New Insights for BPIFB4 in Cardiovascular Therapy.

Aging is the most relevant risk factor for cardiovascular diseases which are the main cause of mortality in industrialized countries. In this context, there is a progressive loss of cardiovascular homeostasis that translates in illness and death. The study of long living individuals (LLIs), which show compression of morbidity toward the end of their life, is a valuable approach to find the key to delay aging and postpone associate cardiovascular events. A contribution to the age-related decline of cardiovascular system (CVS) comes from the immune system; indeed, it is dysfunctional during aging, a process described as immunosenescence and comprises the combination of several processes overpowering both innate and adaptative immune system. We have recently discovered a longevity-associated variant (LAV) in bactericidal/permeability-increasing fold-containing family B member 4 (BPIFB4), which is a secreted protein able to enhance endothelial function through endothelial nitric oxide synthase (eNOS) activation and capable to protect from hypertension, atherosclerosis, diabetic cardiopathy, frailty, and inflammaging. Here, we sum up the state of the art of the mechanisms involved in the main pathological processes related to CVD (atherosclerosis, aging, diabetic cardiopathy, and frailty) and shed light on the therapeutic effects of LAV-BPIFB4 in these contexts.

A Model of Evolutionary Selection: The Cardiovascular Protective Function of the Longevity Associated Variant of BPIFB4.

Evolutionary forces select genetic variants that allow adaptation to environmental stresses. The genomes of centenarian populations could recapitulate the evolutionary adaptation model and reveal the secrets of disease resistance shown by these individuals. Indeed, longevity phenotype is supposed to have a genetic background able to survive or escape to age-related diseases. Among these, cardiovascular diseases (CVDs) are the most lethal and their major risk factor is aging and the associated frailty status. One example of genetic evolution revealed by the study of centenarians genome is the four missense Single Nucleotide Polymorphisms (SNPs) haplotype in bactericidal/permeability-increasing fold-containing family B, member 4 (BPIFB4) locus that is enriched in long living individuals: the longevity associated variant (LAV). Indeed, LAV-BPIFB4 is able to improve endothelial function and revascularization through the increase of endothelial nitric oxide synthase (eNOS) dependent nitric oxide production. This review recapitulates the beneficial effects of LAV-BPIFB4 and its therapeutic potential for the treatment of CVDs.

Serum BPIFB4 levels classify health status in long-living individuals.

BACKGROUND: People that reach extreme ages (Long-Living Individuals, LLIs) are object of intense investigation for increase/decrease of genetic variant frequencies, genetic methylation levels, protein abundance in serum and tissues. The aim of these studies is the discovery of the mechanisms behind LLIs extreme longevity and the identification of markers of well-being. We have recently associated a BPIFB4 haplotype (LAV) with exceptional longevity under a homozygous genetic model, and identified that CD34(+) of LLIs subjects express higher BPIFB4 transcript as compared to CD34(+) of control population. It would be of interest to correlate serum BPIFB4 protein levels with exceptional longevity and health status of LLIs. METHODS: Western blots on cellular medium to detect BPIFB4 secretion in transfected HEK293T cells with plasmid carrying BPIFB4 and ELISA on LLIs serum to detect BPIFB4 levels. RESULTS: Here we show that BPIFB4 is a secreted protein and its levels are increased in serum of LLIs, and high BPIFB4 levels classify their health status. CONCLUSIONS: Serum BPIFB4 protein levels classify longevity and health status in LLIs. Further studies are required to evaluate the possible role of BPIFB4 in monitoring disease progression.

The longevity-associated BPIFB4 gene guarantees vascular homeostasis and immune protection through platelets.

Beyond their activity in hemostasis and thrombosis, recent advances attribute platelets a pro-youthful role capable to attenuate immune senescence and age-related neuroinflammation. Previous studies from our group associated a polymorphic haplotype variant in the BPIFB4 gene (LAV-BPIFB4) with exceptional longevity. Transfer of the LAV-BPIFB4 in preclinical models has proved strategic to cope with frailty conditions, aging-related events, e.g., cardiovascular ones, and immune dysfunction mainly through a favorable conditioning of the immune system. However, whether platelets participate in LAV-BPIFB4 therapeutic action is currently unknown. Herein, we discovered that platelets were instrumental in boosting the favorable health outcomes of the systemic AAV-LAV-BPIFB4 gene transfer in vivo, as the α-CD42b platelet depletion completely abolished the vascular protective action of LAV-BPIFB4 and suppressed its pro-resolutive CD206 + anti-/CD86 + pro-inflammatory Ly6C + monocyte skewing to LPS stimulation. Of note, this is associated with a huge drop in the protective levels of BPIFB4 in the plasma of AAV-LAV-BPIFB4-injected C57BL/6 mice, indicating that plasma circulating platelets may be a reservoir of the BPIFB4 protein. Indeed, we noticed that BPIFB4 was released by human platelets, a process that is amplified in LAV-allele carrier donors. Accordingly, lentivirus-mediated overexpression of human LAV-BPIFB4 isoform, but not WT-BPIFB4 isoform was able in leading differentiated megakaryocytes to release more platelet-like-particles enriched for BPIFB4. In addition, in vitro, the M2 macrophage polarization increased when releasate from platelets, and even more from LAV pre-stimulated once, was added in monocyte cell culture. Our data suggest that platelet release of BPIFB4 and of yet-to-be-determined unidentified factors mediates the therapeutic efficacy of LAV-BPIFB4 treatment.

Transfer of a human gene variant associated with exceptional longevity improves cardiac function in obese type 2 diabetic mice through induction of the SDF-1/CXCR4 signalling pathway.

AIMS: Homozygosity for a four-missense single-nucleotide polymorphism haplotype of the human BPIFB4 gene is enriched in long-living individuals. Delivery of this longevity-associated variant (LAV) improved revascularisation and reduced endothelial dysfunction and atherosclerosis in mice through a mechanism involving the stromal cell-derived factor-1 (SDF-1). Here, we investigated if delivery of the LAV-BPIFB4 gene may attenuate the progression of diabetic cardiomyopathy. METHODS AND RESULTS: Compared with age-matched lean controls, diabetic db/db mice showed altered echocardiographic indices of diastolic and systolic function and histological evidence of microvascular rarefaction, lipid accumulation, and fibrosis in the myocardium. All these alterations, as well as endothelial dysfunction, were prevented by systemic LAV-BPIFB4 gene therapy using an adeno-associated viral vector serotype 9 (AAV9). In contrast, AAV9 wild-type-BPIFB4 exerted no benefit. Interestingly, LAV-BPIFB4-treated mice showed increased SDF-1 levels in peripheral blood and myocardium and up-regulation of the cardiac myosin heavy chain isoform alpha, a contractile protein that was reduced in diabetic hearts. SDF-1 up-regulation was instrumental to LAV-BPIFB4-induced benefit as both haemodynamic and structural improvements were inhibited by an orally active antagonist of the SDF-1 CXCR4 receptor. CONCLUSIONS: In mice with type-2 diabetes, LAV-BPIFB4 gene therapy promotes an advantageous remodelling of the heart, allowing it to better withstand diabetes-induced stress. These results support the viability of transferring healthy characteristics of longevity to attenuate diabetic cardiac disease.

Multi-Omics Analysis of Diabetic Heart Disease in the db/db Model Reveals Potential Targets for Treatment by a Longevity-Associated Gene.

Characterisation of animal models of diabetic cardiomyopathy may help unravel new molecular targets for therapy. Long-living individuals are protected from the adverse influence of diabetes on the heart, and the transfer of a longevity-associated variant (LAV) of the human BPIFB4 gene protects cardiac function in the db/db mouse model. This study aimed to determine the effect of LAV-BPIFB4 therapy on the metabolic phenotype (ultra-high-performance liquid chromatography-mass spectrometry, UHPLC-MS) and cardiac transcriptome (next-generation RNAseq) in db/db mice. UHPLC-MS showed that 493 cardiac metabolites were differentially modulated in diabetic compared with non-diabetic mice, mainly related to lipid metabolism. Moreover, only 3 out of 63 metabolites influenced by LAV-BPIFB4 therapy in diabetic hearts showed a reversion from the diabetic towards the non-diabetic phenotype. RNAseq showed 60 genes were differentially expressed in hearts of diabetic and non-diabetic mice. The contrast between LAV-BPIFB4- and vehicle-treated diabetic hearts revealed eight genes differentially expressed, mainly associated with mitochondrial and metabolic function. Bioinformatic analysis indicated that LAV-BPIFB4 re-programmed the heart transcriptome and metabolome rather than reverting it to a non-diabetic phenotype. Beside illustrating global metabolic and expressional changes in diabetic heart, our findings pinpoint subtle changes in mitochondrial-related proteins and lipid metabolism that could contribute to LAV-BPIFB4-induced cardio-protection in a murine model of type-2 diabetes.

LAV-BPIFB4 associates with reduced frailty in humans and its transfer prevents frailty progression in old mice.

BACKGROUND: There is an increasing concern about age-related frailty because of the growing number of elderly people in the general population. The Longevity-Associated Variant (LAV) of the human BPIFB4 gene was found to correct endothelial dysfunction, one of the mechanisms underlying frailty, in aging mice whereas the RV-BPIFB4 variant induced opposite effects. Thus, we newly hypothesize that, besides being associated with life expectancy, BPIFB4 polymorphisms can predict frailty.Aim and Results: Here we investigated if the BPIFB4 haplotypes, LAV, wild-type (WT) and RV, differentially associate with frailty in a cohort of 237 elderly subjects from Calabria region in Southern Italy. Moreover, we studied the effect of systemic adeno-associated viral vector-mediated LAV-BPIFB4 gene transfer on the progression of frailty in aging mice. We found an inverse correlation of the homozygous LAV-BPIFB4 haplotype with frailty in elderly subjects. Conversely, carriers of the RV-BPIFB4 haplotype showed an increase in the frailty status and risk of death. Moreover, in old mice, LAV-BPIFB4 gene transfer delayed frailty progression. CONCLUSIONS: These data indicate that specific BPIFB4 haplotypes could represent useful genetic markers of frailty. In addition, horizontal transfer of a healthy gene variant can attenuate frailty in aging organisms.

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 (PKCα). Indeed, the overexpression of LAV-BPIFB4 in human endothelial cells enhanced ATP-induced Ca2+ mobilization and the translocation of PKCα to the plasma membrane. Coherently, pharmacological inhibition of PKCα blunted the positive effect of LAV-BPIFB4 on eNOS and endothelial function. In addition, although LAV-BPIFB4 lost the ability to activate PKCα 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 PKCα 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.

The expression of the BPIFB4 and CXCR4 associates with sustained health in long-living individuals from Cilento-Italy.

The study of the health status in long-living individuals (LLIs) may help identifying health-span and life-span determinants. BPI-Fold-Containing-Family-B-Member-4 (BPIFB4) protein is higher in healthy vs. non-healthy (frail) LLIs serum and its longevity-associated variant forced expression improves cardiovascular outcomes in ischemia mice models. Thus, we tested the association of BPIFB4 and ischemia-responding HIF-1α pathway components (i.e. CXCR4, AK3, ALDO-C, ADM, VEGF-A, GLUT-1 and miR-210) with human life-span and health-span by analyzing mRNA expression in circulating mononuclear cells (MNCs) of LLIs (N=14 healthy; N=31 frail) and young controls (N=63).ALDO-C, ADM, VEGF-A and GLUT-1 significantly decreased and miR-210 increased in LLIs vs. CONTROLS: Only VEGF-A and GLUT-1 showed further significant reduction in healthy-LLIs vs. frail-LLIs comparison. While BPIFB4 and CXCR4 were similar between LLIs and controls, BPIFB4 was significantly higher and CXCR4 lower in healthy- versus frail-LLIs. On a new set of LLIs (N=7 healthy and N=5 non-healthy) we assessed a potentially correlated function with low CXCR4 expression. Healthy donors' MNCs showed efficient migration ability toward CXCR4 ligand SDF-1α/CXCL12 and high percentage of migrated CXCR4pos cells which inversely correlated with CXCR4 RNA expression. In conclusion, BPIFB4 and CXCR4 expression classify LLIs health status that correlates with maintained MNCs migration.