Targeting gliovascular connexins prevents inflammatory blood-brain barrier leakage and astrogliosis

Abstract

The blood-brain barrier is formed by capillary endothelial cells expressing Cx37, Cx40 and Cx43, and is joined by closely apposed astrocytes expressing Cx43 and Cx30. We investigated whether connexin-targeting peptides could limit barrier leakage triggered by LPS-induced systemic inflammation in mice. Intraperitoneal LPS increased endothelial and astrocytic Cx43 expression, elevated TNFα, IL1β, IFNγ and IL6 in plasma and IL6 in the brain, and induced barrier leakage recorded over 24h. Barrier leakage was largely prevented by global Cx43 knockdown and Cx43/Cx30 double-knockout in astrocytes, slightly diminished by endothelial Cx43 knockout and not protected by global Cx30 knockout. Intravenous administration of Gap27 or Tat-Gap19 just before LPS also prevented barrier leakage, and intravenous BAPTA-AM to chelate intracellular calcium was equally effective. Patch-clamp experiments demonstrated LPS-induced Cx43 hemichannel opening in endothelial cells, which was suppressed by Gap27, Gap19 and BAPTA.LPS additionally triggered astrogliosis that was prevented by intravenous Tat-Gap19 or BAPTA-AM. Cortically applied Tat-Gap19 or BAPTA-AM to primarily target astrocytes, also strongly diminished barrier leakage. In vivo dye uptake and in vitro patch-clamp showed Cx43 hemichannel opening in astrocytes that was induced by IL6 in a calcium-dependent manner. We conclude that targeting endothelial and astrocytic connexins is a powerful approach to limit barrier failure and astrogliosis.

Inhibiting astrocyte connexin‐43 hemichannels blocks radiation‐induced vesicular VEGF‐A release and blood–brain barrier dysfunction

“…Moreover, VEGF is a major factor in the development of late complications (>3–6 months) (Lan et al, 2022; Zhou et al, 2019), which are mitigated by anti‐VEGF treatment as demonstrated in patient studies with bevacizumab, resulting in decreased BBB leakage and radio‐necrosis lesion size (Stegmayr et al, 2017; Vaios et al, 2022; Xue et al, 2021). Our present findings show that Cx43 HC inhibition with TATGap19 prevents VEGF‐A transport to astrocytic endfeet and subsequent BBB leakage; TATGap19 also prevents astrocytic GFAP upregulation in astrocytes triggered by peripheral inflammation (De Bock et al, 2022). Both GFAP and VEGF are crucial instigators of radiation‐induced complications, making the targeting of the early Cx43 HC opening event a potentially more direct approach relative to bevacizumab intervention that targets the end‐point of the signaling cascade reported here and summarized below:…”

“…TATGap19 was injected 15 min prior to irradiation whereas BAPTA‐AM/Fluo3‐AM were injected 30 min before irradiation. The BAPTA‐AM dose (15.7 μmol/kg) was based on previous work where 12 mg/kg was used and found to not negatively affect survival (100% survival at the 24 h endpoint of the study (De Bock et al, 2022). The Fluo3‐AM dose was further reduced to 10,6 μmol/kg (2/3 of the BAPTA‐AM dose).…”

“…In addition to GJ function, X‐ray exposure also increases HC opening in vascular ECs, including BBB ECs (Hoorelbeke et al, 2020; Ramadan et al, 2019; Ramadan et al, 2020). Astrocytic HCs open in response to blood‐borne inflammation or brain ischemia and contribute to increased Ca 2+ signaling and astrogliosis (De Bock et al, 2022; Freitas‐Andrade et al, 2019; Meunier et al, 2017). X‐rays strongly activate cellular Ca 2+ signaling (Decrock et al, 2017; Hoorelbeke et al, 2020; Wu et al, 2020), which may stimulate vesicular transport, especially endocytosis, vesicle guidance and exocytosis (Hay, 2007).…”

“…feet and subsequent BBB leakage; TATGap19 also prevents astrocytic GFAP upregulation in astrocytes triggered by peripheral inflammation(De Bock et al, 2022). Both GFAP and VEGF are crucial instigators of radiation-induced complications, making the targeting of the early Cx43 HC opening event a potentially more direct approach relative to bevacizumab intervention that targets the end-point of the signaling cascade reported here and summa-rized below: 5 | CONCLUSIONS We conclude that early post-irradiation Cx43 HC opening crucially triggers reorganization of astrocytic vesicular transport, steering VEGF-A/VAMP3 vesicles to endfeet extensions from where it is released and triggers barrier leakage.…”

A year in review: brain barriers and brain fluids research in 2022

“…Astrocytes and brain endothelial cells express connexins important for cell:cell communication. De Bock et al [ 25 ] have shown the importance of connexin 43 in both cell types in BBB disruption following lipopolysaccharide (LPS) administration. Kim et al [ 26 ] have examined the pathways by which inflammatory mediators induce a reactive astrocyte phenotype to then induce BBB disruption.…”