Macrophage-Derived Angiopoietin-Like Protein 2 Exacerbates Brain Damage by Accelerating Acute Inflammation after Ischemia-Reperfusion

Fig 1. The number of infiltrated ANGPTL2-positive macrophages increases in ischemic brain. (A) Representative image of cerebral blood flow measured by laser Doppler flowmeter in the vicinity of the middle cerebral artery during transient middle cerebral artery occlusion (MCAO) surgery. MCA, middle cerebral artery. A laser Doppler flowmetry probe was used to trace cerebral blood flow (CBF). After ligation of the right common carotid artery (CCAO), we observed a 40% drop in CBF. MCA occlusion promoted a further decrease to less than 80–90% of baseline CBF. After 30 min MCAO, the suture was removed and the right common carotid artery was permanently ligated to prevent bleeding, resulting in a slow return of CBF to approximately 60% of baseline levels. CBF tracing was comparable in WT and Angptl2 KO mice during MCAO and subsequent reperfusion. (B) Angptl2 mRNA levels in the ischemic hemisphere 24 hours after transient MCAO (n = 5). Values indicate Angptl2 expression relative to Tbp mRNA. Results are expressed as means ± s.e.m. *P<0.05. (C) ANGPTL2 immunostaining in mouse brain. (Panel 1) Representative image showing ANGPTL2 expression in the contralateral cerebral hemisphere. (Panels 2, 3)…

Fig 2. Ischemic injury is attenuated in the brain of Angptl2-deficient mice in a mouse transient MCAO model. (A) Neurological scores assessed 24 and 72 hours after transient MCAO (day 1: WT = 6 and KO = 6; day 3: WT = 8 and KO = 7) (Garcia scale, S3 Appendix). Results are expressed as means ± s.e.m. (B) Representative triphenyl tetrazolium chloride (TTC)-stained coronal sections from ischemic brain 24 hours after transient MCAO. Scale bars: 2mm. (C) Infarct volume as measured by TTC-staining of serial coronal sections 24 and 72 hours after transient MCAO (day 1: WT = 6 and KO = 6; day 3: WT = 8 and KO = 7). Results are expressed as means ± s.e.m. *P<0.05, **P<0.001.

Fig 3. Acute brain inflammation after ischemic stroke is attenuated in Angptl2-deficient mice. (A and B) Relative expression of Il1b, Tnfa, and Il10 mRNAs in cortex (sham: WT = 7 and KO = 7; day 1: WT = 6 and KO = 7; day 3: WT = 5 and KO = 6) (A) and ipsilateral caudate putamen (sham: WT = 5 and KO = 8; day 1: WT = 7 and KO = 6; day 3: WT = 7 and KO = 5) (B). mRNA levels were normalized to those of Tbp. Values are expressed as fold-increases relative to values in WT, which were set at “1” in each case. (C) Representative images showing MAC-3 immunostaining in the ipsilateral cerebral hemisphere 24 hours after sham or transient MCAO treatment. (Panel 1) Sham-treated wild-type (WT) mice; (Panel 2) transient MCAO-treated wild-type mice; (Panel 3) transient MCAO-treated Angptl2 KO mice. Scale bar: 100μm. (D) (1) Number of MAC-3-positive cells in the ipsilateral hemisphere of transient MCAO mice 24 hours after transient MCAO (sham = 7; WT = 7; KO = 6). Results are expressed as means ± s.e.m. (2, 3) Relative Cd68 mRNA expression normalized to Tbp mRNA in cortex (sham = 6; WT = 8; KO = 8) (2) and ipsilateral caudate putamen (sham = 6; WT = 8; KO = 8) (3) 24 hours after transient MCAO. Values…

Fig 4. Bone marrow-derived ANGPTL2 functions in progression of neuronal damage following brain ischemia. (A) Schema showing relationships between donors and recipients in the bone marrow transplantation (BMT) experiment. (B) Schema showing the time course of BMT, surgery and sacrifice in the mouse transient MCAO model. (C) Graph showing neurological scores assessed by the 18-point neurological severity scale 24 and 72 hours after MCAO treatment (Day1: WT→WT = 6; KO→WT = 5; WT→KO = 7; KO→KO = 7. Day3: WT→WT = 8; KO→WT = 8; WT→KO = 12; KO→KO = 11). Results are expressed as means ± s.e.m. (D) Representative images showing TTC-stained brain sections 24 hours after transient MCAO in bone marrow chimeric mice. Scale bar: 2mm. (E) Infarct volumes as assessed by TTC-staining of brain sections 24 and 72 hours after stroke onset in bone marrow chimeric mice (Day1: WT→WT = 6; KO→WT = 5; WT→KO = 7; KO→KO = 7. Day3: WT→WT = 8; KO→WT = 8; WT→KO = 12; KO→KO = 11). Results are expressed as means ± s.e.m. *P<0.05, and **P<0.01.

Fig 5. ANGPTL2 increases transcription of genes encoding pro-inflammatory cytokines in macrophages. mRNA expression levels of (left) Il1b, (middle) Tnfa, and (right) Il10 following either vehicle or recombinant ANGPTL2 (rANGPTL2) treatment of RAW 264.7 cells (n = 5). mRNAs were harvested at 0, 3, 6, 12, 24 hours after cells were treated with 5 μg/mL rANGPTL2. Data are normalized to Actb levels. mRNA expression in vehicle-treated cells was set to 1 for each transcript assessed. Results are expressed as means ± s.e.m. *P< 0.01.

Fig 6. Macrophage-derived ANGPTL2 exacerbates neuronal injury in the acute phase of ischemic stroke. Schematic diagram showing ANGPTL2 function in ischemic stroke. In normal conditions, brain resident cells express little ANGPTL2. However, following ischemic stroke, bone marrow-derived macrophages infiltrate the brain interstitium and secrete abundant ANGPTL2. Subsequently, ANGPTL2 from activated macrophages induces those same macrophages to increase expression of pro-inflammatory cytokines, including TNF-α or IL-1, in an autocrine or paracrine manner. Pro-inflammatory cytokines damage neurons or supporting cells and worsen functional neurological deficits induced by brain infarction.