A first update on mapping the human genetic architecture of COVID-19

peer reviewe

Heat shock protein 27 activity is linked to endothelial barrier recovery after proinflammatory GPCR-induced disruption

Vascular inflammation causes endothelial barrier disruption and tissue edema. Several inflammatory mediators act through G protein–coupled receptors (GPCRs), including protease-activated receptor-1 (PAR1), to elicit inflammatory responses. The activation of PAR1 by its ligand thrombin stimulates proinflammatory, p38 mitogen-activated protein kinase (MAPK) signaling that promotes endothelial barrier disruption. Through mass spectrometry phosphoproteomics, we identified heat shock protein 27 (HSP27), which exists as a large oligomer that binds to actin, as a promising candidate for the p38-mediated regulation of barrier integrity. Depletion of HSP27 by siRNA enhanced endothelial cell barrier permeability and slowed recovery after thrombin stimulation. We further showed that two effector kinases of p38 MAPK, MAPKAPK2 (MK2) and MAPKAPK3 (MK3), differentially phosphorylated HSP27 at Ser15, Ser78, and Ser82. Whereas inhibition of thrombin-stimulated p38 activation blocked HSP27 phosphorylation at all three sites, inhibition of MK2 reduced the phosphorylation of only Ser15 and Ser78. Inhibition of both MK2 and MK3 was necessary to attenuate Ser82 phosphorylation. Thrombin-stimulated p38-MK2-MK3 signaling induced HSP27 oligomer disassembly. However, a phosphorylation-deficient mutant of HSP27 exhibited defective oligomer disassembly and altered the dynamics of barrier recovery after thrombin stimulation. Moreover, blocking HSP27 oligomer reassembly with the small-molecule inhibitor J2 enhanced endothelial barrier permeability in vitro and vascular leakage in vivo in response to PAR1 activation. These studies reveal the distinct regulation of HSP27 phosphorylation and function induced by the GPCR-stimulated p38-MK2-MK3 signaling axis that controls the dynamics of endothelial barrier recovery in vitro and vascular leakage in vivo

Phosphoproteomic analysis of protease-activated receptor-1 biased signaling reveals unique modulators of endothelial barrier function.

Thrombin, a procoagulant protease, cleaves and activates protease-activated receptor-1 (PAR1) to promote inflammatory responses and endothelial dysfunction. In contrast, activated protein C (APC), an anticoagulant protease, activates PAR1 through a distinct cleavage site and promotes anti-inflammatory responses, prosurvival, and endothelial barrier stabilization. The distinct tethered ligands formed through cleavage of PAR1 by thrombin versus APC result in unique active receptor conformations that bias PAR1 signaling. Despite progress in understanding PAR1 biased signaling, the proteins and pathways utilized by thrombin versus APC signaling to induce opposing cellular functions are largely unknown. Here, we report the global phosphoproteome induced by thrombin and APC signaling in endothelial cells with the quantification of 11,266 unique phosphopeptides using multiplexed quantitative mass spectrometry. Our results reveal unique dynamic phosphoproteome profiles of thrombin and APC signaling, an enrichment of associated biological functions, including key modulators of endothelial barrier function, regulators of gene transcription, and specific kinases predicted to mediate PAR1 biased signaling. Using small interfering RNA to deplete a subset of phosphorylated proteins not previously linked to thrombin or APC signaling, a function for afadin and adducin-1 actin binding proteins in thrombin-induced endothelial barrier disruption is unveiled. Afadin depletion resulted in enhanced thrombin-promoted barrier permeability, whereas adducin-1 depletion completely ablated thrombin-induced barrier disruption without compromising p38 signaling. However, loss of adducin-1 blocked APC-induced Akt signaling. These studies define distinct thrombin and APC dynamic signaling profiles and a rich array of proteins and biological pathways that engender PAR1 biased signaling in endothelial cells

Phosphoproteomic analysis of thrombin- and p38 MAPK-regulated signaling networks in endothelial cells.

Endothelial dysfunction is a hallmark of inflammation and is mediated by inflammatory factors that signal through G protein-coupled receptors including protease-activated receptor-1 (PAR1). PAR1, a receptor for thrombin, signals via the small GTPase RhoA and myosin light chain intermediates to facilitate endothelial barrier permeability. PAR1 also induces endothelial barrier disruption through a p38 mitogen-activated protein kinase-dependent pathway, which does not integrate into the RhoA/MLC pathway; however, the PAR1-p38 signaling pathways that promote endothelial dysfunction remain poorly defined. To identify effectors of this pathway, we performed a global phosphoproteome analysis of thrombin signaling regulated by p38 in human cultured endothelial cells using multiplexed quantitative mass spectrometry. We identified 5491 unique phosphopeptides and 2317 phosphoproteins, four distinct dynamic phosphoproteome profiles of thrombin-p38 signaling, and an enrichment of biological functions associated with endothelial dysfunction, including modulators of endothelial barrier disruption and a subset of kinases predicted to regulate p38-dependent thrombin signaling. Using available antibodies to detect identified phosphosites of key p38-regulated proteins, we discovered that inhibition of p38 activity and siRNA-targeted depletion of the p38α isoform increased basal phosphorylation of extracellular signal-regulated protein kinase 1/2, resulting in amplified thrombin-stimulated extracellular signal-regulated protein kinase 1/2 phosphorylation that was dependent on PAR1. We also discovered a role for p38 in the phosphorylation of α-catenin, a component of adherens junctions, suggesting that this phosphorylation may function as an important regulatory process. Taken together, these studies define a rich array of thrombin- and p38-regulated candidate proteins that may serve important roles in endothelial dysfunction

Markers of Successful Extubation in Extremely Preterm Infants, and Morbidity After Failed Extubation

To identify variables associated with successful elective extubation, and to determine neonatal morbidities associated with extubation failure in extremely preterm neonates. This study was a secondary analysis of the National Institute of Child Health and Human Development Neonatal Research Network's Surfactant, Positive Pressure, and Oxygenation Randomized Trial that included extremely preterm infants born at 240/7 to 276/7 weeks' gestation. Patients were randomized either to a permissive ventilatory strategy (continuous positive airway pressure group) or intubation followed by early surfactant (surfactant group). There were prespecified intubation and extubation criteria. Extubation failure was defined as reintubation within 5 days of extubation. Of 1316 infants in the trial, 1071 were eligible; 926 infants had data available on extubation status; 538 were successful and 388 failed extubation. The rate of successful extubation was 50% (188/374) in the continuous positive airway pressure group and 63% (350/552) in the surfactant group. Successful extubation was associated with higher 5-minute Apgar score, and pH prior to extubation, lower peak fraction of inspired oxygen within the first 24 hours of age and prior to extubation, lower partial pressure of carbon dioxide prior to extubation, and non-small for gestational age status after adjustment for the randomization group assignment. Infants who failed extubation had higher adjusted rates of mortality (OR 2.89), bronchopulmonary dysplasia (OR 3.06), and death/ bronchopulmonary dysplasia (OR 3.27). Higher 5-minute Apgar score, and pH prior to extubation, lower peak fraction of inspired oxygen within first 24 hours of age, lower partial pressure of carbon dioxide and fraction of inspired oxygen prior to extubation, and nonsmall for gestational age status were associated with successful extubation. Failed extubation was associated with significantly higher likelihood of mortality and morbidities. ClinicalTrials.gov: NCT00233324

Growth Rates of Infants Randomized to Continuous Positive Airway Pressure or Intubation After Extremely Preterm Birth.

Objective To evaluate the effects of early treatment with continuous positive airway pressure (CPAP) on nutritional intake and in-hospital growth rates of extremely preterm (EPT) infants. Study design EPT infants (240/7-276/7 weeks of gestation) enrolled in the Surfactant Positive Airway Pressure and Pulse Oximetry Trial (SUPPORT) were included. EPT infants who died before 36 weeks of postmenstrual age (PMA) were excluded. The growth rates from birth to 36 weeks of PMA and follow-up outcomes at 18-22 months corrected age of EPT infants randomized at birth to either early CPAP (intervention group) or early intubation for surfactant administration (control group) were analyzed. Results Growth data were analyzed for 810 of 1316 infants enrolled in SUPPORT (414 in the intervention group, 396 in the control group). The median gestational age was 26 weeks, and the mean birth weight was 839 g. Baseline characteristics, total nutritional intake, and in-hospital comorbidities were not significantly different between the 2 groups. In a regression model, growth rates between birth and 36 weeks of PMA, as well as growth rates during multiple intervals from birth to day 7, days 7-14, days 14-21, days 21-28, day 28 to 32 weeks PMA, and 32-36 weeks PMA did not differ between treatment groups. Independent of treatment group, higher growth rates from day 21 to day 28 were associated with a lower risk of having a Bayley-III cognitive score Conclusions EPT infants randomized to early CPAP did not have higher in-hospital growth rates than infants randomized to early intubation

COVID-19 Host Genetics Initiative. A first update on mapping the human genetic architecture of COVID-19

The COVID-19 pandemic continues to pose a major public health threat, especially in countries with low vaccination rates. To better understand the biological underpinnings of SARS-CoV-2 infection and COVID-19 severity, we formed the COVID-19 Host Genetics Initiative1. Here we present a genome-wide association study meta-analysis of up to 125,584 cases and over 2.5 million control individuals across 60 studies from 25 countries, adding 11 genome-wide significant loci compared with those previously identified2. Genes at new loci, including SFTPD, MUC5B and ACE2, reveal compelling insights regarding disease susceptibility and severity.</p