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. 2019 Feb 22:12:48.
doi: 10.3389/fnmol.2019.00048. eCollection 2019.

Omi/HtrA2 Protease Associated Cell Apoptosis Participates in Blood-Brain Barrier Dysfunction

Yueyu Hu  1 , Yong Bi  1 , Danhua Yao  2 , Pengfei Wang  2 , Yousheng Li  2
Affiliations

Omi/HtrA2 Protease Associated Cell Apoptosis Participates in Blood-Brain Barrier Dysfunction

Yueyu Hu et al. Front Mol Neurosci. .

Abstract

Background: Omi/HtrA2 is a proapoptotic mitochondrial serine protease involved in caspase-dependent cell apoptosis, translocating from mitochondria to the cytosol after an apoptotic insult. Our previous study indicated pre-treatment with UCF-101, a specific inhibitor of Omi/HtrA2, could significantly reduce neuronal apoptosis and attenuate sepsis-induced cognitive dysfunction. Various hypotheses involving blood-brain-barrier (BBB) disruption have been proposed to account for sepsis-associated encephalopathy (SAE). Here, we attempted to explore whether interference of Omi/HtrA2 by RNA interference or UCF-101 pre-treatment can improve sepsis-induced disruption of BBB using human cerebral microvascular endothelial cell line (hCMEC/D3) in vitro and if so, to explore mechanisms involved Omi/HtrA2 protease mediates BBB disruption in SAE. Methods: hCMEC/D3 cell monolayers were intervened by different concentrations of LPS (0-50 μg/mL) over experimental period. Pharmacological or gene interventions (by silencing RNA of Omi/HtrA2) were used to study molecular mechanisms involved in sepsis-associated Omi/HtrA2 translocation, cell apoptosis and BBB dysfunction. BBB function was assessed by trans-endothelial electrical resistance (TEER) and permeability to labeled dextrans (FITC-4kDa). Tight junction (TJ) integrity was assessed by immunofluorescence, western blotting and transmission electron microscopic (TEM) analyses. Apoptosis was determined using flow cytometry and TUNEL assay. Mitochondrial membrane potential (MMP) and oxidative stress were also investigated. Results: LPS affects hCMEC/D3 TJ permeability in a concentration- and time-dependent manner. LPS intervention resulted in a significant disruption of BBB, as manifested by decreased TEER (by ~26%) and a parallel increased paracellular permeability to FITC- (4kDa) dextrans through hCMEC/D3 monolayers. The inhibition of Omi/HtrA2 by UCF-101 or Omi/HtrA2 shRNA reduced LPS-induced brain endothelial cell apoptosis, and resulted in significant improvement on LPS-induced BBB disruption as well as decreased occludin, claudin-5 and ZO-1 expressions. Omi/HtrA2 manipulated endothelial cell apoptosis by shifting into cytosol and inducing X-linked inhibitor of apoptosis protein (XIAP) degradation. UCF-101 administration or Omi/HtrA2 shRNA intervention did attenuate the degradation of XIAP, Poly ADP-ribose polymerase (PARP) cleavage, and caspase-3 cleavage. However, only UCF-101 partly prevented the mobilization of Omi/HtrA2 from the mitochondria to the cytosol after LPS intervention. That abrogation of Omi/HtrA2 by UCF-101 or Omi/HtrA2 shRNA resulted in a significant improvement on LPS-induced decrease of MMP. Oxidative stress was significantly increased in the LPS treated group compared to the control or NC-shRNA group. However, abrogation of Omi/HtrA2 by UCF-101 or Omi/HtrA2 shRNA did not significantly improve oxidative injury. Conclusions: Our study indicated an important role of Omi/HtrA2 in manipulating LPS-induced cell apoptosis and BBB integrity by translocating from mitochondria into cytosol in brain endothelial cells. Omi/HtrA2 induced mitochondrial pathway apoptosis, which involves inhibition of an important antiapoptotic protein XIAP and influence on MMP. Therapeutic methods that inhibit Omi/HtrA2 function may provide a novel therapeutic measure to septic encephalopathy.

Keywords: Omi/HtrA2; UCF-101; blood-brain barrier; hCMEC/D3 cells; sepsis.

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Figures

Figure 1
Figure 1
LPS induces human cerebral microvascular endothelial cell line (hCMEC/D3) cell apoptosis and affects blood-brain-barrier (BBB) permeability in a dose- and time-dependent manner. (A) The effects of different concentrations of LPS (0–50 μg/mL) on BBB integrity was determined in vitro by measuring trans-endothelial electrical resistance (TEER) over a 24-h experimental period. (B) Effects of 10 μg/mL LPS on TEER after 18-h treatment. (C) Effects of 10 μg/mL LPS on paracellular permeability to FITC- (4kDa) dextrans across hCMEC/D3 monolayers after 18-h treatment. (D) Effects of LPS on early cell apoptosis as determined by flow cytometry; quantitative analysis of the percentage of apoptotic cells was calculated. (E) Effects of LPS on cell apoptosis as determined by TUNEL assay. Apoptotic cells appear brown and the nucleus appear blue. Scale bar, 200 μm; quantitative analysis of the percentage of apoptotic cells was calculated. *P < 0.01, vs. control.
Figure 2
Figure 2
Omi/HtrA2 regulates LPS-induced brain endothelial cell apoptosis in LPS-treated hCMEC/D3 monolayers. (A) Fluorescent images of silencing Omi/HtrA2 gene HCMEC/D3 cell. Scale bar, 25 μm. (B) Omi/HtrA2 knock-down efficiency as determined by Rt-PCR or (C) western blot analyses in hCMEC/D3 cells transfected with Omi/HtrA2 shRNA lentivirus. (D) Effects of inhibition of Omi/HtrA2 by UCF-101 or Omi/HtrA2 shRNA on cell apoptosis as determined by flow cytometry; quantitative analysis of the percentage of apoptotic cells. (E) Effects of inhibition of Omi/HtrA2 by UCF-101 or Omi/HtrA2 shRNA on cell apoptosis as determined by TUNEL assay. Apoptotic cells appear brown and the nucleus appear blue. Scale bar, 200 μm; The percentage of apoptotic cells was calculated. *P < 0.05, vs. control or NC-shRNA, **P < 0.01, vs. control or NC-shRNA, ++P < 0.01, vs. control + LPS or NC-shRNA + LPS.
Figure 3
Figure 3
Omi/HtrA2 regulates LPS-induced loss of brain endothelial barrier integrity and tight junction (TJ) protein expression. (A) Effects of inhibition of Omi/HtrA2 by UCF-101 or Omi/HtrA2 shRNA on TEER in LPS-treated hCMEC/D3 monolayers over a 24-h experimental period. (B) Effects of inhibition of Omi/HtrA2 by UCF-101 or Omi/HtrA2 shRNA on TEER after 18-h treatment. (C) Effects of inhibition of Omi/HtrA2 by UCF-101 or Omi/HtrA2 shRNA on paracellular permeability to FITC- (4kDa) dextrans across hCMEC/D3 monolayers after 18-h treatment. (D,E) Effects of inhibition of Omi/HtrA2 by UCF-101 or Omi/HtrA2 shRNA on occludin, claudin-5 and ZO-1 expressions as determined by western blot analyses. Respective bands with GAPDH as loading control were shown. *P < 0.05, vs. control or NC-shRNA, **P < 0.01, vs. control or NC-shRNA. (F) Effects of abrogation of Omi/HtrA2 by Omi/HtrA2 shRNA on BBB ultrastructures in LPS-treated hCMEC/D3 monolayers as determined by transmission electron microscopic (TEM). Arrows show TJ. Scale bar, 10 μm.
Figure 4
Figure 4
Effects of abrogation of Omi/HtrA2 by UCF-101 (A) or Omi/HtrA2 shRNA (B) on TJ protein occludin expression in LPS-treated hCMEC/D3 monolayers as determined by immunofluorescence. Red staining show TJ. Nucleus (blue) was labeled with DAPI. Scale bar, 25 μm.
Figure 5
Figure 5
Effects of abrogation of Omi/HtrA2 by UCF-101 (A) or Omi/HtrA2 shRNA (B) on TJ protein claudin-5 expression in LPS-treated hCMEC/D3 monolayers as determined by immunofluorescence. Red staining show TJ. Nucleus (blue) was labeled with DAPI. Scale bar, 25 μm.
Figure 6
Figure 6
Effects of abrogation of Omi/HtrA2 by UCF-101 (A) or Omi/HtrA2 shRNA (B) on TJ protein ZO-1 expression in LPS-treated hCMEC/D3 monolayers as determined by immunofluorescence. Red staining show TJ. Nucleus (blue) was labeled with DAPI. Scale bar, 25 μm.
Figure 7
Figure 7
Omi/HtrA2 regulates LPS-induced endothelial cell apoptosis by translocating from mitochondria into cytosol via proteolytic degradation of proteins that can normally inhibit caspase activity. (A) Effects of Omi/HtrA2 protease activity inhibition by UCF-101 or (B) Omi/HtrA2 silencing by Omi/HtrA2 shRNA on Omi release into the cytoplasm in LPS-treated hCMEC/D3 monolayers. Respective bands with β-actin as loading control were shown. (C) Effects of Omi/HtrA2 inhibition by UCF-101 or (D) Omi/HtrA2 silencing by Omi/HtrA2 shRNA on degradation of X-linked inhibitor of apoptosis protein (XIAP), Poly ADP-ribose polymerase (PARP) cleavage, and caspase-3 cleavage in LPS-treated hCMEC/D3 monolayers. Respective bands with GAPDH as loading control were shown. *P < 0.05, vs. control or NC-shRNA, **P < 0.01, vs. control or NC-shRNA, +P < 0.05, vs. control + LPS or NC-shRNA + LPS, ++P < 0.01, vs. control + LPS or NC-shRNA + LPS.
Figure 8
Figure 8
Effects of inhibition of Omi/HtrA2 by UCF-101 or Omi/HtrA2 shRNA on mitochondrial membrane potential (MMP) in LPS-treated hCMEC/D3 monolayers as determined by JC-1 staining using fluorescence microscope. Cells stained with red color indicates high MMP and green color indicates low MMP. Scale bar, 25 μm.
Figure 9
Figure 9
Effects of inhibition of Omi/HtrA2 by UCF-101 or Omi/HtrA2 shRNA on MMP in LPS-treated hCMEC/D3 monolayers as determined by JC-1 staining using flow cytometric detection. The results are expressed as the ratio of J-monomers to aggregates. **P < 0.01, vs. pre-LPS, ##P < 0.01, vs. control, @@P < 0.01, vs. NC-shRNA.
Figure 10
Figure 10
Oxidative stress did not play a key role in alleviating apoptosis by Omi/HtrA2 intervention. (A–D) Effects of Omi/HtrA2 inhibition by UCF-101 or (E–H) Omi/HtrA2 shRNA on GSH, malondialdehyde (MDA), catalase (CAT) and myeloperoxidase (MPO) levels in LPS-treated hCMEC/D3 monolayers. *P < 0.05, vs. control or NC-shRNA, **P < 0.01, vs. control or NC-shRNA.
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References

    1. Aborehab N. M., El Bishbishy M. H., Refaiy A., Waly N. E. (2017). A putative Chondroprotective role for IL-1β and MPO in herbal treatment of experimental osteoarthritis. BMC Complement. Altern. Med. 17:495. 10.1186/s12906-017-2002-y - DOI - PMC - PubMed
    1. Althaus J., Siegelin M. D., Dehghani F., Cilenti L., Zervos A. S., Rami A. (2007). The serine protease Omi/HtrA2 is involved in XIAP cleavage and in neuronal cell death following focal cerebral ischemia/reperfusion. Neurochem. Int. 50, 172–180. 10.1016/j.neuint.2006.07.018 - DOI - PubMed
    1. Banks W. A. (2005). Blood-brain barrier transport of cytokines: a mechanism for neuropathology. Curr. Pharm. Des. 11, 973–984. 10.2174/1381612053381684 - DOI - PubMed
    1. Cardoso F. L., Kittel A., Veszelka S., Palmela I., Tóth A., Brites D., et al. . (2012). Exposure to lipopolysaccharide and/or unconjugated bilirubin impair the integrity and function of brain microvascular endothelial cells. PLoS One 7:e35919. 10.1371/journal.pone.0035919 - DOI - PMC - PubMed
    1. Chaudhry N., Duggal A. K. (2014). Sepsis associated encephalopathy. Adv. Med. 2014:762320. 10.1155/2014/762320 - DOI - PMC - PubMed