Cell proliferation and IL-6-type cytokine gene expression, rather than astrocyte hypertrophy, characterize early pressure-induced ONH injury.

Lysyl oxidase is an extracellular enzyme critical for the normal biosynthesis of collagens and elastin. In addition, lysyl oxidase reverts ras-mediated transformation, and lysyl oxidase expression is down-regulated in human cancers. Since suramin inhibits growth factor signaling pathways and induces lysyl oxidase in ras-transformed NIH3T3 cells (RS485 cells), we sought to investigate the effects of suramin on the phenotype of transformed cells and the role of lysyl oxidase in mediating these effects. Suramin treatment resulted in a more normal phenotype as judged by growth rate, cell cycle parameters, and morphology. ␤-aminopropionitrile, the selective inhibitor of lysyl oxidase enzyme activity, was remarkably unable to block suramin-induced reversion. By contrast, ectopic antisense lysyl oxidase demonstrated that lysyl oxidase gene expression mediated phenotypic reversion. Since lysyl oxidase is synthesized as a 50 kDa precursor and processed to a 30 kDa active enzyme and 18 kDa propeptide, the effects of these two products on the transformed phenotype of RS485 cells were then directly assessed in the absence of suramin. Here we report, for the first time, that the lysyl oxidase propeptide, and not the lysyl oxidase enzyme, inhibits ras-dependent transformation as determined by effects on cell proliferation assays, growth in soft agar, and Aktdependent induction of NF-B activity. Thus, the lysyl oxidase propeptide, which is released during extracellular proteolytic processing of pro-lysyl oxidase, functions to inhibit ras-dependent cell transformation.Lysyl oxidase catalyzes oxidative deamination of peptidyl lysine and hydroxylysine residues in collagens, and peptidyl lysine residues in elastin. The resulting peptidyl aldehydes spontaneously condense and undergo oxidation reactions to form the lysine-derived covalent cross-links required for the normal structural integrity of the extracellular matrix (1-3). Lysyl oxidase is synthesized as a 48 -50 kDa proenzyme, secreted into the extracellular environment where it is then processed by proteolytic cleavage to a functional 30 kDa enzyme and an 18 kDa propeptide (4). Evidence supports that 30 kDa lysyl oxidase is active whereas the 50 kDa proenzyme is enzymatically inactive (5-7). Procollagen C-proteinases are active in processing pro-lysyl oxidase and are products of the Bmp1 gene and the structurally related Tll1 and Tll2 genes (6 -8).Lysyl oxidase gene expression was found to inhibit the transforming activity of ras and was hence named the "ras recision gene" (rrg) (9, 10). Lysyl oxidase is down-regulated in rastransformed cells and in many cancer cell lines. Reduced lysyl oxidase levels are also observed in human cancers (9, 11-15), whereas in spontaneous revertants or upon induced phenotypic reversion higher normal levels of lysyl oxidase are again seen (9,14). Conversely stable phenotypic revertants of ras-transfected NIH3T3 cells return to a transformed phenotype upon transfection with an antisense lysyl oxidase vector (9, 10, 16). Antisense lysyl oxidase t...

Differentiation of phenotypically normal osteoblast cultures leads to formation of a bone-like extracellular matrix in vitro. Maximum collagen synthesis occurs early in the life of these cultures, whereas insoluble collagen deposition occurs later and is accompanied by a diminished rate of collagen synthesis. The mechanisms that control collagen deposition seem likely to include regulation of extracellular collagen biosynthetic enzymes, but expression patterns of these enzymes in differentiating osteoblasts has received little attention. The present study determined the regulation of lysyl oxidase as a function of differentiation of phenotypically normal murine MC3T3-E1 cells at the level of RNA and protein expression and enzyme activity. In addition, the regulation of BMP-1/mTLD mRNA levels that encodes procollagen C-proteinases was assayed. The role of lysyl oxidase in controlling insoluble collagen accumulation was further investigated in inhibition studies utilizing beta-aminopropionitrile, a specific inhibitor of lysyl oxidase enzyme activity. Results indicate that lysyl oxidase is regulated as a function of differentiation of MC3T3-E1 cells, and that the maximum increase in lysyl oxidase activity precedes the most efficient phase of insoluble collagen accumulation. By contrast BMP-1/mTLD is more constitutively expressed. Inhibition of lysyl oxidase in these cultures increases the accumulation of abnormal collagen fibrils, as determined by solubility studies and by electron microscopy. Taken together, these data support that regulation of lysyl oxidase activity plays a key role in the control of collagen deposition by osteoblast cultures.

The neurotrophin (NT) hypothesis proposes that the obstruction of retrograde transport at the optic nerve head results in the deprivation of neurotrophic support to retinal ganglion cells (RGC) leading to apoptotic cell death in glaucoma. An important corollary to this concept is the implication that appropriate enhancement of neurotrophic support will prolong the survival of injured RGC indefinitely. This hypothesis is, perhaps, the most widely recognized theory to explain RGC loss resulting from exposure of the eye to elevated intraocular pressure (IOP). Recent studies of NT signaling using rat glaucoma models, have examined the endogenous responses of the retina to pressure exposure as well as studies designed to augment NT signaling in order to rescue RGC from apoptosis following pressure-induced injury. The examination of these studies in this review reveals a number of consistent observations and provides direction for further investigations of this hypothesis.

Lysyl oxidase is required for the normal biosynthesis and maturation of collagen and elastin. It is expressed by vascular smooth muscle cells, and its increased expression has been previously found in atherosclerosis and in models of balloon angioplasty. The lysyl oxidase propeptide (LOX-PP) has more recently been found to have biological activity as a tumor suppressor, and it inhibits Erk1/2 Map kinase activation. We reasoned that LOX-PP may have functions in normal non-transformed cells. We, therefore, investigated its effects on smooth muscle cells, focusing on important biological processes mediated by Erk1/2-dependent signaling pathways including proliferation and matrix metalloproteinase-9 (MMP-9) expression. In addition, we investigated whether evidence for accumulation of LOX-PP could be found in vivo in a femoral artery injury model. Recombinant LOX-PP was expressed and purified, and was found to inhibit primary rat aorta smooth muscle cell proliferation and DNA synthesis by more than 50%. TNF-α-stimulated MMP-9 expression and Erk1/2 activation were both significantly inhibited by LOX-PP. Immunohistochemistry studies carried out with affinity purified anti-LOX-PP antibody showed that LOX-PP epitopes were expressed at elevated levels in vascular lesions of injured arteries. These novel data suggest that LOX-PP may provide a feedback control mechanism that serves to inhibit properties associated with the development of vascular pathology.Tumor necrosis factor-α (TNF-α) contributes to the development of atherosclerotic lesions [15]. Important among its many activities, TNF-α promotes the proliferation of smooth muscle cells (SMCs) [15] and it stimulates matrix metalloproteinase (MMP) production [6]. Thinning of the fibrous cap by MMPs is undesirable due to the release of sequestered factors that leads to thrombosis [15]. TNF-α regulation of MMP-9 is of particular importance in atherosclerotic lesion development and in fibrous cap thinning [3,20]. Thus, up-regulation of MMP-9 expression by TNF-α can promote development of atherosclerotic lesions by multiple mechanisms that include stimulation of SMC proliferation and MMP-9 production.