Set up of an in vitro ischemia model using immortalized-human brain microvascular endothelial cells (i-hBMECs) to study the activation of complement lectin pathway in stroke

Introduction Mannose-binding lectin (MBL) is a protein of the lectin pathway (LP) complement cascade that plays a key role in ischemic stroke pathophysiology. Stroke patients with MBL deficiency show a more favorable outcome than the general population and MBL knock-out mice are protected from ischemic injury. Moreover pharmacological inhibition of MBL leads to neuroprotection in mice. However the exact detrimental mechanisms of MBL are not clear. Our group demonstrated that MBL deposits on brain ischemic endothelial cells after ischemic stroke in mice and that MBL deposition is significantly decreased after a protective treatment with an MBL inhibitor (Orsini et al. 2012). Thus the efficacy of MBL inhibitors may result from modulation of MBL deposition on ischemic vessels. Here we set up an in vitro stroke model using immortalized human brain microvascular endothelial cells (i-hBMECs) exposed to oxygen and glucose deprivation (OGD) to mimic ischemia, and evaluated: 1) the complement activation; 2) the effect of the MBL inhibitor Polyman9. Methods ihBMECs were seeded at 7500 cells/cm2 and, 8 days after seeding,they were exposed to OGD (5% CO2, 0.1% O2, no glucose) for 5 or 24 h. Cells were then reoxygenated for 6, 24 or 48 h. Control cells did not receive OGD. Cell mortality was measured through propidium iodide (PI) and/or alamar blue staining. C3 deposition, as a measure of complement activation, was measured by quantitative immunofluorescence. Cells exposed or not to 24 h OGD followed by 6 h reoxygenation in presence of human serum at different percentages (10, 30, 50%), were assessed for mortality and C3 deposition. In a separate set of experiments, using the same OGD protocol, OGD and control cells were incubated with different concentrations of the MBL inhibitor Polyman9 during reoxygenation. Cell mortality and C3 deposition were measured. Results Twenty-four hour OGD followed by 6 h reoxygenation induced a satisfactory degree of injury (cell mortality: +74%). C3 deposition on i-hBMECs significantly increased after OGD compared to control (normoxic condition) in presence of serum: +75% with 50% human serum, + 152% with 30% human serum, + 257% with 10% human serum. Polyman9 reduced cell mortality regardless of the concentrations (-75% at 900µM, -55% at 300µM, -71% at 100 µM). Polyman9 treatment did not affect C3 deposition on i-hBMECs. Conclusions These data indicate that i-hBMECs exposed to OGD are a useful in vitro model to study the activation of LP in ischemic stroke. Importantly, this model represents a tool to assess the ability of molecules to inhibit MBL or other complement factors involved in the ischemic stroke.