Investigating the effect of hemoglobin-based complexes on macrophages for use in chronic wound healing

Chronic skin wounds are hypoxic and are stalled in a pro-inflammatory state. Hemoglobin(Hb)-based oxygen carriers have shown promise in increasing local oxygen delivery to aid in healing. Less considered in these previous studies is the ability of macrophages to take up extracellular Hb. There is evidence that macrophages that internalize Hb have an anti-inflammatory phenotype due to activation of the heme oxygenase 1 (HO-1) pathway, which may be beneficial to wound healing. Because free extracellular Hb is not stable, here we investigated two novel Hb-based oxygen carriers with respect to their ability to modulate macrophage function, and in turn promote wound healing. These include: 1) crosslinked polymerized hemoglobins (PolyHbs), and 2) Hb and haptoglobin (Hp) complexes. PolyHbs were manufactured by polymerizing Hb in its relaxed (R) or tense (T) quaternary state, and by varying the ratio of glutaraldehyde crosslinking agent to Hb. Alternatively, Hp, Hb, or Hb-Hp complexes were tested. In our studies, primary human macrophages were stimulated with lipopolysaccharide (LPS) to create a highly-inflammatory environment, and incubated with experimental treatments. The resulting secretion profiles consisting of 27 cytokines and growth factors related to inflammation were analyzed within a wound healing context. For the PolyHb studies, Hb decreased the secretion of most measured factors. PolyHb treatment resulted in generally similar secretion profiles to one-another, however Hb had more similar trends with R-state rather than T-state. Ingenuity Pathway Analysis (IPA) using secretion data predicted positive outcomes in wound healing and angiogenesis for T-state PolyHb made with a 30:1 glutaraldehyde:Hb polymerization ratio. When tested in vivo in diabetic mouse wounds, T-state PolyHb resulted in the most improved wound healing response, as evidenced by greatest epidermal thickness and vascular endothelial CD31 staining. For the Hb-Hp studies, unexpectedly, Hp treatment decreased a majority of inflammatory factors; Hb increased many; and Hb-Hp had intermediate trends; indicating that Hp attenuated overall inflammation to the greatest extent. From this data, IPA software identified High motility group box 1 (HMGB1) as a key canonical pathway— strongly downregulated from Hp, strongly upregulated from Hb, and slightly activated from Hb-Hp. HMGB1 measurements in macrophage supernatants confirmed this trend. Taken together, our studies further the characterization and investigation of Hb-based therapies on macrophages in highly-inflammatory, wound healing relevant applications. The effects of PolyHb on macrophages depended upon polymerization ratio and state, and T-state PolyHb yielded secretion profiles that were most beneficial in angiogenesis and wound healing. In addition, Hp —and not Hb-Hp, which is known to be superior in non-inflammatory conditions—reduced inflammation in LPS-stimulated macrophages, and HMGB1 signaling was also implicated. Future work includes thorough analysis of the HO-1 pathway over time, by varying LPS and treatment concentrations, and measuring resulting expression of HMGB1, HO-1, IL-10, and other inflammatory factors. Future studies will also consider the oxygen delivery capability of these Hb-based therapies, particularly in hypoxic conditions that are more representative of the chronic wound environment.Ph.D.Includes bibliographical reference