Biphasic regulation of plasminogen activator/inhibitor by LDL in mesangial cells

Chi Young Song, Bong Cho Kim, Hye Kyoung Hong, Byoung Kwon Kim, Young Sook Kim, and Hyun Soon Lee

1 Department of Pathology, Seoul National University College of Medicine, Seoul 110-799, Korea


    ABSTRACT
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Lipid abnormalities and dysregulation of the plasminogen activator (PA)/plasmin system may be involved in the development of glomerulosclerosis. We investigated the effects of low-density lipoprotein (LDL) on PA inhibitor-1 (PAI-1), urokinase-type PA (uPA), and tissue-type PA (tPA) in relationship to protein kinase C (PKC) in cultured human mesangial cells (HMC). LDL (200 µg/ml) induced two peaks of PKC activation at hours 0.25 and 6, with translocation of PKC-alpha , -beta 1, and -delta from cytosol to the membrane. The second increase in PKC activity gradually decreased to the control value by hour 18. LDL downregulated 2.4-kb PAI-1, uPA, and tPA mRNA expression within 6 h of incubation with HMC. On the other hand, after 12-48 h, LDL-treated cells showed a significant increase in PAI-1, tPA, and uPA mRNA levels. LDL induced up to a twofold increase in PAI-1 antigen levels in the extracellular matrix of HMC after 24-48 h as well as increased PA inhibitory activity in the culture medium. Analysis of the adhesion plaques from cells incubated with LDL for 48 h by zymography showed increased intensity of lysis near molecular weights of ~55,000 and 100,000. LDL slightly increased tPA release at hours 24 and 48 but did not increase PA activity in culture medium. The stimulatory effects of LDL on PAI-1, tPA, and uPA gene regulation in HMC were blocked by the inhibition of PKC using GF-109203X 12 h after treatment with LDL or downregulation of PKC using phorbol myristate acetate. In summary, LDL regulates PAI-1, uPA, and tPA in biphasic patterns in HMC, and the upregulation of PAI-1, uPA, and tPA after long-term LDL exposure seems to be mediated by a delayed PKC activation associated with an increased PA inhibitory activity. These results suggest that LDL, after prolonged incubations with HMC, causes a PA/inhibitor imbalance favoring accumulation of matrix.

glomerulosclerosis; lipids; extracellular matrix; plasmin; protein kinase C; low-density lipoprotein


    INTRODUCTION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

ACCUMULATION OF EXTRACELLULAR MATRIX (ECM) in the glomerular mesangium leading to glomerulosclerosis is an important feature of progressive glomerular injury. Glomerular mesangial cells, which are often compared with vascular smooth muscle cells (VSMC), are a major site of ECM synthesis in chronic glomerular disease. Hypercholesterolemia in nephrosis may play a role in the progression of original glomerular injury to progressive glomerulosclerosis (9, 19). Hypercholesterolemia is mainly due to the increased level of low-density lipoprotein (LDL). LDL stimulates the synthesis of ECM in cultured human mesangial cells (HMC) (14, 15).

The ECM can be degraded, and a distortion of the balance between ECM synthesis and turnover may result in an abnormal ECM accumulation in renal disease. Matrix metalloproteinases and the plasminogen activator (PA)/plasmin system are known to play a key role in matrix degradation (2, 18, 24, 29). Cultured HMC produce urokinase-type PA (uPA) (2, 6), tissue-type PA (tPA), and PA inhibitor-1 (PAI-1) (7, 13, 23). Plasminogen is activated to plasmin by the enzymatic activity of uPA and tPA, and plasmin mediates the degradation of ECM by cultured mesangial cells (2, 32). The rate of plasmin production is primarily regulated by PAI-1 (28), which also inhibits the plasmin-dependent activation of matrix metalloproteinases (20).

Protein kinase C (PKC) is activated by the physiological second messenger diacylglycerol (DAG), a product of phosphatidylinositol. On stimulation of cell surface receptors, there is an immediate and transient increase in DAG levels, often followed by the second phase of sustained DAG elevation (21). Tumor-promoting phorbol esters, such as 12-phorbol 13-myristate acetate (PMA), possess a molecular structure that is similar to that of DAG, cause a prolonged activation of PKC, and downregulate the enzyme. Oxidized LDL stimulated phosphoinositide turnover in cultured VSMC, which was inhibited by pretreatment of cells with PMA (25). In cultured HMC, PMA induced a marked increase in PAI-1 and tPA release over a period of 24 h (8, 23).

Despite the possible relationship between plasmin system and LDL-induced mesangial matrix accumulation, no study has explored the effects of LDL on the PA/plasmin system in HMC. Enhanced effects of oxidized LDL on PAI-1 synthesis have only been described in cultured VSMC (5). Prolonged incubation of native LDL with cultured HMC has been shown to result in oxidative modification (14). Here, we have attempted to determine whether LDL regulates PAI-1, tPA, and uPA according to incubation times in HMC and whether PKC activation could be involved in LDL-induced PA/PAI-1 imbalance.


    MATERIALS AND METHODS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

Reagents. Human tPA was obtained from Calbiochem (La Jolla, CA). PAI-1 and tPA ELISA kits were obtained from Biopool (Umea, Sweden). Purified human fibrinogen and plasminogen, bovine thrombin, synthetic substrate D-Val-Leu-Lys pNA (S2251), and amiloride were purchased from Sigma (St. Louis, MO). Other reagents came from the sources previously reported (14, 15).

Culture of HMC. HMC were obtained from adult nephrectomy specimens, as previously described (11, 16). The culture medium that was used was made of DMEM supplemented with 20% fetal calf serum, 200 mM L-glutamine, and antibiotics. For the present experiments, cells between passages 5 and 7 were used.

LDL isolation. Human LDL (density: 1.019-1.063) and lipoprotein-deficient serum (density >1.215) were isolated from the plasma of normal volunteers by the method of sequential ultracentrifugation, as previously described (16). Isolated LDL was dialyzed for 24 h at 4°C against buffer A, which contained 0.15 M NaCl and 0.24 mM EDTA at pH 7.4. After dialysis, LDL was stored at 4°C under nitrogen and was used within 14 days.

Experimental conditions. HMC were grown to confluency. The cells were synchronized to quiescence in serum-free DMEM containing 5 µg insulin-transferrin-selenite/ml for 48 h. After synchronization, cells were treated with 50-200 µg/ml LDL for a time period varying from 15 min to 48 h. In a particular experiment, simultaneous control monolayers were treated with serum-free DMEM alone. The possibility that prolonged treatment with GF-109203X (GFX) could be toxic to cells was excluded by trypan blue exclusion.

Assay of PKC activity. Quiescent HMC in six-well plates were exposed to 200 µg/ml LDL for 15 min to 24 h, and the PKC activity was assayed by using QKRPSQRSKYL as a specific substrate peptide. In brief, the cell homogenates were centrifuged and the pellets (membrane) were resuspended. Samples were added to a microcentrifuge tube containing a substrate cocktail, inhibitor cocktail, and lipid activator. After the addition of an [gamma -32P]ATP mixture, the tube was incubated at 30°C for 10 min, and an aliquot was transferred onto the phosphocellulose paper. The radioactivity was determined, and the PKC-specific activity (pmol phosphate incorporated into substrate peptide/min) was calculated as described (15).

Cell fractionation and PKC immunoblots. Quiescent HMC in 100-mm plates were stimulated with 200 µg/ml LDL for 6 h. Cells were harvested, sonicated, and centrifuged as described (15). The supernatants were then retained as the cytosolic fraction. The pellets were resuspended, sonicated, and centrifuged. The supernatants were collected and used as the membrane fraction. Aliquots were then electrophoretically resolved by using a 10% polyacrylamide gel in an SDS buffer and then transferred onto nitrocellulose membranes. The blots were incubated with rabbit anti-PKC-alpha (1:1,000) or -beta 1 (1:200), or mouse anti-PKC-delta (1:250) at 4°C overnight. Bound primary antibody was visualized after incubation with horseradish peroxidase-conjugated secondary antibody using an enhanced chemiluminescence system kit (Amersham).

Quantification of ECM-associated PAI-1 by ELISA. At the end of the incubation period, conditioned medium was collected, centrifuged, and stored at -70°C until it was assayed. The cell monolayer was washed twice with culture medium and then treated with 0.5 ml of 0.02 M Tris, 0.15 M NaCl, at pH 7.4, containing 0.5% Triton X-100. After incubation for 45 min at room temperature on a rotating plate, the resulting cell extract was removed by aspiration, and the Triton X-100-insoluble ECM was washed twice with Tris buffer. ECM-associated PAI-1 was then brought into solution by the addition of 0.3 ml of 1 µg/ml tPA and incubated for 2 h at 37°C. This treatment resulted in a >80% release of PAI-1 as a PAI-1/tPA complex (27). A quantitative measurement of PAI-1 antigen was carried out utilizing ELISA kits according to the manufacturer's instructions.

Preparation of adhesion plaques. After the experiments, adhesion plaques (composed of ventral membranes and ECM) were prepared as previously described (1). Briefly, cells were rinsed in 50 mM HEPES buffer containing 3 mM EGTA and 5 mM MgCl2, pH 7.2, and exposed for 2 min to the above buffer containing 1 mM ZnCl2. Then adhesion plaques were prepared by shearing with vigorously pipetted buffer (25 mM HEPES containing 3 mM MgCl2, pH 7.3).

Fibrinolytic activity and inhibitor activity assay. Fibrinolytic activity was measured by a chromogenic assay as previously described (13). Culture supernatants or adhesion plaques (80 µl) were added to 20 µl of Tris · HCl buffer, pH 7.4, containing 0.4 µM plasminogen and 2 mM S2251, which is a synthetic chromogenic substrate of plasmin. The optical density at 405 nm was measured in a microELISA reader after 1 h incubation at 37°C. Inhibitor activity using a conditioned medium was assayed as follows. The medium was incubated with the same volume of Tris buffer, pH 7.4, 0.02% SDS, and 0.2% Triton X-100 for 15 min. Exogenous tPA (2.5-5 U/ml) was incubated for 15 min at 37°C with the sample. Twenty microliters of plasminogen and S2251 were then added to measure the residual tPA activity.

Zymographic analysis. PAs in culture supernatants and adherent plaques were analyzed on gels as described (10). In brief, 11% SDS-polyacrylamide gels were prepared containing 0.12% fibrinogen, thrombin (1 NIH unit/ml), and plasminogen (1 U/ml). N,N,N',N'-tetramethylethylenediamine was added to the gel solution to a final concentration of 0.028%. Samples, which were dissolved in electrophoresis sample buffer without mercaptoethanol, were applied to the gel and separated electrophoretically. The gels were washed in 2.5% Triton X-100, incubated overnight at 37°C, and then stained with Coomassie blue.

Northern blot analysis. Isolated RNA samples were transferred onto nylon filters as previously described (14). A cDNA probe for human uPA detected a transcript of a 1.5-kb PstI fragment (31). A cDNA probe for the human tPA detected a transcript of a 1.6-kb EcoRI fragment (4), and one for the human PAI-1 detected a 2.9-kb EcoRI fragment (22). The cDNA probes for uPA and tPA were obtained from the American Type Culture Collection (Manassas, VA). The PAI-1 cDNA probe was kindly donated by Dr. D. J. Loskutoff (Scripps Clinic, La Jolla, CA). The blotted membranes were incubated with the specific 32P-labeled cDNA probes. The filters were dried and exposed at -70°C using Agfa film (Agfa-Gevaert, Hortsel, Belgium). The mRNA levels for uPA, tPA, and PAI-1 were expressed as a ratio of the optical density units for uPA, tPA, or PAI-1 to beta -actin.

Statistical analyses. Results were expressed as means ± SD. Results were analyzed by two-way analysis of variance for three groups or by Wilcoxon rank sum test between two groups. A P value of <0.05 was considered to be significant.


    RESULTS
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

LDL modulates PAI-1, uPA, and tPA mRNA expression according to incubation times. Starting 1 h after incubation with HMC, LDL at concentrations of 200 µg/ml significantly decreased 2.4-kb PAI-1, uPA, and tPA mRNA synthesis until hour 6. No significant difference in the levels of 3.4-kb PAI-1 mRNA was found between LDL-treated cells and controls. At hour 12, LDL caused a slight but significant increase in both 2.4- and 3.4-kb PAI-1 mRNA levels in HMC. LDL also stimulated uPA and tPA mRNA synthesis at this incubation time. At hours 24 and 48, LDL induced a more than twofold increase in PAI-1, uPA, and tPA mRNA levels in HMC (Fig. 1).


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Fig. 1.   Northern blot analysis of plasminogen activator (PA) inhibitor-1 (PAI-1), urokinase-type PA (uPA), and tissue-type PA (tPA) mRNA in human mesangial cells (HMC). Cells were incubated with serum-free DMEM alone (-) or with the addition of 200 µg/ml low-density lipoprotein (LDL; +) for 6, 12, or 24 h. The blots were hybridized with [32P]-labeled cDNAs for PAI-1 (A), uPA (B), tPA (C), and beta -actin (D). E: quantitative expression of PAI-1, uPA, and tPA mRNA abundance after correcting for the beta -actin signal. The PAI-1, uPA, and tPA mRNA levels of treated HMC are expressed as percentage increases above the mRNA levels of untreated controls. Values are means ± SD of 3 separate experiments.

LDL increases ECM-associated PAI-1 antigen and fibrinolytic inhibitory activity in culture medium after prolonged incubation times. LDL induced a slight decrease in the ECM-associated PAI-1 antigen 1-6 h after incubation with HMC. After 12 h of culture with LDL, a slight but significant increase in ECM-associated PAI-1 antigen was found. LDL caused a 2.1- and 1.8-fold increase in the ECM-associated PAI-1 antigen at hours 24 and 48 (Fig. 2).


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Fig. 2.   Effects of LDL on extracellular matrix (ECM)-associated PAI-1 antigen levels. HMC were incubated with 200 µg of LDL for 1, 6, 12, 24, and 48 h. ECM-associated PAI-1 was brought into solution by the addition of tPA, and PAI-1 antigen was measured by ELISA. Values are means ± SD of 3 separate experiments. *P < 0.05 vs. controls.

Preincubation of exogenous tPA with conditioned medium, which was obtained after incubation of HMC with LDL for 24 h, resulted in a decrease of its enzymatic activity. Inhibitory activity against tPA increased in proportion to the concentration of LDL. Its maximal effect was shown when the concentration of LDL was 200 µg/ml (Fig. 3).


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Fig. 3.   Line plots showing concentration-dependent effects of LDL on inhibition of PA activity by conditioned medium of HMC exposed to LDL for 24 h. tPA (2.5-5 U/ml) was incubated for 15 min, followed by the addition of plasminogen and S2251, a synthetic chromogenic substrate of plasmin. Results are expressed in optical density (O.D.). Values are means ± SD of 3 separate experiments.

LDL increases uPA activity in adhesion plaques and tPA release in culture medium after prolonged incubation times. Zymographic analysis of adhesion plaques prepared from control cells and cells treated with LDL of 200 µg/ml for 48 h showed zones of lysis near relative molecular weights (Mr) of ~55,000, 70,000, and 100,000. There was a significant increase in the intensity of the ~100,000- and ~55,000-Mr bands after LDL treatment (Fig. 4A). The ~55,000-Mr band appears to be a monomeric uPA, whereas the ~100,000-Mr band may be a complex of uPA with PAI-1 (6). In addition, the ~70,000-Mr band seems to be tPA. When zymography was performed in the presence of amiloride, which blocks uPA activity but not tPA (30), fibrinolytic activity near Mr ~55,000 and 100,000 disappeared (Fig. 4B).


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Fig. 4.   A: zymography showing PA activity in adhesion plaques and culture media from LDL-treated (+) or untreated (-) cells after 48-h incubation. B: in the presence of amiloride, uPA, but not tPA, activity is blocked in adhesion plaques.

LDL induced up to a 1.3-fold increase in the tPA antigen present in the medium 24 and 48 h after incubation with HMC, when it was measured utilizing ELISA kits (Table 1). On the other hand, zymography of the medium from HMC incubated with LDL showed no differences in band intensity compared with controls (Fig. 4A).

                              
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Table 1.   Effects of LDL on tPA release in HMC

LDL induces two PKC peaks in HMC. As previously shown by our laboratory (15), incubation of HMC with LDL induced a rapid PKC activation by 15 min. This decreased to control values by hour 2. Total PKC activity again increased to 2.9 ± 1.1 times the control level 6 h after the addition of LDL. At hours 9, 12, and 15, PKC activity was still significantly higher in the LDL-treated group than in controls. After 18 h, this effect had disappeared (Fig. 5).


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Fig. 5.   Effects of LDL on protein kinase C (PKC) activity of HMC. Data are expressed as density units, calculated as the ratio of PKC-specific activity of the sample to that of normal control. Values are means ± SD of 3 comparable experiments. *P < 0.05 vs. controls.

At basal conditions, PKC-alpha , -beta 1, and -delta were predominantly found in cytosol with only a minor portion in the membrane fraction. LDL increased membrane content of PKC-alpha , -beta 1, and -delta to 250-400% of the basal level and showed a decreased cytosol content at 6 h (Fig. 6).


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Fig. 6.   Effects of LDL on expression of PKC-alpha , -beta 1, and -delta in HMC as monitored by Western blot analysis. Cells were incubated with serum-free DMEM alone or with addition of 200 µg/ml LDL for 6 h. PKC isoforms in cytosol and membrane fractions were separately measured.

PKC downregulation or inhibition attenuates the LDL-induced PAI-1, uPA, and tPA mRNA expression. The role played by PKC in causing an LDL-induced increase in PAI-1, uPA, and tPA gene expression was assessed by analysis of the inhibiting enzymatic activity of the kinase in HMC. Pretreatments of HMC with GFX 30 min before the start of the LDL stimulation blocked the LDL-induced increases in PAI-1, uPA, and tPA mRNA expression. Prior exposure of HMC to PMA for 24 h before exposure to LDL also blocked the stimulatory effect of LDL on the mRNA expression (Fig. 7). Addition of GFX at 5 h, which was 1 h before the second peak, and at 12 h also prevented the PAI-1, uPA, and tPA mRNA overproduction induced by LDL (Fig. 8). On the other hand, the addition of GFX at 18 h after the initiation of LDL treatment did not significantly alter LDL-induced PAI-1, uPA, and tPA mRNA generation (data not shown).


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Fig. 7.   Northern blot analysis of PAI-1, uPA, and tPA mRNAs in HMC incubated for 24 h with serum-free DMEM alone (lane 1), with the addition of 200 µg/ml LDL (lane 2), pretreated with GF-109203X (GFX; lane 3), or pretreated with phorbol myristate acetate (PMA; lane 4) and subsequently exposed to LDL for 24 h. The blots were hybridized with [32P]-labeled cDNAs for PAI-1 (A), uPA (B), tPA (C), and beta -actin (D). E: quantitative expression of PAI-1, uPA, and tPA mRNA abundance after correcting for the beta -actin signal. Values are means ± SD of 3 separate experiments. *P < 0.05 vs. LDL only at respective mRNA.



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Fig. 8.   Northern blot analysis of PAI-1, uPA, and tPA mRNAs in HMC incubated for 24 h with serum-free DMEM alone (lane 1), with the addition of 200 µg/ml LDL in the absence of GFX (lane 2) or in the presence of GFX at 5 h (lane 3) or at 12 h after the initiation of LDL treatment (lane 4). The blots were hybridized with [32P]-labeled cDNAs for PAI-1 (A), uPA (B), tPA (C), and beta -actin (D). E: quantitative expression of PAI-1, uPA, and tPA mRNA abundance after correcting for the beta -actin signal. Values are means ± SD of 3 separate experiments. *P < 0.05 vs. LDL only at respective mRNA.

Also, the addition of GFX to HMC exposed to LDL for 6 h inhibited LDL-induced downregulation of PAI-1 mRNA expression (Fig. 9), suggesting that the early effects of LDL with a decrease in PAI-1 mRNA may be mediated by PKC.


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Fig. 9.   Northern blot analysis of PAI-1 (A) and beta -actin (B) mRNA in HMC incubated for 6 h with serum-free DMEM alone (lane 1), with the addition of 200 µg/ml LDL in the absence of GFX (lane 2), or in the presence of GFX (lane 3).

Antioxidant attenuates the LDL-induced PAI-1 mRNA expression. The addition of vitamin E (50 µM) to HMC exposed to LDL for 48 h markedly reduced PAI-1 mRNA expression (Fig. 10).


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Fig. 10.   Northern blot analysis of PAI-1 (A) and beta -actin (B) mRNA in HMC incubated for 48 h with serum-free DMEM alone (lane 1), with the addition of 200 µg/ml LDL in the absence of vitamin E (lane 2), or in the presence of vitamin E (lane 3).


    DISCUSSION
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

These studies demonstrate that LDL either downregulated or upregulated PAI-1, uPA, and tPA in HMC according to incubation times. They also show that the upregulation of PAI-1, uPA, and tPA after long-term LDL exposure could be mediated by a delayed PKC activation and that LDL-induced PA inhibitory activity was greater than PA activity.

We show that LDL induced a second PKC activation at 6 h after the start of LDL stimulation, in addition to the previously described first peak at 15 min (15). In contrast to the transient first PKC activation, the second one persisted for over 9 h and gradually decreased to basal level at hour 18 after the start of LDL administration. Translocation of PKC-alpha , -beta 1, and -delta from cytosol to the membrane was induced by LDL at hour 6. Lipid peroxidation products were detected in culture medium even after a 3-h incubation of LDL with HMC (14). Thus the second PKC activation by LDL in the present study might be mediated through the scavenger receptor (16) rather than through the LDL receptor in HMC.

In this study, LDL promoted both 2.4- and 3.4-kb PAI-1 mRNA expression in HMC after prolonged incubation times, but short incubation times with LDL rather decreased 2.4-kb PAI-1 mRNA levels. In parallel with PAI-1 mRNA levels, LDL increased ECM-associated PAI-1 antigen levels over a 12- to 48-h incubation period. Moreover, PA inhibitory activity in culture medium was increased by LDL, as shown by chromogenic assay for residual tPA activity.

It is tempting to speculate that LDL's liability to peroxidation might be responsible for biphasic regulation of PAI-1 by LDL. Prolonged incubation of native LDL with cultured HMC has been shown to result in oxidative modification (14). In the present study, an antioxidant, vitamin E, attenuated the LDL-induced PAI-1 mRNA synthesis, suggesting that PAI-1 overproduction in HMC after long-term LDL exposure might be mediated by oxidation products. In contrast, initial downregulation of PAI-1 after short-term LDL exposure could be related to native LDL effect on PAI-1 expression and, therefore, may have physiological relevance. In support of our hypothesis, Dichtl et al. (5) demonstrated that the effect of native LDL on PAI-1 expression was not consistent in cultured VSMC and that augmentation of LDL levels in rats led to LDL oxidation at later time frames with increased vascular expression of PAI-1.

We also found that LDL-treated HMC showed a significant increase in uPA mRNA expression at late incubation times, despite a rapid and transient decrease in uPA mRNA at earlier time frames. Adhesion plaques from HMC treated with LDL for 48 h also exhibited an increase in the intensity of possible monomeric uPA and complex of uPA with PAI-1 compared with the controls. Furthermore, the zymography performed in the presence of amiloride showed no fibrinolytic activity, thus confirming the uPA nature of lysis.

The level of tPA mRNA was also markedly decreased by LDL in earlier time frames but increased after prolonged incubation times. Despite a distinctive increase in tPA mRNA expression at hours 24 and 48, LDL enhanced the tPA release only to a slight degree. Furthermore, increased tPA activity was not clearly demonstrated by zymography of the medium.

In our study, both the downregulation of PKC by PMA and inhibition of PKC by GFX resulted in the abolition of the stimulatory effects of LDL on PAI-1, uPA, and tPA gene expression. These results suggest that LDL after long-term incubation stimulates the expression of the PAI-1, uPA, and tPA mRNAs in HMC mediated by PKC. Early PKC activation may not mediate the LDL-induced overproduction of these mRNAs in HMC, because the addition of GFX at 12 h after the administration of LDL or 6 h after the second PKC peak still effectively prevented the mRNA synthesis. The addition of GFX at 18 h after initiation of LDL stimulation failed to inhibit the overexpression of these mRNAs. Thus the delayed activation of PKC occurring between 6 and 15 h after the administration of LDL may signal the upregulation of PAI-1, uPA, and tPA expression in HMC.

PAI-1 has been implicated in renal disease as being a mediator of ECM accumulation (24) and as a feedback mechanism to limit vascular fibrinolysis (17, 26). Our results describing the LDL-induced increase in PAI-1 synthesis as well as increased PA inhibitory activity at later time frames suggest that persistent hypercholesterolemia in chronic renal disease could lead to a mesangial ECM accumulation and to eventual renal fibrosis partly linked to an impaired matrix degradation.

Abnormal ECM accumulation is often preceded or combined with an increased expression of ECM-degrading enzymes (29), suggesting that increased proteolytic activity is required for the degradation of the normal ECM before its replacement by the abnormal ECM (24). Similarly, LDL-induced enhanced tPA or uPA synthesis, though minor, may help PAI-1 to be accumulated in ECM by degrading the normal mesangial matrix.

In the presence of fibrin, the fibrinolytic activity of tPA is much higher than that of uPA. Recent animal studies suggest that uPA may not have an important role in preventing glomerular fibrin deposition (12). uPA, after binding to the uPA receptor, can activate cell-signaling pathways involved in cell adhesion, cell migration, and chemotaxis, independently of its proteolytic activity (3). Whether an LDL-induced increase in uPA can also activate the mesangial cell-signaling pathways needs to be clarified further.

In summary, LDL regulates PAI-1, uPA, and tPA in biphasic patterns in HMC, and the upregulation of PAI-1, uPA, and tPA after long-term LDL exposure seems to be mediated by a delayed PKC activation associated with increased PA inhibitory activity. These results suggest that prolonged incubations of LDL with HMC lead to a PA/inhibitor imbalance that favors accumulation of matrix. This effect of LDL might have a pathophysiological function in the pathogenesis of glomerulosclerosis.


    ACKNOWLEDGEMENTS

We thank Dr. David J. Loskutoff (Department of Immunology, Scripps Clinic, La Jolla, CA) for providing the PAI-1 cDNA probe. We also thank Dr. Eric Rondeau (Hôpital Tenon, Paris, France) for helpful comments and discussion.


    FOOTNOTES

This study was supported by grants from the Ministry of Health and Welfare (Korea).

Address for reprint requests and other correspondence: H. S. Lee, Dept. of Pathology, Seoul National Univ. College of Medicine, Chongno-gu, Yongon-dong 28, Seoul 110-799, Korea (E-mail: hyunsoon{at}plaza.snu.ac.kr).

The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

10.1152/ajprenal.00304.2001

Received 25 September 2001; accepted in final form 19 March 2002.


    REFERENCES
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ABSTRACT
INTRODUCTION
MATERIALS AND METHODS
RESULTS
DISCUSSION
REFERENCES

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