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

No abstract

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-polymerase chain 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 ␣⌴-and ␣X-subunits of ␤2 integrin, and was absent from SSc MVECs. The cleaved uPAR was not associated with ␤2 integrins or with actin. ␤3 integrins were associated with both the full-size and cleaved uPAR at focal contacts. The uncoupling of uPAR from ␤2 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 ␤2/␤3 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 ␤2 integrinmediated connection of uPAR with the actin cytoskeleton in these ECs.Angiogenic signals promote endothelial cell (EC) proliferation as well as EC resistance to apoptosis, changes in proteolytic balance, cytoskeletal reorganization, migration, and formation of a vascular lumen (1).

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.