SARS-CoV-2 infection of endothelial cells, dependent on flow-induced ACE2 expression, drives hypercytokinemia in a vascularized microphysiological system

Abstract

Background: Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), responsible for COVID-19, has caused nearly 7 million deaths worldwide. Severe cases are marked by an aggressive inflammatory response known as hypercytokinemia, contributing to endothelial damage. Although vaccination has reduced hospitalizations, hypercytokinemia persists in breakthrough infections, emphasizing the need for disease models mimicking this response. Using a 3D microphysiological system (MPS), we explored the vascular role in SARS-CoV-2-induced hypercytokinemia.

Methods: The vascularized micro-organ (VMO) MPS, consisting of human-derived primary endothelial cells (ECs) and stromal cells within an extracellular matrix, was used to model SARS-CoV-2 infection. A non-replicative pseudotyped virus fused to GFP was employed, allowing visualization of viral entry into human ECs under physiologic flow conditions. Expression of ACE2, TMPRSS2, and AGTR1 was analyzed, and the impact of viral infection on ACE2 expression, vascular inflammation, and vascular morphology was assessed.

Results: The VMO platform facilitated the study of COVID-19 vasculature infection, revealing that ACE2 expression increased significantly in direct response to shear stress, thereby enhancing susceptibility to infection by pseudotyped SARS-CoV-2. Infected ECs secreted pro-inflammatory cytokines, including IL-6 along with coagulation factors. Cytokines released by infected cells were able to activate downstream, non-infected EC, providing an amplification mechanism for inflammation and coagulopathy.

Discussion: Our findings highlight the crucial role of vasculature in COVID-19 pathogenesis, emphasizing the significance of flow-induced ACE2 expression and subsequent inflammatory responses. The VMO provides a valuable tool for studying SARS-CoV-2 infection dynamics and evaluating potential therapeutics.

Keywords: COVID-19; endothelial dysfunction; hypercytokinemia; microphysiological systems; shear stress.

Figure 1

Development of a vascularized micro-organ microphysiological system and SARS-CoV-2 pseudotyped virus. (A) Schematic of an individual microfluidic device unit. Hydrostatic pressure drives medium from the inlet (MI) through the microfluidic channels and tissue chamber (T1–T3) to the medium outlet (MO). Hydrogel is loaded via the gel loading inlet (GLI) and outlet (GLO) with the pressure regulator (PR) to prevent leak into the microfluidic channel in the case of over-pressure during injection. (B) Development of the vasculature over time. EC labeled red; fibroblasts unlabeled. (C) Perfusion of 70 kDa FITC-Dextran through the vasculature (EC eRFP) at day 8. (D) COMSOL modeling of the shear stress (dynes/cm²) through each tissue chamber. (E) Quantification of fluid flow through each chamber (m/s). (F) Schematic of SARS-CoV-2 pseudotyped virus. SARS-CoV-2 spike protein is inserted into the membrane of an HIV-1 envelope with a Gag-iGFP construct. (G) Schematic showing infectivity of the pseudotyped virus into EC using ACE2, leading to GFP expression.

Figure 2

Upregulated ACE2 expression in the vascularized micro-organ supports SARS-CoV-2 pseudotyped infectivity. qPCR analysis of (A) ACE2, (B) AGTR1, and (C) TMPRSS2, which are necessary for SARS-CoV-2 infectivity, comparing monolayer normal human lung fibroblasts (NHLF), monolayer EC, the vascularized micro-organ (VMO) before (D4) and after (D8) the formation of perfusable vasculature. (D) Immunofluorescence staining of a D8 VMO showing CD31/PECAM1 (red), DAPI (blue), and localization of ACE2 (green) and TMPRSS2 (purple) to the endothelium. Showing nonspecificity of secondary antibodies alone. (E) Infectivity efficiency (mean fluorescent intensity) of SARS-CoV-2 pseudotyped virus compared to a lentivirus expressing green fluorescent protein (GFP). (F) Titering of GFP SARS-CoV-2 pseudotyped virus in a perfused VMO. Infectivity is measured as an increase in the mean fluorescent intensity of the GFP channel. (G) Subset of a D8 VMO EC (mCherry) and background fluorescence or fluorescence induced by infection with SARS-CoV-2 pseudotyped virus (GFP). Ctrl, control; PV, pseudotyped virus. (H) qPCR analysis of ACE2 expression induced by shear stress in an ibidi microfluidic chip. *<0.05, **<0.01, ***<0.001.

Figure 3

Modeled SARS-CoV-2 infection alters VMO transcriptomic profiles. In the VMO, qPCR analysis of pro-inflammatory markers (A) ICAM-1, (B) VCAM-1, and (C) IL-6 for perfused vascularized micro-organ (VMO) that have been treated with SARS-CoV-2 pseudotyped virus (pseudovirus), recombinant angiotensin II (rAngII) or both for 48 h. *<0.05, **<0.01, ***<0.001.

Figure 4

Modeled SARS-CoV-2 infection alters vessel morphometry. (A) Representative images of perfused VMOs treated with SARS-CoV-2 pseudotyped virus (pseudovirus) and/or recombinant angiotensin II (rAngII) for 48 h. Arrow 1: Under control conditions, vasculature is continuous and extends to the edges of the tissue chamber. Arrow 2: Discontinuous vessels, likely undergoing pruning and retraction. Arrow 3: Larger regions of perivascular space emerge due to vessel pruning. (B–G) Quantification of morphometry of devices showing changes in (B) vessel diameter, (C) total vessel length, (D) mean lacunarity, (E) vessel coverage percentage, and (F) total number of end points. *<0.05, **<0.01, ***<0.001, ****<0.0001. Control n = 11, Pseudovirus + rAngII n = 14 (See methods for more detail about replicates).

Figure 5

Modeled SARS-CoV-2 infection induces cytokine production. ELISAs from effluent collected from VMOs treated with SARS-CoV-2 pseudotyped virus (pseudovirus) and/or recombinant angiotensin II (rAngII) for 48 h. (A) Factor VIII, (B) IL-1β, (C) IL-6, (D) sICAM-1 protein content (pg/mL). *<0.05, **<0.01, ***<0.001.

Figure 6

Pharmacological intervention inhibits SARS-CoV-2 pseudotyped virus entry and reduces downstream nFκB activation. (A) Recombinant ACE2 (rACE2) prevented pseudotyped infectivity through competitive binding of the SARS-CoV-2 spike protein. (B) VMOs were treated with SARS-CoV-2 pseudotyped virus and rACE2 or camostat mesylate (camostat). The mean fluorescent intensity of the GFP pseudotyped virus was measured. Camostat mesylate had minimal protective ability, and rACE2 prevented infection. (C) Effluent from non-treated and treated devices was placed on EC transduced with an NFκB induced mCherry reporter. The mean fluorescent intensity of mCherry was measured. *<0.05, **<0.01, ***<0.001.

Figure 7

Generation of pseudotyped virus with omicron spike protein leads to infectivity and inflammatory response. (A) Mean fluorescent intensity of GFP signal for pseudotyped-virus with omicron variant spike protein. The virus can infect EC in the VMO. (B) ELISA of effluent from omicron-infected VMOs shows a significantly increased production of IL-6. ***<0.001.