Erratum: Young Bone Marrow Sca-1 Cells Rejuvenate the Aged Heart by Promoting Epithelial-to-Mesenchymal Transition: Erratum.

[This corrects the article DOI: 10.7150/thno.22788.].

Aging impairs human bone marrow function and cardiac repair following myocardial infarction in a humanized chimeric mouse.

Ventricular remodeling following myocardial infarction (MI) is a major cause of heart failure, a condition prevalent in older individuals. Following MI, immune cells are mobilized to the myocardium from peripheral lymphoid organs and play an active role in orchestrating repair. While the effect of aging on mouse bone marrow (BM) has been studied, less is known about how aging affects human BM cells and their ability to regulate repair processes. In this study, we investigate the effect aging has on human BM cell responses post-MI using a humanized chimeric mouse model. BM samples were collected from middle aged (mean age 56.4 ± 0.97) and old (mean age 72.7 ± 0.59) patients undergoing cardiac surgery, CD34+/- cells were isolated, and NOD-scid-IL2rγnull (NSG) mice were reconstituted. Three months following reconstitution, the animals were examined at baseline or subjected to coronary artery ligation (MI). Younger patient cells exhibited greater repopulation capacity in the BM, blood, and spleen as well as greater lymphoid cell production. Following MI, CD34+ cell age impacted donor and host cellular responses. Mice reconstituted with younger CD34+ cells exhibited greater human CD45+ recruitment to the heart compared to mice reconstituted with old cells. Increased cellular responses were primarily driven by T-cell recruitment, and these changes corresponded with greater human IFNy levels and reduced mouse IL-1ß in the heart. Age-dependent changes in BM function led to significantly lower survival, increased infarct expansion, impaired host cell responses, and reduced function by 4w post-MI. In contrast, younger CD34+ cells helped to limit remodeling and preserve function post-MI.

Age-related defects in autophagy alter the secretion of paracrine factors from bone marrow mononuclear cells.

Bone marrow mononuclear cell therapy improves cardiac repair after myocardial infarction (MI), in-part through signaling to resident cardiac cells, such as fibroblasts, which regulate scar formation. The efficacy of cell therapy declines with age, as aging of both donor and recipient cells decreases repair responses. Autophagy regulates the microenvironment by both extracellular vesicle (EV)-dependent and independent secretion pathways. We hypothesized that age-related autophagy changes in bone marrow cells (BMCs) alter paracrine signaling, contributing to lower cell therapy efficacy. Here, we demonstrate that young Sca-1+ BMCs exhibited a higher LC3II/LC3I ratio compared to old Sca-1+ BMCs, which was accentuated when BMCs were cultured under hypoxia. To examine the effect on paracrine signaling, old cardiac fibroblasts were cultured with conditioned medium (CM) from young and old Sca-1+ BMCs. Young, but not old CM, enhanced fibroblast proliferation, migration, and differentiation, plus reduced senescence. These beneficial effects were lost when autophagy or EV secretion in BMCs was blocked pharmacologically, or by siRNA knockdown of Atg7. Therefore, both EV-dependent and -independent paracrine signaling from young BMCs is responsible for paracrine stimulation of old cardiac fibroblasts. In vivo, bone marrow chimerism of old mice with young BMCs increased the number of LC3b+ cells in the heart compared to old mice reconstituted with old BMCs. These data suggest that the deterioration of autophagy with aging negatively impacts the paracrine effects of BMCs, and provide mechanistic insight into the age-related decline in cell therapy efficacy that could be targeted to improve the function of old donor cells.

Delineating the relationship between immune system aging and myogenesis in muscle repair.

Recruited immune cells play a critical role in muscle repair, in part by interacting with local stem cell populations to regulate muscle regeneration. How aging affects their communication during myogenesis is unclear. Here, we investigate how aging impacts the cellular function of these two cell types after muscle injury during normal aging or after immune rejuvenation using a young to old (Y-O) or old to old (O-O) bone marrow (BM) transplant model. We found that skeletal muscle from old mice (20 months) exhibited elevated basal inflammation and possessed fewer satellite cells compared with young mice (3 months). After cardiotoxin muscle injury (CTX), old mice exhibited a blunted inflammatory response compared with young mice and enhanced M2 macrophage recruitment and IL-10 expression. Temporal immune and cytokine responses of old mice were partially restored to a young phenotype following reconstitution with young cells (Y-O chimeras). Improved immune responses in Y-O chimeras were associated with greater satellite cell proliferation compared with O-O chimeras. To identify how immune cell aging affects myoblast function, conditioned media (CM) from activated young or old macrophages was applied to cultured C2C12 myoblasts. CM from young macrophages inhibited myogenesis while CM from old macrophages reduced proliferation. These functional differences coincided with age-related differences in macrophage cytokine expression. Together, this study examines the infiltration and proliferation of immune cells and satellite cells after injury in the context of aging and, using BM chimeras, demonstrates that young immune cells retain cell autonomy in an old host to increase satellite cell proliferation.

The conductive function of biopolymer corrects myocardial scar conduction blockage and resynchronizes contraction to prevent heart failure.

Myocardial fibrosis, resulting from ischemic injury, increases tissue resistivity in the infarct area, which impedes heart synchronous electrical propagation. The uneven conduction between myocardium and fibrotic tissue leads to dys-synchronous contraction, which progresses towards ventricular dysfunction. We synthesized a conductive poly-pyrrole-chitosan hydrogel (PPY-CHI), and investigated its capabilities in improving electrical propagation in fibrotic tissue, as well as resynchronizing cardiac contraction to preserve cardiac function. In an in vitro fibrotic scar model, conductivity increased in proportion to the amount of PPY-CHI hydrogel added. To elucidate the mechanism of interaction between myocardial ionic changes and electrical current, an equivalent circuit model was used, which showed that PPY-CHI resistance was 10 times lower, and latency time 5 times shorter, compared to controls. Using a rat myocardial infarction (MI) model, PPY-CHI was injected into fibrotic tissue 7 days post MI. There, PPY-CHI reduced tissue resistance by 30%, improved electrical conduction across the fibrotic scar by 33%, enhanced field potential amplitudes by 2 times, and resynchronized cardiac contraction. PPY-CHI hydrogel also preserved cardiac function at 3 months, and reduced susceptibility to arrhythmia by 30% post-MI. These data demonstrated that the conductive PPY-CHI hydrogel reduced fibrotic scar resistivity, and enhanced electrical conduction, to synchronize cardiac contraction.

Considering Cause and Effect of Immune Cell Aging on Cardiac Repair after Myocardial Infarction.

The importance of the immune system for cardiac repair following myocardial infarction is undeniable; however, the complex nature of immune cell behavior has limited the ability to develop effective therapeutics. This limitation highlights the need for a better understanding of the function of each immune cell population during the inflammatory and resolution phases of cardiac repair. The development of reliable therapies is further complicated by aging, which is associated with a decline in cell and organ function and the onset of cardiovascular and immunological diseases. Aging of the immune system has important consequences on heart function as both chronic cardiac inflammation and an impaired immune response to cardiac injury are observed in older individuals. Several studies have suggested that rejuvenating the aged immune system may be a valid therapeutic candidate to prevent or treat heart disease. Here, we review the basic patterns of immune cell behavior after myocardial infarction and discuss the autonomous and nonautonomous manners of hematopoietic stem cell and immune cell aging. Lastly, we identify prospective therapies that may rejuvenate the aged immune system to improve heart function such as anti-inflammatory and senolytic therapies, bone marrow transplant, niche remodeling and regulation of immune cell differentiation.

Knock-out of MicroRNA 145 impairs cardiac fibroblast function and wound healing post-myocardial infarction.

Prevention of infarct scar thinning and dilatation and stimulation of scar contracture can prevent progressive heart failure. Since microRNA 145 (miR-145) plays an important role in cardiac fibroblast response to wound healing and cardiac repair after an myocardial infarction (MI), using a miR-145 knock-out (KO) mouse model, we evaluated contribution of down-regulation of miR-145 to cardiac fibroblast and myofibroblast function during adverse cardiac remodelling. Cardiac function decreased more and the infarct size was larger in miR-145 KO than that in WT mice after MI and this phenomenon was accompanied by a decrease in cardiac fibroblast-to-myofibroblast differentiation. Quantification of collagen I and α-SMA protein levels as well as wound contraction revealed that transdifferentiation of cardiac fibroblasts into myofibroblasts was lower in KO than WT mice. In vitro restoration of miR-145 induced more differentiation of fibroblasts to myofibroblasts and this effect involved the target genes Klf4 and myocardin. MiR-145 contributes to infarct scar contraction in the heart and the absence of miR-145 contributes to dysfunction of cardiac fibroblast, resulting in greater infarct thinning and dilatation. Augmentation of miR-145 could be an attractive target to prevent adverse cardiac remodelling after MI by enhancing the phenotypic switch of cardiac fibroblasts to myofibroblasts.

Rectification of radiotherapy-induced cognitive impairments in aged mice by reconstituted Sca-1+ stem cells from young donors.

BACKGROUND: Radiotherapy is widely used and effective for treating brain tumours, but inevitably impairs cognition as it arrests cellular processes important for learning and memory. This is particularly evident in the aged brain with limited regenerative capacity, where radiation produces irreparable neuronal damage and activation of neighbouring microglia. The latter is responsible for increased neuronal death and contributes to cognitive decline after treatment. To date, there are few effective means to prevent cognitive deficits after radiotherapy. METHODS: Here we implanted hematopoietic stem cells (HSCs) from young or old (2- or 18-month-old, respectively) donor mice expressing green fluorescent protein (GFP) into old recipients and assessed cognitive abilities 3 months post-reconstitution. RESULTS: Regardless of donor age, GFP+ cells homed to the brain of old recipients and expressed the macrophage/microglial marker, Iba1. However, only young cells attenuated deficits in novel object recognition and spatial memory and learning in old mice post-irradiation. Mechanistically, old recipients that received young HSCs, but not old, displayed significantly greater dendritic spine density and long-term potentiation (LTP) in CA1 neurons of the hippocampus. Lastly, we found that GFP+/Iba1+ cells from young and old donors were differentially polarized to an anti- and pro-inflammatory phenotype and produced neuroprotective factors and reactive nitrogen species in vivo, respectively. CONCLUSION: Our results suggest aged peripherally derived microglia-like cells may exacerbate cognitive impairments after radiotherapy, whereas young microglia-like cells are polarized to a reparative phenotype in the irradiated brain, particularly in neural circuits associated with rewards, learning, and memory. These findings present a proof-of-principle for effectively reinstating central cognitive function of irradiated brains with peripheral stem cells from young donor bone marrow.

Cellular senescence contributes to age-dependent changes in circulating extracellular vesicle cargo and function.

Extracellular vesicles (EVs) have emerged as important regulators of inter-cellular and inter-organ communication, in part via the transfer of their cargo to recipient cells. Although circulating EVs have been previously studied as biomarkers of aging, how circulating EVs change with age and the underlying mechanisms that contribute to these changes are poorly understood. Here, we demonstrate that aging has a profound effect on the circulating EV pool, as evidenced by changes in concentration, size, and cargo. Aging also alters particle function; treatment of cells with EV fractions isolated from old plasma reduces macrophage responses to lipopolysaccharide, increases phagocytosis, and reduces endothelial cell responses to vascular endothelial growth factor compared to cells treated with young EV fractions. Depletion studies indicate that CD63+ particles mediate these effects. Treatment of macrophages with EV-like particles revealed that old particles increased the expression of EV miRNAs in recipient cells. Transfection of cells with microRNA mimics recapitulated some of the effects seen with old EV-like particles. Investigation into the underlying mechanisms using bone marrow transplant studies revealed circulating cell age does not substantially affect the expression of aging-associated circulating EV miRNAs in old mice. Instead, we show that cellular senescence contributes to changes in particle cargo and function. Notably, senolytic treatment of old mice shifted plasma particle cargo and function toward that of a younger phenotype. Collectively, these results demonstrate that senescent cells contribute to changes in plasma EVs with age and suggest a new mechanism by which senescent cells can affect cellular functions throughout the body.

A conductive cell-delivery construct as a bioengineered patch that can improve electrical propagation and synchronize cardiomyocyte contraction for heart repair.

Cardiac tissue engineering is of particular importance in the combination of contracting cells with a biomaterial scaffold, which serves as a cell-delivery construct, to replace cardiomyocytes (CMs) that are lost as a result of an infarction, to restore heart function. However, most biomaterial scaffolds are nonconductive and may delay regional conduction, potentially causing arrhythmias. In this study, a conductive CM-delivery construct that consists of a gelatin-based gelfoam that is conjugated with a self-doped conductive polymer (poly-3-amino-4-methoxybenzoic acid, PAMB) is proposed as a cardiac patch (PAMB-Gel patch) to repair an infarcted heart. A nonconductive plain gelfoam (Gel patch) is used as a control. The electrical conductivity of the PAMB-Gel patch is approximately 30 times higher than that of the Gel patch; as a result, the conductive PAMB-Gel patch can substantially increase electrical conduction between distinct clusters of beating CMs, facilitating their synchronous contraction. In vivo epicardial implantation of the PAMB-Gel patch that is seeded with CMs (the bioengineered patch) in infarcted rat hearts can significantly enhance electrical activity in the fibrotic tissue, improving electrical impulse propagation and synchronizing CM contraction across the scar region, markedly reducing its susceptibility to cardiac arrhythmias. Echocardiography shows that the bioengineered conductive patch has an important role in the restoration of cardiac function, perhaps owing to the synergistic effects of its conductive construct and the synchronously beating CMs. These experimental results reveal that the as-proposed bioengineered conductive patch has great potential for repairing injured cardiac tissues.

A self-doping conductive polymer hydrogel that can restore electrical impulse propagation at myocardial infarct to prevent cardiac arrhythmia and preserve ventricular function.

Following myocardial infarction (MI), necrotic cardiomyocytes (CMs) are replaced by fibroblasts and collagen tissue, causing abnormal electrical signal propagation, desynchronizing cardiac contraction, resulting in cardiac arrhythmia. In this work, a conductive polymer, poly-3-amino-4-methoxybenzoic acid (PAMB), is synthesized and grafted onto non-conductive gelatin. The as-synthesized PAMB-G copolymer is self-doped in physiological pH environments, making it an electrically active material in biological tissues. This copolymer is cross-linked by carbodiimide to form an injectable conductive hydrogel (PAMB-G hydrogel). The un-grafted gelatin hydrogel is prepared in a similar manner as a control. Both test hydrogels not only provide an optimal matrix for CM adhesion and growth but also maintain CM morphology and functional proteins. The conductivity of PAMB-G hydrogel is ca. 12 times higher than that of gelatin hydrogel. Microelectrode array analyses reveal that a heart placed on the PAMB-G hydrogel has a higher field potential amplitude than that placed on the gelatin hydrogel and can pass current from one heart to excite another heart at a distance. The injection of PAMB-G hydrogel into the scar zone following an MI in a rat heart improves electrical impulse propagation over that in a heart that has been treated with gelatin hydrogel, and synchronizes heart contraction, leading to preservation of the ventricular function and reduction of cardiac arrhythmia, demonstrating its potential for use in treating MI.

CD34+ Stem Cells: Promising Roles in Cardiac Repair and Regeneration.

Cell therapy has received significant attention as a novel therapeutic approach to restore cardiac function after injury. CD34-positive (CD34+) stem cells have been investigated for their ability to promote angiogenesis and contribute to the prevention of remodelling after infarct. However, there are significant differences between murine and human CD34+ cells; understanding these differences might benefit the therapeutic use of these cells. Herein we discuss the function of the CD34 cell and highlight the similarities and differences between murine and human CD34 cell function, which might explain some of the differences between the animal and human evolutions. We also summarize the studies that report the application of murine and human CD34+ cells in preclinical studies and clinical trials and current limitations with the application of cell therapy for cardiac repair. Finally, to overcome these limitations we discuss the application of novel humanized rodent models that can bridge the gap between preclinical and clinical studies as well as rejuvenation strategies for improving the quality of old CD34+ cells for future clinical trials of autologous cell transplantation.

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 (p16INK4a , P19ARF , p27Kip1 ) and proteins (p16INK4a , p27Kip1 ) 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.

Knockout of Canopy 2 activates p16INK4a pathway to impair cardiac repair.

BACKGROUND: Cardiac repair depends on angiogenesis and cell proliferation. Previously we identified Canopy 2 (CNPY2) as a secreted angiogenic growth factor which promotes neovascularization. We investigated the role of CNPY2 in cardiac repair following myocardial infarction (MI) and the possible mediators involved using Cnpy2 knockout (KO) mice and human cardiac tissue. METHODS AND RESULTS: Cardiac tissue from patients with end-stage heart failure had significantly lower endogenous CNPY2 expression compared to samples from control patients. CNPY2 expression in mouse hearts significantly decreased following MI. Significantly less leukocyte and endothelial cell proliferation was found in Cnpy2 KO than wild-type (WT) mice post MI which contributed to impaired angiogenesis, tissue repair, and decreased cardiac function (fractional shortening: WT: 21.1 ±â€¯2.1% vs. KO: 16.4 ±â€¯1.6%, p < .01 at day 28 post MI). RT-qPCR revealed significantly increased p16INK4a expression in Cnpy2 KO mouse hearts (WT: 1.0 ±â€¯0.04 vs. KO: 2.33 ±â€¯0.11 [relative expression of p16 INK4a], p < .01) which was confirmed by immunostaining (WT: 8.47 ±â€¯1.22 vs. KO: 12.9 ±â€¯1.22 [% total cells], p < .05) for the p16INK4a protein. Expression of cell cycle-related proteins, cyclin D1, cyclin-dependent kinases 4 and 6, and phosphorylated retinoblastoma protein (pRb) was significantly decreased in Cnpy2 KO mouse hearts. The up-regulation of the p16INK4a/cyclin D1/Rb pathway by knockout of Cnpy2 was accompanied by attenuation of PDK1/Akt phosphorylation. MI exacerbated the detrimental effects of p16INK4a on tissue repair in Cnpy2 KO mice. Overexpression of CNPY2 in the cardiac tissue of transgenic mice reversed the inhibition of cell proliferation through suppression of the p16INK4a pathway. CONCLUSIONS: Cardiac injury and progressive heart failure were associated with decreased CNPY2 levels in both humans and mice. Knockout of Cnpy2 resulted in up-regulation of p16INK4a which impaired cardiac function and tissue repair. These data suggest that CNPY2 is an important regulator of p16INK4a and promotes cell proliferation and tissue repair through inhibition of the p16INK4a pathway. CNPY2 treatment may offer a new approach to restore cardiac function after an MI.

Novel mediators of aneurysm progression in bicuspid aortic valve disease.

Bicuspid aortic valve (BAV) disease is a congenital abnormality that is associated with ascending aortic aneurysm yet many of the molecular mechanisms remain unknown. To identify novel molecular mechanisms of aneurysm formation we completed microarray analysis of the proximal (severely dilated) and distal (less dilated) regions of the ascending aorta from five patients with BAV. We identified 180 differentially expressed genes, 40 of which were validated by RT-qPCR. Most genes had roles in inflammation and endothelial cell function including cytokines and growth factors, cell surface receptors and the Activator Protein 1 (AP-1) transcription factor family (FOS, FOSB and JUN) which was chosen for further study. AP-1 was differentially expressed within paired BAV aneurysmal samples (n = 8) but not Marfan patients (n = 5). FOS protein was significantly enriched in BAV aortas compared to normal aortas but unexpectedly, ERK1/2 activity, an upstream regulator of FOS was reduced. ERK1/2 activity was restored when BAV smooth muscle cells were cultured in vitro. An mRNA-miRNA network within paired patient samples identified AP-1 as a central hub of miRNA regulation. FOS knockdown in BAV SMCs increased expression of miR-27a, a stretch responsive miRNA. AP-1 and miR-27a were also dysregulated in a mouse model of aortic constriction. In summary, this study identified a central role for AP-1 signaling in BAV aortic dilatation by using paired mRNA-miRNA patient sample. Upstream analysis of AP-1 regulation showed that the ERK1/2 signaling pathway is dysregulated and thus represents a novel chain of mediators of aortic dilatation in BAV which should be considered in future studies.