miR-126 regulates the proliferation, migration, invasion, and apoptosis of non-small lung cancer cells via AKT2/HK2 axis.

This study tended to clarify the role of miR-126 in non-small cell lung cancer (NSCLC) cell biological behaviors in vitro, containing cell proliferation, migration, invasion, and apoptosis. miRNA expression microarray related to NSCLC was accessed from gene expression omnibus (GEO) database and subjected to differential analysis using the "limma" package. Real-time quantitative PCR was conducted to assess the expression of miR-126 in NSCLC cell lines. wIn vitro experiments including 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide (MTT), wound healing assay, Transwell, and flow cytometry assay were used for evaluating the effect of miR-126 on cell proliferation, migration, invasion, and apoptosis. Additionally, target mRNA for miR-126 was predicted and further validated by bioinformatics analysis and dual-luciferase reporter assay, respectively. It suggested that miR-126 was significantly down-regulated in NSCLS based on the expression microarray, and similar expression trend was exhibited in cancer cell lines. In the meantime, overexpression of miR-126 was found to result in inhibition of cell proliferation, migration, and invasion while promotion of cell apoptosis, with reductions in protein expression of AKT2 and phosphorylated HK2 (p-HK2) as well. AKT2, identified to be a direct target of miR-126 in NSCLC as judged by dual-luciferase reporter assay. Additionally, overexpression of AKT2 was observed to have the ability of elevating p-HK2 protein expression and reversing the effect of miR-126 on NSCLC cell proliferation, migration, and invasion. Given the above findings, we can see that miR-126 exerts its role in NSCLC cell proliferation, migration, invasion, and apoptosis with the aid of AKT2/HK2 axis.

MiR-129-2-3p Inhibits Esophageal Carcinoma Cell Proliferation, Migration, and Invasion via Targeting DNMT3B.

PURPOSE: The study aims to explore the regulatory mechanism of miR-129-2-3p underlying esophageal carcinoma (EC) cell progression and generate new ideas for targeted treatment of EC. METHODS: Mature miRNA expression data and total RNA sequencing data of EC in the TCGAESCA dataset were utilized to explore differentially expressed miRNAs (DEmiRNAs). StarBase database was then utilized to predict targets of miRNA. MiR-129-2-3p and DNMT3B expression in EC cell lines was assayed through qRT-PCR and Western blot. CCK-8, scratch healing, and transwell assays were conducted to assess the impact of miR-129-2-3p on EC cell phenotypes. In addition, a dual-luciferase assay was completed to identify the binding relationship between DNMT3B and miR-129-2-3p. RESULTS: MiR-129-2-3p was noticeably less expressed in EC cell lines, while DNMT3B was highly expressed. MiR-129-2-3p could bind to DNMT3B. Furthermore, in vitro functional experiments uncovered that overexpressed miR-129-2-3p repressed EC cell progression while further overexpressing DNMT3B would restore the above inhibitory effect. CONCLUSION: MiR-129-2-3p is a cancer repressor in EC cells, and it could target DNMT3B, thus hampering the progression of EC cells.

Factors influencing the development of bone starvation syndrome after total parathyroidectomy in patients with renal hyperparathyroidism.

Purpose: To investigate the factors affecting the development of bone starvation syndrome (HBS) after total parathyroidectomy in patients with renal hyperparathyroidism (SHPT). Patients and methods: The clinical data and perioperative indices of 141 patients who underwent PTX for SHPT were retrospectively analyzed. The patients were divided into HBS and non-HBS groups based on postoperative minimum blood calcium <1.87 mmol/L. The differences in general clinical data and perioperative related indices between the two groups were compared; logistic regression analysis was performed to analyze the risk factors influencing HBS occurrence after surgery. Multiple linear regression method was used to analyze the factors influencing the maintenance time of intravenous calcium supplementation and total amount of calcium supplementation during intravenous calcium supplementation. The threshold value for the diagnosis of HBS was analyzed using the ROC subjects' working curve. Results: HBS occurred in 46 (32.6%) patients. Univariate analysis showed statistically significant differences in dialysis age, preoperative calcitonin, preoperative parathyroid hormone, preoperative blood phosphorus, and preoperative alkaline phosphatase between both groups (P < 0.05). Logistic regression analysis using stepwise entry method concluded that preoperative alkaline phosphatase was an independent factor for the development of HBS after surgery. Preoperative parathyroid hormone was an independent factor for the duration of intravenous calcium supplementation and total calcium supplementation during intravenous calcium supplementation in the HBS group. Based on the ROC curve, for postoperative HBS, the cut-off ALP value was 199.5 U/L, with a sensitivity of 80.85% and specificity of 82.61%. Conclusion: Preoperative serum ALP may be an independent factor for HBS occurrence after surgery. When preoperative ALP > 199.5 U/L, patients with SHPT are prone to HBS after surgery, and the higher the preoperative ALP, the higher the incidence of HBS, and vice versa. In addition, preoperative PTH may be the factor in the timing of postoperative intravenous calcium supplementation and the total amount of calcium supplementation during intravenous calcium supplementation in patients with HBS.

Identification of a dysregulated ceRNA network modulated by copy number variation-driven lncRNAs in lung squamous cell carcinoma.

Lung cancer is primarily responsive for cancer death, and its progression is aggressively affected by copy number variation (CNV). Through bioinformatics approach, a ceRNA network of CNV-driven lncRNAs in lung squamous cell carcinoma (LUSC) patients was constructed. Data on normal and LUSC tumor tissue from The Cancer Genome Atlas (TCGA)-LUSC dataset were subjected to differential analysis, and differentially expressed lncRNAs (DElncRNAs), DEmiRNAs, and DEmRNAs were obtained. Based on TCGA-LUSC, CNVs of normal and tumor tissue samples were then compared using a Chi-square test, and lncRNAs were intersected based on their CNVs and expression alternation. In combination with the Kruskal-Wallis test, CNV-driven lncRNAs were acquired. Afterwards, miRNAs and mRNAs that interacted with CNV-driven lncRNAs were obtained based on databases (LncBase, starBase, miRDB, mirDIP and TargetScan), DElncRNAs, DEmiRNAs and DEmRNAs, and correlation analysis. The acquired lncRNAs, miRNAs and mRNAs were subjected to Cytoscape software to construct a CNV-driven ceRNA network, which involved 5 lncRNAs (MIR143HG, LINC00702, MIR22HG, RP11-180 N14.1, RP11-473 M20.9), 6 miRNAs (miR-3200-3p, miR-1301-3p, miR-93-3p, miR-96-5p, miR-96-5p, miR-130b-5p, miR-205-5p) and 80 mRNAs. Kyoto Encyclopedia of Genes and Genomes and Gene Ontology enrichment analyses indicated that downstream mRNAs were mainly correlated with blood vessel development and T cell-mediated immunity. In summary, we devoted to analyzing CNV-related lncRNAs, mRNAs, and miRNAs in LUSC, thus clarifying 5 lncRNAs that may influence the malignant progression of LUSC. The ceRNA network regulated by these lncRNAs may be the novel pathogenesis of LUSC.

EZH2 affects malignant progression and DNA damage repair of lung adenocarcinoma cells by regulating RAI2 expression.

BACKGROUND: Lung adenocarcinoma (LUAD) is featured in high morbidity and mortality. Aberrant activation of the histone methyltransferase EZH2 has close association with cancer progression. This research aimed to deeply dive into the role and possible molecular mechanisms of EZH2 and its downstream genes in malignant progression and DNA damage repair of LUAD cells. METHODS: Expression of EZH2 in LUAD cells was analyzed by qRT-PCR, and the effects of EZH2 on proliferation, and apoptosis of LUAD cells were examined by CCK-8, colony formation and flow cytometry assays. The downstream targets of EZH2 were predicted by bioinformatics analysis. Then, the targeting relationship between EZH2 and RAI2 was examined by CHIP and luciferase reporter assays. Rescue assay were used to further validate the effect of EZH2/RAI2 on the malignant progression of LUAD cells. The expression levels of EZH2, RAI2 and p53 were examined by Western blot. RESULTS: Upregulation of EZH2 was identified in LUAD tissues and cells. RAI2 was a downstream target gene of EZH2, and the two were negatively correlated. Silencing EZH2 suppressed proliferation of LUAD cells, promoted expression of p53, cell cycle arrest and apoptosis. While silencing RAI2 could reverse the above-mentioned effects caused by EZH2 silencing. CONCLUSION: These results demonstrated that EZH2 promoted malignant progression and DNA damage repair of LUAD cells by targeting and negatively regulating RAI2.

Evaluation of the diagnostic performance of contrast-enhanced ultrasound combined with BRAF V600E gene detection in nodules of unclear significance by thyroid fine-needle aspiration.

BACKGROUND: Our study aims to test the diagnostic performance of contrast-enhanced ultrasound (CEUS) combined with the detection of serine/threonine-protein kinase V600E (BRAF V600E) in nodules of unclear significance by thyroid fine-needle aspiration (FNA). METHODS: From January 2015 to December 2019, 244 patients were subjected to ultrasonic strain imaging and elastography, CEUS, and BRAF V600E gene detection at Lishui Hospital of Zhejiang University. Thyroid FNA does not confirm the benignancy and malignancy of the thyroid nodules. With postoperative pathology as the gold standard, the diagnostic value of CEUS, BRAF V600E detection, and the combination in differentiating benign and malignant thyroid nodules were evaluated. The negative predictive value (NPV) and accuracy of CEUS, BRAF V600E detection, and the combination were calculated along with sensitivity, specificity, and positive predictive value (PPV). RESULTS: In this study, the sensitivity, specificity, PPV, NPV, accuracy, and AUC of CEUS alone in predicting benign and malignant thyroid nodules were 69.8%, 94.9%, 98.6%, 37.4%, 73.8% and 0.884, respectively. The sensitivity, specificity, PPV, NPV, accuracy and AUC of BRAF V600E detection alone were 65.4%, 100%, 100%, 35.5%, 70.9% and 0.827, respectively. The sensitivity, specificity, PPV, NPV, accuracy and AUC of the combination were 73.2%, 94.9%, 98.7%, 40.2%, 76.6% and 0.923, respectively. CONCLUSIONS: Therefore, compared with CEUS or BRAF V600E gene detection alone, the combination of CEUS and BRAF V600E gene detection has a higher sensitivity, NPV, and accuracy in the diagnosis of thyroid nodules.

MiR-196b Promotes the Invasion and Migration of Lung Adenocarcinoma Cells by Targeting AQP4.

OBJECTIVE: We aimed to investigate the mechanism of the regulatory axis of miR-196b/AQP4 underlying the invasion and migration of lung adenocarcinoma (LUAD) cells. METHODS: LUAD miRNA and mRNA expression profiles were downloaded from TCGA database and then differential analysis was used to identify the target miRNA. Target gene for the miRNA was obtained via prediction using 3 bioinformatics databases and intersection with the differentially expressed mRNAs searched from TCGA-LUAD. Then, qRT-PCR and western blot were used to validate the expression of miR-196b and AQP4. Dual-luciferase reporter assay was performed to confirm the targeting relationship between miR-196b and AQP4. Transwell assay was used to investigate the migration and invasion of LUAD cells. RESULTS: MiR-196b was screened out by differential and survival analyses, and the downstream target gene AQP4 was identified. In LUAD, miR-196b was highly expressed while AQP4 was poorly expressed. Besides, overexpression of miR-196b promoted cell invasion and migration, while overexpression of AQP4 had negative effects. Moreover, the results of the dual-luciferase reporter assay suggested that AQP4 was a direct target of miR-196b. In addition, we also found that overexpressing AQP4 could suppress the promotive effect of miR-196b on cancer cell invasion and migration. CONCLUSION: MiR-196b promotes the invasion and migration of LUAD cells by down-regulating AQP4, which helps us find new molecular targeted therapies for LUAD.

Correlation between thyroid autoantibodies and the risk of thyroid papillary carcinoma.

BACKGROUND: To investigate the correlation of thyroglobulin antibody (TgAb) and thyroid peroxidase antibody (TPOAb) with the risk of papillary thyroid carcinoma (PTC). METHODS: The clinical data of 322 patients with pathologically confirmed thyroid nodules who underwent surgical treatment in Lishui Hospital of Zhejiang University from January 2018 to December 2019 were enrolled in this study. The enrolled patients were divided into a benign nodule group and a PTC group according their pathological results. Comparison was drawn based around the difference of thyroid autoantibody distribution between groups and its correlation with the risk of PTC. RESULTS: The positive rate of TgAb in the PTC group was significantly higher than that in the benign nodule group (P<0.05). The incidence of PTC was significantly higher in TgAb positive patients in the presence of negative TPOAb (P<0.05). Further regression analysis revealed positive TgAb to be a risk factor of PTC (OR =3.097, P<0.05), while age ≥55 years old (OR =0.188, P<0.05) and nodule diameter ≥10 mm (OR =0.064, P<0.05) reduced the risk of PTC. Simultaneously, positive TgAb was also a risk factor for PTC in females (OR =3.532, P<0.05), but not in males (P>0.05). The risk of PTC in females was not associated with further increase in the titer of TgAb. CONCLUSIONS: TgAb may be associated with an increased risk of PTC in females, but there is no clear correlation between the risk of PTC and higher antibody titer in these patients.

Erratum to laparoscopic needle catheter jejunostomy by using a double semipurse string suture method in minimally invasive Ivor Lewis esophagectomy.

[This corrects the article DOI: 10.21037/jtd.2020.01.53.].

Laparoscopic needle catheter jejunostomy by using a double semipurse string suture method in minimally invasive Ivor Lewis esophagectomy.

BACKGROUND: To investigate the safety and effectiveness of a double semipurse string suture method for jejunum fixation in laparoscopic needle catheter jejunostomy in minimally invasive Ivor Lewis esophagectomy (MIILE). METHODS: Two hundred and six esophageal cancer patients continuously receiving MIILE from March 2014 to February 2018 were enrolled. In all patients, the double semipurse string suture method was applied for jejunum fixation in laparoscopic needle catheter jejunostomy. The methods and details of this technique are introduced herein. General information, clinical data, postoperative complications and follow-up results were retrospectively analyzed, and the complication causes and treatment methods are discussed. RESULTS: Laparoscopic needle catheter jejunostomy-using the double semipurse string suture method was successfully performed in 206 patients. The operative time of laparoscopic needle catheter jejunostomy was 10.56±2.04 min. No conversion to laparotomy or postoperative death or serious infection associated with the jejunostomy tube occurred. The incidence of complications associated with the jejunostomy tube was 16.50% (34/206), and most of the complications were mild. Severe complications occurred in 2 cases (0.97%), which were cured after reoperation, without serious consequence. CONCLUSIONS: The double semipurse string suture method is safe, simple and feasible for the jejunum fixation in laparoscopic needle catheter jejunostomy in MIILE. It is worth popularization and clinical application.

Thoracoscopic surgery for management of pleuroperitoneal communication complicating continuous ambulatory peritoneal dialysis: A case report.

RATIONALE: Pleuroperitoneal communication (PPC) has been reported to complicate continuous ambulatory peritoneal dialysis (CAPD). However, cases of patients in whom the results of the methylene blue dye test and peritoneopleural scintigraphy were negative and treatment was thoracoscopic surgery have been rarely reported. PATIENT CONCERNS: A 58-year-old man with end-stage chronic renal failure who underwent CAPD presented with massive right-sided hydrothorax. The pleural fluid glucose level was high. Results of both the methylene blue dye test and peritoneopleural scintigraphy were negative. DIAGNOSIS: The presence of end-stage chronic renal failure and diaphragm defects amenable to repair, which were identified during thoracoscopic surgery, indicated a definite diagnosis of PPC complicating CAPD. INTERVENTIONS AND OUTCOMES: CAPD was performed twice after the defects were repaired during thoracoscopic surgery. There was no evidence that the repaired sites were leaking again, and the patient did not complain of any discomfort during the second CAPD. LESSON: Although special methods such as the methylene blue dye test and peritoneopleural scintigraphy may not be useful in some cases, thoracoscopic surgery is still effective and reliable in diagnosing and repairing diaphragmatic defects.

Endothelial Cells Regulate Physiological Cardiomyocyte Growth via VEGFR2-Mediated Paracrine Signaling.

BACKGROUND: Heart failure, which is a major global health problem, is often preceded by pathological cardiac hypertrophy. The expansion of the cardiac vasculature, to maintain adequate supply of oxygen and nutrients, is a key determinant of whether the heart grows in a physiological compensated manner or a pathological decompensated manner. Bidirectional endothelial cell (EC)-cardiomyocyte (CMC) cross talk via cardiokine and angiocrine signaling plays an essential role in the regulation of cardiac growth and homeostasis. Currently, the mechanisms involved in the EC-CMC interaction are not fully understood, and very little is known about the EC-derived signals involved. Understanding how an excess of angiogenesis induces cardiac hypertrophy and how ECs regulate CMC homeostasis could provide novel therapeutic targets for heart failure. METHODS: Genetic mouse models were used to delete vascular endothelial growth factor (VEGF) receptors, adeno-associated viral vectors to transduce the myocardium, and pharmacological inhibitors to block VEGF and ErbB signaling in vivo. Cell culture experiments were used for mechanistic studies, and quantitative polymerase chain reaction, microarrays, ELISA, and immunohistochemistry were used to analyze the cardiac phenotypes. RESULTS: Both EC deletion of VEGF receptor (VEGFR)-1 and adeno-associated viral vector-mediated delivery of the VEGFR1-specific ligands VEGF-B or placental growth factor into the myocardium increased the coronary vasculature and induced CMC hypertrophy in adult mice. The resulting cardiac hypertrophy was physiological, as indicated by preserved cardiac function and exercise capacity and lack of pathological gene activation. These changes were mediated by increased VEGF signaling via endothelial VEGFR2, because the effects of VEGF-B and placental growth factor on both angiogenesis and CMC growth were fully inhibited by treatment with antibodies blocking VEGFR2 or by endothelial deletion of VEGFR2. To identify activated pathways downstream of VEGFR2, whole-genome transcriptomics and secretome analyses were performed, and the Notch and ErbB pathways were shown to be involved in transducing signals for EC-CMC cross talk in response to angiogenesis. Pharmacological or genetic blocking of ErbB signaling also inhibited part of the VEGF-B-induced effects in the heart. CONCLUSIONS: This study reveals that cross talk between the EC VEGFR2 and CMC ErbB signaling pathways coordinates CMC hypertrophy with angiogenesis, contributing to physiological cardiac growth.

Three-port single-intercostal versus multiple-intercostal thoracoscopic lobectomy for the treatment of lung cancer: a propensity-matched analysis.

BACKGROUND: In this retrospective study, we aimed to demonstrated that three-port single-intercostal (SIC) thoracoscopic lobectomy is an effective choice for lung cancer by comparing the perioperative outcomes of patients with non-small-cell lung cancer treated with three-port SIC and conventional multiple-intercostal (MIC) thoracoscopic lobectomy. METHODS: From January 2013 to January 2018, 642 non-small-cell lung cancer patients underwent thoracoscopic lobectomy via a three-port SIC or MIC technique. Propensity-matched analysis incorporating preoperative clinical variables was used to compare the perioperative outcomes between the two groups. RESULTS: The first 20 patients were excluded to account for the learning curve effect in the SIC group. Propensity matching yielded 186 patients in each group. A small percentage of patients had major morbidity, including 4.8% in the SIC group and 6.5% in the MIC group; there was no significant difference between the two groups. Although the total number of lymph nodes harvested (25.3 vs. 23.8, p = 0.160) and stations removed (6.5 vs. 6.7, p = 0.368) were similar between the two groups, more subcarinal lymph nodes were removed (6.9 vs. 5.2, p < 0.001) in the SIC group than in the MIC group. Furthermore, other perioperative outcomes in the SIC group were not fewer than those in the MIC group. CONCLUSIONS: Both techniques are acceptable for the treatment of non-small-cell lung cancer. Three-port SIC VATS lobectomy can provide an alternative procedure in thoracoscopic surgery.

Distribution and sources of oxygenated non-hydrocarbons in topsoil of Beijing, China.

The oxygenated non-hydrocarbon compounds are widely distributed in soil. To investigate the distribution and origin of these compounds in topsoil of Beijing, their contents and compositions were measured in topsoil from 62 sites in Beijing. The research results showed that oxygenated non-hydrocarbons were composed primarily of C6∼C28 n-fatty acids, C12∼C28 n-fatty alcohols, n-fatty acid methyl esters, phthalates, sterols, and dehydroabietic acid in the topsoil of Beijing. The contents and compositions of these compounds varied with the sampling site. The concentrations of n-fatty acids and phthalate esters were the highest at all sites, followed by sterols, n-fatty acid methyl esters, fatty alcohols, and dehydroabietic acid in order. The n-fatty acids had a main peak of C16, followed by C18. An odd or even carbon number predominance was not observed in the low-molecular-weight n-fatty acids, indicating a fossil fuel or organic matter source. However, some high-molecular-weight n-fatty acids with an even carbon predominance may derive from a biomass. The n-fatty alcohols showed a main peak of C22 and were predominated by an even carbon number, suggesting plant, microbial, or other natural origins. Phthalates, including diethyl phthalate (DEP), diisobutyl phthalate (DIBP), dibutyl phthalate (DBP), diethylhexyl phthalate (DEHP), and dimethylphthalate (DMP), were detected. The content of phthalate esters was higher in the samples collected from dense human activity areas. The concentrations of DBP, DEHP, and DIBP were relatively high, indicating an anthropogenic source. The sterols (predominantly ß-sitosterol) originated from biological sources, especially plants. The n-fatty acid methyl esters and dehydroabietic acid in topsoil showed apparent even carbon predominance with the former mainly derived from microorganisms or plants and the latter from cork combustion products.

Distribution and sources of phthalate esters in the topsoils of Beijing, China.

Phthalate esters in the topsoil samples collected from Beijing were determined by derivatization and gas chromatography-mass spectrometry techniques. The results showed that diethyl phthalate (DEP), diisobutyl phthalate ester (DIBP), dibutyl phthalate ester (DBP), dibutyl (2-ethyl hexyl) phthalate (DEHP), and dimethyl phthalate (DMP) were found in the topsoils. The total concentrations of the five phthalate esters varied from 2.30 to 24.71 µg g(-1). According to phthalate esters (PAEs) control standards in soil of the USA, the standard exceeding rates of DMP, DBP, and DEHP were 100 %, 100 %, and 4.84 % in soils of Beijing, respectively. The rate of DBP exceeding soil remediation standard was 12.9 %. Overall, concentrations of PAEs in Beijing were at a high level in China. The concentrations of DBP, DEHP, and DIBP were high, and the total concentrations of all the phthalate esters were higher in the areas with intensive human activities than in the other areas, which may be related to the use of phthalate compounds (such as the use of plastic products). The total and individual concentrations of phthalate compounds were relatively low in the areas that used plastic films compared with other samples due to the diffusion of atmospheric motion, categories, and amounts of plastic products and other factors. The greatest contributor may be the usage amount of plastic products in people's daily lives.

Intravenous glial growth factor 2 (GGF2) isoform of neuregulin-1ß improves left ventricular function, gene and protein expression in rats after myocardial infarction.

AIMS: Recombinant Neuregulin (NRG)-1ß has multiple beneficial effects on cardiac myocytes in culture, and has potential as a clinical therapy for heart failure (HF). A number of factors may influence the effect of NRG-1ß on cardiac function via ErbB receptor coupling and expression. We examined the effect of the NRG-1ß isoform, glial growth factor 2 (GGF2), in rats with myocardial infarction (MI) and determined the impact of high-fat diet as well as chronicity of disease on GGF2 induced improvement in left ventricular systolic function. Potential mechanisms for GGF2 effects on the remote myocardium were explored using microarray and proteomic analysis. METHODS AND RESULTS: Rats with MI were randomized to receive vehicle, 0.625 mg/kg, or 3.25 mg/kg GGF2 in the presence and absence of high-fat feeding beginning at day 7 post-MI and continuing for 4 weeks. Residual left ventricular (LV) function was improved in both of the GGF2 treatment groups compared with the vehicle treated MI group at 4 weeks of treatment as assessed by echocardiography. High-fat diet did not prevent the effects of high dose GGF2. In experiments where treatment was delayed until 8 weeks after MI, high but not low dose GGF2 treatment was associated with improved systolic function. mRNA and protein expression analysis of remote left ventricular tissue revealed a number of changes in myocardial gene and protein expression altered by MI that were normalized by GGF2 treatment, many of which are involved in energy production. CONCLUSIONS: This study demonstrates that in rats with MI induced systolic dysfunction, GGF2 treatment improves cardiac function. There are differences in sensitivity of the myocardium to GGF2 effects when administered early vs. late post-MI that may be important to consider in the development of GGF2 in humans.

Disruption of a GATA4/Ankrd1 signaling axis in cardiomyocytes leads to sarcomere disarray: implications for anthracycline cardiomyopathy.

Doxorubicin (Adriamycin) is an effective anti-cancer drug, but its clinical usage is limited by a dose-dependent cardiotoxicity characterized by widespread sarcomere disarray and loss of myofilaments. Cardiac ankyrin repeat protein (CARP, ANKRD1) is a transcriptional regulatory protein that is extremely susceptible to doxorubicin; however, the mechanism(s) of doxorubicin-induced CARP depletion and its specific role in cardiomyocytes have not been completely defined. We report that doxorubicin treatment in cardiomyocytes resulted in inhibition of CARP transcription, depletion of CARP protein levels, inhibition of myofilament gene transcription, and marked sarcomere disarray. Knockdown of CARP with small interfering RNA (siRNA) similarly inhibited myofilament gene transcription and disrupted cardiomyocyte sarcomere structure. Adenoviral overexpression of CARP, however, was unable to rescue the doxorubicin-induced sarcomere disarray phenotype. Doxorubicin also induced depletion of the cardiac transcription factor GATA4 in cardiomyocytes. CARP expression is regulated in part by GATA4, prompting us to examine the relationship between GATA4 and CARP in cardiomyocytes. We show in co-transfection experiments that GATA4 operates upstream of CARP by activating the proximal CARP promoter. GATA4-siRNA knockdown in cardiomyocytes inhibited CARP expression and myofilament gene transcription, and induced extensive sarcomere disarray. Adenoviral overexpression of GATA4 (AdV-GATA4) in cardiomyocytes prior to doxorubicin exposure maintained GATA4 levels, modestly restored CARP levels, and attenuated sarcomere disarray. Interestingly, siRNA-mediated depletion of CARP completely abolished the Adv-GATA4 rescue of the doxorubicin-induced sarcomere phenotype. These data demonstrate co-dependent roles for GATA4 and CARP in regulating sarcomere gene expression and maintaining sarcomeric organization in cardiomyocytes in culture. The data further suggests that concurrent depletion of GATA4 and CARP in cardiomyocytes by doxorubicin contributes in large part to myofibrillar disarray and the overall pathophysiology of anthracycline cardiomyopathy.

Circulating vascular growth factors and central hemodynamic load in the community.

Mean and pulsatile components of hemodynamic load are related to cardiovascular disease. Vascular growth factors play a fundamental role in vascular remodeling. The links between growth factors and hemodynamic load components are not well described. In 3496 participants from the Framingham Heart Study third generation cohort (mean age: 40±9 years; 52% women), we related 4 tonometry-derived measures of central arterial load (carotid femoral pulse wave velocity and forward pressure wave, mean arterial pressure, and the global reflection coefficient) to circulating concentrations of angiopoietin 2, its soluble receptor; vascular endothelial growth factor, its soluble receptor; hepatocyte growth factor; insulin-like growth factor 1; and its binding protein 3. Using multivariable linear regression models, adjusted for standard cardiovascular risk factors, serum insulin-like growth factor 1 concentrations were negatively associated with carotid femoral pulse wave velocity, mean arterial pressure, and reflection coefficient (P≤0.01 for all), whereas serum vascular endothelial growth factor levels were positively associated with carotid femoral pulse wave velocity and mean arterial pressure (P<0.04). Serum insulin-like growth factor binding protein 3 and soluble angiopoietin 2 receptor levels were positively related to mean arterial pressure and to forward pressure wave, respectively (P<0.05). In our cross-sectional study of a large community-based sample, circulating vascular growth factor levels were related to measures of mean and pulsatile hemodynamic load in a pattern consistent with the known physiological effects of insulin-like growth factor 1 and vascular endothelial growth factor.

ErbB4 localization to cardiac myocyte nuclei, and its role in myocyte DNA damage response.

The intracellular domain of ErbB4 receptor tyrosine kinase is known to translocate to the nucleus of cells where it can regulate p53 transcriptional activity. The purpose of this study was to examine whether ErbB4 can localize to the nucleus of adult rat ventricular myocytes (ARVM), and regulate p53 in these cells. We demonstrate that ErbB4 does locate to the nucleus of cardiac myocytes as a full-length protein, although nuclear location occurs as a full-length protein that does not require Protein Kinase C or γ-secretase activity. Consistent with this we found that only the non-cleavable JM-b isoform of ErbB4 is expressed in ARVM. Doxorubicin was used to examine ErbB4 role in regulation of a DNA damage response in ARVM. Doxorubicin induced p53 and p21 was suppressed by treatment with AG1478, an EGFR and ErbB4 kinase inhibitor, or suppression of ErbB4 expression with small interfering RNA. Thus ErbB4 localizes to the nucleus as a full-length protein, and plays a role in the DNA damage response induced by doxorubicin in cardiac myocytes.

Circulating neuregulin-1ß levels vary according to the angiographic severity of coronary artery disease and ischemia.

BACKGROUND: Coronary artery disease (CAD) is the leading killer in the United States. Patients with severe CAD and ischemia have worse prognosis. Therefore expansion of biomarker research, to identify at-risk individuals and explain the complex biology between cardiovascular growth factors and atherosclerosis is needed. Neuregulin-1ß (NRG-1ß) is a myocardial stress activated growth and survival factor released from endocardial and endothelial cells. NRG-1ß is essential for cardiovascular development and a regulator of angiogenesis. We postulated that plasma and serum levels of NRG-1ß would vary in relation to CAD severity and the presence of stress-induced ischemia. METHODS: We measured serum and plasma levels of NRG-1ß and vascular endothelial growth factor (VEGF) in 60 patients undergoing cardiac catheterization. CAD severity was calculated from angiographic results using a modified Duke jeopardy score. RESULTS: Serum NRG-1ß (sNRG-1ß), plasma NRG-1ß (pNRG-1ß), serum VEGF, and plasma VEGF were detectable in the majority of patients. The pNRG-1ß levels were approximately two-fold higher than sNRG-1ß. Both sNRG-1ß and pNRG-1ß correlated inversely with CAD severity. pNRG-1ß levels were statistically higher in patients with stress-induced ischemia denoted by a positive myocardial perfusion imaging study that correlated with angiographic findings (P=0.02). CONCLUSION: Both sNRG-1ß and pNRG-1ß correlated inversely with angiographic severity of CAD. pNRG-1ß levels were two-fold higher than serum and were higher in patients with stress-induced ischemia. Therefore we conclude that plasma is the optimal source for the further exploration of the biological significance of NRG-1ß as a biomarker of CAD severity and ischemia.