The Role Of Angiotensin Ⅱ In The Brain Derived Microvascular Endothelial Cell Injury And The Protective Effect Of EXP-2528,a Novel AT₁ Receptor Blocker

The endothelium of the cerebral microvessels, a major component of the blood brain barrier (BBB), responds dynamically to the changes of local environment, including substance flow, free radical exposure, and cytokine generation. In particular, microvascular changes in the brain are the notable cause of cerebral edema and ischemic injury. A number of studies suggest that angiotensin (Ang) â…¡, the main effector of the renin-angiotensin system, may be involved in the initiation and regulation of processes occurring in brain ischemia partly by injuring the Cerebrovascular endothelial cells. The selective AT₁ receptor antagonists may contribute to prevention and treatment of brain ischemia. The aim of this study was to investigate the possible mechanism of Ang â…¡ -induced rat brain derived microvascular endothelial cell (BMEC) injury and evaluate the protective effects of EXP-2528, a novel Ang â…¡ type 1 (AT₁) receptor blocker.The experiment was performed in cultured rat BMEC. Cell viability was assessed by methylthiazol tetrazolium (MTT) assay and cell injury by the rate oflactate dehydrogenase (LDH) release. The result showed that 10⁶mol/L Ang II increased lactate dehydrogenase (LDH) leakage and time-dependently inhibited the viability of BMEC. Pretreatment with the ATi receptor specific blocker losartan or EXP-2528 or losartan plus the AT2 receptor blocker PD123319 prevented Ang II - induced BMEC injury. However, pretreatment with PD 123319 alone did not inhibit the injury and there were no significant difference between losartan group and losartan pluse PD 123319 group.The content of malondialdehyde (MDA) and the activity of antioxidant enzymes including superoxide dismutase (SOD) and glutathione peroxidase (GSH-Px) were determined by biochemical methods to identify the oxidative stress in BMEC. Incubation with 10'6 mol/L Ang II for 48 hours increased the intracellular and extracellular medium MDA content, decreased the activity of intracellular SOD and GSH-Px significantly. Pretreatment with the ATi receptor specific blocker losartan or EXP-2528 or losartan plus the AT2 receptor blocker PD123319 reduced the MDA formation induced by Ang II, meanwhile increased the activity >f SOD and GSH-Px. However, pretreatment with PD122M9 alone did not prr-iuce significant effect and there were no significant difference between losartan group and losartan pluse PD123319 group.Leukocytes interaction with endothelium via the endothelial expression of adhesion molecules (AMs) may contribute to increasing BBB permeability and extending the area of infarction. Transcription factor nuclear factor-KB (NF-kB) has been proposed to serve as a unifying signaling system for inflammatory stimuli and involve in ischemic insult. Because Ang II may be involved in this process, we investigated its role on the signaling cascade leading to AMs expression in BMEC.The mRNA and protein expression of AMs including intercellular adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 (VCAM-1) and endothelial- Leukocyte adhesion molecule-1 (E-selectin) in BMEC was analyzed by RT-PCR and western blotting respectively. Ang II stimulated mRNA andprotein expression of ICAM-1, VCAM-1 and E-seletin in BMEC. These effects were inhibited by the selective ATi receptor antagonist losartan. The novel ATi receptor antagonist EXP-2528 also significantly inhibited the Ang II- induced AMs expression in BMEC. In contrast, pretreatment with the selective AT2 receptor antagonist PD123319 did not affect Ang II - induced AMs expression in BMEC, and there was no significant difference between losartan and losartan + PD 123319 groups.NF-icB/p65 and IicBa content were measured by Western Blot; DNA binding activity of NF-kB was monitored by electrophoretic mobility shift assay. Ang II treatment resulted in the degradation of IicBa and increase of NF-kB p65 subunit in the nucleus as well as the nuclear translocation of NF-kB. The activation of NF-kB and degradation of IkBo. induced by Ang II were abolished by pretreatment with the selective ATi receptor antagonists losartan and EXP-2528, or losartan plus the AT2 receptor antagonist PD 123319, but not modified by incubation with PD123319 alone. Moreover, there were no significant differences between the losartan and losartan plus PD 123319 groups.We further investigated whether EXP-2528 has protective effect on brain injury after focal cerebral ischemia reperfusion in rats. Experiment was carried out in the normotensive male Wistar Rats. Focal cerebral ischemia was induced by the middle cerebral artery occlusion of (MCAO) lasting for 2 h followed by 24 h reperfusion. The selective ATi antagonists losartan (5mg/kg/d), EXP-2528 (2.5mg/kg/d or 5mg/kg/d), was infused intragastrically over a 14-day period before the induction of ischemia. 24 hours after reperfusion, the behavioral tests, the infarct volume by 2,3,5-triphenyltetrazolium chloride (TTC) staining and routine pathological examination were used to evaluate the damage to central nervous system; the plasma Ang II and endothelin (ET) levels were determined by radioimmunoassay. The results showed that pretreatment with losartan (5mg/kg/d), or EXP-2528 (2.5, 5 mg/kg/d) significantly decreased neurological deficit scores reduced the percentage of infarction in ipsilateral hemisphere andalleviate the histophathological damage in hippocampal CA1 region compare with the model group. The plasma Ang II and ET level increased markedly after focal cerebral ischemia/reperfusion injury, pretreatment with losartan or different doses of EXP-2528 deceased the endothelin level, but increased Ang II level compared with the model group.In summary, the present study suggested that Ang II induced cellular oxidative, activated NF-kB, upregulated ICAM-1, VCAM-1 and E-selectin expression, and injured BMEC mainly via ATi receptor pathway. Novel ATj receptor blocker, EXP-2528, the same as losartan could protect against Ang II -induced BMEC injury by blocking ATi receptor. Pretreatment with EXP-2528 efficiently reduced brain injury after focal cerebral ischemia followed by reperfusion in rats.Our findings may provide a mechanistic basis for the benefits of selective ATi receptor blockade in preserving the integrity of blood brain barrier in cerebrovascular disease. We also provide some basic materials for novel ATi receptor blocker, EXP-2528.