Impacts of the COVID-19 Pandemic on Pedestrian Push-Button Utilization and Pedestrian Volume Model Accuracy in Utah.

This work investigated the impacts of COVID-19 on pedestrian behavior, answering two research questions using pedestrian push-button data from Utah traffic signals: How did push-button utilization change during the early pandemic, owing to concerns over disease spread through high-touch surfaces? How did the accuracy of pedestrian volume estimation models (developed pre-COVID based on push-button traffic signal data) change during the early pandemic? To answer these questions, we first recorded videos, counted pedestrians, and collected push-button data from traffic signal controllers at 11 intersections in Utah in 2019 and 2020. We then compared changes in push-button presses per pedestrian (to measure utilization), as well as model prediction errors (to measure accuracy), between the two years. Our first hypothesis of decreased push-button utilization was partially supported. The changes in utilization at most (seven) signals were not statistically significant; yet, the aggregate results (using 10 of 11 signals) saw a decrease from 2.1 to 1.5 presses per person. Our second hypothesis of no degradation of model accuracy was supported. There was no statistically significant change in accuracy when aggregating across nine signals, and the models were actually more accurate in 2020 for the other two signals. Overall, we concluded that COVID-19 did not significantly deter people from using push-buttons at most signals in Utah, and that the pedestrian volume estimation methods developed in 2019 probably do not need to be recalibrated to work for COVID conditions. This information may be useful for public health actions, signal operations, and pedestrian planning.

Impact of COVID-19 on Traffic Signal Systems: Survey of Agency Interventions and Observed Changes in Pedestrian Activity.

The COVID-19 pandemic, the most significant public health crisis since the 1918-1919 influenza epidemic, is the first such event to occur since the development of modern transportation systems in the twentieth century. Many states across the U.S. imposed lockdowns in early spring 2020, which reduced demand for trips of various types and affected transportation systems. In urban areas, the shift resulted in a reduction in traffic volumes and an increase in bicycling and walking in certain land use contexts. This paper seeks to understand the changes occurring at signalized intersections as a result of the lockdown and the ongoing pandemic, as well as the actions taken in response to these impacts. The results of a survey of agency reactions to COVID-19 with respect to traffic signal operations and changes in pedestrian activity during the spring 2020 lockdown using two case study examples in Utah are presented. First, the effects of placing intersections on pedestrian recall (with signage) to stop pedestrians from pushing the pedestrian button are examined. Next, the changes in pedestrian activity at Utah signalized intersections between the first 6 months of both 2019 and 2020 are analyzed and the impact of land use characteristics is explored. Survey results reveal the importance of using technologies such as adaptive systems and automated traffic signal performance measures to drive decisions. While pedestrian pushbutton actuations decreased in response to the implementation of pedestrian recalls, many pedestrians continued to use the pushbutton. Pedestrian activity changes were also largely driven by surrounding land uses.

Pedestrians and the Built Environment during the COVID-19 Pandemic: Changing Relationships by the Pandemic Phases in Salt Lake County, Utah, U.S.A.

The COVID-19 pandemic has dramatically altered people's travel behavior, in particular outdoor activities, including walking. Their behavior changes may have prolonged effects after the pandemic, and such changes vary by the context and are related to the characteristics of the built environment. But empirical studies about the relationships between pedestrians and the built environment during the pandemic are lacking. This study explores how COVID-19 and related travel restrictions have affected the relationship between pedestrian traffic volume and the built environment. We estimate daily pedestrian volumes for all signalized intersections in Salt Lake County, Utah, U.S.A., from pedestrian push-button log data between January 2019 and October 2020. Multilevel spatial filtering models show that the COVID-19 pandemic has altered the relationship between pedestrian traffic volume and the built environment. During the pandemic, the higher the number of COVID-19 cases, the less (or more negative) the effects of density, street connectivity, and destination accessibility on pedestrian volume being observed. The exception is access to urban parks, as it became more significant in increasing pedestrian activities during the pandemic. The models also highlight the negative impacts of the pandemic in economically disadvantaged areas. Our findings could help urban and transportation planners find effective interventions to promote active transportation and physical activity amid the global pandemic.

Novel Mechanism for Nicotinamide Phosphoribosyltransferase Inhibition of TNF-α-mediated Apoptosis in Human Lung Endothelial Cells.

Nicotinamide phosphoribosyltransferase (NAMPT) exists as both intracellular NAMPT and extracellular NAMPT (eNAMPT) proteins. eNAMPT is secreted into the blood and functions as a cytokine/enzyme (cytozyme) that activates NF-κB signaling via ligation of Toll-like receptor 4 (TLR4), further serving as a biomarker for inflammatory lung disorders such as acute respiratory distress syndrome. In contrast, intracellular NAMPT is involved in nicotinamide mononucleotide synthesis and has been implicated in the regulation of cellular apoptosis, although the exact mechanisms for this regulation are poorly understood. We examined the role of NAMPT in TNF-α-induced human lung endothelial cell (EC) apoptosis and demonstrated that reduced NAMPT expression (siRNA) increases EC susceptibility to TNF-α-induced apoptosis as reflected by PARP-1 cleavage and caspase-3 activation. In contrast, overexpression of NAMPT served to reduce degrees of TNF-α-induced EC apoptosis. Inhibition of nicotinamide mononucleotide synthesis by FK866 (a selective NAMPT enzymatic inhibitor) failed to alter TNF-α-induced human lung EC apoptosis, suggesting that NAMPT-dependent NAD+ generation is unlikely to be involved in regulation of TNF-α-induced EC apoptosis. We next confirmed that TNF-α-induced EC apoptosis is attributable to NAMPT secretion into the EC culture media and subsequent eNAMPT ligation of TLR4 on the EC membrane surface. Silencing of NAMPT expression, direct neutralization of secreted eNAMPT by an NAMPT-specific polyclonal antibody (preventing TLR4 ligation), or direct TLR4 antagonism all served to significantly increase EC susceptibility to TNF-α-induced EC apoptosis. Together, these studies provide novel insights into NAMPT contributions to lung inflammatory events and to novel mechanisms of EC apoptosis regulation.

Transactivation of the receptor-tyrosine kinase ephrin receptor A2 is required for the low molecular weight hyaluronan-mediated angiogenesis that is implicated in tumor progression.

Angiogenesis or the formation of new blood vessels is important in the growth and metastatic potential of various cancers. Therefore, understanding the mechanism(s) by which angiogenesis occurs can have important therapeutic implications in numerous malignancies. We and others have demonstrated that low molecular weight hyaluronan (LMW-HA, ∼2500 Da) promotes endothelial cell (EC) barrier disruption and angiogenesis. However, the mechanism(s) by which this occurs is poorly defined. Our data indicate that treatment of human EC with LMW-HA induced CD44v10 association with the receptor-tyrosine kinase, EphA2, transactivation (tyrosine phosphorylation) of EphA2, and recruitment of the PDZ domain scaffolding protein, PATJ, to the cell periphery. Silencing (siRNA) CD44, EphA2, PATJ, or Dbs (RhoGEF) expression blocked LMW-HA-mediated angiogenesis (EC proliferation, migration, and tubule formation). In addition, silencing EphA2, PATJ, Src, or Dbs expression blocked LMW-HA-mediated RhoA activation. To translate our in vitro findings, we utilized a novel anginex/liposomal targeting of murine angiogenic endothelium with either CD44 or EphA2 siRNA and observed inhibition of LMW-HA-induced angiogenesis in implanted Matrigel plugs. Taken together, these results indicate LMW-HA-mediated transactivation of EphA2 is required for PATJ and Dbs membrane recruitment and subsequent RhoA activation required for angiogenesis. These results suggest that targeting downstream effectors of LMW-HA could be a useful therapeutic intervention for angiogenesis-associated diseases including tumor progression.

The mu opioid receptor: A new target for cancer therapy?

Mu opioids are among the most widely used drugs for patients with cancer with both acute and chronic pain as well as in the perioperative period. Several retrospective studies have suggested that opioid use might promote tumor progression and as a result negatively impact survival in patients with advanced cancer; however, in the absence of appropriate prospective validation, any changes in recommendations for opioid use are not warranted. In this review, the authors present preclinical and clinical data that support their hypothesis that the mu opioid receptor is a potential target for cancer therapy because of its plausible role in tumor progression. The authors also propose the hypothesis that peripheral opioid antagonists such as methylnaltrexone, which reverses the peripheral effects of mu opioids but maintains centrally mediated analgesia and is approved by the US Food and Drug Administration for the treatment of opioid-induced constipation, can be used to target the mu opioid receptor.

Critical role of S1PR1 and integrin ß4 in HGF/c-Met-mediated increases in vascular integrity.

Vascular endothelial cell (EC) barrier integrity is critical to vessel homeostasis whereas barrier dysfunction is a key feature of inflammatory disorders and tumor angiogenesis. We previously reported that hepatocyte growth factor (HGF)-mediated increases in EC barrier integrity are signaled through a dynamic complex present in lipid rafts involving its receptor, c-Met. We extended these observations to confirm that S1PR1 (sphingosine 1-phosphate receptor 1) and integrin ß4 (ITGB4) are essential participants in HGF-induced EC barrier enhancement. Immunoprecipitation experiments demonstrated HGF-mediated recruitment of c-Met, ITGB4 and S1PR1 to caveolin-enriched lipid rafts in human lung EC with direct interactions of c-Met with both S1PR1 and ITGB4 accompanied by c-Met-dependent S1PR1 and ITGB4 transactivation. Reduced S1PR1 expression (siRNA) attenuated both ITGB4 and Rac1 activation as well as c-Met/ITGB4 interaction and resulted in decreased transendothelial electrical resistance. Furthermore, reduced ITGB4 expression attenuated HGF-induced c-Met activation, c-Met/S1PR1 interaction, and effected decreases in S1P- and HGF-induced EC barrier enhancement. Finally, the c-Met inhibitor, XL880, suppressed HGF-induced c-Met activation as well as S1PR1 and ITGB4 transactivation. These results support a critical role for S1PR1 and ITGB4 transactivation as rate-limiting events in the transduction of HGF signals via a dynamic c-Met complex resulting in enhanced EC barrier integrity.

Novel role for non-muscle myosin light chain kinase (MLCK) in hyperoxia-induced recruitment of cytoskeletal proteins, NADPH oxidase activation, and reactive oxygen species generation in lung endothelium.

We recently demonstrated that hyperoxia (HO) activates lung endothelial cell NADPH oxidase and generates reactive oxygen species (ROS)/superoxide via Src-dependent tyrosine phosphorylation of p47(phox) and cortactin. Here, we demonstrate that the non-muscle ~214-kDa myosin light chain (MLC) kinase (nmMLCK) modulates the interaction between cortactin and p47(phox) that plays a role in the assembly and activation of endothelial NADPH oxidase. Overexpression of FLAG-tagged wild type MLCK in human pulmonary artery endothelial cells enhanced interaction and co-localization between cortactin and p47(phox) at the cell periphery and ROS production, whereas abrogation of MLCK using specific siRNA significantly inhibited the above. Furthermore, HO stimulated phosphorylation of MLC and recruitment of phosphorylated and non-phosphorylated cortactin, MLC, Src, and p47(phox) to caveolin-enriched microdomains (CEM), whereas silencing nmMLCK with siRNA blocked recruitment of these components to CEM and ROS generation. Exposure of nmMLCK(-/-) null mice to HO (72 h) reduced ROS production, lung inflammation, and pulmonary leak compared with control mice. These results suggest a novel role for nmMLCK in hyperoxia-induced recruitment of cytoskeletal proteins and NADPH oxidase components to CEM, ROS production, and lung injury.

Reprint of: Exploring tourists' motivations, constraints, and negotiations regarding outdoor recreation trips during COVID-19 through a focus group study.

The current COVID-19 outbreak has duly influenced tourists' psychology and subsequently their behavior and decision making to participate in outdoor activities. The purpose of this paper is to illuminate tourists' motivations, perceived constraints, and negotiation strategies to participate in outdoor recreation trips, within the current COVID-19 context. To explore and categorize motivating factors, constraints, and negotiation strategies, we employed a qualitative approach via semi-structured online focus group discussion with 16 tourists (mostly residents of Utah, United States) during late summer 2020. First, COVID-19 related restrictions and fewer opportunities to go outdoors were found to encourage outdoor recreation, for novelty-seeking and experiencing normalcy. Through content analysis, we found that tourists experience a blend of personal, social, practical, and ethical constraints. Additionally, we identified how tourists negotiate their constraints through different ways: by extensive planning and information searching, avoiding crowds, and changing leisure aspirations. Finally, we discuss theoretical and managerial implications of the study, followed by recommendations for future research. Management implications: Understanding of tourists' motivations, constraints, and negotiation strategies-relevant to outdoor recreation trips-provides several managerial implications to destination managers and marketers, as outlined below:•Lack of centralized and reliable information was frequently cited as a constraint in the focus group discussions. In order to provide adequate and timely information to potential participations, we proposed a novel website template including details about information to be presented.•As our study sheds light on tourists' companionship preferences, activity choice, and evaluation of a destination's COVID-related operational practices, we propose several advertising strategies and destination operational guidelines to attract tourists.

Sphingosine-1-phosphate receptor-3 is a novel biomarker in acute lung injury.

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.

Overexpression of the µ-opioid receptor in human non-small cell lung cancer promotes Akt and mTOR activation, tumor growth, and metastasis.

BACKGROUND: Recent epidemiologic studies suggesting that there were differences in cancer recurrence contingent on anesthetic regimens have raised the possibility that µ-opioid agonists can influence cancer progression. Based on our previous studies indicating the µ-opioid receptor (MOR) is up-regulated in several types of non-small cell lung cancer, this study examined the functional significance of MOR overexpression to elucidate a possible mechanism for the epidemiologic findings. METHODS: Stable vector control and MOR1 overexpressing human bronchioloalveolar carcinoma cells were evaluated using immunoblot analysis, proliferation and transendothelial extravasation assays with or without Akt inhibitor, mTOR inhibitor (temsirolimus), or the peripheral MOR antagonist, methylnaltrexone. In human lung cancer xenograft models, primary tumor growth rates and lung metastasis were analyzed using consecutive tumor volume measurements and nestin immunoreactivity in lungs of the nude mouse model. RESULTS: The authors provide evidence that MOR is an important regulator of lung cancer progression. MOR overexpression increased Akt and mTOR activation, proliferation, and extravasation in human bronchioloalveolar carcinoma cells. In vivo, overexpression of MOR in human bronchoalveolar carcinoma cells increased primary tumor growth rates in nude mice by approximately 2.5-fold and lung metastasis by approximately 20-fold compared with vector control cells (n = 4 per condition). CONCLUSIONS: The overexpression data suggest a possible direct effect of MOR on Akt and mTOR activation and lung cancer progression. Such an effect provides a plausible explanation for the epidemiologic findings. The authors' observations further suggest that exploration of MOR in non-small cell lung carcinoma merits further study both as a diagnostic and therapeutic option.

Shedding light on the pedestrian safety crisis: An analysis across the injury severity spectrum by lighting condition.

OBJECTIVE: Pedestrian fatalities in the United States increased 51% from 2009 to 2019. During that time, pedestrian fatalities occurring at night increased by 63.7%, compared to a 17.6% increase for pedestrian fatalities occurring during daylight conditions. Have there also been increases in serious, minor, and possible pedestrian injuries (i.e., have all pedestrian collisions been occurring more frequently)? Have pedestrian collisions been getting more severe (i.e., are there now higher proportions of more severe injuries)? Have trends differed between night and day? What role does street lighting play in the nighttime trends? METHODS: We analyzed pedestrian fatalities, serious injuries, minor injuries, and possible injuries that occurred in California, North Carolina, and Texas from 2010 to 2019 using linear regressions to explore the strength and statistical significance of trends. We then parsed these trends by lighting condition, exploring outcomes during the day and night and with and without street lighting. RESULTS: Findings suggest that increases in daytime minor (7.9%) and possible (7.5%) injuries closely mirrored increases in population (9.8%). Increases in daytime fatal/serious injuries were significantly higher (43.1% and 35.1%, respectively), suggesting worsening severities during the day. Increases in nighttime minor/possible injuries (31.9% and 27.6%, respectively) were significantly larger than those during the day, suggesting that pedestrian collisions are occurring more frequently at night. Substantial increases in nighttime fatal/serious injuries (78.0% and 74.7%, respectively) likely reflect a combination of worsening severity (seen throughout the day) and increasing frequency (seen particularly at night). A pedestrian injured in the dark was found to be 5.0 times more likely to be killed than a pedestrian injured during the day. While a lack of street lighting does not seem to be the cause of the disproportionate increase in pedestrian injuries at night, pedestrians struck without a street light were 2.4 times more likely to be killed than those struck in the presence of a street light. CONCLUSIONS: As we find ourselves in the midst of a pedestrian safety crisis, understanding that severities have increased throughout the entire day and frequencies have increased particularly at night helps illuminate a path forward.

Joint position statement of the International Federation of Sports Medicine (FIMS) and European Federation of Sports Medicine Associations (EFSMA) on the IOC framework on fairness, inclusion and non-discrimination based on gender identity and sex variations.

The IOC recently published its framework on fairness, inclusion and non-discrimination based on gender identity and sex variations. This framework is drafted mainly from a human rights perspective, with less consideration for medical/scientific issues. The framework places the onus for gender eligibility and classification entirely on the International Federations (IFs), even though most will not have the capacity to implement the framework. The position of no presumption of advantage is contrary to the 2015 IOC consensus. Implementation of the 2021 framework will be a major challenge for IFs that have already recognised the inclusion of trans and women athletes with differences of sexual development (DSD) using a scientific/medical solution. The potential consequences for sports that need to prioritise fairness or safety could be one of two extremes (1) exclusion of all transgender or DSD athletes on the grounds of advantage or (2) self-identification that essentially equates to no eligibility rules. Exclusion of all transgender or DSD athletes is contrary to the Olympic charter and unlawful in many countries. While having no gender eligibility rules, sport loses its meaning and near-universal support. Athletes should not be under pressure to undergo medical procedures or treatment to meet eligibility criteria. However, if an athlete is fully informed and consents, then it is their free choice to undergo carefully considered or necessary interventions for gender classification for sport to compete fairly and safely in their chosen gender. Free choice is a fundamental human right, but so is the right to fair and safe competition.

Non-muscle myosin light chain kinase isoform is a viable molecular target in acute inflammatory lung injury.

Acute lung injury (ALI) and mechanical ventilator-induced lung injury (VILI), major causes of acute respiratory failure with elevated morbidity and mortality, are characterized by significant pulmonary inflammation and alveolar/vascular barrier dysfunction. Previous studies highlighted the role of the non-muscle myosin light chain kinase isoform (nmMLCK) as an essential element of the inflammatory response, with variants in the MYLK gene that contribute to ALI susceptibility. To define nmMLCK involvement further in acute inflammatory syndromes, we used two murine models of inflammatory lung injury, induced by either an intratracheal administration of lipopolysaccharide (LPS model) or mechanical ventilation with increased tidal volumes (the VILI model). Intravenous delivery of the membrane-permeant MLC kinase peptide inhibitor, PIK, produced a dose-dependent attenuation of both LPS-induced lung inflammation and VILI (~50% reductions in alveolar/vascular permeability and leukocyte influx). Intravenous injections of nmMLCK silencing RNA, either directly or as cargo within angiotensin-converting enzyme (ACE) antibody-conjugated liposomes (to target the pulmonary vasculature selectively), decreased nmMLCK lung expression (∼70% reduction) and significantly attenuated LPS-induced and VILI-induced lung inflammation (∼40% reduction in bronchoalveolar lavage protein). Compared with wild-type mice, nmMLCK knockout mice were significantly protected from VILI, with significant reductions in VILI-induced gene expression in biological pathways such as nrf2-mediated oxidative stress, coagulation, p53-signaling, leukocyte extravasation, and IL-6-signaling. These studies validate nmMLCK as an attractive target for ameliorating the adverse effects of dysregulated lung inflammation.

EphA2 mutation in lung squamous cell carcinoma promotes increased cell survival, cell invasion, focal adhesions, and mammalian target of rapamycin activation.

Non-small cell lung cancer (NSCLC) has a poor prognosis and improved therapies are needed. Expression of EphA2 is increased in NSCLC metastases. In this study, we investigated EphA2 mutations in NSCLC and examined molecular pathways involved in NSCLC. Tumor and cell line DNA was sequenced. One EphA2 mutation was modeled by expression in BEAS2B cells, and functional and biochemical studies were conducted. A G391R mutation was detected in H2170 and 2/28 squamous cell carcinoma patient samples. EphA2 G391R caused constitutive activation of EphA2 with increased phosphorylation of Src, cortactin, and p130(Cas). Wild-type (WT) and G391R cells had 20 and 40% increased invasiveness; this was attenuated with knockdown of Src, cortactin, or p130(Cas). WT and G391R cells demonstrated a 70% increase in focal adhesion area. Mammalian target of rapamycin (mTOR) phosphorylation was increased in G391R cells with increased survival (55%) compared with WT (30%) and had increased sensitivity to rapamycin. A recurrent EphA2 mutation is present in lung squamous cell carcinoma and increases tumor invasion and survival through activation of focal adhesions and actin cytoskeletal regulatory proteins as well as mTOR. Further study of EphA2 as a therapeutic target is warranted.

Role of hyaluronan and hyaluronan-binding proteins in lung pathobiology.

Hyaluronan (HA) has diverse functions in normal lung homeostasis and pulmonary disease. HA constitutes the major glycosaminoglycan in lung tissue, with HA degradation products, produced by hyaluronidase enzymes and reactive oxygen species, being implicated in several lung diseases, including acute lung injury, asthma, chronic obstructive pulmonary disease, and pulmonary hypertension. The differential activities of HA and its degradation products are due, in part, to regulation of multiple HA-binding proteins, including cluster of differentiation 44 (CD44), Toll-like receptor 4 (TLR4), HA-binding protein 2 (HABP2), and receptor for HA-mediated motility (RHAMM). Recent research indicates that exogenous administration of high-molecular-weight HA can serve as a novel therapeutic intervention for lung diseases, including lipopolysaccharide (LPS)-induced acute lung injury, sepsis/ventilator-induced lung injury, and airway hyperreactivity. This review focuses on the regulatory role of HA and HA-binding proteins in lung pathology and discusses the capacity of HA to augment and inhibit various lung diseases.

Akt-mediated transactivation of the S1P1 receptor in caveolin-enriched microdomains regulates endothelial barrier enhancement by oxidized phospholipids.

Endothelial cell (EC) barrier dysfunction results in increased vascular permeability, leading to increased mass transport across the vessel wall and leukocyte extravasation, the key mechanisms in pathogenesis of tissue inflammation and edema. We have previously demonstrated that OxPAPC (oxidized 1-palmitoyl-2-arachidonoyl-sn-glycero-3-phosphocholine) significantly enhances vascular endothelial barrier properties in vitro and in vivo and attenuates endothelial hyperpermeability induced by inflammatory and edemagenic agents via Rac and Cdc42 GTPase dependent mechanisms. These findings suggested potential important therapeutic value of barrier-protective oxidized phospholipids. In this study, we examined involvement of signaling complexes associated with caveolin-enriched microdomains (CEMs) in barrier-protective responses of human pulmonary ECs to OxPAPC. Immunoblotting from OxPAPC-treated ECs revealed OxPAPC-mediated rapid recruitment (5 minutes) to CEMs of the sphingosine 1-phosphate receptor (S1P(1)), the serine/threonine kinase Akt, and the Rac1 guanine nucleotide exchange factor Tiam1 and phosphorylation of caveolin-1, indicative of signaling activation in CEMs. Abolishing CEM formation (methyl-beta-cyclodextrin) blocked OxPAPC-mediated Rac1 activation, cytoskeletal reorganization, and EC barrier enhancement. Silencing (small interfering RNA) Akt expression blocked OxPAPC-mediated S1P(1) activation (threonine phosphorylation), whereas silencing S1P(1) receptor expression blocked OxPAPC-mediated Tiam1 recruitment to CEMs, Rac1 activation, and EC barrier enhancement. To confirm our in vitro results in an in vivo murine model of acute lung injury with pulmonary vascular hyperpermeability, we observed that selective lung silencing of caveolin-1 or S1P(1) receptor expression blocked OxPAPC-mediated protection from ventilator-induced lung injury. Taken together, these results suggest Akt-dependent transactivation of S1P(1) within CEMs is important for OxPAPC-mediated cortical actin rearrangement and EC barrier protection.

Hyaluronic Acid binding protein 2 is a novel regulator of vascular integrity.

OBJECTIVE: The disruption of the endothelial cell barrier is a critical feature of inflammation and an important contributing factor to acute lung injury (ALI), an inflammatory condition that is a major cause of morbidity and mortality in critically ill patients. We evaluated the role of the extracellular serine protease, hyaluronic acid binding protein 2 (HABP2), in vascular barrier regulation. METHODS AND RESULTS: By using immunoblot and immunohistochemical analysis, we observed that lipopolysaccharide (LPS) induces HABP2 expression in murine lung endothelium in vivo and in human pulmonary microvascular endothelial cells (ECs) in vitro. High-molecular-weight hyaluronan (HMW-HA, approximately 1x10(6) Da) decreased HABP2 protein expression in human pulmonary microvascular ECs and decreased purified HABP2 enzymatic activity, whereas low-molecular-weight HA (LMW-HA, approximately 2500 Da) increased these activities. The effects of LMW-HA, but not HMW-HA, on HABP2 activity were inhibited with a peptide of the polyanion-binding domain of HABP2. Silencing (small interfering RNA) HABP2 expression augmented HMW-HA-induced EC barrier enhancement and inhibited LPS and LMW-HA-mediated EC barrier disruption, results that were reversed with overexpression of HABP2. Silencing protease-activated receptor 1 and 3, RhoA, or Rho kinase expression attenuated LPS-, LMW-HA-, and HABP2-mediated EC barrier disruption. By using murine models of acute lung injury, we observed that LPS- and ventilator-induced pulmonary vascular hyperpermeability was significantly reduced with vascular silencing (small interfering RNA) of HABP2. CONCLUSIONS: HABP2 negatively regulates vascular integrity via activation of protease-activated receptor/RhoA/Rho kinase signaling and represents a potentially useful therapeutic target for syndromes of increased vascular permeability.