Longevity-associated Variant Research Articles

Healthy longevity-associated protein improves cardiac function in murine models of cardiomyopathy with preserved ejection fraction

AimsAging is influenced by genetic determinants and comorbidities, among which diabetes increases the risk for heart failure with preserved ejection fraction. There is no therapy to prevent heart dysfunction in aging and diabetic individuals. In previous studies, a single administration of the longevity-associated variant (LAV) of the human BPIFB4 gene halted heart decline in older and type 2 diabetic mice. Here, we asked whether orally administered LAV-BPIFB4 protein replicates these benefits.Materials and MethodsIn two controlled, randomized studies, 18-month-old male C57BL/6 J mice and 9-week-old C57BLKS/J-Leprdb/Leprdb/Dock7 + [db/db] mice of both sexes underwent baseline echocardiography. They then received a recombinant purified LAV-BPIFB4 protein (3 µg/animal, every three days) or vehicle by gavage. After 30 days, the animals underwent echocardiography, and the hearts were collected post-termination for histology.ResultsAll the animals completed the study except one female diabetic mouse, which was culled prematurely because tooth malocclusion caused eating problems. There was no effect of the LAV-BPIFB4 protein on body weight in the two studies or glycosuria in the diabetic study. In aging mice, LAV-BPIFB4 increased myocardial Bpifb4 expression, improving heart contractility and capillarity while reducing perivascular fibrosis and senesce. In male diabetic mice, LAV-BPIFB4 therapy improved systolic function, microvascular density, and senescence, whereas the benefit was limited to systolic function in females.ConclusionsThis study shows the feasibility and efficacy of a variant protein associated with human longevity in contrasting pivotal risk factors for heart failure in animal models. The diabetic study revealed that sex influences the treatment efficacy.

Longevity-associated BPIFB4 gene counteracts the inflammatory signaling

BackgroundIncreased levels of pro-inflammatory proteins in plasma can be detected in older individuals and associate with the so called chronic low-grade inflammation, which contributes to a faster progression of aged-related cardiovascular (CV) diseases, including frailty, neurodegeneration, gastro-intestinal diseases and disorders reflected by alterations in the composition of gut microbiota. However, successful genetic programme of long-living individuals alters the trajectory of the ageing process, by promoting an efficient immune response that can counterbalance deleterious effects of inflammation and the CV complications. This is the case of BPIFB4 gene in which, homozygosity for a four single-nucleotide polymorphism (SNP) haplotype, the Longevity-Associated Variant (LAV) correlates with prolonged health span and reduced risk of CV complications and inflammation. The relation between LAV-BPIFB4 and inflammation has been proven in different experimental models, here we hypothesized that also human homozygous carriers of LAV-BPIFB4 gene may experience a lower inflammatory burden as detected by plasma proteomics that could explain their favourable CV risk trajectory over time. Moreover, we explored the therapeutic effects of LAV-BPIFB4 in inflammatory disease and monolayer model of intestinal barrier.ResultsWe used high-throughput proteomic approach to explore the profiles of circulating proteins from 591 baseline participants selected from the PLIC cohort according to the BPIFB4 genotype to identify the signatures and differences of BPIFB4 genotypes useful for health and disease management. The observational analysis identified a panel of differentially expressed circulating proteins between the homozygous LAV-BPIFB4 carriers and the other alternative BPIFB4 genotypes highlighting in the latter ones a higher grade of immune-inflammatory markers. Moreover, in vitro studies performed on intestinal epithelial organs from inflammatory bowel disease (IBD) patients and monolayer model of intestinal barrier demonstrated the benefit of LAV-BPIFB4 treatment.ConclusionsHomozygosity for LAV-BPIFB4 results in the attenuation of inflammation in PLIC cohort and IBD patients providing preliminary evidences for its therapeutic use in inflammatory disorders that need to be further characterized and confirmed by independent studies.

Human platelets and megakaryocytes express the BPIFB4 protein and their stimulation with the recombinant Longevity-associated variant LAV-BPIFB4 displays anti-aggregating activity

Abstract Platelets are not only the main players in primary haemostasis and cardiovascular disease, but recent advances attribute them a key role in immune defence mechanisms via the release of plasma mediators of potential clinical relevance. The bactericidal/permeability-increasing fold-containing family B, member 4 (BPIFB4) belongs to a family protein participating in the innate mechanism of host defence. It is a secreted protein and its plasma levels inversely correlated with COVID-19 and coronary artery disease (CAD) severity and the degree of carotid stenosis and intima media thickness in human patient cohorts. Interestingly, Long Living Individuals (LLIs) displayed high BPIFB4 circulating levels and an enrichment of the 4 SNPs missense haplotype at BPIFB4 locus, the Longevity-Associated Variant (LAV). LAV-BPIFB4 genotype correlate with higher BPIFB4 plasma levels and the transfer of LAV-BPIFB4 gene delayed major age-related diseases, mainly rescuing the immunesenescence and the endothelial and cardiac dysfunctions, yet the critical assessment of the BPIFB4 protein as a platelet release product is lacking. Herein we observed BPIFB4 release by calcium activated human platelets, a process that is amplified in LAV allele carriers donors. Furthermore, LAV gene transfer resulted in an enhanced protein enrichment in bone marrow megakaryocyte precursor cells and in a more efficient process of platelets formation in vitro. At functional level, LAV genotype was mimicked by the rhLAV-BPIFB4 platelet stimulation that induced endogenous BPIFB4 release similarly to calcium stimuli. In a small cohort of treatment-naive patients with type 2 diabetes, while ADP and Collagene agonist stimulation induced high aggregation, the co-treatment with the rhLAV-BPIFB4 significantly reduced the activation capacity of patients’ platelets. Accordingly, an administration of rhLAV-BPIFB4 24 hours before tromboembolism induction partly protected from mortality in a murine model of lethal pulmonary thromboembolism in vivo. As a result of reduced platelets’ activation, lower level of circulating leukocyte-platelets aggregates (LPA) was found in rhLAV-BPIFB4-treated mice compared to control. In summary, our data report for the first time that both, human platelets and megakaryocytes, can express BPIFB4 and unravel a novel rhLAV-BPIFB4 therapeutic action in platelet iperaggregation and thrombocytopenia.

BPIFB4 and its longevity-associated haplotype protect from cardiac ischemia in humans and mice

Long-living individuals (LLIs) escape age-related cardiovascular complications until the very last stage of life. Previous studies have shown that a Longevity-Associated Variant (LAV) of the BPI Fold Containing Family B Member 4 (BPIFB4) gene correlates with an extraordinarily prolonged life span. Moreover, delivery of the LAV-BPIFB4 gene exerted therapeutic action in murine models of atherosclerosis, limb ischemia, diabetic cardiomyopathy, and aging. We hypothesize that downregulation of BPIFB4 expression marks the severity of coronary artery disease (CAD) in human subjects, and supplementation of the LAV-BPIFB4 protects the heart from ischemia. In an elderly cohort with acute myocardial infarction (MI), patients with three-vessel CAD were characterized by lower levels of the natural logarithm (Ln) of peripheral blood BPIFB4 (p = 0.0077). The inverse association between Ln BPIFB4 and three-vessel CAD was confirmed by logistic regression adjusting for confounders (Odds Ratio = 0.81, p = 0.0054). Moreover, in infarcted mice, a single administration of LAV-BPIFB4 rescued cardiac function and vascularization. In vitro studies showed that LAV-BPIFB4 protein supplementation exerted chronotropic and inotropic actions on induced pluripotent stem cell (iPSC)-derived cardiomyocytes. In addition, LAV-BPIFB4 inhibited the pro-fibrotic phenotype in human cardiac fibroblasts. These findings provide a strong rationale and proof of concept evidence for treating CAD with the longevity BPIFB4 gene/protein.

Effects of Human LAV-BPIFB4 Gene Therapy on the Epigenetic Clock and Health of Aged Mice

The homozygous genotype of the Longevity-Associated Variant (LAV) in Bactericidal/Permeability-Increasing Fold-Containing Family B member 4 (BPIFB4) is enriched in long-living individuals of three independent populations and its genetic transfer in C57BL/6J mice showed a delay in frailty progression and improvement of several biomarkers of aging and multiple aspects of health. The C57BL/6J strain is a suitable model for studying therapies aimed at extending healthy aging and longevity due to its relatively short lifespan and the availability of aging biomarkers. Epigenetic clocks based on DNA methylation profiles are reliable molecular biomarkers of aging, while frailty measurement tools are used to evaluate overall health during aging. In this study, we show that the systemic gene transfer of LAV-BPIFB4 in aged C57BL/6J mice was associated with a significant reduction in the epigenetic clock-based biological age, as measured by a three CpG clock method. Furthermore, LAV-BPIFB4 gene transfer resulted in an improvement of the Vitality Score with a reduction in the Frailty Index. These findings further support the use of LAV-BPIFB4 gene therapy to induce beneficial effects on epigenetic mechanisms associated with aging and frailty in aged mice, with potential implications for future therapies to prevent frailty in humans.

The longevity-associated BPIFB4 gene supports cardiac function and vascularization in ageing cardiomyopathy.

The ageing heart naturally incurs a progressive decline in function and perfusion that available treatments cannot halt. However, some exceptional individuals maintain good health until the very late stage of their life due to favourable gene-environment interaction. We have previously shown that carriers of a longevity-associated variant (LAV) of the BPIFB4 gene enjoy prolonged health spans and lesser cardiovascular complications. Moreover, supplementation of LAV-BPIFB4 via an adeno-associated viral vector improves cardiovascular performance in limb ischaemia, atherosclerosis, and diabetes models. Here, we asked whether the LAV-BPIFB4 gene could address the unmet therapeutic need to delay the heart's spontaneous ageing. Immunohistological studies showed a remarkable reduction in vessel coverage by pericytes in failing hearts explanted from elderly patients. This defect was attenuated in patients carrying the homozygous LAV-BPIFB4 genotype. Moreover, pericytes isolated from older hearts showed low levels of BPIFB4, depressed pro-angiogenic activity, and loss of ribosome biogenesis. LAV-BPIFB4 supplementation restored pericyte function and pericyte-endothelial cell interactions through a mechanism involving the nucleolar protein nucleolin. Conversely, BPIFB4 silencing in normal pericytes mimed the heart failure pericytes. Finally, gene therapy with LAV-BPIFB4 prevented cardiac deterioration in middle-aged mice and rescued cardiac function and myocardial perfusion in older mice by improving microvasculature density and pericyte coverage. We report the success of the LAV-BPIFB4 gene/protein in improving homeostatic processes in the heart's ageing. These findings open to using LAV-BPIFB4 to reverse the decline of heart performance in older people.

Longevity-Associated Variant of BPIFB4 Confers Neuroprotection in the STHdh Cell Model of Huntington Disease.

Huntington's disease (HD) is caused by the production of mutant Huntingtin (mHTT), characterized by long polyglutamine repeats with toxic effects. There are currently no clinically validated therapeutic agents that slow or halt HD progression, resulting in a significant clinical unmet need. The striatum-derived STHdh cell line, generated from mHTT knock-in mouse embryos (STHdhQ111/Q111), represents a useful model to study mechanisms behind pathogenesis of HD and to investigate potential new therapeutic targets. Indeed, these cells show susceptibility to nucleolar stress, activated DNA damage response and apoptotic signals, and elevated levels of H3K9me3 that all together concur in the progressive HD pathogenesis. We have previously shown that the adeno-associated viral vector-mediated delivery of the longevity-associated variant (LAV) of BPIFB4 prevents HD progression in a mouse model of HD. Here, we show that LAV-BPIFB4 stably infected in STHdhQ111/Q111 cells reduces (i) nucleolar stress and DNA damage through the improvement of DNA repair machinery, (ii) apoptosis, through the inhibition of the caspase 3 death signaling, and (iii) the levels of H3K9me3, by accelerating the histone clearance, via the ubiquitin-proteasome pathway. These findings pave the way to propose LAV-BPIFB4 as a promising target for innovative therapeutic strategies in HD.

Role of BPIFB4 in Immune System and Cardiovascular Disease: the Lesson from Centenarians

Recent discoveries have shed light on the participation of the immune system in the physio pathology of the cardiovascular system underpinning the importance of keeping the balance of the first to preserve the latter. Aging, along with other risk factors, can challenge such balance triggering the onset of cardiovascular diseases. Among several mediators ensuring the proper cross-talk between the two systems, bactericidal/permeability-increasing fold-containing family B member 4 (BPIFB4) has been shown to have a pivotal role, also by sustaining important signals such as eNOS and PKC-alpha. In addition, the Longevity-associated variant (LAV), which is an haplotype allele in BPIFB4characterized by 4 missense polymorphisms, enriched in homozygosity in Long Living Individuals (LLIs), has been shown to be efficient, if administered systemically through gene therapy, in improving many aspects of cardiovascular diseases (CVDs). This occurs mainly through a fine immune system remodeling across: 1) a M2 macrophage polarizing effect, 2) a favorable redistribution of the circulating monocyte cell subsets and 3) the reduction of T-cell activation. Furthermore, LAV-BPIFB4 treatment induced a desirable recovery of the inflammatory balance by mitigating the pro-inflammatory factor levels and enhancing the anti-inflammatory boost through a mechanism that is partially dependent on SDF-1/CXCR4 axis. Importantly, the remarkable effects of LAV-BPIFB4 treatment, which translates in increased BPIFB4 circulating levels, mirror what occurs in long-living individuals (LLIs) in whom the high circulating levels of BPIFB4 are protective from age-related and CVDs and emphasize the reason why LLIs are considered a model of successful aging. Here, we review the mechanisms by which LAV-BPIFB4 exerts its immunomodulatory activity in improving the cardiovascular-immune system dialogue that might strengthen its role as a key mediator in CVDs.

The Longevity-Associated Variant of BPIFB4 Reduces Senescence in Glioma Cells and in Patients' Lymphocytes Favoring Chemotherapy Efficacy.

Glioblastoma (GBM) is the most common primary brain cancer with the median age at diagnosis around 64 years, thus pointing to aging as an important risk factor. Indeed, aging, by increasing the senescence burden, is configured as a negative prognostic factor for GBM stage. Furthermore, several anti-GBM therapies exist, such as temozolomide (TMZ) and etoposide (ETP), that unfortunately trigger senescence and the secretion of proinflammatory senescence-associated secretory phenotype (SASP) factors that are responsible for the improper burst of (i) tumorigenesis, (ii) cancer metastasis, (iii) immunosuppression, and (iv) tissue dysfunction. Thus, adjuvant therapies that limit senescence are urgently needed. The longevity-associated variant (LAV) of the bactericidal/permeability-increasing fold-containing family B member 4 (BPIFB4) gene previously demonstrated a modulatory activity in restoring age-related immune dysfunction and in balancing the low-grade inflammatory status of elderly people. Based on the above findings, we tested LAV-BPIFB4 senotherapeutic effects on senescent glioblastoma U87-MG cells and on T cells from GBM patients. We interrogated SA-β-gal and HLA-E senescence markers, SASP factors, and proliferation and apoptosis assays. The results highlighted a LAV-BPIFB4 remodeling of the senescent phenotype of GBM cells, enhancement of their sensitivity to temozolomide and a selective reduction of the T cells’ senescence from GBM patients. Overall, these findings candidate LAV-BPIFB4 as an adjuvant therapy for GBM.

Transfer of the longevity-associated variant of BPIFB4 gene rejuvenates immune system and vasculature by a reduction of CD38+ macrophages and NAD+ decline

As we age, our body experiences chronic, systemic inflammation contributing to the morbidity and mortality of the elderly. The senescent immune system has been described to have a causal role in driving systemic aging and therefore may represent a key therapeutic target to prevent pathological consequences associated with aging and extend a healthy lifespan. 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 halted the progression of cardiovascular diseases (CVDs) and frailty by counterbalancing chronic inflammation. In the present study, we aimed to delineate the action of systemic adeno-associated viral vector-mediated LAV-BPIFB4 gene transfer (AAV-LAV-BPIFB4) on the deleterious age-related changes of the immune system and thereby the senescence-associated events occurring in C57BL/6J mice aged 26 months. Our in vivo data showed that 26-months-old mice had a higher frequency of CD45+SA-beta Gal+ immune cells in peripheral blood than young (4-months-old) C57BL/6J mice. Notably, AAV-LAV-BPIFB4 gene transfer in aged mice reduced the pool of peripheral immunosenescent cells that were shown to be enriched in the spleen. In addition, the proper tuning of the immune secretory phenotype (IL1βlow, IL6low, IL10high) associated with a significant reduction in SA-beta Gal-positive area of aorta from AAV-LAV treated mice. At the functional level, the reduction of senescence-associated inflammation ensured sustained NAD+ levels in the plasma of AAV-LAV-BPIFB4 old mice by preventing the NADase CD38 increase in F4/80+ tissue-resident macrophages and Ly6Chigh pro-inflammatory monocytes of the spleen and bone marrow. Finally, to validate the clinical implication of our findings, we showed that Long-living-individuals (LLIs, >95 years), which delay CVDs onset, especially if LAV-carriers, were characterized by high NAD+ levels. In conclusion, the new senotherapeutic action of LAV-BPIFB4 may offer a valuable therapeutic tool to control aging and reduce the burden of its pathophysiological disorders, such as CVDs.

Analysis of Cell and Molecular Phenotypes of a Highly Replicated Longevity-Associated FOXO3 Variant in Older Adults

Aging demographics in the US, and other industrialized nations, are resulting in rapidly increasing health care costs from age-related diseases. New therapeutic interventions to extend healthspan in older adults requires understanding connections between basic aging biology and human longevity factors. Using clinical samples from the Kuakini Honolulu Heart Program (HHP) and their Offspring, we are examining potential links between molecular and cellular mechanisms of aging and the longevity associated FOXO3 genotype (carriage of SNP rs2802292 “G” allele). Telomere dynamics in leucocytes (LTL) have shown strong correlation with multiple lifestyle and health factors. We previously demonstrated a significant protective relation between FOXO3 longevity genotype and LTL in a cross-sectional study. Now we are assessing a longitudinal relation, at three time points over 20+ years, in older men. We are also exploring stem cell frequency and differentiation capacity in neurological and peripheral blood samples to assess FOXO3 genotype and human cell dynamics.

BPIFB4 Circulating Levels and Its Prognostic Relevance in COVID-19.

Aging and comorbidities make individuals at greatest risk of COVID-19 serious illness and mortality due to senescence-related events and deleterious inflammation. Long-living individuals (LLIs) are less susceptible to inflammation and develop more resiliency to COVID-19. As demonstrated, LLIs are characterized by high circulating levels of BPIFB4, a protein involved in homeostatic response to inflammatory stimuli. Also, LLIs show enrichment of homozygous genotype for the minor alleles of a 4 missense single-nucleotide polymorphism haplotype (longevity-associated variant [LAV]) in BPIFB4, able to counteract progression of diseases in animal models. Thus, the present study was designed to assess the presence and significance of BPIFB4 level in COVID-19 patients and the potential therapeutic use of LAV-BPIFB4 in fighting COVID-19. BPIFB4 plasma concentration was found significantly higher in LLIs compared to old healthy controls while it significantly decreased in 64 COVID-19 patients. Further, the drop in BPIFB4 values correlated with disease severity. Accordingly to the LAV-BPIFB4 immunomodulatory role, while lysates of SARS-CoV-2-infected cells induced an inflammatory response in healthy peripheral blood mononuclear cells in vitro, the co-treatment with recombinant protein (rh) LAV-BPIFB4 resulted in a protective and self-limiting reaction, culminating in the downregulation of CD69 activating-marker for T cells (both TCD4+ and TCD8+) and in MCP-1 reduction. On the contrary, rhLAV-BPIFB4 induced a rapid increase in IL-18 and IL-1b levels, shown largely protective during the early stages of the virus infection. This evidence, along with the ability of rhLAV-BPIFB4 to counteract the cytotoxicity induced by SARS-CoV-2 lysate in selected target cell lines, corroborates BPIFB4 prognostic value and open new therapeutic possibilities in more vulnerable people.

The Role of BPIFB4 in Immune System and Cardiovascular Disease: The Lesson from Centenarians.

Recent discoveries have shed light on the participation of the immune system in the physio pathology of the cardiovascular system underpinning the importance of keeping the balance of the first to preserve the latter. Aging, along with other risk factors, can challenge such balance triggering the onset of cardiovascular diseases. Among several mediators ensuring the proper cross-talk between the two systems, bactericidal/permeability-increasing fold-containing family B member 4 (BPIFB4) has been shown to have a pivotal role, also by sustaining important signals such as eNOS and PKC-alpha. In addition, the Longevity-associated variant (LAV), which is an haplotype allele in BPIFB4 characterized by 4 missense polymorphisms, enriched in homozygosity in Long Living Individuals (LLIs), has been shown to be efficient, if administered systemically through gene therapy, in improving many aspects of cardiovascular diseases (CVDs). This occurs mainly through a fine immune system remodeling across: 1) a M2 macrophage polarizing effect, 2) a favorable redistribution of the circulating monocyte cell subsets and 3) the reduction of T-cell activation. Furthermore, LAV-BPIFB4 treatment induced a desirable recovery of the inflammatory balance by mitigating the pro-inflammatory factor levels and enhancing the anti-inflammatory boost through a mechanism that is partially dependent on SDF-1/CXCR4 axis. Importantly, the remarkable effects of LAV-BPIFB4 treatment, which translates in increased BPIFB4 circulating levels, mirror what occurs in long-living individuals (LLIs) in whom the high circulating levels of BPIFB4 are protective from age-related and CVDs and emphasize the reason why LLIs are considered a model of successful aging. Here, we review the mechanisms by which LAV-BPIFB4 exerts its immunomodulatory activity in improving the cardiovascular-immune system dialogue that might strengthen its role as a key mediator in CVDs.

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.

September 2020 at a glance: focus on heart failure with preserved ejection fraction and medical therapy.

Type 2 diabetes mellitus and obesity increase heart failure (HF) risk.1-4 Dang et al.5 showed that delivery of longevity-associated variant of the human BPIFB4 gene in obese mice with diabetes improves cardiac function. Usefulness of biomarker measurements may be lower in patients with HF and preserved ejection fraction (HFpEF).6, 7 Henkens et al.8 performed a systematic review of studies investigating the diagnostic value of novel biomarkers for HFpEF. The risk of bias was high in all studies. The main limitations were use of case-control designs, poor diagnosis of HFpEF, absence of pre-specified cut-off points, lack of external validation, inadequate and variable reference standards. An analysis of TOPCAT Americas showed that spironolactone treatment was associated with a reduction in loop diuretic and thiazide doses. The reduction in the risk of HF hospitalizations with spironolactone (hazard ratio 0.77, 95% confidence interval 0.62–0.96; P = 0.021) was, however, independent of multiple variables, including loop diuretic dose and serum creatinine, thus showing it was not mediated by its diuretic effects.9 Elderly age, renal dysfunction and hyperkalaemia are major causes of spironolactone discontinuation.10 An analysis of TOPCAT Americas confirmed these data and also showed that these patients received lower spironolactone, but not placebo, doses. Spironolactone reduced the primary endpoint also in these subgroups while its withdrawal was associated with a two to four-fold higher risk of events. Thus, a low-dose strategy should be preferred instead of discontinuation.11 Larsen et al.12 showed that metformin can improve myocardial efficiency in insulin-resistant patients with HFrEF without diabetes. Sacubitril/valsartan improves outcome in HFrEF.13 An analysis of PARADIGM-HF showed that liver dysfunction is an independent predictor of outcomes in HFrEF patients and sacubitril/valsartan improved liver function, compared with enalapril.14 Oxidative stress and inflammation have a key role in HF.15 Vericiguat treatment was associated with reduction in inflammatory- and oxidative stress-related markers, such as high-sensitivity C-reactive protein and uric acid, and this may have a role for its beneficial effects.16 Inotropic drugs did not improve survival in HF.17, 18 Better results can be expected from drugs not acting through an increase in intracellular calcium. Danicamtiv, a novel cardiac myosin activator, improved stroke volume, global longitudinal and circumferential strain and left atrial function in experimental models and in a phase 2a trial in patients with HFrEF.19 In patients hospitalized for acute HF, istaroxime had beneficial effects on left ventricular function, without hypotension, tachycardia, or serious cardiac adverse events.20 Lung congestion is a leading cause of dyspnoea and exercise intolerance.21 Transient receptor potential vanilloid 4 (TRPV4) channel regulates fluid exchange in the lungs. A novel transient TRPV4 antagonist (GSK2798745) was tested in a pilot randomized, placebo-controlled trial aimed at the reduction of lung water content. The new drug was well tolerated and showed a trend towards improved gas transfer.22

No Association Between a Genetic Variant of FOXO3 and Risk of Type 2 Diabetes Mellitus in the Elderly Population of North India.

Aging can be considered an evolutionary process that is modulated by various genetic and biochemical processes. Therefore the genetic variants may interplay a role in human longevity as well as age related illness. Forkhead Box O (FOXO) gene is one of the major defensive genes that are known for ameliorating lifespan. FOXO proteins act as nuclear transcription factors that facilitate the action of insulin or insulin-like growth factor (IGF-1) in various physiological processes. The rationale of our study is to find out association between genetic variant rs2253310 of FOXO3 and risk of Type 2 Diabetes Mellitus (T2DM) in elderly population. This case control study involved 172 age sex matched elderly subjects while patients were recruited as per IDF criteria. Clinical, biochemical, ELISA methods were employed for assesement of clinical samples while Taqman method was used for genotyping analysis. Our results revealed that there was no significant difference in genotypic and allelic frequencies for the tested SNP (p > 0.05) between elderly T2DM patients and controls. The SNP rs2253310 was not associated with risk of T2DM in any genetic model. Also no association was found among the studied group between FOXO3 variant and HOMA-IR, HOMA-B index and Fasting plasma glucose. Serum level of inflammatory markers like CRP and TNF-α was significantly higher in patients but its not associated with SNP rs2253310. Our study concluded that, this intronic longevity-associated variant rs2253310 in FOXO3 is not associated with type 2 diabetes in geriatric patients of northern India.

The longevity-associated variant of BPIFB4 improves a CXCR4-mediated striatum\u2013microglia crosstalk preventing disease progression in a mouse model of Huntington\u2019s disease

The longevity-associated variant (LAV) of the bactericidal/permeability-increasing fold-containing family B member 4 (BPIFB4) has been found significantly enriched in long-living individuals. Neuroinflammation is a key player in Huntington’s disease (HD), a neurodegenerative disorder caused by neural death due to expanded CAG repeats encoding a long polyglutamine tract in the huntingtin protein (Htt). Herein, we showed that striatal-derived cell lines with expanded Htt (STHdh Q111/111) expressed and secreted lower levels of BPIFB4, when compared with Htt expressing cells (STHdh Q7/7), which correlated with a defective stress response to proteasome inhibition. Overexpression of LAV-BPIFB4 in STHdh Q111/111 cells was able to rescue both the BPIFB4 secretory profile and the proliferative/survival response. According to a well-established immunomodulatory role of LAV-BPIFB4, conditioned media from LAV-BPIFB4-overexpressing STHdh Q111/111 cells were able to educate Immortalized Human Microglia—SV40 microglial cells. While STHdh Q111/111 dying cells were ineffective to induce a CD163 + IL-10high pro-resolving microglia compared to normal STHdh Q7/7, LAV-BPIFB4 transduction promptly restored the central immune control through a mechanism involving the stromal cell-derived factor-1. In line with the in vitro results, adeno-associated viral-mediated administration of LAV-BPIFB4 exerted a CXCR4-dependent neuroprotective action in vivo in the R6/2 HD mouse model by preventing important hallmarks of the disease including motor dysfunction, body weight loss, and mutant huntingtin protein aggregation. In this view, LAV-BPIFB4, due to its pleiotropic ability in both immune compartment and cellular homeostasis, may represent a candidate for developing new treatment for HD.

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.

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.

AimsHomozygosity 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 resultsCompared 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.ConclusionsIn 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.

P746Protective role of the longevity associated variant of BPIFB4 in chronic ischemia

Abstract Background A rare Longevity Associated Variant (LAV) of the gene BPIFB4, whose protein product can be secreted, is recessively associated with extreme longevity, positively impacting the cardiovascular system and promoting therapeutic angiogenesis in an hindlimb ischemia model. Aim Aim of this work is to verify if LAV-BPIFB4 may temper the phenotype of chronic ischemia in humans, thus suggesting its therapeutic potential. Methods and results 39 hearts explanted from patients affected by end-stage ischemic heart disease were studied. Of these, 7 were homozygous for BPIFB4-LAV. Homozygous and non-homozygous patients did not differ in sex, age and in the prevalence of comorbidities or risk factors. However, the time elapsed from myocardial infarction to transplantation was significantly longer for LAV homozygous with respect to LAV heterozygous patients (159±95 vs 58±27 months, p=0.045). Histologically, while BPIFB4 levels did not differ in accordance with the genotype, the fraction of Tunel+and 53BP1+apoptotic and senescent myocytes was significantly lower in homozygous patients (1.2±0.4% vs 23.8±21.7%, p=0.026 and 31.5±6.7% vs 44.4±6.1%, p=0.031, respectively). Significantly lower levels of lipoperoxides and higher levels of Parkin were observed in homozygous hearts too. Next, we directly tested the role of BPIFB4 on PDGFRb+ NG2+Tbx18+cardiac pericytes/mural cells (PM) cultured from normal atria (PMnorm, n=10) and from ischemic failing hearts (PMfail, n=7). BPIFB4 gene transcript was significantly more expressed in PMnorm than PMfail. Silencing BPIFB4 expression in PMnorm significantly increased the proportion of senescent gH2AX+Ki67-cells (3.6±2.9% vs 13.6±9.5%, p=0.049). Conversely, addition of recombinant LAV BPIFB4 protein to PMfail cultures significantly reduced the rate of senescent cells (21.1±9.4% vs 6.0±4.8%, p=0.03), an effect that was not observed with the administration of WT BPIFB4. PMfail cultures exposed to LAV BPIFB4 significantly reversed alterations observed with pathology, such as the accumulation of both lipofuscins and of very elongated and interconnected mitochondria in the cell cytoplasm, as well as elevated mitochondrial superoxide anion levels. Finally, a larger fraction of PMnorm cells showed, with respect to PMfail, the nuclear translocation of vitamin D receptor (74.6±6.1% vs 50.1±14.4, p=0.04), coupled with a significant upregulation of its, longevity associated, target gene Klotho. Importantly, LAV BPIFB4 significantly increased the fraction of PMfail with vitamin D receptor nuclear localization (55.8±17.6% vs 85.1±8.2%, p=0.005). Conclusion LAV BPIFB4 could attenuate the progression of ischemic heart disease to heart failure. Part of its protective effect may be due to its ability to reduce mitochondrial oxidative stress and to revert the senescent phenotype of cardiac derived cells. Acknowledgement/Funding CARIPLO foundation