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Heparan sulfate (HS) and heparin contain imprinted "sulfation codes", which dictate their diverse physiological and pathological functions. A group of orchestrated biosynthetic enzymes cooperate in polymerizing and modifying HS chains. The biotechnological development of enzymes that can recreate this sulfation pattern on synthetic heparin is challenging, primarily due to the paucity of quantitative data for sulfotransferase enzymes. Herein, we identified critical structural characteristics that determine substrate specificity and shed light on the catalytic mechanism of sugar sulfation of two HS sulfotransferases, 2-O-sulfotransferase (HS2ST) and 6-O-sulfotransferase (HS6ST). Two sets of molecular clamps in HS2ST recognize appropriate substrates; these clamps flank the acceptor binding site on opposite sides. The hexuronic epimers, and not their puckers, have a critical influence on HS2ST selectivity. In contrast, HS6ST recognizes a broader range of substrates. This promiscuity is granted by a conserved tryptophan residue, W210, that positions the acceptor within the active site for catalysis by means of strong electrostatic interactions. Lysines K131 and K132 act in concert with a second tryptophan, W153, shedding water molecules from within the active site, thus providing HS6ST with a binding preference toward 2-O-sulfated substrates. QM/MM calculations provided valuable mechanistic insights into the catalytic process, identifying that the sulfation of both HS2ST and HS6ST follows a SN2-like mechanism. When they are taken together, our findings reveal the molecular basis of how these enzymes recognize different substrates and catalyze sugar sulfation, enabling the generation of enzymes that could create specific heparin epitopes.

Corneal transparency relies on the precise arrangement and orientation of collagen fibrils, made of mostly Type I and V collagen fibrils and proteoglycans (PGs). PGs are essential for correct collagen fibrillogenesis and maintaining corneal homeostasis. We investigated the spatial and temporal distribution of glycosaminoglycans (GAGs) and PGs after a chemical injury. The chemical composition of chondroitin sulfate (CS)/dermatan sulfate (DS) and heparan sulfate (HS) were characterized in mouse corneas 5 and 14 days after alkali burn (AB), and compared to uninjured corneas. The expression profile and corneal distribution of CS/DSPGs and keratan sulfate (KS) PGs were also analyzed. We found a significant overall increase in CS after AB, with an increase in sulfated forms of CS and a decrease in lesser sulfated forms of CS. Expression of the CSPGs biglycan and versican was increased after AB, while decorin expression was decreased. We also found an increase in KS expression 14 days after AB, with an increase in lumican and mimecan expression, and a decrease in keratocan expression. No significant changes in HS composition were noted after AB. Taken together, our study reveals significant changes in the composition of the extracellular matrix following a corneal chemical injury.

Background: Current research suggests that the glial scar surrounding penetrating brain injuries is instrumental in preserving the surrounding uninjured tissue by limiting the inflammatory response to the injury site. We recently showed that tumor necrosis factor (TNF)-stimulated gene-6 (TSG-6), a well-established anti-inflammatory molecule, is present within the glial scar. We hereby investigate the role of TSG-6 within the glial scar.Methods: TSG-6 null and littermate control mice were subjected to penetrating brain injuries (2 mm puncture wound to the frontal cortex), after which both the injury site and remaining injured hemisphere were analyzed. The presence of activated astrocytes, inflammatory markers and glial scar components was evaluated by real-time PCR and immunofluorescence. Results: Our findings show that mice lacking TSG-6 present a more severe inflammatory response after injury, which is correlated with an enlarged area of astrogliosis beyond the injury site.Conclusion: Our data provides evidence that TSG-6 has an anti-inflammatory role within the glial scar.

Studies have estimated that currently 344 million people worldwide and 16.4 million adults in the US have some form of dry eye disease (DED). It is believed that approximately 70% of DED cases are due to some form of evaporative dry eye, for which Meibomian gland dysfunction (MGD) is the major cause. Unfortunately, currently there is no effective treatment for MGD, and solely palliative care is available. Given the importance of MGD in DED, there has been a growing interest in studying Meibomian gland development, homeostasis and pathology, and, also, in developing therapies for treating and/or preventing MGD. For such, animal models have shown to be a vital tool. Much of what is known today about the Meibomian gland and MGD was learnt from these important animal models. In particular, canine and rabbit models have been essential for studying the physiopathology and progression of DED, and the mouse model, which includes different knockout strains, has enabled the identification of specific pathways potentially involved in MGD. Herein, we provide a bibliographic review on the various animal models that have been used to study Meibomian gland development, Meibomian gland homeostasis and MGD, primarily focusing on publications between 2000 and 2020.

Mesenchymal stem cells (MSCs) are a group of fibroblast-like multipotent mesenchymal stromal cells that have the ability to differentiate into osteoblasts, adipocytes, and chondrocytes. Recent studies have demonstrated that MSCs possess a unique ability to exert suppressive and regulatory effects on both adaptive and innate immunity in an autologous and allogeneic manner. A vital step in stem cell transplantation is overcoming the potential graft-versus-host disease, which is a limiting factor to transplantation success. Given that MSCs attain powerful differentiation capabilities and also present immunosuppressive properties, which enable them to survive host immune rejection, MSCs are of great interest. Due to their ability to differentiate into different cell types and to suppress and modulate the immune system, MSCs are being developed for treating a plethora of diseases, including immune disorders. Moreover, in recent years, MSCs have been genetically engineered to treat and sometimes even cure some diseases, and the use of MSCs for cell therapy presents new perspectives for overcoming tissue rejection. In this review, we discuss the potential extrinsic and intrinsic mechanisms that underlie MSCs’ unique ability to modulate inflammation, and both innate and adaptive immunity.

Proteoglycans (PGs) are a diverse family of proteins that consist of one or more glycosaminoglycan (GAG) chains, covalently linked to a core protein. PGs are major components of the extracellular matrix (ECM) and play critical roles in development, normal function and damage-response of the central nervous system (CNS). GAGs are classified based on their disaccharide subunits, into the following major groups: chondroitin sulfate (CS), heparan sulfate (HS), heparin (HEP), dermatan sulfate (DS), keratan sulfate (KS) and hyaluronic acid (HA). All except HA are modified by sulfation, giving GAG chains specific charged structures and binding properties. While significant neuroscience research has focused on the role of one PG family member, chondroitin sulfate proteoglycan (CSPG), there is ample evidence in support of a role for the other PGs in regulating CNS function in normal and pathological conditions. This review discusses the role of all the identified PG family members (CS, HS, HEP, DS, KS and HA) in normal CNS function and in the context of pathology. Understanding the pleiotropic roles of these molecules in the CNS may open the door to novel therapeutic strategies for a number of neurological conditions.

Purpose Establishing the dynamics of corneal wound healing is of vital importance to better understand corneal inflammation, pathology, and corneal regeneration. Numerous studies have made great strides in investigating multiple aspects of corneal wound healing; however, some aspects remain to be elucidated. This study worked toward establishing (1) if epithelial limbal stem cells (LSCs) are necessary for healing all corneal wounds, (2) the mechanism by which epithelial cells migrate toward the wound, and (3) if centrifugal epithelial cell movement exists. Methods To establish different aspects of corneal epithelial wound healing we subjected mice lacking hyaluronan synthase 2 (previously shown to lack LSCs) and wild-type mice to different corneal debridement injury models. Results Our data show that both LSCs and corneal epithelial cells contribute toward closure of corneal wounds. In wild-type mice, removal of the limbal rim delayed closure of 1.5-mm wounds, and not of 0.75-mm wounds, indicating that smaller wounds do not rely on LSCs as do larger wounds. In mice shown to lack LSCs, removal of the limbal rim did not affect wound healing, irrespective of the wound size. Finally, transient amplifying cells and central epithelial cells move toward a central corneal wound in a centripetal manner, whereas central epithelial cells may move in a centrifugal manner to resurface peripheral corneal wounds. Conclusions Our findings show the dimensions of the corneal wound dictate involvement of LSCs. Our data suggest that divergent findings by different groups on the dynamics of wound healing can be in part owing to differences in the wounding models used.

Heparan sulfate (HS) is a sulfated polysaccharide that plays a key role in morphogenesis, physiology and pathogenesis. The biosynthesis of HS takes place in the Golgi apparatus by a group of enzymes that polymerize, epimerize and sulfate the sugar chain. This biosynthetic process introduces varying degrees of sulfate substitution, which are tightly regulated and directly dictate binding specificity to different cytokines, morphogens and growth factors. Here, we report the use of molecular dynamics simulations to investigate the dynamics of substrate recognition of two glycosaminoglycan (GAG) sulfotransferases, N-deacetylase-N-sulfotransferase and 2-O-sulfotransferase to the HS chain during the biosynthetic process. We performed multiple simulations of the binding of the sulfotransferase domains to both the HS oligosaccharide substrate and sulfate donor, 3'-phosphoadenosine-5'-phosphosulfate. Analysis of extended simulations provide detailed and useful insights into the atomic interactions that are at work during oligosaccharide processing. The fast information matching method was used to detect the enzyme global dynamics and to predict the pairwise contact of residues responsible for GAG-enzyme binding and unbinding. The correlation between HS displacement and the location of the modified GAG chain were calculated, indicating a possible route for HS and heparin during sulfotransferase processing. Our data also show sulfotransferases contain a conserved interspaced positively charged amino acid residues that form a patch which controls the protein-GAG binding equilibrium. Together, our findings provide further understanding on the fine-tuned complex mechanism of GAG biosynthesis. Our findings can also be extrapolated to other systems for calculating rates of protein-GAG binding.

University of Houston Mizutani Foundation National Institutes of Health (NIH)/National Eye Institute PURPOSE. Limbal epithelial stem cells (LSCs), located in the basal layer of the corneal epithelium in the corneal limbus, are vital for maintaining the corneal epithelium. LSCs have a high capacity of self-renewal with increased potential for error-free proliferation and poor differentiation. To date, limited research has focused on unveiling the composition of the limbal stem cell niche, and, more important, on the role the specific stem cell niche may have in LSC differentiation and function. Our work investigates the composition of the extracellular matrix in the LSC niche and how it regulates LSC differentiation and function. METHODS. Hyaluronan (HA) is naturally synthesized by hyaluronan synthases (HASs), and vertebrates have the following three types: HAS1, HAS2, and HAS3. Wild-type and HAS and TSG-6 knockout mice-HAS1(-/-) HAS3(-/-), HAS2(Delta/Delta CorEpi), TSG-6(-/-) -were used to determine the importance of the HA niche in LSC differentiation and specification. RESULTS. Our data demonstrate that the LSC niche is composed of a HA rich extracellular matrix. HAS1(-/-) HAS3(-/-), HAS2(Delta/Delta CorEpi), and TSG-6(-/-) mice have delayed wound healing and increased inflammation after injury. Interestingly, upon insult the HAS knock-out mice upregulate HA throughout the cornea through a compensatory mechanism, and in turn this alters LSC and epithelial cell specification. CONCLUSIONS. The LSC niche is composed of a specialized HA matrix that differs from that present in the rest of the corneal epithelium, and the disruption of this specific HA matrix within the LSC niche leads to compromised corneal epithelial regeneration. Finally, our findings suggest that HA has a major role in maintaining the LSC phenotype. Univ Fed São Paulo, São Paulo, Brazil Univ Houston, Coll Optometry, 4901 Calhoun Rd, Houston, TX 77204 USA Sanford Burnham Med Res Inst, Sanford Childrens Hlth Res Ctr, La Jolla, CA USA Chang Gung Univ, Chang Gung Mem Hosp, Dept Ophthalmol, Coll Med, Linkou, Taiwan Cleveland Clin, Cleveland, OH 44106 USA Univ Fed São Paulo, São Paulo, Brazil NIH: P30 EY07551 Web of Science

We have developed a 2D heterotypic co-culture technique between fibroblasts and cancer cells that enables the study of the stromal reaction. For such, stromal cells are seeded and cultured immediately around a tumour cell line, and the cells establish cell-cell contacts, as well as a gradient of soluble factors throughout the stromal cells, similar to that found in tissues. Thus, this system also enables the researcher to distinguish between events that are caused by direct cell-cell contact and secreted factors.

NIH/NEI grants Research to Prevent Blindness Ohio Eye Research Foundation Lumican, a small leucine rich proteoglycan (SLRP), is a component of extracellular matrix which also functions as a matrikine regulating multiple cell activities. In the cornea, lumican maintains corneal transparency by regulating collagen fibrillogenesis, promoting corneal epithelial wound healing, regulating gene expression and maintaining corneal homeostasis. We have recently shown that a peptide designed from the 13 C-terminal amino acids of lumican (LumC13) binds to ALK5/TGFBR1 (type1 receptor of TGF beta) to promote wound healing. Herein we evaluate the mechanism by which this synthetic C-terminal amphiphilic peptide (LumC13), binds to ALK5. These studies clearly reveal that LumC13-ALK5 form a stable complex. In order to determine the minimal amino acids required for the formation of a stable lumican/ALK5 complex derivatives of LumC13 were designed and their binding to ALK5 investigated in silico. These LumC13 derivatives were tested both in vitro and in vivo to evaluate their ability to promote corneal epithelial cell migration and corneal wound healing, respectively. These validations add to the therapeutic value of LumC13 (Lumikine) and aid its clinical relevance of promoting the healing of corneal epithelium debridement. Moreover, our data validates the efficacy of our computational approach to design active peptides based on interactions of receptor and chemokine/ligand. Univ Cincinnati, Dept Ophthalmol, Cincinnati, OH 45267 USA Univ Fed Sao Paulo, Dept Bioquim, Sao Paulo, Brazil Univ Houston, Coll Optometry, Ocular Surface Inst, Houston, TX 77204 USA Univ Fed Sao Paulo, Dept Bioquim, Sao Paulo, Brazil NIH/NEI grants: RO1 EY011845 NIH/NEI grants: R01 021768 Web of Science

Umbilical cord mesenchymal stem cells (UMSCs) have unique immunosuppressive properties enabling them to evade host rejection and making them valuable tools for cell therapy. We previously showed that human UMSCs survive xenograft transplantation and successfully correct the corneal clouding defects associated with the mouse model for the congenital metabolic disorder mucopolysaccharidosis VII. However, the precise mechanism by which UMSCs suppress the immune system remains elusive. This study aimed to determine the key components involved in the ability of the UMSCs to modulate the inflammatory system and to identify the inflammatory cells that are regulated by the UMSCs. Our results show that human UMSCs transplanted into the mouse stroma 24 h after an alkali burn suppress the severe inflammatory response and enable the recovery of corneal transparency within 2 weeks. Furthermore, we demonstrated in vitro that UMSCs inhibit the adhesion and invasion of inflammatory cells and also the polarization of M1 macrophages. UMSCs also induced the maturation of T-regulatory cells and led to inflammatory cell death. Moreover, UMSCs exposed to inflammatory cells synthesize a rich extracellular glycocalyx composed of the chondroitin sulfate-proteoglycan versican bound to a heavy chain (HC)-modified hyaluronan (HA) matrix (HC-HA). This matrix also contains TNFα-stimulated gene 6 (TSG6), the enzyme that transfers HCs to HA, and pentraxin-3, which further stabilizes the matrix. Our results, both in vivo and in vitro, show that this glycocalyx confers the ability for UMSCs to survive the host immune system and to regulate the inflammatory cells.

Purpose: Corneal lymphangiogenesis and angiogenesis leads to the loss of corneal transparency. We have recently shown that in the cornea hyaluronan (HA) is present primarily in the limbal region and plays a key role regulating the limbal stem cell phenotype. Given the HA receptor LYVE-1 is highly expressed in corneal lymphatic vessels we investigated whether HA could play a role in regulating corneal lymphangiogenesis. Methods: Wild-type (wt) and hyaluronan synthase (HAS) knockout mice - specifically combined Has1-/- and Has3 -/- null mice (HAS1-/-;HAS3-/-) and conditional Has2 knock-out mice (HAS2D/DCorEpi), were used. The mice were subjected to injury, alkali burn or suture placement, to investigate the role of HA on corneal lymphangiogenesis. Corneal buttons were also obtained from different developmental time-points to study the role of HA in lymphatic vessel development. The corneas were analyzed by whole mount immunohistochemistry and entire corneas were imaged under an LSM 800 confocal microscope using the both the z-stack and tiling mode. Primary lymphatic vessel endothelial cells from human dermis (hDLECs) and lymph node (hLLECs) were used for tube formation assay and cell proliferation assay in vitro. Results: After injury both wild-type and HAS1-/-;HAS3-/- mice presented both an increase in HA expression and lymphangiogenesis. Interestingly, lymphatic vessels extended exclusively into HA rich areas. In stark contrast, HAS2D/DCorEpi mice did not upregulate HA synthesis after injury and, in turn, did not present lymphangiogenesis. Our developmental studies revealed first HA is expressed in the corneal limbus and thereafter lymphatic vessels invade this region. Our in vitro studies corroborated our in vivo data, with both HA increasing the proliferation and tube formation ability of hDLECs and hLLECs. Conclusions: HA regulates corneal lymphangiogenesis, both during development and after injury. These findings raise the possibility that therapeutic blockade of HA-mediated lymphangiogenesis could be used to reduce corneal scarring and also prevent rejection after corneal transplantation.

We have developed methods for isolating proteoglycans and glycosaminoglycans from archaeological bones and teeth. These methods have been previously published (Coulson- Thomas et al., 2015 ) and are described here in more detail. In the case of glycosaminoglycans, the method was a previously described method ( Nader et al., 1999 ) which we optimized for archeological samples.

Mesenchymal stem cells isolated from connective tissues are pluripotent and differentiate into phenotypes of connective tissue cell lineages (osteoblasts, chondrocytes, and adipocytes) in vitro and in vivo. They have been used to treat mouse models of connective tissue disease such as lumican-null (Lum) and mucopolysaccharidosis (Gusb) mice. Mesenchymal stem cells have unique immunosuppressive properties allowing evasion of host rejection; thus, they are valuable tools for cell therapy of congenital and acquired diseases involving immune dysfunction of multiple tissues including ocular surface tissues (cornea). We previously showed that human umbilical mesenchymal stem cells (UMSCs) modulated host immune responses, enabling them to survive xenograft transplantation. In vitro, UMSCs modulated inflammatory cells by inhibiting adhesion and invasion, and inducing cell death. UMSCs also regulated M1/M2 macrophage polarization and induced T-regulatory cell maturation from naive intraperitoneal cavity lavage cells. UMSCs exposed to inflammatory cells synthesized a rich extracellular glycocalyx composed of hyaluronan (HA) bound to the heavy chains (HCs) of inter-alpha-trypsin inhibitor (HC-HA), which contains tumor necrosis factor-α-stimulated gene 6 (TSG6) that catalyzes the transfer of HCs to HA, versican, and pentraxin-3. Our in vivo and in vitro results showed that the glycocalyx regulated inflammatory cells, allowing UMSCs to survive host immune rejection. Administration of antibodies against glycocalyx constituents or digestion with hyaluronidase and chondroitinase ABC abolished the UMSCs' ability to modulate immune responses. Treatment with anti-CD44 antibodies also diminished modulation of M2 macrophages by UMSCs, indicating that cell surface CD44 is required for correct UMSC glycocalyx assembly to modulate inflammatory cells.

We have developed a 3D co-culture system composed of fibroblasts and colorectal cancer cells that enables us to study the desmoplastic reaction. This method also enables us to study the influence of the desmoplastic reaction on the migration of colorectal cancer cells through the surrounding stroma. This protocol has been previously published (Coulson- Thomas et al., 2011 ) and is described here in more detail.

Syndecans are heparan sulfate proteoglycans characterized as transmembrane receptors that act cooperatively with the cell surface and extracellular matrix proteins. Syn4 knockdown was performed in order to address its role in endothelial cells (EC) behavior. Normal EC and shRNA-Syn4-EC cells were studied comparatively using complementary confocal, super-resolution and non-linear microscopic techniques. Confocal and super-resolution microscopy revealed that Syn4 knockdown alters the level and arrangement of essential proteins for focal adhesion, evidenced by the decoupling of vinculin from F-actin filaments. Furthermore, Syn4 knockdown alters the actin network leading to filopodial protrusions connected by VE-cadherin-rich junction. shRNA-Syn4-EC showed reduced adhesion and increased migration. Also, Syn4 silencing alters cell cycle as well as cell proliferation. Moreover, the ability of EC to form tube-like structures in matrigel is reduced when Syn4 is silenced. Together, the results suggest a mechanism in which Syndecan-4 acts as a central mediator that bridges fibronectin, integrin and intracellular components (actin and vinculin) and once silenced, the cytoskeleton protein network is disrupted. Ultimately, the results highlight Syn4 relevance for balanced cell behavior.

N-Deacetylase-N-sulfotransferase 1 (Ndst1) catalyzes the initial modification of heparan sulfate and heparin during their biosynthesis by removal of acetyl groups from subsets of N-acetylglucosamine units and subsequent sulfation of the resulting free amino groups. in this study, we used a phage display library to select peptides that interact with Ndst1, with the aim of finding inhibitors of the enzyme. the phage library consisted of cyclic random 10-mer peptides expressed in the phage capsid protein pIII. Selection was based on the ability of engineered phage to bind to recombinant murine Ndst1 (mNdst1) and displacement with heparin. Peptides that were enriched through multiple cycles of binding and disassociation displayed two specific sequences, CRGWRGEKIGNC and CNMQALSMPVTC. Both peptides inhibited mNdst1 activity in vitro, however, by distinct mechanisms. the peptide CRGWRGEKIGNC presents a chemokine-like repeat motif (BXX, where B represents a basic amino acid and X is a noncharged amino acid) and binds to heparan sulfate, thus blocking the binding of substrate to the enzyme. the peptide NMQALSMPVT inhibits mNdst1 activity by direct interaction with the enzyme near the active site. the discovery of inhibitory peptides in this way suggests a method for developing peptide inhibitors of heparan sulfate biosynthesis. Universidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, Brazil Universidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, Brazil Univ Texas MD Anderson Canc Ctr, David H Koch Ctr, Houston, TX 77030 USA Univ Mogi das Cruzes, Ctr Interdisciplinar Invest Bioquim, BR-05508000 São Paulo, Brazil Univ Calif San Diego, Dept Cellular & Mol Med, La Jolla, CA 92093 USA Univ Calif San Diego, Biomed Sci Grad Program, San Diego, CA USA Universidade Federal de São Paulo, Dept Bioquim, BR-04044020 São Paulo, Brazil Universidade Federal de São Paulo, Dept Biofis, BR-04044020 São Paulo, Brazil Web of Science

Glycosaminoglycans (GAGs) play important roles in cell behavior and have the ability to bind and modulate cytokines. Using primary cultured fibroblasts from hereditary gingival fibromatosis (HGF), normal gingiva (NG), and NG treated with cyclosporin-A (NGc) we show changes in the expression and structural characteristics of GAGs as well as in the expression of enzymes involved in their biosynthesis and degradation. In addition, we show the over-expression of TGF-beta1 and TGF-beta type II receptor in HGF and NGc. There is an increase in the GAGs retained in the cellular fraction, and the fine structure of galactosaminoglycans show a decrease in alpha-l-iduronic acid content in HGF and NGc. Elevated extracellular levels of low molecular weight hyaluronan (HA) are found in HGF due to increase in the expression of HA synthase 3 and hyaluronidases 1 and 2. The results bring new insights to the accumulation of extracellular matrix related to TGF-beta over-expression.

Injury to the CNS of vertebrates leads to the formation of a glial scar and production of inhibitory molecules, including chondroitin sulphate proteoglycans. Various studies suggest that the sugar component of the proteoglycan is responsible for the inhibitory role of these compounds in axonal regeneration. By degrading chondroitin sulphate chains with specific enzymes, denominated chondroitinases, the inhibitory capacity of these proteoglycans is decreased. Chondroitinase administration involves frequent injections of the enzyme at the lesion site which constitutes a rather invasive method. We have produced a vector containing the gene for Flavobacterium heparinum chondroitinase AC for expression in adult bone marrow-derived cells which were then transplanted into an injury site in the CNS. The expression and secretion of active chondroitinase AC was observed in vitro using transfected Chinese hamster ovarian and gliosarcoma cells and in vivo by immunohistochemistry analysis which showed degraded chondroitin sulphate coinciding with the location of transfected bone marrow-derived cells. Immunolabelling of the axonal growth-associated protein GAP-43 was observed in vivo and coincided with the location of degraded chondroitin sulphate. We propose that bone marrow-derived mononuclear cells, transfected with our construct and transplanted into CNS, could be a potential tool for studying an alternative chondroitinase AC delivery method.

Heparan sulphate (HS) is ubiquitously expressed and is formed of repeating glucosamine and glucuronic/iduronic acid units which are generally highly sulphated. HS is found in tissues bound to proteins forming HS proteoglycans (HSPGs) which are present on the cell membrane or in the extracellular matrix. HSPGs influence a variety of biological processes by interacting with physiologically important proteins, such as morphogens, creating storage pools, generating morphogen gradients and directly mediating signalling pathways, thereby playing vital roles during development. This review discusses the vital role HS plays in the development of tissues from the ectodermal lineage. The ectodermal layer differentiates to form the nervous system (including the spine, peripheral nerves and brain), eye, epidermis, skin appendages and tooth enamel.

Bone tissue is mineralized dense connective tissue consisting mainly of a mineral component (hydroxyapatite) and an organic matrix comprised of collagens, non-collagenous proteins and proteoglycans (PGs). Extracellular matrix proteins and PGs bind tightly to hydroxyapatite which would protect these molecules from the destructive effects of temperature and chemical agents after death. DNA and proteins have been successfully extracted from archaeological skeletons from which valuable information has been obtained; however, to date neither PGs nor glycosaminoglycan (GAG) chains have been studied in archaeological skeletons. PGs and GAGs play a major role in bone morphogenesis, homeostasis and degenerative bone disease. The ability to isolate and characterize PG and GAG content from archaeological skeletons would unveil valuable paleontological information. We therefore optimized methods for the extraction of both PGs and GAGs from archaeological human skeleto ns. PGs and GAGs were successfully extracted from both archaeological human bones and teeth, and characterized by their electrophoretic mobility in agarose gel, degradation by specific enzymes and HPLC. The GAG populations isolated were chondroitin sulfate (CS) and hyaluronic acid (HA). In addition, a CSPG was detected. The localization of CS, HA, three small leucine rich PGs (biglycan, decorin and fibromodulin) and glypican was analyzed in archaeological human bone slices. Staining patterns were different for juvenile and adult bones, whilst adolescent bones had a similar staining pattern to adult bones. The finding that significant quantities of PGs and GAGs persist in archaeological bones and teeth opens novel venues for the field of Paleontology.

Morphological and ultrastructural data from archaeological human bones are scarce, particularly data that have been correlated with information on the preservation of molecules such as DNA. Here we examine the bone structure of macroscopically well-preserved medieval human skeletons by transmission electron microscopy and immunohistochemistry, and the quantity and quality of DNA extracted from these skeletons. DNA technology has been increasingly used for analyzing physical evidence in archaeological forensics; however, the isolation of ancient DNA is difficult since it is highly degraded, extraction yields are low and the co-extraction of PCR inhibitors is a problem. We adapted and optimised a method that is frequently used for isolating DNA from modern samples, Chelex(®) 100 (Bio-Rad) extraction, for isolating DNA from archaeological human bones and teeth. The isolated DNA was analysed by real-time PCR using primers targeting the sex determining region on the Y chromosome (SRY) and STR typing using the AmpFlSTR(®) Identifiler PCR Amplification kit. Our results clearly show the preservation of bone matrix in medieval bones and the presence of intact osteocytes with well preserved encapsulated nuclei. In addition, we show how effective Chelex(®) 100 is for isolating ancient DNA from archaeological bones and teeth. This optimised method is suitable for STR typing using kits aimed specifically at degraded and difficult DNA templates since amplicons of up to 250bp were successfully amplified.

PURPOSE:Heparan sulfate (HS) is a highly modified glycosaminoglycan (GAG) bound to a core protein to form heparan sulfate proteoglycans (HSPGs) that are vital in many cellular processes ranging from development to adult physiology, as well as in disease, through interactions with various protein ligands. This study aimed to elucidate the role of HS in corneal epithelial homeostasis and wound healing. METHODS:An inducible quadruple transgenic mouse model was generated to excise Ext1 and Ndst1, which encode the critical HS chain elongation enzyme and N-deacetylase/N-sulfotransferase, respectively, in keratin 14-positive cells upon doxycycline induction. RESULTS:EXT(Δ/ΔCEpi) mice (deletion of Ext1 in corneal epithelium) induced at P20 presented progressive thinning of the corneal epithelium with a significant loss in the number of epithelial layers by P55. EXT(Δ/ΔCEpi) mice presented tight junction disruption, loss of cell-basement membrane adhesion complexes, and impaired wound healing. Interestingly, EXT(Δ/ΔCEpi) and NDST(Δ/ΔCEpi) mice presented an increase in cell proliferation, which was assayed by both Ki67 staining and 5-ethynyl-2'-deoxyuridine (EdU) incorporation. Moreover, EXT(Δ/ΔCEpi) mice presented compromised epithelial stratification 7 days after a debridement wound. The conditional knockout of HS from keratocytes using the keratocan promoter led to no corneal abnormalities or any disruption in wound healing. CONCLUSIONS:Corneal epithelial cells require HS for maintaining corneal homeostasis, and the loss of epithelial HS leads to both impaired wound healing and impaired corneal stratification.

Hair follicle (HF) morphogenesis and cycling are a result of intricate autonomous epithelial-mesenchymal interactions. Once the first HF cycle is complete it repeatedly undergoes cyclic transformations. Heparan sulfate (HS) proteoglycans are found on the cell surface and in the extracellular matrix where they influence a variety of biological processes by interacting with physiologically important proteins, such as growth factors. Inhibition of heparanase (an HS endoglycosidase) in in vitro cultured HFs has been shown to induce a catagen-like process. Therefore, this study aimed to elucidate the precise role of HS in HF morphogenesis and cycling. An inducible tetratransgenic mouse model was generated to excise exostosin glycosyltransferase 1 (Ext1) in keratin 14-positive cells from P21. Interestingly, EXT1(StEpiΔ/StEpiΔ) mice presented solely anagen HFs. Moreover, waxing the fur to synchronize the HFs revealed accelerated hair regrowth in the EXT1(StEpiΔ/StEpiΔ) mice and hindered cycling into catagen. The ablation of HS in the interfollicular epidermal cells of mature skin led to the spontaneous formation of new HFs and an increase in Sonic Hedgehog expression resembling wild-type mice at P0, thereby indicating that the HS/Sonic Hedgehog signaling pathway regulates HF formation during embryogenesis and prevents HF formation in mature skin. Finally, the knock-out of HS also led to the morphogenesis and hyperplasia of sebaceous glands and sweat glands in mature mice, leading to exacerbated sebum production and accumulation on the skin surface. Therefore, our findings clearly show that an intricate control of HS levels is required for HF, sebaceous gland, and sweat gland morphogenesis and HF cycling.

The stroma surrounding tumors can either restrict or promote tumor growth and progression, and both the cellular and non-cellular components of the stroma play an active role. The cellular components in the surrounding stroma include tumor-associated fibroblasts, host tissue cells and immune cells. The non-cellular components, which form the extracellular matrix (ECM) scaffold, include proteoglycans, collagen, proteinases, growth factors and cytokines. For tumorigenesis to occur it is necessary for tumor cells to modify the surrounding stroma. Tumor cells have mechanisms for achieving this, such as co-opting fibroblasts and modifying the ECM they produce, degrading the surrounding ECM and/or synthesizing a favorable ECM to support invasion. Proteoglycans are an important component of the ECM and play an active role in tumor growth and progression. The expression and glycosylation patterns of proteoglycans are altered in the stroma surrounding tumors and these molecules may support or restrict tumor growth and progression depending on the type and stage of tumor. In the present review we discuss the difference between the tumor promoting and restricting stromal reactions surrounding tumors and the role proteoglycans play.

The structurally diverse polysaccharide lyase enzymes are distributed from plants to animals but share common catalytic mechanisms. One, heparinase I (F. heparinum), is employed in the production of the major anticoagulant drug, low molecular weight heparin, and is a mainstay of cell surface proteoglycan analysis. We demonstrate that heparinase I specificity and efficiency depend on the cationic form of the substrate. Ca(2+)-heparin, in which α-L-iduronate-2-O-sulfate residues adopt (1)C4 conformation preferentially, is a substrate, while Na(+)-heparin is an inhibitor. His and Tyr residues are identified in the catalytic step and a model based on molecular dynamics and docking is proposed, in which deprotonated His203 initiates β-elimination by abstracting the C5 proton of the α-L-iduonate-2-O-sulfate residue in the substrate, and protonated Tyr357 provides the donor to the hexosamine leaving group.

Mucopolysaccharidosis (MPS) are a family of related disorders caused by a mutation in one of the lysosomal exoglycosidases which leads to the accumulation of glycosaminoglycans (GAGs). MPS VII, caused by a mutation in β-glucuronidase, manifests hepatomegaly, skeletal dysplasia, short stature, corneal clouding, and developmental delay. Current treatment regimens for MPS are not effective for treating corneal clouding and impaired mental development. We hypothesized that human umbilical mesenchymal stem cells (UMSCs) transplanted into the corneal stroma could participate in the catabolism of GAGs providing a means of cell therapy for MPS. For such treatment, human UMSCs were intrastromally transplanted into corneas of MPS VII mice. UMSC transplantation restored the dendritic and hexagonal morphology of host keratocytes and endothelial cells, respectively, and in vivo confocal microscopy (HRT-II) revealed reduced corneal haze. Immunohistochemistry using antibodies against heparan sulfate and chondroitin sulfate chains as well as lysosomal-associated membrane protein 2 revealed a decrease in GAG content and both lysosomal number and size in the treated corneas. Labeling UMSC intracellular compartments prior to transplantation revealed the distribution of UMSC vesicles throughout the corneal stroma and endothelium. An in vitro coculture assay between skin fibroblasts isolated from MPS VII mice and UMSC demonstrated that neutral vesicles released by the UMSC are taken up by the fibroblasts and proceed to fuse with the acidic lysosomes. Therefore, transplanted UMSCs participate both in extracellular GAG turnover and enable host keratocytes to catabolize accumulated GAG products, suggesting that UMSC could be a novel alternative for treating corneal defects associated with MPS and other congenital metabolic disorders.

The stromal reaction surrounding tumors leads to the formation of a tumor-specific microenvironment, which may play either a restrictive role or a supportive role in the growth and progression of the tumors. Lumican, a small leucine-rich proteoglycan (SLRP) of the extracellular matrix (ECM), regulates collagen fibrillogenesis. Recently, lumican has also been shown to regulate cell behavior during embryonic development, tissue repair and tumor progression. The role of lumican in cancer varies according to the type of tumor. In this study we analyze the role of lumican in the pathogenesis of prostate cancer both in vivo and in vitro. Overall lumican up-regulation was observed in the primary tumors analyzed through both real-time PCR and immunostaining. The increase in lumican expression was observed in the reactive stroma surrounding prostate primary tumors with fibrotic deposition surrounding the acinar glands. In vitro analysis demonstrated that lumican inhibited both the migration and invasion of metastatic prostate cancer cells isolated from lymph node, bone and brain. Moreover, prostate cancer cells seeded on lumican presented a decrease in the formation of cellular projections, lamellipodia detected by a decreased rearrangement in ZO-1, keratin 8/18, integrin β1 and MT1-MMP, and invadopodia detected by disruption of α-smooth muscle actin, cortactin and N-WASP. Moreover, a significant increase in prostate cancer cell invasion was observed through the peritoneum of lumican knockout mice, further demonstrating the restrictive role lumican present in the ECM has on prostate cancer invasion. In conclusion, lumican present in the reactive stroma surrounding prostate primary tumors plays a restrictive role on cancer progression, and we therefore postulate that lumican could be a valuable marker in prostate cancer staging.

Lumican (Lum), a small leucine-rich proteoglycan (SLRP) family member, has multiple matricellular functions both as an extracellular matrix component and as a matrikine regulating cell proliferation, gene expression and wound healing. To date, no cell surface receptor has been identified to mediate the matrikine functions of Lum. This study aimed to identify a perspective receptor that mediates Lum effects on promoting wound healing. Transforming growth factor-β receptor 1 (ALK5) was identified as a potential Lum-interacting protein through in silico molecular docking and molecular dynamics. This finding was verified by biochemical pull-down assays. Moreover, the Lum function on wound healing was abrogated by an ALK5-specific chemical inhibitor as well as by ALK5 shRNAi. Finally, we demonstrated that eukaryote-specific post-translational modifications are not required for the wound healing activity of Lum, as recombinant GST-Lum fusion proteins purified from E. coli and a chemically synthesized LumC13 peptide (the last C-terminal 13 amino acids of Lum) have similar effects on wound healing in vitro and in vivo.

Fundação de Amparo à Pesquisa do Estado de São Paulo (FAPESP) Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) Tumor cell invasion is vital for cancer progression and metastasis. Adhesion, migration, and degradation of the extracellular matrix are important events involved in the establishment of cancer cells at a new site, and therefore molecular targets are sought to inhibit such processes. the effect of a plant proteinase inhibitor, Enterolobium contortisiliquum trypsin inhibitor (EcTI), on the adhesion, migration, and invasion of gastric cancer cells was the focus of this study. EcTI showed no effect on the proliferation of gastric cancer cells or fibroblasts but inhibited the adhesion, migration, and cell invasion of gastric cancer cells; however, EcTI had no effect upon the adhesion of fibroblasts. EcTI was shown to decrease the expression and disrupt the cellular organization of molecules involved in the formation and maturation of invadopodia, such as integrin beta 1, cortactin, neuronal Wiskott-Aldrich syndrome protein, membrane type 1 metalloprotease, and metalloproteinase-2. Moreover, gastric cancer cells treated with EcTI presented a significant decrease in intracellular phosphorylated Src and focal adhesion kinase, integrin-dependent cell signaling components. Together, these results indicate that EcTI inhibits the invasion of gastric cancer cells through alterations in integrin-dependent cell signaling pathways. Universidade Federal de São Paulo, Escola Paulista Med, Dept Biochem & Mol Biol, BR-04044020 São Paulo, Brazil Universidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Immunol & Parasitol, BR-04044020 São Paulo, Brazil Universidade Federal de São Paulo, Escola Paulista Med, Dept Biochem & Mol Biol, BR-04044020 São Paulo, Brazil Universidade Federal de São Paulo, Escola Paulista Med, Dept Microbiol Immunol & Parasitol, BR-04044020 São Paulo, Brazil FAPESP: 2009/53766-5 Web of Science

During cancer cell growth many tumors exhibit various grades of desmoplasia, unorganized production of fibrous or connective tissue, composed mainly of collagen fibers and myofibroblasts. The accumulation of an extracellular matrix (ECM) surrounding tumors directly affects cancer cell proliferation, migration and spread; therefore the study of desmoplasia is of vital importance. Stromal fibroblasts surrounding tumors are activated to myofibroblasts and become the primary producers of ECM during desmoplasia. The composition, density and organization of this ECM accumulation play a major role on the influence desmoplasia has upon tumor cells. In this study, we analyzed desmoplasia in vivo in human colorectal carcinoma tissue, detecting an up-regulation of collagen I, collagen IV and collagen V in human colorectal cancer desmoplastic reaction. These components were then analyzed in vitro co-cultivating colorectal cancer cells (Caco-2 and HCT116) and fibroblasts utilizing various co-culture techniques. Our findings demonstrate that direct cell-cell contact between fibroblasts and colorectal cancer cells evokes an increase in ECM density, composed of unorganized collagens (I, III, IV and V) and proteoglycans (biglycan, fibromodulin, perlecan and versican). The desmoplastic collagen fibers were thick, with an altered orientation, as well as deposited as bundles. This increased ECM density inhibited the migration and invasion of the colorectal tumor cells in both 2D and 3D co-culture systems. Therefore this study sheds light on a possible restricting role desmoplasia could play in colorectal cancer invasion.

Growth and survival of tumors at a site of metastasis involve interactions with stromal cells in the surrounding environment. Stromal cells aid tumor cell growth by producing cytokines as well as by modifying the environment surrounding the tumor through modulation of the extracellular matrix (ECM). Small leucine-rich proteoglycans (SLRPs) are biologically active components of the ECM which can be altered in the stroma surrounding tumors. The influence tumor cells have on stromal cells has been well elucidated. However, little is understood about the effect metastatic cancer cells have on the cell biology and behavior of the local stromal cells. Our data reveal a significant down-regulation in the expression of ECM components such as collagens I, II, III, and IV, and the SLRPs, decorin, biglycan, lumican, and fibromodulin in stromal cells when grown in the presence of two metastatic prostate cancer cell lines PC3 and DU145. Interestingly, TGF-β down-regulation was observed in stromal cells, as well as actin depolymerization and increased vimentin and α5β1 integrin expression. MT1-MMP expression was upregulated and localized in stromal cell protrusions which extended into the ECM. Moreover, enhanced stromal cell migration was observed after cross-talk with metastatic prostate tumor cells. Xenografting metastatic prostate cancer cells together with "activated" stromal cells led to increased tumorigenicity of the prostate cancer cells. Our findings suggest that metastatic prostate cancer cells create a metastatic niche by altering the phenotype of local stromal cells, leading to changes in the ECM.

The physiology and transparency of the cornea are dependent on corneal endothelial function. The role of temperature sensitive ion channels in maintaining such activity is unknown. This study was undertaken to probe for the functional expression of such pathways in human corneal endothelial cells (HCEC). We used HCEC-12, an immortalized population derived from whole corneal endothelium, and two morphologically distinct clonal cell lines derived from HCEC-12 (HCEC-H9C1, HCEC-B4G12) to probe for gene expression and function of transient receptor potential (TRP) channels of the vanilloid (V) isoform subfamily (i.e. TRPV1-3) in these cell types. Expression of TRPV isotypes 1, 2 and 3 were detected by RT-PCR. Protein expression of TRPV1 in situ was confirmed by immunostaining of corneoscleral remnants after keratoplasty. TRPV1-3 functional activity was evident based on capsaicin-induced Ca(2+) transients and induction of these responses through rises in ambient temperature from 25 degrees C to over 40 degrees C. The currents underlying Ca(2+) transients were characterized with a novel high throughput patch-clamp system. The TRPV1 selective agonist, capsaicin (CAP) (10-20 microM) increased non-selective cation whole-cell currents resulting in calcium increases that were fully blocked by either the TRPV1 antagonist capsazepine (CPZ) or removal of extracellular calcium. Similarly, heating from room temperature to over 40 degrees C increased the same currents resulting in calcium increases that were significantly reduced by the TRP channel blockers lanthanum chloride (La(3+)) (100 microM) and ruthenium-red (RuR) (10 microM), respectively. Moreover, application of the TRPV channel opener 2-aminoethoxydiphenyl borate (2-APB) (400 microM) led to a reversible increase in intracellular Ca(2+) indicating putative TRPV1-3 channel activity. Taken together, TRPV activity modulation by temperature underlies essential homeostatic mechanisms contributing to the support of corneal endothelial function under different ambient conditions.

Mesenchymal stem cells (MSC) have become a promising tool for cell therapy in regenerative medicine. They are readily available, demonstrate powerful differentiation capabilities and present immunosuppressive properties that aid them in surviving from host immune rejection for its great potential use in allograft. Currently clinical trials are underway using MSC, both culture-expanded allogeneic and autologous, for the treatment of a range of diseases not treatable by conventional therapies. A vast array of studies has dedicated towards the use of MSC for treating corneal diseases with very promising outcomes. MSC have successfully differentiated into keratocytes both in vitro and in vivo, and corneal epithelial cells in vitro, but it is uncertain if MSC can assume corneal epithelial cells in vivo. However, to date few studies have unequivocally established the efficacy of MSC for treating corneal endothelial defects. Currently, the diversity in protocols of the isolation and expansion of MSC are hindering to the assessment of cell treatment ability and the further development of treatment regimens. Therefore, future studies should develop international standards for MSC isolation and characterization. In this review, we discuss recent advances in MSC for treating ocular surface diseases.

Purpose The Meibomian gland (MG) produces the lipid layer of the tear film, and changes to the MG that lead to a decrease or alteration in lipid quality/content may lead to MG dysfunction, a major cause of evaporative dry eye disease with prevalence ranging from 39% to 50%. Little is known about the developmental cues that regulate MG morphogenesis and homeostasis. Our study investigates the role of hyaluronan (HA), a major extracellular matrix component, in eyelid formation and MG development and function. Methods Hyaluronan synthase (Has) knockout mice were used to determine the role of HA in the eyelid and MG. Eyelids were obtained during different developmental stages and MG morphology was analyzed. Tet-off H2B-GFP/K5tTA mice and 5-ethynyl-2'-deoxyurdine (EdU) incorporation were used to determine the role of HA in maintaining slow-cycling and proliferating cells within the MG, respectively. Data were confirmed using an in vitro proliferation assay, differentiation assay and spheroid cultures. Results Has knockout mice present precocious MG development, and adult mice present MG hyperplasia and dysmorphic MGs and eyelids, with hyperplastic growths arising from the palpebral conjunctiva. Our data show that a highly organized HA network encompasses the MG, and basal cells are embedded within this HA matrix, which supports the proliferating cells. Spheroid cultures showed that HA promotes acini formation. Conclusions HA plays an important role in MG and eyelid development. Our findings suggest that Has knockout mice have abnormal HA synthesis, which in turn leads to precocious and exacerbated MG morphogenesis culminating in dysmorphic eyelids and MGs.