Proteoglycan Research Articles

Chondroprotective effect of curcumin and lecithin complex in human chondrocytes stimulated by IL-1β via an anti-inflammatory mechanism.

A complex of curcumin and lecithin developed to improve the solubility of curcumin, enhanced its chondroprotective effect via an anti-inflammatory mechanism. In macrophage, proinflammatory cytokines, such as tumor necrosis factor (TNF)-α, interleukin (IL)-1β, IL-6, prostaglandin E2 (PGE2), and nitric oxide (NO) were quantified. In addition, the activity of nuclear factor (NF)-κB was examined. With chondrocytes, inflammatory mediators were assessed by measuring the secretion levels of IL-6, IL-8, and PGE2, also the mRNA expression of cyclooxygenase-2 (COX-2) and inducible nitric oxide synthase (iNOS). Metalloproteinases (MMPs), tissue inhibitor of metalloprotease (TIMP)-1, type II collagen (COL2), proteoglycan (PG), and hyaluronic acid (HA) were measured with respect to the articulation surface. The complex promoted the anti-inflammatory effect by the inhibition of inflammatory mediators. In addition, mRNA expression levels ameliorated. Furthermore, it was effective in decreasing extracellular secretion of polypeptides, also corresponding intracellular MMPs and TIMP-1. In conclusion, the complex may be developed as a functional supplement to maintain articulation health.

Extraculuar material promoted C3H10T1/2 chondrogenic differentiation than Pellet cultured in vitro

Objective In order to understand the chondrogenesis differentiation of mesenchymal stem cells in either hydrogel or pellet culture, we applied the two methods and reveal the possible mechanism and for further investigation. Methods In C3H10T1/2 chondrogenic differentiation, we apply extracellular matrix hydrogel mixed the cell suspensions of freshly prepared (including scaling chondroitin sulfate, sodium hyaluronate synthesis and cross-linking agent) co-culture system and high cell density pellet formed by centrifugation. Chondrogenic differentiation of C3H10T1/2 was induced by treatment with TGF-β3 (10 ng/ml), dexamethasone (100 nmol/L), ascorbic acid (50 ug/ml), 1∶100 dilution ITS+Premix and high glucose-DMEM medium with 0.2 volume fraction fetal bovine serum. And high glucose-DMEM medium with 0.2 volume fraction fetal bovine serum is for control group. Histochemistry staining was utilized to identify extracellular proteoglycan and real-time PCR was performed to assess gene expression of SOX9, collagen IIa1/Xa1 and aggrecan for the 1st, 2nd and 3rd week respectively. Results In the hydrogel model for 3 weeks chondrogenic differentiation, the expression of master transcription factor SOX9 was upregulated in both culture models. While the marker genes of collagen IIa1 and collagen Xa1 were all promoted in hydrogel culture, the aggrecan gene expression was peaked in pellet culture. In addition, immunocytochemistry analysis of the hydrogel and pellet for 3 week illustrated the expression of extracellular matrix and more obviously in the hydrogel model. Conclusion In compared with pellet culture, the MSCs in the hydrogel were more likely promoted chondrogenesis leading to the eventual expression of marker genes. And the hydrogel would be applied in regeneration of cartilage injury. Key words: Mesenchymal stem cells; Cellular microenvironment; Chondrocytes

Cell surface proteoglycan-mediated uptake and accumulation of the Alzheimer's disease peptide Aβ(1–42)

Proteoglycans (PGs) have been found in Alzheimer's disease amyloid-β (Aβ) plaques and their glycosaminoglycan chains reportedly influence Aβ aggregation, neurotoxicity and intracellular accumulation in cell and animal models, but their exact pathophysiological role(s) remain unclear. We have studied the cellular uptake of fluorescently labelled Aβ(1–42) and Aβ(1–40) peptides in normal CHO cells (K1) and the mutant cell line (pgsA-745) which lacks all protein-attached heparan and chondroitin sulfate chains. After 24 h of incubation, CHO-K1 accumulates more Aβ(1–42) and Aβ(1–40) compared with CHO-pgsA-745, consistent with the suggested role of PGs in Aβ uptake. However, after short incubation times (≤3 h) there was no difference; moreover, the time evolution of Aβ(1–42) accumulation in CHO-K1 followed an unusual sigmoidal-like trend, indicating a possible involvement of PG-mediated peptide aggregation in Aβ endocytosis. Neither Aβ(1–42) nor Aβ(1–40) could stimulate uptake of a 10 kDa dextran (a general endocytosis marker) suggesting that Aβ-induced upregulation of endocytosis does not occur. CHO-K1 cells contained a higher number of Aβ(1–42)-positive vesicles, but the intensity difference per vesicle was only marginal suggesting that the superior accumulation of Aβ(1–42) stems from a higher number of endocytic events. FRET imaging support that intracellular Aβ(1–42) is aggregated in both cell types. We also report that CHO-pgsA-745 cells perform less endocytosis than CHO-K1 and, albeit this does not explain their difference in Aβ internalisation, we discuss a general method for data compensation. Altogether, this study contributes new insights into the mechanisms of PG-mediated Aβ uptake that may be relevant for our understanding of their role in AD pathology.

Structure-activity relationship study of hypoxia-activated prodrugs for proteoglycan-targeted chemotherapy in chondrosarcoma

Due to an abundant chondrogenic, poorly vascularized and particularly hypoxic extracellular matrix, chondrosarcoma, a malignant cartilaginous tumour, is chemo- and radio-resistant. Surgical resection with wide margins remains the mainstay of treatment. To address the lack of therapy, our strategy aims to increase anticancer drugs targeting and delivery in the tumour, by leveraging specific chondrosarcoma hallmarks: an extensive cartilaginous extracellular matrix, namely the high negative fixed charge density and severe chronic hypoxia. A dual targeted therapy for chondrosarcoma was investigated by conjugation of a hypoxia-activated prodrug (HAP) to quaternary ammonium (QA) functions which exhibit a high affinity for polyanionic sites of proteoglycans (PGs), the major components of the chondrosarcoma extracellular matrix. Based on preclinical results, an imidazole prodrug, ICF05016, was identified and provided the basis for a lead optimization study. A series of 27 QA-phosphoramide mustard conjugates, differing by the type of QA function and the length of the alkyl linker, was yielded by a common multi-step sequence involving phosphorylation of a key 2-nitroimidazole alcohol. Then, a screening was realized by surface plasmon resonance technology to assess biomolecular interactions between QA derivatives and aggrecan, the most abundant PG in chondrosarcoma. Results revealed that affinity depends more on the type of QA function, than on the linker length. Moreover, the presence of a benzyl group enhanced affinity to aggrecan. Twelve compounds were shortlisted and evaluated for antiproliferative activity (i.e., growth inhibiting concentration 50), under normoxic and hypoxic conditions using the human extraskeletal myeloid chondrosarcoma cell line (HEMC-SS). For all prodrugs, hypoxic selectivity was maintained and even increased, compared with the lead. From this study, compound 31f emerged as the most effective PG-targeted HAPs with a dissociation constant of 2.10 μM in the SPR experiment, a hypoxia cytotoxicity ratio of 24 and an efficient reductive cleavage under chemical and enzymatic conditions.

Glycans and glycosaminoglycans in neurobiology: key regulators of neuronal cell function and fate.

The aim of the present study was to examine the roles of l-fucose and the glycosaminoglycans (GAGs) keratan sulfate (KS) and chondroitin sulfate/dermatan sulfate (CS/DS) with selected functional molecules in neural tissues. Cell surface glycans and GAGs have evolved over millions of years to become cellular mediators which regulate fundamental aspects of cellular survival. The glycocalyx, which surrounds all cells, actuates responses to growth factors, cytokines and morphogens at the cellular boundary, silencing or activating downstream signaling pathways and gene expression. In this review, we have focused on interactions mediated by l-fucose, KS and CS/DS in the central and peripheral nervous systems. Fucose makes critical contributions in the area of molecular recognition and information transfer in the blood group substances, cytotoxic immunoglobulins, cell fate-mediated Notch-1 interactions, regulation of selectin-mediated neutrophil extravasation in innate immunity and CD-34-mediated new blood vessel development, and the targeting of neuroprogenitor cells to damaged neural tissue. Fucosylated glycoproteins regulate delivery of synaptic neurotransmitters and neural function. Neural KS proteoglycans (PGs) were examined in terms of cellular regulation and their interactive properties with neuroregulatory molecules. The paradoxical properties of CS/DS isomers decorating matrix and transmembrane PGs and the positive and negative regulatory cues they provide to neurons are also discussed.

Salmon cartilage proteoglycan attenuates allergic responses in mouse model of papain‑induced respiratory inflammation.

Proteoglycan (PG) is a complex glycohydrate, which is widely distributed in the extracellular matrix. It has been reported that daily oral administration of PG (extracted from salmon nasal cartilage) modulates the severity of proinflammatory cytokine responses in mouse experimental colitis, autoimmune encephalomyelitis, collagen‑induced arthritis and obesity‑induced inflammation. The present study investigated the effect of salmon nasal cartilage PG on allergic responses using a mouse model of papain‑induced respiratory inflammation. Low titers of immunoglobulin E were identified in the sera of the PG‑administered mice. Oral administration of PG attenuated eosinophil infiltration in the lung. In the acute model of papain‑induced allergic inflammation, PG‑administered mice exhibited low titers of epithelium‑derived and T helper 2‑associated cytokines. The results of the present study demonstrated that salmon cartilage PG has an immunomodulatory effect on intranasally delivered papain. These results suggest a potential role for PG as a prophylactic agent which may attenuate allergic respiratory inflammation.

The Role of heparan sulfate proteoglycans in PCSK9-induced LDL receptor degradation

Aim: PCSK9 binds the LDL receptor (LDLR) and induces its degradation. The result is reduced clearance of plasma cholesterol, and inhibitors preventing the binding of PCSK9 to LDLR are of immense clinical interest. However, the affinity of PCSK9 for LDLR is low, and it is an enigma why PCSK9 targets LDLR in the liver, but not in other tissues, all suggesting the requirement of a liver-specific co-receptor. We have found that PCSK9 binds heparin, a mimic of Heparan Sulfate (HS) proteoglycans (PG), and have investigated the role of this interaction in PCSK9 biology.

Spatial correlation of native and engineered cartilage components at micron resolution.

Tissue engineering (TE) approaches are being widely investigated for repair of focal defects in articular cartilage. However, the amount and/or type of extracellular matrix (ECM) produced in engineered constructs does not always correlate with the resultant mechanical properties. This could be related to the specifics of ECM distribution throughout the construct. Here, we present data on the amount and distribution of the primary components of native and engineered cartilage (i.e., collagen, proteoglycan (PG), and water) using Fourier transform infrared imaging spectroscopy (FT-IRIS). These data permit visualization of matrix and water at 25μm resolution throughout the tissues, and subsequent colocalization of these components using image processing methods. Native and engineered cartilage were cryosectioned at 80μm for evaluation by FT-IRIS in the mid-infrared (MIR) and near-infrared (NIR) regions. PG distribution correlated strongly with water in native and engineered cartilage, supporting the binding of water to PG in both tissues. In addition, NIR-derived matrix peaks correlated significantly with MIR-derived collagen peaks, confirming the interpretation that these absorbances arise primarily from collagen and not PG. The combined use of MIR and NIR permits assessment of ECM and water spatial distribution at the micron level, which may aid in improved development of TE techniques.

Ultrastructure and 3D transmission electron tomography of collagen fibrils and proteoglycans of swollen human corneal stroma.

The transparency of the cornea is regulated by the unique organization of collagen fibrils (CFs) which is maintained by proteoglycans (PGs). The interlacing of CF lamellae in the anterior stroma provides the biomechanical properties of the cornea. To investigate the alterations of CFs and PGs in the swollen cornea, with special reference to the anterior stroma by using electron microscopy and 3D ultrastructural tomography. Nine healthy normal scleral corneal rings (age from 40 to 65 years) were hydrated individually in deionised water to induce swelling in the cornea. Three of them were hydrated for 2hr whereas the other three were hydrated for 48hr. The remaining three scleral normal corneal rings were used as a control.The corneas were processed for electron microscopy (EM) to study the CFs and PGs. Ultrathin sections were observed using transmission electron microscopy (JOEL 1400) and digital images of CFs, PGs and lamellae were captured using a bottom mounted Quemesa camera and iTEM Soft Imaging System. The software program 'Composer-x64, version 3.4.2.0' was used to construct individual 3D images from 120 digital images taken from -60 to + 60 degree angles. The 3D tomography showed the degeneration of microfibrils within the CFs of the swollen cornea. The CF diameter was significantly reduced and the interfibrillar spacing significantly increased in both the 2hr and 48hr hydrated corneas compared to the normal cornea. Within the hydrated corneas, the CF diameter was smaller and the interfibrillar spacing was increased in the middle and posterior stroma compared to the anterior stroma. The PG area in both the 2hr and the 48hr hydrated cornea was reduced in the anterior stroma, whereas it was increased in middle and posterior stroma compared to the normal cornea. The density of the PGs in both the 2hr and the 48hr samples, was reduced compared to the density of PGs in the normal cornea. The CFs, PGs and lamellae had degenerated, caused by swelling. 3D imaging demonstrated that the impairment of the microfibrils and PGs within the CF, is caused by the excessive hydration or swelling in the anterior as well as in the middle and posterior stroma. The lamellae of the anterior stroma which provides the biomechanical strength in the normal cornea, had degenerated in the swollen corneas due to the presence of the damaged CFs and PGs.

Collagen cross-linking impact on keratoconus extracellular matrix.

BackgroundKeratoconus (KC) is a common multifactorial ectatic corneal disease with unknown onset. KC most commonly appears in adolescence and affects approximately 1:400 people worldwide. Treatment options, for advanced KC cases, are collagen cross-linking (CXL) and corneal transplants. CXL is a new KC treatment that helps arrest the disease. Unfortunately, only a fraction of KC patients will qualify for CXL treatment. Our goal, in this study, was to begin to understand how CXL affects the corneal microenvironment and pave the way towards a more patient-driven CXL treatment.MethodsPrimary human corneal fibroblasts from healthy and KC donors were plated on transwell polycarbonate membranes and stimulated by a stable vitamin C. At 4 weeks, riboflavin was added followed by UVA irradiation. Transmission Electron Microscopy (TEM) and western blots were used to assess the effect of CXL on the extracellular matrix (ECM) and the resident cells, pre- and post CXL.ResultsData shows CXL improved lamellar organization showing more organized collagen fibrils decorated with proteoglycans (PGs). The distribution of the collagen fibrils and interfibrillar spacing was also visibly improved, post-CXL. Lumican, mimecan, and decorin were the dominant PGs and were significantly upregulated in post-CXL cultures. ECM degradation proteins, matrix metalloproteinases (MMPs), MMP-1, -3, and -9, but not MMP-2, were significantly downregulated post-CXL. TIMP-1 and -2 were not modulated by CXL.ConclusionThe unknown effects of CXL on the human corneal microenvironment have hampered our ability to make CXL available to all KC patients. Our current study provides a deeper understanding on CXL activity, using our unique 3D in vitro model.

Structural Basis for LAR-RPTP-Mediated Synaptogenesis.

Leukocyte common antigen-related protein tyrosine phosphatases (LAR-RPTPs) are cellular receptors of heparan sulfate (HS) and chondroitin sulfate (CS) proteoglycans that regulate neurite outgrowth and neuronal regeneration. LAR-RPTPs have also received particular attention as the major presynaptic hubs for synapse organization through selective binding to numerous postsynaptic adhesion partners. Recent structural studies on LAR-RPTP–mediated trans-synaptic adhesion complexes have provided significant insight into the molecular basis of their specific interactions, the key codes for their selective binding, as well as the higher-order clustering of LAR-RPTPs necessary for synaptogenic activity. In this review, we summarize the structures of LAR-RPTPs in complex with various postsynaptic adhesion partners and discuss the molecular mechanisms underlying LAR-RPTP–mediated synaptogenesis.

ApoB-100 Lipoprotein Complex Formation with Intima Proteoglycans as a Cause of Atherosclerosis and Its Possible Ex Vivo Evaluation as a Disease Biomarker.

Experimental and clinical data indicate that the initiation and progress of atherosclerosis and its clinical manifestations are first caused by circulating apoB-100 lipoproteins that enter and are retained in the arterial intima. Extracellular sulfated proteoglycans (PGs) of the intima are the retention agents. The PGs also initiate physical and biochemical lipoprotein degradation with the production of bioactive, lipid products that trigger an inflammatory response that leads to atherosclerosis. There are many simple methods for measuring abnormalities of circulating lipoproteins and their relation to atherosclerotic cardiovascular disease (ACVD). However, limited research aims to evaluate procedures that could report quantitatively about the contribution of the interaction of apoB-100 lipoprotein-arterial intima PGs to clinical manifestation of ACVD. In the present review we discuss observations indicating that simple ex vivo evaluation of the affinity of apoB-100 lipoproteins for arterial PGs and glycosaminoglycans (GAGs) can give an indication of its association with clinical manifestations of atherosclerosis. In addition, we discuss molecular and cellular aspects of the apoB-100 lipoproteins association with arterial PGs that are related to atherogenesis and that support the experimental framework behind the current “Response-to-Retention” hypothesis of atherosclerosis.

Software for Peak Finding and Elemental Composition Assignment for Glycosaminoglycan Tandem Mass Spectra

Glycosaminoglycans (GAGs) covalently linked to proteoglycans (PGs) are characterized by repeating disaccharide units and variable sulfation patterns along the chain. GAG length and sulfation patterns impact disease etiology, cellular signaling, and structural support for cells. We and others have demonstrated the usefulness of tandem mass spectrometry (MS2) for assigning the structures of GAG saccharides; however, manual interpretation of tandem mass spectra is time-consuming, so computational methods must be employed. In the proteomics domain, the identification of monoisotopic peaks and charge states relies on algorithms that use averagine, or the average building block of the compound class being analyzed. Although these methods perform well for protein and peptide spectra, they perform poorly on GAG tandem mass spectra, because a single average building block does not characterize the variable sulfation of GAG disaccharide units. In addition, it is necessary to assign product ion isotope patterns to interpret the tandem mass spectra of GAG saccharides. To address these problems, we developed GAGfinder, the first tandem mass spectrum peak finding algorithm developed specifically for GAGs. We define peak finding as assigning experimental isotopic peaks directly to a given product ion composition, as opposed to deconvolution or peak picking, which are terms more accurately describing the existing methods previously mentioned. GAGfinder is a targeted, brute force approach to spectrum analysis that uses precursor composition information to generate all theoretical fragments. GAGfinder also performs peak isotope composition annotation, which is typically a subsequent step for averagine-based methods. Data are available via ProteomeXchange with identifier PXD009101.

Nanostructural and mechanical changes in the sclera following proteoglycan depletion

The mechanical properties of ocular tissues, such as the sclera, have a major impact on healthy eye function, and are governed by the properties and composition of the microstructural components. For example, biomechanical degradation associated with myopia occurs alongside a reduction of proteoglycans (PGs). In this study, the role of PG degradation in the nanomechanical properties of the porcine sclera is explored. In-vitro enzymatic degradation of PGs was conducted with α-amylase and chondroitinase ABC enzymes. Collagen fibril morphology and nanomechanical stiffness were measured with atomic force microscopy (AFM). The elastic modulus of the tissue was reduced in all enzyme-treated samples relative to controls. In addition, collagen fibril organization was disrupted by PG depletion. Our data demonstrate that PGs play an important role in determining not only the mechanical properties at these length scales, but also collagen fibril arrangement.

Detection of Glycan Shedding in the Blood: New Class of Multiple Sclerosis Biomarkers?

Multiple sclerosis (MS) is a devastating autoimmune disease, afflicting people in the prime of their lives. Presently, after initial clinical presentation, there are no reliable markers for whether a patient will develop MS, or whether their prognosis will be aggressive or relapsing-remitting. Furthermore, many MS patients do not respond to treatment. Thus, markers for diagnosis, prognosis, and treatment-responsiveness are lacking for a disease, where a precision medicine approach would be valuable. The glycocalyx (GLX) is the carbohydrate-rich outer surface of the blood vessel wall and is the first interaction between the blood and the vessel. We hypothesized that cleavage of the GLX may be an early stage predictor of immune attack, blood-brain barrier (BBB) breakdown, and disease severity in MS. Two experimental models of MS, experimental autoimmune encephalitis (EAE), were included in this study. EAE was induced in C57BL/6J mice and Lewis rats, which were monitored for weight loss and clinical presentation in comparison to healthy controls. Plasma samples were obtained longitudinally from mice until peak disease severity and at peak disease severity in rats. Soluble GLX-associated glycosaminoglycans (GAG) and proteoglycans (PG) were detected in plasma samples. All animals receiving EAE emulsion developed fulminant EAE (100% penetrance). Increased plasma levels of chondroitin sulfate were detected before the onset of clinical symptoms and remained elevated at peak disease severity. Hyaluronic acid was increased at the height of the disease, whereas heparan sulfate was transiently increased during early stages only. By contrast, syndecans 1, 3, and 4 were detected in EAE samples as well as healthy controls, with no significant differences between the two groups. In this study, we present data supporting the shedding of the GLX as a new class of biomarker for MS. In particular, soluble, sugar-based GLX components are associated with disease severity in two models of MS, molecules that would not be detected in proteomics-based screens of MS patient samples. Patient studies are presently underway.

Proteoglycans in normal physiology and carcinogenesis

Malignant transformation of any cell is associated with numerous physiological and morphological disorders at both genomic and protein levels, a variety of macromolecules being involved in. However, the tumour development and metastasis depends on not only the molecular characteristics of the tumour cell but also its interaction with the surrounding extracellular matrix (ECM), which is an important and necessary part of any tissue. An important role in this process belongs to the complex protein-carbohydrate molecules – proteoglycans (PG), which are one of the main component of ECM and cell surface of any tissue and are tightly involved in cell-cell and cell-matrix interactions and signaling. During carcinogenesis, significant changes in the PG structure and composition occur both at the surface of tumour cells and surrounding ECM, resulting in the transformation of normal ECM into a tumour microenvironment and deterioration of cell-cell and cell-matrix communication. Further, the tumorigenic niche contributes to active proliferation of the cancer cells, tumour development and metastasis. At present, many key PG are identified as possible diagnostic and prognostic molecular markers and target molecules for the creation of new antitumor drugs. The review describes the main PG types, their structure, localisation, functional role in normal cell and tissue physiology and participation in molecular mechanisms of carcinogenesis.

Subverting the mechanisms of cell death: flavivirus manipulation of host cell responses to infection.

Viruses exploit host metabolic and defence machinery for their own replication. The flaviviruses, which include Dengue (DENV), Yellow Fever (YFV), Japanese Encephalitis (JEV), West Nile (WNV) and Zika (ZIKV) viruses, infect a broad range of hosts, cells and tissues. Flaviviruses are largely transmitted by mosquito bites and humans are usually incidental, dead-end hosts, with the notable exceptions of YFV, DENV and ZIKV. Infection by flaviviruses elicits cellular responses including cell death via necrosis, pyroptosis (involving inflammation) or apoptosis (which avoids inflammation). Flaviviruses exploit these mechanisms and subvert them to prolong viral replication. The different effects induced by DENV, WNV, JEV and ZIKV are reviewed. Host cell surface proteoglycans (PGs) bearing glycosaminoglycan (GAG) polysaccharides - heparan/chondroitin sulfate (HS/CS) - are involved in initial flavivirus attachment and during the expression of non-structural viral proteins play a role in disease aetiology. Recent work has shown that ZIKV-infected cells are protected from cell death by exogenous heparin (a GAG structurally similar to host cell surface HS), raising the possibility of further subtle involvement of HS PGs in flavivirus disease processes. The aim of this review is to synthesize information regarding DENV, WNV, JEV and ZIKV from two areas that are usually treated separately: the response of host cells to infection by flaviviruses and the involvement of cell surface GAGs in response to those infections.

Quantitative Dual Contrast CT Technique for Evaluation of Articular Cartilage Properties.

Impact injuriesof cartilage may initiate post-traumatic degeneration, making early detection of injury imperative for timely surgical or pharmaceutical interventions. Cationic (positively-charged) CT contrast agents detect loss of cartilage proteoglycans (PGs) more sensitively than anionic (negatively-charged) or non-ionic (non-charged, i.e., electrically neutral) agents. However, degeneration related loss of PGs and increase in water content have opposite effects on the diffusion of the cationic agent, lowering its sensitivity. In contrast to cationic agents, diffusion of non-ionic agents is governed only by steric hindrance and water contentof cartilage. We hypothesize that sensitivity ofan iodine(I)-based cationic agent may be enhanced by simultaneous use of a non-ionic gadolinium(Gd)-based agent. We introduce a quantitative dual energy CT technique (QDECT) for simultaneous quantification of two contrast agents in cartilage. We employ this technique to improve the sensitivity of cationic CA4+ (q =+4) by normalizing its partition in cartilage with that of non-ionic gadoteridol. The technique was evaluated with measurements of contrast agent mixtures of known composition and human osteochondral samples (n = 57) after immersion (72h) in mixture of CA4+ and gadoteridol. Samples were arthroscopically graded and biomechanically tested prior to QDECT (50/100kV). QDECT determined contrast agent mixture compositions correlated with the true compositions (R2= 0.99, average error = 2.27%). Normalizing CA4+ partition in cartilage with that of gadoteridol improved correlation with equilibrium modulus (from ρ = 0.701 to 0.795). To conclude, QDECT enables simultaneous quantification of I and Gd contrast agents improving diagnosis of cartilage integrity and biomechanical status.

Comparative proteomic analysis of fluoride treated rat bone provides new insights into the molecular mechanisms of fluoride toxicity

Long-term excessive intake of fluoride (F) could lead to chronic fluorosis. To explore the underlying molecular mechanisms, present study is designed to elucidate the effect of fluoride on proteome expression of bone in sodium fluoride (NaF)-treated SD rats. Hematoxylin and eosin (H&E) staining was used to determine the severity of osteofluorosis, and bone samples were submitted for iTRAQ analysis. The results showed that the cortical thickness and trabecular area of femur bone in medium- and high-dose groups were higher than in control group. Contrary to this, trabecular area was reduced in the low-dose group, indicating that the bone mass was increased in medium- and high-dose groups, and decreased in the low-dose group. Thirteen (13), 35, and 34 differentially expressed proteins were identified in low-, medium-, and high-dose group, respectively. The medium- and high-dose groups shared a more similar protein expression pattern. These proteins were mainly associated with collagen metabolism, proteoglycans (PGs), matrix metalloproteinases (MMPs), etc. The results suggested that the effect of NaF on SD rats is in a dose-dependent manner. Some key proteins found here may be involved in affecting the bone tissues and bone marrow or muscle, and account for the complex pathology and clinical symptoms of fluorosis.

Endothelial Glycocalyx as a Shield Against Diabetic Vascular Complications: Involvement of Hyaluronan and Hyaluronidases.

The endothelial glycocalyx (EG), which covers the apical surface of the endothelial cells and floats into the lumen of the vessels, is a key player in vascular integrity and cardiovascular homeostasis. The EG is composed of PGs (proteoglycans), glycoproteins, glycolipids, and glycosaminoglycans, in particular hyaluronan (HA). HA seems to be implicated in most of the functions described for EG such as creating a space between blood and the endothelium, controlling vessel permeability, restricting leukocyte and platelet adhesion, and allowing an appropriate endothelial response to flow variation through mechanosensing. The amount of HA in the EG may be regulated by HYAL (hyaluronidase) 1, the most active somatic hyaluronidase. HYAL1 seems enriched in endothelial cells through endocytosis from the bloodstream. The role of the other main somatic hyaluronidase, HYAL2, in the EG is uncertain. Damage to the EG, accompanied by shedding of one or more of its components, is an early sign of various pathologies including diabetes mellitus. Shedding increases the blood or plasma concentration of several EG components, such as HA, heparan sulfate, and syndecan. The plasma levels of these molecules can then be used as sensitive markers of EG degradation. This has been shown in type 1 and type 2 diabetic patients. Recent experimental studies suggest that preserving the size and amount of EG HA in the face of diabetic insults could be a useful novel therapeutic strategy to slow diabetic complications. One way to achieve this goal, as suggested by a murine model of HYAL1 deficiency, may be to inhibit the function of HYAL1. The same approach may succeed in other pathological situations involving endothelial dysfunction and EG damage.