Neogenesis of β-cells is the process that allows the endocrine pancreas to meet the increasing demands in insulin secretion occurring with pregnancy and obesity, or following partial pancreatectomy. ...The critical question concerns the origin of insulin secreting cells. First, we determined the origin of pancreatic cells in a transgenic mouse model in which the tamoxifen-inducible MerCreMer protein was targeted to the Kit locus. These mice were cross-bred with an R-GFP reporter line to irreversibly label with GFP any cell that expressed c-kit at the time of tamoxifen administration. After tamoxifen for 6-8 months, acinar cells were intensely positive for GFP. Additionally, GFP-labeled β-cells were consistently found within the islets of Langerhans. Another fate mapping model was tested, the Kit mouse, in which a different region of the c-kit promoter was targeted. Following tamoxifen for 2-3 months, large regions of the mouse pancreas were positive for GFP, amylase and insulin, indicating that in both mice these specialized cells derived from c-kit-positive cells. For the detection of distinct intracellular proteins, a group of mice was treated with tamoxifen for 6 weeks; this protocol required tissue digestion, fixation and immunolabeling of the isolated cells and the analysis of the various compartments by flow cytometry. The freshly isolated pancreas showed large areas positive for native GFP. Importantly, the pool size of freshly isolated non-fixed cells showing native GFP was comparable to that present in fixed pancreatic cells displaying immunolabeled GFP. Within the entire compartment of pancreatic cells, the fraction of insulin, C-peptide and glucagon positive cells expressing GFP was 6%, 20% and 14%, respectively. The large percentage of non-endocrine cells expressing GFP may reflect the pool of acinar cells synthesizing amylase. Subsequently, the frequency of insulin-positive, C-peptide-positive and glucagon-positive cells labeled by GFP within each cell class was measured. The fraction of double positive cells averaged approximately 20% in all cases. In conclusion, these results strongly suggest that the mammalian pancreas contains a pool of c-kit-positive stem cells which are multipotent and may regulate organ homeostasis.
Diabetes is a major cause of human heart failure, and despite of the progress made in the control of this illness, a cure able to reverse the process and interfere with its systemic consequences is ...lacking. The recognition of a human pancreatic stem cell (hPSC) that regulates β-cell turnover and regeneration would provide, theoretically, a novel therapeutic target for this devastating disease. The c-kit epitope has been employed previously for the identification of hematopoietic, cardiac and lung stem cells, raising the possibility that this receptor tyrosine kinase may be expressed in other classes of tissue specific adult stem cells. Based on this simple hypothesis, we tested whether the presence of c-kit uncovered a pool of hPSCs, which, by analogy to stem cells in other organs, are self-renewing, clonogenic and multipotent. Twenty-nine samples of normally appearing human pancreas discarded at surgery were studied. Histologically, c-kit-positive tryptase-negative putative hPSCs were identified in all cases examined. They were located within the islets, in proximity of acinar cells, or within the epithelium of pancreatic ducts. Following isolation and in vitro expansion, c-kit-positive cells were FACS-sorted, seeded individually in single wells of 96-well-plates, and in 3 weeks, multi-cellular clones were obtained. Clonal cells had the ability to commit to endocrine β-cells and α-cells, and exocrine acinar-cells, generating the corresponding hormones and enzyme. qRT-PCR and NanoString technology were employed to determine whether clonal hPSCs possessed a transcriptional profile consistent with the undifferentiated and multipotent state of established pancreatic progenitors. Clonal hPSCs expressed c-kit, together with Pdx1, Sox9, Ngn3 and Nkx6.1, major determinants of progenitor cell fate. During differentiation, the components of the Notch pathway were downregulated pointing to their acquisition of the β-cell lineage. Thus, we suggest a new paradigm of pancreas biology in which hPSCs are critical determinants of endocrine and exocrine cell renewal. Understanding the mechanisms of β-cell formation may provide the opportunity to potentiate this naturally occurring process offering a novel strategy for the management of human diabetes.
Chronic obstructive pulmonary disease (COPD) is characterized by chronic inflammation, enlargement of bronchioles and alveoli, destruction of the alveolar walls, and ultimately respiratory failure. ...The recognition that the distal airways contain a pool of c-kit-positive lung stem cells (LSCs) suggests that the adverse evolution of COPD may be mediated by a decrease in LSCs and their ability to restore organ integrity. To test this hypothesis, the number and phenotypic properties of LSCs from control (N=13) and COPD (N=7) lungs were determined to establish a potential relationship between stem cell growth and differentiation and the pathobiology of the disease. We identified that the compartment of c-kit-positive LSCs contains a cell population that does not express the mesenchymal epitopes CD44/CD73/CD105, i.e., non-mesenchymal LSCs (non-mLSCs) and a cell class that is positive for these epitopes, i.e., mesenchymal-like LSCs (mLSCs). In control lungs, 77% of c-kit-positive cells were non-mLSCs and 23% were mLSCs. With COPD, however, the proportion of non-mLSCs, 49%, and mLSCs, 51%, was comparable, resulting in an increase in the pool of mLSCs and a decrease in the fraction of non-mLSCs; the loss of this balance may contribute to lung pathology. Importantly, non-mLSCs generated type-1 and type-2 alveolar epithelial cells and capillary endothelial cells, while mLSCs failed to form these specific cell types. The components of the gas exchange units appear to be under the control of a multipotent non-mLSC which, in a coordinated manner, regulates the structures responsible for the gas transfer across the epithelial-endothelial barrier. Because of the controversy in the field, the relevance of lung c-kit-positive cells was ascertained by lineage tracing in mice. The fate mapping strategy clearly documented that c-kit-positive cells differentiate into type-1 and type-2 alveolar epithelial cells and capillary endothelial cells, strengthening the observations in humans. Thus, the c-kit epitope distinguishes a class of stem/progenitor cells which differentiate into structures of endodermal and mesodermal origin, and the delivery of this class of non-mLSCs may have therapeutic importance in patients with COPD and hypoxemic respiratory failure.
A very late thrombosis in a bioresorbable vascular scaffold Giacchi, Marco; Pedrazzini, Giovanni; Moccetti, Tiziano ...
EuroIntervention : journal of EuroPCR in collaboration with the Working Group on Interventional Cardiology of the European Society of Cardiology
11, Številka:
7
Journal Article
Recent evidence suggests that cardiac progenitor cells (CPCs) may improve cardiac function after injury. The underlying mechanisms are indirect, but their mediators remain unidentified. Exosomes and ...other secreted membrane vesicles, hereafter collectively referred to as extracellular vesicles (EVs), act as paracrine signalling mediators. Here, we report that EVs secreted by human CPCs are crucial cardioprotective agents.
CPCs were derived from atrial appendage explants from patients who underwent heart valve surgery. CPC-conditioned medium (CM) inhibited apoptosis in mouse HL-1 cardiomyocytic cells, while enhancing tube formation in human umbilical vein endothelial cells. These effects were abrogated by depleting CM of EVs. They were reproduced by EVs secreted by CPCs, but not by those secreted by human dermal fibroblasts. Transmission electron microscopy and nanoparticle tracking analysis showed most EVs to be 30-90 nm in diameter, the size of exosomes, although smaller and larger vesicles were also present. MicroRNAs most highly enriched in EVs secreted by CPCs compared with fibroblasts included miR-210, miR-132, and miR-146a-3p. miR-210 down-regulated its known targets, ephrin A3 and PTP1b, inhibiting apoptosis in cardiomyocytic cells. miR-132 down-regulated its target, RasGAP-p120, enhancing tube formation in endothelial cells. Infarcted hearts injected with EVs from CPCs, but not from fibroblasts, exhibited less cardiomyocyte apoptosis, enhanced angiogenesis, and improved LV ejection fraction (0.8 ± 6.8 vs. -21.3 ± 4.5%; P < 0.05) compared with those injected with control medium.
EVs are the active component of the paracrine secretion by human CPCs. As a cell-free approach, EVs could circumvent many of the limitations of cell transplantation.
Abstract
Aims
Cell therapy trials using cardiac-resident progenitor cells (CPCs) and bone marrow-derived mesenchymal stem/progenitor cells (BMCs) in patients after myocardial infarction have provided ...encouraging results. Exosomes, nanosized extracellular vesicles of endosomal origin, figure prominently in the bioactivities of these cells. However, a head-to-head comparison of exosomes from the two cell types has not been performed yet.
Methods and results
CPCs and BMCs were derived from cardiac atrial appendage specimens and sternal bone marrow, respectively, from patients (n = 20; age, 69.9 ± 10.9) undergoing heart surgery for aortic valve disease and/or coronary artery disease. Vesicles were purified from cell conditioned media by centrifugation/filtration and ultracentrifugation. Vesicle preparations were predominantly composed of exosomes based on particle size and marker expression (CD9, CD63, CD81, Alix, and TSG-101). CPC-secreted exosomes prevented staurosporine-induced cardiomyocyte apoptosis more effectively than BMC-secreted exosomes. In vivo, CPC-secreted exosomes reduced scar size and improved ventricular function after permanent coronary occlusion in rats more efficiently than BMC-secreted exosomes. Both types of exosomes stimulated blood vessel formation. CPC-secreted exosomes, but not BMC-derived exosomes, enhanced ventricular function after ischaemia/reperfusion. Proteomics profiling identified pregnancy-associated plasma protein-A (PAPP-A) as one of the most highly enriched proteins in CPC vs. BMC exosomes. The active form of PAPP-A was detected on CPC exosome surfaces. These vesicles released insulin-like growth factor-1 (IGF-1) via proteolytic cleavage of IGF-binding protein-4 (IGFBP-4), resulting in IGF-1 receptor activation, intracellular Akt and ERK1/2 phosphorylation, decreased caspase activation, and reduced cardiomyocyte apoptosis. PAPP-A knockdown prevented CPC exosome-mediated cardioprotection both in vitro and in vivo.
Conclusion
These results suggest that CPC-secreted exosomes may be more cardioprotective than BMC-secreted exosomes, and that PAPP-A–mediated IGF-1 release may explain the benefit. They illustrate a general mechanism whereby exosomes may function via an active protease on their surface, which releases a ligand in proximity to the transmembrane receptor bound by the ligand.
Oral P2Y12 receptor antagonists exhibit delayed onset of platelet inhibition in patients with acute myocardial infarction (AMI). Selatogrel is a potent, highly selective, and reversible P2Y12 ...receptor antagonist with a rapid onset and short duration of action.
This study sought to assess inhibition of platelet aggregation following subcutaneous administration of selatogrel in patients with AMI.
Patients with AMI were randomized to a single subcutaneous dose of selatogrel of 8 or 16 mg. The primary endpoint was response to treatment (P2Y12 reaction units <100; measured by VerifyNow) at 30 min post-dose. Safety was assessed up to 48 h post-injection.
Forty-seven patients received selatogrel 8 mg (n = 24) or 16 mg (n = 23) followed by ticagrelor (n = 43) or clopidogrel (n = 1). The proportion of responders 30 min post-dose was 91% (one-sided 97.5% confidence interval CI: 80% to 100%) and 96% (97.5% CI: 87% to 100%) with 8 and 16 mg, respectively (p values for responders >85% target; p = 0.142 and p = 0.009, respectively). Response rates were independent from type of AMI presentation, age, or sex. A similar response rate was observed at 15 min (8 mg: 75% 97.5% CI: 58% to 100%; 16 mg: 91% 97.5% CI: 80% to 100%), which was sustained at 60 min post-dose (8 mg: 75% 97.5% CI: 58% to 100%; 16 mg: 96% 97.5% CI: 87% to 100%). At 15 min, median P2Y12 reaction units was 51 (range: 4 to 208) for 8 mg and 9 (range: 2 to 175) for 16 mg. Selatogrel was well tolerated, without major bleeding complications.
Single-dose subcutaneous administration of selatogrel in patients with AMI was safe and induced a profound, rapid, and dose-related antiplatelet response. (A Medical Research Study to Evaluate the Effects of ACT-246475 in Adults With Heart Attack; NCT03487445, 2018-000765-36 EudraCT)
Display omitted
PDF
