The use of therapeutic gene eNOS delivered via a fibrin scaffold enhances wound healing in a compromised wound model
Introduction
Wound repair results from a complex and highly ordered cellular and biochemical response to tissue injury, including the synthesis of the bioregulatory molecule nitric oxide (NO) [1]. Normal wound healing is best organized into three phases of healing: the inflammatory phase, the proliferative phase, and the maturation phase [2]. Chronic wounds are the result of an interruption in the normal reparative process, in one or all phases of healing [3]. As a result, the healing process is prolonged and incomplete, with lack of restoration of integrity [3]. NO bioactivity is key to wound healing, and is dependant on synthesis by endothelial nitric oxide synthase (eNOS), constitutively expressed by endothelial cells [4]. eNOS is one of the three enzymes that facilitate the conversion of l-arginine to nitric oxide [5], [6]. NO is postulated to be critical to wound collagen accumulation and acquisition of mechanical strength [7]. NO promotes angiogenesis, endothelial and epithelial cell proliferation and migration [1].
Diabetic patients have a reduced capacity to carry out tissue repair processes, and are more susceptible to chronic wounds, such as leg ulcers and diabetic foot ulcers often leading to lower limb amputations [8], [9], [10]. Fifteen percent of patients with diabetes mellitus develop foot ulcers and 70% of diabetic foot ulcers reoccur within 5 years, placing a heavy load in terms of cost on health services [11]. Diabetes mellitus is characterised by chronic hyperglycaemia, which is known to increase vascular superoxide production (O2−) [4]. Superoxide reacts rapidly with nitric oxide (NO), reducing its bioactivity, and may also alter the activity of endothelial NO synthase (NOS) activity [4], [6]. Studies have shown NO deficiency at the diabetic wound site, and impaired wound healing secondary to diabetes mellitus is associated with markedly reduced eNOS protein expression [12], [13]. Mice lacking the eNOS gene exhibit delayed wound closure and decreased wound breaking strength as well as impaired capillary ingrowth in vitro [6]. A single topical administration of adenovirus encoding eNOS has been shown to be effective in reversing impaired healing in diabetic mice [13]. However, adenovirus has a short duration of transgene expression and induces an inflammatory response [14], which precludes high doses and re-administration [15]. To further increase the duration of transgene expression, a tissue engineered fibrin scaffold for the delivery of an adenoviral vector has been described by our group [16]. The fibrin scaffold composition has been optimised for sustained release of adenovirus in vitro and it is has been shown that adenovirus binds to the fibrinogen component of the scaffold [17]. In addition, an in vivo wound healing study has shown that the optimised fibrin scaffold enhances the delivery of adenovirus encoding a marker gene to a normal wound site for up to 7 days, compared to topical application of adenovirus alone [18].
This study hypothesises that fibrin scaffold will enhance the delivery of adenovirus encoding eNOS (AdeNOS) compared with direct administration of AdeNOS alone, resulting in improved epithelialisation and enhanced angiogenesis. To this end, an in vivo study was carried out using an alloxan rabbit ear ulcer model of healing. This impaired healing model was validated using methods of stereology, which have previously been described in evaluating tissue response [19].
Section snippets
Vectors
Adenovirus encoding eNOS was manufactured and purified as previously described [20]. The final stock titre was 3 × 1010 pfu/ml. Ten microlitres of titre 3 × 109 pfu/ml were used in the in vivo study as described below.
Fibrin scaffold fabrication
Fibrin kits (Baxter Healthcare, Vienna, Austria) and fibrinogen dilution buffer were kindly donated by Baxter Healthcare, Vienna. Fibrin scaffold containing adenovirus was fabricated as previously described [16]. Briefly, fibrinogen was reconstituted with aprotonin (3000 KIU), and
In vivo model
Animals became hyperglycaemic within 48 h, and remained in this state until the end of the study. Glucose levels ranged between 20 and 28 mmol/l, compared with normal of 3–10 mmol/l.
eNOS expression
Immunohistochemistry for eNOS showed positive staining for the protein in all groups (Fig. 2). The volume fraction of eNOS expressing cells was quantified (Fig. 3). It can be seen that there was no difference in eNOS expression between groups at 7 days. At 14 days, however, the fibrin containing AdeNOS groups showed a
Discussion
We have previously shown that fibrin scaffold enhances the delivery of an adenoviral vector to a wound site in vivo [18]. It was the aim of this study to determine whether AdeNOS delivered via a fibrin scaffold would result in enhanced wound healing in an impaired healing model. Several studies have probed the importance of eNOS and NO in wound healing [6], [12], and efforts have been made to deliver eNOS to a diabetic wound site using an adenoviral vector [13]. No study to date has examined
Conclusions
It is evident that fibrin scaffold provides an enhanced method of gene transfer of adenovirus encoding eNOS, compared with direct delivery of AdeNOS alone and fibrin scaffold alone, resulting in augmented nitric oxide production and improved wound healing, as seen in rate of epithelialisation, maturation of angiogenesis and inflammatory response. We propose that this is due to the ability of fibrin to retain the vector at the wound site, and present the adenovirus to infiltrating cells involved
Acknowledgements
Tisseel™ kits were kindly donated by Baxter Healthcare, Vienna. This research was funded by the Health Research Board Program Grant. The Faculty of Engineering, NUI Galway supported Ailish Breen with a scholarship. The authors would also like to express gratitude to Dr. Martina Harte, Charles McHale, Dr. Ariella Magee and Yolanda Garcia for their assistance in the in vivo studies.
References (35)
- et al.
Calcium-dependent nitric oxide synthesis in endothelial cytosol is mediated by calmodulin
FEBS Lett
(1990) - et al.
Nitric oxide and wound repair
Surg Clin North Am
(2003) - et al.
Diabetes-impaired healing and reduced wound nitric oxide synthesis: a possible pathophysiologic correlation
Surgery
(1997) - et al.
Determinants and estimation of healing times in diabetic foot ulcers
J Diabetes Complicat
(2002) - et al.
Apoptosis is increased in a model of diabetes-impaired wound healing in genetically diabetic mice
Int J Biochem Cell Biol
(1997) - et al.
Diabetes and peripheral vascular disease
J Vasc Surg
(1999) - et al.
Regulation of eNOS in normal and diabetes-impaired skin repair: implications for tissue regeneration
Nitric Oxide
(2002) - et al.
Gene therapy in wound healing
Surg Clin North Am
(2003) - et al.
Stereological methods to assess tissue response for tissue-engineered scaffolds
Biomaterials
(2007) - et al.
The quantification of vascular beds: a stereological approach
Exp Mol Pathol
(2007)
FGF2-targeted adenovirus encoding platelet-derived growth factor-B enhances de novo tissue formation
Mol Ther
Delivery of FGF genes to wound repair cells enhances arteriogenesis and myogenesis in skeletal muscle
Mol Ther
Reversal of impaired wound repair in iNOS-deficient mice by topical adenoviral-mediated iNOS gene transfer
J Clin Invest
Regulation of wound healing by growth factors and cytokines
Physiol Rev
Wound healing, chronic wounds
Plast Surg (Wound Healing)
Diabetes and vascular disease: pathophysiology, clinical consequences, and medical therapy: part I
Circulation
Complications of diabetes: screening for retinopathy, management of foot ulcers
Eff Health Care Bull
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2020, Principles of Tissue EngineeringTherapeutic strategies for enhancing angiogenesis in wound healing
2019, Advanced Drug Delivery ReviewsCitation Excerpt :Endothelial nitric oxide synthase (eNOS) can be delivered to increase NO production and enhance wound healing. Delivery of adenoviral eNOS (3 x 107 pfu) in a fibrin scaffold (60 mg/mL) led to a sustained production of eNOS for two weeks compared to delivery of the free virus in a diabetic rabbit ear excisional wound model [223,224]. Additionally, the fibrin scaffold yielded a reduction in the volume of inflammatory cells by approximately half from the 7-day time point to the 14-day time point [223].
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2019, Encyclopedia of Biomedical EngineeringWound healing using plasma modified collagen
2018, Clinical Plasma MedicineNanomedicines and gene therapy for the delivery of growth factors to improve perfusion and oxygenation in wound healing
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