Background The heart rejuvenating effects of circulating growth differentiation factor 11 (GDF11)... more Background The heart rejuvenating effects of circulating growth differentiation factor 11 (GDF11), a TGF-β superfamily member that shares 90% homology with myostatin (MSTN), remains controversial. Here, we aimed to probe the role of GDF11 in acute myocardial infarction (MI), a frequent cause of heart failure and premature death during ageing. Methods Young (3-4 months) and old (22-24 months) C57Bl/6 mice were subjected to either systemic recombinant GDF11 (rGDF11; 0.1 mg/kg BW daily) or control treatment over 30 days before myocardial ischaemia-reperfusion (I/R) injury was induced. Dissected hearts underwent in-depth profiling that ranged from histological workup and the assessment of inflammatory pathways to cardiomyocyte viability testing and IPA-guided -omics. Key regulatory mechanisms were studied in vitro using human adult stromal cardiac-specific mesenchymal cells (cMSCs) and HL-1 cardiomyocytes. By harnessing liquid chromatography–tandem mass spectrometry (LC-MS/MS), plasma l...
Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) constitute a mixed popula... more Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) constitute a mixed population of ventricular-, atrial-, nodal-like cells, limiting the reliability for studying chamber-specific disease mechanisms. Previous studies characterised CM phenotype based on action potential (AP) morphology, but the classification criteria were still undefined. Our aim was to use in silico models to develop an automated approach for discriminating the electrophysiological differences between hiPSC-CM. We propose the dynamic clamp (DC) technique with the injection of a specific IK1 current as a tool for deriving nine electrical biomarkers and blindly classifying differentiated CM. An unsupervised learning algorithm was applied to discriminate CM phenotypes and principal component analysis was used to visualise cell clustering. Pharmacological validation was performed by specific ion channel blocker and receptor agonist. The proposed approach improves the translational relevance of the ...
Cardiomyocyte renewal represents an unmet clinical need for cardiac regeneration. Stem cell parac... more Cardiomyocyte renewal represents an unmet clinical need for cardiac regeneration. Stem cell paracrine therapy has attracted increasing attention to resurge rescue mechanisms within the heart. We previously characterized the paracrine effects that human amniotic fluid–derived stem cells (hAFSC) can exert to provide cardioprotection and enhance cardiac repair in preclinical models of myocardial ischemia and cardiotoxicity. Here, we analyze whether hAFSC secretome formulations, namely, hAFSC conditioned medium (hAFSC-CM) over extracellular vesicles (hAFSC-EVs) separated from it, can induce cardiomyocyte renewal. c-KIT+ hAFSC were obtained by leftover samples of II trimester prenatal amniocentesis (fetal hAFSC) and from clinical waste III trimester amniotic fluid during scheduled C-section procedures (perinatal hAFSC). hAFSC were primed under 1% O2 to enrich hAFSC-CM and EVs with cardioactive factors. Neonatal mouse ventricular cardiomyocytes (mNVCM) were isolated from cardiac tissue of...
Inflammatory response following SARS-CoV-2 infection results in substantial increase of amounts o... more Inflammatory response following SARS-CoV-2 infection results in substantial increase of amounts of intravascular pro-coagulant extracellular vesicles (EVs) expressing tissue factor (CD142) on their surface. CD142-EV turned out to be useful as diagnostic biomarker in COVID-19 patients. Here we aimed at studying the prognostic capacity of CD142-EV in SARS-CoV-2 infection. Expression of CD142-EV was evaluated in 261 subjects admitted to hospital for pneumonia and with a positive molecular test for SARS-CoV-2. The study population consisted of a discovery cohort of selected patients (n = 60) and an independent validation cohort including unselected consecutive enrolled patients (n = 201). CD142-EV levels were correlated with post-hospitalization course of the disease and compared to the clinically available 4C Mortality Score as referral. CD142-EV showed a reliable performance to predict patient prognosis in the discovery cohort (AUC = 0.906) with an accuracy of 81.7%, that was confirmed in the validation cohort (AUC = 0.736). Kaplan-Meier curves highlighted a high discrimination power in unselected subjects with CD142-EV being able to stratify the majority of patients according to their prognosis. We obtained a comparable accuracy, being not inferior in terms of prediction of patients' prognosis and risk of mortality, with 4C Mortality Score. The expression of surface vesicular CD142 and its reliability as prognostic marker was technically validated using different immunocapture strategies and assays. The detection of CD142 on EV surface gains considerable interest as risk stratification tool to support clinical decision making in COVID-19.
Background: Inflammatory response following SARS-CoV-2 infection results in substantial increase ... more Background: Inflammatory response following SARS-CoV-2 infection results in substantial increase of amounts of intravascular pro-coagulant extracellular vesicles (EV) expressing the tissue factor (CD142) on their surface. CD142-EV not only can act as clotting initiation agent, contributing to disease severity, but it turned out to be useful as diagnostic biomarker in COVID-19 patients. Here we aimed at studying the prognostic capacity of CD142-EV in SARS-CoV-2 infection.Methods: Level of expression of CD142-EV was evaluated in 261 subjects admitted to hospital for pneumonia and with a positive molecular test for SARS-CoV-2. The study population consisted of a discovery cohort of selected patients (n=60) and an independent validation cohort including unselected consecutive enrolled patients (n=201). CD142-EV biomarker performances were correlated with post-hospitalization course of the disease and compared to the clinically available 4C Mortality Score as referral.Findings: CD142-EV showed a reliable performance to predict patient prognosis in the discovery cohort (AUC=0.906) with an accuracy of 81.7%, that was confirmed in the validation cohort (AUC=0.736). Kaplan-Meier curves further highlighted a high discrimination power in unselected subjects with CD142-EV being able to stratify the majority of patients according to their prognosis. We obtained a comparable accuracy, being not inferior in terms of prediction of patients’ prognosis and risk of mortality with 4C Mortality Score.Interpretation: The detection of CD142 on the surface of EV is a cost-effective and rapid test that can be available at time of admission by using conventional flow cytometer. Such analysis gains considerable interest as risk stratification tool to support clinical decision making in COVID-19.Declaration of Interest: None to declare. Ethical Approval: The study was approved by the Ethical Committee of the Ticino Canton, Switzerland (2020-00795, 96 Rif CE TI 3616).
Second trimester foetal human amniotic fluid‐derived stem cells (hAFS) have been shown to possess... more Second trimester foetal human amniotic fluid‐derived stem cells (hAFS) have been shown to possess remarkable cardioprotective paracrine potential in different preclinical models of myocardial injury and drug‐induced cardiotoxicity. The hAFS secretome, namely the total soluble factors released by cells in their conditioned medium (hAFS‐CM), can also strongly sustain in vivo angiogenesis in a murine model of acute myocardial infarction (MI) and stimulates human endothelial colony‐forming cells (ECFCs), the only truly recognized endothelial progenitor, to form capillary‐like structures in vitro. Preliminary work demonstrated that the hypoxic hAFS secretome (hAFS‐CMHypo) triggers intracellular Ca2+ oscillations in human ECFCs, but the underlying mechanisms and the downstream Ca2+‐dependent effectors remain elusive. Herein, we found that the secretome obtained by hAFS undergoing hypoxic preconditioning induced intracellular Ca2+ oscillations by promoting extracellular Ca2+ entry through Transient Receptor Potential Vanilloid 4 (TRPV4). TRPV4‐mediated Ca2+ entry, in turn, promoted the concerted interplay between inositol‐1,4,5‐trisphosphate‐ and nicotinic acid adenine dinucleotide phosphate‐induced endogenous Ca2+ release and store‐operated Ca2+ entry (SOCE). hAFS‐CMHypo‐induced intracellular Ca2+ oscillations resulted in the nuclear translocation of the Ca2+‐sensitive transcription factor p65 NF‐κB. Finally, inhibition of either intracellular Ca2+ oscillations or NF‐κB activity prevented hAFS‐CMHypo‐induced ECFC tube formation. These data shed novel light on the molecular mechanisms whereby hAFS‐CMHypo induces angiogenesis, thus providing useful insights for future therapeutic strategies against ischaemic‐related myocardial injury.
Although a small number of cardiomyocytes may reenter the cell cycle after injury, the adult mamm... more Although a small number of cardiomyocytes may reenter the cell cycle after injury, the adult mammalian heart is incapable of a robust cardiomyocyte proliferation. Periostin, a secreted extracellular matrix protein, has been implicated as a regulator of cardiomyocyte proliferation; however, this role remains controversial. Alternative splicing of the human periostin gene results in 6 isoforms lacking sequences between exons 17 and 21, in addition to full-length periostin. We previously showed that exosomes (Exo) secreted by human cardiac explant-derived progenitor cells (CPC) carried periostin. Here, we aimed to investigate their cell cycle activity. Methods: CPC were derived as the cellular outgrowth of ex vivo cultured cardiac atrial explants. Exo were purified from CPC conditioned medium using size exclusion chromatography. Exosomal periostin was analyzed by Western blotting using a pair of antibodies (one raised against aa 537-836, and one raised against amino acids mapping at exon 17 of human periostin), by ELISA, and by cryo-EM with immune-gold labeling. Cell cycle activity was assessed in neonatal rat cardiomyocytes, in human induced pluripotent stem cell (iPS)-derived cardiomyocytes, and in adult rat cardiomyocytes after myocardial infarction. The role of periostin in cell cycle activity was investigated by transfecting donor CPC with a siRNA against this protein. Results: Periostin expression in CPC-secreted exosomes was detected using the antibody raised against aa 537-836 of the human protein, but not with the exon 17-specific antibody, consistent with an isoform lacking exon 17. Periostin was visualized on vesicle surfaces by cryo-EM and immune-gold labeling. CPC-derived exosomes induced cell proliferation in neonatal rat cardiomyocytes both in vitro and in vivo, in human iPS-derived cardiomyocytes, and in adult rat cardiomyocytes after myocardial infarction. Exo promoted phosphorylation of focal adhesion kinase (FAK), actin polymerization, and nuclear translocation of Yes-associated protein (YAP) in cardiomyocytes. Knocking down of periostin or YAP, or blocking FAK phosphorylation with PF-573228 nullified Exo-induced proliferation. A truncated human periostin peptide (aa 22-669), but not recombinant human full-length periostin, mimicked the pro-proliferative activity of exosomes. Conclusions: Our results show, for the first time, that CPC-secreted exosomes promote cardiomyocyte cell cycle-reentry via a short periostin isoform expressed on their surfaces, whereas recombinant full-length periostin does not. These findings highlight isoform-specific roles of periostin in cardiomyocyte proliferation.
We previously reported that c-KIT+ human amniotic-fluid derived stem cells obtained from leftover... more We previously reported that c-KIT+ human amniotic-fluid derived stem cells obtained from leftover samples of routine II trimester prenatal diagnosis (fetal hAFS) are endowed with regenerative paracrine potential driving pro-survival, anti-fibrotic and proliferative effects. hAFS may also be isolated from III trimester clinical waste samples during scheduled C-sections (perinatal hAFS), thus offering a more easily accessible alternative when compared to fetal hAFS. Nonetheless, little is known about the paracrine profile of perinatal hAFS. Here we provide a detailed characterization of the hAFS total secretome (i.e., the entirety of soluble paracrine factors released by cells in the conditioned medium, hAFS-CM) and the extracellular vesicles (hAFS-EVs) within it, from II trimester fetal- versus III trimester perinatal cells. Fetal- and perinatal hAFS were characterized and subject to hypoxic preconditioning to enhance their paracrine potential. hAFS-CM and hAFS-EV formulations were a...
Abstract Cardiovascular disease is mainly related to inefficient cardioprotection, defective repa... more Abstract Cardiovascular disease is mainly related to inefficient cardioprotection, defective repair, and lack of myocardial renewal, following cardiac injury and/or aging. Recent work has demonstrated an endogenous restorative program within the heart based on cardiac progenitor cell activation and resident cardiomyocyte proliferation. Although these mechanisms are widely responsive in the neonatal life, they become dormant during adulthood, leaving the heart with limited repair potential in pathological situations. Therefore a working strategy is strongly needed to restore in full the potential for both cardiac repair and regeneration. The so-called “paracrine effect” has been proposed as a possible working strategy, with increasing interest toward the comprehensive analysis of the stem cell “secretome” as the whole of trophic molecules produced by cells via paracrine secretion. In this scenario, different sources of stem cells have been tested; among them, fetal stem cells such as amniotic fluid–derived stem cells may represent an appealing therapeutic option.
Mesenchymal stromal cells (MSCs) are characterized by a regulatory phenotype and respond promptly... more Mesenchymal stromal cells (MSCs) are characterized by a regulatory phenotype and respond promptly to the environmental signals modulating their secretory activity. An appropriate preconditioning may induce MSCs to release secretomes with an enhanced regenerative potential. However, it fails to take into account that secretomes are composed by both soluble factors and extracellular vesicles (EVs), whose functions could be altered differently by the preconditioning approach. Here we demonstrate that the MSC secretome is strongly modulated by the simultaneous stimulation with hypoxia and pro-inflammatory cytokines, used to mimic the harsh environment present at the site of injury. We observed that the environmental variations strongly influenced the angiogenic potential of the different secretome fractions. Upon inflammation, the pro-angiogenic capacity of the soluble component of the MSC secretome was strongly inhibited, regardless of the oxygen level, while the EV-encapsulated component was not significantly affected by the inflammatory stimuli. These effects were accompanied by the modulation of the secreted proteins. On one hand, inflammation-activated MSCs release proteins mainly involved in the interaction with innate immune cells and in tissue remodeling/repair; on the other hand, when MSCs are not exposed to an inflamed environment, they respond to the different oxygen levels modulating the expression of proteins involved in the angiogenic process. The cargo content (in terms of miRNAs) of the corresponding EV fractions was less sensitive to the influence of the external stimuli. Our findings suggest that the therapeutic efficacy of MSC-based therapies could be enhanced by selecting the appropriate preconditioning approach and carefully discriminating its effects on the different secretome components.
Background: Coronavirus-2 (SARS-CoV-2) infection causes an acute respiratory syndrome accompanied... more Background: Coronavirus-2 (SARS-CoV-2) infection causes an acute respiratory syndrome accompanied by multi-organ damage that implicates a prothrombotic state leading to widespread microvascular clots. The causes of such coagulation abnormalities are unknown. The receptor tissue factor, also known as CD142, is often associated with cell-released extracellular vesicles (EV). In this study, we aimed to characterize surface antigens profile of circulating EV in COVID-19 patients and their potential implication as procoagulant agents. Methods: We analyzed serum-derived EV from 67 participants who underwent nasopharyngeal swabs molecular test for suspected SARS-CoV-2 infection (34 positives and 33 negatives) and from 16 healthy controls (HC), as referral. A sub-analysis was performed on subjects who developed pneumonia (n = 28). Serumderived EV were characterized for their surface antigen profile and tested for their procoagulant activity. A validation experiment was performed pre-treating EV with anti-CD142 antibody or with recombinant FVIIa. Serum TNF-a levels were measured by ELISA. Findings: Profiling of EV antigens revealed a surface marker signature that defines circulating EV in COVID-19. A combination of seven surface molecules (CD49e, CD209, CD86, CD133/1, CD69, CD142, and CD20) clustered COVID (+) versus COVID (-) patients and HC. CD142 showed the highest discriminating performance at both multivariate models and ROC curve analysis. Noteworthy, we found that CD142 exposed onto surface of EV was biologically active. CD142 activity was higher in COVID (+) patients and correlated with TNF-a serum levels. Interpretation: In SARS-CoV-2 infection the systemic inflammatory response results in cell-release of substantial amounts of procoagulant EV that may act as clotting initiation agents, contributing to disease severity.
Cystic fibrosis (CF) is a genetic disease characterized by progressive decline of lung function a... more Cystic fibrosis (CF) is a genetic disease characterized by progressive decline of lung function and chronic airway inflammation. Adiponectin and leptin exert several effects on energy metabolism and inflammatory processes. This study evaluated the levels of adiponectin and leptin in adult healthy subjects, in patients with CF and their correlation with long-term physical activity. CF patients were divided into two groups (sedentary versus active) based on their regular physical activity over 3 years. Anthropometric and serum biochemical profiles of CF patients and controls were evaluated and compared. Total serum adiponectin and leptin levels were measured by ELISA; adiponectin oligomeric profiles were analysed by western blot. Adiponectin levels were significantly higher while leptin levels were lower in patients with CF than in healthy controls. Furthermore, adiponectin was significantly lower in active compared to sedentary CF, (p = 0.047). leptin was slightly increased in active compared to sedentary CF. In addition, C-reactive protein levels were significantly lower in active than in sedentary CF patients, (p = 0.048). Interestingly, only in the active group adiponectin levels were inversely correlated with forced expiratory volume (FEV)1% decrease/year and FEV1% decrease. Moreover, adiponectin levels negatively correlated with lipid profiles. Our findings indicated that regular, long-term physical activity in CF improves respiratory function, metabolism, and inflammation status. These improvements in patients' conditions are associated with immunometabolic processed involving adiponectin and leptin levels. Therefore, we propose that both adipokines may be a useful biomarker in the evaluation of metabolic and inflammatory status in patients with CF.
treatment reproduced the same effects in all cell types, but to a lower extent. Considering the f... more treatment reproduced the same effects in all cell types, but to a lower extent. Considering the future exposure to low but constant levels of glyphosate, it is of pivotal importance to elucidate its mechanism of actions in mammalian cells. In light of our results, we hypothesize an involvement of mitochondria in the effects of glyphosate. Different amount of mitochondria and antioxidant enzymes, in the different cell types, will be investigated as a prosecution of the study.
The adult human heart has limited regenerative capacity; hence, stem cell therapy has been invest... more The adult human heart has limited regenerative capacity; hence, stem cell therapy has been investigated as a potential approach for cardiac repair. However, a large part of the benefit of the injection of stem and progenitor cells into injured hearts is mediated by secreted factors. Exosomes—nano-sized secreted extracellular vesicles of endosomal origin—have emerged as key signaling organelles in intercellular communication, and are now viewed as the key regenerative constituent of the secretome of stem and progenitor cells. Exosomes released from mesenchymal stem cells, cardiac-derived progenitor cells, embryonic stem cells, induced pluripotent stem cells (iPSCs), and iPSC-derived cardiomyocytes exhibit cardioprotective, immunomodulatory, and reparative abilities. This concise review discusses the therapeutic benefit of exosomes secreted by stem and progenitor cells in preclinical models of ischemic heart disease.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2019
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Data in brief 25 (2019) 104324 Cardiac progenitor cells Neonatal cardiomyocytes Endothelial proge... more Data in brief 25 (2019) 104324 Cardiac progenitor cells Neonatal cardiomyocytes Endothelial progenitors Proliferation Cardioprotection Angiogenesis of the human amniotic fluid stem cell secretome is provided as well. This data can provide useful insights in regenerative medicine as demonstrating the in vitro cardioprotective and proliferative secretory paracrine potential of human fetal stem cells.
A variety of paracrine signals create networks within the myocardium and mediate intercellular co... more A variety of paracrine signals create networks within the myocardium and mediate intercellular communications. Indeed, paracrine stimulation of the endogenous regenerative program of the heart, mainly based on resident cardiac progenitor cell (CPC) activation together with cardiomyocyte proliferation, has become increasingly relevant for future cardiac medicine. In the last years, it has been shown that extracellular vesicles (EV), including exosomes (Ex), are powerful conveyors of relevant biological effects. EV have been proposed not only as promising therapeutic tool for triggering cardiac regeneration and improving repair, but also as means of better understanding the physiological and pathological relationships between specific cardiac components, including cardiomyocytes and fibroblasts. Actually, EV from different kinds of exogenous stem cells have been shown to mediate beneficial effects on the injured myocardium. Moreover, endogenous cells, like CPC can instruct cardiovascular cell types, including cardiomyocytes, while cardiac stromal cells, especially fibroblasts, secrete EV that modulate relevant aspects of cardiomyocyte biology, such as hypertrophy and electrophysiological properties. Finally, cardiomyocytes too may release EV influencing the function of other cardiac cell types. Therefore, EV-based crosstalk is thought to be important in both physiology and pathology, being involved in the responses of the heart to noxious stimuli. In this review we will discuss the role of EV in both regulating cardiac homeostasis and driving heart regeneration. In particular, we will address their role in: (i) providing cardio-protection and enhancing cardiac repair mechanisms; (ii) CPC biology; and (iii) influencing adult cardiomyocyte behavior.
Background: The adult mammalian heart retains residual regenerative capability via endogenous car... more Background: The adult mammalian heart retains residual regenerative capability via endogenous cardiac progenitor cell (CPC) activation and cardiomyocyte proliferation. We previously reported the paracrine cardioprotective capacity of human amniotic fluid-derived stem cells (hAFS) following ischemia or cardiotoxicity. Here we analyse the potential of hAFS secretome fractions for cardiac regeneration and future clinical translation. Methods: hAFS were isolated from amniotic fluid leftover samples from prenatal screening. hAFS conditioned medium (hAFS-CM) was obtained following hypoxic preconditioning. Anti-apoptotic, angiogenic and proliferative effects were evaluated on rodent neonatal cardiomyocytes (r/mNVCM), human endothelial colony forming cells (hECFC) and human CPC. Mice undergoing myocardial infarction (MI) were treated with hAFS-CM, hAFSextracellular vesicles (hAFS-EV), or EV-depleted hAFS-CM (hAFS-DM) by single intra-myocardial administration and evaluated in the short and long term. Results: hAFS-CM improved mNVCM survival under oxidative and hypoxic damage, induced Ca 2+ -dependent angiogenesis in hECFC and triggered hCPC and rNVCM proliferation. hAFS-CM treatment after MI counteracted scarring, supported cardiac function, angiogenesis and cardiomyocyte cell cycle progression in the long term. hAFS-DM had no effect. hAFS-CM and hAFS-EV equally induced epicardium WT1+ CPC reactivation. Although no CPC cardiovascular differentiation was observed, our data suggests contribution to local angiogenesis by paracrine modulation. hAFS-EV alone were able to recapitulate all the beneficial effects exerted by hAFS-CM, except for stimulation of vessel formation. Conclusions: hAFS-CM and hAFS-EV can improve cardiac repair and trigger cardiac regeneration via paracrine modulation of endogenous mechanisms. While both formulations are effective in sustaining myocardial renewal, hAFS-CM retains higher pro-angiogenic potential, while hAFS-EV particularly enhances cardiac function.
Background The heart rejuvenating effects of circulating growth differentiation factor 11 (GDF11)... more Background The heart rejuvenating effects of circulating growth differentiation factor 11 (GDF11), a TGF-β superfamily member that shares 90% homology with myostatin (MSTN), remains controversial. Here, we aimed to probe the role of GDF11 in acute myocardial infarction (MI), a frequent cause of heart failure and premature death during ageing. Methods Young (3-4 months) and old (22-24 months) C57Bl/6 mice were subjected to either systemic recombinant GDF11 (rGDF11; 0.1 mg/kg BW daily) or control treatment over 30 days before myocardial ischaemia-reperfusion (I/R) injury was induced. Dissected hearts underwent in-depth profiling that ranged from histological workup and the assessment of inflammatory pathways to cardiomyocyte viability testing and IPA-guided -omics. Key regulatory mechanisms were studied in vitro using human adult stromal cardiac-specific mesenchymal cells (cMSCs) and HL-1 cardiomyocytes. By harnessing liquid chromatography–tandem mass spectrometry (LC-MS/MS), plasma l...
Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) constitute a mixed popula... more Human induced pluripotent stem cell (hiPSC)-derived cardiomyocytes (CM) constitute a mixed population of ventricular-, atrial-, nodal-like cells, limiting the reliability for studying chamber-specific disease mechanisms. Previous studies characterised CM phenotype based on action potential (AP) morphology, but the classification criteria were still undefined. Our aim was to use in silico models to develop an automated approach for discriminating the electrophysiological differences between hiPSC-CM. We propose the dynamic clamp (DC) technique with the injection of a specific IK1 current as a tool for deriving nine electrical biomarkers and blindly classifying differentiated CM. An unsupervised learning algorithm was applied to discriminate CM phenotypes and principal component analysis was used to visualise cell clustering. Pharmacological validation was performed by specific ion channel blocker and receptor agonist. The proposed approach improves the translational relevance of the ...
Cardiomyocyte renewal represents an unmet clinical need for cardiac regeneration. Stem cell parac... more Cardiomyocyte renewal represents an unmet clinical need for cardiac regeneration. Stem cell paracrine therapy has attracted increasing attention to resurge rescue mechanisms within the heart. We previously characterized the paracrine effects that human amniotic fluid–derived stem cells (hAFSC) can exert to provide cardioprotection and enhance cardiac repair in preclinical models of myocardial ischemia and cardiotoxicity. Here, we analyze whether hAFSC secretome formulations, namely, hAFSC conditioned medium (hAFSC-CM) over extracellular vesicles (hAFSC-EVs) separated from it, can induce cardiomyocyte renewal. c-KIT+ hAFSC were obtained by leftover samples of II trimester prenatal amniocentesis (fetal hAFSC) and from clinical waste III trimester amniotic fluid during scheduled C-section procedures (perinatal hAFSC). hAFSC were primed under 1% O2 to enrich hAFSC-CM and EVs with cardioactive factors. Neonatal mouse ventricular cardiomyocytes (mNVCM) were isolated from cardiac tissue of...
Inflammatory response following SARS-CoV-2 infection results in substantial increase of amounts o... more Inflammatory response following SARS-CoV-2 infection results in substantial increase of amounts of intravascular pro-coagulant extracellular vesicles (EVs) expressing tissue factor (CD142) on their surface. CD142-EV turned out to be useful as diagnostic biomarker in COVID-19 patients. Here we aimed at studying the prognostic capacity of CD142-EV in SARS-CoV-2 infection. Expression of CD142-EV was evaluated in 261 subjects admitted to hospital for pneumonia and with a positive molecular test for SARS-CoV-2. The study population consisted of a discovery cohort of selected patients (n = 60) and an independent validation cohort including unselected consecutive enrolled patients (n = 201). CD142-EV levels were correlated with post-hospitalization course of the disease and compared to the clinically available 4C Mortality Score as referral. CD142-EV showed a reliable performance to predict patient prognosis in the discovery cohort (AUC = 0.906) with an accuracy of 81.7%, that was confirmed in the validation cohort (AUC = 0.736). Kaplan-Meier curves highlighted a high discrimination power in unselected subjects with CD142-EV being able to stratify the majority of patients according to their prognosis. We obtained a comparable accuracy, being not inferior in terms of prediction of patients' prognosis and risk of mortality, with 4C Mortality Score. The expression of surface vesicular CD142 and its reliability as prognostic marker was technically validated using different immunocapture strategies and assays. The detection of CD142 on EV surface gains considerable interest as risk stratification tool to support clinical decision making in COVID-19.
Background: Inflammatory response following SARS-CoV-2 infection results in substantial increase ... more Background: Inflammatory response following SARS-CoV-2 infection results in substantial increase of amounts of intravascular pro-coagulant extracellular vesicles (EV) expressing the tissue factor (CD142) on their surface. CD142-EV not only can act as clotting initiation agent, contributing to disease severity, but it turned out to be useful as diagnostic biomarker in COVID-19 patients. Here we aimed at studying the prognostic capacity of CD142-EV in SARS-CoV-2 infection.Methods: Level of expression of CD142-EV was evaluated in 261 subjects admitted to hospital for pneumonia and with a positive molecular test for SARS-CoV-2. The study population consisted of a discovery cohort of selected patients (n=60) and an independent validation cohort including unselected consecutive enrolled patients (n=201). CD142-EV biomarker performances were correlated with post-hospitalization course of the disease and compared to the clinically available 4C Mortality Score as referral.Findings: CD142-EV showed a reliable performance to predict patient prognosis in the discovery cohort (AUC=0.906) with an accuracy of 81.7%, that was confirmed in the validation cohort (AUC=0.736). Kaplan-Meier curves further highlighted a high discrimination power in unselected subjects with CD142-EV being able to stratify the majority of patients according to their prognosis. We obtained a comparable accuracy, being not inferior in terms of prediction of patients’ prognosis and risk of mortality with 4C Mortality Score.Interpretation: The detection of CD142 on the surface of EV is a cost-effective and rapid test that can be available at time of admission by using conventional flow cytometer. Such analysis gains considerable interest as risk stratification tool to support clinical decision making in COVID-19.Declaration of Interest: None to declare. Ethical Approval: The study was approved by the Ethical Committee of the Ticino Canton, Switzerland (2020-00795, 96 Rif CE TI 3616).
Second trimester foetal human amniotic fluid‐derived stem cells (hAFS) have been shown to possess... more Second trimester foetal human amniotic fluid‐derived stem cells (hAFS) have been shown to possess remarkable cardioprotective paracrine potential in different preclinical models of myocardial injury and drug‐induced cardiotoxicity. The hAFS secretome, namely the total soluble factors released by cells in their conditioned medium (hAFS‐CM), can also strongly sustain in vivo angiogenesis in a murine model of acute myocardial infarction (MI) and stimulates human endothelial colony‐forming cells (ECFCs), the only truly recognized endothelial progenitor, to form capillary‐like structures in vitro. Preliminary work demonstrated that the hypoxic hAFS secretome (hAFS‐CMHypo) triggers intracellular Ca2+ oscillations in human ECFCs, but the underlying mechanisms and the downstream Ca2+‐dependent effectors remain elusive. Herein, we found that the secretome obtained by hAFS undergoing hypoxic preconditioning induced intracellular Ca2+ oscillations by promoting extracellular Ca2+ entry through Transient Receptor Potential Vanilloid 4 (TRPV4). TRPV4‐mediated Ca2+ entry, in turn, promoted the concerted interplay between inositol‐1,4,5‐trisphosphate‐ and nicotinic acid adenine dinucleotide phosphate‐induced endogenous Ca2+ release and store‐operated Ca2+ entry (SOCE). hAFS‐CMHypo‐induced intracellular Ca2+ oscillations resulted in the nuclear translocation of the Ca2+‐sensitive transcription factor p65 NF‐κB. Finally, inhibition of either intracellular Ca2+ oscillations or NF‐κB activity prevented hAFS‐CMHypo‐induced ECFC tube formation. These data shed novel light on the molecular mechanisms whereby hAFS‐CMHypo induces angiogenesis, thus providing useful insights for future therapeutic strategies against ischaemic‐related myocardial injury.
Although a small number of cardiomyocytes may reenter the cell cycle after injury, the adult mamm... more Although a small number of cardiomyocytes may reenter the cell cycle after injury, the adult mammalian heart is incapable of a robust cardiomyocyte proliferation. Periostin, a secreted extracellular matrix protein, has been implicated as a regulator of cardiomyocyte proliferation; however, this role remains controversial. Alternative splicing of the human periostin gene results in 6 isoforms lacking sequences between exons 17 and 21, in addition to full-length periostin. We previously showed that exosomes (Exo) secreted by human cardiac explant-derived progenitor cells (CPC) carried periostin. Here, we aimed to investigate their cell cycle activity. Methods: CPC were derived as the cellular outgrowth of ex vivo cultured cardiac atrial explants. Exo were purified from CPC conditioned medium using size exclusion chromatography. Exosomal periostin was analyzed by Western blotting using a pair of antibodies (one raised against aa 537-836, and one raised against amino acids mapping at exon 17 of human periostin), by ELISA, and by cryo-EM with immune-gold labeling. Cell cycle activity was assessed in neonatal rat cardiomyocytes, in human induced pluripotent stem cell (iPS)-derived cardiomyocytes, and in adult rat cardiomyocytes after myocardial infarction. The role of periostin in cell cycle activity was investigated by transfecting donor CPC with a siRNA against this protein. Results: Periostin expression in CPC-secreted exosomes was detected using the antibody raised against aa 537-836 of the human protein, but not with the exon 17-specific antibody, consistent with an isoform lacking exon 17. Periostin was visualized on vesicle surfaces by cryo-EM and immune-gold labeling. CPC-derived exosomes induced cell proliferation in neonatal rat cardiomyocytes both in vitro and in vivo, in human iPS-derived cardiomyocytes, and in adult rat cardiomyocytes after myocardial infarction. Exo promoted phosphorylation of focal adhesion kinase (FAK), actin polymerization, and nuclear translocation of Yes-associated protein (YAP) in cardiomyocytes. Knocking down of periostin or YAP, or blocking FAK phosphorylation with PF-573228 nullified Exo-induced proliferation. A truncated human periostin peptide (aa 22-669), but not recombinant human full-length periostin, mimicked the pro-proliferative activity of exosomes. Conclusions: Our results show, for the first time, that CPC-secreted exosomes promote cardiomyocyte cell cycle-reentry via a short periostin isoform expressed on their surfaces, whereas recombinant full-length periostin does not. These findings highlight isoform-specific roles of periostin in cardiomyocyte proliferation.
We previously reported that c-KIT+ human amniotic-fluid derived stem cells obtained from leftover... more We previously reported that c-KIT+ human amniotic-fluid derived stem cells obtained from leftover samples of routine II trimester prenatal diagnosis (fetal hAFS) are endowed with regenerative paracrine potential driving pro-survival, anti-fibrotic and proliferative effects. hAFS may also be isolated from III trimester clinical waste samples during scheduled C-sections (perinatal hAFS), thus offering a more easily accessible alternative when compared to fetal hAFS. Nonetheless, little is known about the paracrine profile of perinatal hAFS. Here we provide a detailed characterization of the hAFS total secretome (i.e., the entirety of soluble paracrine factors released by cells in the conditioned medium, hAFS-CM) and the extracellular vesicles (hAFS-EVs) within it, from II trimester fetal- versus III trimester perinatal cells. Fetal- and perinatal hAFS were characterized and subject to hypoxic preconditioning to enhance their paracrine potential. hAFS-CM and hAFS-EV formulations were a...
Abstract Cardiovascular disease is mainly related to inefficient cardioprotection, defective repa... more Abstract Cardiovascular disease is mainly related to inefficient cardioprotection, defective repair, and lack of myocardial renewal, following cardiac injury and/or aging. Recent work has demonstrated an endogenous restorative program within the heart based on cardiac progenitor cell activation and resident cardiomyocyte proliferation. Although these mechanisms are widely responsive in the neonatal life, they become dormant during adulthood, leaving the heart with limited repair potential in pathological situations. Therefore a working strategy is strongly needed to restore in full the potential for both cardiac repair and regeneration. The so-called “paracrine effect” has been proposed as a possible working strategy, with increasing interest toward the comprehensive analysis of the stem cell “secretome” as the whole of trophic molecules produced by cells via paracrine secretion. In this scenario, different sources of stem cells have been tested; among them, fetal stem cells such as amniotic fluid–derived stem cells may represent an appealing therapeutic option.
Mesenchymal stromal cells (MSCs) are characterized by a regulatory phenotype and respond promptly... more Mesenchymal stromal cells (MSCs) are characterized by a regulatory phenotype and respond promptly to the environmental signals modulating their secretory activity. An appropriate preconditioning may induce MSCs to release secretomes with an enhanced regenerative potential. However, it fails to take into account that secretomes are composed by both soluble factors and extracellular vesicles (EVs), whose functions could be altered differently by the preconditioning approach. Here we demonstrate that the MSC secretome is strongly modulated by the simultaneous stimulation with hypoxia and pro-inflammatory cytokines, used to mimic the harsh environment present at the site of injury. We observed that the environmental variations strongly influenced the angiogenic potential of the different secretome fractions. Upon inflammation, the pro-angiogenic capacity of the soluble component of the MSC secretome was strongly inhibited, regardless of the oxygen level, while the EV-encapsulated component was not significantly affected by the inflammatory stimuli. These effects were accompanied by the modulation of the secreted proteins. On one hand, inflammation-activated MSCs release proteins mainly involved in the interaction with innate immune cells and in tissue remodeling/repair; on the other hand, when MSCs are not exposed to an inflamed environment, they respond to the different oxygen levels modulating the expression of proteins involved in the angiogenic process. The cargo content (in terms of miRNAs) of the corresponding EV fractions was less sensitive to the influence of the external stimuli. Our findings suggest that the therapeutic efficacy of MSC-based therapies could be enhanced by selecting the appropriate preconditioning approach and carefully discriminating its effects on the different secretome components.
Background: Coronavirus-2 (SARS-CoV-2) infection causes an acute respiratory syndrome accompanied... more Background: Coronavirus-2 (SARS-CoV-2) infection causes an acute respiratory syndrome accompanied by multi-organ damage that implicates a prothrombotic state leading to widespread microvascular clots. The causes of such coagulation abnormalities are unknown. The receptor tissue factor, also known as CD142, is often associated with cell-released extracellular vesicles (EV). In this study, we aimed to characterize surface antigens profile of circulating EV in COVID-19 patients and their potential implication as procoagulant agents. Methods: We analyzed serum-derived EV from 67 participants who underwent nasopharyngeal swabs molecular test for suspected SARS-CoV-2 infection (34 positives and 33 negatives) and from 16 healthy controls (HC), as referral. A sub-analysis was performed on subjects who developed pneumonia (n = 28). Serumderived EV were characterized for their surface antigen profile and tested for their procoagulant activity. A validation experiment was performed pre-treating EV with anti-CD142 antibody or with recombinant FVIIa. Serum TNF-a levels were measured by ELISA. Findings: Profiling of EV antigens revealed a surface marker signature that defines circulating EV in COVID-19. A combination of seven surface molecules (CD49e, CD209, CD86, CD133/1, CD69, CD142, and CD20) clustered COVID (+) versus COVID (-) patients and HC. CD142 showed the highest discriminating performance at both multivariate models and ROC curve analysis. Noteworthy, we found that CD142 exposed onto surface of EV was biologically active. CD142 activity was higher in COVID (+) patients and correlated with TNF-a serum levels. Interpretation: In SARS-CoV-2 infection the systemic inflammatory response results in cell-release of substantial amounts of procoagulant EV that may act as clotting initiation agents, contributing to disease severity.
Cystic fibrosis (CF) is a genetic disease characterized by progressive decline of lung function a... more Cystic fibrosis (CF) is a genetic disease characterized by progressive decline of lung function and chronic airway inflammation. Adiponectin and leptin exert several effects on energy metabolism and inflammatory processes. This study evaluated the levels of adiponectin and leptin in adult healthy subjects, in patients with CF and their correlation with long-term physical activity. CF patients were divided into two groups (sedentary versus active) based on their regular physical activity over 3 years. Anthropometric and serum biochemical profiles of CF patients and controls were evaluated and compared. Total serum adiponectin and leptin levels were measured by ELISA; adiponectin oligomeric profiles were analysed by western blot. Adiponectin levels were significantly higher while leptin levels were lower in patients with CF than in healthy controls. Furthermore, adiponectin was significantly lower in active compared to sedentary CF, (p = 0.047). leptin was slightly increased in active compared to sedentary CF. In addition, C-reactive protein levels were significantly lower in active than in sedentary CF patients, (p = 0.048). Interestingly, only in the active group adiponectin levels were inversely correlated with forced expiratory volume (FEV)1% decrease/year and FEV1% decrease. Moreover, adiponectin levels negatively correlated with lipid profiles. Our findings indicated that regular, long-term physical activity in CF improves respiratory function, metabolism, and inflammation status. These improvements in patients' conditions are associated with immunometabolic processed involving adiponectin and leptin levels. Therefore, we propose that both adipokines may be a useful biomarker in the evaluation of metabolic and inflammatory status in patients with CF.
treatment reproduced the same effects in all cell types, but to a lower extent. Considering the f... more treatment reproduced the same effects in all cell types, but to a lower extent. Considering the future exposure to low but constant levels of glyphosate, it is of pivotal importance to elucidate its mechanism of actions in mammalian cells. In light of our results, we hypothesize an involvement of mitochondria in the effects of glyphosate. Different amount of mitochondria and antioxidant enzymes, in the different cell types, will be investigated as a prosecution of the study.
The adult human heart has limited regenerative capacity; hence, stem cell therapy has been invest... more The adult human heart has limited regenerative capacity; hence, stem cell therapy has been investigated as a potential approach for cardiac repair. However, a large part of the benefit of the injection of stem and progenitor cells into injured hearts is mediated by secreted factors. Exosomes—nano-sized secreted extracellular vesicles of endosomal origin—have emerged as key signaling organelles in intercellular communication, and are now viewed as the key regenerative constituent of the secretome of stem and progenitor cells. Exosomes released from mesenchymal stem cells, cardiac-derived progenitor cells, embryonic stem cells, induced pluripotent stem cells (iPSCs), and iPSC-derived cardiomyocytes exhibit cardioprotective, immunomodulatory, and reparative abilities. This concise review discusses the therapeutic benefit of exosomes secreted by stem and progenitor cells in preclinical models of ischemic heart disease.
Biochimica et Biophysica Acta (BBA) - Molecular Cell Research, 2019
This is a PDF file of an article that has undergone enhancements after acceptance, such as the ad... more This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Data in brief 25 (2019) 104324 Cardiac progenitor cells Neonatal cardiomyocytes Endothelial proge... more Data in brief 25 (2019) 104324 Cardiac progenitor cells Neonatal cardiomyocytes Endothelial progenitors Proliferation Cardioprotection Angiogenesis of the human amniotic fluid stem cell secretome is provided as well. This data can provide useful insights in regenerative medicine as demonstrating the in vitro cardioprotective and proliferative secretory paracrine potential of human fetal stem cells.
A variety of paracrine signals create networks within the myocardium and mediate intercellular co... more A variety of paracrine signals create networks within the myocardium and mediate intercellular communications. Indeed, paracrine stimulation of the endogenous regenerative program of the heart, mainly based on resident cardiac progenitor cell (CPC) activation together with cardiomyocyte proliferation, has become increasingly relevant for future cardiac medicine. In the last years, it has been shown that extracellular vesicles (EV), including exosomes (Ex), are powerful conveyors of relevant biological effects. EV have been proposed not only as promising therapeutic tool for triggering cardiac regeneration and improving repair, but also as means of better understanding the physiological and pathological relationships between specific cardiac components, including cardiomyocytes and fibroblasts. Actually, EV from different kinds of exogenous stem cells have been shown to mediate beneficial effects on the injured myocardium. Moreover, endogenous cells, like CPC can instruct cardiovascular cell types, including cardiomyocytes, while cardiac stromal cells, especially fibroblasts, secrete EV that modulate relevant aspects of cardiomyocyte biology, such as hypertrophy and electrophysiological properties. Finally, cardiomyocytes too may release EV influencing the function of other cardiac cell types. Therefore, EV-based crosstalk is thought to be important in both physiology and pathology, being involved in the responses of the heart to noxious stimuli. In this review we will discuss the role of EV in both regulating cardiac homeostasis and driving heart regeneration. In particular, we will address their role in: (i) providing cardio-protection and enhancing cardiac repair mechanisms; (ii) CPC biology; and (iii) influencing adult cardiomyocyte behavior.
Background: The adult mammalian heart retains residual regenerative capability via endogenous car... more Background: The adult mammalian heart retains residual regenerative capability via endogenous cardiac progenitor cell (CPC) activation and cardiomyocyte proliferation. We previously reported the paracrine cardioprotective capacity of human amniotic fluid-derived stem cells (hAFS) following ischemia or cardiotoxicity. Here we analyse the potential of hAFS secretome fractions for cardiac regeneration and future clinical translation. Methods: hAFS were isolated from amniotic fluid leftover samples from prenatal screening. hAFS conditioned medium (hAFS-CM) was obtained following hypoxic preconditioning. Anti-apoptotic, angiogenic and proliferative effects were evaluated on rodent neonatal cardiomyocytes (r/mNVCM), human endothelial colony forming cells (hECFC) and human CPC. Mice undergoing myocardial infarction (MI) were treated with hAFS-CM, hAFSextracellular vesicles (hAFS-EV), or EV-depleted hAFS-CM (hAFS-DM) by single intra-myocardial administration and evaluated in the short and long term. Results: hAFS-CM improved mNVCM survival under oxidative and hypoxic damage, induced Ca 2+ -dependent angiogenesis in hECFC and triggered hCPC and rNVCM proliferation. hAFS-CM treatment after MI counteracted scarring, supported cardiac function, angiogenesis and cardiomyocyte cell cycle progression in the long term. hAFS-DM had no effect. hAFS-CM and hAFS-EV equally induced epicardium WT1+ CPC reactivation. Although no CPC cardiovascular differentiation was observed, our data suggests contribution to local angiogenesis by paracrine modulation. hAFS-EV alone were able to recapitulate all the beneficial effects exerted by hAFS-CM, except for stimulation of vessel formation. Conclusions: hAFS-CM and hAFS-EV can improve cardiac repair and trigger cardiac regeneration via paracrine modulation of endogenous mechanisms. While both formulations are effective in sustaining myocardial renewal, hAFS-CM retains higher pro-angiogenic potential, while hAFS-EV particularly enhances cardiac function.
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Papers by Carolina Balbi