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Ann Thorac Surg 2003;75:S674-S677
© 2003 The Society of Thoracic Surgeons

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I: Pathophysiology of ischemic-reperfusion injury

Cardiac cell transplantation: closer to bedside

^a Division of Cardiac Surgery, Toronto General Hospital, University Health Network, Toronto, Ontario, Canada

* Address reprint requests to Vivek Rao, MD, PhD, 200 Elizabeth St, 14-222, Toronto, ON, Canada.
e-mail: vivek.rao{at}uhn.on.ca

Presented at the 3^rd International Symposium on Myocardial Protection From Surgical Ischemic-Reperfusion Injury, Asheville, NC, June 2–6, 2002.

Abstract

The current treatment for heart failure is inadequate for a large number of patients who do not qualify for heart transplantation or innovative surgical therapy. Cellular augmentation of damaged myocardium has been actively investigated in the past decade. Cells best suited for the task are skeletal myoblasts and bone morrow mesenchymal stem cells. Both cell types are autologous, abundant, and easy to harvest. The focus of early human trials will be to establish the safety of an effective cellular dose. Trials conducted with an inadequate cellular dose may discredit cell therapy because of lack of effect but, more importantly, may provide a false sense of safety because of a lack of adverse events secondary to a small inoculating dose.

Myocardial infarction (MI) is one of the leading causes of cardiac mortality and morbidity [1]. Left ventricular (LV) dysfunction may develop acutely because of complete loss of the contractile function in the infarct region. In survivors, ventricular dysfunction may also result from the process of remodeling which leads to thinning and dilation of the infarct related area, affecting the rest of the ventricle as well [2]. Significant ventricular dysfunction causes symptoms of heart failure (HF). Patients with HF carry a poor prognosis [3]. Cellular death during an MI leads to HF because the adult human heart has a limited ability to regenerate cardiomyocytes [4]. The infarct area is replaced by fibrocytes and collagen fibers, ie, scar tissue. The nonelastic scar eventually thins and dilates, thereby worsening the HF [5].

In 2001, an estimated 1,100,000 Americans had a new or recurrent coronary attack (defined as MI or fatal coronary artery disease) [6]. About 650,000 of these were first attacks and 450,000 were recurrent attacks. More than 40% of persons who experience a coronary attack in a given year die. Among the survivors, 22% of men and 46% of women are disabled with HF. The 5-year mortality rate of HF is approximately 50% [6]. In Canada, the cost directly related to cardiovascular disease in 1993 was C$7.3 billion. Interestingly, the cost due to resulting disability (indirect expenditure) was C$12.4 billion [1].

Studies conducted in the past three decades have shown that patients who have significant multivessel coronary artery disease (CAD) are at high risk for MI and require revascularization. Patients who receive complete revascularization demonstrate the most benefit [7]. In North America alone, 400,000 patients have undergone conventional revascularization in 1999 [8]. Conventional revascularization, either by coronary artery bypass grafting or by percutaneous coronary interventions, has two major limitations: it requires the target area to have suitable vessels, and also viable myocardium. Lack ofeither one renders conventional revascularization impossible or ineffective. Patients who lack suitable vessels in all areas of the heart and patients in whom conventional revascularization results in "incomplete revascularization" need a new treatment to overcome these limitations. Moreover, patients who have an established area of nonviable myocardium are not expected to significantly regain function in that area or prevent it from thinning and dilating by conventional revascularization alone.

The cell therapy concept

Adult heart cells have a limited ability to regenerate infarcted myocardium [4]. Animal studies suggest that implanting cells into scar tissue results in at least two important events. First, it induces angiogenesis and vasculogenesis that may help reincorporate hibernating cardiomyocytes; and second, it improves elasticity of the scar, thereby preventing dilation and thinning. Moreover, the new cells may be able to communicate to native heart cells with cell-to-cell gap junctions and achieve electromechanical coupling (ie, the ability to synchronize their contraction with the electrical current generated in the native myocardium).

Animal studies of cell transplantation

Cell transplantation has been investigated since the early 1990s [9]. Fetal cardiomyocytes, smooth muscle cells, skeletal muscle cells, and bone marrow stem cells have been extensively evaluated. In 1996, Li and colleagues [10] initially demonstrated that fetal cardiomyocyte transplantation improved heart function in a rat model of cryoinjury (muscle necrosis induced by application of liquid nitrogen). The transplanted cells increased ventricular wall thickness in the area of scarring and decreased left ventricular chamber volumes, resulting in lowered wall tension in the left ventricle and improved function. Furthermore, in a separate study, Sakai and colleagues [11] compared three different fetal cell types: cardiomyocytes, enteric smooth muscle cells, and skin fibroblasts. All three cell types were able to form tissue within the scar. Fetal cardiomyocytes transplanted into myocardial scar provided greater contractility and relaxation than fetal smooth muscle cells or fetal fibroblasts. The contractile and elastic properties of transplanted cells determine the degree of improvement in ventricular function achievable with cell transplantation. In a third study using fetal smooth muscle cells, Li and colleagues [12] found that in addition to improved global LV function, the cells induced angiogenesis in the scar area and prevented regional thinning and dilation. The main disadvantage to fetal or neonatal cells is the ethical concern with the use of these cells. In addition, the transplanted allogeneic cells may be eliminated due to immune-mediated rejection. Sakai and colleagues [13] then focused their attention to autologous adult cells. Using the rat cryoinjury model, they harvested atrial cardiomyocytes and expanded them in culture. In rats transplanted with 2x10⁶ cells, the area of the scar was smaller (p = 0.0003) and its thickness greater (p = 0.0003) than control hearts and the volume left ventricular chamber volume was smaller in the transplant group (p = 0.043). Although harvesting heart cells in adult humans is possible and may be safe, it requires the services of a catheterization laboratory. Culture expansion of cells requires 2 to 3 weeks, it may not be practical and feasible to schedule catheterization and surgery within that time frame.

The bone marrow is home to mesenchymal stem cells (MSCs) that are able to differentiate into many different cell types [14]. When MSCs are exposed to 5-azacytidine (a cytosine analogue capable of altering expression of myogenic genes) during culture, they differentiate into cardiomyocyte-like cells that have the ability to beat spontaneously. Seeking to determine whether MSCs were able to revive LV scar as did fetal and adult heart cells, Tomita and colleagues [15] transplanted MSCs into rats with cryoinjury scars and found that transplantation of MSCs induced angiogenesis. Moreover, MSCs cultured with 5-azacytidine differentiated into cardiac-like muscle cells both in culture (Fig 1) and in vivo in ventricular scar tissue, and improved myocardial function. Wang and associates [16], in Montreal, also used MSCs. Four weeks after transplantation, MSCs demonstrate myogenic differentiation with the expression of sarcomeric myosin heavy chain and organized contractile proteins. Positive staining for connexin 43 indicates formation of gap junctions, which suggests that cells derived from marrow stromal cells, as well as native cardiomyocytes, are connected by intercalated disks. In an appropriate microenvironment, MSCs will exhibit cardiomyogenic phenotypes and may replace native cardiomyocytes lost by necrosis or apoptosis.

View larger version (108K): [in this window] [in a new window]   Fig 1. (A) Bone marrow cells cultured with 10-µL 5-azacytidine (magnification x200). Bone marrows cells were cultured for 10 days with 5-aza added to the medium only on day 3. Only the cells cultured with 5-aza formed a network of myotubules. Occasional adipocytes were present. (B) Cultured bone marrow cells imuunohistochemically stained for troponin I (magnification x400). Cultured bone marrow cells were treated with 10 µL 5-aza for 24 hours on day 3 of a 10-day culture. Myotubular cells, but not the other cell types, stained positively for troponin I.

Bone marrow derived cells are autologous, abundant, and relatively easy to harvest; they do not require immune suppression, and they are associated with less ethical concern than are fetal or neonatal heart cells. These characteristics make bone marrow–derived cells uniquely suited to the task of restoring structure and function in the wake of a myocardial infarction [18].

Skeletal muscle precursor cells and smooth muscle cells have also been used to revive myocardial scar tissue [19]. The transplanted cells differentiated to muscle tissue and improved ventricular function in infarcted or injured hearts. The authors concluded that transplanted muscle cells increase regional elasticity and prevent expansion of the infarcted myocardium, which prevents ventricular dilation. Jain and associates [20] studied the effects of skeletal myoblast transplantation on ventricular remodeling. They concluded that cell transplantation after an MI induced by coronary ligation in rats, attenuated ventricular dilation. Current clinical application of skeletal muscle cells demonstrated the safety and efficacy of autologous cell transplantation. Although transplantation of skeletal muscle cells is autologous, there are several difficulties with skeletal myoblast transplantation. First, the number of satellite cells (myoblasts) is inversely related to the patient’s age. Most patients who experience a myocardial infarction are over the age of 50 years. The population of satellite cells in the skeletal muscle tissue of these patients is expected to be small. Second, generation of cells sufficient for transplantation may depend on large muscle biopsies. The challenge is to grow several hundred million to 1 billion cells for transplantation. Uniquely, satellite cells must be implanted in an undifferentiated stage (not left in culture until confluent). It is exhaustive work to generate enough cells for transplantation because of this technical problem.

In a porcine model of MI induced by inserting a coil into the LAD, Tomita and colleagues [21] were able to show that MSCs labeled with BrdU had cardiomyocyte specific troponin I (a very specific marker for heart muscle). The cells also induced significant angiogenesis in the scar area. The angiogenic effect was detectable by Tc-sestamibi single-photon emission computed tomography in the cell transplant group compared to controls undergoing transplantation with media alone (Fig 2). The difference between the two groups in improvement in regional perfusion (expressed as percent Tc-sestamibi activity) was 5% ± 2%. The hearts transplanted with cells also showed improved global LV function measured by preload recruitable stroke work index [21]. The beneficial effect of MSC transplantation on LV function was also demonstrated using sonomicrometry, a technique that uses small piezoelectric crystals implanted in the myocardium and connected to the exterior with electrical leads. This method enabled the investigators to accurately measure LV thickness in the scar area. The LV thickness was found to be significantly higher in cell-transplanted animals than in media-transplanted controls [22].

View larger version (85K): [in this window] [in a new window]   Fig 2. Representative three-dimensional views of reconstructed hearts from gated Single Photon Emission Tomography with Sestamibi (SPECT–MIBI) scan. Animals in control (a) and transplant (b) groups underwent scanning at 4 weeks after cell transplantation. (a) In control group, end-diastolic volume (EDV) was 82 mL, end-systolic volume (ESV) was 52 mL, and ejection fraction (EF) was 37%. (b) In transplant group, end-diastolic volume was 72 mL, end-systolic volume was 40 mL, and ejection fraction was 50%.

Transplantation of cardiomyocytes transfected with vascular endothelial growth factor (VEGF) induces greater angiogenesis than transplantation of unmodified cells. Combined gene transfer and cell transplantation strategies may improve postinfarction LV perfusion and function [24]. Modifying the cells by gene transfection may improve their angiogenic or myogenic properties; however, it may also alter their growth pattern in unexpected ways. Their safety may be more difficult to establish.

Phase I human studies

Preliminary small human studies of cell transplantation are underway around the world. Menasché et al. [25] used skeletal myoblasts cultured from skeletal muscle and reported the first patient in 2001. Since the initial patient, 10 patients have received this treatment [26]. There was an average improvement in global LV ejection fraction of 13% (range 5% to 24%). The most important adverse outcome noted has been the occurrence of nonsustained ventricular arrhythmias in patients who underwent transplantation with more than 600 million cells; no other serious adverse outcomes related to cell therapy have been noted. One patient who had preoperative severe heart failure requiring hemodynamic support and urgent surgery died.

Diacrin Inc (Charlestown, MA) is conducting two phase 1 clinical trials treating patients with damaged heart muscle. One clinical trial involves treating patients at the same time that they receive a ventricular assist device, whereas the other entails treating patients as they undergo coronary bypass surgery. In both trials, the maximum dose of cells is 300 million cells. The relatively small number of cells is not expected to improve perfusion or function. Moreover, it may provide a false sense of safety if subsequent larger trials use higher doses of cells.

Future directions

The ability to track the transplanted cells in vivo over a prolonged period in animals as well as in humans will provide valuable information about the fate of transplanted cells. New magnetic resonance cell markers have been developed [27]. The cells are labeled by the addition of a macromolecule with an iron motif during cell culture. The label does not attenuate the growth or differentiation of mesenchymal stem cells. After transplantation, the labeled cells can be tracked by clinical magnetic resonance systems.

Cell transplantation may prove to be an important addition to the armamentarium against coronary heart disease. However, it is essential to quantify carefully the magnitude of additional benefit that this novel treatment may have, and to balance that against potential adverse effects.

Acknowledgments

Osman Al-Radi is supported by an educational scholarship from King Abdulaziz University, Jeddah, Saudi Arabia. This research was partly supported by the physicians of Ontario through The Physicians Services Incorporated (PSI) Foundation.

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