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. 2019 Apr 13;9(9):2678-2693.
doi: 10.7150/thno.31884. eCollection 2019.

Synechococcus elongatus PCC7942 secretes extracellular vesicles to accelerate cutaneous wound healing by promoting angiogenesis

Hao Yin  1   2 , Chun-Yuan Chen  1   2 , Yi-Wei Liu  2   3 , Yi-Juan Tan  2 , Zhi-Li Deng  4   5 , Fei Yang  6 , Fei-Yu Huang  6 , Cong Wen  6 , Shan-Shan Rao  2   7 , Ming-Jie Luo  2   7 , Xiong-Ke Hu  2 , Zheng-Zhao Liu  2   3   4   8 , Zhen-Xing Wang  2 , Jia Cao  2 , Hao-Ming Liu  2 , Jiang-Hua Liu  1   2 , Tao Yue  1   2 , Si-Yuan Tang  7 , Hui Xie  1   2   3   4   9   8
Affiliations

Synechococcus elongatus PCC7942 secretes extracellular vesicles to accelerate cutaneous wound healing by promoting angiogenesis

Hao Yin et al. Theranostics. .

Abstract

Poor wound healing affects millions of people worldwide each year and needs better therapeutic strategies. Synechococcus elongatus PCC 7942 is a naturally occurring photoautotrophic cyanobacterium that can be easily obtained and large-scale expanded. Here, we investigated the therapeutic efficacy of this cyanobacterium in a mouse model of acute burn injury and whether the secretion of extracellular vesicles (EVs), important mediators of cell paracrine activity, is a key mechanism of the cyanobacterium-induced regulation of wound healing. Methods: The effects of Synechococcus elongatus PCC 7942 on burn wound healing in mice under light or dark conditions were evaluated by measuring wound closure rates, histological and immunofluorescence analyses. A series of assays in vivo and in vitro were conducted to assess the impact of the cyanobacterium on angiogenesis. GW4869 was used to interfere with the secretion of EVs by the cyanobacterium and the abilities of the GW4869-pretreated and untreated Synechococcus elongatus PCC 7942 to regulate endothelial angiogenesis were compared. The direct effects of the cyanobacterium-derived EVs (S. elongatus-EVs) on angiogenesis, wound healing and expressions of a class of pro-inflammatory factors that have regulatory roles in wound healing were also examined. Results: Synechococcus elongatus PCC 7942 treatment under light and dark conditions both significantly promoted angiogenesis and burn wound repair in mice. In vitro, the cyanobacterium enhanced angiogenic activities of endothelial cells, but the effects were markedly blocked by GW4869 pretreatment. S. elongatus-EVs were capable of augmenting endothelial angiogenesis in vitro, and stimulating new blood vessel formation and burn wound healing in mice. The expression of interleukin 6 (IL-6), which has an essential role in angiogenesis during skin wound repair, was induced in wound tissues and wound healing-related cells by S. elongatus-EVs and Synechococcus elongatus PCC 7942. Conclusion: Synechococcus elongatus PCC 7942 has the potential as a promising strategy for therapeutic angiogenesis and wound healing primarily by the delivery of functional EVs, not by its photosynthetic activity. The promotion of IL-6 expression may be a mechanism of the cyanobacterium and its EVs-induced pro-angiogenic and -wound healing effects.

Keywords: Synechococcus elongatus PCC 7942; angiogenesis; extracellular vesicles; wound healing.

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Conflict of interest statement

Competing Interests: The authors have declared that no competing interest exists.

Figures

Figure 1
Figure 1
S. elongatus PCC 7942 accelerates the healing of cutaneous burn wounds. (A) Representative images of wounds treated with PBS, S. elongatus PCC 7942 in the light (S. elongatus-light) and S. elongatus PCC 7942 in the dark (S. elongatus-dark) at days 3, 7 and 12 post-wounding. Scale bar: 2 mm. (B) The rate of wound closure at the indicated times. n = 10 per group. (C) Representative H&E staining images of wound sections at day 12 post-wounding. The black double-headed arrows indicate the edges of scars. Ep: epithelium. Scale bar: 500 μm. (D) Quantification of the rate of re-epithelialization and scar widths. n = 3 per group. (E) Representative images of Masson's trichrome stained-wound sections at day 12 post-wounding. Scale bar: 100 μm (top) or 50 μm (bottom). (F) Quantification of the mean intensity of Masson- stained areas. n = 3 per group. (G) Representative images of ki67-stained wound sections at day 12 post-wounding. Scale bar: 50 μm. (H) Quantification of the number of ki67-positive cells. n = 3 per group. Data are plotted as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 2
Figure 2
S. elongatus PCC 7942 enhances angiogenesis in vivo and in vitro. (A) Gross view of wounds treated with PBS, S. elongatus-light and S. elongatus-dark at day 12 post-wounding from the undersurface. Scale bar: 2 mm. (B) Representative images of vascular marker CD31-stained wound sections at day 12 post-wounding. Scale bar: 50 μm. (C) Quantification of the number of CD31-positive vessels. n = 3 per group. (D) CCK-8 analysis of proliferation of human microvascular endothelial cells (HMECs). n = 4 per group. (E-F) Representative images of transwell migration assay in HMECs (E) and quantification of the migrated cells (F). Scale bar: 100 μm. n = 3 per group. (G-H) Representative images of wound healing assay in HMECs (G) and quantification of the migration rates (H). Scale bar: 200 μm. n = 3 per group. (I) Representative images of HMECs' tube formation. Scale bar: 200 μm. (J) Quantification of the total tube length, total branching points and total loops. n = 3 per group. Data are plotted as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 3
Figure 3
EVs secretion is required for the pro-angiogenic effects of S. elongatus PCC 7942. (A) Total protein contents of S. elongatus-EVs. n = 3 per group. (B) CCK-8 analysis of HMECs' proliferation. n = 4 per group. S: S. elongatus PCC 7942. (C-D) Representative images of transwell migration assay in HMECs (C) and quantitative analysis of the migrated cells (D). Scale bar: 100 μm. n = 3 per group. (E-F) Representative images (E) and quantification (F) of wound healing assay for HMECs. Scale bar: 200 μm. n = 3 per group. (G-H) Representative images (G) and quantification (H) of HMECs' tube formation. Scale bar: 200 μm. n = 3 per group. Data are plotted as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 4
Figure 4
S. elongatus-EVs augment the angiogenic activities of endothelial cells. (A) Morphology of S. elongatus-EVs under transmission electron microscopy. Scale bar: 50 nm. (B) Size distribution analysis of S. elongatus-EVs by dynamic light scattering. (C) Fluorescence microscopy analysis of the internalization of PKH67-labeled S. elongatus-EVs by HMECs. Scale bar: 50 μm. (D) CCK-8 analysis of HMECs' proliferation. n = 4 per group. (E-F) Representative images of transwell migration assay in HMECs (E) and quantitative analysis of the migrated cells (F). Scale bar: 100 μm. n = 3 per group. (G-H) Representative images (G) and quantification (H) of wound healing assay for HMECs. Scale bar: 200 μm. n = 3 per group. (I-J) Representative images (I) and quantification of HMECs' tube formation (J). Scale bar: 200 μm. n = 3 per group. Data are plotted as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 5
Figure 5
S. elongatus-EVs accelerate the healing of cutaneous burn wounds. (A-B) Representative images (A) and closure rate (B) of wounds treated with PBS, S. elongatus and S. elongatus-EVs at days 3, 7 and 12 post-wounding. Scale bar: 2 mm. n = 10 per group. (C) Representative images of H&E-stained wound sections at day 12 post-wounding. Scale bar: 500 μm. (D) Quantification of the rate of re-epithelialization and scar widths. n = 3 per group. (E) Representative images of Masson's trichrome stained-wound sections at day 12 post-wounding. Scale bar: 100 μm (top) or 50 μm (bottom). (F) Quantification of the mean intensity of Masson- stained areas. n = 3 per group. (G-H) Representative images (G) and quantification (H) of skin cell proliferation by ki67 immunofluorescence staining. Scale bar: 50 μm. n = 3 per group. (I) Representative images of immunohistochemistry staining for α-SMA. Scale bar: 100 μm. (J) Quantitative analysis of the mean intensity for the α-SMA positively stained areas. n = 3 per group. Data are plotted as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
Figure 6
Figure 6
S. elongatus-EVs enhance angiogenesis in the wound sites and IL-6 expression in vivo and in vitro. (A) Gross view of wounds treated with vehicle (PBS), S. elongatus and S. elongatus-EVs at day 12 post-wounding from the undersurface. Scale bar: 2 mm. (B-C) Representative images (B) and quantification (C) of CD31-stained blood vessels in the wound sites. Scale bar: 50 μm. n = 3 per group. (D) ELISA of the concentrations of IL-1α, IL-1β, IL-6 and TNF-α in skin tissue homogenate from mice treated with vehicle, S. elongatus and S. elongatus-EVs at day 7 post-wounding. n = 3 per group. (E) qRT-PCR analysis of the expression levels of IL-6 in mouse epidermal JB6 cells, NIH3T3 fibroblasts and bEnd.3 endothelial cells receiving different treatments for 24 h. n = 3 per group. Data are plotted as mean ± SD. *P < 0.05, **P < 0.01, ***P < 0.001.
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