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 (); b) these increases persist after indexing the regions of wall thickness for the LD of the vessel, and after accounting for animal age, weight, and the total cumulative amount of DOX received ( and ); c) when duration of treatment is assessed, those animals receiving 2.5 mg/kg/week for ≤4 weeks experience greater amounts of arterial thickening than those surviving for ≥10 weeks of treatment; and d) after adjustment for age and DOX dose, lower doses of DOX (1.5 mg/kg/week) trend toward an increase in the total wall thickness of coronary arterial segments (). This trend is primarily related to trends in an increase in the thickening of the adventitia ().
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 
. One-half of all patients exposed to anthracyclines, including DOX, demonstrate some degree of cardiac dysfunction 10 to 20 years after chemotherapy, and 5% of them develop overt CHF 
. To date, much of the research into the primary mechanism of anthracycline cardiomyopathy has focused on the direct effect of anthracyclines on the left ventricular myocytes 
, with prior histologic studies (including one from our group) demonstrating evidence of disorganized myofibrillar morphology, myocellular vacuolization and necrosis, and interstitial fibrosis 
In addition to CHF, however, anthracycline exposure has been found to increase the incidence of vascular events including MI and stroke 
. This issue has become highly relevant as the number of cancer survivors has increased significantly over the past 20 years 
. For example, among 10-year survivors of breast cancer, data from 1970 through 1986 indicate that women having received adjuvant anthracycline-based chemotherapy experience an increase in MI (hazard ratio [HR]
2.55, 95% confidence interval [CI]
<0.001), and stroke (HR
1.85, 95% CI
0.002) relative to women without cancer or the prior receipt of an anthracycline 
. As recently recognized by the National Cancer Institute, important trends are emerging regarding cancer treatment: as advanced therapeutics are reducing cancer-related illness, some of this benefit is offset by an adverse rise in morbidity and mortality from CV disease 
. This paper presents new data that describes the effect of DOX exposure on the thickening of the walls of the coronary arterioles in hopes of determining whether acute exposure to DOX influences the coronary arteriolar microcirculation.
As demonstrated in , animals in this study developed an unhealthy appearance, a decrease in weight, and a drop in LVEF upon receipt of DOX. These findings are similar to those observed in prior studies 
. As shown in , total microcirculatory arteriolar wall thickness increased after the 2.5 mg/kg/week dose of DOX. This increase in arterial wall thickness was accounted for primarily by an increase in the medial component of the wall ( and ), and remained present after indexing the size of the arteriolar segments for the diameter of the vessel lumen (). The results after indexing indicate that the increases in wall thickness were not confined to smaller or larger arterioles and not related to potential differences in vessel sizes between our treatment groups (saline, low and high dose DOX).
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 , increases in wall thickness after high DOX exposure persisted after accounting for the age and weight of the animals as well as the cumulative dose of DOX received. Although not statistically significant, strong trends were respectively observed (p<0.06 to 0.09) in increases in adventitial and total wall thickness in animals receiving lower doses of DOX after accounting for age, weight and cumulative DOX dose.
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 
. Anthracyclines are known to increase free radical production via effects on NADPH oxidase 
. Adventitial inflammation with increased NADPH oxidase activity causes significant adventitial fibrosis and smooth muscle hypertrophy 
. Inflammation in the adventitial layer can exhibit strong paracrine effects on both the media and intima, and as a result, stimulate smooth muscle hypertrophy within the walls of the arterial segments 
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 
. In this study, we did not measure blood pressure, nor did we routinely assess renal function; thus we do not have data that can address this issue.
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 
. Bar-Joseph, et al, demonstrated vascular dysfunction acutley after exposure to DOX, and Murata, et al, demonstrated effects of DOX on cultured endothelial cells 
. Inflammation from injury to the tunica media precipitates intimal hyperplasia 
, which in other studies has preceded acute coronary artery syndromes 
. In addition, medial layers with increased thickness are not able to respond to nitric oxide to augment blood flow 
. As such, increased medial and adventitial thickening may contribute to abnormal myocardial perfusion. Our results indicate that increased coronary artery wall thickening occurs early after exposure to the DOX, and suggests further studies are warranted to investigate the mechanism of this association and whether this increase in wall thickening is associated with reduced vasodilator reserve, premature atherosclerosis, or increased myocardial ischemia in the setting of CV stress.
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.