Biology Contribution
Inhibition of Protease-activated Receptor 1 Ameliorates Intestinal Radiation Mucositis in a Preclinical Rat Model

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Purpose

To determine, using a specific small-molecule inhibitor of protease-activated receptor 1 (PAR1) signaling, whether the beneficial effect of thrombin inhibition on radiation enteropathy development is due to inhibition of blood clotting or to cellular (PAR1-mediated) thrombin effects.

Methods and Materials

Rats underwent fractionated X-irradiation (5 Gy × 9) of a 4-cm small-bowel segment. Early radiation toxicity was evaluated in rats receiving PAR1 inhibitor (SCH602539, 0, 10, or 15 mg/kg/d) from 1 day before to 2 weeks after the end of irradiation. The effect of PAR1 inhibition on development of chronic intestinal radiation fibrosis was evaluated in animals receiving SCH602539 (0, 15, or 30 mg/kg/d) until 2 weeks after irradiation, or continuously until termination of the experiment 26 weeks after irradiation.

Results

Blockade of PAR1 ameliorated early intestinal toxicity, with reduced overall intestinal radiation injury (P=.002), number of myeloperoxidase-positive (P=.03) and proliferating cell nuclear antigen-positive (P=.04) cells, and collagen III accumulation (P=.005). In contrast, there was no difference in delayed radiation enteropathy in either the 2- or 26-week administration groups.

Conclusion

Pharmacological blockade of PAR1 seems to reduce early radiation mucositis but does not affect the level of delayed intestinal radiation fibrosis. Early radiation enteropathy is related to activation of cellular thrombin receptors, whereas platelet activation or fibrin formation may play a greater role in the development of delayed toxicity. Because of the favorable side-effect profile, PAR1 blockade should be further explored as a method to ameliorate acute intestinal radiation toxicity in patients undergoing radiotherapy for cancer and to protect first responders and rescue personnel in radiologic/nuclear emergencies.

Introduction

The intestine is an important dose-limiting organ during abdominal and pelvic radiotherapy. Despite advances in treatment delivery techniques, intestinal radiation injury (radiation enteropathy) remains an important obstacle to cancer cures and continues to adversely affect the quality of life of many cancer survivors. Clinically, intestinal radiation toxicity occurs as early (acute) or delayed (chronic) radiation enteropathy, with related but different underlying mechanisms. Pathologically, early radiation enteropathy (“radiation mucositis”) is characterized by epithelial barrier breakdown and mucosal inflammation, whereas delayed radiation enteropathy exhibits prominent vascular sclerosis and intestinal wall fibrosis.

Radiation enteropathy is the result of complex interplay among many dose- and time-dependent pathophysiologic processes, including inflammation, epithelial regeneration, tissue remodeling, and collagen deposition, as well as activation of the coagulation system and, notably, endothelial dysfunction. Endothelial cells are mechanistically involved in early and delayed radiation responses in many normal tissues, including the intestine 1, 2, 3. Exposure to ionizing radiation elicits profound changes in the endothelium, commonly referred to as “endothelial dysfunction.” Loss of thromboresistance due to down-regulation of thrombomodulin and expression of tissue factor, resulting in increased formation of thrombin, is a hallmark feature of radiation-induced endothelial dysfunction 1, 3, 4.

Thrombin is a multifunctional serine protease that plays a central role in blood clotting by converting fibrinogen to fibrin and activating platelets. In addition, thrombin also regulates cell proliferation, inflammation, and tissue remodeling through activation of protease-activated receptors (PARs) (5), a family of 4 G-protein coupled receptors: PAR1, PAR2, PAR3, and PAR4. Protease-activated receptor 1 is the most biologically relevant in inflammation and fibrosis and is prominently up-regulated in irradiated intestine, suggesting a role in radiation enteropathy (3).

Preclinical and clinical studies performed in our and other laboratories demonstrate that so-called “endothelial-oriented” interventions can ameliorate radiation toxicity in several different normal tissues. We have also shown that there is increased thrombin formation, fibrin deposition, and up-regulation of PAR1 in irradiated intestine 3, 4. Although direct inhibition of thrombin ameliorates both early and delayed intestinal radiation injury (4), it is not known to what extent this benefit is due to suppression of thrombin-mediated blood clotting (conversion of fibrinogen to fibrin and stimulation of platelet aggregation) and/or to suppression of PAR1-mediated cellular effects. Because PAR1 is absent in rat platelets (6), this question was addressed using a specific small-molecule PAR1 inhibitor, SCH602539, in a preclinical rat model of radiation enteropathy. The results suggest that, although PAR1 is indeed involved in the development of early radiation toxicity (radiation mucositis), non-PAR1-mediated thrombin effects seem to be important in the mechanisms of development of chronic intestinal radiation fibrosis.

Section snippets

Experimental design

A total of 120 male Sprague-Dawley rats, 43-49 days of age (Harlan, Indianapolis, IN) were housed in conventional cages with free access to tap drinking water and standard mouse chow. All experimental protocols were approved by the University of Arkansas for Medical Sciences Institutional Animal Care and Use Committee.

The rat surgical model for localized small-bowel irradiation was prepared as described previously (7). Briefly, rats underwent bilateral orchiectomy, and a loop of distal ileum

Effect of PAR1 antagonist on early and delayed radiation enteropathy in rats

Radiation-induced histopathologic changes in the vehicle (control) group were similar to those observed in other studies performed in our laboratory (8). Early alterations (2 weeks) consisted mainly of mucosal injury (as measured by mucosal surface area), reactive intestinal wall thickening (as measured by intestinal wall thickening), and inflammatory cell infiltration (as measured by number of myeloperoxidase-positive cells). The delayed changes (26 weeks) included loss of mucosal surface

Discussion

Our data strongly suggest that cellular PAR1 signaling is involved in early radiation enteropathy development (ie, radiation mucositis) but not in delayed injury (ie, fibrosis). These findings have substantial translational significance because (1) PAR1 inhibition has substantially fewer side effects than direct thrombin inhibition (12), and (2) PAR1 blockade inhibits tumor growth and metastasis (reviewed in reference 13), and thus blocking PAR1 to reduce normal tissue toxicity would not be

Conclusions

SCH602539, a selective, small-molecule, nonpeptide antagonist of PAR1, seems to attenuate early intestinal radiation mucositis. This protective effect is likely due to inhibition of the cellular, receptor-mediated thrombin effects rather than to inhibition of blood clotting. The underpinnings of the lack of efficacy of SCH602539 in ameliorating delayed radiation fibrosis need further investigation. Protease-activated receptor 1 blockade has antitumor effects and, compared with direct thrombin

Acknowledgements

This research is supported by the National Institutes of Health (Grant CA-71382), Merck & Company, Inc, and the Veterans Healthcare Administration.

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Conflict of interest: Madhu Chintala is an employee of Merck & Company, Inc. Merck produces SCH602359.

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