The inflamed lung exhibits oxidative and nitrative modifications of multiple target proteins, potentially reflecting disease severity and progression. We identified sphingosine-1-phosphate receptor-3 (S1PR3), a critical signaling molecule mediating cell proliferation and vascular permeability, as a nitrated plasma protein in mice with acute lung injury (ALI). We explored S1PR3 as a potential biomarker in murine and human ALI. In vivo nitrated and total S1PR3 concentrations were determined by immunoprecipitation and microarray studies in mice, and by ELISA in human plasma. In vitro nitrated S1PR3 concentrations were evaluated in human lung vascular endothelial cells (ECs) or within microparticles shed from ECs after exposure to barrier-disrupting agonists (LPS, low-molecular-weight hyaluronan, and thrombin). The effects of S1PR3-containing microparticles on EC barrier function were assessed by transendothelial electrical resistance (TER). Nitrated S1PR3 was identified in the plasma of murine ALI and in humans with severe sepsis-induced ALI. Elevated total S1PR3 plasma concentrations (> 251 pg/ml) were linked to sepsis and ALI mortality. In vitro EC exposure to barrier-disrupting agents induced S1PR3 nitration and the shedding of S1PR3-containing microparticles, which significantly reduced TER, consistent with increased permeability. These changes were attenuated by reduced S1PR3 expression (small interfering RNAs). These results suggest that microparticles containing nitrated S1PR3 shed into the circulation during inflammatory lung states, and represent a novel ALI biomarker linked to disease severity and outcome.

The vascular endothelium serves as a semi-selective barrier between the circulating contents of the blood and the tissues through which they flow. Disruption of this barrier results in significant organ dysfunction during devastating inflammatory syndromes such as sepsis and acute lung injury (ALI). Sphingosine 1-phosphate (S1P) is an endogenous lipid regulator of endothelial permeability that produces potent barrier enhancement via actin and junctional protein rearrangement and resultant cytoskeletal changes. A key effector protein in this S1P response is focal adhesion kinase (FAK), a highly conserved cytoplasmic tyrosine kinase involved in the engagement of integrins and assembly of focal adhesions (FA) through the catalysis of multiple downstream signals. After stimulation by S1P, endothelial FAK undergoes specific tyrosine phosphorylation that results in activation of the kinase and dynamic interactions with other effector molecules to improve the endothelial barrier. FAK participates in peripheral actin cytoskeletal rearrangement as well as cell-matrix (FA) and cell-cell (adherens junction) junctional complex strengthening that combine to decrease vascular permeability. This review summarizes the current knowledge of the role of FAK in mediating enhanced endothelial barrier function by S1P.

The pulmonary endothelial cell forms a critical semi-permeable barrier between the vascular and interstitial space. As part of the blood-gas barrier in the lung, the endothelium plays a key role in normal physiologic function and pathologic disease. Changes in endothelial cell shape, defined by its plasma membrane, determine barrier integrity. A number of key cytoskeletal regulatory and effector proteins including non-muscle myosin light chain kinase, cortactin, and Arp 2/3 mediate actin rearrangements to form cortical and membrane associated structures in response to barrier enhancing stimuli. These actin formations support and interact with junctional complexes and exert forces to protrude the lipid membrane to and close gaps between individual cells. The current knowledge of these cytoskeletal processes and regulatory proteins are the subject of this review. In addition, we explore novel advancements in cellular imaging that are poised to shed light on the complex nature of pulmonary endothelial permeability.

The genetic mechanisms underlying the susceptibility to acute respiratory distress syndrome (ARDS) are poorly understood. We previously demonstrated that sphingosine 1-phosphate (S1P) and the S1P receptor S1PR3 are intimately involved in lung inflammatory responses and vascular barrier regulation. Furthermore, plasma S1PR3 protein levels were shown to serve as a biomarker of severity in critically ill ARDS patients. This study explores the contribution of single nucleotide polymorphisms (SNPs) of the S1PR3 gene to sepsis-associated ARDS. S1PR3 SNPs were identified by sequencing the entire gene and tagging SNPs selected for case-control association analysis in African- and ED samples from Chicago, with independent replication in a European case-control study of Spanish individuals. Electrophoretic mobility shift assays, luciferase activity assays, and protein immunoassays were utilized to assess the functionality of associated SNPs. A total of 80 variants, including 29 novel SNPs, were identified. Because of limited sample size, conclusive findings could not be drawn in African-descent ARDS subjects; however, significant associations were found for two promoter SNPs (rs7022797 -1899T/G; rs11137480 -1785G/C), across two ED samples supporting the association of alleles -1899G and -1785C with decreased risk for sepsis-associated ARDS. In addition, these alleles significantly reduced transcription factor binding to the S1PR3 promoter; reduced S1PR3 promoter activity, a response particularly striking after TNF-α challenge; and were associated with lower plasma S1PR3 protein levels in ARDS patients. These highly functional studies support S1PR3 as a novel ARDS candidate gene and a potential target for individualized therapy.

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Pulmonary endothelial cell (EC) barrier dysfunction and recovery is critical to the pathophysiology of acute respiratory distress syndrome. Cytoskeletal and subsequent cell membrane dynamics play a key mechanistic role in determination of EC barrier integrity. Here, we characterizAQe the actin related protein 2/3 (Arp 2/3) complex, a regulator of peripheral branched actin polymerization, in human pulmonary EC barrier function through studies of transendothelial electrical resistance (TER), intercellular gap formation, peripheral cytoskeletal structures and lamellipodia. Compared to control, Arp 2/3 inhibition with the small molecule inhibitor CK-666 results in a reduction of baseline barrier function (1,241 ± 53 vs 988 ± 64 ohm; p < 0.01), S1P-induced barrier enhancement and delayed recovery of barrier function after thrombin (143 ± 14 vs 93 ± 6 min; p < 0.01). Functional changes of Arp 2/3 inhibition on barrier integrity are associated temporally with increased intercellular gap area at baseline (0.456 ± 0.02 vs 0.299 ± 0.02; p < 0.05) and thirty minutes after thrombin (0.885 ± 0.03 vs 0.754 ± 0.03; p < 0.05). Immunofluorescent microscopy reveals reduced lamellipodia formation after S1P and during thrombin recovery in Arp 2/3 inhibited cells. Individual lamellipodia demonstrate reduced depth following Arp 2/3 inhibition vs vehicle at baseline (1.83 ± 0.41 vs 2.55 ± 0.46 µm; p < 0.05) and thirty minutes after S1P treatment (1.53 ± 0.37 vs 2.09 ± 0.36 µm; p < 0.05). These results establish a critical role for Arp 2/3 activity in determination of pulmonary endothelial barrier function and recovery through formation of EC lamellipodia and closure of intercellular gaps.

Disruption of the pulmonary endothelial barrier and subsequent vascular leak is a hallmark of acute lung injury. Dynamic rearrangements in the endothelial cell (EC) peripheral membrane and underlying cytoskeleton are critical determinants of barrier function. The cytoskeletal effector protein non-muscle myosin light chain kinase (nmMLCK) and the actin-binding regulatory protein cortactin are important regulators of the endothelial barrier. In the present study we functionally characterize a proline-rich region of nmMLCK previously identified as the possible site of interaction between nmMLCK and cortactin. A mutant nmMLCK construct deficient in proline residues at the putative sites of cortactin binding (amino acids 973, 976, 1019, 1022) was generated. Co-immunoprecipitation studies in human lung EC transfected with wild-type or mutant nmMLCK demonstrated similar levels of cortactin interaction at baseline and after stimulation with the barrier-enhancing agonist, sphingosine 1-phosphate (S1P). In contrast, binding studies utilizing recombinant nmMLCK fragments containing the wild-type or proline-deficient sequence demonstrated a two fold increase in cortactin binding (p<0.01) to the mutant construct. Immunofluorescent microscopy revealed increased stress fiber density in ECs expressing GFP-labeled mutant nmMLCK at baseline (p=0.02) and after thrombin (p=0.01) or S1P (p=0.02) when compared to wild-type. Mutant nmMLCK demonstrated an increase in kinase activity in response to thrombin (p<0.01). Kymographic analysis demonstrated increased EC membrane retraction distance and velocity (p<0.01) in response to the barrier disrupting agent thrombin in cells expressing the mutant vs. wild-type nmMLCK construct. These results provide evidence that critical prolines within nmMLCK (amino acids 973, 976, 1019, 1022) regulate cytoskeletal and membrane events associated with pulmonary endothelial barrier function.