Systemic administration of anti-urokinase plasminogen activator receptor monoclonal antibodies induces hepatic fibrin deposition in tissue-type plasminogen activator deficient mice.

BACKGROUND: Degradation of extracellular matrix proteins, such as fibrin, is pivotal to tumor invasion. Inhibition of the interaction between urokinase plasminogen activator (u-PA) and its receptor (u-PAR), and hence pro-u-PA activation, is an attractive approach to anti-invasive cancer therapy. A number of inhibitors exist for the human system, but because of species specificity none of these are efficient in mice. We have recently generated an inhibitory monoclonal antibody (mAb) against mouse u-PAR (mR1) by immunization of u-PAR-deficient mice. OBJECTIVES: To evaluate the effect of mR1 in vivo in a physiological setting sensitive to deregulated fibrinolysis, we have administered mR1 systemically and quantitated the effect on liver fibrin accumulation. METHODS: Wild-type and tissue-type plasminogen activator (t-PA) deficient mice were administered with mR1, or control antibody, during 6 weeks. Thereafter, the livers were retrieved and the amount of liver fibrin measured by unbiased morphometrical analysis of immunofluorescence signal. RESULTS: Systemic administration of mR1 caused significantly increased fibrin signal in anti-u-PAR treated t-PA-deficient mice compared to mock-treated, which mimics the phenotype of u-PAR;t-PA double-deficient mice. Fibrin and fibronectin accumulated within the sinusoidal space and was infiltrated by inflammatory cells. Analysis of small and rare hepatic fibrin plaques observed in t-PA-deficient mice showed infiltrating macrophages that, contrary to surrounding Kuppfer cells, expressed u-PAR. CONCLUSION: We show that u-PAR-expressing macrophages are involved in cell-mediated fibrinolysis of liver fibrin deposits, and that the antimouse-u-PAR mAb is effective in vivo and thus suited for studies of the effect of targeting the u-PA/u-PAR interaction in mouse cancer models.

Protein kinase C epsilon-Src modules direct signal transduction in nitric oxide-induced cardioprotection: complex formation as a means for cardioprotective signaling.

An essential role for protein kinase C epsilon (PKCepsilon) has been shown in multiple forms of cardioprotection; however, there is a distinct paucity of information concerning the signaling architecture that is responsible for the manifestation of a protective phenotype. We and others have recently shown that signal transduction may proceed via the formation of signaling complexes (Circ Res. 2001;88:59-62). In order to understand if the assembly of multiprotein complexes is the manner by which signaling is conducted in cardioprotection, we designed a series of experiments to characterize the associations of Src tyrosine kinase with PKCepsilon in a conscious rabbit model of nitric oxide (NO)-induced late preconditioning. Our data demonstrate that PKCepsilon and Src can form functional signaling modules in vitro: PKCepsilon interacts with Src; the association with PKCepsilon activates Src; and adult cardiac cells receiving recombinant adenoviruses encoding PKCepsilon exhibit increased Src activity. Furthermore, our results show that NO-induced late preconditioning involved PKCepsilon-Src module formation and enhanced the enzymatic activity of PKCepsilon-associated Src. Inhibition of PKC blocked cardioprotection, module formation, and PKCepsilon-associated Src activity, providing direct evidence for a functional role of the PKCepsilon-Src module in the orchestration of NO-induced cardioprotection in conscious rabbits.

Accumulation of platelets in rat syngeneic lung transplants: a potential factor responsible for preservation-reperfusion injury.

BACKGROUND: Platelets are known to play an important role in the pathogenesis of adult respiratory distress syndrome as well as preservation-reperfusion injury of liver allografts. However, the role of platelets in pulmonary preservation-reperfusion injury is unknown. In this study, we examined whether the extent of platelet accumulation in the preserved and subsequently reperfused lungs correlated with the degree of preservation-reperfusion injury in a rat lung isotransplant model. METHODS: Heart-lung blocks from donor rats were flushed with and preserved in modified Euro-Collins solution at 4 degrees C for 0 hr (n=5), 6 hr (n=6), and 24 hr (n=6). The left lung was divided from the heart-lung block, transplanted into the recipient rat, and reperfused for 1 hr. Lung injury was evaluated by the left-to-right pulmonary blood flow ratio, the weight gain of the isograft, and the scores for histological categories of lung injury (intra-alveolar edema, intra-alveolar hemorrhage, and capillary congestion). Small portions of the lung isograft were excised and stained with an antibody specific for rat platelets. A scoring system was developed to semiquantitate the intensity of antibody staining in isografts. RESULTS: Lung isografts were injured and platelets accumulated in the capillaries in proportion to the length of preservation endured before transplantation. The extent of platelet accumulation evaluated by our morphological scoring system correlated significantly with the degree of lung injury assessed by the blood flow ratio (P<0.001), the weight gain (P<0.001), and the histological scores for intra-alveolar hemorrhage (P<0.05) and for capillary congestion (P<0.001). CONCLUSIONS: The results of this study suggest that platelet accumulation is a potential factor responsible for preservation-reperfusion injury of lung isografts in the rat.

Perivascular delivery of a nitric oxide donor inhibits neointimal hyperplasia in vein grafts implanted in the arterial circulation.

OBJECTIVE: Nitric oxide has been reported to reduce intimal hyperplasia as a response to arterial injury. This study was designed to assess the possible effect of perivascular application of a nitric oxide donor on neointimal proliferation occurring in veins exposed to the dynamics of the arterial circulation in a hypercholesterolemic rabbit model. METHODS: Autologous jugular vein grafts were implanted in the carotid circulation of 20 hypercholesterolemic rabbits. A mixture of a biodegradable polymer and the nitric oxide donor, spermine/nitric oxide, which releases nitric oxide with a half-life of 39 minutes, was applied periadventitially at the time of implantation. Controls were veins bathed in saline solution, polymer alone, and polymer plus the carrier vehicle spermine without nitric oxide. Animals (n = 5 in each group) were put to death on day 28 for morphometric analysis, cell count, and immunohistochemical staining. RESULTS: Treatment with perivascular nitric oxide donor significantly decreased wall thickness (126 +/- 24 microm vs 208 +/- 45 microm, p = 0.0017) and area (124 +/- 22 microm2/microm vs 211 +/- 37 microm2/microm, p = 0.005). With the carrier vehicle spermine alone, there was a trend toward reduced intimal thickness, but the change was not statistically significant. In the grafts treated with nitric oxide donor, expression of insulin-like growth factor, fibroblast growth factor, thrombospondins, fibronectin, and tenascin was reduced. CONCLUSION: The periadventitial delivery of nitric oxide donor produces a reduction of neointimal hyperplasia in veins implanted in the arterial circulation. The mechanism of action is not entirely clear, but the reduction cannot be explained on the basis of decreased cell proliferation alone. Other possibilities are modulation of protein synthesis of vascular smooth muscle cells and production of extracellular matrix components.

Endothelial cell calcium mobilization to acetylcholine is attenuated in copper-deficient rats.

Dietary copper deficiency significantly attenuates nitric oxide (NO)-mediated vascular smooth muscle relaxation and vasodilation. There is evidence for both increased inactivation of the NO radical by superoxide anion, and oxidative damage to the endothelium where NO is produced. The current study was designed to examine the NO synthetic pathway in the endothelium during copper deficiency. Male weanling rats were fed a copper-adequate (CuA, 6.4 mg Cu/kg diet) or copper-deficient (CuD, 0.4 mg Cu/kg diet) diet for four weeks. Cremasteric arterioles (approximately 100 microm diameter) were isolated and used for the experiments. Western blot analysis of the arteriole endothelial nitric oxide synthase (eNOS) concentration did not show a difference between dietary groups. Acetylcholine (Ach)-induced vasodilation was significantly reduced in the CuD group both before and after pretreatment with the eNOS substrate L-arginine. Endothelial intracellular calcium ([Ca2+]i) stimulated by 10(-6) M Ach was significantly inhibited in the arterioles from CuD rats. Coincident with the inhibition of [Ca2+]i and vasodilation was a depression of vascular Cu/Zn-SOD activity and an increase in plasma peroxynitrite activity. These data suggest that endothelial Ca2+ signaling and agonist-stimulated NO-mediated vascular dilation are likely reduced by increased oxidative damage in copper-deficient rats.

Pre-transplant donor-specific transfusions induce allograft rejection and IL-2 gene expression in the WKY-->F344 functional tolerance model of rat lung transplantation.

Our previous studies have shown that a spontaneous functional tolerance develops in a rat model of lung transplantation (WKY-->F344). The tolerance observed in this model may be due to the minor histocompatible differences in this combination, however, the possibility of a tolerogenic effect related specifically to the lung allograft must be considered. To further examine this model, the effect of pre-transplant donor-specific spleen cell transfusions (DSTs) was examined on the functional tolerance state seen in this model. F344 rats received WKY spleen cells on days -45 and -30 before lung transplantations. Control F344 rats received lung transplants without DSTs. Recipients in both groups were killed on day 7, 14, 21 and 49 post-transplant, and allograft rejection (AR) was graded histologically (stage 0-IV). Intragraft cytokine gene transcripts were examined on day 7 and 14 post-transplant using reverse transcriptase-polymerase chain reaction (RT-PCR) techniques to investigate the underlying immunological events occurring in each group. In addition, allogeneic (WKY) and third party (BN) skin grafts were placed on lung recipients at day 35 post-transplant to evaluate the development of systemic tolerance. It was seen that control animals showed moderate to severe lymphocytic infiltrations (stage II-III AR) in the first 3 weeks followed by spontaneous recovery with stage I-II AR on day 49. In marked contrast, DST-treated animals showed more aggressive AR with severe lymphocytic infiltration and haemorrhagic infarction (stage III-IV AR) by day 14-21, without any evidence of recovery on day 49. WKY skin grafts showed prolonged survival in control animals, but were promptly rejected in DST-treated animals. Intragraft cytokine gene expression in control animals was characterized by no or weak expression of IL-2 and high IL-10, while DST-treated animals showed high levels of IL-2 transcripts. IL-2:IL-10 and IL-2:IL-4 ratios were significantly increased in DST-treated animals compared with controls on day 7 post-transplant. It was concluded that pre-transplant DSTs did not enhance allograft survival, but actually induced AR and ablated any immunological benefit of the lung allograft on induction of tolerance in the WKY-->F344 lung transplant model. It was found that the DST-induced AR was associated with a deviation of cytokine immune responses from a predominant Th2 to Th1 profile characterized by increased IL-2 gene expression in the allografts. We also conclude that factors other than the degree of histocompatibility matching, such as the route and timing of alloantigen exposure, and the amount or nature of alloantigens associated specifically with lung allografts, are involved in deviating native immune responses toward acceptance or rejection of lung allografts in this model of lung transplantation.

Protein kinases and kinase-modulated effectors in the late phase of ischemic preconditioning.

In the 15 years since the first publication on ischemic preconditioning (PC), our knowledge of this phenomenon has increased exponentially. While the original studies described the early phase of ischemic PC, we now know that a second or late phase of ischemic PC also exists. In particular, the late phase of PC is triggered by factors such as adenosine, opioids, radicals, and nitric oxide. These factors in turn initiate a molecular chain reaction, which includes activation of serine/threonine kinases, tyrosine kinases, and mitogen-activated protein kinases. Subsequently, this cascade of reactions modulates a plethora of cardioprotective proteins including heat shock proteins, KATP channels, nitric oxide synthase, cyclooxygenase-2, and antioxidants. However, despite this phenomenal amount of information, the construction of a unifying hypothesis describing the signaling mechanism of late PC has proved challenging. The purpose of this article, therefore, is to review the current literature and hypothesis regarding the signaling system in the late phase of ischemic PC, to tackle areas of controversy within this model, and to address potential future directions of investigation that will hopefully promote the generation of a unifying paradigm.

Generation of antibodies to the urokinase receptor (uPAR) by DNA immunization of uPAR knockout mice: membrane-bound uPAR is not required for an antibody response.

The urokinase receptor (uPAR) is a glycolipid-anchored cell surface glycoprotein that plays a central role in extracellular proteolysis during tissue remodeling processes including cancer invasion. Furthermore, uPAR is found on the surface of both dendritic cells (DCs) and T cells, and has been proposed to play a role in DC-induced T-cell activation and, therefore, in the induction of an immune response. In order to investigate the possibility of using DNA immunization for the generation of poly- and monoclonal antibodies to uPAR, we injected wild-type mice and mice deficient in uPAR (uPAR knockouts) intramuscularly with plasmid DNA encoding a carboxy-terminal truncated soluble form of the human uPAR. Multiple injections of 100 micro g of DNA resulted in a strong and specific antibody response in all mice irrespective of genotype. Antisera with a maximum titre of 32,000 were obtained, comparable with that obtained after immunization with recombinant uPAR. The subclass distribution of uPAR-specific antibodies in the sera demonstrated the induction of a mixed TH1/TH2 response, irrespective of the genotype of the mice. Our results demonstrate the possibility of generating high titre antibodies to uPAR by DNA immunization of wild-type as well as uPAR knockout mice, and that cell surface uPAR is not indispensable for the generation of a humoral immune response.

Accuracy of presumptive criteria for culture diagnosis of Neisseria gonorrhoeae in low-prevalence populations of women.

The accuracy of presumptive criteria for identification of Neisseria gonorrhoeae was assessed in two separate populations of women with a low prevalence of gonorrhea. Of the presumptively positive cervical isolates available for confirmation, 98.5% were identified as N. gonorrhoeae. Of 25 isolates that could not be confirmed, 20 failed to grow on subculture, and of the remaining 5, only 2 (0.6% of all viable recoveries) were identified as nongonococcal isolates (N. meningitidis). Our study confirms earlier findings that meningococcal isolates are rarely found in endocervical specimens. The benefits from early treatment and counselling of women for gonorrhea on the basis of presumptive criteria outweight the risks occasioned by the rarely encountered nongonococcal isolate.

PKCepsilon activation induces dichotomous cardiac phenotypes and modulates PKCepsilon-RACK interactions and RACK expression.

Receptors for activated C kinase (RACKs) have been shown to facilitate activation of protein kinase C (PKC). However, it is unknown whether PKC activation modulates RACK protein expression and PKC-RACK interactions. This issue was studied in two PKCepsilon transgenic lines exhibiting dichotomous cardiac phenotypes: one exhibits increased resistance to myocardial ischemia (cardioprotected phenotype) induced by a modest increase in PKCepsilon activity (228 +/- 23% of control), whereas the other exhibits cardiac hypertrophy and failure (hypertrophied phenotype) induced by a marked increase in PKCepsilon activity (452 +/- 28% of control). Our data demonstrate that activation of PKC modulates the expression of RACK isotypes and PKC-RACK interactions in a PKCepsilon activity- and dosage-dependent fashion. We found that, in mice displaying the cardioprotected phenotype, activation of PKCepsilon enhanced RACK2 expression (178 +/- 13% of control) and particulate PKCepsilon-RACK2 protein-protein interactions (178 +/- 18% of control). In contrast, in mice displaying the hypertrophied phenotype, there was not only an increase in RACK2 expression (330 +/- 33% of control) and particulate PKCepsilon-RACK2 interactions (154 +/- 14% of control) but also in RACK1 protein expression (174 +/- 10% of control). Most notably, PKCepsilon-RACK1 interactions were identified in this line. With the use of transgenic mice expressing a dominant negative PKCepsilon, we found that the changes in RACK expression as well as the attending cardiac phenotypes were dependent on PKCepsilon activity. Our observations demonstrate that RACK expression is dynamically regulated by PKCepsilon and suggest that differential patterns of PKCepsilon-RACK interactions may be important determinants of PKCepsilon-dependent cardiac phenotypes.

Urokinase-catalysed cleavage of the urokinase receptor requires an intact glycolipid anchor.

Urokinase (uPA) has the striking ability to cleave its receptor, uPAR, thereby inactivating the binding potential of this molecule. Here we demonstrate that the glycosylphosphatidylinositol (GPI) anchor of uPAR, which is attached to the third domain, is an important determinant in governing this reaction, even though the actual cleavage occurs between the first and second domains. Purified full-length GPI-anchored uPAR (GPI-uPAR) proved much more susceptible to uPA-mediated cleavage than recombinant truncated soluble uPAR (suPAR), which lacks the glycolipid anchor. This was not a general difference in proteolytic susceptibility since GPI-uPAR and suPAR were cleaved with equal efficiency by plasmin. Since the amino acid sequences of GPI-uPAR and suPAR are identical except for the C-terminal truncation, the different cleavage patterns suggest that the two uPAR variants differ in the conformation or the flexibility of the linker region between domains 1 and 2. This was supported by the fact that an antibody to the peptide AVTYSRSRYLE, amino acids 84-94 in the linker region, recognizes GPI-uPAR but not suPAR. This difference in the linker region is thus caused by a difference in a remote hydrophobic region. In accordance with this model, when the hydrophobic lipid moiety was removed from the glycolipid anchor by phospholipase C, low concentrations of uPA could no longer cleave the modified GPI-uPAR and the reactivity to the peptide antibody was greatly decreased. Naturally occurring suPAR, purified from plasma, was found to have a similar resistance to uPA cleavage as phospholipase C-treated GPI-uPAR and recombinant suPAR.

Enhanced PKC beta II translocation and PKC beta II-RACK1 interactions in PKC epsilon-induced heart failure: a role for RACK1.

Recent investigations have established a role for the beta II-isoform of protein kinase C (PKC beta II) in the induction of cardiac hypertrophy and failure. Although receptors for activated C kinase (RACKs) have been shown to direct PKC signal transduction, the mechanism through which RACK1, a selective PKC beta II RACK, participates in PKC beta II-mediated cardiac hypertrophy and failure remains undefined. We have previously reported that PKC epsilon activation modulates the expression of RACKs, and that altered epsilon-isoform of PKC (PKC epsilon)-RACK interactions may facilitate the genesis of cardiac phenotypes in mice. Here, we present evidence that high levels of PKC epsilon activity are commensurate with impaired left ventricular function (dP/dt = 6,074 +/- 248 mmHg/s in control vs. 3,784 +/- 269 mmHg/s in transgenic) and significant myocardial hypertrophy. More importantly, we demonstrate that high levels of PKC epsilon activation induce a significant colocalization of PKC beta II with RACK1 (154 +/- 7% of control) and a marked redistribution of PKC beta II to the particulate fraction (17 +/- 2% of total PKC beta II in control mice vs. 49 +/- 5% of total PKC beta II in hypertrophied mice), without compensatory changes of the other eight PKC isoforms present in the mouse heart. This enhanced PKC beta II activation is coupled with increased RACK1 expression and PKC beta II-RACK1 interactions, demonstrating PKC epsilon-induced PKC beta II signaling via a RACK1-dependent mechanism. Taken together with our previous findings regarding enhanced RACK1 expression and PKC epsilon-RACK1 interactions in the setting of cardiac hypertrophy and failure, these results suggest that RACK1 serves as a nexus for at least two isoforms of PKC, the epsilon-isoform and the beta II-isoform, thus coordinating PKC-mediated hypertrophic signaling.