key: cord-0894621-jw34jwad
authors: Alba, George A.; Samokhin, Andriy O.; Wang, Rui‐Sheng; Wertheim, Bradley M.; Haley, Kathleen J.; Padera, Robert F.; Vargas, Sara O.; Rosas, Ivan O.; Hariri, Lida P.; Shih, Angela; Thompson, Boyd Taylor; Mitchell, Richard N.; Maron, Bradley A.
title: Pulmonary endothelial NEDD9 and the prothrombotic pathophenotype of acute respiratory distress syndrome due to SARS‐CoV‐2 infection
date: 2022-05-11
journal: Pulm Circ
DOI: 10.1002/pul2.12071
sha: bc09c959302f5b0ef60760da9bb61550226afda6
doc_id: 894621
cord_uid: jw34jwad

The pathobiology of in situ pulmonary thrombosis in acute respiratory distress syndrome (ARDS) due to severe acute respiratory syndrome coronavirus‐2 (SARS‐CoV‐2) infection is incompletely characterized. In human pulmonary artery endothelial cells (HPAECs), hypoxia increases neural precursor cell expressed, developmentally downregulated 9 (NEDD9) and induces expression of a prothrombotic NEDD9 peptide (N9(P)) on the extracellular plasma membrane surface. We hypothesized that the SARS‐CoV‐2–ARDS pathophenotype involves increased pulmonary endothelial N9(P). Paraffin‐embedded autopsy lung specimens were acquired from patients with SARS‐CoV‐2–​​​​​​ARDS (n = 13), ARDS from other causes (n = 10), and organ donor controls (n = 5). Immunofluorescence characterized the expression of N9(P), fibrin, and transcription factor 12 (TCF12), a putative binding target of SARS‐CoV‐2 and known transcriptional regulator of NEDD9. We performed RNA‐sequencing on normal HPAECs treated with normoxia or hypoxia (0.2% O(2)) for 24 h. Immunoprecipitation‐liquid chromatography‐mass spectrometry (IP‐LC‐MS) profiled protein–protein interactions involving N9(P) relevant to thrombus stabilization. Hypoxia increased TCF12 messenger RNA significantly compared to normoxia in HPAECs in vitro (+1.19‐fold, p = 0.001; false discovery rate = 0.005), and pulmonary endothelial TCF12 expression was increased threefold in SARS‐CoV‐2–ARDS versus donor control lungs (p < 0.001). Compared to donor controls, pulmonary endothelial N9(P)‐fibrin colocalization was increased in situ in non‐SARS‐CoV‐2–ARDS and SARS‐CoV‐2–ARDS decedents (3.7 ± 1.2 vs. 10.3 ± 3.2 and 21.8 ± 4.0 arb. units, p < 0.001). However, total pulmonary endothelial N9(P) was increased significantly only in SARS‐CoV‐2–ARDS versus donor controls (15 ± 4.2 vs. 6.3 ± 0.9 arb. units, p < 0.001). In HPAEC plasma membrane isolates, IP‐LC‐MS identified a novel protein–protein interaction between NEDD9 and the β(3)‐subunit of the α(v)β(3)‐integrin, which regulates fibrin anchoring to endothelial cells. In conclusion, lethal SARS‐CoV‐2–ARDS is associated with increased pulmonary endothelial N9(P) expression and N9(P)‐fibrin colocalization in situ. Further investigation is needed to determine the pathogenetic and potential therapeutic relevance of N9(P) to the thrombotic pathophenotype of SARS‐CoV‐2–ARDS.

Acute respiratory distress syndrome (ARDS) is a highly morbid cause of acute hypoxemic respiratory failure characterized by inflammatory lung injury, in situ pulmonary vascular thrombosis, and microcirculatory dysfunction. 1 Approximately 10%-20% of patients who develop severe infection with severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), the virus responsible for coronavirus disease 2019 (COVID- 19) , develop ARDS. [2] [3] [4] Pulmonary vascular dysfunction in ARDS is an independent predictor of mortality. 5-8 ARDS due to SARS-CoV-2 infection is distinguished by increased pulmonary arteriolar microthrombi and intraluminal fibrin deposition when compared to other forms of ARDS, including pandemic H1N1 influenza. [9] [10] [11] However, studies of thrombotic complications in SARS-CoV-2-ARDS have focused mainly on nonspecific coagulation proteins, 12 thereby hampering insight into pathobiological mechanisms and disease-specific pathogenetic targets.

Neural precursor cell expressed, developmentally downregulated 9 (NEDD9) is a scaffolding protein involved in several important protein-protein interactions. 13 In human pulmonary artery endothelial cells (HPAECs), NEDD9 is profibrotic, 14 and hypoxia-inducible factor-1 α (HIF1α) signaling induces expression of a specific NEDD9 peptide (N9 P ) that ligands activated platelets to promote pathogenic platelet-endothelial adhesion. 15 Additionally, increased pulmonary endothelial NEDD9 expression is observed in human lung diseases characterized by hypoxia and pulmonary vascular thrombosis, such as pulmonary embolism and chronic thromboembolic pulmonary hypertension. 15 These observations suggest that increased pulmonary endothelial N9 P expression may be unrecognized in the SARS-CoV-2-ARDS thrombotic pathophenotype.

This study included only excess human lung tissue and was approved by the institutional review board at our institution (IRB #2020P001386, IRB# P00010717) with a waiver of informed consent. Pathologic diagnoses were adjudicated independently by board-certified pathologists (R. F. P., L. P. H., S. O. V., A. S., and R. N. M.). Human lung tissue was obtained from (1) patients with diffuse alveolar damage due to SARS-CoV-2 ("COVID-19 ARDS") and alternative etiologies ("non-COVID-19 ARDS") (R. F. P., L. P. H., A. S., and R. N. M.) at the time of autopsy, (2) patients with pulmonary arterial hypertension (PAH) or idiopathic pulmonary fibrosis (IPF) at the time of lung explant or undergoing open lung biopsy for malignancy (I. O. R. and S. O. V.), and (3) discarded donor lung explants provided by the New England Organ Bank (K. J. H.). All human tissue samples were deidentified before analysis. A boardcertified pulmonologist (G. A. A.) confirmed each subject with diffuse alveolar damage met the Berlin definition of ARDS. 16 Clinical details of the COVID-19 ARDS and non-COVID-19 ARDS cohorts are summarized in Table 1 and Tables S1 and S2. The nadir partial pressure of oxygen (PaO 2 ) to a fraction of inspired oxygen (FiO 2 ) ratio was determined based on the lowest PaO 2 (and corresponding FiO 2 ) within the first 24 h after intubation and admission to the intensive care unit.

Impaired ventilation, as measured by pulmonary dead space, is an important predictor of adverse outcomes in ARDS. 8 The ventilatory ratio was developed as a simple surrogate for dead space fraction that can be calculated at the bedside 17,18 by comparing actual measurements of minute ventilation and PaCO 2 with predicted values of minute ventilation and PaCO 2, as defined by the following equation:

predicted body weight (kg) × 100 × 37 .5 . 2 To calculate the ventilatory ratio, we used the PaCO 2 (mmHg) from an arterial blood gas measured within the first 24 h of intubation and admission to the intensive care unit matched with the corresponding tidal volume (ml) and respiratory rate (breaths per minute) set on the ventilator to calculate the minute ventilation (ml/min). Predicted body weight (PBW) was calculated using the ARDSnet PBW calculator using the formula PBW (kg) = 50 + 2.3 (height in in.) − 60 for men or PBW (kg) = 45.5 + 2.3 (height in in.) − 60) for women. 19 Histology and immunohistochemistry Paraffin-embedded lung tissue was cut in cross-section (5 μm thickness). Deparaffinized slides were stained with hematoxylin and eosin (Sigma) for general histologic analysis and Masson's trichome (Fisher Scientific) to visualize collagen in the distal pulmonary arterioles (diameter, 20-100 μm). To profile key intermediaries involved in NEDD9 signaling, thrombosis, and endothelial dysfunction, immunofluorescence was performed on lung sections using a commercial antibody against the endothelial plasma membrane protein CD31 (Novus Biologicals; NB100-2284), fibrin (Millipore Sigma; MABS2155), and transcription factor 12 (TCF12) (Thermo Fisher Scientific; OTI4D6). Pulmonary microthrombi were defined in this study as collections of fibrin, confirmed by immunofluorescence, that filled the lumens of small pulmonary arteries or capillaries. These data were quantitated as: total number of pulmonary arterioles or capillaries with pulmonary microthrombi (N)/total number of vessels visualized at 100 × (N) expressed as a percentage.

To visualize the prothrombotic N9 P , we used a custommade antibody, as reported recently. 14 Images were acquired using a Confocal Laser Scanning Microscope (ZEISS LSM 800 with Airyscan), and Zen software (ZEISS) was used to visualize images and quantify immunofluorescence intensity including colocalization of two different immunofluorescence signals according to previously published methods. 14, 15 The Z-stack images were acquired at 0.16 µm intervals for at least 2.4 µm at a data depth of 12 bits with a ×20-63 objective that corresponds to a minimum pixel size of 0.08 µm and processed in Superresolution (3D, Auto) Airyscan mode. The three-dimensional reconstruction images were created using Zen 2 (Blue Edition) software. 14, 15 All quantitative analyses were performed blinded to the etiology of ARDS.

The RNA-Seq results reported in this study are extrapolated from data reported previously by our laboratory, detailed in reference 14 . Briefly, using the RNeasy Mini Kit (Qiagen), messenger RNA (mRNA) was isolated from cultured HPAECs treated with normoxia or hypoxia (0.2% O 2 ) for 24 h. Briefly, the total RNA per sample was quantified using an Agilent 2100 Bioanalyzer instrument with a corresponding Agilent Bioanalyzer RNA Nano assay. The resulting RNA integrity number (RIN) scores and concentrations were considered for qualifying samples to proceed (9.85 ± 0.37 [8.5-10.0 min, max] RIN, n = 18). The samples were then normalized to 200 ng of input in 50 μl (4 ng/μl), and the mRNA was captured using oligo-dT beads as part of the KAPA mRNA HyperPrep workflow. Purified libraries were run on an Agilent 4200 Tapestation instrument with a corresponding Agilent High Sensitivity D1000 ScreenTape assay. The pool was denatured and loaded onto an Illumina NextSeq. 500 instrument, with a High-Output 150-Cycle Kit to obtain Paired-End 75 bp reads. The basecall files were demultiplexed and the resulting FASTQ files were used in subsequent analyses. The read count-based gene expression data were normalized based on library complexity and gene variation using the R package EdgeR. Genes were considered significantly differentially expressed if the p value was <0.05 and the false discovery rate (FDR) was <0.05. The FDR was calculated using the Benjamini-Hochberg method. Results are publicly available (GSE163827). 15 Immunoprecipitation-liquid chromatography-mass spectrometry (IP-LC-MS)

For IP-LC-MS, we isolated plasma membrane fractions of HPAEC lysates using a Plasma Membrane Protein Extraction Kit (Abcam, ab65400) according to the manufacturer's instructions. Magnetic beads (Bio-Rad Protein G SureBeads) were resuspended in 100 µl solution (1 mg at 10 mg/ml), magnetized, and serially washed with 1 ml phosphatebuffered saline (PBS) + 0.1% Tween-20 (PBS-T). NEDD9 antibody (Abcam; 18056) was added to the resuspended beads in a final volume of 200 µl and rotated at room temperature for 10 min. The beads were then magnetized and serially washed with 1 ml PBS-T before incubation with the antigen-containing HPAEC plasma membrane lysate (250-500 µl) for 1 h at room temperature. The beads were then serially washed with 1 ml PBS-T, magnetized, incubated with 40 µl 1× Laemmli buffer (Bio-Rad) for 10 min at 70°C, and magnetized. The eluent was transferred to a new vial before loading sodium dodecyl sulfate-polyacrylamide gel electrophoresis. 20 After staining the gel with Coomassie blue (Bio-Rad), the target bands were excised and prepared for LC-MS to identify protein-protein interactions with plasma membrane NEDD9 according to previously published methods. 15 Peptide sequences (and hence protein identity) were determined by matching protein databases with the acquired fragmentation pattern by the software program Sequest (Thermo Fisher Scientific). For this targeted method, the instrument only activates an ms/ms fragmentation event if one of the m/z values is found within a 6-ppm mass window. All databases include a reversed version of all the sequences and the data were filtered to between a 1%-2% peptide FDR.

All continuous variables are reported as mean ± standard deviation or median and interquartile range [1st-3rd] for normally and non-normally distributed data, respectively. All categorical data are reported as number (%). Continuous variables were analyzed by Student's t test or Mann-Whitney U test according to their distributions based on results of the Shapiro-Wilks test. Categorical variables were analyzed by Fisher's exact test. One-way analysis of variance (ANOVA) was used to compare differences between more than two groups. Post hoc analysis was performed by Tukey's method. Pearson's and Spearman's correlation coefficients are presented for linear regression analyses involving normally and nonnormally distributed data, respectively. A p < 0.05 and FDR < 0.05 were considered significant. All data analyses were performed using OriginPro 2021b (v9.85).

Individual data points for each result are provided in the Supplemental Data File.

Decedents with confirmed ARDS due to SARS-CoV-2 infection (n = 13) had a median age of 61 [57-68] years, were predominantly male (n = 9, 69%), and had a median nadir PaO 2 /FiO 2 ratio of 166 (90-222). Of these patients, n = 3 (24%), n = 5 (38%), and n = 5 (38%) had mild, moderate, and severe ARDS, respectively, when using the nadir PaO 2 /FiO 2 ratio for classification (Table 1) . Decedents with confirmed ARDS due to alternative causes (n = 10) had a median age of 64 [62-67] years, were predominantly male (n = 9, 90%), and had a median nadir PaO 2 /FiO 2 ratio of 147 (89.5-226). Of these patients, n = 3 (30%), n = 5 (50%), and n = 2 (20%) had mild, moderate, and severe ARDS, respectively, based on nadir PaO 2 /FiO 2 ratio. Causes of ARDS among the non-SARS-CoV-2 control group included bacterial pneumonia (n = 1, 10%), influenza A pneumonia (n = 2, 20%), sepsis (n = 2, 20%), acute exacerbation of interstitial lung disease (n = 3, 30%), drug toxicity (n = 1, 10%), and pancreatitis (n = 1, 10%). There was no statistically significant difference in age, sex, ARDS severity, nadir PaO 2 /FiO 2 ratio, or time (in days) between ARDS diagnosis and death between the two cohorts.

A qualitative comparison in vascular phenotype between controls, SARS-CoV-2-ARDS, ARDS from other causes, and PAH/IPF is presented in Figure 1a . Compared to non-SARS-CoV-2-ARDS lungs, SARS-CoV-2-ARDS lungs demonstrated an increased percentage of microthrombi (69 ± 0.2 vs. 55 ± 0.2% thrombosis, p < 0.001) (Figure 1b) . Pulmonary endothelial N9 P expression, quantified by N9 p -CD31 colocalization, was increased significantly in SARS-CoV-2-ARDS pulmonary arterioles compared to non-SARS-CoV-2-ARDS and control lungs (15 ± 4.2 vs. 8.6 ± 3.5 vs. 6.3 ± 0.9 arb. units, respectively, p = 0.004 by ANOVA with post hoc analysis by Tukey's method) (Figure 2a) . Within pulmonary arteriolar microthrombi, we observed that pulmonary endothelial N9 P -fibrin colocalization was increased in SARS-CoV-2-ARDS compared to non-SARS-CoV-2-ARDS and control lungs (22 ± 4.0 vs. 10 ± 1.1 vs. 3.7 ± 1.2 arb. units, respectively, p < 0.001) (Figure 2b ). (Figure 3a ). In patients with ARDS due to SARS-CoV-2 infection, we observed a trend toward a positive correlation between pulmonary endothelial N9 P expression and the ventilatory ratio (r = +0.53, p = 0.05) (Figure 3b ). However, we did not observe a significant association between pulmonary endothelial N9 P expression and the ventilatory ratio in patients with ARDS due to other causes (r = −0.09, p = 0.8).

Data from computational analyses in silico identify TCF12 as one of four HPAEC protein binding targets of the SARS-CoV-2 RNA-dependent RNA polymerase protein, Nsp12, 21 which is part of the viral replicase complex. 22 Additionally, the TCF family has been shown to bind NEDD9 promoter to increase mRNA transcription. 23 Here, RNA-Seq analysis demonstrated that hypoxia increased TCF12 mRNA compared to normoxia in HPAECs in vitro (+1.19-fold, p = 0.001; FDR = 0.005) (Figure 4a ). Additionally, compared to control lungs, SARS-CoV-2-ARDS lung samples expressed increased endothelial TCF12 in HPAECs in situ (0.2 ± 0.1 vs. 0.9 ± 0.2 arb. units, p < 0.001); however, there was no significant difference in TC12 expression between SARS-CoV-2 and non-SARS-CoV-2-ARDS lung samples (Figure 4b ). Taken together with our immunofluorescence data, these observations imply a common pathway that involves hypoxia-TCF12-NEDD9 signaling regulated by hypoxia in HPAECs across all causes of ARDS assessed in this study, but differential regulation of N9 P in association with increased TCF12 in SARS-CoV-2-ARDS. We have also shown recently that hypoxia regulates important transcriptional responses in HPAECs that affect NEDD9-dependent platelet-endothelial adhesion and collagen III transcription, which are each implicated in thrombosis. 14, 15, 24 Microthrombus stabilization, however, requires protein-protein interactions involving integrin anchors. To explore this further, HPAEC plasma membrane fractions were analyzed using IP-LC-MS. From this approach, we identified a novel protein-protein interaction between NEDD9 and the β 3subunit of the α v β 3 -integrin, which is known to regulate fibrin tethering to endothelial cells (Figure 4c) . 25 

In this study of lung tissue from decedents with ARDS, we found that ARDS due to SARS-CoV-2 is associated with increased pulmonary endothelial N9 P expression and N9 P -fibrin colocalization in pulmonary arterial lumens. Additionally, we observed an association between pulmonary endothelial N9 P expression and ventilatory ratio, suggesting a clinically relevant correlation to our histopathophenotypic data linking pulmonary vascular endothelial dysfunction with dead space fraction in SARS-CoV-2 ARDS. Further, we identify a novel pathway by which NEDD9 may regulate pulmonary microthrombosis: through the formation of a protein-protein complex with β 3 -subunit of the α v β 3integrin. Overall, findings from this study establish an initial (but much-needed) molecular framework to understand the thrombotic pathophenotype of SARS-CoV-2.

One important finding in our study is the significant increase in the ventilatory ratio among patients with ARDS due to SARS-CoV-2 compared to alternative etiologies. Ventilatory ratio is a simple bedside index to approximate dead space fraction and has been validated in ARDS cohorts. 17, 18 Pulmonary dead-space fraction is one of few lung-specific independent predictors of mortality in ARDS, 8 but is underutilized clinically. In patients with ARDS due to SARS-CoV-2 infection, increased ventilatory ratios have been observed in patients with elevated D-dimer levels. 26 Here, we show that increased ventilatory ratio in patients with lethal ARDS due to SARS-CoV-2 infection may associate with pulmonary endothelial end-organ damage manifested by elevated N9 P expression and N9 P colocalization with intraluminal fibrin. Future studies are necessary to validate this observation in larger cohorts and determine if specific ARDS subgroups can be identified readily using noninvasive measures of pulmonary endothelial dysfunction, such as plasma NEDD9, to select patients more precisely for antithrombotic therapies or, possibly, emerging anti-NEDD9 therapeutics. 15 We demonstrated previously that in HPAECs, NEDD9 is profibrotic, 14 and hypoxia-HIF-1 α signaling induces expression of a specific N9 P that ligands activated platelets to promote pathogenic platelet-endothelial adhesion. 15 In this study, we demonstrate that N9 P is increased in the pulmonary arteriolar endothelium and intraluminally in patients with lethal ARDS due to SARS-CoV-2 infection compared to patients with other causes of ARDS. The severity of hypoxemia was similar between the two cohorts, but differential patterns emerged in NEDD9 expression between ARDS due to SARS-CoV-2 and ARDS due to other causes. To account for this observation, we focused on TCF12, which is a proposed binding target of the SARS-CoV-2 RNA-dependent RNA polymerase protein, Nsp12. 22, 23 We demonstrated TCF12 is upregulated by hypoxia in vitro and was consistently increased in the pulmonary endothelium of all patients with ARDS, regardless of etiology. This finding implicates TCF12 in hypoxia signaling in ARDS, but does not explain the increased endothelial N9 P expression observed in ARDS due to SARS-CoV-2 infection, suggesting but not proving that the SARS-CoV-2 viral replicase complex per se may be an important driver of N9 P expression. This observation is, in turn, broadly consistent with other transcriptomic data delineating molecular targets of SARS-CoV-2 from other coronaviruses. 9 immunoprecipitation-mass spectrometry was performed using plasma membrane lysates from cultured human pulmonary artery endothelial cells. The MS2 spectra were obtained at a retention time of 28.65 min and a mass/charge ratio (m/z) value of 766.86863 for the amino acid sequence: GSGDSSQVTQVSPQR, which corresponds to the β 3 -subunit of the α v β 3 -integrin. Overall, we identified n = 4 sequences for the β 3 -subunit of the α v β 3 in a single biological replicate (see Table S3 for details). ARDS, acute respiratory distress syndrome; FDR, false discovery rate; mRNA, messenger RNA; NEDD9, neural precursor cell expressed, developmentally downregulated 9; SARS-CoV-2, severe acute respiratory syndrome coronavirus-2; TCF12, transcription factor 12.

The sample size of this study was small, and most of the patients were male; therefore, generalizing our findings to other COVID-19 populations requires further investigation. Nonetheless, data from affected tissue of COVID-19 patients and ARDS controls has not been widely reported. We did not establish the molecular mechanism by which SARS-CoV-2 may upregulate NEDD9 through TCF12. Because of this, the precise circumstances leading to N9 P bioactivity in patients infected with SARS-CoV-2 are not addressed in this study. Furthermore, most COVID-19 patients do not develop ARDS, although some present clinically with pneumonia and hypoxemia. Since the current study does not address pathobiological factors that explain incident ARDS in COVID-19 patients, it is important to state the true importance of N9 P to the SARS-CoV-2 spectrum of acute lung injury is not established by the current work.

These data suggest that the SARS-CoV-2-ARDS pulmonary vascular pathophenotype is associated with increased N9 P expression and N9 P -fibrin colocalization in HPAECs in situ and increased pulmonary dead-space fraction as approximated by the ventilatory ratio. Increased hypoxia signaling or SARS-CoV-2-mediated regulation of TCF12 are two potential mechanisms by which to explain these findings. These observations suggest that N9 P may represent a novel therapeutic target in patients with pulmonary vascular thrombosis due to SARS-CoV-2-ARDS. However, further investigation is needed to determine the pathogenetic, functional, and therapeutic relevance of N9 P in SARS-CoV-2-mediated pulmonary thrombosis.

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SUPPORTING INFORMATION Additional supporting information can be found online in the Supporting Information section at the end of this article. How to cite this article

This study is dedicated to the patients and families affected by the COVID- 19 

This study included only excess human lung tissue and was approved by the institutional review board at our institution (IRB #2020P001386, IRB# P00010717) with a waiver of informed consent.

George A. Alba https://orcid.org/0000-0002-6876-7836