Department of Biochemistry, College of Life Science and Biotechnology, Yonsei University, Seoul, 03722, South Korea.
Department of Neurobiology, School of Medicine, Kangwon National University, Chuncheon, 24341, South Korea.
J Neuroinflammation. 2020 Feb 4;17(1):48. doi: 10.1186/s12974-020-1727-6.
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.
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.
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.
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.
缺血性中风是主要的死亡原因。血脑屏障 (BBB) 破坏似乎在缺血性中风患者的炎症中发挥关键作用，并加速脑损伤。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，尽管存在不良反应，但血管内皮生长因子 (VEGF)-A 和血管内皮生长因子受体 (VEGFR)-2 的调节是一个关键靶点。我们之前的研究发现，C 型凝集素家族 14 成员 A (CLEC14A) 的缺失会激活血管生成中的 VEGF-A/VEGFR-2 信号通路。在这里，我们评估了 CLEC14A 缺乏引起的血脑屏障损伤在缺血再灌注损伤中的作用。
体外用异硫氰酸荧光素 (FITC)-葡聚糖通透性、跨内皮电阻 (TEER) 测定和免疫染色评估内皮完整性。用 Evans 蓝染料和 FITC-葡聚糖注射评估 Clec14a (CLEC14A-KO) 小鼠和野生型小鼠的 BBB 通透性。通过大脑中动脉闭塞手术和行为评估评估神经损伤。通过免疫荧光染色、Western blot 和脑样本定量逆转录聚合酶链反应确认紧密连接蛋白、粘附分子、促炎细胞因子和小胶质细胞的变化。
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；随着 VEGF 处理，这种效应的严重程度增加。免疫荧光染色显示 CLEC14A 敲低的内皮细胞中紧密连接蛋白减弱。与体外结果一致，出生后第 8 天注射 Evans 蓝染料的 CLEC14A-KO 小鼠出现脑血管渗漏；野生型小鼠无渗漏。我们使用大脑中动脉闭塞模型发现，CLEC14A-KO 小鼠的梗死脑组织严重且神经功能缺损，并伴有 VEGFR-2 表达上调。缺血再灌注后 CLEC14A-KO 小鼠出现 FITC-葡聚糖渗漏，紧密连接分子数量明显减少。CLEC14A 的缺失通过粘附分子表达增加了促炎反应，并激活了神经胶质细胞。
这些结果表明，CLEC14A-KO 小鼠中 VEGFR-2 的激活通过加重缺血再灌注损伤后的脑血管渗漏和增加神经元炎症，加重了缺血性中风。
