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. 2010 Feb 9;121(5):675-83.
doi: 10.1161/CIRCULATIONAHA.109.902221. Epub 2010 Jan 25.

Juvenile exposure to anthracyclines impairs cardiac progenitor cell function and vascularization resulting in greater susceptibility to stress-induced myocardial injury in adult mice

Chengqun Huang  1 , Xiaoxue Zhang, Jennifer M Ramil, Shivaji Rikka, Lucy Kim, Youngil Lee, Natalie A Gude, Patricia A Thistlethwaite, Mark A Sussman, Roberta A Gottlieb, Asa B Gustafsson
Affiliations

Juvenile exposure to anthracyclines impairs cardiac progenitor cell function and vascularization resulting in greater susceptibility to stress-induced myocardial injury in adult mice

Chengqun Huang et al. Circulation. .

Abstract

Background: The anthracycline doxorubicin is an effective chemotherapeutic agent used to treat pediatric cancers but is associated with cardiotoxicity that can manifest many years after the initial exposure. To date, very little is known about the mechanism of this late-onset cardiotoxicity.

Methods and results: To understand this problem, we developed a pediatric model of late-onset doxorubicin-induced cardiotoxicity in which juvenile mice were exposed to doxorubicin, using a cumulative dose that did not induce acute cardiotoxicity. These mice developed normally and had no obvious cardiac abnormalities as adults. However, evaluation of the vasculature revealed that juvenile doxorubicin exposure impaired vascular development, resulting in abnormal vascular architecture in the hearts with less branching and decreased capillary density. Both physiological and pathological stress induced late-onset cardiotoxicity in the adult doxorubicin-treated mice. Moreover, adult mice subjected to myocardial infarction developed rapid heart failure, which correlated with a failure to increase capillary density in the injured area. Progenitor cells participate in regeneration and blood vessel formation after a myocardial infarction, but doxorubicin-treated mice had fewer progenitor cells in the infarct border zone. Interestingly, doxorubicin treatment reduced proliferation and differentiation of the progenitor cells into cells of cardiac lineages.

Conclusions: Our data suggest that anthracycline treatment impairs vascular development as well as progenitor cell function in the young heart, resulting in an adult heart that is more susceptible to stress.

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Figures

Figure 1
Figure 1
Establishment of a juvenile mouse model of late onset anthracycline-mediated cardiotoxicity. A. Mouse pups were injected with saline or doxorubicin (DOX) at 5, 10, 15, and 20 days after birth. B. H & E staining of heart sections prepared from 21 days old mice. C. TUNEL staining of heart sections prepared from 21 days old mice (n=4). D. H & E staining of adult heart sections. E. Electron micrographs of adult hearts from saline and doxorubicin mice.
Figure 2
Figure 2
Assessment of blood flow and vascular structure in adult mouse hearts exposed to juvenile saline or DOX treatments. A. Measurement of blood flow in adult hearts using fluorescent microspheres (n=6). B. Microfil polymer injection to visualize coronary branching in hearts. C. Representative CD31 staining of adult heart sections at Day 60 and quantitation of capillary density at 11, 21, 40 and 60 days after birth (n=4). D. Western analysis of VEGF expression in heart. E. Quantitation of VEGF in heart lysates (n=4).
Figure 3
Figure 3
Juvenile doxorubicin treatment increases susceptibility to exercise in adulthood. A. Swimming for 21 days induces cardiac hypertrophy in doxorubicin-treated mice as measured by the ratio of heart weight (HW) to body weight (BW) (n=9). B. H & E staining of heart sections after swimming. C. H & E staining of DOX heart sections after swimming. D. Masson’s trichrome staining of heart sections after swimming. E. Measurement of ventricular end diastolic pressure (mmHg) in sedentary or swimming mice (n=5-8).
Figure 4
Figure 4
Juvenile doxorubicin treatment increases susceptibility to myocardial infarction in adulthood. A. Kaplan-Meier analysis of survival after myocardial infarction. B. TTC staining of heart sections. C. Infarct size measurement by TTC staining 24 h post MI (n=5). D. Area at risk in saline and DOX hearts. E. Masson’s trichrome staining of heart sections 7 days after myocardial infarction (n=6).
Figure 5
Figure 5
Evaluation of vascular changes in adult hearts after myocardial infarction. A. Representative CD31 staining of heart sections 4 days post-MI and quantitation of capillary density (n=3). B. Representative α-SMA staining of heart sections 4 days post-MI and quantitation of relative fluorescence (RFU, n=3) C. Microfil polymer injection and corrosion casting of hearts 10 days post-MI. Arrow marks the site of ligation. Infarct (IN) and border zone (BZ).
Figure 6
Figure 6
Juvenile doxorubicin exposure results in reduced number of progenitor cells in the border zone (BZ) of an infarct in the adult heart. Hearts sections prepared 4 days after ligation of the LAD were stained with anti-tropomyosin (red) and anti-c-kit (green). Nuclei were counterstained with Hoechst 33342 (blue). A. Dead myocytes in the border zone of an infarct stain negative for tropomyosin. B. Detection of c-kit positive cells in the BZ. C. Quantitation of c-kit+ progenitor cells in the BZ (n=4).
Figure 7
Figure 7
Doxorubicin treatment causes a reduction in c-kit+ progenitor cells in juvenile hearts. A. Mouse pups were injected with saline or 1 mg/kg doxorubicin at 5 and 10 days after birth and heart sections, prepared at 12 days after birth, were stained with anti-c-kit (green), anti-tropomyosin (red), and Hoechst 33342 (blue). B. Quantitation of c-kit+ cells in heart sections (n=3). C. Heart sections from 12 days old mice were stained with anti-c-kit and anti-p16INK4a. Nuclei were counterstained with Hoechst 33342. D. Quantitation of p16INK4a positive progenitor cells in tissue sections (n=3). E. C-kit+ progenitor cells isolated from saline or doxorubicin injected mice at Day 12 had reduced BrdU incorporation in vitro (n=4).
Figure 8
Figure 8
Doxorubicin treatment inhibits proliferation of isolated CPCs. Cells were treated with doxorubicin for 72 h and then assessed for (A) cell proliferation by MTT assay (n=3), and (B) cell death by Trypan Blue exclusion assay (n=3). C. Cells were treated with 100 nM doxorubicin and proliferation was determined by BrdU incorporation (n=4). D. Cells were exposed to vehicle or 100 nM DOX for 72 h or 96 h and then assayed for telomerase activity using the TRAP assay (n=3). E. Isolated CPCs were treated with 100 nM DOX for 72 h and then fixed and stained for the presence of c-kit and p16INK4a. F. Quantitation of p16INK4a positive cells (n=4).
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