Endothelial cell culture
HCAECs were purchased from Cambrex Bioscience (Walkersville, MD). Cells were used between passages 6 and 7 for experiments to avoid effects of in vitro cell aging. Cells were grown in endothelial growth media-2 (EGM-2; Cambrex) supplemented with growth factors and 5% (v/v) fetal bovine serum to 70-80% confluency. Cells were cultured in a humidified incubator at 37°C with a 5% CO2 atmosphere and the medium was changed every second day. To determine the effect of high glucose or TNF-α on cell apoptosis and death receptor expression, cells were incubated with both zero growth factors and zero hydrocortisone for 30 h, and they were then cultured for 24 h under different concentrations of glucose (5.6, 11.1 and 16.7 mM).
Reverse transcription PCR (RT-PCR)
Total RNA was isolated from cells using standard procedure, including ISOGEN (Nippon Gene, Tokyo, Japan). The RNA was reverse-transcribed in a 20-μl reaction mixture that contained 5 U ReverScript® III (Wako, Osaka, Japan) and 0.5 μg oligo(dT)12-18 primer (Invitrogen, Carlsbad, CA) at 42°C for 1 h, followed by 51°C for 30 min. The primer pairs for TNF-R1 (forward, 5'-GAG AGG CCA TAG CTG TCT GG-3'; reverse, 5'-GTT CCT TTG TGG CAC TTG GT-3'), for Fas (forward, 5'-TCA GTA CGG AGT TGG GGA AG-3'; reverse, 5'-CAG GCC TTC CAA GTT CTG AG-3'), for DR4 (forward, 5'-AGA GAG AAG TCC CTG CAC CA-3'; reverse, 5'-GTC ACT CCA GGG CGT ACA AT-3'), and for DR5 (forward, 5'-CAC CAG GTG TGA TTC AGG TG-3'; reverse, 5'-CCC CAC TGT GCT TTG TAC CT-3') yielded a 218-, 207-, 154-, and 221-bp PCR product, respectively. Human β-actin (forward, 5'-GGA CTT CGA GCA AGA GAT GG-3'; reverse, 5'-AGC ACT GTG TTG GCG TAC AG-3') was used as a reference gene. PCR was performed using an iCycler (Bio-Rad, Tokyo, Japan). The reaction mixture consisted of template cDNA, 0.2 μM of each primer, 300 μM dNTPs, 3 mM MgCl2, and 0.05 U/μl ExTaq DNA polymerase (Takara Shuzo, Ohtsu, Japan). PCR was performed for 32 cycles for each gene with denaturation at 95°C for 15 s, annealing at 61°C for 15 s, and extension at 72°C for 20 s. PCR products were quantified using NIH Image.
Quantitative real-time PCR
Real-time PCR was performed in a final volume of 20 μl containing cDNA template and primers using Takara RNA PCR kit (Takara Shuzo) as described in the manufacture's manual. PCR amplification was carried out on the genomic DNA using the following primers: TNF-R1, forward 5'-AAC AGA ACA CCG TGT GCA CCT-3' and reverse 5'-AGT CCT CAG TGC CCT TAA CAT TCT C-3'; FAS, forward 5'-AGA GTA AAT GCA GTG GCA TGC TAA G-3' and reverse 5'-GGG TTA GCC TGT GGA TAG ACA TTT G-3'; DR4, forward 5'-GGA ACA CAG CAT GTC AGT GCA A-3' and reverse 5'-TGT CAC TCC AGG GCG TAC AAT C-3'; DR5, forward 5'-CAT CTA TGG ACA GGC TGG GAC A-3' and reverse 5'-CCC AAA CAG GGC TCA AGT TCA-3'; and actin, forward 5'-TGG CAC CCA GCA CAA TGA A-3' and reverse 5'-CTA AGT CAT AGT CCG CCT AGA AGC A-3'. The PCR program consisted of 95°C for 30 s for initial denaturation of DNA, followed by 40 cycles of 95°C for 5 s, 60°C for 34 s for annealing of primers, and 95°C for 1 min for elongation. The PCR product size for TNF-R1, FAS, DR4, DR5, and actin was 141, 150, 130, 96, and 186 bp, respectively; they were visualized after agarose gel electrodes and ethidium bromide staining. Actin served as an internal control for normalization.
Immunocytochemistry and TUNEL staining
After being fixed with 4% paraformaldehyde for 15 min at room temperature, cells were kept in PBS at 4°C until staining. Unspecific binding sites were blocked in PBS containing 10% normal goat sera. The samples were treated with rabbit anti-human von Willebrand factor (vWF) antibody (Chemicon, Temecula, CA) at 1:100 dilution overnight at 4°C, carefully washed and stained with Alexa goat anti-rabbit secondary antibody (Invitrogen) at 1:500 dilution overnight at 4°C. For visualization of endothelial cells, cells were incubated with biotinylated Griffonia simplicifolia isolectin-B4 (Vector Laboratories, Burlingame, CA) overnight at 4°C, followed by an overnight incubation at 4°C with streptavidin (Alexa Fluor® 488 conjugate; Invitrogen). The nucleus was counterstained with Hoechst 33258 (Nakalai Tesque, Kyoto, Japan). Immunofluorescent images were observed under a Leica TCS-SP5 confocal sytem.
The terminal deoxynucleotide transferase-mediated dUTP nick end labeling (TUNEL) technique was conducted to detect apoptotic endothelial cells. The fluorescein in situ cell death detection kit (DeadEnd™ Fluorometric TUNEL System; Promega, Madison, WI) was used according to the manufacturer's instructions. The fluorosein-labeled cells undergoing apoptosis were recognized as a green fluorescent nucleus. The samples were analyzed using a fluorescence microscope.
DNA fragmentation assay
To determine the extent of apoptosis, DNA fragmentation was assessed following the method by Arai et al. . In brief, the cells were lysed in a lysis buffer (10 mM Tris, 1 mM EDTA, 0.2% Triton X-100, pH 7.5) and centrifuged at 13,000 × g for 10 min. Subsequently, each DNA sample in the supernatant and the pellet was precipitated in 12.5% trichloroacetic acid at 4°C and quantified using a diphenylamine reagent after hydrolysis in 5% trichloroacetic acid at 90°C for 20 min. After the absorbance of the supernatant and the pellet was determined spectrophotometrically at a wavelength of 600 nm, the percentage of fragmented DNA in each sample was calculated as the amount of DNA in the supernatant divided by the total DNA for that sample (supernatant plus pellet). The average percentage of fragmented DNA in control samples was 2.7%.
Flow cytometry analysis
Cells were washed with FACS buffer (PBS, 1% bovine serum albumin, 0.2% NaN3), stained with FITC-labeled anti-human TNF-R1 antibody (clone MABTNFR1-B1; BD Pharmingen, San Diego, CA) for 30 min at 4°C, followed by phycoerythrin-labeled goat polyclonal anti-mouse IgG (clone ab74490, Abcam, Cambridge, England). Propidium Iodide (Sigma-Aldrich, St. Louis, MO) was used to discriminate between dead and live cells. Flow cytometric analysis was performed using FACSCalibur and Cell QuestPro software version 6.0 (BD Biosciences, Franklin Lakes, NJ). TNF-R1 expression was determined using mean fluorescence intensity (MFI), with vehicle-treated cells as the baseline for all comparisons.
Enzyme immunoassay for TNF-α
TNF-α concentrations were determined in cell culture supernatants by enzyme-linked immunosorbent assay (ELISA) (BioVender, Modřice, Česká republika). Sensitivity of the assay was 4 pg/ml for TNF-α. No cross reactivity was observed with other human inflammatory cytokines including various interleukins. Optical density was measured at 450 nm by use of a microplate reader. Plasma levels of TNF-α were measured by the use of a commercially available enzyme-linked assay kit (Shibayagi Co. Ltd., Shibukawa, Japan) according to the manufacture's instructions.
Mouse model of type 2 diabetes
The animal study was carried out as approved by the Animal Care and Use Committee of University of Toyama. Male ICR mice, 5-weeks old, received an intraperitoneal injection of 1.5 g/kg body weight of nicotinamide dissolved in saline 15 min before a tail-vein injection of streptozotocin (200 mg/kg; Sigma-Aldrich) dissolved in a citrate buffer solution (0.1 M citric acid and 0.2 M sodium phosphate, pH 4.5), following the protocol previously reported [31, 32]. Control mice received the vehicles of both substances. The experimental model prepared here was devised more than a decade ago . Its diabetic syndrome appears to share a number of features with human type 2 diabetes [31–33]. Animals were diabetic for 9 weeks before heart harvest. Only animals with glycemia levels above 300 mg/dl were used for experiments.
Immunofluorescence and confocal analysis
Mouse hearts were harvested, fixed with 4% buffered formalin solution, immersed in sucrose solutions, dipped into OCT compound (Sakura Finetechnical, Tokyo, Japan), and frozen at -20°C. The embedded tissues were then sectioned at a thickness of 30 μm and air dried. For immunohistochemical detection of the target molecules, the tissue sections were exposed to the fluorescent secondary antibody after incubation with the suitable primary antibody according to the method in our previous study with minor modification . Thus, rehydrated sections were incubated with the primary antibody, anti-mouse CD31 (BD Pharmingen, Franklin Lakes, NJ), anti-mouse TNF-R1 (BD Pharmingen), or anti-mouse Fas (Merck KGaA, Darmstadt, Germany), overnight at 4°C. The antibodies were used at 1:100 dilution, followed by extensive washes with PBS and incubation with the secondary antibodies conjugated to high quality fluophores, including Alexa Fluor 568, Alexa Fluor 488, and DyLight 488, at 1:500 dilution overnight at 4°C. The nucleus was counterstained with Hoechst 33258 (Nacalai Tesque, Kyoto, Japan). Immunofluorescent images were observed under Leica TCS-SP-5 confocal system (Leica, Wetzlar, Germany).
All experiments were performed at least three times. Data are presented as means ± SE. Statistical assessment of the data was made by one- or two-way ANOVA, and then differences among groups were analyzed by Turkey's multiple comparison test, with level of significance set at P < 0.05.