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Purpose: Cellular senescence associated with ageing and disease hampers the functional properties of human c-Kit+ Cardiac Stem Cells (CSC) in vitro. Aim of the study was to identify molecular mechanisms associated with CSC senescence and to pharmacologically modulate this latter, at the aim to improve the therapeutic potential of CSC in vivo.Methods: c-Kit+ CSC were isolated from atrial specimens of end-stage failing hearts (E-CSC, n=20) and hearts donated for transplantation (D-CSC, n=14). D- and E-CSC were compared in vitro for the expression of senescence markers and secretome. The ability of CSC's secretome to protect rat adult cardiomyocytes (CM) in vitro from a simulated ischemia-reperfusion injury was analysed. 3 molecular signalling pathways associated with cell senescence were investigated. A 3-days pharmacologic treatment with a cocktail of 10nM Rapamycin and 0.5μM Resveratrol was employed to revert E-CSC senescence in vitro. The functional recovery of SCID-Beige mice infarcted hearts injected with Vehicle (n=17), D-CSC (n=17), E-CSC (n=18) or drug-conditioned-E-CSC (n=18) (300,000 cells/heart) was compared 14 days post-transplantation.Results: with respect to D-CSC, E-CSC show, in vitro, higher levels of senescence markers (p16, γH2AX), a blunted proliferation and a secretome strongly enriched in the pro-inflammatory IL-1β, unable to protect rat CM from apoptosis and senescence in vitro. Neutralisation of IL-1β restored E-CSC's secretome protective effect. At the molecular level, E-CSC are characterized by an hyper-activation of the canonical NFB pathway and of Caspase1, an increased activity of the TORC1 complex, an impairment of the authophagic flux and a reduction of AMPK, Akt and CREB activation. All these alterations could be successfully reverted employing a cocktail of Rapamycin and Resveratrol. The drug treatment reduced the fraction of senescent E-CSC and the amount of IL-1β secreted in vitro, thus restoring the protective effect on rat CM. Last, D-CSC but not E-CSC were able to enhance myocardial healing when injected in a mouse infarcted heart. However, the ex vivo preconditioning of E-CSC with Rapamycin and Resveratrol, prior to the in vivo transplantation, restored their reparative potential to D-CSC levels.Conclusions: we first demonstrate that senescent c-Kit+ CSC resident in human failing hearts display an impaired in vivo reparative ability; importantly, senescent CSC can be rejuvenated in vitro with a short pharmacologic conditioning, finally boosting the in vivo cardiac regeneration. These findings open new avenues to improve autologous CSC therapy in heart failure. [ABSTRACT FROM PUBLISHER]

Purpose: to compare the regenerative potential of Saphenous Vein-derived Pericytes (SVPs) with that of c-Kit-pos Cardiac Progenitor Cells (CPCs) in a mouse model of myocardial infarction (MI), and to investigate if the simultaneus transplantation of the cells produces additive improvements.Methods: CPCs were isolated from discarded atrial specimens of transplanted hearts; SVPs were immune-sorted from vein leftovers of CABG patients. Cells were compared in vitro according to their surface-phenotype and differentiation ability toward the 3 cardiac lineages. The functional recovery of SCID-Beige mice infarcted hearts injected with Vehicle (n=6), CPCs (n=6), SVPs (n=6) or CPCs+SVPs (n=6) (300,000 cells of each type/heart) was compared, both 14 and 42 days post-MI. Interaction between SVPs and CPCs, secretomes, and paracrine effects were investigated in vitro.Results: both SVPs and CPCs express mesenchymal markers (CD44/CD90/CD105), are negative for endothelial and hematopoietic antigens and express the stemness markers Sox2. In addition, SVPs express the pericyte markers NG2 and PDGFRb. Both cell types secrete similar paracrine factors (HGF, VEGF, FGF, SCF), supplemented by Ang-1 and -2 regarding SVPs. Importantly, co-culture of cells for 48h increased the secretion of SDF-1. CPCs differentiate in endothelial and vascular muscle cells, while both cell types acquire cardiomyocyte markers when exposed for 21 days to an inductive culture medium. Cell transplantation similarly improved volume, contractility and pressure indexes at 14 and 42 days post-MI compared to vehicle, with no additive effect by combined therapy. Similarly, myocardial healing was stimulated by both the cell types, protecting cardiomyocytes from apoptosis and increasing the density of endogenous CPCs; the combined therapy did not add further improvements.When exposed to SVP conditioned media, CPCs tend to increase migration compared to plain media. This response is not abrogated or diminished by RNAase or tyrosine and serine/threonine kinase receptor inhibitors, or a blocking antibodies cocktail (HGF, FGF, VEGF, SCF, and Tie2). Proteinase K pretreatment was able to abrogate the response, differently from the heating denaturation. Fractioning SVP conditioned medium by molecular weight showed that secreted chemoattractant factor is >10 kDa.Conclusions: SVPs are therapeutically equipotent to CPCs. The more accessible source makes SVPs an optimal alternative to CPCs in cell therapy for myocardial repair. Moreover, the attractant effect on CPCs in the site of injury favours the endogenous cardiac regeneration. [ABSTRACT FROM PUBLISHER]

Aims: 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., Methods and Results: 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., Conclusions: 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., Competing Interests: Conflict of interest: A.A.P. and C.V. own shares of LGV1 Inc. and have filed a patent. All the other authors declare that there is no conflict of interest., (© The Author(s) 2023. Published by Oxford University Press on behalf of the European Society of Cardiology.)

Aims: Methylation of non-histone proteins is emerging as a central regulatory mechanism in health and disease. The methyltransferase SETD7 has shown to methylate and alter the function of a variety of proteins in vitro; however, its function in the heart is poorly understood. The present study investigates the role of SETD7 in myocardial ischaemic injury., Methods and Results: Experiments were performed in neonatal rat ventricular myocytes (NRVMs), SETD7 knockout mice (SETD7-/-) undergoing myocardial ischaemia/reperfusion (I/R) injury, left ventricular (LV) myocardial samples from patients with ischaemic cardiomyopathy (ICM), and peripheral blood mononuclear cells (PBMCs) from patients with ST-elevation MI (STEMI). We show that SETD7 is activated upon energy deprivation in cultured NRVMs and methylates the Hippo pathway effector YAP, leading to its cytosolic retention and impaired transcription of antioxidant genes manganese superoxide dismutase (MnSOD) and catalase (CAT). Such impairment of antioxidant defence was associated with mitochondrial reactive oxygen species (mtROS), organelle swelling, and apoptosis. Selective pharmacological inhibition of SETD7 by (R)-PFI-2 restored YAP nuclear localization, thus preventing mtROS, mitochondrial damage, and apoptosis in NRVMs. In mice, genetic deletion of SETD7 attenuated myocardial I/R injury, mtROS, and LV dysfunction by restoring YAP-dependent transcription of MnSOD and CAT. Moreover, in cardiomyocytes isolated from I/R mice and ICM patients, (R)-PFI-2 prevented mtROS accumulation, while improving Ca2+-activated tension. Finally, SETD7 was up-regulated in PBMCs from STEMI patients and negatively correlated with MnSOD and CAT., Conclusion: We show a methylation-dependent checkpoint regulating oxidative stress during myocardial ischaemia. SETD7 inhibition may represent a valid therapeutic strategy in this setting., Competing Interests: Conflict of interest: None declared., (© The Author(s) 2022. Published by Oxford University Press on behalf of the European Society of Cardiology. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Aims: Diabetes impinges upon mechanisms of cardiovascular repair. However, the biochemical adaptation of cardiac stem cells to sustained hyperglycaemia remains largely unknown. Here, we investigate the molecular targets of high glucose-induced damage in cardiac progenitor cells (CPCs) from murine and human hearts and attempt safeguarding CPC viability and function through reactivation of the pentose phosphate pathway., Methods and Results: Type-1 diabetes was induced by streptozotocin. CPC abundance was determined by flow cytometry. Proliferating CPCs were identified in situ by immunostaining for the proliferation marker Ki67. Diabetic hearts showed marked reduction in CPC abundance and proliferation when compared with controls. Moreover, Sca-1(pos) CPCs isolated from hearts of diabetic mice displayed reduced activity of key enzymes of the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD), and transketolase, increased levels of superoxide and advanced glucose end-products (AGE), and inhibition of the Akt/Pim-1/Bcl-2 signalling pathway. Similarly, culture of murine CPCs or human CD105(pos) progenitor cells in high glucose inhibits the pentose phosphate and pro-survival signalling pathways, leading to the activation of apoptosis. In vivo and in vitro supplementation with benfotiamine reactivates the pentose phosphate pathway and rescues CPC availability and function. This benefit is abrogated by either G6PD silencing by small interfering RNA (siRNA) or Akt inhibition by dominant-negative Akt., Conclusion: We provide new evidence of the negative impact of diabetes and high glucose on mechanisms controlling CPC redox state and survival. Boosting the pentose phosphate pathway might represent a novel mechanistic target for protection of CPC integrity.