Total Sulfated Glycosaminoglycan Research Articles

Mechanical stretch implications for vascular endothelial cells: Altered extracellular matrix synthesis and remodeling in pathological conditions

AimsCardiovascular diseases such as hypertension, thrombosis and atherosclerosis are responses to mechanical forces applied to the endothelium. Endothelial cells respond to hemodynamic mechanical forces such as cellular mechanical stretching. We investigated the expression of glycosaminoglycans, proteoglycans and other extracellular matrix molecules in endothelial cells subjected to various mechanical stimuli. Main methodsEndothelial cells were subjected to mechanical stretch in a vacuum system FlexCell™ to 5% (physiological condition) and 15% (pathological condition), for 4 h or 24 h. Culture plates not subjected to strain were used as controls. Subsequently, ECs were subjected to immunofluorescence, real-time PCR, PCR array, glycosaminoglycans biosynthesis using metabolic radiolabeling with 35S-sulfate and cell behavior assays (adhesion, migration and capillary tube formation). Key findingsMechanical stretch induced changes in endothelial cell morphology. Pathological consequences of mechanical stretch included inhibited migration in 2-fold and capillary-like tube formation in 2-fold, when compared to physiological condition after 4 h of ECs exposure; it also reduced total sulfated glycosaminoglycans synthesis thereabout 1.5-fold. Pathological mechanical stretch conditions induced higher expression after 24 h of ECs exposure to mechanical stretch of syndecan-4 (3.5-fold), perlecan (9.1-fold), decorin (5.7-fold), adhesive proteins as fibronectin (5.6-fold) and collagen III α1 (2.2-fold) and growth factors, including VEGF-A (7.3-fold) and TGFβ-1 (14.6-fold) and TGFβ-3 (4.3-fold). SignificanceExposure of endothelial cells to mechanical stretch influenced remodeling of the extracellular matrix as well as cell-matrix interactions. These studies improve understanding of how vascular biology is affected by mechanical forces and how these molecules behave in cardiovascular diseases.

Effects of a 15-month anti-TNF-\u03b1 treatment on plasma levels of glycosaminoglycans in women with rheumatoid arthritis

BackgroundIn this study, the effect of 15-month anti-tumor necrosis factor alpha (TNF-α) treatment on circulating levels of plasma sulfated glycosaminoglycans (GAGs) and the nonsulfated GAG hyaluronic acid (HA) in female rheumatoid arthritis (RA) patients was assessed.MethodsPlasma was obtained from healthy subjects and RA women treated with TNF-α antagonists (etanercept or adalimumab or certolizumab pegol) in combination with methotrexate. GAGs were isolated from plasma samples using ion exchange low-pressure liquid chromatography. Total sulfated GAGs were quantified using a hexuronic acid assay. Plasma levels of keratan sulfate (KS) and HA were measured using immunoassay kits.ResultsTotal sulfated GAGs and HA levels were higher in female RA patients before treatment in comparison to healthy subjects. KS levels did not differ between RA women and controls. Anti-TNF-α treatment resulted in normalization of plasma total GAG and HA levels in RA patients, without any effect on KS levels.ConclusionsOur results suggest that anti-TNF-α therapy has a beneficial effect on extracellular matrix remodeling in the course of RA.

Substrate accumulation and extracellular matrix remodelling promote persistent upper airway disease in mucopolysaccharidosis patients on enzyme replacement therapy.

IntroductionMucopolysaccharide diseases are a group of lysosomal storage disorders caused by deficiencies of hydrolase enzymes, leading to pathological glycosaminoglycan accumulation. A number of mucopolysaccharidosis (MPS) types are characterised by severe airway disease, the aetiology of which is poorly understood. There is ongoing evidence of significant clinical disease in the long-term despite disease modifying therapeutic strategies, including enzyme-replacement therapy (ERT). To provide a better understanding of this aspect of disease, we have characterised extracellular matrix (ECM) and inflammatory alterations in adenotonsillar tissue samples from 8 MPS patients.MethodsAdenotonsillar samples from MPS I, IVA and VI ERT treated patients and from a single enzyme naïve MPS IIIA individual were compared to non-affected control samples using quantitative immunohistochemistry, qPCR and biochemical analysis.ResultsSignificantly increased lysosomal compartment size and total sulphated glycosaminoglycan (p = 0.0007, 0.02) were identified in patient samples despite ERT. Heparan sulphate glycosaminoglycan was significantly elevated in MPS I and IIIA (p = 0.002), confirming incomplete reversal of disease. Collagen IV and laminin α-5 (p = 0.002, 0.0004) staining demonstrated increased ECM deposition within the reticular and capillary network of MPS samples. No significant change in the expression of the pro-inflammatory cytokines IL-1α, IL-6 or TNF-α was seen compared to control.ConclusionThis study suggests a role for ECM remodelling contributing to the obstructive phenotype of airway disease in MPS. Current therapeutic strategies with ERT fail to normalise these pathological alterations within adenotonsillar samples. Our findings lend novel insight into the pathological cascade of events, with primarily structural rather than inflammatory changes contributing to the continuing phenotype seen in patients despite current therapeutic regimes.

Dermal Interstitial Alterations in Patients With Heart Failure and Reduced Ejection Fraction: A Potential Contributor to Fluid Accumulation?

Large networks of interstitial glycosaminoglycans help to regulate water and electrolyte homeostasis. The relation between dermal interstitial alterations and occurrence of edema in heart failure patients with reduced ejection fraction (HFrEF) is unknown. We hypothesize that in HFrEF patients (1) interstitial glycosaminoglycan density is increased, (2) changes in the interstitial glycosaminoglycan network are associated with interstitial fluid accumulation, and (3) there is a link between the interstitial glycosaminoglycan network and the renin-angiotensin-aldosterone system. Two punch biopsies of the skin were obtained in healthy subjects (n=18) and HFrEF patients (n=29). Alcian blue staining and immunostaining for the angiotensin II type 1 receptor was performed. After obtaining tissue water content, total interstitial glycosaminoglycan (uronic acid) and sulfated glycosaminoglycan were quantified. A venous blood sample, clinical examination, and echocardiography were obtained. A significantly higher interstitial glycosaminoglycan content was observed in HFrEF patients compared with healthy subjects (uronic acid: 13.0±4.2 versus 9.6±1.6 μg/mg; P=0.002; sulfated glycosaminoglycan: 14.1 [11.7; 18.1] versus 10.0 [9.1; 10.8] μg/mg; P<0.001). Uronic acid and sulfated glycosaminoglycan density were strongly associated with tissue water content and peripheral edema (uronic acid: ρ=0.66; P<0.0001 and sulfated glycosaminoglycan: τ=0.58; P<0.0001). Expression of the angiotensin II type 1 receptor was found on dermal cells, although use of angiotensin-converting enzyme inhibitors/angiotensin receptor blocker was associated with significantly lower levels of interstitial glycosaminoglycans in HFrEF patients. Interstitial glycosaminoglycan concentration is significantly increased in HFrEF patients compared with healthy subjects and correlated with tissue water content and clinical signs of volume overload. A better appreciation of the interstitial compartment might improve management of volume overload in HF.

Release of pro-inflammatory cytokines from muscle and bone causes tenocyte death in a novel rotator cuff in vitro explant culture model

ABSTRACTPurpose: Tendinopathy is a significant clinical problem thought to be associated with altered mechanical loading. Explant culture models allow researchers to alter mechanical loading in a controlled in vitro environment while maintaining tenocytes in their native matrix. However, current models do not accurately represent commonly injured tendons, ignoring contributions of associated musculature and bone, as well as regional collagen structure. This study details the characterization of amouse rotator cuff explant culture model, including bone, tendon, and muscle (BTM). Materials and Methods: Following harvest, BTM explants were maintained in stress-deprived culture for one week and tendon was then assessed for changes in cell viability, metabolism, matrix structure and content. Results: Matrix turnover occurred throughout culture as manifested in both gene expression and biosynthesis, but this did not translate to net changes in total collagen or sulfated glycosaminoglycan content. Furthermore, tendon structure was not significantly altered throughout culture. However, we found significant cell death in BTM tendons after 3 days in culture, which we hypothesize is cytokine-induced. Using a targeted multiplex assay, we found high levels of pro-inflammatory cytokines released to the culture medium from muscle and bone, levels that did cause cell deathin tendon-alone controls. Conclusions: Overall, this model presents an innovative approach to understandingrotator cuff injury and tenocyte mechanobiology in a clinically-relevant tendon structure. Our model can be a powerful tool to investigate how mechanical and biological stimuli can alter normal tendon health and lead to tendon degeneration, and may provide a testbed for therapeutics for tendon repair.

Biochemical and biomechanical characterisation of equine cervical facet joint cartilage.

The equine cervical facet joint is a site of significant pathology. Located bilaterally on the dorsal spine, these diarthrodial joints work in conjunction with the intervertebral disc to facilitate appropriate spinal motion. Despite the high prevalence of pathology in this joint, the facet joint is understudied and thus lacking in viable treatment options. The goal of this study was to characterise equine facet joint cartilage and provide a comprehensive database describing the morphological, histological, biochemical and biomechanical properties of this tissue. Descriptive cadaver studies. A total of 132 facet joint surfaces were harvested from the cervical spines of six skeletally mature horses (11 surfaces per animal) for compiling biomechanical and biochemical properties of hyaline cartilage of the equine cervical facet joints. Gross morphometric measurements and histological staining were performed on facet joint cartilage. Creep indentation and uniaxial strain-to-failure testing were used to determine the biomechanical compressive and tensile properties. Biochemical assays included quantification of total collagen, sulfated glycosaminoglycan and DNA content. The facet joint surfaces were ovoid in shape with a flat articular surface. Histological analyses highlighted structures akin to articular cartilage of other synovial joints. In general, biomechanical and biochemical properties did not differ significantly between the inferior and superior joint surfaces as well as among spinal levels. Interestingly, compressive and tensile properties of cervical facet articular cartilage were lower than those of articular cartilage from other previously characterised equine joints. Removal of the superficial zone reduced the tissue's tensile strength, suggesting that this zone is important for the tensile integrity of the tissue. Facet surfaces were sampled at a single, central location and do not capture the potential topographic variation in cartilage properties. This is the first study to report the properties of equine cervical facet joint cartilage and may serve as the foundation for the development of future tissue-engineered replacements as well as other treatment strategies.

Mutant Fibulin-3 Causes Proteoglycan Accumulation and Impaired Diffusion Across Bruch's Membrane.

PurposeThe mutation R345W in EFEMP1 (fibulin-3) causes macular degeneration. This study sought to determine whether proteoglycan content and diffusion across Bruch's membrane are altered in Efemp1ki/ki mice carrying this mutation or in Efemp1−/− mice.MethodsProteoglycans in mouse Bruch's membranes were stained with Cupromeronic Blue (CB). Heparan sulfated proteoglycan (HSPG) and chondroitin/dermatan sulfate proteoglycan (C/DSPG) distributions were visualized following treatments with chondroitinase ABC (C-ABC) or nitrous acid. Total sulfated glycosaminoglycans (sGAGs) in Bruch's membrane/choroid (BrM/Ch) were measured with dimethylmethylene blue (DMMB). Matrix metalloprotease (MMP)-2, MMP-9, and tissue inhibitor of metalloproteinase (TIMP)-3 were examined by immunofluorescence and quantified using Image J. Molecules with different Stokes radius (Rs) were allowed simultaneously to diffuse through mouse BrM/Ch mounted in a modified Ussing chamber. Samples were quantified using gel exclusion chromatography.ResultsHSPGs and C/DSPGs were markedly increased in Efemp1ki/ki Bruch's membrane, and MMP-2 and MMP-9 were decreased, but TIMP-3 was increased. Diffusion across Efemp1ki/ki Bruch's membrane was impaired. In contrast, the proteoglycan amount in Efemp1−/− Bruch's membrane was not significantly different, but the size of proteoglycans was much larger. MMP-2, MMP-3, and TIMP-3 levels were similar to that of Efemp1+/+ mice, but they were localized diffusely in retinal pigment epithelium (RPE) cells instead of Bruch's membrane. Diffusion across Efemp1−/− Bruch's membrane was enhanced.ConclusionsMutant fibulin-3 causes proteoglycan accumulation, reduction of MMP-2 and MMP-9, but increase of TIMP-3, and impairs diffusion across Bruch's membrane. Fibulin-3 ablation results in altered sizes of proteoglycans, altered distributions of MMP-2, MMP-9, and TIMP-3, and enhances diffusion across Bruch's membrane.

Hypoxia-mediated regulation of the secretory properties of mitral valve interstitial cells.

The sophisticated function of the mitral valve depends to a large extent on its extracellular matrix (ECM) and specific cellular components. These are tightly regulated by a repertoire of mechanical stimuli and biological pathways. One potentially important stimulus is hypoxia. The purpose of this investigation is to determine the effect of hypoxia on the regulation of mitral valve interstitial cells (MVICs) with respect to the synthesis and secretion of extracellular matrix proteins. Hypoxia resulted in reduced production of total collagen and sulfated glycosaminoglycans (sGAG) in cultured porcine MVICs. Increased gene expression of matrix metalloproteinases-1 and -9 and their tissue inhibitors 1 and 2 was also observed after incubation under hypoxic conditions for up to 24 h. Hypoxia had no effect on MVIC viability, morphology, or phenotype. MVICs expressed hypoxia-inducible factor (HIF)-1α under hypoxia. Stimulating HIF-1α chemically caused a reduction in the amount of sGAG produced, similar to the effect observed under hypoxia. Human rheumatic valves had greater expression of HIF-1α compared with normal or myxomatous degenerated valves. In conclusion, hypoxia affects the production of certain ECM proteins and expression of matrix remodeling enzymes by MVICs. The effects of hypoxia appear to correlate with the induction of HIF-1α. This study highlights a potential role of hypoxia and HIF-1α in regulating the mitral valve, which could be important in health and disease.NEW & NOTEWORTHY This study demonstrates that hypoxia regulates extracellular matrix secretion and the remodeling potential of heart valve interstitial cells. Expression of hypoxia-induced factor-1α plays a role in these effects. These data highlight the potential role of hypoxia as a physiological mediator of the complex function of heart valve cells.

Braided and Stacked Electrospun Nanofibrous Scaffolds for Tendon and Ligament Tissue Engineering.

Tendon and ligament injuries are a persistent orthopedic challenge given their poor innate healing capacity. Nonwoven electrospun nanofibrous scaffolds composed of polyesters have been used to mimic the mechanics and topographical cues of native tendons and ligaments. However, nonwoven nanofibers have several limitations that prevent broader clinical application, including poor cell infiltration, as well as tensile and suture-retention strengths that are inferior to native tissues. In this study, multilayered scaffolds of aligned electrospun nanofibers of two designs-stacked or braided-were fabricated. Mechanical properties, including structural and mechanical properties and suture-retention strength, were determined using acellular scaffolds. Human bone marrow-derived mesenchymal stem cells (MSCs) were seeded on scaffolds for up to 28 days, and assays for tenogenic differentiation, histology, and biochemical composition were performed. Braided scaffolds exhibited improved tensile and suture-retention strengths, but reduced moduli. Both scaffold designs supported expression of tenogenic markers, although the effect was greater on braided scaffolds. Conversely, cell infiltration was superior in stacked constructs, resulting in enhanced cell number, total collagen content, and total sulfated glycosaminoglycan content. However, when normalized against cell number, both designs modulated extracellular matrix protein deposition to a similar degree. Taken together, this study demonstrates that multilayered scaffolds of aligned electrospun nanofibers supported tenogenic differentiation of seeded MSCs, but the macroarchitecture is an important consideration for applications of tendon and ligament tissue engineering.

Extracellular matrix regenerative graft attenuates the negative impact of polypropylene prolapse mesh on vagina in rhesus macaque

The use of wide pore lightweight polypropylene mesh to improve anatomical outcomes in the surgical repair of prolapse has been hampered by mesh complications. One of the prototype prolapse meshes has been found to negatively impact the vagina by inducing a decrease in smooth muscle volume and contractility and the degradation of key structural proteins (collagen and elastin), resulting in vaginal degeneration. Recently, bioscaffolds derived from extracellular matrix have been used to mediate tissue regeneration and have been widely adopted in tissue engineering applications. Here we aimed to: (1) define whether augmentation of a polypropylene prolapse mesh with an extracellular matrix regenerative graft in a primate sacrocolpopexy model could mitigate the degenerative changes; and (2) determine the impact of the extracellular matrix graft on vagina when implanted alone. A polypropylene-extracellular matrix composite graft (n= 9) and a 6-layered extracellular matrix graft alone (n= 8) were implanted in 17 middle-aged parous rhesus macaques via sacrocolpopexy and compared to historical data obtained from sham (n= 12) and the polypropylene mesh (n= 12) implanted by the same method. Vaginal function was measured in passive (ball-burst test) and active (smooth muscle contractility) mechanical tests. Vaginal histomorphologic/biochemical assessments included hematoxylin-eosin and trichrome staining, immunofluorescent labeling of α-smooth muscle actin and apoptotic cells, measurement of total collagen, collagen subtypes (ratio III/I), mature elastin, and sulfated glycosaminoglycans. Statistical analyses included 1-way analysis of variance, Kruskal-Wallis, and appropriate post-hoc tests. The host inflammatory response in the composite mesh-implanted vagina was reduced compared to that following implantation with the polypropylene mesh alone. The increase in apoptotic cells observed with the polypropylene mesh was blunted in the composite (overall P < .001). Passive mechanical testing showed inferior parameters for both polypropylene mesh alone and the composite compared to sham whereas the contractility and thickness of smooth muscle layer in the composite were improved with a value similar to sham, which was distinct from the decreases observed with polypropylene mesh alone. Biochemically, the composite had similar mature elastin content, sulfated glycosaminoglycan content, and collagen subtype III/I ratio but lower total collagen content when compared to sham (P= .011). Multilayered extracellular matrix graft alone showed overall comparable values to sham in aspects of the biomechanical, histomorphologic, or biochemical endpoints of the vagina. The increased collagen subtype ratio III/I with the extracellular matrix graft alone (P= .033 compared to sham) is consistent with an ongoing active remodeling response. Mesh augmentation with a regenerative extracellular matrix graft attenuated the negative impact of polypropylene mesh on the vagina. Application of the extracellular matrix graft alone had no measurable negative effects suggesting that the benefits of this extracellular matrix graft occur when used without a permanent material. Future studies will focus on understanding mechanisms.

Abstract 697: Decline in arylsulfatase B leads to increase in chondroitin sulfate proteoglycan 4 (CSPG4) and invasiveness in melanoma cell lines

Abstract The proteoglycan CSPG4 [chondroitin sulfate proteoglycan 4; also known as melanoma-associated chondroitin sulfate proteoglycan (MCSP) or neuron-glial antigen 2 (NG2)] has been associated with progression of melanoma, and intervention to reduce CSPG4 has been considered as treatment of melanoma. In recent work, we have reported that decline in the enzyme arylsulfatase B (ARSB, N-acetylgalactosamine 4-sulfatase) is associated with malignancy, including prostate, mammary, and colon. ARSB is the enzyme that removes 4-sulfate groups from the N-acetylgalactosamine 4-sulfate residue at the non-reducing end of chondroitin 4-sulfate (C4S) or dermatan sulfate (DS), and thereby regulates the degradation of the sulfated glycosaminoglycan (GAG) chain. Hence, the absence of ARSB leads to the accumulation of more highly sulfated C4S or DS. In prior published work, decline in ARSB and the associated increase in C4S led to transcriptional events, mediated through a decline in galectin-3 binding to the more highly sulfated C4S present. Transcription factors activator protein 1 (AP-1) and Specificity protein 1 (Sp1) interacted with galectin-3 to mediate the increased transcription of versican and Wnt9A. In this study, the mechanism by which decline in ARSB increased expression of CSPG4 and. increased the invasiveness of melanoma cell lines was addressed. Techniques included: cell culture of normal melanocytes (ATCC PCS 200-013) and melanoma cell lines obtained from radial or vertical growth phase [WM1552C, WM1552C/mock (mock CSPG4 transfection), WM1552C/MCSP (CSPG4 transfected), WM1552C/MCSPÄCD (transfected with CSPG4 without the cytoplasmic domain), WM35] and from metastatic melanoma (1205 Lu); QPCR of CSPG4; ARSB activity assay using the exogenous substrate 4-methylumbelliferylsulfate; silencing of ARSB by siRNA; invasion assay; measurements of total sulfated glycosaminoglycans and C4S using 1,9-dimethylmethylene blue dye; and measurements of matrix metalloproteinase (MMP) activity. The results show that ARSB activity was reduced in the metastatic melanoma cells to 51% of the level in the normal melanocytes. Silencing of ARSB was associated with increase to 2.8 times the baseline level in the mRNA expression of chondroitin sulfate proteoglycan 4 (CSPG4) in the normal melanocytes. Invasiveness of the melanoma cells increased by 70% when ARSB was silenced, with corresponding increase in MMP activity. Since CSPG4 has C4S attachments, the effects of ARSB on CSPG4 expression may affect the availability of C4S GAG chains to interact with extracellular matrix components and affect invasiveness. The findings indicate that decline in ARSB and the associated increase in C4S have profound effects on the invasive potential of melanoma and that increased attention to ARSB and C4S may yield new therapeutic interventions. Citation Format: Sumit Bhattacharyya, Leo Feferman, Kaoru Terai, Arkadiusz A. Dudek, Joanne K. Tobacman. Decline in arylsulfatase B leads to increase in chondroitin sulfate proteoglycan 4 (CSPG4) and invasiveness in melanoma cell lines. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 697.

Effect of CFTR modifiers on arylsulfatase B activity in cystic fibrosis and normal human bronchial epithelial cells

BackgroundThe enzyme Arylsulfatase B (ARSB; N-acetylgalactosamine 4-sulfatase), is required for degradation of sulfated glycosaminoglycans (GAGs) which accumulate in cystic fibrosis. ARSB is reduced in cystic fibrosis cells and increases when defective CFTR is repaired by insertion of the normal gene. This study was undertaken to determine if modification of CFTR by small molecule correctors or potentiators could also increase ARSB and reduce the accumulation of chondroitin 4-sulfate (C4S). MethodsCF bronchial epithelial cells homozygous for the F508 deletion (ACD#14071) and normal human bronchial epithelial cells (BEC) were grown and differentiated following an established protocol. Cells were treated with either VRT-532, a CFTR potentiator, or VRT-534, a CFTR corrector, or vehicle control. The impact on ARSB activity, protein and mRNA expression, C4S and total sulfated glycosaminoglycan content, Interleukin-8 and Interleukin-6 secretion, and neutrophil chemotaxis was determined by specific assays. ResultsThe CFTR potentiator, but not the corrector, increased ARSB activity and expression to the level in the normal bronchial epithelial cells (BEC). Concomitantly, total sulfated glycosaminoglycans and C4S declined, secreted IL-8 increased, secreted IL-6 declined, and neutrophil chemotaxis to the spent media obtained from the potentiator-treated CF cells increased. ConclusionThe CFTR potentiator increased ARSB activity and expression and associated effects. This suggests that a critical interaction between CFTR and ARSB is related to CFTR function in regulation of a ligand-gated anion channel at the cell membrane, rather than to CFTR processing and intracellular trafficking.

ANTIOXIDANT AND IMMUNOSTIMULATING ACTIVITY OF POLYSACCHARIDES EXTRACTED FROM CUBED-SNAILFISH (&lt;i&gt;Liparis tessellatus&lt;/i&gt; Gilbert &amp; Burke, 1912) EGGS

An optimized enzymatic extraction using protamexTM was established for polysaccharides from Liparis tesellatus eggs (PLE). Total sugars, sulfated glycosaminoglycans, uronic acids, and hexosamines content of PLE were 43.2%, 9.0%, 23.9%, and 1.73%, respectively. Superoxide radical (SOx*) scavenging activity and 2,2-diphenyl-1-picrylhydrazyl (DPPH*) scavenging activity of PLE was tested to proof its antioxidant properties. Results on SOx* scavenging activity revealed that PLE has antioxidant activity although it’s not surpass the activity of Ascorbic acid as standard. DPPH* scavenging activity showed that PLE has activity increasing at a concentration-dependent manner. However, at 1.25 mg/ml, the anti-DPPH activities of PLE lowered, possibly due to the pro-oxidant action of the sample. PLE also has effects on NO production of RAW 264.7 cells, suggestive of their possible immunostimulating potential in the human body. On human keratinocyte (HaCaT cell line), HT29 and AGS cells lines, PLE was proofed has no toxicity and showed proliferative effects. This effect showed that PLE could have potency as wound healing promoter. An inference can be made that, PLE has indispensable antioxidant potencies as well as cell proliferating activities on specific cell lines which are deemed necessary in the field of medicine and their utilization as functional food supplements. Keywords: Antioxidant, Fish eggs, Immunostimulator, Liparis tessellatus, Polysaccharides

Decline in arylsulfatase B and Increase in chondroitin 4‐sulfotransferase combine to increase chondroitin 4‐sulfate in traumatic brain injury

In an established rat model of penetrating ballistic-like brain injury (PBBI), arylsulfatase B (ARSB; N-acetylgalactosamine 4-sulfatase) activity was significantly reduced at the ipsilateral site of injury, but unaffected at the contralateral site or in sham controls. In addition, the ARSB substrate chondroitin 4-sulfate (C4S) and total sulfated glycosaminoglycans increased. The mRNA expression of chondroitin 4-sulfotransferase 1 (C4ST1; CHST11) and the sulfotransferase activity rose at the ipsilateral site of injury (PBBI-I), indicating contributions from both increased production and reduced degradation to the accumulation of C4S. In cultured, fetal rat astrocytes, following scratch injury, the ARSB activity declined and the nuclear hypoxia inducible factor-1α increased significantly. In contrast, sulfotransferase activity and chondroitin 4-sulfotransferase expression increased following astrocyte exposure to TGF-β1, but not following scratch. These different pathways by which C4S increased in the cell preparations were both evident in the response to injury in the PBBI-I model. Hence, findings support effects of injury because of mechanical disruption inhibiting ARSB and to chemical mediation by TGF-β1 increasing CHST11 expression and sulfotransferase activity. The increase in C4S following traumatic brain injury is because of contributions from impaired degradation and enhanced synthesis of C4S which combine in the pathogenesis of the glial scar. This is the first report of how two mechanisms contribute to the increase in chondroitin 4-sulfate (C4S) in TBI. Following penetrating ballistic-like brain injury in a rat model and in the scratch model of injury in fetal rat astrocytes, Arylsulfatase B activity declined, leading to accumulation of C4S. TGF-β1 exposure increased expression of chondroitin 4-sulfotransferase. Hence, the increase in C4S in TBI is attributable to both impaired degradation and enhanced synthesis, combining in the pathogenesis of the glial scar.

Hematopoietic effect of deer antler extract fermented by Bacillus subtilis on murine marrow cells.

BACKGROUND/OBJECTIVESWe examined the chemical composition and the effect of fermented deer antler on hematopoietic factors in bone marrow cells.MATERIALS/METHODSFor the preparation of fermented deer antler extract (FAB), fermentation was carried out using Bacillus subtilis at 30℃ for 7 days. The hematopoietic effect of FAB was investigated hematopoietic factors in marrow cells.RESULTSThe contents of total sugar, sulfated glycosaminoglycans, and uronic acid and the dry weight gradually increased with fermentation time. The sialic acid content (from 0.14 mg/mL to 0.54 mg/mL) was the highest on the 4th day of fermentation after which it decreased. The proliferating activity of bone marrow cells increased with fermentation times. The levels of various hematopoietic growth factors were determined to verify the beneficial effect of deer antler extract fermented by B. subtilis on hematopoiesis. FAB increased the number of stem cell factors and granulocyte colony-stimulating factor in bone marrow cells. In addition, FAB augmented the burst-forming unit erythroid and total colonies in splenocyte-conditioned medium compared with non-fermented antler extract (NFA). However, FAB did not affect the mRNA levels of erythropoietin, an important factor for erythropoiesis.CONCLUSIONSFAB, like NFA, did not directly affect hematopoiesis, but contributed to hematopoiesis by stimulating the production of hematopoietic factors.

Wound healing potential of total sulfated glycosaminoglycan (GAG) from malaysian sea cucumber, Stichopus vastus coelomic fluid

Wound healing potential of total sulfated glycosaminoglycan (GAG) from malaysian sea cucumber, Stichopus vastus coelomic fluid

In vitro characterization of self-assembled anterior cruciate ligament cell spheroids for ligament tissue engineering.

Tissue engineering of an anterior cruciate ligament (ACL) implant with functional enthesis requires site-directed seeding of different cell types on the same scaffold. Therefore, we studied the suitability of self-assembled three-dimensional spheroids generated by lapine ACL ligament fibroblasts for directed scaffold colonization. The spheroids were characterized in vitro during 14 days in static and 7 days in dynamic culture. Size maintenance of self-assembled spheroids, the vitality, the morphology and the expression pattern of the cells were monitored. Additionally, we analyzed the total sulfated glycosaminoglycan, collagen contents and the expression of the ligament components type I collagen, decorin and tenascin C on protein and for COL1A1, DCN and TNMD on gene level in the spheroids. Subsequently, the cell colonization of polylactide-co-caprolactone [P(LA-CL)] and polydioxanone (PDS) polymer scaffolds was assessed in response to a directed, spheroid-based seeding technique. ACL cells were able to self-assemble spheroids and survive over 14 days. The spheroids decreased in size but not in cellularity depending on the culture time and maintained or even increased their differentiation state. The area of P[LA-CL] scaffolds, colonized after 14 days by the cells of one spheroid, was in average 4.57 ± 2.3 mm(2). Scaffolds consisting of the polymer P[LA-CL] were more suitable for colonization by spheroids than PDS embroideries. We conclude that ACL cell spheroids are suitable as site-directed seeding strategy for scaffolds in ACL tissue engineering approaches and recommend the use of freshly assembled spheroids for scaffold colonization, due to their balanced proliferation and differentiation.

Semi-interpenetrating networks of hyaluronic acid in degradable PEG hydrogels for cartilage tissue engineering

Hydrolytically biodegradable poly(ethylene glycol) (PEG) hydrogels offer a promising platform for chondrocyte encapsulation and tuning degradation for cartilage tissue engineering, but offer no bioactive cues to encapsulated cells. This study tests the hypothesis that a semi-interpenetrating network of entrapped hyaluronic acid (HA), a bioactive molecule that binds cell surface receptors on chondrocytes, and crosslinked degradable PEG improves matrix synthesis by encapsulated chondrocytes. Degradation was achieved by incorporating oligo (lactic acid) segments into the crosslinks. The effects of HA molecular weight (MW) (2.9×104 and 2×106Da) and concentration (0.5 and 5mgg−1) were investigated. Bovine chondrocytes were encapsulated in semi-interpenetrating networks and cultured for 4weeks. A steady release of HA was observed over the course of the study with 90% released by 4weeks. Incorporation of HA led to significantly higher cell numbers throughout the culture period. After 8days, HA increased collagen content per cell, increased aggrecan-positive cells, while decreasing the deposition of hypertrophic collagen X, but these effects were not sustained long term. Measuring total sulfated glycosaminoglycan (sGAG) and collagen content within the constructs and released to the culture medium after 4weeks revealed that total matrix synthesis was elevated by high concentrations of HA, indicating that HA stimulated matrix production although this matrix was not retained within the hydrogels. Matrix-degrading enzymes were elevated in the low-, but not the high-MW HA. Overall, incorporating high-MW HA into degrading hydrogels increased chondrocyte number and sGAG and collagen production, warranting further investigations to improve retention of newly synthesized matrix molecules.

Reciprocal actions of Wnt9A and BMP4 on chondroitin‐4‐sulfotransferase (CHST11) expression in relation to arylsulfatase B activity (1005.2)

Initial experiments in human colonic epithelial cell lines and in the arylsulfatase B (N‐acetylgalactosamine‐4‐sulfatase; ARSB)‐deficient mouse showed that reduction in ARSB and the resultant increase in chondroitin‐4‐sulfate (C4S) were associated with reduction in chondroitin‐4‐sulfotransferase expression and ~50% reduction in sulfotransferase activity with overall increase in total sulfated glycosaminoglycans and C4S. Our experiments have also shown that decline in ARSB leads to increase in Wnt9A expression in human colonic epithelial cells and mouse intestine. Since modeling of the extracellular matrix by Wnt and BMP4 involves their interactions with sulfated glycosaminoglycans, including chondroitin sulfate, we evaluated the interactions among ARSB, Wnt9A, BMP4, and chondroitin‐4‐sulfotransferase (CHST11). Following silencing of Wnt9A by siRNA, the CHST11 expression increased to ~3 times baseline and the sulfotransferase activity increased to ~1.6 times the baseline. In contrast, when BMP4 was neutralized by blocking antibody, the CHST11 expression declined to ~50% of the baseline level. Exogenous administration of BMP4 increased the CHST11 expression to 2‐3 times the baseline level. In ARSB‐deficient mice, the BMP4 expression was ~50% of the value in the wild‐type control, and consistent with a decline in CHST11 expression. Both BMP4 and Wnt exert crucial effects in modeling of the extracellular environment, and these observed opposing effects on CHST11 expression and in relation to ARSB activity may be fundamental to their interaction and to their structural and functional effects.

Differential effects of estrogen exposure on arylsulfatase B, galactose-6-sulfatase, and steroid sulfatase in rat prostate development

Sulfatase enzymes remove sulfate groups from sulfated steroid hormones, including estrone-sulfate and dehydroepiandrosterone-sulfate, and from sulfated glycosaminoglycans (GAGs), including chondroitin sulfates and heparan sulfate. The enzymes N-acetylgalactosamine-4-sulfatase (arylsulfatase B; ARSB) and N-acetylgalactosamine-6-sulfatase (GALNS), which remove sulfate groups from the sulfated GAGs chondroitin 4-sulfate (C4S) and chondroitin 6-sulfate, respectively, have not been studied in prostate development previously. In this report, the endogenous variation and the impact of exogenous estradiol benzoate on the immunohistochemistry and activity of ARSB and GALNS in post-natal (days 1–30) ventral rat prostate are presented, as well as measurements of steroid sulfatase activity (STS), C4S, total sulfated GAGs, and versican, an extracellular matrix proteoglycan with chondroitin sulfate attachments on days 5 and 30. Findings demonstrate distinct and reciprocal localization of ARSB and GALNS, with ARSB predominant in the stroma and GALNS predominant in the epithelium. Control ARSB activity increased significantly between days 5 and 30, but following estrogen exposure (estradiol benzoate 25μg in 25μl sesame oil subcutaneously on days 1, 3, and 5), activity was reduced and the observed increase on day 30 was inhibited. However, estrogen treatment did not inhibit the increase in GALNS activity between days 5 and 30, and reduced STS activity by 50% on both days 5 and 30 compared to vehicle control. Sulfated GAGs, C4S, and the extracellular matrix proteoglycan versican declined between days 5 and 30 in the control, but these declines were inhibited following estrogen. Study findings indicate distinct variation in expression and activity of sulfatases, sulfated GAGs, C4S, and versican in the process of normal prostate development, and disruption of these events by exogenous estrogen.