Adrenomedullin and adrenomedullin binding protein-1 attenuate vascular endothelial cell apoptosis in sepsis

Abstract

Objective: To determine whether vascular endothelial cell apoptosis occurs in the late stage of sepsis and, if so, whether administration of a potent vasodilatory peptide adrenomedullin and its newly reported specific binding protein (AM/AMBP-1) prevents sepsis-induced endothelial cell apoptosis.

Summary background data: Polymicrobial sepsis is characterized by an early, hyperdynamic phase followed by a late, hypodynamic phase. Our recent studies have shown that administration of AM/AMBP-1 delays or even prevents the transition from the hyperdynamic phase to the hypodynamic phase of sepsis, attenuates tissue injury, and decreases sepsis-induced mortality. However, the mechanisms responsible for the beneficial effects of AM/AMBP-1 in sepsis remain unknown.

Methods: Polymicrobial sepsis was induced by cecal ligation and puncture in adult male rats. Human AMBP-1 (40 microg/kg body weight) was infused intravenously at the beginning of sepsis for 20 minutes and synthetic AM (12 microg/kg body weight) was continuously administered for the entire study period using an Alzert micro-osmotic pump, beginning 3 hours prior to the induction of sepsis. The thoracic aorta and pulmonary tissues were harvested at 20 hours after cecal ligation and puncture (ie, the late stage of sepsis). Apoptosis was determined using TUNEL assay, M30 Cytodeath immunostaining, and electromicroscopy. In addition, anti-apoptotic Bcl-2 and pro-apoptotic Bax gene expression and protein levels were assessed by RT-PCR and Western blot analysis, respectively.

Results: Vascular endothelial cells underwent apoptosis formation at 20 hours after cecal ligation and puncture as determined by three different methods. Moreover, partial detached endothelial cell in the aorta was observed. Bcl-2 mRNA and protein levels decreased significantly at 20 hours after the onset of sepsis while Bax was not altered. Administration of AM/AMBP-1 early after sepsis, however, significantly reduced the number of apoptotic endothelial cells. This was associated with significantly increased Bcl-2 protein levels and decreased Bax gene expression in the aortic and pulmonary tissues.

Conclusion: The above results suggest that vascular endothelial cell apoptosis occurs in late sepsis and the anti-apoptotic effects of AM/AMBP-1 appear to be in part responsible for their beneficial effects observed under such conditions.

Figures

FIGURE 1. TUNEL staining in aortic tissues at 20 hours after CLP or sham operation with or without AM/AMBP-1 treatment. The vascular endothelial cell apoptosis was observed after CLP (B, arrows) as compared with sham-operated animals (A). AM/AMBP-1 treatment attenuated endothelial cell apoptosis under such conditions (C). A: Sham 20 hours. B: CLP 20 hours. C: CLP 20 hours with AM/AMBP-1 treatment. D: Negative control. a, b, c, and d: same area as A, B, C, and D, respectively. The aortic tissues were stained with propidium iodide (PI) showing nuclei. Bar = 50 μm.

FIGURE 2. TUNEL staining in pulmonary tissues at 20 hours after CLP or sham operation with or without AM/AMBP-1 treatment. The microvascular endothelial cell apoptosis was observed after CLP (B, arrows) as compared with sham-operated animals (A). AM/AMBP-1 treatment attenuated endothelial cell apoptosis (C). A: Sham 20 hours. B: CLP 20 hours. C: CLP 20 hours with AM/AMBP-1 treatment. D: Negative control. Bar = 50 μm.

FIGURE 3. M30 immunohistochemical staining in aortic tissues at 20 hours after CLP or sham operation with or without AM/AMBP-1 treatment. The vascular endothelial cell apoptosis was observed after CLP (B, arrows). AM/AMBP-1 treatment attenuated endothelial cell apoptosis under such conditions (C). A: Sham 20 hours. B: CLP 20 hours. C: CLP 20 hours with AM/AMBP-1 treatment. D: Negative control. Bar = 50 μm.

FIGURE 4. Effects of AM/AMBP-1 on endothelial cells apoptosis in aortic tissues at 20 hours after CLP as determined by TUNEL assay (A) and M30 staining (B). The data are expressed as the number of apoptotic cells per high power field of 5 animals in each group. Eight to 10 high power fields were randomly taken from each slide and then averaged. Values are presented as mean ± SE and compared by one-way ANOVA and Tukey's test: *P < 0.05 versus sham; #P < 0.05 versus CLP.

FIGURE 5. Representative electron-microscopy photographs of aortic tissues at 20 hours after CLP or sham operation (n = 3 or 4 rats/group). The condensed chromosome can be observed in the endothelial cell at 20 hours after CLP (B, arrow). Moreover, the endothelial cell was partially detached from the basal membrane. A: Sham 20 hours; B: CLP 20 hours. Bar = 2 μm.

FIGURE 6. Gene expression of Bcl-2 and the housekeeping gene G3PDH in aortic tissues at 20 hours after CLP or sham operation with or without AM/AMBP-1 treatment. The representative PCR gel (A) and the ratio of Bcl-2/G3PDH (B) are presented. Values (n = 4−6/group) are presented as mean ± SE and compared by one-way ANOVA and Student-Newman-Keuls test: *P < 0.05 versus sham; #P < 0.05 versus CLP 20 hours.

FIGURE 7. Alteration in Bcl-2 protein levels in aortic tissues at 20 hours after CLP or sham operation with or without AM/AMBP-1 treatment. The representative Western blot (A) and the optical densities as a percentage of sham (B) are presented. Values (n = 4−6/group) are presented as mean ± SE and compared by one-way ANOVA and Tukey's test: *P < 0.05 versus sham; #P < 0.05 versus CLP 20 hours.

FIGURE 8. Gene expression of Bax and the housekeeping gene G3PDH in aortic tissues at 20 hours after CLP or sham operation with or without AM/AMBP-1 treatment. The representative PCR gel (A) and the ratio of Bax/G3PDH (B) are presented. Values (n = 4−6/group) are presented as mean ± SE and compared by one-way ANOVA and Student-Newman-Keuls test: #P < 0.05 versus CLP 20 hours.

FIGURE 9. Alteration in Bax protein levels in aortic tissues at 20 hours after CLP or sham operation with or without AM/AMBP-1 treatment. The representative Western blot (A) and the optical densities as a percentage of sham (B) are presented. Values (n = 4−6/group) are presented as mean ± SE.