Abstracts

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Day 1: Friday, November 4, 2011

Scientific Introduction

Cardiovascular Defense Challenges at the Basic, Clinical and Population Levels
Valentin Fuster, MD, PhD, Mount Sinai Medical Center and Centro Nacional de Investigaciones Cardiovasculares

At a basic level, to understand and promote vascular health, we must reduce the aggression to the vessel wall and enhance the physiologic mechanisms leading to restoration of vessel wall function. Three main defense mechanisms are responsible for maintaining cardiovascular homeostasis: the regenerative production of endothelial progenitor cells, vessel wall angiogenesis, and macrophage-mediated reverse cholesterol transport. At a clinical level, cardiovascular disease (CVD) has become the most common cause of mortality worldwide. Obesity, insufficient physical exercise, diabetes, and advancing age are major risk factors for developing cardiovascular disease that are currently increasing in prevalence. Nevertheless, significant progress has recently been made in the treatment of complex cardiovascular and coronary artery disease (CAD), with pharmacological management set to assume an increasingly important role. At a global level, other timely factors, such as the development of the polypill and high-level medical and political interest in advancing cardiovascular health, are driving forces that may help to make inroads into the global cardiovascular disease burden. In this talk, I will critically review the key challenges that we face in the coming decade as we strive to transition and apply our growing knowledge of complex CAD to promoting global cardiovascular health.

 

Session I: Arterial Challenges at the Basic Level

Vascular Endothelium in Health and Disease: New Insights into Its Pathobiology
Michael A. Gimbrone, Jr., MD, Brigham & Women's Hospital, Harvard Medical School

The vascular endothelial lining of the cardiovascular system comprises a vital, multifunctional interface in health, and its dysfunction can contribute to chronic inflammation, hypertension, thrombosis, and atherosclerosis. Endothelial dysfunction can be elicited by humoral stimuli, such as proinflammatory cytokines, bacterial endotoxins, advanced glycation endproducts, or components of oxidized lipoproteins. Recent work suggests that biomechanical forces generated by the pulsatile flow of blood (wall shear stresses, cyclic strains, hydrostatic pressures) can also directly influence endothelial phenotype at the level of gene regulation. We have applied hi-throughput molecular genetic strategies, such as genome-wide expression profiling via cDNA microarrays and bioinformatic analyses, to compare the patterns of endothelial gene expression induced by humoral stimuli, such as proinflammatory cytokines, and biomechanical stimuli, such as laminar and disturbed blood flow. This approach has defined distinct and reproducible patterns of gene regulation that are associated with pathophysiologically relevant endothelial activation states, and has identified novel genes encoding cell surface receptors, ion transporters, signaling molecules and transcription factors that have relatively selective endothelial expression. Bioinformatic "pathway" analyses have also revealed key transcription factors that serve as nodal regulators in molecular genetic networks that help maintain homeostatic balance across a spectrum of pathophysiologic states. This approach should provide fresh insights into the mechanisms of cardiovascular disease, and, hopefully, reveal novel molecular targets for therapeutic interventions, as well as biomarkers useful in assessing disease risk and prevention.
 

The Adventitia and the Media Vasa Vasorum: Defense vs. Betrayal or a War in Progress
Pedro R. Moreno, MD, FACC, Mount Sinai Medical Center, New York

Vasa vasorum-derived microvessels nurture the atherosclerotic plaque, with an organized system regulated by sympathetic and hormonal stimuli. They also provide a permanent communication between the systemic circulation and the atheroma, increasing leukocyte, albumin, and RBC extravasation, leading to ROS generation and tissue damage mediated by the potent oxidative effects of free Hb. Recent studies strengthen the concept that the intraplaque hemorrhage are events that could play a major role in plaque progression and leucocyte infiltration, and may also serve as a measure of risk for the development of future events. The recent advances in our understanding of intra-plaque Hemorrhage as a critical event in triggering acute clinical events, and may have important implications for clinical research and possibly future clinical practice. Furthermore, microvessels may play a role in plaque regression, as suggested by a dramatic reduction of intima-medial blood flow after regression in atherosclerotic monkeys. These results are in agreement with human data showing reduced microvessels in fibrocalcific plaques compared with lipid-rich and ruptured plaques.
 
Recent experimental data suggest that statins preserve the adventitial vasa vasorum architecture and prevent neovascularization development in hypercholesterolaemic pigs, independently of cholesterol lowering. Statins could also influence the consequences of microbleeding due to their ability to limit the cholesterol content of RBC membranes. Furthermore, Plaque neovessels may be suitable for in vivo evaluation with the use of molecular imaging.
 
Finally, there is a potential role for treatment of plaque neovascularization with angiogenesis inhibitors. Impressive reductions of atherosclerosis in apolipoprotein E knockout mice were obtained using endostatin and TNP-470, respectively. Nevertheless, plaque angiogenesis allows for macrophage trafficking with potential for reverse cholesterol transfer, and plaque regression. Inhibiting this defense mechanism may be responsible for the recent, unexpected reports showing that antiangiogenic therapy for cancer or age-related macular degeneration could increase the risk of cardiovascular disease.
 

The Intimal LDL-C & HDL-C: Inflammatory Resolution vs Thrombotic Chaos
Lina Badimon, PhD, Cardiovascular Research Center, Catalan Institute of Cardiovascular Sciences, Barcelona, Spain

Low Density Lipoprotein (LDL) cholesterol levels in plasma are associated with the presentation of clinical cardiovascular events. Reduction of plasma LDL, by behavioural and pharmacological interventions, has shown to be highly effective in the prevention of cardiovascular disease. Despite the successful treatment of LDL, residual cardiovascular risk still exists as shown by the presentation of events in the LDL-treated patients. Because of the long standing epidemiological understanding on the beneficial effects of High Density Lipoprotein (HDL) cholesterol, the rationale of trying to increase HDL plasma levels, by behavioural and pharmacological means, is the target of the latest investigations. Experimental studies have shown that LDL and HDL have opposed effects in the vessel wall; thus, the pro-atherogenic effects of LDL are counteracted by the anti-atherogenic effects of HDL when both types of micelles are in the appropriate equilibrium in plasma and the vessel wall.
 
Both LDL and HDL are complex particles that transport proteins, enzymes and, even nucleic acids involved in several functions in the organism; therefore, the need of characterizing their composition and unveiling their structure-function and biological activities surpasses the cardiovascular system. HDL particles are more heterogeneous than LDL and just measuring their levels may not predict their function. Late clinical studies on pharmacological agents designed to raise HDL have shown unsuccessful results proving the fact that HDL levels should not only be raised but that the raised HDL should have the appropriate characteristic for anti-atherosclerotic effects. The proinflammatory and proatherosclerotic effects of intimal accumulation of LDL can be counteracted by physiological HDL that are able to remove cholesterol by reverse cholesterol transport; however, the unopposed accumulation of LDL in the intima without the counteracting effects of HDL will destine the atherosclerotic lesion to thrombotic chaos and clinical event presentation.

 

Session II: Myocardial Challenges at the Basic Level

Evolving Role of Imaging for New Understanding
Jagat Narula, MD, PhD, Mount Sinai School of Medicine

More often than not acute coronary events occur as the first manifestation of disease. Therefore, the importance of identification of culprit lesions cannot be overemphasized. Histological examination of plaques retrieved from victims of acute coronary events demonstrates thinned fibrous caps associated with the plaques that are more than 75% obstructive to the luminal diameter. Intravascular optical coherence tomography has been successfully employed for the assessment of fibrous cap thickness, which measures less than 55 microns in patients presenting clinically with an acute event. However, since invasive assessment is not always possible, after exclusion of the cap thickness from computation selects the extent of inflammation and the necrotic core area as the best discriminators that are discernable by noninvasive imaging.
 
Necrotic cores have been identified as low attenuation plaque areas on coronary computed tomography angiography imaging in positively remodeled vascular segments. Such plaques in asymptomatic subjects are associated with a 22.5% adverse event rate over a 2-year follow-up, compared to <0.5% in the lesions that lack these characteristics. Although inflammation has been indirectly estimated by an increase in systemic biomarkers, such as high-sensitivity C-reactive proteins, localization of metabolically active macrophage infiltration in plaques has become undertaken by FDG-based positron emission tomography imaging.
 

Detection of the High-risk Atherosclerotic Plaque — the Role of PET/CT Imaging
James H. F. Rudd, MD, PhD, MRCP, University of Cambridge, Cambridge, UK

Atherosclerosis remains the leading cause of death in the Western world. Despite significant advances in identification of risk factors and therapies over the last three decades, the identification of patients at risk of future cardiovascular events remains a challenge. I will discuss the role of non-invasive atherosclerosis imaging, for this indication, in relation to the pathology of atherosclerosis. I will highlight the role that it is likely to play over the next 10 years for detecting those at high risk, as well as for evaluating the efficacy of novel cardiovascular drugs and devices.
 

Tissue Regeneration: Bone Marrow Cell–Cell Interaction & Release
Simón Méndez-Ferrer, PhD, Centro Nacional de Investigaciones Cardiovasculares, Madrid, Spain

Recently, emerging evidence points towards a key role for mesenchymal stem cells in the regulation of the traffic of hematopoietic stem cells and monocytes. Hematopoietic stem cell traffic is key for bone marrow transplantation, since hematopoietic stem cell homing and engraftment in the bone marrow is critical for the success of transplantations, whereas their mobilization allows for non-invasive harvest of peripheral blood hematopoietic stem cells for life-saving transplantation procedures. Mobilization regimes only exacerbate the poorly characterized steady-state hematopoietic stem cell exit from the bone marrow, which follows circadian oscillations, peaking during the resting phase.
 
Bone marrow-resident monocytes traffic into the bloodstream upon localized inflammation in the periphery. Until very recently it has remained unclear how focal inflammation is able to trigger monocyte emigration from remote bone marrow compartments.
 
Mesenchymal stem cells have been recently shown to directly sense and integrate signals that either keep or attract hematopoietic stem cells and monocytes in the bone marrow, or direct their physiological egress and also their enforced mobilization to the bloodstream. Monocytes in turn modulate the capacity of mesenchymal stem cells to keep hematopoietic stem cells in the bone marrow compartment and might also regulate other mesenchymal stem cell functions.
 
These interactions among bone marrow cells have broad implications in tissue regeneration because they impact the number of circulating hematopoietic stem cells and monocytes during the resting phase (when organs undergo physiological repair) and during inflammatory and stress situations. Elucidation of these interactions could lead to new pharmacological approaches to stimulate tissue regeneration.
 

Gene Therapy for the Treatment of Heart Failure
Roger J. Hajjar, MD, Mount Sinai School of Medicine

Congestive heart failure remains a progressive disease with a desperate need for innovative therapies to reverse the course of ventricular dysfunction. Recent advances in understanding the molecular basis of myocardial dysfunction, together with the evolution of increasingly efficient gene transfer technology have placed heart failure within reach of gene-based therapies. One of the key abnormalities in both human and experimental HF is a defect in sarcoplasmic reticulum (SR) function, which is responsible for abnormal intracellular Ca2⁺ handling. Deficient SR Ca2⁺ uptake during relaxation has been identified in failing hearts from both humans and animal models and has been associated with a decrease in the activity of the SR Ca2⁺-ATPase (SERCA2a). Over the last ten years we have undertaken a program of targeting important calcium cycling proteins in experimental models of heart by somatic gene transfer. This has led to the completion of a first-in-man phase 1 clinical trial of gene therapy for heart failure using adeno-associated vector (AAV) type 1 carrying SERCA2a. In this Phase I trial, there was evidence of clinically meaningful improvements in functional status and/or cardiac function which were observed in the majority of patients at various time points. The safety profile of AAV gene therapy along with the positive biological signals obtained from this phase 1 trial has led to the initiation and recent completion of a phase 2 trial of AAV1.SERCA2a in NYHA class III/IV patients. In the phase 2 trial, gene transfer of SERCA2a was found to be safe and associated with benefit in clinical outcomes, symptoms, functional status, NT-proBNP and cardiac structure.

 

Data-Blitz Session

Adrenergic Receptor Stimulation Protects The Heart From Ischemia / Reperfusion Injury
David Sanz-Rosa, Centro Nacional de Investigaciones Cardiovasculares (CNIC)

β3 adrenergic receptors (β3AR) have been recently identified in the heart. Catecholamine signaling via β3AR results in nitric oxide (NO) production and negative inotropic effect. β3AR stimulation has been newly shown to have beneficial effects in models of heart failure; however, the role of β3AR stimulation on ischemia/reperfusion (I/R) has not been explored to date, being the objective of our work.
 
Methods and results: Cardiomyocytes (HL1 cell line) were in vitro exposed to 6/18h of hypoxia/reoxygenation, observing a significant decrease in cell viability (propidium iodine staining in flow cytometry), explained in part by a significant increase of apoptotic cell death (flow cytometry, western blot and fluorescence microscopy for cleaved-Caspase 3). Pre-treatment of cardiomyocytes with the β3AR agonist (BRL-37344; 25–100µM) resulted in a dramatic ≈25% increase in cell viability and a consistent ≈20% reduction of apoptotic cell death.
 
The cardioprotection afforded by β3AR stimulation is explained, to a big extent, by NO production, since the co-treatment of cardiomyocytes with the β3AR agonist plus the NO inhibitor L-NAME (1mM), partially reverted the cardioprotective effects of β3AR agonist alone. Subsequently C57/bl6 mice were subjected to I/R by 45 min coronary ligation followed by 24h of reperfusion. Ten min before reperfusion a β3AR agonist (BRL-37344, 5µ/kg) or matching placebo was administered intravenously. Preliminary analyses showed that infarct size (normalized to area at risk) was significantly smaller in animals receiving the β3AR agonist before reperfusion.
 
Conclusion: β3AR stimulation results in a significant cardioprotection during I/R, representing a novel promising target to be tested in relevant larger animals models.
 
Coauthors: Jaime García-Prieto¹, Alberto Osuna¹, Valentín Fuster^1,3, and Borja Ibañez^1,2.
 
1. Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid.
2. Hospital Clínico San Carlos, Madrid.
3. Mount Sinai School of Medicine, New York.
 

Engineering Physiological Models of Arterial Bifurcation to Expedite Treatments
Mercedes Balcells, PhD, Massachusetts Institute of Technology and Institut Químic de Sarria, Barcelona

Shear stress gradients along the vessel wall have been linked to endothelial dysfunction, leading to loss of vasoreactivity, high levels of platelet aggregation, and induction of tissue factor expression. In vitro and computational models that mimic arterial conditions are cheaper, more reproducible and easier to handle than animal models. They constitute an alternative platform to design cost-effective and high throughput assays in the early stages of development of new therapies to combat arterial disease.
 
We have developed an automated platform to manufacture biocompatible polymeric arterial bifurcations that allow for layer-by-layer cell assembly and closely recapitulate the arterial trilaminate architecture. Scaffolds were first coated with human adventitial fibroblasts and then with human smooth muscle cells followed by a monolayer of human coronary artery endothelial cells that layered the lumen. Computational simulations were performed in parallel as a predictive tool to map zones of flow stagnation and high shear stress regions along the coronary and carotid geometries under study. We applied the simulated shear stress maps to guide in vitro experiments performed using the above described trilaminate cell-seeded scaffolds after exposure to defined flow regimes in a perfusion bioreactor developed in our laboratory. Our results confirmed that in areas of flow separation, where cells were exposed to stagnant and oscillatory flow regimes, endothelial cells doubled their Ox-LDL absorption, expression of ICAM-1 and VCAM-1, and monocyte attachment levels in comparison to endothelial cells in areas of steady high shear stress. In addition, injured models where vascular smooth muscle cells were directly exposed to oscillatory flow showed a 2.5-fold increase of tissue factor expression compared to those exposed to steady flow.
 
This platform may serve to answer fundamental cell physiology questions that link flow with health and disease, but also with stent performance, re-endothelialization and late stent thrombosis. Only integrated approaches, computational, in vitro and in vivo will enable us to bridge the gap between scientific findings and clinical applications.
 
Coauthors: Jordi Martorell^1,2, José Javier Molins², Andrés. A. García-Granada², José Antonio Bea³, Elazer R. Edelman^1,4.
 
1. Massachusetts Institute of Technology.
2. Institut Químic de Sarria, Barcelona.
3. Universidad de Zaragoza, Zaragoza.
4. Brigham and Women's Hospital, Boston.
 

In vivo Non-Invasive Bioluminescence Imaging Monitoring of Ctni Gene Expression in Cardiac Adipose Tissue-derived Progenitor Cells (Atdpcs) Implanted in a Mouse Model of Myocardial Infarction
Carolina Soler-Botija, PhD, Hospital Universitari Germans Trias i Pujol, Badalona

Purpose: The population of progenitor cells isolated from human cardiac adipose tissue (cardiac ATDPCs) proved to be a valid cell source for cardiac regeneration in rodent models of myocardial infarction. These cells, however, do not express cardiac troponin I (cTnI) in basal conditions in culture, although de novo expression was achieved in co-culture with neonatal cardiomyocytes. Whether cTnI expression is upregulated in vivo after cell delivery in cardiac regeneration protocols is unclear. The purpose of this study was to monitor in vivo cTnI gene expression of cardiac ATDPCs delivered through a fibrin patch in the murine model of myocardial infarction by means of non-invasive Bioluminiscence Imaging (BLI).
 
Methods: ATDPCs of cardiac and subcutaneous origin were transduced with two lentiviral vectors for bioluminescence and fluorescence monitoring: CMV-Rluc-RFP-ttk (constitutive expression) and cTnIpr-Pluc-eGFP (human-specific cTnI expression). Next, cells were loaded in a 3-D fibrin patch (bioimplant) and transplanted covering injured myocardium in a mouse model of myocardial infarction. Sham-operated animals (cells implantation and no infarction) were also performed. In vivo bioluminescent images were obtained and light was quantified at 0, 1, 2 and 3 weeks post-implantation.
 
Results: BLI quantification results indicated that de novo expression of cTnI was already induced one week post-implantation in both cardiac and subcutaneous ATDPCs. However, bioluminescence images revealed a 38-fold increase of cTnI expression in cardiac ATDPCs, while only a 4.8-fold increase was found in subcutaneous ATDPCs (p=0.018). Although the cTnI levels in cardiac ATDPCs tended to decrease over time, in all time points analyzed they were superior to those of subcutaneous origin.
 
Conclusions: Our work indicates that de novo expression of cTnI in cardiac ATDPC implanted into a mouse infarcted myocardium already occurred one week post-implantation. Comparative analysis showed that cardiac ATDPCs had a greater capability to express cardiac marker such as cTnI than subcutaneous ATDPCs.
 
Coauthors: Juli R. Bagó², Aida Llucià-Valldeperas¹, Jerónimo Blanco², Santiago Roura¹, Núria Rubio², Carolina Gálvez-Montón¹, Cristina Prat-Vidal¹, and Antoni Bayés-Genís¹.
 
1. Hospital Universitari Germans Trias i Pujol, Badalona.
2. Cardiovascular Research Center (CSICICCC), CIBER BN, Barcelona.
 

Human Umbilical Cord Blood-derived Mesenchymal Stem Cells Demonstrate Promising Angiogenic and Vasculogenic Potential for Heart Function Recovery
Santiago Roura, PhD, Institute Germans Trias i Pujol (IGTP), Badalona

Stem cell therapies open up a hopeful possibility to restore function following heart failure. Since stem cell-mediated revascularization might improve myocardial blood supply with functional enhancement of remaining cardiac muscle in near future therapeutic interventions, we here examined whether umbilical cord blood-derived mesenchymal stem cells (UCBMSCs) demonstrated angiogenic and/or vasculogenic potential.
 
Cells were differentiated in endothelial cell (EC)-specific growth medium (EGM-2). Gene and protein expression were assessed by RT-PCR, immunofluorescence and Western blot. Migration and formation of capillary-like structures were analyzed in cell invasion assays and in Matrigel, respectively. Chemotaxis of peripheral endothelial progenitor cells (EPCs) was tested in Transwells. Experiments using an in vivo angiogenesis murine model based on the co-implantation of Matrigel and UCBMSCs transfected with a CD31-promoter reporter construct were also carried out. CD31-promoter activation was monitored over time using non-invasive bioluminescence, and Matrigel plugs were finally removed to analyze microvessel growth following fluorescent angiography.
 
Expression of EC, angiogenic and vasculogenic markers as well as migratory capacity (p=0.004) were promoted in EGM-2. Differentiated cells developed capillary-like networks in Matrigel. Moreover, UCBMSC-derived ECs positively attracted EPCs (p=0.02) in a SDF-1 -dependent manner (p=0.016). In vivo, both activation of a CD31 promoter-luciferase reporter (p<0.001) and growth of mature microvessels were detected within Matrigel plugs containing UCBMSCs.
 
Our results demonstrate that UCBMSCs differentiate into EC both in vitro and in vivo. These cells could also activate vasculogenesis through EPC recruitment.
 
Coauthors: Juli Rodríguez Bagó², Carolina Gálvez-Montón¹, Cristina Prat-Vidal¹, Carolina Soler-Botija¹, Aida Llucià-Valldeperas¹, Jerónimo Blanco², and Antoni Bayes-Genis^1,3,4.
 
1. Institute Germans Trias i Pujol (IGTP), Badalona.
2. Cardiovascular Research Center, CSIC-ICCC, CIBERBBN, Barcelona.
3. Hospital Universitari Germans Trias i Pujol, Badalona.
4. Universitat Autònoma de Barcelona, Barcelona.

 

Session III: Development & Regenerative Challenges at the Basic Level

Cell Competition during Heart Development
Miguel Torres, PhD, Centro Nacional de Investigaciones Cardiovasculares (CNIC), Madrid, Spain

Cell competition is a tissue homeostasis mechanism described in Drosophila that promotes the expansion of the fittest cells through the apoptotic elimination of suboptimal but otherwise viable cells. Recent evidence suggests that cell competition is a general strategy used in metazoan development, tissue homeostasis and regeneration. Myc is an important promoter of cell competition ability through its action on the cell anabolic machinery. Using a new inducible genetic mosaic approach we have manipulated Myc levels and apoptosis patterns in the mouse embryo. Our results indicate that epiblast cells actively compete for survival and that the level of Myc expression is an essential determinant of endogenous and induced cell competition. To explore whether cell competition remains an active process during cardiogenesis, we induced Myc overexpression mosaics in differentiating cardiomyocytes during embryogenesis. Our results indicate that mosaic hearts are enriched at birth in Myc-overexpressing versus WT cardiomyocytes. Apoptosis inhibition in WT cardiomyocytes prevents this enrichment, indicating that the Myc-overexpressing population displaces the WT cardiomyocytes by inducing their death. Our study shows that the mammalian epiblast optimizes its cellular composition by eliminating the less active cells through cell competition. The differentiated embryonic cardiomyocyte population is as well able to eliminate less active cells through cell competition, indicating that the phenomenon is not restricted to embryonic stem cell populations. We are currently exploring the ability of cell competition to induce the displacement of less active postmitotic cardiomyocyte populations in homeostatic and injured adult hearts.
 

Regenerating a New Heart
Andre Terzic, MD, PhD, Mayo Clinic

Regenerative paradigms offer a "disruptive innovation" poised to transform medicine and surgery by providing the prospect of definitive solutions for our patients. The decisive goal of regenerative medicine is to advance care from palliation to cure. From pioneering success with bone marrow transplants to breakthroughs in neorganogenesis, regenerative strategies emerge as a promising core component of medical and surgical practice. Aimed at repair of heart disease pathobiology and restoration of organ function, forthcoming regenerative applications encompass unparalleled patient-specific diagnostic algorithms and reconstructive treatments. Across a spectrum, from congenital conditions to acquired age-related cardiovascular pathologies, personalized regenerative medicine products promise significant human health benefit. Stem cell-based regenerative strategies refer to engraftment of progenitor cells that through growth and lineage-specification establish repair outcome within the host environment by supplementing and recruiting resident progenitor pools, facilitating reconstruction of damaged tissues. Stem cells are thus a fundamental tool in the rapidly advancing regenerative medicine toolkit. Rigorous translation of the regenerative science vanguard is now pivotal to address implementation and validation of repair paradigms from principles to practice.
 

Driving Heart Progenitor Fate in vivo with Modified mRNA
Kenneth R. Chien MD PhD, Harvard Stem Cell Institute

A family of islet-1 and other multipotent heart progenitors are responsible for the diversification and expansion of distinct cardiac muscle, vascular smooth muscle, and endothelial cell lineages during murine and human cardiogenesis. Although a rare number of heart progenitors are found in the post-natal heart, previous cell or molecular based approaches to activate these or other endogenous cardiac progenitors following cardiac injury have had limited success. Toward this goal, we have identified VEGF-A as a key switch in the human fetal heart that expands the vascular progenitor pool in the family of Islet-1 human heart progenitors during ES cell cardiogenesis, and have utilized modified RNA to transiently express the corresponding protein in cardiac muscle in vitro and in vivo. In vitro, both neonatal and adult mouse cardiomyocytes, as well as human fetal cardiomyocytes, can be highly efficiently transfected (70–90%) with a transient, non-immunogenic, modified mRNA (MOD RNA), with minimal toxicity or triggering of innate immunity, and enhanced protein production vs non modified RNA. In vivo, direct intramuscular injection of a luciferase reporter MOD RNA in either cardiac or skeletal muscle results in a time and dose dependent expression detectable within 3 hours, persisting over several days, and returning to negligible levels. In addition, injection of a Cre MOD-RNA into the hearts of Rosa26R-LacZ indicator mice revealed that the transfection could drive expression in a wide region, well beyond the initial site of injection. Transfection of human or murine cardiomyocytes with hVEGF-A MOD RNA induced endothelial-like cells to form tube-like structures which were CD31+, VE-cadherin+ and vWF+, whereas most cardiac cells treated with vehicle were vimentin+. By injecting hVEGF and Cre MOD RNA into the Rosa26R-LacZ hearts after myocardial infarction, we have shown that most of the cells in the injected area were positive for LacZ, and marked proliferation of a population of endothelial, smooth muscle, and cardiac myocytes, providing direct evidence of an in vivo regenerative response. Moreover, new proliferating vessels which are LacZ+ were easily visualized on the epicardial surface from the initial site of injection, documenting that the transfected cells within the heart were adopting both a cardiac myogenic and vasculogenic fate. In summary, MOD RNA is a new platform to allow the rapid in vivo assay of known or novel paracrine protein factors that drive endogenous heart progenitor mobilization following heart injury. Further studies are warranted to evaluate whether this approach can be extended to other organ systems and whether hVEGF MOD RNA is a new therapeutic paradigm to achieve the recruitment and subsequent differentiation of endogenous heart progenitors for cardiovascular regeneration.