Cristhiaan D. Ochoa

Pseudomonas aeruginosa exotoxin Y is a promiscuous cyclase that increases endothelial Tau phosphorylation and permeability

Background: ExoY induces inter-endothelial gaps, although the mechanisms by which this occurs are poorly understood. Results: ExoY synthesized cAMP and cGMP, which caused endothelial Tau hyperphosphorylation, accumulation of insoluble Tau, inter-endothelial cell gaps, and increased permeability. Conclusion: ExoY is a promiscuous cyclase and an edema factor. Significance: Acute Pseudomonas infections cause a pathophysiological sequela in endothelium previously recognized only in chronic neurodegenerative diseases. Exotoxin Y (ExoY) is a type III secretion system effector found in ∼ 90% of the Pseudomonas aeruginosa isolates. Although it is known that ExoY causes inter-endothelial gaps and vascular leak, the mechanisms by which this occurs are poorly understood. Using both a bacteria-delivered and a codon-optimized conditionally expressed ExoY, we report that this toxin is a dual soluble adenylyl and guanylyl cyclase that results in intracellular cAMP and cGMP accumulation. The enzymatic activity of ExoY caused phosphorylation of endothelial Tau serine 214, accumulation of insoluble Tau, inter-endothelial cell gap formation, and increased macromolecular permeability. To discern whether the cAMP or cGMP signal was responsible for Tau phosphorylation and barrier disruption, pulmonary microvascular endothelial cells were engineered for the conditional expression of either wild-type guanylyl cyclase, which synthesizes cGMP, or a mutated guanylyl cyclase, which synthesizes cAMP. Sodium nitroprusside stimulation of the cGMP-generating cyclase resulted in transient Tau serine 214 phosphorylation and gap formation, whereas stimulation of the cAMP-generating cyclase induced a robust increase in Tau serine 214 phosphorylation, gap formation, and macromolecular permeability. These results indicate that the cAMP signal is the dominant stimulus for Tau phosphorylation. Hence, ExoY is a promiscuous cyclase and edema factor that uses cAMP and, to some extent, cGMP to induce the hyperphosphorylation and insolubility of endothelial Tau. Because hyperphosphorylated and insoluble Tau are hallmarks in neurodegenerative tauopathies such as Alzheimer disease, acute Pseudomonas infections cause a pathophysiological sequela in endothelium previously recognized only in chronic neurodegenerative diseases.

New Developments in Lung Endothelial Heterogeneity: von Willebrand Factor, P-Selectin, and the Weibel-Palade Body

Quiescent pulmonary endothelium establishes an antithrombotic, anti-inflammatory surface that promotes blood flow. However, the endothelium rapidly responds to injury and inflammation by promoting thrombosis and enabling the directed transmigration of inflammatory cells, such as neutrophils, into the alveolar airspace. Although the endothelial cell signals responsible for establishing a prothrombotic surface are distinct from those responsible for recognizing circulating neutrophils, these processes are highly interrelated. Von Willebrand factor (VWF)-stimulated secretion plays an important role in thrombus formation, and P-selectin surface expression plays a key role in neutrophil binding necessary for transmigration. Both VWF and P-selectin are located within Weibel-Palade bodies in pulmonary arteries and arterioles, yet Weibel-Palade bodies are absent in capillaries. Despite the absence of the Weibel-Palade bodies, pulmonary capillaries express both VWF and P-selectin. The physiological and pathophysiological significance of these observations is unclear. In this review, we address some anatomical and physiological features that distinguish pulmonary artery, capillary, and vein endothelium. In addition, we review our current understanding regarding the stimulated secretion of VWF and P-selectin in pulmonary artery and capillary endothelium. This information is considered in the context of vasculitis and pneumonia, two pathophysiological processes to which the stimulated secretion of VWF and P-selectin contribute.

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.

Cold exposure reveals two populations of microtubules in pulmonary endothelia

Microtubules are composed of α-tubulin and β-tubulin dimers. Microtubules yield tubulin dimers when exposed to cold, which reassemble spontaneously to form microtubule fibers at 37°C. However, mammalian neurons, glial cells, and fibroblasts have cold-stable microtubules. While studying the microtubule toxicity mechanisms of the exotoxin Y from Pseudomonas aeruginosa in pulmonary microvascular endothelial cells, we observed that some endothelial microtubules were very difficult to disassemble in the cold. As a consequence, we designed studies to test the hypothesis that microvascular endothelium has a population of cold-stable microtubules. Pulmonary microvascular endothelial cells and HeLa cells (control) were grown under regular cell culture conditions, followed by exposure to an ice-cold water bath and a microtubule extraction protocol. Polymerized microtubules were detected by immunofluorescence confocal microscopy and Western blot analyses. After cold exposure, immunofluorescence revealed that the majority of HeLa cell microtubules disassembled, whereas a smaller population of endothelial cell microtubules disassembled. Immunoblot analyses showed that microvascular endothelial cells express the microtubule cold-stabilizing protein N-STOP (neuronal stable tubule-only polypeptides), and that N-STOP binds to endothelial microtubules after cold exposure, but not if microtubules are disassembled with nocodazole before cold exposure. Hence, pulmonary endothelia have a population of cold-stable microtubules.

Pseudomonas aeruginosa Exotoxin Y-Mediated Tau Hyperphosphorylation Impairs Microtubule Assembly in Pulmonary Microvascular Endothelial Cells

Pseudomonas aeruginosa uses a type III secretion system to introduce the adenylyl and guanylyl cyclase exotoxin Y (ExoY) into the cytoplasm of endothelial cells. ExoY induces Tau hyperphosphorylation and insolubility, microtubule breakdown, barrier disruption and edema, although the mechanism(s) responsible for microtubule breakdown remain poorly understood. Here we investigated both microtubule behavior and centrosome activity to test the hypothesis that ExoY disrupts microtubule dynamics. Fluorescence microscopy determined that infected pulmonary microvascular endothelial cells contained fewer microtubules than control cells, and further studies demonstrated that the microtubule-associated protein Tau was hyperphosphorylated following infection and dissociated from microtubules. Disassembly/reassembly studies determined that microtubule assembly was disrupted in infected cells, with no detectable effects on either microtubule disassembly or microtubule nucleation by centrosomes. This effect of ExoY on microtubules was abolished when the cAMP-dependent kinase phosphorylation site (Ser-214) on Tau was mutated to a non-phosphorylatable form. These studies identify Tau in microvascular endothelial cells as the target of ExoY in control of microtubule architecture following pulmonary infection by Pseudomonas aeruginosa and demonstrate that phosphorylation of tau following infection decreases microtubule assembly.

Heterogeneity of pulmonary endothelial cyclic nucleotide response to Pseudomonas aeruginosa ExoY infection.

Here, we tested the hypothesis that a promiscuous bacterial cyclase synthesizes purine and pyrimidine cyclic nucleotides in the pulmonary endothelium. To test this hypothesis, pulmonary endothelial cells were infected with a strain of the Gram-negative bacterium Pseudomonas aeruginosa that introduces only exoenzyme Y (PA103 ΔexoUexoT::Tc pUCPexoY; ExoY(+)) via a type III secretion system. Purine and pyrimidine cyclic nucleotides were simultaneously detected using mass spectrometry. Pulmonary artery (PAECs) and pulmonary microvascular (PMVECs) endothelial cells both possess basal levels of four different cyclic nucleotides in the following rank order: cAMP > cUMP ≈ cGMP ≈ cCMP. Endothelial gap formation was induced in a time-dependent manner following ExoY(+) intoxication. In PAECs, intercellular gaps formed within 2 h and progressively increased in size up to 6 h, when the experiment was terminated. cGMP concentrations increased within 1 h postinfection, whereas cAMP and cUMP concentrations increased within 3 h, and cCMP concentrations increased within 4 h postinfection. In PMVECs, intercellular gaps did not form until 4 h postinfection. Only cGMP and cUMP concentrations increased at 3 and 6 h postinfection, respectively. PAECs generated higher cyclic nucleotide levels than PMVECs, and the cyclic nucleotide levels increased earlier in response to ExoY(+) intoxication. Heterogeneity of the cyclic nucleotide signature in response to P. aeruginosa infection exists between PAECs and PMVECs, suggesting the intracellular milieu in PAECs is more conducive to cNMP generation.

Pseudomonas aeruginosa exoenzymes U and Y induce a transmissible endothelial proteinopathy

We tested the hypothesis that Pseudomonas aeruginosa type 3 secretion system effectors exoenzymes Y and U (ExoY and ExoU) induce release of a high-molecular-weight endothelial tau, causing transmissible cell injury characteristic of an infectious proteinopathy. Both the bacterial delivery of ExoY and ExoU and the conditional expression of an activity-attenuated ExoU induced time-dependent pulmonary microvascular endothelial cell gap formation that was paralleled by the loss of intracellular tau and the concomitant appearance of high-molecular-weight extracellular tau. Transfer of the high-molecular-weight tau in filtered supernatant to naïve endothelial cells resulted in intracellular accumulation of tau clusters, which was accompanied by cell injury, interendothelial gap formation, decreased endothelial network stability in Matrigel, and increased lung permeability. Tau oligomer monoclonal antibodies captured monomeric tau from filtered supernatant but did not retrieve higher-molecular-weight endothelial tau and did not rescue the injurious effects of tau. Enrichment and transfer of high-molecular-weight tau to naïve cells was sufficient to cause injury. Thus we provide the first evidence for a pathophysiological stimulus that induces release and transmissibility of high-molecular-weight endothelial tau characteristic of an endothelial proteinopathy.

Estimating the magnitude of near-membrane PDE4 activity in living cells

Recent studies have demonstrated that functionally discrete pools of phosphodiesterase (PDE) activity regulate distinct cellular functions. While the importance of localized pools of enzyme activity has become apparent, few studies have estimated enzyme activity within discrete subcellular compartments. Here we present an approach to estimate near-membrane PDE activity. First, total PDE activity is measured using traditional PDE activity assays. Second, known cAMP concentrations are dialyzed into single cells and the spatial spread of cAMP is monitored using cyclic nucleotide-gated channels. Third, mathematical models are used to estimate the spatial distribution of PDE activity within cells. Using this three-tiered approach, we observed two pharmacologically distinct pools of PDE activity, a rolipram-sensitive pool and an 8-methoxymethyl IBMX (8MM-IBMX)-sensitive pool. We observed that the rolipram-sensitive PDE (PDE4) was primarily responsible for cAMP hydrolysis near the plasma membrane. Finally, we observed that PDE4 was capable of blunting cAMP levels near the plasma membrane even when 100 μM cAMP were introduced into the cell via a patch pipette. Two compartment models predict that PDE activity near the plasma membrane, near cyclic nucleotide-gated channels, was significantly lower than total cellular PDE activity and that a slow spatial spread of cAMP allowed PDE activity to effectively hydrolyze near-membrane cAMP. These results imply that cAMP levels near the plasma membrane are distinct from those in other subcellular compartments; PDE activity is not uniform within cells; and localized pools of AC and PDE activities are responsible for controlling cAMP levels within distinct subcellular compartments.

Accuracy of remote chest X-ray interpretation using Google Glass technology

OBJECTIVES We sought to explore the accuracy of remote chest X-ray reading using hands-free, wearable technology (Google Glass, Google, Mountain View, California). METHODS We compared interpretation of twelve chest X-rays with 23 major cardiopulmonary findings by faculty and fellows from cardiology, radiology, and pulmonary-critical care via: (1) viewing the chest X-ray image on the Google Glass screen; (2) viewing a photograph of the chest X-ray taken using Google Glass and interpreted on a mobile device; (3) viewing the original chest X-ray on a desktop computer screen. One point was given for identification of each correct finding and a subjective rating of user experience was recorded. RESULTS Fifteen physicians (5 faculty and 10 fellows) participated. The average chest X-ray reading score (maximum 23 points) as viewed through the Google Glass, Google Glass photograph on a mobile device, and the original X-ray viewed on a desktop computer was 14.1±2.2, 18.5±1.5 and 21.3±1.7, respectively (p<0.0001 between Google Glass and mobile device, p<0.0001 between Google Glass and desktop computer and p=0.0004 between mobile device and desktop computer). Of 15 physicians, 11 (73.3%) felt confident in detecting findings using the photograph taken by Google Glass as viewed on a mobile device. CONCLUSION Remote chest X-ray interpretation using hands-free, wearable technology (Google Glass) is less accurate than interpretation using a desktop computer or a mobile device, suggesting that further technical improvements are needed before widespread application of this novel technology.

ROS signaling and ER stress in cardiovascular disease

The endoplasmic reticulum (ER) produces the vast majority of all proteins secreted into the extracellular space, including hormones and cytokines, as well as cell surface receptors and other proteins which interact with the environment. Accordingly, this organelle controls essentially all vital links to a cells external milieu, responding to systemic metabolic, inflammatory, endocrine, and mechanical stimuli. The central role the ER plays in meeting protein synthetic and quality control requirements in the face of such demands is matched by an extensive and versatile ER stress response signaling network. ROS mediate several critical aspects of this response. Nox4, an ER resident capable of producing ROS, acts as a proximal signaling intermediate to transduce ER stress-related conditions to the unfolded protein response, a homeostatic corrective mechanism. However, chronic ER stress caused by unrelenting internal or external demands produces a secondary rise in ROS, generally resulting in cell death. Sorting out the involvement of ROS at different levels of the ER stress response in specific cell types is key to understanding the molecular basis for chronic diseases such as atherosclerosis, hypertension, and diabetes. Here, we provide an overview of ER stress signaling with an emphasis on the role of ROS.