Non-canonical cyclic AMP SMAD1/5/8 signalling in human granulosa cells.

Development of mammalian ovarian follicles is promoted by the combined action of endocrine cues and paracrine factors. Follicle stimulating hormone (FSH), through the action of cAMP drives follicular growth and development. The oocyte secretes powerful growth factors such as bone morphogenetic protein 15 (BMP15) to regulate granulosa cell proliferation, metabolism, steroidogenesis and differentiation through the activation of SMAD1/5/8. This study investigated the role of the cAMP signalling pathway on SMAD1/5/8 action in human granulosa cells. Cyclic AMP enhanced BMP15-induction of a SMAD1/5/8-specific BRE reporter. Moreover, in the absence of BMP ligand, cAMP also activated SMAD1/5/8-induced BRE activity. Cyclic AMP increased canonical downstream targets of BMP signalling such as inhibitor of differentiation (ID) mRNA expression. The observed effects were not mediated by secretion of BMPs as cAMP did not promote BMP ligand mRNA expression and a BMP extracellular antagonist, the BMP type II receptor ectodomain, did not affect cAMP-induced ID mRNA expression. Finally, the ERK1/2 pathway was shown to be required for the maintenance of cAMP-induced SMAD1/5/8 activity. Together our results suggest a novel and non-canonical pathway for cAMP signalling in human granulosa cells. Cyclic AMP appears to promote SMAD1/5/8 pathway activity intracellularly and has the ability to activate canonical SMAD1/5/8 downstream targets. Our results add another layer of complexity to the interactions between endocrine signalling and oocyte-secreted BMP ligands during folliculogenesis. Given the importance of both cAMP and SMAD1/5/8 pathways in follicular development, these interactions are likely required for the fine-tuning of oocyte paracrine signalling by endocrine stimuli.

Pulmonary arterial hypertension - progress in understanding the disease and prioritizing strategies for drug development.

Pulmonary arterial hypertension (PAH), at one time a largely overlooked disease, is now the subject of intense study in many academic and biotech groups. The availability of new treatments has increased awareness of the condition. This in turn has driven a change in the demographics of PAH, with an increase in the mean age at diagnosis. The diagnosis of PAH in more elderly patients has highlighted the need for careful phenotyping of patients and for further studies to understand how best to manage pulmonary hypertension associated with, for example, left heart disease. The breadth and depth of expertise focused on unravelling the molecular pathology of PAH has yielded novel insights, including the role of growth factors, inflammation and metabolic remodelling. The description of the genetic architecture of PAH is accelerating in parallel, with novel variants, such as those reported in potassium two-pore domain channel subfamily K member 3 (KCNK3), adding to the list of more established mutations in genes associated with bone morphogenetic protein receptor type 2 (BMPR2) signalling. These insights have supported a paradigm shift in treatment strategies away from simply addressing the imbalance of vasoactive mediators observed in PAH towards tackling more directly the structural remodelling of the pulmonary vasculature. Here, we summarize the changing clinical and molecular landscape of PAH. We highlight novel drug therapies that are in various stages of clinical development, targeting for example cell proliferation, metabolic, inflammatory/immune and BMPR2 dysfunction, and the challenges around developing these treatments. We argue that advances in the treatment of PAH will come through deep molecular phenotyping with the integration of clinical, genomic, transcriptomic, proteomic and metabolomic information in large populations of patients through international collaboration. This approach provides the best opportunity for identifying key signalling pathways, both as potential drug targets and as biomarkers for patient selection. The expectation is that together these will enable the prioritization of potential therapies in development and the evolution of personalized medicine for PAH.

Phenotypic differentiation is associated with divergent sexual selection among closely related barn swallow populations.

Sexual selection plays a key role in the diversification of numerous animal clades and may accelerate trait divergence during speciation. However, much of our understanding of this process comes from phylogenetic comparative studies, which rely on surrogate measures such as dimorphism that may not represent selection in wild populations. In this study, we assess sexual selection pressures for multiple male visual signals across four barn swallow (Hirundo rustica) populations. Our sample encompassed 2400 linear km and two described subspecies: European H. r. rustica (in the Czech Republic and Romania) and eastern Mediterranean H. r. transitiva (in Israel), as well as a potential area of contact (in Turkey). We demonstrate significant phenotypic differentiation in four sexual signalling axes, despite very low-level genomic divergence and no comparable divergence in an ecological trait. Moreover, the direction of phenotypic divergence is consistent with differences in sexual selection pressures among subspecies. Thus, H. r. transitiva, which have the darkest ventral plumage of any population, experience directional selection for darker plumage. Similarly, H. r. rustica, which have the longest tail feathers of any population, experience directional selection for elongated tail feathers and disruptive selection for ventral plumage saturation. These results suggest that sexual selection is the primary driver of phenotypic differentiation in this species. Our findings add to growing evidence of phenotypic divergence with gene flow. However, to our knowledge, this is the first study to relate direct measures of the strength and targets of sexual selection to phenotypic divergence among closely related wild populations.

Genome-wide differentiation in closely related populations: the roles of selection and geographic isolation.

Population divergence in geographic isolation is due to a combination of factors. Natural and sexual selection may be important in shaping patterns of population differentiation, a pattern referred to as 'isolation by adaptation' (IBA). IBA can be complementary to the well-known pattern of 'isolation by distance' (IBD), in which the divergence of closely related populations (via any evolutionary process) is associated with geographic isolation. The barn swallow Hirundo rustica complex comprises six closely related subspecies, where divergent sexual selection is associated with phenotypic differentiation among allopatric populations. To investigate the relative contributions of selection and geographic distance to genome-wide differentiation, we compared genotypic and phenotypic variation from 350 barn swallows sampled across eight populations (28 pairwise comparisons) from four different subspecies. We report a draft whole-genome sequence for H. rustica, to which we aligned a set of 9493 single nucleotide polymorphisms (SNPs). Using statistical approaches to control for spatial autocorrelation of phenotypic variables and geographic distance, we find that divergence in traits related to migratory behaviour and sexual signalling, as well as geographic distance, together explain over 70% of genome-wide divergence among populations. Controlling for IBD, we find 42% of genomewide divergence is attributable to IBA through pairwise differences in traits related to migratory behaviour and sexual signalling alone. By (i) combining these results with prior studies of how selection shapes morphological differentiation and (ii) accounting for spatial autocorrelation, we infer that morphological adaptation plays a large role in shaping population-level differentiation in this group of closely related populations.

The role of mesenchymal stem cells in veterinary therapeutics - a review.

Adult mammalian tissue contains a population of cells known as mesenchymal stem cells (MSC), that possess the capability to secrete regenerative cytokines and to differentiate into specialised cell types. When transplanted to a site of injury MSC embed in damaged tissue and repair and regenerate the tissue by secreting cytokines. The immuno-privileged and immuno-regulatory capabilities of MSC enhance their therapeutic potential not only in autologous but also allogeneic recipients. Studies have demonstrated the beneficial effects of MSC in the treatment of a variety of clinical conditions including osteoarthritis, tendon injuries, and atopic dermatitis in domestic animals. Studies using animal models have shown promising results following MSC or MSC secretion therapy for induced injury in musculoskeletal and nervous systems and some organ diseases. This review describes the stem cell types relevant to regenerative medicine and the procedures used for isolation, identification, expansion, enrichment and differentiation of these cells. We also review the use of MSC in animal models of disease as well as diseases in the clinical veterinary setting.

Pulmonary hypertension: the science behind the disease spectrum.

Pulmonary hypertension (PH) is a complex, multifactorial disorder divided into five major subtypes according to pathological, pathophysiological and therapeutic characteristics. Although there are distinct differences between the PH categories, a number of processes are common to the pathology of all subtypes. Vasoconstriction, as a result of endothelial dysfunction and an imbalance in the levels of vasoactive mediators, is a well-characterised contributory mechanism. Excessive cell proliferation and impaired apoptosis in pulmonary vessels leading to structural remodelling is most evident in pulmonary arterial hypertension (PAH), and several factors have been implicated, including mitochondrial dysfunction and mutations in bone morphogenetic protein receptor type 2. Inflammation plays a key role in the development of PH, with increased levels of many cytokines and chemokines in affected patients. Exciting insights into the role of angiogenesis and bone marrow-derived endothelial progenitor cells in disease progression have also recently been revealed. Furthermore, there is increasing interest in changes in the right ventricle in PH and the role of metabolic abnormalities. Despite considerable progress in our understanding of the molecular mechanisms of PH, further research is required to unravel and integrate the molecular changes into a better understanding of the pathophysiology of PH, particularly in non-PAH, to put us in a better position to use this knowledge for improved treatments.

Iron deficiency in pulmonary arterial hypertension: a potential therapeutic target.

Iron deficiency is known to be common and detrimental in chronic left heart failure, where parenteral iron treatment has been shown to improve exercise capacity, New York Heart Association functional class and patient wellbeing. There is now increasing interest in the role of iron in the natural history of pulmonary arterial hypertension (PAH). Iron availability influences the pulmonary vasoconstrictor response to hypoxia and accumulating evidence indicates that iron deficiency is prevalent in idiopathic and heritable forms of PAH, iron status being related to exercise capacity, symptoms and poorer survival in patients with idiopathic PAH (IPAH). Potential mechanisms behind iron deficiency in IPAH include inhibition of dietary iron uptake by the master iron regulator hepcidin. High hepcidin levels underlie the anaemia of chronic disease. Possible stimuli of the observed high levels of hepcidin in IPAH include dysfunctional bone morphogenetic protein receptor type II signalling and inflammation. Iron status may influence outcomes through modulation of the pulmonary circulation as well as myocardial and skeletal muscle function. Two parallel studies, from our centre (Hammersmith Hospital, London, UK) and others in the UK and Amsterdam (the Netherlands), investigating the safety and potential benefit of iron supplementation in patients with PAH are currently under way.

Tetrahydrobiopterin and the regulation of hypoxic pulmonary vasoconstriction.

Tetrahydrobiopterin (BH(4)) is an essential cofactor for nitric oxide synthases (NOS). This study investigated the effect of increasing BH(4) levels on hypoxia-induced pulmonary vasoconstriction (HPV). Sprague Dawley rats and hph-1 (BH(4) deficient) mice were given BH(4) before and during HPV in an isolated perfused lung preparation. BH(4) inhibited HPV in a concentration-dependent manner and increased NO metabolites in the perfusate. Bradykinin-induced reductions in HPV were blunted in hph-1 mice and pre-administration of BH(4) restored the response. The effect of BH(4) was attenuated by l-NAME (NOS inhibitor), PTIO (NO scavenger), and catalase (H(2)O(2) catalyser) administered prior to HPV but enhanced by MnTMPyP (superoxide dismutase mimetic). The effect of BH(4) on HPV was partially recapitulated by NH(4), a stereoisomer that shares antioxidant properties with BH(4) but is not a NOS cofactor. The bioavailability of BH(4) is an important determinant of the pulmonary vascular response to hypoxia. Its effects are mediated via nitric oxide, hydrogen peroxide and its antioxidant properties, and are attenuated by oxidant stress. Pharmacological administration of BH(4) may have therapeutic potential in pulmonary hypertension.

Simvastatin and sildenafil combine to attenuate pulmonary hypertension.

Statins have been proposed to be a potential treatment for pulmonary arterial hypertension. If introduced into clinical practice, the statin would have to be used in conjunction with established therapy. We investigated the effects of combining simvastatin with a phosphodiesterase type-5 inhibitor, sildenafil, in the rat model of hypoxia-induced pulmonary hypertension. Rats were allocated to either: 1) a prevention protocol, to receive simvastatin 20 mg x kg(-1) x day(-1) by intraperitoneal injection or sildenafil 75 mg x kg(-1) x day(-1) orally or the combination (or vehicle) for 2 weeks beginning at the start of exposure to hypoxia (10% inspired oxygen); or 2) a treatment protocol, where the same agents were administered in the last 2 weeks of a 4-week period of hypoxia. In both protocols, the combination of sildenafil and simvastatin lowered pulmonary artery pressure and produced a significantly greater reduction in right ventricular hypertrophy and pulmonary vascular muscularisation than either drug alone. Moreover, the combination augmented significantly endothelial nitric oxide synthase expression and cGMP levels in the lung and right ventricle above that produced by either drug independently and resulted in greater inhibition of RhoA activity. These data suggest that simvastatin can be usefully combined with sildenafil in the treatment of pulmonary arterial hypertension to achieve greater therapeutic benefit.

Spectacle use after routine cataract surgery.

AIMS: To measure spectacle dependence following bilateral monofocal intraocular lens (IOL) implantation and assess how it is predicted by postoperative refraction. METHODS: 300 cataract patients had bilateral phacoemulsification surgery with monofocal IOL implantation. A spherical equivalent of 0 to -0.5 D was targeted. Three months after surgery, patients answered a questionnaire and had a spectacle refraction. Refractions were converted into vector notation. Logistic regression was used to evaluate whether spectacle dependence for near and distance was related to overall refractive error, spherical error, signed spherical error and astigmatic error. RESULTS: 169 patients attended for assessment. 38 wore distance glasses, and 160 wore reading glasses either some or all of the time. The mean right spherical equivalent was -0.03 D, and the mean right cylinder was -0.64 D. Left outcomes were similar. Patients were 34 times more likely to always use distance glasses per dioptre of astigmatic error in the better eye (p<0.003), but there was no significant increase in the likelihood of wearing distance glasses with spherical error (odds ratio = 3.85, p>0.15). Similar effects were seen for both the better and worse eyes. Near-spectacle use was not dependent on astigmatic error (odds ratio = 0.22, p>0.12). It was only related to the signed spherical error in the worse eye with hypermetropic patients 6.74 times more likely to always wear spectacles per dioptre of positive spherical error (p<0.005). CONCLUSIONS: Following bilateral monofocal intraocular lens implantation, small levels of overall refractive error, in either eye, particularly astigmatism, predict distance-spectacle dependence, whereas spherical ammetropia in the range of +/-1.0 D does not. Hypermetropia in the worse eye, but not astigmatism, predicts reading-spectacle dependence.

Hypoglycaemic and amylase inhibitory activities of leaves of Spondias mombin Linn.

Suppressing the production of glucose by inhibiting a-amylase / a-glucosidase activity is one of the therapeutic approaches for decreasing postprandial hyperglycaemia and a strategy for evaluating antidiabetic activity. We investigated leaves of Spondias mombin because our previous ethnobotanical survey showed that it is used by traditional healers to manage diabetes in South West Nigeria. We report a bioactivity-guided study of S. mombin using glucose loading (1 g/kg) alloxan-induced diabetic rats and inhibition of a-amylase as basis for isolation of active constituents. Hyperglycaemia was induced in albino rats and blood glucose levels monitored for 180 mins using a glucometer. Powdered leaves were macerated with 80% Methanol. The active extract was fractionated on column chromatography packed with silica gel G6OA eluting with gradient mixtures of pet. ether and ethylacetate. The most active a-amylase inhibiting fraction was purified on thin layer chromatography (TLC) and pure compound identified by spectroscopy. Peak decrease in blood glucose of 41.4% (p < 0.05) was recorded after 60 mins. This activity-guided study produced an active TLC band (69.8% amylase inhibition, p < 0.05) from which a-sitosterol was characterized as the main inhibitor. This is first report of hypoglycaemic and amylase inhibitory activities of S. mombin. The role of phytosterols in control of diabetes mellitus is discussed. This study justifies the ethnopharmacological use of this species in recipes for management of diabetes mellitus.

Phosphodiesterase inhibitors for the treatment of pulmonary hypertension.

The pulmonary vascular bed is both a source of and target for a number of vasoactive factors. Among the most important for pulmonary vascular homeostasis are factors that utilise cyclic guanosine monophosphate (cGMP) as an intracellular second messenger. These include nitric oxide and the natriuretic peptide family (atrial, brain and C-type natriuretic peptides). In the search for therapeutic strategies that engage the cGMP signalling pathway for the treatment of pulmonary arterial hypertension (PAH), inhibition of cGMP metabolism by phosphodiesterase type 5 (PDE5)-targeted compounds has proven most successful to date. One PDE5 inhibitor, sildenafil, has been shown to improve pulmonary haemodynamics and exercise capacity in patients with PAH and is now an approved treatment. Others are under investigation. An interesting, although still tentative, observation is the potential of sildenafil to reduce pulmonary vascular resistance without adversely affecting ventilation-perfusion matching. Another is the expression of phosphodiesterase type 5 in the hypertrophied right ventricle. These data suggest that phosphodiesterase type 5 inhibitors may have effects that distinguish them from other treatments for pulmonary hypertension and merit further study.

Noninvasive and invasive evaluation of pulmonary arterial pressure in highlanders.

The purpose of the present study was to evaluate Doppler echocardiography for the detection of pulmonary hypertension in high-altitude inhabitants. In total, 60 (55 male) patients aged 18-71 yrs were recruited from an ECG screening programme applied to 1,430 inhabitants living at an altitude of 2,500-3,600 m in Kyrgyzstan. Of these, 44 met ECG criteria for right ventricular hypertrophy. All underwent Doppler echocardiography followed by a cardiac catheterisation within 7 days of arrival in Bishkek (Kyrgyzstan; altitude 760 m). Pulmonary flow acceleration time and the maximum velocity of tricuspid regurgitation were measured. Sufficient quality tricuspid regurgitant jets were recovered in only 28% of the patients. Therefore, pulmonary artery pressure was estimated from the pulmonary flow acceleration time, which was recovered in 100% of the patients. It was found that 37 (62%) of the patients had pulmonary hypertension on echocardiography. Pulmonary hypertension was confirmed in 29 patients on catheterisation. Pulmonary hypertension was detected with 70% sensitivity and 88% specificity by echocardiography, as compared to 59% sensitivity and 81 % specificity by ECG. The correlation coefficient between echocardiography and catheterisation studies was r(2) = 0.78. It is concluded that a combination of ECG and echocardiography may be useful for screening high-altitude pulmonary hypertension.

Pointwise linear progression criteria and the detection of visual field change in a glaucoma trial.

BACKGROUND: Current pointwise linear regression (PLR) change criteria for visual field analysis are largely empirical. METHODS: Two independent sets of Humphrey Field Analyzer fields were analysed using PLR. Set i, 56 patients, and set ii, 97 patients, were followed over 16 months. Criteria were tested against set i, and then validated using set ii. Each criterion specified a fixed critical slope of 1 dB/year and with a range of significance from P<0.001 to 0.05. The criteria were varied by altering location number, cluster arrangement, and by requiring points to show change over both 12 and 16 months. True glaucomatous change was differentiated from noise by looking for exclusive progression (EP), the detection of progression without detection of improvement. RESULTS: Set i required 1 point to have a slope of 1 dB/year and P<0.05 labelled 64% progressing and 58% improving, whereas several stricter criteria were capable of detecting EP. Two points in a perimetric nerve fibre bundle (PNFB) cluster gave optimal EP detection, labelling 8.9% progressing in set i and 7.2% progressing in set ii with a cutoff P-value of 0.026 inset i and 0.013 inset ii. CONCLUSION: Lax PLR criteria detect large amounts of change. Validating criteria using two data sets allow selection of better criteria, capable of detecting EP. The criterion involving 2 points changing in a PNFB cluster offers the best option for exclusively detecting progression.

Phosphodiesterase type 5 and high altitude pulmonary hypertension.

BACKGROUND: This study explored phosphodiesterase type 5 (PDE5) inhibition as a strategy for treating high altitude pulmonary arterial hypertension (HAPH). METHODS: 689 subjects (313 men) of mean (SD) age 44 (0.6) years living above 2500 m were screened for HAPH by medical examination and electrocardiography, and 188 (27%) met the criteria for right ventricular hypertrophy. 44 underwent cardiac catheterisation and 29 (66%) had a resting mean pulmonary artery pressure (PAP) above 25 mmHg. 22 patients with a raised mean PAP were randomised to receive sildenafil (25 or 100 mg) or matching placebo taken 8 hourly for 12 weeks. RESULTS: At 3 months, patients on sildenafil 25 mg 8 hourly (n = 9) had a significantly (p = 0.018) lower mean PAP (-6.9 mmHg) at the end of the dosing interval than those on placebo (n = 8) (95% CI -12.4 to -1.3). The treatment effect for sildenafil 100 mg 8 hourly (n = 5) compared with placebo was -6.4 mm Hg (95% CI -12.9 to 0.1). Both doses improved 6 minute walk distance, the lower dose by 45.4 m (95% CI 11.5 to 79.4; p = 0.011) and the higher dose by 40.0 m (95% CI 0.2 to 79.8; p = 0.049). Sildenafil was well tolerated. Necroscopic lung specimens from three subjects with HAPH showed abundant PDE5 in the muscular coat of remodelled pulmonary arterioles. CONCLUSIONS: PDE5 is an attractive drug target for the treatment of HAPH and a larger study of the long term effects of PDE5 inhibition in HAPH is warranted.

Warfarin: a case history in pharmacogenetics.

Both hereditary and environmental factors contribute to inter-individual variability in drug response. The considerable interest in the role of genes has to be balanced with the contribution of external influences. Warfarin provides a useful case study of the need to integrate both genetic and non-genetic information when selecting the right dose for a patient. This article discusses the latest data on genotype and warfarin sensitivity and the efforts to incorporate this information into normograms. Exploring the genetics of warfarin response will lead not only to safer prescribing but elucidate the mechanism of action of warfarin and enable the development of new anticoagulant drugs.

Developments in therapeutics for pulmonary arterial hypertension.

For many years, the management of pulmonary hypertension has been frustrated by an inadequate understanding of its pathology and limited therapeutic options, but this is changing rapidly. Recently, novel insight into the pathogenesis of primary pulmonary hypertension (PPH) has been provided by the demonstration of mutations in BMPR2 and ALK-1 genes in a significant number of patients with the condition. These genes encode members of the TGF-b receptor superfamily and their integrity is important in the maintenance of normal pulmonary vascular structure and function. At the same time, there has been a major advance in the treatment of the condition due to development of 2 orally active pharmacological agents, bosentan and sildenafil, which demonstrate some selectivity for the pulmonary vasculature. This review examines how the management of PPH and severe pulmonary hypertension in associated diseases has changed and looks at exciting future developments.

What do we want from proteomics in the detection and avoidance of adverse drug reactions.

The incidence of severe adverse drug reactions is approximately 7% in hospital patients. In many cases the adverse event is difficult to predict or even explain on the basis of the known pharmacology of the causative agent. It is not infrequent in the context of multiple drug therapy, which complicates identification of the culprit. This can present a major problem in the management of chronic diseases such as tuberculosis or epilepsy. Pharmacogenetics offers one approach to reducing the incidence of drug-induced adverse reactions but has recognised limitations as a predictive tool and little role in diagnosis. Proteomics may have some predictive value but is likely to be of greater use in diagnosis-for example by recognising a drug signature in an accessible tissue. This may be possible on a blood sample or biopsy taken at presentation. Alternatively an in vitro assay that replaced rechallenging the patient with a drug would be helpful. The goal is to identify the causative drug permitting resumption of treatment with a safer alternative.