Exosomes from Cardiomyocyte Progenitor Cells and Mesenchymal Stem Cells Stimulate Angiogenesis Via EMMPRIN.

To date, cellular transplantation therapy has not yet fulfilled its high expectations for cardiac repair. A major limiting factor is lack of long-term engraftment of the transplanted cells. Interestingly, transplanted cells can positively affect their environment via secreted paracrine factors, among which are extracellular vesicles, including exosomes: small bi-lipid-layered vesicles containing proteins, mRNAs, and miRNAs. An exosome-based therapy will therefore relay a plethora of effects, without some of the limiting factors of cell therapy. Since cardiomyocyte progenitor cells (CMPC) and mesenchymal stem cells (MSC) induce vessel formation and are frequently investigated for cardiac-related therapies, the pro-angiogenic properties of CMPC and MSC-derived exosome-like vesicles are investigated. Both cell types secrete exosome-like vesicles, which are efficiently taken up by endothelial cells. Endothelial cell migration and vessel formation are stimulated by these exosomes in in vitro models, mediated via ERK/Akt-signaling. Additionally, these exosomes stimulated blood vessel formation into matrigel plugs. Analysis of pro-angiogenic factors revealed high levels of extracellular matrix metalloproteinase inducer (EMMPRIN). Knockdown of EMMPRIN on CMPCs leads to a diminished pro-angiogenic effect, both in vitro and in vivo. Therefore, CMPC and MSC exosomes have powerful pro-angiogenic effects, and this effect is largely mediated via the presence of EMMPRIN on exosomes.

Circulating Extracellular Vesicles Contain miRNAs and are Released as Early Biomarkers for Cardiac Injury.

Plasma-circulating microRNAs have been implicated as novel early biomarkers for myocardial infarction (MI) due to their high specificity for cardiac injury. For swift clinical translation of this potential biomarker, it is important to understand their temporal and spatial characteristics upon MI. Therefore, we studied the temporal release, potential source, and transportation of circulating miRNAs in different models of ischemia reperfusion (I/R) injury. We demonstrated that extracellular vesicles are released from the ischemic myocardium upon I/R injury. Moreover, we provided evidence that cardiac and muscle-specific miRNAs are transported by extracellular vesicles and are rapidly detectable in plasma. Since these vesicles are enriched for the released miRNAs and their detection precedes traditional damage markers, they hold great potential as specific early biomarkers for MI.

MicroRNA-1 enhances the angiogenic differentiation of human cardiomyocyte progenitor cells.

Instigated by the discovery of adult cardiac progenitor cells, cell replacement therapy has become a promising option for myocardial repair in the past decade. We have previously shown that human-derived cardiomyocyte progenitor cells (hCMPCs) can differentiate into cardiomyocyte-, endothelial-, and smooth muscle-like cells in vitro, and in vivo after transplantation in a mouse model of myocardial infarction, resulting in preservation of cardiac function. However, to allow successful repopulation of the injured myocardium, it is of key importance to restore myocardial perfusion by the formation of new vasculature. Several studies have shown that microRNAs regulate vascular differentiation of different stem/progenitor cells. Here, we show that miR-1 is upregulated in hCMPCs during angiogenic differentiation. Upregulation of miR-1 enhanced the formation of vascular tubes on Matrigel and within a collagen matrix, and also increased hCMPC motility, as shown by planar and transwell migration assays. By western blot, qRT-PCR and luciferase reporter assays, miR-1 was found to directly target and inhibit the expression of sprouty-related EVH1 domain-containing protein 1 (Spred1). Knocking down Spred1 phenocopies the functional effect seen for miR-1 upregulation. Using a systems biology approach, we found that in hCMPCs, miR-1 is proposed to control a network of genes predominantly involved in angiogenesis-related processes, including the Spred1 pathway. Our data shows that by upregulation of miR-1, the angiogenic differentiation of hCMPCs can be enhanced, which may be used as a new therapeutic approach to improve the efficiency of cell-based therapy for cardiac regeneration by enhancing the formation of new vasculature.

Transendocardial cell injection is not superior to intracoronary infusion in a porcine model of ischaemic cardiomyopathy: a study on delivery efficiency.

Stem cell therapy is a new strategy for chronic ischaemic heart disease in patients. However, no consensus exists on the most optimal delivery strategy. This randomized study was designed to assess cell delivery efficiency of three clinically relevant strategies: intracoronary (IC) and transendocardial (TE) using electromechanical mapping guidance (NOGA) compared to surgical delivery in a chronic pig model of ischaemic cardiomyopathy. Twenty-four animals underwent delivery of 10(7) autologous Indium-oxine-labelled bone marrow-derived mesenchymal stem cells (MSC) 4 weeks after infarction and were randomized to one of three groups (n = 8 each group): IC, TE or surgical delivery (reference group). Primary endpoint was defined as percentage (%) of injected dose per organ and assessed by in vivo gamma-emission counting. In addition, troponin and coronary flow were assessed before and after MSC injection. Blinded endpoint analysis showed no significant difference in efficiency after surgical (16 ± 4%), IC (11 ± 1%) and TE (11 ± 3%) (P = 0.52) injections. IC showed less variability in efficiency compared with TE and surgical injection. Overall, TE injection showed less distribution of MSC to visceral organs compared with other modalities. Troponin rise and IC flow did not differ between the percutaneous groups. This randomized study showed no significant difference in cell delivery efficiency to the myocardium in a clinically relevant ischaemic large animal model between IC and TE delivery. In addition, no differences in safety profile were observed. These results are important in view of the choice of percutaneous cell delivery modality in future clinical stem cell trials.

MiR-155 inhibits cell migration of human cardiomyocyte progenitor cells (hCMPCs) via targeting of MMP-16.

Undesired cell migration after targeted cell transplantation potentially limits beneficial effects for cardiac regeneration. MicroRNAs are known to be involved in several cellular processes, including cell migration. Here, we attempt to reduce human cardiomyocyte progenitor cell (hCMPC) migration via increasing microRNA-155 (miR-155) levels, and investigate the underlying mechanism. Human cardiomyocyte progenitor cells (hCMPCs) were transfected with pre-miR-155, anti-miR-155 or control-miR (ctrl-miR), followed by scratch- and transwell-assays. These functional assays displayed that miR-155 over-expression efficiently inhibited cell migration by 38 ± 3.6% and 59 ± 3.7% respectively. Conditioned medium from miR-155 transfected cells was collected and zymography analysis showed a significant decrease in MMP-2 and MMP-9 activities. The predicted 3'-UTR of MMP-16, an activator of MMP-2 and -9, was cloned into the pMIR-REPORT vector and luciferase assays were performed. Introduction of miR-155 significantly reduced luciferase activity which could be abolished by cotransfection with anti-miR-155 or target site mutagenesis. By using MMP-16 siRNA to reduce MMP-16 levels or by using an MMP-16 blocking antibody, hCMPC migration could be blocked as well. By directly targeting MMP-16, miR-155 efficiently inhibits cell migration via a reduction in MMP-2 and -9 activities. Our study shows that miR-155 might be used to improve local retention of hCMPCs after intramyocardial delivery.

Intrauterine growth restriction affects the maturation of myelin.

Intrauterine growth-restriction (IUGR) can lead to adverse neurodevelopmental sequelae in postnatal life. Our objective was to determine whether IUGR, induced by chronic placental insufficiency (CPI) in the guinea pig results in long-term deficits in brain myelination and could therefore contribute to altered neural function. CPI was induced by unilateral ligation of the uterine artery at mid-gestation (term~67 days of gestation; dg), producing growth-restricted (GR) foetuses (60 dg), neonates (1 week) and young adults (8 week); controls were from the unligated horn or sham-operated animals. In GR foetuses (n=8) and neonates (n=7), white matter (WM) volume was reduced (p<0.05); this reduction did not persist in young adults (n=11) however the corpus callosum width was reduced (p<0.05). Immunoreactivity (IR) for myelin basic protein (MBP), myelin-associated glycoprotein (MAG) and myelin proteolipid protein (PLP), all markers of myelinating oligodendrocytes (OL), was reduced in GR foetuses compared to controls. MBP was the most markedly affected with an abnormal retention of protein in the OL soma and a reduction of its incorporation into the myelin sheath. MAG-IR OL density was reduced (p<0.05), while the density of OLs immunoreactive for Olig-2, a transcription factor expressed throughout the entire OL lineage, was increased (p<0.05). MBP-, MAG- and PLP-IR recovered to control levels postnatally. These results suggest that IUGR transiently delays OL maturation and myelination in utero but that myelination and WM volume are restored to control levels postnatally. Long-term deficits in myelination are therefore unlikely to be the major factor underlying the altered neurological function which can be associated with IUGR.

Non-surgical stem cell delivery strategies and in vivo cell tracking to injured myocardium.

Heart failure is a major economic and public health problem. Despite the recent advances in drug therapy and coronary revascularization, the lost cardiomyocytes due to necrosis and apoptosis are not replaced by new myocardial tissue. Cell therapy is an interesting therapeutic option as it potentially improves contractility and restores regional ventricular function. Early clinical data demonstrated that cell transplantation, mainly delivered through non-surgical methods, is safe and feasible. However, several important issues need to be elucidated. This includes, next to determining the best cell type, the optimal delivery strategy, the biodistribution and the survival of implanted stem cells after transplantation. In this view, pre-clinical animal experiments are indispensable. Reporter genes, magnetic or radioactive labeling of stem cells have been developed to observe the fate and the distribution of transplanted cells using non-invasive imaging techniques. Several studies have demonstrated that these direct and non-direct labeling techniques may become an important tool in cell therapy. Integration of cell delivery and cell tracking will probably be a key for the success of cell therapy in patients. This review will provide a comprehensive overview on the various cell tracking and non-surgical cell delivery techniques, which are highly important in view of experimental and clinical studies.

MicroRNA-155 prevents necrotic cell death in human cardiomyocyte progenitor cells via targeting RIP1.

To improve regeneration of the injured myocardium, cardiomyocyte progenitor cells (CMPCs) have been put forward as a potential cell source for transplantation therapy. Although cell transplantation therapy displayed promising results, many issues need to be addressed before fully appreciating their impact. One of the hurdles is poor graft-cell survival upon injection, thereby limiting potential beneficial effects. Here, we attempt to improve CMPCs survival by increasing microRNA-155 (miR-155) levels, potentially to improve engraftment upon transplantation. Using quantitative PCR, we observed a 4-fold increase of miR-155 when CMPCs were exposed to hydrogen-peroxide stimulation. Flow cytometric analysis of cell viability, apoptosis and necrosis showed that necrosis is the main cause of cell death. Overexpressing miR-155 in CMPCs revealed that miR-155 attenuated necrotic cell death by 40 ± 2.3%via targeting receptor interacting protein 1 (RIP1). In addition, inhibiting RIP1, either by pre-incubating the cells with a RIP1 specific inhibitor, Necrostatin-1 or siRNA mediated knockdown, reduced necrosis by 38 ± 2.5% and 33 ± 1.9%, respectively. Interestingly, analysing gene expression using a PCR-array showed that increased miR-155 levels did not change cell survival and apoptotic related gene expression. By targeting RIP1, miR-155 repressed necrotic cell death of CMPCs, independent of activation of Akt pro-survival pathway. MiR-155 provides the opportunity to block necrosis, a conventionally thought non-regulated process, and might be a potential novel approach to improve cell engraftment for cell therapy.

Stimulation of homology-directed gene targeting at an endogenous human locus by a nicking endonuclease.

Homologous recombination (HR) is a highly accurate mechanism of DNA repair that can be exploited for homology-directed gene targeting. Since in most cell types HR occurs very infrequently (approximately 10(-6) to 10(-8)), its practical application has been largely restricted to specific experimental systems that allow selection of the few cells that become genetically modified. HR-mediated gene targeting has nonetheless revolutionized genetics by greatly facilitating the analysis of mammalian gene function. Recent studies showed that generation of double-strand DNA breaks at specific loci by designed endonucleases greatly increases the rate of homology-directed gene repair. These findings opened new perspectives for HR-based genome editing in higher eukaryotes. Here, we demonstrate by using donor DNA templates together with the adeno-associated virus (AAV) Rep78 and Rep68 proteins that sequence- and strand-specific cleavage at a native, predefined, human locus can also greatly enhance homology-directed gene targeting. Our findings argue for the development of other strategies besides direct induction of double-strand chromosomal breaks to achieve efficient and heritable targeted genetic modification of cells and organisms. Finally, harnessing the cellular HR pathway through Rep-mediated nicking expands the range of strategies that make use of AAV elements to bring about stable genetic modification of human cells.