Manuela Martins-Green - US grants

The 9E3/CEF4 gene is a member of a recently discovered group of genes, sometimes referred to as the gro family, whose products are secreted proteins that have strong homologies to inflammatory mediators and are evolutionarily conserved. A clear correlation between the expression of these genes and cell growth has been demonstrated in culture. Building on these studies, I have investigated the 9E3 gene in vivo. It is expressed in normal tissues that grow by cell division, is not expressed in tissues that do not grow by cell division, and is transiently expressed early in the cell cycle when cells leave the resting stage. 9E3 expression is enhanced upon wounding, during wound healing (especially in areas of neovascularization), and in association with tumors. Cell damage in culture and specific growth factors known to be angiogenic in vivo also stimulate the expression of this gene. My observations extend the correlation between expression of the gro genes and growth and suggest a role in autocrine or paracrine growth regulation. Moreover, the elevated expression upon wounding and the high stimulation of expression by wound factors point to additional roles in wound response and/or angiogenesis. Therefore, discovering the specific function(s) of the products of these genes may be important for the understanding of response to injury, wound healing, development of tumors, and aspects of growth regulation. To initiate functional studies of the 9E3 gene, I have developed a polyclonal antibody that recognizes the protein in situ and immunoprecipitates it from cultured cells. My working hypothesis is that this gene plays an important role in wound healing and in cell growth in vivo. To test this hypothesis, I propose to: (1) Purify and characterize the 9E3 protein biochemically; (2) assess the role of the secreted forms of the protein during injury, wound healing and development of tumors; (3) determine if the protein plays a role in cell growth; (4) determine the kinetics and pathway of protein secretion. These experiments will establish whether or not 9E3 stimulates or represses growth, has chemoattractant or angiogenic properties in vivo, and affects wound healing an/or tumor growth.

[unreadable] DESCRIPTION (provided by applicant): The overall objective of this project is to generate a transgenic mouse line that expresses the human CXCR1 gene in a Cre-recombinase-driven tissue-specific manner as a tool to study CXCR1-dependent chemokine functions. Chemokines are important molecules playing many roles in physiological and pathological conditions, lnterteukine-8 is one of the chemokines that has been intensively studied because it functions in inflammation, wound healing, angiogenesis and tumorigenesis. Much work has been performed to elucidate the functions of this chemokine but progress has been impaired by the lack of an animal model system that can be genetically manipulated. In humans, IL-8 interacts with two receptors, CXCR1 and CXCR2, hence in order to determine how IL-8 functions it is necessary to study the role of each of these receptors in response to activation by hlL-8. These receptors and r|L-8 are highly homologous to the human proteins and rabbits express both receptors. However, it is very difficult (if at all possible) and expensive to perform gene manipulations in rabbits. Mice, on the other hand, do not have the CXCR1 gene or IL-8, hence cannot be used to fully study the functions of IL-8 in humans. We propose here a plan to create a human CXCR1 transgenic mouse line that can serve as an in vivo experimental system to elucidate the functions of IL-8. The specific aims of the work are: 1) To build and test the DMA construct for the transgenic animal. 2) To produce the founder transgenic mice. 3) To test the transgenic mouse line for tissue-specific Cre- recombinase activation. We will first generate a LoxP-based conditional transgenic founder line in which hCXCRI expression is suppressed in all tissues of the mouse. To do so, a DNA transgenic construct will be prepared by molecular cloning methods, tested in a cell culture system to confirm its functionality, including the transgene hCXCRI expression, and Cre-recombinase mediated recombination. We will then supply the DNA transgenic construct to Xenogen Biological that will create the founder transgenic mice. Finally, we will acquire specific Cre-recombinase expressing mice and cross them with our founder mice to achieve hCXCRI transgene activation in the skin to test the functionality of the mouse transgenic line in vivo. The mice expressing hCXCRI in specific tissues will serve as powerful tools to perform research related to human interieukin-8 because delivered hlL-8 will be able to interact with hCXCRI and mCXCR2 (already shown to occur) in the pertinent tissues, providing insight into the molecular mechanisms of the functions of IL-8 in humans. In general, the transgenic animal we are proposing to build will be a useful tool that brings versatility to our research and those of others for understanding the functions of hlL-8. [unreadable] [unreadable]

DESCRIPTION (provided by applicant): Inflammation is a key process in normal wound repair. Upon wounding, cytokines are activated and chemoattract leukocytes, including macrophages, to the wound site. Macrophages are key for the proper formation of granulation tissue because they clean up dead cells in the wound and produce a plethora of cytokines that initiate formation of the healing tissue. While many detailed mechanisms involved early in inflammation are known, those involved in resolution of inflammation are poorly understood. Understanding how inflammation ends is as important as understanding its beginning because prolongation of inflammation leads to impaired healing and chronic inflammatory conditions. VEGF is a key factor in wound healing and is primarily known for its role in angiogenesis. However, recently VEGF is emerging as a regulator of immunity and inflammation. We have discovered that VEGF contributes to resolution of macrophage- induced inflammation during wound healing and that it stimulates macrophage apoptosis in culture. We have also shown that VEGF stimulates the expression of TNFSF14/LIGHT in human macrophages. LIGHT is a member of the TNF superfamily of cytokines known to regulate co-stimulation of T cells as well as apoptosis in mucosal tumors. Our findings point to a novel cytokine-modulated pathway involved in resolution of the inflammatory response. In the work proposed here we address the consequences of these newly discovered functions of VEGF in resolution of inflammation during wound healing. We hypothesize that a novel function of VEGF in the healing process is modulation of macrophage survival in the damaged tissue and that LIGHT is a critical mediator of this process. Specifically, we will: (1) Determine whether VEGF induces macrophage apoptosis in vivo and stimulates LIGHT expression during healing and (2) Determine the relationship between VEGF-induced macrophage cell death and LIGHT. We will use a combination of cultured human macrophages, normal and genetically-modified mice, coupled with agonists/antagonists of VEGF- and LIGHT-dependant pathways. Histochemical and immunological techniques, ELISA, multiplex RT-PCR and siRNA technology will be used. The work proposed is novel because it reveals previously unknown functions of VEGF and it is important because it may facilitate the development of new therapies for poorly-healing wounds as well as other conditions characterized by excessive inflammation and for tumors in which VEGF is upregulated. Because resolution of inflammation occurs poorly or not at all in most abnormal healing situations, this line of investigation is significant for health because it identifies a novel strategy for improving impaired healing, a critical medical area. PUBLIC HEALTH RELEVANCE: Inflammation is a key process in normal wound repair. Upon wounding, cytokines are activated and chemoattract leukocytes, including macrophages, to the wound site. Macrophages are key for the proper formation of granulation tissue because they clean up dead cells in the wound and produce a plethora of cytokines that initiate formation of the healing tissue. Understanding how inflammation ends is as important as understanding its beginning because prolongation of inflammation leads to impaired healing and chronic inflammatory conditions. We have discovered that VEGF contributes to resolution of macrophage-induced inflammation during wound healing and that it stimulates macrophage death. The studies proposed here are to test the possibility that VEGF is a player in resolution of inflammation and that LIGHT mediates the VEGF effects on resolution of inflammation. Because resolution of inflammation occurs poorly or not at all in most abnormal healing situations, this line of investigation is significant for heath because it identifies a potentially novel strategy for improving impaired healing, a critical medical area.

Abstract Chronic wounds impact ~6.5M people and cost ~$25B per year in the US alone. Despite significant effort, understanding the mechanisms involved in development of chronic wounds in humans has met with limited success, primarily because we cannot experiment in humans and because current animal models are inadequate. The PI and team have developed a novel mouse model for diabetic chronic wounds that closely mimics those of humans. Low levels of oxidative stress (OS) are important for proper healing; however, when OS levels are high wound healing does not occur. Human chronic wounds have high levels of OS, but it is not known if OS is critical for developing chronicity. Using diabetic mice, we can generate chronic wounds 100% of the time by creating high levels of OS immediately after wounding by treating with inhibitors specific to two antioxidant enzymes. The wounds become fully chronic within 20 days after treatment and remain chronic until the mouse dies, sometimes >100 days. The wounds in the mouse model feature all of the same problems observed in human chronic diabetic wounds: high levels of OS lead to DNA damage, gene deregulation, protein and lipid damage, cell death, impaired keratinocyte migration (potentially inhibiting re- epithelialization), chronic inflammation, and lack of proper angiogenesis and deposition of matrix resulting in poor development of the healing new tissue. Equally important, the chronic wounds in the mouse model develop a biofilm from the bacteria present on the skin by elimination of non-biofilm-forming bacteria in favor of the biofilm-forming species. These biofilm-forming bacteria are also present on human skin and appear in human diabetic chronic wounds. All of these characteristics indicate that the PI's mouse model mimics all key aspects of human chronic wounds. The PI hypothesizes that high levels of OS induce changes in the dynamics of the commensal microbiome resulting in a transition to a biofilm-forming microbiome and development of the biofilm. This hypothesis will be investigated through the following studies: Aim #1: Identify the composition of the microbiome to characterize biofilm initiation and progression and develop a longitudinal statistical classifier that enables prediction of biofilm-forming bacteria in chronic wounds. Aim #2: Determine whether OS is necessary and sufficient for biofilm development. This project will use a novel model of chronic wounds developed in the PI's laboratory that has all the tissue characteristics present in human chronic wounds, contains the major bacterial pathogens present in human chronic wounds and develops a biofilm naturally, i.e. without the need to introduce bacteria from external sources. Therefore, this model will allow, for the first time, studies of the natural progression and dynamics of biofilm development in vivo. The results from these exploratory studies will serve as the basis for future mechanistic studies to determine how OS induces biofilm development. These studies will impact the field by providing, for the first time, a better understanding of whether specific bacteria can be used as prognostic tools for development of wound chronicity in mice. This work will provide insight into whether the microbiome can be exploited as a marker for healing/non-healing after debridement and in the future help with the design of proof-of-concept experiments using human specimens.

Chronic wounds impact ~6.5M people and cost ~$25B/year in the US alone. Despite significant effort, understanding the mechanisms involved in development of chronic wounds in humans has met with limited success, primarily because we cannot experiment in human chronic wounds and because current animal models are inadequate. We have developed a novel mouse model for diabetic chronic wounds that closely mimics those of humans. High levels of oxidative stress (OS) are important for chronic wound development. Human chronic wounds have high levels of OS. Using diabetic mice, we can generate chronic wounds 100% of the time by creating high levels of OS immediately after wounding by treating with inhibitors specific to two antioxidant enzymes. The wounds become fully chronic within 20 days after treatment and remain chronic until the mouse dies, sometimes >100 days. The wounds in the mouse model feature all of the same problems observed in human chronic diabetic wounds: high levels of OS lead to DNA damage, gene deregulation, protein and lipid damage, cell death, impaired keratinocyte migration (potentially inhibiting re-epithelialization), chronic inflammation, lack of proper angiogenesis and matrix deposition. Equally important, the chronic wounds in the mouse model develop a biofilm from the bacteria present on the skin microbiome by elimination of non-biofilm-forming bacteria in favor of the biofilm-forming species. These biofilm-forming bacteria are also present on human skin and appear in human diabetic chronic wounds. All of these characteristics indicate that the PI's mouse model mimics key aspects of human chronic wounds. We hypothesize that high OS levels affect the microenvironment of the wound resulting in expression of genes that combat OS, that are involved in adhesin and expression of quorum sensing molecules and virulent factors that favor biofilm development by P. aeruginosa. To test this hypothesis, we will: Aim#1: Isolate a pure culture of PA from the biofilms in our chronic wound mouse model and sequence its genome. We already isolated PA from one such wound. Aim#2: Using RNAseq, perform experiments in sterile wounds infected with the isolated P.A alone in the presence or absence of high OS and: A. Determine whether P.A genes known to be involved in response to high levels of OS, adhesion to surfaces, production of quorum sensing molecules and virulence factors in vitro are also expressed in in vivo in the CW bed during the transition of PA from non-biofilm-forming in the skin microbiome to biofilm-forming in the CW. B. Identify new genes that are expressed by PA in the wound bed versus abiotic surfaces, and if time permits or with future funding determine whether they may be important in the transition of PA to biofilm forming. Our proposal is significant and innovative because with the use of our novel db/db-/- chronic wound model, we will determine how P.A becomes biofilm-forming in the high OS environment of a chronic wound. We will also identify P.A molecules that contribute to biofilm development by this bacterium in the wound bed. Most importantly, our work will impact health care because it will potentially identify biomarkers that are critical for initiation of biofilm development by P.A in diabetic wounds. Such biomarkers have the potential, when verified in humans, to objectively guide treatment after debridement to prevent return of biofilm. Currently, wound bed assessment is subjective.