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Connexin 43 Hemichannel Activity Promoted by Pro-Inflammatory Cytokines and High Glucose Alters Endothelial Cell Function.

ABSTRACT: The present work was done to elucidate whether hemichannels of a cell line derived from endothelial cells are affected by pro-inflammatory conditions (high glucose and IL-1?/TNF-?) known to lead to vascular dysfunction. We used EAhy 926 cells treated with high glucose and IL-1?/TNF-?. The hemichannel activity was evaluated with the dye uptake method and was abrogated with selective inhibitors or knocking down of hemichannel protein subunits with siRNA. Western blot analysis, cell surface biotinylation, and confocal microscopy were used to evaluate total and plasma membrane amounts of specific proteins and their cellular distribution, respectively. Changes in intracellular Ca2+ and nitric oxide (NO) signals were estimated by measuring FURA-2 and DAF-FM probes, respectively. High glucose concentration was found to elevate dye uptake, a response that was enhanced by IL-1?/TNF-?. High glucose plus IL-1?/TNF-?-induced dye uptake was abrogated by connexin 43 (Cx43) but not pannexin1 knockdown. Furthermore, Cx43 hemichannel activity was associated with enhanced ATP release and activation of p38 MAPK, inducible NO synthase, COX2, PGE2 receptor EP1, and P2X7/P2Y1 receptors. Inhibition of the above pathways prevented completely the increase in Cx43 hemichannel activity of cells treated high glucose and IL-1?/TNF-?. Both synthetic and endogenous cannabinoids (CBs) also prevented the increment in Cx43 hemichannel opening, as well as the subsequent generation and release of ATP and NO induced by pro-inflammatory conditions. The counteracting action of CBs also was extended to other endothelial alterations evoked by IL-1?/TNF-? and high glucose, including increased ATP-dependent Ca2+ dynamics and insulin-induced NO production. Finally, inhibition of Cx43 hemichannels also prevented the ATP release from endothelial cells treated with IL-1?/TNF-? and high glucose. Therefore, we propose that reduction of hemichannel activity could represent a strategy against the activation of deleterious pathways that lead to endothelial dysfunction and possibly cell damage evoked by high glucose and pro-inflammatory conditions during cardiovascular diseases.

SUBMITTER: Saez JC

PROVIDER: S-EPMC6104120 | biostudies-literature | 2018

REPOSITORIES: biostudies-literature

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Connexin 43 Hemichannel Activity Promoted by Pro-Inflammatory Cytokines and High Glucose Alters Endothelial Cell Function.

Sáez Juan C JC Contreras-Duarte Susana S Gómez Gonzalo I GI Labra Valeria C VC Santibañez Cristian A CA Gajardo-Gómez Rosario R Avendaño Beatriz C BC Díaz Esteban F EF Montero Trinidad D TD Velarde Victoria V Orellana Juan A JA

Frontiers in immunology 20180815

The present work was done to elucidate whether hemichannels of a cell line derived from endothelial cells are affected by pro-inflammatory conditions (high glucose and IL-1β/TNF-α) known to lead to vascular dysfunction. We used EAhy 926 cells treated with high glucose and IL-1β/TNF-α. The hemichannel activity was evaluated with the dye uptake method and was abrogated with selective inhibitors or knocking down of hemichannel protein subunits with siRNA. Western blot analysis, cell surface biotiny ...[more]

PMID: 30158937

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Project description:The endothelium has been established to generate intercellular stresses and suggested to transmit these intercellular stresses through cell-cell junctions, such as VE-Cadherin and ZO-1, for example. Although the previously mentioned molecules reflect the appreciable contributions both adherens junctions and tight junctions are believed to have in endothelial cell intercellular stresses, in doing so they also reveal the obscure relationship that exists between gap junctions and intercellular stresses. Therefore, to bring clarity to this relationship we disrupted expression of the endothelial gap junction connexin 43 (Cx43) by exposing confluent human umbilical vein endothelial cells (HUVECs) to a low (0.2 ?g/mL) and high (2 ?g/mL) concentration of 2,5-dihydroxychalcone (chalcone), a known Cx43 inhibitor. To evaluate the impact Cx43 disruption had on endothelial cell mechanics we utilized traction force microscopy and monolayer stress microscopy to measure cell-substrate tractions and cell-cell intercellular stresses, respectively. HUVEC monolayers exposed to a low concentration of chalcone produced average normal intercellular stresses that were on average 17% higher relative to control, while exposure to a high concentration of chalcone yielded average normal intercellular stresses that were on average 55% lower when compared to control HUVEC monolayers. HUVEC maximum shear intercellular stresses were observed to decrease by 16% (low chalcone concentration) and 66% (high chalcone concentration), while tractions exhibited an almost 2-fold decrease under high chalcone concentration. In addition, monolayer cell velocities were observed to decrease by 19% and 35% at low chalcone and high chalcone concentrations, respectively. Strain energies were also observed to decrease by 32% and 85% at low and high concentration of chalcone treatment, respectively, when compared to control. The findings we present here reveal for the first time the contribution Cx43 has to endothelial biomechanics.

Dataset Information

Connexin 43 Hemichannel Activity Promoted by Pro-Inflammatory Cytokines and High Glucose Alters Endothelial Cell Function.

Publications

Connexin 43 Hemichannel Activity Promoted by Pro-Inflammatory Cytokines and High Glucose Alters Endothelial Cell Function.

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