Clare E. Hughes

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.

Chondroitin sulfate (CS) is an important component of the extracellular matrix in multiple biological tissues. In cornea, the CS glycosaminoglycan (GAG) exists in hybrid form, whereby some of the repeating disaccharides are dermatan sulfate (DS). These CS/DS GAGs in cornea, through their presence on the proteoglycans, decorin and biglycan, help control collagen fibrillogenesis and organization. CS also acts as a regulatory ligand for a spectrum of signaling molecules, including morphogens, cytokines, chemokines, and enzymes during corneal growth and development. There is a growing body of evidence that precise expression of CS or CS/DS with specific sulfation motifs helps define the local extracellular compartment that contributes to maintenance of the stem cell phenotype. Indeed, recent evidence shows that CS sulfation motifs recognized by antibodies 4C3, 7D4, and 3B3 identify stem cell populations and their niches, along with activated progenitor cells and transitional areas of tissue development in the fetal human elbow. Various sulfation motifs identified by some CS antibodies are also specifically located in the limbal region at the edge of the mature cornea, which is widely accepted to represent the corneal epithelial stem cell niche. Emerging data also implicate developmental changes in the distribution of CS during corneal morphogenesis. This article will reflect upon the potential roles of CS and CS/DS in maintenance of the stem cell niche in cornea, and will contemplate the possible involvement of CS in the generation of eye-like tissues from human iPS (induced pluripotent stem) cells.

The umbilical artery lumen closes rapidly at birth, preventing neonatal blood loss, whereas the umbilical vein remains patent longer. Here, analysis of umbilical cords from humans and other mammals identified differential arterial-venous proteoglycan dynamics as a determinant of these contrasting vascular responses. The umbilical artery, but not the vein, has an inner layer enriched in the hydrated proteoglycan aggrecan, external to which lie contraction-primed smooth muscle cells (SMC). At birth, SMC contraction drives inner layer buckling and centripetal displacement to occlude the arterial lumen, a mechanism revealed by biomechanical observations and confirmed by computational analyses. This vascular dimorphism arises from spatially regulated proteoglycan expression and breakdown. Mice lacking aggrecan or the metalloprotease ADAMTS1, which degrades proteoglycans, demonstrate their opposing roles in umbilical vascular dimorphism, including effects on SMC differentiation. Umbilical vessel dimorphism is conserved in mammals, suggesting that differential proteoglycan dynamics and inner layer buckling were positively selected during evolution.

The umbilical artery lumen occludes rapidly at birth, preventing blood loss, whereas the umbilical vein remains patent, providing the newborn with a placental infusion. Here, we identify differential arterial-venous proteoglycan dynamics as a determinant of these contrasting vascular responses. We show that the umbilical artery, unlike the vein, has an inner layer enriched in the hydrated proteoglycan aggrecan, external to which lie contraction-primed smooth muscle cells (SMC). At birth, SMC contraction drives inner layer buckling and centripetal displacement to occlude the arterial lumen, a mechanism elicited by biomechanical and computational analysis. Vascular dimorphism arises from spatially regulated proteoglycan expression and breakdown in umbilical vessels. Mice lacking aggrecan or the metalloprotease ADAMTS1, which degrades proteoglycans, demonstrated their opposing roles in umbilical cord arterial-venous dimorphism and contrasting effects on SMC differentiation. Umbilical vessel dimorphism is conserved in mammals, suggesting that their differential proteoglycan dynamics were a positive selection step in mammalian evolution.

The ability of a polymer therapeutic to access the appropriate subcellular location is crucial to its efficacy and is defined to a large part by the many and complex cellular biological and biochemical barriers such that a construct must traverse. It is shown here that model dextrin conjugates are able to pass through a cartilaginous extracellular matrix into chondrocytes, with little perturbation of the matrix structure, indicating that targeting of potential therapeutics through a cartilaginous extracellular matrix should be proven possible. Rapid chondrocytic targeting of drugs which require intra cellularisation for their activity and uniform extracellular concentrations of drugs with an extracellular target, is thus enabled though polymer conjugation.

We compared the immunohistochemical distribution of (1) the novel chondroitin sulfate (CS) sulfation motifs 7D4, 4C3, and 3B3[−], (2) native heparan sulfate (HS) and Δ-HS “stubs” generated by heparitinase III digestion and (3) the HS-proteoglycan (PG), perlecan, in the fetal human elbow joint. Putative stem cell populations associated with hair bulbs, humeral perichondrium, humeral and ulnar rudiment stromal/perivascular tissues expressed the CS motifs 4C3, 7D4, and 3B3[−] along with perlecan in close association but not colocalized. Chondrocytes in the presumptive articular cartilage of the fetal elbow expressed the 4C3 and 7D4 CS sulfation motifs consistent with earlier studies on the expression of these motifs in knee cartilage following joint cavitation. This study also indicated that hair bulbs, skin, perichondrium, and rudiment stroma were all perlecan-rich progenitor cell niches that contributed to the organization and development of the human fetal elbow joint and associated connective tissues. One of the difficulties in determining the precise role of stem cells in tissue development and repair processes is their short engraftment period and the lack of specific markers, which differentiate the activated stem cell lineages from the resident cells. The CS sulfation motifs 7D4, 4C3, and 3B3[−] decorate cell surface PGs on activated stem/progenitor cells and thus can be used to identify these cells in transitional areas of tissue development and in repair tissues and may be applicable to determining a more precise mode of action of stem cells in these processes. Isolation of perlecan from 12 to 14 week gestational age fetal knee rudiments demonstrated that perlecan in these fetal tissues was a HS-CS hybrid PG further supporting roles for CS in tissue development.

Objective: Glucosamine hydrochloride (GH) and chondroitin sulfate (CS) are commonly used for the treatment of osteoarthritis (OA). The aim of this study was to assess their effects, alone and in combination, on preventing aggrecan degradation and inflammation in an in vitro model of OA. Design: To test the effects of GH and/or CS as a preventative treatment, cartilage explants were pretreated with the compound(s) using concentrations that showed no detrimental effect on chondrocyte viability. Interleukin-1α (IL-1α) was added to induce cartilage degradation, supernatant and explants were analyzed for proteoglycan degradation products, aggrecanase mRNA expression and activity, and for the release of inflammatory markers. Results: Following treatment with IL-1α, 2 mg/mL dose of GH pretreatment was associated with a reduction of glycosaminoglycan release, reduced generation of the pathological interglobular domain aggrecan catabolites, decreased mRNA levels of ADAMTS-4 and -5 and reduced activity of ADAMTS-4. In contrast, CS alone did not have a significant effect on IL-1α-induced cartilage degradation and the addition of 0.4 mg/mL CS to 2 mg/mL GH did not further inhibit IL-1α-induced activity. Pretreatment with 2 mg/mL GH also reduced the release of inflammatory markers, prostaglandin E₂ and nitric oxide induced by IL-1α while CS did not have a significant effect. Conclusions: The results suggest that GH prevents cartilage degradation mediated by aggrecanases ADAMTS-4 and -5, and may also reduce inflammation. This could be part of the mechanisms by which GH is effective in maintaining joint integrity and function, and preventing or delaying early symptoms of OA.

Kashin–Beck disease (KBD) is a chronic, deforming endemic osteoarticular disease with altered metabolism of the cartilage matrix. Matrix metalloproteinases (MMPs), aggrecanases (ATAMTSs), and their inhibitors (TIMPs) play important roles in cartilage formation and matrix degradation. This study investigated these proteases and inhibitors in young KBD cartilage. The percentages of chondrocytes staining for MMP‐1/‐13 and MMP‐generated DIPEN neoepitope, aggrecanase‐generated ITEGE neoepitope in aggrecan in KBD patients were significantly higher than in controls. However, TIMP‐1 was significantly less numerous than in controls in the superficial and middle zones of KBD samples, the percentage of chondrocytes staining for the TIMP‐2 was significantly higher than in controls. Staining for MMP‐1/‐13 and, TIMP‐1/‐2 in KBD patients was prominent in the superficial zone and the middle zone of articular cartilage. Staining for ITEGE and DIPEN neoepitopes in KBD samples was prominent in the superficial zone and the middle zone of articular cartilage. The strongest staining for the MMP and aggrecanase‐generated neoepitopes was adjacent to areas of chondronecrosis. These results indicated that KBD cartilage destruction depends on collagen‐ and aggrecan‐degrading proteases such as collagenases (MMP‐1/‐13), as well as aggrecanases. Increased TIMP‐2 level adjacent to necrotic areas suggest that attempted repair mechanism are also activated. © 2014 Orthopaedic Research Society. Published by Wiley Periodicals, Inc. J Orthop Res 33:47–55, 2015.

Objective: We previously described a messenger RNA variant of ADAMTS4 (ADAMTS4_v1) in human synovial cell cocultures obtained from patients with osteoarthritis (OA). This RNA message has been found only in OA synovium and, if translated, would result in a protein identical to ADAMTS‐4, except that the C‐terminal spacer domain would be different. The purpose of this study was to determine whether ADAMTS4_v1 is translated into a protein, is expressed in vivo, and acts as a functional aggrecanase.Methods: Polyclonal antibodies were raised against unique C‐terminal sequences of ADAMTS‐4_v1. An immunohistochemical study of human OA synovium was performed. A mammalian expression vector coding for FLAG‐tagged human ADAMTS4 was mutated to contain the different sequences of ADAMTS4_v1, and the resultant plasmid was used to transfect HEK 293 cells. ADAMTS‐4_v1 produced by these cells was purified via the FLAG epitope, and the ability of this recombinant protein to cleave aggrecan, biglycan, and decorin was investigated.Results: An antibody specific for ADAMTS‐4_v1 was found to bind to the synovial membrane surface on cryosections, and the protein was detected in cell lysates from synovium obtained from OA patients. The recombinant ADAMTS‐4_v1 demonstrated enzyme activity toward the target substrate in a commercial aggrecanase 1 enzyme‐linked immunosorbent assay and was also found to cleave aggrecan at the pathologically important Glu^373↓374Ala aggrecanase site.Conclusion: ADAMTS‐4_v1 is expressed as a protein in vivo in human OA synovium, functions as an aggrecanase, and cleaves other proteoglycan substrates. This splice variant may be a major contributor to loss of aggrecan from the superficial zone of OA cartilage.