Î²-Catenin Mediates the Anti-Apoptotic Effects of NO in Endothelial Cells
Apoptosis is implicated in a number of cardiovascular diseases. Increased endothelial cell apoptosis is associated with the development of atherosclerotic plaques. Strategies to promote endothelial cell survival may therefore represent a novel therapeutic approach in cardiovascular disease.
Nitric oxide (NO) and β²-catenin have both been shown to promote cell survival. Recently we showed that pharmacological activation of the endothelial nitric oxide synthase (eNOS), acting through cGMP, can promote nuclear translocation and transcriptional activity of Î²-catenin.
Using an orbital shaker system to generate shear stress, we investigated the physiological role of β²-catenin as a mediator of NO-induced cell survival in endothelial cells. Human umbilical vein endothelial cells (HUVEC) exposed to flow for 72 hour at 150 rpm on an orbital shaker exhibited different degrees of apoptosis between the undisturbed flow (UF) zone in the periphery of the well and the central disturbed flow (DF) zone in the centre of the well (1.54%±0.26 (mean± SEM) cleaved caspase positive cells in DF zone compared to 0.26%±0.07 in UF zone; n=3 p<0.01). The degree of apoptosis increased in both UF and DF areas when cells were treated with an inhibitor of beta-catenin transcriptional ?activity, with 4.45%±0.86 apoptotic cells in the DF zone compared to 0.57%±0.11 in the UF zone (n=3 p<0.05 for non-treated to inhibitor treated in the DF zone).
We confirmed expression of eNOS and beta-catenin in both UF and DF zones by quantitative-PCR and immunostaining, as well as the interaction between eNOS and beta-catenin in both regions by proximity ligation assay. We then studied the expression of several pro-survival and anti-apoptotic genes by q-PCR in HUVEC exposed to flow for 72 hours. We observed that the expression of Bcl-2 and survivin were downregulated in UF exposed cells compared to static conditions (62%±0.08 downregulation of Bcl-2% and 67%±0.08 downregulation of survivin, n=3 p<0.01 in both cases); and using specific beta-catenin/TCF-LEF inhibitors we identified survivin as an anti-apoptotic gene regulated by beta-catenin (89%±0.03 downregulation in inhibitor treated samples compared to non-treated, n=3 p<0.001) in endothelial cells under flow.
We also investigated the reciprocal effects on eNOS of activation of Wnt signalling and beta-catenin in HUVECs. Using Wnt3a and LiCl, that lead to the accumulation of beta-catenin in the cytoplasm, we found that phosphorylation of eNOS at Ser1177 increased 4 fold after 2–5 min (n=3 p<0.01) leading to enzyme activation. Phosphorylation of eNOS at Ser633 and Ser114 was also observed both in HUVEC exposed to UF flow for 72 hour in an orbital shaker and in HUVEC stimulated by Wnt3a.
Together our results indicate that beta-catenin is a key mediator of flow-induced anti-apoptotic effects, both through transcriptional regulation and through activation of eNOS phosphorylation in endothelial cells.
This work has been supported by a grant from the British Heart Foundation.
- Nitric oxide synthase
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