Follow-up of a major linkage peak on chromosome 1 reveals suggestive QTLs associated with essential hypertension: GenNet study

Essential hypertension is a major cardiovascular risk factor and a large proportion of this risk is genetic. Identification of genomic regions consistently associated with hypertension has been difficult in association studies to date as this requires large sample sizes.We previously published a large genome-wide linkage scan in Americans of African (AA) and European (EA) descent in the GenNet Network of the Family Blood Pressure Program (FBPP). A highly significant linkage peak was identified on chr1q spanning a region of 100 cM. In this study, we genotyped 1569 SNPs under this linkage peak in 2379 individuals to identify whether common genetic variants were associated with blood pressure (BP) at this locus.Our analysis, using two different family-based association tests, provides suggestive evidence (P≤2 × 10⁻⁵) for a collection of single nucleotide polymorphisms (SNPs) associated with BP. In EAs, using diastolic BP as a quantitative phenotype, three variants located in or near the GPA33, CD247, and F5 genes, emerge as our top hits; for systolic BP, variants in GPA33, CD247, and REN are our best findings. No variant in AAs came close to suggestive evidence after multiple-test corrections (P≥8 × 10⁻⁵).In summary, we show that systematic follow-up of a linkage signal can help discover candidate variants for essential hypertension that require a follow-up in yet larger samples. The failure to identify common variants is either because of low statistical power or the existence of rare coding variants in specific families or both, which require additional studies to clarify.     

Luminescent and Ionochromic Polyiminofluorene with Conjugated Terpyridine Substituent Groups

Poly(N‐(4‐(2,2′:6,2″‐terpyridyl)phenyl)imino‐2,7‐fluorenylene P1‐2 was prepared upon palladium‐catalyzed polycondensation of 2,7‐dibromo‐9,9‐di‐n‐hexylfluorene 1 and 4′‐(4‐aminophenyl)‐2,2′:6,2″‐terpyridine 2 with 80% yield. MALDI–TOF spectroscopy indicated a maximum degree of polymerization of 14, the most abundant species being the pentamer and the hexamer. P1‐2 is highly fluorescent in solution. The emission maximum in chloroform is 510 nm, and the quantum yield Φ_f is 55%. UV/Vis titration of P1‐2 with zinc acetate and iron(II) perchlorate indicates formation of metal–terpyridine (tpy) 1:2 (bis)complexes. Since formation of the (bis)complex is accompanied by cross‐linking, the polymer slowly precipitates, especially in non‐polar solvents. Comparative titration of monomer 2 with zinc acetate proceeds under sharp increase of photoluminescence intensity until a metal–tpy 2:1 ratio is reached. We assume the formation of two zinc (mono)complexes, one with the tpy and the other one with the amino group of 2 . CV analysis of P1‐2 in acetonitrile indicates three quasi‐reversible oxidation processes with mid‐potentials of 0.45, 0.50 and 0.95 V versus SCE due to oxidation of the backbone nitrogen atoms, while the reduction at ?2.6 V is irreversible. The strong ionochromism renders P1‐2 useful as highly sensitive and specific fluorimetric chemosensor.

     

Analogs of methyllycaconitine as novel noncompetitive inhibitors of nicotinic receptors: pharmacological characterization, computational modeling, and pharmacophore development

As a novel approach to drug discovery involving neuronal nicotinic acetylcholine receptors (nAChRs), our laboratory targeted nonagonist binding sites (i.e., noncompetitive binding sites, negative allosteric binding sites) located on nAChRs. Cultured bovine adrenal cells were used as neuronal models to investigate interactions of 67 analogs of methyllycaconitine (MLA) on native alpha3beta4* nAChRs. The availability of large numbers of structurally related molecules presents a unique opportunity for the development of pharmacophore models for noncompetitive binding sites. Our MLA analogs inhibited nicotine-mediated functional activation of both native and recombinant alpha3beta4* nAChRs with a wide range of IC(50) values (0.9-115 microM). These analogs had little or no inhibitory effects on agonist binding to native or recombinant nAChRs, supporting noncompetitive inhibitory activity. Based on these data, two highly predictive 3D quantitative structure-activity relationship (comparative molecular field analysis and comparative molecular similarity index analysis) models were generated. These computational models were successfully validated and provided insights into the molecular interactions of MLA analogs with nAChRs. In addition, a pharmacophore model was constructed to analyze and visualize the binding requirements to the analog binding site. The pharmacophore model was subsequently applied to search structurally diverse molecular databases to prospectively identify novel inhibitors. The rapid identification of eight molecules from database mining and our successful demonstration of in vitro inhibitory activity support the utility of these computational models as novel tools for the efficient retrieval of inhibitors. These results demonstrate the effectiveness of computational modeling and pharmacophore development, which may lead to the identification of new therapeutic drugs that target novel sites on nAChRs.     

Farnesoid X receptor activation improves erectile function in animal models of metabolic syndrome and diabetes

IntroductionThe farnesoid X receptor (FXR) is critically involved in the regulation of the hepato‐biliary system. Recent data suggest a role for FXR in modulating other metabolic pathways and vascular function.AimTo investigate whether long‐term administration of the selective FXR agonist INT‐747 ameliorates erectile function, we tested it in two animal models of metabolic derangements: a rabbit model of high‐fat diet (HFD)‐induced metabolic syndrome (MetS) and a rat model of streptozotocin (STZ)‐induced type 1 diabetes.MethodsHFD rabbit or STZ rats with or without chronic INT‐747 dosing (10 mg/kg/day for 12 weeks). INT‐747 addition to rabbit penile smooth muscle cells (rpSMCs).Main Outcome MeasureEffects of INT‐747 on metabolic features and erectile function in animal models and clarification of mechanism of action in isolated cells.ResultsINT‐747 dosing normalized visceral adiposity and glucose intolerance in HFD rabbits. INT‐747 increased penile FXR expression and partially restored endothelial nitric oxide synthase and dimethylarginine dimethylaminohydrolase 1 expression as well as impaired nitric oxide (NO)‐dependent relaxation (improved responsiveness to acetylcholine and electrical field stimulation). INT‐747 was also effective in regulating NO downstream events, as shown by increased sodium nitroprusside‐induced relaxation. Because phosphodiesterase type 5 and protein kinase G (PKG) were unaltered by INT‐747, we analyzed the calcium‐sensitizing RhoA/ROCK pathway. HFD increased, and INT‐747 normalized, RhoA membrane translocation/activation. RhoA/ROCK signaling inhibition by INT‐747 was confirmed in rpSMCs by confocal microscopy, MYPT1‐phosphorylation, cytoskeleton remodeling, cell migration, and smooth muscle‐related genes expression. In STZ rats, FXR penile expression was not altered but was significantly upregulated by INT‐747 dosing. In this model, INT‐747 improved penile erection induced by electrical stimulation of cavernous nerve and hypersensitivity to intracavernous injection of a ROCK‐inhibitor, Y‐27632, without improving hyperglycemia.ConclusionIn HFD rabbits, INT‐747 dosing improved glucose sensitivity and MetS‐associated erectile dysfunction, via upregulation of NO transmission and inhibition of RhoA/ROCK pathway. In STZ rats, INT‐747 restored in vivo penile erection and sensitivity to ROCK inhibition, independently of effects on glycemia. Vignozzi L, Morelli A, Filippi S, Comeglio P, Chavalmane AK, Marchetta M, Toce M, Yehiely‐Cohen R, Vannelli GB, Adorini L, and Maggi M. Farnesoid X receptor activation improves erectile function in animal models of metabolic syndrome and diabetes.     

Atorvastatin but not elocalcitol increases sildenafil responsiveness in spontaneously hypertensive rats by regulating the RhoA/ROCK pathway

Spontaneously hypertensive rats (SHR) are characterized by impaired erectile function and overactivity of the procontractile RhoA/Rho-associated, coiled-coil-containing protein kinase (RhoA/ROCK) pathway, as compared with their normotensive counterpart, Wistar-Kyoto rats. By measuring the intracavernous pressure:mean arterial pressure (ICP:MAP) ratio after electrostimulation of the cavernous nerve, we confirmed these findings and showed that responsiveness to sildenafil (25 mg/kg by oral gavage) also is hampered in SHR. A 2-week treatment with atorvastatin (5 and 30 mg/kg) improved the sildenafil-induced ICP:MAP increase and normalized RhoA and ROCK2 overexpression in SHR corpora cavernosa (CC). Conversely, other genes, neuronal nitric oxide synthase (NOS), endothelial NOS, and phosphodiesterase 5, were unaffected. In human fetal smooth muscle cells derived from CC (hfPSMC), atorvastatin inhibited RhoA membrane translocation and ROCK activity, as well as RhoA-dependent biologic functions like cell migration and cell proliferation. Atorvastatin's effect on migration was rescued in a dose-dependent manner by geranylgeranyl pyrophosphate, suggesting the involvement of RhoA geranylgeranylation. In hfPSMC, atorvastatin decreased the expression of RhoA-dependent genes such as ROCK2, desmin, alpha-smooth muscle actin, SM22alpha, and myocardin. In contrast to atorvastatin, elocalcitol, a vitamin D analog that also interferes with RhoA activation in SHR bladder, was unable to restore penile responsiveness to sildenafil. In conclusion, atorvastatin, but not elocalcitol, ameliorates sildenafil-induced penile erections in SHR, likely by interfering with RhoA/ROCK signaling within the penis.     

Renal tubular function in protein energy malnutrition

Renal tubular handling of sodium, potassium, calcium, magnesium, phosphate and aminoacids in children suffering from marasmus and kwashiorkor revealed wastage of phosphate and aminoacids. In the malnourished children aminoaciduria could contribute significantly to the negative nitrogen balance. Exact cause of this tubular functional defect is not known.     

Combined ventricular endocardial and epicardial substrate mapping using a sonomicrometry-based electroanatomical mapping system

BACKGROUND: Substrate mapping using a magnetic electroanatomical mapping system (MEAM) has been shown to accurately delineate the location/extent of scarred myocardium. This study examined the ability of a sonomicrometry-based electroanatomic mapping system (SEAM) to render endocardial and epicardial substrate maps of infarcted ventricular myocardium. METHODS AND RESULTS: In 7 swine with healed myocardial infarctions, combined epicardial and endocardial left ventricular (LV) substrate maps were created with both SEAM and MEAM mapping systems using 246+/-68 and 244+/-44 points respectively. Scarred myocardium was identified based upon bipolar electrogram amplitude < 1.5 mV, and radiofrequency ablation lesions were delivered to the scar border as defined by the sonomicrometry mapping system. The LV endocardial chamber volume as defined by SEAM (125+/-46 ml) correlated well with that defined by the MEAM (137+/-45 ml, r=0.77, p < 0.05). The area of infarcted tissue as determined by SEAM was highly correlated with that determined by gross pathology (r=0.96 for endocardial scar and r=0.92 for epicardial scar p < 0.05). The scar area calculated by the SEAM system also correlated well with the scar area determined by the MEAM system (0.91 for endocardial scar and 0.90 for epicardial scar p < 0.05). Finally, the sonomicrometry-based system was able to guide the placement of radiofrequency ablation lesions to the borders of the scar. CONCLUSIONS: This study demonstrates that the sonomicrometry-based mapping can accurately reconstruct three-dimensional voltage maps of the endocardial and epicardial ventricular surfaces and guide the placement of ablation lesions along the scar border zone.