Influence of Endothelial Cells on Vascular Smooth Muscle Cells Phenotype after Irradiation
2006; Elsevier BV; Volume: 169; Issue: 4 Linguagem: Inglês
10.2353/ajpath.2006.060116
ISSN1525-2191
AutoresFabien Milliat, Agnès François, Muriel Isoir, Éric Deutsch, Radia Tamarat, Georges Tarlet, Azeddine Atfi, Pierre Validire, Jean Bourhis, J.C. Sabourin, Marc Benderitter,
Tópico(s)TGF-β signaling in diseases
ResumoDamage to vessels is one of the most common effects of therapeutic irradiation on normal tissues. We undertook a study in patients treated with preoperative radiotherapy and demonstrated in vivo the importance of proliferation, migration, and fibrogenic phenotype of vascular smooth muscle cells (VSMCs) in radiation-induced vascular damage. These lesions may result from imbalance in the cross talk between endothelial cells (ECs) and VSMCs. Using co-culture models, we examined whether ECs influence proliferation, migration, and fibrogenic phenotype of VSMCs. In the presence of irradiated ECs, proliferation and migration of VSMCs were increased. Moreover, expressions of α-smooth muscle actin, connective tissue growth factor, plasminogen activator inhibitor type 1, heat shock protein 27, and collagen type III, alpha 1 were up-regulated in VSMCs exposed to irradiated ECs. Secretion of transforming growth factor (TGF)-β1 was increased after irradiation of ECs, and irradiated ECs activated the Smad pathway in VSMCs by inducing Smad3/4 nuclear translocation and Smad-dependent promoter activation. Using small interferring RNA targeting Smad3 and a TGFβ-RII neutralizing antibody, we demonstrate that a TGF-β1/TGF-β-RII/Smad3 pathway is involved in the fibrogenic phenotype of VSMCs induced by irradiated ECs. In conclusion, we show the importance of proliferation, migration, and fibrogenic phenotype of VSMCs in patients. Moreover, we demonstrate in vitro that ECs influence these fundamental mechanisms involved in radiation-induced vascular damages. Damage to vessels is one of the most common effects of therapeutic irradiation on normal tissues. We undertook a study in patients treated with preoperative radiotherapy and demonstrated in vivo the importance of proliferation, migration, and fibrogenic phenotype of vascular smooth muscle cells (VSMCs) in radiation-induced vascular damage. These lesions may result from imbalance in the cross talk between endothelial cells (ECs) and VSMCs. Using co-culture models, we examined whether ECs influence proliferation, migration, and fibrogenic phenotype of VSMCs. In the presence of irradiated ECs, proliferation and migration of VSMCs were increased. Moreover, expressions of α-smooth muscle actin, connective tissue growth factor, plasminogen activator inhibitor type 1, heat shock protein 27, and collagen type III, alpha 1 were up-regulated in VSMCs exposed to irradiated ECs. Secretion of transforming growth factor (TGF)-β1 was increased after irradiation of ECs, and irradiated ECs activated the Smad pathway in VSMCs by inducing Smad3/4 nuclear translocation and Smad-dependent promoter activation. Using small interferring RNA targeting Smad3 and a TGFβ-RII neutralizing antibody, we demonstrate that a TGF-β1/TGF-β-RII/Smad3 pathway is involved in the fibrogenic phenotype of VSMCs induced by irradiated ECs. In conclusion, we show the importance of proliferation, migration, and fibrogenic phenotype of VSMCs in patients. Moreover, we demonstrate in vitro that ECs influence these fundamental mechanisms involved in radiation-induced vascular damages. About half of people with cancer are treated with radiation therapy either alone or in combination with other types of cancer treatments. However, normal tissue toxicity still remains a dose-limiting factor in clinical radiation therapy.1Stone HB Coleman CN Anscher MS McBride WH Effects of radiation on normal tissue: consequences and mechanisms.Lancet Oncol. 2003; 4: 529-536Abstract Full Text Full Text PDF PubMed Scopus (682) Google Scholar Vascular injury is one of the most common effects of radiotherapy on normal tissues. Damage to blood vessels and subsequent hypoxia and ischemia are known to contribute to severe tissue injury such as fibrosis and/or necrosis. Vascular fibrosis after radiotherapy contributes to severe normal tissue damage and, in some cases, may be a vital prognosis in patients.2Dorresteijn LD Kappelle AC Boogerd W Klokman WJ Balm AJ Keus RB van Leeuwen FE Bartelink H Increased risk of ischemic stroke after radiotherapy on the neck in patients younger than 60 years.J Clin Oncol. 2002; 20: 282-288Crossref PubMed Scopus (294) Google Scholar The endothelium is known to play a critical role in radiation-induced vascular injury. Irradiated endothelial cells (ECs) acquire a proinflammatory, procoagulant, and prothrombotic phenotype. Up-regulation of endothelial cell adhesion molecules such as vascular cell adhesion molecule-1, intercellular adhesion molecule-1,3Molla M Gironella M Miquel R Tovar V Engel P Biete A Pique JM Panes J Relative roles of ICAM-1 and VCAM-1 in the pathogenesis of experimental radiation-induced intestinal inflammation.Int J Radiat Oncol Biol Phys. 2003; 57: 264-273Abstract Full Text Full Text PDF PubMed Scopus (74) Google Scholar, 4Panes J Anderson DC Miyasaka M Granger DN Role of leukocyte-endothelial cell adhesion in radiation-induced microvascular dysfunction in rats.Gastroenterology. 1995; 108: 1761-1769Abstract Full Text PDF PubMed Scopus (107) Google Scholar P-selectin,5Molla M Gironella M Salas A Miquel R Perez-del-Pulgar S Conill C Engel P Biete A Pique JM Panes J Role of P-selectin in radiation-induced intestinal inflammatory damage.Int J Cancer. 2001; 96: 99-109Crossref PubMed Scopus (37) Google Scholar and platelet-endothelial cell adhesion molecule-16Quarmby S Kumar P Wang J Macro JA Hutchinson JJ Hunter RD Kumar S Irradiation induces upregulation of CD31 in human endothelial cells.Arterioscler Thromb Vasc Biol. 1999; 19: 588-597Crossref PubMed Scopus (43) Google Scholar after irradiation leads to the increase of leukocyte/EC interactions and leukocyte transmigration. Moreover, irradiation increases the interactions of platelets with the endothelium7Mouthon MA Vereycken-Holler V Van der Meeren A Gaugler MH Irradiation increases the interactions of platelets with the endothelium in vivo: analysis by intravital microscopy.Radiat Res. 2003; 160: 593-599Crossref PubMed Scopus (29) Google Scholar and decreases expression of the anticoagulant thrombomodulin.8Wang J Zheng H Ou X Fink LM Hauer-Jensen M Deficiency of microvascular thrombomodulin and up-regulation of protease-activated receptor-1 in irradiated rat intestine: possible link between endothelial dysfunction and chronic radiation fibrosis.Am J Pathol. 2002; 160: 2063-2072Abstract Full Text Full Text PDF PubMed Scopus (136) Google Scholar If radiation-induced EC activation and changes in the physiological properties of the vascular endothelium have been well documented, less is known about dysfunction of the entire vessel wall. Cell-cell communications play a fundamental role in vascular remodeling after injury. ECs, inflammatory cells, and vascular smooth muscle cells (VSMCs) are involved in the pathogenesis of vascular diseases. The interactions between ECs and VSMCs are known to play a key role in the structure and function of the vessel. VSMC migration, proliferation, and differentiation are critical processes involved in vascular injury observed in vascular pathologies such as atherosclerosis,9Ross R Cell biology of atherosclerosis.Annu Rev Physiol. 1995; 57: 791-804Crossref PubMed Scopus (893) Google Scholar intimal hyperplasia, and hypertension. If there is still debate as to whether or not radiation-induced vascular lesions are similar to those cited above, parallels may be drawn to offer some clues to the comprehension of vascular radiation damage. It has been shown that fibrogenic cytokines and growth factors are involved in mechanisms of vascular fibrosis, ie, migration and proliferation of smooth muscle cells, the increase of collagen expression, and the alteration of matrix remodeling. However, molecular mechanisms involved in radiation-induced vascular fibrosis are still unclear and may also result from imbalance in the cross talk between ECs and VSMCs. First, we performed a retrospective study in patients treated with preoperative radiotherapy for rectal cancer to analyze radiation-induced vascular damages. The second purpose of our work was to study, in an EC-VSMC co-culture model, the influence of paracrine factors released by ECs on VSMC proliferation, migration, and fibrogenic phenotype after irradiation. Moreover, molecular mechanisms involved in the fibrogenic phenotype of VSMCs in the presence of ECs were investigated. Thirty-eight patients treated for rectal adenocarcinoma with preoperative radiotherapy (45 Gy; 2 or 1.8 Gy by fraction) were included in this study. Tumors were surgically resected 5 to 7 weeks after treatment. For each patient, specimens of normal tissue were taken in the irradiated field adjacent to the tumor and from microscopically normal mucosa distant from the tumor. Slides were colored by Meyer's hemalum, Masson's trichrome, and elastin coloration (Varehoeff-Van Gieson). Radiation injury scores determined by a pathologist (J.-C.S.) were measured in all patients (at least two slides per patient, normal and pathological). We first identified the distinct compartments in each slide, ie, mucosa, muscularis mucosa, submucosa, muscularis propria, serosa, and mesentery. Then, we scored in these different compartments several alterations that contribute to the global features of radiation-induced intestinal damages. The individual abnormalities were assessed as normal or abnormal, ranked according to severity as described in Table 1. For every slide, a score in each compartment was determined for parameters described in Table 1. Finally, for one slide the sum of the scores for each parameter in all compartments (the retrieved sum to 100) constituted the radiation injury score. Radiation injury score and morphometric measurements were determined independently by two authors, and discrepancies were resolved in conference. Vessel wall thickness was determined by the ratio between luminal surface and outer surface (between 15 to 25 vessels by section) using an imaging analysis system interfaced with the Visiolab 2000 software (Biocom, Les Ulis, France). For immunohistochemistry, 5-μm sections were used to immunolocalize α-smooth muscle actin (α-SMA; Sigma, St. Quentin Fallavie, France), calponin (DAKO, Glostrup, Denmark), proliferating cell nuclear antigen (PCNA; DAKO), collagen I and collagen III (Sigma), transforming growth factor-βTGF-β (R&D Systems, Minneapolis, MN), and phospho-(ser433/435) Smad2/3 (Santa Cruz Biotechnology, Santa Cruz, CA). Biotinylated rabbit anti-mouse IgG and streptavidin/biotinylated-peroxidase kit (DAKO) were used before revelation by Vector NovaRED substrate kit (Bio-Valley, Marne la Vallée, France) and counterstained with hematoxylin. Moreover, radiation-induced vascular damages were studied in irradiated lung (55 to 60 Gy), uterus (60 Gy), and skin (50 Gy). Three patients for each organ were included. Specimens of normal tissue samples were taken in an irradiated field and from normal tissue at a large distance from the tumor. Slides were colored by Meyer's hemalum.Table 1Semiquantitative Histopathological Scoring SystemMucosaNormal: 0Moderately abnormal with slight inflammation: 1Markedly abnormal with strong inflammation: 2Severely abnormal with severe inflammation and loss of epithelium (ulceration): 3Muscularis mucosaNormal: 0Markedly abnormal with slight matrix deposition and/or slight dystrophy: 1Severely abnormal with strong matrix deposition and/or severe dystrophy: 2SubmucosaNormal: 0Moderately abnormal with inflammation: 1Moderately abnormal with edema: 1Markedly abnormal with slight matrix deposition: 2Severely abnormal with fibrosis: 3Submucosal vesselNormal: 0Markedly abnormal with slight matrix deposition and/or slight dystrophy: 1Severely abnormal with strong matrix deposition and/or severe dystrophy: 2Muscularis propriaNormal: 0Markedly abnormal with slight matrix deposition and/or dystrophy: 1Severely abnormal with strong matrix deposition and/or dystrophy: 2MesenteryNormal: 0Markedly abnormal with slight matrix deposition: 1Severely abnormal with fibrosis: 2Mesenteric vesselNormal: 0Markedly abnormal with slight matrix deposition and/or slight dystrophy: 1Severely abnormal with strong matrix deposition and/or severe dystrophy: 2SerosaNormal: 0Markedly abnormal with slight matrix deposition: 1Severely abnormal with fibrosis: 2Histopathologic scoring of radiation injury in each compartment (mucosa, muscularis mucosa, submucosa, muscularis propria, serosa, and mesentery, as well as submucosal and mesenteric vessels) were assessed independently by two authors in a blinded manner. The individual abnormalities were assessed as normal (score = 0) or abnormal, graded according to severity (0 to 2 or 0 to 3). Finally, for every slide, the sum of the scores for each parameter in all compartments (the retrieved sum to 100) constitutes the radiation injury score. Open table in a new tab Histopathologic scoring of radiation injury in each compartment (mucosa, muscularis mucosa, submucosa, muscularis propria, serosa, and mesentery, as well as submucosal and mesenteric vessels) were assessed independently by two authors in a blinded manner. The individual abnormalities were assessed as normal (score = 0) or abnormal, graded according to severity (0 to 2 or 0 to 3). Finally, for every slide, the sum of the scores for each parameter in all compartments (the retrieved sum to 100) constitutes the radiation injury score. ECs (dermal human microvascular endothelial cells) and VSMCs (aortic human smooth muscle cells) were purchased from Cambrex (Verviers, Belgium) and cultured respectively in EGM-2 MV and SmGM-2 culture mediums. ECs were grown in transwell (Falcon 0.4-μm PET cell culture inserts; Becton Dickinson Labware, Le Pont de Claix, France). Cells were irradiated with a 137Cs source (IBL 637, dose rate 1 Gy min−1). Viable cells were counted using trypan blue exclusion method and a standard hemacytometer. Cell cycle analyses were performed by flow cytometry. In brief, trypsinized cells were fixed with ice-cold 70% ethanol, treated with 0.01 mg/ml RNase A and 50 μg/ml propidium iodide, and analyzed using a FACSort flow cytometer (Becton Dickinson). VSMC migration was determined by the scratch injury model.10Sonveaux P Brouet A Havaux X Gregoire V Dessy C Balligand JL Feron O Irradiation-induced angiogenesis through the up-regulation of the nitric oxide pathway: implications for tumor radiotherapy.Cancer Res. 2003; 63: 1012-1019PubMed Google Scholar VSMCs were fixed and colored with a methanol solution containing 3% paraformaldehyde and 0.25% crystal violet from 1 to 4 days after irradiation. To integrate radiation-induced cell death, the quantitative analysis of the migration index was calculated by the ratio between the density of migrating cells in the center of the scratched zone and in a size-matched area of the unscratched region. The contribution of VSMC proliferation was assessed by cycling cell labeling (Ki-67, see below). Total RNA was prepared with the total RNA isolation kit (Rneasy Mini Kit; Qiagen, Valencia, CA). Total RNA quantification and integrity was analyzed using Agilent 2100 Bioanalyzer, and 1 μg of RNA was used for RT with SuperScript II (Invitrogen Life Technologies, Carlsbad, CA) and random hexamer to generate first strand cDNA. The following primers were used (F, forward; R, reverse): CTGF (F, 5′-TGTGTGACGAGCCCAAGGA-3′; R, 5′-TCTGGGCCAAACGTGTCTTC-3′; 5′-carboxyfluorescein-CTGCCCTCGCGGCTTACCGA-3′), PAI-1 (F, 5′-GCACAACCCCACAGGAACAG-3′; R, 5′-GTCCCAGATGAAGGCGTCTTT-3′), HSP27 (F, 5′-AGGATGGCGTGGTGGAGAT-3′; R, 5′-GTGTATTTCCGCGTGAAGCA-3′), COL3A1 (F, 5′-CCAATCCTTTGAATGTTCCACGG-3′; R, 5′-CCATTCCCCAGTGTGTTTCGTGC-3′), COL1A2 (F, 5′-TGAAAACATCCCAGCCAAGAA-3′; R, 5′-AAACTGGCTGCCAGCATTG-3′), SMAD3 (F, 5′-CGAGCCCCAGAGCAATATTC-3′; R, 5′-CTGTGGTTCATCTGGTGGTCACT-3′), and α-SMA (gene expression assay Hs00426835-g1; Applied Biosystems, Foster City, CA). Thermal cycling conditions were 10 minutes at 95°C followed by 40 cycles of 95°C for 15 seconds and 60°C for 1 minute on an ABI PRISM 7700 Sequence detection system (Applied Biosystems). Significant PCR fluorescent signals were normalized to a PCR fluorescent signal obtained from the housekeeping gene GAPDH (Pre-developed Taqman Assay; Applied Biosystems) for each sample. Relative mRNA quantitation was performed by using the comparative ΔΔCT method. Cells were grown on glass coverslips and fixed for 30 minutes with 0.5% paraformaldehyde. After permeabilization and saturation, cells were incubated overnight with primary antibodies anti-Ki-67 (DAKO), anti-Smad3 (Zymed Laboratories, South San Francisco, CA), and anti-Smad4 (Santa Cruz Biotechnology). Cells were then incubated with a goat anti-mouse or rabbit IgG tagged with Alexa Fluor 488 (Molecular Probes), rinsed, and incubated in RNase A/propidium iodide solution. Cells were analyzed on Bio-Rad MRC 1024 ES confocal imaging system (Bio-Rad, Hercules, CA). VSMCs were transiently cotransfected with (CAGA)9-Lux reporter and pRL-TK plasmids using FuGENE 6 (Roche Diagnostics, Meylan, France) as transfection reagent. Cells extracts were prepared for the Dual-Luciferase reporter assay system according the manufacturer's instructions (Promega, Charbonnières, France). Relative luciferase activity was measured using a Mithras luminometer (Berthold Technologies, Bad Wildbad, Germany). The following protein-specific primary antibodies were used: anti-α-SMA (Sigma), anti-HSP27 (Stressgen Biotechnologies, Victoria, BC, Canada), anti-PAI-1 (Novocastra Laboratories Ltd., Newcastle, UK), anti-CTGF (R&D Systems), and anti-glyceraldehyde-3-phosphate dehydrogenase (Biodesign, Saco, ME). Proteins were separated by SDS-polyacrylamide gel electrophoresis before transfer onto nitrocellulose membranes. The membranes were blotted with primary antibodies followed by incubation with secondary antibody HRP-conjugated (Amersham, Orsay, France). Blots were developed using the enhanced chemiluminescence method (Amersham). Membranes were then dehybridized and reprobed with anti-glyceraldehyde-3-phosphate dehydrogenase antibody to detect glyceraldehyde-3-phosphate dehydrogenase expression as control loading. TGF-β1 production in the supernatants of ECs and VSMCs was determined by ELISA assay (Promega) with (total form) and without (active form) acid treatment according to the manufacturer's instructions. The sequence of small interferring RNAs (siRNAs) designed to specifically target Smad3 is 5′-ACCUAUCCCCGAAUCCGAUdTdT-3′. The efficiency of silencing was determined by RT real-time PCR using specific primers and Western blot (anti-Smad3; Zymed Laboratories). Data are given as mean ± SEM. Statistical analyses were performed by analysis of variance or Student's t-test with a level of significance of P < 0.05. We undertook a retrospective study in 38 patients treated with preoperative radiotherapy for rectal cancer. Radiation injury score was determined as well as vessels morphometric measurements. Radiation-induced tissue damage was appreciated by a semiquantitative histopathological scoring system (Table 1) of mucosal injury, submucosal edema and inflammation, dystrophy, and extracellular matrix remodeling in the submucosa, muscularis mucosa, muscularis propria, serosa, and mesentery. A correlation between vascular thickening and radiation injury score was observed (P < 0.001, n = 83 slides, Figure 1A). Radiotherapy treatment is associated with several kinds of vascular damage: vascular dystrophy and hypertrophy (Figure 1B, B–D), vascular and perivascular fibrosis (Figure 1B, F–H), and intimal hyperplasia associated with luminal narrowing (Figure 1B, C,F,J). Immunolabeling of collagen I and collagen III revealed a strong increase of immunoreactivity in vessels from pathological (Figure 1B, N–P, R–T) compared with normal tissues (Figure 1B, M,Q). PCNA labeling (Figure 1B, U–X) showed proliferation of VSMC in hypertrophic vessels (Figure 1B, V) compared with normal (Figure 1B, U). In areas of neointimal hyperplasia, α-SMA (Figure 1B, K,K′), calponin (Figure 1B, L,L′), and PCNA-positive cells (Figure 1B, V–X) in a rich collagen matrix (Figure 1B, F,P) are also observed demonstrating migration and proliferation of VSMC. Interestingly, vascular dystrophy, hypertrophy, and intimal hyperplasia were observed in irradiated lung, uterus, and skin, illustrating that radiation-induced vascular damage is not organ-dependent (Figure 1C). The effect of ECs on VSMC proliferation after irradiation was investigated using cell counting and cell cycling distribution analyses (Figure 2). Interestingly, proliferation of VSMCs decreased in presence of ECs, irradiated or not. However, the number of nonirradiated VSMCs was higher in presence of irradiated ECs compared with nonirradiated ECs (Figure 2, A–B). Irradiation inhibits the proliferation of VSMCs, and this effect is decreased in presence of irradiated ECs. Analyses of cell cycle distribution showed that irradiation induces a classic G1 arrest in VSMCs, which was not affected by the presence of ECs, irradiated or not. Twenty-four hours after irradiation, irradiated ECs increased the percentage of nonirradiated VSMCs in S phase compared with VSMCs alone or VSMCs with nonirradiated ECs. Moreover, 24 to 72 hours after irradiation, the number of irradiated VSMCs in S phase increased in presence of ECs, and this effect was more pronounced in presence of irradiated ECs (Figure 2C). Irradiation did not influence the ability of VSMCs to colonize a wounded area (Figure 3). Moreover, migration index was increased in VSMCs in the presence of irradiated ECs. Migration of irradiated VSMCs was stimulated in the presence of ECs, and this effect was further improved in the presence of irradiated ECs. We next analyzed the ability of irradiated ECs to affect the fibrogenic phenotype of VSMCs. In vivo, both cell types are irradiated, so we therefore performed co-culture of irradiated ECs in the presence of irradiated VSMCs at the same dose (2 or 10 Gy; Figure 4A). In the presence of irradiated ECs, mRNA expression of CTGF, PAI-1, collagen type I, alpha 2 (COL1A2), and COL3A1 increased in irradiated VSMCs. Variations at protein levels were confirmed by Western blot for α-SMA, CTGF, PAI-1, and heat shock protein 27 (HSP27). To be sure that in this case we observed paracrine effects of ECs and not direct effects of irradiation, VSMCs were irradiated alone (Figure 4B). In the absence of ECs, irradiation decreases the mRNA and protein levels of α-SMA. In contrast, the other target genes were unaffected. These results suggest that changes in VSMC phenotype observed in the presence of ECs were not due to direct radiation effects and that irradiated ECs produced paracrine factors that subsequently induced VSMC fibrogenic phenotype. To confirm the paracrine effects of ECs, irradiated ECs were cultured in the presence of nonirradiated VSMCs. As shown in Figure 4C, expression of α-SMA, CTGF, PAI-1, HSP27, and COL3A1 increased in VSMCs exposed to irradiated ECs. TGF-β1 is a well-known growth factor involved in VSMC fibrogenic phenotype. TGF-β1 secretion was measured in supernatants of ECs and VSMCs by ELISA assay. Interestingly, secretion of total and active forms of TGF-β1 increased after irradiation of ECs (Figure 5). No difference in the secretion of TGF-β1 was observed in supernatant of irradiated VSMCs. Immunofluorescence labeling of Smad3 and Smad4 in VSMCs was performed to determine whether ECs activate the Smad pathway (Figure 6A). In 24-hour serum-starved VSMCs, Smad3 and Smad4 were localized in the cytosol. In the presence of irradiated ECs, Smad3 and Smad4 were translocated to the nucleus of irradiated and nonirradiated VSMCs. Transient transfection of VSMCs with the (CAGA)9Lux vector showed that irradiated ECs activate Smad-dependent gene transcription in VSMCs (Figure 6B). Knock-down of Smad3 in VSMCs was performed to investigate the role of this protein in fibrogenic phenotype of VSMCs induced by irradiated ECs. Twenty-four hours after Smad3 siRNA transfection in VSMCs, Smad3 mRNA and protein levels decreased by 80 and 90%, respectively (Figure 7A). In the presence of irradiated ECs, α-SMA, HSP27, CTGF, PAI-1, COL1A2, and COL3A1 mRNA levels decreased in siRNA (si)-Smad3-transfected irradiated VSMCs compared with control-irradiated VSMCs (Figure 7B). To inhibit the TGF-β1 pathway, VSMCs were incubated with a neutralizing antibody directed against TGF-β-RII. The efficiency of TGF-β-RII neutralizing antibody was investigated by its ability to affect Smad nuclear translocation. Twenty-four hours after irradiation, the translocation of Smad3 and Smad4 induced by irradiated ECs decreased in the presence of TGF-β-RII antibody (Figure 8A). Next, the fibrogenic phenotype of nonstarved irradiated VSMCs in the presence of irradiated ECs (10 Gy) was investigated (Figure 8B). Results showed that the expressions of α-SMA, PAI-1, COL1A2, and COL3A1 decreased in the presence of TGF-β-RII antibody, demonstrating that a TGF-β1/TGF-β-RII mechanism is involved in the fibrogenic phenotype of VSMCs induced by ECs. To support in vitro results, we investigated whether radiation-induced vascular damages are associated with overexpression of TGF-β and P-Smad 2/3 in patients treated by radiotherapy (Figure 9). Immunohistochemical staining showed that TGF-β expression increased in irradiated rectum and, in particular, in endothelium. Moreover, a strong increase of P-Smad 2/3 in VSMCs was observed in pathological vessels compared with normal vessels. These in vivo results demonstrate the physiological relevance of an up-regulation of TGF-β expression in endothelium and an activation of Smad signaling in VSMCs. We demonstrate here the importance of proliferation, migration, and fibrogenic phenotype of VSMCs in patients treated with radiotherapy. This study shows that ECs influence these fundamental mechanisms involved in the initiation and progression of vascular damages. The main results of this work are that, in vitro, ECs promote VSMC proliferation, migration, and fibrogenic phenotype after irradiation. The vascular wound healing process is characterized by the proliferative response of VSMCs after injury. Deregulation of VSMC proliferation contributes to the restenotic lesion, atherosclerosis, vascular hypertrophy, and vascular remodeling after hypertension.11Dzau VJ Braun-Dullaeus RC Sedding DG Vascular proliferation and atherosclerosis: new perspectives and therapeutic strategies.Nat Med. 2002; 8: 1249-1256Crossref PubMed Scopus (720) Google Scholar Our results obtained in patients underline the importance of proliferation of VSMCs in radiation-induced vascular damages after radiotherapy. We first showed that nonirradiated ECs have an antiproliferative effect on VSMCs. This is in line with Peiró et al12Peiro C Redondo J Rodriguez-Martinez MA Angulo J Marin J Sanchez-Ferrer CF Influence of endothelium on cultured vascular smooth muscle cell proliferation.Hypertension. 1995; 25: 748-751Crossref PubMed Google Scholar who previously demonstrated that bovine aortic endothelial cells inhibit proliferation of rat VSMCs in a co-culture model. Moreover, our results show that irradiated ECs can stimulate proliferation of VSMCs and/or that irradiated ECs fail to inhibit VSMC proliferation. This is in contrast with de Crom et al13de Crom R Wulf P van Nimwegen H Kutryk MJ Visser P van der Kamp A Hamming J Irradiated versus nonirradiated endothelial cells: effect on proliferation of vascular smooth muscle cells.J Vasc Interv Radiol. 2001; 12: 855-861Abstract Full Text Full Text PDF PubMed Scopus (2) Google Scholar who showed that a very high-dose radiation (40 Gy) of ECs did not affect the proliferation of VSMCs. The strong differences in radiation dose ranges could explain this discrepancy. Cell cycle analyses revealed that irradiated ECs influence cell cycle progression of VSMCs. We can postulate that at the same time ECs produce growth promoters and growth inhibitors that may modulate VSMC growth, ie, platelet-derived growth factor, vascular endothelial growth factor, or basic fibroblast growth factor, but also molecules with short half-lives such as nitric oxide. Nitric oxide inhibits VSMC proliferation by altering the activation of CDK2 and the expression of cyclin A.14Tanner FC Meier P Greutert H Champion C Nabel EG Luscher TF Nitric oxide modulates expression of cell cycle regulatory proteins: a cytostatic strategy for inhibition of human vascular smooth muscle cell proliferation.Circulation. 2000; 101: 1982-1989Crossref PubMed Scopus (193) Google Scholar In an interesting way, a lack of endothelial nitric-oxide synthase was observed in irradiated human cervical arteries from patients treated by radiotherapy for neck cancer15Sugihara T Hattori Y Yamamoto Y Qi F Ichikawa R Sato A Liu MY Abe K Kanno M Preferential impairment of nitric oxide-mediated endothelium-dependent relaxation in human cervical arteries after irradiation.Circulation. 1999; 100: 635-641Crossref PubMed Scopus (109) Google Scholar and in the rabbit ear central artery 2 weeks after an irradiation of 45 Gy,16Zhang XH Matsuda N Jesmin S Sakuraya F Gando S Kemmotsu O Hattori Y Normalization by edaravone, a free radical scavenger, of irradiation-reduced endothelial nitric oxide synthase expression.Eur J Pharmacol. 2003; 476: 131-137Crossref PubMed Scopus (22) Google Scholar whereas an up-regulation of endothelial nitric-oxide synthase was described in bovine aortic endothelial cells.10Sonveaux P Brouet A Havaux X Gregoire V Dessy C Balligand JL Feron O Irradiation-induced angiogenesis through the up-regulation of the nitric oxide pathway: implications for tumor radiotherapy.Cancer Res. 2003; 63: 1012-1019PubMed Google Scholar Further studies are needed to understand molecular mechanisms involved in the control of VSMC proliferation by ECs after irradiation and nitric oxide pathway could be an attractive target. We also f
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