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. 2023 May 24;20(9):3586–3598. doi: 10.1111/iwj.14235

Roxadustat promotes hypoxia‐inducible factor‐1α/vascular endothelial growth factor signalling to enhance random skin flap survival in rats

Qicheng Lan 1,2, Kaitao Wang 1, Zhefeng Meng 1, Hang Lin 2, Taotao Zhou 1, Yi Lin 1, Zhikai Jiang 1, Jianpeng Chen 1, Xuao Liu 1, Yuting Lin 2, Dingsheng Lin 1,✉
PMCID: PMC10588316  PMID: 37225176

Abstract

Random skin flaps have limited clinical application as a broad surgical reconstruction treatment because of distal necrosis. The prolyl hydroxylase domain‐containing protein inhibitor roxadustat (RXD) enhances angiogenesis and reduces oxidative stress and inflammation. This study explored the function of RXD in the survival of random skin flaps. Thirty‐six male Sprague–Dawley rats were randomly divided into low‐dose RXD group (L‐RXD group, 10 mg/kg/2 day), high‐dose RXD group (H‐RXD group, 25 mg/kg/2 day), and control group (1 mL of solvent, 1:9 DMSO:corn oil). The proportion of surviving flaps was determined on day 7 after surgery. Angiogenesis was assessed by lead oxide/gelatin angiography, and microcirculation blood perfusion was evaluated by laser Doppler flow imaging. Specimens in zone II were obtained, and the contents of superoxide dismutase (SOD) and malondialdehyde (MDA) were measured as indicators of oxidative stress. Histopathological status was evaluated with haematoxylin and eosin staining. The levels of hypoxia‐inducible factor‐1α (HIF‐1α), vascular endothelial growth factor (VEGF), and the inflammatory factors interleukin (IL)‐1β, IL‐6, and tumour necrosis factor‐α (TNF‐α) were detected by immunohistochemistry. RXD promoted flap survival and microcirculatory blood perfusion. Angiogenesis was detected distinctly in the experimental group. SOD activity increased and the MDA level decreased in the experimental group. Immunohistochemistry indicated that the expression levels of HIF‐1α and VEGF were increased while the levels of IL‐6, IL‐1β, and TNF‐α were decreased after RXD injection. RXD promoted random flap survival by reinforcing vascular hyperplasia and decreasing inflammation and ischaemia‐reperfusion injury.

Keywords: angiogenesis, HIF‐1α, inflammation, necrosis, random skin flap, roxadustat, VEGF

1. INTRODUCTION

Random skin flaps are widely used in reconstructive surgery 1 as an effective therapeutic practice for excising tumours, superficial wounds, burns, trauma, and congenital defects. 2 Compared with axial‐pattern flaps, random flaps lack axial vascularization. The advantage this brings is the small number of axial blood vessels and exiguous damage to the body. 3 As transplantation results in poor blood perfusion, clinical applications are limited because of flap necrosis. 4 Thus, the mechanism underlying the necrosis of a distal flap and means of improving angiogenesis have been the focus of fundamental research.

Low tolerance to ischaemia, hypoxia, 5 and inflammation, 6 as well as lack of blood flow, 7 are the three main reasons for necrosis of a random flap. The optimal flap ratio is 1.5 to 2:1 to ensure blood perfusion in a distal flap. 8 The onset of oxidative stress and inflammation caused by ischaemia/reperfusion (I/R) after flap transplantation can drastically damage flap cells, leading to vascular injury and an increased risk of skin flap necrosis. 5 Hence, promoting angiogenesis and improving the local blood supply, while reducing complications, play a vital role in enhancing the survival of random skin flaps. The molecular pathways supporting angiogenesis are activated in a variety of ways; among these, hypoxia‐inducible factor‐1α/vascular endothelial growth factor (HIF‐1α/VEGF) signalling is a critical pathway. 9

Roxadustat (RXD) is an orally administered HIF prolyl hydroxylase inhibitor. In 2018 and 2019, it was used clinically to treat patients with anaemia because of dialysis‐dependent chronic kidney disease (CKD) or non‐dialysis‐dependent CKD, as well as patients with myelodysplastic syndrome. 10 Apart from being effective in treating kidney disease, RXD is also efficacious in other diseases, including hypertension 11 and retinopathy of prematurity. 12 Data from animal studies have indicated that the potential anti‐ischaemic effects of RXD are helpful at the cellular and molecular levels. RXD is often used to decrease ischaemia in myocardial and renal cells. 13 , 14 RXD also plays an important role in protecting against I/R‐induced acute kidney injury. 15 In addition, RXD significantly accelerates cutaneous wound healing in diabetic rats and promotes epidermal upregulation of pulmonary angiogenesis. 16

However, the effects of RXD on transplanted skin flaps and the relevant molecular pathway have not been clarified. In this study, we explored the effects of RXD on flap survival and the underlying mechanisms.

2. MATERIALS AND METHODS

2.1. Animals

Ethical approval (approval no. WYDW2022‐0511) was provided by the Laboratory Animal Ethics Committee of Wenzhou Medical University, Wenzhou, China (Chairperson, Prof. Shengwei Jin) on May 11, 2022. Thirty‐six male Sprague–Dawley rats (200‐250 g each, 2‐3 months old) were obtained from the Wenzhou Medical University Laboratory Animal Center. They were separated in cages in a controlled room at 50 ± 10% humidity, a temperature of 22°C to 26°C, and a 12‐h light/dark cycle. The room was well ventilated and all rats had easy access to food and water. RXD (160 mg) was dissolved in 8 mL of DMSO. A 1‐mL aliquot of the solution was dissolved in 9 mL of corn oil, which contained 2 mg/mL of RXD. A random number table was used to divide the rats into three groups: the H‐RXD (25 mg/kg/2 day) group, the L‐RXD (10 mg/kg/2 day) group, and the control group (1 mL of solvent, 1:9 DMSO:corn oil) following previous studies. 16 , 17 All rats were injected at 4 pm every 2 days based on a previous study and a phase‐3 clinical study. 16 , 18 , 19 Each group included 12 rats.

Pentobarbital sodium (1%) was injected intraperitoneally (40 mg/kg) as an anaesthetic. An additional dose was used if necessary. The hair in the middle of the back of each rat was shaved. The iliac crests were the connection sites, and the dorsal spine was the symmetry axis. The skin was disinfected with an iodophor, and a modified rectangular McFarlane (3 × 9 cm) flap was constructed (Figure 1A). The two iliac arteries were amputated, and the subdermal capillary network persisted (Figure 1B). The flap was completely separated from the deep fascia and seamed with non‐absorbable 4‐0 nylon sutures. The flap was dabbed with erythromycin ointment to prevent infection. Sterile conditions were maintained throughout the surgery. To prevent postoperative self‐injury, the rats were dressed in headgear designed by the members. All procedures were performed by the same operator.

FIGURE 1.

FIGURE 1

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(A) Modified McFarlane (3 × 9 cm) flap on each rat. The area was divided into Zone I, Zone II, and Zone III. (B) Disconnection of the two iliac vessels.

Three equal sections, zones I to III, were designed from proximal to distal for better observation. After surgery, the rats were fed separately in 36 cages. All experimental surgeries were completed by the same operator to reduce human error.

2.2. Reagents and antibodies

RXD (ASP1517 purity ≥98%) was purchased from Good Laboratory Practice Bioscience (Montclair, NJ, USA). Antibodies against VEGF (AF5131), IL‐1β (AF5103), IL‐6 (DF6087), and TNF‐α (AF7014) were purchased from Affinity Biosciences (Cincinnati, OH, USA). Malondialdehyde (MDA) assay kits (A003‐1‐2) and superoxide dismutase (SOD) assay kits (A001‐3‐2) were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). A haematoxylin and eosin (H&E) staining kit (G1120) was obtained from Solarbio Science & Technology (Beijing, China).

2.3. Assessment of macroscopic flap survival

Each flap was examined macroscopically on postoperative days 1, 3, 5, and 7. The following changes were recorded: blackened colour, stiff texture, shrunken tissue, reduced elasticity, and the absence of bleeding after tissue puncture, which was defined as flap necrosis. On postoperative day 7, the surviving and total areas of each flap were measured, respectively, using weighing paper and an electronic scale (Mettler Toledo, Shanghai, China). The following formula was used to assess the surviving area: flap surviving area (%) = paperweight of the flap surviving area/paperweight of the total flap surface area × 100.

2.4. SOD and MDA contents

On postoperative day 7, specimens (0.5 × 0.5 cm) were obtained from zone II of six rats in each group. Xanthine oxidase was used to detect the SOD level. The samples were homogenised and diluted 1:9 (w:v) to obtain a 10% solution in ice‐cold 0.9% saline. The protein content was analysed with a BCA protein assay kit. Reagents were added following the manufacturer's instructions. Distilled water was used in the control group. The solution was placed in a 37°C isothermal water bath (Shanghai Yuejin Medical Optical Equipment Factory, Shanghai, China) for 40 min. Then, the colour developer was added. After 10 min of standing at room temperature, the absorbance was detected to measure SOD activity. The thiobarbituric acid (TBA) method was used to determine the MDA content. A 0.15‐mL aliquot of supernatant was added to all tubes and mixed with the same amount of standard solution. Another 0.15 mL of distilled water was then added. After adding 2.5 mL of TBA to each tube, the samples were incubated (Verder Shanghai Instruments and Equipment, Shanghai, China) at 100°C for 1 hour. Then, after cooling, the samples were centrifuged to separate the precipitates. Finally, a colorimetric assay was used to test the suspended substance at 532 nm. The MDA content was determined based on the absorbance value.

2.5. Laser Doppler flowmetry

On postoperative day 7, six rats were randomly selected from each group and placed in a rectangular area in prone position under anaesthesia. A laser Doppler imaging system (Moor Instruments, Axminster, UK) was used to scan blood perfusion in each area (zones I–III). Microcirculatory blood flow in the local tissues was expressed as perfusion units (PU). 20 Moor LDI Review V6.1 software (Moor Instruments, Axminster, UK) was used to measure the colour Doppler images of each skin flap.

2.6. Gelatin/lead oxide angiography

Six randomly selected rats were anaesthetised on postoperative day 7. Arterial access was established after puncturing the common carotid artery at the near cardiac end. Subsequently, 37°C isotonic saline was injected to wash the blood vessels and then drained. Body temperature was monitored. A gelatin/lead oxide solution (100 mL/kg) was perfused and stopped when the limbs, ears, and corneas changed colour. The rats were held at −25°C for 24 hours to accumulate the lead oxide‐gelatin in the flaps. The peeled flaps were exposed to X‐ray angiography (Bucky Diagnost CS, Philips Medical Systems DMC GmbH, Germany) the following day.

2.7. Histopathological examination

All rats were euthanised with a lethal dose of anaesthetic on postoperative day 7. Tissue samples (1 × 1 cm) were sliced from all zones, kept in 4% paraformaldehyde for 24 hours, sectioned, and stained with H&E. Under a light microscope at ×100 magnification (VHX‐7000 Milton Keynes, Keyence, UK), we evaluated the degree of necrosis, tissue oedema, and infiltration by neutrophils. Angiogenic aggregation was determined using the microvascular density (MVD) value obtained from five random visual fields. The density of the microvessels in the section per unit area (/mm2) was counted under high magnification (×200) as the MVD standard.

2.8. Immunohistochemistry

The prepared sections were placed in a 60°C incubator (Verder Shanghai Instruments and Equipment, Shanghai, China) for 12 hours. Then, the dewaxing agent was mixed with distilled water at a 1:9 ratio. This mixture was placed in a 60°C incubator for 30 minutes. The sections were placed in the mixture and heated for another 30 minutes at the same temperature. Later, the sections were washed. EDTA antigen retrieval solution was diluted with double‐distilled water at 1:49. Flap samples were placed in this solution and heat‐induced antigen retrieval was performed at 210°C for 3 minutes. Then, the specimens were removed and cooled to room temperature. An endogenous peroxidase‐blocking solution was added for 20 minutes. The primary antibody dilutions were VEGF (1:160), HIF‐1α (1:100), IL‐1β (1:130), IL‐6 (1:150), and TNF‐α (1:100). The antibodies were added and incubated overnight at 4°C in a freezer (Haier Smart Home, Qingdao, China). The next day, the sections were incubated at 37°C for 50 minutes. The specimens were incubated with secondary antibodies at room temperature for 1 hour, washed three times in phosphate‐buffered saline for 5 minutes each, restained with haematoxylin, and imaged under a microscope at ×200 magnification (VHX‐7000 Milton Keynes, Keyence, UK). The photographs were analysed using Image Pro Plus (6.0.0.260; Media Cybernetics, Rockville, MD, USA).

2.9. Statistical analysis

Statistical analyses were performed using IBM SPSS 25.0 software (IBM Corp., Armonk, NY, USA). Data are presented as the mean ± SE. The mean values of the groups were compared by a one‐way analysis of variance. P values <.05 were considered significant.

3. RESULTS

3.1. RXD significantly promotes skin flap survival

Necrosis developed gradually in the distal area after the operation, and a boundary partition between the necrotic and surviving areas was observed. All of the proximal flaps were in good condition with a light red colour, elastic cortex, and no crispation (Figure 2A). Dark‐coloured necrosis was seen in zones II and III, with a tight texture and irregular surface (Figure 2A). The necrotic areas of the control flaps were the largest and exceeded more than half of the flap; complete necrosis was detected in zone III, with partial survival in zone II. No blood was lost in this group, and a distinct demarcation divided the necrotic and surviving areas. Most of zone III in the L‐RXD group was necrotic, whereas zone III was alive. The black dead area decreased, and the surviving area increased. The necrosis in zone III was much smaller (33% of the area) in the H‐RXD group. In contrast, the survival ratio of zone II was 100%. The survival rate in the H‐RXD group was 77.50 ± 6.20%, which was significantly higher than that in the L‐RXD group (62.86 ± 5.02%, P = .000152) and the control group (46.82 ± 3.58%, P < .0001). The rate in the L‐RXD group was significantly higher than that in the control group (P < .0001) (Figure 2B).

FIGURE 2.

FIGURE 2

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(A) Records by digital photographs of flaps in three different zones. General observations of the surviving and necrotic parts of the flaps. (B) Flap survival rate on day 7. Roxadustat promoted the survival rate of skin flaps.**P < .01.

3.2. RXD enhances blood perfusion

Laser Doppler blood flow imaging demonstrated that the blood perfusion rates in zone II of the H‐RXD, L‐RXD, and control groups were 269.72 ± 46.33, 191.82 ± 37.20, and 49.20 ± 12.41 PU, respectively. The blood perfusion rate in the H‐RXD group was significantly higher than that in the control group (P < .0001) and the L‐RXD group (P = .001573). The blood perfusion rate in the L‐RXD group was significantly higher than that in the control group (P < .0001) (Figure 3).

FIGURE 3.

FIGURE 3

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(A) Laser Doppler flowmetry angiography demonstrating blood perfusion on day 7. (B) Records of blood perfusion in Zone II. Roxadustat enhanced blood perfusion.**P < .01.

3.3. RXD improves angiogenesis

X‐ray angiography verified that the H‐RXD and L‐RXD groups generated more new blood vessels in zone II as well as in the margin than did the control group. The density of newborn microvessels in zone I increased after RXD treatment compared with the control group. A certain number of blood vessels was observed in zone III of the H‐RXD group while fewer were found in the remaining groups (Figure 4).

FIGURE 4.

FIGURE 4

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X‐ray angiography of flaps in three groups on day 7. Roxadustat increased the number of flap vessels.

3.4. RXD decreases histopathological damage

Various degrees of inflammation were detected in the H&E‐stained paraffin sections, including oedema, neutrophil infiltration, and structural damage. The H‐RXD and L‐RXD groups contained larger areas of subcutaneous structure and less inflammatory cell infiltration and oedema than the control group (Figure 5A).

FIGURE 5.

FIGURE 5

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(A) Histopathological features of the flaps were examined with haematoxylin and eosin staining on day 7. Roxadustat (RXD) decreased histopathological damage. (B) Neutrophil density in Zone II. RXD reduced the infiltration of neutrophil. (C) The microvascular density in Zone II on day 7. RXD promoted microvessel density. **P < .01,*P < .1.

A total of 50.50 ± 6.12/mm2 of neutrophils were detected in the H‐RXD group, which was significantly lower than that in the L‐RXD group (113.33 ± 11.27/mm2, P < .0001) and the control group (154.67 ± 9.31/mm2, P < .0001). The number of infiltrating neutrophils was also lower in the L‐RXD group than in the control group (P < .0001) (Figure 5B).

The MVD in the H‐RXD group (33.26 ± 5.65/mm2) was significantly higher than that in the L‐RXD group and the control group (16.84 ± 3.86 versus 8.84 ± 0.94/mm2, respectively; P < .0001). The MVD in the L‐RXD group was significantly higher than that in the control group (P = .008) (Figure 5C).

3.5. RXD inhibits I/R injury

MDA and SOD are universally used as indicators of oxidative stress in flaps. The SOD level in the H‐RXD group was 65.24 ± 3.03 units mg−1 protein−1, which was significantly higher than that in the L‐RXD group (49.74 ± 3.42 units mg−1 protein−1, P < .0001) and the control group (27.23 ± 5.14 units mg−1 protein−1, P < .0001). The SOD level in the L‐RXD group was significantly higher than that in the control group (P < .0001) (Figure 6A).

FIGURE 6.

FIGURE 6

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(A) Mean activity of superoxide dismutase (SOD) on day 7. (B) Mean activity of malondialdehyde (MDA) on day 7. Roxadustat promoted SOD activity and inhibited MDA activity. **P < .01.

The MDA content in the H‐RXD group was 25.06 ± 2.91 nmol/mg protein, which was significantly lower than that in the L‐RXD group (34.81 ± 4.23 nmol/mg protein, P < .0001) and the control group (67.04 ± 3.94 nmol/mg protein, P < .0001). The MDA content in the L‐RXD group was also significantly lower than that in the control group (P < .0001) (Figure 6B).

3.6. RXD attenuates inflammation

According to the immunohistochemical results, the IL‐6 levels in the H‐RXD group (1119.00 ± 313.98 IA, P < .0001) and the L‐RXD group (1812.03 ± 134.09 IA, P < .0001) were significantly lower than that in the control group (3547.62 ± 742.50 IA), indicating that RDX decreased inflammation. The IL‐6 level in the H‐RXD group was significantly lower than that in the L‐RXD group (P = .02) (Figure 7).

FIGURE 7.

FIGURE 7

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(A) Magnification after immunohistochemical staining with different pro‐inflammatory cytokines on day 7. (B) Expression of interleukin (IL)‐6, IL‐1β, and tumour necrosis factor‐α (TNF‐α). Roxadustat attenuated the expression of IL‐6, IL‐1β, and TNF‐α.**P < .01, *P < .1.

The IL‐1β levels were significantly lower in the RXD groups (993.30 ± 334.85 IA in the H‐RXD group and 1733.36 ± 337.68 IA in the L‐RXD group) than those in the control group (3853.91 ± 599.24 IA, P < .0001) (Figure 7).

The TNF‐α level in the H‐RXD group was 397.00 ± 154.03 IA, which was significantly lower than that in the L‐RXD and control groups (1009.23 ± 197.85 IA, P = .000515 and 2280.00 ± 641.34 IA, P = .001753, respectively). The TNF‐α level in the L‐RXD group was also lower than that in the control group (P = .011) (Figure 7).

3.7. RXD improves HIF‐1 α and VEGF expression

Immunohistochemical analysis showed that the highest expression of HIF‐1α was detected in the H‐RXD group (3283.35 ± 405.99 IA), followed by the L‐RXD group (2146.53 ± 157.24 IA) and the control group (1268.26 ± 160.87 IA; all P < .0001) (Figure 8B).

FIGURE 8.

FIGURE 8

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(A) Magnification after immunohistochemical staining of hypoxia‐inducible factor‐1α (HIF‐1α) and vascular endothelial growth factor (VEGF) on day 7. (B) Expression of HIF‐1α on day 7. Roxadustat (RXD) improved the expression of HIF‐1α. (C) Expression of VEGF on day 7. RXD improved the expression of VEGF. **P < .01,***P < .001.

The VEGF expression levels were significantly higher in the H‐RXD and L‐RXD groups than those in the control group (3642.43 ± 384.08 IA, P < .0001; 1586.02 ± 271.37 IA, P = .000769; 801.48 ± 304.17 IA) (Figure 8C), indicating that RXD increased the expression of HIF‐1α and VEGF.

4. DISCUSSION

RXD is a first‐class oral HIF prolyl hydroxylase domain (PHD) inhibitor and analog of 2‐oxoglutarate. 21 It is believed that by promoting erythropoiesis, which is reduced during treatment with conventional erythropoiesis‐stimulating agents, RXD decreases hormones and has a potential toxic effect of anaemia. 19 In addition to the basic treatment, RXD contributes to various hypoxia‐related diseases. A previous study indicated that RXD significantly attenuates acute kidney injury by suppressing the levels of macrophages and neutrophils as well as the inflammatory factors TNF‐α and IL‐1β. 22 Another study showed that RXD is cardioprotective in a murine model of I/R, as it reduced the infarct size and suppressed plasma creatinine kinase activity. 13 Compared with other PHDs, RXD is an orally administered medication that raises haemoglobin levels in patients with chronic inflammation. 23 RXD is cost‐effective and is accepted on the willingness‐to‐pay threshold according to the Chinese medical system. 24 These findings indicate that RXD inhibits inflammation, oxidative stress, and I/R, thus enhancing the distal random flap survival rate. Considering these functions, we speculate that RXD could be a potential inhibitor in the treatment after flap transplantation. In this study, intragastric administration of RXD to rats significantly promoted the survival rate of random flaps with improved angiogenesis. Many newborn blood vessels were observed on gelatin/lead oxide angiography. The SOD content was increased, while the MDA content was decreased. The expression of inflammatory factors such as IL‐1β, IL‐6, and TNF‐α decreased while the expression of VEGF and HIF‐1α increased.

Transplanting a flap is one of the most effective methods to restore regional defects and injuries, 25 prevent postoperative infections, 26 and promote aesthetic value. 27 Flap necrosis, the most dreaded complication following surgery, is primarily caused by inadequate blood supply, greatly limiting the broad application of flaps. 28 , 29 After long‐range tissue hypoxia, the reperfusion of blood causes devastating secondary damage, including apoptosis, autophagy, necrosis, and necroptosis. 30 Rapid reconstruction of capillaries leads to a shorter gap in tissue ischaemia, lower production of inflammatory factors, and less I/R injury (IRI). Therefore, promoting angiogenesis is a priority related to increasing the survival rate. In this study, the survival of flaps treated with RXD was significantly enhanced compared with those in the control group. Furthermore, the MVD increased after RXD treatment. Furthermore, laser Doppler flowmetry showed that RXD enhanced blood perfusion and improved microcirculation. The flap survival rate was significantly higher after high‐dose treatment than that after low‐dose treatment.

IRI and inflammation exacerbate flap necrosis and postpone angiogenesis. Ischaemia occurs in parallel with the inflammatory response when the newborn vessels are reconstructed and the ischaemic tissue is reperfused, and both are accelerated and augmented. 31 The accumulation of lactate and other toxic ischaemic metabolites stimulates the activation of chemical mediators and enzymes, including phospholipase A2 and lysozymes. Phospholipids on the cell membrane are converted to arachidonic acid mediated by phospholipase A2, leading to inflammatory mediators such as leukotrienes and prostaglandins. 32 Reactive oxygen species (ROS) are also produced after IRI, which oxidise a large number of biological molecules, including proteins, lipids, and DNA. 33 MDA, which directly reflects membrane lipid peroxidation, 34 is produced after ROS damage cells. As an antioxidant metalloenzyme, SOD is a synonym for the degree of antioxidation. Pre‐treating rat skeletal muscle with hypoxia decreases inflammation and protects skeletal muscle after acute IRI. 35 The aggravation and persistence of inflammation generate programmed cell death and contribute to IRI and organ injury. 36 Thus, many studies have been performed to reduce inflammation induced by I/R and to restrict tissue damage. Targeted by inflammation and oxidative stress, cordycepin reduces IRI. 37 An alcoholic liver disease study showed that RXD decreased alcohol‐induced ROS generation in a fatty liver model by downregulating cytochrome P450 2E1 and promoting SOD1 expression. 38 Inflammation, vascular injury, and compromised oxygen availability are hallmarks of an immunological reaction to tissue damage and infection. The main characteristic of distal flap necrosis is neutrophil infiltration, as supported by a previous study showing that the extent of necrosis occurs in parallel with the inflammation level. 39 The levels of Bcl‐2 and SOD2 changed with the increase in HIF‐1α, indicating that RXD protects against doxorubicin‐induced cardiotoxicity. 40 Another study showed that RXD protects against ischaemia‐induced acute kidney injury by enhancing the level of CD73 and decreasing the activation of the AIM2 inflammasome. 41 Our results show that RXD upregulated the expression of SOD and downregulated the MDA content. Neutrophil infiltration was also downregulated, as well as the expression of IL‐6, IL‐1β, and TNF‐α. These effects were dose‐dependent. We conclude that RXD prevented IRI and reduced inflammation.

HIF‐1 is a nuclear transcription factor that regulates the oxygen level. Consisting of an alpha and a beta subunit, HIF‐1 participates in various physiological processes, including vascularization, cartilage development, neural tissue formation in developing embryos, and tumour formation. 42 , 43 , 44 The alpha subunit is regulated by oxygen and the beta subunit is continuously expressed. HIF‐1α is one of three isoforms (HIF‐1α, HIF‐2α, and HIF‐3α) in which expression is protected under hypoxic conditions. 45 The three PHD subtypes (PHD1, PHD2, and PHD3) are oxygen‐sensitive enzymes that regulate HIF‐1α activity. 46 Under normoxic conditions, HIF‐1α is degenerated by von Hippel–Lindau tumour protein (VHL), catalysed by PHD via the ubiquitin‐proteasome system. 47 , 48 The function of PHD is limited under hypoxic conditions, which prevents the degeneration of HIF‐1α. 49 Thus, HIF‐1α dimerizes with HIF‐1β. The heterodimer translocates to the nucleus, and after binding with p300, the transcriptional activation complex acts at the DNA site and target genes (eg, VEGF and angiogenin) are activated by transcription of HIF‐1α. 50 RXD alleviates hyperoxia‐induced lung injury in newborn mice by enhancing the expression of proangiogenetic factor and stabilising HIF‐1α. 51 RXD accelerates wound healing in diabetic rats by promoting angiogenesis through HIF‐1α/VEGF signalling. 16 Our results also demonstrate that RXD promoted the expression of HIF‐1α dose‐dependently.

VEGF is a protein family consisting of several members, including VEGF‐A, VEGF‐B, VEGF‐C, VEGF‐D, VEGF‐E, placental growth factor, and endocrine gland‐derived vascular endothelial growth factor. 52 VEGF‐A is considered a therapeutic target in various cancers based on its profound effects, 53 in cardiovascular diseases, 54 and in tendon injuries. 55 VEGF‐A is a hypoxia‐inducible protein 56 secreted by fibroblasts, 57 platelets, 58 neutrophils, 59 and endothelial cells. 60 VEGF‐A regulates its tyrosine kinase receptors to stimulate the pathway. The receptors are homodimeric peptides with an extracellular domain, a transmembrane domain, and an intracellular domain, 61 including VEGFR‐1, VEGFFR‐2, and VEGFR‐3. 62 VEGFR‐1 has a higher affinity than VEGFR‐2 but lower kinase activity 63 ; thus, it likely participates in the differentiation of endothelial cells. VEGFR‐2 regulates blood and lymphatic vessels and is mainly detected in the endothelial cells of those vessels. 62 Many treatments have been designed based on the function of VEGFR‐2 in regulating normal and pathological angiogenesis. Apatinib is a selective inhibitor used to inhibit the growth of osteosarcomas by targeting the VEGFR2/STAT3/BCL‐2 signalling pathway. 64 In a pulmonary hypertension rat model with knock‐in phenylalanine, transudative fluid was found with an overactivated VEGF‐A/VEGFR2 Y949 signalling axis. 65 VEGFR‐3 is mainly expressed in the endothelium of lymph and high endothelial venules. 66 Our study also demonstrates that higher VEGF expression led to the generation of more newborn blood vessels, which enhanced the survival of the random flaps.

More studies are necessary to refine the injection dose and to detail the relevant molecular signalling. The target pathway will be blocked to re‐examine the hypothesis. This study was an experimental investigation performed exclusively in rats, so more studies are needed on human skin flap transplantation. Our results indicate that RXD should be considered a potential therapeutic option for flap therapy.

5. CONCLUSION

Our experiments demonstrate that RXD promoted flap survival by enhancing angiogenesis and restricting the vicious cycle of inflammation and IRI in the HIF‐1α/VEGF signalling pathway (Figure 9). Furthermore, the effects were dose‐dependent. Therefore, RXD is a potential treatment for clinical use in random flap transplantation.

FIGURE 9.

FIGURE 9

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Roxadustat promotes hypoxia‐inducible factor‐1α/vascular endothelial growth factor signalling to enhance random skin flap survival.

CONFLICT OF INTEREST STATEMENT

The authors declare no potential conflict of interest.

ACKNOWLEDGEMENTS

This study was supported by Project of Zhejiang Province Traditional Chinese Medicine Science and Technology Research (2022ZB216), Project of Wenzhou Science and Technology Bureau (Y20210054), and Zhejiang Provincial Medical and Health Science and Technology Program (2021KY210).

Lan Q, Wang K, Meng Z, et al. Roxadustat promotes hypoxia‐inducible factor‐1α/vascular endothelial growth factor signalling to enhance random skin flap survival in rats. Int Wound J. 2023;20(9):3586‐3598. doi: 10.1111/iwj.14235

Qicheng Lan and Kaitao Wang contributed equally to this work and share first authorship.

DATA AVAILABILITY STATEMENT

Data available on request due to privacy/ethical restrictions.

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