Response to Letter Regarding Article, “Norrin Protected Blood–Brain Barrier via Frizzled-4/β-Catenin Pathway After Subarachnoid Hemorrhage in Rats”
We appreciate Zuo and Xu1 for their interest in our study and their concerns about the frizzled-4 small interfering RNA (siRNA) efficacy in our experiments. We concur that the magnitude of suppression of siRNA in vivo was less than that achieved in vitro, therefore we used a pool of 3 different frizzled-4 siRNA duplexes to improve the knockdown efficiency. As presented in Figure IV in the online-only Data Supplement,2 frizzled-4 siRNA pretreatment significantly inhibited frizzled-4 receptor expression in ipsilateral hemisphere 48 hours after intraventricular siRNA injection. The average protein level of frizzled-4 was reduced ≈50% by siRNA than scrambled siRNA. Most importantly, the frizzled-4 siRNA did reverse its ligand norrin’s neuroprotective effects on blood–brain barrier integrity and early brain injury after subarachnoid hemorrhage.
Furthermore, we investigated the possible mechanism of how norrin performed these protective effects on blood–brain barrier and neurological functions and found norrin acted via frizzled-4/β-catenin pathway to enhance the expressions of tight junction proteins zona occludens (ZO)-1, occludin and adherent junction protein vascular endothelial-cadherin.2 However, Zuo and Xu, with reliable speculation, proposed that norrin/frizzled-4/β-catenin pathway could also enhance the expression of gap junction protein connexin-43. Although Connexin-43 is colocalized and coprecipitated with several tight junction proteins, it mainly forms ion channels and allows intercellular trafficking of ions and small signaling molecules through perivascular astrocyte networks. Nagasawa et al3 demonstrated that blocking gap junction did not influence the expression of occludin, claudin-5, ZO-1, or their subcellular localization in the blood–brain barrier endothelial cells. With connexin-43 knockout condition, even astrocyte end-feet enlarged, the endothelial cells were structurally normal in appearance, including their tight junctions, which was only vulnerable when subjected to elevated hydrostatic pressure and sheer stress.4 In addition, claudin-5, occludin, and vascular endothelial-cadherin recovered from the second day after blood nerve barrier injury, whereas connexin-43 was redetected on the fifth day.5 These results indicate that norrin/frizzled-4/β-catenin pathway could preserve tight junction proteins and adherent junction proteins in the early phase after subarachnoid hemorrhage to repair the mechanical barrier of blood–brain barrier and alleviate early brain injury.
Currently, most efforts on the blood–brain barrier and brain edema focus on vasogenic edema after subarachnoid hemorrhage6; Drs Zuo and Xu raised the potential cytotoxic edema in their letter, which is an extremely important question; however, cytotoxic edema is mostly verified by an MRI and which is not in the scope of our published study. But we agree with Drs Zuo and Xu that cytotoxic edema needs to be studied in similar experimental conditions after subarachnoid hemorrhage.
Yujie Chen, MD
Hua Feng, MD, PhD
Department of Neurosurgery, Southwest Hospital
Third Military Medical University
John H. Zhang, MD, PhD
Department of Physiology and Pharmacology
Loma Linda University
Sources of Funding
This study is supported by a grant from National Institutes of Health NS081740 to Dr Zhang.
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- © 2015 American Heart Association, Inc.
- Zuo X,
- Xu E.
- Chen Y,
- Zhang Y,
- Tang J,
- Liu F,
- Hu Q,
- Luo C,
- et al