Effect of neuron‐derived neurotrophic factor on rejuvenation of human adipose‐derived stem cells for cardiac repair after myocardial infarction

The decline of cell function caused by ageing directly impacts the therapeutic effects of autologous stem cell transplantation for heart repair. The aim of this study was to investigate whether overexpression of neuron‐derived neurotrophic factor (NDNF) can rejuvenate the adipose‐derived stem cells in the elderly and such rejuvenated stem cells can be used for cardiac repair. Human adipose‐derived stem cells (hADSCs) were obtained from donors age ranged from 17 to 92 years old. The effects of age on the biological characteristics of hADSCs and the expression of ageing‐related genes were investigated. The effects of transplantation of NDNF over‐expression stem cells on heart repair after myocardial infarction (MI) in adult mice were investigated. The proliferation, migration, adipogenic and osteogenic differentiation of hADSCs inversely correlated with age. The mRNA and protein levels of NDNF were significantly decreased in old (>60 years old) compared to young hADSCs (<40 years old). Overexpression of NDNF in old hADSCs significantly improved their proliferation and migration capacity in vitro. Transplantation of NDNF‐overexpressing old hADSCs preserved cardiac function through promoting angiogenesis on MI mice. NDNF rejuvenated the cellular function of aged hADSCs. Implantation of NDNF‐rejuvenated hADSCs improved angiogenesis and cardiac function in infarcted mouse hearts.     

A single-nucleotide polymorphism in the human p27<Superscript>kip1</Superscript>gene (-838C>A) affects basal promoter activity and the risk of myocardial infarction

Background  

Excessive proliferation of vascular smooth muscle cells and leukocytes within the artery wall is a major event in the development of atherosclerosis. The growth suppressor p27^kip1 associates with several cyclin-dependent kinase/cyclin complexes, thereby abrogating their capacity to induce progression through the cell cycle. Recent studies have implicated p27^kip1 in the control of neointimal hyperplasia. For instance, p27^kip1 ablation in apolipoprotein-E-null mice enhanced arterial cell proliferation and accelerated atherogenesis induced by dietary cholesterol. Therefore, p27^kip1 is a candidate gene to modify the risk of developing atherosclerosis and associated ischaemic events (i.e., myocardial infarction and stroke).     

Loss of Sirt3 Limits Bone Marrow Cell-Mediated Angiogenesis and Cardiac Repair in Post-Myocardial Infarction

Sirtuin-3 (Sirt3) has a critical role in the regulation of human aging and reactive oxygen species (ROS) formation. A recent study has identified Sirt3 as an essential regulator of stem cell aging. This study investigated whether Sirt3 is necessary for bone marrow cell (BMC)-mediated cardiac repair in post-myocardial infarction (MI). In vitro, BMC-derived endothelial progenitor cells (EPCs) from wild type (WT) and Sirt3KO mice were cultured. EPC angiogenesis, ROS formation and apoptosis were assessed. In vivo, WT and Sirt3 KO mice were subjected to MI and BMCs from WT and Sirt3 KO mice were injected into ischemic area immediately. The expression of VEGF and VEGFR2 was reduced in Sirt3KO-EPCs. Angiogenic capacities and colony formation were significantly impaired in Sirt3KO-EPCs compared to WT-EPCs. Loss of Sirt3 further enhanced ROS formation and apoptosis in EPCs. Overexpression of Sirt3 or treatment with NADPH oxidase inhibitor apocynin (Apo, 200 and 400 microM) rescued these abnormalities. In post-MI mice, BMC treatment increased number of Sca1⁺/c-kit⁺ cells; enhanced VEGF expression and angiogenesis whereas Sirt3KO-BMC treatment had little effects. BMC treatment also attenuated NADPH oxidase subunits p47^phox and gp91^phox expression, and significantly reduced ROS formation, apoptosis, fibrosis and hypertrophy in post-MI mice. Sirt3KO-BMC treatment did not display these beneficial effects. In contrast, Sirt3KO mice treated with BMCs from WT mice attenuated myocardial apoptosis, fibrosis and improved cardiac function. Our data demonstrate that Sirt3 is essential for BMC therapy; and loss of Sirt3 limits BMC-mediated angiogenesis and cardiac repair in post-MI.     

VEGF improves survival of mesenchymal stem cells in infarcted hearts

Bone marrow-derived mesenchymal stem cells (MSC) are a promising source for cell-based treatment of myocardial infarction (MI), but existing strategies are restricted by low cell survival and engraftment. We examined whether vascular endothelial growth factor (VEGF) improve MSC viability in infracted hearts. We found long-term culture increased MSC-cellular stress: expressing more cell cycle inhibitors, p16^INK, p21 and p19^ARF. VEGF treatment reduced cellular stress, increased pro-survival factors, phosphorylated-Akt and Bcl-xL expression and cell proliferation. Co-injection of MSCs with VEGF to MI hearts increased cell engraftment and resulted in better improvement of cardiac function than that injected with MSCs or VEGF alone. In conclusion, VEGF protects MSCs from culture-induce cellular stress and improves their viability in ischemic myocardium, which results in improvements of their therapeutic effect for the treatment of MI.     

Long‐term repopulation of aged bone marrow stem cells using young Sca‐1 cells promotes aged heart rejuvenation

Reduced quantity and quality of stem cells in aged individuals hinders cardiac repair and regeneration after injury. We used young bone marrow (BM) stem cell antigen 1 (Sca‐1) cells to reconstitute aged BM and rejuvenate the aged heart, and examined the underlying molecular mechanisms. BM Sca‐1⁺ or Sca‐1^? cells from young (2–3 months) or aged (18–19 months) GFP transgenic mice were transplanted into lethally irradiated aged mice to generate 4 groups of chimeras: young Sca‐1⁺, young Sca‐1^?, old Sca‐1⁺, and old Sca‐1^?. Four months later, expression of rejuvenation‐related genes (Bmi1, Cbx8, PNUTS, Sirt1, Sirt2, Sirt6) and proteins (CDK2, CDK4) was increased along with telomerase activity and telomerase‐related protein (DNA‐PKcs, TRF‐2) expression, whereas expression of senescence‐related genes (p16^INK4a, P19^ARF, p27^Kip1) and proteins (p16^INK4a, p27^Kip1) was decreased in Sca‐1⁺ chimeric hearts, especially in the young group. Host cardiac endothelial cells (GFP^?CD31⁺) but not cardiomyocytes were the primary cell type rejuvenated by young Sca‐1⁺ cells as shown by improved proliferation, migration, and tubular formation abilities. C‐X‐C chemokine CXCL12 was the factor most highly expressed in homed donor BM (GFP⁺) cells isolated from young Sca‐1⁺ chimeric hearts. Protein expression of Cxcr4, phospho‐Akt, and phospho‐FoxO3a in endothelial cells derived from the aged chimeric heart was increased, especially in the young Sca‐1⁺ group. Reconstitution of aged BM with young Sca‐1⁺ cells resulted in effective homing of functional stem cells in the aged heart. These young, regenerative stem cells promoted aged heart rejuvenation through activation of the Cxcl12/Cxcr4 pathway of cardiac endothelial cells.     

Transplantation of marrow-derived cardiac stem cells carried in designer self-assembling peptide nanofibers improves cardiac function after myocardial infarction

Progress in stem cell transplantation for the treatment of myocardial infarction is hampered by the poor retention and survival of the implanted cells. To enhance cell survival and differentiation and thereby improve the efficiency of stem cell therapy, we constructed a novel self-assembling peptide by attaching an RGDSP cell-adhesion motif to the self-assembling peptide RADA16. c-kit^pos/Nkx2.5^low/GATA4^low marrow-derived cardiac stem cells (MCSCs), which have a specific potential to differentiate into cardiomyocytes, were isolated from rat bone marrow. The cytoprotective effects of RGDSP scaffolds were assessed by exposure of MCSCs to anoxia in vitro. The efficacy of transplanting MCSCs in RGDSP scaffolds was evaluated in a female rat MI model. The designer self-assembling peptide self-assembled into RGDSP nanofiber scaffolds under physiological conditions. RGDSP scaffolds were beneficial for the growth of MCSCs and protected them from apoptosis and necrosis caused by anoxia. In a rat MI model, cardiac function was improved and collagen deposition was markedly reduced in the group receiving MCSCs in RGDSP scaffolds compared with groups receiving MCSCs alone, RGDSP scaffolds alone or MCSCs in RADA16 scaffolds. There were more surviving MCSCs in the group receiving MCSCs in RGDSP scaffolds than in the groups receiving MCSCs alone or MCSCs in RADA16 scaffolds. Most of the Y chromosome-positive cells expressed cardiac troponin T and connexin43 (Cx-43). These results suggest that RGDSP scaffolds provide a suitable microenvironment for the survival and differentiation of MCSCs. RGDSP scaffolds enhanced the efficacy of MCSC transplantation to repair myocardium and improve cardiac function.     

Hepatocyte growth factor (HGF) inhibits skeletal muscle cell differentiation: a role for the bHLH protein twist and the cdk inhibitor p27

Hepatocyte growth factor (HGF) plays a crucial role in regulating the differentiation of both fetal and adult skeletal myoblasts. This study aimed at defining the intracellular factors that mediate the effect of HGF on adult myoblast differentiation. HGF increased Twist expression while decreasing p27(kip1) protein levels and not affecting the induction of p21(Cip1/Waf1) in satellite cells. Like HGF, overexpression of Twist did not affect p21 expression while inhibiting muscle-specific proteins. Both ectopic Twist-antisense (Twist-AS) and p27 partially rescued the effects of HGF on bromodeoxyuridine (BrdU) incorporation and myosin heavy chain (MHC) expression in muscle satellite cells; the two plasmids together effected full rescue, suggesting that HGF independently regulates these two factors to mediate its effects. Ectopic p27 promoted differentiation in the presence of HGF by blocking the induction of Twist. Using Twist-AS to lower Twist levels restored the HGF-dependent reduction of p27 and MHC. In the presence of ectopic HGF, satellite cells formed thin mononuclear myotubes. Neither ectopic p27, Twist-AS, or their combination reversed this change in cell morphology, suggesting that HGF acts through additional mediators to inhibit downstream events during myogenesis. Taken together, the results suggest that the effects of HGF on muscle cell proliferation and differentiation are mediated through changes in the expression levels of the myogenic-inhibitory basic helix-loop-helix (bHLH) protein Twist and the cell-cycle inhibitor p27.     

MicroRNA-1 transfected embryonic stem cells enhance cardiac myocyte differentiation and inhibit apoptosis by modulating the PTEN/Akt pathway in the infarcted heart

microRNAs (miRs) have emerged as critical modulators of various physiological processes including stem cell differentiation. Indeed, miR-1 has been reported to play an integral role in the regulation of cardiac muscle progenitor cell differentiation. However, whether overexpression of miR-1 in embryonic stem (ES) cells (miR-1-ES cells) will enhance cardiac myocyte differentiation following transplantation into the infarcted myocardium is unknown. In the present study, myocardial infarction (MI) was produced in C57BL/6 mice by left anterior descending artery ligation. miR-1-ES cells, ES cells, or culture medium (control) was transplanted into the border zone of the infarcted heart, and 2 wk post-MI, cardiac myocyte differentiation, adverse ventricular remodeling, and cardiac function were assessed. We provide evidence demonstrating enhanced cardiac myocyte commitment of transplanted miR-1-ES cells in the mouse infarcted heart as compared with ES cells. Assessment of apoptosis revealed that overexpression of miR-1 in transplanted ES cells protected host myocardium from MI-induced apoptosis through activation of p-AKT and inhibition of caspase-3, phosphatase and tensin homolog, and superoxide production. A significant reduction in interstitial and vascular fibrosis was quantified in miR-1-ES cell and ES cell transplanted groups compared with control MI. However, no statistical significance between miR-1-ES cell and ES cell groups was observed. Finally, mice receiving miR-1-ES cell transplantation post-MI had significantly improved heart function compared with respective controls (P < 0.05). Our data suggest miR-1 drives cardiac myocyte differentiation from transplanted ES cells and inhibits apoptosis post-MI, ultimately giving rise to enhanced cardiac repair, regeneration, and function.     

Bone marrow‐derived cells can acquire cardiac stem cells properties in damaged heart

Experimental data suggest that cell‐based therapies may be useful for cardiac regeneration following ischaemic heart disease. Bone marrow (BM) cells have been reported to contribute to tissue repair after myocardial infarction (MI) by a variety of humoural and cellular mechanisms. However, there is no direct evidence, so far, that BM cells can generate cardiac stem cells (CSCs). To investigate whether BM cells contribute to repopulate the Kit⁺ CSCs pool, we transplanted BM cells from transgenic mice, expressing green fluorescent protein under the control of Kit regulatory elements, into wild‐type irradiated recipients. Following haematological reconstitution and MI, CSCs were cultured from cardiac explants to generate ‘cardiospheres’, a microtissue normally originating in vitro from CSCs. These were all green fluorescent (i.e. BM derived) and contained cells capable of initiating differentiation into cells expressing the cardiac marker Nkx2.5. These findings indicate that, at least in conditions of local acute cardiac damage, BM cells can home into the heart and give rise to cells that share properties of resident Kit⁺ CSCs.     

Effects of hepatocyte growth factor overexpressed bone marrow‐derived mesenchymal stem cells on prevention from left ventricular remodelling and functional improvement in infarcted rat hearts

Bone marrow‐derived mesenchymal stem cells (BM‐MSCs ) transplantation has been reported to be a promising therapy for myocardial infarction (MI). However, low survival rate of BM‐MSCs in infarcted heart is one of the major limitations for the perspective clinical application. In this study, we aimed to investigate the effect of hepatocyte growth factor (HGF) on left ventricular function improvement of HGF gene‐modified BM‐MSCs (HGF‐MSCs) after its delivery into the infarcted rat hearts. BM‐MSCs were isolated with fibroblast‐like morphology and expressed CD44+CD29+CD90+/CD34‐CD45‐CD31‐CD11a. After 5‐azacytidine induction in vitro, 20%–30% of the cells were positively stained for desmin, cardiac‐specific cardiac troponin I and connexin‐43. Histological staining revealed that 2 weeks after MI is an optimal time point with decreased neutrophil infiltration and increased vascular number. Minimal infarct size and best haemodynamic analysis were also observed after cell injection at 2 weeks compared with that of 1 h, 1 week or 4 weeks. Echocardiogram confirmed that transplantation with HGF‐MSCs significantly improved left ventricular function compared with other groups in rat MI models. MSCs and HGF‐MSCslabelled with DAPI were detected 4 weeks after MI in the infarcted area. Decreased infarcted scar area and increased angiogenesis formation could be found in HGF‐MSCs group than in other groups as demonstrated by hematoxylin and eosin (H&E) staining and factor VIII staining. These results indicate that HGF‐MSCs transplantation could enhance the contractile function and attenuate left ventricular remodelling efficiently in rats with MI. Copyright © 2012 John Wiley & Sons, Ltd.     

Depletion of histone demethylase KDM2A inhibited cell proliferation of stem cells from apical papilla by de-repression of p15INK4B and p27Kip1

Mesenchymal stem cells (MSCs) are a reliable resource for tissue regeneration; although, the molecular mechanisms of their differentiation and proliferation are not clearly understood, which restricts the applications of MSCs. The histone demethylase, lysine (K)-specific demethylase 2A (KDM2A), and the mammalian paralog, lysine (K)-specific demethylase 2B (KDM2B), are evolutionarily conserved and ubiquitously expressed members of the JmjC-domain-containing histone demethylase family. A previous study determined that KDM2A and KDM2B can regulate the differentiation of MSCs, and KDM2B has been implicated in cell cycle regulation by de-repressing p15^INK4B (cyclin-dependent kinase inhibitor 2B). It is not known whether KDM2A is involved in the cell proliferation of MSCs. Here, we show that depletion of KDM2A by short hairpin RNAs can inhibit cell proliferation and arrest cell cycle progression at the G1/S-phase in human stem cells from apical papilla (SCAPs). The effect of KDM2A on cell proliferation was found to be mediated through de-repression of the cyclin-dependent kinase inhibitors, p15^INK4B and p27^Kip1 (cyclin-dependent kinase inhibitor 1B), in KDM2A knock-down SCAPs. Furthermore, chromatin immunoprecipitation assays demonstrated that silencing of KDM2A increased histone H3 Lysine 4 (H3K4) trimethylation at the p15^INK4B and p27^Kip1 loci and regulated its expression. Together, our results indicate that KDM2A is a H3K4 demethylase that regulates cell proliferation through p15^INK4B and p27^Kip1 in SCAPs.     

Simultaneous Deletion of p21Cip1/Waf1 and Caspase-3 Accelerates Proliferation and Partially Rescues the Differentiation Defects of Caspase-3 Deficient Hematopoietic Stem Cells

Specialized blood cells are generated through the entire life of an organism by differentiation of a small number of hematopoietic stem cells (HSC). There are strictly regulated mechanisms assuring a constant and controlled production of mature blood cells. Although such mechanisms are not completely understood, some factors regulating cell cycle and differentiation have been identified. We have previously shown that Caspase-3 is an important regulator of HSC homeostasis and cytokine responsiveness. p21^cip1/waf1 is a known cell cycle regulator, however its role in stem cell homeostasis seems to be limited. Several reports indicate interactions between p21^cip1/waf1 and Caspase-3 in a cell type dependent manner. Here we studied the impact of simultaneous depletion of both factors on HSC homeostasis. Depletion of both Caspase-3 and p21^cip1/waf1 resulted in an even more pronounced increase in the frequency of hematopoietic stem and progenitor cells. In addition, simultaneous deletion of both genes revealed a further increase of cell proliferation compared to single knock-outs and WT control mice, while apoptosis or self-renewal ability were not affected in any of the genotypes. Upon transplantation, p21^cip1/waf1-/- bone marrow did not reveal significant alterations in engraftment of lethally irradiated mice, while Caspase-3 deficient HSPC displayed a significant reduction of blood cell production. However, when both p21^cip1/waf1 and Caspase-3 were eliminated this differentiation defect caused by Caspase-3 deficiency was abrogated.     

Transplantation of expanded bone marrow‐derived very small embryonic‐like stem cells (VSEL‐SCs) improves left ventricular function and remodelling after myocardial infarction

Adult bone marrow‐derived very small embryonic‐like stem cells (VSEL‐SCs) exhibit a Sca‐1⁺/Lin^–/CD45^– phenotype and can differentiate into various cell types, including cardiomyocytes and endothelial cells. We have previously reported that transplantation of a small number (1 × 10⁶) of freshly isolated, non‐expanded VSEL‐SCs into infarcted mouse hearts resulted in improved left ventricular (LV) function and anatomy. Clinical translation, however, will require large numbers of cells. Because the frequency of VSEL‐SCs in the marrow is very low, we examined whether VSEL‐SCs can be expanded in culture without loss of therapeutic efficacy. Mice underwent a 30 min. coronary occlusion followed by reperfusion and, 48 hrs later, received an intramyocardial injection of vehicle (group I, n= 11), 1 × 10⁵ enhanced green fluorescent protein (EGFP)‐labelled expanded untreated VSEL‐SCs (group II, n= 7), or 1 × 10⁵ EGFP‐labelled expanded VSEL‐SCs pre‐incubated in a cardiogenic medium (group III, n= 8). At 35 days after myocardial infarction (MI), mice treated with pre‐incubated VSEL‐SCs exhibited better global and regional LV systolic function and less LV hypertrophy compared with vehicle‐treated controls. In contrast, transplantation of expanded but untreated VSEL‐SCs did not produce appreciable reparative benefits. Scattered EGFP⁺ cells expressing α‐sarcomeric actin, platelet endothelial cell adhesion molecule (PECAM)‐1, or von Willebrand factor were present in VSEL‐SC‐treated mice, but their numbers were very small. No tumour formation was observed. We conclude that VSEL‐SCs expanded in culture retain the ability to alleviate LV dysfunction and remodelling after a reperfused MI provided that they are exposed to a combination of cardiomyogenic growth factors and cytokines prior to transplantation. Counter intuitively, the mechanism whereby such pre‐incubation confers therapeutic efficacy does not involve differentiation into new cardiac cells. These results support the potential therapeutic utility of VSEL‐SCs for cardiac repair.     

Bmi‐1 high‐expressing cells enrich cardiac stem/progenitor cells and respond to heart injury

Bmi‐1 gene is well recognized as an oncogene, but has been recently demonstrated to play a role in the self‐renewal of tissue‐specific stem cells. By using Bmi‐1^GFP/+ mice, we investigated the role of Bmi‐1 in cardiac stem/progenitor cells and myocardial repair. RT‐PCR and flow cytometry analysis indicated that the expression of Bmi‐1 was significantly higher in cardiac side population than the main population from CD45^?Ter119^?CD31^? heart cells. More Sca‐1⁺ cardiac stem/progenitor cells were found in Bmi‐1 GFP^hi subpopulation, and these Bmi‐1 GFP^hi heart cells showed the potential of differentiation into SMM⁺ smooth muscle‐like cells and TnT⁺ cardiomyocyte‐like cells in vitro. The silencing of Bmi‐1 significantly inhibited the proliferation and differentiation of heart cells. Otherwise, myocardial infarction induced a significantly increase (2.7‐folds) of Bmi‐1 GFP^hi population, mainly within the infarction and border zones. These preliminary data suggest that Bmi‐1^hi heart cells are enriched in cardiac stem/progenitor cells and may play a role in myocardial repair.     

Correction to: Computational analysis of viable parameter regions in models of synthetic biological systems

Background

Myocardial infarction (MI) is a common cause of mortality in people. Mesenchymal stem cell (MSC) has been shown to exert therapeutic potential to treat myocardial infarction (MI). However, in patients with diabetes, the diabetic environment affected MSCs activity and could impair the efficacy of treatment. Interleukin-10 (IL-10) has been shown to attenuate MI by suppressing inflammation. In current study, the combination of MSC transplantation with IL-10 was evaluated in a diabetic mice model with MI.

Methods

We engineered bone marrow derived MSCs (BM-MSCs) to overexpress IL-10 by using CRISPR activation. We established the diabetic mice model with MI and monitored the IL-10 expression after BM-MSCs transplantation. We also evaluated the effects of BM-MSCs transplantation on inflammatory response, cell apoptosis, cardiac function and angiogenesis.

Results

CRISPR activation system enabled overexpression of IL-10 in BM-MSCs. Transplantation of BM-MSCs overexpressing IL-10 resulted in IL-10 expression in heart after transplantation. Transplantation of BM-MSCs overexpressing IL-10 inhibited inflammatory cell infiltration and pro-inflammatory cytokines production, improved cardiac functional recovery, alleviated cardiac injury, decreased apoptosis of cardiac cells and increased angiogenesis.

Conclusion

In summary, we have demonstrated the therapeutic potential of IL-10 overexpressed BM-MSCs in the treatment of MI in diabetic mice.

     

Attenuation of cell cycle regulator p27<Superscript>Kip1</Superscript> expression in vertebrate epithelial cells mediated by extracellular signals in vivo and in vitro

Cell cycle arrest in potentially dividing cells is often mediated by inhibitors of G1/S-phase cyclin-dependent kinases. The cyclin E/CDK2-inhibitor p27^Kip1 has been implicated in this context in epithelial cells. We cloned and sequenced p27^Kip1 of ducklings (Anas platyrhynchos) and used an in vitro assay system to study the mechanism of p27^Kip1 downregulation in the nasal gland which precedes an increase in proliferation rate upon initial exposure of the animals to osmotic stress. Western blot studies revealed that p27^Kip1 is downregulated during 24 h of osmotic stress in ducklings with the steepest decline in protein levels between 5 and 8 h. As indicated by the results of Northern blot and semi-quantitative PCR studies, protein downregulation is not accompanied by similar changes in mRNA levels indicating that Kip1 is regulated mainly at the translational (synthesis) or posttranslational level (degradation). Using recombinant duck Kip1 protein expressed in E. coli, we showed that Kip1 is subject to polyubiquitinylation by cytosolic enzymes from nasal gland cells indicating that loss of Kip1 may be regulated, at least in part, by acceleration of protein degradation. In cultured nasal gland tissue, attenuation of Kip1 expression could be induced by activation of the muscarinic acetylcholine receptor indicating that mAChR-receptor signalling may play a role in the re-entry of quiescent gland cells into the cell cycle.