17beta-estradiol mediated protection against vascular leak after hemorrhagic shock: role of estrogen receptors and apoptotic signaling

Abstract

Vascular hyperpermeability is a clinical complication associated with hemorrhagic shock (HS) and occurs mainly because of the disruption of the adherens junctional complex. The objective of this study was to understand the role of 17beta-estradiol in HS-induced hyperpermeability particularly focusing on estrogen receptors. In male Sprague-Dawley rats, HS was induced by withdrawing blood to reduce the mean arterial pressure to 40 mmHg for 1 hour followed by 1 hour of resuscitation to 90 mmHg. The study groups were 17beta-estradiol, tamoxifen, fulvestrant plus 17beta-estradiol, propyl pyrazole triol plus 17beta-estradiol, and diarylpropionitrile plus 17beta-estradiol. Intravital microscopy was used to study changes in mesenteric postcapillary venules. Mitochondrial reactive oxygen species formation was studied in vivo using dihydrorhodamine 123. The mitochondrial transmembrane potential was studied using the fluorescent cationic probe 5,5',6,6'tetrachloro-1,1',3,3'tetraethylbenzimidazolyl carbocyanine iodide (JC-1). The mesenteric microvasculature was analyzed for cytochrome c levels by enzyme-linked immunosorbent assay and caspase-3 activity by a fluorometric assay. Our results demonstrated that 17beta-estradiol attenuated HS-induced hyperpermeability. Fulvestrant reversed this protective effect (P < 0.05). Tamoxifen 5 mg/kg attenuated HS-induced hyperpermeability, whereas 10 mg/kg induced permeability (P < 0.05). Both alpha and beta estrogen receptor agonists inhibited HS-induced hyperpermeability (P < 0.05). 17beta-Estradiol decreased HS-induced reactive oxygen species formation and restored mitochondrial transmembrane potential. 17beta-Estradiol decreased both cytosolic cytochrome c level and activation of caspase-3 (P < 0.05). These findings suggest that 17beta-estradiol protects the microvasculature after HS, and that this protection may be mediated through the alpha and beta estrogen receptors.

Figure-1

17β-Estradiol inhibits hemorrhagic shock (HS)-induced vascular leak in rat mesentery post-capillary venules. Intravital microscopy images show FITC -albumin extravasation in shock but not in pre-shock condition (Panels A and B). 17β-Estradiol treatment leads to a decrease in FITC-albumin extravasation in shock (Panel C). The estrogen receptor antagonist fulvestrant inhibits the protective effect of 17β-estradiol (Panel D). The treatments were given 10 minutes prior to the hemorrhagic shock period and image taken after 60 minutes of HS and 60 minutes of resuscitation.

Figure-2

17β-Estradiol inhibits hemorrhagic shock-induced vascular leak in rat mesentery post-capillary venules. Hemorrhagic shock induces significant increase in FITC-albumin extravasation. 17β-Estradiol treatment significantly decreases this effect. † Significant difference vs. shock group (p < 0.05). Estrogen receptor antagonist fulvestrant inhibits the protective effect of 17β-estradiol. * Significant difference vs. 117β-estradiol + hemorrhagic shock group (p < 0.05).

Figure-3

17β-Estradiol treatment reduced the amount of resuscitation fluid required to achieve a MAP of 40 mmHg. * Significant difference vs. hemorrhagic shock group and 17β-estradiol + hemorrhagic shock groups (p <0.05).

Figure-4

Estrogen receptor modulator tamoxifen induces differential effects on vascular leak in rat mesentery post capillary venules. Hemorrhagic shock induces significant increase in FITC-albumin extravasation. Tamoxifen 5 mg/kg inhibits HS-induced vascular leak whereas 10 mg/kg induces leak. † Significant difference vs. shock group (p <0.05). * Significant difference vs. sham group (p < 0.05).

Figure-5

Estrogen receptor alpha agonist PPT and beta agonist DPN attenuate vascular leak following hemorrhagic shock in rat mesentery post capillary venules. Hemorrhagic shock induces significant increase in FITC -albumin extravasation. PPT and DPN DPN groups (p <0.05). * Significant difference vs. shock group (p <0.05).

Figure-6

(A and B) 17β-Estradiol prevents hemorrhagic shock induced ROS formation in rat mesentery. The images shown are of mesenteric post-capillary venules of sham, hemorrhagic shock for 1 hour MAP 40 mm Hg followed by 60 minutes of resuscitation and estradiol treatment 10 minutes prior to hemorrhagic shock (17β-estradiol + Shock) are shown. Increased ROS formation is observed following hemorrhagic shock whereas 173-estradiol treatment decreased the ROS formation. (B) ROS formation is expressed based on the changes in fluorescent intensity. Hemorrhagic shock induces ROS formation compared to sham-control group. 17β-Estradiol treatment prevents HS-induced ROS formation compared with hemorrhagic shock group without 17β-estradiol. * Significant difference vs. sham group (p <0.05). Significant difference vs. shock group (p <0.05).

Figure-7

17β-Estradiol prevents hemorrhagic shock-induced decrease in mitochondrial transmembrane potential. In sham-control, the JC-1 fluoresced the mitochondria red (Cy3) and the cytoplasm green (FITC), indicating intact mitochondria. Hemorrhagic shock for 1 hour MAP 40 mm Hg followed by 60 minutes of resuscitation induced the collapse of membrane potential allowing diffusion of the red (Cy3) fluorescence into the cytoplasm. 17β-estradiol treatment 10 minutes prior to hemorrhagic shock prevents the decrease in membrane potential (leakage of JC- 1).

Figure-8

17β-Estradiol inhibits hemorrhagic shock-induced cytochrome c release in mesenteric vasculature. Cytosolic cytochrome c levels increased significantly 60 minutes after resuscitation compared with sham-control. 17β-Estradiol inhibited hemorrhagic shock- induced increase in cytochrome c levels. * Significant difference vs. sham group (p < 0.05). Significant difference vs. shock group (p < 0.05). 173-Estradiol treated group shows no significant change in vascular leak compared with sham group.

Figure-9

17β-Estradiol inhibits hemorrhagic shock-induced caspase-3 activation in mesenteric vasculature. Caspase-3 activity increased significantly after shock at 60 minutes after resuscitation compared with sham-control. 17β-Estradiol inhibited hemorrhagic shock-induced caspase-3 activation. * Significant difference vs. sham group (p < 0.05; n = 5). Significant difference vs. shock group (p < 0.05). 17β-Estradiol treated group shows no significant change in permeability compared with sham group.