Study On Effects Of Vitronectin On Radiation-Induced Lung Fibrosis

Radiation therapy is one of the main treatment strategies for thoracic tumor. Although the increase in radiation dosage can help increase local control and patient survival rate, the radiation that normal tissues receive during radiation treatment is quite inevitable, and as the dosage of radiation increase, the occurance rate and degree of radiation-induced lung injury also increase. Acute radiation-induced lung injury is an acute complilcation which may happen within 3 months of radiation therapy; after 3 months, patients having received thoracic radiation treatment could be facing the late complication of radiation-induced lung fibrosis. Radiation-induced lung fibrosis is mainly caused by the persistant inflammation after initial injury, which leads to an excessive repair process, characterized by large amounts of inflammatory factors, cytokines and chemokines, causing the accumulation of extracellular components, including hyaluronic acid, fibronectin, proteoglycans, and collagens.Our research group in previous endeavors have screened proteins of predictive value to radiation-induced lung injury by proteomic analysis of plasma proteins in patients before receiving radiation therapy. By comparing patients who have and haven’t developed grade 2 radiation-induced lung toxicity (RILT), vitronectin was found to be higher in the plasma of patients who have developed a grade 2 or higher RILT (P=0.02). This study shows that elevated basal expression of vitronectin is related to radiation-induced lun fibrosis, indicating that vitronectin could play an important role in the process of radiation-induced fibrosis. In addition, many studies have shown that vitronectin is elevated in the circulation or the lung of interstitial lung disease patients, indicating vitronectin could be a regulatory factor in the course of lung fibrosis.In this study, we first irradiated lung fibroblasts in vitro with different doses to observe the change in level of expression of vitronectin and collagen, thus choosing a preferable experimental condition for producing radiation-induced fibrosis in fibroblasts. Subsequently, we constructed lentivirus vector that could either overexpress vitronectin or inhibit vitronectin expression to achieve control of vitronectin expression in fibroblasts, in order to observe the change in degree of radiation-induced fibrosis in fibroblasts with different level of vitronectin expression. At last. we constructed animal models with different basal vitronectin expression by infecting C57BL mice with vitronectin overexpressing or inhibiting lentivirus vectors, thus observing the effect of vitronectin expression level on radiation-induced lung fibrosis in mice.Objective To analyze the changes in the expression of vitronectin and collagen in fibroblasts at different doses post-irradiation in association with time, so as to evaluate the potential of vitronectin as a biomarker for radiation-induced lung fibrosis. To construct human vitronectin (VTN) gene overexpressing and RNA interfering recombinant lentivirus vectors, and achieve stable transduction in human embryo lung fibroblast WI-38 cells, assess vitronectin expression levels and interfering effectiveness, thus providing the research tools and experimental basis for future investigations into the functionalities of vitronectin. To investigate the effects of increased or decreased vitronectin controlled by lentivirus vectors on cultured lung fibroblast cells in radiation-induced fibrosis process.Methods The human fibroblast WI-38 and IMR-90 cells were irradiated with 137Cs at doses of 0 (control),4,6,8,10 and 12 Gy, respectively. The cells and supernatant were collected at 6,12,24,36,48 and 60 h post-irradiation. The expression of vitronectin and collagen Ⅰ and Ⅲ were analyzed by Western Blot, PCR and ELISA. To construct the vitronectin overexpressing lentivirus, the vitronectin gene was amplified using the PCR technique, inserted into the vector pCDH, which went through initial bacterial colony PCR filtering, double digestion, and gene sequencing assessment, together with the packaging vectors, was used to prepare lentivirus solutions by co-transfection of 293T cells with liposome. To construct vitronectin interfering lentivirus, three shRNA sequences targeted at vitronectin were designed and synthesized, inserted into pLVX vector to construct vitronectin interfering lentivirus. The vitronectin overexpressing and interfering lentivirus vectors were transfected into WI-38 cells, and the relative expression levels of vitronectin were assessed using real-time PCR and Western Blotting, and the most efficient interfering lentivirus was assessed. Vitronectin overexpressing or interfering lentivirus vectors were transfected into lung fibroblast WI-38 cells. After transfection, the cells were irradiated with 8 Gy of 137Cs in a single dose. At 48 h post-irradiation, cells and culture supernatant were collected to assess the expression levels of vitronectin and collagen I and III, as well as several proteins involved in the fibrosis regulatory network, including TGF-β1, PI3K, AKT1, ERK1 and JNK. Vitronectin overexpressing lentivirus, vitronectin interfering lentivirus, empty lentivirus (negative control) or saline was given to C57BL mice via tail vein injection to construct mouse models with different levels of basic vitronectin expression. At 48 h after transfection, the 4 groups of mice received a single dose of 8 Gy irradiation to the whole thorax with 6 MeV linear accelerator, constructing the radiation-induced pulmonary fibrosis animal models. At 8 w and 12 w post-irradiation, mice were sacrificed to obtain lung tissue for pathological, real-time PCR, ELISA and Western Blot analysis for qualitative and quantitative assessment of the degree of pulmonary fibrosis in mice.Results 1. After irradiation, the expression of vitronectin and collagen I and III were positively correlated (r=0.40-0.79, P<0.05) and were all significantly higher than that in control group (t>3.04, P<0.05). Vitronectin and collagen reached highest expression at 48 h after receiving 8-10 Gy of irradiation (t>2.92, P<0.05). Real-time PCR analysis and ELISA analysis showed that expression of vitronectin in mRNA and supernatant levels are significantly affected by radiation dose (F=27.09-42.62, P<0.05).2. After transfection of overexpressing lentivirus pCDH-VTN into WI-38 cells, vitronectin expression in both mRNA and protein levels were elevated (t=13.49, P<0.05). Among the 3 lentivirus vectors designed to interfere with vitronectin expression, the lentivirus with the most effective inhibitory effect on vitronectin expression had an inhibitory effect of 75.7%(t=50.1, p<0.05) in mRNA level compared to the negative control lentivirus. The lentivirus after concentration had a titer of over 1×107TU/ml.3. Collagen Ⅰ and Ⅲ expression in vitronectin-overexpressing lentivirus infected WI-38 cells were significantly higher than that in the control group (P<0.05), and the assessed proteins involved in fibrosis regulatory network also showed a significant increase both in protein expression levels and in phosphorylation levels (P<0.05); in contrast, in vitronectin-interfering lentivirus infected WI-38 cells, collagen Ⅰ and Ⅲ showed a significantly lower expression level (P<0.05), and the expression levels and phosphorylation levels of fibrosis regulatory network also showed a significant decrease (P<0.05).4. At 8-12 w after a single dose of 12 Gy irradiation to the mice whole thorax, the levels of collagen Ⅰ, Ⅲ, hydroxyproline, α-SMA and TGF-β1 in the lungs of mice in the vitronectin overexpressing group were significantly higher than that in the control group (P<0.05), the degree of pulmonary fibrosis was also significantly higher. In contrast, the above mentioned proteins in the lungs of mice in the vitronectin inhibitory group were significantly lower than that in the control group, the degree of pulmonary fibrosis is also lower microscopically.Conclusion 1. The fibroblast expression of vitronectin in cellular, supernatant and mRNA levels may be a potential biomarker for fibrosis post-irradiation. The experimental condition of choice for producing radiation-induced fibrosis in fibroblasts are 8 Gy irradiation and 8 h post-irradiation.2. pCDH-VTN and pLVX-shRNA-VTN lentivirus vectors are successfully constructed. The transduction of the vectors into WI-38 cells have proven to produce a higher lever or a repressed level of vitronectin expression. This has provided a useful research tool for further investigations into the functions of vitronectin.3. The expression of vitronectin can enhance the fibrosis changes in cultured fibroblasts, and the inhibition of vitronectin expression can alleviate such changes.4. In vivo study showed that high vitronectin expression levels achieved by lentivirus transfection will lead to a higher degree of radiation-induced pulmonary fibrosis, whereas an inhibited vitronectin expression level achieved by vitronectin interfering lentivirus can alleviate the degree of radiation-induced pulmonary fibrosis.