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. 2023 Dec 1;18(12):e0294566. doi: 10.1371/journal.pone.0294566

Silencing of topical proline hydroxylase domain 2 promotes the healing of rat diabetic wounds by phosphorylating AMPK

Defu Xie 1,2, Mengchang Liu 1,2, Yingxi Lin 1,2, Xingke Liu 1,2, Hong Yan 1,2,*
Editor: Kanhaiya Singh3
PMCID: PMC10691724  PMID: 38039326

Abstract

Background

For diabetic ulcers, the impaired response to hypoxia is a key feature associated with delayed healing. In the early phase of hypoxia, hypoxic signaling activates the AMPK system through direct phosphorylation of the PHD2 pathway, producing a significant endogenous hypoxic protective effect.

Methods

Twenty Sprague-Dawley (SD) rats were randomly divided into two groups: treatment (sh-PHD2) and control (sh-Control). Using lentiviral encapsulation of PHD2-shRNA and transfection, the silencing efficiency of PHD2 expression was verified in rat dermal fibroblasts (RDF) and in rat aortic endothelial cells (RAECs). Changes in the ability of RDF and RAECs to proliferate, migrate, and in the rate of ATP production were observed and then tested after inhibition of AMPK phosphorylation using dorsomorphin. The lentiviral preparation was injected directly into the wounds of rats and wound healing was recorded periodically to calculate the healing rate. Wounded tissues were excised after 14 days and the efficiency of PHD2 silencing, as well as the expression of growth factors, was examined using molecular biology methods. Histological examination was performed to assess CD31 expression and therefore determine effects on angiogenesis.

Results

Lentiviral-encapsulated PHD2-sh-RNA effectively suppressed PHD2 expression and improved the proliferation, migration, and ATP production rate of RDF and RAEC, which were restored to their previous levels after inhibition of AMPK. The rate of wound healing, vascular growth, and expression of growth factors were significantly improved in diabetic-model rats after local silencing of PHD2 expression.

Conclusion

Silencing of PHD2 promoted wound healing in diabetic-model SD rats by activating AMPK phosphorylation.

Introduction

The latest data from the International Diabetes Federation (IDF) show that in 2017, there were approximately 425 million patients diagnosed with diabetes in the world, of which 114 million were located in China, ranking first in the world [1]. Microvascular and macrovascular lesions are one of the main complications in patients with diabetes and are considered an important risk factor for death [2, 3]. It is also an intrinsic cause of impaired wound healing [4, 5]. This is because the high glucose environment and the local inflammatory environment of the wound can lead to a decrease in the number and function of endothelial progenitor cells, thus downregulating the expression of vascular endothelial growth factor (VEGF) and its receptors and inhibiting the formation of new blood vessels [6, 7]. In addition, vascular rupture and the inflammatory response increases the oxygen consumption of tissue cells, further worsening hypoxia in the injured area. Therefore, for patients with diabetes, one of the key factors for wound healing is whether they can timely and effectively respond to the hypoxic state.

Prolyl hydroxylase domain protein 2 (PHD2) is widely recognized as a fine oxygen sensor, a central regulator of cellular oxygen homeostasis, and a major inducer of the adaptive response to hypoxia. In the presence of oxygen, PHD2 hydroxylates an oxygen-sensitive in vivo-specific proline residue HIF-1α, which is then marked for degradation by the proteasome [8]. However, under hypoxic conditions, the catalytic activity of PHD2 hydroxylation decreases, and thus maintains the stability of HIF-1α and activates the transcription of its target genes in the nucleus. However, current studies have shown that not only does PHD2 play the role of an "HIF regulator" in the oxygen detection signal cascade, it also includes other protective anti-hypoxia responses that do not depend on HIF [9, 10]. More critically, activation of the PHD2 signaling pathway modulates various cellular responses to hypoxia and preconditioning stimuli [9]. Under hypoxic conditions, cell survival depends on the ability to efficiently maintain intracellular ATP levels in the presence of restricted mitochondrial oxidative metabolism and the lack of bioenergetic substrates. Therefore, the state of cellular energy is a decisive factor in cell survival and maintaining cellular metabolic homeostasis under hypoxia is particularly important for promoting cellular biological activity. AMP-activated protein kinase (AMPK) is a master regulator of metabolic homeostasis and a cellular energy sensor. It is a heterotrimeric serine/threoninase consisting of 1 catalytic subunit (α1 or rα2) and 2 regulatory subunits (β1 or rβ2 and dγ1, γ2, or γ3) [11, 12]. AMPK is activated under conditions of elevated AMP/ATP, induced by glucose deprivation, muscle contraction, and hypoxia [13], which lays a critical role in maintaining cellular energy homeostasis and adaptive responses [14]. After activation, it can directly phosphorylate downstream proteins or indirectly influence the expression of genes that reduce additional consumption of ATP and increase the tolerance of cells to hypoxia [15]. Combining the role of PHD2 in regulating cellular responses to hypoxic stimulation in the hypoxic state, we speculate that local silencing of PHD2 can promote wound healing in diabetic rats by activating the AMPK pathway.

Methods

Animals, cell lines, and lentiviral vector

Male SD rats (180 g) were purchased from the Southwest Medical University Experimental Animal Centre (Sichuan,China). The rats were housed in a standard environment at room temperature (24±2°C, relative humidity (50±5% =, and a light/dark cycle of 12-h/12-h. All animal operations were performed according to protocols approved by the Animal Care and Use Committee of Southwest Medical University. Methods of sacrifice: The animals were euthanised by intraperitoneal injection of 150 mg/kg of sodium pentobarbital. Efforts to alleviate suffering: Through improving animal facilities, breeding management and experimental conditions, selecting experimental animals, technical routes and experimental means, optimising experimental operation techniques, minimising damage to the animal organism during the experimental process, and reducing the pain and stress suffered by the animals. Rat aortic endothelial cells (RAECs) were purchased from Meisen Chinese Tissue Culture Collections (Meisen CTCC, China). Rat dermal fibroblasts (RDF) were purchased from Procell Life Science & Technology Co., Ltd. (Wuhan, China). Cells were cultured in Dulbecco’s Modified Eagle Medium(DMEM) (Gibco, USA) containing 10% fetal bovine serum, 100 IU/mL penicillin and 100 μg/mL streptomycin in an incubator at 37°C with 5% CO2. The medium was changed every 2 to 3 days. The lentiviral vector expressing rat PHD2-shRNA (pLV.U6.shRNA2(rPHD2).CMVcopGFP-2A-Puro.WPRE) was constructed by PackGene Biotech Co., Ltd. (Guangzhou, China). The lentiviral vector expressing only GFP (pLV.U6.NCshRNA-1.CMV.copGFP-2A-Puro.WPRE) was used as a control. The viruses were produced using Gene-Copoeia protocols (https://www.genecopoeia.com). According to the Gene-Copoeia protocol, lentiviral particles were generated using lipofectamine-mediated lentiviral-expressing plasmids, the packaging plasmid psPAX2 and the envelope plasmid pMD2.G in CAF, and lentiviral particles were collected to transduce target cells.

Lentiviral vector transfection

Cells (RDF, RACE) were seeded in 6-well plates (5×104 cells/well) and cultured overnight. When the cells reached 50–60% confluency, the culture medium was removed. The cells were then transfected with virus-containing diluted medium with 8 μg/mL polybrene for 4 h. Then, the medium was replaced with serum-enriched medium, and the cells were cultured for an additional 96 h. Transfected cells were pooled and treated with puromycin for 1 week. Subsequently, transfected cells were collected and processed for follow-up experiments. These cells were divided into three groups: sh-PHD2 (sh-PHD2 infection group), sh-Control (control group expressing GFP alone), and sh-PHD2+8 μM dorsomorphin (after sh-PHD2 infection, cells were treated with 8 μM dorsomorphin were treated for 24 hours). Dorsomorphin was purchased from AbMole (China) (Cat. No. M2238), a selective reversible inhibitor of AMPK.

Scratch test

One day in advance, infected cells were inoculated in a six-well plate according to 2x105/well, and the 20 μL pipette tip was used to quickly draw a scratch along the middle of the plate. After washing with PBS, 2 mL of serum-free medium was added and the plates were imaged after 0, 6, 12, 24 h of cell migration with an inverted microscope to calculate the migration area.

Detection of cell proliferation and cell viability using the CCK8 method

The proliferation of RDFs and RAECs was detected using the Shanghai Ethan Cell Counting Kit 8 (CCK8). Each group of cells was seeded in 96-well plates at 2000 cells/well. Each well contained 100 μL of complete medium and 10 μL of CCK8 reagent was added after 24, 48, 72 and 96 h, and the absorbance at 450 nm was measured after 4 h of incubation.

Real-time ATP rate assay

The day before the experiment, cells were seeded in Seahorse XFe 24-well microplates at 15,000 cells/well, and the probe plates were hydrated overnight in a 37°C CO2-free incubator. On the day of the experiment, the detection solution was prepared in advance by adding 10 mmol/L XF (Extrancellular Flux) glucose, 1 mmol/L XF pyruvate, and 2 mmol/L XF glutamine to 100Ml Seahorse XF DMEM pH 7.4) and preheated to 37°C. The microplate was then prepared by first washing the microplate with detection solution and then adding 500 μL of freshly preheated detection solution. Next, a 15 μmol/L oligomycin and 5 μmol/L the rotenone/antimycin A mixture were prepared and added to the appropriate wells. Absorbance was then read using the Seahorse XFe24 Analyzer (Agilent Technologies, Sana Clara, USA) according to the manufacturer’s instructions.

Preparation of the SD rat diabetic wound model

After 3 days of adaptive feeding, rats were randomly divided into two groups: the treatment group (sh-PHD2) (in the diabetes model rats, the wound was injected with the PHD2-shRNA lentivirus) (n = 10) and the control group (sh-Control) (in the diabetes model rats, the wound was injected with the sh-Control lentivirus) (n = 10). The diabetic rat model was established by a one-time injection of streptozotocin (50 mg/kg) into the tail vein (AbMole, China). The fasting blood glucose level of each rat was detected 72 h after streptozotocin injection: fasting blood glucose values were ≥16.70 mmol/L, and were indicative of the successful preparation of the diabetes model. After ensuring the diabetes model had been achieved, all rats (including the control group) were used to establish wound models. A circular area with a diameter of 2 cm was marked in the middle of the back, and the skin surface was prepared and disinfected. Using a scalpel and ophthalmic scissors, a full thickness skin defect was created on the marked area, and a sterile dressing was applied to the wound and surrounding areas after injecting the corresponding drugs.

Wound healing rate

On days 0, 3, 7, 14, and 21, digital cameras were used to collect photos of back wounds under fixed focal length and pixel conditions, respectively. The wound area was calculated using special ImageJ software, and the degree of wound healing was calculated according to the change in the wound area using the following formula: wound healing rate = (wound area—wound area at each time point) / wound area × 100%.

Western blotting analysis

To determine the level of expression of growth factors (VEGF, fibroblast growth factor 2 [FGF-2]) in wound tissue and the inhibition efficiency of PHD2 in the wound, samples of wound tissue and surrounding areas were collected on day 14. Protein expression was determined using mouse anti-rat PHD2 antibody (Abcam, USA) and mouse anti-rat VEGF antibody (Abcam, USA) and mouse anti-rat FGF-2 antibody (Abcam, USA).

Reverse transcription polymerase chain reaction (RT-PCR)

Rat PHD2, GAPDH, VEGF, and FGF-2 gene sequences were recovered from PubMed/Negyl GenBank, with GAPDH as the reference gene. Primers were designed using Primer Express 5.0 software as follows: PHD2 sequence: forward: 5’-TTGATAGACTGCTGTTTTTCTGG-3’; reverse: 5’-CCTCACACCTTTTTCACCTGTTA-3’. GAPDH sequence: forward: 5’-CCTGGAGAAACCTGCCAAGT-3’; reverse: 5’-TAGCCCAGGATGCCCTTTAG-3’ [10, 16]. VEGF sequence: forward: 5’-GTCACCGTCGACAGAACAGT-3’; reverse: 5’-GACCCAAAGTGCTCCTCGAA-3’.FGF-2 sequence: forward: 5’-TCCATCAAGGGAGTGTGTGC-3’; reverse: 5’-GGACTCCAGGCGTTCAAAGA-3’. Total RNA extraction was performed after RAECs were successfully infected and treated with puromycin for 1 week. mRNA quantification was performed by real-time quantitative RT-PCR.

CD31 immunohistochemistry

On day 14 after injury, the wound surface and surrounding 2-mm thick uninjured skin were excised to prepare paraffin sections. Vascular endothelial cell density was evaluated by CD31 immunohistochemical staining. Rat liver sections were stained as the positive control group (PCG). Paraffin-embedded sections were prepared using normal rat wounds, and PBS was used instead of the primary antibody as the negative control group (NCG).

Statistical analysis

Statistical analysis was performed using SPSS for Windows ver. 13.0 (SPSS, inc., Chicago, IL, USA).A two-sided P<0.05 was considered statistically significant and is represented in figures as *P<0.05, **P<0.01, ***P<0.001.

Results

PHD2 expression decreased in RAECs after infection with the PHD2-shRNA lentivirus

Fluorescence detection of PHD2 expression performed one week after virus infection to evaluated virus infection. After RAECs were successfully infected and treated with puromycin for 1-week, quantitative real-time fluorescence RT-PCR was performed to detect the mRNA expression of PHD2. After treatment, the expression of PHD2 mRNA decreased to 16.6%, indicating the effectiveness of virus-induced silencing (P<0.001) (Fig 1A).

Fig 1.

Fig 1

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a:PHD2 mRNA expression was significantly reduced in RAECs after successful PHD2-shRNA infection compared with controls (p<0.001). b,c:Western-blot analysis of the expression levels of PHD2 protein and the phosphorylation level of AMPK protein in different groups.

Western blotting analysis

The western blotting analysis showed that the relative density of PHD2 in the sh-PHD2 group was significantly lower than in the sh-Control group, and there was no significant change in PHD2 expression after the addition of dorsomorphin (P<0.05); differences in the remaining groups were not statistically significant. The difference in relative AMPK density between the groups was not statistically significant. Since AMPK is phosphorylated prior to exerting its functional activity, we further explored the expression of phosphorylated-AMPK (p-AMPK) and found that its relative density was significantly higher in the sh-PHD2 group (P<0.05), while the difference was not statistically significant in the remaining groups (Fig 1B). Using ImageJ quantitative analysis, the expression of PHD2 was reduced by 62.7% ± 2.3% in the experimental group, while AMPK phosphorylation levels increased by 47.95% ± 2.635% (Fig 1C).

PHD2 enhanced RDF and RAEC cell proliferation and viability, and this effect disappeared when AMPK phosphorylation was inhibited

After four consecutive days of absorbance measurements, we compared the value-added rate of each group of cells for the following three days using the absorbance of the first day as a benchmark. Cell proliferation in the sh-PHD2 group was significantly higher than that in the sh-Control group, indicating that PHD2 silencing could enhance the proliferative capacity of both cell lines. The proliferative capacity decreased significantly after the addition of dorsomorphin (Fig 2A), which indicates that inhibition of AMPK phosphorylation can effectively inhibit the enhanced proliferative capacity brought about by silencing PHD2. Similarly, the absorbance of the cells in each group measured at 72 h, using the sh-Control group as a benchmark for cell viability, were consistent with the above trend (Fig 2B).

Fig 2.

Fig 2

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a: Changes in the proliferative capacity of cells in different groups at days 1, 2, 3, and 4. b: Changes in the viability of cells in different groups at 72h.

PHD2 improved the migration capacity of RDF and RAEC, and this effect was abolished by inhibition of AMPK phosphorylation

Using scratch assays, the migration ability of RDFs in the PHD2-shRNA group was significantly enhanced after effective silencing of PHD2. Compared to the control group, the width of the scratch was significantly reduced at 12 and 24 h and the healing rate was significantly faster (P<0.05) (Fig 3A). We then quantified the migration ability of the RAECs using a Transwell assay. The migration ability of endothelial cells in the PHD2-shRNA group was significantly higher than that of the control group and the difference was statistically significant (P<0.05) (Fig 3B). After inhibiting AMPK phosphorylation, the above-mentioned effects were significantly reduced.

Fig 3.

Fig 3

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a: Scratch test was used to detect the migration ability of RDFs in different groups.b: Transwell experiment was used to detect the migration ability of RAECs.

PHD2 enhanced the overall ATP production rate of RAEC and RDF cells, and the effect was lost when AMPK phosphorylation was inhibited

We used the Seahorse XFe24 extracellular flux analyzer to quantify real-time rates of ATP synthesis of RAECs and RDFs. The rate of glycoATP production represents the rate of ATP synthesis associated with the conversion of glucose to lactate via the glycolytic pathway, and the rate of mitoATP production represents the rate of ATP synthesis associated with mitochondrial oxidative phosphorylation, and these are summed to obtain the total ATP production rate. We found that the total rate of ATP production of both RAEC (Fig 4A) and RDF (Fig 4B) was significantly higher than that of the control group after effective silencing of PHD2, implying that the total metabolic capacity of both cells was enhanced. We then calculated the ratio of mito-ATP production to glyco-ATP production for both cell lines. The ratio in the Sh-PHD2 group was significantly lower than that in the Sh-Control group (Fig 4C and 4D), which indicated that the Sh PHD2 group achieved a higher glyco-ATP production rate, and also implied that its aerobic glycolytic capacity was improved.

Fig 4.

Fig 4

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Total ATP Production Rate of RAECs (a) and RDFs (b) and the ratio of mito-ATP Production Rate to glyco-ATP Production Rate of RAECs (c)and RDFs(d).

Topical PHD2 silencing promoted wound repair in diabetic rats

Gross observation of wounds on days 0, 3, 7, 10, 14, and 21 revealed a significant increase in wound healing rate in the treatment group (Fig 5A). From day 3 onwards, there was a significant increase in wound closure at each time point relative to the control group (Fig 5B), with wounds in the treated group starting to heal on day 7 (74%), while only 47% of wounds in the control group healed at the same time point, and this relationship continued until wound closure was achieved in the experimental group.

Fig 5.

Fig 5

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a: Wound healing of treatment group (sh-PHD2) and control group (sh-Control) on days 0, 3, 7, 10, 14, and 21.b: Wound healing rates of two groups of rats at different time points.

Silencing of PHD2 expression promoted angiogenesis in wounds

Wound angiogenesis was characterized and quantified by the detection of CD31 by immunohistochemical staining. There was an increase in endothelial cell density in rat wounds following silencing of PHD2 on day 14 (Fig 6A). Quantitative analysis of the staining results of the rat wound sections using Image J showed that the staining density of CD31 increased by 26.13% ± 2.38% in the experimental group compared to the control group (Fig 6B). These results suggested that silencing of PHD2 exerted a promotional effect on angiogenesis in the diabetic rat wound model.

Fig 6.

Fig 6

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a,b: Results of traumatic tissues of two groups of animals on day 14 after staining for CD31 by immunohistochemistry and after quantification of their CD31 expression levels.

Western blot analysis of tissues

Western blotting analysis revealed that the relative density of PHD2 was significantly lower in both treatment groups on day 14 (P<0.05), while the relative densities of both VEGF and FGF-2 increased significantly (P<0.05) (Fig 7).

Fig 7. The expression levels of PHD2, VEGFA, and FGF-2 in the wounds of the two groups of rats were analyzed by Western-blot.

Fig 7

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Discussion

For patients with diabetes, the neovascularization ability of a wound is significantly altered, thus diabetic wounds are undoubtedly hypoxic [17–21]. The proteolytic hydroxylase domain (PHD1-3) is an oxygen-sensitive enzyme that acts as an oxygen sensor in the cell, providing a direct link between oxygen availability and cellular adaptation to hypoxia. Among the three distinct members of the PHD family, PHD2 has been shown to be the most abundant and critical hydroxylase in most cell lines studied to date [22]. Although its most recognized role is that of a ’HIF regulator’, we previously found that PHD2 can activate calmodulin-dependent protein kinase (CaMKK) β upstream of AMP-activated protein kinase (AMPK) and thereby protect against hypoxia-induced effects in cardiomyocytes; phosphorylated AMPK is a key factor in cell survival as it regulates energy metabolism [15, 23]. This hypothesis has also been confirmed by other researchers studying systemic iron homeostasis [24]. Thus, we speculated that in diabetic wounds, PHD2 may promote wound repair by regulating AMPK. Therefore, we first performed local silencing of PHD2 expression in wound tissue of diabetic-model rats using lentivirus coated PHD2-shRNA, and the efficiency of wound repair increased significantly, as expected. We then quantified angiogenesis by evaluating the presence of CD31-labeled vascular endothelial cells [25]. Our results also indicated that silencing PHD2 was beneficial for stimulation of angiogenesis within the wound. To demonstrate the effectiveness of local silencing in the wound region, we also performed a quantitative analysis of wound tissue proteins and found that the density of PHD2 expression in the treatment group was significantly lower than in the control group. The growth factors VEGF and FGF-2 have a synergistic effect on angiogenesis and are significantly more effective than cytokines alone [26–29]. None of the clinical trials to date testing VEGF or FGF-2 treatments alone have demonstrated significant clinical efficacy [27, 30, 31]. We also quantified VEGF and FGF-2 levels, both of which showed a marked increase expression in tissues in harboring cells without PHD2 expression.

Having determined that silencing of PHD2 in wound tissue is beneficial for wound repair, we next needed to explore whether activation of AMPK was also involved in this response. Due to the rapid activity of AMPK inhibitors on the wound surface and the instability of phosphorylated-AMPK itself, we selected two cell lines that were closely related to wound repair to discuss this mechanism. Impaired wound healing in patients with diabetes is a well-known phenomenon, and fibroblast dysfunction may be involved in this process [32–34], while endothelial cells are an integral part of angiogenesis. We first demonstrated the effectiveness of inhibition of PHD2 expression, as demonstrated by the RT-PCR and western blotting results following lentiviral silencing PHD2. Next, we determined that silencing of PHD2, was associated with a significant increase in the phosphorylation of AMPK, and this effect could be completely reversed by exposure to dorsomorphin (an AMPK inhibitor) without altering PHD2 expression. Based on this evidence, we explored changes in the proliferation and migration abilities of these two types of cells and found that cells in which the expression of PHD2 was silenced, there was an enhancement of proliferation and migration, with the effects disappearing completely following exposure to dorsomorphin.

It is well known that AMPK is the main regulator of metabolic homeostasis and as a cellular energy sensor, it is the guardian of the energy state of the cell [24, 35]. AMPK is a key regulator of energy homeostasis, and coordinates adaptive responses in metabolic states depleting ATP such as hypoxia, ischemia/reperfusion, and exercise [36]. Therefore, to explore the processes through which energy changes are necessary for living organisms, we further demonstrated that silencing of PHD2 could enhance the total ATP production rate of these two cells, and the effect can still be inhibited by AMPK inhibitors. Furthermore, the ratio of mito-ATP production to glyco-ATP production was calculated to confirm that the aerobic glycolytic capacity, which showed that in both cell lines ATP production was also significantly enhanced after silencing of PHD2.

Although we found that a decrease in PHD2 expression promotes an increase in the phosphorylation level of AMPK, it is undeniable that it also brings about alterations in the expression of other proteins, the most classical and widely recognised of which is the regulation of hif-1α. When oxygen is sufficient, in the co-presence of iron and 2-oxoglu- tarate (2-OG), PHD2 can hydroxylate specific proline residues (Pro402、Pro564) [37] and asparagine residues of hif-1α, which leads to their binding to von HippelLindau protein (pVHL)-E3 ubiquitin ligase complex and finally to proteasomal degradation mediated by E2 ubiquitin ligand [38]. Thus, when PHD2 expression is reduced, hif1-α is unquestionably increased in vivo and the physiological responses it dominates are enhanced. Current studies have shown that the genes downstream of hif-1α mainly include vascular endothelial growth factor (VEGF), erythropoietin (EPO), GLUT1 glucose trans-porter, glycolytic enzymes [39]. The increased expression of these genes also had a positive effect on diabetic wound repair, which suggests to us that perhaps there is a synergistic effect between the increased phosphorylation of AMPK and the increased expression of hif-1α, and we will also further study and investigate the relationship between the two pathways afterwards.

Conclusions

Local silencing of PHD2 in wound tissue can activate a series of adaptive responses of hypoxia-diabetic wounds via phosphorylation of AMPK, thus promoting angiogenesis and accelerating wound repair.

Supporting information

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Acknowledgments

The pilot research was conducted in National Key Clinical Construction Specialty, Wound Repair and Regeneration Laboratory.

Data Availability

All relevant data are within the paper and its Supporting Information files.

Funding Statement

The authors who received the award is Mr Yan. The full name of the funder is (Luzhou-Medical University) Cooperation Project,the grant number is No.2019LZXNYDZ08.The URL of funder website is http://luzhou.tccxfw.com. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.

References

  • 1.da Rocha Fernandes J, Ogurtsova K, Linnenkamp U, Guariguata L, Seuring T, Zhang P, et al. IDF Diabetes Atlas estimates of 2014 global health expenditures on diabetes. Diabetes Res Clin Pract. 2016;117:48–54. doi: 10.1016/j.diabres.2016.04.016 [DOI] [PubMed] [Google Scholar]
  • 2.Solini A, Zoppini G, Orsi E, Fondelli C, Trevisan R, Vedovato M, et al. Resistant hypertension in patients with type 2 diabetes: clinical correlates and association with complications. J Hypertens. 2014;32(12):2401–10; discussion 10. doi: 10.1097/HJH.0000000000000350 [DOI] [PubMed] [Google Scholar]
  • 3.Smith-Palmer J, Brandle M, Trevisan R, Orsini Federici M, Liabat S, Valentine W. Assessment of the association between glycemic variability and diabetes-related complications in type 1 and type 2 diabetes. Diabetes Res Clin Pract. 2014;105(3):273–84. doi: 10.1016/j.diabres.2014.06.007 [DOI] [PubMed] [Google Scholar]
  • 4.Kato T, Inoue T, Node K. Postprandial endothelial dysfunction in subjects with new-onset type 2 diabetes: an acarbose and nateglinide comparative study. Cardiovasc Diabetol. 2010;9:12. doi: 10.1186/1475-2840-9-12 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 5.Baltzis D, Eleftheriadou I, Veves A. Pathogenesis and treatment of impaired wound healing in diabetes mellitus: new insights. Adv Ther. 2014;31(8):817–36. doi: 10.1007/s12325-014-0140-x [DOI] [PubMed] [Google Scholar]
  • 6.Li W, Kandhare AD, Mukherjee AA, Bodhankar SL. Hesperidin, a plant flavonoid accelerated the cutaneous wound healing in streptozotocin-induced diabetic rats: Role of TGF-ss/Smads and Ang-1/Tie-2 signaling pathways. EXCLI J. 2018;17:399–419. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 7.Rufaihah AJ, Johari NA, Vaibavi SR, Plotkin M, Di Thien DT, Kofidis T, et al. Dual delivery of VEGF and ANG-1 in ischemic hearts using an injectable hydrogel. Acta Biomater. 2017;48:58–67. doi: 10.1016/j.actbio.2016.10.013 [DOI] [PubMed] [Google Scholar]
  • 8.Maxwell PH, Ratcliffe PJ. Oxygen sensors and angiogenesis. Semin Cell Dev Biol. 2002;13(1):29–37. doi: 10.1006/scdb.2001.0287 [DOI] [PubMed] [Google Scholar]
  • 9.Elvidge GP, Glenny L, Appelhoff RJ, Ratcliffe PJ, Ragoussis J, Gleadle JM. Concordant regulation of gene expression by hypoxia and 2-oxoglutarate-dependent dioxygenase inhibition: the role of HIF-1alpha, HIF-2alpha, and other pathways. J Biol Chem. 2006;281(22):15215–26. doi: 10.1074/jbc.M511408200 [DOI] [PubMed] [Google Scholar]
  • 10.Chen B, Meinertzhagen IA, Shaw SR. Circadian rhythms in light-evoked responses of the fly’s compound eye, and the effects of neuromodulators 5-HT and the peptide PDF. J Comp Physiol A. 1999;185(5):393–404. doi: 10.1007/s003590050400 [DOI] [PubMed] [Google Scholar]
  • 11.Herzig S, Shaw RJ. AMPK: guardian of metabolism and mitochondrial homeostasis. Nat Rev Mol Cell Biol. 2018;19(2):121–35. doi: 10.1038/nrm.2017.95 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 12.Hardie DG, Ross FA, Hawley SA. AMPK: a nutrient and energy sensor that maintains energy homeostasis. Nat Rev Mol Cell Biol. 2012;13(4):251–62. doi: 10.1038/nrm3311 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 13.Lin SC, Hardie DG. AMPK: Sensing Glucose as well as Cellular Energy Status. Cell Metab. 2018;27(2):299–313. doi: 10.1016/j.cmet.2017.10.009 [DOI] [PubMed] [Google Scholar]
  • 14.Hardie DG. Sensing of energy and nutrients by AMP-activated protein kinase. Am J Clin Nutr. 2011;93(4):891S–6. doi: 10.3945/ajcn.110.001925 [DOI] [PubMed] [Google Scholar]
  • 15.Yan H, Zhang D-X, Shi X, Zhang Q, Huang Y-S. Activation of the prolyl-hydroxylase oxygen-sensing signal cascade leads to AMPK activation in cardiomyocytes. Journal of Cellular and Molecular Medicine. 2012;16(9):2049–59. doi: 10.1111/j.1582-4934.2011.01500.x [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 16.Li Z, Xing YQ, Cui W, Lu Q. Expression and effect of proline hydroxylase domain 2 in retina of diabetic rats. Int J Ophthalmol. 2016;9(3):357–62. doi: 10.18240/ijo.2016.03.05 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 17.Tepper OM, Galiano RD, Capla JM, Kalka C, Gagne PJ, Jacobowitz GR, et al. Human endothelial progenitor cells from type II diabetics exhibit impaired proliferation, adhesion, and incorporation into vascular structures. Circulation. 2002;106(22):2781–6. doi: 10.1161/01.cir.0000039526.42991.93 [DOI] [PubMed] [Google Scholar]
  • 18.Abaci A, Oguzhan A, Kahraman S, Eryol NK, Unal S, Arinc H, et al. Effect of diabetes mellitus on formation of coronary collateral vessels. Circulation. 1999;99(17):2239–42. doi: 10.1161/01.cir.99.17.2239 [DOI] [PubMed] [Google Scholar]
  • 19.Shan K, Liu C, Liu BH, Chen X, Dong R, Liu X, et al. Circular Noncoding RNA HIPK3 Mediates Retinal Vascular Dysfunction in Diabetes Mellitus. Circulation. 2017;136(17):1629–42. doi: 10.1161/CIRCULATIONAHA.117.029004 [DOI] [PubMed] [Google Scholar]
  • 20.Simons M, Annex BH, Laham RJ, Kleiman N, Henry T, Dauerman H, et al. Pharmacological treatment of coronary artery disease with recombinant fibroblast growth factor-2: double-blind, randomized, controlled clinical trial. Circulation. 2002;105(7):788–93. doi: 10.1161/hc0802.104407 [DOI] [PubMed] [Google Scholar]
  • 21.Liu R, Bal HS, Desta T, Behl Y, Graves DT. Tumor necrosis factor-alpha mediates diabetes-enhanced apoptosis of matrix-producing cells and impairs diabetic healing. Am J Pathol. 2006;168(3):757–64. doi: 10.2353/ajpath.2006.050907 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 22.Appelhoff RJ, Tian YM, Raval RR, Turley H, Harris AL, Pugh CW, et al. Differential function of the prolyl hydroxylases PHD1, PHD2, and PHD3 in the regulation of hypoxia-inducible factor. J Biol Chem. 2004;279(37):38458–65. doi: 10.1074/jbc.M406026200 [DOI] [PubMed] [Google Scholar]
  • 23.Liu L, Liu X, Liu M, Xie D, Yan H. Proline hydroxylase domain-containing enzymes regulate calcium levels in cardiomyocytes by TRPA1 ion channel. Exp Cell Res. 2021;407(2):112777. doi: 10.1016/j.yexcr.2021.112777 [DOI] [PubMed] [Google Scholar]
  • 24.Wang C, Zhang W, Xu W, Liu Z, Huang K. AMP-activated protein kinase alpha1 phosphorylates PHD2 to maintain systemic iron homeostasis. Clin Transl Med. 2022;12(5):e854. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 25.Kalantzopoulos GN, Lundvall F, Checchia S, Lind A, Wragg DS, Fjellvag H, et al. In Situ Flow MAS NMR Spectroscopy and Synchrotron PDF Analyses of the Local Response of the Bronsted Acidic Site in SAPO-34 during Hydration at Elevated Temperatures. Chemphyschem. 2018;19(4):519–28. [DOI] [PubMed] [Google Scholar]
  • 26.Cao R, Brakenhielm E, Pawliuk R, Wariaro D, Post MJ, Wahlberg E, et al. Angiogenic synergism, vascular stability and improvement of hind-limb ischemia by a combination of PDGF-BB and FGF-2. Nat Med. 2003;9(5):604–13. doi: 10.1038/nm848 [DOI] [PubMed] [Google Scholar]
  • 27.Bai Y, Leng Y, Yin G, Pu X, Huang Z, Liao X, et al. Effects of combinations of BMP-2 with FGF-2 and/or VEGF on HUVECs angiogenesis in vitro and CAM angiogenesis in vivo. Cell Tissue Res. 2014;356(1):109–21. doi: 10.1007/s00441-013-1781-9 [DOI] [PubMed] [Google Scholar]
  • 28.Kano MR, Morishita Y, Iwata C, Iwasaka S, Watabe T, Ouchi Y, et al. VEGF-A and FGF-2 synergistically promote neoangiogenesis through enhancement of endogenous PDGF-B-PDGFRbeta signaling. J Cell Sci. 2005;118(Pt 16):3759–68. doi: 10.1242/jcs.02483 [DOI] [PubMed] [Google Scholar]
  • 29.Sassi R, Bond RR, Cairns A, Finlay DD, Guldenring D, Libretti G, et al. PDF-ECG in clinical practice: A model for long-term preservation of digital 12-lead ECG data. J Electrocardiol. 2017;50(6):776–80. doi: 10.1016/j.jelectrocard.2017.08.001 [DOI] [PubMed] [Google Scholar]
  • 30.Henry TD, Annex BH, McKendall GR, Azrin MA, Lopez JJ, Giordano FJ, et al. The VIVA trial: Vascular endothelial growth factor in Ischemia for Vascular Angiogenesis. Circulation. 2003;107(10):1359–65. doi: 10.1161/01.cir.0000061911.47710.8a [DOI] [PubMed] [Google Scholar]
  • 31.Lee SH, Schloss DJ, Swain JL. Maintenance of vascular integrity in the embryo requires signaling through the fibroblast growth factor receptor. J Biol Chem. 2000;275(43):33679–87. doi: 10.1074/jbc.M004994200 [DOI] [PubMed] [Google Scholar]
  • 32.Desta T, Li J, Chino T, Graves DT. Altered fibroblast proliferation and apoptosis in diabetic gingival wounds. J Dent Res. 2010;89(6):609–14. doi: 10.1177/0022034510362960 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 33.Siqueira MF, Li J, Chehab L, Desta T, Chino T, Krothpali N, et al. Impaired wound healing in mouse models of diabetes is mediated by TNF-alpha dysregulation and associated with enhanced activation of forkhead box O1 (FOXO1). Diabetologia. 2010;53(2):378–88. doi: 10.1007/s00125-009-1529-y [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 34.Lerman OZ, Galiano RD, Armour M, Levine JP, Gurtner GC. Cellular Dysfunction in the Diabetic Fibroblast. The American Journal of Pathology. 2003;162(1):303–12. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 35.Srivastava S, Toraldo G, Weitzmann MN, Cenci S, Ross FP, Pacifici R. Estrogen decreases osteoclast formation by down-regulating receptor activator of NF-kappa B ligand (RANKL)-induced JNK activation. J Biol Chem. 2001;276(12):8836–40. doi: 10.1074/jbc.M010764200 [DOI] [PubMed] [Google Scholar]
  • 36.Kahn BB, Alquier T, Carling D, Hardie DG. AMP-activated protein kinase: ancient energy gauge provides clues to modern understanding of metabolism. Cell Metab. 2005;1(1):15–25. doi: 10.1016/j.cmet.2004.12.003 [DOI] [PubMed] [Google Scholar]
  • 37.Forristal CE, Levesque J-P. Targeting the Hypoxia-Sensing Pathway in Clinical Hematology. Stem Cells Translational Medicine. 2014;3(2):135–40. doi: 10.5966/sctm.2013-0134 [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 38.Wu Y, Jiang Z, You Q, Zhang X. Application of in-vitro screening methods on hypoxia inducible factor prolyl hydroxylase inhibitors. Bioorg Med Chem. 2017;25(15):3891–9. doi: 10.1016/j.bmc.2017.05.026 [DOI] [PubMed] [Google Scholar]
  • 39.Semenza GLWaGL. Desferrioxamine Induces Erythropoietin Gene Expression and Hypoxia-Inducible Factor 1 DNA-Binding Activity: Implications for Models of Hypoxia Signal Transduction. Blood. 1993:3610–5. [PubMed] [Google Scholar]

Decision Letter 0

Kanhaiya Singh

31 Aug 2023

PONE-D-23-10650Silencing of topical proline hydroxylase domain 2 promotes the healing of rat diabetic wounds by phosphorylating AMPKPLOS ONE

Dear Dr. Yan,

Thank you for submitting your manuscript to PLOS ONE. After careful consideration, we feel that it has merit but does not fully meet PLOS ONE’s publication criteria as it currently stands. Therefore, we invite you to submit a revised version of the manuscript that addresses the points raised during the review process.

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We look forward to receiving your revised manuscript.

Kind regards,

Kanhaiya Singh, Ph.D

Academic Editor

PLOS ONE

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3. Thank you for stating the following financial disclosure: 

"Tthe authors who received the award is Mr Yan . The  full name of the funder is (Luzhou-Medical University) Cooperation Project,the grant number is No. 2019LZXNYDZ08.The URL of funder website is http://luzhou.tccxfw.com .The sponsor was involved in the design of this study."

Please state what role the funders took in the study.  If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript." 

If this statement is not correct you must amend it as needed. 

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

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"Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

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We will update your Data Availability statement to reflect the information you provide in your cover letter.

5. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels. 

  

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6. Please upload a copy of Supporting Information Figure/Table/etc. Fig.1-7 which you refer to in your text on page 22.

7. Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Additional Editor Comments:

Although the reviewers have found this study of interest, they have suggested to have additional validation of the data presented.

[Note: HTML markup is below. Please do not edit.]

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

2. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: In the present study, the authors demonstrate the critical role of the PHD2 pathway in the hypoxic signaling cascade. Hypoxic signaling activates the AMPK system by directly phosphorylating the PHD2 pathway, leading to a significant endogenous hypoxic protective effect. The study's objective and rationale are well-described, and the authors provide a clear explanation of the methods used. The results obtained are significant.

Here are a few minor suggestions for improvement:

The abbreviation "DMEM" is not explained on page 4, line 86.

The abbreviation "XF" is not explained on page 6, line 126.

In the References section, the word "References" should be written before listing the references.

In reference number 7, the journal name is written in all capital letters. It should be formatted similarly to the other references.

In Figure 4, the labels for "sh-PHD2" and "sh-PDH2+8μM Dorsomorphin" are not written properly in the histogram.

Reviewer #2: In current study, Xie et al. did a detail study of Silencing of topical proline hydroxylase domain 2 promotes the healing of rat diabetic wounds by phosphorylating AMPK. In my opinion, the study design is good and well-illustrated. However following points should be taken into consideration in the present form of this manuscript.

1. To achieve effective suppression of PHD2, authors should possess a comprehensive understanding of transcriptomics through thorough and high-throughput study.

2. The authors should emphasize the distinct pathways that undergo changes due to sh-PHD2. These pathways should be elaborated upon in greater detail within the discussion section.

3. The authors should verify that the figures in the panel maintain an optimal resolution for the imaging of Figure 3, Figure 5, and Figure 6.

4. Authors should ensure that the gene names should be written in italics.

**********

6. PLOS authors have the option to publish the peer review history of their article (what does this mean?). If published, this will include your full peer review and any attached files.

If you choose “no”, your identity will remain anonymous but your review may still be made public.

Do you want your identity to be public for this peer review? For information about this choice, including consent withdrawal, please see our Privacy Policy.

Reviewer #1: Yes: Rajnesh Kumari Yadav

Reviewer #2: No

**********

[NOTE: If reviewer comments were submitted as an attachment file, they will be attached to this email and accessible via the submission site. Please log into your account, locate the manuscript record, and check for the action link "View Attachments". If this link does not appear, there are no attachment files.]

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Attachment

Submitted filename: Paper.pdf

Click here for additional data file. (86.9KB, pdf)
PLoS One. 2023 Dec 1;18(12):e0294566. doi: 10.1371/journal.pone.0294566.r002

Author response to Decision Letter 0

4 Oct 2023

Dear Editor, Dear reviewers,

Thank you for your letter dated September 1. We were pleased to know that our work was rated as potentially acceptable for publication in Journal, subject to adequate revision. We thank the reviewers for the time and effort that they have put into reviewing the previous version of the manuscript. Their suggestions have enabled us to improve our work. Based on the instructions provided in your letter, we uploaded the file of the revised manuscript. Accordingly, we have uploaded a copy of the original manuscript with all the changes highlighted by using the track changes mode in Word. Appended to this letter is our point-by-point response to the comments raised by the reviewers. The comments are reproduced and our responses are given directly afterward in a different color (red). We would like also to thank you for allowing us to resubmit a revised copy of the manuscript.

We hope that the revised manuscript is accepted for publication in“PLOS ONE” .

Sincerely, Hong Yan

Responses to journals:

1. Please ensure that your manuscript meets PLOS ONE's style requirements, including those for file naming. The PLOS ONE style templates can be found at

https://journals.plos.org/plosone/s/file?id=wjVg/PLOSOne_formatting_sample_main_body.pdf and https://journals.plos.org/plosone/s/file?id=ba62/PLOSOne_formatting_sample_title_authors_affiliations.pdf

Responses:We have revised the manuscript according to your template standard when we started to submit the manuscript, if there is any deficiency, we hope that you can clearly point it out, and we will make changes to it as soon as possible.

2.To comply with PLOS ONE submissions requirements, in your Methods section, please provide additional information regarding the experiments involving animals and ensure you have included details on (1) methods of sacrifice, (2) efforts to alleviate suffering.

Responses:We have added methods of sacrifice and efforts to alleviate sufferingon page 4, line 85 of the manuscript. if there are any deficiencies, we hope that you will let us know and we will perfect them at the first opportunity.

3. Thank you for stating the following financial disclosure:

"Tthe authors who received the award is Mr Yan . The full name of the funder is (Luzhou-Medical University) Cooperation Project,the grant number is No. 2019LZXNYDZ08.The URL of funder website is http://luzhou.tccxfw.com .The sponsor was involved in the design of this study."

Please state what role the funders took in the study. If the funders had no role, please state: "The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript."

If this statement is not correct you must amend it as needed.

Please include this amended Role of Funder statement in your cover letter; we will change the online submission form on your behalf.

Responses:For contributions made by funders, we have added information in Cover Latter.

4. In your Data Availability statement, you have not specified where the minimal data set underlying the results described in your manuscript can be found. PLOS defines a study's minimal data set as the underlying data used to reach the conclusions drawn in the manuscript and any additional data required to replicate the reported study findings in their entirety. All PLOS journals require that the minimal data set be made fully available. For more information about our data policy, please see http://journals.plos.org/plosone/s/data-availability.

"Upon re-submitting your revised manuscript, please upload your study’s minimal underlying data set as either Supporting Information files or to a stable, public repository and include the relevant URLs, DOIs, or accession numbers within your revised cover letter. For a list of acceptable repositories, please see http://journals.plos.org/plosone/s/data-availability#loc-recommended-repositories. Any potentially identifying patient information must be fully anonymized.

Important: If there are ethical or legal restrictions to sharing your data publicly, please explain these restrictions in detail. Please see our guidelines for more information on what we consider unacceptable restrictions to publicly sharing data: http://journals.plos.org/plosone/s/data-availability#loc-unacceptable-data-access-restrictions. Note that it is not acceptable for the authors to be the sole named individuals responsible for ensuring data access.

We will update your Data Availability statement to reflect the information you provide in your cover letter.

Responses:We have uploaded the minimum dataset as support information.

5. PLOS ONE now requires that authors provide the original uncropped and unadjusted images underlying all blot or gel results reported in a submission’s figures or Supporting Information files. This policy and the journal’s other requirements for blot/gel reporting and figure preparation are described in detail at https://journals.plos.org/plosone/s/figures#loc-blot-and-gel-reporting-requirements and https://journals.plos.org/plosone/s/figures#loc-preparing-figures-from-image-files. When you submit your revised manuscript, please ensure that your figures adhere fully to these guidelines and provide the original underlying images for all blot or gel data reported in your submission. See the following link for instructions on providing the original image data: https://journals.plos.org/plosone/s/figures#loc-original-images-for-blots-and-gels.

In your cover letter, please note whether your blot/gel image data are in Supporting Information or posted at a public data repository, provide the repository URL if relevant, and provide specific details as to which raw blot/gel images, if any, are not available. Email us at plosone@plos.org if you have any questions.

Responses:We have uploaded all gel results as support information.

6.Please upload a copy of Supporting Information Figure/Table/etc. Fig.1-7 which you refer to in your text on page 22.

Responses:We have uploaded all the information corresponding to Fig.1-7 as supporting information.

7.Please review your reference list to ensure that it is complete and correct. If you have cited papers that have been retracted, please include the rationale for doing so in the manuscript text, or remove these references and replace them with relevant current references. Any changes to the reference list should be mentioned in the rebuttal letter that accompanies your revised manuscript. If you need to cite a retracted article, indicate the article’s retracted status in the References list and also include a citation and full reference for the retraction notice.

Responses:In the subsequent discussion, we have added further information on the impact of altered PHD2 on other relevant pathways, and therefore cited three additional papers, references 37, 38, and 39.All references are complete and correct, and all references are formatted and inserted in the format required by the journals.

Responses to reviewer 1:

1、The abbreviation "DMEM" is not explained on page 4, line 86.

Responses:We have added to the description of "DMEM" on page 4, line 86 of the manuscript, with the following explanation: Dulbecco's Modified Eagle Medium Thank you for your suggestion!

2、The abbreviation "XF" is not explained on page 6, line 126.

Responses:We have explained "XF" on page 6, line 126 of the manuscript, specifically: Extrancellular Flux .Thank you for your suggestion!

3、In the References section, the word "References" should be written before listing the references.

Responses:We've added this detail before the reference, thanks for the suggestion!

4、In reference number 7, the journal name is written in all capital letters. It should be formatted similarly to the other references.

Responses:We have re-corrected the formatting of all references as required by the journal, thank you for your suggestion!

5、In Figure 4, the labels for "sh-PHD2" and "sh-PDH2+8μM Dorsomorphin" are not written properly in the histogram.

Responses:As Figure 4 is not standardised enough, we have recreated Figure 4 and re-uploaded it, thank you for your suggestion.

Responses to reviewer 2:

1.To achieve effective suppression of PHD2, authors should possess a comprehensive understanding of transcriptomics through thorough and high-throughput study.

Responses:Due to the time problem, we can't improve the PHD2 related high throughput research for the time being, in the future our group will continue to improve the related research for PHD2, thank you for your suggestion!

2.The authors should emphasize the distinct pathways that undergo changes due to sh-PHD2. These pathways should be elaborated upon in greater detail within the discussion section.

Responses:On page 16, line 350 of the manuscript, we provide additional elaboration in response to the other pathways that are altered due to sh-PHD2, and thank you for the suggestion!

3.The authors should verify that the figures in the panel maintain an optimal resolution for the imaging of Figure 3, Figure 5, and Figure 6.

Responses:When uploading Figure 3, Figure 5, and Figure 6., we are sure that it is already the best resolution, we are not sure why the resolution has dropped in the finished manuscript, we will upload all the figures again, if the result is still not good, we hope that you can contact us, thank you for your suggestion!

4.Authors should ensure that the gene names should be written in italics.

Responses:We have gone through the manuscript again and changed all gene names to italics, specifically: manuscript page 8, line 174; manuscript page 17, line 360.

Attachment

Submitted filename: Response to Reviewers.docx

Click here for additional data file. (16.7KB, docx)

Decision Letter 1

Kanhaiya Singh

5 Nov 2023

Silencing of topical proline hydroxylase domain 2 promotes the healing of rat diabetic wounds by phosphorylating AMPK

PONE-D-23-10650R1

Dear Dr. Yan,

We’re pleased to inform you that your manuscript has been judged scientifically suitable for publication and will be formally accepted for publication once it meets all outstanding technical requirements.

Within one week, you’ll receive an e-mail detailing the required amendments. When these have been addressed, you’ll receive a formal acceptance letter and your manuscript will be scheduled for publication.

An invoice for payment will follow shortly after the formal acceptance. To ensure an efficient process, please log into Editorial Manager at http://www.editorialmanager.com/pone/, click the 'Update My Information' link at the top of the page, and double check that your user information is up-to-date. If you have any billing related questions, please contact our Author Billing department directly at authorbilling@plos.org.

If your institution or institutions have a press office, please notify them about your upcoming paper to help maximize its impact. If they’ll be preparing press materials, please inform our press team as soon as possible -- no later than 48 hours after receiving the formal acceptance. Your manuscript will remain under strict press embargo until 2 pm Eastern Time on the date of publication. For more information, please contact onepress@plos.org.

Kind regards,

Kanhaiya Singh, Ph.D

Academic Editor

PLOS ONE

Additional Editor Comments (optional):

Reviewers' comments:

Reviewer's Responses to Questions

Comments to the Author

1. If the authors have adequately addressed your comments raised in a previous round of review and you feel that this manuscript is now acceptable for publication, you may indicate that here to bypass the “Comments to the Author” section, enter your conflict of interest statement in the “Confidential to Editor” section, and submit your "Accept" recommendation.

Reviewer #1: All comments have been addressed

Reviewer #2: All comments have been addressed

**********

2. Is the manuscript technically sound, and do the data support the conclusions?

The manuscript must describe a technically sound piece of scientific research with data that supports the conclusions. Experiments must have been conducted rigorously, with appropriate controls, replication, and sample sizes. The conclusions must be drawn appropriately based on the data presented.

Reviewer #1: Yes

Reviewer #2: Yes

**********

3. Has the statistical analysis been performed appropriately and rigorously?

Reviewer #1: Yes

Reviewer #2: Yes

**********

4. Have the authors made all data underlying the findings in their manuscript fully available?

The PLOS Data policy requires authors to make all data underlying the findings described in their manuscript fully available without restriction, with rare exception (please refer to the Data Availability Statement in the manuscript PDF file). The data should be provided as part of the manuscript or its supporting information, or deposited to a public repository. For example, in addition to summary statistics, the data points behind means, medians and variance measures should be available. If there are restrictions on publicly sharing data—e.g. participant privacy or use of data from a third party—those must be specified.

Reviewer #1: Yes

Reviewer #2: Yes

**********

5. Is the manuscript presented in an intelligible fashion and written in standard English?

PLOS ONE does not copyedit accepted manuscripts, so the language in submitted articles must be clear, correct, and unambiguous. Any typographical or grammatical errors should be corrected at revision, so please note any specific errors here.

Reviewer #1: Yes

Reviewer #2: Yes

**********

6. Review Comments to the Author

Please use the space provided to explain your answers to the questions above. You may also include additional comments for the author, including concerns about dual publication, research ethics, or publication ethics. (Please upload your review as an attachment if it exceeds 20,000 characters)

Reviewer #1: Author successfully addressed all the comments.

Author successfully addressed all the comments given by me as a reviewer.

Reviewer #2: (No Response)

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Reviewer #1: Yes: Rajnesh Kumari Yadav

Reviewer #2: No

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Acceptance letter

Kanhaiya Singh

16 Nov 2023

PONE-D-23-10650R1

Silencing of topical proline hydroxylase domain 2 promotes the healing of rat diabetic wounds by phosphorylating AMPK

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