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Protein levels and polymorphisms of p[22.sup.phox] have been suggested to modulate vascular NAD(P)H oxidase activity and vascular production of reactive oxygen species (ROS). We sought to determine whether increasing p[22.sup.phox] expression would alter vascular ROS production and hemodynamics by targeting p[22.sup.phox] expression to smooth muscle in transgenic (Tg) mice. Aortas of [Tg.sup.p22smc] mice had increased p[22.sup.phox] and Nox1 protein levels and produced more superoxide and [H.sub.2][O.sub.2]. Surprisingly, endothelium-dependent relaxation and blood pressure in [Tg.sup.p22smc] mice were normal. Aortas of [Tg.sup.p22smc] mice produced twofold more nitric oxide (NO) at baseline and sevenfold more NO in response to calcium ionophore as detected by electron spin resonance. Western blot analysis revealed a twofold increase in endothelial NO synthase (eNOS) protein expression in [Tg.sup.p22smc] mice. Both eNOS expression and NO production were normalized by infusion of the glutathione peroxidase mimetic ebselen or by crossing [Tg.sup.p22smc] mice with mice overexpressing catalase. We have previously found that NO stimulates extracellular superoxide dismutase (ecSOD) expression in vascular smooth muscle. In keeping with this, aortic segments from [Tg.sup.p22smc] mice expressed twofold more ecSOD, and chronic treatment with the NOS inhibitor [N.sup.G]-nitro-L-arginine methyl ester normalized this, suggesting that NO regulates ecSOD protein expression in vivo. These data indicate that chronic oxidative stress caused by excessive [H.sub.2][O.sub.2] production evokes a compensatory response involving increased eNOS expression and NO production. NO in turn increases ecSOD protein expression and counterbalances increased ROS production leading to the maintenance of normal vascular function and hemodynamics. transgenic animals; oxidant stress; endothelium; nitric oxide

Molecular alterations that confer phenotypic advantages to tumors can also expose specific therapeutic vulnerabilities. To search for potential treatments that would selectively affect metastatic cells, we examined the sensitivity of lineage-related human bladder cancer cell lines with different lung colonization abilities to chloroquine (CQ) or bafilomycin A1, which are inhibitors of lysosome function and autophagy. Both CQ and bafilomycin A1 were more cytotoxic in vitro to highly metastatic cells compared with their less metastatic counterparts. Genetic inactivation of macroautophagy regulators and lysosomal proteins indicated that this was due to greater reliance on the lysosome but not upon macroautophagy. To identify the mechanism underlying these effects, we generated cells resistant to CQ in vitro. Surprisingly, selection for in vitro CQ resistance was sufficient to alter gene expression patterns such that unsupervised cluster analysis of whole-transcriptome data indicated that selection for CQ resistance alone created tumor cells that were more similar to the poorly metastatic parental cells from which the metastatic cells were derived; importantly, these tumor cells also had diminished metastatic ability in vivo. These effects were mediated in part by differential expression of the transcriptional regulator ID4 (inhibitor of DNA binding 4); depletion of ID4 both promoted in vitro CQ sensitivity and restored lung colonization and metastasis of CQ-resistant cells. These data demonstrate that selection for metastasis ability confers selective vulnerability to lysosomal inhibitors and identify ID4 as a potential biomarker for the use of lysosomal inhibitors to reduce metastasis in patients. [ABSTRACT FROM AUTHOR]

When platelets are strongly stimulated, a procoagulant platelet subpopulation is formed that is characterized by phosphati-dylserine (PS) exposure and epitope modulation of integrin αIIbβ3 or a loss of binding of activation-dependent antibodies. Mitochondrial permeability transition pore (mPTP) formation, which is essential for the formation of procoagulant platelets, is impaired in the absence of cyclophilin D (CypD). Here we investigate the mechanisms responsible for these procoagulant platelet-associated changes in integrin αIIbβ3 and the physiologic role of procoagulant platelet formation in the regulation of platelet aggregation. Among strongly stimulated adherent platelets, integrin αIIbβ3 epitope changes, mPTP formation, PS exposure, and platelet rounding were closely associated. Furthermore, platelet mPTP formation resulted in a decreased ability to recruit additional platelets. In the absence of CypD, integrin αIIbβ3 function was accentuated in both static and flow conditions, and, in vivo, a prothrombotic phenotype occurred in mice with a platelet- specific deficiency of CypD. CypD-dependent proteolytic events, including cleavage of the integrin β3 cytoplasmic domain, coincided closelywith integrin αIIbβ3 inactivation.Calpain inhibition blocked integrin3 cleavage and inactivation but not mPTP formation or PS exposure, indicating that integrin inactivation and PS exposure are mediated by distinct pathways subsequent to mPTP formation. mPTP-dependent alkalinization occurred in procoagulant platelets, suggesting a possible alternative mechanism for enhancement of calpain activity in procoagulant platelets. Together, these results indicate that, in strongly stimulated platelets, mPTP formation initiates the calpain-dependent cleavage of integrin β3 and associated regulatory proteins, resulting in integrin αIIbβ3 inactivation, and demonstrate a novel CypD-dependent negative feedback mechanism that limits platelet aggregation and thrombotic occlusion. [ABSTRACT FROM AUTHOR]

Protein levels and polymorphisms of p22phox have been suggested to modulate vascular NAD(P)H oxidase activity and vascular production of reactive oxygen species (ROS). We sought to determine whether increasing p22phox expression would alter vascular ROS production and hemodynamics by targeting p22phox expression to smooth muscle in transgenic (Tg) mice. Aortas of Tgp22smc mice had increased p22phox and Nox 1 protein levels and produced more superoxide anti H2O2. Surprisingly, endothelium-dependent relaxation and blood pressure in Tgp22smc mice were normal. Aortas of Tgp22smc mice produced twofold more nitric oxide (NO) at baseline and sevenfold more NO in response to calcium ionophore as detected by electron spin resonance. Western blot analysis revealed a twofold increase in endothelial NO synthase (eNOS) protein expression in Tgp22smc mice. Both eNOS expression and NO production were normalized by infusion of the glutathione peroxidase mimetic ebselen or by crossing Tgp22smc mice with mice overexpressing catalase. We have previously found that NO stimulares extracellular superoxide dismutase (ecSOD) expression in vascular smooth muscle. In keeping with this, aortic segments from Tgp22smc mice expressed twofold more ecSOD, and chronic treatment with the NOS inhibitor NG-nitro-L-arginine methyl ester normalized this, suggesting that NO regulates ecSOD protein expression in vivo. These data indicate that chronic oxidative stress caused by excessive H2O2 production evokes a compensatory response involving increased eNOS expression and NO production. NO in turn increases ecSOD protein expression and counterbalances increased ROS production leading to the maintenance of normal vascular function and hemodynamics. [ABSTRACT FROM AUTHOR]