Endothelial Heterogeneity in the Response to Autophagy Drives Small Vessel Muscularization in Pulmonary Hypertension.

BACKGROUND: Endothelial cell (EC) apoptosis and proliferation of apoptosis-resistant cells is a hallmark of pulmonary hypertension (PH). Yet, why some ECs die and others proliferate and how this contributes to vascular remodeling is unclear. We hypothesized that this differential response may: (1) relate to different EC subsets, namely pulmonary artery (PAECs) versus microvascular ECs (MVECs); (2) be attributable to autophagic activation in both EC subtypes; and (3) cause replacement of MVECs by PAECs with subsequent distal vessel muscularization. METHODS: EC subset responses to chronic hypoxia were assessed by single-cell RNA sequencing of murine lungs. Proliferative versus apoptotic responses, activation, and role of autophagy were assessed in human and rat PAECs and MVECs, and in precision-cut lung slices of wild-type mice or mice with endothelial deficiency in the autophagy gene Atg7 (Atg7EN-KO). Abundance of PAECs versus MVECs in precapillary microvessels was assessed in lung tissue from patients with PH and animal models on the basis of structural or surface markers. RESULTS: In vitro and in vivo, PAECs proliferated in response to hypoxia, whereas MVECs underwent apoptosis. Single-cell RNA sequencing analyses support these findings in that hypoxia induced an antiapoptotic, proliferative phenotype in arterial ECs, whereas capillary ECs showed a propensity for cell death. These distinct responses were prevented in hypoxic Atg7EN-KO mice or after ATG7 silencing, yet replicated by autophagy stimulation. In lung tissue from mice, rats, or patients with PH, the abundance of PAECs in precapillary arterioles was increased, and that of MVECs reduced relative to controls, indicating replacement of microvascular by macrovascular ECs. EC replacement was prevented by genetic or pharmacological inhibition of autophagy in vivo. Conditioned medium from hypoxic PAECs yet not MVECs promoted pulmonary artery smooth muscle cell proliferation and migration in a platelet-derived growth factor-dependent manner. Autophagy inhibition attenuated PH development and distal vessel muscularization in preclinical models. CONCLUSIONS: Autophagic activation by hypoxia induces in parallel PAEC proliferation and MVEC apoptosis. These differential responses cause a progressive replacement of MVECs by PAECs in precapillary pulmonary arterioles, thus providing a macrovascular context that in turn promotes pulmonary artery smooth muscle cell proliferation and migration, ultimately driving distal vessel muscularization and the development of PH.

Carbonic Anhydrase IX and Hypoxia Promote Rat Pulmonary Endothelial Cell Survival during Infection.

Low tidal volume ventilation protects the lung in mechanically ventilated patients. The impact of the accompanying permissive hypoxemia and hypercapnia on endothelial cell recovery from injury is poorly understood. CA (carbonic anhydrase) IX is expressed in pulmonary microvascular endothelial cells (PMVECs), where it contributes to CO2 and pH homeostasis, bioenergetics, and angiogenesis. We hypothesized that CA IX is important for PMVEC survival and that CA IX expression and release from PMVECs are increased during infection. Although the plasma concentration of CA IX was unchanged in human and rat pneumonia, there was a trend toward increasing CA IX in the bronchoalveolar fluid of mechanically ventilated critically ill patients with pneumonia and a significant increase in CA IX in the lung tissue lysates of pneumonia rats. To investigate the functional implications of the lung CA IX increase, we generated PMVEC cell lines harboring domain-specific CA IX mutations. By using these cells, we found that infection promotes intracellular (IC) expression, release, and MMP (metalloproteinase)-mediated extracellular cleavage of CA IX in PMVECs. IC domain deletion uniquely impaired CA IX membrane localization. Loss of the CA IX IC domain promoted cell death after infection, suggesting that the IC domain has an important role in PMVEC survival. We also found that hypoxia improves survival, whereas hypercapnia reverses the protective effect of hypoxia, during infection. Thus, we report 1) that CA IX increases in the lungs of pneumonia rats and 2) that the CA IX IC domain and hypoxia promote PMVEC survival during infection.

cAMP signaling primes lung endothelial cells to activate caspase-1 during Pseudomonas aeruginosa infection.

Activation of the inflammasome-caspase-1 axis in lung endothelial cells is emerging as a novel arm of the innate immune response to pneumonia and sepsis caused by Pseudomonas aeruginosa. Increased levels of circulating autacoids are hallmarks of pneumonia and sepsis and induce physiological responses via cAMP signaling in targeted cells. However, it is unknown whether cAMP affects other functions, such as P. aeruginosa-induced caspase-1 activation. Herein, we describe the effects of cAMP signaling on caspase-1 activation using a single cell flow cytometry-based assay. P. aeruginosa infection of cultured lung endothelial cells caused caspase-1 activation in a distinct population of cells. Unexpectedly, pharmacological cAMP elevation increased the total number of lung endothelial cells with activated caspase-1. Interestingly, addition of cAMP agonists augmented P. aeruginosa infection of lung endothelial cells as a partial explanation underlying cAMP priming of caspase-1 activation. The cAMP effect(s) appeared to function as a priming signal because addition of cAMP agonists was required either before or early during the onset of infection. However, absolute cAMP levels measured by ELISA were not predictive of cAMP-priming effects. Importantly, inhibition of de novo cAMP synthesis decreased the number of lung endothelial cells with activated caspase-1 during infection. Collectively, our data suggest that lung endothelial cells rely on cAMP signaling to prime caspase-1 activation during P. aeruginosa infection.

Development of an endothelial cell-restricted transgenic reporter rat: a resource for physiological studies of vascular biology.

Here, we report the generation of a Cre-recombinase (iCre) transgenic rat, where iCre is driven using a vascular endothelial-cadherin (CDH5) promoter. The CDH5 promoter was cloned from rat pulmonary microvascular endothelial cells and demonstrated ~60% similarity to the murine counterpart. The cloned rat promoter was 2,508 bp, it extended 79 bp beyond the transcription start site, and it was 22,923 bp upstream of the translation start site. The novel promoter was cloned upstream of codon-optimized iCre and subcloned into a Sleeping Beauty transposon vector for transpositional transgenesis in Sprague-Dawley rats. Transgenic founders were generated and selected for iCre expression. Crossing the CDH5-iCre rat with a tdTomato reporter rat resulted in progeny displaying endothelium-restricted fluorescence. tdTomato fluorescence was prominent in major arteries and veins, and it was similar in males and females. Quantitative analysis of the carotid artery and the jugular vein revealed that, on average, more than 50% of the vascular surface area exhibited strong fluorescence. tdTomato fluorescence was observed in the circulations of every tissue tested. The microcirculation in all tissues tested displayed homogenous fluorescence. Fluorescence was examined across young (6-7.5 mo), middle (14-16.5 mo), and old age (17-19.5 mo) groups. Although tdTomato fluorescence was seen in middle- and old-age animals, the intensity of the fluorescence was significantly reduced compared with that seen in the young rats. Thus, this endothelium-restricted transgenic rat offers a novel platform to test endothelial microheterogeneity within all vascular segments, and it provides exceptional resolution of endothelium within-organ microcirculation for application to translational disease models.NEW & NOTEWORTHY The use of transgenic mice has been instrumental in advancing molecular insight of physiological processes, yet these models oftentimes do not faithfully recapitulate human physiology and pathophysiology. Rat models better replicate some human conditions, like Group 1 pulmonary arterial hypertension. Here, we report the development of an endothelial cell-restricted transgenic reporter rat that has broad application to vascular biology. This first-in-kind model offers exceptional endothelium-restricted tdTomato expression, in both conduit vessels and the microcirculations of organs.

Unleashing shear: Role of intercellular traction and cellular moments in collective cell migration.

In the field of endothelial biology, the term "shear forces" is tied to the forces exerted by the flowing blood on the quiescent cells. But endothelial cells themselves also exert physical forces on their immediate and distant neighbors. Specific factors of such intrinsic mechanical signals most relevant to immediate neighbors include normal (Fn) and shear (Fs) components of intercellular tractions, and those factors most relevant to distant neighbors include contractile or dilatational (Mc) and shear (Ms) components of the moments of cytoskeletal forces. However, for cells within a monolayer, Fn, Fs, Mc, and Ms remain inaccessible to experimental evaluation. Here, we present an approach that enables quantitative assessment of these properties. Remarkably, across a collectively migrating sheet of pulmonary microvascular endothelial cells, Fs was of the same order of magnitude as Fn. Moreover, compared to the normal components (Fn, Mc) of the mechanical signals, the shear components (Fs, Ms) were more distinctive in the cells closer to the migration front. Individual cells had an innately collective tendency to migrate along the axis of maximum contractile moment - a collective migratory process we referred to as cellular plithotaxis. Notably, larger Fs and Ms were associated with stronger plithotaxis, but dilatational moment appeared to disengage plithotactic guidance. Overall, cellular plithotaxis was more strongly associated with the "shear forces" (Fs, Ms) than with the "normal forces" (Fn, Mc). Finally, the mechanical state of the cells with fast migration speed and those with highly circular shape were reminiscent of fluid-like and solid-like matter, respectively. The results repeatedly pointed to neighbors imposing shear forces on a cell as a highly significant event, and hence, the term "shear forces" must include not just the forces from flowing fluid but also the forces from the substrate and neighbors. Collectively, these advances set the stage for deeper understanding of mechanical signaling in cellular monolayers.

Infection-induced endothelial amyloids impair memory.

Patients with nosocomial pneumonia exhibit elevated levels of neurotoxic amyloid and tau proteins in the cerebrospinal fluid (CSF). In vitro studies indicate that pulmonary endothelium infected with clinical isolates of either Pseudomonas aeruginosa, Klebsiella pneumoniae, or Staphylococcus aureus produces and releases cytotoxic amyloid and tau proteins. However, the effects of the pulmonary endothelium-derived amyloid and tau proteins on brain function have not been elucidated. Here, we show that P. aeruginosa infection elicits accumulation of detergent insoluble tau protein in the mouse brain and inhibits synaptic plasticity. Mice receiving endothelium-derived amyloid and tau proteins via intracerebroventricular injection exhibit a learning and memory deficit in object recognition, fear conditioning, and Morris water maze studies. We compared endothelial supernatants obtained after the endothelia were infected with P. aeruginosa possessing an intact [P. aeruginosa isolated from patient 103 (PA103) supernatant] or defective [mutant strain of P. aeruginosa lacking a functional type 3 secretion system needle tip complex (ΔPcrV) supernatant] type 3 secretion system. Whereas the PA103 supernatant impaired working memory, the ΔPcrV supernatant had no effect. Immunodepleting amyloid or tau proteins from the PA103 supernatant with the A11 or T22 antibodies, respectively, overtly rescued working memory. Recordings from hippocampal slices treated with endothelial supernatants or CSF from patients with or without nosocomial pneumonia indicated that endothelium-derived neurotoxins disrupted the postsynaptic synaptic response. Taken together, these results establish a plausible mechanism for the neurologic sequelae consequent to nosocomial bacterial pneumonia.-Balczon, R., Pittet, J.-F., Wagener, B. M., Moser, S. A., Voth, S., Vorhees, C. V., Williams, M. T., Bridges, J. P., Alvarez, D. F., Koloteva, A., Xu, Y., Zha, X.-M., Audia, J. P., Stevens, T., Lin, M. T. Infection-induced endothelial amyloids impair memory.

Mechanical signaling in a pulmonary microvascular endothelial cell monolayer.

The mechanical microenvironment of an endothelial cell includes a stable protein scaffold on the basal side, flowing blood on the apical side and contractile cells on the lateral sides. Interaction with the protein scaffold and flowing blood modulates the ability of endothelial cells to migrate, align and maintain barrier function. Interaction with neighbors provides the endothelial monolayer unique "collective" properties. However, the nature of local mechanical signaling - i.e., the local functional consequence of a cell interacting with its contractile neighbors - remains unclear. Using an advancing sheet of pulmonary microvascular endothelial cells, here we examine the mechanical properties of an individual cell and its neighboring region. By combining Monolayer Stress Microscopy (MSM) with a novel analysis, we assessed several mechanical properties of an individual cell and its neighboring region. Across the monolayer, mechanical properties of the neighboring region defined multicellular "subdivisions" wherein constituent cells were exposed to a similar mechanical microenvironment. Adjacent subdivisions were separated by a narrow interface where adjoining cells were exposed to remarkably different mechanical microenvironments. Comparison of temporal fluctuations in mechanical properties of individual cells and those of their neighboring regions suggested three distinct intercellular mechanical signaling processes. These processes indicated that change in size, shape and speed of individual cells is associated with change in contractile forces in their neighboring regions. In summary, we present a novel approach to assess the mechanical interactions of individual cells with their contractile neighbors and identify potential functional consequences of such interactions.

Caspase-1 inhibition by VX-765 administered at reperfusion in P2Y12 receptor antagonist-treated rats provides long-term reduction in myocardial infarct size and preservation of ventricular function.

Patients with acute myocardial infarction receive a P2Y12 receptor antagonist prior to reperfusion, a treatment that has reduced, but not eliminated, mortality, or heart failure. We tested whether the caspase-1 inhibitor VX-765 given at reperfusion (a requirement for clinical use) can provide sustained reduction of infarction and long-term preservation of ventricular function in a pre-clinical model of ischemia/reperfusion that had been treated with a P2Y12 receptor antagonist. To address, the hypothesis open-chest rats were subjected to 60-min left coronary artery branch occlusion/120-min reperfusion. Vehicle or inhibitors were administered intravenously immediately before reperfusion. With vehicle only, 60.3 ± 3.8% of the risk zone suffered infarction. Ticagrelor, a P2Y12 antagonist, and VX-765 decreased infarct size to 42.8 ± 3.3 and 29.2 ± 4.9%, respectively. Combining ticagrelor with VX-765 further decreased infarction to 17.5 ± 2.3%. Similar to recent clinical trials, combining ticagrelor and ischemic postconditioning did not result in additional cardioprotection. VX-765 plus another P2Y12 antagonist, cangrelor, also decreased infarction and preserved ventricular function when reperfusion was increased to 3 days. In addition, VX-765 reduced infarction in blood-free, isolated rat hearts indicating at least a portion of injurious caspase-1 activation originates in cardiac tissue. While the pro-drug VX-765 only protected isolated hearts when started prior to ischemia, its active derivative VRT-043198 provided the same amount of protection when started at reperfusion, indicating that even in blood-free hearts, caspase-1 appears to exert its injury only at reperfusion. Moreover, VX-765 decreased circulating IL-1ß, prevented loss of cardiac glycolytic enzymes, preserved mitochondrial complex I activity, and decreased release of lactate dehydrogenase, a marker of pyroptosis. Our results are the first demonstration of a clinical-grade drug given at reperfusion providing additional, sustained infarct size reduction when added to a P2Y12 receptor antagonist.

Caspase-1 Activation Protects Lung Endothelial Barrier Function during Infection-Induced Stress.

Dysregulated activation of the inflammasome-caspase-1-IL-1ß axis elicits damaging hyperinflammation during critical illnesses, such as pneumonia and sepsis. However, in critical illness models of Salmonella infection, burn, or shock, caspase-1 inhibition worsens outcomes. These paradoxical effects suggest that caspase-1 drives novel protective responses. Whether the protective effects of caspase-1 activation involve canonical immune cell and/or nonimmune cell responses is unknown. The objective of this study was to test the hypothesis that, in addition to its recognized proinflammatory function, caspase-1 initiates protective stress responses in nonimmune cells. In vivo, lung epithelial and endothelial barrier function and inflammation were assessed in mice infected with Pseudomonas aeruginosa in the presence or absence of a caspase-1 inhibitor. Lung endothelial barrier function was assessed ex vivo in isolated, perfused rat lungs infected with P. aeruginosa in the presence or absence of a caspase-1 inhibitor. Endothelial barrier function during P. aeruginosa infection was assessed in vitro in cultured rat wild-type pulmonary microvascular endothelial cells (PMVECs) or recombinant PMVECs engineered to decrease caspase-1 expression. We demonstrated in vivo that caspase-1 inhibition in P. aeruginosa-infected mice ameliorated hyperinflammation, but, counterintuitively, increased pulmonary edema. Ex vivo, caspase-1 inhibition increased pulmonary permeability in P. aeruginosa-infected isolated rat lungs. To uncouple caspase-1 from its canonical inflammatory role, we used cultured rat PMVECs in vitro and discovered that genetic knockdown of caspase-1 accelerated P. aeruginosa-induced barrier disruption. In conclusion, caspase-1 is a sentinel stress-response regulator that initiates proinflammatory responses and also initiates novel response(s) to protect PMVEC barrier function during pneumonia.

The Pseudomonas aeruginosa exoenzyme Y impairs endothelial cell proliferation and vascular repair following lung injury.

Exoenzyme Y (ExoY) is a Pseudomonas aeruginosa toxin that is introduced into host cells through the type 3 secretion system (T3SS). Once inside the host cell cytoplasm, ExoY generates cyclic nucleotides that cause tau phosphorylation and microtubule breakdown. Microtubule breakdown causes interendothelial cell gap formation and tissue edema. Although ExoY transiently induces interendothelial cell gap formation, it remains unclear whether ExoY prevents repair of the endothelial cell barrier. Here, we test the hypothesis that ExoY intoxication impairs recovery of the endothelial cell barrier following gap formation, decreasing migration, proliferation, and lung repair. Pulmonary microvascular endothelial cells (PMVECs) were infected with P. aeruginosa strains for 6 h, including one possessing an active ExoY (PA103 exoUexoT::Tc pUCPexoY; ExoY(+)), one with an inactive ExoY (PA103ΔexoUexoT::Tc pUCPexoY(K81M); ExoY(K81M)), and one that lacks PcrV required for a functional T3SS (ΔPcrV). ExoY(+) induced interendothelial cell gaps, whereas ExoY(K81M) and ΔPcrV did not promote gap formation. Following gap formation, bacteria were removed and endothelial cell repair was examined. PMVECs were unable to repair gaps even 3-5 days after infection. Serum-stimulated growth was greatly diminished following ExoY intoxication. Intratracheal inoculation of ExoY(+) and ExoY(K81M) caused severe pneumonia and acute lung injury. However, whereas the pulmonary endothelial cell barrier was functionally improved 1 wk following ExoY(K81M) infection, pulmonary endothelium was unable to restrict the hyperpermeability response to elevated hydrostatic pressure following ExoY(+) infection. In conclusion, ExoY is an edema factor that chronically impairs endothelial cell barrier integrity following lung injury.

Amyloid-ß and caspase-1 are indicators of sepsis and organ injury.

Background: Sepsis is a life-threatening condition that results from a dysregulated host response to infection, leading to organ dysfunction. Despite the prevalence and associated socioeconomic costs, treatment of sepsis remains limited to antibiotics and supportive care, and a majority of intensive care unit (ICU) survivors develop long-term cognitive complications post-discharge. The present study identifies a novel regulatory relationship between amyloid-ß (Aß) and the inflammasome-caspase-1 axis as key innate immune mediators that define sepsis outcomes. Methods: Medical ICU patients and healthy individuals were consented for blood and clinical data collection. Plasma cytokine, caspase-1 and Aß levels were measured. Data were compared against indices of multiorgan injury and other clinical parameters. Additionally, recombinant proteins were tested in vitro to examine the effect of caspase-1 on a functional hallmark of Aß, namely aggregation. Results: Plasma caspase-1 levels displayed the best predictive value in discriminating ICU patients with sepsis from non-infected ICU patients (area under the receiver operating characteristic curve=0.7080). Plasma caspase-1 and the Aß isoform Aßx-40 showed a significant positive correlation and Aßx-40 associated with organ injury. Additionally, Aß plasma levels continued to rise from time of ICU admission to 7 days post-admission. In silico, Aß harbours a predicted caspase-1 cleavage site, and in vitro studies demonstrated that caspase-1 cleaved Aß to inhibit its auto-aggregation, suggesting a novel regulatory relationship. Conclusions: Aßx-40 and caspase-1 are potentially useful early indicators of sepsis and its attendant organ injury. Additionally, Aßx-40 has emerged as a potential culprit in the ensuing development of post-ICU syndrome.

Bariatric surgery rapidly improves mitochondrial respiration in morbidly obese patients.

BACKGROUND: Obesity and its attendant comorbidities are an emerging epidemic. Chronic metabolic inflammation (metainflammation) is thought to precipitate obesity-associated morbidities; however, its mechanistic progression is poorly understood. Moreover, although interventions such as diet, exercise, and bariatric surgery can control body weight, their effects on metainflammation are also poorly understood. Recently, metainflammation and the pathobiology of obesity have been linked to mitochondrial dysfunction. Herein we examined the effects of bariatric surgery on mitochondrial respiration as an index of resolving metainflammation in morbidly obese patients. METHODS: This institutional review board-approved study involved morbidly obese patients (body mass index > 35 kg/m(2)) undergoing sleeve gastrectomy or Roux-en-Y gastric bypass. Mitochondrial respiration was assessed in peripheral blood monocytes and in skeletal muscle samples before surgery and at 12 weeks after surgery. Patient biometrics, homeostasis model assessment-estimated insulin resistance (HOMA-IR) score, C-reactive protein, and lipid profile were analyzed. RESULTS: Twenty patients were enrolled and showed an average percent excess body weight loss of 30.3% weight loss at 12 weeks after surgery. Average HOMA-IR score decreased from 3.0 to 1.2 in insulin-resistant patients. C-reactive protein, an index of metainflammation, showed a modest decrease. Lipid profile remained stable. Intriguingly, mitochondrial basal and maximal respiration rates in peripheral blood monocytes increased after surgery. Basal rates of skeletal muscle mitochondrial respiration were unchanged, but the maximal respiration rate trended toward an increase after surgery. CONCLUSIONS: Cellular and tissue mitochondrial respiration increased in a morbidly obese patient cohort after laparoscopic bariatric surgery. These changes were consistent in patients with postsurgical weight loss. Importantly, no significant changes or improvements occurred in canonical indices used to assess recovery after bariatric surgery over this short time course. Thus, increased mitochondrial respiration may represent a novel biomarker of early improvement and positive outcome after surgery in morbidly obese patients.

An approach for characterizing and comparing hyperspectral microscopy systems.

Hyperspectral imaging and analysis approaches offer accurate detection and quantification of fluorescently-labeled proteins and cells in highly autofluorescent tissues. However, selecting optimum acquisition settings for hyperspectral imaging is often a daunting task. In this study, we compared two hyperspectral systems-a widefield system with acoustic optical tunable filter (AOTF) and charge coupled device (CCD) camera, and a confocal system with diffraction gratings and photomultiplier tube (PMT) array. We measured the effects of system parameters on hyperspectral image quality and linear unmixing results. Parameters that were assessed for the confocal system included pinhole diameter, laser power, PMT gain and for the widefield system included arc lamp intensity, and camera gain. The signal-to-noise ratio (SNR) and the root-mean-square error (RMS error) were measured to assess system performance. Photobleaching dynamics were studied. Finally, theoretical sensitivity studies were performed to estimate the incremental response (sensitivity) and false-positive detection rates (specificity). Results indicate that hyperspectral imaging assays are highly dependent on system parameters and experimental conditions. For detection of green fluorescent protein (GFP)-expressing cells in fixed lung tissues, a confocal pinhole of five airy disk units, high excitation intensity and low detector gain were optimal. The theoretical sensitivity studies revealed that widefield hyperspectral microscopy was able to detect GFP with fewer false positive occurrences than confocal microscopy, even though confocal microscopy offered improved signal and noise characteristics. These studies provide a framework for optimization that can be applied to a variety of hyperspectral imaging systems.

Terminal sialic acids are an important determinant of pulmonary endothelial barrier integrity.

The surface of vascular endothelium bears a glycocalyx comprised, in part, of a complex mixture of oligosaccharide chains attached to cell-surface proteins and membrane lipids. Importantly, understanding of the structure and function of the endothelial glycocalyx is poorly understood. Preliminary studies have demonstrated structural differences in the glycocalyx of pulmonary artery endothelial cells compared with pulmonary microvascular endothelial cells. Herein we begin to probe in more detail structural and functional attributes of endothelial cell-surface carbohydrates. In this study we focus on the expression and function of sialic acids in pulmonary endothelium. We observed that, although pulmonary microvascular endothelial cells express similar amounts of total sialic acids as pulmonary artery endothelial cells, the nature of the sialic acid linkages differs between the two cell types such that pulmonary artery endothelial cells express both α(2,3)- and α(2,6)-linked sialic acids on the surface (i.e., surficially), whereas microvascular endothelial cells principally express α(2,3)-linked sialic acids. To determine whether sialic acids play a role in endothelial barrier function, cells were treated with neuraminidases to hydrolyze sialic acid moieties. Disruption of cell-cell and cell-matrix adhesions was observed following neuraminidase treatment, suggesting that terminal sialic acids promote endothelial barrier integrity. When we measured transendothelial resistance, differential responses of pulmonary artery and microvascular endothelial cells to neuraminidase from Clostridium perfringens suggest that the molecular architecture of the sialic acid glycomes differs between these two cell types. Collectively our observations reveal critical structural and functional differences of terminally linked sialic acids on the pulmonary endothelium.

Integrative Toolkit to Analyze Cellular Signals: Forces, Motion, Morphology, and Fluorescence.

Quantitative assessment of cellular forces and motion advanced considerably over the last four decades. These advancements provided the framework to examine insightful mechanical signaling processes in cell culture systems. However, the field currently faces three problems: lack of quality standardization of the acquired data, technical errors in data analysis and visualization, and perhaps most importantly, the technology remains largely out of reach for common cell biology laboratories. To overcome these limitations, we developed a new experimental platform - Integrative Toolkit to Analyze Cellular Signals (iTACS). iTACS consists of two components: Acquisition and Training Module (AcTrM) and Analysis and Visualization Module (AnViM). AcTrM is based on µManager - an NIH-ImageJ-based microscope control software - and facilitates user self-training and automation of common image acquisition protocols. AnViM is based on NIH-ImageJ and facilitates user-friendly automation of data analysis and insightful visualization of results. These experiments involve culturing adherent cells on hydrogels, imaging fiducial markers embedded in the hydrogel, and finally extracting from these images a comprehensive mechanical characterization of the cells. Currently, iTACS enables the user to analyze and track a wide array of properties, including morphology, motion, cytoskeletal forces, and fluorescence of individual cells and their neighboring region. The quality standardization issue was addressed in AcTrM with, a reference image-guided refocusing technique. The technical issues in data analysis were addressed in AnViM with a multi-pronged image segmentation procedure, a user-friendly approach to identify boundary conditions, and a novel cellular property-based data visualization. AcTrM is designed to facilitate the straightforward transformation of basic fluorescence microscopes into experimental cell mechanics rigs, and AnViM is equipped to enable users to measure cellular mechanical signals without requiring an engineering background. iTACS will be available to the research community as an open-source suite with community-driven development capabilities.

ExoU Induces Lung Endothelial Cell Damage and Activates Pro-Inflammatory Caspase-1 during Pseudomonas aeruginosa Infection.

The Gram-negative, opportunistic pathogen Pseudomonas aeruginosa utilizes a type III secretion system to inject exoenzyme effectors into a target host cell. Of the four best-studied exoenzymes, ExoU causes rapid cell damage and death. ExoU is a phospholipase A2 (PLA2) that hydrolyses host cell membranes, and P. aeruginosa strains expressing ExoU are associated with poor outcomes in critically ill patients with pneumonia. While the effects of ExoU on lung epithelial and immune cells are well studied, a role for ExoU in disrupting lung endothelial cell function has only recently emerged. Lung endothelial cells maintain a barrier to fluid and protein flux into tissue and airspaces and regulate inflammation. Herein, we describe a pulmonary microvascular endothelial cell (PMVEC) culture infection model to examine the effects of ExoU. Using characterized P. aeruginosa strains and primary clinical isolates, we show that strains expressing ExoU disrupt PMVEC barrier function by causing substantial PMVEC damage and lysis, in a PLA2-dependent manner. In addition, we show that strains expressing ExoU activate the pro-inflammatory caspase-1, in a PLA2-dependent manner. Considering the important roles for mitochondria and oxidative stress in regulating inflammatory responses, we next examined the effects of ExoU on reactive oxygen species production. Infection of PMVECs with P. aeruginosa strains expressing ExoU triggered a robust oxidative stress compared to strains expressing other exoenzyme effectors. We also provide evidence that, intriguingly, ExoU PLA2 activity was detectable in mitochondria and mitochondria-associated membrane fractions isolated from P. aeruginosa-infected PMVECs. Interestingly, ExoU-mediated activation of caspase-1 was partially inhibited by reactive oxygen species scavengers. Together, these data suggest ExoU exerts pleiotropic effects on PMVEC function during P. aeruginosa infection that may inhibit endothelial barrier and inflammatory functions.

Heterogeneous activation of p19Arf in pulmonary artery smooth muscle cells.

p19(ARF) is a tumor suppressor that leads to cell cycle arrest or apoptosis by stabilizing p53. p19(ARF) is not critical for cell cycle regulation under normal conditions, but loss of p19(ARF) is seen in many human cancers, and a murine p19(Arf) knockout model leads to malignant proliferation and tumor formation; its role in controlling nonmalignant proliferation is less defined. To examine this question, pulmonary artery smooth muscle cells (PASMC) were expanded in culture from a transgenic mouse in which the coding sequence of the p19(Arf) gene was replaced with a cDNA encoding green fluorescent protein (GFP), leaving the promoter intact. During the first 10 days in culture, wild-type, heterozygous, and knockout PASMC grew similarly, but, by day 14, p19(Arf)-deficient PASMC proliferated faster than p19(Arf) heterozygous or wild-type cells; reexpression of p19(Arf) prevented the increased proliferation. This time course correlated with activation of the p19(Arf) promoter, as indicated by the appearance of GFP positivity in p19(Arf)-deficient PASMC. By day 42, ∼80% of p19(Arf)-deficient cells were GFP-positive. When GFP-positive, p19(Arf)-deficient cells were sorted and subcultured separately, they remained GFP-positive, indicating that once cells had activated the p19(Arf) promoter, the promoter remained active in those and all subsequent daughter cells. In contrast, GFP-negative p19(Arf)-deficient cells gave rise to a combination of GFP-positive and -negative daughter cells over time. These results suggest that a subpopulation of PASMC are resistant to the signals that activate the p19(Arf) promoter, an event that would normally target these cells for arrest or cell death.

Monitoring physiology trainee needs to focus professional society responses: the APS Trainee Needs Surveys.

In 2004 and 2007, the American Physiological Society (APS) Trainee Advisory Committee (TAC) conducted surveys of graduate students, postdoctoral fellows, and new investigators in physiology to identify topics and issues important to those trainees. Two major trends emerged from the data. First, trainees in 2007 expressed somewhat greater interest in professional development information than did those in 2004. Second, needs expressed by trainees in both years were closely related to their specific career development stage. Survey findings guided the TAC and other APS committees and groups to focus their efforts toward the issues that were of the greatest interest to trainees. It also led to improved communication with trainees and increased involvement of trainees in APS governance.

Engineering Functional Vasculature in Decellularized Lungs Depends on Comprehensive Endothelial Cell Tropism.

Tissue engineering using decellularized whole lungs as matrix scaffolds began as a promise for creating autologous transplantable lungs for patients with end-stage lung disease and can also be used to study strategies for lung regeneration. Vascularization remains a critical component for all solid organ bioengineering, yet there has been limited success in generating functional re-endothelialization of most pulmonary vascular segments. We evaluated recellularization of the blood vessel conduits of acellular mouse scaffolds with highly proliferating, rat pulmonary microvascular endothelial progenitor cells (RMEPCs), pulmonary arterial endothelial cells (PAECs) or microvascular endothelial cells (MVECs). After 8 days of pulsatile perfusion, histological analysis showed that PAECs and MVECs possessed selective tropism for larger vessels or microvasculature, respectively. In contrast, RMEPCs lacked site preference and repopulated all vascular segments. RMEPC-derived endothelium exhibited thrombomodulin activity, expression of junctional genes, ability to synthesize endothelial signaling molecules, and formation of a restrictive barrier. The RMEPC phenotype described here could be useful for identifying endothelial progenitors suitable for efficient vascular organ and tissue engineering, regeneration and repair.