Macrophage contribution to the survival of transferred expanded skin flap through angiogenesis

Abstract

Background: Despite the application of tissue expansion in the reconstruction of significant tissue defects, complications with expanded random-pattern skin flaps remain a major challenge. Insufficient angiogenesis is one of the keys factors in flap ischemia and dysfunction. Macrophages play a key role in promoting tissue angiogenesis, but their effects on expanded flap angiogenesis and the survival of the transferred skin flap are still unknown.

Methods: A rat scalp expansion model was established to evaluate the dynamic changes of macrophages in expanded skin. Clodronate liposomes (Clo-lipo) were injected into the expanded scalps to deplete the macrophages, and the expanded scalp flaps with macrophage depletion were orthotopically transferred. The remaining expanded rat scalp flaps were treated with either a macrophage-colony stimulating factor (M-CSF) alone or M-CSF in combination with Clo-lipo and transferred. The number of macrophages, blood perfusion, microvascular densities (MVDs), flap survival, histological changes, and gene expression related to macrophage polarization and angiogenesis were determined with immunofluorescence (IF) staining, full-field laser perfusion imager, hematoxylin and eosin (HE) staining, and quantitative real-time polymerase chain reaction.

Results: The number of pan-macrophages significantly increased in the expanded scalp on days 14 and 21 after expander placement. The depletion rate after treatment with Clo-lipo was 29.06%, and the number of macrophages was significantly reduced in the group that underwent Clo-lipo treatment on day 14 before flap transfer (P<0.05). Macrophage depletion resulted in decreased blood perfusion, reduced MVDs, lower expression of factors, and poor survival rate. The recruitment of macrophages with a M-CSF led to higher blood perfusion, increased MVDs, greater expression of angiogenic factors, and better flap survival after flap transfer.

Conclusions: Alternatively activated macrophages in the expanded flap could significantly promote angiogenesis, improve blood perfusion, and ultimately increase the flap survival rate. Modulating alternatively activated macrophages may provide a key therapeutic strategy to promote expanded skin flap survival. Our study has provided a basis for clinically improving random-pattern skin flap survival.

Keywords: Tissue expansion; angiogenesis; macrophages; random-pattern flap; survival.

Figure 1

The number of macrophages in the scalps remained high during scalp expansion. (A) Injection schedule. Dates of saline injections for expanded scalp () are displayed at the top of the schedule. (B) Representative graphs of the scalp expansion model. (C) The hematoxylin and eosin staining images of rat scalp sections on days 7, 14, and 21. (D) Data from one experiment with 6 rats per time point. The thickness of the expanded scalp was thinner than the sham expanded scalp on days 14 and 21 (6 rats per time point in each group). (E) Skin sections stained with CD68⁺ (red) were used to visualize macrophages in the expanded and sham expanded scalps. (F) The total number of macrophages in the scalp was quantified. The expanded scalp had higher total macrophage levels than did the sham expanded scalp. *, P<0.05; **, P<0.01; ***, P<0.001. DAPI, 4',6-diamidino-2-phenylindole.

Figure 2

Macrophage depletion inhibited transferred scalp flap survival. (A) Model construction: macrophage depletion following clodronate-liposome injection during expansion and gross appearance after flap transfer. Dates of saline injections for expanding tissue and biopsy () and scalp flap transfer in situ () are displayed in the schedule. The bottom showed Clo-lipo or PBS-liposome control (PBS-lipo groups) (50 µg/mL, ) and time points. (B,C) Representative images of CD68⁺ macrophages (IF staining) and quantitative analysis of CD68⁺ cells. (D,E) Survived area of transferred flaps and quantitative analysis of survival rate. (F,G) Representative hematoxylin and eosin staining images of transferred expanded scalp flap and thickness analysis of the epidermis and dermis. *, P<0.05. IF staining, immunofluorescence staining; DAPI, 4',6-diamidino-2-phenylindole; PBS, phosphate-buffered saline; Clo-lipo, clodronate-liposome.

Figure 3

The changes of macrophage subtypes in the expanded scalp flap 7 days after flap transfer (day 21) by IF staining. (A) The pan-macrophage marker CD68⁺ (red) cells in the transferred flap. (B) M2 marker CD206⁺ (green) CD68⁺ (red) cells (M2 macrophages). (C) iNOS⁺ (green) CD68⁺ (red) cells (M1 macrophages). Insets show high-magnification (×40) images of the M2 or M1 macrophages. (D) The total number of macrophages (CD68⁺), M2 macrophages (CD206⁺/CD68⁺), and M1 macrophages (iNOS⁺/CD68⁺) per field. The expression of pan-macrophage marker CD68 (E) and M2 macrophage marker CSF-1R (F) dramatically decreased in the Clo-lipo group. (G) M1 macrophage marker iNOS showed no significant difference between the Clo-lipo (n=6) and PBS-lipo (n=5) groups. *, P<0.05; **, P<0.01. IF staining, immunofluorescence staining; PBS, phosphate-buffered saline; DAPI, 4',6-diamidino-2-phenylindole; Clo-lipo, clodronate-liposome; iNOS, inducible nitric oxide synthase; CSF-1R, colony stimulating factor 1 receptor.

Figure 4

Macrophage depletion attenuated blood perfusion, MVDs, and the expression of angiogenic factors. (A) Representative photographs of flap blood perfusion. Red boxes indicate the transferred expanded flaps. (B) Compared with the PBS-lipo group (n=6), significantly lower blood perfusion was detected in the Clo-lipo group (n=5). (C) Representative photographs of CD34⁺ microvessels (red) in the transferred expanded scalp flap (IF staining). The white arrows indicate CD34⁺ microvessels. (D) Quantitative analysis of CD34⁺ MVDs. The number of CD34⁺ MVDs in the PBS-lipo group was higher than that in the Clo-lipo group per field. (E) Angiogenic factors, PDGFR and FGF, showed decreased expression in the Clo-lipo group versus the PBS-lipo group, while the angiostatic factor, CXCL10, showed increased expression in the Clo-lipo group versus the PBS-lipo group. No significant difference was found in VEGFα between the 2 groups. *, P<0.05; ***, P<0.001. IF staining, immunofluorescence staining; PBS, phosphate-buffered saline; Clo-lipo, clodronate-liposome; DAPI, 4',6-diamidino-2-phenylindole; MVD, microvascular density; PDGFR, platelet-derived growth factor receptor; FGF, fibroblast growth factor; VEGFα, vascular endothelial growth factor α; CXCL10, C-X-C motif chemokine ligand 10.

Figure 5

The survival and thickness of the expanded scalp flap after M-CSF treatment or combined M-CSF and Clo-lipo treatment. (A) Study design: dates of saline injections for expanding tissue (), scalp flap transfer in situ () and biopsy () are displayed at the top of the schedule. Dates of Clo-lipo/PBS-lipo injections (50 µg/mL, ) and M-CSF/PBS injections (20 ng/µL, ) are displayed at the bottom of schedule. (B,C) Survived area of transferred flaps and quantitative analysis of survival rate. Increased average flap survival rates were observed in the M-CSF group on day 21 (1-way ANOVA, P<0.001), whereas this effect of flap survival rate (1-way ANOVA, P<0.05) was reversed by M-CSF + Clo. (D,E) Representative HE staining images of transferred expanded scalp flap and thickness analysis of the epidermis and dermis. A thicker epidermis (1-way ANOVA, P<0.001) and dermis (1-way ANOVA, P<0.05) was observed in the M-CSF group, whereas this effect of epidermis (1-way ANOVA, P<0.001) and dermis (1-way ANOVA, P<0.01) was reversed by M-CSF + Clo (E). No significant difference in thickness was observed between PBS and M-CSF + Clo groups. *, P<0.05; **, P<0.01; ***, P<0.001. PBS, phosphate-buffered saline; M-CSF, macrophage-colony stimulating factor; Clo-lipo, clodronate-liposome; HE staining, hematoxylin eosin staining; ANOVA, analysis of variance.

Figure 6

The number of macrophages of different subtypes in the survived flaps after transfer with PBS, M-CSF, and M-CSF + Clo-lipo treatment. (A-C) Representative IF staining images of pan-macrophages, and M2 and M1 macrophages. (A) CD68⁺ cells (red). (B) iNOS⁺ (green) CD68⁺ (red) cells (M1 macrophages). (C) Insets show high-magnification (×40) images of the M1 or M2 macrophage cells. (D) Quantitative analysis of macrophages. M-CSF group rats showed significantly higher numbers of M1, M2, and total macrophages than did the PBS group rats (1-way ANOVA, total and M2: P<0.001; M1: P<0.01). Furthermore, M-CSF + Clo group rats had significantly fewer numbers of M1, M2, and total macrophages than did the M-CSF group rats (1-way ANOVA, total and M1: P<0.001; M2: P<0.01). Total macrophages (CD68⁺), M2 macrophages (CD206⁺CD68⁺), and M1 macrophages (iNOS⁺CD68⁺). PBS group (n=6), M-CSF group (n=5) and M-CSF + Clo group (n=5). **, P<0.01; ***, P<0.001. PBS, phosphate-buffered saline; M-CSF, macrophage-colony stimulating factor; Clo-lipo, clodronate-liposome; ANOVA, analysis of variance; iNOS, inducible nitric oxide synthase.

Figure 7

The effects of M-CSF treatment on the angiogenesis of scalp flap and blood perfusion, MVDs, and the expression of angiogenic factors after flap transfer. (A) Representative photographs of flap blood perfusion. Red boxes indicate the transferred expanded flaps. (B) Compared with the PBS group, significantly higher blood perfusion was observed in the M-CSF group, whereas the M-CSF + Clo group showed lower blood perfusion than did the M-CSF group. (C) Representative IF staining photographs of CD34⁺ microvessels (red) in the transferred flaps. The white arrows indicate CD34⁺ microvessels. (D) Quantitative analysis of CD34⁺ MVDs. The total number of CD34⁺ MVDs in the M-CSF group was higher than that in the PBS group, whereas M-CSF + Clo group showed a marked decrease in CD34⁺ MVDs compared with M-CSF group (P<0.05). (E) Increased expression of PDGFR (P<0.05), FGF (P<0.01), and VEGFα (P<0.001) was detected in the M-CSF group versus the control group. The M-CSF + Clo group showed lower expression of PDGFR than did the M-CSF group (P<0.01). Decreased expression of CXCL10 was found in the M-CSF group versus the PBS group (P<0.01), whereas CXCL10 in the M-CSF + Clo group showed a higher expression than did the M-CSF group (P<0.05). PBS group, n=6; M-CSF group, n=5; M-CSF + Clo group, n=5. *, P<0.05; **, P<0.01; ***, P<0.001. IF staining, immunofluorescence staining; PBS, phosphate-buffered saline; M-CSF, macrophage-colony stimulating factor; Clo, clodronate; PDGFR, platelet-derived growth factor receptor; FGF, fibroblast growth factor; VEGFα, vascular endothelial growth factor α; CXCL10, C-X-C motif chemokine ligand 10; MVD, microvascular density; ANOVA, analysis of variance.