Institute of Molecular Cardiology, University of Louisville, Louisville, Kentucky
The discovery of preconditioning (PC) has arguably been the single most important development in the field of ...ischemic biology in the past 20 years. The significance of this phenomenon transcends cardiovascular medicine, since it is ubiquitously observed in virtually every tissue of the body. This article reviews the pathophysiology and molecular basis of myocardial PC, with particular emphasis on the late phase of this cardioprotective adaptation. The article also discusses the exploitation of late PC for the development of novel gene therapy strategies aimed at inducing a permanently preconditioned cardiac phenotype (prophylactic cardioprotection). Besides its conceptual interest, deciphering the mechanism of late PC has considerable therapeutic reverberations, since transfer of the genes that underlie late PC would be expected to emulate the salubrious effects of this response of the heart to stress.
nitric oxide synthase; protection; cyclooxygenase-2
Address for reprint requests and other correspondence: R. Bolli, Division of Cardiology, Univ. of Louisville, 550 S. Jackson St., ACB, 3rd floor, Louisville, KY 40292 (E-mail: rbolli{at}louisville.edu )
Unlike the early phase of preconditioning (PC), which lasts 2 to 3 hours and protects against infarction but not against stunning, the late phase of PC lasts 3 to 4 days and protects against both ...infarction and stunning, suggesting that it may have greater clinical relevance. It is now clear that late PC is a polygenic phenomenon that requires the simultaneous activation of multiple stress-responsive genes. Chemical signals released by a sublethal ischemic stress (such as NO, reactive oxygen species, and adenosine) trigger a complex cascade of signaling events that includes the activation of protein kinase C, Src protein tyrosine kinases, and nuclear factor κB and culminates in increased synthesis of inducible NO synthase, cyclooxygenase-2, aldose reductase, Mn superoxide dismutase, and probably other cardioprotective proteins. An analogous sequence of events can be triggered by a variety of stimuli, such as heat stress, exercise, and cytokines. Thus, late PC appears to be a universal response of the heart to stress in general. Importantly, the cardioprotective effects of late PC can be reproduced pharmacologically with clinically relevant agents (eg, NO donors, adenosine receptor agonists, endotoxin derivatives, or opioid receptor agonists), suggesting that this phenomenon might be exploited for therapeutic purposes. The purpose of this review is to summarize current information regarding the pathophysiology and mechanism of late PC.
Over the past decade, an enormous number of studies (>100) have focused on the role of nitric oxide (NO) in myocardial ischemia. It is important to distinguish the function of NO in unstressed ...(non-preconditioned) myocardium from its function in preconditioned myocardium (i.e. myocardium that has shifted to a defensive phenotype in response to stress). Of the 92 studies that have examined the role of NO in modulating the severity of ischemia/reperfusion injury in non-preconditioned myocardium, the vast majority 67 (73%) have concluded that NO (either endogenous or exogenous) has a protective effect and only 11 (12%) found a detrimental effect. The proportion of studies supporting a cytoprotective role of NO is similar in vivo35 (71%) out of 49 and in vitro32 (74%) out of 43. With regard to the delayed acquisition of tolerance to ischemia late preconditioning (PC), overwhelming evidence indicates a critical role of NO in this phenomenon. Specifically, enhanced biosynthesis of NO by eNOS is essential to trigger the late phase of ischemia-induced and exercise-induced PC, and enhanced NO production by iNOS is obligatorily required to mediate the anti-stunning and anti-infarct actions of late PC elicited by five different stimuli (ischemia, adenosine A1agonists, opioidδ1 agonists, endotoxin derivatives and exercise). Thus, NO plays a dual role in the pathophysiology of the late phase of PC, acting initially as the trigger and subsequently as the mediator of this adaptive response (“NO hypothesis of late PC”). The diversity of the PC stimuli that converge on iNOS implies that the upregulation of this enzyme is a central mechanism whereby the myocardium protects itself from ischemia. The NO hypothesis of late PC has thus revealed a cytoprotective function of iNOS in the heart, a novel paradigm which has recently been extended to other tissues, including kidney and intestine. Other corollaries of this hypothesis are that the heart responds to stress in a biphasic manner, utilizing eNOS as an immediate but short-term response and iNOS as a delayed but long-term defense, and that the fundamental difference between non-preconditioned and late preconditioned myocardium is the tissue level of iNOS-derived NO, which is tonically higher in the latter compared with the former. Hence, late PC can be viewed as a state of enhanced NO synthesis. The NO hypothesis of late PC has important therapeutic implications. In experimental animals, administration of NO donors in lieu of ischemia can faithfully reproduce the molecular and functional aspects of ischemia-induced late PC, indicating that NO is not only necessary but also sufficient to induce late PC. The recent demonstration that nitroglycerin also induces late PC in patients provides proof-of-principle for the concept that nitrates could be used as a PC-mimetic therapy for the prophylaxis of ischemic injury in the clinical arena. This novel application of nitrates could be as important as, or perhaps even more important than, their current use as antianginal and preload-reducing agents. In addition, gene transfer of either eNOS or iNOS has been shown to replicate the infarct-sparing actions of ischemic PC, suggesting that NOS gene therapy could be an effective strategy for alleviating ischemia/reperfusion injury. Ten years of research have demonstrated that NO plays a fundamental biological role in protecting the heart against ischemia/reperfusion injury. The time has come to translate this enormous body of experimental evidence into clinically useful therapies by harnessing the cytoprotective properties of NO.
...the cells were delivered transendocardially with the NOGA system for electroanatomic mapping (Biosense Webster, Inc., Diamond Bar, California) as opposed to the transepicardial route (6), which is ...not feasible or practical in most patients. ...the cells were transplanted 3 months after myocardial infarction (MI) as opposed to 2 weeks after MI; that is, cell therapy was performed in a setting that simulates chronic ischemic cardiomyopathy rather than acute or subacute MI.
Myocardial infarction is a prevalent major cardiovascular event that arises from myocardial ischemia with or without reperfusion, and basic and translational research is needed to better understand ...its underlying mechanisms and consequences for cardiac structure and function. Ischemia underlies a broad range of clinical scenarios ranging from angina to hibernation to permanent occlusion, and while reperfusion is mandatory for salvage from ischemic injury, reperfusion also inflicts injury on its own. In this consensus statement, we present recommendations for animal models of myocardial ischemia and infarction. With increasing awareness of the need for rigor and reproducibility in designing and performing scientific research to ensure validation of results, the goal of this review is to provide best practice information regarding myocardial ischemia-reperfusion and infarction models. Listen to this article's corresponding podcast at ajpheart.podbean.com/e/guidelines-for-experimental-models-of-myocardial-ischemia-and-infarction/.
RATIONALE:The effects of c-kit cardiac progenitor cells (CPCs, and adult cell therapy in general) on left ventricular (LV) function have been regarded as modest or inconsistent.
OBJECTIVE:To ...determine whether 3 CPC infusions have greater efficacy than 1 infusion.
METHODS AND RESULTS:Rats with a 30-day-old myocardial infarction received 1 or 3 CPC infusions into the LV cavity, 35 days apart. Compared with vehicle-treated rats, the single-dose group exhibited improved LV function after the first infusion (consisting of CPCs) but not after the second and third (vehicle). In contrast, in the multiple-dose group, regional and global LV function improved by a similar degree after each CPC infusion, resulting in greater cumulative effects. For example, the total increase in LV ejection fraction was approximately triple in the multiple-dose group versus the single-dose group (P<0.01). The multiple-dose group also exhibited more viable tissue and less scar, less collagen in the risk and noninfarcted regions, and greater myocyte density in the risk region.
CONCLUSIONS:This is the first demonstration that repeated CPC administrations are markedly more effective than a single administration. The concept that the full effects of CPCs require repeated doses has significant implications for both preclinical and clinical studies; it suggests that the benefits of cell therapy may be underestimated or even overlooked if they are measured after a single dose, and that repeated administrations are necessary to evaluate the effectiveness of a cell product properly. In addition, we describe a new method that enables studies of repeated cell administrations in rodents.
Although treatment with stem/progenitor cells is a promising approach to heart disease, enthusiasm for cell therapy has been dampened by the inconsistent, modest, borderline, or undetectable benefits ...reported in clinical trials (all of which have used one dose of cells)
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. As a result, clinical translation has not occurred (no cell-based therapy is close to being approved for heart disease), and a rising tide of skepticism has bedeviled the field,
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leading some critics even to question whether clinical studies should continue. Here I propose that a major reason for the modest, borderline, or disappointing results is the administration of only one dose of cells, which causes the benefits of cell therapy to be underestimated. Iargue that just as most pharmacologic agents are ineffective when given once but can be highly effective when given repeatedly, so a cell product may be ineffective, or modestly effective, when given as a single treatment, but may turn out to be quite efficacious if given repeatedly. This concept constitutes a major paradigm shift, with potentially vast implications for the entire field of reparative medicine.
BACKGROUND—Relevant preclinical models are necessary for further mechanistic and translational studies of c-kit+ cardiac stem cells (CSCs). The present study was undertaken to determine whether ...intracoronary CSCs are beneficial in a porcine model of chronic ischemic cardiomyopathy.
METHODS AND RESULTS—Pigs underwent a 90-minute coronary occlusion followed by reperfusion. Three months later, autologous CSCs (n=11) or vehicle (n=10) were infused into the infarct-related artery. At this time, all indices of left ventricular (LV) function were similar in control and CSC-treated pigs, indicating that the damage inflicted by the infarct in the 2 groups was similar; 1 month later, however, CSC-treated pigs exhibited significantly greater LV ejection fraction (echocardiography) (51.7±2.0% versus 42.9±2.3%, P<0.01), systolic thickening fraction in the infarcted LV wall, and maximum LV dP/dt, as well as lower LV end-diastolic pressure. Confocal microscopy showed clusters of small α-sarcomeric actin–positive cells expressing Ki67 in the scar of treated pigs, consistent with cardiac regeneration. The origin of these cycling myocytes from the injected cells was confirmed in 4 pigs that received enhanced green fluorescent protein –labeled CSCs, which were positive for the cardiac markers troponin I, troponin T, myosin heavy chain, and connexin-43. Some engrafted CSCs also formed vascular structures and expressed α-smooth muscle actin.
CONCLUSIONS—Intracoronary infusion of autologous CSCs improves regional and global LV function and promotes cardiac and vascular regeneration in pigs with old myocardial infarction (scar). The results mimic those recently reported in humans (Stem Cell Infusion in Patients with Ischemic CardiOmyopathy SCIPIO trial) and establish this porcine model of ischemic cardiomyopathy as a useful and clinically relevant model for studying CSCs.
Summary Background c-kit-positive, lineage-negative cardiac stem cells (CSCs) improve post-infarction left ventricular (LV) dysfunction when administered to animals. We undertook a phase 1 trial ...(Stem Cell Infusion in Patients with Ischemic cardiOmyopathy SCIPIO) of autologous CSCs for the treatment of heart failure resulting from ischaemic heart disease. Methods In stage A of the SCIPIO trial, patients with post-infarction LV dysfunction (ejection fraction EF ≤40%) before coronary artery bypass grafting were consecutively enrolled in the treatment and control groups. In stage B, patients were randomly assigned to the treatment or control group in a 2:3 ratio by use of a computer-generated block randomisation scheme. 1 million autologous CSCs were administered by intracoronary infusion at a mean of 113 days (SE 4) after surgery; controls were not given any treatment. Although the study was open label, the echocardiographic analyses were masked to group assignment. The primary endpoint was short-term safety of CSCs and the secondary endpoint was efficacy. A per-protocol analysis was used. This study is registered with ClinicalTrials.gov , number NCT00474461. Findings This study is still in progress. 16 patients were assigned to the treatment group and seven to the control group; no CSC-related adverse effects were reported. In 14 CSC-treated patients who were analysed, LVEF increased from 30·3% (SE 1·9) before CSC infusion to 38·5% (2·8) at 4 months after infusion (p=0·001). By contrast, in seven control patients, during the corresponding time interval, LVEF did not change (30·1% 2·4 at 4 months after CABG vs 30·2% 2·5 at 8 months after CABG). Importantly, the salubrious effects of CSCs were even more pronounced at 1 year in eight patients (eg, LVEF increased by 12·3 ejection fraction units 2·1 vs baseline, p=0·0007). In the seven treated patients in whom cardiac MRI could be done, infarct size decreased from 32·6 g (6·3) by 7·8 g (1·7; 24%) at 4 months (p=0·004) and 9·8 g (3·5; 30%) at 1 year (p=0·04). Interpretation These initial results in patients are very encouraging. They suggest that intracoronary infusion of autologous CSCs is effective in improving LV systolic function and reducing infarct size in patients with heart failure after myocardial infarction, and warrant further, larger, phase 2 studies. Funding University of Louisville Research Foundation and National Institutes of Health.
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