Intracoronary administration of cardiac progenitor cells alleviates left ventricular dysfunction in rats with a 30-day-old infarction

Abstract

Background: Administration of cardiac progenitor cells (CPCs) 4 hours after reperfusion ameliorates left ventricular function in rats with acute myocardial infarction (MI). Clinically, however, this approach is not feasible, because expansion of autologous CPCs after acute MI requires several weeks. Therefore, we sought to determine whether CPCs are beneficial in the more clinically relevant setting of an old MI (scar).

Methods and results: One month after coronary occlusion/reperfusion, rats received an intracoronary infusion of vehicle or enhanced green fluorescent protein-labeled CPCs. Thirty-five days later, CPC-treated rats exhibited more viable myocardium in the risk region, less fibrosis in the noninfarcted region, and improved left ventricular function. Cells that stained positive for enhanced green fluorescent protein that expressed cardiomyocyte, endothelial, and vascular smooth muscle cell markers were observed only in 7 of 17 treated rats and occupied only 2.6% and 1.1% of the risk and noninfarcted regions, respectively. Transplantation of CPCs was associated with increased proliferation and expression of cardiac proteins by endogenous CPCs.

Conclusions: Intracoronary administration of CPCs in the setting of an old MI produces beneficial structural and functional effects. Although exogenous CPCs can differentiate into new cardiac cells, this mechanism is not sufficient to explain the benefits, which suggests paracrine effects; among these, the present data identify activation of endogenous CPCs. This is the first report that CPCs are beneficial in the setting of an old MI when given by intracoronary infusion, the most widely applicable therapeutic approach in patients. Furthermore, this is the first evidence that exogenous CPC administration activates endogenous CPCs. These results open the door to new therapeutic applications for the use of autologous CPCs in patients with old MI and chronic ischemic cardiomyopathy.

Figure 1. Echocardiographic assessment of LV function

(A) Representative M-mode echocardiographic images from a vehicle-treated and a CPC-treated rat recorded at baseline (BSL), at 30 d after MI (before vehicle or CPC treatment) (MI), and at 35 d after treatment (35 d). (B) Quantitative echocardiographic parameters including systolic thickness of the anterior (infarcted) wall, thickening fraction in the anterior (infarcted) wall, LV fractional shortening, and LV ejection fraction. Data are means ± SEM.

Figure 2. Hemodynamic assessment of LV function at 35 d after treatment

(A) Representative pressure-volume loops from a normal, a vehicle-treated, and a CPC-treated rat recorded during preload manipulation by a brief period of inferior vena cava occlusion. (B) Quantitative analysis of hemodynamic variables including LV end-diastolic pressure, dP/dt, ejection fraction, end-systolic elastance (E_es), preload-adjusted maximal power, and preload-recruitable stroke work. Data are means ± SEM.

Figure 3. Morphometric analysis

(A) Representative Masson’s trichrome-stained myocardial sections from a vehicle-treated and a CPC-treated rat. Scar tissue and viable myocardium are identified in blue and red, respectively. (B) Quantitative analysis of LV morphometric parameters (for definition, see supplemental Fig. 11). Data are means ± SEM.

Figure 4. Myocardial collagen content

Representative microscopic images of a normal, a vehicle-treated, and a CPC-treated heart obtained using regular (A) and polarized (B) light. (C) Collagen content expressed as percent of total area in the scarred and noninfarcted region. LV sections were stained with picrosirius red and collagen content was quantitated under polarized light. Data are means ± SEM.

Figure 5. Myocardial content and differentiation of transplanted CPCs

(A) Representative confocal microscopic images from a CPC-treated rat showing presence of transplanted CPCs in the risk (infarcted) and noninfarcted regions, as evinced from immunoreactivity for EGFP (green). Some EGFP^pos cells also express α-sarcomeric actin (red). (B) Quantitation of EGFP^pos cells in the risk and noninfarcted region, expressed as percent EGFP^pos myocardial area and as total calculated number of EGFP^pos cells/heart. The number of EGFP^pos cells/heart was estimated by multiplying the number of EGFP^pos cells per unit area by the estimated number of EGFP^pos cells present through the thickness of each slice in which EGFP^pos cells were found (estimated 100 cells per 2-mm thickness of slice). (C) Representative confocal microscopic images showing colocalization of EGFP and α-sarcomeric actin in several cells in the border zone (the area in the white box is magnified in the three panels on the right). Yellow arrows indicate EGFP^pos cells that do not expressα-sarcomeric actin. (D) Quantitative analysis of α-sarcomeric actin^pos/EGFP^pos cells. Data are means ± SEM.

Figure 6. Proliferation of transplanted CPCs

(A) Representative confocal microscopic images from a CPC-treated rat showing BrdU uptake in the infarcted region during the last 2 weeks of life. Yellow arrows indicate EGFP^pos cells that are BrdU positive; white arrowheads EGFP^pos cells that are BrdU negative; and white arrows EGFP^neg cells that are BrdU positive. (B, C, and D) Quantitative analysis of BrdU^pos cells in the risk and noninfarcted regions. Panel B shows the number of BrdU^pos cells in vehicle-treated and CPC-treated groups; panel C shows the same data except that the CPC-treated group is subdivided into the subgroups that did or did not exhibit EGFP^pos cells (in the seven hearts with EGFP^pos cells, BrdU^pos cells were counted in the entire region examined, both in the EGFP^pos and in the EGFP^neg areas). (D) Colocalization of BrdU and EGFP immunoreactivity in the seven CPC-treated hearts that exhibited EGFP^pos cells. Data are means ± SEM.

Figure 7. Effect of CPC transplantation on c-kit^pos cells

(A) Representative confocal microscopic images from a vehicle- treated and a CPC-treated rat showing c-kit^pos cells (red) and BrdU^pos cells (green) in the risk region. Yellow arrows indicate c-kit^pos cells that are BrdU positive; white arrows c-kit^neg cells that are BrdU positive; (B) Quantitative analysis of c-kit^pos cells and double positive (c-kit^pos/BrdU^pos) cells in normal, vehicle-treated, and CPC-treated hearts (the last group is subdivided into two subgroups, that with EGFP^pos cells [EGFP^pos] and that without EGFP^pos cells [EGFP^neg]). (C) Quantitative analysis of c-kit^pos cells in risk and noninfarcted regions in normal, vehicle-treated, and CPC-treated hearts [as in (B), the last group is subdivided into EGFP^pos and EGFP^neg subgroups]. (D) Quantitative analysis of double positive (c-kit^pos/BrdU^pos) cells expressed as a percent of all c-kit^pos cells. (E) Correlation between the number of c-kit^pos cells and the percent of viable myocardium in the risk region (r=0.59, P<0.01). Data are means ± SEM.

Figure 8. Cardiac commitment of endogenous CPCs after trasplantation of exogenous CPCs

The commitment of endogenous CPCs (c-kit^pos/EGFP^neg cells) to cardiac lineage was assessed by quantitating endogenous CPCs that expressed Nkx2.5 and MHC in vehicle- and CPC-treated hearts. New myocytes formed in the last 2 weeks of life were identified by measuring α-sarcomeric actin^pos and BrdU^pos cells. (A) Representative confocal microscopic image of a c-kit^pos/EGFP^neg/Nkx2.5^pos/MHC^pos cell obtained in serial sections of a CPC-treated heart. (B) Quantitative analysis of c-kit^pos/EGFP^neg/Nkx2.5^pos/MHC^pos cells in the risk and noninfarcted regions of vehicle- and CPC-treated hearts (the CPC-treated group is subdivided into two subgroups, one with EGFP^pos cells [EGFP^pos] and one without EGFP^pos cells [EGFP^neg]). The number of c-kit^pos/EGFP^neg/Nkx2.5^pos/MHC^pos cells is normalized to both number of cells (10⁴ nuclei, upper panels) and area (mm², lower panels). Because cell density in CPC-treated, EGFP^pos hearts was higher (due to presence of clusters of EGFP^pos cells), the magnitude of the increase in endogenous CPCs in CPC-treated, EGFP^pos hearts was greater when the number of cells was normalized to area (mm²) than to number of nuclei. (C) Representative confocal microscopic image of α-sarcomeric actin^pos/BrdU^pos cells in a CPC-treated heart. (D) Quantitative analysis of the number of α-sarcomeric actin^pos/BrdU^pos cells in the risk and noninfarcted regions of vehicle- and CPC-treated hearts (subdivided into EGFP^neg and EGFP^pos subgroups). Data are means ± SEM.