T1R3 Anchors ACE2 at the Pulmonary Endothelial Cell Surface to Attenuate Vascular Injury Following SARS ‐ CoV ‐2 Spike 1 Protein Exposure In Vitro

Figures

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  FIGURE 1. Sp1 peptide significantly disrupts pulmonary endothelial barrier function with increased oxidative stress and inflammation. HLMVEC were exposed to Sp1 at concentrations from 0 to 100 nM for 24 h or media control. Cell viability (a), gap formation (b) and monolayer permeability (c) were measured using MTT, XPert and FITC‐monolayer flux assay respectively. VE‐cadherin phosphorylation (d) was measured with Western blotting with representative blot (i) and quantification (ii). VE‐cadherin (e) and ICAM‐1 (f) endothelial cell surface expression was assessed with whole‐cell ELISA. ROS accumulation (g) and cytokine release (h) were measured using DCFDA and Quantikine kits. Data are presented as mean ± SEM. n = 6. *p < 0.05 versus vehicle‐treated (0 μM) cells.

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  FIGURE 2. The T1R3 agonist, T1R3‐A, significantly attenuates Sp1‐induced barrier disruption and oxidative stress, but not inflammation, in the pulmonary endothelium in vitro. HLMVEC were treated with Sp1 (10 nM) or vehicle (media) with T1R3‐A (sucralose, 100 μM) or vehicle for sucralose (H2O) for 24 h. Gap formation (a) and monolayer permeability (b) were assessed using the XPert and FITC‐flux assay respectively. VE‐cadherin (c) and ICAM‐1 (d) surface expression was measured using whole‐cell ELISA, whilst ROS accumulation (e) and cytokine release of TGF‐β, IL‐6 and IL‐8 (f) was analysed with DCFDA and Quantikine kits, respectively. Data are presented as mean ± SEM. n = 6–7. *p < 0.05 versus vehicle for Sp1, # p < 0.05 versus vehicle for T1R3‐A.

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  FIGURE 3. T1R3‐A attenuates Sp1‐induced internalisation of ACE2 at the pulmonary endothelial cell surface through intracellular trafficking. HLMVEC were treated with Sp1 (10 nM) for up to 12 h, or vehicle (media), with T1R3‐A (sucralose, 100 μM) or vehicle for sucralose (H2O). For panel (d), Dynasore was added to cell treatment as an inhibitor of trafficking. ACE2 (a, c, e and g) and TMPRSS2 (b, d, f) expression was assessed with Western blotting (a, b, e, f) of endothelial cell membrane preparations (a and b) and whole lysate (e and f) and by whole cell ELISA (c, d, g). For Western blots, a representative blot (i) and quantification (ii) are included. Data are presented as mean ± SEM. n = 5–6. *p < 0.05 versus vehicle for Sp1.

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  FIGURE 4. T1R3‐A maintains ACE2 expression at the endothelial cell surface by promoting T1R3‐ACE2 interaction. Panels a–e: HLMVEC were exposed to siRNA specific to T1R3, or a non‐specific (ns) scrambled control, for 44 h prior to treatment with Sp1 (10 nM) for 4 h, or vehicle (media), with T1R3‐A (sucralose, 100 μM) or vehicle for sucralose (H2O) in the presence or absence of the T1R3 inhibitor, lactisole (3 μM) (e). mRNA expression of TAS1R3 (a) and ACE2 (b) was measured using RT‐PCR. ACE2 protein expression in whole lysate (c) and cell surface (d and e) was assessed using Western blotting and whole cell ELISA respectively. T1R3 cell surface expression was measured with whole cell ELISA (f). Immunocomplexes from ACE2 were probed with antibodies specific to T1R3 (g) with representative blot (i) and quantification (ii) included. Data are presented as mean ± SEM. n = 6–7. *p < 0.05 versus ns siRNA vehicle; # p < 0.05 versus vehicle for Sp1.