Functional and structural phenotyping of cardiomyocytes in the 3D organization of embryoid bodies exposed to arsenic trioxide.

Chronic exposure to environmental pollutants threatens human health. Arsenic, a world-wide diffused toxicant, is associated to cardiac pathology in the adult and to congenital heart defects in the foetus. Poorly known are its effects on perinatal cardiomyocytes. Here, bioinformatic image-analysis tools were coupled with cellular and molecular analyses to obtain functional and structural quantitative metrics of the impairment induced by 0.1, 0.5 or 1.0 µM arsenic trioxide exposure on the perinatal-like cardiomyocyte component of mouse embryoid bodies, within their 3D complex cell organization. With this approach, we quantified alterations to the (a) beating activity; (b) sarcomere organization (texture, edge, repetitiveness, height and width of the Z bands); (c) cardiomyocyte size and shape; (d) volume occupied by cardiomyocytes within the EBs. Sarcomere organization and cell morphology impairment are paralleled by differential expression of sarcomeric α-actin and Tropomyosin proteins and of acta2, myh6 and myh7 genes. Also, significant increase of Cx40, Cx43 and Cx45 connexin genes and of Cx43 protein expression profiles is paralleled by large Cx43 immunofluorescence signals. These results provide new insights into the role of arsenic in impairing cytoskeletal components of perinatal-like cardiomyocytes which, in turn, affect cell size, shape and beating capacity.

Expression of NEDD9 and connexin-43 in neoplastic and stromal cells of gastric adenocarcinoma.

Gastric cancer is related to high mortality rates and advanced disease stage at the time of diagnosis. Its carcinogenesis is extensively studied and is associated with genetic and epigenetic changes, changed the interaction between tumor and adjacent stromal cells, and changes in the microenvironment molecule status. Neural precursor cell-expressed developmentally down-regulated 9 (NEDD9) affects different signaling proteins and pathways, apoptosis, adhesion, cell migration, and invasiveness. Connexin-43 (Cx43) also assists in intercellular communications and has several channel-independent functions. Aberrant expression of those two gap junction proteins plays an essential role in metastatic processes. Our scope was to detect the expression of Cx43 and NEDD9 in epithelial and stromal gastric cancer compartments and its relation to tumor progression and lymph node metastases. Cancer tissue from 53 cases of node-negative and 55 cases of node-positive primary gastric carcinoma patients was analyzed for Cx43 and NEDD9 expression by immunohistochemical assay, and the results were correlated with the remaining clinical and pathological findings and survival. In our cohort of patients with lymph node metastases, we detected higher expression of epithelial Cx43 in the primary tumor and stromal Cx43 expression correlated with both epithelial NEDD9 (rho = 0.453) and stromal NEDD9 (rho = 0.484). Higher epithelial Cx43 and NEDD9 expression were associated with higher mortality (HR 1.54, 95% CI 1.01-2.37, p = 0.048). Epithelial Cx43 expression, both epithelial and stromal NEDD9 expression, T and N status were all independently associated with shorter survival. In summary, our findings suggest that increased expression of both epithelial and stromal NEDD9 and epithelial Cx43 could potentially be used as prognostic gastric cancer biomarkers.

Expression of connexin 43 by atypical fibroxanthoma.

INTRODUCTION: Connexins are transmembrane channel proteins that interconnect adjacent cells and allow the exchange of signaling molecules between cells and the extracellular milieu. They have been investigated in many tumors to obtain information about tumor nature, behavior, and prognosis. METHODS: Herein, we present a study on the immunohistochemical expression of connexin (Cx) 43 in 16 cases of atypical fibroxanthoma (AFX). For the immunohistochemical staining, a tissue array was obtained from the paraffin-embedded blocks. RESULTS: The expression was membranous and cytoplasmic in all cases. Thirteen cases (81.25%) showed strong staining. In the other three cases (18.75%), the staining was medium. None of the cases showed nuclear staining. Fifteen out of 16 cases showed a diffuse pattern, and only one case showed a focal pattern. CONCLUSIONS: Our results suggest that Cx43 may play an important role in the natural behavior of AFX.

Connexin43 Expression and Associated Chronic Inflammation Presages the Development of Cerebral Radiation Necrosis.

Cerebral radiation necrosis (CRN) is a delayed complication of radiosurgery that can result in severe neurological deficits. The biological changes leading to necrotic damage may identify therapeutic targets for this complication. Connexin43 expression associated with chronic inflammation may presage the development of CRN. A mouse model of delayed CRN was used. The left hemispheres of adult female mice were irradiated with single-fraction, high-dose radiation using a Leksell Gamma Knife. The brains were collected 1 and 4 days, and 1-3 weeks after the radiation. The expression of connexin43, interleukin-1ß (IL-1ß), GFAP, isolectin B-4, and fibrinogen was evaluated using immunohistochemical staining and image analysis. Compared with the baseline, the area of connexin43 and IL-1ß staining was increased in ipsilateral hemispheres 4 days after radiation. Over the following 3 weeks, the density of connexin43 gradually increased in parallel with progressive increases in GFAP, isolectin B-4, and fibrinogen labeling. The overexpression of connexin43 in parallel with IL-1ß spread into the affected brain regions first. Further intensified upregulation of connexin43 was associated with escalated astrocytosis, microgliosis, and blood-brain barrier breach. Connexin43-mediated inflammation may underlie radiation necrosis and further investigation of connexin43 hemichannel blockage is merited for the treatment of CRN.

An Alternatively Translated Connexin 43 Isoform, GJA1-11k, Localizes to the Nucleus and Can Inhibit Cell Cycle Progression.

Connexin 43 (Cx43) is a gap junction protein that assembles at the cell border to form intercellular gap junction (GJ) channels which allow for cell-cell communication by facilitating the rapid transmission of ions and other small molecules between adjacent cells. Non-canonical roles of Cx43, and specifically its C-terminal domain, have been identified in the regulation of Cx43 trafficking, mitochondrial preconditioning, cell proliferation, and tumor formation, yet the mechanisms are still being explored. It was recently identified that up to six truncated isoforms of Cx43 are endogenously produced via alternative translation from internal start codons in addition to full length Cx43, all from the same mRNA produced by the gene GJA1. GJA1-11k, the 11kDa alternatively translated isoform of Cx43, does not have a known role in the formation of gap junction channels, and little is known about its function. Here, we report that over expressed GJA1-11k, unlike the other five truncated isoforms, preferentially localizes to the nucleus in HEK293FT cells and suppresses cell growth by limiting cell cycle progression from the G0/G1 phase to the S phase. Furthermore, these functions are independent of the channel-forming full-length Cx43 isoform. Understanding the apparently unique role of GJA1-11k and its generation in cell cycle regulation may uncover a new target for affecting cell growth in multiple disease models.

Expression and functional regulation of gap junction protein connexin 43 in dermal mesenchymal stem cells from psoriasis patients.

BACKGROUND: Psoriasis is a chronic recurrent inflammatory disease. Mesenchymal stem cells (MSCs) can regulate the inflammatory microenvironment, thereby controlling the proliferation, differentiation, and migration of immune cells. Connexin 43(Cx43), a key gap junction protein, has been shown to form gap junctions for communication between neighboring cells. OBJECTIVE: We investigated the expression of Cx43 in dermal mesenchymal stem cells (DMSCs) derived from psoriasis patients and explored the relationship between the Cx43-mediated gap junction intercellular communication (GJIC) and DMSCs. METHODS: Human DMSCs were isolated and propagated in adherent culture. Quantitative real-time reverse transcription PCR and western blot and immunofluorescence were used to detect the expression and localization of Cx43 in DMSCs. Fluorescence redistribution after photobleaching was performed to assess adjacent DMSCs GJIC. CCK8 was used to detect the proliferation of DMSCs before and after gap junction blocker (18α-glycyrrhetinic acid; AGA) treatment. Cell energy metabolism was analyzed with an energy metabolism analyzer. RESULTS: Cx43 was located in the cytoplasm and cytomembrane, as well as partially in the nucleus of DMSCs. The expression of Cx43 in psoriasis DMSCs was higher than that in control samples and the gap junction function was enhanced. In addition, the glycolysis and mitochondrial respiration of psoriasis DMSCs were also enhanced. However, AGA inhibited the expression of Cx43, attenuated GJIC function, and inhibited the proliferation of DMSCs. CONCLUSIONS: Our results indicated that the expression of Cx43 in DMSCs from psoriasis lesions is increased and that the inhibition of Cx43 leads to the inhibition of both GJIC and DMSCs proliferation.

Connexin 43: A novel ginsenoside Rg1-sensitive target in a rat model of depression.

Our previous studies have shown that ginsenoside Rg1 (Rg1) exerts antidepressant-like effects in animal models of depression, accompanied by an improvement of astrocytic gap junction functions. However, whether connexin 43 (Cx43), the major connexin forming gap junctions between astrocytes, is the key regulator of Rg1-induced antidepressant-like effects is still unknown. In this study, we examine in vitro and in vivo the involvement of Cx43 in the antidepressant effects of Rg1. Corticosterone was used to establish an in vitro rat model of depression. Treatment with Rg1 1 h prior to corticosterone significantly improved the cell viability of astrocytes, which was significantly inhibited by carbenoxolone, a widely used gap junction inhibitor. Moreover, Rg1 treatment significantly ameliorated antidepressant-sensitive behaviours induced by infusion of carbenoxolone or Gap26, a selective inhibitor of Cx43, into the prefrontal cortex of the animals. Rg1 treatment increased the expression of Cx43 compared with Gap26 group. According to these results, the antidepressant-like effects of Rg1 were mainly mediated by Cx43-formed gap junctions.

Expression of Connexin 43 in 32 Cases of Merkel Cell Carcinoma.

INTRODUCTION: Connexins (Cxs) are channel proteins that allow direct connection among cells and between cells and the extracellular space. There is very little information in the literature on the expression of Cxs by Merkel cell carcinoma (MCC). MATERIALS AND METHODS: Thirty-two cases of MCC were recovered from our archives and studied immunohistochemically for Cx43. RESULTS: All our cases expressed several neuroendocrine markers. Most cases showed nonimmunohistochemically perceptible staining for Cx43. There was no difference between Merkel cell polyomavirus (MCPyV)-positive and MCPyV-negative cases. One case could not be evaluated. Only 2 cases showed a focal (10% of the tumor) membranous staining of Cx43. One of these cases was MCPyV-negative and, in the other, CM2B4 could not be evaluated. CM2B4 was positive in 18 cases and negative in 13 cases, and it could not be evaluated in 1 case. CONCLUSIONS: MCC shows a low Cx43 level, with no differences between MCPyV-positive and MCPyV-negative cases. Therefore, this opens the door for Cx43 targeting in therapeutic approaches to MCC.

Overexpression of miR-27b-3p Targeting Wnt3a Regulates the Signaling Pathway of Wnt/ß-Catenin and Attenuates Atrial Fibrosis in Rats with Atrial Fibrillation.

MicroRNAs (miRNAs) are regarded as a potential method for the treatment of atrial fibrillation (AF) although its molecular mechanism remains unknown. We found in our previous study that the level of peripheral blood miR-27b-3p and the expression of atrial tissue CX43 were both significantly downregulated in AF patients. In the present study, we propose and test this hypothesis that overexpression of miR-27b-3p attenuates atrial fibrosis, increases CX43 expression, and regulates the signaling pathway of Wnt/ß-Catenin by targeting Wnt3a. miR-27b-3p overexpression was induced by rat tail vein injection of adeno-associated virus. Two weeks after transfection of adeno-associated virus, the rat AF model was established by tail vein injection of acetylcholine- (ACh-) CaCl2 for 7 days, and 1 ml/kg was injected daily. The incidence and duration of AF were recorded with an electrocardiogram. Cardiac function was monitored by cardiac ultrasound. Serum cardiac enzyme was detected by ELISA. The expression of atrial miR-27b-3 and Wnt3a was assayed by quantitative RT-PCR. Atrial fibrosis was determined by Masson's trichrome staining. Expression of atrial Collagen-I and Collagen-III was tested by the immunohistochemical method. Expression of CX43 was measured by immunofluorescence. The expression of Collagen-I, a-SMA, Collagen-III, TGF-ß1, CX43, Wnt3a, ß-Catenin, and p-ß-Catenin was assayed by western blot. Our results showed that miR-27b-3p overexpression could reduce the incidence and duration of AF, alleviate atrial fibrosis, increase atrial CX43 expression, and decrease the expression of Collagen-I, a-SMA, Collagen-III, TGF-ß1, Wnt3a, and p-ß-Catenin. In addition, the results of luciferase activity assay showed that Wnt3a is a validated miR-27b-3p target in HEK 293T cells. Our results provide a new evidence that miR-27b-3p regulates the signaling pathway of Wnt/ß-Catenin by targeting Wnt3a, which may play an important role in the development of atrial fibrosis and AF.

A Logistic Regression Model for Detecting the Presence of Malignant Progression in Atypical Meningiomas.

OBJECTIVE: To develop a method to distinguish atypical meningiomas (AMs) with malignant progression (MP) from primary AMs without a clinical history. METHODS: The clinical, radiologic, and pathologic data of 33 previously Simpson grade I resected (if any) as well as no radiotherapy treated intracranial AMs between January 2008 and December 2015 were reviewed. Immunohistochemical staining for connexin 43 (Cx43) and Ki-67 was performed. Descriptive analysis and univariate and multivariate logistic regression analyses were used to explore independent predictors of MP. A multivariable logistic model was developed to estimate the risk of MP, and its diagnostic value was determined from a receiver operating characteristic curve. RESULTS: There were 11 AMs (33.3%) with histopathologically confirmed MP from benign meningiomas. The other 22 (66.7%) were initially diagnosed AMs with no histopathologically confirmed MP during a median 60.5 months (range, 42-126 months) of follow-up. Univariate and multivariate logistic analyses showed that irregular tumor shape (P = 0.010) and low Cx43 expression (P = 0.010) were independent predictors of the presence of MP, and the predicted probability was calculated by the following formula: P = 1/[1+exp.{1.218-(3.202×Shape)+(3.814×Cx43)}]. P > 0.5 for an irregularly shaped (score 1) AM with low Cx43 expression (score 0) indicated a high probability of MP. The sensitivity, specificity, positive predictive value, negative predictive value, and overall predictive accuracy were 63.6, 95.6, 87.5, 84.0, and 84.8%, respectively. CONCLUSIONS: Low Cx43 expression and irregular tumor shape were independent predictors of the presence of MP. The relevant logistic regression model was found to be effective in distinguishing MP-AMs from primary AMs.

Expression of Connexin 43 (Cx43) in Benign Cutaneous Tumors With Follicular Differentiation.

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.

Suppression of connexin 43 expression by miR-106a promotes melanoma cell proliferation.

OBJECTIVE: Connexin 43 (Cx43), a vital gap junction protein is reported to be involved in melanoma progression. The aim of the study is to investigate the regulatory role of Cx43 in melanoma. MATERIALS AND METHODS: Western blot assay was used to detect the protein expression of Cx43 in melanoma cells and the human epidermal melanocytes (HEMn). MTT cell proliferation and cell colony formation assays were used to assess cell proliferation. Bioinformatics prediction, luciferase reporter assay, Western blot and qRT-PCR assays were applied to demonstrate that Cx43 was a direct target of miR-106a in melanoma cells. RESULTS: Connexin 43 (Cx43) was lower expressed in melanoma cells compared with human epidermal melanocytes (HEMn). Cx43 overexpression significantly inhibited melanoma cell proliferation and colony formation ability in vitro. However, knockdown of Cx43 had opposite effects on cell proliferation and colony formation. Bioinformatics prediction and luciferase reporter assays demonstrated that miR-106a targeted the 3' untranslated region (3'UTR) of Cx43 and regulated its mRNA and protein expression levels in melanoma cells. MiR-106a was upregulated in melanoma cells, and its overexpression attenuated the effects caused by upregulating Cx43 expression. CONCLUSIONS: Thus, our results indicated that Cx43 was downregulated in melanoma cells and may be a potential target of melanoma treatment.

Connexin 43 is an independent predictor of patient outcome in breast cancer patients.

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.

mTOR- and SGK-Mediated Connexin 43 Expression Participates in Lipopolysaccharide-Stimulated Macrophage Migration through the iNOS/Src/FAK Axis.

Connexin 43 (Cx43) deficiency was found to increase mortality in a mouse model of bacterial peritonitis, and Cx43 is upregulated in macrophages by LPS treatment. In this study, we characterized a novel signaling pathway for LPS-induced Cx43 expression in RAW264.7 cells and thioglycolate-elicited peritoneal macrophages (TGEMs). LPS alone or LPS-containing conditioned medium (CM) upregulated Cx43. Overexpression or silencing of Cx43 led to the enhancement or inhibition, respectively, of CM-induced TGEM migration. This response involved the inducible NO synthase (iNOS)/focal adhesion kinase (FAK)/Src pathways. Moreover, CM-induced migration was compromised in TGEMs from Cx43+/- mice compared with TGEMs from Cx43+/+ littermates. Cx43 was upregulated by a serum/glucocorticoid-regulated kinase 1 (SGK) activator and downregulated, along with inhibition of CM-induced TGEM migration, by knockdown of the SGK gene or blockade of the SGK pathway. LPS-induced SGK activation was abrogated by Torin2, whereas LPS-induced Cx43 was downregulated by both Torin2 and rapamycin. Analysis of the effects of FK506 and methylprednisolone, common immunosuppressive agents following organ transplantation, suggested a link between these immunosuppressive drugs and impaired macrophage migration via the Cx43/iNOS/Src/FAK pathway. In a model of Escherichia coli infectious peritonitis, GSK650349-, an SGK inhibitor, or Torin2-treated mice showed less accumulation of F4/80+CD11b+ macrophages in the peritoneal cavity, with a delay in the elimination of bacteria. Furthermore, following pretreatment with Gap19, a selective Cx43 hemichannel blocker, the survival of model mice was significantly reduced. Taken together, our study suggested that Cx43 in macrophages was associated with macrophage migration, an important immune process in host defense to infection.

TGF-ß1 and connexin-43 expression in neurogenic bladder from rats with sacral spinal cord injury.

AIMS: Sacral spinal cord injury (SCI) could induce underactive bladder (UAB). Malfunction of connexin 43 (CX43) regulated by TGF-ß1 might involve in urinary bladder dysfunction. We studied the changes of CX43 and TGF-ß1/Smad3 signaling in detrusor of neurogenic bladder (NB) in sacral SCI rats. METHODS: Sacral SCI was produced by hemisection (SSCH) or transection (SSCT) of spinal cord between L4 and L5 in female Wistar rats. BBB scores, residual urine volume and bladder weight as well as characteristic cystometric parameters at 6th week were used to confirm the successful establishment of NB. Western blotting and qRT-PCR were used to exam the protein and mRNA expression levels of CX43, CX45, TGF-ß1, and Smad3 in detrusor. RESULTS: BBB scores were significantly decreased, with the lowest in SSCT rats (P < 0.01). The residual urine volume, mean bladder weight, and cystometric parameters were increased, with the highest in SSCT rats. CX43 and phospho-CX43 protein levels were significantly decreased, but those of TGF-ß1, Smad3, and phospho-Smad3 were significantly increased. It was the protein and mRNA levels of CX43 but not those of CX45 which were decreased in negative accordance with those of TGF-ß1 and Smad3. Those changes were more significant in SSCT than in SSCH rats. CONCLUSIONS: This study indicates that voiding dysfunction is related to the decreased CX43 function in detrusor from NB. TGF-ß1/Smad3 signaling might be involved in the down-regulation of CX43 in SCI rats. Early regulation of CX43 might be beneficial to patients with voiding dysfunction.

Effects of Pinocembrin Pretreatment on Connexin 43 (Cx43) Protein Expression After Rat Myocardial Ischemia-Reperfusion and Cardiac Arrhythmia.

BACKGROUND Cardiac infarction frequently leads to arrhythmia and ischemia/reperfusion (I/R) aggravates cardiac injury. Pinocembrin can resist cerebral ischemia and decrease cardiac infarction area. This study thus generated a rat myocardial I/R model to assess the effect on ventricular rhythm and expression of gap junction connexin (Cx43). MATERIAL AND METHODS Male SD rats were randomly assigned into sham, model, and pinocembrin (30 mg/kg) pretreatment groups (N=15 each). The I/R model was generated by ligation of the left anterior descending coronary artery for 30 min. The pinocembrin group received intravenous injection 10 min before surgery. Heart rate (HR), mean artery pressure (MAP), rate pressure product (RPP), and arrhythmia were observed at 10 min before ischemia, 30 min after ischemia, and at 30, 60, and 120 min after reperfusion. ELISA was used to assess serum CK-MB and cTnI levels. Na+-K+ATPase and Ca+-Mg2+ATPase levels were quantified by spectrometry, followed by HE staining, IHC approach for Cx43 expression, and Western blot for Kir2.1 protein expression. RESULTS Model rats had significantly lower HR, MAP, and RPP than in the sham group, and the pinocembrin pretreatment group had higher serum indexes. Arrhythmia index, CK-MB, and cTnI were higher in the model and pinocembrin groups, while Na+-K+ATPase, Ca+-Mg2+ATPase, Cx43, and Kir2.1 proteins were lower (p<0.05). CONCLUSIONS Pinocembrin alleviated ventricular arrhythmia in I/R rats via enhancing Na+-K+ATPase and Ca+-Mg2+ATPase activity and upregulating Cx43 and Kir2.1 protein expression.

Lysophosphatidic acid accelerates development of porcine embryos by activating formation of the blastocoel.

Culture media modifications, including the addition of various factors, are important for the in vitro production of oocytes and embryos. In this study, we investigated the effects of lysophosphatidic acid (LPA) on porcine embryo development. Porcine parthenogenetic embryos were cultured with 0, 0.1, 1, and 10 µM LPA for 7 days, or cultured in basic medium until Day 4 and then treated with LPA from Days 4 to 7. No difference in the in vitro development of embryos cultured with LPA for 7 days was observed. Conversely, rates of blastocyst and over-expanded blastocyst formation were higher in the 0.1 and 1 µM LPA-treated versus the other groups of embryos treated from Days 4 to 7. Moreover, formation of early blastocysts occurred earlier and embryo size was larger in LPA-treated compared to control embryos. Expression of Connexin 43 and gap junction and cell adhesion-related genes (GJC1 and CDH1, respectively) was also higher in LPA-treated compared to control embryos. Despite no difference in the blastocyst total cell number between groups, the apoptotic index was lower in the LPA-treated group than in the control group; indeed, BCL2L1 (B-cell lymphoma 2-like protein 1) expression increased while BAK (Bcl-2 homologous antagonist killer) decreased in the LPA-treated group. Thus, addition of LPA to the medium from Days 4 to 7 of culture improves blastocyst formation and aids the development of preimplantation embryos.

Telmisartan reduces arrhythmias through increasing cardiac connexin43 by inhibiting IL-17 after myocardial infarction in rats.

OBJECTIVE: To observe the expressions of myocardial connexin43 (Cx43) and interleukin-17 (IL-17) in acute myocardial infarction (AMI) rats and investigate its possible mechanism of telmisartan in the prevention and treatment of arrhythmia in AMI. MATERIALS AND METHODS: Sprague Dawley (SD) rats were selected and myocardial infarction model was established. After the successful modeling, the rats were randomly divided into three groups: Sham group, MI group, Telm group. Ventricular arrhythmias was induced by the programmed electrical stimulation at 2, 4, 8 weeks. After 8 weeks, rats were sacrificed and heart tissues were collected for immunohistochemistry and Western blot detection. RESULTS: Telmisartan reduced the induction rate of ventricular arrhythmia after myocardial infarction in rats. Telmisartan increased the Cx43 expression while reduced the IL-17 expression in myocardial infarction in rats. Moreover, there was a negative correlation between the expressions of Cx43 and IL-17 after myocardial infarction. CONCLUSIONS: Telmisartan can reduce the occurrence rate of malignant arrhythmias after myocardial infarction, whose mechanism may be increasing the Cx43 expression through inhibition of IL-17 expression.

SRC3 expressed in BMSCs promotes growth and migration of multiple myeloma cells by regulating the expression of Cx43.

Interactions between bone marrow stromal cells (BMSCs) and multiple myeloma cells significantly contribute to the progression of multiple myeloma (MM). However, little is known about the molecular mechanisms that regulate these interactions. Connexin-43 (Cx-43) has been implicated in the interplay between BMSCs and MM cells. In this study, we hypothesized that the steroid receptor co-activator-3 (SRC3) expressed in BMSCs regulates the expression of Cx-43 to promote the proliferation and migration of myeloma cells. To address this, we co-cultured a human multiple myeloma cell line, RPMI-8226 transfected with either control BMSCs or sh-SRC3-BMSCs. We found that knocking down SRC3 expression in BMSCs inhibited the proliferation and migration of RPMI-8226 cells. In addition, we found that co-culturing RPMI 8266 cells with BMSCs increased Cx43 expression, while knocking down SRC3 expression in BMSCs decreased Cx43 expression. Moreover, our work revealed that SRC3 in BMSCs regulates Cx43 expression via the mitogen-activated protein kinase (MAPK) pathway. To validate this result in vivo, we knocked down SRC3 expression in BMSCs in nude mice and found that tumor growth and cell apoptosis were significantly decreased. In addition, mice treated with either RPMI 8266 cells overexpressing Cx43 or with a P38 MAPK inhibitor (SB202190) exhibited increased intratumoral leukocyte populations and promoted cell apoptosis in tumor tissue. Our findings demonstrate how SRC3 and Cx43 regulation between BMSCs and myeloma cells mediate cell growth and disease progression, with potential implications for prognosis and therapeutic interventions.

Adrenomedullin improves fertility and promotes pinopodes and cell junctions in the peri-implantation endometrium.

Implantation is a complex event demanding contributions from both embryo and endometrium. Despite advances in assisted reproduction, endometrial receptivity defects persist as a barrier to successful implantation in women with infertility. We previously demonstrated that maternal haploinsufficiency for the endocrine peptide adrenomedullin (AM) in mice confers a subfertility phenotype characterized by defective uterine receptivity and sparse epithelial pinopode coverage. The strong link between AM and implantation suggested the compelling hypothesis that administration of AM prior to implantation may improve fertility, protect against pregnancy complications, and ultimately lead to better maternal and fetal outcomes. Here, we demonstrate that intrauterine delivery of AM prior to blastocyst transfer improves the embryo implantation rate and spacing within the uterus. We then use genetic decrease-of-function and pharmacologic gain-of-function mouse models to identify potential mechanisms by which AM confers enhanced implantation success. In epithelium, we find that AM accelerates the kinetics of pinopode formation and water transport and that, in stroma, AM promotes connexin 43 expression, gap junction communication, and barrier integrity of the primary decidual zone. Ultimately, our findings advance our understanding of the contributions of AM to uterine receptivity and suggest potential broad use for AM as therapy to encourage healthy embryo implantation, for example, in combination with in vitro fertilization.