The aetiology and pathogenesis of IBD remains obscure, although consecutive phases of epithelial ulceration and regeneration are known to occur. Neo-angiogenesis is part of the pathology of IBD, as this study confirmed, but it is unclear whether such an angiogenic process is the cause or the consequence of IBD.
Recent studies have indicated that serum VEGF concentrations are increased in patients with IBD,2,3
suggesting that this angiogenic factor is overproduced within the context of an intense angiogenic activity. However, we did not find high amounts of VEGF in our immunohistochemical study, where VEGF expression was absent in patients with CD, and only weakly positive in UC, specifically in the epithelial cells, a finding that was also true in normal intestinal tissues. Furthermore, inflammatory cells (lymphocytes and macrophages), another putative source of VEGF, also failed to react with the VG1 monoclonal antibody (anti-VEGF), and only focally reacted with the 11B5 monoclonal antibody (anti-VEGF–KDR complex). VEGF–KDR activity in IBD may reflect the upregulation of KDR receptor expression rather than VEGF, because the affinity of 11B5 for VEGF is low.10
In agreement with our findings, Kanazawa et al
, investigating IBD tissues, found that VEGF reactivity was present in endothelial cells but not in epithelial or inflammatory cells.2
“Angiogenesis is probably not a vascular endothelial growth factor dependent process in inflammatory bowel disease”
The lack of VEGF staining in our material seems to contrast with the results of Griga et al
These investigators found that the spontaneous production of VEGF was higher in cultures produced from patients with IBD than in those derived from patients with irritable bowel syndrome. The degree of VEGF production, either stimulated or unstimulated, did not differ between cultures derived from apparently normal mucosa of patients with IBD and cultures derived from controls. However, the high standard error and range of VEGF measurements obtained in their study, together with the limited number of patients recruited, do not allow reliable conclusions to be made. In addition, the mechanism that suppresses VEGF expression in patients with IBD in vivo may be absent in ex vivo situations, which could explain the findings of Griga et al
Our results with regard to the VEGF–KDR activation status of the neovasculature are very interesting. Indeed, although the vessel density was significantly higher in UC and CD than in normal mucosa/submucosa, the activated VEGF–KDR reactivity was only slightly increased (10% in UC and CD v
5% in normal vasculature) and, certainly, not within areas of intense inflammation. This low VEGF–KDR activation status indicates that angiogenesis is probably not a VEGF dependent process in IBD. In contrast, the consistent expression of TP in the stromal fibroblasts of both CD and UC and, to a lesser extent, its detection in epithelial cells, the vascular endothelium, and the inflammatory component, strongly suggests that TP is an important member of the molecular cascade stimulating angiogenesis in these diseases. The increased amounts of 2-deoxyribose-1-phosphate, released from epithelial and stromal cells with high TP activity, provide a potent chemotactic stimulus for endothelial cells14
and, at the same time, exert a strong oxidative stress on surrounding cells15
; this may account for the increased angiogenesis and the upregulation of HIF found in all cellular components of the intestine in patients with IBD.
This discrepancy between immunohistochemical and serological findings raises the question of whether serum VEGF values are informative. Indeed, platelets are the major source of VEGF in the human body,16,17
and during ex vivo platelet aggregation VEGF is released into the supernatant.18
Therefore, serum VEGF concentrations do not reflect VEGF production by the intestinal epithelium or the related inflammation. Plasma concentrations of VEGF are several times lower than those obtained from the sera of patients.19
Because platelet counts are high, especially in active IBD,20
it is possible that serum VEGF concentrations reflect VEGF of platelet origin rather than that produced by the intestine. In a recently submitted study of ours, not only were plasma concentrations of VEGF not increased in patients with IBD, but they were also reduced in patients with active CD (Koukourakis et al
, 2002, unpublished data).
The observed upregulation of HIFα molecules in IBD raises two main questions regarding (1) the cause, and (2) the pathological relevance of this event. Certainly, HIFα molecules are hypoxia regulated proteins. However, in the case of quiescent IBD, the intestinal mucosa seems histologically similar to normal tissue and the high degree of vascularisation of the mucosa in active disease does not support an obvious causative link between HIF upregulation and protracted hypoxia of vascular origin. Indeed, HIF expression was not related to the presence of necrosis. Although vasoconstriction or microthrombosis of the intestinal vessels (a theory supported by the high platelet counts, the prevalence of activated platelets, and the increased thrombopoietin values seen in patients with IBD2,20,21
) may cause focal hypoxia and HIF upregulation, other mechanisms may also contribute towards this observation. HIFα proteins are also induced by cytokines, released by the inflammatory cell component.22
The upregulation of TP expression by stromal fibroblasts may also occur as a consequence of cytokine stimulation,23
and the oxidative stress produced by 2-deoxyribose may directly upregulate the expression of HIFα molecules or even contribute to the release of cytokines from fibroblasts, which further induce the expression of HIFα molecules. A strong association between TP expression and the accumulation of HIF2α has been also confirmed in previous studies of ours in various human carcinomas.24,25
The pathological relevance of HIFα overexpression in IBD should be examined in relation to the lack of VEGF reactivity observed. Both HIF1α and HIF2α are potent inducers of VEGF gene expression.26,27
The rather focal expression of HIF1α in the intestinal mucosal and submucosal cells is compatible with the lack of VEGF upregulation. The diffuse expression of HIF2α by all cellular components in CD, including the muscular layer and serosa, conforms with an intensively activated HIF2α pathway, which nonetheless fails to induce VEGF. Therefore, the eventual disruption of the HIF2α–VEGF pathway is probably part of the pathogenesis of CD. Whether upregulation of the recently discovered HIF inhibitory protein (IPAS), or the decreased individual ability to express VEGF as a result of a specific VEGF gene polymorphism, accounts for this event remains to be investigated.28,29
Unlike CD, overexpression of HIF2α in UC affects mainly the inflammatory component and not the intestinal cell population. This HIF2α prevalence in CD may reflect the increased severity of the inflammatory process, which extends throughout the intestinal wall and is not necessarily confined to the surface.
“The reduced ability of a tissue to produce vascular endothelial growth factor (VEGF) or to respond to VEGF upregulation may result in reduced endothelial and epithelial cell viability”
Alternatively, upregulation of HIF2α in CD may represent the trend of the intestinal tissues to induce the production of VEGF, which, apart from being angiogenic, is a major survival factor inhibiting the apoptosis of both endothelial and epithelial cells.30,31
Constant, although low, amounts of VEGF production may be essential to maintain tissue integrity by regulating the delicate balance between proliferation and apoptosis. This has been shown in studies on lung embryogenesis and physiology.32,33
After an insult (inflammatory, chemical, or physical), VEGF upregulation may be required to facilitate tissue regeneration and repair. Certainly, this occurs during oesophageal mucosa healing, following radiation damage.34
Therefore, the reduced ability of a tissue to produce VEGF or to respond to VEGF upregulation may result in reduced endothelial and epithelial cell viability. The discordance between the expression of HIF2α and VEGF found in our study, predominantly concerning CD, suggests an inherent deficiency of the intestine to respond to various stresses by inducing VEGF. The precise mechanism of the disrupted HIF–VEGF intercommunication in IBD pathology requires further investigation.