High glucose-induced IL-7/IL-7R upregulation of dermal fibroblasts inhibits angiogenesis in a paracrine way in delayed diabetic wound healing

Cell and culture

Human dermal fibroblasts (HDFs) were bought from ScienCell (California, USA) and cultured in a fibroblast Medium (FM). Human umbilical vein endothelial cells (HUVECs) and human epidermal keratinocytes (HEKs) were purchased from American Type Culture Collection (ATCC) (Manassas, USA), cultured in Dulbecco’s Modified Eagle’s medium (DMEM, Gibco) containing 10% fetal bovine serum (FBS, Gibco). HDFs, HEKs and HUVECs were cultured at 37 °C with 5% CO2.

Normal glucose (NG) conditions consisted of DMEM containing 5.5 mM glucose, and high glucose (HG) conditions comprised of DMEM containing 30 mM glucose. The concentration of HG was achieved by adding sterile dextrose solution (4 g glucose in 20 g DEPC water, filter sterilization). D-mannitol was added in NG to elevate osmotic pressure. Different concentrations of rhIL-7 (0, 20, 40, 80 ng/mL) were added into the medium, while rMuIL-7 (100 ng/ml) was added in PBS.

Materials and regents

Recombinant Human IL-7 (rhIL-7, Cat#: P5147-10 μg) and recombinant murine IL-7 (rMuIL-7 Cat#: P5930-10 μg) were bought from Beyotime. Mouse Angptl4 neutralizing monoclonal Antibodies were bought from Lexicon Pharmaceuticals.Human Angptl4-neutralizing monoclonal antibody was bought from Regeneron. Rabbit anti-IL-7 antibody (Cat:1650, for humans and mice) was bought from ABclonal. Rabbit anti-IL-7R antibody (Cat: 7626-1-AP, for human and mouse), rabbit anti-CD31 antibody (Cat: 28083-1-AP, for human and mouse), rabbit anti-ANGPTL4 antibody (Cat: 18374-1-AP, for human and mouse), rabbit anti-GAPDH antibody (Cat: 10494-1-AP, for human and mouse), rabbit IgG control antibody (Cat: 30000-0-AP), goat anti-rabbit IgG(H + L) HRP-conjugated Affinipure (Cat: SA00001-2), coralite488—conjugates affinipure goat anti-rabbit IgG(H + L), coralite594–conjugates affinipure goat anti-mouse IgG(H + L) were from proteintech.

Mouse wound model and treatment

Male C57BL/6J mice (6 weeks old) were obtained from Vital River Laboratories, Beijing, and raised at the Center of Experimental Animals, Tongji Medical College, Huazhong University of Science and Technology.

Diabetic mice were established as follows: maleC57BL/6J mice (6 weeks old) were fed a high-fat diet for 4 weeks. Then the mice were fasted for 24 h and administered an intraperitoneal injection of freshly prepared streptozotocin (100 mg/kg body weight) dissolved in citric acid- sodium citrate buffer. Next, mice that exhibited polydipsia, polyuria and weight loss with blood glucose over 16 mM from the tail vein for three consecutive days indicated successful modeling (Zhang et al. 2021).

Intraperitoneal pentobarbital sodium (50 mg/kg) was used to anesthetize animals. Then the hair in the treatment region was shaved, and two 1.0 × 1.0 cm full-thickness excision skin wounds were generated on the dorsum to evaluate the effects of IL-7 or ANGPTL4 neutralizing antibody on wound healing. Digital images of the wound were captured on the day of surgery and postoperative day 1 and 3 and every other day after the third day until the wound healed. Wound areas were measured by ImageJ software (National Institutes of Health), and the wound healing rate was identified as the percentage of the initial area.

On days 7 (Fig. 2) or 11 (Fig. 8), wounded mice were euthanized, and wound tissues were paraffin-embedded, followed by staining for CD31 staining. A 15 min antigen retrieval step in citrate buffer was first conducted, and then, samples were blocked for 30 min with goat serum. Next, an anti-CD31 antibody (1:100; proteintech, Wuhan) was used to stain samples overnight at 4 °C, followed by washing with PBS and development with DAB prior to hematoxylin counterstaining.

A microscope was used to image sections, and CD31 staining was performed to assess micro-vascularization at the wound sites via quantification of the number of CD31 + cells in five random fields of view. Vessels with a 2–10 µm diameter were counted as individual vessels.

To evaluate the effects of IL-7, the normal mice received 30 µl PBS on one side and 30 µl 100 μg/ml IL-7 (dissolved in PBS) on the other side on days 0, 3, 5, 7, and 9. Moreover, the tissues were harvested on day 7 for CD31 staining.

To evaluate the effects of ANGPTL4, diabetic mice were administered IgG (0.5 mg/ml) on one side and anti-mouse ANGPTL4 (0.5 mg/ml) antibody on the other side on days 0, 3, 5, 7, 9, 11, and 13. The tissues were harvested on day 11 for CD31 staining. To establish STZ-induced diabetic models, mice were fed high-fat feed (Cat: D12108C, from New Brunswick, NJ 08,901 USA) for 4 weeks and were intraperitoneally injected with STZ (100 mg/kg) dissolved in citric acid-sodium citrate buffer once except for those in the control group. After 3 days, blood glucose levels were measured, and the mice whose blood glucose levels kept steadily over 16.7 mM for two weeks were considered diabetic (Dinh 2005) and used for the wound experiments to evaluate the effects of anti-ANGPTL4 antibody.

The Institution Animal Care and Use Committee of the Tongji Medical College, Huazhong University of Science and Technology, approved all animal studies.

Western blotting

Cell samples or homogenized skin tissues were lysed on ice with RIPA lysis buffer according to routine procedures for protein extraction. After being centrifuged at 12,000×g for 10 min at 4 °C, the cleared lysate was collected, and a BCA protein assay kit was used to determine the total protein concentrations.

The collected protein was subjected to 10% or 12.5%SDS-PAGE for protein separation and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The membranes were blocked in a rapid blocking solution for 20 min, followed by incubation with respective primary antibodies at 4℃ overnight. After washing with TBST (0.1%Tween-20), the membranes were incubated with the corresponding secondary antibody for 1 h at room temperature and visualized using Bio-rad Odyssey Infrared Imager.

RNA extraction and real-time PCR

Total RNA was isolated from cells or homogenized skin tissues using TRIzol reagent following the manufacturer’s instructions. The mRNA was reverse-transcribed into cDNA using PrimeScript RT Master Mix for RT-PCR (TaKaRa). Quantitative real-time RT-PCR (qRT-PCR) was performed in triplicates on BIO-RADT100 PCR Detection System using the SYBR Green PCR Master Mix. The housekeeping gene GAPDH was used for the normalization of mRNA expression. Analysis was carried out using the 2 − ΔΔCT method. The sequences of primer pairs used are shown in Table 1.

Table 1 The sequences of primer pairs used in qPCRImmunofluorescent staining and immunohistochemical analysis

The mice were randomly divided into groups and euthanized on day 9 or day 13 after wounding, and harvested wound edge tissues were fixed in 4% paraformaldehyde and subsequently embedded with paraffin or flash-frozen in liquid nitrogen for protein expression analysis.

For immunofluorescent staining analysis, the skin sections were dewaxed, hydrated and antigen restored, washed with PBS and blocked in 2% BSA for 1 h. Then, the tissue was incubated with the primary antibody (Rabbit anti-IL-7 antibody 1:100, Rabbit anti-IL-7R antibody, 1:100) at 4°Covernight and then washed with 0.1% Tween-20 in TBS (TBST) three times for 5 min each followed by staining with Alexa 488 secondary antibodies (proteintech, Wuhan) for 1 h at room temperature in the dark. After washing, the slides were treated with 1 mg/mL DAPI (Beyotime) in PBS for 5 min at room temperature to visualize the nuclei. The collected images (at least three views for each sample) were then measured using ImageJ software to analyze the fluorescence intensity of IL-7 or IL-7R in different groups relative to DAPI as an internal reference.

For the immunohistochemical assay, the sections were dewaxed, hydrated and quenched the endogenous peroxidase activity with 3% H2O2 for 10 min, and then antigen was restored. After washing with PBS three times, the slices were blocked with 2% BSA for 1 h and incubated with primary CD31 antibody (1:100) overnight at 4 °C. Subsequently, after incubating with horseradish peroxidase (HRP)–conjugated secondary antibody at room temperature for 1 h, the slices were colored with DAB substrate solution. (Risau 1997) Images were captured using an Olympus FluoView FV3000 confocal microscope (Tokyo, Japan).

Scratch wound migration test

HUVECs were grown to confluence in a 6-well plate, and a scratch wound migration test was performed by using a 1 mL pipette tip to scratch on the surface of cells in the middle of the 6-well plate. Cells were washed with PBS, and the corresponding culture medium was added. The wound-healing process was photographed at 0, 12, 24, and 36 h after wounding. The migration rate was quantified by calculating the wound closure area versus that of the primordial wound and quantified using ImageJ software.

Cell counting kit 8 (CCK8)

For the CCK-8 assay, 5 × 103 HUVECs were added to 96-well plates and cultured for 0, 24, 48, or 72 h. Then, 10ul/well CCK-8 reagent was added for 2 h, and absorbance measurements were taken at 450 nm.

Tube formation assay

HUVECs (2 × 104 per well) were added to Matrigel-coated 96-well plates, and cells were assigned to groups. Cells were cultured for 6 h, then three random fields of view were imaged with an inverted microscope. The total tube length, the number of master segments and the number of meshes were quantified using ImageJ software.

Sandwich ELISA for detecting ANGPTL4

HDFs were cultured with rhIL-7(0 and 40 ng/ml) for 24 h, and the sample was centrifuged for 20 min at 1000 g. After adding 100 µL of each standard and sample to the appropriate wells, the plate was incubated for 2 h at 37 °C in a humid environment. After washing 4 times, 300 µL of 1 × Wash Buffer was added per well, followed by 100 µL of 1 × Detection Antibody solution and incubated for 1 h at 37 °C in a humid environment. After washing, 100 µL of 1 × HRP-conjugated antibody was added to each well. The plate was sealed with a cover and incubated for 40 min at 37 °C in a humid environment. After washing, 100 µL of TMB substrate solution was added to each well and incubated for 20 min at 37 °C in the dark. A blue color indicated a positive reaction. 100 µL of Stop Solution was added to each well in the same order as the addition of the TMB substrate leading to a color change from blue to yellow. Immediately after adding the Stop solution, the absorbance on a microplate reader was read at a wavelength of 450 nm. The average of the duplicate readings for each standard and sample was obtained and subtracted from the average zero standard absorbance (obtained from the average of the “sd0” readings). The best-fit standard curve was determined by regression analysis using a four-parameter logistic curve fit (4-PL). As an alternative, a standard curve was constructed by plotting the mean absorbance for each standard on the y-axis against the concentration on the x-axis and drawing a best-fit curve through the points on the graph.

RNA sequencingSample preparation and total RNA extraction

RNA-Seq was performed in primary HDFs exposed to either Control or IL-7 (80 ng/ml) medium to investigate transcriptome abundance for 24 h. RNAs from cultivated HDF cells were extracted using RNAiso Plus (Cat # 9108, TaKaRa). The extracted RNA was measured and checked for concentration, purity, and integrity of RNA on a Fragment Analyzer Bioanalyzer using a standard sensitivity RNA Analysis Kit. RNA-Seq was then performed on a commercially available service (service ID # F21FTSCCKF8180_HUMacnwT, BGI, Wuhan, China).

Quality control and alignment of sequencing datasets

Sequencing of cDNA libraries generated over 41 to 53 million paired reads per sample. Low-quality reads of FASTQ files were filtered using SOAPnuke software v1.5.2 to obtain clean reads. After filtering, the high-quality reads were aligned against the human reference genome (GRCh38.p12) using an RNA-Seq-spliced read mapper HISAT2 (version 2.0.4). FPKM values for gene expression levels were calculated for annotated RefSeq genes using RSEM (version 1.2.8). To estimate the heterogeneity of our samples, we performed Pearson correlation coefficient analysis and PCA using R software to map clusters within the gene expression profile from all 18 samples. Normalization and differential expression analyses were estimated from count data using DESeq2. The heat map analysis was performed with the “pheatmap” R package with default parameters selecting the DEGs after FDR-corrected p values (q values ≤ 0.05) and fold changes ≥ 1.2

Functional enrichment analysis

Gene Ontology (GO) and Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of DEGs was performed using the Dr. TOM approach, an online database of BGI. The hypergeometric distribution was applied during GO and KEGG functional enrichment to find significantly enriched GO terms and KEGG pathways. Subsequently, the q value was obtained. We generated bubble plots to show the significantly enriched KEGG signaling pathway and GO terms. The x-axis represents the Rich Ratio (the ratio of the selected gene number annotated to a particular item to the total gene number annotated in this item), calculated using the following formula: Rich Ratio = Candidate Term Gene Num/Term Gene Num. The y-axis represents the GO term or KEGG Pathway. The GO terms and KEGG pathways with q value ≤ 0.05 were significantly enriched.

Statistical analysis

Results were presented as means ± SEMs with at least three independent experiments calculated using GraphPad Prisms 8.0 software. The differences among treatment groups were then assessed by student’s t-test to compare the means of two groups or one-way ANOVA for more than two groups. A P-value < 0.05 was statistically significant.

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