Spontaneous lung and lymph node metastasis in transgenic breast cancer is independent of the urokinase receptor uPAR.

Urokinase-type plasminogen activator (uPA) is an extracellular protease that plays a pivotal role in tumor progression. uPA activity is spatially restricted by its anchorage to high-affinity uPA receptors (uPAR) at the cell surface. High tumor tissue expression of uPA and uPAR is associated with poor prognosis in lung, breast, and colon cancer patients in clinical studies. Genetic deficiency of uPA leads to a significant reduction in metastases in the murine transgenic MMTV-PyMT breast cancer model, demonstrating a causal role for uPA in cancer dissemination. To investigate the role of uPAR in cancer progression, we analyze the effect of uPAR deficiency in the same cancer model. uPAR is predominantly expressed in stromal cells in the mouse primary tumors, similar to human breast cancer. In a cohort of MMTV-PyMT mice [uPAR-deficient (n = 31) or wild type controls (n = 33)], tumorigenesis, tumor growth, and tumor histopathology were not significantly affected by uPAR deficiency. Lung and lymph node metastases were also not significantly affected by uPAR deficiency, in contrast to the significant reduction seen in uPA-deficient mice. Taken together, our data show that the genetic absence of uPAR does not influence the outcome of the MMTV-PyMT cancer model.

Interleukin 20 protein locates to distinct mononuclear cells in psoriatic skin.

We previously demonstrated that mRNA for the pro-inflammatory cytokine interleukin 20 (IL-20) is expressed in suprapapillary keratinocytes of lesional psoriatic skin (LS). Here, we describe the distribution of IL-20 protein and the identity of the IL-20-positive cells in LS. We found that the main part of IL-20 immunoreactivity is present in mononuclear cells of the dermal papillae, and that the IL-20-positive cells located in the papillae were langerin+, CD1a+, CD4+ and CD303+. These cells might be immature dendritic cell. In situ hybridization for IL-20 mRNA on non-LS, ex vivo stimulated with IL-1ß revealed a colocalization between IL-20 mRNA and the keratinocyte marker CK14. No IL-20 mRNA was detected in the dermal mononuclear cells. Our results suggest that IL-20 is produced by keratinocytes, released into the epidermis and then possibly taken up by papillary mononuclear cells. Our study supports that IL-20 is involved in the pathogenesis of psoriasis.

Ultrasound colour Doppler is associated with synovial pathology in biopsies from hand joints in rheumatoid arthritis patients: a cross-sectional study.

OBJECTIVES: Little is known regarding the association between ultrasound-determined pathological synovial blood flow and synovial pathology in rheumatoid arthritis (RA). We therefore examined the association between colour Doppler ultrasound imaging and synovitis assessed by histopathology and specific cell markers by immunohistochemistry in patients with RA. METHODS: 81 synovial sites from wrist and finger joints from 29 RA patients were evaluated by ultrasound colour Doppler and subsequently biopsied by needle arthroscopy. The association between ultrasound colour fraction and an overall synovitis score and immunohistochemical staining for CD3, CD68, Ki67 and von Willebrand factor was investigated, including repeated samples from the same patients. The overall synovitis score (total 0-9) assessed synovial lining hyperplasia (0-3), stromal activation (0-3) and inflammatory infiltration (0-3). Data were clustered within patients, thus a linear mixed model was applied for the statistical tests. Parsimony in the statistical models was achieved omitting covariates from the model in the case of what was judged no statistical significance (p>0.1). RESULTS: Doppler colour fraction showed an association with the overall synovitis score (approximated Spearman, approximately r=0.43, p=0.003). The density of all immunohistochemical stainings showed a significant association with Doppler colour fraction: von Willebrand factor (approximately r=0.44, p=0.01), CD68 (approximately r=0.53, p=0.02), Ki67 (approximately r=0.57, p=0.05) and CD3 (approximately r=0.57, p=0.0003). CONCLUSIONS: Colour Doppler activity is associated with the extent of inflammation present in the synovial biopsies from RA patients. However, synovial pathology was also seen in biopsies taken from Doppler negative sites.

Spontaneous metastasis in congenic mice with transgenic breast cancer is unaffected by plasminogen gene ablation.

Plasminogen (Plg) plays a central role in tissue remodeling during ontogeny, development, and in pathological tissue remodeling following physical injury, inflammation and cancer. Plg/plasmin is, however, not critical for these processes, as they all occur to a varying extent in its absence, suggesting that there is a functional redundancy with other proteases. To explore this functional overlap in the transgenic MMTV-PyMT breast cancer metastasis model, we have combined Plg deficiency and a pharmacological metalloprotease inhibitor, which is known to reduce metastasis in this model, and has been shown to synergistically inhibit other tissue remodeling events in Plg-deficient mice. While metalloprotease inhibition dramatically reduced metastasis, we found no effect of Plg deficiency on metastasis, either independently or in combination with metalloprotease inhibition. We further show that Plg gene deficiency is of no significant consequence in this metastasis model, when analyzed in two different congenic strains: the FVB strain, and a F1 hybrid of the FVB and C57BL/6J strains. We suggest that the extensive backcrossing performed prior to our studies has eliminated the confounding effect of a known polymorphic metastasis modifier gene region located adjacent to the Plg gene.

Concomitant lack of MMP9 and uPA disturbs physiological tissue remodeling.

Urokinase-type plasminogen activator (uPA) and matrix metalloproteinase-9 (MMP9, gelatinase B) have separately been recognized to play important roles in various tissue remodeling processes. In this study, we demonstrate that deficiency for MMP9 in combination with ablation of either uPA- or tissue-type plasminogen activator (tPA)-catalyzed plasminogen activation is critical to accomplish normal gestation in mice. Gestation was also affected by simultaneous lack of MMP9 and the uPA receptor (uPAR). Interestingly, uPA-deficiency additionally exacerbated the effect of MMP9-deficiency on bone growth and an additive effect caused by combined lack in MMP9 and uPA was observed during healing of cutaneous wounds. By comparison, MMP9-deficiency combined with absence of either tPA or uPAR resulted in no significant effect on wound healing, indicating that the role of uPA during wound healing is independent of uPAR, when MMP9 is absent. Notably, compensatory upregulation of uPA activity was seen in wounds from MMP9-deficient mice. Taken together, these studies reveal essential functional dependency between MMP9 and uPA during gestation and tissue repair.

Neutralisation of uPA with a monoclonal antibody reduces plasmin formation and delays skin wound healing in tPA-deficient mice.

BACKGROUND: Proteolytic degradation by plasmin and metalloproteinases is essential for epidermal regeneration in skin wound healing. Plasminogen deficient mice have severely delayed wound closure as have mice simultaneously lacking the two plasminogen activators, urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA). In contrast, individual genetic deficiencies in either uPA or tPA lead to wound healing kinetics with no or only slightly delayed closure of skin wounds. METHODOLOGY/PRINCIPAL FINDINGS: To evaluate the therapeutic potential in vivo of a murine neutralizing antibody directed against mouse uPA we investigated the efficacy in skin wound healing of tPA-deficient mice. Systemic administration of the anti-mouse uPA monoclonal antibody, mU1, to tPA-deficient mice caused a dose-dependent delay of skin wound closure almost similar to the delayed kinetics observed in uPA;tPA double-deficient mice. Analysis of wound extracts showed diminished levels of plasmin in the mU1-treated tPA-deficient mice. Immunohistochemistry revealed that fibrin accumulated in the wounds of such mU1-treated tPA-deficient mice and that keratinocyte tongues were aberrant. Together these abnormalities lead to compromised epidermal closure. CONCLUSIONS/SIGNIFICANCE: Our findings demonstrate that inhibition of uPA activity with a monoclonal antibody in adult tPA-deficient mice mimics the effect of simultaneous genetic ablation of uPA and tPA. Thus, application of the murine inhibitory mU1 antibody provides a new and highly versatile tool to interfere with uPA-activity in vivo in mouse models of disease.

Skin wound healing in MMP2-deficient and MMP2 / plasminogen double-deficient mice.

During healing of incisional skin wounds, migrating keratinocytes dissect their way under the crust to re-epithelialize the wounded area. The efficiency of this tissue remodelling process depends on the concomitant activity of several extracellular proteases, including members of the plasminogen activation (PA) system and the matrix metalloproteinase (MMP) family. Treatment with the broad spectrum MMP inhibitor, galardin, delays wound healing in wildtype mice and completely arrest wound healing in plasminogen (Plg)-deficient mice, indicating a functional overlap between plasmin- and galardin-sensitive MMPs during wound healing. To address whether MMP2 is accountable for the galardin-induced healing deficiency in wildtype and Plg-deficient mice, incisional skin wounds were generated in MMP2 single-deficient mice and in MMP2/Plg double-deficient mice and followed until healed. Alternatively, tissue was isolated 7 days post wounding for histological and biochemical analyses. No difference was found in the time from wounding to overt gross restoration of the epidermal surface between MMP2-deficient and wildtype control littermate mice. MMP2/Plg double-deficient mice were viable and fertile, and displayed an unchallenged general phenotype resembling that of Plg-deficient mice, including development of rectal prolapses. MMP2/Plg double-deficient mice displayed a slight increase in the wound length throughout the healing period compared with Plg-deficient mice. However, the overall time to complete healing was not significantly different between Plg-deficient and MMP2/Plg double-deficient mice. These results show that MMP2 activity is not essential for wound healing and indicate that lack of MMP2 only marginally potentiates the effect of Plg deficiency.

Pancreatic beta-cell overexpression of the glucagon receptor gene results in enhanced beta-cell function and mass.

In addition to its primary role in regulating glucose production from the liver, glucagon has many other actions, reflected by the wide tissue distribution of the glucagon receptor (Gcgr). To investigate the role of glucagon in the regulation of insulin secretion and whole body glucose homeostasis in vivo, we generated mice overexpressing the Gcgr specifically on pancreatic beta-cells (RIP-Gcgr). In vivo and in vitro insulin secretion in response to glucagon and glucose was increased 1.7- to 3.9-fold in RIP-Gcgr mice compared with controls. Consistent with the observed increase in insulin release in response to glucagon and glucose, the glucose excursion resulting from both a glucagon challenge and intraperitoneal glucose tolerance test (IPGTT) was significantly reduced in RIP-Gcgr mice compared with controls. However, RIP-Gcgr mice display similar glucose responses to an insulin challenge. beta-Cell mass and pancreatic insulin content were also increased (20 and 50%, respectively) in RIP-Gcgr mice compared with controls. When fed a high-fat diet (HFD), both control and RIP-Gcgr mice developed similar degrees of obesity and insulin resistance. However, the severity of both fasting hyperglycemia and impaired glucose tolerance (IGT) were reduced in RIP-Gcgr mice compared with controls. Furthermore, the insulin response of RIP-Gcgr mice to an IPGTT was twice that of controls when fed the HFD. These data indicate that increased pancreatic beta-cell expression of the Gcgr increased insulin secretion, pancreatic insulin content, beta-cell mass, and, when mice were fed a HFD, partially protected against hyperglycemia and IGT.

Spontaneous metastasis in matrix metalloproteinase 3-deficient mice.

Matrix metalloproteinases (MMPs) have been linked to the metastatic potential of tumor cells due to their ability to degrade the extracellular matrix. MMP-3 (stromelysin-1) is upregulated in a wide variety of human tumors. We used the MMTV-PyMT breast cancer model to determine if MMP-3 is involved in tumorigenesis and metastatic growth. In this model the stromal expression of MMP-3 mRNA resembles the predominant MMP-3 expression pattern observed in human ductal breast carcinomas. We studied a cohort of 63 PyMT transgenic mice, either deficient for MMP-3 or wild-type controls. The degree of metastasis did not differ significantly between the two groups of mice, although the median lung metastasis volume was more than threefold increased in MMTV-PyMT mice deficient in MMP-3. Likewise, primary tumor growth rate and lymph node metastasis were not significantly affected by MMP-3-deficiency. By comparing mRNA levels in MMP-3-deficient PyMT tumors with PyMT wild-type tumors we excluded compensatory transcriptional changes of other MMPs or their specific inhibitors. Thus, we conclude that genetic ablation of MMP-3 does not significantly affect tumor growth and metastasis in the MMTV-PyMT model.

Metastasis is strongly reduced by the matrix metalloproteinase inhibitor Galardin in the MMTV-PymT transgenic breast cancer model.

Matrix metalloproteinases (MMP) have several roles that influence cancer progression and dissemination. However, low molecular weight metalloproteinase inhibitors (MPI) have not yet been tested in transgenic/spontaneous metastasis models. We have tested Galardin/GM6001, a potent MPI that reacts with most MMPs, in the MMTV-PymT transgenic breast cancer model. We followed a cohort of 81 MMTV-PymT transgenic mice that received Galardin, placebo, or no treatment. Galardin treatment was started at age 6 weeks with 100 mg/kg/d, and all mice were killed at age 13.5 weeks. Galardin treatment significantly reduced primary tumor growth. Final tumor burden in Galardin-treated mice was 1.69 cm3 compared with 3.29 cm3 in placebo-treated mice (t test, P = 0.0014). We quantified the total lung metastasis volume in the same cohort of mice. The median metastasis volume was 0.003 mm(3) in Galardin-treated mice compared with 0.56 mm(3) in placebo-treated mice (t test, P < 0.0001). Thus, metastasis burden was reduced more than 100-fold, whereas primary tumor size was reduced only 2-fold. We also found that primary tumors from Galardin-treated mice exhibited a lower histopathologic tumor grade, increased collagen deposition, and increased MMP-2 activity. MMPs are known to have tumor-promoting and tumor-inhibitory effects, and several clinical trials of broad-spectrum MPIs have failed to show promising effects. The very potent antimetastatic effect of Galardin in the MMTV-PymT model does, however, show that it may be possible to find broad-spectrum MPIs with favorable inhibition profiles, or perhaps combinations of monospecific MPIs, for future clinical application.

Antibody-mediated targeting of the urokinase-type plasminogen activator proteolytic function neutralizes fibrinolysis in vivo.

Urokinase-type plasminogen activator (uPA) plays a central role in tissue remodeling processes. Most of our understanding of the role of uPA in vivo is derived from studies using gene-targeted uPA-deficient mice. To enable in vivo studies on the specific interference with uPA functionality in mouse models, we have now developed murine monoclonal antibodies (mAbs) directed against murine uPA by immunization of uPA-deficient mice with the recombinant protein. Guided by enzyme-linked immunosorbent assay, Western blotting, surface plasmon resonance, and enzyme kinetic analyses, we have selected two highly potent and inhibitory anti-uPA mAbs (mU1 and mU3). Both mAbs recognize epitopes located on the B-chain of uPA that encompasses the catalytic site. In enzyme activity assays in vitro, mU1 blocked uPA-catalyzed plasminogen activation as well as plasmin-mediated pro-uPA activation, whereas mU3 only was directed against the first of these reactions. We additionally provide evidence that mU1, but not mU3, successfully targets uPA-dependent processes in vivo. Hence, systemic administration of mU1 (i) rescued mice treated with a uPA-activable anthrax protoxin and (ii) impaired uPA-mediated hepatic fibrinolysis in tissue-type plasminogen activator (tPA)-deficient mice, resulting in a phenotype mimicking that of uPA;tPA double deficient mice. Importantly, this is the first report demonstrating specific antagonist-directed targeting of mouse uPA at the enzyme activity level in a normal physiological process in vivo.

Expression of the GLP-1 receptor in mouse, rat, and human pancreas.

We studied the intra-islet localization of the glucagon-like peptide 1 receptor (GLP-1R) by colocalization studies of the GLP-1R mRNA and protein with islet cell hormones in mice, rats, and humans. In contrast to previous reports, we show that the GLP-1R is selectively located on the beta cells. The localization of GLP-1R in islets and ducts was studied using ISH and double and triple fluorescence microscopy. In normal pancreatic tissue from mice and rats, GLP-1R mRNA was only detectable in the beta cells. Double and triple immunofluorescence using two different GLP-1R antisera and combinations of insulin, glucagon, pancreatic polypeptide, and somatostatin showed that GLP-1R protein is almost exclusively colocalized with insulin. The same pattern was observed in human pancreas, but the GLP-1R expression was more heterogeneous, with populations of insulin immunoreactive cells with high and low expression. This is the first time that the GLP-1R has been localized in human islets. Furthermore, GLP-1R immunoreactivity was found in the pancreatic ducts in mouse, rat, and human pancreas. As an important confirmation of the specificity of our methods, we found no signals for GLP-1R mRNA or protein in pancreatic tissue from gene-targeted GLP-1R-deficient mice. In conclusion, our data suggest that the GLP-1 receptor is restricted to the pancreatic beta cells and the lack of receptor immunoreactivity on delta cells cannot be explained suitably to correspond with published in vivo and in vitro data. This manuscript contains online supplemental material at http://www.jhc.org. Please visit this article online to view these materials.

Profibrinolytic effects of metalloproteinases during skin wound healing in the absence of plasminogen.

Genetic ablation of plasminogen (Plg) and pharmacological inhibition of metalloproteinase activity by galardin delay skin wound healing in mice, whereas the combined inhibition of these two enzyme systems completely prevents healing. In this study, the impact of plasmin and metalloproteinases as profibrinolytic enzymes has been investigated by comparing skin wound healing in the absence and presence of fibrin. Plg deficiency impairs skin wound healing kinetics, but this delay is only partially restored in the absence of fibrin. This suggests that plasmin-mediated fibrinolysis is the primary, but not the exclusive, requirement for healing of wounds in these mice. In addition, we observe that lack of fibrin reduces Plg activation significantly during wound healing. The profibrinolytic role of metalloproteinases is revealed by the finding that lack of fibrin partially restores the otherwise arrested healing of Plg-deficient wounds after metalloproteinase inhibition. In conclusion, the residual impairment of skin wound healing in the absence of fibrin suggests the existence of a fibrin-independent substrate(s) for plasmin and metalloproteinases. Furthermore, these in vivo data reveal that galardin-sensitive metalloproteinases mediate compensatory fibrinolysis to facilitate wound healing in the absence of plasmin.

Sealing of gastrointestinal anastomoses with a fibrin glue-coated collagen patch: a safety study.

Sealing of anastomoses has previously been tested with several methods, including sealing with liquid fibrin glue. Sealing with a collagen patch coated with fibrin glue components has never been systematically examined. The aim of the present study was to determine the safety of sealing gastrointestinal anastomoses with a collagen patch coated with fibrin glue. The study is a prospective, experimental animal study comparing sealed and unsealed gastrointestinal anastomoses. Laparotomy was performed in 11 pigs under general anesthesia. In each pig two anastomoses were performed on the small intestine. One of the anastomoses was sealed with a collagen patch coated with fibrin glue components (TachoSil). The other anastomosis contained no sealing. The pigs were observed for 1 to 6 weeks. The observation period was followed by in vivo examination under general anesthesia and included observation for anastomotic leakage, signs of present or former peritonitis, abscess, adhesions to the anastomoses, and signs of intestinal obstruction. In addition, the anastomotic diameter was measured with barium and radiography. Finally, bursting pressure was measured in each segment. After the pigs were sacrificed, the bowel segments were microscopically examined. There were no differences between the sealed and the unsealed anastomoses with respect to abdominal pathology, in vivo bursting pressure, or degree of stenosis. The collagen fleeces were in situ in all anastomoses. Microscopically, we found no difference in healing or signs of infection.

Murine monoclonal antibodies against murine uPA receptor produced in gene-deficient mice: inhibitory effects on receptor-mediated uPA activity in vitro and in vivo.

Binding of urokinase plasminogen activator (uPA) to its cellular receptor, uPAR, potentiates plasminogen activation and localizes it to the cell surface. Focal plasminogen activation is involved in both normal and pathological tissue remodeling processes including cancer invasion. The interaction between uPA and uPAR therefore represents a potential target for anti-invasive cancer therapy. Inhibitors of the human uPA-uPAR interaction have no effect in the murine system. To enable in-vivo studies in murine cancer models we have now generated murine monoclonal antibodies (mAbs) against murine uPAR (muPAR) by immunizing uPAR-deficient mice with recombinant muPAR and screened for antibodies, which inhibit the muPA-muPAR interaction. Two of the twelve mAbs obtained, mR1 and mR2, interfered with the interaction between muPAR and the amino-terminal fragment of muPA (mATF) when analyzed by surface plasmon resonance. The epitope for mR1 is located on domain I of muPAR, while that of mR2 is on domains (II-III). In cell binding experiments using radiolabelled mATF, the maximal inhibition obtained with mR1 was 85% while that obtained with mR2 was 50%. The IC(50) value for mR1 was 0.67 nM compared to 0.14 nM for mATF. In an assay based on modified anthrax toxins, requiring cell-bound muPA activity for its cytotoxity, an approximately 50% rescue of the cells could be obtained by addition of mR1. Importantly, in-vivo efficacy of mR1 was demonstrated by the ability of mR1 to rescue mice treated with a lethal dose of uPA-activatable anthrax toxins.

Extracellular proteolysis in transgenic mouse models of breast cancer.

Growth and invasion of breast cancer require extracellular proteolysis in order to physically restructure the tissue microenvironment of the mammary gland. This pathological tissue remodeling process depends on a collaboration of epithelial and stromal cells. In fact, the majority of extracellular proteases are provided by stromal cells rather than cancer cells. This distinct expression pattern is seen in human breast cancers and also in transgenic mouse models of breast cancer. The similar expression patterns suggest that transgenic mouse models are ideally suited to study the role of extracellular proteases in cancer progression. Here we give a status report on protease intervention studies in transgenic models. These studies demonstrate that proteases are involved in all stages of breast cancer progression from carcinogenesis to metastasis. Transgenic models are now beginning to provide vital mechanistic insight that will allow us to combat breast cancer invasion and metastasis with new protease-targeted drugs.

The plasminogen fibrinolytic pathway is required for hematopoietic regeneration.

Hematopoietic stem cells within the bone marrow exist in a quiescent state. They can differentiate and proliferate in response to hematopoietic stress (e.g., myelosuppression), thereby ensuring a well-regulated supply of mature and immature hematopoietic cells within the circulation. However, little is known about how this stress response is coordinated. Here, we show that plasminogen (Plg), a classical fibrinolytic factor, is a key player in controlling this stress response. Deletion of Plg in mice prevented hematopoietic stem cells from entering the cell cycle and undergoing multilineage differentiation after myelosuppression, leading to the death of the mice. Activation of Plg by administration of tissue-type plasminogen activator promoted matrix metalloproteinase-mediated release of Kit ligand from stromal cells, thereby promoting hematopoietic progenitor cell proliferation and differentiation. Thus, activation of the fibrinolytic cascade is a critical step in regulating the hematopoietic stress response.

Lack of plasminogen leads to milk stasis and premature mammary gland involution during lactation.

The extracellular serine protease, plasmin, is activated from its precursor, plasminogen (Plg), by the urokinase-type and tissue-type Plg activators (uPA and tPA respectively). One of the main plasmin substrates, fibrin, is formed from fibrinogen via thrombin activity. We have previously shown that mice deficient for Plg are strikingly less able to support a litter during lactation compared to wild type mice. Here we suggest a mechanism responsible for this lactation defect. Reduced epithelial content and increased apoptosis are observed in Plg-deficient mammary glands at lactation day 7. Immunofluorescence analysis reveals the presence of fibrin(ogen) in the stroma surrounding mammary alveoli and adipocytes and identifies fibrin(ogen) as a component of breast milk in both wild type and Plg-deficient mice. Furthermore, a large accumulation of fibrin(ogen) together with apoptotic epithelial cells is observed in the lactating mammary alveoli and ducts of some Plg-deficient mice. This suggests that fibrin plays a key role in the malfunction of mammary glands in the absence of Plg, possibly through blockade of mammary ducts inducing milk stasis, inhibiting milk expulsion and thereby inducing premature apoptosis and involution.

Plasminogen activation independent of uPA and tPA maintains wound healing in gene-deficient mice.

Simultaneous ablation of the two known activators of plasminogen (Plg), urokinase-type (uPA) and the tissue-type (tPA), results in a substantial delay in skin wound healing. However, wound closure and epidermal re-epithelialization are significantly less impaired in uPA;tPA double-deficient mice than in Plg-deficient mice. Skin wounds in uPA;tPA-deficient mice treated with the broad-spectrum matrix metalloproteinase (MMP) inhibitor galardin (N-[(2R)-2-(hydroxamido-carbonylmethyl)-4-methylpentanoyl]-L-tryptophan methylamide) eventually heal, whereas skin wounds in galardin-treated Plg-deficient mice do not heal. Furthermore, plasmin is biochemically detectable in wound extracts from uPA;tPA double-deficient mice. In vivo administration of a plasma kallikrein (pKal)-selective form of the serine protease inhibitor ecotin exacerbates the healing impairment of uPA;tPA double-deficient wounds to a degree indistinguishable from that observed in Plg-deficient mice, and completely blocks the activity of pKal, but not uPA and tPA in wound extracts. These findings demonstrate that an additional plasminogen activator provides sufficient plasmin activity to sustain the healing process albeit at decreased speed in the absence of uPA, tPA and galardin-sensitive MMPs and suggest that pKal plays a role in plasmin generation.

Antitumor efficacy of a urokinase activation-dependent anthrax toxin.

Previously, we have generated a potent prodrug consisting of modified anthrax toxins that is activated by urokinase plasminogen activator (uPA). The cytotoxicity of the drug, PrAg-U2 + FP59, is dependent on the presence of receptor-associated uPA activity. Local intradermal administration of PrAg-U2 + FP59 adjacent to the tumor nodules in mice with transplanted solid tumors had a potent antitumor effect. In succession of these experiments, we have now investigated the systemic antitumor efficacy of PrAg-U2 + FP59. C57Bl/6J mice bearing syngenic tumors derived from B16 melanoma, T241 fibrosarcoma, or Lewis lung carcinoma cells were treated with different mass ratios and doses of PrAg-U2 + FP59. Tumor volumes were recorded daily by caliper measurements. In some experiments, dexamethasone was coadministered. Our data show a significant antitumor effect of systemic administration of PrAg-U2 + FP59 in three syngenic tumor models. Optimal antitumor effect and low toxicity was obtained with a 25:1 mass ratio between the two components (PrAg-U2 and FP59). The experiments show that PrAg-U2 + FP59 displays a clear dose-response relationship with regard to both antitumor efficacy and systemic toxicity. Dose-limiting toxicity seemed to be due to activation of the prodrug by uPA and its receptor in the intestinal mucosa. Concurrent treatment with dexamethasone was found to prevent dose-limiting toxicity. Taken together, these data indicate that uPA-activated toxins may be promising candidates for targeted therapy of human cancers that overexpress uPA and its receptor.