Identification of a new way to induce differentiation of dermal fibroblasts into vascular endothelial cells

Animals

A total of 20 eight-week-old and pathogen‑free BALB/C nude mice with a mean weight of 20 g were housed at 20‑24˚C with 40–60% humidity and with a regular light–dark cycle. All animal experiments were performed according to Institutional Animal Care and Use Committee guidelines. All efforts were made to minimize animal suffering.

Antibodies

Antibodies against CD31 (sc-1506), PDGF-BB (sc-7878), VEGF (sc-7269), FGF-2 (sc-271847) and CD133 (sc-30219) were from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Antibodies against Vimentin (10,366–1-AP) were from Proteintech group (Wuhan, China). The antibody against β-actin was from Sigma-Aldrich (St. Louis, MO, USA). Horseradish peroxidase-conjugated secondary antibodies were from Jackson Immunoresearch (West Grove, PA, USA). The secondary antibody used for immunofluorescence was donkey anti-rabbit IgG Alexa Fluor-546 (A-11037; Invitrogen, Carlsbad, CA, USA).

Cell culture

Human primary HDFs were derived from adult foreskins, and were isolated according to our previous publication [19]. HDFs were cultured in DMEM Basic medium (C11995500BT, Gibco, Grand Island, NY, USA) supplemented with 10% (v/v) bovine calf serum. HDFs were cultured in a humidified incubator at 37 °C in a 5% CO2 atmosphere. Cells were seeded in appropriate dishes (35,000 cells/ml), and all cell lines were authenticated by DNA short tandem repeat (STR) profiling and were confirmed to be mycoplasma negative.

Cell morphology

Morphological changes of HDFs were examined using an inverted phase contrast microscope (Eclipse TS-100; Nikon, Tokyo, Japan) after 10 days of treatment with CPP at the indicated concentrations.

Cell viability assay

HDFs were seeded in 96-well plates and were then treated with 0.1% DMSO (as a control) or with CPP at the indicated concentrations for 48 h. Cell viability was determined using a sulforhodamine B (SRB) assay (L109288, Aladdin, Shanghai, China) according to the manufacturer’s instructions.

Western blot analysis

Cell lysates (30 μg protein per lane) were separated by SDS-PAGE, after which the proteins were transferred to polyvinylidene difluoride membranes. At room temperature, the membranes were blocked with 5% non-fat milk in TBST (TBS containing 0.05% Tween-20) for 1 h. After that, the membranes were incubated with the primary antibody at 4 °C overnight, then were washed with TBST three times for 5 min each. Each membrane was incubated with the secondary antibody at room temperature for 1 h, and then washed with TBST 3 times, for 5 min each time. Antibodies bound to proteins were detected using an enhanced chemiluminescence detection kit (34,080, Thermo Fisher, Waltham, MA, USA). Relative quantities of specific bands were analyzed by Image J software and were normalized to loading controls.

Quantitative real-time PCR

RNA was extracted from the whole-cell fraction by the Trizol reagent method (Takara, Tokyo, Japan), and extracted total RNAs were reverse transcribed using the primer sequences of the target genes. Reverse transcription was performed using the PrimeScript RT reagent kit with gDNA Eraser (Takara). PCR reactions involved the use of SYBR Premix Ex Taq (Tli RNaseH Plus, Takara) and levels of expressed genes were measured by the 2−ΔΔCt method with MxPro 4.00 (Stratagene, La Jolla, CA, USA). The following primers were used: VEGF: 5′-ATCGAGTACATCTTCAAGCCAT-3′ (forward) and 5′-GTGAGGTTTGATCCGCATAATC-3′ (reverse); FGF-2: 5′-CATCAAGCTACAACTTCAAGCA-3′ (forward) and 5′-CCGTAACACATTTAGAAGCCAG-3′ (reverse); PDGF-BB: 5′-ACCGCACCAACGCCAACTTC-3′ (forward) and 5′-TCTTCCGCACAATCTCGATCTTTCTC-3′ (reverse); CD31: 5′-TCAGACGTGCAGTACACGGA-3′ (forward) and 5′-CTTTCCACGGCATCAGGGAC-3′ (reverse); CD133: 5′-GTGGCGTGTGCGGCTATGAC-3′ (forward) and 5′-CCAACTCCAACCATGAGGAAGACG-3′ (reverse); Vimentin: 5′-GGTGGACCAGCTAACCAACG-3′ (forward) and 5′-TTGCAGGGTGTTTTCGGCTT-3′ (reverse); Actin: 5′-CCTGGCACCCAGCACAAT-3′ (forward) and 5′-GCCGATCCACACGGAGTACT-3′ (reverse); CDH5: 5′- AAAGAATCCATTGTGCAAGTCC-3′ (forward) and 5′-CGTGTTATCGTGATTATCCGTG-3′ (reverse); ERG: 5′-GGAGTGGGCGGTGAAAGAATATGG-3′ (forward) and 5′-GAGAAGGATGTCGGCGTTGTAGC-3′ (reverse); KDR: 5′-GGAGCTTAAGAATGCATCCTTG-3′ (forward) and 5′-GATGCTTTCCCCAATACTTGTC-3′ (reverse); TEK: 5′-CGTGATTGACACTGGACATAAC-3′ (forward) and 5′-GAGTTCATATTCTGTCCGAGGT-3′ (reverse); vWF: 5′-CCTGTTACTATGACGGTGAGAT-3′ (forward) and 5′-CATGAAGCCATCCTCACAGTAG-3′ (reverse).

Matrigel assays

Aliquots of Matrigel were stored at − 80 °C and were melted in ice overnight immediately prior to use. After mixing the culture medium and Matrigel (3:1), 300 μl Matrigel was added to each well in 24-well plates. The 24-well plates were cultured in a humidified incubator at 37 °C in a 5% CO2 atmosphere for 30 min. Cells were digested with trypsin and were then resuspended in culture medium and seeded in the 24-well plates at a concentration of 4 × 10,000 cells/ml. Morphological changes of HDFs were observed using an inverted phase contrast microscope (Eclipse TS-100; Nikon). The lengths of renal tubules were analyzed by Image J software and were normalized to the control group.

Immunofluorescence microscopy

HDFs were seeded onto confocal dishes (20 mm) (SPL, Korea) and treated with CPP for 10 days. Next, the cells were fixed in 4% paraformaldehyde for 20 min. After washing with 1 × PBS three times, permeated cells with 0.2% TritonX-100 for 2 min, then washed and blocked with donkey serum (1:30 dilution in 0.1 M PBS) at room temperature for 30 min. Cells were incubated with primary antibodies at 4℃ overnight. On the second day, the cells were washed with PBS three times, and then incubated with secondary antibodies (1:200) at 37 ℃ for 1 h. Fluorescence was detected by confocal fluorescence microscopy Zeiss LSM700 (Germany).

Flow cytometry

HDFs were treated with 0.1% DMSO (as a control) or with CPP for 10 days. Next, cells were digested into single cells by 0.25% trypsin (Sangon Biotech) and collected into 15-mL centrifuge tubes. Centrifugation at 300 g for 15 min was performed, then the supernatant was discarded. Cells were washed twice with 1 × PBS and suspended in 1 × PBS supplemented with 2% (v/v) FBS by centrifugation at 300 g for 15 min each time. We discarded the supernatant. Cells were resuspended in 1 × PBS with 2% (v/v) FBS and incubated at 4℃for 1 h with antibodies as indicated below. After staining, the cells were analyzed on a flow cytometer (ImageStreamX MarkII, Merck, Billerica, MA, USA). Antibodies used included: Alexa Fluor® 488 anti-human KDR (VEGFR2) Antibody (359,914, Biolegend, San Diego, CA, USA); PE anti-human CDH5 (VE-Cadherin) Antibody (348,506, Biolegend, San Diego, CA, USA).

Acetylated-LDL uptake assay

HDFs were treated with 0.1% DMSO (as a control) or with CPP for 10 days, cells were incubated with DiI-Ac-LDL (L3484, Invitrogen, Carlsbad, CA, USA) at 10 μg/ml in growth media for 4 h. Next, cells were fixed with 4% Paraformaldehyde (w/v) at room temperature for 20 min and washed with 1 × PBS three times. Finally, cells were rinsed and stained with DAPI and monitored by a laser scanning confocal microscope (Zeiss, Germany). Randomly select the field of view for cell count in each group (total cell count: about 200; The percentage of cells uptake Acetylated-LDL = (the cell counts of Acetylated-LDL uptake/the total cell counts) × 100%).

Angiogenesis assay of chick embryo chorioallantoic membrane (CAM)

Fertilized chicken eggs were incubated at 37 °C with 60% relative humidity. HDFs were treated with 0.1% DMSO (as a control) or with CPP for 10 days, and then cells were digested into single cells by 0.25% trypsin (Sangon Biotech) and collected into 15-mL centrifuge tubes. Centrifugation at 1000 rpm for 8 min was performed. The supernatant was discarded, and the cells were incubated in the HBSS with 1.5 μM CM-DiI (C7000, Invitrogen, Carlsbad, CA, USA) for 5 min, and then for an additional 15 min at 4 ℃. After labeling, wash cells with phosphate-buffered saline (PBS). On embryonic day 7, eight million labeled HDFs or HUVECs in 20 μl of medium were seeded into chick embryo chorioallantoic membrane (CAM). After one week, 1 ml 4% paraformaldehyde was placed on the CAM and incubated for 30 min. Fluorescence was detected by the laser scanning confocal microscope Zeiss LSM900 (Germany). Randomly select the field of view, and calculate the number of cells around the blood vessel by ImageJ software.

Hindlimb ischemia model

Hindlimb ischemia was generated in 8-week-old BALB/C nude mice as previously described [20, 21]. Briefly, the femoral artery was ligated, then it was cut off. Twenty nude mice with hindlimb ischemia were randomly divided into four groups, five in each group. HDFs were treated with 0.1% DMSO (as a control) or with CPP for 10 days, one million CPP-treated HDFs, DMSO-treated HDFs or HUVECs in 100 μl PBS or the same volume PBS without cells were intramuscularly injected into the ischemic hindlimb of BALB/C nude mice. Laser Doppler imaging was conducted to quantitatively measure hindlimb blood flow of nude mice using a PeriCam PSupporting Information System (Perimed AB, Sweden) every week up to the 4th week. After sacrificed for tissue harvest, changes of muscle in the ischemic hindlimb of nude mice were assessed by histology analysis.

Histological analysis

BALB/C nude mice were sacrificed at four weeks, hindlimb muscle tissues were fixed for 24 h in 4% paraformaldehyde, embedded in paraffin after dehydration, and cut into 5-μm slices. Hematoxylin and eosin-stained (H&E staining) paraffin sections were conducted to analyze muscle changes in the ischemic hindlimb of nude mice. For the calculation of necrotic area, the total area and necrotic area of H&E slices were calculated by Caseviewer software. Fixed and equilibrated tissues were embedded in Optimal Cutting Temperature (OCT) compound (Sakura Finetek USA, Torrance, CA, USA), snap-frozen overnight in -80 °C refrigerator, and cut at 10 μm. Frozen sections were incubated with fluorescein-labeled Griffonia (Bandeiraea) Simplicifolia Lectin I (BSL1, Vector Laboratory Inc.) at 37 °C to stain functional endothelial cells in blood vessels. Fluorescence was detected by the laser scanning confocal microscope Zeiss LSM900 (Germany).

Statistical analysis

Data are reported as means ± SE from at least three independent experiments. Student’s t-test was performed to compare the mean between two groups. One-way ANOVA followed by multiple comparisons was used for comparison between more than two groups. For the hindlimb ischemia study, repeated measures ANOVA was used for comparison with LSD. Images were processed by GraphPad Prism 5 (GraphPad Software, USA) and Adobe Photoshop CC 2015 (Adobe, USA). Statistical significance was set at p < 0.05 using SPSS 17.0 (SPSS Inc., Chicago, IL, USA). For cell counts, randomly select the field of view for cell count in each group: the percentage of CD31-positive cells = (the count of CD31-positive cells/total cells) × 100%; The percentage of cells uptake Acetylated-LDL = (the cell count of Acetylated-LDL uptake/the total cell count) × 100%

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