Chinese Medical Journal 2004;117(10):1443-1448
Improvement of cardiac function after transplantation of autologous bone marrow mesenchymal stem cells in patients with acute myocardial infarction

CHEN Shao-liang 陈绍良,  FANG Wu-wang 方五旺,  QIAN Jun 钱 钧,  YE Fei 叶 飞,  LIU Yu-hao 刘煜昊,  SHAN Shou-jie 单守杰,  ZHANG Jun-jie 张俊杰,  LIN Song 林 松,  LIAO Lian-ming 廖联明,  Robert Chun Hua ZHAO 赵春华

CHEN Shao-liang 陈绍良 (Department of Cardiology, Nanjing First Municipal Hospital, Nanjing Medical University, Nanjing 210006, China)

FANG Wu-wang 方五旺 (Department of Cardiology, Nanjing First Municipal Hospital, Nanjing Medical University, Nanjing 210006, China)

QIAN Jun 钱 钧 (Department of Cardiology, Maanshan Municipal People’s Hospital, Maanshan 240058, China)

YE Fei 叶 飞 (Department of Cardiology, Nanjing First Municipal Hospital, Nanjing Medical University, Nanjing 210006, China)

LIU Yu-hao 刘煜昊 (Tissue and Engineering Centre, Chinese Academy of Medical Sciences, Beijing 100036, China)

SHAN Shou-jie 单守杰 (Department of Cardiology, Nanjing First Municipal Hospital, Nanjing Medical University, Nanjing 210006, China)

ZHANG Jun-jie 张俊杰 (Department of Cardiology, Nanjing First Municipal Hospital, Nanjing Medical University, Nanjing 210006, China)

LIN Song 林 松 (Department of Cardiology, Nanjing First Municipal Hospital, Nanjing Medical University, Nanjing 210006, China)

LIAO Lian-ming 廖联明 (Tissue and Engineering Centre, Chinese Academy of Medical Sciences, Beijing 100036, China)

Robert Chun Hua ZHAO 赵春华 (Tissue and Engineering Centre, Chinese Academy of Medical Sciences, Beijing 100036, China)

Correspondence to:Chen Shao-liang,Department of Cardiology, Nanjing First Municipal Hospital, Nanjing Medical University, Nanjing 210006, China (Tel: 86-25-52208048. Fax:86-25-52208048. E-mail:chmengx@126.com)
Keywords
acute myocardial infarction;bone marrow mesenchymal stem cell;cell transplantation
Abstract
Background The infarct size determines the long-term prognosis of patients with acute myocardial infarction (AMI). There is a growing interest in repairing scar area by transplanting bone marrow stem cells. However, effectiveness of intracoronary injection of bone marrow mesenchymal stem cells (BMSCs) in patients with AMI still remains unclear.
Methods Sixty-nine patients with AMI after percutaneous coronary intervention (PCI) were randomly divided into intracoronary injection of BMSCs (n=34) and saline (control group, n=35) groups. Serial single positron emission computer tomography (SPECT), cardiac echo and cardiac electromechanical mapping were done at the designed time intervals until six months after transplantation of BMSCs or injection of saline.
Results The proportion with functional defect decreased significantly in the BMSCs patients after three months [(13±5)%] compared with that pre-transplantation [(32±11)%] and the control group [(28±10)%] at three month follow-up (P<0.05, respectively). Wall movement velocity over the infracted region increased significantly in the BMSCs group [(4.2±2.5) cm/s vs (2.2±1.3) cm/s, P<0.05], but not in the control group [(2.2±1.5) cm/s vs (2.7±1.7) cm/s, P>0.05]. Left ventricular ejection fraction (LVEF) three months after transplantation in BMSCs group increased significantly compared with that pre-implantation and with that of the control group at three months post-injection [(67±11)% vs (49±9)% and (53±8)%, P<0.05 respectively]. SPECT scan results showed that perfusion defect was improved significantly in BMSCs group at three-month follow-up compared with that in the control group [(134±66)cm2 vs (185±87)cm2, P<0.01]. At the same time, left ventricular end-diastolic volume [(136±31) ml vs (162±27) ml, P<0.05] and end-systolic volume [(63±20) ml vs (88±19) ml, P<0.05] decreased synchronously. The ratio of end-systolic pressure to end-systolic volume [Psyst/ESV, (2.84±1.30) mmHg/ml vs (1.72±1.23) mmHg/ml, P<0.05] increased significantly. Cardiac electromechnical mapping demonstrated significant improvement at three months after implantation of BMSCs compared with that pre-injection in both cardiac mechanical capability as left line local shorting [LLS, (11.29±1.64)% vs (7.32±1.86)%, P<0.05] and electrical property as left ventricular endocardial unipolar voltage [UV, (10.38±1.12) mV vs (7.61±1.09) mV, P<0.01]; perfusion defect decreased from (36.2±6.2) % to (20.3±5.31)% (P<0.01). Twenty-four-hour electrocardiographic monitoring demonstrated no arrhythmias occurred at three-months follow-up.
Conclusions The transplantation of BMSCs might improve the cardiac function and it is safe and feasible with no deaths or malignant arrhythmias.
The process of left ventricular remodeling after acute myocardial infarction (AMI) involves the acute and chronic transformation of both the necrotic infarct region and peri-infarct tissue.[1] Conventional approaches, including medication (beta-blocker and/or angiotensin converting enzyme inhibitors), coronary stenting, and coronary artery bypass grafting surgery, have the limitations in preventing ventricular remodeling and improving cardiac function. Several methods have been attempted to repair the infracted myocardium.[2] Of these, the most important is ‘biointerventional therapy’, which name indicates the main role of bone marrow-derived stem cells.

Bone marrow contains multipotent adult stem cells which still have a high capacity for differentiation to myocardium.[3] Although many other bone marrow-related cell types may participate in organ repair after infarctions, bone marrow mesenchymal stem cells (BMSCs) have retained the greatest capacity for myocardial repair.[4] BMSCs have been considered as ‘second class marrow citizens’ for a long time. However, recently it becomes clear that a small population of BMSCs in the bone marrow is a putative adult stem cell population with important functional features including capacity for rapid self-renewal: giving rise to a wide variety of connective tissues including bone, cartilage, muscle, fat, haemopoiesis-supporting stroma and, potentially, also neuronal cells.[5,6] Several aspects of BMSCs have already been elucidated.[7] BMSCs occur at the very low frequency of two to five BMSCs per million mononuclear cells in bone marrow harvests. However, rapid proliferation of these cells in vitro allows expansion of this population by a factor of 10(3) within 14 to 21 days of culture. Growth rate of BMSCs depends on the cell density of the culture. Unless cultured for prolonged periods, the BMSCs do not show senescence and apoptosis. Thus, BMSCs can be tremendously multiplied within a reasonable time period.

Furthermore, two reports have stated that cultured bone marrow mononuclear cells implantation by intracoronary injection improves the left ventricular function.[8,9] Therefore, BMSCs may show superior penetration into myocardium from coronary vessel and initiate marvellous myocardial regeneration. Taking all the above into account, we designed the present randomized study to confirm the effectiveness of intracoronary injection of BMSCs for patients with AMI.

METHODS

Patients
From November 2002 to May 2003, 78 patients with acute transmural myocardial infarction, according to WHO criteria, within twelve hours from the onset of continuous chest pain were enrolled and underwent emergency angiography/angioplasty. The mean time from onset of myocardial infarction (MI) to angiography and angioplasty was (8.0±3.7) hours.

The infarct-related artery was recanalized by balloon angioplasty only (n=7) or subsequent stent deployment (n=71) after right and left catheterization with residual stenosis <20%. All patients were briefed in detail about the significance and exact procedure of BMSCs implantation immediately after percutaneous coronary intervention (PCI) procedure. Formal consents from patient’s relatives were gained and the study protocol was approved by the ethics committees of Jiangsu Provincial Health Bureau and the Nanjing First Hospital. The enrolment criteria for BMSCs implantation included age <70 years, without cardiac shock and cardiac block, stable haemodynamics and no severe comorbidity. Finally, 69 of 78 patients were candidates for BMSCs transplantation and were randomly divided into true BMSCs group (n=34) and false BMSCs group (PCI only group, n=35) as controls.

BMSCs preparation and intracoronary transplantation
Sixty milliliters of autologous bone marrow were aspirated under local anaesthesia from ileum of all the 69 patients during the morning of the 8th day after the PCI procedure and cultured for 7 days to 10 days. BMSCs was cultured and harvested by the method of Jaiswal et al.[5] Several attempts were used to control BMSCs under 15 generations. BMSCs was harvested and washed three or four times with heparinized saline. The BMSCs suspension was mixed with heparin, filtered and prepared for implantation just two hours before implantation. Each milliliter of the final BMSCs suspension had over 8×10(9) cells.

The infarct-related coronary artery was occluded just at the proximal edge of former angioplasty as described by Strauer et al.[8] Each milliliter of the BMSCs suspension containing 8×10(9)-10×10(9) cells was directly injected through an inflated over-the-wire (OTW) balloon catheter central lumen at high pressure (1 MPa) into the target coronary artery. The balloon was inflated for at least two minutes continuously to occlude the anterior blood flow just before the beginning of BMSCs injection.

Standard saline rather than BMSCs was injected through the coronary in false BMSCs group patients by the same method as above for the BMSCs group patients, and the harvested BMSCs were collected as basic research only. This procedure was approved by the Ethics Committee of Jiangsu Provincial Health Bureau.

Myocardial viability and cardiac function tests
Single positron emission computer tomography (SPECT), cardiac catheterization and cardiac echo were performed on all the 69 patients at the day of transplantation, and three months and six months after BMSCs transplantation. Myocardial viability and cardiac function were recorded as base indices. The contractility index, the ratio of end-systolic pressure to end-systolic volume (Psyst/ESV), was calculated. A perfusion defect was calculated by scintigraphic bull’s eye technique.[10] Sheehan’s method was used to quantify infarction wall movement velocity by cardiac echo and 5 measurements were made perpendicular to the long axis in the main akinetic or dyskinetic segment of the ventricular wall.[10] Lengths at systole and diastole were measured, and the mean difference was divided by the systolic duration.

Electromechanical mapping (EMM)[10] was performed for 15 patients in BMSCs group and 8 patients in the control group on the day before and three month after transplantation. Briefly, a 45 cm, 8F sheath was put into the right femoral artery. Systemic heparin (100 U/kg) was administered through the side-arm of the sheath. An electromechanical mapping catheter (8F) was inserted into the sheath and directly onto the aortic valve after standard ventriculography at right oblique 30° and left oblique 30°. The operator advanced the handle to curve the distal mapping catheter for easy advance into left ventricle and points were acquired when the catheter tip was stable on the endocardium; this occurred after noting local activation time stability, location stability, loop stability and cycle-length stability. An interpolation threshold of 40 mm was set between adjacent points. Continuous mapping of left ventricle was maintained at right oblique 30° and left oblique 30°. For each patient, colour-coded, linear shortening maps were generated. EMM images were divided into 14 segments corresponding to SPECT images. The long axis was defined as the line connecting the apex and the centre of mass. Unipolar voltage was recorded automatically at the target area and compared with that at normal tissue. No pericardial effusion occurred during the procedure.

Follow-up
All patients were cared for 24 hours after intracoronary injection and NOGA procedure, and went into clinical follow-up for at least six months. Cardiac echo was recorded once monthly, and SPECT was performed at the third and sixth months after implantation procedure for all patients. Twenty-four-hour electrocardiographic monitoring was performed at the third month after procedure. An optional repeat electromechanical mapping was performed on some patients.

Statistical analysis
All data were expressed as mean±standard deviation (SD). Discrete variables were compared as rates, and comparisons were tested by χ2 analysis. Intra-individual comparison between the baseline and follow-up continuous variables was performed with a paired t test. Comparison of nonparametric data between the two groups was performed with Wilcoxon test and Mann-Whitney test. Statistical analysis was performed with SPSS 10.1. All indices were collected and analysed by three statisticians who did not know the aims of the present study. A value of P<0.05 was considered statistically significant.

RESULTS

Clinical characteristics
Nine patients were excluded from this study. Four of the 9 were >70 years of age, 1 with cardiogenic shock, 2 with complete left bundle branch block and 2 without thrombolysis in myocardial infarction (TIMI) flow grade 3 immediately after PCI procedure. All the other patients had TIMI 3 grade after PCI. Baseline clinical characteristics between the two groups did not differ significantly. The level of creatinine kinase-MB was slightly higher, but not significantly so, in BMSCs group than that in the control group ( Table 1 ). No deaths occurred during six-month follow-up.

Left ventricular hemodynamics
Left ventricular dynamics according to left ventriculogram demonstrated several significant differences between the two groups ( Table 2 ). The functional defect as a percentage of hypokinetic, akinetic and dyskinetic segments decreased significantly in BMSCs patients at the third month compared with that before BMSCs transplantation [(13±5)% vs (32±11)%, P<0.01]. It was also smaller compared with that in the control group at three-month follow-up [(13±5)% vs (28±10)%, P<0.05]. Wall movement velocity over the infarcted-region increased significantly in BMSCs group [(4.2±2.5) cm/s vs (2.2±1.3) cm/s, P<0.05] but not in the control group [(2.2±1.5) cm/s vs (2.7±1.7) cm/s, P>0.05]. Left ventricular ejection fraction (LVEF) at the third months after transplantation in BMSCs group increased significantly compared to pre-implantation [(67±11)% vs (49±9)%, P<0.05] and the control group [(67±11)% vs (53±8)%, P<0.05]. LVEF showed no change between the third and the sixth month after transplantation in BMSCs group.

Parameters of cardiac function
As shown in Table 3 , perfusion defect detected by SPECT was improved significantly in BMSCs group at three-month follow-up compared with that in the control group [(134±66) cm2 vs (185±87) cm2, P<0.01]. Cardiac size by cardiac echo decreased significantly as shown by left ventricular end-diastolic volume [(136±31) ml vs (162±27) ml, P<0.05] and end-systolic volume [(63±20) ml vs (88±19) ml, P<0.05]. Psyst/ESV increased significantly [(2.84±1.30) mmHg/ml vs (1.72±1.23) mmHg/ml, P<0.01], which indicated the significant improvement of cardiac function. Twenty-four-hour electrocardiographic monitoring demonstrated no arrhythmias occurred at three-month follow-up.

Real-time cardiac electromechnical mapping demonstrated significant improvement at the third month after implantation compared with preinjection in both cardiac mechanical capability as left line local shorting [LLS, (11.29±1.64) % vs (7.32±1.86)%, P<0.05] and electrical property as left ventricular endocardial unipolar voltage [UV, (10.38±1.12) mV vs (7.61±1.09) mV, P<0.01]. Perfusion defect as a percentage decreased from (36.2±6.2)% to (20.3±5.31)% (P<0.01, Table 4 ).

DISCUSSION

Ventricular remodeling and ultimately heart failure are the inexorable consequences of substantial MI and correlate with infarct size.[11] In recent years, the understanding that regenerative processes exist at the level of the myocardium has placed stem cell research at forefront of cardiology.[12] The concept of ‘growing’ heart muscle and vascular tissue has revolutionized the approach to treating MI.[13] Autologous bone marrow stem cell, without the property of immune rejection, has the ability to differentiate into myocardium, and plays a central role in modern “cell therapy” of MI. However, the stem cell therapy gives rise to more questions than answers, such as “What type of stem cell is suitable for patients?” and “When is the best time for transplantation?”.[14] The crucial questions we have to address before designing this trial are the follows: Which type of cell population should we deliver? When should the cells be transplanted? and How to keep detecting the viability of cells transplanted?

The microenvironment plays a fundamental role in the differentiation of stem cells.[15] Skeletal myoblasts have invariably succeeded in reconstituting heart muscle structure, i.e. myocardium and coronary vessels. However, this type of cell has several disadvantages: too large to inject through coronary artery, risk of thrombo-embolism, the need for open chest and cardiac improvement until 5 months afterward.[16] Therefore, considerable attention has been paid to BMSCs. BMSCs is considered as “the second class marrow citizens” for a long time. However, recent studies have demonstrated that BMSCs have several useful features including a potential for rapid-renewal.[5,6] Intracoronary injection of bone marrow stem cells supplies the simplest approach clinically.[8] Theoretically, BMSCs has the highest potentiality for adhesion to vessel walls and may infiltrate into the infarct zone even after the balloon has been deflated and anterior blood flow restored. We find experimentally that anterior blood flow sweep away the bone marrow mononuclear cells very quickly and a large amount of these cells infiltrated into the distal less infracted zone compared with BMSCs injected (in print). Our detecting by NOGA system confirms this phenomenon clinically and finds that BMSCs transplanted into the infracted zone along the vessel from the proximal edge at the site of injection. This contributes to the BMSCs growth in the heart and significant improvement of cardiac function. Hence, we choose BMSCs as the target stem cell for transplantation.

Wakitani et al[17] first discovered that BMSCs could differentiate into skeletal muscles and supply the basis for clinical research. Fukuda et al[18] undertook animal research with cultured BMSCs preconditioned with 5-azacytidine and transplanted into heart. The results reveal BMSCs might differentiate into myocardium. This process of differentiation is characterized by automatic control within one week and by myocardial special gene expression as expressing atrial natriuretic peptide and troponin T. However, the optimum time window is the key problem for transplantation of BMSCs in patients with MI and remains undetermined. Strauer et al[8] determined that the best transplanting time should be between 7 days and 14 days after the AMI. Li et al[19] reported the only animal research aimed to identify the optimum time for cardiomyocyte transplantation after left ventricular injury. Adult rat hearts were cryoinjured immediately, 2 and 4 weeks later. The inflammatory process was strongest in the first day, and best results were seen after 2 weeks which may due to mild scar expansion. Bolognese et al[20] reported three types of the left ventride remodeling after AMI: early (24 hours to one month), late (one month to six months) and progressive (one day to six months) dilation of ventricular chamber although no correlation occurred between remodeling types and worse outcomes. The results indicate that more aggressive prevention of the left ventricle remodeling by cell therapy during late dilation phase is practical because cell therapy is impossible at early phase of remodeling. Therefore, we extend this study to detect the effect of delayed BMSCs transplantation on nearly 3-week-old infarction. The time interval is nearly 18 days from PCI to BMSCs transplantation which definitely exclude the effect of PCI on outcomes of BMSCs transplantation. The final results demonstrate that BMSCs implantation significantly improves the cardiac function which confirms BMSCs improve the remodeling of the left ventricle via supplying the working cardiomyocytes.

Nuclear perfusion imaging including SPECT, has been considered the gold standard for detection of viable myocardium.[21] From the clinical and practical point, a real-time detecting device should be the best. For example, we need the real-time and accurate answer during angiography about the viability of myocardium in the area perfusioned by a chronic occluded coronary artery. Cardiac electromechanical mapping resolves this problem and has significantly correlated with SPECT reported by others.[22] This is the first study detecting the viability of BMSCs and cardiac function with NOGA. The results show that cardiac function is viable with high LLS and UV in the infarcted-area three months after BMSCs transplantation, and cardiac functional parameters are increased as demonstrated by cardiac echo. These results encourage our further study, resolving the assessment of viability of implanted BMSCs clinically and confirming that BMSCs work together with host that cardiomyocytes.

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