Neuropeptides: Title and subTitle BreakMediators of Inflammation and Tissue Repair? FREE

Successful repair of injured tissues requires diverse interactions between cells, biochemical mediators, and the cellular microenvironment.^{1 - 3} Much has been learned about the individual events that are involved in this process, but their integration is clearly far more complex than has been imagined, and the important role of neurogenic stimuli is only recently being recognized.

Neurogenic stimuli profoundly affect cellular events that are involved in inflammation, proliferation, and matrix, as well as cytokine and growth factor synthesis. Immune cells regulated by neuropeptides include lymphocyte subsets, macrophages, and mast cells. In addition, neuropeptides may affect the proliferative and synthetic activity of epithelial, vascular, and connective tissue cells. Furthermore, a close interaction between the nervous and the immune systems has become obvious.⁴

The peripheral nervous system (PNS), acting through neuropeptides, not only relays sensory information to the central nervous system (CNS) but also plays an effector role in the inflammatory, proliferative, and reparative processes after injury. These effects range from growth factor and cytokine responses to control of local blood flow. Neuropeptides mediate many of the actions important in tissue–nervous system communication.

We review the accumulated knowledge about the role of neuropeptides in inflammation as it pertains to tissue repair.

Neuropeptides constitute one of the largest families of extracellular messengers, having a long phylogenetic history. They can act as neurotransmitters, hormones, and paracrine factors.

In contrast to the classic low molecular weight neurotransmitters, neuropeptides are exclusively produced in the cell soma without local synthesis in nerve endings.⁵ In most instances, several different neuropeptides are encoded by a single continuous messenger RNA (mRNA), which is translated into 1 large protein precursor (polyprotein). Like other secretory proteins, neuropeptides or their precursors are processed in the endoplasmatic reticulum and then move to the Golgi apparatus to be processed further. They leave the Golgi apparatus within secretory granules and are transferred to terminals by fast axonal transport.⁶

In the PNS, neuropeptides occur in the perivascular terminals of noradrenergic (sympathetic) and cholinergic nerve fibers, as well as in the free nerve endings of primary afferent neurons.^{7 - 8}

Numerous neuropeptides are localized in nociceptive afferent nerve fibers, including thinly myelinated Aδ pain fibers and unmyelinated C fibers.⁹ Antidromic stimulation of these fibers induces the release of the stored neuropeptides, resulting in vasodilation, increased vascular permeability, and edema (neurogenic inflammation).^{8 ,10} These effects are not only restricted to the point of the initial stimulus but also can be observed in the surrounding area, indicating that the nerve impulses travel not only centrally but at the collateral branches they also pass antidromically to unstimulated nerve endings to cause release of neuropeptides (axon reflex).⁸

Two neuropeptides playing an essential role in repair mechanisms are introduced in more detail below. An overview of the most important neuropeptides of the PNS is given in Table 1.

Substance P (SP), an 11–amino acid peptide, is a member of a family of structurally related peptides called tachykinins, which are characterized by a conserved carboxyl terminal sequence of Phe-X-Gly-Leu-Met-NH₂ (in the mammalian forms of these peptides, X represents Phe or Val).¹⁸ Mammalian tachykinins are encoded by 2 distinct genes: the preprotachykinin (PPT)-A gene and the PPT-B gene. The PPT-A gene is transcribed to an mRNA precursor that undergoes alternative splicing to give rise to at least 4 forms, of which α– and δ–PPT-A mRNAs encode SP only, whereas β– and γ–PPT-A mRNAs encode both SP and neurokinin (NK) A.^{19 - 20} The PPT-B gene, which encodes NKB only, is expressed in the CNS but not in sensory neurons.²¹

Substance P is present in many areas of the CNS and PNS. In the periphery, SP is located especially in areas of immunologic importance, such as the skin, gastrointestinal tract, and respiratory tract.²² Substance P is synthesized in the dorsal root ganglia, from which it migrates centrally to the dorsal horn of the spinal cord and peripherally to nerve terminals of sensory neurons.²³

The tachykinins bring about their actions mainly by activating 3 primary types of receptors: NK1, NK2, and NK3. Each receptor has been defined pharmacologically by the rank order of potencies of tachykinins in bioassays and in radioligand binding studies.^{24 - 25} The pharmacological definition of these 3 receptors has been confirmed by the molecular cloning and heterologous expression of the genes encoding each receptor type. All 3 receptors are members of the superfamily of receptors coupled to G-regulatory proteins. Receptor stimulation leads to the activation of phospholipase C and thus to the generation of inositol triphosphate and diacylglycerol and to the release of Ca²⁺ from internal stores.^{26 - 27}

Substance P and other tachykinins are able to cause vasodilation because of direct actions on vascular smooth muscle and enhanced production of nitric oxide by the endothelium.^{28 - 29} In addition, SP can initiate increased vascular permeability and protein extravasation after tissue injury.^{22 ,30 - 31} Many of the inflammatory actions of SP, such as plasma leakage, are mediated by NK1 receptors, which are rapidly desensitized after exposure to agonists and then gradually become resensitized.^{11 ,32} The receptors are internalized after ligand binding, which may be a limiting factor in the inflammatory response.³³

Neurokinin 1 tachykinin receptors are expressed by neurons and glia in the CNS, neurons within the mesenteric plexus, smooth muscle cells, acinar cells, endothelial cells, fibroblasts, keratinocytes, and various types of circulating immune cells and inflammation-activated immune cells.^{26 ,34 - 35}

Calcitonin gene-related peptide (CGRP), a 37–amino acid peptide, is known to exist in 2 forms, α and β. In humans, they differ from each other by 3 amino acid residues.³⁶ α–Calcitonin gene-related peptide is encoded by the calcitonin gene. The expression of either CGRP in the CNS or calcitonin in the thyroid is tissue related. In contrast, β-CGRP is the sole biologically active product of a separate gene.^{36 - 37}

Binding sites for CGRP with properties consistent with those of receptors are present in central and peripheral tissue. Stimulation of CGRP receptors in various cells and tissue has been shown to increase intracellular cyclic adenosine monophosphate concentration and to activate adenylate cyclase.^{38 - 39} Pharmacologically, a division into CGRP₁ and CGRP₂ receptor subtypes has been proposed.^{40 - 41} Recently, 2 proposed CGRP receptors have been cloned.^{42 - 43} Conceivably, these findings will promote studies of how the CGRP receptors are to be classified.

Calcitonin gene-related peptide is present mainly in small sensory neurons partially colocalized with SP.^{8 ,44} Peripheral secretion of CGRP causes prolonged increases in blood flow.⁴⁵ Unlike SP, CGRP is not capable of enhancing vascular permeability on its own but potentiates the protein extravasation induced by tachykinins.^{46 - 47}

It has been recognized since the early part of the century that stimulation of afferent nerve fibers is associated with peripheral inflammatory responses such as vasodilation and plasma extravasation.⁴⁸ This observation has led to the notion that afferent neurons not only serve a sensory role but also take part in local effector systems that are involved in inflammatory responses to tissue irritation and injury.⁸ The hypothesis that neuropeptides act as a link between the immune and nervous systems has been supported by the demonstration of (1) a direct peptidergic innervation of primary and secondary lymphoid organs, (2) a close proximity between sensory nerve endings and immune cells, and (3) specific neuropeptide receptors on immune effector cells.^{4 ,49} It has become clear that neuropeptides are capable of interacting with virtually all components of the immune system.

A host inflammatory response is necessary to orchestrate tissue repair following injury.³ There is increasing evidence that neuropeptides participate in many of the inflammatory processes that are crucial for normal wound healing.

Polymorphonuclear leukocytes (PMNs) are the first inflammatory cells to enter the wound space from the intact microcirculation at the edge of the wound, peaking at 24 to 48 hours.^{2 ,50} Their main function seems to be the phagocytosis of bacteria and cellular debris to prevent wound infection. The presence of PMNs does not seem to be essential for normal healing of uncontaminated wounds.⁵¹

Adhesion to endothelial cells is an initial step in the recruitment of leukocytes to sites of inflammation. Tachykinins are capable of inducing adhesion of PMNs to the endothelium.^{12 ,52 - 53} Neurokinin 1 receptors are present on the endothelial cells of capillaries that become leaky in response to tachykinins.^{33 ,54} Stimulation of these receptors causes the rapid mobilization of adhesion molecules for PMNs (eg, P selectin) to the cell surface, presumably by increasing the intracellular Ca²⁺ concentration.⁵⁵ Furthermore, neuropeptides have the capacity to affect neutrophil transendothelial migration. Substance P has been shown to exert direct chemotactic actions on PMNs.^{56 - 58} There are conflicting data concerning the chemotactic capacity of vasoactive intestinal peptide (VIP) and somatostatin. These neuropeptides have been reported to both inhibit and stimulate neutrophil chemotaxis.^{59 - 60} In contrast, Carolan and Casale⁵⁶ were unable to show that VIP and somatostatin had any direct chemotactic effects. These neuropeptides may not directly affect neutrophil chemotaxis, but they do inhibit neutrophil chemotaxis induced via inflammatory mediators.

Monocytes appear at the site of injury within 48 to 96 hours.¹ In the wound, they mature into wound macrophages. Initially, macrophages participate in the inflammatory process and débridement; later, they play a pivotal role in regulating the proliferative phase through the release of growth factors and cytokines.⁵⁰

Several studies have been carried out to elucidate the actions of neuropeptides on monocyte and macrophage functions. Somatostatin and CGRP were found to prevent macrophage activation and to profoundly inhibit the ability of macrophages to produce hydrogen peroxide.^{61 - 62} Using allogenic monocytes as stimulator cells, Fox and coworkers⁶³ demonstrated that CGRP has the ability to inhibit the proliferation of peripheral blood mononuclear cells, suggesting that CGRP exerts a direct effect on the monocyte stimulator population. Somatostatin has been shown to have direct inhibitory effects on tumor necrosis factor α (TNF-α), interleukin (IL)-1β, and IL-6 secretion by lipopolysaccharide-activated monocytes.⁶⁴

Substance P seems to exhibit proinflammatory actions, including activation of arachidonic acid metabolism, chemotaxis, and oxidative burst.⁶⁵

There are contradictory reports concerning the potential of SP to affect the synthesis and release of cytokines in mononuclear phagocytes. In 1988, Lotz and coworkers⁶⁶ demonstrated that SP induced the release of IL-1, IL-6, and TNF-α from human monocytes. Similar results were obtained by Rameshwar et al,⁶⁷ who showed that SP mediated the release of IL-1 and IL-6 by bone marrow mononuclear cells. In contrast, Bahl and Foreman⁶⁸ demonstrated that SP did not cause either the release or the accumulation of IL-1 from murine peritoneal macrophages. Similarly, Lieb and coworkers⁶⁹ showed that SP and other neuropeptides were unable to induce the synthesis of IL-1 and IL-6 in human peripheral blood monocytes. These authors⁶⁹ suggested that undetected levels of endotoxin or lipopolysaccharide in the culture medium may have been primarily responsible for results suggesting an inductive effect of neuropeptides on monocytes and macrophages. However, the cited studies used different cell types and species. Possibly, there are differences in the activation requirements and in the sensitivity of mononuclear cells from different species and different tissues in their response to neuropeptides. Furthermore, the stage of differentiation and maturation of mononuclear phagocytes may be important for the ability of neuropeptides to render the cells sensitive to secondary stimulation, eg, by lipopolysaccharide, and determines to what extent monocyte and macrophage subpopulations contribute to inflammatory responses in vivo. In addition, the physical state of the animal from which the cells are recovered may have a profound affect on macrophage function in vitro. Chancellor-Freeland and coworkers⁷⁰ were able to show that stress alters macrophage functions and induces the expression of SP binding sites in peritoneal macrophages.

Results of investigations of the biochemical properties of the SP binding sites revealed that monocytes express a specific non–NK receptor for SP, which is functionally coupled to a glutamyl transpeptidase binding protein of the G_iclass.^{71 - 72} Triggering of this receptor results in stimulation of mitogen-activated protein kinase, mobilization of calcium, and activation of phospholipase D.⁷²

T lymphocytes, the second arm of the cellular immune system, appear in significant numbers in wound sites on about the fifth day.⁷³ They affect wound healing through the release of numerous chemical mediators.^{1 - 2}

Neuropeptides specifically bind to and modulate the function of lymphocytes. Substance P promotes T-lymphocyte endothelial cell adhesion by preferentially up-regulating lymphocyte function–associated antigen-1 and intercellular adhesion molecule-1 interactions.⁷⁴ In human T lymphocytes, SP and its C-terminal fragment SP_4-11 stimulate [³ H]-thymidine and [³ H]-leucine uptake in the presence and absence of other mitogens.⁷⁵ Proliferative responses of lymphocytes from spleen, mesenteric lymph nodes, and Peyer patches are enhanced by SP, whereas VIP and somatostatin significantly decrease DNA synthesis.⁷⁶ Furthermore, CGRP and VIP exert an inhibitory effect on the proliferative response of CD4⁺ and CD8⁺ T-murine lymphocytes and induce a rapid and dose-dependent increase in intracellular cyclic adenosine monophosphate.⁷⁷ Vasoactive intestinal peptide also inhibits the production of IL-2 and IL-4 in murine thymocytes.⁷⁸ In contrast, SP can act as a cosignal to enhance the expression of specific IL-2 mRNA and IL-2 secretion in T cells.^{79 - 80} Substance P also increases synthesis of immunglobulins from mixed lymphocyte cultures, the major effect being on IgA synthesis.⁷⁶

Mast cells play an important role in a variety of biological responses. They are critical effector cells in certain forms of IgE-dependent hypersensitivity reactions in which mediators such as histamine, proteoglycans, prostaglandin D2, proteases, and acid hydrolases are released.⁸¹ Furthermore, they participate in the modulation of late-phase inflammatory responses, including the augmentation of vascular permeability, fibrin deposition, tissue swelling, and leukocyte infiltration.⁸² In addition to immediate hypersensitivity mediators such as histamine, mast cells express a number of multifunctional cytokines, including IL-1, IL-3, IL-4, IL-6, and TNF-α.^{83 - 84}

Mast cells have been suggested as one of the principal effector cells responding to neuropeptides because nerve cell stimulation caused degranulation of mast cells and histamine release.⁸⁵ Peptidergic nerve fibers and mast cells are associated anatomically in several tissues, and IgE-activated mast cell mediators may amplify an inflammatory response by directly stimulating nerve terminals and initiating an axon reflex.^{86 - 89} Several neuropeptides (eg, SP, VIP, and neuropeptide Y) can bind to and activate mast cells, resulting in degranulation and histamine release.^{90 - 92} Furthermore, Ansel and coworkers⁹³ demonstrated that mast cell TNF-α mRNA is selectively up-regulated by SP in a dose-dependent manner. Substance P increased TNF-α secreted from cloned murine mast cells and freshly isolated peritoneal mast cells.⁹³

Mast cells, however, are not just passive responders to neuropeptides. Mast cell secretory products also have been implicated to excite various portions of the nervous system. For example, tryptase, the most abundant secretory granule protein in all subsets of human mast cells, has been shown to activate proteinase-activated receptor (PAR)–2.⁹⁴ Proteinase-activated receptor–2 belongs to a growing subfamily of G-protein–coupled receptors that are activated by proteolysis. The first PAR described, PAR-1, is a receptor for thrombin.⁹⁵ Trypsin, mast cell tryptase, and probably other trypsinlike proteases activate PAR-2. Proteases cleave within the extracellular N-termini of PARs, exposing tethered ligand domains that bind to and activate the cleaved receptors. Proteolytic activation of these receptors represents a new concept in receptor signaling mechanisms because the ligand is physically attached to its own receptor. In other words, unlike traditional soluable ligand-to-receptor interactions, PARs are activated by removal of part of the receptor protein.

Proteinase-activated receptors are expressed in many different tissues and cell types. In addition to its central role in coagulation, thrombin has numerous biological functions that are related to inflammation, tissue remodeling, and wound healing. Many of these effects are mediated by PAR-1. Proteinase-activated receptor–2 is also widely distributed. It is expressed in the kidney, pancreas, intestine, stomach, prostate, eye, spleen, heart, and several cell types. The proteases that activate PAR-2 in these tissues and cells and the biological functions of PAR-2 remain to be clarified. As mentioned above, mast cell tryptase represents 1 possibility. It has recently been shown that a large proportion of myenteric neurons express PAR-1 and PAR-2. Thrombin and mast cell tryptase have been shown to excite PAR-1 and PAR-2, respectively, on myenteric neurons.⁹⁶ Thus, during trauma and inflammation, when prothrombin is activated and mast cells degranulate, thrombin and tryptase may excite myenteric neurons by cleaving and triggering PAR-1 and PAR-2, respectively, perhaps contributing to the neuro-inflammatory response.⁹⁴ The consequences of this unique observation are not yet clear, but they promise to provide a direct path from mast cells to the sympathetic nervous system. This is critical to wound healing in several ways, not the least being regulation of blood flow in injured tissue.⁹⁴

The diverse actions of neuropeptides in injury and inflammation are under the control of a complex pattern of a variety of chemical mediators that operate together in either antagonistic or synergistic manners (Table 2). The intricate regulatory processes that have to be orchestrated comprise generation, release, and metabolism of neuropeptides, as well as expression of neuropeptide receptors on target cells. The precise spatial and temporal interweaving of the reaction pathways involved is still poorly understood.

The biological actions of classic neurotransmitters such as acetylcholine are terminated by enzymatic degradation, re-uptake into nerve endings, or diffusion away from target cells.³⁴ Because no re-uptake mechanisms seem to operate for neuropeptides, enzymatic catabolism represents the major mechanism by which biological activity of neuropeptides is regulated.^{5 ,97} Accumulating evidence suggests that neuropeptides are degraded and inactivated mainly by cell surface peptidases.

The best-studied membrane-bound surface peptidase, neutral endopeptidase (EC 3.4.24.11; NEP), also known as enkephalinase or CD10, was identified as the main degrading enzyme for several neuroactive peptides. Neutral endopeptidase, which has been localized on the surface of epithelial, endocrine, and connective tissues; Schwann cells; subpopulations of neurons; immunocytes; and smooth muscle cells and fibroblasts, cleaves 5– to 37–amino acid residue peptides at bonds involving preferentially hydrophobic residues (eg, tachykinins, enkephalins, somatostatin, VIP, and CGRP).^{13 ,98 - 101}

Neutral endopeptidase activity in the trachea is reduced by up to 50% by infection,¹⁰² and NEP is down-regulated by intestinal inflammation.¹⁰³ Consequently, inhibition of NEP by specific inhibitors (phosphoramidon and thiorphan) has been shown to potentiate tracheal neurogenic inflammation.¹⁰⁴ Glucocorticoids are able to stimulate expression of NEP¹⁰⁵ and to down-regulate NK receptor,¹⁰⁶ and these actions may constitute additional mechanisms by which corticosteroids exert anti-inflammatory effects.

By now, several membrane-bound surface peptidases (eg, dipeptidylaminopeptidase IV; EC 3.4.14.5, aminopeptidase N; and EC 3.4.11.2 [EC signifies enzyme classification]) have been implicated in the metabolism of bioactive peptides in various tissues.^{94 - 95} In addition, Jackman and coworkers¹⁰⁷ discovered an enzyme, released from human platelets by thrombin, that deamidizes protected peptides, such as SP and other tachykinins.

Peptidases and their cleavage sites may be potential targets for the development of inhibitors as vasoactive drugs or of metabolically stable peptide agonists.

Kinins are a group of small peptides formed in blood and biological fluids by the action of proteolytic enzymes (kallikreins) on α₂-globulins (kininogens). When an appropriate physiologic or pathophysiological stimulus activates the kallikreins, the nonapeptide bradykinin is formed in the blood from high molecular weight kininogens (plasma pathway). Similarly, the decapeptide kallidin (lysyl bradykinin) is released in tissues by the actions of the kallikreins on low molecular weight kininogens (tissue pathway).¹⁰⁸

Kinins are among the naturally occuring agents involved in inflammatory reactions, eg, vasodilation, increase of vascular permeability, and mobilization of blood and tissue cells.¹⁰⁸ There is increasing evidence that these kinin effects are, at least in part, mediated by tachykinins released from sensory nerve endings. Blockade of the kinin B₂ receptors with a selective antagonist had an inhibitory effect on plasma extravasation in the trachea and nasal mucosa caused by antigen challenge in the guinea pig.^{109 - 110} A similar effect was seen when a tachykinin receptor antagonist was used,¹⁰⁹ suggesting that kinins and tachykinins may use a common final pathway. Pharmacological and biochemical evidence confirms that kinins released by the anaphylactic reaction and tissue injury are powerful stimulants of sensory nerves and exert at least part of their actions through the release of sensory neuropeptides.¹¹¹

The opioid peptide family comprises many distinct peptides with opioid activity. All of these so-called endogenous opiates contain a common sequence of Tyr-Gly-Gly Phe and bind to the same cell-surface receptors as morphine. Triggering of cell-surface opiate receptors activates G_iproteins that inhibit adenylate cyclase and thereby cause a decrease in intracellular cyclic adenosine monophosphate levels.¹¹²

Opioid antinociception usually has been associated with the activation of opioid systems within the CNS. Recently, however, evidence has accumulated that opioid peptides and receptors in the periphery may play an important role in such phenomena. Endogenous opioids can interact with opiate receptors located on primary afferent neurons in inflamed tissue, resulting in antinociception and decreased release of neuropeptides.¹¹³ Furthermore, it seems that these opioid peptides are released from immunocompetent cells (T and B lymphocytes and monocytes and macrophages) infiltrating the inflamed tissue.^{114 - 116} Consequently, opioids may alter inflammatory processes by inhibiting the release of neuropeptides from sensory nerve terminals. Receptors for opioid peptides are also present on lymphocytes and macrophages, and, thus, opioids are capable of directly modulating immune cell responses such as chemotaxis, proliferation, cytokine production, and cytotoxicity.^{117 - 120} The observed effects, however, are highly diverse, and further studies have to be undertaken to elucidate the role of opioids in immunoregulation. The effects of opioids in enhancing local blood flow that has been impaired because of vasoconstriction is, however, well known.

Nerve growth factor (NGF), a 118-acid polypeptide hormone, is the best-characterized member of the neurotrophin family.¹²¹ The role of NGF in the development and maintenance of peripheral sympathetic and nociceptive sensory neurons is well established.^{122 - 123} Nerve growth factor binds to the high-affinity receptor tyrosine kinase on neurons, and, after internalization, it is transported retrogradely to the cell body. Through the activation of second messenger signals and changes in transcription factor expression, NGF controls the survival, growth, and phenotype of immature neurons.^{124 - 128} In addition to this specific neurotrophic action during development, a constant supply of NGF from the periphery may be important for the maintenance of normal phenotype in tyrosine kinase receptor–expressing nociceptive adult sensory neurons.¹²²

A variety of cell types are capable of producing NGF. The main cellular source in normal skin seems to be keratinocytes.^{14 ,129} In addition, NGF may be synthesized by immunocompetent cells (lymphocytes, macrophages, and mast cells), fibroblasts, smooth muscle cells, and Schwann cells.^{130 - 134} Nerve growth factor levels have been found to be elevated in inflammatory exudates, inflamed skin, and the nerves innervating inflamed tissue.^{135 - 137}

Nerve growth factor may directly or indirectly affect tissue immune reactivity. Indirect effects of NGF may result from its cytokinelike actions, including stimulation of the release of inflammatory mediators from lymphocytes^{138 - 139} and degranulation of mast cells.¹⁴⁰ Furthermore, NGF is capable of directly modulating neuropeptide expression in tyrosine kinase–expressing neurons.^{132 ,136} After inflammation, synthesis of SP and CGRP has been shown to be up-regulated in dorsal root ganglion cells by NGF.^{135 ,141 - 144} We are just beginning to understand, however, the molecular mechanisms that allow neuropeptide genes to respond to transsynaptic, humoral, and trophic stimuli.¹⁴⁵

The elevation of NGF during the inflammatory response is mediated by several different cytokines and growth factors.¹⁴³ There is strong evidence that 2 cytokines, TNF-α and IL-1β, are necessary intermediates leading to the production of NGF in inflammation.^{132 ,136} As mentioned before, SP can stimulate the release of these mediators from inflammatory cells, which, in turn, increases NGF levels in inflamed tissues. Thus, it is conceivable that a positive feedback mechanism exists in which SP induces synthesis of NGF by cytokines, leading to increased production of SP in the dorsal root of ganglion cells (Figure 1).

Injury induces a sequence of neuropeptide responses in wounds. The initial ones involve vasomotor activity through nociceptive influences and the initial events in inflammation. Vasomotor tone lies in the balance, and in this balance, resistance to infection, collagen deposition, and angiogenesis are regulated through the supply of blood and, in particular, oxygen. These influences continue in more subtle ways, for instance, injury induces a reversible sprouting of peptidergic nerve fibers adjacent to the injury site, which increases in proportion to the severity of the injury.^{146 - 148} The significance of this sprouting has not yet been fully determined. The ability to affect proliferation of various types of target cells³⁴ and to improve healing of experimentally malperfused tissues^{149 - 150} suggest a regulatory role of neuropeptides in tissue repair.

Substance P has been shown to act through the NK receptor to stimulate proliferation of cultured keratinocytes.^{151 - 152} Vasoactive intestinal peptide can exert both stimulatory and inhibitory effects on the proliferation of keratinocytes.¹⁵³ There is increasing evidence that neuropeptides play an important role in angiogenesis, including formation of new vessels in inflammation and wound healing. Substance P stimulates DNA synthesis in cultured arterial smooth muscle cells.¹⁵⁴ Similarly, CGRP increases both cell number and DNA synthesis in cultured endothelial cells.¹⁵⁵ In vivo, SP, CGRP, and VIP have been shown to stimulate angiogenesis.^{156 - 158} Using an in vitro model, Wiedermann and coworkers¹⁵⁹ showed that SP stimulated endothelial cell differentiation into capillarylike structures. Furthermore, SP and CGRP exert potent proliferative stimuli on cultured fibroblasts.^{154 ,160 - 161} These data suggest that neuropeptides released from peripheral nerve endings in association with tissue injury may not only affect vasodilation and the inflammatory response but may also stimulate proliferation of epithelial, vascular, and connective tissue cells (Figure 2).

In vivo experiments support the hypothesis that tissue–nervous system interactions promote healing. More than 70 years ago, it was noted that damage to the PNS may alter skin repair, leading to chronic wounds at the affected area.¹⁶² Experimentally, denervation of skin was shown to decrease mechanical strength and collagen content of incisional wounds in rabbits.¹⁶³ Capsaicin-induced depletion of neuropeptides in corneas delayed cornea wound healing.¹⁶⁴ Also, rats treated with capsaicin showed greater severity of experimentally induced skin ulcers.¹⁶⁵ After a scalding injury of a dog's paw, an increase in SP immunoreactivity was demonstrated, indicative of SP release after trauma.¹⁶⁶ In healing ligamentous tissue, more SP- and CGRP-containing nerve fibers were found compared with normal ligaments.¹⁶⁷ Similarly, elevated neuropeptide levels were found in healing bones by others.^{147 ,168} In wounded rat skin, however, diminished content of SP, CGRP, and somatostatin was reported.¹⁶⁹ Whether this decrease in neuropeptide levels represented an increased release from terminals to the wound site, decreased synthesis and transport to terminals, or loss of terminals remained unclear. In a different study,¹⁷⁰ when rats were treated with SP, their skin wounds healed more rapidly. Clearly, in wound healing there is an increased tissue–nervous system interaction that may modulate repair mechanisms and thus affect the outcome of healing.

Last, neurogenic mechanisms that govern local blood perfusion and oxygenation may literally govern the rate of healing. The affected processes include angiogenesis, collagen deposition, bacterial killing, and epithelialization.

Complex mechanisms controlling neuropeptide activity include not only their synthesis and secretion but also their tissue availability and interactions with receptors on target cells and their degradation by peptidases. The rate of tissue reinnervation in healing critically affects availability of neuropeptides.

Recent advances in neuropeptide research, including the cloning of neuropeptide receptors and the introduction of highly potent and specific receptor antagonists, have provided new insights into the pathogenesis of inflammatory diseases and chronic wounds. Clinically, diseases and injuries with impaired tissue–nervous system integrity are known to be associated with poor outcome of healing (eg, patients with diabetes, herpes zoster, or spinal cord injury). The rapidly accumulating body of knowledge on the pathophysiological role of neuropeptides in tissue repair may open new therapeutic options to treat clinical states of impaired tissue–nervous system interaction.

Reprints: Thomas K. Hunt, MD, Department of Surgery, University of California at San Francisco, 513 Parnassus Ave, HSW-1652, San Francisco, CA 94143-0522.