In a previous experiment we have shown that mast cells, in contrast to what has been the prevailing assumption, play a predominantly protective role in radiation-induced heart disease. As shown previously, and again in the current study, cardiac mast cell numbers are reduced in the first weeks after irradiation but increase thereafter. Recent evidence suggests that the endothelin (ET) system may mediate the protective effect of mast cells in certain disorders. Upon mast cell activation by ETA, mast cells may reduce the toxicity of ET1 by the release of proteases that are able to degrade ET1 (16
). A recent study showed that acute increases in cardiac mast cell density due to chronic volume overload in a rat model were prevented by bosentan, demonstrating that cardiac mast cells are responsive to ET1 in vivo
). In the current study, left ventricular mRNA levels of ET1 were increased by radiation at all times investigated but only in mast cell-competent (+/+) rats. Although the sample size in this experiment was small (n
= 3), these results may suggest interactions between mast cells and the cardiac ET system in radiation-induced heart disease. To investigate the role of ET1 in radiation-induced heart disease, this study was focused on examining the effects of the ET1 receptor antagonist bosentan on cardiac radiation injury in rats.
This study showed that a single dose of radiation locally to the heart induced sustained up-regulation of ET1 mRNA together with an increase in cardiac mast cell numbers in Sprague-Dawley rats. The exact mechanisms responsible for these sustained increases in mast cell numbers and ET1 mRNA are not known. Recently, it has been shown that ionizing radiation induces long-term oxidative stress (35
). Moreover, oxidative stress is known to affect mast cell function (37
). Hence it may be speculated that sustained oxidative stress is involved in mast cell activation and up-regulation of ET1. In addition, ionizing radiation is known to induce prolonged endothelial dysfunction (loss of thromboresistance and increased expression of chemokines and cytokines) (39
), which is likely involved in sustained changes in tissues after localized irradiation. A wide variety of cell types in the heart, including myocytes, fibroblasts, endothelial cells and mast cells, are capable of synthesizing ET1 (12
). In a previous study using mast cell-deficient (Ws/Ws) and mast cell-competent (+/+) rats to examine cardiac transplantation, myocytes and mast cells were found to be the main locations of ET1 protein in +/+ rat hearts (8
). Unequivocal identification of the source of ET1 mRNA and relative contributions by cardiac endothelial cells, myocytes, fibroblasts or mast cells may be done by in situ
hybridization, similar to what has been performed previously (40
This study examined the effects of the dual ETA/ETB antagonist bosentan on functional, structural and molecular changes in the Sprague-Dawley rat heart after irradiation. The cellular effects of ETA and ETB are highly diverse and are sometimes opposing. While activation of ETA on smooth muscle cells induces the well-known vasoconstrictive effects of ET1, for instance, ETB may be involved in both vasoconstriction and vasodilation (41
). Both receptors, on the other hand, seem to promote a fibrogenic response in cardiac fibroblasts (43
). Therefore, selective ET1 receptor antagonists do not always reduce experimental myocardial fibrosis (44
). Because of the complexity of the ET system, a dual ETA/ETB antagonist was chosen for this initial study.
Bosentan reduced systolic pressure and +dP/dtmax
, which suggest reduced contractility of the left ventricle. Interestingly, bosentan induced reductions of these parameters in irradiated as well as sham-irradiated hearts. Few studies have reported the results of isolated perfused heart preparations after long-term treatment with bosentan or other dual ETA/ETB receptor antagonists in otherwise untreated or healthy animals. In a study on the effects of bosentan on cardiovascular function in diabetic rats, 7 weeks of bosentan treatment reduced developed pressure against left atrial filling pressure in isolated working hearts of all rats, including control rats (45
). Long-term inhibition of ET1 receptors not only may directly inhibit the positive inotropic effects of ET1 on the heart but also may lead to activation of compensatory mechanisms in an attempt to reverse the cardiac effects of ET1 receptor inhibition.
This study used computerized image analysis for quantitative assessment of immunohistochemistry results. We have previously done extensive quality testing of our quantitative immunohistochemistry readings, including testing of batch-to-batch variability and variability among observers (46
). Our technique showed to be highly reproducible. With this technique, the area of staining is considered and not the intensity of staining. This is because studies have shown that staining intensity does not reflect protein levels (47
). However, one may debate whether quantitative assessment of immunoreactivity is a true measure of protein expression. For instance, in the TGF-β immunostaining, no distinction is made between the latent and active form of the protein. One should keep these considerations in mind when interpreting the quantitative immunohistochemistry results.
Even though bosentan administration resulted in altered cardiac gene expression of several pro-inflammatory and pro-fibrogenic mediators, radiation-induced collagen deposition was not affected. This is a somewhat surprising result, considering the well-known anti-fibrotic properties of bosentan (48
). Several explanations may account for these results. It is possible that ET1 simply is not involved in radiation-induced fibrosis in the heart. Although ET1 has been shown to be involved in microvessel dysfunction after exposure to ionizing radiation (11
), to our knowledge a role for ET1 in radiation-induced late-occurring adverse tissue remodeling has not been reported. On the other hand, a dual ETA/ETB antagonist may not reveal the role of each receptor subtype, since the effects of ETA and ETB are diverse and are sometimes opposing. Also, a dual ETA/ETB antagonist such as bosentan may block clearance of ET1 by the ETB receptor, which could result in an increase in circulating ET1 levels (49
). The use of selective antagonists of ETA and ETB may give further insight into the role of each receptor subtype. Finally, future studies may be designed in which the dose of bosentan is increased to at least 100 mg/kg per day, analogous to the dose that reduced myocardial fibrosis in chronic heart failure, induced by left coronary artery ligation in rats (50
Interestingly, a trend toward increased expression of CGRP
was found both in +/+ rats and in Sprague-Dawley rats. Indeed, levels of CGRP, a potent vasodilator, often correlate with levels of the vasoconstrictor ET1 (26
). The fact that CGRP mRNA was not up-regulated in Ws/Ws rats suggests that mast cells were involved in regulation of cardiac CGRP levels after irradiation. The role of CGRP, a neuropeptide expressed and released by sensory nerves, has been studied in radiation-induced intestinal injury (51
). Like radiation-induced up-regulation in the heart, intestinal CGRP is up-regulated after localized irradiation. Administration of full-length CGRP ameliorated radiation-induced intestine injury in rats, while a CGRP antagonist exacerbated injury (51
). Since CGRP is known to play a protective role in myocardial injury (28
), its role in the heart after irradiation deserves further investigation.
During localized heart irradiation in the current rat model, parts of the spinal cord and the lungs are included in the radiation field. Changes in these tissues may influence the radiation response in the heart. Levels of ET1 in rat spinal cords, for instance, are increased up to 6 months after a single dose of 15 Gy (11
). Data on levels of circulating ET1, however, have not been reported. Therefore, it is not known whether sufficient quantities of ET1 would be released into the circulation to have effects in the heart. In our study, the animals did not show paralysis at any time after local heart irradiation, although this observation does not entirely rule out spinal cord injury. However, because irradiated volumes and radiation doses to these tissues were similar in each of the experimental groups, responses of these tissues likely do not have a significant impact on the comparisons made in the heart.
In conclusion, mast cell-competent but not mast cell-deficient hearts exhibited postirradiation up-regulation of ET1, suggesting the possibility of interactions between mast cells and the cardiac ET system. The dual ETA/ETB receptor antagonist, bosentan, induced reductions in left ventricular systolic pressure, developed pressure, and +dP/dtmax but did not affect radiation-induced cardiac remodeling. Moreover, bosentan did not significantly alter the cardiac radiation response in Sprague-Dawley rats in the current study. Further studies are needed to clarify the role of ET1 and each of its two receptors in radiation-induced heart disease.