Angiopoietin-Like4 Is a Novel Marker of COVID-19 Severity

Abstract

Vascular dysfunction and capillary leak are common in critically ill COVID-19 patients, but identification of endothelial pathways involved in COVID-19 pathogenesis has been limited. Angiopoietin-like 4 (ANGPTL4) is a protein secreted in response to hypoxic and nutrient-poor conditions that has a variety of biological effects including vascular injury and capillary leak.

Objectives: To assess the role of ANGPTL4 in COVID-19-related outcomes.

Design setting and participants: Two hundred twenty-five COVID-19 ICU patients were enrolled from April 2020 to May 2021 in a prospective, multicenter cohort study from three different medical centers, University of Washington, University of Southern California and New York University.

Main outcomes and measures: Plasma ANGPTL4 was measured on days 1, 7, and 14 after ICU admission. We used previously published tissue proteomic data and lung single nucleus RNA (snRNA) sequencing data from specimens collected from COVID-19 patients to determine the tissues and cells that produce ANGPTL4.

Results: Higher plasma ANGPTL4 concentrations were significantly associated with worse hospital mortality (adjusted odds ratio per log₂ increase, 1.53; 95% CI, 1.17-2.00; p = 0.002). Higher ANGPTL4 concentrations were also associated with higher proportions of venous thromboembolism and acute respiratory distress syndrome. Longitudinal ANGPTL4 concentrations were significantly different during the first 2 weeks of hospitalization in patients who subsequently died compared with survivors (p for interaction = 8.1 × 10^-5). Proteomics analysis demonstrated abundance of ANGPTL4 in lung tissue compared with other organs in COVID-19. ANGPTL4 single-nuclear RNA gene expression was significantly increased in pulmonary alveolar type 2 epithelial cells and fibroblasts in COVID-19 lung tissue compared with controls.

Conclusions and relevance: ANGPTL4 is expressed in pulmonary epithelial cells and fibroblasts and is associated with clinical prognosis in critically ill COVID-19 patients.

Keywords: COVID-19; angiopoietin-like 4; endothelial dysfunction; intensive care unit; sepsis.

Figure 1.

Association of plasma angiopoietin-like 4 (ANGPTL4) concentrations with hospital mortality. The smooth spline estimates the odds ratio (OR) of hospital mortality according to ICU admission plasma ANGPTL4 concentrations. Analyses are adjusted for age, sex, and body mass index. Dotted lines represent 95% CIs. Below the spline is the histogram of the distribution of ANGPTL4 to indicate the range of the data.

Figure 2.

Longitudinal plasma angiopoietin-like 4 (ANGPTL4) measurements are associated with hospital mortality. Plasma ANGPTL4 markers measured on days 1, 7, and 14 after ICU admission stratified by hospital mortality. The dots represent the mean ANGPTL4 concentrations and the n provides the total number of patients with ANGPTL4 concentrations measured on a given day. The dashed lines provide the bootstrap 95% CI of the mean.

Figure 3.

Upregulation of angiopoietin-like 4 (ANGPTL4) gene expression in human lung alveolar type 2 epithelial cells and fibroblasts. A, Single-cell RNA sequencing data comparing ANGPTL4 gene expression in 20 COVID-19 patients to seven without COVID-19. Transcript counts are cell specific. Analysis demonstrates significant upregulation of ANGPTL4 in alveolar type 2 epithelial cells and fibroblasts and downregulation in monocytes (Mono) and macrophages (Mac). Cells classified as airway epithelial cells demonstrated high expression of dynein axonemal heavy chain 9 gene expression (DNAH9), whose product is essential for ciliary function. B, ANGPTL4 gene expression in immortalized human small airway epithelial cells infected with severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2). These cells gene expression pattern was consistent with distal respiratory/alveolar epithelium (no DNAH9 expression and high podoplanin, a gene expressed by type 1 alveolar epithelial cells. A two-way analysis of variance revealed there was a significant interaction between the effects of SARS-CoV-2 and time on ANGPTL4 expression (p = 0.0027). Simple main effects analysis showed that SARS-CoV-2 did have a statistically significant effect on ANGPTL4 expression (p = 0.04). FDR = false discovery rate; LSMean = Least squares mean; B = B cells; T and NK = T and Natural Killer Cells; 2^-(ΔΔCt) =Cycle thresholds.