key: cord-0728337-03dxeo4f
authors: Leisman, Daniel E.; Mehta, Arnav; Thompson, B. Taylor; Charland, Nicole C.; Gonye, Anna L. K.; Gushterova, Irena; Kays, Kyle R.; Khanna, Hargun K.; LaSalle, Thomas J.; Lavin-Parsons, Kendall M.; Lilley, Brendan M.; Lodenstein, Carl L.; Manakongtreecheep, Kasidet; Margolin, Justin D.; McKaig, Brenna N.; Rojas-Lopez, Maricarmen; Russo, Brian C.; Sharma, Nihaarika; Tantivit, Jessica; Thomas, Molly F.; Parry, Blair Alden; Villani, Alexandra-Chloé; Sade-Feldman, Moshe; Hacohen, Nir; Filbin, Michael R.; Goldberg, Marcia B.
title: Alveolar, Endothelial, and Organ Injury Marker Dynamics in Severe COVID-19
date: 2021-12-08
journal: American journal of respiratory and critical care medicine
DOI: 10.1164/rccm.202106-1514oc
sha: 1df530934d05842688a5353e1458bc17f60d57f4
doc_id: 728337
cord_uid: 03dxeo4f

RATIONALE: Alveolar and endothelial injury may be differentially associated with coronavirus disease (COVID-19) severity over time. OBJECTIVES: To describe alveolar and endothelial injury dynamics and associations with COVID-19 severity, cardiorenovascular injury, and outcomes. METHODS: This single-center observational study enrolled patients with COVID-19 requiring respiratory support at emergency department presentation. More than 40 markers of alveolar (including receptor for advanced glycation endproducts [RAGE]), endothelial (including angiopoietin-2), and cardiorenovascular injury (including renin, kidney injury molecule-1, and troponin-I) were serially compared between invasively and spontaneously ventilated patients using mixed-effects repeated-measures models. Ventilatory ratios were calculated for intubated patients. Associations of biomarkers with modified World Health Organization scale at Day 28 were determined with multivariable proportional-odds regression. MEASUREMENTS AND MAIN RESULTS: Of 225 patients, 74 (33%) received invasive ventilation at Day 0. RAGE was 1.80-fold higher in invasive ventilation patients at Day 0 (95% confidence interval [CI], 1.50–2.17) versus spontaneous ventilation, but decreased over time in all patients. Changes in alveolar markers did not correlate with changes in endothelial, cardiac, or renal injury markers. In contrast, endothelial markers were similar to lower at Day 0 for invasive ventilation versus spontaneous ventilation, but then increased over time only among intubated patients. In intubated patients, angiopoietin-2 was similar (fold difference, 1.02; 95% CI, 0.89–1.17) to nonintubated patients at Day 0 but 1.80-fold higher (95% CI, 1.56–2.06) at Day 3; cardiorenovascular injury markers showed similar patterns. Endothelial markers were not consistently associated with ventilatory ratios. Endothelial markers were more often significantly associated with 28-day outcomes than alveolar markers. CONCLUSIONS: Alveolar injury markers increase early. Endothelial injury markers increase later and are associated with cardiorenovascular injury and 28-day outcome. Alveolar and endothelial injury likely contribute at different times to disease progression in severe COVID-19.

Severe coronavirus disease (COVID- 19) pneumonia frequently involves progression to hypoxemic respiratory failure and acute respiratory distress syndrome (ARDS). ARDS is a heterogenous syndrome (1, 2) . In patients with a direct insult etiology (e.g., pneumonia, aspiration), ARDS is associated with higher plasma levels of pulmonary epithelial injury markers (3), whereas with indirect insults (e.g., sepsis, trauma, etc.), ARDS displays higher endothelial injury markers (3) . Given that severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) causes pneumonia, an alveolar injury-predominant phenotype might be expected, yet the high prevalence of venous thromboembolism and shunt physiology (4) (5) (6) (7) and reported postmortem pulmonary endothelialitis (8) suggest significant endothelial dysfunction.

Whether epithelial and endothelial markers are indicative of clinical progression, extrapulmonary organ dysfunction, and/or patient outcomes in SARS-CoV-2 infection is unknown. Direct comparisons of epithelial and endothelial injury patterns are limited, and longitudinal analyses are lacking. Understanding their dynamics over time would facilitate biological interpretation of clinical trial results. We investigated the evolution of SARS-CoV-2 illness using serially measured pulmonary epithelial, endothelial, and organ injury markers in a cohort of prospectively enrolled patients with COVID-19 presenting with respiratory distress. We hypothesized these markers would display distinct associations with clinical variables and would vary over time in ways that reflect the dynamics of underlying disease biology.

Some of the results have been previously reported in the form of an abstract (9) .

We performed a secondary analysis of a prospectively enrolled observational cohort of patients with severe COVID-19, with three objectives: 1) describe epithelial versus endothelial markers over time by level of respiratory support; 2) determine whether these markers correlate with markers of systemic inflammation, renin-angiotensin system (RAS) activation, and nonpulmonary organ dysfunction; and 3) determine whether epithelial and endothelial markers associate with 28-day patient outcomes. The study was designed after enrollment had completed but without consideration of the results of the primary cohort analysis.

Patients were enrolled in the emergency department of an urban academic hospital in Boston, Massachusetts during the peak of the initial COVID-19 surge (March 24 to April 30, 2020) as described (10) . The institutional review board waived informed consent. Inclusion criteria were age > 18 years, clinical concern for COVID-19, and one or more of the following: 1) respirations > 22 breaths/min; 2) oxygen saturation as measured by pulse oximetry (Sp O 2 ) < 92% on room air; 3) supplemental oxygen requirement; or 4) positive-pressure ventilation. For this study, we excluded enrolled patients without COVID-19 subsequently confirmed by PCR or not requiring respiratory support, defined as supplemental oxygen or invasive mechanical ventilation. Per hospital policy during the study period, no patients received noninvasive mechanical ventilation because of aerosolization concerns.

Subjects contributed dedicated research blood samples with their initial clinical blood draw on Day 0 and, if still hospitalized, at Day 3 and Day 7. Clinical course was followed for 28 days after enrollment or until discharge, whichever occurred later. Recorded clinical data included demographics, comorbidities, home medications, presenting symptoms, serial vital signs, laboratory values, Sequential Organ Failure Assessment (SOFA) score, and World Health Organization (WHO) scale. Organ dysfunction criteria are described in the online supplement.

Patients were categorized by respiratory support level based on WHO scale. Among intubated patients, dead space ventilation was estimated by ventilatory ratio (11) found that alveolar injury markers peaked early, whereas endothelial injury markers increased later and were associated with renin-angiotensin system dysfunction, cardiorenovascular injury, and 28-day outcome. These results suggest alveolar and endothelial injury contribute at different times to disease progression in severe COVID-19. weight 3 100 3 37.5). Ventilatory ratios >2.0 were considered high (11) .

Specimen processing, banking, and assay were as described (10) . Plasma biomarkers were measured using the Olink proximity extension assay, an oligonucleotide-labeled antibody assay for high-specificity high-dimensional multiplex that allows measurement of low-abundance proteins. Because the signal is amplified by PCR, measurements are expressed in normalized protein expression units, reflecting relative abundance on a log2 scale, rather than absolute concentration.

This assay allows within-analyte comparisons between different samples but not between-analyte comparisons. For example, angiopoietin-2 levels between two different patients can be compared, but angiopoietin-2 level cannot be directly compared with IL-8 level in the same patient. Analytical performance validation for each protein assay is available at https://www.olink.com/.

We a priori selected markers from the Olink protein library based on prior literature (12) and hypothesized importance. Markers were selected without consideration of the results of subsequently published analyses of this cohort. To facilitate interrogation of alveolar, club cell, and endothelial injury; systemic inflammation; RAS activation; and nonpulmonary organ injury, the selected analytes included cell-type-specific markers (see Table E1 in the online supplement). To confirm specificity, biomarkers were crossreferenced against the Human Protein Atlas: Tissue and Blood Atlases (www.proteinatlas. org) (13) .

Detailed methods are in the online supplement. Briefly, to study how respiratory illness severity influenced each marker's 7-day course, we constructed mixed-effects repeated measures models, adjusting for age, sex, and chronic heart, lung, and kidney disease. To account for within-subject correlation, models included a random effect for subject with study day treated categorically as a repeated measure. Class variables for respiratory support level and interaction terms for day and support level were included.

To ensure results were robust and consistent, each biomarker was analyzed with three different approaches to specifying the respiratory support variable. First, a binary variable was used for whether the patient was invasively ventilated or deceased at that time point versus not invasively ventilated and alive (strategy-1). Second, respiratory support was treated as a categorical variable with three levels: invasively ventilated or deceased, alive and requiring supplemental oxygen, or alive without respiratory support (strategy-2). Third, the respiratory status variable was kept fixed based on Day 0 status: invasively ventilated or requiring supplemental oxygen (strategy-3). As additional sensitivity analyses, we evaluated representative markers under complete-case analysis and when excluding patients who received steroids or tocilizumab.

C-statistics were calculated to measure marker discrimination for intubation or death at Days 0 and 3.

To determine the association of each marker at Days 0 and 3 with Day 28 clinical status, we used multivariable proportionalodds regression, with clinical status a threelevel categorical variable: 1) died; 2) invasive ventilation; and 3) off invasive respiratory support. Covariates were age, sex, Day 0 SOFA, and chronic heart, lung, and kidney disease. Models assessing Day 3 biomarker levels' association with clinical status included an adjustment for the Day 0 biomarker level. The proportional-odds assumption was assessed graphically in each case and found to be reasonable. We report models in ascending format, such that a higher odds ratio (OR) indicates higher probability of worse outcome at Day 28.

Analyses were done in SAS (SAS Institute). Figures were produced in SAS or Prism (GraphPad Inc.).

Among 384 enrolled patients, 274 required respiratory support at Day 0. Forty-nine of these proved to not have COVID-19, yielding 225 for the current longitudinal analysis ( Figure E1 ). At Day 0, 151 (67%) subjects received supplemental oxygen only, and 74 (33%) received invasive ventilation (Table 1 ). Attrition at Day 3 was minimal: 10 (4%) patients died and 10 (4%) were discharged ( Figure 1A ). By Day 28, 37 (16%) patients had died, 37 (16%) continued receiving invasive ventilation, and 148 (68%) had been discharged ( Figure 1A ). Severe hypoxemia was most prevalent on Day 0, whereas severe abnormalities of renal, coagulation, and circulatory function became more prevalent over time (Figure 1B-1F; missing data prevalence in Tables E2 and E3) .

Among patients intubated on Day 0, hypoxemia generally improved over time. Although ventilatory ratios also increased over time (Figure 2 ), suggesting increasing dead space ventilation, a minority of patients reached a high ventilatory ratio (>2.0), increasing from 7 (9%) on Day 0 to 23 (31%) by Day 7.

Day 0 alveolar injury markers were generally higher in patients requiring invasive ventilation than those requiring only supplemental oxygen (Figures 3, 4 , E2, and E3). Although remaining higher among invasively ventilated patients, these markers decreased over time in both groups ( Figure 4 ; modeling strategy-2), and the differences between the two became less pronounced ( Figure 3 ; modeling strategy-1). This pattern was consistent across the alveolar injury markers receptor for advanced glycation endproducts (RAGE) and surfactant proteins A1 and A2, but not surfactant protein D or lysosome-associated membrane protein-3 (LAMP3). Alveolar markers had moderate to poor discrimination for invasive ventilation at Day 0 (Table E4) . C-statistics ranged between a maximum of 0.76 (95% confidence interval [CI], 0.69-0.83) for RAGE to a minimum of 0.60 (95% CI, 0.51-0.68) for LAMP3. At Day 3, the C-statistic was <0.72 for all alveolar injury markers.

Results were similar across all three modeling strategies (Figures 3, 4, and E4; strategies 1, 2, and 3, respectively.).

The dynamics of endothelial injury markers differed from those of alveolar markers (Figures 3, 4C , 4D, E3, and E4). At Day 0, endothelial injury markers were comparable for all respiratory support levels ( Figures 3, 4 , and E4). Unlike alveolar markers, after Day 0, endothelial markers increased (Figure 4 ), and by Day 3, most were significantly higher in invasively ventilated patients than nonintubated patients (Figures 3, 4 , and E3). The endothelial markers tPA (tissue-type plasminogen activator), PAI-1 (plasminogen activator inhibitor 1), tissue factor, and protein C were exceptions, demonstrating significantly more anomalous levels in intubated patients at all times. ADAMTS13 (a disintegrin and metalloproteinase with thrombospondin motifs 13), which is consumed during endothelial activation, showed an inverse pattern, where levels were significantly lower in invasively ventilated patients (P interaction , 0.0001). All three methods of specifying respiratory severity, complete-case analysis, and analysis excluding patients who received steroids or tocilizumab yielded similar results (Figures 3, 4 , and E4-E7).

For nearly all endothelial injury markers, discrimination for invasive ventilation was initially poor but markedly increased over time (Table E4) . Day 0 C-statistics were less than 0.60 for angiopoitein-2 (0.58; 95% CI, 0.50-0.66), endocan (0.59; 95% CI, 0.51-0.67), and Among intubated patients, higher endothelial injury marker levels were not reliably associated with higher ventilatory ratios ( Figure E8 ).

Club cell proteins followed the pattern of endothelial rather than alveolar markers ( Figures 3, 4 , E2, and E3). Club cell secretory protein (CC16) poorly discriminated respiratory severity at Day 0 (C-statistic, 0.59) but improved by Day 3 (C-statistic, 0.72; D c , 0.13) (Table E4) . Moreover, changes in club cell markers showed greater correlation with endothelial markers (Table E5) .

In multivariable regression, in invasively ventilated subjects, Day 0 alveolar injury markers were consistently higher in patients with COVID-19 than control subjects without COVID, while most endothelial injury markers were not ( Figure E9 ). COVID-19-negative patients were not followed past Day 0.

To explore whether endothelial abnormalities reflect transitions from predominantly pulmonary injury to dysregulated systemic responses, we examined markers of cardiac, renal, and vascular disturbances. Among these, markers of RAS activation, cardiac injury, and kidney injury showed dynamics similar to the endothelial pattern (Figures 3  and 5) , with similarities between intubated and nonintubated patients at Day 0, yet at Days 3 and 7, significant and progressively higher levels in intubated versus nonintubated patients. Further suggestive of an increasingly systemic response, changes in cardiorenovascular injury markers were closely correlated with changes in endothelial, RAS activation, and club cell markers but not alveolar markers (Table E5 and E6).

Changes in soluble tumor necrosis factor receptor 1 (P interaction , 0.0001) and D-dimer (P interaction = 0.0001) levels mirrored the endothelial pattern and were more strongly correlated with cardiac and renal injury markers than IL-6, IL-8, or C-reactive protein. The latter, as described (14-17), were persistently higher among ventilated patients (Figures E10-E12 and Tables E7 and  E8) .

Among patients on supplemental oxygen at Day 0, 17 (11%) were intubated or deceased by Day 7. Their biomarker course more closely resembled patients intubated on Day 0 than initially hypoxemic patients who were never intubated ( Figure E13 ).

In multivariable proportional-odds analysis, worse 28-day outcome was significantly associated with Day 0 values of protein C, but not the other 11 endothelial markers, and none of the 7 epithelial markers ( Figure 6 and 

In this cohort of 225 patients with COVID-19 requiring respiratory support at presentation, we observed distinct and consistent patterns in the dynamics of alveolar and endothelial injury markers: 1) elevations in alveolar injury markers diminish over time in both mechanically ventilated patients receiving lung-protective ventilation and spontaneously breathing patients; 2) elevations in endothelial markers are delayed, are limited to invasively ventilated patients, and correlate with nonpulmonary organ injury; and 3) among intubated patients, despite severe hypoxemia, ventilatory ratios are not prominently elevated early.

First, the finding that RAGE and surfactant proteins peak at Day 0 suggests that alveolar injury represents an early insult in COVID-induced respiratory failure. This is consistent with our prior proteomic analyses showing strong association of Day 0 RAGE levels with disease severity (10). Other P values indicate the statistical hypothesis tests from the multivariable mixed-effects repeated measures models for class differences by time in cohorts also report higher RAGE levels in COVID-19 versus non-COVID ARDS (18, 19) . The similar decrease in alveolar markers between intubated and nonintubated patients over time could indicate that lungprotective ventilation allows for alveolar recovery to a similar degree as spontaneous breathing. In the context of the controversy surrounding early intubation in COVID-19 and the opposing concerns of ventilator versus self-inflicted lung injuries, we note institutional practice during the study period was early intubation; noninvasive mechanical ventilation was prohibited. However, decreasing alveolar injury markers clearly do not equate with clinical recovery, as many patients required prolonged intubation despite downward trajectories in alveolar markers. Second, the delayed peak in endothelial markers among intubated patients suggests that unlike alveolar injury, endothelial activation and injury are prominent features of later severe disease. Consistent with our findings, patients with COVID-19 display conspicuous pulmonary vasodilation (5), glycocalyx damage (20, 21) , postmortem endothelialitis (8), and elevated von Willebrand factor with low levels of ADAMTS13 (22, 23) . Comparisons of COVID-19 and non-COVID-19 ARDS report comparable to lower initial levels of endothelial injury markers, such as angiopoietin-2, in COVID-19 ARDS (18, 19) .

The shift from alveolar-to endothelialpredominant injury may alternatively, or additionally, indicate that the "natural" course of COVID-19 respiratory failure involves a transition from a primarily lunglocalized pathology to a systemic one. Herein, increased endothelial injury markers coincided with increased extrapulmonary organ injury and dysfunction and were more strongly associated with 28-day outcome than alveolar markers. In ARDS, trajectories of endothelial markers vary (24, 25) , but higher levels consistently portend worse organ dysfunction and outcomes (25) (26) (27) (28) (29) . In COVID-19, endothelial injury markers are associated with sustained elevations in viral mRNA (30) . Therefore, the delayed increase in endothelial markers we observed might in part reflect increased systemic viral-induced injury.

Third, ventilatory ratios increased over time but were infrequently elevated and not consistently associated with endothelial injury. In contrast, Sp O 2 /FI O 2 was lowest at Day 0 and increased over time. A simple explanation might be imprecision in the ventilatory ratio as an estimate of dead space. However, intubated patients with COVID-19 undergoing right heart catheterization show increased cardiac output with elevated pulmonary capillary wedge pressure but atypically low pulmonary vascular resistance and comparable intrapulmonary shunt versus control subjects with non-COVID-19 ARDS (31). These observations suggest excess pulmonary vasodilation may contribute to severe COVID-19 hypoxemia, at least early on. This hypothesis is consistent with prominent early alveolar injury with minimal endothelial injury and does not necessarily invoke increased dead space ventilation. We note ventilatory ratios were infrequently elevated in our study but increased over time. This could simply reflect aggressive low-tidal-volume ventilation and permissive hypercapnia. Alternatively, it could suggest intrapulmonary thrombosis accrues over time.

Together, these results may explain the seemingly contradictory findings of the Accelerating COVID-19 Therapeutic Interventions and Vaccines 4 ACUTE (ACTIV-4A) trial. In ACTIV4A, full-dose heparin improved mortality in hospitalized nonintubated patients (32), but for intubated patients it was not beneficial and suggested harm (33) . Heparin-treated critically ill patients showed no mortality benefit despite a nearly twofold reduction in major thrombotic events (5.7% vs. 10.3%) and similar frequency of (clinically detected) major bleeding (3.1% vs. 2.4%). Alveolar hemorrhage is common at autopsy in COVID-19 (7) . Therapeutic anticoagulation likely exacerbates risk of alveolar hemorrhage in patients with severe alveolar injury. We found alveolar injury markers were highest on Day 0 and among intubated patients. The delayed endothelial injury occurring among intubated patients could reflect alveolar bleeding and vessel injury, rather than thrombosis alone, an interpretation supported by the lack of association between endothelial markers and ventilatory ratios. Both intubated and nonintubated patients in ACTIV4A were enrolled at presentation (our study's Day 0). Therefore, our results suggest anticoagulation of intubated patients in ACTIV4A was initiated when alveolar bleeding risk was high, and thrombosis was not a significant contributor to hypoxemia. In contrast, at Day 0, nonintubated patients display less severe alveolar injury and similar levels of endothelial injury, such that these patients would be expected to have lower risk of alveolar bleeding and therefore be more likely to benefit from anticoagulation.

We included patients requiring both invasive and noninvasive respiratory support, reasoning their common insult (i.e., COVID-19 pneumonia) made them an ideal biological comparator. Extending validity to this approach, "inflammatory" ARDS phenotypes are validated in patients at risk of ARDS who do not yet meet Berlin criteria (25) . Some patients on supplemental oxygen might have met Berlin ARDS criteria had they received positive end-expiratory pressure (PEEP); institutional policy prohibited noninvasive positive-pressure ventilation during the study period. In addition, not all patients had an arterial blood-gas on Day 0. For this reason, we used modified Berlin criteria (34) that did not consider PEEP and that used Sp O 2 /FI O 2 when Pa O 2 data were missing to designate ARDS.

The implications of the late increase in club cell markers are unclear. Injury of distal airways, the site of club cells, occurring after alveolar damage seems unlikely given that SARS-CoV-2 reaches alveoli via the airways. In vitro, club cells are particularly susceptible to SARS-CoV-2 infection, which induces inflammatory cytokine secretion (35) . Although higher club cell markers were associated herein with greater disease severity, lower levels of club cell secretory protein have been previously associated with non-COVID-19 ARDS, albeit not with outcome (36, 37) .

RAS activation markers displayed marked elevation, with dynamics similar to endothelial injury. They correlated closely with cardiac and renal injury, albeit only in Figure 5 . Plasma levels of renin-angiotensin system activation, cardiac injury, and renal injury markers over time show patterns similar to endothelial markers. Representative markers of (A) renin-angiotensin system activation, and (B and C) cardiorenal injury and dysfunction over the study period by level of respiratory support. P values indicate the results of the statistical hypothesis tests from the multivariable intubated patients, consistent with trials showing RAS antagonism does not modify disease course in moderate and mild COVID-19 (38) . Because our study did not measure angiotensin-II or ACE1, we cannot discriminate primary hyperreninemia (i.e., true RAS activation) from a compensatory response to a relative hypo-RAS state, as described in distributive shock (39) . Suggesting the latter are high levels of plasma ACE2, which suppresses RAS and is catalytically active in COVID-19 (40, 41) , and decreases in angiotensin-II levels over time in COVID-19 ARDS (42) . The close correlations we observed between renin and markers of renal and cardiac injury further support attenuated RAS effects, as described in sepsis and distributive shock and after cardiac surgery (43) (44) (45) (46) (47) (48) . Alternatively, renin elevations could reflect responses to prolonged sedation-induced vasodilatation or hypovolemia following diuresis, representing the balance in ARDS management between protecting the lung and protecting the kidney. Regardless of mechanism, these findings indicate that renin's utility as a marker of hypoperfusion (49) and prognosticator of cardiovascular and renal injury (39, 48) in severe COVID-19 and ARDS should be explored. This work differs from the previously published analysis of this cohort (10) based on 1) prespecified biomarker selection rather than unbiased discovery analysis; 2) focus on patients presenting with hypoxemia rather than all comers; and 3) specific interrogation of lung epithelial, endothelial, and nonpulmonary organ injury markers. These tissue-specific injury markers may inform disease biology more than characterizations of nonspecific inflammatory markers alone. Important limitations of this study include its single-center observational design, which facilitates thorough description but precludes causal evaluation of any markers under investigation. Specifically, we cannot definitively discern whether chronological differences between intubated and nonintubated patients result from Figure 5 . (Continued). mixed-effects repeated measures models for class differences by time in study (day), level of respiratory support (status), and whether respiratory support level is an effect modifier for time (interaction). Each dot represents an individual patient. Box, 25th to 75th percentiles; whiskers, 5th to 95th percentiles. Dashed lines connect the means at each time point. Normalized protein expression (NPX) units are on a log2 scale (i.e., a 1-unit increase corresponds to a doubling in level). ACE-2 = angiotensin-converting enzyme-2; NT-proBNP = N-terminal pro-brain natriuretic peptide. . The response is coded in ascending order such that a higher odds ratio indicates worse clinical status at Day 28. All models adjusted for age, sex, body mass index, initial Sequential Organ Failure Assessment (SOFA) score, heart failure, chronic kidney disease, and chronic obstructive pulmonary disease. Error bars, 95% confidence intervals. The right-hand columns display the model estimated P value (P) and the P value after a false-discovery rate correction (P adj ). ADAMTS13 = a disintegrin and metalloproteinase with thrombospondin motifs 13; CC-16 = club cell secretory protein; ICAM-1 = intercellular adhesion molecule 1; LAMP3 = lysosome-associated membrane glycoprotein 3; PAI-1 = plasminogen activator inhibitor 1; PnSP1 = pneumocyte secretory protein 1; RAGE = receptor for advanced glycation endproducts; SP-A1 = pulmonary surfactant-associated protein A1; SP-A2 = pulmonary surfactant-associated protein A2; SP-D = pulmonary surfactantassociated protein D; sTM = soluble thrombomodulin; tPA = tissue-type plasminogen activator; VCAM-1 = vascular cell adhesion protein 1; vWF = von Willebrand factor. COVID-19 severity or mechanical ventilation itself. In addition, because we enrolled subjects before the approval of remdesivir and dexamethasone treatment for COVID-19, these drugs were not administered to most subjects. Although this may limit extrapolation to current practice, it presents a valuable opportunity to observe biology unconfounded by the pleiotropic effects of steroids. Furthermore, although proximity extension assays increase assay sensitivity and enable detection of plasma proteins that cannot be detected by other methods, normalized protein expression units cannot be translated to ELISA-based readouts, which precluded applying validated models for ARDS phenotype identification (27) . Multiple comparison adjustments were applied only to biomarker associations with patient outcomes. Finally, as with all biomarker studies, protein levels in plasma variably reflect those in tissue compartments.

Alveolar injury markers peak early in severe COVID-19 and decrease among both spontaneously breathing and invasively ventilated patients. Endothelial injury markers increase with delayed kinetics and are significantly associated with evidence of systemic inflammation, renin-angiotensin system activation, extrapulmonary organ injury, and 28-day outcome. In severe COVID-19, alveolar and endothelial injury likely contribute at different times to disease progression.

Author disclosures are available with the text of this article at www.atsjournals.org.

Acute respiratory distress syndrome

ARDS Subphenotypes: understanding a Heterogeneous Syndrome

Distinct molecular phenotypes of direct vs indirect ARDS in singlecenter and multicenter studies

Coagulopathy of coronavirus disease 2019

Pulmonary vascular dilatation detected by automated transcranial Doppler in COVID-19 pneumonia

Prevalence of thrombotic complications in ICU-treated patients with coronavirus disease 2019 detected with systematic CT scanning

Autopsy findings and venous thromboembolism in patients with COVID-19: a prospective cohort study

Pulmonary vascular endothelialitis, thrombosis, and angiogenesis in Covid-19

Trajectories of pulmonary epithelial and endothelial injury markers in COVID-19 patients requiring respiratory support at presentation

Longitudinal proteomic analysis of severe COVID-19 reveals survivalassociated signatures, tissue-specific cell death, and cell-cell interactions

Physiologic analysis and clinical performance of the ventilatory ratio in acute respiratory distress syndrome

Biomarkers of ARDS: what's new?

Proteomics: tissue-based map of the human proteome

Cytokine elevation in severe and critical COVID-19: a rapid systematic review, meta-analysis, and comparison with other inflammatory syndromes

Prevalence of phenotypes of acute respiratory distress syndrome in critically ill patients with COVID-19: a prospective observational study

Weathering the COVID-19 storm: lessons from hematologic cytokine syndromes

Inflammatory response and phenotyping in severe acute respiratory infection from the Middle East respiratory syndrome coronavirus and other etiologies

Markers of endothelial and epithelial pulmonary injury in mechanically ventilated COVID-19 ICU patients

Comparison of host endothelial, epithelial and inflammatory response in ICU patients with and without COVID-19: a prospective observational cohort study

Injury to the endothelial glycocalyx in critically ill patients with COVID-19

Endothelial injury and glycocalyx degradation in critically ill coronavirus disease 2019 patients: implications for microvascular platelet aggregation

von Willebrand factor/ADAMTS13 axis and venous thromboembolism in moderate-to-severe COVID-19 patients

von Willebrand factor multimer formation contributes to immunothrombosis in coronavirus disease 2019

Plasma angiopoietin-2 in clinical acute lung injury: prognostic and pathogenetic significance

Host-response subphenotypes offer prognostic enrichment in patients with or at risk for acute respiratory distress syndrome

Subphenotypes in acute respiratory distress syndrome: latent class analysis of data from two randomised controlled trials

Development and validation of parsimonious algorithms to classify acute respiratory distress syndrome phenotypes: a secondary analysis of randomised controlled trials

Acute respiratory distress syndrome subphenotypes and differential response to simvastatin: secondary analysis of a randomised controlled trial

d-dimer and death in critically ill patients with coronavirus disease 2019

SARS-CoV-2 viremia is associated with distinct proteomic pathways and predicts COVID-19 outcomes

Haemodynamic characteristics of COVID-19 patients with acute respiratory distress syndrome requiring mechanical ventilation: an invasive assessment using right heart catheterization

ATTACC Investigators; ACTIV-4a Investigators

Therapeutic anticoagulation with heparin in noncritically ill patients with Covid-19

Therapeutic anticoagulation with heparin in critically ill patients with Covid-19

ARDS Definition Task Force. Acute respiratory distress syndrome: the Berlin Definition

Single cell resolution of SARS-CoV-2 tropism, antiviral responses, and susceptibility to therapies in primary human airway epithelium

Clara cell protein (CC16), a marker of lung epithelial injury, is decreased in plasma and pulmonary edema fluid from patients with acute lung injury

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Continuation versus discontinuation of reninangiotensin system inhibitors in patients admitted to hospital with COVID-19: a prospective, randomised, open-label trial

Renin and survival in patients given angiotensin II for catecholamineresistant vasodilatory shock: a clinical trial

Plasma ACE2 activity is persistently elevated following SARS-CoV-2 infection: implications for COVID-19 pathogenesis and consequences

Plasma ACE2 predicts outcome of COVID-19 in hospitalized patients

Efficacy of serum angiotensin II levels in prognosis of patients with coronavirus disease 2019

Hyperreninemic hypoaldosteronism: a possible etiological factor of septic shock-induced acute renal failure

The dissociation of renin and aldosterone during critical illness

Outcomes in patients with vasodilatory shock and renal replacement therapy treated with intravenous angiotensin II

Impaired angiotensin II type 1 receptor signaling contributes to sepsis-induced acute kidney injury

Physiologic response to angiotensin II treatment for coronavirus disease 2019-induced vasodilatory shock: a retrospective matched cohort study

Kinetic changes of plasma renin concentrations predict acute kidney injury in cardiac surgery patients

Renin as a marker of tissue-perfusion and prognosis in critically ill patients

Acknowledgment: The authors thank Arthur, Sandra, and Sarah Irving for a gift that enabled this study and funded the David P. Ryan, M.D., Endowed Chair in Cancer Research (N.H.). They also thank Olink Proteomics Inc. for providing in-kind all proteomics assays presented in this work, without which our findings would not have been possible.