X­irradiation induces acute and early term inflammatory responses in atherosclerosis­prone ApoE­/­ mice and in endothelial cells.

Thoracic radiotherapy is an effective treatment for many types of cancer; however it is also associated with an increased risk of developing cardiovascular disease (CVD), appearing mainly ≥10 years after radiation exposure. The present study investigated acute and early term physiological and molecular changes in the cardiovascular system after ionizing radiation exposure. Female and male ApoE­/­ mice received a single exposure of low or high dose X­ray thoracic irradiation (0.1 and 10 Gy). The level of cholesterol and triglycerides, as well as a large panel of inflammatory markers, were analyzed in serum samples obtained at 24 h and 1 month after irradiation. The secretion of inflammatory markers was further verified in vitro in coronary artery and microvascular endothelial cell lines after exposure to low and high dose of ionizing radiation (0.1 and 5 Gy). Local thoracic irradiation of ApoE­/­ mice increased serum growth differentiation factor­15 (GDF­15) and C­X­C motif chemokine ligand 10 (CXCL10) levels in both female and male mice 24 h after high dose irradiation, which were also secreted from coronary artery and microvascular endothelial cells in vitro. Sex­specific responses were observed for triglyceride and cholesterol levels, and some of the assessed inflammatory markers as detailed below. Male ApoE­/­ mice demonstrated elevated intercellular adhesion molecule­1 and P­selectin at 24 h, and adiponectin and plasminogen activator inhibitor­1 at 1 month after irradiation, while female ApoE­/­ mice exhibited decreased monocyte chemoattractant protein­1 and urokinase­type plasminogen activator receptor at 24 h, and basic fibroblast growth factor 1 month after irradiation. The inflammatory responses were mainly significant following high dose irradiation, but certain markers showed significant changes after low dose exposure. The present study revealed that acute/early inflammatory responses occurred after low and high dose thoracic irradiation. However, further research is required to elucidate early asymptomatic changes in the cardiovascular system post thoracic X­irradiation and to investigate whether GDF­15 and CXCL10 could be considered as potential biomarkers for the early detection of CVD risk in thoracic radiotherapy­treated patients.

Connexin43 Hemichannel Targeting With TAT-Gap19 Alleviates Radiation-Induced Endothelial Cell Damage.

BACKGROUND: Emerging evidence indicates an excess risk of late occurring cardiovascular diseases, especially atherosclerosis, after thoracic cancer radiotherapy. Ionizing radiation (IR) induces cellular effects which may induce endothelial cell dysfunction, an early marker for atherosclerosis. In addition, intercellular communication through channels composed of transmembrane connexin proteins (Cxs), i.e. Gap junctions (direct cell-cell coupling) and hemichannels (paracrine release/uptake pathway) can modulate radiation-induced responses and therefore the atherosclerotic process. However, the role of endothelial hemichannel in IR-induced atherosclerosis has never been described before. MATERIALS AND METHODS: Telomerase-immortalized human Coronary Artery/Microvascular Endothelial cells (TICAE/TIME) were exposed to X-rays (0.1 and 5 Gy). Production of reactive oxygen species (ROS), DNA damage, cell death, inflammatory responses, and senescence were assessed with or without applying a Cx43 hemichannel blocker (TAT-Gap19). RESULTS: We report here that IR induces an increase in oxidative stress, cell death, inflammatory responses (IL-8, IL-1ß, VCAM-1, MCP-1, and Endothelin-1) and premature cellular senescence in TICAE and TIME cells. These effects are significantly reduced in the presence of the Cx43 hemichannel-targeting peptide TAT-Gap19. CONCLUSION: Our findings suggest that endothelial Cx43 hemichannels contribute to various IR-induced processes, such as ROS, cell death, inflammation, and senescence, resulting in an increase in endothelial cell damage, which could be protected by blocking these hemichannels. Thus, targeting Cx43 hemichannels may potentially exert radioprotective effects.

Cx43 channels and signaling via IP3/Ca2+, ATP, and ROS/NO propagate radiation-induced DNA damage to non-irradiated brain microvascular endothelial cells.

Radiotherapeutic treatment consists of targeted application of radiation beams to a tumor but exposure of surrounding healthy tissue is inevitable. In the brain, ionizing radiation induces breakdown of the blood-brain barrier by effects on brain microvascular endothelial cells. Damage from directly irradiated cells can be transferred to surrounding non-exposed bystander cells, known as the radiation-induced bystander effect. We investigated involvement of connexin channels and paracrine signaling in radiation-induced bystander DNA damage in brain microvascular endothelial cells exposed to focused X-rays. Irradiation caused DNA damage in the directly exposed area, which propagated over several millimeters in the bystander area. DNA damage was significantly reduced by the connexin channel-targeting peptide Gap26 and the Cx43 hemichannel blocker TAT-Gap19. ATP release, dye uptake, and patch clamp experiments showed that hemichannels opened within 5 min post irradiation in both irradiated and bystander areas. Bystander signaling involved cellular Ca2+ dynamics and IP3, ATP, ROS, and NO signaling, with Ca2+, IP3, and ROS as crucial propagators of DNA damage. We conclude that bystander effects are communicated by a concerted cascade involving connexin channels, and IP3/Ca2+, ATP, ROS, and NO as major contributors of regenerative signal expansion.

Blocking connexin43 hemichannels protects mice against tumour necrosis factor-induced inflammatory shock.

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.

Single and fractionated ionizing radiation induce alterations in endothelial connexin expression and channel function.

Radiotherapy is an effective treatment for most tumor types. However, emerging evidence indicates an increased risk for atherosclerosis after ionizing radiation exposure, initiated by endothelial cell dysfunction. Interestingly, endothelial cells express connexin (Cx) proteins that are reported to exert proatherogenic as well as atheroprotective effects. Furthermore, Cxs form channels, gap junctions and hemichannels, that are involved in bystander signaling that leads to indirect radiation effects in non-exposed cells. We here aimed to investigate the consequences of endothelial cell irradiation on Cx expression and channel function. Telomerase immortalized human Coronary Artery/Microvascular Endothelial cells were exposed to single and fractionated X-rays. Several biological endpoints were investigated at different time points after exposure: Cx gene and protein expression, gap junctional dye coupling and hemichannel function. We demonstrate that single and fractionated irradiation induce upregulation of proatherogenic Cx43 and downregulation of atheroprotective Cx40 gene and protein levels in a dose-dependent manner. Single and fractionated irradiation furthermore increased gap junctional communication and induced hemichannel opening. Our findings indicate alterations in Cx expression that are typically observed in endothelial cells covering atherosclerotic plaques. The observed radiation-induced increase in Cx channel function may promote bystander signaling thereby exacerbating endothelial cell damage and atherogenesis.

Species-dependent extracranial manifestations of a brain seeking breast cancer cell line.

PURPOSE: Metastatic brain tumors pose a severe problem in the treatment of patients with breast carcinoma. Preclinical models have been shown to play an important role in unraveling the underlying mechanisms behind the metastatic process and evaluation of new therapeutic approaches. As the size of the rat brain allows improved in vivo imaging, we attempted to establish a rat model for breast cancer brain metastasis that allows follow-up by 7 tesla (7T) preclinical Magnetic Resonance Imaging (MRI). PROCEDURES: Green fluorescent protein-transduced (eGFP) MDA-MB-231br breast cancer cells were labeled with micron-sized particles of iron oxide (MPIOs) and intracardially injected in the left ventricle of female nude rats and mice. 7T preclinical MRI was performed to show the initial distribution of MPIO-labeled cancer cells and to visualize metastasis in the brain. Occurrence of potential metastasis outside the brain was evaluated by 2-deoxy-2-[18F]fluoro-D-glucose ([18F]FDG) positron emission tomography (PET)/computed tomography (CT) and potential bone lesions were assessed using [18F]sodium fluoride ([18F]NaF) PET/CT. RESULTS: The first signs of brain metastasis development were visible as hyperintensities on T2-weighted (T2w) MR images acquired 3 weeks after intracardiac injection in rats and mice. Early formation of unexpected bone metastasis in rats was clinically observed and assessed using PET/CT. Almost no bone metastasis development was observed in mice after PET/CT evaluation. CONCLUSIONS: Our results suggest that the metastatic propensity of the MDA-MB-231br/eGFP cancer cell line outside the brain is species-dependent. Because of early and abundant formation of bone metastasis with the MDA-MB-231br/eGFP cancer cell line, this rat model is currently not suitable for investigating brain metastasis as a single disease model nor for evaluation of novel brain metastasis treatment strategies.

Calcium, a pivotal player in photodynamic therapy?

Photodynamic therapy combines three non-toxic components: light, oxygen and a photosensitizer to generate singlet oxygen and/or other ROS molecules in order to target destruction of cancer cells. The damage induced in the targeted cells can furthermore propagate to non-exposed bystander cells thereby exacerbating the damage. Ca2+ signaling is strongly intertwined with ROS signaling and both play crucial roles in cell death. In this review we aimed to review current knowledge on the role of Ca2+ and ROS signaling, their effect on cell-cell propagation via connexin-linked mechanisms and the outcome in terms of cell death. In general, photodynamic therapy results in an increased cytosolic Ca2+ concentration originating from Ca2+ entry or Ca2+ release from internal stores. While photodynamic therapy can certainly induce cell death, the outcome depends on the cell type and the photosensitizer used. Connexin channels propagating the Ca2+ signal, and presumably regenerating ROS at distance, may play a role in spreading the effect to neighboring non-exposed bystander cells. Given the various cell types and photosensitizers used, there is currently no unified signaling scheme to explain the role of Ca2+ and connexins in the responses following photodynamic therapy. This article is part of a Special Issue entitled: Calcium signaling in health, disease and therapy edited by Geert Bultynck and Jan Parys.

Noninvasive Whole-Body Imaging of Phosphatidylethanolamine as a Cell Death Marker Using 99mTc-Duramycin During TNF-Induced SIRS.

Systemic inflammatory response syndrome (SIRS) is an inflammatory state affecting the whole body. It is associated with the presence of pro- and antiinflammatory cytokines in serum, including tumor necrosis factor (TNF). TNF has multiple effects and leads to cytokine production, leukocyte infiltration, and blood pressure reduction and coagulation, thereby contributing to tissue damage and organ failure. A sterile mouse model of sepsis, TNF-induced SIRS, was used to visualize the temporal and spatial distribution of damage in susceptible tissues during SIRS. For this, a radiopharmaceutical agent, 99mTc-duramycin, that binds to exposed phosphatidylethanolamine on dying cells was longitudinally visualized using SPECT/CT imaging. Methods: C57BL/6J mice were challenged with intravenous injections of murine TNF or vehicle, and necrostatin-1 was used to interfere with cell death. Two hours after vehicle or TNF treatment, mice received 99mTc-duramycin intravenously (35.44 ± 3.80 MBq). Static whole-body 99mTc-duramycin SPECT/CT imaging was performed 2, 4, and 6 h after tracer injection. Tracer uptake in different organs was quantified by volume-of-interest analysis using PMOD software and expressed as SUVmean After the last scan, ex vivo biodistribution was performed to validate the SPECT imaging data. Lastly, terminal deoxynucleotidyl-transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) staining was performed to correlate the obtained results to cell death. Results: An increased 99mTc-duramycin uptake was detected in mice injected with TNF, when compared with control mice, in lungs (0.55 ± 0.1 vs. 0.34 ± 0.05), intestine (0.75 ± 0.13 vs. 0.56 ± 0.1), and liver (1.03 ± 0.14 vs. 0.64 ± 0.04) 4 h after TNF and remained significantly elevated until 8 h after TNF. The imaging results were consistent with ex vivo γ-counting results. Significantly increased levels of tissue damage were detected via TUNEL staining in the lungs and intestine of mice injected with TNF. Interestingly, necrostatin-1 pretreatment conferred protection against lethal SIRS and reduced the 99mTc-duramycin uptake in the lungs 8 h after TNF (SUV, 0.32 ± 0.1 vs. 0.51 ± 0.15). Conclusion: This study demonstrated that noninvasive 99mTc-duramycin SPECT imaging can be used to characterize temporal and spatial kinetics of injury and cell death in susceptible tissues during TNF-induced SIRS, making it useful for global, whole-body assessment of tissue damage during diseases associated with inflammation and injury.

Inhibition of connexin hemichannels alleviates non-alcoholic steatohepatitis in mice.

While gap junctions mediate intercellular communication and support liver homeostasis, connexin hemichannels are preferentially opened by pathological stimuli, including inflammation and oxidative stress. The latter are essential features of non-alcoholic steatohepatitis. In this study, it was investigated whether connexin32 and connexin43 hemichannels play a role in non-alcoholic steatohepatitis. Mice were fed a choline-deficient high-fat diet or normal diet for 8 weeks. Thereafter, TAT-Gap24 or TAT-Gap19, specific inhibitors of hemichannels composed of connexin32 and connexin43, respectively, were administered for 2 weeks. Subsequently, histopathological examination was carried out and various indicators of inflammation, liver damage and oxidative stress were tested. In addition, whole transcriptome microarray analysis of liver tissue was performed. Channel specificity of TAT-Gap24 and TAT-Gap19 was examined in vitro by fluorescence recovery after photobleaching analysis and measurement of extracellular release of adenosine triphosphate. TAT-Gap24 and TAT-Gap19 were shown to be hemichannel-specific in cultured primary hepatocytes. Diet-fed animals treated with TAT-Gap24 or TAT-Gap19 displayed decreased amounts of liver lipids and inflammatory markers, and augmented levels of superoxide dismutase, which was supported by the microarray results. These findings show the involvement of connexin32 and connexin43 hemichannels in non-alcoholic steatohepatitis and, simultaneously, suggest a role as potential drug targets in non-alcoholic steatohepatitis.

Connexin hemichannel inhibition reduces acetaminophen-induced liver injury in mice.

Historically, connexin hemichannels have been considered as structural precursors of gap junctions. However, accumulating evidence points to independent roles for connexin hemichannels in cellular signaling by connecting the intracellular compartment with the extracellular environment. Unlike gap junctions, connexin hemichannels seem to be mainly activated in pathological processes. The present study was set up to test the potential involvement of hemichannels composed of connexin32 and connexin43 in acute hepatotoxicity induced by acetaminophen. Prior to this, in vitro testing was performed to confirm the specificity and efficacy of TAT-Gap24 and TAT-Gap19 in blocking connexin32 and connexin43 hemichannels, respectively. Subsequently, mice were overdosed with acetaminophen followed by treatment with TAT-Gap24 or TAT-Gap19 or a combination of both after 1.5h. Sampling was performed 3, 6, 24 and 48h following acetaminophen administration. Evaluation of the effects of connexin hemichannel inhibition was based on a series of clinically relevant read-outs, measurement of inflammatory cytokines and oxidative stress. Subsequent treatment of acetaminophen-overdosed mice with TAT-Gap19 only marginally affected liver injury. In contrast, a significant reduction in serum alanine aminotransferase activity was found upon administration of TAT-Gap24 to intoxicated animals. Furthermore, co-treatment of acetaminophen-overdosed mice with both peptides revealed an additive effect as even lower serum alanine aminotransferase activity was observed. Blocking of connexin32 or connexin43 hemichannels individually was found to decrease serum quantities of pro-inflammatory cytokines, while no effects were observed on the occurrence of hepatic oxidative stress. This study shows for the first time a role for connexin hemichannels in acetaminophen-induced acute liver failure.

The BH4 domain of Bcl-2 orthologues from different classes of vertebrates can act as an evolutionary conserved inhibitor of IP3 receptor channels.

Ca2+ signalling plays an important role in various physiological processes in vertebrates. In mammals, the highly conserved anti-apoptotic B-cell lymphoma-2 (Bcl-2) protein is an important modulator of the inositol 1,4,5-trisphosphate receptor (IP3R), i.e. the main intracellular Ca2+ - release channel located at the endoplasmic reticulum (ER). The Bcl-2 Homology (BH) 4 domain of Bcl-2 (BH4-Bcl-2) is a critical determinant for inhibiting IP3Rs, by directly targeting a region in the modulatory domain of the receptor (domain 3). In this paper, we aimed to track the evolutionary history of IP3R regulation by the BH4 domain of Bcl-2 orthologues from different classes of vertebrates, including Osteichthyes, Amphibia, Reptilia, Aves and Mammalia. The high degree of conservation of the BH4 sequences correlated with the ability of all tested peptides to bind to the domain 3 of mouse IP3R1 in GST-pull downs and their overall ability to inhibit IP3-induced Ca2+ release (IICR) in permeabilized cells. Nevertheless, the BH4 domains differed in their potency to suppress IICR. The peptide derived from X. laevis was the least potent inhibitor. We identified a critical residue in BH4-Bcl-2 from H. sapiens, Thr7, which is replaced by Gly7 in X. laevis. Compared to the wild type X. laevis BH4-Bcl-2, a "humanized" version of the peptide (BH4-Bcl-2 Gly7Thr), displayed increased IP3R-inhibitory properties. Despite the differences in the inhibitory efficiency, our data indicate that the BH4 domain of Bcl-2 orthologues from different classes of vertebrates can act as a binding partner and inhibitor of IP3R channels.

Calcium, oxidative stress and connexin channels, a harmonious orchestra directing the response to radiotherapy treatment?

Although radiotherapy is commonly used to treat cancer, its beneficial outcome is frequently hampered by the radiation resistance of tumor cells and adverse reactions in normal tissues. Mechanisms of cell-to-cell communication and how intercellular signals are translated into cellular responses, have become topics of intense investigation, particularly within the field of radiobiology. A substantial amount of evidence is available demonstrating that both gap junctional and paracrine communication pathways can propagate radiation-induced biological effects at the intercellular level, commonly referred to as radiation-induced bystander effects (RIBE). Multiple molecular signaling mechanisms involving oxidative stress, kinases, inflammatory molecules, and Ca2+ are postulated to contribute to RIBE. Ca2+ is a highly versatile and ubiquitous second messenger that regulates diverse cellular processes via the interaction with various signaling cascades. It furthermore provides a fast system for the dissemination of information at the intercellular level. Channels formed by transmembrane connexin (Cx) proteins, i.e. hemichannels and gap junction channels, can mediate the cell-to-cell propagation of increases in intracellular Ca2+ by ministering paracrine and direct cell-cell communication, respectively. We here review current knowledge on radiation-induced signaling mechanisms in irradiated and bystander cells, particularly focusing on the contribution of oxidative stress, Ca2+ and Cx channels. By illustrating the tight interplay between these different partners, we provide a conceptual framework for intercellular Ca2+ signaling as a key player in modulating the RIBE and the overall response to radiation.

Blocking connexin channels improves embryo development of vitrified bovine blastocysts.

Connexins (Cxs) are required for normal embryo development and implantation. They form gap junctions (GJs) connecting the cytoplasm of adjacent cells and hemichannels (HCs), which are normally closed but open in response to stress conditions. Excessive HC opening is detrimental for cell function and may lead to cell death. We found that hatching of in vitro-produced bovine embryos, matured in serum-containing conditions, was significantly improved when vitrification/warming was done in the presence of Gap26 that targets GJA1 (Cx43) and GJA4 (Cx37). Further work showed that HCs from blastocysts produced after oocyte maturation in the presence of serum were open shortly after vitrification/warming, and this was prevented by Gap26. Gap26, applied for the exposure times used, inhibited Cx43 and Cx37 HCs while it did not have an effect on GJs. Interestingly, Gap26 had no effect on blastocyst degeneration or cell death. We conclude that blocking HCs protects embryos during vitrification and warming by a functional effect not linked to cell death.

At the cross-point of connexins, calcium, and ATP: blocking hemichannels inhibits vasoconstriction of rat small mesenteric arteries.

AIMS: Connexins form gap-junctions (GJs) that directly connect cells, thereby coordinating vascular cell function and controlling vessel diameter and blood flow. GJs are composed of two hemichannels contributed by each of the connecting cells. Hemichannels also exist as non-junctional channels that, when open, lead to the entry/loss of ions and the escape of ATP. Here we investigated cross-talk between hemichannels and Ca2+/purinergic signalling in controlling blood vessel contraction. We hypothesized that hemichannel Ca2+ entry and ATP release contributes to smooth muscle cell (SMC) Ca2+ dynamics, thereby influencing vessel contractility. We applied several peptide modulators of hemichannel function and inhibitors of Ca2+ and ATP signalling to investigate their influence on SMC Ca2+ dynamics and vessel contractility. METHODS AND RESULTS: Confocal Ca2+ imaging studies on small mesenteric arteries (SMAs) from rat demonstrated that norepinephrine-induced SMC Ca2+ oscillations were inhibited by blocking IP3 receptors with xestospongin-C and by interfering with hemichannel function, most notably by the specific Cx43 hemichannel blocking peptide TAT-L2 and by TAT-CT9 that promotes Cx43 hemichannel opening. Evidence for hemichannel involvement in SMC function was supported by the fact that TAT-CT9 significantly increased SMC resting cytoplasmic Ca2+ concentration, indicating it facilitated Ca2+ entry, and by the observation that norepinephrine-triggered vessel ATP release was blocked by TAT-L2. Myograph tension measurements on isolated SMAs showed significant inhibition of norepinephrine-triggered contractility by the ATP receptor antagonist suramin, but the strongest effect was observed with TAT-L2 that gave ∼80% inhibition at 37 °C. TAT-L2 inhibition of vessel contraction was significantly reduced in conditional Cx43 knockout animals, indicating the effect was Cx43 hemichannel-dependent. Computational modelling suggested these results could be explained by the opening of a single hemichannel per SMC. CONCLUSIONS: These results indicate that Cx43 hemichannels contribute to SMC Ca2+ dynamics and contractility, by facilitating Ca2+ entry, ATP release, and purinergic signalling.

Pannexin1 as mediator of inflammation and cell death.

Pannexins form channels at the plasma membrane surface that establish a pathway for communication between the cytosol of individual cells and their extracellular environment. By doing so, pannexin signaling dictates several physiological functions, but equally underlies a number of pathological processes. Indeed, pannexin channels drive inflammation by assisting in the activation of inflammasomes, the release of pro-inflammatory cytokines, and the activation and migration of leukocytes. Furthermore, these cellular pores facilitate cell death, including apoptosis, pyroptosis and autophagy. The present paper reviews the roles of pannexin channels in inflammation and cell death. In a first part, a state-of-the-art overview of pannexin channel structure, regulation and function is provided. In a second part, the mechanisms behind their involvement in inflammation and cell death are discussed.

Connexins and their channels in inflammation.

Inflammation may be caused by a variety of factors and is a hallmark of a plethora of acute and chronic diseases. The purpose of inflammation is to eliminate the initial cell injury trigger, to clear out dead cells from damaged tissue and to initiate tissue regeneration. Despite the wealth of knowledge regarding the involvement of cellular communication in inflammation, studies on the role of connexin-based channels in this process have only begun to emerge in the last few years. In this paper, a state-of-the-art overview of the effects of inflammation on connexin signaling is provided. Vice versa, the involvement of connexins and their channels in inflammation will be discussed by relying on studies that use a variety of experimental tools, such as genetically modified animals, small interfering RNA and connexin-based channel blockers. A better understanding of the importance of connexin signaling in inflammation may open up towards clinical perspectives.

Electroporation Loading and Dye Transfer: A Safe and Robust Method to Probe Gap Junctional Coupling.

Intercellular communication occurring via gap junction channels is considered a key mechanism for synchronizing physiological functions of cells and for the maintenance of tissue homeostasis. Gap junction channels are protein channels that are situated between neighboring cells and that provide a direct, yet selective route for the passage of small hydrophilic biomolecules and ions. Here, an electroporation method is described to load a localized area within an adherent cell monolayer with a gap junction-permeable fluorescent reporter dye. The technique results in a rapid and efficient labeling of a small patch of cells within the cell culture, without affecting cellular viability. Dynamic and quantitative information on gap junctional communication can subsequently be extracted by tracing the intercellular movement of the dye via time-lapse microscopy.

Into rather unexplored terrain-transcellular transport across the blood-brain barrier.

Efficient neuronal signaling in the central nervous system strictly depends on a well-balanced microenvironment around glial cells, synapses, and axons. Unique features of the blood-brain barrier (BBB) endothelium largely determine the composition of this micro-milieu and are dependent on the tight interplay with surrounding astrocytes and pericytes. BBB endothelial cells are endowed with a highly restrictive junctional complex that occludes the intercellular cleft, thereby preventing paracellular diffusion. The paracellular pathway is subject to extensive research as integrity loss of the junctional complex is associated with many neuropathologies, inflammation, and edema. Another important feature of the BBB endothelium is the low prevalence of nonspecific, transcytotic events, including (macro)pinocytosis, clathrin-dependent and caveolin-dependent endocytosis and the subsequent trafficking of vesicles to the opposite membrane. Although less studied, evidence is accruing that this pathway importantly contributes to increased BBB permeability, often when the junctional complex remains intact. Here, we review current knowledge on the contribution of the transcellular pathway to the BBB leak observed in different pathologic conditions. In addition, we hypothesize that nonselective, large pore connexin and pannexin channels may contribute to transcellular transport, either by providing a direct diffusion pathway across the endothelial monolayer, or indirectly, by exerting control over intracellular levels of the signaling ion Ca(2+) that is involved in many steps of the vesicular pathway. We conclude that transcytotic events at the BBB, despite being less acknowledged, cannot be simply dismissed as done in the past, but actively contribute to BBB leakage in many different pathologies. GLIA 2016;64:1097-1123.

Intracellular Cleavage of the Cx43 C-Terminal Domain by Matrix-Metalloproteases: A Novel Contributor to Inflammation?

The coordination of tissue function is mediated by gap junctions (GJs) that enable direct cell-cell transfer of metabolic and electric signals. GJs are formed by connexin (Cx) proteins of which Cx43 is most widespread in the human body. Beyond its role in direct intercellular communication, Cx43 also forms nonjunctional hemichannels (HCs) in the plasma membrane that mediate the release of paracrine signaling molecules in the extracellular environment. Both HC and GJ channel function are regulated by protein-protein interactions and posttranslational modifications that predominantly take place in the C-terminal domain of Cx43. Matrix metalloproteases (MMPs) are a major group of zinc-dependent proteases, known to regulate not only extracellular matrix remodeling, but also processing of intracellular proteins. Together with Cx43 channels, both GJs and HCs, MMPs contribute to acute inflammation and a small number of studies reports on an MMP-Cx43 link. Here, we build further on these reports and present a novel hypothesis that describes proteolytic cleavage of the Cx43 C-terminal domain by MMPs and explores possibilities of how such cleavage events may affect Cx43 channel function. Finally, we set out how aberrant channel function resulting from cleavage can contribute to the acute inflammatory response during tissue injury.

Connexin and pannexin signaling pathways, an architectural blueprint for CNS physiology and pathology?

The central nervous system (CNS) is composed of a highly heterogeneous population of cells. Dynamic interactions between different compartments (neuronal, glial, and vascular systems) drive CNS function and allow to integrate and process information as well as to respond accordingly. Communication within this functional unit, coined the neuro-glio-vascular unit (NGVU), typically relies on two main mechanisms: direct cell-cell coupling via gap junction channels (GJCs) and paracrine communication via the extracellular compartment, two routes to which channels composed of transmembrane connexin (Cx) or pannexin (Panx) proteins can contribute. Multiple isoforms of both protein families are present in the CNS and each CNS cell type is characterized by a unique Cx/Panx portfolio. Over the last two decades, research has uncovered a multilevel platform via which Cxs and Panxs can influence different cellular functions within a tissue: (1) Cx GJCs enable a direct cell-cell communication of small molecules, (2) Cx hemichannels and Panx channels can contribute to autocrine/paracrine signaling pathways, and (3) different structural domains of these proteins allow for channel-independent functions, such as cell-cell adhesion, interactions with the cytoskeleton, and the activation of intracellular signaling pathways. In this paper, we discuss current knowledge on their multifaceted contribution to brain development and to specific processes in the NGVU, including synaptic transmission and plasticity, glial signaling, vasomotor control, and blood-brain barrier integrity in the mature CNS. By highlighting both physiological and pathological conditions, it becomes evident that Cxs and Panxs can play a dual role in the CNS and that an accurate fine-tuning of each signaling mechanism is crucial for normal CNS physiology.