Signalling Underlying Wound Healing

Abstract

Wound healing is critical for normal tissue function and the survival of most multicellular organisms. Understanding the biochemical and cellular mechanisms underlying constructive physiological wound healing is crucial for developing therapies to aid in repair and regeneration. Wound healing involves multiple systems including blood, vasculature, somatosensory, immune system and the skin. Following vascular damage, coagulation system activation occurs resulting in clot formation and cell signalling events in both cells of the vasculature (e.g. endothelial cells) as well as circulating cells (e.g. platelets, leukocytes) in order to drive cessation of blood loss. Pain signalling is initiated in part by surrounding inflammatory cues and is used to prevent further damage to the wound. Following these initial responses to the wound, healing/regeneration returns the damaged tissue to a functional state. This occurs by engaging the fibrin network to create a new microenvironment architecture. A broad overview is presented of how the different organ systems are integrated to repair tissue after a wound.

Key Concepts

  • Blood coagulation is driven by exposure of the subendothelial receptor tissue factor that drives activation of downstream clotting factors, including activation of the central protease thrombin. Platelet activation is mediated by thrombin cleavage of cell surface protease‐activated receptors, tight receptor‐mediated binding to collagen or activation of the purinergic receptors (i.e. P2Y1, P2Y12). In addition to platelet activation, thrombin catalyses the conversion of fibrinogen into fibrin, the primary structural component of the blood clot.
  • Immune cells like mast cells and macrophages are recruited and activated once a wound occurs, releasing cytokines and histamine. This inflammatory response aids in promoting pain signalling and eventually aids in the wound healing process.
  • In response to the wound and release of inflammatory factors (e.g. cytokines, histamine, bradykinin, NGF and PGE2) sensory neurons become hypersensitive. This hypersensitisation (hyperalgesia) leads to innocuous tactile information to be interpreted as painful by both the peripheral nervous system (PNS) and central nervous system (CNS).
  • Numerous stem cell populations derived from the dermis and hair follicle contribute to wound remodelling. These stem cells differentiate into cell types like myofibroblasts, which either repopulate the site of the wound and/or deposit new extracellular matrix proteins to form a fibrotic scar.
  • A balance between Wnt and TGF‐β signalling guides regenerative and healing processes, respectively. Wnt signalling leads to cell proliferation and the reappearance of hair follicles inducing wounds to heal without a scar. TGF‐β signalling induces reticular dermis stem cells to differentiate into myofibroblasts, which leads to scar tissue.

Keywords: wound healing; cell signalling; haemostasis; vascular repair; pain; inflammation; nociceptors; scar tissue; fibrosis; regeneration

Figure 1. Wound healing overview. Wounds spontaneously heal by stopping blood‐flow (a), inducing pain and inflammation (b), and finally remodelling the wound site (c). Clotting factors, neuropeptides, cytokines, and numerous stem cell populations play key roles in this process.
Figure 2. An overview of platelet binding and activation. Platelets bind to extravascular collagen and serum‐derived VWF, activating integrins which bind to fibrin(ogen), stabilising platelet adhesion. The release of clotting factors from platelets following aggregation drives rapid, local fibrinogen hydrolysis by thrombin.
Figure 3. Noxious stimuli induce the release of chemical factors (e.g. cytokines, NGF, histamine and bradykinin) from immune cells (e.g. mast cells and macrophages), which bind to the first order neuron in the PNS. The first order neuron synapses with the second‐order neuron in the dorsal horn of the spinal cord, releasing excitatory neurotransmitters (e.g. glutamate and substance P). This transmits pain information from the spinal cord to the brain, resulting in hyperalgesia and inflammation.
Figure 4. First Responders: First Order Neuron. (a) When mast cells are activated, they release histamine, which can act in an autocrine manner by stimulated more mast cells, and paracrine manner by stimulating nerve endings (Gupta and Harvima, ). There are four main G‐protein coupled receptors responsible for histamine binding, H1R‐H4R, and once activated, stimulate the influx of Ca2+ into the neuron (Bell et al., ). TNFα binds to TNFR1/2, upregulating the COX‐2 pathway, leading to more PGE2. IL‐1β is expressed in nociceptive neurons and can increase the production of substance P and prostaglandin E2 (PGE2), aiding in neural sensitisation and the transmission of pain to the brain (Zhang and An, ). This interleukin has a specific receptor antagonist, IL‐1ra, that will bind to the same receptor but won't transduce any signals, blocking IL‐1β from inducing inflammation. (b) Once NGF binds to Tropomyosin receptor kinase A (TrkA), a receptor tyrosine kinase located on the nerve ending of the first order neuron, there are two different pathways that lead to TrpV1 sensitisation; through PI3 kinase (PI3K) activation and through phospholipase Cγ (PLCγ)activation (Zhang et al., ). PI3K helps to activate Src downstream, which phosphorylates TrpV1. PLCγ activates PKCϵ, which also phosphorylates TrpV1. Once activated, TrpV1 pumps Ca2+ into the neuron, leading to an increase in substance P and glutamate. (c) Once bradykinin binds to B1, phospholipase Cβ (PLCβ) is activated triggering PKCϵ, which then sensitises TrpV1 resulting in hyperalgesia (Djouhri et al., ; Okuse, ). Along with activating PLCβ, B1 will also activate phospholipase A2 (PLA2) (Okuse, ). This phospholipase will help produce arachidonic acid (AA) from the phospholipid bilayer. AA is converted to prostanoids like prostaglandin E2 (PGE2) by the COX‐2 pathway. Once released, PGE2 binds to G‐protein coupled exchange protein receptors (EP2), which then activates adenylyl cyclase (AC) (Huang and Gu, ). This will then catalyse the conversion of ATP to cAMP which normally leads to the activation of protein kinase A (PKA). With the increase of all of these proinflammatory factors, there is an increase in pain and inflammation in the body.
Figure 5. Damage to C and Aδ fibres lead to the release of glutamate, a major excitatory neurotransmitter, and results in neuropathic pain (Jiang et al., ). Once released from the first order neuron, glutamate acts on postsynaptic glutamate receptors, releasing sodium ions into the second order neuron (Okuse, ). This results in the pain signal finally travelling up the neuron through the spinal cord, and into the brain, causing central sensitisation. Substance P (SP) participates in the neurotransmission of noxious stimuli in the form of pain and can be found in first‐order neurons in the PNS (Widgerow, ; O'Connor et al., ). SP acts by binding to the neurokinin‐1 receptor (NK‐1R) in the synapse between the first and second order neurons in dorsal horn of the spinal cord (O'Connor et al., ). SP can also induce neovascularisation and vasodilation by stimulating the release of histamine and cytokines from mast cells and macrophages, respectively (Widgerow, ). The inflammatory response can be amplified through SP‐induced release of certain inflammatory mediators like cytokines (TNF‐α and IL‐1β), arachidonic acid derivatives (PGE2) and histamine (O'Connor et al., ). Once the ascending pain pathway garners enough action potential to go from peripheral sensitisation to central sensitisation, a negative feedback loop is activated. The descending pain pathway can act as an inhibitory pathway, shutting off the pain signalling if necessary, or increasing the pain output. One way that the pain signal is inhibited is through gamma‐aminobutyric acid (GABA) release (Jiang et al., ). The inhibitory interneuron releases GABA, which binds to the GABA receptor on the second order neuron. This drives Cl into the cell, which blocks Na+ from increasing the action potential of the neuron, aiding to shut down the pain transmission to the brain.
Figure 6. The role of Wnt signalling and TGF‐β1 in wound remodelling. (a) Cells from the papillary dermis primarily participate in Wnt signalling, which promotes regeneration of healing wounds. β‐catenin is constantly degraded by a destruction complex when Wnt signals are not present (Komiya and Habas, ). The destruction complex is composed of two adaptor proteins, axin and adenomatosis polyposis coli (APC), which localise two protein kinases, CK1 and GSK‐3. With no Wnt, the kinases phosphorylate β‐catenin, targeting it for ubiquitination and degradation at the proteasome. Wnt binds to the Frizzled receptor (Fz), a seven‐pass transmembrane protein and lipoprotein receptor‐related co‐receptor (LRP), a single‐pass transmembrane protein. LRP is phosphorylated by either CK1 or GSK‐3. Axin binds to phosphorylated LRP. Dishevelled protein (Dsh) binds Fz and axin upon Wnt binding and inhibits GSK‐3 (Mlodzik, ). The transduction of the Wnt signal both inactivates the destruction complex and colocalises its components to the cell membrane. β‐catenin then evades degradation and enter the nucleus, causing transcription. (b) Cells from the reticular dermis are involved in TGF‐β1 signalling, which promotes scar formation and fibrosis. The receptor, type‐II TGF‐β receptor (TGF‐βrII) dimerises in the presence of TGF (Border and Noble, ). TGF‐βrII dimerises, recruiting a second TGF‐βr (TGF‐βrI) that also has an intracellular serine/threonine kinase domain. TGF‐βrII phosphorylates the TGF‐βrI, recruiting SMAD. SMAD is oriented by the lipid‐anchored chaperone, SARA (SMAD Anchor for Receptor Activation). Phosphorylated SMAD binds to another SMAD protein, referred to as the co‐SMAD, which then enters the nucleus to perform transcription.
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Cook, Sophie R, Hart, William S, Zatorski, Jonathan M, Flick, Matthew J, and Deppmann, Christopher D(Sep 2019) Signalling Underlying Wound Healing. In: eLS. John Wiley & Sons Ltd, Chichester. http://www.els.net [doi: 10.1002/9780470015902.a0028825]