Serum BPIFB4 levels classify health status in long-living individuals

In the third millennium average life expectancy is 78 years for males and 83 years for females in industrialized countries, while at the beginning of the 20th century it was almost 20 years shorter [1]. Thus, the Long-Living Individuals (LLIs, >95 years of age) recruited in the 21st century reached around 30 years more than the average life expectancy of their cohorts. Among the LLIs some were able to escape illness (unaffected LLIs) while others survived to age-related diseases (cancer, diabetes, cardiovascular disease, or stroke) got at younger ages (affected LLIs) [2]. One of the principal factor in determining health during ageing is the state of the vascular system that is the responsible for provisioning all the body areas with nutrients and oxygen [3]. The aging process is associated with a reduction in NO availability in the endothelial district and this decrement triggers a progressive decline of the entire vascular system [4].

Our recent multi-step genetic analysis of Italian (the screening set),[5] and US and German LLIs (replication sets) and relative control populations ,[6‐8] identified a variant in BPIFB4, which transcript showed to be down-regulated during aging and high in CD34⁺ of LLIs and the codified protein (LAV-BPIFB4) to be a powerful boost for endothelial vasorelaxation and revascularization, two functions lost during aging and cause of human frailty [9]. Furthermore, we have demonstrated that BPIFB4 is beneficial to cellular homeostasis by showing that: a) LAV-BPIFB4 potentiates eNOS activation; b) BPIFB4 isoforms cellular overexpression activates stress response (upregulation of heat shock proteins) and proteostasis (translation, ribosomal biogenesis and snoRNA/scaRNAs involved in genomic integrity), as also shown by reduction of eIF2alpha phosphorylation, thus activating protein synthesis.

Based on our previous results, we envisioned that serum levels of BPIFB4 could correlate with the ability to reach extreme ages and with LLIs health status.

To evaluate if BPIFB4 was secreted and detectable in serum, we first evaluated the medium of HEK293T cells transfected with BPIFB4-encoding plasmid (Fig. 1). Indeed, we found abundant BPIFB4 in the medium, confirming its extracellular secretion.

We then used ELISA to evaluate the amount of the protein in sera from middle-aged individuals (age range: 45–59 years, n = 32) and from LLIs (>94 years, n = 30) (see Table 1 for details). We found the serum BPIFB4 level to be significantly higher in LLIs compared to controls (median ELISA quantifications in LLIs = 823.76, IQR = 69.91–1146.35 vs. median ELISA quantifications in CTRLs = 132.74, IQR = 11.25–751.32, p - value = 0.004, Fig. 2), pointing to BPIFB4 as a protein associated with longevity.

Then we looked into the LLIs population to test whether BPIFB4 serum levels could discriminate between their healthy statuses. We analyzed different variables, which included age, familiarity for longevity trait, affection status (defined as presence or absence of medical history for cancer, diabetes, cardiovascular disease, or stroke) and gender. Of the analyzed variables, ELISA quantifications were significantly lower in affected LLIs (or frail LLIs, F-LLIs, n = 7) with respect to the rest of the LLIs (or healthy aged individuals, HA-LLIs, n = 23) (median ELISA quantifications in F-LLIs = 31.21, IQR = 20.56–82.78 vs. median ELISA quantifications in HA-LLIs = 865.22, IQR = 672.30–1236.43, p = 0.004, Bonferroni adjusted p = 0.01, Fig. 2). No statistically significant difference in terms of age, familiarity for longevity trait or gender was observed between F-LLIs and HA-LLIs (p > 0.05). We further observed that the protein concentration was significantly higher in HA-LLIs compared to CTRLs (median ELISA quantifications in HA-LLIs = 865.22, IQR = 672.30–1236.43 vs. median ELISA quantifications in CTRLs = 132.74, IQR = 11.25–751.32, p = 0.0002, Bonferroni adjusted p = 0.0005). No statistically significant difference was observed in terms of protein concentration between F-LLIs and CTRLs (median ELISA quantifications in F-LLIs = 31.21, IQR = 20.56–82.78vs. median ELISA quantifications in CTRLs = 132.74, IQR = 11.25–751.32, p = 0.570, Bonferroni adjusted p = 1).

Figure 3 shows the ROC curve and the AUROC corresponding to the ELISA quantification in discriminating LLIs from CTRLs (AUROC = 0.71, 95 % CI = 0.58–0.84) and F-LLIs from HA-LLIs (AUROC = 0.86, 95 % CI = 0.72–1).

No other variable was associated to statistically significant variations in terms of ELISA quantifications (p > 0.05 Fig. 4).

The power of the ELISA quantifications and of the combination of ELISA quantifications plus other clinical variables (age, gender and familiarity for longevity trait) in discriminating F-LLIs from HA-LLIS were assessed by stepwise logistic regression coupled with a Leave One Out (LOO) cross validation schema [10] on a subset of 29 LLIs showing complete data for all covariates (n F-LLIS = 7, n H-LLIS = 22). According to this resampling strategy, three models (clinical covariates based model (age + gender + familiarity); clinical covariates and ELISA quantifications based model (ELISA quantifications + age + gender + familiarity) and ELISA quantifications based model (ELISA quantifications)) were learned on the each training set using the LLIs disease status as outcome (F-LLIs were coded 1, HA-LLIs were coded 0) and the generalization capability of the set of features selected by the stepwise regression tested on the external test set. Results are reported in Table 2 and they confirm that ELISA quantification was the strongest parameter to correctly discriminate F-LLIs from HA-LLIs, since no other combination of clinical covariates or clinical covariates plus ELISA quantifications was able to outperform its classification capability. However, these results should be cautiously interpreted due to the small sample size of our analysis and due to the lack of an independent cohort of individuals to be used to confirm the generalization capability of the BPIFB4 serum levels as marker of healthy aging.

We have recently found a potential link between BPIFB4 and exceptional longevity by analyzing more than 3000 LLIs individuals and 2000 controls of different recruitment efforts (Italian, US and German). We have identified a homozygous enrichment of the longevity associated variant haplotype (LAV) in LLIs indicating a possible protective role of this genotype [9, 11]. Indeed, further studies have shown that the BPIFB4 transcript was high in CD34⁺ cells of LLIs, corroborating a possible protective role of the protein. Such a role was further investigated in vitro by overexpressing BPIFB4 variants and in vivo by injecting mice and rats with AAV carrying different isoforms of the BPIFB4 gene. In vitro studies pointed to a role of the protein in survival processes, such as stress response, proteostasis, genomic integrity, and in vivo experiments of role of LAV in potentiating eNOS activation, endothelial function, revascularization after induced ischemia. All these aspects are lost during aging and we hypothesize that can be kept active in LLIs by higher expression of the BPIFB4 protein. Indeed, here we describe that BPIFB4 is secreted and ELISA detected higher BPIFB4 levels in serum of LLIs as compared to young controls, that BPIFB4 serum levels can be used to classify the LLIs health status (affected versus unaffected) and that classification was not improved by including in the analysis familiarity for longevity and age. Recently, Heyn et al. [12] generated methylation profiles of 485,577 highly informative CpG sites (Illumina Infinium Human Methylation 450 BeadChip) and showed a list of CpG hypomethylated (N = 1920), which included cg04087207 in the first exon of BPIFB4, indicating its possible overexpression. Thus, the analysis, which was performed in CD4⁺ cells of 19 LLIs in comparison to 19 newborns (NBs), would support our observation that BPIFB4 is more abundant in LLIs as compared to young controls.

Our results, which describe the detection of high levels of BPIFB4 protein in LLIs serum, further support the hypothesis of an important role of the protein in exceptional longevity. BPIFB4 levels could be adopted as biomarkers of health status in LLIs.

Further studies will evaluate the potential use of BPIFB4 levels for following health of individuals with pathologies.