Division of Hematology/Oncology, Department of Internal Medicine (R.B.P., N.D., B.S., M.G., P.D., T.B., M.K., A.K.C.), University of Iowa, Iowa City.
Departments of Neurology, Neurosurgery and Epidemiology (E.C.L.), University of Iowa, Iowa City.
Stroke. 2023 Sep;54(9):2409-2419. doi: 10.1161/STROKEAHA.123.042714. Epub 2023 Jul 14.
Obesity-induced hyperglycemia is a significant risk factor for stroke. Integrin α9β1 is expressed on neutrophils and stabilizes adhesion to the endothelium via ligands, including Fn-EDA (fibronectin containing extra domain A) and tenascin C. Although myeloid deletion of α9 reduces susceptibility to ischemic stroke, it is unclear whether this is mediated by neutrophil-derived α9. We determined the role of neutrophil-specific α9 in stroke outcomes in a mice model with obesity-induced hyperglycemia.
α9 (α9MRP8Cre) and littermate control α9 (α9MRP8Cre) mice were fed on a 60% high-fat diet for 20 weeks to induce obesity-induced hyperglycemia. Functional outcomes were evaluated up to 28 days after stroke onset in mice of both sexes using a transient (30 minutes) middle cerebral artery ischemia. Infarct volume (magnetic resonance imaging) and postreperfusion thrombo-inflammation (thrombi, fibrin, neutrophil, phospho-nuclear factor kappa B [p-NFκB], TNF [tumor necrosis factor]-α, and IL [interleukin]-1β levels, markers of neutrophil extracellular traps) were measured post 6 or 48 hours of reperfusion. In addition, functional outcomes (modified Neurological Severity Score, rota-rod, corner, and wire-hanging test) were measured for up to 4 weeks.
Stroke upregulated neutrophil α9 expression more in obese mice (<0.05 versus lean mice). Irrespective of sex, deletion of neutrophil α9 improved functional outcomes up to 4 weeks, concomitant with reduced infarct, improved cerebral blood flow, decreased postreperfusion thrombo-inflammation, and neutrophil extracellular traps formation (NETosis) (<0.05 versus α9 obese mice). Obese α9 mice were less susceptible to thrombosis in FeCl injury-induced carotid thrombosis model. Mechanistically, we found that α9/cellular fibronectin axis contributes to NETosis via ERK (extracellular signal-regulated kinase) and PAD4 (peptidyl arginine deiminase 4), and neutrophil α9 worsens stroke outcomes via cellular fibronectin-EDA but not tenascin C. Obese wild-type mice infused with anti-integrin α9 exhibited improved functional outcomes up to 4 weeks (<0.05 versus vehicle).
Genetic ablation of neutrophil-specific α9 or pharmacological inhibition improves long-term functional outcomes after stroke in mice with obesity-induced hyperglycemia, most likely by limiting thrombo-inflammation.
肥胖引起的高血糖是中风的一个重要危险因素。整合素 α9β1 表达于中性粒细胞上，并通过包括 Fn-EDA（含有额外结构域 A 的纤连蛋白）和 tenascin C 在内的配体稳定与内皮细胞的黏附。尽管髓样细胞中 α9 的缺失会降低对缺血性中风的易感性，但尚不清楚这是否是由中性粒细胞衍生的 α9 介导的。我们在一种肥胖诱导高血糖的小鼠模型中确定了中性粒细胞特异性 α9 在中风结局中的作用。
α9（α9MRP8Cre）和同窝对照 α9（α9MRP8Cre）小鼠接受 60%高脂肪饮食喂养 20 周，以诱导肥胖诱导的高血糖。使用短暂（30 分钟）大脑中动脉缺血，在雌雄两性小鼠中风发作后长达 28 天评估功能结局。在再灌注后 6 或 48 小时，通过磁共振成像测量梗死体积和再灌注后血栓形成-炎症（血栓、纤维蛋白、中性粒细胞、磷酸核因子 kappa B [p-NFκB]、肿瘤坏死因子-α [TNF-α]和白细胞介素-1β [IL-1β]水平，中性粒细胞胞外陷阱的标志物）。此外，在长达 4 周的时间内，测量了功能结局（改良神经严重程度评分、转棒、角落和悬线试验）。
中风在肥胖小鼠中上调中性粒细胞 α9 的表达更为明显（<0.05 与瘦小鼠相比）。无论性别如何，中性粒细胞 α9 的缺失均可改善长达 4 周的功能结局，同时减少梗死、改善脑血流、减少再灌注后血栓形成-炎症和中性粒细胞胞外陷阱形成（NETosis）（<0.05 与肥胖 α9 小鼠相比）。肥胖 α9 小鼠在 FeCl 诱导的颈动脉血栓形成模型中不易发生血栓形成。从机制上讲，我们发现 α9/细胞纤连蛋白轴通过 ERK（细胞外信号调节激酶）和 PAD4（肽基精氨酸脱亚氨酶 4）促进 NETosis，而中性粒细胞 α9 通过细胞纤连蛋白-EDA 而非 tenascin C 加重中风结局。肥胖野生型小鼠输注抗整合素 α9 可改善长达 4 周的功能结局（<0.05 与载体相比）。
在肥胖诱导高血糖的小鼠中，中性粒细胞特异性 α9 的基因缺失或药物抑制可改善中风后的长期功能结局，这很可能是通过限制血栓形成-炎症来实现的。
