The gastrointestinal mucosa is a richly perfused vascular bed directly juxtaposed with the anaerobic and nonsterile lumen of the gut. As such, intestinal epithelial cells, which line the mucosa, experience a uniquely steep physiologic oxygen gradient in comparison with other cells of the body. Inflammation associated with a loss of epithelial barrier function and unregulated exposure of the mucosal immune system to luminal antigens leads to inflammatory bowel disease (IBD), a relatively common disorder with severe morbidity and a limited therapeutic repertoire. During IBD, increased tissue metabolism and vasculitis renders the chronically inflamed mucosa and particularly the epithelium hypoxic, giving rise to the activation of the hypoxia-responsive transcription factor hypoxia-inducible factor (HIF). Recent studies utilizing conditional intestinal epithelial hif1a-null mice have revealed a protective role for epithelial HIF-1α in murine models of IBD. Such protection occurs, at least in part, through HIF-dependent induction of barrier-protective genes in the epithelium. More recently, studies employing pharmacologic activation of HIF via inhibition of HIF prolyl hydroxylases revealed a profoundly protective effect of these agents in murine models of colitis. In this paper, we review this pathway in detail and examine the therapeutic potential for targeting HIF hydroxylases in intestinal mucosal inflammatory disease.
KeywordsHypoxia Inflammation Gastroenterology
Tissue oxygenation in the gastrointestinal tract
The primary functions of the gastrointestinal tract are the processing and absorption of ingested nutrients, waste removal, fluid homeostasis, and the development of oral tolerance to nonpathogenic luminal antigens. The last of these functions involves the intestinal mucosa being unique among tissues as it is in a constant state of controlled inflammation . This occurs as the mucosal immune system is constantly exposed to new food-borne material in the lumen, which is processed to avoid inappropriate inflammatory reactions to harmless ingested antigens .
A critical cell type in the maintenance of intestinal homeostasis is the epithelial cell. The intestinal epithelium is a monolayer of cells that covers an area of approximately 250–300 m2 in an adult human and forms a critical barrier between the external (luminal) and internal (vascular) compartments. This dynamic barrier is maintained primarily by the existence of regulated intercellular tight junctions. As well as being a critical barrier, the epithelium is responsible for the absorption of approximately 9 l of fluid from consumed liquids and secreted digestive fluids per day. This fluid transport function is carried out through coordinated ion transport events and the subsequent regulation of salt and water transport between the lumen of the gut and the bloodstream. Importantly, both the barrier and absorptive functions of the intestinal epithelium can be physiologically regulated by oxygen [2–4].
Hypoxia and mucosal inflammation
Inflammatory bowel disease (IBD) is an umbrella term for a range of disorders including ulcerative colitis and Crohn’s disease, which are characterized by a breakdown in the intestinal epithelial barrier with subsequent unregulated exposure of the mucosal immune system to luminal antigenic material leading to inflammation and further barrier breakdown. Thus, a self-perpetuating cycle of inflammation is initiated leading to severe pathology [5–7]. Because of the limited number of current therapeutic options available, treatment often ultimately resorts to surgical resection of significant amounts of chronically inflamed intestinal tissue.
Active inflammation is characterized by dramatic shifts in tissue metabolism and perfusion. These changes include diminished availability of oxygen (hypoxia) [8–10] with subsequent lactate accumulation and resultant metabolic acidosis. Such shifts in tissue metabolism result, at least in part, from profound recruitment of inflammatory cells, in particular myeloid cells such as neutrophils (polymorphonuclear cells) and monocytes. The vast majority of inflammatory cells are not resident cells but are recruited to inflammatory lesions . As such, it is important to understand the interactions between microenvironmental metabolic changes (e.g., hypoxia) as they relate to molecular mechanisms of leukocyte recruitment and intestinal epithelial dysfunction during inflammation. More importantly, it is imperative to define whether mechanisms initiated by hypoxia might serve as potential therapeutic targets.
A number of studies have implicated the occurrence of hypoxia in mucosal inflammatory diseases such as IBD . Surgical specimens from patients with IBD have revealed prominent hypoxia-inducible factor (HIF)-1 and HIF-2 activation associated with increased vascular density in diseased areas . Other studies in humans have revealed that a number of microvascular abnormalities may contribute to diminished blood flow to the intestine in IBD, including the loss of endothelial nitric oxide generation and enhanced tissue vasoconstrictor production . Moreover, Vascular endothelial growth factor-dependent angiogenesis appears to be an integral part of human IBD . In support of these hypotheses, studies in murine models have identified the epithelium as the central target of hypoxia during active mucosal inflammation . As part of our ongoing work, we have confirmed the existence of mucosal hypoxia in murine models of IBD using 2-nitroimidazole dyes, a class of compounds known to undergo intracellular metabolism depending on the availability of oxygen within tissue (Fig. 1). Nitroimidazoles enter viable cells where they undergo a single electron reduction, to form a reactive intermediate species. In the presence of normal oxygen levels, the molecule is immediately reoxidized and diffuses out of the cell. In the absence of adequate oxygen concentrations, the molecule is incompletely reoxidized, and the highly reactive reduced form associates with intracellular proteins, forming adducts that can be localized with antibodies .
Localization of hypoxia utilizing these 2-nitroimidazole dyes revealed two interesting observations. First, in the small intestine and especially the colon, “physiologic hypoxia” appears to predominate. Indeed, accumulation of nitroimidazole adducts were readily evident in epithelial cells lining the lumenal aspect of the intestine. This was not the case in other tissues (e.g., lung and liver, unpublished observation), confirming previous studies that the resting pO2 in the intestinal epithelium is quite low, likely because of the steep gradient of oxygen across the lumenal aspect. Second, these imaging studies revealed that cells overlying mucosal lesions are considerably more hypoxic. Accumulation of nitroimidazole adducts, particularly in the epithelium, were as intense as those observed in some tumors, suggesting the existence of intense foci of hypoxia associated with these inflammatory lesions. While we do not yet know the basis for such inflammatory hypoxia, some evidence suggests that tissue vasculitis could predispose epithelia toward diminished oxygen delivery .
HIF is protective for mucosal inflammation
To more fully understand the physiologic implications of intestinal epithelial HIF, Karhausen et al.  generated two mouse lines with intestinal epithelial-targeted expression of either mutant Hif1a (constitutive repression of HIF-1) or mutant von Hippel-Lindau gene (Vhlh, constitutive overexpression of HIF, which includes HIF-1 and HIF-2). Studies of colitis in these mice revealed that the loss of epithelial HIF-1 correlated with more severe clinical symptoms (mortality, weight loss, colon length, intestinal epithelial permeability), whereas an increase in epithelial HIF was protective for these individual parameters. These studies clearly demonstrated that HIF-1α plays a critical role in barrier maintenance and provide evidence for our initial hypothesis of a HIF-1-controlled apical gene cluster. The role of HIF-2α in inflammatory lesions in the intestine remains less clear. However, given the differences in both tissue distribution patterns and target gene preferences between HIF-1α and HIF-2α, it is likely that this isoform plays a distinct role in IBD. Future studies will address this important question.
Further evidence in support of a protective role for HIF in mucosal disease are provided by studies directed at HIF prolyl hydroxylase (PHD) inhibitors [22, 23]. These enzymes were identified on the principle that other mammalian PHDs such as those which target extracellular collagen were 2-oxoglutarate dependent , and it was predicted that the HIF PHDs would also belong to this family of enzymes. Based on conserved structural features , a candidate molecular approach was used to define HIF-modifying enzymes. This approach identified the HIF PHDs as the products of genes related to C.elegans egl-9, a gene that was first described in the context of an egg-laying abnormal phenotype . In mammalian cells, three PHD isoforms were identified (PHD 1–3), and shown to hydroxylate HIF-α in vitro [26–27]. These enzymes have an absolute requirement for oxygen as the substrate. The overall reaction results in insertion of one oxygen atom into the HIF-α peptide substrate at the proline residue, with the other oxygen molecule generating succinate from 2-OG with the release of CO2. Reactions conducted in a limited oxygen environment have revealed that the activity of the purified enzyme is strikingly sensitive to diminished levels of oxygen in vitro [26–27]. The three enzymes have different tissue distributions and, at least under conditions of overexpression, have distinct patterns of subcellular localization [15, 19]. PHD1 mRNA is expressed in many tissues, with especially high expression in the testis. Likewise, PHD2 mRNA is widely expressed, with particularly abundant expression in adipose tissue [24, 28]. PHD3 mRNA is also expressed in many tissues but is most abundant in the heart and placenta [24, 28]. In mouse intestinal mucosal tissue, we have found expression all three isoforms of PHDs with a distribution of PHD1 < PHD2 = PHD3 [22–23].
The discovery of HIF-selective PHDs as central regulators of HIF expression has now provided the basis for potential development of PHD-based molecular tools and therapies [29–30]. Pharmacological inactivation of the PHDs by 2-OG analogues is sufficient to stabilize HIF-α , but this action is nonspecific with respect to individual PHD isoforms. In vitro studies suggest significant differences in substrate specificity. For example, comparison of enzyme activity in vitro showed that the HIF ODD sequence is hydroxylated most efficiently by PHD2 [24, 28]. These observations have generated interest in identifying enzyme-modifying therapeutics. Indeed, a number of PHD inhibitors have been described, including direct inhibitors of the PHDs [31–32], analogs of naturally occurring cyclic hydroxamates , as well as antagonists of α-keto-glutarate . As such, we hypothesized that pharmacologic activation of HIF would provide a protective adaptation to murine colitic disease. For these purposes, we have used PHD inhibitors that stabilize HIF-α and subsequently drive the expression of downstream HIF target genes. Our results show that the PHD inhibition provides an overall beneficial influence on clinical symptoms (weight loss, colon length, tissue tumor necrosis factor-α/interferon-γ) in multiple murine models of colitis. These effects are most likely due to their barrier-protective function and enhancement of wound healing at the site of inflammation [22–23]. Taken together, these findings emphasize the role of epithelial HIF-1α during inflammatory diseases in the colon and may provide the basis for a therapeutic use of PHD inhibitors in inflammatory mucosal disease.
Signaling interactions between hypoxia and inflammation
Conclusions and perspectives
The gastrointestinal mucosa provides a unique setting to study tissue oxygenation and changes in disease states. The relatively low baseline pO2 coupled with high blood flow and energy demand against a background of physiologic inflammatory activity identify this mucosal surface as having high potential for targeted HIF-based therapy. Results from animal models of IBD have demonstrated an overall beneficial impact of hydroxylase inhibition. Key issues remaining to be elucidated include identification of the critical gene targets involved, determination of the relative roles of HIF and NF-κB pathways, identification of tissue-specific expression of HIF PHD isoforms, and elucidation of the role of HIF-2α in this protective response. In summary, the endogenous adaptive pathways activated in response to hypoxia represent potentially important new windows of therapeutic opportunity in IBD.