BACKGROUND Ischemic stroke is a main cause of mortality. Blood-brain barrier (BBB) breakdown appears to play a critical role in inflammation in patients with ischemic stroke and acceleration of brain injury. The BBB has a protective function and is composed of endothelial cells, pericytes, and astrocytes. In ischemic stroke treatments, regulation of vascular endothelial growth factor (VEGF)-A and vascular endothelial growth factor receptor (VEGFR)-2 is a crucial target despite adverse effects. Our previous study found that loss of C-type lectin family 14 member A (CLEC14A) activated VEGF-A/VEGFR-2 signaling in developmental and tumoral angiogenesis. Here, we evaluate the effects of BBB impairment caused by CLEC14A deficiency in ischemia-reperfusion injury. METHODS In vitro fluorescein isothiocyanate (FITC)-dextran permeability, transendothelial electrical resistance (TEER) assay, and immunostaining were used to evaluate endothelial integrity. BBB permeability was assessed using Evans blue dye and FITC-dextran injection in Clec14a-/- (CLEC14A-KO) mice and wild-type mice. Middle cerebral artery occlusion surgery and behavioral assessments were performed to evaluate the neurologic damage. The change of tight junctional proteins, adhesion molecules, pro-inflammatory cytokines, and microglial were confirmed by immunofluorescence staining, Western blotting, and quantitative reverse transcription polymerase chain reaction of brain samples. RESULTS In endothelial cells, knockdown of CLEC14A increased FITC-dextran permeability and decreased transendothelial electrical resistance; the severity of this effect increased with VEGF treatment. Immunofluorescence staining revealed that tight junctional proteins were attenuated in the CLEC14A knockdown endothelial cells. Consistent with the in vitro results, CLEC14A-KO mice that were injected with Evans blue dye had cerebral vascular leakage at postnatal day 8; wild-type mice had no leakage. We used a middle cerebral artery occlusion model and found that CLEC14A-KO mice had severe infarcted brain and neurological deficits with upregulated VEGFR-2 expression. FITC-dextran leakage was present in CLEC14A-KO mice after ischemia-reperfusion, and the numbers of tight junctional molecules were significantly decreased. Loss of CLEC14A increased the pro-inflammatory response through adhesion molecule expression, and glial cells were activated. CONCLUSIONS These results suggest that activation of VEGFR-2 in CLEC14A-KO mice aggravates ischemic stroke by exacerbating cerebral vascular leakage and increasing neuronal inflammation after ischemia-reperfusion injury.

中文翻译：

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CLEC14A缺乏症通过增加血脑屏障通透性和炎症而加剧神经元丢失。

背景技术缺血性中风是死亡的主要原因。血脑屏障（BBB）的破坏似乎在缺血性中风和脑损伤加速患者的炎症中起关键作用。BBB具有保护功能，由内皮细胞，周细胞和星形胶质细胞组成。在缺血性中风治疗中，尽管有不良反应，调节血管内皮生长因子（VEGF）-A和血管内皮生长因子受体（VEGFR）-2仍是关键目标。我们先前的研究发现，C型凝集素家族14成员A（CLEC14A）的缺失在发育和肿瘤血管生成中激活了VEGF-A / VEGFR-2信号传导。在这里，我们评估由CLEC14A缺乏引起的BBB损伤在缺血再灌注损伤中的作用。方法体外荧光素异硫氰酸酯（FITC）-葡聚糖渗透性，经皮内皮电阻（TEER）测定和免疫染色用于评估内皮完整性。使用伊文思蓝染料和FITC-右旋糖酐注射液在Clec14a-/-（CLEC14A-KO）小鼠和野生型小鼠中评估BBB通透性。进行大脑中动脉闭塞手术和行为评估以评估神经系统损害。通过免疫荧光染色，蛋白质印迹和定量反转录聚合酶链反应对脑样本进行证实，紧密连接蛋白，粘附分子，促炎性细胞因子和小胶质细胞的变化。结果在内皮细胞中，CLEC14A的敲低增加了FITC-葡聚糖的通透性并降低了跨内皮电阻。该效应的严重性随着VEGF治疗而增加。免疫荧光染色显示紧密连接蛋白在CLEC14A敲低的内皮细胞中减弱。与体外结果一致，注射伊文思蓝染料的CLEC14A-KO小鼠在出生后第8天出现脑血管渗漏；而在出生后第8天，脑血管渗漏。野生型小鼠没有泄漏。我们使用大脑中动脉闭塞模型，发现CLEC14A-KO小鼠具有严重的脑梗塞和神经功能缺损，VEGFR-2表达上调。缺血再灌注后CLEC14A-KO小鼠中存在FITC-右旋糖酐泄漏，紧密连接分子的数量显着减少。CLEC14A的缺失通过粘附分子表达增强了促炎反应，并且胶质细胞被激活。