LAV-BPIFB4 isoform modulates eNOS signalling through Ca2+/PKC-alpha-dependent mechanism.

Aims Ageing is associated with impairment of endothelial nitric oxide synthase (eNOS) and progressive reduction in endothelial function. A genetic study on long-living individuals-who are characterized by delays in ageing and in the onset of cardiovascular disease-previously revealed I229V (rs2070325) in bactericidal/permeability-increasing fold-containing-family-B-member-4 (BPIFB4) as a longevity-associated variant (LAV); the LAV protein enhanced endothelial NO production and vasorelaxation through a protein kinase R-like endoplasmic reticulum kinase/14-3-3/heat shock protein 90 signal. Here, we further characterize the molecular mechanisms underlying LAV-BPIFB4-dependent enhancement of vascular function. Methods and results LAV-BPIFB4 upregulated eNOS function via mobilization of Ca2+ and activation of protein kinase C alpha (PKC alpha). Indeed, the overexpression of LAV-BPIFB4 in human endothelial cells enhanced ATP-induced Ca2+ mobilization and the translocation of PKC alpha to the plasma membrane. Coherently, pharmacological inhibition of PKC alpha blunted the positive effect of LAV-BPIFB4 on eNOS and endothelial function. In addition, although LAV-BPIFB4 lost the ability to activate PKC alpha and eNOS in ex vivo vessels studied in an external Ca2+-free medium and in vessels from eNOS(-/-)mice, it still potentiated endothelial activity, recruiting an alternative mechanism dependent upon endothelium-derived hyperpolarizing factor (EDHF). Conclusions We have identified novel molecular determinants of the beneficial effects of LAV-BPIFB4 on endothelial function, showing the roles of Ca2+ mobilization and PKCa in eNOS activation and of EDHF when eNOS is inhibited. These results highlight the role LAV-BPIFB4 can have in restoring signals that are lost during ageing.