Simvastatin inhibits goblet cell hyperplasia and lung arginase in a mouse model of allergic asthma: a novel treatment for airway remodeling?

Abstract

Airway remodeling in asthma contributes to airway hyperreactivity, loss of lung function, and persistent symptoms. Current therapies do not adequately treat the structural airway changes associated with asthma. The statins are cholesterol-lowering drugs that inhibit the enzyme 3-hydroxy-3-methyl-glutaryl-CoA reductase, which is the rate-limiting step of cholesterol biosynthesis in the mevalonate (MA) pathway. These drugs have been associated with improved respiratory health, and ongoing clinical trials are testing their therapeutic potential in asthma. We hypothesized that simvastatin treatment of ovalbumin (OVA)-exposed mice would attenuate early features of airway remodeling by a mevalonate-dependent mechanism. BALB/c mice initially were sensitized to OVA and then exposed to 1% OVA aerosol for 2 weeks after sensitization for 6 exposures. Simvastatin (40 mg/kg) or simvastatin plus MA (20 mg/kg) were injected intraperitoneally before each OVA exposure. Treatment with simvastatin attenuated goblet cell hyperplasia, arginase-1 protein expression, and total arginase enzyme activity, but it did not alter airway hydroxyproline content or transforming growth factor-β1. Inhibition of goblet cell hyperplasia by simvastatin was mevalonate-dependent. No appreciable changes to airway smooth muscle cells were observed in any control or treatment groups. In conclusion, in an acute mouse model of allergic asthma, simvastatin inhibited early hallmarks of airway remodeling, which are indicators that can lead to airway thickening and fibrosis. Statins are potentially novel treatments for airway remodeling in asthma. Additional studies using subchronic or chronic allergen exposure models are needed to extend these initial findings.

Figure 1. A) Fraction of Exhaled Nitric Oxide (FeNO), B) Whole Lung Homogenate Nitrate/Nitrite Levels

A). The effect of simvastatin on in vivo FeNO measurements as described in Methods. Simvastatin (40 mg/kg) treatment decreased FeNO by 61.7% (*p<0.0019 by Kruskal-Wallis test) down to air control levels. Simvastatin and MA (20 mg/kg) co-treatment did not reverse the inhibitory statin effect (p=NS, Kruskal-Wallis test). Each treatment group had between 5–8 mice. B) Treatment with simvastatin did not alter whole lung nitrate/nitrite content, an indicator of NO production (p=NS by 1-way ANOVA). Each treatment group had between 6–9 mice.

Figure 2. Airway goblet cell hyperplasia

i) Treatment with simvastatin (40 mg/kg) reduced the mean goblet cell index (see Methods) by 33% (*p<0.005 by 1-way ANOVA). Co-treatment with MA (20 mg/kg) reversed this inhibitory statin effect (**p<0.05 by 1-way ANOVA). The air controls are not shown since there were virtually no goblet cells visualized (see Fig 2ii image D). Each treatment group had between 4–6 mice. ii) The four different treatment groups seen at an original magnification of x400 were stained with Alcian Blue-PAS to show goblet cells in the airway epithelium: A) 6OVA+EtOH, B) 6OVA+Sim, C) 6OVA+Sim+MA, and D) FA+PBS. The OVA group treated with simvastatin (6OVA+Sim) has significantly less goblet cell staining corresponding to the 33% decrease in mean goblet cell index as described above in i).

Figure 3. A) Arginase-1 (Arg1) protein expression in whole lung homogenate by Western blot, B) Total arginase enzyme activity in whole lung homogenate, and C) Correlation between total arginase enzyme activity and Arg1 protein expression

A) Simvastatin (40 mg/kg) reduced Arg1 protein expression by 59.4% (*p<0.05 by 1-way ANOVA). Co-treatment with MA (20 mg/kg) reversed the inhibitory statin effect on Arg1 expression, but this was not statistically significant (p=NS by 1-way ANOVA, p=0.057 by t-test). Each treatment group had between 8–12 mice. B) Simvastatin (40 mg/kg) reduced total arginase enzyme activity by 57% in the OVA group (*p<0.005 by 1-way ANOVA). Co-treatment with MA (20 mg/kg) reversed the statin effect (not significant by 1-way ANOVA, but significant by t-test (^#p=0.0275)). Each treatment group had between 6–9 mice. C) Arg1 protein expression is represented by protein band intensity (measured by Western blots of whole lung homogenates). Total arginase enzyme activity correlated positively with Arg1 protein expression (r²=0.70, p<0.0001). The dotted lines above and below the best fit line represent the 95% confidence interval. Each dot represents one mouse.

Figure 4. Immunohistochemical staining of Arginase-1 (Arg1)

The four different treatment groups are seen at an original magnification of x100, where Arg1 stains brown. We evaluated the epithelial and subepithelial compartments, of which the latter contains the airway smooth muscle cells, inflammatory cells, and extracellular space. There was no Arg1 staining of airway smooth muscle cells in any of the treatment groups. A) 6OVA+EtOH. Arg1 stains dark brown and localizes to the subepithelial space and in macrophages (red arrow heads). There is little-to-no Arg1 staining visualized in airway epithelial cells. B) 6OVA+Sim. Simvastatin treatment (40 mg/kg) of OVA-exposed mice attenuated Arg1 staining in the subepithelial space. No differences were seen compared to 6OVA+EtOH in macrophage Arg1 staining by this qualitative assessment. C) 6OVA+Sim+MA. Co-treatment with MA (20 mg/kg) partially abrogated the simvastatin effect on Arg1 staining in the subepithelial compartment. No significant differences were seen with respect to macrophage Arg1 staining. D) FA+PBS. There is no Arg1 staining noted in this air control group.

Figure 5. Correlations between Th2 Cytokines and Arginase-1 (Arg1) protein expression

Arg1 protein expression is represented by protein band intensity (equivalent to RLU) as measured by Western blots of whole lung homogenates. The dotted lines above and below the best fit line represent the 95% confidence interval. Each dot represents one mouse. A) The whole lung lavage IL-4 levels correlated positively with increased Arg1 protein expression in whole lung (r²=0.78, p<0.0001). B) The whole lung lavage IL-13 levels correlated positively with increased Arg1 protein expression in whole lung (r²=0.56, p=0.0003).

Figure 6. Correlations between lung physiology and Arginase-1 (Arg1) protein expression

Arg1 protein expression is represented by protein band intensity (equivalent to RLU) as measured by Western blots of whole lung homogenates. The dotted lines above and below the best fit line represent the 95% confidence interval. Each dot represents one mouse. A) The percent increase in respiratory system resistance (Rrs) correlated positively with increased Arg1 protein expression in whole lung (r²=0.45, p=0.0044). B) The percent decrease in dynamic lung compliance (Cdyn) correlated positively with increased Arg1 protein expression in whole lung (r²=0.78, p<0.0001).

Figure 7. A) Concentration of TGFβ1 in whole lung lavage, B) Whole lung lavage Absolute Macrophage Counts, and C) TGFβ1 immunohistochemical staining of lung and inflammatory cells

A) The group 6OVA+EtOH had significantly higher TGFβ1 concentration than any of the FA controls (*p<0.005 by 1-way ANOVA). Simvastatin (40 mg/kg) reduced TGFβ1 concentration in lung lavage by 60.4% in the OVA group (p=NS by 1-way ANOVA or t-test). Co-treatment with MA (20 mg/kg) did not reverse this inhibitory statin effect on TGFβ1. Each treatment group had between 5–6 mice. B) Simvastatin (40 mg/kg) treatment decreased lung lavage absolute macrophage count by 48.3% (*p<0.005 by 1-way ANOVA) in OVA exposed animals. Co-treatment with MA (20 mg/kg) reversed the simvastatin inhibitory effect (p=NS by 1-way ANOVA, but ^#p=0.0029 by t-test). Each treatment group had between 10–17 mice. C) By qualitative immunohistochemical assessment, macrophages appear to be the main inflammatory cells responsible for TGFβ1 production under OVA-induced allergic inflammation (image shows the 6OVA+EtOH treatment group: representative slide where TGFβ1 stains brown seen at x400 magnification). Arrows represent inflammatory cells with little-to-no staining for TGFβ1 (likely eosinophils or lymphocytes). Arrow heads represent macrophages that stain densely brown for TGFβ1.

Figure 8. TGFβ1 immunohistochemical staining and scoring in different lung compartments

The groups illustrated in the histology images are seen at an original magnification of x400, where TGFβ1 stains brown: A) 6OVA+EtOH, B) 6OVA+Sim, C) 6OVA+Sim+MA, and D) FA+PBS. Simvastatin (40 mg/kg) had no statistically significant effect on total TGFβ1 content in the different lung compartments evaluated. Each treatment group had between 4–6 mice. All analyses were done by 1-way ANOVA. i) In the proximal airway epithelium (ProxEpi), the air controls (FA+Sim, FA+PBS) had greater TGFβ1 content compared to the OVA groups, specifically 6OVA+EtOH (*p<0.05). ii) Histology corresponding to i) where TGFβ1 stains brown most intensely in the epithelium of the FA+PBS group. iii) In the lung parenchyma, all three air control groups had much lower TGFβ1 content compared to the 6OVA+EtOH group (*p<0.005). The treatment group 6OVA+Sim+MA showed less TGFβ1 content than the 6OVA+EtOH group (^#p<0.005). iv) Histology corresponding to iii) shows the highest intensity TGFβ1 staining in group A) 6OVA+EtOH, greater than the other OVA groups, and significantly greater than all of the air controls. v) In the distal airway epithelium (DistEpi), there were no statistically significant differences amongst all six groups (p=NS). vi) In the basement membrane (BM), the air controls had significantly less TGFβ1 content than the OVA groups, specifically 6OVA+EtOH (*p<0.05) and 6OVA+Sim (^#p<0.05).

Scheme 1. The mevalonate (MA) pathway and statin mechanism of action

Simvastatin inhibits the rate-limiting enzyme hydroxymethylglutaryl (HMG)-CoA reductase to prevent the conversion of HMG-CoA to mevalonate (MA). Reversal of observed statin effects with MA co-treatment indicates that HMG-CoA reductase is the likely target enzyme of the statin.