HMEC-1 cell culture
HMEC-1 were purchased from American Type Culture Collection (ATCC, Rockefeller of Maryland, the certificate and STR test result are supplemented in additional file 1,2). According to the previous experimental practice of our research group [11]. The cell culture medium was prepared in MCDB131 medium containing 10% fetal bovine serum (FBS, Gibco, USA), 1% antibiotics (including streptomycin 0.1mg/mL, and penicillin 100U/mL, Gibco), 2mM glutamine(25030, Gibco) and 1μg/ml hydrocortisone (M3451, Abmole, USA) [12]. 10mM heme (51280, Sigma, USA) was prepared in 20 mM NaOH (PBS dilution) for standby [9]. HMEC-1 were cultured in vitro and divided into normoxia and hyperoxia groups. Normoxia group maintained in an incubator containing 5% CO2, 21% O2 and 74% N2, and hyperoxia group maintained in an incubator containing 5% CO2, 40% O2 and 55% N2. Both of them were kept at 37 ℃ and 90% humidity [13,14]. Meanwhile, different concentrations of heme, blank (medium) or negative control (vehicle:20mM NaOH) were added into the culture medium according to groups. The cells used in the experiment were from the 5 to 10th generations.
HMEC-1 proliferation CCK8 assay
Cell proliferation was measured using CCK8 (42830, MCE, USA) according to the supplier’s protocol. Cells were seeded in 96-well plates (5×103/well) for 12 hours and then adhered to the wall under normoxia condition. In dose effect test, the culture medium was changed according to the heme concentration (9 groups:0, 5, 10, 20, 40, 80, 160, 320μM and negative control), and then cultured under normoxia or hyperoxia conditions for 48 hours (Change the medium every 24 hours). Subsequently, add 100μL medium mixed with CCK8 reagent (1:10) into each hole, set blank control, incubate at 37 ℃ for 1 hour. Finally, the cells were measured on the microplate reader of enzyme-linked immunosorbent assay (Gene Company Limited, ELX800ux, Hong Kong) at a wavelength of 450nm [15]. The Cell proliferation value OD450 = ODgroup-ODblank. In time effect test, 4 groups were set up: normoxia control group(21%O2+0μM heme), normoxia heme group (21%O2+20μM heme), hyperoxia control group (40%O2+0μM heme) and hyperoxia heme group (40%O2+20μM heme), OD450 values were measured by CCK8 method at 0, 12, 24, 48, 72 and 96 hour after changing culture medium respectively. Four multiple holes were measured each experiment, the experiment was repeated three times.
HMEC-1 proliferation activity EDU assay
Cell proliferation activity was analyzed by EDU assay (RiboBio, Guangzhou, China) according to the manufacturer’s instructions. Cells were seeded in Millicell (1.5×104/well, 200μL) for 12 hours and then adhered to the wall under normoxia condition. According to the heme concentration group (0μM heme, 20μM heme or vehicle), the culture medium was changed and cultured under normoxia or hyperoxia conditions respectively for 48 hours (Change the medium every 24 hours). Then, the cell culture medium was added to 200 µL of reagent A (1:1000) for 2 hours at 37℃. Step by step, cells were fixed for 30 minutes with 4% paraformaldehyde, destained by 2 mg/mL glycine decolorization for 5 minutes, washed with PBS for 5 minutes, permeabilized with 0.5% Triton for 10 minutes. Other reagents, B to F, were mixed together according to the instructions. Then washed with PBS for 5 minutes, cell nucleus were stained with DAPI (1:1000, 4083, CST, USA) for 5 minutes, washed with PBS and sealed with anti fluorescence quenching [16]. Three regions in each well were randomly selected and imaged under a laser confocal microscope (Leica, SP8, Germany), the number of proliferative cells were counted by Image J software (National Institutes of Health, Bethesda, Germany). Each experiment was repeated at least three times.
HMEC-1 migration assay
HMEC-1 were seeded in 6-well plates (1×105/well) under normoxia condition, change the medium every 24 hours until cells filled with the bottom of the well. The cells were then wounded with 200μL pipette tips and washed with PBS. According to the heme concentration group (0μM heme, 20μM heme or vehicle), the culture medium was changed and cultured under normoxia or hyperoxia conditions respectively. Using an inverted phase contrast microscope (Zeiss, Primo Vert, Germany) equipped with a 5×objective and CCD cameras to take photos at 0, 12and 24 hours respectively, the central area of the scratch of the 6-well plate was selected [17]. After taking pictures, firstly, a solid line was drawn along both sides of the cell scratch on the photo at 0h, and a dotted line was drawn along the cell migration front at 12/24h by drawing software (Photoshop 8.0, adobe, USA), then the scratch area was calculated by Image J software. The area between solid lines is S0h Scratch area, and the area between dotted lines is S12/24h Scratch area, SMigration area=S0h Scratch area-S12/24h Scratch area. The experiment was repeated independently for three times.
HMEC-1 capillary-like tube formation assay
Tube formation was assessed as previously described [18]. HMEC-1 were seeded in 6-well plates (1×105/well) for 12 hours and then adhered to the wall under normoxia condition. According to the heme concentration group (0μM heme, 20μM heme or vehicle), the culture medium was changed and cultured under normoxia or hyperoxia conditions respectively for 48 hours (change the medium every 24 hours). Matrigel (356234, Corning, USA) was added to the precooled millicell (200μL/well) and polymerized for 40 minutes at 37℃. Then, the cultured cells in each group were digested by trypsin, adjusting the cell concentration to 3.5x105/mL with corresponding medium. Finally, adding the adjusted cells into the millicell contained polymerized Matrigel (200μL/well). Incubating for 4 hours under normoxia or hyperoxia condition. Wash once with Hank's Balanced Salt Solution (HBSS), adding 8μg/mL (HBSS dilution, 200μL/well) Calcein AM Fluorescent Dye (354216, Corning), incubating for 4 hours under normoxia or hyperoxia condition. Wash twice with the HBSS, using a laser confocal microscope (Leica, SP8) to take pictures, three regions in each well were randomly selected and imaged. The Angiotool Software was used to measure the number of junctions, length and area of the tubular-like structures [19]. Three independent experiments were performed.
BACH1 and VEGF protein expression by Western blot (WB)
Refered to previous method [20], HMEC-1 were seeded in 6-well plates (1×105/well) for 12 hours and then adhered to the wall under normoxia condition. According to the heme concentration group (0μM heme, 20μM heme or vehicle), the culture medium was changed and cultured under normoxia or hyperoxia conditions respectively for 48 hours (change the medium every 24 hours). Washing with PBS, and then incubating them in 100μL lysis buffer for 30 minutes, scrape the cells with 1ml sterile pipette. Finally, the samples were collected, and completely disrupted by ultrasound for 5 minutes. The lysates were centrifuged at 12000g for 15 minutes at 4°C and total protein was extracted from the supernatant. The protein concentration was detected by the double octanoic acid protein quantitative kit (Beyotime, ShangHai, China) and equaled with loading buffer, boiled for 10 minutes. Gel electrophoresis, and transferred to a 0.45μm polyvinylidene fluoride membrane. After blocking with 5% skimmed milk for 2 hours, the membranes were incubated successively with primary antibodies overnight at 4°C and secondary antibodies for 2 hours at room temperature. The primary antibodies and the dilutions used were: anti- BACH1(1:1000, ab49657, Abcam, UK), anti-VEGF (1:1000, ab46154, Abcam), anti-β-actin (1:10000, BS6007M, Bioworld, Minnesota), and were diluted by Western blot-specific diluent (Beyotime). The secondary antibodies and the dilutions used were: goat anti-rabbit (1:5000, BS13278, Bioworld), goat anti-mouse (1:5000, BS12478, Bioworld), and were diluted by TBST. Immunoreactive bands were developed by Immobilon Western Chemiluminescent Horseradish Peroxidase Substrate (Vilber-Lourmat, France) according to the manufacturer 's instructions. All bands were quantified by Image J software, and normalized with respect to the β-actin values. Three independent experiments were performed.
PECAM-1, BACH1 and VEGF expressions were detected by immunofluorescence (IF)
Refered to previous method [11], HMEC-1 were seeded in Millicell (1.5×104/well, 200μL) for 12 hours and then adhered to the wall under normoxia condition. According to the heme concentration group (0μM heme, 20μM heme or vehicle), the culture medium was changed and cultured under normoxia or hyperoxia condition respectively for 48 hours (change the medium every 24 hours). Then, cells were washed with PBS three times for 5minutes, fixed with 4% paraformaldehyde for 30minutes, permeabilized with 0.3% Triton for 20minutes, sealed with 5% goat serum for 30 minutes. Next, the cells were treated with anti-BACH1 (1:100, ab49657, Abcam), anti-VEGF (1:250, ab1316, Abcam), or anti-PECAM-1 (1:100, sc-133091, SANTA, USA), overnight at 4°C. After washing with PBS three times, cells were incubated for 2 hours at 37°C with goat anti-rabbit IgG/Alexa Flour 488 (1:300, bs-0295G-AF488, CST) and goat anti-mouse IgG/Alexa Flour 594 (1:300, bs-0296G-AF594, CST). After incubation, cells were washed with PBS again and the nucleus were stained with DAPI (1:1000, 4038, CST) for 5 minutes. Finally, washed with PBS and sealed with anti fluorescence quenching. Three regions in each well were randomly selected and imaged under a laser confocal microscope, the fluorescence intensity was counted by Image J software, in order to reduce the error, we use the system automatic default value when setting the threshold value. Each experiment was repeated at least three times. Negative control was set in the experiment, including omission of the primary antibody and use of an irrelevant polyclonal or isotypematched monoclonal primary antibody. In all cases, negative controls showed only weak staining.
Statistical analysis
SPSS 20.0 (IBM, Armonk, USA) statistical software was used for statistical analysis. All experiments were performed at least three times. Results are expressed as the mean ± SD. One-way ANOVA or multivariate analysis of variance was used in cases with homogeneity of variance and normal distribution, and for all other cases, a nonparametric test was used. P<0.05 was considered to indicate statistical significance.