The role of angiotensin II type 1a receptor on intestinal epithelial cells following small bowel resection in a mouse model

Abstract

Aim: We have previously shown that inhibition of angiotensin converting enzyme (ACE) significantly reduced intestinal epithelial cell (EC) apoptosis and improved morphometric intestinal adaptation in a mouse model of massive small-bowel resection (SBR). This study attempted to further examine the downstream signaling factors in this system by blocking the action of angiotensin II (ATII), hypothesizing that this would lead to similar improvement of intestinal adaptation after SBR.

Method: Two groups of mice (C57BL/6J) underwent either a 60% mid-intestinal resection (SBR group) or a transection/re-anastomosis (Sham group). Because real-time PCR studies showed that only ATII receptor type 1a (ATII-1a) expression was significantly increased after SBR, compared to SHAM mice, we decided to use the specific ATII-1a receptor antagonist Losartan to block this signaling pathway. An additional two groups of mice received daily i.p. injections of Losartan (SBR + Losartan and Sham + Losartan group). At 7 days, the adaptive response was assessed in the remnant gut including villus height, crypt depth, EC apoptosis (TUNEL staining) and proliferation (BrdU incorporation). The apoptotic and proliferation signaling pathways were addressed by analysis of EC mRNA expression.

Result: SBR (with and without Losartan) led to a significant increase in villus height and crypt depth. Losartan treatment did not significantly change EC proliferation, but did significantly reduce EC apoptosis rates as compared to the non-treated SBR group. Losartan treatment was associated with a significant reduction of the bax-to-bcl-2 ratio and TNF-alpha expression after SBR compared to non-treated groups. Interestingly, Losartan-treated groups showed a tremendous increase in proliferation of signaling factors EGFR, KGFR and IL7R, which may indicate an expanded potential for further intestinal adaptation also beyond 7 days after SBR.

Conclusion: This study showed that the ATII-1a receptor may be of crucial importance for the modulation of intestinal EC apoptosis, and for regulating the post-resectional EC adaptive response.

Figure 1

mRNA expression of bcl-2 and bax derived from epithelial cell samples in each group and measured by real time PCR. Result are expressed as 2 ^{−(−ΔΔCt)} in relation to β-actin expression. Expression of both bcl-2 and bax were significantly increased in the SBR group compared to the Sham group. Administration of Losartan (expressed in figure as ATII-1a) to SBR mice significantly reduced the expression of bcl-2 and bax; however, the reduction in bax fell to levels not significantly different than non-treated Sham mice. Results are shown as the mean±SD. Statistical comparisons are made using ANOVA with post hoc Bonferroni test.

Figure 2

mRNA expression of the extrinsic apoptotic pathways as detected by real time PCR. Result are expressed as 2 ^{−(−ΔΔCt)} in relation to β-actin expression. Note that gene expression of all tested factors were increased in SBR mice, regardless of whether mice received Losartan (expressed in figure as ATII-1a) or not. Results are expressed as mean ± SD. Statistical comparisons are made using ANOVA with post hoc Bonferroni test.

Figure 3

mRNA expression of EGF-R, KGFR₁, IL-7R. Note the significantly higher expression of these receptors in the Losartan (expressed in figure as ATII-1a) treated mice. Statistical comparisons are made using ANOVA with post hoc Bonferroni test.