Abstract
Background:
Whether aging modifies mesenchymal stem cell (MSC) properties is unknown.
Aim:
To compare the differentiation capacity of human CD105+ MSCs obtained from young and elderly ...donors.
Methods and results:
Cells were obtained from young (n = 10, 24 ± 6.4 years) and elderly (n = 9, 77 ± 8.4 years) donors. Cell senescence was assessed by telomere length assays and lipofuscin accumulation. Cell pluripotentiality was analysed by adipogenic and osteogenic induction media, and myocyte phenotype was attempted with 5-azacytidine (5-AZ). Immunofluorescence, Western blot, transmission electron microscopy and fluo-4 confocal imaging were used to analyse the sarcomere, gap junctions and Ca2+ dynamics. Cells obtained from young and elderly donors showed no significant differences in relative telomere length (40.1 ± 6.4% and 40.3 ± 3.6%, p = 0.9) and lipofuscin accumulation. Adipogenic and osteogenic potential of CD105+ MSCs was demonstrated. 5-AZ induced increased expression of sarcomeric proteins without complete sarcomere organization. Treated cells also showed increased presence of connexin-43 both in young and old donor-derived cells. Intercellular communications were verified by the observation of gap junctions and passage of Ca2+ between neighbouring cells. Spontaneous Ca2+ raises did not significantly increase after 5-AZ treatment in both age groups.
Conclusion:
Age does not influence the adipogenic and myogenic differentiation potential of human CD105+ MSCs.
Mechanical conditioning is incompletely characterized for stimulating therapeutic cells within the physiological range. We sought to unravel the mechanism of action underlying mechanical conditioning ...of adipose tissue-derived progenitor cells (ATDPCs), both in vitro and in silico. Cardiac ATDPCs, grown on 3 different patterned surfaces, were mechanically stretched for 7 days at 1 Hz. A custom-designed, magnet-based, mechanical stimulator device was developed to apply ~10% mechanical stretching to monolayer cell cultures. Gene and protein analyses were performed for each cell type and condition. Cell supernatants were also collected to analyze secreted proteins and construct an artificial neural network. Gene and protein modulations were different for each surface pattern. After mechanostimulation, cardiac ATDPCs increased the expression of structural genes and there was a rising trend on cardiac transcription factors. Finally, secretome analyses revealed upregulation of proteins associated with both myocardial infarction and cardiac regeneration, such as regulators of the immune response, angiogenesis or cell adhesion. To conclude, mechanical conditioning of cardiac ATDPCs enhanced the expression of early and late cardiac genes in vitro. Additionally, in silico analyses of secreted proteins showed that mechanical stimulation of cardiac ATDPCs was highly associated with myocardial infarction and repair.
Our hypothesis was that overexpression of certain lipoprotein receptors might be related to lipid accumulation in the human ischemic myocardium. Intramyocardial lipid overload contributes to ...contractile dysfunction and arrhythmias in cardiomyopathy. Thus, the purpose of this study was to assess the effect of hypercholesterolemic LDL and hypertrigliceridemic VLDL dose on LRP1 expression in cardiomyocytes, as well as the potential correlation between LRP1 expression and neutral lipid accumulation in the left ventricle tissue from ischemic cardiomyopathy patients. Cell culture experiments include control and LRP1-deficient cardiomyocytes exposed to lipoproteins under normoxic and hypoxic conditions. Explanted hearts from 18 ICM patients and eight non-diseased hearts (CNT) were included. Low density lipoprotein receptor-related protein 1 (LRP1), very low density lipoprotein receptor (VLDLR) and low density lipoprotein receptor (LDLR) expression was analyzed by real time PCR and Western blotting. Cholesteryl ester (CE), triglyceride (TG) and free cholesterol (FC) content was assess by thin layer chromatography following lipid extraction. Western blotting experiments showed that protein levels of LRP1, VLDLR and HIF-1α were significantly upregulated in ischemic hearts. Immunohistochemistry and confocal microscopy analysis showed that LRP1 and HIF-1α were upregulated in cardiomyocytes of ICM patients. In vitro studies showed that VLDL, LDL and hypoxia exerted an upregulatory effect on LRP1 expression and that LRP1 played a major role in cholesteryl ester accumulation from lipoproteins in cardiomyocytes. Myocardial CE accumulation strongly correlated with LRP1 levels in ischemic hearts. Taken together, our results suggest that LRP1 upregulation is key for myocardial cholesterol ester accumulation in ischemic human hearts and that LRP1 may be a target to prevent the deleterious effects of myocardial cholesterol accumulation in ischemic cardiomyopathy.
Objective
To assess the arrhythmic safety profile of the adipose graft transposition procedure (AGTP) and its electrophysiological effects on post-myocardial infarction (MI) scar.
Background
...Myocardial repair is a promising treatment for patients with MI. The AGTP is a cardiac reparative therapy that reduces infarct size and improves cardiac function. The impact of AGTP on arrhythmogenesis has not been addressed.
Methods
MI was induced in 20 swine. Contrast-enhanced magnetic resonance (ce-MRI), electrophysiological study (EPS), and left-ventricular endocardial high-density mapping were performed 15 days post-MI. Animals were randomized 1:1 to AGTP or sham-surgery group and monitored with ECG-Holter. Repeat EPS, endocardial mapping, and ce-MRI were performed 30 days post-intervention. Myocardial SERCA2, Connexin-43 (Cx43), Ryanodine receptor-2 (RyR2), and cardiac troponin-I (cTnI) gene and protein expression were evaluated.
Results
The AGTP group showed a significant reduction of the total infarct scar, border zone and dense scar mass by ce-MRI (
p
= 0.04), and a decreased total scar and border zone area in bipolar voltage mapping (
p
< 0.001). AGTP treatment significantly reduced the area of very-slow conduction velocity (<0.2 m/s) (
p
= 0.002), the number of deceleration zones (
p
= 0.029), and the area of fractionated electrograms (
p
= 0.005). No differences were detected in number of induced or spontaneous ventricular arrhythmias at EPS and Holter-monitoring. SERCA2, Cx43, and RyR2 gene expression were decreased in the infarct core of AGTP-treated animals (
p
= 0.021,
p
= 0.018,
p
= 0.051, respectively).
Conclusion
AGTP is a safe reparative therapy in terms of arrhythmic risk and provides additional protective effect against adverse electrophysiological remodeling in ischemic heart disease.
Extracellular vesicles (EVs) include a variety of nanosized vesicles released to the extracellular microenvironment by the vast majority of cells transferring bioactive lipids, proteins, mRNA, miRNA ...or non-coding RNA, as means of intercellular communication. Remarkably, among other fields of research, their use has become promising for immunomodulation, tissue repair and as source for novel disease-specific molecular signatures or biomarkers. However, a major challenge is to define accurate, reliable and easily implemented techniques for EV isolation due to their nanoscale size and high heterogeneity. In this context, differential ultracentrifugation (dUC) has been the most widely used laboratory methodology, but alternative procedures have emerged to allow purer EV preparations with easy implementation. Here, we present and discuss the most used of the different EV isolation methods, focusing on the increasing impact of size exclusion chromatography (SEC) on the resulting EV preparations from in vitro cultured cells-conditioned medium and biological fluids. Comparatively, low protein content and cryo-electron microscopy analysis show that SEC removes most of the overabundant soluble plasma proteins, which are not discarded using dUC or precipitating agents, while being more user friendly and less time-consuming than gradient-based EV isolation. Also, SEC highly maintains the major EVs’ characteristics, including vesicular structure and content, which guarantee forthcoming applications. In sum, together with scaling-up possibilities to increase EV recovery and manufacturing following high-quality standards, SEC could be easily adapted to most laboratories to assist EV-associated biomarker discovery and to deliver innovative cell-free immunomodulatory and pro-regenerative therapies.
The optimal cell lineage for cardiac-regeneration approaches remains mysterious. Additionally, electrical stimulation promotes cardiomyogenic differentiation of stimulated cells. Therefore, we ...hypothesized that electrical conditioning of cardiomyocyte progenitor cells (CMPCs) might enrich their cardiovascular potential. CMPCs were isolated from human adult atrial appendages, characterized, and electrically stimulated for 7 and 14 days. Electrical stimulation modulated CMPCs gene and protein expression, increasing all cardiac markers. GATA-binding protein 4 (GATA4) early transcription factor was significantly overexpressed (P = 0.008), but also its coactivator myocyte enhancer factor 2A (MEF2A) was upregulated (P = 0.073) under electrical stimulation. Moreover, important structural proteins and calcium handling-related genes were enhanced. The cardioregeneration capability of CMPCs is improved by electrical field stimulation. Consequently, short-term electrical stimulation should be a valid biophysical approach to modify cardiac progenitor cells toward a cardiogenic phenotype, and can be incorporated into transdifferentiation protocols. Electrostimulated CMPCs may be best-equipped cells for myocardial integration after implantation.
Background:
Ultrastructural findings of idiopathic dilated cardiomyopathy (IDCM) include myocyte atrophy and myofilament loss, yet little is known about the vascular abnormalities present in IDCM.
...Methods and results:
Patients with IDCM and controls underwent multi-slice CT to examine length and diameter of epicardial vasculature. The levels of mobilizing cytokines and circulating EPCs were assessed by endothelial colony formation assay and flow cytometry. Immunohistochemistry and Western blot were used to examine microvessel density and expression of HIF-1α and β-catenin.
Main epicardial coronary arteries were shorter and smaller, and microvascular density was reduced in the epicardium in IDCM. Epicardial vessel paucity was associated with increased numbers of HIF-1α+ cells (46.8±13.1% vs. 19.4±9.4%, p=0.006) indicating local epicardial hypoxia and elevation of circulating VEGF-A (394pg/mL vs. 22pg/mL, p=0.001). The number of mobilized progenitors CD133+/VEGF-R2+ was 21-fold higher in IDCM compared with controls (6.5±3.3% vs. 0.3±0.2%; p<0.001). Moreover, this defective vascularization was associated with reduced myocardial expression of vascular β-catenin, an important angiogenic regulator.
Conclusions:
This study shows defective vascularization and impaired vasculogenesis (the de novo vascular organization of mobilized endothelial progenitors) and angiogenesis (by which new blood vessels are formed from pre-existing mature endothelial cells) in human IDCM.
The present study evaluates the safety and efficacy of the Adipose Graft Transposition Procedure (AGTP) as a biological regenerative innovation for patients with a chronic myocardial scar.
This ...prospective, randomized single-center controlled study included 10 patients with established chronic transmural myocardial scars. Candidates for myocardial revascularization were randomly allocated into two treatment groups. In the control arm (n=5), the revascularizable area was treated with CABG and the non-revascularizable area was left untouched. Patients in the AGTP-treated arm (n=5) were treated with CABG and the non-revascularizable area was covered by a biological adipose graft. The primary endpoint was the appearance of adverse effects derived from the procedure including hospital admissions and death, and 24-hour Holter monitoring arrhythmias at baseline, 1week, and 3 and 12months. Secondary endpoints of efficacy were assessed by cardiac MRI.
No differences in safety were observed between groups in terms of clinical or arrhythmic events. On follow-up MRI testing, participants in the AGTP-treated arm showed a borderline smaller left ventricular end systolic volume (LVESV; p=0.09) and necrosis ratio (p=0.06) at 3months but not at 12months. The AGTP-treated patient with the largest necrotic area and most dilated chambers experienced a noted improvement in necrotic mass size (−10.8%), and ventricular volumes (LVEDV: −55.2mL and LVESV: −37.8mL at one year follow-up) after inferior AGTP.
Our results indicate that AGTP is safe and may be efficacious in selected patients. Further studies are needed to assess its clinical value. (ClinicalTrials.org NCT01473433, AdiFlap Trial).
•The use of an autologous vascular adipose graft may be a safe alternative in patients with a chronic myocardial scar.•The Adipose Graft Transposition Procedure (AGTP) has autologous nature and is easy to implement.•Its translation to clinical practice should not be hampered by technical, economic, or ethical/social considerations.
The Adipose Graft Transposition Procedure (AGTP) is a new surgical technique for reducing the scar after a heart attack. It has been evaluated in 10 patients for the first time and has been proved to be safe. This technique consists on the use of the existing fat surrounding the heart that has beneficial properties. The procedure is easy for the surgeons to perform. The next step will be to test the efficacy in a larger population.
Idiopathic dilated cardiomyopathy (IDCM) is defined as myocardial dilatation and dysfunction in the absence of overt coronary heart disease. Myocardial injury and genetic or environmental factors can ...lead to the development of IDCM, which was historically characterized by marked abnormalities in the function and integrity of cardiomyocytes. However, cardiac endothelial dysfunction has also been shown to be associated with progression and poor prognosis of IDCM. Moreover, marked vascular derangements and impaired vasculogenic and angiogenic responses have been reported in patients with IDCM. On the basis of these data we re-examine IDCM pathophysiology as a downstream complication of vascular derangements that contribute to myocyte damage. Animal models closely resembling the marked vascular alterations found in patients with IDCM will be of paramount importance for further enhancing our comprehension of disease progression and for testing new drugs and stem-cell or gene-based therapies.
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