Human Cardiac Progenitor Cells Research Articles

Extracellular vesicles selective capture by peptide-functionalized hollow fiber membranes

Recently, membrane devices and processes have been applied for the separation and concentration of subcellular components such as extracellular vesicles (EVs), which play a diagnostic and therapeutic role in many pathological conditions. However, the separation and isolation of specific EV populations from other components found in biological fluids is still challenging. Here, we developed a peptide-functionalized hollow fiber (HF) membrane module to achieve the separation and enrichment of highly pure EVs derived from the culture media of human cardiac progenitor cells. The strategy is based on the functionalization of PSf HF membrane module with BPt, a peptide sequence able to bind nanovesicles characterized by highly curved membranes. HF membranes were modified by a nanometric coating with a copoly azide polymer to limit non-specific interactions and to enable the conjugation with peptide ligand by click chemistry reaction. The BPt-functionalized module was integrated into a TFF process to facilitate the design, rationalization, and optimization of EV isolation. This integration combined size-based transport of species with specific membrane sensing ligands. The TFF integrated BPt-functionalized membrane module demonstrated the ability to selectively capture EVs with diameter < 200 nm into the lumen of fibers while effectively removing contaminants such as albumin. The captured and released EVs contain the common markers including CD63, CD81, CD9 and syntenin-1. Moreover, they maintained a round shape morphology and structural integrity highlighting that this approach enables EVs concentration and purification with low shear stress. Additionally, it achieved the removal of contaminants such as albumin with high reliability and reproducibility, reaching a removal of 93%.

Study on the Lupine-hulls Based Cellular Scaffolds for Human Cardiac Tissue Regeneration

It's becoming more popular to develop new biomaterials enabling in vitro cell cultivation with microbial environments of biological structures. The novel strategies for creating native cellular environments that facilitate the growth of human cardiac progenitor cells (hCPCs) (Sca-1pos) and scaffolding more closely resemble authentic cell function microenvironments with offer a wide range of appealing in vivo and in vitro uses. Both the biologically active substances and the mechanical properties of the hull scaffold were studied. SEM analysis of the hull's microstructure reveals that the inside surface shows its large and naturalistic activity, while the outside surface displays the celular-structures. Tensile properties, high-performance liquid chromatography (HPLC), and fourier transform infrared spectroscopy (FTIR) spectra are further characteristics of the lupine-hulls. Scaffolds made from lupine-hulls are cheap, simple to make, and from a renewable material. The MTT assay for cell proliferation served to evaluate the impact of the hulls that are solubility substance on the Sca-1pos. The cell lines utilized in this study were able to multiply and maintain high viability even after being cultured for 7 straight days. Results also demonstrate that the nuclei of the cells were visible under fluorescence microscopy and remained viable within the cellular structures of the hulls in vitro. This research showed that scaffold made from lupine hulls are being used as biological scaffolds for in vitro cell growth, which may play a role in the tissue engineering of hCPCs.

Sunitinib malate induces cell death in adult human cardiac progenitor cells

Sunitinib malate is known to cause cardiotoxicity in a sub-population of patients, with heart failure seen in more severe cases. Cardiac progenitor cells (CPCs) have been identified in adult human myocardium and contribute to overall tissue maintenance, with previous work identifying negative impacts of sunitinib on these cells. This study aimed to characterise the toxic effects of sunitinib in human CPCs, applying sunitinib concentrations equivalent to clinical plasma levels to these cells in vitro. Cell viability was reduced by 26.5 ± 6.6 % by 2 μM sunitinib for 24 h (p < 0.01); this concentration also induced fold-change increases in gene expression of: calpain (3.1 ± 0.73, p < 0.05), FAS (2.3 ± 0.8, p < 0.05) and BAX (1.9 ± 0.2, p < 0.05), and a decrease in BCL-2 (3.5 ± 0.0, p < 0.001), vs. control (1.0 ± 0.0). This was affirmed by sunitinib inducing fold changes in protein expression of: calpain-1 (2.5 ± 0.5, p < 0.05); FAS receptor (2.1 ± 0.2, p < 0.05) and BAX (2.1 ± 0.2, p < 0.05) vs. control (1.0 ± 0.0). These results indicated that sunitinib induced apoptosis in CPCs, but negative annexin V staining and lack of protection by caspase inhibitors indicated this was not the cell death pathway activated. Further investigation found sunitinib was concentrated in the lysosomes and autophagosomes within CPCs, but did not induce accumulation of acidic organelles. In conclusion, these data confirm that cell death is caused by sunitinib in CPCs at concentrations equivalent to clinical plasma levels, inducing cell death pathway signals that lead to non-apoptotic cell death.

Genetic Engineering of Therapeutic Cells with the Sodium Iodide Symporter (NIS) to Enable Noninvasive In Vivo Therapy Tracking.

Noninvasive long-term imaging of therapeutic cells in preclinical models can be achieved through introducing a reporter gene into the cells of interest. Despite important recent developments such as gene editing, cell engineering based on lentiviruses remains a mainstream tool for gene transfer applicable to a variety of different cell types.In this chapter, we describe how to use lentivirus-based genetic engineering to render different candidate cell therapies in vivo traceable by radionuclide imaging. We illustrate this reporter gene technology using the sodium iodide symporter (NIS), which is compatible with both positron emission tomography (PET) and single-photon emission computed tomography (SPECT). For preclinical experimentation, we fused NIS with a suitable fluorescent protein such as monomeric GFP or RFP to streamline cell line generation and downstream analyses of ex vivo tissue samples. We present protocols for reporter gene engineering of human cardiac progenitor cells, regulatory T cells, and effector T cells as well as for the characterization experiments required to validate NIS-fluorescent protein reporter function in these candidate therapeutic cells.

Impact of Isolation Methods on Extracellular Vesicle Functionality In Vitro and In Vivo.

This study compares the impact of two isolation methods, ultracentrifugation (UC) and size exclusion chromatography (SEC), on small extracellular vesicles (sEVs) from primary human cardiac mesenchymal-derived progenitor cells (CPCs). sEV_UC and sEV_SEC exhibit similar size, marker expression, and miRNA cargo, but sEV_UC contains notably higher total protein levels. In vitro assays show that sEV_UC, despite an equal particle count, induces more robust ERK phosphorylation, cytoprotection, and proliferation in iPS-derived cardiomyocytes (iPS-CMs) compared to sEV_SEC. sEV_UC also contains elevated periostin (POSTN) protein levels, resulting in enhanced focal adhesion kinase (FAK) phosphorylation in iPS-CMs. Importantly, this effect persists with treatment with soluble free-sEV protein fraction from SEC (Prote_SEC), indicating that free proteins like POSTN in sEV_UC enhance FAK phosphorylation. In vivo, sEV contamination with soluble proteins doesn't affect cardiac targeting or FAK phosphorylation, underscoring the intrinsic tissue targeting properties of sEV. These findings emphasize the need for standardized sEV isolation methods, as the choice of method can impact experimental outcomes, particularly in vitro.

Intracoronary delivery of extracellular vesicles from human cardiac progenitor cells reduces infarct size in porcine acute myocardial infarction.

Intracoronary delivery of extracellular vesicles from human cardiac progenitor cells reduces infarct size in porcine acute myocardial infarction.

Cardiac Progenitor Cell Exosomal miR-935 Protects against Oxidative Stress.

Oxidative stress-induced myocardial apoptosis and necrosis are critically involved in ischemic infarction, and several sources of extracellular vesicles appear to be enriched in therapeutic activities. The central objective was to identify and validate the differential exosome miRNA repertoire in human cardiac progenitor cells (CPC). CPC exosomes were first analyzed by LC-MS/MS and compared by RNAseq with exomes of human mesenchymal stromal cells and human fibroblasts to define their differential exosome miRNA repertoire (exo-miRSEL). Proteomics demonstrated a highly significant representation of cardiovascular development functions and angiogenesis in CPC exosomes, and RNAseq analysis yielded about 350 different miRNAs; among the exo-miRSEL population, miR-935 was confirmed as the miRNA most significantly up-regulated; interestingly, miR-935 was also found to be preferentially expressed in mouse primary cardiac Bmi1+high CPC, a population highly enriched in progenitors. Furthermore, it was found that transfection of an miR-935 antagomiR combined with oxidative stress treatment provoked a significant increment both in apoptotic and necrotic populations, whereas transfection of a miR-935 mimic did not modify the response. Conclusion. miR-935 is a highly differentially expressed miRNA in exo-miRSEL, and its expression reduction promotes oxidative stress-associated apoptosis. MiR-935, together with other exosomal miRNA members, could counteract oxidative stress-related apoptosis, at least in CPC surroundings.

The Protective Role of Glutathione against Doxorubicin-Induced Cardiotoxicity in Human Cardiac Progenitor Cells.

This study investigated the protective effect of glutathione (GSH), an antioxidant drug, against doxorubicin (DOX)-induced cardiotoxicity. Human cardiac progenitor cells (hCPCs) treated with DOX (250 to 500 nM) showed increased viability and reduced ROS generation and apoptosis with GSH treatment (0.1 to 1 mM) for 24 h. In contrast to the 500 nM DOX group, pERK levels were restored in the group co-treated with GSH and suppression of ERK signaling improved hCPCs' survival. Similarly to the previous results, the reduced potency of hCPCs in the 100 nM DOX group, which did not affect cell viability, was ameliorated by co-treatment with GSH (0.1 to 1 mM). Furthermore, GSH was protected against DOX-induced cardiotoxicity in the in vivo model (DOX 20 mg/kg, GSH 100 mg/kg). These results suggest that GSH is a potential therapeutic strategy for DOX-induced cardiotoxicity, which performs its function via ROS reduction and pERK signal regulation.

38-OR: Insulin Prevents Palmitate-Induced Stress Kinase Activation, Autophagy, and Apoptosis in Human Cardiac Progenitor Cells

Abnormal accumulation of saturated fatty acids in the heart results in insulin resistance, stress kinase activation, and increased cardiovascular risk in humans. The viability of human cardiac progenitor cells (CPC) is essential for myocardium homeostasis. This study investigates the ability of palmitate, a saturated fatty acid, to induce apoptosis, autophagy, and stress kinase activation in human CPC isolated from right auricle biopsies of nondiabetic, non-obese subjects, and the potential protective effects of insulin on palmitate-induced abnormalities. Human CPC expressed both IR-A and IR-B, and IR-A was more expressed than IR-B, as assessed by quantitative RT-PCR and immunoblotting. Exposure of human CPC to 100 nM insulin for 15 minutes resulted in increased Akt (S473) and p44/p42 MAPK (T202/Y204) phosphorylation (p&amp;lt;0.05), as evidenced by immunoblotting. Treatment of human CPC with palmitate 0.25 mM for 16 h induced apoptosis (p&amp;lt;0.05), as assessed by caspase-3 cleavage and ELISA assay, and increased S63-phosphorylation of c-Jun (p&amp;lt;0.05), a downstream transcription factor of JNK 1/2, as assessed by immunoblotting. Exposure of human CPC to 0.25 mM palmitate for 16 h resulted also in increased autophagy, evidenced by light chain 3-II immunoblotting (p&amp;lt;0.05). Pretreatment with 20 µM SP600125, a JNK inhibitor, for 1 h inhibited palmitate-induced apoptosis (p&amp;lt;0.05), but not autophagy. Similarly, pretreatment with 10 mM 3-methyladenine, an autophagy inhibitor, for 1 h decreased palmitate-induced apoptosis (p&amp;lt;0.05). Interestingly, palmitate effects on apoptosis, autophagy, and on stress kinase activation, were prevented when human CPC were pretreated with 100 nM insulin for 1 h (p&amp;lt;0.05). In conclusion, insulin prevents palmitate-induced apoptosis by inhibition of JNK signaling and autophagy in human CPC. Hence, preserving insulin signaling in human CPC might protect from lipotoxicity-induced metabolic alterations in the heart. Disclosure I. Calderoni: None. R. Doria: None. C. Caccioppoli: None. V. Genchi: None. G. Palma: None. G. Santarpino: None. A.D. Milano: None. A. Leonardini: None. A. Natalicchio: Other Relationship; AstraZeneca, Novo Nordisk, Sanofi, Boehringer-Ingelheim, Lilly. S. Perrini: None. A. Cignarelli: Speaker's Bureau; Eli Lilly and Company, Novo Nordisk, Sanofi. F. Giorgino: Research Support; Lilly, Roche Diabetes Care. Consultant; Novo Nordisk, Lilly. Speaker's Bureau; Abbott, Boehringer-Ingelheim, Lilly. Advisory Panel; Boehringer-Ingelheim, Amarin Corporation, Medtronic, Roche Diabetes Care, Sanofi. Speaker's Bureau; Sanofi. Advisory Panel; Bayer Inc., Novo Nordisk. Research Support; AlfaSigma. Speaker's Bureau; Medscape. L. Laviola: Speaker's Bureau; Abbott. Advisory Panel; A. Menarini Diagnostics, Boehringer Ingelheim Inc., Eli Lilly and Company, Novo Nordisk, Roche Diabetes Care. Speaker's Bureau; Terumo Corporation. Other Relationship; Medtronic. Advisory Panel; Sanofi.

246-OR: Dapagliflozin Counteracts the Proapoptotic Effects of the Secretome of Visceral Adipose Cells from Obese Subjects in Human Cardiac Progenitor Cells via the SGLT2 Cotransporter

Dapagliflozin (DAPA), an SGLT2 inhibitor, has been shown to counteract heart failure outcomes in subjects with obesity and diabetes. We investigated the mechanisms of the protective action of DAPA in human cardiac progenitor cells (hCPC) exposed to the conditioned medium (CM) from abdominal visceral (AV) and epicardial (E) adipose stem cells (ASC) and from AV mature adipocytes from obese subjects. ASC and mature adipocytes were isolated from AV and E adipose tissue biopsies of 15 obese (Ob) and 13 non-Ob subjects (n-Ob), respectively. hCPC were isolated from right auricle biopsies of healthy non-Ob donors. Exposure of hCPC to the CM of adipose cells from Ob, but not from non-Ob subjects, induced apoptosis, c-Jun phosphorylation, and impairment of actin filaments, as assessed by ELISA assay and immunofluorescence, respectively, while these effects were not observed when the hCPC were pretreated with DAPA. The CM of adipose cells from Ob compared to n-Ob subjects displayed a different pattern of pro-inflammatory and anti-inflammatory cytokines. The levels of pro-inflammatory cytokines such as RANTES and MIP1β were increased in the CM from AV-ASC with higher BMI (p&amp;lt;0.05), while the levels of anti-inflammatory factors such as GCSF in the CM of E-ASC were inversely correlated with BMI (p&amp;lt;0.05). SGLT2 was found to be expressed as both mRNA and protein in the hCPC, and silencing of SGLT2 with a specific siRNA attenuated the capacity of DAPA to counteract the pro-apoptotic effects of the CM. In conclusion, we show that: i) in human obesity, the CM of both AV- and E-ASC and mature adipocytes is characterized by pro-inflammatory cytokines that induce stress kinase activation and apoptosis in the hCPC; ii) hCPC express SGLT2 mRNA and protein; iii) DAPA prevents the hCPC damage induced by the CM through an SGLT2-dependent mechanism. Disclosure G.Palma: None. A.Natalicchio: Other Relationship; AstraZeneca, Novo Nordisk, Sanofi, Boehringer-Ingelheim, Lilly. L.Laviola: Advisory Panel; A. Menarini Diagnostics, Boehringer Ingelheim Inc., Eli Lilly and Company, Novo Nordisk, Roche Diabetes Care, Sanofi, Other Relationship; Medtronic, Speaker's Bureau; Abbott, Terumo Corporation. A.Pezzolla: None. F.Giorgino: Advisory Panel; Boehringer-Ingelheim, Amarin Corporation, Medtronic, Roche Diabetes Care, Sanofi, Bayer Inc., Novo Nordisk, Consultant; Novo Nordisk, Lilly, Research Support; Lilly, Roche Diabetes Care, AlfaSigma, Speaker's Bureau; Abbott, Boehringer-Ingelheim, Lilly, Sanofi, Medscape. S.Perrini: None. C.Caccioppoli: None. R.Doria: None. V.Genchi: None. I.Calderoni: None. A.Braun: None. G.Santarpino: None. A.D.Milano: None. A.Cignarelli: Speaker's Bureau; Eli Lilly and Company, Novo Nordisk, Sanofi.

Dapagliflozin counteracts the pro-apoptotic effects of the secretome of visceral adipose cells from obese subjects in human cardiac progenitor cells via the SGLT2 co-transporter

Aim: Dapagliflozin (DAPA), an SGLT2 inhibitor, has been shown to counteract heart failure outcomes in subjects with obesity and diabetes. We investigated the protective mechanisms of DAPA in human cardiac progenitor cells (hCPC) exposed to the conditioned medium (CM) from abdominal visceral (AV) and epicardial (E) adipose stem cells (ASC) and from AV mature adipocytes from obese subjects. Methods: ASC and mature adipocytes were isolated from AV adipose tissue biopsies of 27 obese (Ob) and 19 non-Ob subjects (n-Ob), and from E adipose tissue biopsies of 9 Ob and 10 non-Ob subjects, respectively. hCPC were isolated from right auricle biopsies of 10 healthy non-Ob donors. Results: Exposure of hCPC to the CM of adipose cells from Ob, but not from non-Ob subjects, induced apoptosis, c-Jun phosphorylation, and impairment of actin filaments, while these effects were not observed when hCPC were pretreated with DAPA. The CM of adipose cells from Ob compared to n-Ob subjects displayed a different pattern of cytokines. The levels of pro-inflammatory cytokines RANTES and MIP1β were increased in the CM from AV-ASC with higher BMI (p&lt;0.05), while the levels of the cardioprotective factor GCSF in the CM of E-ASC were inversely correlated with BMI (p&lt;0.05). SGLT2 was found to be expressed as both mRNA and protein in hCPC, and silencing of SGLT2 with a specific siRNA abrogated the capacity of DAPA to counteract the pro-apoptotic effects of the CM. Conclusions: In human obesity, the CM of both AV- and E-ASC and mature adipocytes is characterized by pro-inflammatory cytokines that induce stress kinase activation and apoptosis in hCPC. DAPA prevents the hCPC damage induced by the CM through an SGLT2-dependent mechanism.

Overexpression of GREM1 Improves the Survival Capacity of Aged Cardiac Mesenchymal Progenitor Cells via Upregulation of the ERK/NRF2-Associated Antioxidant Signal Pathway.

Ischemic heart disease is the leading cause of mortality in the United States. Progenitor cell therapy can restore myocardial structure and function. However, its efficacy is severely limited by cell aging and senescence. Gremlin-1 (GREM1), a member of the bone morphogenetic protein antagonist family, has been implicated in cell proliferation and survival. However, GREM1's role in cell aging and senescence has never been investigated in human cardiac mesenchymal progenitor cells (hMPCs). Therefore, this study assessed the hypothesis that overexpression of GREM1 rejuvenates the cardiac regenerative potential of aging hMPCs to a youthful stage and therefore allows better capacity for myocardial repair. We recently reported that a subpopulation of hMPCs with low mitochondrial membrane potential can be sorted from right atrial appendage-derived cells in patients with cardiomyopathy and exhibit cardiac reparative capacity in a mouse model of myocardial infarction. In this study, lentiviral particles were used to overexpress GREM1 in these hMPCs. Protein and mRNA expression were assessed through Western blot and RT-qPCR. FACS analysis for Annexin V/PI staining and lactate dehydrogenase assay were used to assess cell survival. It was observed that cell aging and cell senescence led to a decrease in GREM1 expression. In addition, overexpression of GREM1 led to a decrease in expression of senescence genes. Overexpression of GREM1 led to no significant change in cell proliferation. However, GREM1 appeared to have an anti-apoptotic effect, with an increase in survival and decrease in cytotoxicity evident in GREM1-overexpressing hMPCs. Overexpressing GREM1 also induced cytoprotective properties by decreasing reactive oxidative species and mitochondrial membrane potential. This result was associated with increased expression of antioxidant proteins, such as SOD1 and catalase, and activation of the ERK/NRF2 survival signal pathway. Inhibition of ERK led to a decrease in GREM1-mediated rejuvenation in terms of cell survival, which suggests that an ERK-dependent pathway may be involved. Taken altogether, these results indicate that overexpression of GREM1 can allow aging hMPCs to adopt a more robust phenotype with improved survival capacity, which is associated with an activated ERK/NRF2 antioxidant signal pathway.

Identification of SALL4 Expressing Islet-1+ Cardiovascular Progenitor Cell Clones.

The utilization of cardiac progenitor cells (CPCs) has been shown to induce favorable regenerative effects. While there are various populations of endogenous CPCs in the heart, there is no consensus regarding which population is ideal for cell-based regenerative therapy. Early-stage progenitor cells can be differentiated into all cardiovascular lineages, including cardiomyocytes and endothelial cells. Identifying an Islet-1+ (Isl-1+) early-stage progenitor population with enhanced stemness, multipotency and differentiation potential would be beneficial for the development of novel regenerative therapies. Here, we investigated the transcriptome of human neonatal Isl-1+ CPCs. Isl-1+ human neonatal CPCs exhibit enhanced stemness properties and were found to express Spalt-like transcription factor 4 (SALL4). SALL4 plays a role in embryonic development as well as proliferation and expansion of hematopoietic progenitor cells. SALL4, SOX2, EpCAM and TBX5 are co-expressed in the majority of Isl-1+ clones isolated from neonatal patients. The pre-mesendodermal transcript TFAP2C was identified in select Isl-1, SALL4, SOX2, EpCAM and TBX5 expressing clones. The ability to isolate and expand pre-mesendodermal stage cells from human patients is a novel finding that holds potential value for applications in regenerative medicine.

Abstract 1886: Human Cardiac Progenitor Cells Require ALK1 for Paracrine Signaling in Angiogenesis

Abstract 1886: Human Cardiac Progenitor Cells Require ALK1 for Paracrine Signaling in Angiogenesis

Imatinib Mesylate Induces Necroptotic Cell Death and Impairs Autophagic Flux in Human Cardiac Progenitor Cells.

The receptor tyrosine kinase inhibitor imatinib improves patient cancer survival but is linked to cardiotoxicity. This study investigated imatinib’s effects on cell viability, apoptosis, autophagy, and necroptosis in human cardiac progenitor cells in vitro. Imatinib reduced cell viability (75.9 ± 2.7% vs. 100.0 ± 0.0%) at concentrations comparable to peak plasma levels (10 µM). Imatinib reduced cells’ TMRM fluorescence (74.6 ± 6.5% vs. 100.0 ± 0.0%), consistent with mitochondrial depolarisation. Imatinib increased lysosome and autophagosome content as indicated by LAMP2 expression (2.4 ± 0.3-fold) and acridine orange fluorescence (46.0 ± 5.4% vs. 9.0 ± 3.0), respectively. Although imatinib increased expression of autophagy-associated proteins and also impaired autophagic flux, shown by proximity ligation assay staining for LAMP2 and LC3II (autophagosome marker): 48 h of imatinib treatment reduced visible puncta to 2.7 ± 0.7/cell from 11.3 ± 2.1 puncta/cell in the control. Cell viability was partially recovered by autophagosome inhibition by wortmannin, with the viability increasing 91.8 ± 8.2% after imatinib-wortmannin co-treatment (84 ± 1.5% after imatinib). Imatinib-induced necroptosis was associated with an 8.5 ± 2.5-fold increase in mixed lineage kinase domain-like pseudokinase activation. Imatinib-induced toxicity was rescued by RIP1 inhibition: 88.6 ± 3.0% vs. 100.0 ± 0.0% in the control. Imatinib applied to human cardiac progenitor cells depolarises mitochondria and induces cell death through necroptosis, recoverable by RIP1 inhibition, with a partial role for autophagy.

Systemic GDF11 replenishment ignites myocardial injury through diminishing anti-apoptotic activity of cardiac progenitor cells

Abstract Background Tissue damage due to acute myocardial infarction is caused by both the ischemic insult and subsequent reperfusion injury (I/R). Restoration of coronary blood flow accelerates cardiomyocyte death, a phenomenon referred to as reperfusion injury, the extent of which is partly modulated by cardiac progenitor cells (CPC). Development of novel therapies to reduce infarct size, the main determinant of outcome, represent a huge unmet medical need (1). Systemic levels of growth differentiation factor 11 (GDF11), a TGF-β superfamily member that shares 90% homology with myostatin, decline with age, and GDF11 replenishment by heterochronic parabiosis or systemic recombinant GDF11 (rGDF11) delivery was postulated to have rejuvenating effects (2). Purpose We aimed to probe the effects of systemic GDF11 replenishment on I/R injury and deepen insights into the molecular mechanisms involved. Methods We designed a vehicle-controlled study in which young (3–4 months) and old (22–24 months) C57Bl/6 mice were randomly assigned to either daily systemic rGDF11 or control treatment over 30 days before myocardial I/R injury was induced. Dissected hearts were subjected to in-depth profiling followed by IPA-guided -omics to identify key regulatory mechanisms. Finally, in vitro experiments on human CPCs and HL-1 cardiomyocytes were performed. Results Myocardial Gdf11 expression declined with age, whereas myostatin (Mstn) showed an opposing expression pattern (Fig. 1A), a trend similarly observed upon I/R (Fig. 1B). Surprisingly, after the 30-day study period (Fig. 1C), young and aged rGDF11-treated mice showed higher I/R-induced infarct size and serum cardiac troponin I levels than controls, despite comparable areas at risk (Fig. 1D). Importantly, while proxies of necroptosis/pyroptosis remained unchanged, rGDF11-treated animals showed reduced cardiomyocyte viability irrespective of their age (Fig. 2A). Targeted transcriptomics applied on cardiac tissues of both groups identified the CPC-marker Nkx2–5 to be differentially regulated (Fig. 2B-C), an expression pattern validated in an independent cohort at both mRNA and protein levels (Fig. 2D). In the adult myocardium, the expression of both Nkx2–5 and its cofactor Gata4 is mainly confined to CPCs; indeed, similar reductions in Nkx2–5 and Gata4 expression were observed in CPCs exposed to rGDF11 (Fig. 2E) which coincided with accelerated cardiomyocyte death if cultured in conditioned media obtained from CPCs treated with rGDF11 (Fig. 2F), pointing toward a paracrine signalling pathway. Conclusions Myocardial expression of GDF11 declines with age, and is blunted upon I/R injury, thereby opposing the expression pattern of myostatin. Surprisingly, however, systemic GDF11 replenishment by rGDF11 supplementation enhances rather than reduces myocardial infarct size through augmented apoptosis, a phenomenon mediated by diminished cardioprotective function of CPCs. Funding Acknowledgement Type of funding sources: Foundation. Main funding source(s): Foundation for Cardiovascular Research – Zurich Heart House

Extracellular nucleophosmin is upregulated in psoriasis and correlates with determinants of cardiovascular diseases

Abstract Introduction We previously showed that genotoxic stress induced an active extracellular release of nucleophosmin (NPM) in human cardiac mesenchymal progenitor cells, and that serum deprivation provokes NPM secretion from human endothelial cells, eliciting inflammation via nuclear factor kappa B (NF-kB) transcriptional activation. Purpose The aim of this study was to determine whether NPM was modulated in skin and plasma of psoriatic patients (Pso). Methods 29 Pso were compared to 29 control subjects (Ctrl) age- and sex-matched. All Pso had a severe psoriasis, defined as Psoriasis Area and Severity Index (PASI) &amp;gt;10 and one of the following: at least two systemic psoriasis treatments, psoriasis onset &amp;lt;40 years of age, disease duration &amp;gt;10 years. Exclusion criteria were: diabetes, cardiovascular events, psoriatic arthritis. Total RNA was extracted from plasma and miR-200c levels assayed by quantitative real-time PCR. NPM levels were assayed by ELISA. The clinical parameters were similar between the two groups except for Total Cholesterol (mg/dl) (Ctrl 193.3±6.2; Pso 213.2±6.9; P&amp;lt;0.05). Blood pressure measurement, wave reflection analysis and pulse wave velocity (PWV) were similar between groups, echocardiographic parameters were different for left ventricular (LV) mass index (g/m2) (Ctrl 84.2±5.2; Pso 102.5±4.7; P&amp;lt;0.05) and relative wall thickness (RWT) (Ctrl 0.4±0.0; Pso 0.48±0.0; P&amp;lt;0.01). Total RNA was extracted from biopsies of nonlesional (NLS) and lesional (LS) of 6 Pso and 6 healthy subject skin (HS) from which human fibroblasts (HFs) and primary Keratinocytes (KCs) were extracted. Results NPM was upregulated in LS (1.9+0.3 fold; p=0.05) vs NLS biopsies (2.9+0.1 fold; p&amp;lt;0.01) and was also increased at the transcriptional level in LS vs NLS (8.6+ 1.7fold; p&amp;lt;0.05). Both the innate stimuli, such as lipopolysaccharides and Poly I:C and adaptive stimuli, i.e. cytokine mix, were able to induce extracellular release of NPM in immortalized KCs and HFs. Interestingly, NPM interacts with Toll like receptor (TLR)4 and activates a NF-kB-dependent inflammatory pathway upregulating interleukin IL-6 and COX-2 gene expression. Circulating NPM was increased in the plasma of 29 Pso (1.1+0.1ng/ml; p&amp;lt;0.001) compared to 29 healthy controls (0.78+ 0.03ng/ml) and positively correlates with PASI (Rs=0.44; p=0.05) and with determinants of cardiovascular diseases (CVDs), i.e. PWV (Rs=0.5; p&amp;lt;0.008), PASis (Rs= 0.4; P&amp;lt;0.05) and LV mass (Rs=0.49; p&amp;lt;0.05). Further, NPM positively correlates with miR-200c circulating levels (Rs=0.29; p&amp;lt;0.05), that we previously showed to increase in Pso and to correlate with CVD progression. We found that circulating miR-200c physically associated with NPM, that most probably is responsible for its extracellular release and protection upon cytokine mix via a TLR4-mechanism. Conclusions NPM is increased in psoriasis both in skin and in plasma and could be a novel biologic target to counteract chronic inflammation associated with CVD risk. Funding Acknowledgement Type of funding sources: Public grant(s) – EU funding. Main funding source(s): Italian Ministry of Health and AFM Telethon 22522 grant to AM

Receptor tyrosine kinase inhibitors negatively impact on pro-reparative characteristics of human cardiac progenitor cells

Receptor tyrosine kinase inhibitors improve cancer survival but their cardiotoxicity requires investigation. We investigated these inhibitors’ effects on human cardiac progenitor cells in vitro and rat heart in vivo. We applied imatinib, sunitinib or sorafenib to human cardiac progenitor cells, assessing cell viability, proliferation, stemness, differentiation, growth factor production and second messengers. Alongside, sunitinib effects were assessed in vivo. Inhibitors decreased (p < 0.05) cell viability, at levels equivalent to ‘peak’ (24 h; imatinib: 91.5 ± 0.9%; sunitinib: 83.9 ± 1.8%; sorafenib: 75.0 ± 1.6%) and ‘trough’ (7 days; imatinib: 62.3 ± 6.2%; sunitinib: 86.2 ± 3.5%) clinical plasma levels, compared to control (100% viability). Reduced (p < 0.05) cell cycle activity was seen with imatinib (29.3 ± 4.3% cells in S/G2/M-phases; 50.3 ± 5.1% in control). Expression of PECAM-1, Nkx2.5, Wnt2, linked with cell differentiation, were decreased (p < 0.05) 2, 2 and 6-fold, respectively. Expression of HGF, p38 and Akt1 in cells was reduced (p < 0.05) by sunitinib. Second messenger (p38 and Akt1) blockade affected progenitor cell phenotype, reducing c-kit and growth factor (HGF, EGF) expression. Sunitinib for 9 days (40 mg/kg, i.p.) in adult rats reduced (p < 0.05) cardiac ejection fraction (68 ± 2% vs. baseline (83 ± 1%) and control (84 ± 4%)) and reduced progenitor cell numbers. Receptor tyrosine kinase inhibitors reduce cardiac progenitor cell survival, proliferation, differentiation and reparative growth factor expression.

441-P: Oleate Prevents Palmitate-Induced Abnormalities in Insulin Signaling in Human Cardiac Progenitor Cells by Inhibiting p38 MAPK and c-Jun Phosphorylation

Elevated saturated fatty acid deposition in the heart results in insulin resistance, stress kinase activation, and increased cardiovascular risk in humans. The viability of human cardiac progenitor cells (hCPC) is essential for myocardium homeostasis. This study investigates the ability of palmitate, a saturated fatty acid, to impair insulin signaling in hCPC isolated from right auricle biopsies, and the potential protective effects of oleate, a mono-unsaturated fatty acid. hCPC were found to express both insulin receptor (IR) isoforms, IR-A and IR-B, with higher IR-A:IR-B ratio. Exposure of hCPC to 100 nM insulin for 15 min induced Akt (S473) phosphorylation (p&amp;lt;0.05) . However, treatment with 0.25 mM palmitate, but not with 0.1 mM oleate, for 24 h resulted in impaired insulin-induced Akt phosphorylation (p&amp;lt;0.05) with no effect on total Akt protein levels. Palmitate, but not oleate, also induced p38 MAPK (T180/Y182) and c-Jun (S63) phosphorylation (p&amp;lt;0.05) . Pretreatment for 1 h with 15 µM SB202190, a p38 MAPK inhibitor, or with 20 µM SP600125, a JNK inhibitor, significantly inhibited the ability of palmitate to impair insulin-induced Akt phosphorylation (p&amp;lt;0.05) . Interestingly, co-incubation of palmitate with oleate abolished the palmitate-induced p38 MAPK and c-Jun phosphorylation (p&amp;lt;0.05) and inhibition of insulin-induced Akt phosphorylation (p&amp;lt;0.05) . In addition, palmitate, but not oleate, was associated with downregulation of IR protein levels (p&amp;lt;0.05) , although with increased expression of total IR mRNA, IR-A, IR-B, and increased IR-A/IR-B ratio (p&amp;lt;0.05) . Co-incubation with oleate also prevented the palmitate-induced changes in IR (p&amp;lt;0.05) . In conclusion, oleate prevents the palmitate-induced abnormalities in insulin signaling in hCPC, largely by counteracting p38 MAPK and JNK activation. Hence, oleate supplementation might limit lipotoxicity, thus contributing to cardiac protection. Disclosure R.Doria: None. F.Giorgino: Advisory Panel; AstraZeneca, Boehringer Ingelheim International GmbH, Novo Nordisk, Consultant; Lilly Diabetes, Sanofi, Research Support; Lilly Diabetes, Roche Diabetes Care, Takeda Pharmaceutical Company Limited. L.Laviola: Advisory Panel; Lilly Diabetes, Novo Nordisk, Roche Diabetes Care, Sanofi, Speaker's Bureau; A. Menarini Diagnostics, Abbott Diabetes, Medtronic, Terumo Corporation. I.Calderoni: None. C.Caccioppoli: None. V.Genchi: None. G.Santarpino: None. A.Leonardini: None. A.Natalicchio: Consultant; Novo Nordisk, Other Relationship; AstraZeneca, Lilly Diabetes, Sanofi. S.Perrini: None. A.Cignarelli: None.

1414-P: Dapagliflozin Protects Human Cardiac Progenitor Cells from Deleterious Effects of the Secretome from Visceral Adipose Cells of Obese Subjects

Dysfunction of visceral and epicardial adipose tissue may alter heart performance and promote adverse cardiovascular outcomes in obesity and diabetes. We assessed whether the secretome from abdominal visceral (AV) and epicardial (E) adipose stem cells (ASC) and from AV mature adipocytes might affect the viability of human cardiac progenitor cells (hCPC) in human obesity, and the effects of dapagliflozin (DAPA) , a selective SGLT-2 inhibitor. ASC and mature adipocytes were isolated from AV and E adipose tissue biopsies of obese (Ob) and non-obese subjects, respectively. hCPC were isolated from right auricle biopsies of non-Ob, non-diabetic donors. hCPC were pretreated with uM DAPA for 24 h or left untreated, and then exposed to the conditioned media (CM) of AV-ASC, E-ASC, and AV mature adipocytes. The CM of AV-ASC, E-ASC, and AV mature adipocytes induced apoptosis, assessed by ELISA for cytoplasmic oligonucleosomes, in hCPC after 24 h (p&amp;lt;0.05) . Exposure of hCPC to the CM of AV-ASC, E-ASC, and AV mature adipocytes for 24 h also resulted in impaired organization of the hCPC actin filaments, assessed by indirect immunofluorescence (p&amp;lt;0.05) . In addition, the CM of AV-ASC, E-ASC, and AV mature adipocytes induced c-Jun phosphorylation, a measure of JNK activation, after 4 h and 8 h (p&amp;lt;0.05) . Importantly, these effects were not observed when the hCPC were exposed to the CM of AV-ASC, E-ASC, and AV mature adipocytes from non-Ob subjects. Pretreatment of hCPC with DAPA resulted in reduced apoptosis and less impairment of actin filaments following exposure to the CM of AV-ASC, E-ASC, and AV mature adipocytes (p&amp;lt;0.05) , as well as in reduced c-Jun phosphorylation (p&amp;lt;0.05) . In conclusion, in human obesity, the secretome of both AV and E ASC and mature adipocytes induces stress kinase activation in hCPC and impairs their viability and functionality, and these effects can be counteracted by SGLT-2 inhibitors directly acting on the hCPC. Disclosure G.Palma: None. F.Giorgino: Advisory Panel; AstraZeneca, Boehringer Ingelheim International GmbH, Novo Nordisk, Consultant; Lilly Diabetes, Sanofi, Research Support; Lilly Diabetes, Roche Diabetes Care, Takeda Pharmaceutical Company Limited. S.Perrini: None. C.Caccioppoli: None. R.Doria: None. V.Genchi: None. G.Santarpino: None. A.Cignarelli: None. A.Natalicchio: Consultant; Novo Nordisk, Other Relationship; AstraZeneca, Lilly Diabetes, Sanofi. L.Laviola: Advisory Panel; Lilly Diabetes, Novo Nordisk, Roche Diabetes Care, Sanofi, Speaker's Bureau; A. Menarini Diagnostics, Abbott Diabetes, Medtronic, Terumo Corporation. A.Pezzolla: None.