- Email: [email protected]
Differential effect of radiation on endothelial cell function in rectal cancer and normal rectum
Differential Effect of Radiation on Endothelial Cell Function in Rectal Cancer and Normal Rectum Konrad K. Richter, MD, Louis M. Fink, MD, Bradley M. Hughes, BS, Hasan M. Shmaysani, MD, Ching-Ching Sung, MS, Martin Hauer-Jensen, MD, Little Rock, Arkansas
BACKGROUND: Chronic radiation injury of the intestine is associated with significant underexpression of a potent physiological anticoagulant, endothelial cell thrombomodulin (TM). This study compared early and late radiation-induced changes in endothelial TM, urokinase plasminogen activator (uPA), and transforming growth factor b (TGF-b) in normal rectum and tumors. METHODS: Rectal resection specimens from 27 patients were analyzed: Nine patients underwent primary resection of rectal cancer, 11 tumors were resected after neo-adjuvant radiotherapy, and 7 because of local recurrence after prior resection and adjuvant radiotherapy. TM, uPA, and extracellular matrix-associated TGF-b immunoreactivity were assessed using computerized image analysis. RESULTS: Multivariate analysis revealed that tumors had more TM-positive vessels (P 5 0.003), more uPA-positive cells (P <0.001), and higher TGF-b immunoreactivity levels (P <0.001) than normal rectum. Preoperative irradiation was associated with decreased proportions of TM-positive vessels in tumors (P 5 0.003) and normal rectum (P <0.001). Irradiated tumors had fewer uPA-positive cells (P 5 0.003) and less TGF-b immunoreactivity (P 5 0.001) than unirradiated tumors. The proportion of TM-positive vessels in irradiated rectum from patients with recurrence was decreased (P 5 0.03), whereas the recurrent (ie, unirradiated) tumors did not differ from primary tumors in terms of TM, TGF-b, or uPA immunoreactivity. CONCLUSIONS: The results support a role for endothelial dysfunction in the pathogenesis of radiation proctitis. Maintaining endothelial cell anticoagulant function may be a potential method to optimize the therapeutic ratio of adjuvant radio-
From the Departments of Surgery and Pathology, University of Arkansas for Medical Sciences and John L. McClellan Veterans Affairs Medical Center, Little Rock, Arkansas. This work was supported by a grant from the American Cancer Society (RPG-93-008-04 EDT). Requests for reprints should be addressed to Martin HauerJensen, MD, PhD, University of Arkansas for Medical Sciences, 4301 West Markham, Slot 725, Little Rock, Arkansas 72205. Presented at the 50th Annual Meeting of the Southwestern Surgical Congress, San Antonio, Texas, April 19 –22, 1998.
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© 1998 by Excerpta Medica, Inc. All rights reserved.
therapy of rectal cancer. Am J Surg. 1998;176: 642– 647. © 1998 by Excerpta Medica, Inc.
T
he risk of injury to the normal intestine (radiation enteropathy) is a major dose-limiting factor during radiation therapy for colorectal tumors. Radiation enteropathy is characterized by microvascular injury, fibrosis, and intestinal dysfunction. However, the underlying molecular mechanisms have not been identified. We have shown previously that chronic small bowel radiation injury is associated with significant upregulation of the fibrogenic cytokine, transforming growth factor b (TGF-b), and downregulation of the endothelial cell surface protein, thrombomodulin (TM).1–3 TM plays a pivotal role in the protein C pathway and is essential for maintaining the normal coagulation-anticoagulation homeostasis. Radiation-induced TM downregulation may initiate and maintain local microvascular injury, inflammation, and fibrosis. This may affect the response of the tumor to treatment, as well as the development of normal tissue radiation toxicity. The present study, therefore, compared changes in TM, TGF-b, and urokinase plasminogen activator (uPA) in normal rectum and rectal tumors at early and late times after irradiation. The results suggest that maintaining endothelial function during and after treatment may be a promising approach to enhance the efficacy and safety of adjuvant pelvic radiotherapy.
MATERIALS AND METHODS Rectal resection specimens from 27 patients (25 men, 2 women; median age 66 years, range 46 to 79) were obtained from the archives of the Department of Pathology: Nine patients had undergone low anterior or abdominoperineal resection of rectal cancer (“unirradiated” group). Eleven patients had been resected 7 weeks (median, range 21 to 61 days) after neo-adjuvant radiotherapy (“early” group). Seven patients underwent resection of local recurrence, 12 months (median, range 102 to 841 days) after prior resection and adjuvant radiotherapy (“late” group). Patients in the early and late groups received adjuvant therapy either as 30 Gy in 10 fractions or as 45 Gy in 25 fractions with concomitant 5-fluorouracil. The histopathologic diagnosis was reviewed and confirmed by a pathologist (LMF) using hematoxylin-eosinstained slides. Paraffin-sections of tumor and nonmalignant rectal tissue from each patient were stained with antibodies against TM (DakoTM 1009; Dako, Calenteria, California), TGF-b (AB-100-NA; R&D Systems, Minneapolis, Minnesota), and uPA (#3689; American Diagnostica, Green0002-9610/98/$19.00 PII S0002-9610(98)00280-3
RADIATION EFFECT ON RECTAL ENDOTHELIAL CELL FUNCTION/RICHTER ET AL
Figure 1. Proportion of thrombomodulin (TM)-positive vessels in normal tissue (left) and tumor (right) in rectal resection specimens from patients undergoing primary resection (no XRT), patients resected after neo-adjuvant radiotherapy (“early”), and patients resected for recurrence after a previous resection followed by postoperative adjuvant radiotherapy (“late”). The box plots show median, interquartile range (IQR), and upper and lower adjacent values.
wich, Connecticut) using appropriate negative and positive controls and standard immunohistochemical techniques as described elsewhere.1 Interactive computer-assisted image analysis of immunoreactivity was performed using the Samba 4000 System (Dynatech Laboratories, Chantilly, Virginia) as reported previously.1–3 Vessels with diameters between 5 and 70 mm in the submucosa of normal rectum and in a highly vascular area in the tumor sections were assessed for TM immunoreactivity according to a predefined grid pattern. Identification and counting of tumor vessels were performed using a modified Folkman assay.4 Twenty fields per slide were analyzed, and the mean proportion of TM-positive vessels in each section was considered a single value for statistical purposes. The area (measured in pixel units) positive for extracellular matrix-associated TGF-b was assessed in 20 fields per specimen according to a predefined grid pattern. The number of uPA-positive cells was assessed in 20 fields per slide and graded using an adaptation of the method described by Mulcahy et al:5 1 5 no positive cells, 2 5 few scattered positive cells, 3 5 abundant positive cells grouped in few clusters, and 4 5 abundant positive cells in clusters in multiple areas. Differences in the relative and total number of TMpositive vessels, TGF-b immunoreactivity, and uPA-positive cells were assessed with fixed-factor ANOVA using NCSS 6 for Windows (NCSS, Kaysville, Utah). Post-hoc testing was performed using StatXact 3 for Windows (Cytel Software, Cambridge, Massachusetts), a software package for exact nonparametric inference. Two-sided tests were used, and P values less than 0.05 were considered statistically significant.
RESULTS All three antibodies exhibited specific immunoreactivity to TM, TGF-b, and uPA, respectively, and qualitative staining patterns as reported previously by our laboratory and others.1,6 TM-positive vessels exhibited well-defined endothelial staining, both in tumor specimens and in normal rectum. Extracellular matrix-associated TGF-b staining was present in connective tissue in normal rectal specimens and in areas around malignant glands in tumors. Immunoreactivity for uPA was found in clusters of fibroblast-like cells, mononuclear cells, and leukocytes surrounding invading malignant glands. In normal rectum, uPA staining was restricted to few, scattered inflammatory cells. Analysis of variance revealed that tumors exhibited a significantly higher proportion of TM-positive vessels (P 5 0.003), higher TGF-b immunoreactivity levels (P ,0.001), and more uPA-positive cells (P ,0.001) than normal rectal tissue. Preoperative irradiation (early group) was associated with a decrease in the proportion of TMpositive vessels, both in tumors (P 5 0.003) and in normal, irradiated rectum (P ,0.001). The proportion of TM-positive vessels in irradiated rectum from the late group was also smaller than in the unirradiated primary resection specimens (P 5 0.03; Figure 1). Irradiated tumors exhibited less extracellular matrix-associated TGF-b immunoreactivity (P 5 0.001) than unirradiated tumors, whereas the differences in TGF-b immunoreactivity among the groups of nonmalignant rectum (unirradiated, early, and late) did not reach statistical significance (Figure 2). The number of uPA-positive cells was significantly lower in irradiated tumors than in nonirradiated primary tumors (P 5 0.003; Figure 3). Recurrent (ie, not
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Figure 2. Extracellular matrix-associated transforming growth factor b (TGF-b) immunoreactivity in normal tissue (left) and tumor (right) in rectal resection specimens. Patient groups and box plot attributes as for Figure 1.
Figure 3. Grading of urokinase plasminogen activator (uPA)-positive cells in normal tissue (left) and tumor (right) in rectal resection specimens. Patient groups as for Figure 1. 1 5 no positive cells, 2 5 few scattered positive cells, 3 5 abundant positive cells grouped in few clusters, 4 5 abundant positive cells in clusters in multiple areas.
previously irradiated) tumors did not differ significantly from primary tumors in terms of TM, TGF-b, or uPA immunoreactivity levels.
COMMENTS Vascular injury is a prominent and characteristic feature of chronic radiation injury in most organs. Initial endothelial cell damage increases vascular permeability and causes leakage of plasma proteins and subsequent development of obliterative vascular sclerosis and tissue fibrosis. However, the molecular links between endothelial function and chronic radiation toxicity have not yet been established in vivo. In the normal vasculature in most organs, TM acts as an important “natural anticoagulant” by changing the sub644
strate specificity of thrombin, so that thrombin no longer converts fibrinogen to fibrin, but instead activates protein C.7 Reduced endothelial TM levels may contribute to hypercoagulation with increased fibrin formation, platelet aggregation, and subsequent upregulation or release of inflammatory and fibrogenic cytokines, such as TGF-b. The results from the present study confirm and extend our previous clinical and experimental observations, and point toward the microvasculature as a potential target for differential modulation of the radiation response in tumors and normal tissues. For example, we have shown experimentally that endothelial TM in rat small bowel decreases during ongoing radiation therapy.8 Furthermore, decreased TM in lung capillaries during pneumonia is associated with fibrin deposition (unpublished data). Hence, it is conceiv-
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able that maintaining endothelial TM levels would preserve microvascular patency and prevent clot formation. In the clinical situation, this may prevent an increase in the fraction of hypoxic tumor cells during therapy and, at the same time, protect the surrounding normal tissue. Conversely, it is possible that a relative TM deficiency in tumor vessels after irradiation may contribute to tumor regression, in addition to the direct radiation-induced mitotic cell death and apoptosis. Our finding of similar TM levels in vessels in primary tumors and (unirradiated) tumors that recurred in an irradiated tumor bed shows that tumor angiogenesis is associated with de novo synthesis of TM. In normal (nonmalignant) rectal submucosa, the proportion of TM-positive vessels was somewhat greater in the late group than in the early group (26% versus 5%). Preclinical studies from our laboratory indicate that significant recovery of TM in irradiated vessels is unlikely.8 However, further studies are needed to determine whether the apparent moderate increase in the late group relative to the early group found in the present clinical material represents partial recovery in irradiated vessels. It is also possible that there is angiogenesis with associated TM synthesis, a differential endothelial response in the preoperative relative to the postoperative irradiation setting, or that the severity of radiation injury determines whether TM production can recover or not. Fibrosis and vascular sclerosis in radiation enteropathy are associated with overexpression of the fibrogenic cytokine TGF-b.9,10 TGF-b is found at high levels in a-granules of platelets and is thus released at sites of radiationinduced vascular injury. TGF-b regulates a diverse array of biological functions, including cell migration, cell differentiation, and immune function, and affects both tissue remodeling and fibrosis in normal tissue injury and the progression of malignancy.11–13 The in vivo significance of the reciprocal regulation of TM and TGF-b after irradiation requires further elucidation. TGF-b is a potent downregulator of TM. On the other hand, decreased TM leads to clotting and increased local release of TGF-b from platelets and autoinduction of TGF-b synthesis. This may initiate a self-perpetuating cycle that may be responsible for the chronicity of radiation toxicity. Our finding of increased TGF-b immunoreactivity in tumors relative to normal rectum agrees with previous reports of increased TGF-b protein and TGF-b mRNA in colorectal cancer.14,15 Similarly, increased production of TGF-b by tumor-associated stromal cells has been reported in breast cancer.16 The decrease in tumor TGF-b immunoreactivity after radiotherapy found in the present study likely reflects the response of the tumor to the treatment. In contrast, normal (unirradiated) intestine exhibits low TGF-b immunoreactivity levels, and the postradiation increase reflects inflammatory and fibrogenic processes.1,3 TGF-b has several tumor growth promoting properties, including stimulation of angiogenesis, suppression of immune response, and stimulation of invasion and metastatic cell spread through induction of adhesion molecule receptors and increased protease activity.12,17,18 However, TGF-b also inhibits epithelial cell growth and reduces proteolysis by suppression of protease transcription.19 This apparent contradiction may be explained by in vitro data
suggesting that TGF-b promotes the growth of cancer cells with microsatellite instability by indirect proteolytic alteration of the TGF-b type II receptor.20 The predominant role of tissue plasminogen activator (t-PA) relates to fibrinolysis, whereas uPA is also a major regulator of tissue remodeling, tumor invasion, and metastasis. The distribution of uPA-positive cells in tumors and normal rectum in the present study is in accordance with other reports.6,21 It is conceivable that the reduction of uPA-positive cells found in rectal tumors after radiotherapy indicates reduced invasive capacity. There are several biologically important interactions among TM, TGF-b, and uPA. Local plasmin activity depends on the balance between plasminogen activators and their inhibitors, as well as on regulation by other factors, including TM and TGF-b. TM greatly enhances the normal inhibition by thrombin of the conversion of pro-uPA to active uPA.22 Although radiation significantly reduced the number of cells producing uPA in the present study, the decrease in TM may actually increase uPA activation, and subsequently increase local plasmin levels and enhance TGF-b activation. Therefore, radiation-induced TM-depression may lead to a relative increase in uPA activity and promote tumor invasiveness. Active TGF-b decreases plasmin levels through inhibition of uPA production by endothelial cells and increased plasminogen activator inhibitor levels. Conversely, plasmin acts as an important regulator of TGF-b activity by activating latent TGF-b once it is bound to the mannose-6-phosphate/insulin-like growth factor II receptor on the cell membrane.23 The results from the present study suggest that the development of strategies to preserve or enhance endothelial anticoagulant function during and after radiation treatment may increase the therapeutic ratio of radiation. Maintaining TM in tumors may prevent the development hypoxia and resulting radioresistance during treatment. TM may also contribute to reducing tumor invasiveness through reduced activation of uPA. Moreover, maintaining TM in normal tissues may prevent endothelial dysfunction and local increase in TGF-b release, and thereby abrogate long-term normal tissue injury. Further studies using synthetic or biologic modulators of coagulation and endothelial function are needed to test this hypothesis.
REFERENCES 1. Richter KK, Fink LM, Hughes BM, et al. Is the loss of endothelial thrombomodulin involved in the mechanism of chronicity in late radiation enteropathy? Radiother Oncol. 1997;44:65–71. 2. Richter KK, Langberg CW, Sung C-C, Hauer-Jensen M. Increased transforming growth factor b (TGF-b) immunoreactivity is independently associated with chronic injury in both consequential and primary radiation enteropathy. Int J Radiat Oncol Biol Phys. 1997;39:187–195. 3. Richter KK, Sung C-C, Langberg CW, Hauer-Jensen M. Association of transforming growth factor b (TGF-b) immunoreactivity with specific histopathologic lesions in subacute and chronic experimental radiation enteropathy. Radiother Oncol. 1996;39:201– 302. 4. Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis— correlation in invasive breast carcinoma. NEJM. 1991;324:1– 8. 5. Mulcahy HE, Duffy MJ, Gibbons D, et al. Urokinase-type plas-
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minogen activator and outcome in Dukes’ B colorectal cancer. Lancet. 1994;344:583–584. 6. Sier CFM, Fellbaum C, Verspaget HW, et al. Immunolocalization of urokinase-type plasminogen activator in adenomas and carcinomas of the colorectum. Histopathology. 1991;19:231–237. 7. Dittman WA, Majerus PW. Structure and function of thrombomodulin: a natural anticoagulant. Blood. 1990;75:329 – 336. 8. Wang J, Richter KK, Sung C-C, Hauer-Jensen M. Association of chronic endothelial dysfunction with sustained transforming growth factor-b (TGF-b) overexpression and progression of experimental radiation enteropathy. Radiation Res Soc. 1997;45:194. 9. Langberg CW, Hauer-Jensen M, Sung C-C, Kane CJM. Expression of fibrogenic cytokines in rat small intestine after fractionated irradiation. Radiother Oncol. 1994;32:29 –36. 10. Canney PA, Dean S. Transforming growth factor beta: a promotor of late connective tissue injury following radiotherapy? Br J Radiol. 1990;63:620 – 623. 11. Lahm H. Role of transforming growth factor b in colorectal cancer. Growth Factors. 1993;9:1–9. 12. Ueki N, Nakazato M, Ohkawa T, et al. Excessive production of transforming growth factor b1 can play an important role in the development of tumorigenesis by its action for angiogenesis: validity of neutralizing antibodies to block tumor growth. Biochim Biophys Acta. 1992;1137:189 –196. 13. Wrana JL, Attisano L, Wieser R, et al. Mechanism of activation of the TGF-beta receptor. Nature. 1994;370:341–347. 14. Friedman E, Gold LI, Klimstra D, et al. High levels of transforming growth factor b1 correlate with disease progression in human colon cancer. Cancer Epidemiol Biomark Prevent. 1995;4: 549 –554. 15. Tsushima H, Kawata S, Tamura S, et al. High levels of trans-
forming growth factor b1 in patients with colorectal cancer: association with disease progression. Gastroenterology. 1996;110:375– 382. 16. Kong F-M, Anscher MS, Murase T, et al. Elevated plasma transforming growth factor beta1 levels in breast cancer patients decrease after surgical removal of the tumor. Ann Surg. 1995;222: 155–162. 17. Li XF, Takiuchi H, Zou JP, et al. Transforming growth factor-b (TGF-b)-mediated immunosuppression in the tumor-bearing state: enhanced production of TGF-b and a progressive increase in TGF-b susceptibility of anti-tumor CD41 T cell function. Jpn/J Cancer Res. 1993;84:315–325. 18. Samuel SK, Hurta RAR, Kondaiah P, et al. Autocrine induction of tumor protease production and invasion by a metallothionine-regulated TGF-b1. EMBO J. 1992;11:1599 –1605. 19. Barnard JA, Beauchamp RD, Coffey RJ, Moses HL. Regulation of intestinal epithelial growth by transforming growth factor type b. Proc Natl Acad Sci USA. 1989;86:1578 –1582. 20. Markowitz S, Wang J, Myeroff L, et al. Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability. Science. 1995;268:1336 –1338. 21. Pyke C, Kristensen P, Ralfkiaer E, et al. Urokinase-type plasminogen activator is expressed in stromal cells and its receptor in cancer cells at invasive foci in human colon adenocarcinomas. Am J Pathol. 1991;138:1059 –1067. 22. Wilhelm S, Wilhelm O, Schmitt M, Graeff H. Inactivation of receptor-bound pro-urokinase-type plasminogen activator (prouPA) by thrombin and thrombin/thrombomodulin complex. Biol Chem Hoppe-Seyler. 1994;375:603– 608. 23. Nunes I, Munger JS, Harpel JG, et al. Structure and activation of the large latent transforming growth factor-beta complex. Int J Obes Relat Metab Disord. 1996;20(suppl 3):S4 – 8.
DISCUSSION
thrombomodulin levels would preserve microvascular patency and prevent clot formation. This is important in your hypothesis, and I wondered if you could expand on the basis for this statement? Secondly, while it didn’t achieve statistical significance in your figure indicating the results with TGF beta, it appears that there are increases in normal tissue but decreases in the tumor, particularly during the early phase after radiation. And this obviously is different than the results that you see with thrombomodulin. I would ask you to comment on why there appeared to be differences in the response of these two agents in response to the stimulus? And finally, I would like to ask you why you feel that there’s a difference in rectal tumors compared with normal tissue and what implications that might have for the response to radiation therapy. Carey P. Page, MD (San Antonio, Texas): Could you speculate on the modulation of thrombomodulin with local nutrients, such as short-chain fatty acids and glutamine? Tim Nelson, MD (Albuquerque, New Mexico): You postulate that thrombomodulin would cause the problems of radiation enteritis, but what about the potential benefit of down-regulating thrombomodulin to tumor growth or suppression?
Jon S. Thompson, MD (Omaha, Nebraska): This work points the way to a new era in our understanding of radiation-induced gastrointestinal problems, and, as reported by Dr. Richter and his colleagues, represents the clinical application of information that they’ve learned from carefully performed animal studies and previous clinical studies that have identified an association between changes in cytokines and radiation exposure that could explain many of the changes observed in our patients who received radiation to the GI tract, either as part of their therapy or as a side effect of that therapy. The need for this work was identified by Dr. Lang, who reviewed the literature over the past several years on radiation enteritis. When you look at all the different theories that have been postulated for what causes it, and all the different therapies that have been applied to this problem, including alterations in diet, binding bile salts, and a variety of other anti-inflammatory type programs, this information clearly indicates that there’s a need for better understanding of the causes of this condition so that we can help prevent or ameliorate the problem. This paper, then, represents a beginning of our understanding of radiation enteritis and radiation proctitis at the cellular level. These new approaches hopefully will provide new avenues for prevention as well as to improve the benefits from radiation therapy by manipulating these critical elements responsible for the changes observed in radiation enteritis. In the manuscript you state that maintaining endothelial 646
CLOSING Konrad K. Richter, MD: In response to the first question, maintaining microvascular patency in rectal cancer and normal rectum is a good thing for tumor control because it increases tumor oxygenation and therefore in-
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creases radiosensitivity. It may also be better to maintain microvascular patency in normal intestine, since endothelial dysfunction is an important part of the pathogenesis of chronic intestinal injury. This may reduce vascular sclerosis and hypercoagulation and thrombosis, and the factors that eventually lead to fibrosis and other problems. In response to the question that TGFb was up and thrombomodulin was down, if there is clot formation because of endothelial damage and down-regulation of thrombomodulin, there will be an increase of TGFb. Since TGFb is
found in high amounts in platelets, it will be released at the site of injury. Regarding the question how we can modulate thrombomodulin, taking our results into account, one option would be to stimulate thrombomodulin synthesis in endothelial cells. We didn’t look at glutamine and short-chain fatty acids, but there are ways to stimulate thrombomodulin synthesis. Other options are to influence the coagulation system, for example, by using specific thrombin inhibitors or increasing anticoagulant properties.
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BACKGROUND: Chronic radiation injury of the intestine is associated with significant underexpression of a potent physiological anticoagulant, endothelial cell thrombomodulin (TM). This study compared early and late radiation-induced changes in endothelial TM, urokinase plasminogen activator (uPA), and transforming growth factor b (TGF-b) in normal rectum and tumors. METHODS: Rectal resection specimens from 27 patients were analyzed: Nine patients underwent primary resection of rectal cancer, 11 tumors were resected after neo-adjuvant radiotherapy, and 7 because of local recurrence after prior resection and adjuvant radiotherapy. TM, uPA, and extracellular matrix-associated TGF-b immunoreactivity were assessed using computerized image analysis. RESULTS: Multivariate analysis revealed that tumors had more TM-positive vessels (P 5 0.003), more uPA-positive cells (P <0.001), and higher TGF-b immunoreactivity levels (P <0.001) than normal rectum. Preoperative irradiation was associated with decreased proportions of TM-positive vessels in tumors (P 5 0.003) and normal rectum (P <0.001). Irradiated tumors had fewer uPA-positive cells (P 5 0.003) and less TGF-b immunoreactivity (P 5 0.001) than unirradiated tumors. The proportion of TM-positive vessels in irradiated rectum from patients with recurrence was decreased (P 5 0.03), whereas the recurrent (ie, unirradiated) tumors did not differ from primary tumors in terms of TM, TGF-b, or uPA immunoreactivity. CONCLUSIONS: The results support a role for endothelial dysfunction in the pathogenesis of radiation proctitis. Maintaining endothelial cell anticoagulant function may be a potential method to optimize the therapeutic ratio of adjuvant radio-
From the Departments of Surgery and Pathology, University of Arkansas for Medical Sciences and John L. McClellan Veterans Affairs Medical Center, Little Rock, Arkansas. This work was supported by a grant from the American Cancer Society (RPG-93-008-04 EDT). Requests for reprints should be addressed to Martin HauerJensen, MD, PhD, University of Arkansas for Medical Sciences, 4301 West Markham, Slot 725, Little Rock, Arkansas 72205. Presented at the 50th Annual Meeting of the Southwestern Surgical Congress, San Antonio, Texas, April 19 –22, 1998.
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© 1998 by Excerpta Medica, Inc. All rights reserved.
therapy of rectal cancer. Am J Surg. 1998;176: 642– 647. © 1998 by Excerpta Medica, Inc.
T
he risk of injury to the normal intestine (radiation enteropathy) is a major dose-limiting factor during radiation therapy for colorectal tumors. Radiation enteropathy is characterized by microvascular injury, fibrosis, and intestinal dysfunction. However, the underlying molecular mechanisms have not been identified. We have shown previously that chronic small bowel radiation injury is associated with significant upregulation of the fibrogenic cytokine, transforming growth factor b (TGF-b), and downregulation of the endothelial cell surface protein, thrombomodulin (TM).1–3 TM plays a pivotal role in the protein C pathway and is essential for maintaining the normal coagulation-anticoagulation homeostasis. Radiation-induced TM downregulation may initiate and maintain local microvascular injury, inflammation, and fibrosis. This may affect the response of the tumor to treatment, as well as the development of normal tissue radiation toxicity. The present study, therefore, compared changes in TM, TGF-b, and urokinase plasminogen activator (uPA) in normal rectum and rectal tumors at early and late times after irradiation. The results suggest that maintaining endothelial function during and after treatment may be a promising approach to enhance the efficacy and safety of adjuvant pelvic radiotherapy.
MATERIALS AND METHODS Rectal resection specimens from 27 patients (25 men, 2 women; median age 66 years, range 46 to 79) were obtained from the archives of the Department of Pathology: Nine patients had undergone low anterior or abdominoperineal resection of rectal cancer (“unirradiated” group). Eleven patients had been resected 7 weeks (median, range 21 to 61 days) after neo-adjuvant radiotherapy (“early” group). Seven patients underwent resection of local recurrence, 12 months (median, range 102 to 841 days) after prior resection and adjuvant radiotherapy (“late” group). Patients in the early and late groups received adjuvant therapy either as 30 Gy in 10 fractions or as 45 Gy in 25 fractions with concomitant 5-fluorouracil. The histopathologic diagnosis was reviewed and confirmed by a pathologist (LMF) using hematoxylin-eosinstained slides. Paraffin-sections of tumor and nonmalignant rectal tissue from each patient were stained with antibodies against TM (DakoTM 1009; Dako, Calenteria, California), TGF-b (AB-100-NA; R&D Systems, Minneapolis, Minnesota), and uPA (#3689; American Diagnostica, Green0002-9610/98/$19.00 PII S0002-9610(98)00280-3
RADIATION EFFECT ON RECTAL ENDOTHELIAL CELL FUNCTION/RICHTER ET AL
Figure 1. Proportion of thrombomodulin (TM)-positive vessels in normal tissue (left) and tumor (right) in rectal resection specimens from patients undergoing primary resection (no XRT), patients resected after neo-adjuvant radiotherapy (“early”), and patients resected for recurrence after a previous resection followed by postoperative adjuvant radiotherapy (“late”). The box plots show median, interquartile range (IQR), and upper and lower adjacent values.
wich, Connecticut) using appropriate negative and positive controls and standard immunohistochemical techniques as described elsewhere.1 Interactive computer-assisted image analysis of immunoreactivity was performed using the Samba 4000 System (Dynatech Laboratories, Chantilly, Virginia) as reported previously.1–3 Vessels with diameters between 5 and 70 mm in the submucosa of normal rectum and in a highly vascular area in the tumor sections were assessed for TM immunoreactivity according to a predefined grid pattern. Identification and counting of tumor vessels were performed using a modified Folkman assay.4 Twenty fields per slide were analyzed, and the mean proportion of TM-positive vessels in each section was considered a single value for statistical purposes. The area (measured in pixel units) positive for extracellular matrix-associated TGF-b was assessed in 20 fields per specimen according to a predefined grid pattern. The number of uPA-positive cells was assessed in 20 fields per slide and graded using an adaptation of the method described by Mulcahy et al:5 1 5 no positive cells, 2 5 few scattered positive cells, 3 5 abundant positive cells grouped in few clusters, and 4 5 abundant positive cells in clusters in multiple areas. Differences in the relative and total number of TMpositive vessels, TGF-b immunoreactivity, and uPA-positive cells were assessed with fixed-factor ANOVA using NCSS 6 for Windows (NCSS, Kaysville, Utah). Post-hoc testing was performed using StatXact 3 for Windows (Cytel Software, Cambridge, Massachusetts), a software package for exact nonparametric inference. Two-sided tests were used, and P values less than 0.05 were considered statistically significant.
RESULTS All three antibodies exhibited specific immunoreactivity to TM, TGF-b, and uPA, respectively, and qualitative staining patterns as reported previously by our laboratory and others.1,6 TM-positive vessels exhibited well-defined endothelial staining, both in tumor specimens and in normal rectum. Extracellular matrix-associated TGF-b staining was present in connective tissue in normal rectal specimens and in areas around malignant glands in tumors. Immunoreactivity for uPA was found in clusters of fibroblast-like cells, mononuclear cells, and leukocytes surrounding invading malignant glands. In normal rectum, uPA staining was restricted to few, scattered inflammatory cells. Analysis of variance revealed that tumors exhibited a significantly higher proportion of TM-positive vessels (P 5 0.003), higher TGF-b immunoreactivity levels (P ,0.001), and more uPA-positive cells (P ,0.001) than normal rectal tissue. Preoperative irradiation (early group) was associated with a decrease in the proportion of TMpositive vessels, both in tumors (P 5 0.003) and in normal, irradiated rectum (P ,0.001). The proportion of TM-positive vessels in irradiated rectum from the late group was also smaller than in the unirradiated primary resection specimens (P 5 0.03; Figure 1). Irradiated tumors exhibited less extracellular matrix-associated TGF-b immunoreactivity (P 5 0.001) than unirradiated tumors, whereas the differences in TGF-b immunoreactivity among the groups of nonmalignant rectum (unirradiated, early, and late) did not reach statistical significance (Figure 2). The number of uPA-positive cells was significantly lower in irradiated tumors than in nonirradiated primary tumors (P 5 0.003; Figure 3). Recurrent (ie, not
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Figure 2. Extracellular matrix-associated transforming growth factor b (TGF-b) immunoreactivity in normal tissue (left) and tumor (right) in rectal resection specimens. Patient groups and box plot attributes as for Figure 1.
Figure 3. Grading of urokinase plasminogen activator (uPA)-positive cells in normal tissue (left) and tumor (right) in rectal resection specimens. Patient groups as for Figure 1. 1 5 no positive cells, 2 5 few scattered positive cells, 3 5 abundant positive cells grouped in few clusters, 4 5 abundant positive cells in clusters in multiple areas.
previously irradiated) tumors did not differ significantly from primary tumors in terms of TM, TGF-b, or uPA immunoreactivity levels.
COMMENTS Vascular injury is a prominent and characteristic feature of chronic radiation injury in most organs. Initial endothelial cell damage increases vascular permeability and causes leakage of plasma proteins and subsequent development of obliterative vascular sclerosis and tissue fibrosis. However, the molecular links between endothelial function and chronic radiation toxicity have not yet been established in vivo. In the normal vasculature in most organs, TM acts as an important “natural anticoagulant” by changing the sub644
strate specificity of thrombin, so that thrombin no longer converts fibrinogen to fibrin, but instead activates protein C.7 Reduced endothelial TM levels may contribute to hypercoagulation with increased fibrin formation, platelet aggregation, and subsequent upregulation or release of inflammatory and fibrogenic cytokines, such as TGF-b. The results from the present study confirm and extend our previous clinical and experimental observations, and point toward the microvasculature as a potential target for differential modulation of the radiation response in tumors and normal tissues. For example, we have shown experimentally that endothelial TM in rat small bowel decreases during ongoing radiation therapy.8 Furthermore, decreased TM in lung capillaries during pneumonia is associated with fibrin deposition (unpublished data). Hence, it is conceiv-
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able that maintaining endothelial TM levels would preserve microvascular patency and prevent clot formation. In the clinical situation, this may prevent an increase in the fraction of hypoxic tumor cells during therapy and, at the same time, protect the surrounding normal tissue. Conversely, it is possible that a relative TM deficiency in tumor vessels after irradiation may contribute to tumor regression, in addition to the direct radiation-induced mitotic cell death and apoptosis. Our finding of similar TM levels in vessels in primary tumors and (unirradiated) tumors that recurred in an irradiated tumor bed shows that tumor angiogenesis is associated with de novo synthesis of TM. In normal (nonmalignant) rectal submucosa, the proportion of TM-positive vessels was somewhat greater in the late group than in the early group (26% versus 5%). Preclinical studies from our laboratory indicate that significant recovery of TM in irradiated vessels is unlikely.8 However, further studies are needed to determine whether the apparent moderate increase in the late group relative to the early group found in the present clinical material represents partial recovery in irradiated vessels. It is also possible that there is angiogenesis with associated TM synthesis, a differential endothelial response in the preoperative relative to the postoperative irradiation setting, or that the severity of radiation injury determines whether TM production can recover or not. Fibrosis and vascular sclerosis in radiation enteropathy are associated with overexpression of the fibrogenic cytokine TGF-b.9,10 TGF-b is found at high levels in a-granules of platelets and is thus released at sites of radiationinduced vascular injury. TGF-b regulates a diverse array of biological functions, including cell migration, cell differentiation, and immune function, and affects both tissue remodeling and fibrosis in normal tissue injury and the progression of malignancy.11–13 The in vivo significance of the reciprocal regulation of TM and TGF-b after irradiation requires further elucidation. TGF-b is a potent downregulator of TM. On the other hand, decreased TM leads to clotting and increased local release of TGF-b from platelets and autoinduction of TGF-b synthesis. This may initiate a self-perpetuating cycle that may be responsible for the chronicity of radiation toxicity. Our finding of increased TGF-b immunoreactivity in tumors relative to normal rectum agrees with previous reports of increased TGF-b protein and TGF-b mRNA in colorectal cancer.14,15 Similarly, increased production of TGF-b by tumor-associated stromal cells has been reported in breast cancer.16 The decrease in tumor TGF-b immunoreactivity after radiotherapy found in the present study likely reflects the response of the tumor to the treatment. In contrast, normal (unirradiated) intestine exhibits low TGF-b immunoreactivity levels, and the postradiation increase reflects inflammatory and fibrogenic processes.1,3 TGF-b has several tumor growth promoting properties, including stimulation of angiogenesis, suppression of immune response, and stimulation of invasion and metastatic cell spread through induction of adhesion molecule receptors and increased protease activity.12,17,18 However, TGF-b also inhibits epithelial cell growth and reduces proteolysis by suppression of protease transcription.19 This apparent contradiction may be explained by in vitro data
suggesting that TGF-b promotes the growth of cancer cells with microsatellite instability by indirect proteolytic alteration of the TGF-b type II receptor.20 The predominant role of tissue plasminogen activator (t-PA) relates to fibrinolysis, whereas uPA is also a major regulator of tissue remodeling, tumor invasion, and metastasis. The distribution of uPA-positive cells in tumors and normal rectum in the present study is in accordance with other reports.6,21 It is conceivable that the reduction of uPA-positive cells found in rectal tumors after radiotherapy indicates reduced invasive capacity. There are several biologically important interactions among TM, TGF-b, and uPA. Local plasmin activity depends on the balance between plasminogen activators and their inhibitors, as well as on regulation by other factors, including TM and TGF-b. TM greatly enhances the normal inhibition by thrombin of the conversion of pro-uPA to active uPA.22 Although radiation significantly reduced the number of cells producing uPA in the present study, the decrease in TM may actually increase uPA activation, and subsequently increase local plasmin levels and enhance TGF-b activation. Therefore, radiation-induced TM-depression may lead to a relative increase in uPA activity and promote tumor invasiveness. Active TGF-b decreases plasmin levels through inhibition of uPA production by endothelial cells and increased plasminogen activator inhibitor levels. Conversely, plasmin acts as an important regulator of TGF-b activity by activating latent TGF-b once it is bound to the mannose-6-phosphate/insulin-like growth factor II receptor on the cell membrane.23 The results from the present study suggest that the development of strategies to preserve or enhance endothelial anticoagulant function during and after radiation treatment may increase the therapeutic ratio of radiation. Maintaining TM in tumors may prevent the development hypoxia and resulting radioresistance during treatment. TM may also contribute to reducing tumor invasiveness through reduced activation of uPA. Moreover, maintaining TM in normal tissues may prevent endothelial dysfunction and local increase in TGF-b release, and thereby abrogate long-term normal tissue injury. Further studies using synthetic or biologic modulators of coagulation and endothelial function are needed to test this hypothesis.
REFERENCES 1. Richter KK, Fink LM, Hughes BM, et al. Is the loss of endothelial thrombomodulin involved in the mechanism of chronicity in late radiation enteropathy? Radiother Oncol. 1997;44:65–71. 2. Richter KK, Langberg CW, Sung C-C, Hauer-Jensen M. Increased transforming growth factor b (TGF-b) immunoreactivity is independently associated with chronic injury in both consequential and primary radiation enteropathy. Int J Radiat Oncol Biol Phys. 1997;39:187–195. 3. Richter KK, Sung C-C, Langberg CW, Hauer-Jensen M. Association of transforming growth factor b (TGF-b) immunoreactivity with specific histopathologic lesions in subacute and chronic experimental radiation enteropathy. Radiother Oncol. 1996;39:201– 302. 4. Weidner N, Semple JP, Welch WR, Folkman J. Tumor angiogenesis and metastasis— correlation in invasive breast carcinoma. NEJM. 1991;324:1– 8. 5. Mulcahy HE, Duffy MJ, Gibbons D, et al. Urokinase-type plas-
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minogen activator and outcome in Dukes’ B colorectal cancer. Lancet. 1994;344:583–584. 6. Sier CFM, Fellbaum C, Verspaget HW, et al. Immunolocalization of urokinase-type plasminogen activator in adenomas and carcinomas of the colorectum. Histopathology. 1991;19:231–237. 7. Dittman WA, Majerus PW. Structure and function of thrombomodulin: a natural anticoagulant. Blood. 1990;75:329 – 336. 8. Wang J, Richter KK, Sung C-C, Hauer-Jensen M. Association of chronic endothelial dysfunction with sustained transforming growth factor-b (TGF-b) overexpression and progression of experimental radiation enteropathy. Radiation Res Soc. 1997;45:194. 9. Langberg CW, Hauer-Jensen M, Sung C-C, Kane CJM. Expression of fibrogenic cytokines in rat small intestine after fractionated irradiation. Radiother Oncol. 1994;32:29 –36. 10. Canney PA, Dean S. Transforming growth factor beta: a promotor of late connective tissue injury following radiotherapy? Br J Radiol. 1990;63:620 – 623. 11. Lahm H. Role of transforming growth factor b in colorectal cancer. Growth Factors. 1993;9:1–9. 12. Ueki N, Nakazato M, Ohkawa T, et al. Excessive production of transforming growth factor b1 can play an important role in the development of tumorigenesis by its action for angiogenesis: validity of neutralizing antibodies to block tumor growth. Biochim Biophys Acta. 1992;1137:189 –196. 13. Wrana JL, Attisano L, Wieser R, et al. Mechanism of activation of the TGF-beta receptor. Nature. 1994;370:341–347. 14. Friedman E, Gold LI, Klimstra D, et al. High levels of transforming growth factor b1 correlate with disease progression in human colon cancer. Cancer Epidemiol Biomark Prevent. 1995;4: 549 –554. 15. Tsushima H, Kawata S, Tamura S, et al. High levels of trans-
forming growth factor b1 in patients with colorectal cancer: association with disease progression. Gastroenterology. 1996;110:375– 382. 16. Kong F-M, Anscher MS, Murase T, et al. Elevated plasma transforming growth factor beta1 levels in breast cancer patients decrease after surgical removal of the tumor. Ann Surg. 1995;222: 155–162. 17. Li XF, Takiuchi H, Zou JP, et al. Transforming growth factor-b (TGF-b)-mediated immunosuppression in the tumor-bearing state: enhanced production of TGF-b and a progressive increase in TGF-b susceptibility of anti-tumor CD41 T cell function. Jpn/J Cancer Res. 1993;84:315–325. 18. Samuel SK, Hurta RAR, Kondaiah P, et al. Autocrine induction of tumor protease production and invasion by a metallothionine-regulated TGF-b1. EMBO J. 1992;11:1599 –1605. 19. Barnard JA, Beauchamp RD, Coffey RJ, Moses HL. Regulation of intestinal epithelial growth by transforming growth factor type b. Proc Natl Acad Sci USA. 1989;86:1578 –1582. 20. Markowitz S, Wang J, Myeroff L, et al. Inactivation of the type II TGF-beta receptor in colon cancer cells with microsatellite instability. Science. 1995;268:1336 –1338. 21. Pyke C, Kristensen P, Ralfkiaer E, et al. Urokinase-type plasminogen activator is expressed in stromal cells and its receptor in cancer cells at invasive foci in human colon adenocarcinomas. Am J Pathol. 1991;138:1059 –1067. 22. Wilhelm S, Wilhelm O, Schmitt M, Graeff H. Inactivation of receptor-bound pro-urokinase-type plasminogen activator (prouPA) by thrombin and thrombin/thrombomodulin complex. Biol Chem Hoppe-Seyler. 1994;375:603– 608. 23. Nunes I, Munger JS, Harpel JG, et al. Structure and activation of the large latent transforming growth factor-beta complex. Int J Obes Relat Metab Disord. 1996;20(suppl 3):S4 – 8.
DISCUSSION
thrombomodulin levels would preserve microvascular patency and prevent clot formation. This is important in your hypothesis, and I wondered if you could expand on the basis for this statement? Secondly, while it didn’t achieve statistical significance in your figure indicating the results with TGF beta, it appears that there are increases in normal tissue but decreases in the tumor, particularly during the early phase after radiation. And this obviously is different than the results that you see with thrombomodulin. I would ask you to comment on why there appeared to be differences in the response of these two agents in response to the stimulus? And finally, I would like to ask you why you feel that there’s a difference in rectal tumors compared with normal tissue and what implications that might have for the response to radiation therapy. Carey P. Page, MD (San Antonio, Texas): Could you speculate on the modulation of thrombomodulin with local nutrients, such as short-chain fatty acids and glutamine? Tim Nelson, MD (Albuquerque, New Mexico): You postulate that thrombomodulin would cause the problems of radiation enteritis, but what about the potential benefit of down-regulating thrombomodulin to tumor growth or suppression?
Jon S. Thompson, MD (Omaha, Nebraska): This work points the way to a new era in our understanding of radiation-induced gastrointestinal problems, and, as reported by Dr. Richter and his colleagues, represents the clinical application of information that they’ve learned from carefully performed animal studies and previous clinical studies that have identified an association between changes in cytokines and radiation exposure that could explain many of the changes observed in our patients who received radiation to the GI tract, either as part of their therapy or as a side effect of that therapy. The need for this work was identified by Dr. Lang, who reviewed the literature over the past several years on radiation enteritis. When you look at all the different theories that have been postulated for what causes it, and all the different therapies that have been applied to this problem, including alterations in diet, binding bile salts, and a variety of other anti-inflammatory type programs, this information clearly indicates that there’s a need for better understanding of the causes of this condition so that we can help prevent or ameliorate the problem. This paper, then, represents a beginning of our understanding of radiation enteritis and radiation proctitis at the cellular level. These new approaches hopefully will provide new avenues for prevention as well as to improve the benefits from radiation therapy by manipulating these critical elements responsible for the changes observed in radiation enteritis. In the manuscript you state that maintaining endothelial 646
CLOSING Konrad K. Richter, MD: In response to the first question, maintaining microvascular patency in rectal cancer and normal rectum is a good thing for tumor control because it increases tumor oxygenation and therefore in-
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creases radiosensitivity. It may also be better to maintain microvascular patency in normal intestine, since endothelial dysfunction is an important part of the pathogenesis of chronic intestinal injury. This may reduce vascular sclerosis and hypercoagulation and thrombosis, and the factors that eventually lead to fibrosis and other problems. In response to the question that TGFb was up and thrombomodulin was down, if there is clot formation because of endothelial damage and down-regulation of thrombomodulin, there will be an increase of TGFb. Since TGFb is
found in high amounts in platelets, it will be released at the site of injury. Regarding the question how we can modulate thrombomodulin, taking our results into account, one option would be to stimulate thrombomodulin synthesis in endothelial cells. We didn’t look at glutamine and short-chain fatty acids, but there are ways to stimulate thrombomodulin synthesis. Other options are to influence the coagulation system, for example, by using specific thrombin inhibitors or increasing anticoagulant properties.
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