What do a scraped knee, a paper cut, or any form of surgery have in common?
The short answer is a wound in need of healing – but the long answer lies in a series of biological activities that allow tissues to repair themselves.
Questions about wound healing have been asked for more than 1,000 years. Now, scientists have begun decoding how signaling proteins are intimately involved in a process that transforms an open wound into remodeled healthy tissue.
While knowing more about simple wounds is important, the research underway at Virginia Commonwealth University also has a larger aim – helping to solve extraordinarily complex medical problems.
Inflammatory disorders such as sepsis, anaphylaxis and trauma-induced coagulopathy might eventually be aided by research into wound repair. Trauma-induced coagulopathy refers to the blood’s inability to coagulate – form clots.
The impairment, caused by severe trauma, can lead to prolonged or excessive bleeding, which can prove fatal.
Wounds heal through a dynamic process that involves various molecular pathways and cell types that are coordinated to facilitate healing in a timely and orderly manner, Dr. Charles E. Chalfant and colleagues write in the journal Science Signaling.
“One of the key players in this orchestra is a group of molecules called eicosanoids, which are derived from fatty acids, and control both inflammation and the migration of cells around the wound site,” reported Chalfant, who heads the lipid signaling research laboratory at Virginia Commonwealth University.
Eicosanoids not only are fatty-acid-derived molecules, as Chalfant and his colleagues have found, they are also signaling proteins. Signaling molecules are critical components of cells that allow short and long distance communications. Imagine a microscopic network that is roughly comparable to a telephone system through which calls are relayed and received.
As it turns out, eicosanoids steer two key stages of wound repair, Chalfant his team of investigators discovered in a groundbreaking investigation that has implications for surgical patients, accident victims, and possibly one day for those with potentially deadly inflammatory disorders. H. Patrick MacKnight, a colleague of Chalfant at Virginia Commonwealth University and the molecular biology division of the University of South Florida in Tampa, served as first author on the new report.
Wound repair, the team revealed, advances through distinct phases, which includes inflammation followed by proliferation and tissue remodeling. The team reported in Science Signaling that fibroblasts infiltrate the wound, proliferate, and secrete collagen, which is remodeled as the epithelium regenerates.
In their observations of laboratory animal models – mice that were assessed on a variety of wound-repair parameters – the scientists were able to document each step. They also noted the importance of eicosanoid signaling, which is crucial to the repair process.
Eicosanoids have to be produced by the body, according to Chalfant and his colleagues. They say the sphingolipid, ceramide 1-phosphate, binds to and activates group IVA cytosolic phospholipase to stimulate the production of eicosanoids.
“Because eicosanoids are important in wound healing, we examined the repair of skin wounds in knockout mice,” Chalfant wrote. The knockout (KO) mice were compared with their “knock-in” (KI) counterparts. The knockouts lacked the protein cPLA2α, but the knock-ins were endowed with it.
The wound closure rate was not affected in the KO or KI mice, but wound maturation was enhanced in the KI mice. Their wounds displayed increased infiltration of dermal fibroblasts, increased wound tensile strength and several other noteworthy differences compared with their KO counterparts.
The research is part of active area of inquiry in Chalfant’s laboratory, which is examining how lessons learned about inflammatory processes in wound repair can help shed new light on more complex medical conditions.
Eicosanoids, for example, are known mediators of inflammatory responses. The molecules underlie the pathogenesis of sepsis and other major medical disorders.
With sepsis mortality exceeding more than 200,000 people in the United States annually, it is time to look at it from a fresh perspective, according to Chalfant’s laboratory mission statement. Knowledge gained from wound repair research is a step in a new direction, he says.
Structure and Function of the Skin
The skin provides a life-protective barrier between the body and the external environment against physical damage, pathogens, fluid loss, and has immune-neuroendocrine functions that contribute to the maintenance of body homeostasis .
Its structure is composed of two layers: the epidermis and the dermis. The epidermis contains keratinocytes, melanocytes, dendritic cells, Langerhans cells and other immune cells, sensory axons, and the epidermal-dermal basement membrane [2, 3].
The dermis has the skin appendages, mast cells, fibroblasts, antigen presenting dermal cells, resident and circulating immune cells .
Additionally, the dermis includes the extracellular matrix complex that provides support to intercellular connections, cellular movement, and regulates cytokine and growth factors’ functions.
Skin innervation consists of a dense network of sensory and autonomic fibers that form tight junctions with keratinocytes and transmit sensations of pain, temperature, pressure, vibration, and itch .
Skin circulation is composed of parallel arterial-venous thermoregulatory shunt circulation controlled by tonic adrenergic sympathetic vasoconstrictor and vasodilator nerves that give origin to a subepidermal capillary network that provide oxygen and nutrients to the epidermis and remove CO2 and waste products .
The lymphatic vessels of the skin consist of lymph capillaries that run horizontally under the epidermis, followed by precollector vessels located deeper in the dermis and lymph collecting vessels in the subcutaneous fat layer. Lymph vessels are connected to the skin local draining lymph nodes, and lymph vessels that exit these lymph nodes converge to the regional sentry lymph nodes before reaching the thoracic duct [7, 8].
The Healing Process
After injury, skin integrity must be promptly restored in order to maintain its functions. In this process, peripheral blood mononuclear cells, resident skin cells, extracellular matrix, cytokines, chemokines, growth factors, and regulatory molecules participate in the wound healing process.
The intricate skin repair process has been organized in three sequential and overlapping steps: the inflammatory phase, the proliferative phase, and the remodelling phase. The inflammatory phase includes cutaneous neurogenic inflammation and hemostasis; these early events start in the first seconds after injury and last approximately 1 hour. Followed by the fast recruitment of neutrophils to the injured tissue during the first 24 hours and its posterior decline during the subsequent week.
The progressive infiltration of inflammatory monocytes-macrophages to the wound starts the second day after injury and continues to increase, reaching its maximum during the proliferative phase, starting its decline during the following two weeks, becoming the dominant mononuclear cell in the tissue repair process. Circulating lymphocytes migrate to the skin early after injury reaching a plateau by day 4 and their presence continues for two more weeks before declining.
The last phase starts in the second week after injury and includes remodeling the tissue previously formed in the proliferation phase and the organization of a scar in order to restore the skin integrity. This last stage could last for months. This review provides present day information regarding the central role of the resident and peripheral immune cells as well as the microenvironment and their interactions during the wound healing process.
The Inflammatory Phase (Alarm and Stop the Damage)
Cutaneous Neurogenic Inflammation
The peripheral nervous system is among the first to respond to a skin injury. Skin cell damage activates transient receptor potential channels TPRV1 and TPRA1 present in primary sensory neuron endings and in other cells such as keratinocytes, mast cells, dendritic cells, and endothelial cells which act as nociceptive receptors .
Injury stimulation of sensory neurons generates action potentials that travel orthodromically to the spinal cord initiating pain. Action potentials start the axon reflex by traveling antidromically in other axonal branches of sensory nerve endings promoting the release of substance P and calcitonin gene-related peptide from sensory nerve endings .
These neuropeptides have three targets: (a) in blood vessels, CGRP act on microvascular smooth muscle fibers promoting vasodilation and increased blood flow, (b) SP causes vascular permeability, edema, and recruitment of inflammatory leukocytes, and (c) SP stimulates mast cells degranulation with discharge of histamine, serotonin, proteases, and other mediators [9, 11–13], promoting increased microvascular permeability of the blood vessels encircling the wound (redness and warmth)  and facilitating the extravasation of fibrinogen and other plasma derived factors that serve as chemoattractants for the influx of inflammatory cells into the wound (swelling) [9, 15, 16].
Additionally, the release of histamine from mast cells triggers the release of substance P and CGRP from sensory nerve endings, implementing the bidirectional link of cutaneous neurogenic inflammation  (see Figure 1). The peripheral nervous system continues to have regulatory interactions with mast cells , monocyte-macrophages [18, 19], Langerhans cells , and lymphocytes [21, 22], as well as microvascular, and other local skin cells during the distinct phases of skin wound healing .
There are about 160,000-400,000/μl blood platelets, being the second most abundant cells after erythrocytes. An average healthy adult produces 1011 platelets per day that circulate around 10 days. Platelets retain many of the RNA metabolic processes of nucleated cells. They contain large amounts of noncoding RNAs, including microRNAs and long noncoding RNAs, and utilize postranscriptional mechanisms to preserve its proteome of approximately 4000 proteins .
After they are released into the blood, the progressive degradation of the antiapoptotic protein Bcl-xL determines the lifespan of platelets in the blood, and at the end of their life, they are removed from the circulation in the liver and spleen [25, 26]. Under normal physiological conditions, platelets do not interact with the endothelial surface. Blood constituents tend to migrate toward the center of the blood flow but, given the small size of platelets, they are forced to circulate marginally toward the wall, where the glycocalyx barrier impedes their contact with the endothelial surface [27–29].
Vascular injury exposes the basement membrane proteins and the macromolecules of the extracellular matrix . Platelet membrane surface receptors bind to collagen, activating platelets and producing thrombin that catalyze the initiation of the coagulation cascade . Platelet integrins binding to fibrinogen give origin to fibrin [32, 33] that accumulates with the interstitial collagen, trapping neutrophils, erythrocytes, and other blood components forming the clot [34, 35]. A provisional extracellular matrix is formed by fibrin monomers forming fibrin protofibrils that are stabilized by intermolecular links through the action of Factor XIIIa.
In vitro studies suggest that fibrin fibers connect to native collagen type I fibers with cells through αVβ3 integrins, and this extracellular provisional matrix is used by fibroblasts and endothelial cells to migrate and to promote protomyofibroblast-mediated contraction of the provisional extracellular matrix [36–38]. This initial extracellular matrix is further remodeled by metalloproteinases released from fibroblasts  and macrophages,  forming a new provisional extracellular matrix to support neutrophil and monocyte migration [41, 42].
Besides hemostasis, degranulation of alpha granules from platelets releases TGF-β that acts as an important chemoattractant for the recruitment of various types of immune cells including neutrophils and macrophages . Platelet cell surface receptors participate in cell-cell interaction and microbial recognition and in the release of growth factors such as PDGF, TGF-β1, FGF, and VEGF that interact with endothelial cells, neutrophils monocytes, dendritic cells, B and T cells, and natural killer cells, promoting neutrophil activation, pathogen detection, trapping, and modulation of the innate and adaptive immune responses [43, 44].
Conclusion and Perspectives
Experimental work of the last two decades has revealed the general steps of the wound healing process. All cells, tissues, cytokines, chemokines, and growth factors of the skin participate in the wound healing process, revealing redundant and pleiotropic functions and interactions in many of the cellular and extracellular participants in wound repair. Further understanding of this complex network will elucidate how skin cell interaction with the changing tissue microenvironment defines their phenotype in every stage of tissue repair. Present knowledge has revealed that when cells are healthy, the inflammatory phase is well orchestrated, lasting only a few days, and the following stages of tissue repair: reepithelialization of the wound, granulation tissue formation, wound contraction, and scar formation, proceed normally. However, when cells are dysfunctional, as in diabetes, the inflammatory process is extended, the integrity of the skin is not restored, and ulcer or pathological fibrosis occurs. Macrophages are the dominant cells present in all phases of tissue repair. They have an essential regulatory role and are therefore seen as important therapeutic targets to control the wound healing process in the future.
More information: H. Patrick MacKnight, et al. The interaction of ceramide 1-phosphate with group IVA cytosolic phospholipase A2 coordinates acute wound healing and repair, Science Signaling (2019) DOI: 10.1126/scisignal.aav5918