Recent advances in vasoactive intestinal peptide physiology and pathophysiology: focus on the gastrointestinal system

Introduction

Vasoactive intestinal peptide (VIP) is a 28-residue amino acid peptide first characterized in 1970 that was initially isolated from porcine duodenum¹. A member of the secretin/glucagon hormone superfamily^1,2, VIP is evolutionarily well conserved with sequence similarity among fish, frogs, and humans³; among mammals, except for guinea pigs and chickens⁴, the sequence similarity is at least 85%⁵. VIP was initially discovered owing to its potent vasodilatory effects (as its name implies). VIP is widely distributed in the central and peripheral nervous system as well as in the digestive, respiratory, reproductive, and cardiovascular systems as a neurotransmitter and neuroendocrine releasing factor^5,6. These effects contribute to an extensive range of physiological and pathological processes related to development, growth, and the control of neuronal, epithelial, and endocrine cell function. VIP has also been implicated in the regulation of carcinogenesis, immune responses, and circadian rhythms⁷. Here, we focus on current findings related to VIP and its signals in the gastrointestinal (GI) tract with regard to its effects on secretion, intestinal barrier function, and mucosal immunology.

Historical background

In the late 1960s, Dr. Sami I. Said at the Medical College of Virginia reported that systemic injection of extracts of mammalian lungs produced generalized vasodilation and hypotension. Together with Dr. Viktor Mutt from Karolinska University, Stockholm, Sweden, Dr. Said turned his search from the lung to duodenal extracts, which were more readily available, based on the premise that the same peptide might be present in other organs. They soon discovered that peptide fractions from porcine duodenum indeed contained a component with vasodilatory activity⁸, supporting Bayliss and Starling’s assumption (made in 1902 during their discovery of secretin) that a “vasodepressor principle” was present in intestinal extracts⁹.

A few years later, VIP was identified in the central and peripheral nervous systems¹⁰ and has since been recognized as a widely distributed neuropeptide, acting as a neurotransmitter or neuromodulator in many organs and tissues, including the heart, lung, thyroid gland, kidney, immune system, urinary tract, and genital organs³. VIP’s presence across numerous locations is related to its participation in a vast number of biological events¹¹.

Structure and classification

The three-dimensional structure of VIP is similar to that of other members of the glucagon and secretin family², in which the structure, function, and signaling activity of pituitary adenylyl cyclase-activating peptide (PACAP) is the most closely related peptide to VIP, sharing 68% sequence homology¹¹. VIP is cleaved from a ~9 kb precursor molecule, prepro-VIP, located in the chromosomal region 6q24 containing seven exons⁶, each encoding a functional domain. The signal peptidase located in the endoplasmic reticulum cleaves the signal peptide from the 170-amino-acid prepro-VIP, then forms a 149-amino-acid precursor peptide termed pro-VIP, which is then cleaved by prohormone convertases to a form of VIP precursor containing the internal cleave-amidation site Gly–Lys–Arg (GKR) (VIP–GKR; prepro-VIP_125–155)¹² (Figure 1). The KR residues of VIP-GKR are then cleaved by carboxypeptidase B-like enzymes to VIP-G¹³, which is then metabolized by peptidyl-glycine alpha-amidating monooxygenase (PAM) to VIP, which has an amidated C-terminus¹¹ (Figure 1). Prepro-VIP also contains a bioactive hormone, peptide histidine methionine (PHM) in humans or peptide histidine isoleucine (PHI) in other mammals; PHM/PHI are less potent than VIP¹⁴. VIP varies its conformation depending on the environment. Most notably, its α-helical forms are present when VIP is in the presence of an anionic lipid bilayer or liposomes when bound to receptors⁵.

Figure 1. Processing of prepro-VIP to VIP.

PHI, peptide histidine isoleucine; PHM, peptide histidine methionine; VIP, vasoactive intestinal peptide; VIP–GKR, VIP precursor containing the internal cleave-amidation site Gly–Lys–Arg.

VIP and its receptors

The two receptors that recognize VIP, termed VPAC1 and VPAC2, are class B of G-protein-coupled receptors (GPCRs), also known as the secretin receptor family, which includes receptors for VIP, PACAP, secretin, glucagon, glucagon-like peptide (GLP)-1 and -2, calcitonin, gastric inhibitory peptide (GIP), corticotropin-releasing factor (CRF)-1 and -2, and parathyroid hormone (PTH). VPAC1 and VPAC2 are activated by VIP and PACAP¹⁵, whereas PACAP has its own specific receptor, named PAC1, for which VIP has very low affinity¹⁶. Through these receptors, VIP can mediate an extensive number of GI functions such as regulating gastric acid secretion, intestinal anion secretion, enzyme release from the pancreas, cellular motility, vasodilation, and intestinal contractility^17–19. The localization of VIP, VPAC1, and VPAC2 is closely related to their physiological and pathological functions, which are also discussed under the heading “Functions in the GI tract”.

Functions in the GI tract

VIP and irritable bowel syndrome

Irritable bowel syndrome (IBS) is a chronic symptomatic GI disorder characterized by abdominal pain with altered bowel function, typically constipation and/or diarrhea. IBS with diarrhea (IBS-D) correlates with increased mast cell function and VIP release. Mast cell number and tissue immunoreactivity for substance P and VIP are greater in IBS-D patients, especially in women⁸⁷. A recent study shows that female IBS patients have higher plasma VIP and higher mast cell tryptase content and mast cell number in colonic biopsies compared to data from controls⁸⁸. Furthermore, colonic biopsies show greater transcellular bacterial passage and a higher percentage of mast cells that express VPAC1 than do biopsies from controls. Bacterial passage through the colonic biopsies was inhibitable with anti-VPAC antibodies or with the mast cell stabilizer ketotifen⁸⁸. These data suggest that mast cells and VIP are key modifiers of bacterial translocation in the colonic mucosa of IBS patients. However, the observations of colonic mucosal barrier function and the roles of VIP and mast cells in colonic biopsies require confirmation in patients with IBS in vivo.

Stress is a key factor in IBS pathogenesis. One of the stress-induced hormones is corticotropin-releasing factor (CRF), which is an important bioactive molecule not only in the central nervous system but also in the peripheral enteric nervous system. Stress-induced defecation and diarrhea in rodents is induced by peripheral administration of CRF via CRF1 receptor activation⁸⁹. Peripheral CRF-induced defecation and diarrhea involves VIP signals via the activation of CRF1-positive VIPergic submucosal neurons⁹⁰, suggesting that stress-induced diarrhea observed in IBS-D patients can be treated with VPAC1 antagonists that reduce the volume and frequency of bowel movements⁵⁸.

VIP and immunity

The GI mucosa is the largest immune system in the body, likely owing to its status as the largest area of interface with the outside world. The GI tract contains luminal microbiota and numerous immune cells in the epithelium, lamina propria mucosa, and lymphoid follicles⁹¹. VIP, as an anti-inflammatory mediator, downregulates the abundance of pro-inflammatory cytokines and mediators such as TNF-α, IL-6, IL-12, nitric oxide, and chemokines⁹². VIP, which is also produced by type 2 lymphocytes (Th2), could also be classified as a Th2 cytokine^31,92. The potent anti-inflammatory effects of VIP may result from its promotion of Th cell differentiation toward a “Th2” phenotype¹¹. Moreover, VIP also increases regulatory T cell production while inhibiting macrophage pro-inflammatory actions, all contributing to its anti-inflammatory effects.

VIP maintains immunological tolerance and homeostasis in the gut primarily by regulation of T cell responses and Toll-like receptor (TLR)-mediated innate immune responses. VPAC1 is primarily expressed on T cells, whereas VPAC2 expression is induced by inflammation⁹². The anti-inflammatory effects of VIP are principally mediated via VPAC2 activation, which suppresses Th1 and Th17 functions and induces Th2 and regulatory T cells, resulting in immunosuppression⁸⁶. Therefore, the immunomodulatory actions of VIP expand its abilities to treat acute and chronic inflammatory and autoimmune diseases, including sepsis⁹³, multiple sclerosis⁹⁴, Crohn’s disease⁹⁵, and type 1 diabetes⁹⁶.

VIP and inflammatory bowel diseases

VIP was proposed as a biomarker for inflammatory bowel disease (IBD) such as Crohn’s disease and ulcerative colitis in a study reporting elevated VIP plasma concentrations during the active inflammatory disease phase⁹⁷. A recent study also reported that VIP content is higher in plasma and in ileal or colonic tissues resected from Crohn’s disease or ulcerative colitis patients, respectively, than those from healthy subjects⁹⁸. Furthermore, the anti-inflammatory properties of VIP on Th1 immunity, which is involved in autoimmune diseases including IBD, suggest that VIP is involved in the pathogenesis of IBD and may be a therapeutic target. Nevertheless, the connection of VIP with animal models of colitis related to IBD has yet to be fully elucidated. The contribution of VIP towards the pathogenesis of dextran sulfate sodium (DSS)-induced and 2,4,6-trinitrobenzene sulfonic acid (TNBS)-induced models of colitis in mice is controversial¹⁸.

The first report regarding VIP and colitis is that exogenous VIP improves TNBS-induced colitis in BALB/c mice, most likely via VPAC1 activation with anti-inflammatory and Th1–Th2 switching effects of VIP⁹⁹. Notably, higher doses of VIP likely aggravate the colitis⁹⁹. Later, another group reported that VIP administration by constant infusion enhanced the severity of TNBS-induced colitis¹⁰⁰. Subsequently, genetically modified animal models have been used to clarify the contributions of endogenous VIP and its receptors to the pathogenesis of colitis. In the DSS-induced colitis model, VPAC1 KO mice are resistant to DSS-induced colitis, whereas colitis is exacerbated in VPAC2 KO mice; PKA inhibitors reverse the impairment of DSS colitis in VPAC2 KO mice, suggesting that enhanced VPAC1 activity in VPAC2 KO mice may aggravate DSS colitis⁵⁶ or is alternatively explained by the protective effects of VPAC2 during the development of DSS-induced colitis, since VPAC2 activation inhibits Th1 signals¹¹.

In VIP KO mice, DSS treatment had no effect on colitis in males, compared to wild-type males, whereas body weight loss and disease activity index in females was less frequently observed in VIP KO subjects⁴⁰, suggesting that VIP may have enhanced pro-inflammatory functions in females. Furthermore, male VIP KO mice or wild-type mice treated with a pan-VIP receptor antagonist (VIP-hybrid¹⁰¹) or the selective VPAC1 antagonist (PG97-269)¹⁵ are resistant to DSS-induced colitis with reduced levels of colonic inflammatory mediators and cytokines¹⁰², suggesting that VIP acts as a pro-inflammatory mediator. In TNBS-colitis, VIP KO mice are resistant to colitis with lower levels of TNF-α and IL-6¹⁰³. Similar resistant phenotypes are observed in a VIP KO with LPS-induced endotoxemia model, where LPS induced less mortality in VIP KO mice³⁶, and with the experimental autoimmune encephalomyelitis (EAE) model, where clinical scores were less in VIP KO mice¹⁰⁴. Nevertheless, VIP KO mice develop more severe colitis in the DNBS- or DSS-induced colitis models, which is rescued by exogenous VIP treatment³⁷. Most recently, a recombinant stable VIP analog (rVIPa) was reported to ameliorate TNBS-induced colonic injury and inflammation, effectively preserving intestinal mucosal barrier function in rats¹⁰⁵, most likely owing to increased stability of the VIP analog.

These discrepancies between anti-inflammatory and pro-inflammatory effects of VIP on chemically induced colitis models may reflect the differences between endogenous and exogenous effects of VIP due to dose effects and peptide stability in the tissues and circulation because VIP is rapidly degraded by dipeptidyl peptidase 4 (DPP4), similar to the incretins¹⁰⁶, and by other peptidases. Furthermore, genetic deficiency of VIP or VPAC irreversibly alters epithelial, neural, and immune responses during development. Another possibility is that targets of VIP may induce opposite effects on inflammation; VPAC2 activation on T cells shifts Th1 to Th2 differentiation as anti-inflammatory, whereas VPAC1 activation of epithelial cells increases anion and water secretion, with resultant diarrhea, which may affect the disease activity of colitis. VPAC2 activation of GI smooth muscles increases GI motility, whereas impaired motility in VIP KO or VPAC2 KO may affect GI transit, affecting the exposure time to luminal toxic chemicals such as DSS in drinking water. Therefore, cell-specific, conditional knockout will clarify these contradictory results.

VIP/PACAP and diabetes

The metabolic syndrome, including type 2 diabetes and obesity, is also a GI-related disorder, since insulinotropic hormones, termed incretins, including GLP-1 and GIP, are secreted from enteroendocrine L and K cells, respectively. As mentioned above, vagal stimulation increases the release of PACAP and VIP in pancreatic islets, suggesting that PACAP and VIP modulate insulin secretion from β cells through the activation of cognate receptors.

Pancreatic islet β cells express PAC1 and VPAC2 with less VPAC1²⁸. Selective VPAC2 agonists are insulinotropic, similar to PACAP and GLP-1, amplifying glucose-induced insulin secretion¹⁰⁷. VIP KO mice exhibit elevated plasma glucose, insulin, and leptin levels with no change in islet mass³⁸, probably due to the compensatory effect of PACAP. In VPAC2 KO mice, glucose-induced insulin secretion is decreased with no change in glucose tolerance. VPAC1 KO mice show growth retardation, intestinal obstruction, and hypoglycemia⁴⁸, suggesting that VPAC1 is also involved in glucagon secretion, which counteracts the hypoglycemic effects of insulin. In isolated perfused pancreas, PAC1 KO mice exhibit a 50% reduction of the PACAP-induced insulin secretory response, whereas VIP-induced insulin secretion is unchanged¹⁰⁸, suggesting that the insulinotropic action of PACAP is partially mediated by PAC1. Therefore, VPAC2 agonists and PAC1 agonists are candidates for the therapy of type 2 diabetes.

VIP/PACAP and cancers

Human cancers including bladder, breast, colon, liver, lung, pancreatic, prostate, thyroid, and uterine cancers often overexpress VPAC1, whereas VPAC2 is limited in stromal tumors such as gastric leiomyomas, sarcomas, and neuroendocrine tumors¹⁰⁹. Since VPAC1 is normally expressed in the epithelium and VPAC2 in smooth muscle in the GI tract, these expression profiles may reflect their tumor expression with VPAC1 in adenocarcinoma and VPAC2 in stromal tumors. PAC1 is also expressed in diverse tumors including brain, breast, colon, lung, neuroendocrine, pancreas, pituitary, and prostate tumors as well as neuroblastomas¹¹⁰. This suggests that VIP/PACAP may affect tumor growth and differentiation. VIP and PACAP stimulate the growth of several cancer cell lines in vitro¹¹⁰, supporting this hypothesis.

Regarding the GI tract, colon cancer tissue overexpresses VPAC1: in 35% of well-differentiated, 65% of moderately differentiated, and 87% of poorly differentiated colon cancers¹¹¹, predicting tumor differentiation can be accomplished by measuring VPAC1 levels. Therefore, VPAC1 can be a target for anti-cancer drugs, since VPAC1 antagonists inhibit the growth of colonic cancer cell lines in vitro¹¹².

Overexpression of VPAC and PAC1 in tumors can be used for imaging and targeting tumors using radiolabeled VIP analogues. Clinical studies show that radiolabeled VIP analogues localize breast cancer, pancreatic cancer, intestinal adenocarcinomas, neuroendocrine tumors, and colorectal cancer using a combination of positron emission tomography (PET) and computed tomography (CT) scans^110,113. Furthermore, VIP-conjugated nanoparticles have been developed to deliver the cytotoxic drug to tumor cells overexpressing VPAC¹¹⁴.

Therapeutic potential of VIP

Since VIP contributes to important physiological functions including anion secretion, the regulation of permeability of epithelial tight junctions, mucosal inflammation, glycemic control, Th1–Th2 balance, and tumor growth, VIP has been suggested to be a therapeutic target for diseases such as diarrhea⁵⁸, IBD⁹⁵, diabetes²⁸, autoimmune diseases¹¹⁵, neurodegenerative disorders¹¹⁶, lung disease^117,118, sarcoidosis¹¹⁹, and cancers¹¹⁴. Although VIP has well-studied anti-inflammatory and other therapeutic potential, VIP-based drug design has not been entirely successful because rapid degradation of the peptide limits its bioavailability and delivery. Furthermore, multiple cellular targets that bind VIP at high affinity may cause undesirable adverse effects. Therefore, synthesis of a stable VIP analog, or the targeted delivery of VIP or its analogs via nanoparticles are desirable options.

Recent advances in the field include the synthesis of stable analogs such as lipophilic or peptide VIP derivatives that mimic the activity of native VIP¹²⁰. Another strategy is VIP self-associated with sterically stabilized micelles, which protects VIP from degradation and inactivation¹¹⁵. Injection of VIP-induced regulatory dendritic cells ameliorates TNBS-induced colitis models in mice⁹⁵. VIP gene transfer using lentivirus is also useful to induce immunosuppression in the murine arthritis model¹²¹. Finally, VIP-tagged nanoparticles may be a useful strategy for selective drug delivery to VPAC-overexpressing tumor cells and immune cells^114,122.

Summary and conclusions

Since its discovery in 1970, VIP has been studied in numerous organ systems including the gastrointestinal, respiratory, cardiovascular, immune, endocrine, and central and peripheral nervous systems, where it exerts numerous important effects (Figure 2). Nevertheless, owing to its protean and widespread effects on numerous organ systems combined with its inherent instability, VIP has been challenging to clearly discern and analyze regarding its influence on isolated pathophysiological functions. Specifically, in the gut, VIP has therapeutic potential for a variety of inflammatory disorders such as IBD. The recent progress of VIP-related medicine is aimed at improvement of its stability, selectivity, and efficacy with reduced adverse effects. In order to make optimal therapeutic use of, it is essential to further study its localization and actions, working towards selective targeting or individual effects.

Figure 2. Broad multiple functions of vasoactive intestinal peptide in various organs.

Number in parenthesis represents the corresponding reference number.