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Background and Objectives: Head and neck squamous cell carcinomas (HNSCCs) vary significantly in terms of invasiveness, growth rate, and metastatic potential. This study aimed to investigate the expression of several prognostic biomarkers (Ki67, p53, EGFR, COX-2, Cx43, and p16) in HNSCC from various anatomical regions and to correlate these expressions with clinicopathological parameters. Materials and Methods : We performed immunohistochemistry on 91 histologically verified HNSCC cases from the County Emergency Hospital, Targu Mures. Biomarker expression for Ki67, COX-2, and Cx43 was assessed using a standard immunoexpression scoring system: S1: 0-10%, S2: 11-25%, S3: 26-50%, S4 > 50%; EGFR was scored based on membrane staining intensity: 0, 1+, 2+, 3+; we classified p16 as positive or negative; p53 was grouped into mutant and wild-type; and we compared these across histopathological types, tumor grades, anatomical locations, gender, and different age groups. We performed a comparative analysis of Cx43 expression levels in relation to the expression of the rest of the markers. Statistical analysis was conducted using GraphPad InStat 3 software, version 3.06 (GraphPad Software Inc., San Diego, USA). Results : The majority of tumors were in males (95.6%) aged 51-60 years. Mutant p53 expression was prevalent in most cases. Elevated Ki67 and EGFR expression were associated with more aggressive tumors. COX-2 levels varied, with a higher proportion of moderate and high immunoexpression (S3 + S4) observed in patients under 70 years old. Cx43 expression was generally low, especially in extralaryngeal tumors. Conclusions : HNSCC primarily affects older males, with the larynx being the most common site. High levels of Ki-67 and EGFR suggest more aggressive tumors, while low COX-2 levels reflect varying prognoses. Women may develop more aggressive tumors, and extralaryngeal tumors often present with more challenging prognoses. Low Cx43 expression may be more likely to coincide with higher Ki67 and COX-2 levels, possibly indicating a link with more aggressive tumor behavior.

Background and Objectives : The Connexin43 ( Cx43 ) gene is a suspected tumor suppressor gene, as re-expressed wild-type Cx43 genes reduce the malignancy potential of tumor cells. However, the role of Cx43 gene expression in human lung tumorigenesis remains unclear. Materials and Methods: Tumor tissues from 165 primary lung cancer patients were collected to study Cx43 protein expression and gene mutations using immunohistochemistry and direct DNA sequencing. In addition, Cx43 genes with or without mutations were transfected to CL-3 human lung cancer cells to confirm the function of these mutant forms of the Cx43 gene. Results: Aberrant localization of Cx43 protein in the nucleus and cytoplasm of tumor cells was detected in 14 out of 165 non-small cell lung cancer (NSCLC) patients. Mutations in the Cx43 gene were also found in patients with aberrant Cx43 localization, and transfection of these mutant genes into lung cancer cells enhanced their proliferation. Conclusions: To our knowledge, this is the first study to demonstrate Cx43 gene mutations in human lung neoplasm, supporting the hypothesis that Cx43 may function as a tumor suppressor in some lung cancer patients. Additionally, the findings suggest an association between aberrant localization of Cx43 protein expression and tumor progression.

Connexins are essential gap junction proteins that play pivotal roles in intercellular communication in various organs of mammals. Connexin-43 (Cx43) is expressed in various components of the immune system, and there is extensive evidence of its participation in inflammation responses. The involvement of Cx43 in macrophage functionality involves the purinergic signaling pathway. Macrophages contribute to defenses against inflammatory reactions such as bacterial sepsis and peritonitis. Several assays can identify the presence and activity of Cx43 in macrophages. Real-time polymerase chain reaction (PCR) can measure the relative mRNA expression of Cx43, whereas western blotting can detect protein expression levels. Using immunofluorescence assays, it is possible to analyze the expression and observe the localization of Cx43 in cells or tissues. Moreover, connexin-mediated gap junction intercellular communication can be evaluated using functional assays such as microinjection of fluorescent dyes or scrape loading-dye transfer. The use of selective inhibitors contributes to this understanding and reinforces the role of connexins in various processes. Here, we discuss these methods to evaluate Cx43 and macrophage gap junctions., (Copyright © 2024. Published by Elsevier B.V.)

Background and Objectives : Connexin 43 (Cx43) is involved in the transfer of small signaling molecules between neighboring cells, thereby exerting a major influence on the initiation and progression of tumorigenesis. However, there is a lack of systematic research on Cx43 expression and its predictive role in clinical diagnosis and prognosis in pan-cancer. Materials and Methods : Several biological databases were used to evaluate the expression levels of GJA1 (encoding Cx43) and its diagnostic and prognostic significance in pan-cancer. We targeted kidney renal clear cell carcinoma (KIRC) and investigated the relationship between GJA1 expression and different clinical features of KIRC patients. Then, we performed cell-based experiments to partially confirm our results and predicted several proteins that were functionally related to Cx43. Results : The expression of GJA1 has a high level of accuracy in predicting KIRC. High GJA1 expression was remarkably correlated with a favorable prognosis, and this expression was reduced in groups with poor clinical features in KIRC. Cell experiments confirmed the inhibitory effects of increased GJA1 expression on the migratory capacity of human renal cancer (RCC) cell lines, and protein-protein interaction (PPI) analysis predicted that CDH1 and CTNNB1 were closely related to Cx43. Conclusions : GJA1 could be a promising independent favorable prognostic factor for KIRC, and upregulation of GJA1 expression could inhibit the migratory capacity of renal cancer cells.

Background: Interneural gap junctional coupling represents neural development that decreases during the postnatal period. The decrease of gap junction function coincides with the main period of chemical synapse creation and increment of synaptic activity during postnatal weeks 1 to 3., Methods: Here, we have assessed the role of chemical synapses on connexin (Cx) expression in neurons and glial cells of hippocampal and cortical neurons. We characterized the impact of NMDA receptors blockade on the expression of Cx36 and Cx43 proteins by western blot analysis in postnatal day (PND)14 and PND28. MK801 was injected subcutaneously from the first day of birth until 14 or 28 days, depending on the experimental groups. Saline was injected in the same volumes in the control group., Results: Early postnatal blockade of the NMDA subtype of glutamate receptors by the non-competitive antagonist dizocilpine maleate (MK801) arrested the developmental reduction in gap junctions during the initial postnatal weeks. Expression of Cx43 declined in PND28 compared to PND14 in visual cortex (VC) neurons. Also, we found that the expression of Cx36 and Cx43 augmented in the rats' VC in PND28 following the blockade of NMDA receptors. Expression of Cx36 declined in PND28 compared to PND14 in hippocampal neurons. Also, we found that the expression of Cx36 augmented in the rats' hippocampal neurons in PND14 and PND28 following a blockade of NMDA receptors., Conclusion: These results suggest that the postnatal enhancement in glutamatergic synaptic activity is associated with the loss of gap junctional connections and downregulation of Cx36 and Cx43 between developing neurons and glial cells., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Inflammation is a critical mediator of renal ischemia-reperfusion (I/R) injury (IRI), and T lymphocytes exert a key role in the renal IRI-induced inflammation. Connexin 43 (Cx43) is related to the maintenance of T lymphocyte homeostasis. Various preclinical researches have reported that estrogen is a renoprotective agent based on its anti-inflammatory potential. The present research is aimed at studying the role of T lymphocytes activated by Cx43 in 17 β -estradiol-mediated protection against renal IRI. Female rats were classified into six groups: control rats, I/R rats, ovariectomized rats, ovariectomized I/R rats, and ovariectomized rats treated with 17 β -estradiol or gap27. Levels of serum creatinine (Scr) and blood urea nitrogen (BUN) and Paller scoring were dramatically increased in I/R rats, especially in ovariectomized rats. By contrast, these indicators were markedly decreased by administering estradiol or gap27. Immunofluorescence staining revealed that CD4 + T cells infiltrated kidney tissues in the early stage of IRI. In both peripheral blood and renal tissue, the proportion of CD3 + CD4 + T cells and ratio of CD4 + to CD8 + were high in I/R rats, especially in ovariectomized rats. The proportion of CD3 + CD8 + T cells was low in peripheral blood but high in renal tissues. Administration of estrogen or Gap27 reversed these effects. IL-17 levels in both serum and tissue homogenate were significantly increased in ovariectomized rats subjected to I/R but significantly decreased in estrogen or gap 27 treated rats. The opposite trend was observed for IL-10 levels. Correlation analysis demonstrated that IL-17 was correlated positively with BUN, Scr, and Paller scores, while IL-10 was negatively correlated with these indicators. Western blot showed that Cx43 expression was markedly increased in the peripheral blood T lymphocytes of I/R rats, especially ovariectomized rats. After intervention with estrogen and gap27, Cx43 expression was significantly downregulated. These findings indicate that Cx43 may participate in the regulation of Th17/Treg balance by estrogen against renal IRI., Competing Interests: The authors declare that there is no conflict of interest regarding the publication of this article., (Copyright © 2022 Yang Zhang et al.)

Background: Connexins are transmembrane proteins forming gap junctions between the cells, which allow intercellular communication. Significance of gap junctions and connexins in lung carcinoma is not yet understood. The objective of the study was to investigate immunohistochemical expression and the localization of connexin-43 (Cx43) in primary lung carcinoma and its lymphatic metastases., Methods: Surgical specimens of excised tumors from 88 patients (45 men and 43 women, 61.9 ± 7.4 years) with lung carcinoma (52 adenocarcinoma (AC), 36 squamous cell carcinoma (SqCC)) who were operated on at the University Hospital "Bezanijska Kosa" in a five-year period (2012-2016) were used. We conducted immunohistochemical staining for Cx43 and measured the degree of expression (percentage of positive cells and staining intensity) as well as localization of Cx43 in primary tumor and in lymphatic metastases., Results: Immunohistochemical analysis of the primary tumors revealed that SqCC showed significantly higher percentage of tumor cells expressing Cx43 as well as higher staining intensity than AC ( p  < 0.001). Almost 70% of samples with SqCC showed high Cx43 expression, whereas AC showed no expression in more than 50% of cases. Localization of Cx43 expression was most often cytoplasmic (AC and SqCC) and combined membranous and cytoplasmic (SqCC) with very rare instances of nuclear localization (AC). Almost the same pattern in distribution, intensity, and localization of Cx43 expression was observed in the lymph node metastases; however, almost a third of AC cases changed the pattern of Cx43 expression in the metastasis compared to primary tumor., Conclusion: The results of this study showed that lung carcinomas express Cx43 in more than 65% of cases and that it was aberrantly localized (not membranous localization). We highlighted that SqCC expressed Cx43 more than did AC, both in primary tumor and lymphatic metastases. Further research is needed to establish whether Cx43 could be used as a prognostic biomarker in lung carcinoma., Competing Interests: The authors declare there are no competing interests., (©2022 Savic et al.)

Background: Connexins (Cxs) are gap junction proteins involved in the communication between acute myeloid leukemia (AML) and stromal cells. They consist of intercellular channels termed "connexions", which can cause uncontrolled cell proliferation if dysregulated. This study aimed to evaluate the expression levels of the Cx32 and Cx43 genes and their correlations with other prognostic markers in patients with AML., Methods: This cross sectional study was performed on peripheral blood samples from 60 newly diagnosed patients with AML and 40 healthy control subjects at Kasr Alainy School of Medicine, Cairo University, from June 2016 to December 2017. The quantitative real-time polymerase chain reaction (qRT-PCR) test was used to examine the relative expression level of Cx43 and Cx32 genes in the patients and the control subjects. The Chi square test or the Fisher exact test was employed to examine the relationship between qualitative variables, while the independent t test or the Mann-Whitney test was employed for quantitative data. All the tests were two-tailed, and a P value of less than 0.05 was considered significant., Results: Among the patients with AML, 65% had a high Cx32 expression level, whereas 63.3% had a low Cx43 expression level. There was a statistically significant difference in the fold change values of Cx32 and Cx43 expression between the patient group and the control group (P=0.009 vs P=0.013, respectively). There was a remarkable association between both Cxs and CD34 and HLA-DR cells., Conclusion: Cx expression in samples may add to the diagnostic workup of AML. Although we found a negative correlation between Cx43 expression and the peripheral blood blast percentage, the response after the first induction of chemotherapy showed no significant relationship with Cx43 and Cx32 ., (Copyright: © Iranian Journal of Medical Sciences.)

Drug-induced organ toxicity/injury, especially in the liver, kidney, and gastrointestinal tract, is a systematic disorder that causes oxidative stress formation and inflammation resulting in cell death and organ failure. Current therapies target reactive oxygen species (ROS) scavenging and inhibit inflammatory factors in organ injury to restore the functions and temporary relief. Organ cell function and tissue homeostasis are maintained through gap junction intercellular communication, regulating connexin hemichannels. Mis-regulation of such connexin, especially connexin (Cx) 43, affects a comprehensive process, including cell differentiation, inflammation, and cell death. Aim to describe knowledge about the importance of connexin role and insights therapeutic targeting. Cx43 misregulation has been implicated in recent decades in various diseases. Moreover, in recent years there is increasing evidence that Cx43 is involved in the toxicity process, including hepatic, renal, and gastrointestinal disorders. Cx43 has the potential to initiate the immune system to cause cell death, which has been activated in the acceleration of apoptosis, necroptosis, and autophagy signaling pathway. So far, therapies targeting Cx43 have been under inspection and are subjected to clinical trial phases. This review elucidates the role of Cx43 in drug-induced vital organ injury, and recent reports compromise its function in the major signaling pathways., (Copyright © 2021 Elsevier Inc. All rights reserved.)

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.

Purpose: High LET including alpha radiation-based approaches have been proved as a promising mode for cancer therapy owing to their biophysical and radiobiological advantages compared to photon beams. Studies pertaining to effect of α-radiation on cancer cells are limited to cytotoxic high doses., Materials and Methods: In this study, human lung adenocarcinoma (A549) cells were α-irradiated using 241 Am α-irradiator and effects of low dose of alpha radiation on these cells was studied under in vitro and in vivo conditions., Results: Clonogenic and other assays showed increased cellular proliferation at lower doses (1.36 and 6.8 cGy) but killing at higher doses (13.6-54.4 cGy). Further studies at low dose of alpha (1.36 cGy) showed increased TGF-β1 in the conditioned medium (CM) at early time point (24 h) but CM replacement did not affect the clonogenic survival. In these cells, increased phosphorylation of connexin 43 was correlated with decrease in gap-junction communication observed by dye transfer co-culture experiment. A decrease in caveolin-1 but increase in survivin expression was observed in low dose α-irradiated cells. An increase in cyclinD1 and decrease in Bcl-2, the target proteins of survivin, was observed in these cells. Low dose α-irradiated cancer cells transplanted in SCID mice showed significantly higher tumor volume, which was accompanied with an increased fraction of mitotic and PCNA/Ki67 positive cells in these tumor tissues., Conclusions: Taken together, our results suggest an increase in proliferation and tumor volume at in vitro and in vivo levels, respectively, when A549 cells were irradiated with low dose of α-radiation. These findings may be relevant for a better understanding of radiobiological processes during high LET-based cancer radiotherapy.

Background: Scientific research on atrial fibrosis in atrial fibrillation (AF) has mainly focused on quantitative or molecular features. The purpose of this study was to perform a clinicoarchitectural/structural investigation of fibrosis to provide one key to understanding the electrophysiological/clinical aspects of AF., Methods: We characterized the fibrosis (amount, architecture, cellular components, and ultrastructure) in left atrial biopsies from 121 patients with persistent/long-lasting persistent AF (group 1; 59 males; 60±11 years; 91 mitral disease-related AF, 30 nonmitral disease-related AF) and from 39 patients in sinus rhythm with mitral valve regurgitation (group 2; 32 males; 59±12 years). Ten autopsy hearts served as controls., Results: Qualitatively, the fibrosis exhibited the same characteristics in all cases and displayed particular architectural scenarios (which we arbitrarily subdivided into 4 stages) ranging from isolated foci to confluent sclerotic areas. The percentage of fibrosis was larger and at a more advanced stage in group 1 versus group 2 and, within group 1, in patients with rheumatic disease versus nonrheumatic cases. In patients with AF with mitral disease and no rheumatic disease, the percentage of fibrosis and the fibrosis stages correlated with both left atrial volume index and AF duration. The fibrotic areas mainly consisted of type I collagen with only a minor cellular component (especially fibroblasts/myofibroblasts; average value range 69-150 cells/mm 2 , depending on the areas in AF biopsies). A few fibrocytes-circulating and bone marrow-derived mesenchymal cells-were also detectable. The fibrosis-entrapped cardiomyocytes showed sarcolemmal damage and connexin 43 redistribution/internalization., Conclusions: Atrial fibrosis is an evolving and inhomogeneous histological/architectural change that progresses through different stages ranging from isolated foci to confluent sclerotic zones which-seemingly-constrain impulse conduction across restricted regions of electrotonically coupled cardiomyocytes. The fibrotic areas mainly consist of type I collagen extracellular matrix and, only to a lesser extent, mesenchymal cells.

The changes in the myocardial proteome and metabolome associated with left ventricular assist device (LVAD) therapy in patients with ischemic cardiomyopathy (ICM) are poorly characterized. We investigated the impact of mechanical unloading following LVAD therapy on the myocardial proteome and metabolome. Matched samples of 5 patients' myocardial tissue, harvested at the time of LVAD implant ("pre-LVAD") or heart transplant ("post-LVAD"), were studied by quantitative proteomics and metabolomics as well as being probed for T-tubule structure and connexin-43 distribution. Moreover, pre-LVAD proteome profiles of ICM context were bioinformatically compared to pre-LVAD proteome profiles of dilated cardiac myopathy (DCM). More than 2120 proteins were reliably identified and quantified in paired patient samples. LVAD therapy led to proteomic remodeling, including reduced levels of α-1-antichymotrypsin together with an overall decrease of immune response proteins and an increase of proteins involved in membrane biology. Metabolomics highlighted increased glucose and glucose-6-phosphate levels in the left ventricle upon LVAD therapy. Wheat germ agglutinin staining demonstrated improved T-tubule structure. Connexin-43 displayed a trend for more pronounced intercalated disc localization. In comparing pre-LVAD proteome profiles of ICM context with pre-LVAD proteome profiles of dilated cardiac myopathy (DCM), we noticed an overrepresentation in ICM of proteins associated with humoral immune response. Our findings underline an impact of LVAD therapy on left ventricular biology in ICM. The proteomic, metabolomic, and structural alterations described here are typically associated with cardiac recovery. On the molecular level, our findings indicate the possibility of cardiac remodeling under LVAD therapy in ICM., (© 2019 The Authors. Artificial Organs published by International Center for Artificial Organ and Transplantation (ICAOT) and Wiley Periodicals, Inc.)

Cardiac pathologies associated with arrhythmic activity are often accompanied by inflammation. The contribution of inflammatory cells to the electrophysiological properties of injured myocardium is unknown. Myocardial scar cell types and intercellular contacts were analyzed using a three-dimensional reconstruction from serial blockface scanning electron microscopy data. Three distinct cell populations were identified: inflammatory, fibroblastic and endocardial cells. While individual fibroblastic cells interface with a greater number of cells, inflammatory cells have the largest contact area suggesting a role in establishing intercellular electrical connections in scar tissue. Optical mapping was used to study the electrophysiological properties of scars in fetal liver chimeric mice generated using connexin43 knockout donors (bmpKO). Voltage changes were elicited in response to applied current pulses. Isopotential maps showed a steeper pattern of decay with distance from the electrode in scars compared with uninjured regions, suggesting reduced electrical coupling. The tissue decay constant, defined as the distance voltage reaches 37% of the amplitude at the edge of the scar, was 0.48 ± 0.04 mm (n = 11) in the scar of the bmpCTL group and decreased 37.5% in the bmpKO group (n = 10). Together these data demonstrate inflammatory cells significantly contribute to scar electrophysiology through coupling mediated at least partially by connexin43 expression.

Purpose: To evaluate that Connexin (Cx43) plays a role in lesions after hepatic ischemia/reperfusion (IR) injury., Methods: We use Cx43 deficient model (heterozygotes mice) and compared to a wild group. The groups underwent 1 hour ischemia and 24 hours reperfusion. The heterozygote genotype was confirmed by PCR. We analyzed the hepatic enzymes (AST, ALT, GGT) and histology., Results: The mice with Cx43 deficiency showed an ALT mean value of 4166 vs. 307 in the control group (p<0.001); AST mean value of 7231 vs. 471 in the control group (p<0.001); GGT mean value of 9.4 vs. 1.7 in the control group (p=0.001); histology showed necrosis and inflammation in the knockout group., Conclusions: This research demonstrated that the deficiency of Cx43 worses the prognosis for liver injury. The topic is a promising target for therapeutics advancements in liver diseases and procedures.

Introduction: Benign cutaneous tumors with follicular differentiation are alleged to differentiate toward parts of the hair follicle. Connexin 43 (Cx43) is a gap junction protein, the tumoral role of which has been investigated in several types of tumors., Objective: To study the pattern of expression of Cx43 in benign cutaneous tumors with follicular differentiation and to compare it with that shown by their alleged anatomical counterparts of the hair follicle., Materials and Methods: Five cases each of trichofolliculoma, trichilemmoma, fibrofolliculoma/trichodiscoma, trichoblastoma, trichoepithelioma, pilomatrixoma, and proliferating trichilemmal tumor, 3 cases of pilar sheath acanthoma, and 1 case of tumor of the follicular infundibulum were examined. Anti-Cx43 antibody was used., Results: Cx43 was expressed by all follicular tumors studied. Comparisons between trichoblastoma and trichoepithelioma and their respective normal counterparts could not be made. In 3 tumors (trichofolliculoma, pilomatrixoma, and the spectrum fibrofolliculoma/trichodiscoma), there was a parallelism between their Cx43 expression pattern and that of their alleged anatomical counterparts. In pilar sheath acanthoma, trichilemmoma, and the tumor of the follicular infundibulum, we only found partial similarities in Cx43 expression. Only the proliferating trichilemmal tumor showed a discordant pattern of expression., Conclusions: Cx43 expression is preserved in benign cutaneous tumors with follicular differentiation and the patterns of Cx43 expression in benign cutaneous tumors with follicular differentiation parallel those of their alleged anatomical counterparts in 5 types (either totally or partially). This preservation might be related to the good behavior of the entities studied.

In order to evaluate the influence of reproductive activity on the functional role of the epididymal epithelium in the Egyptian dromedary camel, Connexin-43 (Cx-43), vascular endothelial growth factor (VEGF), and androgen receptor (AR) immunoreactivity in the epididymal epithelium and the fine structure of the principal, dark, basal, apical, and halo cells were investigated. The secretory activity of the principal cells was amplified in the breeding season, while its endocytotic function became more active in the nonbreeding season. This was evidenced by punctate strong immunoreactive signals for Cx-43, which appeared to be more intense in the apical region of these epithelial cells, and the extremely long slender stereocilia (microvilli) with multiple junctional complexes. The nonbreeding principal cells revealed granular immunoreactive signals for VEGF scattered in the apical and basal cytoplasm. Ultrastructurally, both extreme vacuolation and several multivesicular inclusion bodies were observed in their cytoplasm. Dark cell size greatly diminished in the nonbreeding season and their nuclear morphology greatly changed from oval to lobulated shape. The plasma membrane of the apical cells expressed several infoldings (microvilli) in the breeding season. However, it was almost smooth in the nonbreeding season except for a small microvillus that appeared as a bleb-like projection. In some regions, a strong dense immunoreactivity for VEGF could be recognized in the cytoplasm of the apical cells and some basal ones. Halo cells with numerous multivesicular inclusions occupying most of the cytoplasm and a lobulated eccentric nucleus were detected in the nonbreeding season. In conclusion, these findings indicate that the reproductive activity has a significant impact on the immunohistochemical and ultrastructural profiles of the epithelial cells lining the Egyptian dromedary camel epididymis.

Renal fibrosis is an important component of chronic kidney disease, an incurable pathology with increasing prevalence worldwide. With a lack of available therapeutic options, end-stage renal disease is currently treated with renal replacement therapy through dialysis or transplantation. In recent years, many efforts have been made to identify novel targets for therapy of renal diseases, with special focus on the characterization of unknown mediators and pathways participating in renal fibrosis development. Using experimental models of renal disease and patient biopsies, we identified four novel mediators of renal fibrosis with potential to constitute future therapeutic targets against kidney disease: discoidin domain receptor 1, periostin, connexin 43, and cannabinoid receptor 1. The four candidates were highly upregulated in different models of renal disease and were localized at the sites of injury. Subsequent studies showed that they are centrally involved in the underlying mechanisms of renal fibrosis progression. Interestingly, inhibition of either of these proteins by different strategies, including gene deletion, antisense administration, or specific blockers, delayed the progression of renal disease and preserved renal structure and function, even when the inhibition started after initiation of the disease. This review will summarize the current findings on these candidates emphasizing on their potential to constitute future targets of therapy.

Purpose: Gap junctions are specialized membrane structures that form channels between adjacent cells allowing cell communication. Gap junctions and specifically Connexin 43 (Cx43) are down-regulated in cancer; however, there are contrasting reports on how this effects breast cancer patient survival. This paper is the first large-scale tissue microarray analysis of Cx43 expression in breast cancer patients with an associated clinical long-term follow-up., Methods: Using a validated TMA of 1118 primary breast cancers, coupled to a comprehensive database of clinicopathological variables, the expression levels and subcellular localisation of Cx43 was assessed by immunohistochemistry. Its impact in terms of survival, distant metastasis-free survival, and clinicopathological variables was determined., Results: Patients whose tumors expressed high levels of Cx43 had significantly better survival (p < 0.001) than patients with low levels. High Cx43 expression within tumors was associated with an 18-month survival advantage. Loss of Cx43 expression was associated with markers of poor prognosis, namely large tumor size, high grade, high proliferation status, high pleomorphism, high mitosis, poor Nottingham Prognostic Index (NPI), and triple negative tumors. Cx43 expression was independent of tumor size, grade, stage and ER-status in predicting poor survival on multivariate analysis (p = 0.004)., Conclusion: Connexin 43 (Cx43) is an independent predictor of breast cancer survival and distant metastasis-free survival. High expression of Cx43 was seen in only 13% of tumors, suggesting that drugs to increase Cx43 expression may result in prolonged patients survival.

It is well recognized that osteocytes communicate with each other via gap junctions and that connxin43 (Cx43) shows its great potential in gap junction for the contribution enabling transmission of small molecules and operating in an autocrine/a paracrine manner. Fibroblast growth factors (FGFs) play significant roles in new bone formation and adult bone remodeling, and FGF signaling is regulated by the precise spatiotemporal approaches. However, the influence of FGF7 on osteocyte cell processes is not well elucidated. In this study, we aimed to examine the impact of FGF7 on osteocyte cell processes by characterizing the expression of Cx43 and to reveal the underlying mechanism regulating this cell process. We first found that the mRNA level of FGF7 was higher relative to other FGF family members both in osteocytes cell line (MLO-Y4) and bone tissue. We then demonstrated that FGF7 could increase the expression of Cx43 in osteocytes and promote the cell processes in the form of gap junctions between osteocytes. This modulation was due to the FGF7-induced cytoplasmic accumulation and resultant nuclear translocation of β-catenin. Our results could help us to further understand the importance of FGF7 on bone cell behavior and bone physiology and even pathology.

Background: Imbalances in circulating T lymphocytes play critical roles in the pathogenesis of hypertension-mediated inflammation. Connexins (Cxs) in immune cells are involved in the maintenance of homeostasis of T lymphocytes. However, the association between Cxs in peripheral blood T lymphocytes and hypertension-mediated inflammation remains unknown. This study was designed to investigate the role of Cxs in T lymphocytes in hypertension-mediated inflammation in spontaneously hypertensive rats (SHRs)., Methods: The systolic blood pressure (SBP) in Wistar-Kyoto (WKY) rats and SHRs was monitored using the tail-cuff method. The serum cytokine level was determined using ELISA. The proportions of different T-lymphocyte subtypes in the peripheral blood, the expressions of Cx40/Cx43 in the T-cell subtypes, and the gap junctional intracellular communication (GJIC) of peripheral blood lymphocytes were measured using flow cytometry (FC). The accumulations of Cx40/Cx43 at the plasma membrane and/or in the cytoplasm were determined using immunofluorescence staining. The in vitro mRNA levels of cytokines and GJIC in the peripheral blood lymphocytes were respectively examined using real-time PCR and FC after treatment with Gap27 and/or concanavalin A (Con A)., Results: The percentage of CD4 + T cells and the CD4 + /CD8 + ratio were high, and the accumulation or expressions of Cx40/Cx43 in the peripheral blood lymphocytes in SHRs were higher than in those of WKY rats. The percentage of CD8 + and CD4 + CD25 + T cells was lower in SHRs. The serum levels of IL-2, IL-4 and IL-6 from SHRs were higher than those from WKY rats, and the serum levels of IL-2 and IL-6 positively correlated with the expression of Cx40/Cx43 in the peripheral blood T lymphocytes from SHRs. The peripheral blood lymphocytes of SHRs exhibited enhanced GJIC. Cx43-based channel inhibition, which was mediated by Gap27, remarkably reduced GJIC in lymphocytes, and suppressed IL-2 and IL-6 mRNA expressions in Con A stimulated peripheral blood lymphocytes., Conclusions: Our data suggest that Cxs may be involved in the regulation of T-lymphocyte homeostasis and the production of cytokines. A clear association was found between alterations in Cxs expression or in Cx43-based GJIC and hypertension-mediated inflammation., Competing Interests: Xin-zhi Li is from the Department of Pathophysiology of the Medical College of Shihezi University. The other authors are from the Department of Physiology of the Medical College of Shihezi University.This manuscript does not report on studies involving human participants, human data or human tissue. The protocol of the study was approved by Institutional Animal Care and Use Committees (IACUC) of the Medical College of Shihezi University, and all animal handling and experimental procedures were performed in accordance with guidelines for the Care and Use of Laboratory Animals published by the US National Institutes of Health (Public Health Service Policy on Humane Care and Use of Animals, DHEW Publication No. 96–01, PHS Policy revised in 2002).Not applicableThe authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as potential conflicts of interest.Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.

Aims: Ageing-related tau astrogliopathy (ARTAG) appears in subependymal, subpial, perivascular, white matter (WM) and grey matter (GM) locations. Physical effects, blood-brain barrier dysfunction and blood- or vessel-related factors have been considered as aetiology. As connexin-43 (Cx43) and aquaporin-4 (AQP4) are related to these, we hypothesized that their immunoreactivity (IR) varies with ARTAG in a location-specific manner., Methods: We performed a morphometric immunohistochemical study measuring the densities of IR of Cx43, AQP4, AT8 (phospho-tau) and glial fibrillar acidic protein (GFAP). We analysed the amygdala and hippocampus in age-matched cases with (n = 19) and without (n = 20) ARTAG in each of the locations it aggregates., Results: We show a dramatic increase (>6-fold; P < 0.01) of Cx43 density of IR in ARTAG cases correlating strongly with AT8 density of IR, irrespective of the presence of neuronal tau pathology or reactive gliosis measured by GFAP density of IR, in the GM. In contrast, AQP4 density of IR was increased only in the WM and GM, and was associated with increased AT8 density of IR only in WM and perivascular areas., Discussion: Our study reveals distinctive astroglial responses in each of the locations associated with ARTAG. Our observations support the concept that factors related to brain-fluid interfaces and water-ion imbalances most likely play a role in the generation of ARTAG. As Cx43 is crucial for maintaining neuronal homeostasis, the ARTAG-dependent increase of Cx43 density of IR suggests that the development of ARTAG in the GM most likely indicates an early response to the degeneration of neurons., (© 2017 British Neuropathological Society.)

Atrial fibrillation (AF) is the most common arrhythmia reported in clinical practice. Connexin 43 (Cx43) is a member of the connexin protein family, which serves important roles in signal transduction in vivo. The aim of the present study was to investigate the role of Cx43 in the induction and maintenance of atrial fibrillation by using an animal model of sympathomimetic atrial fibrillation. Cx43 was successfully knocked down in the myocardium with gene‑specific small interfering (si)RNA via lentiviral infection. A total of 25 dogs were randomly and evenly divided into five groups: Normal (N), rapid atrial pacing (RAP), isoproterenol (ISO) + RAP, RAP + Cx43 siRNA and ISO + RAP + Cx43 siRNA. The mRNA and protein levels, as well as the distribution of Cx43 on the cell membrane, were gradually decreased in each group compared with the N group following treatment (P<0.05). The induction rate of the atrial effective refractory period was not significantly affected in the RAP and RAP + Cx43 siRNA groups, whereas it was significantly reduced in the ISO + RAP and ISO + RAP + Cx43 siRNA groups compared with the N group (P<0.05). The induction rate of AF was gradually increased in the RAP + Cx43 siRNA, ISO + RAP and ISO + RAP + Cx43 siRNA groups compared with the N group (P<0.05). The expression of nerve growth factor (NGF) and tyrosine hydroxylase (TH) was gradually increased in the ISO + RAP and ISO + RAP + Cx43 siRNA groups compared with their respective controls (RAP and RAP + Cx43 siRNA groups, respectively). However, no significant difference in the levels of NGF and TH was observed between the RAP, RAP + Cx43 siRNA, ISO + RAP and ISO + RAP + Cx43 siRNA groups. The mitochondrial morphology in each group was notably altered compared with the N group. The mitochondrial reactive oxygen species production and apoptotic index were gradually increased in each group compared with the N group (P<0.05). The results of the present study suggest that Cx43 reduces susceptibility to AF. Downregulation of Cx43 mediates the induction and maintenance of sympathetic AF.

Background and Purpose: Moyamoya disease has a high incidence of cerebral vascular accident in children and adolescents, which can endanger the physical and mental health of children and adults seriously. However, the etiology and the pathogenesis of moyamoya disease remain unclear. Connexin43 (Cx43) is a predominant intercellular gap junction protein that plays an important role in the normal function of arteries and the development of several cardiovascular diseases. We aimed to preliminarily investigate pathological changes and the expression of Cx43 in cerebral arteries of patients with moyamoya disease., Materials and Methods: This study collected 10 experimental cerebral artery specimens from patients with moyamoya disease and 10 control cerebral artery specimens from patients without moyamoya disease during surgery, then pathological changes and change in Cx43 expression of cerebral artery specimens were investigated in the 2 groups by hematoxylin and eosin staining and immunofluorescence., Results: The intima of cerebral arteries was thin with monolayer endothelial cells in the control group but had asymmetrical thickening for the cerebral arteries in the experimental group. The mean ± standard deviation of the mean optical density of Cx43 in the experimental group was .065 ± .011 (range, .045-.081), whereas that in the control group was .035 ± .005 (range, .028-.042). The expression of Cx43 in the experimental group was statistically significantly higher in comparison with the control group (P < .01)., Conclusion: The abnormal expression of Cx43 in the cerebral arteries may play an important role in the formation of vascular intima thickening in patients with moyamoya disease., (Copyright © 2018 National Stroke Association. Published by Elsevier Inc. All rights reserved.)

Connexins are widely supported as tumor suppressors due to their downregulation in cancers, nevertheless, more recent evidence suggests roles for connexins in facilitating tumor progression in later stages, including metastasis. One of the key factors regulating the expression, modification, stability, and localization of connexins is hormone receptors in hormone-dependent cancers. It is reasonable to consider that hormones/hormone receptors may modulate connexins expression and play critical roles in the cellular control of connexins during breast cancer progression. In estrogen receptor (ER)-positive breast cancers, tamoxifen and fulvestrant are widely used therapeutic agents and are considered to alter ER signaling. In this present study, we investigated the effects of fulvestrant and tamoxifen in Cx43 expression, and we also explored the role of Cx43 in ER-positive breast cancer migration and the relationship between Cx43 and ER. The involvement of estrogen/ER in Cx43 modulation was further verified by administering tyrosine kinase inhibitors and chemotherapeutic agents. We found that inhibition of ER promoted the binding of E3 ligase Nedd4 to Cx43, leading to Cx43 ubiquitination. Furthermore, inhibition of ER by fulvestrant and tamoxifen phosphorylated p38 MAPK, and inhibition of Rac, MKK3/6, and p38 reversed fulvestrant-reduced Cx43 expression. These findings suggest that Cx43 expression which may positively regulate cell migration is ER-dependent in ER-positive breast cancer cells., (Copyright © 2017 Elsevier Inc. All rights reserved.)

Background/aims: This experimental study aims to observe whether the protective effect of propofol against renal ischemia-reperfusion injury (IRI) in the rat interlobar artery occurs through altered expression of the gap junction protein connexin 43 (Cx43)., Methods: This study randomly divided male Sprague Dawley (SD) rats into an untreated control group, a sham-operated control group (sham group), an ischemia-reperfusion group (IR group), a propofol group (propofol+IR group) and a fat emulsion group (Intralipid group). The ischemia/reperfusion model was prepared through resection of the right kidney and noninvasive arterial occlusion of the left kidney. Forty-five minutes after renal ischemia-reperfusion, an automatic biochemical analyzer was employed to measure blood urea nitrogen (BUN) and serum creatinine (SCr); changes in renal tissue pathology were observed using hematoxylin and eosin (HE) staining, and the vasomotor activity of the interlobar artery was detected using a pressure mechanogram technique. The protein expression of Cx43 in renal artery cross-sections was determined through western blotting., Results: The experimental study confirmed that the BUN and SCr of rats markedly increased after ischemia-reperfusion injury; additionally, we observed some coagulation necrosis and shedding of cells, some solidification of nuclear chromatin, degeneration of cytoplasmic vacuoles, high renal interstitial vascular congestion and obvious inflammatory cell infiltration, characterized by focal hemorrhages. Furthermore, the contraction activity of the renal interlobar artery greatly decreased, and the tension of the arteries in the renal lobe increased remarkably. After the gap junction blocking agents 2-APB and Gap27 were applied, the systolic velocity of blood vessels and the vascular contraction rate both decreased. In addition, the expression of Cx43 in kidney tissues increased markedly. The damage was more severe after 24 h of ischemic reperfusion than after only 4 h. However, after pretreatment with propofol, regardless of whether ischemia-reperfusion was applied for 4 h or 24 h, the previously increased expression of Cx43 decreased obviously, and all forms of renal damage were reversed., Conclusion: Our research suggests new ways for propofol to relieve ischemia-reperfusion injury by decreasing the abnormal expression of the gap junction protein Cx43. This study reveals a novel mechanism for the action of propofol against IRI, and we hope this finding will lead to new treatments for IRI., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Background/aims: Changes in cell-to-cell communication have been linked to several secondary complications of diabetes, but the mechanism by which connexins affect disease progression in the kidney is poorly understood. This study examines a role for glucose-evoked changes in the beta1 isoform of transforming growth factor (TGFβ1), on connexin expression, gap-junction mediated intercellular communication (GJIC) and hemi-channel ATP release from tubular epithelial cells of the proximal renal nephron., Methods: Biopsy material from patients with and without diabetic nephropathy was stained for connexin-26 (CX26) and connexin-43 (CX43). Changes in expression were corroborated by immunoblot analysis in human primary proximal tubule epithelial cells (hPTECs) and model epithelial cells from human renal proximal tubules (HK2) cultured in either low glucose (5mmol/L) ± TGFβ1 (2-10ng/ml) or high glucose (25mmol/L) for 48h or 7days. Secretion of the cytokine was determined by ELISA. Paired whole cell patch clamp recordings were used to measure junctional conductance in control versus TGFβ1 treated (10ng/ml) HK2 cells, with carboxyfluorescein uptake and ATP-biosensing assessing hemi-channel function. A downstream role for ATP in mediating the effects of TGF-β1 on connexin mediated cell communication was assessed by incubating cells with ATPγS (1-100µM) or TGF-β1 +/- apyrase (5 Units/ml). Implications of ATP release were measured through immunoblot analysis of interleukin 6 (IL-6) and fibronectin expression., Results: Biopsy material from patients with diabetic nephropathy exhibited increased tubular expression of CX26 and CX43 (P<0.01, n=10), data corroborated in HK2 and hPTEC cells cultured in TGFβ1 (10ng/ml) for 7days (P<0.001, n=3). High glucose significantly increased TGFβ1 secretion from tubular epithelial cells (P<0.001, n=3). The cytokine (10ng/ml) reduced junctional conductance between HK2 cells from 4.5±1.3nS in control to 1.15±0.9nS following 48h TGFβ1 and to 0.42±0.2nS after 7days TGFβ1 incubation (P<0.05, n=5). Acute (48h) and chronic (7day) challenge with TGFβ1 produced a carbenoxolone (200µM)-sensitive increase in carboxyfluorescein loading, matched by an increase in ATP release from 0.29±0.06μM in control to 1.99±0.47μM after 48hr incubation with TGFβ1 (10ng/ml; P<0.05, n=3). TGF-β1 (2-10ng/ml) and ATPγs (1-100µM) increased expression of IL-6 (P<0.001 n=3) and fibronectin (P<0.01 n=3). The effect of TGF-β1 on IL-6 and fibronectin expression was partially blunted when preincubated with apyrase (n=3)., Conclusion: These data suggest that chronic exposure to glucose-evoked TGFβ1 induce an increase in CX26 and CX43 expression, consistent with changes observed in tubular epithelia from patients with diabetic nephropathy. Despite increased connexin expression, direct GJIC communication decreases, whilst hemichannel expression/function and paracrine release of ATP increases, changes that trigger increased levels of expression of interleukin 6 and fibronectin. Linked to inflammation and fibrosis, local increases in purinergic signals may exacerbate disease progression and highlight connexin mediated cell communication as a future therapeutic target for diabetic nephropathy., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Background/aims: Osteocytes can sense and respond to extracellular stimuli, including biochemical factors throughout the cell body, dendritic processes, and cilia bending. However, further exploration is required of osteocyte function in response to substrate stiffness, an important passive mechanical cue at the interface between osteocytes and the extracellular matrix, and the deep bio-mechanism in osteocytes involving mechanosensing of cell behavior., Methods: We fabricated silicon-based elastomer polydimethylsiloxane substrates with different stiffnesses but with the same surface topologies. We then seeded osteocytes onto the substrates to examine their responses. Methodologies used included scanning electron microscopy (SEM) for cell morphology, confocal laser scanning microscopy (CLSM) for protein distribution, western blot for protein levels, co-immunoprecipitation for protein interactions, and quantitative real-time polymerase chain reaction for gene expression., Results: SEM images revealed that substrate stiffness induced a change in osteocyte morphology, and CLSM of F-actin staining revealed that substrate stiffness can alter the cytoskeleton. These results were accompanied by changes in focal adhesion capacity in osteocytes, determined via characterization of vinculin expression and distribution. Furthermore, on the exterior of the cell membrane, fibronectin was altered by substrate stiffness. The fibronectin then induced a change in paxillin on the inner membrane of the cell via protein-protein interaction through transmembrane processing. Paxillin led to changes in connexin 43 via protein-protein binding, thereby influencing osteocyte gap junction elongation., Conclusion: This process -from mechanosensing and mechanotransduction to cell function - not only indicates that the effects of mechanical factors on osteocytes can be directly sensed from the cell body, but also indicates the involvement of paxillin transduction., (© 2018 The Author(s). Published by S. Karger AG, Basel.)

Connexin 43 (Cx43) is the main astrocytic connexin and forms the basis of the glial syncytium. The morphology of connexin-expressing cells can be best studied in transgenic mouse lines expressing cytoplasmic fluorescent reporters, since immunolabeling the plaques can obscure the shapes of the individual cells. The Cx43kiECFP mouse generated by Degen et al. (FASEBJ 26:4576, 2012) expresses cytosolic ECFP and has previously been used to establish that Cx43 may not be expressed by all astrocytes within a population, and this can vary in a region-dependent way. To establish this mouse line as a tool for future astrocyte and connexin research, we sought to consolidate reporter authenticity, studying cell types and within-region population heterogeneity. Applying anti-GFP, all cell types related to astroglia were positive-namely, protoplasmic astrocytes in the hippocampus, cortex, thalamus, spinal cord, olfactory bulb, cerebellum with Bergmann glia and astrocytes also in the molecular layer, and retinal Müller cells and astrocytes. Labeled cell types further comprise white matter astrocytes, olfactory ensheathing cells, radial glia-like stem cells, retinal pigment epithelium cells, ependymal cells, and meningeal cells. We furthermore describe a retinal Cx43-expressing amacrine cell morphologically reminiscent of ON-OFF wide-field amacrine cells, representing the first example of a mammalian CNS neuron-expressing Cx43 protein. In double staining with cell type-specific markers (GFAP, S100ß, glutamine synthetase), Cx43 reporter expression in the hippocampus and cortex was restricted to GFAP + astrocytes. Altogether, this mouse line is a highly reliable tool for studies of Cx43-expressing CNS cells and astroglial cell morphology. © 2017 Wiley Periodicals, Inc., (© 2017 Wiley Periodicals, Inc.)

A fast and robust procedure for the quantification of GFP-tagged membrane proteins in cell homogenates was developed employing capillary gel electrophoresis coupled to laser-induced fluorescence detection (CGE-LIF). The new method was found to be highly sensitive and applicable to structurally diverse membrane proteins including synaptic vesicle protein 2A (SV2A), adenosine A 2A receptor (A 2A AR), and connexin 43 (Cx43). Quantification of SV2A and A 2A AR using radioligand binding assays confirmed the results obtained with CGE-LIF. The CGE-LIF method showed significantly higher sensitivity as compared to fluorimetric measurement in a microplate. Importantly, CGE-LIF involves separation of the target proteins and their degradation products prior to quantification and thereby ensures specificity. We anticipate broad applicability of the method for any fluorophore-tagged protein.

Objective: We developed an in vitro model to examine whether trauma induces connexin 43 (Cx43) expression and collagen organisation in the amniotic membrane (AM) of fetal membrane (FM) defects., Method: Term human FM was traumatised in vitro. Cell morphology and Cx43 were examined in the wound edge AM by immunofluorescence (IMF) confocal microscopy and compared to control AM. Collagen microstructure was examined by second harmonic generation (SHG) imaging. Cell viability was assessed with calcein and ethidium staining., Results: After trauma, the AM showed a dense region of cells, which had migrated towards the wound edge. In wound edge AM, Cx43 puncta was preferentially distributed in mesenchymal cells compared to epithelial cells with significant expression in the fibroblast layer than epithelial layer (p < 0.001). In the fibroblast layer, the collagen fibres were highly polarised and aligned in parallel to the axis of the wound edge AM. There was an absence of cell migration across the defect with no healing after 168 h. Cell viability of the FM after trauma was maintained during culture., Conclusion: Cx43 overexpression in wounded AM drives structural changes in collagen that slows down efficacy of cell migration across the FM defect. © 2017 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd., (© 2017 The Authors. Prenatal Diagnosis published by John Wiley & Sons, Ltd.)

Background: The dietary limitation during pregnancy influences the growth and development of the fetus and offspring and their health into adult life. The mechanisms underlying the adverse effects of gestational protein restriction (GPR) in the development of the offspring hearts are not well understood., Objectives: The aim of this study was to evaluate the effects of GPR on cardiac structure in male rat offspring at day 60 after birth (d60)., Methods: Pregnant Wistar rats were fed a normal-protein (NP, 17% casein) or low-protein (LP, 6% casein) diet. Blood pressure (BP) values from 60-day-old male offspring were measured by an indirect tail-cuff method using an electro sphygmomanometer. Hearts (d60) were collected for assessment of connexin 43 (Cx43) mRNA expression and morphological and morphometric analysis., Results: LP offspring showed no difference in body weight, although they were born lighter than NP offspring. BP levels were significantly higher in the LP group. We observed a significant increase in the area occupied by collagen fibers, a decrease in the number of cardiomyocytes by 104 µm2, and an increase in cardiomyocyte area associated with an increased Cx43 expression., Conclusion: GPR changes myocardial levels of Cx43 mRNA in male young adult rats, suggesting that this mechanism aims to compensate the fibrotic process by the accumulation of collagen fibers in the heart interstitium.

The aims of this study were to investigate the effects of medium replacement system (experiment I) and of FSH presentations (homeopathic - FSH 6cH and allopathic FSH - rFSH; experiment II) on the in vitro development, hormone production and gene expression of isolated ovine preantral follicles cultured for 6 days. In experiment I, secondary follicles were cultured in the α-MEM + supplemented with FSH 6cH (0.05 fg/ml) or recombinant bovine FSH (100 ng/ml) without/with daily medium addition. The homeopathic FSH treatments with/without medium addition improved (p < .05) follicular development compared to rFSH100 treatment without addition. FSH 6cH with addition showed the highest (p < .05) estradiol production. To verify whether the effects of homeopathic FSH were not due to its vehicle, experiment II was performed. The α-MEM + was supplemented or not with alcohol (0.2% grain ethanol, v/v), FSH 6cH or rFSH100 with daily medium addition. Surprisingly, we found that all treatments improved follicular development compared to the α-MEM + (p < .05). Moreover, homeopathic FSH was similar to the other treatments including its vehicle. In conclusion, its vehicle (ethanol) causes the effect of homeopathic FSH on in vitro development of isolated ovine preantral follicles., (© 2016 Wiley Periodicals, Inc.)

Ethnopharmacological Relevance: Tongxinluo (TXL) is a multifunctional traditional Chinese medicine and has been widely used in the treatment of cardiovascular and cerebrovascular diseases. Numerous studies demonstrate that TXL is a novel neuroprotective drug, however, the mechanisms are largely unknown., Aim of the Study: we aimed to demonstrate the protective effect of TXL on cerebral ischemia/reperfusion (I/R) injury and provide the evidence for the involvement of Connexin 43/Calpain II/ Bax/Caspase-3 pathway in TXL-mediated neuroprotection., Methods: Focal cerebral I/R injury were induced by transient middle cerebral artery occlusion (MCAO, for 90min) in adult male Sprague-Dawley rats. We estimated the effects of TXL on I/R injury including neurological deficit assessment and cerebral infarct volume measurement via TTC staining, and detected the protein expression of Connexin 43 (Cx43) by western blot. Furthermore, after the intracerebroventricular injection of carbenoxolone (CBX, the inhibitor of Cx43) at 30min before MCAO surgery, Calpain II, Bax and cleaved Caspased-3 immunoreactivity in ischemic penumbra region was detected by immunofluorescent staining, and cell apoptosis was detected by TUNEL staining., Results: TXL treatment greatly improved neurological deficit and reduced the infarction volume compared to MCAO with buffer treatment (P<0.05), and TXL pre-post treatment showed better results than TXL pre-treatment. TXL pre-post treatment significantly up-regulated Cx43 protein expression at 3d, 7d and 14d post-injury compared to MCAO with buffer treatment (P<0.05). Meanwhile, the immunoreactivity of Calpain II, Bax and cleaved Caspase-3 in ischemic penumbra region was obviously decreased by TXL pre-post treatment compared to MCAO group (P<0.05). However, with the treatment of the Cx43 inhibitor, CBX, the down-regulated effect of TXL on Calpain II, Bax and cleaved Caspase-3 immunoreactivity was abolished (P<0.05). Moreover, the protective effect of TXL against neuron apoptosis in penumbra region was conteracted by CBX (P<0.05)., Conclusions: TXL could effectively protect against I/R injury and reduced cell death via Cx43/Calpain II/Bax/Caspase-3 pathway, which contribute to I/R injury prevention and therapy., (Copyright © 2017 Elsevier Ireland Ltd. All rights reserved.)

The aim of the report is to evaluate the prognostic and predictive role of Connexin 43 (Cx43) expression in laryngeal squamous cell carcinomas. Eighty-seven previously untreated patients submitted to laryngectomy ± neck dissection ± radiotherapy were enrolled in this retrospective study. The original primary tumor slides were reassessed, tumor grade and stage reviewed, and Cx43 immunohistochemical analysis performed: only cytoplasmic membranous staining of Cx43 has been shown. Neither significant correlation has been showed for clinical T (p = 0.75) and N (p = 0.81), while significant correlation has been found with grading (p < 0.0001) and pathological N (p < 0.0001). Five year overall survival (OS) of the 87 patients was 54 %; 5 year OS was 59.6 % in Cx43 positive patients and 37.1 % in Cx43 negative patients, but also this difference did not reach statistical significance (p = 0.058). Our best findings were: poorly differentiated carcinomas had low or negative Cx43 expression; moderately differentiated tumors without node metastasis and no radiotherapy but with Cx43 expression had a better outcome; moderately differentiated tumors without node metastasis and no radiotherapy but without Cx43 expression had a worse outcome; moderately differentiated tumors with node metastasis and radiotherapy but without Cx43 expression had a better outcome. Interestingly, in G2 head and neck squamous cell carcinomas with lymph node metastasis at the time of diagnosis, Cx43 aberrant overexpression could identify a subset of patients with poor prognosis, far less responsive to radio/chemotherapy.

Background and Aims: Being goalkeepers of liver homeostasis, gap junctions are also involved in hepatotoxicity. However, their role in this process is ambiguous, as gap junctions can act as both targets and effectors of liver toxicity. This particularly holds true for drug-induced liver insults. In the present study, the involvement of connexin26, connexin32 and connexin43, the building blocks of liver gap junctions, was investigated in acetaminophen-induced hepatotoxicity., Methods: C57BL/6 mice were overdosed with 300mg/kg body weight acetaminophen followed by analysis of the expression and localization of connexins as well as monitoring of hepatic gap junction functionality. Furthermore, acetaminophen-induced liver injury was compared between mice genetically deficient in connexin43 and wild type littermates. Evaluation of the toxicological response was based on a set of clinically relevant parameters, including protein adduct formation, measurement of alanine aminotransferase activity, cytokines and glutathione., Results: It was found that gap junction communication deteriorates upon acetaminophen intoxication in wild type mice, which is associated with a switch in mRNA and protein production from connexin32 and connexin26 to connexin43. The upregulation of connexin43 expression is due, at least in part, to de novo production by hepatocytes. Connexin43-deficient animals tended to show increased liver cell death, inflammation and oxidative stress in comparison with wild type counterparts., Conclusion: These results suggest that hepatic connexin43-based signaling may protect against acetaminophen-induced liver toxicity., (Copyright © 2016 Elsevier B.V. All rights reserved.)

Background: Present study was designed to establish a causal connection between changes in the cell-cell junction protein expression at the blood-testis barrier and alterations in the adult rat testis histology following an anti-androgen flutamide exposure. Particular emphasis was placed on the basal ectoplasmic specialization (ES) in the seminiferous epithelium and expression of gap junction protein, connexin 43 (Cx43)., Methods: Flutamide (50 mg/kg body weight) was administered to male rats daily from 82 to 88 postnatal day. Testes from 90-day-old control and flutamide-exposed rats were used for all analyses. Testis morphology was analyzed using light and electron microscopy. Gene and protein expressions were analyzed by real-time RT-PCR and Western blotting, respectively, protein distribution by immunohistochemistry, and steroid hormone concentrations by radioimmunoassay., Results: Seminiferous epithelium of both groups of rats displayed normal histology without any loss of germ cells. In accord, no difference in the apoptosis and proliferation level was found between control and treated groups. As shown by examination of semi-thin and ultrathin sections, cell surface occupied by the basal ES connecting neighboring Sertoli cells and the number of gap and tight junctions coexisting with the basal ES were apparently reduced in flutamide-treated rats. Moreover, the appearance of unconventional circular ES suggests enhanced internalization and degradation of the basal ES. These changes were accompanied by decreased Cx43 and ZO-1 expression (p < 0.01) and a loss of linear distribution of these proteins at the region of the blood-testis barrier. On the other hand, Cx43 expression in the interstitial tissue of flutamide-treated rats increased (p < 0.01), which could be associated with Leydig cell hypertrophy. Concomitantly, both intratesticular testosterone and estradiol concentrations were elevated (p < 0.01), but testosterone to estradiol ratio decreased significantly (p < 0.05) in flutamide-treated rats compared to the controls., Conclusions: Short-term treatment with flutamide applied to adult rats exerts its primary effect on the basal ES, coexisting junctional complexes and their constituent proteins Cx43 and ZO-1, without any apparent morphological alterations in the seminiferous epithelium. In the interstitial compartment, however, short-term exposure leads to both histological and functional changes of the Leydig cells.

Aim: Argirein (rhein-arginine) is a derivative of rhein isolated from Chinese rhubarb (Rheum Officinale Baill.) that exhibits antioxidant and anti-inflammatory activities. In the present study we investigated the effects of argirein on stress-induced (hypergonadotrophic) and diabetic (hypogonadotrophic) hypogonadism in male rats., Methods: Stress-induced and diabetic hypogonadism was induced in male rats via injection of isoproterenol (ISO) or streptozotocin (STZ). ISO-injected rats were treated with argirein (30 mg·kg(-1)·d(-1), po) or testosterone replacement (0.5 mg·kg(-1)·d(-1), sc) for 5 days, and STZ-injected rats were treated with argirein (40-120 mg·kg(-1)·d(-1), po) or aminoguanidine (100 mg·kg(-1)·d(-1), po) for 4 weeks. After the rats were euthanized, blood samples and testes were collected. Serum hormone levels were measured, and the expression of endothelin receptor A (ETA), connexin 43 (Cx43) and other proteins in testes was detected. For in vitro experiments, testis homogenate was prepared from normal male rats, and incubated with ISO (1 μmol/L) or high glucose (27 mmol/L)., Results: ISO injection induced hyper-gonadotrophic hypogonadism characterized by low testosterone and high FSH and LH levels in the serum, whereas STZ injection induced hypogonadotrophic hypogonadism as evidenced by low testosterone and low FSH and LH levels in the serum. In the testes of ISO- and STZ-injected rats, the expression of ETA, MMP-9, NADPH oxidase and pPKCε was significantly increased, and the expression of Cx43 was decreased. Administration of argirein attenuated both the abnormal serum hormone levels and the testis changes in ISO- and STZ-injected rats, and aminoguanidine produced similar actions in STZ-injected rats; testosterone replacement reversed the abnormal serum hormone levels, but did not affect the testis changes in ISO-injected rats. Argirein (0.3-3 μmol/L) exerted similar effects in testis homogenate incubated with ISO or high glucose in vitro., Conclusion: Two types of hypogonadism of male rats exhibit increased expression of ETA and depressed expression of Cx43 in testes, despite different patterns of serum FSH and LH. Argirein alleviates the two types of male hypogonadism via normalizing ETA and Cx43 in testes.

Aim: Atrial fibrosis, one of the most striking features in the pathology of atrial fibrillation (AF), is promoted by local and systemic inflammation. Electrophilic fatty acid nitroalkenes, endogenously generated by both metabolic and inflammatory reactions, are anti-inflammatory mediators that in synthetic form may be useful as drug candidates. Herein we investigate whether an exemplary nitro-fatty acid can limit atrial fibrosis and AF., Methods and Results: Wild-type C57BL6/J mice were treated for 2 weeks with angiotensin II (AngII) and vehicle or nitro-oleic acid (10-nitro-octadec-9-enoic acid, OA-NO2, 6 mg/kg body weight) via subcutaneous osmotic minipumps. OA-NO2 significantly inhibited atrial fibrosis and depressed vulnerability for AF during right atrial electrophysiological stimulation to levels observed for AngII-naive animals. Left atrial epicardial mapping studies demonstrated preservation of conduction homogeneity by OA-NO2. The protection from fibrotic remodelling was mediated by suppression of Smad2-dependent myofibroblast transdifferentiation and inhibition of Nox2-dependent atrial superoxide formation., Conclusion: OA-NO2 potently inhibits atrial fibrosis and subsequent AF. Nitro-fatty acids and possibly other lipid electrophiles thus emerge as potential therapeutic agents for AF, either by increasing endogenous levels through dietary modulation or by administration as synthetic drugs., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

Aims: The progression of pathological left ventricular remodelling leads to cardiac dysfunction and contributes to the occurrence of malignant arrhythmias and sudden cardiac death. The underlying molecular mechanisms remain unclear, however. Our aim was to examine the role of the renin-angiotensin system (RAS) in the mechanism underlying arrhythmogenic cardiac remodelling using a transgenic mouse expressing a cardiac-specific dominant-negative form of neuron-restrictive silencer factor (dnNRSF-Tg). This mouse model exhibits progressive cardiac dysfunction leading to lethal arrhythmias., Methods and Results: Subcutaneous administration of aliskiren, a direct renin inhibitor, significantly suppressed the progression of pathological cardiac remodelling and improved survival among dnNRSF-Tg mice while reducing arrhythmogenicity. Genetic deletion of the angiotensin type 1a receptor (AT1aR) similarly suppressed cardiac remodelling and sudden death. In optical mapping analyses, spontaneous ventricular tachycardia (VT) and fibrillation (VF) initiated by breakthrough-type excitations originating from focal activation sites and maintained by functional re-entry were observed in dnNRSF-Tg hearts. Under constant pacing, dnNRSF-Tg hearts exhibited markedly slowed conduction velocity, which likely contributes to the arrhythmogenic substrate. Aliskiren treatment increased conduction velocity and reduced the incidence of sustained VT. These effects were associated with suppression of cardiac fibrosis and restoration of connexin 43 expression in dnNRSF-Tg ventricles., Conclusion: Renin inhibition or genetic deletion of AT1aR suppresses pathological cardiac remodelling that leads to the generation of substrates maintaining VT/VF and reduces the occurrence of sudden death in dnNRSF-Tg mice. These findings demonstrate the significant contribution of RAS activation to the progression of arrhythmogenic substrates., (Published on behalf of the European Society of Cardiology. All rights reserved. © The Author 2015. For permissions please email: journals.permissions@oup.com.)

Background: Ventricular arrhythmia is an important cause of late death in patients with repaired tetralogy of Fallot (rTOF). By using an rTOF canine model, we investigated the role of repolarization alternans and its electrophysiological mechanisms., Methods and Results: Six dogs received right ventricular outflow tract (RVOT) transannular patch, pulmonary valve destruction, and right bundle branch ablation to simulate rTOF. After 1 year, we performed high-resolution dual-voltage and calcium optical mapping to record action potentials and calcium transients on the excised right ventricular outflow tract wedges. Another 6 dogs without operation served as control. The rTOF group was more susceptible to action potential duration alternans (APD-ALT) and spatially discordant APD-ALT than control (threshold for APD-ALT: 516±36 vs 343±36 ms; P=0.017; threshold for discordant APD-ALT: 387±30 vs 310±14 ms; P=0.046). We detected 2 episodes of ventricular tachycardia in the rTOF group, but none in the control. Expressions of Kv4.3 and KChIP2 decreased in the rTOF group. Expression of connexin 43 also decreased in the rTOF group with a corresponding decrease of conduction velocity and might contribute to spatially discordant APD-ALT. We also found distinct electrophysiological features of the RVOT, including biphasic relationship between magnitude of APD-ALT and pacing cycle length, uncoupling of APD-ALT, and calcium transients alternans, and shortened APD, but unchanged, APD restitution in rTOF., Conclusions: We demonstrated novel electrophysiological properties of the RVOT. In an rTOF model, the RVOT exhibits increased susceptibility to temporal and spatially discordant APD-ALT, which was not totally dependent on calcium transient alternans., (© 2015 The Authors. Published on behalf of the American Heart Association, Inc., by Wiley Blackwell.)

Objective: The purpose of this study was to investigate the expression of A-kinase anchor protein 95 (AKAP95), cell cycle protein E1 (cyclinE1) and D1 (cyclinD1), and gap junction protein connexin 43 (Cx43) in ovarian cancer tissues, the relationship between four proteins and clinicopathologic parameters, and the correlation between these proteins., Methods: The expression of proteins in 54 cases of ovarian cancer tissues was detected by immunohistochemical method., Results: The positive expression rates of AKAP95, cyclinD1 and cyclinE1 in ovarian cancer tissues were 72.22%, 66.67% and 79.63%, respectively, which were higher than that of ovarian pericarcinoma tissues expressing as 33.33%, 25% and 8.30% (P<0.05). The positive expression rate of Cx43 in ovarian cancer tissues was 40.74%, which was lower than that of ovarian pericarcinoma tissues expressing as 75%; respectively, and the difference was statistically significant between groups (P<0.05). The expression of cyclinD1 in ovarian cancer tissues was related to the histologic type (P<0.05) while it showed no correlation with the degree of differentiation (P>0.05). Additionally, the expression of AKAP95, Cx43 and cyclinE1 in ovarian cancer tissues showed no correlation with the degree of differentiation or the histologic type (P>0.05). Protein expressions of AKAP95, Cx43 and cyclinE1 were correlated with each other (P<0.05), and the expressions of cyclinD1, cyclinE1 and Cx43 were also correlated with each other (P<0.05). However, AKAP95 and cyclinD1 showed no correlation (P>0.05)., Conclusion: AKAP95, cyclinD1 and cyclinE1 play an important role in promoting the process of ovarian cancer formation. The tumor inhibitory effects of Cx43 protein on the pathogenesis of ovarian cancer were weakened. The expression of cyclinD1 in ovarian cancer tissues is related to the histologic type while it shows no correlation with the degree of differentiation. Additionally, the expression of AKAP95, Cx43 and cyclinE1 in ovarian cancer tissues shows no correlation with the degree of differentiation or the histologic type. AKAP95 expression is correlated with Cx43 and cyclinE1 expression; Cx43 expression is correlated with AKAP95, cyclinD1 and cyclinE1 expression; cyclinE1 expression is correlated with AKAP95, Cx43, cyclinD1 expression; cyclinD1 expression is correlated with Cx43 and cyclinE1 expression, while AKAP95 and cyclinD1 show no correlation.

Objectives: To evaluate the protein expression of connexin 43 (Cx43) in primary urothelial bladder cancer and test its association with the histopathological characteristics and clinical outcome., Methods: A tissue microarray containing 348 tissue samples from 174 patients with primary urothelial carcinomas of the bladder was immunohistochemically stained for Cx43. The intensity of staining was semiquantitatively evaluated (score 0, 1+, 2+), and the association with clinicopathological features was assessed. Univariable and multivariable analyses were performed to identify predictors for progression-free survival (PFS)., Results: Membranous Cx43 immunoreactivity was detected in 118 (67.8%) of 174 analysable urothelial carcinomas, of which 31 (17.8%) showed even a strong (score 2+) and mainly homogeneous staining. Strong expression levels of Cx43 (score 2+) were associated with higher tumour grade, multiplicity and increased proliferation (all p<0.05). In the subgroup of patients with stage pTa and pT1 bladder tumours (n=158), strong Cx43 expression (p<0.001), solid growth pattern (p<0.001) and increased Ki-67 proliferation fraction (p<0.05) were significantly associated with shorter PFS in an univariable Cox regression analysis. In multivariable Cox regression models, Cx43 immunoreactivity and histological growth pattern remained highly significant and adverse risk factors for PFS., Conclusions: The expression levels of Cx43 are frequent in non-muscle invasive bladder cancer (NMIBC), with high expression levels being associated with poor prognosis. Routine assessment of Cx43 expression may improve the identification of high-risk NMIBC., (Published by the BMJ Publishing Group Limited. For permission to use (where not already granted under a licence) please go to http://group.bmj.com/group/rights-licensing/permissions.)

Objective: To design a biphasic pulsed electrical stimulation (BPES) system for cardiac tissue engineering and investigate the effect of BPES on the cardiomyocyte-like differentiation of adipose-derived stem cells (ADSCs) in vitro., Methods: ADSCs were isolated and cultivated from Sprague Dawley rats. Surface protein expression was detected by flow cytometry to identify the cell phenotype. The multi-differentiation potential of ADSCs was verified by adipogenic and osteogenic inducers. The cells of the third passage were randomly divided into 2 groups: BPES group was subjected to BPES 24 hours after the cells were plated in 6-well culture plates, and BPES was set as 2 ms square pulses delivered at 2 Hz, 2 V voltage amplitude, and lasted 2 hours per day, for 3, 4 or 7 days. The culture medium was replaced every 3 days. Control group was cultured with the same condition but without BPES. The cell morphology was observed under an inverted phase-contrast microscope. Cell proliferation was evaluated by the MTT assay. The expression of cardiac-specific homeobox Nkx-2.5 and connexin 43(CX43) was determined by immunofluorescence cytochemistry., Results: The flow cytometry proved that the isolated cells were ADSCs. Oil red O staining showed the fat droplets in the cytoplasm of ADSCs after adipogenic induction. alkaline phosphatase and Von Kossa staining showed calcium nodes in the ADSCs after osteogenic induction. No obvious changes were found in the proliferation of ADSCs. The levels of Nkx-2.5 and CX43 proteins were significantly higher in the BPES group than in the control group., Conclusion: BPES system we designed for tissue engineering could promote the differentiation of ADSCs into cardiomyocyte-like cells in vitro.

The Sertoli cell (SC)-specific knockout (KO) of connexin43 (Cx43) results in spermatogenic arrest at the level of spermatogonia and/or SC-only syndrome. Histology of the interstitial compartment suggests Leydig cell (LC) hyperplasia. Our aim has been to investigate possible effects of the SC-specific KO of Cx43 (SCCx43KO) on interstitial LC. We therefore counted LC via the optical dissector method (per microliter of testicular tissue and per testis) and found LC to be significantly increased in SCCx43KO(-/-) compared with wild-type mice. Semiquantitative western blot together with Cx43 and 3β-hydroxysteroid dehydrogenase immunohistochemistry showed that Cx43 protein was significantly reduced and barely detectable in LC in adult SCCx43KO(-/-) mice. This reduction of Cx43 protein was accompanied by a reduction of Cx43 mRNA as analyzed by laser-assisted microdissection of interstitial cells and subsequent quantitative real-time polymerase chain reaction (PCR). Interestingly, Cx45, another recently detected connexin in LC, was also downregulated. Preliminary qualitative data of LC differentiation markers (Thb2, Hsd3b6) and a steroidogenic marker (Hsd17b3) obtained by reverse transcription plus PCR revealed no obvious differences. Thus, the loss of Cx43 in SC also provokes the downregulation of connexins in interstitial LC at the transcriptional and translational levels. Moreover, SCCx43KO leads to alterations in LC numbers. Despite these alterations, steroidogenesis seems not to be impaired. Further studies, including ultrastructural analysis of the tissue as well as quantitative examination of additional LC markers and testosterone, and functional in vitro experiments, should provide more information about LC differentiation and function in SCCx43KO(-/-) mice.

Aims: The objective of this study was to explore the potential involvement of connexin43 (Cx43) and connexin32 (Cx32), two vital members of the connexin families, in the pathogenesis of keratocystic odontogenic tumours (KCOT)., Methods and Results: The expression levels of Cx43 and Cx32 in human KCOT and normal oral mucosa (OM) tissues were measured using immunohistochemistry and real-time quantitative polymerase chain reaction (qPCR). The relationship between Cx43 and Cx32 expression and markers of proliferation [proliferating cell nuclear antigen (PCNA), cyclin D1], anti-apoptosis [B cell lymphoma 2 (Bcl-2)] and autophagy [light chain 3 (LC3), Sequestosome 1 p62 (p62)] was then investigated in the KCOT samples. The results showed that Cx43 and Cx32 expression was down-regulated significantly in KCOT samples relative to OM samples. Meanwhile, the expression levels of Cx43 and Cx32 were correlated negatively with the expression levels of PCNA, cyclin D1, Bcl-2, LC3 and p62, as confirmed further by double-labelling immunofluorescence analyses., Conclusions: This study reveals for the first time that Cx43 and Cx32 are down-regulated in KCOT and suggests an association with growth regulation, anti-apoptosis and autophagy in KCOT., (© 2014 John Wiley & Sons Ltd.)

Erythropoietin (EPO) has protective effects on many neurological diseases, including cerebral ischemia. Here, we aimed to test EPO's effects on ischemic neurovascular unit (NVU) injuries and examine whether the effects were dependent on connexin43 (Cx43) mediated gap junctional intercellular communication (GJIC). We detected the expression of Cx43 and phosphorylation of Cx43 (p-Cx43) at 1 d, 3 d, and 7 d after middle cerebral artery occlusion (MCAO). Meanwhile, we examined the effects of EPO on NVU injuries including neuronal survival, astrocyte activation and regeneration of endothelial cells as well as whether the effects were Cx43 dependent by using multiple inhibitors. We found EPO highly increased p-Cx43, but not total Cx43 at all chosen times. Importantly, EPO led to neurological and blood-brain barrier functions improvement by associating with promotion of angiogenesis as well as reduction of neuronal death, astrocyte activation and neurotoxic substances levels. Moreover, these effects were significantly weakened by the inhibitors blocking GJIC, Cx43 communicative function, phosphorylation and expression, only Cx43 redistribution inhibitor excluded. Our data suggest the protective effects of EPO on NUV injuries are highly associated with the increase of p-Cx43, which improves GJIC to reduce neurotoxic substances., (Copyright © 2015 Elsevier Inc. All rights reserved.)

Background: The biologic factors associated with shoulder osteoarthritis (OA) have not been elucidated. The purpose of this study was to investigate osteoarthritic biomarkers of the shoulder. To our knowledge, this is the first study to analyze shoulder cartilage for OA-associated genes and to examine human shoulder cartilage for a possible biomarker, connexin 43 (Cx43)., Materials and Methods: Cartilage from 16 osteoarthritic and 10 nonosteoarthritic humeral heads was assessed for expression of the following genes by real-time polymerase chain reaction: types I, II, and X collagen; matrix metalloproteinases (MMPs); tissue inhibitors of MMP (TIMPs); interleukins; versican; cyclooxygenase 2 (Cox-2); inducible nitric oxide synthase (iNOS); tumor necrosis factor α (TNF-α); aggrecanase 2 (ADAMTS5); and Cx43., Results: In osteoarthritic shoulders, Cx43, Cox-2, versican, collagen type I, ADAMTS5, MMP-3, and TNF-α expressions were significantly increased compared with controls. TIMP-3 and iNOS trended toward significance, with robust expression in osteoarthritic shoulders and low expression in nonosteoarthritic shoulders. In osteoarthritic shoulders, gene expression of Cx43, ADAMTS5, collagen type I, Cox-2, versican, and TIMP-3 showed predominance (85-, 33-, 13-, 12-, 11.5-, and 3-fold increases, respectively) relative to nonosteoarthritic controls. Spearman correlation analysis showed significant correlations between Cx43 and collagen (types I, II, and X), MMP-9, TIMP-2 and TIMP-3, versican, Cox-2, iNOS, and ADAMTS5., Conclusions: Certain genes are markedly upregulated in osteoarthritic shoulders compared with nonosteoarthritic shoulders, with Cx43, Cox-2, versican, collagen type I, ADAMTS5, MMP-3, and TNF-α expression being significantly increased. These genes might be useful biomarkers for examining shoulder OA., Clinical Relevance: Identification of osteoarthritic biomarkers can help us better understand shoulder OA and build the foundation for future research on disease progression and treatments., (Copyright © 2015 Journal of Shoulder and Elbow Surgery Board of Trustees. Published by Elsevier Inc. All rights reserved.)

Objective: To explore the expression of A-kinase anchor protein 95 (AKAP95), Cyclin D1, Cyclin E1, and Connexin43 (Cx43) in rectal cancer tissues and assess the associations between each of the proteins and pathological parameters, as well as their inter-relationships., Methods: AKAP95, Cyclin D1, Cyclin E1, and Cx43 protein expression rates were evaluated by immunohistochemistry in 50 rectal cancer specimens and 16 pericarcinoma tissues., Results: The positive rates of AKAP95, Cyclin E1, and Cyclin D1 proteins were 54.00 vs. 18.75%, 62.00 vs. 6.25%, and 72.00 vs. 31.25% in rectal cancer specimens and pericarcinoma tissues, respectively, representing statistically significant differences (P < 0.05). The positive rate of Cx43 protein expression in rectal cancer tissues was 44.00% and 62.50% in pericarcinoma tissues, and the difference between them was not significant (P > 0.05). No significant associations were found between protein expression of AKAP95, Cyclin E1, Cyclin D1, and Cx43, and the degree of differentiation, histological type, and lymph node metastasis of rectal cancer (P > 0.05). However, significant correlations were obtained between the expression rates of AKAP95 and Cyclin E1, Cyclin E1 and Cyclin D1, Cyclin E1 and Cx43 protein, and Cyclin D1 and Cx43, respectively (P < 0.05)., Conclusion: AKAP95, Cyclin E1, and Cyclin D1 protein expression rates were significantly higher in rectal cancer tissues compared with pericarcinoma samples, suggesting an association between these proteins and the development and progression of rectal cancer. In addition, the significant correlations between the proteins (AKAP95 and Cyclin E1, Cyclin E1 and Cyclin D1, Cyclin E1 and Cx43 protein, and Cyclin D1 and Cx43) indicate the possible synergistic effects of these factors in the development and progression of rectal cancer.

The use of carbamazepine (CBZ) during pregnancy increases cardiovascular anomalies. In this study CBZ developmental cardiotoxic effects were evaluated using chick cardiomyocyte micromass (MM) culture and mouse embryonic stem cells derived cardiomyocyte (ESDC) systems. In MM culture, CBZ only inhibited the cardiomyocyte contractile activity, while in ESDC it completely ceased the contractile activity at 200 μM with decreased cell viability and protein content. The antioxidant superoxide dismutase (SOD) supplement in MM and ascorbic acid (AA) in ESDC showed protective effects on CBZ toxicity, but elevated levels of reactive oxygen species (ROS) production were recorded with CBZ treatment only in ESDC. CBZ has also affected cardiac connexin 43 expression in both in vitro systems. Our results indicated CBZ induced ROS stress as mechanism of developmental cardiotoxicity at early stage of cardiogenesis in ESDC system compared to MM system's differentiated cells. These toxic effects can be negated by using antioxidant agent., (Copyright © 2014 Elsevier Inc. All rights reserved.)