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EXPERIMENTAL STUDY

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

Manuscript received March 5, 2002; revised manuscript received May 23, 2002, accepted June 7, 2002.

^* Reprint requests and correspondence: Dr. Claudio Napoli, Department of Medicine, University of Naples, PO Box, Naples 80131, Italy or Dr. Claudio Napoli, Department of Medicine-0682, University of California at San Diego, 9500 Gilman Drive, MTF110, La Jolla, California 92093.
claunap{at}tin.it
cnapoli{at}ucsd.edu

OBJECTIVES: We examined the hypothesis that low-density lipoprotein (LDL) that is both oxidized and glycosylated potently downregulates the expression of endothelial nitric oxide synthase III (NOSIII) in human coronary endothelial cells.

BACKGROUND: Diabetes mellitus is accompanied by both oxidation and glycosylation of LDL, but the potential interaction of these processes or the pathophysiologic effects of these modified lipoproteins on arteries are poorly understood.

METHODS: Low-density lipoprotein was glycoxidized in vitro, and Western and Northern blot analyses were used to investigate NOSIII expression in human coronary endothelial cells. Nitric oxide (NO) bioactivity was represented by both basal and bradykinin-stimulated cellular cyclic guanosine monophosphate accumulation and L-citrulline conversion from L-arginine. Nuclear run-on experiments were performed to study the transcription rate of nascent NOSIII messenger ribonucleic acid (mRNA).

RESULTS: Data showed a significant decrease in NOSIII expression after 24-h treatment with glycosylated low-density lipoprotein (glycLDL) and oxidized low-density lipoprotein (oxLDL). Accordingly, we observed a significant dose-dependent reduction in NO bioactivity (p < 0.05 to p < 0.001 vs. untreated cells, native low density lipoprotein [nLDL], glycLDL, and oxLDL). Glyc-oxLDL did not reduce the half-life of NOSIII mRNA or significantly enhance L-citrulline conversion. Nuclear run-on experiments showed that high doses of glyc-oxLDL can reduce the transcription rate of nascent NOSIII mRNA (densitometric analysis revealed a reduction of 25% [p < 0.05 vs. untreated cells, nLDL, and glycLDL] after treatment of cells with 300 µg/ml glyc-oxLDL). The effects of glyc-oxLDL are not related to the higher levels of oxidative compounds in comparison to those of oxLDL.

CONCLUSIONS: These results indicate that glyc-oxLDL, per se, may influence signal transduction pathways involving NO-mediated regulatory signals and NOSIII activity in human endothelial cells. This phenomenon can adversely influence the evolution of clinical vascular complications, coronary heart disease, and atherogenesis in diabetic patients.

Abbreviations and Acronyms   AGEP   advanced glycosylation end products   cDNA   complementary deoxyribonucleic acid   cGMP   cyclic guanosine monophosphate   EDTA   ethylenediamine-tetraacetic acid   EMSA   electrophoretic mobility shift assay   glycLDL   glycosylated low-density lipoprotein   LDL   low-density lipoprotein   mRNA   messenger ribonucleic acid   nLDL   native low-density lipoprotein   NO   nitric oxide   NOSIII   endothelial nitric oxide synthase III   oxLDL   oxidized low-density lipoprotein   RNA   ribonucleic acid   SRE   sterol-responsive element   TBARS   thiobarbituric acid-reactive substances   TNBSA   trinitrobenzenesulfonic acid

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