P enos

Protein extracts of HUVEC and HMEC‐1 cells that were exposed to HG or OxLDL alone or in combination were prepared by RIPA lysis buffer (Millipore) containing protease and phosphatase inhibitor. The cell lysates were first resolved on SDS‐PAGE gels and transferred to polyvinyldene difluoride membranes by electroblotting. Membranes were incubated with 1:500 diluted monoclonal antibodies against eNOS, phosphorylated eNOS (p‐eNOS) (Santa Cruz), ECE, ETA, ETB, Nrf2, pNrf2 and CD31 (Abcam) in PBS for one hour at room temperature. The membranes were then washed thoroughly in Tris‐buffered saline contained 0.1% (v/v) Tween 20 before a second incubation for one hour at room temperature with a 1:1000 diluted horseradish peroxidase‐conjugated secondary antibody (Santa Cruz). An antibody against β‐actin (Santa Cruz) was used to normalize protein loading. The resultant bands were quantified by densitometry.

Osteoblasts were seeded on coverslips in a 24-well plate and further cultured for 24 h before staining. After washing in PBS and fixing in 4% paraformaldehyde for 15 min, the samples were incubated with alkaline phosphatase (ALP), collagenase 1 (COL-1) and osteocalcin (OCN) (1∶200; R&D Systems Inc., Minneapolis, MN) for 2 h and subsequently incubated with fluorescein isothiocyanate (FITC)- or Rhodamine-conjugated antirabbit secondary antibodies. eNOS and P-eNOS (1∶100; Santa Cruz Biotechnology, Santa Cruz, CA) immunocytochemical staining was performed on osteoblasts that had been cultured in DMEM containing 10% FCS with 5.5 mM or 16.5 mM glucose. The osteoblasts were cultured in medium containing 5.5 mM or 16.5 mM glucose for 24 h and then changed to medium containing 10 µM LY294002 for 120 min. After being fixed in 4% paraformaldehyde, samples were incubated with e-NOS (1∶100; Santa Cruz) for 2 h and subsequently incubated with FITC-conjugated antirabbit secondary antibodies. Positive cells were observed under a fluorescence microscope (Olympus Optical, Tokyo, Japan). Each experiment was repeated at least three times.

Aortic tissue and cell homogenates (protein of 30–50 μg) were separated using 10% SDS-polyacrylamide gel electrophoresis (PAGE) and transferred onto nitrocellulose membranes. Blots were then blocked by 5% Bovine Serum Albumin (BSA) (GenDEPOT, Katy, TX, USA) powder in Tris-bufferd saline (TBS) for 1 h, and incubated with the antibodies against ICAM-1, VCAM-1, E-selectin, MMP-2, MMP-9, eNOS, p-eNOS, Akt, p-AKT, and GTPCH (Santa Cruz Biotechnology, INC, Dallas, TX, USA) (1:1000 dilution in 0.05%TBS-T (Tween 20)). Subsequently, the membrane was then incubated with a secondary antibody of goat anti rabbit IgG or goat anti mouse IgG conjugated to horseradish peroxidase (Enzo Life Sciences, Farmingdale, NY, USA) (1:5000 dilution in 0.05%TBS-T), and the bands were detected with EzWestLumi plus solution (Cat. no. WSE-7120, Atto Corporation) using a ChemiDoc (Bio-Rad Laboratories, Hercules, CA, USA). Densitometry analysis of protein bands was conducted with the ImageJ (NIH, Bethesda, MD, USA) program.