Blocking 5-HT2B receptor provides a therapeutic target for fibrotic diseases caused by activated platelet release of serotonin during vascular damage.

In situ and perfused rat livers showed a spon- .) The existence of such light emission, which should be termed "low-level chemiluminescence" to differentiate it from the more effective photoemission of the luciferin/luciferase systems (5, 6), was soon related to oxygendependent chain reactions involving biological lipids (3-5). This early work lay fallow for years, notwithstanding the reports by Stauff and Ostrowski on the chemiluminescence of mitochondria (7) and Howes and Steele on the chemiluminescence of microsomes (8, 9), both isolated from rat liver. The more recent reports by Nakano et al. (10) and Sugioka and Nakano (11) of light emission during lipid peroxidation and other oxidative reactions (12) in microsomes revived interest in the phenomenon and suggested chemiluminescence as a tool for the investigation of the radical reactions of lipid peroxidation under physiological conditions. We have recently reported that maximal light emission in isolated mitochondria and microsomes (13) and in submitochondrial particles (14) requires an electron transfer system, hydroperoxide, and oxygen, and that hydroperoxide-supplemented cytochrome c provides a chemiluminescent model system suitable for the elucidation of some of the molecular mechanisms responsible for light emission (15). On the other hand, isolated cells such as amoebae (16) and phagocytizing leukocytes (17) also have been found to be effective chemiluminescent sources.The most important aspect of the organ chemiluminescence is that it gives readily detectable, continuously monitorable, noninvasive signals of oxidative metabolism. This article explores the possibility of continuously monitoring the metabolism of exposed or fiberoptic probed organs in vivo by the chemiluminescent technique. In this paper we report the spontaneous and hydroperoxide-induced chemiluminescence of the in situ and perfused rat liver, as well as a partial spectral analysis of the chemiluminescence of the perfused liver. Light emission seems to indicate the generation of shkrt-lived free radicals and excited states derived from the side reactions of the free radical process of lipid peroxidation. A preliminary report on light emission has been published elsewhere (13). MATERIALS AND METHODSPhoton Counting. A single-photon-counting apparatus was used (Fig. 1). Both an EMI 9658 photomultiplier, responsive in the range 300-900 nm, with an applied potential of -1.2 kV (dark current: 20-30 counts per second), and an RCA 8850 photomultiplier, responsive in the range 300-650 nm, with an applied potential of -1.8 kV (dark current: 300-400 counts per second) were used. Phototube output was connected to an amplifier-discriminator (model 1121; Princeton Applied Res., Princeton, NJ) adjusted for single photon counting and connected to both a frequency counter (Heathkit IB 1100, Heath, Benton Harbor, MI) and a recorder. The EMI phototube, cooled .down to -400C by a thermoelectric cooler (EMI-Gencom, Plainview, NY) and the RCA phototube were placed in an Ortec housing, sealed and su...

Purpose: Aurora kinaseA (AURKA/STK15/BTAK) encodes a serine/threonine kinase associated with chromosomal distribution and its up-regulation induces chromosomal instability, thereby leading to aneuploidy and cell transformation in several types of cancer. In this study, we investigated the role of AURKA in head and neck squamous cell carcinoma (HNSCC). Experimental Design: The mRNA expression levels of AURKA were compared in tumor tissues of 66 HNSCC patients with those in corresponding normal squamous epithelium by real-time quantitative reverse transcriptase-PCR. In addition, the association between AURKA mRNA and protein expression, centrosome abnormalities, and aneuploidy was studied in a subset of cases (n = 34). All molecular variables were correlated to histomorphologic findings and clinical followup data of the patients. Results: AURKA mRNA up-regulation was significantly associated with tumor stage and the occurrence of regional lymph node, as well as distant metastasis (P < 0.0001 for all). Similarly, a correlation was found for protein expression and the occurrence of regional lymph node (P = 0.0183) and distant metastasis (P = 0.03).The mRNA was positively associated with protein expression (P = 0.003) and centrosome abnormalities (P = 0.03). Cox regression analysis revealed that AURKA mRNA up-regulation correlated with disease-free survival of the patients (P = 0.03) as well as shorter overall survival (P < 0.001). Conclusions:We conclude that the up-regulation of AURKA mRNA may play a critical role in the tumor progression of HNSCC and provides useful information as a prognostic factor for HNSCC patients.Head and neck squamous cell carcinoma (HNSCC) is the sixth most common cancer worldwide and survival rates are not improving (1). Therapeutic decisions are usually based on clinical and histopathologic variables like tumor-node-metastasis stage and tumor grading, which, however, often fail to predict patient outcome. Therefore, there is a need to better understand HNSCC development and progression on the molecular level. This should lead to an improved stratification between higherrisk and lower-risk patients, which can be treated in a more selective and individualized manner.DNA gains on chromosome 20q are recurrent findings in HNSCC (2, 3) and are associated with lymph node metastasis, as recently shown by array-based comparative genomic hybridization (4). Aurora kinase A (AURKA/BTAK/AIK1/ STK15) maps close to the critical region of this DNA gain and is localized on 20q13.2 (5). AURKA is a member of the Aurora/Ipl1p family of cell cycle -regulating serine/threonine kinases and is localized at interphase and mitotic centrosomes and at the spindle poles where it regulates proper chromosome segregation and cytokinesis (6). Recent studies have shown that the ectopic expression of Aurora-A in mouse NIH 3T3 cells and Rat-1 fibroblasts causes centrosome amplification and transformation in vitro as well as tumorigenesis in vivo (7,8). Furthermore, the up-regulation of AURKA leads to abnormal cent...

Nonsyndromic cleft lip with/without cleft palate (nsCL/P) and nonsyndromic cleft palate only (nsCPO) are the most frequent subphenotypes of orofacial clefts. A common syndromic form of orofacial clefting is Van der Woude syndrome (VWS) where individuals have CL/P or CPO, often but not always associated with lower lip pits. Recently, ∼5% of VWS-affected individuals were identified with mutations in the grainy head-like 3 gene (GRHL3). To investigate GRHL3 in nonsyndromic clefting, we sequenced its coding region in 576 Europeans with nsCL/P and 96 with nsCPO. Most strikingly, nsCPO-affected individuals had a higher minor allele frequency for rs41268753 (0.099) than control subjects (0.049; p = 1.24 × 10(-2)). This association was replicated in nsCPO/control cohorts from Latvia, Yemen, and the UK (pcombined = 2.63 × 10(-5); ORallelic = 2.46 [95% CI 1.6-3.7]) and reached genome-wide significance in combination with imputed data from a GWAS in nsCPO triads (p = 2.73 × 10(-9)). Notably, rs41268753 is not associated with nsCL/P (p = 0.45). rs41268753 encodes the highly conserved p.Thr454Met (c.1361C>T) (GERP = 5.3), which prediction programs denote as deleterious, has a CADD score of 29.6, and increases protein binding capacity in silico. Sequencing also revealed four novel truncating GRHL3 mutations including two that were de novo in four families, where all nine individuals harboring mutations had nsCPO. This is important for genetic counseling: given that VWS is rare compared to nsCPO, our data suggest that dominant GRHL3 mutations are more likely to cause nonsyndromic than syndromic CPO. Thus, with rare dominant mutations and a common risk variant in the coding region, we have identified an important contribution for GRHL3 in nsCPO.

Nonsyndromic orofacial clefts are common birth defects with multifactorial etiology. The most common type is cleft lip, which occurs with or without cleft palate (nsCLP and nsCLO, respectively). Although genetic components play an important role in nsCLP, the genetic factors that predispose to palate involvement are largely unknown. In this study, we carried out a meta-analysis on genetic and clinical data from three large cohorts and identified strong association between a region on chromosome 15q13 and nsCLP (P = 8.13×10−14 for rs1258763; relative risk (RR): 1.46, 95% confidence interval (CI): 1.32–1.61)) but not nsCLO (P = 0.27; RR: 1.09 (0.94–1.27)). The 5 kb region of strongest association maps downstream of Gremlin-1 (GREM1), which encodes a secreted antagonist of the BMP4 pathway. We show during mouse embryogenesis, Grem1 is expressed in the developing lip and soft palate but not in the hard palate. This is consistent with genotype-phenotype correlations between rs1258763 and a specific nsCLP subphenotype, since a more than two-fold increase in risk was observed in patients displaying clefts of both the lip and soft palate but who had an intact hard palate (RR: 3.76, CI: 1.47–9.61, Pdiff<0.05). While we did not find lip or palate defects in Grem1-deficient mice, wild type embryonic palatal shelves developed divergent shapes when cultured in the presence of ectopic Grem1 protein (P = 0.0014). The present study identified a non-coding region at 15q13 as the second, genome-wide significant locus specific for nsCLP, after 13q31. Moreover, our data suggest that the closely located GREM1 gene contributes to a rare clinical nsCLP entity. This entity specifically involves abnormalities of the lip and soft palate, which develop at different time-points and in separate anatomical regions.

Background Idiopathic pulmonary fibrosis (IPF) has a poor prognosis and limited responsiveness to available treatments. It is characterised by epithelial cell injury, fibroblast activation and proliferation and extracellular matrix deposition. Serotonin (5-hydroxytryptamine; 5-HT) induces fibroblast proliferation via the 5-HTR 2A and 5-HTR 2B receptors, but its pathophysiological role in IPF remains unclear. A study was undertaken to determine the expression of 5-HT receptors in IPF and experimental lung fibrosis and to investigate the effects of therapeutic inhibition of 5-HTR 2A/B signalling on lung fibrosis in vivo and in vitro.Methods and results Quantitative RT-PCR showed that the expression of 5-HTR 1A/B and 5-HTR 2B was significantly increased in the lungs of patients with IPF (n¼12) and in those with non-specific interstitial pneumonia (NSIP, n¼6) compared with transplant donors (n¼12). The expression of 5-HTR 2A was increased specifically in IPF lungs but not in NSIP lungs. While 5-HTR 2A protein largely localised to fibroblasts, 5-HTR 2B localised to the epithelium. To assess the effects of 5HTR 2A/B inhibition on fibrogenesis in vivo, mice were subjected to bleomycin-induced lung fibrosis and treated with the 5-HTR 2A/B antagonist terguride (or vehicle) in a therapeutic approach (days 14e28 after bleomycin). Terguride-treated mice had significantly improved lung function and histology and decreased collagen content compared with vehicle-treated mice. Functional in vitro studies showed that terguride is a potent inhibitor of transforming growth factor b 1 -or WNT3a-induced collagen production. Conclusion The studies revealed an increased expression of 5-HTR 2A specifically in IPF. Blockade of 5-HTR 2A/B signalling by terguride reversed lung fibrosis and is thus a promising therapeutic approach for IPF.

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Pulmonary arterial hypertension (PAH) is a life-threatening disease characterised by vasoconstriction and remodelling of the pulmonary vasculature. The serotonin (5-hydroxytryptamine (5-HT)) pathway has been shown to play a major role in the pathogenesis of PAH, but pharmacological modulation of this pathway for treatment of PAH is, to date, at a preclinical level. Terguride is a 5-HT receptor (5-HTR) antagonist that is well tolerated and clinically approved for ovulation disorders.Immunohistochemistry against 5-HTR 2A/B on human lungs revealed their localisation to the vascular smooth muscle layer and quantitative RT-PCR showed 5-HTR 2B upregulation in pulmonary artery smooth muscle cells (PASMC) isolated from PAH patients. Proliferation and migration of cultured primary human PASMC were dose-dependently blocked by terguride. Therapeutic 5-HT signalling inhibition was 1) demonstrated in isolated, ventilated and perfused rat lungs and 2) by chronic terguride treatment of rats with monocrotaline (MCT)-induced pulmonary hypertension in a preventive or curative approach.Terguride inhibited proliferation of PASMCs and abolished 5-HT-induced pulmonary vasoconstriction. Chronic terguride treatment prevented dose-dependently the development and progression of MCT-induced PAH in rats. Thus, terguride represents a valuable novel therapeutic approach in PAH.KEYWORDS: Collagen, experimental therapeutics, inflammation, pulmonary hypertension, smooth muscle cells, vascular remodelling P ulmonary arterial hypertension (PAH) is a life-threatening disease characterised by an increase of pulmonary artery pressure resulting from endothelial injury, proliferation and hypercontraction of vascular smooth muscle cells [1]. When untreated, the disease finally results in right ventricular (RV) failure and death. Several important signalling systems have been shown to be dysregulated in pulmonary hypertension (PH). In patients with idiopathic PAH (IPAH), a reduced excretion of prostaglandin I 2 and an enhanced excretion of thromboxane metabolites have been noted [2]. Moreover, enhanced expression of phosphodiesterase (PDE)-5, which hydrolyses the NO-induced second messenger cyclic guanosine monophosphate, was observed in PH [3]. In addition, the vasoconstrictor endothelin is upregulated in PAH [4] and correlates with the degree of the disease [5]. Furthermore, an epidemic of PH in patients using anorexic agents implied a role of serotonin (5-hydroxytryptamine (5-HT)) in the pathogenesis of PH [6]. Expression analysis of lung tissues from PAH patients undergoing lung transplantation revealed an increased expression of 5-HT transporter (5-HTT) and an enhanced proliferative growth response of isolated pulmonary arterial smooth muscle cells (PASMC) to 5-HT [7]. While most of these pathways are currently addressed clinically for treatment of PAH, e.g. by infusion or inhalation of prostanoids [8,9], oral application of PDE-5 inhibitors [10,11] and endothelin antagonists [12], the 5-HT pathway is still studied only on a preclinical ...