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1.
J Anat ; 243(4): 697-705, 2023 10.
Artículo en Inglés | MEDLINE | ID: mdl-37222261

RESUMEN

The aim of this study is to correlate small dot hyper-reflective foci (HRF) observed in spectral domain optical coherence tomography (SD-OCT) scans of an animal model of hyperglycaemia with focal electroretinography (fERG) response and immunolabelling of retinal markers. The eyes of an animal model of hyperglycaemia showing signs of diabetic retinopathy (DR) were imaged using SD-OCT. Areas showing dot HRF were further evaluated using fERG. Retinal areas enclosing the HRF were dissected and serially sectioned, stained and labelled for glial fibrillary acidic protein (GFAP) and a microglial marker (Iba-1). Small dot HRF were frequently seen in OCT scans in all retinal quadrants in the inner nuclear layer or outer nuclear layer in the DR rat model. Retinal function in the HRF and adjacent areas was reduced compared with normal control rats. Microglial activation was detected by Iba-1 labelling and retinal stress identified by GFAP expression in Müller cells observed in discrete areas around small dot HRF. Small dot HRF seen in OCT images of the retina are associated with a local microglial response. This study provides the first evidence of dot HRF correlating with microglial activation, which may allow clinicians to better evaluate the microglia-mediated inflammatory component of progressive diseases showing HRF.


Asunto(s)
Diabetes Mellitus , Retinopatía Diabética , Hiperglucemia , Ratas , Animales , Retinopatía Diabética/diagnóstico por imagen , Tomografía de Coherencia Óptica , Retina/diagnóstico por imagen , Inflamación/diagnóstico por imagen
2.
Int J Mol Sci ; 24(4)2023 02 15.
Artículo en Inglés | MEDLINE | ID: mdl-36835288

RESUMEN

Diabetic retinopathy (DR), a microvascular complication of diabetes, is associated with pronounced inflammation arising from the activation of a nucleotide-binding and oligomerization domain-like receptor (NLR) protein 3 (NLRP3) inflammasome. Cell culture models have shown that a connexin43 hemichannel blocker can prevent inflammasome activation in DR. The aim of this study was to evaluate the ocular safety and efficacy of tonabersat, an orally bioavailable connexin43 hemichannel blocker, to protect against DR signs in an inflammatory non-obese diabetic (NOD) DR mouse model. For retina safety studies, tonabersat was applied to retinal pigment epithelial (ARPE-19) cells or given orally to control NOD mice in the absence of any other stimuli. For efficacy studies, either tonabersat or a vehicle was given orally to the inflammatory NOD mouse model two hours before an intravitreal injection of pro-inflammatory cytokines, interleukin-1 beta, and tumour necrosis factor-alpha. Fundus and optical coherence tomography images were acquired at the baseline as well as at 2- and 7-day timepoints to assess microvascular abnormalities and sub-retinal fluid accumulation. Retinal inflammation and inflammasome activation were also assessed using immunohistochemistry. Tonabersat did not have any effect on ARPE-19 cells or control NOD mouse retinas in the absence of other stimuli. However, the tonabersat treatment in the inflammatory NOD mice significantly reduced macrovascular abnormalities, hyperreflective foci, sub-retinal fluid accumulation, vascular leak, inflammation, and inflammasome activation. These findings suggest that tonabersat may be a safe and effective treatment for DR.


Asunto(s)
Benzamidas , Conexina 43 , Retinopatía Diabética , Animales , Ratones , Conexina 43/antagonistas & inhibidores , Retinopatía Diabética/tratamiento farmacológico , Modelos Animales de Enfermedad , Inflamasomas/metabolismo , Inflamación/metabolismo , Ratones Endogámicos NOD , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Administración Oral , Benzamidas/administración & dosificación , Benzamidas/farmacología
3.
Int J Mol Sci ; 24(24)2023 Dec 14.
Artículo en Inglés | MEDLINE | ID: mdl-38139284

RESUMEN

Multiple sclerosis (MS) is a neurodegenerative disease marked by chronic neuroinflammation thought to be mediated by the inflammasome pathway. Connexin 43 (Cx43) hemichannels contribute to the activation of the inflammasome through the release of adenosine triphosphate (ATP) inflammasome activation signals. The objective of the study was to evaluate if the Cx43 hemichannel blocker, tonabersat, is effective in modulating the inflammatory response and reducing disability in the myelin oligodendrocyte glycoprotein 35-55-induced experimental autoimmune encephalomyelitis (MOG35-55 EAE) model of MS. Here, we show that the Cx43 hemichannel blocking drug, tonabersat, significantly reduced expression of neuroinflammatory markers for microglial activation (ionized calcium-binding adapter molecule 1 (Iba1)) and astrogliosis (glial fibrillary acidic protein (GFAP)) while preserving myelin basic protein (MBP) expression levels in the corpus callosum, motor cortex, and striatum regions of the brain in MOG35-55 EAE mice. Reduced NOD-like receptor protein 3 (NLRP3) inflammasome complex assembly and Caspase-1 activation confirmed the drug's mode of action. MOG35-55 EAE mice showed clinical signs of MS, but MOG35-55 EAE mice treated with tonabersat retained behavior closer to normal. These data suggest that clinical trial phase IIb-ready tonabersat may merit further investigation as a promising candidate for MS treatment.


Asunto(s)
Encefalomielitis Autoinmune Experimental , Esclerosis Múltiple , Enfermedades Neurodegenerativas , Ratones , Animales , Esclerosis Múltiple/tratamiento farmacológico , Conexina 43/metabolismo , Inflamasomas/metabolismo , Progresión de la Enfermedad , Ratones Endogámicos C57BL , Modelos Animales de Enfermedad
4.
Exp Eye Res ; 215: 108911, 2022 02.
Artículo en Inglés | MEDLINE | ID: mdl-34958779

RESUMEN

The inflammasome pathway is a fundamental component of the innate immune system, playing a key role especially in chronic age-related eye diseases (AREDs). The inflammasome is of particular interest because it is a common disease pathway that once instigated, can amplify and perpetuate itself leading to chronic inflammation. With aging, it becomes more difficult to shut down inflammation after an insult but the common pathway means that a shared solution may be feasible that could be effective across multiple disease indications. This review focusses on the NLRP3 inflammasome, the most studied and characterized inflammasome in the eye. It describes the two-step signalling required for NLRP3 inflammasome complex activation, and provides evidence for its role in AREDs. In the final section, the article gives an overview of potential NLRP3 inflammasome targeting therapies, before presenting evidence for connexin hemichannel regulators as upstream blockers of inflammasome activation. These have shown therapeutic efficacy in multiple ocular disease models.


Asunto(s)
Oftalmopatías , Inflamasomas , Animales , Enfermedad Crónica , Conexinas , Humanos , Inflamasomas/metabolismo , Inflamación/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo
5.
Cell Biol Int ; 46(2): 323-330, 2022 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-34719065

RESUMEN

Epithelial-mesenchymal transition (EMT) occurs when polarised epithelial cells change to a mesenchymal phenotype. EMT plays a role in several chronic conditions, including ocular diseases with retinal pigment epithelium (RPE) EMT associated with retinal diseases such as diabetic retinopathy (DR). Here, EMT results in breakdown of the blood-retinal barrier (BRB) leading to sub-retinal fluid deposition and retinal detachment. Previous studies have shown that blocking connexin43 (Cx43) hemichannels can protect against RPE BRB breakdown, but the underlying mechanism is unknown. To determine whether open Cx43 hemichannels may enable EMT of RPE cells and thus result in BRB breakdown, ARPE-19 cells were either challenged with high glucose plus the inflammatory cytokines IL-1ß and TNF-α (HG + Cyt) to simulate DR or treated with the Cx43 hemichannel blocker tonabersat alongside the HG + Cyt challenge. HG + Cyt induced a morphological change in RPE cells to a fibroblastic phenotype with a corresponding decrease in epithelial zonular occludens-1 and an increase in the fibroblastic marker α-SMA. The HG + Cyt challenge also induced loss of transepithelial electrical resistance while increasing dye passage between RPE cells. All of these changes were significantly reduced with tonabersat treatment, which also prevented HG + Cyt-induced transforming growth factor-ß2 (TGF-ß2) release. In conclusion, Cx43 hemichannel block with tonabersat attenuated both TGF-ß2 release and RPE EMT under disease-mimicking conditions, offering the potential to ameliorate the progression of EMT-associated retinal diseases.


Asunto(s)
Transición Epitelial-Mesenquimal , Factor de Crecimiento Transformador beta2 , Conexina 43/metabolismo , Células Epiteliales/metabolismo , Humanos , Epitelio Pigmentado de la Retina/metabolismo , Factor de Crecimiento Transformador beta2/metabolismo , Factor de Crecimiento Transformador beta2/farmacología , Regulación hacia Arriba
6.
Exp Cell Res ; 407(2): 112823, 2021 10 15.
Artículo en Inglés | MEDLINE | ID: mdl-34506760

RESUMEN

Cell transdifferentiation is the conversion of a cell type to another without requiring passage through a pluripotent cell state, and encompasses epithelial- and endothelial-mesenchymal transition (EMT and EndMT). EMT and EndMT are well defined processes characterized by a loss of epithelial/endothelial phenotype and gain in mesenchymal spindle shaped morphology, which results in increased cell migration and decreased apoptosis and cellular senescence. Such cells often develop invasive properties. Physiologically, these processes may occur during embryonic development and can resurface, for example, to promote wound healing in later life. However, they can also be a pathological process. In the eye, EMT, EndMT and cell transdifferentiation have all been implicated in development, homeostasis, and multiple diseases affecting different parts of the eye. Connexins, constituents of connexin hemichannels and intercellular gap junctions, have been implicated in many of these processes. In this review, we firstly provide an overview of the molecular mechanisms induced by transdifferentiation (including EMT and EndMT) and its involvement in eye diseases. We then review the literature for the role of connexins in transdifferentiation in the eye and eye diseases. The evidence presented in this review supports the need for more studies into the therapeutic potential for connexin modulators in prevention and treatment of transdifferentiation related eye diseases, but does indicate that connexin channel modulation may be an upstream and unifying approach for regulating these otherwise complex processes.


Asunto(s)
Transdiferenciación Celular , Conexinas/metabolismo , Transición Epitelial-Mesenquimal , Oftalmopatías/patología , Factor de Crecimiento Transformador beta/metabolismo , Animales , Oftalmopatías/metabolismo , Humanos , Transducción de Señal
7.
J Cell Physiol ; 236(2): 911-920, 2021 02.
Artículo en Inglés | MEDLINE | ID: mdl-32592185

RESUMEN

Connexin31.1 (Cx31.1) is a gap junction protein associated with apoptosis. In the skin, apoptosis is modulated by diabetes. A HaCaT skin model investigated whether normal (NGI) and high glucose and insulin (HGI; diabetic) conditions altered Cx31.1 expression, and if these were apoptosis linked. Cx31.1 was found in HaCaT and HeLa Ohio cells, with HaCaT Cx31.1 protein increased in HGI conditions, and around apoptotic cells. HeLa Cx31.1 channels were noncommunicative. Post scrape-wounding, Cx31.1 increased at wound edges. Caspase 3/7 in scrape-wounds media (containing cells) elevated in HGI. UV exposure raised Cx31.1, and caspase 3/7, in NGI and HGI. UV reduced cell viability in NGI cells, although not significantly in HGI. Cx31.1 is modulated during HaCaT cell wound closure, and associated with 'diabetic' conditions. Cx31.1 expression matched apoptosis levels, higher in HGI cultures. Cx31.1 is noncommunicating, modulated after wounding, linked to apoptosis, and may be associated with tissue turn-over around diabetic wounds.


Asunto(s)
Conexinas/metabolismo , Heridas y Lesiones/metabolismo , Apoptosis/fisiología , Línea Celular , Línea Celular Tumoral , Supervivencia Celular/fisiología , Células HaCaT , Células HeLa , Humanos , Insulina/metabolismo
8.
Exp Eye Res ; 202: 108384, 2021 01.
Artículo en Inglés | MEDLINE | ID: mdl-33285185

RESUMEN

Diabetic retinopathy (DR), the most common ocular complication associated with diabetes, is a chronic vascular and inflammatory disease that leads to vision loss. The inflammasome pathway, a key part of the innate immune system, is required to activate chronic inflammation in DR. Unfortunately, current therapies for DR target pathological signs that are downstream of the inflammasome pathway, making them only partly effective in treating the disease. Using in vitro and in vivo DR models, it was discovered that connexin43 hemichannel blockers can inhibit activation of the inflammasome pathway. However, those studies were conducted using in vitro cell culture and in vivo animal disease models that are predictive but do not, of course, like any model, completely replicate the human condition. Here, we have developed an addition to our armamentarium of useful models, an ex vivo human organotypic retinal culture model of DR by exposing human donor retinal explants to a combination of high glucose (HG) and pro-inflammatory cytokines, interleukin-1 beta (IL-1ß) and tumour necrosis factor alpha (TNF-α). We hypothesized that in this model, connexin43 hemichannel block would protect against NLRP3 inflammasome complex assembly which would in turn decrease signs of inflammation characteristic of DR. To test our hypothesis, molecular changes in the inflammatory and inflammasome pathway were assessed using immunohistochemistry and a Luminex cytokine release assay. Our results showed that the human retinal explant DR model was associated with increased inflammation and activation of the inflammasome pathway, characteristic of the human condition. Furthermore, we showed that by blocking connexin43 hemichannels with the hemichannel modulator, tonabersat, we were able to prevent NLRP3 inflammasome complex assembly, Müller cell activation, as well as release of pro-inflammatory cytokines and VEGF. This further supports the possible use of connexin43 hemichannel blockers as potential new therapies for DR.


Asunto(s)
Benzamidas/farmacología , Benzopiranos/farmacología , Conexina 43/metabolismo , Retinopatía Diabética/tratamiento farmacológico , Inflamasomas/metabolismo , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Anciano , Anciano de 80 o más Años , Retinopatía Diabética/metabolismo , Retinopatía Diabética/patología , Femenino , Humanos , Microscopía Confocal , Persona de Mediana Edad
9.
Int J Mol Sci ; 22(4)2021 Feb 10.
Artículo en Inglés | MEDLINE | ID: mdl-33578721

RESUMEN

Dysregulation of retinal function in the early stages of light-induced retinal degeneration involves pannexins and connexins. These two types of proteins may contribute to channels that release ATP, leading to activation of the inflammasome pathway, spread of inflammation and retinal dysfunction. However, the effect of pannexin channel block alone or block of both pannexin channels and connexin hemichannels in parallel on retinal activity in vivo is unknown. In this study, the pannexin channel blocker probenecid and the connexin hemichannel blocker tonabersat were used in the light-damaged rat retina. Retinal function was evaluated using electroretinography (ERG), retinal structure was analyzed using optical coherence tomography (OCT) imaging and the tissue response to light-induced injury was assessed immunohistochemically with antibodies against glial fibrillary acidic protein (GFAP), Ionized calcium binding adaptor molecule 1 (Iba-1) and Connexin43 (Cx43). Probenecid did not further enhance the therapeutic effect of connexin hemichannel block in this model, but on its own improved activity of certain inner retina neurons. The therapeutic benefit of blocking connexin hemichannels was further evaluated by comparing these data against results from our previously published studies that also used the light-damaged rat retina model. The analysis showed that treatment with tonabersat alone was better than probenecid alone at restoring retinal function in the light-damaged retina model. The results assist in the interpretation of the differential action of connexin hemichannel and pannexin channel therapeutics for potential treatment of retinal diseases.


Asunto(s)
Benzamidas/uso terapéutico , Benzopiranos/uso terapéutico , Conexinas/antagonistas & inhibidores , Proteínas del Tejido Nervioso/antagonistas & inhibidores , Probenecid/uso terapéutico , Retina/efectos de los fármacos , Enfermedades de la Retina/tratamiento farmacológico , Animales , Benzamidas/farmacología , Benzopiranos/farmacología , Conexina 43/análisis , Femenino , Inflamación/tratamiento farmacológico , Inflamación/etiología , Inflamación/patología , Luz/efectos adversos , Masculino , Probenecid/farmacología , Ratas , Ratas Sprague-Dawley , Retina/patología , Retina/efectos de la radiación , Enfermedades de la Retina/etiología , Enfermedades de la Retina/patología
10.
Int J Mol Sci ; 22(7)2021 Mar 31.
Artículo en Inglés | MEDLINE | ID: mdl-33807408

RESUMEN

Chronic Kidney Disease (CKD) is associated with sustained inflammation and progressive fibrosis, changes that have been linked to altered connexin hemichannel-mediated release of adenosine triphosphate (ATP). Kidney fibrosis develops in response to increased deposition of extracellular matrix (ECM), and up-regulation of collagen I is an early marker of renal disease. With ECM remodeling known to promote a loss of epithelial stability, in the current study we used a clonal human kidney (HK2) model of proximal tubular epithelial cells to determine if collagen I modulates changes in cell function, via connexin-43 (Cx43) hemichannel ATP release. HK2 cells were cultured on collagen I and treated with the beta 1 isoform of the pro-fibrotic cytokine transforming growth factor (TGFß1) ± the Cx43 mimetic Peptide 5 and/or an anti-integrin α2ß1 neutralizing antibody. Phase microscopy and immunocytochemistry observed changes in cell morphology and cytoskeletal reorganization, whilst immunoblotting and ELISA identified changes in protein expression and secretion. Carboxyfluorescein dye uptake and biosensing measured hemichannel activity and ATP release. A Cytoselect extracellular matrix adhesion assay assessed changes in cell-substrate interactions. Collagen I and TGFß1 synergistically evoked increased hemichannel activity and ATP release. This was paralleled by changes to markers of tubular injury, partly mediated by integrin α2ß1/integrin-like kinase signaling. The co-incubation of the hemichannel blocker Peptide 5, reduced collagen I/TGFß1 induced alterations and inhibited a positive feedforward loop between Cx43/ATP release/collagen I. This study highlights a role for collagen I in regulating connexin-mediated hemichannel activity through integrin α2ß1 signaling, ahead of establishing Peptide 5 as a potential intervention.


Asunto(s)
Colágeno Tipo I/metabolismo , Conexina 43/metabolismo , Túbulos Renales Proximales/metabolismo , Adenosina Trifosfato/metabolismo , Calcio/metabolismo , Adhesión Celular , Línea Celular , Células Cultivadas , Colágeno Tipo I/fisiología , Conexina 43/fisiología , Conexinas/metabolismo , Citocinas , Células Epiteliales/metabolismo , Humanos , Integrina alfa2beta1/metabolismo , Integrina alfa2beta1/fisiología , Insuficiencia Renal Crónica/metabolismo , Insuficiencia Renal Crónica/fisiopatología , Transducción de Señal , Factor de Crecimiento Transformador beta1/metabolismo
11.
Cell Commun Signal ; 18(1): 79, 2020 05 25.
Artículo en Inglés | MEDLINE | ID: mdl-32450899

RESUMEN

BACKGROUND: Tubulointerstitial fibrosis represents the key underlying pathology of Chronic Kidney Disease (CKD), yet treatment options remain limited. In this study, we investigated the role of connexin43 (Cx43) hemichannel-mediated adenosine triphosphate (ATP) release in purinergic-mediated disassembly of adherens and tight junction complexes in early tubular injury. METHODS: Human primary proximal tubule epithelial cells (hPTECs) and clonal tubular epithelial cells (HK2) were treated with Transforming Growth Factor Beta1 (TGF-ß1) ± apyrase, or ATPγS for 48 h. For inhibitor studies, cells were co-incubated with Cx43 mimetic Peptide 5, or purinergic receptor antagonists Suramin, A438079 or A804598. Immunoblotting, single-cell force spectroscopy and trans-epithelial electrical resistance assessed protein expression, cell-cell adhesion and paracellular permeability. Carboxyfluorescein uptake and biosensing measured hemichannel activity and real-time ATP release, whilst a heterozygous Cx43+/- mouse model with unilateral ureteral obstruction (UUO) assessed the role of Cx43 in vivo. RESULTS: Immunohistochemistry of biopsy material from patients with diabetic nephropathy confirmed increased expression of purinergic receptor P2X7. TGF-ß1 increased Cx43 mediated hemichannel activity and ATP release in hPTECs and HK2 cells. The cytokine reduced maximum unbinding forces and reduced cell-cell adhesion, which translated to increased paracellular permeability. Changes were reversed when cells were co-incubated with either Peptide 5 or P2-purinoceptor inhibitors. Cx43+/- mice did not exhibit protein changes associated with early tubular injury in a UUO model of fibrosis. CONCLUSION: Data suggest that Cx43 mediated ATP release represents an initial trigger in early tubular injury via its actions on the adherens and tight junction complex. Since Cx43 is highly expressed in nephropathy, it represents a novel target for intervention of tubulointerstitial fibrosis in CKD. Video Abstract In proximal tubular epithelial cells (PTECs), tight junction proteins, including zona occuludens-1 (ZO-1), contribute to epithelial integrity, whilst the adherens junction protein epithelial (E)-cadherin (ECAD) maintains cell-cell coupling, facilitating connexin 43 (Cx43) gap junction-mediated intercellular communication (GJIC) and the direct transfer of small molecules and ions between cells. In disease, such as diabetic nephropathy, the pro-fibrotic cytokine transforming growth factor beta1 (TGF-ß1) binds to its receptor and recruits SMAD2/3 signalling ahead of changes in gene transcription and up-regulation of Cx43-mediated hemichannels (HC). Uncoupled hemichannels permit the release of adenosine triphosphate (ATP) in to the extracellular space (↑[ATP]e), where ATP binds to the P2X7 purinoreceptor and activates the nucleotide-binding domain and leucine-rich repeat containing (NLR) protein-3 (NLRP3) inflammasome. Inflammation results in epithelial-to-mesenchymal transition (EMT), fibrosis and tubular injury. A major consequence is further loss of ECAD and reduced stickiness between cells, which can be functionally measured as a decrease in the maximum unbinding force needed to uncouple two adherent cells (Fmax). Loss of ECAD feeds forward to further lessen cell-cell coupling exacerbating the switch from GJIC to HC-mediated release of ATP. Reduction in ZO-1 impedes tight junction effectiveness and decreases trans-epithelial resistance (↓TER), resulting in increased paracellular permeability.


Asunto(s)
Adenosina Trifosfato/metabolismo , Conexina 43/fisiología , Túbulos Renales , Insuficiencia Renal Crónica/metabolismo , Animales , Adhesión Celular , Línea Celular , Humanos , Túbulos Renales/metabolismo , Túbulos Renales/patología , Ratones , Persona de Mediana Edad
12.
Pharmacol Rev ; 69(4): 396-478, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28931622

RESUMEN

Connexins are ubiquitous channel forming proteins that assemble as plasma membrane hemichannels and as intercellular gap junction channels that directly connect cells. In the heart, gap junction channels electrically connect myocytes and specialized conductive tissues to coordinate the atrial and ventricular contraction/relaxation cycles and pump function. In blood vessels, these channels facilitate long-distance endothelial cell communication, synchronize smooth muscle cell contraction, and support endothelial-smooth muscle cell communication. In the central nervous system they form cellular syncytia and coordinate neural function. Gap junction channels are normally open and hemichannels are normally closed, but pathologic conditions may restrict gap junction communication and promote hemichannel opening, thereby disturbing a delicate cellular communication balance. Until recently, most connexin-targeting agents exhibited little specificity and several off-target effects. Recent work with peptide-based approaches has demonstrated improved specificity and opened avenues for a more rational approach toward independently modulating the function of gap junctions and hemichannels. We here review the role of connexins and their channels in cardiovascular and neurovascular health and disease, focusing on crucial regulatory aspects and identification of potential targets to modify their function. We conclude that peptide-based investigations have raised several new opportunities for interfering with connexins and their channels that may soon allow preservation of gap junction communication, inhibition of hemichannel opening, and mitigation of inflammatory signaling.


Asunto(s)
Enfermedades Cardiovasculares/fisiopatología , Conexinas/antagonistas & inhibidores , Conexinas/fisiología , Enfermedades del Sistema Nervioso/fisiopatología , Animales , Enfermedades Cardiovasculares/tratamiento farmacológico , Fenómenos Fisiológicos Cardiovasculares/efectos de los fármacos , Humanos , Enfermedades del Sistema Nervioso/tratamiento farmacológico , Fenómenos Fisiológicos del Sistema Nervioso/efectos de los fármacos
13.
Int J Mol Sci ; 22(1)2020 Dec 30.
Artículo en Inglés | MEDLINE | ID: mdl-33396676

RESUMEN

This study was undertaken to evaluate the connexin hemichannel blocker tonabersat for the inhibition of inflammasome activation and use as a potential treatment for diabetic retinopathy. Human retinal pigment epithelial cells (ARPE-19) were stimulated with hyperglycemia and the inflammatory cytokines IL-1ß and TNFα in order to mimic diabetic retinopathy molecular signs in vitro. Immunohistochemistry was used to evaluate the effect of tonabersat treatment on NLRP3, NLRP1, and cleaved caspase-1 expression and distribution. A Luminex cytokine release assay was performed to determine whether tonabersat affected proinflammatory cytokine release. NLRP1 was not activated in ARPE-19 cells, and IL-18 was not produced under disease conditions. However, NLRP3 and cleaved caspase-1 complex formation increased with hyperglycemia and cytokine challenge but was inhibited by tonabersat treatment. It also prevented the release of proinflammatory cytokines IL-1ß, VEGF, and IL-6. Tonabersat therefore has the potential to reduce inflammasome-mediated inflammation in diabetic retinopathy.


Asunto(s)
Benzamidas/farmacología , Benzopiranos/farmacología , Conexina 43/metabolismo , Retinopatía Diabética/metabolismo , Células Epiteliales/efectos de los fármacos , Inflamasomas/efectos de los fármacos , Epitelio Pigmentado de la Retina/metabolismo , Caspasa 1/metabolismo , Línea Celular , Citocinas/metabolismo , Citocinas/farmacología , Retinopatía Diabética/fisiopatología , Células Epiteliales/metabolismo , Glucosa/farmacología , Humanos , Inflamasomas/metabolismo , Mediadores de Inflamación/metabolismo , Activación del Canal Iónico/efectos de los fármacos , Proteína con Dominio Pirina 3 de la Familia NLR/metabolismo , Epitelio Pigmentado de la Retina/citología
14.
Int J Mol Sci ; 21(18)2020 Sep 04.
Artículo en Inglés | MEDLINE | ID: mdl-32899855

RESUMEN

Perinatal hypoxia-ischemia is associated with disruption of cortical gamma-aminobutyric acid (GABA)ergic interneurons and their surrounding perineuronal nets, which may contribute to persisting neurological deficits. Blockade of connexin43 hemichannels using a mimetic peptide can alleviate seizures and injury after hypoxia-ischemia. In this study, we tested the hypothesis that connexin43 hemichannel blockade improves the integrity of cortical interneurons and perineuronal nets. Term-equivalent fetal sheep received 30 min of bilateral carotid artery occlusion, recovery for 90 min, followed by a 25-h intracerebroventricular infusion of vehicle or a mimetic peptide that blocks connexin hemichannels or by a sham ischemia + vehicle infusion. Brain tissues were stained for interneuronal markers or perineuronal nets. Cerebral ischemia was associated with loss of cortical interneurons and perineuronal nets. The mimetic peptide infusion reduced loss of glutamic acid decarboxylase-, calretinin-, and parvalbumin-expressing interneurons and perineuronal nets. The interneuron and perineuronal net densities were negatively correlated with total seizure burden after ischemia. These data suggest that the opening of connexin43 hemichannels after perinatal hypoxia-ischemia causes loss of cortical interneurons and perineuronal nets and that this exacerbates seizures. Connexin43 hemichannel blockade may be an effective strategy to attenuate seizures and may improve long-term neurological outcomes after perinatal hypoxia-ischemia.


Asunto(s)
Conexina 43/efectos de los fármacos , Hipoxia-Isquemia Encefálica/tratamiento farmacológico , Péptidos/farmacología , Animales , Biomimética/métodos , Isquemia Encefálica/tratamiento farmacológico , Infarto Cerebral/tratamiento farmacológico , Conexina 43/antagonistas & inhibidores , Conexina 43/metabolismo , Conexinas/antagonistas & inhibidores , Conexinas/metabolismo , Matriz Extracelular/metabolismo , Femenino , Feto/metabolismo , Hipoxia/fisiopatología , Infusiones Intraventriculares , Interneuronas/metabolismo , Masculino , Parvalbúminas/metabolismo , Péptidos/administración & dosificación , Embarazo , Convulsiones/tratamiento farmacológico , Convulsiones/fisiopatología , Convulsiones/prevención & control , Ovinos
15.
Crit Rev Biochem Mol Biol ; 51(6): 413-439, 2016.
Artículo en Inglés | MEDLINE | ID: mdl-27387655

RESUMEN

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.


Asunto(s)
Conexinas/inmunología , Uniones Comunicantes/inmunología , Inflamación/inmunología , Animales , Comunicación Celular , Humanos , Transducción de Señal
16.
Semin Cell Dev Biol ; 50: 49-58, 2016 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-26688335

RESUMEN

It is 45 years since gap junctions were first described. Universities face increasing commercial pressures and declining federal funding, with governments and funding foundations showing greater interest in gaining return on their investments. This review outlines approaches taken to translate gap junction research to clinical application and the challenges faced. The need for commercialisation is discussed and key concepts behind research patenting briefly described. Connexin channel roles in disease and injury are also discussed, as is identification of the connexin hemichannel as a therapeutic target which appears to play a role in both the start and perpetuation of the inflammasome pathway. Furthermore connexin hemichannel opening results in vascular dieback in acute injury and chronic disease. Translation to human indications is illustrated from the perspective of one connexin biotechnology company, CoDa Therapeutics, Inc.


Asunto(s)
Conexinas/metabolismo , Investigación Biomédica Traslacional , Animales , Industria Farmacéutica , Humanos , Modelos Biológicos , Terapia Molecular Dirigida , Patentes como Asunto
17.
Biochim Biophys Acta Biomembr ; 1860(1): 224-236, 2018 Jan.
Artículo en Inglés | MEDLINE | ID: mdl-28347700

RESUMEN

A common cause of mortality and long-term adult disability, cerebral ischemia or brain ischemia imposes a significant health and financial burden on communities worldwide. Cerebral ischemia is a condition that arises from a sudden loss of blood flow and consequent failure to meet the high metabolic demands of the brain. The lack of blood flow initiates a sequelae of cell death mechanisms, including the activation of the inflammatory pathway, which can ultimately result in irreversible brain tissue damage. In particular, Connexins and Pannexins are non-selective channels with a large pore that have shown to play time-dependent roles in the perpetuation of ischaemic injury. This review highlights the roles of Connexin and Pannexin channels in cell death mechanisms as a promising therapeutic target in cerebral ischemia, and in particular connexin hemichannels which may contribute most of the ATP release as a result of ischemia as well as during reperfusion. This article is part of a Special Issue entitled: Gap Junction Proteins edited by Jean Claude Herve.


Asunto(s)
Adenosina Trifosfato/metabolismo , Isquemia Encefálica/metabolismo , Corteza Cerebral/metabolismo , Conexinas/metabolismo , Canales Iónicos/metabolismo , Animales , Isquemia Encefálica/patología , Corteza Cerebral/patología , Humanos
18.
Biochim Biophys Acta Gen Subj ; 1862(3): 385-393, 2018 Mar.
Artículo en Inglés | MEDLINE | ID: mdl-29158134

RESUMEN

BACKGROUND: Connexin43 hemichannels have been implicated in many inflammatory diseases including diabetic retinopathy (DR). Particularly, hemichannel-mediated ATP release has been associated with inflammasome pathway activation. Using an in vitro cell culture model, we evaluated hemichannel roles in response to inflammatory cytokines under high glucose (HG) conditions and propose a mechanism by which a connexin43 hemichannel-mediated autocrine ATP feedback loop augments chronic inflammatory disease. METHODS: Retinal pigment epithelial cells were exposed to HG, 10ng/mL pro-inflammatory cytokines IL-1ß and TNF-α, or a combination of both. Quantitative Cytometric Bead Array analysis was used to measure the release of inflammatory cytokines IL-6, IL-8, MCP-1, and sICAM-1, as well as VEGF and ATP. To determine the role of connexin43 hemichannels in the disease process, changes in cytokine and ATP release were evaluated following treatment with Peptide5, a connexin43 hemichannel blocker. Immunohistochemistry was used to compare NLRP3 inflammasome assembly under control and treatment conditions. RESULTS: Co-application of HG and cytokines increased the secretion of IL-6, IL-8, MCP-1, sICAM-1, VEGF and ATP, to significantly higher levels compared to cytokines alone. Peptide5 prevented cytokine release and prevented the increase in ATP release following co-application of HG and cytokines. Adding exogenous ATP negated Peptide5-mediated protection against inflammatory cytokine release in injury conditions. CONCLUSIONS: Our findings show that connexin43 hemichannels play an important role in the amplification and perpetuation of inflammation by mediating an ATP autocrine feedback loop in the inflammasome/inflammation cycle. GENERAL SIGNIFICANCE: Targeting connexin43 hemichannels offers a potential therapeutic strategy to break the inflammatory cycle in diseases such as DR, but also other chronic inflammatory indications.


Asunto(s)
Adenosina Trifosfato/metabolismo , Conexina 43/fisiología , Retinopatía Diabética/metabolismo , Inflamasomas/metabolismo , Epitelio Pigmentado de la Retina/metabolismo , Adenosina Trifosfato/farmacología , Comunicación Autocrina , Línea Celular , Conexina 43/antagonistas & inhibidores , Citocinas/metabolismo , Citocinas/farmacología , Células Epiteliales/efectos de los fármacos , Células Epiteliales/metabolismo , Uniones Comunicantes/fisiología , Glucosa/farmacología , Humanos , Hiperglucemia/metabolismo , Inflamación/metabolismo , Epitelio Pigmentado de la Retina/citología , Factor A de Crecimiento Endotelial Vascular/metabolismo
19.
Biochim Biophys Acta Gen Subj ; 1861(2): 68-78, 2017 Feb.
Artículo en Inglés | MEDLINE | ID: mdl-27816754

RESUMEN

BACKGROUND: Non-selective Connexin43 hemichannels contribute to secondary lesion spread. The hemichannel blocking peptidomimetic Peptide5, derived from the second extracellular loop of the human Connexin43 protein, prevents lesion spread and reduces vascular permeability in preclinical models of central nervous system injury. The molecular mode of action of Peptide5, however, was unknown and is described here. METHODS: Human cerebral microvascular endothelial cells and APRE-19 cells were used. Scrape loading was used to assess gap junction function and hypoxic, acidic ion-shifted Ringer solution induced ATP release used to assess hemichannel function. Peptide modifications, including amino acid substitutions and truncations, and competition assays were used to demonstrate Peptide5 functional specificity and site of action respectively. RESULTS: Peptide5 inhibits Connexin43 hemichannel-mediated ATP release by acting on extracellular loop two of Connexin43, adjacent to its matching sequence within the protein. Precise sequence specificity is important for hemichannel block, but less so for uncoupling of gap junction channels (seen only at high concentrations). The SRPTEKT motif is central to Peptide5 function but on its own is not sufficient to inhibit hemichannels. Both the SRPTEKT motif and Peptide5 reduce gap junction communication, but neither uncoupling below 50%. CONCLUSIONS: Reduced gap junction coupling at high peptide concentrations appears to be relatively non-specific. However, Peptide5 at low concentrations acts upon extracellular loop two of Connexin43 to block hemichannels in a precise, sequence specific manner. GENERAL SIGNIFICANCE: The concentration dependent and sequence specific action of Peptide5 supports its development for the treatment of retinal injury and chronic disease, as well as other central nervous system injury and disease conditions.


Asunto(s)
Cerebro/efectos de los fármacos , Conexina 43/metabolismo , Células Endoteliales/efectos de los fármacos , Isquemia/tratamiento farmacológico , Péptidos/farmacología , Daño por Reperfusión/tratamiento farmacológico , Adenosina Trifosfato/metabolismo , Línea Celular , Enfermedades del Sistema Nervioso Central/tratamiento farmacológico , Enfermedades del Sistema Nervioso Central/metabolismo , Cerebro/metabolismo , Células Endoteliales/metabolismo , Uniones Comunicantes/efectos de los fármacos , Uniones Comunicantes/metabolismo , Humanos , Canales Iónicos/metabolismo , Isquemia/metabolismo , Daño por Reperfusión/metabolismo , Enfermedades de la Retina/tratamiento farmacológico , Enfermedades de la Retina/metabolismo
20.
Exp Brain Res ; 235(10): 3033-3048, 2017 10.
Artículo en Inglés | MEDLINE | ID: mdl-28725925

RESUMEN

Systemic administration of a Connexin43 mimetic peptide, Peptide5, has been shown to reduce secondary tissue damage and improve functional recovery after spinal cord injury (SCI). This study investigated safety measures and potential off-target effects of Peptide5 systemic administration. Rats were subjected to a mild contusion SCI using the New York University impactor. One cohort was injected intraperitoneally with a single dose of fluorescently labelled Peptide5 and euthanised at 2 or 4 h post-injury for peptide distribution analysis. A second cohort received intraperitoneal injections of Peptide5 or a scrambled peptide and was culled at 8 or 24 h post-injury for the analysis of connexin proteins and systemic cytokine profile. We found that Peptide5 did not cross the blood-spinal cord barrier in control animals, but reached the lesion area in the spinal cord-injured animals without entering non-injured tissue. There was no evidence that the systemic administration of Peptide5 modulates Connexin43 protein expression or hemichannel closure in the heart and lung tissue of SCI animals. The expression levels of other major connexin proteins including Connexin30 in astrocytes, Connexin36 in neurons and Connexin47 in oligodendrocytes were also unaltered by systemic delivery of Peptide5 in either the injured or non-injured spinal cords. In addition, systemic delivery of Peptide5 had no significant effect on the plasma levels of cytokines, chemokines or growth factors. These data indicate that the systemic delivery of Peptide5 is unlikely to cause any off-target or adverse effects and may thus be a safe treatment option for traumatic SCI.


Asunto(s)
Materiales Biomiméticos/farmacología , Conexina 43/farmacología , Traumatismos de la Médula Espinal/tratamiento farmacológico , Animales , Materiales Biomiméticos/administración & dosificación , Materiales Biomiméticos/efectos adversos , Materiales Biomiméticos/farmacocinética , Conexina 43/administración & dosificación , Conexina 43/efectos adversos , Conexina 43/farmacocinética , Modelos Animales de Enfermedad , Femenino , Ratas , Ratas Sprague-Dawley
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