Glycosaminoglycans compositional analysis of Urodele axolotl (Ambystoma mexicanum) and Porcine Retina.

Retinal degenerative diseases, such as age-related macular degeneration (AMD) and retinitis pigmentosa (RP), are major causes of blindness worldwide. Humans cannot regenerate retina, however, axolotl (Ambystoma mexicanum), a laboratory-bred salamander, can regenerate retinal tissue throughout adulthood. Classic signaling pathways, including fibroblast growth factor (FGF), are involved in axolotl regeneration. Glycosaminoglycan (GAG) interaction with FGF is required for signal transduction in this pathway. GAGs are anionic polysaccharides in extracellular matrix (ECM) that have been implicated in limb and lens regeneration of amphibians, however, GAGs have not been investigated in the context of retinal regeneration. GAG composition is characterized native and decellularized axolotl and porcine retina using liquid chromatography mass spectrometry. Pig was used as a mammalian vertebrate model without the ability to regenerate retina. Chondroitin sulfate (CS) was the main retinal GAG, followed by heparan sulfate (HS), hyaluronic acid, and keratan sulfate in both native and decellularized axolotl and porcine retina. Axolotl retina exhibited a distinctive GAG composition pattern in comparison with porcine retina, including a higher content of hyaluronic acid. In CS, higher levels of 4- and 6- O-sulfation were observed in axolotl retina. The HS composition was greater in decellularized tissues in both axolotl and porcine retina by 7.1% and 15.4%, respectively, and different sulfation patterns were detected in axolotl. Our findings suggest a distinctive GAG composition profile of the axolotl retina set foundation for role of GAGs in homeostatic and regenerative conditions of the axolotl retina and may further our understanding of retinal regenerative models.

Pharmaceutical grade chondroitin sulfate: Structural analysis and identification of contaminants in different commercial preparations.

The aim of the present study was to characterize 16 pharmaceutical grade chondroitin sulfate (CS) samples, concerning the structure and presence of contaminants, in comparison to USP and analytical grade CS. Agarose gel electrophoresis has shown that only 5 samples were >90% CS, while 11 contained less than 15% CS. FACE (fluorophore-assisted carbohydrate electrophoresis) revealed that maltodextrin was the main contaminant in nine of them, and lactose in two. Raman spectroscopy corroborated these results. Concerning the structure of the CS present in the five CS-rich samples, the ratios 4-sulfated:6-sulfated disaccharides varied from 0.9 to 1.7, and their modal molecular weight was 20-29 kDa. Also, they were all contaminated by small amounts of keratan sulfate (<1%). In conclusion, our findings indicate that the composition of CS preparations not always corresponds to the manufacturers' descriptions, and indicate that further characterization should be required for the registry and license of pharmaceutical grade CS.

Residual keratan sulfate in chondroitin sulfate formulations for oral administration.

Chondroitin sulfate is a biomedical glycosaminoglycan (GAG) mostly used as a dietary supplement. We undertook analysis on some formulations of chondroitin sulfates available for oral administration. The analysis was based on agarose-gel electrophoresis, strong anion-exchange chromatography, digestibility with specific GAG lyases, uronic acid content, NMR spectroscopy, and size-exclusion chromatography. Keratan sulfate was detected in batches from shark cartilage, averaging ∼16% of the total GAG. Keratan sulfate is an inert material, and hazardous effects due to its presence in these formulations are unlikely to occur. However, its unexpected high percentage compromises the desired amounts of the real ingredient specified on the label claims, and forewarns the pharmacopeias to update their monographs. The techniques they recommended, especially cellulose acetate electrophoresis, are inefficient in detecting keratan sulfate in chondroitin sulfate formulations. In addition, this finding also alerts the manufacturers for improved isolation procedures as well as the supervisory agencies for better audits. Analysis based on strong anion-exchange chromatography is shown to be more reliable than the methods presently suggested by standard pharmacopeias.

Airway proteoglycans are differentially altered in fatal asthma.

It has been suggested that airway remodelling is responsible for the persistent airway obstruction and decline in lung function observed in some asthmatic patients. The small airways are thought to contribute significantly to this functional impairment. Proteoglycans (PGs) are important components of the extracellular matrix (ECM) in the lungs. Besides controlling biophysical properties of the ECM, they play important roles in the regulation of some cytokines. Increased subepithelial PG deposition in the airways of mild asthmatics has been reported. However, there are no data on the PG content in small airways in asthma. This study has compared the content and distribution of PGs in large and small airways of patients who died of asthma with those in control lungs. Immunohistochemistry and image analysis were used to determine the content of lumican, decorin, biglycan, and versican in large (internal perimeter >6 mm) and small (internal perimeter < or =6 mm) airways of 18 patients who had died of asthma (A) and ten controls (C). The results were expressed as PG area (microm2)/epithelial basement membrane length (microm). The main differences between asthmatics and controls were observed in the small airways. There was a significant decrease in decorin and lumican contents in the external area of small airways in asthmatics (decorin: A = 1.05 +/- 0.27 microm, C = 3.97 +/- 1.17 microm, p = 0.042; lumican: A = 1.97 +/- 0.37 microm, C = 5.66 +/- 0.99 microm, p = 0.002). A significant increase in versican content in the internal area of small and large airways in asthmatics was also observed (small: A = 7.48 +/- 0.84 microm, C = 5.16 +/- 0.61 microm, p = 0.045; large: A = 18.38 +/- 1.94 microm, C = 11.90 +/- 2.86 microm, p = 0.028). The results show that PGs are differentially expressed in the airways of fatal asthma and may contribute to airway remodelling. These data reinforce the importance of the small airways in airway remodelling in asthma.

Small leucine-rich proteoglycans (SLRPs) in uterine tissues during pregnancy in mice.

Remodelling of the extracellular matrix (ECM) occurs during decidualization of the endometrium in mice. Previously we have documented the appearance of large-diameter collagen fibrils around mature decidual cells between day 5 and day 7 of pregnancy. Proteoglycans are important in the regulation of collagen fibrillogenesis, and the present study analysed four members (decorin, biglycan, lumican and fibromodulin) of the family of small leucine-rich proteoglycans (SLRPs) in the uterus from day 1 to day 7 of pregnancy. Decorin was present together with lesser amounts of lumican in the stroma before the onset of decidualization, whereas biglycan and fibromodulin were almost absent. Biglycan and, less significantly, lumican were expressed in decidualized regions of the endometrium, but decorin was absent. Fibromodulin was weakly expressed in the non-decidualized stroma, but only after implantation. Decorin and lumican were strongly expressed in the undifferentiated interimplantation site stroma, whereas biglycan and fibromodulin were expressed only weakly. These results indicate that the SLRP profile of the uterine ECM alters with differentiation of endometrial stromal cells. The large decidual collagen fibrils are thought to arise by lateral association of smaller diameter fibrils. As decorin has been shown to inhibit lateral association of collagen fibrils, its disappearance between day 2 and day 5 of pregnancy may be a prerequisite for the formation of large fibrils in decidua in mice.

Eggshells are shaped by a precise spatio-temporal arrangement of sequentially deposited macromolecules.

The avian eggshell is a composite bioceramic which is formed by a controlled interaction of an organic and an inorganic phase. The organic phase contains, among other constituents, type X collagen and proteoglycans, mainly keratan and dermatan sulfate. Understanding the principles governing the synthesis and temporo-spatial distribution of such macromolecules, and their influence on the organization of the crystalline phase, is an essential aspect of establishing the biological basis of the quality of eggshell, both as an embryonic chamber and as a natural food package. In the present study, we have examined the process of eggshell formation by immunohistochemistry, scanning electron microscopy and energy dispersive X-ray microanalysis. Precise sites and timing of secretion were established for the deposition of particular macromolecules. Type X collagen is detected at the very first moment of shell membrane formation. The appearance of keratan sulfate coincides with the appearance of mammillae, while dermatan sulfate is deposited later, coincident with shell matrix deposition. We propose that keratan sulfate, due to its precise localization, temporal appearance and calcium-binding affinity, relates to the maintenance of calcium reserve bodies, the primary source of calcium for the embryo. On the other hand, dermatan sulfate may control crystal growth, resulting in a preferential orientation of calcite crystals within the palisade layer.