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 ).