Glycoxidized low-density lipoprotein downregulates endothelial nitricoxide synthase in human coronary cells
Claudio Napoli, MD, PhD, FACA*, ,*,
Lilach O. Lerman, MD, PhD ,
Filomena de Nigris, PhD*,,
Joseph Loscalzo, MD, PhD and
Louis J. Ignarro, PhD||
* Department of Medicine-0682, University of California, San Diego, California, USA
Division of Hypertension, Department of Medicine, Mayo Clinic, Rochester, Minnesota, USA
Evans Department of Medicine and Whitaker Cardiovascular Institute, Boston University, Boston, Massachusetts, USA
|| Department of Molecular and Medical Pharmacology, University of California, Los Angeles, California, USA
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Figure 1 (Upper) Cyclic guanosine monophosphate (cGMP) levels in human endothelial cells after 24-h exposure to: native low-density lipoprotein (nLDL) (100 µg/ml), glycosylated low-density lipoprotein (glcLDL) (100 µg/ml), oxidized low-density lipoprotein (oxLDL) (100 µg/ml), glc-oxLDL (30 µg/ml), glc-oxLDL (100 µg/ml), glc-oxLDL (300 µg/ml), glc-oxLDL (second form) (100 µg/ml), and oxLDL (second form) (100 µg/ml). The 100- and 300-µg/ml doses of glc-oxLDL reduced significantly cGMP activity. *p < 0.05 vs. untreated cells; $p < 0.01 vs. untreated cells, nLDL and glcLDL; #p < 0.05 vs. oxLDL; **p < 0.001 vs. glc-oxLDL at 30 and 100 µg/ml, second form of glc-oxLDL and second form of ox-LDL; p < 0.0001 vs. untreated cells, nLDL and glcLDL. (Lower) The cGMP levels in cells stimulated with bradykinin and exposed for 24 h to the same lipoproteins as in the upper panel. *p < 0.001 vs. untreated cells; @p < 0.01 vs. nLDL; ¶p < 0.001 vs. nLDL and glcLDL; **p < 0.001 vs. oxLDL and glc-oxLDL at 30 µg/ml and second form of ox-LDL; p < 0.0001 vs. untreated cells, glcLDL, oxLDL, glc-oxLDL at 30 µg/ml.
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Figure 2 (Top) Western blot of endothelial nitric oxide synthase III (NOSIII) in cells exposed to the following conditions: A = untreated cells; B = native low-density lipoprotein; C = glycosylated low-density lipoprotein (glycLDL); D = oxidized low-density lipoprotein (oxLDL); E = glyc-oxLDL; F = oxLDL (second form); and G = glyc-oxLDL (second form). All concentrations are 100 µg/ml. (Bottom) Densitometric analysis of endothlelial NOSIII levels (mean ± SD) representative of six separate experiments. Endothelial NOSIII was significantly decreased in C (*p < 0.01 vs. B) and E (#p < 0.0001 vs. B and C). Similarly, G was significantly decreased compared with F (**p < 0.0004 vs. F).
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Figure 3 Northern blot of endothelial nitric oxide synthase III (NOSIII) in human endothelial cells. A = untreated cells; B = nLDL; C = glycLDL; D = oxLDL; E = glyc-oxLDL; F = oxLDL (second form); and G = glyc-oxLDL (second form). All concentrations are 100 µg/ml. GAPDH = glyceraldehyde-3-phosphate dehydrogenase; mRNA = messenger ribonucleic acid. Other abbreviations as in Figure 2.
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Figure 4 Cells treated with 3 nM of actinomycin D (to inhibit further transcription) in the absence (–) or presence (+) of glyc-oxLDL (300 µg/ml) for 24 h before harvesting ribonucleic acid. A representative Northern blot hybridizated with a specific probe for NOSIII and GAPDH is shown. The relative percentage of NOSIII signal is plotted against time. The results did not show any difference between the treatments. Each point represents the mean value ± SEM for three experiments. Abbreviations as in Figures 2 and 3.
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Figure 5 In vitro elongation of nascent ribonucleic acid (run-on assay) performed in human endothelial cells exposed to following conditions: 100 µg/ml of nLDL, glycLDL, or oxLDL; glyc-oxLDL at 30, 100, and 300 µg/ml; and 100 µg/ml of the second forms of glyc-oxLDL and oxLDL. Densitometric analysis revealed a 25% reduction in NOSIII transcript after treatment with 300 µg/ml of glyc-oxLDL (*p < 0.05 vs. all other forms). Abbreviations as in Figures 2 and 3.
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Figure 6 Analysis of putative sterol-responsive element (SRE) present in the NOSIII promoter by electrophoretic mobility shift assay (EMSA). (Upper) Representative EMSA was performed with a 32P end-labeled probe containing the SRE element and nuclear extracts from cells treated for 24 h with nLDL (100 µg/ml), glycLDL (100 µg/ml), oxLDL (100 µg/ml), glyc-oxLDL (30 µg/ml), glyc-oxLDL (100 µg/ml), glyc-oxLDL (300 µg/ml), glyc-oxLDL (100 µg/ml, second form), and glyc-oxLDL (100 µg/ml, second form). (Lower) The EMSA was performed with the Oct-1 element used as a control agent to normalize the results (as described in [55]) and with a nuclear extract from cells treated for 24 h with the same lipoproteins. Abbreviations as in Figure 2.
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