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

Abstract

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 (PKCδ) as a critical regulator of the inflammatory response and demonstrated that pharmacologic inhibition of PKCδ by a peptide inhibitor (PKCδ-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 PKCδ in the control of individual steps in neuroinflammation during sepsis.MethodsThe role of PKCδ 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 (B3C) 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 B3C. Confocal imaging and staining were used to confirm tight junction and lumen formation. Confluent HBMVEC were pretreated with TNF-α (10 U/ml) for 4 h in the absence or presence of PKCδ-i (5 μM) to quantify neutrophil adhesion and migration in the B3C. Permeability was measured using a 40-kDa fluorescent dextran in vitro and Evans blue dye in vivo.ResultsDuring sepsis, PKCδ is activated in the rat brain resulting in membrane translocation, a step that is attenuated by treatment with PKCδ-i. Similarly, TNF-α-mediated activation of PKCδ and its translocation in HBMVEC are attenuated by PKCδ-i in vitro. PKCδ inhibition significantly reduced TNF-α-mediated hyperpermeability and TEER decrease in vitro in activated HBMVEC and rat brain in vivo 24 h after CLP induced sepsis. TNF-α-treated HBMVEC showed interrupted tight junction expression, whereas continuous expression of tight junction protein was observed in non-treated or PKCδ-i-treated cells. PKCδ inhibition also reduced TNF-α-mediated neutrophil adhesion and migration across HBMVEC in B3C. Interestingly, while PKCδ 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 PKCδ in regulating neutrophil transmigration.ConclusionsThe BBB on-a-chip (B3C) in vitro assay is suitable for the study of BBB function as well as screening of novel therapeutics in real-time. PKCδ activation is a key signaling event that alters the structural and functional integrity of BBB leading to vascular damage and inflammation-induced tissue damage. PKCδ-TAT peptide inhibitor has therapeutic potential for the prevention or reduction of cerebrovascular injury in sepsis-induced vascular damage.

Protein kinase C‐delta inhibition is organ‐protective, enhances pathogen clearance, and improves survival in sepsis

“…Sepsis was induced by the cecal ligation and puncture (CLP) method as described previously. [14][15][16][17][18][19] Sham controls underwent a laparotomy without cecal ligation or puncture. Following CLP or Sham surgery, the abdominal incision was closed and the animals were orally intubated with a 16-gauge intravenous cannula and randomized to receive either the PKCδ TAT inhibitory peptide (200 µg/kg in 200 µL of PBS) or a | 2499 LIVERANI Et AL.…”

“…As previously described, [12][13][14][15][16][17][18][19] PKCδ activity was selectively inhibited by a peptide antagonist that consisted of a peptide derived from the first unique region (V1) of PKCδ (SFNSYELGSL: amino acids [8][9][10][11][12][13][14][15][16][17] coupled to a membrane permeant peptide sequence in the HIV TAT gene product (YGRKKRRQRRR: amino acids 47-57 of TAT). 28 Extensive in vitro and in vivo studies demonstrate that, when taken up by cells, the PKCδ TAT peptide produces a unique dominant-negative phenotype that effectively inhibits activation of PKCδ, but not other PKC isotypes.…”

“…We identified Protein Kinase C-delta (PKCδ) as a critical regulator of the inflammatory response. [10][11][12][13][14][15][16][17][18][19][20] Inflammatory mediators involved in the septic response including LPS, TNF and IL-1 activate PKCδ. [21][22][23] Studies with PKCδ −/− mice and PKCδ inhibitors indicate a role for PKCδ in regulating neutrophil trafficking to the lung in response to inflammation triggered by asbestos, stroke/reperfusion injury, LPS, or pancreatitis.…”

A specific small-molecule inhibitor of protein kinase CδI activity improves metabolic dysfunction in human adipocytes from obese individuals

“…PKC␦, a member of serine/threonine kinase family, is involved in regulation of cellular differentiation, growth, and apoptosis (6,7). PKC␦ has also been shown to regulate inflammatory responses, neurotoxicity, and B cell tolerance; thus, PKC␦ plays pivotal roles in normal cellular processes and in diseases such as diabetes, sepsis, neurodegenerative diseases, obesity-related metabolic dysfunction, cancer, and stroke (5,(8)(9)(10)(11)(12)(13)(14)(15)(16)(17). The primary amino acid structure of PKC␦ can be divided into conserved regions (C1-C4) separated by the variable regions (V1-V5).…”

“…PKC␦I has been shown to mediate inflammation in several cell types, including adipocytes, macrophages, vascular smooth muscle cells, and hepatocytes (14,(21)(22)(23)(24)(25). PKC␦I is proteolytically cleaved by caspase-3 at its hinge region, separating the regulatory domain from the catalytic domain (26 -28).…”