SARS-CoV-2 spike protein induces endothelial inflammation via ACE2 independently of viral replication, 2023, Montezano et al.

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SARS-CoV-2 spike protein induces endothelial inflammation via ACE2 independently of viral replication
Montezano, Augusto C.; Camargo, Livia L.; Mary, Sheon; Neves, Karla B; Rios, Francisco J; Stein, Ross; Lopes, Rheure A.; Beattie, Wendy; Thomson, Jacqueline; Herder, Vanessa; Szemiel, Agnieszka M.; McFarlane, Steven; Palmarini, Massimo; Touyz, Rhian M.

COVID‑19, caused by SARS‑CoV‑2, is a respiratory disease associated with inflammation and endotheliitis. Mechanisms underling inflammatory processes are unclear, but angiotensin converting enzyme 2 (ACE2), the receptor which binds the spike protein of SARS‑CoV‑2 may be important.

Here we investigated whether spike protein binding to ACE2 induces inflammation in endothelial cells and determined the role of ACE2 in this process. Human endothelial cells were exposed to SARS‑CoV‑2 spike protein, S1 subunit (rS1p) and pro‑inflammatory signaling and inflammatory mediators assessed. ACE2 was modulated pharmacologically and by siRNA. Endothelial cells were also exposed to SARS‑CoV‑2. rSP1 increased production of IL‑6, MCP‑1, ICAM‑1 and PAI‑1, and induced NFkB activation via ACE2 in endothelial cells. rS1p increased microparticle formation, a functional marker of endothelial injury. ACE2 interacting proteins involved in inflammation and RNA biology were identified in rS1p‑treated cells. Neither ACE2 expression nor ACE2 enzymatic function were affected by rSP1. Endothelial cells exposed to SARS‑CoV‑2 virus did not exhibit viral replication.

We demonstrate that rSP1 induces endothelial inflammation via ACE2 through processes that are independent of ACE2 enzymatic activity and viral replication. We define a novel role for ACE2 in COVID‑19‑ associated endotheliitis.

Link | PDF (Nature Scientific Reports)
 
Endotheliitis has been described in COVID-19 patients, and molecular mechanisms involved are still under debate. Here we demonstrate that SARS-CoV-2 is a potent inducer of pro-inflammatory molecules, such as cytokines (IL-6, TNFα), chemokines (MCP-1), PAI-1 and adhesion molecules (ICAM-1, VCAM-1), and that it activates pro-inflammatory signalling through NFκB and ERK1/2 in endothelial cells.

Our findings propose another paradigm where ACE2 acts as a receptor-like protein, since pharmacological inhibition, as well as downregulation of ACE2 inhibited SARS-CoV-2 spike-induced inflammation, without any changes in ACE2 activity after exposure of human endothelial cells to the recombinant spike protein.

Glucose-regulated protein 78 (GRP78), also known as the endoplasmic reticulum stress regulator BIP, has been described as another receptor for SARS-CoV-2 spike protein. Under cell stress, GRP78 whereby it acts as a danger associated molecular patter (DAMP) activating toll-like receptors. In addition, TLR activation itself may be an additional mechanism of spike protein-induced inflammation.
 
Glucose-regulated protein 78 (GRP78), also known as the endoplasmic reticulum stress regulator BIP, has been described as another receptor for SARS-CoV-2 spike protein. Under cell stress, GRP78 whereby it acts as a danger associated molecular patter (DAMP) activating toll-like receptors. In addition, TLR activation itself may be an additional mechanism of spike protein-induced inflammation.

GRP78 / BiP is important in holding back or activating the ER stress response. It binds to the 3 trans-membrane ER stress sensors (IRE1⍺, PERK, ATF6⍺). However, it preferentially binds to mis-folded proteins in the ER lumen and this competes with the stress sensors. So, once dissociated from BiP, eg PERK is phosphorylated, which leads to phosphorylation of eIF2⍺, which activates that arm of the unfolded protein response (UPR). That has two mechanisms: phosphorylation of eIF2⍺ ceases ribosomal translation of mRNA into proteins (to reduce the ongoing ER protein overload); but also upregulates ATF4 which induces CHOP which upregulates ER chaperones and foldases to try and clear the misfolded junk already in the ER lumen.

Phew. See Endoplasmic Reticulum ER Stress Response Failure in Diseases (2020).

Screenshot 2023-08-29 at 9.43.03 PM Large.jpeg

In WASF3 disrupts mitochondrial respiration and may mediate exercise intolerance in myalgic encephalomyelitis/chronic fatigue syndrome (2023) —

Given the importance of the interaction between the ER and mitochondria for muscle function, we reasoned that ER stress [...] may regulate WASF3 in muscle cells. Indeed, the ER stress marker PERK was significantly higher while BiP was lower in the ME/CFS muscle samples. This discordance between PERK and BiP levels in ME/CFS samples suggested impairment of the canonical ER stress pathway, termed “ER Stress Response Failure”

Increased ER stress would normally be expected to turn down protein translation, but intriguingly, the inhibitory phosphorylation of protein translation factor eIF2α, a target of PERK kinase activity, was unexpectedly lower (i.e., activated) in patient S1 cells. This finding suggested that there was “ER stress response failure” in patient S1 cells as also observed in the ME/CFS muscle biopsy samples

Back to Endoplasmic Reticulum (ER) Stress Response Failure in Diseases (2020) —

During severe ER stress conditions in metabolic diseases, the activated UPR elements may not trigger downstream signaling, as evidence is accumulating in response to IRE1 downstream signaling sXBP1, which is termed ER stress response failure

If I'm following this correctly, and if something similar to the ME/CFS findings is happening in LC, my questions would be:

if spike protein can bind to GRP78 / BiP, can this occur in the ER? And could this lead to the equivalent of a severe ER stress condition, where BiP is preferentially binding to spike + any misfolded proteins and overloading the UPR activation, leading to downstream failure? (ie ER stress response failure).
 
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If I'm following this correctly, and if something similar to the ME/CFS findings is happening in LC, my questions would be:

if spike protein can bind to GRP78 / BiP, can this occur in the ER? And could this lead to the equivalent of a severe ER stress condition, where BiP is preferentially binding to spike + any misfolded proteins and overloading the UPR activation, leading to downstream failure? (ie ER stress response failure).

See also Identification of a Druggable Site on GRP78 at the GRP78-SARS-CoV-2 Interface and Compounds to Disrupt that Interface (2023, Preprint: BioRxiv)
 
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