Figure Legends
Figure 1: CS exposure causes endothelial dysfunction in the thoracic aorta. Cumulative concentration response curves to (A ) acetylcholine and (B ) sodium nitroprusside (1x10-8M to 1x10-5M) to assess both endothelial-dependent and smooth muscle-dependent vasodilatory responses in mouse thoracic aorta (n=8/group) following either chronic CS or sham exposure, respectively. Results are expressed as mean percentage relaxation relative to precontraction ± SEM. * indicates statistical significance (p<0.05 ) between data sets by two-way ANOVA with Tukey’s multiple comparisons.
Figure 2: Chronic CS exposure increases BAL fluid cellularity and enhances both lung pro-inflammatory and oxidative stress mediator gene expression. The lungs of mice exposed to CS were lavaged for the assessment of total cells (A), macrophages (B), neutrophils (C) and lymphocytes (D) (n=10). Whole lungs excised from mice were then used to measure mRNA expression by RT-qPCR of TNFα (E) (n=10), IL-6 (F) (n=10) and NOX-2 (G) (n=9). Gene expression data are expressed as fold change relative to the sham group. All data are expressed as mean + SEM and were analysed by students unpaired t-test with significance being denoted by * (p<0.05) between treatment groups.
Figure 3 Immunofluorescent staining of endothelial nitric oxide synthase and 3-nitrotyrosine in the thoracic aorta of mice exposed to 8 weeks of CS or room air. Immunofluorescent quantification of eNOS and 3-NT expression in either sham or 8-week CS-exposed mice. Green staining indicates the presence of eNOS (A) or 3-NT (B) and blue staining denotes the nuclear counterstain, DAPI (4’, 6-diamidino-2- phenylindole, dilactate). Representative photographs of immunofluorescent staining in (A) sham-exposed or (B) CS-exposed mice. Expression of eNOS (n=5-6 mice per group) and 3-NT (n=5 mice per group) both normalised to the relative negative control and expressed as fold percentage change relative to the sham-treated group. Scale bar represents 50µM. Data expressed as mean + SEM and analysed by students unpaired t-test with significance being denoted by; * indicating p<0.05 .
Figure 4 Ebselen prevents CS-induced endothelial dysfunction.Cumulative concentration response curves to acetylcholine (A) and sodium nitroprusside (B) (1x10-8M to 1x10-5M) to assess endothelial and smooth muscle-dependent vasodilation in mouse thoracic aorta (n=6) following either chronic CS or sham exposure in either ebselen or vehicle-treated mice, respectively. Results are expressed as mean percentage relaxation relative to pre-constriction ± SEM. * indicates statistical significance (p<0.05 ) between data sets by two-way ANOVA with Tukey’s multiple comparisons.
Figure 5: Effect of chronic CS and ebselen treatment on BAL fluid cellularity and pulmonary pro-inflammatory and oxidative stress gene expression. The lungs of mice exposed to CS were lavaged for the assessment of total cells (A), macrophages (B), neutrophils (C) and lymphocytes (D) (n=10). Whole lungs excised from mice were then used to measure mRNA expression by RT-qPCR of TNFα (E), NOX-2 (F) and GPX-1(G) (n=10). Responses are expressed as fold change relative to the sham + vehicle-treated group, post normalisation to GAPDH (housekeeping gene). All data are expressed as mean + SEM and were analysed by 2-way ANOVA and Tukey’s post hoc analysis with significance being denoted by * (p <0.05) between treatment groups.
Figure 6: Immunofluorescent staining of 3-Nitrotyrosine in the thoracic aorta of mice exposed to chronic CS and treated with ebselen.Immunofluorescent quantification of 3-NT expression in either sham or 8-week CS-exposed mice that were treated with either vehicle or ebselen. Green staining indicates the presence of 3-NT specific and blue staining denotes the nuclear counterstain, DAPI (4’, 6-diamidino-2- phenylindole, dilactate). Representative photographs of immunofluorescent staining in (A) sham-exposed vehicle treated (n=5), (B) sham-exposed ebselen treated (n=5), (C) CS-exposed vehicle treated (n=4) and (D) CS-exposed ebselen treated mice (n=6). Expression of 3-NT normalised to the negative control and expressed as fold percentage change relative to the sham vehicle-treated group. Scale bar represents 50µM. Data expressed as mean + SEM and analysed by two-way ANOVA with Tukey’s multiple comparisons; significance is denoted by; * indicating p<0.05 .
Figure 7: Immunofluorescent staining of endothelial nitric oxide synthase in the thoracic aorta of mice exposed chronically to CS and treated with ebselen Immunofluorescent staining of eNOS in either sham or 8-week CS-exposed mice with or without ebselen administration. Green staining detects the presence of eNOS and Blue staining denotes the nuclear counterstain, DAPI (4’, 6-diamidino-2- phenylindole, dilactate). Representative photographs of immunofluorescent staining in (A) sham-exposed vehicle treated (n=4), (B) sham-exposed ebselen treated (n=4), (C) CS-exposed vehicle treated (n=7) and (D) CS-exposed ebselen treated mice (n=7). Expression of eNOS normalised to the negative control and expressed as fold percentage change relative to the sham vehicle-treated group. Scale bar represents 50µM. Data expressed as mean ± SEM and analysed by two-way ANOVA with Tukey’s multiple comparisons; significance is denoted by * (p<0.05 ).