
==== Front
Int J Biol Sci
Int J Biol Sci
ijbs
International Journal of Biological Sciences
1449-2288
Ivyspring International Publisher Sydney

10.7150/ijbs.98856
ijbsv20p4532
Research Paper
The Essential Role of Angiogenesis in Adenosine 2A Receptor Deficiency-mediated Impairment of Wound Healing Involving c-Ski via the ERK/CREB Pathways
Peng Yan
Xiong Renping
Wang Bo
Chen Xing
Ning Yalei
Zhao Yan
Yang Nan
Zhang Jing
Li Changhong
Zhou Yuanguo
Li Ping ✉
Department of Army Occupational Disease, State Key Laboratory of Trauma, Burn and Combined Injury, Daping Hospital, Army Medical University (Third Military Medical University), 10 Changjiang Zhilu, Chongqing 400042, People's Republic of China.
✉ Corresponding author: Ping Li, Department of Army Occupational Disease, State Key Laboratory of Trauma, Burn and Combined Injury, Daping Hospital, Army Medical University (Third Military Medical University), 10 Changjiang Zhilu, Chongqing 400042, People's Republic of China. E-mail addresses: ping__ping0074@sina.com; liping123123@tmmu.edu.cn.
Competing Interests: The authors have declared that no competing interest exists.

2024
19 8 2024
20 11 45324550
24 5 2024
7 8 2024
© The author(s)
2024
https://creativecommons.org/licenses/by/4.0/ This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/). See http://ivyspring.com/terms for full terms and conditions.
Adenosine receptor-mediated signaling, especially adenosine A2A receptor (A2AR) signaling, has been implicated in wound healing. However, the role of endothelial cells (ECs) in A2AR-mediated wound healing and the mechanism underlying this effect are still unclear. Here, we showed that the expression of A2AR substantially increased after wounding and was especially prominent in granulation tissue. The delaying effects of A2AR knockout (KO) on wound healing are due mainly to the effect of A2AR on endothelial cells, as shown with A2AR-KO and EC-A2AR-KO mice. Moreover, the expression of c-Ski, which is especially prominent in CD31-positive cells in granulation tissue, increased after wounding and was decreased by both EC-A2AR KO and A2AR KO. In human microvascular ECs (HMECs), A2AR activation induced EC proliferation, migration, tubule formation and c-Ski expression, whereas c-Ski depletion by RNAi abolished these effects. Mechanistically, A2AR activation promotes the expression of c-Ski through an ERK/CREB-dependent pathway. Thus, A2AR-mediated angiogenesis plays a critical role in wound healing, and c-Ski is involved mainly in the regulation of angiogenesis by A2AR via the ERK/CREB pathway. These findings identify A2AR as a therapeutic target in wound repair and other angiogenesis-dependent tissue repair processes.

Wound healing
A2AR
Angiogenesis
c-Ski
Endothelial cell
==== Body
pmcIntroduction

The enhancement of wound healing has been a goal of medical practitioners for thousands of years. Therapeutic interventions that activate the innate repair mechanism of native tissue and promote healing are of particular interest. Adenosine, a potent endogenous physiological mediator, and its receptors play key roles in tissue repair and wound healing1-4. Moreover, caffeine, a major component of many beverages, such as coffee and tea, is an adenosine receptor antagonist and has inhibitory effects on tissue repair and wound healing5-7. Therefore, exploration of the mechanisms of adenosine and its receptors in wound repair is very important.

The adenosine A2A receptor (A2AR), a subtype of G protein-coupled adenosine receptor, is broadly expressed on most cell types involved in wound healing, including macrophages, fibroblasts and microvascular endothelial cells (ECs)8. We and others have demonstrated that the topical application of A2AR agonists promotes healing of normal wounds9-11 and diabetic wounds8, 12, 13, and one such agonist, sonedenoson, is currently being evaluated as a therapy for diabetic foot ulcers8, 11. In addition, polydeoxyribonucleotides have been used to improve wound healing in clinical studies through A2AR activation14. Although A2AR activation reportedly accelerates the wound healing process by affecting ECs and stimulating angiogenesis15, relieving inflammation15, 16, and affecting fibroblasts and epithelial cells13, 17, 18, the roles of these regulatory effects in A2AR-mediated healing have not been fully elucidated. Specifically, the regulation of ECs in A2AR-mediated wound healing and the mechanism underlying this effect are still unclear.

The cellular Sloan-Kettering Institute (c-Ski) is an intracellular homolog of the virus oncogene v-ski and is involved in various physiological and pathological processes, such as the proliferation of hematopoietic cells, muscle regeneration, bone and nervous system development, synaptic projection, and tumorigenesis19-23. Further research has shown that c-Ski is involved in the repair of injured skeletal muscle, liver and other tissues24, 25. In recent years, we demonstrated that c-Ski is a wound repair factor that can accelerate wound healing in skin tissue and the brain26, 27. These effects are related to c-Ski's role as a multifunctional transcription regulator28, 29 that is involved in cell proliferation, transformation, secretion of the extracellular matrix and the inflammatory response. In particular, c-Ski is expressed on ECs30, and its overexpression promotes the proliferation of ECs31, 32. However, whether A2AR regulates wound healing through c-Ski's effects on ECs is unclear.

The extracellular signal-regulated kinase (ERK)/cAMP response element binding protein (CREB) pathway, an upstream signal, can transform extracellular stimulation into intracellular responses to promote the proliferation and differentiation of cells33. Moreover, the ERK/CREB pathway has a regulatory effect on angiogenesis34-36 and is also the canonical pathway of A2AR regulation37-39. Additionally, it has been reported that the expression of c-Ski can be regulated by the ERK/CREB pathway40. Thus, we speculated that A2AR may regulate c-Ski expression through the ERK/CREB pathway.

In this study, we explored the effect and mechanism of A2AR in regulating skin wound healing and its relationship with c-Ski. First, a full-thickness excisional wound model in mice was used to observe the expression of A2AR and explore its role in angiogenic regulation during wound healing via genetic [A2AR knockout (A2AR KO) and EC-specific A2AR knockout (EC-A2AR KO) mice] blockade of A2AR. Furthermore, we examined the effects of A2AR on the expression of c-Ski to explore its role in A2AR-mediated angiogenesis and wound healing by using A2AR KO and EC-A2AR KO mice. Next, we analyzed the effects of A2AR on the regulation of angiogenesis in human microvascular ECs (HMECs) in vitro and the role of c-Ski in this process by RNAi. Finally, the ERK1/2 antagonist PD98059 was used to confirm that A2AR regulates the expression of c-Ski through the ERK/CREB pathway in vitro.

Materials and methods

Animals

A2AR knockout (KO) mice (A2AR-/-) and their wild-type (WT) controls were bred on a C57BL/6 background. The A2AR+/- breeding pairs were backcrossed to C57BL/6 mice for 10 generations, resulting in a congenic C57BL/6 genetic background. Heterozygous interbreedings (A2AR+/- × A2AR+/-) were used so that the global A2AR homozygous KO mice (A2AR-/-) and their WT littermates were generated from the same breeding pairs 41. Mice with a 'floxed' adenosine A2AR gene (A2Aflox/flox mice) have been described previously42 and were provided by Dr. Chen. TeK-Cre transgenic mice (Tek, a strain that targets ECs)43, 44 were obtained from Shanghai Biomodel Organism Science & Technology Development Co., Ltd. (No. NMX-TG-192000, Shanghai, China). Global A2AR knockout mice, A2Aflox/flox mice and TeK-Cre transgenic mice were backcrossed for 10 to 12 generations to C57BL/6 mice. TeK-Cre transgenic mice were then crossed with A2Aflox/flox mice to generate TeK-A2AR KO mice and TeK-A2AR WT littermates, which were injected with 120 mg/kg tamoxifen (10540-29-1, Sigma Aldrich, St. Louis, MO, USA) in oil via intraperitoneal injection once every 48 hours 5 times in total, followed by a 14-day waiting period between the final injection and full-thickness wounding. The mice were used at 90 days of age. The animals were treated in accordance with the guidelines of the Animal Ethical and Welfare Committee of the Army Medical University and according to the protocol approved by the Administration of Affairs Concerning Experimental Animals Guidelines of The Army Medical University (No. AMUWEC20172073).

Reagents

We purchased MCDB 131 medium (10372019), fetal bovine serum (FBS) (10099141C), TrypLE™ Express (12605028), penicillin‒streptomycin (15140122), and L-glutamine (25030081) from Fisher Scientific (Thermo Fisher, MA, USA). Hydrocortisone (M3451) was obtained from Abmole (Abmole, TX, USA), and recombinant mouse EGF protein (ab126695) was obtained from Abcam (Abcam, Cambridge, UK). The A2AR agonist 2-p-[2-carboxyethyl]phenethyl-amino-5'-N-ethylcarboxamido-adenosine (CGS21680) (1063) and the MEK inhibitor PD98059 (1213) were purchased from Tocris (Bio-Techne, MN, USA). Hydrocortisone, CGS21680, and PD98059 were prepared as stock solutions in dimethyl sulfoxide (DMSO), aliquoted, and stored at -80°C.

Model and observation of wound healing

A mouse model of wound healing was generated as previously described with some modifications26. Briefly, to minimize discomfort and pain, mice were anesthetized with intraperitoneal injection of 50 mg/kg sodium pentobarbital, and two 1 cm diameter, full-thickness circular skin flaps were cut from the buttocks of the mice (at least six animals in each group). The wounds were covered with a piece of sterile gauze. The mice were then returned to their cages and maintained separately to avoid any further wound damage under a 12:12 h light/dark cycle, and supplied with unrestricted food and water.

Wound healing was observed as described previously26, 45. At 0, 3, 6, 9 and 15 d after wounding, wound closure was traced in each group using transparent film. And, percentage wound closure at each time point was derived by the formula: [1- (current wound area/original wound area)]×100%. The scar areas were calculated every 2 weeks using image processing software.

Histological and quantitative image analysis

After perfusion, the mice were anesthetized with intraperitoneal injection of 50 mg/kg sodium pentobarbital and perfused with ice-cold normal saline and then with 4% paraformaldehyde solution. Next, the entire wound and its associated normal skin was excised and then was fixed overnight in 4% paraformaldehyde solution overnight for further fixation at 3, 6 and 9 d postwounding. After routine dehydration and paraffin embedding, the tissue sections (4 mm) were stained with hematoxylin and eosin (H&E) for morphological assessment. Re-epithelialization and granulation tissue formation were assessed as previously described26, 45.

Immunofluorescence was performed as previously described46, 47. Briefly, after dewaxing and antigen repair, primary antibodies were incubated with the samples overnight at 4°C in 0.01% Triton X-100 and 10% goat serum. The primary antibodies included goat anti-A2AR (1:200, Frontier Institute, AB_2571655), rabbit anti-CD31 (1:100, Servicebio, GB113151), rabbit anti-Ski (1:100, Santa Cruz, sc-9140), rabbit anti-CD11b (1:2000, Abcam, ab133357), rabbit anti-vimentin (1:400, Abway, CY5134), and rabbit anti-F4/80 (1:400, Cell Signaling, 70076). The tissue sections were washed with phosphate-buffered saline (PBS) and then treated with an Alexa Fluor 488-conjugated goat anti-rabbit antibody (1:500, Abcam, ab150077) and/or a Cy3-conjugated donkey anti-goat (1:500, Abcam, ab6949) antibody for 50 min at room temperature. The nuclei were stained with DAPI (Solarbio, C0060), and an autofluorescence quenching kit (Vector Laboratories, SP-8400) was used to remove unwanted fluorescence.

The results were captured by laser scanning confocal microscopy (TCS-SP8, Leica), and the quantitative analysis was performed independently by two observers who were blinded to the treatment conditions. The number of CD31-positive cells was counted and normalized to the total number of cells in each high-power field (HPF, 400×). The ratio of the integrated density (IntDen) to the total number of cells was determined with ImageJ software, and three fields were randomly chosen from each slice (three fields per section, three sections per mouse, five mice from each analyzed group).

RNA isolation and RT‒PCR

At 0, 6, 9, and 15 d postwounding, total RNA was extracted from the full-thickness skin of the mouse wounds using a TRIzol Reagent RNA Extraction Kit (Invitrogen, 15596026) according to the manufacturer's instructions and then reverse transcribed to cDNA using a kit (Promega, A2791). A qPCR master mix kit (Promega, A600A) was used for quantitative PCR. The methods used were previously reported48. The primers for c-Ski were as follows: 5′-TCAACTCGGTGTGCGATG-3′ and 5′-CGTCCGTCTTGGTGATGAG-3′. The primers for GAPDH were as follows: 5′-AGGTTGTCTCCTGCGACTT-3′ and 5′-TGGTCCAGGGTTTCTTACTCC-3′. The samples were denatured by heating at 95°C for 30 s, followed by 40 cycles of 95°C for 30 s, 58°C for 30 s and 72°C for 30 s, and the relative abundance of the target gene was calculated via normalization to GAPDH.

Western blotting

Western blotting was performed as previously described46. Briefly, at 0, 6, 9, and 15 d postwounding, the tissues were lysed in RIPA lysis buffer (Beyotime, P0013B) containing a protease and phosphatase inhibitor cocktail (Thermo Fisher, 78440). The total protein concentration was measured using a BCA kit (Aidlab, PP0102). The samples were resolved on 10% SDS‒PAGE and then transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, IPVH00010). After the membranes were blocked with 5% bovine serum albumin (Solarbio, A8020) in Tris-buffered saline (pH 7.6) containing 0.1% Tween-20 (TBS-T) at room temperature for 1 hour, they were probed with the following primary antibodies overnight at 4°C: anti-Ski (1:100, Santa Cruz, sc-9140), anti-A2A adenosine receptor (1:1000, Abcam, ab3461), and anti-GAPDH (1:5000, Abcam, ab9485). After 3 washes with TBS-T incubation for 1 h at room temperature with HRP-conjugated goat anti-mouse IgG or goat anti-rabbit IgG secondary antibodies (1:8000, Abways, AB0035), the membranes were visualized using Clarity Western ECL Substrate (Beyotime, P0018). The band intensity was quantified by ImageJ software, and the relative quantity of the target protein was normalized to that of GAPDH or the nonphosphorylated protein for three independent experiments.

Cell culture and treatment

HMEC-1 cells were provided by Dr. Lan49 and were characterized by Feiouer Biotechnology Co., Ltd. (Chengdu, China) using short tandem repeat (STR) markers (Fig. S9). The cells were maintained at 37°C at 5% CO2 in MCDB 131 medium supplemented with 10% FBS, 10 ng/mL recombinant mouse EGF protein, 1 μg/mL hydrocortisone, 1% penicillin‒streptomycin, and L-glutamine.

To determine the possible signaling mechanism, the cells were treated with 20 nM, 100 nM, or 500 nM of the A2AR agonist CGS21680, which was dissolved in DMSO, for 24 h or were pretreated with 100 µM of the MEK inhibitor PD98059 for 60 min before 100 nM CGS21680 treatment according to the protocol previously described. DMSO was used as the vehicle control.

Lentivirus infection

Short hairpin RNA (shRNA)-knockdown lentiviral particles for mouse c-Ski (sh c-Ski) and short hairpin negative control (sh Nc) lentiviruses were purchased from Shanghai Jikai Company (Shanghai, China). For c-Ski knockdown experiments, HMEC-1 cells were transfected with 5 nM, 10 nM, or 20 nM sh c-Ski for 24 h following the manufacturer's instructions before 100 nM CGS21680 treatment for 24 h.

Cell proliferation and scratch assays

A Cell Counting Kit (CCK)-8 assay (Beyotime, C0038) was used to assess cell proliferation47. Briefly, HMEC-1 cells were trypsinized using TrypLE™ Express and centrifuged at 1000g for 5 min at room temperature and then were seeded in 96-well plates with 100 μL of medium at a density of 8,000 cells/well. After various treatments, 10 µL of CCK-8 solution was added to each well and incubated for 2 h at 37°C and 5% CO2. The optical density (OD) values were measured at 450 nm using a microplate reader (Gene, ELX800ux). Wells without cells served as blank controls. Each experiment was performed in triplicate.

The scratch assay was performed as previously described49. Briefly, HMEC-1 cells were seeded in 6-well plates. The monolayer was scratched with a 200 μL pipette tip and rinsed with PBS after various treatments. Images were taken under an inverted phase contrast microscope (Zeiss, Primo Vert) with CCD cameras at 0, 24, 48, and 72 h after scratching. The experiment was repeated independently three times.

Tube formation

Matrigel (Corning, 356,234) was added to a precooled Millicell EZ SLIDE 8-well glass slide (200 μL/well) and polymerized for 40 min at 37°C. HMEC-1 cells were grown to 80% confluence and trypsinized using TrypLE™ Express. 1 × 104 cells in each treatment group were seeded on the Matrigel, incubated for 4 h in the corresponding medium at 37°C during tubule formation and then and washed with Hank's balanced salt solution (HBSS). For CGS21680 treatment, the cells were treated with calcein AM fluorescent dye (1:300, Corning, 354,216) for 4h. For CGS21680 and c-Ski RNAi treatment, the cells were treated with DiIC12(3) fluorescent dye (1:1000, Corning, 354,218). The results were captured by laser scanning confocal microscopy (TCS-SP8, Leica), and the number of junctions of the tubular-like structures was determined via Angiotool software 50. Three independent experiments were performed.

In vitro immunofluorescence and western blotting

In vitro immunofluorescence and western blot assays were performed according to previously described methods. Briefly, for immunofluorescence analyses, HMEC-1 cells were fixed with 4% paraformaldehyde for 10 minutes and then incubated with the primary antibodies goat anti-A2AR (1:200, Frontier Institute, AB_2571655), rabbit anti-Ski (1:100, Santa Cruz, sc-9140), anti-p-PKA (1:500, Abcam, ab32390), anti-p-ERK1/2 (1:200, Cell Signaling, 9101), and anti-p-CREB (1:500, Cell Signaling, 9198) at 4°C overnight. The cells were then rinsed with phosphate-buffered saline (PBS) and treated with an Alexa Fluor 488-conjugated goat anti-rabbit antibody or a Cy3-conjugated donkey anti-goat antibody for 1 h at 37°C. The nuclei were stained with DAPI. The results were captured by laser scanning confocal microscopy (TCS-SP8, Leica), and the quantitative analysis was performed independently by two observers who were blinded to the treatment conditions. The ratio of the integrated density (IntDen) to the total number of cells was determined via ImageJ software, and three fields were randomly chosen from each well (five wells per group and 3 independent experiments).

For western blot assays, HMEC-1 cells were seeded into 25 cm2 vented cap flasks. After various treatments, the whole-cell lysates were collected and then separated on a 10% SDS‒polyacrylamide gel. After blocking with 5% BSA in Tris-buffered saline (pH 7.6) containing 0.1% Tween 20 (TBS-T) at room temperature for 1 hour, the PVDF membranes were probed with the following primary antibodies overnight at 4°C: anti-Ski (1:100, Santa Cruz, sc-9140), anti-A2A adenosine receptor (1:1000, Abcam, ab3461), anti-PKA (1:1000, Cell Signaling, 4782), anti-p-PKA (1:1000, Abcam, ab32390), anti-CREB (1:300, Cell Signaling, 9197), anti-p-CREB (1:1000, Cell Signaling, 9198), anti-ERK1/2 (1:1000, Cell Signaling, 9102), anti-p-ERK1/2 (1:1000, Cell Signaling, 9101), and anti-GAPDH (1:5000, Abcam, ab9485). After incubation with HRP-conjugated goat anti-mouse IgG or goat anti-rabbit IgG secondary antibodies (1:8000, Abways, AB0035), the membranes were washed with TBS-T 3 times and visualized using Clarity Western ECL Substrate (Beyotime, P0018). The band intensity was quantified by ImageJ software, and the relative quantity of the target protein was normalized to that of GAPDH or the nonphosphorylated protein for three independent experiments.

Statistical analysis

All procedures and analyses were performed by an experienced researcher who was blinded to all groups. All of the results are expressed as the means ± standard errors of the means. Statistical analyses were performed using GraphPad Prism 6 software (San Diego, CA, USA). Two-group comparisons were performed using Student's t test. One-way and two-way analysis of variance (ANOVA) was used for data with one variable and multiple conditions, followed by Tukey's multiple comparisons test. A value of P < 0.05 was considered statistically significant.

Results

A2AR expression in full-thickness excisional wounds

To study the role of A2AR in wound healing, we assessed the expression profile of A2AR in mouse dermal excisional wounds (Fig. 1A). Western blot analyses revealed that A2AR expression began to increase after the injury, peaked at 6 days, and was maintained at a high level during the late stages of wound healing (Fig. 1B). Next, immunofluorescence staining revealed that A2AR was widely distributed in the undamaged epidermis and subcutaneous tissue around the wound, resulting in a hole-like morphology (Fig. 1C), similar to our previous results39, 42. Moreover, A2AR expression increased in the epidermis and granulation tissue in the wound at 9 days postwounding and was especially prominent in granulation tissue (Fig. 1C-D). To characterize the expression of A2AR in granulation tissue further, we performed double immunofluorescence staining and revealed that A2AR was widely expressed in repair cells; was especially prominent in CD31+ ECs, vimentin+fibroblasts and F4/80+ macrophages; and was expressed at low levels in CD11b+ inflammatory cells (Fig. 3E-G). Thus, changes in the expression of A2AR during wound healing and its localization in repair cells suggest that this molecule is involved in the regulation of healing.

Both A2AR KO and EC-A2AR KO delay full-thickness excisional wound healing and inhibit granulation tissue formation

To directly confirm the role of A2AR in angiogenic regulation during wound healing through ECs, we used A2AR-KO mice and A2AR flox+/+ Tek-Cre+ mice (EC-A2AR KO and Tek-Cre+ mice were bred with A2ARflox/flox mice for a generation). Western blot analysis revealed that A2AR protein was not detected in the A2AR-KO mice (Fig. S1A), and its level was slightly lower in the EC-A2AR-KO mice compared with the A2AR flox+/+ Tek-Cre- control (EC-A2AR control) mice (Fig. S1B). Furthermore, immunofluorescence staining revealed that the A2AR protein level in CD31-positive cells was not significantly different between the wild-type (WT) and EC-A2AR control mice in undamaged subcutaneous tissue or in the granulation tissue of the wound at 9 days post-wounding (Fig. S1C and S2). In contrast, A2AR protein was not detected in any cells from the A2AR-KO mice and was found only in CD31-positive cells from the EC-A2AR-KO mice (Fig. S1A-C and S2). Collectively, these results suggest that the A2AR-KO and EC-A2AR-KO mice were successfully established.

Next, we assessed the effects of A2AR-KO and EC-A2AR KO on wound healing using full-thickness excisional wounds as described in a previous experiment26, 45 (Fig. 2A). Comparison of the wound size revealed a clear delay in wound healing in the A2AR-KO and EC-A2AR-KO mice compared with the WT and EC-A2AR control mice, particularly at 9 days postwounding (Fig. 2B). Moreover, there were no significant differences in the rate of wound closure or healing time between the WT and EC-A2AR control mice, while both the A2AR-KO and EC-A2AR-KO mice presented a decreased rate of wound closure and delayed healing time (Fig. 2C-D). Furthermore, there was no significant difference in the rate of wound closure or healing time between the A2AR-KO and EC-A2AR-KO mice (Fig. 2C-D). Collectively, these results demonstrate that the delayed wound healing induced by A2AR KO is due mainly to the endothelial A2AR effect and suggest that A2AR-mediated angiogenesis plays a critical role in wound healing.

Additionally, H&E-stained sections revealed that granulation tissue grew significantly slower in both the A2AR-KO and EC-A2AR-KO mice than in the WT and EC-A2AR control mice at 6 and 9 days post wounding (Fig. 2E-F). Moreover, there was no significant difference in granulation tissue formation between the A2AR-KO and EC-A2AR-KO mice or the WT and EC-A2AR control mice (Fig. 2E-F). Conversely, there was no significant difference in re-epithelialization at 6 and 9 days postwounding in any group, including the WT, A2AR-KO, EC-A2AR control and EC-A2AR-KO mice (Fig. 2E and G). However, there was an obvious decrease in tube-like structures in both A2AR-KO and EC-A2AR-KO mice (Fig. 2E). Taken together, these results suggest that A2AR plays an important role in granulation tissue formation, but not epithelialization, and it is possible that A2AR affects granulation tissue formation mainly by regulating angiogenesis.

Both A2AR KO and EC-A2AR KO decrease angiogenesis in the granulation tissue of full-thickness excisional wounds and the expression of c-Ski

Previous studies have indicated that adenosine receptor-mediated stimulation of angiogenesis promotes wound closure in mice treated with the adenosine receptor agonist CGS21680 and A2AR-KO mice15, 51. We first used EC-A2AR KO mice to examine the vascularity of wounds in total KO, EC-specific A2AR KO and control mice (Fig.3A and S4A). We observed a nearly twofold decrease in CD31-positive cells in both the A2AR-KO and EC-A2AR-KO mice compared with the WT and EC-A2AR control mice at 6 and 9 days postwounding (Fig. 3B-D, S2, S3A-B, S4B), but the differences between the A2AR-KO and EC-A2AR-KO mice or the WT and EC-A2AR control mice were no longer significant (Fig. 3B-D, S2, S4B). Furthermore, the number of CD31-positive cells in the A2AR-KO or EC-A2AR-KO mice decreased to a certain extent between 6 and 9 days postwounding but did not significantly differ (Fig. 3B-D). Additionally, the number of CD31-positive cells in the undamaged subcutaneous tissue of these four groups was not significantly different (Fig. 3B-D). These results suggest that A2AR affects granulation tissue formation mainly by regulating angiogenesis.

To determine whether the deletion of A2AR affects angiogenesis during wound healing through c-Ski, we analyzed the effects of A2AR on the expression of c-Ski in A2AR-KO and EC-A2AR-KO mice (Fig.4A and S5A). Consistent with our previous results39, 42, the level of c-Ski in the wound area was greater than that in the surrounding area (Fig. 4B and S5B), and c-Ski expression significantly increased after injury and was maintained at a high level during the late stages of wound healing (Fig. 4D-E). Furthermore, immunofluorescence revealed that the c-Ski level in CD31-positive cells was greater than that in non-CD31-positive cells at 9 days post wounding (Fig. 4C and S5B). Moreover, c-Ski was colocalized with A2AR in both undamaged subcutaneous tissue and the granulation tissue of the wound (Fig. S5C). In contrast to WT mice, the expression and level of c-Ski were significantly reduced in A2AR-KO mice after wounding (Fig. 4D-E). Immunofluorescence staining further confirmed that the c-Ski protein level in CD31-positive cells was not significantly different between the WT and EC-A2AR control mice (Fig. 4F-G), whereas c-Ski protein expression was significantly decreased in all cells of the A2AR-KO mice and was found only in CD3-positive cells of the EC-A2AR-KO mice (Fig. 4F-G and S6). Collectively, these results suggest that A2AR regulates angiogenesis through c-Ski.

A2AR activation induces a proangiogenic effect, as shown by the application of an A2AR agonist in vitro

After confirming the essential role of angiogenesis in A2A R deficiency-mediated impairment of wound healing involving c-Ski, we further verified the proangiogenic effect of A2A R in vitro, laying the foundation for future exploration of the role of c-Ski in this effect. Previous studies have indicated that adenosine, which acts through A2AR, promotes angiogenesis in pulmonary ECs52-54, vascular ECs55, 56, retinal ECs57, umbilical vein ECs58, lymphatic ECs59, and human EA.hy926 ECs60. However, the activation of A2AR does not affect angiogenesis in bone fracture medium55 or human lung microvascular ECs61. We used HMECs, a critical component of granulation tissue involved in wound healing, to verify the proangiogenic effect induced by A2AR activation via cell proliferation, migration and tube formation assays, which are often used to evaluate angiogenesis in vitro53, 62, 63. Immunofluorescence staining revealed that A2AR was expressed in HMECs and exhibited a hole-like morphology (Fig. 5A). Moreover, application of the A2AR agonist CGS21680 promoted cell proliferation in a dose-dependent manner (Fig. 5B). In the scratch assay, cell migration was obviously greater in the CGS21680 group than in the DMEM and DMSO control groups (Fig. 5C). Moreover, CGS21680 treatment resulted in a significant increase in the tube formation of HMECs compared with that of the DMEM and DMSO control groups in angiogenesis assays in vitro (Fig. 5D-F). Consistent with other studies53, 54, 56, 64, the activation of A2AR by CGS21680 significantly promoted the proliferation, migration and angiogenesis of HMECs.

A2AR regulates angiogenesis by regulating c-Ski, as shown by RNAi of c-Ski expression in HMECs

To determine whether c-Ski is required for A2AR-mediated angiogenesis, we used CGS21680 to activate A2AR and RNAi to inhibit the expression of c-Ski in HMECs. Transfection with sh c-Ski successfully reduced the protein level of c-Ski in a dose-dependent manner (Fig. S8A-B), and CGS21680 treatment increased the c-Ski protein level in a dose-dependent manner (Fig. 6A). Immunofluorescence staining revealed that the c-Ski protein level increased after CGS21680 treatment, and c-Ski clearly aggregated in the nucleus (Figs. 6B and S7A). Interestingly, the c-Ski protein level was greater in dividing cells (Fig. S7B), suggesting an important role of c-Ski in cell proliferation. RNAi not only significantly reduced the level of the c-Ski protein compared with that of the control but also significantly reduced the increase in the c-Ski protein induced by CGS21680 treatment (Fig. 6C). Similarly, c-Ski depletion reduced the proliferation and tube formation of HMECs and abolished the effects of CGS21680 treatment on promoting cell proliferation and tube formation (Fig. 6D-F). Overall, these cell proliferation and tube formation assays strongly indicate that c-Ski plays an essential role in A2AR-mediated angiogenesis in HMECs.

A2AR regulates the expression of the c-Ski protein through the ERK/CREB pathway in HMECs

To characterize the underlying mechanisms by which A2AR regulates the expression of the c-Ski protein, we surveyed the ERK/CREB signaling pathway. As shown in Fig. 7A-B, CGS21680 treatment significantly increased the PKA, ERK and CREB phosphorylation levels in HMECs. Similar results were obtained by immunofluorescence staining at 24 h after CGS21680 treatment, indicating that activation of A2AR by CGS21680 can enhance the PKA/ERK/CREB signaling pathways in HMECs (Fig. 7C-F).

To further verify that A2AR regulates c-Ski protein expression through the ERK/CREB pathway in HMECs, we used CGS21680 to activate A2AR and the MEK1/2 inhibitor PD9805940, 65 to inhibit ERK activity in HMECs. Compared with the lack of effect on p-PKA (Fig. 8A-B), PD98059 alone significantly reduced p-ERK, p-CREB and c-Ski levels and abrogated the CGS21680 treatment-induced increases in p-ERK, p-CREB and c-Ski levels (Fig. 8A and C-E). Similarly, immunofluorescence staining revealed that PD98059 not only decreased the fluorescence intensity of c-Ski but also abrogated the CGS21680 treatment-induced increase in the fluorescence intensity of c-Ski (Fig. 8F). Moreover, compared with DMSO, PD98059 not only decreased the tube formation of HMECs but also abolished the CGS21680 treatment-induced increase in the tube formation of HMECs in angiogenesis assays in vitro (Fig.8G-H). Taken together, these results suggest that A2AR regulates c-Ski protein expression in HMECs through the ERK/CREB pathway.

Discussion

Some studies have shown that A2AR is expressed on most cell types involved in wound healing, including macrophages, fibroblasts and microvascular ECs, in vitro8, 18, 61, 66. We first explored the types of expression in these repair-associated cells in vivo. According to the histochemical results of full-thickness excisional wounds, A2A R was expressed on most repair cells, including epidermal cells, ECs, macrophages and fibroblasts (Fig. 1), suggesting that it regulates wound healing. Interestingly, the increase was greater in the granulation tissue of the wound than in the epidermis relative to the undamaged side, suggesting that regulating granulation tissue formation is more important than re-epithelialization in A2AR regulation of wound healing, which is consistent with the findings of some studies15, 54. Importantly, the peak expression level of A2AR during wound healing occurred at 6 days postwounding (Fig. 1), which is also the fast period of wound healing (Fig. 2)67-69. In contrast, A2AR protein levels are lower at 7 days postwounding than at 3 days during the healing of refractory wounds in diabetic mice70. Taken together, these results suggest that A2AR may have a wound-promoting effect. A2AR agonists can promote skin wound healing and radiation-impaired wound healing8-13, whereas A2AR gene knockout delays the course of skin wound healing10, 15, 71, which also indicates the promotion of healing by A2AR.

Similar to the findings of a previous experiment15, 54, our study revealed that A2AR KO delayed wound healing compared with WT mice. Importantly, there was no significant difference in the rate of wound closure or healing time between the A2AR-KO and EC-A2AR-KO mice, suggesting that the effects of A2AR on wound healing are mainly mediated through endothelial cells. However, the mechanism by which A2AR regulates wound healing has not been established and is likely multifactorial, as it affects inflammation15, 16, fibroblasts and epithelial cells 13, 17, 18. On the one hand, these findings regarding the effects on fibroblasts and epithelial cells are based on the use of agonists and in vitro experiments2, 17, 72, 73, and there are specific concerns about the effects of agonists and their widespread regulation compared with those of exogenous substances. Moreover, regarding the effect of A2AR on fibroblasts, researchers have focused mainly on its role in fibrotic diseases71, 74. On the other hand, for the regulation of inflammatory cells, an induced deficiency in A2AR is not associated with a defect in the acute inflammatory response or with a difference in the number or functional capacity of A2AR-deficient leukocytes15, and convincing in vivo experimental evidence for a role of A2AR in wound healing through inflammatory reactions is lacking75. In addition, some research has shown that A2AR regulates inflammation through ECs76. Thus, A2AR's effects on ECs play a critical role in the regulation of wound healing. However, further studies are needed to determine the role of A2AR in other cells during wound healing.

Interestingly, we also found that the deficit in granulation tissue was obvious at 6 and 9 days postwounding, whereas the extent of re-epithelialization did not differ between the WT controls and the A2AR KO mice, which is consistent with the findings of a previous experiment15, 54. Moreover, EC-A2AR KO mice presented similar results and were not significantly different from A2AR KO mice, strongly suggesting that A2AR regulates granulation tissue formation, but not epithelialization, during wound healing. Previous studies have indirectly demonstrated that A2A R is involved in the angiogenic effects of ECs by total A2AR KO or the application of agonists or antagonists13, 15, 51, which critically affects the formation of granulation tissue, and these results are consistent with the observed defects in granulation tissue formation. Importantly, our study directly demonstrated that A2AR affects granulation tissue formation mainly by regulating angiogenesis by using EC-A2AR KO mice, further indicating that the angiogenic effects of A2AR on ECs play a critical role in the regulation of wound healing. Similarly, Liu et al. demonstrated that A2AR is crucial for pathological angiogenesis in proliferative retinopathies using EC-A2AR-KO mice57.

Angiogenesis promotes growth, development, and wound healing through the formation of granulation tissue77. Among the many cells involved in the angiogenic process, ECs play an essential role in new vessel formation via an increase in cell proliferation, migration, and tube formation capacity78. Although cell proliferation, migration, and tube formation capacity were assessed in vitro in this study, only the number of CD31-positive cells was determined in vivo. In future experiments, we should use other parameters to clarify this process in vivo. In addition to the above effects, further exploration is needed to determine whether A2AR regulates angiogenesis through other mechanisms or can regulate angiogenesis by influencing other cellular effects (for example, inflammatory cells).

However, previous reports have shown that the angiogenic effect of A2AR activation is likely mediated by increased expression of VEGF in ECs79-81. Other studies have also reported that A2AR stimulation increases angiogenesis through the antiangiogenic matrix proteins thrombospondin 164, annexin A256, and MyD8816 and macrophage exosomes55.

We demonstrated that A2AR regulates angiogenesis through c-Ski. c-Ski was significantly expressed on ECs during wound healing and was significantly decreased after A2AR KO and EC-A2AR KO. A2AR activation induced a proangiogenic effect and increased the expression of c-Ski, whereas c-Ski depletion abolished the proangiogenic effects of A2AR activation. In addition, similar to our previous research26, 82, the expression of c-Ski increased after wounding, peaked at 6 days and then significantly decreased, which is consistent with the changes in A2AR expression after wounding. Furthermore, c-Ski is expressed on ECs30, and reduced c-Ski expression in the wound results in significantly slower wound healing26, similar to the results of A2A R KO. Taken together, these results strongly indicate that c-Ski plays an essential role in A2AR-mediated angiogenesis. More importantly, c-Ski depletion reduced the proliferation and tube formation of HMECs and abolished the promotive effects on cell proliferation and tube formation caused by A2AR activation in vitro, further demonstrating the important role of c-Ski in A2AR-mediated angiogenesis.

Although there is little research on the role of c-Ski in angiogenesis or EC regulation, as a multieffector factor, this molecule can regulate the proliferation and migration of fibroblasts83, vascular smooth muscle cells84, cardiomyocytes85, astrocytes86, and tumor cells87, 88. These results indicate that c-Ski may regulate angiogenesis through these effects. Moreover, the overexpression of c-Ski was shown to promote the proliferation of ECs31, 32. In addition, an analysis of the regulated signal transduction pathway revealed that c-Ski not only synergizes with factors such as activator protein-1 (AP-1)89 and signal transducer and activator of transcription 3 (STAT-3)90, which upregulate the expression of the essential angiogenic factor VEGF91, 92, but can also reduce the expression of the antiangiogenic factor thrombospondin-1 (TSP-1)93, suggesting that c-Ski plays an important role in angiogenesis. Interestingly, A2AR can regulate angiogenesis not only through VEGF18,19,20 but also through TSP164, further indicating that A2AR can regulate angiogenesis through c-Ski. Nevertheless, the mechanism of the proangiogenic effect of c-Ski remains to be further explored.

As reported in previous studies, the ERK/CREB pathway is an important pathway for regulating c-Ski expression40. Our study revealed that MEK inhibition markedly reduced c-Ski expression and abrogated A2AR-mediated promotion of c-Ski expression with CGS21680 in HMECs, indicating that A2AR regulates c-Ski protein expression in HMECs through the ERK1/2 pathway. Our results demonstrated that c-Ski expression is regulated by A2AR in ECs through this pathway, and the results of other studies support this finding. Several studies have demonstrated that the ERK pathway is important in angiogenesis94, 95 and A2AR activation can activate the ERK pathway in ECs52, 96-98. In particular, Liu et al. reported that A2AR activation promotes pathological angiogenesis via ERK-dependent translational activation57. In addition, our study revealed that A2AR activation by CGS21680 enhanced the ERK1/2 pathway through PKA, which is consistent with the findings of several previous reports99, 100. However, further in vivo experimental verification of A2AR activation inducing the expression of c-Ski through the ERK/CREB pathway is needed.

Conclusions

Taken together, our results demonstrate that the delayed effects of A2AR KO on wound healing are due mainly to the effects of A2AR on endothelial cells, indicating that A2AR-mediated angiogenesis plays a critical role in wound healing. Moreover, our results demonstrate a novel cellular and molecular mechanism whereby endothelial A2AR deficiency delays wound healing and decreases angiogenesis through c-Ski deficiency. A2AR regulates the expression of the c-Ski protein through the ERK/CREB pathway in HMECs. Thus, these findings provide new insights into a previously unrecognized effect of A2AR on angiogenesis by ECs in wounds and highlight the mechanism of A2AR in wound repair, which not only identifies A2AR as a therapeutic target in wound repair but also highlights other angiogenesis-dependent tissue repair processes.

Supplementary Material

Supplementary figures.

We would like to thank AJE (www.aje.com) for English language editing.

Funding

This work was supported by the National Natural Science Foundation of China (Grant No: 81701915) and the projects of Chongqing (cstc2021yszx-jcyjX0001).

Author contributions

PL, YP, and YZ designed the experiments; YP, RX, XC, and BW performed the experiments and acquired the data; YZ, BW, YN, YZ, NY, JZ, and CL reviewed and interpreted the data; PL and YP wrote the first draft of the paper; and all of the authors contributed to the final manuscript before submission.

Availability of data and materials

Raw data supporting the conclusions of this article are available from the corresponding author upon reasonable request.

Abbreviations

A2AR adenosine A2A receptor

ECs endothelial cells

c-Ski Cellular Sloan-Kettering Institute

A2AR KO A2AR knockout

EC-A2AR KO EC-specific A2AR knockout

HMECs human microvascular ECs

ERK1/2 signal-regulated kinase 1/2

WT wild-type

H&E hematoxylin and eosin

CGS21680 2-p-[2-carboxyethyl]phenethyl-amino-5'-N-ethylcarboxamido-adenosine

DMSO dimethyl sulfoxide

AP-1 activator protein-1

STAT-3 signal transducer and activator of transcription 3

TSP-1 thrombospondin-1

FBS fetal bovine serum

PBS phosphate-buffered saline

IntDen integrated density

STR short tandem repeat

HBSS Hank's balanced salt solution

OD optical density

Figure 1 Localization and expression of A2AR in full-thickness excisional wounds of WT mice. (A) Experimental procedure. (B) A2AR levels were detected by western blotting at 0, 6, 9 and 15 days postwounding. **p < 0.01, *p <0.05 (n=3); NS, not significant. Immunohistochemistry for A2AR (red, white arrow) in the wound and surrounding area at 9 days postwounding (C) and its quantitative analysis (D) in WT mice. **p < 0.01 (n=5); NS, not significant. The lower panel shows higher magnification sections of the white squares in the upper panel. Scale bar, 50 µm. Immunohistochemistry for CD31 (E), vimentin (F), CD11b (G), F4/80 (H) (green) and A2AR (red) in wound granulation tissue at 9 days postwounding in WT mice. The right panel shows higher magnification sections of the white squares on the left, and the white arrow indicates that the green fluorescent-labeled cells colocalized with the red fluorescent-labeled cells. Short scale bar, 50 µm; long scale bar, 50 µm.

Figure 2 Wound healing and histopathological characteristics of A2AR KO and EC-A2AR KO mice after full-thickness wounding. (A) Experimental procedure. (B) Photographs of representative wounds of mice at 0, 3 and 9 days postwounding. (C) Wound healing time for each experimental and control group (n = 7 for each group), **p < 0.01. (D) Time course of wound closure for each experimental and control group (n = 9 for each group). **p <0.01, compared with the appropriate controls; ##p <0.01, compared with the previous adjacent time point. (E) Histopathological observation of wound healing at 6 and 9 days postwounding. Short scale bar, 200 µm; long scale bar, 50 µm. (F) Quantitative analysis of granulation tissue thickness at 6 and 9 days postwounding. **p < 0.01 (n = 5); NS, not significant. (G) Quantitative analysis of the crawling distance at 6 and 9 days postwounding; **p < 0.01 (n = 5); NS, not significant.

Figure 3 Changes in angiogenesis in A2AR KO and EC-A2AR KO mice after full-thickness wounding. (A) Experimental procedure. (B) Immunohistochemistry for CD31 in A2AR KO and EC-A2AR KO mice at 6 and 9 days postwounding. Scale bar, 50 µm. (C) Quantitative analysis of CD31-positive cells in each group. **p < 0.01 (n = 3); NS, not significant.

Figure 4 The expression of c-Ski in A2AR KO and EC-A2AR KO mice after full-thickness wounding. (A) Experimental procedure. (B) Immunohistochemistry for c-Ski (green, white arrow) in the granulation tissue of the wound and subcutaneous tissue of the surrounding wound at 9 days postwounding. The lower panel shows higher magnification sections of the black squares in the upper panel. Scale bar, 50 µm. (C) Double-label immunofluorescence for c-Ski (red) and CD31 (green) in the granulation tissue of the wound at 9 days postwounding revealed the colocalization of c-Ski and CD31 (white arrow). The surrounding area is a higher magnification section of the white square. Short scale bar, 50 µm; long scale bar, 50 µm. Expression of c-Ski detected by (D) real-time PCR (n = 9) and (E) western blot analysis (n = 3) at 0, 6, 9 and 15 days postwounding. **p < 0.01; NS, not significant. (F) Double-label immunofluorescence for c-Ski (red) and CD31 (green) in the granulation tissue of the wound at 9 days postwounding in each group. The white arrow indicates the colocalization of c-Ski and CD31. Scale bar, 50 µm. (G) Quantitative analysis of (F). **p < 0.01 (n=5); NS, not significant.

Figure 5 Changes in the proangiogenic effect in HMECs by the application of an A2AR agonist in vitro. (A) Immunohistochemistry of A2AR (red) in HMECs. Scale bar, 50 µm. (B) Cell proliferation was measured by the CCK-8 assay 24 h after treatment with different concentrations of CGS21680. **p < 0.01 (n=9); NS, not significant. (C) Representative images of cell migration evaluated by scratch wound healing assays at 0, 24, 48 and 72 h after CGS21680 treatment. Scale bar, 50 µm. (D) Representative images of tube formation 24 h after treatment with CGS21680. Scale bar, 50 µm. (E) Quantitative analysis of (D). **p < 0.01 (n=5); NS, not significant.

Figure 6 Effects of c-Ski on the proangiogenic effect of A2AR activation in HMECs. (A) Levels of c-Ski detected by western blots 24 h after treatment with different concentrations of CGS21680. ** p < 0.01, ##p <0.01, compared with the previous adjacent group (n = 3); NS, not significant. (B) Immunohistochemistry for c-Ski (green) in HMECs 24 h after CGS21680 treatment. Scale bar, 50 µm. (C) Changes in the CGS21680-induced increase in the intracellular protein level of c-Ski in HMECs after 24 h of c-Ski RNAi. ** p < 0.01, *p <0.05 (n = 3); NS, not significant. (D) Changes in CGS21680-induced cell proliferation for 24 h in HMECs after c-Ski RNAi. ** p < 0.01 (n = 9); NS, not significant. (E) Representative images of CGS21680-induced tube formation in HMECs after c-Ski RNAi treatment for 24 h. Scale bar, 50 µm. (F) Quantitative analysis of (E). **p < 0.01, *p <0.05 (n=5); NS, not significant.

Figure 7 Changes in the ERK/CREB signaling pathways after A2AR activation in HMECs. (A) Representative immunoblot images of the levels of PKA, p-PKA, ERK, p-ERK, CREB and p-CREB in HMECs 24 h after treatment with CGS21680. (B) Quantitative analysis of (A). **p < 0.01 (n=3); Immunohistochemistry for p-PKA (C), p-ERK (D) and p-CREB (E) in HMECs 24 h after treatment with CGS21680. Scale bar, 50 µm. (F) Quantitative analysis of (C-E). **p < 0.01, *p <0.05 (n=5).

Figure 8 Effects of the ERK/CREB signaling pathways on A2AR-induced expression of c-Ski in HMECs. Representative immunoblot images of the CGS21680-induced changes in the intracellular protein levels of PKA, p-PKA, ERK, p-ERK, CREB and p-CREB in HMECs after ERK/CREB inhibition. (B-E) Quantitative analysis of (A). **p < 0.01 (n=3); NS, not significant. (F) Immunohistochemistry for c-Ski (green) induced by treatment with CGS21680 in HMECs after ERK/CREB inhibition. Scale bar, 50 µm. (G) Representative images of tube formation 24 h after treatment with CGS21680. Scale bar, 50 µm. (H) Quantitative analysis of (G). **p < 0.01 (n=5); NS, not significant.
==== Refs
1 Hoque J Zeng Y Newman H Gonzales G Lee C Varghese S Microgel-Assisted Delivery of Adenosine to Accelerate Fracture Healing ACS Biomater Sci Eng 2022 8 4863 72 36266245
2 Kim J Shin JY Choi YH Lee SY Jin MH Kim CD Adenosine and Cordycepin Accelerate Tissue Remodeling Process through Adenosine Receptor Mediated Wnt/beta-Catenin Pathway Stimulation by Regulating GSK3b Activity Int J Mol Sci 2021 22 5571 34070360
3 Cheng X Yin C Deng Y Li Z Exogenous adenosine activates A2A adenosine receptor to inhibit RANKL-induced osteoclastogenesis via AP-1 pathway to facilitate bone repair Mol Biol Rep 2022 49 2003 14 34846650
4 Shaikh G Cronstein B Signaling pathways involving adenosine A2A and A2B receptors in wound healing and fibrosis Purinergic Signal 2016 12 191 7 26847815
5 Ojeh N Stojadinovic O Pastar I Sawaya A Yin N Tomic-Canic M The effects of caffeine on wound healing Int Wound J 2016 13 605 13 25041108
6 Bezerra JP de Siqueira A Pires AG Marques MR Duarte PM Bastos MF Effects of estrogen deficiency and/or caffeine intake on alveolar bone loss, density, and healing: a study in rats J Periodontol 2013 84 839 49 22873654
7 Supit T Susilaningsih N Prasetyo A Najatullah Effects of Caffeine Consumption on Autologous Full-Thickness Skin Graft Healing in an Animal Model Indian J Plast Surg 2021 54 314 20 34667517
8 Valls MD Cronstein BN Montesinos MC Adenosine receptor agonists for promotion of dermal wound healing Biochem Pharmacol 2009 77 1117 24 19041853
9 Montesinos MC Desai-Merchant A Cronstein BN Promotion of Wound Healing by an Agonist of Adenosine A2A Receptor Is Dependent on Tissue Plasminogen Activator Inflammation 2015 38 2036 41 25991438
10 Dai SS Xiong RP Yang N Li W Zhu PF Zhou YG [Different effects of adenosine A2A receptors in the models of traumatic brain injury and peripheral tissue injury] Sheng Li Xue Bao 2008 60 254 8 18425315
11 Victor-Vega C Desai A Montesinos MC Cronstein BN Adenosine A2A receptor agonists promote more rapid wound healing than recombinant human platelet-derived growth factor (Becaplermin gel) Inflammation 2002 26 19 24 11936752
12 Altavilla D Squadrito F Polito F Irrera N Calo M Lo Cascio P Activation of adenosine A2A receptors restores the altered cell-cycle machinery during impaired wound healing in genetically diabetic mice Surgery 2011 149 253 61 20570301
13 Montesinos MC Gadangi P Longaker M Sung J Levine J Nilsen D Wound healing is accelerated by agonists of adenosine A2 (G alpha s-linked) receptors J Exp Med 1997 186 1615 20 9348321
14 Galeano M Pallio G Irrera N Mannino F Bitto A Altavilla D Polydeoxyribonucleotide: A Promising Biological Platform to Accelerate Impaired Skin Wound Healing Pharmaceuticals (Basel) 2021 14 1103 34832885
15 Montesinos MC Desai A Chen JF Yee H Schwarzschild MA Fink JS Adenosine promotes wound healing and mediates angiogenesis in response to tissue injury via occupancy of A(2A) receptors Am J Pathol 2002 160 2009 18 12057906
16 Macedo L Pinhal-Enfield G Alshits V Elson G Cronstein BN Leibovich SJ Wound healing is impaired in MyD88-deficient mice: a role for MyD88 in the regulation of wound healing by adenosine A2A receptors Am J Pathol 2007 171 1774 88 17974599
17 Allen-Gipson DS Wong J Spurzem JR Sisson JH Wyatt TA Adenosine A2A receptors promote adenosine-stimulated wound healing in bronchial epithelial cells Am J Physiol Lung Cell Mol Physiol 2006 290 L849 55 16361356
18 Perez-Aso M Fernandez P Mediero A Chan ES Cronstein BN Adenosine 2A receptor promotes collagen production by human fibroblasts via pathways involving cyclic AMP and AKT but independent of Smad2/3 FASEB J 2014 28 802 12 24200882
19 Pearson-White S Deacon D Crittenden R Brady G Iscove N Quesenberry PJ The ski/sno protooncogene family in hematopoietic development Blood 1995 86 2146 55 7662963
20 Berk M Desai SY Heyman HC Colmenares C Mice lacking the ski proto-oncogene have defects in neurulation, craniofacial, patterning, and skeletal muscle development Genes Dev 1997 11 2029 39 9284043
21 Atanasoski S Notterpek L Lee HY Castagner F Young P Ehrengruber MU The protooncogene Ski controls Schwann cell proliferation and myelination Neuron 2004 43 499 511 15312649
22 Fumagalli S Doneda L Nomura N Larizza L Expression of the c-ski proto-oncogene in human melanoma cell lines Melanoma Res 1993 3 23 7 8471834
23 Rashidian J Le Scolan E Ji X Zhu Q Mulvihill MM Nomura D Ski regulates Hippo and TAZ signaling to suppress breast cancer progression Sci Signal 2015 8 ra14 25670202
24 Soeta C Suzuki M Suzuki S Naito K Tachi C Tojo H Possible role for the c-ski gene in the proliferation of myogenic cells in regenerating skeletal muscles of rats Dev Growth Differ 2001 43 155 64 11284965
25 Macias-Silva M Li W Leu JI Crissey MA Taub R Up-regulated transcriptional repressors SnoN and Ski bind Smad proteins to antagonize transforming growth factor-beta signals during liver regeneration J Biol Chem 2002 277 28483 90 12023281
26 Li P Liu P Xiong RP Chen XY Zhao Y Lu WP Ski, a modulator of wound healing and scar formation in the rat skin and rabbit ear J Pathol 2011 223 659 71 21341267
27 Zhai Y Ye SY Wang QS Xiong RP Fu SY Du H Overexpressed ski efficiently promotes neurorestoration, increases neuronal regeneration, and reduces astrogliosis after traumatic brain injury Gene Ther 2022 30 75 87 35132206
28 Liao HY Da CM Wu ZL Zhang HH Ski: Double roles in cancers Clin Biochem 2021 87 1 12 33188772
29 Bonnon C Atanasoski S c-Ski in health and disease Cell Tissue Res 2012 347 51 64 21647564
30 Wang J Li H Lv Z Luo X Deng W Zou T The miR-214-3p/c-Ski axis modulates endothelial-mesenchymal transition in human coronary artery endothelial cells in vitro and in mice model in vivo Hum Cell 2022 35 486 97 34978047
31 Sun B Huang Z Yang H Zhao X MicroRNA-195-5p inhibits the progression of hemangioma via targeting SKI Exp Ther Med 2022 23 165 35069846
32 O TM Tan M Tarango M Fink L Mihm M Ma Y Differential expression of SKI oncogene protein in hemangiomas Otolaryngol Head Neck Surg 2009 141 213 8 19643254
33 Li X Sun C Chen J Ma JF Pan YH ERK-CREB pathway is involved in HSPB8-mediated glioma cell growth and metastatic properties Exp Mol Pathol. 2021: 104653
34 Rao J Li H Zhang H Xiang X Ding X Li L Periplaneta Americana (L.) extract activates the ERK/CREB/BDNF pathway to promote post-stroke neuroregeneration and recovery of neurological functions in rats J Ethnopharmacol 2024 321 117400 37952730
35 Xin Y Roh K Cho E Park D Whang W Jung E Isookanin Inhibits PGE(2)-Mediated Angiogenesis by Inducing Cell Arrest through Inhibiting the Phosphorylation of ERK1/2 and CREB in HMEC-1 Cells Int J Mol Sci 2021 22 6466 34208772
36 Zhang Y Zheng D Zhou T Song H Hulsurkar M Su N Androgen deprivation promotes neuroendocrine differentiation and angiogenesis through CREB-EZH2-TSP1 pathway in prostate cancers Nat Commun 2018 9 4080 30287808
37 Trinh PNH Baltos JA Hellyer SD May LT Gregory KJ Adenosine receptor signalling in Alzheimer's disease Purinergic Signal 2022 18 359 81 35870032
38 Fredholm BB Chern Y Franco R Sitkovsky M Aspects of the general biology of adenosine A2A signaling Prog Neurobiol 2007 83 263 76 17804147
39 Li P Rial D Canas PM Yoo JH Li W Zhou X Optogenetic activation of intracellular adenosine A2A receptor signaling in the hippocampus is sufficient to trigger CREB phosphorylation and impair memory Mol Psychiatry 2015 20 1339 49 25687775
40 Li P Liu P Peng Y Zhang ZH Li XM Xiong RP The ERK/CREB pathway is involved in the c-Ski expression induced by low TGF-beta1 concentrations during primary fibroblast proliferation Cell Cycle 2018 17 1319 28 29950153
41 Chen JF Huang Z Ma J Zhu J Moratalla R Standaert D A(2A) adenosine receptor deficiency attenuates brain injury induced by transient focal ischemia in mice J Neurosci 1999 19 9192 200 10531422
42 Xu Y Ning Y Zhao Y Peng Y Luo F Zhou Y Caffeine Functions by Inhibiting Dorsal and Ventral Hippocampal Adenosine 2A Receptors to Modulate Memory and Anxiety, Respectively Front Pharmacol 2022 13 807330 35185566
43 Zeng Q Wu Z Duan H Jiang X Tu T Lu D Impaired tumor angiogenesis and VEGF-induced pathway in endothelial CD146 knockout mice Protein Cell 2014 5 445 56 24756564
44 Wang Z Chai Q Zhu M Differential Roles of LTbetaR in Endothelial Cell Subsets for Lymph Node Organogenesis and Maturation J Immunol 2018 201 69 76 29760194
45 Peng Y Li P Zhao ZA Chen L Zhao XG Chen X Comparative evaluation of the wound-healing potency of recombinant bFGF and ski gene therapy in rats Growth Factors 2016 34 119 27 27418111
46 Zhang ZH Xu YW Peng Y Chen X Li P Zhou YG Expression of a short antibody heavy chain peptide effectively antagonizes adenosine 2A receptor in vitro and in vivo Expert Opin Ther Targets 2020 24 707 17 32308059
47 Peng Y Xiong RP Zhang ZH Ning YL Zhao Y Tan SW Ski promotes proliferation and inhibits apoptosis in fibroblasts under high-glucose conditions via the FoxO1 pathway Cell Prolif 2021 54 e12971 33349993
48 Li P Wang QS Zhai Y Xiong RP Chen X Liu P Ski mediates TGF-beta1-induced fibrosarcoma cell proliferation and promotes tumor growth J Cancer 2020 11 5929 40 32922535
49 Jian L Mei Y Xing C Rongdi Y Haem relieves hyperoxia-mediated inhibition of HMEC-1 cell proliferation, migration and angiogenesis by inhibiting BACH1 expression BMC Ophthalmol 2021 21 104 33632168
50 Zudaire E Gambardella L Kurcz C Vermeren S A computational tool for quantitative analysis of vascular networks PLoS One 2011 6 e27385 22110636
51 Montesinos MC Shaw JP Yee H Shamamian P Cronstein BN Adenosine A(2A) receptor activation promotes wound neovascularization by stimulating angiogenesis and vasculogenesis Am J Pathol 2004 164 1887 92 15161625
52 Ahmad A Schaack JB White CW Ahmad S Adenosine A2A receptor-dependent proliferation of pulmonary endothelial cells is mediated through calcium mobilization, PI3-kinase and ERK1/2 pathways Biochem Biophys Res Commun 2013 434 566 71 23583199
53 Ahmad A Ahmad S Glover L Miller SM Shannon JM Guo X Adenosine A2A receptor is a unique angiogenic target of HIF-2alpha in pulmonary endothelial cells Proc Natl Acad Sci U S A 2009 106 10684 9 19541651
54 Troncoso F Herlitz K Acurio J Aguayo C Guevara K Castro FO Advantages in Wound Healing Process in Female Mice Require Upregulation A(2A)-Mediated Angiogenesis under the Stimulation of 17beta-Estradiol Int J Mol Sci 2020 21 7145 32998232
55 Wang D Wang J Zhou J Zheng X The Role of Adenosine Receptor A2A in the Regulation of Macrophage Exosomes and Vascular Endothelial Cells During Bone Healing J Inflamm Res 2021 14 4001 17 34429631
56 Valls MD Soldado M Arasa J Perez-Aso M Williams AJ Cronstein BN Annexin A2-Mediated Plasminogen Activation in Endothelial Cells Contributes to the Proangiogenic Effect of Adenosine A(2A) Receptors Front Pharmacol 2021 12 654104 33986681
57 Liu Z Yan S Wang J Xu Y Wang Y Zhang S Endothelial adenosine A2a receptor-mediated glycolysis is essential for pathological retinal angiogenesis Nat Commun 2017 8 584 28928465
58 Escudero C Roberts JM Myatt L Feoktistov I Impaired adenosine-mediated angiogenesis in preeclampsia: potential implications for fetal programming Front Pharmacol 2014 5 134 24926270
59 Allard B Cousineau I Allard D Buisseret L Pommey S Chrobak P Adenosine A2a receptor promotes lymphangiogenesis and lymph node metastasis Oncoimmunology 2019 8 1601481 31413909
60 Bonyanian Z Walker M Du Toit E Rose'Meyer RB Multiple adenosine receptor subtypes stimulate wound healing in human EA.hy926 endothelial cells Purinergic Signal 2019 15 357 66 31254200
61 Feoktistov I Goldstein AE Ryzhov S Zeng D Belardinelli L Voyno-Yasenetskaya T Differential expression of adenosine receptors in human endothelial cells: role of A2B receptors in angiogenic factor regulation Circ Res 2002 90 531 8 11909816
62 Ling X Jiang X Li Y Han W Rodriguez M Xu Z Sequential Treatment of Bioresponsive Nanoparticles Elicits Antiangiogenesis and Apoptosis and Synergizes with a CD40 Agonist for Antitumor Immunity ACS Nano 2021 15 765 80 33347262
63 Wilhelm EN Gonzalez-Alonso J Parris C Rakobowchuk M Exercise intensity modulates the appearance of circulating microvesicles with proangiogenic potential upon endothelial cells Am J Physiol Heart Circ Physiol 2016 311 H1297 H310 27638881
64 Desai A Victor-Vega C Gadangi S Montesinos MC Chu CC Cronstein BN Adenosine A2A receptor stimulation increases angiogenesis by down-regulating production of the antiangiogenic matrix protein thrombospondin 1 Mol Pharmacol 2005 67 1406 13 15673602
65 Huang K Chen Y Zhang R Wu Y Ma Y Fang X Honokiol induces apoptosis and autophagy via the ROS/ERK1/2 signaling pathway in human osteosarcoma cells in vitro and in vivo Cell Death Dis 2018 9 157 29410403
66 Khoa ND Montesinos MC Reiss AB Delano D Awadallah N Cronstein BN Inflammatory cytokines regulate function and expression of adenosine A(2A) receptors in human monocytic THP-1 cells J Immunol 2001 167 4026 32 11564822
67 Scrima M Melito C Merola F Iorio A Vito N Giori AM Evaluation of Wound Healing Activity of Salvia haenkei Hydroalcoholic Aerial Part Extract on in vitro and in vivo Experimental Models Clin Cosmet Investig Dermatol 2020 13 627 37
68 Brett E Zielins ER Chin M Januszyk M Blackshear CP Findlay M Isolation of CD248-expressing stromal vascular fraction for targeted improvement of wound healing Wound Repair Regen 2017 25 414 22 28464475
69 Milan PB Lotfibakhshaiesh N Joghataie MT Ai J Pazouki A Kaplan DL Accelerated wound healing in a diabetic rat model using decellularized dermal matrix and human umbilical cord perivascular cells Acta Biomater 2016 45 234 46 27591919
70 Zhu W Dong Y Xu P Pan Q Jia K Jin P A composite hydrogel containing resveratrol-laden nanoparticles and platelet-derived extracellular vesicles promotes wound healing in diabetic mice Acta Biomater 2022 154 212 30 36309190
71 Chan ES Fernandez P Merchant AA Montesinos MC Trzaska S Desai A Adenosine A2A receptors in diffuse dermal fibrosis: pathogenic role in human dermal fibroblasts and in a murine model of scleroderma Arthritis Rheum 2006 54 2632 42 16871530
72 Tian Z Zhang H Dixon J Traphagen N Wyatt TA Kharbanda K Cigarette Smoke Impairs A(2A) Adenosine Receptor Mediated Wound Repair through Up-regulation of Duox-1 Expression Sci Rep 2017 7 44405 28337995
73 Dignass AU Becker A Spiegler S Goebell H Adenine nucleotides modulate epithelial wound healing in vitro Eur J Clin Invest 1998 28 554 61 9726036
74 Zhang J Corciulo C Liu H Wilder T Ito M Cronstein B Adenosine A(2a) Receptor Blockade Diminishes Wnt/beta-Catenin Signaling in a Murine Model of Bleomycin-Induced Dermal Fibrosis Am J Pathol 2017 187 1935 44 28667836
75 Pasquini S Contri C Borea PA Vincenzi F Varani K Adenosine and Inflammation: Here, There and Everywhere Int J Mol Sci 2021 22 7685 34299305
76 Sands WA Martin AF Strong EW Palmer TM Specific inhibition of nuclear factor-kappaB-dependent inflammatory responses by cell type-specific mechanisms upon A2A adenosine receptor gene transfer Mol Pharmacol 2004 66 1147 59 15286208
77 Mendez-Valdes G Gomez-Hevia F Lillo-Moya J Gonzalez-Fernandez T Abelli J Cereceda-Cornejo A Endostatin and Cancer Therapy: A Novel Potential Alternative to Anti-VEGF Monoclonal Antibodies Biomedicines 2023 11 718 36979697
78 Stryker ZI Rajabi M Davis PJ Mousa SA Evaluation of Angiogenesis Assays Biomedicines 2019 7 37 31100863
79 Takagi H King GL Robinson GS Ferrara N Aiello LP Adenosine mediates hypoxic induction of vascular endothelial growth factor in retinal pericytes and endothelial cells Invest Ophthalmol Vis Sci 1996 37 2165 76 8843903
80 Leibovich SJ Chen JF Pinhal-Enfield G Belem PC Elson G Rosania A Synergistic up-regulation of vascular endothelial growth factor expression in murine macrophages by adenosine A(2A) receptor agonists and endotoxin Am J Pathol 2002 160 2231 44 12057925
81 Acurio J Herlitz K Troncoso F Aguayo C Bertoglia P Escudero C Adenosine A(2A) receptor regulates expression of vascular endothelial growth factor in feto-placental endothelium from normal and late-onset pre-eclamptic pregnancies Purinergic Signal 2017 13 51 60 27696086
82 Liu X Zhang E Li P Liu J Zhou P Gu DY Expression and possible mechanism of c-ski, a novel tissue repair-related gene during normal and radiation-impaired wound healing Wound Repair Regen 2006 14 162 71 16630105
83 Zhang H Wang JS Chen XG Kang L Lin MB Overexpression of c-Ski promotes cell proliferation, invasion and migration of gastric cancer associated fibroblasts Kaohsiung J Med Sci 2019 35 214 21 30896889
84 Feng M Zhou Q Tu W Wang Y Du Y Xu K ATF4 promotes brain vascular smooth muscle cells proliferation, invasion and migration by targeting miR-552-SKI axis PLoS One 2022 17 e0270880 35857794
85 Ling J Cai Z Jin W Zhuang X Kan L Wang F Silencing of c-Ski augments TGF-b1-induced epithelial-mesenchymal transition in cardiomyocyte H9C2 cells Cardiol J 2019 26 66 76 29570207
86 Liao HY Wang ZQ Da CM Zhou KS Zhang HH Ski regulates proliferation and migration of reactive astrocytes induced by lipopolysaccharide (LPS) through PI3K/Akt pathway J Neuroimmunol 2022 364 577807 35007896
87 Chen D Lin Q Box N Roop D Ishii S Matsuzaki K SKI knockdown inhibits human melanoma tumor growth in vivo Pigment Cell Melanoma Res 2009 22 761 72 19845874
88 Zhao X Fang Y Wang X Yang Z Li D Tian M Knockdown of Ski decreases osteosarcoma cell proliferation and migration by suppressing the PI3K/Akt signaling pathway Int J Oncol 2020 56 206 18 31746363
89 Xu W Angelis K Danielpour D Haddad MM Bischof O Campisi J Ski acts as a co-repressor with Smad2 and Smad3 to regulate the response to type beta transforming growth factor Proc Natl Acad Sci U S A 2000 97 5924 9 10811875
90 Makino Y Yoon JH Bae E Kato M Miyazawa K Ohira T Repression of Smad3 by Stat3 and c-Ski/SnoN induces gefitinib resistance in lung adenocarcinoma Biochem Biophys Res Commun 2017 484 269 77 28115165
91 Shih SC Claffey KP Role of AP-1 and HIF-1 transcription factors in TGF-beta activation of VEGF expression Growth Factors 2001 19 19 34 11678207
92 Hwang S Seong H Ryu J Jeong JY Kang TS Nam KY Phosphorylation of STAT3 and ERBB2 mediates hypoxia-induced VEGF release in ARPE-19 cells Mol Med Rep 2020 22 2733 40 32945388
93 Kiyono K Suzuki HI Morishita Y Komuro A Iwata C Yashiro M c-Ski overexpression promotes tumor growth and angiogenesis through inhibition of transforming growth factor-beta signaling in diffuse-type gastric carcinoma Cancer Sci 2009 100 1809 16 19594546
94 Pages G Milanini J Richard DE Berra E Gothie E Vinals F Signaling angiogenesis via p42/p44 MAP kinase cascade Ann N Y Acad Sci 2000 902 187 200 10865838
95 Song YY Liang D Liu DK Lin L Zhang L Yang WQ The role of the ERK signaling pathway in promoting angiogenesis for treating ischemic diseases Front Cell Dev Biol 2023 11 1164166 37427386
96 Wyatt AW Steinert JR Wheeler-Jones CP Morgan AJ Sugden D Pearson JD Early activation of the p42/p44MAPK pathway mediates adenosine-induced nitric oxide production in human endothelial cells: a novel calcium-insensitive mechanism FASEB J 2002 16 1584 94 12374781
97 Ribe D Sawbridge D Thakur S Hussey M Ledent C Kitchen I Adenosine A2A receptor signaling regulation of cardiac NADPH oxidase activity Free Radic Biol Med 2008 44 1433 42 18206127
98 Thakur S Du J Hourani S Ledent C Li JM Inactivation of adenosine A2A receptor attenuates basal and angiotensin II-induced ROS production by Nox2 in endothelial cells J Biol Chem 2010 285 40104 13 20940302
99 Ouyang X Ghani A Malik A Wilder T Colegio OR Flavell RA Adenosine is required for sustained inflammasome activation via the A(2)A receptor and the HIF-1alpha pathway Nat Commun 2013 4 2909 24352507
100 Germack R Dickenson JM Characterization of ERK1/2 signalling pathways induced by adenosine receptor subtypes in newborn rat cardiomyocytes Br J Pharmacol 2004 141 329 39 14751870
