In Vivo Inhibition of miR-155 Promotes Recovery after Experimental Mouse Stroke

Abstract

A multifunctional microRNA, miR-155, has been recently recognized as an important modulator of numerous biological processes. In our previous in vitro studies, miR-155 was identified as a potential regulator of the endothelial morphogenesis. The present study demonstrates that in vivo inhibition of miR-155 supports cerebral vasculature after experimental stroke. Intravenous injections of a specific miR-155 inhibitor were initiated at 48 h after mouse distal middle cerebral artery occlusion (dMCAO). Microvasculature in peri-infarct area, infarct size, and animal functional recovery were assessed at 1, 2, and 3 weeks after dMCAO. Using in vivo two-photon microscopy, we detected improved blood flow and microvascular integrity in the peri-infarct area of miR-155 inhibitor-injected mice. Electron microscopy revealed that, in contrast to the control group, these animals demonstrated well preserved capillary tight junctions (TJs). Western blot analysis data indicate that improved TJ integrity in the inhibitor-injected animals could be associated with stabilization of the TJ protein ZO-1 and mediated by the miR-155 target protein Rheb. MRI analysis showed significant (34%) reduction of infarct size in miR-155 inhibitor-injected animals at 21 d after dMCAO. Reduced brain injury was confirmed by electron microscopy demonstrating decreased neuronal damage in the peri-infarct area of stroke. Preservation of brain tissue was reflected in efficient functional recovery of inhibitor-injected animals. Based on our findings, we propose that in vivo miR-155 inhibition after ischemia supports brain microvasculature, reduces brain tissue damage, and improves the animal functional recovery.

Inhibition of MicroRNA‐155 Supports Endothelial Tight Junction Integrity Following Oxygen‐Glucose Deprivation

“…These data suggest that stabilization of ZO‐1 in the OGD/I group of cells was not mediated via miR‐155 direct targets Rheb and RhoA. Taken together, analyses of the cell junction proteins revealed that miR‐155 inhibition in the OGD‐subjected HBMECs led to ZO‐1 stabilization, which is in agreement with previous studies on neuroinflammation and experimental ischemia 18, 29…”

“…ZO‐1 upregulation was also reported after the in vivo inhibition of miR‐155. In these studies, we proposed that ZO‐1 stabilization was mediated by miR‐155 direct target protein Ras homolog enriched in brain (Rheb) 18, 27. However, in the present study, Rheb levels were not affected by miR‐155 inhibition or overexpression (which probably means that in vivo experiments detected the combined Rheb levels in the endothelial cells, as well as microglia and astrocytes).…”

“…For more accurate monitoring of the inhibition efficiency, we checked the expression levels of miR‐155 direct target proteins Rictor and SMAD‐1. These proteins were upregulated following the in vivo inhibition of miR‐155 in our previous studies 18. Accordingly, Western blot analysis of OGD/I cell lysates revealed statistically significant upregulation of Rictor (by 40%) and SMAD‐1 (by 84%), as compared with control OGD/IC samples (Figure 1E).…”

“…No significant differences in cell viability were detected between OGD/I, OGD/IC, OGD/M, and OGD/MC samples. Another possible regulator of the endothelial integrity, endothelial NO synthase, is directly targeted by miR‐155 16, 18. NO is involved in protection of the BBB from hypoxia/reoxygenation‐mediated injury 22.…”

“…In addition to these findings, we also demonstrated that in vivo inhibition of miR‐155 following experimental cerebral ischemia results in preservation of microvascular integrity in the peri‐infarct area of the mouse brain. Improved TJ integrity in the inhibitor‐injected animals was accompanied by increased levels of the TJ protein ZO‐1 in brain tissue extracts 18. In addition, we proposed that miR‐155 inhibition–induced stabilization of TJs could be associated with altered poststroke inflammatory response 19.…”