key: cord-0895925-0ujrayer authors: Price, D. R.; Benedetti, E.; Hoffman, K.; Gomez-Escobar, L.; Alvarez-Mulett, S.; Capili, A.; Sarwath, H.; Parkhurst, C. N.; LaFond, E.; Weidman, K.; Ravishankar, A.; Cheong, J. G.; Batra, R.; Buyukozkan, M.; Chetnik, K.; Easthausen, I.; Schenck, E. J.; Racanelli, A. C.; Outtz Reed, H.; Laurence, J. C.; Josefowicz, S. Z.; Lief, L.; Choi, M. E.; Rafii, S.; Schmidt, F.; Borczuk, A. C.; Krumsiek, J.; Choi, A. M. K. title: The maladaptive vascular response in COVID-19 acute respiratory distress syndrome and recovery date: 2021-05-24 journal: nan DOI: 10.1101/2021.05.20.21257542 sha: 41e64fc52e5e989638862528fa84d6fe37cdfc2d doc_id: 895925 cord_uid: 0ujrayer Vascular injury is a menacing element of acute respiratory distress syndrome (ARDS) pathogenesis. To better understand the role of vascular injury in COVID-19 ARDS, we used lung autopsy immunohistochemistry and blood proteomics from COVID-19 subjects at distinct timepoints in disease pathogenesis, including a hospitalized cohort at risk of ARDS development ("at risk", N=59), an intensive care unit cohort with ARDS ("ARDS", N=31), and a cohort recovering from ARDS ("recovery", N=12). COVID-19 ARDS lung autopsy tissue revealed an association between vascular injury and platelet-rich microthrombi. This link guided the derivation of a protein signature in the at risk cohort characterized by lower expression of vascular proteins in subjects who died, an early signal of vascular limitation termed the maladaptive vascular response. These findings were replicated in COVID-19 ARDS subjects, as well as when bacterial and influenza ARDS patients (N=29) were considered, hinting at a common final pathway of vascular injury that is more disease (ARDS) then cause (COVID-19) specific, and may be related to vascular cell death. Among recovery subjects, our vascular signature identified patients with good functional recovery one year later. This vascular injury signature could be used to identify ARDS patients most likely to benefit from vascular targeted therapies. Vascular injury has been linked to COVID-19 acute respiratory distress syndrome (ARDS) 73 10 abundance (P<0.001, Supplementary Figure 5B) . Notably, plasma ANGPT2 was higher in the 168 low mean protein abundance cluster (P=0.001, Figure 4C and Supplementary Figure 4E) , 169 linking low vascular protein abundance and plasma ANGPT2 in diverse ARDS subjects. 170 Interestingly, when COVID-19 ARDS was considered alone (Supplementary Figure 4) , 171 this higher vascular injury signature was present in 39% (12 of 31) of COVID-19 ARDS subjects, 172 yet when all three infection types were considered (Figure 4) , only 13% (4 of 31) of COVID-19 173 ARDS were in the higher vascular injury cluster compared to 58% (14 of 24) of bacterial sepsis 174 ARDS and 80% (3 of 4) of influenza ARDS subjects, demonstrating that vascular injury may be 175 relative to the causative infection, with COVID-19 ARDS overall being associated with less 176 vascular injury than bacterial sepsis and influenza related ARDS. This finding is supported by a 177 lower ventilator ratio in COVID-19 ARDS subjects compared to non-COVID-19 (Supplementary 178 Table 1 ) a physiologic surrogate for vascular injury in ARDS (18). This is also consistent with 179 previous investigations showing higher platelet counts and less platelet consumption in COVID-180 19 compared to bacterial sepsis ARDS (19) . 181 Having 182 validated our vascular injury signature in diverse ARDS populations, we assessed whether ARDS 183 vascular injury could be associated with genetically regulated necrotic cell death, known as 184 necroptosis. We first demonstrated increased expression of plasma RIPK3, a vital necroptosis 185 protein (20), in ARDS subjects with higher vascular injury (P=0.020, Figure 5A ). Plasma RIPK3 186 was also correlated with plasma ANGPT2 (r=0.40, P=0.003, Figure 5B . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 24, 2021. In this study, we traced a maladaptive vascular response through the natural history of 215 COVID-19 ARDS from hospital admission to either recovery or death. Reflected in both the lung 216 tissue and blood proteome, we demonstrated the clinical relevance of the low abundance of 217 circulating vascular proteins with known vascular functions and implied a link with vascular cell 218 death, and in particular specialized necroptotic cell death. 219 This vascular phenotype is notably present in certain COVID-19 subjects prior to ICU 220 admission. While vascular injury spans the COVID-19 disease continuum from asymptomatic blue 221 toes to catastrophic thromboembolic disease and ARDS-associated microangiopathy, our The role of activated platelets in vascular injury and repair is also apparent in our data. 235 Activated platelets amplify immune responses in early ARDS but also play an essential role in 236 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 24, 2021. 13 vascular repair. The consistently low platelet levels across our cohorts and the extensive 237 microthrombi observed in our autopsy subjects implies a circulating milieu of platelet 238 consumption. This milieu of platelet consumption is supported by a blood signature of ongoing 239 thrombolysis (high UPA and low PAI) and low levels of platelet derived proteins (low SELP, and 240 GP6) in our high vascular injury subjects. Relative loss of ADAMTS13, linked to secondary 241 microangiopathy in COVID-19 (32), is similarly deficient in our higher vascular injury subjects, 242 linking platelet consumption with microangiopathy in severe COVID-19. Low platelets have 243 previously been linked to ARDS mortality (33) and our data suggest this may be related to 244 depletion in platelet related angiogenic (34-36) and junctional barrier factors (37-40). 245 Consistently low circulating angiogenic (low PDGFA and PDGFB) and barrier protein (low 246 ANGPT1) in our higher vascular injury and low platelet subjects imply limitations in these 247 essential reparative processes. 248 The validation of our vascular phenotype across diverse causes of ARDS broadens the 249 relevance of our findings. In linking low platelets, vascular function, and mortality in COVID-19, 250 bacterial sepsis, and influenza ARDS, we hint at a common final pathway of vascular injury that 251 is more disease-(ARDS) than cause-(COVID-19) specific. Of note is that this vascular injury 252 pattern may be related to a reduced baseline vascular resilience in our high vascular injury subjects. The identification of this severe vascular phenotype across infectious causes of ARDS also 257 presents an opportunity for targeted vascular therapies in ARDS, including those that have shown 258 promise in COVID-19 (45), ARDS generally (46), and in exciting preclinical (47, 48) and early 259 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 24, 2021. ; 14 human experimental therapies, including ANGPT1 supplementation trial currently underway in 260 COVID-19 subjects (49). And while a ANGPT2 neutralizing antibody study in hospitalized patient 261 with COVID-19 was stopped for futility in October 2020 (50), our data could improve patient 262 selection for similar trials in the future, including the use of platelet levels to identify subjects with 263 vascular limitation. 264 Finally, our identification of a vascular recovery proteome is novel. An estimated 2 million 265 patients have been hospitalized in the United States since the start of the COVID-19 pandemic, 266 with the overwhelming majority recovering (51). But even in recovery, patients remain at risk for 267 disease related morbidity and mortality (52). We demonstrate that a stable circulating vascular 268 proteome is important for functional recovery. This association between vascular stability, platelet 269 levels, and functional recovery could also support platelet levels as a novel biomarker in ARDS 270 recovery. Larger studies will be needed to validate this observation. 271 In summary, we identify an early vascular injury signal in COVID-19 ARDS that has 272 predictive value in early disease through to recovery and well as in bacterial sepsis and influenza 273 ARDS and could improve patient selection and timing of vascular targeted therapies in ARDS. 274 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. (which was not certified by peer review) The copyright holder for this preprint this version posted May 24, 2021. This study enrolled COVID-19 subjects at New . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 24, 2021. ; https://doi.org/10.1101/2021.05.20.21257542 doi: medRxiv preprint The study was approved by the institutional review board (IRB) at WCM (20-05022072, 296 20-0302168, 20-03021681, and 1811019771). Written informed consent was received from all 297 participants prior to inclusion in the study. 298 299 In the at risk cohort, between 1 and 3 consecutive daily samples were obtained from our 301 central lab after routine processing to obtain serum. To obtain serum, blood collected in serum 302 separator tubes (SST) was processed within 2 hours of venipuncture. Whole blood was centrifuged 303 at 1,500 g for 7 minutes. The serum layer was aliquoted and stored at -80°C. These samples were 304 obtained with a waiver of informed consent. In this cohort, samples collected after patient 305 intubation were excluded from the analysis. In the ARDS and recovery cohorts, plasma was 306 isolated from study subjects according to our existing plasma isolation protocol (53) (54) (55) (56) . To obtain 307 plasma, blood collected in EDTA tubes was processed within 6 hours of venipuncture. Whole 308 blood was centrifuged at 490 g for 10 minutes. The plasma layer was removed in 200 uL aliquots 309 and stored at -80. 310 Baseline clinical parameters and outcomes were extracted from the electronic medical 313 record (EMR) as described previously (57, 58). Baseline comorbidities were manually abstracted 314 from the EMR. Baseline clinical data (labs, severity of illness, ventilator data) were measured at 315 time of blood sampling in both the at risk cohort and ARDS cohort. Severity of illness was defined 316 by the sequential organ failure assessment score (SOFA) (59). ARDS was determined according 317 to the Berlin definition with ARDS severity capped at mild for subjects on non-invasive ventilation 318 . CC-BY-NC-ND 4.0 International license It is made available under a is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 24, 2021. ; https://doi.org/10.1101/2021.05.20.21257542 doi: medRxiv preprint (60). Two critical care investigators independently adjudicated the ARDS diagnosis. In all study 319 subjects, COVID-19 was diagnosed if a subject had a syndrome compatible with COVID-19 and 320 a nasopharangeal (NP) swab positive for SARS-CoV-2 by reverse transcriptase polymerase chain 321 reaction (RT-PCR). 322 323 Recovery subjects were assessed for recovery using the EuroQol-5D-3L (EQ-5D-3L) 325 questionnaire (61) at 12 months after ICU admission. The EQ-5D-3L is a self-assessment of the 326 patient recovery, and considers 5 distinct domains, namely mobility, self-care, usual activities, 327 pain or discomfort, and anxiety or depression (62). Each domain was scored 0, 1, or 2 depending 328 on whether the patient reported no, some, or extensive limitations in each respective domain. For 329 each patient, a final score was defined as the sum of the scores across the five domains and treated 330 as an ordinal variable in the statistical analysis. Maximal functional limitation would have a score 331 of (2*5=)10 while an optimal recovery would be scored 0. is the author/funder, who has granted medRxiv a license to display the preprint in perpetuity. The copyright holder for this preprint this version posted May 24, 2021. an Aperio slide scanner with a resolution of 0.24 μm/pixel. Control tissue was from non-diseased 342 sections of lung taken during clinically indicated lung biopsies. Quantification of ANGPT2 and 343 CD61 was performed on four random 20X images selected using a random overlay of points and 344 excluding fields with large vessels or airway. All twenty autopsies were analyzed using 345 Immunohistochemistry profiler (63) as a plugin for Image J (National Institutes of Health, USA). 346 High, intermediate, low, and overall percent positive was averaged over the four 347 measurements. The median ANGPT2 quantification was used to define the high (>median) and 348 low (