Materials and Methods

Reagents and Cells

Intestinal and nasal polyp ECs were obtained from the University of Oslo (1). Two of the HUVEC samples were provided by the J. Swain lab (Stanford University). All other ECs were from Cambrex Corporation. The cells were thawed and propagated in EGM-2MV (Clonetics). After the cells grew to 60-70 % confluency, we changed the media 13 hours before harvesting the mRNA with FastTrack (Invitrogen). The cells were harvested between the 3rd-5th passages (10-16 generations in culture). Antibodies against cytokeratins (C-11, Sigma), desmin (Ab-1, NeoMarkers), glial fibrillary acidic protein (ab-7, NeoMarkers), vimentin (V9, Sigma), CD31 (Ab-2, Neomarkers or Pharmingen) were obtained from the indicated sources. (Note: The endothelial cell samples from nasal polyps were tested later as positive for mycoplasma. The impact of infection on the gene expression profile is currently unknown.)

Microarray Procedure and data analysis

Human DNA microarray production (of the Stanford Functional Genomic Facility (SFGF)) and hybridization, scanning and analysis with the Stanford Microarray Database (SMD) (2) were performed as previously described (3). For the Hey2 expression study, total RNAs were purified with Trizol reagents and amplified using a linear amplification method. Human common RNA reference (Strategene) was used in all experiments as the standard reference. Hierarchical clustering with weighted average linkage clustering (4) was performed as described. To identify genes that showed significant variations in expression between large vessels vs. microvascular ECs and artery vs. vein ECs, a Wilcoxon rank sum test was performed using p<0.005 as a threshold (5). To identify genes with tissue specific expression, we used multi-class Significance Analysis of Microarrays (SAM) (6) to analyze variations in expression in ECs from different tissues. The tissue-specific gene list was selected to have a false discovery rate (FDR) of 0.2 %, using 100 iterations. For detailed procedures and complete data, please see the accompanied web supplements.

Retroviral vector production and infection of HUVEC

The Hey2 cDNA (ATCC) was cloned into pMIGR (gift of W. Pear, U. Pennsylvania) (7) and used to transfect amphotropic Phoenix cells (gift of Gary Nolan, Stanford University) to generate retrovirus containing either GFP or Hey_GFP to infect HUVEC by spin infection (protocol detailed in http://www.stanford.edu/group/nolan/). The HUVECs were analyzed and collected with a cell sorter 48 hours after retroviral infection.

Real-time quantitative PCR.

mRNAs (10 ng) were reverse transcribed at 42°C for 60 min in a 50-µl reaction mixture with MultiScribe reverse transcriptase. Specific primers and fluorogenic probe for human Hey2 (Fw, 5'-GCTCTTGCCATGGACTTCATG-3'; Rev, 5'-CGCAAGTGCTGAGATGAGACA-3'; probe, 5'-TTGCGCGGTACCTGAGCTCCGT-3') and C17 (Fw, 5'-CCCAGGCTGTACCTGGACATAC-3'; Rev, 5'-AGGAATCTACCTGGGCCACTTT-3'; probe, 5'-CAAGCTGCGGGACTTTGTGGCC-3') were designed using Primer Express 1.0 software. The probes were labeled by FAM as reporters and TAMRA as quenchers.  Amplification of the GAPDH gene was used to standardize the amount of RNA in each reaction mixture (Taqman GAPDH control reagents). PCR was performed using an ABI Prism 7900HT sequence detector with 40 cycle amplifications of 95°C for 15 s, 60°C for 1min followed by enzyme activation at 95°C for 10 min.  All reagents for real-time PCR were purchased from Applied Biosystems.


Flow Cytometry

The indicated cells were detached with trypsin-free buffer and stain with 1:30 dilution of PE-antibodies against human CD44 (clone G44-26), macrophage mannose receptor(clone 19) and CD49a (BD pharmingen) and analyzed by FacScan. The data were deposited into Stanford FACS facility and analyzed with Flow Jo.






1. Haraldsen, G., Rugtveit, J., Kvale, D., Scholz, T., Muller, W. A., Hovig, T. & Brandtzaeg, P. (1995) Gut 37, 225-34.
2. Sherlock, G., Hernandez-Boussard, T., Kasarskis, A., Binkley, G., Matese, J. C., Dwight, S. S., Kaloper, M., Weng, S., Jin, H., Ball, C. A., Eisen, M. B., Spellman, P. T., Brown, P. O., Botstein, D. & Cherry, J. M. (2001) Nucleic Acids Res 29, 152-5.
3. Alizadeh, A. A., Eisen, M. B., Davis, R. E., Ma, C., Lossos, I. S., Rosenwald, A., Boldrick, J. C., Sabet, H., Tran, T., Yu, X., Powell, J. I., Yang, L., Marti, G. E., Moore, T., Hudson, J., Jr., Lu, L., Lewis, D. B., Tibshirani, R., Sherlock, G., Chan, W. C., Greiner, T. C., Weisenburger, D. D., Armitage, J. O., Warnke, R., Staudt, L. M. & et al. (2000) Nature 403, 503-11.
4. Eisen, M. B., Spellman, P. T., Brown, P. O. & Botstein, D. (1998) Proc Natl Acad Sci U S A 95, 14863-8.
5. Troyanskaya, O. G., Garber, M. E., Brown, P. O., Botstein, D. & Altman, R. B. (2002) Bioinformatics 18, 1454-61.
6. Tusher, V. G., Tibshirani, R. & Chu, G. (2001) Proc Natl Acad Sci U S A 98, 5116-21.
7. Pear, W. S., Miller, J. P., Xu, L., Pui, J. C., Soffer, B., Quackenbush, R. C., Pendergast, A. M., Bronson, R., Aster, J. C., Scott, M. L. & Baltimore, D. (1998) Blood 92, 3780-92.