In the present study, we have expanded upon prior observations of increased expression of a pro-angiogenic chemokine (CXCL5) within intestinal segments during the adaptation response to massive SBR. We have targeted increased CXCL5 expression to the villus mesenchymal core, specifically within newly formed capillaries. Further, we have demonstrated that CXCL5 expression within model endothelial cells in vitro is regulated at least in part, by EGFR signaling. This observation is strengthened by the evidence of increased CXCL5 expression in mice that have enhanced EGF production within the intestine at baseline. Taken together, these findings suggest that one possible mechanism for EGFR regulation of resection-induced villus growth involves CXCL5 induction of angiogenesis.
In the current experiments, we confirmed that CXCL5 expression was elevated after SBR (2.39-fold), but we were not able to demonstrate the same magnitude of change as in our prior study (14-18-fold). It is possible that the dissimilar intestinal wall components accounted for the differences since we previously measured CXCL5 expression within the entire bowel wall devoid of epithelial cells whereas CXCL5 expression has now been recorded in LCM-isolated lamina propria. As such, it is possible that a large amount of CXCL5 expression occurs within the intestinal smooth muscle cell layers and larger blood vessels. Since we had already shown increased capillary density in the villus core, we felt it was most logical to measure CXCL5 expression in this specific region. Another explanation for the disparate findings between the two studies may be the conditions for mRNA isolation.
Intestinal villus units are composed of an epithelial lining and underlying lamina propria. The capillaries of the lamina propria carry the digested nutrients from the intestine via the portal circulation to the liver. Since the entire villus unit grows during resection-induced adaptation, it is logical to assume that there is also growth of new capillaries. This was confirmed in a prior study by our laboratory whereby capillary villus density was elevated at 7 days following SBR 6
. This 7-day time point was preceded by an increase in CXCL5 expression (3rd
postoperative day) as well as two other pro-angiogenic peptides interleukin-1β and placental growth factor. We chose to focus the present study on CXCL5, since this was the pro-angiogenic gene whose expression increased to the greatest extent. For the first time, we were able to demonstrate that CXCL5 expression co-localizes with villus endothelial cells. This finding supports a local role for CXCL5 to act within the villus capillaries to induce new capillary growth during adaptation.
Our laboratory has previously established a critical role for EGFR signaling as a major mediator of resection-induced adaptation 9
. In experiments whereby the EGFR is stimulated by either administration of various EGFR ligands or transgenic EGF overexpression, the magnitude of adaptation (increased villus growth) is augmented. Alternatively, adaptive responses are attenuated when the EGFR is inhibited by several different paradigms, which include removal of the major source of endogenous EGF (submandibular gland excision), administration of an EGFR inhibitor, or performing SBR procedures in mutant mice with perturbed EGFR signaling capacity. The mechanisms for EGFR regulation of adaptation have been assumed to be due to the effects of this receptor on the kinetics of enterocyte turnover via stimulation of proliferation and inhibition of apoptosis. In addition to this consideration, the findings of this study would suggest that EGFR signaling may have separate, but complementary effects, depending on the cell type.
A known ligand of the EGFR, heparin binding EGF-like factor (HB-EGF), has been shown to induce endothelial cell migration and vascular network formation in HUVECs and to be involved in the angiogenic response that occurs after ischemia/reperfusion injury 10-12
. After ischemia/reperfusion injury, mice with a defective HB-EGF gene showed delayed wound healing and decreased angiogenesis 12
. These studies further support a role for EGFR signaling in the angiogenic response to injury, and possibly adaptation, in the intestine.
Since EGFR stimulation has also been demonstrated to enhance angiogenesis in other tissues and organs 13,14
our finding of increased angiogenesis in the adapting intestine logically directed us to test the hypothesis that this receptor may also be a key player in this important response. The results of this study would support the concept that EGFR stimulation is associated with angiogenesis via regulation of CXCL5 within villus endothelial cells. It is possible that CXCL5 expression in human umbilical endothelial cells may not reflect the expression profile of intestinal endothelial cells. As such, a more specific intestinal endothelial cell line 15
may be a more suitable model to test the role of EGFR regulation of CXCL5 expression.
Although we have established increased CXCL5 expression in endothelial cells in response to EGFR stimulation, the effect of CXCL5 on angiogenesis in vitro and in vivo has not yet been established. Further, we recognize that the relationship that we have demonstrated between CXCL5 mRNA and post-operative villus height is only a correlation and does not imply that the increase in villus height is caused directly by CXCL5. While outside the scope of this study, one approach to interrogate this relationship would be to perform experiments with transgenic mice that have the receptor for CXCL5 knocked out. These mice would allow a more direct interrogation of CXCL5 signaling and resection induced angiogenesis and its importance in adaptation.