THE ROLE OF CONNEXINS, PANNEXINS AND THEIR CHANNELS IN ACUTE LIVER FAILURE

A homeostase tecidual é controlada por uma série de mecanismos de comunicação. Ao nível intercelular, as junções gap permitem o tráfego de mensageiros pequenos e hidrofílicos, menores do que 1.5 kilodalton, entre duas células adjacentes. As junções gap são formadas por dois hemicanais, que por sua vez são compostos por seis proteínas denominadas conexinas (Cx). No fígado, os hepatócitos produzem principalmente Cx32, enquanto as células hepáticas não parenquimatosas contem predominantemente Cx43. As junções gap hepáticas são conhecidas por serem essenciais em diversas funções específicas do fígado, tais como secreção de albumina e metabolismo de xenobióticos. Na última década, tem-se tornado claro que os hemicanais de conexinas não são apenas precursores das junções gap, mas eles também podem participar da via de comunicação, contudo entre o citoplasma de células individuais e seus ambientes extracelulares. Além disso, foi descrita uma nova classe de proteínas semelhantes às conexinas, as panexinas (Panx), das quais a Panx1 é expressa no fígado. As pannexins se reúnem em uma configuração que lembra os hemicanais de conexinas e também participam da sinalização extracelular. Acredita-se que tanto os hemicanais de conexinas como os canais de pannexinas se tornam ativados preferencialmente em condições patológicas e, deste modo, promovam a morte celular e inflamação. O presente projeto de doutorado foi estabelecido para verificar se esta hipótese também é verdadeira nas doenças hepáticas, em particular na insuficiência hepática aguda. Inicialmente, o padrão de expressão das conexinas hepáticas foi totalmente caracterizado em um modelo murino de insuficiência hepática aguda induzida pelo paracetamol (APAP). Isto revelou uma mudança da Cx32 para Cx43 após a hepatotoxicidade induzida por APAP, com expressão de novo de Cx43 nos hepatócitos. Subsequentemente, a relevância funcional da expressão aumentada de Cx43 na insuficiência hepática aguda foi testada utilizando camundongos deficientes em Cx43. Verificou-se que a Cx43 tem um efeito protetor, uma vez que a lesão hepática induzida por APAP se agravou após a ablação genética da Cx43. Em paralelo, foi realizado um estudo semelhante utilizando camundongos knockout para Cx32, não mostrando diferenças significativas na extensão da hepatotoxicidade induzida pelo APAP em comparação aos animais selvagens. Uma vez que os animais deficientes em conexina não permitem diferenciar entre a comunicação via junções gap e a sinalização pelos hemicanais de conexina, uma série de experimentos seguintes baseou-se na utilização de inibidores dos hemicanais de Cx32 e Cx43, chamados de TAT-Gap24 e TAT-Gap19, respectivamente. Após os testes in vitro quanto a sua especificidade e eficiência in vitro, ambos os peptídeos foram administrados em camundongos com overdose pelo APAP. Enquanto TAT-Gap24 reduziu claramente a morte celular e inflamação, TAT-Gap19 apresentou apenas efeitos discretos sobre a lesão hepática. Além disso, o co-tratamento 12 de camundongos intoxicados pelo APAP com ambos os peptídeos revelou um efeito aditivo na redução da lesão hepática. Um estudo final foi focado na elucidação da expressão hepática e função da Panx1 na hepatotoxicidade induzida pelo APAP, utilizando o peptídeo inibidor da Panx1, 10Panx1. Verificou-se que a inibição dos canais de Panx1 minimiza as características clínicas da hepatotoxicidade desencadeada pelo APAP, em particular a relacionada com morte celular e inflamação. Em geral, este projeto de doutorado demonstra, pela primeira vez, o envolvimento dos hemicanais de conexina e canais de pannexina na insuficiência hepática aguda induzida pelo APAP, o que sugere um papel como alvos terapêuticos para fármacos.

Tissue homeostasis is controlled by a plethora of communication mechanisms. At the intercellular level, gap junctions allow the trafficking of small and hydrophilic messengers of less than 1.5 kilodalton between two adjacent cells. Gap junctions are built up by two hemichannels, which in turn are composed of six connexin (Cx) proteins. In liver, hepatocytes mainly produce Cx32, while non-parenchymal hepatic cells predominantly harbor Cx43. Hepatic gap junctions are known to underlie several liver-specific functions, such as albumin secretion and xenobiotic metabolism. In the last decade, it has become clear that the connexin hemichannels are not only precursors of gap junctions, but that they can also provide a communication pathway, albeit between the cytosol of individual cells and their extracellular environment. In addition, a novel class of connexin-like proteins has been identified, the pannexins (Panx), of which Panx1 is expressed in the liver. Pannexins gather in a configuration reminiscent of connexin hemichannels and also support extracellular signaling. Both connexin hemichannels and pannexin channels are believed to become preferably activated in pathological conditions and thereby to promote cell death and inflammation. The present doctoral project was set up to verify the hypothesis that this also holds true for liver disease, in particular acute liver failure. In a first instance, the hepatic connexin expression pattern was fully characterized in a mouse model of acute liver failure induced by acetaminophen (APAP). This revealed a switch from Cx32-to-Cx43 upon APAP-induced hepatotoxicity with de novo expression of Cx43 in hepatocytes. Subsequently, the functional relevance of enhanced Cx43 expression in acute liver failure was tested using Cx43-deficient mice. It was found that Cx43 has a protective effect, since liver APAP-induced injury became aggravated upon genetic ablation of Cx43. In parallel, a similar study was set up using Cx32 knock-out mice, showing no major differences in the extent of APAP-induced hepatotoxicity compared to wild type animals. Since connexin-deficient animals do not allow to discriminate between gap junction communication and connexin hemichannel signaling, a next set of experiments relied on the use of claimed peptide-based inhibitors of Cx32 and Cx43 hemichannels, namely TAT-Gap24 and TAT-Gap19, respectively. Following testing of their specificity and efficiency in vitro, both peptides were administered to APAPoverdosed mice. While TAT-Gap24 clearly reduced liver cell death and inflammation, TAT-Gap19 only had marginal effects on liver injury. Furthermore, co-treatment of APAPintoxicated mice with both peptides revealed an additive effect in lowering liver injury. A final study was focused on elucidating the hepatic expression and role of Panx1 in APAPinduced hepatotoxicity using the Panx1 inhibiting peptide 10Panx1. It was found that inhibition of Panx1 channels counteracts the clinical features of APAP-triggered hepatotoxicity, in particular related to cell death and inflammation. Overall, this doctoral 10 project shows for the first time the involvement of connexin hemichannels and pannexin channels in APAP-induced acute liver failure, which suggests a role as therapeutic drug targets.

Effects of Deletion of the Connexin 32 gene on pro and Anti-inflammatories Aspects in the Ethidium Bromide Toxic Demyelination Model

Gap junctions are cellular structures that allow transit of molecules between cells, allowing intercellular signaling and transportation. They are formed by proteins denominated connexins and represent key structures in highly complex and integrated tissues, such as the central nervous system (CNS). The present study evaluates the effects of connexin 32 (Cx32) deletion upon CNS inflammation and regeneration/repair after 1, 3, 7, 10 and 20 days after intracerebral injection of ethidium bromide in Cx32 Knock Out and normal mice. To accomplish so, Real Time PCR gene expression quantification was performed upon Tumour Necrosis Factor alpha (TNFα), Transforming Growth Factor beta 1 (TGFß1), Metalloproteinase 3 (MMP3), Metalloproteinase 9 (MMP9) and Tissue Inhibitor of Metalloproteinases 1 (TIMP1) genes. Results indicate varying differences in the expression pattern, including difference in expression of all evaluated genes in the 3 days post injection period, apex of the acute inflammation mechanisms. These results suggest that Cx32 may perform important functions on molecular, inflammatory and regenerative/repair signalling in the CNS.(AU)

Expression of Connexins 43, 26 and 32 in normal, hyperplastic and neoplastic perianal dog glands

Connexin (Cx) expression is reportedly altered in neoplasms. This study aimed to investigate the expression of Cx43, 26 and 32 in normal and pathological canine perianal glands. Thirty perianal glands bearing pathological processes and ten normal canine perianal glands were submitted to immunohistochemistry to search for presence of Cx43, Cx26 and Cx32. Both Cx43 and Cx26 expressions were observed in normal, hyperplastic glands, and in well and moderately differentiated adenomas. However, in poorly differentiated adenomas, expressions were reduced, and they were absent in carcinomas. Cx26 was located in the cytoplasm of normal, hyperplastic perianal gland cells, and in well and moderately differentiated adenomas. Cx32 was not observed in any neoplasm neither in normal or hyperplastic glands. Our results show that Cx43 and Cx26 expressions are altered in more aggressive canine perianal gland neoplasms, and we conclude that they may be related to the perianal gland carcinogenesis process

Expression of Connexins 43, 26 and 32 in normal, hyperplastic and neoplastic perianal dog glands

Connexin (Cx) expression is reportedly altered in neoplasms. This study aimed to investigate the expression of Cx43, 26 and 32 in normal and pathological canine perianal glands. Thirty perianal glands bearing pathological processes and ten normal canine perianal glands were submitted to immunohistochemistry to search for presence of Cx43, Cx26 and Cx32. Both Cx43 and Cx26 expressions were observed in normal, hyperplastic glands, and in well and moderately differentiated adenomas. However, in poorly differentiated adenomas, expressions were reduced, and they were absent in carcinomas. Cx26 was located in the cytoplasm of normal, hyperplastic perianal gland cells, and in well and moderately differentiated adenomas. Cx32 was not observed in any neoplasm neither in normal or hyperplastic glands. Our results show that Cx43 and Cx26 expressions are altered in more aggressive canine perianal gland neoplasms, and we conclude that they may be related to the perianal gland carcinogenesis process (AU)

EXPRESSÃO DAS CONEXINAS 37 E 43 EM FOLÍCULOS OVARIANOS OVINOS, FRESCOS, VITRIFICADOS OU CULTIVADOS IN VITRO

Alguns estudos têm mostrado que crioinjúrias observadas durante a vitrificação levam à perda da integridade de proteínas membranárias que constituem as comunicações intercelulares. Dentre as proteínas de membranas que formam essas comunicações, destacam-se as conexinas 37 (Cx37) e 43 (Cx43), as quais são importantes na interação entre oócito e células foliculares. Este estudo teve como principal objetivo avaliar o efeito da vitrificação do tecido ovariano ovino utilizando o ovarian tissue cryosystem (OTC), associado ao cultivo in vitro de folículos secundários isolados, sobre o padrão de expressão gênica e protéica das Cx37 e Cx43. Para isso, seis diferentes tratamentos foram avaliados: 1) tecido ovariano fresco (FOT); 2) tecido ovariano vitrificado (VOT); 3) folículos secundários frescos isolados de tecido ovariano fresco (FIF); 4) folículos secundários cultivados in vitro, isolados do tecido ovariano fresco (CIF); 5) folículos secundários isolados do tecido ovariano vitrificado (VIF) e 6) folículos secundários cultivados in vitro, isolados de tecido ovariano vitrificado (CVIF). O tecido ovariano fresco ou vitrificado, bem como os folículos isolados de todos os tratamentos foram avaliados quanto à expressão gênica e protéica para as Cx37 e Cx43. Adicionalmente, folículos secundários foram analisados para a integridade e crescimento folicular, morte por apoptose e proliferação celular, e folículos secundários somente cultivados in vitro (CIF e CVIF) foram avaliados para morfologia (folículos extrusos), formação de antro e viabilidade. O percentual de folículos intactos no tratamento CVIF foi significativamente superior (P < 0,05), enquanto que a formação de antro, taxa de extrusão e viabilidade folicular foram inferiores (P < 0,05) aos folículos do tratamento CIF. O teste TUNEL mostrou que folículos frescos (FIF) não apresentaram apoptose, enquanto os folículos nos demais tratamentos (CIF, VIF e CVIF) mostraram marcação positiva. A proliferação celular nos tratamentos VIF e CVIF foi mais intensa do que nos folículos do FIF. Em relação à expressão gênica, nos folículos secundários apenas a Cx43 apresentou alterações nos níveis de expressão de mRNA, sendo que os folículos do tratamento CVIF apresentaram níveis de RNAm significativamente inferiores, comparados aos folículos dos demais tratamentos. A Cx37 e Cx43 foram predominantemente imunolocalizadas nas células da granulosa e oócitos, respectivamente. Em conclusão, folículos secundários ovinos podem ser isolados com sucesso após a vitrificação do tecido ovariano e são capazes de manter a integridade da membrana após o cultivo in vitro. Embora a expressão gênica e protéica da Cx37 não varie após a vitrificação do tecido ovariano, a Cx43 é alterada nos folículos secundários após vitrificação e cultivo in vitro.

Previous studies have demonstrated that cryoinjuries showed through vitrification leads to a loss of integrity of membrane proteins that constitutes the intercellular communications. Among these membrane proteins, it is prominent the role of connexins 37 (Cx 37) and 43 (Cx 43). Thus, this study aimed evaluate the effect of vitrification of ovine ovarian tissue using ovarian tissue cryosystem (OTC), associated to in vitro culture of isolated secondary follicles, under genetic and protein expression pattern of Cx 37 and Cx 43. Ovarian fragments were distributed into six different treatments: 1) fresh ovarian tissue (FOT); 2) vitrified ovarian tissue (VOT); 3) isolated secondary follicles of fresh ovarian tissue (FIF); 4) secondary follicles cultured in vitro, follow by isolation of ovarian tissue (CIF); 5) isolated secondary follicles of vitrified ovarian tissue (VIF) and 6) secondary follicles cultured in vitro, and then isolated of vitrified ovarian tissue (CVIF). Fresh or vitrified ovarian tissue as well as isolated follicles of all treatments were evaluated to genetic and protein expression for Cx 37 and Cx 43. Additionally, secondary follicles were analyzed to integrity, follicular growth, apoptosis, and cell proliferation, while only cultured secondary follicles (CIF and CVIF) were evaluated by morphology (extruded follicles), antrum formation and viability. The percentage of intact follicles on CVIF treatment had higher (P < 0.05) while antrum formation, extruded rate and follicle viability were lower (P < 0.05) to follicles on treatment CIF. TUNEL assay demonstrates that in fresh follicles (FIF) did not show apoptosis, while follicles from other treatments had a positive labelling. A cell proliferation on VIF and CVIF treatments had more intensity of follicles from FIF. In relation to genetic expression on secondary follicles, only Cx43 alters mRNA levels, and follicles of CVIF treatment had lower (P < 0.05) mRNA levels when compared to follicles of other treatments. Cx37 and Cx43 had an immunolabelling mainly on granulosa cells and oocytes, respectively. In conclusion, the isolation of ovine secondary follicles occurs successfully after vitrification of ovarian tissue and be able to maintain the integrity of membrane after in vitro culture. Although the genetic and protein expression of Cx37 did not varies after vitrification of ovarian tissue, Cx 43 is altered on secondary follicles after vitrification and in vitro culture. Keywords:

Efeito da circulação extracorpórea na expressão da conexina 43 no miocárdio

As conexinas são proteínas essenciais e estão diretamente relacionadas à propagação do impulso elétrico no coração, à velocidade de condução bem como à gênese de muitas afecções cardíacas. Em face da circulação extracorpórea (CEC) ainda ser utilizada de forma inconsistente na medicina veterinária e devido aos poucos estudos observados na literatura sobre os efeitos provocados pelo emprego da CEC na expressão da conexina 43 (Cx43) no miocárdio, objetivou-se avaliá-la em 15 animais da espécie canina, distribuidos em três grupos (C, CEC-1 e CEC -2) sendo, respectivamente, antes de realizada a CEC, com 60 minutos após esta e 60 minutos de CEC seguida de 30 minutos de restauração da perfusão espontânea. Avaliou-se a Cx43 pelas técnicas de imunofluorescência, western blot e RT-PCR em tempo real no tecido muscular cardíaco de regiões correspondentes aos átrios direito (AD) e esquerdo (AE), ventrículos direito (VD) e esquerdo (VE) e septo transverso. Os resultados indicaram a presença da Cx43 em todas as regiões do miocárdio nos grupos C, CEC-1 e CEC-2. A expressão da Cx43 variou significativamente em CEC-2 no AE e VD em relação ao grupo C (p<0,05). A expressão gênica de Gja1 (gene da Cx43) não apresentou diferença significativa entre os grupos estudados. Em CEC-2 identificou-se a presença de vacuolização na túnica média de artérias de pequeno calibre do miocárdio. Concluiu-se também que a canulação da aorta bem como a instalação do circuito de CEC no modo aorto-bicaval constitui técnica exequível.

Connexins are essential proteins that are directly associated with electrical impulse propagation and speed of propagation in the heart. They also play a major role in numerous heart conditions. The use of cardiopulmonary bypass (CPB) in veterinary medicine is inconsistent and few studies describe the effect of cardiopulmonary bypass on the expression of connexin 43 (Cx43) in the myocardium. The objective of this study was to evaluate myocardial expression of Cx43. Connexin 43 of 15 dogs was assessed at 3 moments: prior to CPB (Group C); 60 minutes after CPB (Group CPB1); and 60 minutes after CPB followed by 30 minutes of spontaneous perfusion (Group CPB2). Assessment of Cx43 included immunofluorescence, western blot and RT-PCR real time of heart tissue samples from the right atrium (RA), left atrium (LA), right ventricle (LV), right ventricle (RV) and transverse septum. Our results showed the presence of Cx43 in all 5 areas of the myocardium in groups C, CPB1 and CPB2. A significant variation on the expression of Cx43 was observed when CPB2 LA and CPB2 RV were compared to group C (p<0,05). Expression of Gja1 (gene for Cx43) did not vary significantly among the study groups. Group CBP2 presented vacuolation of the tunica media of small myocardial arteries. We conclude that cannulation of the aorta and aorto-bicaval setup of the CPB circuit is feasible technique.

Gap junctions and communication within the nervous system

Gap junctions are sites on the cellular membrane with intercellular channels build up by twelve protein subunits called connexins. Each connected cell contributes with a hemichannel made up by six connexins subunits. This kind of connection represents and efficient way of intercellular communication in most tissues, including the nervous system. It works as a passage for ions, secondary messenger and metabolites exchange between the cells. In a complex tissue like the nervous tissue they are particularly important because they connect the various cellular types composing a panglial syncytium that performs neuronal protection and tissue homeostasis. The expression of connexins and the intercellular communication through gap junctions are crucial to regulate vital functions as cellular motility, proliferations and survival; changes in the conformational expression of connexins may be involved in diseases as Alzheimer's disease, neoplasms, bacterial and parasitic infections, or even affect cellular groups when they occur as genetic mutations leading to functional defects of the nervous system as demyelination in the PNS (Charcot-Marie-Tooth disease), hereditary epilepsy, non-syndromic deafness and senile cataract.

Gap junctions and communication within the nervous system

Gap junctions are sites on the cellular membrane with intercellular channels build up by twelve protein subunits called connexins. Each connected cell contributes with a hemichannel made up by six connexins subunits. This kind of connection represents and efficient way of intercellular communication in most tissues, including the nervous system. It works as a passage for ions, secondary messenger and metabolites exchange between the cells. In a complex tissue like the nervous tissue they are particularly important because they connect the various cellular types composing a panglial syncytium that performs neuronal protection and tissue homeostasis. The expression of connexins and the intercellular communication through gap junctions are crucial to regulate vital functions as cellular motility, proliferations and survival; changes in the conformational expression of connexins may be involved in diseases as Alzheimer´s disease, neoplasms, bacterial and parasitic infections, or even affect cellular groups when they occur as genetic mutations leading to functional defects of the nervous system as demyelination in the PNS (Charcot-Marie-Tooth disease), hereditary epilepsy, nonsyndromic deafness and senile cataract.

Effects of deletion of the connexin 32 gene on pro and anti-inflammatories aspects in the ethidium bromide toxic demyelination model

Gap junctions are cellular structures that allow transit of molecules between cells, allowing intercellular signaling and transportation. They are formed by proteins denominated connexins and represent key structures in highly complex and integrated tissues, such as the central nervous system (CNS). The present study evaluates the effects of connexin 32 (Cx32) deletion upon CNS inflammation and regeneration/repair after 1, 3, 7, 10 and 20 days after intracerebral injection of ethidium bromide in Cx32 Knock Out and normal mice. To accomplish so, Real Time PCR gene expression quantification was performed upon Tumour Necrosis Factor alpha (TNFa), Transforming Growth Factor beta 1 (TGFb1), Metalloproteinase 3 (MMP3), Metalloproteinase 9 (MMP9) and Tissue Inhibitor of Metalloproteinases 1 (TIMP1) genes. Results indicate varying differences in the expression pattern, including difference in expression of all evaluated genes in the 3 days post injection period, apex of the acute inflammation mechanisms. These results suggest that Cx32 may perform important functions on molecular, inflammatory and regenerative/repair signalling in the CNS.