Human L5 study
LDL was isolated from the plasma of asymptomatic, middle-aged men and age-matched, premenopausal women who did (MetS patients, n = 30; 15 men and 15 women) or did not (control subjects, n = 26; 11 men and 15 women) meet 3 of the criteria for MetS according to the National Cholesterol Education Program-Adult Treatment Panel III guidelines . Patients who were taking medications that may affect the electronegativity of LDL such as statins, angiotensin-converting enzyme inhibitors, and angiotensin receptor blockers were excluded . All participants gave informed consent for the use of their plasma, and the study was conducted according to the principles in the Declaration of Helsinki. Patient characteristics, including waist circumference, systolic blood pressure, diastolic blood pressure, and levels of glucose, triglyceride, total cholesterol, high-density lipoprotein cholesterol (HDL-C), and LDL cholesterol (LDL-C) were determined according to standard operating procedures. LDL samples were further resolved into subfractions L1-L5 by using fast protein liquid chromatography (GE Healthcare, Pittsburgh, PA) with an anion-exchange UnoQ12 column (BioRad, Hercules, CA).
Biochemical analysis of mouse blood plasma
All animal experiments were approved by the China Medical University Institutional Animal Care and Use Committee and were performed in accordance with the Guide for the Care and Use of Laboratory Animals published by the US National Institutes of Health (NIH Publication No. 85–23, revised 1996). Whole blood was drawn from mice by means of intubation via the common carotid artery, and plasma was obtained by using centrifugation. Using an automated Biochemical Analyzer (SP-4430, Spotchem EZ, Arkray), we examined plasma levels of glucose, total cholesterol, total triglyceride, HDL-C, and LDL-C. LDL (d = 1.030-1.063 g/mL) was isolated by using sequential ultracentrifugation as previously described . Protein concentrations were determined by using the Lowry method . The charge of LDL was analyzed by subjecting LDL samples to gel electrophoresis in 0.7% agarose, and the composition of delipidated LDL was analyzed by subjecting samples to SDS-PAGE. Relative mobility (Rf) was quantified as a measure of the distance migrated by a band divided by the distance migrated by the dye front. For Western blot analysis, proteins were transferred to PVDF membrane and probed by using antibodies against apolipoprotein CIII (ApoCIII) and ApoB100 (Academy Biomedical Company, Inc., Houston, TX). Images were acquired and analyzed by using the G-box imaging system (Syngene, Frederick, MD). To quantify the protein expression of ApoCIII, we normalized the band intensity with that of ApoB100 (internal control).
Analysis of cellular senescence and DNA damage in mouse aortas
Male and female homozygous leptin receptor knock-out mice (db/db) and wild-type C57B6/J littermates (controls) were purchased from The Jackson Laboratory (Bar Harbor, ME) and were maintained on a 12-hour light/dark cycle with free access to food and water. After 6 months on a normal chow diet, all animals were anesthetized by means of 2% isoflurane inhalation, and the aorta of each mouse was removed for histologic examination.
For the detection of cellular senescence in the aortic endothelium, aortas were fixed for 10 minutes at room temperature in 2% formaldehyde/0.2% glutaraldehyde solution and were incubated at 37°C for 24 hours with fresh senescence-associated–β-galactosidase (SA-β-gal) staining solution (Cell Signaling Technology, Inc., Danvers, MA). Using images acquired with a Nikon D300s digital camera, we identified senescent cells marked by a blue color produced by the enzymatic reaction.
For the detection of DNA damage in the aortic endothelium, thoracic descending aortas were paraffin-embedded, and serial cross-sections were obtained from each sample. Slides were immunostained with anti–phospho-histone H2AX (anti-γH2AX) antibody (Cell Signaling Technology, Inc.) and analyzed by using an Olympus IX70 inverted microscope (Japan) with OPTRONICS digital microscope cameras (Goleta, CA).
To examine telomerase enzyme activity, aortas or subconfluent bovine aortic endothelial cells (BAECs) were homogenized in 3-[3-(cholamidopropyl) diethylammonio]-1-propane sulfonate (CHAPS) buffer. Homogenized tissues were then used to perform telomerase activity assays by using a modified telomeric repeat amplification protocol (TRAPeze RT Telomerase Detection Kit; Chemicon, Billerica, MA) according to the manufacturer’s instructions. Real-time PCR amplification and detection was performed with StepOnePlus Real-Time PCR Systems (Applied Biosystems, Grand Island, NY). Threshold cycle (Ct) measurements were obtained for determining telomerase activity. Values for Ct, which are inversely related to telomerase activity, were then adjusted with the standard curve by using the commercial TSR8 template.
Analysis of cellular senescence in cultured BAECs
Subconfluent BAECs were incubated with a subapoptotic concentration (30 μg/mL) of LDL from male or female db/db or wild-type mice for 5 consecutive days. Cells were then either lysed in radio-immunoprecipitation assay lysis buffer (Sigma-Aldrich Co., St. Louis, MO) containing protease inhibitor cocktail and 0.1% Triton X-100 for Western blot analysis or were stained with SA-β-gal for the detection of senescent cells. A total of 500 cells were scored in 10 randomly selected fields to determine the percentage of SA-β-gal–positive cells.
To investigate the protective effects of estrogen against cell senescence induced by LDL from db/db mice, BAECs were pretreated with 17β-estradiol (10 nM; Biorbyt Ltd., UK) or its isoflavone analogue genistein (100 nM; LC Laboratories, MA), followed by exposure with 30 μg/mL LDL from male or female db/db or wild-type mice. For the detection of cell senescence, cells were stained with SA-β-gal as described above.
Western blot analysis of lectin-like oxidized LDL receptor-1 (LOX-1) and tumor necrosis factor-α (TNF-α)
Using Western blot analysis, we examined the protein expression of LOX-1 (antibody from R&D Systems, Minneapolis, MN) and TNF-α (antibody from GeneTex Inc., Irvine, CA) in BAECs treated with 30 μg/mL LDL from male db/db mice or PBS in the presence or absence of pretreatment with 17β-estradiol (10 nM) or genistein (100 nM), as described previously .