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.

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.

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.

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.

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.

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.

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.

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.

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.

Ticagrelor Does Not Protect Isolated Rat Hearts, Thus Clouding Its Proposed Cardioprotective Role Through ENT 1 in Heart Tissue.

P2Y12 receptor-blocking drugs given at reperfusion offer protection against myocardial infarction in animal models of transient coronary occlusion. Two recent reports concluded that ticagrelor was more cardioprotective than clopidogrel and attributed this to ticagrelor's unique ability to raise tissue adenosine by blocking the equilibrative nucleoside transporter 1. Indeed, an adenosine receptor blocker attenuated ticagrelor's protection. The related P2Y12 inhibitor cangrelor, which does not block the transporter, protects hearts only when platelets are in the perfusate, while adenosine is known to protect equally in situ blood-perfused and crystalloid-perfused isolated hearts. We, therefore, tested whether ticagrelor liberates a sufficient amount of adenosine to protect a Krebs buffer-perfused isolated rat heart subjected to 40 minutes of global ischemia followed by 2 hours of reperfusion. In untreated hearts, 77.6% ± 4.0% of the ventricle was infarcted as measured by triphenyltetrazolium staining. Ischemically preconditioned hearts had only 32.7% ± 3.6% infarction ( P < .001 vs untreated), indicating that our model could be protected by preconditioning which is known to involve adenosine. Strikingly, hearts treated with 10 µmol/L ticagrelor in the buffer throughout the reperfusion period had 77.5% ± 2.4% infarction comparable to unprotected controls ( P = NS vs untreated). These data strongly suggest that ticagrelor was unable to release sufficient adenosine from the crystalloid-perfused rat heart to protect it against infarction. Our previous studies have found no difference in the anti-infarct potency among clopidogrel, cangrelor, and ticagrelor in open-chest rats and rabbits, and surprisingly adenosine receptor antagonists block protection from all 3 drugs. We have no explanation why ticagrelor is more protective in the pig than clopidogrel but suspect a species or perhaps a treatment schedule difference that may or may not involve adenosine.

Analysis of pulmonary vascular injury and repair during Pseudomonas aeruginosa infection-induced pneumonia and acute respiratory distress syndrome.

Herein we describe lung vascular injury and repair using a rodent model of Pseudomonas aeruginosa pneumonia-induced acute respiratory distress syndrome (ARDS) during: 1) the exudative phase (48-hour survivors) and 2) the reparative/fibro-proliferative phase (1-week survivors). Pneumonia was induced by intratracheal instillation of P. aeruginosa strain PA103, and lung morphology and pulmonary vascular function were determined subsequently. Pulmonary vascular function was assessed in mechanically ventilated animals in vivo (air dead space, PaO2, and lung mechanics) and lung permeability was determined in isolated perfused lungs ex vivo (vascular filtration coefficient and extravascular lung water). At 48 hours post infection, histological analyses demonstrated capillary endothelial disruption, diffuse alveolar damage, perivascular cuffs, and neutrophil influx into lung parenchyma. Infected animals displayed clinical hallmarks of ARDS, including increased vascular permeability, increased dead space, impaired gas exchange, and decreased lung compliance. Overall, the animal infection model recapitulated the morphological and functional changes typically observed in lungs from patients during the exudative phase of ARDS. At 1 week post infection, there was lung histological and pulmonary vascular functional evidence of repair when compared with 48 hours post infection; however, some parameters were still impaired when compared with uninfected controls. Importantly, lungs displayed increased fibrosis and cellular hyperplasia reminiscent of lungs from patients during the fibro-proliferative phase of ARDS. Control, sham inoculated animals showed normal lung histology and function. These data represent the first comprehensive assessment of lung pathophysiology during the exudative and reparative/fibro-proliferative phases of P. aeruginosa pneumonia-induced ARDS, and position this pre-clinical model for use in interventional studies aimed at advancing clinical care.

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.

Re-endothelialization of rat lung scaffolds through passive, gravity-driven seeding of segment-specific pulmonary endothelial cells.

Effective re-endothelialization is critical for the use of decellularized scaffolds for ex vivo lung engineering. Current approaches yield insufficiently re-endothelialized scaffolds that haemorrhage and become thrombogenic upon implantation. Herein, gravity-driven seeding coupled with bioreactor culture facilitated widespread distribution and engraftment of endothelial cells throughout rat lung scaffolds. Initially, human umbilical vein endothelial cells were seeded into the pulmonary artery by either gravity-driven, variable flow perfusion seeding or pump-driven, pulsatile flow perfusion seeding. Gravity seeding evenly distributed cells and supported cell survival and re-lining of the vascular walls while perfusion pump-driven seeding led to increased cell fragmentation and death. Using gravity seeding, rat pulmonary artery endothelial cells and rat pulmonary vein endothelial cells attached in intermediate and large vessels, while rat pulmonary microvascular endothelial cells deposited mostly in microvessels. Combination seeding of these cells led to positive vascular endothelial cadherin staining. In addition, combination seeding improved barrier function as assessed by serum albumin extravasation; however, leakage was observed in the distal portions of the re-endothelialized tissue suggesting that recellularization of the alveoli is necessary to complete barrier function of the capillary-alveolar network. Overall, these data indicate that vascular recellularization of rat lung scaffolds is achieved through gravity seeding. Copyright © 2016 John Wiley & Sons, Ltd.

The Highly Selective Caspase-1 Inhibitor VX-765 Provides Additive Protection Against Myocardial Infarction in Rat Hearts When Combined With a Platelet Inhibitor.

Use of ischemic postconditioning and other related cardioprotective interventions to treat patients with acute myocardial infarction (AMI) has failed to improve outcomes in clinical trials. Because P2Y12 inhibitors are themselves postconditioning mimetics, it has been postulated that the loading dose of platelet inhibitors routinely given to patients treated for AMI masks the anti-infarct effect of other intended cardioprotective interventions. To further improve outcomes of patients with AMI, an intervention must be able to provide additive protection in the presence of a P2Y12 platelet inhibitor. Previous studies reported an anti-infarct effect using a peptide inhibitor of the pro-inflammatory caspase-1 in animal models of AMI. Herein we tested whether a pharmacologic caspase-1 inhibitor can further limit infarct size in open-chest, anesthetized rats treated with a P2Y12 inhibitor. One hour occlusion of a coronary branch followed by 2 hours of reperfusion was used to simulate clinical AMI and reflow. One group of rats received an intravenous bolus of 16 mg/kg of the highly selective caspase-1 inhibitor VX-765 30 minutes prior to onset of ischemia. A second group received a 60 µg/kg intravenous bolus of the P2Y12 inhibitor cangrelor 10 minutes prior to reperfusion followed by 6 µg/kg/min continuous infusion. A third group received treatment with both inhibitors as above. Control animals received no treatment. Infarct size was measured by tetrazolium stain and volume of muscle at risk by fluorescent microspheres. In untreated hearts, 73.7% ± 4.1% of the ischemic zone infarcted. Treatment with either cangrelor or VX-765 alone reduced infarct size to 43.8% ± 2.4% and 39.6% ± 3.6% of the ischemic zone, respectively. Combining cangrelor and VX-765 was highly protective, resulting in only 14.0% ± 2.9% infarction. The ability of VX-765 to provide protection beyond that of a platelet inhibitor alone positions it as an attractive candidate therapy to further improve outcomes in today's patients with AMI.

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.

Evaluation of Microvascular Perfusion and Resuscitation after Severe Injury.

Achieving adequate perfusion is a key goal of treatment in severe trauma; however, tissue perfusion has classically been measured by indirect means. Direct visualization of capillary flow has been applied in sepsis, but application of this technology to the trauma population has been limited. The purpose of this investigation was to compare the efficacy of standard indirect measures of perfusion to direct imaging of the sublingual microcirculatory flow during trauma resuscitation. Patients with injury severity scores >15 were serially examined using a handheld sidestream dark-field video microscope. In addition, measurements were also made from healthy volunteers. The De Backer score, a morphometric capillary density score, and total vessel density (TVD) as cumulative vessel area within the image, were calculated using Automated Vascular Analysis (AVA3.0) software. These indices were compared against clinical and laboratory parameters of organ function and systemic metabolic status as well as mortality. Twenty severely injured patients had lower TVD (X = 14.6 ± 0.22 vs 17.66 ± 0.51) and De Backer scores (X = 9.62 ± 0.16 vs 11.55 ± 0.37) compared with healthy controls. These scores best correlated with serum lactate (TVD R(2) = 0.525, De Backer R(2) = 0.576, P < 0.05). Mean arterial pressure, heart rate, oxygen saturation, pH, bicarbonate, base deficit, hematocrit, and coagulation parameters correlated poorly with both TVD and De Backer score. Direct measurement of sublingual microvascular perfusion is technically feasible in trauma patients, and seems to provide real-time assessment of microcirculatory perfusion. This study suggests that in severe trauma, many indirect measurements of perfusion do not correlate with microvascular perfusion. However, visualized perfusion deficiencies do reflect a shift toward anaerobic metabolism.

Bariatric surgery improves the circulating numbers and biological activity of late outgrowth endothelial progenitor cells.

BACKGROUND: Although the salutary effects of bariatric surgery as a treatment for excess weight and type 2 diabetes are established, there is scant evidence for effects on other contributors to cardiovascular diseases such as repair of endothelial dysfunction. This study evaluates outcomes of bariatric surgery on late outgrowth endothelial progenitor cells (LOEPCs), a cell phenotype essential for endothelial repair. METHODS: Patients with a body mass index >35 kg/m(2) and type 2 diabetes were enrolled into either medical or bariatric surgical arms. Primary outcomes included analysis of isolated LOEPCs from peripheral blood for growth, function, and mitochondrial respiration. Plasma was used for metabolic profiling. RESULTS: Medical arm patients showed no improvement in any of the parameters tested. Bariatric surgical arm patients showed a 24% reduction in body mass index as early as 3 months postintervention and resolution of type 2 diabetes at 24 months postintervention (HbA1c 31% reduction; fasting glucose 29% reduction). Bariatric surgery increased the numbers of LOEPCs 8-fold and increased LOEPC network formation 3-fold at 24 months postintervention. The increased numbers and activity of LOEPCs in the bariatric surgical arm correlated with improvements in body mass index, insulin, and triglyceride levels only at 24 month postintervention. LOEPC mitochondrial respiration displayed a trend toward improvement compared with baseline as evidenced by an increase (36%) at 24 months in the bariatric arm. CONCLUSION: Bariatric surgery increases LOEPC levels and activity, which correlates with weight loss and improved metabolic profile at 24 months postintervention.

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.