Biodistribution and Efficacy of Targeted Pulmonary Delivery of a Protein Kinase C-δ Inhibitory Peptide: Impact on Indirect Lung Injury.

Sepsis and sepsis-induced lung injury remain a leading cause of death in intensive care units. We identified protein kinase C-δ (PKCδ) as a critical regulator of the acute inflammatory response and demonstrated that PKCδ inhibition was lung-protective in a rodent sepsis model, suggesting that targeting PKCδ is a potential strategy for preserving pulmonary function in the setting of indirect lung injury. In this study, whole-body organ biodistribution and pulmonary cellular distribution of a transactivator of transcription (TAT)-conjugated PKCδ inhibitory peptide (PKCδ-TAT) was determined following intratracheal (IT) delivery in control and septic [cecal ligation and puncture (CLP)] rats to ascertain the impact of disease pathology on biodistribution and efficacy. There was negligible lung uptake of radiolabeled peptide upon intravenous delivery [<1% initial dose (ID)], whereas IT administration resulted in lung retention of >65% ID with minimal uptake in liver or kidney (<2% ID). IT delivery of a fluorescent-tagged (tetramethylrhodamine-PKCδ-TAT) peptide demonstrated uniform spatial distribution and cellular uptake throughout the peripheral lung. IT delivery of PKCδ-TAT at the time of CLP surgery significantly reduced PKCδ activation (tyrosine phosphorylation, nuclear translocation and cleavage) and acute lung inflammation, resulting in improved lung function and gas exchange. Importantly, peptide efficacy was similar when delivered at 4 hours post-CLP, demonstrating therapeutic relevance. Conversely, spatial lung distribution and efficacy were significantly impaired at 8 hours post-CLP, which corresponded to marked histopathological progression of lung injury. These studies establish a functional connection between peptide spatial distribution, inflammatory histopathology in the lung, and efficacy of this anti-inflammatory peptide.

Pulmonary endothelial protein kinase C-delta (PKCδ) regulates neutrophil migration in acute lung inflammation.

Excessive neutrophil migration across the pulmonary endothelium into the lung and release of oxidants and proteases are key elements in pathogenesis of acute lung injury. Previously, we identified protein kinase C-delta (PKCδ) as an important regulator of proinflammatory signaling in human neutrophils and demonstrated that intratracheal instillation of a TAT-conjugated PKCδ inhibitory peptide (PKCδ-TAT) is lung protective in a rat model of sepsis-induced indirect pulmonary injury (cecal ligation and puncture). In the present study, intratracheal instillation of this PKCδ inhibitor resulted in peptide distribution throughout the lung parenchyma and pulmonary endothelium and decreased neutrophil influx, with concomitant attenuation of sepsis-induced endothelial ICAM-1 and VCAM-1 expression in this model. To further delineate the role of PKCδ in regulating neutrophil migration, we used an in vitro transmigration model with human pulmonary microvascular endothelial cells (PMVECs). Consistent with in vivo findings, inhibition of PMVEC PKCδ decreased IL-1ß-mediated neutrophil transmigration. PKCδ regulation was stimulus-dependent; PKCδ was required for transmigration mediated by IL-1ß and fMLP (integrin-dependent), but not IL-8 (integrin-independent). PKCδ was essential for IL-1ß-mediated neutrophil adherence and NF-κB-dependent expression of ICAM-1 and VCAM-1. In PMVECs, IL-1ß-mediated production of ROS and activation of redox-sensitive NF-κB were PKCδ dependent, suggesting an upstream signaling role. Thus, PKCδ has an important role in regulating neutrophil-endothelial cell interactions and recruitment to the inflamed lung.

Protein kinase C and acute respiratory distress syndrome.

The acute respiratory distress syndrome (ARDS) is a major public health problem and a leading source of morbidity in intensive care units. Lung tissue in patients with ARDS is characterized by inflammation, with exuberant neutrophil infiltration, activation, and degranulation that is thought to initiate tissue injury through the release of proteases and oxygen radicals. Treatment of ARDS is supportive primarily because the underlying pathophysiology is poorly understood. This gap in knowledge must be addressed to identify urgently needed therapies. Recent research efforts in anti-inflammatory drug development have focused on identifying common control points in multiple signaling pathways. The protein kinase C (PKC) serine-threonine kinases are master regulators of proinflammatory signaling hubs, making them attractive therapeutic targets. Pharmacological inhibition of broad-spectrum PKC activity and, more importantly, of specific PKC isoforms (as well as deletion of PKCs in mice) exerts protective effects in various experimental models of lung injury. Furthermore, PKC isoforms have been implicated in inflammatory processes that may be involved in the pathophysiologic changes that result in ARDS, including activation of innate immune and endothelial cells, neutrophil trafficking to the lung, regulation of alveolar epithelial barrier functions, and control of neutrophil proinflammatory and prosurvival signaling. This review focuses on the mechanistic involvement of PKC isoforms in the pathogenesis of ARDS and highlights the potential of developing new therapeutic paradigms based on the selective inhibition (or activation) of specific PKC isoforms.