key: cord-0769086-18l906wl
authors: Kaltschmidt, Barbara; Fitzek, Antonia D.E.; Schaedler, Julia; Förster, Christine; Kaltschmidt, Christian; Hansen, Thorsten; Steinfurth, Fabian; Windmöller, Beatrice A.; Pilger, Christian; Kong, Cihang; Singh, Kashika; Nierhaus, Axel; Wichmann, Dominic; Sperhake, Jan; Püschel, Klaus; Huser, Thomas; Krüger, Martin; Robson, Simon C.; Wilkens, Ludwig; Schulte am Esch, Jan
title: Hepatic vasculopathy and regenerative responses of the liver in fatal cases of COVID-19
date: 2021-01-29
journal: Clin Gastroenterol Hepatol
DOI: 10.1016/j.cgh.2021.01.044
sha: 85a6bc1bfee97f52cc444e3e761e98a5f4eb76f7
doc_id: 769086
cord_uid: 18l906wl

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infects the nasopharynx and lungs and causes coronavirus disease-2019 (COVID-19). It may impact the heart, brain, kidney, and liver.1 Although functional impairment of the liver has been correlated with worse clinical outcomes, little is known about the pathophysiology of hepatic injury and repair in COVID-19.2,3 Histologic evaluation has been limited to small numbers of COVID-19 cases with no control subjects2,4 and demonstrated largely heterogeneous patterns of pathology.2,3.

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) infects the nasopharynx and lungs and causes coronavirus disease-2019 . It may impact the heart, brain, kidney and liver [1] .

Although functional impairment of the liver has been correlated with worse clinical outcomes, little is known about the pathophysiology of hepatic injury and repair in COVID-19 [2, 3] . Histological evaluation has been limited to small numbers of COVID-19 cases with no controls [2, 4] and demonstrated largely heterogeneous patterns of pathology [2, 3] .

Liver tissues of 60 patients who died of COVID-19 pneumonia were obtained from complete autopsies performed in Hamburg between March and June 2020. A cohort of 13 patients with fatal pneumonia in the absence of SARS-CoV-2 infection served as controls.

Hematoxylin and eosin staining and immunohistochemistry were applied to comprehensively evaluate the pathophysiology and regeneration aspects at the level of the hepatic microarchitecture. Label-free coherent-Raman-scattering-and second-harmonic-generation-imaging visualized major morphological changes, revealing steatosis and dilatation of sinusoids.

There were no significant differences in the patient characteristics except for pre-existing neurological conditions (p=0.04; Table S1 ). Only 5 patients with COVID-19 and 1 control had history of liver disease. A minority of patients in the COVID-19-group (17%) received intensive care at the time of death with a trend within that group towards home or nursing home care, when compared with controls (Table S1) .

qRT-PCR and in situ hybridization revealed viral RNA and replicative intermediates in liver tissues. Viral nucleocapsid-protein was detected in hepatic stem/progenitor cells (HSPC), cholangiocytes and hepatocytes (Fig. 1A) . SARS-CoV-2 was detected at the RNA-and/or protein level in 25% of livers in COVID-19 patients (table S1) . qRT-PCR demonstrated two out of five bile samples positive for SARS-CoV-2 (data not shown).

Sinusoidal platelet-aggregates were predominantly observed in the hepatic microvasculature of COVID-19 patients when compared to controls (70% vs. 30%; p=0.032; Fig. 1B ). Likewise, sinusoidal (p=0.024; Fig. 1B ) and portal dilatation were observed (p=0,002; Table S1 ). Hepatic microvascular thrombosis in COVID-19 patients was predominantly observed in non-hospitalized patients (32%; not receiving anti-coagulant therapy), when compared to general in-patients (3%) and those requiring intensive care (10%) (p=0.016; data not shown).

COVID-19 patients demonstrated more hepatic steatosis (p=0.046), mainly the microvesicular variant (p=0.01), when compared to SARS-CoV-2-negative controls (Fig. 1C) . Severe intrahepatic injury was associated with the activation of the intrahepatic stem cell niche along the canal of Hering (Fig. 1D) , resulting in regenerative clusters of EpCAM + HSPC, hepatobiliary intermediate cells and premature hepatocytes (Fig. 1D) . Increased numbers of EpCAM + HSPC were observed alone, and in J o u r n a l P r e -p r o o f regenerative clusters, in patients with COVID-19, when compared to control livers (>25 HSPC/10 field views: 66% vs 22%; p=0.020; Fig. 1D ).

We show here secretion of virus into bile and that replication of SARS-CoV-2 occurs in liver tissues during COVID-19. Viral infection of HSPC was observed, comparable to that noted in lung alveolocyteprogenitors in the first variant of SARS-CoV-infections [5] .

The major pathological finding is that microvascular changes and platelet rich thromboembolic phenomena reflect disordered thromboregulation and vascular insults as described previously [4, 6] .

As noted with other prothrombotic features of SARS-CoV-2 infections [4, 7] , our observation of microthrombotic pathology predominantly occurring at time of death in non-hospitalized patients suggests that early initiation of anticoagulant therapy in those hospitalized may attenuate microvascular disease of the liver.

Platelet activation, sinusoidal injury and parenchymal damage result in necrosis, which may be combined with predominantly microvesicular hepatocyte steatosis. Previous reports on post-mortem evaluations have demonstrated hepatic steatosis to variable degrees in COVID-19 and appear to demonstrate macrovesicular over microvesicular forms of steatosis [3, 4] .

We demonstrate that vascular injury is accompanied by activation of the intrahepatic stem cell compartment showing features of aberrant regeneration. Similar scenarios of HSPC activation, correlating with the extent of liver cell necrosis, as noted here, have been also shown in hepatitis Bmediated liver injury [8] .

We conclude that SARS-CoV-2 infects the liver as part of systemic illness and results in substantial microvascular thrombotic disease, organ injury and attempts at regeneration.

J o u r n a l P r e -p r o o f

Figure 1: Microvascular pathology and regenerative responses in livers of Covid-19 patients

SARS-CoV-2 replication, histopathology and regeneration responses of the liver in fatal COVID-19

Coherent Raman scattering (CRS) microscopy provided label-free contrast for tissue structure (grey) and lipid droplet identification (yellow), while second harmonic generation (SHG) provided contrast for collagen fibers (blue). Panel C: Liver injury: High levels of hepatocyte steatosis in Covid-19 patients, in comparison to controls, as detected by HE staining and CRS/SHG (lipid droplets in yellow and collagen in blue). White arrows indicate macrovesicular steatosis, whereas black arrows show microvesicular steatosis. Panel D: Intra-hepatic stem cell niche: Intra-hepatic stem cell niche: Cartoon of the bi-potent intra-hepatic stem cell compartment which is located in the Canal of Hering. Upon severe hepatic injury HSPC emerge and expand

HSPC, hepatobiliary intermediate cells and premature hepatocytes, which are adjacent to the canal of

Stem cell marker EpCam (red) is also expressed by numerous HSPC and intermediates of hepatocytes /cholangiocytes as well as regenerative clusters of periportal hepatocytes of irregular size and shape. HSPC-frequency in column-diagram is representing numbers of HSPC per 10 field views