Apelin

We examined these multidimensional biomarkers to determine the underlying pathophysiological mechanisms, including P3NP, HtrA1, apelin, and Hsp72. According to the manufacturer’s instructions, plasma P3NP (Wuhan Fine Biotech Co., Ltd., Wuhan. China), HtrA1 (Cloud-Clone Corp., Houston, TX, USA), apelin (Phoenix Pharmaceuticals, Belmont, CA, USA, and Hsp72 (Enzo Life Science Inc., Farmingdale, NY, USA) were performed using a competitive enzyme-linked immunoassay (ELISA). The lower detection limit was 156 pg/mL for P3NP, 31.2 pg/mL for HtrA1, 70 pg/mL for apelin and 200 pg/mL for Hsp72. During the sample analysis period, the variation of these assays ranged from 5 to 10%.

HepG2 cells were obtained from American Type Culture Collection (ATCC, Manassas, VA, USA). This immortalized, stable cell line can be repeatedly frozen, thawed and propagated. HepG2 cells were seeded (2×10⁶ cells/well) in vented T-75 flasks and grown to confluence in Dulbecco’s Modified Eagle Medium (DMEM), supplemented with 50 U/ml penicillin, 50 µg/ml streptomycin and 10% of fetal calf serum (FCS). Thereafter, cells were switched to 1% FCS and incubated (37°C) under normoxic (21% O₂, 5% CO₂) or hypoxic conditions (5% O₂, 5% CO₂) in a controlled O₂ water-jacketed CO₂ incubator (Forma Scientific Series II, 3131, Marietta, OH) or treated with TNF-α (10 ng/ml, Sigma, St Louis, MO), lipopolysaccharide (LPS, 10 ng/ml, Sigma), AII (80 pM, Sigma), Endothelin-1 (2 nM, Sigma), Apelin (100 nM, Phoenix Pharmaceuticals, Burlingane, Ca), Fibronectin (10 ng/ml, Sigma), Interleukin-1β (20 ng/ml, Sigma) and TGF-β (10 ng/ml, R&D Systems, Minneapolis, Mn). All experiments carried out in cell lines were reproduced three times in at least 2 independent assays. Conditioned media were harvested, concentrated (80∶1) using 3000 MW Amicon Ultra centrifugal filters (Millipore Corp) and the presence of the fibrinogen α C-chain was assessed by SELDI-TOF-MS as described above.

Groups of three C57BL/6 mice were injected subcutaneously with 0.25 ml Matrigel (BD Biosciences) containing PBS, 0.25 μg bFGF (Eubio), or 0.25 μg apelin (Phoenix Pharmaceuticals). One week after implantation, the plug was removed, and cryosections (5 μm) were prepared for immunohistochemistry. After labeling with rabbit anti-mouse LYVE-1 and rhodamine-conjugated goat anti-rabbit IgG (purchased from ReliaTech, Braunschweig, Germany and Jackson ImmunoResearch Inc., West Grove, PA, respectively), sections were examined using a Nikon Eclipse 80i microscope and digital images were captured using a SPOT digital camera (Diagnostic Instruments, Sterling Heights, MI). Quantitative analyses of the LYVE-1-positive lymphatic vessels in the Matrigels were performed using ImageJ software.