Pulmonary and Systemic Pathology in COVID-19: Holistic Pathological Analyses

Background: The COVID-19 pandemic is the third worldwide coronavirus-associated disease outbreak in the past 20 years. Lung involvement, with acute respiratory distress syndrome (ARDS) in severe cases, is the main clinical feature of this disease; the cardiovascular system, the central nervous system, and the gastrointestinal tract can also be affected. The pathophysiology of both pulmonary and extrapulmonary organ damage was almost completely unknown when the pandemic began.

Methods: This review is based on pertinent publications retrieved by a selective search concerning the structural changes and pathophysiology of COVID-19, with a focus on imaging techniques.

Results: Immunohistochemical, electron-microscopic and molecular pathological analyses of tissues obtained by autopsy have improved our understanding of COVID-19 pathophysiology, including molecular regulatory mechanisms. Intussusceptive angiogenesis (IA) has been found to be a prominent pattern of damage in the affected organs of COVID-19 patients. In IA, an existing vessel changes by invagination of the endothelium and formation of an intraluminal septum, ultimately giving rise to two new lumina. This alters hemodynamics within the vessel, leading to a loss of laminar flow and its replacement by turbulent, inhomogeneous flow. IA, which arises because of ischemia due to thrombosis, is itself a risk factor for the generation of further microthrombi; these have been detected in the lungs, heart, liver, kidneys, brain, and placenta of COVID-19 patients.

Conclusion: Studies of autopsy material from various tissues of COVID-19 patients have revealed ultrastructural evidence of altered microvascularity, IA, and multifocal thrombi. These changes may contribute to the pathophysiology of post-acute interstitial fibrotic organ changes as well as to the clinical picture of long COVID.

COVID-19 (1) is the third global coronavirus-associated disease outbreak in 20 years – following the severe acute respiratory syndrome (SARS) in 2002–2004 (e1) and the Middle East Respiratory Syndrome (MERS) in 2012–2015 (e2). Infection with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) is associated with a broad spectrum of clinical presentations, ranging from mild upper airway infection to life-threatening viremia with multi-organ involvement.

Besides the lungs (2), other organ systems may also be involved, such as the cardiovascular system (3), the central nervous system (4) and the gastrointestinal tract (5). Initially, the pathophysiology of both pulmonary and extrapulmonary organ damage was almost completely unknown, making therapeutic interventions as well as predictions of the clinical course and prognosis of the disease extremely difficult. The first groundbreaking studies to shed light on the pathophysiology of COVID-19 were all tissue-based; initially examining lung tissue of patients who had died of COVID-19 (5, 6, e3, e4, e5).

The acute respiratory distress syndrome in COVID-19

In the majority of the cases with severe disease, acute respiratory distress syndrome (ARDS) was the most prominent clinical feature (7). This condition is characterized by impaired pulmonary gas exchange so that many of these patients required mechanical ventilation and/or extracorporeal membrane oxygenation (ECMO). Radiography shows a “white” lung with a diffuse, bilateral increase in parenchymal density (Figure 1, eFigure 1). Morphologically, the so-called diffuse alveolar wall damage is characterized by epithelial necrosis and intra-alveolar fibrin deposition (Figure 2) (8). However, neither the radiographic nor the morphological pattern of injury is pathognomonic for COVID-19 pneumonia, as they may also be seen in patients with ARDS due to other infectious (influenza) or non-infectious (shock, adverse drug reaction, toxic inhalation) causes. Thus, novel and above all cross-method approaches are required to accomplish a more precise description of the cause and pathogenesis of the lung damage.

Figure 1

Clinicoradiographic pattern of lung damage in COVID-19 and scaling down to the microscopic level using hierarchical phase-contrast tomography (HIP-CT)

Figure 2

Histological changes in the lungs in COVID-19 over time

eFigure 1

Clinicoradiographic pattern of lung damage in COVID-19 and scaling down to the microscopic level using hierarchical phase-contrast tomography (HIP-CT)

Radiographic evidence of systemic organ involvement

Despite limited specificity, computed tomography (CT) remains the main imaging modality of the COVID-19 pandemic. A CT scan is sensitive, widely available, and quick to perform and interpret; in addition, it can confirm the presence of pneumonia in symptomatic patients, classify its severity and rule out differential diagnoses (9). Rather typical CT patterns of COVID-19 damage to the lungs include (9, e6):

• In the early phase, ground-glass opacities (GGOs), commonly with patchy and peripheral distribution pattern
• Interstitial changes with inter- and intralobular septal thickening, as well as
• Alveolar ground-glass opacities with “crazy paving pattern” (superimposed irregular reticulation pattern) (Figure 1, eFigure 1).

Besides the bowels, the diagnostic work-up of the non-pulmonary organ systems focuses on the brain and the heart. Early on, potential signs of COVID-19-related vascular changes were noted.

Whereas changes in macro-circulation can be well detected on conventional CT after contrast administration, the in-vivo detection of microvascular changes requires the use of newer technologies: dual- and multi-energy CT, or magnetic resonance imaging (MRI) (e7, 10, 11) Figure 1, eFigure 1). With this approach, cerebral ischemic damage patterns and microhemorrhages can be detected on special diffusion-weighted or susceptibility-weighted sequences (12, e8). However, the findings obtained with these novel and often complex imaging techniques are also not specific to COVID-19. They can occur in a comparable form with other infections too, for example in the case of severe systemic inflammation associated with influenza (13).

COVID-19 as a vascular system disease

With the adoption of immunohistochemistry, electron microscopy and molecular pathology methods, modern pathology has advanced beyond purely morphological description. It thus provides a bridge from macroscopy to the regulation of the affected individual cell, in line with a contemporary approach to the concept of cellular pathology developed by Rudolf Virchow (14). The sequential use of these methods on autopsy tissues has led to a better understanding of the pathophysiology of COVID-19, including the molecular regulatory mechanisms (Figure 2, eFigure 2) and targeted treatment approaches (15).

Figure 3

Multi-resolution imaging of a COVID-19 autopsy lung, using hierarchical phase contrast tomography (HiP-CT)

eFigure 3

Multi-resolution imaging of a COVID-19 autopsy lung, using hierarchical phase contrast tomography (HiP-CT)

eFigure 2

Histological and molecular pathology changes in the lungs in COVID-19 over time

Injury to the protective endothelial barrier results in significant functional impairments, such as hypercoagulability and abnormal vascular tone. The three-dimensional visualization of these alterations as well as microvascular pulmonary changes were for the first time achieved by means of micro-CT-based ex-vivo imaging (e9, 15) (eFigure 4). Besides the known metabolic risk factors, such as arterial hypertension and dyslipidemia, numerous viral diseases result in injury to endothelial cells, either indirectly (e.g., influenza or HIV) or directly (SARS-CoV-2, parvovirus B19) (e10, 16).

Figure 4

Scanning electron microscope images show changes in alveolar lung architecture in COVID-19 (b) in comparison to the physiological architecture of the lungs (a)

eFigure 4

In patients with COVID-19, the endothelial injury triggers an angiocentric inflammatory reaction (e3) and ultimately leads to repair mechanisms, which, in case of COVID-19, include a particular, functionally only to a certain extent favorable variant of angiogenesis, the so-called intussusceptive angiogenesis (IA) (Figure 4b, eFigure 4, eFigure 5). In patients with severe COVID-19, IA features were documented in histological lung preparations with a density of 60.7 ± 11.8 per section; in influenza patients of 22.5 ± 6.9 and in controls of 2.1 ± 0.6 (p<0.001) (e3). In addition, one study reported that in a total of 24 patients, seven of whom had COVID-19, the extent of IA formation in COVID-19 patients—but not in influenza patients— correlated with the length of hospital stay. Besides IA, severe endothelial damage with intracellular SARS-CoV-2 viruses and numerous thromboses with microangiopathy and occlusion of alveolar capillaries was observed.

eFigure 5

COVID-19 in other organs

Direct virus-induced cytopathic effects alone could not be responsible for the capillary microthrombi observed, because significantly less microthrombi are seen, for example, in patients with pneumonia due to SARS or influenza. By immunophenotyping the inflammatory infiltrate and by means of mRNA expression analyses, it was shown that in the hypoxic microenvironment associated with COVID-19 initially specific CD11+/TIE2+ macrophages are recruited.

In COVD-19 cases with multi-organ involvement, macrophages dominate the acute picture of inflammatory infiltration of the lungs (eFigure 2), heart (eFigure 5), kidney, liver, and muscles (17). By releasing and activating prothrombotic factors and immunothrombosis formation, so-called neutrophil extracellular traps (NETs) (18), these macrophages first trigger a self-sustaining vicious circle of thrombosis formation. Initial therapeutic approaches were based on inhibiting inflammation and modulating hypercoagulability.

However, the association between the occurrence of macrophages and the presence of capillary microthrombi could not be adequately explained by the methods used in conventional histology and immunohistology. It was only when transmission electron microscopy (TEM) was used in combination with novel methods of vascular imaging—which are based on the injection of a polyurethane polymer into the vascular bed—that it became possible to demonstrate the presence of IA (e11). IA is a form of angiogenesis (19) already known for some time. Beyond intrauterine organ development, it has so far been described in cancer (e12, 20), colitis (21) and interstitial lung disease (22). In contrast to classic sprouting angiogenesis, IA involves the splitting of an existing vessel by intussusception of the endothelium and formation of an intraluminal septum (e11). The septum is then elongated by embedment of angiogenic precursor cells, a process that ultimately leads to the creation of two new lumina. Compared to sprouting neoangiogenesis, IA is a potentially rapid response of tissue to hypoxia. However, these advantages of IA come at the price of more pronounced changes in hemodynamics, including the loss of laminar flow and resulting turbulent inhomogeneous flow (23) which is, in turn, associated with an increased risk of microthrombi formation. Meanwhile, IA has been found in COVID-19 patients, and not only in the lungs, but also in heart, liver, kidneys, brain, and placenta, sometimes even long after a COVID-19 infection had cleared. Thus, IA may be a potential link to structural organ changes associated with long COVID.

Cardiac involvement in COVID-19

Acute myocardial involvement is found in approximately 15% to 35% of hospitalized patients with COVID-19 (24). Even though serological detection of increased troponin levels is a robust biomarker of cardiac involvement, such increases are common among ICU patients regardless of the primary disease and cardiac symptoms such as acute heart failure, reduced ejection fraction and arrhythmia are rarely the main clinical concern in patients with COVID-19 (25). Radiographic evidence of active myocarditis can be obtained using advanced MRI techniques, such as late gadolinium-enhanced inversion-recovery sequences (26).

The pathophysiology and development of SARS-CoV-2-mediated heart damage are as yet poorly understood, but direct and indirect mechanisms of injury are being discussed:

• Direct cell damage due to entry of SARS-CoV-2 into the myocardium via angiotensin-converting-enzyme 2 (27)
• Infection of endothelial cells by SARS-CoV-2 and resulting injury to the tissue supplied by the blood vessel
• Indirect cell damage due to impaired microcirculation along with hypercoagulability as well as microthrombi formation with impairment of cardiac capillary flow
• Indirect cell damage due to systemic release of proinflammatory cytokines (such as IL-1, IL-6, TNF-α, and IFN-γ) with subsequent cytokine storm (28).

Currently, however, the assumptions are solely based on autopsy studies in the form of small case series of patients with severe disease (29). In conventional histopathology, only a discrete infiltration by interstitial macrophages is observed; according to current Dallas criteria, this is classified as a borderline myocarditis. Nevertheless, in the heart this macrophage population, although small in numbers, seems to trigger a vasculocentric inflammatory response with prominent IA and change in hemodynamics, and is thus to be regarded as the cause of the clinical symptoms (30). In the hearts of deceased COVID-19 patients, ultrastructural evidence of changes in microvascularity, increased IA as well as multifocal thrombi was found; none of these were visible in conventional light microscopy. Such changes were not found in patients with non-COVID-19-associated myocarditis. As in the lungs, a clearly divergent inflammatory reaction on the level of gene expression was revealed between patients with COVID-19 and patients with influenza. To what extent this vascular damage and remodeling (eFigure 4, eFigure 5) could trigger interstitial fibrosis and thus long-term damage to the heart, is the subject of current research.

Synchrotron-based phase-contrast tomography

The techniques of histology and electron microscopy used previously only supported the analysis of small and very small tissue samples, leaving the distribution and the actual extent of the (vascular) changes in the affected organs unknown. With the help of the extremely brilliant source (EBS) of the European Synchrotron Radiation Facility (ESRF), for the first time whole organs could be analyzed non-destructively in three dimensions on the single-cell level (Figure 3, eFigure 3, eBox) (31, e13, e14, e15).

eBox

Synchrotron-based phase-contrast tomography (SPT)

This paved the way for a new approach to deciphering pathophysiological processes of many diseases across system boundaries. Meanwhile, the Human Organ Atlas has been established, an online database, providing scientists worldwide with free access to the data of synchrotron-scanned organs (33). Long-COVID syndrome

Given the evidence of vascular remodeling described in the context of acute COVID-19 infection, the question arises whether this is also functionally related to the clinically described pulmonary and cardiac long-term effects of COVID 19. A variety of clinical symptoms are grouped together as long-COVID syndrome, including persistent dyspnea, chest pain, palpitations, fatigue, nausea and vomiting, among others (34). Such disease courses have been described not only for ARDS survivors but also for patients with milder disease, albeit with lower frequency.

The fibrovascular interface tissue, composed of vascular (endothelial cells, macrophages) and (myo-) fibroblast components, plays a key role here (e3, e16). The altered vascular network post-COVID-19 shows impaired microcirculation as well as altered signal transmission in the extracellular matrix, triggering fibroblast activation and progressive fibrosis of the interstitium (eFigure 2). The resulting local hypoxemia with matrix densification contributes to the structural fixation of organ dysfunction and could trigger a functional downward spiral by further promoting IA.

In addition to the described vascular injury sequence, autoimmune responses are held responsible for the cardiac (long-term) effects in particular—based on findings of noninvasive imaging, such as MRI (e17, 34). Especially in the context of re-exposure of macrophages to the spike protein of SARS-CoV-2, a hyperinflammatory syndrome has been described, similar to the response to BCG vaccine or infection with mycobacterium tuberculosis (35). Macrophages show similarities in their activation on the transcriptome, miRNA and epigenetic levels and exhibit immune memory: In mononuclear cells of COVID-19 convalescents, rapid inflammasome activation was demonstrated following stimulation with S protein of SARS-CoV-2 (36). This rapid activation of the immune response, even months after recovery from the infection, is based on SARS-CoV-2-induced epigenetic changes in macrophages (37). Similar effects were described in individuals vaccinated with the BCG (tuberculosis) live vaccine, in whom a reduction in the number of cases of severe COVID-19 disease was documented (36). In line with the proposed autoimmune component, initial observational studies showed a positive effect of COVID-19 vaccination on pre-existing long COVID symptoms (38).

German COVID-19 autopsy registry

Tissue from autopsies on COVID-19 deaths with appropriate clinical annotation is indispensable for COVID-19 research. The available studies are substantiated, among others, based on samples of the German Network for Autopsies in Pandemics (DEFEAT PANDEMIcs). The foundation of DEFEAT PANDEMIcs is the German Registry of COVID-19 Autopsies, DeRegCOVID. In close collaboration and with support from the German Society of Pathology (DGP, Deutsche Gesellschaft für Pathologie), the Federal Association of German Pathologists (BDP, Bundesverband Deutscher Pathologen), the German Society of Neuropathology and Neuroanatomy (DGNN, Deutsche Gesellschaft für Neuropathologie und Neuroanatomie), and the German Society of Legal Medicine (DGRM, Deutsche Gesellschaft für Rechtsmedizin), a central registry—the German Registry of COVID-19 Autopsies (DeRegCOVID)—was established (39, e18, e19). Internationally, it is the first and currently only registry of its kind and serves the following purposes:

• Recording of autopsies of patients who died of/with COVID-19.
• Recording of autopsies of patients who died in temporal association with COVID-19 vaccination
• Recording of biological samples (for example, tissue and liquid samples, formalin samples and cryo samples, storage in special media) and corresponding clinical data (for example, leading organ manifestation, medication, periods of hospitalization)
• Central coordination and support of research projects, as well as support of the public health system and the specialist societies.

With almost 1300 recorded autopsies from 32 German autopsy units (as of year-end 2021), the DeRegCOVID is currently the largest multicenter autopsy study worldwide.

Building on the DeRegCOVID in the framework of the National University Network (NUM, Netzwerk Universitätsmedizin), the German Network for Autopsies in Pandemics (DEFEAT PANDEMIcs) was developed, which is one of the fundamental pillars of this research.

Conclusion

The early and systematic use of autopsies and the analysis of autopsy tissue samples have proven to be an essential and efficient tool to improve our understanding of COVID-19. This approach revealed the nature of COVID-19 infection as an angiocentric systemic disease, enabling the introduction of treatments derived from this insight. Autopsy is the basis of all examination techniques presented in this article. Pathology is founded on autopsy and continues to rely on it to address future epidemics and pandemics in a timely and targeted manner. Quite in the spirit of the Director-General of the World Health Organization, Tedros Adhanom Ghebreyesus, who stated on 7 September 2020: “This will not be the last pandemic. . […] But when the next pandemic comes, the world must be ready – more ready than it was this time.“ (40).

Acknowledgement

This article is the expression of an international research consortium with many brains and hands. The following are mentioned in particular, in alphabetical order: by name Prof. Dr. med. Gustavo Baretton, Prof. Dr med. Johann Bauersachs, Prof. Dr. med. Peter Boor, PhD; Dr. med. Peter Braubach, Dr. med. Roman Bülow, Prof. Dr. Dr. h.c. A. Axel Haverich, Prof. Dr. med. Marius Hoeper, Dr. med. Jan Christopher Kamp, Prof. Dr. med. Hans-Heinrich Kreipe, Michael Krisch, PhD; PD Dr. rer nat. Mark Kühnel, Prof. Dr. med. Bruno Märkl, Dr. med. Lavinia Neubert, Dr. med. Berenice Rath, Dr. rer nat. Harald Reichert, Dr. rer nat. Marius Reichardt, Dr. Jan-Lucas Robertus, Prof. Dr. rer nat. Tim Salditt, Dr. med. Saskia von Stillfried, Paul Tafforeau, PhD, Stijn Verleden, PhD; Willi Wagner, Claire Walsh, PhD; Prof. Dr. med. Tobias Welte, as well as the European Synchrotron Radiation Facility (ESRF) in Grenoble and the German Registry of COVID-19 Autopsies (DeRegCOVID). This paper was supported by the National Autopsy Network (NATON), grant number 01KX2121. The authors thank Caja Boekhoff, Lennart Brandt, Edwin Lennart Busch, Regina Engelhardt, Nicole Krönke, Annette Müller-Brechlin, and Christina Petzold-Mügge for their excellent technical and administrative support.

Conflict of interest statement
Prof. Lee received financial support from the Royal Academy of Engineering (PDL – CiET1819/10), the UK Medical Research Council (MR/R025673/1), grant number 2020–225394 from the Chan Zuckerberg Initiative DAF, an advised fund of Silicon Valley Community Foundation (grant number CZIF2021–006424) from the Chan Zuckerberg Initiative Foundation, and the European Synchrotron Research Facility, proposals md1252 and md1290.

The remaining authors declare that they have no conflicts of interest.

Manuscript received on 22 March 2022; revised version accepted on 10 May 2022

Translated from the original German by Ralf Thoene, MD.

Corresponding author
Prof. Dr. med. Danny Jonigk, FRCPath
Institut für Pathologie, Medizinische Hochschule Hannover
Carl-Neuberg-Straße 1, 30625 Hannover, Germany

Cite this as:
Jonigk D, Werlein C, Lee PD, Kauczor HU, Länger F, Ackermann M: Pulmonary and systemic pathology in COVID-19—holistic pathological analyses. Dtsch Arztebl Int 2022; 119: 429–35. DOI: 10.3238/arztebl.m2022.0231

►Supplementary material
eReferences, eBox and eFigures:
www.aerzteblatt-international.de/m2022.0231

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