Cultured Human Mesangial Cells Produce both Type 1 and Type 2 Plasminogen Activator Inhibitors

Summary

Cultured human mesangial cells (HMC) derived from normal kidneys have been shown to synthesize tissue-type plasminogen activator (t-PA) and excess amounts of PA inhibitor type 1 (PAI-1). Conflicting results have been obtained concerning the production of urokinase-type PA (u-PA) and efforts to show PA inhibitor 2 (PAI-2) met with failure. We evaluated the fibrinolytic profile of cultured HMC lines obtained from 12 patients with renal carcinoma and one cadaveric kidney donor. Subconfluent cells (third passage) were incubated overnight in serum-free medium. t-PA, u-PA, PAI-1 and PAI-2 antigens were assayed by ELISA methods and PA and PAI activities by amidolytic methods both in conditioned medium (CM) and cell extracts (CE). Besides PAI-1, PAI-2 antigen was detected in all but one HMC lines. At variance with the former, which was largely released in the culture medium, PAI-2 was mainly cell-associated. t-PA antigen was found in all but two cell lines while u-PA antigen was detected in relatively high concentrations in 8 cell lines. PA activity, identified as u-PA by functional and immunological criteria, was measured in CM of six of the eight u-PA producing cell lines, whereas PAI activity was undetectable or very low in CM of all cell lines, suggesting that PAI-1 was largely inactive. Functional assays of cell extracts demonstrated the presence of PA activity, again identified as u-PA, only in samples (five lines) containing u-PA antigen in excess over PAI-2. PAI activity was found instead in the extracts in which the inhibitor was higher than the activator (six lines) and was identified as PAI-2, as it inhibited u-PA but not single-chain t-PA and was neutralized by a polyclonal anti-PAI-2 antibody. The heterogeneous fibrinolytic pattern of HMC lines was confirmed by mRNA analysis of three representative lines. Results were similar when HMC lines at passage five were used, except that the u-PA content was significantly reduced both in CM and CE. These findings indicate that the fibrinolytic profile of cultured HMC is more complex than previously reported. The production of large amounts of PAI-2 may represent an additional control mechanism of proteinase activity.

• References

• 1 Hancock W, Atkins R. Activation of coagulation pathways and fibrin deposition in human glomerulonephritis. Semin Nephrol 1985; 5: 69-77
• 2 Takemura T, Yoshioka K, Akano N, Miyamoto H, Matsumoto K, Maki S. Glomerular deposition of cross-linked fibrin in human kidney diseases. Kidney Int 1987; 32: 102-111
• 3 Sraer JD, Kanfer A, Rondeau E, Lacave R. Glomerular hemostasis in normal and pathological conditions. Adv Nephrol 1988; 17: 27-55
• 4 Wiggins RC, Njoku N, Sedor JR. Tissue factor production by cultured rat mesangial cells. Stimulation by TNFα and lipopolysaccharide Kidney Int 1990; 37: 1281-1285
• 5 Lacave R, Rondeau E, Ochi S, Delarue F, Schleuning WD, Sraer JD. Characterization of a plasminogen activator and its inhibitor in human mesangial cells. Kidney Int 1989; 35: 806-811
• 6 Wong AP, Cortez SL, Baricos WH. Role of plasmin and gelatinase in extracellular matrix degradation by cultured rat mesangial cells. Am J Physiol 1992; 263: F112-118
• 7 Reid FJ, Wilson HM, Moore NR, Power DA, Haites NE, Booth NA. Cultured human mesangial cells produce urokinase (μ-PA) and plasminogen activator inhibitor 1 (PAI-1). Fibrinolysis 1992; 6 suppl (Suppl. 02) 173
• 8 Schonermark M, Kramer MD, Klaus R, Hansch GM. The terminal complement components C5b-9 induce the synthesis of collagenase type IV (MMP-2) in mesangial cells (GMC) and promote generation of its potential activator plasmin. J Am Soc Nephrol 1992; 3: 614
• 9 Glass WF II, Kreisberg JI, Troyer DA. Two-chain urokinase, receptor, and type 1 inhibitor in cultured human mesangial cells. Am J Physiol 1993; 264: F532-539
• 10 Ritchie H, Jamieson A, Booth NA. Thrombin modulates synthesis of plasminogen activator inhibitor type 2 by human peripheral blood monocytes. Blood. in press
• 11 Quadracci LJ, Striker GE. Growth and maintenance of glomerular cells in vitro. Proc Soc Exp Biol Med 1970; 135: 974-979
• 12 Colucci M, Páramo JA, Collen D. Generation in plasma of a fast acting inhibitor of plasminogen activator in response to endotoxin stimulation. J Clin Invest 1985; 75: 818-824
• 13 Loskutoff DJ, Mussoni L. Interaction between fibrin and the plasminogen activators produced by cultured endothelial cells. Blood 1983; 62: 62-68
• 14 Chomczynski P, Sacchi N. Single-step method of RNA isolation by acid guanidinium thiocyanate-phenol-chloroform extraction. Anal Biochem 1987; 162: 156-161
• 15 Lecander I, Roblin R, Astedt B. Differential inhibition of two molecular forms of melanoma cell plasminogen activator by a placental inhibitor. Br Med J. 1984; 57: 407-412
• 16 Meulders Q, He CJ, Adida C, Peraldi MN, Schleuning WD, Sraer JD, Rondeau E. Tumor necrosis factor a increases antifibrinolytic activity of cultured human mesangial cells. Kidney Int 1992; 42: 327-334
• 17 Andreasen PA, Georg B, Lung LR, Riccio A, Stasey SN. Plasminogen activator inhibitors: hormonally regulated serpins. Mol Cell Endocrinol 1990; 68: 1-19
• 18 Loskutoff DJ, Sawdey M, Mimuro J. Type 1 plasminogen activator inhibitor. In: Progress in Hemostasis and Thrombosis. Coller BS. ed WB Saunders Company; Philadelphia, PA: 1989. pp 87-115
• 19 Kruithof EK O, Tran-Thang C, Ransijn A, Bachmann F. Demonstration of a fast-acting inhibitor of plasminogen activators in human plasma. Blood 1984; 64: 907-913
• 20 De Munk GA W, Rijken DC. Fibrinolytic properties of single-chain urokinase-type plasminogen activator (pro-urokinase). Fibrinolysis 1990; 4: 1-9
• 21 Rondeau E, Ochi S, Lacave R, He C-J, Medcalf R, Delarue F, Sraer JD. Urokinase synthesis and binding by glomerular epithelial cells in culture. Kidney Int 1989; 36: 593-600
• 22 Iwamoto T, Nakashima Y, Sueishi K. Secretion of plasminogen activator and its inhibitor by glomerular epithelial cells. Kidney Int 1990; 37: 1466-1476
• 23 Wilson HM, Reid FJ, Brown PA J, Power DA, Haites NE, Booth NA. Effect of transforming growth factor-β¹on plasminogen activator and plasminogen activator inhibitor-1 in renal glomerular cells. Exp Nephrol 1993; 52: 1-6
• 24 Brown PA J, Wilson HM, Reid FJ, Booth NA, Simpson JG, Morrison L, Power DA, Haites NE. Urokinase-plasminogen activator is synthesized in vitro by human glomerular epithelial cells but not by mesangial cells. Kidney Int 1994; 45: 43-47
• 25 Vassalli J-D, Sappino A-P, Belin D. The plasminogen activator/plasmin system. J Clin Invest 1991; 88: 1067-1072
• 26 van Hinsbergh VW M, Kooistra T, Emeis JJ, Koolwijk P. Regulation of plasminogen activator production by endothelial cells: role in fibrinolysis and local proteolysis. Int J Radiat Biol 1991; 60: 261-272
• 27 Hamilton JA, Cheung D, Filonzi EL, Piccoli DS, Wojta J, Gallichio M, McGrath K, Last K. Independent regulation of plasminogen activator inhibitor 2 and plasminogen activator inhibitor 1 in human synovial fibroblasts. Arthritis Rheum 1992; 35: 1526-1534
• 28 van Hinsbergh VW M, Kooistra T, Scheffer MA, van Bockel JH, van Muijen GN P. Characterization and fibrinolytic properties of human omental tissue mesothelial cells. Comparison with endothelial cells. Blood 1990; 75: 1490-1497
• 29 Zoellner H, Wojta J, Gallicchio M, McGrath K, Hamilton JA. Cytokine regulation of the synthesis of plasminogen activator inhibitor-2 by human vascular endothelial cells. Comparison with plasminogen activator inhibitor-1 synthesis. Thromb Haemost 1993; 69: 135-140
• 30 Kumar S, Baglioni C. Protection from tumor necrosis factor-mediated cytolysis by overexpression of plasminogen activator inhibitor type-2. J Biol Chem 1991; 266: 20960-20964