Disruption of the lung endothelial barrier is a hallmark of acute respiratory distress syndrome (ARDS), for which no effective pharmacologic treatments exist. Prior work has demonstrated that FTY720 S-phosphonate (Tys), an analog of sphingosine-1-phosphate (S1P) and FTY720, exhibits potent endothelial cell (EC) barrier protective properties. In this study, we investigated the in vitro and in vivo efficacy of Tys against methicillin-resistant Staphylococcus aureus (MRSA), a frequent bacterial cause of ARDS. Tys-protected human lung EC from barrier disruption induced by heat-killed MRSA (HK-MRSA) or staphylococcal α-toxin and attenuated MRSA-induced cytoskeletal changes associated with barrier disruption, including actin stress fiber formation and loss of peripheral VE-cadherin and cortactin. Tys-inhibited Rho and myosin light chain (MLC) activation after MRSA and blocked MRSA-induced NF-κB activation and release of the proinflammatory cytokines, IL-6 and IL-8. In vivo, intratracheal administration of live MRSA in mice caused significant vascular leakage and leukocyte infiltration into the alveolar space. Pre- or posttreatment with Tys attenuated MRSA-induced lung permeability and levels of alveolar neutrophils. Posttreatment with Tys significantly reduced levels of bronchoalveolar lavage (BAL) VCAM-1 and plasma IL-6 and KC induced by MRSA. Dynamic intravital imaging of mouse lungs demonstrated Tys attenuation of HK-MRSA-induced interstitial edema and neutrophil infiltration into lung tissue. Tys did not directly inhibit MRSA growth or viability in vitro. In conclusion, Tys inhibits lung EC barrier disruption and proinflammatory signaling induced by MRSA in vitro and attenuates acute lung injury induced by MRSA in vivo. These results support the potential utility of Tys as a novel ARDS therapeutic strategy.

Cortactin (CTTN) is an actin-binding and cytoskeletal protein that is found in abundance in the cell cortex and other peripheral structures of most cell types. It was initially described as a target for Src-mediated phosphorylation at several tyrosine sites within CTTN, and post-translational modifications at these tyrosine sites are a primary regulator of its function. CTTN participates in multiple cellular functions that require cytoskeletal rearrangement, including lamellipodia formation, cell migration, invasion, and various other processes dependent upon the cell type involved. The role of CTTN in vascular endothelial cells is particularly important for promoting barrier integrity and inhibiting vascular permeability and tissue edema. To mediate its functional effects, CTTN undergoes multiple post-translational modifications and interacts with numerous other proteins to alter cytoskeletal structures and signaling mechanisms. In the present review, we briefly describe CTTN structure, post-translational modifications, and protein binding partners and then focus on its role in regulating cellular processes and well-established functional mechanisms, primarily in vascular endothelial cells and disease models. We then provide insights into how CTTN function affects the pathophysiology of multiple lung disorders, including acute lung injury syndromes, COPD, and asthma.

Lung endothelial dysfunction is a key feature of acute lung injury (ALI) and clinical acute respiratory distress syndrome (ARDS). Previous studies have identified the lipid-generating enzyme, group V phospholipase A2 (gVPLA₂), as a mediator of lung endothelial barrier disruption and inflammation. The current study aimed to determine the role of gVPLA₂ in mediating lung endothelial responses to methicillin-resistant Staphylococcus aureus (MRSA, USA300 strain), a major cause of ALI/ARDS. In vitro studies assessed the effects of gVPLA₂ inhibition on lung endothelial cell (EC) permeability after exposure to heat-killed (HK) MRSA. In vivo studies assessed the effects of intratracheal live or HK-MRSA on multiple indices of ALI in wild-type (WT) and gVPLA₂-deficient (KO) mice. In vitro, HK-MRSA increased gVPLA₂ expression and permeability in human lung EC. Inhibition of gVPLA₂ with either the PLA2 inhibitor, LY311727, or with a specific monoclonal antibody, attenuated the barrier disruption caused by HK-MRSA. LY311727 also reduced HK-MRSA-induced permeability in mouse lung EC isolated from WT but not gVPLA₂-KO mice. In vivo, live MRSA caused significantly less ALI in gVPLA₂ KO mice compared to WT, findings confirmed by intravital microscopy assessment in HK-MRSA-treated mice. After targeted delivery of gVPLA₂ plasmid to lung endothelium using ACE antibody-conjugated liposomes, MRSA-induced ALI was significantly increased in gVPLA₂-KO mice, indicating that lung endothelial expression of gVPLA₂ is critical in vivo. In summary, these results demonstrate an important role for gVPLA₂ in mediating MRSA-induced lung EC permeability and ALI. Thus, gVPLA₂ may represent a novel therapeutic target in ALI/ARDS caused by bacterial infection.

Cigarette smoke (CS) is the primary cause of Chronic Obstructive Pulmonary Disease (COPD), and an important pathophysiologic event in COPD is CS-induced apoptosis in lung endothelial cells (EC). Cortactin (CTTN) is a cytoskeletal actin-binding regulatory protein with modulation by Src-mediated tyrosine phosphorylation. Based upon data demonstrating reduced CTTN mRNA levels in the lungs of smokers compared to non-smokers, we hypothesized a functional role for CTTN in CS-induced mitochondrial ROS generation and apoptosis in lung EC. Exposure of cultured human lung EC to CS condensate (CSC) led to the rearrangement of the actin cytoskeleton and increased CTTN tyrosine phosphorylation (within hours). Exposure to CS significantly increased EC mitochondrial ROS generation and EC apoptosis. The functional role of CTTN in these CSC-induced EC responses was explored using cortactin siRNA to reduce its expression, and by using a blocking peptide for the CTTN SH3 domain, which is critical to cytoskeletal interactions. CTTN siRNA or blockade of its SH3 domain resulted in significantly increased EC mitochondrial ROS and apoptosis and augmented CSC-induced effects. Exposure of lung EC to e-cigarette condensate demonstrated similar results, with CTTN siRNA or SH3 domain blocking peptide increasing lung EC apoptosis. These data demonstrate a novel role for CTTN in modulating lung EC apoptosis induced by CS or e-cigarettes potentially providing new insights into COPD pathogenesis.

E-cigarettes (E-cigs) are increasing in popularity and are considered as a potentially safer alternative to traditional cigarettes. However, prior studies have demonstrated that inhalation of nicotine-containing e-cigs can cause substantial pathophysiologic changes, and “vaping” of some substances has led to severe lung damage. Our group recently described the role of cortactin (CTTN), a cytoskeletal actin-binding regulatory protein, in mediating cigarette smoke (CS) and E-cig-induced lung endothelial apoptosis and mitochondrial dysfunction. In the current study, we advance this work by characterizing the effects of E-cig on lung endothelial nanomechanical properties and barrier function. Lung EC exposure to E-cig extract (50 µg/ml) resulted in disruption of endothelial barrier properties as assessed by Electric Cell Substrate Impedance (ECIS). Since excess mitochondrial ROS (mitoROS) is an important marker of mitochondrial dysfunction, we next assessed the effect of Mito-TEMPO (10 µM, 3 h), a cell-permeable antioxidant, on E-cig-induced endothelial permeability. Pretreatment with Mito-TEMPO provided EC barrier protection after E-cig challenge, suggesting a key role of mitoROS in E-cig-induced EC permeability. E-cig exposure induces cytoskeleton rearrangement leading to gap formation in lung EC and significantly alters EC elastic properties as assessed by atomic force microscopy (AFM). Reduction in CTTN expression by siRNA further augmented the injurious effects of E-cig on EC permeability and elastic properties. This is the first study to explore the role of CTTN in evaluating the effect of E-cigarette exposure on lung endothelium using AFM and provides novel mitochondrial and biophysical characterization of the effects of E-cig exposure on human lung EC. This work advances our understanding of the pathophysiologic effects of E-cig exposure.

E-cigarettes (E-cigs) are increasing in popularity and are considered a potentially safer alternative to traditional cigarettes. However, prior studies have demonstrated that inhalation of nicotine-containing e-cigs can cause substantial pathophysiologic changes, and “vaping” of some substances has led to severe lung damage. Our group recently described the role of cortactin (CTTN), a cytoskeletal actin-binding regulatory protein, in mediating cigarette smoke (CS) and E-cig-induced lung endothelial apoptosis and mitochondrial dysfunction. In the current study, we advance this work by characterizing the effects of E-cig on lung endothelial nanomechanical properties and barrier function. Lung EC exposure to E-cig extract (50 µg/mL) resulted in disruption of endothelial barrier properties as assessed by Electric Cell–Substrate Impedance Sensing (ECIS). Since excess mitochondrial reactive oxygen species (mitoROS) is an important marker of mitochondrial dysfunction, we next assessed the effect of Mito-TEMPO (10 µM, 3 h), a cell-permeable antioxidant, on E-cig-induced endothelial permeability. Pretreatment with Mito-TEMPO provided EC barrier protection after E-cig challenge, suggesting a key role of mitoROS in E-cig-induced EC permeability. E-cig exposure induces cytoskeleton rearrangement, leading to gap formation in lung EC, and significantly alters EC elastic properties as assessed by atomic force microscopy (AFM). Reduction in CTTN expression by siRNA further augmented the injurious effects of E-cig on EC permeability and elastic properties. This is the first study to explore the role of CTTN in evaluating the effect of E-cigarette exposure on the lung endothelium using AFM and provides novel mitochondrial and biophysical characterization of the effects of E-cig exposure on human lung EC. This work advances our understanding of the pathophysiologic effects of E-cig exposure.