RESUMEN
Genomic variants in both ADTRP and TFPI genes are associated with risk of coronary artery disease (CAD). ADTRP regulates TFPI expression and endothelial cell functions involved in the initiation of atherosclerotic CAD. ADTRP also specifies primitive myelopoiesis and definitive hematopoiesis by upregulating TFPI expression. However, the underlying molecular mechanism is unknown. Here we show that transcription factor POU1F1 is the key by which ADTRP regulates TFPI expression. Luciferase reporter assays, chromatin-immunoprecipitation (ChIP) and electrophoretic mobility shift assay (EMSA) in combination with analysis of large and small deletions of the TFPI promoter/regulatory region were used to identify the molecular mechanism by which ADTRP regulates TFPI expression. Genetic association was assessed using case-control association analysis and phenome-wide association analysis (PhenGWA). ADTRP regulates TFPI expression at the transcription level in a dose-dependent manner. The ADTRP-response element was localized to a 50 bp region between -806 bp and -756 bp upstream of TFPI transcription start site, which contains a binding site for POU1F1. Deletion of POU1F1-binding site or knockdown of POU1F1 expression abolished ADTRP-mediated transcription of TFPI. ChIP and EMSA demonstrated that POU1F1 binds to the ADTRP response element. Genetic analysis identified significant association between POU1F1 variants and risk of CAD. PhenGWA identified other phenotypic traits associated with the ADTRP-POU1F1-TFPI axis such as lymphocyte count (ADTRP), waist circumference (TFPI), and standing height (POU1F1). These data identify POU1F1 as a transcription factor that regulates TFPI transcription in response to ADTRP, and link POU1F1 variants to risk of CAD for the first time.
Asunto(s)
Enfermedad de la Arteria Coronaria/metabolismo , Lipoproteínas/biosíntesis , Proteínas de la Membrana/metabolismo , Factor de Transcripción Pit-1/metabolismo , Aterosclerosis/genética , Estudios de Casos y Controles , Línea Celular , Inmunoprecipitación de Cromatina/métodos , Enfermedad de la Arteria Coronaria/genética , Bases de Datos Genéticas , Células Endoteliales/metabolismo , Genes Homeobox , Células HeLa , Humanos , Lipoproteínas/genética , Lipoproteínas/metabolismo , Proteínas de la Membrana/genética , Proteínas de la Membrana/fisiología , Regiones Promotoras Genéticas , Elementos de Respuesta , Sitio de Iniciación de la Transcripción , Transcripción GenéticaRESUMEN
Low androgen levels are associated with an increased risk of coronary artery disease (CAD), thrombosis and myocardial infarction (MI), suggesting that androgen has a protective role. However, little is known about the underlying molecular mechanism. Our genome-wide association study identified the ADTRP gene encoding the androgen-dependent TFPI regulating protein as a susceptibility gene for CAD and MI. The expression level of ADTRP was regulated by androgen, but the molecular mechanism is unknown. In this study, we identified the molecular mechanism by which androgen regulates ADTRP expression and tested the hypothesis that androgen plays a protective role in cardiovascular disease by activating ADTRP expression. Luciferase assays with an ADTRP promoter luciferase reporter revealed that androgen regulated ADTRP transcription in a dose- and time-dependent manner, and the effect was abolished by three different androgen inhibitors, including pyrvinium pamoate, bicalutamide, and cyproterone acetate. Chromatin-immunoprecipitation showed that the androgen receptor bound to a half androgen response element (ARE, TGTTCT) located at +324bp from the ADTRP transcription start site. The ARE is required for concentration-dependent transcriptional activation of ADTRP. HL-60 monocyte adhesion to EAhy926 endothelial cells (ECs) and transmigration across the EC layer, the two processes critical to development of CAD and MI, were inhibited by androgen, but the effect was rescued by ADTRP siRNA and exacerbated by overexpression of ADTRP and its downstream genes PIK3R3 and MIA3. These data suggest that one molecular mechanism by which androgen confers protection against CAD is stimulation of ADTRP expression.
Asunto(s)
Andrógenos/farmacología , Aterosclerosis/metabolismo , Enfermedad de la Arteria Coronaria/metabolismo , Regulación de la Expresión Génica/efectos de los fármacos , Proteínas de la Membrana/biosíntesis , Elementos de Respuesta , Transcripción Genética/efectos de los fármacos , Aterosclerosis/genética , Aterosclerosis/patología , Técnicas de Cocultivo , Enfermedad de la Arteria Coronaria/genética , Enfermedad de la Arteria Coronaria/patología , Células Endoteliales/metabolismo , Estudio de Asociación del Genoma Completo , Células HL-60 , Células HeLa , Humanos , Proteínas de la Membrana/genética , Monocitos/metabolismo , Monocitos/patología , Migración Transendotelial y Transepitelial/efectos de los fármacosRESUMEN
Human neutrophil elastase (HNE) is a key driver of inflammation in many cardiopulmonary and systemic inflammatory and autoimmune conditions. Overshooting high HNE activity is the consequence of a disrupted protease-antiprotease balance. Accordingly, there has been an intensive search for potent and selective HNE inhibitors with suitable pharmacokinetics that would allowing oral administration in patients. Based on the chemical probe BAY-678 and the clinical candidate BAY 85-8501 we explored further ring topologies along the equator of the parent pyrimidinone lead series. Novel ring systems were annulated in the east, yielding imidazolo-, triazolo-, and tetrazolopyrimidines in order to ensure additional inhibitor-HNE contacts beyond the S1 and the S2 pocket of HNE. The western annulation of pyridazines led to the polar pyrimidopyridazine BAY-8040, which combines excellent potency and selectivity with a promising pharmacokinetic profile. In vivo efficacy with regard to decreasing cardiac remodeling and amelioration of cardiac function was shown in a monocrotaline-induced rat model for pulmonary arterial hypertension. This demonstrated in vivo proof of concept in animals.
Asunto(s)
Hipertensión Pulmonar/tratamiento farmacológico , Elastasa de Leucocito/antagonistas & inhibidores , Proteínas Inhibidoras de Proteinasas Secretoras/química , Proteínas Inhibidoras de Proteinasas Secretoras/farmacología , Piridazinas/química , Piridazinas/farmacología , Pirimidinas/química , Pirimidinas/farmacología , Animales , Modelos Animales de Enfermedad , Relación Dosis-Respuesta a Droga , Humanos , Hipertensión Pulmonar/metabolismo , Elastasa de Leucocito/metabolismo , Modelos Moleculares , Estructura Molecular , Proteínas Inhibidoras de Proteinasas Secretoras/síntesis química , Piridazinas/síntesis química , Pirimidinas/síntesis química , Ratas , Relación Estructura-ActividadRESUMEN
Human neutrophil elastase (HNE) is a key protease for matrix degradation. High HNE activity is observed in inflammatory diseases. Accordingly, HNE is a potential target for the treatment of pulmonary diseases such as chronic obstructive pulmonary disease (COPD), acute lung injury (ALI), acute respiratory distress syndrome (ARDS), bronchiectasis (BE), and pulmonary hypertension (PH). HNE inhibitors should reestablish the protease-anti-protease balance. By means of medicinal chemistry a novel dihydropyrimidinone lead-structure class was identified. Further chemical optimization yielded orally active compounds with favorable pharmacokinetics such as the chemical probe BAY-678. While maintaining outstanding target selectivity, picomolar potency was achieved by locking the bioactive conformation of these inhibitors with a strategically positioned methyl sulfone substituent. An induced-fit binding mode allowed tight interactions with the S2 and S1 pockets of HNE. BAY 85-8501 ((4S)-4-[4-cyano-2-(methylsulfonyl)phenyl]-3,6-dimethyl-2-oxo-1-[3-(trifluoromethyl)phenyl]-1,2,3,4-tetrahydropyrimidine-5-carbonitrile) was shown to be efficacious in a rodent animal model related to ALI. BAY 85-8501 is currently being tested in clinical studies for the treatment of pulmonary diseases.
Asunto(s)
Congelación , Elastasa de Leucocito/antagonistas & inhibidores , Enfermedades Pulmonares/enzimología , Proteínas Inhibidoras de Proteinasas Secretoras/farmacología , Pirimidinonas/farmacología , Sulfonas/farmacología , Relación Dosis-Respuesta a Droga , Humanos , Elastasa de Leucocito/metabolismo , Conformación Molecular , Proteínas Inhibidoras de Proteinasas Secretoras/química , Pirimidinonas/química , Relación Estructura-Actividad , Sulfonas/químicaRESUMEN
BACKGROUND: Approved therapies for pulmonary arterial hypertension can induce oxygen desaturation when administered to patients with secondary forms of pulmonary hypertension (PH), probably due to an increase in ventilation/perfusion mismatch. Thus, so far these treatments have largely failed in secondary forms of PH. METHODS: We established an animal model of heterogeneous lung ventilation to evaluate the desaturation potential of mechanistically distinct vasoactive drugs launched or currently in clinical development for the treatment of PH. Single-lung ventilation was induced in five groups (N = 6) of anesthetized minipigs (7 weeks, 4 to 5 kg BW), and their hemodynamic parameters were monitored before and after intravenous injection of control (vehicle only), endothelin antagonist (bosentan; 0.3, 1, 3, 10 mg/kg), phosphodiesterase type 5 inhibitor (sildenafil; 3, 10, 30, 100 µg/kg), and soluble guanylate cyclase stimulators (BAY 41-8543 and riociguat; 1, 3, 10, 30 µg/kg). Cumulative doses were administered before successive unilateral ventilation cycles. The doses were chosen to achieve equal effect on blood pressure by the different pharmacologic principles. RESULTS: Single-lung ventilation resulted in transient increases in mean pulmonary artery pressure (mPAP) and desaturation. In contrast to control, all drugs dose-dependently decreased hypoxic mPAP (a positive treatment effect) and increased area under the arterial hemoglobin saturation curve (unwanted desaturation effect). Riociguat and bosentan reduced hypoxic mPAP to the greatest extent, while the soluble guanylate cyclase stimulators riociguat and BAY 41-8543 lowered arterial oxygen saturation of hemoglobin the least. CONCLUSIONS: Future investigations will be required to confirm these findings in clinical settings.
Asunto(s)
Presión Sanguínea/efectos de los fármacos , Hemodinámica/efectos de los fármacos , Hipertensión Pulmonar , Arteria Pulmonar , Vasodilatadores/farmacología , Animales , Modelos Animales de Enfermedad , Hipertensión Pulmonar/metabolismo , Hipertensión Pulmonar/patología , Hipertensión Pulmonar/fisiopatología , Arteria Pulmonar/metabolismo , Arteria Pulmonar/patología , Arteria Pulmonar/fisiopatología , Porcinos , Porcinos EnanosRESUMEN
Nitric oxide (NO) plays an important role in protection against the onset and progression of various cardiovascular disorders. Therefore, the NO/guanosine 3',5'-cyclic monophosphate (cGMP) pathway has gained considerable attention and has become a target for new drug development. We have established a rapid, homogeneous, cell-based, and highly sensitive reporter assay for NO generated by endothelial nitric oxide synthase (eNOS). In a coculture system, NO production is indirectly monitored in living cells via soluble guanylyl cyclase (sGC) activation and calcium influx mediated by the olfactory cyclic nucleotide-gated (CNG) cation channel CNGA2, acting as the intracellular cGMP sensor. Using this NO reporter assay, we performed a fully automated high-throughput screening campaign for stimulators of NO synthesis. The coculture system reflects most aspects of the natural NO/cGMP pathway, namely, Ca(2+)-dependent and Ca(2+)-independent regulation of eNOS activity by G protein-coupled receptor agonists, oxidative stress, phosphorylation, and cofactor availability as well as NO-mediated stimulation of cGMP synthesis by sGC activation. The NO reporter assay allows the real-time detection of NO synthesis within living cells and makes it possible to identify and characterize activators and inhibitors of enzymes involved in the NO/cGMP signaling pathway.