The investigation was approved by the local ethics committee, the Institutional Review Board of Ewha Womans University, and was in accordance with the Declaration of Helsinki; the Animal Care Guidelines of Ewha Womans University, Medical School; and the National Institutes of Health Guide for the Care and Use of Laboratory Animals.

Mice were anesthetized with an intra-peritoneal injection of pentobarbital sodium (300 mg/kg body weight) plus 500 U heparin. The aorta, branches of the superior mesenteric artery (SMA), and the cerebral artery from the circle of Willis (CA) were removed and placed in Ca²⁺-free phosphate-buffered saline (PBS). Periadventitial fats and connective tissues around the vessels were carefully cleaned under a dissecting microscope using forceps and iris scissors. Matrigel (BD Biosciences, San Jose, CA) was added to 24-well plates (about 250 µl in each well) and polymerized at 37℃ for 30 min.

The aorta was cut into 8~10 small pieces and opened longitudinally. These pieces were placed with the intima side down on the Matrigel in the wells (4~5 pieces in each well). Next, a small amount of culture media was added to keep the explants moist but not submerged. The explants were placed in an incubator at 37℃ in a 5% CO₂ atmosphere, and cells migrated from the aortic segments. After 5~7 days, the aortic pieces were removed, and 2 ml culture medium was added to the wells.

Branches of SMA or CA were placed into the Matrigel in 24-well plates, and cut into very small pieces using iris scissors in the Matrigel. Then, culture medium was added, and the explants were placed in an incubator at 37℃ in a 5% CO₂ atmosphere. Cells migrated from the vessel segments after a few days, and the migrated cells were allowed to grow to confluence over 3~4 weeks.

When the cells reached confluence, the medium was removed, and the cells were incubated for 60~90 min at 37℃ in 2 ml/10 cm² (100 U) dispase (BD Biosciences, San Jose, CA) to detach the cultured cells from the Matrigel. Thereafter, culture medium was added to stop the dispase activity via dilution. Cells were then harvested and centrifuged for five minutes at 1000 rpm. After the supernatant was decanted, small samples of the pellet were transferred to six-well plates (passage 1). When the cells had grown to confluence, the medium was removed, and the cell pellet obtained was washed twice with Ca²⁺-free PBS containing 0.1 mM EDTA. After removing PBS, cells were passaged as described by Suh et al. [10] (passage 2).

The culture medium (100 ml) was composed of 80 ml Dulbecco's modified Eagle's medium (DMEM, GIBCO BRL 41965), 10 ml fetal calf serum (FCS, GIBCO BRL 10270), 7.5 mg ECGS (Sigma E-2759), 200 µl heparin (10 U/ml final), 2 ml penicillin/streptomycin (100 U/ml final, GIBCO BRL 15070), 1 ml L-glutamine (100×, GIBCO BRL 25030- 024), and 1 ml Minimal Essential Amino acids (100×, GIBCO BRL 11140-035). ECGS in the culture medium promotes EC growth, and heparin prevents vascular SMC growth.

The cells (passages 2-5), seeded on 1% gelatine-coated glass cover slips, were incubated in growth medium containing 10 µg/ml DiI-Ac-LDL (Biomedical Technologies Inc., Stoughton, MA) at 37℃ for 4 h. After washing twice with PBS to remove free DiI-Ac-LDL, cells were fixed with 3.7% paraformaldehyde in PBS for 20 min at room temperature. Next, the cells were counterstained with 4',6-diamidino-2-phenylindole dihydrochloride (DAPI) and mounted, and images were observed with a fluorescence microscope (Axiovert 200; Carl Zeiss Co., Oberkochen, Germany).

After washing once in ice-cold PBS, cells were lysed in a protein extraction solution (passage 2). Electrophoresis and transfer to nitrocellulose were carried out according to the manufacturer's instructions (Bio-Rad, Hercules, CA) using SDS-PAGE gels (10~12%). The membrane was blocked for 2 h in TBST (10 mM Tris-HCl, 150 mM NaCl, and 1% Tween 20, pH 7.6) containing 5% bovine serum albumin. After incubation with anti-eNOS antibody for 3 h and three washes in TBST (each 10 min), the membrane was incubated with a horse-radish peroxidase-conjugated anti-mouse antibody for 1 h. Bands were visualized by chemiluminescence, and data collection and processing were performed using an LAS-3000 luminescent image analyzer and IMAGE GAUSE software (Fujifilm, Tokyo, Japan).

RNA isolation from cells and RT-PCR analysis for vWF expression performed using the RNeasy mini kit (Qiagen, Valencia, CA) and OneStep RT-PCR kit (Qiagen), according to the manufacturer's instructions. Primers for mouse vWF were 5'-CAGCATCTCTGTGGTCCTGA-3' (sense) and 5'-GATGTTGTTGTGGCAAGTGG-3' (antisense). vWF mRNA expression was normalized to the mouse house-keeping gene mouse GAPDH 5'-AACTTTGGCATTGTGGAAGG-3' (sense) and 5'-ACACATTGGGGGTAGGAACA-3' (antisense). MOVAS (CRL-2797) and NIH-3T3 (CRL-1658) were used as a positive control for SMC and fibroblasts, respectively.

When the cells were approximately 80% confluent, DiI-Ac-LDL (5 µg/ml) was loaded into the culture media. The unloaded DiI-Ac-LDL (5 µg/ml) cells were prepared as an autofluorescent blank. The following day, the cells were detached with trypsin and resuspended in PBS up to 1×10⁶ cells/ml. The cells were kept in a tube on ice until they were sorted with a flow cytometer (Beckman Coulter, Fullerton, CA), as described previously [12]. Ten thousand cells were analyzed in each sample. Autofluorescence signals from unlabeled cells were used as negative controls in each experiment.

We used immunohistochemical techniques to detect vWF and eNOS. For immunostaining, mouse EC of passages 2~4 were washed with Tris (hydroxymethyl)aminomethane (TRIS)-buffered saline (TBS, 50 mM TRIS-HCl, 150 mM NaCl, pH 7.5). After fixation with a 7:3 mixture of methanol/acetone (20 min at -20℃), cells were permeabilized with 0.25% Triton X 100 (in TBS) for 30 min and blocked with 5% bovine serum albumin (BSA, in TBS) for 1 h. Rabbit anti-human vWF antibody (DAKO A0082; 1:300 in TBS/1%BSA) or rat anti-mouse CD31 (PECAM-1) monoclonal antibody (PharMingen, Cat. No. 01951; 1:50 in TBS/1% BSA) was added and allowed to process overnight (4℃). Subsequently, cells were washed with TBS containing 0.1% Tween and incubated with either an alkaline phosphatase-conjugated, monoclonal anti-rabbit immunoglobulin G (IgG) (Sigma, A-2556; 1:200 in TBS/1%BSA) or goat anti-rat Ig-specific polyclonal antibody (PharMingen, Cat. No. 12113; 1:20 in TBS/1%BSA) for 1 h at room temperature. After an additional TBS wash (3'), cells were stained using nitro blue tetrazolium/5-bromo-4-chloro-3-indolylphosphate-p-toluidine (NBT, Sigma N-6876; BCIP, Roth Art. No. 6368.1), as substrates in alkaline phosphatase buffer (100 mM TRIS-HCl, pH 9.5; 100 mM NaCl; 5 mM MgCl₂).

Data are presented as mean±SEM, and significant differences were detected using Student's t-test (p<0.05).

Two to seven days after mouse vascular explants attached to Matrigel matrix, cells with large and round nuclei started to migrate from the edges of the explants. Matrigel matrix was gradually filled with proliferating cells, and the cells from the explants formed tube-like structures in the Matrigel (Fig. 1), suggesting that the cells were EC. When cells in the Matrigel were passaged by treatment with dispase, we collected about 1.5×10⁶ cells (passage 1) from aortic explants from one mouse. We obtained better cell yield by using Matrigel with high protein content (>9.5 mg/ml) compared to Matrigel with low protein content (<8 mg/ml). To confirm the origin of the cells at passages 1~5, we performed a Dil-Ac-LDL uptake test, since active uptake of Dil-Ac-LDL is a well-known characteristic of EC (Fig. 2) [13,14,15]. When the test was conducted in cells at passage 1, the ratio of Dil-Ac-LDL uptake was greater than 90%, but the ratio decreased with each additional passage. The ratio decreased to about 81% in cells at passage 2, 65% in cells at passage 3, and 35% in cells at passage 5, suggesting that growth of cell types other than EC, SMC or fibroblasts, increased with passage cycle. We, thus, examined whether SMC overgrew in the experimental setting. Among cells at passage 5, we could not find characteristics of SMC, such as α-smooth muscle actin (α-SMA) (Fig. 6F); the absence of SMC might be caused by heparin in the culture medium, because heparin inhibits SMC growth [10]. Thus, the decrease in the ratio might be caused by overgrowth of fibroblasts.

Cells at passage 1 proliferated to form a monolayer, whereas cells at passage 2 or later started to form a multilayer; the number of cells in the multilayer gradually increased with each passage. Since EC grow to form a monolayer, cells growing to form a multilayer might not be EC. Thus, we tried to eliminate the cells in the multilayer, in order to increase the purity of EC among the cultured cells. Cells at passage 2 or 3 were pooled into a six-well plate, and anti-fibroblast antibody (100 mg/ml) (1:200) was applied via the culture medium. Then, the plates were gently rocked at very low speed (less than 50 rpm) in an incubator at 37℃ in a 5% CO₂ atmosphere. The combined treatment with the antibody and gentle rocking eliminated the formation of a multilayer (Fig. 3). Almost all of the cells in the multilayer disappeared after 5~7 days of the combined treatment, at which time we stopped applying the treatments. When the cells reached confluence, we passaged the cells using the standard passage procedure for subculture. The passaged cells formed a monolayer and showed a typical endothelial morphology such as cobblestone appearance and contact-inhibited growth (Fig. 4). We performed a Dil-Ac-LDL uptake test to confirm that the proliferating cells in the monolayer were EC and compared the Dil-Ac-LDL uptake ratio with that of human umbilical vein endothelial cells (HUVEC); HUVEC showed characteristics of EC after passaging and were used as a positive control. Mouse aortic endothelial cells (MAEC) and HUVEC formed a monolayer with a Dil-Ac-LDL uptake ratio greater than 95% (Fig. 5). The ratio did not decrease after passaging. These results suggest that almost all of the cells were EC. When FCS in the culture medium was replaced with platelet-derived serum (PDS), the cells in the multilayer were more easily removed by the combined treatment (data not shown).

It took about 21 days to complete the procedure from starting explants to the selection of EC. At passage 3, EC were prepared for the next characterization step or frozen in a liquid nitrogen tank for future studies (Scheme 1).

Western blotting or RT-PCR analysis for an EC-specific gene or protein was employed to profile characteristics of EC. vWF was dominantly expressed in MAEC at the gene level, whereas it was hardly observed in MOVAS (10%) or NIH-3T3 (Fig. 6A). We showed the presence of vWF in cells from branches of SMA or CA by using immunocytochemistry (Fig. 6B). In addition, we used immunocytochemistry to show that eNOS was expressed in MAEC at the protein level (Fig. 6C) and in cells from branches of SMA or CA (Fig. 6D). We then examined whether fibroblast- or SMC-specific protein was expressed in MAEC. The fibroblast-specific protein, fibroblast surface protein (FSP), was not observed in MAECs or MOVAS (Fig. 6E). In addition, α-SMA was observed in MOVAS, but not in MAEC or NIH-3T3 (Fig. 6F). Our observations suggest that this method can be used to isolate high purity EC.