ABSTRACT
Beta-2 Glycoprotein I (ß2-GPI) is the main target of anti-phospholipid antibodies (aPL) in the autoimmune anti-phospholipid syndrome, characterized by increased risk of stroke. We here investigated the antibody independent role of ß2-GPI after ischemia/reperfusion, modeled in vivo by transient middle cerebral artery occlusion (tMCAo) in male C57Bl/6J mice; in vitro by subjecting immortalized human brain microvascular endothelial cells (ihBMEC) to 16 h hypoxia and 4 h re-oxygenation. ApoH (coding for ß2-GPI) was upregulated selectively in the liver at 48 h after tMCAo. At the same time ß2-GPI circulating levels increased. ß2-GPI was detectable in brain parenchyma and endothelium at all time points after tMCAo. Parenchymal ß2-GPI recognized apoptotic neurons (positive for annexin V, C3 and TUNEL) cleared by CD68+ brain macrophages. Hypoxic ihBMEC showed increased release of IL-6, over-expression of thrombomodulin and IL-1α after re-oxygenation with ß2-GPI alone. ß2-GPI interacted with mannose-binding lectin in mouse plasma and ihBMEC medium, potentially involved in formation of thrombi. We show for the first time that brain ischemia triggers the hepatic production of ß2-GPI. ß2-GPI is present in the ischemic endothelium, enhancing vascular inflammation, and extravasates binding stressed neurons before their clearance by phagocytosis. Thus ß2-GPI may be a new mediator of brain injury following ischemic stroke.
Subject(s)
Brain Ischemia/pathology , Neurons/metabolism , Vascular System Injuries/pathology , beta 2-Glycoprotein I/metabolism , Animals , Brain/metabolism , Brain/pathology , Brain Ischemia/etiology , Complement System Proteins/metabolism , Disease Models, Animal , Endothelial Cells/cytology , Endothelial Cells/metabolism , Humans , Interleukin-6/metabolism , Liver/metabolism , Liver/pathology , Macrophages/cytology , Macrophages/immunology , Macrophages/metabolism , Male , Mannose-Binding Lectin/metabolism , Mice , Mice, Inbred C57BL , Microscopy, Confocal , Neurons/cytology , Phagocytosis , Protein Binding , Vascular System Injuries/complications , beta 2-Glycoprotein I/bloodABSTRACT
Mannose-binding lectin (MBL), an initiator of the lectin pathway, is detrimental in ischemic stroke. MBL deposition on the ischemic endothelium indicates the beginning of its actions, but downstream mechanisms are not clear yet.We investigated MBL interactions with the ischemic endothelium by exposing human brain microvascular endothelial cells (hBMECs) to protocols of ischemia. Cells were exposed to hypoxia or oxygen-glucose deprivation (OGD), and re-oxygenated with human serum (HS) or recombinant MBL (rhMBL). Hypoxic hBMECs re-oxygenated with HS showed increased complement system activation (C3c deposition, +59%) and MBL deposition (+93%) than normoxic cells. Super-resolution microscopy showed MBL internalization in hypoxic cells and altered cytoskeletal organization, indicating a potential MBL action on the endothelial structure. To isolate MBL effect, hBMECs were re-oxygenated with rhMBL after hypoxia/OGD. In both conditions, MBL reduced viability (hypoxia: -25%, OGD: -34%) compared to conditions without MBL, showing a direct toxic effect. Ischemic cells also showed greater MBL deposition (hypoxia: +143%, OGD: +126%) than normoxic cells. These results were confirmed with primary hBMECs exposed to OGD (increased MBL-induced cell death: +226%, and MBL deposition: +104%). The present findings demonstrate that MBL can exert a direct deleterious effect on ischemic brain endothelial cells in vitro, independently from complement activation.
Subject(s)
Brain Ischemia/metabolism , Endothelial Cells/drug effects , Endothelium, Vascular/drug effects , Mannose-Binding Lectin/metabolism , Brain Ischemia/pathology , Cell Hypoxia/drug effects , Cell Survival/drug effects , Cells, Cultured , Complement Pathway, Mannose-Binding Lectin/drug effects , Endothelial Cells/metabolism , Endothelial Cells/pathology , Endothelium, Vascular/metabolism , Endothelium, Vascular/pathology , Glucose/metabolism , Humans , Mannose-Binding Lectin/genetics , Mannose-Binding Lectin/pharmacology , Oxygen/metabolism , Primary Cell Culture , Serum/metabolismABSTRACT
Mannose-binding lectin (MBL), an initiator of the lectin pathway (LP) of complement activation, is detrimental in ischemic stroke, as shown in clinical studies and rodent models. Whereas humans have one functional MBL protein, rodents have two isoforms, MBL-A and MBL-C, whose functions relative to that of human MBL are unknown. To permit the clinical translation of preclinical data, we aimed to define the specific contributions of MBL-A and MBL-C to brain ischemia. We subjected mice with double (MBL-/-) or single (MBL-A-/- or MBL-C-/-) MBL isoform depletion to transient middle cerebral artery occlusion (tMCAo). MBL-/- mice had fewer neurological deficits and smaller ischemic lesions than WT mice. MBL-A-/- mice had smaller lesions than WT mice and exhibited no significant behavioral defects, whereas MBL-C-/- mice did not differ from WT mice. The induction of Mbl1 and Mbl2 (the MBL-A and MBL-C genes) expression 48 h after tMCAo was similar across genotypes. The time course of Mbl1 and Mbl2 expression in WT ischemic mice showed that Mbl1 activation occurred earlier (24 h) than Mbl2 activation (48 h). The plasma levels of MBL-A and MBL-C in MBL-C-/- and MBL-A-/- mice, respectively, were similar to those in WT mice both at baseline and at 48 h after tMCAo. At 48 h, MBL-A-/- ischemic mice showed higher MBL-C levels in the brain than WT mice. WT and MBL-C-/- ischemic mice had higher LP activity in plasma and, accordingly, higher levels of C3 deposition in the brain than MBL-A-/- and MBL-/- mice. In conclusion, mice with depletion of both MBL isoforms exhibited strong protection from ischemia/reperfusion injury. MBL-A was the main contributor to injury, likely owing to its earlier activation after ischemia and more efficient activation of the complement system than MBL-C.
Subject(s)
Brain Ischemia/immunology , Brain/immunology , Mannose-Binding Lectin/immunology , Reperfusion Injury/immunology , Animals , Brain/pathology , Brain Ischemia/genetics , Brain Ischemia/pathology , Humans , Male , Mannose-Binding Lectin/genetics , Mice , Mice, Knockout , Protein Isoforms/genetics , Protein Isoforms/immunology , Reperfusion Injury/genetics , Reperfusion Injury/pathologyABSTRACT
We explored the involvement of the lectin pathway of complement in post-traumatic brain injury (TBI) pathophysiology in humans. Brain samples were obtained from 28 patients who had undergone therapeutic contusion removal, within 12 h (early) or from >12 h until five days (late) from injury, and from five non-TBI patients. Imaging analysis indicated that lectin pathway initiator molecules (MBL, ficolin-1, ficolin-2 and ficolin-3), the key enzymes MASP-2 and MASP-3, and the downstream complement components (C3 fragments and TCC) were present inside and outside brain vessels in all contusions. Only ficolin-1 was found in the parenchyma of non-TBI tissues. Immunoassays in brain homogenates showed that MBL, ficolin-2 and ficolin-3 increased in TBI compared to non-TBI (2.0, 2.2 and 6.0-times) samples. MASP-2 increased with subarachnoid hemorrhage and abnormal pupil reactivity, two indicators of structural and functional damage. C3 fragments and TCC increased, respectively, by 3.5 - and 4.0-fold in TBI compared to non-TBI tissue and significantly correlated with MBL, ficolin-2, ficolin-3, MASP-2 and MASP-3 levels in the homogenates. In conclusion, we show for the first time the direct presence of lectin pathway components in human cerebral contusions and their association with injury severity, suggesting a central role for the lectin pathway in the post-traumatic pathophysiology of human TBI.
