Skewing cPLA2α activity toward oxoeicosanoid production promotes neutrophil N2 polarization, wound healing, and the response to sepsis.

Uncontrolled inflammation is linked to poor outcomes in sepsis and wound healing, both of which proceed through distinct inflammatory and resolution phases. Eicosanoids are a class of bioactive lipids that recruit neutrophils and other innate immune cells. The interaction of ceramide 1-phosphate (C1P) with the eicosanoid biosynthetic enzyme cytosolic phospholipase A2 (cPLA2) reduces the production of a subtype of eicosanoids called oxoeicosanoids. We investigated the effect of shifting the balance in eicosanoid biosynthesis on neutrophil polarization and function. Knockin mice expressing a cPLA2 mutant lacking the C1P binding site (cPLA2αKI/KI mice) showed enhanced and sustained neutrophil infiltration into wounds and the peritoneum during the inflammatory phase of wound healing and sepsis, respectively. The mice exhibited improved wound healing and reduced susceptibility to sepsis, which was associated with an increase in anti-inflammatory N2-type neutrophils demonstrating proresolution behaviors and a decrease in proinflammatory N1-type neutrophils. The N2 polarization of cPLA2αKI/KI neutrophils resulted from increased oxoeicosanoid biosynthesis and autocrine signaling through the oxoeicosanoid receptor OXER1 and partially depended on OXER1-dependent inhibition of the pentose phosphate pathway (PPP). Thus, C1P binding to cPLA2α suppresses neutrophil N2 polarization, thereby impairing wound healing and the response to sepsis.

Ceramide kinase regulates acute wound healing by suppressing 5-oxo-ETE biosynthesis and signaling via its receptor OXER1.

The sphingolipid, ceramide-1-phosphate (C1P), has been shown to promote the inflammatory phase and inhibit the proliferation and remodeling stages of wound repair via direct interaction with group IVA cytosolic phospholipase A2, a regulator of eicosanoid biosynthesis that fine-tunes the behaviors of various cell types during wound healing. However, the anabolic enzyme responsible for the production of C1P that suppresses wound healing as well as bioactive eicosanoids and target receptors that drive enhanced wound remodeling have not been characterized. Herein, we determined that decreasing C1P activity via inhibitors or genetic ablation of the anabolic enzyme ceramide kinase (CERK) significantly enhanced wound healing phenotypes. Importantly, postwounding inhibition of CERK enhanced the closure rate of acute wounds, improved the quality of healing, and increased fibroblast migration via a "class switch" in the eicosanoid profile. This switch reduced pro-inflammatory prostaglandins (e.g., prostaglandin E2) and increased levels of 5-hydroxyeicosatetraenoic acid and the downstream metabolite 5-oxo-eicosatetraenoic acid (5-oxo-ETE). Moreover, dermal fibroblasts from mice with genetically ablated CERK showed enhanced wound healing markers, while blockage of the murine 5-oxo-ETE receptor (oxoeicosanoid receptor 1) inhibited the enhanced migration phenotype of these cell models. Together, these studies reinforce the vital roles eicosanoids play in the wound healing process and demonstrate a novel role for CERK-derived C1P as a negative regulator of 5-oxo-ETE biosynthesis and the activation of oxoeicosanoid receptor 1 in wound healing. These findings provide foundational preclinical results for the use of CERK inhibitors to shift the balance from inflammation to resolution and increase the wound healing rate.

What Makes Wounds Chronic.

Chronic wounds present a unique therapeutic challenge to heal. Chronic wounds are colonized with bacteria and the presence of a biofilm that further inhibits the normal wound healing processes, and are locked into a very damaging proinflammatory response. The treatment of chronic wounds requires a coordinated approach, including debridement of devitalized tissue, minimizing bacteria and biofilm, control of inflammation, and the use of specialized dressings to address the specific aspects of the particular nonhealing ulcer.

The interaction of ceramide 1-phosphate with group IVA cytosolic phospholipase A2 coordinates acute wound healing and repair.

The sphingolipid ceramide 1-phosphate (C1P) directly binds to and activates group IVA cytosolic phospholipase A2 (cPLA2α) to stimulate the production of eicosanoids. Because eicosanoids are important in wound healing, we examined the repair of skin wounds in knockout (KO) mice lacking cPLA2α and in knock-in (KI) mice in which endogenous cPLA2α was replaced with a mutant form having an ablated C1P interaction site. Wound closure rate was not affected in the KO or KI mice, but wound maturation was enhanced in the KI mice compared to that in wild-type controls. Wounds in KI mice displayed increased infiltration of dermal fibroblasts into the wound environment, increased wound tensile strength, and a higher ratio of type I:type III collagen. In vitro, primary dermal fibroblasts (pDFs) from KI mice showed substantially increased collagen deposition and migration velocity compared to pDFs from wild-type and KO mice. KI mice also showed an altered eicosanoid profile of reduced proinflammatory prostaglandins (PGE2 and TXB2) and an increased abundance of certain hydroxyeicosatetraenoic acid (HETE) species. Specifically, an increase in 5-HETE enhanced dermal fibroblast migration and collagen deposition. This gain-of-function role for the mutant cPLA2α was also linked to the relocalization of cPLA2α and 5-HETE biosynthetic enzymes to the cytoplasm and cytoplasmic vesicles. These findings demonstrate the regulation of key wound-healing mechanisms in vivo by a defined protein-lipid interaction and provide insights into the roles that cPLA2α and eicosanoids play in orchestrating wound repair.

Network proteomics of human dermal wound healing.

OBJECTIVE: The healing of wounds is critical in protecting the human body against environmental factors. The mechanisms involving protein expression during this complex physiological process have not been fully elucidated. APPROACH: Here, we use reverse-phase protein microarrays (RPPA) involving 94 phosphoproteins to study tissue samples from tubes implanted in healing dermal wounds in seven human subjects tracked over two weeks. We compare the proteomic profiles to proteomes of controls obtained from skin biopsies from the same subjects. MAIN RESULTS: Compared to previous proteomic studies of wound healing, our approach focuses on wound tissue instead of wound fluid, and has the sensitivity to go beyond measuring only highly abundant proteins. To study the temporal dynamics of networks involved in wound healing, we applied two network analysis methods that integrate the experimental results with prior knowledge about protein-protein physical and regulatory interactions, as well as higher-level biological processes and associated pathways. SIGNIFICANCE: We uncovered densely connected networks of proteins that are up- or down-regulated during human wound healing, as well as their relationships to microRNAs and to proteins outside of our set of targets that we measured with proteomic microarrays.

Human as the Ultimate Wound Healing Model: Strategies for Studies Investigating the Dermal Lipidome.

PURPOSE OF REVIEW: Educate the reader of the multiple roles undertaken by the human epidermal lipidome and the experimental techniques of measuring them. RECENT FINDINGS: Damage to skin elicits a wound healing process that is capped by the recreation of the lipid barrier. In addition to barrier function, lipids also undertake an active signaling role during wound healing. Achievement of these multiple functions necessitates a significant complexity and diversity in the lipidome resulting in a composition that is unique to the human skin. As such, any attempts to delineate the function of the lipidome during the wound healing process in humans need to be addressed via studies undertaken in humans. SUMMARY: The human cutaneous lipidome is unique and play a functionally significant role in maintaining barrier and regulating wound healing. Modern mass spectrometry and Raman spectroscopy based methods enable the investigation epidermal lipidome with respect to those functions.

Modeling the effects of systemic mediators on the inflammatory phase of wound healing.

The normal wound healing response is characterized by a progression from clot formation, to an inflammatory phase, to a repair phase, and finally, to remodeling. In many chronic wounds there is an extended inflammatory phase that stops this progression. In order to understand the inflammatory phase in more detail, we developed an ordinary differential equation model that accounts for two systemic mediators that are known to modulate this phase, estrogen (a protective hormone during wound healing) and cortisol (a hormone elevated after trauma that slows healing). This model describes the interactions in the wound between wound debris, pathogens, neutrophils and macrophages and the modulation of these interactions by estrogen and cortisol. A collection of parameter sets, which qualitatively match published data on the dynamics of wound healing, was chosen using Latin Hypercube Sampling. This collection of parameter sets represents normal healing in the population as a whole better than one single parameter set. Including the effects of estrogen and cortisol is a necessary step to creating a patient specific model that accounts for gender and trauma. Utilization of math modeling techniques to better understand the wound healing inflammatory phase could lead to new therapeutic strategies for the treatment of chronic wounds. This inflammatory phase model will later become the inflammatory subsystem of our full wound healing model, which includes fibroblast activity, collagen accumulation and remodeling.

Ceramide kinase is required for a normal eicosanoid response and the subsequent orderly migration of fibroblasts.

In these studies, the role of ceramide-1-phosphate (C1P) in the wound-healing process was investigated. Specifically, fibroblasts isolated from mice with the known anabolic enzyme for C1P, ceramide kinase (CERK), ablated (CERK(-/-) mice) and their wild-type littermates (CERK(+/+)) were subjected to in vitro wound-healing assays. Simulation of mechanical trauma of a wound by scratching a monolayer of fibroblasts from CERK(+/+) mice demonstrated steadily increasing levels of arachidonic acid in a time-dependent manner in stark contrast to CERK(-/-) fibroblasts. This observed difference was reflected in scratch-induced eicosanoid levels. Similar, but somewhat less intense, changes were observed in a more complex system utilizing skin biopsies obtained from CERK-null mice. Importantly, C1P levels increased during the early stages of human wound healing correlating with the transition from the inflammatory stage to the peak of the fibroplasia stage (e.g., proliferation and migration of fibroblasts). Finally, the loss of proper eicosanoid response translated into an abnormal migration pattern for the fibroblasts isolated from CERK(-/-) As the proper migration of fibroblasts is one of the necessary steps of wound healing, these studies demonstrate a novel requirement for the CERK-derived C1P in the proper healing response of wounds.

A Network Approach to Wound Healing.

OBJECTIVE: The wound healing process is well-understood on the cellular and tissue level; however, its complex molecular mechanisms are not yet uncovered in their entirety. Viewing wounds as perturbed molecular networks provides the tools for analyzing and optimizing the healing process. It helps to answer specific questions that lead to better understanding of the complexity of the process. What are the molecular pathways involved in wound healing? How do these pathways interact with each other during the different stages of wound healing? Is it possible to grasp the entire mechanism of regulatory interactions in the healing of a wound? APPROACH: Networks are structures composed of nodes connected by links. A network describing the state of a cell taking part in the healing process may contain nodes representing genes, proteins, microRNAs, metabolites, and drug molecules. The links connecting nodes represent interactions such as binding, regulation, co-expression, chemical reaction, and others. Both nodes and links can be weighted by numbers related to molecular concentration and the intensity of intermolecular interactions. Proceeding from data and from molecular profiling experiments, different types of networks are built to characterize the stages of the healing process. Network nodes having a higher degree of connectivity and centrality usually play more important roles for the functioning of the system they describe. RESULTS: We describe here the algorithms and software packages for building, manipulating and analyzing networks proceeding from information available from a literature or database search or directly extracted from experimental gene expression, metabolic, and proteomic data. Network analysis identifies genes/proteins most differentiated during the healing process, and their organization in functional pathways or modules, and their distribution into gene ontology categories of biological processes, molecular functions, and cellular localization. We provide an example of how network analysis can be used to reach better understanding of regulation of key wound healing mediators and microRNAs that regulate them. INNOVATION: Univariate statistical tests widely used in clinical studies are not enough to improve understanding and optimize the processes of wound healing. Network methods of analysis of patients "omics" data, such as transcriptoms, proteomes, and others can provide a better insight into the healing processes and help in development of better treatment practices. We review several articles that are examples of this emergent approach to the study of wound healing. CONCLUSION: Network analysis has the potential to considerably contribute to the better understanding of the molecular mechanisms of wound healing and to the discovery of means to control and optimize that process.

Structural Stereochemistry of Androstene Hormones Determines Interactions with Human Androgen, Estrogen, and Glucocorticoid Receptors.

DHEA, 17α-AED, 17ß-AED, and 17ß-AET exhibit strong biological activity that has been attributed to androgenic, estrogenic, or antiglucocorticoid activity in vivo and in vitro. This study compared DHEA, 17α-AED, 17ß-AED, and 17ß-AET for their ability to activate the human AR, ER, and GR and determine the relative androgenicity, estrogenicity, and glucocorticoid activity. The results show that, at the receptor level, these androstene hormones are weak AR and even weaker ER activators. Direct androstene hormone activation of the human AR, ERα, and ERß may not be essential for their biological function. Similarly, these hormones indirectly activated the human GR, only in the presence of high dexamethasone concentrations. These results underscore the major difference between androstene hormone interactions with these nuclear receptors and their biological effects.

A differential equation model of collagen accumulation in a healing wound.

Wound healing is a complex biological process which involves many cell types and biochemical signals and which progresses through multiple, overlapping phases. In this manuscript, we develop a model of collagen accumulation as a marker of wound healing. The mathematical model is a system of ordinary differential equations which tracks fibroblasts, collagen, inflammation and pathogens. The model was validated by comparison to the normal time course of wound healing where appropriate activity for the inflammatory, proliferative and remodeling phases was recorded. Further validation was made by comparison to collagen accumulation experiments by Madden and Peacock (Ann. Surg. 174(3):511-520, 1971). The model was then used to investigate the impact of local oxygen levels on wound healing. Finally, we present a comparison of two wound healing therapies, antibiotics and increased fibroblast proliferation. This model is a step in developing a comprehensive model of wound healing which can be used to develop and test new therapeutic treatments.

Systemic central venous oxygen saturation is associated with clot strength during traumatic hemorrhagic shock: A preclinical observational model.

BACKGROUND: Clot strength by Thrombelastography (TEG) is associated with mortality during trauma and has been linked to severity of tissue hypoperfusion. However, the optimal method for monitoring this important relationship remains undefined. We hypothesize that oxygen transport measurements will be associated with clot strength during traumatic shock, and test this hypothesis using a swine model of controlled traumatic shock. METHODS: N = 33 swine were subjected to femur fracture and hemorrhagic shock by controlled arterial bleeding to a predetermined level of oxygen debt measured by continuous indirect calorimetry. Hemodynamics, oxygen consumption, systemic central venous oxygenation (ScvO2), base excess, lactate, and clot maximal amplitude by TEG (TEG-MA) as clot strength were measured at baseline and again when oxygen debt = 80 ml/kg during shock. Oxygen transport and metabolic markers of tissue perfusion were then evaluated for significant associations with TEG-MA. Forward stepwise selection was then used to create regression models identifying the strongest associations between oxygen transport and TEG-MA independent of other known determinants of clot strength. RESULTS: Multiple markers of tissue perfusion, oxygen transport, and TEG-MA were all significantly altered during shock compared to baseline measurements (p < 0.05). However, only ScvO2 demonstrated a strong bivariate association with TEG-MA measured during shock (R = 0.7, p < 0.001). ScvO2 measured during shock was also selected by forward stepwise selection as an important covariate in linear regression models of TEG-MA after adjusting for the covariates fibrinogen, pH, platelet count, and hematocrit (Whole model R² = 0.99, p ≤ 0.032). CONCLUSIONS: Among multiple measurements of oxygen transport, only ScvO2 was found to retain a significant association with TEG-MA during shock after adjusting for multiple covariates. ScvO2 should be further studied for its utility as a clinical marker of both tissue hypoxia and clot formation during traumatic shock.

Wound healing primer.

Surgeons often care for patients with conditions of abnormal wound healing, which include conditions of excessive wound healing, such as fibrosis, adhesions, and contractures, as well as conditions of inadequate wound healing, such as chronic nonhealing ulcers, recurrent hernias, and wound dehiscences. Despite many recent advances in the field, which have highlighted the importance of adjunct therapies in maximizing the healing potential, conditions of abnormal wound healing continue to cause significant cost, morbidity, and mortality. To understand how conditions of abnormal wound healing can be corrected, it is important to first understand the basic principles of wound healing.

An in silico approach to the analysis of acute wound healing.

The complex interactions that characterize acute wound healing have stymied the development of effective therapeutic modalities. The use of computational models holds the promise to improve our basic approach to understanding the process. By modifying an existing ordinary differential equation model of systemic inflammation to simulate local wound healing, we expect to improve the understanding of the underlying complexities of wound healing and thus allow for the development of novel, targeted therapeutic strategies. The modifications in this local acute wound healing model include: evolution from a systemic model to a local model, the incorporation of fibroblast activity, and the effects of tissue oxygenation. Using these modifications we are able to simulate impaired wound healing in hypoxic wounds with varying levels of contamination. Possible therapeutic targets, such as fibroblast death rate and rate of fibroblast recruitment, have been identified by computational analysis. This model is a step toward constructing an integrative systems biology model of human wound healing.

Androstenediol reverses steroid-inhibited wound healing.

It is well recognized that stress of any nature will cause a delay in the wound healing response. This delayed healing response appears closely associated with immune regulators. In this study, CD-1 mice were injected with a long acting form of methyl prednisolone to cause a steroid-induced immune suppression. After 24 hours, two 6-mm full thickness wounds were placed on the animals' backs and one group of animals received the immune-regulating hormone, androstenediol. Wound contraction was quantified by planimetry for the subsequent 14 days. Animals that were stressed with methyl prednisolone but receiving androstenediol contracted their open wounds at faster rates compared with methyl prednisolone-stressed animals treated with the vehicle alone. These findings suggest that restoration of immune regulation by androstenediol can reverse the delayed open wound contraction secondary to steroid stress.

Primary cultured fibroblasts derived from patients with chronic wounds: a methodology to produce human cell lines and test putative growth factor therapy such as GMCSF.

BACKGROUND: Multiple physiologic impairments are responsible for chronic wounds. A cell line grown which retains its phenotype from patient wounds would provide means of testing new therapies. Clinical information on patients from whom cells were grown can provide insights into mechanisms of specific disease such as diabetes or biological processes such as aging. The objective of this study was 1) To culture human cells derived from patients with chronic wounds and to test the effects of putative therapies, Granulocyte-Macrophage Colony Stimulating Factor (GM-CSF) on these cells. 2) To describe a methodology to create fibroblast cell lines from patients with chronic wounds. METHODS: Patient biopsies were obtained from 3 distinct locations on venous ulcers. Fibroblasts derived from different wound locations were tested for their migration capacities without stimulators and in response to GM-CSF. Another portion of the patient biopsy was used to develop primary fibroblast cultures after rigorous passage and antimicrobial testing. RESULTS: Fibroblasts from the non-healing edge had almost no migration capacity, wound base fibroblasts were intermediate, and fibroblasts derived from the healing edge had a capacity to migrate similar to healthy, normal, primary dermal fibroblasts. Non-healing edge fibroblasts did not respond to GM-CSF. Six fibroblast cell lines are currently available at the National Institute on Aging (NIA) Cell Repository. CONCLUSION: We conclude that primary cells from chronic ulcers can be established in culture and that they maintain their in vivo phenotype. These cells can be utilized for evaluating the effects of wound healing stimulators in vitro.

Biologic therapeutics and molecular profiling to optimize wound healing.

Non-healing wounds represent a significant cause of morbidity and mortality for a large portion of the adult population. Wounds that fail to heal are entrapped in a self-sustaining cycle of chronic inflammation leading to the destruction of the extracellular matrix. Among cancer patients, malnutrition, radiation, physical dehabilitation, chemotherapy, and the malignancy itself increase the likelihood of chronic wound formation, and these co-morbidity factors inhibit the normal wound healing process. Current wound treatments are aimed at some, but not all, of the underlying causes responsible for delayed healing of wounds. These impediments block the normal processes that allow normal progression through the specific stages of wound healing. This review summarizes the current information regarding the management and treatment of complex wounds that fail to heal normally and offers some insights into novel future therapies [Menke N, Ward KR, Diegelmann R. Non-healing wounds. Emerg Med Rep 2007; 28(4).,Menke NB, Ward KR, Witten TM, Bonchev DG, Diegelmann RF. Impaired wound healing. Clin Dermatol 2007;25:19-25].

Androstenetriol immunomodulation improves survival in a severe trauma hemorrhage shock model.

BACKGROUND: Traumatic shock activates the hypothalamic-pituitary-adrenal axis (HPA) to mediate a cascade of defensive mechanisms that often include overwhelming inflammatory response and immunosuppression, which may lead to multiple organ failure. Androstenetriol (5 androstene, 3beta, 7beta, 17beta triol-AET) is a metabolite of dehydroepiandrosterone that markedly up regulates host immune response, prevents immune suppression, modulates inflammation and improves survival after lethal infections by pathogens and lethal radiation. HYPOTHESIS: AET-induced immune modulation will improve survival in a conscious rodent model of traumatic shock. METHODS: A relevant traumatic shock rodent model that applies to both combat and civilian sectors was used. After creation of a midline laparotomy (soft tissue trauma), animals were hemorrhaged to a mean arterial pressure of 35-40 mm Hg. Resuscitation was initiated sixty minutes later with crystalloid fluid and packed red blood cells and animals were observed for two days. In a randomized and blinded fashion, AET or vehicle was administered subcutaneously at the beginning of resuscitation. RESULTS: In the vehicle group 5 out of 16 animals survived, (31%). In contrast, 9 out of 13 animals who received AET survived (69%), (Fisher Exact Test p < 0.05). Survival in the AET treatment group was associated with reduced levels of IL-6, IL-10, and IL-18, and enhanced IFN-gamma and IL-2 levels. CONCLUSION: : The results indicate that AET provides a significant protective effect and improves survival in a clinically relevant model of traumatic hemorrhagic shock. AET protective effects are associated with an elevation of Th1 and reduction of Th2 cytokines.

Regulation of fibroblast functions by lysophospholipid mediators: potential roles in wound healing.

The bioactive lysophospholipids, primarily lysophosphatidic acid (LPA) and sphingosine-1-phosphate (S1P), are recent additions to the list of potent mediators of tissue repair and wound healing. In this review, we highlight the diverse actions of LPA and S1P on many types of cells involved in the wound healing process, with special emphasis on their regulation of fibroblasts. The effects of LPA and S1P are principally mediated via specific cell surface receptors. Important signaling pathways downstream of these receptors and the importance of TGFbeta and S1P cross-talk for wound healing are also discussed. Moreover, specific agonists and antagonists of the lysophospholipid receptors may be useful for the treatment of wounds and abnormal wound healing.