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VIP
Chapter 209
VIP Sami I. Said, Valerie Chappe and Sayyed A. Hamidi
ABSTRACT The neuropeptide vasoactive intestinal peptide (VIP) is widely distributed in the central and peripheral nervous systems and is expressed in all pulmonary structures, including airway epithelial cells, airway and pulmonary vascular smooth muscle cells, secretory glands, and immune and inflammatory cells. VIP actions in the lung, mediated predominantly by the adenylyl cyclase–cAMP pathway and by receptors shared with PACAP, include relaxation of airway and vascular smooth muscle and inhibition of their proliferation, modulation of inflammatory and immune responses, protection against cell injury, and stimulation of water and electrolyte transport. Strong evidence suggests that physiological roles of VIP in the lung include cotransmission of airway and pulmonary vascular smooth muscle relaxation, modulation of immune and inflammatory responses, and guarding against the pathogenesis of bronchial asthma, pulmonary hypertension, and CFTR dysfunction in cystic fibrosis. Therefore, it seems probable that VIP has strong therapeutic potential in a variety of clinical disorders.
HOW WAS VIP DISCOVERED? In 1967, as a pulmonary physician in Richmond, Virginia, I (S.I.S.) wondered why serious acute events in the lung, such as severe pneumonia and the Acute Respiratory Distress Syndrome (ARDS), are often complicated by systemic hypotension. One possibility that appealed to me was that injured lung tissue releases into the circulation one or more vasodilator compounds. Preliminary experiments confirmed that supernatants of lung extracts, when injected into other animals, reduced systemic arterial blood pressure. This hypotension was not fully explained by the presence in lung extracts of the then-known vasodilators histamine, prostaglandins, and kallikreins, suggesting that one or more vasodilator peptides contributed to the phenomenon.16 To seek validation of this explanation, and identification of such a peptide, I decided to spend a sabbatical year at the Karolinska Institute, in Stockholm, Sweden, with Professor Viktor Mutt, a leading peptide biochemist, as my guide and mentor. Handbook of Biologically Active Peptides. http://dx.doi.org/10.1016/B978-0-12-385095-9.00209-8 Copyright © 2013 Elsevier Inc. All rights reserved.
Although we soon confirmed the presence of strong vasodilator activity in extracts of porcine lung, isolation of a pure peptide with this property was progressing too slowly, because of the difficulty in securing sufficient quantities of fresh lungs.22 Although lung extracts were in short supply, extracts of upper small intestine were plentiful in Viktor’s laboratory, because he was preparing the gastrointestinal hormones secretin and cholecystokinin on a large scale. One day, as I wished we were searching for our peptide in the intestine instead of in the lung, I recalled that both upper intestine and lungs originated from the same embryonic bud, the foregut, and decided to look for vasodilator activity in intestinal extracts. The search proved fruitful, culminating in the isolation of the “vasoactive intestinal peptide,” so named because, at that point, we did not know whether it also existed outside the intestine or whether it had any other biological activity. During its purification, the dominant biological activity of VIP was closely associated with that of secretin.23,24 Reading the original articles on the discovery of secretin, I noted that Bayliss and Starling observed some “vasodepressor” activity in the secretin-containing preparations, which they attributed to the presence of an unidentified compound that, in all likelihood, was VIP! Soon after VIP became available in pure form, its amino acid composition and sequence were identified,12 and it was successfully synthesized, by Miklos Bodanszky.1 After the development of a radioimmunoassay for the measurement of VIP content in plasma and tissues, and reports that the “watery diarrhea” or “pancreatic cholera” syndrome was attributable to excessive production of VIP by certain tumors, we began to receive biological samples from around the country and abroad, from patients suspected of having that disorder. Those assays revealed that the highest levels of VIP were associated with neurogenic tumors, especially neuroblastomas.21 In addition, analysis of neuroblastoma cell lines showed them to be rich in immunoreactive VIP. As I looked into the biology of these cells, I learned that they share many features with normal neurons, so I wondered whether VIP could also be produced by normal neurons. 1535
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With neurologist colleagues, we searched for the peptide in the human brain and peripheral nerves and found it to be widely and selectively present. VIP was thus rediscovered as a neuropeptide and a probable neurotransmitter.27
VIP IS A MEMBER OF A FAMILY OF PEPTIDES24 VIP is a 28-amino acid residue peptide (Fig. 1) that belongs to a family of structurally and functionally related peptides, including secretin, glucagon, gastric inhibitory peptide or glucose-dependent insulinotropic peptide (GIP), the peptide with N-terminal histidine and C-terminal isoleucine amide (PHI) and its counterpart in human tissues, the peptide with N-terminal histidine and C-terminal methionine amide (PHM), corticotropin-releasing hormone (CRH), growth hormone-releasing hormone (GHRH), sauvagine, urotensin I, helodermin, and the pituitary adenylate cyclase-activating polypeptides (PACAP), with which VIP shares the greatest degree of homology (Fig. 2).
TISSUE DISTRIBUTION AND CELLULAR LOCALIZATION The expression of VIP in different cells and organs has been defined by immunofluorescence, radioimmunoassay,
and more recently, by measurement of VIP mRNA. In the brain, the peptide is found in the highest concentrations in the cerebral cortex, suprachiasmatic nucleus, hippocampus, amygdala, and striatum, but it is also present in the midbrain periaqueductal gray and the sacral spinal cord. In the peripheral nervous system, VIP is localized in the sympathetic ganglia, the vagus nerve, predominantly motor nerves such as the sciatic nerve, and nerves supplying exocrine glands, blood vessels, and nonvascular smooth muscle.3–5,25 In the lungs, VIP-containing nerve fibers and nerve terminals are principally localized in the smooth muscle layer of the airways from the trachea through the small bronchioles, around submucosal mucous and serous glands, and in the walls of both pulmonary and bronchial arteries. Immunoreactive VIP is also present in neuronal cell bodies forming microganglia that provide a source of intrinsic innervation of pulmonary structures. In addition to its neuronal localization, VIP is richly present in the immune system, including inflammatory cells such as mast cells, eosinophils, and B and T lymphocytes.
COLOCALIZATION WITH OTHER PEPTIDES AND NEUROTRANSMITTERS VIP coexists with acetylcholine in most cholinergic neurons, both central and peripheral. In exocrine glands, this colocalization serves the dual functional advantages of promoting both blood flow and glandular secretion. VIP may also be colocalized with other peptides, including PHI (or PHM, its human counterpart), peptides that are synthesized from the same precursor molecule, and substance P. Other neurotransmitters that may coexist with VIP include nitric oxide (nitric oxide synthase) and the excitatory transmitter glutamic acid.
FIGURE 1 Amino acid sequence of VIP.
FIGURE 2 Some members of the VIP family of peptides. Amino acid homologies are noted by shading.
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BIOLOGICAL ACTIONS IN THE LUNG Airways Airway Smooth Muscle Tone5,6,11,15,17 VIP relaxes airway smooth muscle, both in vitro and in vivo, in guinea pigs, rats, mice, rabbits, dogs, and humans. VIP also counteracts airway smooth muscle contraction induced by a variety of bronchoconstrictor agents, including histamine, prostaglandin F2α, kallikrein, leukotriene D4, neurokinin A, serotonin, and endothelin (Fig. 3). Relative to most other bronchodilators, VIP-induced airway relaxation is longer lasting and is unaffected by blockade of adrenergic or cholinergic receptors or of cyclooxygenase activity. In limited trials in human asthmatics, however, VIP, given by inhalation, is generally less effective than expected in relieving bronchoconstriction, probably because of its degradation by proteases in airway secretions.
Airway Smooth Muscle Proliferation VIP inhibits human airway smooth muscle proliferation,9 an action that is potentially beneficial in reducing airway remodeling, which contributes to increased airway resistance in bronchial asthma.
Tracheobronchial Secretion VIP stimulates water and ion transport in canine tracheal epithelium and may also promote macromolecular secretion. The coexistence of VIP and acetylcholine in cholinergic neurons brings together the blood flow-promoting and secretion-promoting effects of VIP and acetylcholine, respectively.
Pulmonary Circulation6,13,15,20 VIP dilates the vessels supplying the nose, upper airways, trachea, and bronchi, as well as pulmonary vessels. As a
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pulmonary vasodilator, VIP is more potent than isoproterenol, acetylcholine, or prostacyclin, and its action is independent of endothelium. Given intravenously in sufficient concentrations, VIP reduces both pulmonary and systemic vascular resistance. Delivered as an aerosol, however, it acts as a selective pulmonary vasodilator. As with airway smooth muscle, VIP suppresses human pulmonary vascular smooth muscle proliferation, a major feature of pulmonary hypertensive disease.
Immune and Inflammatory Responses2,28,30 VIP modulates several aspects of the immune and inflammatory responses: (1) It has a moderate inhibitory effect on antigen-induced release of histamine from guinea pig lung. Because the peptide is normally present in mast cells, the peptide may act as a natural modulator of mast-cell degranulation, an important mechanism underlying acute asthmatic and anaphylactic reactions. (2) VIP also modulates T-cell activation and inhibits the expression of proinflammatory cytokines (TNF-α, IL-6, IL-12, IL-18) and chemokines (e.g. RANTES); inducible nitric oxide synthase (i NOS), and proteases (MMP-2 gelatinase), while upregulating the expression of anti-inflammatory cytokine IL-10 and promoting the generation of tolerogenic dendritic and regulatory T-cells.
Anti-Injury and Cell-Protective Effects18,19,30 Acting by a number of actions, VIP prevents or attenuates acute lung injury in a variety of experimental models. These actions include antiapoptotic, prosurvival effects that are mediated by 3 complementary and synergistic mechanisms: Inhibition of the activation of caspases, key effectors of apoptosis; upregulation of the antiapoptotic protein bcl2; and suppression of cytoplasmic translocation of cytochrome c, a critical step in mitochondrial-mediated apoptosis.
RECEPTORS AND SIGNAL TRANSDUCTION PATHWAYS
FIGURE 3 VIP as a tracheal smooth muscle relaxant. Isolated strip of guinea pig trachea was strongly contracted in response to endothelin 1. Application of VIP promptly and completely restored tracheal tension to resting level.
Specific, high-affinity receptors for VIP have been identified in membrane preparations of normal lungs and human lung tumor cells. These receptors, localized immunocytochemically and by the increased cyclic (c)AMP levels resulting from activation by VIP, exist in a variety of pulmonary cells. Molecular and pharmacologic studies have led to the identification, characterization, and cloning of at least three receptors for VIP and the related PACAP peptides (see the chapter in this section of the book by Mario Delgado). These receptors, known as VPAC receptors, belong to a distinct family of seven transmembrane-domain receptors coupled to G proteins. The three VPAC receptors are VPAC1 and VPAC2, both of which respond to VIP and PACAP with
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comparable affinity, and PAC1, which has greater affinity for PACAP than for VIP. The actions of VIP are mediated principally by adenylyl cyclase stimulation and cAMP production. VIP biosynthesis itself is promoted by higher cAMP levels, and thus, the peptide is potentially capable of stimulating its own generation. In some VIP actions, additional second messengers and signal transduction pathways are involved, for example, phospholipase C activation in the case of the PACAP-preferring PAC 1 receptor. Some actions of VIP, such as the relaxation of airway and vascular smooth muscle cells, are mediated partly by NO, released from neurons or smooth muscle cells. Through its activation of a constitutive NOS in neural elements and smooth muscle, leading to activation of cytosolic guanylyl cyclase, VIP can stimulate intracellular cyclic GMP formation. The contribution of neuronal NOS to VIP-induced relaxation of the trachea was examined in neuronal NOS-knockout mice and estimated to account for a significant proportion of the relaxation. It is now believed that both NO and carbon monoxide (CO) mediate VIP-induced relaxation of airway, gastrointestinal, and other smooth muscle. The combined activation of cyclic AMP and GMP pathways ensures greater smooth muscle relaxation, inhibition of mitogenesis, and smooth muscle cell proliferation.26
PHYSIOLOGICAL ROLES The physiological significance of VIP is suggested by 3 lines of evidence: 1. It is widely present, in a conserved structure, throughout the animal kingdom. 2. Deletion of the VIP gene alters the expression of almost 27% of all other genes. 3. VIP KO mice express the phenotypes of at least 4 disorders, which are correctable with VIP.7 Deletion of the VIP gene results in (i) bronchial asthma, manifested by airway hyperresponsiveness and airway inflammation; (ii) pulmonary arterial hypertension, with vascular remodeling, right ventricular hypertrophy, and perivascular inflammation; (iii) cystic fibrosis (CF)-like features, including dysfunctional CFTR, associated with structural abnormalities in the lung and gastrointestinal tract; and (iv) increased susceptibility to death from endotoxemia. Several lines of evidence suggest the following physiological roles for VIP, with special reference to the lungs: 1. Mediation of vascular and nonvascular smooth muscle relaxation: Together with NO and CO, VIP is a cotransmitter of neurogenic, noncholinergic, nonadrenergic smooth muscle relaxation. 2. Modulation of smooth muscle cell proliferation.
3. Modulation of inflammatory responses. 4. Modulation of immune responses and promotion of immune tolerance. 5. Enhancement of cell survival, and defense against injury and apoptosis: VIP exerts these effects by attenuating inflammation and reducing apoptotic cell death. For example, VIP protects lung cells against oxidant stress resulting from: xanthine + xanthine oxidase, paraquat, excitotoxicity (glutamate toxicity), and ischemia-reperfusion injury; as well as platelet-activating factor and endotoxin lipopolysaccharide. In addition, VIP also protects neuronal cells against injury, as by oxidants, for example, H2O2 and glutamate; as well as by envelope protein of HIV, nutrient or nerve growth factor deprivation, tetrodotoxin, and β-amyloid (Aβ). Further, as discussed in the chapter by Paul Gressens, VIP promotes embryonic brain growth and differentiation. 6. Stimulation of water and electrolyte transport: Among the earliest biological actions of VIP to be recognized is its ability to promote water and electrolyte transport. This is true for Cl− in bronchial and intestinal epithelium, and for HCO3− in pancreatic secretion. As earlier mentioned, mice lacking the VIP gene express CFlike pathophysiological features, and the role of VIP in the pathogenesis and possible modulation of the human disease is further discussed later. Finally, excessive stimulation of H2O/Cl− transport, in the presence of VIP-secreting tumors (VIPomas), results in the “watery diarrhea syndrome.”
VIP IN THE PATHOGENESIS AND MODULATION OF PULMONARY DISEASE Bronchial Asthma17,31 A close link between VIP deficiency and the pathogenesis of asthma is suggested by 3 sets of findings: 1. VIP-immunoreactive nerves are absent in airways of a group of severely asthmatic subjects who died accidentally. 2. Mice with targeted deletion of the VIP gene exhibit airway hyperresponsiveness and histopathologic features of airway inflammation. 3. As an airway smooth muscle cell relaxant, with antiinflammatory and antiproliferative activities, VIP is well qualified to counteract the pathophysiological features of bronchial asthma, including airway constriction, hyperresponsiveness, airway inflammation, and, in severe cases, smooth muscle proliferation. Despite its demonstrated effectiveness in animal models in vivo, however, the results of several clinical trials in asthmatics are less impressive than anticipated. The inhalation
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route would seem to be ideally suited for delivering VIP to asthmatics. Unfortunately, success of this approach seems to be limited by degradation of the inhaled peptide by peptidases in airway secretions. This problem may be solved by either of 2 alternate solutions, neither of which has been put to the test in clinical trials: (a) Instead of VIP itself, use a VIP-like analog that is more resistant to enzymatic degradation, such as the C-terminally extended helodermin (Fig. 2) or (b) combining VIP with one or more enzyme inhibitors that are particularly effective against VIP-selective peptidases, notably dipeptidyl peptidase (DPP IV) and neutral endopeptidase (enkephalinase).
Pulmonary Arterial Hypertension (PAH) Strong experimental evidence and 2 clinical studies suggest a useful therapeutic potential for VIP in PAH: 1. As noted above, in the absence of the VIP gene, mice express a PAH phenotype, with pulmonary vascular remodeling, inflammation, and right ventricular hypertrophy.29 RT-PCR arrays in these mice show the upregulated expression of pathways that promote vasoconstriction, smooth muscle and collagen proliferation, and inflammation, together with the downregulation of key pathways that promote vasodilation, and suppress vascular proliferation, including endothelial NOS (eNOS) and prostacyclin (PGI2). 2. VIP relaxes pulmonary vascular smooth muscle cells in vitro and in vivo.26 Further, it suppresses pulmonary vascular smooth muscle cell proliferation, and corrects pulmonary vascular remodeling, inflammation, and RV pathology in experimental PAH induced by (a) VIP gene deletion in mice, (b) monocrotaline in rats (Fig. 4), and (c) chronic hypoxia in mice. 3. In patients with Idiopathic Pulmonary Hypertension (IPAH), serum VIP concentrations are below normal, and VIP-containing nerves are absent in pulmonary arterial walls, whereas VIP receptors VPAC1 and VPAC2 are upregulated in pulmonary arterial smooth muscle cells.13
(A)
(B)
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Lung Cancer VIP suppresses the growth of small-cell lung cancer cells in culture and in athymic nude mice in vivo.10 These results suggest that VIP may offer an effective and less toxic alternative to conventional chemotherapy in the management of this highly malignant form of cancer, but no clinical trials have been conducted to validate this possibility.
Acute Lung Injury (ALI) and Related Conditions19,30 1. VIP protects the lungs and airways in at least 13 experimental models (Table 1). This protection is attributable to a combination of anti-inflammatory, antioxidant, and antiapoptotic properties. 2. Based on these encouraging data, a phase I clinical trial of VIP, funded by the Orphan Drug Program of the FDA, was conducted in 8 patients with relatively severe ARDS, with or without Multiorgan Dysfunction Syndrome (MODS), in the setting of sepsis. VIP was given by continuous iv infusion, for 6 or 12 h. The study confirmed the safety of the peptide under these conditions: of the 8 patients, only 2 died, with neither death being drug related. A follow-up, multicenter clinical trial to evaluate efficacy is being planned.
Inflammatory and Autoimmune Diseases2 Based on its anti-inflammatory and immune-modulating properties, VIP has undergone early trials in a number of clinical disorders and experimental models of such disorders, including pulmonary sarcoidosis, COPD, rheumatoid arthritis, experimental autoimmune encephalomyelitis, inflammatory bowel disease, and type I diabetes. It is still too early to draw firm conclusions from these studies.
(C)
FIGURE 4 VIP corrects pulmonary vascular and RV pathology in monocrotaline (MCT)-induced PAH in rats: (A) H&E section of normal control lung, showing alveoli, bronchus, and small PA. No evidence of inflammation or vascular thickening. (B) Lung section 3 weeks after injection of MCT, showing vascular thickening and inflammatory cell infiltrates. (C) 3 weeks after VIP treatment, almost complete reversal of MCT-induced pathology. See color plate 58.
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TABLE 1 Experimental Models of Acute Lung and Airway Injury Prevented by VIP (A) In isolated lung 1. HCl intratracheally 2. Platelet-activating factor into the PA 3. Xanthine + xanthine oxidase into the PA 4. Phospholipase C into the PA 5. Prolonged perfusion ex vivo 6. Paraquat into the PA 7. Capsaicin into the airways 8. Anaphylaxis 9. Eosinophil-mediated injury of cultured bronchial epithelial cells (B) In vivo: 10. Platelet-activating factor i.v. 11. Cobra venom factor 12. Endotoxemia 13. Excitotoxicity (NMDA receptor activation)
VIP and the NFAT Transcription Factor In considering the role of VIP in disease, it is important to keep in mind the relationship of VIP to transcription factor nuclear factor of activated T-cells (NFAT). Activation of calcineurin–NFAT signaling is triggered by increased concentration of intracellular Ca2+, which leads to dephosphorylation (i.e. activation) of the phosphatase enzyme calcineurin (Fig. 5). In its resting, inactive state, this enzyme is located in the cytosol; on activation, it enters the nucleus to act on its target genes. In addition to T-cell activation, activation of this pathway results in inflammation, smooth muscle cell contraction and proliferation, and autoimmune responses. Inhibition of calcineurin-mediated effects, as by cyclosporine A and the peptide VIVIT, can prevent organ transplant rejection. With special reference to VIP, mice lacking the VIP gene show activation of NFAT, and strong evidence suggests that VIP is a physiological modulator of this pathway.
CYSTIC FIBROSIS (CF) CF, a fatal autosomal recessive disease, is characterized by abnormal ion transport across epithelia, viscous secretions, chronic bacterial infections, and airway inflammation. Over 25 years ago, we first observed that normal skin had a rich network of VIP-immunoreactive nerves around sweat gland acini and ducts, in contrast to absent or minimal VIP innervation of CF sweat glands. We wondered (1) whether deficient VIP innervation is responsible for the chloride ion abnormality in CF; and (2) to what extent is VIP involved in the pathogenesis, or possible correction, of CF pathology?8
We now know that the basic molecular defect in CF is a deficiency or absence of the cystic fibrosis transmembrane conductance regulator (CFTR) from the apical membrane of epithelial cells, due to mutations in the CFTR gene.
CFTR and Its Mutations The most common (>90%) mutation in CF, a deletion of phenylalanine 508 (ΔF508), causes improper folding of the CFTR protein, resulting in its retention in the endoplasmic reticulum and proteosomal degradation. Major CF research efforts are devoted to the attempt to rescue ΔF508-CFTR defective trafficking to restore normal epithelial function. We have investigated the potential rescue and stability at the cell membrane of ΔF508-CFTR, by VIP treatment, in human nasal epithelial cells JME/CF15, derived from a patient with this homozygous mutation. When the cells, maintained at 37 °C, are treated with VIP for 1 or 2 h, mature ΔF508-CFTR proteins are observed: Immunostaining confirms localization at the cell membrane, and functional assays confirm that CFTR-dependent chloride secretion has been restored after VIP treatment. In these cells, which express the VPAC1 receptor, we found that VIP-dependent rescue of ΔF508-CFTR trafficking is mediated by the PKA-dependent signaling pathway, whereas membrane stability by prolonged VIP treatment involves the Gαq and PKCε signaling cascade. Evidence from airway cells thus shows that the VIP mechanisms of regulating CFTR activity involve both PKA and PKC signaling in a synergistic manner to rescue defective trafficking of mutant CFTR, activate CFTR gating through direct phosphorylation of its regulatory domain, and most importantly, stabilize CFTR channels at the cell surface by reducing their internalization rate, thus optimizing CFTR-dependent secretions.
In Vivo Confirmation of the Role of VIP14 As earlier mentioned above, VIP knockout mice express several features of the absence of the VIP gene, including an asthma-like phenotype, a PAH phenotype, and increased vulnerability to endotoxemia. In addition, they also show CF-like pathological changes: the lungs show significant lymphocyte accumulation, increased airway secretion, alveolar thickening, and edema; the pancreas also shows increased secretion, and inflammatory cell infiltrates around the ducts. These pathological changes could be reversed, close to a wild-type phenotype, by treatment with VIP (15 µg) intraperitoneally, every other day, for 3 weeks. In this model, whereas normal control tissues show CFTR predominantly at the apical membrane of epithelial cells, CFTR distribution in VIP KO tissues is mainly observed intracellularly. VIP treatment restores strong CFTR membrane localization, confirming the important role of chronic
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FIGURE 5 Calcineurin–NFAT signaling and its inhibition by VIP. In resting cells, NFAT resides in the cytoplasm, where it is maintained in a phosphorylated, inactive state. Stimulation of cell-surface receptors coupled to phospholipase C (PLC) results in calcium mobilization, initially from intracellular stores, followed by influx of Ca2+ through specialized voltage-dependent Ca2+ channels (VDCC) and Ca2+ release-activated Ca2+ channels (CRAC). On activation by Ca2+, the phosphatase calcineurin dephosphorylates NFAT, triggering its activation and nuclear translocation. In the nucleus, NFAT interacts with other transcription factors, including activator protein (AP)-1, to stimulate gene expression. When NFAT is rephosphorylated, it is exported back to the cytoplasm. The immunosuppressive drug cyclosporin A inhibits calcineurin interactions with all its substrates, whereas VIVIT selectively inhibits NFAT activation. VIP seems to inhibit this pathway, but its mechanism of action is yet to be determined. IP3: inositol-1,4,5-triphosphate. See color plate 59.
VIP exposure tin maintaining CFTR channels at the membrane, and for exocrine epithelial tissues integrity. We conclude that inflammation and damage observed in VIP KO tissues can be attributed, at least in part, to the lack of CFTR-dependent secretions that ultimately depend on VIP stimulation, both for acute and long-term regulation of CFTR function. These observations also provide evidence of the molecular link between early observations of deficient VIP-containing fibers innervation of epithelial layers of exocrine organs in CF tissues and the absence of CFTRdependent secretions. The results set VIP, or its analogs, as a potential candidate for the treatment of CF as it corrects many features of this disease including its molecular basis. Ivacaftor, an investigational drug targeting the second most common CFTR genetic mutation, G551D, found in 4–5% of CF patients, boosts lung function in patients with CF with that mutation. If the ongoing studies with VIP are equally successful, the result could be an even bigger prize.
REFERENCES 1. Bodanszky M, Klausner YS, Lin CY, Mutt V, Said SI. Synthesis of the vasoactive intestinal peptide (VIP). J Am Chem Soc 1974;96:4973–8. 2. Delgado M, Pozo D, Ganea D. The significance of vasoactive intestinal peptide in immunomodulation. Pharmacol Rev 2004;56:249–90. 3. Fuxe K, Hökfelt T, Said SI, Mutt V. Vasoactive intestinal polypeptide and the nervous system: immunohistochemical evidence of localization in central and peripheral neurons, particularly intracortical neurons of the cerebral cortex. Neurosci Lett 1977;5:241–6. 4. Giachetti A, Said SI, Reynolds RC, Koniges FC. Vasoactive intestinal polypeptide (VIP) in brain: localization in, and release from, isolated nerve terminals. Proc Natl Acad Sci U S A 1977;74:3424–8. 5. Goyal RK, Rattan S, Said SI. VIP as a possible neurotransmitter of non-cholinergic non-adrenergic inhibitory neurones. Nature 1980;288:378–80. 6. Hamasaki Y, Mojarad M, Said SI. Relaxant action of VIP on cat pulmonary artery: comparison with acetylcholine, isoproterenol and PGE1. J Appl Physiol Respir Environ Exerc Physiol 1983;54:1607–11. 7. Hamidi SA, Szema AM, Lyubsky S, Dickman KG, Degene A, Mathew SM, Waschek JA, Said SI. Clues to VIP function from knockout mice. Ann NY Acad Sci 2006;1070:5–9.
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8. Heinz-Erian P, Flux M, Dey RD, Said SI. Deficient vasoactive intestinal peptide innervation in sweat glands of cystic fibrosis patients. Science 1985;229:1407–8. 9. Maruno K, Absood A, Said SI. VIP inhibits basal and histaminestimulated proliferation of human airway smooth muscle cells. Am J Physiol 1995;268:L1047–51:(Lung Cell Mol Physiol 12). 10. Maruno K, Absood A, Said SI. Vasoactive intestinal peptide inhibits human small-cell lung cancer proliferation in vitro and in vivo. Proc Natl Acad Sci U S A 1998;95:14373–8. 11. Matsuzaki Y, Hamasaki Y, Said SI. Vasoactive intestinal peptide: a possible transmitter of non-adrenergic relaxation of guinea pig airways. Science 1980;210:1252–3. 12. Mutt V, Said SI. Structure of the porcine vasoactive intestinal octacosapeptide: the amino acid sequence. Use of kallikrein in its determination. Eur J Biochem 1974;42:581–9. 13. Petkov V, Mosgoeller W, Ziesche R, Raderer M, Stiebellehner L, Vonbank K, et al. Vasoactive intestinal peptide as a new drug for treatment of primary pulmonary hypertension. J Clin Invest 2003;111:1339–46. 14. Rafferty S, Alcolado N, Norez C, Chappe F, Pelzer S, Becq F, et al. Rescue of functional F508del CFTR by VIP in the human nasal epithelial cell line JME/CF15. J Pharmacol Exp Ther 2009;331:2–13. 15. Saga T, Said SI. Vasoactive intestinal peptide relaxes isolated strips of human bronchus, pulmonary artery, and lung parenchyma. Trans Assoc Am Physicians 1984;97:304–10. 16. Said SI. Vasoactive substances in the lung. In: Proceedings of Tenth Aspen Emphysema Conference, Aspen, CO, June 7–10, 1967. U.S. Public Health Service Publication. 1967;1787:223–8. 17. Said SI. Vasoactive intestinal polypeptide and asthma (Editorial). New Engl J Med 1989;320:1271–3. 18. Said SI. Molecules that protect: the defense of neurons and other cells (Editorial). J Clin Invest 1996;97:2163–4. 19. Said SI. The Viktor Mutt Memorial Lecture: protection by VIP and related peptides against cell death and tissue injury. Ann NY Acad Sci 2000;921:264–74.
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20. Said SI. The VIP gene is a key modulator of pulmonary vascular remodeling and inflammation. Ann N Y Acad Sci 2008;1144:148–53. 21. Said SI, Faloona GR. Elevated plasma and tissue levels of vasoactive intestinal polypeptide in the watery diarrhea syndrome due to pancreatic, bronchogenic and other tumors. N Engl J Med 1975;293: 155–60. 22. Said SI, Mutt V. Long acting vasodilator peptide from lung tissue. Nature 1969;224:699–700. 23. Said SI, Mutt V. Polypeptide with broad biological activity: isolation from small intestine. Science 1970;169:1217–8. 24. Said SI, Mutt V. Isolation from porcine intestinal wall of a vasoactive octacosapeptide related to secretin and to glucagon. Eur J Biochem 1972;28:199–204. 25. Said SI, Porter JC. Vasoactive intestinal polypeptide: release into hypophyseal portal blood. Life Sci 1979;24:227–30. 26. Said SI, Rattan S. The multiple mediators of neurogenic smooth muscle relaxation. Trends Endocrinol Metab 2004;15:189–91. 27. Said SI, Rosenberg RN. Vasoactive intestinal polypeptide: abundant immunoreactivity in neural cell lines and normal nervous tissues. Science 1976;192:907–8. 28. Said SI, Berisha HI, Pakbaz H. Excitotoxicity in lung: N-methylD-aspartate-induced, nitric oxide-dependent, pulmonary edema is attenuated by vasoactive intestinal peptide and by inhibitors of poly (ADP-ribose) polymerase. Proc Natl Acad Sci U S A 1996;93: 4688–92. 29. Said SI, Hamidi SA, Dickman KG, Szema AM, Lyubski S, Lin RZ, et al. Moderate pulmonary arterial hypertension in male mice lacking the vasoactive intestinal peptide gene. Circulation 2007;115:1260–8. 30. Said SI, Dickman KG. Pathways of inflammation and cell death in the lung: modulation by vasoactive intestinal peptide. Regul Pept 2000;93:21–9. 31. Szema AM, Hamidi SA, Lyubsky S, Dickman KG, Mathew S, Abdel-Razek T, et al. Mice lacking the VIP gene show airway hyperresponsiveness and airway inflammation, partially reversible by VIP. Am J Physiol Lung Cell Mol Physiol 2006;291:L880–6.
VIP Sami I. Said, Valerie Chappe and Sayyed A. Hamidi
ABSTRACT The neuropeptide vasoactive intestinal peptide (VIP) is widely distributed in the central and peripheral nervous systems and is expressed in all pulmonary structures, including airway epithelial cells, airway and pulmonary vascular smooth muscle cells, secretory glands, and immune and inflammatory cells. VIP actions in the lung, mediated predominantly by the adenylyl cyclase–cAMP pathway and by receptors shared with PACAP, include relaxation of airway and vascular smooth muscle and inhibition of their proliferation, modulation of inflammatory and immune responses, protection against cell injury, and stimulation of water and electrolyte transport. Strong evidence suggests that physiological roles of VIP in the lung include cotransmission of airway and pulmonary vascular smooth muscle relaxation, modulation of immune and inflammatory responses, and guarding against the pathogenesis of bronchial asthma, pulmonary hypertension, and CFTR dysfunction in cystic fibrosis. Therefore, it seems probable that VIP has strong therapeutic potential in a variety of clinical disorders.
HOW WAS VIP DISCOVERED? In 1967, as a pulmonary physician in Richmond, Virginia, I (S.I.S.) wondered why serious acute events in the lung, such as severe pneumonia and the Acute Respiratory Distress Syndrome (ARDS), are often complicated by systemic hypotension. One possibility that appealed to me was that injured lung tissue releases into the circulation one or more vasodilator compounds. Preliminary experiments confirmed that supernatants of lung extracts, when injected into other animals, reduced systemic arterial blood pressure. This hypotension was not fully explained by the presence in lung extracts of the then-known vasodilators histamine, prostaglandins, and kallikreins, suggesting that one or more vasodilator peptides contributed to the phenomenon.16 To seek validation of this explanation, and identification of such a peptide, I decided to spend a sabbatical year at the Karolinska Institute, in Stockholm, Sweden, with Professor Viktor Mutt, a leading peptide biochemist, as my guide and mentor. Handbook of Biologically Active Peptides. http://dx.doi.org/10.1016/B978-0-12-385095-9.00209-8 Copyright © 2013 Elsevier Inc. All rights reserved.
Although we soon confirmed the presence of strong vasodilator activity in extracts of porcine lung, isolation of a pure peptide with this property was progressing too slowly, because of the difficulty in securing sufficient quantities of fresh lungs.22 Although lung extracts were in short supply, extracts of upper small intestine were plentiful in Viktor’s laboratory, because he was preparing the gastrointestinal hormones secretin and cholecystokinin on a large scale. One day, as I wished we were searching for our peptide in the intestine instead of in the lung, I recalled that both upper intestine and lungs originated from the same embryonic bud, the foregut, and decided to look for vasodilator activity in intestinal extracts. The search proved fruitful, culminating in the isolation of the “vasoactive intestinal peptide,” so named because, at that point, we did not know whether it also existed outside the intestine or whether it had any other biological activity. During its purification, the dominant biological activity of VIP was closely associated with that of secretin.23,24 Reading the original articles on the discovery of secretin, I noted that Bayliss and Starling observed some “vasodepressor” activity in the secretin-containing preparations, which they attributed to the presence of an unidentified compound that, in all likelihood, was VIP! Soon after VIP became available in pure form, its amino acid composition and sequence were identified,12 and it was successfully synthesized, by Miklos Bodanszky.1 After the development of a radioimmunoassay for the measurement of VIP content in plasma and tissues, and reports that the “watery diarrhea” or “pancreatic cholera” syndrome was attributable to excessive production of VIP by certain tumors, we began to receive biological samples from around the country and abroad, from patients suspected of having that disorder. Those assays revealed that the highest levels of VIP were associated with neurogenic tumors, especially neuroblastomas.21 In addition, analysis of neuroblastoma cell lines showed them to be rich in immunoreactive VIP. As I looked into the biology of these cells, I learned that they share many features with normal neurons, so I wondered whether VIP could also be produced by normal neurons. 1535
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With neurologist colleagues, we searched for the peptide in the human brain and peripheral nerves and found it to be widely and selectively present. VIP was thus rediscovered as a neuropeptide and a probable neurotransmitter.27
VIP IS A MEMBER OF A FAMILY OF PEPTIDES24 VIP is a 28-amino acid residue peptide (Fig. 1) that belongs to a family of structurally and functionally related peptides, including secretin, glucagon, gastric inhibitory peptide or glucose-dependent insulinotropic peptide (GIP), the peptide with N-terminal histidine and C-terminal isoleucine amide (PHI) and its counterpart in human tissues, the peptide with N-terminal histidine and C-terminal methionine amide (PHM), corticotropin-releasing hormone (CRH), growth hormone-releasing hormone (GHRH), sauvagine, urotensin I, helodermin, and the pituitary adenylate cyclase-activating polypeptides (PACAP), with which VIP shares the greatest degree of homology (Fig. 2).
TISSUE DISTRIBUTION AND CELLULAR LOCALIZATION The expression of VIP in different cells and organs has been defined by immunofluorescence, radioimmunoassay,
and more recently, by measurement of VIP mRNA. In the brain, the peptide is found in the highest concentrations in the cerebral cortex, suprachiasmatic nucleus, hippocampus, amygdala, and striatum, but it is also present in the midbrain periaqueductal gray and the sacral spinal cord. In the peripheral nervous system, VIP is localized in the sympathetic ganglia, the vagus nerve, predominantly motor nerves such as the sciatic nerve, and nerves supplying exocrine glands, blood vessels, and nonvascular smooth muscle.3–5,25 In the lungs, VIP-containing nerve fibers and nerve terminals are principally localized in the smooth muscle layer of the airways from the trachea through the small bronchioles, around submucosal mucous and serous glands, and in the walls of both pulmonary and bronchial arteries. Immunoreactive VIP is also present in neuronal cell bodies forming microganglia that provide a source of intrinsic innervation of pulmonary structures. In addition to its neuronal localization, VIP is richly present in the immune system, including inflammatory cells such as mast cells, eosinophils, and B and T lymphocytes.
COLOCALIZATION WITH OTHER PEPTIDES AND NEUROTRANSMITTERS VIP coexists with acetylcholine in most cholinergic neurons, both central and peripheral. In exocrine glands, this colocalization serves the dual functional advantages of promoting both blood flow and glandular secretion. VIP may also be colocalized with other peptides, including PHI (or PHM, its human counterpart), peptides that are synthesized from the same precursor molecule, and substance P. Other neurotransmitters that may coexist with VIP include nitric oxide (nitric oxide synthase) and the excitatory transmitter glutamic acid.
FIGURE 1 Amino acid sequence of VIP.
FIGURE 2 Some members of the VIP family of peptides. Amino acid homologies are noted by shading.
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BIOLOGICAL ACTIONS IN THE LUNG Airways Airway Smooth Muscle Tone5,6,11,15,17 VIP relaxes airway smooth muscle, both in vitro and in vivo, in guinea pigs, rats, mice, rabbits, dogs, and humans. VIP also counteracts airway smooth muscle contraction induced by a variety of bronchoconstrictor agents, including histamine, prostaglandin F2α, kallikrein, leukotriene D4, neurokinin A, serotonin, and endothelin (Fig. 3). Relative to most other bronchodilators, VIP-induced airway relaxation is longer lasting and is unaffected by blockade of adrenergic or cholinergic receptors or of cyclooxygenase activity. In limited trials in human asthmatics, however, VIP, given by inhalation, is generally less effective than expected in relieving bronchoconstriction, probably because of its degradation by proteases in airway secretions.
Airway Smooth Muscle Proliferation VIP inhibits human airway smooth muscle proliferation,9 an action that is potentially beneficial in reducing airway remodeling, which contributes to increased airway resistance in bronchial asthma.
Tracheobronchial Secretion VIP stimulates water and ion transport in canine tracheal epithelium and may also promote macromolecular secretion. The coexistence of VIP and acetylcholine in cholinergic neurons brings together the blood flow-promoting and secretion-promoting effects of VIP and acetylcholine, respectively.
Pulmonary Circulation6,13,15,20 VIP dilates the vessels supplying the nose, upper airways, trachea, and bronchi, as well as pulmonary vessels. As a
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pulmonary vasodilator, VIP is more potent than isoproterenol, acetylcholine, or prostacyclin, and its action is independent of endothelium. Given intravenously in sufficient concentrations, VIP reduces both pulmonary and systemic vascular resistance. Delivered as an aerosol, however, it acts as a selective pulmonary vasodilator. As with airway smooth muscle, VIP suppresses human pulmonary vascular smooth muscle proliferation, a major feature of pulmonary hypertensive disease.
Immune and Inflammatory Responses2,28,30 VIP modulates several aspects of the immune and inflammatory responses: (1) It has a moderate inhibitory effect on antigen-induced release of histamine from guinea pig lung. Because the peptide is normally present in mast cells, the peptide may act as a natural modulator of mast-cell degranulation, an important mechanism underlying acute asthmatic and anaphylactic reactions. (2) VIP also modulates T-cell activation and inhibits the expression of proinflammatory cytokines (TNF-α, IL-6, IL-12, IL-18) and chemokines (e.g. RANTES); inducible nitric oxide synthase (i NOS), and proteases (MMP-2 gelatinase), while upregulating the expression of anti-inflammatory cytokine IL-10 and promoting the generation of tolerogenic dendritic and regulatory T-cells.
Anti-Injury and Cell-Protective Effects18,19,30 Acting by a number of actions, VIP prevents or attenuates acute lung injury in a variety of experimental models. These actions include antiapoptotic, prosurvival effects that are mediated by 3 complementary and synergistic mechanisms: Inhibition of the activation of caspases, key effectors of apoptosis; upregulation of the antiapoptotic protein bcl2; and suppression of cytoplasmic translocation of cytochrome c, a critical step in mitochondrial-mediated apoptosis.
RECEPTORS AND SIGNAL TRANSDUCTION PATHWAYS
FIGURE 3 VIP as a tracheal smooth muscle relaxant. Isolated strip of guinea pig trachea was strongly contracted in response to endothelin 1. Application of VIP promptly and completely restored tracheal tension to resting level.
Specific, high-affinity receptors for VIP have been identified in membrane preparations of normal lungs and human lung tumor cells. These receptors, localized immunocytochemically and by the increased cyclic (c)AMP levels resulting from activation by VIP, exist in a variety of pulmonary cells. Molecular and pharmacologic studies have led to the identification, characterization, and cloning of at least three receptors for VIP and the related PACAP peptides (see the chapter in this section of the book by Mario Delgado). These receptors, known as VPAC receptors, belong to a distinct family of seven transmembrane-domain receptors coupled to G proteins. The three VPAC receptors are VPAC1 and VPAC2, both of which respond to VIP and PACAP with
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comparable affinity, and PAC1, which has greater affinity for PACAP than for VIP. The actions of VIP are mediated principally by adenylyl cyclase stimulation and cAMP production. VIP biosynthesis itself is promoted by higher cAMP levels, and thus, the peptide is potentially capable of stimulating its own generation. In some VIP actions, additional second messengers and signal transduction pathways are involved, for example, phospholipase C activation in the case of the PACAP-preferring PAC 1 receptor. Some actions of VIP, such as the relaxation of airway and vascular smooth muscle cells, are mediated partly by NO, released from neurons or smooth muscle cells. Through its activation of a constitutive NOS in neural elements and smooth muscle, leading to activation of cytosolic guanylyl cyclase, VIP can stimulate intracellular cyclic GMP formation. The contribution of neuronal NOS to VIP-induced relaxation of the trachea was examined in neuronal NOS-knockout mice and estimated to account for a significant proportion of the relaxation. It is now believed that both NO and carbon monoxide (CO) mediate VIP-induced relaxation of airway, gastrointestinal, and other smooth muscle. The combined activation of cyclic AMP and GMP pathways ensures greater smooth muscle relaxation, inhibition of mitogenesis, and smooth muscle cell proliferation.26
PHYSIOLOGICAL ROLES The physiological significance of VIP is suggested by 3 lines of evidence: 1. It is widely present, in a conserved structure, throughout the animal kingdom. 2. Deletion of the VIP gene alters the expression of almost 27% of all other genes. 3. VIP KO mice express the phenotypes of at least 4 disorders, which are correctable with VIP.7 Deletion of the VIP gene results in (i) bronchial asthma, manifested by airway hyperresponsiveness and airway inflammation; (ii) pulmonary arterial hypertension, with vascular remodeling, right ventricular hypertrophy, and perivascular inflammation; (iii) cystic fibrosis (CF)-like features, including dysfunctional CFTR, associated with structural abnormalities in the lung and gastrointestinal tract; and (iv) increased susceptibility to death from endotoxemia. Several lines of evidence suggest the following physiological roles for VIP, with special reference to the lungs: 1. Mediation of vascular and nonvascular smooth muscle relaxation: Together with NO and CO, VIP is a cotransmitter of neurogenic, noncholinergic, nonadrenergic smooth muscle relaxation. 2. Modulation of smooth muscle cell proliferation.
3. Modulation of inflammatory responses. 4. Modulation of immune responses and promotion of immune tolerance. 5. Enhancement of cell survival, and defense against injury and apoptosis: VIP exerts these effects by attenuating inflammation and reducing apoptotic cell death. For example, VIP protects lung cells against oxidant stress resulting from: xanthine + xanthine oxidase, paraquat, excitotoxicity (glutamate toxicity), and ischemia-reperfusion injury; as well as platelet-activating factor and endotoxin lipopolysaccharide. In addition, VIP also protects neuronal cells against injury, as by oxidants, for example, H2O2 and glutamate; as well as by envelope protein of HIV, nutrient or nerve growth factor deprivation, tetrodotoxin, and β-amyloid (Aβ). Further, as discussed in the chapter by Paul Gressens, VIP promotes embryonic brain growth and differentiation. 6. Stimulation of water and electrolyte transport: Among the earliest biological actions of VIP to be recognized is its ability to promote water and electrolyte transport. This is true for Cl− in bronchial and intestinal epithelium, and for HCO3− in pancreatic secretion. As earlier mentioned, mice lacking the VIP gene express CFlike pathophysiological features, and the role of VIP in the pathogenesis and possible modulation of the human disease is further discussed later. Finally, excessive stimulation of H2O/Cl− transport, in the presence of VIP-secreting tumors (VIPomas), results in the “watery diarrhea syndrome.”
VIP IN THE PATHOGENESIS AND MODULATION OF PULMONARY DISEASE Bronchial Asthma17,31 A close link between VIP deficiency and the pathogenesis of asthma is suggested by 3 sets of findings: 1. VIP-immunoreactive nerves are absent in airways of a group of severely asthmatic subjects who died accidentally. 2. Mice with targeted deletion of the VIP gene exhibit airway hyperresponsiveness and histopathologic features of airway inflammation. 3. As an airway smooth muscle cell relaxant, with antiinflammatory and antiproliferative activities, VIP is well qualified to counteract the pathophysiological features of bronchial asthma, including airway constriction, hyperresponsiveness, airway inflammation, and, in severe cases, smooth muscle proliferation. Despite its demonstrated effectiveness in animal models in vivo, however, the results of several clinical trials in asthmatics are less impressive than anticipated. The inhalation
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route would seem to be ideally suited for delivering VIP to asthmatics. Unfortunately, success of this approach seems to be limited by degradation of the inhaled peptide by peptidases in airway secretions. This problem may be solved by either of 2 alternate solutions, neither of which has been put to the test in clinical trials: (a) Instead of VIP itself, use a VIP-like analog that is more resistant to enzymatic degradation, such as the C-terminally extended helodermin (Fig. 2) or (b) combining VIP with one or more enzyme inhibitors that are particularly effective against VIP-selective peptidases, notably dipeptidyl peptidase (DPP IV) and neutral endopeptidase (enkephalinase).
Pulmonary Arterial Hypertension (PAH) Strong experimental evidence and 2 clinical studies suggest a useful therapeutic potential for VIP in PAH: 1. As noted above, in the absence of the VIP gene, mice express a PAH phenotype, with pulmonary vascular remodeling, inflammation, and right ventricular hypertrophy.29 RT-PCR arrays in these mice show the upregulated expression of pathways that promote vasoconstriction, smooth muscle and collagen proliferation, and inflammation, together with the downregulation of key pathways that promote vasodilation, and suppress vascular proliferation, including endothelial NOS (eNOS) and prostacyclin (PGI2). 2. VIP relaxes pulmonary vascular smooth muscle cells in vitro and in vivo.26 Further, it suppresses pulmonary vascular smooth muscle cell proliferation, and corrects pulmonary vascular remodeling, inflammation, and RV pathology in experimental PAH induced by (a) VIP gene deletion in mice, (b) monocrotaline in rats (Fig. 4), and (c) chronic hypoxia in mice. 3. In patients with Idiopathic Pulmonary Hypertension (IPAH), serum VIP concentrations are below normal, and VIP-containing nerves are absent in pulmonary arterial walls, whereas VIP receptors VPAC1 and VPAC2 are upregulated in pulmonary arterial smooth muscle cells.13
(A)
(B)
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Lung Cancer VIP suppresses the growth of small-cell lung cancer cells in culture and in athymic nude mice in vivo.10 These results suggest that VIP may offer an effective and less toxic alternative to conventional chemotherapy in the management of this highly malignant form of cancer, but no clinical trials have been conducted to validate this possibility.
Acute Lung Injury (ALI) and Related Conditions19,30 1. VIP protects the lungs and airways in at least 13 experimental models (Table 1). This protection is attributable to a combination of anti-inflammatory, antioxidant, and antiapoptotic properties. 2. Based on these encouraging data, a phase I clinical trial of VIP, funded by the Orphan Drug Program of the FDA, was conducted in 8 patients with relatively severe ARDS, with or without Multiorgan Dysfunction Syndrome (MODS), in the setting of sepsis. VIP was given by continuous iv infusion, for 6 or 12 h. The study confirmed the safety of the peptide under these conditions: of the 8 patients, only 2 died, with neither death being drug related. A follow-up, multicenter clinical trial to evaluate efficacy is being planned.
Inflammatory and Autoimmune Diseases2 Based on its anti-inflammatory and immune-modulating properties, VIP has undergone early trials in a number of clinical disorders and experimental models of such disorders, including pulmonary sarcoidosis, COPD, rheumatoid arthritis, experimental autoimmune encephalomyelitis, inflammatory bowel disease, and type I diabetes. It is still too early to draw firm conclusions from these studies.
(C)
FIGURE 4 VIP corrects pulmonary vascular and RV pathology in monocrotaline (MCT)-induced PAH in rats: (A) H&E section of normal control lung, showing alveoli, bronchus, and small PA. No evidence of inflammation or vascular thickening. (B) Lung section 3 weeks after injection of MCT, showing vascular thickening and inflammatory cell infiltrates. (C) 3 weeks after VIP treatment, almost complete reversal of MCT-induced pathology. See color plate 58.
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TABLE 1 Experimental Models of Acute Lung and Airway Injury Prevented by VIP (A) In isolated lung 1. HCl intratracheally 2. Platelet-activating factor into the PA 3. Xanthine + xanthine oxidase into the PA 4. Phospholipase C into the PA 5. Prolonged perfusion ex vivo 6. Paraquat into the PA 7. Capsaicin into the airways 8. Anaphylaxis 9. Eosinophil-mediated injury of cultured bronchial epithelial cells (B) In vivo: 10. Platelet-activating factor i.v. 11. Cobra venom factor 12. Endotoxemia 13. Excitotoxicity (NMDA receptor activation)
VIP and the NFAT Transcription Factor In considering the role of VIP in disease, it is important to keep in mind the relationship of VIP to transcription factor nuclear factor of activated T-cells (NFAT). Activation of calcineurin–NFAT signaling is triggered by increased concentration of intracellular Ca2+, which leads to dephosphorylation (i.e. activation) of the phosphatase enzyme calcineurin (Fig. 5). In its resting, inactive state, this enzyme is located in the cytosol; on activation, it enters the nucleus to act on its target genes. In addition to T-cell activation, activation of this pathway results in inflammation, smooth muscle cell contraction and proliferation, and autoimmune responses. Inhibition of calcineurin-mediated effects, as by cyclosporine A and the peptide VIVIT, can prevent organ transplant rejection. With special reference to VIP, mice lacking the VIP gene show activation of NFAT, and strong evidence suggests that VIP is a physiological modulator of this pathway.
CYSTIC FIBROSIS (CF) CF, a fatal autosomal recessive disease, is characterized by abnormal ion transport across epithelia, viscous secretions, chronic bacterial infections, and airway inflammation. Over 25 years ago, we first observed that normal skin had a rich network of VIP-immunoreactive nerves around sweat gland acini and ducts, in contrast to absent or minimal VIP innervation of CF sweat glands. We wondered (1) whether deficient VIP innervation is responsible for the chloride ion abnormality in CF; and (2) to what extent is VIP involved in the pathogenesis, or possible correction, of CF pathology?8
We now know that the basic molecular defect in CF is a deficiency or absence of the cystic fibrosis transmembrane conductance regulator (CFTR) from the apical membrane of epithelial cells, due to mutations in the CFTR gene.
CFTR and Its Mutations The most common (>90%) mutation in CF, a deletion of phenylalanine 508 (ΔF508), causes improper folding of the CFTR protein, resulting in its retention in the endoplasmic reticulum and proteosomal degradation. Major CF research efforts are devoted to the attempt to rescue ΔF508-CFTR defective trafficking to restore normal epithelial function. We have investigated the potential rescue and stability at the cell membrane of ΔF508-CFTR, by VIP treatment, in human nasal epithelial cells JME/CF15, derived from a patient with this homozygous mutation. When the cells, maintained at 37 °C, are treated with VIP for 1 or 2 h, mature ΔF508-CFTR proteins are observed: Immunostaining confirms localization at the cell membrane, and functional assays confirm that CFTR-dependent chloride secretion has been restored after VIP treatment. In these cells, which express the VPAC1 receptor, we found that VIP-dependent rescue of ΔF508-CFTR trafficking is mediated by the PKA-dependent signaling pathway, whereas membrane stability by prolonged VIP treatment involves the Gαq and PKCε signaling cascade. Evidence from airway cells thus shows that the VIP mechanisms of regulating CFTR activity involve both PKA and PKC signaling in a synergistic manner to rescue defective trafficking of mutant CFTR, activate CFTR gating through direct phosphorylation of its regulatory domain, and most importantly, stabilize CFTR channels at the cell surface by reducing their internalization rate, thus optimizing CFTR-dependent secretions.
In Vivo Confirmation of the Role of VIP14 As earlier mentioned above, VIP knockout mice express several features of the absence of the VIP gene, including an asthma-like phenotype, a PAH phenotype, and increased vulnerability to endotoxemia. In addition, they also show CF-like pathological changes: the lungs show significant lymphocyte accumulation, increased airway secretion, alveolar thickening, and edema; the pancreas also shows increased secretion, and inflammatory cell infiltrates around the ducts. These pathological changes could be reversed, close to a wild-type phenotype, by treatment with VIP (15 µg) intraperitoneally, every other day, for 3 weeks. In this model, whereas normal control tissues show CFTR predominantly at the apical membrane of epithelial cells, CFTR distribution in VIP KO tissues is mainly observed intracellularly. VIP treatment restores strong CFTR membrane localization, confirming the important role of chronic
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FIGURE 5 Calcineurin–NFAT signaling and its inhibition by VIP. In resting cells, NFAT resides in the cytoplasm, where it is maintained in a phosphorylated, inactive state. Stimulation of cell-surface receptors coupled to phospholipase C (PLC) results in calcium mobilization, initially from intracellular stores, followed by influx of Ca2+ through specialized voltage-dependent Ca2+ channels (VDCC) and Ca2+ release-activated Ca2+ channels (CRAC). On activation by Ca2+, the phosphatase calcineurin dephosphorylates NFAT, triggering its activation and nuclear translocation. In the nucleus, NFAT interacts with other transcription factors, including activator protein (AP)-1, to stimulate gene expression. When NFAT is rephosphorylated, it is exported back to the cytoplasm. The immunosuppressive drug cyclosporin A inhibits calcineurin interactions with all its substrates, whereas VIVIT selectively inhibits NFAT activation. VIP seems to inhibit this pathway, but its mechanism of action is yet to be determined. IP3: inositol-1,4,5-triphosphate. See color plate 59.
VIP exposure tin maintaining CFTR channels at the membrane, and for exocrine epithelial tissues integrity. We conclude that inflammation and damage observed in VIP KO tissues can be attributed, at least in part, to the lack of CFTR-dependent secretions that ultimately depend on VIP stimulation, both for acute and long-term regulation of CFTR function. These observations also provide evidence of the molecular link between early observations of deficient VIP-containing fibers innervation of epithelial layers of exocrine organs in CF tissues and the absence of CFTRdependent secretions. The results set VIP, or its analogs, as a potential candidate for the treatment of CF as it corrects many features of this disease including its molecular basis. Ivacaftor, an investigational drug targeting the second most common CFTR genetic mutation, G551D, found in 4–5% of CF patients, boosts lung function in patients with CF with that mutation. If the ongoing studies with VIP are equally successful, the result could be an even bigger prize.
REFERENCES 1. Bodanszky M, Klausner YS, Lin CY, Mutt V, Said SI. Synthesis of the vasoactive intestinal peptide (VIP). J Am Chem Soc 1974;96:4973–8. 2. Delgado M, Pozo D, Ganea D. The significance of vasoactive intestinal peptide in immunomodulation. Pharmacol Rev 2004;56:249–90. 3. Fuxe K, Hökfelt T, Said SI, Mutt V. Vasoactive intestinal polypeptide and the nervous system: immunohistochemical evidence of localization in central and peripheral neurons, particularly intracortical neurons of the cerebral cortex. Neurosci Lett 1977;5:241–6. 4. Giachetti A, Said SI, Reynolds RC, Koniges FC. Vasoactive intestinal polypeptide (VIP) in brain: localization in, and release from, isolated nerve terminals. Proc Natl Acad Sci U S A 1977;74:3424–8. 5. Goyal RK, Rattan S, Said SI. VIP as a possible neurotransmitter of non-cholinergic non-adrenergic inhibitory neurones. Nature 1980;288:378–80. 6. Hamasaki Y, Mojarad M, Said SI. Relaxant action of VIP on cat pulmonary artery: comparison with acetylcholine, isoproterenol and PGE1. J Appl Physiol Respir Environ Exerc Physiol 1983;54:1607–11. 7. Hamidi SA, Szema AM, Lyubsky S, Dickman KG, Degene A, Mathew SM, Waschek JA, Said SI. Clues to VIP function from knockout mice. Ann NY Acad Sci 2006;1070:5–9.
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8. Heinz-Erian P, Flux M, Dey RD, Said SI. Deficient vasoactive intestinal peptide innervation in sweat glands of cystic fibrosis patients. Science 1985;229:1407–8. 9. Maruno K, Absood A, Said SI. VIP inhibits basal and histaminestimulated proliferation of human airway smooth muscle cells. Am J Physiol 1995;268:L1047–51:(Lung Cell Mol Physiol 12). 10. Maruno K, Absood A, Said SI. Vasoactive intestinal peptide inhibits human small-cell lung cancer proliferation in vitro and in vivo. Proc Natl Acad Sci U S A 1998;95:14373–8. 11. Matsuzaki Y, Hamasaki Y, Said SI. Vasoactive intestinal peptide: a possible transmitter of non-adrenergic relaxation of guinea pig airways. Science 1980;210:1252–3. 12. Mutt V, Said SI. Structure of the porcine vasoactive intestinal octacosapeptide: the amino acid sequence. Use of kallikrein in its determination. Eur J Biochem 1974;42:581–9. 13. Petkov V, Mosgoeller W, Ziesche R, Raderer M, Stiebellehner L, Vonbank K, et al. Vasoactive intestinal peptide as a new drug for treatment of primary pulmonary hypertension. J Clin Invest 2003;111:1339–46. 14. Rafferty S, Alcolado N, Norez C, Chappe F, Pelzer S, Becq F, et al. Rescue of functional F508del CFTR by VIP in the human nasal epithelial cell line JME/CF15. J Pharmacol Exp Ther 2009;331:2–13. 15. Saga T, Said SI. Vasoactive intestinal peptide relaxes isolated strips of human bronchus, pulmonary artery, and lung parenchyma. Trans Assoc Am Physicians 1984;97:304–10. 16. Said SI. Vasoactive substances in the lung. In: Proceedings of Tenth Aspen Emphysema Conference, Aspen, CO, June 7–10, 1967. U.S. Public Health Service Publication. 1967;1787:223–8. 17. Said SI. Vasoactive intestinal polypeptide and asthma (Editorial). New Engl J Med 1989;320:1271–3. 18. Said SI. Molecules that protect: the defense of neurons and other cells (Editorial). J Clin Invest 1996;97:2163–4. 19. Said SI. The Viktor Mutt Memorial Lecture: protection by VIP and related peptides against cell death and tissue injury. Ann NY Acad Sci 2000;921:264–74.
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20. Said SI. The VIP gene is a key modulator of pulmonary vascular remodeling and inflammation. Ann N Y Acad Sci 2008;1144:148–53. 21. Said SI, Faloona GR. Elevated plasma and tissue levels of vasoactive intestinal polypeptide in the watery diarrhea syndrome due to pancreatic, bronchogenic and other tumors. N Engl J Med 1975;293: 155–60. 22. Said SI, Mutt V. Long acting vasodilator peptide from lung tissue. Nature 1969;224:699–700. 23. Said SI, Mutt V. Polypeptide with broad biological activity: isolation from small intestine. Science 1970;169:1217–8. 24. Said SI, Mutt V. Isolation from porcine intestinal wall of a vasoactive octacosapeptide related to secretin and to glucagon. Eur J Biochem 1972;28:199–204. 25. Said SI, Porter JC. Vasoactive intestinal polypeptide: release into hypophyseal portal blood. Life Sci 1979;24:227–30. 26. Said SI, Rattan S. The multiple mediators of neurogenic smooth muscle relaxation. Trends Endocrinol Metab 2004;15:189–91. 27. Said SI, Rosenberg RN. Vasoactive intestinal polypeptide: abundant immunoreactivity in neural cell lines and normal nervous tissues. Science 1976;192:907–8. 28. Said SI, Berisha HI, Pakbaz H. Excitotoxicity in lung: N-methylD-aspartate-induced, nitric oxide-dependent, pulmonary edema is attenuated by vasoactive intestinal peptide and by inhibitors of poly (ADP-ribose) polymerase. Proc Natl Acad Sci U S A 1996;93: 4688–92. 29. Said SI, Hamidi SA, Dickman KG, Szema AM, Lyubski S, Lin RZ, et al. Moderate pulmonary arterial hypertension in male mice lacking the vasoactive intestinal peptide gene. Circulation 2007;115:1260–8. 30. Said SI, Dickman KG. Pathways of inflammation and cell death in the lung: modulation by vasoactive intestinal peptide. Regul Pept 2000;93:21–9. 31. Szema AM, Hamidi SA, Lyubsky S, Dickman KG, Mathew S, Abdel-Razek T, et al. Mice lacking the VIP gene show airway hyperresponsiveness and airway inflammation, partially reversible by VIP. Am J Physiol Lung Cell Mol Physiol 2006;291:L880–6.