The Antimicrobial Peptide Esculentin-1a(1–21)NH₂ Stimulates Wound Healing by Promoting Angiogenesis through the PI3K/AKT Pathway

Abstract

Delayed wound healing is a persistent medical problem mainly caused by decreased angiogenesis. Esculentin-1a(1–21)NH₂ [Esc-1a(1–21)NH₂], has broad-spectrum antibacterial properties which comes from frog skins. It has shown promise as a treatment for wound healing. However, its effects on angiogenesis as well as the mechanism by which esc-1a(1–21)NH₂ enhanced wound healing remained unclear. In this study, we analyzed the structural properties and biocompatibility of esc-1a(1–21)NH₂ and evaluated its effect on wound closure using a full-thickness excision model in mice. Our results showed that esc-1a(1–21)NH₂ significantly accelerated wound healing by increasing collagen deposition and angiogenesis, characterized by elevated expression levels of platelet, endothelial cell adhesion molecule-1 (CD31) and proliferating cell nuclear antigen (PCNA). Furthermore, the angiogenic activity of esc-1a(1–21)NH₂ was confirmed in vitro by various assays. Esc-1a(1–21)NH₂ significantly promoted cell migration and cell proliferation in human umbilical vein vascular endothelial cells (HUVECs) via activation of the phosphatidylinositol 3′-kinase (PI3K)/protein kinase B (AKT) pathway, and upregulated the expression of CD31 at both mRNA and protein levels. The effect of esc-1a(1–21)NH₂ on angiogenesis was diminished by LY294002, a PI3K pathway inhibitor. Taken together, this study demonstrates that esc-1a(1–21)NH₂ accelerates wound closure in mice by promoting angiogenesis via the PI3K/AKT signaling pathway, suggesting its effective application in the treatment of wound healing.

INTRODUCTION

Delayed wound healing has been a major medical problem and is causing the rising cost of healthcare.¹⁾ Wound healing is a finely regulated and coordinated process. In general, it involves four stages, namely hemostasis, inflammation, proliferation, and shaping.²⁾ Among them, impaired angiogenesis is the most important underlying reasons for delayed wound healing.³⁾ It is well known that, the normal healing process is characterized by well-formed granulation, which has a pink granular appearance and is filled with new microvessels.^4,5) However, reduced angiogenesis results in insufficient nutrient and oxygen delivery to the wound bed and poor granulation formation. In addition, early development of a functional neovascular system is associated with enhanced maintenance of the extracellular matrix, which prevents scarring and subsequent wound site contraction.⁶⁾ Recently, accumulating evidence has shown that dysfunctional angiogenesis leads to healing failure.⁷⁾ Therefore, new treatments that enhancing angiogenesis should be explored to accelerate wound healing.

Antimicrobial peptides (AMPs) are one of the important components of the innate immune system in various species. They tend to be a class of amphiphilic peptides of no more than 50 amino acids. At physiological pH, most AMPs have a net positive charge between +2 and +9. The AMPs possess broad-spectrum antimicrobial activity, and many of them can promote wound healing.^8,9) LL-37, the only member of the cathelicidin family of human antimicrobial peptides, has been reported to play an integral role in skin immune defense and healing.¹⁰⁾ It has not only chemotactic effects, such as attracting neutrophils, monocytes, mast cells, and T cells, but also regulates the apoptosis of different skin cells. In addition, it activates the transactivation of epidermal growth factor receptor (EGFR) and thereby promotes the migration of keratinocytes. Furthermore, LL-37 promotes angiogenesis by activating formyl peptide receptor-like 1 (FPRL1).¹¹⁾ Esculentin-1a(1–21)NH₂ [Esc-1a(1–21)NH₂], is a truncated N-terminal derived peptide of Esculentin-1a, which is an AMP in frog (Rana esculenta) skin. It consists of 21 amino acids, with the sequence GIFSKLAGKKIKNLISGLKG-NH₂.¹²⁾ Notably, esc-1a(1–21)NH₂ has been shown to possess compelling antimicrobial activity against Pseudomonas aeruginosa, the most common serious infectious bacterium isolated from chronic wound infections.^13,14) Recently, esc-1a(1–21)NH₂ was discovered to considerably accelerate the migration of human keratinocytes (HaCaT) by activating the epidermal growth factor receptor, and the pro-migration activity of esc-1a(1–21)NH₂ was stronger than that of LL-37.¹⁵⁾ As a result, esc-1a(1–21)NH₂ has emerged as a promising candidate as a healing promoter. However, it is unknown whether esc-1a(1–21)NH₂ has the same pro-angiogenic activity as LL-37 in promoting wound healing, and the underlying mechanisms remain unclear.

In this article, we aimed to evaluate the role of esc-1a(1–21)NH₂ in promoting wound healing in a mouse model of full-thickness resection. Next, we investigated the angiogenic activity of esc-1a(1–21)NH₂ through a series of in vitro and in vivo assays and explored the possible mechanism by which esc-1a(1–21)NH₂ stimulated the angiogenic processes.

MATERIALS AND METHODS

Bioinformatics Analysis

The chemical and physical properties of esc-1a(1–21)NH₂ were displayed using the ProtParam online program (https://web.expasy.org/protparam/). The PyMOL free software was used to draw a mock structure diagram of esc-1a(1–21)NH₂. The spiral wheel diagram of esc-1a(1–21)NH₂ was plotted using the online site (https://heliquest.ipmc.cnrs.fr/). The hydrophilic and hydrophobic properties of esc-1a(1–21)NH₂ were showed using the ProtScale online program (https://web.expasy.org/protscale/). The NPS@BioTools online tool (https://npsa-prabi.ibcp.fr/cgi-bin/npsa automat.pl?page=npsa sopma.html) was made to estimate the secondary structure and folding type of esc-1a(1–21)NH₂.

Biocompatibility Assays

The cytotoxicity of esc-1a(1–21)NH₂ was assessed using human umbilical vein endothelial cells (HUVECs) (ATCC, U.S.A.). The prepared HUVECs (1 × 10⁴ cells/mL) cell suspension was added to the 96-well plate. After the cells are adhered overnight, the supernatant is removed and the cells are treated with 100 µL of drug per well for 24 h. The cell viability was ascertained with the Cell Counting Kit-8 (CCK8) assay as the instructions of the manufacturer described.¹⁶⁾ The absorbance at 450 nm was measured with a microplate reader. Three independent experiments were conducted.

The hemolysis rate of esc-1a(1–21)NH₂ was determined using the previously described method.¹⁷⁾ The samples of venous blood were collected from the male Sprague-Dawley (SD) rat (7-week-old, 200–250 g). The 3% erythrocyte suspension was diluted in physiological saline (0.9% NaCl, pH = 7.4). Next, 150 µL of 3% erythrocyte suspension and 150 µL of esc-1a(1–21)NH₂ (0.1–500 µg/mL) were blended and incubated at 37 °C for 30 min. As negative and positive controls, physiological saline and 1% TritonX-100 were mixed with equal volumes of erythrocyte suspensions. Subsequently, the supernatant was collected by centrifugation at 12000 rpm for 10 min while the absorbance was detected at 545 nm.

Experimental Animal and Cell Cultures

Male BALB/c mice (6-week-old, 18–22 g) were acquired from the Guangdong Medical Laboratory Animal Center (SCXK 2018-0002). These mice were reared in a specific pathogen-free room with a 12 h/12 h light–dark cycle. The Animal Ethics Committee of Guangdong Pharmaceutical University authorized all of the animal research stated (Approval No. gdpulacspf 2017567). In addition, HUVECs were bought from iCell Bioscience (iCell-h110, Shanghai, China). At 37 °C in 5% CO₂, the HUVECs were grown in endothelial cell medium supplemented with 1% penicillin and streptomycin (P/S), 5% fetal bovine serum (FBS), and 1% Endothelial Cell Growth Supplement (ECGS).

Wound Healing Assay and Histological Analysis

BALB/c mice (6-week-old) were split into three groups at random: the control group (treated with 20 µL of normal saline per wound site), the esc-1a(1–21)NH₂ group (treated with 20 µL of esc-1a(1–21)NH₂, 200 µg/mL), and the positive control group (treated with 20 µL of 100 ng/mL vascular endothelial growth factor (VEGF)).¹⁸⁾ Esc-1a(1–21)NH_2, saline, and VEGF were administered for 7 d after modeling once a day. Two 8 mm full-thickness round wounds were created on both sides of the back using a biopsy perforator under aseptic conditions. The wound area was covered with a three-millage dressing (3M, Maplewood, NJ, U.S.A.). The wound images were taken by a digital device on days 1, 3, 5, 7, 9, 11, and 14 and the healing rate was computed using Image J software (NIH, Baltimore, MD, U.S.A.). On days 7 after surgery, mice were sacrificed and their tissues of wounds were excised and fixed in 4% paraformaldehyde.

Tissues were cut into 4 µm sections. After dewaxing and hydration, these sections were stained with hematoxylin–eosin (H&E) for blue nuclei and red cytoplasm.¹⁹⁾ According to the result of pathological section, re-epithelialization is assessed by detecting the length of the epidermis and the thickness of the granulation tissue is measured at the wound centre using Image J software.²⁰⁾ Masson and Sirius Red were used to stain collagen.²¹⁾

The blocking solution with 5% bovine serum albumin (BSA) in phosphate-buffered saline containing Tween 20 (PBS-T) was used to block these sections for an hour. There were three types of primary antibodies that were used: anti-CD31 (1 : 100; Abcam, U.K.), anti-proliferating cell nuclear antigen (PCNA) (1 : 100; Abcam), and anti-endothelial nitric oxide synthase (eNOS) (1 : 100; Abcam). Then the sections were incubated with the antibodies overnight at 4 °C. The next day, slices were incubated for 60 min at room temperature with HRP-coupled secondary antibodies (1 : 1000, Beyotime, Shanghai, China). Then all sections were stained with diaminobenzidine (DAB) and re-stained with hematoxylin. Finally, images were captured under a microscope (Leica-DM6000B, Germany).

Tube Formation Assay, Chick Embryo Chorioallantoic Membrane (CAM) Assay and Rat Aortic Ring Assay

Tube Formation Assay: In each hole of 96-well plates, each hole was filled with 50 µL of BD Matrigel (354234, Corning, NY, U.S.A.) and then placed at 37 °C for 30 min to 2 h until it solidified. HUVECs (2 × 10⁵ cells/mL) seeded onto 96-well plates and treated for 6 h with various doses of esc-1a(1–21)NH₂ (0, 1.56, 6.25, 25 µg/mL) or VEGF (10 ng/mL). The tubular formations were then observed using an inverted microscope, with five view spots in each well chosen at random. Analysis was conducted using Image J software (version 6.0).

Chick Embryo Chorioallantoic Membrane Assay: Fertilized eggs were hatched at 38 °C with 60–70% humidity for 8 d. A window was made on the egg shell near the chick embryo, and a round filter paper with a diameter of 5 mm was placed near the branches of the large vessels. The filter paper was treated with 100 µL of esc-1a(1–21)NH₂ (0, 1.56, 6.25, 25 µg/mL) or 100 µL of VEGF (10 ng/chick embryo), and the eggs were hatched until the embryos were 12 d old. Then formaldehyde: acetone = 1 : 1 was added dropwise to the allantoic membrane and fixed for 20 min. Then, the membrane was carefully removed and the CAM vascular plexus was photographed with a camera (Canon, China). Vessel density (the percentage of vessels/area within the field of view) was analyzed using Image J software (version 6.0).²²⁾

Rat Aortic Ring Assay: Briefly, after euthanasia, the aorta of 7-week-old SD rats was rapidly dissected and the adventitia was stripped in the endothelial cell medium. The isolated aorta was sliced into 1 mm rings. These rings were cultivated for 30 min at 37 °C with 5% CO₂ in a 96-well plate containing 50 µL Matrigel. Every other day, 100 µL of fresh endothelial cell medium with esc-1a(1–21)NH₂ was changed in each well. Then, the pictures of the aortic rings samples were photographed with an inverted microscope 7 d later. The number of microvessels growing from the aortic rings was calculated using the Image J software (version 6.0).

Flow Cytometry and Cell Proliferation Assay

The cell cycle assay kit was used to detect the cell cycle of HUVECs (Beyotime). HUVECs (2 × 10⁵ cells/well) were seeded into 24-well plates and incubated with Esc-1a(1–21)NH₂ for 24 h. HUVECs were then washed twice in PBS before being digested with trypsin. The cells were centrifuged and then treated with 75% ethanol at 4 °C for 16 h. The next day, the cells were rinsed in PBS, resuspended, and treated with ribonuclease (RNase) A (20 µg/mL) and propidium iodide (PI) for 30 min at 37 °C in the dark, as directed by the manufacturer. Flow cytometry (BD FACSArray system) was used to identify cell cycle characteristics, which were then analyzed using Modift software.

In 96-well plates, HUVECs (1 × 10⁵ cells/well) were incubated overnight. HUVECs were cultured for 24 h with serially diluted esc-1a(1–21)NH₂ at a final concentration of 1.56–100 µg/mL. Then, in each well, add 10 µL of CCK8 and incubate for 1 h at 37 °C. The absorbance value of the supernatant at 490 nm was read.

Transwell Assay and Scratch Wound Healing Assay

HUVECs (1 × 10⁴ cells/well) were incubated in the 8-µm-sized upper chamber (353097, FALCON) and inserted into a 24-well plate for 16 h at 37 °C, 5% CO₂. Cotton swabs were used to carefully clean off the cells sticking to the top surface of the transwell membrane. The cells that moved to the chamber’s bottom were then fixed for 30 min with 4% paraformaldehyde and stained for 10 min with 0.1% crystal violet. Finally, these transwell membranes were photographed using a camera after being placed on slides. The number of cells in five random fields per chamber were counted to determine migration capability.

In 6-well plates, HUVECs (2 × 10⁵ cells/well) were seeded. The cells were starved for 12 h in media lacking 10% FBS before being scraped vertically with a 200-µL pipette tip. Eventually, cells were imaged at 0 and 24 h using an inverted phase-contrast microscope (Leica-DM6000B, Germany).

Western Blotting and Quantitative Real-Time PCR (qRT-PCR)

HUVECs treated with LY294002 (10 µM) for 1 h, esc-1a(1–21)NH₂ (6.25 µg/mL) for 3 h, or their combination, respectively.²³⁾ HUVECs were added into 6-well plates and incubated with 80 µL of radio immunoprecipitation assay (RIPA) lysis buffer supplemented with phosphatase inhibitors (Beyotime). The bicinchoninic acid (BCA) test kit was applied to determine the total protein content (Beyotime). The 12% sodium dodecyl sulfate (SDS)-polyacrylamide gels were used for isolating the samples (Millipore, PVH00010, U.S.A.). Polyvinylidene difluoride (PVDF) membranes were first activated with methanol and then covered with the gels. After the transfer, the membranes were blocked with 5% skim milk powder for half 60 min and then washed three times with TBST (TBS + Tween20) for five minutes. Subsequently, the membranes were completely covered with primary antibodies and incubated overnight at 4 °C in a refrigerator. Platelet-endothelial cell adhesion molecule CD31 (1 : 1000, ab9498, Abcam), phosphatidylinositol 3′-kinase (PI3K) (1 : 1000, ab180967, Abcam), phosphorylation phosphatidylinositol 3′-kinase (p-PI3K) (1 : 500, ab182651, Abcam), AKT (1 : 1000, 4691, CST), p-AKT (1 : 1000, 13038, CST). The primary antibody is then recovered. Membranes were covered with goat anti-rabbit or goat anti-mouse horseradish peroxidase affinity immunoglobulin G (IgG) secondary antibodies for one hour on a shaker. Protein bands were detected using a fluorescence-based gel scanner (Tannon, Shanghai, China).

Total RNA was extracted with Trizol reagent (10606ES60, YEASEN). The RT First Strand cDNA Synthesis Kit (G3330-50, Servicebio, China) was then used to reverse transcribe cDNA, and SYBR Green (11195ES, YEASEN, China) was utilized for real-time quantitative PCR of target genes. The Bio-Rad CFX program was used to calculate the relative fold change. The primer sequence was as belows: CD31 (forward, 5′-AGAGAGGCTGCTGTCATTGC-3′; reverse, 5′-GGCCCCTCAGAAGACAACAT-3′); eNOS (forward, 5′-GAGGGGCACGTGGCTATG-3′; reverse, 5′-GCCGGCTGTAACTTCCTT-3′).

Statistical Analysis

All data were analyzed with the Graphpad Prism software (version 8.0, U.S.A.), and the results were expressed as mean ± standard deviation (S.D.). Differences between the two groups were compared using the Student’s t-test. One-way ANOVA was applied to examine differences between several groups. p < 0.05 was used as the basis for statistical analysis of significant differences.

RESULTS

The Structural, Cytotoxic, and Hemolytic Properties of Esc-1a(1–21)NH₂

Since the structural characterization and biocompatibility of esc-1a(1–21)NH₂ are closely related to its biological activity and safety, we carried out a series of assays to investigate the structural and hemolytic properties of esc-1a(1–21)NH₂. The mock structure diagram of esc-1a(1–21)NH₂ was drawn with PyMOL software (Fig. 1A). We analyzed the physicochemical properties of esc-1a(1–21)NH₂ with the ProtParam online tool. The molecular weight and theoretical isoelectric point of esc-1a(1–21)NH₂ were 2190 Da and 8.86, respectively. The instability index of esc-1a(1–21)NH₂ was −18.78 and the grand average value of hydropathicity (GRAVY) was 0.338 (Table 1). The helical wheel diagram showed that esc-1a(1–21)NH₂ is a linear cationic peptide (Fig. 1B). The Protscale website predicted that Asn at position 83 of the peptide chain was the most hydrophilic (−2.878) and Cys at position 116 was the most hydrophobic (1.489) (Fig. 1C).

Fig. 1. The Structural, Cytotoxic and Hemolytic Properties of Esc-1a(1–21)NH₂

(A) Mock structure diagram of esc-1a(1–21)NH₂. (B) Helical wheel diagram of esc-1a(1–21)NH₂. (C) Hydrophilic analysis of esc-1a(1–21)NH₂. Positive numbers represent hydrophobicity and negative numbers represent hydrophilicity. (D) Secondary structure analysis of esc-1a(1–21)NH₂, including α-helix, β-turning angle, extended chain, and irregular coiling. (E) The cytotoxicity of esc-1a(1–21)NH₂ in HUVEC in a range of 0.1–500 µg/mL. (F) Hemolytic activity of esc-1a(1–21)NH₂ in, saline and 1% TritonX-100 were used as negative and positive controls, respectively. **** p < 0.0001.

Table 1. Amino Acid Sequences and Physicochemical Properties of Antimicrobial Peptide Esc-1a(1–21)NH₂

Peptide	Sequence	Molecular weight	Instability index	Aliphatic index	GRAVY
Esc-1a(1–21)NH2	GIFSKLAGKKIKNLLISGLKG-NH2	2190	−18.78	134.76	0.338

The secondary structure of esc-1a(1–21)NH₂ was mainly ɑ-helix (47.62%), random coil (30.0%), extended chains (19.05%), and β-sheet (19.05%) according to SOPMA analysis (Fig. 1D). We also evaluated the effects of esc-1a(1–21)NH₂ on cell viability and hemocompatibility using CCK8 and hemolysis assays, respectively. Esc-1a(1–21)NH₂ did not affect HUVEC proliferation at doses up to 100 µg/mL (Fig. 1E). Esc-1a(1–21)NH₂ caused a maximum hemolysis rate of 2.6% at the maximum tested concentration of 250 µg/mL, showing good biocompatibility. (Fig. 1F). These findings imply that esc-1a(1–21)NH₂ has low cytotoxicity and has potential for application in wound therapy.

Esc-1a(1–21)NH₂ Promoted Wound Healing and Granulation Tissue Formation in Mice

We established a full-thickness excision model in mice to test the effects of esc-1a(1–21)NH₂ on wound cloure in vivo. In comparison to the control group, we found that topical treatment of esc-1a(1–21)NH₂ dramatically accelerated wound closure, and the greatest difference occurred in the period from day 3 to day 7 (Figs. 2A, B). The healing effect of esc-1a(1–21)NH₂ was equivalent to that of VEGF. The control wounds closed in 15 d (± 1 d), which was 4 d (± 1 d) later than esc-1a(1–21)NH₂-treated wounds (Fig. 2C). Esc-1a(1–21)NH₂-treated wounds had greater re-epithelialization and higher granulation tissue development on day 7 than untreated wounds, according to a H&E staining experiment (Figs. 2D–F). Furthermore, esc-1a(1–21)NH₂ boosted collagen deposition and induced more regular collagen textile fiber organization in wound sites on day 7 post-injury, according to Masson and Sirius Red staining (Figs. 2G, H). Overall, esc-1a(1–21)NH₂ therapy promotes wound healing in vivo by triggering the proliferative phase. Granulation tissue formation is widely established as a necessary event for effective wound healing. In fact, granulation tissue is made up of proliferating fibroblasts, which produced extracellular matrix, and new capillaries, as well as inflammatory cells. Therefore, we performed immunohistochemical staining of CD31 and PCNA in wound tissue on day 7 post-injury and found the expression levels of CD31 and PCNA increased remarkably in the esc-1a(1–21)NH₂-treated group, suggesting better granulation tissue formation (Figs. 3A–D). Since angiogenesis in wound healing can be regulated by nitric oxide and eNOS, eNOS has an important role in angiogenesis. We examined eNOS in tissues on day 7 after injury using immunohistochemical staining and found that the expression level of eNOS was significantly increased in the esc-1a(1–21)NH₂-treated group, indicating that esc-1a(1–21)NH₂ has a facilitative effect on angiogenesis (Figs. 3E, F). Meanwhile, we examined the expression levels of eNOS in the wounds using qRT-PCR, and the results showed that esc-1a(1–21)NH₂ treatment group was significantly higher compared with the control group (Fig. 3G). Collectively, our findings suggest that esc-1a(1–21)NH₂ may promote wound healing by promoting granulation formation.

Fig. 2. Esc-1a(1–21)NH₂ Accelerates Wound Healing in Mice

(A) Representative images of wounds in a mouse model at days 0, 3, 5, 7, and 14 (n = 8). VEGF was used as a positive control. (B) Wound closure rates were quantified by the Image J software (C) Healing time in mice. (D) HE stains of the wound tissue at day 7 (scale bar = 100 µm, 400×; scale bar = 400 µm, 100×). (E, F) Percentage (%) of re-epithelialization and granulation tissue formation in the HE staining. (G) Masson staining of the wound tissue at day 7 (scale bar = 100 µm, 400×; scale bar = 400 µm, 100×). (H) Collagen fibers of skin tissue stained with Sirius Red staining of the wound tissue at day 7 (scale bar = 100 µm, 400×; scale bar = 400 µm, 100×). * p < 0.05, ** p < 0.01 compared to control.

Fig. 3. Esc-1a(1–21)NH₂ Promote Granulation Tissue Formation

(A) Immunohistochemistry of CD31 in the wound tissue at day 7 (scale bar = 100 µm, 400×; scale bar = 400 µm, 100×). (B) Immunohistochemistry of PCNA in the wound tissue at day 7 (scale bar = 100 µm, 400×; scale bar = 400 µm, 100×). (C, D) The expression levels of PCNA and CD31 were quantified by Image J software, respectively. (E) Immunohistochemistry of eNOS in the wound tissue at day 7 (scale bar = 100 µm, 400×; scale bar = 400 µm, 100×).(F) The expression levels of eNOS were quantified by Image J software. (G) The mRNA expression levels of eNOS was measured by the qRT-PCR assay. * p < 0.05 vs. control. Red arrows indicate CD31, PCNA, and eNOS, respectively. *** p < 0.001, **** p < 0.0001 vs. control.

Esc-1a(1–21)NH₂ Enhanced Angiogenesis by the Tube Formation Assay, CAM and the Rat Aortic Ring Assay

Considering that esc-1a(1–21)NH₂ increases the microvessel density in granulation tissue, we investigated the pro-angiogenic ability of esc-1a(1–21)NH₂ by a series of assays. The tube formation assay showed that HUVECs treated with 6.25 µg/mL esc-1a(1–21)NH₂ for 6 h could form a more obvious network structure, which was comparable to that of VEGF (10 ng/mL) (Fig. 4A). Compared with the control group, the esc-1a(1–21)NH₂-treated group showed significant differences in the number of junctions, length, and branches in the vascular network (Figs. 4B–D). Additionally, the CAM assay showed more neovascularization around the medicated filter paper after 5 d of esc-1a(1–21)NH₂ treatment (Fig. 4E). Meanwhile, esc-1a(1–21)NH₂ increased vessel density and the length of the 2nd and 3rd vessels (Figs. 4G, H). We also found that esc-1a(1–21)NH₂ (6.25 µg/mL), like VEGF (10 ng/mL), could significantly increase the number of vascular sprouts through the rat aortic ring experiments. (Figs. 4F, I). These findings showed that esc-1a(1–21)NH₂ had proangiogenic activity in vitro, implying that it might be useful in wound healing research.

Fig. 4. Proangiogenic Activity of Esc-1a(1–21)NH₂ in Vitro

(A) Esc-1a(1–21)NH₂ induces tube formation in HUVECs. (B–D) Quantification of the capillary network measured by Image J software. (E) Esc-1a(1–21)NH₂ promotes angiogenesis in chicken embryo chorioallantoic membrane assay. Chicken embryos were cultured until day 8, each chicken embryo filter paper was incubated with 100 µL of antimicrobial peptide dropwise until day 12, and the allantoic membrane was fixed 1 : 1 with methanol:acetone (n = 10/group). (F) Esc-1a(1–21)NH₂ promotes angiogenesis in rat aortic rings assay. (n = 5/group). (G, H) The density of blood vessels on the allantoic membrane of chicken embryos was measured by Image J software. (I) The number of vascular outgrowths from the aortic rings. * p < 0.05, ** p < 0.01, *** p < 0.001 vs. control.

Esc-1a(1–21)NH₂ Significantly Promoted Proliferation and Migration of HUVECs

Since formation of new blood vessels depends on proliferation and migration of endothelial cells, we investigated the activity of esc-1a(1–21)NH₂ on endothelial cells. Cell cycle analysis showed that esc-1a(1–21)NH₂ could significantly increase the S-phase ratio of HUVECs, especially the S-phase ratio of HUVECs reached the peak at the doses of 6.25 µg/mL (Figs. 5A, B).

Fig. 5. Esc-1a(1–21)NH₂ Promotes Cell Proliferation and Cell Migration in HUVECs

(A) Cell cycle of HUVECs treated with esc-1a(1–21)NH₂. (B) The display of cell cycle data was by Modfit software. (C) Cell proliferation in HUVECs treated with esc-1a(1–21)NH₂. (D) Esc-1a(1–21)NH₂ promotes endothelial cell migration in a dose-dependent manner (scale bar = 50 µm). (E) Esc-1a(1–21)NH₂ promotes cell migration by scratch assay. (scale bar = 200 µm). (F, G) Cell migration data were quantified by Image J software. 10 ng/mL VEGF was used as a positive control. Data are expressed as mean ± S.D. from three independent experiments. * p < 0.05, ** p < 0.01, *** p < 0.001, **** p < 0.0001 compared to control.

Furthermore, the CCK8 assay revealed that at 6.25 µg/mL, esc-1a(1–21)NH₂ had the greatest proliferative impact on HUVECs (Fig. 5C). This was in line with the cell cycle experiment’s findings. An in vitro wound healing test and a transwell assay were used to study the effect of esc-1a(1–21)NH₂ on cell migration. The cell scratch assay showed that HUVECs treated with 6.25 µg/mL esc-1a(1–21)NH₂ for 24 h exhibited the fastest significant closure (Figs. 5E, G). Consistently, the transwell results showed that 6.25 µg/mL esc-1a(1–21)NH₂ had the most pro-migratory effect on HUVECs (Figs. 5D, F). These findings suggest that esc-1a(1–21)NH₂ can promote HUVECs’ proliferation, migration, and angiogenesis.

The PI3K/AKT Pathway Was Activated and CD31 Expression Was Upregulated in HUVECs by Esc-1a(1–21)NH₂

Because PI3K/AKT pathway activation is a critical signaling event for angiogenesis, we investigated whether esc-1a(1–21)NH₂ increased angiogenesis in HUVECs by raising PI3K and AKT phosphorylation. We found that phosphorylation of PI3K and AKT was considerably increased in esc-1a(1–21)NH₂-treated cells (Figs. 6A–C), along with the enhanced expression level of CD31 (Figs. 6D, E). LY294002, an inhibitor of PI3K, tended to blocked the activity of esc-1a(1–21)NH₂ to promote PI3K and AKT phosphorylation in HUVECs, but the difference was not statistically significant. Correspondingly, the tube formation assay showed that LY294002 also abolished the proangiogenic activity of esc-1a(1–21)NH₂ on HUVECs (Fig. 6F), showing fewer junctions, lengths, and branches (Figs. 6G–I). In general, esc-1a(1–21)NH₂ increased angiogenesis in HUVECs during healing via activation of the PI3K/AKT pathway.

Fig. 6. Esc-1a(1–21)NH₂ Promotes Angiogenesis by Activating the PI3K/AKT Signaling Pathway

(A) Inhibition of the PI3K/AKT pathway by LY294002 diminished the effects on angiogenesis caused by esc-1a(1–21)NH₂ in HUVECs. (B–D) Quantification of p-PI3K/PI3K, p-AKT/AKT, and CD31 measured by Western blot assay. (E) The mRNA expression levels of PI3K, AKT, and CD31 were measured by the qRT-PCR assay. (F) Esc-1a(1–21)NH₂ stimulated in vitro angiogenesis via the PI3K/AKT pathway. (G–I)The vascular junctions, length, and branches were quantified by Image J software. Data are expressed as mean ± S.D. from three independent experiments. Compared with control, * p < 0.05, ** p < 0.01, *** p < 0.001. n.s. means compared with the esc-1a(1–21)NH₂ group.

DISCUSSION

The structure of the antimicrobial peptide esc-1a(1–21)NH₂ consisted mainly of α-helix, β-fold, and 21 amino acids (the first 20 amino acids were followed by an N-terminal amidated glycine residue).²⁴⁾ For enhanced bactericidal activity, it must have a stable helical structure and a minimum length of 20 amino acids for interaction with the phospholipid bilayer.²⁵⁾ The positive charge and amphiphilicity allow esc-1a(1–21)NH₂ to be rapidly bactericidal within 15 min, achieving 99.9% killing of P. aeruginosa at concentrations of 0.5 to 1 µM.²⁶⁾ Topically applied by AMP, its activity was limited by the wound environment, resulting in reduced stability and biological activity.²⁷⁾ The esc-1a(1–21)NH₂ synthetic peptide used in this study is an aminoacetylated peptide.²⁸⁾ The N-terminal acetylated peptide is resistant to hydrolysis by proteases, while the C-terminal amidation increases the antimicrobial activity and decreases the hemolytic activity. Amphiphilic hydrophobic peptides have the potential to form oligomers. Similar to the antimicrobial peptide LL-37, esc-1a(1–21)NH₂ is an amphiphilic hydrophobic peptide. The optimal concentration of LL-37 to exert its pro-angiogenic activity is 5 µg/mL. At higher concentrations, LL-37 may form oligomers.²⁹⁾ We found that esc-1a(1–21)NH₂, like LL-37, may affect its pro-angiogenic activity. The study of these structural properties can provide strong support for future modification with esc-1a(1–21)NH₂ as a template.

We tried several concentrations and found that 200 µg/mL of esc-1a(1–21)NH₂ was the most effective concentration to promote wound healing in mice. Similarly, the human antimicrobial peptide LL-37 was also administered in vivo at 200 µg/mL.³⁰⁾ The process of wound healing involves several important events, including angiogenesis, development of granulation tissue, extracellular matrix formation, and re-epithelialization.³¹⁾ AMPs can promote wound tissue regeneration in multiple ways.³²⁾ For example, Boparai et al. found that Tylotoin directly enhanced formation of vascular endothelial cell tubules, thus accelerating angiogenesis at the wound site.³³⁾ Simonetti et al. demonstrated that LL-37 significantly improved granulation tissue formation and re-epithelialization in an ob/ob mouse excisional wound model.³⁴⁾ Huang et al. showed that Epinecidin-1 heals wounds by increasing the synthesis of extracellular matrix (e.g., collagen) at the wound site.^35,36) Overall, our results show that esc-1a(1–21)NH₂ may increase granulation tissue development, collagen deposition, and re-epithelialization in a full-thickness excision model in mice via boosting angiogenesis.

Endothelial cell migration, proliferation, and angiogenesis are required for tissue renewal.³⁷⁾ The PI3K/AKT pathway is one of the key angiogenesis signaling mechanisms.³⁸⁾ Previous research has found that activating the PI3K/AKT pathway in vascular endothelial cells promotes proliferation, migration, and tube formation while simultaneously inhibiting reactive oxygen species (ROS) and members of the Bcl-2 family that promote apoptosis include Bad, Bax, and caspase-3.³⁹⁾ AKT increased nitric oxide (NO) production in endothelial cell, which then stimulated endothelial cell proliferation, increased vascular permeability, and promoted angiogenesis. During angiogenesis, both VEGF and eNOS are upregulated through PI3K activation. PI3K/AKT can also activate interleukin-8 (IL-8).⁴⁰⁾ Therefore, we cannot exclude that the observed angiogenic effects are mediated through VEGF and IL-8.⁴¹⁾ In addition, PI3K/AKT significantly increased the expression of matrix metalloproteinase-2 (MMP-2), which is a degradative enzyme that aids in cell invasion and metastasis.⁴²⁾ Our findings imply that esc-1a(1–21)NH₂ stimulates granulation tissue development, collagen deposition, and re-epithelialization in HUVECs via the PI3K/AKT signal pathway. Grazia et al. found that in the presence of 10% fresh human serum after 5 h of incubation at 37 °C, the stability of esc-1a(1–21)NH₂ was still retained at 44.4%. This stabilization time is sufficient to activate signaling pathways to promote angiogenic activity.⁴³⁾ In conclusion, we discovered that esc-1a(1–21)NH₂ boosted wound healing by activating the PI3K/AKT signaling pathway in HUVECs, allowing them to proliferate, migrate, and form blood vessels.

Acknowledgments

This work was supported by the Natural Science Foundation of Guangdong Province, China (Grant No.2021A1515011003) and the Key Projects of Traditional Chinese Medicine Bureau of Guangdong Province (No.20193001).

Author Contributions

Juan Shen conributed to the study conception and design. Qiong Hu and Chujun Chen performed the experiments. Zhenming Lin, Liyao Zhang and Sujiuan Guan were responsible for the data analysis. Juan Shen, Guangfu Dong and Qiong Hu wrote the manuscript. Xiaoyan Zhuang supplemented the experiments and participated in revising the manuscript. All authors read and approved the final manuscript.

Conflict of Interest

The authors declare no conflict of interest.

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