Gabriella Fibbi

Interaction of urokinase with specific receptors stimulates mobilization of bovine adrenal capillary endothelial cells

On the basis of 125I-labeled plasminogen activator binding analysis we have found that bovine adrenal capillary endothelial cells have specific receptors for human urinary-type plasminogen activator on the cell membrane. Each cell exposes about 37,000 free receptors with a Kd of 0.8958 x 10(-9) M [corrected]. A monoclonal antibody against the 17,500 proteolytic fragment of the A chain of the plasminogen activator, not containing the catalytic site of the enzyme, impaired the specific binding, thus suggesting the involvement of a sequence present on the A chain in the interaction with the receptor, as previously shown in other cell model systems. Both the native molecule and the A chain are able to stimulate endothelial cell motility in the Boyden chamber, when used at nanomolar concentrations. The use of the same monoclonal antibody that can inhibit ligand-receptor interaction can impair the plasminogen activator and A-chain-induced endothelial cell motility, suggesting that under the conditions used in this in vitro model system, the motility of bovine adrenal capillary endothelial cells depends on the specific interaction of the ligand with free receptors on the surface of endothelial cells.

Cell Invasion Is Affected by Differential Expression of the Urokinase Plasminogen Activator/Urokinase Plasminogen Activator Receptor System in Muscle Satellite Cells from Normal and Dystrophic Patients

The aim of this study was to evaluate the differential expression and the function in cell movement and proliferation of the urokinase plasminogen activator (u-PA) system in muscle satellite cells (MSC) of normal individuals and patients with Duchenne muscular dystrophy (DMD). By immunoenzymatic, zymographic, and radioligand binding methods and by quantitative polymerase chain reaction of the specific mRNA we have shown that both normal and DMD MSC produce u-PA and the plasminogen activator inhibitor-1 and express u-PA receptors (u-PAR). During the proliferation phase of their growth-differentiation program, MSC from DMD patients show more u-PAR than their normal counterpart, produce more plasminogen activator inhibitor-1, and release low amounts of u-PA into the culture medium. By Boyden chamber Matrigel invasion assays we have shown that normal MSC are more prone than DMD cells to spontaneous invasion but, when subjected to a chemotactic gradient of u-PA, DMD MSC sense the ligand much better and to a greater extent than normal MSC. u-PA also stimulates proliferation of MSC, but no difference is observable between normal and DMD patients. Antagonization of u-PA/u-PAR interaction with specific anti–u-PA and anti–u-PAR monoclonal antibodies and with antisense oligonucleotides inhibiting u-PAR expression indicates that u-PA/u-PAR interaction is required in spontaneous and u-PA–induced invasion, as well as in u-PA–induced proliferation.

Endothelial progenitor cell–dependent angiogenesis requires localization of the full-length form of uPAR in caveolae

Endothelial urokinase-type plasminogen activator receptor (uPAR) is thought to provide a regulatory mechanism in angiogenesis. Here we studied the proangiogenic role of uPAR in endothelial colony-forming cells (ECFCs), a cell population identified in human umbilical blood that embodies all of the properties of an endothelial progenitor cell matched with a high proliferative rate. By using caveolae-disrupting agents and by caveolin-1 silencing, we have shown that the angiogenic properties of ECFCs depend on caveolae integrity and on the presence of full-length uPAR in such specialized membrane invaginations. Inhibition of uPAR expression by antisense oligonucleotides promoted caveolae disruption, suggesting that uPAR is an inducer of caveolae organization. Vascular endothelial growth factor (VEGF) promoted accumulation of uPAR in ECFC caveolae in its undegraded form. We also demonstrated that VEGF-dependent ERK phosphorylation required integrity of caveolae as well as caveolar uPAR expression. VEGF activity depends on inhibition of ECFC MMP12 production, which results in impairment of MMP12-dependent uPAR truncation. Further, MMP12 overexpression in ECFC inhibited vascularization in vitro and in vivo. Our data suggest that intratumor homing of ECFCs suitably engineered to overexpress MMP12 could have the chance to control uPAR-dependent activities required for tumor angiogenesis and malignant cells spreading.

Antisense oligodeoxynucleotides for urokinase-plasminogen activator receptor have anti-invasive and anti-proliferative effects in vitro and inhibit spontaneous metastases of human melanoma in mice

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 106 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.

The Urokinase Receptor System, A Key Regulator at the Intersection between Inflammation, Immunity, and Coagulation

The urokinase plasminogen activator (uPA) and its receptor (uPAR) provide a cell surface integrated multimolecular complex that exerts pleiotropic functions influencing the development of inflammatory, immune, coagulation and fibrinolytic responses. Here we review the evidences indicating a role of the uPA/uPAR system in the regulation of the innate immune system in the inflammation process, of the adaptive immune response, as well as the role of fibrin and fibrin degradation products at the cross-road between coagulation and inflammation. Comparative studies have clearly highlighted the notion that coagulation and immunity are co-regulated and intertwined. The implication is that the vertebrate blood clotting system is evolutionarily by product of the innate immune system, where the blood clotting proteases have diverged from those comprising the complement system. Differences have emerged gradually, as shown by the acquisition of unique protein structures, such as kringle domains and gla (glutammic acid) domains, in order to comply with the increasingly complex vertebrate systems and to defend higher organisms against a range of infections and injuries. Plasminogen activation also controls the formation of complement anaphylotoxins (responsibe for vasodilatation, increase of venular permeability and leukocyte chemotaxis) and of bradykinin (which accounts for vasodilatation, increase of venular permeability and pain) by regulating the plasma contact system. The urokinase plasminogen activator and its cellular receptor, expressed on the surface of human leukocytes, provide a functional unit that, by regulating interaction of leukocytes with extracellular matrix, as well as its degradation, is critical for the migration of leukocytes and for their movement in the damaged tissues.

Involvement of glycosaminoglycans in detachment of early myeloid precusors from bone-marrow stromal cells

Fibroblast-like cells were obtained by in vitro cultivation of needle aspirations of human bone-marrow. These cells show a unique composition of coat-associated glycosaminoglycans: 10% chondroitin 4-sulfate, 30% hyaluronic acid and 60% heparan sulfate which were resolved and characterized by electrophoresis, nitrous acid treatment and enzymatic degradation. Chondroitin 4-sulfate is the only glycosaminoglycan detectable on the surface of mature granulocytes, whereas the immature cells do not seem to possess surface glycosaminoglycans. Immature hemopoietic cells can adhere on to marrow-derived fibroblast cells, whereas mature granulocytes cannot. Treatment with mucopolysaccharidases of both mature leukocytes and marrow stromal cells can interfere in these adhesive relationships, suggesting a role of glycosaminoglycans in regulating short-range interactions during hematopoiesis.

Modulation of the angiogenic phenotype of normal and systemic sclerosis endothelial cells by gain-loss of function of pentraxin 3 and matrix metalloproteinase 12.

OBJECTIVE Studies have shown that in systemic sclerosis (SSc) endothelial cells, overproduction of matrix metalloproteinase 12 (MMP-12) and pentraxin 3 (PTX3) is associated with defective angiogenesis. This study was undertaken to examine whether overexpression of the relevant molecules could inhibit angiogenesis of normal microvascular endothelial cells (MVECs), and whether silencing of these molecules in SSc MVECs could restore the lost angiogenic properties of the cells in vitro and in vivo. METHODS Transient transfection of MVECs with human MMP12 and PTX3 was performed by electroporation. Silencing of MMP12 and PTX3 was obtained by treatment with small interfering RNA, and treatment effects were validated by Western blotting with specific antibodies and a fluorimetric assay. In vitro cell migration and capillary morphogenesis were studied on Matrigel substrates. In vivo angiogenesis was studied using a Matrigel sponge assay in mice. RESULTS Transfection of MMP12 and PTX3 in normal MVECs resulted in loss of proliferation, invasion, and capillary morphogenesis in vitro, attributed to truncation of the urokinase-type plasminogen activator receptor by MMP12 and to the anti-fibroblast growth factor 2/anti-vascular endothelial growth factor activity of PTX3. These effects were particularly evident in mixed populations of transfected normal MVECs (50% transfected with MMP12 and 50% with PTX3). Silencing of the same molecules in SSc MVECs increased their invasion in Matrigel. Single-gene silencing did not increase the capillary morphogenesis of SSc MVECs, whereas double-gene-silenced cells showed a burst of capillary tube formation. Culture medium of silenced SSc MVECs stimulated angiogenesis in assays of Matrigel sponge invasion in mice. CONCLUSION Overexpression of either MMP12 or PTX3 in normal MVECs blunts their angiogenic properties. Loss of function of MMP12 and PTX3 in SSc MVECs restores the ability of the cells to produce capillaries in vitro and induces vascularization in vivo on a Matrigel sponge.

Endothelial cells and normal breast epithelial cells enhance invasion of breast carcinoma cells by CXCR-4-dependent up-regulation of urokinase-type plasminogen activator receptor (uPAR, CD87) expression.

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

Systemic sclerosis fibroblasts inhibit in vitro angiogenesis by MMP-12-dependent cleavage of the endothelial cell urokinase receptor

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 β1, 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. Copyright

EphA2-mediated mesenchymal-amoeboid transition induced by endothelial progenitor cells enhances metastatic spread due to cancer-associated fibroblasts.

Tumor progression is deeply influenced by epigenetic changes induced by tumor stroma. Cancer-associated fibroblasts (CAFs) have been reported to promote epithelial–mesenchymal transition in cancer cells, thereby enhancing their aggressiveness and stem-like properties. As CAFs are able to recruit endothelial progenitor cells (EPCs) to tumor site, we aim to investigate their interplay for prostate carcinoma progression. Both prostate CAFs and cancer cells actively recruit EPCs, known to affect tumor progression through increased vasculogenesis. EPCs synergize with CAFs to further promote epigenetic plasticity of cancer cells, through a mesenchymal-to-amoeboid transition. Indeed, after fibroblasts have engaged epithelial–mesenchymal transition in cancer cells, a further shift towards amoeboid motility is promoted by EPCs through contact-mediated triggering of the bidirectional ephrinA1/EphA2 signaling. The activation of ephrinA1 reverse pathway enhances EPC-induced neo-vascularization, thus promoting tumor growth, while EphA2 forward signaling elicits mesenchymal–amoeboid transition in cancer cells, favoring their adhesion to endothelium, transendothelial migration, and lung metastatic colonization. We therefore underscore that the metastatic advantage given by tumor microenvironment embraces different motility strategies and propose EphA2-targeted tools as useful adjuvants in anti-metastatic treatments.