Endothelial-to-mesenchymal transition drives atherosclerosis progression (original) (raw)

Cell culture and reagents. HUVECs (passage 5–10; CC-2517, Lonza) were cultured in endothelial basal medium-2, supplemented with the EGM-2MV BulletKit (CC-3202, Lonza), or in M199 with ECGS, supplemented with 20% FBS (Sigma-Aldrich), l-glutamine (Gibco), and penicillin/streptomycin (Gibco). Human coronary aortic SMCs (HCASMCs) (C-017-5C), media (M231-500), and supplements (SMGS, S-007-25; SMDS, S-008-5) were purchased from Life Technologies. The cells were grown at 37°C, 5% CO2, in Medium 231 (Life Technologies), supplemented with smooth muscle growth supplement (SMGS containing 4.9% FBS, 2 ng/ml FGF2, 0.5 ng/ml EGF, 5 ng/ml heparin, 2 μg/ml IGF-1, and 0.2 μg/ml BSA). For SMC differentiation, HCASMCs were incubated with Medium 231 containing smooth muscle differentiation supplement (SMDS containing 1% FBS and 30 g/ml heparin) for 8 days. For different cytokine treatment, HUVECs or HCASMCs were treated with human IFN-γ (300-02, PeproTech), human TNF-α (300-01A, Peprotech), or human IL-1β (200-01B, Peprotech) in complete growth medium or in differentiation medium.

Fluid shear stress. HUVECs were seeded on tissue culture plastic slides coated with 10 μg/ml fibronectin and grown to confluence in M199 culture media supplemented with 20% FBS, ECGS, 100 mg/l heparin, and penicillin/streptomycin. Cells were starved in M199 media supplemented with 8% FBS 4 hours prior stimulation. Slides were then clamped into a parallel plate flow chamber and sheared at 12 dynes/cm2 (laminar flow) or 1 ± 4 dynes/cm2 (oscillatory flow) for 16 or 48 hours. Cells were then washed in PBS and further processed for immunostaining or mRNA extraction.

SMAD2/3 translocation assay. Cells were fixed with 4% formaldehyde for 15 minutes and then permeabilized with 1% Triton X-100 for 15 minutes. After permeabilization, cells were blocked with StartingBlock Blocking Buffer (Thermo Fisher Scientific) for 15 minutes and incubated overnight at 4°C with a SMAD2/3 primary antibody diluted in StartingBlock Blocking Buffer (1:500, Cell Signaling). After 3 washing steps, cells were probed with DAPI and Alexa Fluor 647–conjugated secondary antibody (1:500, Molecular Probes) diluted in StartingBlock Blocking Buffer for 2 hours at room temperature. Cells were then mounted with Fluoromount-G Mounting Media (Southern Biotech). Fifty images were then captured with a ×20 objective mounted on a Perkin Elmer spinning disk confocal microscope equipped with a motorized stage (Prior Scientific). Images were then analyzed with a proprietary MATLAB function as described previously (35). Briefly, masks of the images were made using a combination of an adaptive histogram equalization algorithm with intensity and size thresholding. Cell orientation was calculated by taking the masks of the cell nuclei (determined from DAPI images), fitting them to an ellipse, and determining the angle between the flow direction and the major axis of the ellipse. Nuclei eccentricity was measured on the basis of the eccentricity of the fitted ellipse. Nuclear translocation was computed by taking the mask of the transcription factor stain (SMAD2/3), calculating the product of both the area and the intensity of the stain present in the nucleus, and dividing it by the product of the total stain area and the total intensity of the stain. This yields a unitless metric we termed the translocation factor, which equals 1 in cells with complete nuclear translocation and 0 in cells with no translocation.

Generation of lentiviruses. The human FGFR1 shRNA lentiviral construct was purchased from Sigma-Aldrich and the human FRS2α shRNA lentiviral construct was purchased from Open Biosystems. For the production of shRNA lentivirus, 3.7 μg Δ8.2, 0.2 μg VSVG, and 2.1 μg pLKO.1 carrying the control, FGFR1, and FRS2α shRNA were cotransfected into 293T cells using X-tremeGENE 9 DNA Transfection Reagent (06365787001, Roche). Forty-eight hours later, the medium was harvested, cleared by a 0.45-μm filter (PN4184, PALL Life Sciences), mixed with polybrene (5 μg/ml) (H9268, Sigma-Aldrich), and applied to the cells. After 6 hours of incubation, the virus-containing medium was replaced by fresh medium. Virus supernatants were tittered using the QuickTiter Lentivirus Titer Kit (Lentivirus-Associated HIV p24, VPK-107, Cell Biolabs).

RNA isolation and quantitative real-time PCR. Cells were suspended in TRIzol Reagent (15596018, Invitrogen), and total RNA (74134, QIAGEN) was isolated according to the manufacturer’s instructions. Reverse transcriptions were performed by using the iScript cDNA Synthesis Kit (170-8891, Bio-Rad). Quantitative real-time PCR (qRT-PCR) was performed using the Bio-Rad CFX94 by mixing equal amount of cDNAs, iQ SYBR Green Supermix (170-8882, Bio-Rad), and gene-specific primers from SABiosciences (a QIAGEN company) (FGFR1 [PPH00372F], VE-cadherin [PPH00668F], N-cadherin [PPH00636F], eNOS [PPH01298F], TGF-βR1 [PPH00237C], PAI-1 [PPH00215F], ICAM-1 [PPH00640F], VCAM-1 [PPH00623E], MCP-1 [PPH00192F], ZEB2 [PPH09021B], Slug [PPH02475A], and Snail [PPH02459A]) or the following primers for detecting mRNA: TWIST1, actggcctgcaaaaccatag and tgcattttaccatgggtcct; ACTA2, gccacagcagcttcctcttc and tgaattccagcggactccat; NOTCH3, ctcatccgaaaccgctctac and tcttccaccatgccctctac; fibronectin 1, gttgtgtccggcttgactcc and agtgtcagggtttgcctcca; and collagen 1A, tgtggcccagaagaactggt and caggaaggtcagctggatgg. All reactions were done in a 25 μl reaction volume in duplicate. Individual mRNA expression was normalized in relation to expression of endogenous β-actin. PCR amplification consisted of 10 minutes of an initial denaturation step at 95°C, followed by 46 cycles of PCR at 95°C for 15 seconds and 60°C for 30 seconds.

Antibodies used for immunodetection of proteins. We used the following antibodies for immunoblotting (IB), immunofluorescence (IF), or immunohistochemistry (IHC): CD31 (sc-1506, Santa Cruz; IHC for human and mouse paraffin samples), CD31 (sc-8306, Santa Cruz; IB), CD31 (M0823, Dako; IHC for human frozen samples), collagen 1 (600-401-103S, Rockland; IB), collagen 1 (NB600-408, Novus Biologicals; IHC), p44/42 MAPK (ERK1/2) (Thr202/Tyr204) (4370, Cell Signaling; IHC), F4/80 (ab6640, abcam; IHC), fibronectin (F0916, Sigma-Aldrich; IB), fibronectin (F3648, Sigma-Aldrich; IHC), FGFR1 (2144-1, Epitomics; IB), FGFR1 (ab10646, abcam; IHC for mouse and human paraffin samples), FGFR1 (FB817; IHC for human paraffin samples) (14), FRS2α (ab10425, abcam; IHC), FRS2 (sc-8318, Santa Cruz; IB), GAPDH (2118, Cell Signaling; IB), HSP90 (SAB4300541, Sigma-Aldrich; IB), ICAM-1 (MCA532, AbD Serotec; IHC for human tissue), ICAM-1 (116102, BioLegend; IHC for mouse tissue), ICAM-1 (3482-1, Epitomics; IB), N-cadherin (610920, BD; IB), eNOS (610297, BD; IB), NOTCH3 (5276, Cell Signaling; IB), NOTCH3 (ab23426, abcam; IHC), PAI-1 (612024, BD; IB), SM22α (ab14106, abcam; IB, IHC), smooth muscle–MHC (M7786, Sigma-Aldrich; IB), p-SMAD2 (Ser465/467) (3101, Cell Signaling; IHC for human paraffin samples), p-SMAD2 (Ser465/467) (AB3849, Millipore; IHC for mouse paraffin samples), p-SMAD2 (Ser465/467) (3108, Cell Signaling; IB), SMAD2 (3122, Cell Signaling; IB), SMAD2/3 (8685, Cell Signaling; IF), smooth muscle α-actin (A2547, Sigma-Aldrich; IB), smooth muscle α-actin–allophycocyanin (IC1420A, R&D Systems; IHC), TGF-βR1 (3712, Cell Signaling; IB), TGF-βR1 (sc-398, Santa Cruz; IB), β-tubulin (T7816, Sigma-Aldrich; IB), VCAM-1 (NBP1-47491, Novus Biologicals; IHC for human tissue), VCAM-1 (ab19569, abcam; IHC for mouse tissue), VCAM-1 (3540-1, Epitomics; IB), VE-cadherin (sc-6458, Santa Cruz; IB), VEGFR2 (2479, Cell Signaling; IB), and vimentin (V6630, Sigma-Aldrich; IB).

Generation of mice. Frs2afl/fl mice were previously described (36). Frs2afl/fl mice were crossed to the Rosa26 fluorescent reporter mT/mG reporter line (JAX SN:007676) and then crossed to C57BL/6 Apoe–/– mice (JAX SN:002052). Frs2afl/fl Apoe–/– mT/mG offspring were bred with mice expressing Cre recombinase under the Cdh5 promoter (gift from R.H. Adams, Max Planck Institute, Münster, Germany). _Apoe_–/– and FRS2αECKO _Apoe_–/– male mice were fed a Western diet (40% kcal% fat, 1.25% cholesterol, 0% cholic acid) for 4 or 16 weeks (product D12108, Research Diets), starting at the age of 8 weeks. Their body weight and blood lipid profile were indistinguishable from those of Apoe–/– mice (Supplemental Figure 5, D and E). PCR genotyping analysis was done using the following primers: Frs2afl/fl (5′-GAGTGTGCTGTGATTGGAAGGCAG-3′ and 5′-GGCACGAGTGTCTGCAGACACATG-3′), mT/mG (5′-CTCTGCTGCCTCCTGGCTTCT-3′, 5′-CGAGGCGGATCACAAGCAATA-3′, and 5′-TCAATGGGCGGGGGTCGTT-3′), Cdh5-CreERT2 (5′-GCCTGCATTACCGGTCGATGCAACGA-3′ and 5′-GTGGCAGATGGCGCGGCAACACCATT-3′), Apoe (5′-GCCTAGCCGAGGGAGAGCCG-3′, 5′-GTGACTTGGGAGCTCTGCAGC-3′, and 5′-GCCGCCCCGACTGCATCT-3′).

Immunohistochemical staining. Blocks were sectioned at 5-μm intervals using a Microm cryostat (for frozen blocks) or a Paraffin Microtome (for paraffin blocks). For frozen tissue sections, slides were fixed in acetone for 10 minutes at –20°C. For paraffin sections, slides were dewaxed in xylene, boiled for 20 minutes in citrate buffer (10 mM, pH 6.0) for antigen retrieval, and rehydrated. After washing 3 times with PBS, tissue sections were incubated with primary antibodies diluted in blocking solution (10% BSA and horse serum in PBS) overnight at 4°C in a humidified chamber. Sections were washed 3 times with Tris-buffered saline; incubated with appropriate Alexa Fluor 488–, Alexa Fluor 594–, or Alexa Fluor 647–conjugated secondary antibodies diluted 1:1,000 in blocking solution for 1 hour at room temperature; washed again 3 times, and mounted on slides with ProLong Gold Mounting Reagent with DAPI (P36935, Life Technologies). All immunofluorescence micrographs were acquired using a Axiovert 200M microscopy system (Carl Zeiss MicroImaging). Images were captured using Velocity software, and quantifications were performed using ImageJ software (NIH).

Histological analysis of atherosclerotic lesions. The animals were euthanized and perfusion fixed with 4% paraformaldehyde (18814, Polysciences Inc.) via the left ventricle for 5 minutes. The lesions located in the aorta, aortic roots, brachiocephalic artery, and abdominal aorta were analyzed using Oil Red O staining. To measure lesions in the aorta, the whole aorta, including the ascending arch and thoracic and abdominal segments, was dissected, gently cleaned of adventitial tissue, and stained with Oil Red O (O0625, Sigma-Aldrich) as previously described (37). The surface lesion area was quantified with ImageJ software (NIH). To measure lesions in the aortic root, the heart and proximal aorta were excised, and the apex and lower half of the ventricles were removed.

Serum lipid analysis. Serum was obtained through centrifugation of the blood for 2 minutes at 9,391 g at 4°C and stored at –80°C until each assay was performed. Measurement of total cholesterol, triglyceride, and HDL cholesterol levels was performed in the Yale Mouse Metabolic Phenotyping Center.

Western blot analysis. Cells were lysed with HNTG lysis buffer (20 mM HEPES [pH 7.4], 150 mM NaCl, 10 % glycerol, 1 % Triton X-100, 1.5 mM MgCl2, 1.0 mM EGTA) containing complete Mini EDTA-free Protease Inhibitors (11836170001, Roche) and Phosphatase Inhibitors (04906837001, Roche). 20 μg of total protein from each sample was resolved on Criterion TGX Precast Gels (567-1084, Bio-Rad) with Tris/Glycin/SDS Running Buffer (161-0772, Bio-Rad), transferred to nitrocellulose membranes (162-0094, Bio-Rad), and then probed with various antibodies. Chemiluminescence measurements were performed using SuperSignal West Pico Chemiluminescent Substrate (34080, Thermo Fisher Scientific).

Patient population. Human coronary arteries were obtained from the explanted hearts of transplant recipients or cadaver organ donors.

Specimen collection. Investigators were on call with the surgical team and collected the heart at the time of explant. To minimize ex vivo artifacts, an approximately 5- to 20-mm segment of the left main coronary artery was removed within the operating room (Supplemental Figure 8A) and immediately processed as frozen sections (n = 43) in Optimal Cutting Temperature Medium (Sakura Finetek USA Inc.), and, when of sufficient length, an additional segment (n = 29) was also fixed in formalin for later embedding, sectioning, and staining. The majority of analyses were in frozen specimens, although detection by certain antibodies (FGFR1 and p-SMAD2) was more optimal in paraffin-embedded specimens.

Histology and morphometric analysis. Sections of left main coronary arteries were stained with H&E and Movat. Digital H&E- and Movat-stained photographs of one section from each block were projected at final magnifications of ×40. ImageJ software (NIH) was used for morphometric analyses. As described in Supplemental Figure 8B, measurements were made of the intima and media thickness. The ratio of I/M thickness and plaque stages (38) were used to grade the severity of atherosclerosis. We averaged the results for these parameters from 4 different areas for each specimen to obtain mean values. Left main coronary arteries with an I/M ratio of less than 0.2 were considered as having no disease or mild disease; those with an I/M ratio of between 0.2 and 1 were considered as having moderate disease; those with an I/M ratio of greater than 1 or with calcification were considered as having severe disease.

Statistics. All graphs were created using GraphPad Prism software, and statistical analyses were calculated using GraphPad Prism. The significance of the differences between the controls and the experimental groups was determined using a 2-tailed Student’s t test. For multiple comparisons, 1-way ANOVA with Newman-Keuls test was used. Correlation analyses between variables were performed using the Pearson rank correlation test. A P value of less than 0.05 was considered significant. All results were confirmed by at least 3 independent experiments. Error bars represent mean ± SEM.

Study approval. All experiments involving animals were reviewed and approved by the Yale University Institutional Animal Care and Use Committee. The procedures related to human subjects were approved by the Institutional Review Boards of Yale University and the New England Organ Bank. A waiver for consent was approved for surgical patients, and written informed consent was obtained from a member of the family for deceased organ donors.