Non-sulphated Glycosaminoglycans Research Articles

Effects of mucopolysaccharide polysulphate on tight junction barrier in human epidermal keratinocytes.

Tight junctions (TJs) play important roles in epidermal barrier function and their dysfunction is involved in the pathogenesis of various skin diseases, including atopic dermatitis (AD). Mucopolysaccharide polysulphate (MPS) is the active ingredient of a moisturizing agent used to treat xerosis in patients with AD; however, its mechanism of action on TJ barrier function remains unclear. To elucidate the effects of MPS on TJs, adult human epidermal keratinocyte (HEKa) cells were exposed to MPS, subjected to Western blotting and quantitative PCR analyses for the investigation of TJ-related factors. MPS treatment significantly increased the mRNA and protein expression of claudin-1 (CLDN1) and zonula occludens-1, and significantly increased transepithelial electrical resistance (TEER), which indicates TJ integrity. Conversely, the sulphated and non-sulphated glycosaminoglycans, chondroitin sulphate and hyaluronic acid, respectively, had little effect on TEER or the expression of mRNAs or TJ-related proteins. Interestingly, MPS treatment also inactivated the extracellular signal-regulated kinase signalling pathway, which is known to negatively regulate CLDN1 expression. Furthermore, MPS notably improved the reduction in CLDN1 expression and TEER caused by histamine, which is upregulated in the skin of patients with AD and is known to disrupt the TJ barrier function. Taken together, these findings demonstrate that treatment with the moisturizing agent, MPS, can repair TJ dysfunction and could therefore represent a new therapeutic option for treating patients with AD.

P–045 A retrospective study on Hyaluronic acid binding sperm selection in ICSI cycles

Abstract Study question Does Hyaluronic acid(HA) binding sperm selection prior ICSI produce better outcome compare to PVP? Summary answer No significant difference in fertilization rate, blastocyst utilization rate, pregnancy rate and miscarriage rate in HA vs PVP group. What is known already HA is natural, non-sulphated glycosaminoglycan secretion that abundantly found in the cervical mucus and the cumulus oophorus complex (COC). In in-vivo, HA binding sites on the sperm plasma membrane indicate sperm maturity, mature sperm reaching HA-coated COC can bind and digest HA and subsequent hyperactivation further facilitate fertilization. In IVF, the use of HA tries to mimic in-vivo to select mature sperm with high DNA integrity prior to ICSI. In HA, movement of mature sperm is ‘slowed’ thus allow the selection of sperm to be used in ICSI. Sperm immaturity is known to associated with aneuploidy incidence in the embryos. Study design, size, duration ICSI cycle using HA (N = 83) was adapted from January to December 2020 while ICSI cycle using PVP (N = 133) was adapted from January 2018 to December 2019. Mean age of patient were 35.64±4.33 vs 34.15±4.75 for HA vs PVP group respectively. Fertilization rate, blastocyst utilization rate, pregnancy rate and miscarriage rate were recorded. This study included all ICSI cycle and both frozen and fresh embryo transfer data. Surgical retrieved sperm was excluded from this study. Participants/materials, setting, methods A 1.5ul of treated spermatozoa suspension was connected with a pipette tip to a 5ul droplet of fresh holding medium (Cook Gamete Buffer). Simultaneously, a 5ul droplet of HA medium (Origio SpermSlow) was connected to the 5ulL droplet of holding medium in a ICSI dish (Sparmed Oosafe) covered with oil (Vitrolife Ovoil). Sperm which were ‘slowed’ in the Sperm slow droplets with normal morphology according to WHO 2010 guideline were selected for ICSI at 400X. Main results and the role of chance The fertilization rate of the HA vs PVP- binding sperm are 68.6% vs 66.2%. As for the blastocyst utilisation rate is 61.6% vs 73.22% for HA vs PVP- binding sperm group. Pregnancy and miscarriage rate for HA vs PVP are 42.3% vs 51.5% and 19.4% vs 26.2% respectively. There was no significant difference in the fertilization rates, blastocysts utilisation rate, pregnancy rate and miscarriage rate between the HA vs PVP- binding sperm groups (P < 0.05). However, a trend of higher pregnancy (51.5% vs 42.3%; P = 0.279) and miscarriage rate were observed in the PVP group (26% vs 19%; P = 0.545) as compared to the HA group, but the difference was not statistically significant. The reason behind this might be the HA assist to select the mature sperm with higher DNA integrity and low frequency of chromosomal aneuploidies which contribute to the lower miscarriage rate in the study. Limitations, reasons for caution This is a retrospective study on HA binding sperm selection vs PVP prior to ICSI. Further research which includes a large number of RCT sample size should be warranted. Wider implications of the findings: The HA- sperm binding selection ICSI might only be beneficial to certain group of patients (high- DNA fragmentation sperm). A larger RCT study may be necessary to establish a relationship between HA-sperm binding selection vs aneuploidy rate via PGT analysis. Trial registration number Not applicable

Hyaluronan (HA)-inspired glycopolymers as molecular tools for studying HA functions.

Hyaluronic acid (HA), the only non-sulphated glycosaminoglycan, serves numerous structural and biological functions in the human body, from providing viscoelasticity in tissues to creating hydrated environments for cell migration and proliferation. HA is also involved in the regulation of morphogenesis, inflammation and tumorigenesis through interactions with specific HA-binding proteins. Whilst the physicochemical and biological properties of HA have been widely studied for decades, the exact mechanisms by which HA exerts its multiple functions are not completely understood. Glycopolymers offer a simple and precise synthetic platform for the preparation of glycan analogues, being an alternative to the demanding synthetic chemical glycosylation. A library of homo, statistical and alternating HA glycopolymers were synthesised by reversible addition–fragmentation chain transfer polymerisation and post-modification utilising copper alkyne–azide cycloaddition to graft orthogonal pendant HA monosaccharides (N-acetyl glucosamine: GlcNAc and glucuronic acid: GlcA) onto the polymer. Using surface plasmon resonance, the binding of the glycopolymers to known HA-binding peptides and proteins (CD44, hyaluronidase) was assessed and compared to carbohydrate-binding proteins (lectins). These studies revealed potential structure-binding relationships between HA monosaccharides and HA receptors and novel HA binders, such as Dectin-1 and DEC-205 lectins. The inhibitory effect of HA glycopolymers on hyaluronidase (HAase) activity was also investigated suggesting GlcNAc- and GlcA-based glycopolymers as potential HAase inhibitors.

Changes in human intervertebral disc biochemical composition and bony end plates between middle and old age.

ObjectiveThis study evaluates molecular, nutritional and biochemical alterations in human intervertebral discs between middle and old age.MethodsTwenty-eight human lumbar intervertebral discs from donors were evaluated and separated into two groups: Middle-aged (35–50 years old, relatively non-degenerate discs of Pfirrmann grades 1–3, n = 15) and Old-aged (≥80 years old, all degenerate Pfirrmann grade 4 or 5, n = 13). Parameters which might be expected to to be related to nutrient supply and so the health of disc cells (eg the porosity of the vertebral endplate, cell viability and cell density) and to disc extracellular composition (ie quantification of glycosaminoglycan disaccharides and hyaluronic acid molecular weight) and collagen organization, were analyzed. Three regions of the intervertebral disc (anterior annulus fibrosus, nucleus pulposus, and posterior annulus fibrosus) were examined.ResultsThe old-aged group showed a decrease in content of sulphated and non-sulphated glycosaminoglycans relative to middle-aged and there were also alterations in the proportion of GAG disaccharides and a decrease of collagen fiber size. Hyaluronic acid molecular weight was around 200 kDa in all regions and ages studied. The anterior annulus differed from the posterior annulus particularly in relation to cell density and GAG content. Additionally, there were changes in the bony endplate, with fewer openings observed in the caudal than cranial endplates of all discs in both groups.ConclusionsResults show the cranial vertebral endplate is the main vascular source for the intervertebral discs. Hylauronic acid molecular weight is the same through the intervertebral disc after age of 50 years.

Hyaluronan in the experimental injury of the cartilage: biochemical action and protective effects.

Our knowledge of extracellular matrix (ECM) structure and function has increased enormously over the last decade or so. There is evidence demonstrating that ECM provides signals affecting cell adhesion, shape, migration, proliferation, survival, and differentiation. ECM presents many domains that become active after proteolytic cleavage. These active ECM fragments are called matrikines which play different roles; in particular, they may act as potent inflammatory mediators during cartilage injury. A major component of the ECM that undergoes dynamic regulation during cartilage damage and inflammation is the non-sulphated glycosaminoglycan (GAG) hyaluronan (HA). In this contest, HA is the most studied because of its different activity due to the different polymerization state. In vivo evidences have shown that low molecular weight HA exerts pro-inflammatory action, while high molecular weight HA possesses anti-inflammatory properties. Therefore, the beneficial HA effects on arthritis are not only limited to its viscosity and lubricant action on the joints, but it is especially due to a specific and effective anti-inflammatory activity. Several in vitro experimental investigations demonstrated that HA treatment may regulate different biochemical pathways involved during the cartilage damage. Emerging reports are suggesting that the ability to recognize receptors both for the HA degraded fragments, whether for the high-polymerized native HA involve interaction with integrins, toll-like receptors (TLRs), and the cluster determinant (CD44). The activation of these receptors induced by small HA fragments, via the nuclear factor kappa-light-chain enhancer of activated B cell (NF-kB) mediation, directly or other different pathways, produces the transcription of a large number of damaging intermediates that lead to cartilage erosion. This review briefly summarizes a number of findings of the recent studies focused on the protective effects of HA, at the different polymerization states, on experimental arthritis in vitro both in animal and human cultured chondrocytes.

Regulation and roles of the hyaluronan system in mammalian reproduction.

Hyaluronan (HA) is a non-sulphated glycosaminoglycan polymer naturally occurring in many tissues and fluids of mammals, including the reproductive system. Its biosynthesis by HA synthase (HAS1-3) and catabolism by hyaluronidases (HYALs) are affected by ovarian steroid hormones. Depending upon its molecular size, HA functions both as a structural component of tissues in the form of high-molecular-weight HA or as a signalling molecule in the form of small HA molecules or HA fragments with effects mediated through interaction with its specific cell-membrane receptors. HA is produced by oocytes and embryos and in various segments of the reproductive system. This review provides information about the expression and function of members of the HA system, including HAS, HYALs and HA receptors. We examine their role in various processes from folliculogenesis through oocyte maturation, fertilisation and early embryo development, to pregnancy and cervical dilation, as well as its application in assisted reproduction technologies. Particular emphasis has been placed upon the role of the HA system in pre-implantation embryo development and embryo implantation, for which we propose a hypothetical sequential model.

Hyaluronic acid auto-crosslinked polymer (ACP): Reaction monitoring, process investigation and hyaluronidase stability

Hyaluronic Acid (HA) is a non-sulphated glycosaminoglycan that, despite its high molecular weight, is soluble in water and is not resistant to enzymatic degradation, the latter of which hinders its wider application as a biomedical material. Auto-crosslinked polymer (ACP) gels of HA are fully biocompatible hydrogels that exhibit improved viscoelastic properties and prolonged in vivo residence times compared to the native polymer. Crosslinking is achieved through a base-catalysed reaction consisting of the activation of HA carboxyl groups by 2-chloro-1-methylpyridinium iodide (CMPI) and subsequent nucleophilic acyl substitution by the hydroxyl groups of HA in organic solvent.In this study, a number of ACP hydrogels have been obtained via reactions using varying ratios of CMPI to HA. The crosslinking reaction was monitored by rheological measurements in organic solvents during CMPI addition to the reaction mixture. The ACP intermediates, powders and hydrogels were characterized, helping to elucidate the crosslinking process. A two-step mechanism was proposed to explain the observed trends in viscosity and particle size. Syntheses were carried out by varying the reaction temperature, respectively at 0 °C, 25 °C and 45 °C in N-Methyl-2-Pyrrolidone (NMP), as well as the solvent respectively in NMP, DMSO and DMF at 25 °C. Interestingly, varying these parameters did not substantially affect the degree of crosslinking but likely did influence the intra/inter-molecular crosslinking ratio and, therefore, the viscoelastic properties. A wide range of crosslinking densities was confirmed through ESEM analysis.Finally, a comparative hyaluronidase degradation assay revealed that the ACPs exhibited a higher resistance toward enzymatic cleavage at low elastic modulus compared to other more chemically resistant, crosslinked HAs. These observations demonstrated the importance of crosslinking density of matrix structures on substrate availability.

AB0996 Increased release time of diclofenac loaded into a cross-linked hyaluronic acid hydrogel

BackgroundHyaluronic acid (HA) is a natural linear and unbranched polysaccharide belonging to the class of non-sulphated glycosaminoglycans and it is composed of beta-1,3-N-acetyl glucosamine and beta-1,4-glucuronic acid repeating disaccharide units....

Time-dependent mechanical properties of aortic valve cusps: Effect of glycosaminoglycan depletion

Aortic valve (AV) performance is closely linked to its structural components. Glycosaminoglycans (GAGs) are thought to influence the time-dependent properties of living tissues. This study investigates the effect of GAGs on the viscoelastic behaviour of the AV. Fresh porcine AV cusps were either treated enzymatically to remove GAGs or left untreated (control). The specimens were tested for stress relaxation and tensile properties under equibiaxial load conditions. The stress relaxation curves were fitted using a double exponential decay equation and the early relaxation constant (τ1) and late relaxation constant (τ2) calculated for each specimen. Immunohistochemistry confirmed the successful depletion of both sulphated and non-sulphated GAGs from the AV cusps. A statistical increase in τ1 was found for both the radial and circumferential directions between the control and –GAGs group (radial, control 17.37s vs. –GAGs 25.65s; circumferential, control 21.47s vs. –GAGs 27.37s). It was also found that τ1 differed between the two directions for the control group but not after GAG depletion (control, radial 17.37s vs. circumferential 21.47s; –GAGs, radial 25.65s vs. circumferential 27.37s). No effect on stiffness was found. The results showed that the presence of GAGs influences the mechanical viscoelastic properties of the AV but has no effect on the stiffness. The natural anisotropy, which reflects the relaxation kinematics, is lost after GAG depletion.

Age- and gender-related alteration in plasma advanced oxidation protein products (AOPP) and glycosaminoglycan (GAG) concentrations in physiological ageing

The authors studied the role of increased oxidative stress in the development of oxidative protein damage and extracellular matrix (ECM) components in ageing. The age- and gender-associated disturbances in connective tissue metabolism were evaluated by the plasma chondroitin sulphated glycosaminoglycans (CS-GAG) and non-sulphated GAG-hyaluronan (HA) measurements. Plasma concentration of advanced oxidation protein products (AOPP) was analysed in order to assess oxidative protein damage and evaluate the possible deleterious role of oxidative phenomenon on tissue proteoglycans' metabolism during the physiological ageing process. Sulphated and non-sulphated GAGs as well as AOPP were quantified in plasma samples from 177 healthy volunteers. A linear age-related decline of plasma CS-GAG level was found in this study (r=-0.46; p<0.05). In contrast, HA concentrations rise gradually with age (r=0.44; p<0.05) in plasma samples. For both ECM components, the observed differences were not gender-specific. A strong age-dependent relationship has been shown in regard to AOPP. AOPP levels significantly increased with age (r=0.63; p<0.05), equally strongly in both men (r=0.69; p<0.05) and women (r=0.57; p<0.05) during physiological ageing. A significant correlation was found between the concentrations of AOPP and both CS-GAG (r=-0.31; p<0.05) and HA (r=0.33; p<0.05). Proceeding with age changes in the ECM are reflected by CS-GAG and HA plasma levels. Strong correlations between AOPP and ECM components indicate that oxidative stress targets protein and non-protein components of the connective tissue matrix during human ageing.

In vitro evaluation of antiviral and virucidal activity of a high molecular weight hyaluronic acid

Backgroundhyaluronic acid (HA), a non-sulphated glycosaminoglycan, is present in synovial fluid, vitreous humour serum and many connective tissues. Pharmaceutical preparations of HA are used in clinical practice for wound healing, joint pain, kerato-conjunctivitis, asthma, mouth care, oesophageal-reflux, and gastritis. Moreover, it is used as a filler to counteract ageing and facial lipoatrophy. Our study aims at investigating the in vitro antiviral activity of a high molecular weight HA.Methodsthe MTT test was used to rule out the potential toxic effects of HA on the different cell lines used in the antiviral assays. The antiviral activity of HA against Coxsackievirus B5, Herpes Simplex Virus-1, Mumps Virus, Adenovirus-5, Influenza Virus A/H1N1, Human Herpesvirus-6, Porcine Parvovirus, Porcine Reproductive and Respiratory Syndrome Virus was assessed by virus yield assays.Resultsthe most effective inhibition was observed against Coxsackievirus B5, with 3Log reduction of the virus yield at 4 mg/ml, and a reduction of 3.5Log and 2Log, at 2 mg/ml and 1 mg/ml, respectively: the selectivity index was 16. Mumps virus was highly inhibited too showing a reduction of 1.7Log at 1 mg/ml and 1Log at 4 mg/ml and 2 mg/ml (selectivity index = 12). The selectivity index for Influenza Virus was 12 with the highest inhibition (1Log) observed at 4 mg/ml. Herpes Simplex Virus-1 and Porcine Parvovirus were mildly inhibited, whereas no antiviral activity was observed with respect to Adenovirus-5, Human Herpesvirus-6, Porcine Reproductive and Respiratory Syndrome Virus. No HA virucidal activity was ever observed against any of the viruses tested. Kinetic experiments showed that both Coxsackievirus B5 and Herpes simplex virus-1 replication were consistently inhibited, not influenced by the time of HA addition, during the virus replication cycle.Conclusionsthe spectrum of the antiviral activity exhibited by HA against both RNA and DNA viruses, known to have different structures (with or without envelope) and replication strategies, suggests a non specific mechanism of action, probably involving cell membrane-virus interaction steps. The results of the kinetic experiments support this hypothesis.

Hyaluronan and Human Endothelial Cell Behavior

Hyaluronan (HA) is the only nonsulphated glycosaminoglycan of extracellular matrix. In mammals HA is synthesised by three homologues HA synthases: HAS1, HAS2, and HAS3. The HA is daily catabolized by the hyaluronidase enzymes to either oligosaccharides or larger polymer. Despite its simple structure, HA is involved in a great number of biological functions, such as cell proliferation and migration, morphogenesis, wound healing, inflammation, angiogenesis, and tumor growth. Moreover, an important biological role is related to HA oligosaccharides that stimulate cytokine secretion and endothelial cell proliferation. Nevertheless no data about HA presence in endothelium are reported in literature. Several studies underline HA involvement in endothelial cell proliferation, migration, new vessels formation, and leucocytes recruitment. We review the role of HA in endothelial cell in normal condition and during vascular injury.

Regulation of hyaluronan and versican deposition by growth factors in fibrosarcoma cell lines

Versican, a large chondroitin sulphate proteoglycan and hyaluronan (HA), a non-sulphated glycosaminoglycan are major constituents of the pericellular matrix. In many neoplastic tissues, changes in the expression of versican and HA affect tumour progression. Here, we analyse the synthesis of versican and hyaluronan by fibrosarcoma cells, and document how the latter is affected by PDGF-BB, bFGF and TGFB2, growth factors endogenously produced by these cells. Fibrosarcoma cell lines B6FS and HT1080 were utilised and compared with normal lung fibroblasts (DLF). The major versican isoforms expressed by DLF and B6FS cells were V0 and V1. Treatment of B6FS cells with TGFB2 showed a significant increase of V0 and V1 mRNAs. Versican expression in HT1080 cells was not significantly affected by any of the growth factors. In addition, TGFB2 treatment increased versican protein in DLF cells. HA, showed approximately a 2-fold and a 9-fold higher production in DLF cells compared to B6FS and HT1080 cells, respectively. In HT1080 cells, HA biosynthesis was significantly increased by bFGF, whereas, in B6FS cells it was increased by TGFB2 and PDGF-BB. Furthermore, analysis of HA synthases (HAS) expression indicated that HT1080 expressed similar levels of all three HAS isoforms in the following order: HAS2> HAS3> HAS1. bFGF shifted that balance by increasing the abundance of HAS1. The major HAS isoform expressed by B6FS cells was HAS2. PDGF-BB and TGFB2 showed the most prominent effects by increasing both HAS2 and HAS1 isoforms. In conclusion, these growth factors modulated, through upregulation of specific HAS isoforms, HA synthesis, secretion and net deposition to the pericellular matrix.

Bench-to-bedside review: The role of glycosaminoglycans in respiratory disease

The extracellular matrix (ECM) plays a significant role in the mechanical behaviour of the lung parenchyma. The ECM is composed of a three-dimensional fibre mesh that is filled with various macromolecules, among which are the glycosaminoglycans (GAGs). GAGs are long, linear and highly charged heterogeneous polysaccharides that are composed of a variable number of repeating disaccharide units. There are two main types of GAGs: nonsulphated GAG (hyaluronic acid) and sulphated GAGs (heparan sulphate and heparin, chondroitin sulphate, dermatan sulphate, and keratan sulphate). With the exception of hyaluronic acid, GAGs are usually covalently attached to a protein core, forming an overall structure that is referred to as proteoglycan. In the lungs, GAGs are distributed in the interstitium, in the sub-epithelial tissue and bronchial walls, and in airway secretions. GAGs have important functions in lung ECM: they regulate hydration and water homeostasis; they maintain structure and function; they modulate the inflammatory response; and they influence tissue repair and remodelling. Given the great diversity of GAG structures and the evidence that GAGs may have a protective effect against injury in various respiratory diseases, an understanding of changes in GAG expression that occur in disease may lead to opportunities to develop innovative and selective therapies in the future.

Glycosaminoglycans of Normal Veins and Their Alterations in Varicose Veins and Varicose Veins Complicated by Thrombophlebitis

The aim of the study was to examine the content and molecular differentiation of glycosaminoglycans (GAGs) in the wall of varicose veins. The studied material consisted of normal, varicose veins and varicose veins complicated by thrombophlebitis collected during operations on 26 patients. In the wall of varicose veins the mean GAGs’ content as well as the content of sulphated GAGs, except heparan sulphate was increased, whereas the amount of hyaluronic acid was decreased. Furthermore, the increased quantitative ratio between sulphated and nonsulphated GAGs was demonstrated. The results indicate an evident extracellular matrix remodelling in the wall of varicose veins particularly those complicated by thrombophlebitis, that is characterised by alterations in the content and molecular differentiation of GAGs.

Production of Glycosaminoglycans by the Porcine Oviduct in Relation to Sperm Storage

ContentsFollicular and oviductal fluids have shown modulatory effects on sperm capacitation of boar spermatozoa in vitro, implicating glycosaminoglycans (GAGs) in the process. Capacitation in pigs is believed to occur in vivo during storage in the sperm reservoir, where spermatozoa maintain viability and membrane stability awaiting ovulation. Exposure to the nonsulphated GAG hyaluronan induces capacitation in vitro (chlortetracycline‐monitored) without eliciting acrosome exocytosis, indicating that it exerts a protective role on sperm viability. Oviductal fluids (isthmus and ampulla) of cycling sows were collected in vivo to disclose temporal changes in concentrations of both sulphated and nonsulphated GAGs between late pro‐oestrus and meta‐oestrus. Further, heparan‐sulphate and hyaluronan were immunohistochemically localized in the oviductal tissues of uninseminated and inseminated sows, around the period of ovulation. Concentrations of sulphated GAGs were two‐fold higher than hyaluronan. All tubal GAGs increased significantly from late pro‐oestrus and were highest during the pre‐ovulatory period before beginning to decrease postovulation, the exception being the ampullar hyaluronan, whose levels remained high towards meta‐oestrus. Heparan sulphate (syndecans) and hyaluronan were both immunohistochemically detected in the endosalpinx. Syndecan‐immunolabelling was present in the epithelium and stroma along the tube, whereas hyaluronan was always localized in the lamina propria and most of the lining epithelium was negative, during the stages studied. The only exception was the sperm reservoir (deep crypts and furrows of the Utero‐tubal Junction UTJ/isthmus) where sperm clusters were seen close to the epithelial HA‐immunolabelling in the inseminated sows, but only pre‐ovulatory. The presence of GAGs in the tubal fluid and the conspicuous location of hyaluronan suggest they play a role in the formation and function of the sperm reservoir.

Anatomy and development of the sinoatrial valves in the dogfish Scyliorhinus canicula

ABSTRACT Background: We describe the adult anatomy and the development of the cardiac sinoatrial valves in the dogfish (Scyliorhinus canicula). Methods We use scanning electron microscopy, histological and histochemical techniques in 39 hearts from embryos and adult specimens. Results The sinoatrial valvular set of the adult dogfish is composed of two transverse valves laterally attached to the sinoatrial junction at their bases. Both valves are composed of two muscular layers, the sinusal and the atrial, whose histological features are similar to the cardiac wall which they face. Collagen bundles, elastic fibers and fibroblasts are present between the muscular layers. The extracellular matrix between the valvular layers also contains sulphated and non-sulphated glycosaminoglycans. The sinoatrial valves develop from two lateral infoldings of the cardiac wall. The left fold is deeper than the right, causing a shift of the sinoatrial communication to the right. The epicardium progressively covers the outer sinoatrial groove and the space between the folds becomes populated by mesenchymal cells. The posterior atrioventricular endocardial cushion is in contact with the base of the left fold until the embryo has about 40 mm TL. Conclusions The sinoatrial valves, in the dogfish, develop from lateral infoldings of the cardiac wall. This origin results in histological and histochemical differences between the two muscular layers which constitute the valves of the adult. The comparison of the sinoatrial valve morphogenesis between the dogfish and some higher vertebrates suggests that the right sinoatrial valve, but not the left, is homologous throughout the vertebrate phylogeny. Anat. Rec. 248:224-232, 1997. © 1997 Wiley-Liss, Inc.

Anatomy and development of the sinoatrial valves in the dogfish (Scyliorhinus canicula).

We describe the adult anatomy and the development of the cardiac sinoatrial valves in the dogfish (Scyliorhinus canicula). We use scanning electron microscopy, histological and histochemical techniques in 39 hearts from embryos and adult specimens. The sinoatrial valvular set of the adult dogfish is composed of two transverse valves laterally attached to the sinoatrial junction at their bases. Both valves are composed of two muscular layers, the sinusal and the atrial, whose histological features are similar to the cardiac wall which they face. Collagen bundles, elastic fibers and fibroblasts are present between the muscular layers. The extracellular matrix between the valvular layers also contains sulphated and non-sulphated glycosaminoglycans. The sinoatrial valves develop from two lateral infoldings of the cardiac wall. The left fold is deeper than the right, causing a shift of the sinoatrial communication to the right. The epicardium progressively covers the outer sinoatrial groove and the space between the folds becomes populated by mesenchymal cells. The posterior atrioventricular endocardial cushion is in contact with the base of the left fold until the embryo has about 40 mm TL. The sinoatrial valves, in the dogfish, develop from lateral infoldings of the cardiac wall. This origin results in histological and histochemical differences between the two muscular layers which constitute the valves of the adult. The comparison of the sinoatrial valve morphogenesis between the dogfish and some higher vertebrates suggests that the right sinoatrial valve, but not the left, is homologous throughout the vertebrate phylogeny.

Production of hyaluronan by glomerular mesangial cells in response to fibronectin and platelet-derived growth factor

The formation of the non-sulphated glycosaminoglycan hyaluronan by cells of the renal glomerulus in diabetes may contribute to altered matrix composition. We describe an increased production of hyaluronan from mesangial cell-enriched glomerular cores from diabetic animals, and further show that increased hyaluronan production follows the exposure of non-diabetic and diabetic preparations to fibronectin and to platelet-derived growth factor in vitro. Hyaluronan production appeared dependent on protein kinase C activity, and could not be shown after prolonged phorbol ester preincubation. Stimulation by fibronectin was wholly dependent on cyclooxygenase activity and prior prostaglandin production, while the effect of platelet-derived growth factor showed only a partial dependence.

Retinoic acid induces changes in the rhombencephalic neural crest cells migration and extracellular matrix composition in chick embryos.

Chick embryos at 9-10 stages (Hamburger and Hamilton: J Morphol 88:49-82, 1951) have been treated with all-trans retinoid acid (RA) (0.5 microgram, 1.5 micrograms, and 2.5 micrograms) to determine the pattern and mechanism of RA-induced effects on early cephalic development. We found that while 0.5 microgram RA did not produce any significant dysmorphogenesis, 2.5 micrograms RA elicited wide malformation of both cephalic and trunk regions. However, 1.5 micrograms RA produced selective and specific changes at the cephalic level, which consisted of morphological alterations, changes in neural crest cells (NCC) migration and extracellular matrix (ECM) composition. Morphological alterations included hypoplasia of the first three branchial arches, swelling of either anterior cardinal veins or dorsal aortae, and atrophy of branchial arch arteries. Concurrently NCC did not migrate away, remaining clustered on the dorsal surface of the rhombencephalon, and in some cases they shifted into the neural tube cavity. Accordingly, the second branchial arch showed a reduction of the mesenchymal cellular population. The extracellular matrix in RA-injected embryos showed changes in glycosaminoglycans (GAGs) concentration as compared with controls, that is, an increase in the non-sulphated GAGs, stained with alcian blue 8GX at 2.5 pH, and a decrease in the sulphated GAGs stained with alcian blue 8GX at 1 pH. These quantitative changes reflected alterations in the pattern of distribution and composition of the GAGs within the cephalic ECM, which specifically consisted in an increase of the hyaluronic acid and a decrease of the chondroitin sulphate. Our findings indicate that RA is involved in abnormal cephalic development, suggesting that RA may effect neural crest cell migration via changes in the GAGs of the ECM.