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. 2020 Apr 13;41(1):151–162. doi: 10.1007/s10571-020-00842-1

CD151 Alleviates Early Blood–Brain Barrier Dysfunction After Experimental Focal Brain Ischemia in Rats

Wendeng Xu 1, Ceshu Gao 1, Jian Wu 1,✉
PMCID: PMC11448708  PMID: 32285246

Abstract

Preservation of the blood–brain barrier (BBB) function is a potential protective strategy against cerebral ischemic injuries. CD151 has a beneficial effect in maintaining vascular stability and plays a role in pro-angiogenesis. Both vascular stability and angiogenesis can affect BBB function. Therefore, we aimed to examine the action of CD151 in regulating BBB permeability after cerebral ischemic injury in the present study. Using a transient focal cerebral ischemia (tFCI) rat model, we established that CD151 overexpression in the brain mitigated the leakage of endogenous IgG at 6–24 h after tFCI in vivo. Moreover, we found that CD151 can decrease the diffusion of macromolecules through monolayer brain microvessel endothelial cells (BMVECs) after glucose and oxygen deprivation (OGD)–reoxygenation in vitro. Furthermore, overexpression of CD151 in BMVECs suppressed OGD–reoxygenation-induced F-actin formation and RhoA activity. However, while preserving BBB integrity after tFCI, CD151 overexpression did not affect the post-stroke outcomes. Taken together, the present study demonstrated that CD151 overexpression in the brain protects BBB permeability at early phase after tFCI. CD151 may be a potential target for early BBB protection in ischemic stroke.

Electronic supplementary material

The online version of this article (10.1007/s10571-020-00842-1) contains supplementary material, which is available to authorized users.

Keywords: CD151, Blood–brain barrier, Transient focal cerebral ischemia, Brain microvessel endothelial cells

Introduction

Stroke occupies the 5th position among all causes of death and ranks first among all causes of adult disability worldwide (Benjamin et al. 2019). Of all stroke events, ischemic stroke contributes to 87% (Benjamin et al. 2017). Despite advances in its prevention, ischemic stroke remains a significant global health burden (GBD 2016 Neurology Collaborators 2019). Currently, effective therapies are dependent largely on recanalization by using recombinant thrombolytic drugs or performing intra-arterial thrombectomy in appropriate patients. However, few patients benefit from these therapies owing to their threatening complications and narrow time window.

The blood–brain barrier (BBB) disruption plays a key role in stroke progresses, including the promotion of brain edema and hemorrhagic transformation which restricts the use of recanalization therapy, leading to unfavorable patient prognosis (Shi et al. 2016a). Moreover, a prior study has suggested that BBB disruption in the acute phase of brain ischemia is reversible (Kaur et al. 2009; Simpkins et al. 2016), and early BBB breakdown is a cause leading to neuron injury (Shi et al. 2016b). Hence, BBB-targeted protection in the initial phase after ischemia may extend the time window of recanalization therapy and improve the prognosis of ischemic stroke.

CD151, a member of the transmembrane four superfamily (Hemler 2005; Zöller 2009), mediates signal transduction events that play a part in regulating cell development, activation, motility, and growth. Currently, a few studies have shown that CD151 has various vascular functions (Bailey et al. 2011), including regulating endothelial cell (EC) migration, maintaining vascular stability (Zhang et al. 2011), and pro-angiogenic role (Huang et al. 2016). In vivo studies have suggested that CD151 overexpression exhibits a protective effect in ischemic animal models, including myocardial infarction (Zheng and Liu 2006; Yang et al. 2016) and hind limb ischemia models (Lan et al. 2005; Yang et al. 2016).

Based on these reports, CD151 can maintain vascular stability (Zhang et al. 2011) and exhibit a protective effect under ischemic conditions (Lan et al. 2005; Zuo et al. 2009). We hypothesized that CD151 overexpression on brain microvessel endothelial cells (BMVECs) alleviate BBB permeability in the early phase after cerebral ischemia–reperfusion injury. We tested this hypothesis by determining the role of CD151 overexpression on BMVECs in BBB function at different time points of reperfusion after transient focal cerebral ischemia (tFCI) in vivo and in vitro models (Fig. 1).

Fig. 1.

Fig. 1

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Schematic diagram of the experimental design. Briefly, adult Sprague–Dawley (SD) rats were transfected with Lenti-CD151/Lenti. Then, these rats were used to prepare tFCI models. CD151 expression pattern, BBB permeability, and stroke outcome (infarct size and neurologic function) were assessed. Moreover, primary BMVECs were extracted from SD rats. The cells transfected with Lenti-CD151/Lenti were used to generate an in vitro BBB model and was then subjected to OGD–reoxygenation insult. The effect of CD151 on the permeability of BBB was assessed in vitro. At the molecular level, the expression of tight junctional proteins, F-actin, and RhoA activity was assessed

Methods

Animals

SD rats (male, 7–8-week, 240-260 g) were purchased from Beijing Vital River Laboratory. The rats were housed in an animal center with a cycle of the 12-h light/12-h dark at 20–25 °C in humidified 50–60%. Water and food were provided ad libitum. The animal protocol was approved by the University of Tsinghua Institutional Animal Care and Use Committee.

Lentiviral Transfection Experiments and tFCI Ischemia Model

Rats were transfected with lentivirus-CD151 (Lenti-CD151)/lentivirus (lenti) via lateral cerebral ventricle injection. 5ul of lenti-CD151/lenti was injected into the right lateral cerebral ventricle. Rats were anesthesia with 10% chloral hydrate (30 mg/kg) by intraperitoneal injection. Lenti-CD151/lenti were slowly injected at a rate of 0.5 ul/min into the right lateral ventricle with the coordinates of 1.0 mm posterior to the bregma, 1.5 mm lateral to the midline, and 4.0 mm ventral to the surface of the skull with the aid of small animal stereo locator. Transfection efficiency was evaluated via Western blot and immunofluorescence at 4-day and 7-day after Lentiviral transfection, respectively.

Wild type rats and rats transfected with lenti-CD151/lenti were used to induce tFCI model by the method of intraluminal thread, and this has been detailed in the supplementary material.

Determination of BBB Permeability After tFCI

Endogenous IgG leakage was measured for assessing BBB permeability. Briefly, rats were transcardially perfused with normal saline, followed by 4% paraformaldehyde in phosphate-buffered saline (PBS) under deep anesthesia. Brains were obtained and dehydrated in 30% sucrose in PBS. Frozen coronal Sections (25 µm-thick) were prepared using a freezing microtome (Leica, German). We blocked sections in 5% bovine serum albumin (BSA) and incubated with anti-rat IgG antibody, Alexa Fluor® 584 conjugate (1:250; Jackson ImmunoResearch, USA) at 25 °C for 60 min, respectively. Sections were scanned with a four objective lens and then subjected to image mosaicking using a laser confocal microscopic (Nikon, Japan). Alexa Fluor® 584 immunofluorescences (IF) integrated density was obtained. Three non-continuos sections (the distance between two sections about 1.5 mm) encompassing whole striatum (bregma 2.52-bregma -2.04) per rat brain were used to analyze in our research, and BBB permeability was assessed by the ipsilateral IF intensity normalized by contralateral IF intensity.

Neurological Function Score

The severity of rat injury after tFCI was assessed by Longa five-point score at 24 h of reperfusion after tFCI (Longa et al. 1989)and by modified Bederson scale (Bederson et al. 1986) at 72 h of reperfusion after tFCI, detailed in the supplementary material.

Isolation and Purification of Rat BMVECs and Lentivirus Transfection

Primary rat BMVECs were obtained following the methods previously reported (Abbott et al. 1992; Xue et al. 2013); this has been detailed in the supplementary material. CD151 overexpression recombinant lentivirus (Lenti-CD151) and empty carrier recombinant lentivirus (Lenti) with a green fluorescent protein (GFP) tag were constructed by Genepharma (Shanghai, China). BMVECs were infected with Lenti-CD151 or lenti at a multiplicity of infection (MOI) of 50. Transfection efficiency of the lentiviruses was assessed by determining GFP expression by immunofluorescence, and CD151 expression was determined by western blotting (Supplementary Fig).

OGD Reoxygenation and In Vitro BBB Model

To prepare an in vitro ischemia model, BMVECs were treated with transient OGD (95% N2 and 5% CO2 in a glucose-free medium) in a modular incubator chamber for 60 min. Then, BMVECs were reoxygenation in a complete medium for a time period based on the experiment requirement.Transwell membranes with 0.4 μm pore (Corning, USA) and monolayer BMVECs were used to construct the vitro BBB model. Briefly, the membrane was coated with collagen and fibronectin at a concentration of 15 mg/ml and 30 mg/ml respectively at 2 h before seeding BMVECs. Approximately 2*105 BMVECs were grown on each membrane for 4 days until fusion. The BMVECs were then treated with OGD reoxygenation. Each luminal chamber was added 1 mg 3-kDa fluorescein isothiocyanate (FITC)–dextran and 1 mg 70-kDa tetramethylrhodamine (TRITC)–dextran in 500 μl medium. 25 µl medium from the lower chamber was collected for measuring fluorescence intensity using a Varioskan Flash (Thermo Scientific, US) at 1 h intervals for 6-h after OGD reoxygenation. The flux of the two macromolecules was used to determine paracellular permeability.

TTC

Triphenyl tetrazolium chloride (TTC) staining is commonly applied for measuring infarct size (Kramer et al. 2010). Brain coronal Sections (2 mm-thick) were prepared using a rodent brain matrix. Sections were immersed in 1.5% TTC in PBS at 37 °C for 15 min. The formula used to determine the infarct degree was as follows: related infarct area = ipsilateral total infarct area/total contralateral area.

Western Blotting

Lysis buffer (1 ml of radioimmunoprecipitation assay lysis buffer, 5 μl of phosphatase inhibitor, 5 μl of phenylmethylsulphonyl fluoride, and 1 μl of proteinase inhibitor mixture) was used to extract protein from BMVECs and brain tissue. Lysates were assayed for protein concentrations using a bicinchoninic acid protein assay kit. Twenty micrograms of protein sample per lane were analyzed following a standard protocol of sodium dodecyl sulfate (SDS)-polyacrylamide gel electrophoresis (PAGE); resultant bands were transferred onto polyvinylidene difluoride membranes. After blocking using 4% nonfat dried milk (BD, USA) in PBS, membranes were incubated using the primary antibodies anti-F-actin (1:1000; Bioss, China), anti-occludin (1:4000; Abcam, USA), anti-ZO-1 (1:1000; Proteintech, China), anti-CD151 (1:1000; Bioss, China) and anti-VE-cadherin (1:1000; Abcam, USA) overnight at 4 °C. After washing with Tris-Buffered Saline Tween (TBST), the membranes were incubated in peroxidase-conjugated anti-mouse IgG (1:5000) or anti-rabbit IgG (1:5000) for 1 h at 25 °C. Then, the membranes were treated with chemiluminescence solution (Millipore, USA). Signals were detected using a biomolecule imager (LAS4000, GE; USA). The blots were then analyzed by densitometry using the Image J software. Western blotting results were normalized by β-actin.

Immunofluorescence

Coronal frozen sections of 25-μm thickness and cell slides were chosen for immunofluorescence staining. Non-specific binding sites of slides were blocked with 5% donkey serum in PBS at 25 °C for 1 h. Primary antibodies against CD151 (1:100; Bioss, China) and CD31 (1:100; Abcam, USA) diluted in PBS were used to incubate slices overnight at 4 °C. Then, the slides were incubated at 25 °C for 60 min with donkey anti-mouse IgG secondary antibody Alexa Fluor® 584 conjugate (1:250; Jackson ImmunoResearch, USA) and donkey anti-rabbit IgG secondary antibody Cy5 conjugate (1:250; Bioss, China). F-actin staining was performed using Alexa Fluor 594 conjugated phalloidin (1:1000; Abcam, USA). The slides were scanned using a laser confocal microscopic (Nikon, Japan).

RhoA Activity Assay

RhoA activity was determined using a commercially available G-LISA RhoA Activity assay Biochem kit (Cytoskeleton Inc, USA) following the manufacturer’s instructions. Briefly, cell lysates were prepared using a cell lysis buffer (the kit supplied), and then the lysate protein concentration was measured using a Precision RedTM Advanced Protein Assay Reagent (the kit supplied). Twenty-five micrograms of protein sample per well were used to perform GLISA assay. Absorbance was read at 490 nm using a Varioskan Flash (Thermo Scientific, US).

Statistical Analyses

All data were performed statistical analyses using GraphPad Prism software. We presented the results as the means ± SD. Kolmogorov–Smirnov tests and Shapiro–Wilk’s were performed to establish and confirm the normality of parameters. Statistical significance between groups were analysed using unpaired Student’s t tests, and among multiple groups were analysed using one-way analysis of variance (ANOVA) with post hoc Bonferroni test, as appropriate. We considered p values < 0.05 as significant.

Results

Cerebral Ischemic Injury Increases CD151 Expression on BMVECs

CD151 is expressed on various cells and upregulated under ischemic conditions in the hind limb model (Lan et al. 2005). To investigate CD151 expression features in the brain under homeostatic conditions, we adopted CD151/CD31 dual-IF. CD151 was primarily expressed in ECs in brain vessels under homeostatic conditions (Fig. 2a). Meanwhile, we also measured CD151 expression after Lenti-CD151 transfection. There was a significant upregulation of CD151 in the Lenti-CD151 transfected group (p < 0.05; Fig. 2b–d). Then, we characterized CD151 expression under ischemic conditions in vivo. CD151 expression was upregulated at 3 h after tFCI, and there was a significant difference at 24 h after tFCI (Fig. 2d, e).

Fig. 2.

Fig. 2

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CD151 expression pattern under various conditions. a Representative photograph of CD151 expression in the rat brain under homeostatic condition. White arrow corresponds to CD151+ and CD31+ cells. Scale bar, 20 µm. b Representative photographs of CD151 expression in rat brain transfected with Lenti/Lenti-CD151. Scale bar, 20 µm. c Quantification of CD151 expression level in the brain of rats transfected with Lenti/Lenti-CD151. Transfecting with Lenti-CD151 significantly increased CD151 expression in the brain. **p < 0.01. d Western blotting analysis of the brain tissue lysates with anti-CD151 antibody at 7-day after Lentiviral transfection. *p < 0.05. e Representative photograph of CD151 expression in the rat brain under tFCI conditions. Scale bar, 20 µm. f Quantification of CD151 expression level in rat brain under tFCI conditions. tFCI increases CD151 expression at 3, 6, and 24 h after tFCI.IF intensity was normalized by sham *p < 0.05

CD151 Overexpression Can Alleviate BBB Permeability After tFCI

To explore the effect of CD151 upregulation on BBB function under ischemic condition, we assessed BBB permeability by measuring the leakage of endogenous plasma IgG from blood to brain parenchyma. We observed that endogenous plasma IgG leakage between the Lenti and Lenti-CD151 group was comparable at 3 h after tFCI (Fig. 3). However, at 6 and 24 h after tFCI, endogenous plasma IgG leakage in the Lenti group rats was significantly higher than that in Lenti-CD151 group rats (p < 0.05; Fig. 3). These data showed that CD151 overexpression in the brain could mitigate BBB injury in the acute phase after ischemia–reperfusion injury.

Fig. 3.

Fig. 3

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The leakage of endogenous IgG in rat brain after tFCI. a Representative photograph of the leakage of endogenous IgG from blood to brain parenchyma at 3, 6 and 24 h of reperfusion after tFCI. Scale bar, 2000 µm. b Quantification of the leakage of endogenous IgG. Rats transfected with Lenti-CD151 exhibited decreased leakage of endogenous IgG at 6 and 24 h of reperfusion after tFCI. n = 5–6 rats/per group. *p < 0.05, ***p < 0.01

CD151 Overexpression has no Effects on Post-stroke Outcome

The above data suggested that CD151 overexpression in the brain can protect BBB function in rats after tFCI. Hence, we continued to explore the effects of CD151 upregulation on post-stroke outcomes. The extent of infarction after tFCI was comparative between the Lenti and Lenti-CD151 group rats at 24 h after tFCI (Fig. 4a, b). Moreover, there were no significant differences in Longa score at 24 h after tFCI and Bederson score at 72 h after tFCI in the two groups (Fig. 4c, d). These data suggested that in addition to maintaining BBB permeability, CD151 overexpression has other potential mechanisms that offset its beneficial effect.

Fig. 4.

Fig. 4

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Effect of CD151 overexpression on post-stroke outcome a Representative photograph of infarcts stained with TTC. b Relative infarct area. Rats transfected with Lenti-CD151 showed comparable relative infarct area at 1 d of reperfusion after tFCI compared to that in rats transfected with Lenti. n = 6–7 rats/per group. c Box graph represents Bederson score. Bederson score showed no significant difference between rats transfected with Lenti-CD151 and Lenti. n = 6 rats/per group. d Box graph represents Longa score. Longa score showed no significant difference between rats transfected with Lenti-CD151 and Lenti. n = 6 rats/per group

CD151 Overexpression in BMVECs Alleviates BBB Permeability in an In Vitro Model

In vivo study, we observed that CD151 overexpression had a protective effect on BBB dysfunction after tFCI but no effect on stroke outcome. An in vitro BBB model was used to further address the protective effect of CD151 overexpression on BBB function. It consisted of a monolayer of rat primary BMVECs in a Transwell insert (Shi et al. 2017). Transportation of both small (3-KDa FITC–dextran) and large (70-KDa TRITC–dextran) macromolecules significantly decreased across the monolayer Lenti-CD151 BMVECs than those in Lenti-BMVECs after OGD–reoxygenation (Fig. 5). Approximately, 3 h after OGD reoxygenation transportation of the small macromolecule (3-KDa FITC–dextran), began to show differences in the two kinds of cell monolayer; the time was delayed to 6 h for the transportation of the large macromolecule (70-KDa TRITC–dextran).

Fig. 5.

Fig. 5

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The effect of CD151 overexpression on permeability of the in vitro BBB model after OGD–reoxygenation. a Line chart showing the diffusion of 3-KDa FITC–dextran. Monolayer Lenti-CD151 BMVECs showed decreased permeability for the macromolecule 3-KDa FITC–dextran at 3–6 h of reoxygenation after OGD. *p < 0.05. b Line chart showing the diffusion of 70-KDa TRITC–dextran. Monolayer Lenti-CD151 BMVECs showed decreased permeability for 70-KDa TRITC–dextran at 6 h of reoxygenation after OGD. *p < 0.05

CD151 Overexpression Decreases Stress Fiber Formation and RhoA Activation After OGD–Reoxygenation

To explore the role of CD151 in rat BMVECs on junctional protein expression ischemia–reperfusion conditions, we assayed the expression level of tight junctional proteins (Fig. 6a). At 3 h and 4 h of reoxygenation after OGD, there was no significant difference in the level of occludin, VE-cadherin and ZO-1 in the two types of cells. Therefore, CD151 mitigating BBB dysfunction after OGD may be in a junctional protein degradation-independent way.

Fig. 6.

Fig. 6

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Impact of CD151 overexpression on junctional proteins, stress fibers, and RhoA activity. a Western blotting analysis of the indicated lysates with anti-occludin, ZO-1, and VE-cadherin antibodies Pre-OGD and at 3 h and 4 h of reoxygenation after OGD. b Western blotting analysis of the indicated lysates with anti-F-actin antibody Pre-OGD and at 3 h of reoxygenation after OGD. *p < 0.05, ** < .0.01. c Quantification of ZO-1expression level in wild-type-(WT) BMVECs, Lenti-BMVECs and Lenti-CD151 BMVECs Pre-OGD and at 3 h and 4 h of reoxygenation after OGD. d Quantification of VE-cadherin expression level in WT-BMVECs, Lenti-BMVECs and Lenti-CD151 BMVECs Pre-OGD and at 3 h and 4 h of reoxygenation after OGD. e Quantification of Occludin expression level in WT-BMVECs, Lenti-BMVECs and Lenti-CD151 BMVECs Pre-OGD and at 3 h and 4 h of reoxygenation after OGD. f Representative photograph of stained BMVECs to visualize F-actin (red) and nucleus (blue) pre OGD and at 3 h of reoxygenation after OGD. Scale bar, 50 µm. g Quantification of F-actin expression level in wild-type-(WT) BMVECs, Lenti-BMVECs and Lenti-CD151 BMVECs. Lenti-CD151 BMVECs showed less F-actin expression levels than Lenti-BMVECs at 3 h of reoxygenation after OGD. *p < 0.05. h Bar graph represents RhoA activity. RhoA activity of Lenti-CD151 BMVECs was less compared to that of Lenti-BMVECs and WT-BMVECs at 3 h of reoxygenation after OGD. *p < 0.05

Junctional protein translocation is one of the primary reasons underlying increased permeability after brain ischemia (Shi et al. 2017; Jiang et al. 2018). To explore the potential mechanism of CD151 in maintaining BBB integrity, firstly, we assessed stress fiber formation in BMVECs after ischemic insult. After 3 h of OGD, rat BMVECs transfected with Lenti-CD151 exhibited significantly decreased accumulation of F-actin (p < 0.05; Fig. 6b–d). Moreover, we investigated RhoA activity, which is crucial in the regulation cytoskeleton (Feng et al. 2018). At 3 h of reperfusion after OGD, Lenti-CD151 BMVECs showed less RhoA activation than Lenti-BMVECs (p < 0.05; Fig. 6 e).

Discussion

The present study explored the effect of CD151 overexpression in BMVECs on BBB function after tFCI in vivo and in vitro models. Our findings revealed CD151 overexpression in BMVECs has a protective role in BBB dysfunction at the early stage of reperfusion after tFCI, presumably by decreasing RhoA activity and stress fiber formation (Fig. 7).

Fig. 7.

Fig. 7

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Schematic diagram of the proposed effect of CD151 overexpression on BBB after tFCI. Our results suggested that CD151 overexpression reduced RhoA activation and stress fiber formation (F-actin) but had no effect on the level of whole-cell junctional protein expression when BMVECs were subjected to ischemic injury

In our study, we investigated the pattern and degree of CD151 expression in the brain under homeostatic and ischemic conditions. Consistent with previous research, CD151 was primarily expressed in ECs in the brain under homeostatic conditions (Sincock et al. 1997). Previous studies also showed a transient upregulation of CD151 expression in the brain after chronic hypoxia/ischemia, which may promote angiogenesis (Welser-Alves et al. 2016; Yang et al. 2017). Our study showed that CD151 overexpression has a beneficial effect on maintaining BBB function after tFCI, but self-upregulation of CD151 under ischemic conditions may not have the same capacity due to the delay of CD151 expression upregulation, which is approximately at 24 h after tFCI.

As CD151 is necessary for maintaining EC stability (Zhang et al. 2011), our observations raised the possibility that CD151 overexpression can protect BBB permeability after ischemia–reperfusion injury. Under homeostatic conditions, CD151 maintains vascular stability by balancing cell adhesion and cytoskeletal tension (Zhang et al. 2011). Moreover, studies have suggested that rapid endothelial cytoskeletal reorganization contributes to early BBB disruption after ischemia–reperfusion injury (Shi et al. 2016b; Jiang et al. 2018). Furthermore, earlier studies have revealed that preventing or reversing cytoskeletal reorganization protects against BBB breakdown after ischemic injury (Eira et al. 2016; Shi et al. 2017). Hence, the possible protection of BBB function by CD151 after ischemia may result from its ability to prevent or reverse cytoskeletal reorganization, but the underlying mechanism needs to be studied further.

In our study, we found no significant difference in IgG extravasation at 3 h of reperfusion after ischemia between the Lenti-CD151 and Lenti groups. The reason may result from the molecular weight of IgG, which is approximately 70 kDa. Previous studies have suggested that leakage of small-sized molecules occurs in early BBB dysfunction, and leakage of larger-sized molecules occurs after 3 h of tFCI, which is MMP9-dependent (Shi et al. 2016b). We did not measure the effect of CD151 on small size molecule extravasation in vivo. However, we found that CD151 overexpression in ECs initially decreased smaller size molecule leakage from OGD–reoxygenation insult. Moreover, CD151 contributes to MMP expression, which contributes to tight junctional protein degradation (Hong et al. 2006; Shi et al. 2010); thus, we speculate that CD151 overexpression in ECs can protect against early BBB dysfunction in an MMP9-independent manner.

The RhoA/RHO-associated kinase (ROCK)-signaling pathway plays a vital role in regulating the permeability of vascular ECs (Sladojevic et al. 2017; Feng et al. 2018). It has been previously reported that OGD reoxygenation increases RhoA activity of BMVECs (Chen et al. 2019). In our study, pretreatment with a CD151 overexpression vector significantly decreased RhoA activity of BMVECs insulted with OGD reoxygenation. In a previous in vitro study, CD151-knockdown human umbilical vein endothelial cells (HUVECs) showed increased RhoA activity (Zhang et al. 2011). Moreover, RNAi-mediated silencing of CD151 in epidermal carcinoma cells also showed elevated RhoA activity (Johnson et al. 2009). Hence, the beneficial effects of CD151 overexpression on BBB function may result from decreased RhoA activity.

Our data suggested that CD151 overexpression can alleviate BBB dysfunction after ischemia–reperfusion injury. However, it did not improve post-stroke outcomes in our study, implying that CD151 has other effects, which are contradictory to its protective effects on BBB function after ischemia. As mentioned above, CD151 can upregulate MMP9 expression, which can aggravate the ischemia–reperfusion injury. Moreover, CD151 can promote endothelial–leukocyte adhesion by interaction with VCAM-1 (Ley and Zhang 2008; Bailey et al. 2011; Wadkin et al. 2017), which plays a crucial role in secondary damage after ischemia. Therefore, the effects of CD151 on inflammation after ischemic–reperfusion injury are worth exploring in the future.

In our study, CD151 overexpression decreased RhoA activation after OGD–reoxygenation. Increasing evidence has suggested that ROCK inhibition has potential neuroprotective effects (Mueller et al. 2005; Shibuya et al. 2005; Sladojevic et al. 2017; Feng et al. 2018). However, as a critical regulating signal, nonspecific inhibition of RhoA/ROCK signaling may cause untoward side effects (Sladojevic et al. 2017). Therefore, RhoA activity inhibition in a CD151-dependent way may be more specific to the pathological process of an ischemic stroke.

The mechanisms underlying the CD151-mediated decrease of RhoA activity are still uncertain. The ROCK signalling pathway can be activated by secreted bioactive molecules or integrin (Inoue and Tanihara 2013). As an integrin partner, CD151 associates tightly with the integrins and modulates integrin-dependent signalling. Moreover, the modulatory impact of tetraspanins in integrin signalling can be both negative and positive (Berditchevski and Odintsova 1999). In vitro, we found that CD151 overexpression on rat BMVECs decreased RhoA activity. Hence, we speculated that CD151 might be a negative regulator of integrin-mediated RhoA activation. However, this hypothesis needs to be tested.

Our study had several limitations. First, we upregulate CD151 expression via systemic delivery of the CD151 lentiviral particles. Hence, we cannot exclude the potential effects of CD151 over-expression on other non-endothelial cells in vivo. Second, we only measured the endogenous IgG leakage in BBB permeability assay in vivo. Endogenous IgG combined with different sized, visualizable dextrans leakage seems to be the more appropriate approach for qualitative and quantitative assessment of barrier integrity. Third, our research could not show the detailed mechanism underlying the CD151-mediated BBB protection after cerebral ischemia injury. Conditional transgenic animals and comprehensive methods to detect blood–brain barrier permeability will be needed to obtain a better understanding of the underlying mechanism.

In conclusion, the current study provides a previously undetermined role of CD151 in reducing BBB breakdown after tFCI by modulating RhoA activity. Furthermore, remission of BBB damage by CD151 overexpression may be a novel approach to protect patients from ischemia–reperfusion injury, particularly in combination with recanalization therapy. However, the specific mechanisms underlying the CD151-mediated decrease of RhoA activity in BMVECs following insult with OGD reperfusion require further inspection.

Electronic supplementary material

Below is the link to the electronic supplementary material.

Supplementary file1 (PDF 369 kb) (369.3KB, pdf)

Acknowledgements

This work was supported by the Beijing Municipal Health Bureau project (2013-2-034). Thanks are due to Yi Shen, Chenming Wei and Qiang Huang for valuable discussion and to Ping Liang and Wei Liu for assistance with the experiments.

Author Contributions

All authors contributed to the study conception and design. Material preparation, experiments, data collection and analysis were performed by WX and CG. The first draft of the manuscript was written by WX and all authors commented on previous versions of the manuscript. JW contributed to writing, review and editing. All authors read and approved the final manuscript.

Funding

This study was funded by the Beijing Municipal Health Bureau project (2013-2-034).

Compliance with Ethical Standards

Conflict of interest

The authors declare that they have no conflict of interest.

Ethical Approval

All procedures performed in studies involving animals were in accordance with the ethical standards of the University of Tsinghua Institutional Animal Care and Use Committee (SYXK20190037).

Footnotes

Publisher's Note

Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

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