Purification of an Acidic Polysaccharide with Anticoagulant Activity from the Marine Sponge Sarcotragus spinosulus.

Thromboembolic conditions are the most common cause of death in developed countries. Anticoagulant therapy is the treatment of choice, and heparinoids and warfarin are the most adopted drugs. Sulphated polysaccharides extracted from marine organisms have been demonstrated to be effective alternatives, blocking thrombus formation by inhibiting some factors involved in the coagulation cascade. In this study, four acidic glycan fractions from the marine sponge Sarcotragus spinosulus were purified by anion-exchange chromatography, and their anticoagulant properties were investigated through APTT and PT assays and compared with both standard glycosaminoglycans and holothurian sulphated polysaccharides. Moreover, their topographic localization was assessed through histological analysis, and their cytocompatibility was tested on a human fibroblast cell line. A positive correlation between the amount of acid glycans and the inhibitory effect towards both the intrinsic and extrinsic coagulation pathways was observed. The most effective anticoagulant activity was shown by a highly charged fraction, which accounted for almost half (about 40%) of the total hexuronate-containing polysaccharides. Its preliminary structural characterization, performed through infrared spectroscopy and nuclear magnetic resonance, suggested that it may consist of a fucosylated chondroitin sulphate, whose unique structure may be responsible for the anticoagulant activity reported herein for the first time.

Searching for Atherosclerosis Biomarkers by Proteomics: A Focus on Lesion Pathogenesis and Vulnerability.

Plaque rupture and thrombosis are the most important clinical complications in the pathogenesis of stroke, coronary arteries, and peripheral vascular diseases. The identification of early biomarkers of plaque presence and susceptibility to ulceration could be of primary importance in preventing such life-threatening events. With the improvement of proteomic tools, large-scale technologies have been proven valuable in attempting to unravel pathways of atherosclerotic degeneration and identifying new circulating markers to be utilized either as early diagnostic traits or as targets for new drug therapies. To address these issues, different matrices of human origin, such as vascular cells, arterial tissues, plasma, and urine, have been investigated. Besides, proteomics was also applied to experimental atherosclerosis in order to unveil significant insights into the mechanisms influencing atherogenesis. This narrative review provides an overview of the last twenty years of omics applications to the study of atherogenesis and lesion vulnerability, with particular emphasis on lipoproteomics and vascular tissue proteomics. Major issues of tissue analyses, such as plaque complexity, sampling, availability, choice of proper controls, and lipoproteins purification, will be raised, and future directions will be addressed.

A Method for Urinary Trypsin Inhibitor (UTI) Purification Combining Anion-Exchange Chromatography Enrichment and Preparative SDS-PAGE.

This chapter describes a method for the purification of urinary trypsin inhibitor (UTI), a small chondroitin sulfate proteoglycan with Ser-proteinase inhibitory activity, excreted at high levels into urine following an inflammatory condition. The method consists of two fractionation steps: an anion-exchange chromatography and a sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) followed by Coomassie Brilliant Blue G-250 gel staining. Several UTI bands are excised from gel, minced, destained, and dehydrated for extraction with SDS-containing buffer, at 60 °C for 24 h. This allows for obtaining a highly purified UTI sample useful for both structural and functional studies.

Molecular and pathobiological insights of bikunin/UTI in cancer.

Bikunin is a small chondroitin sulfate proteoglycan (PG) with Ser-protease inhibitory activity that plays pleiotropic roles in health and disease. It is involved in several physiological processes including stabilization of the extracellular matrix (ECM) of connective tissues and key reproductive events. Bikunin is also implicated in both acute and chronic inflammatory conditions and represents a non-invasive circulating and/or urinary (as Urinary Trypsin Inhibitor or UTI) biomarker. It exerts inhibitory effects on urokinase-type plasminogen activator (uPA) and its receptor (uPAR) mediating tumor invasiveness by a down-regulation of uPA mRNA expression, thus representing an anti-metastatic agent. However, only limited data on its potential as a diagnostic and/or prognostic marker of cancer have been reported so far. Recent technological advances in mass spectrometry-based proteomics have provided researchers with a huge amount of information allowing for large-scale surveys of the cancer proteome. To address such issues, we analyzed bikunin expression data across several types of tumors, by using UALCAN proteogenomic analysis portal. In this article we critically review the roles of bikunin in human pathobiology, with a special focus on its inhibitory effects and mechanisms in cancer aggressiveness as well as its significance as cancer circulating biomarker.

Molecular Characterization of Plasma HDL, LDL, and VLDL Lipids Cargos from Atherosclerotic Patients with Advanced Carotid Lesions: A Preliminary Report.

Carotid atherosclerosis represents a relevant healthcare problem, since unstable plaques are responsible for approximately 15% of neurologic events, namely transient ischemic attack and stroke. Although statins treatment has proven effective in reducing LDL-cholesterol and the onset of acute clinical events, a residual risk may persist suggesting the need for the detection of reliable molecular markers useful for the identification of patients at higher risk regardless of optimal medical therapy. In this regard, several lines of evidence show a relationship among specific biologically active plasma lipids, atherosclerosis, and acute clinical events. We performed a Selected Reaction Monitoring-based High Performance Liquid Chromatography-tandem Mass Spectrometry (SRM-based HPLC-MS/MS) analysis on plasma HDL, LDL, and VLDL fractions purified, by isopycnic salt gradient ultracentrifugation, from twenty-eight patients undergoing carotid endarterectomy, having either a "hard" or a "soft" plaque, with the aim of characterizing the specific lipidomic patterns associated with features of carotid plaque instability. One hundred and thirty lipid species encompassing different lipid (sub)classes were monitored. Supervised multivariate analysis showed that lipids belonging to phosphatidylethanolamine (PE), sphingomyelin (SM), and diacylglycerol (DG) classes mostly contribute to discrimination within each lipoprotein fraction according to the plaque typology. Differential analysis evidenced a significant dysregulation of LDL PE (38:6), SM (32:1), and SM (32:2) between the two groups of patients (adj. p-value threshold = 0.05 and log2FC ≥ |0.58|). Using this approach, some LDL-associated markers of plaque vulnerability have been identified, in line with the current knowledge of the key roles of these phospholipids in lipoprotein metabolism and cardiovascular disease. This proof-of-concept study reports promising results, showing that lipoprotein lipidomics may present a valuable approach for identifying new biomarkers of potential clinical relevance.

Collagenic architecture and morphotraits in a marine basal metazoan as a model for bioinspired applied research.

In some Porifera (Demospongiae: Keratosa), prototypes of the connective system are almost exclusively based on collagenic networks. We studied the topographic distribution, spatial layout, microtraits, and/or morphogenesis of these collagenic structures in Ircinia retidermata (Dictyoceratida: Irciniidae). Analyses were carried out on a clonal strain from sustainable experimental mariculture by using light and scanning electron microscopy. Histology revealed new insights on the widely diversified and complex hierarchical assemblage of collagenic structures. Key evolutionary novelties in the organization of sponge connective system were found out. The aquiferous canals are shaped as corrugate-like pipelines conferring plasticity to the water circulation system. Compact clusters of elongated cells are putatively involved in a nutrient transferring system. Knob-ended filaments are characterized by a banding pattern and micro-components. Ectosome and outer endosome districts are the active fibrogenetic areas, where exogenous material constitutes an axial condensation nucleus for the ensuing morphogenesis. The new data can be useful to understand not only the evolutionary novelties occurring in the target taxon but also the morpho-functional significance of its adaptive collagenic anatomical traits. In addition, data may give insights on both marine collagen sustainable applied researches along with evolutionary and phylogenetic analyses, thus highlighting sponges as a key renewable source for inspired biomaterials. Therefore, we also promote bioresources sustainable exploitation with the aim to provide new donors of marine collagen, thereby supporting conservation of wild populations/species.

Apolipoprotein Signature of HDL and LDL from Atherosclerotic Patients in Relation with Carotid Plaque Typology: A Preliminary Report.

In the past years, it has become increasingly clear that the protein cargo of the different lipoprotein classes is largely responsible for carrying out their various functions, also in relation to pathological conditions, including atherosclerosis. Accordingly, detailed information about their apolipoprotein composition and structure may contribute to the revelation of their role in atherogenesis and the understanding of the mechanisms that lead to atherosclerotic degeneration and toward vulnerable plaque formation. With this aim, shotgun proteomics was applied to identify the apolipoprotein signatures of both high-density and low-density lipoproteins (HDL and LDL) plasma fractions purified from healthy volunteers and atherosclerotic patients with different plaque typologies who underwent carotid endarterectomy. By this approach, two proteins with potential implications in inflammatory, immune, and hemostatic pathways, namely, integrin beta-2 (P05107) and secretoglobin family 3A member 2 (Q96PL1), have been confirmed to belong to the HDL proteome. Similarly, the list of LDL-associated proteins has been enriched with 21 proteins involved in complement and coagulation cascades and the acute-phase response, which potentially double the protein species of LDL cargo. Moreover, differential expression analysis has shown protein signatures specific for patients with "hard" or "soft" plaques.

Glycosaminoglycans: From Vascular Physiology to Tissue Engineering Applications.

Cardiovascular diseases represent the number one cause of death globally, with atherosclerosis a major contributor. Despite the clinical need for functional arterial substitutes, success has been limited to arterial replacements of large-caliber vessels (diameter > 6 mm), leaving the bulk of demand unmet. In this respect, one of the most challenging goals in tissue engineering is to design a "bioactive" resorbable scaffold, analogous to the natural extracellular matrix (ECM), able to guide the process of vascular tissue regeneration. Besides adequate mechanical properties to sustain the hemodynamic flow forces, scaffold's properties should include biocompatibility, controlled biodegradability with non-toxic products, low inflammatory/thrombotic potential, porosity, and a specific combination of molecular signals allowing vascular cells to attach, proliferate and synthesize their own ECM. Different fabrication methods, such as phase separation, self-assembly and electrospinning are currently used to obtain nanofibrous scaffolds with a well-organized architecture and mechanical properties suitable for vascular tissue regeneration. However, several studies have shown that naked scaffolds, although fabricated with biocompatible polymers, represent a poor substrate to be populated by vascular cells. In this respect, surface functionalization with bioactive natural molecules, such as collagen, elastin, fibrinogen, silk fibroin, alginate, chitosan, dextran, glycosaminoglycans (GAGs), and growth factors has proven to be effective. GAGs are complex anionic unbranched heteropolysaccharides that represent major structural and functional ECM components of connective tissues. GAGs are very heterogeneous in terms of type of repeating disaccharide unit, relative molecular mass, charge density, degree and pattern of sulfation, degree of epimerization and physicochemical properties. These molecules participate in a number of vascular events such as the regulation of vascular permeability, lipid metabolism, hemostasis, and thrombosis, but also interact with vascular cells, growth factors, and cytokines to modulate cell adhesion, migration, and proliferation. The primary goal of this review is to perform a critical analysis of the last twenty-years of literature in which GAGs have been used as molecular cues, able to guide the processes leading to correct endothelialization and neo-artery formation, as well as to provide readers with an overall picture of their potential as functional molecules for small-diameter vascular regeneration.

Circulating Heparan Sulfate Proteoglycans as Biomarkers in Health and Disease.

Cell-surface heparan sulfate proteoglycans (HSPGs) play key roles in regulating cell behavior, cell signaling, and cell matrix interactions in both physiological and pathological conditions. Their soluble forms from glycocalyx shedding are not merely waste products, but, rather, bioactive molecules, detectable in serum, which may be useful as diagnostic and prognostic markers. In addition, as in the case of glypican-3 in hepatocellular carcinoma, they may be specifically expressed by pathological tissue, representing promising targets for immunotherapy. The primary goal of this comprehensive review is to critically survey the main findings of the clinical data from the last 20 years and provide readers with an overall picture of the diagnostic and prognostic value of circulating HSPGs. Moreover, issues related to the involvement of HSPGs in various pathologies, including cardiovascular disease, thrombosis, diabetes and obesity, kidney disease, cancer, trauma, sepsis, but also multiple sclerosis, preeclampsia, pathologies requiring surgery, pulmonary disease, and others will be discussed.

HDAC1 inhibition ameliorates TDP-43-induced cell death in vitro and in vivo.

TDP-43 pathology is a disease hallmark that characterizes both amyotrophic lateral sclerosis (ALS) and frontotemporal lobar degeneration (FTLD-TDP). TDP-43 undergoes several posttranslational modifications that can change its biological activities and its aggregative propensity, which is a common hallmark of different neurodegenerative conditions. New evidence is provided by the current study pointing at TDP-43 acetylation in ALS cellular models. Using both in vitro and in vivo approaches, we demonstrate that TDP-43 interacts with histone deacetylase 1 (HDAC1) via RRM1 and RRM2 domains, that are known to contain the two major TDP-43 acetylation sites, K142 and K192. Moreover, we show that TDP-43 is a direct transcriptional activator of CHOP promoter and this activity is regulated by acetylation. Finally and most importantly, we observe both in cell culture and in Drosophila that a HDCA1 reduced level (genomic inactivation or siRNA) or treatment with pan-HDAC inhibitors exert a protective role against WT or pathological mutant TDP-43 toxicity, suggesting TDP-43 acetylation as a new potential therapeutic target. HDAC inhibition efficacy in neurodegeneration has long been debated, but future investigations are warranted in this area. Selection of more specific HDAC inhibitors is still a promising option for neuronal protection especially as HDAC1 appears as a downstream target of both TDP- 43 and FUS, another ALS-related gene.

Plasma vitronectin is reduced in patients with myasthenia gravis: Diagnostic and pathophysiological potential.

Myasthenia gravis (MG) is an autoimmune disease leading to varying degrees of skeletal muscle weakness. It is caused by specific antibodies directed against definite components in the postsynaptic membrane at the neuromuscular junction (NMJ), such as the acetylcholine receptor (AChR) and the muscle-specific kinase (MUSK) receptor. In clinical practice, MG patients may be classified into three main subgroups based on the occurrence of serum autoantibodies directed against AChR or MUSK receptor or antibody-negative. As the MG subgroups differ in terms of clinical characteristics, disease pathogenesis, prognosis, and response to therapies, they could benefit from targeted treatment as well as the detection of other possible disease biomarkers. We performed proteomics on plasma fractions enriched in low-abundance proteins to identify potential biomarkers according to different autoimmune responses. By this approach, we evidenced a significant reduction of vitronectin in MG patients compared to healthy controls, irrespective of the autoantibodies NMJ target. The obtained results were validated by mono- and two-dimensional Western blotting analysis. Vitronectin is a multifunctional glycoprotein involved in the regulation of several pathophysiological processes, including complement-dependent immune response, coagulation, fibrinolysis, pericellular proteolysis, cell attachment, and spreading. The pathophysiological significance of the reduction of plasma vitronectin in MG patients has yet to be fully elucidated. It could be related either to a possible deposition of vitronectin at NMJ to counteract the complement-mediated muscle damage at this level or to a parallel variation of this glycoprotein in the muscle extracellular matrix with secondary induced alteration in clustering of AChRs at NMJ, as it occurs with variation in concentrations of agrin, another extracellular matrix component. The clinical value of measuring plasma vitronectin has yet to be defined. According to present findings, significantly lower plasma values of this glycoprotein might be indicative of an impaired complement-dependent immune response.

Glycosaminoglycan functionalization of electrospun scaffolds enhances Schwann cell activity.

Nerve fibers of the peripheral nervous system (PNS) have a remarkable ability to regenerate up to an almost complete recovery of normal function following a crush or a Sunderland Type II injury. This process is governed by glial cells, known as Schwann cells, through their unique capacity to dedifferentiate into cells that drive the healing process. Despite that many progresses have occurred in restorative medicine and microsurgery, the regenerative process after a severe lesion of a major nerve trunk (e.g., Sunderland Types III-V) is often incomplete and functional recovery is unsatisfactory. In this aspect, it is known that glycosaminoglycans (GAGs) of the extracellular matrix are involved in proliferation, synaptogenesis, neural plasticity, and regeneration of the PNS. Here, we developed poly(caprolactone) (PCL) fibrous scaffolds functionalized with GAGs, which allowed us to assess their influence on the adhesion, proliferation, and differentiation of Schwann cells. We found that both aligned and random fiber scaffolds functionalized with GAGs resulted in increased cell proliferation on day 1. In addition, aligned functionalized scaffolds also resulted in increased GAG presence on day 1, probably because of cell extracellular matrix (ECM) formation and an increased syndecan-4 expression on day 7. A different modification and activation of Schwann cells in the presence of GAG versus no-GAG scaffolds was underlined by proteomic comparative analysis, where a general downregulation of the expression of intracellular/structural and synthetic proteins was shown on day 7 for GAG-functionalized scaffolds with regard to the nonfunctionalized ones. In conclusion, we have shown that GAG-functionalized scaffolds are effective in modulating Schwann cell behavior in terms of adhesion, proliferation, and differentiation and should be considered in strategies to improve PNS repair. STATEMENT OF SIGNIFICANCE: Nerve fibers functional recovery following a severe trauma of the Peripheral Nervous System (PNS) still represents a huge challenge for neurosurgery nowadays. In this respect, tissue engineering is committed to develop new constructs able to guide Schwann cells by mimicking the natural extracellular matrix environment. To this purpose, we successfully fabricated polycaprolactone (PCL) scaffolds with two well-defined fiber deposition patterns, functionalized with glycosaminoglycans (GAGs) and assessed for their potential as support for Schwann cells adhesion, growth and differentiation, by both classical biochemistry and LC-MS-based proteomic profiling. By this way, we showed that PCL-GAGs scaffolds could represent a promising artificial substrate that closely mimics the recently established pattern of Schwann cells migration into the regenerating nerve and, therefore, it should be considered in strategies to improve PNS repair.

Levels of Urinary Trypsin Inhibitor and Structure of Its Chondroitin Sulphate Moiety in Type 1 and Type 2 Diabetes.

BACKGROUND: Diabetes mellitus is a global health problem representing the fifth leading cause of mortality and a major risk factor for cardiovascular diseases. In the last years, we reported an association among urinary trypsin inhibitor (UTI), a small proteoglycan that plays pleiotropic roles in many inflammatory processes, and both type 1 and 2 diabetes and developed a method for its direct quantitation and structural characterization. METHODS: Urine from 39 patients affected by type 1 diabetes, 32 patients with type 2 diabetes, and 52 controls were analysed. UTI was separated from the main glycosaminoglycans physiologically present in urine by anion exchange chromatography, treated for chondroitin sulphate (CS) chain complete depolymerisation, and analysed for both UTI content and CS structure. UTI identification was performed by nano-LC-MS/MS analysis. RESULTS: We evidenced increased UTI levels, as well as reduced sulphation of its CS moiety in association with diabetes, regardless of both age and medium-term glycaemic control. Furthermore, no association between UTI and albumin excretion rate was found. CONCLUSIONS: Evidences suggest that UTI levels are not directly correlated with renal function or, otherwise, that they may increase before the onset of renal impairment in diabetes, representing a potential marker for the underlying inflammatory condition.

Plasma PP13 and urinary GAGs/PGs as early markers of pre-eclampsia.

PURPOSE: To evaluate at 11-13 weeks' gestation biochemical markers that may predict complications of pregnancy such as pre-eclampsia, proteinuria, and hypertension. METHODS: Analyses were performed on first-morning urine and plasma samples from first trimester pregnant women with increased risk of developing pre-eclampsia such as positive personal or family history of cardiovascular disease and diabetes mellitus. A total of 62 women were enrolled, 24 of them presented complications such as pre-eclampsia, proteinuria, and hypertension during pregnancy. The remaining 38 women had a physiological course of pregnancy and formed the reference group. Urine glycosaminoglycans/proteoglycans (GAGs/PGs) distribution was determined by electrophoresis on cellulose acetate strips. Urinary N-acetyl-ß-glucosaminidase was estimated kinetically. Plasma levels of placental protein 13 (PP13) were measured by enzyme-linked immunosorbent assay. RESULTS: No significant differences in total GAG excretion and N-acetyl-ß-glucosaminidase (NAG) concentration were observed between the two groups of pregnant women, whereas we detected increased relative content of total urinary trypsin inhibitor (UTI plus low-sulfated chondroitin sulfate) (p = 0.001) and reduced excretion of heparan sulfate (p = 0.007) and chondroitin sulfate (p = 0.011) in women presenting with pregnancy complications respect to controls. Plasma levels of PP13 were significantly reduced in the group of women who went on to develop complications compared with controls (p = 0.022). CONCLUSIONS: The reduced plasma levels of PP13 and the alteration of the relative content of urinary GAGs and PGs observed in our study could be a promising tool for the prediction of pre-eclampsia in an early stage of pregnancy.

Identification of differentially expressed plasma proteins in atherosclerotic patients with type 2 diabetes.

Besides hyperglycaemia and insulin resistance, several factors are associated with a higher cardiovascular risk in type 2 diabetes mellitus (T2DM), many of them being closely related to each other owing to common origins or pathways. The pathophysiological mechanisms underlying vascular dysfunctions in diabetes include reduced bioavailability of nitric oxide, increased ROS and prothrombotic factors production, as well as activation of receptors for advanced glycation end-products. These alterations contribute to create a pro-inflammatory/thrombotic state that ultimately leads to plaque formation and complication. This study aimed at identifying differentially expressed plasma proteins between T2DM and non-diabetic patients undergoing carotid endarterectomy, by means of two-dimensional electrophoresis coupled with LC-MS/MS. Before analysis, plasma samples were enriched in low-expression proteins through combinatorial hexapeptide ligand libraries. Both mono- and two-dimensional western blotting were performed for data validation. Differentially expressed proteins were mapped onto STRING v10 to build a protein-protein interaction network. Sixteen differentially expressed spots were identified with a high score. Among them, there were fibrinogen beta and gamma chains, complement C1r, C3 and C4-B subcomponents, alpha-1-antitrypsin (AAT), vitronectin and CD5 antigen-like. Protein-Protein interaction analysis evidenced a network among differentially expressed proteins in which vitronectin seems to represent a potentially pivotal node among fibrinolysis, complement dependent immune responses and inflammation in accordance with a number of in vitro and in vivo evidences for a contributory role of these proteins to the development of diabetic atherosclerosis.

Evaluation of Early Markers of Nephropathy in Patients with Type 2 Diabetes Mellitus.

Aims. T2DM often remains undiagnosed for many years because hyperglycemia develops gradually and may not produce any symptoms. As patients with T2DM are at increased risk of microvascular and macrovascular complications, the preclinical diagnosis of the state is the key point of the disease management. Methods. We evaluated parameters such as GAGs/PGs, NAG, and NGAL in urine samples from 43 normoalbuminuric T2DM patients and 31 apparently healthy control subjects. Results. The total urinary GAG excretion showed no significant differences between patients and controls. The electrophoretic analysis evidenced the presence of UTI and its degradation products (LSC and SM-LSC), CS, and HS. We observed modifications of HS and total UTI (including UTI and its degradation products) relative contents in T2DM patients compared with controls whereas no differences in CS percentage were found. NGAL levels were significantly increased in T2DM patients and were positively correlated with both NAG (r = 0.606, p < 0.0001) and the presence of hypertension (r = 0.352, p < 0.05). Conclusions. These data suggest that the assessed molecules could represent useful markers to detect early renal impairment in patients with T2DM.

Human serum albumin Cys34 oxidative modifications following infiltration in the carotid atherosclerotic plaque.

OBJECTIVES: To evaluate if the prooxidant environment present in atherosclerotic plaque may oxidatively modify filtered albumin. METHODS: Fluorescein-5-maleimide labelled plasma samples and plaque extracts from 27 patients who had undergone carotid endarterectomy were analysed through nonreducing SDS-PAGE for albumin-Cys(34) oxidation. Furthermore, degree and pattern of S-thiolation in both circulating and plaque-filtered albumin were assayed. RESULTS: Albumin filtered in the atherosclerotic plaque showed higher levels of Cys(34) oxidative modifications than the corresponding circulating form as well as different patterns of S-thiolation. CONCLUSIONS: Data indicate that the circulating albumin, once filtered in plaque, undergoes Cys(34) oxidative modifications and demonstrate for the first time that albumin is a homocysteine and cysteinylglycine vehicle inside the plaque environment.

Development of a method for urine bikunin/urinary trypsin inhibitor (UTI) quantitation and structural characterization: Application to type 1 and type 2 diabetes.

Bikunin is a plasma proteinase inhibitor often associated with inflammatory conditions. It has a half-life of few minutes and it is rapidly excreted into urine as urinary trypsin inhibitor (UTI). UTI levels are usually low in healthy individuals but they can increase up to tenfold in both acute and chronic inflammatory diseases. This article describes a sensitive method for both direct UTI quantitation and structural characterization. UTI purification was performed by anion exchange micro-chromatography followed by SDS-PAGE. A calibration curve for protein quantitation was set up by using a purified UTI fraction. UTI identification and structural characterization was performed by Nano-LC-MS/MS analysis. The method was applied on urine samples from 9 patients with type 1 diabetes, 11 patients with type 2 diabetes, and 28 healthy controls, matched for age and sex with patients, evidencing higher UTI levels in both groups of patients with respect to controls (p < 0.001 and p = 0.001, respectively). Spearman's correlation tests highlighted no association between UTI levels and age in each group tested. Owing to the elevated sensitivity and specificity, the described method allows UTI quantitation from very low quantities of specimen. Furthermore, as UTI concentration is normalized for creatinine level, the analysis could be also performed on randomly collected urine samples. Finally, MS/MS analysis prospects the possibility of characterizing PTM sites potentially able to affect UTI localization, function, and pathophysiological activity. Preliminary results suggest that UTI levels could represent a useful marker of chronic inflammatory condition in type 1 and 2 diabetes.

Urine bikunin as a marker of renal impairment in Fabry's disease.

Fabry's disease is a rare lysosomal storage disorder caused by the deficiency of α -galactosidase A that leads to the accumulation of neutral glycosphingolipids in many organs including kidney, heart, and brain. Since end-stage renal disease represents a major complication of this pathology, the aim of the present work was to evaluate if urinary proteoglycan/glycosaminoglycan excretion could represent a useful marker for monitoring kidney function in these patients at high risk. Quali-quantitative and structural analyses were conducted on plasma and urine from 24 Fabry's patients and 43 control subjects. Patients were sorted for presence and degree of renal impairment (proteinuria/renal damage). Results showed that levels of urine bikunin, also known as urinary trypsin inhibitor (UTI), are significantly higher in patients with renal impairment than in controls. In this respect, no differences were evidenced in plasma chondroitin sulfate isomers level/structure indicating a likely direct kidney involvement. Noteworthy, urine bikunin levels are higher in patients since early symptoms of renal impairment occur (proteinuria). Overall, our findings suggest that urine bikunin level, as well as proteinuria, could represent a useful parameter for monitoring renal function in those patients that do not present any symptoms of renal insufficiency.

Protein sulfhydryl group oxidation and mixed-disulfide modifications in stable and unstable human carotid plaques.

OBJECTIVES: Oxidative stress has been implicated in the outcome of atherosclerotic plaques. However, at present, no data are available neither on the degree of plaque protein sulfhydryl groups oxidation nor on its relationship with plaque vulnerability. We investigated the entity of protein-SH oxidative modifications, focusing on low molecular weight thiols adduction, in human carotid plaque extracts in relation to plaque stability/instability. METHODS: Plaque stability/instability was histologically assessed. The extent of protein-SH oxidative modifications was established by a differential proteomic approach on fluorescein-5-maleimide-labeled plaque extracts and corresponding plasma samples from 48 endarterectomized patients. The analysis on protein thiolation was performed by capillary zone electrophoresis. RESULTS: We observed a higher protein-SH oxidation of both plasma-derived and topically expressed proteins in unstable plaques, partly due to higher levels of S-thiolation. Conversely, in plasma, none of the investigated parameters discriminated among patients with stable and unstable plaques. CONCLUSIONS: Our results suggest the presence of a more pronounced oxidative environment in unstable plaques. Identifying specific oxidative modifications and understanding their effects on protein function could provide further insight into the relevance of oxidative stress in atherosclerosis.