Matrix metalloproteinases (MMPs) are of central importance in the proteolytic remodeling of matrix and the generation of biologically active molecules. MMPs are distinguished by a conserved catalytic domain containing a zinc ion, as well as a prodomain that regulates enzyme activation by modulation of a cysteine residue within that domain. Because nitric oxide (NO) and derived reactive nitrogen species target zinc ions and cysteine thiols, we assessed the ability of NO to regulate MMPs. A dose-dependent, biphasic regulatory effect of NO on the activity of MMPs (MMP-9, -1, and -13) secreted from murine macrophages was observed. Low exogenous NO perturbed MMP/tissue inhibitor of metalloproteinase (TIMP)-1 levels by enhancing MMP activity and suppressing the endogenous inhibitor TIMP-1. This was cGMP-dependent, as confirmed by the cGMP analog 8-bromo-cGMP, as well as by the NO-soluble guanylyl cyclase-cGMP signaling inhibitor thrombospondin-1. Exposure of purified latent MMP-9 to exogenous NO demonstrated a concentration-dependent activation and inactivation of the enzyme, which occurred at higher NO flux. These chemical reactions occurred at concentrations similar to that of activated macrophages. Importantly, these results suggest that NO regulation of MMP-9 secreted from macrophages may occur chemically by reactive nitrogen species-mediated protein modification, biologically through soluble guanylyl-cyclase-dependent modulation of the MMP-9/TIMP-1 balance, or proteolytically through regulation of MMP-1 and -13, which can cleave the prodomain of MMP-9. Furthermore, when applied in a wound model, conditioned media exhibiting peak MMP activity increased vascular cell migration that was MMP-9-dependent, suggesting that MMP-9 is a key physiologic mediator of the effects of NO in this model. tissue inhibitor of metalloproteinase-1 ͉ thrombospondin-1

Traumatic brain injury (TBI) is a silent epidemic affecting approximately 1.4 million Americans annually, at an estimated annual cost of $60 billion in the United States alone. Despite an increased understanding of the pathophysiology of closed head injury, there remains no pharmacological intervention proven to improve functional outcomes in this setting. Currently, the existing standard of care for TBI consists primarily of supportive measures. Apolipoprotein E (apoE) is the primary apolipoprotein synthesized in the brain in response to injury, where it modulates several components of the neuroinflammatory cascade associated with TBI. We have previously demonstrated that COG133, an apoE mimetic peptide, improved functional outcomes and attenuated neuronal death when administered as a single intravenous injection at 30 min post-TBI in mice. Using the principles of rational drug design, we developed a more potent analog, COG1410, which expands the therapeutic window for the treatment of TBI by a factor of four, from 30 min to 2 h. Mice that received a single intravenous injection of COG1410 at 120 min post-TBI exhibited significant improvement on a short term test of vestibulomotor function and on a long term test of spatial learning and memory. This was associated with a significant attenuation of microglial activation and neuronal death in the hippocampus, the neuroanatomical substrate for learning and memory. Rationally derived apoE mimetic peptides have been demonstrated to exert neuroprotective and anti-inflammatory effects in vitro and in clinically relevant models of brain injury. This represents a novel therapeutic strategy in the treatment of TBI.

The low affinity IgE receptor, Fc⑀RII (CD23), is both a positive and negative regulator of IgE synthesis. The proteinase activity that converts the membrane-bound form of CD23 into a soluble species (sCD23) is an important regulator of the function of CD23 and may be an important therapeutic target for the control of allergy and inflammation. We have characterized the catalytic activity of ADAM (a disintegrin and metalloproteinase) 10 toward human CD23. We found that ADAM10 efficiently catalyzes the cleavage of peptides derived from two distinct cleavage sites in the CD23 backbone. Tissue inhibitors of metalloproteinases and a specific prodomain-based inhibitor of ADAM10 perturb the release of endogenously produced CD23 from human leukemia cell lines as well as primary cultures of human B-cells. Expression of a mutant metalloproteinase-deficient construct of ADAM10 partially inhibited the production of sCD23. Similarly, small inhibitory RNA knockdown of ADAM10 partially inhibited CD23 release and resulted in the accumulation of the membrane-bound form of CD23 on the cells. ADAM10 contributes to CD23 shedding and thus could be considered a potential therapeutic target for the treatment of allergic disease.The low affinity IgE receptor Fc⑀RII (CD23) is a 46-kDa type II membrane protein that is expressed on B-cells and cells of the myeloid lineage (1). CD23 has multiple functions. It is both a positive and negative regulator of IgE synthesis (2). It facilitates IgE-dependent antigen presentation through its binding of IgEantigen complexes (3,4). Moreover, the release of proinflammmatory cytokines from macrophages is stimulated by CD23 binding to CD18/11b and /11c (complement receptors 3 and 4) (5-7).In humans, two isoforms of CD23 that differ by only seven amino acids in the short N-terminal cytoplasmic domain are observed (8). CD23a is expressed only on B-cells. Stimulation of B-cells and cells of the myeloid lineage with interleukin-4 (IL-4) 3 induces the expression of CD23b. The C-terminal extracellular domain consists of a globular fold that has homology to the C-type lectin family (9). This globular domain has been shown to contain two distinct binding sites, one for IgE and a second that recognizes CD21 (complement receptor 2) (10, 11). At the cell surface CD23 self-assembles to form homotrimers that have a higher affinity for IgE than the CD23 monomer (12, 13). The self-association is driven by a leucine zipper-like domain (14) that connects the N-terminal cytoplasmic and transmembrane domains to the C-terminal globular domain.Homotrimeric CD23 molecules exhibit a 15-nm ␣-helical coiled coil stalk that extends the globular C-terminal domains from the plasma membrane (15). Cleavage in the stalk region by a membrane-associated endoproteolytic activity generates soluble fragments of CD23 (sCD23) that possess apparent molecular masses of 37, 33, and 29 kDa (16). All three of these sCD23 fragments exist as homotrimers (15). Smaller fragments of CD23 (25 and 16 kDa) are known. However, these are thought to be form...