A time-resolved proteomic and prognostic map of COVID-19.

COVID-19 is highly variable in its clinical presentation, ranging from asymptomatic infection to severe organ damage and death. We characterized the time-dependent progression of the disease in 139 COVID-19 inpatients by measuring 86 accredited diagnostic parameters, such as blood cell counts and enzyme activities, as well as untargeted plasma proteomes at 687 sampling points. We report an initial spike in a systemic inflammatory response, which is gradually alleviated and followed by a protein signature indicative of tissue repair, metabolic reconstitution, and immunomodulation. We identify prognostic marker signatures for devising risk-adapted treatment strategies and use machine learning to classify therapeutic needs. We show that the machine learning models based on the proteome are transferable to an independent cohort. Our study presents a map linking routinely used clinical diagnostic parameters to plasma proteomes and their dynamics in an infectious disease

A time-resolved proteomic and prognostic map of COVID-19

COVID-19 is highly variable in its clinical presentation, ranging from asymptomatic infection to severe organ damage and death. We characterized the time-dependent progression of the disease in 139 COVID-19 inpatients by measuring 86 accredited diagnostic parameters, such as blood cell counts and enzyme activities, as well as untargeted plasma proteomes at 687 sampling points. We report an initial spike in a systemic inflammatory response, which is gradually alleviated and followed by a protein signature indicative of tissue repair, metabolic reconstitution, and immunomodulation. We identify prognostic marker signatures for devising risk-adapted treatment strategies and use machine learning to classify therapeutic needs. We show that the machine learning models based on the proteome are transferable to an independent cohort. Our study presents a map linking routinely used clinical diagnostic parameters to plasma proteomes and their dynamics in an infectious disease

Regulation and Dysregulation of Endothelial Permeability during Systemic Inflammation

Systemic inflammation can be triggered by infection, surgery, trauma or burns. During systemic inflammation, an overshooting immune response induces tissue damage resulting in organ dysfunction and mortality. Endothelial cells make up the inner lining of all blood vessels and are critically involved in maintaining organ integrity by regulating tissue perfusion. Permeability of the endothelial monolayer is strictly controlled and highly organ-specific, forming continuous, fenestrated and discontinuous capillaries that orchestrate the extravasation of fluids, proteins and solutes to maintain organ homeostasis. In the physiological state, the endothelial barrier is maintained by the glycocalyx, extracellular matrix and intercellular junctions including adherens and tight junctions. As endothelial cells are constantly sensing and responding to the extracellular environment, their activation by inflammatory stimuli promotes a loss of endothelial barrier function, which has been identified as a hallmark of systemic inflammation, leading to tissue edema formation and hypotension and thus, is a key contributor to lethal outcomes. In this review, we provide a comprehensive summary of the major players, such as the angiopoietin-Tie2 signaling axis, adrenomedullin and vascular endothelial (VE-) cadherin, that substantially contribute to the regulation and dysregulation of endothelial permeability during systemic inflammation and elucidate treatment strategies targeting the preservation of vascular integrity

TRP Channels as Sensors of Aldehyde and Oxidative Stress

The transient receptor potential (TRP) cation channel superfamily comprises more than 50 channels that play crucial roles in physiological processes. TRP channels are responsive to several exogenous and endogenous biomolecules, with aldehydes emerging as a TRP channel trigger contributing to a cellular cascade that can lead to disease pathophysiology. The body is not only exposed to exogenous aldehydes via tobacco products or alcoholic beverages, but also to endogenous aldehydes triggered by lipid peroxidation. In response to lipid peroxidation from inflammation or organ injury, polyunsaturated fatty acids undergo lipid peroxidation to aldehydes, such as 4-hydroxynonenal. Reactive aldehydes activate TRP channels via aldehyde-induced protein adducts, leading to the release of pro-inflammatory mediators driving the pathophysiology caused by cellular injury, including inflammatory pain and organ reperfusion injury. Recent studies have outlined how aldehyde dehydrogenase 2 protects against aldehyde toxicity through the clearance of toxic aldehydes, indicating that targeting the endogenous aldehyde metabolism may represent a novel treatment strategy. An addition approach can involve targeting specific TRP channel regions to limit the triggering of a cellular cascade induced by aldehydes. In this review, we provide a comprehensive summary of aldehydes, TRP channels, and their interactions, as well as their role in pathological conditions and the different therapeutical treatment options

Protein Z Exerts Pro-Angiogenic Effects and Upregulates CXCR4

<div><p>Objective</p><p>Protein Z (PZ) is a vitamin K-dependent coagulation factor without catalytic activity. Evidence points towards PZ as an independent risk factor for the occurrence of human peripheral arterial disease. However, the role of PZ in ischemia-driven angiogenesis and vascular healing processes has not been elucidated so far.</p><p>Approach</p><p>Angiogenic potency of PZ was assessed in established <i>in vitro</i> assays using endothelial cells. PZ-deficient (PZ^−/−) mice and their wild-type littermates (PZ^+/+) were subjected to hindlimb ischemia. Furthermore, PZ^−/− mice were exposed to PZ expressing adenovirus (AdV-PZ) or control adenovirus (AdV-GFP). In an additional set of animals, PZ^−/− mice were exposed to AdV-PZ and AdV-GFP, each in combination with the CXCR4 antagonist AMD3100.</p><p>Results</p><p><i>In vitro</i>, PZ stimulated migratory activity and capillary-like tube formation of endothelial cells comparable to SDF-1. PZ^−/− mice exhibited diminished hypoxia-driven neovascularization and reperfusion in post-ischemic hindlimbs, which was restored by adenoviral gene transfer up to levels seen in PZ^+/+ mice. The stimulatory impact of PZ on endothelial cells <i>in vitro</i> was abolished by siRNA targeting against PZ and PZ was not able to restore reduced migration after knock-down of CXCR4. The increased surface expression of CXCR4 on PZ-stimulated endothelial cells and the abrogated restoration of PZ^−/− mice via AdV-PZ after concomitant treatment with the CXCR4 antagonist AMD3100 supports the idea that PZ mediates angiogenesis via a G-protein coupled pathway and involves the SDF-1/CXCR4 axis. This is underlined by the fact that addition of the G-protein inhibitor PTX to PZ-stimulated endothelial cells abolished the effect of PZ on capillary-like tube formation.</p><p>Conclusions</p><p>The results of the current study reveal a role of PZ in ischemia-induced angiogenesis, which involves a G-protein coupled pathway and a raised surface expression of CXCR4. Our findings thereby extend the involvement of PZ from the coagulation cascade to a beneficial modulation of vascular homeostasis.</p></div

Efficiency and safety of inhalative sedation with sevoflurane in comparison to an intravenous sedation concept with propofol in intensive care patients: study protocol for a randomized controlled trial

Abstract Background State of the art sedation concepts on intensive care units (ICU) favor propofol for a time period of up to 72 h and midazolam for long-term sedation. However, intravenous sedation is associated with complications such as development of tolerance, insufficient sedation quality, gastrointestinal paralysis, and withdrawal symptoms including cognitive deficits. Therefore, we aimed to investigate whether sevoflurane as a volatile anesthetic technically implemented by the anesthetic-conserving device (ACD) may provide advantages regarding ‘weaning time’, efficiency, and patient’s safety when compared to standard intravenous sedation employing propofol. Method/Design This currently ongoing trial is designed as a two-armed, monocentric, randomized prospective phase II study including intubated intensive care patients with an expected necessity for sedation exceeding 48 h. Patients are randomly assigned to either receive intravenous sedation with propofol or sevoflurane employing the ACD. Primary endpoint is the comparison of the ‘weaning time’ defined as the time required from discontinuation of the sedating agent until sufficient spontaneous breathing occurs. Moreover, sedation depth evaluated by Richmond Agitation Sedation Scale and parameters of patient’s safety (that is, vital signs, laboratory monitoring of organ function) as well as the duration of mechanical ventilation and overall stay on the ICU are analyzed and compared. An intention-to-treat analysis will be carried out with all patients for whom it will be possible to define a wake-up time. In addition, a per-protocol analysis is envisaged. Completion of patient recruitment is expected by the end of 2012. Discussion This clinical study is designed to evaluate the impact of sevoflurane during long-term sedation of critically ill patients on ‘weaning time’, efficiency, and patient’s safety compared to the standard intravenous sedation concept employing propofol. Trial registration EudraCT2007-006087-30; ISCRTN90609144</p

Targeting PZ and CXCR4 with siRNA results in decreased migratory capacity and endothelial tube formation <i>in vitro</i>.

<p>PZ and CXCR4 knock-down in unstimulated HUVECs as well as CXCR4 knock-down in PZ and SDF-1 stimulated HUVECs resulted in migration capacity comparable to that of ctrl after both 8 (<b>A</b>) and 24 hours (<b>B</b>). Data are given as box plots indicating the median with the 25^th and 75^th percentiles. Ø corresponds to unstimulated cells. ANOVA on ranks; $p<0.05 vs. ctrl; n = 4 independent experiments. Representative images (<b>C</b>) and quantitative analysis (<b>D</b>) of capillary-like tube formation. HUVECs targeted with siRNA against PZ or CXCR4 showed significantly reduced capacity for capillary-like tube formation compared to ctrl. Addition of PZ or SDF-1 to CXCR4 siRNA targeted cells failed to restore endothelial tube formation. Data are given as box plots indicating the median with the 25^th and 75^th percentiles. Ø corresponds to unstimulated cells. ANOVA on ranks;$ p<0.05 vs. ctrl; n = 4 independent experiments.</p

PZ promotes the migration and capillary-like tube formation of endothelial cells <i>in vitro</i>.

<p><b>A</b>, Representative images of scratch-wound closures after 8 and 24 hours of incubation with SDF-1 or PZ, ctrl corresponds to untreated cells. 100-fold magnification. Exposure of endothelial cells to PZ (3 µg/ml) led to a significantly higher wound closure after 8 (<b>B</b>) and 24 hours (<b>C</b>) comparable to stimulation with SDF-1 (50 ng/ml). Data are given as box plots indicating the median with the 25^th and 75^th percentiles. ANOVA on ranks; $p<0.05 vs. ctrl; n = 4–7 independent experiments. <b>D</b>, Representative images of tubular networks after 8 hours of incubation in matrigel angiogenesis assay in the presence of SDF-1 (50 ng/ml), PZ (3 µg/ml), PZ+PTX and PTX alone (100 ng/ml), ctrl are untreated cells. 100-fold magnification. <b>E</b>, HUVECs showed significantly enhanced formation of capillary-like tubular structures on Matrigel when incubated with SDF-1 and PZ compared with ctrl. Coincubation of endothelial cells with PZ and PTX abolished the PZ-mediated increase in tube formation, while incubation with PTX alone had no effect on tube formation. Data are given as box plots indicating the median with the 25^th and 75^th percentiles. ANOVA on ranks;$ p<0.05 vs. ctrl; ß p<0.05 vs. PZ; n = 6 independent experiments.</p

PZ upregulates CXCR4 surface expression on endothelial cells <i>in vitro</i> and mediates its angiogenic effects via CXCR4 <i>in vivo</i>.

<p>Flow cytometric analysis of CXCR4 surface expression on HUVECs after 8 and 24 hours of incubation with PZ (red line), displayed in representative histograms (<b>A</b> and <b>C</b>). Quantitative analysis (<b>B</b> and <b>D</b>) of CXCR4 expression. Stimulation with PZ resulted in an 1.4-fold (B) and almost 2-fold (D) increase of CXCR4 expression vs. unstimulated cells (ctrl, black line). Data are given as box plots indicating the median with the 25^th and 75^th percentiles. ANOVA on ranks; p<0.05; $vs. ctrl; n = 4–8 independent experiments. ANOVA on ranks; p<0.05;$ vs. ctrl; n = 4–8 independent experiments. <b>E</b>, Representative images of confocal laser scanning microscopy of HUVECs stimulated for 8 or 24 hours with SDF-1 (50 ng/ml) or PZ (3 µg/ml), displaying an increased surface expression of CXCR4 after stimulation with both substances. <b>F</b>, Representative pictures of thermal imaging of mice hindlimbs. <b>G</b>, Quantitative summary of pad temperature differences pre OP, post OP and on POD 21. In both groups treated with AMD3100 no significant change in temperature difference on POD 21 was detectable. Data are given as box plots indicating the median with the 25^th and 75^th percentiles. ANOVA on ranks repeated measures; * p<0.05 vs. pre OP; n = 3–6. <b>H</b>, Representative pictures of M. gastrocnemius after immunofluorescent staining for CD31 (red) or cell nuclei (DAPI, blue). <b>I</b>, Quantitative summary of enumbered CD31/DAPI double positive cells revealed no significant increase after induction of ischemia in both groups. Data are given as box plots indicating the median with the 25^th and 75^th percentiles. ANOVA on ranks; n = 3–6.</p