Cardiac Progenitor Cell Function Research Articles

β-Adrenergic Regulation of Cardiac Progenitor Cell Death Versus Survival and Proliferation

Short-term β-adrenergic stimulation promotes contractility in response to stress but is ultimately detrimental in the failing heart because of accrual of cardiomyocyte death. Endogenous cardiac progenitor cell (CPC) activation may partially offset cardiomyocyte losses, but consequences of long-term β-adrenergic drive on CPC survival and proliferation are unknown. We sought to determine the relationship between β-adrenergic activity and regulation of CPC function. Mouse and human CPCs express only β2 adrenergic receptor (β2-AR) in conjunction with stem cell marker c-kit. Activation of β2-AR signaling promotes proliferation associated with increased AKT, extracellular signal-regulated kinase 1/2, and endothelial NO synthase phosphorylation, upregulation of cyclin D1, and decreased levels of G protein-coupled receptor kinase 2. Conversely, silencing of β2-AR expression or treatment with β2-antagonist ICI 118, 551 impairs CPC proliferation and survival. β1-AR expression in CPC is induced by differentiation stimuli, sensitizing CPC to isoproterenol-induced cell death that is abrogated by metoprolol. Efficacy of β1-AR blockade by metoprolol to increase CPC survival and proliferation was confirmed in vivo by adoptive transfer of CPC into failing mouse myocardium. β-adrenergic stimulation promotes expansion and survival of CPCs through β2-AR, but acquisition of β1-AR on commitment to the myocyte lineage results in loss of CPCs and early myocyte precursors.

ATP-Binding Cassette G-Subfamily Transporter 2 Regulates Cell Cycle Progression and Asymmetric Division in Mouse Cardiac Side Population Progenitor Cells

After cardiac injury, cardiac progenitor cells are acutely reduced and are replenished in part by regulated self-renewal and proliferation, which occurs through symmetric and asymmetric cellular division. Understanding the molecular cues controlling progenitor cell self-renewal and lineage commitment is critical for harnessing these cells for therapeutic regeneration. We previously have found that the cell surface ATP-binding cassette G-subfamily transporter 2 (Abcg2) influences the proliferation of cardiac side population (CSP) progenitor cells, but through unclear mechanisms. To determine the role of Abcg2 on cell cycle progression and mode of division in mouse CSP cells. Herein, using CSP cells isolated from wild-type and Abcg2 knockout mice, we found that Abcg2 regulates G1-S cell cycle transition by fluorescence ubiquitination cell cycle indicators, cell cycle-focused gene expression arrays, and confocal live-cell fluorescent microscopy. Moreover, we found that modulation of cell cycle results in transition from symmetric to asymmetric cellular division in CSP cells lacking Abcg2. Abcg2 modulates CSP cell cycle progression and asymmetric cell division, establishing a mechanistic link between this surface transporter and cardiac progenitor cell function. Greater understanding of progenitor cell biology and, in particular, the regulation of resident progenitor cell homeostasis is vital for guiding the future development of cell-based therapies for cardiac regeneration.

Boosting the pentose phosphate pathway restores cardiac progenitor cell availability in diabetes.

Diabetes impinges upon mechanisms of cardiovascular repair. However, the biochemical adaptation of cardiac stem cells to sustained hyperglycaemia remains largely unknown. Here, we investigate the molecular targets of high glucose-induced damage in cardiac progenitor cells (CPCs) from murine and human hearts and attempt safeguarding CPC viability and function through reactivation of the pentose phosphate pathway. Type-1 diabetes was induced by streptozotocin. CPC abundance was determined by flow cytometry. Proliferating CPCs were identified in situ by immunostaining for the proliferation marker Ki67. Diabetic hearts showed marked reduction in CPC abundance and proliferation when compared with controls. Moreover, Sca-1(pos) CPCs isolated from hearts of diabetic mice displayed reduced activity of key enzymes of the pentose phosphate pathway, glucose-6-phosphate dehydrogenase (G6PD), and transketolase, increased levels of superoxide and advanced glucose end-products (AGE), and inhibition of the Akt/Pim-1/Bcl-2 signalling pathway. Similarly, culture of murine CPCs or human CD105(pos) progenitor cells in high glucose inhibits the pentose phosphate and pro-survival signalling pathways, leading to the activation of apoptosis. In vivo and in vitro supplementation with benfotiamine reactivates the pentose phosphate pathway and rescues CPC availability and function. This benefit is abrogated by either G6PD silencing by small interfering RNA (siRNA) or Akt inhibition by dominant-negative Akt. We provide new evidence of the negative impact of diabetes and high glucose on mechanisms controlling CPC redox state and survival. Boosting the pentose phosphate pathway might represent a novel mechanistic target for protection of CPC integrity.

Abstract 77: Fibronectin Signals Through Pim-1 and β1 Integrin in Cardiac Progenitor Cells to Induce Proliferation and Protection

Background: Cardiac Progenitor Cells (CPC) are pivotally involved in cardiac repair. Fibronectin (FN), an extracellular matrix protein, is highly expressed during cardiac development. In adult heart FN is a component of the cardiac stem cell niche and re-appears after myocardial infarction (MI). The role of FN signaling in CPC function and cardiac remodeling following MI has not been elucidated. We demonstrate here proliferative and protective effects of FN signaling in CPC as mediated by β1 integrin receptor and cardioprotective serine/threonine kinase Pim1. Methods: Cell death and proliferation of CPCs was measured using propidium iodide and CyQuant assays. Signaling pathways were analyzed by immunoblotting, qRT-PCR and siRNA depletion of targets. FN was localized in heart sections by immunohistochemistry and cardiac function assessed by echocardiography in control and conditional FN knockout (KO) mouse hearts following MI. Results: FN inhibits starvation and staurosporine induced cell death in CPCs and promotes proliferation in conjunction with induction of Pim1 expression. Protective and pro-proliferative effects of FN are abrogated by inhibition of Pim1 or deletion of β1 integrin receptor. In vivo CPC expansion correlates with FN expression following MI, and CPC localize to regions of up-regulated FN protein in the infarct. Cardiac function in control and KO mice remains equivalent up to 2 weeks post MI, however by 4 weeks KO heart function worsens compared to control as evidenced by ejection fraction values measuring 16.4 +/- 1.5% vs 26.6 +/- 1.8% at 12 weeks post MI in KO and control hearts, respectively. Conclusion: FN provides pro-survival and pro-proliferative effects to CPCs in a Pim1 kinase and β1 integrin dependent manner. CPCs and FN colocalize in vivo in an infarction injury model, while conditional KO of FN in mice leads to further impairment of cardiac function after MI. Taken together these results indicate previously unidentified cardioprotective and regenerative roles for FN in pathologically challenged heart.

Abstract P008: Fibronectin Enhances Survival and Proliferation via Pim-1 and β1 Integrin in Cardiac Progenitor Cells

Background: Cardiac Progenitor Cells (CPC) are pivotally involved in cardiac repair. Extracellular matrix (ECM) components also contribute decisively in cardiac remodeling after myocardial infarction (MI). However, impact of ECM on CPC function and the involved signaling pathways have not been clearly elucidated. This study examines the relationship of ECM to cardioprotective signaling mediated by Pim-1, a serine/threonine kinase downstream of Akt, in CPC. Methods: Isolated mouse CPCs were plated on fibronectin (FN) or albumin coated dishes. FACS analysis and fluorescence based cell quantification have been used to determine viability and proliferation under stress and growth conditions. Signaling pathways were analyzed by immunoblotting, qRT-PCR and siRNA. Immunhistochemistry (IHC) has been used for in vivo studies after MI in mice. Results: FN inhibits starvation and staurosporine induced cell death in CPCs and promotes proliferation in conjunction with induction of Pim-1 expression. Protective and pro-proliferative effects of FN are abrogated by inhibition of Pim-1. Fibronectin signaling operates through the β1 integrin receptor that is crucial for FN-mediated signaling. Ongoing studies correlate these observations with in vivo responses to cardiomyopathic injury such as infarction challenge. Conclusion: FN provides pro-survival and pro-proliferative effects in CPCs in a Pim-1 kinase and β1 integrin dependent manner. Involved molecules colocalize in vivo in an infarction injury model. The causal contribution of FN in regeneration and wound healing after MI is to be confirmed by studies of a conditional FN knock-out mouse that is under construction.

Abstract P031: Enhanced Cardiac Progenitor Cell Function and Differentiation in a Naturally Derived Extracellular Matrix

Cardiovascular disease, including myocardial infarction, is a leading cause of death worldwide. Though several pharmacological treatments for severe dysfunction exist, much recent work has focused on the transplantation of adult-derived stem and progenitor cells. Early acute functional improvements have been noted, however long-term clinical efficacy is hampered by poor cell survival and engraftment. While the current treatment is infusion into the coronary artery, biomaterials may play an important role in modulating implanted cell function. This work aims to establish the role that a naturally derived extracellular matrix plays in the differentiation of cardiac progenitor cells (CPCs) and their potentially protective enzymatic systems. To test this hypothesis, we cultured rat CPCs in a naturally derived porcine ECM (pECM) and compared it to Collagen I. Quantitative real-time PCR was used to assess expression of cardiac, endothelial and smooth muscle markers. Additionally, angiotensin receptor (AT1R and AT2R) and antioxidant gene expressions were evaluated to determine the protective qualities of pECM. Preliminary data at 2 days following LIF removal demonstrate an increase in the expression of cardiac lineage markers (Nkx-2.5, Gata-4, α-MHC, and troponin I) in pECM compared to collagen. Smooth muscle markers, smooth muscle α-actin and sm22α as well as the endothelial marker Flk1 were also increased in pECM samples. Increased expression was also seen for antioxidant genes GPX1, SOD1, SOD2 and catalase in pECM cultured cells. Culturing in pECM for 7 days demonstrated an increase in Flt-1 and α-myosin heavy chain, indicating a potential increase in cardiogenesis. Moreover a 60% reduction in AT1R gene expression was observed with no significant change in AT2R expression. Our data demonstrate that culturing CPCs in naturally derived matrices may provide protection and enhance differentiation compared to collagen (present in high amounts in scarred myocardium). Future work will further elucidate this protective effect of AT1R downregulation and antioxidant increases in functional studies. In conclusion, pECM may be a potential cell delivery scaffold in post-MI treatment given its protective nature and improved differentiation influence.

New insights into paracrine mechanisms of human cardiac progenitor cells

Cardiac progenitor cells (CPCs) have been shown to promote cardiac regeneration in vivo. Understanding the function of CPCs is essential for further implementation of these cells in the treatment of cardiac diseases. The present study tested the hypothesis that adult CPC exert paracrine effects that lead to an improvement in the functional characteristics of cardiomyocytes. This study also investigated whether aging (we included patients aged between 4 months and 81 years) has any effect on the paracrine mechanisms of CPC. The supernatant of CPC generated both from human and rat hearts-so called 'conditioned cardiosphere medium' improved the contractile behaviour of isolated adult cardiomyocytes in a concentration-dependent manner after incubation for 24 h and increased the SERCA/NCX ratio. The observed positive effects on contractile behaviour were independent of the CPC donors' age. Conditioned cardiosphere media also normalized angiotensin II-induced contractile dysfunction. Cytokines released by CPC into the media were detected by cytokine arrays. The observed diversity of cytokines released by CPC needs to be further elucidated in detail. Nevertheless, CPC are a promising therapeutic approach in the field of cardiac disease. The methods described allow investigation of the underlying paracrine mechanisms in a standardized in vitro situation.

Juvenile Exposure to Anthracyclines Impairs Cardiac Progenitor Cell Function and Vascularization Resulting in Greater Susceptibility to Stress-Induced Myocardial Injury in Adult Mice

The anthracycline doxorubicin is an effective chemotherapeutic agent used to treat pediatric cancers but is associated with cardiotoxicity that can manifest many years after the initial exposure. To date, very little is known about the mechanism of this late-onset cardiotoxicity. To understand this problem, we developed a pediatric model of late-onset doxorubicin-induced cardiotoxicity in which juvenile mice were exposed to doxorubicin, using a cumulative dose that did not induce acute cardiotoxicity. These mice developed normally and had no obvious cardiac abnormalities as adults. However, evaluation of the vasculature revealed that juvenile doxorubicin exposure impaired vascular development, resulting in abnormal vascular architecture in the hearts with less branching and decreased capillary density. Both physiological and pathological stress induced late-onset cardiotoxicity in the adult doxorubicin-treated mice. Moreover, adult mice subjected to myocardial infarction developed rapid heart failure, which correlated with a failure to increase capillary density in the injured area. Progenitor cells participate in regeneration and blood vessel formation after a myocardial infarction, but doxorubicin-treated mice had fewer progenitor cells in the infarct border zone. Interestingly, doxorubicin treatment reduced proliferation and differentiation of the progenitor cells into cells of cardiac lineages. Our data suggest that anthracycline treatment impairs vascular development as well as progenitor cell function in the young heart, resulting in an adult heart that is more susceptible to stress.

Enhancement of Myocardial Regeneration Through Genetic Engineering of Cardiac Progenitor Cells Expressing Pim-1 Kinase

Despite numerous studies demonstrating the efficacy of cellular adoptive transfer for therapeutic myocardial regeneration, problems remain for donated cells with regard to survival, persistence, engraftment, and long-term benefits. This study redresses these concerns by enhancing the regenerative potential of adoptively transferred cardiac progenitor cells (CPCs) via genetic engineering to overexpress Pim-1, a cardioprotective kinase that enhances cell survival and proliferation. Intramyocardial injections of CPCs overexpressing Pim-1 were given to infarcted female mice. Animals were monitored over 4, 12, and 32 weeks to assess cardiac function and engraftment of Pim-1 CPCs with echocardiography, in vivo hemodynamics, and confocal imagery. CPCs overexpressing Pim-1 showed increased proliferation and expression of markers consistent with cardiogenic lineage commitment after dexamethasone exposure in vitro. Animals that received CPCs overexpressing Pim-1 also produced greater levels of cellular engraftment, persistence, and functional improvement relative to control CPCs up to 32 weeks after delivery. Salutary effects include reduction of infarct size, greater number of c-kit(+) cells, and increased vasculature in the damaged region. Myocardial repair is significantly enhanced by genetic engineering of CPCs with Pim-1 kinase. Ex vivo gene delivery to enhance cellular survival, proliferation, and regeneration may overcome current limitations of stem cell-based therapeutic approaches.