Articular cartilage undergoes matrix degradation and loss of mechanical properties when stimulated with proinflammatory cytokines such as interleukin-1 (IL-1). Aggrecanases and matrix metalloproteinases (MMPs) are thought to be principal downstream effectors of cytokine-induced matrix catabolism, and aggrecanase- or MMP-selective inhibitors reduce or block matrix destruction in several model systems. The objective of this study was to use metalloproteinase inhibitors to perturb IL-1-induced matrix catabolism in bovine cartilage explants and examine their effects on changes in tissue compression and shear properties. Explanted tissue was stimulated with IL-1 for up to 24 days in the absence or presence of inhibitors that were aggrecanase-selective, MMP-selective, or non-selective. Analysis of conditioned media and explant digests revealed that aggrecanase-mediated aggrecanolysis was delayed to varying extents with all inhibitor treatments, but that aggrecan release persisted. Collagen degradation was abrogated by MMP- and non-selective inhibitors and reduced by the aggrecanase inhibitor. The inhibitors delayed but did not reduce loss of the equilibrium compression modulus, whereas the losses of dynamic compression and shear moduli were delayed and reduced. The data suggest that non-metalloproteinase mechanisms participate in IL-1-induced matrix degradation and loss of tissue material properties.

A novel series of TNF inhibitors was identified based on the screening of existing MMP inhibitor libraries. Further SAR optimization led to the discovery of a novel lead compound. Its synthesis, efficacy in experimental animal models, and pharmacokinetic data are discussed.

Pulmonary fibrosis is a progressive and largely untreatable group of disorders that affects up to 100,000 people on any given day in the United States. To elucidate the molecular mechanisms that lead to end-stage human pulmonary fibrosis we analyzed samples from patients with histologically proven pulmonary fibrosis (usual interstitial pneumonia) by using oligonucleotide microarrays. Gene expression patterns clearly distinguished normal from fibrotic lungs. Many of the genes that were significantly increased in fibrotic lungs encoded proteins associated with extracellular matrix formation and degradation and proteins expressed in smooth muscle. Using a combined set of scoring systems we determined that matrilysin (matrix metalloproteinase 7), a metalloprotease not previously associated with pulmonary fibrosis, was the most informative increased gene in our data set. Immunohistochemisry demonstrated increased expression of matrilysin protein in fibrotic lungs. Furthermore, matrilysin knockout mice were dramatically protected from pulmonary fibrosis in response to intratracheal bleomycin. Our results identify matrilysin as a mediator of pulmonary fibrosis and a potential therapeutic target. They also illustrate the power of global gene expression analysis of human tissue samples to identify molecular pathways involved in clinical disease.

BACKGROUND:Mycophenolic acid (MPA), a selective inhibitor of inosine monophosphate dehydrogenase, is the active agent of the immunosuppressive drug, mycophenolate mofetil (MMF). Previous studies have shown that MPA inhibits DNA synthesis in T and B lymphocytes by blocking de novo guanosine synthesis, and that MPA induces monocyte differentiation. MMF is being used for prevention of organ graft rejection and has also shown efficacy in rheumatoid arthritis trials. This study was designed to determine if apoptosis also plays a role in the immunosuppressive and anti-inflammatory effects of MMF.METHODS:Cultured human T lymphocytic (MOLT-4) and monocytic (THP-1 and U937) cell lines were treated with MPA. Apoptosis, cell viability, DNA content, lipid content, cell volume, and lysosomes were measured by a variety of microscopic, flow cytometric, and biochemical techniques.RESULTS:MPA inhibits proliferation, arrests cell cycle in S phase, and increases apoptosis in all three cell lines. Exogenous guanosine added within 24 hr of MPA treatment, but not later, partially reversed MPA-induced apoptosis in MOLT-4 cells. MPA increased lipid droplets in all three cell lines and increased both cell volumes and numbers of lysosomes in the monocytic cell lines. In both monocytic cell lines, MPA also reduced the number of nuclei containing nucleoli and greatly increased neutral lipids, primarily triacylglycerols, suggesting that these cells were differentiating.CONCLUSIONS:Increased apoptosis and terminal differentiation of both lymphocytes and monocytes may promote the antiproliferative, immunosuppressive, and anti-inflammatory effects of MMF seen clinically in transplantation and rheumatoid arthritis.