Effects of adriamycin and candesartan on the collagen and elastin of the aorta in rats
© Uhm et al.; licensee BioMed Central Ltd. 2014
Received: 27 March 2014
Accepted: 10 September 2014
Published: 25 September 2014
It has been reported that the chemotherapeutic agent, adriamycin, not only has an effect on the myocardium but also on the arteries. The aim of this study is to elucidate effects of adriamycin and an angiotensin receptor blocker, candesartan, on collagen and elastin of the aorta in rats.
Twenty four male 8-week-old Wistar-Kyoto rats were divided into four groups: control (C) group, adriamycin-treated (AD) group, candesartan-treated (CA) group, and adriamycin- and candesartan-treated (AD + CA) group. Adriamycin of 2.5 mg/kg/wk was administered intraperitoneally one time per week for 6 weeks, and candesartan of 10 mg/kg/day was administered orally everyday for 6 weeks. After 6 weeks, the rats were sacrificed and the aortas were harvested. Hematoxylin-eosin staining, Verhoff’s elastic, and Goldner’s trichrome staining were performed for histopathologic analyses. Tunica media thickness, collagen, and elastic area fractions were measured quantitatively with a computerized digital image analyzer.
Tunica media thickness in the CA and AD + CA groups was significantly lesser than that in the C and AD groups, respectively. The AD and AD + CA groups had a tendency of lower elastin area fraction than the C and CA groups, respectively. Collagen area fraction in the AD + CA group was significantly lower than that in the AD group. There were no significant differences of collagen/elastin ratio between groups.
These findings suggest that adriamycin has a tendency of decreasing the quantity of elastin fibers and candesartan cannot mitigate the effects of adriamycin on elastin fibers.
KeywordsDoxorubicin Aorta Candesartan Collagen Elastin
The anthracycline chemotherapeutic agent, adriamycin, is widely used and effective in many malignancies . However, adriamycin has an important adverse effect, cardiotoxicity . In practice, oncologists cannot often continue with the administration of adriamycin because of adriamycin-induced cardiomyopathy, though adriamycin is effective in inducing remission of cancer.
The aorta has the same embryologic origin as the heart, and it is anatomically and hemodynamically directly connected to the heart. The heart and the aorta often share the same pathophysiology of the diseases including hypertensive cardiovascular disease. Some researchers proposed that adriamycin has adverse effects not only on the heart but also on the arteries [3–5]. However, there have been not many studies on vascular pathology by adriamycin, and changes of aorta microstructure by adriamycin have not been known in detail. In addition, it has been reported that angiotensin-converting enzyme inhibitors or angiotensin receptor blockers can alleviate the fibrotic process . This was a pilot study to investigate whether adriamycin can have an effect on collagen and elastin of the aorta in rats and whether candesartan can mitigate the effects of adriamycin.
Staining and light microscopic examination
After the extracted aorta was irrigated with normal saline, the descending thoracic aorta was cut into small pieces. These pieces were fixed in 10% neutral-buffered formalin solution. After the dehydration process, the aortic tissue sample was embedded into a paraffin block and cut with a microtome. Hematoxylin-eosin staining, Verhoff’s elastin staining, and Goldner’s trichrome staining were performed for light microscopic examination. Elastin was stained black using the Verhoff’s elastin staining, and collagen was stained green using the Goldner’s trichrome staining. Photos of the aorta tissue were taken from six different areas per a rat at a magnification of × 400 with a light microscope (Zeiss, Jena, Germany). The photo images were quantitatively analyzed with Image-Pro Plus (MediaCybernetics, Silver Spring, MD, USA), a computerized digital image analyzer. Tunica media thickness was measured with a digital ruler and a microscopic scale in hematoxylin-eosin-stained section. The area in black was semi-automatically measured with Image-Pro Plus in Verhoff’s elastin-stained sections for measurement of the elastin area. The area in green was semi-automatically measured with Image-Pro Plus in Goldner’s trichrome-stained section for measurement of the collagen area. Elastin area fraction was calculated by dividing the elastin area by the total sampling area, and collagen area fraction was calculated by dividing the collagen area by the total sampling area. Collagen/elastin ratio was calculated by dividing the collagen area by the elastin area.
Electron microscopic examination
The aortic tissue was fixed in 2.5% phosphate-buffered glutaraldehyde for 2 h and fixed in 1% osmium tetroxide and then dehydrated in serial alcohol. The aortic tissue was embedded in araldite and sectioned. After staining with toluidine blue, the aortic tissue sections were observed under microscope for precise location to cut for ultrathin sections. The endothelial and medial areas of the aorta were observed with an electron microscope (EM, Zeiss) at magnifications of × 8,000 and × 16,000, respectively.
Because of nonnormal distribution of data, the results were expressed as median (interquartile range). Kruskal-Wallis test was used for comparison of baseline characteristics of continuous variables between the four groups. For post hoc analysis, we used Mann–Whitney U test with Dunnett comparisons between two groups:  the C group and the AD group, between the C group and the CA group, between the AD group and the AD + CA group, between the CA group and the AD + CA group, and between the C group and the AD + CA group. A p value and Dunnett-corrected p value (pDun) <0.05 was considered statistically significant. The data were analyzed using the IBM SPSS ver. 20.0 (IBM Co., Armonk, NY, USA) and R Package ver. 2.0 (R Foundation for Statistical Computing, Vienna, Austria).
Changes of body weight before and after administration of drugs
ΔBW/BW1 × 100 (%)
AD + CA
This pilot study demonstrated that the adriamycin-treated groups had a tendency of lower elastin area fraction than the adriamycin-untreated groups. And candesartan decreased the thickness of tunica media in the aorta in adriamycin-treated and adriamycin-untreated rats and the quantity of collagen fibers in the aorta of adriamycin-treated rats. According to the results of the present study, adriamycin had a tendency of reduced quantity of elastin fibers in the aorta. As a result, elastance of the aorta could decrease and aortic stiffness could increase . Decreased elastance and increased stiffness of the aorta mean pathologic processes and structural remodeling of the aorta. It could aggravate hypertension and cause target organ damage including left ventricular hypertrophy [10, 11]. Decrement of aortic elastance could result in aggravation of adriamycin-induced cardiotoxicity by myocardial fibrosis and structural remodeling. In addition, candesartan decreased the tunica media thickness and the quantity of collagen fibers in the aorta in the present study. It was because, generally, angiotensin receptor blockers have the effects of mitigating fibrosis by decreasing thickness of tunica media and collagenogenesis. Reduction of the quantity of collagen fibers means mitigation of fibrosis and reverse of structural remodeling of the aorta. Decrease of tunica media thickness could be relative to reverse of structural remodeling of the aorta. However, candesartan could not show the mitigation of decrement of elastin fibers by adriamycin.
Because fibrosis plays an important role in cardiomyopathy and vasculopathy, drugs that have an effect on the fibrotic process are essential for the treatment of cardiomyopathy and vasculopathy. Angiotensin converting enzyme inhibitors or angiotensin receptor blockers can regulate the renin-angiotensin-aldosterone system and ameliorate the fibrotic process . In addition, it was reported that spironolactone, an aldosterone receptor blocker, and torasemide, a loop diuretic, can reduce fibrosis [12, 13].
Because the aorta is directly connected to the heart, the heart and aorta often share the same pathophysiologic processes. It has been reported that adriamycin induces aortic toxicity . However, the mechanisms of adriamycin-induced aortopathy are not known in detail. The suggested mechanisms of adriamycin-induced vasculopathy were endothelial dysfunction [15–18], suppression of endothelin-1 in endothelial cells , degeneration of endothelial structures , apoptosis of the endothelial cells , enhancement of procoagulant activity , smooth muscle dysfunction, and dysregulation of its calcium contents [10, 22]. In addition, adriamycin could induce not only cardiovascular toxicity but also nephrotoxicity by alteration of glomerular endothelial cells [16, 23].
Usually, injection of adriamycin induces retardation of weight gain . In this study, adriamycin- or candesartan-treated rats reduced weight gain compared with rats in control group, although there was no statistical significance. In adriamycin-treated rats, it could be because adriamycin has systemic toxicity. It has reported that incidence of gastrointestinal adverse effects of candesartan was 1% in human study . In adriamycin-treated rats, reduced weight gain might be due to gastrointestinal adverse effects of candesartan.
We have shown in the previous report that collagen in the myocardium significantly increased in adriamycin-induced cardiomyopathy rat model . However, collagen area fraction between the C group and the AD group was not significantly different in the present study. It could be because the mechanisms of aortopathy could be different from those of cardiomyopathy and because the numbers of rats and the duration from the drug administration to sacrifice could be short.
In this study, ultramicroscopic structural changes were not obvious. It might be because the adriamycin-induced microscopic changes in the aorta were slower than those in cardiomyopathy due to the extracellular matrix of the aorta. It might be because the rats in the present study were too young to present the microscopic pathologic processes including atherosclerotic changes.
We had some limitations of the present study. We could not confirm the development of cardiomyopathy because we did not perform echocardiography in the rats. We could not show microscopic and ultramicroscopic changes of endothelial cells by adriamycin and candesartan because the endothelial layers were damaged in the processes of fixation and staining.
Adriamycin had a tendency of decreasing the quantity of elastin fibers, and candesartan cannot mitigate the effects of adriamycin on elastin fibers. In oncology clinic, physicians should be cautious about not only adriamycin-induced cardiotoxicity but also adriamycin-induced vascular changes. Further studies are necessary to evaluate the effects of angiotensin-converting enzyme inhibitors or angiotensin receptor blockers on the adriamycin-induced vascular changes.
- P Dun :
Dunnett-corrected p value.
This work was supported by the funds of Korean Society of Hypertension and Research Foundation of Internal Medicine, The Catholic University of Korea in 2010.
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