A. van der Laarse

On-line detection of cholesterol and calcification by catheter based Raman spectroscopy in human atherosclerotic plaque ex vivo

Background: Raman spectroscopy has the unique potential to detect and quantify cholesterol and calcification in an atherosclerotic plaque in vivo. Objective: To evaluate the sensitivity and specificity of this technique for detecting cholesterol or calcification in human coronary artery and aorta specimens ex vivo, using a compact clinical fibreoptic based Raman system developed for in vivo applications. Design: From nine coronary arteries and four aorta specimens, 114 sites were evaluated for the presence of cholesterol and calcification by Raman spectroscopy and standard histology. Raman spectra were acquired and evaluated on-line in around five seconds. Results: The correlation between Raman spectroscopy and histology was r = 0.68 for cholesterol and r = 0.71 calcification in the plaque (p < 0.0001). Sensitivity and specificity for detecting cholesterol and calcification were excellent: receiver operating characteristic (ROC) analysis for each of the components revealed areas under the curves of > 0.92 (p < 0.0001). At the optimal cut-off values determined by ROC analysis, positive predictive values of > 80% and negative predictive values of > 90% were obtained. Conclusions: On-line real time catheter based Raman spectroscopy detects accumulation of cholesterol and calcification in atherosclerotic plaque with high sensitivity and specificity.

Rapid gene transcription induced by stretch in cardiac myocytes and fibroblasts and their paracrine influence on stationary myocytes and fibroblasts.

Functional adaptation of cardiac cells in response to haemodynamic load requires dynamic alteration of gene expression. In this study, we examined early changes in gene expression following stretch in myocytes and fibroblasts isolated from neonatal rat hearts. In the first hour of biaxially applied static stretch, the changes in expression of immediate-early genes, such as c-fos, c-jun and fra-1, were quantified. The expression of the atrial natriuretic peptide (ANP) gene in myocytes was measured as an indication of the hypertrophic response. In stretched myocytes, expression of c-fos and ANP increased transiently to 227% and 176% respectively after 30xa0min stretch, whereas c-jun and fra-1 expression decreased in the 1stxa0hour of stretch. In stretched fibroblasts the expression of c-fos and fra-1 increased transiently to maxima of 145% and 146% respectively after 30xa0min stretch, whereas c-jun expression did not change significantly. To study the indirect effects of stretch, as an indication of cross-talk between cardiac cells, stationary myocytes and fibroblasts were incubated with stretch-conditioned medium (CM) from stretched (0–60xa0min) myocytes and fibroblasts. CM from stretched myocytes reduced c-fos and induced c-jun expression in myocytes and fibroblasts, reduced fra-1 expression in myocytes but induced fra-1 expression in fibroblasts. CM from stretched fibroblasts induced c-fos expression and had little effect on c-jun expression in myocytes and fibroblasts, induced the fra-1 expression in myocytes but had little effect on fra-1 expression in fibroblasts. CM from myocytes and CM from fibroblasts induced ANP expression in myocytes to 206% and 120% respectively after 45xa0min stretch. Static stretch of myocytes and fibroblasts appears to stimulate, within 1xa0h, secretion of cell type-specific factors that participate in the regulation of proto-oncogene and ANP expression of stationary myocytes and fibroblasts. These early changes in gene transcription suggest that stretch of the myocardium initiates intracellular gene expression as well as cross-talk between the cell types.

Altered calcium handling at normal contractility in hypertrophied rat heart

Left or right ventricular hypertrophy was induced by banding of the aorta or pulmonary artery in different groups of rats. After 5 to 10 weeks the degree of hypertrophy was about 15% in left and 80-160% in right ventricles, as determined by weight of the ventricle or by myocyte size. Action potentials and force-interval relationships were measured in papillary muscles isolated from either ventricle. As compared to muscles from control and SHAM-operated rats, hypertrophied papillary muscles showed: (1) Marked prolongation of the action potential and greater degree of post-extrasystolic potentiation. This indicates enhanced influx of Ca2+ probably via Ica; (2) Delayed relaxation of isometric force and faster decay of potentiation, which indicates reduced sequestration of Ca2+ by the sarcoplasmic reticulum; (3) Minor changes in steady-state peak force under standard conditions, which is explained from the opposite inotropic effects of enhanced Ca2+ influx and impaired function of the reticulum. Myocyte volume in the normal left ventricle was almost two times larger than in the normal right ventricle, and this was associated with a longer action potential and greater degree of post-extrasystolic potentiation in left as compared with right ventricular muscles. The rate of decay of potentiation, however, was not different. This might indicate that depressed function of the sarcoplasmic reticulum occurs with pressure-overload hypertrophy and not with normal age-dependent growth.

In Vitro Assessment of Cardiac Performance after Irradiation Using an Isolated Working Rat Heart Preparation

The effect of irradiation on cardiac function was assessed using an isolated working rat heart preparation. The animals were given single doses of X-rays in the range 15-30 Gy to their hearts. Cardiac output (CO = aortic flow + coronary flow), heart weight and body weight were followed for a period of 10 months after treatment. Irradiation led to a decrease in cardiac function. This reduction was dose-dependent and progressive with time after treatment. The shape of the Frank-Starling curves constructed for irradiated hearts suggests a loss of contractile function of the myocardium. Coronary flow rates measured in working hearts and in Langendorff hearts were not significantly changed by the irradiation treatment. The isolated working rat heart preparation proved to be a simple and suitable animal model for the investigation of irradiation-induced cardiotoxicity.

The (ISO)enzyme activities of lactate dehydrogenase, α-hydroxybutyrate dehydrogenase, creatine kinase and aspartate aminotransferase in human myocardial biopsies and autopsies

In a study on enzyme activities in normal human myocardial tissue the activities of lactate dehydrogenase (LDH), alpha-hydroxybutyrate dehydrogenase (HBDH), creatine kinase (CK), and aspartate aminotransferase (AST), and the activities of the isoenzymes of LDH (1--5) and AST (c and m) were measured in myocardial specimens obtained from live patients (18 biopsies) and postmortem (14 autopsies). Comparison of measurements in 4 right ventricular biopsies from 1 patient with those in 9 right ventricular biopsies from 9 patients shows that (1) the enzymes studied are homogeneously distributed throughout the right ventricular myocardium, (2) errors in the measurement of the activities of the enzymes studied are within 5%, and (3) inter-individual differences in myocardial enzyme activities are quite considerable (10--17%). No significant differences in the activities of the enzymes studied were found between left and right ventricular myocardium. The effect of autolysis on the activities of the enzymes studied in myocardial specimens obtained postmortem is small, amounting to -1% to -6% in the first 10 h, irrespective of whether a linear or an exponential decay of enzyme activity is assumed. Comparison of myocardial enzyme activities in biopsies with those in autopsies, the latter being corrected for autolysis-induced inactivation, reveals significant differences with respect to CK, AST and mAST. At present it is impossible to conclude whether these differences were due to the different conditions existing before the myocardial specimens were frozen at -20 degrees C or to the different types of pathology present in the hearts from which the specimens were taken.

Interference of the measurement of lactate dehydrogenase (LDH) activity in human serum and plasma by LDH from blood cells

This study was performed to find a reliable method to measure lactate dehydrogenase (LDH) activity in blood samples. Human plasma contains thrombocytes whose number depends on the speed of centrifugation applied to the freshly-drawn blood sample. They do not disturb the measurement of LDH in plasma, provided the plasma sample has not been subjected to procedures that may destroy the integrity of the thrombocytes, e.g. standing overnight at 4 degrees C, freezing, or treatment with ultrasonic waves. Centrifugation of plasma at a minimum of 1174 x g eliminates the presence of thrombocytes. The serum of healthy human subjects invariably contains higher LDH activity than the plasma: the difference is 30 +/- 18 U/l (mean +/- S.D.). LDH-isoenzyme analysis shows that a large part of the LDH activity difference is caused by lysis of erythrocytes in the clot. Liberation of 30 U LDH per litre is not associated with visible hemolysis.

Integrin stimulation induces calcium signalling in rat cardiomyocytes by a NO-dependent mechanism

The myocardial stretch-induced increase in intracellular [Ca2+] ([Ca2+]i) is considered to be caused by integrin stimulation. Myocardial stretch is also associated with increased nitric oxide (NO) formation. We hypothesised that NO is implicated in calcium signalling following integrin stimulation. Integrins of neonatal rat cardiomyocytes were stimulated with a pentapeptide containing the Arg-Gly-Asp (RGD) sequence. [Ca2+]i was measured with Fura2, [NO]i was measured with DAF2 and phosphorylation of focal adhesion kinase (FAK) was monitored with immunofluorescence techniques. Integrin stimulation increased both [NO]i and [Ca2+]i, the latter response being inhibited by ryanodine receptor-2 (RyR2) blockers and by NG-monomethyl-L-arginine (L-NMMA), an inhibitor of NOS, but resistant to GdCl3, diltiazem and wortmannin. Integrin-induced intracellular Ca2+ release thus appears to be independent of the influx of extracellular Ca2+ and phosphatidylinositol-3 kinase activity. In addition, integrin stimulation induced phosphorylation of FAK. Our results provide evidence for an integrin-induced Ca2+ release from RyR2 which is mediated by NO formation, probably via FAK-induced NOS activation.

Plasma lipoproteins in familial dysbetalipoproteinemia associated with apolipoproteins E2(Arg158-->Cys), E3-Leiden, and E2(Lys146-->Gln), and effects of treatment with simvastatin.

Using a density-gradient ultracentrifugation technique, we analyzed in detail the plasma lipoprotein profiles of 18 patients with familial dysbetalipoproteinemia (FD) who had apolipoprotein (apo) E2(Arg158-->Cys) homozygosity (the E2-158 variant, n = 6), apoE3-Leiden heterozygosity (the E3-Leiden variant, n = 6), or apoE2(Lys146-->Gln) heterozygosity (the E2-146 variant, n = 6), with average plasma cholesterol concentrations of 8.99 +/- 1.34 mmol/L, 9.29 +/- 1.55 mmol/L, and 8.46 +/- 1.10 mmol/L, respectively. No significant differences in sex, age, body mass index, dietary habits, and standard laboratory tests between the three groups were observed. The lipoprotein profiles of all FD patients were characterized by higher concentrations of very-low-density lipoprotein (VLDL) 1, VLDL2, and intermediate-density lipoprotein (IDL) and a higher cholesteryl ester content of VLDL1 and VLDL2 than in 6 normolipidemic control subjects with an average plasma cholesterol concentration of 5.90 +/- 0.53 mmol/L. Major differences between the plasma lipoprotein profiles of patients with the E2-158 variant, the E3-Leiden variant, and the E2-146 variant and the normolipidemic control subjects were in IDL cholesterol concentration (1.70 +/- 0.26, 1.50 +/- 0.26, 1.05 +/- 0.36, and 0.47 +/- 0.14 mmol/L, respectively), LDL cholesterol concentration (1.83 +/- 0.50, 3.09 +/- 0.32, 3.79 +/- 0.76, and 3.77 +/- 0.56 mmol/L, respectively), and the molar ratio of IDL cholesterol to LDL cholesterol (0.98 +/- 0.28, 0.48 +/- 0.04, 0.28 +/- 0.09, and 0.12 +/- 0.03, respectively). After 10 weeks of simvastatin treatment the concentrations of plasma cholesterol, VLDL2 cholesterol, IDL cholesterol, and LDL cholesterol in 3 patients with the E2-158 variant fell significantly, by 46%, 56%, 53%, and 48%, respectively; they also fell in 3 patients with the E3-Leiden variant, by 48%, 54%, 57%, and 52%, respectively, and in 3 patients with the E2-146 variant, by 38%, 55%, 46%, and 35%, respectively. Simvastatin therapy lowered plasma activity of cholesteryl ester transfer protein but had no significant effect on plasma activity of lecithin:cholesterol acyltransferase. It is concluded that patients with FD due to various apoE variants have different lipoprotein profiles, mainly with regard to IDL and LDL levels, although they have a number of similar features of dysbetalipoproteinemia. Simvastatin therapy effectively reduced the plasma concentrations of total cholesterol, VLDL2 cholesterol, IDL cholesterol, and LDL cholesterol in the three groups of patients studied. It is proposed that apoE-dependent defects of the conversion of IDL to LDL may be an important mechanism in the pathophysiology of FD.

Time dependent changes in myocardial norepinephrine concentration and adrenergic receptor density following x-irradiation of the rat heart

The hearts of 9 to 12-weeks-old Sprague-Dawley rats were locally irradiated with a single dose of 20 Gy. The effects on myocardial norepinephrine concentrations and on alpha-adrenergic and beta-adrenergic receptor densities was examined up to 16 months post-treatment. Myocardial norepinephrine concentrations were reduced (to 50% of control values between 8 and 16 months) after irradiation. Receptor binding studies using radioactive ligands demonstrated that alpha-adrenergic receptor density was increased to maximally 210% of control values and that beta-adrenergic receptor density was increased to maximally 150% of control values, both measured at 8 months posttreatment. The affinities of both receptor types were not changed after irradiation. An inverse correlation was found between the myocardial norepinephrine concentration and the alpha-adrenergic receptor density. Myocardial norepinephrine concentration was not correlated to the beta-adrenergic receptor density. The changes in myocardial norepinephrine concentration and receptor density observed after irradiation suggest that even 16 months after irradiation overt cardiac failure was not occurring as the radiation-induced alterations differ considerably from those reported for failing hearts.

Effects of in vivo heart irradiation on myocardial energy metabolism in rats

To investigate the effect of in vivo heart irradiation on myocardial energy metabolism, we measured myocardial adenosine nucleotide concentrations and mitochondrial oxygen consumption in left ventricular tissue of rats 0-16 months after local heart irradiation (20 Gy). At 24 h and 2 months no difference in myocardial adenosine nucleotide concentration was apparent between irradiated and control hearts. The total myocardial adenosine nucleotide concentrations in irradiated hearts compared to those of nonirradiated controls tended to be lower from 4 months onward. The rate of oxidative energy production (state 3 respiration) in irradiated hearts was significantly reduced compared with that of age-matched controls from 2 months onward. Moreover, as a result of aging, a time-dependent decrease in the rate of oxidative energy production was observed in both irradiated and control hearts (P < 0.001). The respiratory control index (RCI = oxygen consumption in state 3/oxygen consumption in state 4) in irradiated hearts was not different from the RCI measured in age-matched control animals. During the period of study the RCI diminished significantly with age in both groups (P < 0.005). The number of oxygen atoms used per molecule of ADP phosphorylated (P/O ratio) was not influenced by the irradiation. The P/O ratio for the NAD(+)-linked substrates remained unchanged at a value of about 3 during the period studied. At 6 months after irradiation activities of myocardial enzymes such as lactate dehydrogenase, creatine kinase, citrate synthase, and cytochrome c oxidase were reduced. The reduction in myocardial energy production and the changes in energy supplies provide a mechanism to explain impaired contractility after local heart irradiation.