Effect of angiotensin-converting enzyme inhibitor and angiotensin receptor blocker initiation on organ support-free days in patients hospitalized with COVID-19

IMPORTANCE Overactivation of the renin-angiotensin system (RAS) may contribute to poor clinical outcomes in patients with COVID-19. Objective To determine whether angiotensin-converting enzyme (ACE) inhibitor or angiotensin receptor blocker (ARB) initiation improves outcomes in patients hospitalized for COVID-19. DESIGN, SETTING, AND PARTICIPANTS In an ongoing, adaptive platform randomized clinical trial, 721 critically ill and 58 non–critically ill hospitalized adults were randomized to receive an RAS inhibitor or control between March 16, 2021, and February 25, 2022, at 69 sites in 7 countries (final follow-up on June 1, 2022). INTERVENTIONS Patients were randomized to receive open-label initiation of an ACE inhibitor (n = 257), ARB (n = 248), ARB in combination with DMX-200 (a chemokine receptor-2 inhibitor; n = 10), or no RAS inhibitor (control; n = 264) for up to 10 days. MAIN OUTCOMES AND MEASURES The primary outcome was organ support–free days, a composite of hospital survival and days alive without cardiovascular or respiratory organ support through 21 days. The primary analysis was a bayesian cumulative logistic model. Odds ratios (ORs) greater than 1 represent improved outcomes. RESULTS On February 25, 2022, enrollment was discontinued due to safety concerns. Among 679 critically ill patients with available primary outcome data, the median age was 56 years and 239 participants (35.2%) were women. Median (IQR) organ support–free days among critically ill patients was 10 (–1 to 16) in the ACE inhibitor group (n = 231), 8 (–1 to 17) in the ARB group (n = 217), and 12 (0 to 17) in the control group (n = 231) (median adjusted odds ratios of 0.77 [95% bayesian credible interval, 0.58-1.06] for improvement for ACE inhibitor and 0.76 [95% credible interval, 0.56-1.05] for ARB compared with control). The posterior probabilities that ACE inhibitors and ARBs worsened organ support–free days compared with control were 94.9% and 95.4%, respectively. Hospital survival occurred in 166 of 231 critically ill participants (71.9%) in the ACE inhibitor group, 152 of 217 (70.0%) in the ARB group, and 182 of 231 (78.8%) in the control group (posterior probabilities that ACE inhibitor and ARB worsened hospital survival compared with control were 95.3% and 98.1%, respectively). CONCLUSIONS AND RELEVANCE In this trial, among critically ill adults with COVID-19, initiation of an ACE inhibitor or ARB did not improve, and likely worsened, clinical outcomes. TRIAL REGISTRATION ClinicalTrials.gov Identifier: NCT0273570

Correction: Phosphoproteomic analysis reveals Smad protein family activation following Rift Valley fever virus infection.

[This corrects the article DOI: 10.1371/journal.pone.0191983.]

Phosphoproteomic analysis reveals Smad protein family activation following Rift Valley fever virus infection

<div><p>Rift Valley fever virus (RVFV) infects both ruminants and humans leading to a wide variance of pathologies dependent on host background and age. Utilizing a targeted reverse phase protein array (RPPA) to define changes in signaling cascades after <i>in vitro</i> infection of human cells with virulent and attenuated RVFV strains, we observed high phosphorylation of Smad transcription factors. This evolutionarily conserved family is phosphorylated by and transduces the activation of TGF-β superfamily receptors. Moreover, we observed that phosphorylation of Smad proteins required active RVFV replication and loss of NSs impaired this activation, further corroborating the RPPA results. Gene promoter analysis of transcripts altered after RVFV infection identified 913 genes that contained a Smad-response element. Functional annotation of these potential Smad-regulated genes clustered in axonal guidance, hepatic fibrosis and cell signaling pathways involved in cellular adhesion/migration, calcium influx, and cytoskeletal reorganization. Furthermore, chromatin immunoprecipitation confirmed the presence of a Smad complex on the interleukin 1 receptor type 2 (IL1R2) promoter, which acts as a decoy receptor for IL-1 activation.</p></div

ChIP analysis of possible Smad-dependent transcripts.

<p>Lysates from mock, TGF-β activated, and MP12 infected were utilized for Smad4 (A) or methylated histone H3 lysine 4 (mH3K4; B) immunoprecipitations. Relative DNA abundance from immunoprecipitations was determined by qPCR. Signal over background normalization and fold enrichment was calculated for each condition. Bars represent means and standard deviations of four replicates. C) Levels of IL1R2, IL1RL1, and VAV3 RNA are analyzed by qRT-PCR. RNA lysates from HSAECs infected with MP12 (blue) or ZH548 (red) RVFV at an MOI 5 were utilized. Bars represent means and standard deviations of four replicates.</p

Summary of RPPA results.

<p>Summary of RPPA results.</p

Potential Smad-regulated genes selected for ChIP analysis.

<p>Potential Smad-regulated genes selected for ChIP analysis.</p

Promoter analysis of RVFV RNASeq yields potential Smad-dependent promoters.

<p>A) Schematic depicting data-mining of RVFV RNASeq dataset for Smad-dependent promoters. B) Top ten significantly altered canonical pathways from IPA are shown for the virulent RVFV strain at 18hpi. The top axis indicates the statistical significance calculated using the right-tailed Fisher exact test. Threshold line represents significance cut-off at <i>p = 0</i>.<i>05</i>. Bars represent the level of significance, with orange and blue bars indicating whether the pathway is predicted to be activated or inhibited, respectively (z-score). Pathways where no prediction can be made are colored gray. The ratio (bottom axis) represents the number of molecules identified in a given pathway divided by total number of molecules that constitute that pathway.</p

siRNA knockdown of Smad1, -2 and 4 does not impact RVFV replication.

<p>HSAECs transfected with media alone (Mock) or siRNAs (75nM) directed against a nontargeting control (NTC), Smad1, -2, or -4 were infected with MP12 virus (MOI 0.1). Both extracellular media supernatants (A) and protein lysates (B,C) were harvested at 9 and 24hpi. A) Infectious viral titers were determined by plaque assay. Data plotted as a bar graph of the mean with standard deviation of four replicates. Protein lysates from single (B) and double knockdowns (C) were analyzed by western blot for actin, total Smad1, -2, and -4 expression.</p

Viral kinetics of virulent and attenuated RVFV viruses.

<p>A) Schematic depicting infection and harvest conditions for comparative analysis. HSAECs were infected with attenuated (MP12) or virulent (ZH548) RVFV at an MOI 5 using conditioned media. RVFV RNA replication (B), percentage of RVFV NP positivity (C), and infectious viral titers (D) were determined at the indicated time points (hours post infection (hpi)). The data are plotted as means with standard deviations: for panel (B) n = 3, (C) n = 6 and (D) n = 9. ** = P≤0.01 and **** = P≤0.0001.</p

ChIP analysis of possible Smad-dependent transcripts.

<p>Lysates from mock, TGF-β activated, and MP12 infected were utilized for Smad4 (A) or methylated histone H3 lysine 4 (mH3K4; B) immunoprecipitations. Relative DNA abundance from immunoprecipitations was determined by qPCR. Signal over background normalization and fold enrichment was calculated for each condition. Bars represent means and standard deviations of four replicates. C) Levels of IL1R2, IL1RL1, and VAV3 RNA are analyzed by qRT-PCR. RNA lysates from HSAECs infected with MP12 (blue) or ZH548 (red) RVFV at an MOI 5 were utilized. Bars represent means and standard deviations of four replicates.</p