Cardioprotective c-kit+
cells are from the bone marrow and regulate the myocardial balance of angiogenic cytokines ([original](https://doi.org/10.1172%2FJCI27019)) ([raw](?raw))c-kit+ cells increase in infarcted myocardium. The kinetics of EPC distribution after MI was assessed after coronary ligation in wild-type mice. Fibronectin-adherent endothelial-like cell numbers increased in both the bone marrow and the peripheral circulation within the first week, with a rapid decline thereafter (Figure 1A). As anticipated, the dynamics of VEGFR2+ cell redistribution in the bone marrow and the PBMCs closely matched these data (Figure 1A). Cell surface phenotyping of the VEGFR2+ cells showed that more than 95% of VEGFR2+ cells also expressed Sca-1 and c-kit (Figure 1B). Thus, coronary ligation leads to a robust increase in the number of peripheral blood VEGFR2+c-kit+Sca-1+ cells, a phenotype that is consistent with previously reported phenotype of EPCs.
c-kit+ cells increase in infarcted myocardium. (A) Quantification of EPCs over a time course after MI in wild-type mice. Upperpanels, flow cytometry for VEGFR2+ cells. Lower panels, in vitro splenocyte fibronectin (Fib.) adhesion, acetylated LDL uptake (Ac-LDL), and lectin-binding assay results. Total number of cells was calculated by multiplying percentage of positive cells by total number of cells isolated from both tibia and femur of 1 mouse. n = 3–5 per time point. *P < 0.05 compared to day 0 values. (B) MI causes an increase in VEGFR2+c-kit+Sca-1+ subset of PBMCs. (C) Increase of c-kit+ cells is specific to the injured myocardium (arrowhead). Seven days after MI, c-kit+ cells were not detected in other organs and the peripheral circulation. Gray, isotype control; red, c-kit. (D and E) The c-kit+ cells detected after MI in the heart were homogeneously VEGFR2+ but heterogeneous with respect to CD45 expression. –ve con, negative control. (F) Quantification of the number of c-kit+ cells. Lowerpanel shows that the majority of the c-kit+ cells in the heart were CD45–. n = 3 per time point. (G–J) Confocal microscopic images. (G) c-kit+ cells visualized at the infarct border zone both as isolated cells (arrowheads) and in clusters (arrow). Scale bar: 100 μm. (H) The majority of the c-kit+ cells did not express CD45 (arrowhead) although some of the clusters contained CD45-expressing cells (arrow). Scale bar: 50 μm. (I) c-kit+ cells present in the clusters are shown to express Ki67 cell cycling–associated nuclear antigen. Scale bar: 10 μm. (J) c-kit+ cells in the cell cycle (arrowhead) did not coexpress CD45 (arrow). Scale bar: 10 μm.
After MI, c-kit+ cells could readily be detected in the myocardium that was subtended by the ligated coronary artery (Figure 1, C–F). Seven days after MI, c-kit+ cells could not be detected in noninjured organs: lung, liver, kidney, and the peripheral blood (Figure 1C). Of the c-kit+ cells in infarcted myocardium, more than 95% expressed VEGFR2 (Figure 1D), but only 30% expressed CD45 (Figure 1, E and F). The CD45-expressing cells had 1.2 ± 0.2 fluorescent log units lower CD45 levels than c-kit+ cells in the bone marrow (P <0.001). This finding was independent of the treatment required to release single cells from the heart for flow cytometry (Supplemental Figure 1; supplemental material available online with this article; doi:10.1172/JCI27019DS1). Whether this phenomenon represented preferential proliferation or recruitment of CD45– cells or CD45 downregulation by CD45+ cells is not clear.
Microscopy extended observations made by flow cytometry. The c-kit+ cells were rarely detected in the heart prior to MI (2 cells in 8 coronal sections from 4 mice). Visualization of the c-kit+ cells in infarcted myocardium showed cells in isolation and in clusters (Figure 1G). Within the clusters, 85% of the c-kit+ cells did not express CD45 (Figure 1H). Nearly 10% of the c-kit+ cells in the clusters also expressed the cell cycle–associated antigen Ki67 (Figure 1I). Of the 26 c-kit+ clusters comprising 134 c-kit+ cells, 21 c-kit+CD45+ cells were scanned, none of which expressed Ki67 cycling antigen (Figure 1J). This data is consistent with the notion that there is preferential proliferation of c-kit+ CD45– cells. None of the c-kit+ cells within the clusters showed metachromasia with toluidine blue staining or cytoplasmic granular staining with avidin, suggesting that the c-kit+ cells within the c-kit+ clusters are not mast cells. In fact, toluidine blue staining of infarcted myocardium 7 days after coronary ligation showed that at most 9.6 ± 0.7 mast cells were present per 8 μm coronal myocardial section (Supplemental Figure 2). Thus, MI leads to an increase in c-kit+ VEGFR2+ cells, first in the blood and then specifically within the infarcted myocardium, and the majority of these c-kit+ cells are not mast cells.
c-kit+ cells in infarcted myocardium are from the bone marrow. To address whether the c-kit+ cells in infarcted myocardium are from the bone marrow, we engineered GFP bone marrow chimeric mice to track GFP-expressing bone marrow cells. After stable reconstitution, 69% ± 0.4% of bone marrow cells, 94% ± 0.7% of PBMCs, 84% ± 4% of peripheral EPCs (Figure 2A), 70% ± 6% of PBMC VEGFR2+ cells, and 74% ± 1.2% of c-kit+ cells in the bone marrow expressed GFP (Figure 2B and Table 1). Control C57BL/6 mice did not have GFP-expressing cells in the bone marrow, blood, spleen, or heart. Seven days after MI, c-kit+ cells could be identified in the hearts of C57BL/6 mice, and none expressed GFP. In the GFP chimeric mouse, 74% ± 0.6% of the c-kit+ cells in the heart expressed GFP (Figure 1B and Table 1). Confocal microscopy confirmed that c-kit+ cells in the heart also had GFP expression in the chimeric mice (Figure 2C). Of 7 mast cells identified in sections from 2 mice by avidin staining, none showed GFP staining. Thus, the majority of the non–mast cell c-kit+ cells that infiltrate the infarcted heart are from the bone marrow.
c-kit+ cells in infarcted myocardium are from the bone marrow. (A) EPCs from bone marrow chimeric mice carry the GFP transgene. (B) Dual-colored flow cytometry of C57BL/6 (C57) or C57BL/6-GFP bone marrow chimeric mice (C57-GFP) for GFP on the x axis and c-kit on the y axis. In the bone marrow preparation, 74% of c-kit+ cells in the C57-GFP chimeric mice expressed GFP; 74% of c-kit+ cells in the infarcted myocardium in the C57-GFP chimeric mice also expressed GFP. Representative flow cytometry data from 5 independent experiments is shown with results summarized in Table 1. Iso. con, isotype control (C) Confocal micrograph confirming that c-kit+ cells in infarcted myocardium also expressed GFP in chimeric mice (arrow). A GFP+ cell that did not express c-kit (arrowhead) is also visualized in this micrograph. Scale bar: 50 μm. (D) Engraftment of bone marrow–derived cells was minimal when evaluated at 28 days after MI in the bone marrow chimeric mice. Scale bar: 100 μm.
c-kit+ cells in infarcted myocardium are from the bone marrow
Analysis of 28-day engraftment of bone marrow cells in infarcted myocardium in 4 GFP chimeric mice showed that 30 cells of total 3029 nuclei expressed GFP within the infarcted area and the surrounding border zone. None of these GFP+ cells were cardiomyocytes (Figure 2D). Thus, although bone marrow c-kit+ cells traffic to the infarcted heart, they do not significantly engraft over the long term or give rise to new cardiomyocytes in our current model.
c-kit dysfunction is associated with dilated cardiomyopathy after MI. To examine the functional importance of c-kit–expressing cells in cardiac repair, we examined c-kit mutant _KitW/Kit_W–v mice and their congenic Kit+/+ wild-type littermates. Coronary ligation excluded a similar volume of myocardium from the circulation immediately after ligation (Figure 3A). Similarly, at 24 hours, no difference in the volume of infarcted myocardium was present (Figure 3A). In spite of the similarity within the first 24 hours, _KitW/Kit_W–v mice had a 2-fold increase in mortality compared with Kit+/+ mice (Figure 3B). Serial echocardiography demonstrated that cardiac systolic function was reduced and the left ventricle was dilated in _KitW/Kit_W–v mice (Figure 3C), with nearly maximal differences emerging within 14 days. Invasive pressure-volume measurements (Figure 3D) showed greater end-systolic and end-diastolic volumes and lower ejection fraction in _KitW/Kit_W–v mice. Reduced first derivative of pressure during isovolumic contraction (dP/dt max) and relaxation (dP/dt min) coupled with prolonged time constant of isovolumic relaxation (t) suggested abnormalities in myocardial energetics in _KitW/Kit_W–v mice (see Supplemental Table 1 for detailed invasive hemodynamic measurements). Both end-systolic elastance (Ees) and preload recruitable stroke work (PRSW) analysis demonstrated a marked rightward shift of the volume intercept in _KitW/Kit_W–v mice (Ees: 2.1 ± 5 μl versus 20.3 ± 5 μl, P =0.036; PRSW: 14.0 ± 3 μl versus 26.0 ± 4 μl, P =0.03), suggesting that the worse cardiac function in _KitW/Kit_W–v mice was to a large degree because of exaggerated left ventricular dilation after MI.
c-kit dysfunction is associated with dilated cardiomyopathy after MI. (A)Coronary ligation results in a similar volume of myocardium being at risk for infarction and a similar volume becoming necrotic within 24 hours. n =3–4 per group. (B) Actuarial survival is worse in the _KitW/Kit_W–v mice after coronary ligation. n =86 per group. (C) Echocardiography shows rapid decline in cardiac systolic function (fractional area contraction) and rapid ventricular dilation (left ventricular end diastolic diameter in the mutant mice). n =5 per group. *P <0.05. (D) Representative pressure-volume loops of uninfarcted (D0) and infarcted (day 14, D14) Kit+/+ and _KitW/Kit_W–v mice. n =5 per group. Volume is indicated on the x axis and pressure on the y axis. (E) Representative perfusion-fixed hearts before (D0) and 6 weeks after (D42) coronary ligation. Note the ventricular dilation in the _KitW/Kit_W–v mouse. n =5 per group. (F) Representative H&E-stained mid-papillary transverse myocardial sections depicting the pronounced ventricular dilation and larger infarct size in the _KitW/Kit_W–v mice. n =5 per group.
Computerized planimetry of explanted hearts fixed at physiologic pressures at 6 weeks also showed 1.8-fold greater left ventricular dilation mainly because of a 2.8-fold greater scar surface area in the _KitW/Kit_W–v mice (Figure 3, E and F). The heart to body weight ratio was also 1.2-fold higher in the _KitW/Kit_W–v mice, suggesting greater myocyte hypertrophy, which was confirmed by histologic evaluation of cardiomyocyte cross sectional diameter (see Supplemental Table 2 for detailed histomorphometric measurements). These measurements are consistent with end-stage cardiac failure in the _KitW/Kit_W–v mice after MI. Interestingly, nonischemic wound healing in the ear was not affected in _KitW/Kit_W–v mice (Supplemental Figure 3). Taken together, these data indicate abnormalities in ischemic cardiac repair but not in generalized wound healing in the KitW/KitW–v mice.
Acute c-kit dysfunction recapitulates the cardiomyopathic phenotype. To ascertain whether the _KitW/Kit_W–v cardiomyopathic phenotype is independent of its anemia, we examined the impact of acute c-kit inhibition in mice treated with Gleevec and in mice rendered acutely anemic by normovolumic hemodilution. Hemodilution nonsignificantly reduced dP/dt max and ejection fraction but did not contribute to ventricular dilation. Treatment with Gleevec markedly reduced dP/dt max and ejection fraction and led to rapid ventricular dilation after MI. Gleevec also reduced myocardial VEGF and proliferating cell nuclear antigen (PCNA) levels (Supplemental Figure 4). Thus, acute inhibition of c-kit partially recapitulates the functional defects seen in the KitW/KitW–v mice.
c-kit dysfunction causes hyporesponsiveness in c-kit+ cells. To confirm the attenuated c-kit function in _KitW/Kit_W–v mice in vitro, we harvested bone marrow cells from _KitW/Kit_W–v and Kit+/+ mice and cultured them in the absence or presence of SCF. Whereas the Kit+/+ cell number and size increased over the 7 days in the presence of SCF, the _KitW/Kit_W–v bone marrow cells showed neither an increase in cellularity nor an increase in cell size (Figure 4A). Consistent with the in vitro data, although MI increased myocardial SCF expression in both groups of mice (Figure 4B), the few c-kit+ cells detected in the _KitW/Kit_W–v did not express the Ki67 cycling antigen as frequently as those in wild-type mice (Figure 4C).
c-kit dysfunction increases apoptosis and decreases mitogenesis. (A) Incubation of bone marrow cells from Kit+/+ mice with 50 ng/ml of recombinant SCF causes cell proliferation. _KitW/Kit_W–v bone marrow cells had no response to SCF. (B) After coronary ligation, myocardial SCF levels increased in both Kit+/+ and _KitW/Kit_W–v mice. Representative immunoblot of 5 independent experiments is shown. (C) Consistent with the in vitro data, the recruited c-kit+ cells in _KitW/Kit_W–v mice had lower index of proliferation as assessed by Ki67 and c-kit staining and visualized by confocal microscopy of 10 random ×400 fields. n =3 per group. *P <0.05. (D) The total number of c-kit+ cells in _KitW/Kit_W–v mice was lower than in Kit+/+ mice. (E) Quantification of the total number of c-kit–expressing cells in the infarcted myocardium. n =3 per time point per group. (F) The Kit+/+ mice had more CD45– c-kit+ cells than _KitW/Kit_W–v mice. n =3 per group. (G) Nonmyocyte mitogenesis was markedly higher in Kit+/+ mice in the infarct border zone. (H) Quantification of general mitogenesis by region and over a time course. n =3 per time point per group. #P <0.01. BZ, border zone. (I) The number of infiltrating c-kit–expressing cells correlated with the number of cycling cells (r =0.78). (J) Differences in apoptotic cell death as quantified in K were smaller than differences in mitogenesis. n =3 per time point per group. *P <0.05 versus Kit+/+. Magnification, ×200.
c-kit dysfunction increases apoptosis and decreases mitogenesis. Next, to understand the mechanism of cardiac failure in _KitW/Kit_W–v mice, we evaluated surrogate measures of cellular proliferation and apoptosis. Nearly 15% of nuclei in wild-type mice expressed Ki67 in the infarct border zone on day 7 after MI (Figure 4, G and H). Previous studies have suggested that the majority of the cycling cells in infarcted myocardium are cardiac endothelial cells and myofibroblasts but not the recruited leukocytes (22). Interestingly, we observed that the percentage of cells that expressed the cycling antigen Ki67 in the border zone and the remote myocardium was significantly diminished in the _KitW/Kit_W–v mice (Figure 4, G and H). At most, only 0.14% of cardiomyocytes coexpressed Ki67 in Kit+/+ mice, ruling out the possibility that absent cardiomyocyte regeneration played a potential role in the cardiomyopathic phenotype of _KitW/Kit_W–v mice. Plotting the number of infiltrating c-kit+ cells against the number of cells expressing Ki67 in the border zone showed a linear correlation (Figure 4I). Evaluation of the rate of apoptotic cell death by a TUNEL assay also showed a difference at day 7, with a lower apoptosis rate in the _Kit+/+_mice (Figure 4, J and K). Thus, greater c-kit+ cell infiltration was associated with increased mitogenesis and decreased apoptosis in particular in the infarct border zone 7 days after MI.
c-kit dysfunction is associated with abnormal EPC mobilization and function. To trace back the diminished c-kit+ cell infiltration in the _KitW/Kit_W–v mice to the bone marrow, we evaluated the impact of MI on mobilization of c-kit+ cells into the peripheral circulation. We used 3 independent assays that included quantification of the number of hematopoietic progenitor cells by a semisolid methylcellulose CFU assay, quantification of the number of VEGFR2+ cells by flow cytometry, and quantification of the number of EPCs by the fibronectin adhesion assay. MI in Kit+/+ mice rapidly increased the number of circulating VEGFR2+ cells, circulating EPCs, and circulating hematopoietic CFCs (Figure 5, A–C). In _KitW/Kit_W–v mice, no increase in the number of such cells was observed (P <0.05 for all 3 experiments) (Figure 5, A–C).
c-kit dysfunction is associated with abnormal EPC mobilization and function. (A–C) c-kit function is required for the mobilization of hematopoietic progenitor cells (HPC), VEGFR2+ PBMCs, and EPCs after MI (n =5 per group). **P <0.05 versus Day 0 values; #P < 0.05 versus Kit+/+. (D) RT-PCR reaction for VEGF, angiopoietin-1 (Ang-1), and angiopoietin-2 from bone marrow cells of Kit+/+ or _KitW/Kit_W–v mice cultured for 7 days in the absence or presence of recombinant SCF. SCF led to marked-up regulation of VEGF mRNA only in _Kit+/+_mice. Angiopoietin-2 levels were higher in Kit+/+ mice. (E–G) Quantification of VEGF by ELISA and angiopoietin-2 and angiopoietin-1 levels by immunoblotting and densitometry from cell supernatant described above. SCF caused increased VEGF and higher angiopoietin-2/angiopoietin-1 ratio only in Kit+/+ mice. The values are from 3 independent experiments quantified in triplicate. **P <0.05 versus no SCF values; #P <0.05 versus Kit+/+.
To test the c-kit–dependent proangiogenic functionality of bone marrow cells isolated from Kit+/+ and _KitW/Kit_W–v mice, we cultured whole bone marrow cells from the 2 types of mice in the presence or absence of SCF. In Kit+/+ cells, coincubation with SCF resulted in marked upregulation of the VEGF mRNA (Figure 5D). Angiopoietin-1 was expressed by both groups and was not induced by incubation with SCF (Figure 5D). Angiopoietin-2 was expressed at higher levels in the Kit+/+ bone marrow cells (Figure 5D). Enzyme-linked immunosorbent measurement of VEGF levels in the supernatant confirmed that Kit+/+ cells incubated with SCF produced highest levels of VEGF (Figure 5E). Immunoblotting also showed greater angiopoietin-2 production in the Kit+/+ and greater angiopoietin-1 production in the _KitW/Kit_W–v bone marrow cells (Figure 5, F and G). Thus, proangiogenic cytokine oscillations may be induced by SCF in bone marrow cells only if the c-kit receptor is functional, and c-kit functionality is critical for the mobilization of c-kit+ cells to the blood and heart after MI.
c-kit dysfunction limits myocardial angiogenesis and formation of repair tissue. We next examined the balance of angiogenic cytokines within the infarcted heart to evaluate the influence of bone marrow c-kit+ cell mobilization on endothelial mitogenesis and angiogenesis. Seven days after MI, Kit+/+ hearts had an upregulation of VEGF that was principally immunolocalized to the border zone (Figure 6, A–C). Concomitantly, myocardial levels of angiopoietin-2 were increased relative to angiopoietin-1 (Figure 6, D–F). In the _KitW/Kit_W–v mice, myocardial VEGF levels were high at baseline, but there was no further increase after coronary ligation (Figure 6A), and no targeting of VEGF to the infarct border zone was evident (Figure 6, B and C). In contrast to Kit+/+ mice, which had a pattern similar to that of angiopoietin expression in other studies (23), the angiopoietin-2 levels were decreased relative to angiopoietin-1 in the _KitW/Kit_W–v mice (Figure 6, D–F). Thus, reversal of angiopoietin ratios in favor of angiopoietin-2, which is necessary for release of the endothelial cells from quiescence (24), and the microenvironmental increase in border-zone VEGF (25) were both absent in KitW/KitW–v mice.
c-kit dysfunction limits myocardial angiogenesis. (A) VEGF upregulation by total heart ELISA following MI is abrogated in _KitW/Kit_W–v mice. n =5 per group. (B) The VEGF in the _KitW/Kit_W–v mouse myocardium is diffusely present and is not localized to the border zone, as quantified in C. n =4 per group. (C) Integrated density value determined by random sampling in 3 ×400 fields per animal. n =4 per group. (D and E) Kit+/+ responds to MI by increasing the ratio of angiopoietin-2 to angiopoietin-1 whereas _KitW/Kit_W–v responds in the opposite fashion. Representative immunoblot is shown. Data are quantified by immunoblotting and densitometry from 4 independent experiments in triplicates. (F) Angiopoietin-2/angiopoietin-1 ratio. **P <0.05 versus day 0 (D0) values; #P <0.05 versus Kit+/+. (G) Number of endothelial cells (blue is CD31) in the cell cycle (red is Ki67) was quantified using confocal microscopy (actin is green) in 5 random ×400 fields in the border zone. n =3 per group per time point. Number of cycling endothelial cells, which appear magenta in color because of overlap of blue CD31 and red Ki67 staining, was higher in the Kit+/+ mice. *P <0.05. hpf, high-power field. (H) Blood vessel density was assessed by CD31 immunohistochemistry in the border zone. (I) Quantification of the number of CD31+ structures from 5 random ×400 fields converted to mm2 showing diminished angiogenic response in _KitW/Kit_W–v mice. ##P <0.01. (J) Blood vessel size quantification showing the _KitW/Kit_W–v mice vessels to be fewer and of larger caliber. n =5 per group.
Considering the proangiogenic milieu established in the Kit+/+ border zone compared with the _KitW/Kit_W–v mice, we evaluated endothelial mitogenesis and found that a greater number of endothelial cells appeared to be in the cell cycle in the Kit+/+ than the _KitW/Kit_W–v mice by double immunofluorescence quantification of CD31+ cells that expressed Ki67 in their nuclei (Figure 6G). Accordingly, after a day 1 nadir following ligation, the number of border-zone blood vessels was increased in the Kit+/+ mice as compared with the _KitW/Kit_W–v group (Figure 6, H and I). Morphometry demonstrated that the Kit+/+ blood vessels in the border zone were smaller in caliber and more similar to capillaries, suggestive of sprouting angiogenesis, in contrast to the markedly enlarged and fewer blood vessels in the _KitW/Kit_W–v mice (Figure 6J).
The onset of angiogenesis was closely correlated with repopulation of the infarcted segment with α-SMA–expressing myofibroblasts in Kit+/+ mice (Figure 7, A–C). Specifically, the percentage of the scar occupied by α-SMA–expressing cells was 30.1% ± 4.1% in Kit+/+ mice compared with 16.1% ± 5.1% in the _KitW/Kit_W–v mice (P <0.01) (Figure 7D) 7 days after MI. Thus, the formation of granulation tissue composed of myofibroblasts and blood vessels, which is a critical aspect of cardiac repair after MI, was significantly attenuated when c-kit was dysfunctional.
c-kit dysfunction limits the formation of repair tissue. (A) Representative composite images constructed from 15–20 ×10 magnification images demonstrates less α-SMA per infarct area in _KitW/KitW–v_7 days after MI when compared with _Kit+/+_mice. (B) Higher magnification images of the border zone and scar area demonstrate that the majority of α-SMA–positive cells are localized in the scar region. (C) Quantitative morphometrical analysis shows a significant difference between the strains. n = 3 per group per time point. **P < 0.05versus day 0 values; #P < 0.01 versus Kit+/+.
Bone marrow rescue also rescues the cardiomyopathic phenotype. Lastly, given that the majority of the c-kit+ cells in the heart appeared to be from the bone marrow in our chimeric experiments, we evaluated whether the cardiomyopathic phenotype of the c-kit mutant could be rescued solely by bone marrow rescue of the mutant mice with wild-type bone marrow cells. Chimeric _KitW/Kit_W–v mice were engineered whose bone marrow cells were of Kit+/+ origin. As a control for the irradiation conditioning protocol used for bone marrow replacement, a subgroup of Kit+/+ mice were also irradiated and reinjected with Kit+/+ bone marrow cells. Two weeks after coronary ligation, transthoracic echocardiography and invasive hemodynamic measurements confirmed that bone marrow rescue led to the rescue of the dilated cardiomyopathic phenotype in the KitW/Kit_W–v mice. We observed near complete restoration of systolic function and prevention of exaggerated ventricular dilation (Figure 8, A–C). No significant impact of the irradiation protocol was evident in the Kit+/+→_Kit+/+bone marrow transplantation.
Bone marrow rescue also rescues the cardiomyopathic phenotype. (A) Representative M-mode echocardiographic images in Kit+/+, _KitW/Kit_W–v, and _KitW/Kit_W–v mice whose bone marrow was rescued by Kit+/+ bone marrow after lethal irradiation (Kit+/+ → _KitW/Kit_W–v) and Kit+/+ mice who received the same dose of irradiation and whose bone marrow was reconstituted from other Kit+/+ mice (Kit+/+ → Kit+/+), showing the prevention of ventricular dilation in Kit+/+ → _KitW/Kit_W–v mice. (B) Quantification of echocardiographic parameters: left ventricular end-diastolic diameter (LVEDD), left ventricular end-systolic diameter (LVESD), fractional shortening (FS), and fractional area contraction (FAC). n =5 per group. Bone marrow rescue prevented ventricular dilation and better preserved ventricular systolic function. (C) Invasive hemodynamic measurements 2 weeks after coronary ligation. Bone marrow rescue also rescued parameters of systolic function, such as ejection fraction and dP/dt maximum (max), and prevented ventricular dilation, but did not affect dP/dt minimum (min). n =5–7 per group. *P <0.05 versus Kit+/+; **P <0.05 versus _KitW/Kit_W–v. (D) Bone marrow rescue results in higher myocardial VEGF levels and greater cell cycle activity (PCNA expression). The immunoblots are representative of 4 independent experiments. (E) Quantification of myocardial VEGF from 4 independent experiments performed in triplicate. (F) Bone marrow rescue increases border-zone blood vessels. n =5–7 per group. *P <0.05 versus _KitW/Kit_W–v (E and F).(G) Recruitment of c-kit+ cells from the bone marrow to the injured region of the heart is cardioprotective because it regulates the myocardial balance of angiogenic cytokines.
Furthermore, bone marrow rescue increased myocardial VEGF levels to 1.3-fold above those of nonrescued _KitW/Kit_W–v mice (P =0.01) (Figure 8, D and E). Similarly, myocardial PCNA expression increased 1.2-fold above that of nonrescued _KitW/Kit_W–v mice. Blood vessel count in the infarct border zone showed that bone marrow rescue also resulted in a 1.4-fold increase in the number of blood vessels in the _KitW/Kit_W–v border zone 7 days after MI (P <0.01) (Figure 8F). Thus, reconstitution of _KitW/Kit_W–v bone marrow with Kit+/+ bone marrow rescued the _KitW/Kit_W–v cardiomyopathic phenotype at the biochemical, histological, morphometric, and fuctional levels.