Oxidized LDL Stimulates Matrix Metalloproteinase-1 Expression in Human Vascular Endothelial Cells

Cell Culture

Human umbilical vein endothelial cells (HUVECs), human aortic endothelial cells (HAECs), and culture medium were purchased from Cascade Biologics, Inc (Portland, Ore). The cells were cultured in a 5% CO₂ atmosphere in medium 200 (Cascade Biologics) containing 2% FBS, 0.17 nmol/L human fibroblast growth factor, 1.6 nmol/L human epidermal growth factor, 2.76 μmol/L hydrocortisone, and antibiotics. The flasks were precoated with 0.1% gelatin. The medium was changed every 2 or 3 days. Cell monolayers in the second to sixth passage were used in the experiments.

Lipoprotein Isolation and Oxidation

LDL (1.019 to 1.063 g/mL) was separated from the plasma of normal volunteers by sequential ultracentrifugation at 60 000 rpm for 24 hours at 10°C in a 60-Ti rotor (Beckman). The isolated LDL was washed and concentrated by ultracentrifugation in a SW41 rotor (Beckman) spun at 40 000 rpm for 24 hours at 10°C. LDL was then dialyzed against a 0.16 mol/L NaCl solution containing 300 μmol/L EDTA, pH 7.4, sterilized by passage through a 0.45-μm filter (Gelman Sciences) and stored under N₂ at 4°C. LDL was oxidized with Cu²⁺ as described previously.⁸ In brief, EDTA was removed by passing LDL over a PD-10 column. LDL (1.5 mg/mL) was mixed with 10 μmol/L Cu²⁺ and incubated at 37°C for 18 hours. EDTA (300 μmol/L) and 40 μmol/L BHT were added to stop the reaction. The oxidized LDL was dialyzed against a 0.16 mol/L NaCl solution containing 300 μmol/L EDTA, pH 7.4, by using dialysis tubing with a molecular weight cutoff of 12 000 kDa. The degree of LDL oxidation was determined by the thiobarbituric acid–reactive substances assay,⁹ by measurements of conjugated dienes¹⁰ and fluorescent compounds,¹¹ and by electrophoretic mobility assay on agarose gel in barbital buffer at pH 8.6.¹² The formation of conjugated dienes and fluorescent compounds, as surrogates for oxidation of lipid and protein moieties, respectively, in oxidized LDL was 5.4-fold and 13-fold of those in native LDL, respectively (the Table⇓). The endotoxin level in oxidized LDL preparations was measured using an endotoxin assay kit (Sigma), and the level was below the lower limit of detection (0.015 U/mL).

Immunoblotting Analysis of Secreted MMP-1

Immunoblotting analysis of secreted MMP-1 in conditioned medium was performed with anti–MMP-1 monoclonal antibodies (Oncogene Research Products) according to the instructions from the manufacturer. In brief, conditioned medium was 10-fold concentrated by lyophilization, and an aliquot containing 25 μg of protein was electrophoresed in a 10% SDS polyacrylamide gel and then transferred to a polyvinylidene membrane (NEN Life Science Products). After the transfer, the membrane was incubated with a blocking buffer containing 20 mmol/L Tris, pH 7.6, 130 mmol/L NaCl, 0.1% Tween-20, and 5% nonfat dry milk for 1 hour at room temperature. The membrane was washed and then incubated at 4°C overnight with anti–MMP-1 monoclonal antibodies, followed by incubation with a horseradish peroxidase–conjugated rabbit anti-mouse IgG for 1 hour at room temperature. MMP-1 was detected by incubating the membrane with chemiluminescence reagents (NEN Life Science Products) for 1 minute and exposing it to x-ray film for 30 seconds. The film was scanned by densitometry for quantitation of MMP-1.

ELISA of Secreted MMP-1

Secreted MMP-1 by endothelial cells was quantitated using an ELISA kit (Oncogene) according to the protocol from the manufacturer. One hundred microliters of conditioned medium were pipetted into each well of the plate coated with monoclonal anti-human MMP-1 antibodies, and incubation was carried out at room temperature for 2 hours. After the incubation, the plate was washed 5 times with PBS and second monoclonal anti-human MMP-1 antibodies conjugated with horseradish peroxidase were added. After incubation at room temperature for 1 hour, the plate was washed exhaustively, followed by addition of tetramethylbenzidine. The plate was incubated in the dark at room temperature for 30 minutes, and the reaction was stopped by addition of 2.5N H₂SO₄. The absorbance at 450 nm was measured by a spectrophotometric plate reader. A standard curve was obtained using purified MMP-1 as the antigen supplied with the kit.

Zymography Analysis of Secreted MMP-1

Ten microliters of HUVEC-conditioned medium was electrophoresed on 12% precast zymogram gels (Novex) containing 1 mg/mL casein under nonreducing conditions. The gel was washed with Tris-buffered saline buffer containing 2.5% Triton X-100 and then incubated with developing buffer containing 50 mmol/L Tris, pH 7.5, 200 mmol/L NaCl, 5 mmol/L CaCl₂, and 0.02% Brij-35 at 37°C for 24 hours in the presence or absence of 0.3 mmol/L 1,10-phenanthroline, a specific MMP inhibitor.¹³ After the incubation, the gel was stained for 3 hours with Coomassie blue and destained.

Preparation of Radiolabeled MMP-1 cDNA Probe

A plasmid clone of human MMP-1 was purchased from the American Type Culture Collection (Manassas, Va). A 2.2-kb MMP-1 cDNA fragment was released from the plasmid by digestion of the plasmid with XhoI. The fragments were purified from agarose gel and radiolabeled with [α-³²P]ATP by using the Prime-a-Gene Labeling System (Promega). Human glyceraldehyde-3-phosphate dehydrogenase (GAPDH) cDNA was also purchased from the American Type Culture Collection and radiolabeled as described above.

Northern Blot Analysis

Total cellular RNA was isolated from control and stimulated endothelial cells with the Ultraspec RNA isolation reagent according to the instructions from the manufacturer (Biotecx Laboratories, Inc). Twenty micrograms of total RNA for each sample was electrophoresed in a 1% agarose gel and transferred to a nylon membrane in a 20× saline–sodium citrate (SSC) buffer. The nylon membrane was prehybridized at 42°C for 3 hours in a buffer containing 50% formamide and then hybridized with the ³²P-labeled MMP-1 or GAPDH cDNA for 18 hours at 42°C in the presence of 50% formamide. After hybridization, the membrane was washed at 50°C for 1 hour in a buffer containing 2× SSC and 0.1% SDS and for 0.5 hours in a buffer containing 0.1× SSC and 0.1% SDS. The membrane was then exposed to x-ray film at −70°C for 8 to 18 hours. The relative amounts of MMP-1 or GAPDH mRNA were estimated by densitometry scanning of the films.

Nuclear Run-on Assay

Nuclear run-on transcription assay was performed using a method described by Greenberg and Bender,¹⁴ with slight modifications. Endothelial cells were dislodged from culture dishes by scraping with a rubber policeman. The cells were then centrifuged and lysed with a lysis buffer containing 10 mmol/L Tris-HCl, 10 mmol/L NaCl, 3 mmol/L MgCl₂, and 0.5% NP-40. The nuclei were spun down at 500g at 4°C and the supernatant was removed. The nuclear pellet was resuspended in a buffer containing 50 mmol/L Tris-HCl, pH 8.3, 40% glycerol, 5 mmol/L MgCl₂, and 0.1 mmol/L EDTA and stored at −70°C. To perform the nuclear run-on assay, 20×10⁶ nuclei from each treatment group were incubated in buffer containing 5 mmol/L Tris-HCl, pH 8.0; 2.5 mmol/L MgCl₂; 150 mmol/L KCl; 2.5 mmol/L each of ATP, GTP, CTP, and [α-³²P]UTP (100 μCi, Amersham); and 12.5 mmol/L DTT for 30 minutes at 37°C with shaking. Radiolabeled transcripts were isolated using Ultraspec RNA (Biotecx) and dissolved in diethylpyrocarbonate-treated water.

One microgram of MMP-1 and GAPDH cDNA was boiled for 5 minutes, chilled quickly on ice, and then applied to a nylon membrane mounted in a dot-blot apparatus (Schleicher & Schuell, Inc). The immobilized cDNAs were hybridized with the radiolabeled transcripts obtained from the nuclear run-on reaction for 48 hours at 45°C in buffer containing 50 mmol/L PIPES, 500 mmol/L NaCl, 33% formamide, 0.1% SDS, 2 mmol/L EDTA, and 5 mg/mL salmon sperm DNA. The nylon membrane was washed twice with 1× SSC with 0.1% SDS at 50°C for 30 minutes each. Dried membrane was exposed to x-ray film.

Collagenase Activity Assay

This assay was modified from the procedure of Fisher et al.¹⁵ Type I collagenase activity in the conditioned medium of endothelial cells was determined using [³H]type I collagen (DuPont-NEN) as a substrate, according to the procedures provided by the manufacturer. In brief, the ³H-labeled type I collagen was mixed with unlabeled collagen (1 mg/mL) in 0.01N acetic acid (specific activity 0.68 nCi/μg). Formation of collagen fibrils was induced by mixing 20 μL of the above collagen with 30 μL of a buffer containing 20 mmol/L Tris-HCl, pH 7.5, 50 mmol/L NaCl, and 10 mmol/L CaCl₂ and incubating the mixture at 37°C for 2 hours. After the incubation, 100 μL of the endothelial cell–conditioned medium was added to the mixture, and the incubation was continued at 25°C for 3 hours. The reaction was stopped by chilling the samples on ice and then centrifuged at 12 000g for 5 minutes. The amount of degraded type I collagen was determined by counting the radioactivity in the supernatant. Radioactivity of the nonclotted collagen that was present in the supernatant before the addition of the conditioned medium was predetermined and subtracted from the radioactivity of the supernatant obtained after the incubation. The protein content of the cultured endothelial cells was determined by the assay of Lowry et al,¹⁶ and collagenase activity in the conditioned medium was adjusted according to the cell protein content.

Stimulation of MMP-1 Secretion From HUVECs by Oxidized LDL

The secretion of MMP-1 by endothelial cells exposed to oxidized LDL was determined by immunoblotting analysis of the conditioned medium by using an anti–MMP-1 monoclonal antibody. The blot (Figure 1⇓) showed a single band of 52 kDa, which corresponds to the latent form of MMP-1. Results showed that cells incubated with either native or oxidized LDL at doses of 50 or 100 μg/mL increased MMP-1 secretion to a greater extent than in cells incubated with medium alone. MMP-1 secretion by cells stimulated with 100 μg/mL oxidized LDL was, however, 2-fold higher than that obtained from cells stimulated with the same amount of native LDL, indicating that the oxidized LDL is more potent than native LDL in stimulating MMP-1 secretion by endothelial cells. As a positive control, 100 nmol/L phorbol 12-myristate 13-acetate (PMA) increased MMP-1 secretion markedly. In contrast, stimulation of the cells with IL-1β led only to a slight increase in MMP-1 secretion.

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Figure 1.

MMP-1 secretion from HUVECs exposed to native or oxidized LDL. HUVECs were incubated at 37°C for 24 hours with culture medium alone or with culture medium containing 100 nmol/L PMA, 10 U/mL IL-1β, 50 μg/mL native (n) LDL, 50 μg/mL oxidized (ox) LDL, 100 μg/mL native LDL, or 100 μg/mL oxidized LDL. Immunoblotting analysis (A) of the conditioned medium was performed to identify MMP-1. The MMP-1 band was visualized by incubating the membrane with Renaissance chemiluminescence reagent for 60 seconds and exposing it to x-ray film for 30 seconds. The amount of MMP-1 was semiquantitated by densitometry scanning of the film (B). The data presented are representative of 3 experiments with similar results.

To quantitate MMP-1 in endothelial cell–conditioned medium, we performed an enzyme immunoassay by using a sandwich ELISA plate. The results (Figure 2⇓) showed that oxidized LDL stimulated MMP-1 secretion by 3.5-fold over control level, which is significantly higher than that stimulated by native LDL (2-fold). PMA stimulated MMP-1 secretion by 4.5-fold. These results are consistent with the data observed from the immunoblotting as described above.

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Figure 2.

Quantitation of secreted MMP-1 from HUVECs by ELISA. HUVECs were incubated at 37°C for 24 hours with culture medium alone (control) or with the culture medium containing 100 nmol/L PMA, 100 μg/mL native LDL, or 100 μg/mL oxidized (Ox) LDL. The conditioned medium was collected for measurement of MMP-1 by using an ELISA kit. After incubation with the medium at room temperature for 2 hours, the ELISA plate was incubated with anti-human MMP-1 antibodies conjugated with horseradish peroxidase for 1 hour, followed by incubation with substrate as described in Methods. Absorbance was measured by an ELISA reader at a wavelength of 450 nm. The amount of MMP-1 secreted by the cells was determined using a standard curve generated with purified MMP-1. MMP-1 secreted by unstimulated cells (278 ng · mg⁻¹ · h⁻¹) was designated as 100%. Data are mean±SEM.

Concentration-Dependent Stimulation of MMP-1 Secretion by Oxidized LDL

HUVECs were incubated with increasing concentrations of oxidized LDL (0 to 125 μg/mL) for 24 hours, and the conditioned medium was collected and used to perform immunoblotting analysis of secreted MMP-1 as described above. Our results showed that oxidized LDL induced MMP-1 release in a concentration-dependent manner, and the maximal stimulation was obtained with 100 μg/mL oxidized LDL, which led to a 4-fold increase over the level released by untreated cells (Figure 3⇓). Therefore, we selected 100 μg/mL oxidized LDL as the optimal concentration to stimulate cells in the subsequent experiments.

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Figure 3.

Concentration-dependent stimulation of MMP-1 release by HUVECs exposed to oxidized (Ox) LDL. The cells were incubated for 24 hours with increasing concentrations of oxidized LDL as indicated, and the conditioned medium was collected and subjected to immunoblotting analysis to determine the amount of secreted MMP-1.

Casein Substrate Zymography Analysis of Secreted MMP-1

Immunoblotting data only showed a single band with a molecular weight of 52 kDa, which corresponds to the latent form of MMP-1 (Figure 1⇑). The absence of cleaved MMP-1 in the immunoblot could be due to the following reasons: (1) the monoclonal antibody used in the immunoblotting only recognizes the latent form of MMP-1 or (2) the MMP-1 secreted from endothelial cells was not cleaved in this in vitro system. To determine whether or not the MMP-1 secreted from endothelial cells was cleaved, we performed casein substrate zymography analysis. After treatment of the cells with medium alone (control) or with medium containing 100 μg/mL oxidized LDL at 37°C for 24 hours, 10 μL of conditioned medium was loaded onto 12% precast zymogram gels containing 1 mg/mL casein, and zymography was performed. Two bands with molecular weights of 52 and 46 kDa were observed, and the latter was the major band (Figure 4⇓), suggesting that most of the secreted MMP-1 was cleaved. We can therefore conclude the possibility that cells stimulated by oxidized LDL secreted both latent and cleaved forms of MMP-1 to a much higher level than did cells incubated with medium alone. The caseinolytic activity was completely inhibited when the gel was incubated in the presence of 0.3 mmol/L 1,10-phenanthroline, an MMP inhibitor,¹³ indicating that MMPs were responsible for the activity. The above results indicate that the monoclonal antibody used in immunoblotting recognizes only latent MMP-1.

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Figure 4.

Casein substrate zymography analysis of MMP-1. HUVECs were incubated with medium alone (a) or with 100 μg/mL oxidized LDL (b) at 37°C for 24 hours. Ten microliters of conditioned medium was electrophoresed on 12% polyacrylamide gel containing 1 mg/mL casein under nonreducing condition. The gel was incubated in developing buffer containing 50 mmol/L Tris, pH 7.5, 200 mmol/L NaCl, 5 mmol/L CaCl₂, and 0.02% Brij-35 at 37°C for 24 hours in the presence or absence of 0.3 mmol/L 1,10-phenanthroline. The gel was stained with Coomassie blue.

Oxidized LDL Increased Steady-State MMP-1 mRNA Level

The effect of oxidized LDL on cellular steady-state levels of MMP-1 mRNA was determined by Northern blotting. HUVECs were incubated with or without oxidized LDL for 24 hours, and RNA was then isolated for Northern blot analysis. PMA was used as a positive control, because it had been previously shown that PMA activates MMP-1 transcription.¹⁷ To determine whether the MMP-1 expression was dependent on the degree of LDL oxidation, the cells were treated with 100 μg/mL native LDL, 100 μg/mL oxidized LDL that was copper oxidized for 12 hours, and 100 μg/mL oxidized LDL that was copper oxidized for 21 hours. Results showed that both native LDL and oxidized LDL increased the MMP-1 mRNA level. However, oxidized LDL stimulated MMP-1 mRNA to a significantly greater extent compared with native LDL (Figure 5⇓). The degree of LDL oxidation influenced MMP-1 mRNA expression considerably, as our data showed that LDL oxidized for 21 hours induced more MMP-1 mRNA than did LDL oxidized for 12 hours. PMA induced a remarkable increase in MMP-1 mRNA.

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Figure 5.

Effect of oxidized LDL on MMP-1 mRNA expression by HUVECs. HUVECs were incubated for 18 hours with medium alone (control, a) or with the medium containing 100 nmol/L PMA (b), 100 μg/mL native (n) LDL (c), 100 μg/mL oxidized LDL (12-hour oxidation, d), or 100 μg/mL oxidized LDL (21-hour oxidation, e). RNA was isolated after the incubation, and 20 μg of RNA for each sample was used to perform Northern blot analysis of MMP-1 mRNA (A). MMP-1 and GAPDH cDNAs were radiolabeled with [³²P]ATP and hybridized with the immobilized MMP-1 and GAPDH mRNA, respectively, as described in Methods. The amounts of MMP-1 and GAPDH mRNAs were quantitated by densitometry scanning (B). The MMP-1 to GAPDH ratio of the relative intensity was calculated. The data are presented as percent of control. oxLDL1, LDL oxidized in the presence of copper for 12 hours; oxLDL2, LDL oxidized in the presence of copper for 21 hours.

Time-Dependent Stimulation of MMP-1 Expression by Oxidized LDL

The time course of MMP-1 expression by HUVECs exposed to oxidized LDL was also determined by Northern blot analysis. The cells were incubated with 100 μg/mL oxidized LDL for 2, 4, 10, and 22 hours, and RNA was isolated and run on a Northern blot. The results showed that oxidized LDL stimulated MMP-1 mRNA expression in a time-dependent manner (Figure 6⇓). A 3- and a 24-fold increase in MMP-1 mRNA over the control level was observed by 2 and 22 hours of stimulation, respectively, showing that the cellular MMP-1 mRNA level increased quickly and steadily in response to oxidized LDL.

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Figure 6.

Time course of oxidized LDL–stimulated MMP-1 expression in HUVECs. HUVECs were incubated with medium containing 100 μg/mL oxidized LDL for 2 hours (a), 4 hours (b), 10 hours (c), and 22 hours (d) or with medium alone for 22 hours as control (e). After the incubation, RNA was isolated and 20 μg of RNA from each sample was electrophoresed on a 1% agarose gel and transferred to a nylon membrane. The immobilized MMP-1 and GAPDH mRNAs were hybridized with radiolabeled MMP-1 and GAPDH cDNA, respectively, and detected by autoradiography (A). The amounts of MMP-1 and GAPDH mRNAs were quantitated by densitometry scanning of the film, and the MMP-1 to GAPDH radio of the relative intensity was calculated. The data are presented as percent of the control (B).

Stimulation of MMP-1 Secretion by Oxidized LDL Is Due to Transcriptional Activation

The increase in MMP-1 mRNA in cells stimulated by oxidized LDL could be due to transcriptional activation of the MMP-1 gene or to an increased stability of MMP-1 mRNA. To determine whether or not oxidized LDL activates MMP-1 transcription, HUVECs were incubated with oxidized LDL in the absence or presence of actinomycin D, an inhibitor of transcription.¹⁸ After the incubation, the MMP-1 secreted from the cells was determined by immunoblotting analysis. Our results showed that 2 μg/mL actinomycin D completely inhibited oxidized LDL–stimulated MMP-1 secretion (Figure 7⇓), suggesting that oxidized LDL–stimulated MMP-1 secretion is transcription-dependent. To further confirm that oxidized LDL activates MMP-1 transcription, nuclear run-on analysis was performed. Results showed higher hybridization signals of MMP-1 after using transcription products from nuclei of oxidized LDL–treated endothelial cells, compared with those from nuclei of control cells (Figure 8⇓), indicating that oxidized LDL induces MMP-1 gene activation.

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Figure 7.

The effect of actinomycin (Act.) D on the oxidized (Ox) LDL–stimulated MMP-1 secretion. HUVECs were incubated with 100 μg/mL oxidized LDL in the absence or presence of actinomycin D (1 to 5 μg/mL) at 37°C for 24 hours. The conditioned medium was collected and immunoblotting performed as described in Figure 1⇑ to determine secreted MMP-1.

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Figure 8.

Nuclear run-on analysis of MMP-1 transcription activated by oxidized (Ox) LDL. Radiolabeled nascent RNA transcribed in nuclei, isolated from endothelial cells treated with medium alone (Control) or from the cells treated with 100 μg/mL oxidized LDL), were hybridized to 1 μg of MMP-1 and GAPDH cDNAs immobilized on a dot-blotted nylon membrane for 48 hours at 45°C. The nylon membrane was washed twice with 1× SSC with 0.1% SDS at 50°C for 30 minutes each before exposure to x-ray film.

Stimulation of MMP-1 Expression in HAECs by Oxidized LDL

In this study, HAECs was employed to determine whether oxidized LDL also stimulates MMP-1 expression in arterial endothelial cells. The results from immunoblotting study showed that oxidized LDL also stimulated MMP-1 secretion from HAECs more effectively than did native LDL (Figure 9A⇓). Northern blot showed that oxidized LDL increased more MMP-1 mRNA than did native LDL (Figures 9B⇓ and 9C⇓). These data indicated that oxidized LDL stimulated the expression of MMP-1 in both arterial and venous endothelial cells.

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Figure 9.

Stimulation of MMP-1 secretion and expression by HAECs exposed to oxidized LDL. A, HAECs were incubated at 37°C for 24 hours with medium alone (a) or with the medium containing 100 nmol/L PMA (b), 10 U/mL IL-1β (c), 100 μg/mL native LDL (d), or 100 μg/mL oxidized LDL (e). The conditioned medium was subjected to immunoblotting to determine the secreted MMP-1 as described above. B, HAECs were incubated at 37°C for 24 hours with medium alone (a) or with the medium containing 100 μg/mL native LDL (b), 100 μg/mL oxidized LDL (c), or 10 U/mL IL-1β (d). RNA was isolated and subjected to Northern blotting to determine MMP-1 mRNA level as described above. C, The amounts of MMP-1 and GAPDH mRNAs were quantitated by densitometry scanning of the film and the MMP-1 to GAPDH radio of the relative intensity was calculated.

Stimulation of Collagenase Activity by Oxidized LDL

In addition to the zymography analysis, collagenase activity in conditioned medium was further determined using ³H-labeled type I collagen as a substrate. Our results (Figure 10⇓) showed that the collagenase activity in the medium conditioned by exposure of the cells to oxidized LDL was significantly higher than that observed in the medium collected from untreated cells. Although native LDL also increased collagenase activity, the extent of the increase was lower than that present in medium conditioned by exposure of the cells to oxidized LDL. Interestingly, medium conditioned by exposure of the cells to PMA did not have increased collagenase activity, suggesting that regardless of the fact that PMA stimulated MMP-1 release from HUVECs, it may also induce release of a comparable amount of MMP-1 inhibitors, such as tissue inhibitors of MMP, which prevent an increase in MMP-1 activity. The collagenase activity stimulated by oxidized LDL was inhibited by 1 mmol/L 1,10-phenanthroline, indicating that MMPs were responsible for the collagenase activity in the cell-conditioned medium.

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Figure 10.

Collagenase activity in medium conditioned by exposure of HUVECs to oxidized LDL.³H-labeled type I collagen (1 mg/mL, specific activity 0.68 nCi/μg) was mixed with a buffer containing 20 mmol/L Tris-HCl (pH 7.5), 50 mmol/L NaCl, and 10 mmol/L CaCl₂ and incubated at 37°C for 2 hours. After the incubation, endothelial cell–conditioned medium was added into the mixture, and the incubation was continued at 25°C for 3 hours in the presence or absence of 1 mmol/L 1,10-phenanthroline. After centrifugation to pellet the collagen fibril, the radioactivity in the supernatant was measured. Radioactivity that presented in the supernatant before the addition of the conditioned medium was subtracted from that of the supernatant obtained after the incubation with the conditioned medium. The collagenase activity was adjusted by cell protein content. The data presented are representative of 3 experiments with similar results.