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. 2016 Sep 15;5(9):e003615.
doi: 10.1161/JAHA.116.003615.

MKEY, a Peptide Inhibitor of CXCL4-CCL5 Heterodimer Formation, Protects Against Stroke in Mice

Yifang Fan  1 , Xiaoxing Xiong  2 , Yongming Zhang  3 , Dongmei Yan  3 , Zhihong Jian  2 , Baohui Xu  4 , Heng Zhao  5
Affiliations

MKEY, a Peptide Inhibitor of CXCL4-CCL5 Heterodimer Formation, Protects Against Stroke in Mice

Yifang Fan et al. J Am Heart Assoc. .

Abstract

Background: MKEY, a synthetic cyclic peptide inhibitor of CXCL4-CCL5 heterodimer formation, has been shown to protect against atherosclerosis and aortic aneurysm formation by mediating inflammation, but whether it modulates neuroinflammation and brain injury has not been studied. We therefore studied the role of MKEY in stroke-induced brain injury in mice.

Methods and results: MKEY was injected into mice after stroke with 60 minutes of middle cerebral artery occlusion. Infarct volume and neurological deficit scores were measured. Protein levels of CCL5 and its receptor CCR5 were detected by Western blot and fluorescence-activated cell sorting (FACS), respectively. Numbers of microglia-derived macrophages (MiMΦs) and monocyte-derived MΦs (MoMΦs) in the brain, and their subsets, based on the surface markers CD45, CD11b, CCR2, CX3CR1, and Ly6C, were analyzed by FACS. MΦs and neutrophil infiltration in the ischemic brain were stained with CD68 and myeloperoxidase (MPO), respectively, and assessed by immunofluorescent confocal microscopy. The results showed that expressions of CCL5 and its receptor CCR5, were increased in the ischemic brain after stroke. MKEY injection significantly reduced infarct sizes and improved neurological deficit scores measured 72 hours after stroke. In addition, MKEY injection inhibited the number of MoMΦs, but not MiMΦs, in the ischemic brain. Furthermore, MKEY inhibited protein expression levels of Ly6C,CCR2, and CX3CR1 on MoMΦs. Lastly, the confocal study also suggests that the number of CD68-positive MΦs and MPO-positive neutrophils was inhibited by MKEY injection.

Conclusions: MKEY injection protects against stroke-induced brain injury, probably by inhibiting MoMΦ-mediated neuroinflammation.

Keywords: cerebrovascular disease/stroke; immunology; infarct or infarction; inflammation.

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Figures

Figure 1
Figure 1
The effect of MKEY injection on infarction and neurological scores measured 72 hours after stroke. A, Left: Representative TTC staining images for infarction in mice treated with vehicle and MKEY. Right: Infarct volumes expressed as a percentage of the nonischemic hemisphere. B, Neurological scores in mice treated with vehicle and MKEY. n=9 to 11/group. *P<0.05 compared to vehicle.
Figure 2
Figure 2
CCL5 expression was increased and measured 72 hours poststroke. A, Representative Western blots for CCL5 at 10.2 kDa in sham surgery and stroke brain. B, Quantification of CCL5 protein levels in sham and stroke brain. n=3/group. *P=0.05 vs sham.
Figure 3
Figure 3
CCR5 expression was increased in both MiMΦs and MoMΦs. A, Representative FACS gates for MiMΦs (CD45int CD11b+ cells) and MoMΦs (CD45hi CD11b+) are shown. B, Representative FACS plots for CCR5 and CX3CR1 expression on gated MiMΦs isolated from sham and stroke brain are shown. C, Representative FACS histograms for CCR5 expression on peripheral blood or brain CD45hi CD11b+ cells in mice with sham surgery or stroke.
Figure 4
Figure 4
MKEY injection reduced numbers of MoMΦs and their subsets, but not those of MiMΦs, in the ischemic brain 72 hours after stroke. A, Gating strategies to identify MiMΦs and MoMΦs, as well as their subsets, CD45hi CD11b+ CCR2+, CD45hi CD11b+Ly6C+, CD45hi CD11b+ CX3CR1+, CD45int CD11b+ CCR2+, and CD45int CD11b+ CX3CR1+ in the ischemic brain. B, Quantification of MΦ numbers and their subsets in mice treated with vehicle or MKEY, with sham or stroke surgery. *, **, ***, vs sham surgery, P<0.05, 0.01, 0.001, respectively; #, ##, between the 2 indicated groups, P<0.05, 0.01, respectively.
Figure 5
Figure 5
MKEY treatment poststroke reduced the infiltration of CD68+ and MPO + cells. A, A square area in a coronal brain section stained with cresyl violet was predefined and used for counting immunostained cells. B, Representative immunofluorescence staining for CD68, MPO, and DAPI (control) from vehicle and MKEY‐treated animals euthanized 72 hours after stroke. C, Quantification of CD68‐ and MPO‐positive cells in the predefined area. N=5/group. Bar: 20 μm. *P<0.05 compared to vehicle.
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