Histamine Potentiates SARS-CoV-2 Spike Protein Entry Into Endothelial Cells

Abstract

Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) which causes coronavirus disease (COVID-19) is one of the most serious global health crises in recent history. COVID-19 patient symptoms range from life-threatening to mild and asymptomatic, which presents unique problems in identifying, quarantining, and treating the affected individuals. The emergence of unusual symptoms among survivors, now referred to as "Long COVID", is concerning, especially since much about the condition and the treatment of it is still relatively unknown. Evidence so far also suggests that some of these symptoms can be attributed to vascular inflammation. Although famotidine, the commonly used histamine H2 receptor (H2R) blocker, was shown to have no antiviral activity, recent reports indicate that it could prevent adverse outcomes in COVID-19 patients. Histamine is a classic proinflammatory mediator, the levels of which increase along with other cytokines during COVID-19 infection. Histamine activates H2R signaling, while famotidine specifically blocks H2R activation. Investigating the effects of recombinant SARS-CoV-2 spike protein S1 Receptor-Binding Domain (Spike) on ACE2 expression in cultured human coronary artery endothelial cells, we found that the presence of histamine potentiated spike-mediated ACE2 internalization into endothelial cells. This effect was blocked by famotidine, protein kinase A inhibition, or by H2 receptor protein knockdown. Together, these results indicate that histamine and histamine receptor signaling is likely essential for spike protein to induce ACE2 internalization in endothelial cells and cause endothelial dysfunction and that this effect can be blocked by the H2R blocker, famotidine.

Keywords: SARS-CoV-2; angiotensin-converting enzyme-2; famotidine; histamine; spike.

FIGURE 1

Spike-induced ACE2 internalization is enhanced in the presence of histamine. (A) Representative Western blotting after surface biotinylation of intact endothelial cells with or without spike treatment for 30 min. (B) Mean data. (C) Representative Western blot after surface biotinylation showing that increased incubation time after spike treatment induced ACE2 internalization and degradation. (D) Mean data. *p < 0.05 vs. surface band intensity of the untreated control. (E) Representative Western blot after surface biotinylation showing the effect of bafilomycin A1 on spike-induced ACE2 degradation. (F) Mean data, *p < 0.05 vs. untreated, #p < 0.05 vs. spike treated. (G) Representative Western blot after surface biotinylation showing that histamine potentiates spike-induced ACE2 internalization within 30 min of treatment. (H) Mean data, *p < 0.05 vs. surface band intensity of untreated control. n = 4 for all the experimental sets. I- indicates intracellular fraction, S-denotes surface or plasma membrane fraction.

FIGURE 2

Histamine H2 receptor signaling is involved in histamine potentiating spike–ACE2 internalization. (A) Representative Western blot after surface biotinylation of the intact endothelial cells showing the effect of famotidine on the potentiating effect of histamine on spike-ACE2 internalization. (B) Mean data. (C) Representative Western blot after surface biotinylation showing the effect of the protein kinase A inhibitor, PKI, in preventing histamine-induced spike–ACE2 internalization. (D) Mean data. *p < 0.05 vs. the untreated control, #p < 0.05 vs. spike + histamine. (E) Representative Western blot after surface biotinylation showing the effect of H2 receptor protein knockdown on spike + histamine treatment. Scrm-scrambled siRNA. (F) Mean data. *p < 0.05 vs. untreated control, #p < 0.05 vs. spike + histamine scrambled control. n = 4 for all the experimental sets. I- indicates intracellular fraction, S-denotes surface or plasma membrane fraction.