Micrometer scale guidance of mesenchymal stem cells to form structurally oriented large-scale tissue engineered cartilage.

Current clinical methods to treat articular cartilage lesions provide temporary relief of the symptoms but fail to permanently restore the damaged tissue. Tissue engineering, using mesenchymal stem cells (MSCs) combined with scaffolds and bioactive factors, is viewed as a promising method for repairing cartilage injuries. However, current tissue engineered constructs display inferior mechanical properties compared to native articular cartilage, which could be attributed to the lack of structural organization of the extracellular matrix (ECM) of these engineered constructs in comparison to the highly oriented structure of articular cartilage ECM. We previously showed that we can guide MSCs undergoing chondrogenesis to align using microscale guidance channels on the surface of a two-dimensional (2-D) collagen scaffold, which resulted in the deposition of aligned ECM within the channels and enhanced mechanical properties of the constructs. In this study, we developed a technique to roll 2-D collagen scaffolds containing MSCs within guidance channels in order to produce a large-scale, three-dimensional (3-D) tissue engineered cartilage constructs with enhanced mechanical properties compared to current constructs. After rolling the MSC-scaffold constructs into a 3-D cylindrical structure, the constructs were cultured for 21days under chondrogenic culture conditions. The microstructure architecture and mechanical properties of the constructs were evaluated using imaging and compressive testing. Histology and immunohistochemistry of the constructs showed extensive glycosaminoglycan (GAG) and collagen type II deposition. Second harmonic generation imaging and Picrosirius red staining indicated alignment of neo-collagen fibers within the guidance channels of the constructs. Mechanical testing indicated that constructs containing the guidance channels displayed enhanced compressive properties compared to control constructs without these channels. In conclusion, using a novel roll-up method, we have developed large scale MSC based tissue-engineered cartilage that shows microscale structural organization and enhanced compressive properties compared to current tissue engineered constructs. STATEMENT OF SIGNIFICANCE: Tissue engineered cartilage constructs made with human mesenchymal stem cells (hMSCs), scaffolds and bioactive factors are a promising solution to treat cartilage defects. A major disadvantage of these constructs is their inferior mechanical properties compared to the native tissue, which is likely due to the lack of structural organization of the extracellular matrix of the engineered constructs. In this study, we developed three-dimensional (3-D) cartilage constructs from rectangular scaffold sheets containing hMSCs in micro-guidance channels and characterized their mechanical properties and metabolic requirements. The work led to a novel roll-up method to embed 2-D microscale structures in 3-D constructs. Further, micro-guidance channels incorporated within the 3-D cartilage constructs led to the production of aligned cell-produced matrix and enhanced mechanical function.

Micrometer scale guidance of mesenchymal stem cells to form structurally oriented cartilage extracellular matrix.

Tissue engineering is a possible method for long-term repair of cartilage lesions, but current tissue-engineered cartilage constructs have inferior mechanical properties compared to native cartilage. This problem may be due to the lack of an oriented structure in the constructs at the microscale that is present in the native tissue. In this study, we utilize contact guidance to develop constructs with microscale architecture for improved chondrogenesis and function. Stable channels of varying microscale dimensions were formed in collagen-based and polydimethylsiloxane membranes via a combination of microfabrication and soft-lithography. Human mesenchymal stem cells (MSCs) were selectively seeded in these channels. The chondrogenic potential of MSCs seeded in these channels was investigated by culturing them for 3 weeks under differentiating conditions, and then evaluating the subsequent synthesized tissue for mechanical function and by type II collagen immunohistochemistry. We demonstrate selective seeding of viable MSCs within the channels. MSC aligned and produced mature collagen fibrils along the length of the channel in smaller linear channels of widths 25-100 µm compared to larger linear channels of widths 500-1000 µm. Further, substrates with microchannels that led to cell alignment also led to superior mechanical properties compared to constructs with randomly seeded cells or selectively seeded cells in larger channels. The ultimate stress and modulus of elasticity of constructs with cells seeded in smaller channels increased by as much as fourfolds. We conclude that microscale guidance is useful to produce oriented cartilage structures with improved mechanical properties. These findings can be used to fabricate large clinically useful MSC-cartilage constructs with superior mechanical properties.

How HLA-DM works: recognition of MHC II conformational heterogeneity.

Helper T cells respond to peptide antigens derived from exogenous sources presented by MHC II on antigen presenting cells. Antigens from pathogens are internalized by professional antigen presenting cells (APC) and processed for presentation. Certain epitopes are selected during processing as the final peptides for stimulation of T cells and are termed "immunodominant". Understanding how selection of immunodominant epitopes takes place has been a difficult task because of the complexity of the mechanisms governing both antigen processing and T cell recognition. In this review, we discuss our current understanding of HLA-DM function in peptide exchange and selection and its relevance to epitope immunodominance.

Induction of angiogenic immediate early genes by activation of FP prostanoid receptors in cultured human ciliary smooth muscle cells.

PURPOSE: Agonists of the F prostanoid receptor for prostaglandin F2alpha exert. exert an ocular hypotensive effect that has been attributed to increased aqueous humor outflow through the uveoscleral pathway. Although tissue remodeling of the ciliary muscle has been described, the signaling mechanisms that link activation of the FP receptor to remodeling of the ciliary muscle are poorly understood. Herein, we describe the identification of novel signaling mechanisms that may contribute to this process. MATERIALS AND METHODS: Cultures of human ciliary smooth muscle cells were established from fetal eye tissue explants. The cells were validated by their expression of alpha-smooth muscle-actin and functional FP receptors. Cultures were treated with prostaglandin F(2 alpha) and examined for the induction of three immediate early genes related to tissue remodeling using Western blot analysis, quantitative real-time polymerase chain reaction, and reporter gene assays. RESULTS: Human ciliary smooth muscle cells express functional FP receptors whose activation up-regulates the expression of early growth response factor-1 and connective tissue growth factor at the mRNA and protein levels. Prostaglandin F(2 alpha) stimulation also increases the protein expression of hypoxia-inducible factor-1 alpha and activates luciferase reporter plasmids under the control of the hypoxia response element. CONCLUSIONS: Early growth response factor-1 and hypoxia-inducible factor-1 alpha are important transcriptional activators of downstream genes involved in tissue remodeling and angiogenesis, whereas connective tissue growth factor is a secreted growth factor that also contributes to these processes. Thus, stimulation of FP receptors in human ciliary smooth muscle cells up-regulates the expression of immediate early genes that may coordinate the remodeling of the ciliary muscle, thereby facilitating aqueous outflow.

EP1 prostanoid receptor coupling to G i/o up-regulates the expression of hypoxia-inducible factor-1 alpha through activation of a phosphoinositide-3 kinase signaling pathway.

The EP1 prostanoid receptor is one of four subtypes whose cognate physiological ligand is prostaglandin-E2 (PGE(2)). It is in the family of G-protein-coupled receptors and is known to activate Ca(2+) signaling, although relatively little is known about other aspects of E-type prostanoid receptor (EP) 1 receptor signaling. In human embryonic kidney (HEK) cells expressing human EP1 receptors, we now show that PGE(2) stimulation of the EP1 receptor up-regulates the expression of hypoxia-inducible factor-1 alpha (HIF-1 alpha), which can be completely blocked by pertussis toxin, indicating coupling to G(i/o). This up-regulation of HIF-1 alpha occurs under normoxic conditions and could be inhibited with wortmannin, Akt inhibitor, and rapamycin, consistent with the activation of a phosphoinositide-3 kinase/Akt/mammalian target of rapamycin (mTOR) signaling pathway, respectively. In contrast to the hypoxia-induced up-regulation of HIF-1 alpha, which involves decreased protein degradation, the up-regulation of HIF-1 alpha by the EP1 receptor was associated with the phosphorylation of ribosomal protein S6 (rpS6), suggesting activation of the ribosomal S6 kinases and increased translation. Stimulation of endogenous EP1 receptors in human HepG2 hepatocellular carcinoma cells recapitulated the normoxic up-regulation of HIF-1 alpha observed in HEK cells, was sensitive to pertussis toxin, and involved the activation of mTOR signaling and phosphorylation of rpS6. In addition, treatment of HepG2 cells with sulprostone, an EP1-selective agonist, up-regulated the mRNA expression of vascular endothelial growth factor-C, a HIF-regulated gene. HIF-1 alpha is known to promote tumor growth and metastasis and is often up-regulated in cancer. Our findings provide a potential mechanism by which increased PGE(2) biosynthesis could up-regulate the expression of HIF-1 alpha and promote tumorigenesis.

Conformational heterogeneity of MHC class II induced upon binding to different peptides is a key regulator in antigen presentation and epitope selection.

T cells bearing alphabeta receptors recognize antigenic peptides bound to class I and class II glycoproteins encoded in the major histocompatibility complex (MHC). Cytotoxic and helper T cells respond respectively to peptide antigens derived from endogenous sources presented by MHC class I, and exogenous sources presented by MHC II, on antigen presenting cells. Differences in the MHC class I and class II structures and their maturation pathways have evolved to optimize antigen presentation to their respective T cells. A main focus of our laboratory is on efforts to understand molecular events in processing of antigen for presentation by MHC class II. The different stages of MHC class II-interactions with molecular chaperons involved in folding and traffic from the ER through the antigen-loading compartments, peptide exchange, and transport to the cell surface have been investigated. Through intense research on biophysical and biochemical properties of MHC class II molecules, we have learned that the conformational heterogeneity of MHC class II induced upon binding to different peptides is a key regulator in antigen presentation and epitope selection, and a determinant of the ability of MHC class II to participate in peptide association or dissociation and interaction with the peptide editor HLA-DM.

HLA-DM mediates peptide exchange by interacting transiently and repeatedly with HLA-DR1.

The peptide editor HLA-DM (DM) catalyzes the exchange of peptides bound to MHC class II molecules within antigen presenting cells by generating a "peptide-receptive" MHC class II conformation (MHC(receptive)) to which peptides readily bind and rapidly unbind. While recent work has uncovered the determinants of DM recognition and effector functions, the nature of MHC(receptive) and its interaction with DM remains unclear. Here, we show that DM induces but does not stabilize MHC(receptive) in the absence of peptides. We demonstrate that DM is out-competed by certain superantigens, and increasing solvent viscosity inhibits DM-induced peptide association. We suggest that DM mediates peptide exchange by interacting transiently and repeatedly with MHC class II molecules, continually generating MHC(receptive). The simultaneous presence of peptide and DM in the milieu is thus crucial for the efficient generation of specific peptide-MHC class II complexes over time.

Activation of EP2 prostanoid receptors in human glial cell lines stimulates the secretion of BDNF.

Prostaglandin E(2) (PGE(2)) is produced at high levels in the injured central nervous system, where it is generally considered a cytotoxic mediator of inflammation. The cellular actions of PGE(2) are mediated by G-protein signaling activated by prostanoid receptors termed EP(1), EP(2), EP(3) and EP(4). Recent studies have implicated the EP(2) prostanoid receptor to be in apparently conflicting roles promoting neuronal death in some model systems and the survival of neurons in others. Here we show that treatment of immortalized human microglia and CCF-STTG1 astrocytes with either PGE(2) or the EP(2) selective agonist butaprost stimulates the release of brain-derived neurotrophic factor (BDNF). Both cell lines express mRNA for the EP(2) receptor, whereas transcripts for the other subtypes are not detected. Pharmacological studies using PGE(2) and modulators of cyclic AMP signaling implicate this pathway in PGE(2)-stimulated BDNF release. These results indicate that EP(2) prostanoid receptor activation induces BDNF secretion through stimulation of cyclic AMP dependent signaling. Our findings provide a mechanism by which endogenous PGE(2) might contribute to either neurotoxicity or neuroprotection in the injured brain via the induction of BDNF release from microglial cells and astrocytes.

PGF(2alpha) stimulates FP prostanoid receptor mediated crosstalk between Ras/Raf signaling and Tcf transcriptional activation.

Prostaglandin-F(2alpha) (PGF(2alpha)) is a product of the cyclooxygenase pathway and is a local signaling molecule that activates a G-protein coupled prostanoid receptor named FP. FP receptors can stimulate T-cell factor (Tcf) transcriptional activation by stabilization of beta-catenin and can upregulate the expression of mRNA encoding cysteine-rich protein 61 (Cyr61), a secreted extracellular matrix protein that stimulates angiogenesis. We now show in both HEK cells and human microglial cells that the induction of Cyr61 protein expression by the human FP receptor utilizes a novel mechanism involving the activation of Ras and Raf followed by a MEK/ERK independent activation of Tcf signaling. The upregulation of Cyr61 in microglial cells may contribute to glioma tumorigenesis and could be a potential therapeutic target.

FP prostanoid receptor-mediated induction of the expression of early growth response factor-1 by activation of a Ras/Raf/mitogen-activated protein kinase signaling cascade.

FP prostanoid receptors are G-protein-coupled receptors whose physiological activator is prostaglandin-F(2alpha) (PGF(2alpha)). PGF(2alpha) has been implicated in wound healing and cardiac hypertrophy, which are both known to involve the induction of the immediate-early response gene, early growth response factor-1 (EGR-1). We hypothesized that activation of the human FP receptor by PGF(2alpha) could induce the expression of EGR-1 and found that 1 muM PGF(2alpha) produced a time-dependent induction of both mRNA and protein expression for EGR-1. This FP receptor-mediated induction of EGR-1 expression involved activation of the small GTPase Ras followed by activation of C-Raf and the mitogen-activated protein (MAP) kinase kinases 1 and 2 (MEK1/2). Thus, induction of EGR-1 expression by PGF(2alpha) was blocked using dominant-negative constructs of Ras and C-Raf and the Raf kinase inhibitor 4-(4-(3-(4-chloro-3-trifluoromethylphenyl)ureido)phenoxy)-pyridine-2-carboxyllic acid methyamide-4-methylbenzenesulfonate (BAY43-9006). Likewise, the MEK1/2 inhibitor 2'-amino-3'-methoxyflavone (PD98059) blocked the induction of EGR-1 expression by PGF(2alpha). FP receptor stimulation by PGF(2alpha) induced the phosphorylation of C-Raf, MEK1/2, and extracellular signal-regulated kinases 1 and 2, consistent with the activation of a MAP kinase signaling cascade. PGF(2alpha) was also found to induce the expression of EGR-1 in rat cardiomyocytes through the activation of endogenous FP receptors. This induction of EGR-1 expression in cardiomyocytes also involved the activation of Raf and MAP kinase signaling and was dependent on the activation of protein kinase C. This is the first report to show the regulation of EGR-1 expression after PGF(2alpha) activation of FP receptors and suggests that this could be an early event involved in wound healing and cardiac hypertrophy.

Short peptide sequences mimic HLA-DM functions.

HLA-DM (DM) plays a critical role in Ag presentation to CD4 T cells by catalyzing the exchange of peptides bound to MHC class II molecules. It is known that DM interaction with MHC II involves conformational changes in the MHC II molecule leading to the disturbance of H-bonds formed between the bound peptide and the MHC II groove leading to peptide dissociation. The specific region of the DM molecule that induces this peptide dissociation is not defined. In this study, we describe three short peptides (helper peptides) that accelerate DM-catalyzed peptide exchange. Kinetic studies presented here demonstrate that these peptides act similarly to DM in; (a) enhancing peptide binding to HLA-DR1; (b) dissociation of complexes of peptide-DR1; and (c) maintaining a receptive conformation of empty DR1. We further report that helper peptides are effective in increasing peptide binding to DR1 expressed on B cells in vitro, and, when mixed with peptide and adjuvant, cause enhanced T cell priming in HLA-DR1 Tg mice. We suggest that helper peptides might interact with the same critical residues on MHC class II that is targeted by DM.

HLA-DM targets the hydrogen bond between the histidine at position beta81 and peptide to dissociate HLA-DR-peptide complexes.

The peptide editor HLA-DM (DM) mediates exchange of peptides bound to major histocompatibility (MHC) class II molecules during antigen processing; however, the mechanism by which DM displaces peptides remains unclear. Here we generated a soluble mutant HLA-DR1 with a histidine-to-asparagine substitution at position 81 of the beta-chain (DR1betaH81N) to perturb an important hydrogen bond between MHC class II and peptide. Peptide-DR1betaH81N complexes dissociated at rates similar to the dissociation rates of DM-induced peptide-wild-type DR1, and DM did not enhance the dissociation of peptide-DR1betaH81N complexes. Reintroduction of an appropriate hydrogen bond (DR1betaH81N betaV85H) restored DM-mediated peptide dissociation. Thus, DR1betaH81N might represent a 'post-DM effect' conformation. We suggest that DM may mediate peptide dissociation by a 'hit-and-run' mechanism that results in conformational changes in MHC class II molecules and disruption of hydrogen bonds between betaHis81 and bound peptide.

Synaptic PDZ domain-mediated protein interactions are disrupted by inhalational anesthetics.

Anesthetics exert multiple effects on the central nervous system through altering synaptic transmission, but the mechanisms for this process are poorly understood. PDZ domain-mediated protein interactions play a central role in organizing signaling complexes around synaptic receptors for efficient signal transduction. We report here that clinically relevant concentrations of inhalational anesthetics dose-dependently and specifically inhibit the PDZ domain-mediated protein interaction between PSD-95 or PSD-93 and the N-methyl-d-aspartate receptor or neuronal nitric-oxide synthase. These inhibitory effects are immediate, potent, and reversible and occur at a hydrophobic peptide-binding groove on the surface of the second PDZ domain of PSD-95 in a manner relevant to anesthetic action. These findings reveal the PDZ domain as a new molecular target for inhalational anesthetics.