To explore whether or not inhibition of protein kinase C βII (PKC βII) stimulates angiogenesis as well as prevents excessive NETosis in diabetics thus accelerating wound healing. Streptozotocin (STZ, 60 mg/kg/day for 5 days, i.p.) was injected to induce type I diabetes in male ICR mice. Mice were treated with ruboxistaurin (30 mg/kg/day, orally) for 14 consecutive days. Wound closure was evaluated by wound area and number of CD31-stained capillaries. Peripheral blood flow cytometry was done to evaluate number of circulating endothelial progenitor cells (EPCs). NETosis assay and wound tissue immunofluorescence imaging were done to evaluate the percentage of neutrophils undergoing NETosis. Furthermore, the expression of PKC βII, protein kinase B (Akt), endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF), and histone citrullation (H3Cit) were determined in the wound by Western blot analysis. Ruboxistaurin accelerated wound closure and stimulated angiogenesis in diabetic mice. The number of circulating EPCs was increased significantly in ruboxistaurin-treated diabetic mice. Moreover, ruboxistaurin treatment significantly decreases the percentages of H3Cit+ cells in both peripheral blood and wound areas. This prevented excess activated neutrophils forming an extracellular trap (NETs) formation. The expressions of phospho-Akt (p-Akt), phospho-eNOS (p-eNOS), and VEGF increased significantly in diabetic mice on ruboxistaurin treatment. The expressions of PKC βII and H3Cit+, on the other hand, decreased with ruboxistaurin treatment. The results of the present study suggest that ruboxistaurin by inhibiting PKC βII activation, reverses EPCs dysfunction as well as prevents exaggerated NETs formation in a diabetic mouse model; thereby accelerating the wound healing process.

Introduction

Diabetic foot ulcers (DFUs) are a major end-stage complication of diabetes mellitus (DM). It is characterized by impaired wound healing resulting in considerable morbidity and mortality [1]. It is believed that foot ulcers in diabetic patients are difficult to heal due to low grade chronic infection of the wound bed. This low chronic inflammation has been suggested to be brought on by several factors such as neuropathy, improper oxygenation, insufficient vascular supply to extremities, and bacterial infections [2]. With regard to insufficient vascular supply to the wound site, impaired angiogenesis has been identified as one of the major reasons in diabetic patients [3].

In addition to insufficient blood supply, more recently, chronicity of the wound in diabetic patients as brought on by exaggerated neutrophil response to injury has also been believed to delay wound healing. Exaggerated neutrophil response to injury has been thought to cause further tissue damage leading to chronic inflammation [4,5]. It is recognized that activated neutrophils form an extracellular trap (NET) by releasing granules of protein and chromatin to form extracellular fiber to bind and kill bacteria. This process is followed by a type of cell death (NETosis) from the release of nuclear materials within NETs [6,7]. Several pathological conditions may predispose neutrophils to undergo excessive or unnecessary NETosis which, in turn, might also damage normal tissue.

More interestingly, past studies found that both angiogenesis and NETosis pathways are driven by a common protein; protein kinase C βII (PKC βII) [8–11]. Studies have shown PKC βII to be involved in down-regulation of the Akt/endothelial nitric oxide synthase (eNOS) angiogenic pathway under diabetic conditions [8,9]. Furthermore, recent studies have shown that PKC hyperactivity, especially βII, predisposes neutrophils to undergo NETosis in diabetic conditions [10,12,13]. PKC βII level is markedly increased in diabetic patients due to excess glycolytic and diacylglycerol (DAG) generation in these patients [4]. This excess PKC βII concentration in diabetic patients not only impairs angiogenesis, but it also primes neutrophils to undergo NETosis. This then affects the healing process as a whole (Figure 1). Ruboxistaurin mesylate (LY333531) is a macrocyclic bisindolylmaleimide compound that specifically inhibits the β-isoform of PKC. It is a competitive inhibitor interacting with the adenosine triphosphate (ATP) binding site of PKC isoforms. By competitively binding to the ATP site of PKC isoform, ruboxistaurin inhibits isolated enzymes PKCβI and PKCβII with a half-maximal inhibitory constant of 4.5 and 5.9 nM, respectively, whereas inhibition of other PKC isoforms required 250-times higher concentrations [14]. Therefore, in the present study, we used a specific PKC β inhibitor, ruboxistaurin, to inhibit PKC βII production. We then analyzed whether or not its inhibition affected angiogenesis and the NETosis process. This eventually assesses the effect on wound healing process under diabetic conditions.

Schematic diagram of PKC pathway under hyperglycemic conditions

Figure 1
Schematic diagram of PKC pathway under hyperglycemic conditions

Hyperglycemia induced increased PKC level stimulates neutrophil to form NET. PKC overactivity also down-regulates Akt-eNOS pathway leading to EPC dysfunction. Abbreviation: EPC, endothelial progenitor cell.

Figure 1
Schematic diagram of PKC pathway under hyperglycemic conditions

Hyperglycemia induced increased PKC level stimulates neutrophil to form NET. PKC overactivity also down-regulates Akt-eNOS pathway leading to EPC dysfunction. Abbreviation: EPC, endothelial progenitor cell.

Materials and methods

Animal model and treatment

Male ICR mice (6 weeks in age; 25–30 g) were included in the present study as an animal model. We induced type I diabetes to these mice by injecting streptozotocin (STZ) (60 mg/kg/day for 5 days, i.p.) dissolved in 0.1 mM sodium citrate buffer (pH 4.5). The mice were declared STZ-induced diabetic when random blood glucose levels, as measured by glucose monitoring system from tail vein on 21 days, post-STZ injection, were ≥ 300.0 mg/dl. These diabetic mice were then divided into two groups; a diabetic group (STZ group) and a treatment group (STZ + ruboxistaurin). All mice in the treatment group were treated with ruboxistaurin (LY333531) (MedChem Express; catalog number HY-10195B) orally by gastric lavage. The dosage of ruboxistaurin in the mouse for the present study was chosen on the basis of their bioavailability and metabolic experiments results reported previously [15]. Treatment was started from day 1 post-wounding of the mice. On day 14 post wounding, the mice were killed for final wound healing evaluation (Figure 2).

Ethical approval

All animal experiments in the present study comply with the ARRIVE guidelines and were conducted in accordance with the National Institutes of Health Guide for the Care and Use of Laboratory Animals and approved by the Biological Research Ethics Committee of the Chinese Academy of Sciences.

Illustration of experimental protocols

Figure 2
Illustration of experimental protocols

STZ (60 mg/kg/day for 5 days, i.p.) was given to induce diabetes in male ICR mice, and blood glucose was monitored. Ruboxistaurin (30 mg/kg/day for 14 days, p.o.) was given for 14 consecutive days. Wound healing, NETosis, and angiogenesis experiments were then conducted.

Figure 2
Illustration of experimental protocols

STZ (60 mg/kg/day for 5 days, i.p.) was given to induce diabetes in male ICR mice, and blood glucose was monitored. Ruboxistaurin (30 mg/kg/day for 14 days, p.o.) was given for 14 consecutive days. Wound healing, NETosis, and angiogenesis experiments were then conducted.

Wounding of the mouse

By using a 6-mm disposable sterile punch biopsy, a 6-mm wound was generated on the dorsum of the mice after anesthetizing them with ketamine (100 mg/kg; i.p.). Before wounding, the skin on the dorsum of the mice was shaved with razor and cleaned with 70% ethanol and betadine. Wounded areas were covered with bi-occlusive transparent dressing (Johnson & Johnson, Arlington, TX, U.S.A.).

Evaluation of wound healing

Wound areas were measured every 2 days until the 12th day to assess the closure of the wound.

Histological assessment of wound healing

On the 14th post-wounding day, wounds were cut in half, fixed overnight in zinc fixative, and embedded in paraffin. The tissues were then sectioned at 10 μm and stained with Hematoxylin and Eosin (H&E), and with Masson’s trichrome in accordance with the protocols of the manufacturer (Cyagen Biosciences Inc.) to detect the re-epithelialization/granulation tissue formation and collagen deposition, respectively.

Evaluation of angiogenesis

Capillary density in the wounds

Capillary densities in the healing wounds were quantitated by histological analysis. Wound samples were fixed with zinc chloride fixative (BD) for 24 h, then embedded in paraffin and sectioned at 4-µm intervals. Slides were de-paraffinized and hydrated. The slides were then placed in tris-buffered saline (TBS) (pH 7.5) for 5 min for pH adjustment. Endogenous peroxidase was blocked by 3% hydrogen peroxide/methanol bath for 20 min and followed by a distilled H2O rinse. Slides were blocked with 10% goat serum for 30 min at 37°C. The slides were then incubated for 60 min at room temperature with primary antibodies against CD31 (1:500 rabbit anti-rat CD31) and vascular endothelial growth factor (VEGF) 165 (VEGF165) (1:250; rabbit anti-rat VEGF) (both from Abcam, Cambridge, MA, U.S.A.). After primary antibody incubation, the sections were reacted with a secondary antibody (1:1000; goat anti-rat; Abcam). All the sections were counterstained with Hematoxylin.

Circulating endothelial progenitor cells in peripheral blood

Approximately 0.5 ml of mouse peripheral blood was obtained through cardiac puncture. Each sample was added into 1 ml Histopaque 1083 (Sigma–Aldrich) and centrifuged at (800×g, 4°C) 3000 rpm for 30 min to isolate mononuclear cells. The mononuclear cells were then extracted and centrifuged (400×g, 4°C). Red blood cells were lysed with ammonium chloride solution (Stem Cell Technologies). The samples were placed in polypropylene tubes with 100 µl phosphate buffered saline (PBS) with 1% albumin. They were then stained with FITC-conjugated CD34 and PE-conjugated Flk-1 for 1 h at 4°C and then washed three times in PBS with 1% albumin. Quantitation of FITC CD34/PE-Flk-1 double-positive cells was performed with a BD Vantage Flow cytometer.

Evaluation of NETosis

NETosis assay

Neutrophils were isolated from blood sample using Histopaque-1119 (Sigma) and Percoll Plus (GE Healthcare) gradients as described [16], a method that causes minimal activation of neutrophils during isolation. Red blood cell contamination was eliminated by hypotonic lysis, and final cell concentration was determined by hemacytometer. Neutrophil purity was established to be routinely >90% as determined by Wright–Giemsa staining. Isolated neutrophils were then re-suspended in RPMI medium (without glutamine) supplemented with 10 mM HEPES and plated at 15000 cells per well in a 96-well glass-bottomed plates. Neutrophils were then stimulated with 25 µg/ml of Klebsiella pneumoniae LPS (Sigma) for 2.5 h at 37°C. After 2.5 h of incubation, the medium containing reagents was removed and the remaining cells were washed with PBS followed by fixation with 2% (vol/vol) paraformaldehyde for 15 min. Fixed cells were washed with PBS and permeabilized (0.1% Triton X-100, 0.1% sodium citrate) for 10 min at 4°C. Samples were then blocked with 3% (wt/vol) bovine serum albumin (BSA) for 90 minutes at 37°C, rinsed, and then stained with 0.3 µg/ml rabbit polyclonal of anti-histone citrullation (H3Cit) (Abcam, catalog number ab5103), diluted 1:1000, in antibody dilution buffer (0.3% BSA and 0.05% Tween-20 in PBS), for 1 h at 37°C. After removal of unbound antibodies, the specimens were next stained with 1.5 µg/ml of Alexa Fluor 488-conjugated anti-rabbit secondary antibody (Alexa Fluor 488 goat anti-rabbit IgG (H + L); Invitrogen, catalog number A11008), diluted 1:1500, in antibody dilution buffer, for 1 h at 37°C. After washing with PBS, the specimens were finally mounted with 1 µg/ml of Hoechst 33342 (Invitrogen, catalog number H3570) which was diluted to 1:10000, with 0.3% BSA in PBS.

Evaluation of NETosis in the wound

Immunofluorescence microscopy was carried out to localize H3Cit+ neutrophils in the wounds. The wound tissues were dissected and embedded in optimal cutting temperature compound (OCT) instantly. Then, the tissues were cryosectioned into sections for immunofluorescence microscopy. These sections were fixed with zinc fixative (100 mM Tris/HCl, 37 mM zinc chloride, 23 mM zinc acetate, and 3.2 mM calcium acetate). Post fixation the sections were permeabilized and blocked with 0.1% Triton X-100 in PBS with 5% BSA for 20 min at 4°C. The sections were then incubated with primary antibodies against H3Cit (Abcam, catalog number ab5103) diluted 1:1000 and Ly6G (BD Pharmingen, catalog number 551459) diluted 1:500 in an antibody dilution buffer (0.3% BSA and 0.05% Tween-20 in PBS) at 4°C overnight. These sections were then incubated with Alexa Fluor–conjugated secondary antibodies; Alexa Fluor 488 goat anti-rabbit IgG (H + L) (Invitrogen, catalog number A11008), and Alexa Fluor 555 goat anti-rat IgG (H + L), (Invitrogen, catalog number A21434) for 2 h at room temperature. Both secondary antibodies were diluted 1:1500 in dilution buffer. Finally, Hoechst 33342 (Invitrogen, catalog number H3570) diluted to 1:10000 concentration was used to stain for DNA.

Western blot analysis

Furthermore, to verify the effect of ruboxistaurin on angiogenesis and NETosis, the expressions of the proteins levels involved in the respective pathways were evaluated in the wounded tissues by using Western blot analysis. For the angiogenesis pathway, proteins were extracted from the wound tissue and transferred on to a polyvinylidene fluoride (PVDF) membrane. After incubation in 5% non-fat milk in PBS-Tween 20, the membranes were incubated with primary antibodies against PKC βII (1:500), p-PKC βII (Thr641) (1:250), Akt (1:1000), p-Akt (Ser473) (1:500), eNOS (1:1000), p-eNOS (Ser1117) (1:500), and VEGF165 (1:250) overnight at 4°C.

For NETosis, H3Cit level in the mouse wound was quantitated. Mice wound tissues were collected, frozen, and homogenized in RIPA buffer supplemented with protease inhibitor cocktails (Sigma) on ice. The samples were then centrifuged at 20000×g for 20 min at 4°C. The protein content of the supernant was determined by bicinchoninic acid (BCA) protein assay. Then, an equal amount of protein per sample resolved on gradient gels (4–20% Tris-Glycine gels, Lonza or Bolt 4–12% Bis/Tris Plus gels, Life Technologies) was transferred on to PVDF membranes (Millipore, Marlborough, MA, U.S.A.). The blotted membrane was blocked by incubation in 5% defatted dry milk in PBS-Tween 20 and hybridized with primary antibodies (rabbit polyclonal anti-H3Cit, 1:1000, Abcam, catalog number ab5103; rabbit polyclonal anti-H3, 1:6000, Abcam, catalog number ab1791; and rat monoclonal anti-mouse Ly6G, 1:500, BD Pharmingen, catalog number 551459) at 4°C overnight and then subsequently with appropriate HRP-conjugated secondary antibodies (1:10000, goat anti-rabbit IgG (H + L)-HRP conjugate, Bio–Rad, catalog number 170-6515; 1:10000, goat anti-mouse IgG (H + L)-HRP conjugate, Bio–Rad, catalog number 170-6516; and 1:5000, goat anti-rat IgG (H + L)-HRP conjugate, Invitrogen, catalog number A10549) for 2 h at room temperature. The blots were developed with enhanced chemiluminescence (ECL) substrate (Thermo Scientific, catalog number 32106). Equal loading was confirmed by probing for glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (1:40000, Ambion, catalog number AM4300).

Statistical analysis

All statistical analyses were performed using GraphPad Prism version 5.0 (GraphPad Software Inc., San Diego, California). Continuous variables were reported as means ± S.D., and categorical variables as percentages of at least two independent experiments. Continuous variables were compared using two-tailed Student’s t test (unpaired), and categorical variables were compared using Fisher’s exact test, followed by repeated-measure ANOVA with Bonferroni’s post test, where appropriate. A P-value of <0.05 was considered significant for all statistical analyses.

Results

Body weight and blood glucose changes in diabetic mice

Blood glucose in STZ-induced diabetic mice was significantly increased (388 ± 10 mg/dl compared with 159 ± 10 mg/dl, P<0.05; Figure 3A), while body weight was significantly decreased when compared with the control group after STZ injection on day 20 (42.4 ± 0.6 g compared with 30.1 ± 0.9 g, P<0.05; Figure 3B). However, ruboxistaurin treatment for 14 consecutive days did not alter either blood glucose levels or the body weights of the mice when compared with group having STZ-induced diabetes (P>0.05; Figure 3C,D).

Establishment of STZ-induced diabetic mice

Figure 3
Establishment of STZ-induced diabetic mice

Compared with the control, mice treated with STZ (60.0 mg/kg/day for 5 days, i.p.) displayed higher blood glucose (A) and lower body weight (B), ruboxistaurin (30.0 mg/kg/day for 14 days, p.o.) treatment did not alter the blood glucose levels (C) or body weights (D) in the STZ-induced diabetic mice. The data are expressed as the means ± S.E.M. (*P<0.05 compared with control, n=10 per group).

Figure 3
Establishment of STZ-induced diabetic mice

Compared with the control, mice treated with STZ (60.0 mg/kg/day for 5 days, i.p.) displayed higher blood glucose (A) and lower body weight (B), ruboxistaurin (30.0 mg/kg/day for 14 days, p.o.) treatment did not alter the blood glucose levels (C) or body weights (D) in the STZ-induced diabetic mice. The data are expressed as the means ± S.E.M. (*P<0.05 compared with control, n=10 per group).

PKC inhibition accelerated wound closure and angiogenesis in diabetic mice

To assess the effects of ruboxistaurin on wound healing in mice with STZ-induced diabetes, the wound closure percentage was calculated every 2 days until day 12 as 1 − (average wound area on day X/average wound area on day 0) × 100. The data demonstrated that the wound closure in STZ-induced diabetic mice was significantly delayed compared with that of control group (P<0.05). Ruboxistaurin treatment significantly accelerated wound closure in the diabetic mice (Figure 4A,B).

Ruboxistaurin accelerated wound healing in diabetic mice

Figure 4
Ruboxistaurin accelerated wound healing in diabetic mice

A 6.0-mm diameter wound was made by punch biopsy, and the closure of the wound area was measured every 2 days until day 12; ruboxistaurin accelerated the wound closure in STZ-induced diabetic mice (A,B). H&E staining of wound sections showed better dermal re-epithelialization as well as thicker granulation tissues in mice treated with ruboxistaurin compared with STZ group mice (C,D). Collagen deposition assessed by Masson’s trichrome staining showed more intense blue staining in ruboxistaurin-treated mice than in the STZ group, suggesting that ruboxistaurin treatment accelerated collagen deposition in the granulation tissues. Scale bar: 50.0 μm (E,F). **P<0.01, *P<0.05 compared with Control; #P<0.05 compared with STZ. The data are expressed as the means ± S.E.M. (n=10 per group).

Figure 4
Ruboxistaurin accelerated wound healing in diabetic mice

A 6.0-mm diameter wound was made by punch biopsy, and the closure of the wound area was measured every 2 days until day 12; ruboxistaurin accelerated the wound closure in STZ-induced diabetic mice (A,B). H&E staining of wound sections showed better dermal re-epithelialization as well as thicker granulation tissues in mice treated with ruboxistaurin compared with STZ group mice (C,D). Collagen deposition assessed by Masson’s trichrome staining showed more intense blue staining in ruboxistaurin-treated mice than in the STZ group, suggesting that ruboxistaurin treatment accelerated collagen deposition in the granulation tissues. Scale bar: 50.0 μm (E,F). **P<0.01, *P<0.05 compared with Control; #P<0.05 compared with STZ. The data are expressed as the means ± S.E.M. (n=10 per group).

Histological assessment of wound healing

Histological analysis of the wound bed was performed to further evaluate the healing of the ulcers. The histological observation demonstrated that the tissue regeneration was much greater in the group treated with ruboxistaurin compared with the STZ group. H&E stained skin sections from the wound bed on day 14 after treatment showed that wounds were not fully re-epithelialized in the STZ group; however, nearly complete re-epithelialization was achieved in the ruboxistaurin-treated group (Figure 4C). Furthermore, the granulation of tissue in the ruboxistaurin-treated group was significantly thicker than that in the STZ group (276  ± 13.1 μm compared with 190 ± 15.4 μm; P <0 .05) (Figure 4C,D). Collagen formation in the wounded areas on day 14 after treatment was assessed by Masson’s trichrome staining which used computer-assisted morphometric analysis. Figure 4E,F show the presence of a large amount of collagen deposition organized in aligned fibers in the ruboxistaurin-treated group, but relatively far less in the STZ group. The mean density value of the ruboxistaurin-treated group was 0.45  ±  0.04, and for the control group was 0.31  ±  0.05 (P <0 .05).

Angiogenesis assessment

Capillary density in the wounds

The number of CD31 and VEGF-positive tubular structures in the wounds and surrounding skin was calculated to further evaluate the role of ruboxistaurin on neovascularization. CD31 and VEGF immunostaining demonstrated large and dense vessels growing widely in wound granulation tissues of the control ruboxistaurin-treated group. However, only small and narrow new vessels at the wound bed were presented in the STZ group (Figure 5A,B). Density of CD31-positive capillary lumen was significantly worse in STZ-induced diabetic mice when compared with control group. However, ruboxistaurin significantly improved the capillary formation in diabetic mice (P<0.05, Figure 5C).

Ruboxistaurin stimulated angiogenesis in diabetic mice.
Figure 5
Ruboxistaurin stimulated angiogenesis in diabetic mice.

Typical photographs of CD31-positive staining (A), quantitation of the CD31+ capillaries by digital image analysis (B), and VEGF-positive staining (C) on the wounded area obtained on day 12 showed increased capillary formation stimulated by ruboxistarin. **P<0.01, *P<0.05 compared with Control; #P<0.05 compared with STZ. The data are expressed as the means ± S.E.M. (n=10 per group).

Figure 5
Ruboxistaurin stimulated angiogenesis in diabetic mice.

Typical photographs of CD31-positive staining (A), quantitation of the CD31+ capillaries by digital image analysis (B), and VEGF-positive staining (C) on the wounded area obtained on day 12 showed increased capillary formation stimulated by ruboxistarin. **P<0.01, *P<0.05 compared with Control; #P<0.05 compared with STZ. The data are expressed as the means ± S.E.M. (n=10 per group).

Circulating endothelial progenitor cells

The numbers of CD34+ Flk-1+ cells in peripheral blood were measured to determine whether ruboxistaurin stimulated bone marrow endothelial progenitor cells (EPCs). The number of circulating EPCs in STZ-induced diabetic mice decreased significantly compared with that in the control group (P<0.01). Ruboxistaurin treatment increased the number of circulating EPCs (P<0.05; Figure 6A,B).

Flow cytometry analysis of mouse circulating EPCs
Figure 6
Flow cytometry analysis of mouse circulating EPCs

(A) Typical histograms of FITC-CD34+/PE-Flk-1+ analysis for EPCs (A,B), ruboxistaurin treatment increased circulating EPCs in STZ‐induced diabetic mice. **P<0.01 compared with Control; #P<0.05 compared with STZ. The data are expressed as the means ± S.E.M. (n=10 per group).

Figure 6
Flow cytometry analysis of mouse circulating EPCs

(A) Typical histograms of FITC-CD34+/PE-Flk-1+ analysis for EPCs (A,B), ruboxistaurin treatment increased circulating EPCs in STZ‐induced diabetic mice. **P<0.01 compared with Control; #P<0.05 compared with STZ. The data are expressed as the means ± S.E.M. (n=10 per group).

Susceptibility of diabetic mouse model to undergo NETosis

NETs formation in peripheral blood

Immunostaining of fresh blood cells from STZ-induced diabetic mice revealed a significant increase in H3Cit+ neutrophils compared with that of the control group. LPS further stimulated more neutrophils from the STZ-induced diabetic mice to be H3Cit+. Moreover, a significantly high percentage of neutrophils produced NETs after incubation in vitro with as well as without LPS stimulation in the STZ group compared with the control group. Ruboxistaurin treatment significantly decreases H3Cit+ neutrophils as well as percentages of neutrophils producing NETs as compared with the STZ group. (Figure 7A–C).

Representative immunofluorescence images of isolated neutrophils from mice
Figure 7
Representative immunofluorescence images of isolated neutrophils from mice

Neutrophils were exposed to LPS (25 g/ml) for 2.5 h. White arrowheads indicate NETs (A). Quantitation of hypercitrullinated H3-stained neutrophils following LPS stimulation (B). Quantitation of NET-forming neutrophils following LPS stimulation (C). NETs were identified as H3Cit+ cells with spread nuclear morphology as visualized by Hoechst 33342 stain. **P<0.01 compared with control; #P<0.05 compared with STZ.

Figure 7
Representative immunofluorescence images of isolated neutrophils from mice

Neutrophils were exposed to LPS (25 g/ml) for 2.5 h. White arrowheads indicate NETs (A). Quantitation of hypercitrullinated H3-stained neutrophils following LPS stimulation (B). Quantitation of NET-forming neutrophils following LPS stimulation (C). NETs were identified as H3Cit+ cells with spread nuclear morphology as visualized by Hoechst 33342 stain. **P<0.01 compared with control; #P<0.05 compared with STZ.

NETs formation in wound tissue

Immunofluorescence images of wounds 3 days after injury showed that H3Cit+ neutrophils were significantly higher in the STZ-induced diabetic mice as compared with those in the control group suggesting hyperglycemic environment to prime neutrophils to undergo exaggerated NETs formation. Ruboxistaurin treatment significantly decreased H3Cit+ neutrophils in the wound suggesting inhibition of PKC βII preventing neutrophils to undergo NETs formation (Figure 8).

Representative immunofluorescence microscopic images of wound bed 3 days after injury
Figure 8
Representative immunofluorescence microscopic images of wound bed 3 days after injury

LyG6 and H3Cit levels were at the maximum in the STZ group as compared with control and ruboxistaurin-treated group. Ruboxistaurin treatment significantly decreased the H3Cit and LyG6 level in the wound bed compared with untreated STZ group.

Figure 8
Representative immunofluorescence microscopic images of wound bed 3 days after injury

LyG6 and H3Cit levels were at the maximum in the STZ group as compared with control and ruboxistaurin-treated group. Ruboxistaurin treatment significantly decreased the H3Cit and LyG6 level in the wound bed compared with untreated STZ group.

Western blot analysis

Effect of ruboxistaurin on p-Akt and p-eNOS and VEGF expression

We determined whether high glucose levels had any effect on phosphorylation of Akt and eNOS as well as VEGF expression in wound tissue. Western blot analysis demonstrated that p-Akt and p-eNOS expressions along with VEGF expression in wound tissue were decreased in STZ-induced diabetic mice compared with the control group. Ruboxistaurin increased p-Akt and p-eNOS as well as VEGF expressions in wound tissue of the diabetic mice (P<0.05; Figure 9A–D).

Ruboxistaurin inhibits PKC βII mediated downregulation of VEGF dependent Akt/eNOS pathway.
Figure 9
Ruboxistaurin inhibits PKC βII mediated downregulation of VEGF dependent Akt/eNOS pathway.

Western blot analysis showing protein expression level of p-Akt over total Akt (A,B), p-eNOS over total (eNOS) (A,C) and total VEGF over total GAPDH (A,D), in wounds 12 days post-wounding. **P<0.01 compared with Control; #P<0.05 compared with STZ. The data are expressed as the means ± S.E.M. (n=10 per group).

Figure 9
Ruboxistaurin inhibits PKC βII mediated downregulation of VEGF dependent Akt/eNOS pathway.

Western blot analysis showing protein expression level of p-Akt over total Akt (A,B), p-eNOS over total (eNOS) (A,C) and total VEGF over total GAPDH (A,D), in wounds 12 days post-wounding. **P<0.01 compared with Control; #P<0.05 compared with STZ. The data are expressed as the means ± S.E.M. (n=10 per group).

Effect of ruboxistaurin on PKC activation and NETs formation

Furthermore, we determined whether PKC βII activation was induced by high glucose levels and if there was any effect of NETs on wound healing. Western blot analysis of wound tissues revealed increased p-PKC βII expression in STZ group compared with control group suggesting hyperglycemic conditions stimulate PKC βII formation. With ruboxistaurin treatment, PKC βII expression was found to decrease significantly as compared with STZ group suggesting inhibitory effect of ruboxistaurin on PKC βII production (Figure 10A,B). Moreover, the ratio of H3Cit to H3 was significantly higher in the STZ group compared with that of the control group. However, on ruboxistaurin treatment, the ratio of H3Cit to H3 was found to decrease significantly suggesting decreased NETs formation on inhibition of PKC βII (Figure 10A,C).

Effect of Ruboxistaurin on the protein expression of PKC βII and H3Cit in wounded tissues.
Figure 10
Effect of Ruboxistaurin on the protein expression of PKC βII and H3Cit in wounded tissues.

Western blot analysis showing protein expression level of p-PKC βII over total PKC βII (A,B) and total H3Cit over total H3 (A,C) in wounds 12 days post wounding. **P<0.01, *P<0.05 compared with Control; #P<0.05 compared with STZ. The data are expressed as the means ± S.E.M. (n=10 per group).

Figure 10
Effect of Ruboxistaurin on the protein expression of PKC βII and H3Cit in wounded tissues.

Western blot analysis showing protein expression level of p-PKC βII over total PKC βII (A,B) and total H3Cit over total H3 (A,C) in wounds 12 days post wounding. **P<0.01, *P<0.05 compared with Control; #P<0.05 compared with STZ. The data are expressed as the means ± S.E.M. (n=10 per group).

Discussion

The present study demonstrates that ruboxistaurin, a selective PKC βII inhibitor, not only stimulates angiogenesis but also prevents neutrophils to form excess NETs; thereby accelerating wound healing. It is believed that hyperglycemia is one of the most important metabolic factors in development of both micro- and macrovascular complications in diabetic patients. Hyperglycemia causes glycolytic overload as well as excess production of diacylglycerol (DAG). These in turn cause overactivation of PKC, a family of enzymes that are involved in controlling the function of other proteins [17]. The present study also found that diabetic mice had higher level of PKC βII in wound tissue when compared with normal glycemia controlled mice. Furthermore, it is known by now that the EPCs recruitment and functions are fundamentally impaired in diabetic patients affecting the neo-angiogenesis process, and one of the major culprit is PKC βII. PKC βII hyperactivity results in reactive oxygen species (ROS)-induced EPC apoptosis as well as decreased EPC motility by down-regulating the VEGF which eventually decreases NO bioavailability [18–20]. Recent studies have also shown that PKC βII activation decreases the PI3-kinase/Akt-dependent eNOS activation in response to VEGF or insulin stimulation [8,9]. eNOS is one of the major players in mobilizing EPCs into circulation initiating the angiogenesis process. Bone marrow stromal cells sense the signal molecules, such as VEGF-A, released by injured tissue. eNOS within the bone marrow stromal cells is then activated by VEGF-A and other mobilizing stimuli. eNOS induces production of NO which then S-nitrosylates by paracrine mechanisms and activates and/or maintains matrix metallopeptidase 9 (MMP-9) activity. This results in cleavage of soluble kit ligand (sKitL) from the membrane-bound kit ligand (mKitL) to mobilize c-Kit+ EPCs from the bone marrow niche into the circulation [21–25]. This stipulation is verified in the present study showing significantly decreased level of p-Akt and p-eNOS expression in wound tissue and consequently decreased density of capillaries in the wounded tissue as observed by CD31 staining in the diabetic mice when compared with normal glycemic controlled mice. Furthermore, the number of circulating EPCs was significantly reduced in the diabetic mice. The present study further demonstrates that ruboxistaurin treatment inhibits PKC βII activation as well as increases the level of p-Akt and p-eNOS in the wounded tissue. Moreover, capillary density in the wound tissues and EPC number in the peripheral blood increase in the ruboxistaurin-treated mice. Thus, the present study demonstrates that Ruboxistaurin treatment is associated with increased angiogenesis which is attributed to inhibition of PKC βII mediated down-regulation of the VEGF-dependent Akt/eNOS pathway.

More recently, NETosis-led chronic infection has been proposed as one of the main obstacle in diabetic wound healing. Under diabetic conditions, neutrophils produce more superoxide, cytokines, and tumor necrosis factor α, which prime neutrophils for NETosis. Local infection is very common in diabetic food ulcers (DFUs). Hyperglycemic condition provides suitable environment for pathogens to survive and reproduce. These local infections in DFU trigger neutrophil activation which, in turn, release their nuclear and granular content. These nuclear and granular contents work together to form extracellular traps, which use their sticky extracellular network loaded with bactericidal proteins, to contain and kill bacteria. These NETs after extruding their nuclear material die by a process termed as NETosis [26]. A recent report by Wong et al. [6] found strikingly higher expression of peptidylarginine deiminase 4 (PAD4) enzyme which is essential for chromatin decondensation (one of the step in NETosis) in neutrophils of diabetic patients. Wong et al. [6], also reported increased NETting neutrophils in the wounded tissue of diabetic mice. In fact, they found that the wound healing was accelerated in both diabetic and non-diabetic PAD4-deficient mice, which were protected from NETosis, suggesting important role of NETosis in delaying wound healing. Consistent with Wong et al. [6], the present study shows a higher percentage of neutrophils isolated from diabetic mice were H3Cit3+ when compared with mice in the control group. Moreover, diabetic mice produced large quantities of NETs in wound tissues and had significantly decreased wound healing rates as compared with normal glycemic controlled mice. Besides PAD4 and few other mediators, PKC has also been identified as one of the key players for inducing NETosis. Research performed in recent years has shown that NET formation is enabled through the activation of PKC [10,12,27]. Moreover, they emphasize a significant role of isoenzyme PKC β in the regulation of this phenomenon. In fact, Gray et al. [10], blocked NET formation with the use of Pan PKC inhibitor as well as specific PKC βII inhibitor. In the present study also, inhibition of PKC βII by ruboxistaurin, which was proven by decreased expression of PKC βII in wound tissue, demonstrated the presence of less H3Cit3+ neutrophils in peripheral blood as well as decreased quantities of NETs in wounds when compared with non-treated diabetic mice. Furthermore, wound healing was found to be accelerated significantly as compared with non-treated diabetic mice.

It is noteworthy that, although, ruboxistaurin has shown promising results in preclinical studies for several diabetic complications, results from clinical trials are mixed and largely negative. Apart from non-proliferative diabetic retinopathy [28–31] and diabetic nephropathy to some extent [32], where the drug showed some positive results, ruboxistaurin has failed to show significant positive outcomes in other diabetic complications. The manufacturer, Eli Lilly Co., has received an approval letter from the Food and Drug Administration (FDA) for the prevention of vision loss in patients with diabetic retinopathy with ruboxistaurin. However, the results of additional clinical trials for this indication is still pending. No clinical trial using ruboxistaurin for treatment of DFU has been conducted. Nevertheless, the involvement of PKC pathway in these and several other pathological conditions cannot be denied. Similarly, in the context of the present study as well it is evident that the inhibition of PKC βII would accelerate wound healing in diabetic patients. This, therefore, might warrant further studies to develop more potent specific isoform inhibitors. More recently, a novel series of pyrrolopyrazole-based PKC βII inhibitors have been identified which has shown superior results (Ki = 29 nM) against PKC βII [33]. Moreover, Mochley–Rosen laboratory has developed separation of function inhibitors (SoF inhibitors) which are peptides that are designed to selectively inhibit the phosphorylation of one particular substrate of an individual PKC isozyme [34].

In summary, the present study demonstrates that hyperglycemic condition leads to the activation of PKC βII which in turn not only predisposes neutrophils to undergo NETosis but also impairs angiogenesis by down-regulating the VEGF-dependent Akt/eNOS pathway. Moreover, the inhibition of PKC βII by a selective PKC βII inhibitor, ruboxistaurin, accelerates wound healing in diabetic mice by preventing neutrophils from undergoing excessive NETosis prompting the angiogenesis process to reverse impaired EPC functions.

We thank Morgan A. McClure, medical editor at North Sichuan Medical College, for English grammar correction and revision.

Competing interests

The authors declare that there are no competing interests associated with the manuscript.

Funding

This work was supported by the National Natural Science Foundation of China [grant number 81671793].

Author contribution

S.K.D. designed the study, researched and analyzed data, and wrote the manuscript. Y.F.Y. researched and analyzed data. M.Q.L. provided helpful suggestions on experimental design, reviewed and edited the manuscript, and contributed to discussion.

Abbreviations

     
  • Akt

    protein kinase B

  •  
  • ARRIVE

    animal research: reporting of in vivo experiments

  •  
  • DAG

    diacylglycerol

  •  
  • DFU

    diabetic foot ulcer

  •  
  • eNOS

    endothelial nitric oxide synthase

  •  
  • EPC

    endothelial progenitor cell

  •  
  • FITC

    fluorescein isothiocynate

  •  
  • Flk-1

    fetal liver kinase-1

  •  
  • H3Cit

    histone citrullation

  •  
  • H&E

    hematoxylin and eosin

  •  
  • HRP

    horseradish peroxidase

  •  
  • ICR mice

    Swiss albino mice (Institute of Cancer Research)

  •  
  • i.p.

    intra-peritoneal

  •  
  • LPS

    lipopolysaccharides

  •  
  • NET

    activated neutrophil form an extracellular trap

  •  
  • PE

    phycoerythrin

  •  
  • PI3-kinase

    phosphatidylinositol 3-kinase

  •  
  • PKC βII

    protein kinase C βII

  •  
  • RIPA

    radio immunoprecipitation assay

  •  
  • STZ

    streptozotocin

  •  
  • VEGF

    vascular endothelial growth factor

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