Mannose-binding lectin has a direct deleterious effect on ischemic brain microvascular endothelial cells

Abstract

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.

Targeted deletions of complement lectin pathway genes improve outcome in traumatic brain injury

“…However, as previously reported in models of ischemia/reperfusion injury, recognition molecules can have detrimental effects independently from the LP cascade activation 16,38 . In particular, MBL itself has been shown to promote vascular injury on endothelial cells having undergone hypoxic stimulus by direct action -including structural damage to the endothelial cell cytoskeletonwithout requiring LP activation 16 .…”

“…However, as previously reported in models of ischemia/reperfusion injury, recognition molecules can have detrimental effects independently from the LP cascade activation 16,38 . In particular, MBL itself has been shown to promote vascular injury on endothelial cells having undergone hypoxic stimulus by direct action -including structural damage to the endothelial cell cytoskeletonwithout requiring LP activation 16 . Consistent with this finding, genetic deletion of MBL or its pharmacological inhibition were sufficient to obtain significant protection in experimental models of ischemic stroke, where vascular dysfunction is critical 21,24,39 .…”

“…Depending on the signals, complement activation may occur through three different pathways, the classical, the alternative and the lectin pathway (LP), each composed of specific initiators and effector enzymes. Carbohydrate structures or acetylated proteins exposed on the surface of damaged cells, including apoptotic or necrotic cells and stressed endothelium 16 , are typical DAMPs recognized by the LP. The LP is initiated by recognition molecules such as mannose-binding lectin (MBL, rodents have two isoforms, MBL-A and MBL-C), collectin-11 (CL-11) and ficolins (in humans: ficolin-1, 2 and 3; in rodents: ficolin-A and ficolin-B), acting as soluble receptors.…”

“…Genetic deletion of LP sub-components (MBL or MASP-2) [21][22][23] or their pharmacological targeting 21,22 are protective in experimental models of brain ischemia. In addition to a key role of the enzyme MASP-2, the recognition subcomponent MBL itself was shown to possess direct activity, driving platelet-dependent inflammation and vascular damage following ischemic injury independent of LP activation 16,24 . Of note, TBI shares blood perfusion deficits and metabolic derangements with ischemic injury 25 , thus suggesting that similar mechanisms might be involved in the traumatic pericore tissue, an area subjected to post-injury hypoxia 26 .…”

This was the year that was: brain barriers and brain fluid research in 2019

“…Now, super-resolution microscopy techniques [9] have opened the possibility of using light microscopy to examine cells, including live cells, at much greater resolution and these methods can be applied to the cerebral endothelium. Examples are structured illumination microscopy (SIM) to examine the actin cytoskeleton [10], and stochastic optical reconstruction microscopy (STORM) for changes in endothelial junction organization [11] and glycocalyx molecular structure [12]. Imaging using magnetic resonance (MR) to quantify brain metabolites and to measure dynamic changes is expanding.…”