Identification of hypoxiaâresponsive messengers expressed in human microvascular endothelial cells using differential display RTâPCR
2000
In order to identify new genes overexpressed in endothelial cells exposed to hypoxia, differential display RTâPCR 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 metalloproteinaseâ1, prostate tumor inducing factorâ1, enolaseâα and prothymosinâα 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 hypoxiaâresponsive genes.
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