A Low Molecular Weight Hyaluronic Acid Derivative Accelerates Excisional Wound Healing by Modulating Pro-Inflammation, Promoting Epithelialization and Neovascularization, and Remodeling Collagen

Abstract

Recent knowledge of the cellular and molecular mechanisms underlying cutaneous wound healing has advanced the development of medical products. However, patients still suffer from the failure of current treatments, due to the complexity of healing process and thus novel therapeutic approaches are urgently needed. Previously, our laboratories produced a range of low molecular weight hyaluronic acid (LMW-HA) fragments, where a proportion of the glucosamine moieties were chemically N-acyl substituted. Specifically, N-butyrylation results in anti-inflammatory properties in a macrophage system, and we demonstrate the importance of N-acyl substituents in modulating the inflammatory response of LMW-HA. We have set up an inter-institutional collaborative program to examine the biomedical applications of the N-butyrylated LMW-HA (BHA). In this study, the potentials of BHA for dermal healing are assessed in vitro and in vivo. Consequently, BHA significantly promotes dermal healing relative to a commercial wound care product. By contrast, the "parent" partially de-acetylated LMW-HA (DHA) and the re-acetylated DHA (AHA) significantly delays wound closure, demonstrating the specificity of this N-acylation of LMW-HA in wound healing. Mechanistic studies reveal that the BHA-mediated therapeutic effect is achieved by targeting three phases of wound healing (i.e., inflammation, proliferation and maturation), demonstrating the significant potential of BHA for clinical translation in cutaneous wound healing.

Keywords: N-butyrylation; angiogenesis; anti-inflammation; hyaluronan; lymphangiogenesis.

Figure 1

Representative images of incisional wounds in rats after treatments. Healing rate (%) when compared with the wound area on Day 0 (n = 6). # p < 0.05 and ## p < 0.01 relative to the untreated control group; & p < 0.05 and && p < 0.01 relative to Blank-Gel; * p < 0.05 and ** p < 0.01 relative to carboxymethyl chitosan (CMC).

Figure 2

In representative hematoxylin-eosin (H&E) staining images (×100, bar in the lower right corner = 50 μm), green, blue, red and black arrows represent pro-inflammatory cells, epidermal hyperplasia, new blood vessel and intact epidermal structure, respectively. The pro-inflammatory cells, epidermal hyperplasia, and new blood vessel were quantified (n = 6). ## p < 0.01 relative to the untreated control group; && p < 0.01 relative to Blank-Gel; ** p < 0.01 relative to CMC.

Figure 3

The mRNA and protein levels of (a) tumor necrosis factor-α (TNF-α), (b) IL-6 and (c) interleukin-1β (IL-1β) in wounds are determined by reverse transcription polymerase chain reaction (RT-PCR) and enzyme-linked immunosorbent assay (ELISA), (n = 6). # p < 0.05 and ## p < 0.01 relative to the untreated control group; & p < 0.05 and && p < 0.01 relative to Blank-Gel; * p < 0.05 and ** p < 0.01 relative to CMC.

Figure 4

The protein level of (a) TGF-β activated kinase 1 (TAK-1) and phosphorylated TAK-1, (b) nuclear-p65, (c) p38 and phosphorylated p38 is determined using western blotting and quantified (n = 6). ## p < 0.01 relative to the untreated control group; && p < 0.01 relative to Blank-Gel; * p < 0.05 and ** p < 0.01 relative to CMC.

Figure 5

(a) The transforming growth factor beta 1 (TGF-β1) mRNA and protein levels in wounds are determined by RT-PCR and western blotting, respectively (n = 6). (b) The mRNA level of Smad2, Smad3 and Smad7 is determined by RT-PCR (n = 6). # p < 0.05 and ## p < 0.01 relative to untreated control group; & p < 0.05 and && p < 0.01 relative to Blank-Gel; * p < 0.05 and ** p < 0.01 relative to CMC.

Figure 6

In representative Masson’s trichrome staining images (×100, bar in the lower right corner = 50 μm) (red = keratin, muscle fibers or cytoplasm, blue = collagen), the collagen deposition was quantified (n = 6). ## p < 0.01 relative to the untreated control group; && p < 0.01 relative to Blank-Gel; ** p < 0.01 relative to CMC.

Figure 7

(a) The potential of N-butyrylated LMW-HA (BHA) to promote angiogenesis was assessed in Human Umbilical Vein Endothelial Cells (HUVEC), using an endothelial cell tube formation assay (×100, Bar in the lower right corner = 50 μm). (b) The potential of BHA to promote migration was assessed in HUVEC using the scratch assay (×40, Bar in the lower right corner = 200 μm; ×100, Bar in the lower right corner = 50 μm). (c) The vascular endothelial growth factor (VEGF), endothelial nitric oxide synthase (eNOS), E-selectin and Integrin-β3 mRNA levels were determined by RT-PCR (n = 6). # p < 0.05 and ## p < 0.01 relative to untreated control group; & p < 0.05 and && p < 0.01 relative to Blank-Gel; * p < 0.05 and ** p < 0.01 relative to CMC.

Figure 8

In representative immunohistochemical staining images (100×, bar in the lower right corner = 50 μm), the expression of CD31 was quantified (n = 6). For clarity, the expression of CD31 in the blood vessels and capillaries was not indicated by arrows in the images. ## p < 0.01 relative to untreated control group; && p < 0.01 relative to Blank-Gel; ** p < 0.01 relative to CMC.

Figure 9

In representative immunohistochemical staining images (100×, bar in the lower right corner = 50 μm), the expression of lymph vessel endothelial hyaluronan receptor-1 (LYVE-1) on lymph vessels (indicated by arrows) was quantified (n = 6). ## p < 0.01 relative to untreated control group; && p < 0.01 relative to Blank-Gel; ** p < 0.01 relative to CMC.

Figure 10

The mRNA (a) and protein (b) levels of collagen I, collagen III and β-actin were determined using RT-PCR and western blotting and quantified (n = 6). ## p < 0.01 relative to untreated control group; && p < 0.01 relative to Blank-Gel; * p < 0.05 and ** p < 0.01 relative to CMC.

Figure 11

In representative immunohistochemical staining images (×100, bar in the lower right corner = 50 μm), the expression of CD44 was quantified (n = 6). ## p < 0.01 relative to the untreated control group; && p < 0.01 relative to Blank-Gel; ** p < 0.01 relative to CMC.