Angiopoietin-like 4 Promotes Angiogenesis And Neurogenesis In Ischemic Stroke And Attenuates Brain Edema And Neurologic Deficits In Intracerebral Hemorrhage

Objective:This study aims to explore whether angiopoietin-like 4 (ANGPTL4) can promote angiogenesis and neurogenesis to enhance cerebralvascular reserve capacity, reduce infarct volume.Methods:In this study, C57BL/6 mice were divided into normal salin-treated group and ANGPTL4-treated group and were injected via tail vein with normal saline or ANGPTL4 injection 5 min before surgical procedures. Then, animals were subjected to distal middle cerebral artery occlusion (dMCAO). MRI examination was performed to check whether ischemic stroke was successful on the 1st day after ischemic stroke. The infarct volume difference between both groups were measured via TTC staining on the 1st day after stroke. Angiogenesis and neurogenesis were evaluated by immunofluorescence co-labelling bromodeoxyuridine (BrdU) with von Willebrand factor (vWF) in subventricular zone (SVZ) and ischemic penumbra, with doublecortin (DCX) in SVZ and subgranular zone (SGZ). Proliferation and activation of microglia evaluated by immunofluorescence co-labelling Iba-Iwith BrdU on the 7th day after stroke. Immunocytochemistry were performed to assess expression level of MPO on the 1st,3rd day and to count the number of neuron in ischemic penumbra on the 7th day. The amount of AKT, p-AKT, VEGF, MPO (myeloperoxidase), Fas and FasL were measured via western blot assays on the 1st day after stroke.Results:(1) The infarct volume was significantly decreased in ANGPTL4-treated group compared with normal salin-treated group. (2) The number of BrdU+ and BrdU+/vWF+ cells in ischemic penumbra and SVZ in the ANGPTL4-treated group were significantly higher than those of the normal saline-treated group. (3) The number of BrdU+/DCX+ showed a significant increase in SVZ and SGZ of ANGPTL4-treated mice compared with controls. The number of Neuron in ischemic penumbra in the ANGPTL4-treated mice were significantly higher than control group. (4) The expression level of MPO in ischemic penumbra in the ANGPTL4-treated mice were significantly higher than control group. (5) The number of Iba-1+/BrdU+ cells in ischemic penumbra in the ANGPTL4-treated mice were significantly lower than control group. (6) Western blot analysis showed that increased relative amount of p-AKT and the ratio of phosphor-AKT to Total-AKT, decreased relative amount of MPO in ANGPTL4-treated mice, the relative amount of VEGF and Total-AKT in both groups was no significant difference on the 1st day after ischemic stroke. (7) The expression level of MPO, Fas, FasL in ANGPTL4-treated mice was significantly lower than control group.Conclusions:ANGPTL4 can promote angiogenesis and neurogenesis via enhancing AKT kinase activity in C57BL/6 mice model of ischemic stroke. Meanwhile, it significantly reduce neuronal death and inhibit acute and chronic phase inflammatory in ischemic stroke, which resulted from the inhibition of Fasl/Fas expression and its downstream pathway.Objective:Brain edema after intracerebral hemorrhage (ICH) is highly associated with its poor outcome. Hence, this study aimed to evaluate the effect of angiopoietin-like 4 (ANGPTL4) to prevent the formation of brain edema and promote the recovery of neurological function following ICH.Methods:168 C57BL/6J mice were subjected to sham surgery or ICH induction via bacterial collagenase (0.2 U). Animals were randomly assigned to receive single i.v. injection in the tail vein of either normal saline or rhANGPTL4 (40 ug/kg body weight) 5 min before surgical procedures. The behavioral tests used were rotarod, modified neurologic severity score (mNSS) and corner turn test. Mice were tested before surgery and at 1,3,7,14,21, and 28 days after ICH. Brain edema and hematoma volume were separately examined via the wet weight/dry weight method and hematoxylin-eosin staining. Integrity of tight and adherens junctions was quantified via immunofluorescence and the ultrastructure of blood brain barrier (BBB) was examined via transmission electron microscopy. The relative amount of VE-cadherin, claudin-5, Src and phospho-Src were measured via western blot assays.Results:(1) The battery of tests indicated that there were marked neurological deficits by day 1 after ICH, with progressive recovery of function over 4 weeks in compared with the control mice. (2) Both of brain edema and hematoma volume were significantly decreased in ANGPTL4-treated mice. (3) ANGPTL4-treated mice displayed increased Quantification of VE-cadherin and claudin-5-positive areas normalized to the total EC surface area (Isolectin B4 staining) in infarcted hemispheres of ANGPTL4-treated mice was significant higher than contralateral hemispheres of control mice. (4) The ultrastructure of BBB in perihematoma in ANGPTL4-treated mice was better than control group. (5) Western blot analysis showed that increased relative amount of VE-cadherin and claudin-5, decreased relative amount of Src and phospho-Src and the ratio of phosphor-Src to Src in ANGPTL4-treated mice.Conclusions:These finding demonstrated ANGPTL4 can prevent BBB from being destroyed induced by ICH and promote the recovery of neurological function. The reduction of Src kinase activity in ANGPTL-treated mice was paralleled by a decrease in vascular permeability and edema formation.