BACKGROUND

Identify genes probably associated with chronic heart failure and predict potential target genes for dilated cardiomyopathy using bioinformatics analyses.

METHODS

Gene expression profiles (series number GSE3585 and GSE42955) of cardiomyopathy patients and healthy controls were downloaded from the Expression Omnibus Gene (GEO) database. Differential expression of genes (DEGS) between the two groups of total 14 cardiomyopathy patients and 10 healthy controls were subsequently identified by limma package of R. Database for Annotation, Visualization, and Integrated Discovery (DAVID Tool), which is an analysis of enriched biological processes. Search Tool for the Retrieval Interacting Genes (STRING) was used as well for the analysis of protein-protein interaction network (PPI). Prediction of the potential drugs was suggested based on the preliminarily identified genes using Connectivity Map (CMap).

RESULTS

Eighty-nine DEGs were identified (57 up-regulated and 32 down-regulated). The most enrichment Gene Ontology (GO) terms (P < 0.05) contain genes involved in extracellular matrix (ECM) and biological adhesion signal pathways (P < 0.05, ES>> 1.5) such as ECM-receptors, focal adhesion and transforming growth factor beta (TGF-β), etc. Fifty-one differentially expressed genes were found to encode interacting proteins. Eleven key genes along with related transcription factors were identified including CTGF, POSTN, CORIN, FIGF, etc. CONCLUSION: Bioinformatics-based analyses reveal the targeted genes probably associated with cardiomyopathy, which provide clues for pharmacological therapies aiming at the targets.

Wound healing evaluation is important in forensic pathology, in which angiogenesis plays an important role. We have already shown that vascular endothelial growth factor A (VEGF) is produced in the rat skin incision wounds by neutrophils, endothelial cells, and fibroblasts. In this study, we assessed the changes in the mRNA expressions of various factors possibly involved in angiogenesis including angiopoietin (ANGPT) 1 and 2, cadherin 5 (CDH5), granulocyte-macrophage colony stimulating factor (CSF2/GM-CSF), granulocyte colony stimulating factor (CSF3/G-CSF), chemokine (C-X-C motif) ligand 2 (CXCL2), chemokine (C-X-C motif) ligand12 (CXCL12/SDF1), endothelin 1 (ET1), fibroblast growth factor 1 (FGF 1), hepatocyte growth factor (HGF), hypoxia inducible factor 1 alpha (HIF1a), leptin, matrix metallopepitidase 9 (MMP9), serpine/plasminogen activator inhibitor1 (PAI1), platelet-derived growth factor-A (PDGF-A), transforming growth factor alpha and beta 1 (TGFa and b1), tenomodulin (TNMD), and troponin I type 2 (TNNI2) in the early stage of the rat skin incision wounds by real time RT-PCR. Factors reported to be involved in lymphangiogenesis such as fibroblast growth factor 2 (FGF 2), c-fos induced growth factor (FIGF/VEGF-D), forkhead box C2 (FOXC2), and prospero homeobox 1 (PROX1) were also studied. One and 3 days after the dorsal skin incisions, wounds on male Sprague-Dawley rats showed the statistically significant increases in the mRNA expressions for CXCL2, CSF3, MMP9, PAI1, and CSF2, whereas TGFa, TNNI2, FGF1, TNMD, leptin, and CXCL12 showed the statistically significant decreases. Interestingly, lymphgangiogenic factors FOXC2, PROX1, and FGF2 also showed the statistically significant decreases. In situ hybridization and immunohistochemistry showed the mRNA and protein positivity in endothelial cells, fibroblasts, and some leukocytes at the bottom of the wound tissue for PAI1, CSF3, and MMP9, 1 day after the skin incisions. Our novel findings show the possible involvement of several factors involved in angiogenesis and lymphangiogenesis in the early stage of wound healing process, which may be useful for forensic wound evaluations.

We have recently described a competitive binding assay for rat insulin-like growth factor-binding protein-3 (IGFBP-3) based on the ability of IGFBP-3 to form a ternary complex with the acid-labile subunit (ALS) in the presence of IGF-I. Using this assay we studied groups of male (n = 6) and female rats (n = 6) at 20, 30, 40, 50, 60, 80, and 130 days of age. Nonfasting serum levels of IGFBP-3 were compared with those of total (extractable) IGF-I (tIGF-I) and ALS as well as IGFBP-3 determined by ligand blotting. Additionally, we studied the relationship between ultrafiltered free IGF-I (fIGF-I) and immunoassayable IGFBP-1. IGFBP-3 was dependent on age only (P < 0.0001), but tended to be higher in males than in females (P = 0.06); between 20-130 days levels increased from 6.5 +/- 1.7 to 73.6 +/- 7.2 nmol/liter in males and from 5.4 +/- 1.6 to 51.3 +/- 8.0 nmol/liter in females. IGFBP-3 correlated positively with tIGF-I (r = 0.90; P < 0.0001), ALS (r = 0.92; P < 0.0001), and IGFBP-3, as determined by ligand blotting (r = 0.88; P < 0.0001). The molar ratio of IGFBP-3 to tIGF-I increased from 0.23 +/- 0.04 to 0.76 +/- 0.04 (P < 0.0001) without any sex dependence. An age- and sex-dependent decrease in IGFBP-1 was observed (P < 0.0001), from 10.9 +/- 2.5 to 1.2 +/- 0.2 nmol/liter in females and from 8.9 +/- 0.7 to 0.2 +/- 0.04 nmol/liter in males. Free IGF-I (fIGF-I) increased with age (from 0.7 +/- 0.2 to 7.1 +/- 0.5 nmol/liter; P < 0.0001), and levels were inversely correlated with IGFBP-1 (r = -0.80; P < 0.0001). In young rats, IGFBP-1 circulated in a 10-fold molar excess over the level of fIGF-I, whereas in older rats, fIGF-I exceeded IGFBP-1 by an average of 9-fold in females and by up to almost 60-fold in males. We conclude that in rats 1) IGFBP-3 and fIGF-I are strongly age dependent; 2) IGFBP-3 correlates positively with ALS and tIGF-I; and 3) fIGF-I and IGFBP-1 are inversely correlated. This is in accordance with clinical findings. However, in humans the adult level of fIGF-I rarely exceeds 0.3 nmol/liter, and IGFBP-1 usually circulates in excess of fIGF-I. Thus, our results also imply species differences in the IGF systems of humans and rats.

Despite the well-accepted notion that early maternal influences persist beyond fetal life and may underlie many adult diseases, the risks imposed by the maternal environment on breast cancer development and underlying biological mechanisms remain poorly understood. In this study, we investigated whether early exposure to blueberry (BB) via maternal diet alters oncogene Wnt1-induced mammary tumorigenesis in offspring. Wnt1-transgenic female mice were exposed to maternal Casein (CAS, control) or blueberry-supplemented (CAS + 3%BB) diets throughout pregnancy and lactation. Offspring were weaned to CAS and mammary tumor development was followed until age 8 months. Tumor incidence and latency were similar for both groups; however, tumor weight at killing and tumor volume within 2 weeks of initial detection were lower (by 50 and 60%, respectively) in offspring of BB- versus control-fed dams. Dietary BB exposure beginning at weaning did not alter mammary tumor parameters. Tumors from maternal BB-exposed offspring showed higher tumor suppressor (Pten and Cdh1) and lower proproliferative (Ccnd1), anti-apoptotic (Bcl2) and proangiogenic (Figf, Flt1 and Ephb4) transcript levels, and displayed attenuated microvessel density. Expression of Pten and Cdh1 genes was also higher in mammary tissues of maternal BB-exposed offspring. Mammary tissues and tumors of maternal BB-exposed offspring showed increased chromatin-modifying enzyme Dnmt1 and Ezh2 transcript levels. Body weight, serum insulin and serum leptin/adiponectin ratio were lower for maternal BB-exposed than control tumor-bearing offspring. Tumor weights and serum insulin were positively correlated. Results suggest that dietary influences on the maternal environment contribute to key developmental programs in the mammary gland to modify breast cancer outcome in adult progeny.

Pulmonary lymphangioleiomyomatosis (LAM), a rare progressive lung disease associated with mutations of the tuberous sclerosis complex 2 (Tsc2) tumor suppressor gene, manifests by neoplastic growth of LAM cells, induction of cystic lung destruction, and respiratory failure. LAM severity correlates with upregulation in serum of the prolymphangiogenic vascular endothelial growth factor D (VEGF-D) that distinguishes LAM from other cystic diseases. The goals of our study was to determine whether Tsc2 deficiency upregulates VEGF-D, and whether axitinib, the Food and Drug Administration-approved small-molecule inhibitor of VEGF receptor (VEGFR) signaling, will reduce Tsc2-null lung lesion growth in a mouse model of LAM. Our data demonstrate upregulation of VEGF-D in the serum and lung lining in mice with Tsc2-null lesions. Progressive growth of Tsc2-null lesions induces recruitment and activation of inflammatory cells and increased nitric oxide production. Recruited cells isolated from the lung lining of mice with Tsc2-null lesions demonstrate upregulated expression of provasculogenic Vegfa, prolymphangiogenic Figf, and proinflammatory Nos2, Il6, and Ccl2 genes. Importantly, axitinib is an effective inhibitor of Tsc2-null lesion growth and inflammatory cell recruitment, which correlates with reduced VEGF-D levels in serum and lung lining. Our data demonstrate that pharmacological inhibition of VEGFR signaling with axitinib inhibits Tsc2-null lesion growth, attenuates recruitment and activation of inflammatory cells, and reduces VEGF-D levels systemically and in the lung lining. Our study suggests a potential therapeutic benefit of inhibition of VEGFR signaling for treatment of LAM.

BACKGROUND

Invasive lobular carcinoma (ILC) comprises approximately ~10-20% of breast cancers. In general, multifocal/multicentric (MF/MC) breast cancer has been associated with an increased rate of regional lymph node metastases. Tumor heterogeneity between foci represents a largely unstudied source of genomic variation in those rare patients with MF/MC ILC.

METHODS

We characterized gene expression and copy number in 2 or more foci from 11 patients with MF/MC ILC (all ER+, HER2-) and adjacent normal tissue. RNA and DNA were extracted from 3x1.5 mm cores from all foci. Gene expression (730 genes) and copy number (80 genes) were measured using Nanostring PanCancer and Cancer CNV panels. Linear mixed models were employed to compare expression in tumor versus normal samples from the same patient, and to assess heterogeneity (variability) in expression among multiple ILC within an individual.

RESULTS

35 and 34 genes were upregulated (FC>2) and down-regulated (FC<0.5) respectively in ILC tumor relative to adjacent normal tissue, q<0.05. 9/34 down-regulated genes (FIGF, RELN, PROM1, SFRP1, MMP7, NTRK2, LAMB3, SPRY2, KIT) had changes larger than CDH1, a hallmark of ILC. Copy number changes in these patients were relatively few but consistent across foci within each patient. Amplification of three genes (CCND1, FADD, ORAOV1) at 11q13.3 was present in 2/11 patients in both foci. We observed significant evidence of within-patient between-foci variability (heterogeneity) in gene expression for 466 genes (p<0.05 with FDR 8%), including CDH1, FIGF, RELN, SFRP1, MMP7, NTRK2, LAMB3, SPRY2 and KIT.

CONCLUSIONS

There was substantial variation in gene expression between ILC foci within patients, including known markers of ILC, suggesting an additional level of complexity that should be addressed.

To date, there have been no detailed studies on the lymphatic system in the primate corpus luteum (CL); early reports suggested that the presence of this "secondary circulation" in luteal tissue is species-dependant. Therefore, studies were designed to determine if (a) lymphatic vessels exist, and (b) recently discovered lymphangiogenic factors and their receptor are expressed in the macaque CL during the menstrual cycle. Immunohistochemistry (IHC) detected the lymphatic endothelial cell marker, lymphatic vessel endothelial hyaluronan receptor 1 (LYVE1), in some endothelial cells and vessels within the ovarian stroma and theca layer of preovulatory follicles and in the CL. Dual fluorescent IHC demonstrated that LYVE1 co-localized with another lymphatic endothelial cell marker D2-40, but a blood vascular endothelial cell marker (von Willebrand Factor, VWF) was in different cells. The numbers and staining intensity of LYVE1-positive cells in the CL appeared to increase from early to mid luteal phase, and remained elevated thereafter. RT-PCR detected cDNA fragments for mRNAs encoding VEGFC, FIGF, and their receptor FLT4 in CL. Real-time PCR analyses revealed similar patterns of VEGFC and FLT4 expression during the luteal lifespan; mRNA levels increased (p < 0.05) from early to mid luteal phase and decreased (p < 0.05) by late luteal phase. In contrast, FIGF levels were elevated initially, declined (p < 0.05) at mid luteal phase, and then increased (p < 0.05) to very late luteal phase. The data strongly suggest that lymphatic vessels are present in the primate CL, and that the VEGFC/FIGF-FLT4 system regulates lymphangiogenesis and luteal structure-function during the menstrual cycle.

BACKGROUND

Prostatic inflammation is an important factor in development and progression of BPH/LUTS. This study was performed to characterize the normal development and vascular anatomy of the mouse prostate and then examine, for the first time, the effects of prostatic inflammation on the prostate vasculature.

METHODS

Adult mice were perfused with India ink to visualize the prostatic vascular anatomy. Immunostaining was performed on the E16.5 UGS and the P5, P20, and adult prostate to characterize vascular development. Uropathogenic E. coli 1677 was instilled transurethrally into adult male mice to induce prostate inflammation. RT-PCR and BrdU labeling was performed to assay anigogenic factor expression and endothelial proliferation, respectively.

RESULTS

An artery on the ventral surface of the bladder trifurcates near the bladder neck to supply the prostate lobes and seminal vesicle. Development of the prostatic vascular system is associated with endothelial proliferation and robust expression of pro-angiogenic factors Pecam1, Tie1, Tek, Angpt1, Angpt2, Fgf2, Vegfa, Vegfc, and Figf. Bacterial-induced prostatic inflammation induced endothelial cell proliferation and increased vascular density but surprisingly decreased pro-angiogenic factor expression.

CONCLUSIONS

The striking decrease in pro-angiogenic factor mRNA expression associated with endothelial proliferation and increased vascular density during inflammation suggests that endothelial response to injury is not a recapitulation of normal development and may be initiated and regulated by different regulatory mechanisms.

In previous studies Sulf2 has been evidenced to play an important role in tumor progression through editing sulfate moieties on heparan sulfate proteoglycans (HSPGs) and modulating heparin binding growth factors. However, the role of Sulf2 in breast cancer progression is still poorly understood. In the present study, we hypothesized that Sulf2 promoted breast cancer progression. Two different breast cancer cell lines, MCF-7 and MDA-MB-231, were chosen for this study because of high and low Sulf2 expression levels. We also altered their Sulf2 expression by establishing Sulf2 knockdown and overexpressing breast cancer cell lines MCF-7 shSulf2 and MDA-MB-231 Sulf2. To evaluate the functions of Sulf2, cell proliferation, apoptosis, cell cycle, invasion, mobility and adhesion of these cell lines were measured in vitro, and xenograft formation, invasion and metastasis ability were examined in vivo. Furthermore, expression of related genes were screened and were certified in these cell lines. We found that Sulf2 increased breast cancer proliferation, invasion, mobility and adhesion both in vitro and in vivo. Sulf2 also decreased cisplatin inducing breast cancer apoptosis without affecting the cell cycle. Sulf2 upregulated c-fos induced growth factor (FIGF) and nuclear receptor subfamily 4 group A member 3 (NR4A3) expression and downregulated the cluster of differentiation 82 (CD82) and platelet-derived growth factor C (PDGFC) expression in breast cancer. Our data confirmed that Sulf2 promoted breast cancer progression and regulated the expression of tumor-related genes in breast cancer.

OBJECTIVE

Right ventricular failure (RVF) varies significantly from the more common left ventricular failure (LVF). This study was undertaken to determine potential molecular pathways that are important in human right ventricular (RV) function and may mediate RVF.

METHODS

We analyzed mRNA of human non-failing LV and RV samples and RVF samples from patients with pulmonary arterial hypertension (PAH), and post-LVAD implantation. We then performed transcript analysis to determine differential expression of genes in the human heart samples. Immunoblot quantification was performed followed by analysis of non-failing and failing phenotypes.

RESULTS

Inflammatory pathways were more commonly dysregulated in RV tissue (both non-failing and failing phenotypes). In non-failing human RV tissue we found important differences in expression of FIGF, TRAPPAC, and CTGF suggesting that regulation of normal RV and LV function are not the same. In failing RV tissue, FBN2, CTGF, SMOC2, and TRAPP6AC were differentially expressed, and are potential targets for further study.

CONCLUSIONS

This work provides some of the first analyses of the molecular heterogeneity between human RV and LV tissue, as well as key differences in human disease (RVF secondary to pulmonary hypertension and LVAD mediated RVF). Our transcriptional data indicated that inflammatory pathways may be more important in RV tissue, and changes in FIGF and CTGF supported this hypothesis. In PAH RV failure samples, upregulation of FBN2 and CTGF further reinforced the potential significance that altered remodeling and inflammation play in normal RV function and failure.

Renal cell carcinoma (RCC) consists of the most lethal common urological cancer and the clinical practice has shown that resistant RCC to commons therapies is extremely high. Berberine is an isoquinoline alkaloid, presents in different kinds of plants and it has long been used in Chinese medicine. It has several properties, such as antioxidant, anti-inflammatory, anti-diabetic, anti-microbial and anti-cancer. Moreover, berberine has photosensitive characteristics and its association with photodynamic therapy (PDT) is effective against tumor cells. This study aimed to evaluate the effects of berberine associated with PDT in renal carcinoma cell lines. The cellular viability assay showed increased cytotoxicity in concentration and time-dependent manner. Berberine presented efficient internalization in all cell lines analyzed. In addition, after treatment with berberine associated with PDT, it was observed a high phototoxicity eff ;ect with less than 20 % of viable cells. In this study we observed that the increase of reactive oxygen species (ROS) levels was accompanied by an increase of autophagy levels and apoptosis by caspase 3 activity, suggesting cell death by both mechanisms. Additionally, three target genes of anti-cancer drugs were differentially expressed in 786-O cells, being that Vascular Endothelial Growth Factor-D (FIGF) and Human Telomerase Reverse Transcriptase (TERT) gene presented low expression and Polo Like Kinase 3 (PLK3) presented overexpression after treatment with berberine associated with PDT. In this study, the proposed treatment triggered metabolites changes related to cell proliferation, tumorigenesis and angiogenesis. Thus, it was possible to suggest that berberine has promising potential as a photosensitizing agent in a photodynamic therapy, because it induced significant anticancer effects on renal carcinoma cells.

Chemotherapeutics are sometimes administered with drugs, like antiangiogenic compounds, to increase their effectiveness. Melatonin exerts antitumoral actions through antiangiogenic actions. We studied if melatonin regulates the response of HUVECs to chemotherapeutics (docetaxel and vinorelbine). The inhibition that these agents exert on some of the processes involved in angiogenesis, such as, cell proliferation, migratory capacity or vessel formation, was enhanced by melatonin. Regarding to estrogen biosynthesis, melatonin impeded the negative effect of vinorelbine, by decreasing the activity and expression of aromatase and sulfatase. Docetaxel and vinorelbine increased the expression of VEGF-A, VEGF-B, VEGF-C, VEGFR-1, VEGFR-3, ANG1 and/or ANG-2 and melatonin inhibited these actions. Besides, melatonin prevented the positive actions that docetaxel exerts on the expression of other factors related to angiogenesis like JAG1, ANPEP, IGF-1, CXCL6, AKT1, ERK1, ERK2, MMP14 and NOS3 and neutralized the stimulating actions of vinorelbine on the expression of FIGF, FGFR3, CXCL6, CCL2, ERK1, ERK2, AKT1, NOS3 and MMP14. In CAM assay melatonin inhibited new vascularization in combination with chemotherapeutics. Melatonin further enhanced the chemotherapeutics-induced inhibition of p-AKT and p-ERK and neutralized the chemotherapeutics-caused stimulatory effect on HUVECs permeability by modifying the distribution of VE cadherin. Our results confirm that melatonin blocks proangiogenic and potentiates antiangiogenic effects induced by docetaxel and vinorelbine enhancing their antitumor effectiveness.

We have studied the effects of insulin on the bioavailability of insulin-like growth factor (IGF) I in insulin-resistant patients after surgery. Serum levels of total IGF-I (tIGF-I), free IGF (fIGF)-I, fIGF-II, and IGF-binding protein (IGFBP) 1 and IGFBP-3 proteolytic activity (IGFBP-3-PA), determined on the day before surgery and on the 1st postoperative day, were related to insulin sensitivity measured by a hyperinsulinemic, normoglycemic clamp. Before surgery, the decreased tIGF-I (P < 0.05) in response to insulin infusion was accompanied by an 18% reduction of IGFBP-1 (P < 0.001), while IGFBP-3-PA remained unchanged. Levels of fIGF-I and fIGF-II were not changed by insulin infusions. After surgery, IGFBP-3-PA increased (P < 0.05) during insulin infusion, and this was associated with an increase in tIGF-I (P < 0.001) and fIGF-I (P < 0.01), while no significant change was found in fIGF-II. The reduction in IGFBP-1 in response to insulin infusion was not affected by surgery. The change in IGFBP-3-PA during insulin infusion after surgery was related to the corresponding change in fIGF-I (r(2) = 0.26, P < 0.05) and postoperative insulin sensitivity (r(2) = -0.22, P < 0.05). These data suggest that increased IGFBP-3-PA during insulin infusion after surgery governs the increased levels of fIGF-I, while insulin-induced suppression of IGFBP-1 was not affected by surgery. We propose that, in catabolic, postoperative patients, increased levels of insulin from exogenous or, possibly, endogenous sources (nutritionally induced) may be a signal to increase IGF-I bioavailability by increased expression of IGFBP-3-PA to counteract further deterioration in glucose metabolism.

Muscle characteristics such as myofiber diameter, density, and total number are important traits in broiler breeding and production. In the present study, 19 SNP of 13 major genes, which are located in the vicinity of quantitative trait loci affecting breast muscle weight, including INS, IGF2, PIK3C2A, AKT3, PRKAB2, PRKAG3, VEGFA, RPS6KA2/3, FIGF, and TGF-β1/2/3, were chosen to be genotyped by high-throughput matrix-assisted laser desorption/ionization time-of-flight mass spectrometry in a broiler population. One hundred twenty birds were slaughtered at 6 wk of age. Body weight, breast muscle weight, myofiber diameter, density, and total number were determined for each bird. Six SNP with a very low minor allele frequency (<1%) were excluded for further analysis. The remaining 13 SNP were used for the association study with muscle characteristics. The results showed that SNP in TGF-β1/2/3 had significant effects on myofiber diameter. A SNP in PRKAG3 had a significant effect on myofiber density (P < 0.05). A C>> G mutation in FIGF was strongly associated with total fiber number (P < 0.05). Additionally, birds with the GG genotype of the C>> G mutation in AKT3 had significantly larger myofiber numbers (P < 0.05) than birds with the CC or GC genotype. The SNP identified in the present study might be used as potential markers in broiler breeding.

The main circulating insulin-like growth factor-binding protein (IGFBP) in human, IGFBP-3, markedly decreases during pregnancy, as determined by Western ligand blotting, and its decrease is due to an endogenous pregnancy-related serum IGFBP-3 proteolytic activity. Proteolysis of IGFBP-3 may result in the free form of IGF-I (fIGF-I) being liberated from or weakly bound to IGFBP-3 fragments. The purpose of this study was to determine the plasma concentration of fIGF-I and the mechanism of fIGF-I regulation during normal human pregnancy. We studied 19 normal pregnant women at 6-8 weeks gestation, 24 normal pregnant women at 28-30 weeks gestation, and 25 nonpregnant women. We measured plasma fIGF-I using a recently established immunoradiometric assay. We also measured plasma total IGF-I (free plus complexed forms of IGF-I) using immunoradiometric assay, and IGFBP-3 proteolytic activity using a IGFBP-3 protease assay. fIGF-I levels at 6-8 weeks gestation (4.11 +/- 0.23 ng/mL) and at 28-30 weeks gestation (3.67 +/- 0.21 ng/mL) were significantly higher than that in nonpregnant women (2.48 +/- 0.12 ng/mL; P < 0.001 in both). Total IGF-I levels at 6-8 weeks gestation (168.0 +/- 9.1 ng/mL) and at 28-30 weeks gestation (241.5 +/- 19.0 ng/mL) were significantly lower than that in nonpregnant women (283.9 +/- 12.3 ng/mL; P < 0.001 at 6-8 weeks gestation and P < 0.05 at 28-30 weeks gestation). All pregnant serum studied had IGFBP-3 proteolytic activity. These data indicate that during normal human pregnancy, circulatory fIGF-I increases, probably due to IGFBP-3 proteolytic activity.

It has been found that Arabian and Thoroughbred horses differ in muscle fiber structure and thus in physiological changes occurring in muscles during exercise. The aim of the present study was to identify the global gene expression modifications that occur in skeletal muscle following a training regime to prepare for flat racing. Whole transcriptomes of muscle (gluteus medius) were compared between three time points of tissue collection: T0 (untrained horses), T1 (horses after intense gallop phase), and T2 (horses at the end of racing season), 23 samples in total. The numerous groups of exercise-regulated differentially expressed genes (DEGs) were related to muscle cell structure and signaling and included insulin-like growth factor 1 receptor (IGF1R), insulin receptor (INSR), transforming growth factor beta receptors 1 and 2 (TGFBR1, TGFBR2), vascular endothelial growth factor B (VEGFB); epidermal growth factor (EGF), hepatocyte growth factor (HGF), and vascular endothelial growth factor D (FIGF). In Arabian horses, exercise modified the expression of genes belonging to the PPAR signaling pathway (e.g., PPARA, PPARD, and PLIN2), calcium signaling pathway, and pathways associated with metabolic processes (e.g., oxidative phosphorylation, fatty acid metabolism, glycolysis/gluconeogenesis, and citrate cycle). According to detected gene expression modifications, our results suggested that in Arabian horses, exercise switches energy generation toward fatty acid utilization and enhances glycogen transport and calcium signaling. The use of the RNA-Seq approach in analyzing the skeletal muscle transcriptome allowed for the proposal of a panel of new candidate genes potentially related to body homeostasis maintenance and racing performance in Arabian horses.

The signal regulatory network involved in colorectal cancer metastasis is complicated and thus the search for key control steps in the network is of great significance for unraveling colorectal cancer metastasis mechanism and finding drug-target site. Previous studies suggested that CREB5 (cAMP responsive element binding protein 5) might play key role in the metastatic signal network of colorectal cancer. Through colorectal cancer expression profile and enriching analysis of the effect of CREB5 gene expression levels on colorectal cancer molecular events, we found that these molecular events are correlated with tumor metastasis. Based on the feature that CREB5 could combine with c-Jun to form heterodimer, together with enriched binding sites for transcription factor AP-1, we identified 16 genes which were up-regulated in the CREB5 high-expression group, contained AP-1 binding sites, and participated in cancer pathway. The molecular network involving these 16 genes, in particular, CSF1R, MMP9, PDGFRB, FIGF and IL6, regulates cell migration. Therefore, CREB5 might accelerate the metastasis of colorectal cancer by regulating these five key genes.

Enucleation-induced rapid proliferation of adrenocortical cells and restoration of adrenals structure requires formation of new blood vessels. The performed studies aimed to select from around 30,000 transcripts, identified by means of Affymetrix(®) Rat Gene 1.1 ST Array, the genes involved in angiogenesis in the course of enucleation-induced adrenal regeneration and to characterize their expression levels in regenerating gland between days 1 and 15 after surgery. At day 1 of regeneration almost 2000 genes showed more than 2-fold up/down-regulation. At days 1-3 after surgery the highest expression demonstrated genes involved in the development of inflammation and blood clot formation. From around 2000 genes we selected genes involved in angiogenesis. During the regeneration 62 genes involved in angiogenesis were identified as up- or down-regulated. Some data were also validated by QPCR. Levels of Vegfa and Kdr (Vegfr-2) mRNAs were very low at day 1 of regeneration and remained unchanged thereafter. The highest expression of Figf gene was found at day 5 while that of Vwf gene at days 1 and 2 after surgery. Levels of Thy1 mRNA increased notably between days 2 and 5 of the experiment. In comparison to control rats, Mc2r (ACTH receptor) expression was lowered at day 1 of the experiment and remained unchanged thereafter. This suggests that enucleation-induced adrenal neoangiogenesis does not require elevated expression of ACTH receptor. Results of our studies strongly suggest that enucleation-induced adrenal regeneration is an angiogenesis-dependent process. Moreover, immunohistochemistry suggests that regenerating adrenal parenchymal cells release numerous angiogenic factors which paracrinally may regulate formation of new vessels.

Insulin receptor (IR) and IGF-I receptor (IGF-IR) are structurally and functionally related and belong to the tyrosine kinase receptor family. In teleosti such as salmonids and turbot, occurrence of multiple IR and IGF-IR members has been reported, but the structures of a complete set of both IR and IGF-IR members in a single teleost species have not yet been characterized. In this study, we cloned and analysed four distinct cDNA clones for IR and IGF-IR members from the liver and kidney of the Japanese flounder (Paralichthys olivaceus). Deduced amino acid sequence analyses and phylogenetic analysis have revealed that two of them (fIR-1 and fIR-2) belong to IR members and the other two (fIGF-IR-1 and fIGF-IR-2) are IGF-IRs. fIR-1 and fIR-2 comprised 1369 and 1368 amino acid residues respectively, and fIGF-IR-1 and fIGF-IR-2 comprised 1412 and 1418 residues respectively. All the receptor proteins contained cysteine-rich domains in their alpha-subunits, and conserved each transmembrane and tyrosine kinase domains in their beta-subunits. The amino acid sequences of fIRs and fIGF-IRs showed more than 90% sequence identity with turbot IR and IGF-IR respectively. When compared with their mammalian homologues, fIGF-IR-1 and fIGF-IR-2 proteins contained large insertions at their C-termini, as was observed in the corresponding region of turbot IGF-IR. Occurrence of multiple species of mRNA for each IR and IGF-IR was suggested by Northern blot analyses. A ribonuclease protection assay revealed diverse expressions of four receptor mRNAs in a wide range of tissues including heart, liver, ovary, testis, brain, gill arch, kidney, skeletal muscle, intestine, stomach, spleen and eye of the flounder.

Melioidosis is an underreported infectious disease, caused by the Gram-negative bacterium Burkholderia pseudomallei. Understanding the disease susceptibility and pathogenesis is crucial for developing newer diagnostic and therapeutic strategies for this life-threatening infection. In this study, we aimed to analyze the gene expression levels of important cytokines in melioidosis patients and establish useful correlates with disease biomarkers compared to cases of sepsis infection caused by other pathogens and healthy individuals. A Qiagen common human cytokines array profiling the gene expression of 84 important cytokines by real-time quantitative PCR (RT-qPCR) was used. We analyzed 26 melioidosis cases, 5 healthy controls, and 10 cases of sepsis infection caused by other pathogens. Our results showed consistently upregulated expression of interleukins (IL) interleukin-4 (IL-4), interleukin-17 alpha (IL-17A), IL-23A, and IL-24, interferons (IFN) interferon alpha 1 (IFNA1) and interferon beta 1 (IFNB1), tumor necrosis factor (TNF) superfamily 4 (TNFSF4), transforming growth factor (TGF) superfamily, bone morphogenetic proteins 3 and 6 (BMP3 and BMP6), transforming growth factor beta 1 (TGFB1), and other growth factors, including macrophage colony-stimulating factor (M-CSF), C-fos-induced growth factor (FIGF), and platelet-derived growth factor alpha (PDGFA) polypeptide, in melioidosis patients compared to their expression in other sepsis cases, irrespective of comorbidities, duration of fever/clinical symptoms, and antibiotic treatment. Our findings indicate a dominant Th2- and Th17-type-cytokine response, suggesting that their dysregulation at initial stages of infection may play an important role in disease pathogenesis. IL-1A, interleukin-1 beta (IL-1B), and IL-8 were significantly downregulated in septicemic melioidosis patients compared to their expression in other sepsis cases. These differentially expressed genes may serve as biomarkers for melioidosis diagnosis and targets for therapeutic intervention and may help us understand immune response mechanisms. IMPORTANCE Melioidosis is a life-threatening infectious disease caused by a soil-associated Gram-negative bacterium, B. pseudomallei. Melioidosis is endemic in Southeast Asia and northern Australia; however, the global distribution of B. pseudomallei and the disease burden of melioidosisis are still poorly understood. Melioidosis is difficult to treat, as B. pseudomallei is intrinsically resistant to many antibiotics and requires a long course of antibiotic treatment. The mortality rates remain high in areas of endemicity, with reoccurrence being common. Therefore, it is imperative to diagnose the disease at an early stage and provide vital clinical care to reduce the mortality rate. With limitations in treatment and lack of a vaccine, it is crucial to study the immune response mechanisms to this infection to get a better understanding of disease susceptibility and pathogenesis. Therefore, this study aimed to analyze the gene expression levels of important cytokines to establish useful correlations for diagnostic and therapeutic purposes.

Prenatal stress (PS) exposure is known to increase the risk of developing emotional disorders like major depression in later life. However, some individuals do not succumb to adversity following developmental stress exposure, a phenomenon referred to as resilience. To date, the molecular mechanisms explaining why some subjects are vulnerable and others more resilient to PS are far from understood. Recently, we have shown that the serotonin transporter (5-HTT) gene may play a modulating role in rendering individuals susceptible or resilient to PS. However, it is not clear which molecular players are mediating the interaction between PS and the 5-Htt genotype in the context of vulnerability and resilience to PS. For this purpose, we performed a microarray study with the help of Affymetrix GeneChip® Mouse Genome 430 2.0 Array, in which we separated wild-type and heterozygous 5-Htt-deficient (5-Htt+/-) PS offspring into susceptible and resilient offspring according to their performance in the forced swim test. Performance-oriented LIMMA analysis on the mRNA expression microarray data was followed by subsequent Spearman's correlation analysis linking the individual qRT-PCR mRNA expression data to various anxiety- and depression-related behavioral and neuroendocrine measures. Results indicate that, amongst others, Fos-induced growth factor (Figf), galanin receptor 3 (Galr3), growth hormone (Gh) and prolactin (Prl) were differentially expressed specifically in resilient offspring when compared to controls, and that the hippocampal expression of these genes showed several strong correlations with various measures of the hypothalamus-pituitary-adrenal axis (re)activity. In conclusion, there seems to be an intricate interplay between the expression of Figf, Galr3, Gh and Prl and neuroendocrine regulation, which may be critical in mediating resilience to PS exposure. More insight into the exact role of these molecular players may significantly enhance the development of new treatment strategies for stress-related emotional disorders.

BACKGROUND

20(S)-hydroxycholesterol (20(S)) potentially reduces adipogenesis in mammalian cells. The role of this oxysterol and molecular mechanisms underlying the adipogenesis of preadipocytes from laying hens have not been investigated. This study was conducted to 1. Analyze genes differentially expressed between preadipocytes treated with an adipogenic cocktail (DMIOA) containing 500 nM dexamethasone, 0.5 mM 3-isobutyl-1-methylxanthine, 20 μg/mL insulin and 300 μM oleic acid (OA) and control cells and 2. Analyze genes differentially expressed between preadipocytes treated with DMIOA and those treated with DMIOA + 20(S) using Affymetrix GeneChip® Chicken Genome Arrays.

RESULTS

In experiment one, where we compared the gene expression profile of non-treated (control) cells with those treated with DMIOA, out of 1,221 differentially expressed genes, 755 were over-expressed in control cells, and 466 were over-expressed in cells treated with DMIOA. In experiment two, where we compared the gene expression profile of DMIOA treated cells with those treated with DMIOA+20(S), out of 212 differentially expressed genes, 90 were over-expressed in cells treated with DMIOA, and 122 were over-expressed in those treated with DMIOA+20(S). Genes over-expressed in control cells compared to those treated with DMIOA include those involved in cell-to-cell signaling and interaction (IL6, CNN2, ITGB3), cellular assembly and organization (BMP6, IGF1, ACTB), and cell cycle (CD4, 9, 38). Genes over-expressed in DMIOA compared to control cells include those involved in cellular development (ADAM22, ADAMTS9, FIGF), lipid metabolism (FABP3, 4 and 5), and molecular transport (MAP3K8, PDK4, AGTR1). Genes over-expressed in cells treated with DMIOA compared with those treated with DMIOA+20(S) include those involved in lipid metabolism (ENPP2, DHCR7, DHCR24), molecular transport (FADS2, SLC6A2, CD36), and vitamin and mineral metabolism (BCMO1, AACS, AR). Genes over-expressed in cells treated with DMIOA+20(S) compared with those treated with DMIOA include those involved in cellular growth and proliferation (CD44, CDK6, IL1B), cellular development (ADORA2B, ATP6VOD2, TNFAIP3), and cell-to-cell signaling and interaction (VCAM1, SPON2, VLDLR).

CONCLUSIONS

We identified important adipogenic regulators and key pathways that would help to understand the molecular mechanism of the in vitro adipogenesis in laying hens and demonstrated that 20(S) is capable of suppressing DMIOA-induced adipogenesis.

The aim of the present study was to explore the toxic effects and underlying mechanisms of nickel ions during therapeutic nickel‑based alloy‑treatment in congenital heart disease by investigating the metal‑induced cytotoxicity to the human monocyte‑derived macrophage cell line THP‑1. THP‑1 cells were treated with NiCl2·6H2O (25, 50, 100, 200, 400 and 800 µM) for 24, 48 and 72 h, respectively. MTT was applied to detect THP‑1 cell proliferation following NiCl2 treatment. Apoptosis of THP‑1 cells was quantified using flow cytometry. Illumina sequencing was used for screening the associated genes, whose mRNA expression levels were further confirmed by quantitative real‑time polymerase chain reaction. High concentrations of nickel ions had a significant suppressive effect on cell proliferation at the three concentrations investigated (200, 400 and 800 µM). Treatment with nickel ions (25‑400 µM) for 48 h reduced cell viability in a dose‑dependent manner. The mRNA expression levels of RELB, FIGF, SPI‑1, CXCL16 and CRLF2 were significantly increased following nickel treatment. The results of the present study suggested that nickel ions exert toxic effects on THP‑1 cell growth, which may indicate toxicity of the nickel ion during treatment of congenital heart disease. The identification of genes modified by the toxic effects of nickel on THP‑1 cells (EPOR, RELB, FIGF, SPI‑1, TGF‑β1, CXCL16 and CRLF2) may aid in the development of interventional measures for the treatment/prevention of nickel ion‑associated toxic effects during the treatment of congenital heart disease.

BACKGROUND

Ranibizumab (Lucentis®) is a Fab-antibody fragment developed from Bevacizumab, a full-length anti-VEGF antibody. Both compounds are used for treating age-related macular degeneration (AMD). The influence of bevacizumab and ranibizumab on genes involved in signal transduction and cell signaling downstream of VEGF were compared in order to detect possible differences in their mode of action, which are not related to their Fab-antibody fragments.

METHODS

Human umbilical vein cell lines (EA.hy926) and retinal pigment epithelial cells (ARP-19) were exposed to oxidative stress. The cells were treated with therapeutic concentrations of bevacizumab (0.25 mg/mL) and ranibizumab (125 mg/mL) for 24 hours prior to all experiments, and their effects on gene expressions were determined by RT- PCR.

RESULTS

After exposure to bevacizumab, more genes in the endothelial cells were up-regulated (KDR, NFATc2) and down-regulated (Pla2g12a, Rac2, HgdC, PRKCG) compared to non-treated controls. After exposure to ranibizumab, fewer genes were up-regulated (PTGS2) and down-regulated (NOS3) compared to controls. In comparison between drugs, more genes were up-regulated (NFATc2 and KDR) and more were down-regulated (Pla2g12a, Pla2g1b, Ppp3r2, Rac2) by bevacizumab than by ranibizumab. In RPE cells, NOS3 and PGF were up-regulated and Pla2g12b was down-regulated after exposure to ranibizumab, while PIK3CG was up-regulated and FIGF was down-regulated after exposure to bevacizumab, but the differences in gene expression were minor between drugs (PIK3CGand PGF were down-regulated more by ranibizumab than by bevacizumab).

CONCLUSIONS

The different gene expressions after exposure to ranibizumab and bevacizumab in endothelial and RPE cells may indicate a somewhat different biological activity of the two compounds.