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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.

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.

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., (© 2022. The Author(s).)

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., (© 2023. The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature.)

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 log 2 FC ≥ |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.

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.

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., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Lepedda, Nieddu, Formato, Baker, Fernández-Pérez and Moroni.)

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., Competing Interests: None declared., (Thieme. All rights reserved.)

Purpose: Female reproductive events, including ovulation, menstruation, implantation, and delivery, are physiologically characterized by deep tissue remodeling and display hallmark signs of inflammation. This review discusses the pleiotropic roles played by bikunin in human reproduction., Methods: A comprehensive literature search of the Medline/PubMed database was performed on the following topics: bikunin structure, roles in pathophysiological conditions and involvement in human reproduction, and usefulness as a marker of gestational complications or as a drug to improve pregnancy outcomes., Results: Bikunin is a small chondroitin sulfate proteoglycan found in blood, urine, and amniotic and cerebrospinal fluids, known for its anti-inflammatory and anti-proteolytic activities. Its levels are usually low, but they can increase several-fold in both acute and chronic inflammatory diseases. Bikunin plays key roles in reproductive events, such as cumulus-oocyte complex formation, pregnancy, and delivery. Its levels have been associated with the most common pregnancy complications such as preterm delivery, pre-eclampsia, and gestational diabetes mellitus. Finally, its intravaginal administration has been reported to reduce the risk of preterm delivery and to improve neonatal outcomes., Conclusions: Because of its pleiotropic roles in several reproductive events and its association with some life-threatening pathological conditions of pregnancy, bikunin may represent a non-invasive marker for improving follow-up and early diagnosis. Studies showing its usefulness as a drug for reducing the risk of preterm delivery and improving neonatal outcomes have yielded interesting results that deserve to be investigated through further research.

Although oxidative stress has been long associated with the genesis and progression of the atherosclerotic plaque, scanty data on its in situ effects on protein sulfhydryl group modifications are available. Within the arterial wall, protein sulfhydryls and low-molecular-weight (LMW) thiols are involved in the cell regulation of both Reactive Oxygen Species (ROS) and Reactive Nitrogen Species (RNS) levels and are a target for several posttranslational oxidative modifications that take place inside the atherosclerotic plaque, probably contributing to both atherogenesis and atherosclerotic plaque progression towards complicated lesions. Advanced carotid plaques are characterized by very high intraplaque GSH levels, due to cell lysis during apoptotic and/or necrotic events, probably responsible for the altered equilibrium among protein sulfhydryls and LMW thiols. Some lines of evidence show that the prooxidant environment present in atherosclerotic tissue could modify filtered proteins also by protein-SH group oxidation, and demonstrate that particularly albumin, once filtered, represents a harmful source of homocysteine and cysteinylglycine inside the plaque. The oxidative modification of protein sulfhydryls, with particular emphasis to protein thiolation by LMW thiols and its association with atherosclerosis, is the main topic of this review., Competing Interests: The authors declare that they have no conflicts of interest., (Copyright © 2020 Antonio Junior Lepedda and Marilena Formato.)

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., Competing Interests: Declaration of Conflicting Interests: The author(s) declared no potential conflicts of interest with respect to the research, authorship, and/or publication of this article., (© The Author(s) 2019.)

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.

Because of the high incidence of kidney disease in diabetic patients, the early diagnosis of renal impairment is a key point for intervention and management. Although urinary albumin excretion currently represents the accepted standard to assess both diabetic nephropathy and cardiovascular risk, it has some limitations as structural changes in the glomerular basement membrane may occur before the onset of microalbuminuria. It is therefore important to identify urinary markers that may provide greater sensitivity, earlier detection, and greater predictive power for diabetes complications. In this respect, urinary glycosaminoglycans/proteoglycans (GAGs/PGs) have been long associated with several kidney diseases as well as diabetic nephropathies as their levels increase more readily than albuminuria. In particular, heparan sulfate, a key component of the glomerular basement membrane responsible for its charge-dependent permeability, is excreted into urine at higher concentrations during the early kidney remodeling events caused by the altered glucose metabolism in diabetes. Over the past few years, also urinary trypsin inhibitor has been linked to a chronic inflammatory condition in both type 1 and 2 diabetes. The underlying mechanisms of such increase are not completely known since either a systemic inflammatory condition or a more localized early renal impairment could play a role. Nevertheless, the association with other inflammatory markers and a detailed urinary trypsin inhibitor structural characterization in diabetes remain to be elucidated. This review will discuss a great deal of information on the association between urinary GAGs/PGs and type 1 and 2 diabetes, with particular emphasis on renal involvement, and their potential as markers useful in screening, diagnosis and follow up to be associated with the current standard tests., (Copyright © 2017. Published by Elsevier Inc.)

The growing interest in the use of recyclable and biodegradable natural materials has become a relevant topic in pharmaceutics. In this work, we suggest the use and valorization of natural horny skeleton of marine sponges (Porifera, Dictyoceratida) as bio-based dressing for topical drug delivery. Biomaterial characterization focusing on morpho-functional traits, swelling behavior, fluid uptake performances, glycosaminoglycans content and composition and microbiological quality assessment was carried out to investigate the collagenic skeleton properties. After grinding and sieving processes, l-cysteine hydrochloride-loaded formulations were designed in form of powder or polymeric film by testing various drug concentrations and different drying parameters. Drug content, SEM analyses and in vitro permeation studies were performed to test the suitability of skeleton-based formulations. To this respect, drying time and temperature are key parameters for skeleton-mediated drug crystallization. Consequently, this behavior seems to influence drug loading and permeation profiles of formulations. The high percentages of drug are found after absorption into sponge powder and in vitro permeation studies demonstrate that cysteine is released more slowly than the pure drug within 1h. Such a system is attractive because it combines the known healing properties of cysteine with the advantageous potentials of the collagen/proteoglycan network, which can act as biocompatible carrier able to absorb the excess of the wound exudate while releasing the drug. Furthermore, due to its glycosaminoglycans content, natural sponge skeletal scaffold might act as bioactive-biomimetic carrier regulating the wound healing processes., (Copyright © 2016. Published by Elsevier B.V.)

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-β-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-β-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.

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., (Copyright © 2016 Elsevier Inc. All rights reserved.)

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.

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.

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., (© 2013 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Allograft rejection during the first year after renal transplantation can lead to persistent allograft dysfunction and reduced long-term graft survival. Thus, it is important to define early predictors of kidney damage, less invasive than allograft biopsy. Urinary glycosaminoglycan/proteoglycan concentration and distribution, N-acetyl-β-(D)-glucosaminidase (NAG), and monokine induced by IFN-γ (MIG) levels were evaluated in the immediate post-transplant and during a 1-year follow-up. We observed increased urinary levels of MIG, urinary trypsin inhibitor and its degradation products, the lack of urinary heparan sulfate excretion, and the decreased chondroitin sulfate relative content at day 1 post-transplant in most patients who developed complications in the postoperative period. Moreover, urinary MIG levels showed significant correlations with NAG, C-reactive protein, and GFR at day 1 post-transplant. The monitoring of glycosaminoglycan/proteoglycan urinary pattern and the levels of urine MIG could serve as useful markers for predicting possible complications of transplantation, unraveling an early inflammatory state, on whose basis the immunosuppressive therapy could be appropriately modified.

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.

Apolipoproteins are very heterogeneous protein family, implicated in plasma lipoprotein structural stabilization, lipid metabolism, inflammation, or immunity. Obtaining detailed information on apolipoprotein composition and structure may contribute to elucidating lipoprotein roles in atherogenesis and to developing new therapeutic strategies for the treatment of lipoprotein-associated disorders. This study aimed at developing a comprehensive method for characterizing the apolipoprotein component of plasma VLDL, LDL, and HDL fractions from patients undergoing carotid endarterectomy, by means of two-dimensional electrophoresis (2-DE) coupled with Mass Spectrometry analysis, useful for identifying potential markers of plaque presence and vulnerability. The adopted method allowed obtaining reproducible 2-DE maps of exchangeable apolipoproteins from VLDL, LDL, and HDL. Twenty-three protein isoforms were identified by peptide mass fingerprinting analysis. Differential proteomic analysis allowed for identifying increased levels of acute-phase serum amyloid A protein (AP SAA) in all lipoprotein fractions, especially in LDL from atherosclerotic patients. Results have been confirmed by western blotting analysis on each lipoprotein fraction using apo AI levels for data normalization. The higher levels of AP SAA found in patients suggest a role of LDL as AP SAA carrier into the subendothelial space of artery wall, where AP SAA accumulates and may exert noxious effects.

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.

Cardiac valves are dynamic structures, exhibiting a highly specialized architecture consisting of cells and extracellular matrix with a relevant proteoglycan and glycosaminoglycan content, collagen and elastic fibers. Biological valve substitutes are obtained from xenogenic cardiac and pericardial tissues. To overcome the limits of such non viable substitutes, tissue engineering approaches emerged to create cell repopulated decellularized scaffolds. This study was performed to determine the glycosaminoglycans content, distribution, and disaccharides composition in porcine aortic and pulmonary valves and in pericardium before and after a detergent-based decellularization procedure. The fine structural characteristics of galactosaminoglycans chondroitin sulfate and dermatan sulfate were examined by FACE. Furthermore, the mechanical properties of decellularized pericardium and its propensity to be repopulated by in vitro seeded fibroblasts were investigated. Results show that galactosaminoglycans and hyaluronan are differently distributed between pericardium and valves and within heart valves themselves before and after decellularization. The distribution of glycosaminoglycans is also dependent from the vascular district and topographic localization. The decellularization protocol adopted resulted in a relevant but not selective depletion of galactosaminoglycans. As a whole, data suggest that both decellularized porcine heart valves and bovine pericardium represent promising materials bearing the potential for future development of tissue engineered heart valve scaffolds.

Several studies have evidenced variations in plasma glycosaminoglycans content in physiological and pathological conditions. In normal human plasma GAGs are present mainly as undersulfated chondroitin sulfate (CS). The aim of the present study was to evaluate possible correlations between plasma CS level/structure and the presence/typology of carotid atherosclerotic lesion. Plasma CS was purified from 46 control subjects and 47 patients undergoing carotid endarterectomy showing either a soft or a hard plaque. The concentration and structural characteristics of plasma CS were assessed by capillary electrophoresis of constituent unsaturated fluorophore-labeled disaccharides. Results showed that the concentration of total CS isomers was increased by 21.4% (P < 0.01) in plasma of patients, due to a significant increase of undersulfated CS. Consequently, in patients the plasma CS charge density was significantly reduced with respect to that of controls. After sorting for plaque typology, we found that patients with soft plaques and those with hard ones differently contribute to the observed changes. In plasma from patients with soft plaques, the increase in CS content was not associated with modifications of its sulfation pattern. On the contrary, the presence of hard plaques was associated with CS sulfation pattern modifications in presence of quite normal total CS isomers levels. These results suggest that the plasma CS content and structure could be related to the presence and the typology of atherosclerotic plaque and could provide a useful diagnostic tool, as well as information on the molecular mechanisms responsible for plaque instability.

Evaluation of the physiological performance of biological scaffolds for tissue engineering applications has been mostly based on biophysical and morphological methods, with limited attention paid to the quantitative contribution of the main structural components to native and/or treated valve assemblies. In the present study quantitation addressed the porcine leaflet, sinus and adjacent wall of aortic and pulmonary valved conduits before and after detergent-based cell removal. Collagen, elastin, glycosaminoglycan, lipid and water contents were expressed in terms of relative concentration and volume fraction in order to assess their effective contribution to the native tissue and to changes following decellularization procedures. The main findings were recognition of unexpectedly large water and underestimated collagen contents, differential distribution of elastin between the sectors and of glycosaminoglycan along the conduits and pulmonary scaffold destabilization upon cell removal, not found in the aortic case. Simultaneous investigations allowed consistent comparisons between native and decellularized tissues and added analytical knowledge crucial for designing realistic constitutive models. We have provided a quantitative structural foundation for earlier biomechanical findings in pulmonary leaflets and the basis for validation of theoretical assumptions still lacking the support of experimental evidence in both conduits. Future insights into the distribution of load-bearing components in human conduits are likely to provide indications important to optimize the surgical positioning of valvular grafts., (Copyright © 2010 Acta Materialia Inc. Published by Elsevier Ltd. All rights reserved.)

Phosphorylation of erythrocyte membrane proteins has been previously documented following infection and intracellular growth of the malarial parasite, Plasmodium falciparum in red cells. Much of this data dealt with phosphorylation of serine residues. In this study, we report detailed characterization of phosphorylation of serine and tyrosine residues of red cell membrane proteins following infection by P falciparum. Western blot analysis using anti-phosphotyrosine and anti-phosphoserine antibodies following 2-DE in conjunction with double channel laser-induced infrared fluorescence enabled accurate assessment of phosphorylation changes. Tyrosine phosphorylation of band 3 represented the earliest modification observed during parasite development. Band 3 tyrosine phosphorylation observed at the ring stage appears to be under the control of Syk kinase. Serine and tyrosine phosphorylation of additional cytoskeletal, trans-membrane and membrane associated proteins was documented as intracellular development of parasite progressed. Importantly, during late schizont stage of parasite maturation, we observed widespread protein dephosphorylation. In vitro treatments that caused distinct activation of red cell tyrosine and serine kinases elicited phosphorylative patterns similar to what observed in parasitized red blood cell, suggesting primary involvement of erythrocyte kinases. Identification of tyrosine phosphorylations of band 3, band 4.2, catalase and actin which have not been previously described in P. falciparum infected red cells suggests new potential regulatory mechanisms that could modify the functions of the host cell membrane.

Purpose: To assess the plasma levels of the inflammatory markers such as C-reactive protein (CRP), leptin, and glycosaminoglycans (GAGs) during the menstrual cycle., Methods: Eighteen 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)., Results: In 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., Conclusions: There 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.

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.

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.

Atherosclerosis is a multifactorial disease in which hypertension, diabetes, hyperlipidemia and other risk factors are thought to play a role. However, the molecular processes underlying plaque formation and progression are not yet completely known. In the last years some researchers applied proteomics technologies for the comprehension of biochemical pathways of atherogenesis and to search new cardiovascular biomarkers to be utilized either as early diagnostic traits or as targets for new drug therapies. Due to its intrinsic complexity, the problem has been approached by different strategies, all of which have some limitations. In this review, we summarize the most common critical experimental variables in two-dimensional electrophoresis-based techniques and recent data obtained by applying proteomic approaches in the study of atherosclerosis.

We describe a new ultra-rapid capillary electrophoresis method with UV detection for analysis of the disaccharides obtained after enzymatic depolymerization of plasma chondroitin sulfates. The free reducing groups of the released carbohydrate molecules are derivatized with 2-aminoacridone by reductive amination in the presence of cyanoborohydride. The fluorotagged products can be separated by short-end injection capillary electrophoresis in a capillary with an effective length of 10.2 cm. The migration times of Delta di-0S and Delta di-4S were 0.95 and 1.81 min, respectively. We compared the proposed method with UV detection to a reference CE-LIF assay by measuring plasma chondroitin sulfate in 94 subjects. The described assay for total plasma CS measurement may, owing to the high throughput and the fast analytical times, be a good tool for routine studies both in research and in clinical applications.

The report describes a rapid and simple CE method using LIF detection for the analysis of unsaturated disaccharides obtained from enzymatic depolymerization of plasma chondroitin sulfate (CS) isomers. The disaccharide reducing groups were labeled with 2-aminoacridone (AMAC). The fluorotagged products can be separated by reversed-polarity CE using a sodium acetate buffer, pH 3.8, in the presence of 0.05% methylcellulose. The choice of the appropriate electrophoretic conditions was performed after a deep analysis of the most important parameters affecting analyte separation. In particular, the effect of both run buffer concentration and pH on resolution, efficiency, migration times, and peak area was evaluated. The selected electrophoretic conditions allowed us to separate the CS isomers-derived Delta-disaccharides in less than 12 min, also resolving the nonsulfated disaccharides released from CS isomers from those released from hyaluronan (HA). Moreover, these conditions gave a good reproducibility of both the migration times (CV%, 0.25) and the peak areas (CV%, 1.4). Intra- and interassay CV were 5.37 and 7.23%, respectively, and analytical recovery was about 86%. The applicability of the above method to the quantitative and structural disaccharide analyses of plasma CS isomers was investigated. Data obtained from 44 healthy human subjects were compared with those obtained by a fluorophore-assisted carbohydrate electrophoresis (FACE) reference assay, by using the Passing and Bablok regression and Bland-Altman tests. The developed method could represent a good tool for an ultrasensitive analysis of CS isomers in biological samples from different sources, particularly when samples are available in very low amounts.