Direct cardiac injection of G-CSF mobilized bone-marrow stem-cells improves ventricular function in old myocardial infarction

Life Sciences 78 (2005) 279 – 283 www.elsevier.com/locate/lifescie

Direct cardiac injection of G-CSF mobilized bone-marrow stem-cells improves ventricular function in old myocardial infarction Abel Archundia a, Jose´ Luis Aceves a, Manuel Lo´pez-Herna´ndez b, Martha Alvarado b, Emma Rodriguez c, Guillermo Dı´az Quiroz a, Araceli Pa´ez c, Felipe Masso Rojas c,*, Luis Felipe Montan˜o d a

c

Departamento de Cirugı´a Cardiovascular, CMN 20 de Noviembre, ISSSTE, Me´xico DF, Me´xico b Departamento de Hematologı´a, CMN 20 de Noviembre, ISSSTE, Me´xico DF, Me´xico Seccio´n de Biologı´a Celular, Instituto Nacional de Cardiologı´a ‘‘Ignacio Cha´vez’’, Juan Badiano 1, Col. Seccio´n XVI, Tlalpan 14080, Me´xico DF, Me´xico d Laboratorio de Inmunologı´a, Departamento de Bioquı´mica, Facultad de Medicina, UNAM, Me´xico DF, Me´xico Received 22 November 2004; accepted 21 April 2005

Abstract Autologous transplant of bone marrow stem cells (BMSC), although extremely useful after acute myocardial events, has not been evaluated in patients with old (> one-year-old) myocardial infarction. Our aim was to determine if CD34+-enriched peripheral-blood cells, obtained by apheresis, injected directly into the severely damaged myocardium of five patients with old myocardial infarction could restore depressed myocardial function. We found that 28 weeks after revascularization and peri-infarction injection of the enriched CD34+ peripheral mononuclear cells, ventricular hemodynamic parameters that included left ventricular ejection fraction, left ventricular diastolic volume, ventricular systolic volume and left ventricular diastolic diameter approximated normal values and there was no restenosis; two patients have been followed for > 52 weeks and their parameters are within normal values. In conclusion, intramyocardial injection of easily obtained CD34+ enriched peripheral blood cells represent an encouraging procedure for patients with severely scarred and dysfunctional myocardium. D 2005 Elsevier Inc. All rights reserved. Keywords: Stem cells; Myocardial infarction; CD34+ cells

Introduction Cardiomyocyte death, fibrotic scar development and loss of the ventricular function are considered an irreversible event of acute and chronic myocardial infarction, which may cause sudden cardiac death and heart failure. It was believed that adult cardiac myocytes do not replicate but improvement in the methodological approach to the analysis of tissue sections has defeated the dogma in the adult heart (Kajstura et al., 2004). Most importantly, primitive and progenitor cells, that express stem cell markers and telomerase, have been identified in the human heart (Timmermans et al., 2003; Urbanek et al., 2003). Intracoronary infusion of autologous bone marrow-derived progenitor cells into the infarct artery in acute myocardial infarction patients has been shown to improve regional wall * Corresponding author. Tel.: +52 5573 2911x1337; fax: +52 5573 0926. E-mail address: [email protected] (F.M. Rojas). 0024-3205/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.lfs.2005.04.080

motion in the infarct zone and to reduce end-systolic left ventricular volumes (Assmus et al., 2002), as opposed to the intracoronary injection of mesenchymal stromal cells that produce microinfarcts (Vulliet et al., 2004) or the intracoronary infusion of stem cells together with an intracoronary stent that induce restenosis in humans (Kang et al., 2004). In humans, the intracoronary or peri-infarct injection of BMSC in acute or sub-acute lesions (less than 3 months after the infarct) with viable tissue has shown a marked therapeutic effect as demonstrated by improved hemodynamic criteria (Kajstura et al., 2004; Stamm et al., 2003; Strauer et al., 2002). Nevertheless, the patient with old myocardial infarct is usually treated more conservatively despite the need, in some of them, of heart transplant. Our aim was to evaluate if the peri-infarction injection of circulating autologous stem cells, mobilized from the bone marrow by granulocyte colony stimulating factor treatment, could improve ventricular function in patients with old (> 1 year) myocardial infarction.

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Materials and methods Patients Five men (mean age of 51 years old; range 35 –61) with ischemic cardiomyopathy and more than one-year-old myocardial infarction were eligible for inclusion into the study. These patients had a serum cholesterol level of 250 mg/dl or higher and high blood pressure (diastolic values of >90 mm Hg and systolic values of 140 –160 mm Hg) for more than two years before the infarct. They all had left ventricular failure as determined by the low left ventricular ejection fraction (LVEF) values and were considered as NYHA-II/III functional class and were being treated with appropriate doses of angiotension converting enzyme inhibitors, AT1R blockers and statins. As an internal reference group we selected 10 patients matched for ejection fraction, infarct localization, and infarct size with our study population. The presence and extent of the infarct was determined by ECG (old anterior apical infarcted zone), angiography (C type proximal injury in the left anterior descending coronary artery and akinesia in the anterior wall and apical heart zones), echocardiogram (akinesia in the apex and the anterior myocardial side) and radionuclide myocardial perfusion imaging (old transmural infarction in the anterior wall and apical zone without evidence of viable tissue according to rest, stress and 24 h images). The ethics review board of the Hospital approved the protocol in January 2003, and the study was conducted in accordance with the Declaration of Helsinki. Written informed consent was obtained from each patient. The criteria used for allocating patients to either surgical procedure (CD34-enriched cell fraction or control) was based on the lack of viable tissue in the anterior wall of the heart together with the presence of ischemic areas in the lateral and/or inferior heart wall and ongoing clinical manifestations of angina. Mobilization of stem cells The patient were treated with 300 mg of granulocyte colony stimulating factor (G-CSF; Filgistrin, Neupogeni) subcutaneously for five days, in order to enhance the percentage of circulating stem cells derived from the bone marrow according to a protocol widely used in leukemia patients (Gyger et al., 2000; Jansen et al., 2002). Harvest, characterization and cryopreservation of mononuclear cells One day after Neupogen treatment was finished the patient’s right subclavian vein was catheterized (Mahurkar catheter) and mononuclear cells were harvested with a close circuit apheresis machine (Baxter) in a volume of 500 ml. The percentage of CD34+ cells was estimated by cytofluorometry with a FACScalibur cytofluorometer (Becton Dickinson) with a specific FITC-labeled anti human CD34+ monoclonal antibody (Becton Dickinson). Cryopreservation of CD34+ enriched mononuclear cells was made with a mixture of dimethylsulf-

oxide 11.25%, albumin 32.5%, and hydroxyethyl starch 6% (all from Sigma-Aldrich, USA), in RPMI medium at 1 : 1 patient plasma : mixture ratio at 80 -C in an ultra freezer (REVCO); cells were preserved until surgery. Surgery and CD34+ enriched mononuclear cells injection The surgical procedure was performed two days after obtaining the CD34-enriched cell preparation in order to permit the patient to stabilize after the apheresis procedure and to be in optimal pre-surgical conditions. All patients were subjected to conventional open thorax revascularization, with continuous trans- and post-surgery electrocardiographic monitoring. An anastomosis from the left internal thoracic artery to the left anterior descending coronary artery was made with the intention of giving blood flow to the old infarcted zone as well as to nourish the transplanted cells. Prior to the surgical procedure the frozen cells were thawed at 37 -C, in a water bath with gentle shaking, for 30 m. In order to remove the DMSO and the hydroxyethyl starch, cells were washed with sterile saline solution at 800 rpm for 3 min, three times. After the revascularization procedure was finished, we injected 20 ml of the CD34+ enriched mononuclear cell preparation which contained 2  107 mononuclear cells/ml including 6  105 CD34+ cells/ml with a cell viability > 90%, distributed in 10  2 ml radial injections from the center of the scar to the periphery at the border zone between healthy and infarcted tissue, in the five patients that had been assigned to the protocol. Control group patients did not receive an injection of either saline solution or culture medium at the time of coronary reperfusion because of the risk of inducing arrhythmia. None of the patients in either group, developed arrhythmia during or after the surgical procedure. Left ventricular function monitorization After surgery the patients were monitored by echocardiography at 8, 16 and 28 weeks. The hemodynamic parameters that were evaluated included heart motility, left ventricular ejection fraction, left ventricular end diastolic volume, left ventricular end systolic volume, left-ventricular end diastolic cavital diameter and left ventricular thickness. Tc99 SPECT was made after 8 and 28 weeks post-surgery. Statistical analysis. The results between the two groups were analyzed by the non-parametric Student’s T test, using the SPSS v.11 software. A p value < 0.05 was considered statistically significant. The values in Table 2 represent the maximal and minimal values. Results None of the patients showed post surgical complications. Similarly, none of the patients receiving G-CSF presented secondary manifestations attributable to the administration of filgistrin. All the patients, experimental and control, left the hospital seven to ten days later. Table 1 summarizes the presurgery values of the five CD34-transplanted patients. The

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Table 1 Pre-surgery values in CD34+-treated patients Age Motility LVEF LVDV LVSV LVDD Time elapsed (> 55%) (<90 ml) (< 50 ml) (<60 mm) between heart infarct and surgery 35 45 55 60 61

Akinesia Akinesia Akinesia Akinesia Akinesia

38% 40% 45% 39% 40%

170 165 150 177 199

ml ml ml ml ml

108 ml 110 ml 109 ml 113 ml 115 ml

68 60 69 55 66

mm mm mm mm mm

52 53 56 68 76

weeks weeks weeks weeks weeks

Pre-surgical myocardial functional values in the five patients treated with autologous CD34+-enriched mononuclear cell transplantation. Numbers inside brackets correspond to the normal values. LVEF = Left Ventricular Ejection Fraction; LVDV = Left Ventricular Diastolic Volume; LVSV = Left Ventricular Systolic Volume; LVDD = Left Ventricular Diastolic Diameter.

echocardiographic study eight weeks after surgery showed an important improvement in hemodynamic parameters even though heart motility showed only a discrete improvement. Sixteen weeks after the surgical procedure the five patients treated with mobilized bone marrow stem cells showed enhanced heart motility, and the other hemodynamic parameters had progressed towards normal values reaching statistical significance in comparison with the pre-surgery values (Table 2). Two of the patients have been followed for fifty-two weeks and their echocardiograms and ventricle contractility show practically normal values (Table 2). Myocardial muscular thickness in this two patients progressed to normal values (10 and 11 mm vs. 5 and 6 mm, before cellular transplantation, respectively) during ventricular systole. It was interesting to observe in these two patients, that eight weeks after the intervention the Tc99 SPECT demonstrated an improved perfusion in the infarcted zone which reached normal values 28 weeks post-surgery (Fig. 1). Both patients are now considered as class I NYHA functional status. One of the remaining three patients that has been followed for 28 weeks is now considered a class I NYHA functional status whereas the remaining two are cataloged as class II. Control patients showed a very weak engrossing of the myocardium during ventricular systole (6 –7 mm vs. 5– 6 mm before surgery) after 28 weeks of follow up and most importantly, the surgical procedure did not improve their ventricular function. Discussion Stent application has diminished the need for bypass surgery in acute myocardial infarct individuals. From a clinical standpoint, despite undoubted progress, there remains a large

Fig. 1. Tecnesium-99 SPECT images. Sequence of events in the 60-year-old patient. A and B show the transversal and longitudinal images, respectively, before the cellular transplant. The white arrows show the apical and anterior wall perfusion defect. C and D show the same areas 28 weeks after the cell transplant. There is a clear improvement in Tc-99 capture.

population of survivors of extensive infarctions with diminished cardiac reserve, which comprise a reservoir of patients who will develop congestive heart failure. The concept that such patients could be treated by myocardial regeneration is logical since many of them require aortic-coronary bypass and eventually, depending on the status of their ventricular function, a heart transplant. As mobilized peripheral blood stem cells (CD34+) are increasingly used for clinical cell transplantation, it is becoming clear that proteolytic degradation of the chemokine, stromal cell-derived factor (SDF-1), and its receptor, CXCR-4, on stem cells could be an important step in stem-cell release and homing (Le´vesque et al., 2003a,b). It is known that stem cells transplant and endogenous stem-cell mobilization restore depressed myocardial function in acute infarcted myocardium, in the first post-infarct week (Askari et al., 2003; Franz et al., 2003). The possible benefits of these treatments in people with extensive vast scar tissue from an old heart infarction episode, is unknown. The concept that such patients could be treated by myocardial regeneration is logical. CD34+ cells are bone marrow-derived vascular progenitor cells which include a hematopoietic stem cell population (Asahara and Kawamoto, 2004). Administration of granulocyte colony-stimulating factor, activates neutrophil elastases, which then cleave the membrane-bound SDF-1 of stromal cells of the

Table 2 Post-surgery parameter evolution values in infarcted and control patients Motility

LVEF (<55%) LVDV (<90 ml) LVSV (<50 ml) LVDD (<60 mm)

Basal n = 5

8 weeks n = 5

16 weeks n = 5

28 weeks n = 5

52 weeks n = 2

Controls 28 weeks n = 10

Akinesia

Hypokinesia

Hypokinesia

Normal

Normal

Akinesia

38 – 45% 150 – 199 108 – 115 55 – 69

48 – 50% 69 – 77 38 – 70 50 – 55

55 – 60% 60 – 70 35 – 40 56 – 47

55 – 60%* 53 – 70* 32 – 40** 55 – 47*

58 – 60% 52 – 60 33 – 35 47 – 55

35 – 45% 150 – 217 90 – 147* 58 – 63

Evolution of the echocardiograph values in the CD34+-enriched mononuclear cell transplanted patients (n = 5) and controls (n = 10). The numbers represent the maximal and minimal values. * p < 0.005 and ** p < 0.001 in comparison with basal pre-surgery values. Numbers inside brackets correspond to the normal values. Only two patients have been followed for more than 28 weeks.

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bone marrow and provoke an efflux of stem cells that express CXCR-4, releasing stem cells to the peripheral circulation (Le´vesque et al., 2003a,b; Carion et al., 2003), and causing a rapid and large but transient increases in the number of circulating CD34+ cells (Shier et al., 2004). Because of their properties, we evaluated the usefulness of direct intramyocardial injection of an autologous CD34+-enriched mononuclear blood cell preparation in five patients with old myocardial infarct. We chose the direct intramyocardial approach in open heart surgery because stem cells are found in low quantities in old infarcts. Another reason was that intracoronary perfusion does not guarantee the arrival of adequate number of cells into the damaged zone, due to the coronary obstruction produced by the infarct, and can induce restenosis (Kang et al., 2004). The anastomosis was performed in order to enhance the blood flow to the old infarcted zone and consequently, nourish the transplanted cells. Extensive regeneration of myocardial infarcts has been reported after direct stem cell injection (Deb et al., 2003). Similarly, transdifferentiation of CD34+ adult human peripheral blood cells into cardiomyocytes, has also been reported (Yeh et al., 2003). Nevertheless, two recent studies have unambiguously shown that bone marrow cells do not transdifferentiate (Balsam et al., 2004; Murry et al., 2004). Even so, they may have functional benefits, mediated by a limitation of ventricular dilatation, paracrine induction of angiogenesis, or churning out growth factors that encourage cell proliferation and/or survival (Menasche, 2004; Schneider, 2004). It has recently been demonstrated that the direct injection of bone marrow cells into an organ is associated with a much greater seeding efficiency (Castello et al., 2004). The pre-implant perfusion images in our patients showed areas, especially in the anterior wall and the apex of the heart, lacking movement and Tc99 capture; these images were no longer observable after 28 weeks of follow up, suggesting the presence of viable and functional cells. It has been suggested that improvement in cardiac contractility occurs via effects on Starling forces. In such cases, bone marrow-derived cells played an important therapeutic role, albeit not via cell replacement or transdifferentiation (Daley et al., 2003). A recent study performed in patients with stable and severe angina pectoris, a situation that resembles in many ways an old infarct, who received direct intramyocardial plasmid VEGF-A165 gene therapy [0.5 mg distributed in 10 injections] showed an increase in plasma CD34+ stem cells three weeks after treatment and improved regional wall motion after three months (Kastrup et al., 2005). Natural stem-cell mobilization to the heart (Jackson et al., 2001) is extremely slow after acute and chronic myocardial infarction consequently the in situ enrichment of CD34+ stem cells in myocardial necrotic areas is a much faster alternative, nevertheless it remains to bee seen if enhanced stem-cell homing due to other peripheral blood mononuclear cells present in our CD34+-enriched cell suspension, such as T lymphocytes, that produce, secrete and express chemokines and chemokine receptors, contributed to the success we achieved in our patients. It is well known that statins mobilize progenitor stem cells following ischemic injury (Landmesser et

al., 2004). Because all the patients, transplanted and control, were receiving statins the differences observed between both groups can only be attributed to the effect of the CD34+enriched cell suspension. One of most interesting results was the important myocardial muscle thickening obtained after the procedure which traduces into a better myocardial function. The values we registered of 10 –11 mm are similar to those obtained with the use of Dobutamine (Rahimtoola, 1996) in patients with hibernating life tissue. The fact that control individuals did not thicken their myocardium, demonstrates that autologous CD34+-enriched cell implant, induces a better contractile performance, as suggested by the slight increase in the diastolic diameter of the left ventricle. A similar observation has been made in patients with end-stage ischemic heart disease that received autologous bone marrow mononuclear cells (Perin et al., 2004). Conclusion We believe that direct intramyocardial injection of autologous CD34+-enriched mononuclear cells mobilized by G-CSF pre-treatment is safe and constitutes a viable long-term alternative for patients with severely damaged myocardium. Acknowledgments This work was financed by CONACyT-Mexico grant SALUD-2002-C01-7630. References Asahara, T., Kawamoto, A., 2004. Endothelial progenitor cells for postnatal vasculogenesis. American Journal of Physiology. Cell Physiology 287 (3), C572 – C579. Askari, A.T., Unzek, S., Popovic, Z.B., Goldman, C.K., Forudi, F., Kiedrowski, M., Rovner, A., Ellis, S.G., Thomas, J.D., DiCorleto, P.E., Topol, E.J., Penn, M.S., 2003. Effect of stromal-cell-derived factor 1 on stem-cell homing and tissue regeneration in ischaemic cardiomyopathy. Lancet 362 (9385), 697 – 703. Assmus, B., Schachinger, V., Teupe, C., Britten, M., Lehmann, R., Dobert, N., Grunwald, F., Aicher, A., Urbich, C., Martin, H., Hoelzer, D., Dimmeler, S., Zeiher, A.M., 2002. Transplantation of progenitor cells and regeneration enhancement in acute myocardial infarction (TOPCARE-AMI). Circulation 106 (24), 3009 – 3017. Balsam, L.B., Wagers, A.J., Christensen, J.L., Kofidis, T., Weissman, I.L., Robbins, R.C., 2004. Haematopoietic stem cells adopt mature haematopoietic fates in ischaemic myocardium. Nature 428 (6983), 668 – 673. Carion, A., Benboubker, L., Herault, O., Roingeard, F., Degenne, M., Senecal, D., Desbois, I., Colombat, P., Charbord, P., Binet, C., Domenech, J., 2003. Stromal-derived factor 1 and matrix metalloproteinase 9 levels in bone marrow and peripheral blood of patients mobilized by granulocyte colonystimulating factor and chemotherapy. Relationship with mobilizing capacity of haematopoietic progenitor cells. British Journal of Haematology 122 (6), 918 – 926. Castello, S., Podesta, M., Menditto, V.G., Ibatici, A., Pitto, A., Figari, O., Scarpati, D., Magrassi, L., Bacigalupo, A., Piaggio, G., Frassoni, F., 2004. Intra-bone marrow injection of bone marrow and cord blood cells: an alternative way of transplantation associated with a higher seeding efficiency. Experimental Hematology 32 (8), 782 – 787. Daley, G.Q., Goodell, M.A., Snyder, E.Y., 2003. Realistic prospects for stem cell therapeutics. Hematology (Education Program Book) 2003, 398 – 418.

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