A productive angiogenic response must couple to the survival machinery of endothelial cells to preserve the integrity of newly formed vessels. Angiopoietin-1 (Ang-1) is an endothelium-specific ligand essential for embryonic vascular stabilization, branching morphogenesis, and post-natal angiogenesis, but its contribution to endothelial cell survival has not been completely elucidated. Here we show that Ang-1 acting via the Tie 2 receptor induces phosphorylation of the survival serinethreonine kinase, Akt (or protein kinase B). This is associated with up-regulation of the apoptosis inhibitor, survivin, in endothelial cells and protection of endothelium from death-inducing stimuli. Moreover, dominant negative survivin negates the ability of Ang-1 to protect cells from undergoing apoptosis. The activation of antiapoptotic pathways mediated by Akt and survivin in endothelial cells may contribute to Ang-1 stabilization of vascular structures during angiogenesis, in vivo.During angiogenesis, endothelial cells receive cues from growth factors to initiate mitosis, migration, and organization of endothelial cells into primitive angiotubes and patent vascular networks (1, 2). These processes critically depend on preservation of endothelial cell viability. Disruption of endothelial cell-matrix contacts or interference with extracellular survival signals is sufficient to initiate caspase-dependent apoptosis in endothelium, culminating with rapid involution of vascular structures (3, 4). Unlike most angiogenic regulators, including fibroblast growth factor or vascular endothelial growth factor (VEGF), 1 angiopoietin-1 (Ang-1) does not stimulate endothelial cell growth but rather promotes stabilization of vascular networks and branching morphogenesis in vivo and in vitro (5-8). Little is known about the signaling requirements of these responses, and the mechanism(s) of Ang-1-induced cytoprotection are unknown (7, 9). The major goal of this paper was to elucidate a potential link between endothelial cell viability and maintenance of angiogenesis by examining the ability of Ang-1 to activate the antiapoptotic serine-threonine kinase, Akt (or protein kinase B). Moreover, we examined the relationship between Ang-1, Akt activation, and the expression of the anti-apoptotic genes, bcl-2 and survivin, in cultured microvascular endothelial cells (MVEC). MATERIALS AND METHODSCell Culture and Reagents-Bovine MVEC (Vec Technologies, Rensselaer, NY) were cultured in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum, L-glutamine, and antibiotics (penicillin and streptomycin). Cells (up to passage 12) were used for the experiments. In experiments examining endogenous survivin expression, human umbilical vein endothelial cells (HUVEC) were used, because the survivin antibody recognized human survivin better than bovine survivin. HUVEC were cultured on gelatin-coated tissue culture flasks in M199 medium containing 20% fetal bovine serum, 50 g/ml endothelial cell growth supplement (a commercial preparation that contains main...

Recent efforts to identify treatments for myocardial ischemia reperfusion injury have resulted in the discovery of a novel series of highly potent α,α-disubstituted amino acid-based arginase inhibitors. The lead candidate, (R)-2-amino-6-borono-2-(2-(piperidin-1-yl)ethyl)hexanoic acid, compound 9, inhibits human arginases I and II with IC50s of 223 and 509 nM, respectively, and is active in a recombinant cellular assay overexpressing human arginase I (CHO cells). It is 28% orally bioavailable and significantly reduces the infarct size in a rat model of myocardial ischemia/reperfusion injury. Herein, we report the design, synthesis, and structure-activity relationships (SAR) for this novel series of inhibitors along with pharmacokinetic and in vivo efficacy data for compound 9 and X-ray crystallography data for selected lead compounds cocrystallized with arginases I and II.

1 Vasodilator responses to bradykinin (BK) in the rat heart are reported to be independent of NO and cyclo-oxygenase/lipoxygenase products of arachidonic acid (AA).2 We verified that inhibition of NO synthase with L-nitroarginine (50 [LM) and cyclo-oxygenase with indomethacin (2.8 AM) were without effect on vasodilator responses to BK (10-1000 ng) in the Langendorff rat heart preparation. 3 L-Nitroarginine elevated perfusion pressure, signifying a crucial role of NO in the maintenance of basal vasculature tone. 4 In hearts treated with L-nitroarginine to eliminate NO and elevate perfusion pressure, vasodilator responses were reduced by inhibitors of cytochrome P450 (P450), clotrimazole (1 pM) and 7-ethoxyresorufin (1 JLM). 17-Octadecynoic acid (17-ODYA 2 pM), a mechanism based inhibitor of P450-dependent metabolism of fatty acids, also reduced vasodilator responses to BK. 5 These results confirm that NO and prostaglandins do not mediate vasodilator responses to BK in the rat heart but suggest a major role for a P450-dependent mechanism via AA metabolism.

SUMMARY: reduces injury both in vivo and in vitro, but the mechanisms are unknown. Stimulation of serum-and growth factor-deprived HUVEC with IL-11 increased survivin mRNA and protein expression levels in a dosedependent manner, with maximal induction at 50 to 100 ng/ml of IL-11. Survivin mRNA expression peaked after 3 to 6 hours of IL-11 treatment and decreased by 24 hours. Survivin protein expression was maximal at 6 hours of treatment and remained elevated through 24 hours. Survivin induction may be mediated by activation of protein kinase B/Akt, but IL-11 failed to activate this pathway in HUVEC. IL-11 did activate signal transducer and activator of transcription (STAT)-3 and IL-11 failed to induce survivin expression in HUVEC transduced with a dominant-negative STAT3 mutant, whereas control-transduced HUVEC responded normally. An IL-11 transgene caused increased survivin mRNA expression in mice compared with control littermates. Intradermal injection of IL-11 (500 ng) into human skin xenografts on immunodeficient mice up-regulated survivin protein in microvascular endothelium and epithelial keratinocytes. We conclude that IL-11 induces expression of survivin, an antiapoptotic protein, in vitro and in vivo, and identify STAT3 as a critical mediator of this response. (Lab Invest 2001, 81:327-334).

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