Cardiac angiotensin II formation in the clinical course of heart failure and its relationship with left ventricular function.

In 76 patients with heart failure (HF) (New York Heart Association [NYHA] classes I through IV) and in 15 control subjects, cardiac angiotensin II (Ang II) generation and its relationship with left ventricular function were investigated by measuring aorta-coronary sinus concentration gradients of endogenous angiotensins and in a part of patients by studying (125)I-labeled Ang I kinetics. Gene expression and cellular localization of the cardiac renin-angiotensin system components, the density of AT(1) and AT(2) on membranes and isolated myocytes, and the capacity of isolated myocytes for synthesizing the hypertrophying growth factors insulin-like growth factor-I (IGF-I) and endothelin (ET)-1 were also investigated on 22 HF explanted hearts (NYHA classes III and IV) and 7 nonfailing (NF) donor hearts. Ang II generation increased with progression of HF, and end-systolic wall stress was the only independent predictor of Ang II formation. Angiotensinogen and angiotensin-converting enzyme mRNA levels were elevated in HF hearts, whereas chymase levels were not, and mRNAs were almost exclusively expressed on nonmyocyte cells. Ang II was immunohistochemically detectable both on myocytes and interstitial cells. Binding studies showed that AT(1) density on failing myocytes did not differ from that of NF myocytes, with preserved AT(1)/AT(2) ratio. Conversely, AT(1) density was lower in failing membranes than in NF ones. Ang II induced IGF-I and ET-1 synthesis by isolated NF myocytes, whereas failing myocytes were unable to respond to Ang II stimulation. This study demonstrates that (1) the clinical course of HF is associated with progressive increase in cardiac Ang II formation, (2) AT(1) density does not change on failing myocytes, and (3) failing myocytes are unable to synthesize IGF-I and ET-1 in response to Ang II stimulation.

Early sequence of cardiac adaptations and growth factor formation in pressure- and volume-overload hypertrophy.

To investigate the time sequence of cardiac growth factor formation, echocardiographic and hemodynamic measurements were performed at scheduled times, and mRNAs for angiotensinogen, prepro-endothelin-1 (ppET-1), and insulin-like growth factor I (IGF-I) were quantified with RT-PCR and localized with in situ hybridization in pigs (fluothane anesthesia) by use of pressure or volume overload (aortic banding and aorta-cava fistula, respectively). Relative peptide formation was also measured by radioimmunoassay. In pressure overload, angiotensinogen and ppET-1 mRNA overexpression on myocytes (13 times vs. sham at 3 h and 112 times at 6 h, respectively) was followed by recovery (12 h) of initially decreased (0.5-6 h) myocardial contractility. In volume overload, contractility was not decreased, the angiotensinogen gene was slightly upregulated at 6 h (6.7 times), and ppET-1 was not overexpressed. IGF-I mRNA was overexpressed on myocytes (at 24 h) in both volume and pressure overload (14 times and 37 times, respectively). In the latter setting, a second ppET-1 overexpression was detectable on myocytes at 7 days. In conclusion, acute cardiac adaptation responses involve different growth factor activation over time in pressure versus volume overload; growth factors initially support myocardial contractility and thereafter induce myocardial hypertrophy.

Selective upregulation of cardiac endothelin system in patients with ischemic but not idiopathic dilated cardiomyopathy: endothelin-1 system in the human failing heart.

Only scarce information is available on the activity and modifications of the cardiac endothelin (ET)-1 system in heart failure due to ischemic (ICM) or idiopathic dilated (DCM) cardiomyopathy. The activity of the ET-1 system was investigated by measuring cardiac ET-1 and big ET-1 formation and quantifying cardiac mRNA for prepro-ET-1 (ppET-1), ET-converting enzyme-1, and ET(A) and ET(B) receptors both in myocardium and in isolated myocytes using Northern blot, reverse transcription-polymerase chain reaction, and in situ hybridization in 22 patients with DCM and 20 with ICM who underwent cardiac transplantation and in 7 potential heart transplant donors (nonfailing hearts). Notwithstanding a similar increase of plasma ET-1 in the 2 groups, cardiac ET formation, mRNA levels for ppET-1, and ET(A) and ET(B) receptors were higher on both the myocardium and isolated myocytes from ICM than on those from DCM hearts (P<0.001 for all). ppET-1 and ET-converting enzyme-1 mRNAs were expressed on myocytes and endothelial and interstitial cells in ICM, whereas in DCM and nonfailing hearts they were mainly expressed on nonmyocyte cells. In both ICM and DCM, the ET(A) mRNA signal was expressed on both myocytes and nonmyocyte cells, whereas ET(B) mRNA was almost exclusively localized on nonmyocyte cells. ET(A)- and ET(B)-specific receptor binding was increased on both myocytes and cardiac membranes, showing a positive correlation with left ventricular ejection fraction in ICM (r=0.78 and 0.70) but not in DCM patients. The present results show that human ventricular myocytes express all of the components of the ET-1 system, which is selectively upregulated in ICM patients and appears to be functionally important in the maintenance of cardiac function.

Characterization of endothelin-1 receptor subtypes in isolated human cardiomyocytes.

On cardiac membranes and isolated cardiomyocytes from the human heart, cell-type distribution and functional activities of endothelin-1 (ET-1) receptor subtypes were investigated by using binding methods and messenger RNA (mRNA) in situ hybridization. The ET-receptor antagonist BMS-182874 selectively and competitively inhibits ET(A) receptors both on isolated myocytes and ventricular membranes with approximately 1,300 times greater affinity for ET(A) than ET(B) subtypes. The [125I]-ET-1 specific binding revealed 42.851+/-2,546 receptors/myocyte with a prevalent proportion of ET(A)-receptor subtypes on both myocytes (84+/-2%) and ventricular membranes (66+/-3%). In situ hybridization studies revealed that mRNA for ET(A) receptors was expressed on both myocytes and nonmyocyte cells, whereas mRNA for ET(B) receptors was almost exclusively expressed on fibroblasts and endothelial cells. Specific binding of [125I]-ET-1 to both myocytes and ventricular membranes in the presence of specific ET(A) (BMS-182874) and ET(B) (BQ-788)-receptor antagonists showed a displacement of [125I]-ET-1 by unlabeled ET-1, which were significantly faster from ET(B) than from ET(A). This suggests a clearance function of ventricular ET(B) receptors.

Cardiac growth factors in human hypertrophy. Relations with myocardial contractility and wall stress.

The aim of the present study was to investigate whether and which cardiac growth factors are involved in human hypertrophy, whether growth factor synthesis is influenced by overload type and/or by the adequacy of the hypertrophy, and the relationships between cardiac growth factor formation and ventricular function. Cardiac growth factor formation was assessed by measuring aorta-coronary sinus concentration gradient in patients with isolated aortic stenosis (n=26) or regurgitation (n=15) and controls (n=12). Gene expression and cellular localization was investigated in ventricular biopsies using reverse transcriptase-polymerase chain reaction and in situ hybridization. Cardiac hypertrophy with end-systolic wall stress <90 kdyne/cm2 was associated with a selective increased formation of insulin-like growth factor (IGF)-I in aortic regurgitation and of IGF-I and endothelin (ET)-1 in aortic stenosis. mRNA levels for IGF-I and preproET-1 were elevated and mainly expressed in cardiomyocytes. At stepwise analysis, IGF-I formation was correlated to the mean velocity of circumferential fiber shortening (r=0.86, P<0.001) and ET-1 formation to relative wall thickness (r=0.82, P<0. 001). When end-systolic wall stress was >90 kdyne/cm2, IGF-I and ET-1 synthesis by cardiomyocytes was no longer detectable, and only angiotensin (Ang) II was generated, regardless of the type of overload. The mRNA level for angiotensinogen was high, and the mRNA was exclusively expressed in the interstitial cells. Ang II formation was positively correlated to end-systolic stress (r=0.89, P<0.001) and end-diastolic stress (r=0.84, P<0.001). Multivariate stepwise analysis selected end-systolic stress as the most predictive variable and left ventricular end-diastolic pressure as the independent variable for Ang II formation (r=0.93, P<0.001). In conclusion, the present results indicate that the course of human left ventricular hypertrophy is characterized by the participation of different cardiac growth factors that are selectively related both to the type of hemodynamic overload and to ventricular function.

Effect of low-dose heparin on fibrinogen levels in patients with chronic ischemic heart disease.

Several prospective studies have demonstrated that high plasma fibrinogen levels are associated with an increased risk of ischemic heart disease. Since in most patients an increased thrombin generation has been reported, we investigated whether the control of thrombin generation could affect plasma fibrinogen levels. Forty male outpatients (20 asymptomatic with previous myocardial infarction and 20 with stable effort angina) were enrolled in a randomized medium-term (6 months) cross-over study. Clottable fibrinogen, according to Clauss, prothrombin fragment 1 + 2, thrombin-antithrombin complex, and fibrinopeptide A were evaluated in relation to treatment with low-dose heparin. After a 15-day wash-out period, during which patients had been treated only with nitrates if needed, patients were allocated to two sequential periods of treatment with standard heparin (12,500 U, subcutaneously daily) plus antianginal treatment or antianginal treatment alone, separated by a second 15-day wash-out period. At the end of the treatment period with low-dose heparin significant decreases in the plasma fibrinogen (2.5 +/- 0.6 g/l vs. 3.3 +/- 0.5 g/l, P < 0.001), prothrombin fragment 1 + 2 (1.4 +/- 0.5 nmol/l vs. 1.9 +/- 0.7 nmol/l, P < 0.001), thrombinantithrombin (4.5 +/- 2.4 ng/ml vs. 9.7 +/- 3.6 ng/ml, P < 0.001), and fibrinopeptide A (2.1 +/- 1.1 ng/ml vs. 3.5 +/- 2.1 ng/ml, P < 0.001) were observed compared with the period without heparin. The present results indicate that low-dose heparin can effectively control the increased abnormal thrombin generation and elevated fibrinogen levels in patients with ischemic heart disease, possibly decreasing the risk of cardiovascular death.

Effect of medium-term supplementation with a moderate dose of n-3 polyunsaturated fatty acids on blood pressure in mild hypertensive patients.

Several studies have shown that n-3 polyunsaturated fatty acids (n-3 PUFA) are able to lower blood pressure (BP) in humans, but large doses of fish oils have been often used. Moreover, most of the studies available in the literature were not able to evaluate the specific effects of n-3 PUFA because they employed fish oils which contain, together with n-3 PUFA, many other different components. The aim of this preliminary study was to evaluate if medium-term supplementation with a moderate dose of highly purified eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) ethyl esters is able to reduce BP in mild hypertensive patients. Sixteen mild essential hypertensive (diastolic BP: 95-104 mm Hg), non-diabetic, normolipidemic male outpatients and 16 normotensive male controls were recruited to participate in the study. Both hypertensive and control subjects were randomly assigned to receive either EPA and DHA ethyl esters (2.04 g EPA and 1.4 g DHA) as active treatment or olive oil (4 g/day) as a placebo for a period of 4 months. These subjects were followed up with 24-hour ambulatory BP monitoring and blood chemistry analyses at 2 and 4 months of treatment and 2 months after its discontinuation. The intake of n-3 PUFA was checked by red blood cell (RBC) phosphatidylcholine (PC) fatty acid composition. The effect of n-3 PUFA on BP in the active group was maximum after 2 months. Both systolic (-6 mm Hg, p<0.05) and diastolic (-5 mm Hg, p<0.05) BP significantly decreased during the n-3 PUFA ethyl ester supplementation. No further effect was observed at 4 months with a return to baseline values during the recovery period. These data indicate that 4 g/day of highly purified EPA + DHA ethyl esters are able to favorably affect BP in mild hypertensives.

Evaluation of clotting and fibrinolytic activation after protracted physical exercise.

The behavior of hemostatic system activation during protracted physical exercise is well known, but the duration of its modification is not yet defined. In order to evaluate the time of hemostatic system activation after prolonged strenuous endurance physical exercise (typical marathon race: 42.195 km, v=15.35 km/h; mean length of time run 2.45+/-0.15 hours) 12 well-trained long-distance male runners (mean age: 35+/-7, range 25-47 years) were investigated. Blood samples were drawn in the morning on the day before the performance, immediately after the race, and 24 hours and 48 hours after the end of run. With respect of baseline, immediately after the race, a significant decrease of fibrinogen (-25%) and significant increases of prothrombin fragment 1+2 (+633%) and thrombin-antithrombin complex (+848%) were observed. A significant acceleration of euglobulin lysis time (-41%), and rises of plasma levels of tissue plasminogen activator antigen (+361%), plasminogen activator inhibitor type 1 antigen (+235%), d-dimer (+215%), and plasma fibrinogen degradation products (+1200%) were also found. Only a slight, yet not significant, decrease in plasminogen activator inhibitor type 1 activity was observed. One day after the end of marathon different parameters were still unchanged. Forty-eight hours after the competition all parameters investigated returned to baseline values. These results indicate a persistence of clotting as well as fibrinolysis activation up to 24 hours after the end of the race.

Relationship between plasma and platelet phospholipid fatty acid composition in healthy subjects.

In order to furtherly clarify the mechanisms regulating the fatty acid composition of platelet phospholipids the relationships between the fatty acid composition of major phospholipid fractions from plasma and platelets were investigated in 30 healthy male subjects. Strict correlations between all but two plasma and platelet fatty acids were observed for phosphatidylcholine (PC) (r = 0.51-0.95, at least P < 0.01), whereas only poor correlations were found for the other fractions. These results suggest that the direct transfer of PC molecules from plasma to platelet membrane is a pivotal mechanism for renovation of platelet PC fatty acids, while other mechanisms appear to play a major role for renovation of other phospholipid fractions of the platelet membrane.

Fibrin generation and digestion in patients with angina pectoris.

Fibrin generation and lysis were studied in 28 patients with angina pectoris (14 with active disease and 14 with inactive disease) and in 14 normal controls. The fibrinolytic response was evaluated by comparing the ratio between the plasma levels of fibrinopeptide A and fibrin degradation products. Levels of both were higher in patients than in controls (P < 0.001), with higher levels in active than in inactive disease (P < 0.001). The fibrinopeptide A/fibrin degradation products ratio was much higher (P < 0.001) in the active group than in other groups. Thus, in patients with angina pectoris, especially in the active state, the increased thrombin generation is not paralleled by an equivalent increase in fibrinolytic activity.

Euglobulin lysis time in fresh and stored samples.

Euglobulin lysis time is a global test for the study of fibrinolysis. The aim of this study was to evaluate the influence of storage of plasma and euglobulin precipitates on euglobulin lysis time, by testing samples stored in different conditions. In 20 healthy subjects, euglobulin lysis time was measured by (1) euglobulin precipitates prepared within 90 minutes from blood withdrawal (reference euglobulin lysis time); (2) euglobulin precipitates obtained from platelet-poor plasma stored for 24 hours at either -80 degrees C or at -20 degrees C; (3) euglobulin precipitates frozen for 24 hours at either -80 degrees C or at -20 degrees C; (4) euglobulin precipitates dissolved in Owren's buffer and frozen for 24 hours at either -80 degrees C or at -20 degrees C. Euglobulin lysis time measured on euglobulin precipitates dissolved in Owren's buffer and stored at -20 degrees C and at -80 degrees C, and euglobulin lysis time measured on platelet-poor plasma stored at -20 degrees C were significantly longer than the reference euglobulin lysis time (at least P < .05). On the contrary, no changes were observed in euglobulin lysis time measured on platelet-poor plasma stored at -80 degrees C, and on euglobulin precipitates undissolved and stored at -20 degrees C and at -80 degrees C versus reference euglobulin lysis time. The pattern was similar in samples obtained both before and after venous occlusion. These data indicate that the freezing of samples of platelet-poor plasma or euglobulin precipitates at -80 degrees C and of euglobulin precipitates at -20 degrees C makes the simultaneous determination of a large number of samples collected at different times the previous day possible.

No changes in PAI-1 levels after four-month n-3 PUFA ethyl ester supplementation in healthy subjects.

Recent studies have indicated that diets rich in fish or supplemented with fish oils may increase PAI-1 plasma levels. However, this finding has not been consistent and could be related, at least in part, to the type of supplementation. Aim of this study was to investigate the effects of medium-term treatment with n-3 polyunsaturated fatty acid (PUFA) ethyl esters on fibrinolysis. Twenty normolipemic healthy male subjects (age 27 to 41 yrs) were randomly assigned to receive either 4 x 1 g capsules of n-3 PUFA ethyl esters (ESAPENT, Farmitalia-Carlo Erba, Milan, Italy) or 4 x 1 g capsules of olive oil (as placebo) for 4 months in a double blind study. Blood samples for lipid and hemostatic studies were obtained at 0, 2, and 4 months of treatment and 1, 2 and 3 months of wash-out. Plasma lipids, fibrinolytic system, lipoprotein (a)-Lp(a)-, fibrinogen (Fbg) and prothrombin activation fragment 1+2 (F1+2) were assayed. No changes in these parameters were observed in the group of ten subjects treated with olive oil. After n-3 PUFA supplementation no significant alterations were found in plasma lipids, even if a trend to lower triglyceride and Lp(a) levels was detectable. No changes in either PAI-1 activity or PAI-1 antigen levels or F1+2 plasma levels were observed. A trend to lower Fbg levels was found after n-3 PUFA, but changes were not statistically significant. The results of this study indicate that a 4-month treatment with 4 g daily n-3 PUFA ethyl esters does not affect PAI-1 plasma levels.