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Experimental Therapeutics |
Pharmacology and Therapeutics Unit (FATH 5349) [P. S., A. B., C. D., J-L. B., O. F.], Cardiovascular Pathology Unit [X. H.], and Radiobiology and Radioprotection Unit [V. G.], University of Louvain Medical School, B-1200 Brussels, Belgium
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ABSTRACT |
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 Top ABSTRACT INTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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INTRODUCTION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Among the potential targets of irradiation on ECs, we chose to focus on the isoform of NOS (eNOS) expressed in these cells for two major reasons. First, NO is a key mediator of the proangiogenic effects of different growth factors, including VEGF (11) . Second, in the context of cardiovascular diseases, transcriptional and posttranslational regulations of eNOS have been reported on exposure to ROS (12, 13, 14) . ROS are produced in large amounts at the time of irradiation through chain reactions initiated by water radiolysis. Moreover, beside this regulation of the enzyme activity, NO and ROS are known to scavenge each other and thereby mutually modulate their biological effects (15) .
Therefore, we set up a series of expressional and functional experiments, with emphasis on the regulation of NO, to evaluate the effects of irradiation in various models including EC cultures, isolated tumor microvessels, mouse-implanted Matrigel (basement membrane matrix) plugs and tumors as well as human tumor biopsies. Our data reveal that low-dose irradiation can induce NO-mediated pathways leading to EC migration, colonization of host tissues, and organization in vascular network. These findings underline a hitherto downplayed but distinctive angiogenic response of tumors to irradiation and emphasize the need for associating radiotherapy with antiangiogenic approaches to enhance its overall therapeutic efficacy.
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MATERIALS AND METHODS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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Irradiation.
Cells and anesthetized mice were irradiated using the RT-250 device (Philips) for a dose delivery of 0.86 and 0.76 Gy/min, respectively. For the in vivo experiments, mice were anesthetized with ketamine/xylazine, and the tumor was centered in a 3-cm-diameter circular irradiation field.
Immunoblotting, IP, and NO Determination.
Immunoblotting and IPs were carried out, as described previously (18
, 19)
, with antibodies directed against caveolin-1, eNOS, and Akt (Becton Dickinson, Lexington, KY) and with phospho-specific antibodies against phospho-Ser1177-eNOS and phospho-Ser473-Akt (NEB Cell Signaling Technologies, Beverly, MA). The determination of NO level, e.g., the 24-h accumulation of NO derivatives in the cell-bathing (serum-deprived) medium, was carried out using the Nitric Oxide Colorimetric Assay (Roche Diagnostics, Mannheim, Germany).
In Vitro Migration and Angiogenesis Assays.
To determine the ability of irradiated ECs to migrate, the scratch injury model (e.g., scraping of a 0.5-mm-wide line across confluent, serum-starved bovine aortic endothelial cells) was used as described previously (20)
. For the quantitative analysis, a migration index was defined as the ratio (expressed as a percentage) of the density of migrating cells in the center of the scratch zone versus the density of surviving (e.g., nondetached) cells in a size-matched area of the unwounded region; this allowed us to integrate cell death in the evaluation of the process of irradiation-induced migration. Of note, under our experimental conditions, cell detachment occurred in the first 24 h after irradiation, and the calculated LD37 value (i.e., the dose reducing survival to 37% of cells) amounted to 2.1 Gy. To assess the formation of capillary-like tubes, an in vitro angiogenesis assay (e.g., plating of human umbilical vein endothelial cells on GFR-Matrigel) was used as reported previously (18)
. Cell migration and tube formation, respectively, were observed using an inverted phase-contrast microscope and quantified by analysis of images randomly captured by a video camera system.
Matrigel Plug Assay.
Anesthetized C57Bl/6J mice received a s.c. injection of 1 ml of GFR-Matrigel (Becton Dickinson, Bedford, MA) supplemented with 10 µg of VEGF and 100 µg of heparin. The plugs were allowed to be partially colonized by host cells for 10 days and then locally irradiated (6 Gy). All mice were sacrificed 5 days later, and paraffin-embedded 5-µm-thick sections of the plugs were immunostained with rabbit polyclonal anti-eNOS (Calbiochem, San Diego, CA), anti-von Willebrand factor (Sigma, St. Louis, MO), and rat monoclonal anti-CD31 antibodies (BD Biosciences PharMingen, Erembodegem, Belgium). Endogenous peroxidase activity was inhibited by 0.3% H2O2 in PBS, and Envision system (Dako, Glostrup, Denmark) was used for revelation. Sections were counterstained with Mayer’s hematoxylin and mounted with the aqueous Faramount mounting medium (Dako).
Tumor Angiogenesis Assays.
Mice received an i.m. injection of 106 LLc cells or 105 TLT cells in the posterior right leg. When the tumors reached 4.0 ± 0.5 mm in diameter, they were locally irradiated (1 x 6 Gy for single-dose radiotherapy or 5 x 6 Gy each 24 h for fractionated therapy). For ex vivo assays, tumor-bearing mice were locally irradiated (or not) and sacrificed 24 h later to isolate tumor arterioles (under a stereoscopic microscope). The collected vessels were directly seeded in GFR-Matrigel with or without 5 mM L-NAME, and angiogenic sprouting was evaluated 3 days later. For in vivo assays, mice were sacrificed either 1 day or 5 days after the last dose delivery of the fractionated protocol, and cryo-slices of the tumors were immunostained with rat monoclonal CD31 IgG2a antibodies. Rabbit polyclonal antirat IgG peroxidase-conjugated antibodies (Jackson Immunoresearch Laboratories, West Grove, PA) and AEC substrate system (Dako) were used for revelation; sections were finally counterstained with Mayer’s hematoxylin. Quantitative image analyses were performed with AnalySIS software (Soft Imaging System, Münster, Germany).
Real-Time Quantitative PCR from Human Tumor Biopsies.
Total RNA was isolated using Tripure reagent (Roche, Mannheim, Germany) from human tumor (oropharyngeal and oral cavity squamous cell carcinoma) biopsies collected before and 24 h after local irradiation (2 Gy); this radiation dose was the first treatment received by these patients. Informed consent was obtained from each patient before surgery under local anesthesia. cDNA was synthesized from total RNA using random hexamers, and PCR amplification was performed using SYBR Green reagent (Applied Biosystems, Warrington, United Kingdom). The specific primers were as follows: eNOS sense, 5'-ctcatgggcacggtgatg-3'; eNOS antisense, 5'-accacgtcatactcatccatacac-3'; GAPDH sense, 5'-cctgttcgacagtcagccg-3'; GAPDH antisense, 5'-cgaccaaatccgttgactcc-3'; CD31 sense, 5'-gatccatatgcagacctcagaatct-3'; and CD31 antisense, 5'-caagagtgaactggtcaccgtgacgg-3'. Fluorescence data were analyzed, after PCR completion, with the Abi PRISM 5700 Sequence Detection System instrument (Applied Biosystems). Results were expressed as Ct (number of cycles needed to generate a fluorescent signal above a predefined threshold). Relative quantitation for a given gene, expressed as fold variation over control, was calculated using the 2-
Ct formula after normalization to GAPDH (Ct) and determination of the difference in Ct (Ct) between control and irradiated tumors.
Statistical Analyses.
Data are normalized for the amounts of protein in the dish (or for the number of cells engaged) and presented for convenience as mean ± SE. Statistical analyses were performed using Student’s t test or one-way ANOVA where appropriate.
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RESULTS |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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and dose-dependent (Fig. 1B
, top row) increase in eNOS expression (n = 3–4). Of note, under these experimental conditions (as well as in all other models used in this study), we did not detect any induction of inducible NOS expression after irradiation.
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, middle row). In fact, the dose-dependent increase in the abundance of eNOS protein corresponded to an increased caveolin-free fraction of the enzyme, as shown by the amounts of eNOS left behind in the caveolin IP SN (Fig. 1B
, bottom row and bar graph). Of note, the bar graph accompanying the blots in Fig. 1B
gives the exact proportion of caveolin-free and -bound eNOS because it integrates the dilution factor (
16x) of SN samples and the differences in film exposures.
Fig. 1C
(left panels) shows that the extent of eNOS phosphorylation dose-dependently increases with irradiation. Importantly, when the data were normalized for the amounts of eNOS protein, the extents of eNOS phosphorylation at 2 and 6 Gy reached 125 ± 9% and 168 ± 21% of control levels (n = 4–10), respectively. We also found that the PI3K/Akt signaling pathway was involved in the process of irradiation-induced eNOS phosphorylation. Indeed, irradiation led to a significant increase in the amounts of the Ser-473-phosphorylated (activated) form of Akt (n = 4–10; Fig. 1C
, right panels), and LY294002, the inhibitor of PI3K (an upstream activator of Akt), completely prevented eNOS phosphorylation in 6-Gy-irradiated ECs (94 ± 3% of control levels; n = 3; Fig. 1D
).
Finally, to examine whether changes in eNOS expression and posttranslational regulations had a real impact on the activity of the enzyme, we measured the production of nitrites and nitrates (the decomposition products of NO) in ECs exposed to ionizing radiations; these measurements were repeated in the presence of the NOS inhibitor L-NAME (5 mM). Accordingly, low-dose irradiations (2 and 6 Gy) led to a significant increase (+65% and +72%, respectively) in L-NAME-sensitive NO production (P < 0.05; n = 6).
Irradiation Promotes NO-dependent EC Migration and Tube Formation.
We then examined whether the up-regulation of the NO pathway was associated with functional changes in the ability of ECs to migrate and form capillary-like networks.
To evaluate migration, we used the scratch injury test based on the ability of ECs to sprout into the wounded area. Preliminary studies revealed that under the experimental conditions used in this assay, proliferation was almost absent [as also validated by others (20)
], and only migration could account for the colonization of the scratched area. Of note, the cells found in the scratched area consistently presented the typical migratory spindle shape (see Fig. 2A
).
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reveals that a 6-Gy dose dramatically stimulated the ability of ECs to migrate toward the center of the wound. At 72 h, the scratched area was almost entirely repopulated. However, the evaluation of the promigratory effects of irradiation needed to take into account the fact that a proportion of ECs did not survive to the X-ray exposure. Accordingly, the migration index, which is corrected for cell death (see "Materials and Methods"), revealed a maximal 5.2-fold higher capacity of ECs to sprout 48 h after irradiation (Fig. 2B)
. Conversely, this migration index (n = 3–11) was not significantly different from controls when cells were exposed to the NOS inhibitor L-NAME after irradiation. Importantly, this promigratory impact of ionizing radiations was dose dependent because the effects were detected with doses as low as 2 Gy and increased progressively up to a dose of 20 Gy (data not shown).
We then examined whether irradiation could also lead to the reorganization of ECs in tubes or precapillary structures when cultured on extracellular matrices. Accordingly, ECs were irradiated at 6 and 20 Gy and plated 24 h later on Matrigel. Fig. 3A
shows that contrary to nonirradiated cells, cells preexposed to X-rays rapidly formed tubes or precapillary structures. Because of the use of GFR-Matrigel, the irradiation-induced network then progressively faded. In fact, a bell-shaped effect was observed in the time course of network formation and disassembly (see Fig. 3B
). Importantly, when the cells were treated with the NOS inhibitor L-NAME (n = 4), tube formation was completely prevented (Fig. 3B)
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When tumor microarteries were collected from TLT-bearing mice and cultured in GFR-Matrigel, the tumor vessels isolated from locally irradiated mice showed the sprouting of a vascular network (Fig. 4)
; von Willebrand labeling (data not shown) confirmed the endothelial nature of >90% of the outgrowing cells. When microvessels were grown in the presence of L-NAME, they did not lead to tube formation when incubated in Matrigel, confirming the key role of NO in this proangiogenic process (Fig. 4)
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. As found in the ex vivo experiments, the irradiation-induced proangiogenic process was completely abolished when mice received L-NAME (500 mg/liter) from day 9 to sacrifice (Fig. 5A
, right panel). Of note, hypercellular areas were clearly visible at the host tissue-Matrigel plug interface. In the Matrigel plugs exposed to ionizing radiations, this compact cell rim was much larger and gave rise to infiltrating septa positive for von Willebrand factor staining (Fig. 5B)
. L-NAME treatment repressed both the the interface thickening and the cellular path formation (Fig. 5B
, right panel).
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, the extent of EC infiltration was 7-fold higher than that in nonirradiated tumors. Interestingly, this irradiation-induced EC recruitment in tumors was repressed when mice were treated with L-NAME (from day -1 to sacrifice; Fig. 5C
, right panel). Moreover, the tumor-host interface appeared to be a privileged zone for EC recruitment because irradiated host muscular tissues distant from the tumor were not colonized by ECs (Fig. 5C
, middle panel, inset). These findings were confirmed by using other EC markers including eNOS and the von Willebrand factor (data not shown).
Irradiation Induces eNOS Expression in Human Tumors.
To determine whether irradiation also increases eNOS expression in human tumors, we used real-time PCR to determine the abundance of eNOS mRNA in squamous cell carcinoma biopsies collected before and after a 2-Gy irradiation. Because of the intrinsic variability of vascular content in tumors, eNOS gene expression was normalized by the levels of CD31 mRNA transcript in the corresponding samples. In experiments using cultured ECs, the expression of this vascular marker did not appear to be influenced by irradiation (contrary to eNOS). Also, to exclude amplification from contaminating genomic DNA, samples of RNA that had not been reverse-transcribed were run in parallel PCR reactions. These controls always remained negative.
Fig. 6
represents the changes in eNOS expression, e.g., the ratio of eNOS:CD31 gene expression, in repetitive biopsies collected before and after irradiation in five patients (n = 14 biopsies). These data indicate that a 2-Gy irradiation was sufficient to induce a significant increase in eNOS expression in this human tumor type.
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DISCUSSION |
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TopABSTRACTINTRODUCTIONMATERIALS AND METHODSRESULTSDISCUSSIONREFERENCES |
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. Furthermore, the activability of this increased pool of eNOS did not appear to be counterbalanced by a higher level of interaction with its functional, allosteric inhibitor caveolin-1 (see Fig. 1B
). Second, the level of eNOS phosphorylation was also enhanced (to a larger extent than the up-regulation of eNOS expression itself), suggesting a higher ability of these cells to exhibit a sustained production of NO (see Fig. 1, C and D
). Accordingly, such increases in both abundance and activation of eNOS led to a robust increase in NO release compatible with the angiogenic phenotype shift.
It is important to stress that although irradiation produces large amounts of ROS known to react with NO and neutralize its action, in our study the blocking effects of the NOS inhibitor L-NAME on functional read-outs such as EC migration (Fig. 2)
and tube formation (Fig. 3)
demonstrate that NO production reached sufficient levels to overcome the scavenging effects of ROS. Nevertheless, ROS production is likely to be involved in the transcriptional and posttranslational regulation of eNOS. Several investigators have indeed reported that ROS can induce AP-1 activation, which in turn promotes eNOS gene transcription (12
, 21)
, whereas others have documented that cell exposure to ROS can lead to Akt activation (14)
, which, in turn, phosphorylates eNOS on serine 1177 (19)
. This Akt pathway is also likely to account, at least in part, for the survival of a proportion of ECs exposed to ionizing radiations, as recently documented by Edwards et al. (22)
.
Importantly, our results do not refute the existence of cytotoxic effects of irradiation on ECs, as reported previously to participate in the antitumor treatment (23)
. In fact, our results support the nonexclusive proposition that a proportion of ECs did survive the radiation stress and that this selected cell population underwent the phenotypic changes observed. Doses and schemes of radiation administration are likely to influence the outcomes of ECs exposed to X-rays, e.g., the proapoptotic or prosurvival signaling pathways (24)
. Interestingly, in the clinical context of fractionated radiotherapy as administered nowadays, we consistently found an increase in eNOS mRNA transcript abundance in tumor biopsies collected after local exposure to a 2-Gy irradiation (Fig. 6)
. These data support the paradigm of the rapid induction of the NO pathway as elicited in our experimental models. Together with recent reports documenting the X-ray-induced increase in VEGF (2
, 10)
, an upstream activator of eNOS signaling (11)
, our findings strongly suggest that an angiogenic phenotype shift is also likely to occur in human tumors. Furthermore, our data obtained with mouse-implanted Matrigel plug and tumors suggest that irradiation induced an increase in total density of ECs. The origin of ECs lining tumor vessels is still under debate. Recent findings suggest that hematopoietic and/or circulating endothelial progenitor cells participate in tumor neovascularization (25)
. In our mouse models, the hypercellularity and the presence of vascular structures (observed after irradiation) at the interface between host tissues and tumor or Matrigel (Fig. 5, A and B)
suggest the participation of an inflammatory process in the initiation of the angiogenic pathway as observed in other models (26)
. Interestingly, the inhibitory effect of L-NAME on EC colonization of the implanted Matrigel plugs and tumors strongly suggests that NO is not only involved in the network reorganization of ECs but also participates in their recruitment. Additional experiments on slow-growing tumors (e.g., which are more angiogenesis dependent for their growth than the tumor models used in this study) are required to evaluate whether this NO-mediated process originates from endothelial progenitor cells recruitment and/or EC pruning from existing or inflammation-induced local vascularization.
Beside the mechanistic dissection of the irradiation-induced up-regulation of the NO pathway in the tumor vessel ECs, our data provide an additional rationale for associating antiangiogenic strategies and radiotherapy. Indeed, various antiangiogenic agents have been demonstrated to enhance the response of tumors to radiotherapy (2, 3, 4, 5, 6, 7, 8, 9
, 27, 28, 29, 30, 31, 32)
. Although different mechanisms underlying these effects were identified (such as the inhibition of protection against radiation damage or of the damage repairs themselves), the consensus view from these studies is that the radiosensitization of both tumor and ECs is the main trigger of the beneficial effects of the cotherapy. In the current study, we showed that irradiation may be endowed with collateral effects conferring radioresistance to ECs, e.g., through the NO-induced proangiogenic pathway. Of interest, the concept that irradiation itself can activate signaling cascades that allow tumors to escape radiation-induced damages has already been reported in the context of tumor cells per se (33)
. Also, several studies have documented that drugs targeting these irradiation-evoked pathways exert cooperative effects in tumor eradication. For instance, antibodies directed against epidermal growth factor receptor have been shown to abrogate the prosurvival effects of transforming growth factor 
(a specific activator of the mitogenic epidermal growth factor receptor/ras/raf/mitogen-activated protein kinase pathway), which is released on tumor cell exposure to ionizing radiations (34
, 35)
. With regard to irradiation-induced angiogenesis, drugs interacting with the NO pathway may be good candidates as adjuvant to radiotherapy.
The scheduling of the antiangiogenic treatments still needs to be optimized in the context of association with radiotherapy. Indeed, contrary to antivascular drugs such as tubulin-binding agents that induce O2 starvation and therefore better fit a therapeutic scheme in which radiation precedes their use (1) , the best timing for administrating antiangiogenic drugs is still under debate and investigation (36) . Indeed, the question of the exact relationship between tumor vascularization and resistance to treatments or metastasis dissemination is largely unresolved. For instance, although we report here that radioresistance may come, in part, from the chronic induction by radiations of NO-mediated angiogenic pathways, we recently reported that the same source of NO can acutely contribute to a better efficacy of fractionated radiotherapy (37) . In this latter study, as documented previously with various pharmacological agents targeting tumor vessel functionality (38 , 39) , other biological effects of NO, including vasodilation and regulation of mitochondrial respiration, account for the observed radiosensitization through a better tumor perfusion and reoxygenation. This suggests that the already poorly efficient tumor vasculature should initially be spared to maximize radiotherapy, and, in a second time, the antiangiogenic strategies should be combined with radiotherapy to block the provascular effects of radiations. However, several authors have also documented an increase in tumor pO2 (3 , 27 , 40) or a transient normalization of tumor vasculature (41) on treatment with antiangiogenic compounds, suggesting that the administration of the latter should instead precede radiotherapy. In conclusion, the variety of tumors and experimental models will probably not lead to a single response for the coordinated use of different antitumor therapies. More likely, noninvasive methods aiming to evaluate local changes in pO2 in a tumor should allow the clinician to choose in the future between various combinations of treatments and administration schemes on an individual basis.
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ACKNOWLEDGMENTS |
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FOOTNOTES |
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1 Supported by grants from the Fonds de la Recherche Scientifique Médicale, the Belgian Federation Against Cancer, the Fortis Cancerology Research Fund, the J. Maisin Foundation, the Association Sportive Contre le Cancer, and the Pôle d’Attraction Interuniversitaire P5/02. C. D. and O. F. are Fonds National de la Recherche Scientifique (FNRS) Research Associates. P. S. is a FNRS Research Assistant, and A. B. is the recipient of a FNRS-Télévie grant. 
2 To whom requests for reprints should be addressed, at University of Louvain Medical School, Pharmacology and Therapeutics Unit (FATH 5349), 53 Ave E. Mounier, B-1200 Brussels, Belgium. Phone: 32-2-764-5349; Fax: 32-2-764-9322; E-mail: feron{at}mint.ucl.ac.be
3 The abbreviations used are: VEGF, vascular endothelial growth factor; NOS, nitric oxide synthase; eNOS, endothelial nitric oxide synthase; EC, endothelial cell; ROS, reactive oxygen species; TLT, transplantable liver tumor; LLc, Lewis lung carcinoma; GFR-Matrigel, growth factor-reduced Matrigel; IP, immunoprecipitation; SN, supernatant; L-NAME, N
-nitro-L-arginine methyl ester; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; PI3K, phosphatidylinositol 3'-kinase.
Received 9/18/02. Accepted 1/ 6/03.
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), three biopsies were obtained before and after irradiation: only the mean values (±SE) are presented.