Nitroglycerin upregulates matrix metalloproteinase expression by human macrophages
Alison K. Death, PhD*,
Shirley Nakhla, BSc ,
Kristine C. Y. McGrath, BSc (Hons)*,
Sally Martell, BSc (Hons) ,
Dennis K. Yue, MBBS, PhD, FRACP*,
Wendy Jessup, PhD and
David S. Celermajer, MBBS, PhD, FRACP* ,*
* Department of Medicine, University of Sydney, Sydney, Australia
Heart Research Institute, Camperdown, Australia
Departments of Endocrinology, Camperdown, Australia
Cardiology, Royal Prince Alfred Hospital, Camperdown, Australia
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Figure 1 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) zymography of media conditioned by human monocyte-derived macrophages after 24-h exposure to nitroglycerin (NTG). (a) Cells were exposed for 24 h to glycerol 200 pmol (Lanes 1 and 2) or NTG 200 pmol (Lanes 3 and 4). Aliquots of conditioned media, normalized to 15 µg total protein, were run on SDS-PAGE (10%[w/v] gel) supplemented with 1 mg/ml gelatin substrate under nonreducing conditions. Zones of clearing at approximately 90 kDa (as measured by simultaneous electrophoresis of protein standards, Lane 5) indicate gelatinolytic activity of both latent (heavier) and active (lighter) matrix metalloproteinase (MMP)-9. Band densities are: Lanes 1 and 2, glycerol (latent 162,160; 167,492: active 176,945; 173,336) and Lanes 3 and 4, NTG 200 pmol (latent 29,515; 22,223: active 39,664; 31,960). (b) Aliquots of conditioned media, normalized to 15 µg total protein, were treated with 1 mM p-aminophenyl mercuric acetate (APMA) for 30 min at 37°C. Treated samples were then run on SDS-PAGE (10%[w/v] gel) supplemented with 1 mg/ml gelatin substrate under nonreducing conditions. Zones of clearing at approximately 90 kDa (as measured by simultaneous electrophoresis of protein standards, Lane 5) indicate gelatinolytic activity of both latent and active MMP-9. Band densities are Lanes 1 and 2, glycerol (active 269,957; 276,765) and Lanes 3 and 4, NTG 200 pmol (active 321,321; 302,633). MW = molecular weight.
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Figure 2 Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) zymography of media conditioned by human monocyte-derived macrophages exposed to nitroglycerin (NTG) for 4 h. (a) Cells were exposed for 4 h to control media (Lanes 1 and 2), glycerol (GLY) 200 pmol (Lanes 3 and 4), GLY 2,000 pmol (Lanes 5 and 6), NTG 200 pmol (Lanes 7 and 8), NTG 2,000 pmol (Lanes 9 and 10), 3-morpholinosydnoimine hydrochloride 200 pmol (Lanes 11 and 12) or S-nitroso-N-acetylopenicillamine 200 pmol (Lanes 13 and 14). Aliquots of conditioned media, normalized to 4.5 µg total protein, were run on SDS-PAGE (10%[w/v]gel) supplemented with 1 mg/ml gelatin substrate under nonreducing conditions. Zones of clearing indicate gelatinolytic activity of latent matrix metalloproteinase (MMP)-9. (b) Scanning analysis of MMP-9 secreted by human MDMs. Results represent the mean ± SEM for analysis of four independent experiments and do not represent the analysis in (a) alone. *p < 0.05 compared with control cells. **p < 0.005 compared with control cells. ***p < 0.0001 compared to control cells.
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Figure 3 The effect of nitroglycerin (NTG) on matrix metalloproteinase (MMP)-9 messenger ribonucleic acid (mRNA) levels in human monocyte-derived macrophages (MDMs). Cells were exposed to control (CTRL) media (a), NTG 200 pmol (b) or NTG 2,000 pmol (c) for 4 h. Matrix metalloproteinase-9 mRNA levels were measured by competitive reverse transcription-polymerase chain reaction (described in Methods). Competitor mRNA concentrations were 8.56 (Lane 1), 17.13 (Lane 2), 34.25 (Lane 3), 68.5 (Lane 4) and 137 (Lane 5) attomoles. Arrows represent MMP-9 target (lower band) versus competitor (upper band) equivalence points for each condition. (d) Quantification of RT-PCR bands was determined by gel densitometry. Results represent the mean ± SEM for analysis of four independent experiments and do not represent the analysis in (a to c) alone. **p < 0.005 compared with control cells. ***p < 0.001 compared with control cells.
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Figure 4 The effect of nitroglycerin (NTG) on matrix metalloproteinase (MMP)-2 and MMP-7 messenger ribonucleic acid (mRNA) levels in human monocyte-derived macrophages (MDMs). Cells were exposed to control media, NTG 200 pmol or NTG 2,000 pmol for 4 h. Total ribonucleic acid was extracted and competitive reverse transcription-polymerase chain reaction (RT-PCR) was performed to measure MMP-2 (solid bars) or MMP-7 (open bars) as described in the Methods section, and each PCR reaction mix was subjected to electrophoresis on a 2% (w/v) agarose gel. Quantification of RT-PCR bands was determined by gel densitometry. Results represent the mean ± SEM for analysis of three independent experiments. *p < 0.05 and **p < 0.005 compared with control cells.
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Figure 5 The effect of nitroglycerin (NTG) on tissue inhibitor of metalloproteinase (TIMP)-1 expression in human monocyte-derived macrophages. Monocyte-derived macrophages were exposed to control media, NTG 200 pmol or NTG 2,000 pmol for 4 h. Ribonucleic acid was extracted and competitive reverse transcription-polymerase chain reaction performed to measure TIMP-1 messenger ribonucleic acid (mRNA) levels (solid bars) Y-axis represents mRNA levels as % of control. Aliquots of conditioned media were collected and TIMP-1 protein levels measured by enzyme-linked immunosorbent assay (open bars). Y-axis represents protein concentration (ng/ml). Results represent the mean ± SEM for analysis of three independent experiments. **p < 0.005 compared with control cells.
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