The Role Of Integrin αVβ3 In Ventilator-Induced Lung Injury And Its Underlying Mechanism

Objective Mechanical ventilation (MV) is a necessary supportive method for patients with respiratory failure without alternatives, it is also a common way to manage respiration during anesthesia and operations. However, ventilator induced lung injury (VILI) is a common complication of MV and aggravates previous lung injury. Although lung protective MV with low tidal volume (VT) can attenuate lung injury degree of VILI, it is still needed to find effective adjuvant pharmacological therapy for prevention from VILI in addition to lung protective ventilation because the necessity to guarantee sufficient gas exchange frequently limits a further substantial reduction of tidal volumes. The main pathology of VILI is alveolar inflammation response and hyperpermeability of pulmonary capillary, but the mechanism is not clear yet. Therefore, model of rats exposed to mechanical ventilation with high VT was constructed to investigate alveolar inflammation and activation of integrinαVβ3 and nuclear factor kappa B (NF-κB), and effect of synthesized RGDS peptide (non-specific blocker of integrins) on activation of integrinαVβ3 and inhibitor of NF-κB (I-κB), and then to evaluate the role of integrinαVβ3 mediated NF-κB activation in alveolar inflammation of VILI in rats. In addition, model of human pulmonary microvascular endothelial cells (HPMVEC) exposed to mechanical stretch was constructed to investigate the effect of mechanical stretch on monolayer permeability and integrinαVβ3 activity, and to analyze the relationship between integrinαVβ3 activity and monolayer permeability, and then to assess the effect of integrinαVβ3 in endothelium permeability. Taken all together, this study is aimed to discover the role of integrinαVβ3 in the development of VILI and its underlying mechanism, and to find a new target of prevention from VILI.MethodsAnimal experiment: Anesthetized and intratracheal intubation male Sprague Dawley (SD) rats were randomly divided into 3 groups: control group (Control), mechanical ventilating with high tidal volume (H_VT) group, RGDS pretreatment group (H_VT +RGDS). Each group had 7 rats. Rats kept spontaneous respiration in Control group; rats in H_VT group were mechanical ventilated with high tidal volme of 35 ml/kg and frequency of 50 per minutes; rats in H_VT +RGDS group were pretreated with RGDS peptide 5 mg/kg, i.p. 30 min before mechanical ventilation, and the parameters of mechanical ventilation were as same as H_VT group. Rats were killed after 4 h mechanical ventilation, and bronchoalveolar lavaged fluid (BALF) and lung tissues were collected. BALF was centrifuged to get supernant and deposit. Cytokines of tumor necrosis factor alpha (TNF-α), interleukin-6 (IL-6) in supernant were detected with enzyme linked immunoabsorbance assay (ELISA); total protein in supernant was detected with bicinchoninic acid (BCA) protein assay kit. Cell counts and classification were examined with deposit slides and Wright's staining. Pathological changes of lung tissue were detected with hematoxylin and eosin (HE) staining; expression of integrinαVβ3, I-κB and their phosphataion were detected with Western Blot.Cellular experiment: Primary HPMVEC were purchased and passaged, and the passages 4 to 8 were used in this study. Cells were randomly divided into 3 groups: control group (group Control), mechanical stretching group (group Streth), and ML9 pretreatment group (group Stretch+ML9). Cells in group Control were cultured in static condition without mechanical stretch; cells in group Stretch were exposed to mechanical stretch for 2 h; cells in group Stretch+ML9 were pretreated with 50μM ML9 2 h before mechanical stretch. Mechanical stretch was produced with magnetic twisting stimulation (MTS); monolayer permeability was examined with fluorescein isothiocyanate marked dextran (FITC-dextran) in Transwell model, permeability was evaluated by the quantity of dextran through monolayer HPMVEC and expressed as optical density (OD); distribution of integrinαVβ3 and actin was investigated by immunofluorescence staining; expression of integrinαVβ3 was detected with Western Blot; intracellular tension was measured with magnetic twisting cytometry (MTC).All data were expressed as mean±S.D. (standard deviation), difference was tested by ANOVA with SPSS software,P values less than or equal to 0.05 was considered significant.ResultsAnimal experiment: (1) There was no pathological changes in lung tissues of Control group; in, significant pathological changes in lung tissue was observed including leukocyte recruitment, alveolar edema and structure damage of alveolar; the pathological changes in lung tissue of H_VT+RGDS group was less than H_VT group. (2) Compared with Control group, polynuclear cells, mononuclear cells and total protein in BALF increased significantly in H_VT group; compared with H_VT group, they decreased significantly in H_VT+RGDS group (P<0.05). (3) Compared with Control group, IL-6 and TNF-αin BALF increased significantly in H_VT group; compared with H_VT group, they decreased significantly in H_VT+RGDS group (P<0.05). (4) Compared with Control group, phosphorylation of integrinαVβ3 and NF-κB in lung tissues increased significantly in H_VT group; compared with H_VT group, they decreased significantly in H_VT+RGDS group (P<0.05).Cellular experiment: (1) Compared with Control group, permeability of monolayer HPMVEC increased significantly in Stretch group (P<0.05), while no significant change was observed in Stretch+ML9 group. (2) Actin was distributed at the border of HPMVEC in control group; in Stretch group, actin polymerized into stress fiber in plasma; in Stretch+ML9 group, actin distributed similarly with Control group. (3) IntegrinαVβ3 distributed evenly on the HPMVEC surface in Control group, while integrinαVβ3 clustered significantly in Stretch group, and its clustering decreased in Stretch+ML9 group. (4) Compared with Control group, intracellular tension increased significantly in Stretch group (P<0.05), while no significant change was observed in Stretch+ML9 group.Conclusions (1) IntegrinαVβ3 activation takes part in pulmonary inflammation response and hyperpermeability of capillary in VILI. (2) IntegrinαVβ3 mediated NF-κB activation is one of the mechanisms of pulmonary inflammation in VILI. (3) Stress fiber formation and integrinαVβ3 clustering may be the mechanism of mechanical stretch induced monolayer hyperpermeability in HPMVEC. (4) It is a promising method to inhibit integrin activity for prevention of VILI.