No abstract

Inactivators of cysteine proteinases (CPs) were tested as inhibitors of bone resorption in vitro and in vivo. The following four CP inactivators were tested: Ep475, a compound with low membrane permeability which inhibits cathepsins B, L, S, H, and calpain; Ep453, the membrane-permeant prodrug of Ep475; CA074, a compound with low membrane permeability which selectively inactivates cathepsin B; and CA074Me, the membrane-permeant prodrug of CA074. The test systems consisted of 1) monitoring the release of radioisotope from prelabelled mouse calvarial explants and 2) assessing the extent of bone resorption in an isolated osteoclast assay using confocal laser microscopy. Ep453, Ep475, and CA074Me inhibited both stimulated and basal bone resorption in vitro while CA074 was without effect; the inhibition was reversible and dose dependent. None of the inhibitors affected protein synthesis, DNA synthesis, the PTH-enhanced secretion of beta-glucuronidase, and N-acetyl-beta-glucosaminidase, or the spontaneous release of lactate dehydrogenase. Ep453, Ep475, and CA074Me dose-dependently inhibited the resorptive activity of isolated rat osteoclasts cultured on bone slices with a maximal effect at 50 microM. The number of resorption pits and their mean volume was reduced, whilst the mean surface area remained unaffected. Again, CA074 was without effect. Ep453, Ep475, and CA074Me, but not CA074, when administered subcutaneously at a dose of 60 micrograms/g body weight inhibited bone resorption in vivo as measured by an in vivo/in vitro assay, by about 20%. This study demonstrates that cathepsins B, L, and/or S are involved in bone resorption in vitro and in vivo. Whilst cathepsin L and/or S act extracellularly, and possibly intracellularly, cathepsin B mediates its effects intracellularly perhaps through the activation of other proteinases involved in subosteoclastic collagen degradation.

We explored the physiologic role of endothelial cell apoptosis during development by generating mouse embryos lacking the inhibitor of apoptosis protein (IAP) survivin in endothelium. This was accomplished by intercrossing survivin lox/lox mice with mice expressing cre recombinase under the control of the endothelial cell specific tie1 promoter (tie1-cre mice). Lack of endothelial cell survivin resulted in embryonic lethality. Mutant embryos had prominent and diffuse hemorrhages from embryonic day 9.5 (E9.5) and died before E13.5. Heart development was strikingly abnormal. Survivin-null endocardial lineage cells could not support normal epithelial-mesenchymal transformation (EMT), resulting in hypoplastic endocardial cushions and in utero heart failure. In addition, 30% of mutant embryos had neural tube closure defects (NTDs) that were not caused by bleeding or growth retardation, but were likely due to alterations in the release of soluble IntroductionEmbryonic development depends on the establishment of a complex network of blood vessels to meet the functional demands of each organ system. 1,2 During vasculo/angiogenesis, factors that regulate endothelial survival are balanced to provide resistance to exogenous stresses, to facilitate vascular regression during vessel remodeling, and to promote endothelial proliferation and migration during sprouting. Prominent among these factors is vascular endothelial cell growth factor (VEGF). VEGF stimulates angiogenesis by promoting endothelial growth, migration, and survival via interactions with VEGF receptors, PI3 kinase (PI3K), beta-catenin, and VE-cadherin, which in turn leads to activation of Akt and up-regulation of antiapoptotic proteins such as nitric oxide, Bcl-2, Bcl-XL, XIAP, and survivin. 3 Withdrawal of VEGF results in vessel regression, and inactivation of a single VEGF allele causes profound defects with endothelial apoptosis. 4 Other factors that modulate endothelial survival include angiopoietin-1 and -2, which promote survival and apoptosis, respectively. 5,6 Inhibitors of angiogenesis, such as endostatin, 7 interleukin-12, 8 and cyclo-oxygenase-2 inhibitors 9 induce apoptosis via suppression of Bcl-2 and Bcl-XL. Remarkably, mice in which Bcl-2, Bcl-XL, or XIAP have been inactivated [10][11][12] do not exhibit obvious angiogenic defects, and thus the in vivo significance of these pathways in angiogenesis is raised. Indeed, the physiologic relevance of endothelial apoptosis is poorly documented due to lack of genetic models in which regulators of apoptosis have specifically been inactivated in the endothelium. Delineating the role of endothelial apoptosis, however, may lead to elucidation of clinically relevant endothelial-derived signal pathways. Furthermore, studies indicating that organogenesis is regulated by endothelial factors [13][14][15] additionally underscores the importance of better characterizing functional properties of the endothelium during and after fetal development.Survivin is an inhibitor of apoptosis protein (IAP) and a regulator o...