Soluble form of the urokinase-type plasminogen activator receptor (suPAR) is markedly increased in biological fluids during different inflammatory conditions. It has previously been observed that the highest suPAR concentrations in inflammatory exudates tend to be associated with the presence of high number of neutrophils. Guided by this observation and our recent finding that activated neutrophils release suPAR we investigated whether neutrophils can be a source of suPAR during the inflammatory response in vivo. To address this question we conducted the comparative analysis of neutrophils isolated from the paired samples of synovial fluid (SF) and peripheral blood (PB) of rheumatoid arthritis patients. Freshly isolated SF neutrophils released significantly (p < 0.01) higher amounts of suPAR compared with PB neutrophils. We demonstrated that neutrophils from both sources release predominantly the truncated D2D3 form of suPAR. Migration of formyl peptide receptor-like 1 (FPRL1)-transfected human embryonic kidney (HEK) 293 cells toward the supernatants harvested from in vitro cultured SF neutrophils was significantly diminished when D2D3 form of suPAR was immunodepleted from the supernatants. Taken together, these data demonstrate that neutrophils, first, contribute to or are responsible for the generation of the increased suPAR levels during the inflammatory response and, second, release the chemotactically active form of suPAR that might be involved in the recruitment of formyl peptide receptors-expressing leukocytes into the inflamed tissues.

Objective-We showed previously that increased urokinase plasminogen activator (uPA) expression contributes to vascular smooth muscle cell (VSMC) proliferation and neointima formation after injury. Proliferation of cultured rat aortic VSMCs induced by uPA was inhibited by the antioxidant ebselen. Because increases in VSMC reactive oxygen species (ROS) contribute to VSMC proliferation, we hypothesized that uPA increases ROS generation by regulating expression or activity of cellular oxidases. Methods and Results-uPA stimulated ROS production to levels equivalent to angiotensin II as measured by electron spin resonance and fluorescent redox indicators (dichlorofluorescein diacetate, lucigenin, and hydroethidine). The increase in ROS was biphasic, with the first peak at 30 minutes and the second peak at 4 hours. uPA increased expression of the NAD(P)H oxidases Nox1 and Nox4 as measured by RT-PCR and Western blot analysis. Knockdown of Nox1 and Nox4 expression with small interfering RNA showed that both isoforms (Nox1ϾNox4) contributed significantly to uPA-stimulated ROS production and VSMC proliferation. Transfection of VSMCs with uPA cDNA to increase endogenous uPA expression enhanced ROS production dramatically, suggesting that autocrine uPA production may be an important mechanism for uPA-mediated VSMC events. Conclusion-These data show that uPA is an autocrine VSMC growth factor that increases ROS generated by both Nox1 and Nox4 oxidases. Key Words: urokinase Ⅲ superoxide Ⅲ VSMC proliferation Ⅲ arterial remodeling P lasminogen activators, their inhibitors, and receptors are the main components of the fibrinolytic (or plasminogen/ plasmin) system that, together with the blood coagulation system, determine the balance between the formation and dissolution of blood clots. 1 In addition, much evidence suggests that the fibrinolytic system also participates in vascular remodeling processes such as atherosclerosis and postangioplasty restenosis. [2][3][4][5][6] The predominant processes that contribute to vessel remodeling in cardiovascular diseases are proliferation and migration of vascular smooth muscle cells (VSMCs), extracellular matrix deposition, adhesion of inflammatory cells, invasion into the vessel wall, and proliferation. These processes are regulated by growth factors and the components of the fibrinolytic or plasminogen system. 7,8 The plasminogen system is composed of an inactive proenzyme, plasminogen, that is converted to active plasmin by 2 physiological plasminogen activators: tissue-type plasminogen activator (tPA) and urokinase type plasminogen activator (uPA). 1,9 VSMCs use proteinases to degrade the extracellular matrix that encages them, releasing them to migrate into the wound. 10 Plasmin may trigger this process because it can directly degrade fibrin and matrix and also activate other matrix-degrading proteinases, including the metalloproteinases. Plasmin has been presumed to play a role in tissue remodeling via proteolysis of extracellular matrix components and activation of growth factors. ...

Tissue regeneration requires coordinated "teamwork" of growth factors, proteases, progenitor and immune cells producing inflammatory cytokines. Mesenchymal stem cells (MSC) might play a pivotal role by substituting cells or by secretion of growth factors or cytokines, and attraction of progenitor and inflammatory cells, which participate in initial stages of tissue repair. Due to obvious impact of inflammation on regeneration it seems promising to explore whether inflammatory factors could influence proangiogenic abilities of MSC. In this study we investigated effects of TNF-α on activity of adipose-derived stem cells (ADSC). We found that treatment with TNF-α enhances ADSC proliferation, F-actin microfilament assembly, increases cell motility and migration through extracellular matrix. Exposure of ADSC to TNF-α led to increased mRNA expression of proangiogenic factors (FGF-2, VEGF, IL-8, and MCP-1), inflammatory cytokines (IL-1β, IL-6), proteases (MMPs, uPA) and adhesion molecule ICAM-1. At the protein level, VEGF, IL-8, MCP-1, and ICAM-1 production was also up-regulated. Pre-incubation of ADSC with TNF-α-enhanced adhesion of monocytes to ADSC but suppressed adherence of ADSC to endothelial cells (HUVEC). Stimulation with TNF-α triggers ROS generation and activates a number of key intracellular signaling mediators known to positively regulate angiogenesis (Akt, small GTPase Rac1, ERK1/2, and p38 MAP-kinases). Pre-treatment with TNF-α-enhanced ADSC ability to promote growth of microvessels in a fibrin gel assay and accelerate blood flow recovery, which was accompanied by increased arteriole density and reduction of necrosis in mouse hind limb ischemia model. These findings indicate that TNF-α plays a role in activation of ADSC angiogenic and regenerative potential.

Urokinase-type plasminogen activator (uPA) is suggested to exert its proliferatory, migratory and invasive action through binding with its membrane receptor, promoting pericellular proteolysis and mediating cell signal transduction. One of the possible actions of urokinase can be related to the regulation of activity and/or the expression of proteolytic enzymes participating in extracellular matrix degradation. In the present study, the role of uPA in regulating matrix metalloproteinase (MMP) expression and release by the monocyte cell line THP-1 was investigated. Recombinant uPA induced the release of MMP9/gelatinase B, as detected by zymography and Western blotting, and this release was abolished by actinomycin D and cycloheximide (inhibitors of DNA transcription and protein synthesis) and partially suppressed by monensin (an inhibitor of secretion). Proteolytically inactive urokinase with substitution of His(204) for Gln was able to reproduce about 70% of the effect induced by the wild-type recombinant uPA. The reverse transcription-PCR and Northern blot data indicated that the action of r-uPA on THP-1 cells resulted in formation of MMP9 mRNA, which depended on time, within 6-48 h, of the cell incubation with r-uPA. These results suggest that urokinase upregulates MMP9 expression in monocytes via MMP9 gene transcription and protein biosynthesis.