Airway Vascular Angiogenesis And Remodeling In A Murine Model Of Bronchial Asthma

Study objectiveAsthma is a complex disease characterized by airway inflammation, airway hyperresponsiveness and airway remodeling. Airway vascular angiogenesis and remodeling are main contributor to these process. As while, vascular endothelial growth factor(VEGF) and platelet derived growth factor(PDGF) play important roles in vascular angiogenesis and remodeling. VEGF induces vascular permeability, vascular leakage and enhances angiogenesis, but deficiency of PDGF, especially PDGFB, will induce mural cells dysfunction, endothelial cells dysfunction and vascular dilatation, leakage and haemorrhage. The detailed results of vascular angiogenesis and remodeling and the role of VEGF and PDGF in the mouse model of asthma were elucidated in this study.Materials and methods1,AnimalsMale A/J mice, three groups, were initially immunized intraperitoneally four times with 10μg ovalbumin(OVA)+2mg aluminum hydroxide gel(on days 0, 28, 42, 56). Subsequently, 20mg/mL OVA inhalations were done for 20 minutes everyday for 1 weeks(group B: acute asthma) or 5 weeks(group C: chronic asthma), using the ultrasonic nebulizer. Group A(normal control) inhaled 0.9% sodium chloride. 2,Airway hypersensitivityAfter pentobarbital anesthesia, the mice were tracheostomized, The animals were placed in the whole-body plethysmograph to measure airway resistance(Raw). Data are expressed as percent Raw:(Raw after inhalation of ACh/Raw before inhalation)×100(%).3,Vascular perfusion, sacrifice of mice, sample preparation.Cell numbers in BALF were detected. Total IgE and OVA-specific IgE in serum, and IL-4 and IL-5 in BALF were measured using ELISA kits.4,HE and immunofuorescence staining of frozen slicesFrozen blocks were sectioned into 4 to 6μm using cryostat machine and stained with HE methods and taken pictures using light microscope.Briefly, double and triple immunofluorescence staining: frozen sections were fixed with acetone for 10 minutes, incubated in 10% normal goat serum(Sigma, MO, USA) for 20 minutes at room temperature to block nonspecific antibody binding. Next, the sections were incubated overnight at 4℃in humidified chamber of primary antibodies. After several rinses with PBS, specimens were incubated for 2 hours at room temperature with fluorescent secondary antibodies.Triple immunofluorescence staining: After incubated with the first and secondary antibodies(Same as above), specimens were incubated for 0.5 hour at room temperature with TOTO-3 for nuclear staining. Finally after several rinses with PBS, specimens were mounted in 10% glycerine in PBS. We observed and took pictures by a laser scanning confocal microscope.5,RT-PCR analysisTotal RNAs were extracted using the RNeasy Mini Kit(Qiagen; Hilden, Germany) with DNase treatment. cDNA was synthesized from total RNA with oligo(dT) as a primer using Superscriptâ…¡RNase H-Reverse Transcriptase(Gibco BRL, Rockville, MD, USA) at 37℃for 1.5 h. PCR was carried out using 1 ul of the mixed cDNA aliquot. cDNA products were electrophoresed on 2% agarose gels and visualized with SYBR Greenâ… (Molecular Probes; Eugene, OR) and band images were captured using Molecular Imager FX, and normalized to or against glyceraldehyde-3-phosphate dehydrogenase(GAPDH) signal intensity. GAPDH was used as an internal control and detected at uniform levels. The analyses were done using NIH Image Software.6,Western blot methods to detect protein levelAfter extraction and quantification of protein from tissues, the samples were electrophoresed with SDS-PAGE. The samples were cultured with primary and secondary antibodies and detected using NIH Image Software.7,Statistical analysisValues are expressed as means±SEM(SE). The significance of differences between means was assessed by the t-test for comparisons, with statistical significance judged as p<0.05.Results1,This murine model showed following characteristics: they induced airway hyperrsponsiveness to acetylcholine; they increased serum total IgE and OA-specific IgE; increased both the number of total cells and the fraction of eosinophils in BALF; histologically, airway ciliated cells were replaced by mucus-secreting cells and many inflammatory cells accumulated into the sub-epithelial area by HE staining; they induced inflammatory cytokines expression such as IL-4 and IL-5 in the lung. Namely, they mimicked human asthma physiologically, serologically, immunologically, pathologically and molecular biologically.2,The results of FITC-Dextran infusion sections showed that enhanced vascularity and vasodilatation in the early and late phases as compared to the control, as well as twisting of the capillaries, dilatation and leakage were observed in the tracheal wall vessels in the late phase. Vascularity in tracheal and bronchial wall vessels were detected by morphometric measurements of area densities(% of total tissue area). The result showed a significantly enhanced vascularity in the tracheal walls in the late phase.3,The phenotype of vascular endothelial cells and mural cells(vascular smooth muscle cells and pericytes) changed, the function of these cells may also be changed. Proliferation of endothelial cells occurred mainly in small sized vessels in late phase. Smooth muscle cells decreased in large vessels in early phase, however, they returned to nearly normal in chronic stage, suggesting that vascular remodeling occurred not only in small vessels but also in large vessels and it was reversible.4,Expression of VEGF mRNA isoforms were detected by reverse transcription-polymerase chain reaction(RT-PCR). The rare isoform VEGF₁₄₄ could only be detected in lung tissue. The expression of diffusable VEGF₁₆₄ mRNA and VEGF₁₈₈ mRNA was highly upregulated in late phase asthma at a statistically significant level in tracheal tissues and in lung tissues respectively. The densities of VEGFR1 mRNA didn't show different expressions in asthma tracheal and lung tissues. VEGFR2 was not detected in normal tracheal tissue, but it was increased in trachea in asthma. It didn't show different expressions in asthma lung tissues compared with those in normal tissues. The results of protein levels using westernblot method were consistent with mRNA expression.5,PDGFB and PDGFRβmRNA were not detected in normal and acute tracheal tissue. But they were increased in trachea in chronic asthma. They showed enhanced expressions in asthma lung tissues compared with those in normal tissues, especially in chronic phase. The results of protein levels using westernblot method were consistent with mRNA expression. Conclusion1,On the basis of asthma model, we observed the vascular angiogenesis and remodeling in asthma murine model: increased vascularity, vascular dilatation, leakage; phonotype and number of vascular endothelial cells and mural cells changed, and these kind of changes were related with vessels size and disease process.2,VEGF/VEGFR play an important role in vascular angiogenesis and remodeling in asthma murine model. Different isoforms of VEGF expressed in different level and play different roles in asthma in tracheal and lung tissues. VEGFR-2 is increased in trachea in asthma. It is the major mediator of the mitogenic, angiogenic, and permeability-enhancing effects of VEGF.3,PDGFB and PDGFRβmRNA and protein showed enhanced expressions in asthma tracheal and lung tissues compared with those in normal tissues, especially in chronic phase. That suggested us PDGFB and PDGFRp play an important role in vascular angiogenesis and remodeling in asthma murine model.