Inhibitory effects of vasostatin-1 against atherogenesis

Abstract

Vasostatin-1, a chromogranin A (CgA)-derived peptide (76 amino acids), is known to suppress vasoconstriction and angiogenesis. A recent study has shown that vasostatin-1 suppresses the adhesion of human U937 monocytes to human endothelial cells (HECs) via adhesion molecule down-regulation. The present study evaluated the expression of vasostatin-1 in human atherosclerotic lesions and its effects on inflammatory responses in HECs and human THP-1 monocyte-derived macrophages, macrophage foam cell formation, migration and proliferation of human aortic smooth muscle cells (HASMCs) and extracellular matrix (ECM) production by HASMCs, and atherogenesis in apolipoprotein E-deficient (ApoE−/−) mice. Vasostatin-1 was expressed around Monckeberg’s medial calcific sclerosis in human radial arteries. Vasostatin-1 suppressed lipopolysaccharide (LPS)-induced up-regulation of monocyte chemotactic protein-1 (MCP-1), vascular cell adhesion molecule-1 (VCAM-1), and E-selectin in HECs. Vasostatin-1 suppressed inflammatory M1 phenotype and LPS-induced interleukin-6 (IL-6) secretion via nuclear factor-κB (NF-κB) down-regulation in macrophages. Vasostatin-1 suppressed oxidized low-density lipoprotein (oxLDL)-induced foam cell formation associated with acyl-CoA:cholesterol acyltransferase-1 (ACAT-1) and CD36 down-regulation and ATP-binding cassette transporter A1 (ABCA1) up-regulation in macrophages. In HASMCs, vasostatin-1 suppressed angiotensin II (AngII)-induced migration and collagen-3 and fibronectin expression via decreasing ERK1/2 and p38 phosphorylation, but increased elastin expression and matrix metalloproteinase (MMP)-2 and MMP-9 activities via increasing Akt and JNK phosphorylation. Vasostatin-1 did not affect the proliferation and apoptosis in HASMCs. Four-week infusion of vasostatin-1 suppressed the development of aortic atherosclerotic lesions with reductions in intra-plaque inflammation, macrophage infiltration, and SMC content, and plasma glucose level in ApoE−/− mice. These results indicate the inhibitory effects of vasostatin-1 against atherogenesis. The present study provided the first evidence that vasostatin-1 may serve as a novel therapeutic target for atherosclerosis.

β-Endorphin Mediates the Development and Instability of Atherosclerotic Plaques

“…Cells were incubated at 37°C in a 5% CO 2 humidified incubator for 3 days in RPMI-1640 medium containing 10% fetal bovine serum (FBS), 0.05 mg/mL streptomycin, 50 U/mL penicillin, and the indicated concentrations of β-endorphin in the presence of 150 ng/mL TPA to induce differentiation into macrophages. Subsequently, THP-1 monocyte-derived macrophages were incubated for 3 days in the absence of TPA prior to immunoblotting [2,[29][30][31][32][33][34].…”

“…en, THP-1 monocyte-derived macrophages were further incubated for 2 days in the renewal medium with the same concentrations of β-endorphin along with 50 μg/mL oxLDL and 100 μM [ 3 H]oleate (PerkinElmer, Yokohama, Japan) conjugated to bovine serum albumin [2,[29][30][31][32][33][34]. Cellular lipids were extracted, and the radioactivity of cholesterol-[ 3 H]oleate was determined with thin-layer chromatography [2,[29][30][31][32][33][34].…”

“…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].…”