脑损伤
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Figure 1｜Effect of ECs-MKP-1 overexpression on ischemic brain injury.
To elucidate the specific role of MKP-1 expression in ECs, lentiviral vector encoding MKP-1 under the control of the endothelial promotor Tie1 was injected into the ischemic side of the brain. Five days after injection, isolated microvessel homogenate was prepared to validate MKP-1 overexpression by western blot assay. We found that MKP-1 expression was significantly enhanced in the Tie1-MKP-1 group compared with the control vector group (P < 0.01; Figure 1A). MKP-1 overexpression was confirmed by immunofluorescence staining, as an increase in MKP-1 expression in microvessels (lectin+) was observed in the Tie1-MKP-1 group compared with the control group (P < 0.05; Figure 1B). To evaluate the effect of MKP-1 on brain injury, infarct size was measured 3 days after tMCAO. As expected, MKP-1 overexpression significantly reduced infarct size compared with the control vector group (P < 0.05; Figure 1C).
Figure 3｜Effect of EC-MKP-1 overexpression on stroke-induced cerebrovascular destruction.
Cerebral microvessel destruction and BBB impairment after stroke are the key factors causing secondary brain injury (Arai et al., 2011). Therefore, to investigate the role of EC-specific MKP-1 expression in vascular integrity, we visualized microvessels by intravenous injection of lectin conjugated with DyLight 594 and quantified MKP-1 and lectin coexpression 3 days after tMCAO. We found that ischemia induced blood vessel destruction in the peri-infarct zone, as evidenced by a reduction in lectin signal intensity compared with the sham group (P < 0.01; Figure 3A). EC-specific MKP-1 overexpression protected against vessel destruction compared with the control vector group (P < 0.05). Next, the effect of EC-specific MKP-1 on BBB leakage was investigated. Ischemia increased BBB permeability, which was reflected by the increase in IgG signal intensity compared with the sham group (P < 0.001; Figure 3B). As expected, EC-specific MKP-1 overexpression ameliorated stroke-induced BBB leakage compared with the control vector group (P < 0.01).
Circulating neutrophils and macrophages can infiltrate through the disrupted BBB within hours of an ischemic event and produce pro-inflammatory cytokines, such as IL-1, IL-6, and TNF-α. This induces the release of chemokines such as CCL-2 from brain tissue (An et al., 2014; Jayaraj et al., 2019), leading to expansion of the infarct area. We hypothesized that the protective effect of EC-specific MKP-1 overexpression on BBB integrity would alleviate this inflammatory reaction. We measured a panel of inflammatory cytokines and chemokines in ischemic brain parenchyma 3 days after tMCAO. Indeed, tMCAO led to a significant increase in the expression of markers of inflammation, including IL-1β, IL-6, TNF-α, and CCL-2 (all P < 0.001; Figure 3C). EC-specific MKP-1 overexpression alleviated the increase in IL-1β, IL-6, and CCL-2 expression compared with the control vector group (all P < 0.05). However, TNF-α expression levels were not altered by MKP-1 overexpression.
Figure 5｜Effect of inhibiting MKP-1-ERK1/2 signaling on BBB permeability in vitro.
Given that Tie1 has been detected in human and rat platelets (Fujikawa et al., 1999), it is possible that the Tie1-MKP-1 vector-mediated BBB protection that we observed in vivo may be due to MKP-1 expression within platelets. To rule out this possibility, we confirmed the role of EC-specific MKP-1 expression in an in vitro BBB model. An EC monolayer was subjected to 60-minute OGD, and the transit of fluorescent tracers (fluorescein isothiocyanate-dextran, 70 kDa) through the monolayer was evaluated. Compared with normoxic conditions, OGD induced significant leakage through the monolayer, as evidenced by an increase in the concentration of the fluorescent tracer in the abluminal medium 6 hours after OGD (P < 0.001; Figure 5A). Importantly, perfusion with 1 μM of the MKP-1 inhibitor BCI progressively worsened barrier leakage compared with vehicle control group (P < 0.01 at 3 hours, P < 0.001 at 6 hours), which is in line with our in vivo results. We then explored pathways downstream of MKP-1. ERK1/2 inhibition with 10 nM ravoxertinib significantly alleviated monolayer leakage (P < 0.01 at 6 hours). In addition, occludin expression was measured 12 hours after OGD by immunofluorescence assay. The OGD-induced reduction in occludin expression was worsened by BCI (P < 0.01; Figure 5B) and alleviated by ravoxertinib (P < 0.05) compared with the vehicle groups. Interestingly, co-incubating cells with MKP-1 and ERK1/2 inhibitors had the same effect on barrier permeability and occludin expression as treatment with ERK1/2 inhibitor alone, suggesting that MKP-1 protects against BBB leakage by deactivating ERK1/2.
Figure 6｜Effect of inhibiting MKP-1-ERK1/2 signaling on HBMEC viability in vitro.
We next asked whether MKP-1-ERK1/2 signaling regulates EC viability. Neither 1 μM BCI nor 10 nM ravoxertinib altered cell viability under normoxic conditions, as determined by CCK-8 assay (Figure 6A), suggesting that MKP-1 and ERK1/2 inhibition were not cytotoxic. Similar to a previous study (Bu et al., 2019), OGD induced significant cell death (P < 0.001; Figure 6A) as well as caspase-3 cleavage, an essential sign of programmed cell death (Porter and J?nicke, 1999) (P < 0.001; Figure 6B) at 12 hours after OGD. Importantly, incubation of ECs with BCI further reduced cell viability compared with the vehicle group (CCK-8: P < 0.001; cleaved caspase-3: P < 0.01), whereas ravoxertinib significantly salvaged cells (CCK-8: P < 0.05; cleaved caspase-3: P < 0.05). There was no significant difference between cells treated with both BCI and ravoxertinib and cell treated with ravoxertinib alone.