Andrew Kimball

The Emerging Role of NETs in Venous Thrombosis and Immunothrombosis.

Venous thrombosis (VT), a leading cause of morbidity and mortality worldwide, has recently been linked to neutrophil activation and release of neutrophil extracellular traps (NETs) via a process called NETosis. The use of various in vivo thrombosis models and genetically modified mice has more precisely defined the exact role of NETosis in the pathogenesis of VT. Translational large animal VT models and human studies have confirmed the presence of NETs in pathologic VT. Activation of neutrophils, with subsequent NETosis, has also been linked to acute infection. This innate immune response, while effective for bacterial clearance from the host by formation of an intravascular bactericidal “net,” also triggers thrombosis. Intravascular thrombosis related to such innate immune mechanisms has been coined immunothrombosis. Dysregulated immunothrombosis has been proposed as a mechanism of pathologic micro- and macrovascular thrombosis in sepsis and autoimmune disease. In this focused review, we will address the dual role of NETs in the pathogenesis of VT and immunothrombosis.

Macrophage-Mediated Inflammation in Normal and Diabetic Wound Healing

The healing of cutaneous wounds is dependent on the progression through distinct, yet overlapping phases of wound healing, including hemostasis, inflammation, proliferation, and resolution/remodeling. The failure of these phases to occur in a timely, progressive fashion promotes pathologic wound healing. The macrophage (MΦ) has been demonstrated to play a critical role in the inflammatory phase of tissue repair, where its dynamic plasticity allows this cell to mediate both tissue-destructive and -reparative functions. The ability to understand and control both the initiation and the resolution of inflammation is critical for treating pathologic wound healing. There are now a host of studies demonstrating that metabolic and epigenetic regulation of gene transcription can influence MΦ plasticity in wounds. In this review, we highlight the molecular and epigenetic factors that influence MΦ polarization in both physiologic and pathologic wound healing, with particular attention to diabetic wounds.

Notch regulates macrophage-mediated inflammation in diabetic wound healing

Macrophages are essential immune cells necessary for regulated inflammation during wound healing. Recent studies have identified that Notch plays a role in macrophage-mediated inflammation. Thus, we investigated the role of Notch signaling on wound macrophage phenotype and function during normal and diabetic wound healing. We found that Notch receptor and ligand expression are dynamic in wound macrophages during normal healing. Mice with a myeloid-specific Notch signaling defect (DNMAMLfloxedLyz2Cre+) demonstrated delayed early healing (days 1–3) and wound macrophages had decreased inflammatory gene expression. In our physiologic murine model of type 2 diabetes (T2D), Notch receptor expression was significantly increased in wound macrophages on day 6, following the initial inflammatory phase of wound healing, corresponding to increased inflammatory cytokine expression. This increase in Notch1 and Notch2 was also observed in human monocytes from patients with T2D. Further, in prediabetic mice with a genetic Notch signaling defect (DNMAMLfloxedLyz2Cre+ on a high-fat diet), improved wound healing was seen at late time points (days 6–7). These findings suggest that Notch is critical for the early inflammatory phase of wound healing and directs production of macrophage-dependent inflammatory mediators. These results identify that canonical Notch signaling is important in directing macrophage function in wound repair and define a translational target for the treatment of non-healing diabetic wounds.

The Histone Methyltransferase, MLL1, Directs Macrophage-Mediated Inflammation in Wound Healing and is Altered in a Murine Model of Obesity and Type 2 Diabetes

Macrophages are critical for the initiation and resolution of the inflammatory phase of wound repair. In diabetes, macrophages display a prolonged inflammatory phenotype in late wound healing. Mixed-lineage leukemia-1 (MLL1) has been shown to direct gene expression by regulating nuclear factor-κB (NF-κB)–mediated inflammatory gene transcription. Thus, we hypothesized that MLL1 influences macrophage-mediated inflammation in wound repair. We used a myeloid-specific Mll1 knockout (Mll1f/fLyz2Cre+) to determine the function of MLL1 in wound healing. Mll1f/fLyz2Cre+ mice display delayed wound healing and decreased wound macrophage inflammatory cytokine production compared with control animals. Furthermore, wound macrophages from Mll1f/fLyz2Cre+ mice demonstrated decreased histone H3 lysine 4 trimethylation (H3K4me3) (activation mark) at NF-κB binding sites on inflammatory gene promoters. Of note, early wound macrophages from prediabetic mice displayed similarly decreased MLL1, H3K4me3 at inflammatory gene promoters, and inflammatory cytokines compared with controls. Late wound macrophages from prediabetic mice demonstrated an increase in MLL1, H3K4me3 at inflammatory gene promoters, and inflammatory cytokines. Prediabetic macrophages treated with an MLL1 inhibitor demonstrated reduced inflammation. Finally, monocytes from patients with type 2 diabetes had increased Mll1 compared with control subjects without diabetes. These results define an important role for MLL1 in regulating macrophage-mediated inflammation in wound repair and identify a potential target for the treatment of chronic inflammation in diabetic wounds.

Dysfunctional Wound Healing in Diabetic Foot Ulcers: New Crossroads

Purpose of ReviewDiabetic foot ulcerations (DFU) affect 25% of patients with diabetes mellitus during their lifetime and constitute a major health problem as they are often recalcitrant to healing due to a constellation of both intrinsic and extrinsic factors. The purpose of this review is to (1) detail the current mechanistic understanding of DFU formation and (2) highlight future therapeutic targets.Recent FindingsFrom a molecular perspective, DFUs exhibit a chronic inflammatory predisposition. In addition, increased local hypoxic conditions and impaired cellular responses to hypoxia are pathogenic factors that contribute to delayed wound healing. Finally, recent evidence suggests a role for epigenetic alterations, including microRNAs, in delayed DFU healing due to the complex interplay between genes and the environment.SummaryIn this regard, notable progress has been made in the molecular and genetic understanding of DFU formation. However, further studies are needed to translate preclinical investigations into clinical therapies.

Ly6CHi Blood Monocyte/Macrophage Drive Chronic Inflammation and Impair Wound Healing in Diabetes Mellitus

Objective— Wound monocyte-derived macrophage plasticity controls the initiation and resolution of inflammation that is critical for proper healing, however, in diabetes mellitus, the resolution of inflammation fails to occur. In diabetic wounds, the kinetics of blood monocyte recruitment and the mechanisms that control in vivo monocyte/macrophage differentiation remain unknown. Approach and Results— Here, we characterized the kinetics and function of Ly6CHi [Lin− (CD3−CD19−NK1.1−Ter-119−) Ly6G−CD11b+] and Ly6CLo [Lin− (CD3−CD19−NK1.1−Ter-119−) Ly6G−CD11b+] monocyte/macrophage subsets in normal and diabetic wounds. Using flow-sorted tdTomato-labeled Ly6CHi monocyte/macrophages, we show Ly6CHi cells transition to a Ly6CLo phenotype in normal wounds, whereas in diabetic wounds, there is a late, second influx of Ly6CHi cells that fail transition to Ly6CLo. The second wave of Ly6CHi cells in diabetic wounds corresponded to a spike in MCP-1 (monocyte chemoattractant protein-1) and selective administration of anti-MCP-1 reversed the second Ly6CHi influx and improved wound healing. To examine the in vivo phenotype of wound monocyte/macrophages, RNA-seq–based transcriptome profiling was performed on flow-sorted Ly6CHi [Lin−Ly6G−CD11b+] and Ly6CLo [Lin−Ly6G−CD11b+] cells from normal and diabetic wounds. Gene transcriptome profiling of diabetic wound Ly6CHi cells demonstrated differences in proinflammatory and profibrotic genes compared with controls. Conclusions— Collectively, these data identify kinetic and functional differences in diabetic wound monocyte/macrophages and demonstrate that selective targeting of CD11b+Ly6CHi monocyte/macrophages is a viable therapeutic strategy for inflammation in diabetic wounds.

Murine macrophage chemokine receptor CCR2 plays a crucial role in macrophage recruitment and regulated inflammation in wound healing

Macrophages play a critical role in the establishment of a regulated inflammatory response following tissue injury. Following injury, CCR2+ monocytes are recruited from peripheral blood to wound tissue, and direct the initiation and resolution of inflammation that is essential for tissue repair. In pathologic states where chronic inflammation prevents healing, macrophages fail to transition to a reparative phenotype. Using a murine model of cutaneous wound healing, we found that CCR2‐deficient mice (CCR2−/−) demonstrate significantly impaired wound healing at all time points postinjury. Flow cytometry analysis of wounds from CCR2−/− and WT mice revealed a significant decrease in inflammatory, Ly6CHi recruited monocyte/macrophages in CCR2−/− wounds. We further show that wound macrophage inflammatory cytokine production is decreased in CCR2−/− wounds. Adoptive transfer of mT/mG monocyte/macrophages into CCR2+/+ and CCR2−/− mice demonstrated that labeled cells on days 2 and 4 traveled to wounds in both CCR2+/+ and CCR2−/− mice. Further, adoptive transfer of monocyte/macrophages from WT mice restored normal healing, likely through a restored inflammatory response in the CCR2‐deficient mice. Taken together, these data suggest that CCR2 plays a critical role in the recruitment and inflammatory response following injury, and that wound repair may be therapeutically manipulated through modulation of CCR2.

PC200 A Deacetylase Enzyme, Sirtuin 3, Plays a Major Role in Macrophage Inflammation and Wound Healing

Objectives: Diabetic patients suffer an increased risk of restenosis and late stent thrombosis after angioplasty, complications that are related to a defective re-endothelialization. Dipeptidyl peptidase-4 inhibitors have been suggested to exert direct effect on endothelial and smooth muscle cells (SMCs). Therefore, the objective was to study whether the dipeptidyl peptidase-4 inhibitor linagliptin could influence vascular repair and accelerate re-endothelialization after arterial injury in healthy and diabetic animals. Methods: Diabetic Goto-Kakizaki and healthy Wistar rats were subjected to arterial injury and treated with linagliptin or vehicle. Vessel wall healing was monitored noninvasively using ultrasound imaging and upon sacrifice with Evans blue staining and immunohistochemistry. The effect of linagliptin on SMCs was also studied in vitro. Results: We observed a delay in the healing response in the diabetic animals compared with the Wistar controls. Goto-Kakizaki rats had more pronounced intimal hyperplasia, which affected the lumen diameter. We found that linagliptin reduced the proliferation and dedifferentiation of SMCs in vitro and modulated the inflammatory response in the SMCs after arterial injury in vivo. However, these effects of linagliptin did not affect the neointima formation or the re-endothelialization under normal and diabetic conditions. Conclusions: Although linagliptin did not influence vessel wall healing, it appears to possess a desirable antiproliferative influence on SMCs in vitro and an anti-inflammatory effect in vivo. These pharmacologic properties might carry a potential significance for favorable outcome after vascular interventions in diabetic patients.