Anti-inflammatory effects of haptoglobin on LPS-stimulated macrophages: Role of HMGB1 signaling and implications in chronic wound healing.

Nonhealing wounds possess elevated numbers of pro-inflammatory M1 macrophages, which fail to transition to anti-inflammatory M2 phenotypes that promote healing. Hemoglobin (Hb) and haptoglobin (Hp) proteins, when complexed (Hb-Hp), can elicit M2-like macrophages through the heme oxygenase-1 (HO-1) pathway. Despite the fact that nonhealing wounds are chronically inflamed, previous studies have focused on non-inflammatory systems, and do not thoroughly compare the effects of complexed vs individual proteins. We aimed to investigate the effect of Hb/Hp treatments on macrophage phenotype in an inflammatory, lipopolysaccharide (LPS)-stimulated environment, similar to chronic wounds. Human M1 macrophages were cultured in vitro and stimulated with LPS. Concurrently, Hp, Hb, or Hb-Hp complexes were delivered. The next day, 27 proteins related to inflammation were measured in the supernatants. 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, Ingenuity Pathway Analysis software identified high motility group box 1 (HMGB1) as a key canonical pathway-strongly down-regulated from Hp, strongly up-regulated from Hb, and slightly activated from Hb-Hp. HMGB1 measurements in macrophage supernatants confirmed this trend. In vivo results in diabetic mice with biopsy punch wounds demonstrated accelerated wound closure with Hp treatment, and delayed wound closure with Hb treatment. This work specifically studied Hb/Hp effects on macrophages in a highly inflammatory environment relevant to chronic wound healing. Results show that Hp-and not Hb-Hp, which is known to be superior in noninflammatory conditions-reduces inflammation in LPS-stimulated macrophages, and HMGB1 signaling is also implicated. Overall, Hp treatment on M1 macrophages in vitro reduced the inflammatory secretion profile, and also exhibited benefits in in silico and in vivo wound-healing models.

Macrophage modulation by polymerized hemoglobins: Potential as a wound-healing therapy.

Chronic skin wounds are hypoxic and are stalled in a pro-inflammatory state. Hemoglobin (Hb)-based oxygen carriers have shown potential in increasing oxygen delivery to aid wound healing. Macrophages also take up Hb, thus altering their phenotype and the regulation of inflammation. Herein, we compared the effect of Hb and polymerized Hbs (PolyHbs) on the phenotype of human macrophages. Macrophages were incubated with Hb or different forms of PolyHbs, and the inflammatory secretion profile was analyzed. PolyHbs were produced by polymerizing Hb in the relaxed (R) or tense (T) quaternary state and by varying the molar ratio of the glutaraldehyde crosslinking agent to Hb. Hb decreased the secretion of most measured factors. PolyHb treatment led to generally similar secretion profiles; however, Hb had more similar trends to R-state PolyHb. Ingenuity pathway analysis predicted positive outcomes in wound healing and angiogenesis for T-state PolyHb prepared with a 30:1 (glutaraldehyde:Hb) polymerization ratio. When tested in diabetic mouse wounds, T-state PolyHb resulted in the greatest epidermal thickness and vascular endothelial CD31 staining. Thus, the effects of PolyHb on macrophages are affected by the polymerization ratio and the quaternary state, and T-state PolyHb yields secretion profiles that are most beneficial in wound healing.

The Role of Macrophages in Acute and Chronic Wound Healing and Interventions to Promote Pro-wound Healing Phenotypes.

Macrophages play key roles in all phases of adult wound healing, which are inflammation, proliferation, and remodeling. As wounds heal, the local macrophage population transitions from predominantly pro-inflammatory (M1-like phenotypes) to anti-inflammatory (M2-like phenotypes). Non-healing chronic wounds, such as pressure, arterial, venous, and diabetic ulcers indefinitely remain in inflammation-the first stage of wound healing. Thus, local macrophages retain pro-inflammatory characteristics. This review discusses the physiology of monocytes and macrophages in acute wound healing and the different phenotypes described in the literature for both in vitro and in vivo models. We also discuss aberrations that occur in macrophage populations in chronic wounds, and attempts to restore macrophage function by therapeutic approaches. These include endogenous M1 attenuation, exogenous M2 supplementation and endogenous macrophage modulation/M2 promotion via mesenchymal stem cells, growth factors, biomaterials, heme oxygenase-1 (HO-1) expression, and oxygen therapy. We recognize the challenges and controversies that exist in this field, such as standardization of macrophage phenotype nomenclature, definition of their distinct roles and understanding which phenotype is optimal in order to promote healing in chronic wounds.

The growing role of precision and personalized medicine for cancer treatment.

Cancer is a devastating disease that takes the lives of hundreds of thousands of people every year. Due to disease heterogeneity, standard treatments, such as chemotherapy or radiation, are effective in only a subset of the patient population. Tumors can have different underlying genetic causes and may express different proteins in one patient versus another. This inherent variability of cancer lends itself to the growing field of precision and personalized medicine (PPM). There are many ongoing efforts to acquire PPM data in order to characterize molecular differences between tumors. Some PPM products are already available to link these differences to an effective drug. It is clear that PPM cancer treatments can result in immense patient benefits, and companies and regulatory agencies have begun to recognize this. However, broader changes to the healthcare and insurance systems must be addressed if PPM is to become part of standard cancer care.

PRO-RESOLUTION POTENCY OF RESOLVINS D1, D2 AND E1 ON NEUTROPHIL MIGRATION AND IN DERMAL WOUND HEALING.

An exuberant inflammatory response may exacerbate the primary tissue damage caused by injuries to the skin due to burns, surgery, excessive pressure, and other etiologies, thus increasing the time to heal. We hypothesized that application of factors that decrease inflammation would allow the skin to more quickly restore its barrier function, and promote the return to homeostasis. Resolvins are endogenous, pro-resolving lipid mediators derived from omega-3 fatty acids that serve to inhibit neutrophil migration and enhance macrophage phagocytosis, thus promoting the resolution of inflammation and the beginning of the proliferative phase of wound healing. Resolvins are derived either from docosahexaenoic (D-series) or eicosapentaenoic (E-series) acid. Herein, we compare the effects of resolvins D1 (RvD1), D2 (RvD2) and E1 (RvE1) on their abilities to inhibit neutrophil migration in vitro and to promote wound healing in vivo. In Transwell experiments, all resolvins inhibited neutrophil migration, with RvE1 being the most effective at a 2000nM concentration. In an in vivo murine excisional wound (1cm × 1cm) healing model, topically applied resolvins accelerated wound closure. RvE1-treated wounds healed by 19.4 ± 1.5 days post-wounding, which was significantly shorter than the RvD2-treated and RvD1-treated groups (p<0.05), which closed by an average of 22.8 ± 1.8 and 24.4 ± 2.2 days, respectively. Furthermore, all resolvin-treated groups healed faster than vehicle controls (p<0.05), which closed at 28.6 ± 1.5 days. There was a strong linear correlation (R2=0.9384) between each resolvin's potency in inhibiting neutrophil migration in vitro versus accelerating wound healing in vivo. Furthermore, upon histological analysis, the RvE1-treated group exhibited more mature collagen organization and reepithelialization.

Hypoxia Impairs Mesenchymal Stromal Cell-Induced Macrophage M1 to M2 Transition.

The transition of macrophages from the pro-inflammatory M1 to the anti-inflammatory M2 phenotype is crucial for the progression of normal wound healing. Persistent M1 macrophages within the injury site may lead to an uncontrolled macrophage-mediated inflammatory response and ultimately a failure of the wound healing cascade, leading to chronic wounds. Mesenchymal stromal cells (MSCs) have been widely reported to promote M1 to M2 macrophage transition; however, it is unclear whether MSCs can drive this transition in the hypoxic environment typically observed in chronic wounds. Here we report on the effect of hypoxia (1% O2) on MSCs' ability to transition macrophages from the M1 to the M2 phenotype. While hypoxia had no effect on MSC secretion, it inhibited MSC-induced M1 to M2 macrophage transition, and suppressed macrophage expression and production of the anti-inflammatory mediator interleukin-10 (IL-10). These results suggest that hypoxic environments may impede the therapeutic effects of MSCs.

Mechanosensing can result from adhesion molecule dynamics.

When plated onto a substrate, cells spread. Many cell types, including smooth muscle cells and fibroblasts, spread more effectively on stiffer substrates, and a simple empirical relationship has been determined that relates a cell's spread area to the substrate stiffness. In addition, some crawling cells when plated onto a stiff substrate will not migrate onto soft substrate, a process called "durotaxis". Here we show that the resistive force predicted previously for dynamic adhesion molecules can account for both of these mechanosensory effects.