Carbon monoxide (CO) can arrest cellular respiration, but paradoxically, it is synthesized endogenously by heme oxygenase type 1 (Ho-1) in response to ischemic stress. Ho-1-deficient (Hmox1-/-) mice exhibited lethal ischemic lung injury, but were rescued from death by inhaled CO. CO drove ischemic protection by activating soluble guanylate cyclase and thereby suppressed hypoxic induction of the gene encoding plasminogen activator inhibitor-1 (PAI-1) in mononuclear phagocytes, which reduced accrual of microvascular fibrin. CO-mediated ischemic protection observed in wild-type mice was lost in mice null for the gene encoding PAI-1 (Serpine1). These data establish a fundamental link between CO and prevention of ischemic injury based on the ability of CO to derepress the fibrinolytic axis. These data also point to a potential therapeutic use for inhaled CO.

The PREDIMED (PREvención con DIeta MEDiterránea) multicenter, randomized, primary prevention trial assessed the long-term effects of the Mediterranean diet (MeDiet) on clinical events of cardiovascular disease (CVD). We randomized 7447 men and women at high CVD risk into three diets: MeDiet supplemented with extra-virgin olive oil (EVOO), MeDiet supplemented with nuts, and control diet (advice on a low-fat diet). No energy restriction and no special intervention on physical activity were applied. We observed 288 CVD events (a composite of myocardial infarction, stroke or CVD death) during a median time of 4.8years; hazard ratios were 0.70 (95% CI, 0.53-0.91) for the MeDiet+EVOO and 0.70 (CI, 0.53-0.94) for the MeDiet+nuts compared to the control group. Respective hazard ratios for incident diabetes (273 cases) among 3541 non-diabetic participants were 0.60 (0.43-0.85) and 0.82 (0.61-1.10) for MeDiet+EVOO and MeDiet+nuts, respectively versus control. Significant improvements in classical and emerging CVD risk factors also supported a favorable effect of both MeDiets on blood pressure, insulin sensitivity, lipid profiles, lipoprotein particles, inflammation, oxidative stress, and carotid atherosclerosis. In nutrigenomic studies beneficial effects of the intervention with MedDiets showed interactions with several genetic variants (TCF7L2, APOA2, MLXIPL, LPL, FTO, M4CR, COX-2, GCKR and SERPINE1) with respect to intermediate and final phenotypes. Thus, the PREDIMED trial provided strong evidence that a vegetable-based MeDiet rich in unsaturated fat and polyphenols can be a sustainable and ideal model for CVD prevention.

During development of TGF-β1-initiated fibroproliferative disorders, NADPH oxidases (NOX family members) generate reactive oxygen species (ROS) resulting in downstream transcription of a subset genes encoding matrix structural elements and profibrotic factors. Prominent among the repertoire of disease-implicated genes is the TGF-β1 target gene encoding the potent profibrotic matricellular protein plasminogen activator inhibitor-1 (PAI-1 or SERPINE1). PAI-1 is the major physiologic inhibitor of the plasmin-based pericellular cascade and a causative factor in the development of vascular thrombotic and fibroproliferative disorders. ROS generation in response to TGF-β1 stimulation is rapid and precedes PAI-1 induction; engagement of non-SMAD (e.g., EGFR, Src kinase, MAP kinases, p53) and SMAD2/3 pathways are both required for PAI-1 expression and are ROS-dependent. Recent findings suggest a novel role for p53 in TGF-β1-induced PAI-1 transcription that involves ROS generation and p53/SMAD interactions. Targeting ROS and ROS-activated cellular events is likely to have therapeutic implications in the management of fibrotic disorders, particularly in the context of prolonged TGF-β1 signaling.

Cancer hallmark genes are responsible for the most essential phenotypic characteristics of malignant transformation and progression. In this study, our aim was to estimate the prognostic effect of the established cancer hallmark genes in multiple distinct cancer types. RNA-seq HTSeq counts and survival data from 26 different tumor types were acquired from the TCGA repository. DESeq was used for normalization. Correlations between gene expression and survival were computed using the Cox proportional hazards regression and by plotting Kaplan-Meier survival plots. The false discovery rate was calculated to correct for multiple hypothesis testing. Signatures based on genes involved in genome instability and invasion reached significance in most individual cancer types. Thyroid and glioblastoma were independent of hallmark genes (61 and 54 genes significant, respectively), while renal clear cell cancer and low grade gliomas harbored the most prognostic changes (403 and 419 genes significant, respectively). The eight genes with the highest significance included BRCA1 (genome instability, HR 4.26, p < 1E-16), RUNX1 (sustaining proliferative signaling, HR 2.96, p = 3.1E-10) and SERPINE1 (inducing angiogenesis, HR 3.36, p = 1.5E-12) in low grade glioma, CDK1 (cell death resistance, HR = 5.67, p = 2.1E-10) in kidney papillary carcinoma, E2F1 (tumor suppressor, HR 0.38, p = 2.4E-05) and EREG (enabling replicative immortality, HR 3.23, p = 2.1E-07) in cervical cancer, FBP1 (deregulation of cellular energetics, HR 0.45, p = 2.8E-07) in kidney renal clear cell carcinoma and MYC (invasion and metastasis, HR 1.81, p = 5.8E-05) in bladder cancer. We observed unexpected heterogeneity and tissue specificity when correlating cancer hallmark genes and survival. These results will help to prioritize future targeted therapy development in different types of solid tumors.

BACKGROUND

A recently developed probe-based technology, the NanoString nCounter™ gene expression system, has been shown to allow accurate mRNA transcript quantification using low amounts of total RNA. We assessed the ability of this technology for mRNA expression quantification in archived formalin-fixed, paraffin-embedded (FFPE) oral carcinoma samples.

RESULTS

We measured the mRNA transcript abundance of 20 genes (COL3A1, COL4A1, COL5A1, COL5A2, CTHRC1, CXCL1, CXCL13, MMP1, P4HA2, PDPN, PLOD2, POSTN, SDHA, SERPINE1, SERPINE2, SERPINH1, THBS2, TNC, GAPDH, RPS18) in 38 samples (19 paired fresh-frozen and FFPE oral carcinoma tissues, archived from 1997-2008) by both NanoString and SYBR Green I fluorescent dye-based quantitative real-time PCR (RQ-PCR). We compared gene expression data obtained by NanoString vs. RQ-PCR in both fresh-frozen and FFPE samples. Fresh-frozen samples showed a good overall Pearson correlation of 0.78, and FFPE samples showed a lower overall correlation coefficient of 0.59, which is likely due to sample quality. We found a higher correlation coefficient between fresh-frozen and FFPE samples analyzed by NanoString (r = 0.90) compared to fresh-frozen and FFPE samples analyzed by RQ-PCR (r = 0.50). In addition, NanoString data showed a higher mean correlation (r = 0.94) between individual fresh-frozen and FFPE sample pairs compared to RQ-PCR (r = 0.53).

CONCLUSIONS

Based on our results, we conclude that both technologies are useful for gene expression quantification in fresh-frozen or FFPE tissues; however, the probe-based NanoString method achieved superior gene expression quantification results when compared to RQ-PCR in archived FFPE samples. We believe that this newly developed technique is optimal for large-scale validation studies using total RNA isolated from archived, FFPE samples.

Systems biology offers considerable promise in uncovering novel pathways by which viruses and other microbial pathogens interact with host signaling and expression networks to mediate disease severity. In this study, we have developed an unbiased modeling approach to identify new pathways and network connections mediating acute lung injury, using severe acute respiratory syndrome coronavirus (SARS-CoV) as a model pathogen. We utilized a time course of matched virologic, pathological, and transcriptomic data within a novel methodological framework that can detect pathway enrichment among key highly connected network genes. This unbiased approach produced a high-priority list of 4 genes in one pathway out of over 3,500 genes that were differentially expressed following SARS-CoV infection. With these data, we predicted that the urokinase and other wound repair pathways would regulate lethal versus sublethal disease following SARS-CoV infection in mice. We validated the importance of the urokinase pathway for SARS-CoV disease severity using genetically defined knockout mice, proteomic correlates of pathway activation, and pathological disease severity. The results of these studies demonstrate that a fine balance exists between host coagulation and fibrinolysin pathways regulating pathological disease outcomes, including diffuse alveolar damage and acute lung injury, following infection with highly pathogenic respiratory viruses, such as SARS-CoV.

OBJECTIVE

Severe acute respiratory syndrome coronavirus (SARS-CoV) emerged in 2002 and 2003, and infected patients developed an atypical pneumonia, acute lung injury (ALI), and acute respiratory distress syndrome (ARDS) leading to pulmonary fibrosis and death. We identified sets of differentially expressed genes that contribute to ALI and ARDS using lethal and sublethal SARS-CoV infection models. Mathematical prioritization of our gene sets identified the urokinase and extracellular matrix remodeling pathways as the most enriched pathways. By infecting Serpine1-knockout mice, we showed that the urokinase pathway had a significant effect on both lung pathology and overall SARS-CoV pathogenesis. These results demonstrate the effective use of unbiased modeling techniques for identification of high-priority host targets that regulate disease outcomes. Similar transcriptional signatures were noted in 1918 and 2009 H1N1 influenza virus-infected mice, suggesting a common, potentially treatable mechanism in development of virus-induced ALI.

A functional promoter variant of the gene encoding the MET receptor tyrosine kinase alters SP1 and SUB1 transcription factor binding, and is associated with autism spectrum disorder (ASD). Recent analyses of postmortem cerebral cortex from ASD patients revealed altered expression of MET protein and three transcripts encoding proteins that regulate MET signaling, hepatocyte growth factor (HGF), urokinase plasminogen activator receptor (PLAUR) and plasminogen activator inhibitor-1 (SERPINE1). To address potential risk conferred by multiple genes in the MET signaling pathway, we screened all exons and 5' promoter regions for variants in the five genes encoding proteins that regulate MET expression and activity. Identified variants were genotyped in 664 families (2,712 individuals including 1,228 with ASD) and 312 unrelated controls. Replicating our initial findings, family-based association test (FBAT) analyses demonstrated that the MET promoter variant rs1858830 C allele was associated with ASD in 101 new families (P=0.033). Two other genes in the MET signaling pathway also may confer risk. A haplotype of the SERPINE1 gene exhibited significant association. In addition, the PLAUR promoter variant rs344781 T allele was associated with ASD by both FBAT (P=0.006) and case-control analyses (P=0.007). The PLAUR promoter rs344781 relative risk was 1.93 (95% confidence interval [CI]: 1.12-3.31) for genotype TT and 2.42 (95% CI: 1.38-4.25) for genotype CT compared to genotype CC. Gene-gene interaction analyses suggested a significant interaction between MET and PLAUR. These data further support our hypothesis that genetic susceptibility impacting multiple components of the MET signaling pathway contributes to ASD risk.

Broad-spectrum inhibitors of HDACs are therapeutic in many inflammatory disease models but exacerbated disease in a mouse model of atherosclerosis. HDAC inhibitors have anti- and proinflammatory effects on macrophages in vitro. We report here that several broad-spectrum HDAC inhibitors, including TSA and SAHA, suppressed the LPS-induced mRNA expression of the proinflammatory mediators Edn-1, Ccl-7/MCP-3, and Il-12p40 but amplified the expression of the proatherogenic factors Cox-2 and Pai-1/serpine1 in primary mouse BMM. Similar effects were also apparent in LPS-stimulated TEPM and HMDM. The pro- and anti-inflammatory effects of TSA were separable over a concentration range, implying that individual HDACs have differential effects on macrophage inflammatory responses. The HDAC1-selective inhibitor, MS-275, retained proinflammatory effects (amplification of LPS-induced expression of Cox-2 and Pai-1 in BMM) but suppressed only some inflammatory responses. In contrast, 17a (a reportedly HDAC6-selective inhibitor) retained anti-inflammatory but not proinflammatory properties. Despite this, HDAC6(-/-) macrophages showed normal LPS-induced expression of HDAC-dependent inflammatory genes, arguing that the anti-inflammatory effects of 17a are not a result of inhibition of HDAC6 alone. Thus, 17a provides a tool to identify individual HDACs with proinflammatory properties.

Tumor-associated inflammation can induce various molecules expressed from the tumors themselves or surrounding cells to create a microenvironment that potentially promotes cancer development. Inflammation, particularly chronic inflammation, is often linked to cancer development, even though its evolutionary role should impair nonself objects including tumors. The inflammation amplifier, a hyperinducer of chemokines in nonimmune cells, is the principal machinery for inflammation and is activated by the simultaneous stimulation of NF-κB and STAT3. We have redefined inflammation as local activation of the inflammation amplifier, which causes an accumulation of various immune cells followed by dysregulation of local homeostasis. Genes related to the inflammation amplifier have been genetically associated with various human inflammatory diseases. Here, we describe how cancer-associated genes, including interleukin (IL)-6, Ptgs2, ErbB1, Gas1, Serpine1, cMyc, and Vegf-α, are strongly enriched in genes related to the amplifier. The inflammation amplifier is activated by the stimulation of cytokines, such as TNF-α, IL-17, and IL-6, resulting in the subsequent expression of various target genes for chemokines and tumor-related genes like BCL2L11, CPNE7, FAS, HIF1-α, IL-1RAP, and SOD2. Thus, we conclude that inflammation does indeed associate with the development of cancer. The identified genes associated with the inflammation amplifier may thus make potential therapeutic targets of cancers.

BACKGROUND

TGF-β has potent profibrotic activity in vitro and has long been implicated in systemic sclerosis (SSc), as expression of TGF-β-regulated genes is increased in the skin and lungs of patients with SSc. Therefore, inhibition of TGF-β may benefit these patients.

METHODS

Patients with early, diffuse cutaneous SSc were enrolled in an open-label trial of fresolimumab, a high-affinity neutralizing antibody that targets all 3 TGF-β isoforms. Seven patients received two 1 mg/kg doses of fresolimumab, and eight patients received one 5 mg/kg dose of fresolimumab. Serial mid-forearm skin biopsies, performed before and after treatment, were analyzed for expression of the TGF-β-regulated biomarker genes thrombospondin-1 (THBS1) and cartilage oligomeric protein (COMP) and stained for myofibroblasts. Clinical skin disease was assessed using the modified Rodnan skin score (MRSS).

RESULTS

In patient skin, THBS1 expression rapidly declined after fresolimumab treatment in both groups (P = 0.0313 at 7 weeks and P = 0.0156 at 3 weeks), and skin expression of COMP exhibited a strong downward trend in both groups. Clinical skin disease dramatically and rapidly decreased (P < 0.001 at all time points). Expression levels of other TGF-β-regulated genes, including SERPINE1 and CTGF, declined (P = 0.049 and P = 0.012, respectively), and a 2-gene, longitudinal pharmacodynamic biomarker of SSc skin disease decreased after fresolimumab treatment (P = 0.0067). Dermal myofibroblast infiltration also declined in patient skin after fresolimumab (P < 0.05). Baseline levels of THBS1 were predictive of reduced THBS1 expression and improved MRSS after fresolimumab treatment.

CONCLUSIONS

The rapid inhibition of TGF-β-regulated gene expression in response to fresolimumab strongly implicates TGF-β in the pathogenesis of fibrosis in SSc. Parallel improvement in the MRSS indicates that fresolimumab rapidly reverses markers of skin fibrosis.

BACKGROUND

Clinicaltrials.gov NCT01284322.

We previously observed association between variants in the plasmacytoma variant translocation 1 gene (PVT1) and end-stage renal disease (ESRD) attributed to both type 1 and type 2 diabetes, and demonstrated PVT1 expression in a variety of renal cell types. While these findings suggest a role for PVT1 in the development of ESRD, potential mechanisms for involvement remain unknown. The goal of this study was to identify possible molecular mechanisms by which PVT1 may contribute to the development and progression of diabetic kidney disease. We knocked-down PVT1 expression in mesangial cells using RNA interference, and analyzed RNA and protein levels of fibronectin 1 (FN1), collagen, type IV, alpha 1 (COL4A1), transforming growth factor beta 1 (TGFB1) and plasminogen activator inhibitor-1 (SERPINE1 or PAI-1) by qPCR and ELISA, respectively. PVT1 expression was significantly upregulated by glucose treatment in human mesangial cells, as were levels of FN1, COL4A1, TGFB1, and PAI-1. Importantly, PVT1 knockdown significantly reduced mRNA and protein levels of the major ECM proteins, FN1 and COL4A1, and two key regulators of ECM proteins, TGFB1 and PAI-1. However, we observed a higher and more rapid reduction in levels of secreted FN1, COL4A1, and PAI-1 compared with TGFB1, suggesting that at least some of the PVT1 effects on ECM proteins may be independent of this cytokine. These results indicate that PVT1 may mediate the development and progression of diabetic nephropathy through mechanisms involving ECM accumulation.

Our previous work has demonstrated that the Tudor domain of the 'survival of motor neuron' protein and the Tudor domain-containing protein 3 (TDRD3) are highly similar and that they both have the ability to interact with arginine-methylated polypeptides. TDRD3 has been identified among genes whose overexpression has a strong predictive value for poor prognosis of estrogen receptor-negative breast cancers, although its precise function remains unknown. TDRD3 is a modular protein, and in addition to its Tudor domain, it harbors a putative nucleic acid recognition motif and a ubiquitin-associated domain. We report here that TDRD3 localizes predominantly to the cytoplasm, where it co-sediments with the fragile X mental retardation protein on actively translating polyribosomes. We also demonstrate that TDRD3 accumulates into stress granules (SGs) in response to various cellular stresses. Strikingly, the Tudor domain of TDRD3 was found to be both required and sufficient for its recruitment to SGs, and the methyl-binding surface in the Tudor domain is important for this process. Pull down experiments identified five novel TDRD3 interacting partners, most of which are potentially methylated RNA-binding proteins. Our findings revealed that two of these proteins, SERPINE1 mRNA-binding protein 1 and DEAD/H box-3 (a gene often deleted in Sertoli-cell-only syndrome), are also novel constituents of cytoplasmic SGs. Taken together, we report the first characterization of TDRD3 and its functional interaction with at least two proteins implicated in human genetic diseases and present evidence supporting a role for arginine methylation in the regulation of SG dynamics.

Hypovitaminosis D is an important public health problem. Serum 25-hydroxyvitamin D (25-OHD) is now recognized as an independent predictor for cardiovascular and related diseases (CVD) as well as other chronic medical conditions. However, the biologic pathways through which these effects are mediated remain poorly understood. We hypothesized that exposing mesenchymal multipotent cells (MMCs) to the active form of vitamin D would increase the expression of selected antifibrotic factors that in turn would ameliorate the progression of chronic diseases. MMCs were primed with 5'-azacytidine to induce a fibrotic phenotype and then treated with active vitamin D (1,25D) or ethanol <0.1% as vehicle in a time course manner (30 min, 1, 5, and 24 h, and for 4 and 7 days). The addition of 1,25D to MMCs promotes: a) increased expression and nuclear translocation of the vitamin D receptor; b) decreased expression of TGFB1 and plasminogen activator inhibitor (SERPINE1), two well-known profibrotic factors; c) decreased expression of collagen I, III and other collagens isoforms; and d) increased expression of several antifibrotic factors such as BMP7 a TGFB1 antagonist, MMP8 a collagen breakdown inducer and follistatin, an inhibitor of the profibrotic factor myostatin. In conclusion, the addition of 1,25D to differentiated MMCs displays a decreased profibrotic signaling pathway and gene expression, leading to decrease in collagen deposition. This study highlights key mechanistic pathways through which vitamin D decreases fibrosis, and provides a rationale for studies to test vitamin D supplementation as a preventive and/or early treatment strategy for CVD and related fibrotic disorders.

As glucocorticoid use increased in acute lymphoblastic leukemia, osteonecrosis became an increasingly frequent complication. Besides increased age, host risk factors are poorly defined. We tested whether 12 polymorphisms were associated with osteonecrosis among patients 10 years and older treated on the CCG1882 protocol. Candidate genes (TYMS, MTHFR, ABCB1, BGLAP, ACP5, LRP5, ESR1, PAI-1, VDR, PTH, and PTHR) were chosen based on putative mechanisms underlying osteonecrosis risk. All children received dexamethasone, with doses varying by treatment arm. A PAI-1 polymorphism (rs6092) was associated with risk of osteonecrosis in univariate (P = .002; odds ratio = 2.79) and multivariate (P = .002; odds ratio = 2.89) analyses (adjusting for gender, age, and treatment arm). Overall, 21 of 78 (26.9%) children with PAI-1 GA/AA genotypes, versus 25 of 214 (11.7%) children with GG genotype, developed osteonecrosis. PAI-1 polymorphisms and PAI-1 serum levels have previously been associated with thrombosis. We conclude that PAI-1 genetic variation may contribute to risk of osteonecrosis.

Oral squamous cell carcinoma (OSCC) is a common malignancy, the incidence of which is particularly high in some Asian countries due to the geographically linked areca quid (AQ) chewing habit. In this study, array-based comparative genomic hybridization was used to screen microdissected OSCCs for genome-wide alterations. The highest frequencies of gene gain were detected for TP63, Serpine1, FGF4/FGF3, c-Myc and DMD. The highest frequencies of deletion were detected for Caspase8 and MTAP. Gained genes, classified by hierarchical clustering, were mainly on 17q21-tel; 20q; 11q13; 3q27-29 and the X chromosome. Among these, gains of EGFR at 7p, FGF4/FGF3, CCND1 and EMS1 at 11q13, and AIB1 at 20q were significantly associated with lymph node metastasis. The genomic profiles of FHIT and EXT1 in AQ-associated and non-AQ-associated OSCCs exhibited the most prominent differences. RT-PCR confirmed the significant increase of TP63 and Serpine1 mRNA expression in OSCC relative to non-malignant matched tissue. A significant increase in Serpine1 immunoreactivity was observed from non-malignant matched tissue to OSCC. However, there was no correlation between the frequent genomic loss of Caspase 8 and a significant decrease in Caspase8 expression. These data demonstrate that genomic profiling can be useful in analysing pathogenetic events involved in the genesis or progression of OSCC.

Background and Objective: Despite striking advances in multimodality management, gastric cancer (GC) remains the third cause of cancer mortality globally and identifying novel diagnostic and prognostic biomarkers is urgently demanded. The study aimed to identify potential key genes associated with the pathogenesis and prognosis of GC. Methods: Differentially expressed genes between GC and normal gastric tissue samples were screened by an integrated analysis of multiple gene expression profile datasets. Key genes related to the pathogenesis and prognosis of GC were identified by employing protein-protein interaction network and Cox proportional hazards model analyses. Results: We identified nine hub genes (TOP2A, COL1A1, COL1A2, NDC80, COL3A1, CDKN3, CEP55, TPX2, and TIMP1) which might be tightly correlated with the pathogenesis of GC. A prognostic gene signature consisted of CST2, AADAC, SERPINE1, COL8A1, SMPD3, ASPN, ITGBL1, MAP7D2, and PLEKHS1 was constructed with a good performance in predicting overall survivals. Conclusion: The findings of this study would provide some directive significance for further investigating the diagnostic and prognostic biomarkers to facilitate the molecular targeting therapy of GC.

Plasminogen activator inhibitor-1 (PAI-1, also known as SERPINE1) is a member of the serine protease inhibitor (SERPIN) superfamily and is the primary physiological regulator of urokinase-type plasminogen activator (uPA) and tissue-type plasminogen activator (tPA) activity. Although the principal function of PAI-1 is the inhibition of fibrinolysis, PAI-1 possesses pleiotropic functions besides haemostasis. In the quarter century since its discovery, a number of studies have focused on improving our understanding of PAI-1 functions in vivo and in vitro. The use of Serpine1-deficient mice has particularly enhanced our understanding of the functions of PAI-1 in various physiological and pathophysiological conditions. In this review, the results of recent studies on PAI-1 and its role in clinical conditions are discussed.

Obesity is characterized by excessive adipose tissue mass and associated with type 2 diabetes and cardiovascular diseases. To fight obesity and its sequels, elucidating molecular events that govern adipocyte differentiation and function is of key importance. MicroRNAs (miRNAs) are a novel class of non-coding, regulatory RNAs that have been shown to regulate crucial cellular processes, including differentiation. Several studies have already assigned miRNAs to distinct functions in murine adipocyte differentiation but only a few studies did so for humans. Here, we investigated the function of miR-30c in human adipogenesis. miR-30c expression was increased during adipogenesis of human multipotent adipose-derived stem (hMADS) cells, and miR-30c overexpression enforced adipocyte marker gene induction and triglyceride accumulation. miRNA target prediction revealed two putative direct targets of miR-30c, PAI-1 (SERPINE1) and ALK2 (ACVR1, ACTRI), both inversely regulated to miR-30c during adipogenesis and responsive to miR-30c overexpression. Luciferase reporter assays confirmed PAI-1 and ALK2 as direct miR-30c targets. Moreover, reciprocal expression between miR-30c and PAI-1 could also be demonstrated in white adipose tissue of obesity mouse models, suggesting a potential physiological role of miR-30c for PAI-1 regulation in the obese state. Validating PAI-1 and ALK-2 as miR-30c mediators in adipogenesis revealed that not single silencing of PAI-1 or ALK2, but only co-silencing of both phenocopied the pro-adipogenic miR-30c effect. Thus, miR-30c can target two, so far not interconnected genes in distinct pathways, supporting the idea that miRNAs might coordinate larger regulatory networks than previously anticipated.

We conducted a genome-wide association study to identify novel associations between genetic variants and circulating plasminogen activator inhibitor-1 (PAI-1) concentration, and examined functional implications of variants and genes that were discovered. A discovery meta-analysis was performed in 19 599 subjects, followed by replication analysis of genome-wide significant (P < 5 × 10(-8)) single nucleotide polymorphisms (SNPs) in 10 796 independent samples. We further examined associations with type 2 diabetes and coronary artery disease, assessed the functional significance of the SNPs for gene expression in human tissues, and conducted RNA-silencing experiments for one novel association. We confirmed the association of the 4G/5G proxy SNP rs2227631 in the promoter region of SERPINE1 (7q22.1) and discovered genome-wide significant associations at 3 additional loci: chromosome 7q22.1 close to SERPINE1 (rs6976053, discovery P = 3.4 × 10(-10)); chromosome 11p15.2 within ARNTL (rs6486122, discovery P = 3.0 × 10(-8)); and chromosome 3p25.2 within PPARG (rs11128603, discovery P = 2.9 × 10(-8)). Replication was achieved for the 7q22.1 and 11p15.2 loci. There was nominal association with type 2 diabetes and coronary artery disease at ARNTL (P < .05). Functional studies identified MUC3 as a candidate gene for the second association signal on 7q22.1. In summary, SNPs in SERPINE1 and ARNTL and an SNP associated with the expression of MUC3 were robustly associated with circulating levels of PAI-1.

BACKGROUND

The steady increase in the incidence of obesity among adults has been paralleled with higher levels of obesity-associated breast cancer. While recent studies have suggested that adipose stromal/stem cells (ASCs) isolated from obese women enhance tumorigenicity, the mechanism(s) by which this occurs remains undefined. Evidence suggests that increased adiposity results in increased leptin secretion from adipose tissue, which has been shown to increased cancer cell proliferation. Previously, our group demonstrated that ASCs isolated from obese women (obASCs) also express higher levels of leptin relative to ASCs isolated from lean women (lnASCs) and that this obASC-derived leptin may account for enhanced breast cancer cell growth. The current study investigates the impact of inhibiting leptin expression in lnASCs and obASCs on breast cancer cell (BCC) growth and progression.

METHODS

Estrogen receptor positive (ER+) BCCs were co-cultured with leptin shRNA lnASCs or leptin shRNA obASCs and changes in the proliferation, migration, invasion, and gene expression of BCCs were investigated. To assess the direct impact of leptin inhibition in obASCs on BCC proliferation, MCF7 cells were injected alone or mixed with control shRNA obASCs or leptin shRNA obASCs into SCID/beige mice.

RESULTS

ER+ BCCs were responsive to obASCs during direct co-culture, whereas lnASCs were unable to increase ER(+) BCC growth. shRNA silencing of leptin in obASCs negated the enhanced proliferative effects of obASC on BCCs following direct co-culture. BCCs co-cultured with obASCs demonstrated enhanced expression of epithelial-to-mesenchymal transition (EMT) and metastasis genes (SERPINE1, MMP-2, and IL-6), while BCCs co-cultured with leptin shRNA obASCs did not display similar levels of gene induction. Knockdown of leptin significantly reduced tumor volume and decreased the number of metastatic lesions to the lung and liver. These results correlated with reduced expression of both SERPINE1 and MMP-2 in tumors formed with MCF7 cells mixed with leptin shRNA obASCs, when compared to tumors formed with MCF7 cells mixed with control shRNA obASCs.

CONCLUSIONS

This study provides mechanistic insight as to how obesity enhances the proliferation and metastasis of breast cancer cells; specifically, obASC-derived leptin contributes to the aggressiveness of breast cancer in obese women.

Mixed pathology, with both Alzheimer's disease and vascular abnormalities, is the most common cause of clinical dementia in the elderly. While usually thought to be concurrent diseases, the fact that changes in cerebral blood flow are a prominent early and persistent alteration in Alzheimer's disease raises the possibility that vascular alterations and Alzheimer pathology are more directly linked. Here, we report that aged tau-overexpressing mice develop changes to blood vessels including abnormal, spiraling morphologies; reduced blood vessel diameters; and increased overall blood vessel density in cortex. Blood flow in these vessels was altered, with periods of obstructed flow rarely observed in normal capillaries. These changes were accompanied by cortical atrophy as well as increased expression of angiogenesis-related genes such as Vegfa, Serpine1, and Plau in CD31-positive endothelial cells. Interestingly, mice overexpressing nonmutant forms of tau in the absence of frank neurodegeneration also demonstrated similar changes. Furthermore, many of the genes we observe in mice are also altered in human RNA datasets from Alzheimer patients, particularly in brain regions classically associated with tau pathology such as the temporal lobe and limbic system regions. Together these data indicate that tau pathological changes in neurons can impact brain endothelial cell biology, altering the integrity of the brain's microvasculature.

Overexpression of plasminogen activator inhibitor-1 (SERPINE1, PAI-1), the major physiological inhibitor of pericellular plasmin generation, is a significant causative factor in the progression of vascular disorders (e.g. arteriosclerosis, thrombosis, perivascular fibrosis) as well as a biomarker and a predictor of cardiovascular-disease associated mortality. PAI-1 is a temporal/spatial regulator of pericellular proteolysis and ECM accumulation impacting, thereby, vascular remodeling, smooth muscle cell migration, proliferation and apoptosis. Within the specific context of TGF-beta1-initiated vascular fibrosis and neointima formation, PAI-1 is a member of the most prominently expressed subset of TGF-beta1-induced transcripts. Recent findings implicate EGFR/pp60c-src->>MEK/ERK1/2 and Rho/ROCK->>SMAD2/3 signaling in TGF-beta1-stimulated PAI-1 expression in vascular smooth muscle cells. The EGFR is a direct upstream regulator of MEK/ERK1/2 while Rho/ROCK modulate both the duration of SMAD2/3 phosphorylation and nuclear accumulation. E-box motifs (CACGTG) in the PE1/PE2 promoter regions of the human PAI-1 gene, moreover, are platforms for a MAP kinase-directed USF subtype switch (USF-1->>USF-2) in response to growth factor addition suggesting that the EGFR->>MEK/ERK axis impacts PAI-1 expression, at least partly, through USF-dependent transcriptional controls. This paper reviews recent data suggesting the essential cooperativity among the EGFR->>MAP kinase cascade, the Rho/ROCK pathway and SMADs in TGF-beta1-initiated PAI-1 expression. The continued clarification of mechanistic controls on PAI-1 transcription may lead to new targeted therapies and clinically-relevant options for the treatment of vascular diseases in which PAI-1 dysregulation is a major underlying pathogenic feature.

A distinct structural change in the trabecular meshwork (TM) of patients with primary open-angle glaucoma (POAG) is the increase in fibrillar extracellular matrix (ECM) in the juxtacanalicular region of the TM. Transforming growth factor (TGF)-beta2 signaling may be involved, as TGF-beta2 is significantly increased in the aqueous humor of patients with POAG. In cultured human TM cells, TGF-beta2 causes an increase in ECM deposition, an effect that is blunted or prevented, if BMP7 is added in combination with TGF-beta2. In order to know more about the signaling network that is induced in HTM cells treated with BMP7, TGF-beta2 or the combination of both factors, we identified differentially regulated genes by microarray analysis, and confirmed selected genes by quantitative RT-PCR, Western blotting, or immunohistochemistry. We observed multiple effects of both TGF-beta2 and BMP7 on the expression of a considerable number of genes involved in growth factor signaling, ECM structure and turnover, and modification of the cytoskeleton. Among the genes that were found to be regulated were CAPZA1, CDC42BPB, EFEMP1, FGF5, FSTL3, HBEGF, LTBP1, LTBP2, MATN2, NRP1, SERPINE1, SH3MD1, SMTN, SMAD7, TFPI2, TNFAIP6, and VEGF. Since SMAD7 encodes for Smad7, an inhibitory Smad that acts in a negative-feedback loop to inhibit TGF-beta activity, we silenced Smad7 mRNA in cultured human TM cells by a specific small interfering RNA. Silencing of its mRNA caused a substantial knock down of Smad7 in TM cells. Following combined BMP7/TGF-beta2 treatment, the antagonizing effect of BMP7 on TGF-beta2-induced CTGF expression was abolished. We conclude that Smad7 is the key molecular switch that inhibits TGF-beta2 signaling, and mediates the blunting effects of BMP7 on TGF-beta2 in TM cells. A therapeutic modulation of Smad7 might be a promising approach to influence ECM turnover in the TM and to treat POAG.

OBJECTIVE

Cathepsin and plasmin may favor cancer cell invasion degrading extracellular matrix. Plasmin formation from plasminogen is regulated by plasminogen activator inhibitor type-1 (PAI-1). ARNTL2 activates the promoters of the PAI-1 gene, officially called SERPINE1, driving the circadian variation in circulating PAI-1 levels.

METHODS

We evaluated ARNTL2 and SERPINE1 expression in 50 colorectal cancer specimens and adjacent normal tissue and in colon cancer cell lines.

RESULTS

We found up-regulation of ARNTL2 (P = 0.004) and SERPINE1 (P = 0.002) in tumor tissue. A statistically significant association was found between high ARNTL2 mRNA levels and vascular invasion (P < 0.0001), and between high SERPINE1 mRNA levels and microsatellite instability (MSI-H and MSI-L, P = 0.025). Sorting the subjects into quartile groups, a statistically significant association was found between high ARNTL2 expression and lymph node involvement (P < 0.001), between high SERPINE1 expression and grading (P < 0.001) and between high SERPINE1 expression and MSI H-L (P < 0.0001). In SW480 cells, a more proliferative model compared to CaCo2 cells, there were higher mRNA levels of ARNTL2 (P < 0.001) and SERPINE1 (P = 0.001).

CONCLUSIONS

ARNTL2 and SERPINE1 expression is increased in colorectal cancer and in a highly proliferative colon cancer cell line and is related to tumor invasiveness and aggressiveness.