- Email: [email protected]
Hyaluronan oligosaccharides promote diabetic wound healing by increasing angiogenesis
Accepted Manuscript Title: Hyaluronan Oligosaccharides Promote Diabetic Wound Healing by Increasing Angiogenesis Author: Yi Wang Guanying Han Bin Guo Jianhua Huang PII: DOI: Reference:
S1734-1140(16)30088-3 http://dx.doi.org/doi:10.1016/j.pharep.2016.07.001 PHAREP 528
To appear in: Received date: Revised date: Accepted date:
26-4-2016 21-6-2016 14-7-2016
Please cite this article as: Yi Wang, Guanying Han, Bin Guo, Jianhua Huang, Hyaluronan Oligosaccharides Promote Diabetic Wound Healing by Increasing Angiogenesis, http://dx.doi.org/10.1016/j.pharep.2016.07.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Hyaluronan Oligosaccharides Promote Diabetic Wound Healing by Increasing Angiogenesis Yi Wang1,2, Guanying Han3, Bin Guo3, Jianhua Huang3 1. Graduated School of Liaoning Medical University, Jinzhou, China. 2. School of Pharmaceutical Science, Liaoning Medical University, Jinzhou, China. 3. First Affiliated Hospital of Liaoning Medical University, Jinzhou, China. Correspondence
to
Jianhua
Huang
Phone:
+86-416-419-7073;
Fax:
+86-416-419-7074; E-mail address: [email protected] or Guanying Han Phone: +86-416-419-7073; Fax: +86-416-419-7074; E-mail address: [email protected]
Abstract Background: Hyaluronan (also known as hyaluronic acid) oligosaccharides (O-HA) can promote angiogenesis and wound healing; however, there are few reports on whether O-HA also plays a role in healing wounds of diabetic patients. Methods: In this study, we prepared a special ointment containing a mixture of hyaluronan fragments from 2 to 10 disaccharide units and investigated its effects on healing the wounds of diabetic rats. Results: We found that O-HA significantly increases proliferation, migration, and tube formation of endothelial cells under high glucose conditions, and topical administration of O-HA ointment promotes wound healing by increasing angiogenesis in the wounded area of the skin. The underlying mechanisms are that O-HA increases the phosphorylation of Src and ERK, and expression of TGF beta1, thereby increasing angiogenesis. Conclusions: This suggests that topical application of O-HA could be a useful method by which to treat diabetic wounds in clinical practice. Key
words:
Angiogenesis
Hyaluronan
oligosaccharides,
Diabetic
wound
healing,
Introduction Skin is the first barrier that protects the human body against microorganisms and physical and chemical damage. When there is damage to the skin, a series of biological and molecular events occur to help repair the damage, such as hemostasis, inflammation, proliferation, vascularization, and production and remodeling of the extracellular matrix (ECM)[1]; however, in diabetic patients, wound healing is often impaired[2]. Insufficient blood supply to the wound area of a diabetic patient is one of the most important factors that impairs wound healing[3,4]. It is estimated that 15% of diabetic patients have foot ulcerations, that result in prolonged hospitalization and even amputation[5], because current interventions and therapies on the diabetic wound are unsatisfactory. Looking for the new ways to increase angiogenesis is crucial for promoting diabetic wound healing in clinical practice. Hyaluronan (also known as hyaluronic acid, HA) is a naturally occurring nonsulfated, linear glycosaminoglycan consisting of repeating units of (β,1–4)-D-glucuronic acid-(β,1–3)-N-acetyl-D-glucosamine [6]. HA is found in its native state as a high molecular–mass polymer (>106 kDa) in the ECM of nearly all animal tissues and in significant amounts in the skin. HA plays an important role in maintaining tissue homeostasis, including angiogenesis. Native high molecular–weight HA is anti-angiogenic[7], whereas products of degradation that are a specific size (e.g., 3–10 disaccharide units; O-HA) are pro-angiogenic[8]. Thus, HA/O-HA might play important roles in diabetic wound healing. Although there are many in vitro studies related to O-HA and angiogenesis, few studies on the role O-HA plays in vivo, especially in healing wounds in diabetic animal models, have been reported. In the present study, we prepared a special slow-releasing ointment that contained a mixture of HA fragments from 2 to 10 disaccharides, investigated the effects of O-HA on endothelial cells in a high glucose environment, and
further studied the role that O-HA plays and its mechanisms in wound healing in a diabetic rat model. Materials and Methods Preparation of HA oligosaccharide ointment HA oligosaccharides were prepared as described previously[9]. Briefly, native high molecular–weight HA (Sigma-Aldrich Corporation, St. Louis MO, USA) was digested by hyaluronidase, and oligomers were fractionated by size exclusion chromatography using a Bio-Gel P-10 column (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Fragments from 2–10 disoligosaccharide units (4–20 mers) were pooled. An endotoxin activity check was performed using a Limulus amebocyte lysate assay. HA oligosaccharide–containing ointment was prepared by emulsification. Briefly, the oil phase was made by mixing 2.4 g vaseline, 2.4 g stearic acid, 1.6 g glycerol monostearate, and 2.5 g liquid paraffin wax. The mixture was melted at 75oC. The water phase was made by adding 2.4 g glycerin sodium, 0.2 g dodecyl sulfate, and 0.02 g methylparaben to 9.6 mL purified water and heating to 85oC to dissolve the agents. After cooling the water phase to 35–40oC, 0.03 g HA oligosaccharides was added and completely dissolved. The solution was slowly poured into the oil phase and stirred to a uniform paste. The final concentration of HA oligosaccharide was 0.15%. Cell proliferation assay Human umbilical vein endothelial cells (HUVECs) were plated at a density of 1.0 × 104 cells/well in complete medium containing 10% fetal bovine serum (FBS) in 96-well plates. After attachment of HUVECs, the medium was removed and replaced with complete medium containing 33 mmol/L glucose either with or without 10 μg/mL O-HA. The cells were then cultured for 72 h, after which 20 μL methylthiazolyldiphenyl-tetrazolium bromide (MTT) reagent
were added to each well, and the plates incubated for 4.0 h at 37°C. The supernatants were discarded and 150 μL dimethyl sulfoxide were added to each well. The 96-well plate was shaken in oscillators at room temperature for 10 min. The wells without cells were used as the zero point of absorbance. The absorbance was measured using a microplate reader at 570 nm. Tube formation assay Two hundred microliters Matrigel were polymerized in the wells of a 24-well plate at 37°C for 30 min. HUVECs (1.0 × 105) were dispensed into each well in 1000 μL complete medium containing 33 mM glucose, with or without 10 μg/mL O-HA. Cord morphogenesis of HUVECs was assessed by phase-contrast microscopy. Tubule structure was photographed with a microscope 8.0 h after cell seeding. Vascular cross points were counted in five randomly selected fields under the microscope in a blinded manner. In vitro assay of wound recovery HUVECs were plated at a density of 4.0 × 105 cells/well in complete medium containing 10% FBS in six-well plates. After 90% confluence of cultured cells, the cell layer was rinsed with phosphate buffered saline (PBS), wounded using a mechanical wounder, rinsed again in PBS to remove loose and dislodged cells, and placed into a fresh medium containing 30 mM glucose either in the presence or absence of 10 μg/mL O-HA. Cells were cultured again for 24 h. Movement of cells into the denuded area was quantified using a Seescan computerized image analysis system (Seescan Ltd., Cambridge, UK). Animals Male Sprague-Dawley (SD) rats weighing 200–250 g were provided by the Experimental Animal Center of our university. All experimental procedures were carried out in accordance with the recommended guidelines for the care and use of laboratory animals issued by the Chinese Council on Animal Research.
Diabetes was induced in the male SD rats (n = 20) by a one-time tail-vein injection of streptozotocin (STZ, Sigma-Aldrich, St Louis, MO, USA) dissolved in 100 mmol/L sodium citrate buffer (pH = 4.5) at a dose of 60 mg/kg body weight. For the preparation of citrate buffer, 3g Na3C6H5O7.2H2O and 0.4g C6H8O7.H2O were mixed and dissolved in 100 mL double distilled water, the final concentration is 100 mmol/L, pH is about 4.5. For this study, a diabetic model was defined as an experimental model characterized by a non-fasting blood glucose measurement of 16.7 mmol/L or higher, detected by three consecutive measurements. A total of 16 rats complied with the standard. After 7 days of streptozotocin administration, the diabetic rats were randomly and equally divided into two groups as follows: control (ointment) and treatment (ointment + O-HA). After anesthesia using an intramuscular injection of 40 mg/kg ketamine and 4.0 mg/kg xylazine, the hair was shaved, and a 2.0-cm full-thickness wound was made on the dorsal surface of the right flank. The ointment with or without O-HA was smeared on the surface of the wound twice a day during the whole experimental process. Measurement of blood flow Immediately after the wound was made and every 7.0 d during the wound-healing period, the blood flow around the wound area was measured using laser Doppler (Perimed , PSI, Perimed). Histological examination After 14 d, the skins around the wound were harvested, skin specimens were fixed in 10% neutral buffered formalin; after fixation, the tissues were embedded in paraffin. Five micrometer thick sections were mounted on glass slides, de-waxed, rehydrated to distilled water and stained with haematoxylin and eosin. All slides were examined by a pathologist without knowledge of the previous treatment, and the granulation tissue and re-epithelialization were evaluated. Capillary density measurement
After 14 d, the skins around the wound were harvested, snap frozen in liquid nitrogen, and 10-μm cryosections were prepared. Endothelial cells were stained with mouse monoclonal anti-CD31 primary antibody (Catalogue#555027, BD Pharmingen, SD, CA, USA) at a dilution of 1:200, followed by biotinylated anti-mouse IgG secondary antibody, and an avidin-HRP conjugate for color reaction (DAB paraffin IHC staining module, Ventana Medical Systems, Inc., Tucson, AZ, USA). Hemotoxilin was used for counter staining. The sections were analyzed by microscopy with five high-power fields randomly selected for each section. CD31-positive cells were counted in a blinded manner. The number of CD 31-positive cells in each field was used as an index of capillary density. Western blot The tissues from the wound area were washed twice with cold PBS and resuspended in cold lysis buffer containing 20 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 1.0 mmol/L ethylenediaminetetraacetic acid, 0.5% Triton X-100, and protease inhibitors (Roche). Similar quantities of total protein (20 μg) were separated by electrophoresis using sodium dodecyl sulfate polyacrylamide gel, transferred onto polyvinylidene fluoride membranes, and blocked overnight in blocking solution at 4.0oC. To detect p-Src and p-ERK, and TGF beta1, the membranes were incubated for 1.0 h with a rabbit polyclonal antibodies raised against p-Src (Catalogue#44-662G, Invitrogen Corporation, Carlsbad, CA, USA, dilution
1:1000),
a
mouse
monoclonal
antibody
against
p-ERK
(Catalogue#4696, Cell Signaling Technology, Danvers, MA, USA, dilution 1:1000),
and
a
rabbit
polyclonal
antibody
against
TGF-beta1(Catalogue#ab92486, Abcam, dilution 1:1000) respectively, followed by anti-mouse or anti-rabbit secondary antibodies conjugated to horseradish peroxidase (Zymed, Inc., South San Francisco, CA, USA) for 1.0 h at room temperature. The ECL Plus system (Amersham Biosciences UK, Little Chalfont,
UK) was used to reveal the signals. The total Src and ERK, and actin were detected as control respectively. Statistical analyses Data are presented as the mean ± standard deviation (SD). Statistical comparisons were performed using analysis of variance followed by Bonferroni/Dunn tests. p< 0.05 was considered statistically significant. Results O-HA promotes the proliferation of HUVECs under high glucose conditions To determine the effect of O-HA on the proliferation of HUVECs under high glucose conditions, an MTT assay was performed. The concentration of O-HA used was 10 μg/mL, which proved to be effective in increasing the proliferation of endothelial cells under normal glucose levels[10]. The results showed that after 72 h of treatment with O-HA, endothelial cell proliferation increased more significantly than that in the control (p <0.05, Fig.1A). O-HA promotes wound healing in vitro under high glucose conditions To determine the effect of O-HA on the migration of endothelial cells, an in vitro wound recovery assay was performed. After creating a wound on the sample plate, 10 μg/mL O-HA were added to the culture medium. The results showed that after 24 h of treatment with O-HA, the wound had recovered more significantly than that of the control (p < 0.01, Fig1B and C). O-HA promotes tube formation of endothelial cells under high glucose conditions To determine the effect of O-HA on angiogenesis in vitro, tube formation of endothelial cells was assessed. O-HA (10 μg/mL) was added to the culture medium. After 8 h of culturing endothelial cells in Matrigel, endothelial cell tube formation was increased more significantly than that of the control (p <0.01, Fig1D and C).
Topical application of O-HA promotes wound healing in diabetic rats To determine the effect of O-HA on wound healing in vivo, 16 diabetic rats were randomly divided into 2 groups with 8 rats in each group, and used for the wound model. Our preliminary data showed that the ointment containing 0.15% O-HA was effective in decreasing wound size in rats (data not shown). The ointment with 0.15% O-HA was topically applied to the wound at twice a day and the wound size was measured every 7.0 d during the wound recovery process. The results showed that topical use of the ointment containing O-HA significantly decreased wound size (p <0.05, Fig2A and B). Haematoxylin-eosin staining of wound sections at 14 days post-wounding showed that O-HA increases granulation the tissue and re-epithelizlization of the wound (Fig 2C). O-HA improves of blood flow in wounds in diabetic rats To determine whether O-HA increases the blood supply to the wound area of diabetic rats, blood flow around wound area was measured using laser Doppler every 7.0 d during the wound recovery process. The average blood flow–intensity scale was compared between the groups treated with and without O-HA. The results showed that O-HA improved blood supply to the wound area more significantly than that of the control (p < 0.05, Fig 3A and B). O-HA increases capillary density in wounds in diabetic rats To determine the effects of O-HA on angiogenesis in vivo, the skin tissues around the wound area were harvested on day 14 and immunohistochemistry was performed to detect CD31 expression. The results showed that O-HA significantly increased the capillary density in the wound area of the diabetic rats (p<0.01, Fig. 3C and D). O-HA increases the expression of p-Src and p-ERK, and TGF-beta1 in wound area To determine the mechanisms by which O-HA improves angiogenesis, we focused on the effects of O-HA on the downstream signaling pathway of CD44
receptor. The results showed that O-HA did not affect the expression of total Src and Erk, but increased the phosphorylation of Src and ERK in the tissue of diabetic wound (Fig 4A and B). Furthermore, O-HA was found to increased expression of TGF-beta1 (Fig 4C). Discussion O-HA has been shown to increase angiogenesis in vitro and in vivo. Although there are many in vitro studies related to O-HA and angiogenesis, few studies on the role of O-HA in vivo, particularly those of diabetic wound healing in animal models, have been reported. This prompted us to test the effects of O-HA on diabetic wound healing. In this study, we investigated the effects of O-HA on angiogenesis under diabetic conditions and found that O-HA promotes diabetic wound healing by increasing angiogenesis. A wound on a diabetic patient is difficult to treat in clinical practice and has a high rate of morbidity. Although new methods, such as stem cell therapy, are being used to deal with this issue and show encouraging results, there are concerns about the safety of using stem cell therapy in clinical practice[11]. Traditional treatment methods, such as drug therapy, still play important roles in the treatment of diabetic wounds. In this study, O-HA was mixed in a topical ointment and applied directly onto the wound surface. The O-HA was slowly released from the ointment and had a positive effect on the wound-healing process. Because O-HA is the final digested product of HA and contains no protein, there are no concerns about the potential health risks, such as those associated with some new treatment methods (e.g., stem cell therapy). HA is synthesized by HA synthases and degraded by hyaluronidases in vivo. The synthesis and degradation of HA maintain equilibrium in organic tissue. Under normal physiological conditions, hyaluronidases enable HA turnover at a rate of nearly 33% of totally body HA content per day[12]; short (<20 monomers) HA fragments (O-HA) can be generated as products of HA
degradation by the hyaluronidase isoform HYAL-1[13]. Under pathologic conditions, such as an injury, the production of O-HA is enhanced[14] and interacts with the surrounding cells, causing a series of pathologic changes of the cells in the wound, including angiogenesis[15]. It was found that in diabetic mice, the level of HA decreases, which might influence the production of O-HA[16]. Thus, in the diabetic wound, supplementing with O-HA has a beneficial effect on healing. Our topical use of ointment containing O-HA significantly promoted wound healing in diabetic rats, indicating the feasibility of its use as a therapeutic tool. O-HA functions in the body by binding to surface receptors, resulting in activation of signal transducers and mitogenesis[17]. In vascular endothelial cells, both CD44 and RHAMM have been identified as the targets for transduction of O-HA-induced angiogenesis[18-20]. Savani et al.[21] demonstrated that RHAMM–ligand interaction dictates endothelial cell motility, and CD44–ligand interaction dictates endothelial cell proliferation. Both these receptors work in tandem to facilitate formation of new blood vessels. O-HA induces a rapid CD44-dependent activation of multiple isoforms of PKC, Raf-1 kinase, MEK-1, and ERK1/2, resulting in proliferation of endothelial cells[18]. O-HA also binds to the RHAMM receptor and induces tyrosine phosphorylation of p125FAK, paxillin, p42/44, and extracellular ERK1/2, resulting in cell proliferation[20]. Recently, Guo et al. demonstrated that O-HA causes the upregulation of eNOS and procollagen-1 and downregulation of MMP-9 and MMP-13, resulting in an increase in angiogenesis[10]. This means that O-HA application also affects angiogenetic-related cytokine production in the wound. We found that O-HA increases expression of TGF beta1 in the wound, which is in accordance with the study by Galeano M, in which systemic administration of HA was shown to increase diabetic wound healing by increasing the expression of TGF beta1[22]. Src family kinases are signaling enzymes that have long been recognized to regulate critical cellular processes such as
proliferation, survival, migration and metastasis[23]. While the generic mitogen-activated protein kinases(signaling pathway is shared by four distinct cascades, including the extracellular signal-related kinases(ERK). MAPK/ERK signaling pathway plays important roles in cell proliferation, differentiation, migration senescence and apoptosis[24]. In this study, we found that O-HA administration increases phosphorylation of Src and ERK in a diabetic wound, which indicates that by initiating Src/ERK transduction signaling, O-HA plays an important role in promoting angiogenesis in diabetic mellitus. In addition to promoting angiogenesis, O-HA stimulates abundant collagen deposits of endothelial cells in dermal tissue[10]. Although collagen deposits will accelerate the wound-healing process, it raises concerns that rapid closure of an open wound by O-HA treatment increases scar formation, which in turn, inferior the quality of the acute open wound[8]; however, in the treatment of a diabetic wound, fast collagen production is needed to accelerate healing and, as such, might be more acceptable. Further studies are needed to investigate the effects of O-HA on collagen production in the diabetic wound-healing process. Conclusion In summary, O-HA increases endothelial cell proliferation, migration, and tube formation under high glucose conditions. Topical administration of ointment containing O-HA promotes wound healing in diabetic rats by increasing angiogenesis. The mechanisms are in part by increasing the phosphorylation of Src and ERK, and expression of TGF beta1. This would provide a new treatment method for diabetic wounds in clinical practice.
Funding This work was supported in part by a grant to JH from the Key Laboratory Program
of
Liaoning
Provincial
Department
Ministration(LZ2015051). Conflict of interest The authors declare no conflict of interest.
of
Education
References 1. Reinke JM, Sorg H. Wound repair and regeneration. Eur Surg Res. 2012;49(1):35–43. 2. Xu F, Zhang C, Graves DT. Abnormal cell responses and role of TNF-α in impaired diabetic wound healing.Biomed Res Int. 2013;2013:754802. 3. Martin A, Komada MR, Sane DC. Abnormal angiogenesis in diabetes mellitus. Med Res Rev. 2003;23(2):117–45. 4. O'Loughlin A, Kulkarni M, Creane M, Vaughan EE, Mooney E, Shaw G,et al.Topical administration of allogeneic mesenchymal stromal cells seeded in a collagen scaffold augments wound healing and increases angiogenesis in the diabetic rabbit ulcer. Diabetes. 2013;62(7):2588-94. 5. American Diabetes Association. Economic costs of diabetes in the U.S. in 2007. Diabetes Care. 2008;31(13):596–615. 6. Brinck J, Heldin P.Expression of recombinant hyaluronan synthase (HAS) isoforms in CHO cells reduces cell migration and cell surface CD44. Exp Cell Res. 1999;252(2):342-51. 7. Feinberg RN, Beebe DC.Hyaluronate in vasculogenesis. Science. 1983 ;220(4602):1177-9 8. Slevin M, Krupinski J, Gaffney J, Matou S, West D, Delisser H, et al.Hyaluronan-mediated angiogenesis in vascular disease: uncovering RHAMM and CD44 receptor signaling pathways. Matrix Biol. 2007;26(1):58-68. 9. Gao F, Liu Y, He Y, Yang C, Wang Y, Shi X,et al.Hyaluronan oligosaccharides promote excisional wound healing through enhanced angiogenesis.Matrix Biol. 2010;29(2):107-16. 10. Gao F, Yang CX, Mo W, Liu YW, He YQ.Hyaluronan oligosaccharides are potential stimulators to angiogenesis via RHAMM mediated signal pathway in wound healing. Clin Invest Med. 2008;31(3):E106-16 11. Yang M, Sheng L, Zhang TR, Li Q.Stem cell therapy for lower extremity diabetic ulcers: where do we stand? Biomed Res Int. 2013;2013:462179. 12. Stern R.Hyaluronan catabolism: a new metabolic pathway. Eur J Cell Biol. 2004;83(7):317-25 13. West DC, Hampson IN, Arnold F, Kumar S. Angiogenesis induced by degradation products of hyaluronic acid. Science. 1985(4705) ;228:1324-6. 14. Slevin M, West D, Kumar P, Rooney P, Kumar S.Hyaluronan, angiogenesis and malignant disease. Int J Cancer. 2004;109(5):793-4; author reply 795-6.
15. Rooney P, Kumar S, Ponting J, Wang M.The role of hyaluronan in tumour neovascularization (review). Int J Cancer. 1995;60(5):632-6. 16. Galeano M, Polito F, Bitto A, Irrera N, Campo GM, Avenoso A,et al.Systemic administration of high-molecular weight hyaluronan stimulates wound healing in geneticallydiabetic mice.Biochim Biophys Acta. 2011;1812(7):752-9. 17. Hall CL, Lange LA, Prober DA, Zhang S, Turley EA.pp60(c-src) is required for cell locomotion regulated by the hyaluronanreceptor RHAMM. Oncogene. 1996 ;13(10):2213-24. 18. Slevin M, Krupinski J, Kumar S, Gaffney J.Angiogenic oligosaccharides of hyaluronan induce protein tyrosine kinase activity in endothelial cells and activate a cytoplasmic signal transduction pathway resulting in proliferation. Lab Invest. 1998;78(8):987-1003. 19. Nandi A, Estess P, Siegelman MH. Hyaluronan anchoring and regulation on the surface of vascular endothelial cells is mediated through the functionally active form of CD44. J Biol Chem. 2000;275(20):14939-48. 20. Lokeshwar VB, Selzer MG.Differences in hyaluronic acid-mediated functions and signaling in arterial, microvessel, and vein-derived human endothelial cells. J Biol Chem. 2000 ;275(36):27641-9. 21. Savani RC, Cao G, Pooler PM, Zaman A, Zhou Z, DeLisser HM. Differential involvement of the hyaluronan (HA) receptors CD44 and receptor for HA-mediated motility in endothelial cell function and angiogenesis. J Biol Chem. 2001;276(39):36770-8. 22. Galeano M, Polito F, Bitto A, Irrera N, Campo GM, Avenoso A, et al. Systemic administration of high-molecular weight hyaluronan stimulates wound healing in genetically diabetic mice.Biochim Biophys Acta. 2011;1812(7):752-9. 23. Schlessinger J.New roles for Src kinases in control of cell survival and angiogenesis.Cell. 2000 ;100(3):293-6. 24. Sun Y, Liu WZ, Liu T, Feng X, Yang N, Zhou HF.Signaling pathway of MAPK/ERK in cell proliferation, differentiation, migration, senescence and apoptosis.J Recept Signal Transduct Res. 2015;35(6):600-4.
Figure legends Figure 1. O-HA promotes proliferation, migration, and tube formation of endothelial cells under high glucose conditions in vitro. A) Effect of O-HA on the proliferation of endothelial cells. Methylthiazolyldiphenyl-tetrazolium bromide assay shows that O-HA significantly increases the proliferation of endothelial cells after 72 h of cell culture under high glucose condition (p < 0.05). B) Effect of O-HA on migration of endothelial cells. Upper panel, phosphate buffered saline (PBS)–treated group, lower panel, O-HA-treated group. C) O-HA significantly improves migration of endothelial cells after 24 h of cell culture of endothelial cells (p < 0.01). D) Effect of O-HA on endothelial cell tube formation. Left panel, PBS-treated group; right panel, O-HA-treated group. E) O-HA significantly increases tube formation after 8 h of cell culture in Matrigel (p <0.01). Figure 2. Topical administration of ointment containing O-HA decreases wound size in diabetic rats. A) Upper panel, diabetic wound treated with ointment with O-HA; lower panel, diabetic wound treated with ointment without O-HA. B) O-HA significantly decreases wound size in diabetic rats compared with that in the controls (n = 8, p <0.01). C) Haematoxylin-eosin staining of wound sections at 14 days post-wounding. The double-headed arrows indicate the edges of the scar. O-HA increases granulation the tissue and re-epithelizlization of the wound (Bar = 100 μm). Figure 3. O-HA increases blood flow in the wounded area of diabetic rats. A) Laser Doppler imaging of blood flow in the wound area. Upper panel, wound treated with ointment containing O-HA; lower panel, wound treated with ointment without O-HA. B) O-HA significantly increases blood flow in the wound area of diabetic rats compared with that of the control (n = 8, p < 0.01). C) Immunostaining with CD31 of tissue from wound area. Left panel, tissue
from the wound treated with ointment without O-HA; right panel, tissue from the wound treated with ointment with O-HA (Bar = 100 μm). D) O-HA significantly increases capillary density in the wound area of diabetic rats compared with that of the controls (n = 8, p < 0.01). Figure 4. O-HA increases the phosphorylation of Srk and ERK, and expression of TGF beta1 in the wound area of diabetic rats. A) Phosphorylation of Srk in a diabetic wound. Lane 1, wound treated with ointment without O-HA; lane 2, wound treated with ointment with O-HA. B) Phosphorylation of ERK in diabetic wound. Lane 1, wound treated with ointment without O-HA; lane2, wound treated with ointment with O-HA. C) Expression of TGF beta1 in diabetic wound, Lane 1, wound treated with ointment without O-HA; lane2, wound treated with ointment with O-HA. Each experiment was repeated three times.
S1734-1140(16)30088-3 http://dx.doi.org/doi:10.1016/j.pharep.2016.07.001 PHAREP 528
To appear in: Received date: Revised date: Accepted date:
26-4-2016 21-6-2016 14-7-2016
Please cite this article as: Yi Wang, Guanying Han, Bin Guo, Jianhua Huang, Hyaluronan Oligosaccharides Promote Diabetic Wound Healing by Increasing Angiogenesis, http://dx.doi.org/10.1016/j.pharep.2016.07.001 This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
Hyaluronan Oligosaccharides Promote Diabetic Wound Healing by Increasing Angiogenesis Yi Wang1,2, Guanying Han3, Bin Guo3, Jianhua Huang3 1. Graduated School of Liaoning Medical University, Jinzhou, China. 2. School of Pharmaceutical Science, Liaoning Medical University, Jinzhou, China. 3. First Affiliated Hospital of Liaoning Medical University, Jinzhou, China. Correspondence
to
Jianhua
Huang
Phone:
+86-416-419-7073;
Fax:
+86-416-419-7074; E-mail address: [email protected] or Guanying Han Phone: +86-416-419-7073; Fax: +86-416-419-7074; E-mail address: [email protected]
Abstract Background: Hyaluronan (also known as hyaluronic acid) oligosaccharides (O-HA) can promote angiogenesis and wound healing; however, there are few reports on whether O-HA also plays a role in healing wounds of diabetic patients. Methods: In this study, we prepared a special ointment containing a mixture of hyaluronan fragments from 2 to 10 disaccharide units and investigated its effects on healing the wounds of diabetic rats. Results: We found that O-HA significantly increases proliferation, migration, and tube formation of endothelial cells under high glucose conditions, and topical administration of O-HA ointment promotes wound healing by increasing angiogenesis in the wounded area of the skin. The underlying mechanisms are that O-HA increases the phosphorylation of Src and ERK, and expression of TGF beta1, thereby increasing angiogenesis. Conclusions: This suggests that topical application of O-HA could be a useful method by which to treat diabetic wounds in clinical practice. Key
words:
Angiogenesis
Hyaluronan
oligosaccharides,
Diabetic
wound
healing,
Introduction Skin is the first barrier that protects the human body against microorganisms and physical and chemical damage. When there is damage to the skin, a series of biological and molecular events occur to help repair the damage, such as hemostasis, inflammation, proliferation, vascularization, and production and remodeling of the extracellular matrix (ECM)[1]; however, in diabetic patients, wound healing is often impaired[2]. Insufficient blood supply to the wound area of a diabetic patient is one of the most important factors that impairs wound healing[3,4]. It is estimated that 15% of diabetic patients have foot ulcerations, that result in prolonged hospitalization and even amputation[5], because current interventions and therapies on the diabetic wound are unsatisfactory. Looking for the new ways to increase angiogenesis is crucial for promoting diabetic wound healing in clinical practice. Hyaluronan (also known as hyaluronic acid, HA) is a naturally occurring nonsulfated, linear glycosaminoglycan consisting of repeating units of (β,1–4)-D-glucuronic acid-(β,1–3)-N-acetyl-D-glucosamine [6]. HA is found in its native state as a high molecular–mass polymer (>106 kDa) in the ECM of nearly all animal tissues and in significant amounts in the skin. HA plays an important role in maintaining tissue homeostasis, including angiogenesis. Native high molecular–weight HA is anti-angiogenic[7], whereas products of degradation that are a specific size (e.g., 3–10 disaccharide units; O-HA) are pro-angiogenic[8]. Thus, HA/O-HA might play important roles in diabetic wound healing. Although there are many in vitro studies related to O-HA and angiogenesis, few studies on the role O-HA plays in vivo, especially in healing wounds in diabetic animal models, have been reported. In the present study, we prepared a special slow-releasing ointment that contained a mixture of HA fragments from 2 to 10 disaccharides, investigated the effects of O-HA on endothelial cells in a high glucose environment, and
further studied the role that O-HA plays and its mechanisms in wound healing in a diabetic rat model. Materials and Methods Preparation of HA oligosaccharide ointment HA oligosaccharides were prepared as described previously[9]. Briefly, native high molecular–weight HA (Sigma-Aldrich Corporation, St. Louis MO, USA) was digested by hyaluronidase, and oligomers were fractionated by size exclusion chromatography using a Bio-Gel P-10 column (Bio-Rad Laboratories, Inc., Hercules, CA, USA). Fragments from 2–10 disoligosaccharide units (4–20 mers) were pooled. An endotoxin activity check was performed using a Limulus amebocyte lysate assay. HA oligosaccharide–containing ointment was prepared by emulsification. Briefly, the oil phase was made by mixing 2.4 g vaseline, 2.4 g stearic acid, 1.6 g glycerol monostearate, and 2.5 g liquid paraffin wax. The mixture was melted at 75oC. The water phase was made by adding 2.4 g glycerin sodium, 0.2 g dodecyl sulfate, and 0.02 g methylparaben to 9.6 mL purified water and heating to 85oC to dissolve the agents. After cooling the water phase to 35–40oC, 0.03 g HA oligosaccharides was added and completely dissolved. The solution was slowly poured into the oil phase and stirred to a uniform paste. The final concentration of HA oligosaccharide was 0.15%. Cell proliferation assay Human umbilical vein endothelial cells (HUVECs) were plated at a density of 1.0 × 104 cells/well in complete medium containing 10% fetal bovine serum (FBS) in 96-well plates. After attachment of HUVECs, the medium was removed and replaced with complete medium containing 33 mmol/L glucose either with or without 10 μg/mL O-HA. The cells were then cultured for 72 h, after which 20 μL methylthiazolyldiphenyl-tetrazolium bromide (MTT) reagent
were added to each well, and the plates incubated for 4.0 h at 37°C. The supernatants were discarded and 150 μL dimethyl sulfoxide were added to each well. The 96-well plate was shaken in oscillators at room temperature for 10 min. The wells without cells were used as the zero point of absorbance. The absorbance was measured using a microplate reader at 570 nm. Tube formation assay Two hundred microliters Matrigel were polymerized in the wells of a 24-well plate at 37°C for 30 min. HUVECs (1.0 × 105) were dispensed into each well in 1000 μL complete medium containing 33 mM glucose, with or without 10 μg/mL O-HA. Cord morphogenesis of HUVECs was assessed by phase-contrast microscopy. Tubule structure was photographed with a microscope 8.0 h after cell seeding. Vascular cross points were counted in five randomly selected fields under the microscope in a blinded manner. In vitro assay of wound recovery HUVECs were plated at a density of 4.0 × 105 cells/well in complete medium containing 10% FBS in six-well plates. After 90% confluence of cultured cells, the cell layer was rinsed with phosphate buffered saline (PBS), wounded using a mechanical wounder, rinsed again in PBS to remove loose and dislodged cells, and placed into a fresh medium containing 30 mM glucose either in the presence or absence of 10 μg/mL O-HA. Cells were cultured again for 24 h. Movement of cells into the denuded area was quantified using a Seescan computerized image analysis system (Seescan Ltd., Cambridge, UK). Animals Male Sprague-Dawley (SD) rats weighing 200–250 g were provided by the Experimental Animal Center of our university. All experimental procedures were carried out in accordance with the recommended guidelines for the care and use of laboratory animals issued by the Chinese Council on Animal Research.
Diabetes was induced in the male SD rats (n = 20) by a one-time tail-vein injection of streptozotocin (STZ, Sigma-Aldrich, St Louis, MO, USA) dissolved in 100 mmol/L sodium citrate buffer (pH = 4.5) at a dose of 60 mg/kg body weight. For the preparation of citrate buffer, 3g Na3C6H5O7.2H2O and 0.4g C6H8O7.H2O were mixed and dissolved in 100 mL double distilled water, the final concentration is 100 mmol/L, pH is about 4.5. For this study, a diabetic model was defined as an experimental model characterized by a non-fasting blood glucose measurement of 16.7 mmol/L or higher, detected by three consecutive measurements. A total of 16 rats complied with the standard. After 7 days of streptozotocin administration, the diabetic rats were randomly and equally divided into two groups as follows: control (ointment) and treatment (ointment + O-HA). After anesthesia using an intramuscular injection of 40 mg/kg ketamine and 4.0 mg/kg xylazine, the hair was shaved, and a 2.0-cm full-thickness wound was made on the dorsal surface of the right flank. The ointment with or without O-HA was smeared on the surface of the wound twice a day during the whole experimental process. Measurement of blood flow Immediately after the wound was made and every 7.0 d during the wound-healing period, the blood flow around the wound area was measured using laser Doppler (Perimed , PSI, Perimed). Histological examination After 14 d, the skins around the wound were harvested, skin specimens were fixed in 10% neutral buffered formalin; after fixation, the tissues were embedded in paraffin. Five micrometer thick sections were mounted on glass slides, de-waxed, rehydrated to distilled water and stained with haematoxylin and eosin. All slides were examined by a pathologist without knowledge of the previous treatment, and the granulation tissue and re-epithelialization were evaluated. Capillary density measurement
After 14 d, the skins around the wound were harvested, snap frozen in liquid nitrogen, and 10-μm cryosections were prepared. Endothelial cells were stained with mouse monoclonal anti-CD31 primary antibody (Catalogue#555027, BD Pharmingen, SD, CA, USA) at a dilution of 1:200, followed by biotinylated anti-mouse IgG secondary antibody, and an avidin-HRP conjugate for color reaction (DAB paraffin IHC staining module, Ventana Medical Systems, Inc., Tucson, AZ, USA). Hemotoxilin was used for counter staining. The sections were analyzed by microscopy with five high-power fields randomly selected for each section. CD31-positive cells were counted in a blinded manner. The number of CD 31-positive cells in each field was used as an index of capillary density. Western blot The tissues from the wound area were washed twice with cold PBS and resuspended in cold lysis buffer containing 20 mmol/L HEPES (pH 7.5), 150 mmol/L NaCl, 1.0 mmol/L ethylenediaminetetraacetic acid, 0.5% Triton X-100, and protease inhibitors (Roche). Similar quantities of total protein (20 μg) were separated by electrophoresis using sodium dodecyl sulfate polyacrylamide gel, transferred onto polyvinylidene fluoride membranes, and blocked overnight in blocking solution at 4.0oC. To detect p-Src and p-ERK, and TGF beta1, the membranes were incubated for 1.0 h with a rabbit polyclonal antibodies raised against p-Src (Catalogue#44-662G, Invitrogen Corporation, Carlsbad, CA, USA, dilution
1:1000),
a
mouse
monoclonal
antibody
against
p-ERK
(Catalogue#4696, Cell Signaling Technology, Danvers, MA, USA, dilution 1:1000),
and
a
rabbit
polyclonal
antibody
against
TGF-beta1(Catalogue#ab92486, Abcam, dilution 1:1000) respectively, followed by anti-mouse or anti-rabbit secondary antibodies conjugated to horseradish peroxidase (Zymed, Inc., South San Francisco, CA, USA) for 1.0 h at room temperature. The ECL Plus system (Amersham Biosciences UK, Little Chalfont,
UK) was used to reveal the signals. The total Src and ERK, and actin were detected as control respectively. Statistical analyses Data are presented as the mean ± standard deviation (SD). Statistical comparisons were performed using analysis of variance followed by Bonferroni/Dunn tests. p< 0.05 was considered statistically significant. Results O-HA promotes the proliferation of HUVECs under high glucose conditions To determine the effect of O-HA on the proliferation of HUVECs under high glucose conditions, an MTT assay was performed. The concentration of O-HA used was 10 μg/mL, which proved to be effective in increasing the proliferation of endothelial cells under normal glucose levels[10]. The results showed that after 72 h of treatment with O-HA, endothelial cell proliferation increased more significantly than that in the control (p <0.05, Fig.1A). O-HA promotes wound healing in vitro under high glucose conditions To determine the effect of O-HA on the migration of endothelial cells, an in vitro wound recovery assay was performed. After creating a wound on the sample plate, 10 μg/mL O-HA were added to the culture medium. The results showed that after 24 h of treatment with O-HA, the wound had recovered more significantly than that of the control (p < 0.01, Fig1B and C). O-HA promotes tube formation of endothelial cells under high glucose conditions To determine the effect of O-HA on angiogenesis in vitro, tube formation of endothelial cells was assessed. O-HA (10 μg/mL) was added to the culture medium. After 8 h of culturing endothelial cells in Matrigel, endothelial cell tube formation was increased more significantly than that of the control (p <0.01, Fig1D and C).
Topical application of O-HA promotes wound healing in diabetic rats To determine the effect of O-HA on wound healing in vivo, 16 diabetic rats were randomly divided into 2 groups with 8 rats in each group, and used for the wound model. Our preliminary data showed that the ointment containing 0.15% O-HA was effective in decreasing wound size in rats (data not shown). The ointment with 0.15% O-HA was topically applied to the wound at twice a day and the wound size was measured every 7.0 d during the wound recovery process. The results showed that topical use of the ointment containing O-HA significantly decreased wound size (p <0.05, Fig2A and B). Haematoxylin-eosin staining of wound sections at 14 days post-wounding showed that O-HA increases granulation the tissue and re-epithelizlization of the wound (Fig 2C). O-HA improves of blood flow in wounds in diabetic rats To determine whether O-HA increases the blood supply to the wound area of diabetic rats, blood flow around wound area was measured using laser Doppler every 7.0 d during the wound recovery process. The average blood flow–intensity scale was compared between the groups treated with and without O-HA. The results showed that O-HA improved blood supply to the wound area more significantly than that of the control (p < 0.05, Fig 3A and B). O-HA increases capillary density in wounds in diabetic rats To determine the effects of O-HA on angiogenesis in vivo, the skin tissues around the wound area were harvested on day 14 and immunohistochemistry was performed to detect CD31 expression. The results showed that O-HA significantly increased the capillary density in the wound area of the diabetic rats (p<0.01, Fig. 3C and D). O-HA increases the expression of p-Src and p-ERK, and TGF-beta1 in wound area To determine the mechanisms by which O-HA improves angiogenesis, we focused on the effects of O-HA on the downstream signaling pathway of CD44
receptor. The results showed that O-HA did not affect the expression of total Src and Erk, but increased the phosphorylation of Src and ERK in the tissue of diabetic wound (Fig 4A and B). Furthermore, O-HA was found to increased expression of TGF-beta1 (Fig 4C). Discussion O-HA has been shown to increase angiogenesis in vitro and in vivo. Although there are many in vitro studies related to O-HA and angiogenesis, few studies on the role of O-HA in vivo, particularly those of diabetic wound healing in animal models, have been reported. This prompted us to test the effects of O-HA on diabetic wound healing. In this study, we investigated the effects of O-HA on angiogenesis under diabetic conditions and found that O-HA promotes diabetic wound healing by increasing angiogenesis. A wound on a diabetic patient is difficult to treat in clinical practice and has a high rate of morbidity. Although new methods, such as stem cell therapy, are being used to deal with this issue and show encouraging results, there are concerns about the safety of using stem cell therapy in clinical practice[11]. Traditional treatment methods, such as drug therapy, still play important roles in the treatment of diabetic wounds. In this study, O-HA was mixed in a topical ointment and applied directly onto the wound surface. The O-HA was slowly released from the ointment and had a positive effect on the wound-healing process. Because O-HA is the final digested product of HA and contains no protein, there are no concerns about the potential health risks, such as those associated with some new treatment methods (e.g., stem cell therapy). HA is synthesized by HA synthases and degraded by hyaluronidases in vivo. The synthesis and degradation of HA maintain equilibrium in organic tissue. Under normal physiological conditions, hyaluronidases enable HA turnover at a rate of nearly 33% of totally body HA content per day[12]; short (<20 monomers) HA fragments (O-HA) can be generated as products of HA
degradation by the hyaluronidase isoform HYAL-1[13]. Under pathologic conditions, such as an injury, the production of O-HA is enhanced[14] and interacts with the surrounding cells, causing a series of pathologic changes of the cells in the wound, including angiogenesis[15]. It was found that in diabetic mice, the level of HA decreases, which might influence the production of O-HA[16]. Thus, in the diabetic wound, supplementing with O-HA has a beneficial effect on healing. Our topical use of ointment containing O-HA significantly promoted wound healing in diabetic rats, indicating the feasibility of its use as a therapeutic tool. O-HA functions in the body by binding to surface receptors, resulting in activation of signal transducers and mitogenesis[17]. In vascular endothelial cells, both CD44 and RHAMM have been identified as the targets for transduction of O-HA-induced angiogenesis[18-20]. Savani et al.[21] demonstrated that RHAMM–ligand interaction dictates endothelial cell motility, and CD44–ligand interaction dictates endothelial cell proliferation. Both these receptors work in tandem to facilitate formation of new blood vessels. O-HA induces a rapid CD44-dependent activation of multiple isoforms of PKC, Raf-1 kinase, MEK-1, and ERK1/2, resulting in proliferation of endothelial cells[18]. O-HA also binds to the RHAMM receptor and induces tyrosine phosphorylation of p125FAK, paxillin, p42/44, and extracellular ERK1/2, resulting in cell proliferation[20]. Recently, Guo et al. demonstrated that O-HA causes the upregulation of eNOS and procollagen-1 and downregulation of MMP-9 and MMP-13, resulting in an increase in angiogenesis[10]. This means that O-HA application also affects angiogenetic-related cytokine production in the wound. We found that O-HA increases expression of TGF beta1 in the wound, which is in accordance with the study by Galeano M, in which systemic administration of HA was shown to increase diabetic wound healing by increasing the expression of TGF beta1[22]. Src family kinases are signaling enzymes that have long been recognized to regulate critical cellular processes such as
proliferation, survival, migration and metastasis[23]. While the generic mitogen-activated protein kinases(signaling pathway is shared by four distinct cascades, including the extracellular signal-related kinases(ERK). MAPK/ERK signaling pathway plays important roles in cell proliferation, differentiation, migration senescence and apoptosis[24]. In this study, we found that O-HA administration increases phosphorylation of Src and ERK in a diabetic wound, which indicates that by initiating Src/ERK transduction signaling, O-HA plays an important role in promoting angiogenesis in diabetic mellitus. In addition to promoting angiogenesis, O-HA stimulates abundant collagen deposits of endothelial cells in dermal tissue[10]. Although collagen deposits will accelerate the wound-healing process, it raises concerns that rapid closure of an open wound by O-HA treatment increases scar formation, which in turn, inferior the quality of the acute open wound[8]; however, in the treatment of a diabetic wound, fast collagen production is needed to accelerate healing and, as such, might be more acceptable. Further studies are needed to investigate the effects of O-HA on collagen production in the diabetic wound-healing process. Conclusion In summary, O-HA increases endothelial cell proliferation, migration, and tube formation under high glucose conditions. Topical administration of ointment containing O-HA promotes wound healing in diabetic rats by increasing angiogenesis. The mechanisms are in part by increasing the phosphorylation of Src and ERK, and expression of TGF beta1. This would provide a new treatment method for diabetic wounds in clinical practice.
Funding This work was supported in part by a grant to JH from the Key Laboratory Program
of
Liaoning
Provincial
Department
Ministration(LZ2015051). Conflict of interest The authors declare no conflict of interest.
of
Education
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Figure legends Figure 1. O-HA promotes proliferation, migration, and tube formation of endothelial cells under high glucose conditions in vitro. A) Effect of O-HA on the proliferation of endothelial cells. Methylthiazolyldiphenyl-tetrazolium bromide assay shows that O-HA significantly increases the proliferation of endothelial cells after 72 h of cell culture under high glucose condition (p < 0.05). B) Effect of O-HA on migration of endothelial cells. Upper panel, phosphate buffered saline (PBS)–treated group, lower panel, O-HA-treated group. C) O-HA significantly improves migration of endothelial cells after 24 h of cell culture of endothelial cells (p < 0.01). D) Effect of O-HA on endothelial cell tube formation. Left panel, PBS-treated group; right panel, O-HA-treated group. E) O-HA significantly increases tube formation after 8 h of cell culture in Matrigel (p <0.01). Figure 2. Topical administration of ointment containing O-HA decreases wound size in diabetic rats. A) Upper panel, diabetic wound treated with ointment with O-HA; lower panel, diabetic wound treated with ointment without O-HA. B) O-HA significantly decreases wound size in diabetic rats compared with that in the controls (n = 8, p <0.01). C) Haematoxylin-eosin staining of wound sections at 14 days post-wounding. The double-headed arrows indicate the edges of the scar. O-HA increases granulation the tissue and re-epithelizlization of the wound (Bar = 100 μm). Figure 3. O-HA increases blood flow in the wounded area of diabetic rats. A) Laser Doppler imaging of blood flow in the wound area. Upper panel, wound treated with ointment containing O-HA; lower panel, wound treated with ointment without O-HA. B) O-HA significantly increases blood flow in the wound area of diabetic rats compared with that of the control (n = 8, p < 0.01). C) Immunostaining with CD31 of tissue from wound area. Left panel, tissue
from the wound treated with ointment without O-HA; right panel, tissue from the wound treated with ointment with O-HA (Bar = 100 μm). D) O-HA significantly increases capillary density in the wound area of diabetic rats compared with that of the controls (n = 8, p < 0.01). Figure 4. O-HA increases the phosphorylation of Srk and ERK, and expression of TGF beta1 in the wound area of diabetic rats. A) Phosphorylation of Srk in a diabetic wound. Lane 1, wound treated with ointment without O-HA; lane 2, wound treated with ointment with O-HA. B) Phosphorylation of ERK in diabetic wound. Lane 1, wound treated with ointment without O-HA; lane2, wound treated with ointment with O-HA. C) Expression of TGF beta1 in diabetic wound, Lane 1, wound treated with ointment without O-HA; lane2, wound treated with ointment with O-HA. Each experiment was repeated three times.