Clonality and altered behavior of endothelial cells from hemangiomas (original) (raw)
Patient material. Proliferating-phase hemangioma specimens were obtained from nine female infants. The patients’ ages at the time of resection, the location and size of the lesion(s), any prior treatment, and other relevant clinical information are given in Table 1. In addition, a patient (patient 8) with multiple high-grade hemangioendothelioma (heoma) lesions of variable size was included in the study. Normal skin samples were obtained from age-matched female infants undergoing other surgical procedures in which these tissues would have been discarded. The tissue specimens were identified by age and gender only, in accordance with the protocol approved by the Committee on Clinical Investigation at Children’s Hospital, Boston, and were collected and processed immediately after resection.
Clinical data: ages of patients at the time of resection of each lesion, location and size of the lesions, and other relevant information
Normal blood samples from 40 age-matched female infants, from a pool of samples left over from routine hematological tests, were obtained from the Hematology Laboratory at Children’s Hospital, Boston, in accordance with the protocol approved by the Committee on Clinical Investigation. These samples were identified by the child’s age and gender only.
Isolation, culture, and characterization of hemangioma-derived ECs. ECs were isolated from hemangiomas (hemECs) and normal skin (HFSECs) by the method described previously by Kräling and Bischoff (5) for human dermal microvascular ECs (HDMECs). The only modification was trypsinization of the hemangioma tissue for 4 minutes at 37°C instead of 10 minutes. The cells were resuspended in EBM-A (EBM131 (Clonetics, San Diego, California, USA), 20% heat-inactivated FBS (Hyclone Laboratories, Logan, Utah, USA), 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin (Irvine Scientific, Santa Ana, California, USA), 0.25 μg/ml amphotericin B (Life Technologies Inc., Gaithersburg, Maryland, USA), 0.5 mM N-6,2′-O-dibutyryladenosine 3′:5′-cyclic monophosphate (Sigma Chemical Co., St. Louis, Missouri, USA), 1.0 μg/ml hydrocortisone (Sigma Chemical Co.) and grown overnight at 37°C with 5% CO2, washed vigorously with PBS to remove unattached cells, and fed with fresh EBM-A. These primary cultures were grown to preconfluence (5–7 days). The ECs were purified from the primary culture using Ulex europaeus I lectin–coated (Vector Laboratories Inc., Burlingame, California, USA) magnetic beads (Dynal A.S., Lake Success, New York, USA), as described by Jackson et al. (6). ECs bound to the lectin-coated beads were collected with a magnetic particle concentrator, and any unbound cells were removed with five washes in HBSS wash buffer (5% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin [Irvine Scientific, Santa Ana, California, USA], and 0.25 μg/ml amphotericin B [Life Technologies Inc.]). The ECs and unbound fibroblast-like cells (UBCs) were separately resuspended in EBM-A and grown to confluence on gelatin-coated 60-mm plates. To expand further the purified cultures after P2, the cells were passaged 1:3 into a simplified medium EBM-B (EBM131, 10% heat inactivated FBS, 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, 2 ng/ml bFGF [kindly provided by Scios Inc., Mountain View, California, USA]) (5).
Indirect immunofluorescence staining was performed to characterize the isolated cells. The reagents used included: rabbit polyclonal anti-human vWF (diluted 1/2,000; DAKO Corp., Carpinteria, California, USA) with nonimmune rabbit IgG as a control; mouse monoclonal anti-human E-selectin IgG1 (clone 4B12; 5 μg/ml; kindly provided by D. Hollenbaugh, Bristol-Myers Squibb) (7) mouse monoclonal anti-CD31/PECAM-1 IgG1 (DAKO, Carpinteria, CA), with an isotype-matched control mouse IgG1 (5 μg/ml; Becton Dickinson, Bedford, Massachusetts, USA) as a control; biotinylated goat anti-rabbit IgG1, biotinylated horse anti-mouse IgG, and fluorescein-conjugated avidin (Vector Laboratories Inc.). In addition, we used anti-KDR (Flk1; Santa Cruz Biotechnology Inc., Santa Cruz, California, USA), and mouse anti-human smooth muscle cell α-actin (Sigma Chemical Co.) to stain many of the EC and UBC samples.
For immunostaining, cells were grown in EBM-B on gelatin-coated glass coverslips, washed in PBS containing 1.26 mM CaCl2 and 0.8 mM MgSO4, and fixed with methanol for 10 minutes on ice. Immunostaining was performed at room temperature, and cells were washed three times in PBS after each step. PBS with 1% BSA was used for antibody dilution. Cells were mounted with Fluoromount G (Southern Biotechnology Associates Inc., Birmingham, Alabama, USA) and observed at 400× with an Axiophot II fluorescence microscope (Zeiss, Oberkochen, Germany). Photographs were taken with Kodak TMAX p3200 film (Eastman Kodak Co., Rochester, New York, USA).
DNA isolation and clonality assay. The Puregene DNA isolation Kit (Gentra Systems Inc., Minneapolis, Minnesota, USA) was used to extract DNA, according to the manufacturer’s instructions, from peripheral blood leukocytes (PBLs) of 40 control individuals and from cultured ECs at passages 4–7 from ten hemangioma lesions (1, 4, 5, 10, 12, 13, 17A, 17B, 20, and 21), four hemangioendothelioma lesions (8B, 8C, 8F, and 8G), and five normal skin samples. In addition, DNA was isolated from the cultured unbound fibroblast-like cells (UBCs) from hemangioma lesions 1 and 10.
Paraffin-embedded tissue sections of myometrium and leiomyomata from hysterectomy specimens were provided by B. Quade (Department of Pathology, Brigham and Women’s Hospital, Boston, Massachusetts, USA). These samples, previously shown to be clonal (8), were deparaffinized, proteinase K–treated, and used as a positive controls for the HUMARA assay.
We analyzed these DNA samples for clonality using the X-linked human androgen receptor gene (HUMARA) assay described by Allen et al. (9). Exon 1 of the HUMARA gene contains a highly (90%) polymorphic CAG short tandem repeat (STR) (11–31 copies) with adjacent _Hha_I and _Hpa_I sites, at which methylation correlates with X-chromosome inactivation. In females heterozygous at this allele, STR size polymorphism distinguishes the maternal and paternal X chromosomes, and digestion with methylation-sensitive _Hha_I distinguishes between the unmethylated, active X, which is cut, and the methylated, inactive X, which is not.
A total of 5 μg of DNA was digested in a 50-μl reaction with 20 units of _Hha_I (New England Biolabs Inc., Beverly, Massachusetts, USA) at 37°C for up to 24 hours. For each sample, PCR was performed on 1 μl of the digest and 50 ng of undigested DNA. PCR reactions (50 μl) contained 20 nM each of primers: H1 5′-GCT GTG AAG GTT GCT GTT CCT CAT-3′ and H2 5′-TCC AGA ATC TGT TCC AGA GCG TGC-3′ (9) and 0.5 μl α-33P dATP (1,000–3,000 Ci/mmol, 10 mCi/ml; ICN Radiochemicals Inc., Irvine, California, USA). The amplification was carried out with 0.5 units of Taq DNA Polymerase (QIAGEN Inc., Valencia, California, USA) in a 10X buffer, containing 15 mM MgCl2, supplied by the manufacturer. The PCR conditions were as follows: an initial denaturation at 93°C for 3 minutes, followed by 30 cycles of denaturation at 93°C for 40 seconds, annealing at 65°C for 40 seconds, and extension at 72°C for 40 seconds. These steps were followed by a final extension at 72°C for 5 minutes.
The PCR products were electrophoresed in an 8% nondenaturing polyacrylamide gel (Accugel 29:1; National Diagnostics, Atlanta, Georgia, USA) at 600 V for approximately 10 hours and visualized by exposure to autoradiographic film (Eastman Kodak Co.) or phosphorimaging screens (Fuji Medical Systems Inc., Stamford, Connecticut, USA).
An initial PCR was used to look for informative alleles and homozygous individuals were excluded from the study. The NIH Image (version 1.62) software was used to obtain densitometric scans for each lane on the gels. Defined bands representing the different CAG alleles were seen as major peaks that could be easily distinguished from “stutter” bands, seen as minor peaks slightly out of phase with the main peaks. The intensity of each band and its stutter bands was determined as the total area under the corresponding peaks in the densitometric tracing. The ratio of the least intense to the most intense CAG allele in each heterozygous sample represented the amplification ratio. A ratio of 1.00 represents random X-inactivation; a ratio of 0.00 represents complete skewing and monoclonality. Amplification ratios determined before and after _Hha_I digestion were compared as a measure of clonality.
On the basis of other studies (10, 11) and a comparison with samples of leiomyomata previously shown to be clonal (see earlier discussion here), we defined samples as clonal when there was significant skewing toward one allele after _Hha_I digestion, so that the amplification ratio of the minor allele to the predominant allele was 0.45, corresponding to a ratio between the two alleles of about 30:70.
Cell proliferation assay. ECs were plated onto gelatin-coated 24-well plates at a density of 8,000 cells per 2 cm2 well in EBM131 medium with 5% FBS and 2 mM glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin. The following day, the plating efficiencies of the cells were determined and the proliferation of HDMECs and hemECs were compared after a 48-hour incubation with 1 ng/ml bFGF or 10 ng/ml VEGF (R&D Systems Inc., Minneapolis, Minnesota, USA) at 37°C with 5% CO2. Assays were performed in quadruplicate, and cells were counted in a Coulter counter. The results were expressed as mean ± SD.
Cell migration and endostatin response assays. Migration assays for ECs were performed in a standard 48-well chemotaxis chamber (Neuro Probe Inc., Gaithersburg, Maryland, USA) according to the method described by Yamaguchi et al. (12). HemECs and HDMECs were assayed in EBM131 medium containing 2 mM glutamine, 100 U/ml penicillin, 100 μg/ml streptomycin, and 0.025% BSA. A total of 10,000 cells per well were placed in the upper chamber and their migration across a Nucleopore polyvinylpyrrolidone-free polycarbonate membrane (Corning-Costar Corp., Cambridge, Massachusetts, USA) with 8-μm pores was stimulated with 5 ng/ml VEGF165 (R&D Systems Inc.) in EBM131 with 0.025% BSA, placed in the lower chamber. Assays were performed in quadruplicate for 4–6 hours, and the migrated cells, adhered to the underside of the membrane, were stained with Diff-Quick stain (VWR Scientific Products, Bridgeport, New Jersey, USA); nuclei were counted using a light microscope. Results were plotted as mean ± SD.
EC samples were pretreated with human recombinant endostatin (hES) prepared as described previously (12), at concentrations of 1, 10, and 100 ng/ml, by incubation at 37°C for 30 minutes, and the migration assay was performed as already described here.