Epithelial restitution and wound healing in inflammatory bowel disease

Abstract

Inflammatory bowel disease is characterized by a chronic inflammation of the intestinal mucosa. The mucosal epithelium of the alimentary tract constitutes a key element of the mucosal barrier to a broad spectrum of deleterious substances present within the intestinal lumen including bacterial microorganisms, various dietary factors, gastrointestinal secretory products and drugs. In addition, this mucosal barrier can be disturbed in the course of various intestinal disorders including inflammatory bowel diseases. Fortunately, the integrity of the gastrointestinal surface epithelium is rapidly reestablished even after extensive destruction. Rapid resealing of the epithelial barrier following injuries is accomplished by a process termed epithelial restitution, followed by more delayed mechanisms of epithelial wound healing including increased epithelial cell proliferation and epithelial cell differentiation. Restitution of the intestinal surface epithelium is modulated by a range of highly divergent factors among them a broad spectrum of structurally distinct regulatory peptides, variously described as growth factors or cytokines. Several regulatory peptide factors act from the basolateral site of the epithelial surface and enhance epithelial cell restitution through TGF-β-dependent pathways. In contrast, members of the trefoil factor family (TFF peptides) appear to stimulate epithelial restitution in conjunction with mucin glycoproteins through a TGF-β-independent mechanism from the apical site of the intestinal epithelium. In addition, a number of other peptide molecules like extracellular matrix factors and blood clotting factors and also non-peptide molecules including phospholipids, short-chain fatty acids (SCFA), adenine nucleotides, trace elements and pharmacological agents modulate intestinal epithelial repair mechanisms. Repeated damage and injury of the intestinal surface are key features of various intestinal disorders including inflammatory bowel diseases and require constant repair of the epithelium. Enhancement of intestinal repair mechanisms by regulatory peptides or other modulatory factors may provide future approaches for the treatment of diseases that are characterized by injuries of the epithelial surface.

Key Words: Intestines; Wound healing; Inflammation; Restitution

THE MUCOSAL DEFENSE SYSTEM

The surface of the digestive tract is covered by epithelial cells that constitute an efficient physical barrier between the dietary and enteric flora pathogens found in the intestinal lumen and the individuum, but also allows an exchange between nutrients and the systemic circulation[1]. The epithelial defense mechanism can be categorized into three key components: pre-epithelial, epithelial and post-epithelial, the latter is represented by the lamina propria[2]. The pre-epithelial mucus barrier is composed of mucin associated with other proteins and lipids and forms a continuous gel into which a bicarbonate-rich fluid is secreted, maintaining a neutralizing pH at the epithelial surface. Phosphatidylcholine is the predominant surface bioactive phospholipid found within the gastrointestinal tract[3]. Intestinal epithelial cells secrete mucins and glycocalyx, which contain membrane-anchored negatively charged mucin-like glycoproteins and hydrophobic phospholipids[4]. The tight adherence of mucin to the apical surfaces of epithelia is owed to the existence of the specific complex between mucin oligosaccharides and the mucin binding protein of the apical mucosal membrane[5]. The hydrophobic lining of the luminal surface has an important functional role. It prevents microorganisms from getting into contact with and to adhering to the plasma membrane. It furthermore protects the mucosal epithelium against chemical and mechanical injuries[6]. Epithelial cells provide the second line of the mucosal defense system. Whereas in the upper digestive tract this layer consists of a stratified epithelium, the stomach, small, and large bowel are surfaced with a simple epithelial layer sealed by tight junctions[7]. When intact, the uptake of antigens, macro- and microorganism through this layer is restricted by luminal cell-surface structures. The mucosal surface epithelial cells are rapidly proliferating with a complete turnover every 24 to 96 h[8]. The proliferative compartment of epithelial cells is localized in the crypt region and is segregated from a gradient of increasingly differentiated epithelial cells present along the vertical axis of the functional villus compartment[910].

INTESTINAL WOUND HEALING

Damage and impairment of the intestinal surface barrier are observed in the course of various diseases and may result in an increased penetration and absorption of toxic and immunogenic factors into the body leading to inflammation, uncontrolled immune response, and disequilibrium of the homeostasis of the host. Thus, rapid resealing of the epithelial surface barrier following injuries or physiological damage is essential to preserve the normal homeostasis. Observations over the past several years have demonstrated the ability of the intestinal tract to rapidly reestablish the continuity of the surface epithelium after extensive destruction[11–14]. The continuity of the epithelial surface is reestablished by at least three distinct mechanisms. First, epithelial cells adjacent to the injured surface migrate into the wound to cover the denuded area. Those epithelial cells that migrate into the wound defect dedifferentiate, form pseudopodia-like structures, reorganize their cytoskeleton, and redifferentiate after closure of the wound defect. This process has been termed epithelial restitution and does not require cell proliferation[15]. Intestinal epithelial restitution occurs within minutes to hours both in vivo and in vitro. Secondly, epithelial cell proliferation is necessary to replenish the decreased cell pool. Third, maturation and differentiation of undifferentiated epithelial cells is needed to maintain the numerous functional activities of the mucosal epithelium. The separation of intestinal epithelial wound healing in three distinct processes is rather artificial and simplified. These three wound-healing processes overlap and distinct processes may not be observed in vivo where these processes overlap. The preservation of this barrier following injuries is regulated by a broad spectrum of structurally distinct regulatory factors, including cytokines, growth factors, adhesion molecules, neuropeptides and phospholipids[16–19]. However, this artificial and simplified model provides a tool to better understand the physiology and pathophysiology of intestinal epithelial wound healing. Moreover, deeper lesions or penetrating injuries will require additional repair mechanisms that involve inflammatory processes and non-epithelial cell populations. Inflammatory processes especially may interfere with epithelial cell migration and proliferation and thus modulate intestinal epithelial healing.

IMPORTANT MODULATORS OF THE INTESTINAL EPITHELIAL CELL FUNCTION

The epithelial cell populations of the intestinal mucosa are modulated by a number of factors that are present within the lumen, the epithelium itself or the underlying lamina propria (Table 1). Although the full variety of regulatory factors that play a role in the control of intestinal epithelial and non-epithelial cell populations has not been fully defined yet, there is increasing appreciation of the diversity of these factors in general and the importance of several specific peptide and non-peptide factors produced or released within the intestine. The identification and characterization of numerous regulatory peptide and nonpeptide factors has led to the recognition of a network of interrelated factors within the intestine (Figure 1). The constituents of this network generally possess multiple functional properties and exhibit pleiotropism in their cellular sources and targets. As a result, this network is highly redundant in several dimensions[20]. Regulatory peptides especially seem to play a key role in intestinal epithelial wound repair, as they are abundantly detectable in the intestinal lumen, intestinal epithelium and the underlying lamina propria. Various members of several distinct regulatory peptide families have been recognized to modulate a broad spectrum of intestinal epithelial cell functions including cell migration, proliferation and/or differentiation (Figure 2). As outlined above the latter epithelial cell functions are highly relevant for the modulation of intestinal epithelial wound repair.

Table 1 Important modulators of intestinal epithelial cell function.

Localization	Modulator
Gastrointestinal lumen	Dietary compounds
	Alimentary secretions from salivary glands, stomach, pancreas or intestinal glandular cells
	Secreted regulatory peptides
	Physiological and pathogenic intestinal microflora
	Non-peptides factors (e.g. phospholipids, polyamines, short chain fatty acids and other)
	Drugs
	Other
Epithelium	Regulatory peptides
	IEL
	Local cell-cell interactions
	Non-peptide factors (e.g. phospholipids, adenine nucleotides, polyamines and others)
	Other
Lamina propria and basal lamina	Regulatory peptides expressed by various constituents of the lamina propria
	Extracellular matrix factors
	Neurotransmitters
	Nerval interactions
	Various mediators that are transported via the blood stream
	Non-peptide factors (e.g. phospholipids, adenine nucleotides, polyamines and others)
	Other

Figure 1 Regulatory network within the intestinal mucosa (adopted and modified from Reference 32).

Figure 2 Functional activities of peptide growth factors within the intestinal mucosa (adopted and modified from Reference 32).

THE ROLE OF REGULATORY PEPTIDES FOR INTESTINAL EPITHELIAL WOUND HEALING

A broad spectrum of structurally distinct regulatory peptides is expressed from various cell populations within the mucosa of the intestinal tract. These regulatory peptides, conventionally designated as growth factors and cytokines play an essential role in regulating differential epithelial cell functions in order to preserve normal homeostasis and integrity of the intestinal mucosa[20–25]. The terminology of regulatory peptides is often confusing and arbitrary. The term cytokines is now increasingly used to describe a bunch of regulatory peptides that can be variously identified as regulatory peptides, peptide growth factors, interleukins, interferons and colony stimulating or hematopoetic stem cell factors. For the purpose of simplification, the term regulatory peptide or cytokine will be used in this paper to address all the different classes of regulatory peptide factors. Regulatory peptides can be reasonably classified on the basis of structural homologies and disparities into several discrete families. Peptide growth factor families and selected members with functional activities in the modulation of intestinal wound healing. In addition to growth factor families, a number of regulatory peptide factors, seemingly without structural similarities to other regulatory peptide family members like vascular endothelial cell growth factor (VEGF) and platelet-derived growth factor (PDGF) have been identified to be expressed within the intestinal tract and to modulate wound healing properties within the intestinal mucosa[25]. Furthermore, a countless number of classical cytokines like IL-1, IL-2, IL-15, IL-22 and IFN-γ are expressed within the intestine and modulate numerous intestinal epithelial cell functions[26–30].

The various effects of a number of regulatory peptides on cell adhesion, migration, proliferation, differentiation, intestinal epithelial barrier function and angiogenesis suggest that these peptides are likely relevant factors for intestinal repair mechanisms. Both in-vitro and in-vivo studies have demonstrated that several growth factors and cytokines can enhance epithelial cell restitution[3132]. A spectrum of growth factors and cytokines including EGF, VEGF, HGF, GLP-2, various FGF peptides, IL-1, IL-2 and IFN-γ have been demonstrated to enhance epithelial cell restitution through a TGF-β-dependent pathway[33–36]. Interestingly, it appears that restitution-enhancing cytokines use different mechanisms to modulate TGF-β-peptide levels. While TGF-β, EGF, IL-1, IFN-γ and HGF only increased the concentration of bioactive TGF-β, acidic and basic FGF and also IL-2 enhanced both the bioactivation of TGF-β and the expression of TGF-β mRNA and production of latent TGF-β peptide. This might reflect a different mechanism, by which acidic and basic FGF and also IL-2 modulate the synthesis and bioactivation of TGF-β. In contrast to the above mentioned growth factors and cytokines that are assumed to act from the basolateral site of the epithelial surface and that seem to stimulate intestinal epithelial restitution through a common TGF-β-dependent pathway, various members of the trefoil factor family (TFF Peptide family) appear to stimulate epithelial restitution in conjunction with mucin glycoproteins through a TGF-β-independent mechanism from the apical pole of the epithelium[353738]. In this regard, our group recently also demonstrated that mesalamine promotes intestinal wound healing in vitro through a TGF-β-independent mechanism[39]. Recent studies suggest that modulation of repair mechanisms by trefoil peptides may be mediated by modulation of the E-cadherin/catenin complex[4041]. However, a double-blind, randomized, placebo-controlled study treating 16 patients with mild-to-moderate left sided ulcerative colitis with enemas containing human recombinant trefoil factor family-3 did not reveal any additional benefit above that of adding 5-aminosalicylic acid alone[42].

It has been demonstrated, that keratinocyte growth factor (KGF) has an important function in wound re-epithelialization[43] and KGF expression is strikingly increased in surgical specimens from patients suffering from Crohn’s disease and ulcerative colitis[44]. Furthermore, in an experimental model of colitis, administration of KGF after but not before induction of colitis significantly ameliorated tissue damage demonstrating that exogenous KGF might promote IBD[45]. However, clinical studies treating patients with CD or UC with exogenous KGF have not been performed yet. In addition to their potent effects on epithelial restitution, a number of regulatory peptide factors act also as potent modulators of epithelial cell proliferation[17183246–49]. The most important modulators of intestinal epithelial cell proliferation include EGF and TGF-β which both act as potent stimulators of intestinal epithelial proliferation and TGF-β which inhibits intestinal epithelial cell proliferation and plays an important counterbalancing role in the regulation of intestinal epithelial cell proliferation. TGF-β is the most potent inhibitor of intestinal epithelial cell proliferation overriding the stimulatory effects of other stimulatory factors. The growth stimulating effect of IL-2, FGF peptides, IGF and HGF is rather moderate compared to the effects of EGF and TGF-β that stimulate epithelial cell proliferation five- to ten-fold in several intestinal epithelial cell lines in vitro[33344850]. Thus, it is not astonishing that in a randomized, double-blind clinical trial, after a 2-wk treatment period, patients receiving EGF enemas had a significant lower disease activity score than the control patients[51]. Concerning basic FGF, in a mouse DSS-model of experimental colitis, rectal administration of human recombinant basic FGF ameliorated the inflammation score and suppressed TNF-α gene expression in the colonic tissue[5253]. Recently, the important role of Glucagon-like-peptide-2 (GLP-2), which is secreted from local neuroendocrine epithelial cells and promotes epithelial cell proliferation via stimulation of enteric neurons[54], has received more attention. GLP-2 demonstrating ability to ameliorate murine short bowel syndrome and experimental colitis[5556] has consequently lead to clinical studies, evaluating the effect of GLP-2 in patients with short bowel syndrome.

However, it has also to be considered that cytokines have pleiotropic activities. For example, FGF also induces stricture formation in Crohn’s disease[57] and GLP-2 might have undesired effects in tumorogenesis[58], which might limit their therapeutic use. In addition, it has to be considered that these factors and also various non-peptide factors may act in an additive or even synergistic fashion which may potentiate their single effects. Notably, in addition to TGF-β the TGF-β family member Activin A has been identified to also inhibit epithelial cell proliferation, thus providing an additional mechanism to counterbalance the effects of proliferative factors present within the intestinal mucosa and to inhibit unrestrained cell growth[5960].

MODULATION OF INTESTINAL EPITHELIAL WOUND HEALING BY NON-PEPTIDE FACTORS

In addition to the potent modulation of intestinal epithelial wound healing by regulatory peptides, it is increasingly appreciated that a broad spectrum of non-peptide factors exerts potent effects on intestinal epithelial cell populations and modulates those epithelial cell functions that are involved in the healing of intestinal injury (Table 2). These non-peptide factors encompass a broad spectrum of unrelated factors like phospholipids, nutrients (adenine) nucleotides, polyamines, short chain fatty acids (SCFA), products of the intestinal microflora, trace elements, pharmacological agents and other factors. Some of these factors are released by injured or dying mucosal cell populations (e.g. adenine nucleotides, phospholipids), other reach the intestinal mucosa via the intestinal lumen or the blood stream. These non peptide factors may exert growth factor like activities and exert potent effects on cell growth and differentiation in different cell populations including fibroblasts, vascular smooth muscle cells, endothelial cells and keratinocytes[203561–63]. As some of these non-peptide factors are stable within the gastrointestinal tract despite high concentrations of acid, bile salts, proteases and microorganisms and as they exhibit only limited toxicity in vivo, they may serve as potential future targets to improve the armamentarium for the healing of mucosal epithelial injury.

Table 2 Selected non-peptide factors with relevance for intestinal epithelial wound healing.

Factor	Mechanism of action	References
Lysophosphatidic acid	Stimulates intestinal epithelial restitution and inhibits intestinal epithelial proliferation	[61,67]
Polyamines	Stimulate intestinal epithelial restitution and proliferation	[11,66,75]
Adenine nucleotides	Stimulate intestinal epithelial restitution and inhibit intestinal epithelial proliferation	[59]
Short chain fatty acids	Stimulate intestinal epithelial migration	[63,76]
Glutamine	Stimulates intestinal epithelial proliferation and migration	[76,77]

Especially phospholipids and polyamines seem to be of special interest, as these non-peptide factors can be easily added to the regular diet and their overall content and biological activity can be modulated by various pharmacological agents[6264–66]. Lysophosphatidic acid (LPA) is a key intermediate in the early steps of phospholipid biosynthesis and is rapidly produced and released from thrombin-activated platelets and growth factor stimulated fibroblasts to influence target cells by activating a specific 38-40 kDa G-protein coupled receptor that is expressed in many cells[67]. As a product of the blood-clotting process, LPA is a normal constituent of serum, where it is present in an albumin-bound form in physiologically relevant concentrations[64]. Major sources of LPA in the vicinity of injured epithelial cells are activated platelets, stimulated fibroblasts and presumably injured cells that release LPA due to non-specific phospholipase activation[67]. LPA promotes platelet aggregation and induces cellular tension and cell surface fibronectin assembly[68], which are also important events in wound repair suggesting an important role of LPA in inflammatory disorders. This was confirmed by our group when we demonstrated, that LPA not only promotes epithelial wound healing in vitro by a TGF-β-independent pathway, but also ameliorates experimental colitis in an experimental model of colitis in rats[61]. Interestingly, also lysophosphatidylethanolamine and lisofylline, which decreases lipid peroxidation, significantly reduced the degree of inflammation and necrosis in an experimental colitis model[69], demonstrating that the administration of anti-inflammatory lysophospholipids and suppression of pro-inflammatory lipid metabolites by lisofylline may provide new approaches to ameliorate intestinal inflammation. This beneficial effect could also be demonstrated in other diseases since lisofylline and its analogs reversed autoimmune diabetes in a non-obese diabetic (NOD) mouse model and thus might act as a potential treatment for Type 1 diabetes[7071].

Many studies have reported that bone marrow cells may have the potential to contribute to the repair of many non-hematopoetic tissues, including the intestinal epithelial cells. Bone-marrow derived cells are capable of promoting regeneration of damaged intestinal epithelial cells[72]. However, the underlying effects are not fully understood[18]. Nevertheless, bone-marrow transplantation combined with immune-suppressive therapy improves epithelial wound healing[73] and recombinant granulocyte-macrophage colony-stimulating factor have a therapeutic effect in patients with active Crohn’s disease[74].