Faculty Bibliography

Inflammation plays a critical role in osteoarthritis (OA). It stimulates catabolic events in articular chondrocytes and prevents chondrogenic precursor cells from repairing cartilage lesions, leading to accelerated cartilage degradation. Therefore, the identification of novel factors that reduce catabolic events in chondrocytes and enhances chondrogenic differentiation of precursor cells in an inflammatory environment may provide novel therapeutic strategies for the treatment of OA. The goal of this study was to determine whether a hyaluronan (HA)-binding peptide (P15-1), via interacting with high molecular weight (HMW)HA can enhance the anti-inflammatory properties of HMWHA and decrease catabolic events in interleukin-1beta (IL-1β)-treated human articular chondrocytes. Treatment with P15-1 decreased catabolic events and stimulated anabolic events in articular chondrocytes cultured in an inflammatory environment. P15-1 pre-mixed with HMWHA was more effective in inhibiting catabolic events and stimulating anabolic events than P15-1 or HMWHA alone. Our findings suggest that P15-1 together with HMWHA inhibits catabolic events in articular chondrocytes via the inhibition of p38 mitogen-activated protein kinases (MAPK) and increasing the thickness of the pericellular matrix (PCM) around chondrocytes thereby decreasing catabolic signaling. Finally, conditioned medium from IL-1β and P15-1-treated human articular chondrocytes was less inhibitory for chondrogenic differentiation of precursor cells than conditioned medium from chondrocytes treated with IL-1β alone. In conclusion, P15-1 is proposed to function synergistically with HMWHA to enhance the protective microenvironment for chondrocytes and mesenchymal stem cells during inflammation and regeneration.

Hyaluronan (HA) fragments have been proposed to elicit defensive or pro-inflammatory responses in many cell types. For articular chondrocytes in an inflammatory environment, studies have failed to reach consensus on the endogenous production or effects of added HA fragments. The present study was undertaken to resolve this discrepancy. Cultured primary human articular chondrocytes were exposed to the inflammatory cytokine IL-1β, and then tested for changes in HA content/size in conditioned medium, and for the expression of genes important in HA binding/signaling or metabolism, and in other catabolic/anabolic responses. Changes in gene expression caused by enzymatic degradation of endogenous HA, or addition of exogenous HA fragments, were examined. IL-1β increased the mRNA levels for HA synthases HAS2/HAS3 and for the HA-binding proteins CD44 and TSG-6. mRNA levels for TLR4 and RHAMM were very low and were little affected by IL-1β. mRNA levels for catabolic markers were increased, while type II collagen (α1(II)) and aggrecan were decreased. HA concentration in the conditioned medium was increased, but the HA was not degraded. Treatment with recombinant hyaluronidase or addition of low endotoxin HA fragments did not elicit pro-inflammatory responses. Our findings showed that HA fragments were not produced by IL-1β-stimulated human articular chondrocytes in the absence of other sources of reactive oxygen or nitrogen species, and that exogenous HA fragments from oligosaccharides up to about 40 kDa in molecular mass were not pro-inflammatory agents for human articular chondrocytes, probably due to low expression of TLR4 and RHAMM in these cells.

The average molecular mass of hyaluronan (HA) in most healthy biological fluids and tissues is usually about 6000-8000 kDa, but the biosynthetic mechanism results in a polydisperse mixture of sizes. Subsequent enzymatic degradation, or the action of reactive oxygen and nitrogen species, can further increase polydispersity and decrease the average size. Fragmented HA can be a biomarker of inflammation. In addition, reductions in HA size are associated with tissue remodeling and repair processes. Some cell-surface receptor proteins have been reported to have HA-binding affinities that are size specific, and participate in activation of signaling cascades controlling multiple aspects of cell behavior. Here we describe simple agarose gel electrophoresis protocols for the determination of the molecular mass distribution of HA isolated from tissues and fluids.

OBJECTIVE:Intrapericardial fibrous adhesions increase the risk of sternal reentry. Proteoglycan 4/lubricin (PRG4) is a mucin-like glycoprotein that lubricates tissue compartments and prevents inflammation. We characterized PRG4 expression in human pericardium and examined its effects in vitro on human cardiac myofibroblast fibrotic activity and in vivo as a measure of its therapeutic potential to prevent adhesions. METHODS:Full-length PRG4 expression was determined using Western blot analysis and amplified luminescent proximity homogeneous assay in human pericardial tissues obtained at cardiotomy. The in vitro effects of PRG4 were investigated on human cardiac myofibroblasts for cell adhesion, collagen gel contraction, and cell-mediated extracellular matrix remodeling. The influence of PRG4 on pericardial homeostasis was determined in a chronic porcine animal model. RESULTS:PRG4 is expressed in human pericardial fluid and colocalized with pericardial mesothelial cells. Recombinant human PRG4 prevented human cardiac myofibroblast attachment and reduced myofibroblast activity assessed using collagen gel contraction assay (64.6% ± 8.1% vs 47.1% ± 6.8%; P = .02). Using a microgel assay, human cardiac myofibroblast mediated collagen fiber remodeling was attenuated by PRG4 (1.17 ± 0.03 vs 0.90 ± 0.05; P = .002). In vivo, removal of pericardial fluid alone induced severe intrapericardial adhesion formation, tissue thickening, and inflammatory fluid collections. Restoration of intrapericardial PRG4 was protective against fibrous adhesions and preserved the pericardial space. CONCLUSIONS:For the first time, we show that PRG4 is expressed in human pericardial fluid and regulates local fibrotic myofibroblast activity. Loss of PRG4-enriched pericardial fluid after cardiotomy might induce adhesion formation. Therapeutic restoration of intrapericardial PRG4 might prevent fibrous/inflammatory adhesions and reduce the risk of sternal reentry.

A method for specific quantification of hyaluronan (HA) concentration using AlphaScreen® (Amplified Luminescent Proximity Homogeneous Assay) technology is described. Two types of hydrogel-coated and chromophore-loaded latex nanobeads are employed. The proximity of the beads in solution is detected by excitation of the donor bead leading to the production of singlet oxygen, and chemiluminescence from the acceptor bead upon exposure to singlet oxygen. In the HA assay, the donor bead is modified with streptavidin, and binds biotin-labeled HA. The acceptor bead is modified with Ni(II), and is used to bind a specific recombinant HA-binding protein (such as HABP; aggrecan G1-IGD-G2) with a His-tag. Competitive inhibition of the HA-HABP interaction by free unlabeled HA in solution is used for quantification. The assay is specific for HA, and not dependent on HA molecular mass above the decasaccharide. HA can be quantified over a concentration range of approximately 30-1600 ng/mL using 2.5 μL of sample, for a detectable mass range of approximately 0.08-4 ng HA. This sensitivity of the AlphaScreen assay is greater than existing ELISA-like methods, due to the small volume requirements. HA can be detected in biological fluids using the AlphaScreen assay, after removal of bound proteins from HA and dilution or removal of other interfering proteins and lipids.

Mammary gland morphogenesis begins during fetal development but expansion of the mammary tree occurs postnatally in response to hormones, growth factors and extracellular matrix. Hyaluronan (HA) is an extracellular matrix polysaccharide that has been shown to modulate growth factor-induced branching in culture. Neither the physiological relevance of HA to mammary gland morphogenesis nor the role that HA receptors play in these responses are currently well understood. We show that HA synthase (HAS2) is expressed in both ductal epithelia and stromal cells but HA primarily accumulates in the stroma. HA accumulation and expression of the HA receptors CD44 and RHAMM are highest during gestation when gland remodeling, lateral branch infilling and lobulo-alveoli formation is active. Molecular weight analyses show that approximately 98% of HA at all stages of morphogenesis is >300kDa. Low levels of 7-114kDa HA fragments are also detected and in particular the accumulation of 7-21kDa HA fragments are significantly higher during gestation than other morphogenetic stages (p<0.05). Using these in vivo results as a guide, in culture analyses of mammary epithelial cell lines (EpH4 and NMuMG) were performed to determine the roles of high molecular weight, 7-21kDa (10kDa MWavg) and HA receptors in EGF-induced branching morphogenesis. Results of these assays show that while HA synthesis is required for branching and 10kDa HA fragments strongly stimulate branching, the activity of HA decreases with increasing molecular weight and 500kDa HA strongly inhibits this morphogenetic process. The response to 10kDa HA requires RHAMM function and genetic deletion of RHAMM transiently blunts lateral branching in vivo. Collectively, these results reveal distinct roles for HA polymer size in modulating growth factor induced mammary gland branching and implicates these polymers in both the expansion and sculpting of the mammary tree during gestation.

Semidilute polymer solutions differ greatly from dilute solutions in properties such as viscosity, relaxation time, elastic modulus, colloid osmotic pressure, and light scattering. Previously, Matsuoka and Cowman proposed a single semiempirical expression for the nonideality contribution due to the concentration and intrinsic viscosity dependence, which has no other adjustable parameters, but quantitatively fits data for flexible, semiflexible, and rigid polymers in good solvents. In this report, the excluded volume theory as proposed by Ogston and Laurent is generalized to include mutual crowding between identical polymers based on hydrodynamic volumes, and applied to derive the expression for the nonideality contribution to specific viscosity, colloid osmotic pressure, and light scattering. Additionally, consideration of the contribution of mutual macromolecular crowding to the effective solvent viscosity allows prediction of polymer relaxation time and elastic modulus in semidilute solutions. This theoretical approach now allows the prediction of semidilute polymer solution properties based only on concentration and intrinsic viscosity, and conversely allows intrinsic viscosity (and thus average molecular weight) to be calculated from measurements made on semidilute solutions of known concentration

Introduction: Damage to the articular cartilage is common, especially through a trauma or injury to the knee joint. Because of the lack of intrinsic capacity to heal, chondral defects remain a major challenge to repair. Current methods used for cartilage regeneration generally result in poorly repaired defects leading to early onset of posttraumatic osteoarthritis (PTOA) and subsequently requiring joint replacement (1). The use of mesenchymal stem cells (MSCs) derived from ones own bone marrow or adipose tissues has been suggested to be used for cartilage repair (2). The transplantation of stem cells for tissue repair requires cell settlement, proliferation and differentiation. The local tissue microenvironment or stem cell niche plays a key role for the successful transplantation of stem cells for tissue repair (3). Very little, however, is known about the stem cell niche required for the successful transplantation of stem cells for cartilage repair. In addition, stem cell settlement and chondrogenesis in cartilage repair has to occur in an unfriendly inflammatory environment in response to injury. In this study, we determined how a novel peptide (NP-0100) that binds to hyaluronan (HA) affects MSC attachment, proliferation and chondrogenic differentiation under normal and inflammatory conditions. Previously we have shown that NP-0100 inhibited catabolic events and stimulated the expression of articular cartilage markers in human articular chondrocytes cultured in an inflammatory environment (4). Therefore, we hypothesized that NP-0100 together with high molecular HA (HMWHA) will enhance cartilage repair by optimizing the stem cell niche for precursor cells to repair cartilage and reduce inflammation. Methods: Chondrogenesis of the multipotential murine C3H/10T1/2 cell line was induced in micromass cultures in the presence of BMP-2 (100ng/ml). In addition, the micromass cultures were treated with NP-0100 or cultured in conditioned media from untreated and IL-1beta-treated articular chondrocytes in the absence or presence of NP-0100. Chondrogenesis was determined by alcian blue staining and real time PCR analysis of chondrocyte marker genes. Cell attachment and proliferation was assessed on tissue culture plates or tissue culture plates with immobilized high molecular HA (HMWHA) or HMWHA together with NP- 0100. Cell attachment was determined by DAPI staining. Cell proliferation was determined using the CCK-8 kit. Results: C3H10T1/2 cells better attached to HMWHA-coated tissue culture plates than to uncoated tissue culture plates. The largest number of cells, however, attached to tissue culture plates that were coated with both HMWHA and NP-0100. In addition, cells showed the highest proliferation rate on HMWHA/NP- 0100-coated plates followed by HMWHA-coated plates. The lowest proliferation rate was detected on uncoated tissue culture plates. Furthermore, NP-0100 stimulated the expression of articular cartilage markers (aggrecan and type II collagen) and Sox-9, a master transcription factor that regulates chondrogenesis. NP-0100-treated C3H/10T1/2 micromass cultures also stained more intensely with Alcian blue, which is indicative of increased levels of sulfated proteoglycans than micromass cultures not treated with NP-0100. Chondrogenesis was markedly inhibited when the micromass cultures were cultured in the presence of conditioned media from IL-1beta-treated human articular chondrocytes compared to conditioned media from untreated human articular chondrocytes. However, conditioned media from human articular chondrocytes treated with IL-1beta in combination with NP-0100 showed a reduced inhibition of chondrogenesis compared to conditioned media from IL-1beta-treated human articular chondrocytes. Discussion: Our findings show that a novel peptide NP-0100 in the presence of HMWHA stimulated attachment, proliferation and chondrogenic differentiation of precursor cells, and this suggests a potential therapeutic role for NP-0100 in promoting cartilage repair. The improved attachment and proliferation of precursor cells on tissue culture plates that were coated with HMWHA and NP-0100 rather than HMWHA alone suggests that NP-0100 stabilizes or crosslinks HMWHA to create a more favorable microenvironment (stem cell niche) for the precursor cells to adhere and proliferate. This notion is supported by a previous study showing that a cross-linked HMWHA is required for the formation of the stem cell niche for precursor cells to repair muscle after injury (5). NP-0100 not only supported the formation of the stem cell niche but also stimulated chondrogenesis of C3H10T1/2 cells in high-density micromass culture. Furthermore, the peptide was able to protect the micromass cultures from an inflammatory environment that otherwise inhibited chondrogenesis. Future studies have to determine the mechanisms by which NP-0100 together with HMWHA stimulates chondrogenesis even in an inflammatory environment

The glycosaminoglycan hyaluronan (HA) is a key component of the microenvironment surrounding cells. In healthy tissues, HA molecules have extremely high molecular mass and consequently large hydrodynamic volumes. Tethered to the cell surface by clustered receptor proteins, HA molecules crowd each other, as well as other macromolecular species. This leads to severe nonideality in physical properties of the biomatrix, because steric exclusion leads to an increase in effective concentration of the macromolecules. The excluded volume depends on both polymer concentration and hydrodynamic volume/molecular mass. The biomechanical properties of the extracellular matrix, tissue hydration, receptor clustering, and receptor-ligand interactions are strongly affected by the presence of HA and by its molecular mass. In inflammation, reactive oxygen and nitrogen species fragment the HA chains. Depending on the rate of chain degradation relative to the rates of new synthesis and removal of damaged chains, short fragments of the HA molecules can be present at significant levels. Not only are the physical properties of the extracellular matrix affected, but the HA fragments decluster their primary receptors and act as endogenous danger signals. Bioanalytical methods to isolate and quantify HA fragments have been developed to determine profiles of HA content and size in healthy and diseased biological fluids and tissues. These methods have potential use in medical diagnostic tests. Therapeutic agents that modulate signaling by HA fragments show promise in wound healing and tissue repair without fibrosis.