Nung Rudarakanchana

Dysfunctional Smad Signaling Contributes to Abnormal Smooth Muscle Cell Proliferation in Familial Pulmonary Arterial Hypertension

Mutations in the bone morphogenetic protein type II receptor gene (BMPR2) are the major genetic cause of familial pulmonary arterial hypertension (FPAH). Although smooth muscle cell proliferation contributes to the vascular remodeling observed in PAH, the role of BMPs in this process and the impact of BMPR2 mutation remains unclear. Studies involving normal human pulmonary artery smooth muscle cells (PASMCs) suggest site-specific responses to BMPs. Thus, BMP-4 inhibited proliferation of PASMCs isolated from proximal pulmonary arteries, but stimulated proliferation of PASMCs from peripheral arteries, and conferred protection from apoptosis. These differences were not caused by differential activation of BMP signaling pathways because exogenous BMP-4 led to phosphorylation of Smad1, p38MAPK, and ERK1/2 in both cell types. However, the proproliferative effect of BMP-4 on peripheral PASMCs was found to be p38MAPK/ERK-dependent. Conversely, overexpression of dominant-negative Smad1 converted the response to BMP-4 in proximal PASMCs from inhibitory to proliferative. Furthermore, we confirmed that proximal PASMCs harboring kinase domain mutations in BMPR2 are deficient in Smad signaling and are unresponsive to the growth suppressive effect of BMP-4. Moreover, we show that the pulmonary vasculature of patients with familial and idiopathic PAH are deficient in the activated form of Smad1. We conclude that defective Smad signaling and unopposed p38MAPK/ERK signaling, as a consequence of mutation in BMPR2, underlie the abnormal vascular cell proliferation observed in familial PAH.

Nucleus accumbens dopamine depletion impairs both acquisition and performance of appetitive Pavlovian approach behaviour: implications for mesoaccumbens dopamine function.

The involvement of mesoaccumbens dopamine in adaptive learning and behaviour is unclear. For example, dopamine may act as a teaching signal to enable learning, or more generally modulate the behavioural expression, or selection, of an already-learned response. The present study investigated the involvement of the mesoaccumbens dopamine system in a fundamental form of learning: Pavlovian conditioning. In this case, the temporal association of a previously neutral visual stimulus and a biologically significant unconditioned stimulus (US), subsequently led to the production of the conditioned response (CR) of discriminated approach behaviour directed toward the conditioned stimulus (CS+), relative to a control (CS-) stimulus. 6-hydroxydopamine lesions of the nucleus accumbens (NAcc), leading to approximately 80% reductions in tissue dopamine, were made at varying time points in four experimental groups of rats, either before or subsequent to the acquisition of the CR. NAcc dopamine depletion produced long-term neuroadaptations in dopamine function 2 months after surgery, and profoundly impaired discriminated Pavlovian approach regardless of when the lesion was made. Thus, NAcc dopamine not only plays a role in conditioned behavioural activation, but also in making the appropriate discriminated response i.e. the direction of response. Further, acquisition lesions produced a far greater impact on discriminated approach than performance lesions. This difference in lesion-induced impairment implies that mesoaccumbens dopamine may play differential roles in the learning and performance of preparatory Pavlovian conditioning.

Serotonin Increases Susceptibility to Pulmonary Hypertension in BMPR2-Deficient Mice

Heterozygous germline mutations in the gene encoding the bone morphogenetic protein type II (BMPR-II) receptor underlie the majority (>70%) of cases of familial pulmonary arterial hypertension (FPAH), and dysfunction of BMP signaling has been implicated in other forms of PAH. The reduced disease gene penetrance in FPAH indicates that other genetic and/or environmental factors may also be required for the clinical manifestation of disease. Of these, the serotonin pathway has been implicated as a major factor in PAH pathogenesis. We investigated the pulmonary circulation of mice deficient in BMPR-II (BMPR2+/− mice) and show that pulmonary hemodynamics and vascular morphometry of BMPR2+/− mice were similar to wild-type littermate controls under normoxic or chronic hypoxic (2- to 3-week) conditions. However, chronic infusion of serotonin caused increased pulmonary artery systolic pressure, right ventricular hypertrophy, and pulmonary artery remodeling in BMPR2+/− mice compared with wild-type littermates, an effect that was exaggerated under hypoxic conditions. In addition, pulmonary, but not systemic, resistance arteries from BMPR2+/− mice exhibited increased contractile responses to serotonin mediated by both 5-HT2 and 5-HT1 receptors. Furthermore, pulmonary artery smooth muscle cells from BMPR2+/− mice exhibited a heightened DNA synthesis and activation of extracellular signal-regulated kinase 1/2 in response to serotonin compared with wild-type cells. In vitro and in vivo experiments suggested that serotonin inhibits BMP signaling via Smad proteins and the expression of BMP responsive genes. These findings provide the first evidence for an interaction between BMPR-II-mediated signaling and the serotonin pathway, perturbation of which may be critical to the pathogenesis of PAH.

Effects of Selective Excitotoxic Lesions of the Nucleus Accumbens Core, Anterior Cingulate Cortex, and Central Nucleus of the Amygdala on Autoshaping Performance in Rats

The nucleus accumbens core (AcbC), anterior cingulate cortex (ACC), and central nucleus of the amygdala (CeA) are required for normal acquisition of tasks based on stimulus-reward associations. However, it is not known whether they are involved purely in the learning process or are required for behavioral expression of a learned response. Rats were trained preoperatively on a Pavlovian autoshaping task in which pairing a visual conditioned stimulus (CS+) with food causes subjects to approach the CS+ while not approaching an unpaired stimulus (CS-). Subjects then received lesions of the AcbC, ACC, or CeA before being retested. AcbC lesions severely impaired performance; lesioned subjects approached the CS+ significantly less often than controls, failing to discriminate between the CS+ and CS-. ACC lesions also impaired performance but did not abolish discrimination entirely. CeA lesions had no effect on performance. Thus, the CeA is required for learning, but not expression, of a conditioned approach response, implying that it makes a specific contribution to the learning of stimulus-reward associations.

Failure of bone morphogenetic protein receptor trafficking in pulmonary arterial hypertension: potential for rescue

Heterozygous germline mutations in the gene encoding the bone morphogenetic protein type II receptor cause familial pulmonary arterial hypertension (PAH). We previously demonstrated that the substitution of cysteine residues in the ligand-binding domain of this receptor prevents receptor trafficking to the cell membrane. Here we demonstrate the potential for chemical chaperones to rescue cell-surface expression of mutant BMPR-II and restore function. HeLa cells were transiently transfected with BMPR-II wild type or mutant (C118W) receptor constructs. Immunolocalization studies confirmed the retention of the cysteine mutant receptor mainly in the endoplasmic reticulum. Co-immunoprecipitation studies of Myc-tagged BMPR-II confirmed that the cysteine-substituted ligand-binding domain mutation, C118W, is able to associate with BMP type I receptors. Furthermore, following treatment with a panel of chemical chaperones (thapsigargin, glycerol or sodium 4-phenylbutyrate), we demonstrated a marked increase in cell-surface expression of mutant C118W BMPR-II by FACS analysis and confocal microscopy. These agents also enhanced the trafficking of wild-type BMPR-II, though to a lesser extent. Increased cell-surface expression of mutant C118W BMPR-II was associated with enhanced Smad1/5 phosphorylation in response to BMPs. These findings demonstrate the potential for rescue of mutant BMPR-II function from the endoplasmic reticulum. For the C118W mutation in the ligand-binding domain of BMPR-II, cell-surface rescue leads to at least partial restoration of BMP signalling. We conclude that enhancement of cell-surface trafficking of mutant and wild-type BMPR-II may have therapeutic potential in familial PAH.

Stoichiometric imbalance in the receptor complex contributes to dysfunctional BMPR-II mediated signalling in pulmonary arterial hypertension

Heterozygous germline defects in a gene encoding a type II receptor for bone morphogenetic proteins (BMPR-II) underlie the majority of inherited cases of the vascular disorder known as pulmonary arterial hypertension (PAH). However, the precise molecular consequences of PAH causing mutations on the function of the receptor complex remain unclear. We employed novel enzymatic and fluorescence activity based techniques to assess the impact of PAH mutations on pre-mRNA splicing, nonsense-mediated decay (NMD) and receptor complex interactions. We demonstrate that nonsense and frameshift mutations trigger NMD, providing further evidence that haplo-insufficiency is a major molecular consequence of disease-related BMPR2 mutations. We identified heterogeneous functional defects in BMPR-II activity, including impaired type I receptor phosphorylation, receptor interactions and altered receptor complex stoichiometry leading to perturbation of downstream signalling pathways. Importantly, these studies demonstrate that the intracellular domain of BMPR-II is both necessary and sufficient for receptor complex interaction. Finally and to address the potential for resolution of stoichiometric balance, we investigated an agent that promotes translational readthrough of a BMPR2 nonsense reporter construct without interfering with the NMD pathway. We propose that stoichiometric imbalance, due to either haplo-insufficiency or loss of optimal receptor-receptor interactions impairs BMPR-II mediated signalling in PAH. Taken together, these studies have identified an important target for early therapeutic intervention in familial PAH.

New insights into the pathogenesis and treatment of primary pulmonary hypertension

Primary pulmonary hypertension (PPH) is defined clinically by a sustained elevation of pulmonary arterial pressure (>25 mm Hg at rest or >30 mm Hg during exercise) without a demonstrable cause. Symptoms of PPH are largely non-specific but may include worsening shortness of breath, chest pain, syncope, fatigue, and peripheral oedema. It is a rare disorder with an estimated incidence of 2–3 per million per year.1 PPH shows a female bias with a F:M ratio of 2.3:1. The median age at diagnosis is 36 years but it may occur at any age. PPH is a progressive, often fatal, disease with mean survival from diagnosis of 2.8 years.2 The precise molecular mechanisms underlying PPH have hitherto remained elusive. Clues to the aetiology of the disease have been suggested by environmental stimuli associated with the development of severe pulmonary hypertension, pathologically indistinguishable from PPH. The association with appetite suppressants, in particular the fenfluramine/dexfenfluramine group,3 suggested a role of re-uptake inhibition of serotonin.4 Severe PPH may also follow HIV-1 infection, and PPH is associated with autoimmune thyroid disease and the presence of circulating anti-Ku and antinuclear antibodies.1 Pathologically, PPH is characterised by the obliteration of precapillary pulmonary arteries leading to right ventricular failure. Histologically, small pulmonary arteries show increased wall thickness, distal extension of smooth muscle into normally non-muscular vessels, the formation of a neointima, and plexiform lesions. The nature of the cell type responsible for the intimal obliteration of small pulmonary arteries in PPH has been the subject of considerable debate.5-7 The intimal lesions comprise myofibroblast-like cells, and the expression of endothelium specific markers is confined to cells lining the vascular lumen.5 8 Further debate surrounds the composition of the plexiform lesion,9 although substantial evidence exists for regarding this lesion as a proliferation of endothelial cells supported by a …

Trapping of BMP receptors in distinct membrane domains inhibits their function in pulmonary arterial hypertension

Bone morphogenetic proteins (BMPs) are pleiotrophic growth factors that influence diverse processes such as skeletal development, hematopoiesis, and neurogenesis. They play crucial roles in diseases such as pulmonary arterial hypertension (PAH). In PAH, mutants of the BMP type II receptors (BMPR2) were detected, and their functions were impaired during BMP signaling. It is thought that expression levels of these receptors determine the fate of BMP signaling, with low levels of expression leading to decreased Smad activation in PAH. However, our studies demonstrate, for the first time, that the localization of receptors on the plasma membrane, in this case BMPR2, was misdirected. Three BMPR2 mutants, D485G, N519K, and R899X, which are known to be involved in PAH, were chosen as our model system. Our results show that all three BMPR2 mutants decreased BMP-dependent Smad phosphorylation and Smad signaling. Although the three mutants reached the cell membrane and their expression was lower than that of BMPR2, they formed smaller clusters and associated differently with membrane domains, such as caveolae and clathrin-coated pits. The disruption of these domains restored the Smad signaling of D485G and N519K to the level of wild-type BMPR2, showing that these mutants were trapped in the domains, rather than just expressed at a lower level on the surface. Therefore, new treatment options for PAH should also target receptor localization, rather than just expression level.