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. 2017 Feb 7:7:41550.
doi: 10.1038/srep41550.

Improving the therapeutic efficacy of mesenchymal stromal cells to restore perfusion in critical limb ischemia through pulsed focused ultrasound

Pamela A Tebebi  1   2 , Saejeong J Kim  1 , Rashida A Williams  1 , Blerta Milo  1 , Victor Frenkel  3 , Scott R Burks  1 , Joseph A Frank  1   4
Affiliations

Improving the therapeutic efficacy of mesenchymal stromal cells to restore perfusion in critical limb ischemia through pulsed focused ultrasound

Pamela A Tebebi et al. Sci Rep. .

Abstract

Mesenchymal stem cells (MSC) are promising therapeutics for critical limb ischemia (CLI). Mechanotransduction from pulsed focused ultrasound (pFUS) upregulates local chemoattractants to enhance homing of intravenously (IV)-infused MSC and improve outcomes. This study investigated whether pFUS exposures to skeletal muscle would improve local homing of iv-infused MSCs and their therapeutic efficacy compared to iv-infused MSCs alone. CLI was induced by external iliac arterial cauterization in 10-12-month-old mice. pFUS/MSC treatments were delayed 14 days, when surgical inflammation subsided. Mice were treated with iv-saline, pFUS alone, IV-MSC, or pFUS and IV-MSC. Proteomic analyses revealed pFUS upregulated local chemoattractants and increased MSC tropism to CLI muscle. By 7 weeks post-treatment, pFUS + MSC significantly increased perfusion and CD31 expression, while reducing fibrosis compared to saline. pFUS or MSC alone reduced fibrosis, but did not increase perfusion or CD31. Furthermore, MSCs homing to pFUS-treated CLI muscle expressed more vascular endothelial growth factor (VEGF) and interleukin-10 (IL-10) than MSCs homing to non-pFUS-treated muscle. pFUS + MSC improved perfusion and vascular density in this clinically-relevant CLI model. The molecular effects of pFUS increased both MSC homing and MSC production of VEGF and IL-10, suggesting microenvironmental changes from pFUS also increased potency of MSCs in situ to further enhance their efficacy.

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Conflict of interest statement

The authors declare no competing financial interests.

Figures

Figure 1
Figure 1. Laser Doppler perfusion imaging (LDPI) of feet in critical limb ischemia (CLI) up to 7 weeks post-surgery.
(A) Relative limb perfusion in healthy (Day-1) legs and ischemic legs at 1 day after surgery (Day 1) and at 7 weeks post-surgery (Day 49) (n = 8) (perfusion in ischemic legs is normalized to perfusion of the contralateral leg) (B) Representative LDPI at days -1, 1, and 49 showing regions-of-interest for quantification.
Figure 2
Figure 2. Proteomic responses to critical limb ischemia (CLI) alone and pulsed focused ultrasound (pFUS) in CLI skeletal muscle.
(A) Experimental timeline showing CLI at Day 0. pFUS treatment time points (Days 14, 15, and 16) are indicated by dashed arrows on top of timeline and tissue harvest time points (Days 1, 14, and 16) are indicated by solid arrows on the bottom of timeline. (B) Stack box plot showing significant (p < 0.05; n = 6 mice per group per time point) elevations of cytokines, chemokines, trophic factors, and cell adhesion molecules compared to normal muscle after CLI alone (Days 1 and 14), after 1 course of pFUS (day 14 pFUS 1x), and after 3 daily courses of pFUS (day 14 pFUS 3x). Comparisons were made using one-way ANOVA with Bonferroni corrections; n = 6 mice/time point. (C) Heat map depicting significant differences shown in (B). Red indicates significant elevations, yellow represents no change, and black indicates undetectable protein levels. (see Supplemental Figure 1 for primary data and Supplemental Table 1 for abbreviations).
Figure 3
Figure 3. Mesenchymal stem cell (MSC) homing to critical limb ischemia CLI muscle with and without pulsed focused ultrasound (pFUS).
Mice (n = 5 per group) were given CLI surgery on day 0 and then on day 14, superparamagnetic iron oxide nanoparticle (SPION)-labeled MSC were IV infused (106 per animal) with and without pFUS. Mice were euthanized 24 hr after treatment (day 15) and MSC were counted. MSC were defined as cells positive for both F4/80 and Prussian blue (PB). (A) Statistically greater numbers of MSC homed to CLI muscle compared to healthy (untreated) muscle, but homing is further increased to CLI muscle using pFUS. *Denotes statistical significance (p < 0.05) by one-way ANOVA. (B) Representative microscopy showing PB+ cells (left, blue cells) and F4/80+ cells (right, brown cells) in each group. Scale bar = 200 μm.
Figure 4
Figure 4. Weekly laser Doppler perfusion imaging (LDPI) in the feet of mice.
(A) Mice had critical limb ischemia (CLI) induced on Week 0. Two weeks post-surgery, mice were treated for 3 consecutive days (Day 14, 15, and 16) with: saline (n = 8), pFUS (n = 7), mesenchymal stem cells (MSC 106/day IV, n = 8) and pulsed focused ultrasound (pFUS) + MSC (106/day IV, n = 17) and imaged by LDPI weekly for another 5 weeks (7 weeks post-surgery). Statistical significance (p < 0.05; two-way ANOVA) of the pFUS + MSC groups is indicated by * compared to the saline group, + compared to the MSC only group, # compared to the pFUS only group. (B) Representative LDPI of saline, pFUS, MSC, and pFUS + MSC at 7 weeks post induction of CLI.
Figure 5
Figure 5. CD31 immunostaining in treatment groups 7 weeks post-surgery.
(A) Area of CD31-positive signal for each treatment group. (n = 3 slides per animal; 5 animals per groups) *Indicates significance (p < 0.05) by one-way ANAOVA. (B) Representative images of CD31 staining with CD31 appearing in brown. Scale bar = 200 μm.
Figure 6
Figure 6. Masson Trichrome staining in treatment groups 7 weeks post-surgery.
(A) Percentage area of fibrosis for each treatment group. (n = 3 slides per animal; 5 animals per groups) *Indicates significance (p < 0.05) by one-way ANOVA. (B) Representative images of Masson Trichrome stain. Scale bar = 3 mm.
Figure 7
Figure 7. Immunostaining for human vascular endothelial growth factor (VEGF) and interleukin (IL)-10 reveals greater cytokine production by mesenchymal stem cells (MSC) in homing to muscle previously treated with pulsed focused ultrasound (pFUS).
(A) Relative fluorescence intensity of IL-10 or VEGF in regions-of-interest (ROI) drawn around human MSCs (n = 10 ROI from 5 mice per group). *Indicates significance (p < 0.05) by one-way ANOVA with Bonferroni correction. (B) Representative fluorescence images showing human MSCs (red) and either VEGF or IL-10 expression (green).
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