Connexin Research Articles

Abstract NT-095: ERADICATION OF CHEMORESISTANT METASTATIC OVARIAN CANCER WITH IMMUNOTARGETED EPIGENOMIC NANOSURGERY (IEN)

Abstract INTRODUCTION: Resistance to conventional chemotherapy such as cisplatin, and radiotherapy in ovarian cancer may be caused by deregulated oncogenic miRNA-221/222 inhibiting induction of apoptosis. METHODS: Chemoradioresistant cells overexpressing EphA2 were obtained for developing an ovarian metastatic cancer model, which was treated with anti-EphA2 MAbs immunotargeted pegylated nanosomes with encapsulated paired guide-RNAs targeting 3' and 5' of the precursor microRNA 221/222. RESULTS: Post-treatment, the paired guide-RNAs caused deletions in the targeted pre-microRNA 221/222 genomic-sequences removing the stem-loop hairpins after blockade of RNase-III Drosha and DGCR8 (microprocessor-complex), subsequent transport by exportin-5/RanGTP and cleavage by Dicer inhibiting miRNA221/222 biogenesis, and miRNP circumventing their incorporation in RISC, and subsequent mRNA cleavage of coding genes regulating according to NGS the PTEN, GJA1, SEMA3B, TIMP3, PTPRM, CDKN1C, MGMT, CDKN1B, PUMA, connexin-43 (CX-43), c-Jun, MMP, NF-kB, PI3K, p27/Kip1, PTPμ, NIAP, ICAM-1, STAT1, STAT2, STAT3/Akt, CDK4, CDKN1C/p57, p53, and p57 inhibiting metastatic ovarian cancer cell proliferation, chemo/radioresistance, migration, angiogenesis, invasion, metastasis and maintenance of MET expressing ovarian metastatic cancer stem cells, while inducing cytotoxic immune responses (CTLs), and D2 irreversible stage of apoptosis or programmed cell death type-I leading to a bystander killing effect (BKE) due to phagocytosis of adjacent tumor cells according to transmission electron micrographs. CONCLUSION: Immunotargeted epigenomic nanosurgery may eradicate metastatic ovarian cancer after inhibition of oncogenic miRNA 221/222 expression, and downstream signaling pathways involving interactions with coding RNA genes leading to apoptosis or PCD type I and BKE after the circumvention of chemo/radioresistant mechanisms. Citation Format: John N. Giannios. ERADICATION OF CHEMORESISTANT METASTATIC OVARIAN CANCER WITH IMMUNOTARGETED EPIGENOMIC NANOSURGERY (IEN) [abstract]. In: Proceedings of the 12th Biennial Ovarian Cancer Research Symposium; Sep 13-15, 2018; Seattle, WA. Philadelphia (PA): AACR; Clin Cancer Res 2019;25(22 Suppl):Abstract nr NT-095.

Tumor-suppressor microRNA-139-5p restrains bladder cancer cell line ECV-304 properties via targeting Connexin 43.

Background:In our previous paper, we demonstrated that Connexin 43 (CX43) was highly expressed in bladder cancer (BC) tissues. But the molecular mechanism about microRNAs (miRNAs) regulation upstream of CX43 in BC has not been well elucidated and remains to be further studied. MicroRNA-139-5p (miR-139-5p) is a tumor suppressor in progression of multifarious cancers including BC. Nevertheless, the underlying mechanisms of CX43/miR-139-5p in tumorigenesis of BC are still not well illustrated. The specific objective of our study was to inquiry the effect of CX43/miR-139-5p on BC progression and its underlying mechanism.Methods:The bioinformatics analysis softwares were applied to predict the miRNAs in the upstream of CX43. First, the expression levels of miR-139-5p in BC tissues (tumor) and paracancer tissues (normal) were investigated using the data from The Cancer Genome Atlas database. Quantitative reverse transcription-polymerase chain reaction (qRT-PCR) was used to detect the mRNA expression level of miR-139-5p in three human BC cell lines 5637, T24, ECV-304 and a human bladder epithelial immortalized cell line SV-HUC-1 (normal control). Then si-CX43, si-control, miR-139-5p mimic, and its negative control (NC) were transfected into BC cell line ECV-304. The relationship of miR-139-5p and CX43 was analyzed by dual-luciferase reporter assay. The qRT-PCR and Western blotting were used to test the mRNA and protein expression level of CX43. The proliferation of ECV-304 and T24 cells were examined by cell counting kit-8. The migration and invasion of ECV-304 cells were tested by transwell assay. To determine whether miR-139-5p would affect cell proliferation, migration and invasion by targeting CX43, we executed the rescue assay. The comparison between two groups was analyzed by Student's t test, and comparisons among multiple samples were performed by one-way analysis of variance and a Bonferroni post hoc test.Results:The expression of miR-139-5p was remarkably down-regulated in BC tissues (tumor vs. normal, 2.286 ± 0.017 vs. 3.211 ± 0.034, t = 11.540, P < 0.0001) and cell lines (P < 0.01 in all BC cell lines). Besides, we also indicated that over-expression of miR-139-5p reduced the proliferation of ECV-304 (P = 0.001) and T24 cells (P = 0.005). Moreover, miR-139-5p over-expression weakened the invasion (P = 0.001) and migration (P = 0.001) of ECV-304 cells. Furthermore, the relative luciferase activity of CX43-wild type construct was distinctly lessened by up-regulation of miR-139-5p (miR-139-5p mimic NC vs. miR-139-5p mimic, 0.916 ± 0.063 vs. 0.356 ± 0.048, t = 7.085, P = 0.002), nevertheless the activity of CX43-mutant type construct was untouched (miR-139-5p mimic NC vs. miR-139-5p mimic, 0.918 ± 0.057 vs. 0.878 ± 0.039, t = 0.577, P = 0.595). Finally, the rescue assay revealed that CX43 deletion enhanced the depressor effect of miR-139-5p on ECV-304 cell proliferation (P < 0.01), invasion (P = 0.028), and migration (P = 0.014).Conclusion:MiR-139-5p, as a tumor-suppressor, repressed cell proliferation, invasion, and migration in BC, which might be achieved by regulating CX43.

Effect of sevoflurane on phosphorylation of connexin 43 at ser368 in ventricular myocardium in isolated hearts of diabetic rats

Objective To evaluate the effect of sevoflurane on phosphorylation of connexin 43 (Cx43) at Ser368 (Cx43 Ser368) in ventricular myocardium in isolated hearts of diabetic rats. Methods Twenty-four clean-grade healthy adult male Sprague-Dawley rats, aged 3 months, weighing 180-220 g, were used in this study.The diabetic model was established by intraperitoneally injecting streptozotocin 60 mg/kg.The hearts were rapidly excised and retrogradely perfused with K-H solution saturated with 95% O2-5% CO2 at 37 ℃ in a Langendorff apparatus.Sixteen Langendorff-perfused diabetic hearts were divided into 3 groups (n=8 each) using a random number table method: diabetes mellitus group (group D) and sevoflurane group (group DS). Another 8 Langendorff-perfused normal hearts of rats were selected and served as control group (group C). After 15 min equilibration with K-H solution, the hearts were continuously perfused for 30 min with K-H solution in group C and group D and with K-H solution saturated with 2.5% sevoflurane in group DS.S1S2 program-controlled stimulation was performed at the end of perfusion, the occurrence of induced ventricular arrhythmia (VA) and the maximal pacing cycle length (PCL) of induced VA were recorded, and conduction velocity (CV) was calculated.The expression of phosphorylated Cx43 at Ser368 (p-Cx43 Ser368) in ventricular myocytes was determined by Western blot. Results Compared with group C, the incidence of induced VA was significantly increased, the maximal PCL of induced VA was prolonged, the CV was decreased, and the expression of p-Cx43 Ser368 was up-regulated in group D (P<0.05). Compared with the incidence of induced VA was significantly decreased, the maximal PCL of induced VA was shortened, the CV was increased, and the expression of p-Cx43 Ser368 was down-regulated in group DS (P<0.05). Conclusion The mechanism by which sevoflurane stabilizes the ventricular electrical conduction is associated with decreasing the phosphorylation of Cx43 at Ser368 in diabetic rats. Key words: Anesthetics, inhalation; Diabetes mellitus; Connexins

Hypoxia‑induced internalization of connexin 26 and connexin43 in pulmonary epithelial cells is involved in the occurrence of non‑small cell lung cancer via the P53/MDM2 signaling pathway.

Reports have highlighted an association between connexins (CXs) or gap junction proteins and non-small cell lung cancer (NSCLC). In the present study, it was aimed to elucidate the regulatory mechanism of CX26 and CX43 under hypoxic conditions in NSCLC. Clinical samples were collected for analysis of CX26 and CX43 expression and clinical cancerization followed by quantification of CX26 and CX43 expression. Following the establishment of an in vitro hypoxia model, P53/murine double minute-2 (MDM2) signaling pathway-, proliferation- and epithelial-mesenchymal transition (EMT)-related genes were quantified to evaluate the influence of CX26 and CX43 on the biological functions of pulmonary epithelial cells in NSCLC. In addition, the proliferation and tumorigenicity of cancer cells were assessed by EdU staining and xenograft tumors, respectively. Decreased expression of CX26 and CX43 was found in cancer tissues compared with surrounding normal tissue. Hypoxia was shown to activate the P53/MDM2 axis and stimulate the downregulation, ubiquitination and degradation of CX26 and CX43, which were translocated from the membrane to the cytoplasm. Low levels of CX26 and CX43 were demonstrated to further promote EMT and the induction of the proliferation and tumorigenicity of cancer cells. These results were reflected by decreased E-cadherin expression and increased N-cadherin expression, along with increased cell migration, promoted cell proliferation ability and elevated relative protein expression of Oct4 and Nanog, and accelerated tumor growth, accompanied by a higher number of metastatic nodes. Taken together, the key observations of the present study demonstrate that the internalization of CX26 and CX43 promoted proliferation, EMT and migration and thus induced NSCLC via aberrant activation of the P53/MDM2 signaling pathway under hypoxic conditions.

Role of NL-1 in trafficking of GluR1-containing AMPA receptor to cell membrane in spinal cord dorsal horn during remifentanil-induced hyperalgesia in mice with incisional pain

Objective To evaluate the role of neuroligin 1 (NL-1) in trafficking of GluR1-containing AMPA receptor to cell membrane in spinal cord dorsal horns during remifentanil-induced hyperalgesia in mice with incisional pain. Methods Forty SPF healthy male C57BL/6J mice, aged 8-10 weeks, weighing 18-22 g, were divided into 5 groups (n=8 each) using a random number table method: control group (group C), NL1-shRNA plasmid group (group NL), incisional pain plus remifentanil group (group I+ R), incisional pain plus remifentanil plus blank vector group (group I+ R+ B), and incisional pain plus remifentanil plus NL-1-shRNA plasmid group (group I+ R+ NL). Negative lentivirus was intrathecally injected in group I+ R+ B.In NL and I+ R+ NL groups, 10 μl NL-1-shRNA lentivirus at 1×108 IFU/ml was intrathecally injected once a day for 3 consecutive days.Normal saline 10 μl was intrathecally injected at the same time point in C and I+ R groups.After transfection was stable, normal saline 0.1 ml was injected via the caudal vein for 4 consecutive times at 15 min intervals in C and NL groups.In I+ R, I+ R+ B and I+ R+ NL groups, 0.1 ml remifentanil 10 μg/kg was injected via the caudal vein for 4 consecutive times at 15 min intervals, and the model of incisional pain was established after the first administration.The mechanical paw withdrawal threshold (MWT) and tail-flick latency (TFL) were measured at 24 h before normal saline or remifentanil administration (T0) and at 3, 6, 24 and 48 h after the end of administration (T1-4). The animals were sacrificed after measurement of pain threshold at T4, and L4-6 segments of the spinal dorsal horn were then collected for determination of the expression of NL-1 protein and mRNA and AMPA receptors, and the ratio of AMPA receptor expression in the membrane protein to that in the total protein (m/t ratio) was calculated. Results Compared with group C, the MWT was significantly decreased, and TFL was shortened at T1-4, the expression of NL-1 protein and mRNA and GluR1-containing AMPA receptors in membrane and total proteins was up-regulated, and m/t ratio was increased in I+ R and I+ R+ B groups (P<0.05). Compared with I+ R and I+ R+ B groups, the MWT was significantly increased and TFL was prolonged at T1-4, the expression of NL-1 protein and mRNA and GluR1-containing AMPA receptors in membrane and total proteins was down-regulated, and m/t ratio was decreased in group I+ R+ NL (P<0.05). Conclusion NL-1 in spinal cord dorsal horns can promote the trafficking of GluR1-containing AMPA receptors to cell membrane, which is involved in the development and maintenance of remifentanil-induced hyperalgesia in mice with incisional pain. Key words: Connexins; Pain, postopertive; Piperidines; Hyperalgesia; Spinal cord dorsal horn; Receptors, glutamate; Receptors, AMPA

The prevalence of common genetic deafness related mutation detected by microarray and follow-up in pregnant women in Beijing

Objective To determine the distribution of common deafness gene mutations by microarray-PCR in pregnant women of Beijing, then explore their prevalence and clinical significance. Methods Totally 1 709 pregnant women were prospectively enrolled from the outpatients of Obstetrical Clinical from Peking University Peoples′ Hospital in this study from June 2016 to April 2018. Peripheral blood samples were obtained and DNA templates were extracted from all subjects. The coding region of the GJB2 gene(35del G, 235delC, 176-191del16, 299-300delAT), SLC26A4 gene (IVS7-2 A>G,2168A>G) , 12sRNA gene (1494C>T,1555A>G) and GJB3 gene (538 C>T) were detected by microarray-PCR. Meanwhile, 256 cases firstly recruited in this study were confirmed by Sanger sequencing. The analysis was performed to explore the distribution of deafness gene mutations and the subjects detected to be positive were further followed up until they had given birth to their babies. Results Among 1 709 pregnant women, 89 cases were found to be carrying at least one mutation sites(5.21%).Among them,83 cases were heterozygous mutation(35 cases of GJB2 235delC, 12 cases of GJB2 299-300delAT mutation, one case of GJB2 176-191del 16, 30 cases of SLC26A4 IVS 7-2 A>G, 3 cases of SLC26A4 2168 A>G and two cases of GJB3 538 C>T). There were one case of 235delC homozygous mutation and 2 cases of double mutations(IVS7 -2 A>G/GJB2 299-300delAT, IVS7 -2 A>G/GJB2 235delC). The positive rate of 235delC, 299-300delAT and 176-191del16 was 2.17%, 0.76% and 0.06%, respectively. As to SLC26A4, 1.87% of the pregnant women were carrying IVS 7-2 A>G and 0.18% for 2168 A>G. Two cases were detected carrying GJB3 C>T mutation and three for 12s RNA1555 A>G mutation, respectively. The results of microarray-PCR were identical to those of Sanger sequencing, with the coincidence of 100%.26 spouses of the 56 cases were followed up. Two proved to be carrier of the same gene mutation. One of their babies was born with normal hearing until the end of this research, while another baby was born deafness and implant cochlear when three month old. Conclusions GJB2 235delC and SLC26A4 IVS 7-2 A>G were the most prevalent mutations in pregnant women of Beijing, and it may provide guidance to eugenics and early clinical intervention. Key words: Microarray analysis; Deafness; Connexins; Sulfate transporters; Mutation; Sequence analysis, DNA; Follow-up studies

Progress in research on connexins in diabetic retinopathy

Diabetic retinopathy is the most common microvascular complication of diabetes. It is one of the major blinding diseases in the world. The intercellular communication function of connexins plays a key role in maintaining retinal microvascular homeostasis. The connexins involve in the development of diabetic retinopathy. This article reviews the role of connexins in the pathogenesis of diabetic retinopathy. Key words: Connexin; Gap junction intercellular communication; Diabetic retinopathy

The expression and clinical significance of epithelial cell adhesion molecule and Claudin18 in gastric cancer

Objective To investigate the expression and clinical significance of epithelial cell adhesion molecule(EpCAM)and Claudin-18 in gastric cancer. Methods Surgical specimens of gastric cancer were taken from 64 patients.The histological diagnostic criteria were based on WHO standards.Immunohistochemical staining was used to detect expression levels of EpCAM and Claudin-18 in all samples.The relationship between the expression of EpCAM and Claudin-18 and clinicopathological parameters of gastric cancer was analyzed.Correlations of the prognosis of gastric cancer patients with EpCAM and Claudin-18 expression were analyzed using the Cox proportional hazards regression model. Results The positive expression rate of EpCAM was elevated with the increase in tumor size and the occurrence of distant metastasis(χ2=4.526 and 36.090, P=0.033 and 0.000). There were significant differences in Claudin-18 protein expression among different age, TNM stage, growth pattern, depth of invasion and distant metastasis patients(all P 0.05). Conclusions EpCAM and Claudin-18 are closely related to the invasiveness of gastric cancer, but abnormally high levels of EpCAM and Claudin-18 are not independent risk factors for the prognosis of gastric cancer patients. Key words: Stomach neoplasms; Cell adhesion molecules; Connexins

Advances in corneal endothelial-to-mesenchymal transition

Corneal endothelial cells (CECs) are essential for maintenance of corneal transparency.Proliferative potency of human CECs is poor in vivo, so endothelial dysfunction is irreversible.Many studies have showed that CECs are able to proliferate in vitro.However, endothelial to mesenchymal transition (EndMT) will emerge with CECs acquire a myofibroblastic phenotype and lose their specific markers, which hinders the development of corneal regenerative medicine.EndMT participates in corneal disorders, but its process has not been clarified yet.Now, researchers have found that the mechanism of EndMT may include transforming growth factor-β (TGF-β) signaling pathway, fibroblast growth factors (FGFs), and Notch signaling pathway.Therefore, there are several ways to prevent EndMT, such as inhibiting TGF-β signaling pathway, using anti-phosphatidylinositol 3-kinase antibody, small interfering RNA (siRNA) against p120, Leucine-rich repeat-containing G protein-coupled receptor 5 (LGR5) and LGR5 ligand, inhibiting Notch signaling pathway, making connexin43 knockdown, inhibiting matrix metalloproteinase activity and using Asc-2P.This review elaborated the mechanism of EndMT and ways to prevent or reverse it. Key words: Corneal endothelial cells; Endothelial to mesenchymal transition; Mechanics; Inhibition; Reverse

MiR‐382‐3p suppressed IL‐1β induced inflammatory response of chondrocytes via the TLR4/MyD88/NF‐κB signaling pathway by directly targeting CX43

miR-382-3p has been reported to be upregulated in synovial membrane in knee osteoarthritis (OA). Nevertheless, its role in OA remains largely unknown. The aim of this study was to investigate the specific function and mechanisms of miR-382-3p in the course of OA. In this study, human OA chondrocytes were pretreated with interleukin-1β (IL-1β) at 5 ng/ml for 12 hr to stimulate inflammatory response and matrix metalloproteinases (MMPs) expression in chondrocytes. Meanwhile, miR-382-3p was downregulated in IL-1β-stimulated chondrocytes. In addition, we found that miR-382-3p directly interacts with connexin 43 (CX43) and attenuates the increase of cytochrome c oxidase polypeptide II, inducible nitric oxide synthase, and MMP-1/13 that is induced by IL-1β. Furthermore, our observations indicated that miR-382-3p inhibited the expression of Toll-like receptor 4 (TLR4), Myeloid differentiation primary response 88 (MyD88) and nuclear factor κB (NF-κB) in IL-1β-stimulated chondrocytes, while CX43 overexpression could partly reverse these decreases. In conclusion, miR-382-3p participated in OA may through the TLR4/MyD88/NF-κB signaling pathway by directly targeting CX43.

Quantitative proteomics analysis of differentially expressed proteins in patients with sarco-osteoporosis and osteoporosis

Objective The differential expression of protein mass spectrum of patients with sarco-osteoporosis (SO) and osteoporosis (OP) was constructed to reveal the molecular mechanism of sarcopenia that facilitates the development of osteoporosis. Methods The samples of muscle tissues of patients with sarco-osteoporosis and osteoporosis were collected, and label free quantitative proteomic technique was used to analyze and identify the differentially expressed proteins between the two groups, and differential expressed proteins were analyzed by Gene Ontology (GO), Kyoto Encyclopedia of Genes and Genomes (KEGG). Results There were 1 507 differentially expressed proteins identified. Among them, 53 were significantly dysregulated, including 47 down-regulated proteins and 6 up-regulated ones. The GO analysis showed that differentially expressed proteins in SO group were mainly involved in the redox process, protein hydrolysis and metabolic process, and the main molecular functions were protein binding, adenosine triphosphate (ATP) binding and calcium binding. The most significant cellular structural difference between the SO and OP groups was total components located in the membrane. Based on KEGG pathway analysis, connexin 43 (Cx43), voltage dependent L-type calcium channel (VLCC), superoxide dismutase (SOD) were screened as candidate proteins which may be involved in the mechanism of bone remodeling of osteoporosis. Conclusion Cx43, VLCC, SOD may be associated with bone loss in patients with sarcopenia. Key words: Sarco-osteoporosis; Osteoporosis; Label-free quantitative proteomics; Bioinformatics analysis

Neuregulin-1 protects cardiac electrical conduction through downregulating matrix metalloproteinase-9 and upregulating connexin 43 in a rat myocardial infarction model.

Aims: Neuregulin-1 (NRG-1) is a member of the epidermal growth factor family, and has an important role in cardiomyocyte development and myocardial regeneration. The aim of this study was to determine the protective effect of NRG-1 on cardiac electrical conduction in a rat myocardial infarction (MI) model. Methods: Thirty-three adult male SPF SD rats were randomized into three groups: sham-operated (n=9), acute myocardial infarction (AMI, n=12), and the NRG-1-treated (NRG-1, n=12) groups. All rats were sacrificed on day 8 after inducing MI. The 6-lead electrocardiograms (ECG) were recorded pre-operatively and eight days after operation, and analyzed. The expression levels of matrix metalloproteinase (MMP)-9 and gap junction protein connexin 43 (Cx43) in the infarcted myocardium were measured by Western blotting, and its in-situ distribution was evaluated using immunohistochemistry. Results: The PR, QRS and QT intervals were significantly prolonged in the AMI group compared to the sham operated animals (P<0.05, P<0.01 and P<0.01 respectively), and the PR and QRS intervals were partially restored in the NRG-1-treated rats (P<0.01 and P<0.01 compared to AMI group). Similarly, the increased levels of MMP-9 in the AMI group was restored upon NRG-1 treatment. The myocardial expression of Cx43 was decreased significantly in the AMI group, and was upregulated by NRG-1 treatment. Conclusions: NRG-1 attenuates MI-induced dysfunctional cardiac electrical conduction by downregulating MMP-9 and upregulating Cx43.

Changes in expression of Cx45 and Cx40 in sinoatrial node during dexmedetomidine-induced sinus bradycardia in rats

Objective To evaluate the changes in the expression of Cx45 and Cx40 in the sinoatrial node during dexmedetomidine-induced sinus bradycardia in rats. Methods Forty healthy Sprague-Dawley rats, weighing 250-300 g, were divided into 5 groups (n=8 each) using a random number table method: control group (group C), low-dose dexmedetomidine group (group D1), high-dose dexmedetomidine group (group D2), low-dose dexmedetomidine plus atropin group (group D1A), and high-dose dexmedetomidine plus atropin group (group D2A). Normal saline was intravenously infused in group C. Dexmedetomidine was intravenously infused for 10 min as a loading dose of 20 and 120 μg/kg, followed by an infusion of 10 and 60 μg·kg-1·h-1 for 110 min in D1 and D2 groups, respectively.In D1A and D2A groups, dexmedetomidine was correspondingly given according the method previously described in D1 and D2 groups, and in addition atropin 0.5 mg was intravenously injected at the end of infusing the loading dose of dexmedetomidine. Heart rate(HR), mean arterial pressure(MAP) and SpO2 were recorded before giving dexmedetomidine and at 10, 60 and 120 min after giving dexmedetomidine, and the development of bradycardia was recorded.The sinoatrial node tissues were obtained at the end of administration for determination of the expression of Cx45 and Cx40 protein and mRNA by Western blot and real-time polymerase chain reaction, respectively. Results The incidence of bradycardia was 100% in D1 and D2 groups and 0 after using atropin in D1A and D2A groups.Compared with group C, HR and MAP were significantly decreased in the other four groups, the expression of Cx45 protein and mRNA was up-regulated, the expression of Cx40 protein and mRNA was down-regulated in D1 and D2 groups (P 0.05). Compared with group D1, HR and MAP were significantly increased, the expression of Cx45 protein and mRNA was down-regulated, and the expression of Cx40 protein and mRNA was up-regulated in group D1A (P<0.05). Compared with group D2, HR and MAP were significantly increased, the expression of Cx45 protein and mRNA was down-regulated, and the expression of Cx40 protein and mRNA was up-regulated in group D2A (P<0.05). Conclusion The mechanism by which dexmedetomidine induces sinus bradycardia may be related to up-regulated expression of Cx45 and down-regulated expression of Cx40, and the autonomic nervous activity is involved in dexmedetomidine-induced regulation of Cx45 and Cx40 expression in rats. Key words: Dexmedetomidine; Bradycardia; Sinus node; Connexins

The Use of Foxa2-Overexpressing Adipose Tissue-Derived Stem Cells in a Scaffold System Attenuates Acute Liver Injury.

Background/AimsFor the clinical application of stem cell therapy, functional enhancement is needed to increase the survival rate and the engraftment rate. The purpose of this study was to investigate functional enhancement of the paracrine effect using stem cells and hepatocyte-like cells and to minimize stem cell homing by using a scaffold system in a liver disease model.MethodsA microporator was used to overexpress Foxa2 in adipose tissue-derived stem cells (ADSCs), which were cultured in a poly(lactic-co-glycolic acid) (PLGA) scaffold. Later, the ADSCs were cultured in hepatic differentiation medium for 2 weeks by a 3-step method. For in vivo experiments, Foxa2-overexpressing ADSCs were loaded in the scaffold, cultured in hepatic differentiation medium and later were implanted in the dorsa of nude mice subjected to acute liver injury (thioacetamide intraperitoneal injection).ResultsFoxa2-overexpressing ADSCs showed greater increases in hepatocyte-specific gene markers (alpha fetoprotein [AFP], cytokeratin 18 [CK18], and albumin), cytoplasmic glycogen storage, and cytochrome P450 expression than cells that underwent the conventional differentiation method. In vivo experiments using the nude mouse model showed that 2 weeks after scaffold implantation, the mRNA expression of AFP, CK18, dipeptidyl peptidase 4 (CD26), and connexin 32 (CX32) was higher in the Foxa2-overexpressing ADSCs group than in the ADSCs group. The Foxa2-overexpressing ADSCs scaffold treatment group showed attenuated liver injury without stem cell homing in the thioacetamide-induced acute liver injury model.ConclusionsFoxa2-overexpressing ADSCs applied in a scaffold system enhanced hepatocyte-like differentiation and attenuated acute liver damage in an acute liver injury model without homing effects.

Connexin 26 and 43 play a role in regulating proinflammatory events in the epidermis.

Dysregulation of Connexin (CX) expression and function is associated with a range of chronic inflammatory conditions including psoriasis and nonhealing wounds. To mimic a proinflammatory environment, HaCaT cells, a model human keratinocyte cell line, were challenged with 10 µg/ml peptidoglycan (PGN) isolated from Staphylococcus aureus for 15 min to 24 hr in the presence or absence of CX blockers and/or following CX26, CX43, PANX1 and TLR2 small interfering RNA (siRNA) knockdown (KD). Expression levels of IL-6, IL-8, CX26, CX43, PANX1, TLR2 and Ki67 were assessed by quantitative real-time polymerase chain reaction, western blot analysis and/or immunocytochemistry. Nuclear factor kappa β (NF-κβ) was blocked with BAY 11-7082, CX-channel function was determined by adenosine 5'-triphosphate (ATP) release assays. Enzyme-linked immunosorbent assay monitored IL6 release following PGN challenge in the presence or absence of siRNA or blockers of CX or purinergic signalling. Exposure to PGN induced IL-6, IL-8, CX26 and TLR2 gene expression but it did not influence CX43, PANX1 or Ki67 messenger RNA expression levels. CX43 protein levels were reduced following 24 hr PGN exposure. PGN-induced CX26 and IL-6 expression were also aborted by TLR2-KD and inhibition of NF-κβ. ATP and IL-6 release were stimulated following 15 min and 1-24 hr challenge with PGN, respectively. Release of both agents was inhibited by coincubation with CX-channel blockers, CX26-, CX43- and TLR2-KD. The IL-6 response was also reduced by purinergic blockers. CX-signalling plays a role in the innate immune response in the epidermis. PGN is detected by TLR2, which via NF-κβ, directly activates CX26 and IL-6 expression. CX43 and CX26 maintain proinflammatory signalling by permitting ATP release, however, PANX1 does not participate.

Lipopolysaccharide-induced redistribution of myocardial connexin43 is associated with increased macrophage infiltration in both normotensive and spontaneously hypertensive rats.

We investigated whether changes in gap junction alpha-1 protein (Cx43) expression may be associated with macrophage-induced inflammation in the heart of spontaneously hypertensive rats (SHR). To examine mutual interactions of macrophage infiltration with Cx43 expression and redistribution, we applied a bolus of bacterial lipopolysaccharide (LPS) to SHR and age-matched normotensive Wistar rats. The results demonstrated association of Cx43 downregulation with increased infiltration of cardiac CD-68 macrophages and upregulation of nuclear factor-κB (NFκB) and tumor necrosis factor-α (TNF-α) expression in the heart of SHR. LPS application to SHR caused further degradation and redistribution of Cx43 accompanied with extensively increased macrophage infiltration and NFκB and TNF-α expression. LPS administration to Wistar rats resulted in elevation of cardiac CD-68 macrophages but it did not significantly affect total Cx43 expression. Our results are suggestive of regulation of Cx43 expression with macrophages-related inflammation in the heart of SHR. The data also indicate that SHR can be more sensitive to LPS than are normotensive rats.

Endogenous nociceptin/orphanin FQ affects the occurrence of arrhythmia in acute myocardial ischemia in rats through the regulation of connexin 43 protein

Objective Observing influence of various ventricular arrhythmias induced by acute myocardial ischemia and the change of connexin 43 (Cx43) protein content in myocardium in rats and effects of nociceptin/orphanin FQ (OFQ) receptor antagonist UFP-101 on the expression of arrhythmia and Cx43 protein. Methods Sixty-four male SD rats, weighing 200-250 g, were randomly divided into control group(group C, each group was divided into three subgroups, which named coronary artery occlusion 15 min, coronary artery occlusion 60 min and coronary artery occlusion 120 min, n=8), N/OFQ receptor antagonist group (group U, each group was divided into three subgroups, which named coronary artery occlusion 15 min, coronary artery occlusion 60 min and coronary artery occlusion 120 min, n=8), Sham operation group (group S, each group was divided into two subgroups, which named thread without ligation 15 min and thread without ligation 60 min, n=8). Group U was treated with OFQ antagonist compound UFP-101 in the dosage of 1 μl/g by caudal vein. Group C was treated with equal volume of saline. Group C and group U were administered 5 min before they were subjected to the coronary artery occlusion. Group S only was thread without ligation. Record data of electrocardiogram of group C and group U that from 10 min before the injection to 15, 60 min and 120 min after coronary artery occlusion, analyze data of all kinds of ventricular arrhythmia. Determination of expression levels of phosphorylated Cx43 (p-Cx43) protein and total Cx43 protein in all specimens by Western blot. Results At 15 min, numbers of ventricular premature contraction and ventricular bigeminy or trigeminy, the duration of ventricular tachycardia and ventricular fibrillation of group U were obviously lower than numbers in group C (P<0.05). At 60 min, numbers of ventricular premature contraction, ventricular bigeminy and trigeminy, ventricular tachycardia and ventricular fibrillation of group U were lower than those numbers in group C obviously (P<0.05). Expression of p-Cx43 protein at 15 min after coronary artery occlusion, the expression of p-Cx43 in group C were significantly lower than expression levels in group S (P<0.05), the expression of p-Cx43 in group U was significantly higher than that in group C (P<0.05). At 60 min after coronary artery occlusion, the expression of p-Cx43 in group C and group U were significantly lower than expression levels in group S (P<0.05). Conclusions Endogenous OFQ antagonist UFP-101 can reduce the occurrence of arrhythmia in acute myocardial ischemia by promoting phosphorylation of Cx43 protein in rats. Key words: Endogenous nociceptin/orphanin FQ; Protein Cx43; Phosphorylation; Arrhythmia

Detection of single-nucleotide polymorphism Gap junction protein Beta-2 genes in deaf schoolchildren of javanese population in Surabaya, Indonesia

Background: Genetic factors account for about 50%–75% responsible for hearing loss. The existence of single-nucleotide polymorphism (SNP) as genetic factors that affect hereditary hearing loss. The widely studied SNP was the gap junction protein beta-2 (GJB2) gene encoding the gap junction beta-2 protein (connexin26) that found in cochlea and required to convert sound waves into electrical nerve impulse. This study was aimed to detect the SNP GJB2 gene of hereditary hearing loss patients from Javanese population in Surabaya, Indonesia. Methods: The design of this study was a cross-sectional, analytic observational. The participant was taken randomly among the students from a deaf School in Surabaya. The questionnaire was completed by the parents of the deaf children. Blood sampling was taken from venous peripheral blood. DNA was extracted and amplified on GJB2 gene area by polymerase chain reaction (PCR). The positive results of PCR were processed further for sequencing. The sequencing results were analyzed to detect the GJB2 gene SNP with reference sequence/rs-80338939. Results: A total of 22 children participated in this study; all were profound sensorineural hearing loss (SNHL). The hereditary hearing loss was obtained with fewer in five children (22.73%), who had a history of hearing loss in their family. It was compared to 17 children (77.27%) who had no family history of hearing loss. It was found that the nucleotide variation in nucleotide number 8473 of GJB2 gene as much as 3 (13.64%) out of 22 children in hereditary hearing loss patients in Deaf School Type B Surabaya. Conclusions: This study did not found any SNP GJB2 gene (rs-80338939) of hereditary hearing loss patients from the Javanese population in Surabaya, Indonesia. There was the nucleotide substitution G to A in nucleotide number 8473 of GJB2 gene, which indicated the change of amino acid code genetic code table (valine) to amino acid code genetic code table (isoleucine). It may as the cause of SNHL.

Immunoproteasome Subunit β5i Promotes Ang II (Angiotensin II)-Induced Atrial Fibrillation by Targeting ATRAP (Ang II Type I Receptor-Associated Protein) Degradation in Mice.

Atrial fibrillation (AF) is the most common type of cardiac arrhythmia and increases the risk of stroke, heart failure, and death. Ang II (angiotensin II) triggers AF, mainly through stimulation of the AT1R (Ang II type I receptor). The immunoproteasome is a highly efficient proteolytic machine derived from the constitutive proteasome, but the role it plays in regulating AT1R activation and triggering AF remains unknown. Here, we show that among the catalytic subunits, β5i (PSMB8) expression, and chymotrypsin-like activity were the most significantly upregulated in atrial tissue of Ang II-infused mice or serum from patients with AF. β5i KO (β5i knockout) in mice markedly attenuated Ang II-induced AF incidence, atrial fibrosis, inflammatory response, and oxidative stress compared with WT (wild type) animals, but injection with recombinant adeno-associated virus serotype 9-β5i increased these effects. Moreover, we found that ATRAP (AT1R-associated protein) was a target of β5i. Overexpression of ATRAP significantly attenuated Ang II-induced atrial remodeling and AF in recombinant adeno-associated virus serotype 9-β5i-injected mice. Mechanistically, Ang II upregulated β5i expression to promote ATRAP degradation, which resulted in activation of AT1R-mediated NF-κB signaling, increased NADPH oxidase activity, increased TGF (transforming growth factor)-β1/Smad signaling, and altered the expression of Kir2.1 and CX43 (connexin 43) in the atria, thereby affecting atrial remodeling and AF. In summary, this study identifies β5i as a negative regulator of ATRAP stability that contributes to AT1R activation and to AF, highlighting that targeting β5i activity may represent a potential therapeutic approach for the treatment of hypertensive AF.

The role of TCPTP on leptin effects on astrocyte morphology

Leptin and LPS has been implicated in the development of hypothalamic astrogliosis in rodents. Astrocytes, which are interconnected by gap junction proteins, have emerged as important players in the control of energy homeostasis exerted by the hypothalamus. To investigate the hypothesis of action of T-cell protein tyrosine phosphatase (TCPTP) on the astrocyte morphology, astrocytes from the hypothalamus of one-day-old rats were stimulated with leptin and LPS (used as a positive control). Leptin and LPS induced a marked increase in astrocyte size, an increase in Ptpn2 (TCPTP gene) and gap junction alpha-1 protein, - Gja1 (connexin 43 - CX43 gene) mRNA expression and a decrease in gap junction protein, alpha 6 - Gja6 (CX30 gene) mRNA expression. Remarkably, these effects on astrocytes morphology and connexins were prevented by Ptpn2 siRNA. Astrocytes are known to produce cytokines; here we show that TCPTP acts as an important regulator of the cytokines and it possesses a reciprocal interplay with protein tyrosine phosphatase 1B (PTP1B). Our findings demonstrate that leptin and LPS alter astrocyte morphology by increasing TCPTP, which in turn modulates connexin 30 (CX30) and connexin 43 (CX43) expression. TCPTP and PTP1B seem to act in the regulation of cytokine production in astrocytes.