The authors have declared that no competing interests exist.
Scientific and editorial support: DE FT RR DS JY SW KL. Conceived and designed the experiments: WGH NK. Performed the experiments: NK WGH. Analyzed the data: DME RBS RR RD SW KL WGH. Contributed reagents/materials/analysis tools: NK WGH. Wrote the paper: DME RBS WGH.
Doxorubicin (DOX) is associated with premature cardiovascular events including myocardial infarction. This study was performed to determine if the weekly administration of DOX influenced coronary arteriolar medial and/or adventitial wall thickening.
Thirty-two male Sprague-Dawley rats aged 25.1± 2.4 weeks were randomly divided into three groups and received weekly intraperitoneal injections of normal saline (saline, n = 7), or low (1.5 mg/kg to 1.75 mg/kg, n = 14) or high (2.5 mg/kg, n = 11) doses of DOX. The animals were treated for 2–12 weeks, and euthanized at pre-specified intervals (2, 4, 7, or 10+ weeks) to obtain histopathologic assessments of coronary arteriolar lumen diameter, medial wall thickness, adventitial wall thickness, and total wall thickness (medial thickness + adventitial thickness).
Lumen diameter was similar across all groups (saline: 315±34 µm, low DOX: 286±24 µm, high DOX: 242±27 µm;
In conclusion, these data demonstrate that weekly treatment of rats with higher doses of DOX increases coronary arteriolar medial, adventitial, and total wall thickness. Future studies are warranted to determine if DOX related coronary arteriolar effects are reversible or preventable, exacerbate the known cardiomyopathic effects of DOX, influence altered resting or stress-induced myocardial perfusion, or contribute to the occurrence of myocardial infarction.
Cardiovascular (CV) events including myocardial infarction (MI), stroke, and congestive heart failure (CHF) are the second leading cause of premature morbidity and mortality for those surviving beyond 5 years of their initial diagnosis for breast cancer or a hematologic malignancy such as Hodgkin's disease or Non-Hodgkin's lymphoma
To this end, we investigated the association between the administration of DOX and histopathologic changes in coronary arteriolar microcirculatory arterial segments. To address this issue, we performed a series of studies to determine the relationship between DOX administration and measures of coronary arteriolar wall thickening. In this study, we evaluated 2 doses of DOX in a rat model without coronary arterial atherosclerosis. These doses were selected to mimic doses commonly administered to patients receiving DOX for treatment of breast cancer or lymphoma. We measured the thickening of the media as well as the adventitia of the coronary microcirculatory arteriolar segments.
This study was performed at the Wake Forest University School of Medicine as part of a protocol approved by the Institutional Animal Care and Use Committee (Assurance #A04-137) of Wake Forest University, and was funded by the National Institutes of Health (study identifier R21CA109224). A total of thirty-two male Sprague-Dawley rats (a subset of animals previously reported
At the 2, 4, 7, and 10–12 week intervals, rats from the DOX treatment groups were randomly selected for necropsy and histopathologic examination of the coronary arteriolar and adventitial diameters. Histopathological examination of saline treated animals was conducted at 2 weeks in 1 animal and at the end of the 10 week study in the other 6 animals. During receipt of saline and DOX, rat physical appearance and weights were routinely monitored. If the physical appearance of rats reached a level of 5 or body weight decreased greater than 25% from the start of study (see
Treatment | Weeks in study | Rat weight, gm | Physical Appearance score | EF Measurements |
|
0 | 483±37 (n = 7) | 1.0±0.0 (n = 7) | 77±3 (n = 7) |
2 | 488±35 (n = 7) | 1.3±0.3 (n = 7) | 73±2 (n = 5) | |
4 | 521±30 (n = 6) | 1.0±0.0 (n = 6) | 77±3 (n = 6) | |
7 | 538±29 (n = 6) |
1.0±0.0 (n = 6) | 78±2 (n = 5) | |
10+ | 544±29 (n = 6) |
1.0±0.0 (n = 6) | 77±1 (n = 4) | |
|
0 | 415±12 (n = 14) | 1.0±0.0 (n = 14) | 77±1 (n = 14) |
2 | 411±12 (n = 14) | 1.9±0.1 (n = 14) | 75±1 (n = 12) | |
4 | 401±14 (n = 9) | 2.0±0.0 (n = 9) | 73±3 (n = 6) | |
7 | 389±13 (n = 8) |
3.8±0.4 (n = 8)# | 73±3 (n = 8) |
|
10+ | 368±16 (n = 3) |
3.3±0.3 (n = 3)# | 67±9 (n = 3) |
|
|
0 | 467±17 (n = 11) | 1.0±0.0 (n = 11) | 70±2 (n = 11) |
2 | 460±15 (n = 11) | 1.8±0.1 (n = 11) | 69±2 (n = 9) | |
4 | 418±11 (n = 11) | 3.4±0.2 (n = 11)# | 65±3 (n = 11) | |
7 | 408±10 (n = 5) |
4.4±0.4 (n = 5)# | 55±6 (n = 4) |
|
10+ | 438±0 (n = 1) | 3.0±0.0 (n = 1)# | 74±0 (n = 1) |
Number of animals in treatment group at any given time is indicated within the parentheses.
Average appearance score of animals in study, where 1 = excellent, active; 2 = good: active, slight hair loss; 3 = fair: less active, slight bloating; 4 = poor: reluctant to move, poor appetite and diarrhea; 5 = critical: marked pallor, lethargic.
increase in weight relative to baseline
decrease in weight relative to baseline
#increase in appearance score relative to baseline
decrease in LVEF relative to baseline
decrease in LVEF relative to baseline
After animal euthanasia (ketamine/xylazine 80/12 mg/kg IM followed by CO2 asphyxiation), gross examination of the rats was performed. The hearts were excised, the aorta was cannulated, and then the heart was mounted on a Langendorff apparatus, pressurized to 52 cm H2O, and perfused with paraformaldehyde for 20 minutes. The heart was then transferred to chilled paraformaldehyde for 24 hours followed by a solution of 70% ethanol. Appropriate sections were trimmed to obtain the middle short axis view. As shown in
Schematic representation that illustrates the location, selection, and analysis method of the histological specimens used to measure arteriolar wall thickness. Sectioned histologic specimens were obtained in a short axis plane located ½ the distance between the mitral annulus and the apex of the left ventricle (top left). In the top right panel, a low resolution image of the rat myocardium at 2x with high resolution is shown with an insert of a coronary arteriolar vessel at 20x (bottom right). Values for individual artery lumen, medial and adventitial circumferences (LC, MC, and AC, respectively) were obtained by tracing the region of interest using the digital imaging analysis program NDP.view (indicated in green, bottom right). The bottom left panel shows how the circumference data were used to find the lumen, medial, and adventitial radii (LR, MR, and AR, respectively) in order to calculate average diameters and wall thickness.
Lumen calculations:
Average Lumen Diameter (LD) = LC/π
Average Lumen Radius (LR) = LD/2
Medial calculations:
Average Medial Diameter (MD) = MC/π
Average Medial Radius (MR) = MD/2
Average Medial Thickness (MT) = MR - LR
Adventitial calculations:
Average Adventitial Diameter (AD) = AC/π
Average Adventitial Radius (AR) = AD/2
Average Adventitial Wall Thickness (AT) = AR – MR
Full Thickness (FT):
FT = MT+AT
Lumen diameter (LD), medial wall thickness, adventitial thickness, and full thickness (medial thickness + adventitial thickness) were calculated and presented in the absence (
Treatment | Number of animals in analysis | Lumen Diameter (µm) | Medial Thickness (µm) | Adventitial Thickness (µm) | Total Wall Thickness (µm) |
|
7 | 315±34 | 13±3 | 23±4 | 36±5 |
|
14 | 286±2 | 12±2 | 24±3 | 36±4 |
|
11 | 242±27 | 22±2** | 28±3 | 51±4* |
Low Doxorubicin: 1.5 mg/kg to 1.75 mg/kg
High Doxorubicin: 2.5 mg/kg
Values are expressed as the unadjusted mean ± SEM. All statistical comparisons shown are between different doxorubicin doses to normal saline (*
Treatment | Number of animals in analysis | Medial Thickness/LD | Adventitial Thickness/LD | Total Wall Thickness/LD |
|
7 | 0.04±0.01 | 0.07±0.02 | 0.12±0.03 |
|
14 | 0.04±0.01 | 0.09±0.02 | 0.14±0.02 |
|
11 | 0.10±0.01*** | 0.14±0.02 * | 0.25±0.02** |
Low Doxorubicin: 1.5 mg/kg to 1.75 mg/kg
High Doxorubicin: 2.5 mg/kg
Values are expressed as the unadjusted mean ± SEM. All statistical comparisons shown are between different doxorubicin doses to normal saline (*
Animals within each group were assessed at pre-specified time intervals. All analyses that compared groups at a fixed point in time were performed first using analysis of variance (ANOVA), then using analysis of covariance (ANCOVA) to adjust for age, weight, and the total cumulative dose of DOX. In addition, separate analyses were performed to account for the potential influence of time or duration of treatment on our results. Only when there were overall significant differences in appearance among the 3 groups would pair-wise comparisons of least means squares be tested with Student's t-tests between the saline and the low- and high-DOX groups. Comparisons were made to determine whether the thickness of the adventitia, thickness of the media, and the total thickness of the media-adventitia differed between DOX and saline treatment groups. All values are reported as mean ± the standard error of the mean (SEM), and unless stated otherwise, a value of
Characteristics pertaining to the animals are provided in
Lumen diameter, medial wall thickness, adventitial thickness and total arterial wall thickness are shown in
Coronary arteriolar histology from an animal treated with normal saline (1cc/week) for 7–10 weeks (left) with a coronary arteriole obtained from an animal treated with 2.5 mg/kg/week of doxorubicin for 10 weeks (right). Compared with the normal saline treated artery, the doxorubicin treated artery has increased medial and adventitial thickness.
When normalized to LD (
These data summarize the effect of saline (n = 7), low (1.5 mg/kg to 1.75 mg/kg, n = 14) or high (2.5 mg/kg, n = 11) doses of doxorubicin (DOX) on coronary arteriolar medial, adventitial, and total wall thickness after normalization to lumen diameter and adjusting for animal age, weight, and duration of treatment. Data shown are adjusted mean ± the standard error of the mean (SEM). P-values are provided for comparisons between saline and doxorubicin treated groups (analysis of covariance).
To examine the potential influence of treatment duration on wall thickness values, we categorized duration of exposure into 3 intervals (2 or 4, 7 and 10 weeks). Because of the relatively few numbers of animals available for study of the 2 week interval, the animals within the 2 and 4 week groups were combined. For the high DOX animals, those with the shortest duration of exposure (2 or 4 weeks) had the highest levels of adventitial, medial and total wall thickness relative to those treated for 7 or 10 weeks. This observation was strongest for the adventitial measures (
The results of this study indicate: a) the administration of 2.5 mg/kg/week of DOX increases the thickness of the walls of coronary arterioles of Sprague-Dawley rats, driven primarily by an increase in medial thickening (
Anthracycline-based chemotherapeutic regimens (including those that incorporate DOX) are among the most widely used regimens to treat malignancies. Today, more than 60,000 patients receive anthracyclines each year to treat leukemia and lymphoma, and breast, uterine, ovarian and lung cancer
In addition to CHF, however, anthracycline exposure has been found to increase the incidence of vascular events including MI and stroke
As demonstrated in
Since the animals in the study exhibited differences in age, weight, and total cumulative DOX dose, we also performed analyses to account for these differences. As shown in
There are several mechanisms which may explain the coronary arteriolar wall thickening observed in this study after DOX. Adventitial hypertrophy has been linked with oxidative inflammation, most notably NADPH oxidase
In addition to a primary effect of DOX on the media of the arterioles, other comorbidities may be exacerbated by DOX that indirectly promote increased arteriolar wall thickening. For example, increased medial thickness may result chronically from pressure overload such as elevated blood pressure which may result from increased arterial stiffness
The results of this study do not provide mechanisms to account for the increased risk of subsequent MI after anthracycline exposure. Other study results, however, have demonstrated associations between anthracycline exposure and vascular dysfunction in non-coronary vascular beds
There are several limitations to this study. First, this may be a phenomenon isolated to rats. As such, subsequent studies will need to be employed in other animal models and human participants to document the occurrence. Second, we are uncertain if this response would occur using other anthracycline preparations. Further studies will need to be performed to see if these changes persist with encapsulated DOX, which is used for human treatment. Third, this study focused on the anatomy of the involved coronary arterioles. Future studies need to also describe the physiologic function of coronary arteries as well as the smaller micro vessels exposed to anthracyclines.
In conclusion, early after exposure to anthracycline-based chemotherapy with DOX, microcirculatory coronary arteriolar wall segments develop increased thickening of the medial and adventitial layers leading to an overall increase in microcirculatory arteriolar wall thickness. Further studies are indicated to determine the etiology of this occurrence and whether this finding is permanent and promotes myocardial ischemia in the setting of vasodilator or exercise induced stress.