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1.
Int J Mol Sci ; 22(19)2021 Oct 07.
Artigo em Inglês | MEDLINE | ID: mdl-34639171

RESUMO

Diabetes is a major risk factor for cardiovascular diseases, especially cardiomyopathy, a condition in which the smooth muscles of the heart become thick and rigid, affecting the functioning of cardiomyocytes, the contractile cells of the heart. Uncontrolled elevated glucose levels over time can result in oxidative stress, which could lead to inflammation and altered epigenetic mechanisms. In the current study, we investigated whether hyperglycemia can modify cardiac function by directly affecting these changes in cardiomyocytes. To evaluate the adverse effect of high glucose, we measured the levels of gap junction protein, connexin 43, which is responsible for modulating cardiac electric activities and Troponin I, a part of the troponin complex in the heart muscles, commonly used as cardiac markers of ischemic heart disease. AC16 human cardiomyocyte cells were used in this study. Under hyperglycemic conditions, these cells demonstrated altered levels of connexin 43 and Troponin-I after 24 h of exposure. We also examined hyperglycemia induced changes in epigenetic markers: H3K9me1, Sirtuin-1 (SIRT1), and histone deacetylase (HDAC)-2 as well as in inflammatory and stress-related mediators, such as heat shock protein (HSP)-60, receptor for advanced glycation end products (RAGE), toll-like receptor (TLR)-4, high mobility group box (HMGB)-1 and CXC chemokine receptor (CXCR)-4. Cardiomyocytes exposed to 25mM glucose resulted in the downregulation of HSP60 and SIRT1 after 48 h. We further examined that hyperglycemia mediated the decrease in the gap junction protein CX43, as well as CXC chemokine receptor CXCR4 which may affect the physiological functions of the cardiomyocytes when exposed to high glucose for 24 and 48 h. Upregulated expression of DNA-binding nuclear protein HMGB1, along with changes in histone methylation marker H3K9me1 have demonstrated hyperglycemia-induced damage to cardiomyocyte at 24 h of exposure. Our study established that 24 to 48 h of hyperglycemic exposure could stimulate stress-mediated inflammatory mediators in cardiomyocytes in vitro. These stress-related changes in hyperglycemia-induced cardiomyocytes may further initiate an increase in injury markers which eventually could alter the epigenetic processes. Therefore, epigenetic and inflammatory mechanisms in conjunction with alterations in a downstream signaling pathway could have a direct effect on the functionality of the cardiomyocytes exposed to high glucose during short and long-term exposures.


Assuntos
Biomarcadores/metabolismo , Epigênese Genética , Hiperglicemia/fisiopatologia , Mediadores da Inflamação/metabolismo , Miócitos Cardíacos/patologia , Estresse Fisiológico , Chaperonina 60/genética , Chaperonina 60/metabolismo , Conexina 43/genética , Conexina 43/metabolismo , Proteína HMGB1/genética , Proteína HMGB1/metabolismo , Histona Desacetilase 2/genética , Histona Desacetilase 2/metabolismo , Humanos , Proteínas Mitocondriais/genética , Proteínas Mitocondriais/metabolismo , Miócitos Cardíacos/metabolismo , Receptores CXCR4/genética , Receptores CXCR4/metabolismo , Sirtuína 1/genética , Sirtuína 1/metabolismo
2.
J Agric Food Chem ; 69(40): 11847-11855, 2021 Oct 13.
Artigo em Inglês | MEDLINE | ID: mdl-34609142

RESUMO

Estrogen and its analogues are ubiquitous in agricultural environments, with large biological functions of oocyte development. Gap junction intercellular communications (GJICs) are the structural basis in cumulus-oocyte complexes (COCs) and regulate oocyte maturation and developmental material transport through a number of pathways. This study mainly determines the effect and potential mechanism of estrogen (17ß-estradiol) in regulating GJICs in porcine COCs. In our study, 17ß-estradiol increased porcine nuclear maturation in a time-dependent manner. The analysis revealed that 17ß-estradiol upregulated the autophagy in COCs during in vitro maturation. In contrast with the control, 17ß-estradiol decreased GJICs in a time-dependent manner between cumulus cells and oocytes, while it was consistent with the control group at 24 h. Carbenoxolone (CBX) blocks GJICs as a negative control group used in our system. Autophagy inhibitor autophinib decreased oocyte maturation, and the reduced nuclear maturation treated with autophinib was abolished by 17ß-estradiol. Besides, the upregulation effect of autophinib on GJICs and transzonal projections (TZPs) was decreased by 17ß-estradiol. 17ß-Estradiol could reduce serine 368 phosphorylation of connexin 43 (Cx43) protein by autophinib in porcine COCs. These results were dependent upon the MEK/ERK signaling pathway. Furthermore, 17ß-estradiol-induced GJICs and Cx43 phosphorylation were inhibited by autophinib or the MEK/ERK pathway inhibitors (Trametinib and FR 180204), indicating that 17ß-estradiol regulated GJICs through the MEK/ERK signaling pathway. In conclusion, 17ß-estradiol improves the autophagy-mediated nuclear maturation with downregulating GJICs and TZPs in porcine COCs. Such an effect occurs by phosphorylation of Cx43, which was regulated via the MEK/ERK signaling pathway.


Assuntos
Conexina 43 , Sistema de Sinalização das MAP Quinases , Animais , Autofagia , Conexina 43/genética , Conexina 43/metabolismo , Estradiol/metabolismo , Estradiol/farmacologia , Feminino , Junções Comunicantes/metabolismo , Meiose , Quinases de Proteína Quinase Ativadas por Mitógeno/metabolismo , Oócitos/metabolismo , Fosforilação , Transdução de Sinais , Suínos
3.
Elife ; 102021 10 05.
Artigo em Inglês | MEDLINE | ID: mdl-34608863

RESUMO

The Connexin43 gap junction gene GJA1 has one coding exon, but its mRNA undergoes internal translation to generate N-terminal truncated isoforms of Connexin43 with the predominant isoform being only 20 kDa in size (GJA1-20k). Endogenous GJA1-20k protein is not membrane bound and has been found to increase in response to ischemic stress, localize to mitochondria, and mimic ischemic preconditioning protection in the heart. However, it is not known how GJA1-20k benefits mitochondria to provide this protection. Here, using human cells and mice, we identify that GJA1-20k polymerizes actin around mitochondria which induces focal constriction sites. Mitochondrial fission events occur within about 45 s of GJA1-20k recruitment of actin. Interestingly, GJA1-20k mediated fission is independent of canonical Dynamin-Related Protein 1 (DRP1). We find that GJA1-20k-induced smaller mitochondria have decreased reactive oxygen species (ROS) generation and, in hearts, provide potent protection against ischemia-reperfusion injury. The results indicate that stress responsive internally translated GJA1-20k stabilizes polymerized actin filaments to stimulate non-canonical mitochondrial fission which limits ischemic-reperfusion induced myocardial infarction.


Assuntos
Conexina 43/metabolismo , Mitocôndrias/metabolismo , Dinâmica Mitocondrial/genética , Animais , Conexina 43/genética , Células HEK293 , Humanos , Masculino , Camundongos , Camundongos Endogâmicos C57BL , Dinâmica Mitocondrial/fisiologia , Infarto do Miocárdio , Miócitos Cardíacos/metabolismo , Espécies Reativas de Oxigênio/metabolismo
4.
Nat Commun ; 12(1): 5558, 2021 09 24.
Artigo em Inglês | MEDLINE | ID: mdl-34561429

RESUMO

Cardiac radiotherapy (RT) may be effective in treating heart failure (HF) patients with refractory ventricular tachycardia (VT). The previously proposed mechanism of radiation-induced fibrosis does not explain the rapidity and magnitude with which VT reduction occurs clinically. Here, we demonstrate in hearts from RT patients that radiation does not achieve transmural fibrosis within the timeframe of VT reduction. Electrophysiologic assessment of irradiated murine hearts reveals a persistent supraphysiologic electrical phenotype, mediated by increases in NaV1.5 and Cx43. By sequencing and transgenic approaches, we identify Notch signaling as a mechanistic contributor to NaV1.5 upregulation after RT. Clinically, RT was associated with increased NaV1.5 expression in 1 of 1 explanted heart. On electrocardiogram (ECG), post-RT QRS durations were shortened in 13 of 19 patients and lengthened in 5 patients. Collectively, this study provides evidence for radiation-induced reprogramming of cardiac conduction as a potential treatment strategy for arrhythmia management in VT patients.


Assuntos
Conexina 43/genética , Sistema de Condução Cardíaco/efeitos da radiação , Coração/efeitos da radiação , Canal de Sódio Disparado por Voltagem NAV1.5/genética , Taquicardia Ventricular/radioterapia , Potenciais de Ação/fisiologia , Potenciais de Ação/efeitos da radiação , Animais , Conexina 43/metabolismo , Relação Dose-Resposta à Radiação , Eletrocardiografia , Fibrose Endomiocárdica , Feminino , Regulação da Expressão Gênica , Coração/fisiopatologia , Sistema de Condução Cardíaco/fisiopatologia , Frequência Cardíaca/fisiologia , Frequência Cardíaca/efeitos da radiação , Humanos , Masculino , Camundongos , Canal de Sódio Disparado por Voltagem NAV1.5/metabolismo , Transdução de Sinais , Taquicardia Ventricular/genética , Taquicardia Ventricular/metabolismo , Taquicardia Ventricular/fisiopatologia
5.
Int J Mol Sci ; 22(18)2021 Sep 16.
Artigo em Inglês | MEDLINE | ID: mdl-34576176

RESUMO

It has been established that enhancement of serotonergic transmission contributes to improvement of major depression; however, several post-mortem studies and experimental depression rodent models suggest that functional abnormalities of astrocytes play important roles in the pathomechanisms/pathophysiology of mood disorders. Direct effects of serotonin (5-HT) transporter inhibiting antidepressants on astroglial transmission systems has never been assessed in this context. Therefore, to explore the effects of antidepressants on transmission associated with astrocytes, the present study determined the effects of the selective 5-HT transporter inhibitor, escitalopram, and the 5-HT partial agonist reuptake inhibitor, vortioxetine, on astroglial L-glutamate release through activated hemichannels, and the expression of connexin43 (Cx43), type 1A (5-HT1AR) and type 7 (5-HT7R) 5-HT receptor subtypes, and extracellular signal-regulated kinase (ERK) in astrocytes using primary cultured rat cortical astrocytes in a 5-HT-free environment. Both escitalopram and 5-HT1AR antagonist (WAY100635) did not affect basal astroglial L-glutamate release or L-glutamate release through activated hemichannels. Subchronic (for seven days) administrations of vortioxetine and the 5-HT7R inverse agonist (SB269970) suppressed both basal L-glutamate release and L-glutamate release through activated hemichannels, whereas 5-HT1AR agonist (BP554) inhibited L-glutamate release through activated hemichannels, but did not affect basal L-glutamate release. In particular, WAY100635 did not affect the inhibitory effects of vortioxetine on L-glutamate release. Subchronic administration of vortioxetine, BP554 and SB269970 downregulated 5-HT1AR, 5-HT7R and phosphorylated ERK in the plasma membrane fraction, but escitalopram and WAY100635 did not affect them. Subchronic administration of SB269970 decreased Cx43 expression in the plasma membrane but did not affect the cytosol; however, subchronic administration of BP554 increased Cx43 expression in the cytosol but did not affect the plasma membrane. Subchronic vortioxetine administration increased Cx43 expression in the cytosol and decreased it in the plasma membrane. WAY100635 prevented an increased Cx43 expression in the cytosol induced by vortioxetine without affecting the reduced Cx43 expression in the plasma membrane. These results suggest that 5-HT1AR downregulation probably increases Cx43 synthesis, but 5-HT7R downregulation suppresses Cx43 trafficking to the plasma membrane. These results also suggest that the subchronic administration of therapeutic-relevant concentrations of vortioxetine inhibits both astroglial L-glutamate and Cx43 expression in the plasma membrane via 5-HT7R downregulation but enhances Cx43 synthesis in the cytosol via 5-HT1AR downregulation. This combination of the downregulation of 5-HT1AR, 5-HT7R and Cx43 in the astroglial plasma membrane induced by subchronic vortioxetine administration suggest that astrocytes is possibly involved in the pathophysiology of depression.


Assuntos
Conexina 43/metabolismo , Animais , Astrócitos/efeitos dos fármacos , Astrócitos/metabolismo , Cromatografia Líquida de Alta Pressão , Citalopram/farmacologia , Conexina 43/genética , Depressão/genética , Depressão/metabolismo , Feminino , Immunoblotting , Ratos , Receptores de Serotonina/metabolismo , Vortioxetina/farmacologia
6.
Int J Mol Sci ; 22(15)2021 Jul 25.
Artigo em Inglês | MEDLINE | ID: mdl-34360693

RESUMO

Testicular Connexin43 (Cx43) connects adjacent Sertoli cells (SC) and SC to germ cells (GC) in the seminiferous epithelium and plays a crucial role in spermatogenesis. However, the distinction whether this results from impaired inter-SC communication or between GC and SC is not possible, so far. Thus, the question arises, whether a GC-specific Cx43 KO has similar effects on spermatogenesis as it is general or SC-specific KO. Using the Cre/loxP recombinase system, two conditional KO mouse lines lacking Cx43 in premeiotic (pGCCx43KO) or meiotic GC (mGCCx43KO) were generated. It was demonstrated by qRT-PCR that Cx43 mRNA was significantly decreased in adult pGCCx43KO mice, while it was also reduced in mGCCx43KO mice, yet not statistically significant. Body and testis weights, testicular histology, tubular diameter, numbers of intratubular cells and Cx43 protein synthesis and localization did not show any significant differences in semi-quantitative Western blot analysis and immunohistochemistry comparing adult male KO and WT mice of both mouse lines. Male KO mice were fertile. These results indicate that Cx43 in spermatogonia/spermatids does not seem to be essential for successful termination of spermatogenesis and fertility as it is known for Cx43 in somatic SC, but SC-GC communication might rather occur via heterotypic GJ channels.


Assuntos
Conexina 43/metabolismo , Espermátides/metabolismo , Espermatogênese , Espermatogônias/metabolismo , Testículo/metabolismo , Animais , Conexina 43/genética , Fertilidade , Masculino , Camundongos , Camundongos Knockout , Testículo/anatomia & histologia
7.
Biomolecules ; 11(8)2021 08 11.
Artigo em Inglês | MEDLINE | ID: mdl-34439853

RESUMO

Polyamines (PAs) are polycationic biomolecules containing multiple amino groups. Patients with HIV-associated neurocognitive disorder (HAND) have high concentrations of the polyamine N-acetylated spermine in their brain and cerebral spinal fluid (CSF) and have increased PA release from astrocytes. These effects are due to the exposure to HIV-Tat. In healthy adult brain, PAs are accumulated but not synthesized in astrocytes, suggesting that PAs must enter astrocytes to be N-acetylated and released. Therefore, we tested if Cx43 hemichannels (Cx43-HCs) are pathways for PA flux in control and HIV-Tat-treated astrocytes. We used biotinylated spermine (b-SPM) to examine polyamine uptake. We found that control astrocytes and those treated with siRNA-Cx43 took up b-SPM, similarly suggesting that PA uptake is via a transporter/channel other than Cx43-HCs. Surprisingly, astrocytes pretreated with both HIV-Tat and siRNA-Cx43 showed increased accumulation of b-SPM. Using a novel polyamine transport inhibitor (PTI), trimer 44NMe, we blocked b-SPM uptake, showing that PA uptake is via a PTI-sensitive transport mechanism such as organic cation transporter. Our data suggest that Cx43 HCs are not a major pathway for b-SPM uptake in the condition of normal extracellular calcium concentration but may be involved in the release of PAs to the extracellular space during viral infection.


Assuntos
Astrócitos/metabolismo , Transporte Biológico/efeitos dos fármacos , Conexina 43/metabolismo , Infecções por HIV/metabolismo , Espermina/metabolismo , Animais , Astrócitos/virologia , HIV-1 , Camundongos , Camundongos Endogâmicos C57BL , Cultura Primária de Células
8.
Sci Rep ; 11(1): 16975, 2021 08 18.
Artigo em Inglês | MEDLINE | ID: mdl-34408164

RESUMO

The wound healing capacity of the fetal membranes after spontaneous or iatrogenic membrane rupture is unclear. We examined the healing mechanisms in amniotic membrane (AM) defects after trauma. Traumatised human AM defects were cultured for 4 days. Markers for nuclear (DAPI), cell type (vimentin, αSMA) and healing (Cx43, TGFß1, collagen) were examined by immunofluorescence (IMF) confocal microscopy, Second Harmonic Generation (SHG) imaging and RT-qPCR. After trauma, AMCs and myofibroblasts migrated to the AM wound edge. Within four days, αSMA expressing myofibroblasts showed abundant Cx43 localized in the cytoplasmic processes. The highly contractile spindle-shaped myofibroblasts were present in the defect site and released collagen. In contrast, AMCs expressed vimentin and formed Cx43 plaques between cells found in the outer edges of the wound. Whilst AMCs were absent in the defect site, αSMA expressing myofibroblasts continued to elongate and polarize the collagen fibres. Both TGFß1 and Cx43 gene expression were significantly increased after trauma. Cx43 has differential effects on AM cell populations that increase cellularity, contraction and potentially migration to the wound edge resulting in collagen polarisation in the AM defect site. Establishing how Cx43 regulates AM cell function could be an approach to repair defects in the membranes after trauma.


Assuntos
Âmnio/metabolismo , Colágeno/metabolismo , Conexina 43/metabolismo , Miofibroblastos/metabolismo , Membranas Extraembrionárias/metabolismo , Feminino , Ruptura Prematura de Membranas Fetais/metabolismo , Humanos , Gravidez , Vimentina/metabolismo , Cicatrização/fisiologia
9.
Inflamm Res ; 70(10-12): 1165-1175, 2021 Dec.
Artigo em Inglês | MEDLINE | ID: mdl-34436630

RESUMO

OBJECTIVE: Junctional proteins are the most important component of the blood-testis barrier and maintaining the integrity of this barrier is essential for spermatogenesis and male fertility. The present study elucidated the effect of SARS-CoV-2 infection on the blood-testis barrier (BTB) in patients who died from severe acute respiratory syndrome coronavirus 2 (COVID-19) complications. METHODS: In this study, lung and testis tissue was collected from autopsies of COVID-19 positive (n = 10) and negative men (n = 10) and was taken for stereology, immunocytochemistry, and RNA extraction. RESULTS: Evaluation of the lung tissue showed that the SARS-CoV-2 infection caused extensive damage to the lung tissue and also increases inflammation in testicular tissue and destruction of the testicular blood barrier. Autopsied testicular specimens of COVID-19 showed that COVID-19 infection significantly changes the spatial arrangement of testicular cells and notably decreased the number of Sertoli cells. Moreover, the immunohistochemistry results showed a significant reduction in the protein expression of occluding, claudin-11, and connexin-43 in the COVID-19 group. In addition, we also observed a remarkable enhancement in protein expression of CD68 in the testes of the COVID-19 group in comparison with the control group. Furthermore, the result showed that the expression of TNF-α, IL1ß, and IL6 was significantly increased in COVID-19 cases as well as the expression of occludin, claudin-11, and connexin-43 was decreased in COVID-19 cases. CONCLUSIONS: Overall, the present study demonstrated that SARS-CoV-2 could induce the up-regulation of the pro-inflammatory cytokine and down-regulation of junctional proteins of the BTB, which can disrupt BTB and ultimately impair spermatogenesis.


Assuntos
Barreira Hematotesticular/patologia , COVID-19/patologia , Citocinas/metabolismo , Autopsia , Claudinas/metabolismo , Conexina 43/metabolismo , Humanos , Imuno-Histoquímica , Interleucina-1beta/metabolismo , Interleucina-6/metabolismo , Pulmão/patologia , Masculino , Pessoa de Meia-Idade , Ocludina/metabolismo , RNA Viral/análise , Células de Sertoli/patologia , Testículo/patologia , Fator de Necrose Tumoral alfa/metabolismo
10.
Int J Mol Sci ; 22(13)2021 Jun 30.
Artigo em Inglês | MEDLINE | ID: mdl-34209088

RESUMO

Breast cancer (BC) brain metastases is a life-threatening condition to which accounts the poor understanding of BC cells' (BCCs) extravasation into the brain, precluding the development of preventive strategies. Thus, we aimed to unravel the players involved in the interaction between BCCs and blood-brain barrier (BBB) endothelial cells underlying BBB alterations and the transendothelial migration of malignant cells. We used brain microvascular endothelial cells (BMECs) as a BBB in vitro model, under conditions mimicking shear stress to improve in vivo-like BBB features. Mixed cultures were performed by the addition of fluorescently labelled BCCs to distinguish individual cell populations. BCC-BMEC interaction compromised BBB integrity, as revealed by junctional proteins (ß-catenin and zonula occludens-1) disruption and caveolae (caveolin-1) increase, reflecting paracellular and transcellular hyperpermeability, respectively. Both BMECs and BCCs presented alterations in the expression pattern of connexin 43, suggesting the involvement of the gap junction protein. Myosin light chain kinase and phosphorylated myosin light chain were upregulated, revealing the involvement of the endothelial cytoskeleton in the extravasation process. ß4-Integrin and focal adhesion kinase were colocalised in malignant cells, reflecting molecular interaction. Moreover, BCCs exhibited invadopodia, attesting migratory properties. Collectively, hub players involved in BC brain metastases formation were unveiled, disclosing possible therapeutic targets for metastases prevention.


Assuntos
Barreira Hematoencefálica/citologia , Neoplasias Encefálicas/secundário , Neoplasias da Mama/metabolismo , Redes Reguladoras de Genes , Animais , Barreira Hematoencefálica/metabolismo , Neoplasias Encefálicas/metabolismo , Caveolina 1/metabolismo , Técnicas de Cultura de Células , Linhagem Celular Tumoral , Conexina 43/metabolismo , Feminino , Regulação Neoplásica da Expressão Gênica , Humanos , Camundongos , Fosforilação , Resistência ao Cisalhamento , Migração Transendotelial e Transepitelial , Proteína da Zônula de Oclusão-1/metabolismo , beta Catenina/metabolismo
11.
Int J Mol Sci ; 22(14)2021 Jul 09.
Artigo em Inglês | MEDLINE | ID: mdl-34299018

RESUMO

Connexins (Cx) form gap junctions (GJ) and allow for intercellular communication. However, these proteins also modulate gene expression, growth, and cell migration. The downregulation of Cx43 impairs endothelial cell migration and angiogenetic potential. Conversely, endothelial Cx43 expression is upregulated in an in vivo angiogenesis model relying on hemodynamic forces. We studied the effects of Cx43 expression on tube formation and proliferation in HUVECs and examined its dependency on GJ communication. Expectedly, intercellular communication assessed by dye transfer was linked to Cx43 expression levels in HUVECs and was sensitive to a GJ blockade by the Cx43 mimetic peptide Gap27. The proliferation of HUVECs was not affected by Cx43 overexpression using Cx43 cDNA transfection, siRNA-mediated knockdown of Cx43, or the inhibition of GJ compared to the controls (transfection of an empty vector, scrambled siRNA, and the solvent). In contrast, endothelial tube and sprout formation in HUVECs was minimized after Cx43 knockdown and significantly enhanced after Cx43 overexpression. This was not affected by a GJ blockade (Gap27). We conclude that Cx43 expression positively modulates the angiogenic potential of endothelial cells independent of GJ communication. Since proliferation remained unaffected, we suggest that Cx43 protein may modulate endothelial cell migration, thereby supporting angiogenesis. The modulation of Cx43 expression may represent an exploitable principle for angiogenesis induction in clinical therapy.


Assuntos
Movimento Celular/genética , Proliferação de Células/genética , Conexina 43/metabolismo , Células Endoteliais/metabolismo , Junções Comunicantes/metabolismo , Neovascularização Fisiológica/genética , Comunicação Celular/efeitos dos fármacos , Comunicação Celular/genética , Movimento Celular/efeitos dos fármacos , Proliferação de Células/efeitos dos fármacos , Conexina 43/genética , Conexinas/farmacologia , Células Endoteliais/efeitos dos fármacos , Expressão Gênica , Técnicas de Silenciamento de Genes , Células Endoteliais da Veia Umbilical Humana , Humanos , Técnicas In Vitro , Oligopeptídeos/farmacologia , RNA Interferente Pequeno , Reação em Cadeia da Polimerase em Tempo Real , Regulação para Cima
12.
Cells ; 10(6)2021 06 02.
Artigo em Inglês | MEDLINE | ID: mdl-34199470

RESUMO

Astrocytes are intimately involved in the response to neurodegenerative stress and have become an attractive target for the development of neuroprotective therapies. However, studies often focus on astrocytes as single-cell units. Astrocytes are densely interconnected by gap junctions that are composed primarily of the protein connexin-43 (Cx43) and can function as a broader network of cells. Such networks contribute to a number of important processes, including metabolite distribution and extracellular ionic buffering, and are likely to play an important role in the progression of neurodegenerative disease. This review will focus on the pro-degenerative and pro-survival influence of astrocyte Cx43 in disease progression, with a focus on the roles of gap junctions and hemichannels in the spread of degenerative stress. Finally, we will highlight the specific evidence for targeting these networks in the treatment of glaucomatous neurodegeneration and other optic neuropathies.


Assuntos
Astrócitos/metabolismo , Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Glaucoma/metabolismo , Doenças Neurodegenerativas/metabolismo , Doenças do Nervo Óptico/metabolismo , Astrócitos/patologia , Junções Comunicantes/patologia , Glaucoma/patologia , Humanos , Doenças Neurodegenerativas/patologia , Doenças do Nervo Óptico/patologia
13.
FASEB J ; 35(8): e21762, 2021 08.
Artigo em Inglês | MEDLINE | ID: mdl-34246197

RESUMO

Phase II clinical trials have reported that acute treatment of surgical skin wounds with the therapeutic peptide alpha Connexin Carboxy-Terminus 1 (αCT1) improves cutaneous scar appearance by 47% 9-month postsurgery. While Cx43 and ZO-1 have been identified as molecular targets of αCT1, the mode-of-action of the peptide in scar mitigation at cellular and tissue levels remains to be further characterized. Scar histoarchitecture in αCT1 and vehicle-control treated skin wounds within the same patient were compared using biopsies from a Phase I clinical trial at 29-day postwounding. The sole effect on scar structure of a range of epidermal and dermal variables examined was that αCT1-treated scars had less alignment of collagen fibers relative to control wounds-a characteristic that resembles unwounded skin. The with-in subject effect of αCT1 on scar collagen order observed in Phase I testing in humans was recapitulated in Sprague-Dawley rats and the IAF hairless guinea pig. Transient increase in histologic collagen density in response to αCT1 was also observed in both animal models. Mouse NIH 3T3 fibroblasts and primary human dermal fibroblasts treated with αCT1 in vitro showed more rapid closure in scratch wound assays, with individual cells showing decreased directionality in movement. An agent-based computational model parameterized with fibroblast motility data predicted collagen alignments in simulated scars consistent with that observed experimentally in human and the animal models. In conclusion, αCT1 prompts decreased directionality of fibroblast movement and the generation of a 3D collagen matrix postwounding that is similar to unwounded skin-changes that correlate with long-term improvement in scar appearance.


Assuntos
Diferenciação Celular/efeitos dos fármacos , Cicatriz/metabolismo , Conexina 43/metabolismo , Derme/metabolismo , Fibroblastos/metabolismo , Peptídeos/farmacologia , Animais , Cicatriz/patologia , Matriz Extracelular/metabolismo , Feminino , Cobaias , Humanos , Masculino , Ratos , Ratos Sprague-Dawley
14.
Int J Mol Sci ; 22(11)2021 May 28.
Artigo em Inglês | MEDLINE | ID: mdl-34071707

RESUMO

Cardiac connexins (Cxs) are proteins responsible for proper heart function. They form gap junctions that mediate electrical and chemical signalling throughout the cardiac system, and thus enable a synchronized contraction. Connexins can also individually participate in many signal transduction pathways, interacting with intracellular proteins at various cellular compartments. Altered connexin expression and localization have been described in diseased myocardium and the aim of this study is to assess the involvement of Cx43, Cx26, and some related molecules in ponatinib-induced cardiac toxicity. Ponatinib is a new multi-tyrosine kinase inhibitor that has been successfully used against human malignancies, but its cardiotoxicity remains worrisome. Therefore, understanding its signaling mechanism is important to adopt potential anti cardiac damage strategies. Our experiments were performed on hearts from male and female mice treated with ponatinib and with ponatinib plus siRNA-Notch1 by using immunofluorescence, Western blotting, and proteomic analyses. The altered cardiac function and the change in Cxs expression observed in mice after ponatinib treatment, were results dependent on the Notch1 pathway and sex. Females showed a lower susceptibility to ponatinib than males. The downmodulation of cardiac Cx43, Cx26 and miR-122, high pS368-Cx43 phosphorylation, cell viability and survival activation could represent some of the female adaptative/compensatory reactions to ponatinib cardiotoxicity.


Assuntos
Cardiomiopatias , Conexina 26 , Conexina 43 , Imidazóis , Piridazinas , Fatores Sexuais , Anormalidades Induzidas por Medicamentos , Animais , Antineoplásicos/efeitos adversos , Antineoplásicos/farmacologia , Cardiomiopatias/etiologia , Cardiomiopatias/fisiopatologia , Cardiotoxicidade , Conexina 26/efeitos dos fármacos , Conexina 26/metabolismo , Conexina 43/efeitos dos fármacos , Conexina 43/metabolismo , Modelos Animais de Doenças , Feminino , Junções Comunicantes/efeitos dos fármacos , Expressão Gênica/efeitos dos fármacos , Coração/efeitos dos fármacos , Coração/fisiopatologia , Imidazóis/efeitos adversos , Imidazóis/farmacologia , Masculino , Camundongos , Miocárdio/metabolismo , Miocárdio/patologia , Proteínas Tirosina Quinases/antagonistas & inibidores , Proteômica , Piridazinas/efeitos adversos , Piridazinas/farmacologia , Receptor Notch1/metabolismo , Transdução de Sinais
15.
Int J Mol Sci ; 22(11)2021 May 21.
Artigo em Inglês | MEDLINE | ID: mdl-34063751

RESUMO

The role of immunoproteasome (iP) in astroglia, the cellular component of innate immunity, has not been clarified. The results so far indicate that neuroinflammation, a prominent hallmark of Alzheimer's disease, strongly activates the iP subunits expression. Since omega-3 PUFAs possess anti-inflammatory and pro-resolving activity in the brain, we investigated the effect of DHA and EPA on the gene expression of constitutive (ß1 and ß5) and inducible (iß1/LMP2 and iß5/LMP7) proteasome subunits and proteasomal activity in IL-1ß-stimulated astrocytes. We found that both PUFAs downregulated the expression of IL-1ß-induced the iP subunits, but not the constitutive proteasome subunits. The chymotrypsin-like activity was inhibited in a dose-dependent manner by DHA, and much strongly in the lower concentration by EPA. Furthermore, we established that C/EBPα and C/EBPß transcription factors, being the cis-regulatory element of the transcription complex, frequently activated by inflammatory mediators, participate in a reduction in the iP subunits' expression. Moreover, the expression of connexin 43 the major gap junction protein in astrocytes, negatively regulated by IL-1ß was markedly increased in PUFA-treated cells. These findings indicate that omega-3 PUFAs attenuate inflammation-induced hyperactivity of iPs in astrocytes and have a beneficial effect on preservation of interastrocytic communication by gap junctions.


Assuntos
Astrócitos/efeitos dos fármacos , Ácidos Graxos Ômega-3/farmacologia , Interleucina-1beta/metabolismo , Complexo de Endopeptidases do Proteassoma/metabolismo , Animais , Anti-Inflamatórios/farmacologia , Astrócitos/metabolismo , Proteína alfa Estimuladora de Ligação a CCAAT/metabolismo , Proteína beta Intensificadora de Ligação a CCAAT/metabolismo , Conexina 43/metabolismo , Ácidos Docosa-Hexaenoicos/farmacologia , Regulação para Baixo/efeitos dos fármacos , Ácido Eicosapentaenoico/farmacologia , Ácidos Graxos Insaturados/metabolismo , Junções Comunicantes/efeitos dos fármacos , Junções Comunicantes/metabolismo , Expressão Gênica/efeitos dos fármacos , Inflamação/metabolismo , Ratos , Transdução de Sinais/efeitos dos fármacos
16.
Int J Mol Sci ; 22(11)2021 May 25.
Artigo em Inglês | MEDLINE | ID: mdl-34070699

RESUMO

Recently, accumulating preclinical findings suggest the possibility that functional abnormalities of tripartite synaptic transmission play important roles in the pathophysiology of schizophrenia and affective disorder. Therefore, to explore the novel mechanisms of mood-stabilizing effects associated with tripartite synaptic transmission, the present study determined the effects of mood-stabilizing antipsychotics, clozapine (CLZ), quetiapine (QTP) and brexpiprazole (BPZ), on the astroglial l-glutamate release and expression of connexin43 (Cx43) in the astroglial plasma membrane using cortical primary cultured astrocytes. Neither acute (for 120 min) nor subchronic (for 7 days) administrations of CLZ, QTP and BPZ affected basal astroglial l-glutamate release, whereas both acute and subchronic administration of CLZ, QTP and BPZ concentration-dependently enhanced astroglial l-glutamate release through activated hemichannels. Subchronic administration of therapeutic-relevant concentration of valproate (VPA), a histone deacetylase inhibiting mood-stabilizing antiepileptic drug, enhanced the stimulatory effects of therapeutic-relevant concentration of CLZ, QTP and BPZ on astroglial l-glutamate release through activated hemichannel. Subchronic administration of therapeutic-relevant concentration of CLZ, QTP and BPZ did not affect Cx43 protein expression in the plasma membrane during resting stage. After subchronic administration of VPA, acute and subchronic administration of therapeutic-relevant concentrations of CLZ increased Cx43 protein expression in the plasma membrane. Both acute administrations of therapeutic-relevant concentrations of QTP and BPZ did not affect, but subchronic administrations enhanced Cx43 protein expression in the astroglial plasma membrane. Furthermore, protein kinase B (Akt) inhibitor suppressed the stimulatory effects of CLZ and QTP, but did not affect Cx43 protein expression in the astroglial plasma membrane. These results suggest that three mood-stabilizing atypical antipsychotics, CLZ, QTP and BPZ enhance tripartite synaptic glutamatergic transmission due to enhancement of astroglial Cx43 containing hemichannel activities; however, the Cx43 activating mechanisms of these three mood-stabilizing antipsychotics were not identical. The enhanced astroglial glutamatergic transmission induced by CLZ, QTP and BPZ is, at least partially, involved in the actions of these three mood-stabilizing antipsychotics.


Assuntos
Antipsicóticos/farmacologia , Astrócitos/metabolismo , Córtex Cerebral/metabolismo , Clozapina/farmacologia , Conexina 43/metabolismo , Fumarato de Quetiapina/farmacologia , Quinolonas/farmacologia , Transmissão Sináptica/efeitos dos fármacos , Tiofenos/farmacologia , Animais , Membrana Celular/metabolismo , Ratos , Ratos Sprague-Dawley
17.
Biomed Pharmacother ; 140: 111705, 2021 Aug.
Artigo em Inglês | MEDLINE | ID: mdl-34118598

RESUMO

PURPOSE: The purpose of this study was to explore the effects of a short-term high-calorie diet and the regulation mechanism of Raphanus sativus L. seeds (RSL seeds) on the intestinal motility of young rats. METHODS: We fed 20 Specific Pathogen Free (SPF) 4-week-old male Sprague-Dawley (SD) rats special high-calorie diet for 3 days and then randomized them to a high-calorie diet group (HCG, 10 rats) and an RSL seeds treatment group (TG, 10 rats). Ten rats of the same age served as the control group (CG). HCG and TG rats continued to be fed high-calorie feed. All of the rats were weighed every 2 days. After 3 days of treatment, the effects of RSL seeds on the regulation of intestinal motility in rats consuming a high-calorie diet were examined. RESULTS: After 3 days of consuming a high-calorie diet, body weight was significantly lower in the HCG group than in the control group, and body weight of the HCG group increased slowly with time. Serum substance P (SP) and ghrelin levels were significantly lower, while the nitric oxide (NO) level was significantly higher. There were no differences in hematoxylin-eosin (HE) staining of colon sections between the groups. The expression levels of Cx43 and BDNF protein and mRNA in colon tissue were significantly lower in the HCG group. There were no significant differences in body weight between the CG and TG groups. Serum SP and ghrelin indexes in TG group were higher than those in the HCG group, and the NO index was significantly decreased. The expression levels of Cx43 and BDNF proteins and mRNA in the colon tissue were also significantly greater. CONCLUSION: Consumption of a short-term high-calorie diet may result in intestinal motility dysfunction and reduced intestinal motility. RSL seeds may improve the intestinal motility by regulating the secretion of gastrointestinal motility hormones and the expression of intestinal motility-related proteins, such as Cx43 and BDNF.


Assuntos
Dieta Hiperlipídica , Motilidade Gastrointestinal/efeitos dos fármacos , Preparações de Plantas/farmacologia , Raphanus , Sementes , Animais , Peso Corporal/efeitos dos fármacos , Fator Neurotrófico Derivado do Encéfalo/genética , Fator Neurotrófico Derivado do Encéfalo/metabolismo , Colo/efeitos dos fármacos , Colo/metabolismo , Colo/fisiologia , Conexina 43/genética , Conexina 43/metabolismo , Ingestão de Alimentos/efeitos dos fármacos , Grelina/sangue , Masculino , Óxido Nítrico/sangue , Ratos Sprague-Dawley , Substância P/sangue
18.
J Cancer Res Clin Oncol ; 147(10): 3003-3009, 2021 Oct.
Artigo em Inglês | MEDLINE | ID: mdl-34173871

RESUMO

PURPOSE: The biologic behavior and the therapeutic resistance of diffuse and anaplastic gliomas varies greatly. This may be explained by differences in cell-to-cell communication, determined by the Cx43-associated junctional activity and the microtubules-defined network, in which GAP-43 is the dominant structural component. We assessed the expression of these crucial communication proteins in samples of patients harboring WHO°II and III gliomas, graded according to the current 4th revised WHO classification. METHODS: Tissue of adult patients with WHO°II and III gliomas, who underwent surgery between 2014 and 2018, were selected from our institutional biobank. GAP-43 and Cx43 expression was analyzed using IHC. Routine clinical and neuropathological findings were additionally retrieved from our institutional prospective database. RESULTS: 43 (57%) males and 33 (43%) females with a median age of 47 (IqR: 35-61) years were selected. IDH1 wildtype tumors showed a significantly higher expression of Cx43 (p = 0.014) and a tendency for increased GAP-43 production. Advanced Cx43 expression significantly correlated with lower mitosis rate (p = 0.014): more in IDH1 wildtype (r = - 0.57, p = 0.003) than in mutated gliomas (r = - 0.37, p = 0.019). There was no difference in Cx43 or GAP-43 expression in relation to anaplastic phenotype, Gadolinum-contrasted enhancement (CE) on MRI and advanced EGFR or p53 expression. CONCLUSIONS: Intercellular communication tends to be more relevant in slower proliferating, e.g. lower malignant tumors. They could have more time to establish this network, providing longitudinally acquired resistance against specific oncological therapy. This feature matches the unfavorable IDH1 wildtype status of glioma and supports the noted malignant behavior of these tumors in the upcoming 5th WHO classification of gliomas.


Assuntos
Biomarcadores Tumorais/metabolismo , Neoplasias Encefálicas/patologia , Conexina 43/metabolismo , Proteína GAP-43/metabolismo , Glioma/patologia , Isocitrato Desidrogenase/genética , Mutação , Adulto , Idoso , Idoso de 80 Anos ou mais , Biomarcadores Tumorais/genética , Neoplasias Encefálicas/genética , Neoplasias Encefálicas/metabolismo , Neoplasias Encefálicas/cirurgia , Feminino , Seguimentos , Glioma/genética , Glioma/metabolismo , Glioma/cirurgia , Humanos , Masculino , Pessoa de Meia-Idade , Mitose , Prognóstico , Adulto Jovem
19.
Life Sci Alliance ; 4(7)2021 07.
Artigo em Inglês | MEDLINE | ID: mdl-34078741

RESUMO

The challenge for treating breast cancer (BC) is partly due to long-term dormancy driven by cancer stem cells (CSCs) capable of evading immune response and resist chemotherapy. BC cells show preference for the BM, resulting in poor prognosis. CSCs use connexin 43 (Cx43) to form gap junctional intercellular communication with BM niche cells, fibroblasts, and mesenchymal stem cells (MSCs). However, Cx43 is an unlikely target to reverse BC dormancy because of its role as a hematopoietic regulator. We found N-cadherin (CDH2) and its associated pathways as potential drug targets. CDH2, highly expressed in CSCs, interacts intracellularly with Cx43, colocalizes with Cx43 in BC cells within BM biopsies of patients, and is required for Cx43-mediated gap junctional intercellular communication with BM niche cells. Notably, CDH2 and anti-apoptotic pathways maintained BC dormancy. We thereby propose these pathways as potential pharmacological targets to prevent dormancy and chemosensitize resistant CSCs.


Assuntos
Antígenos CD/metabolismo , Neoplasias da Mama/metabolismo , Caderinas/metabolismo , Conexina 43/metabolismo , Antígenos CD/genética , Medula Óssea/metabolismo , Caderinas/genética , Caderinas/fisiologia , Conexina 43/genética , Resistencia a Medicamentos Antineoplásicos/fisiologia , Feminino , Junções Comunicantes/metabolismo , Junções Comunicantes/patologia , Humanos , Células-Tronco Mesenquimais/metabolismo , Metástase Neoplásica/patologia , Células-Tronco Neoplásicas/metabolismo , Evasão Tumoral/fisiologia
20.
Neuron ; 109(15): 2427-2442.e10, 2021 08 04.
Artigo em Inglês | MEDLINE | ID: mdl-34171291

RESUMO

Astrocytes extensively infiltrate the neuropil to regulate critical aspects of synaptic development and function. This process is regulated by transcellular interactions between astrocytes and neurons via cell adhesion molecules. How astrocytes coordinate developmental processes among one another to parse out the synaptic neuropil and form non-overlapping territories is unknown. Here we identify a molecular mechanism regulating astrocyte-astrocyte interactions during development to coordinate astrocyte morphogenesis and gap junction coupling. We show that hepaCAM, a disease-linked, astrocyte-enriched cell adhesion molecule, regulates astrocyte competition for territory and morphological complexity in the developing mouse cortex. Furthermore, conditional deletion of Hepacam from developing astrocytes significantly impairs gap junction coupling between astrocytes and disrupts the balance between synaptic excitation and inhibition. Mutations in HEPACAM cause megalencephalic leukoencephalopathy with subcortical cysts in humans. Therefore, our findings suggest that disruption of astrocyte self-organization mechanisms could be an underlying cause of neural pathology.


Assuntos
Astrócitos/metabolismo , Moléculas de Adesão Celular Neurônio-Glia/metabolismo , Córtex Cerebral/metabolismo , Proteínas do Tecido Nervoso/metabolismo , Neurogênese/fisiologia , Animais , Conexina 43/metabolismo , Junções Comunicantes/metabolismo , Camundongos , Ratos
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