Protein kinase C-delta inhibition protects blood-brain barrier from sepsis-induced vascular damage

BackgroundNeuroinflammation often develops in sepsis leading to activation of cerebral endothelium, increased permeability of the blood-brain barrier (BBB), and neutrophil infiltration. We have identified protein kinase C-delta (PKC) as a critical regulator of the inflammatory response and demonstrated that pharmacologic inhibition of PKC by a peptide inhibitor (PKC-i) protected endothelial cells, decreased sepsis-mediated neutrophil influx into the lung, and prevented tissue damage. The objective of this study was to elucidate the regulation and relative contribution of PKC in the control of individual steps in neuroinflammation during sepsis.MethodsThe role of PKC in mediating human brain microvascular endothelial (HBMVEC) permeability, junctional protein expression, and leukocyte adhesion and migration was investigated in vitro using our novel BBB on-a-chip ((BC)-C-3) microfluidic assay and in vivo in a rat model of sepsis induced by cecal ligation and puncture (CLP). HBMVEC were cultured under flow in the vascular channels of (BC)-C-3. Confocal imaging and staining were used to confirm tight junction and lumen formation. Confluent HBMVEC were pretreated with TNF- (10U/ml) for 4h in the absence or presence of PKC-i (5M) to quantify neutrophil adhesion and migration in the (BC)-C-3. Permeability was measured using a 40-kDa fluorescent dextran in vitro and Evans blue dye in vivo.ResultsDuring sepsis, PKC is activated in the rat brain resulting in membrane translocation, a step that is attenuated by treatment with PKC-i. Similarly, TNF--mediated activation of PKC and its translocation in HBMVEC are attenuated by PKC-i in vitro. PKC inhibition significantly reduced TNF--mediated hyperpermeability and TEER decrease in vitro in activated HBMVEC and rat brain in vivo 24h after CLP induced sepsis. TNF--treated HBMVEC showed interrupted tight junction expression, whereas continuous expression of tight junction protein was observed in non-treated or PKC-i-treated cells. PKC inhibition also reduced TNF--mediated neutrophil adhesion and migration across HBMVEC in (BC)-C-3. Interestingly, while PKC inhibition decreased the number of adherent neutrophils to baseline (no-treatment group), it significantly reduced the number of migrated neutrophils below the baseline, suggesting a critical role of PKC in regulating neutrophil transmigration.ConclusionsThe BBB on-a-chip ((BC)-C-3) in vitro assay is suitable for the study of BBB function as well as screening of novel therapeutics in real-time. PKC activation is a key signaling event that alters the structural and functional integrity of BBB leading to vascular damage and inflammation-induced tissue damage. PKC-TAT peptide inhibitor has therapeutic potential for the prevention or reduction of cerebrovascular injury in sepsis-induced vascular damage.