Lung epithelial and endothelial damage, loss of tissue repair, inhibition of fibrinolysis, and cellular senescence in fatal COVID-19

Transcripts associated with the reactive oxygen species response were modulated in lung tissue.

“Transcripts associated with the reactive oxygen species (ROS) response were also modulated in lung tissue (Fig. 3D).”

Plasma concentrations of IFNLR1, DDX58, and leukemia inhibitory factor were significantly elevated in patients with COVID-19 compared to normal healthy volunteers.

“Multiplex measurements of immune response proteins in antemortem plasma samples from patients with COVID-19 (n = 6 patients sampled up to four times before death; n = 11 samples total) demonstrated that concentrations of IFN-related proteins IFNLR1, DDX58, and leukemia inhibitory factor were significantly (P < 0.05) elevated compared to normal healthy volunteers (n = 10) (fig. S8B).”

FGFR2 and SOX2 transcripts were decreased in lung tissue from patients with COVID-19.

“However, FGFR2 and SOX2 transcripts were decreased, as were transcripts of other genes involved in stem cell renewal and differentiation (BMP4 and BMPR1A) (30).”

Postmortem lung sections from all 18 cases showed DAD as the predominant histopathologic finding, ranging from acute DAD through organizing and late fibrotic DAD.

“The postmortem lung sections from all 18 cases showed DAD as the predominant histopathologic finding, ranging from acute DAD through organizing and late (fibrotic)–stage DAD (fig. S3 and table S11).”

• AssumptionRests on an assumption: The postmortem lung autopsy findings reflect the antemortem pathophysiological processes of fatal COVID-19.

Cases with longer illness duration displayed a pattern of multiple colocalized fibrin and tissue factor patches or fibrin balls in alveolar spaces that also expressed Col 1 but not αSMA.

“The first pattern featured multiple colocalized fibrin and tissue factor patches or fibrin “balls” in alveolar spaces that also expressed Col 1 but not αSMA (Fig. 3B).”

• AssumptionRests on an assumption: The symptom onset-to-death interval reflects the duration and stage of pulmonary disease progression.

Pulmonary vascular remodeling with excessive vessel wall-associated αSMA and Col 1 expression was prominent in intermediate and long-duration cases.

“Pulmonary vascular remodeling with excessive vessel wall–associated αSMA and Col 1 expression was another prominent finding in intermediate and long-duration cases (fig. S13).”

• AssumptionRests on an assumption: The symptom onset-to-death interval reflects the duration and stage of pulmonary disease progression.

There was a negative correlation between symptom onset-to-death time and SARS-CoV-2 RNA viral load (R = −0.722, P = 0.001).

“and SOTD with SARS-CoV-2 RNA viral load (R = −0.722, P = 0.001).”

Gene ontology analysis of transcripts increased in longer duration cases revealed enrichment of processes linked to fibrosis and wound repair, including fibrosis signaling, epithelial-mesenchymal transition, Wnt and stem cell-related signaling, adipogenesis, and macrophage, fibroblast, and endothelial cell function.

“Gene ontology analysis of transcripts with increased expression in longer duration cases revealed enrichment of processes linked to fibrosis and wound repair, including fibrosis signaling, epithelial-mesenchymal transition, Wnt and stem cell–related signaling, adipogenesis, as well as macrophage, fibroblast, and endothelial cell function (Fig. 4A).”

• AssumptionRests on an assumption: The symptom onset-to-death interval reflects the duration and stage of pulmonary disease progression.

Acute DAD was characterized by marked deposition and colocalization of intra-alveolar fibrin and tissue factor in hyaline membranes.

“Acute DAD was characterized by marked deposition and colocalization of intra-alveolar fibrin and tissue factor in hyaline membranes (Fig. 3A and fig. S3, A to D).”

There was a positive correlation between acute DAD and SARS-CoV-2 viral RNA load (R = 0.565, P = 0.024).

“a positive correlation was calculated between acute DAD and SARS-CoV-2 viral RNA load [correlation coefficient (R) = 0.565, P = 0.024]”

In cases with short SOTD, high viral antigen deposition was identified in AT1 and AT2 cells, bronchiolar epithelial cells including respiratory epithelial basal cells, and less frequently in vascular endothelial cells and adjacent pericytes.

“In cases with a short SOTD, high viral antigen deposition was identified by immunohistochemistry in AT1 and AT2 cells, bronchiolar epithelial cells (including respiratory epithelial basal cells) (Fig. 1B), and, less frequently, in the cytoplasm of vascular endothelial cells and adjacent pericytes of medium and small blood vessels in cases with the highest viral load (Fig. 1C and fig. S5).”

• AssumptionRests on an assumption: The postmortem lung autopsy findings reflect the antemortem pathophysiological processes of fatal COVID-19.

Plasma proteins associated with neutrophil and ROS responses, including IL8, CXCL10, EN-RAGE, IL6, and PRDX1, were elevated in lung tissue from patients with COVID-19.

“Plasma proteins associated with neutrophil and ROS responses including IL8, CXCL10, and EN-RAGE primarily derived from activated neutrophils (37), IL6, and PRDX1 were also elevated in lung tissue from patients with COVID-19 (Fig. 3E).”

A subset of neutrophils showed increased nuclear expression of citrullinated histone H3, an early indicator of neutrophil extracellular trap formation.

“A subset of neutrophils showed increased nuclear expression of citrullinated histone H3 (CitH3), an early indicator of neutrophil extracellular trap (NET) formation (fig. S15A).”

Expression of genes encoding type I and III interferons and other interferon signaling transcripts was increased in lung tissue from patients with COVID-19 compared to normal healthy control lung tissue.

“Expression of genes encoding type I and III IFNs, including IFNA4, IFNA5, IFNA10, IFNA21, IL28, and IL29, and other transcripts associated with IFN signaling was increased in lung tissue from patients with COVID-19 compared to normal healthy control lung tissue (fig. S8A).”

Lung tissue from patients with COVID-19 showed severely diminished and discontinuous claudin-5 and Col IV immunoreactivity.

“In contrast, lung tissue from patients with COVID-19 showed severely diminished and discontinuous claudin-5 and Col IV immunoreactivity.”

Differential expression of genes associated with neutrophil infiltration and activation was observed in lung tissue.

“Consistent with this neutrophil response, differential expression of genes associated with neutrophil infiltration and activation was observed in lung tissue (fig. S16).”

There was a positive correlation between fibrotic DAD and symptom onset-to-death time (R = 0.555, P = 0.017).

“and fibrotic DAD with SOTD (R = 0.555, P = 0.017).”

Cases with longer illness duration displayed a pattern of diffuse interstitial fibrosis with marked expression and colocalization of αSMA and Col 1.

“The second pattern featured diffuse interstitial fibrosis with marked expression and colocalization of αSMA and Col 1 (Fig. 3B).”

• AssumptionRests on an assumption: The symptom onset-to-death interval reflects the duration and stage of pulmonary disease progression.

Prominent CD163-positive macrophage accumulation was identified in cases with extensive fibrotic DAD compared to lower CD163 expression in normal lungs and acute DAD cases.

“Immunofluorescence analyses identified prominent CD163-positive macrophage accumulation in cases with extensive fibrotic DAD compared to lower CD163 expression in normal lungs and acute DAD cases (Fig. 4C and fig. S19).”

BMP4 and BMPR1A transcripts were decreased in lung tissue from patients with COVID-19.

“However, FGFR2 and SOX2 transcripts were decreased, as were transcripts of other genes involved in stem cell renewal and differentiation (BMP4 and BMPR1A) (30).”

There was a negative correlation between acute DAD and fibrotic DAD (R = −0.76, P = 0.00026).

“A negative correlation was calculated between acute DAD and fibrotic DAD (R = −0.76, P = 0.00026)”

Plasma concentrations of FGF2 and stem cell factor were decreased in patients with COVID-19.

“Plasma concentrations of FGF2 and stem cell factor, required for lung repair (31, 33), were also decreased in patients with COVID-19 (Fig. 1E).”

Prominent SARS-CoV-2 nucleocapsid protein staining was mainly detected in patients with shorter clinical illness (SOTD <10 days).

“Prominent NP staining was mainly detected in patients with shorter clinical illness (SOTD <10 days) (Fig. 1, A and B, and fig. S4).”

• AssumptionRests on an assumption: The postmortem lung autopsy findings reflect the antemortem pathophysiological processes of fatal COVID-19.
• AssumptionRests on an assumption: The symptom onset-to-death interval reflects the duration and stage of pulmonary disease progression.

Cases with short SOTDs showed extensive accumulation of MPO-positive neutrophils either trapped in alveolar septal capillaries or extravasated into fibrin-filled alveolar spaces.

“Cases with short SOTDs showed extensive accumulation of MPO-positive neutrophils either trapped in alveolar septal capillaries or extravasated into fibrin-filled alveolar spaces (Fig. 3C and fig. S14).”

• AssumptionRests on an assumption: The symptom onset-to-death interval reflects the duration and stage of pulmonary disease progression.

Plasma concentrations of amphiregulin were elevated in patients with COVID-19.

“Plasma concentrations of amphiregulin, an epidermal growth factor produced by damaged epithelial cells that promotes lung repair (34), were elevated in patients with COVID-19 (Fig. 1E).”

Widespread thrombosis was seen in most cases, ranging from medium-sized vessels to capillary microthrombi.

“Widespread thrombosis was seen in most of the cases, ranging from medium-sized vessels to capillary microthrombi (fig. S3, J to L).”

• AssumptionRests on an assumption: The postmortem lung autopsy findings reflect the antemortem pathophysiological processes of fatal COVID-19.

Expression of NFATc1, THBS1, FGF7, and FGF10 mRNAs was increased in lung tissue from patients with COVID-19.

“Expression of NFATc1, THBS1, FGF7, and FGF10 mRNAs, which are known to activate FGFR2 (31), was increased in lung tissue from patients with COVID-19 (Fig. 1D).”

Gene ontology analysis of transcripts positively correlated with SARS-CoV-2 viral load revealed enrichment for innate immune and antiviral responses, including interferon signaling, IRF activation, JAK/STAT signaling, and pattern recognition receptors.

“Gene ontology analysis of these sequences revealed enrichment for processes related to innate immune responses and antiviral responses, including interferon (IFN) signaling, IFN regulatory factor activation, Janus kinase/signal transducer and activator of transcription signaling, and pattern recognition receptors (Fig. 1F).”

There was a positive correlation between fibrotic DAD and age (R = 0.565, P = 0.015).

“fibrotic DAD with age (R = 0.565, P = 0.015)”

Transcripts negatively correlated with viral load were mainly enriched for cellular metabolism, including carbohydrate/lipid metabolism and molecular transport.

“These transcripts with reduced expression were mainly enriched for processes associated with cellular metabolism, including carbohydrate/lipid metabolism and molecular transport (fig. S9B).”

Released NETs costaining for CitH3, MPO, and cell-free DNA were sporadically detected in damaged lung parenchyma, airway lumens, alveolar spaces, and platelet-rich thrombi.

“Released NETs costaining for CitH3, MPO, and cell-free DNA were sporadically detected in damaged lung parenchyma, airway lumens, alveolar spaces, and platelet-rich thrombi (fig. S15, C to E).”

Acute DAD was the predominant histopathology in cases with the shortest recorded symptom onset-to-death times (<10 days).

“Acute DAD was the predominant histopathology observed in cases with the shortest recorded SOTDs (<10 days).”

• AssumptionRests on an assumption: The postmortem lung autopsy findings reflect the antemortem pathophysiological processes of fatal COVID-19.
• AssumptionRests on an assumption: The symptom onset-to-death interval reflects the duration and stage of pulmonary disease progression.

Reduced Pro-SPC and E-cadherin staining was observed in the alveolar septa of lung tissue from patients with COVID-19 relative to normal lungs, particularly in patients with a short SOTD.

“Reduced Pro-SPC and E-cadherin staining was observed in the alveolar septa of lung tissue from patients with COVID-19 relative to normal lungs, particularly in patients with a short SOTD (Fig. 2A and fig. S10, A and B).”

Patients with COVID-19 with higher viral loads tended to have a shorter symptom onset-to-death time.

“Principal component analysis of these transcripts showed that, in general, patients with COVID-19 with higher viral loads tended to have a shorter SOTD (fig. S7), consistent with a previous report (35).”

• cites [35] Nienhold R, Ciani Y, Koelzer VH, Tzankov A, Haslbauer JD, Menter T, Schwab N, Henkel M, Frank A, Zsikla V, Willi N, Kempf W, Hoyler T, Barbareschi M, Moch H, Tolnay M, Cathomas G, Demichelis F, Junt T, Mertz KD, Two distinct immunopathological profiles in autopsy lungs of COVID-19. Nat. Commun 11, 5086 (2020). 33033248 PMC7546638 sourceNot checked yet

  Not checked in the free run.

In all evaluated cases, the SARS-CoV-2 strains encoded the Spike D614G mutation.

“In all cases evaluated, the SARS-CoV-2 strains encoded this mutation (table S10).”

• AssumptionRests on an assumption: The postmortem lung autopsy findings reflect the antemortem pathophysiological processes of fatal COVID-19.

Processes related to respiratory function, including regulation of surfactant production from AT2 cells, negatively correlated with viral load.

“Processes related to respiratory function also negatively correlated with viral load, including regulation of surfactant production from AT2 cells.”

All 18 fatal COVID-19 autopsy lung tissues were positive for SARS-CoV-2 by qRT-PCR.

“RNA extracted from formalin-fixed, paraffin-embedded (FFPE) autopsy lung tissues were positive by quantitative reverse transcription polymerase chain reaction (qRT-PCR) for SARS-CoV-2 in all 18 cases (Table 1).”

• AssumptionRests on an assumption: The postmortem lung autopsy findings reflect the antemortem pathophysiological processes of fatal COVID-19.

Marked VWF extravasation into alveolar spaces was observed.

“Marked VWF extravasation into alveolar spaces was also observed, potentially because of alveolar-capillary barrier breakdown (Fig. 5, A to C).”

Endothelial and inflammatory cell expression of IL6 and TGFβ1 in pulmonary blood vessels and alveolar septal capillaries paralleled the localization of PAI-1.

“Endothelial and inflammatory cell expression of IL6 and TGFβ1 in pulmonary blood vessels and alveolar septal capillaries also paralleled the localization of PAI-1 (fig. S22A).”

Expression of SIRT1 mRNA was decreased in COVID-19 lung tissue.

“Expression of SIRT1 mRNA, a major inhibitor of oxidative stress–induced senescence in endothelial and epithelial cells as well as stem cells, was also decreased (Fig. 7F).”

This series of lung autopsy samples from patients with fatal SARS-CoV-2 infection showed a wide range of aberrant pulmonary responses to infection that were associated with viral load, immune response, and duration of clinical illness before death.

“This series of lung autopsy samples from patients with fatal SARS-CoV-2 infection showed a wide range of aberrant pulmonary responses to infection that were associated with viral load, immune response, and duration of clinical illness before death.”

Plasma concentrations of tissue factor and urokinase plasminogen activator were elevated in patients with COVID-19.

“Plasma concentrations of tissue factor, the major activator of the extrinsic coagulation pathway, and urokinase plasminogen activator (uPA), which mediates cleavage of plasminogen to plasmin that then breaks down fibrin clots, were also elevated (Fig. 5E).”

Injured or senescent epithelial and endothelial cells showed elevated nuclear expression of γH2A.X, a marker of ROS-induced DNA damage, in lung tissue from patients with COVID-19.

“Injured or senescent epithelial and endothelial cells also showed elevated nuclear expression of phosphorylated histone H2A.X (γH2A.X), a marker of ROS-induced DNA damage, in lung tissue from patients with COVID-19”

Prominent intravascular and parenchymal VWF immunoreactivity was observed in lung tissue from patients with COVID-19 compared to normal lungs.

“Representative immunofluorescence analyses revealed prominent intravascular and parenchymal VWF immunoreactivity in lung tissue from patients with COVID-19 compared to normal lungs (Fig. 5A and fig. S20, A and B).”

Gene expression analysis revealed increased expression of genes that promote senescence, including SERPINE1 (PAI-1), eNOS3, and the cell cycle inhibitors CDKN2A (p16) and CDKN1A (p21), in COVID-19 lung tissue.

“Gene expression analysis revealed increased expression of genes that promote senescence including SERPINE1 (PAI-1), eNOS3, and the cell cycle inhibitors CDKN2A (p16) and CDKN1A (p21).”

Decreased plasma concentrations of ADAMTS13 were observed in patients with COVID-19.

“Decreased plasma concentrations of ADAMTS13, the natural metalloprotease inhibitor of VWF, were also observed (Fig. 5E) (38).”

• cites [38] Bansal R, Nakagawa S, Yazdani S, van Baarlen J, Venkatesh A, Koh AP, Song WM, Goossens N, Watanabe H, Beasley MB, Powell CA, Storm G, Kaminski N, van Goor H, Friedman SL, Hoshida Y, Prakash J, Integrin alpha 11 in the regulation of the myofibroblast phenotype: Implications for fibrotic diseases. Exp. Mol. Med 49, e396 (2017). 29147013 PMC5704196 sourceNot checked yet

  Not checked in the free run.

Expression of genes encoding key inhibitors of fibrinolysis, including SERPINE1, SERPINF1, THBS1, and HRG, was elevated relative to normal lung tissue regardless of clinical illness duration.

“Expression of genes encoding key inhibitors of fibrinolysis, including SERPINE1 [PAI-1 (plasminogen activator inhibitor-1)], SERPINF1 (A2AP), THBS1 (thrombospondin 1), and HRG (histidine-rich glycoprotein) were elevated relative to normal lung tissue regardless of clinical illness duration (Fig. 5D).”

Prominent PAI-1 staining was detected on the endothelium of alveolar septal capillaries and in small- and medium-sized blood vessels containing unresolved clots with tightly packed polyhedrocyte-shaped red blood cells and distinctive entrapped PAI-1-positive cells.

“Prominent PAI-1 staining was also detected on the endothelium of alveolar septal capillaries and in small- and medium-sized blood vessels containing unresolved clots with tightly packed polyhedrocyte-shaped red blood cells and distinctive entrapped PAI-1–positive cells (Fig. 6, A and B).”

The study demonstrated epithelial and endothelial cell senescence in the pathophysiology of COVID-19.

“Our study also demonstrated epithelial and endothelial cell senescence in the pathophysiology of COVID-19, consistent with increased susceptibility and severity of COVID-19 in the elderly and comorbid risk populations.”

Increased nuclear expression of p21 was identified in vascular endothelium and hyperplastic and metaplastic E-cadherin-labeled epithelial cells in lung tissue from patients with COVID-19 compared to normal lungs.

“Immunofluorescence analyses identified increased nuclear expression of the cyclin-dependent kinase inhibitor p21, a marker of senescence, in vascular endothelium and hyperplastic and metaplastic E-cadherin–labeled epithelial cells in lung tissue from patients with COVID-19 compared to endothelial and epithelial cells in normal lungs (Fig. 7, A and B).”

Expression of genes associated with fibrin clot formation (FN1, FGA, and F13A1) was increased in lung tissue from patients with COVID-19.

“Consistent with histological evidence of clot formation with increased staining of VWF and fibrin, expression of genes associated with fibrin clot formation (FN1, FGA, and F13A1) was also increased in lung tissue from patients with COVID-19 (Fig. 5D).”

Cases with the shortest SOTD showed marked viral antigen deposition in alveolar and bronchiolar epithelial cells, including respiratory epithelial basal cells.

“Cases with the shortest SOTD showed marked viral antigen deposition in alveolar and bronchiolar epithelial cells, including respiratory epithelial basal cells.”

PAI-1-labeled neutrophils were negative for cleaved caspase 3 and CitH3.

“These PAI-1–labeled neutrophils were also negative for cleaved caspase 3 and CitH3, suggesting a nonapoptotic and non–NET-producing phenotype, respectively (figs. S21 and S15D).”

Colocalized staining of PAI-1 and TGFβ1 was detected in developing fibrotic lesions.

“Colocalized staining of PAI-1 and TGFβ1 was also detected in developing fibrotic lesions (fig. S22, B and C).”

Reduced alveolar septal expression of sirtuin-1 (Sirt-1) was observed in lung tissue from patients with COVID-19.

“Reduced alveolar septal expression of sirtuin-1 (Sirt-1), a nicotinamide adenine dinucleotide–dependent histone deacetylase involved in regulating inflammation, cellular senescence, and stress resistance was also observed (Fig. 7E).”

Elevated nuclear expression of phosphorylated histone H2A.X (γH2A.X), a marker of ROS-induced DNA damage, was observed in lung tissue from patients with COVID-19.

“elevated nuclear expression of phosphorylated histone H2A.X (γH2A.X), a marker of ROS-induced DNA damage, in lung tissue from patients with COVID-19 (Fig. 7D).”

Plasma concentrations of PAI-1 were increased in patients with COVID-19.

“Plasma concentrations of PAI-1 were also increased in patients with COVID-19 (Fig. 5E).”

Alveolar damage was accompanied by marked early reduction of pulmonary surfactant expression.

“Alveolar damage was accompanied by the marked early reduction of pulmonary surfactant expression, likely mediated by direct SARS-CoV-2 cytopathic effects on lung AT2 cells.”

Increased nuclear and cytoplasmic p21 expression was observed in αSMA-positive cells, presumably myofibroblasts, located in interstitial fibrotic foci in lungs from COVID-19 cases with longer illness duration.

“Increased nuclear and cytoplasmic p21 expression was also observed in αSMA-positive cells, presumably myofibroblasts, located in interstitial fibrotic foci in lungs from COVID-19 cases with longer illness duration (Fig. 7C).”

• AssumptionRests on an assumption: The symptom onset-to-death interval reflects the duration and stage of pulmonary disease progression.

High PAI-1 immunoreactivity was embedded in large fibrin- and MPO-positive blood clots.

“Immunofluorescence analyses revealed high PAI-1 immunoreactivity embedded in large fibrin- and MPO-positive blood clots (Fig. 6A).”

Cells expressing nuclear p21 showed minimal staining for the proliferation marker Ki67, providing further evidence of a senescence-associated growth arrest phenotype.

“Cells expressing nuclear p21 showed minimal staining for the proliferation marker Ki67, providing further evidence of a senescence-associated growth arrest phenotype (fig. S23).”

Large- and medium-sized thrombi containing intense VWF staining colocalized with CD61-positive platelets, MPO-positive neutrophils, and fibrin.

“Large- and medium-sized thrombi containing intense VWF staining colocalized with CD61-positive platelets, MPO-positive neutrophils, and fibrin (Fig. 5, B and C).”

Cases with a longer SOTD frequently showed denuded respiratory epithelium and a lack of lung tissue repair and regeneration.

“Cases with a longer SOTD frequently showed denuded respiratory epithelium and a lack of lung tissue repair and regeneration.”

Marked CitH3 staining was observed in platelet-rich thrombi but not in platelet-poor thrombi.

“Marked CitH3 staining was also observed in these platelet-rich thrombi but not in platelet-poor thrombi (fig. S15, D and E).”

SASP markers were elevated at the protein and gene levels in lung tissue from the COVID-19 autopsy case series, including PAI-1.

“SASP markers were elevated at the protein and gene levels in lung tissue from our COVID-19 autopsy case series, including PAI-1, which is considered both a major marker and mediator of cellular senescence (65).”

• cites [65] Vaughan DE, Rai R, Khan SS, Eren M, Ghosh AK, Plasminogen activator inhibitor-1 is a marker and a mediator of senescence. Arterioscler. Thromb. Vasc. Biol 37, 1446– 1452 (2017). 28572158 PMC5846199 sourceNot checked yet

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Increased nuclear p21 expression in hyperplastic and metaplastic epithelial cells as well as endothelial cells was detected in this COVID-19 lung autopsy case series.

“Increased nuclear p21 expression in hyperplastic and metaplastic epithelial cells as well as endothelial cells was detected in this COVID-19 lung autopsy case series.”

PAI-1-positive neutrophils were often identified in clotted vessels in this study.

“PAI-1–positive neutrophils were often identified in clotted vessels in this study, although it is not clear whether this reflected PAI-1 production by neutrophils or binding of plasma PAI-1 to the outer membrane of neutrophils.”

The study observed increased gene and protein expression of several antifibrinolytic factors including PAI-1, the main inhibitor of tissue-type plasminogen activator (tPA) and uPA.

“We also observed increased gene and protein expression of several antifibrinolytic factors including PAI-1, the main inhibitor of tissue-type plasminogen activator (tPA) and uPA.”

Long-duration cases commonly showed vascular remodeling in medium and large vessels.

“Long-duration cases commonly showed vascular remodeling in medium and large vessels.”

The major reported lung pathologies of severe COVID-19 include diffuse alveolar damage, organizing pneumonia, and chronic interstitial pneumonia.

“The major reported lung pathologies of severe COVID-19 include diffuse alveolar damage (DAD), organizing pneumonia, and chronic interstitial pneumonia (1–3, 5).”

• cites [1] Batah SS, Fabro AT, Pulmonary pathology of ARDS in COVID-19: A pathological review for clinicians. Respir. Med 176, 106239 (2021). 33246294 PMC7674971 sourceNot checked yet

  Not checked in the free run.

• cites [2] Bösmüller H, Matter M, Fend F, Tzankov A, The pulmonary pathology of COVID-19. Virchows Arch. 478, 137– 150 (2021). 33604758 PMC7892326 sourceNot checked yet

  Not checked in the free run.

• cites [3] Polak SB, Van Gool IC, Cohen D, von der Thüsen JH, van Paassen J, A systematic review of pathological findings in COVID-19: A pathophysiological timeline and possible mechanisms of disease progression. Mod. Pathol 33, 2128– 2138 (2020). 32572155 PMC7306927 sourceNot checked yet

  Not checked in the free run.

• cites [5] Sauter JL, Baine MK, Butnor KJ, Buonocore DJ, Chang JC, Jungbluth AA, Szabolcs MJ, Morjaria S, Mount SL, Rekhtman N, Selbs E, Sheng ZM, Xiao Y, Kleiner DE, Pittaluga S, Taubenberger JK, Rapkiewicz AV, Travis WD, Insights into pathogenesis of fatal COVID-19 pneumonia from histopathology with immunohistochemical and viral RNA studies. Histopathology 77, 915– 925 (2020). 32614086 PMC7361244 sourceNot checked yet

  Not checked in the free run.

The small sample size (n = 6 patients) made it difficult to identify potential differences due to the range of clinical illness and sampling time points relative to symptom onset.

“However, the small sample size (n = 6 patients) made it difficult to identify potential differences due to these variables.”

The decrease in plasma ITGA11 may reflect increased binding of ITGA11 to Col 1 in fibrotic lung tissue.

“ITGA11 selectively binds to Col 1 during fibrogenesis (38); thus, the decrease in plasma ITGA11 may reflect the increased binding of ITGA11 to Col 1 in fibrotic lung tissue.”

• cites [38] Bansal R, Nakagawa S, Yazdani S, van Baarlen J, Venkatesh A, Koh AP, Song WM, Goossens N, Watanabe H, Beasley MB, Powell CA, Storm G, Kaminski N, van Goor H, Friedman SL, Hoshida Y, Prakash J, Integrin alpha 11 in the regulation of the myofibroblast phenotype: Implications for fibrotic diseases. Exp. Mol. Med 49, e396 (2017). 29147013 PMC5704196 sourceNot checked yet

  Not checked in the free run.

Loss of surfactant gene expression is likely the result of viral infection and destruction of AT2 cells.

“Loss of surfactant gene expression is likely the result of viral infection and destruction of AT2 cells.”

Endothelial cell staining, while suggestive, does not definitively indicate productive viral infection of these cells.

“Endothelial cell staining while suggestive does not definitively indicate productive viral infection of these cells.”

Detection of nucleocapsid protein in airway basal cells suggests that basal cells may be early targets of cytopathic viral infection.

“Detection of NP in airway basal cells, which play key roles in repair and homeostasis of the respiratory epithelium (28), suggests that basal cells may be early targets of cytopathic viral infection.”

Loss of basal cells precludes regeneration of the airway epithelium, resulting in sustained respiratory compromise.

“Loss of basal cells precludes regeneration of the airway epithelium, resulting in sustained respiratory compromise (41).”

• cites [41] Reynolds SD, Giangreco A, Hong KU, McGrath KE, Ortiz LA, Stripp BR, Airway injury in lung disease pathophysiology: selective depletion of airway stem and progenitor cell pools potentiates lung inflammation and alveolar dysfunction. Am. J. Physiol. Lung Cell. Mol. Physiol 287, L1256– L1265 (2004). 15298853 sourceNot checked yet

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Greater staining of CitH3-positive neutrophils in lung tissue from short-duration cases compared to longer-duration cases may indicate a more active contribution of NETs to early pathological processes relative to later disease stages.

“The greater staining of CitH3-positive neutrophils in lung tissue from short-duration cases compared to longer-duration cases may indicate a more active contribution of NETs to early pathological processes relative to later disease stages.”

The data indicate that COVID-19 coagulopathies may be driven by an imbalance in the regulation of prothrombotic and anti-fibrinolytic processes.

“Together, these data indicate that COVID-19 coagulopathies may be driven by an imbalance in the regulation of prothrombotic and anti-fibrinolytic processes.”

Intra-alveolar neutrophils and injured alveolar epithelial cells are key producers of tissue factor, which directly stimulates fibrin deposition in alveolar spaces, hyaline membranes, and fibrotic foci.

“Intra-alveolar neutrophils and injured alveolar epithelial cells are also key producers of tissue factor, which directly stimulates fibrin deposition in alveolar spaces, hyaline membranes, and fibrotic foci (47, 48).”

• cites [47] Bastarache JA, Wang L, Geiser T, Wang Z, Albertine KH, Matthay MA, Ware LB, The alveolar epithelium can initiate the extrinsic coagulation cascade through expression of tissue factor. Thorax 62, 608– 616 (2007). 17356058 PMC2117249 sourceNot checked yet

  Not checked in the free run.

• cites [48] Kambas K, Markiewski MM, Pneumatikos IA, Rafail SS, Theodorou V, Konstantonis D, Kourtzelis I, Doumas MN, Magotti P, DeAngelis RA, Lambris JD, Ritis KD, C5a and TNF-alpha up-regulate the expression of tissue factor in intra-alveolar neutrophils of patients with the acute respiratory distress syndrome. J. Immunol 180, 7368– 7375 (2008). 18490736 PMC2673518 sourceNot checked yet

  Not checked in the free run.

A spectrum of pulmonary fibrotic disease has been observed in COVID-19 pneumonia from fibrosis associated with organizing pneumonia to severe acute lung injury and widespread fibrosis.

“A spectrum of pulmonary fibrotic disease has been observed in COVID-19 pneumonia from fibrosis associated with organizing pneumonia to severe acute lung injury and widespread fibrosis (52).”

• cites [52] Shi H, Han X, Jiang N, Cao Y, Alwalid O, Gu J, Fan Y, Zheng C, Radiological findings from 81 patients with COVID-19 pneumonia in Wuhan, China: A descriptive study. Lancet Infect. Dis 20, 425– 434 (2020). 32105637 PMC7159053 sourceNot checked yet

  Not checked in the free run.

VWF extravasation into alveolar spaces was potentially because of alveolar-capillary barrier breakdown.

“Marked VWF extravasation into alveolar spaces was also observed, potentially because of alveolar-capillary barrier breakdown (Fig. 5, A to C).”

Excessive and persistent damage to alveolar architecture often precedes dysregulated tissue repair processes and fibrosis.

“The observed loss of alveolar basement membrane integrity indicates excessive and persistent damage to the alveolar architecture, which often precedes dysregulated tissue repair processes and fibrosis (16, 17).”

• cites [16] Yanagi S, Tsubouchi H, Miura A, Matsumoto N, Nakazato M, Breakdown of epithelial barrier integrity and overdrive activation of alveolar epithelial cells in the pathogenesis of acute respiratory distress syndrome and lung fibrosis. Biomed. Res. Int 2015, 1– 12 (2015). PMC4615219 26523279 sourceNot checked yet

  Not checked in the free run.

• cites [17] Lucas A, Yasa J, Lucas M, Regeneration and repair in the healing lung. Clin. Transl. Immunol 9, e1152 (2020). PMC7338595 32665845 sourceNot checked yet

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Basal cells are stem cells that give rise to ciliated and secretory epithelial cells of the pseudostratified respiratory epithelium.

“basal cells are stem cells that give rise to ciliated and secretory epithelial cells of the pseudostratified respiratory epithelium (40).”

• cites [40] Hogan BLM, Barkauskas CE, Chapman HA, Epstein JA, Jain R, Hsia CCW, Niklason L, Calle E, Le A, Randell SH, Rock J, Snitow M, Krummel M, Stripp BR, Vu T, White ES, Whitsett JA, Morrisey EE, Repair and regeneration of the respiratory system: Complexity, plasticity, and mechanisms of lung stem cell function. Cell Stem Cell 15, 123– 138 (2014). 25105578 PMC4212493 sourceNot checked yet

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These observations suggested a temporal framework for disease progression that outlines a natural history and pathogenesis of SARS-CoV-2 infection.

“these observations nevertheless suggested a temporal framework for disease progression that outlines a natural history and pathogenesis of SARS-CoV-2 infection (fig. S17).”

• AssumptionRests on an assumption: Pathologic changes likely occurred asynchronously throughout the lung.

AT2 cell and surfactant loss may correlate clinically with diminished lung compliance and progressive respiratory failure in severe COVID-19.

“This AT2 cell and surfactant loss may correlate clinically with diminished lung compliance and progressive respiratory failure in severe COVID-19.”

Fatal COVID-19 appears to differ from fatal influenza in that direct pulmonary damage due to SARS-CoV-2 infection, and the immune response such damage elicits, is of sufficient severity that it does not require secondary bacterial copathogenesis as seen in influenza.

“Thus, fatal COVID-19 appears to differ from fatal influenza in that direct pulmonary damage due to SARS-CoV-2 infection, and the immune response such damage elicits, is of sufficient severity that it does not require secondary bacterial copathogenesis as seen in influenza.”

Increased TNF and caspase 8 concentrations with minimal evidence of caspase 3-dependent apoptosis suggested a role for inflammatory-driven cell death pathways (e.g., necroptosis).

“Increased TNF and caspase 8 concentrations with minimal evidence of caspase 3–dependent apoptosis suggested a role for inflammatory-driven cell death pathways (e.g., necroptosis), as previously reported (39).”

• cites [39] Li S, Zhang Y, Guan Z, Li H, Ye M, Chen X, Shen J, Zhou Y, Shi ZL, Zhou P, Peng K, SARS-CoV-2 triggers inflammatory responses and cell death through caspase-8 activation. Signal Transduct. Target. Ther 5, 235 (2020). 33037188 PMC7545816 sourceNot checked yet

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Activation of coagulation in fatal COVID-19 appears to coincide with inhibition of fibrinolysis in the lung.

“Activation of coagulation in fatal COVID-19 also appears to coincide with the inhibition of fibrinolysis in the lung.”

The marked early reduction of pulmonary surfactant expression was likely mediated by direct SARS-CoV-2 cytopathic effects on lung AT2 cells.

“Alveolar damage was accompanied by the marked early reduction of pulmonary surfactant expression, likely mediated by direct SARS-CoV-2 cytopathic effects on lung AT2 cells.”

NETs play a role in intra-alveolar thrombosis and the formation or organization of platelet-rich clots.

“NETs have been shown to promote tissue factor activation and fibrin deposition, consistent with our observations, suggesting a role for NETs in intra-alveolar thrombosis and the formation or organization of platelet-rich clots (43, 45).”

The data support a close link between cellular senescence processes, oxidative stress, pulmonary epithelial and endothelial damage, and defective repair in lung tissue from patients with fatal COVID-19.

“Together, these data support a close link between cellular senescence processes, oxidative stress, pulmonary epithelial and endothelial damage, and defective repair in lung tissue from patients with fatal COVID-19.”

Loss of alveolar basement membrane integrity indicates excessive and persistent damage to the alveolar architecture.

“The observed loss of alveolar basement membrane integrity indicates excessive and persistent damage to the alveolar architecture, which often precedes dysregulated tissue repair processes and fibrosis (16, 17).”

The finding of epithelial and endothelial cell senescence is consistent with increased susceptibility and severity of COVID-19 in the elderly and comorbid risk populations.

“Our study also demonstrated epithelial and endothelial cell senescence in the pathophysiology of COVID-19, consistent with increased susceptibility and severity of COVID-19 in the elderly and comorbid risk populations.”

Findings from this study and others indicate that inflammatory responses serve as a primary driver of the progressive and persistent pathophysiologic features of severe COVID-19.

“Findings from this study and others indicate that inflammatory responses serve as a primary driver of the progressive and persistent pathophysiologic features of severe COVID-19 (43–46).”

• cites [43] Borges L, Pithon-Curi TC, Curi R, Hatanaka E, COVID-19 and neutrophils: The relationship between hyperinflammation and neutrophil extracellular traps. Mediat. Inflamm 2020, 1– 7 (2020). PMC7732408 33343232 sourceNot checked yet

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• cites [44] Gustine JN, Jones D, Immunopathology of hyperinflammation in COVID-19. Am. J. Pathol 191, 4– 17 (2021). 32919977 PMC7484812 sourceNot checked yet

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• cites [45] Skendros P, Mitsios A, Chrysanthopoulou A, Mastellos DC, Metallidis S, Rafailidis P, Ntinopoulou M, Sertaridou E, Tsironidou V, Tsigalou C, Tektonidou M, Konstantinidis T, Papagoras C, Mitroulis I, Germanidis G, Lambris JD, Ritis K, Complement and tissue factor-enriched neutrophil extracellular traps are key drivers in COVID-19 immunothrombosis. J. Clin. Invest 130, 6151– 6157 (2020). 32759504 PMC7598040 sourceNot checked yet

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• cites [46] Zuo Y, Yalavarthi S, Shi H, Gockman K, Zuo M, Madison JA, Blair CN, Weber A, Barnes BJ, Egeblad M, Woods RJ, Kanthi Y, Knight JS, Neutrophil extracellular traps in COVID-19. JCI Insight 5, e138999 (2020). 32329756 PMC7308057Not checked yet

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• AssumptionRests on an assumption: Activated neutrophils, along with damaged epithelial and endothelial cells, release cytokines, chemokines, proteases, MMPs, and cytotoxic ROS, leading to DNA damage and perpetuation of alveolar destruction and respiratory dysfunction.

An enhanced prothrombotic environment may result, at least in part, from prominent intravascular expression of VWF caused by endothelial injury and neutrophil-mediated proinflammatory factors as well as by reduced plasma concentrations of the natural VWF inhibitor ADAMTS13.

“An enhanced prothrombotic environment may result, at least in part, from the prominent intravascular expression of VWF caused by endothelial injury and neutrophil-mediated proinflammatory factors as well as by reduced plasma concentrations of the natural VWF inhibitor ADAMTS13.”

Both SARS-CoV-1 and MERS-CoV infections have been associated with long-term fibrotic lung disease.

“Both SARS-CoV-1 and MERS-CoV infections have been associated with long-term fibrotic lung disease (84, 85).”

• cites [84] Das KM, Lee EY, Singh R, Enani MA, al Dossari K, van Gorkom K, Larsson SG, Langer RD, Follow-up chest radiographic findings in patients with MERS-CoV after recovery. Indian J. Radiol. Imaging 27, 342– 349 (2017). 29089687 PMC5644332 sourceNot checked yet

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• cites [85] Zhang P, Li J, Liu H, Han N, Ju J, Kou Y, Chen L, Jiang M, Pan F, Zheng Y, Gao Z, Jiang B, Long-term bone and lung consequences associated with hospital-acquired severe acute respiratory syndrome: A 15-year follow-up from a prospective cohort study. Bone Res. 8, 8 (2020). 32128276 PMC7018717 sourceNot checked yet

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Patients with severe COVID-19 were found to have a high rate of fibrotic lung function abnormalities at hospital release.

“Furthermore, patients with severe COVID-19 were found to have a high rate of fibrotic lung function abnormalities at hospital release, highlighting important implications for long-term health complications (54).”

• cites [54] Mo X, Jian W, Su Z, Chen M, Peng H, Peng P, Lei C, Chen R, Zhong N, Li S, Abnormal pulmonary function in COVID-19 patients at time of hospital discharge. Eur. Respir. J 55, 2001217 (2020). 32381497 PMC7236826 sourceNot checked yet

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The findings link the induction of epithelial and endothelial senescence to oxidative stress-induced DNA damage and modulation of ROS pathways.

“Our findings here also link the induction of epithelial and endothelial senescence to oxidative stress–induced DNA damage and modulation of ROS pathways.”

A review of 107 patients with COVID-19 showed that 7 to 13 days after illness onset is a critical stage in this disease.

“A review of 107 patients with COVID-19 showed that 7 to 13 days after illness onset is a critical stage in this disease (67).”

• cites [67] Wang D, Yin Y, Hu C, Liu X, Zhang X, Zhou S, Jian M, Xu H, Prowle J, Hu B, Li Y, Peng Z, Clinical course and outcome of 107 patients infected with the novel coronavirus, SARS-CoV-2, discharged from two hospitals in Wuhan, China. Crit Care 24, 188 (2020). 32354360 PMC7192564 sourceNot checked yet

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The marked incorporation of PAI-1 in abnormal or persistent clots supports a plausible mechanism that may explain poor clot resolution in COVID-19.

“Microvascular endothelium is a main site of PAI-1 production, and the marked incorporation of PAI-1 in abnormal or persistent clots supports a plausible mechanism that may explain poor clot resolution in COVID-19 (57).”

The time interval between symptom onset and requirement for critical care may represent a unique therapeutic window in which to counter progressive COVID-19 pathologies.

“The time interval between symptom onset and the requirement for critical care may therefore represent a unique therapeutic window in which to counter progressive COVID-19 pathologies.”

The overall number of patients in this case series was small.

“Whereas the present case series allowed for the examination of lung tissue from patients with COVID-19 with short, intermediate, or long clinical illness duration, the overall number of patients was small.”

Correlation analysis of the severity and clinical prognosis of patients with COVID-19 demonstrated that an increase in fibrosis indicators detected at hospital admission, including hyaluronic acid, laminin, and type III procollagen, was predictive of critical illness and poor prognosis.

“Correlation analysis of the severity and clinical prognosis of patients with COVID-19 demonstrated that an increase in fibrosis indicators detected at hospital admission, including hyaluronic acid, laminin, and type III procollagen, was predictive of critical illness and poor prognosis (49).”

• cites [49] Ding M, Zhang Q, Li Q, Wu T, Huang YZ, Correlation analysis of the severity and clinical prognosis of 32 cases of patients with COVID-19. Respir. Med 167, 105981 (2020). 32421546 PMC7167578 sourceNot checked yet

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Endothelial senescence may underlie the enhanced prothrombotic properties of injured endothelium in COVID-19 by shifting the balance between anti- and procoagulant pathways.

“Endothelial senescence may underlie the enhanced prothrombotic properties of injured endothelium in COVID-19 by shifting the balance between anti- and procoagulant pathways (63).”

• cites [63] Nehme J, Borghesan M, Mackedenski S, Bird TG, Demaria M, Cellular senescence as a potential mediator of COVID-19 severity in the elderly. Aging Cell 19, e13237 (2020). 32955770 PMC7576296 sourceNot checked yet

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Increased angiotensin II concentrations mediated by the actions of SARS-CoV-2 on ACE2 receptor-Ang II signaling may up-regulate ROS and cellular senescence pathways.

“Aside from the contribution of neutrophils and other cell types to ROS generation, increased angiotensin (Ang) II concentrations mediated by the actions of SARS-CoV-2 on ACE2 receptor–Ang II signaling may up-regulate ROS and cellular senescence pathways (64).”

• cites [64] Mogi M, Effect of renin-angiotensin system on senescence. Geriatr Gerontol Int 20, 520– 525 (2020). 32346971 sourceNot checked yet

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Clinical studies have demonstrated that tocilizumab lowers all-cause mortality in patients with COVID-19.

“Clinical trials have been conducted with the IL6-blocking agent tocilizumab in patients with COVID-19 (78), and clinical studies have demonstrated efficacy in lowering all-cause mortality (79).”

• cites [79] WHO Rapid Evidence Appraisal for COVID-19 Therapies (REACT) Working Group, Shankar-Hari M, Vale CL, Godolphin PJ, Fisher D, Higgins JPT, Spiga F, Savovic J, Tierney J, Baron G, Benbenishty JS, Berry LR, Broman N, Cavalcanti AB, Colman R, De Buyser SL, Derde LPG, Domingo P, Omar SF, Fernandez-Cruz A, Feuth T, Garcia F, Garcia-Vicuna R, Gonzalez-Alvaro I, Gordon AC, Haynes R, Hermine O, Horby PW, Horick NK, Kumar K, Lambrecht BN, Landray MJ, Leal L, Lederer DJ, Lorenzi E, Mariette X, Merchante N, Misnan NA, Mohan SV, Nivens MC, Oksi J, Perez-Molina JA, Pizov R, Porcher R, Postma S, Rajasuriar R, sourceNot checked yet

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The data presented here highlight key scientific links between these processes and common comorbidities including age, diabetes, and obesity that may help to define important determinants of disease severity and recovery.

“The data presented here highlight key scientific links between these processes and common comorbidities including age, diabetes, and obesity that may help to define important determinants of disease severity and recovery.”

Senescence-mediated loss of progenitor cell capabilities among AT2 and basal cell populations has been implicated in impaired reepithelialization of damaged alveoli and airways.

“Senescence-mediated loss of progenitor cell capabilities among AT2 and basal cell populations has been implicated in the impaired reepithelialization of damaged alveoli and airways (62).”

• cites [62] Kathiriya JJ, Brumwell AN, Jackson JR, Tang X, Chapman HA, Distinct airway epithelial stem cells hide among club cells but mobilize to promote alveolar regeneration. Cell Stem Cell 26, 346– 358.4 (2020). 31978363 PMC7233183 sourceNot checked yet

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Microvascular endothelium is a main site of PAI-1 production.

“Microvascular endothelium is a main site of PAI-1 production, and the marked incorporation of PAI-1 in abnormal or persistent clots supports a plausible mechanism that may explain poor clot resolution in COVID-19 (57).”

• cites [57] Bochenek ML, Schütz E, Schäfer K, Endothelial cell senescence and thrombosis: Ageing clots. Thromb. Res 147, 36– 45 (2016). 27669126 sourceNot checked yet

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Aberrant clotting reactions in COVID-19 are driven by an imbalance in prothrombotic and antifibrinolytic processes.

“Collectively, these observations suggest that aberrant clotting reactions in COVID-19 are driven by an imbalance in prothrombotic and antifibrinolytic processes.”

Fibrosis may occur independently of mechanical ventilation.

“This finding suggests that fibrosis may occur independently of mechanical ventilation.”