Dermatan Sulfate Content Research Articles

Quantitative, compositional, and immunohistochemical analyses of chondroitin sulfate, dermatan sulfate, and hyaluronan in internal organs of deer (Cervus nippon centralis and C. n. yesoensis) and cattle (Bos taurus)

Chondroitin sulfate (CS) + dermatan sulfate (DS) and hyaluronan (HA) concentrations and the sulfation patterns of CS-DS in the cartilaginous tissues and alimentary canals of Honshu Sika deer, Hokkaido Sika deer, and cattle were investigated in the present study. CS + DS concentrations were high in cartilaginous tissues, namely, the trachea and scapular cartilage region (5– 12 g*), and low in the alimentary canal (~0.3 g*). HA concentrations were low in cartilaginous tissues and the alimentary canal (~0.2 g*). All tissues mainly contained A-type [HexAGalNAc(4-sulfate)] and C-type [HexAGalNAc(6-sulfate)] CS + DS. The ratios of A-type/C-type CS + DS were 1.2– 3.1 and 0.9– 16.4 in cartilaginous tissues and the alimentary canal, respectively. CS + DS predominantly comprised β-D-GlcA and α-L-IdoA in cartilaginous tissues and the alimentary canal, respectively. The alimentary canal characteristically contained up to 14 % highly sulfated E-type [HexAGalNAc(4,6-disulfate)] and D-type [HexA(2-sulfate)GalNAc(6-sulfate)] CS + DS. The specific distributions of CS and DS were immunohistochemically confirmed using CS + DS-specific antibodies. Although the omasum of cattle is more likely to have higher concentrations of CS + DS and HA, no significant species differences were observed in the concentrations or sulfation patterns of CS + DS among species for Honshu Sika deer, Hokkaido Sika deer, and cattle. (*per 100 g of defatted dry tissue).

α-L-iduronidase fused with humanized anti-human transferrin receptor antibody (lepunafusp alfa) for mucopolysaccharidosis type I: A phase 1/2 trial

Mucopolysaccharidosis type I (MPS I) causes systemic accumulation of glycosaminoglycans due to a genetic deficiency of α-L-iduronidase (IDUA), which results in progressive systemic symptoms affecting multiple organs, including the central nervous system (CNS). Because the blood-brain barrier (BBB) prevents enzymes from reaching the brain, enzyme replacement therapy is effective only against the somatic symptoms. Hematopoietic stem cell transplantation can address the CNS symptoms, but the risk of complications limits its applicability. We have developed a novel genetically modified protein consisting of IDUA fused with humanized anti-human transferrin receptor antibody (lepunafusp alfa; JR-171), which has been shown in nonclinical studies to be distributed to major organs, including the brain, bringing about systemic reductions in heparan sulfate (HS) and dermatan sulfate concentrations. Subsequently, a first-in-human study was conducted to evaluate the safety, pharmacokinetics, and exploratory efficacy of JR-171 in 18 patients with MPS I. No notable safety issues were observed. Plasma drug concentration increased dose-dependently and reached its maximum approximately 4 hours after the end of drug administration. Decreased HS in the cerebrospinal fluid suggested successful delivery of JR-171 across the BBB, while suppressed urine and serum concentrations of the substrates indicated its somatic efficacy was comparable to that of laronidase.

Enzyme replacement with transferrin receptor-targeted α-L-iduronidase rescues brain pathology in mucopolysaccharidosis I mice

Mucopolysaccharidosis I (MPS I), a lysosomal storage disease caused by dysfunction of α-L-iduronidase (IDUA), is characterized by the deposition of dermatan sulfate (DS) and heparan sulfate (HS) throughout the body, which causes several somatic and central nervous symptoms. Although enzyme-replacement therapy (ERT) is currently available to treat MPS I, it does not alleviate central nervous disorders, as it cannot penetrate the blood-brain barrier. Here we evaluate the brain delivery, efficacy, and safety of JR-171, a fusion protein comprising humanized anti-human transferrin receptor antibody Fab and IDUA, using monkeys and MPS I mice. Intravenously administered JR-171 was distributed in major organs, including the brain, and reduced DS and HS concentrations in the central nervous system and peripheral tissues. JR-171 exerted similar effects on peripheral disorders similar to conventional ERT and further reversed brain pathology in MPS I mice. We found that JR-171 improved spatial learning ability, which was seen to deteriorate in the vehicle-treated mice. Further, no safety concerns were noted in repeat-dose toxicity studies in monkeys. This study provides nonclinical evidence that JR-171 might potentially prevent and even improve disease conditions in patients with neuronopathic MPS I without serious safety concerns.

Quantitative 2D 1H, 13C HSQC NMR Spectroscopy for the Determination of Chondroitin Sulfate and Dermatan Sulfate Content in Danaparoid Sodium.

Danaparoid sodium is a biopolymeric complex drug composed of the most abundant heparan sulfate (HS) followed in descending order by dermatan sulfate (DS) and chondroitin sulfate (CS). This composite nature explains its peculiar antithrombotic and anticoagulant properties and make it particularly advantageous when the risk of heparin-induced thrombocytopenia occurs. A specific control of the danaparoid composition is required by the Ph. Eur. The monograph includes the CS and DS limit contents and describes the method for their quantification through selective enzymatic degradations. In this study, a quantitative two-dimensional nuclear magnetic resonance (NMR) method is proposed as a new method suitable for CS and DS quantification. Statistical comparison of the results provided by the analysis of a series of danaparoid samples with both NMR and enzymatic methods highlights a small systematic difference, likely derived from lyase-resistant sequences bearing oxidized terminals. Some modified structures, whose survival to the enzymatic action was confirmed by mass spectrometry, can be detected and quantified by NMR. The proposed NMR method can serve for the determination of DS and CS contents, is an easy-to-apply method with no dependence from enzymes and standards, and provides extensive structural information on the overall glycosaminoglycans mixture.

A biphasic response of polymerized Type 1 collagen architectures to dermatan sulfate.

Collagen I, the most abundant extracellular matrix (ECM) protein in vertebrate tissues provides mechanical durability to tissue microenvironments and regulates cell function. Its fibrillogenesis in biological milieu is predominantly regulated by dermatan sulfate proteoglycans, proteins conjugated with iduronic acid-containing dermatan sulfate (DS) glycosaminoglycans (GAG). Although DS is known to regulate tissue function through its modulation of Coll I architecture, a precise understanding of the latter remains elusive. We investigated this problem by visualizing the fibrillar pattern of fixed Coll I gels polymerized in the presence of varying concentrations of DS using second harmonic generation microscopy. Measuring mean second harmonic generation signal (which estimates the ordering of the fibrils), and surface occupancy (which estimates the space occupied by fibrils) supported by confocal reflectance microscopy, our observations indicated that the effect on fibril pattern of DS is contextual upon the latter's concentrations: Lower levels of DS resulted in sparse disorganized fibrils; higher levels restore organization, with fibrils occupying greater space. An appropriate change in elasticity as a result of DS levels was also observed through atomic force microscopy. Examination of dye-based GAG staining and scanning electron microscopy suggested distinct constitutions of Coll I gels when polymerized with higher and lower levels of DS. We observed that adhesion of the invasive ovarian cancer cells SKOV3 decreased for lower DS levels but was partially restored at higher DS levels. Our study shows how the Coll I gel pattern-tuning of DS is of relevance for understanding its biomaterial applications and possibly, pathophysiological functions.

A Phase 2/3 Trial of Pabinafusp Alfa, IDS Fused with Anti-Human Transferrin Receptor Antibody, Targeting Neurodegeneration in MPS-II

Pabinafusp alfa (JR-141) is a novel enzyme drug that crosses the blood-brain barrier by transcytosis via transferrin receptors. In order to establish its efficacy and safety, a multicenter, single-arm, open-label phase 2/3 clinical trial was conducted in 28 Japanese patients with mucopolysaccharidosis II (MPS-II, Hunter syndrome) by intravenous administrations of 2.0 mg/kg of pabinafusp alfa for 52 weeks. The primary efficacy endpoint was changes in heparan sulfate (HS) concentrations in the cerebrospinal fluid (CSF). Secondary endpoints included assessments of neurocognitive development for central efficacy, and changes in plasma HS and dermatan sulfate (DS) concentrations for peripheral efficacy. HS concentrations in the CSF significantly decreased from baseline to week 52 (p < 0.001), suggesting continuous inhibition of substrate accumulations in the CNS, i.e., hitherto unaddressed progressive neurodegeneration. Evaluations of neurocognitive developments showed positive changes in 21 of the 28 patients. Serum HS and DS concentrations, liver and spleen volumes, and other assessments suggested the peripheral efficacy of pabinafusp alfa was comparable to that of idursulfase. Drug-related adverse events were mild or moderate in severity, transient, and manageable. The results establish delivery across the BBB of pabinafusp alfa as an effective therapeutic for treating both the CNS and peripheral symptoms of patients with MPS-II.

Intravenously Administered Iduronate-2-Sulfatase Fused with Anti-Human Transferrin Receptor Antibody (Jr-141) Crosses the Blood-Brain Barrier to Address Central Nervous System Disorders in Hunter Syndrome: A Randomised, Open-Label, Phase 1/2 Trial

Background: Hunter syndrome (mucopolysaccharidosis II, MPS II) is an X-linked recessive lysosomal storage disease in which a deficiency of iduronate-2-sulfatase causes an accumulation of glycosaminoglycans, giving rise to multiple systemic as well as central nervous system (CNS) symptoms. The currently available enzyme replacement therapy with idursulfase is ineffective against the associated CNS symptoms, because the blood-brain barrier (BBB) prevents the enzyme from reaching the brain. A novel iduronate-2-sulfatase fused with anti-human transferrin receptor antibody (JR-141) has been shown to penetrate the BBB and ameliorate learning deficits in model mice. This first-in-human translational study evaluated the pharmacokinetics, safety and efficacy of JR-141 in patients with MPS II. Methods: In this phase 1/2 multicentre, open-label, randomised clinical trial, 15 patients with MPS II who were being treated with intravenous idursulfase were recruited in Japan, and treatment was switched to intravenous administration of JR-141. In Part 1 of the study, which was designed to evaluate the safety of JR-141, the dosage was escalated over a period of 4 weeks (from 0.01 to 2.0 mg/kg/week); in Part 2, a 4-week randomised evaluation of the pharmacokinetics, safety and exploratory efficacy of JR-141 (at 1.0 or 2.0 mg/kg/week) was made. Cerebrospinal fluid (CSF) concentrations of heparan sulfate (HS) and dermatan sulfate (DS) were compared at the start of JR-141 administration and after 3 weeks, and plasma and urine concentrations of HS and DS were compared at the start and after 4 weeks. Findings: Between 10th May, 2017 and 25th October, 2017, we recruited a total of 15 male patients from 9 investigational sites in Japan. Of these, 14 were enrolled after 1 was excluded as unsuitable. Two patients without intellectual disabilities were selected for the dose escalation study (Part 1) and were given 0*01, 0*1, 1.0, and 2*0 mg/kg/week of JR-141 intravenously; plasma concentrations of JR-141 were dose-dependent and peaked at 3 h after initiation of each infusion. One patient developed erythema and pyrexia, which abated quickly, and the other exhibited no adverse reactions. In Part 2, the remaining 12 patients were randomly allocated to JR-141 dosing groups of 1*0 or 2*0 mg/kg/week. The plasma concentration profiles of JR-141 did not differ between the first and final administration, indicating no drug accumulation. Plasma and urinary levels of HS and DS were both suppressed by JR-141, and HS concentrations in the CSF had significantly decreased after the last administration (by an average of 25*1% in patients taking 1*0 mg/kg/week, and 31*5% in those taking 2*0 mg/kg/week). Two patients showed some amelioration of neurocognitive and motor symptoms following the 4-week treatment. No drug-related serious adverse events were observed during the study period. Interpretation: Patients with Hunter syndrome who were treated with JR-141 showed significant reductions in neurotoxin levels in the CSF, suggesting the drug successfully penetrated the BBB. Furthermore, JR-141 was no less effective in treating systemic symptoms than conventional enzyme replacement therapy with idursulfase. If JR-141 reduces glycosaminoglycan accumulation in the CSF, it should also be effective in treating CNS symptoms. This will be investigated in future clinical trials. Clinical Trial Number: NCT03128593 Funding Statement: The trial was funded by JCR Pharmaceuticals. Declaration of Interests: Torayuki Okuyama reports research grants from Sanofi, JCR Pharmaceuticals, GC Pharma, Dainippon Sumitomo Pharma, Actelion, Alexion and Anges, along with honoraria from Sanofi, Dainippon Sumitomo Pharma, Actelion and Anges. Yoshikatsu Eto has conducted consultancy for JCR Pharmaceuticals, and he has been awarded grants and research support from Actelion, BioMarin Pharmaceutical Inc. and Sanofi; he has also received honoraria from Actelion, BioMarin Pharmaceutical Inc., Sanofi, Shire and Dainippon Sumitomo Pharma. Norio Sakai has conducted consultancy for JCR Pharmaceuticals, and he has been awarded grant/research support from Sanofi and Dainippon Sumitomo Pharma, and honoraria from Actelion, BioMarin Pharmaceutical Inc., Sanofi, Shire and Dainippon Sumitomo Pharma. Ethics Approval Statement: The study was conducted in 8 hospitals and specialised MPS centres in Japan, and was in compliance with the Declaration of Helsinki. The protocol and patient informed consent were reviewed and approved by the Institutional Review Board at each participating institution.

Urinary metabolic phenotyping of mucopolysaccharidosis type I combining untargeted and targeted strategies with data modeling

BackgroundApplication of metabolic phenotyping could expand the pathophysiological knowledge of mucopolysaccharidoses (MPS) and may reveal the comprehensive metabolic impairments in MPS. However, few studies applied this approach to MPS. MethodsWe applied targeted and untargeted metabolic profiling in urine samples obtained from a French cohort comprising 19 MPS I and 15 MPS I treated patients along with 66 controls. For that purpose, we used ultra-high-performance liquid chromatography combined with ion mobility and high-resolution mass spectrometry following a protocol designed for large-scale metabolomics studies regarding robustness and reproducibility. Furthermore, 24 amino acids have been quantified using liquid chromatography coupled to tandem mass spectrometry (LC-MS/MS). Keratan sulfate, Heparan sulfate and Dermatan sulfate concentrations have also been measured using an LC-MS/MS method. Univariate and multivariate data analyses have been used to select discriminant metabolites. The mummichog algorithm has been used for pathway analysis. ResultsThe studied groups yielded distinct biochemical phenotypes using multivariate data analysis. Univariate statistics also revealed metabolites that differentiated the groups. Specifically, metabolites related to the amino acid metabolism. Pathway analysis revealed that several major amino acid pathways were dysregulated in MPS. Comparison of targeted and untargeted metabolomics data with in silico results yielded arginine, proline and glutathione metabolisms being the most affected. ConclusionThis study is one of the first metabolic phenotyping studies of MPS I. The findings might help to generate new hypotheses about MPS pathophysiology and to develop further targeted studies of a smaller number of potentially key metabolites.

A Fluorescent Probe for Glycosaminoglycans Applied to the Detection of Dermatan Sulfate by a Mix-and-Read Assay.

Glycosaminoglycans are complex biomolecules of great biological and medical importance. The quantification of glycosaminoglycans, in particular in complex matrices, is challenging due to their inherent structural heterogeneity. Heparin Red, a polycationic, fluorescent perylene diimide derivative, has recently emerged as a commercial probe for the convenient detection of heparins by a mix-and-read fluorescence assay. The probe also detects glycosaminoglycans with a lower negative charge density than heparin, although with lower sensitivity. We describe here the synthesis and characterization of a structurally related molecular probe with a higher positive charge of +10 (vs. +8 of Heparin Red). The superior performance of this probe is exemplified by the quantification of low dermatan sulfate concentrations in an aqueous matrix (quantification limit 1 ng/mL) and the detection of dermatan sulfate in blood plasma in a clinically relevant concentration range. The potential applications of this probe include monitoring the blood levels of dermatan sulfate after administration as an antithrombotic drug in the absence of heparin and other glycosaminoglycans.

Sulfated glycosaminoglycans in human vocal fold lamina propria

IntroductionThe distribution, concentration and function of glycosaminoglycans in the various vocal fold tissues are still unclear. ObjectiveTo evaluate the distribution and concentration of sulfated glycosaminoglycans in different layers of the human vocal fold according to gender and age. MethodsWe used 11 vocal folds obtained from cadavers (7 men and 4 women) with no laryngeal lesion, less than 12h after death, and aged between 35 and 98 years. The folds underwent glycosaminoglycans extraction from the cover and ligament, and post-electrophoresis analysis. Data were compared according to the layer, age and gender. ResultsThe concentration of dermatan sulfate was significantly higher in all layers. No differences were observed in the total concentrations of glycosaminoglycans in layers studied according to gender. It is significantly lower in the cover of individuals aged below 60 years. ConclusionDermatan sulfate, chondroitin sulfate, and heparan sulfate were observed in the human vocal folds cover and ligament of both genders, with the concentration of dermatan sulfate being significantly higher in all layers. Glycosaminoglycans concentration on the cover is significantly lower in individuals below 60 years compared with elderly.

The levels of urinary glycosaminoglycans of patients with attenuated and severe type of mucopolysaccharidosis II determined by liquid chromatography-tandem mass spectrometry

Glycosaminoglycans (GAGs) play important roles on the regulation of extracellular signaling, neuronal development, and cartilage maintenance. The extracellular concentration of total GAGs has been used as an established measure for the diagnosis of mucopolysaccharidoses (MPSs). Heparan sulfate (HS), Dermatan sulfate (DS) and chondroitin sulfate are known to be elevated in the GAGs under pathological conditions associated with MPS. Furthermore, the selective accumulation of disease-specific one of, or a combination of, them has also been used for the estimation of subtypes of MPS. A previously developed method [Auray-Blais C et al. Molecular Genetics and Metabolism 102 (2011) 49–56.] measures the concentration of GAGs using liquid chromatography with tandem mass spectrometry (LC-MS/MS) with higher precision. To ask whether the selective accumulation of HS and DS in the urine of MPS II patients discriminate the attenuated and severe type of MPS II, we examined the concentrations of HS and DS by this methodology. Compared to the healthy controls, we found a marked elevation of HS and DS in all of the MPS II-affected patients. Among patients who received ERT with confirmed elevation of antibody titer, the concentrations of HS in the urine of patients with attenuated type were lower than those with severe type of MPS II. In these patients, the concentrations of DS by LC-MS/MS and of total GAG by DMB failed to depend on the accumulation of antibody. These results suggest that the LC-MS/MS method employed in this study might discriminate the subtypes of MPS II in different clinical background.

S&T-51 EXTRACELLULAR MATRIX CHARACTERISTICS OF TUNICA ALBUGINEA IN MEN WITH AND WITHOUT PEYRONIE'S DISEASE

INTRODUCTION AND OBJECTIVES: Peyronie’s disease (PD) is characterized by the formation of fibrous plaques in the tunica albuginea (TA) of the penis, causing pain and penile curvature during erection, with great impact on quality of life of patients. There is a lack of highly effective medical treatments for PD, mainly because it’s pathophysiology is not completely understood. The aim of this study was to compare possible markers related to alterations and remodeling of extracellular matrix which can be involved on the development of the disease in men with and without PD METHODS: Samples of TA of 10 patients with PD were obtained at the moment of surgical correction of penile curvature. Samples of TA of 10 corpses with normal penis at the physical exam were obtained (control group). Tissues were processed and stained with Hematoxilin, submitted to immunohistochemistry (IHQ) with the following primary antibodies: heparanase (HPSE), heparanase2 (HPSE2), metalloproteinases 2 and 9 (MMP2 and MMP9) and TGF-s. IHQ reaction used peroxidase kit LSAB -peroxidase (DakoCytomation , Copenhagen, Dinamarca). Genic expression of heparanase-1 (HPSE), heparanase2 (HPSE2), metalloprotease 9 (MMP9), syndecan 1 (SYN1), interleukin 6 (IL-6), hyaluronic acid sintetase 1 e 2 (HAS1 e HAS2), hyaluronidases 1 e 2 (HYL1 e HYL2), was done by quantitative RTPCR (qRT-PCR). Statystical analyses were done using software Graph PRISM 5 . Project was reviewed and approved by the Research Ethical Committee of Faculdade de Medicina do ABC, (# 242/2010). RESULTS: Histopathological evaluation showed higher number of cells, reduction of apoptosis events, alteration of the distribution of collagen fibers and diminished number of vessels on TA of patients with PD compared to the controls TA. qRT-PCR analysis showed increased concentration of TGF-s and interleucine1 reduction on HSPE-1 expression and decreased concentration of dermatan sulfate and chondroitin sulfate in patients with PD (p<0.05). However, there were no alterations on HPSE2, MMP2 e MMP9 expressions. Those findings suggest a higher remodeling of extracellular matrix in patients with PD. CONCLUSIONS: In comparison with the controls the TA of patients with PD showed lesser number of vessels and apoptosis events; increased concentration of TGF-s and interleukin-1, reduction of heparanase-1 expression and decreased concentration of dermatan sulfate and chondroitin sulfate.

S&T-52 EARLY CHANGES IN SKELETAL MUSCLE AS A STRONG PROGNOSTIC BIOMARKER IN PATIENTS WITH METASTATIC RENAL CELL CARCINOMA

INTRODUCTION AND OBJECTIVES: Peyronie’s disease (PD) is characterized by the formation of fibrous plaques in the tunica albuginea (TA) of the penis, causing pain and penile curvature during erection, with great impact on quality of life of patients. There is a lack of highly effective medical treatments for PD, mainly because it’s pathophysiology is not completely understood. The aim of this study was to compare possible markers related to alterations and remodeling of extracellular matrix which can be involved on the development of the disease in men with and without PD METHODS: Samples of TA of 10 patients with PD were obtained at the moment of surgical correction of penile curvature. Samples of TA of 10 corpses with normal penis at the physical exam were obtained (control group). Tissues were processed and stained with Hematoxilin, submitted to immunohistochemistry (IHQ) with the following primary antibodies: heparanase (HPSE), heparanase2 (HPSE2), metalloproteinases 2 and 9 (MMP2 and MMP9) and TGF-s. IHQ reaction used peroxidase kit LSAB -peroxidase (DakoCytomation , Copenhagen, Dinamarca). Genic expression of heparanase-1 (HPSE), heparanase2 (HPSE2), metalloprotease 9 (MMP9), syndecan 1 (SYN1), interleukin 6 (IL-6), hyaluronic acid sintetase 1 e 2 (HAS1 e HAS2), hyaluronidases 1 e 2 (HYL1 e HYL2), was done by quantitative RTPCR (qRT-PCR). Statystical analyses were done using software Graph PRISM 5 . Project was reviewed and approved by the Research Ethical Committee of Faculdade de Medicina do ABC, (# 242/2010). RESULTS: Histopathological evaluation showed higher number of cells, reduction of apoptosis events, alteration of the distribution of collagen fibers and diminished number of vessels on TA of patients with PD compared to the controls TA. qRT-PCR analysis showed increased concentration of TGF-s and interleucine1 reduction on HSPE-1 expression and decreased concentration of dermatan sulfate and chondroitin sulfate in patients with PD (p<0.05). However, there were no alterations on HPSE2, MMP2 e MMP9 expressions. Those findings suggest a higher remodeling of extracellular matrix in patients with PD. CONCLUSIONS: In comparison with the controls the TA of patients with PD showed lesser number of vessels and apoptosis events; increased concentration of TGF-s and interleukin-1, reduction of heparanase-1 expression and decreased concentration of dermatan sulfate and chondroitin sulfate.

Establishment of chondroitin B lyase-based analytical methods for sensitive and quantitative detection of dermatan sulfate in heparin

Dermatan sulfate (DS) is one of the hardest impurities to remove from heparin products due to their high structural similarity. The development of a sensitive and feasible method for quantitative detection of DS in heparin is essential to ensure the clinical safety of heparin pharmaceuticals. In the current study, based on the substrate specificity of chondroitin B lyase, ultraviolet spectrophotometric and strong anion-exchange high-performance liquid chromatographic methods were established for detection of DS in heparin. The former method facilitated analysis in heparin with DS concentrations greater than 0.1mgmL−1 at 232nm, with good linearity, precision and recovery. The latter method allowed sensitive and accurate detection of DS at concentrations lower than 0.1mgmL−1, exhibiting good linearity, precision and recovery. The linear range of DS detection using the latter method was between 0.01 and 0.5mgmL−1.

Human pulmonary artery endothelial cells in the model of mucopolysaccharidosis VI present a prohypertensive phenotype

BackgroundMucopolysaccharidosis type VI (MPS VI) is an autosomal recessive lysosomal disorder caused by a deficient activity of N-acetylgalactosamine-4-sulfatase (ARSB). Pulmonary hypertension (PH) occurs in MPS VI patients and is a marker of bad prognosis. Malfunction of endothelium, which regulates vascular tonus and stimulates angiogenesis, can contribute to the occurrence of PH in MPS VI. AimThe aim of the study was to establish a human MPS VI cellular model of pulmonary artery endothelial cells (HPAECs) and evaluate how it affects factors that may trigger PH such as proliferation, apoptosis, expression of endothelial nitric oxide synthase (eNOS), natriuretic peptide type C (NPPC), and vascular endothelial growth factor A (VEGFA). ResultsIncreasing concentrations of dermatan sulfate (DS) reduce the viability of the cells in both ARSB deficiency and controls, but hardly influence apoptosis. The expression of eNOS in HPAECs is reduced up to two thirds in the presence of DS. NPPC shows a biphasic expression reaction with an increase at 50μg/mL DS and reduction at 0 and 100μg/mL DS. The expression of VEGFA decreases with increasing DS concentrations and absence of elastin, and increases with increasing DS in the presence of elastin. ConclusionOur data suggest that MPS VI endothelium presents a prohypertensive phenotype due to the reduction of endothelium's proliferation ability and expression of vasorelaxing factors.

Increase of glycosaminoglycans and metalloproteinases 2 and 9 in liver extracellular matrix on early stages of extrahepatic cholestasis.

Cholestasis produces hepatocellular injury, leukocyte infiltration, ductular cells proliferation and fibrosis of liver parenchyma by extracellular matrix replacement. Analyze bile duct ligation effect upon glycosaminoglycans content and matrix metalloproteinase (MMPs) activities. Animals (6-8 weeks; n = 40) were euthanized 2, 7 or 14 days after bile duct ligation or Sham-surgery. Disease evolution was analyzed by body and liver weight, seric direct bilirubin, globulins, gamma glutamyl transpeptidase (GGT), alkaline phosphatase (Alk-P), alanine and aspartate aminotransferases (ALT and AST), tissue myeloperoxidase and MMP-9, pro MMP-2 and MMP-2 activities, histopathology and glycosaminoglycans content. Cholestasis caused cellular damage with elevation of globulins, GGT, Alk-P, ALT, AST. There was neutrophil infiltration observed by the increasing of myeloperoxidase activity on 7 (P = 0.0064) and 14 (P = 0.0002) groups which leads to the magnification of tissue injuries. Bile duct ligation increased pro-MMP-2 (P = 0.0667), MMP-2 (P = 0.0003) and MMP-9 (P<0.0001) activities on 14 days indicating matrix remodeling and establishment of inflammatory process. Bile duct ligation animals showed an increasing on dermatan sulfate and/or heparan sulfate content reflecting extracellular matrix production and growing mitosis due to parenchyma depletion. Cholestasis led to many changes on rats' liver parenchyma, as so as on its extracellular matrix, with major alterations on MMPs activities and glycosaminoglycans content.

Plasmatic dermatan sulfate and chondroitin sulfate determination in mucopolysaccharidoses

The evaluation of plasmatic galactosaminoglycans, dermatan sulfate (DS) and chondroitin sulfate (CS) can be helpful in the early identification of MPS patients, also considering that primary storage of one type of GAG can lead to secondary accumulation of other lysosomal substrates. We explore the possibility to determine plasmatic DS and CS in numerous healthy pediatric (and sometimes adult) subjects depending on age and in patients affected by various forms of MPS. A highly sensitive HPLC separation and fluorescence detection was applied for plasma/serum DS and CS determination after a specific enzymatic treatment able to release their constituent disaccharides. DS and CS content decrease significantly with age in controls having high values in the first year (∼8μg/mL). A highly significant decrease was observed for 1–5-year-old (∼−33%) and 5–10-year-old (∼−65%) healthy subgroups. No further decrease was determined showing a stabilization after 5 years of age. MPS I Scheie and Hurler patients showed rather similar DS and CS content significantly higher than controls matched for age. Similarly, MPS II, III and IV subjects all presented significantly higher plasmatic DS and CS content compared to healthy subjects matched for age. The same trend was determined for the only patient affected by MPS VI. Plasmatic DS and CS analyzed by the present procedure may be a useful diagnostic and screening marker for various forms of MPS.

Determination of galactosamine impurities in heparin sodium using fluorescent labeling and conventional high-performance liquid chromatography

Heparin is a sulfated glycosaminoglycan (GAG), which contains N-acetylated or N-sulfated glucosamine (GlcN). Heparin, which is generally obtained from the healthy porcine intestines, is widely used as an anticoagulant during dialysis and treatments of thrombosis such as disseminated intravascular coagulation. Dermatan sulfate (DS) and chondroitin sulfate (CS), which are galactosamine (GalN)-containing GAGs, are major process-related impurities of heparin products. The varying DS and CS contents between heparin products can be responsible for the different anticoagulant activities of heparin. Therefore, a test to determine the concentrations of GalN-containing GAG is essential to ensure the quality and safety of heparin products. In this study, we developed a method for determination of relative content of GalN from GalN-containing GAG in heparin active pharmaceutical ingredients (APIs). The method validation and collaborative study with heparin manufacturers and suppliers showed that our method has enough specificity, sensitivity, linearity, repeatability, reproducibility, and recovery as the limiting test for GalN from GalN-containing GAGs. We believe that our method will be useful for ensuring quality, efficacy, and safety of pharmaceutical heparins. On July 30, 2010, the GalN limiting test based on our method was adopted in the heparin sodium monograph in the Japanese Pharmacopoeia.

Plasmatic kinetics of dermatan sulfate during enzyme replacement therapy with iduronate-2-sulfatase in a mucopolysaccharidosis II Patient

Enzyme replacement therapy (ERT) is the worldwide standard of care for a number of mucopolysaccharidosis (MPS) diseases. We report a kinetic study of plasmatic dermatan sulfate (DS) in a 3-year-old subject affected by a severe form of MPS II during the first 10months of ERT with Idursulfase. A strong increase in the DS plasmatic concentration was measured immediately after the first enzyme infusion, with a maximum after 3h, followed by a continuous decrease in the 8-15days following the beginning of treatment. After this, a constant plasmatic content of DS concentration was observed. Overall, during the 10-month treatment period, ERT reduced the plasmatic concentration of DS up to ~80-85%, but it was unable to totally remove it from the blood. We can suppose that immediately after the first enzyme administrations, a large amount of abnormal DS is removed from tissues reaching the blood compartment and eliminated via the urine, and thereafter only minimal changes are observed. The persistency of the residual amounts of DS with the actually recommended dosage in our Patient may suggest the opportunity to promote further studies with increased enzyme dosages to completely remove the accumulation of lysosomal DS.

Alterations of fibrin network structure mediated by dermatan sulfate

Dermatan sulfate (DS) is well-known for its anticoagulant activity through binding to heparin cofactor II (HCII) to enhance thrombin inhibition. It has also been reported that DS has a profibrinolytic effect. We have evaluated the effects of DS solutions (4-20 μg/mL) on the formation (by kinetic studies), structure (by electron microscopy and compaction assays) and lysis (with urokinase-type plasminogen activator) of plasma fibrin networks. The results showed that DS significantly prolonged the lag phase and decreased the fibrin formation rate and the optical density of the final networks versus control, in a concentration dependent way. DS-associated networks presented a minor network percentage compared with control, composed of lower number of fibers per field, which resulted significantly thinner and longer. Moreover, DS rendered gels more sensible to rupture by centrifugal force and more susceptible to lysis. When fibrin formation kinetic assays were performed with purified fibrinogen instead of plasma, in the absence of HCII, the optical density of final DS-associated networks was statistically lower than control. Therefore, a direct effect of DS on the thickness of fibers was observed. Since in all in vitro assays low DS concentrations were used, it could be postulated that the fibrin features described above are plausible to be found in in vivo thrombi and therefore, DS would contribute to the formation of less thrombogenic clots.