The Role of Catecholamines in Human Chronic Wounds A

THE ROLE OF CATECHOLAMINES IN HUMAN CHRONIC WOUNDS

A Project

Presented to the faculty of the Department of Biological Sciences

California State University, Sacramento

Submitted in partial satisfaction of the requirements for the degree of

MASTER OF ARTS

Biological Sciences

Anil Azad

SPRING 2018

THE ROLE OF CATECHOLAMINES IN HUMAN CHRONIC WOUNDS

A Project

Anil Azad

Approved by:

______, Committee Chair Thomas R. Peavy, Ph.D.

______, Second Reader Robert Crawford, Ph.D.

______, Third Reader Andrew Reams, Ph.D.

______Date

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Student: Anil Azad

I certify that this student has met the requirements for format contained in the University format manual, and that this project is suitable for shelving in the Library and credit is to be awarded for the project.

______, Graduate Coordinator ______James Baxter, Ph.D. Date

Department of Biological Sciences

Abstract of THE ROLE OF CATECHOLAMINES IN HUMAN CHRONIC WOUNDS by Anil Azad

Catecholamines such as dopamine, norepinephrine, and epinephrine elicit many diverse cellular and physiological responses in mammals. The role of catecholamines in wound healing and their contribution to wound delay is relatively underappreciated. Articles published prior to 2005 have indicated that catecholamine expression appear to be involved in prolonging the wound healing process, leading to chronic wounds such as diabetic foot ulcers, but the specific causes were unknown. Since then, wound healing studies have revealed specific cell types that are affected by catecholamines when bound to their adrenergic receptors on the cell surface and their cellular responses that contribute to delayed healing such as a prolonged inflammatory response. For example, the binding of norepinephrine to β1-adrenergic receptors on macrophages can deter their phagocytic activity, preventing the removal of cellular debris and microbes in the wound environment, thereby stretching out the immune response within the inflammatory phase of wound healing. In this review, up to date research regarding catecholamines and their role in delaying wound healing will be summarized, specifically focusing on research which details the mechanisms by which catecholamines affect each of the major steps and components of wound healing. Catecholamine detection methodology will also be reviewed with respect to automation, ease of use, sample processing, costs, and detection levels. In addition, the connection between catecholamines and microbes infecting wounds with respect to their ability to respond to and produce catecholamines will also be presented. Finally, along with analyzing and synthesizing the

research in this area, future research directions will be postulated. This review will provide a much-needed compilation of what recent findings have been made in the field.

______, Committee Chair Thomas R. Peavy, Ph.D.

______Date

ACKNOWLEDGEMENTS

This paper has been completed in great part to Dr. Thomas Peavy, who’s patience and insight was greatly appreciated by the author. The author would also like to acknowledge Diana Nguyen for her constant moral support, grace, free food and consoling. Without them, the author would not be able to complete this project.

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TABLE OF CONTENTS Page Acknowledgements ...... vii

List of Figures ...... ix

Introduction ...... 1

Catecholamine Detection and Quantification ...... 9

Catecholamines and Chronic Wounds ...... 17

Utilization and Production of Catecholamines by Bacteria ...... 29

Summary ...... 38

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List of Figures Figures Page 1. Catechol Structure ...... 4

2. Catecholamine synthesis in humans ...... 5

Introduction

Current estimates by experts suggest that the “wound-care market” could be worth 18.9 billion dollars (DiPietro and Guo, 2010). This projection is based on several factors, first and foremost is the fact that there has been a significant change in the number of people that are at risk for wounds that exhibit delay in the healing process causing them to need more medical attention. Recent publications assert that up to 6 million individuals in the U.S. are affected by chronic wounds, and a majority of these individuals (85%) are the elderly population. In particular, the elderly and individuals diagnosed with diabetes are prone to chronic wounds due to physiological changes in their health (DiPietro and Guo, 2010). Typically, wounds are treated using dressings and/or topical applications however these standard procedures do not always result in healing of the wound. Wounds that do not heal properly over a period of three months are termed chronic wounds and are a serious problem that can lead to infection, sickness, and amputation. Taken altogether, chronic wounds are a burgeoning medical issue that needs more attention since its incidence is not predicted to decrease due to individuals living longer and the fact that diabetes is becoming more prevalent.

Over the last 20 years, researchers have been working diligently to understand what causes delays in the wound healing process and how best to treat at risk wounds so as to improve the healing rate. This process is complex and entails a myriad of environmental and biological factors. It is important to review the normal wound healing process so that dysfunctional repair can be put into perspective. The entire process can

be broken up into four distinct phases: hemostasis, inflammation, proliferation, and tissue remodeling (Lyte, 1992, 1993). Hemostasis begins immediately after the wound is incurred. The wound area undergoes vasoconstriction and fibrin clot formation to restrict blood flow and loss which is facilitated by thrombocytes in the vicinity. Cytokines and growth factors are released by platelets and cells during clot formation to attract various cell types that will assist in the transition into the inflammatory stage. Inflammation is intended to prevent infection and to clean up cellular debris so that repair of the tissue can occur. During this phase, immune and endocrine factors are released to recruit cell types such as neutrophils, macrophages and lymphocytes to enter the affected area which aid in this clean-up process. One of the first lines of defense are the neutrophils which engulf damaged cells and invading microbes. Macrophages release additional cytokines which recruit leukocytes for further support to remove debris and microbes. Other released cytokines attract keratinocytes, fibroblasts and endothelial cells which help promote the transition to the next stage, the proliferation phase. This phase is where the wound repair begins and is characterized by three hallmarks: wound contraction, re-epithelialization, and angiogenesis. Should inflammation or the proliferation stage be prolonged, then wound healing would be delayed. Tissue remodeling is the last phase which primarily entails the deposition of collagen and other extracellular components by fibroblasts to rebuild the dermal tissue.

Along with a bevy of other elements, hormones play a major role in both positively and negatively affecting the wound healing process, as well as either

lengthening or shortening it. Hormones play a pivotal role in transducing signals throughout the body of any organism. Within the body of a human, a stimulus from either an external source or some shift of the internal status quo induces the formation and propagation of a signal which results in a specific action by target tissues and cells. There are a variety of different types of hormones and their classification is based primarily on their molecular composition. Of the four major types (amine, peptide, protein, and steroid), this paper will address the subgroup of amine-based hormones termed catecholamines which includes the compound epinephrine, otherwise known as adrenaline. Catecholamines generally are utilized in response to stress stimuli and are involved in the “fight or flight” response. As will be discussed, catecholamines play a major role in many mechanisms that affect the efficacy, and the length of wound healing by affecting specific cell types, levels of inflammation, collagen deposition and even growth of foreign bacteria within the wound microenvironment.

The hallmark of catecholamines is the catechol functional group which consists of a benzene ring with two attached hydroxyl groups (Figure 1). The three catecholamines that are regarded as extremely important in biological processes are: epinephrine, norepinephrine, and dopamine. These three catecholamines are synthesized from a single pathway originating from the amino acid tyrosine (Figure 2).

Figure 1. Catechol Structure

Figure 2. Catecholamine synthesis in humans. Enzymes involved are listed above arrows between compounds.

The addition of a hydroxyl group to tyrosine on the meta-carbon of the benzene ring by the enzyme tyrosine hydroxylase is the first step in the process which creates the catechol group and converts tyrosine into the intermediate L-dopa. Following this addition, the enzyme amino acid decarboxylase will then remove the carboxyl group from the alpha carbon creating dopamine, the first of the notable catecholamines.

Dopamine can then be hydroxylated at the former alpha carbon to generate norepinephrine using the enzyme dopamine beta hydroxylase. Finally, epinephrine is formed after its amine group is methylated by the enzyme phenylethanolamine N- methyltransferase.

In addition, bacterial infections can deter wound healing and are responsive to catecholamines (DiPietro and Guo, 2010). Recent studies have indicated that bacterial species can use the catecholamines produced by the human body in order to propagate, and even generate them through their own biochemical pathways which are yet to be determined. Research has also shown that these bacteria can also secrete biofilms

(communities of bacteria which habituate a polysaccharide matrix) which even further delay wound healing by extending the inflammatory phase and providing a level of immunity from antimicrobial treatments and the immune system.

Diabetes has also been shown to confer an improper wound healing phenotype

(DiPietro and Guo, 2010). Aside from neuronal damage and other ailments, high levels of glucose can also lead to stiffening of arteries and prevention of oxygen availability in a wound. This state of poor oxygenation and impairment of blood flow can also stem from

infection by the aforementioned bacteria. Nerve damage or neuropathy is another common diabetic issue which causes a loss of sensation and can lead to ulcerous wounds in the extremities termed diabetic foot ulcers. The immune system of diabetic patients is also typically compromised by exhibiting lower levels of circulating white blood cells, and impairment of neutrophils to perform phagocytosis and chemotaxis. Individually or in combination, these factors can severely hamper wound healing. If left untreated, these chronic wounds can lead to the need to amputate the limb, or even death.

The intent of this review is to synthesize the current literature and evidence indicating that catecholamines are a major factor in delaying wound healing. In doing so, it is also important to address the methodologies used to detect and quantify catecholamines in biological samples when determining which cells are releasing these compounds so as to better understand the strengths and weaknesses of these studies. It is the aim of this article to specifically look at what is commonly utilized in modern laboratories today, the efficacy of these methods, as well as to contribute ideas to further the field. In addition, this article will discuss the research on what is known about how catecholamines interact with major actors in wound healing such as with keratinocytes and fibroblasts and their effects on gene expression. In addition, the article will highlight research indicating that bacterial species are able to recognize, and even utilize catecholamines which are synthesized by humans to increase their survival in a wound environment. A better understanding of the pathways involving catecholamine induced wound delay could allow for researchers to develop more fine-tuned methods to improve

wound healing treatments. With a longer living elderly population and increasing incidence of diabetes, the role of catecholamines in wound healing is a major area of research that needs further study.

Catecholamine Detection and Quantification

In order to appreciate studies that involve the detection and quantification of catecholamines, it is important to understand how this data is collected since this is not a trivial procedure. In some cases, the methodology used will influence the outcome with respect to detection limits and accuracy of the quantitative methods. The overall schema of catecholamine analysis can be broken up into three general steps:

Sample Collection→ Catecholamine extraction → Analysis

With each of these steps there are a variety of methods that can be utilized, however there are caveats that must be considered given the sample’s origins and the reagents used, which can in turn affect the instrumentation utilized, and even sensitivity. Before analyses begin, the sample usually needs be either extracted or enriched from its source

(typically blood or urine), as other biomolecules and contaminants can affect the efficiency of the sensitive detection instruments. Urine collection is the favored method for this purpose for its ease of use in purification. Catecholamine extraction from urine was first documented utilizing alumina resin by Hallman et al. (1978) with an efficiency or yield calculated to be approximately 63% (Zhang et al., 2012). Extraction of catecholamines through the use of resins and affinity matrices has been termed solid phase extraction (SPE). SPE methods utilize a solid matrix where a liquid sample can be passed through, and targeted molecules are retained in the matrix through chemical

interactions. Then, the targeted molecule (catecholamines) can be collected after an elution solvent is added. SPEs are found in conventionally packed columns or spin column formats.

In order to extract catecholamines from human samples such as blood, urine, or tissue, specialized methodologies must be utilized to remove any contaminants or compounds which may degrade catecholamines and prevent any further degradation from environmental factors (heat or light). Since the original alumina extractions, researchers over the last 30 years have produced a variety of different methods to improve on the extraction of catecholamines. SPE columns utilizing alumina are still commonly used, however a variety of different matrix components have been implemented. Phenylboronic acid (PBA) or weak-cation exchange sorbents (WCX) have been shown to be highly efficient at extracting catecholamines (Clark and Frank, 2011; Kumar et al., 2011).

In regards to contemporary methods, molecular imprinted polymers are a novel method for the separation of targeted molecules. With a desired target in mind, monomeric components used for generating the polymer are added together with their target and are then induced to form polymers. The target molecule is then removed thereby leaving an imprint of the target molecule on the polymer (Vasapollo et al., 2011).

MIPs are highly specific as their cavities are conditioned to match the targeted molecule, and within them contain functional groups which interact with the molecule to stabilize its binding. In addition, MIPS have been reported to have a relatively high efficiency of extraction ranging from 87-95%.

Microextraction by packed solvent (MEPS) uses a similar methodology to SPEs and has recently been shown to extract catecholamines from dried urine and plasma samples with a relatively high yield of 85% (Saracino et al., 2014). The ability for catecholamines to be extracted from dried samples serves to simplify sampling from patients and prevent degradation from harmful enzymes. Furthermore, MEPS is less time consuming than the aforementioned SPE, and thus can limit the degradation of catecholamines, as well as serve as a much cheaper alternative.

As mentioned previously, there are other elements to consider when conducting analyses on extracted catecholamines, such as sample volume, effects of the washing solution, binding capacity, and conditioning of the LC column. Each of these variables can lead to poor detection, or even false positives. Li et al. (2016) analyzed each of these variables for the spot extraction from urine samples and showed how careful optimization of SPE procedures improved their analysis of catecholamines and metaphrines

(metabolites of epinephrine and norepinephrine) using liquid chromatography-mass spectrometry (LCMS) for detection. The research group sought to develop and validate a simplified method that required little to no pH adjustment. As previous studies within the field of catecholamine analysis have indicated, pH is an extremely important condition since it affects the binding interactions with SPE sorbents and can be pivotal for the success of the extraction. Maintaining the pH of a solution (i.e. urine) during extraction can often be delicate. In order to address this factor, specific columns were tested in a variety of pH ranges. In the first part of their research, the research group tested the utility

of pentaflurophenyl (PFP) columns. The purpose of substituting PFP columns over the typical C18 column was the ability of PFP columns to better resolve between norepinephrine and epinephrine. Increasing the pH of the mobile phase allowed the group to separate both molecules more efficiently. In order to alleviate the need for pH balancing and save time, the research group hypothesized that lowering the sample volume could limit pH variation from various urine samples. Comparing various sample sizes, the researchers found that extracted amounts of norepinephrine and epinephrine could increase by 10 to 15 percent. It was reasoned that through limiting sample volume, the effect of pH variation of urine was diminished. Samples were eluted via gradient elution with 0.01% acetic acid in water and 0.01% Acetic Acid in methanol. The amount of PBA within the SPE was also tested since this corresponds to its binding capacity.

Testing three concentrations of PBA, it was discovered that having lower concentrations of PBA lead to increased yields of NE. Although the results were interesting to say the least, there was no proposed explanation for the significant change in extraction efficiency. Utilizing their conditions, the researchers were able to describe a simple cost- effective method for catecholamine extraction and that forgoes the need of pH adjustment as well as sample processing that plagues standard methods to increase throughput (Li et al., 2016).

After extraction, there are multiple choices for the separation, and detection of catecholamines. Typical instrumentation used by researchers consist of High

Performance Liquid Chromatography (HPLC), Ultra-performance Liquid Chromatograph

(UPLC), Hydrophilic Interaction Chromatography (HILIC) and Capillary Electrophoresis

(CE). All of these methodologies separate catecholamines (or other target molecules) based on molecular properties such as charge and hydrophobic characteristics.

In HPLC, a pressurized sample is passed through a separation column which is filled with a stationary phase. Typically, the stationary phase has an embedded moiety which allows for separation based on polarity or other characteristics. An 18-carbon chain (C18) is typically the most commonly used column for catecholamine separation as it allows for excellent resolution between epinephrine, norepinephrine and dopamine. The stationary phase is usually silica or other types of polymers. Use of ion pairing agents along with a C18 column allow for the improvement of retention of compounds for separation. Typically, ion pairing agents are used in situations where molecules of interest are ionic compounds and cannot be properly retained by a hydrophobic stationary phase. The ion pairing agent usually has a charged region coupled with a non-polar region, allowing it to be embedded in the stationary phase. Once the agent is added, the sample can be better resolved.

HILIC has been explored as an alternative to the aforementioned HPLC. In using

HILIC, retention of solutes was accomplished with better resolution without the need of an ion pairing reagent and could be dissolved with high concentrations of acetonitrile (a commonly used solvent for HPLC); this particular feature allows for detection by mass spectrometry. Furthermore, use of HILIC granted higher precision allowing for less variability when quantifying catecholamines from patients (Kumar et al., 2011). HPLC

and its alternatives like HILIC allow for rapid and consistent sample analysis through automation, simple maintenance, and ease of use.

Although HPLC is considered a golden standard of catecholamine analysis, other techniques are available which have inherent advantages such as capillary electrophoresis

(CE). CE is considered an attractive alternative since it allows for higher throughput and lower sample volume injections. In CE, a sample is introduced into a capillary tube filled with a silica polymer, and an electric field is induced to allow for migration of the sample from one end of the capillary to the other. CE unlike HPLC does not utilize analyte-resin interactions but instead uses the polymer pores to size separate the molecules. In addition,

CE can be coupled with almost any of the detection techniques that is normally paired with HPLC.

After sufficient separation from other compounds, these instruments must be coupled with a sensitive detector which can be used to quantify their presence in the sample. Typical detectors are ultraviolet (UV), electrochemical detection (ECD), and

Mass spectrometry (MS). Each of these separation methods along with the detector have advantages and disadvantages that researchers must weigh to determine the proper one for their experimental conditions. With regards to UV detection, they are typically not sensitive enough to detect analytes at concentrations relevant to biological levels; UV detection generally ranges from 0.5µM to 0.1nM. ECD functions through the oxidation/reduction of targeted molecules after separation by applying a charge to a flow cell. The gain or loss of electrons causes an electrical current that can be measured and

correlated to concentration using a calibration curve from a known compound. Generally, the sensitivity for ECD ranges from 1nM to 0.5pM. Degradation of the ECD flow cell can occur over time with repetitive use which decreases the lower limit of detectability so electrodes need to maintained or replaced routinely (Lin et al., 2010).

Mass spectrometry is an analytical tool where compounds are fragmented with an electron source. Each compound fragments in a particular way which essentially creates a fingerprint. This method allows for precise identification of compounds provided researchers have prior knowledge of how the compound characteristically fragments. The major disadvantage for such an instrument arrives from when experiments concern samples that have very similar chemical structures or chemical formula. Detection limits for mass spectrometers can vary by manufacturer but can be as low as 0.5nM.

The preferred detector for catecholamine analysis when coupled with CE is laser- induced fluorescence (LIF), or light-emitting diodes (LEDs). Diao et al. (2011) devised a

CE apparatus which implemented both LIF and LED together for an extremely sensitive and accurate instrument termed “in-column fiber-optic light-emitting diode-induced fluorescence detection” (ICFO-LED-IFD) for the use of catecholamine detection in urine.

To prevent any background noise from the LED and the fluorescence signal, Diao et al. used two bandpass filters. Finally, the group utilized the fluorophore fluorescein isothiocyanate (FITC) to derivatize catecholamines within urine. Derivatization of compounds is commonly used when a compound of interest is not easily analyzed.

However, complete derivatization can be an issue and needs to be assessed when

determining yields. Ultimately, with this methodology, detection limits of dopamine, epinephrine and norepinephrine were found to be 1.0, 0.3 and 0.9 nM, respectively.

Catecholamines and Chronic Wounds

Chronic wounds are a complex medical issue and are influenced by a number of biological factors. In order to best combat wound chronicity and prevent further complications, it is important to understand the mechanisms of what cause them. Based on this knowledge, novel treatments can be designed to enhance the wound healing pathway. Although Guo and DiPietro’s review (2010) did outline some of the factors in involved, this section will focus on catecholamines and their role in delaying wound healing.

In particular, diabetic patients have a high incidence of chronic wounds on their extremities such as feet, and catecholamines have been implicated as a factor. The incidence of diabetic foot ulcers (DFUs) is 15% of the 20.8 million diabetic patients within the US population, and shockingly is estimated to rise (Brem and Tomic-Canic,

2007). Diabetic foot ulcers require extensive medical monitoring due to their inability to heal properly. This is due to a variety of factors such as decreased blood flow to the wound, suppressed immune response, inability of wound closure, loss of nerve function/pain sensation, and lack of proper responses from keratinocytes and fibroblasts.

The average size of DFUs is approximately 118 mm2 but can get as massive as 588 mm2.

If not treated properly, the persistent wound can lead to further complications such as amputation (Di Marco et al., 2008). The need for amputation is typically due to bacterial infection, tissue necrosis, and sepsis. It is known that diabetic individuals are under constant physiological stress due to their glycemic condition and exhibit unique

concentrations of catecholamines; studies have shown elevated levels of NE and DOPA from kidney homogenates of diabetic individuals (Di Marco et al., 2008). It is important to understand the mechanism as to how catecholamines contribute towards wound delay which would provide researchers with a basis to design treatments to improve wound healing outcomes.

It is well known that catecholamines bind to adrenergic receptors. The receptors are a class of G-protein coupled receptors that specifically bind norepinephrine and epinephrine. Of these receptors there are two major classifications, alpha and beta, and each are further broken down into sub-classes. Alpha receptors are further broken up into

Alpha-1, and Alpha-2 adrenergic receptors (α1AR and α2AR, respectively) and bind to epinephrine and norepinephrine. Typically, α1ARs control contraction of the bladder, seminal tract, and uterus, as well as modulation of neuronal responses, glycogenolysis, gluconeogenesis and secretion from sweat glands. α2ARs have been associated with the control of insulin release in the pancreas, and thrombocytic aggregation.

Beta-1 adrenergic receptors (β1ARs) also bind to epinephrine and norepinephrine and are responsible for a variety of responses such as increasing heartrate, contracting ventricular cardiac muscle, and releasing amylase from salivary glands. β1ARs have a higher affinity for norepinephrine than epinephrine, whereas in Beta-2-Adrenergic receptors (β2ARs) the opposite is true. β2AR activation has been shown to play a role in maintaining the function of the epidermis and helping to maintain its integrity (Rang,

2003). β1ARs also bind to epinephrine and norepinephrine, but unlike β2ARs, they are

primarily expressed in cardiac tissue. When stimulated, β1Ars can increase cardiac output. It is notable that the binding affinity for epinephrine is stronger in β2ARs compared to β1ARs.

The two beta receptors show similarities in their signaling responses. Both utilize heterotrimeric G-protein coupled receptors that lead to the generation of cyclic adenosine monophosphate (cAMP) by activation of adenylyl cyclase. cAMP then activates cAMP- dependent protein kinase (PKA) which leads to various cellular responses (Hu et al.,

2003). Specifically, in β2ARs, after binding to their primary ligand (e.g. epinephrine or norepinephrine), a signal transduction cascade is triggered that typically turns on adenylyl cyclase, thereby producing cAMP. This secondary messenger leads to the activation of the ERK/MAPK proteins by phosphorylation. Once activated, ERK/MAPK leads to transcription activation and gene expression. Although the beta receptors show similarities in their general signaling pathway, their cellular responses differ. For example, within cardiac myocytes, stimulation of β1ARs increases apoptosis, whereas in

β2ARs, stimulation inhibits apoptosis (Wei-Zhong et al., 2001). β2ARs have been shown to be present of the surface of fibroblasts, keratinocytes and melanocytes and shown to bind to epinephrine and norepinephrine (Sivamani et al., 2007). With the notion that

B2ARs are common to these cell types which are integral to wound healing mechanisms, researchers have been able to narrow down to a possible target for research.

Epinephrine seems to be the primary factor in delaying wound healing, and as such, research has focused on the β2AR pathway as a primary effector in wound

chronicity. Understanding the mechanisms of how β2AR stimulation delays wound healing has been an active area of research that may lead to the development of treatments to speed up the wound healing process, especially in chronic wounds. There are other agonists that can bind to β2ARs such as arrestins, isoproterenol, and fenoterol, however different physiological effects unrelated to the delay of wound healing occur when bound. Drugs that block the receptor’s activity are often used when performing experiments to determine the biological effects of compounds such as epinephrine. In particular, alpha and beta blockers are typically used to block the alpha or beta-adrenergic receptor pathways, respectively, which will be discussed in more detail later.

In addition, in vitro cell culture scratch assays have been utilized quite often to investigate the role of catecholamines on the wound healing progress. To begin with, researchers grow their cells of interest to confluence and then perform a mechanical scratch (e.g. pipet tip or needle) to examine the response time of the cells to close the scratch wound gap. With this experimental method, keratinocytes were found to close the wound gap within 2 hours. However, when epinephrine was added, keratinocytes showed a migration delay that was extended to 4 hours. Keratinocytes treated with epinephrine and the β2AR inhibitor okadaic acid (OA) restored the time to close the gap or heal.

It was found that OA inhibited the downstream β2AR response by preventing desphosphyorylation of ERK/MAPK thereby inhibiting Protein Phosphatase 2 (PP2A) which is a known negative-regulator of the ERK/MAPK signaling pathway (Pullar et al.,

2011). When PP2A activity was further investigated using immunostaining and fluorescence microscopy, it was observed that within non-β2AR-activated keratinocytes, actin and vinculin were polarized in a crescent shape. Normally, vinculin aggregates at the lamellipodial edge and colocalizes with the actin cytoskeleton during cell migration.

As expected, keratinocytes treated with either epinephrine and norepinephrine were shown to have diffuse actin throughout the cell which indicates a non-migratory cell stage. Treating cells with the antagonist OA restores the migratory phenotype with actin concentrated at its leading edge.

These researchers also investigated whether epinephrine or norepinephrine stimulation of β2ARs leads to cell proliferation. Keratinocytes were grown with and without the presence of either epinephrine or norepinephrine and it was shown that the presence of each severely retarded the proliferation of keratinocytes. Incubating keratinocytes with only OA showed no marked change in proliferation levels. Finally, the epithelialization potential for wound closure was measured in the presence of epinephrine. It was shown that stimulation of β2AR by epinephrine led to a decrease in wound closure by 34% as compared to controls after 3 days, demonstrating that β2AR stimulation directly hampers wound healing (Pullar et al., 2011).

Aside from keratinocytes, other cell types which are integral to the wound healing process can also be affected by catecholamines. Neutrophils also exhibit β2ARs on their surface and are another important candidate to consider for understanding how chronic wounds are established. Kim et al. (2008) sought to use an animal model to essentially

mimic chronic catecholamine stress and see how β2AR activation by epinephrine affects neutrophil trafficking. In their study, mice subjected to punch biopsies were constantly delivered epinephrine (at physiological levels) via an osmotic pump to examine the effects. As expected, epinephrine treated mice exhibited delayed wound healing in comparison to saline controls. With this model, researchers then sought to understand how epinephrine affected the dynamics of neutrophil trafficking. Utilizing mice that expressed a lysozyme-EGFP gene within neutrophil cells so as to track these cells by fluorescence imaging, it was shown that after two days of incurring the wound, neutrophil recruitment was drastically increased in epinephrine stressed mice; after eight days neutrophil trafficking persisted. These results indicate that epinephrine plays a major role in neutrophil trafficking and persistence within a wound, which causes a sustained or elevated inflammatory response (Kim et al., 2008).

Further experiments examined whether pro-inflammatory cytokines were released by neutrophils exposed to norepinephrine to account for the inflammatory response (Kim et al., 2008). In examining the levels of IL-1beta, TNF-alpha, IL-1alpha and IL-6 in wound tissue on day five after treatment with norepinephrine, it was found that only IL-6 was increased (140% over control levels). IL-6 serves as a pro-inflammatory cytokine which stimulates the immune response during an infection. A follow up experiment was performed to block IL-6 activity by using an antibody. This antibody treatment rescued wound healing by reducing the number of neutrophils trafficking to the wound site and accelerating wound healing two-fold.

Macrophages were also analyzed since they are known to produce many pro- inflammatory cytokines and also express β2ARs. Macrophages cultured from a skin biopsy were exposed to epinephrine and found to have a 3-fold increase in IL-6 levels indicating that they are the likely source of the influx of IL-6 at the wound site.

Summarily, epinephrine induced stress leads to β2AR activation signaling, which then ultimately causes increased trafficking of pro-inflammatory neutrophils to wounds thereby causing prolonged inflammation and delayed wound healing (Kim et al., 2008).

Fibroblasts are another cell type important to wound healing, as they are responsible for the regeneration of the ECM and wound contraction. Like neutrophils, and keratinocytes, fibroblasts also express β2ARs on their surface. As was shown with keratinocytes and neutrophils, one would expect that stimulation of β2ARs via agonists such as catecholamines could impair the ability of fibroblasts to facilitate proper wound healing. Consistent with this hypothesis, exposure of dermal fibroblasts with the β2AR inhibitor OA resulted in an increase in migratory speed (Pullar, 2006). Although there are indications that epinephrine inhibits proliferation of fibroblasts, evidence of its effects on migratory speed are less well known and are an area where further research is required.

Further studies showed stimulation of β2AR via epinephrine increased proliferation of fibroblasts by 55 percent after six days. The persistence of fibroblasts at the wound site can prolong the inflammatory phase, which can cause overexpression of fibronectin and can cause oxidative tissue damage. This effect was observed, using a range of 10 nM to 10 μM which is within the physiological levels generally found in

humans which is approximately 40 nM (Le Provost and Pullar, 2015). Finally, as β2ARs can interact with cAMP dependent kinases, the research group sought to analyze whether proliferation due to β2AR stimulation was due to cAMP. Thus, fibroblasts were treated with a cAMP analog (sp-cAMP) in order to increase the levels of intracellular cAMP since it has a longer half-life in the cell. Fibroblasts treated with sp-cAMP were then treated with and without epinephrine, and it was found that there was no difference in proliferation levels; however, when cAMP was added before the agonist, the proliferation of fibroblasts was severely decreased, suggesting that β2AR stimulation in fibroblasts leads to a proliferation response through a cAMP mechanism (Le Provost and Pullar,

2015).

Much of the research on catecholamines in relation to wound healing has primarily focused on epinephrine, however norepinephrine has been shown to contribute also. Generally, norepinephrine is released as a response to injury as a means to activate synaptic modulators, or elements which induce changes in synapse efficacy and morphology. Furthermore, studies have also indicated that norepinephrine can induce bacterial growth in wound microenvironments, however this aspect will be covered in a later section.

Macrophages are one of the primary actors in the elimination of infectious bacteria, and if unable to function adequately, bacterial species are able to propagate, and delay wound healing. Thus, phagocytosis has become a prime area of study within the wound healing pathway. As studies have shown that macrophage migration is negatively

affected by NE (Xiu et. al., 2013), researchers have further proposed that the phagocytic efficiency of macrophages could also be affected and thus be unable to remove infectious bacteria. Using macrophages obtained from the spleen of mice, Gosian et al. (2007) sought to test the effectiveness of phagocytosis with and without the modulation of NE with E. coli as the target of phagocytosis. Utilizing pharmacological and physiological concentrations of NE (1uM and 1 nM, respectively), macrophages were shown to have consumed less bacteria. Norepinephrine is primarily a β1AR agonist, but also binds to

Alpha-1, and 2 adrenergic receptors. Thus, alpha and beta blockers were used test to determine which receptor pathway affects phagocytosis. Using alpha blockers, partial impairment was observed whereas complete impairment was noted with beta-blocker treatment (Gosian et al., 2007). Thus, beta adrenergic receptors appear to be the primary receptor for NE.

In addition to phagocytosis, norepinephrine affects cell proliferation of important cell types. Mouse macrophage proliferation assays have shown that the addition of NE at

1 and 100 μM was enough to inhibit macrophage proliferation. The research team further elucidated this experiment by applying NE at different time points of the assay, finding that the effect was most prominent when NE was incubated early within the cell cycle.

Xiu et al. (2013) assayed the expression of C-C chemokine receptor type 2 (CCR2) since it is primarily associated with chemotaxis (cell migration homing to a chemical signal by binding activity). Incubating macrophages with NE led to dose dependent decreases in levels of CCR2 receptor expression; 10nM of NE had no effect, whereas 1

μM had significantly decreased levels of CCR2. Additional experiments revealed that NE stimulation also induced macrophages to approximately double the release of TNF-α

(from 40 to 85%), which is an important cytokine for systemic inflammation and thus could potentially prolong the inflammatory phase (Xiu et al., 2013).

In regards to dopamine, there have been indications that the stimulation of dopamine receptors may actually increase angiogenesis in mouse wounds which may be a method to promote wound healing. Unlike norepinephrine and epinephrine, dopamine binds to a separate receptor group termed Dopamine receptors. Within this group are two families, D1 and D2. Both are also G protein coupled receptors but regulate different pathways. D1 receptors, when activated increase levels of cAMP, whereas D2 receptors inhibit cAMP formation (Beaulieu and Gainetdinov, 2011; Vaughn et al., 2017).

Dopamine receptors are expressed on a variety of cell types, most importantly on many skin cells such as keratinocytes (Tammaro et al., 2012). Even further, observations have also found evidence that keratinocytes may produce enzymes which produce dopamine, however more research and insight is needed to confirm these findings (Fuziwara et al.,

2005).

In the context of wound healing, dopamine has been shown to play a role in angiogenesis. Angiogenesis is mediated by VEGF (vascular endothelial growth factor), a primary cytokine for the formation of blood vessels. Researchers have made observations concerning the activation of dopamine receptors and the promotion or repression of angiogenesis, however research is still ongoing (Ferrera, 2009; Vaughn et al., 2017).

Interestingly opposing observations on the activation of dopamine receptors in regards to

VEGF expression have been noted. Activation of D1 dopamine receptors in diabetic mice lead to a marked increase in VEGF expression which promotes angiogenesis, and decreased wound closure time (Chakroberty et al., 2016). Specifically, researchers observed complete wound closure at 11 days in experimental mice, whereas control mice had only 42% wound closure; a similar effect was also observed in human dermal fibroblasts. These observations show a potential pathway to develop treatments to improve wound healing through stimulation of D1 receptors through a topical application or even injection at the wound site. However, other studies reported that dopamine receptor activation decreased the production of VEGF, further illustrating that more study is required (Ewing et al., 1983; Basu et al., 2001; Vaughn et al., 2017). Conversely, using a D2 family agonist eticlopride, wounds in mice were shown to have augmented wound healing in comparison with controls, highlighting the divergent nature of both receptor types when activated (Shome et al., 2011). Further studies on the downstream pathway of eticlopride antagonism on D2 receptors showed an increase in the transcription factor

HoxD3, a transcription factor integral for angiogenesis (Uyeno et al., 2001). Taken as a whole, these findings demonstrate that dopamine’s activation of these receptors can be pro-wound healing through the expression of important factors such as HoxD3.

Interestingly, other findings have shown that dopamine can affect other aspects of wound repair through different pathways. Dopamine receptor activation has been shown to affect cAMP levels and decrease mitogenic behavior in keratinocytes (Harper and

Flaxman, 1975; Vaughn et al., 2017). Specifically, activation of D2 receptors in skin cells decreased keratinocyte proliferation (lowered cAMP levels) and use of the antagonist L-741626 had an opposite effect (Fuziwara et al., 2005). Conflicting results in regards to promotion of wound healing have been documented. For example, wound closure in epithelial tissue was increased with the activation of D2 receptors but use of agonists such as L-741626 hampered this recovery (Aoyagi et al., 1981). As these results highlight cAMP levels affecting the skin barrier recovery, it has been hypothesized that as increasing cAMP lowered barrier recovery, drugs which lowered cAMP function, turnover or synthesis could restore wound healing (Ramachand et al., 1995; Vaughn et al., 2017).

Taken as a whole, catecholamines have a number of effects in regards to wound healing. Epinephrine causes delayed wound migration for keratinocytes and inhibits neutrophil trafficking to the wound site; whereas norepinephrine has been shown to deter phagocytosis and proliferation in macrophages when bound to B2ARs. Stimulation by both Epi and NE were shown to have generally negative effects on the cell types whereas dopamine’s effects on wound healing were not as clear. When bound to D1 receptors, dopamine was shown to promote angiogenesis through VEGF expression which propels wound healing. However other studies have demonstrated that when bound to D2 receptors, keratinocyte proliferation was decreased, which would delay wound healing.

Further insight is needed to better understand what other roles dopamine has, and whether its binding to β2ARs is negative or positive with regards to wound healing.

Utilization and Production of Catecholamines by Bacteria

The primary focus of this section is to highlight the utilization and potential production of catecholamines by the bacterial species that inhabit chronic wounds, as well as to consider whether or not catecholamines play a role in bacterial survival or propagation. As previously discussed, catecholamines are synthesized in the adrenal gland, in chromaffin cells, and are supplied to the rest of the body. Normal levels of catecholamines can vary; epinephrine for example is present at 10–900 pg/mL in plasma due to the variance of body weight and other factors in humans (Rayamondos et al.,

2000). Levels of catecholamines can be raised by ten times the normal amount during exercise, and most notably, by fifty times during stress (Boyanova, 2017; Robertson et al., 1979). It should also be noted that catecholamines can be toxic in high concentrations, as catecholamines have been shown to degrade neuronal tissue in mice at 25 µM, interestingly this was not related to adrenergic receptor stimulation. Researchers reported that damage due to catecholamine toxicity was reminiscent of hydrogen peroxide damage

(Rosenberg, 1988). In a wound, bacteria have the chance to invade and propagate within the human body, and thus have access to any nutrients or other biomolecules that may be in the vicinity.

A wound provides easy access to valuable nutrients for bacteria. In comparison with other routes of entry, the skin barrier is compromised when a wound is inflicted which allows for the opportunity for bacteria to invade. Since catecholamines are within the wound area, bacteria could interact in some manner that could affect their survival.

Research by various laboratories have shown that E. coli have higher levels of growth in the presence of catecholamines, but how the bacteria utilize the catecholamines was unknown (Lyte and Ernst, 1992; Lyte, 1993) Specifically, with NE and its metabolites dihydroxymandelic acid (DOMA) and Dihydroxyphenylethylene glycol (DOPEG), E. coli experienced a threefold increase in the number of colonies found during growth studies (Lyte, 1993). Furthermore, the Beta-ketoadipate pathway represents a method for bacterial species to break down aromatic structures like catecholamines into beta- ketodiapate suggesting that bacteria do have mechanisms to utilize catecholamines

(Harwood and Parales, 1996).

The response of E. coli growth to catecholamines may to be due to an increased ability of iron uptake by the bacteria. Iron can be used in cellular processes such as ATP generation, or oxygen transport. Bacterial access to iron is limited, as it is usually complexed by host proteins, and is present in extremely low concentrations in its free state. Uptake of free iron allows for E. coli to use the element to drive metabolism and enhance growth. Siderophores are compounds secreted by bacteria which are able to chelate extracellular iron and transport it across their cellular membrane. It has been proposed that free NE stimulates release of iron bound to the host protein transferrin for cellular uptake. Transferrin is responsible for controlling the level of free iron within the host’s biological fluids. In addition to iron release, NE upregulates synthesis of the siderophore enterobactin to then capture the NE-freed iron for E. coli. Enterobactin is one of the strongest siderophores, forming a tight complex with the free iron, then allowing

the iron to be brought into the bacterial cell through active transport. Increased iron uptake through would likely increase bacterial viability. However, further experimental evidence is still needed to support these claims (Burton et al., 2002; Dong et al., 2016).

The ability for bacteria to hijack the host’s biomolecules and nutrients can prevent wound healing through prolonging the inflammatory stage by sustaining the infection. Although E. coli was shown to respond to catecholamines, it was not known if other species shared the same type of response. A study by Belay et al. (2003) observed how gram negative pathogenic species of bacteria responded to catecholamines in vitro.

Bacteria were selected based on their natural occurrence in the GI tract and association with gastrointestinal infections. It was found that with all species in the trial (i.e.

Enterobacter sp., Salmonella enterica ser. Typhimurium, Shigella boydii, Shigella sonnei, Porphyromonas gingivalis and Bacteroides fragilis) there were no significant increases in growth with response to the addition of catecholamines. Interestingly other reports have noted that catecholamines have been able to increase motility and colonization for S. enterica subspecies Choleraesuis. This was also noted to occur in E. coli as well as to elicit an increased expression of Shiga-Toxin, and attachment to eukaryotic cells. An increased production of the toxin would allow for these bacterial species to toxify host cells, thus allowing them to utilize their host resources (Bansal et al., 2007; Bearson et al., 2008; Chen et al., 2003).

Since bacteria can respond to catecholamines, researchers have inquired as to whether or not it is possible for any bacterial species to produce catecholamines. Studies

have examined the gut microbiome of individuals who may likely have an overabundance of stress related compounds such as in individuals that have Attention Deficit

Hyperactivity Disorder (ADHD) (Aarts et al.,2017). In ADHD patients, an upregulation of the bacterial gene encoding cyclohexadienyl dehydratase (CDT) was identified, which is an enzyme involved in the production of phenylalanine (a precursor which can be converted into dopamine, norepinephrine and epinephrine). Furthermore, it was also discovered that the makeup of the microbiome differed between ADHD and non-ADHD individuals. ADHD patients showed a small increase in the amount of the bacterium

Bifidobacterium compared to controls, which has been proposed as an age-related hallmark of the ADHD disorder. In comparison with normal patients, CDT was upregulated 150% percent more in the gut microbiome compared to non-ADHD patients when corrected. The upregulation of CDT and phenylalanine provides a possible indication that dopamine can originate indirectly from bacterial species in that they have the capability to induce production of dopamine in the human host. The authors assert that as CDT expression is upregulated, phenylalanine is produced, (which cannot be produced by humans) and then is able to be utilized in the dopaminergic pathway (Aarts et al., 2017).

If bacterial species produce catecholamines, it is likely they would be found in relatively high levels in the intestinal lumen given the density of bacteria inhabiting this lining. Studies have shown several bacterial species can indeed produce dopamine and norepinephrine such as Bacillus cereus, B. mycoides, B. subtilis, (Kruk, and Pycock,

1990); and E. coli, Tsavkelova et al., 2000). These comparative quantitative studies show the ability of microorganisms to synthesize catecholamine compounds in equal or greater relative concentrations to that found in multicellular organisms. This suggests that microbes and eukaryotes may either share common catecholamine synthesis pathways with eukaryotes or have developed alternative pathways by way of convergent evolution.

However, evidence for the actual mechanisms of synthesis is still lacking, and further research is required (Yano et al., 2015).

In order to better understand the bacterial mechanisms for bacterial catecholamine synthesis, research has focused on bacteria that inhabit the gut microbiome. Asano et al.

(2012) sought to try and find out what role gut bacteria had in catecholamine synthesis using a mouse model. Lumen contents were collected and processed from sacrificed mice and analyzed. Initial results showed that in mice that were kept free of germs (GF), a significantly lower concentration of biologically active catecholamines were identified in comparison with control mice that had bacteria. As the only difference between the two types of mice was the presence of gut bacteria, it was reasoned that the gut bacteria were responsible for the higher levels of catecholamines to be present in the intestine.

However, it is also possible the presence of the bacteria caused the host to produce elevated levels of catecholamines.

With bacteria being the possible culprit, it was hypothesized that the bacterial molecule β-glucuronidase (GUS) could be the contributing factor for the higher levels of catecholamines in the intestine. Members of the GUS family of enzymes are involved in

the breakdown of complex carbohydrates in humans and in bacteria, and also have been associated with deconjugation of catecholamines. Catecholamines can be conjugated to a variety of groups such as sulfates to ensure stability. This deconjugation pathway represents a possible avenue for bacterial species to activate catecholamines for their use.

Significantly higher levels of catecholamines were detected all throughout the intestine of wild-type E. coli inoculated mice than with GUS deficient E. coli (via gene knockout), supporting the notion that bacteria play a critical role in the generation of activated catecholamines in the host intestine. However, these researchers were unable to discern whether these catecholamines were derived from the bacteria or from the host originally.

Based on these observations, researchers have hypothesized the following pathway for deconjugation: 1) catecholamines are absorbed in the gut during food ingestion, 2) catecholamines are then conjugated to compounds in the liver, 3) conjugated catecholamines are then released back into the gut via the bile duct, 4) and finally the catecholamines are then deconjugated by bacterial-derived GUS and absorbed by the bacteria (Asano et al., 2012)

As the lumen is relatively rich in NE and DA (48.7 and 132.2 ng per gram of luminal contents respectively), it has been hypothesized that bacterial species have evolutionarily favored these two catecholamines over epinephrine, since epinephrine has not been reported within the GI tract. Researchers found that E. coli, and Salmonella responded to NE by increasing growth by 40 percent whereas epinephrine has no effect

(Goldstein et al., 2003; Furness, 2006). Summarily, these findings show that bacteria can

respond and utilize catecholamines for survival. It is possible that treatments could target the uptake and use of catecholamines to prevent their persistence in the context of wound healing.

Catecholamines may also cause an increase in horizontal gene transfer (HGT) rates which is a major mechanism of conferring resistance to antibiotics. Peterson et al.

(2011) looked at this very question within Salmonella typhimurium by examining the transfer of plasmids to E. coli. It was observed that in the presence of NE at physiological levels (5 µM), conjugation incidence rose significantly 2-6 hours post exposure. With

HGT rates increasing threefold due to NE exposure, antimicrobial resistance genes or genes involved in the production of catecholamines could be transferred to other bacteria.

HGT could also affect the chronicity of wounds by allowing for bacterial species to thrive by the transfer of any beneficial genes that could improve their chance for survival

(Peterson et al., 2011).

In addition, bacterial biofilms are another factor in promoting chronic wounds.

Biofilms are a mucus-like secretion composed of polysaccharides, proteins, lipids and

DNA. Biofilms are secreted by bacteria adhering to surfaces such as plastic in medical instruments or extracellular matrices such as collagen or hyaluranonan (Birkenhauer et. al., 2014). In a wound that has a secreted biofilm, it has been discovered that two types of bacteria are present; 1) free floating or planktonic bacteria, and 2) sedentary bacteria that produce biofilms once they have adhered to a surface. Factors such as availability of nutrients can influence whether or not a bacterial cell will adhere to a surface or not.

Although there has been speculation on the effects of catecholamines on these secretions, observations and hypotheses indicate that it may be possible that the presence of catecholamines and other environmental factors may induce bacteria to enter a “biofilm forming state”. However, there is much more research that must be done to better understand the interaction of catecholamines and bacteria to support this notion. Research has shown that planktonic bacteria undergo a change in gene expression which prompts them to exit the free-floating state and adhere to surfaces (Williamson et. al., 2012).

Through microarray analysis, it has been confirmed that planktonic and sedentary bacteria differ both on a transcriptional level, and at a metabolic level (Williamson et. al.,

2012). Catecholamines may be able to induce the change from one state to the other, but as stated previously, more research is required to establish this connection.

In addition to being implicated in biofilm production and being present in wounds, S. aureus has also been shown to deter keratinocyte viability, and even inducing keratinocyte apoptosis when in a sedentary mode through the invasion of host cells

(Kirker et al., 2012; Edwards et al., 2011). By being able to invade and kill host cells, such as keratinocytes, they can directly impede wound healing. In addition, the presence of S. aureus biofilms has been shown to induce expression of inflammatory cytokines thereby prolonging wound healing (Kirker et al., 2012).

In summation, it is clear that some bacteria can respond to catecholamines from the host that may exist within the gut and wound environment. Primarily, studies have shown that the presence of catecholamines can induce upregulation of siderophore

production for iron capture to allow for increased survival, increase motility of select species, increase growth rates, and increase the incidence of HGT. In addition, dopamine and norepinephrine can also be produced by select bacteria. Bacteria may also secrete biofilms in response to catecholamines, however further research is required to better understand this interaction.

Summary

Over the last ten years there has been a significant amount of research conducted on the role of catecholamines and their contribution toward delaying wound healing and chronic wounds. This topic represents a dire area that requires even more attention. As the United States has a growing elderly, and diabetic population, the need for a more comprehensive understanding is paramount in order to address the needs for wound treatment, and prevention of chronic wounds. In particular, future studies are needed to address the amount of catecholamines that are found in wounds naturally and where they originate from. There are currently a variety of SPE methodological approaches to purify catecholamines from biological sources and many viable separation and detection methods to tackle these questions. In regards to the separation and detection of catecholamines, the golden standard is still HPLC-ECD due to its ease of use, sensitivity, and automation which can allow for even a small research group to obtain accurate, reliable results in a relatively short amount of time. However, there are other methodological advances such as ICFO-LED-IFD which could help advance the field.

What is known about catecholamines and their involvement in delaying the wound healing process has also advanced over the last decade. The molecular and cellular mechanisms as to how catecholamines bind to adrenergic receptors and influence changes in the behavior of keratinocytes, neutrophils, macrophages and neutrophils has allowed a more comprehensive understanding of the negative effects that epinephrine and norepinephrine can have on the wound healing process. In particular, β2AR stimulation

by epinephrine appears to be a major factor for wound chronicity. Further studies exploring the utility of beta adrenergic blockers in preventing the delay of wound healing will surely be a promising research avenue. Beta blockers could prevent the prolonged inflammatory phase experienced in many chronic wound patients by allowing proper migration of keratinocytes during epithelialization, white blood cell phagocytosis, decreased inflammatory cytokine production, and proliferation of fibroblasts.

There is still more to be understand about all the responses of various cell types that are applicable to wound healing to epinephrine, norepinephrine and dopamine.

Future studies on norepinephrine and dopamine would be helpful in further elucidating their role. In particular, it is not clear as to whether dopamine is pro-healing or contributes to the delay of healing. In addition, it will be also important to further understand the response of bacteria to catecholamines in promoting a chronic wound setting. As mentioned previously, bacteria can respond to catecholamines by proliferating, toxifying host cells, and chelating iron for their own cellular uptake. In addition, it has been shown that NE and DA are produced by select species of bacteria thereby contributing to the chronic wound environment and promoting their survival.

Currently, there is no evidence that bacteria can produce epinephrine. It is also intriguing that catecholamines can stimulate horizontal gene transfer among bacteria which would certainly promote the transfer of genes that could improve their survival such as through the transfer of antibiotic resistance genes. In summary, even though we have learned much about catecholamines in the wound healing process, there is much still to be

understood. In regards to future research, it will be important to more fully understand the cellular and genetic response to catecholamines of cells involved in wound healing.

Current research has shown that epinephrine, norepinephrine and dopamine certainly do affect the wound healing process, but their exact role needs to more fully elucidated. A complete understanding of the role of catecholamines will certainly lead to viable therapeutic approaches to improve healing rates, especially among the growing population of elderly and diabetic patients.

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