Abstract
Upon intravenous injection of tumour necrosis factor (TNF) in mice, a systemic inflammatory response syndrome (SIRS) is initiated, characterized by an acute cytokine storm and induction of vascular hyperpermeability. Connexin43 hemichannels have been implicated in various pathological conditions, e.g. ischemia and inflammation, and can lead to detrimental cellular outcomes. Here, we explored whether targeting connexin43 hemichannels could alleviate TNF-induced endothelial barrier dysfunction and lethality in SIRS. Therefore, we verified whether administration of connexin43-targeting-peptides affected survival, body temperature and vascular permeability in vivo. In vitro, TNF-effects on connexin43 hemichannel function were investigated by single-channel studies and Ca2+-imaging. Blocking connexin43 hemichannels with TAT-Gap19 protected mice against TNF-induced mortality, hypothermia and vascular leakage, while enhancing connexin43 hemichannel function with TAT-CT9 provoked opposite sensitizing effects. In vitro patch-clamp studies revealed that TNF acutely activated connexin43 hemichannel opening in endothelial cells, which was promoted by CT9, and inhibited by Gap19 and intracellular Ca2+-buffering. In vivo experiments aimed at buffering intracellular Ca2+, and pharmacologically targeting Ca2+/calmodulin-dependent protein kinase-II, a known modulator of endothelial barrier integrity, demonstrated their involvement in permeability alterations. Our results demonstrate significant benefits of inhibiting connexin43 hemichannels to counteract TNF-induced SIRS-associated vascular permeability and lethality.
Highlights
Upon intravenous injection of tumour necrosis factor (TNF) in mice, a systemic inflammatory response syndrome (SIRS) is initiated, characterized by an acute cytokine storm and induction of vascular hyperpermeability
Prophylactic administration of Gap[27] (25 mg/kg i.v.), a peptide mimicking a conserved domain on the second extracellular loop of various Cxs and known to first block hemichannels followed by delayed gap junction channels (GJCs) block[26], significantly protected 50% of the mice against TNF-induced hypothermia and mortality (Fig. 1a)
We found that the CaMKII inhibitor KN-93 (10 mg/kg i.v.) decreased TNF-induced renal vascular leakage, suggesting that CaMKII may be implicated in this process (Figs 6d and 7)
Summary
Upon intravenous injection of tumour necrosis factor (TNF) in mice, a systemic inflammatory response syndrome (SIRS) is initiated, characterized by an acute cytokine storm and induction of vascular hyperpermeability. We explored whether targeting connexin[43] hemichannels could alleviate TNF-induced endothelial barrier dysfunction and lethality in SIRS. In vitro patch-clamp studies revealed that TNF acutely activated connexin[43] hemichannel opening in endothelial cells, which was promoted by CT9, and inhibited by Gap[19] and intracellular Ca2+-buffering. Endothelial cells (ECs) are connected by different junctional proteins that mediate vascular barrier function and intercellular communication[5]. Hemichannels provide a passageway between the intra- and extracellular environment, while GJCs connect the cytoplasm of two neighbouring cells, providing a route for direct cell-cell communication. Our in vivo and in vitro approaches demonstrate that Cx43 hemichannels, through TNF-induced Ca2+-increases, lead to renal vascular permeability and animal mortality in SIRS
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