells from LAR rabbits. Similar results were also obtained with [125I]-acLDL in fibroblasts from LAR and HAR rabbits with respect to cellular lipoprotein degradation after PMA pretreatment. Even though the attenuated atherosclerotic response to hypercholesterolemia of LAR rabbits may have multiple underlying causes, the most prominent so far is an apparent difference in inducibility of scavenger receptor in SMC and fibroblasts.

Neuronal network analysis of serum electrophoresis.

AIMS: To advise a system of neuronal networks which can classify the densitometric patterns of serum electrophoresis. METHODS: Digitised data containing 83 normal and 132 pathological serum protein electrophoresis patterns were presented to four neuronal networks containing 1900 neurons. Network 1 evaluates the integrated values of the albumin, alpha 1, alpha 2, beta and gamma fractions together with total protein (Biuret method). Networks 2, 3, and 4 analyse the shape of the albumin, beta and gamma fractions. To increase the sensitivity for the detection of monoclonal gammopathies a Fourier transformation was applied to the beta and gamma fractions. RESULTS: After a learning period of 20 minutes (back-propagation learning algorithm) the system was tested with a set of electrophoresis patterns comprising 446 routinely collected samples. It differentiated between physiological and pathological curves with a sensitivity of 97.5% and a specificity of 98.8%, with 86% correct diagnoses. All monoclonal gammopathies were recognised by the Fourier detector. CONCLUSIONS: Neuronal networks could be useful for certain medical uses. Unlike rule based systems, neuronal networks do not have to be programmed but have the capacity to "learn" quickly.

[Accumulation of lipid particles in Bruch's membrane of LDL receptor knockout mice as a model of age-related macular degeneration]. / Akkumulation von Lipidpartikeln in der Bruch-Membran von LDL-Rezeptor-defizienten Mäusen als Modell für die altersbezogene Makuladegeneration.

BACKGROUND: Atherosclerosis is a suspected risk factor for the development of neovascular age-related macular degeneration (AMD). METHODS: We used a well-established murine knockout model with low-density lipoprotein (LDL) receptor deficiency for atherosclerotic vascular pathogenesis to evaluate changes in Bruch's membrane due to high cholesterol levels. Blood cholesterol levels were modified by the diet fed (standard rodent diet or high-fat diet western type). Animals were sacrificed and plasma cholesterol levels were determined. Eyes were examined by transmission electron microscopy (TEM). RESULTS: Plasma total cholesterol levels were highest in LDL receptor-deficient mice after high-fat diet and elevated in LDL receptor-deficient mice after standard diet compared to control mice with and without special high-fat diet. While Bruch's membranes of control animals did not exhibit any visible changes by TEM even after a high-fat diet, membrane-bound translucent particles were seen in all membranes in knockout mice. The amount of these particles was substantially increased and membranes were thickened in knockout animals following high-fat diet with additional deposits of non-membrane-bound particles. CONCLUSION: LDL receptor-deficient mice exhibited a degeneration of Bruch's membrane with accumulation of lipid particles, which is further increased after fat intake due to elevated blood lipid levels. In our opinion, this animal model is suitable for investigating more aspects in the pathogenesis of neovascular AMD.

Telmisartan improves absolute walking distance and endothelial function in patients with peripheral artery disease.

BACKGROUND: Peripheral artery disease (PAD) is associated with high cardiovascular mortality and a poor quality of life. The AT1-receptor blocker telmisartan has been shown to have pleiotropic effects and it may also improve endothelial function. The aim of this study was to analyze the effects of telmisartan on absolute walking distance (WD) and endothelial function in patients with PAD. METHODS: In a single centre, single-blinded, prospective study, 36 patients with PAD at stage Fontaine II or higher and mild to moderate arterial hypertension were treated with telmisartan 40/80 mg once daily or placebo for 12 months. Primary endpoint was the improvement of the absolute treadmill WD. Flow-mediated vasodilation (FMD), carotid intima-media thickness (IMT), ankle-brachial index (ABI) and disease-related quality of life (DRQL) were examined as well. RESULTS: After 12 months, maximum WD increased by 26% in the telmisartan group (P < 0.001). However, in the placebo group it was comparable to baseline. FMD rose by 40% in the telmisartan group while it deteriorated in the placebo group (P < 0.001). IMT and ABI were comparable in both groups at baseline and did not change considerably after 12 months. In non-diabetic patients (72.2%), the ABI did not change in the placebo group, whereas it increased by 11% in the telmisartan group (P < 0.001). While the DRQL remained stable in the telmisartan group, placebo treatment was associated with a marked deterioration (P < 0.01). CONCLUSION: Telmisartan improves WD and endothelial function, the ABI in non-diabetic patients and it may prevent further loss of quality of life in patients with advanced PAD.

Radial artery thrombosis following transradial coronary angiography: incidence and rationale for treatment of symptomatic patients with low-molecular-weight heparins.

BACKGROUND: Transradial access for diagnostic and therapeutic coronary angiography gains more and more popularity because of its advantages over the femoral approach, enhancing patient comfort, reducing bleeding complications and duration of hospital stay. However, these benefits are overshadowed by an increased rate of radial artery (RA) occlusion. There are little data regarding the exact incidence, potential predictors and outcome of post-procedural RA occlusions. Furthermore, there is no clear evidence for the optimal treatment of this complication. METHODS: In a single-centre prospective observational study, 488 consecutive patients were evaluated by ultrasound the day after transradial cardiac catheterization for signs of RA occlusion. Symptomatic patients with sonographically identified radial artery thrombosis underwent treatment with low-molecular-weight heparin (LMWH) for 4 weeks. Asymptomatic patients did not receive anticoagulation therapy. The primary endpoint was the patency rate of the radial artery at 4 weeks of follow-up. RESULTS: Radial artery thrombosis was found in 51 of 488 (10.5%) patients 1 day after transradial cardiac catheterization. 30 (58.8%) patients showed symptoms on access site, whereas 21 (41.2%) did not show any symptoms. After 4 weeks, 26 (86.7%) of the symptomatic patients showed a partial or complete recanalization of the radial artery after treatment with LMWH, compared with 4 (19.1%) of the asymptomatic patients without anticoagulation (P < 0.001). CONCLUSION: Radial artery thrombosis is a frequent complication after transradial coronary angiography. Incidence of RA occlusion is underestimated due to the often asymptomatic clinical course. Treatment of symptomatic RA occlusion with low-molecular-weight heparins significantly increases patency rates after 4 weeks.

Effect of stress-induced reversible ischemia on serum concentrations of ischemia-modified albumin, natriuretic peptides and placental growth factor.

OBJECTIVE: There is controversy whether new biomarkers are able to identify myocardial ischemia in the absence of myonecrosis. METHOD: We measured NT-pro BNP, NT-pro ANP, ischemia-modified albumin (IMA) and placental growth factor (PlGF) in patients undergoing nuclear stress testing for suspected ischemic heart disease. A thallium scan was used for detection of reversible myocardial ischemia and cardiac troponin T (cTnT) for exclusion of stress-induced myonecrosis. Of 195 patients, 24 with reversible and 62 with no perfusion defect were included in the analysis. Plasma levels were measured before, 18 min and 4 h after stress testing. RESULTS: Of the 86 patients, 52 received an exercise stress and 34 dipyridamol. New myonecrosis indicated by cTnT could be excluded in all patients. Plasma levels of NT-pro BNP and NT-pro ANP before testing were significantly higher in patients who later developed reversible perfusion defects (NT-pro BNP 139.00 (58.25/367.01) pg/mL vs 327.45 (120.50/972.85) pg/mL, p<0.05; NT-pro ANP 732.5 (470.0/1220.0) pg/mL vs 1470.0 (694.0/1910.0) pg/mL, p<0.05). Plasma levels of NT-pro BNP, NT-pro ANP and PIGF did not change significantly after stress testing, IMA levels rose significantly after 4 h in patients with and without reversible perfusion defects. CONCLUSION: The elevation of NTpro BNP and NT-pro ANP at baseline may represent the cumulative effect of repeated bouts of myocardial ischemia. A single brief episode of provoked ischemia does not cause a significant increase of the measured biomarkers beside from IMA after exercise stress test potentially indicating skeletal muscle ischemia.

Hyperlipoprotein(a)aemia in nephrotic syndrome.

The nephrotic syndrome is frequently associated with hyperlipidaemia and hyperfibrinogenaemia, leading to an increased coronary and thrombotic risk, which may be enhanced by high lipoprotein (a) [Lp(a)] concentrations. We followed the quantitative and qualitative pattern of plasma lipoproteins over 18 months in a patient with nephrotic syndrome suffering from premature coronary artery disease and with elevated level of Lp(a) (470 mg dL-1). Analysis of kinetic parameters after heparin-induced extracorporeal plasma apheresis revealed a reduced fractional catabolic rate for both low-density lipoprotein (LDL) and Lp(a). After improvement of the nephrotic syndrome, Lp(a) decreased to 169 mg dL-1 and LDL concentrations were normalized. The decrease of Lp(a) was associated with an increase in plasma albumin concentrations. Analysis of apo(a) isoforms in the patient showed the presence of isoform S2 (alleles 10 and 19). Consequently, the authors' present strategy is to normalize the elevated Lp(a) and fibrinogen levels. For this purpose heparin-mediated extracorporeal LDL precipitation (HELP) apheresis is a promising regimen, helping to reduce the thrombotic risk and prevent coronary and graft atherosclerosis as well as the progression of glomerulosclerosis in our patient.

Biliary cholesterol hypersecretion in gallstone-susceptible mice is associated with hepatic up-regulation of the high-density lipoprotein receptor SRBI.

Enhanced hepatocellular trafficking of cholesterol to the bile canaliculus and cholesterol hypersecretion appears critical for gallstone formation. Therefore, we studied in more detail the hepatic cholesterol transport pathways in a mouse model of cholesterol gallstone disease. Biliary lipid secretion rates, plasma lipoprotein levels, hepatic expression of lipoprotein receptors, lipid regulatory enzymes, and putative cholesterol transporting proteins were analyzed in gallstone-susceptible C57L/J and gallstone-resistant AKR/J mice, which were fed a lithogenic diet. Biliary cholesterol hypersecretion in C57L mice was associated with decreased plasma high-density lipoprotein (HDL) cholesterol levels and significant hepatic induction of the HDL receptor (SRBI) and cholesteryl ester hydrolase. In response to the lithogenic diet, fatty-acid binding protein of liver (FABPL) was markedly induced in both mouse strains. Caveolin 1 was elevated only in plasma membranes of gallstone-susceptible C57L mice, which also failed to down-regulate cholesterol synthesis. These data suggest a role of the reverse cholesterol transport pathway for genetically determined gallstone susceptibility in the mouse.

A locus on chromosome 7 determines myocardial cell necrosis and calcification (dystrophic cardiac calcinosis) in mice.

Dystrophic cardiac calcinosis, an age-related cardiomyopathy that occurs among certain inbred strains of mice, involves myocardial injury, necrosis, and calcification. Using a complete linkage map approach and quantitative trait locus analysis, we sought to identify genetic loci determining dystrophic cardiac calcinosis in an F2 intercross of resistant C57BL/6J and susceptible C3H/HeJ inbred strains. We identified a single major locus, designated Dyscalc, located on proximal chromosome 7 in a region syntenic with human chromosomes 19q13 and 11p15. The statistical significance of Dyscalc (logarithm of odds score 14.6) was tested by analysis of permuted trait data. Analysis of BxH recombinant inbred strains confirmed the mapping position. The inheritance pattern indicated that this locus influences susceptibility of cells both to enter necrosis and to subsequently undergo calcification.

Role of group II secretory phospholipase A2 in atherosclerosis: 1. Increased atherogenesis and altered lipoproteins in transgenic mice expressing group IIa phospholipase A2.

Some observations have suggested that the extracellular group IIa phospholipase A2 (sPLA2), previously implicated in chronic inflammatory conditions such as arthritis, may contribute to atherosclerosis. We have examined this hypothesis by studying transgenic mice expressing the human enzyme. Compared with nontransgenic littermates, the transgenic mice exhibited dramatically increased atherosclerotic lesions when maintained on a high-fat, high-cholesterol diet. Surprisingly, the transgenic mice also exhibited significant atherosclerotic lesions when maintained on a low-fat chow diet. Immunohistochemical staining indicated that sPLA2 was present in the atherosclerotic lesions of the transgenic mice. On both chow and atherogenic diets, the transgenic mice exhibited decreased levels of HDLs and slightly increased levels of LDLs compared with nontransgenic littermates. These data indicate that group IIa sPLA2 may promote atherogenesis, in part, through its effects on lipoprotein levels. These data also provide a possible mechanism for the observation that there is an increased incidence of coronary artery disease in many chronic inflammatory diseases.

Role of group II secretory phospholipase A2 in atherosclerosis: 2. Potential involvement of biologically active oxidized phospholipids.

Secretory nonpancreatic phospholipase A2 (group II sPLA2) is induced in inflammation and present in atherosclerotic lesions. In an accompanying publication we demonstrate that transgenic mice expressing group II sPLA2 developed severe atherosclerosis. The current study was undertaken to determine whether 1 mechanism by which group II sPLA2 might contribute to the progression of inflammation and atherosclerosis is by increasing the formation of biologically active oxidized phospholipids. In vivo measurements of bioactive lipids were performed, and in vitro studies tested the hypothesis that sPLA2 can increase the accumulation of bioactive phospholipids. We have shown previously that 3 oxidized phospholipids derived from the oxidation of 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphorylcholine (PAPC) stimulated endothelial cells to bind monocytes, a process that is known to be an important step in atherogenesis. We now show that these 3 biologically active phospholipids are significantly increased in livers of sPLA2 transgenic mice fed a high-fat diet as compared with nontransgenic littermates. We present in vitro evidence for several mechanisms by which these phospholipids may be increased in sPLA2 transgenics. These studies demonstrated that polyunsaturated free fatty acids, which are liberated by sPLA2, increased the formation of bioactive phospholipids in LDL, resulting in increased ability to stimulate monocyte-endothelial interactions. Moreover, sPLA2-treated LDL was oxidized by cocultures of human aortic endothelial cells and smooth muscle cells more efficiently than untreated LDL. Analysis by electrospray ionization-mass spectrometry revealed that the bioactive phospholipids, compared with unoxidized PAPC, were less susceptible to hydrolysis by human recombinant group II sPLA2. In addition, HDL from the transgenic mice and human HDL treated with recombinant sPLA2 in vitro failed, in the coculture system, to protect against the formation of biologically active phospholipids in LDL. This lack of protection may in part relate to the decreased levels of paraoxonase seen in the HDL isolated from the transgenic animals. Taken together, these studies show that levels of biologically active oxidized phospholipids are increased in sPLA2 transgenic mice; they also suggest that this increase may be mediated by effects of sPLA2 on both LDL and HDL.