Giorgio Finardi

LDL oxidation in patients with severe carotid atherosclerosis. A study of in vitro and in vivo oxidation markers.

Among the various risk factors involved in the development and progression of carotid atherosclerosis, the oxidation of LDL has been proposed to play a relevant role. LDL oxidation has been investigated in 94 patients with severe carotid atherosclerosis undergoing elective carotid artery endarterectomy and in 42 matched control subjects. LDL oxidation was evaluated in all patients as (1) the susceptibility to in vitro oxidation, (2) vitamin E concentration and its efficiency in LDL, and (3) the presence of autoantibodies against oxidatively modified lipoprotein to monitor the occurrence of the oxidative processes taking place in vivo. No difference was detected between control subjects and patients concerning vitamin E concentration and the kinetics of conjugated diene formation in isolated LDL exposed to CuSO4. However, vitamin E efficiency was lower (9.6 +/- 4.2 versus 30.2 +/- 7.6 min/nmol vitamin E) and the duration of the vitamin E-independent lag phase was longer (105.5 +/- 16.5 versus 58 +/- 11.8 minutes) in the patient group. Autoantibodies against oxidatively modified lipoproteins were measured with an ELISA method using native LDL, Cu(2+)-oxidized LDL (oxLDL), or malondialdehyde-derivatized LDL (MDA-LDL) as antigens. To monitor cross-reactivity of the antibodies detected with other oxidatively modified proteins, human serum albumin (HSA) and MDA-derivatized HSA (MDA-HSA) were also employed. The antibody titer was calculated as the ratio of antibodies against modified versus native proteins.(ABSTRACT TRUNCATED AT 250 WORDS)

Impaired circadian modulation of sympathovagal activity in diabetes. A possible explanation for altered temporal onset of cardiovascular disease.

BackgroundDiabetic subjects have a high incidence of cardiovascular accidents, with an altered circadian distribution. Abnormalities in the circadian rhythm of autonomic tone may be responsible for this altered temporal onset of cardiovascular disease. Methods and ResultsTo assess circadian changes of sympathovagal balance in diabetes, we performed 24-hour power spectral analysis of RR interval fluctuations in 54 diabetic subjects (age, 44±2 years) with either normal autonomic function or mild to severe autonomic neuropathy and in 54 age-matched control subjects. The power in the low-frequency (LF, 0.03–0.15 Hz) and high-frequency (HF, 0.18–0.40 Hz) bands was considered an index of relative sympathetic and vagal activity, respectively. Diabetic subjects with autonomic abnormalities showed a reduction in LF compared with control subjects (5.95±0.12 In-msece versus 6.73±0.11, p<0.001) and an even greater reduction in LF, particularly during the night and the first hours after awakening (5.11±0.18 In-msece versus 6.52±0.14, p<0.001). Day-night rhythm in sympathovagal balance was reduced or absent in diabetic subjects compared with control subjects. ConclusionsDiabetic subjects with or without signs of autonomic neuropathy have a decreased vagal activity (and hence a relatively higher sympathetic activity) during night hours and at the same time of the day, during which a higher frequency of cardiovascular accidents has been reported. These observations may provide insight into the increased cardiac risk of diabetic patients, particularly if autonomic neuropathy is present.

Menadione-induced bleb formation in hepatocytes is associated with the oxidation of thiol groups in actin

Incubation of isolated rat hepatocytes with menadione (2-methyl-1,4-naphthoquinone) or the thiol oxidant, diamide (azodicarboxylic acid bis(dimethylamide)), resulted in the appearance of numerous plasma membrane protrusions (blebs) preceding cell death. Analysis of the Triton X-100-insoluble fraction (cytoskeleton) extracted from treated cells revealed a dose- and time-dependent increase in the amount of cytoskeletal protein and a concomitant loss of protein thiols. These changes were associated with the disappearance of actin and formation of large-molecular-weight aggregates, when the cytoskeletal proteins were analyzed by polyacrylamide gel electrophoresis under nonreducing conditions. However, if the cytoskeletal proteins were treated with the thiol reductants, dithiothreitol or beta-mercaptoethanol, no changes in the relative abundance of actin or formation of large-molecular-weight aggregates were detected in the cytoskeletal preparations from treated cells. Moreover, addition of dithiothreitol to menadione- or diamide-treated hepatocytes protected the cells from both the appearance of surface blebs and the occurrence of alterations in cytoskeletal protein composition. Our findings show that oxidative stress induced by the metabolism of menadione in isolated hepatocytes causes cytoskeletal abnormalities, of which protein thiol oxidation seems to be intimately related to the appearance of surface blebs.

Autoantibodies Against Oxidatively Modified Low-Density Lipoproteins in NIDDM

Diabetes is an independent risk factor in the development of atherosclerosis, although the pathophysiological processes underlying this association are poorly understood. The oxidation of low-density lipoprotein (LDL) is considered a key event in the development and progression of atherosclerosis because it generates molecular epitopes that are more atherogenic than parent LDL. A total of 138 patients suffering from non-insulin-dependent diabetes mellitus (NIDDM) and 80 matched control subjects were investigated. LDL oxidation was evaluated as the presence of autoantibodies against oxidatively modified LDL, since they mirror the in vivo occurrence of oxidative processes. NIDDM patients had an antibody ratio (calculated as the ratio of antibodies against modified versus native LDL) significantly higher than control subjects for Cu2+-oxidized LDL (1.88 ± 0.6 vs. 1.05 ± 0.3, P < 0.01, for IgG), malondialdehyde-modified LDL (2.54 ± 0.73 vs. 2.04 ± 0.11, P < 0.01, for IgG and 3.96 ± 1.51 vs. 2.90 ± 0.15, P < 0.01, for IgM), and malondialdehyde-modified human serum albumin (1.79 ± 0.54 vs. 1.46 ± 0.1, P < 0.05 for IgG). The possible role played by glycation in sensitizing LDL to oxidation was investigated by measuring autoantibodies against both glycated LDL (glycLDL) and glycoxydated LDL (glycoxLDL). NIDDM patients had an antibody ratio significantly higher than control subjects for anti-glycLDL and anti-glycoxLDL IgG (1.79 ± 0.38 vs. 1.12 ± 0.23, P < 0.01 and 2.55 ± 1.03 vs. 1.39 ± 0.44, P < 0.01, respectively) but not anti-glycLDL and anti-glycox-LDL IgM. These results demonstrate that in NIDDM patients enhanced LDL oxidation occurs in vivo and that LDL glycation may represent a predisposing event that facilitates subsequent oxidative modifications.

Demonstrable cardiac reinnervation after human heart transplantation by carotid baroreflex modulation of RR interval.

BACKGROUND After heart transplantation, respiration-synchronous fluctuations (0.18 to 0.35 Hz, high frequency [HF]) in RR interval may result from atrial stretch caused by changes in venous return, but slower fluctuations (0.03 to 0.15 Hz, low frequency [LF]) not due to respiration suggest reinnervation. In normal subjects, sinusoidal neck suction selectively stimulates carotid baroreceptors and causes reflex oscillations of RR interval. METHODS AND RESULTS To evaluate the presence of reinnervation, we measured the power of RR-LF and RR-HF in 26 heart transplant recipients and 16 control subjects before and during sinusoidal neck suction at 0.1 Hz and 0.20 Hz (similar to but distinct from that of controlled respiration, 0.25 Hz) and before and during administration of atropine or beta-blocker (esmolol hydrochloride) by spectral analysis. All transplant recipients showed small respiratory HF fluctuations. Nonrespiratory LF fluctuations were present in 13 of 26 transplant recipients and increased with months since transplantation (r = .53, P < .01). HF neck suction induced a 0.20-Hz component in all 16 control subjects and none of the 26 transplant subjects. LF neck suction increased RR-LF (from 0.73 +/- 0.20 to 1.30 +/- 0.26 ln ms2, P < .001), similar to but less than in control subjects (from 6.12 +/- 0.21 to 8.27 +/- 0.21 ln ms2, P < .001). Atropine reduced all fluctuations in control subjects and blocked the HF increase caused by 0.20-Hz neck suction but not the LF increase during 0.10-Hz stimulation. Neck suction-induced changes in LF fluctuations persisted after administration of atropine in transplant recipients but were attenuated by esmolol hydrochloride, suggesting sympathetic rather than vagal reinnervation. CONCLUSIONS The presence of baroreceptor-induced RR oscillations is evidence of functional, although incomplete, autonomic reinnervation.

Cardiovascular autonomic modulation in essential hypertension. Effect of tilting.

To better understand the role played by the autonomic nervous system in essential hypertension, we used autoregressive power spectrum analysis to study the noncasual oscillations in RR interval, blood pressure, and skin blood flow in 40 subjects with mild to moderate hypertension and in 25 age-matched control subjects at low frequency (index of sympathetic activity to the heart and the peripheral circulation) and high frequency, respiratory related (index of vagal tone to the heart). RR interval, respiration, noninvasive systolic blood pressure, and skin arteriolar blood flow were simultaneously and continuously recorded with subjects in the supine position and immediately after tilting. The low-frequency component was not significantly different in the two groups either at the cardiac level (control versus hypertensive subjects: 39.1 +/- 4.3 versus 39.9 +/- 3.7 normalized units [NU]) or at the vascular level (1.52 +/- 0.17 versus 1.69 +/- 0.13 ln mm Hg2). After head-up tilting, the RR interval fluctuations were less in hypertensive subjects (low-frequency components from 39.9 +/- 3.7 to 48.4 +/- 4.1 NU, P < .05; high-frequency components from 53.9 +/- 3.7 to 44 +/- 4 NU, P < .05) than in control subjects (low-frequency components from 39.1 +/- 4.3 to 64.4 +/- 4.9 NU, P < .001; high-frequency components from 56.0 +/- 4.5 to 31.2 +/- 4.6 NU, P < .001); the low-frequency components in systolic blood pressure increased similarly in hypertensive subjects (to 2.43 +/- 0.17 ln mm Hg2, P < .0001) and in control subjects (to 2.44 +/- 0.21 ln mm Hg2, P < .01), but the low-frequency components in skin blood flow increased only in control subjects (from 5.34 +/- 0.45 to 6.55 +/- 0.53 mm Hg2, P < .01), not in hypertensive subjects (from 5.55 +/- 0.34 to 5.60 +/- 0.35 ln mm Hg2). In hypertensive subjects with left ventricular hypertrophy, the low-frequency components in systolic blood pressure did not increase after tilting (from 1.75 +/- 0.33 to 2.05 +/- 0.41 ln mm Hg2). Baroreflex sensitivity, as assessed by spectrum analysis, was significantly lower in hypertensive than in control subjects (5.17 +/- 0.49 versus 13.18 +/- 2.44 ms/mm Hg, P < .001. Power spectrum analysis did not reveal an increased sympathetic activity or reactivity either at the cardiac or at the vascular level. The decreased baroreceptor sensitivity in hypertensive subjects could explain the reduced change in sympathovagal balance in the tilt position at the cardiac level. In hypertensive subjects without left ventricular hypertrophy, cardiopulmonary reflex deactivation induced by tilting and/or amplification of sympathetic nervous tone by arteriolar structural change could have preserved the sympathetic activation at the vascular level.

Specificity of autoantibodies against oxidized LDL as an additional marker for atherosclerotic risk.

BACKGROUND LDL oxidation is a crucial step in the development and progression of atherosclerotic lesions. The detection of an increase in the anti-oxidized LDL antibody titre may thus represent a biological marker of enhanced LDL oxidation in vivo. METHODS The occurrence of anti-oxidized LDL autoantibodies was investigated in control patients, in patients with atherosclerotic coronary artery disease, in those without clinically relevant signs of atherosclerosis, but considered at risk, and in patients with chronic alcohol-related liver disease. RESULTS Anti-oxidized LDL autoantibodies were present in the plasma of the majority of patients with overt coronary atherosclerosis. An increased antibody titre can also be detected well before the onset of clinically relevant signs of the atherosclerotic disease in patients classically considered at risk, indicating the occurrence of in-vivo LDL oxidation during atherosclerosis development. The specificity of molecular targets (LDL) for oxidative modifications is supported by the demonstration that anti-oxidized LDL autoantibodies are absent in the plasma of alcoholic patients who exhibit a marked increase in biological markers of oxidative stress but do not classically develop atherosclerosis. CONCLUSION These data demonstrate that the occurrence of anti-oxidized LDL autoantibodies could be specifically related to the promotion and progression of atherosclerosis and is not a simple epiphenomenon of any oxidative process occurring in vivo.

Low-density lipoprotein oxidation in essential hypertension.

Objectives: To investigate the occurrence of enhanced low-density lipoprotein (LDL) oxidation as an additional factor promoting atherosclerosis progression in hypertensive patients. Design: The oxidation of plasma LDL was investigated in a group of untreated patients with mild-to-moderate essential hypertension without clinically evident target organ damage and in a group of control subjects. Methods: LDL oxidation was evaluated as both the susceptibility to oxidation in vitro and the presence of plasma anti-oxidized LDL antibodies (as an index for oxidation in vivo). Results: LDL from hypertensive subjects exhibited enhanced susceptibility to oxidation in vitro as revealed by early and accelerated generation of conjugated dienes after exposure to CuSO4. Vitamin E concentration in LDL from hypertensive subjects was slightly but significantly decreased and its efficiency in protecting LDL from oxidation was impaired. Furthermore, a higher plasma anti-oxidized LDL titre was found in hypertensive patients. Subclass analysis revealed that the contemporary presence of hypercholesterolaemia did not significantly modify either the increased susceptibility of LDL to oxidation or the presence of plasma anti-oxidized LDL antibodies detected in hypertensive patients. Moreover, no correlation was found between LDL oxidation parameters and blood pressure values. Conclusions: LDL from hypertensive patients is more susceptible to oxidation in vitro and is more promptly oxidized in vivo. These findings suggest a possible participation of LDL oxidation in promoting and accelerating the atherosclerosis that often develops in hypertensive patients.

Presence of autoantibodies against oxidatively modified low-density lipoprotein in essential hypertension: a biochemical signature of an enhanced in vivo low-density lipoprotein oxidation.

Objective We have previously reported that low-density lipoproteins (LDL) isolated from patients with essential hypertension are more susceptible to in vitro oxidation than lipoproteins isolated from normotensive control subjects. In the present study we investigated the occurrence of in vivo LDL oxidation in hypertensive patients. Design The presence of antioxidatively modified LDL autoantibodies was taken as a suitable index of in vivo LDL oxidation because, after oxidative modifications, LDL express antigenic epitopes that elicit an immune response. The antibody titres were measured in plasma from untreated patients with newly diagnosed essential hypertension. Methods An enzyme-linked immunosorbent assay method was employed, using native LDL, Cu2+-oxidized LDL and malondialdehyde-derivatized LDL (MDA-LDL) as antigens. Human serum albumin and MDA human serum albumin were also used to monitor cross-reactivity with other oxidized molecules. The antibody titre was expressed as the ratio between anti-modified and anti-native antigen absolule values. Results The patients with essential hypertension had an antibody ratio significantly higher than control subjects with respect to anti-Cu2+-oxidized LDL immunoglobulins G and M, and with respect to anti-MDA-LDL immunoglobulins G and M. A significant positive correlation was found between anti-MDA-LDL and anti-Cu2+-oxidized LDL antibody titres. The anti-MDA human serum albumin antibody titre was not different in the two groups of palients. An inverse correlation was detected between the anti-MDA-LDL immunoglobulin M titre and the age of the patients. Conclusions The results obtained are consistent with the view that, during the early phases of hypertension development, LDL undergo in vivo oxidation that is mirrored by the generation of autoantibodies against epitopes of oxidized LDL. The oxidation process appears specific for LDL and might be relevant both for the progression of hypertension and for the development of the atherosclerosis that often complicates hypertension itself.

Relationship between phasic changes in human skin blood flow and autonomic tone

Heart rate beat-to-beat oscillations synchronous with respiration and blood pressure waves, have been found to be a marker of sympathovagal interaction in man and animals. Oscillations of heart rate, respiration, and cutaneous blood flow were simultaneously recorded to assess the relationship between autonomic nervous control and cutaneous circulation in a group of 21 healthy subjects and in a group of 6 healthy patients after brachial plexus anesthesia and consequent sympathetic blockade. In the first group, changes in posture were employed to modify autonomic tone. Relative changes in cutaneous blood flow were recorded by laser-Doppler flowmetry. Spectral analysis techniques (cross-correlation) were used to quantify the relationship between oscillations common to the recorded signals. A standing maneuver induced a significant decrease of the cross-correlation between respiratory and heart rate fluctuations (from 4.93 +/- 0.16 to 4.44 +/- 0.16 a.u.; P less than 0.001), and a significant increase of the cross-correlation between heart rate and skin blood flow fluctuations (from 0.64 +/- 0.31 to 1.33 +/- 0.21 a.u.; P less than 0.001), but did not modify the cross-correlation between respiratory and skin blood flow fluctuations (from 2.87 +/- 0.15 to 3.04 +/- 0.14 a.u.; NS). After the standing maneuver the maximum correlation between heart rate and skin blood flow was always due to oscillations in the range of 0.1 Hz (or 10-sec period), similar to the oscillations described in large arteries. Sympathetic blockade reduced significantly the cross-correlation between heart rate and skin blood flow (P less than 0.001). These results suggest that the cross-correlation between skin blood flow and heart rate at 10-sec period fluctuations can be used as an index of the influence of the autonomic tone on skin blood circulation.