Migration towards SDF-1 selects angiogenin-expressing bone marrow monocytes endowed with cardiac reparative activity in patients with previous myocardial infarction
© Ascione et al.; licensee BioMed Central. 2015
Received: 19 June 2014
Accepted: 27 February 2015
Published: 11 April 2015
Chemokine-directed migration is crucial for homing of regenerative cells to the infarcted heart and correlates with outcomes of cell therapy trials. Hence, transplantation of chemokine-responsive bone marrow cells may be ideal for treatment of myocardial ischemia. To verify the therapeutic activity of bone marrow mononuclear cells (BM-MNCs) selected by in vitro migration towards the chemokine stromal cell-derived factor-1 (SDF-1) in a mouse model of myocardial infarction (MI), we used BM-MNCs from patients with previous large MI recruited in the TransACT-1&2 cell therapy trials.
Unfractioned BM-MNCs, SDF-1-responsive, and SDF-1-nonresponsive BM-MNCs isolated by patients recruited in the TransACT-1&2 cell therapy trials were tested in Matrigel assay to evaluate angiogenic potential. Secretome and antigenic profile were characterized by flow cytometry. Angiogenin expression was measured by RT-PCR. Cells groups were also intramyocardially injected in an in vivo model of MI (8-week-old immune deficient CD1-FOXN1nu/nu mice). Echocardiography and hemodynamic measurements were performed before and at 14 days post-MI. Arterioles and capillaries density, infiltration of inflammatory cells, interstitial fibrosis, and cardiomyocyte proliferation and apoptosis were assessed by immunohistochemistry.
In vitro migration enriched for monocytes, while CD34+ and CD133+ cells and T lymphocytes remained mainly confined in the non-migrated fraction. Unfractioned total BM-MNCs promoted angiogenesis on Matrigel more efficiently than migrated or non-migrated cells. In mice with induced MI, intramyocardial injection of unfractionated or migrated BM-MNCs was more effective in preserving cardiac contractility and pressure indexes than vehicle or non-migrated BM-MNCs. Moreover, unfractioned BM-MNCs enhanced neovascularization, whereas the migrated fraction was unique in reducing the infarct size and interstitial fibrosis. In vitro studies on isolated cardiomyocytes suggest participation of angiogenin, a secreted ribonuclease that inhibits protein translation under stress conditions, in promotion of cardiomyocyte survival by migrated BM-MNCs.
Transplantation of bone marrow cells helps post-MI healing through distinct actions on vascular cells and cardiomyocytes. In addition, the SDF-1-responsive fraction is enriched with angiogenin-expressing monocytes, which may improve cardiac recovery through activation of cardiomyocyte response to stress. Identification of factors linking migratory and therapeutic outcomes could help refine regenerative approaches.
Bone marrow mononuclear cells (BM-MNCs) are predominant in cell therapy trials of myocardial infarction (MI) and heart failure . Recent reviews and meta-analyses indicate that BM cell therapy is safe and leads to tangible improvements in cardiac function, ventricular remodeling and clinical outcomes, including incidence of death, recurrent MI and stent thrombosis [2-4].
In spite of these encouraging results, the heterogeneity of BM cell products and complexity of intercellular interactions in the treated myocardium fuels major controversies in the field. Originally supposed to induce de novo cardiomyogenesis , BM cells are now mainly acknowledged as promoters of reparative neovascularization . Paracrine communication of transplanted cells with endothelial cells, resident cardiomyocytes and progenitor cells (PCs) recruited from cardiac or distant niches, but also crosstalk between the different cell types within the applied preparation, boosts vascular repair and also conveys survival cues to cardiac cells in the area at risk [6-10]. The extent to which specific BM cell subfractions participate and possibly synergize to determine distinct therapeutic benefits remains a matter of debate [11-13]. Furthermore, risk factors and comorbidities cause pauperization of BM-PCs and shift to the myeloid lineage, together with reduction of regenerative potential and immune competence [8,14-16]. Therefore, transplantation of unselected autologous preparations bears the risk that presence of useless or even harmful cells may hamper the activity of few regenerative cells. In addition, isolation protocols may variably impact on BM cell viability and functionality, thus calling for introduction of quantity and quality control standards .
Following these considerations, immunomagnetically and antigenically sorted CD133+ or CD34+ PCs have been proposed for cardiovascular cell therapy [18-20]. Another attractive option is to select cells on the basis of their functional qualities. This is supported by the observation that the in vitro migratory activity towards the chemokine stromal cell-derived factor 1 (SDF-1) predicts the outcome of pre-clinical and human BM cell therapy studies [21,22]. Following this logic, we have developed a cell sorting method based on responsiveness to chemotactic cues. We reported that peripheral blood (PB) MNCs that migrate in response to chemoattractants such as SDF-1 or bradykinin are enriched for CD133+ and CD34+ PCs, release higher amounts of pro-angiogenic cytokines, and generate more nitric oxide and less superoxide in comparison with non-migrated PB-MNCs [23,24]. Therefore, the in vitro migration assay not only recapitulates a fundamental pathophysiological mechanism implicated in tissue repair [25,26], but also provides additional insights into the molecular profile of distinct cell types associated with directed cell motility.
The primary objective of this study was to test if migration towards SDF-1 allows isolating of therapeutically valuable cells from the BM of cardiovascular patients with a history of previous MI. To this end, we compared unfractioned total BM-MNCs and SDF-1-responsive or SDF-1-nonresponsive BM-MNCs with regard to antigenic characteristics, in vitro functions, and therapeutic activity in a mouse model of MI. Furthermore, we investigated if differences in secreted pro-angiogenic and pro-survival factors may account for specific therapeutic activities of fractioned cells. In view of clinical translation, BM-MNCs were obtained from coronary artery disease patients undergoing the Bristol-based cell therapy surgical trials TransACT-1 and TransACT-2. Newly results indicate that BM cells from patients with large recent or chronic MI contain populations able to promote cardiac repair by distinct mechanisms involving angiogenesis and cardiomyocyte response to stress.
Detailed Methods are provided in Additional file 1.
The study was performed in accordance with the declaration of Helsinki (1964) and subsequent amendments and approved by the Bristol National Health Service ethical committee. BM specimens were obtained blindly from risk-profiled coronary artery disease patients participating in the Bristol-based TransACT-1&2 autologous BM cell therapy trials (RA; recruitment completed at the Bristol Heart Institute in January 2014). All necessary informed consent, including acceptance to participate in the study, was obtained from any patients involved in the study. Patients were selected according to strict cardiac magnetic resonance imaging criteria identifying a previous large MI, requiring coronary artery bypass grafting (TransACT-1) or left ventricular restoration surgery (TransACT-2). Key inclusion criteria of the TransACT trials and the anonymized cardiovascular risk profile of the BM donors recruited to this study are reported in Additional file 1: Tables S1 and S2. Details of the clinical trials from which the study is generated are available online . In addition, as a “healthy” reference, BM leftover was obtained from two male individuals (aged 56 and 58 years with no evidence of cardiovascular disease) at the occasion of orthopedic surgery for hip transplantation (covered by National Health Service ethical approval, REC number: 14/WA/1005).
Isolation of bone marrow mononuclear cell fractions
MNCs were isolated by density centrifugation of fresh human BM cell suspension (5 mM phosphate-buffered saline (PBS)-ethylenediaminetetraacetic acid (EDTA)) on Histopaque 1077 (Sigma, Poole, UK). Migration was performed as previously described using SDF-1 (100 ng/mL) as chemoattractant [23,24]. In some experiments, bradykinin (100 ng/mL) was also used as a stimulus for cell migration. Migration medium without chemoattractant (vehicle) was employed to control for unspecific cell motility. After 18 hours of migration, non-migrating and migrating populations were obtained separately by washing the upper and lower chambers and respective sides of the migration filter three times with 1 × PBS containing 5 mM EDTA. Cell viability was assessed by trypan blue staining. In selected experiments, BM-MNCs were exposed to SDF-1 or vehicle for the same duration of migration.
Unfractioned, non-migrated (SDF-1non, vehnon) and migrated (SDF-1mig, vehmig) BM-MNCs from 10 individual patients were stained for surface antigen expression and analyzed on a FACS Canto II flow cytometer equipped with FACS Diva software (BD Biosciences, Oxford, UK). Fluorescence minus one staining controls were performed to define positivity.
In vitro network formation by human umbilical vein endothelial cells (HUVECs) was studied using a Matrigel assay in the presence of unfractioned or SDF-1mig BM-MNCs. Analysis of cumulative branch length was performed using ImagePro software (Media Cybernetics, Rockville, MD, USA).
Measurement of growth factors and cytokines
The paracrine activity of unfractioned and SDF-1mig BM-MNCs was quantified by measuring the levels of chemokines (interleukin (IL)-1β, IL-2, IL-3, IL-4, IL-6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, and RANTES), chemoattractant factors (granulocyte colony-stimulating factor, granulocyte macrophage colony-stimulating factor, and monocyte chemoattractant protein (MCP-1)), and molecules associated with inflammation and angiogenesis (macrophage inflammatory protein 1α/β, FASL, tumor necrosis factor α, interferon γ, vascular endothelial growth factor (VEGF), basic fibroblast growth factor (bFGF), and angiogenin in cell conditioned media (flow cytometry bead array; BD Biosciences, Oxford, UK). Nine individual samples were studied in duplicate. Furthermore, we used an enzyme-linked immunosorbent assay (ELISA; R&D, Abingdon, UK) for quantitative measurement of human angiogenin in conditioned media (n = 7 patients).
Angiogenin expression in sorted monocyte subpopulations
BM cells were suspended in PBS-EDTA 5 mM and filtered through a strainer (70 μm sieve). MNCs were isolated by Ficoll Histopaque 1077 (Sigma) gradient. The MNC layer was carefully collected and washed twice in PBS. Cells were stained with PE-conjugated CD3 (MACS Milteny, Bisley, Surrey, United Kingdom), APC-conjugated CD14 (R&D), and PerCP-conjugated CD16 (BioLegend, London, United Kingdom) antibodies for 30 minutes at 4°C. After washing, 5 × 107 MNCs cells were acquired and sorted by Influx™ Cell sorter (BD) into four different groups: CD3−CD14+CD16+, CD3−CD14+CD16−, CD3−CD14−CD16+ and CD3−CD14−CD16− cells. The expression of angiogenin was then verified in the sorted populations by RT-PCR (n = 4 patients).
Cell therapy in a murine myocardial infarction model
Animal experiments were performed in accordance with the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, National Academy of Sciences, Bethesda, MD, USA, 1996) and under ethical licence from the UK Home Office (licence holder PM, University of Bristol). MI was induced in 8-week-old immune deficient CD1-FOXN1nu/nu mice (Charles River, Wilmington, MA, US) by ligation of the left anterior descending coronary artery as described previously [28,29]. This was followed by injection of 3 × 105 unfractioned, SDF-1non or SDF-1mig BM-MNCs, or cell-free EBM-2 (vehicle) at three different sites along the infarct border zone with a final volume of 10 μL at each site.
Echocardiography and hemodynamic measurements
Measurements of dimensional and functional parameters were performed before and 14 days post-MI using a high-frequency, high-resolution echocardiography system (Vevo 770, Visual Sonics, Toronto, Canada) [28,29]. Following the final echocardiography session, intraventricular pressure was measured using a high-fidelity 1.4F transducer tipped catheter (Millar Instruments, Houston, TX, US) inserted into the left ventricle through the right carotid artery. Measurement of echocardiography and pressure indexes was performed in 14 mice per group.
Hearts were stopped in diastole by intramyocardial injection of cadmium chloride, washed free of blood by retrograde perfusion with PBS-2% EDTA solution, followed by fixation with 4% paraformaldehyde solution overnight. Cryosections were made at 10 μm thickness, placed on superfrost ultra plus (Thermo, Braunschweig, Germany) slides, air dried for 30 minutes and immunostained for the assessment of capillary and arteriole densities, infiltration of inflammatory cells, interstitial fibrosis and cardiomyocyte proliferation and apoptosis (see Additional file 1).
Immunocytochemistry of isolated cardiomyocytes
Adult rat cardiomyocytes were isolated using collagenase perfusion and used immediately for experiments. Cells were exposed to hypoxia for 15 hours in cardiomyocyte media mixed with concentrated conditioned media, which were collected from SDF-1non or SDF-1mig or unfractioned BM-MNCs, or EBM-2 basal medium. Synthetic angiogenin (BD, 10 ng/mL) was used as a positive control. In inhibition experiments, recombinant human ribonuclease/angiogenin inhibitor 1 (RNH1; OriGene, Rockville, MD, US) was added to the conditioned medium 1 hour prior to incubation with cardiomyocytes. Cardiomyocytes were fixed and immunostained for the stress granules marker eIF3 (goat polyclonal; Santa Cruz, Dallas, TX, US), α-sarcomeric actinin (mouse monoclonal; Sigma) and 4′,6-diamidino-2-phenylindole to identify nuclei. For cell viability study, cardiomyocytes were incubated for 15 hours in hypoxia with unconditioned medium or conditioned medium from SDF-1mig BM-MNCs, in the presence or absence of RNH1. At the end of the incubation period, rod-shaped cardiomyocytes still attached to the culture-dish were counted as viable cells, while round cells detached from the dish were evaluated as dead cells. Experiments were performed in duplicate. At least 800 cells were analyzed for each condition.
All results are presented as mean ± standard error of the mean, or median and range, from three or more experiments as indicated. For comparison of multiple groups, analysis of variance or Friedman test were used after verification of Gaussian distribution. Statistical significance was then determined by paired or unpaired t-tests as appropriate. Furthermore, best-fit and stepwise regression analyses were performed to verify the predictive value of clinical and laboratory data and cell phenotype on outcome endpoints. Variables were entered into the equation only if they satisfied the following criteria: F-to-enter, default value = 3.84; probability of F-to-enter, default value = 0.05. The cumulative effect of these relationships was illustrated by Radar plot. Survival rate was calculated by Kaplan-Meyer test. A P value <0.05 was considered significant. Stated n values represent biological replicates.
Stromal cell-derived factor 1-induced migration enriches for viable bone marrow mononuclear cells
Evaluation of viability is a fundamental step in the quality control assessment of cell therapy products. An average 5.7% (range 2.0 to 19.1%) of unfractioned BM-MNCs stained positive for trypan blue, a dye that traverses the membrane in a dead cell. This figure did not change after storage of cells in their original medium for the duration of the migration assay (unfractioned post, 7.6%; range 5.2 to 19.0%; Figure 1B). Importantly, trypan blue-positive cells were fewer in the vehmig (3.4%; range 2.0 to 7.9%) and SDF-1mig fractions (2.4%; range 0.7 to 7.4%; P < 0.01 versus unfractioned, SDF-1non or vehnon BM-MNCs; Figure 1B). A similar migration-induced enrichment in viable cells was obtained using bradykinin as a chemoattractant in the in vitro assay (3.0%; range 0 to 7.5%). Altogether, these data indicate that either spontaneous or chemokine-directed migration results in a quality-improved BM cell product.
Antigenic and molecular characteristics of unfractioned and fractioned bone marrow mononuclear cells
Verification of the migration assay in BM cells from subjects without a cardiovascular disease background confirms the depletion of CD3+ lymphocytes and CD34+ and CD133+ PCs along with enrichment of CD14++CD16−, CD14++CD16+ and CD14+CD16++ monocytes in the SDF-1-attracted fraction (Additional file 1: Figure S3).
Support of endothelial tube formation and paracrine factors secreted by unfractioned and fractioned bone marrow mononuclear cells
BM-MNCs secrete growth factors and cytokines that influence angiogenesis in vitro and in vivo . Using a cytometric bead array that measures a variety of cytokines and growth factors implicated in reparative angiogenesis, we found that SDF-1mig BM-MNCs release angiogenin more abundantly than unfractioned BM-MNCs (P < 0.01), while the latter tend to release more VEGF, tumor necrosis factor-α and RANTES (Additional file 1: Figure S4A). Regression analysis revealed that the association of different secreted factors (for example, VEGF, IL-8, angiogenin and MCP-1) predicts the increase in endothelial network formation induced by unfractioned BM-MNCs (P < 0.01). This cooperative interaction is also illustrated by the radar plot generated by connecting the F-to-enter values from regression analyses (Additional file 1: Figure S4B).
In vivo therapeutic activity of unfractioned and migration-fractioned bone marrow mononuclear cells
We next asked if BM cell populations selected by SDF-1-induced migration differ from total BM cells with regard to therapeutic activity in ischemia. To this end, we injected SDF-1non, SDF-1mig or unfractioned BM-MNCs from three donors into the infarct border zone of immunodeficient mice.
Cell therapy improved left ventricular end diastolic pressure, and maximum and minimum changes in developed pressure (P < 0.0001 versus vehicle; Figure 5J,K), with no difference among donors. When examining the effects of distinct cell fractions, we found that injections of unfractioned or SDF-1migr BM-MNCs blunts the post-MI increase in left ventricular end diastolic pressure (P < 0.001 and P < 0.05 versus vehicle, respectively), while SDF-1non BM-MNCs were ineffective (not significant versus vehicle, P < 0.01 versus unfractioned BM-MNCs; Figure 5J). Likewise, SDF-1non BM-MNCs were inferior to total BM-MNCs in preserving the maximum and minimum changes in developed pressure indexes (Figure 5K). Taken together, these data indicate that injections of unfractioned or migrated BM-MNCs improves left ventricular remodeling and contractility to a greater extent than the non-migrated counterparts.
Analysis of the coronary microvasculature showed that mice receiving cell therapy exhibit higher levels of capillaries and arterioles than mice given vehicle (P < 0.01; Figure 6E-S). Post-hoc analysis by Tukey’s test revealed the superior pro-angiogenic activity of unfractioned BM-MNCs versus SDF-1non or SDF-1mig cells, in line with results of in vitro network formation on Matrigel. Histological studies also revealed an effect of cell therapy on the number of infiltrating CD45+ cells (unfractioned, 21 ± 2; SDF-1non, 28 ± 2; SDF-1mig, 25 ± 2; versus vehicle, 52 ± 4 cells/mm2; P < 0.01 for all comparisons), with no difference between cell therapy groups.
Altogether, these data indicate that unfractioned and SDF-1migr BM-MNCs exert similar therapeutic effects as assessed by echocardiography and intraventricular pressure measurements. However, only unfractioned cells have pro-angiogenic activity, whereas SDF-1migr cells reduce infarct size and interstitial fibrosis.
Stromal cell-derived factor 1 migrated bone marrow mononuclear cells induce the formation of stress granules in hypoxic cardiomyocytes through an angiogenin-mediated mechanism
Importantly, the induction of SGs by SDF-1mig cell-conditioned medium (P < 0.05 versus unconditioned medium) was reduced by co-incubation with RNH1, a ribonuclease/angiogenin inhibitor (P < 0.05 versus conditioned medium), thus indicating the involvement of secreted angiogenin in the augmentation of cardiomyocyte stress response (Figure 7C). Additionally, SDF-1mig cell-conditioned medium increases the viability of hypoxic cardiomyocytes, with this effect being reduced by RNH1 (Figure 7D). Altogether, these data indicate a novel cardioprotective effect of migrating BM cells, involving angiogenin-mediated stress response in hypoxic cardiomyocytes.
Results of the present study indicate that migration-based selection separates two distinct fractions from total BM cells of patients with previous MI: the non-migrated component contains an abundance of lymphocytes and CD34+/CD133+ PCs, while the migrated element is more enriched with classical, intermediate and non-classical monocytes. Furthermore, intramyocardial injection of unfractioned or migrated cells induces similar therapeutic benefits in a murine model of MI, but through different mechanisms. As expected, unfractioned cells promote reparative angiogenesis; in contrast, we show for the first time that SDF-1migr cells reduce the infarct size and peri-infarct fibrosis and exert cardiac protection through the release of angiogenin and potentiation of stress response in hypoxic cardiomyocytes. The present study was limited to 14 days post-MI, as the a priori endpoint was to discriminate differences in early reparative responses to BM cell therapy that can be revealed by the migration-based fractioning. More investigation is necessary to determine the impact of the fractioning approach on prevention of late cardiac remodeling and heart failure.
Advances in clinical management over the last decades have led to a marked improvement in the survival rate of patients suffering from acute MI. However, due to the permanent loss of viable myocardium, a large proportion of survivors develop cardiac dysfunction, which calls for novel regenerative treatments. To date, the most successful clinical trials performing autologous BM-MNC transplantation in MI patients achieved an improvement of LVEF by up to 20% and a decrease in infarct size of up to 30% [31-36]. However, when looking at meta-analysis reports, the size of the beneficial effects has varied grossly, often due to methodological differences. Several studies have since attempted further refinement by modulating the composition of the transplanted cells with the aim of selecting for a presumed most efficient cell type [37-41]. However, efforts have been often hindered by functional limitations and weakened regenerative capacity intrinsic to patient-derived cells [15,42-44]. In addition, cell damage can occur as result of harvesting procedures and subsequent processing prior to transplantation. The novel observation that migration fractioning significantly increases the number of viable cells from the total BM preparation and enriches for cells endowed with specific reparative activity has therefore, in our opinion, a relevant translational value.
Cardiovascular risk factors and ageing induce quantitative and functional defects in BM PCs. Consistently, in the present study, the number of CD34+ PCs was influenced by patient’s age, (that is, older patients had lower CD34+ cell counts in total BM-MNCs). Another study showed that age influences the number and colony-forming capacity of circulating CD34+ PCs . Furthermore, we have previously observed that migration towards bradykinin enriches for pro-angiogenic PCs from PB-MNCs of healthy donors. However, migration-based enrichment for PCs was significantly blunted, although not completed abrogated, in acute MI patients. In the present study, conducted in patients with previous MI, SDF-1-induced migration resulted in the depletion of BM CD34+ and CD133+ PCs. The discrepant behavior of BM- and PB-derived PCs is intriguing and may reflect a “migratory immaturity” of BM-resident PCs, as opposed to circulating PCs which are already primed to mobilization. Impaired CD34+ PC migration is reportedly associated with adverse events in patients with MI. The migratory defect might be attributable to the downregulation of receptor expression or the dysfunction of signaling pathways downstream of the receptor, as it has been reported for circulating cells before [46-48]. In addition, increased cleavage of SDF-1 by circulating or cell membrane-bound proteases, like dipeptidylpeptidase IV/CD26, might result in the loss of its signaling capabilities . Other possibilities include the choice of stimulus and the time period allowed for migration. When testing those hypotheses, we did not observe any difference using bradykinin instead of SDF-1. Likewise, similar results were obtained in experiments where migration was performed for 6 or 18 hours (data not shown).
Of note, we show that different cell populations within the total BM-MNC fraction contribute together in stimulation of angiogenesis. Separation of PC and monocyte components by migration resulted in the loss of pro-angiogenic activity. These findings might have high translational value for cell therapy applications, in particular with respect to the choice of delivering BM cell products directly or systemically . They imply that BM-derived cells from MI patients might not be able to provide their key pro-angiogenic effect in the MI area if delivered through a route (like the intracoronary delivery approach, for example) that relies on cell intrinsic migration potential to reach the MI area. We additionally found that lymphocytes are predominantly retained in the non-migrated fraction. There is a lack of consensus on the role played by lymphocytes in reparative processes, as these cells have been reported either to inhibit  or promote angiogenesis , including by shedding of microvesicles, which in turn can stimulate vascular growth . However, the present study did not show any association between lymphocytes and promotion of in vitro angiogenesis by unfractioned BM cells.
Different secreted factors, including VEGF, IL-8, MCP-1 and angiogenin, were complementary in stimulation of in vitro angiogenesis by total BM cells. Surprisingly, however, transplantation of unfractioned BM-MNCs or SDF-1mig BM-MNCs, which are enriched with angiogenin-expressing monocytes, promoted similar functional recovery in a mouse MI model, but only the former improved reparative angiogenesis in vivo. On the other hand, SDF-1mig BM cells induced a significant reduction of the infarct size and interstitial fibrosis, while unfractioned BM-MNCs did not. Interestingly, a recent pre-clinical cell therapy study in rats with acute MI using mesenchymal stem cells (MSCs) transfected with an adenoviral vector carrying the angiogenin gene (MSCAdANG) showed that angiogenin expression confers donor cells with high resistance to oxygen deprivation and with increased capacity to reduce infarct size, left ventricular remodeling and improve cardiac function . At variance with our study, cell therapy with MSCAdANG also induced reparative angiogenesis in the peri-infarct zone . However, it should be noted that MSCAdANG secrete much higher levels of angiogenin (~700 ng/mL) as compared with SDF-1mig BM-MNCs (~100 pg/mL). The activity of angiogenin is relatively low compared with that of other angiogenic factors. Therefore, differences in angiogenin dosage may account for the distinction between MSCAdANG and SDF-1mig BM-MNCs with regard to promotion of reparative angiogenesis.
Emerging evidence indicates that, besides inducing the transcription of various angiogenic genes , angiogenin promotes stress responses instrumental to cell survival under adverse conditions . In particular, angiogenin acts as an RNAase cleaving tRNAs into stress-induced tiRNAs, which in turn suppress the synthesis of housekeeping proteins by inhibiting mRNA translation . Translationally repressed messenger ribonucleoproteins are then transiently stored in SGs, which are dynamic structures that can be recycled as cells recover from stress . Importantly, translation of IRES-dependent mRNAs, many of which are involved in pro-survival and anti-apoptosis, is resistant to tiRNA-dependent inhibition, thereby allowing stressed cells to direct the translational machinery and residual energy to functions instrumental to survival [58,59]. Additional pro-survival mechanisms of angiogenin include an interaction with the p53, p21 and Bax pathway in cancer cells . In line with results on other cell types [57,61], we report for the first time that exogenous angiogenin stimulates the formation of SGs in isolated rat cardiomyocytes. Most importantly, we newly show that SDF-1mig BM-MNCs express and release large amounts of angiogenin and that the conditioned medium of these cells enhances the formation of SGs in rat cardiomyocytes exposed to hypoxia. This was associated with a reduction of cardiomyocyte death. An implication of angiogenin in the formation of SGs in cardiomyocytes exposed to SDF-1migr cell media is confirmed by the finding that this effect is abrogated by an angiogenin inhibitor. Similarly inhibited was the improvement in cardiomyocyte survival. These new data highlight a novel mechanism that may contribute to cardiac protection elicited by the migrating component of BM-MNCs. It should be noted that, in our in vivo study, cell therapy did not produce any significant reduction in cardiomyocyte apoptosis at the level of the peri-infarct zone, as detected by Tunel staining at 2 weeks post-MI. Cardiomyocyte loss is prevalent in the early phase after permanent coronary artery ligation and therefore any benefit induced by cell therapy might have been underestimated in our study. Furthermore, it is not clear whether angiogenin-induced protection derives from inhibition of “programmed, apoptotic” or “accidental, non-apoptotic” cell death. More in-depth investigation is warranted to address this aspect.
Sorting experiments newly show that, within BM-MNCs, CD14+CD16+ monocytes express the highest levels of angiogenin. Experimental evidence indicates that healing of infarcted heart requires monocytes/macrophages. Classical monocytes, recruited in the early phase after an MI, degrade cellular debris and macromolecules at the infarct site, while non-classical monocytes intervene later, modulating the formation of granulation tissue and cardiac remodeling . Previous studies by Nahrendorf and colleagues have shown that Ly-6Clo (mouse analogues of CD14+CD16+ monocytes) promote myocardial healing via myofibroblast accumulation, angiogenesis, and deposition of collagen . Although circulating levels did not change following MI induction, the capacity of Ly-6Clo/CD14+CD16+ monocytes to migrate into ischemic myocardium increased 4.8-fold. This unique homing capacity has been attributed to the expression of different chemokine receptors, including CX3CR1, CCR5 and the SDF-1 receptor CXCR4. Due to their ability to sense migratory cues, angiogenin-expressing monocytes could spread through the peri-infarct area and exert protective effect on stunned cardiomyocytes. Therefore, the expression of chemokine receptors and pro-survival factors may represent a fundamental association for monocyte healing capacity.
This study conducted on BM specimens of TransACT cell therapy trials newly demonstrates that migratory features of BM-MNCs are associated with distinct cardioprotective modalities. These new findings improve current understanding of mechanisms implicated in spontaneous and cell therapy-induced cardiac repair. On the other hand, this research was not designed to make any assumption or prediction of the trial outcomes. The TransAct studies compare the benefit of intramyocardial delivery of BM CD133+ PCs or their medium, in combination with surgical revascularization or ventricular reshaping in patients with previous MI, whereas here we verified the therapeutic effect of BM subfractions in a mouse model of acute MI induced by permanent coronary artery ligation. While additional investigation is necessary to confirm the actions of migrated and non-migrated fractions in models of ischemia-reperfusion or chronic ischemia, this study highlights the mechanistic complexity of current cell therapy and the requirement of standardized cell products in future clinical trials.
bone marrow mononuclear cell
enzyme-linked immunosorbent assay
human umbilical vein endothelial cell
left ventricular ejection fraction
monocyte chemoattractant protein
mesenchymal stem cell
ribonuclease/angiogenin inhibitor 1
stromal cell-derived factor 1
- SDF-1mig :
- SDF-1non :
stress-induced small RNA
vascular endothelial growth factor
- Vehmig :
- Vehnon :
This study was funded by an MRC project grant MRC G0900912 “Function based enrichment of pro-angiogenic cells for cardiac repair”. The study was also supported by grants from the British Heart Foundation and the National Institute for Health Research Bristol Cardiovascular Biomedical Research Unit.
- Burt RK, Loh Y, Pearce W, Beohar N, Barr WG, Craig R, et al. Clinical applications of blood-derived and marrow-derived stem cells for nonmalignant diseases. JAMA. 2008;299:925–36.View ArticlePubMedGoogle Scholar
- Delewi R, Andriessen A, Tijssen JG, Zijlstra F, Piek JJ, Hirsch A. Impact of intracoronary cell therapy on left ventricular function in the setting of acute myocardial infarction: a meta-analysis of randomised controlled clinical trials. Heart. 2013;99:225–32.View ArticlePubMedGoogle Scholar
- Clifford DM, Fisher SA, Brunskill SJ, Doree C, Mathur A, Watt S, et al. Stem cell treatment for acute myocardial infarction. Cochrane Database Syst Rev. 2012;2:CD006536.PubMedGoogle Scholar
- Jeevanantham V, Butler M, Saad A, Abdel-Latif A, Zuba-Surma EK, Dawn B. Adult bone marrow cell therapy improves survival and induces long-term improvement in cardiac parameters: a systematic review and meta-analysis. Circulation. 2012;126:551–68.View ArticlePubMed CentralPubMedGoogle Scholar
- Orlic D, Kajstura J, Chimenti S, Jakoniuk I, Anderson SM, Li B, et al. Bone marrow cells regenerate infarcted myocardium. Nature. 2001;410:701–5.View ArticlePubMedGoogle Scholar
- Popa ER, Harmsen MC, Tio RA, van der Strate BW, Brouwer LA, Schipper M, et al. Circulating CD34+ progenitor cells modulate host angiogenesis and inflammation in vivo. J Mol Cell Cardiol. 2006;41:86–96.View ArticlePubMedGoogle Scholar
- Korf-Klingebiel M, Kempf T, Sauer T, Brinkmann E, Fischer P, Meyer GP, et al. Bone marrow cells are a rich source of growth factors and cytokines: implications for cell therapy trials after myocardial infarction. Eur Heart J. 2008;29:2851–8.View ArticlePubMedGoogle Scholar
- Nahrendorf M, Swirski FK, Aikawa E, Stangenberg L, Wurdinger T, Figueiredo JL, et al. The healing myocardium sequentially mobilizes two monocyte subsets with divergent and complementary functions. J Exp Med. 2007;204:3037–47.View ArticlePubMed CentralPubMedGoogle Scholar
- Orlic D, Kajstura J, Chimenti S, Limana F, Jakoniuk I, Quaini F, et al. Mobilized bone marrow cells repair the infarcted heart, improving function and survival. Proc Natl Acad Sci U S A. 2001;98:10344–9.View ArticlePubMed CentralPubMedGoogle Scholar
- Gnecchi M, Zhang Z, Ni A, Dzau VJ. Paracrine mechanisms in adult stem cell signaling and therapy. Circ Res. 2008;103:1204–19.View ArticlePubMed CentralPubMedGoogle Scholar
- Barclay GR, Tura O, Samuel K, Hadoke PW, Mills NL, Newby DE, et al. Systematic assessment in an animal model of the angiogenic potential of different human cell sources for therapeutic revascularization. Stem Cell Res Ther. 2012;3:23.View ArticlePubMed CentralPubMedGoogle Scholar
- Kawamoto A, Iwasaki H, Kusano K, Murayama T, Oyamada A, Silver M, et al. CD34-positive cells exhibit increased potency and safety for therapeutic neovascularization after myocardial infarction compared with total mononuclear cells. Circulation. 2006;114:2163–9.View ArticlePubMedGoogle Scholar
- Seeger FH, Rasper T, Koyanagi M, Fox H, Zeiher AM, Dimmeler S. CXCR4 expression determines functional activity of bone marrow-derived mononuclear cells for therapeutic neovascularization in acute ischemia. Arterioscler Thromb Vasc Biol. 2009;29:1802–9.View ArticlePubMedGoogle Scholar
- Rossi DJ, Jamieson CH, Weissman IL. Stems cells and the pathways to aging and cancer. Cell. 2008;132:681–96.View ArticlePubMedGoogle Scholar
- Spinetti G, Cordella D, Fortunato O, Sangalli E, Losa S, Gotti A, et al. Global remodeling of the vascular stem cell niche in bone marrow of diabetic patients: implication of the microRNA-155/FOXO3a signaling pathway. Circ Res. 2013;112:510–22.View ArticlePubMed CentralPubMedGoogle Scholar
- Bodi V, Sanchis J, Nunez J, Mainar L, Minana G, Benet I, et al. Uncontrolled immune response in acute myocardial infarction: unraveling the thread. Am Heart J. 2008;156:1065–73.View ArticlePubMedGoogle Scholar
- Seeger FH, Tonn T, Krzossok N, Zeiher AM, Dimmeler S. Cell isolation procedures matter: a comparison of different isolation protocols of bone marrow mononuclear cells used for cell therapy in patients with acute myocardial infarction. Eur Heart J. 2007;28:766–72.View ArticlePubMedGoogle Scholar
- Bartunek J, Vanderheyden M, Vandekerckhove B, Mansour S, De Bruyne B, De Bondt P, et al. Intracoronary injection of CD133-positive enriched bone marrow progenitor cells promotes cardiac recovery after recent myocardial infarction: feasibility and safety. Circulation. 2005;112:I178–83.PubMedGoogle Scholar
- Mansour S, Vanderheyden M, De Bruyne B, Vandekerckhove B, Delrue L, Van Haute I, et al. Intracoronary delivery of hematopoietic bone marrow stem cells and luminal loss of the infarct-related artery in patients with recent myocardial infarction. J Am Coll Cardiol. 2006;47:1727–30.View ArticlePubMedGoogle Scholar
- Losordo DW, Henry TD, Davidson C, Sup Lee J, Costa MA, Bass T, et al. Intramyocardial, autologous CD34+ cell therapy for refractory angina. Circ Res. 2011;109:428–36.View ArticlePubMed CentralPubMedGoogle Scholar
- Walter DH, Haendeler J, Reinhold J, Rochwalsky U, Seeger F, Honold J, et al. Impaired CXCR4 signaling contributes to the reduced neovascularization capacity of endothelial progenitor cells from patients with coronary artery disease. Circ Res. 2005;97:1142–51.View ArticlePubMedGoogle Scholar
- Britten MB, Abolmaali ND, Assmus B, Lehmann R, Honold J, Schmitt J, et al. Infarct remodeling after intracoronary progenitor cell treatment in patients with acute myocardial infarction (TOPCARE-AMI): mechanistic insights from serial contrast-enhanced magnetic resonance imaging. Circulation. 2003;108:2212–8.View ArticlePubMedGoogle Scholar
- Krankel N, Armstrong SP, McArdle CA, Dayan C, Madeddu P. Distinct kinin-induced functions are altered in circulating cells of young type 1 diabetic patients. PLoS One. 2010;5, e11146.View ArticlePubMed CentralPubMedGoogle Scholar
- Krankel N, Katare RG, Siragusa M, Barcelos LS, Campagnolo P, Mangialardi G, et al. Role of kinin B2 receptor signaling in the recruitment of circulating progenitor cells with neovascularization potential. Circ Res. 2008;103:1335–43.View ArticlePubMed CentralPubMedGoogle Scholar
- Abbott JD, Huang Y, Liu D, Hickey R, Krause DS, Giordano FJ. Stromal cell-derived factor-1alpha plays a critical role in stem cell recruitment to the heart after myocardial infarction but is not sufficient to induce homing in the absence of injury. Circulation. 2004;110:3300–5.View ArticlePubMedGoogle Scholar
- Askari AT, Unzek S, Popovic ZB, Goldman CK, Forudi F, Kiedrowski M, et al. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy. Lancet. 2003;362:697–703.View ArticlePubMedGoogle Scholar
- Selected autologous bone marrow cell transplant following trans-mural myocardial infarction in patients undergoing coronary surgery: A prospective, double-blind, randomised controlled trial. http://www.controlled-trials.com/ISRCTN65630838/transact.
- Katare R, Caporali A, Emanueli C, Madeddu P. Benfotiamine improves functional recovery of the infarcted heart via activation of pro-survival G6PD/Akt signaling pathway and modulation of neurohormonal response. J Mol Cell Cardiol. 2010;49:625–38.View ArticlePubMed CentralPubMedGoogle Scholar
- Katare R, Riu F, Mitchell K, Gubernator M, Campagnolo P, Cui Y, et al. Transplantation of human pericyte progenitor cells improves the repair of infarcted heart through activation of an angiogenic program involving micro-RNA-132. Circ Res. 2011;109:894–906.View ArticlePubMed CentralPubMedGoogle Scholar
- Li S, Hu GF. Angiogenin-mediated rRNA transcription in cancer and neurodegeneration. Int J Biochem Mol Biol. 2010;1:26–35.PubMed CentralPubMedGoogle Scholar
- Janssens S, Dubois C, Bogaert J, Theunissen K, Deroose C, Desmet W, et al. Autologous bone marrow-derived stem-cell transfer in patients with ST-segment elevation myocardial infarction: double-blind, randomised controlled trial. Lancet. 2006;367:113–21.View ArticlePubMedGoogle Scholar
- Lunde K, Solheim S, Aakhus S, Arnesen H, Abdelnoor M, Egeland T, et al. Intracoronary injection of mononuclear bone marrow cells in acute myocardial infarction. N Engl J Med. 2006;355:1199–209.View ArticlePubMedGoogle Scholar
- Schachinger V, Assmus B, Britten MB, Honold J, Lehmann R, Teupe C, et al. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction: final one-year results of the TOPCARE-AMI Trial. J Am Coll Cardiol. 2004;44:1690–9.View ArticlePubMedGoogle Scholar
- Schachinger V, Erbs S, Elsasser A, Haberbosch W, Hambrecht R, Holschermann H, et al. Intracoronary bone marrow-derived progenitor cells in acute myocardial infarction. N Engl J Med. 2006;355:1210–21.View ArticlePubMedGoogle Scholar
- Wollert KC, Meyer GP, Lotz J, Ringes-Lichtenberg S, Lippolt P, Breidenbach C, et al. Intracoronary autologous bone-marrow cell transfer after myocardial infarction: the BOOST randomised controlled clinical trial. Lancet. 2004;364:141–8.View ArticlePubMedGoogle Scholar
- Strauer BE, Brehm M, Zeus T, Kostering M, Hernandez A, Sorg RV, et al. Repair of infarcted myocardium by autologous intracoronary mononuclear bone marrow cell transplantation in humans. Circulation. 2002;106:1913–8.View ArticlePubMedGoogle Scholar
- Manginas A, Goussetis E, Koutelou M, Karatasakis G, Peristeri I, Theodorakos A, et al. Pilot study to evaluate the safety and feasibility of intracoronary CD133(+) and CD133(−) CD34(+) cell therapy in patients with nonviable anterior myocardial infarction. Catheter Cardiovasc Interv. 2007;69:773–81.View ArticlePubMedGoogle Scholar
- Klein HM, Ghodsizad A, Marktanner R, Poll L, Voelkel T, Mohammad Hasani MR, et al. Intramyocardial implantation of CD133+ stem cells improved cardiac function without bypass surgery. Heart Surg Forum. 2007;10:E66–9.View ArticlePubMedGoogle Scholar
- Ahmadi H, Baharvand H, Ashtiani SK, Soleimani M, Sadeghian H, Ardekani JM, et al. Safety analysis and improved cardiac function following local autologous transplantation of CD133(+) enriched bone marrow cells after myocardial infarction. Curr Neurovasc Res. 2007;4:153–60.View ArticlePubMedGoogle Scholar
- Mansour S, Roy DC, Bouchard V, Stevens LM, Gobeil F, Rivard A, et al. One-year safety analysis of the COMPARE-AMI trial: comparison of intracoronary injection of CD133 bone marrow stem cells to placebo in patients after acute myocardial infarction and left ventricular dysfunction. Bone Marrow Res. 2011;2011:385124.View ArticlePubMed CentralPubMedGoogle Scholar
- Colombo A, Castellani M, Piccaluga E, Pusineri E, Palatresi S, Longari V, et al. Myocardial blood flow and infarct size after CD133+ cell injection in large myocardial infarction with good recanalization and poor reperfusion: results from a randomized controlled trial. J Cardiovasc Med. 2011;12:239–48.View ArticleGoogle Scholar
- Taraldsrud E, Grogaard HK, Solheim S, Lunde K, Floisand Y, Arnesen H, et al. Age and stress related phenotypical changes in bone marrow CD34+ cells. Scand J Clin Lab Invest. 2009;69:79–84.View ArticlePubMedGoogle Scholar
- Assmus B, Iwasaki M, Schachinger V, Roexe T, Koyanagi M, Iekushi K, et al. Acute myocardial infarction activates progenitor cells and increases Wnt signalling in the bone marrow. Eur Heart J. 2012;33:1911–9.View ArticlePubMedGoogle Scholar
- Wang X, Takagawa J, Lam VC, Haddad DJ, Tobler DL, Mok PY, et al. Donor myocardial infarction impairs the therapeutic potential of bone marrow cells by an interleukin-1-mediated inflammatory response. Sci Transl Med. 2011;3:100ra90.PubMed CentralPubMedGoogle Scholar
- Moresi R, Tesei S, Costarelli L, Viticchi C, Stecconi R, Bernardini G, et al. Age- and gender-related alterations of the number and clonogenic capacity of circulating CD34+ progenitor cells. Biogerontology. 2005;6:185–92.View ArticlePubMedGoogle Scholar
- Fortunato O, Spinetti G, Specchia C, Cangiano E, Valgimigli M, Madeddu P. Migratory activity of circulating progenitor cells and serum SDF-1alpha predict adverse events in patients with myocardial infarction. Cardiovasc Res. 2013;100:192–200.View ArticlePubMedGoogle Scholar
- Vasa M, Fichtlscherer S, Aicher A, Adler K, Urbich C, Martin H, et al. Number and migratory activity of circulating endothelial progenitor cells inversely correlate with risk factors for coronary artery disease. Circ Res. 2001;89:E1–7.View ArticlePubMedGoogle Scholar
- Hill JM, Zalos G, Halcox JP, Schenke WH, Waclawiw MA, Quyyumi AA, et al. Circulating endothelial progenitor cells, vascular function, and cardiovascular risk. N Engl J Med. 2003;348:593–600.View ArticlePubMedGoogle Scholar
- Hosono M, de Boer OJ, van der Wal AC, van der Loos CM, Teeling P, Piek JJ, et al. Increased expression of T cell activation markers (CD25, CD26, CD40L and CD69) in atherectomy specimens of patients with unstable angina and acute myocardial infarction. Atherosclerosis. 2003;168:73–80.View ArticlePubMedGoogle Scholar
- Li SH, Lai TY, Sun Z, Han M, Moriyama E, Wilson B, et al. Tracking cardiac engraftment and distribution of implanted bone marrow cells: comparing intra-aortic, intravenous, and intramyocardial delivery. J Thorac Cardiovasc Surg. 2009;137:1225–33.e1.View ArticlePubMedGoogle Scholar
- Ishida S, Yamashiro K, Usui T, Kaji Y, Ogura Y, Hida T, et al. Leukocytes mediate retinal vascular remodeling during development and vaso-obliteration in disease. Nat Med. 2003;9:781–8.View ArticlePubMedGoogle Scholar
- Stabile E, Burnett MS, Watkins C, Kinnaird T, Bachis A, la Sala A, et al. Impaired arteriogenic response to acute hindlimb ischemia in CD4-knockout mice. Circulation. 2003;108:205–10.View ArticlePubMedGoogle Scholar
- Benameur T, Soleti R, Porro C, Andriantsitohaina R, Martinez MC. Microparticles carrying Sonic hedgehog favor neovascularization through the activation of nitric oxide pathway in mice. PLoS One. 2010;5, e12688.View ArticlePubMed CentralPubMedGoogle Scholar
- Liu XH, Bai CG, Xu ZY, Huang SD, Yuan Y, Gong DJ, et al. Therapeutic potential of angiogenin modified mesenchymal stem cells: angiogenin improves mesenchymal stem cells survival under hypoxia and enhances vasculogenesis in myocardial infarction. Microvasc Res. 2008;76:23–30.View ArticlePubMedGoogle Scholar
- Li S, Hu GF. Emerging role of angiogenin in stress response and cell survival under adverse conditions. J Cell Physiol. 2012;227:2822–6.View ArticlePubMed CentralPubMedGoogle Scholar
- Yamasaki S, Ivanov P, Hu GF, Anderson P. Angiogenin cleaves tRNA and promotes stress-induced translational repression. J Cell Biol. 2009;185:35–42.View ArticlePubMed CentralPubMedGoogle Scholar
- Emara MM, Ivanov P, Hickman T, Dawra N, Tisdale S, Kedersha N, et al. Angiogenin-induced tRNA-derived stress-induced RNAs promote stress-induced stress granule assembly. J Biol Chem. 2010;285:10959–68.View ArticlePubMed CentralPubMedGoogle Scholar
- Kieran D, Sebastia J, Greenway MJ, King MA, Connaughton D, Concannon CG, et al. Control of motoneuron survival by angiogenin. J Neurosci. 2008;28:14056–61.View ArticlePubMedGoogle Scholar
- Sebastia J, Kieran D, Breen B, King MA, Netteland DF, Joyce D, et al. Angiogenin protects motoneurons against hypoxic injury. Cell Death Differ. 2009;16:1238–47.View ArticlePubMedGoogle Scholar
- Sadagopan S, Veettil MV, Chakraborty S, Sharma-Walia N, Paudel N, Bottero V, et al. Angiogenin functionally interacts with p53 and regulates p53-mediated apoptosis and cell survival. Oncogene. 2012;31:4835–47.View ArticlePubMed CentralPubMedGoogle Scholar
- Fu H, Feng J, Liu Q, Sun F, Tie Y, Zhu J, et al. Stress induces tRNA cleavage by angiogenin in mammalian cells. FEBS Lett. 2009;583:437–42.View ArticlePubMedGoogle Scholar
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