These studies provide new insight into the potential therapeutic use of PHD inhibitors on the acute phases of mucosal inflammation. Based on previous work implicating a protective role for HIF-1α in mucosal inflammatory disease, we focused on defining epithelial HIF activation and colitis endpoints. These studies extend previous work into the role of HIF in vivo and provide new insight into the potential use of PHD inhibitors as therapeutic modalities.
HIF protein expression and activity occur primarily through post-translational modification of the alpha subunit of the HIF heterodimer30
. The discovery of HIF PHD's identified targets for the possible pharmacological control of HIF expression, whereby inhibitors activate the stabilization of the alpha subunit of HIF and PHD activators promote the degradation of HIF31
. Our previous work in vitro and in vivo utilizing mucosal inflammatory models have identified HIF-1 as an endogenously protective molecule2
. To extend these findings, we sought to define the functional role of systemic HIF activation. We demonstrate here a potent and specific upregulation of fully functional HIF-1α in vitro and in vivo by administration of a novel class of HIF prolyl hydroxylase inhibitors, FG-4442 and FG4497, confirming the unique regulation of HIF-1α half life via hydroxylation of conserved proline residues7-9
. The capacity of FG-4497 to activate HIF was demonstrated by several means, including the induction of HIF-responsive luciferase constructs, the induction of EPO and EPO-dependent endpoints in vivo, and the lack of HIF response in epithelial conditional hif1a
-null animals. It is currently not known to what extent differences exist between functional PHD-1, PHD-2 or PHD-3 expression in various cells or tissues. The most recent work in this regard utilized monoclonal antibodies directed against the various PHD molecules, and revealed that the PHD-1, -2 and -3 are widely expressed in all tissues and cells examined, including the epithelium32
. Likewise, it is currently not known to what extent differences exist between functional HIF subunits. While it is likely that the influence seen here with colitis is HIF-1α-mediated, our previous studies also suggested that HIF-2α is expressed in the colon2
. Further studies in intact tissue will be necessary to define these principles.
Inflammatory diseases such as ulcerative colitis or Crohn's disease are heavily impacted by changes in energy demand and tissue oxygenation. It is recently appreciated that tissue hypoxia and inflammation occur coincidentally, a condition we have termed “inflammatory hypoxia”33
. Such findings are evidenced by increased localization of oxygen-sensitive (nitroimidazole) dyes within the inflamed tissue2
. Moreover, studies as background for the current work revealed that HIF activation accompanying inflammatory disease subserves the mucosa in a protective manner2
. These studies have resulted in the identification of several targets of inflammation transcriptionally controlled by HIF-1α. Most prominently implicated are genes which promote nonclassical epithelial barrier function and control leukocyte trafficking, including the ecto-5'-nucleotidase (CD73)17
, the adenosine A2B receptor34
, intestinal trefoil factor26
. These findings of HIF activation in murine colitis models are consistent with studies in human patients indicating that HIF-1α expression is consistently induced in both ulcerative colitis36
and in Crohn's disease37
, as well as ischemic colitis38
. Here, we demonstrate both a barrier protective and an anti-inflammatory role for HIF PHD inhibitors in inflammatory mucosal disorders. Indeed, administration of FG-4497 24 h prior to TNBS challenge clearly reduces the initial drop in weight when compared to the respective controls, and thus may be attributable to the concerted upregulation of barrier protective and immunoregulatory genes mutually involved in the adaptive response to decreased oxygen. In support of this notion, we demonstrate a significant decrease (~40%) in baseline permeability when HIF levels are stabilized, correlating with results using genetically engineered for constitutive over-expression of HIF-1alpha (via expression of mutant von Hippel-Lindau)2
. To rule out possible non-HIF mediated drug related effects on barrier, we used conditional hif1a
-null mice, wherein no changes were observed in response to pharmacological PHD inhibition in vivo. Our studies also showed a beneficial influence of PHD inhibition on colon TNFα, a molecule with a clear pathophysiologic role in colitis39
. These studies revealed a dose-dependent decrease in tissue TNFα and IFNγ mRNA levels, with nearly 70% inhibition. In this context, TNFα and IFNγ levels provide a clear indicator of overall colonic inflammation and suggest an anti-inflammatory role for PHD inhibition in inflammatory disorders of the colon.
The intestine, particularly the colon, may be a unique tissue for which to study HIF function. Previous studies, in fact, have established a view of the intestine as relatively hypoxic under normal physiological conditions2, 40, 41
, with a steep oxygen gradient from anaerobic lumen across the highly metabolic epithelium. In this context it is not surprising that the intestinal epithelium is comparatively resistant to hypoxic challenge3
, assuming that “physiologic hypoxia” potentially elevates baseline levels of HIF-regulated gene activity. To this end, it was shown that the expression of HIF-target genes (e.g. CD73) is highest in the colon, with other epithelial tissues (e.g. kidney) expressing nearly two-fold less4, 42
. Remarkable in this context, PHD inhibitors have shown efficacy in epithelial tissues which are considered highly oxygenated. For example, it was recently reported that the PHD inhibitor FG-4095, , promotes VEGF and angiogenesis in human microvascular endothelia and in baboon lung explants43
. Important in this regard, and likely explanatory, FG-4095 s was shown to be effective at even high oxygen concentrations (e.g. 95% O2
). Thus, as a class, the actions of PHD inhibitors are likely to be relatively insensitive to local oxygen concentrations.
As guided by findings of increased histologic re-epithelialization by FG-4497, we explored the possible interplay between HIF and wound healing. For these purposes, we used the established collagen gel model system to measure functional outputs associated with tissue remodeling24
with NIH 3T3 fibroblasts stably expressing a luciferase-linked HRE. These studies revealed a remarkable increase in collagen gel contraction induced by FG-4497. Moreover, HIF activity correlated with the extent of contraction, and thereby linking HIF activity with collagen contraction. We do not currently know how HIF influences collagen contraction. While much is known about HIF and angiogenesis44
, far less is known about the role of HIF in wound healing. It is possible that the same mechanisms which drive angiogenesis also control wound healing, particularly since many of the cytokines and growth factors overlap in the GI tract45
. Alternatively, it was recently shown that HIF-mediated induction of the alpha subunit of heat shock protein 90 (Hsp90α) significantly enhances cell migration and wound closure. Whether these studies explain our findings with FG-4497-mediated collagen contraction await further experimentation. Nonetheless, these findings directly implicate HIF in tissue remodeling and suggest that PHD inhibitors such as FG4497 might significantly enhance mucosal restitution and wound healing.
Taken together, the combined inflammatory parameters of weight loss, colon length, and TNFα/IFNγ expression suggest a protective role for PHD-dependent induction of HIF in murine experimental colitis. These results demonstrate the beneficial impact and regulatory influence of HIF stabilization via PHD inhibition and hence propose a novel therapeutic approach to inflammatory intestinal diseases like ulcerative colitis and Crohn's disease using HIF prolyl hydroxylase inhibitors. Future work will be aimed at better defining these parameters, including gene targets and mechanisms of anti-inflammation, as well as tissue-specific influences of PHD inhibition.