Oxidized LDL differentially regulates MMP-1 and TIMP-1 expression in vascular endothelial cells Yan Huang, Lanxi Song, Shan Wu, Fan Fan, Maria F Lopes-Virella Atherosclerosis Volume 156, Issue 1, Pages 119-125 (May 2001) DOI: 10.1016/S0021-9150(00)00638-9
Fig. 1 Western blot analysis of MMP-1 secreted by HUVECs exposed to oxidized LDL. HUVECs were incubated at 37°C for 24 h with culture medium alone (C) or with culture medium containing 100 nM PMA (P), 100 μg/ml oxLDL (O), 100 μg/ml native LDL (N). The conditioned medium was collected for Western blot analysis of secreted MMP-1 as described in Section 2. Immunoreactive MMP-1 was visualized by incubating the membrane with chemiluminescence reagent for 60 s and exposing it to X-ray film for 15 s. Atherosclerosis 2001 156, 119-125DOI: (10.1016/S0021-9150(00)00638-9)
Fig. 2 Northern blot analysis of TIMP-1 mRNA in HUVECs stimulated with oxLDL. HUVECs were incubated for 24 h with medium alone (C) or with the medium containing 100 μg/ml oxLDL (O), or 100 nM PMA (P). RNA was isolated after the incubation, and 20 μg RNA for each sample was used for Northern blot (A). The amounts of TIMP-1 and GAPDH mRNAs were quantitated by scanning densitometry. The ratios of relative intensity between MMP-1 and GAPDH were calculated. The value for the control cells was designated as 100%. The data are presented as percent of control (B). Atherosclerosis 2001 156, 119-125DOI: (10.1016/S0021-9150(00)00638-9)
Fig. 2 Northern blot analysis of TIMP-1 mRNA in HUVECs stimulated with oxLDL. HUVECs were incubated for 24 h with medium alone (C) or with the medium containing 100 μg/ml oxLDL (O), or 100 nM PMA (P). RNA was isolated after the incubation, and 20 μg RNA for each sample was used for Northern blot (A). The amounts of TIMP-1 and GAPDH mRNAs were quantitated by scanning densitometry. The ratios of relative intensity between MMP-1 and GAPDH were calculated. The value for the control cells was designated as 100%. The data are presented as percent of control (B). Atherosclerosis 2001 156, 119-125DOI: (10.1016/S0021-9150(00)00638-9)
Fig. 3 Effect of oxLDL on MMP-1 expression by HUVECs. HUVECs were incubated for 24 h with medium alone (C) or with the medium containing 100 nM PMA (P), 100 μg/ml oxLDL (O). RNA was isolated after the incubation, and 20 μg RNA for each sample was used to perform Northern blot analysis of MMP-1 mRNA. Atherosclerosis 2001 156, 119-125DOI: (10.1016/S0021-9150(00)00638-9)
Fig. 4 Northern blot analysis of MMP-1 and TIMP-1 mRNA in HAECs stimulated with oxLDL. HAECs were incubated for 24 h with medium alone (C) or with the medium containing 100 μg/ml oxLDL (O) or 100 nM PMA (P). RNA was isolated after the incubation and 20 μg RNA for each sample was used for Northern blot (A). The amounts of MMP-1 and GAPDH mRNAs were quantitated by scanning densitometry. The ratios of relative intensity between MMP-1 and GAPDH were calculated. The ratio for the control cells was designated as 100%. The data are presented as percent of control (B). Atherosclerosis 2001 156, 119-125DOI: (10.1016/S0021-9150(00)00638-9)
Fig. 4 Northern blot analysis of MMP-1 and TIMP-1 mRNA in HAECs stimulated with oxLDL. HAECs were incubated for 24 h with medium alone (C) or with the medium containing 100 μg/ml oxLDL (O) or 100 nM PMA (P). RNA was isolated after the incubation and 20 μg RNA for each sample was used for Northern blot (A). The amounts of MMP-1 and GAPDH mRNAs were quantitated by scanning densitometry. The ratios of relative intensity between MMP-1 and GAPDH were calculated. The ratio for the control cells was designated as 100%. The data are presented as percent of control (B). Atherosclerosis 2001 156, 119-125DOI: (10.1016/S0021-9150(00)00638-9)
Fig. 5 Collagenase activity in culture medium conditioned by oxLDL-stimulated HUVECs. HUVECs were incubated for 24 h with medium alone (control), or medium containing 100 μg/ml oxLDL in the absence or presence of 1 mM of 1,10-phenanthroline. One hundred microliters of the conditioned medium was then mixed with 100 μl of 0.5 mg/ml fluorescein-conjugated type I collagen and incubated at room temperature for 24 h. After the incubation, the intensity of fluorescence in the mixture was measured at 495/515 nm (Ex/Em) by a microplate fluorescence reader. The intensity of fluorescence in the control sample was designated as 100%. The data presented are representative of three experiments with similar results. Atherosclerosis 2001 156, 119-125DOI: (10.1016/S0021-9150(00)00638-9)