J Korean Neurosurg Soc.  2016 Nov;59(6):544-550. 10.3340/jkns.2016.59.6.544.

Proteomic Analysis of a Rat Cerebral Ischemic Injury Model after Human Cerebral Endothelial Cell Transplantation

• Choi TM
• Yun M
• Lee JK
• Park JT
• Park MS
• Kim HS

• KMID: 2417334
• DOI: http://doi.org/10.3340/jkns.2016.59.6.544

Abstract

OBJECTIVE
Cerebral endothelial cells have unique biological features and are fascinating candidate cells for stroke therapy.
METHODS
In order to understand the molecular mechanisms of human cerebral endothelial cell (hCMEC/D3) transplantation in a rat stroke model, we performed proteomic analysis using 2-dimensional electrophoresis and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry. Protein expression was confirmed by quantitative real-time PCR and Western blot.
RESULTS
Several protein spots were identified by gel electrophoresis in the sham, cerebral ischemia (CI), and CI with hCMEC/D3 treatment cerebral ischemia with cell transplantation (CT) groups, and we identified 14 differentially expressed proteins in the CT group. Proteins involved in mitochondrial dysfunction (paraplegin matrix AAA peptidase subunit, SPG7), neuroinflammation (peroxiredoxin 6, PRDX6), and neuronal death (zinc finger protein 90, ZFP90) were markedly reduced in the CT group compared with the CI group. The expression of chloride intracellular channel 4 proteins involved in post-ischemic vasculogenesis was significantly decreased in the CI group but comparable to sham in the CT group.
CONCLUSION
These results contribute to our understanding of the early phase processes that follow cerebral endothelial cell treatment in CI. Moreover, some of the identified proteins may present promising new targets for stroke therapy.

Keyword

Brain; Ischemia; Cell therapy; Proteomics; 2-dimensional electrophoresis

MeSH Terms

Animals
Blotting, Western
Brain
Brain Ischemia
Cell Transplantation
Cell- and Tissue-Based Therapy
Electrophoresis
Endothelial Cells*
Fingers
Humans*
Ischemia
Mass Spectrometry
Neurons
Proteomics
Rats*
Real-Time Polymerase Chain Reaction
Stroke
Transplants

Figure

• 

  Fig. 1 Representative two-dimensional gel electrophoretograms of rat brain tissue. (A) Sham group, (B) Photothrombotic focal cerebral ischemia (CI) group, and (C) CI treated with human cerebral endothelial cells (hCMEC/D3; CT) group. Circles show identified proteins that exhibit differences in average abundance values for each group (n=3 per group).

• 

  Fig. 2 qRT-PCR for selected target genes in the CT group compared with sham and CI groups. The mRNA levels of Ephb1, Zfp90, Prdx6, Spg7, and Clic4 were measured by qRT-PCR and calculated by the ΔΔCt methodology as described in methods. The assays were run in triplicate, and the standard error of the mean (SEM) was determined. Relative expression of Zfp90, Prdx6, and Spg7 were markedly decreased in the CT group compared with the CI group, but changes in Ephb1 did not reach statistical significance (A-D). mRNA for Clic4 was markedly decreased in the CI group but was rescued in the CT group (E; n=5 per group). Data are presented as mean±SEM. *p<0.05, **p<0.01 vs. CT group. qRT-PCR : quantitative real-time PCR, CI : cerebral ischemia, mRNA : messenger ribonucleic acid, EPHB1 : eph receptor B1, ZFP90 : zinc finger protein 90, PRDX6 : peroxiredoxin 6, SPG7 : paraplegin matrix AAA peptidase subunit, CLIC4 : chloride intracellular channel 4, GAPDH : glyceraldehyde 3-phosphate dehydrogenase.

• 

  Fig. 3 Western blot analysis of proteomic and qRT-PCR results from CI and CT groups. A : Cropped bands of the Western blot gel corresponding to EPHB1, ZFP90, PRDX6, SPG7, and CLIC4 by group. B : Proteins that were increased in the CI group were markedly decreased in the CT group. C : Reduced CLIC4 in the CI group was normalized to sham level in the CT group (n= 3 per group). Data are presented as mean±SEM. *p<0.05, **p<0.01 vs. CT group. qRT-PCR : quantitative real-time PCR, CI : cerebral ischemia, EPHB1 : eph receptor B1, ZFP90 : zinc finger protein 90, PRDX6 : peroxiredoxin 6, SPG7 : paraplegin matrix AAA peptidase subunit, CLIC4 : chloride intracellular channel 4, SEM : standard error of the mean.

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