Blood–brain barrier disruption following subarchnoid hemorrhage may be faciliated through PUMA induction of endothelial cell apoptosis from the endoplasmic reticulum
Research highlights
► The activated PUMA protein was involved in BBB breakdown through, ER pathway partly. ► The expressions of PUMA, BAX, BAK, GRP78 and DRP1 were accompanied with apoptosis. ► PUMA siRNA reduced the mortality, brain water, and Evans blue content after SAH.
Introduction
Subarachnoid hemorrhage (SAH) is a devastatingly tragic stroke subtype with a significantly high morbidity and mortality rate. Despite promising therapeutic approaches such as triple-H therapy and calcium channel blockades, curative treatment that can reverse the damages following SAH injury is not available. This is partly attributed to the poor strategic approach when dealing with the impact of the initial bleeding in SAH; this includes a rise in intracranial pressure, decrease in cerebral blood flow, and a disruption of the blood–brain barrier (BBB) causing further brain swelling, edema formation and/or accumulation.
One of the main factors attributed to BBB disruption and subsequent brain edema accumulation is apoptotic cell death—especially among endothelial cells which play a vital role in maintaining an adequate barrier. Recently it has been shown that PUMA (p53 upregulated modulator of apoptosis), a direct transcriptional target of p53, is a an essential mediator of cell death and plays a key functional role in the process of p53-mediated apoptosis (Jeffers et al., 2003; Yan et al., 2008). At times of cell damage, the expression of PUMA is up-regulated and binds to and neutralizes almost all prosurvival members of the the Bcl-2 family (Chipuk et al., 2005). Hence it begs the questions whether PUMA is a key orchestrator of early brain injury following SAH.
As a result, the present study was designed to investigate the role of PUMA following SAH injury in rats. Specifically evaluating whether through the endoplasmic reticulum (ER), PUMA could orchestrate the induction of endothelial cell apoptosis and cause a disruption in the blood–brain barrier integrity. In order to test these aims, we used a PUMA siRNA and compared outcome results of mortality rate, brain edema, BBB disruption, neurobehavioral tests versus those animals not treated with the siRNA. We also used Western blotting techniques to measure the expression of key pro-apoptotic proteins such as BAX, BAK, and DRP1. To support our hypothesis of ER invovlvment, GRP78, a molecular chaperone located in the lumen of the ER, was measured.
Section snippets
Materials and methods
All protocols for this study were evaluated and approved by the Animal Care Committee at Peking University Health Sciences Center and the Guidelines for the Use of Animals in Neuroscience Research by the Society for Neuroscience.
PUMA siRNA reduced mortality and neurobehavioral deficits following SAH injury
Animal mortality was measured 24 hours following SAH injury and was presented in a percentage form (Fig. 1A). In the respective groups, the mortality rates were as follow: SAH group 40.42% (19/47 rats), control siRNA 39.13% (18/46 rats), PUMA siRNA 17.65% (6/34 rats), and 0% (0/28 rats) in the sham group. Statistical analysis revealed that there was a significant difference of mortality between the SAH, control siRNA group and PUMA siRNA group (p < 0.05). However, no statistical significance in
Discussion
The present study was designed to investigate the role of PUMA (p53 upregulated modulator of apoptosis) following SAH injury in rats; specifically evaluating whether through the endoplasmic reticulum, PUMA could orchestrate the induction of endothelial cell apoptosis and cause a disruption in the blood–brain barrier integrity. This study made the following novel observations: (1) SAH-induced brain injury resulted in a significant increase in mortality, brain edema, and neurobehavioral deficits
Conclusion
In conclusion, this study showed that PUMA induced endothelial cell apoptosis may in fact play a significant role in BBB disruption following SAH and its mediation may be through the endoplasmic reticulum. By blocking the activity of PUMA using siRNA, we were able to prevent the accumulation of cerebral edema that occurs following BBB disruption. This translated into a preservation of functional integrity and an improvement in mortality. Obviously, more studies evaluating the exact mechanistic
Acknowledgments
This work was partially supported by the National Natural Science Foundation of China (30901548 and 30971527). We would also like to recognize and thank Professor Han Hongbin and Dr. He Qingyuan for their support with the MRI.
References (25)
- et al.
Differential targeting of pro-survival Bcl-2 proteins by their BH3-only ligands allows complementary apoptotic function
Mol. Cell
(2005) - et al.
Therapeutic application of gene silencing MMP-9 in a middle cerebral artery occlusion-induced focal ischemia rat model
Exp. Neurol.
(2009) - et al.
Puma is an essential mediator of p53-dependent and -independent apoptotic pathways
Cancer Cell
(2003) - et al.
2-Methoxyestradiol reduces cerebral vasospasm after 48 hours of experimental hemorrhage in rats
Exp. Neurol.
(2006) - et al.
The role of p53 in brain edema after 24 h of experimental subarachnoid hemorrhage in a rat model
Exp. Neurol.
(2008) - et al.
Comparison of two neonatal ischemic injury models using magnetic resonance imaging
Pediatr. Res.
(2007) - et al.
Cortical blood flow and cerebral perfusion pressure in a new noncraniotomy model of subarachnoid hemorrhage in the rat
Stroke
(1995) - et al.
Regulation of apoptosis by endoplasmic reticulum pathways
Oncogene
(2003) - et al.
Caspase cleavage product of BAP31 induces mitochondrial fission through endoplasmic reticulum calcium signals, enhancing cytochrome c release to the cytosol
J. Cell Biol.
(2003) - et al.
Multiple effects of 2ME2 and D609 on the cortical expression of HIF-1alpha and apoptotic genes in a middle cerebral artery occlusion-induced focal ischemia rat model
J. Neurochem.
(2007)