A Virus-Derived Immune Modulating Serpin Accelerates Wound Closure with Improved Collagen Remodeling

Abstract

Numerous treatments have been developed to promote wound healing based on current understandings of the healing process. Hemorrhaging, clotting, and associated inflammation regulate early wound healing. We investigated treatment with a virus-derived immune modulating serine protease inhibitor (SERPIN), Serp-1, which inhibits thrombolytic proteases and inflammation, in a mouse excisional wound model. Saline or recombinant Serp-1 were applied directly to wounds as single doses of 1 μg or 2 µg or as two 1 µg boluses. A chitosan-collagen hydrogel was also tested for Serp-1 delivery. Wound size was measured daily for 15 days and scarring assessed by Masson’s trichrome, Herovici’s staining, and immune cell dynamics and angiogenesis by immunohistochemistry. Serp-1 treatment significantly accelerated wound healing, but was blocked by urokinase-type plasminogen activator (uPAR) antibody. Repeated dosing at a lower concentration was more effective than single high-dose serpin. A single application of Serp-1-loaded chitosan-collagen hydrogel was as effective as repeated aqueous Serp-1 dosing. Serp-1 treatment of wounds increased arginase-1-expressing M2-polarized macrophage counts and periwound angiogenesis in the wound bed. Collagen staining also demonstrated that Serp-1 improves collagen maturation and organization at the wound site. Serp-1 has potential as a safe and effective immune modulating treatment that targets thrombolytic proteases, accelerating healing and reducing scar in deep cutaneous wounds.

Local Serpin Treatment via Chitosan-Collagen Hydrogel after Spinal Cord Injury Reduces Tissue Damage and Improves Neurologic Function

“…In prior work neurotrophin-3 (NT3), a neurotrophic growth factor that promotes proliferation of neural stem cells, enhanced functional recovery after SCI in rats when delivered by chitosan-collagen carriers [21,41]. We recently reported that Serp-1 maintains function when released from chitosan-collagen in vivo in a full-thickness wound healing model [42]. Thus, we pursued this mode of delivery for SCI treatment in our model.…”

“…Serp-1 has previously been found to modulate infiltration of both T cells [24,61] and macrophages [3,22,42], dependent on the preclinical model being studied. Immunologically, we found in the current study that CD3-positive T cells, but not macrophages, were reduced more quickly in the area of injury after treatment with Serp-1.…”

“…Shortly before application, the lyophilized product was added to Type I collagen solution (C3867, Sigma Life Science, St. Louis, MO, USA) to a total volume of 500 µL to form a chitosan-collagen/Serp-1 gel at a Serp-1 concentration of 0, 10 µg (low dose), and 100 µg (high dose) per 50 µL gel, respectively. Previous characterization by our group demonstrated that this CCH material functions as a sustained release substrate, with in-tact Serp-1 continuing release after at least 4 days post-reconstitution [42]. Other groups have demonstrated similar chitosan-collagen hydrogel materials to release functional protein for 4 weeks [43].…”

“…The procedure for preparing the chitosan-collagen hydrogel (CCH) was performed as previously described [42]. Briefly, 15 mg of low molecular weight chitosan (448869l, 75%-85% deacetylated, Sigma Life Science, St. Louis, MO, USA) was swelled in 10 mL of deionized water and gently rotated overnight at 4 • C. The excess water was removed by centrifugation at 1000 ×g for 15 min.…”

“…We recently demonstrated that Serp-1 maintained therapeutic efficacy to improve healing in a full-thickness wound mouse model when delivered by a chitosan-collagen hydrogel (CCH) [40]. Chitosan, in a hydrogel combination with the structural stability of collagen, produces a highly biodegradable material with excellent biocompatibility, low antigenicity, and prior success in animal models of SCI [41,42]. Here, we studied whether Serp-1 delivered by chitosan-collagen hydrogel implant provides therapeutic efficacy in a forceps crush-induced spinal cord injury model.…”