18 research outputs found
Hyaluronan Rich Microenvironment in the Limbal Stem Cell Niche Regulates Limbal Stem Cell Differentiation
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.University of HoustonMizutani FoundationNational Institutes of Health (NIH)/National Eye InstituteUniv Fed São Paulo, São Paulo, BrazilUniv Houston, Coll Optometry, 4901 Calhoun Rd, Houston, TX 77204 USASanford Burnham Med Res Inst, Sanford Childrens Hlth Res Ctr, La Jolla, CA USAChang Gung Univ, Chang Gung Mem Hosp, Dept Ophthalmol, Coll Med, Linkou, TaiwanCleveland Clin, Cleveland, OH 44106 USAUniv Fed São Paulo, São Paulo, BrazilNIH: P30 EY07551Web of Scienc
Lumican Peptides: Rational Design Targeting ALK5/TGFBRI
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.NIH/NEI grantsResearch to Prevent BlindnessOhio Eye Research FoundationUniv Cincinnati, Dept Ophthalmol, Cincinnati, OH 45267 USAUniv Fed Sao Paulo, Dept Bioquim, Sao Paulo, BrazilUniv Houston, Coll Optometry, Ocular Surface Inst, Houston, TX 77204 USAUniv Fed Sao Paulo, Dept Bioquim, Sao Paulo, BrazilNIH/NEI grants: RO1 EY011845NIH/NEI grants: R01 021768Web of Scienc
Extracellular matrix deposition and remodeling after corneal alkali burn in mice
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
Novel Peptides with Dual Properties for Treating Pseudomonas aeruginosa Keratitis::Antibacterial and Corneal Wound Healing
The corneal epithelium is a layer in the anterior part of eye that contributes to light refraction onto the retina and to the ocular immune defense. Although an intact corneal epithelium is an excellent barrier against microbial pathogens and injuries, corneal abrasions can lead to devastating eye infections. Among them, Pseudomonas aeruginosa-associated keratitis often results in severe deterioration of the corneal tissue and even blindness. Hence, the discovery of new drugs able not only to eradicate ocular infections, which are often resistant to antibiotics, but also to elicit corneal wound repair is highly demanded. Recently, we demonstrated the potent antipseudomonal activity of two peptides, Esc(1-21) and its diastereomer Esc(1-21)-1c. In this study, by means of a mouse model of P. aeruginosa keratitis and an in vivo corneal debridement wound, we discovered the efficacy of these peptides, particularly Esc(1-21)-1c, to cure keratitis and to promote corneal wound healing. This latter property was also supported by in vitro cell scratch and ELISA assays. Overall, the current study highlights Esc peptides as novel ophthalmic agents for treating corneal infection and injury, being able to display a dual function, antimicrobial and wound healing, rarely identified in a single peptide at the same micromolar concentration range
DNA and bone structure preservation in medieval human skeletons
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 optimized 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 optimized method is suitable for STR typing using kits aimed specifically at degraded and difficult DNA templates since amplicons of up to 250 bp were successfully amplified
The identification of proteoglycans and glycosaminoglycans in archaeological human bones and teeth
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
Anti-inflammatory properties of the glial scar
Univ Cincinnati, Dept Ophthalmol, Cincinnati, OH USAUniv Fed Sao Paulo, Dept Biochem, Sao Paulo, SP, BrazilUniv Houston, Coll Optometry, Houston, TX 77004 USADepartment of Biochemistry, Universidade Federal de São Paulo, São Paulo, SP, BrazilWeb of Scienc
Role of Hyaluronan in Regulating Limbal Stem Cells
Our data indicates that LSCs can be cultured on HA and a subpopulation of CECs can be induced into the LSCs phenotype and used for the reconstitution of corneal epithelium if provided the environment present in the LSC niche.Vision Science
ROS-Mediated Fragmentation Alters the Effects of Hyaluronan on Corneal Epithelial Wound Healing
A buildup of reactive oxygen species (ROS) occurs in virtually all pathological conditions. Hyaluronan (HA) is a major extracellular matrix component and is susceptible to oxidation by reactive oxygen species (ROS), yet the precise chemical structures of oxidized HA products (oxHA) and their physiological properties remain largely unknown. This study characterized the molecular weight (MW), structures, and physiological properties of oxHA. For this, high-molecular-weight HA (HMWHA) was oxidized using increasing molar ratios of hydrogen peroxide (H2O2) or hypochlorous acid (HOCl). ROS lead to the fragmentation of HA, with the oxHA products produced by HOCl exhibiting an altered chemical structure while those produced by H2O2 do not. HMWHA promotes the viability of human corneal epithelial cells (hTCEpi), while low MWHA (LMWHA), ultra-LMWHA (ULMWHA), and most forms of oxHA do not. HMWHA and LMWHA promote hTCEpi proliferation, while ULMWHA and all forms of oxHA do not. LMWHA and some forms of oxHA promote hTCEpi migration, while HMWHA does not. Finally, all native forms of HA and oxHA produced by HOCl promote in vivo corneal wound healing, while oxHA produced by H2O2 does not. Taken together, our results show that HA fragmentation by ROS can alter the physiological activity of HA by altering its MW and structure
Novel fibrinogen hydrogels for cell encapsulation and delivery
Introduction Hydrogels have the potential to deliver cells by injection directly to a site of injury to aid in the regeneration of damaged tissue. We have investigated a novel fibrinogen hydrogel produced using a proprietary fibrinogen polymerisation agent (FPA). When added to fibrinogen, FPA causes the polymerisation of fibrinogen into a multi-branched fibrin-like gel. The porosity of the fibrinogen hydrogels can be modified by changing the FPA concentration during gelation. The aim of this research was to investigate the potential of these novel hydrogels for encapsulation and culture of chondrocytes and mesenchymal stem cells (MSCs).
Materials and Methods Fibrinogen and fibrin hydrogels were formed by mixing solutions of 34 mg/ml fibrinogen with various concentrations of FPA or thrombin. For cell encapsulation, either bovine chondrocytes or MSCs were resuspended in fibrinogen prior to gelation. The cell encapsulated hydrogels were incubated under chondrogenic conditions for up to twenty one days. Cell viability was determined using resazurin dye and extracellular matrix (ECM) formation was assessed using 1,9-dimethylmethylene blue.
Results Chondrocytes and MSCs exhibited good survival and proliferation in all fibrinogen and fibrin hydrogels. Chondrocytes showed deposition of glycosaminoglycan's (GAGs) indicating ECM formation after twenty one days of encapsulation. In contrast, MSCs rapidly degraded and migrated out of all hydrogels after three days. Aprotinin inhibited hydrogel degradation and the deposition of GAGs could be assessed and indicated ECM formation.
Discussion The novel fibrinogen hydrogels supported cell survival and ECM formation for chondrocytes and MSCs. The MSC-induced degradation and their migration from the hydrogels was potentially due to the activation or production of a serine proteinase. This property could allow MSCs to migrate from the hydrogel into the injured tissue, and with the addition of aprotinin to the culture, greater control over the degradation of the hydrogels was provided. The fibrinogen hydrogels were comparable to commercial fibrin hydrogels, but have the additional advantage of modifying the porosity, by altering the concentration of FPAs during gelation thereby optimising the hydrogel for different cell types. In conclusion, this novel fibrinogen hydrogel has strong potential for use in cell encapsulation and delivery for the regeneration of injured tissue