COMP-angiopoietin 1 Gene Transfer Enhances Cutaneous Wound Healing by Promoting Angiogenesis

The closure of cutaneous wounds involves events such as hemorrhage, inflammation, re-epithelialization, granulation tissue formation, and the late remodeling phase of tissue repair. The early and acute phases of repair involve macrophage accumulation, fibroblast ingrowth, matrix deposition, and angiogenesis. These events are triggered by a complex mixture of cytokines and growth factors that are released at the site of injury1.

Angiogenesis is the sprouting of new blood vessels from a pre-existing network, and this process is central to the formation of granulation tissue because the ingrowth of newly formed vessels is needed to ensure the supply of oxygen, nutrients and inflammatory cells to the regenerating tissue1, 2. Previous studies have shown that delayed wound healing such as diabetic ulcers, chronic venous ulcers, pressure ulcers, and cutaneous wounds in the aged, are associated with decreased angiogenesis and altered levels of angiogenic growth factors including vascular endothelial growth factor (VEGF) and angiopoietin (Ang)3, 4, 5. Based on these results, "therapeutic angiogenesis", i.e. supplementation of such recombinant angiogenic growth factors as VEGF, erythropoietin, nerve growth factor, platelet-derived growth factor, fibroblast growth factor2 and granulocyte-machrophage colony stimulating factor, has been used both in animal models and in clinical trials for patients to enhance cutaneous wound healing6, 7, 8.

Ang was discovered as a ligands for the tyrosine kinase with immunoglobulin and epidermal growth factor homology (Tie) 2 that was selectively expressed on the vascular endothelium. There are four definitive members of the Ang family, Ang 1, 2, 3 and 4. Although the functions of Ang and Tie have not been well established, Ang is thought to be involved in the proliferation, maturation, stabilization and remodeling of vessels1, 9. Therefore, Ang 1 could be one of the promising candidate growth factors for therapeutic angiogenesis.

Large-scale production of recombinant Ang 1 is hindered by the aggregation and insolubility of protein. The activity of the purified protein frequently varies. These difficulties are due to its unique structural characteristics. COMP-Ang 1 is a soluble, stable, and potent Ang 1 variant in which the N-terminal portion of Ang 1 is replaced with the short coiled-coil domain of cartilage oligometric matrix protein (COMP). COMP-Ang 1 is more potent than native Ang 1 in phosphorylating Tie 2 and Akt in primary cultured endothelial cells and in angiogenesis in vivo10, 11.

In this study, we investigated the potential benefits of the therapy with adeno-associated systemic COMP-Ang 1 on cutaneous wound healing. The results of the study show that systemic COMP-Ang 1 treatment enhances cutaneous wound healing in vivo by promoting angiogenesis.

The angiogenic effect of Ang 1 has been reported in previous studies. Ang 1 and Tie 2-deficient mice had similar phenotypes that were characterized by embryonic lethality with severe vascular remodeling defects, insufficient vessel stabilization and perturbed vascular maturation12, 13. Promoted angiogenesis has been reported on for transgenic mice that overexpress Ang 1 in the skin14. In addition, there have been reports that exogenous and localized Ang 1 treatment with naked DNA-mediated or adenovirus-mediated Ang 1 enhanced angiogenesis in the gastric ulcer model or the skin flap model15, 16. In this study, we wanted to verify whether a systemic adenovirus-associated COMP-Ang 1 treatment can induce angiogenesis in a cutaneous wound healing model. Our results demonstrated that angiogenesis in the COMP-Ang 1 treated group was significantly increased at day 3, 7 and 14, compared with the control group. Although we did not confirm the expression of COMP-Ang 1 either in the plasma or in the cutaneous wound after systemic injection, the level of systemic circulating COMP-Ang 1 is known to be increased as early as 12 hours after Ade-COMP-Ang 1 injection, peaking at 1 to 2 weeks and gradually declining thereafter. The levels return to the control levels at 6 weeks after systemic treatment in the murine model10. Therefore, we could infer that the enhanced angiogenesis in the COMP-Ang 1 treated group is attributable to the increased level of COMP-Ang 1 in the plasma.

The results of this study showed that adenovirus mediated COMP-Ang 1 therapy significantly enhanced the reduction of wound size at day 3 and 7 after wound surgery; however, there was no significant difference at day 14. These data coincided with previous reports showing the difference of the reduction of the wound size did not reach statistical significance after day 7 or 9 after wounding, in an adenovirus-mediated VEGF treatment model or a cultured autologous fibroblast transplant model, respectively6, 17.

A recent explosion of newly discovered growth factors that act on the vascular endothelium has coincided with the application of powerful new genetic approaches to the problem of vascular development. The vascular endothelium-specific growth factors include five members of the VEGF family, four members of the Ang family, and at least one member of the large ephrin family1. In addition, many other growth factors that are not vascular endothelium-specific are also required for blood vessel formation, and these include the members of the platelet-derived growth factor families or the transforming growth factor families. However, the critical roles on many other systems of the latter limit their clinical application. VEGF has been the most commonly studied and used growth factor for promoting wound healing. The previous studies have shown that VEGF is a more potent growth factor for endothelial proliferation than is Ang 115. However, continual overexpression of VEGF has been found to result in hemangioma-type tumors18, indicating that the VEGF expression or effective dose must be tightly regulated and that the expression must transiently occur only at the early stage of wound healing. Furthermore, the microvessels that develop from the continual overexpression of VEGF were found to be disorganized, sinusoidal and leaky because of VEFG's additional function as a vascular permeability factor19. In contrast, Ang 1 induces maturation, stabilization and remodeling of vessels and overexpression of Ang 1 does not result in vascular leakage20, 21, 22. Therefore, Ang 1 is thought to have a wider therapeutic window and it could be a safer modality for clinical use.

As far as treatment with growth factors is concerned, naive protein treatment in the clinical setting is limited by several factors, such as their short half-lives, their inactivation by wound proteases, their poor bioavailability from the utilized vehicles and consequently the need for daily applications and high initial doses that might become toxic23. An alternative approach that might overcome most of these problems is the delivery of growth factor-encoding genes. Gene therapy could overcome the short-comings of direct application of the growth factor and so promote continual production and release of the growth factor within the blood.

In conclusion, the results of this study suggest that Ade-COMP-Ang 1 gene therapy significantly accelerates acute cutaneous wound healing by promoting angiogenesis at the site of injury. Further study using Ade-COMP-Ang 1 gene therapy in chronic wounds will be needed in the near future.