Myocardial Homing of Nonmobilized Peripheral-Blood CD34 Cells After Intracorona - 《stemcells》干细胞学杂志干细胞之家



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发表于 2009-3-5 00:11 |只看该作者 |倒序浏览 |打印

作者：Didier Blockleta, Michel Toungouzb, Guy Berkenboomc, Micheline Lambermontb, Philippe Ungerc, Nicolas Preumontc, Eric Stoupelc, Dominique Egrisea, Jean-Paul Degautec, Michel Goldmanb, Serge Goldmana作者单位：a Department of Nuclear Medicine and PET/Biomedical Cyclotron Unit;
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      【摘要】
   Granulocyte¨C colony-stimulating factor administered for autologous hematopoietic stem cell isolation from blood may favor restenosis in patients implanted after acute myocardial infarction (AMI). We therefore tested the isolation of peripheral-blood CD34  cells without mobilization in six patients with AMI. After large-volume cytapheresis and positive CD34  cell selection, 3.6 to 27.6 million CD34  cells were obtained. We performed intra-coronary implantation of these cells and recorded no restenosis or arrhythmia. We used positron emission tomography (PET) to assess myocardial-labeled CD34  cell homing, which accounted for 5.5% of injected cells 1 hour after implantation. In conclusion, large amounts of CD34  cells, in the range reported in previous studies, can be obtained from nonmobilized peripheral blood. PET with -fluorodeoxyglucose cell labeling is an efficient imaging method for homing assessment.
      【关键词】 Stem cells Labeling Homing Positron emission tomography Infarction Heart Implantation
   INTRODUCTION' a- R9 ?' u7 |+ \1 u9 W2 f, X8 \
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Adult myocardium has limited capacity to restore contractile function after infarction. Autologous hematopoietic stem cell (HSC) implantation in damaged myocardium has been proposed to prevent remodeling processes leading to cardiac insufficiency. HSC isolation from blood usually requires pretreatment with granulocyte¨Ccolony-stimulating factor (G-CSF). Administration of such factors in patients with acute myocardial infarction (AMI) is a matter of controversy. Several animal studies indicated a potentially beneficial role of G-CSF in the prevention of cardiac remodeling after AMI . In particular, CD34  cells could release growth factors activating cardiac stem cells; they also may differentiate into endothelial cells, leading to enhanced neoangiogenesis. Whatever the mechanism involved, it certainly requires CD34  cell homing in the injured tissue. Evaluations of cell trafficking and homing are therefore essential. They may be studied on a gamma camera after radiopharmaceutical cell labeling, but sensitivity and resolution would be much better with positron emission tomography (PET). Nevertheless, the short half-life of PET radiopharmaceuticals allows trafficking studies for only a few hours. We may therefore take advantage of the longer half-life of tracers used for gamma camera imaging to prolong cell detection for 2 to 3 days.

In this study, we tested the feasibility of peripheral-blood CD34  cell isolation without mobilization and applied PET and gamma camera imaging to assess their homing after intracoronary implantation in patients with AMI./ y' L" I# N; X5 N8 p9 t

MATERIALS AND METHODS4 d& J- L8 a4 `7 R

Patients7 i: f: @! x; w: ^

The selection criteria were a first acute anterior myocardial infarction with severe hypokinesis or akinesis of at least two adjacent segments on the baseline left ventricular echocardiogram and the absence of reperfusion within 6 hours after the onset of symptoms. After approval by the local ethics committee, six patients admitted in our institution gave their written informed consent and were included in the study. All patients were treated by aspirin, clopidogrel, statins, beta-blockers, and angiotensin-converting enzyme inhibitors. At the time of CD34  cell implantation, patients were 7 to 21 days after a documented AMI and 4 to 21 days after culprit coronary artery stenting. At 2 to 3 days before implantation, we assessed regional myocardial viability with PET after the injection of 370 MBq of , dividing the left ventricle into 17 segments to produce a wall motion score index.2 q3 D; }7 r' g
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CD34  Cell Enrichment
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Autologous peripheral blood HSCs were obtained after large-volume cytapheresis (15 to 20 L) performed on a Cobe Spectra cell separator. Positive selection of CD34  cells was performed on a Baxter Isolex 300i medical device. Selected cells were maintained at 4¡ãC for approximately 20 hours and then labeled., y$ \7 `# K' k( B! x: U  V

Cell Labeling and Implantation

Two groups of 2 to 4 million CD34  cells were labeled with 74 MBq of FDG and 7.4 MBq of 111In-oxine, respectively, at the concentration of 2 x 106 cells/ml. Cells were subsequently washed in phosphate-buffered saline (PBS) and resuspended in 1 ml PBS containing 5% human albumin. During coronary angiography, labeled and unlabeled stem cells were injected through the central lumen of an over-the-wire balloon catheter (oversized by 0.5 mm) into the culprit coronary artery as described by Assmus et al. . The balloon was inflated inside the stent with low pressure to completely block the blood flow during 3 minutes, and the CD34  cell suspension (2 ml) was infused distally to the occluding balloon through the central catheter port. After a washout with saline, the balloon was deflated for 3 minutes. The maneuver was repeated two to three times, and labeled stem cells were always used in the first infusion. This procedure allows for high-pressure infusions under stop-flow conditions to facilitate the infiltration of the cells into the infarcted zone and to prevent backflow. Enoxiparin 0.5 mg/kg was used during the implantation procedure.! E- X8 Y% o# Q3 V' E$ U& }. U

Homing Studies- q9 F& i) P  w

One hour after implantation, 20-minute 3D PET images were obtained over the chest. For 111In imaging, 20-minute planar images were acquired on a gamma camera 19 hours and 43 hours after implantation. At 19 hours, tomographic acquisition was also performed.

Follow-Up( p' R' p" D* u+ a! g& o- D) f- u4 ?
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Four months after implantation, patients were reevaluated by FDG-PET, echocardiography, and coronary angiography.4 F- y1 V! b. p+ b5 }; p9 o

RESULTS

CD34  Cell Enrichment

A mean of 32.4 x 109 (range, 22 to 40 x 109) mononuclear cells containing less than 0.1% CD34  cells were collected after treating a mean of 3.5 x total blood volume (range, 2.9 to 5.3). After CD34  selection, a mean of 14.9 x 106 (range, 3.6 to 27.6 x 106) cells containing a mean of 30% (range, 14%¨C35%) CD34  cells were obtained for implantation. Major contaminants were CD19  B-lymphocytes (mean, 42%; range, 29%¨C58%); monocytes were less than 1% of all cells.4 R9 `+ a8 X) ~2 `) U5 n: A& v9 j

Implantation and Homing Studies0 y0 a3 B9 E( p
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Mean (¡À standard deviation) labeling efficiency of implanted cells with 111In-oxine and FDG was 65% ¡À 8% and 6% ¡À 1%, respectively., R/ M+ c6 z  v: @8 T. P

The stem cell implantation was well tolerated, without any complications, especially any early restenosis or arrhythmia (Table 1). One hour after implantation, 5.5% ¡À 2.3% of the administered label remained in the myocardium. The major part of the radioactive label was present in the liver (48% ¡À 35%), spleen (29% ¡À 19%), and bone marrow.# M2 @- X' H. r9 n
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Table 1. Clinical characteristics, CD34  cell preparation, PET imaging, and echocardiography findings in six patients with intra-coronary CD34  cell implantation for acute myocardial infarction
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In four patients, detection of myocardial radioactivity was restricted to the vascular territory of the revascularized vessel, more precisely within the borders of the infarcted zone (Fig. 1). In one patient presenting with a large infarction and a no-reflow pattern, injected cells remained in a linear strip at the surface of the myocardium, suggesting a trapping in the left anterior descending artery. Apart from this observation, neither PET nor 111In imaging demonstrated cell homing in the patients¡¯ vasculature, including the aorta.
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Figure 1. First row: successive transverse chest PET slices of FDG uptake testing the regional myocardial viability evaluation (gray scale). FDG uptake is markedly reduced in the antero-apical region of the left ventricle. Second row: superimposed on the PET-FDG slices presented in the first row, FDG-labeled CD34  cell homing is detected 1 hour after intracoronary injection in the hypometabolic infarcted area, except in the central core of the myocardial lesion (orange-to-yellow scale). Cells detected in the postero-median aspect of the chest correspond to CD34  cell homing in thoracic vertebral bone marrow. Abbreviations: FDG, fluorodeoxyglucose; PET, positron emission tomography.( c0 M4 O1 }  M1 v

One patient exhibited cell homing in the middle part of the anteroseptal region; this patient had small septal arteries emerging at the distal extremity of the stent. At 19 hours after implantation, 111In radioactivity was detected in the liver (71% ¡À 10%), spleen (29% ¡À 10%), and bone marrow. Only one of the six patients had detectable, although faint, activity in the myocardium. At 43 hours, the activity¡¯s distribution was similar.

Patient Follow-Up) n) z' \' W) p2 p9 L7 M2 D) W

No restenosis or arrhythmia occurred in any patient. Echocardiography demonstrated enhancement in wall motion score index (p 5 S3 z. x1 q% Q. i' a

DISCUSSION
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We have shown that large amounts of CD34  cells can be obtained from nonmobilized peripheral blood by large-volume cytapheresis followed by positive selection of CD34  cells. The amounts of CD34  cells available for therapeutic administration were in the range of those reported in previous studies . Our study therefore opens the way to clinical studies testing CD34  cell administration without the confounding effects of G-CSF. This study was not designed to assess the clinical efficacy of the cell-therapy protocol proposed. This would require a study on a larger number of patients and with a longer follow-up period. Such a study would also help confirm the inocuity of the procedure in terms of restenosis and arrhythmia occurrence.

Double radioactive labeling allowed for the assessment of cell trafficking and homing of injected cells. The sensitive and high-resolution PET study demonstrated deposition and early homing of CD34  cells in the myocardium surrounding the necrotic area. One patient exhibited cell homing in the middle part of the antero-septal region; as this patient had small septal arteries emerging just after the distal extremity of the stent, we postulate that labeled cell infusion preferentially occurred in these arteries rather than in the left anterior descending artery. As 4%¨C7% of the labeled cells were detected in the myocardium, we can reasonably conclude that CD34  cells constitute a major part of them because other labeled cells were essentially B-lymphocytes, a cell type that is not prone to adhere to, and migrate through, diseased coronary endothelium (monocytes were

CONCLUSION; f3 q: m! j, n

Large amounts of CD34  cells, in the range reported in previous studies, can be obtained from nonmobilized peripheral blood. PET with FDG cell labeling is an efficient imaging method for homing assessment.0 w2 }2 U9 `- O6 N
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ACKNOWLEDGMENTS
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This study was supported by the FNRS and the Fondation pour le Chirurgie Cardiaque (Belgium)./ x- p+ l6 C; q  e  Z! a

DISCLOSURES
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The authors indicate no potential conflicts of interest.
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