Apelin/APJ signaling suppresses the pressure ulcer formation in cutaneous ischemia-reperfusion injury mouse model

Several studies have demonstrated potential roles for apelin/APJ signaling in the regulation of oxidative stress associated with ischemia-reperfusion (I/R) injury in several organs. Objective was to assess the role of apelin/APJ signaling in the development of pressure ulcers (PUs) formation after cutaneous I/R injury in mice. We identified that cutaneous I/R injury increased the expression of apelin in the skin at I/R site. Administration of apelin significantly inhibited the formation of PUs. The reductions of blood vessels, hypoxic area and apoptosis in I/R site were inhibited by apelin injection. Oxidative stress signals in OKD48 mice and the expressions of oxidative stress related genes in the skin were suppressed by apelin injection. H2O2-induced intracellular ROS and apoptosis in endothelial cells and fibroblasts were suppressed by apelin in vitro. Furthermore, MM07, biased agonist of APJ, also significantly suppressed the development of PUs after cutaneous I/R, and the inhibitory effect of MM07 on PUs formation was higher than that in apelin. We conclude that apelin/APJ signaling may inhibit cutaneous I/R injury-induced PUs formation by protecting the reduction of vascularity and tissue damage via suppression of oxidative stress. Exogenous application of apelin or MM07 might have therapeutic potentials against the development of PUs.


Apelin protected pUs formation after cutaneous i/R in mice model in vivo.
To assess the effect of apelin ([Pyr 1 ]-Apelin-13) on the development of PUs after cutaneous I/R in vivo, we compared wound area after I/R injury in normal C57BL/6 mice treated with subcutaneous injection of apelin or PBS as a control. We used a simple, reproducible and noninvasive experimental mouse model to evaluate the pathogenesis of cutaneous PUs by I/R in vivo 3,19 . Administration of apelin significantly inhibited the formation of PUs ( Fig. 2A,B). The wound area in apelin-injected mice was significantly smaller that in control mice from 1 to 5 days after reperfusion. At 4 days after reperfusion, the size of wound area in the apelin-injected mice was 70% of that in the control mice. Next, we examined the expression of apelin in the wound healing process in I/R mice with or without apelin treatment, and found that administration of apelin did not change the mRNA levels of apelin in I/R site at 4, 12 and 48 hours after cutaneous I/R injury (Fig. 2C). These results demonstrate that apelin partially protected the formation of cutaneous PUs after cutaneous I/R. injection of apelin protected vascular loss by cutaneous i/R injury. We previously identified that the number of blood vessels was reduced after cutaneous I/R injury in mice 20 . Therefore, we investigated the effect of apelin on vascular loss caused by cutaneous I/R injury. At 4 days after reperfusion, the numbers of CD31 + endothelial cells and NG2 + pericytes around I/R areas in apelin-treated mice were significantly higher than those in control mice (Fig. 3A). These results suggest that apelin might prevent the reduction of vascularity after cutaneous I/R injury.
Apelin suppressed hypoxia and apoptosis by cutaneous i/R injury. We next examined the influence of apelin treatment on tissue hypoxia after cutaneous I/R injury in mice. Hypoxic area in the skin tissue was analyzed using the hypoxia marker pimonidazole. At one day after cutaneous I/R, hypoxic area in the I/R site was increased compared with control mice without I/R (Fig. 3B). However, hypoxia staining positive area in I/R site in apelin-treated mice were significantly decreased compared to those in vehicle-treated mice (Fig. 3B). As hypoxia activates cellular apoptosis 21 , we examined the influence of apelin on the number of apoptotic cells in I/R areas in mice. TUNEL staining revealed that administration of apelin into I/R site reduced the increased number of apoptotic cells after I/R injury (Fig. 3C). These results suggest that administration of apelin might suppress hypoxic area and apoptosis in I/R site after cutaneous I/R injury.

Apelin regulated the infiltration of inflammatory cells and the expressions of cytokines and
growth factors after cutaneous i/R. We previously reported that cutaneous I/R injury induced the infiltration of inflammatory cells in I/R site, and the growth factors and cytokines produced by inflammatory cells were key factors following the development of PUs and wound healing process 22,23 . To examine the effect of apelin administration on the dynamics of infiltration of inflammatory cells (MPO + neutrophils, CD68 + macrophages and CD3 + T cells), immunohistochemical studies were performed using skin tissue of I/R site between control and apelin-treated mice at day 0, 1, 4 and 7 after I/R injury. At day 1, the number of MPO + neutrophils was significantly inhibited by apelin treatment (Fig. 4A). However, there were no significant difference between control and apelin-treated mice at day 0, 4 and 7. The numbers of CD68 + macrophages and CD3 + T cells at day1 was significantly inhibited by apelin, and the peak of the numbers of infiltrated CD68 + macrophages and CD3 + T cells were delayed by apelin treatment (Fig. 4A).
Furthermore, we examined the expression levels of growth factors and cytokines expression, including tumor necrosis factor-α (TNF-α), transforming growth factor-β (TGF-β), interleulin-6 (IL-6), IL-10 and basic fibroblast growth factor (bFGF) in I/R site through wound healing process by real-time PCR assay. The expression levels of proinflammatory cytokines (TNF-α, IL-6) were not different between control and apelin-treated mice through day 0 to 7. The mRNA levels of IL-10 and bFGF expressions were significantly increased at day 1 by apelin treatment, and TGF-β expressions was significantly increased at day 1 to 4 by apelin treatment (Fig. 4B). These results suggest that apelin might suppress recruitment of inflammatory cells at early phase of wound healing, and change the expressions of several growth factors and cytokines in the I/R site.
Apelin suppressed oxidative stress after cutaneous i/R. Next, we investigated the effect of apelin on oxidative stress induced by I/R injury by using OKD48 (Keap1-dependent oxidative stress detector, NO-48) mice 24 . OKD48 mice have a transgene encoding a modified Nuclear factor erythroid 2-related factor 2 (Nrf2), The size of the wound area after I/R injury in normal C57BL/6 mice treated with subcutaneous injection of apelin (10 ng/mice) or phosphatebuffered saline as a control. The size of the ulcer in control mice at 4 days after reperfusion was assigned a value of 100% (vehicle: n = 9, apelin: n = 10, for each time point and group). (B) Representative images of the wound after cutaneous I/R in control or apelin treated mice at 0, 4, 8, and 12 days after reperfusion. (C) Comparison of mRNA levels of apelin expression during wound healing in I/R site between control and apelin-treated mice at 4, 12 and 48 hours after I/R injury. n = 5. All values represent mean ± SEM. **P < 0.01, *P < 0.05. The amount of CD31 + ECs and NG2 + pericytes in the cutaneous I/R area at 4 days after reperfusion. (B) The amount of pimonidazole + hypoxic area in cutaneous I/R site at 1 day after reperfusion. Quantification of the pimonidazole + areas in 6 random microscopic fields from the center of I/R area in n = 3 mice per groups was performed using ImageJ software. Positive area in control mice was assigned a value of 1. Scale bar = 20 μm. (C) The number of apoptotic cells in the I/R site at 1 day after reperfusion was determined by counting both TUNEL-and DAPI-positive cells. Values were determined in 6 random microscopic fields in n = 3 mice per group. All values represent mean ± SEM. **P < 0.01, *P < 0.05. (2020) 10:1349 | https://doi.org/10.1038/s41598-020-58452-2 www.nature.com/scientificreports www.nature.com/scientificreports/ cutaneous I/R injury, and apelin injection suppressed those gene expressions (Fig. 5C). These results suggest that the oxidative stress in cutaneous I/R area might be inhibited by apelin injection.
Apelin suppressed RoS production and apoptosis driven by oxidative stress in vitro. Next, we examined whether apelin suppress ROS production and oxidative stress-induced apoptosis of endothelial cells and fibroblasts in vitro. H 2 O 2 -induced ROS productions in endothelial cells was suppressed by apelin treatment (Fig. 6A). In addition, apelin treatment significantly reduced the amount of H 2 O 2 -induced early apoptotic and necrotic cells in fibroblasts (Fig. 6B). These results suggest that apelin might reduce oxidative stress and oxidative stress-induced cell apoptosis in vitro.

MM07 protected PUs formation after cutaneous I/R injury in vivo.
Finally, we investigated the effect of the synthetic biased agonist of APJ, MM07 30,31 , on the development PUs formation after I/R injury in vivo. Since it has been reported that MM07 has greater potential than apelin to increase the forearm blood flow in human 30 , the amount of MM07 for treatment was designed 1/10 with respect to treatment with apelin ( Fig. 2; 10 µg apelin, Fig. 7; 1 µg MM07). Wound area after I/R injury in C57BL/6 mice treated with subcutaneous injection of MM07 or PBS as a control were analyzed. Administration of MM07 significantly inhibited the formation of PUs after cutaneous I/R injury (Fig. 7A,B). At 4 days after reperfusion, the size of the wound area in the MM07-injected mice was 50% of that in the control mice. These results suggest that MM07 might have protective effects on the development of PUs formation after cutaneous I/R injury, and that MM07 might have greater potential than apelin to inhibit PUs formation in vivo.  www.nature.com/scientificreports www.nature.com/scientificreports/

Discussion
At first, we investigated the expression of apelin during cutaneous I/R injury. We found that the expression of apelin was increased by ischemia, and then it was maximized at 4 hours after reperfusion and decreased until 12 hours. After that, it was gradually upregulated again in time dependent manner, suggesting that hypoxia and/ or inflammatory pathways after I/R injury might induce the upregulation of apelin expression in I/R site.
Several studies reported that apelin has protective effect for tissue damage after I/R injury or traumatic injury via suppression of inflammatory cytokines, such as TNF-α, IL-1β and IL-6, oxidative stress, autophagy and N-methyl-D-aspartate (NMDA)-induced excitotoxicity 16,32,33 . However, the role of apelin in cutaneous I/R injury is unknown. Therefore, this is the first study to identify the mechanisms of protective regulation in cutaneous I/R injury by apelin/APJ signaling.
It has been reported that the number of blood vessels in I/R site were reduced after cutaneous I/R injury, indicating that hypoxia-induced ROS might cause cellular damage and apoptosis 7,20 . In this study, we assessed vascularity in I/R areas and identified that the reductions of CD31 + endothelial cells and NG2 + pericytes in I/R areas were inhibited by apelin administration, suggesting that apelin might inhibit the damage of blood vessels induced by cutaneous I/R. Apelin has been described to be an activator of angiogenesis and increase blood flow 30,34,35 , suggesting that administration of apelin might increase blood flow and angiogenesis, leading to the inhibition of I/R injury-induced vascular damages and hypoxic area in I/R site.
In addition, we demonstrated that enhanced oxidative stress caused by cutaneous I/R was reduced by injection of apelin in OKD48 mice. We also identified that oxidative stress related factors in I/R site were also suppressed in apelin-treated mice. Furthermore, we identified the anti-oxidative effects of apelin through the analysis of the production of ROS in endothelial cells and cellular apoptosis/necrosis in fibroblasts under oxidative stress in vitro. These results suggest that administration of apelin might suppress the oxidative stress and apoptosis via the inhibition of ROS production after cutaneous I/R injury. There are several studies regarding the mechanisms of the reduction of ROS production by apelin. For example, Zeng et al. reported that apelin suppressed ROS production and enhanced superoxide dismutase (SOD) activity in primary cultured myocardial cells after I/R injury 17 . SOD is an enzyme that degrades the ROS generated in cells by oxidative stress. Additionally, it has been reported that apelin-APJ signaling activates the AMP-activated protein kinase (AMPK)/Nrf2 signals, and MAP kinase pathway, resulting in the upregulation of anti-oxidant enzyme, including SOD-1 under oxidative stress condition in adipocytes and neuronal cells 36,37 . These mechanisms may be involved in the suppression of ROS production and oxidative stress by apelin in our study, however further investigation is required. www.nature.com/scientificreports www.nature.com/scientificreports/ Resent study demonstrated that the proinflammatory cytokines derived from inflammatory cells, including neutrophils and macrophages, are key factors in the development of PUs 19,22,23 . In this study, we found the numbers of inflammatory cells were suppressed in apelin-treated mice at day 1. It has been reported that IL-10, bFGF and TGF-β prevent tissue damage after I/R injury [38][39][40] . We demonstrated that qPCR analysis revealed that apelin administration enhanced the expression of these tissue protective cytokines and growth factors, such as IL-10, bFGF and TGF-β in I/R site, however, further studies will be needed to clarify the precise mechanisms.
It has been known that chronic administration of apelin induces β-arrestin-mediated internalization of APJ and receptor desensitization, suggesting that there is a limitation for the clinical use of apelin 31 . On the other hand, MM07 is cyclic apelin peptide, and preferentially activates G protein responses with low potency in β-arrestin-mediated receptor internalization 30,31 . Consistent with these findings, it has been reported that intrabrachial infusion of MM07 had greater potential than apelin to increase blood flow in human 30 . Similar to previous results, we identified that inhibitory effect on the development of PUs by MM07 was higher than that by apelin.
Administration of apelin/MM07 suppressed the development of PUs in early phase, however there was no difference in the whole period of wound healing in our study. Since aperin/MM07 has a vasodilatory effect and an oxidative stress inhibitory effect, there is a possibility that it promotes wound healing. However, we considered that there was no difference in the whole period of wound healing because of the strong skin contraction that occurs at the end of wound healing in mice. The limit of this model is that it is highly influenced by the effect of ulcer contraction induced by the fibrosis. This is a so much difference compared by PUs in the humans. Further study will be needed to examine the effect of apelin/MM07 on wound healing.
Taken together, we demonstrate that apelin/APJ signaling suppresses the formation of PUs induced by cutaneous I/R injury by preventing the reduction of blood vessels and the protection of tissue damage through the inhibition of oxidative stress induced by I/R injury. Exogenous apelin or MM07 administration has possible therapeutic potential for cutaneous I/R injury-induced PUs.

Animals. All experiments were approved by the Gunma University Animal Care and Experimentation
Committee. C57BL/6 mice were purchased from the SLC (Shizuoka, Japan). OKD48 (Keap1-dependent oxidative stress detector, NO-48) mice were kindly provided from Dr. T. Iwawaki (Department of Life Science, Kanazawa Medical University, Ishikawa, Japan). Eight-to 12-week-old mice were used for all experiments. Mice were bred and maintained in the Institute of Experimental Animal Research of Gunma University under specific pathogen-free conditions. Mice were handled in accordance with the animal care guidelines of Gunma University.
Antibodies. Antibodies (Abs) and their sources were as follows: rat anti-mouse CD31 monoclonal Ab (mAb) (MEC13.3; BD Bioscience, San Jose, CA), rabbit anti-mouse NG2 polyclonal Ab (pAb) (Millipore, Billerica, MA), rabbit anti-mouse apelin antibody pAb(Santa Cruz Biotechnology). Alexa 488-, Alexa 568-conjugated secondary Abs were obtained from Invitrogen (Carlsbad, CA). HRP-conjugated goat anti-mouse or anti-rabbit secondary Abs were obtained from Dako (Glostrup, Denmark). i/R cycles and analysis. The I/R model that has been previously reported was used 2,3,19,20,22,23,26 . Briefly, mice were anesthetized, and hair was shaved and cleaned with 70% ethanol. The dorsal skin was gently pulled up and trapped between two round ferrite magnetic plates that had a 12-mm diameter (113 mm²) and 5 mm thick, with an average weight of 2.69 g and 1180 G magnetic forces (NeoMag Co, Ichikawa, Japan). Epidermis, dermis, subcutaneous fat layer and subcutaneous loose connective tissue layer, but not muscles, were pinched by magnetic plates. This process creates a compressive pressure of 50 mmHg between the two magnets 2, 3 . It has been demonstrated that an external pressure of 50 mmHg is sufficient to induce skin necrosis and ulcer by reducing blood flow 80% 3 . Dorsal skin was trapped between magnetic palates for 12 hours, and then plates were removed. Mice were not immobilized, and not anesthetized during ischemia. All of the mice developed two round ulcers separated by a bridge of normal skin. To assess the effects of apelin ([[Pyr 1 ]-Apelin-13) (Tocris) or MM07 (cyclo [1][2][3][4][5][6]CRPRLCHKGPMPF; synthesized by Sigma-Aldrich) 30,41 on the development of PUs formation and wound healing after cutaneous I/R injury, 10 µg of apelin or 1 µg of MM07 per 200 µl phosphate buffered salts (PBS) or 200 µl saline as a control were injected into the dermis in the I/R site just after reperfusion. For analysis, each wound sites were digitally photographed at the indicated time points after wounding, and wound areas were measured on photographs using Image J (version1.48, NIH, Bethesda, MD).
Histological examination and immunofluorescence staining. Immunofluorescence staining of frozen sections and analyses were performed as described previously 42 . Murine skins were removed and 4 μm frozen sections were prepared and fixed in 4% PFA in PBS for 30 minutes. After blocking with 3% dry milk-PBS supplemented with 5% normal donkey serum or 5% normal goat serum for 1 hour at room temperature, sections were stained with Abs of interest followed by Alexa 488-, Alexa 568-conjugated secondary Abs. Sections were counterstained with 4,6-diamidino-2-phenylindole (DAPI) to visualize nuclei, mounted in ProLong Gold antifade reagent (Life Technologies).
Assessment of tissue hypoxia. Hypoxic areas after cutaneous I/R injury in I/R site were detected using the Hypoxyprobe-1 TM Omni kit (HPI, Burlington, MA) as previously described 20,43 . Pimonidazole HCl was injected by intraperitoneal (60 mg/kg) 30 min prior to sacrificing the mice. Murine skins were removed and 4μm frozen sections were prepared and fixed cold acetone (4 degrees Celsius) for 10 min. Sections were incubated overnight at 4 degrees Celsius with rabbit anti-pimonidazole antisera PAb2627 diluted 1:20 in PBS containing 0.1% bovine serum albumin and 0.1% Tween 20. Sections were incubated for 1 h Alexa 488-conjugated secondary Abs. Images (6 fields/section) were taken and visualized with a FV10i-DOC confocal laserscanning microscope (Olympus). The positive area was determined by ImageJ (version1.48, NIH, Bethesda, MD) in the field (x600).