Biomedical subjects

S L Cortez

Publications and source records attributed to S L Cortez.

7 recordsLinked to original sources

PubMed1999

Transforming growth factor-beta is a potent inhibitor of extracellular matrix degradation by cultured human mesangial cells.

Accumulation of the glomerular extracellular matrix (ECM) is a pivotal event in the progression from acute glomerular injury to end-stage renal disease. Although enhanced ECM synthesis has been demonstrated to contribute to ECM accumulation, the role of decreased ECM degradation is largely unknown. It was previously shown that glomerular ECM degradation is mediated by a plasminogen activator (PA)/plasmin/matrix metalloproteinase 2 (MMP-2) cascade. However, little information is available regarding the factors that regulate the activity of this degradative cascade in normal or pathologic states. Transforming growth factor-beta1 (TGF-beta1) is shown here to be a potent inhibitor of ECM degradation by cultured human mesangial cells. Using human mesangial cells grown on thin films of 125I-labeled Matrigel, dose-dependent inhibition of ECM degradation in the presence of TGF-beta1 was observed, reaching >90% inhibition with 0.4 ng/ml TGF-beta1. Addition of anti-TGF-beta antibodies (4 microg/ml) in the absence of exogenous TGF-beta increased ECM degradation (1.8+/-0.2-fold versus controls, P<0.05). In contrast, platelet-derived growth factor, at concentrations up to 10 ng/ml, had no effect on ECM degradation. TGF-beta completely blocked the conversion of plasminogen to plasmin and markedly reduced the conversion of latent MMP-2 to active MMP-2. TGF-beta did not significantly alter the levels of tissue PA, total MMP-2, or tissue inhibitor of metalloproteinase-1, but did increase the levels of PA inhibitor- (1.8-fold, P<0.05), the major physiologic inhibitor of PA. These data document that TGF-beta is a potent inhibitor of ECM degradation by cultured human mesangial cells, and they suggest that decreased mesangial matrix degradation, caused by TGF-beta-mediated decreases in the activity of the PA/plasmin/MMP-2 cascade, may contribute to the glomerular matrix accumulation that occurs in progressive renal disease.

Cells, Cultured↗

PubMed1995

Plasminogen activators augment endothelial cell organization in vitro by two distinct pathways.

Endothelial cell differentiation into capillary structures is a complex process that requires the concerted effects of several extracellular matrix proteases, including plasminogen activators. Here, the role of tissue-type plasminogen activator (tPA) and urokinase-type plasminogen activator (uPA) was evaluated in an in vitro model of endothelial morphogenesis involving organization of human umbilical vein endothelial cells into tubular structures when they are cultured on the basement membrane preparation, Matrigel. Both uPA and tPA were detected in HUVEC cultures on Matrigel, and inhibitors of plasminogen activators or of serine proteases decreased the extent of the tube network formed by the cells. The decrease resulting from serine protease inhibitors was additive to that from matrix metalloproteinase inhibitors which have previously been shown to decrease tube formation in this model, suggesting that the two classes of proteases modulate tube formation by distinct mechanisms. Plasminogen activator inhibitor (PAI)-1 decreased tube formation by 50% when added up to 4.5 h after the initiation of an 18 h assay and caused 25% inhibition when added 9.5 h after culture initiation, indicating that the effects of plasminogen activators are not limited to an early event in the differentiation process. Steady-state expression of mRNA for uPA increased during the first several hours of culture on Matrigel, further supporting a role for PA activity throughout the process of tube formation. These findings suggested that PAs may affect multiple events during tube-forming activity. A fucosylated peptide comprising the amino-terminal domain of uPA that binds to the uPA receptor (uPAR) but lacking proteolytic activity enhanced tube formation. In contrast, a defucosylated form of the same peptide had no effect. Since fucosylation of this fragment has been shown to be essential in other models of cell stimulation by uPA-uPAR interaction, these data support the hypothesis that uPA enhances endothelial morphogenesis both through proteolytic activity and via uPAR occupancy. Plasminogen activators could facilitate angiogenesis in vivo.

Capillaries↗

PubMed1995

ECM degradation by cultured human mesangial cells is mediated by a PA/plasmin/MMP-2 cascade.

We examined the role of the plasminogen activator/plasmin system in extracellular matrix (ECM) degradation by human mesangial cells cultured on thin films of 125I-labeled ECM (Matrigel). ECM degradation (release of 125I into the medium) was dependent on exogenous plasminogen, proportional to the number of mesangial cells and amount of plasminogen added, and coincident with the appearance of plasmin in the medium. ECM degradation was completely blocked (P < 0.001) by two plasmin inhibitors, alpha-2-antiplasmin (40 micrograms/ml) and aprotinin (216 KIU/ml), and partially reduced (-33 +/- 1.8%, P < 0.01) by TIMP-1 (40 micrograms/ml), a specific inhibitor of matrix metalloproteinases. Zymography of medium obtained from cells cultured in the absence of plasminogen revealed the presence of latent matrix metalloproteinase-2 (MMP-2) which was converted to a lower molecular weight, active form in the presence of mesangial cells and plasminogen. Northern analysis of poly A+RNA prepared from cultured human mesangial cells revealed mRNA for tissue-type plasminogen activator (tPA), urokinase-type plasminogen activator (uPA), plasminogen activator inhibitor-1 (PAI-1), and uPA receptor (uPAR). The presence of uPA protein in medium obtained from cultured human mesangial cells was demonstrated by Western blotting and ELISA which revealed a large molar excess of PAI-1 (1.2 +/- 0.1 x 10(-9) M) over uPA (1.2 +/- 0.1 x 10(-12) M) and tPA (0.19 +/- 0.04 x 10(-9) M). ECM degradation was reduced by a monoclonal antibody (MAb) against human tPA (-54 +/- 8.6%) or human uPA (-39 +/- 5.2%) compared to cells treated with identical amounts of non-specific monoclonal IgG (P < 0.01).(ABSTRACT TRUNCATED AT 250 WORDS)

Aprotinin↗

PubMed1992

Role of plasmin and gelatinase in extracellular matrix degradation by cultured rat mesangial cells.

We have examined the ability of mesangial cells (MCs) to degrade extracellular matrix (ECM) using cultured rat MCs grown on thin films of radiolabeled Matrigel. ECM degradation by cultured MCs was observed only in presence of exogenously added plasminogen and was a function of plasminogen concentration (0-50 mU), cell number (0-50,000 cells), and length of incubation (0-72 h). A high positive correlation (r > 0.93) was observed between ECM degradation and plasmin activity in medium, suggesting an important role for plasmin in ECM degradation by cultured MCs. This suggestion was confirmed by ability of plasmin inhibitors, alpha 2-antiplasmin (40 micrograms/ml) and aprotinin (216 kallikrein inhibition units/ml), to inhibit (> 90%) ECM degradation. Inhibitors of cysteine proteinases [trans-epoxysuccinyl-L-leucylamido(4-guanidino)butane, 10 microM] and aspartic proteinases (pepstatin, 5.0 micrograms/ml) had no effect on ECM degradation. However, in presence of plasminogen, inhibitors of matrix metalloproteinases, TIMP-1 (40 micrograms/ml) and o-phenanthroline (100 microM), inhibited ECM degradation -42 +/- 4% and -43 +/- 3% (SE), respectively (n = 8-10). Thus, in addition to plasmin, a matrix metalloproteinase(s) is also involved in ECM degradation by cultured rat MCs. Zymography of culture medium obtained from MCs grown on radiolabeled Matrigel films in absence of plasminogen revealed only two closely migrating bands of gelatinase activity, relative mol wt of approximately 70,000-72,000. MCs grown in absence of plasminogen failed to degrade ECM despite presence of gelatinase in medium, indicating that, in absence of plasmin, gelatinase is present in an inactive form, either as a latent proenzyme or as a gelatinase-inhibitor complex.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1991

Evidence suggesting a role for cathepsin L in an experimental model of glomerulonephritis.

We have utilized specific, irreversible inhibitors of cysteine proteinases to examine the role of renal cathepsin B and cathepsin L in the proteinuria which occurs in an experimental model of human glomerular disease. Administration of trans-epoxysuccinyl-L-leucylamido-(3-methyl)butane (Ep475) a specific, irreversible inhibitor of cysteine proteinases, including cathepsins B and L, significantly reduced proteinuria in rats with experimentally induced, neutrophil-independent, anti-GBM antibody disease (controls: 10 +/- 1 mg/24 h, N = 8; anti-GBM antibody disease: 203 +/- 30 mg/24 h, N = 8; anti-GBM antibody disease + Ep475: 112 +/- 13 mg/24 h, mean +/- SEM, N = 6, P less than 0.05). There was a marked reduction in the activity of both cathepsin B and cathepsin L in renal cortices obtained from Ep475-treated rats compared to either saline-treated controls or rats treated with anti-GBM IgG only. Administration of Z-Phe-Tyr(O-t-butyl)CHN2, a specific, irreversible cysteine proteinase inhibitor with a high degree of selectivity toward cathepsin L, also caused a reduction in anti-GBM antibody-induced proteinuria (90 +/- 18 mg/24 h, N = 6, P less than 0.05). This reduction in proteinuria was accompanied by a marked decrease (-84%) in the specific activity of renal cortical cathepsin L in Z-Phe-Tyr(O-t-butyl)CHN2-treated rats. However, cathepsin B activity was unchanged. There was no significant change in the renal anti-GBM antibody uptake, plasma urea nitrogen, or plasma creatinine values in the Z-Phe-Tyr(O-t-butyl)CHN2-treated rats compared to rats treated with anti-GBM IgG only or saline-treated controls. These data document the ability of cysteine proteinase inhibitors to decrease the proteinuria which occurs in a neutrophil-independent model of human anti-GBM antibody disease and suggest an important role for cathepsin L in the pathophysiology of the proteinuria which occurs in this model.

Animals↗

PubMed1990

Glomerular basement membrane degradation by endogenous cysteine proteinases in isolated rat glomeruli.

Recent in vitro and in vivo studies suggest that cysteine proteinases may play an important role in degradation of the glomerular basement membrane (GBM) by renal glomeruli. However, little information is available concerning the cysteine proteinases present in glomeruli, the distribution of cysteine proteinases in other areas of the kidney, or the potential role of endogenous glomerular cysteine proteinases in GBM degradation. Using well characterized fluorogenic substrates, we have documented the presence of the cysteine proteinases, cathepsins B, H, and L, in glomeruli (0.45 +/- 0.06, 0.39 +/- 0.05, and 0.66 +/- 0.14 mU/mg protein, mean +/- SEM, N = 8) and other fractions prepared from normal rat kidney. The presence of cysteine proteinases in glomeruli was verified by fluorescence microscopy. For each proteinase, the activity was: proportional to the amount of tissue protein and time of incubation; dependent on the presence of exogenously added dithiothreitol; and completely inhibited by the specific cysteine proteinase inhibitor, E-64. The pH optimum for cathepsin B (substrate: Z-Arg-Arg-HNMec) and L (substrate: Z-Phe-Arg-HNMec in the presence of Z-Phe-Phe-CHN2) was approximately pH 6.0 for both glomeruli and renal cortex; while that for cathepsin H (substrate: Arg-HNMec) was approximately 6.5. Incubation of sonicated glomeruli with 3H-GBM under conditions optimal for cysteine proteinase activity (pH 4.5, 1 mM EDTA, and 1 mM dithiothreitol, 37 degrees C) resulted in significant GBM degradation as measured by the release of non-sedimentable (10,000 x g, 10 min) radioactivity or hydroxyproline.(ABSTRACT TRUNCATED AT 250 WORDS)

Animals↗

PubMed1988

The cysteine proteinase inhibitor, E-64, reduces proteinuria in an experimental model of glomerulonephritis.

Proteinuria is a major manifestation of glomerular disease (glomerulonephritis, GN). We examined the effect of trans-epoxysuccinyl-L-leucylamido-(4-guanidino)butane (E-64), a specific and irreversible cysteine proteinase inhibitor, on urinary protein excretion in a complement- and neutrophil-independent model of antiglomerular basement membrane (GBM) antibody disease. A single injection of rabbit antirat-GBM IgG produced a marked increase in urinary protein excretion 24hr after injection. In two separate studies using different pools of antiGBM IgG, administration of E-64 (5mg every 6h starting 2hr prior to induction of GN) reduced proteinuria (-45 +/- 7%, and -41 +/- 14%, Mean +/- SEM, n = 6; P less than 0.001) in the 24 hour period following induction of the disease. This reduction in urinary protein excretion was accompanied by a marked decrease in the specific activity of the cysteine proteinases cathepsins B and L in glomeruli (B: -97%; L: -84%) and renal cortex (B: -87%; L: -75%) isolated from the same E-64-treated rats compared to same saline-treated controls. These data, combined with the specificity of E-64 for cysteine proteinases, suggest a potential role for cysteine proteinases in the increased GBM permeability and proteinuria in this experimental model of glomerular disease.

Animals↗