Identification of hypoxia-responsive messengers expressed in human microvascular endothelial cells using differential display RT-PCR.
In order to identify new genes overexpressed in endothelial cells exposed to hypoxia, differential display RT-PCR was performed on total RNA extracted from human microvascular endothelial cells incubated under hypoxia or under normoxic conditions. Northern blot and reverse Northern blot analyses were used to confirm the results. Sequences corresponding to tissue inhibitor of metalloproteinase-1, prostate tumor inducing factor-1, enolase-alpha and prothymosin-alpha were evidenced as overexpressed in hypoxia. These results were confirmed by Western blot and immunofluorescence experiments. Moreover, several elements homologous to partial sequences of cDNA (expressed sequence tag) were also identified, as well as unknown cDNA sequences. The present study suggests that hypoxia can change the expression of numerous genes in endothelial cells, and that mRNA differential display is useful for cloning known and unknown hypoxia-responsive genes.
Roland
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I
Laboratoire de Biochimie et Biologie Cellulaire, Facultés Universitaires Notre-Dame de la Paix, Namur, Belgium.
Minet
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E
Ernest
I
I
Pascal
T
T
Michel
G
G
Remacle
J
J
Michiels
C
C
D015687:Cell Hypoxia / Q000235:genetics*; D004730:Endothelium, Vascular / Q000502:physiology*; D020869:Gene Expression Profiling / Q000379:methods; D005786:Gene Expression Regulation; D006801:Humans; D015513:Oncogene Proteins / Q000235:genetics / Q000378:metabolism; D020648:Peptide Elongation Factor 1; D011498:Protein Precursors / Q000235:genetics / Q000378:metabolism; D012333:RNA, Messenger; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000379:methods; D013947:Thymosin / Q000031:analogs & derivatives / Q000235:genetics / Q000378:metabolism; D019715:Tissue Inhibitor of Metalloproteinase-1 / Q000235:genetics / Q000378:metabolism
C094814:EEF1A1 protein, human; D015513:Oncogene Proteins; D020648:Peptide Elongation Factor 1; D011498:Protein Precursors; D012333:RNA, Messenger; D019715:Tissue Inhibitor of Metalloproteinase-1; C040782:prothymosin alpha; D013947:Thymosin