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

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Over 221 million confirmed severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infections with over 4.2 million coronavirus disease 2019 (COVID-19) deaths have been recorded worldwide as of September 2021. Many individuals suffer asymptomatic or mild-to-moderate symptoms, whereas others develop severe disease characterized by major pulmonary involvement, respiratory distress, systemic thrombosis, and death (14). Viral infection is mediated mainly by the binding of SARS-CoV-2 to the angiotensin-converting enzyme 2 (ACE2) receptor expressed on the surface of lung epithelial cells, endothelium, pericytes, and other cell types (14). The major reported lung pathologies of severe COVID-19 include diffuse alveolar damage (DAD), organizing pneumonia, and chronic interstitial pneumonia (13, 5). Pulmonary and systemic inflammation, vascular-related complications, including pulmonary embolisms, abnormal microthrombi, strokes, and direct blood vessel damage highlight the critical pathogenic involvement of endothelial dysfunction in severe COVID-19 (6, 7). Key risk factors associated with severe COVID-19 include advanced age (>80 years), diabetes, obesity, male gender, and hypertension (812).
Alveolar-capillary barrier dysfunction, which is characterized by impaired gas exchange, vascular leakage, and reduced fluid clearance, is a major cause of the acute respiratory distress syndrome (ARDS) in patients with severe COVID-19. The alveolar-capillary barrier consists of (1) the alveolar epithelium composed of simple squamous alveolar type 1 cells (AT1) and surfactant-producing cuboidal alveolar type 2 cells (AT2); (2) the alveolar capillary endothelium; and (3) the collagen- and laminin-rich alveolar basement membrane separated by a sparse connective tissue interstitium. Permeability across the alveolar epithelium and endothelium is tightly regulated by intercellular junctional complexes, including cadherin-expressing adherens junctions and claudin-rich tight junctions (1316). Loss or destabilization of epithelial or endothelial junctions is often a key feature of ARDS, acute lung injury, and microbial infections (1316). Direct endothelial damage or infection also disrupts vascular tone regulation as well as the anti-inflammatory and anti-thrombogenic properties of the endothelium. Damaged or denuded alveolar epithelium triggers repair processes that are often defective or overwhelmed during infection (16, 17).
Research efforts have advanced the understanding of the pathophysiology of SARS-CoV-2 infection. However, key questions remain regarding the factors associated with severe and fatal disease outcomes. Moreover, delineating the mechanisms underlying the increased disease severity in individuals with advanced age, hypertension, diabetes, and other risk factors remains paramount (811). In the present study, lung autopsy tissue samples from 18 patients with fatal COVID-19 and antemortem plasma samples from 6 of these cases were examined using viral genomics, host genetic susceptibility single-nucleotide polymorphism (SNP) analysis, serum antibody and multiplexed cytokine and chemokine protein measurements, and pulmonary transcriptomic and imaging analyses to better understand the molecular pathological processes underlying COVID-19 respiratory failure.


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. Pulmonary pathologic changes over a wide range of illness durations (SOTDs ranging from 3 to 47 days) were investigated. Although pathologic changes likely occurred asynchronously throughout the lung, and although each case examined resulted in a fatal outcome regardless of SOTD duration, these observations nevertheless suggested a temporal framework for disease progression, that is, a natural history and pathogenesis of SARS-CoV-2 infection (fig. S17).

Pathological observations indicated that both direct virus-induced cytopathic effects and host inflammatory and immune responses led to early pulmonary epithelial and endothelial injury, alveolar-capillary barrier dysfunction, impaired lung tissue repair processes, and widespread vascular thrombosis with reduced fibrinolysis. Furthermore, in cases with a longer disease duration, disease progression led to excessive pulmonary fibrosis, loss of alveoli, and vascular remodeling. 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.

DAD and alveolar-capillary barrier breakdown are prominent pathological features of severe COVID-19 (14). This lung autopsy series showed that pulmonary SARS-CoV-2 infection damaged all three major components of the alveolar-capillary barrier including loss of alveolar epithelial and endothelial junction integrity, marked desquamation of lung AT1 and AT2 cells, and disruption of the collagen IV-expressing alveolar basement membrane. 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). 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). 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. This AT2 cell and surfactant loss may correlate clinically with diminished lung compliance and progressive respiratory failure in severe COVID-19. Cases with the shortest SOTD showed marked viral antigen deposition in alveolar and bronchiolar epithelial cells, including respiratory epithelial basal cells. Cases with a longer SOTD frequently showed denuded respiratory epithelium and a lack of lung tissue repair and regeneration. The acute damage was significant because basal cells are stem cells that give rise to ciliated and secretory epithelial cells of the pseudostratified respiratory epithelium (40). Loss of basal cells precludes regeneration of the airway epithelium, resulting in sustained respiratory compromise (41). Direct viral infection of basal cells in fatal COVID-19 contrasts with fatal influenza viral infection where viral replication does not occur in basal cells but associated secondary bacterial infections result in basal cell loss and lack of tissue repair and regeneration (42). 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 co-pathogenesis as seen in influenza.
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 (4346). In short SOTD cases, alveolar-capillary breakdown coincided with prominent recruitment of MPO-positive neutrophils in damaged alveoli with excessive fibrin and tissue factor deposition, in thickened alveolar interstitial spaces, and along the endothelial lining of injured blood vessels. Activated neutrophils, along with damaged epithelial and endothelial cells, release a host of cytokines, chemokines, proteases, matrix metalloproteinases, and cytotoxic ROS, leading to DNA damage and perpetuation of alveolar destruction and respiratory dysfunction. Neutrophil extracellular traps (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 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. 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).
Prominent pulmonary fibrosis was observed in this lung autopsy series consistent with previous reports (4951), and correlated with SOTD and patient age (fig. S17). 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). A systematic review of 45 studies of chest computed tomography (CT) images of patients with COVID-19 demonstrated pulmonary fibrosis in 17% of patients, which was attributed to impaired healing after viral infection of lung AT2 cells (53). Correlation analysis of the severity and clinical prognosis of COVID-19 patients demonstrated that an increase in fibrosis indicators detected at hospital admission, including hyaluronic acid, laminin, and type III procollagen, were predictive of critical illness and poor prognosis (49). This finding suggests that fibrosis may occur independently of mechanical ventilation. 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 complications (54).
Vascular dysfunction and thrombotic complications are hallmark features of COVID-19 (55). The present study revealed evidence of viral antigen staining in pulmonary vascular endothelial cells in cases with short SOTD and high viral loads, and a high frequency of thrombi in small and medium-sized pulmonary blood vessels, particularly in short and intermediate SOTD cases. Long duration cases commonly showed vascular remodeling in medium and large vessels. Collectively, these observations suggest that SARS-CoV-2 infection is followed by an imbalance in pro-thrombotic and anti-fibrinolytic processes that drives aberrant thrombotic reactions. This has been previously proposed for COVID-19 and other coronaviral diseases including SARS-CoV-1 and MERS-CoV (56). An enhanced prothrombotic environment may result, at least in part, from the prominent endothelial and intravascular expression of von Willebrand factor (VWF) caused by endothelial injury and neutrophil-mediated proinflammatory factors as well as by reduced plasma concentrations of the natural VWF inhibitor ADAMTS13. We also observed increased gene and protein expression of several anti-fibrinolytic factors including PAI-1, the main inhibitor of tissue-type plasminogen activator (tPA) and (uPA). 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). 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. A recent study reported a correlation between elevated PAI-1 plasma concentrations and circulating neutrophils in COVID-19 patients (58). It may be particularly relevant that risk factors for developing severe COVID-19 such as advanced age, obesity, diabetes, and vascular disease are all associated with increased plasma PAI-1 concentrations (59, 60).
Cellular senescence is defined by a stable growth arrest regulated by a family of CDK inhibitors including p21 and p16 (61). 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. Senescence-mediated loss of progenitor cell capabilities among AT2 and basal cell populations has been implicated in the impaired re-epithelialization of damaged alveoli and airways (62). Endothelial senescence may underlie the enhanced prothrombotic properties of injured endothelium in COVID-19 by shifting the balance between anti‐ and pro-coagulant pathways (63). Our findings here also link the induction of epithelial and endothelial senescence to oxidative stress-induced DNA damage and modulation of ROS pathways. Aside from the contribution of neutrophils and other cell types to ROS generation, increased angiotensin II (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). Senescent cells exhibit a senescence-associated secretory phenotype (SASP) characterized by an overproduction of proinflammatory molecules such as IL6, TGFβ, IL8, MCP1, extracellular matrix remodeling enzymes (MMPs), serine/cysteine proteinase inhibitors (Serpins), and tissue inhibitors of metalloproteinases (TIMPs) (61). SASP markers were elevated at the protein and gene level 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). Notably, key clinical risk factors for developing severe COVID-19 such as advanced age, obesity, diabetes, and vascular disease are all characterized by the activation or exacerbation of cellular senescence processes (66).

There are certain limitations to our study. Whereas the present case series allowed for the examination of lung tissue from COVID-19 patients with short, intermediate, or long clinical illness duration, the overall number of patients was small. Larger autopsy studies will be needed to specifically examine mitigating factors including varying patient demographics, comorbidities, and in-hospital interventions (e.g., mechanical ventilatory support). Another potential concern is related to lung tissue sampling, and specifically, whether the available lung specimens used for imaging and transcriptomic analyses were representative of the whole lung. Finally, as all cases in this study were fatal, it is unknown to what extent these pathological processes occur in mild to moderate or hospitalized, non-fatal COVID-19 cases, or in cases with long-term sequelae post-SARS-CoV-2 infection.

Understanding the natural history and pathogenesis of COVID-19 at the cellular and immunological level can potentially provide clues to different therapeutic interventions at different stages of disease progression. Such interventions could reduce the number of patients progressing to severe disease who require intensive care and mechanical ventilation, as well as reducing the number of individuals experiencing long-term impaired pulmonary function. A review of 107 patients with COVID-19 showed that 7-13 days after illness onset is a critical stage in this disease (67). Additional studies found that among patients who developed severe disease, the median time to dyspnea was 5-8 days, the median time to ARDS was 8-12 days, and the median time to ICU admission was 10-12 days after symptom onset (6870). Importantly, 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.
Due to the complexity of COVID-19 pathology, combination treatment with both direct acting antiviral drugs such as remdesivir and therapeutics that modulate damaging host immune responses are likely needed (7174). Treatment with corticosteroids, acting on multiple anti-inflammatory pathways (75), has been shown to have some efficacy in COVID-19 (76). More targeted anti-inflammatory drugs, including IFNβ, mavrilimumab (a monoclonal antibody targeting GM-CSF), modified tetracyclines, and ROS scavengers (e.g., N-acetylcysteine) are also being evaluated. ROS scavengers have shown efficacy in influenza (77) which, as noted, has certain clinical and pathologic similarities to COVID-19. Clinical trials have been conducted with the IL6-blocking agent tocilizumab in COVID-19 patients (78), and clinical studies have demonstrated efficacy in lowering all-cause mortality (79). Others have reported that targeting surfactant deficiency may improve respiratory function in COVID-19 patients (80, 81). Mesenchymal stem cell (MSC) therapy has also been proposed as a treatment for COVID-19 (82, 83). Clinical trials have shown reduced mortality in patients with influenza A/H7N9 virus-induced ARDS treated with transplanted MSCs, and ongoing trials are currently investigating stem cell therapies for treating COVID-19 patients (83).
Another critical concern is the resolution of pulmonary fibrosis secondary to severe COVID-19 in surviving patients. Indeed, both SARS-CoV-1 and MERS-CoV infections have been associated with long-term fibrotic lung disease (84, 85). Clinical trials specifically targeting fibrosis using pirfenidone ( Identifier: NCT04282902, NCT04607928, NCT04653831) and LYT-100 (deupirfenidone) (NCT04652518) are currently underway. The anti-oxidative and anti-inflammatory properties of pirfenidone may also target ROS-mediated and DNA-mediated damage to epithelial and endothelial cell populations. Other inhaled or intravenous fibrinolytic therapies for COVID-19-related alveolar fibrosis and coagulopathies have also received considerable attention (e.g., tPA) (86, 87). Therapeutic targeting of PAI-1 using the inhibitory compound TM5614 is currently being tested in hospitalized patients with severe COVID-19 to treat both fibrotic and thrombotic complications ( Identifier: NCT04634799). Efforts have also focused on using antithrombotic therapy such as heparin and low-molecular-weight heparins in COVID-19, although an incomplete understanding of thrombogenic risk factors and reports of increased pulmonary hemorrhage in certain COVID-19 patients have complicated the establishment of optimal anticoagulation treatments for COVID-19 (8890). Elevated plasma tissue factor and D-dimer concentrations observed in these COVID-19 patients suggest that these mediators may be useful markers of ongoing endothelial dysfunction and hypercoagulability, and warrant further investigation of tissue factor as a possible therapeutic target ( Identifier: NCT04655586). Potential therapeutic approaches targeting cellular senescence to improve epithelial and endothelial cell function include the use of new senolytic compounds, such as ABT-263 (Navitodax), which promote clearance of senescent cells by inhibiting pro-survival pathways (9193). Manipulation of specific components of the SASP represents another potential therapeutic strategy to counter cellular senescence (93).

In summary, the present study provides insight into the pathological, immunological, and host genetic correlates of progressive pulmonary failure and impaired tissue repair in fatal COVID-19. 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. A more complete understanding of the specific interplay between these pulmonary responses and cellular senescence-driven risk factors may prove critical in the development of relevant disease markers and urgently needed therapeutics.



We thank David M. Morens for helpful discussions.

Funding: This project (1ZIAAI001271-01) was funded by the Intramural Research Program of the National Institute of Allergy and Infectious Diseases of the NIH (to JKT), by Intramural funding from the Center for Biologics Evaluation and Research, FDA (to FD), an early postdoctoral mobility fellowship (P2BSP3_188158) awarded by the Swiss National Science Foundation (to SG), and the Intramural Research Program of the National Institute of Biomedical Imaging and Bioengineering (to KS). This article reflects the views of the authors and should not be construed to represent FDA’s views or policies.

Author contributions: FD, KAW, JCK, JKT conceived and designed experiments, analyzed and interpreted data, drafted and revised the article. YX, ZMS, SG, KS, and LAR performed molecular experiments. FD, ZMS, and JKT performed histological and immunohistochemical experiments. FD and JKT analyzed pathological findings. JP, LAR, KS, HK, and MM performed serological analyses. FD, KAW, JCK, CAB, RZ, LG and CB performed statistical analyses. SJO, TDF, TCH, RCL, JCC, MEP, GEO, KJB, AVR, WDT, and SPL contributed autopsy tissue and plasma samples. FD, KAW, YX, JCK, and JKT performed data curation.

Competing interests: SJO has received funding from Agenus, Amgen, Biothera, Bristol Myers Squibb, Exicure, Genocea, Incyte, Merck, Ultimovacs, Viralytics. SJO has consulted for Biothera, Bristol Myers Squibb, BionTech, Exicure, Immunsys, Merck. KS is an inventor on a provisional U.S. patent application no. 63/092,350 entitled “Antibody specific for SARS-CoV-2 receptor binding domain and therapeutic methods.” The other authors declare no competing interests.

Data and materials availability: All data associated with this study are available in the main text or the supplementary materials. The complete MIAME-compliant microarray data set has been deposited in NCBI’s Gene Expression Omnibus ( and is accessible through GEO Series accession number GSE180226.
This work is licensed under a Creative Commons Attribution 4.0 International (CC BY 4.0) license, which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. To view a copy of this license, visit This license does not apply to figures/photos/artwork or other content included in the article that is credited to a third party; obtain authorization from the rights holder before using this material.

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