(19d) Recombinant Serine Protease Inhibitors (serpins) for Tissue Repair

Purpose/Objectives: Cutaneous wound repair is a major global economic and medical burden characterized by several continuous, overlapping phases. Delay in the onset or resolution of any phase leads to impaired healing, as occurs with complex comorbidities such as diabetes. Bioactive treatments to modulate these phases offer the potential to improve healing and to enhance the performance of biomaterial solutions for wound repair. Re-epithelialization and appropriate immune response in excisional and incisional wounds are critical early steps in healing. Re-epithelialization describes the process of epidermal keratinocytes undergoing a partial epithelial-to-mesenchymal transition (EMT) to mobilize and is a well-recognized characteristic of non-healing diabetic wounds and a goal for enhanced tissue regeneration. Sustained and inappropriate immune response to wound healing is also known to convert chronic wounds to acute wounds. There is growing interest in precisely defining the cellular and molecular mechanisms of repair and treatments to specifically augment re-epithelialization and immune response to wounds are under investigation. Serp-1 is a Myxoma virus-derived serine protease inhibitor (serpin) deployed as an immune evasion protein which demonstrates potent immune modulating activities when utilized as a recombinant protein therapeutic. Serpinb3a (paralog of human SerpinB3/B4) is a cross-class serpin canonically associated with cancer EMT, but which has not yet been investigated in wound healing. Here, we investigate the ability for Serp-1 and Serpinb3a to therapeutically augment healing in two different models of wound repair: granulation-mediated (second intention) excisional wound repair and light-activated sealant (LASE) biomaterial-mediated incisional wound closure.

Methodology: For Serpinb3a studies: NCBI Gene Expression Omnibus (GEO) was queried for relevant publicly available datasets and GEO2R was used to evaluate gene expression levels of Serpinb3a in acute and diabetic wounds. Western blot for Serpinb3a and immunohistochemistry for E-cadherin and Slug was performed on healing skin of immunocompetent Balb/c mice with 5-mm full-thickness splinted wounds at 6 hrs and 2, 4 and 7 days post-wounding. Full-length Serpinb3a with a C-terminal 6x-Histag was expressed in E. coli BL21(DE3) and purified using Ni-NTA IMAC and polished by Triton X-114 to endotoxin-free levels. Serpinb3a identity was confirmed by immunoblot and serpin inhibitory function was validated against purified human neutrophil Cathepsin G. In vitro closure was measured by HaCaT scratch assay and compared to EGF. Cell proliferation was measured by PrestoBlue assay. Therapeutic efficacy of Serpinb3a was evaluated in 5-mm full-thickness splinted wounds in C57BL6/J and diabetic and obese BKS.Cg-Dock7^m +/+ Lepr^db/J (db/db) mice. Wound closure rate was evaluated by planimetry. For Serp-1 studies: Light-activated sealants (LASE) were generated by solvent evaporation of aqueous solutions of Bombyx mori cocoon silk fibroin impregnated with indocyanine green dye (ICG). Serp-1 was recombinantly produced in CHO-K1 cells. Incisional wounds were treated with sutures or LASE activated by a handheld 808 nm laser and treated with saline or with recombinant Serp-1. Barrier function recovery was measured by trans-epidermal water loss (TEWL) with a VapoMeter.

Results: NCBI GEO analysis indicated evidence for skin-specific expression of Serpinb3a after dermal wounding, but not mucosal (tongue) wounding, and further suggested that Serpinb3a gene expression was lower in migrating keratinocytes at the wound edge of db/db but not C57BL6/J mice. Immunoblot analysis of acute wounds in Balb/c mice demonstrated high levels of Serpinb3a within 6 hours of wounding, peaking at 2-4 days and resolving by day 7 post-wounding. Importantly, proteins levels of Serpinb3a correlated tightly with immunohistochemical markers of EMT (E-cadherin loss and nuclear localization of Slug) in keratinocytes at the healing wound edge. Treatment of HaCaT cells with Serpinb3a dose-dependently reduced proliferation and significantly accelerated wound closure compared to controls and at levels comparable to EGF. Functional recombinant Serpinb3a was topically applied to the wound bed of healing acute and diabetic wounds. Significant acceleration of early closure, during the epidermal EMT phase of healing, was observed with exogenous Serpinb3a in both C57BL6/J and db/db mice. When co-delivered during laser tissue welding of incisional wounds with ICG-loaded LASE sealants, MYXV Serp-1 enhanced barrier function recovery nearing equivalence to intact tissue as early as 1-day post-incision.

Conclusion/Significance: Serpins represent a novel class of recombinant therapeutic modulators for wound healing and tissue repair. Serpinb3a is a novel endogenous mediator of wound healing in the skin and therapeutic administration accelerates poorly healing wounds. Serp-1 is a potent virus-derived immune modulator which enhances functional recovery of biomaterial-sealed skin injuries. Topical or controlled delivery of these recombinant serpins may provide new treatment approaches for tissue repair and regeneration.