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LETTERS TO THE EDITOR |
Department of Biochemistry and Molecular Biology, Keck School of Medicine, University of Southern California, Los Angeles, CA 90033
In our recent article by Kim et al. (1), we show that CoCl2 treatment induced a severalfold increase in IL-8 mRNA expression in a variety of human primary endothelial cells and established endothelial cell lines (HDMVEC or HMEC-1). IL-8 release was determined for HDMVEC and HPMVEC grown in serum-free medium and induced with CoCl2 (0.5 mM) (1). Basal IL-8 release from HDMVEC was low but increases severalfold in response to CoCl2 (Figs. 2 and 6). The effect is optimal between 0.5 and 1.0 mM CoCl2 and did not affect HDMVEC viability. Consequently, this concentration range was used throughout our studies (1). It is pertinent to note that others (2) observe CoCl2 induction of IL-8 expression in a similar concentration range (0.6–1.2 mM) in tumor cells and not below this threshold concentration. Moreover, exposure of HDMVEC to hypoxia (1% O2, 6 h) resulted in an 
1.7-fold increase (1) in IL-8 release (Fig. 3). HDMVEC transfected with an IL-8 promoter luciferase reporter showed an 8-fold increase in reporter expression in response to 0.5 mM CoCl2. Significantly, this response was attenuated by mutating the hypoxia response element in IL-8 promoter. Increased IL-8 mRNA and protein expression in response to hypoxia were previously observed in HUVEC (3).
In contrast to the literature, Dulak and coworkers show that neither hypoxia (4) nor CoCl2 (0.25 mM) (5) increases expression of IL-8 mRNA or protein in HMEC-1 cells. They show (4) in Fig. 3A that exposure of HMEC-1 to hypoxia (1% O2) for 24 h resulted in an 60% reduction of IL-8 mRNA from basal level, as determined by real-time RT-PCR only. Additionally, their studies (3) in Fig. 3C show an 20% reduction in IL-8 release following hypoxia. These effects are observed at a low concentration of CoCl2 (0.25 mM), which activates hypoxia response element but has no effect on IL-8 gene expression in their system.
All primary endothelial cells and cell lines described in our studies (1) (HDMVEC or MHEC-1) were grown in EBM-2MV medium containing 5% FCS as per the manufacturer’s instructions (Cambrex). IL-8 release was determined in HDMVEC and HPMVEC after culturing in serum-free medium (Materials and Methods). EBM-2MV is a defined medium, most notably containing hydrocortisone, human epidermal growth factor, vascular endothelial growth factor, R3-insulin-like growth factor-1, and ascorbic acid. Dulak et al. (3) used MCB1 medium containing 10% FCS, epidermal growth factor, and hydrocortisone. Now, it is not known whether these media differences (including serum) have any consequence on the discordant CoCl2 response on IL-8 expression between our laboratories in these cells, and clearly, this requires additional investigation. Moreover, future studies should be directed toward elucidating the physiological relevance of hypoxia-inducible factor-1
to chemokine expression in primary endothelial cells and in an animal model.
References
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