Brain Circulation (Jan 2017)

Activated complement protein C5a does not affect brain-derived endothelial cell viability and zonula occludens-1 levels following oxygen-glucose deprivation

  • Aren Khoyetsyan,
  • Rachid Kacimi,
  • Gohar Tsakanova,
  • Anna Boyajyan,
  • Arsen Arakelyan,
  • Midori A Yenari

DOI
https://doi.org/10.4103/2394-8108.203258
Journal volume & issue
Vol. 3, no. 1
pp. 14 – 20

Abstract

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BACKGROUND AND PURPOSE: Ischemic brain injury induces both functional and structural disarray affecting the blood–brain barrier (BBB) which in return aggravates stroke outcomes. Complement system and its bioactive proteins are important molecular responders to ischemia. C5a protein along with its receptor C5a receptor 1 is a key component of this system with potent pro-inflammatory and chemoattractant properties. The purpose of this study is to investigate the role of C5a protein and its receptor which are believed to participate in the inflammatory response that follows ischemic insult. MATERIALS AND METHODS: To mimic an ischemic in vivo event in which C5a may contact brain endothelial cells after injury, we studied oxygen-glucose deprivation (OGD) followed by reperfusion in brain microvascular endothelial cells (b.End. 3) by only added C5a at the time of reperfusion. Cell death and viability were estimated by trypan blue and 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide assays, respectively. Tight junction protein zonula occluden (ZO-1) levels were analyzed by Western blot analysis, and nitric oxide (NO) was assessed using the Griess reagent. RESULTS: Brain-derived endothelial cell was susceptible to OGD-induced injury in a duration-dependent manner as was the presence of ZO-1 protein. However, the addition of C5a protein had no notable effects even when used at high concentrations up to 100 nM. While OGD led to reduction in ZO-1 protein levels, no change was seen following the addition of C5a. Finally, OGD led unexpectedly to small decreases in NO generation, but this was again unaltered by C5a. CONCLUSIONS: Our study suggests that complement system protein C5a may not have a direct role in the disruption of BBB, following brain ischemia. This is in contrary with previous literature that suggests a possible role of this protein in the inflammatory response to ischemia.

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