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Here we show the increase of invasion of three breast cancer cell lines (8701-BC, MDA-MB-231 and SKBR3) upon long-term co-incubation with culture medium of normal microvascular endothelial cells (MVEC) and normal breast epithelial cells (HB2). The enhancement of invasion relied on the interaction of microvascular endothelial cell and normal breast epithelial cell CXCL12 (SDF1) chemokine, whose expression by breast cancer cells was very low, with the cognate CXCR4 receptor of malignant cells, which resulted in over-expression of the urokinase-type plasminogen activator receptor (uPAR) on their surfaces. uPAR over-expression, showed by RT-PCR and Western blotting, was paralleled by increased urokinase-type plasminogen activator (uPA) partitioning on the cell surface with respect to the fluid phase, as demonstrated by zymography. Long-term interaction of SDF1 with CXCR4 stimulated sustained activation of JNK phosphorylation. Blocking antibodies to CXCR4 were able to block the endothelial/epithelial cell-dependent enhancement of invasion, as well as to inhibit SDF1-CXCR4-dependent JNK phosphorylation and uPAR over-expression of malignant cells. We suggest that acquisition of the angiogenic phenotype by breast cancer cells triggers an amplification loop, in which endothelial cells and normal breast epithelial cells of the tumour cooperate to provide facilitated routes to cell invasion and metastasis and to enhance the aggressive phenotype of cancer cells., (Copyright (c) 2008 Pathological Society of Great Britain and Ireland)

Alveolar osteitis (AO) is characterized by excess fibrinolysis, leading to early dissociation of the clot that normally follows tooth extraction. Nonetheless, scarce information is available on the fibrinolytic system in AO. In this study, we report on the differential composition of postextraction wound healing tissue and of peri-alveolar gingival epithelium from normal healing and AO patients in terms of plasminogen activators, plasminogen activator inhibitor-type 1, and urokinase-type plasminogen activator receptor. Plasminogen activators were studied by overlay zymography, western blotting, and enzyme-linked immunosorbent assay (ELISA). Plasminogen activator inhibitor-type 1 and urokinase receptor were measured by ELISA. In AO, the fibrinolytic activity of wound healing tissue was accounted for by an increase ( approximately 85%) of urokinase-type plasminogen activator, whereas tissue-type plasminogen activator was unchanged. Plasminogen activator inhibitor-type 1 showed a 6.7-fold increase in AO. These results point to key roles of urokinase in AO hyper-fibrinolysis and of plasminogen activator inhibitor type-1 in slowing down the healing response. Peri-alveolar gingival epithelium in AO showed an overall decrease of all the components of the fibrinolytic system, including the urokinase receptor, which indicates a decrease of the migration properties of epithelial cells.

Objective: In systemic sclerosis (SSc) microvascular endothelial cells (MVECs), angiogenesis is blocked by matrix metalloproteinase 12-dependent cleavage of domain 1 of the urokinase-type plasminogen activator receptor (uPAR). Since integrins are associated with the invasive activity of uPAR in angiogenesis, this study was undertaken to show whether full-size and truncated uPAR are differentially associated with integrins and with motor components of the cytoskeleton., Methods: SSc and normal MVECs were isolated from human skin biopsy specimens and studied by confocal laser scanning microscopy and immunoprecipitation to assess the mechanisms of association of truncated and full-size uPAR with integrins and the actin cytoskeleton. The integrin composition of the MVECs was studied by reverse transcription-polymerasechain reaction. Cell migration and capillary morphogenesis were studied on fibrinogen substrates. Involvement of Rac and Cdc42 was evaluated by Western blotting., Results: Only full-size uPAR showed a connection with the actin cytoskeleton in ECs. This connection was mediated by the uPAR-associated alphaMu- and alphaX-subunits of beta2 integrin, and was absent from SSc MVECs. The cleaved uPAR was not associated with beta2 integrins or with actin. beta3 integrins were associated with both the full-size and cleaved uPAR at focal contacts. The uncoupling of uPAR from beta2 integrins in SSc MVECs impaired the activation of Rac and Cdc42 (thus inhibiting their mediation of uPAR-dependent cytoskeletal rearrangements and cell motility) and blocked the integrin-engagement-delivered signals to the actin cytoskeleton. Invasion and capillary morphogenesis on fibrinogen-coated substrates indicated that ligation of uPAR by uPA empowers the beta2/beta3 integrin-dependent invasion of fibrinogen, and that this system is impaired in SSc MVECs., Conclusion: The reduced angiogenic properties of SSc MVECs can be explained by the effects of uPAR truncation and the subsequent loss of the beta2 integrin-mediated connection of uPAR with the actin cytoskeleton in these ECs.

Failure of endothelial cells to develop new vessels in response to hypoxia is a distinctive feature of systemic sclerosis (SSc) in the avascular phase. We have previously shown that SSc endothelial cells over-express matrix metalloproteinase-12 (MMP-12), which blocks angiogenesis by cleavage of the endothelial urokinase-type plasminogen activator receptor (uPAR). In the present study, we have investigated whether over-expression of MMP-12 and of angiostatic factors, or hypo-expression of angiogenic factors by SSc fibroblasts, contributes to impaired angiogenesis in SSc. Dermal fibroblasts were isolated from healthy subjects (N-Fb) and patients with diffuse SSc (SSc-Fb). Angiogenesis of target normal human microvascular endothelial cells (H-MVECs) was assayed by Matrigel invasion, cell proliferation, and capillary morphogenesis. uPAR cleavage and MMP-12 activity were evaluated by western blotting. We show that the over-expression of MMP-12 by SSc-Fb determines uPAR cleavage in H-MVECs. Conditioned medium from SSc-Fb impaired H-MVEC proliferation, invasion, and capillary morphogenesis. Anti-MMP-12 antibodies restored such impairment. Altered expression of angiostatic/angiogenic factors, including transforming growth factor beta1, did not account for SSc-Fb-dependent impairment of angiogenesis. The over-expression of MMP-12 by both SSc-Fb and SSc endothelial cells indicates that MMP-12 over-production may have a critical pathogenic role in SSc-associated vascular alterations.

The aggressive and malignant nature of AIDS-associated Kaposi's sarcoma (KS) lesions have largely been ascribed to Tat, the HIV-1 transactivator protein. Among other activities, HIV-Tat induces the migration and invasion of KS and endothelial cells. Since cell invasion is strictly correlated to the activity of lytic enzymes, we elucidated the role of the cell-associated plasminogen activation system in Tat-dependent and in constitutive invasion and proliferation of KS and of microvascular endothelial cells (MVEC). We demonstrate that KS cells and MVEC express the u-PA receptor (u-PAR) and release plasminogen activators and plasminogen activator inhibitor type-1 (PAI-1). The urokinase-type plasminogen activator (u-PA) is chemotactic, chemoinvasive and mitogenic for KS cells and for MVEC. Conditioned medium from KS cells induced invasion and proliferation of MVEC through the u-PA/u-PAR system. Tat is motogenic and mitogenic on KS cells and MVEC, and stimulates morphogenesis of MVEC. These activities were inhibited following antagonization of u-PA and u-PAR, which also reduced constitutive proliferation and invasion of KS cells and MVEC. These data indicate that the u-PA/u-PAR/PAI-1 system is involved in KS-induced endothelial cell invasion, proliferation, and differentiation. Further, exogenous Tat protein could up-regulate the fibrinolytic system, increasing its influence on KS and endothelial cell proliferation and migration, potentially promoting KS progression. These observations suggest the potential for application of u-PA/u-PAR system inhibitors for control of AIDS-associated KS, that has a high risk of recurrence with highly active antiretroviral therapy failure, and of other KS forms.

An important factor implicated in tumor cell predisposition for invasion and metastasis is the malignancy-related upregulation of urokinase plasminogen activator receptor (uPAR). uPAR signals by activating different tyrosine kinases in different cells. We examined the effects of inhibiting uPAR signaling by inhibition of uPAR expression with antisense oligonucleotides (aODNs) in PC3 human prostate cancer cells and evaluated aODN effect in a mouse model of prostate cancer bone metastasis. Following uPAR aODN treatment, PC3 cells exhibited a strong decrease in uPAR expression, evaluated by flow cytometry and by polymerase chain reaction, and of FAK/JNK/Jun phosphorylation. The synthesis of cyclins A, B, D1 and D3 was inhibited, as shown by Western blotting, flow cytometry and polymerase chain reaction, and PC3 cells accumulated in the G2 phase of the cell cycle. PC3 cells' adhesion was unaffected, while proliferation and invasion of Matrigel were impaired. A total of 60 mice were subjected to intracardiac injection of PC3 cells and were randomly assigned to three groups: aODN (treated with 0.5 mg intraperitoneum/mouse/day), dODN (treated with the same amounts of a degenerated ODN) and control (injected with a saline solution). At 28 days after heart injection, mice were subjected to a digital scan of total body radiography, which revealed 80% reduction in mice affected by bone metastasis. The use of uPAR aODNs produced a substantial prophylactic effect against prostate cancer bone metastasis, which has to be ascribed to downregulation of uPAR expression.

Objective: Defective angiogenesis, resulting in tissue ischemia, is particularly prominent in the diffuse form of systemic sclerosis (SSc). The present study was undertaken to identify possible differences between normal and SSc microvascular endothelial cells (MVECs) in the expression of the cell-associated urokinase-type plasminogen activator (uPA)/uPA receptor (uPAR) system, which is critical in the angiogenic process., Methods: MVECs were isolated from the dermis of healthy individuals and from the dermis of patients with diffuse SSc. The uPA/uPAR system was examined at the protein and messenger RNA levels. Angiogenesis was assayed on Matrigel-coated porous filters and plates to evaluate cell proliferation, invasion, and capillary morphogenesis. Cleavage of uPAR and the activity of matrix metalloproteinase 12 (MMP-12) were evaluated by Western blotting., Results: Compared with MVECs from healthy skin, MVECs from SSc patients showed higher expression of uPAR. However, in SSc MVECs, uPAR undergoes truncation between domain 1 and domain 2, as shown by flow cytometry, enzyme-linked immunosorbent assay, and Western blotting, a cleavage that is known to impair uPAR functions. These properties of SSc MVECs were associated with poor spontaneous and uPA-dependent invasion, proliferation, and capillary morphogenesis. The uPAR cleavage occurring in SSc MVECs was associated with overexpression of MMP-12. SSc MVEC-conditioned medium impaired uPA-dependent proliferation and invasion as well as capillary morphogenesis in normal MVECs in vitro. Both a general hydroxamate inhibitor of MMP activity and anti-MMP-12 antibodies restored this SSc MVEC-induced impaired functioning., Conclusion: Overproduction of MMP-12 by SSc MVECs accounts for the cleavage of uPAR and the impairment of angiogenesis in vitro and may contribute to reduced angiogenesis in SSc patients., (Copyright 2004 American College of Rheumatology)

We have targeted the urokinase-type plasminogen activator receptor (uPAR) with phosphorothioate antisense oligonucleotides (aODN) in vitro to evaluate the anti-invasive and anti-proliferative effects of uPAR down-regulation, as well as in vivo to evaluate anti-tumor and anti-metastatic activity. aODN-dependent uPAR downregulation in vitro was induced in cells of human melanoma, mammary carcinoma, ovarian carcinoma and SV-40-transformed embryonic lung fibroblasts. uPAR was determined by an antibody-based assay and by semiquantitative polymerase chain reaction. Cell invasion was evaluated by Matrigel invasion assay and cell proliferation by direct cell counting. aODN reduced uPAR, invasion and proliferation in all the treated cell lines. Following aODN treatment, human melanoma cells exhibited a strong decrease of uPAR-dependent ERK1/2 activation and were used in vivo to control metastasis in CD-1 male nude (nu/nu) mice by uPAR aODN injection. 60 mice were injected in the hind leg muscles with a suspension of 10(6) melanoma cells. After 4 days, when a tumor mass of about 350 mg was evident in all the mice injected, 20 mice were treated i.v. with aODN and 20 with dODN at 0.5 mg/day for 5 consecutive days. Twenty control mice were not treated. A second and third cycle of treatment was administered at 2-day intervals. Treatment with aODN resulted into a 78% reduction of lung metastases and 45% reduction of the primary tumor mass with no loss of body weight. Our results suggest to evaluate the utility of uPAR aODN in controlling the metastatic spreading of human melanoma., (Copyright 2004 Wiley-Liss, Inc.)