Heparan Sulfate Content Research Articles

Method Development and Analysis of Free HS and HS in Proteoglycans from Pre- and Postmenopausal Women: Evidence for Biosynthetic Pathway Changes in Sulfotransferase and Sulfatase Enzymes

Heparan sulfate (HS) is one of the most complex and informative biopolymers found on the cell surface or in the extracellular matrix as either free HS fragments or constituents of HS proteoglycans (HSPGs). Analysis of free HS and HSPG sugar chains in human serum at the disaccharide level has great potential for early disease diagnosis and prognosis; however, the low concentration of HS in human serum, together with the complexity of the serum matrix, limits the information on HS. In this study, we present and validate the development of a new sensitive method for in-depth compositional analysis of free HS and HSPG sugar chains. This protocol involved several steps including weak anion exchange chromatography, ultrafiltration, and solid-phase extraction for enhanced detection prior to LC-MS/MS analysis. Using this protocol, a total of 51 serum samples from 26 premenopausal and 25 postmenopausal women were analyzed. Statistically significant differences in heparin/HS disaccharide profiles were observed. The proportion of N-acetylation and N-sulfation in both free HS and HSPG sugar chains were significantly different between pre- and postmenopausal women, indicating changes in N-deacetylase/N-sulfotransferases (NDSTs), the enzymes involved in the initial step of the biosynthetic pathway. Differences in the proportion of 6-O-sulfation suggest that 6-O-sulfotransferase and/or 6-O-sulfatase enzymes may also be implicated.

Infrared irradiation alters the expression of matrix metalloproteinases and glycosaminoglycans in the cornea and crystalline lens

Prolonged exposure to infrared (IR) radiation is associated with different types of damage to cornea and lens. The aim of our study was to investigate the effect of acute and chronic exposure to IR radiation on the activity of matrix metalloproteinase-2 (MMP-2) and MMP-9 and on the expression of glycosaminoglycans (GAG) in the rabbit cornea and crystalline lens. New Zealand rabbits were subjected to IR radiation for 4 months (chronic exposure to IR) or to normal light (control group). In experiments regarding acute exposure, animals were subjected to IR radiation or normal light for 12 h, in the presence of 0.1% diclofenac sodium (eye drops instilled in the right eye of animals) or saline (instilled in the left eye of animals). The cornea and lens were dissected away and homogenized. The activity of MMP-2 and MMP-9 was assayed by gelatine zymography. Total GAG were isolated from tissue specimens after lipid extraction and extensive digestion with pronase and DNase and characterized by treatment with GAG-degrading enzymes, followed by electrophoresis on cellulose acetate membranes. Acute or chronic exposure to IR radiation induced the activity of MMP-2 in cornea and lens, whereas only acute IR radiation increased the content of heparan sulphate in crystalline lens. Local administration of diclofenac sodium did not prevent the above effects of acute IR radiation. The detrimental effects of excessive or prolonged exposure of the eyes to IR radiation are associated with induced activity of MMP-2 in cornea and lens and alterations in the content of heparan sulphate in lens. Thus, MMP and GAG may offer alternative targets for pharmacological intervention to confront ocular damages associated with IR radiation.

Tissue engineering for cartilage repair: growth and proliferation of hBM-MSCs on scaffolds composed of Collagen I and Heparan Sulphate

Purpose: To evaluate the proliferation and chondrogenic potential of human Bone Marrow Mesenchymal Stem Cells (hBM-MSCs) grown on type I Collagen (Col I) and different concentrations of Heparan Sulfate (HS) scaffolds. We also studied the quality of the neo-synthesized cartilaginous tissue. Methods: hBM-MSCs were cultured over the scaffolds in chondrogenic differentiation medium (Lonza, Biowhittaker, Belgium) or DMEM with 20% FBS (Fetal Bovine Serum) for 15 and 30 days, in both cases plus 100 nM PTHrP (Parathyroid hormone-related protein). Scaffolds without cells were cultured in chondrogenic medium as negative controls. Chondrogenic differentiation and neosythesized cartilage quality were evaluated by histochemical and immunohistochemical analysis, molecular biology techniques and electron microscopy. Culture supernatants were collected every 3-4 days to determine collagen presence by Elisa assays. Results: Hematoxylin-eosin (HE) (fig.1A) and Masson's trichrome (MT) (fig.1B) staining showed that stimulated hBM-MSCs spread throughout the biomaterials in high percentage, showing a good morphology at both times of study as well as a wide distribution of Extracellular Matrix (ECM). These constructs showed positivity for safranin O (SO) (fig.1C), Toluidine Blue (TB), aggrecan (Agg) (fig.1D) and type II collagen (Col II) (fig.1E). Isolated cells were able to proliferate on Col I and various concentrations of HS scaffolds, as they showed high percentages of positivity for Proliferating Cell Nuclear Antigen (PCNA) proliferation marker (fig.1F). hBM-MSCs proliferated better when cultured in chondrogenic medium than in the usual growth medium (DMEM 20% FBS).Degradation of biomaterials was gradual, as fibers were replaced with ECM. Molecular analysis indicated the expression of cartilage-characteristic genes, such as Sox9 [SRY (sex determining region Y)-box 9], Agg and Col II (fig.2) and absence of Col I, a marker of hypertrophic cells. Scanning and transmission electron microscopy confirmed the presence of cells and ECM after 15 and 30 days of culture. Cells attached to the scaffolds and exhibited a rounded morphology (fig.3A-3D). They showed a high number of distended Rough Endoplasmic Reticulum (R.E.R) cisternae (fig.3E), electrodense and bright vesicles (fig.3F, 3G), and great amount of mitochondria, suggesting great cell activity. New fibrils were differentiated surrounding cell surfaces at both times of study, conforming neosynthesized cartilage (fig.3H). Finally, culture supernatants analyses showed the release of collagen in most of the time periods studied (fig.4), confirming the chondrogenic differentiation of the hBM-MSCs. Conclusions: Col I and HS scaffolds were optimal for hBM-MSCs growth and also contributed to their phenotype maintenance. Our supports favour differentiation towards chondrocytes-like cells after both 15 and 30 days in chondrogenic medium. Detection of extracellular matrix characteristic compounds in the constructs confirms chondrogenic differentiation and synthesis of cartilaginous tissue, proving their possible use to regenerate cartilage damages. Acknowledgements: B. Parma (OPOCRIN S.P.A.); P. Esbrit (Fundación Jiménez Díaz), CAM(S2009/MAT-1472); CIBER BBN CB06-01-0040; REDICENT.

Immobilization of Heparan Sulfate on Electrospun Meshes to Support Embryonic Stem Cell Culture and Differentiation *

As our understanding of what guides the behavior of multi- and pluripotent stem cells deepens, so too does our ability to utilize certain cues to manipulate their behavior and maximize their therapeutic potential. Engineered, biologically functionalized materials have the capacity to influence stem cell behavior through a powerful combination of biological, mechanical, and topographical cues. Here, we present the development of a novel electrospun scaffold, functionalized with glycosaminoglycans (GAGs) ionically immobilized onto the fiber surface. Bound GAGs retained the ability to interact with GAG-binding molecules and, crucially, presented GAG sulfation motifs fundamental to mediating stem cell behavior. Bound GAG proved to be biologically active, rescuing the neural differentiation capacity of heparan sulfate-deficient mouse embryonic stem cells and functioning in concert with FGF4 to facilitate the formation of extensive neural processes across the scaffold surface. The combination of GAGs with electrospun scaffolds creates a biomaterial with potent applicability for the propagation and effective differentiation of pluripotent stem cells.

Mild mental retardation and low levels of urinary heparan sulfate in a patient with the attenuated phenotype of mucopolysaccharidosis type IIIA

ObjectivesWe report the case of a 28-year-old female subject affected by the attenuated phenotype of mucopolysaccharidosis type IIIA characterized by moderate slowly evolving mental retardation in which the urinary content of heparan sulfate was demonstrated as being substantially low compared to that found in patients with the severe phenotype. Design and methodsThe specific evaluation of macromolecular heparan sulfate by electrophoresis and the determination of related glucosamine in the urine were performed. ResultsIn our patient, the urinary macromolecular heparan sulfate content (4.2μg/mg creatinine) was ~7.5-times higher than in healthy subjects (0.56μg/mg creatinine±0.9 SD) while it was ~28-times lower compared to the severe mucopolysaccharidosis IIIA group (117μg/mg creatinine±44.8 SD). Furthermore, the urinary glucosamine (86.4μg/mg creatinine) was ~2.4-times greater than in healthy subjects (36.0μg/mg creatinine±18.2 SD) but ~2.4-times lower than in severe subjects (208.1μg/mg creatinine±55.0 SD). ConclusionsThe above data could reflect the reduced heparan sulfate storage in her tissues and organs, and in particular in the brain, consequently explaining her moderate mental retardation. Furthermore, the clinical presentation of patients with an attenuated form of MPS III confirms the need for a specific evaluation of urinary GAGs in all young and adult subjects showing a not well-defined or not particularly severe mental retardation, along with an early MPS diagnosis. Such investigation should also be associated with a more specific characterization of heparan sulfate.

Plasma and urinary levels of dermatan sulfate and heparan sulfate derived disaccharides after long‐term enzyme replacement therapy (ERT) in MPS I: correlation with the timing of ERT and with total urinary excretion of glycosaminoglycans

Mucopolysaccharidosis type I (MPS I) results in a defective breakdown of the glycosaminoglycans (GAGs) heparan sulfate and dermatan sulfate, which leads to a progressive disease. Enzyme replacement therapy (ERT) results in clearance of these GAGs from a range of tissues and can significantly ameliorate several symptoms. The biochemical efficacy of ERT is generally assessed by the determination of the total urinary excretion of GAGs. However, this has limitations. We studied the concentrations of heparan sulfate and dermatan sulfate derived disaccharides (HS and DS, respectively) in the plasma and urine of seven patients and compared these levels with total urinary GAGs (uGAGs) levels. Plasma and urine samples were collected at different time points relative to the weekly ERT for three non-consecutive weeks in seven MPS I patients who had been treated with ERT for at least 2.5years. Heparan and dermatan sulfate in plasma and urine were enzymatically digested into disaccharides, and HS and DS levels were determined by HPLC-MS/MS analysis. uGAGs were measured by the DMB test. The levels of HS and DS were markedly decreased compared with the levels before the initiation of ERT. However, the concentrations of DS in plasma and of both HS and DS in urine remained significantly elevated in all studied patients, while in six patients the level of total uGAGs had normalized. The concentrations of plasma and urinary HS during the weekly ERT followed a U-shaped curve. However, the effect size is small. The concentrations of plasma and urinary DS and uGAGs appeared to be in a steady state. HS and DS are sensitive biomarkers for monitoring the biochemical treatment efficacy of ERT and remain elevated despite long-term treatment. This finding may be related to the labeled dose or antibody status of the patient. The timing of the sample collection is not relevant, at least at the current dose of 100IU/kg/weekly.

Heparan sulfate derived disaccharides in plasma and total urinary excretion of glycosaminoglycans correlate with disease severity in Sanfilippo disease

Sanfilippo disease (Mucopolysaccharidosis III) is a neurodegenerative lysosomal disorder characterized by accumulation of the glycosaminoglycan heparan sulfate (HS). MPS III has a large phenotypic variability and early assessment of disease severity is difficult. We investigated the correlation between disease severity and the plasma concentration of HS (pHS, defined by the sum of the heparan sulfate derived disaccharides obtained after enzymatic digestion) and urinary total GAGs level (uGAGs, measured by the dimethylene blue test) in a cross-sectional cohort of 44 MPS III patients. Disease severity was established on the basis of the age of complete loss of independent walking and of full loss of speech in all patients. Hazard ratios (HR) were obtained with cox-regression analysis. In order to allow prediction of a severe phenotype based on a cut-off value for pHS, patients were divided in two groups (severely affected and less severely affected) based on predictive mutations or on the age of full loss of speech. Receiver operator characteristics (ROC) were obtained for pHS. pHS and uGAGs were independently and linearly associated with an increased risk of speech loss with a HR of 1.8 (95% CI 1.3-2.7) per 500ng/ml increase of HS in plasma (p = 0.002), and a HR of 2.7 (95% CI 1.6-4.4) per 10mg/mmol creatinine increase of uGAGs (p < 0.001). pHS and uGAGS were less strongly associated with loss of walking. The area under the ROC curve for pHS was 0.85, indicating good discrimination. pHS and uGAGs may be useful biomarkers for prediction of severity in MPS III.

Fibroblast EXT1-Levels Influence Tumor Cell Proliferation and Migration in Composite Spheroids

BackgroundStromal fibroblasts are important determinants of tumor cell behavior. They act to condition the tumor microenvironment, influence tumor growth, support tumor angiogenesis and affect tumor metastasis. Heparan sulfate proteoglycans, present both on tumor and stromal cells, interact with a large number of ligands including growth factors, their receptors, and structural components of the extracellular matrix. Being ubiquitously expressed in the tumor microenvironment heparan sulfate proteoglycans are candidates for playing central roles in tumor-stroma interactions. The objective of this work was to investigate the role of heparan sulfate expressed by stromal fibroblasts in modulating the growth of tumor cells and in controlling the interstitial fluid pressure in a 3-D model.Methodology/Principal FindingsWe generated spheroids composed of fibroblasts alone, or composite spheroids, composed of fibroblasts and tumor cells. Here we show that stromal fibroblasts with a mutation in the heparan sulfate elongating enzyme Ext1 and thus a low heparan sulfate content, formed composite fibroblast/tumor cell spheroids with a significant lower interstitial fluid pressure than corresponding wild-type fibroblast/tumor cell composite spheroids. Furthermore, immunohistochemistry of composite spheroids revealed that the cells segregated, so that after 6 days in culture, the wild-type fibroblasts formed an inner core and the tumor cells an outer layer of cells. For composite spheroids containing Ext1-mutated fibroblasts this segregation was less obvious, indicating impaired cell migration. Analysis of tumor cells expressing the firefly luciferase gene revealed that the changes in tumor cell migration in mutant fibroblast/tumor cell composite spheroids coincided with a lower proliferation rate.Conclusions/SignificanceThis is the first demonstration that stromal Ext1-levels modulate tumor cell proliferation and affect the interstitial fluid pressure in a 3-D spheroid model. Learning how structural changes in stromal heparan sulfate influence tumor cells is essential for our understanding how non-malignant cells of the tumor microenvironment influence tumor cell progression.

Serum heparan sulfate concentration in healthy subjects

Heparan sulfate is one of the essential components of extracellular matrices and plasma membrane. They take part in the interaction with protein ligands, which affects the metabolism, transport, information transfer, support and regulation in all organ systems. The aim of the study was to examine the physiological HS level in the serum of the healthy subjects, as well as to check if this level is age- and sex- related. The research was conducted on 30 healthy subjects: 16 women and 14 men, aged from 23 to 65 years, with the average of 30 years. Quantificational determination of HS was performed with the use of Heparan Sulfate ELISA kit. Concentrations of total cholesterol, HDL-cholesterol and triglycerides were measured with the use of standard enzymatic methods. The LDL-cholesterol was analyzed by direct method, and non-HDL-cholesterol was calculated. Based on the conducted research, it can be concluded that higher HS serum concentrations were observed in the group of healthy men compared to women group, and in the older ≥30 year-old group. The determination of serum HS concentration should consider gender and age differences in the result interpretation. Changes in HS serum concentrations are not correlated with the changes in the lipid profile parameters. For complete determining of existing statistical correlations between HS concentration, age, gender and the lipid profile parameters study results require confirmation on a greater number of cases.

Extracellular matrix and breast cancer cells' response to trastuzumab: The role of glycosaminoglycans, heparanases, and syndecan-1.

10568 Background: Trastuzumab is an antibody anti-epidermal growth factor 2 receptor (HER2), which improves disease-free and overall survival in HER2 positive breast cancer. Nevertheless, many patients become resistant to this treatment. Heparanase (HPSE) is an enzyme that is responsible for removal of heparan sulfate (HS) chains from proteoglycans, generating free oligosaccharides that modulate many physiopathological functions, including tumor developing. We have analyzed whether some extracellular matrix components influence trastuzumab efficacy. Methods: Heparanase-1 (HPSE-1) overexpression effect was analyzed using MCF7 cells stable transfected with HPSE-1 cDNA (MCF7-HPSE-1). HPSE-1, HPSE-2, Syndecan-1 (Syn-1) and HER2 expression, HPSE-1 activity and cell viability were evaluated in different breast cancer cells treated or not with trastuzumab. The glycosaminoglycans synthesis and shedding were also evaluated. Trastuzumab and HS binding were analyzed by confocal microscopy and Fluorescence Resonance Energy Transfer (FRET). Results: MCF7 transfected with HPSE-1 cDNA becomes completely resistant to trastuzumab. HS affinity by Trastuzumab was then tested, showing that they bind in high levels and this binding is necessary to antibody activity. In MCF7 cells, trastuzumab decreases HPSE-1, HPSE-2, HER2 and Syn-1 mRNA expression, while in MCF7-HPSE-1 the antibody increases the expression of these molecules. Conclusions: Our results have demonstrated that an ideal concentration of HS in cell surface, regulated by trastuzumab, is necessary to its action, beyond HER2 high levels. High HS concentration at cell surface enhances the antibody amount disposable to interact with HER2 in cell surface, determining breast cancer cells susceptibility to trastuzumab. These new insights could be useful when devising strategies for overcoming trastuzumab resistance in HER2 positive cancers. Supported by FAPESP, CNPq, CAPES.

Ischemia–Reperfusion-Induced Unmeasured Anion Generation and Glycocalyx Shedding: Sevoflurane versus Propofol Anesthesia

ABSTRACTIntroduction: Vascular leakage after ischemia–reperfusion (IR) is largely attributed to the destruction of the endothelial barrier and its associated negatively charged glycocalyx. In vitro, sevoflurane attenuates these changes. Therefore, we compared sevoflurane with propofol with regard to the protection of the glycocalyx and the release of negatively charged substances in vivo. Methods: After surgical preparation under midazolam–fentanyl, nine pigs each received either propofol or sevoflurane. Ischemia of 90 min was induced by a balloon catheter in the thoracic aorta. After 120 min of reperfusion, the anesthetics were changed back to midazolam–fentanyl. Five animals, each without aortic occlusion, served as time controls. Blood electrolyte parameters were measured, from which the strong ion gap (SIG) was calculated. Serum heparan sulfate concentrations and immunohistology served as a marker of glycocalyx destruction. Results: Immediately after reperfusion, SIG increased significantly only in the propofol group (+6.7 mEq/l versus baseline; p < .05), remaining stable in sevoflurane and both time-controlled groups. Initially, heparan sulfate concentration increased comparably in both experimental groups, but after 120 min, it became stable in sevoflurane-anesthetized animals, while increasing further in the propofol group (p < .05). Conclusions: Unmeasured anions, predictive of negative outcome in previous studies, did not increase significantly in sevoflurane-anesthetized animals. Additionally, there was less heparan sulfate shedding over time, signaling less destruction of the glycocalyx. Therefore, in this in-vivo situation, sevoflurane proves to be superior to propofol in protecting the endothelium from IR injury.

Rapid oligomer formation of human muscle acylphosphatase induced by heparan sulfate

Many human diseases are caused by the conversion of proteins from their native state into amyloid fibrils that deposit in the extracellular space. Heparan sulfate, a component of the extracellular matrix, is universally associated with amyloid deposits and promotes fibril formation. The formation of cytotoxic prefibrillar oligomers is challenging to study because of its rapidity, transient appearance and the heterogeneity of species generated. The process is even more complex with agents such as heparan sulfate. Here we have used a stopped-flow device coupled to turbidometry detection to monitor the rapid conversion of human muscle acylphosphatase into oligomers with varying heparan sulfate and protein concentrations. We also analyzed mutants of the 15 basic amino acids of acylphosphatase, identifying the residues primarily involved in heparan sulfate-induced oligomerization of this protein and tracing the process with unprecedented molecular detail.

Heparan Sulfate Chains of Syndecan-1 Regulate Ectodomain Shedding

Matrix metalloproteinases release intact syndecan-1 ectodomains from the cell surface giving rise to a soluble, shed form of the proteoglycan. Although it is known that shed syndecan-1 controls diverse pathophysiological responses in cancer, wound healing, inflammation, infection, and immunity, the mechanisms regulating shedding remain unclear. We have discovered that the heparan sulfate chains present on syndecan core proteins suppress shedding of the proteoglycan. Syndecan shedding is dramatically enhanced when the heparan sulfate chains are enzymatically degraded or absent from the core protein. Exogenous heparan sulfate or heparin does not inhibit shedding, indicating that heparan sulfate must be attached to the core protein to suppress shedding. Regulation of shedding by heparan sulfate occurs in multiple cell types, for both syndecan-1 and syndecan-4 and in murine and human syndecans. Mechanistically, the loss of heparan sulfate enhances the susceptibility of the core protein to proteolytic cleavage by matrix metalloproteinases. Enhanced shedding of syndecan-1 following loss of heparan sulfate is accompanied by a dramatic increase in core protein synthesis. This suggests that in response to an increase in the rate of shedding, cells attempt to maintain a significant level of syndecan-1 on the cell surface. Together these data indicate that the amount of heparan sulfate present on syndecan core proteins regulates both the rate of syndecan shedding and core protein synthesis. These findings assign new functions to heparan sulfate chains, thereby broadening our understanding of their physiological importance and implying that therapeutic inhibition of heparan sulfate degradation could impact the progression of some diseases.

Genistein in Sanfilippo disease: A randomized controlled crossover trial

Sanfilippo disease (mucopolysaccharidosis type III [MPS III]) is a rare neurodegenerative metabolic disease caused by a deficiency of 1 of the 4 enzymes involved in the degradation of heparan sulfate (HS), a glycosaminoglycan (GAG). Genistein has been proposed as potential therapy but its efficacy remains uncertain. We aimed to determine the efficacy of genistein in MPS III. Thirty patients were enrolled. Effects of genistein were determined in a randomized, crossover, placebo-controlled intervention with a genistein-rich soy isoflavone extract (10mg/kg/day of genistein) followed by an open-label extension study for patients who were on genistein during the last part of the crossover. Genistein resulted in a significant decrease in urinary excretion of total GAGs (p = 0.02, slope -0.68 mg GAGs/mmol creatinine/mo) and in plasma concentrations of HS (p = 0.01, slope -15.85 ng HS/ml/mo). No effects on total behavior scores or on hair morphology were observed. Parents or caregivers could not predict correctly during which period of the crossover a patient was on genistein. Genistein at 10mg/kg/day effectively reduces urinary excretion of GAGs and plasma HS concentration in patients with MPS III. However, the absolute reduction in GAGs and in HS is small and values after 12 months of treatment remain within the range as observed in untreated patients. No clinical efficacy was detected. Substantially higher doses of genistein might be more effective as suggested by recent studies in animal models.

Serum Heparan Sulfate Concentration is Correlated with the Failure of Epidermal Growth Factor Receptor Tyrosine Kinase Inhibitor Treatment in Patients with Lung Adenocarcinoma

The epidermal growth factor receptor (EGFR) mutation status is a validated biomarker for the stratification of EGFR-tyrosine kinase inhibitor (EGFR-TKIs) treatment in patients with non-small cell lung cancer (NSCLC); however, its use is limited in patients with wild-type EGFR, and new biomarkers are needed. We hypothesized that the serum concentration of heparan sulfate (HS), which activates oncogenic growth factor receptor signaling through EGFR and non-EGFR signaling pathways, may be a novel glycobiological biomarker for EGFR-TKIs treatment in NSCLC. The pretreatment serum HS concentrations were determined using enzyme-linked immunosorbent assay in 83 patients with stage IV non-small cell lung adenocarcinoma who received EGFR-TKIs treatment. The relationship between the serum HS concentrations and patient characteristics, tumor response, progression-free survival (PFS), and overall survival (OS) were analyzed. Patient sex, performance status, smoking history, and EGFR mutation status were associated with tumor response. The serum HS concentrations were significantly higher among patients with progressive disease than among those without progressive disease (p = 0.003). Furthermore, the serum HS concentrations were strongly associated with a poor PFS and OS in a univariate Cox analysis (p = 0.0022 and p = 0.0003, respectively). A stratified multivariate Cox model according to the EGFR mutation status showed that higher HS concentrations were significantly associated with a shorter PFS and OS (p = 0.0012 and p = 0.0003). We concluded that a high-serum HS concentration was strongly related to a poor treatment outcome of EGFR-TKIs and may be a promising noninvasive and repeatable glycobiological biomarker in cancer treatment.

Ultraviolet-B irradiation induces epidermal up-regulation of heparanase expression and activity

Heparan sulfate (HS) glycosaminoglycans are abundant components of basement membranes and cell surfaces where they are present associated with specific core-proteins to form proteoglycans, mainly perlecan, glypicans and syndecans. They play many roles such as modulation of cell proliferation and differentiation, cell–matrix adhesion and assembly. It was previously shown that HS content decreases during skin aging. This decrease could be explained either by a decrease of HS synthesis or by an increased activity of its degrading enzyme, heparanase (Hpse-1). Since UV-B irradiation is one of the most important factor for skin photo-damage, we decided to study the effects of UV-B irradiation on heparanase expression and activity in human epidermal keratinocytes. Normal human keratinocytes and reconstructed epidermis were submitted to increasing doses of UV-B. HPSE1 mRNA levels were measured using real time PCR and heparanase enzymatic activity was quantified in human keratinocyte cultures using a microtiter-based assay. Expression and distribution of Hpse-1 were also studied in reconstructed epidermis by immunofluorescence. Both HPSE1 mRNA level and heparanase enzymatic activity were increased after UV-B irradiation of keratinocyte cultures in a time and dose-dependent manner. Protein expression of Hpse-1 was also up-regulated with increasing doses of UV-B in reconstructed epidermis. Increase of Hpse-1 expression and activity in the epidermis after UV-B irradiation could contribute to skin photo-aging.

Zebrafish Ext2 is necessary for Fgf and Wnt signaling, but not for Hh signaling

BackgroundHeparan sulfate (HS) biosynthesis is tightly regulated during vertebrate embryo development. However, potential roles for HS biosynthesis in regulating the function of paracrine signaling molecules that bind to HS are incompletely understood.ResultsIn this report we have studied Fgf, Wnt and Hedgehog (Hh) signaling in ext2 mutants, where heparan sulfate content is low. We found that Fgf targeted gene expression is reduced in ext2 mutants and that the remaining expression is readily inhibited by SU5402, an FGF receptor inhibitor. In the ext2 mutants, Fgf signaling is shown to be affected during nervous system development and reduction of Fgf ligands in the mutants affects tail development. Also, Wnt signaling is affected in the ext2 mutants, as shown by a stronger phenotype in ext2 mutants injected with morpholinos that partially block translation of Wnt11 or Wnt5b, compared to injected wild type embryos. In contrast, Hh dependent signaling is apparently unaffected in the ext2 mutants; Hh targeted gene expression is not reduced, the Hh inhibitor cyclopamine is not more affective in the mutants and Hh dependent cell differentiation in the retina and in the myotome are normal in ext2 mutants. In addition, no genetic interaction between ext2 and shha during development could be detected.ConclusionWe conclude that ext2 is involved in Fgf and Wnt signaling but not in Hh signaling, revealing an unexpected specificity for ext2 in signaling pathways during embryonic development. Thus, our results support the hypothesis that regulation of heparan sulfate biosynthesis has distinct instructive functions for different signaling factors.

Fine structural characterization of chondroitin sulfate in urine of bladder pain syndrome subjects

Urothelial glycosaminoglycans (GAGs) are decreased in bladder pain syndrome (BPS), and urinary GAGs are thought to reflect this deficiency. In previous researches, urine GAG levels were found increased, decreased, or similar between BPS and controls. Additionally, no study is available on the structure characterization of urinary chondroitin sulfate (CS) in BPS patients. CS in the urine of BPS-affected patients and controls has been determined by specific electrophoresis, along with total GAGs and heparan sulfate (HS) percentage, and CS disaccharides have been quantified by high-performance liquid chromatography. No significant differences were obtained for total amount of GAGs, absolute content of CS and HS, and their relative percentages. Moreover, no differences were observed for CS structure confirming similar urine CS composition in BPS subjects and controls. This study found no significant differences of BPS and control urine GAG levels and CS structure to allow use of these parameters as diagnostic markers for BPS diagnosis.

An analysis of serum heparan sulfate concentration and EGFR tyrosine kinase inhibitor treatment in patients with non-small cell lung adenocarcinoma.

10601 Background: The EGFR mutation status is a validated biomarker for the stratification of EGFR-tyrosine kinase inhibitor (EGFR-TKIs) treatment in patients with non-small-cell lung cancer (NSCLC); however, its use is limited in patients with wild-type EGFR, and new biomarkers are needed. We hypothesized that the serum concentration of heparan sulfate (HS), which activates oncogenic growth factor receptor signaling through EGFR and non-EGFR signaling pathways, may be a novel glycobiological biomarker for EGFR-TKIs treatment in NSCLC. Methods: The pre-treatment serum HS concentrations were determined using ELISA in 83 patients with stage IV non-small-cell lung adenocarcinoma who received EGFR-TKIs treatment. The relationship between the serum HS concentrations and patient characteristics, tumor response, progression-free survival (PFS) and overall survival (OS) were analyzed. Results: Patient sex, performance status, smoking history, and EGFR mutation status were associated with tumor response. The serum HS concentrations were significantly higher among patients with progressive disease (PD) than among those without PD (P=0.003). Furthermore, the serum HS concentrations were strongly associated with a poor PFS and OS in a univariate Cox analysis (P=0.0022 and P=0.0003, respectively). A stratified multivariate Cox model according to the EGFR mutation status showed that higher HS concentrations were significantly associated with a shorter PFS and OS (P=0.0012 and P=0.0003). Conclusions: We concluded that a high serum HS concentration was strongly related to a poor treatment outcome of EGFR-TKIs and may be a promising non-invasive and repeatable glycobiological biomarker in cancer treatment.

Glycosaminoglycans of Abdominal Skin After Massive Weight Loss in Post-bariatric Female Patients

The number of post-bariatric patients had a significant increase over the last years, and a better understanding of the consequences of massive weight loss on skin is imperative. Despite weight-loss-related changes in collagen and elastin have been reported, less is known about changes in another of the matrix components of the skin, the glycosaminoglycans. The objective of this study is to evaluate abdominal skin glycosaminoglycans concentrations and perlecan and collagen III expression in post-bariatric female patients. Skin tissue samples from the abdomen of lean (n = 19) and post-bariatric (n = 24) female patients were compared. Sulfated glycosaminoglycans and hyaluronic acid were extracted, characterized and quantified. Perlecan and collagen III expression was assessed by immunofluorescence. The major glycosaminoglycans found were dermatan sultafe and hyaluronic acid; the others were found in smaller amounts. The skin of the post-bariatric patients had lower concentrations of heparan sulfate (p = 0.002) while hyaluronic acid, dermatan sulfate, and chondroitin sulfate concentrations were similar to the lean women's skin. Post-bariatric skin showed decreased expression of perlecan and increased expression of collagen III. No correlation was found among glycosaminoglycans concentrations and age, body mass index, frequency of pregnancies, or skin types, but it was observed in higher skin heparan sulfate concentrations in post-bariatric patients who had their weights stabilized for over than 24months (p = 0.000). Abdominal skin of post-bariatric women presented decreased heparan sulfate concentrations and perlecan expression and increased expression of collagen III.