Alberico Borghetti

Natural anti-endothelial cell antibodies (AECA)

Natural AECA constitute a pool of autoantibodies circulating in healthy subjects, which react with a restricted and conserved set of endothelial antigens and establish idiotypic interactions within the immunoglobulin networks. Normal IgG interacts with living endothelial cells and is internalized with a mechanism involving microtubules and resembling that of ligand-receptor internalization. IgG-endothelial cell interaction appears to be dependent on the variable region of antibodies and is followed by modifications of endothelial cell function. Natural AECA increase anti-inflammatory properties of endothelial cells through the selective inhibition of thromboxane A2, endothelin and metalloproteinase-9 secretion, and also through the inhibition of endothelial cell proinflammatory response to TNF-alpha. We have gathered evidence demonstrating that natural AECA constitute a strictly controlled autoantibody pool, interact with living endothelial cells and take part in the regulation of endothelial function, through direct anti-inflammatory effects.

Circadian blood pressure and heart rate changes in patients in a persistent vegetative state after traumatic brain injury.

Alteration of autonomic nervous system regulation is known to be present in the persistent vegetative state after traumatic brain injury, termed the dysautonomic syndrome. This study assessed the circadian blood pressure and heart rate pattern and variability in the persistent vegetative state through noninvasive 24‐hour ambulatory blood pressure monitoring. The study was performed in 20 subjects: 10 patients (six men and four women; mean age, 29.5±9.9 years; range, 19–39 years) in a vegetative state (mean, 27.3±5.6 days after trauma) and 10 healthy subjects as controls (six men and four women; mean age, 28±5.7 years; range, 29–37 years). The patients showed a blood pressure nondipper pattern; 24‐hour, daytime, and nighttime values of blood pressure and heart rate were significantly higher in patients than in controls. The day‐night difference in heart rate and blood pressure was also significantly lower in patients. Finally, SD and variation coefficients were significantly lower in patients. The results show changes in the variability and circadian blood pressure and heart rate patterns in persistent vegetative state patients with dysautonomic syndrome, as an expression of the sympathetic‐parasympathetic activity imbalance in the control of vasomotor tone.

Norepinephrine Reuptake Is Impaired in Skeletal Muscle of Hypertensive Rats In Vivo

Certain forms of experimental hypertension are characterized by organ-specific alterations of catecholaminergic pathways. The purpose of this study was to evaluate, in the same awake and freely moving normotensive Wistar-Kyoto rats (WKY) and spontaneously hypertensive rats (SHR) before and after the development of arterial hypertension, the norepinephrine (NE) turnover and, in particular, the neuronal NE reuptake activity that ends its effects once released from nerve terminals, in subcutaneous adipose tissue and in skeletal muscle, whose sympathetic efferents are respectively independent or dependent from baroreflexes. Plasma and tissue interstitial NE and 3,4-dihydroxyphenylethylene glycol (DHPG), its major deaminated metabolite, were measured before and after blockade of NE reuptake by tissue perfusion of desipramine through microdialysis probes. Arterial pressure and plasma NE in SHR were similar to those in WKY at 5 weeks of age but increased at 16 weeks of age. In contrast, plasma DHPG was already higher in young SHR. Basal interstitial NE and DHPG were increased in both tissues of young and old SHR compared with age-matched WKY. Desipramine induced a higher rise of interstitial NE in SHR of both ages, with a lesser increase in the skeletal muscle of old compared with young SHR. These results indicate an increased NE turnover in prehypertensive and hypertensive SHR in both baroreflex-dependent and -independent tissues, not shown by plasma NE levels in young SHR. In the skeletal muscle, where sympathetic efferents are baroreflex dependent, the reduced interstitial NE reuptake contributes to the higher availability of interstitial NE for postsynaptic effects in old SHR.

Urinary excretion of brush-border antigen and plasma proteins in early stages of diabetic nephropathy

In 109 patients with insulin-dependent diabetes mellitus (IDDM), we measured the urinary excretion of albumin, the low molecular weight proteins (LMWP) retinol-binding protein (RBP) and beta 2-microglobulin (beta 2m), and brush-border antigens (BBA) revealed by monoclonal antibodies. All such markers of kidney damage and/or dysfunction were higher in diabetic patients than in 44 controls. Increased urinary levels of BBA (p = 0.0001) were associated with higher values of albumin (p = 0.0002), RBP (p = 0.0005) and, to a lesser extent, of beta 2m (p = 0.1), different combinations of values above the reference limits being observed. Some 30 and 40% of patients with and without microalbuminuria, respectively, also exhibited signs of tubulopathy. Although under certain circumstances tubular defects may give rise to small increases in albuminuria, the most likely explanation for our findings is the coexistence of glomerular and tubular damage in some patients with IDDM. Neither the prognostic value nor the pathophysiological meaning of tubular damage and/or dysfunction can be assessed by the present study, owing to its cross-sectional design. Tubular markers thus deserve further studies to clarify whether in diabetic patients they indicate a more severe or diffuse kidney impairment.

Myocardial ultrasonic tissue characterization in patients with different types of left ventricular hypertrophy: A videodensitometric approach

Although analysis of the radio frequency signal is the most accurate approach to myocardial tissue characterization, clinical diffusion has been limited because of the complex technology required. Much easier to perform, videodensitometric analysis could represent a valuable alternative. Previous works carried out on radio frequency data have shown that the absolute value of ultrasonic back scatter increases while its diastole-to-systole variation decreases in the hypertrophied myocardium. This study was aimed at clarifying whether alterations in characterization indexes of ultrasonic tissue can be detected by means of a videodensitometric approach, whether a specific type of left ventricular (LV) hypertrophy can be identified with this method, and finally what possible relationships exist between parameters of contractile function and tissue characterization indexes. Myocardial echo intensity (MEI), its cyclic variation (CV), and the dynamic relationship between myocardial signal and wall thickness variations during the cardiac cycle were assessed in 20 healthy subjects, 11 patients with essential hypertension and LV hypertrophy, 15 patients with hypertrophic cardiomyopathy, and 4 patients with primary amyloidosis. The CV was lower in the interventricular septum of patients with cardiac hypertrophy as a group, compared with that of control subjects (13.0% +/- 5.6% versus 18.8% +/- 5.5%, p < 0.001), but it was similar among patients with different types of hypertrophy. In control subjects, a significant inverse correlation was found between the progressive decrease of the myocardial signal and the parallel increase in wall thickness during systole; this correlation was lost in 60% of patients with hypertrophic cardiomyopathy and 50% of those with amyloidosis, but only in 9% of patients with essential hypertension (chi square analysis 12.68, p < 0.01). The CV was associated with systolic wall thickening (r = 0.53, p = 0.0001) and fractional shortening (r = 0.44, p = 0.0014). MEI and its CV per se cannot distinguish among different types of LV hypertrophy; however, the loss of an inverse relationship between the myocardial signal and wall thickness may suggest abnormal myocardial conditions in individual patients with the same disease or comparable wall thickness.

Increased sensitivity to protein kinase C activation in aortas of spontaneously hypertensive rats.

The aortic muscle of spontaneously hypertensive (SHR) and normotensive control (WKY) rats was stimulated with phorbol esters and the contractile response was measured as isometric tension. Phorbol esters are known activators of protein kinase C. The aortas of SHR were characterized by the following distinct alterations in the response to phorbol myristate acetate: (1) increased sensitivity: half-maximal force was achieved at 62 ± 6 nmol/1 phorbol myristate acetate in SHR and 105 ± 8 nmol/l in WKY; (2) increased contractility: the maximal force developed by phorbol myristate acetate was greater in SHR aortas (1.9 ± 0.3 versus 1.6 ± 0.2 g in WKY) compared with the decreased contractility generated with noradrenaline and high levels of potassium; (3) decreased dependency on extracellular calcium for half-maximal tension: in the presence of 3 μmol/l phorbol myristate acetate 50% of maximal force was attained at 21 ± 8 μmol/1 extracellular calcium compared with 49 ± 9 μmol/l in WKY; (4) diminished relaxation in response to excess extracellular calcium: phorbol myristate acetate-precontracted WKY aortas began to relax when calcium was raised above 4 mmol/l in the bath and relaxation reached 51% at 8–10 mmol/l. Relaxation was almost absent in SHR (3–7%). Hence, there is an abnormality in the response to protein kinase C activation by phorbol esters in SHR vascular smooth muscle. Intracellular calcium appears to be involved. Studies of protein kinase C will prove important in understanding vascular smooth muscle function in normal and abnormal states.

Receptor-ligand internalization.

Publisher Summary The chapter presents a discussion on receptor-ligand internalization. Currently, confocal imaging has provided new insights in the observation of fluorescent specimens. The virtual absence of out-of-focus blurring allows a much better definition of probe localization at the subcellular level together with the possibility of exploiting the three-dimensional (3-D) reconstruction capability of most confocal systems. A self-constructed flow chamber to an inverted confocal scanning laser microscope that allows long-term observation of adherent cells, under controlled micro-environmental conditions have been paired. This method provides images of intact, nonfixed cells and allows one to change culture conditions and to observe living cell responses directly or to perform two-step staining to identify subcellular structures involved in the observed processes. The reliability of this procedure has been verified on a well-known model of receptor-ligand internalization, that of insulin and insulin receptor. The same technique has also been applied to studying the interactions between natural human antibodies, circulating in healthy subjects, and living human endothelial cells, fibroblasts, and proximal tubular epithelial cells. The device described in the chapter is simple and inexpensive but fulfills all the described criteria.

Characterization of myocardial hypertrophy in prehypertensive spontaneously hypertensive rats: interaction between adrenergic and nitrosative pathways.

Objective and methods Left ventricular hypertrophy in human and experimental hypertension is not always associated with pressure overload but seems to precede an increase in blood pressure. In this study, performed in male 5-week-old prehypertensive spontaneously hypertensive rats (SHR; n = 65) and age-matched Wistar–Kyoto rats (n = 56), the relationship between myocardial structure and activation of the adrenergic and nitric oxide systems was evaluated. Results Body weight, blood pressure and heart rate were similar in both groups. A higher left ventricle/body weight ratio was found in SHR, as a result of greater mononuclear (+47%) and binuclear (+43%) myocyte volumes, without changes in interstitial collagen. Both adrenergic and nitric oxide pathways were activated in SHR, as expressed by higher myocardial norepinephrine content, tyrosine hydroxylase activity, myocardial nitric oxide synthase 3 expression and protein nitration, indicating greater peroxynitrite (ONOO−) generation from nitric oxide and superoxide. No difference was measured in nitric oxide synthase 1 expression, whereas nitric oxide synthase 2 was undetectable. A positive correlation between myocardial tyrosine hydroxylase activity and protein nitration was observed in SHR (r2 = 0.328; P < 0.01). Early treatment with a superoxide dismutase mimetic, 4-hydroxy-2,2,6,6-tetramethyl piperidinoxyl, from the third to the fifth week of age, reduced ONOO− generation, protein nitration and sympathetic activation in SHR without changes in myocardial structure. Conclusion In prehypertensive SHR, left ventricular hypertrophy is associated with adrenergic and nitrosative imbalance. Early superoxide dismutase mimetic treatment in SHR effectively reduces higher myocardial ONOO− generation, sympathetic activation, and heart rate without affecting the development of myocardial hypertrophy.

Early Proinflammatory Activation of Renal Tubular Cells by Normal and Pathologic IgG

Background/Aims: To verify whether human IgG induces proinflammatory activation of human proximal tubular epithelial cells (PTEC) independent of the metabolic overload of protein reabsorption. Methods: Cultured PTEC were incubated with normal IgG, IgG from systemic lupus erythematosus (SLE) patients, albumin or transferrin. IL-6 secretion and extracellular regulated kinase (ERK) activation (dual-phosphorylated ERK) were measured by ELISA and by Western blotting of PTEC extracts, respectively; renal biopsy specimens from patients with IgG and non-IgG proteinuria were analyzed by immunohistochemistry and in situ hybridization to detect ERK-P and IL-6. Results: Normal and SLE IgG, but not albumin or transferrin, induced an early significant increase in IL-6 secretion by PTECs. Also ERK activation was found after 1-hour incubation of PTEC with IgG, but not with control medium and albumin-treated PTEC. Activated ERK and IL-6 were found to colocalize in tubular cells in the kidney specimens of patients with IgG proteinuria only. Conclusion: IgG-dependent early activation of ERK and increased IL-6 secretion in PTEC suggest that IgG filtered during nonselective proteinuria may play a specific role in tubulointerstitial disease. Such a role could be particularly relevant in diseases associated with abnormal IgG pool compositions, such as SLE. Preliminary results on human renal biopsy specimens suggest that our in vitro observations may also be relevant in vivo.

Myocardial involvement during the early course of type 2 diabetes mellitus: usefulness of Myocardial Performance Index

To evaluate whether myocardial performance index detects a subclinical impairment of left ventricular systolic and diastolic function in patients with early stage of type 2 diabetes, without coronary artery disease, with or without hypertension. Furthermore, to evaluate whether some echocardiographic parameters relate to the metabolic control. Fourty-five consecutive male patients (mean age 52.5 years) with type 2 diabetes mellitus of recent onset (23 hypertensives and 22 normotensives) and 22 age matched healthy controls males were analysed. All participants had normal exercise ECG. All subjects underwent standard and Doppler echocardiography for the assessment of the isovolumic Doppler time interval and Doppler-derived myocardial performance index. In all diabetic patients a glycated haemoglobin test was also performed.No differences were observed in blood pressure, heart rate, and conventional echocardiographic parameters comparing the 2 subgroups of diabetic patients and the controls. Myocardial performance index was significantly higher in diabetic patients independently of the hypertension occurrence, compared to the controls (0.49 and 0.49 diabetic normotensives and hypertensives respectively vs. 0.39, p < 0.01). Myocardial performance index correlated to glycated haemoglobin significantly (r = 0.37, p < 0.01) in both diabetic subgroups. Thus, an early involvement of left ventricular performance was shown by myocardial performance index in patients with type 2 diabetes of recent onset without coronary artery disease, independently of the hypertension presence. These abnormalities can provide a feasible approach to detect a pre-clinical diabetic cardiomyopathy and could be useful for an indirect assessment of the metabolic control.