ABSTRACT
This study was designed to evaluate the additive effects of transforming growth factor-beta3 (TGF-ß3) and hyaluronic acid (HA) on chondrogenic differentiation of human mesenchymal stem cells (hMSCs). The hMSCs were cultured on collagen type I-, HA-, or fibronectin-coated cell culture dishes with or without TGF-ß3 added to the culture medium. Four weeks after cell culture, chondrogenic differentiation of hMSCs was determined by evaluating the expression of cartilage-specific markers using real-time polymerase chain reaction, immunocytochemistry, and Western blot analysis. hMSCs cultured on HA-coated dishes with TGF-ß3 supplementation revealed a prominent increase in collagen type II, aggrecan, and Sox9. When hMSCs were cultured without TGF-ß3 supplementation, only hMSCs cultured on HA-coated dishes showed prominent expression of the cartilage-specific markers. This study shows that chondrogenic differentiation of hMSCs can be enhanced additively by interactions with both a specific cell-adhesion matrix and a soluble growth factor.
Subject(s)
Chondrogenesis , Hyaluronic Acid/pharmacology , Mesenchymal Stem Cells/drug effects , Mesenchymal Stem Cells/metabolism , Tissue Engineering/methods , Transforming Growth Factor beta3/pharmacology , Aggrecans/metabolism , Animals , Biomarkers/metabolism , Cartilage/metabolism , Cells, Cultured , Collagen Type II/metabolism , Extracellular Matrix/metabolism , Gene Expression , Humans , Mesenchymal Stem Cells/cytology , Rats , Rats, Sprague-Dawley , SOX9 Transcription Factor/metabolismABSTRACT
Mitogen-activated protein kinase phosphatase-1 (MKP-1) has proved to be an attractive target for the development of therapeutics for the treatment of cancer. We report the first example for a successful application of the structure-based virtual screening to identify the novel inhibitors of MKP-1. It is shown that the efficiency of virtual screening can be enhanced significantly by the incorporation of a new solvation energy term in the scoring function. The newly found inhibitors have desirable physicochemical properties as a drug candidate and reveal a moderate potency with IC(50) values ranging from 20 to 50 µM. Therefore, they deserve a consideration for further development by structure-activity relationship studies to optimize the inhibitory activities. Structural features relevant to the stabilization of the inhibitors in the active site of MKP-1 are discussed in detail.
Subject(s)
Drug Evaluation, Preclinical , Dual Specificity Phosphatase 1/antagonists & inhibitors , Dual Specificity Phosphatase 1/chemistry , Mitogen-Activated Protein Kinase 1/chemistry , Models, Molecular , Binding Sites , Catalytic Domain , Drug Design , Enzyme Assays , Enzyme Inhibitors/chemistry , Inhibitory Concentration 50 , Mitogen-Activated Protein Kinase 1/antagonists & inhibitors , Molecular Structure , Structure-Activity RelationshipABSTRACT
The L1CAM antibody A10-A3 efficiently reduces tumor growth in a nude mouse model. Here, we describe the crystal structure of the Fab fragment of A10-A3 determined at 2.0 angstrom resolution. The A10-A3 antibody H3 loop reveals a characteristic arrangement of exposed aromatic residues that may play an important role in antigen binding. A structure model of the complex between L1CAM Ig1-4 and A10-A3 Fab indicates that the Fab binds to three small loops outside Ig1 and a residue between Ig1 and Ig2, consistent with an epitope mapping result. The data presented here should contribute to the design of high-affinity antibody for therapeutic purposes as well as to the understanding of neural cell remodeling and cancer progression mechanism mediated by L1CAM.
Subject(s)
Antibodies/immunology , Antigen-Antibody Complex/immunology , Antigens/immunology , Neural Cell Adhesion Molecule L1/immunology , Amino Acid Sequence , Animals , Antibodies/chemistry , Antibodies/metabolism , Antigen-Antibody Complex/chemistry , Antigen-Antibody Complex/metabolism , Antigens/chemistry , Antigens/metabolism , Binding Sites , Crystallization , Epitope Mapping , Epitopes/chemistry , Epitopes/immunology , Epitopes/metabolism , HEK293 Cells , Humans , Immunoglobulin Fab Fragments/chemistry , Immunoglobulin Fab Fragments/immunology , Immunoglobulin Fab Fragments/metabolism , Mice , Models, Molecular , Neural Cell Adhesion Molecule L1/chemistry , Neural Cell Adhesion Molecule L1/metabolism , Protein Binding , Protein Conformation , Protein Structure, Tertiary , X-Ray DiffractionABSTRACT
Long-term release of bone morphogenetic protein-2 (BMP-2) can promote bone regeneration. We developed an injectable system for long-term delivery of BMP-2 by covalently conjugating heparin to fibrinogen. The heparin-conjugated fibrinogen formed an injectable, heparin-conjugated fibrin (HCF) gel when mixed with thrombin. HCF released 89.4 +/- 3.8% of the loaded BMP-2 for 13 days, whereas normal fibrin released 83.7 +/- 7.6% for the initial 3 days. BMP-2 released from HCF significantly increased alkaline phosphatase activity of cultured osteoblasts, whereas BMP-2 released from normal fibrin did not do so, indicating that BMP-2 released from HCF is bioactive and suggesting that long-term delivery of BMP-2 is advantageous over short-term delivery for bone regeneration. HCF, BMP-2-loaded HCF, and BMP-2-loaded normal fibrin containing free heparin were contained in polyester cylindrical tubes and implanted into the hind limb muscle pockets of rats for 8 weeks. Soft X-ray radiography, computed tomography, histomorphometry, calcium assay, and western blot analysis showed that BMP-2-loaded HCF yielded the most extensive bone formation among the groups. Since HCF can deliver BMP-2 over a long term, is an injectable system, and is made of clinically benign materials, this system would have advantages for clinical applications to regenerate bone.
