Low Intensity Shockwave Treatment Modulates Macrophage Functions Beneficial to Healing Chronic Wounds

Jason S Holsapple, Ben Cooper, Susan H Berry, Aleksandra Staniszewska, Bruce M Dickson, Julie A Taylor, Paul Bachoo, Heather M Wilson, Jason S Holsapple, Ben Cooper, Susan H Berry, Aleksandra Staniszewska, Bruce M Dickson, Julie A Taylor, Paul Bachoo, Heather M Wilson

Abstract

Extracorporeal Shock Wave Therapy (ESWT) is used clinically in various disorders including chronic wounds for its pro-angiogenic, proliferative, and anti-inflammatory effects. However, the underlying cellular and molecular mechanisms driving therapeutic effects are not well characterized. Macrophages play a key role in all aspects of healing and their dysfunction results in failure to resolve chronic wounds. We investigated the role of ESWT on macrophage activity in chronic wound punch biopsies from patients with non-healing venous ulcers prior to, and two weeks post-ESWT, and in macrophage cultures treated with clinical shockwave intensities (150-500 impulses, 5 Hz, 0.1 mJ/mm2). Using wound area measurements and histological/immunohistochemical analysis of wound biopsies, we show ESWT enhanced healing of chronic ulcers associated with improved wound angiogenesis (CD31 staining), significantly decreased CD68-positive macrophages per biopsy area and generally increased macrophage activation. Shockwave treatment of macrophages in culture significantly boosted uptake of apoptotic cells, healing-associated cytokine and growth factor gene expressions and modulated macrophage morphology suggestive of macrophage activation, all of which contribute to wound resolution. Macrophage ERK activity was enhanced, suggesting one mechanotransduction pathway driving events. Collectively, these in vitro and in vivo findings reveal shockwaves as important regulators of macrophage functions linked with wound healing. This immunomodulation represents an underappreciated role of clinically applied shockwaves, which could be exploited for other macrophage-mediated disorders.

Keywords: chronic wounds; cytokine; healing; inflammation; macrophage; phagocytosis; shockwave therapy.

Conflict of interest statement

The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest.

Figures

Figure 1
Figure 1
Extracorporeal shock wave therapy (ESWT) improves healing of chronic ulcers. (A) ESWT improved healing in seven out of the nine patients as determined by a decrease in total wound area (cm2) from baseline before ESWT and at 2 weeks post-therapy. (B) Seven out of nine patients showed a decrease in the percentage area of their wound from baseline to 2 weeks post-ESWT, one patient showed no change and one patient a decrease in reduction. Numbers shown on the x-axis (1–9) represent individual patients.
Figure 2
Figure 2
Extracorporeal shock wave therapy (ESWT) improves angiogenic markers in patient wound biopsies. (A) Collagen fiber deposition was quantified and presented as mean blue intensity of Masson‘s Trichrome stain in patient biopsies before and two weeks post-ESWT treatment. (B) Quantification of the CD31 stained area and (C) smooth muscle cell actin (SMC) stained area was determined as a marker of angiogenesis and (D) Ki67 staining per total wound biopsy area represented cell proliferation in individual patients pre- and post-treatment. Graphs represent values from individual patients; n = 9; * represents differences between the mean values before and after ESWT treatment, p < 0.05.
Figure 3
Figure 3
Extracorporeal shock wave therapy (ESWT) alters the number and activation of macrophages in wound biopsies. The number (A) and activation status of macrophages (B–D) was determined by immunohistochemistry in patient biopsies before and after ESWT. Macrophage activation markers were defined as (B) HLA DR and (C) SOCS3 for M1 activated macrophages and (D) CD163 for M2 activated macrophages. * represents differences between values before and after ESWT treatment, p < 0.05.
Figure 4
Figure 4
Shockwave stimulation up-regulates macrophage phagocytosis of apoptotic cells. Macrophages were unstimulated (control) or exposed to shockwave intensities of 150–500; 5 Hz, 0.1 mJ/mm2 then cultured with apoptotic cells for typically 2 h. (A) Percentage uptake and (B) relative percentage uptake of apoptotic cells by J774 macrophages. (C) Percentage uptake and (D) relative percentage uptake of apoptotic cells by human monocyte-derived macrophages. Data plotted represent individual values as shown by each symbol with mean value presented (A,C) and the normalized data with means ± SEM determining the relative percentage of shock wave exposed cells to control cells for each cell preparation (B,D). Data from experiments from 5–7 individual macrophage preparations. * represents p < 0.05; ** represents < 0.001 compared to unstimulated, control macrophages.
Figure 5
Figure 5
Shockwave stimulation does not up-regulate macrophage phagocytosis of the non-specific substrate, polystyrene beads. Macrophages were exposed to shockwave intensities of 150–500; 5 Hz, 0.1 mJ/mm2 then cultured with polystyrene beads. (A) Percentage uptake and (B) relative percentage uptake over control macrophages without shockwave exposure of polystyrene beads by J774 macrophages. (C) Phagocytic index and (D) relative percent phagocytic index over control J774 macrophages without shockwave exposure for uptake of polystyrene beads. Data plotted represent individual values as illustrated by the individual symbols (A,C) and the normalized data determining the relative percentage of shockwave exposed cells to non-exposed control cells for each cell preparation (B,D). (E) Percentage of viable J774 macrophages where symbols represent individual values from different experiments and (F) the normalized values where non-exposed control represents 100%. Data shown as means ± SEM of independent experiments from 4–7 individual macrophage preparations.
Figure 6
Figure 6
Shockwave stimulation selectively enhances macrophage mRNA expression. J774 macrophages were exposed to shockwave intensities of 150–500; 5 Hz, 0.1 mJ/mm2 and after 4 h, macrophage gene expression levels of (A) TNF, (B) IL-1, (C) PDGF, (D) TGFβ, (E) IL-6 and (F) VEGF, were analyzed by qPCR. Results were normalized on the housekeeping gene and expressed as fold enrichment compared to untreated control macrophages (control). Results are shown as mean ± SEM of 3–5 independent experiments, * p < 0.05.
Figure 7
Figure 7
Shockwave treatment alters macrophage morphology and activates ERK signaling. J774 macrophages were exposed to shockwave intensities of 150–500; 5 Hz, 0.1 mJ/mm2 or left unstimulated (control). (A) Cell area and (B) changes in the percentage of J774 macrophages with rounded morphology and (C) elongated morphology as determined by aspect ratios; mean values ± SEM; n = 4 independent experiments; average 100 cells analyzed per preparation. (D) Shockwave stimulation induces ERK phosphorylation. (E) Representative Western blot for pERK and total ERK 30 min post-shockwave stimulation. Quiescent J774 cells were stimulated with shockwaves for 30 min. Total cell lysates were subjected to SDS-PAGE. Phospho-ERK mAbs were used to detect the active form of the kinase. The same blots were re-probed with antibodies against total ERK as loading controls. Quantification of protein phosphorylation levels was performed by scanning densitometry and presented as percentage increase above control non-exposed levels (mean ± SEM, n = 3).

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Source: PubMed

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