Inhibiting Airway Smooth Muscle Contraction Using Pitavastatin: A Role for the Mevalonate Pathway in Regulating Cytoskeletal Proteins

Abstract

Despite maximal use of currently available therapies, a significant number of asthma patients continue to experience severe, and sometimes life-threatening bronchoconstriction. To fill this therapeutic gap, we examined a potential role for the 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) inhibitor, pitavastatin. Using human airway smooth muscle (ASM) cells and murine precision-cut lung slices, we discovered that pitavastatin significantly inhibited basal-, histamine-, and methacholine (MCh)-induced ASM contraction. This occurred via reduction of myosin light chain 2 (MLC2) phosphorylation, and F-actin stress fiber density and distribution, in a mevalonate (MA)- and geranylgeranyl pyrophosphate (GGPP)-dependent manner. Pitavastatin also potentiated the ASM relaxing effect of a simulated deep breath, a beneficial effect that is notably absent with the β2-agonist, isoproterenol. Finally, pitavastatin attenuated ASM pro-inflammatory cytokine production in a GGPP-dependent manner. By targeting all three hallmark features of ASM dysfunction in asthma-contraction, failure to adequately relax in response to a deep breath, and inflammation-pitavastatin may represent a unique asthma therapeutic.

Keywords: asthma; bronchodilation; inflammation; mechanics; mechanopharmacology; mevalonate; statin; stretch.

Figure 1

Pitavastatin inhibits basal- and histamine-induced airway smooth muscle (ASM) contraction. (A) As compared to no treatment (0 µM), statistically significant reductions in contraction occurred at 24 h as follows: pitavastatin (Pit) at 0.4, 2, and 10 µM and simvastatin (Sim) at 0.4 and 10 µM. Under similar experimental conditions, pravastatin had no effect on ASM cell contraction. (B) While both 1 µM Sim and 1 µM Pit reduced ASM contraction time-dependently compared to no-treatment (NT), Pit was significantly more efficacious than Sim at 24 h (indicated by ^#). (C) Compared to NT, both 0.4 μM Pit and 0.4 μM Sim reduced basal ASM contraction to a similar extent after 24-h of treatment. However, while 0.4 μM Pit inhibits histamine (Hist)-induced ASM contraction, 0.4 μM Sim does not. Statistical comparison was performed using the student t-test. (D) The force inhibitory effects of 1 μM Pit was reversed when the wells were resuspended with media without Pit. For (A–D), all experiments were performed using one non-asthmatic primary human ASM donor line. (A, B, D) were performed in serum [10% fetal bovine serum (FBS)]-containing media conditions while (C) was performed under serum-deprived media conditions, imposed for 24 h. Serum deprivation was imposed to enhance the ability of the ASM to contract to histamine (Halayko et al., 1999). In all graphs, ASM contraction is plotted as fold change to the pre-treatment baseline value at 0 h. p-values: *,^#p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001. For each group, n=4–8 separate wells per condition.

Figure 2

Pitavastatin is non-toxic in cells and precision cut lung slices (PCLS). (A) Statin treatment did not induce apoptosis in airway smooth muscle (ASM) cells derived from one human non-asthmatic donor. Digitonin (50 µg/ml) was used as a positive control. The experiment was performed in serum [10% fetal bovine serum (FBS)]-containing media conditions. n=3 wells per condition; n's indicate the number of separate wells of ASM monolayers. (B) As compared to no treatment (0 µM), no reductions in viability were observed in mouse PCLS treated with pitavastatin. 0.01% Triton treatment for 2 h is included as a positive control. For each of the 0, 0.1, and 1 µM pitavastatin treatment groups, we used n=25 slices that were obtained from four mice, with three to eight slices obtained per mouse. For the triton group, we used n=4 slices obtained from two mice, with two slices per mouse.

Figure 3

Pitavastatin inhibits basal contraction of asthmatic human airway smooth muscle (ASM) cells and methacholine (MCh)-induced constriction of murine precision cut lung slices (PCLS). (A) We observed heterogeneity in basal ASM contraction in both asthmatic (D1–D3) and non-asthmatic (D4–D6) donors. (B) Across both asthmatic and non-asthmatic donor cells, pitavastatin dose-dependently inhibited ASM contraction (#p < 0.0001 compared to 0 µM treatment). Cells from each donor were tested using n=4–8 separate wells. All cellular experiments were performed in serum [10% fetal bovine serum (FBS)]-containing media conditions. (C) When PCLS derived from neonatal mice pre-exposed to MCh alone (Ctrl), MCh+vehicle (Veh), or MCh+pitavastatin (Pit) were treated with 500 nM MCh, the airways of the Pit group constricted significantly less (Ctrl=24.9%, Veh=27.2%, Pit=14.2%, *p < 0.05). For each group, we used n=10–13 PCLS that were obtained from 2 to 3 mice.

Figure 4

Pitavastatin inhibits the airway smooth muscle (ASM) cytoskeleton. ASM cells from a non-asthmatic human donor lung were grown in serum [10% fetal bovine serum (FBS)]-containing media in the presence of drug-vehicle (Veh) or 1 μM pitavastatin (Pit) for 24 h. (A–C) Pit treatment significantly reduced basal phospho-MLC2 (pMLC2) expression, but not total-MLC2. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as a protein loading control. Pit treatment also significantly reduced thrombin (2 U/ml, 30 min)-induced pMLC2 enhancement. (D) Immunostaining measurements revealed that pitavastatin significantly reduced F-actin (green) and pMLC2 (red) expression. For these representative images, F-actin and pMLC2 intensities were quantified using the “integrated density” measurement in ImageJ and normalized to Veh. Scale bar=50 μm. p-values: **p < 0.01; ***p < 0.001.

Figure 5

Pitavastatin Inhibits the airway smooth muscle (ASM) cytoskeleton via a mevalonate (MA)- and geranylgeranyl pyrophosphate (GGPP)-dependent mechanism. ASM cells obtained from a non-asthmatic human donor lung were grown in serum [10% fetal bovine serum (FBS)]-containing media in the additional presence of one of the following five supplements: drug-vehicle, 0.1 “or” 1 μM pitavastatin (Pit), 10 μM GGPP, 1 μM Pit+10 μM GGPP, 100 μM MA, 0.1 μM “or” 1 μM Pit+100 μM MA for 24 h. (A) While Pit alone inhibited basal ASM contraction, co-treatment with MA or GGPP abrogated Pit-induced ASM force inhibition. (B) While Pit alone inhibited ASM F-actin expression, co-treatment with GGPP or MA abrogated Pit-mediated F-actin inhibition. Scale bar = 20 μm. For these representative images, F-actin intensity was quantified using the “integrated density” measurement in ImageJ and normalized to the mevalonate (MA) group. (C) Silencing 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR) expression in the non-asthmatic human ASM donor line used in (A, B) for 48 h significantly reduced basal ASM contraction. n=12 separate well of ASM monolayer per condition. All experiments were performed in serum (10% FBS)-containing media conditions. p values: *p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001.

Figure 6

Pitavastatin potentiates the airway smooth muscle (ASM) relaxation effect of a simulated deep breath, a beneficial effect of pitavastatin that is absent for isoproterenol. (A) As compared to untreated controls (n=7), pre-treatment with 1 µM Pit (24 h) (n=7) or 10 µM isoproterenol (Iso, 30 min) (n=6) significantly inhibited basal ASM contraction. Shown are contraction values normalized to the untreated control group. (B) In response to a subsequent single stretch-unstretch maneuver that mimics a deep breath (10% magnitude, 4-s duration, see inset), the ASM cell promptly and dramatically ablates its contraction. The forces then subsequently recover over 180 s. While force ablation was similar across all three groups, the subsequent force recovery was significantly inhibited by Pit treatment. The n's indicate the number of separate wells of ASM monolayers. The experiment was performed in serum [10% fetal bovine serum (FBS)]-containing media conditions. P-values: *p < 0.05; ****p < 0.0001.

Figure 7

Pitavastatin inhibits airway smooth muscle (ASM) inflammation by a geranylgeranyl pyrophosphate (GGPP)-dependent mechanism. ASM cells obtained from a non-asthmatic human donor lung were grown in serum [10% fetal bovine serum (FBS)]-containing media for at least 72 h and were treated with either drug-vehicle, 2 μM Pit, or 10 μM GGPP for a total of 72 h. During the final 18 h of treatment, cells were exposed to a cytokine mixture (CM) comprising interleukin (IL)-13, IL-17, and TNFα, at 10 ng/ml each. (A–D) While Pit alone inhibited CM-induced IL-6, IL-8, and eotaxin-3 production, co-treatment with GGPP abrogated these effects, confirming a GGPP-dependent mechanism. GGPP alone or CM+GGPP had no inducing or inhibiting effect on these cytokines/chemokines (data not shown). p values: *p < 0.05; **p < 0.01; ****p < 0.0001.

Figure 8

Proposed mechanism of action of pitavastatin: pitavastatin inhibits components of the airway smooth muscle (ASM) cytoskeletal apparatus including myosin light chain (MLC) and F-actin. The inhibition of F-actin occurs by a geranylgeranyl pyrophosphate (GGPP)-dependent mechanism. Pitavastatin depletes the pool of available intracellular GGPP by inhibiting 3-hydroxy-3-methylglutaryl-coenzyme A reductase (HMGCR). This schema explains, at least in part, the molecular mechanism by which pitavastatin inhibits human ASM contraction.