LONGEVITY-ASSOCIATED VARIANT (LAV)-BPIFB4 CHARACTERIZATION AND ITS ROLE IN THE MODULATION OF ENOS SIGNALING THROUGH Ca2+/PKC-ALPHA DEPENDENT MECHANISM

Abstract Rationale: Endothelial nitric oxide synthase (eNOS) is a crucial enzyme for vascular physiology and its reduced activity during aging leads to increased cardio and cerebrovascular disease susceptibility1-4. Caloric restriction, which delays aging and increases life-span in all species, doesn’t exert any effect in mice lacking eNOS5. Long Living Individuals (LLIs) have a favourable genetic profile, characterized by an enrichment of alleles that protect from aging and cardiovascular disease6, 7. We have recently shown that LLIs of three populations are enriched for the minor allele rs2070325 (I229V) of the bactericidal/permeability-increasing fold-containing family B member 4 (BPIFB4) and that rs2070325 is part of a four SNPs haplotype that codified for a Wild Type (WT) and a longevity- associated variant (LAV) of BPIFB4, that is able to potentiate eNOS activity. This unique ability is correlated with its higher efficiency, as compared to WT-BPIFB4, in binding 14-3-3 through a BPIFB4 atypical binding site for 14-3-3, which correlates with its level of phosphorylation at serine 75, a phosphorylation site for stress kinase PERK. HSP90 recruitment into the complex is part of the eNOS activation machinery triggered by LAV-BPIFB4. Indeed HSP90 is co-immunoprecipitated together with BPIFB4 and a specific HSP90 inhibitor blocks LAV-BPIFB4 potentiation on endothelial function and eNOS activation. The data so far reported clearly demonstrate that LAV-BPIFB4 activates eNOS function trough 14-3-3 and HSP90 recruitment and, as underlined by William Sessa in its editorial, further characterization is needed to define how LAV-BPIFB4 transduce upstream signals to eNOS Objective: To further characterize the molecular signaling regulated by LAV-BPIFB4 to modulate vascular function. Methods and Results: Here we show that eNOS activation by LAV- BPIFB4 is mediated by Ca2+ mobilization and PKC-alpha activation. In LAV-BPIFB4 transfected HEK293 cells, there was an enhancement of ATP- induced Ca2+ mobilization and PKC-alpha translocation on plasma membrane. In vascular reactivity studies, LAV-BPIFB4 failed to potentiate eNOS and endothelial function upon PKC-alpha inhibition by Gö6976. Moreover, when vessels were exposed to external Ca2+-free conditions, LAV-BPIFB4 lost the ability to activate PKC-alpha and eNOS. In these conditions, though, and in eNOS knockout vessels, LAV-BPIFB4 still potentiated endothelial activity, through mechanisms alternative to eNOS, and this function was blunted by the inhibitors of the endothelium-derived hyperpolarizing factors (EDHF). Notably, peripheral blood mononuclear cells from subjects carrying the a/a genotype for rs2070325 had a significant increase of phosphorylation of PKC-alpha (T497). Conclusions: We have identified novel molecular determinants of the beneficial effects of LAV-BPIFB4 on endothelial function, showing the role of Ca2+ mobilization and PKC-alpha in eNOS activation and of EDHF activation upon eNOS inhibition. These results highlight the role of LAV- BPIFB4.