Arnebin-1, a naphthoquinone derivative, plays a crucial role in the wound healing properties of Zicao (a traditional wound healing herbal medicine). It has been noted that Arnebin-1, in conjunction with vascular endothelial growth factor (VEGF), exerts a synergistic pro-angiogenic effect on human umbilical vein endothelial cells (HUVECs) and accelerates the healing process of diabetic wounds. However, the mechanisms responsible for the pro-angiogenic effect of arnebin-1 on HUVECs and its healing effect on diabetic wounds have not yet been fully elucidated. In this study, in an aim to elucidate these mechanisms of action of arnebin-1, we investigated the effects of arnebin-1 on the VEGF receptor 2 (VEGFR2) and the phosphoinositide 3-kinase (PI3K)-dependent signaling pathways in HUVECs treated with VEGF by western blot analysis. The pro-angiogenic effects of arnebin-1 on HUVECs, including its effects on proliferation and migration, were evaluated by MTT assay, Transwell assay and tube formation assay
In developed countries, a major cause of hospital admissions for patients with diabetes is chronic diabetic foot ulcers, which are a common symptom of diabetes and often result in pain and a lower quality of life (
Relative hypoxia is a critical stimulus for normal wound healing, and the major cause of impaired wound healing in patients with diabetes may be an impaired response to hypoxia (
Diabetic foot ulcers heal slowly due to impaired neovascularization in response to tissue ischemia (
Although some therapeutic methods, such as gene therapy and treatment with recombinant growth factors have been used in an aim to promote angiogenesis, these methods are impeded by limitations, such as safety issues and high costs (
Therefore, the aim of the present study was to investigate the mechanisms responsible for the pro-angiogenic effects of arnebin-1 on human umbilical vein endothelial cells (HUVECs), as well as those responsible for its healing effects on wounds of rats with alloxan-induced diabetes mellitus (DM). The effects of arnebin-1 on the phosphoinositide 3-kinase (PI3K)-dependent signaling pathway, VEGFR2 signaling and on the expression levels of eNOS, VEGF and HIF-1α
Arnebin-1 was purchased from Tokyo Chemical Industry Co. Ltd. (Tokyo, Japan), and recombinant human VEGF was from PeproTech Inc. (Rocky Hill, NJ, USA). Growth factor-reduced Matrigel basement membrane matrix was obtained from BD Biosciences (Bedford, MA, USA). Medium 199 (M199) and fetal bovine serum (FBS) were purchased from Gibco (Carlsbad, CA, USA). LY294002, a PI3K inhibitor, was purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). All other reagents utilized were purchased from Sigma Chemical Co. (St. Louis, MO, USA) unless otherwise specified.
The isolation and culture of the HUVECs was carried out as previously described (
Cell proliferation was examined by mitochondrial MTT tetrazolium assay. The HUVECs were plated at 3×103 cells/well in 96-well plates. Overnight, the HUVECs were pre-treated with or without LY294002 (2
Cell migration assay was performed using Transwell chambers as previously described (
To examine the pro-angiogenic effect Arnebin-1, we used the experimental
The HUVECs were lysed using protein lysis buffer and protease inhibitor cocktail. The protein concentrations of the cell lysates were quantified using a bicinchoninic acid assay (BCA) kit, and equal amounts of protein were separated by SDS-PAGE and then transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked in 5% non-fat dried milk diluted with Tris-Buffered Saline Tween-20 (TBST; in mmol/l: Tris-HCl 20, NaCl 150, pH 7.5 nd 0.1% Tween-20) at room temperature for 1 h and probed overnight at 4°C with a polyclonal rabbit anti-phosphorylated (p-)VEGFR2 (#2478), a polyclonal rabbit anti-VEGFR2 (#9698), a polyclonal rabbit anti-p-Erk1/2, a polyclonal rabbit anti-Erk1/2, a polyclonal rabbit anti-p-FAK (#8556), a polyclonal rabbit anti-FAK (#13009), a polyclonal rabbit anti-p-Src (#5473), a polyclonal rabbit anti-Src (#2109), a polyclonal rabbit anti-PI3K (#4257), a polyclonal rabbit anti-p-PI3K (#3821), a polyclonal rabbit anti-Akt (#4691), a polyclonal rabbit anti-p-Akt (#4060), a polyclonal rabbit anti-p-mammalian target of rapamycin (mTOR; #2983), a polyclonal rabbit anti-p-mTOR (#2971; all from Cell Signaling Technology, Beverly, MA, USA), a polyclonal rabbit anti-proliferating cell nuclear antigen (PCNA; sc-7907), a polyclonal rabbit anti-eNOS (sc-654), a monoclonal rabbit anti-VEGF (sc-152; both from Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) or a monoclonal rabbit anti-HIF-1α antibody (NB-100-479; Novus Biologicals, Littleton, CO, USA), and then incubated for 2 h with anti-rabbit IgG (Santa Cruz Biotechnology Inc.). Incubation with polyclonal mouse β-actin antibody (#3700; 1:3,000 dilution; Cell Signaling Technology) or monoclonal mouse α-tubulin antibody (T5168; 1:1,000 dilution; Sigma) was used as the internal standard control. The proteins were visualized using ECL™ western blotting detection reagents (Amersham Biosciences Corp., Piscataway, NJ, USA). The densitometry of the bands was quantified using ImageJ 1.38X software.
A Quantikine human VEGF ELISA kit (R&D Systems, Minneapolis, MN, USA) was used according to the manufacturer's instructions. Briefly, VEGF standards and the conditioned medium from the HUVECs were placed into wells overlaid with antibody specific for human VEGF. After binding with an VEGF enzyme-linked polyclonal antibody specific for VEGF, the absorbance was measured at 450 nm using a microplate reader. The VEGF concentration was evaluated (in pg/ml) with the standard curve and adjusted for protein concentrations.
All animal procedures were approved by the Laboratory Animal Center of Sun Yat-sen University. As previously described (
As described in a previous study of ours (
As previously described (
As stated above, each diabetic rat had 3 wounds on the dorsal surface and the non-diabetic rats had 1 wound. In the D + V group, only wounds on the top of the dorsal side were treated with only the vehicle base (without the test compound). In the diabetic group, only wounds near the tail were treated with distilled water. In the D + A group, only wounds in the middle were treated with arnebin-1 (0.1% ointment). Thus, in each group of rats, a different wound area was treated. The wounds at the top served as the vehicle controls for the treated wounds. The test compound ointment and the vehicle were applied every other day, in quantities sufficient to cover the wounds with a thin layer. All the treatments were continued until the day of sacrifice. The rats were sacrificed with the use of an intraperitoneal injection of an overdose of barbiturate.
The rats were anesthetized with an overdose of pentobarbital (200 mg/kg, injected intraperitoneally) on day 7 post-wounding. The wound and a margin of approximately 5 mm of unwounded skin was excised. These wound tissues were snap-frozen in liquid nitrogen until they were processed for protein isolation.
In order to measure the levels of PCNA, CD31, HIF-1α, VEGF and eNOS in the tissue, wounds treated with arnebin-1 or the vehicle were harvested on day 7 post-wounding. Following excision, the tissues were homogenized in lysis buffer. The VEGF, eNOS and HIF-1α expression levels were determined by western blot analysis as described above.
Wound samples, taken on day 7, were embedded in paraffin and frozen in liquid nitrogen immediately for immunofluorescence. To assess new blood vessel formation, vessel density was estimated after staining for CD31. Serial 6-
All statistical analyses were performed using GraphPad Prism 5.0 (GraphPad Software, Inc., USA). Data for each study parameter from each group are presented as the means ± standard error of the mean (SEM). Data from each group were statistically analyzed by one-way analysis of variance (ANOVA). Differences were considered statistically significant at P<0.05.
PCNA is a nuclear cell proliferation marker. To determine whether arnebin-1 promotes the proliferation of HUVECs, the PCNA levels were measured by western blot analysis. At concentrations ranging from 1×10−3
It has been reported that VEGFR2 phosphorylation activates extensive downstream signaling substrates that are closely related to endothelial cell proliferation, migration and tube formation (
Subsequently, we investigated the effects of arnebin-1 on the expression levels of eNOS and VEGF in HUVECs. At concentrations ranging from 1×10−3
We also investigated whether arnebin-1 has any effect on the PI3K/Akt/mTOR pathway, which functions upstream of eNOS, VEGF and HIF-1α. Following treatment for 24 h, arnebin-1 induced a marked increase in the protein expression levels of PI3K, Akt and mTOR in the HUVECs in a concentration-dependent manner (
In a previous study (
The mean FBG levels and body weight of the animals are presented in
To investigate the mechanisms through which neovascularization is promoted, following treatment with arnebin-1, we measured the
In
At present, diabetic wounds remain a considerable challenge in clinical practice, and current treatments are inadequate. Only 66% of diabetic wounds ultimately heal, and up to 28% result in amputation (
At present, there is no effective topical drugs which can be applied in routine clinical practice to treat diabetic foot ulcers (
HIF-1α is necessary for the wound healing process, and the entire process of normal wound healing is dependent on its expression (
Arnebin-1 has previously been shown to promote angiogenesis both
It has long been recognized that blood supply is an important factor in wound healing. VEGF plays an essential role in promoting the growth of new blood vessels in certain organ systems (
In our
VEGFR2 signaling is necessary for vascular endothelial cells to function. The main autophosphorylation site of VEGFR2 is tyrosine (Tyr)1175, and its phosphorylation initiates the downstream signaling events in endothelial cells (
eNOS activity has two important functions: it mobilizes endothelial progenitor cells from bone marrow to peripheral blood, and induces ischemia-induced vascularization (
In conclusion, based on the outcomes of the present study, and in conjunction with our previous data (
This study was supported by grants from the Joint Project of National Education Ministry and Guangdong Province (no. 2007B090400089) and (no. 2007A032702001).
(A) Structure of arnebin-1 [5,8-dihydroxy-2-(1′-b,b-dimethylaryoxy-4′-methylpent-3-enyl)-1,4-naphthoquinone]. (B) Human umbilical vein endothelial cells (HUVECs) were treated with arnebin-1 only at various concentrations for 24 h. Cell lysates were subjected to western blot analysis. Upper panel shows representative blots of the protein expression of proliferating cell nuclear antigen (PCNA). Lower panel shows the quantification of the PCNA protein level. (C) The HUVECs were treated with arnebin-1 at various concentrations in the absence or presence of vascular endothelial growth factor (VEGF; 1 ng/ml) for 24 h. Cell lysates were subjected to western blot analysis. Upper panel shows representative blots of the protein expression of PCNA. Lower panel shows the quantification of the PCNA protein level. α-tubulin was used as a loading control. Bars represent the means ± SEM. *P<0.05 vs. control; #P<0.05 vs. VEGF-treated group. Control, vehicle-treated group.
Arnebin-1 promotes vascular endothelial growth factor (VEGF) receptor 2 (VEGFR2) kinase activity and its downstream signaling molecules. (A) Arnebin-1 increased the phosphorylation of VEGFR2 induced by VEGF in human umbilical vein endothelial cells (HUVECs). Total protein was isolated and subjected to western blot analysis. Upper panel shows representative blots of the protein levels of phosphorylated (p-)VEGFR2 and total (t-)VEGFR2 proteins. Lower panel shows the quantification of the p-VEGFR2 protein level. (B–D) Arnebin-1 also increased VEGFR2-mediated protein kinase activation of focal adhesion kinase (FAK), extracellular signal-regulated kinase (Erk) and Src. (B) Upper panel shows representative blots of the protein levels of p-FAK and t-FAK. Lower panel shows the quantification of the p-FAK protein level. (C) Upper panel shows the representative blots of the levels of p-Erk and t-Erk proteins. Lower panel shows the quantification of the p-Erk protein level. (D) Upper panel shows representative blots of the protein levels of p-Src and t-Src proteins. Lower panel shows the quantification of the p-Src protein level. (E) Diagram of signaling pathways involved in arnebin-1-induced angiogenesis. α-tubulin was used as a loading control. Bars represent the means ± SEM. *P<0.05 vs. control; #P<0.05, ##P<0.01 vs. VEGF-treated group. Control, vehicle-treated group.
The expression levels of endothelial nitric oxide synthase (eNOS), vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1α were increased by arnebin-1 in a phosphoinositide 3-kinase (PI3K)-dependent manner. (A–C) Human umbilical vein endothelial cells (HUVECs) were treated with arnebin-1 only at various concentrations (10−3, 10−2 and 10−1
The effect of arnebin-1 on the phosphoinositide 3-kinase (PI3K) pathway. (A–C) Human umbilical vein endothelial cells (HUVECs) were treated with arnebin-1 only, at various concentrations (10−3, 10−2 and 10−1
Hypoxia-inducible factor (HIF)-1α is essential for arnebin-1-induced (A) cell proliferation, (B and C) cell migration and (D–E) tube formation of human umbilical vein endothelial cells (HUVECs) in the presence of vascular endothelial growth factor (VEGF). HUVECs were treated with or without LY294002 (2
Effects of arnebin-1 on the hypoxia-inducible factor (HIF)-1α, vascular endothelial growth factor (VEGF) and endothelial nitric oxide synthase (eNOS) expression levels in diabetic rats. (A–C) Effects of arnebin-1 on the protein expression levels of HIF-1α, VEGF and eNOS. (A) Upper panel shows representative blots of the protein level of HIF-1α. Lower panel shows the quantification of the HIF-1α protein level. (B) Upper panel shows representative blots of the protein level of eNOS. Lower panel shows the quantification of the eNOS protein level. (C) Upper panel shows representative blots of the protein level of VEGF. Lower panel shows the quantification of the VEGF protein level. Bars represent the means ± SEM. *P<0.05, **P<0.01 vs. non-diabetic rats; #P<0.05, ##P<0.01 vs. diabetic rats. n=6 for each group. D+V, diabetic rats treated with the vehicle; D+A, diabetic rats treated with arnebin-1.
Effects of arnebin-1 on proliferation and neovascularization in diabetic rats. (A) Effects of arnebin-1 on the expression levels of proliferating cell nuclear antigen (PCNA), a nuclear cell proliferation marker. Upper panel shows representative blots of the protein level of PCNA. Lower panel shows the quantification of the PCNA protein level. (B) Effects of arnebin-1 on wound vascularity. Wound sections were stained with an anti-CD31 antibody and detected with Cy3 (red). Representative immunofluorescence images of wound samples on day 7 after treatment. Immunostaining for CD31-positive blood vessels (red) was performed to show vasculature in wounds, and nuclei (blue) were counterstained with Hoechst 33342. (C) Quantitative analysis of CD31-positive blood vessels in each section. Results are expressed as the number of vessels per high-power field. (D) Effects of arnebin-1 on the expression levels of CD31. Upper panel shows representative blots of the protein level of CD31. Lower panel shows the quantification of the CD31 protein level. Bars represent the means ± SEM. *P<0.05, **P<0.01 vs. non-diabetic rats; ##P<0.01 vs. diabetic rats. n=6 for each group. D+V, diabetic rats treated with the vehicle; D+A, diabetic rats treated with arnebin-1.
Schematic diagram of the mechanisms through which arnebin-1 promotes vascularization and wound healing. Arnebin-1 treatment leads to the accumulation of hypoxia-inducible factor (HIF)-1α, and the consequent upregulation of VEGF and endothelial nitric oxide synthase (eNOS). The expression of HIF-1α target genes in turn promotes neovascularization in diabetic wounds through angiogenesis and vasculogenesis.
Effects of arnebin-1 on body weight and blood glucose.
Factors | Non-diabetic rats (n=6) | Diabetic rats 3 days after injection (n=6) | Non-diabetic rats 7 days post-wounding (n=6) | Diabetic rats 7 days post-wounding (n=6) |
---|---|---|---|---|
Body weight (g) | 284.1±4.0 | 249.5±4.6 |
310.8±7.4 |
220.0±7.7 |
Blood glucose (mmol/l) | 6.0±0.2 | 23.9±0.9 |
6.4±0.2 | 23.0±1.2 |
Injection refers to an intraperitoneal injection of alloxan monohydrate dissolved in normal saline to induce diabetes. Values are presented as the means ± SEM. FBS levels were measured before and after the experiments. The 'diabetic rats 7 days post-wounding' group indicates the group treated with arnebin-1.
P<0.01 vs. non-diabetic rats.