Cyclooxygenase-2 or tumor necrosis factor-α inhibitors attenuate the mechanotransductive effects of pulsed focused ultrasound to suppress mesenchymal stromal cell homing to healthy and dystrophic muscle

Abstract

Maximal homing of infused stem cells to diseased tissue is critical for regenerative medicine. Pulsed focused ultrasound (pFUS) is a clinically relevant platform to direct stem cell migration. Through mechanotransduction, pFUS establishes local gradients of cytokines, chemokines, trophic factors (CCTF) and cell adhesion molecules (CAM) in treated skeletal muscle that subsequently infused mesenchymal stromal cells (MSC) can capitalize to migrate into the parenchyma. Characterizing molecular responses to mechanical pFUS effects revealed tumor necrosis factor-alpha (TNFα) drives cyclooxygenase-2 (COX2) signaling to locally increase CCTF/CAM that are necessary for MSC homing. pFUS failed to increase chemoattractants and induce MSC homing to treated muscle in mice pretreated with ibuprofen (nonspecific COX inhibitor) or etanercept (TNFα inhibitor). pFUS-induced MSC homing was also suppressed in COX2-knockout mice, demonstrating ibuprofen blocked the mechanically induced CCTF/CAM by acting on COX2. Anti-inflammatory drugs, including ibuprofen, are administered to muscular dystrophy (MD) patients, and ibuprofen also suppressed pFUS-induced homing to muscle in a mouse model of MD. Drug interactions with cell therapies remain unexplored and are not controlled for during clinical cell therapy trials. This study highlights potentially negative drug-host interactions that suppress stem cell homing and could undermine cell-based approaches for regenerative medicine.

Keywords: Cell adhesion molecules; Cyclooxygenase-2; Cytokines; Etanercept; Focused ultrasound; Ibuprofen; MDX; Mechanotransduction; Mesenchymal stromal cell, Mesenchymal stem cell, Cell homing; Nuclear factor kappa-light-chain-enhancer of activated B cells; Tumor necrosis factor alpha; muscle; muscular dystrophy.

© 2014 AlphaMed Press.

Figures

Figure 1. pFUS elicits a transient microenvironmental response that can be utilized on to enhance MSC homing to treated tissue

(a) One hamstring in each mouse (n=5–6/time point) was treated with pFUS. Hamstrings from treated and contralateral control limbs were harvested at various time points for molecular analyses. (b) Time courses of CCTF quantities in muscle homogenates after pFUS (Y axes are in pg/mL; X axis are hours post-pFUS; Time 0 represents sham control). Asterisks indicate significantly elevated levels (p<0.05) identified by ANOVA and Bonferroni post-hoc tests. (c) Heat map depicting fold changes in CCTF and CAMs over time after pFUS to muscle. Protein levels measured by ELISA (shown in [b]) were normalized to sham control values (Time=0). Asterisks indicate significantly elevated levels (also shown in [b]) (d) Stack plot displaying which CCTF and CAM are significantly elevated at each time point. Note the initial increase in TNFα and LIF at 0.17 hrs (i.e.,10 minutes) after pFUS and the biphasic response of CCTF and CAM over time (see Supplementary Table 1 for abbreviations).

Figure 2. MSC home to pFUS-treated muscle when injected prior to pFUS and up to 16 hours post-pFUS

(a) Mice (n=6 per group) were given 106 human MSC intravenously either 45 min before, or 3, 8, or 16 hours post-pFUS to muscle. MSC were quantified with fluorescence immunohistochemistry (fIHC) using an anti-human-mitochondria antibody (HuMito). MSC were counted in 10 high powered fields-of-view from 3 sections per animal. (b) High-magnification images of HuMito staining in muscle from mice that did not receive human MSC (left panel), and HuMito and Isotype staining in mice that received MSC. Scale bars = 20 μm (c) fIHC and (d) quantification of MSC homing after infusing cells at various times before or after pFUS. MSC stained with an anti-human mitochondria antibody are shown in red. Scale bars = 50 μm. The greatest amount of MSC homing was observed when cells were injected either 45 minutes before or 3 hr after pFUS. However, statistically significant increases in MSC homing were also observed when MSC were injected at 8 or 16 hr after pFUS (statistical significance following ANOVA with Bonferroni post-tests is indicated by asterisks and crosses).

Figure 3. pFUS significantly increased expression of COX2 in targeted muscle and pFUS-induced expression is blocked by pretreatment with ibuprofen or etanercept

(a,d) COX2 was significantly elevated as early as 0.5 hours post-pFUS with significant expression declined by 8 hours after pFUS. (b, e) Pretreatment with ibuprofen (30 mg/kg) or etanercept (100 μg) prior to pFUS blocked pFUS-induced COX2 expression. Scale bars = 100 μm. (c) Magnified views of COX2 fIHC showing staining with isotype (left panel) and primary antibody 4 hr post-pFUS (right panel). Scale bars = 10 μm. (f) Western blotting for COX2 4hr post-pFUS compared to untreated contralateral muscle. Statistical significance (p<0.05) is indicated by asterisks after ANOVA or t-test for pairwise comparisons; n=5–6 per time point.

Figure 4. pFUS significantly increased expression of NFκB in targeted muscle and pFUS-induced expression is blocked by pretreatment with ibuprofen or etanercept

(a,d) NFkB was significantly elevated as early as 0.5 hours post-pFUS with significant expression declined by 8 hours after pFUS. (b, e) Pretreatment with ibuprofen (30 mg/kg) or etanercept (100 μg) prior to pFUS blocked pFUS-induced NFkB expression. Scale bars = 100 μm. (c) Magnified views of NFkB fIHC showing staining with isotype (left panel) and primary antibody 4 hr post-pFUS (right panel). Scale bars = 10 μm. (f) Western blotting for NFkB 4hr post-pFUS compared to untreated contralateral muscle. Statistical significance (p<0.05) is indicated by asterisks after ANOVA or t-test for pairwise comparisons; n=5–6 per time point.

Figure 5. Mechanotransductive effects of pFUS are inhibited by ibuprofen or etanercept

(a) Mice (n=6 per time point) were pretreated with either ibuprofen (30 mg/kg orally) 30 minutes prior to pFUS or etanercept (100 mg/kg ip) at 3 days and 1 day prior to pFUS. Muscles were harvested at various time points for molecular analyses. (b) Heat maps depicting the temporal fold changes of CCTF and CAMs following pFUS to muscle when mice were pretreated with either ibuprofen or etanercept. Protein levels were measured by ELISA and normalized to sham control values (See Supplementary Figures 3 and 4 for raw data). No significant increases in expression of any CCTF or CAM were observed at any time point by ANOVAs with Bonferroni post-hoc tests.

Figure 6. Ibuprofen or Etanercept suppress MSC homing to normal pFUS-treated muscle

(a) Mice (n=6 per group) were pretreated with either ibuprofen or etanercept prior to pFUS. Three hours after pFUS, mice received 106 MSC intravenously. Treated and contralateral hamstrings were harvested 24 hr after cell infusions and quantified. (b) MSC homing to pFUS-treated muscle is disrupted when mice are pretreated with ibuprofen or etanercept and MSC do not home to pFUS-treated muscle in COX2-knockout mice. Since ibuprofen is a nonspecific COX inhibitor, MSC homing in COX2-knockout mice (no drug treatments) was also investigated and they were found not to home to pFUS-treated muscle. (c) Quantification of pFUS-induced MSC homing to skeletal muscle in normal mice (pFUS alone), normal mice treated with ibuprofen or etanercept, or COX2-knockout mice that received no drugs. Significant inhibition (p<0.05) of MSC homing to pFUS-treated muscle was observed compared to normal pFUS-treated mice (wild-type without drugs). There were no differences in the numbers of MSC observed in contralateral control muscle from any group. Statistical significance (p<0.05) is indicated by asterisks after ANOVA with Bonferroni post-hoc tests; scale bars = 50 μm.

Figure 7. Ibuprofen suppresses MSC homing to dystrophic pFUS-treated muscle

(a) MDX mice (n=6 per group) were administered pFUS to the hamstring. One group was treated with Ibuprofen 15 min prior to pFUS and the other was not. Three hours after pFUS, MDX mice received 106 MSC intravenously. Treated and contralateral hamstrings were harvested 24 hr after cell infusions and quantified. (b) MSC homing to dystrophic pFUS-treated muscle is disrupted when mice were pretreated with ibuprofen. (c) shows MDX muscle stained with istoype antibodies. (d) Quantification of pFUS-induced MSC homing to dystrophic muscle in MDX mice with and without ibuprofen pretreatment. Significant inhibition (p<0.05) of MSC homing to the pFUS-treated hamstring in ibuprofen-treated was observed compared to pFUS-treated mice without ibuprofen. There were no differences in the numbers of MSC observed in contralateral control muscle from both groups. Statistical significance (p<0.05) is indicated by asterisks after ANOVA with Bonferroni post-hoc tests; scale bars = 50 μm.