Repeated Administrations of Cardiac Progenitor Cells Are Superior to a Single Administration of an Equivalent Cumulative Dose

Figures

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  Figure 1. A, Experimental protocol. BrdU indicates 5‐bromo‐2′‐deoxyuridine; CPCs, c‐kitPOS cardiac progenitor cells; Echo, echocardiogram; first, second, third Rx, first, second, and third treatment; IdU, 5‐iodo‐2′‐deoxyuridine; PBS, Dulbecco's phosphate‐buffered saline; Pre‐Rx, pretreatment (30 days after MI). B, Representative images of echo‐guided injection into the LV cavity. 1, B‐mode short‐axis image showing the LV cavity, the injection needle, the needle tip, and the needle insertion guideline; 2, B‐mode color Doppler image showing the needle tip in the center of the LV cavity before start of injection; 3, B‐mode color Doppler image showing a blue stream of infusate during injection; and 4, B‐mode color Doppler image at the end of the infusion. LV indicates left ventricular.

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  Figure 2. Echocardiographic assessment of LV function. A, Representative B‐mode long‐axis images in systole (left) and M‐mode images (right) obtained before the first treatment (Pre‐Rx) (30 days after MI) and at 35 days after the first (first Rx) and third treatment (third Rx). B, Bar graph illustrating LV EF at baseline (BSL), before the first treatment (Pre‐Rx) (ie, 30 days after myocardial infarction, 35 days after the first treatment (first Rx), 35 days after second treatment (second Rx), and 35 days after the third treatment (third Rx). C through I, Cumulative changes in echocardiographic parameters (absolute units) at 35 days after the first, second, and third treatment vs pretreatment values (Pre‐Rx) values. EF, ejection fraction (C); IWd, infarcted LV wall thickness in diastole (D); IWs, infarcted LV wall thickness in systole (E); IW ThF, infarcted LV wall thickening fraction (F); EDV, end‐diastolic volume (G); ESV, end‐systolic volume (H); SV, stroke volume (I). *P<0.05, **P<0.01 vs vehicle; † P<0.05 vs Pre‐Rx; and § P<0.05 vs Combined dose. Data are means±SEM.

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  Figure 3. Hemodynamic assessment of LV function. Hemodynamic studies were performed with a Millar conductance catheter at 35 days after the third treatment, just before euthanasia. A, Representative pressure‐volume loops recorded during preload manipulation by brief inferior vena cava occlusions. B, Quantitative analysis of hemodynamic variables. EF indicates ejection fraction; EDV, end‐diastolic volume; ESV, end‐systolic volume; EES end‐systolic elastance; LVEDP, left ventricular end‐diastolic pressure; PAMP, preload adjusted maximal power. Data are means±SEM.

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  Figure 4. Morphometric analysis (A and B) and myocardial collagen content (C through E). A, Representative Masson trichrome‐stained myocardial sections. Scar tissue and viable myocardium are identified in blue and red, respectively. B, Quantitative analysis of LV morphometric parameters. The risk region comprises both the border zones and the scarred region. C through E, Representative transmission light (C) and polarized light (D) images of an LV section stained with picrosirius red; note the presence of a transmural infarction. E, Quantitative analysis of polarized light microscopic images showing total collagen content as a percent of the risk and noninfarcted regions. Data are means±SEM. LV indicates left ventricular.

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  Figure 5. Myocardial content of inflammatory cells. A, Representative microscopic images of LV sections stained for CD45 and deoxyribonucleic acid (with 4′,6‐diamidino‐2‐phenylindole [DAPI]; αSA, α‐sarcomeric actin). B, Quantitative analysis of CD45‐positive cells. Values are means±SEM.

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  Figure 6. Analysis of myocyte cross‐sectional area. Myocyte cross‐sectional area and myocyte density were determined in rat hearts stained with wheat germ agglutinin (WGA) (green) and α‐sarcomeric actin (red).; A, Representative epifluorescent microscopic image, sequentially acquired from 18 fields of the infarcted region of a vehicle‐treated rat at a magnification of ×300. Myocytes with round nuclei and clearly defined sarcolemmal borders were selected for analysis of cross‐section area. DAPI indicates 4′,6‐diamidino‐2‐phenylindole. B, Box‐and‐whisker plots showing the quantitative analysis of cardiomyocyte cross‐sectional area in the risk and noninfarcted LV regions of hearts treated with vehicle (n=6, with a total of 536 and 319 cells counted in the risk and noninfarcted region, respectively), combined dose (n=5, 586 and 147 cells, respectively), or repeated doses (n=6, 464 and 162 cells, respectively). Boxes show the median and the first and third quartile (25th and 75th percentile); whiskers show the first and last decile (10th and 90th percentile); dots show the smallest and largest observations. LV indicates left ventricular.

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  Figure 7. Analysis of Y‐chromosomePOS cells (A through C) and proliferation of CPCs (D through G). A, Representative confocal microscopic image acquired from the infarcted region. Green arrowheads indicate Y‐chromosome fluorescent signals (green/cyan) in nuclei. Nuclei are stained with DAPI in blue. Cardiomyocyte (CM) morphology was examined with a confocal transmitted light channel's detector (ChD) in which the pseudocolor selected for myocardial background in the ChD channel was gray white. B, Quantitative analysis of the number of total Y‐chromosomePOS cells and (C) Y‐chromosomePOS matured myocytes at 105 days after CPC transplantation. The risk region comprises both the border zones and the infarcted region. D, Representative confocal microscopic image acquired from the border zone. BrdU (given in the first 35 days after start of treatment) is shown in white and IdU (given on days 70 to 105 after start of treatment) in red. Nuclei are stained with DAPI. Cardiomyocyte (CM) morphology was examined with the confocal transmitted light ChD; the pseudocolor selected for myocardial background in the ChD channel was gray white. E through G, Quantitative analysis of the number of BrdUPOS,…

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  Figure 8. Schematic illustration of different duration of paracrine factor release with single vs multiple cell injections. As proposed previously,1 administration of a single dose of 36×106 CPCs results in a “spike” in myocardial CPC numbers, which then fall precipitously as the cells are cleared from the tissue (A, green line). These changes in CPC numbers are paralleled by similar changes in the myocardial content of paracrine factors secreted by the cells (B, green line). When the 36×106 cells are split into 3 doses of 12×106 each, the peak levels of cells and paracrine factors are lower than with a single dose, but the total period of time during which the tissue is exposed to cells and paracrine factors is much greater (A and B, respectively, 3 green dotted bars vs red dotted bar). According to this paradigm, a longer release of paracrine factors at lower levels is superior to a shorter release at higher levels. A similar paradigm would apply if the factors responsible for the beneficial effects of CPCs are released systemically by CPCs residing in extracardiac tissues rather than by CPCs residing in the heart. CPC indicates cardiac progenitor cells.