Hypoxia inhibits expression of eNOS via transcriptional and posttranscriptional mechanisms

Abstract

Normal blood vessel tone is maintained by a balance of vasoconstrictors and vasodilators produced by endothelial cells in the vasculature. Nitric oxide (NO) is a potent vasodilator that causes vascular smooth muscle cell relaxation by elevating intracellular guanosine 3',5'-cyclic monophosphate (cGMP) levels. The physiological mechanisms regulating NO production in the vasculature are not completely understood. We report here that production of this vasodilator by vascular endothelial cells can be significantly suppressed by hypoxia. Exposing human endothelial cells to low PO2 results in 40-60% reduction in the steady-state mRNA levels of endothelial constitutive NO synthase (eNOS), the major enzyme responsible for NO production in these cells. The lower levels of eNOS mRNA result from decreased transcription of the gene as well as reduced message stability. In endothelial-smooth muscle cell co-culture experiments, hypoxic endothelial cells stimulated significantly less cGMP production by smooth muscle cells than the corresponding normoxic controls. This inhibitory effect of hypoxia on NOS production by endothelial cells occurs after 24 h of hypoxia and persists for at least 48 h. These new findings suggest that hypoxia might cause changes in blood vessel tone through compound mechanisms: by increasing the production of endothelium-derived vasoconstrictors and, as shown here, by suppressing the production of vasodilators like NO.

Hypoxia-inducible Expression of a Natural cis-Antisense Transcript Inhibits Endothelial Nitric-oxide Synthase

“…As a control, the stability of eNOS RNA was also assessed. As we have previously published (9), hypoxia resulted in a decrease in the half-life of eNOS RNA (half-life of eNOS in hypoxia/normoxia ϭ 0.31 Ϯ 0.19; p Ͻ 0.05). These results suggest that sONE transcript stabilization, rather than transcriptional induction, occurs during hypoxia.…”

“…Several models could be put forward for the regulation of eNOS RNA levels by sONE. eNOS mRNA is highly stable in endothelial cells, and changes in RNA stability and adenylation of the eNOS transcript have been implicated in the response of endothelial cells to several physiological and pathological stimuli, including shear stress (63), exposure to inflammatory cytokines (15,64), and hypoxia (9). A multiprotein complex binds to the eNOS 3Ј-UTR in endothelial cells to actively stabilize eNOS RNA (14,15).…”

“…We were interested in assessing whether sONE could be up-regulated in endothelial cells by stimuli that are known to decrease the stability of eNOS mRNA. Therefore, we measured steadystate levels of sONE RNA in hypoxic endothelial cells, since hypoxic stimulation of HUVEC decreases eNOS RNA levels, in part by post-transcriptional mechanisms (9). Hypoxia can be mimicked by treating cells with DFO (26).…”

“…This is much longer than typical mRNAs (13). Hypoxic stimulation of endothelial cells markedly decreases the stability of eNOS mRNAs (9,11). The molecular mechanisms responsible for the long half-life of the eNOS transcript in normoxic endothelial cells are not fully understood but are mediated, at least in part, by protein binding to the 3Ј-UTR of the eNOS mRNA (14 -16).…”

Inhibition of MicroRNA‐155 Supports Endothelial Tight Junction Integrity Following Oxygen‐Glucose Deprivation

“…These expectations were not supported by our in vitro results. We therefore presume that in the in vitro setting, hypoxia induces endothelial dysfunction, which inhibits NO release from the HBMECs 40, 41. This could be associated with: (1) NO trapping by free radicals, and (2) reduced endothelial NO synthase activity (even with its elevated expression).…”