Antiapoptotic action of hypoxia-inducible factor-1 alpha in human endothelial cells.
Hypoxia-inducible factor-1 (HIF-1) is the major transcription factor involved in the adaptive response to hypoxia and consists of HIF-1 alpha and HIF-1 beta subunits. Indirect evidence suggests that HIF-1 alpha may exert both proapoptotic and antiapoptotic actions in response to hypoxia. In this study, we evaluated the effects of RNA interference (RNAi) targeting HIF-1 alpha messenger RNA (mRNA) on apoptosis in primary cultured human umbilical vascular endothelial cells (HUVECs) exposed to anoxia and reoxygenation (A/R). HUVECs were transfected with specific 21-nt small interfering RNA (siRNA) duplexes targeting HIF-1 alpha mRNA sequences or scrambled RNA duplexes and subjected either to normoxia for 251/2 h or to anoxia for 11/2 h, and subsequently normoxia for 24 h (A/R). Control samples were subjected to A/R but not transfected. HUVECs apoptosis was evaluated by Tdt-mediated dUTP nick end-labeling (TUNEL) assay and by activated caspase-3 immunostaining and immunoblotting. The efficacy of RNAi was assessed by knockdown of HIF-1 alpha mRNA and protein expression via in situ hybridization, real-time quantitative PCR, immunohistochemistry, and Western blotting. When compared with normoxic cultures, A/R significantly upregulated HIF- 1 alpha mRNA and protein expression in HUVECs, but did not appreciably alter the percentage of apoptotic cells. In contrast, a significantly greater proportion of HUVECs transfected with specific siRNA duplexes and exposed to A/R demonstrated evidence of apoptosis when compared with nontransfected cells. Transfection with specific siRNA duplexes knocked down HIF-1 alpha mRNA and protein expression in A/R-treated cells by approximately 60%, whereas transfection with scrambled siRNA duplexes had no noticeable effect on HIF-1 alpha expression. These findings strongly suggest that HIF-1 alpha exerts an antiapoptotic role in HUVECs stressed by anoxia.
Yu
Erik Z
EZ
Department of Pathology, Uniformed Services University of the Health Sciences, Bethesda, and Department of Resuscitative Medicine, Naval Medical Research Center, Silver Spring, MD, USA.
Li
Ying-Yue
YY
Liu
Xiu-Huai
XH
Kagan
Elliott
E
McCarron
Richard M
RM
D017209:Apoptosis / Q000502:physiology*; D001483:Base Sequence; D053148:Caspase 3; D020169:Caspases / Q000378:metabolism; D015687:Cell Hypoxia; D002478:Cells, Cultured; D004730:Endothelium, Vascular / Q000166:cytology* / Q000378:metabolism; D006801:Humans; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D034622:RNA Interference; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D034741:RNA, Small Interfering / Q000235:genetics; D014157:Transcription Factors / Q000235:genetics / Q000502:physiology*; D014162:Transfection
C497442:HIF1A protein, human; D051795:Hypoxia-Inducible Factor 1, alpha Subunit; D012333:RNA, Messenger; D034741:RNA, Small Interfering; D014157:Transcription Factors; C505407:CASP3 protein, human; D053148:Caspase 3; D020169:Caspases