ANTICANCER RESEARCH 24: 2941-2946 (2004)
X-rays Affect the Expression of Genes Involved in Angiogenesis C. POLYTARCHOU1, T. GLIGORIS1, D. KARDAMAKIS2, E. KOTSAKI1 and E. PAPADIMITRIOU1 1Laboratory 2Department
of Molecular Pharmacology, Department of Pharmacy and of Radiotherapy, School of Health Sciences, University of Patras, GR 26504, Greece
Abstract. Background: We have previously shown, using the chicken embryo chorioallantoic membrane (CAM) model of in vivo angiogenesis, that X-rays act on the extracellular matrix and enhance normal and tumor-induced angiogenesis. In the present work, we studied the effect of X-rays on the gene expression of three proteins that are important regulators of angiogenesis: vascular endothelial growth factor (VEGF), heparin affin regulatory peptide (HARP) and inducible nitric oxide synthase (iNOS). Materials and Methods: An area of 1 cm2 of the CAM, restricted by a plastic ring, was irradiated at room temperature. The expression of the genes was studied using RT-PCR and the amounts of the mRNAs were quantified using image analysis of the corresponding agarose gels of the RT-PCR products. Results: VEGF mRNA was decreased 6 h after irradiation. However, at later time points, VEGF expression was significantly increased compared with the nonirradiated tissue. Similarly, X-rays down-regulated both HARP and iNOS expression 6 h after irradiation and the effect was reversed at later time points, similarly to the effect of X-rays on VEGF. Conclusion: These data support the notion that X-rays increase the expression of genes that favor angiogenesis. Radiation therapy is considered to inhibit angiogenesis through damage to the endothelium (1-3). However, there are also in vivo studies that show that X-rays induce angiogenesis (4, 5). Since the exact mechanisms of action of ionizing radiation on tissues remain unclear, it has not been easy to explain this discrepancy. We have recently shown, in the in vivo chicken embryo chorioallantoic membrane (CAM) model of angiogenesis, that X-rays influence the expression, deposition and turnover of extracellular matrix
Correspondence to: E. Papadimitriou, Lab. of Molecular Pharmacology, Dept. of Pharmacy, University of Patras, GR 26504 Greece. Tel: +30-2610-969336, Fax: +30-2610-997665, e-mail:
[email protected] Key Words: Angiogenesis, X-rays, vascular endothelial growth factor, heparin affin regulatory peptide, nitric oxide synthase.
0250-7005/2004 $2.00+.40
(ECM) proteins in a way that secondarily favors both normal and tumor-induced angiogenesis (6). Angiogenesis, the formation of new blood vessels from pre-existing ones, is an active process that is dependent upon the balance of positive and negative regulators. Vascular endothelial growth factor (VEFG) is one of the most potent angiogenic growth factors and plays a significant role in both development and homeostasis (7). Besides its role in activation of endothelial cells during the initial steps of angiogenesis, VEGF is also very important for the maintenance of the differentiated state of blood vessels (8). VEGF-induced proliferation, migration, differentiation of endothelial cells and angiogenesis are believed to be at least partly mediated by nitric oxide (NO) (9). NO production is catalyzed by a family of enzymes, the NO synthases (NOS), which exist in three isoforms, neuronal, inducible (iNOS) and endothelial (eNOS) (10). VEGF induces the expression of both eNOS and iNOS in endothelial cells (11). Heparin affin regulatory peptide (HARP), also called pleiotrophin or heparin binding-growth associated molecule, is an 18 kDa secreted protein with high affinity for heparin. It is a secreted polypeptide and is localized in the extracellular matrix where it interacts with glycosaminoglycans. It is highly conserved among species and contains two distinct lysine-rich clusters within both the NH2- and COOH-terminal domains. HARP displays important functions in growth and differentiation processes and may also have important physiological roles during adulthood (12). We have recently shown that HARP induces angiogenesis in several in vitro models of angiogenesis and in vivo, in the chicken embryo CAM (13, 14). The aim of the present work was to study the effect of Xrays on the expression of VEGF, iNOS and HARP in the chicken embryo CAM.
Materials and Methods CAM assay. The in vivo CAM angiogenesis model was used as previously described (6). Leghorn fertilized eggs (Pindos, Greece) were incubated for 4 days at 37ÆC, when a window was opened on
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ANTICANCER RESEARCH 24: 2941-2946 (2004) the egg shell, exposing the CAM. The window was covered with tape and the eggs were returned to the incubator until the 9th day of development, when the area restricted by the ring was irradiated at room temperature, using an RT 50 mobile contact therapy unit (Philips, Amsterdam, The Netherlands), with 20 KV X-rays (0.1mm Al). The SSD was 4 cm and the dose rate 2,600 cGy per min. A single X-ray dose of 10 Gy was used, because inhibition of angiogenesis in the CAM was maximal at this dose without affecting the viability of the embryos (2, 6). The eggs were further incubated at 37ÆC and after different time periods of incubation, CAMs were excised from the eggs and processed as described below. Reverse transcriptase-polymerase chain reaction (RT-PCR) for VEGF, HARP and iNOS. Total cellular RNA isolation from CAMs was performed as previously described (6). The primers used for chicken sequences of iNOS, VEGF and GAPDH have been previously described (15, 16). The primers used for the detection of HARP mRNA were designed according to the chicken sequence (Accession number BI394859) and were: 5’-AGA GAA ACC AGA GAA AAA GG-3’ (sense) and 5’-CAG TCA GCA TTA TGA AGA GC-3’ (antisense), yielding a product of 288 bp. The RT-PCR reactions for iNOS, GAPDH and VEGF were performed in a single step with 200–250 ng of total RNA, using the Access RT-PCR system (Promega, Madison, WI, USA) or the One Step RT-PCR kit (Qiagen, Valencia, CA, USA), as previously described (15, 16). The RT-PCRs for HARP mRNA were performed in a single step using 200 to 250 ng of total RNA and the Access RT-PCR system, as follows: The reverse transcriptase reaction was performed by AMV-RT for 1 h at 48ÆC. After an initial denaturation step for 2 min at 94ÆC, 30 cycles of amplification (94ÆC for 1 min, 55ÆC for 40 sec and 68ÆC for 1.5 min) were performed and ended with a final DNA synthesis step at 68ÆC for 7 min. In all cases, PCR reactions were not in the saturating phase (data not shown). DNA contamination was excluded by performing PCR reactions in the absence of the reverse transcription step. The RT-PCR products were subjected to electrophoresis on 2% agarose gels containing 0.5 mg/ml ethidium bromide and photographed using a digital camera. The electrophoretic bands were quantified using the ImagePC image analysis software and the ratios iNOS or VEGF or HARP/GAPDH of the electrophoretic band values represent the expression of each gene at different time points after irradiation. Statistical analysis. The significance of variability between the results from each group and the corresponding control was determined by unpaired t-test or ANOVA. Each experiment included triplicate measurements for each condition tested. All results are expressed as mean±SEM from at least three independent experiments.
Results The primers used for chicken VEGF amplified two splicing variants of avian VEGF, VEGF190 (456 bp) and VEGF165 (381 bp) (16). As shown in Figure 1, the mRNA levels of both isoforms of VEGF were significantly decreased 6 h after irradiation, while this decrease was reversed at later time points and, 48 h after irradiation, there was a significant increase in the mRNA expression of both VEGF190 and VEGF165.
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Figure 1. Irradiating chicken embryo CAM leads to temporal changes in VEGF190 and VEGF165 mRNA levels. (A) Products of RT-PCRs for the mRNAs of VEGF190, VEGF165 and GAPDH at several time points after irradiation of the chicken embryo CAM. C: control, Ir: irradiated tissue. Representative picture of 4 independent experiments. (B) The mRNA amounts were quantified by densitometric analysis of the corresponding bands and the ratio VEGF isoform/GAPDH mRNA was calculated in each lane. Results are expressed as mean ± S.E.M. of the % change of the mRNA amounts in the irradiated compared to the non-irradiated tissue (control). Asterisks denote a statistically significant difference (unpaired t-test) from the non-irradiated tissue. * p
