The binding of urokinase plasminogen activator (uPA) to its specific receptor (uPAR) facilitates migration of vascular smooth muscle cells (VSMC). However, the signaling cascade utilized by the urokinase receptor is only incompletely understood. We investigated intracellular uPA/uPAR signaling in human aortic VSMC from the cell membrane to the nucleus. uPA binding to VSMC induced a rapid and pronounced increase in tyrosine phosphorylation of several proteins with molecular masses of 53-60, 85-90, and 130 -140 kDa. By using co-immunoprecipitation techniques and in vitro kinase assays, the uPAR-associated proteins were identified as Janus (Jak) and Src non-receptor protein-tyrosine kinases (PTK) Jak1, Tyk2, and p59 fyn , p53/56 lyn , p53/59 hck , and p55fgr . Furthermore, uPA induced a time-dependent reversible translocation of the Stat1 (signal transducer and activator of transcription) protein to the VSMC nuclei, as shown by confocal microscopy studies. Using an electrophoretic mobility shift assay, we then demonstrated that Stat1 is rapidly activated in response to stimulation with uPA and specifically binds to the DNA regulatory elements GAS (interferon-␥ activation site) and ISRE (interferon-stimulated response element). Mobility supershift experiments confirmed DNA-protein complexes containing Stat1 protein. Migration experiments with double immunofluorescence staining revealed polarization of uPAR, and colocalization with Jak1 and Tyk2 to the leading edge of the migrating cells. Under the same conditions, Jak2, Jak3, and the SrcPTKs remained randomly distributed over the entire body of the cells. Our studies therefore suggest that, in VSMC, the uPAR-signaling complex utilizes at least two different mechanisms, a direct signaling pathway utilizing the Jak/Stat cascade and a second signal transduction mechanism via Src-like protein-tyrosine kinases. uPA-induced signaling via Jak/Stat is most likely involved in the regulation of cell migration, while the functional purpose of the uPA-associated Src-PTK activation remains to be elucidated.

The leukocyte adhesion molecule ICAM-1 is implicated in ischemic renal reperfusion injury. We tested the utility of an ICAM-1 antisense oligodeoxyribonucleotide (ODN) with lipofectin, six hours prior to 30 minutes of bilateral renal ischemia in the rat. We measured ICAM-1 expression by immunohistochemistry and Western blot. Our antisense ODN showed a specific ICAM-1 surface expression inhibition in vitro. We then assessed ICAM-1 expression, leukocyte infiltration, serum creatinine, serum urea concentration, and renal histology in rats subjected to renal ischemia and controls. Serum creatinine and urea concentrations 12 and 24 hours post-ischemia were increased in saline treated and reverse ODN treated rats, compared to antisense ODN treated or sham operated rats (P < 0.05). Western blotting showed decreased ICAM-1 protein in antisense ODN-treated kidneys, compared to reverse ODN treated and saline treated ischemic controls (P < 0.05). Antisense ODN also ameliorated the ischemia-induced infiltration of granulocytes and macrophages (P < 0.05), and resulted in less cortical renal damage as assessed by a quantitative pathological grading scale (P < 0.05), compared to reverse ODN or saline treatment. Thus, antisense ODN for ICAM-1 protected the kidney against ischemic renal failure. The clinical applicability of these findings extends beyond ischemic acute renal failure.

Abstract-Endothelial cells demonstrate high urokinase expression and upregulation of urokinase receptors in response to vascular injury. Urokinase receptor binding facilitates endothelial cell migration into an arterial wound; however, the signaling cascade induced by the urokinase receptor in this cell type is incompletely understood. Because the Janus kinase (Jak)/signal transducer and activator of transcription (Stat) pathway seems to be important for vessel function, we investigated the hypothesis that urokinase receptor binding activates Jak/Stat signaling in human vascular endothelial cells. Incubation of endothelial cells with urokinase-type plasminogen activator (uPA,1 nmol/L) induced a rapid and pronounced increase in tyrosine phosphorylation of several proteins with a molecular weight between 80 to 90 and 130 to 140 kDa. The same pattern of tyrosine phosphorylation was found after treatment with 1 nmol/L ATF, the urokinase amino-terminal fragment, which is devoid of proteolytic activity but still binds to the urokinase receptor. Using coimmunoprecipitation techniques, we demonstrated that the activated urokinase receptor is associated with 2 cytoplasmic tyrosine kinases of the Jak family, viz, Jak1 and Tyk2. uPA and ATF induced a time-dependent activation of both kinases, as shown by immunoprecipitation and Western blot analysis. Using electrophoretic mobility shift and supershift assays, we then demonstrated that Stat1 is rapidly activated in endothelial cells in response to uPA and ATF. Furthermore, Stat1 specifically binds to the regulatory elements interferon-␥ activation site/interferon-stimulated response element. The uPA-induced, time-dependent translocation of Stat1 to cell nuclei was confirmed by confocal microscopy study and immunoblotting of nuclear extracts with an anti-Stat1 antibody. This study provides evidence for a novel signaling pathway for uPA in human vascular endothelial cells. Direct activation of the Jak/Stat system via the uPA-receptor complex may be an important mechanism for endothelial cell migration and/or proliferation during angiogenesis and after vascular injury. (Arterioscler Thromb Vasc Biol. 1999;19:290-297.)

A brief antecedent period of myocardial ischemia and reperfusion can delay cellular injury during a subsequent ischemic condition. Recent observations suggest that this protective mechanism depends on the continued activation of adenosine A1 receptors and Gi proteins. During acute myocardial ischemia, sufficient amounts of adenosine for maximal activation of adenosine A1 receptors are released, independent of a preconditioning ischemia. Hence, the protective mechanism of ischemic preconditioning may not exclusively be explained by activation of adenosine A1 receptors. As a working hypothesis, an increased responsiveness of Gi proteins toward receptor-mediated activation, leading to an increased response of Gi-regulated effectors, was tested in this study. In 47 anesthetized dogs, ischemia was induced by proximal ligation of the left anterior descending coronary artery. Animals underwent either a single period of 5 minutes of ischemia (n = 9), a single period of 15 minutes of ischemia (n = 10), 5 minutes of ischemia followed by 15 minutes of reperfusion (n = 8), 15 minutes of ischemia followed by 60 minutes of reperfusion (n = 5), or 5 minutes of ischemia followed by 15 minutes of reperfusion and a second period of 5 minutes of ischemia (n = 15). Sarcolemmal membranes were prepared from the central ischemic area and from the posterior left ventricular wall, which served as the control. During ischemia, carbochol-stimulated GTPase decreased by 38% (control, 33.5 +/- 17.7; ischemia, 24.2 +/- 15 pmol.min-1.mg protein-1; n = 9; P < .001). The decrease in carbachol-stimulated GTPase activity was associated with a 45% decrease in carbachol-mediated inhibition of adenylyl cyclase (control, 28.9 +/- 2.4% maximal inhibition; ischemia, 15.1 +/- 2.6% maximal inhibition; n = 5; P < .001). Prolongation of the ischemic period to 15 minutes did not lead to a further reduction of the Gi-mediated signal transduction. The binding properties of muscarinic receptors were not affected by ischemia. Furthermore, as demonstrated by carbachol-stimulated binding of [gamma-35S]GTP to sarcolemmal membranes, high- and low-affinity binding sites for the muscarinic antagonist carbachol, the EC50 for carbachol-stimulated GTPase activity and the substrate dependency of the high-affinity GTPase, the interaction between muscarinic receptors and inhibitory G proteins, and GTP binding to G proteins were not altered (n = 14). Immunoblotting with alpha 1- and alpha 2-specific antibodies did not indicate a loss of Gi proteins during ischemia that could explain the reduced GTPase activity.(ABSTRACT TRUNCATED AT 400 WORDS)