SARS‐CoV‐2 spike spurs intestinal inflammation via VEGF production in enterocytes

Article19 April 2022Open Access Source DataTransparent process SARS-CoV-2 spike spurs intestinal inflammation via VEGF production in enterocytes Fa-Min Zeng Fa-Min Zeng Guangdong Provincial Key Laboratory of Biomedical Imaging and Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Department of Pathology, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Data curation, Formal analysis, Funding acquisition, ​Investigation, Visualization, Writing - original draft Search for more papers by this author Ying-wen Li Ying-wen Li Guangdong Provincial Key Laboratory of Biomedical Imaging and Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Data curation, Formal analysis, ​Investigation, Visualization, Writing - original draft Search for more papers by this author Zhao-hua Deng Zhao-hua Deng Guangdong Provincial Key Laboratory of Biomedical Imaging and Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Data curation, ​Investigation Search for more papers by this author Jian-zhong He Jian-zhong He Department of Pathology, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Data curation, Formal analysis, ​Investigation Search for more papers by this author Wei Li Wei Li Department of Pathology, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Methodology Search for more papers by this author Lijie Wang Lijie Wang Guangdong Provincial Key Laboratory of Biomedical Imaging and Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Methodology Search for more papers by this author Ting Lyu Ting Lyu Department of Pathology, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Methodology Search for more papers by this author Zhanyu Li Zhanyu Li Department of Pathology, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Methodology Search for more papers by this author Chaoming Mei Chaoming Mei Guangdong Provincial Key Laboratory of Biomedical Imaging and Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Methodology Search for more papers by this author Meiling Yang Meiling Yang Guangdong Provincial Key Laboratory of Biomedical Imaging and Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: ​Investigation, Methodology Search for more papers by this author Yingying Dong Yingying Dong Guangdong Provincial Key Laboratory of Biomedical Imaging and Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: ​Investigation, Methodology Search for more papers by this author Guan-Min Jiang Guan-Min Jiang Department of Clinical Laboratory, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Resources Search for more papers by this author Xiaofeng Li Xiaofeng Li orcid.org/0000-0001-5155-8475 Department of Gastroenterology, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: ​Investigation, Methodology Search for more papers by this author Xi Huang Xi Huang Department of Infectious Diseases, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Resources, ​Investigation Search for more papers by this author Fei Xiao Corresponding Author Fei Xiao [email protected] orcid.org/0000-0001-7353-2700 Guangdong Provincial Key Laboratory of Biomedical Imaging and Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Department of Infectious Diseases, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Conceptualization, Resources, Supervision, Validation, Methodology Search for more papers by this author Ye Liu Corresponding Author Ye Liu [email protected] orcid.org/0000-0001-5214-0134 Department of Pathology, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Resources, Supervision, Validation Search for more papers by this author Hong Shan Corresponding Author Hong Shan [email protected] orcid.org/0000-0001-6640-1390 Guangdong Provincial Key Laboratory of Biomedical Imaging and Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Department of Interventional Medicine, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Validation Search for more papers by this author Huanhuan He Corresponding Author Huanhuan He [email protected] orcid.org/0000-0002-5903-0840 Guangdong Provincial Key Laboratory of Biomedical Imaging and Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Visualization, Project administration, Writing - review & editing Search for more papers by this author Fa-Min Zeng Fa-Min Zeng Guangdong Provincial Key Laboratory of Biomedical Imaging and Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Department of Pathology, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Data curation, Formal analysis, Funding acquisition, ​Investigation, Visualization, Writing - original draft Search for more papers by this author Ying-wen Li Ying-wen Li Guangdong Provincial Key Laboratory of Biomedical Imaging and Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Data curation, Formal analysis, ​Investigation, Visualization, Writing - original draft Search for more papers by this author Zhao-hua Deng Zhao-hua Deng Guangdong Provincial Key Laboratory of Biomedical Imaging and Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Data curation, ​Investigation Search for more papers by this author Jian-zhong He Jian-zhong He Department of Pathology, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Data curation, Formal analysis, ​Investigation Search for more papers by this author Wei Li Wei Li Department of Pathology, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Methodology Search for more papers by this author Lijie Wang Lijie Wang Guangdong Provincial Key Laboratory of Biomedical Imaging and Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Methodology Search for more papers by this author Ting Lyu Ting Lyu Department of Pathology, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Methodology Search for more papers by this author Zhanyu Li Zhanyu Li Department of Pathology, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Methodology Search for more papers by this author Chaoming Mei Chaoming Mei Guangdong Provincial Key Laboratory of Biomedical Imaging and Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Methodology Search for more papers by this author Meiling Yang Meiling Yang Guangdong Provincial Key Laboratory of Biomedical Imaging and Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: ​Investigation, Methodology Search for more papers by this author Yingying Dong Yingying Dong Guangdong Provincial Key Laboratory of Biomedical Imaging and Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: ​Investigation, Methodology Search for more papers by this author Guan-Min Jiang Guan-Min Jiang Department of Clinical Laboratory, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Resources Search for more papers by this author Xiaofeng Li Xiaofeng Li orcid.org/0000-0001-5155-8475 Department of Gastroenterology, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: ​Investigation, Methodology Search for more papers by this author Xi Huang Xi Huang Department of Infectious Diseases, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Resources, ​Investigation Search for more papers by this author Fei Xiao Corresponding Author Fei Xiao [email protected] orcid.org/0000-0001-7353-2700 Guangdong Provincial Key Laboratory of Biomedical Imaging and Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Department of Infectious Diseases, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Conceptualization, Resources, Supervision, Validation, Methodology Search for more papers by this author Ye Liu Corresponding Author Ye Liu [email protected] orcid.org/0000-0001-5214-0134 Department of Pathology, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Resources, Supervision, Validation Search for more papers by this author Hong Shan Corresponding Author Hong Shan [email protected] orcid.org/0000-0001-6640-1390 Guangdong Provincial Key Laboratory of Biomedical Imaging and Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Department of Interventional Medicine, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Conceptualization, Resources, Supervision, Funding acquisition, Validation Search for more papers by this author Huanhuan He Corresponding Author Huanhuan He [email protected] orcid.org/0000-0002-5903-0840 Guangdong Provincial Key Laboratory of Biomedical Imaging and Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China Contribution: Conceptualization, Data curation, Formal analysis, Supervision, Funding acquisition, Validation, Visualization, Project administration, Writing - review & editing Search for more papers by this author Author Information Fa-Min Zeng1,2,†, Ying-wen Li1,†, Zhao-hua Deng1,†, Jian-zhong He2,†, Wei Li2, Lijie Wang1, Ting Lyu2, Zhanyu Li2, Chaoming Mei1, Meiling Yang1, Yingying Dong1, Guan-Min Jiang3, Xiaofeng Li4, Xi Huang5, Fei Xiao *,1,5, Ye Liu *,2, Hong Shan *,1,6 and Huanhuan He *,1 1Guangdong Provincial Key Laboratory of Biomedical Imaging and Guangdong Provincial Engineering Research Center of Molecular Imaging, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China 2Department of Pathology, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China 3Department of Clinical Laboratory, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China 4Department of Gastroenterology, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China 5Department of Infectious Diseases, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China 6Department of Interventional Medicine, The Fifth Affiliated Hospital, Sun Yat-sen University, Zhuhai, China † These authors contributed equally to this work *Corresponding author. Tel: (+86)756-2528024; E-mail: [email protected] *Corresponding author. Tel: (+86)756-2528106; E-mail: [email protected] *Corresponding author. Tel: (+86) 756-2528555; E-mail: [email protected] *Corresponding author. Tel: (+86)756-2526143; E-mail: [email protected] EMBO Mol Med (2022)14:e14844https://doi.org/10.15252/emmm.202114844 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) can cause gastrointestinal (GI) symptoms that often correlate with the severity of COVID-19. Here, we explored the pathogenesis underlying the intestinal inflammation in COVID-19. Plasma VEGF level was particularly elevated in patients with GI symptoms and significantly correlated with intestinal edema and disease progression. Through an animal model mimicking intestinal inflammation upon stimulation with SARS-CoV-2 spike protein, we further revealed that VEGF was over-produced in the duodenum prior to its ascent in the circulation. Mechanistically, SARS-CoV-2 spike promoted VEGF production through activating the Ras-Raf-MEK-ERK signaling in enterocytes, but not in endothelium, and inducing permeability and inflammation. Blockage of the ERK/VEGF axis was able to rescue vascular permeability and alleviate intestinal inflammation in vivo. These findings provide a mechanistic explanation and therapeutic targets for the GI symptoms of COVID-19. Synopsis VEGF is a key factor in vascular permeability and inflammation. A correlation between VEGF and COVID-19-related GI symptoms was established: upon viral spike protein-induced ERK activation, VEGF was produced by the enterocytes, which led to inflamed and leaky gut. VEGF secretion positively correlated with the occurrence of GI symptoms and COVID-19 severity, making it a key factor to predict disease progression. VEGF production was induced by SARS-CoV-2 spike RBD through the Ras-Raf-MEK-ERK pathway in enterocytes. Blocking either ERK or VEGF relieved intestinal inflammation and leakage induced by spike both in vitro and in vivo. The paper explained Problem COVID-19 patients with gastrointestinal (GI) symptoms tend to develop severe disease, but the underlying mechanism is unclear. VEGF is upregulated in the blood of COVID-19 patients, yet its association with the development of GI symptoms has not been explored. Results VEGF level correlated with intestinal edema, GI symptoms, and disease progression in COVID-19 patients. In an animal model mimicking intestinal inflammation upon stimulation with SARS-CoV-2 spike protein, we found that VEGF was over-produced in the duodenum prior to its ascent in the circulation, which led to hyperpermeability and systemic inflammation. Cell experiments demonstrated that the SARS-CoV-2 spike protein activated the Ras-Raf-MEK-ERK-VEGF pathway in enterocytes, which promoted VE-cad-mediated vascular permeability. Blocking the ERK/VEGF axis reversed hyperpermeability and alleviated intestinal inflammation stimulated by SARS-CoV-2 spike protein both in vitro and in vivo. Impact This study identifies a possible route of SARS-CoV-2 spike-induced VEGF production in the GI tract, which leads to vascular permeability and inflammation. Mechanistically, it uncovers the spike-activated ERK/VEGF pathway in enterocytes. Translationally, it provides potential targets for the treatment of GI symptoms, harnessing the disease progression of COVID-19. Introduction The coronavirus disease 2019 (COVID-19), caused by severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2), has become a persistent health emergency since its outbreak in late 2019 (Rader et al, 2020). Since we and others provided initial evidence for gastrointestinal (GI) infection in COVID-19 patients (Jin et al, 2020; Lin et al, 2020; Xiao et al, 2020), the reports of GI manifestations caused by SARS-CoV-2 in clinical cases and nonhuman primate models have escalated (Jiao et al, 2021). Notably, GI symptoms are often correlated with disease severity and systemic inflammation (Jin et al, 2020; Wan et al, 2020). It has been shown that specific insult of GI by SARS-CoV-2 can lead to systemic inflammation due to impaired GI barrier (Jiao et al, 2021). This was further exemplified by the fact that when the GI barrier was repaired by a zonulin antagonist, the systemic inflammation in Multisystem Inflammatory Syndrome in Children (MIS-C) upon SARS-CoV-2 infection or exposure was relieved (Gruber et al, 2020; Yonker et al, 2021). However, the pathogenesis of GI symptoms upon SARS-CoV-2 spike stimulation and how GI barrier impairment contributes to the systemic inflammation are still obscure, leaving treatment options nebulous. Vascular barrier plays a key role in confining antigen spreading and inflammation. Increased inflammatory cytokines from intestine could overflow to bloodstream and cause lung inflammation (Mjösberg & Rao, 2018). Current studies proved that SARS-CoV-2 infection could induce vascular barrier dysfunction in vivo and in vitro (Colunga Biancatelli et al, 2021; Raghavan et al, 2021). However, as a critical component of GI barrier (Bouziat & Jabri, 2015), the role and the mechanism of intestinal vascular barrier in GI inflammation and disease progression are still unclear. Progression of COVID-19 often involves excessive pro-inflammatory cytokines and mediators (Choreño-Parra et al, 2021). VEGF, a key factor involved in vascular permeability and inflammation (Lee et al, 2004), was found skyrocketed in the blood of COVID-19 patients and related to disease severity (Polidoro et al, 2020; Syed et al, 2021). Based on the crucial role of VEGF in pulmonary edema and inflammation upon SARS-CoV-2 infection in the lung, a clinical trial has made its successful debut in treating severe lung injury of COVID-19 patients by targeting VEGF with Bevacizumab (Pang et al, 2021). However, the role of VEGF in SARS-CoV-2-mediated GI inflammation remains enigmatic. The spike protein of SARS-CoV-2 mediates the binding of virus to host cell receptors or co-receptors including angiotensin-converting enzyme 2 (ACE2), transmembrane protease serine 2 (TMPRSS2), and neuropilin-1 (NRP-1) (Hoffmann et al, 2020; Mayi et al, 2021). Besides, the spike protein can alter cellular functions by activating various signaling pathways (Lei et al, 2021; Moutal et al, 2021). Although it has been reported that spike protein could induce lung injury and barrier dysfunction in pulmonary endothelial cells (Colunga Biancatelli et al, 2021), whether and how the viral spike protein alone contributes to the GI inflammation and gut vascular barrier stays unknown. Here, we investigated the underlying mechanisms involved in SARS-CoV-2 spike-induced VEGF production and inflammation in the intestine, which may provide therapeutic targets for the remediation of GI symptoms in patients with COVID-19. Results Serum VEGF level is elevated in COVID-19 patients with GI symptoms and correlated with intestinal edema and disease progression To systemically characterize intestinal infection and inflammation in COVID-19 patients, we evaluated different segments of intestinal tissues from 18 patients who were diagnosed with COVID-19 and presented with GI symptoms (Table EV1). Robust expression of virus receptor ACE2 and virus antigen spike protein was detected in small intestine (Fig EV1A and B), indicating SARS-CoV-2 infection in GI tract, which agreed with previous studies (Du et al, 2020; Zhang et al, 2020). Intriguingly, small intestine displayed the most severe inflammation characterized by inflammatory cell infiltration and signs of vascular damage such as interstitial edema, hemorrhage and vasodilation (Fig 1A and B). Notably, interstitial edema was significantly correlated with disease type, acid reflux, total bilirubin, alanine aminotransferase (ALT), and aspartate aminotransferase (AST) (Table 1 and Table EV1), implying vascular permeability change as a crucial factor in disease progression. Since it has been reported that myriad cytokines were raised in the blood of COVID-19 patients (Mangalmurti & Hunter, 2020), we examined the ones that could potentially affect permeability, including IL-1α, IL-1β, TNF-α, IFN-γ, and VEGF, in the cases with or without GI symptoms. Intriguingly, only VEGF level showed significant elevation in the plasma of patients with GI symptoms compared to those without (Fig 1C). Subsequently, we expanded the serologic analysis of VEGF concentrations in another independent cohort and confirmed the correlation between VEGF level and GI symptoms (Fig EV1C). Not only was VEGF correlated with disease progression (Figs 1D and EV1D, and Table 2), but the VEGF level was even higher at the early stage of the progressed patients (Figs 1E and EV1E), suggesting that VEGF may lead the cytokine cascade and contribute to the inflammation in the early phase of viral infection. To be noticed, the VEGF level was also significantly associated with gut interstitial edema and vasodilation (Fig 1F and G), restating the role of VEGF in intestinal inflammation. Click here to expand this figure. Figure EV1. VEGF level correlates with disease progression of COVID-19 Immunofluorescence analysis of ACE2 and SARS-CoV-2 spike protein in the intestinal tissues of COVID-19 patients. ACE2 staining in green, spike protein staining in red and nuclear staining in blue. Scale bars, 50 µm. The percentage of spike stained positive in different parts of the intestinal tissues from patients with COVID-19. Number of samples for each group as indicated. ELISA analysis of VEGF concentration in plasma of COVID-19 patients with (n = 11) or without (n = 8) GI symptoms. ELISA analysis of VEGF concentration in plasma of COVID-19 patients with (n = 8) or without (n = 11) disease progression. Temporal course of plasma VEGF at the early and late stage of COVID-19 infection by ELISA analysis. Data shown are the levels of plasma VEGF in patients with (n = 8) and without (n = 11) disease progression at the early stage (one to three days after laboratory-confirmed for COVID-19) and late stage (more than three days after laboratory-confirmed for COVID-19). mRNA levels of VEGF-B and VEGF-C in the intestinal tissues from COVID-19 patients (n = 5) or healthy controls (n = 5) by RNA-seq. mRNA levels of VEGFR1, VEGFR2, and VEGFR3 in the intestinal tissues from COVID-19 patients (n = 5) or healthy controls (n = 5) by RNA-seq. Data information: All data are shown as mean ± SD. P values are determined by Student’s t-test. Download figure Download PowerPoint Figure 1. Plasma VEGF level correlates with GI symptoms and disease progression of COVID-19 H&E staining of sections of intestinal tissues obtained from endoscopic biopsy of COVID-19 patients who presented with gastrointestinal (GI) symptoms. The inflammatory infiltrates were indicated by a yellow arrow. The edema area was indicated by a red star. Scale bars, 100 µm. The bar chart shows the degrees of intestinal inflammation in patients with COVID-19. Number of samples for each group as indicated. Cytokine levels in the plasma of COVID-19 patients with (n = 12) or without (n = 6) GI symptoms by cytokine assay. Levels of plasma VEGF in COVID-19 patients with (n = 8) or without (n = 10) disease progression by cytokine assay. Temporal course of plasma VEGF at the early and late stage of COVID-19 infection by cytokine assay. Data shown are the levels of plasma VEGF in patients with (n = 8) and without (n = 10) disease progression at the early stage (one to three days after laboratory-confirmed for COVID-19) and late stage (more than three days after laboratory-confirmed for COVID-19). Levels of plasma VEGF in COVID-19 patients with (n = 6) or without (n = 7) interstitial vasodilation by cytokine assay. Levels of plasma VEGF in COVID-19 patients with (n = 8) or without (n = 5) interstitial edema by cytokine assay. Data information: All data are shown as mean ± SD. P values are determined by Student’s t-test. Source data are available online for this figure. Source Data for Figure 1 [emmm202114844-sup-0002-SDataFig1.zip] Download figure Download PowerPoint Table 1. The correlation between interstitial edema/ vasodilation and GI manifestations in 18 patients with SARS-CoV-2 infection. Interstitial edema Interstitial vasodilation R P R P Disease classification (Non-severe, Severe) 0.544 0.001 0.172 0.322 Symptoms Diarrhea 0.151 0.386 −0.180 0.302 Anorexia −0.187 0.282 −0.124 0.478 Nausea −0.320 0.061 0.180 0.300 Vomit −0.258 0.134 0.241 0.164 Acid reflux 0.375 0.027 0.049 0.779 Epigastric discomfort 0.180 0.302 0.112 0.521 Hepatic function impairment Total bilirubin (μmol/l; normal range 3.0–24.0) 0.374 0.027 0.030 0.866 ALT (U/l; normal range 7–40 in female, 9–50 in male) 0.475 0.004 0.215 0.214 AST (U/l; normal range 13–35 in female, 15–40 in male) 0.487 0.003 0.323 0.059 ALT, alanine aminotransferase; AST, aspartate aminotransferase (value on initial presentation). P < 0.05 was considered statistically significant (in red). Table 2. The correlation between plasma VEGF and clinicopathological features in patients with COVID-19. VEGF (Test cohort) VEGF (Validation cohort) R P R P Disease progression 0.490 0.002 0.756 <0.001 Interstitial edema 0.460 0.018 Interstitial vasodilation 0.439 0.025 Hepatic function impairment Total bilirubin (μmol/l; normal range 3.0–24.0) 0.677 <0.001 0.507 0.027 ALT (U/l; normal range 7–40 in female, 9–50 in male) 0.51 0.011 0.728 <0.001 AST (U/l; normal rage 13–35 in female, 15–40 in male) 0.566 0.004 0.492 0.032 Serologic markers of disease severity CRP (μg/l; normal range 0.068–8.2) 0.280 0.261 0.582 0.004 D-dimers (μg/l; normal range 0–243) −0.104 0.687 0.404 0.086 Procalcitonin 0.236 0.346 0.579 0.009 Nucleic acid test results of fecal samples (Positive or Negative) −0.086 0.733 −0.005 0.983 P < 0.05 was considered statistically significant (in red). VEGF is induced in intestinal tissue upon abdominal stimulation of SARS-CoV-2 spike Next, we asked where plasma VEGF was from with the speculation that it could be induced in the local inflammatory tissue. Indeed, we detected a higher VEGF-A level in the intestinal tissues from COVID-19 patients compared to those from healthy individuals (Fig 2A). To be noted, only VEGF-A was significantly raised, leaving VEGF-B and VEGF-C and VEGF receptors unaltered (Fig EV1F and G). In order to validate whether SARS-CoV-2 was responsible for the VEGF production in intestinal tissue, we generated an animal model to specifically mimic the intestinal inflammation by intraperitoneally injecting the recombinant spike-Fc containing the receptor binding domain (RBD) to C57BL/6J mice (Fig EV2A). Co-localization of murine ACE2 with spike was confirmed by immunofluorescence staining (Fig EV2B–D), which was consistent with previous results (Kuba et al, 2005; Raghavan et al, 2021; Shin et al, 2021). Since intestinal mucosal barrier renders strong protection from the infectious agents (Sharma & Riva, 2020), treating mice with spike RBD alone for a short time only gave rise to mild inflammation (Fig EV2E). Hence, we treated the intestinal epithelium by acid enema for a very short time (2 min) prior to viral challenge adapted from a previous lung infection model (Kuba et al, 2005) (Fig 2B). Similar to the observations in COVID-19 patients, treatment by SARS-CoV-2 spike RBD induced intestinal inflammation with the most severe pathological alterations in the duodenum (Fig 2C and D; Table 3). Overproduced inflammatory factors, such as IL-1α, IL-8, IL-10, and TNF-α, specifically in the duodenum further confirmed the tissue tropism to the sites of inflammation (Figs 2E and EV2F). Since it is still controversial whether spike RBD can bind to murine ACE2 (Wu et al, 2020; Niu et al, 2021), spike-induced hACE2-B6J mice were adopted as confirmatory animal models and similar phenotype was observed (Fig EV3A and B). Intriguingly, consistent with our speculation but more specific, levels of murine VEGF in the duodenum of spike-stimulated C57BL/6J or hACE2-B6J mice were significantly raised (Figs 2F, and EV3C and D). More importantly, plasma VEGF was not elevated initially, but markedly increased after seven days of continual treatment of spike RBD (Figs 2G and EV3E). Moreover, the abdominal vascular hyperpermeability echoed the findings from patient tissues (Figs 2H and EV3F). The above results indicate that the initial VEGF could originate from local production in the small intestine, which may lead to systemic VEGF promotion over long-term inflammation. Figure 2. VEGF is elevated in small intestine upon abdominal stimulation of SARS-CoV-2 spike A. Levels of VEGF-A mRNA in the intestinal tissues from COVID-19 patients (n = 5) or healthy controls (n = 5) by RNA-seq. B. Illustration depicts generation of the animal model with SARS-CoV-2 spike RBD-induced intestinal inflammation. Mice were fasted for 24 h before acetic acid enema. 16 h after acid