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Joslin-Beth Israel Deaconess Foot Center and The Rongxiang Xu, MD, Center for Regenerative Therapeutics, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
Joslin-Beth Israel Deaconess Foot Center and The Rongxiang Xu, MD, Center for Regenerative Therapeutics, Beth Israel Deaconess Medical Center, Harvard Medical School, Boston, MA, USA
Lower extremity ulcerations represent a major complication in diabetes mellitus and involve multiple physiological factors that lead to impairment of wound healing. Neuropeptides are neuromodulators implicated in various processes including diabetic wound healing. Diabetes causes autonomic and small sensory nerve fibers neuropathy as well as inflammatory dysregulation, which manifest with decreased neuropeptide expression and a disproportion in pro- and anti- inflammatory cytokine response. Therefore to fully understand the contribution of autonomic nerve dysfunction in diabetic wound healing it is crucial to explore the implication of neuropeptides. Here, we will discuss recent studies elucidating the role of specific neuropeptides in wound healing.
1. Introduction
The skin is densely innervated by an interconnected system of highly specialized afferent sensory and efferent autonomic nerve fibers (
). Cutaneous autonomic nerve fibers almost completely derive from sympathetic neurons and albeit very effective, they represent only a minority of skin nerve fibers in comparison with sensory nerves. In addition, as opposed to sensory nerves, the distribution of autonomic nerve fibers is restricted to the dermis, innervating blood vessels, lymphatic vessels, erector pili muscles, apocrine and eccrine glands, and hair follicles. Therefore, cutaneous autonomic nerve fibers take part in the modulation of blood circulation, lymphatic function, and skin appendages regulation (
). Diabetic patients' skin exhibits motor, sensory and autonomic fiber denervation: sensory neuropathy restricts the sensations of pain, temperature, pressure and others; autonomic denervation leads to arteriovenous shunting, thereby causing vasodilation in small arteries; motor neuropathy induces weakness and wasting of small intrinsic muscles (
). Importantly, microcirculation is affected in the diabetic neuropathic foot, mainly through impairment of both endothelium dependent and independent vasodilation (
Cutaneous microcirculation in the neuropathic diabetic foot improves significantly but not completely after successful lower extremity revascularization.
). Finally, another consequence of autonomic denervation is sudomotor dysfunction that leads to dry skin and callus formation that play an important role in the development of diabetic foot ulceration.
A growing body of studies in both patients and animal models points to a synergistic role of cutaneous nerve fibers and the immune system in mediating wound healing. The regulation of the healing response is realized through intricate interplay of components of the local immune and nervous system, which is further regulated via endocrine feedback (
). Neuropeptides are neuronal short-chain polypeptides that act as signaling molecules affecting numerous processes. Cutaneous nerve fibers and inflammatory cells such as monocytes, macrophages and eosinophils are known to release neuromodulators including cytokines and neuropeptides that regulate the activity of specific cytokine and neuropeptide receptors on a variety of skin cells including mature T and B cells, Langerhans cells, endothelial cells, mast cells, fibroblasts and keratinocytes resulting to the direct activation of G-protein signaling cascades (
). Fig. 1 summarizes how diabetes and neuropeptide expression dysregulation culminate in aberrant wound healing. Neuropeptide Y (NPY), Substance P (SP) and calcitonin gene related peptide (CRGP) are neuropeptides involved in modulating the immune response and wound healing. Further, other neuropeptides such as Melanocyte Stimulating Hormone (MSH) and Neurotensin are also neuromodulators and could potentially participate in impaired diabetic wound healing. These neuropeptides are released from autonomic nerve fibers as well as from cells within the dermis and the epidermis (
). Furthermore, these neuropeptides regulate the expression and function of numerous cytokines that are implicated and dysregulated in diabetes including IL-1, IL-6, IL-8, IL-10 and TNF-α (
Diabetes mellitus causes autonomic and small sensory nerve fibers neuropathy in the lower extremity as well as inflammatory dysregulation, which manifest with reduced neuropeptide expression and disproportion in pro- and anti- inflammatory cytokine response. Neuropeptides have a direct effect on leukocytes and further contribute to the cytokine imbalance. Also, cytokines and neuropeptides directly influence various skin cells including fibroblasts, keratinocytes and endothelial cells decreasing their proliferation and resulting in irregular angiogenesis, ECM production and reepithelialization. Reduced neovascularization, reepithelialization and dysregulation in remodeling and granulation tissue deposition, also affected by the abnormal cytokine expression profile, lead to impaired cutaneous wound healing.
NPY is a highly conserved 36 amino acid polypeptide involved in dysregulated healing, and is one of the most abundant neurotransmitters in the mammalian central (CNS) and peripheral nervous system (PNS) (
). NPY is mostly studied for its impact on the central nervous system, where it induces conservation of energy and counteracts the effects of Leptin. Thus, most of the NPY diabetes studies focus on its CNS effects (
). In a recent study NPY in the plasma of type 2 diabetic patients was found to be increased; however there is no data on dermal NPY expression for these patients. Baseline expression of NPY remains unchanged in a diabetic rabbit model of cutaneous wound healing (
). Specifically, NPY modulates cell migration, cytokine release from macrophages and helper T cells, antigen presentation as well as activation of natural killer cells and antibody production (
). Platelet lysate derived NPY was recently shown to affect migration and angiogenesis potential of human adipose derived stromal cells and co-localized with endothelial markers CD31 and VEGF in difficult to heal wound samples treated with lysate (
). NPY is mostly known to be associated with tendon and cartilage healing, but through its pro-angiogenic receptors NPY-2R and NPY-5R it also influences cutaneous healing (
Neuropeptides regulate expression of matrix molecule, growth factor and inflammatory mediator mRNA in explants of normal and healing medial collateral ligament.
). Notably, in genetically modified mice where NPY-2R was deleted, a significant delay in cutaneous wound healing with decreased neovascularization was reported (
). The enzyme dipeptidyl peptidase IV (DPP IV) that cleaves NPY into its pro-angiogenic form, which subsequently binds to NPY-2R and NPY-5R receptors, is enriched in aging mice (
). NPY is thus involved in both the inflammatory and angiogenic phases of wound healing. More research is necessary to elucidate the exact role of NPY in diabetic wound healing.
3. Substance P (SP)
A member of the tachykinin neuropeptide family, SP is an 11 amino acid neuropeptide encoded by the TAC1 gene and is one of the main neuropeptides released by C-nociceptive fibers in response to injury (
). In the last two decades, SP has emerged as a potent modulator of cutaneous wound healing among all healing associated neuropeptides. The pro-angiogenic function of SP has been demonstrated in both in vitro and in vivo experiments and importantly SP has been reported to have a critical role in wound site infiltration of polymorphonuclear leukocytes (
). In addition, topical administration of SP on excisional wounds in a db/db mouse model led to increased leukocyte infiltration compared to saline treatment at the early stages post-wounding, suggesting a role for SP involvement during early inflammation in wound healing (
). Moreover, the enzyme that inactivates SP, neutral endopeptidase (NEP) or neprilysin, is increased in diabetes and the use of a NEP inhibitor has been effective in accelerating diabetic wound healing (
). In a rabbit model of diabetic wound-healing, our group has demonstrated reduced SP levels in the diabetic rabbit skin compared to non-diabetic and post-wounding, both NPY and SP gene expression is diminished regardless of diabetic status (
). In endothelial cells, SP is an established vasodilating factor by inducing the production of nitric oxide, consequently enhancing endothelial permeability and leukocyte extravasation into the underlying tissues (
). It has been recently reported to promote the mobilization of endothelial progenitor cells in the wounded tissue of a murine model of type 2 diabetes and increase the amount of Yes-associated protein expression in the dermis (
). Furthermore, it acts as a potent chemoattractant for immune cells, promotes elevated expression of endothelial leukocyte adhesion molecule-1 on human microvascular endothelial cells and leukocyte function-associated antigen-1 (LFA-1) on murine endothelial cells and lymphocytes and can raise the levels of an array of inflammation linked cytokines including TGF-beta, TNF-α, IL-1β, IL-2, IL-8, IL-6 from dendritic and T cells, neutrophils, macrophages and fibroblasts (
Production of tumor necrosis factor-alpha, interleukin 1-beta, interleukin 2, and interleukin 6 by rat leukocyte subpopulations after exposure to substance P.
). Hence by generating a pro-inflammatory environment within the wound site SP plays a crucial role in the inflammatory and angiogenic phases of wound healing.
4. Calcitonin gene related peptide (CGRP)
CGRP is a 37 amino acid neuropeptide produced by an alternative splicing of the calcitonin gene (
). Just like NPY, CGRP is present in both the CNS and the PNS. In the PNS, CGRP is stored and released together with SP from capsaicin sensitive peripheral afferent neurons and is also a potent vasodilator (
Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides.
). Notably, co-application of CGRP and SP to human skin induced long lasting vasodilation in a dose-dependent manner highlighting a synergistic effect of the two neuropeptides (
). Diabetes has been shown to decrease the levels of CGRP in murine hearts, limit CGRP-mediated vasodilation in rats and diminish both CGRP and CGRP receptor expression in a rat model of diabetic cardiomyopathy (
Cardiomyopathy in streptozotocin-induced diabetes involves intra-axonal accumulation of calcitonin gene-related peptide and altered expression of its receptor in rats.
Sensory nerve innervation of epineurial arterioles of the sciatic nerve containing calcitonin gene-related peptide: effect of streptozotocin-induced diabetes.
The effect of long-term streptozotocin-induced diabetes on contractile and relaxation responses of coronary arteries: selective attenuation of CGRP-induced relaxations.
). CGRP is also involved in the wound healing process by promoting neovascularization through elevated VEGF secretion from wound site cells and triggering the cAMP pathway (
). Moreover, CGRP induces release of both IL-1α and IL-8 in keratinocytes, IL-8 in the corneal epithelium, IL-1α, IL-8 and ICAM-1 in airway epithelium, IL-1β and TNF-α in macrophages, IL-1β, IL-6 and TNF-α in dental pulp fibroblasts, and acts as a chemoattractant for T cells, mediates lymphocyte proliferation and inhibits IL-2 expression (
Regulatory peptides modulate adhesion of polymorphonuclear leukocytes to bronchial epithelial cells through regulation of interleukins, ICAM-1 and NF-kappaB/IkappaB.
Acta Biochim. Biophys. Sin. Shanghai.2006; 38: 119-128
Effects of the neuropeptides substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide and galanin on the production of nerve growth factor and inflammatory cytokines in cultured human keratinocytes.
). In animal models of diabetes CGRP was also decreased in tissues such as the heart and the dorsal root ganglion, but not much is known about its cutaneous expression (
The effect of long-term streptozotocin-induced diabetes on contractile and relaxation responses of coronary arteries: selective attenuation of CGRP-induced relaxations.
). In a recent study, vacuum-assisted treated wounds in a diabetic mouse model exhibited a significant increase in dermal and epidermal nerve fiber densities and in SP, CGRP, and nerve growth factor expression. In particular, the cyclical treatment mode correlated with the largest enhancement in granulation tissue production, and a slightly quicker wound closure rate (
). In CGRP-null mice (CGRP−/−), neovascularization and wound healing were impaired in comparison with control wild-type mice, and a reduction in the levels of VEGF from the wound granulation tissue was demonstrated (
). These findings indicate that the association of CGRP in wound healing is modulated through its impact on angiogenesis. Thus, exogenous CGRP addition may promote enhanced angiogenesis and wound healing.
5. Neurotensin (NT)
The 13 amino acid neuropeptide NT is primarily produced in the CNS (mainly hypothalamus, amygdala and nucleus accumbens) and in endocrine cells (N cells) of the ileum and jejunum. NT inhibits CNS dopaminergic pathways and promotes growth of various gastrointestinal tissues as well as adrenal gland, hepatocytes and fibroblasts (
). According to different studies, NT may be involved in the pathogenesis of diabetes. Increased levels and total amounts of NT are found in the pancreas of obese (ob/ob) mice and in the intestine of both ob/ob and diabetic (db/db) mice (
). Nevertheless, in another study, NT expression was comparable between lean and diabetic mice. In addition, research in human patients did not reveal any difference in NT amounts between healthy nondiabetic subjects and lean and obese diabetic patients either pre- or postprandially (
Peptidergic (neurotensin, VIP, substance P) nerve fibres in the skin. Immunohistochemical evidence of an involvement of neuropeptides in nociception, pruritus and inflammation.
). In a recent study, in vitro treatment of keratinocytes and T cells with NT was shown to enhance migration and reduced the expression of TNF-α and IL-8. Interestingly, co-stimulation with SP led to decreased migratory capacity, while the angiogenesis in HUVEC cells was elevated (
). NT also has an effect on cutaneous dendritic cells through downregulation of activation of inflammatory pathways JNK and NF-κB and reduction of expression of inflammatory cytokines IL-6, TNF-α and IL-10 (
). Noteworthy, in two different mouse diabetic wound healing studies, delivery of NT with specially designed biomaterials enhanced wound closure. Collagen dressings functionalized with NT reduced inflammation and accelerated wound healing (
). Therefore, topical delivery of NT could potentially be a promising treatment for diabetic foot ulcers.
6. Alpha-melanocyte-stimulating hormone (a-MSH)
a-MSH is a 13 amino acid hormone and neuropeptide and belongs to the family of melanocortins, a number of structurally related peptides that not only participate in the regulation of pigmentation and cortisol expression but also modulate food intake, energy homeostasis, exocrine gland function, and inflammatory response (
). a-MSH is a proteolytic cleavage product of proopiomelanocortin (POMC) and is predominantly released from the pars intermedia region of the pituitary gland (
). A number of different cutaneous cell types including keratinocytes, fibroblasts, melanocytes and endothelial cells generate a-MSH and express melanocortin receptors (MCRs). Long-term activation of a-MSH decreases body weight and improves glucose metabolism in a model of diet-induced obesity (
). Two diabetic rat studies demonstrated that POMC mRNA in arcuate nucleus, pituitary and the hypothalamus is diminished and cannot be reversed following insulin treatment (
Effects of streptozotocin-induced diabetes and insulin treatment on the hypothalamic melanocortin system and muscle uncoupling protein 3 expression in rats.
), while in human peripheral blood monocytes and cultured monocytes, α-MSH enhances the expression of the anti-inflammatory cytokine IL-10. In septic patients, small concentrations of α-MSH added to LPS-stimulated whole blood samples inhibit TNF-α and IL-1β production and in RAW264.7 mouse macrophages cell line a-MSH inhibits nitric oxide generation induced by LPS and IFN-γ treatment (
Effects of melanocortin peptides on lipopolysaccharide/interferon-gamma-induced NF-kappaB DNA binding and nitric oxide production in macrophage-like RAW 264.7 cells: evidence for dual mechanisms of action.
). Moreover, a-MSH suppresses the expression CD86, a major T cell costimulatory molecule, in activated monocytes and M1 classically activated macrophages and promotes the expression of the anti-inflammatory cytokine IL-10 in human peripheral blood monocytes and cultured human monocytes (
). In endothelial cells, α-MSH causes an increase in the release of IL-8, while in stimulated dermal fibroblasts it reduces IL-8 release and in human keratinocytes increases production of IL-10 (
Human dermal microvascular endothelial cells express the melanocortin receptor type 1 and produce increased levels of IL-8 upon stimulation with alpha-melanocyte-stimulating hormone.
). In murine cutaneous wound healing as well as human burn wounds and hypertrophic scars upregulation of both a-MSH and its receptor was observed. Cells positive for a-MSH were epidermal keratinocytes, fibroblasts and inflammatory cells (
). In a rabbit model of corneal wound healing, topical delivery of the C-terminal tripeptide sequence of a-MSH (a-MSH11–13) ameliorated the healing response (
). Furthermore, intraperitoneal injection of a-MSH prior to injury led to significant reduction of leukocytes and mast cells in the granulation tissue of mice 3 and 7 days post-wounding and reduced scar area and collagen fiber organization on day 40 after injury (
). Hence, it appears that a-MSH influences inflammatory pathways and its presence in the skin and involvement in various functions of different skin cell types makes it an attractive target for additional cutaneous diabetic wound healing studies (
A number of other neuropeptides have also been lately implicated in cutaneous wound healing. Somatostatin was shown to exert an inhibitory effect on keratinocyte migration and proliferation both in vitro and on an ex vivo porcine wound healing model (
). Adrenomedullin topically delivered in a sustained-release ointment formation significantly improved wound closure in pressure ulcer patients through acceleration of granulation tissue formation and enhanced neovascularization (
). In addition, when used in a combination treatment with its binding protein adrenomedullin also promoted faster wound repair in a rat model of cutaneous healing (
). Using topical gene therapy with the angiogenic neuropeptide secretoneurin in mice resulted in accelerated diabetic wound healing with elevated arteriole and capillary densities in the wounded area (
). Lastly, treatment of diabetic mice with the neuropeptide relaxin at the wound site lead to increased angiogenesis, vegf mRNA expression and elevated MMP11 levels (
The functions of diverse neuropeptides have been studied in detail in the brain, but remain underexplored in other densely innervated organs, like the skin. The above studies clearly suggest that the cutaneous nervous system is not only responsible for sensory neurotransmissions to the CNS but plays a crucial role in various skin functions including wound healing. Importantly, they have been associated with impaired diabetic wound healing. More comprehensive investigations of the function of each neuropeptide may assist in determining which neuropeptide is more important in the skin, both in physiological and pathological conditions, and to what extent. Finally, with various positive studies in animal models of wound healing, utilizing neuropeptides for therapeutic interventions of the diabetic foot ulceration could be a promising strategy.
Funding
This work was supported by the National Institutes of Health Grant DP3DK108224 (AV). AV received funding from the National Rongxiang Xu Foundation. GT received a George and Marie Vergottis Foundation Postdoctoral Fellowship award.
References
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HP-228, a novel synthetic peptide, inhibits the induction of nitric oxide synthase in vivo but not in vitro.
Cutaneous microcirculation in the neuropathic diabetic foot improves significantly but not completely after successful lower extremity revascularization.
Cardiomyopathy in streptozotocin-induced diabetes involves intra-axonal accumulation of calcitonin gene-related peptide and altered expression of its receptor in rats.
Effects of the neuropeptides substance P, calcitonin gene-related peptide, vasoactive intestinal polypeptide and galanin on the production of nerve growth factor and inflammatory cytokines in cultured human keratinocytes.
Production of tumor necrosis factor-alpha, interleukin 1-beta, interleukin 2, and interleukin 6 by rat leukocyte subpopulations after exposure to substance P.
Human dermal microvascular endothelial cells express the melanocortin receptor type 1 and produce increased levels of IL-8 upon stimulation with alpha-melanocyte-stimulating hormone.
Peptidergic (neurotensin, VIP, substance P) nerve fibres in the skin. Immunohistochemical evidence of an involvement of neuropeptides in nociception, pruritus and inflammation.
Effects of streptozotocin-induced diabetes and insulin treatment on the hypothalamic melanocortin system and muscle uncoupling protein 3 expression in rats.
Local effector functions of capsaicin-sensitive sensory nerve endings: involvement of tachykinins, calcitonin gene-related peptide and other neuropeptides.
Effects of melanocortin peptides on lipopolysaccharide/interferon-gamma-induced NF-kappaB DNA binding and nitric oxide production in macrophage-like RAW 264.7 cells: evidence for dual mechanisms of action.
Neuropeptides regulate expression of matrix molecule, growth factor and inflammatory mediator mRNA in explants of normal and healing medial collateral ligament.
The effect of long-term streptozotocin-induced diabetes on contractile and relaxation responses of coronary arteries: selective attenuation of CGRP-induced relaxations.
Sensory nerve innervation of epineurial arterioles of the sciatic nerve containing calcitonin gene-related peptide: effect of streptozotocin-induced diabetes.
Regulatory peptides modulate adhesion of polymorphonuclear leukocytes to bronchial epithelial cells through regulation of interleukins, ICAM-1 and NF-kappaB/IkappaB.
Acta Biochim. Biophys. Sin. Shanghai.2006; 38: 119-128
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