Proteoglycan Research Articles

Indole-3-Acetic Acid Alters Intestinal Microbiota and Alleviates Ankylosing Spondylitis in Mice.

Ankylosing spondylitis (AS) is a systemic, chronic, and inflammatory autoimmune disease associated with the disorder of intestinal microbiota. Unfortunately, effective therapies for AS are lacking. Recent evidence has indicated that indole-3-acetic acid (IAA), an important microbial tryptophan metabolite, can modulate intestinal homeostasis and suppress inflammatory responses. However, reports have not examined the in vivo protective effects of IAA against AS. In this study, we investigated the protective effects and underlying mechanisms through which IAA acts against AS. We constructed a proteoglycan (PG)-induced AS mouse model and administered IAA (50 mg/kg body weight) by intraperitoneal injection daily for 4 weeks. The effects of IAA on AS mice were evaluated by examining disease severity, intestinal barrier function, aryl hydrocarbon receptor (AhR) pathway, T-helper 17 (Th17)/T regulatory (Treg) balance, and inflammatory cytokine levels. The intestinal microbiota compositions were profiled through whole-genome sequencing. We observed that IAA decreased the incidence and severity of AS in mice, inhibited the production of pro-inflammatory cytokines (tumor necrosis factor α [TNF-α], interleukin [IL]-6, IL-17A, and IL-23), promoted the production of the anti-inflammatory cytokine IL-10, and reduced the ratios of pro-/anti- inflammatory cytokines. IAA ameliorated pathological changes in the ileum and improved intestinal mucosal barrier function. IAA also activated the AhR pathway, upregulated the transcription factor forehead box protein P3 (FoxP3) and increased Treg cells, and downregulated the transcription factors retinoic acid receptor–related orphan receptor gamma t (RORγt) and signal transducer and activator of transcription 3 (STAT3) and decreased Th17 cells. Furthermore, IAA altered the composition of the intestinal microbiota composition by increasing Bacteroides and decreasing Proteobacteria and Firmicutes, in addition to increasing the abundances of Bifidobacterium pseudolongum and Mucispirillum schaedleri. In conclusion, IAA exerted several protective effects against PG-induced AS in mice, which was mediated by the restoration of balance among the intestinal microbial community, activating the AhR pathway, and inhibiting inflammation. IAA might represent a novel therapeutic approach for AS.

An ultrastructural 3D reconstruction method for observing the arrangement of collagen fibrils and proteoglycans in the human aortic wall under mechanical load

An insight into changes of soft biological tissue ultrastructures under loading conditions is essential to understand their response to mechanical stimuli. Therefore, this study offers an approach to investigate the arrangement of collagen fibrils and proteoglycans (PGs), which are located within the mechanically loaded aortic wall. The human aortic samples were either fixed directly with glutaraldehyde in the load-free state or subjected to a planar biaxial extension test prior to fixation. The aortic ultrastructure was recorded using electron tomography. Collagen fibrils and PGs were segmented using convolutional neural networks, particularly the ESPNet model. The 3D ultrastructural reconstructions revealed a complex organization of collagen fibrils and PGs. In particular, we observed that not all PGs are attached to the collagen fibrils, but some fill the spaces between the fibrils with a clear distance to the collagen. The complex organization cannot be fully captured or can be severely misinterpreted in 2D. The approach developed opens up practical possibilities, including the quantification of the spatial relationship between collagen fibrils and PGs as a function of the mechanical load. Such quantification can also be used to compare tissues under different conditions, e.g., healthy and diseased, to improve or develop new material models. Statement of significanceThe developed approach enables the 3D reconstruction of collagen fibrils and proteoglycans as they are embedded in the loaded human aortic wall. This methodological pipeline comprises the knowledge of arterial mechanics, imaging with transmission electron microscopy and electron tomography, segmentation of 3D image data sets with convolutional neural networks and finally offers a unique insight into the ultrastructural changes in the aortic tissue caused by mechanical stimuli.

Experimental and numerical analysis of electroactive characteristics of scleral tissue

The sclera provides mechanical support to retina and protects internal contents of the eye against external injuries. The scleral extracellular matrix is mainly composed of collagen fibers and proteoglycans (PGs). At physiological pH, collagen molecules are neutral but PGs contain negatively charged glycosaminoglycan chains. Thus, the sclera can be considered as a polyelectrolyte hydrogel and is expected to exhibit mechanical response when subjected to electrical stimulations. In this study, we mounted scleral strips, dissected from the posterior part of porcine eyes, at the center of a custom-designed container between two electrodes. The container was filled with NaCl solution and the bending deformation of scleral strips as a function of the applied electric voltage was measured experimentally. It was found that scleral strips reached to an average bending angle of 3°, 10° and 23° when subjected to 5V, 10V, and 15V, respectively. We also created a chemo-electro-mechanical finite element model for simulating the experimental measurements by solving coupled Poisson-Nernst-Plank and equilibrium mechanical field equations. The scleral fixed charge density and modulus of elasticity were found by fitting the experimental data. The ion concentration distribution inside the domain was found numerically and was used to explain the underlying mechanisms for the scleral electroactive response. The numerical simulations were also used to investigate the effects of various parameters such as the electric voltage and fixed charge density on the scleral deformation under an electric field. Statement of significanceThis manuscript investigates the electroactive response of scleral tissue. It demonstrates that the sclera deforms mechanically when subjected to electrical stimulations. A chemo-electro-mechanical model is also presented in order to numerically capture the electromechanical response of the sclera. This numerical model is used to explain the experimental observations by finding the ion distribution inside the tissue under an electric field. This work is significant because it shows that the sclera is an electroactive polyanionic hydrogel and it provides new information about the underlying mechanisms governing its mechanical and electrical properties.

Synthetic Modifications of the Linkage Region of Proteoglycans and Impact on CSGalNAcT‐1

AbstractWe herein report for the first time a multi‐step synthesis of a library of disaccharides and trisaccharides of the linkage region of proteoglycans (PGs) having a d‐galactose modified at position C‐2, C‐4 or C‐6 by replacing the hydroxyl group by methoxy or deoxy moieties. Efficient radical dehalogenation, Barton‐McCombie reaction, O‐methylation and stereoselective glycosylation are described. We also report preliminary results of the effect of these modifications upon the activity of recombinant chondroitin sulfate N‐acetylgalactosaminyltransferase‐1 (CSGalNAcT‐1), an enzyme involved at the bifurcation step of the biosynthesis of PGs.

The anti-inflammation effect of strontium ranelate on rat chondrocytes with or without IL-1β in vitro.

Temporomandibular joint osteoarthritis (TMJ-OA) is a common disease with a high level of inflammation in the joint micro-environment and cartilage degradation. Anti-inflammation and cartilage regeneration are the key therapies for TMJ-OA, but currently, there are no novel medicines or treatments that can control its pathogenic progression. Strontium ranelate (SrR) is an anti-osteoporosis drug and is now considered a promising anti-OA drug, but the anti-inflammatory effect of SrR remains to be elucidated. In the present study, the anti-inflammatory effect of SrR in a normal or high IL-1β environment was observed. Cell viability under the treatment of SrR was tested using Cell Counting Kit-8. Toluidine blue staining, immunofluorescence staining, hydroxyproline assay, PCR assay and western blotting were used to detect the expression of collagen (Col)II, proteoglycans (PG) and aggrecan as a reflection of extracellular matrix synthesis and MMP-9,13 hydroxyproline was used as an inflammation indicator. IL-1β of 10 ng/ml was added to the culture medium as inflammation environment and the tests of those biomarkers were done again. Then, the changes in β-catenin were also studied by immunofluorescence staining, PCR assay and western blotting to explore the possible involvement of the Wnt/β-catenin pathway. The results showed a significant inhibition of MMP-9, MMP-13, β-catenin and promotion of Col-II, PG and aggrecan in normal chondrocytes. The presence of IL-1β markedly upregulated the expression of MMP-9, MMP-13 and β-catenin while suppressing Col-II and PG and SrR partially reversed this trend. In conclusion, SrR decreased MMPs but promoted Col-II, aggrecan and PG synthesis in rat chondrocytes with or without the presence of IL-1β and SrR attenuated the IL-1β-induced increase in β-catenin, thus reducing the inflammatory reaction.

Ultrastructure of collagen fibrils, proteoglycans and lamellae of the human cornea treated with Iontophoresis ‐ UVA cross‐linking and hypotonic riboflavin solution

AbstractPurposeTo investigate the effect of iontophoresis‐UVA cross‐linking with hypotonic riboflavin solution on the ultrastructural changes in the lamellae, collagen fibrils (CF) and proteoglycans (PGs) in the central and peripheral stroma of the human corneal buttons.MethodsIontophoresis method was used for the trans‐epithelial application of hypotonic riboflavin in ex vivo corneal culture for 5 minutes. The corneas were irradiated using three methods; Group 1 (G1): an UV‐A irradiance of 3 mW/cm2 for 30 minutes; Group 2 (G2): an UV‐A irradiance of 10 mW/cm2 for 9 minutes; Group 3 (G3): without UV‐A irradiation. Three untreated corneas were used as a control group (G0). After CXL procedure, the corneas were processed for electron microscopy. CF diameter and PGs in each sample were analyzed using iTEM program.ResultsThe keratocyte organelles and stromal architecture in the peripheral cornea were better preserved than in the central cornea. The mean CF diameter in the peripheral cornea of the ‘G1 and G2’ was significantly higher compared to those in the central cornea. In G3, the central corneal CF diameter was significantly larger than the peripheral corneal CF diameter. There were also differences in the PGs area size between the central and peripheral cornea in all groups.ConclusionsRiboflavin + UVA application at 3mW2 /cm for 30minutes and 10mW2/cm for 9 minutes is a suitable method of CXL; however, the 10mW2/cm for 9 minutes combination seems to present stronger phototoxic effect and better prognosis for corneal healing. CXL treatment was more effective at the periphery than center.AcknowledgmentsThis research project was funded by National Plan for Science and Technology Program (Grant Number 15MED4229‐02), King Saud University.

Effects of salmon cartilage proteoglycan on obesity in mice fed with a high-fat diet.

This study investigated the effects of salmon nasal cartilage proteoglycan (PG), which shows anti‐inflammatory properties, on obesity induced by high‐fat diet (HFD) in a mouse model. Mice were fed either a HFD or normal diet (ND), with or without PG, for 8–12 weeks. After 12 weeks, the body weight of mice fed with PG‐free HFD was 54.08 ± 4.67 g, whereas that of mice fed with HFD containing PG was 41.83 ± 4.97 g. The results suggest that the increase in body weight was attenuated in mice fed with HFD containing PG. This effect was not observed in mice fed with ND. The PG administration suppressed the elevation of serum lipids (the level of serum lipids ranged between 54% and 69% compared to 100% in mice fed with PG‐free HFD) and the upregulated mRNA expression of sterol regulatory element‐binding protein‐1c (SREBP‐1c), which is a transcription factor that acts as a master regulator of lipogenic gene expression in the liver (the expression level was 77.5% compared to 100% in mice fed with PG‐free HFD). High leptin levels in mice fed with PG‐free HFD were observed during fasting (average at 14,376 ng/ml), and they did not increase after refeeding (average of 14,263 ng/ml), whereas serum leptin levels in mice fed with HFD containing PG were low during fasting (average of 6481 ng/ml) and increased after refeeding (average 13,382 ng/ml). These results suggest that PG feeding has an anti‐obesity effect and that the regulation of SREBP‐1c and leptin secretion play a role in this effect.

Biomechanics in ascending aortic aneurysms correlate with tissue composition and strength

ObjectiveThis study correlates low strain tangential modulus (LTM) and transition zone onset (TZo) stress, biomechanical parameters that occur within the physiological range of stress seen in vivo, with tissue strength and histopathologic changes in aneurysmal ascending aortic tissue. MethodAscending aortic aneurysm tissue samples were collected from 41 patients undergoing elective resection. Samples were subjected to planar biaxial testing to quantify LTM and TZo. These were then correlated with strength assessed from uniaxial testing and with histopathologic quantification of pathologic derangements in elastin, collagen, and proteoglycan (PG). ResultsDecreased LTM and TZo were correlated with reduced strength (P < .05), PG content (P < .05), and elastin content (P < .05). Reduced TZo also was correlated with increased elastin fragmentation (P < .05). ConclusionsLTM and TZo are correlated with common biomechanical and histopathologic alterations in ascending aortic aneurysm tissue that are thought to relate to the risk of acute aortic syndromes. LTM and TZo are measured under conditions approximating in vivo physiology and have the potential to be obtained noninvasively using medical imaging techniques. Therefore, they represent parameters that warrant future study as potential contributors to our growing knowledge of pathophysiology, disease progression, and risk stratification of aortic disease.

Evaluation of enzymatic proteoglycan loss and collagen degradation in human articular cartilage using ultrashort echo time-based biomarkers: A feasibility study.

The objective of the current study was to investigate the feasibility of quantitative 3D ultrashort echo time (UTE)-based biomarkers in detecting proteoglycan (PG) loss and collagen degradation in human cartilage. A total of 104 cartilage samples were harvested for a trypsin digestion study (n = 44), and a sequential trypsin and collagenase digestion study (n = 60), respectively. Forty-four cartilage samples were randomly divided into a trypsin digestion group (tryp group) and a control group (phosphate-buffered saline [PBS] group) (n = 22 for each group) for the trypsin digestion experiment. The remaining 60 cartilage samples were divided equally into four groups (n = 15 for each group) for sequential trypsin and collagenase digestion, including PBS + Tris (incubated in PBS, then Tris buffer solution), PBS + 30 U col (incubated in PBS, then 30 U/ml collagenase [30 U col] with Tris buffer solution), tryp + 30 U col (incubated in trypsin solution, then 30 U/ml collagenase with Tris buffer solution), and tryp + Tris (incubated in trypsin solution, then Tris buffer solution). The 3D UTE-based MRI biomarkers included T1 , multiecho T2 *, adiabatic T1ρ (AdiabT1ρ ), magnetization transfer ratio (MTR), and modeling of macromolecular proton fraction (MMF). For each cartilage sample, UTE-based biomarkers (T1 , T2 *, AdiabT1ρ , MTR, and MMF) and sample weight were evaluated before and after treatment. PG and hydroxyproline assays were performed. Differences between groups and correlations were assessed. All the evaluated biomarkers were able to differentiate between healthy and degenerated cartilage in the trypsin digestion experiment, but only T1 and AdiabT1ρ were significantly correlated with the PG concentration in the digestion solution (p=0.004 and p= 0.0001, respectively). In the sequential digestion experiment, no significant differences were found for T1 and AdiabT1ρ values between the PBS + Tris and PBS + 30 U col groups (p=0.627 and p= 0.877, respectively), but T1 and AdiabT1ρ values increased significantly in the tryp + Tris (p=0.031 and p= 0.024, respectively) and tryp + 30 U col groups (both p<0.0001). Significant decreases in MMF and MTR were found in the tryp + 30 U col group compared with the PBS + Tris group (p=0.002 and p= 0.001, respectively). It was concluded that AdiabT1ρ and T1 have the potential for detecting PG loss, while MMF and MTR are promising for the detection of collagen degradation in articular cartilage, which could facilitate earlier, noninvasive diagnosis of osteoarthritis.

Multiple Irradiation Affects Cellular and Extracellular Components of the Mouse Brain Tissue and Adhesion and Proliferation of Glioblastoma Cells in Experimental System In Vivo.

Intensive adjuvant radiotherapy (RT) is a standard treatment for glioblastoma multiforme (GBM) patients; however, its effect on the normal brain tissue remains unclear. Here, we investigated the short-term effects of multiple irradiation on the cellular and extracellular glycosylated components of normal brain tissue and their functional significance. Triple irradiation (7 Gy*3 days) of C57Bl/6 mouse brain inhibited the viability, proliferation and biosynthetic activity of normal glial cells, resulting in a fast brain-zone-dependent deregulation of the expression of proteoglycans (PGs) (decorin, biglycan, versican, brevican and CD44). Complex time-point-specific (24–72 h) changes in decorin and brevican protein and chondroitin sulfate (CS) and heparan sulfate (HS) content suggested deterioration of the PGs glycosylation in irradiated brain tissue, while the transcriptional activity of HS-biosynthetic system remained unchanged. The primary glial cultures and organotypic slices from triple-irradiated brain tissue were more susceptible to GBM U87 cells’ adhesion and proliferation in co-culture systems in vitro and ex vivo. In summary, multiple irradiation affects glycosylated components of normal brain extracellular matrix (ECM) through inhibition of the functional activity of normal glial cells. The changed content and pattern of PGs and GAGs in irradiated brain tissues are accompanied by the increased adhesion and proliferation of GBM cells, suggesting a novel molecular mechanism of negative side-effects of anti-GBM radiotherapy.

A novel model of a biomechanically induced osteoarthritis-like cartilage for pharmacological in vitro studies.

Excessive pressure or overload induces and aggravates osteoarthritic changes in articular cartilage, but the underlying biomechanical forces are largely ignored in existing pharmacological in vitro models that are used to investigate drugs against osteoarthritis (OA). Here, we introduce a novel in vitro model to perform pathophysiological and pharmacological investigations, in which cartilage explants are subjected to intermittent cyclic pressure, and characterize its ability to mimic OA‐like tissue reactivity. Mechanical loading time‐dependently increased the biosynthesis, content and retention of fibronectin (Fn), whereas collagen metabolism remained unchanged. This protocol upregulated the production and release of proteoglycans (PGs). The release of PGs from explants was significantly inhibited by a matrix metalloproteinase (MMP) inhibitor, suggesting the involvement of such proteinases in the destruction of the model tissue, similar to what is observed in human OA cartilage. In conclusion, the metabolic alterations in our new biomechanical in vitro model are similar to those of early human OA cartilage, and our pharmacological prevalidation with an MMP‐inhibitor supports its value for further in vitro drug studies.

Proteoglycan-depleted regions of annular injury promote nerve ingrowth in a rabbit disc degeneration model.

BackgroundTo assess the effects of proteoglycan-depleted regions of annular disruptions on nerve ingrowth in the injury site in vivo.MethodsNew Zealand white rabbits (n = 18) received annular injuries at L3/4, L4/5, and L5/6. The experimental discs were randomly assigned to four groups: (a) an annular defect was created; (b) an annular defect implanted with a poly lactic-co-glycolic acid (PLGA)/fibrin/PBS plug; (c) an annular defect implanted with a PLGA/fibrin/chondroitinase ABC (chABC) plug; and (d) an uninjured L2/3 disc (control). Disc degeneration was evaluated by radiography, MRI, histology, and analysis of the proteoglycan (PG) content. Immunohistochemical detection of nerve fibers and chondroitin sulfate (CS) was performed.ResultsThe injured discs produced progressive and reliable disc degeneration. In the defective discs, the lamellated appearance of AF (Annulus fibrosus) was replaced by extensive fibrocartilaginous-like tissue formation outside the injured sites. In contrast, newly formed tissue was distributed along small fissures, and small blood vessels appeared in the outer part of the disrupted area in the PLGA/fibrin/PBS discs. More sprouting nerve fibers grew further into the depleted annulus regions in the PLGA/fibrin/chABC discs than in the control discs and those receiving PLGA/fibrin/PBS. In addition, the innervation scores of the PLGA/fibrin/chABC discs were significantly increased compared with those of the PLGA/fibrin/PBS discs and defected discs.ConclusionChABC-based PLGA/fibrin gel showed promising results by achieving biointegration with native annulus tissue and providing a local source for the sustained release of active chABC. Disc-derived PG-mediated inhibition of nerve and blood vessel ingrowth was abrogated by chABC enzymatic deglycosylation in an annular-injured rabbit disc degeneration model.

Metabolic Labeling of Proteoglycans and Analysis of Their Synthesis and Sorting in Filter-Grown and Polarized Epithelial Cells.

Studies of synthesis, turnover, and secretion of macromolecules in cell culture are carried out to address mechanisms of cellular and physiological importance. Culture systems have been developed to mimic the in vivo situation as much as possible. In line with this aim, epithelial and endothelial cells have been grown on filters for more than three decades. Growing such cells on permeable support allows for nutrient uptake via the basolateral membrane of tight epithelial monolayers, from a medium reservoir underneath the filter. While this basolateral medium reservoir resembles the blood supply, the apical medium reservoir resembles the organ lumen. Growing the cells in a polarized manner allows for studies of differential transport and localization of apical and basolateral proteins and of endocytic and secretory transport at both sides of the epithelium. Here we describe how metabolic labeling of proteoglycans (PGs) with 35S-labeled sulfate enables analysis of synthesis of different types of PGs, with respect to size, glycosaminoglycan (GAG) chain length, and charge. We also describe protocols for studies of intracellular PG sorting, in the apical and basolateral direction in polarized epithelial cells, in the absence and presence of inhibitors of synthesis and transport.

Role of Glycosylation in Vascular Calcification.

Glycosylation is an important step in post-translational protein modification. Altered glycosylation results in an abnormality that causes diseases such as malignancy and cardiovascular diseases. Recent emerging evidence highlights the importance of glycosylation in vascular calcification. Two major types of glycosylation, N-glycosylation and O-glycosylation, are involved in vascular calcification. Other glycosylation mechanisms, which polymerize the glycosaminoglycan (GAG) chain onto protein, resulting in proteoglycan (PG), also have an impact on vascular calcification. This paper discusses the role of glycosylation in vascular calcification.

An injectable hydrogel having proteoglycan-like hierarchical structure supports chondrocytes delivery and chondrogenesis

The ECM of cartilage is composed of proteoglycans (PG) that contain glycosaminoglycan (GAG), aggrecan, hyaluronic acid (HA) and other molecular components which play an important role in regulating chondrocyte functions via cell-matrix interactions, integrin-mediated signalling etc. Implantation of chondrocytes encapsulated in scaffolds that mimic the micro-architecture of proteoglycan, is expected to enhance cartilage repair. With an aim to create a hydrogel having macromolecular structure that resembles the cartilage-specific ECM, we constructed a hierarchal structure that mimic the PG. The bottle brush structure of the aggrecan was obtained using chondroitin sulphate and carboxymethyl cellulose which served as GAG and core protein mimic respectively. A proteoglycan-like structure was obtained by cross-linking it with modified chitosan that served as a HA substitute. The physico-chemical characteristics of the above cross-linked injectable hydrogel supported long term human articular chondrocyte subsistence and excellent post-injection viability. The chondrocytes encapsulated in the PMH expressed significant levels of articular cartilage specific markers like collagen II, aggrecan, GAGs etc., indicating the ability of the hydrogel to support chondrocyte differentiation. The biocompatibility and biodegradability of the hydrogels was confirmed using suitable in vivo studies. The results revealed that the PG-mimetic hydrogel could serve as a promising scaffold for chondrocyte implantation.

Lumican in Carcinogenesis-Revisited.

Carcinogenesis is a multifactorial process with the input and interactions of environmental, genetic, and metabolic factors. During cancer development, a significant remodeling of the extracellular matrix (ECM) is evident. Proteoglycans (PGs), such as lumican, are glycosylated proteins that participate in the formation of the ECM and are established biological mediators. Notably, lumican is involved in cellular processes associated with tumorigeneses, such as EMT (epithelial-to-mesenchymal transition), cellular proliferation, migration, invasion, and adhesion. Furthermore, lumican is expressed in various cancer tissues and is reported to have a positive or negative correlation with tumor progression. This review focuses on significant advances achieved regardingthe role of lumican in the tumor biology. Here, the effects of lumican on cancer cell growth, invasion, motility, and metastasis are discussed, as well as the repercussions on autophagy and apoptosis. Finally, in light of the available data, novel roles for lumican as a cancer prognosis marker, chemoresistance regulator, and cancer therapy target are proposed.

Abstract P316: Role Of Scleraxis In Cardiac Valve Development And Disease

Valvular heart disease is one of the major causes of cardiac-related deaths in the US and effective treatment is currently limited to surgical repair or replacement. Mature heart valves are composed of highly organized and stratified layers of extracellular matrix (ECM) regulated by valve interstitial cells (VICs). This stratification that is initiated during embryogenesis and completed during valve maturation after birth, needs to be maintained throughout life for normal valve function. Myxomatous degeneration is a common disease histologically characterized by imbalance in ECM composition and organization resulting in valve biomechanical failure. Yet, key regulators of ECM homeostasis are not well characterized. Scleraxis (Scx) is a bHLH transcription factor that we previously showed to be critical for heart valve development and its loss of function leads to defective VIC maturation and aberrant ECM organization. However, due to lack of efficient tools, the mechanistic function of Scx in valve development and disease in vivo remains to be understood. Herein, we performed temporal analysis of Scx transcript levels during development of murine homeostatic valves and identified that Scx is predominantly expressed in the VICs between E13.5 and P14. We also assessed Scx levels and ECM abnormalities in valve tissues derived from patients with cardiac valve diseases and identified several distinct populations of VICs with high Scx levels that partially colocalized with elevated collagen fragmentation and proteoglycan (PG) deposition in the neighboring ECM. Scx levels were also elevated in valves of Fbn1 C1039G/+ and osteogenesis imperfecta murine ( OIM ) mice that develop myxomatous valve abnormalities signifying a potential role in pathogenesis. To further study the function of Scx in valve development and disease in vivo , we have generated a conditional Scx-transgenic model (Scx-TG) that will allow for overexpression in targeted cell lineages upon Cre recombination. Additionally, we will employ Scx-Cre mouse model expressing Scx-promoter driven Cre -recombinase to perform Scx-expressing cell lineage analyses and high-throughput sequencing analyses to identify direct gene targets and protein-interaction partners of Scx to better understand its mechanistic role in regulating valve ECM homeostasis. Together these in vivo approaches will provide novel insights into the function of Scx in heart development and disease.

A Reinterpretation of Evidence for the Endothelial Glycocalyx Filtration Structure.

The endothelial glycocalyx (eGlx) is thought to be the primary macromolecular filter for fluid flux out of the vasculature. This filter maintains the higher protein concentration within the vessel lumen relative to the tissue. Whilst the arguments for the eGlx being the size filter are convincing the structural evidence has been limited to specialized stains of perfusion fixed tissue, which are further processed for resin embedding for transmission electron microscopy. The staining and processing of the delicate pore structure has left many researchers struggling to interpret the observed surface coat. Previous work has alluded to a 19.5 nm spacing between fibers; however, whilst repeatable it does not give an eGlx pore size consistent with known glycosaminoglycan (GAG) molecular structure due to the required fiber thickness of >10 nm. Here a new interpretation is proposed based on the likelihood that the electron micrographs of are often of collapsed eGlx. The 19.5 nm spacing measured may therefore be the core protein of the proteoglycans (PGs) with the GAGs wrapped up around them rather than in an expanded in vivo state. The concept is explored to determine that this is indeed consistent with experimental measurements of permeability if the syndecans are predominately dimerized. Further an alteration of core protein lattice from hexagonal packing to square packing dramatically changes the permeability which could be facilitated via known mechanisms such as transient actin binding.

Ultrastructural study of collagen fibrils, proteoglycans and lamellae of the cornea treated with iontophoresis - UVA cross-linking and hypotonic riboflavin solution.

To investigate the effects of iontophoresis–ultraviolet A (UVA) cross-linking (CXL) with hypotonic riboflavin solution on the ultrastructural changes in the lamellae, collagen fibrils (CFs), and proteoglycans (PGs) in the central and peripheral stroma of the human corneal buttons. The iontophoresis method was used for the trans-epithelial application of hypotonic riboflavin in ex vivo corneal culture for 5 min. The corneas were irradiated using three methods: Group 1 (G1), a UVA irradiance of 3 mW/cm2 for 30 min; Group 2 (G2), a UVA irradiance of 10 mW/cm2 for 9 min; Group 3 (G3), without UVA irradiation. Three untreated corneas were used as controls (G0). After the CXL procedure, the corneas were processed for electron microscopy. The CF diameter and PGs in each sample were analyzed using the iTEM program. The keratocyte organelles and stromal architecture in the peripheral cornea were better preserved than those in the central cornea. In G1 and G2, the mean CF diameter in the peripheral cornea was significantly higher than that in the central cornea. In G3, the CF diameter in the central cornea was significantly larger than that in the peripheral cornea. Furthermore, differences in PG area size were observed between the central and peripheral corneas in all groups. Riboflavin + UVA application at 3 mW/cm2 for 30 min and 10 mW/cm2 for 9 min was a suitable method of CXL; however, 3 mW/cm2 for 30 min improved the organization and size of the collagen fibrils. CXL treatment applied at the periphery was more effective than that applied at the center.

Chondroitin Sulfate/Dermatan Sulfate-Protein Interactions and Their Biological Functions in Human Diseases: Implications and Analytical Tools.

Chondroitin sulfate (CS) and dermatan sulfate (DS) are linear anionic polysaccharides that are widely present on the cell surface and in the cell matrix and connective tissue. CS and DS chains are usually attached to core proteins and are present in the form of proteoglycans (PGs). They not only are important structural substances but also bind to a variety of cytokines, growth factors, cell surface receptors, adhesion molecules, enzymes and fibrillary glycoproteins to execute series of important biological functions. CS and DS exhibit variable sulfation patterns and different sequence arrangements, and their molecular weights also vary within a large range, increasing the structural complexity and diversity of CS/DS. The structure-function relationship of CS/DS PGs directly and indirectly involves them in a variety of physiological and pathological processes. Accumulating evidence suggests that CS/DS serves as an important cofactor for many cell behaviors. Understanding the molecular basis of these interactions helps to elucidate the occurrence and development of various diseases and the development of new therapeutic approaches. The present article reviews the physiological and pathological processes in which CS and DS participate through their interactions with different proteins. Moreover, classic and emerging glycosaminoglycan (GAG)-protein interaction analysis tools and their applications in CS/DS-protein characterization are also discussed.