THE IMPACT OF NON-ALCOHOLIC FATTY LIVER DISEASE ON OUCOMES OF PATEINTS WITH COVID-19: A PROPENSITY-MATCHED COHORT STUDY.

Cholangiocarcinoma (CCA), a cancer of biliary epithelium is highly aggressive, and shows a high degree of lymphatic infiltration (lymphangiogenesis) in early stages of metastasis. Expansion of the lymphatic network associated with inflammation or infection often precedes metastatic progression and dissemination and contributes to aggressiveness of these CCA tumors. Yet the mechanisms remain unknown. The emergence of a global pandemic with COVID-19 also brought some of these mechanisms to the forefront. Several lines of evidence have implicated COVID-19 as an endothelial disease and the resultant cytokine storm, endothelial dysfunction, cause significant changes in the tissue microenvironment that in turn could be favorable for tumor progression and activate metabolic changes. We hypothesized that SARS-CoV-2 induced alterations in the lymphatic endothelial cells (LECs) contributes to tumor-LEC crosstalk by inducing an inflammatory milieu. LECs and CCA cells were treated with the spike protein (S1) and different pathway specific inhibitors to determine activation of lymphangiogenic and tumor promoting mechanisms. In addition, proliferation, migration, and metabolic assays were carried out. LECs express the angiotensin converting enzyme 2 (ACE2) the well-known receptor for SARS-CoV-2. Our data suggested that S1 protein, significantly increased the cellular proliferation and cellular lactate production with concomitant increase in the expression of the key metabolic genes PFKP, FASN. S1 protein also had a robust impact on lymphatic tube formation and invasion that was associated with alterations in VEGFR3 expression. Interestingly, S1 protein infected LEC produces high level of TGFβ, a well-known factor for liver fibrosis and cholangiocarcinoma. Our data suggested that high level of TGFβ induced the ROS production, endothelial to mesenchymal transition (EndMT), creates an inflammatory milieu and also increased production of CXCL5, that has been demonstrated to actively participate in the HLEC-CCA cross talk through the specific CCR2 receptor expressed by CCA cells. CCA cells treated with the conditioned media (CM) from S1 protein treated HLEC, showed increased migratory potential that was associated with an increase in transcription factors for epithelial to mesenchymal transition (EMT), e.g., Snail1, Snail2, Zeb1, Zeb2 and the EMT genes α-SMA, Vimentin, Fibronectin along with the increase in matrix metalloproteinases, MMP2, MMP21. Taken together, our data proposes a novel axis mediated by SARS-CoV-2 infection increasing CCA-HLEC cross talk via the TGFb-CXCL5-CCR2 axis and ROS production that accelerates the metastatic potential of CCA cells. Hence, targeting the TGFb-ROS axis through small molecule inhibitors can be potential therapeutic option for managing the post COVID susceptibility of CCA progression and expansion of the lymphatic vasculature.

Introduction:
Coronavirus disease 2019 (COVID-19) has become the greatest health threat of the last few years. It is well established that patients with metabolic syndrome have higher prevalence of COVID-19. In addition, metabolic syndrome is associated with increased severity of COVID-19. Non-alcoholic fatty liver disease (NAFLD) represents the hepatic manifestation of metabolic syndrome. Data is limited regarding the COVID-19 related complications in patients with NAFLD. In our study, we aimed to investigate COVID-19 related outcomes in NAFLD.

Methods:
A retrospective cohort study was conducted using TriNetX, a multi-institutional database of more than 70 million patients from 49 healthcare organizations in the U.S between 1/2020 – 11/2022. We compared several COVID-19 related complications in patients with and without NAFLD. 1:1 propensity-score matching was performed for age, gender, race, ethnicity, and all major risk factors of COVID-19 among all cohorts. We assessed COVID-19 outcomes up to 3 months of index date (COVID-19 diagnosis). Adjusted odds ratios (aOR) with 95% confidence interval (CI) were calculated.

Results:
Of COVID-19 patients, we identified 17,937 patients with NAFLD (Cohort 1) and 3,077,926 patients with no NAFLD (Cohort 2). After propensity score matching, 17,936 patients were accounted for in each group (Table 1). Reviewing three-months outcomes of these patients after adjustment, cohort 1 had an increased risk of cardiac complications (aOR 1.64 [1.53-1.75]), VTE (aOR 3.01 [2.76-3.29]), cerebrovascular accident (1.82[1.62-2.03]), AKI (aOR 2.59 [2.37-2.83]) and PVD (aOR 1.48 [1.29-1.71]) compared to cohort 2. Moreover, Cohort 1 patients were at higher risk of acute respiratory distress syndrome (ARDS) (aOR 3.36 [3.12-3.62]), ICU-mechanical ventilation (aOR 3.22 [2.75-3.77]) and shock (aOR 4.35 [3.62-5.23]). In addition, cohort 1 had higher risk of hospitalization (aOR 1.20 [1.15-1.26]) and higher mortality (aOR 2.12 [1.89-2.38]) when compared with cohort 2 patients, table 2.

Discussion:
We found that NAFLD patients infected with COVID-19 have significantly worse COVID-19 related outcomes compared with COVID-19 patients without NAFLD. Targeting NAFLD and metabolic syndrome management could address modifiable risk factors to reduce COVID-19 related morbidity and morbidity.