Annexin A12–26 Treatment Improves Skin Heterologous Transplantation by Modulating Inflammation and Angiogenesis Processes

Skin graft successful depends on reduction of local inflammation evoked by the surgical lesion and efficient neovascularization to nutrition the graft. It has been shown that N-terminal portion of the Annexin A1 protein (AnxA1) with its anti-inflammatory properties induces epithelial mucosa repair and presents potential therapeutic approaches. The role of AnxA1 on wound healing has not been explored and we investigated in this study the effect of the peptide Ac2–26 (N-terminal AnxA1 peptide Ac2–26; AnxA12–26) on heterologous skin scaffolds transplantation in BALB/c mice, focusing on inflammation and angiogenesis. Treatment with AnxA12–26, once a day, from day 3–60 after scaffold implantation improved the take of the implant, induced vessels formation, enhanced gene and protein levels of the vascular growth factor-A (VEGF-A) and fibroblast influx into allograft tissue. It also decreased pro- while increasing anti-inflammatory cytokines. The pro-angiogenic activity of AnxA12–26 was corroborated by topical application of AnxA12–26 on the subcutaneous tissue of mice. Moreover, treatment of human umbilical endothelial cells (HUVECs) with AnxA12–26 improved proliferation, shortened cycle, increased migration and actin polymerization similarly to those evoked by VEGF-A. The peptide treatment instead only potentiated the tube formation induced by VEGF-A. Collectively, our data showed that AnxA12–26 treatment favors the tissue regeneration after skin grafting by avoiding exacerbated inflammation and improving the angiogenesis process.

all samples were assessed for the presence of cells and heterologous DNA content as described previously (Greco et al., 2015), and results revealed the complete removal of cells and nuclear material from the matrix (data not shown).

Heterologous transplantation
To carry out heterologous transplantation using skin scaffolds, mice were anesthetized, shaved and disinfected with povidone solution (10 % w/w Cutaneous Solution -Iodinated Povidone, Videne, Garforth, UK), followed by the removal of 1 cm 2 of skin from the dorsum, along the spine of the recipient animals (Teixeira et al., 2016). To help the "taking" of the skin scaffolds, approximately 1 cm 3 of a preparation of collagen paste (Permacol™ paste; Covidien, Leeds, UK) was applied to the wound and the scaffold was immediately placed on the paste and sutured to the native skin (6-0 nylon). Transplanted mice were subjected to daily administration of either PBS (control -TX) or AnxA1 2-26 (TX + AnxA1) (n = 5 mice/group) and sacrificed at 3, 10, 15 and 60 days after transplantation. Pharmacological treatments started 3 days before heterologous skin transplantation. The AnxA1 2-26 [(5mg/kg·per day) diluted in sterile PBS] was administrated intraperitoneally (Teixeira et al., 2016).

Quantitative reverse transcription polymerase chain reaction (qRT-PCR)
Fluorogenic qRT-PCR-based (TaqMan) assay was used to detect amplification of the target genes. Briefly, total RNA was extracted using a commercially available kit (Qiagen RNeasy Mini Kit; Qiagen, Hilden, Germany), according to the manufacturer's instructions, with the following modifications to minimize RNA degradation by abundant skin RNAses. Samples were homogenized using bead-beating technology (Precellys, Bertin Technologies, Montigny-le-Bretonneux, France). Proteins potentially interfering with RNA isolation were removed by incubating the homogenate in 590 µL distilled water and 5 µL Proteinase K solution 20 mg/mL (Life Technologies, Paisley, UK) at 55 °C for 10 min then centrifuged at room temperature for 3 min. Supernatants were combined with 0.5 volumes of ethanol (96-100 %) into a Rnase-Dnase free tube and RNA was isolated through a RNeasy mini column. The concentration and purity of the RNA was analyzed using the Nandrop ND-1000 (NanoDrop Technologies, Wilmington, DE). Complementary DNA (cDNA) was obtained by reverse transcription (RT) of 1 µg of total RNA, with the Superscript III reverse transcriptase system (Invitrogen, Carlsbad, CA, USA) following the manufacturer's protocol and using oligo(dT)15 as a primer. Real-time PCR was performed with the Eco Real-Time PCR System (Illumina, San Diego, CA, USA). The following amplification profile was used: UDG Incubation 50 o C for 2 min, AmpliTaq Gold 95 o C for 10 min, PCR 40 Cycles -95 o C for 15 sec and 60 o C for 1 min. For each reaction, a total volume of 20 µL was used, which consisted of 9 µL of diluted cDNA (10 ng/µL of RNA), 10 µL of TaqMan Gene Expression Master Mix (2×) and 1 µL of TaqMan Gene Expression Assay (20×) (Applied Biosystems, CA, USA). Commercially available primers (GAPDH, Mm99999915_g1; VEGFA, Mm00437306_m1; bFGF Mm01285715_m1; ASMA Mm00725412_s1; TGFb1 Mm01178820_m1 -Applied Biosystems, CA, USA) were used to probe for target mRNA. mRNA data were normalized relative to GAPDH and then used to calculate expression levels. The comparative Ct method was used to measure the gene transcription in samples. Results are expressed as relative units based on calculation of 2-ΔΔCt, which gives the relative amount of target gene normalized to endogenous control (GAPDH) and to the control (sham-operated) samples with the expression set as 1. Negative controls were either RT without enzyme or PCR without cDNA template.

Multiplex Assays
To quantify the inflammatory mediators IL-1β, IL-6, TNF-α, IL-17 and INF-γ, we used the multiplex instrument LUMINEX xMAP MAGPIX (Millipore Corporation, Billerica, MA, USA). The transplanted tissues were macerated in liquid nitrogen and placed in clean, 1.5 mL tubes to which 500 µL of a solution containing protease inhibitor cocktail (GE Healthcare, Amersham, UK) and Tween 20 (1 µL) (Sigma-Aldrich, Poole, Dorset, UK) was added. The samples were incubated for 1 h at 4 °C under constant agitation and then centrifuged at 21,000 g for 10 min at 4 °C. The protein concentration in the supernatant was measured using a Bradford assay (Biorad, Hemel Hempsted, UK). Antibody beads, controls, wash buffer, serum matrix and standards were prepared following the manufacturer's instructions (MILLIPLEX HCYTOMAG-60K kit). A further 200 µL of wash buffer was added to each well of a magnetic 96-well plate and mixed on a shaker for 10 min. The wash buffer was decanted and 25 µL of standards, controls and samples were added to the wells. Next, 25 µL of assay buffer was added to the samples, and 25 µL of serum matrix was added to the standards. Finally, 25 µL of magnetic beads (coated with a specific capture antibody) was added to all wells and incubated overnight at 4 °C on a shaker. The next day, the plate was washed with wash buffer and incubated with 25 µL of detection antibodies for 1 h on a shaker. Next, 25 µL of streptavidinphycoerythrin was added to each well and incubated for 30 min on a shaker. The plate was washed and incubated with 150 µL of drive fluid for 5 min on a shaker. Finally, the plate was analyzed using MAGPIX with xPONENT software. Tissues were collected from mice were subjected to daily administration of either PBS or AnxA1 2-26 (n=5 animals/group) and sacrificed at 3, 10, 15 and 60 days after transplantation procedure. Pharmacological treatments started 3 days before heterologous skin transplantation. The AnxA1 2-26 (100 µg/day diluted in sterile PBS) was administrated intraperitoneally.

Cell proliferation assay
To investigate the effect of AnxA1 2-26 peptide on cell growth, HUVECs were plated (1 × 10 4 cells/well) and, after cell adhesion, were incubated with PBS (control), AnxA1 2-26 peptide (30 µM) and/or VEGF-A (10 ng/mL) for 24, 48 or 72 h. Later, the number of cells was quantified with Countess ® Automated Cell Counter (Invitrogen, UK). To evaluate the analysis of cell proliferation, the counted cells were compared with a growth curve of cells grown without any treatment. The results are expressed as number of cells × 10 4 .

Cell migration assay
Semiconfluent HUVECs were disrupted with a pipette tip, creating a "groove" in the centre of the well (Bürk, 1973). Afterwards, cells were gently washed and incubated with PBS (control), AnxA1 2-26 peptide (1, 10 or 30 µM) and/or VEGF-A (50 ng/mL) for 12 h. Cell migration was monitored with images obtained before and after treatments, using an Olympus DP73 camera (Olympus, Tokyo, Japan) coupled to a microscope (Olympus, Tokyo, Japan, magnification 50×). The number of cell nuclei that crossed the grove line was determined in three different microscopic fields.

Tube formation assay on Matrigel ®
The tube formation assay was performed on a Matrigel ® layer as previously described by Drewes et al. 2015(Drewes et al., 2015. Briefly, 200 µL of Matrigel ® were added to each well and incubated at 37 ºC for 1 h to form a gel layer. Subsequently, HUVECs (2 × 10 4 cells/well) were incubated with PBS (control), AnxA1 2-26 peptide (1, 10 or 30 µM) and/or VEGF-A (50 ng/mL) for 2 h and cells were plated under the Matrigel ® layer to form capillary-like structures for 6 h. Afterwards, the capillary-like structures formed in the gel were photographed (Axioskop II, Carl Zeiss, Germany) and the number of tubules was quantified using Image J software.

F-actin staining by confocal microscopy assay
HUVECs (1×10 4 cells/well) were plated on a glass-bottom culture dish and once adhered, they were treated with PBS (control), AnxA1 2-26 peptide (30 µM) and/or VEGF-A (50 ng/mL) for 2 h. Immediately after the treatment protocol, cells were stained using an F-actin kit (Cytoskeleton, Inc, Denver, USA). Briefly, cells were fixed with fixative buffer and permeated with a permeability buffer. F-actin of cells present on glass bottom culture dishes were incubated at room temperature with rhodamine-phalloidin (100 nM) for 30 min. Cells were washed three times with a wash buffer and immediately visualized by confocal microscopy (Carl Zeiss LSM 780-NLO, Germany). Representative photographs were obtained in three different fields using a magnification of 63×.

Ultrastructural immunocytochemical analysis
To detect the co-localisation of endogenous ANXA1 with the formyl peptide receptor 1 (FPR1), ultrathin sections (~70 nm) of LR Gold embedded-HUVECs were incubated sequentially with the following reagents at room temperature: i) 0.1 mol/L phosphate buffer containing 1 % egg albumin (PBEA); ii) 0.1 mol/L PBS containing 5 % egg albumin (PBEA) for 30 minutes; iii) the sheep polyclonal antibody anti-ANXA1 (1:200 in PBEA) and rabbit polyclonal antibody anti-FPR2 (1:200 in PBEA) (Santa Cruz Biotechnology) for 2 hours, with normal sheep and rabbit sera as controls; and iv) three washes (5 minutes each) in PBEA containing 0.01 % Tween 20. To detect ANXA1 and FPR2, a donkey anti-sheep and goat anti-rabbit IgG antibody (1:50 in PBEA) conjugated to 20 and 10 nm colloidal gold (British Biocell, Cardiff, UK), respectively, were added. After 1 hour, the sections were washed in PBEA, stained with uranyl acetate and lead citrate and examined using a ZEISS EM900 electron microscope. The area of the cell compartment was determined with Axiovision software. The density of ANXA1 expression was showed as the mean ± SEM of immunogold particles per µm 2 from distinct cells analyzed (n=12-20/group). Figure 2. VEGF-A induced angiogenesis is not altered in knockout AnxA1 mice (AnxA1-/-). Images obtained showed equivalent number of vessels in AnxA1 -/and WT mice, in both basal and VEGF-A-stimulated animals. Mice were topically treated with PBS (10 µL) or VEGF-A in the dorsal skin. The treatments were administrated once per day, every 2 days, resulting three applications in each mouse. Representative images of the microcirculatory network of dorsal skin were obtained before (day 4) and after (day 9) treatments (A). The images in the upper panel represent the stained normal tissue and in the lower panel, the same computational images obtained after inverting the colours are displayed (A). The quantification of vessels is represented in B. The values express the mean ± S.E.M. of five animals per group (ANOVA). * p<0.05, ** p<0.01 vs PBS.