Expression of hBD-2 in Endothelial Cells Associated with Oral Cancer

Human β-defensin 2 (hBD-2) is a small cationic peptide involved in innate immune protection in response to microbial challenges of the oral epithelium. Objectives: To determine in vivo hBD-2 expression in endothelium associated with oral cancer and assess microenvironmental factors that regulate its transcriptional expression in vitro. Methods: Immunofluorescence microscopy was performed on formalin-fixed, paraffin-embedded biopsy sections of oral squamous cell carcinoma. Antigen-retrieved and serum-blocked slides were probed with anti-CD34 and anti-hBD-2 antibodies followed by staining with secondary antibodies conjugated with Alexa Fluor488 and Cy3. The localization and expression of CD34 and hBD-2 were detected with a Zeiss Fluorescence Microscope. Human umbilical vein endothelial cells (HUVECs) cultured in HUVEC-media were treated with TGFβ, IL-1β, IFNα, and PMA for 16 hr, respectively, followed by RT-PCR and quantitative PCR analyses. Cell lysates extracted from HUVECs treated with the different agents were subjected to ELISA to quantify hBD-2 peptide expression in HUVECs. Results: The expression of hBD-2 peptide is co-localized in oral squamous cell carcinoma biopsies to cells that express CD34, an endothelial cell marker, whereas endothelial cells in normal parts of the biopsy do not express hBD-2 peptide. HBD-2 transcript can be induced by PMA, TGFβ1, and IL-1β in cultured HUVECs. IL-1β induces hBD-2 expression by more than 120-fold, while PMA and TGFβ1 induce hBD-2 transcript expression by about 20-fold. The production of hBD-2 peptide is 1.5-2 folds higher in HUVECs treated with TGFβ1 and proinflammatory factors polyI:C and LPS than that in untreated HUVECs. Conclusion: Our novel findings suggest that the expression of hBD-2 in endothelial cells of oral cancer is modulated by factors of the tumor microenvironment, and could implicate the involvement of hBD-2 in tumorigenesis and angiogenesis. Supported by American Heart Association Scientist Development Grant 0535088N and Case Western Reserve University School of Dental Medicine.