Neopterin Counters Vascular Inflammation and Atherosclerosis

Abstract

BackgroundNeopterin, a metabolite of GTP, is produced by activated macrophages and is abundantly expressed within atherosclerotic lesions in human aorta and carotid and coronary arteries. We aimed to clarify the influence of neopterin on both vascular inflammation and atherosclerosis, as neither effect had been fully assessed.Methods and ResultsWe investigated neopterin expression in coronary artery lesions and plasma from patients with coronary artery disease. We assessed the atheroprotective effects of neopterin in vitro using human aortic endothelial cells, human monocyte‐derived macrophages, and human aortic smooth muscle cells. In vivo experiments included a study of aortic lesions in apolipoprotein E–deficient mice. Neopterin expression in coronary artery lesions and plasma was markedly increased in patients with versus without coronary artery disease. In human aortic endothelial cells, neopterin reduced proliferation and TNF‐α (tumor necrosis factor α)–induced upregulation of MCP‐1 (monocyte chemotactic protein 1), ICAM‐1 (intercellular adhesion molecule 1), and VCAM‐1 (vascular cell adhesion molecule 1). Neopterin attenuated TNF‐α–induced monocyte adhesion to human aortic endothelial cells and the inflammatory macrophage phenotype via NF‐κB (nuclear factor‐κB) downregulation. Neopterin suppressed oxidized low‐density lipoprotein–induced foam cell formation associated with CD36 downregulation and upregulation of ATP‐binding cassette transporters A1 and G1 in human monocyte‐derived macrophages. In human aortic smooth muscle cells, neopterin suppressed angiotensin II–induced migration and proliferation via c‐Src/Raf‐1/ERK1/2 downregulation without inducing apoptosis. Exogenous neopterin administration and endogenous neopterin attenuation with its neutralizing antibody for 4 weeks retarded and promoted, respectively, the development of aortic atherosclerotic lesions in apolipoprotein E–deficient mice.ConclusionsOur results indicate that neopterin prevents both vascular inflammation and atherosclerosis and may be induced to counteract the progression of atherosclerotic lesions. Consequently, neopterin could be of use as a novel therapeutic target for atherosclerotic cardiovascular diseases.

β-Endorphin Mediates the Development and Instability of Atherosclerotic Plaques

“…Aliquots of protein extracts derived from THP-1 monocytes, THP-1-derived macrophages, HASMCs, and HUVECs were separated with 10% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and then immunoblotted with antibodies raised against the following proteins: CD36, CD68, ACAT-1, ICAM-1, VCAM-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA), ABCA1, phosphorylated nuclear factor-κB (p-NF-κB), phosphorylated c-jun N-terminal kinase (p-JNK), α-tubulin, arginase-1 (GeneTex, Irvine, CA, USA), E-selectin, MARCO (Bioss, Woburn, MA, USA), peroxisome proliferator-activated receptor-c (PPAR-c; Signalway Antibody, College Park, MD, USA), phosphorylated Akt, phosphorylated extracellular signal-regulated kinase 1/2 (p-ERK1/2), phosphorylated p38 (p-p38), Bax (Cell Signaling Technology, Danvers, MA, USA), Bcl-2 (Abcam, Cambridge, UK), cleaved caspase-3 (R&D Systems, Minneapolis, MN, USA), glyceraldehyde-3phosphate dehydrogenase (GAPDH; Acris-OriGene Technologies, Herford, Germany), and β-actin (Sigma, St. Louis, MO, USA) [2,[29][30][31][32][33][34][35][36][37][38][39][40]. Proteins were visualized by enhanced chemiluminescence western blotting detection reagents (GE Healthcare, Amersham, UK).…”

“…Complementary DNAs were synthesized from isolated RNA templates using a High Capacity cDNA Reverse Transcription Kit (Applied Biosystems, Foster City, CA, USA). e PCR products were visualized with 2% agarose gel electrophoresis [2,[29][30][31][32][33][34][35][36][37][38][39][40].…”

“…Cells were fixed with 4% paraformaldehyde in phosphate-buffered saline. Terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate-biotin nick end labeling (TUNEL) staining was then performed using an In Situ Apoptosis Detection Kit (Takara Bio, Otsu, Japan) as described previously [2,[29][30][31][32][33][34][35][36].…”

“…All samples were then mounted in fluorescence mounting medium (Dako, Glostrup, Denmark) and examined with confocal microscopy (FV1000D, Olympus, Tokyo, Japan). Fluorescence was detected using the excitation wavelengths of 488 nm (TUNEL) and 360 nm (DAPI) [2,[29][30][31][32][33][34][35][36]. e number of TUNEL-positive cells was counted in three fields of view chosen randomly from each sample.…”

“…ese areas of the aortic wall were traced by an investigator blind to the treatment and quantified by image analysis (Adobe Photoshop, San Jose, CA, USA, and NIH ImageJ). In addition, an increased ratio of macrophage contents (μm 2 ) : VSMC contents (μm 2 ) within the atheromatous plaques was regarded as a marker of plaque instability [2,30,[32][33][34][35][36].…”