Gian Mario Cherchi

A proteomic approach to differentiate histologically classified stable and unstable plaques from human carotid arteries

OBJECTIVES By using proteomics we isolated and identified proteins that were expressed/retained in stable and unstable human carotid artery atherosclerotic plaques. METHODS The criteria for plaque instability were the presence of a thin fibrous cap or fissured cap covering the foamy or necrotic core, and the presence of overt, hemorrhagic, ulcerated or thrombotic plaques. Proteins were extracted from finely minced endarterectomy specimens (19 stable and 29 unstable plaques) and separated by two-dimensional gel electrophoresis. Coomassie Blue-stained gels were analysed using PD-Quest software. RESULTS A total of 57 distinct spots corresponding to 33 different proteins were identified by matrix assisted laser desorption/ionization mass spectrometry using the NCBI database. Most of the spots were present in both types of extracts, although significantly (p<0.05) differing in abundance between them. Compared to stable plaque, unstable ones showed reduced abundance of: protective enzymes SOD3 and GST, small heat shock proteins HSP27 and HSP20, annexin A10, and Rho GDI. In unstable plaques the more abundant proteins were: ferritin light subunit, SOD 2 and fibrinogen fragment D. For fibrinogen fragment D, the increased levels in unstable versus stable plaques was confirmed by Western blot analysis. CONCLUSIONS Since many of the differentially expressed proteins are known to have a functional role in inflammation and oxidative stress, we speculate that they may be involved in events relating to plaque stability.

Plasma lipids in beta-thalassemia minor

Because total cholesterol levels have been found to be lower in patients affected by thalassemia major and intermedia, we examined the plasma lipid pattern of 628 beta-thalassemia trait carriers and 4552 controls in order to evaluate whether the plasma lipid impairment is also present in the heterozygous state. Total cholesterol and low density lipoprotein (LDL)-cholesterol levels were significantly lower in beta-thalassemia trait carriers when compared to controls, whereas plasma triglycerides and high density lipoprotein (HDL)-cholesterol levels did not differ between the two groups. We suggest that accelerated erythropoiesis and increased uptake of LDL by macrophages and histiocytes of the reticuloendothelial system are the main determinants of low plasma cholesterol levels in heterozygous thalassemia.

Plasma Lipids and Lipoproteins Pattern in Beta-Thalassemia Major

We studied serum lipids and lipoproteins in 20 patients with beta-thalassemia major, under high transfusion programme and regular chelation therapy, and in 20 control subjects. Total cholesterol, HDL-cholesterol, HDL2-and HDL3-cholesterol, apolipoprotein A and B levels were significantly lower in patients with Cooleys anemia, whereas free cholesterol, triglycerides and the HDL-/HDL3-cholesterol ratio did not differ in the two groups. We think that liver damage plays an important role in determining the altered lipoprotein pattern in beta-thalassemia major. However, other factors may contribute to cause such lipid changes.

Evidence for a Proinflammatory and Proteolytic Environment in Plaques From Endarterectomy Segments of Human Carotid Arteries

ObjectivesBased on previous observations on apolipoprotein(a), apo(a), in human unstable carotid plaques, we explored whether in the inflammatory environment of human atheroma, proteolytic events affect other hepatic and topically generated proteins in relation to the issue of plaque stability. Methods and ResultsForty unstable and 24 stable plaques from endarterectomy segments of affected human carotid arteries were extracted with buffered saline (PBS) and then 6 mol/L guanidine-hydrochloride (GdHCl) to identify loosely and tightly bound products, respectively. The extracts were studied before and after ultracentrifugation at d 1.21 g/mL. In the extracts, the concentrations of interleukin (IL)-6, −8, and −18 were significantly higher in the unstable plaques and correlated to those of MMP-2 and MMP-9. By Western blots, both apoB and apo(a) were highly fragmented and mostly present in the d 1.21 bottom that also contained fragments of apoE (10 and 22 kDa), decorin, biglycan, and versican. Fragmentation was higher in the unstable plaques. In baseline plasmas, concentrations of lipids, lipoproteins, and ILs did not differ between patients with unstable and stable plaques. ConclusionsIn unstable and to a lesser extent in stable plaques, there is a proinflammatory and proteolytic microenvironment with the generation of fragments with potential pathobiological significance that requires investigation.

Plasma levels of C-reactive protein, leptin and glycosaminoglycans during spontaneous menstrual cycle: differences between ovulatory and anovulatory cycles.

PurposeTo assess the plasma levels of the inflammatory markers such as C-reactive protein (CRP), leptin, and glycosaminoglycans (GAGs) during the menstrual cycle.MethodsEighteen healthy volunteers were divided into two groups according to the presence of ovulatory or anovulatory menstrual cycles. Blood samples were collected at different time points: at the menstrual phase (days 2–3), periovulatory phase (days 12–13), and luteal phase (days 23–24). CRP and leptin concentrations were measured by enzyme immunoassay. GAGs were isolated using ion-exchange chromatography on DEAE-Sephacel and quantified as hexuronate. The structural characterization of chondroitin sulfate (CS) isomers was performed by fluorophore-assisted carbohydrate electrophoresis (FACE).ResultsIn the women with ovulatory cycles, plasma GAG levels differed significantly during menstrual cycle, with increased values at the periovulatory with respect to the menstrual phase. No significant differences in CRP and leptin concentrations were observed through the menstrual cycle in both the examined cycles, but inter-group analysis revealed significant differences of CRP and leptin levels between the ovulatory and anovulatory cycles with higher values at periovulatory phase in the ovulatory cycles.ConclusionsThere are no fluctuations of both total GAG concentration and CS isomer content during menstrual cycle in the anovulatory cycles. A significant correlation between CRP and gonadotrophins was found. There is no significant difference in CRP across the menstrual cycle among ovulatory cycles, but there is a trend toward higher CRP at the periovulatory than the other phases, consistent with the significant difference in CRP between ovulatory and anovulatory cycles at the periovulatory phase. Both the trend and the significant result suggest an elevation in CRP with ovulation. These observations provide additional evidences to the hypothesis that the ovulation is an inflammatory-like phenomenon.

Modifications of low density lipoprotein induced by the interaction with human plasma glycosaminoglycan-protein complexes

Glycosaminoglycan (GAG)-protein complexes from human plasma were separated into low charge (LC-GP) and high charge (HC-GP) components. LC-GP and HC-GP differed with respect to GAG and protein composition and to molecular size. The in vitro interaction of both GAG-protein complexes with human LDL was investigated. LC-GP did not precipitate LDL. On the contrary, HC-GP formed insoluble complexes with LDL, following a biphasic behaviour on increasing HC-GP concentration. In the presence of a HC-GP/LDL ratio higher than 0.02 the interaction stoichiometry was shifted towards the formation of soluble complexes. Papain treatment of HC-GP completely prevented LDL precipitation. Moreover, the extent of HC-GP-induced precipitation of LDL was markedly reduced by the simultaneous addition of LC-GP. Data obtained with standard GAGs showed that heparin (HE) and chondroitin-6-sulphate (C6S) were the most effective ligands in precipitating LDL. However, the shape of precipitation curves was markedly different. C6S behaved similarly to HC-GP, suggesting that GAG chains could play an important role in insoluble complex formation with LDL. Steady-state fluorescence anisotropy investigation indicated that HC-GP induced a significant decrease in the microviscosity of LDL hydrophobic region. This effect was no longer detectable after either addition of LC-GP or papain treatment of HC-GP. Differential scanning calorimetry (DSC) demonstrated that both lipid and protein components of LDL were affected by the interaction with HC-GP. The temperature of irreversible thermal unfolding of apo B100 was shifted to a lower value and a second peak appeared in the region of the reversible melting of cholesterol esters. Both the fluorescence anisotropy and the DSC data obtained with standard HE and C6S indicated that GAG chains were directly involved in affecting physico-chemical properties of complexed LDL. These results suggest that the interaction with plasma HC-GP could modify LDL structural properties. However, LC-GP is likely to act as a modulator, probably preventing the interaction between HC-GP and circulating LDL.

Quali-quantitative analysis of urinary glycosaminoglycans for monitoring glomerular inflammatory activity.

Objective. A 2-year follow-up study was carried out in patients with IgA nephropathy (IgAN) in order to verify the possible use of quali-quantitative analysis of urinary glycosaminoglycans (GAGs) as a prognostic index of disease and for drug treatment monitoring. Material and methods. Ten patients with IgAN were evaluated at four time points: baseline, and 6, 9 and 24 months later. GAGs were isolated from 24-h urine using ion-exchange chromatography on diethylaminoethyl–Sephacel, and concentrations were expressed as milligrams of hexuronate per gram of creatinine. GAG composition was determined by cellulose acetate electrophoresis and expressed as relative percentages by means of densitometric scanning of Alcian Blue-stained strips. Results. The relative content of total low-sulphated chondroitin sulphate species decreased significantly during the study period compared to baseline, whereas the relative percentages of heparan sulphate and chondroitin sulphate increased significantly. Moreover, a significant correlation was noted between the relative contents of urinary GAGs, renal function and inflammation indexes. Conclusions. It is likely that the excretion of various types of GAGs may be related to different glomerular pathophysiological conditions. Therefore, the determination of urinary GAG composition may represent a reliable indicator of disease activity.

A longitudinal evaluation of urinary glycosaminoglycan excretion in normoalbuminuric type 1 diabetic patients

Abstract Background: Previously, we found high urinary glycosaminoglycan (GAG) concentration, together with an altered electrophoretic pattern, in normoalbuminuric type 1 diabetic subjects with hemoglobin A1c (HbA1c) ≥8.0%. The purpose of this study was long-term evaluation of GAG excretion variations in these patients compared to those with HbA1c <8.0% at baseline who maintained better metabolic control over time. Methods: We enrolled 26 normotensive, normoalbuminuric type 1 diabetic patients and divided them into two groups according to mean HbA1c levels during the follow-up period. GAGs were isolated from 24-h urine samples on two separate occasions, at baseline and after a mean (±SD) follow-up of 6.8±1.1years. Results: All patients remained normoalbuminuric at follow-up, and had normal urinary α1-microglobulin levels. In patients with HbA1c <8.0%, total GAG levels and low sulfated chondroitin sulfate-proteoglycan/chondroitin sulfate ratio were almost unchanged during the follow-up period. In contrast, these increased in patients with HbA1c≥8.0% and were significantly related to both HbA1c levels and the duration of poor glycemic control. Conclusions: Our results show a strong influence of hyperglycemic environment on GAG metabolism in diabetes and indicate that the distribution pattern of urinary GAGs, besides their total concentration, may be predictive of altered GAG metabolism in type 1 diabetes.

Glycosaminoglycan and transforming growth factor β1 changes in human plasma and urine during the menstrual cycle, in vitro fertilization treatment, and pregnancy

OBJECTIVE To evaluate transforming growth factor beta1 (TGF-beta1) and glycosaminoglycans (GAG) changes in human plasma and urine during the menstrual cycle, IVF-ET, and pregnancy. DESIGN Prospective clinical study. SETTING University hospital. PATIENT(S) Thirteen women with apparently normal menstrual cycle (group 1); 18 women undergoing IVF-ET (group 2); and 14 low-risk pregnant women (group 3). INTERVENTION(S) We assayed plasma and urine concentrations of TGF-beta1, urine content, and distribution of GAG. Blood and urine samples were collected during days 2 to 3, 12 to 13, and 23 to 24 in group 1; in group 2, samples were obtained at menstrual phase, oocyte pick-up day, and 15 days after ET; in group 3, samples were obtained during gestational weeks 10-12, 22-24, and 30-32 and 1 month after delivery. MAIN OUTCOME MEASURE(S) Changes in TGF-beta1 and GAG content. RESULT(S) The mean value of total urinary trypsin inhibitor/chondroitin sulfate (UTI/CS) showed a distinct peak at day 12 of the menstrual cycle in the fertile women in whom we monitored the ovulatory period. In the IVF-ET group, GAG distribution and TGF-beta1 levels showed significant differences during the cycle. We observed increased levels of plasma TGF-beta1 15 days after ET. A significant increase of total UTI/CS value with increasing gestation was detected. CONCLUSION(S) Transforming growth factor beta1 and GAG levels could represent an additional tool to monitor reproductive events and could be useful, noninvasive markers of ovulation and ongoing pregnancy.

A reversed phase HPLC method for the simultaneous determination of all monosaccharides contained in galactosaminoglycan isomers from human aorta proteoglycans

Monosaccharides obtained by reduction and hydrolysis of galactosaminoglycan isomers, are entirely determined as their perbenzoyl derivatives by reversed phase HPLC, without removal of hexosamines prior to benzoylation. The method is suitable for the analysis of arterial proteoglycan constituent galactosaminoglycans, providing specific, precise and reproducible results. Moreover, synthesis and characterization of tri-O-benzoyl-1,6-L-anhydroidose and N-benzoyl-tetra-O-benzoyl-alpha- and -beta-D-galactosamine have been accomplished.