Hepatic Shunting of Eggs and Pulmonary Vascular Remodeling in Bmpr2(+/-) Mice with Schistosomiasis.

RATIONALE: Schistosomiasis is a major cause of pulmonary arterial hypertension (PAH). Mutations in the bone morphogenetic protein type-II receptor (BMPR-II) are the commonest genetic cause of PAH. OBJECTIVES: To determine whether Bmpr2(+/-) mice are more susceptible to schistosomiasis-induced pulmonary vascular remodeling. METHODS: Wild-type (WT) and Bmpr2(+/-) mice were infected percutaneously with Schistosoma mansoni. At 17 weeks postinfection, right ventricular systolic pressure and liver and lung egg counts were measured. Serum, lung and liver cytokine, pulmonary vascular remodeling, and liver histology were assessed. MEASUREMENTS AND MAIN RESULTS: By 17 weeks postinfection, there was a significant increase in pulmonary vascular remodeling in infected mice. This was greater in Bmpr2(+/-) mice and was associated with an increase in egg deposition and cytokine expression, which induced pulmonary arterial smooth muscle cell proliferation, in the lungs of these mice. Interestingly, Bmpr2(+/-) mice demonstrated dilatation of the hepatic central vein at baseline and postinfection, compared with WT. Bmpr2(+/-) mice also showed significant dilatation of the liver sinusoids and an increase in inflammatory cells surrounding the central hepatic vein, compared with WT. This is consistent with an increase in the transhepatic passage of eggs. CONCLUSIONS: This study has shown that levels of BMPR-II expression modify the pulmonary vascular response to chronic schistosomiasis. The likely mechanism involves the increased passage of eggs to the lungs, caused by altered diameter of the hepatic veins and sinusoids in Bmpr2(+/-) mice. Genetically determined differences in the remodeling of hepatic vessels may represent a new risk factor for PAH associated with schistosomiasis.

Fluorinated perhexiline derivative attenuates vascular proliferation in pulmonary arterial hypertension smooth muscle cells.

Increased proliferation and reduced apoptosis of pulmonary artery smooth muscle cells (PASMCs) is recognised as a universal hallmark of pulmonary arterial hypertension (PAH), in part related to the association with reduced pyruvate dehydrogenase (PDH) activity, resulting in decreased oxidative phosphorylation of glucose and increased aerobic glycolysis (Warburg effect). Perhexiline is a well-recognised carnitine palmitoyltransferase-1 (CPT1) inhibitor used in cardiac diseases, which reciprocally increases PDH activity, but is associated with variable pharmacokinetics related to polymorphic variation of the cytochrome P450-2D6 (CYP2D6) enzyme, resulting in the risk of neuro and hepatotoxicity in 'slow metabolisers' unless blood levels are monitored and dose adjusted. We have previously reported that a novel perhexiline fluorinated derivative (FPER-1) has the same therapeutic profile as perhexiline but is not metabolised by CYP2D6, resulting in more predictable pharmacokinetics than the parent drug. We sought to investigate the effects of perhexiline and FPER-1 on PDH flux in PASMCs from patients with PAH. We first confirmed that PAH PASMCs exhibited increased cell proliferation, enhanced phosphorylation of AKTSer473, ERK 1/2Thr202/Tyr204 and PDH-E1αSer293, indicating a Warburg effect when compared to healthy PASMCs. Pre-treatment with perhexiline or FPER-1 significantly attenuated PAH PASMC proliferation in a concentration-dependent manner and suppressed the activation of the AKTSer473 but had no effect on the ERK pathway. Perhexiline and FPER-1 markedly activated PDH (seen as dephosphorylation of PDH-E1αSer293), reduced glycolysis, and upregulated mitochondrial respiration in these PAH PASMCs as detected by Seahorse analysis. However, both perhexiline and FPER-1 did not induce apoptosis as measured by caspase 3/7 activity. We show for the first time that both perhexiline and FPER-1 may represent therapeutic agents for reducing cell proliferation in human PAH PASMCs, by reversing Warburg physiology.

Regulation of neurogenin stability by ubiquitin-mediated proteolysis.

NGN (neurogenin), a proneural bHLH (basic helix-loop-helix) transcription factor, plays a central role in promoting neuronal specification and differentiation in many regions of the central nervous system. NGN activity has been shown extensively to be controlled at the transcriptional level. However, in addition, recent findings have indicated that the levels of NGN protein may also be regulated. In the present study, we have demonstrated that NGN protein stability was regulated in both Xenopus embryos and P19 embryonal carcinoma cells, a mammalian neuronal model system. In both systems, NGN was a highly unstable protein that was polyubiquitinated for destruction by the proteasome. NGN binds to DNA in complex with its heterodimeric E-protein partners E12 or E47. We observed that NGN was stabilized by the presence of E12/E47. Moreover, NGN was phosphorylated, and mutation of a single threonine residue substantially reduced E12-mediated stabilization of NGN. Thus E-protein partner binding and phosphorylation events act together to stabilize NGN, promoting its accumulation when it can be active.

Inhibition of hyaluronan synthesis attenuates pulmonary hypertension associated with lung fibrosis.

BACKGROUND AND PURPOSE: Group III pulmonary hypertension (PH) is a highly lethal and widespread lung disorder that is a common complication in idiopathic pulmonary fibrosis (IPF) where it is considered to be the single most significant predictor of mortality. While increased levels of hyaluronan have been observed in IPF patients, hyaluronan-mediated vascular remodelling and the hyaluronan-mediated mechanisms promoting PH associated with IPF are not fully understood. EXPERIMENTAL APPROACH: Explanted lung tissue from patients with IPF with and without a diagnosis of PH was used to identify increased levels of hyaluronan. In addition, an experimental model of lung fibrosis and PH was used to test the capacity of 4-methylumbeliferone (4MU), a hyaluronan synthase inhibitor to attenuate PH. Human pulmonary artery smooth muscle cells (PASMC) were used to identify the hyaluronan-specific mechanisms that lead to the development of PH associated with lung fibrosis. KEY RESULTS: In patients with IPF and PH, increased levels of hyaluronan and expression of hyaluronan synthase genes are present. Interestingly, we also report increased levels of hyaluronidases in patients with IPF and IPF with PH. Remarkably, our data also show that 4MU is able to inhibit PH in our model either prophylactically or therapeutically, without affecting fibrosis. Studies to determine the hyaluronan-specific mechanisms revealed that hyaluronan fragments result in increased PASMC stiffness and proliferation but reduced cell motility in a RhoA-dependent manner. CONCLUSIONS AND IMPLICATIONS: Taken together, our results show evidence of a unique mechanism contributing to PH in the context of lung fibrosis.

The development of an in vitro screening strategy for topically applied products.

The objective of this study was the pharmaco-toxicological understanding of the constituents of an authenticated herbal mixture. The mixture was prepared by maceration in ethanol and subsequent dilution to produce a topically applied lotion, for which the intended target conditions are psoriasis and eczema. A three-tiered in vitro screening strategy was adopted for evaluating this product, comprising cytotoxicity assays; mutagenicity screening and therapeutic evaluation. Viability assays performed with dilutions of both the herbal concentrate and final product on organotypic cell lines indicated that neither preparation acted as an irritant. Genotoxicity screening using six strains of Salmonella typhimurium showed no mutagenic potential, and furthermore significant anti-microbial activity was evident. Therapeutic evaluation involved assessing the antioxidant potential of the extract, which can be correlated to an anti-inflammatory effect. Nitroblue-tetrazolium (NBT) assay results indicate that the extract can reduce superoxide anion generation by 45%. The extract also increased cell viability on exposure to hydrogen peroxide by 28%, illustrating its dismutation potential. A 3-D skin culture system, EpiDerm, released 3000 microg/ml upon exposure to the extract, implying that the components enhance arachidonic acid metabolism. Overall, it may be concluded that the herbal extract is sufficiently non-toxic for dermal application and possesses anti-inflammatory activity.

Ubiquitylation on canonical and non-canonical sites targets the transcription factor neurogenin for ubiquitin-mediated proteolysis.

Polyubiquitylation targets multiple proteins for degradation by the proteasome. Typically, the first ubiquitin is linked to lysine residues in the substrate for degradation via an isopeptide bond, although rarely ubiquitin linkage to the N-terminal residue has also been observed. We have recently shown that Neurogenin (NGN), a basic helix-loop-helix transcription factor that plays a central role in regulating neuronal differentiation, is degraded by ubiquitin-mediated proteolysis. We have taken a biochemical and mutagenesis approach to investigate sites of ubiquitylation of NGN, initially using extracts of eggs from the frog Xenopus laevis as a source of ubiquitylation and degradation components. NGN can be targeted for destruction by ubiquitylation via lysines or the N terminus. However, we see that a modified NGN, where canonical lysine ubiquitylation and N-terminally linked ubiquitylation are prevented, is nevertheless ubiquitylated and degraded by the proteasome. We show that polyubiquitin chains covalently attach to non-canonical cysteine residues in NGN, and these non-canonical linkages alone are capable of targeting NGN protein for destruction. Importantly, canonical and non-canonical ubiquitylation occurs simultaneously in the native protein and may differ in importance for driving degradation in interphase and mitosis. We conclude that native NGN is ubiquitylated on multiple canonical and non-canonical sites by cellular ubiquitin ligases, and all types of linkage can contribute to protein turnover.

The E3 ubiquitin ligase skp2 regulates neural differentiation independent from the cell cycle.

BACKGROUND: The SCFskp2 complex is an E3 ubiquitin ligase that is known to target a number of cell cycle regulators, including cyclin-dependent kinase inhibitors, for proteolysis. While its role in regulation of cell division has been well documented, additional functions in differentiation, including in the nervous system, have not been investigated. RESULTS: Using Xenopus as a model system, here we demonstrate that skp2 has an additional role in regulation of differentiation of primary neurons, the first neurons to differentiate in the neural plate. Xenopus skp2 shows a dynamic expression pattern in early embryonic neural tissue and depletion of skp2 results in generation of extra primary neurons. In contrast, over-expression of skp2 inhibits neurogenesis in a manner dependent on its ability to act as part of the SCFskp2 complex. Moreover, inhibition of neurogenesis by skp2 occurs upstream of the proneural gene encoding NeuroD and prior to cell cycle exit. We have previously demonstrated that the Xenopus cyclin dependent kinase inhibitor Xic1 is essential for primary neurogenesis at an early stage, and before these cells exit the cell cycle. We show that SCFskp2 degrades Xic1 in embryos and this contributes to the ability of skp2 to regulate neurogenesis. CONCLUSION: We conclude that the SCFskp2 complex has functions in the control of neuronal differentiation additional to its role in cell cycle regulation. Thus, it is well placed to be a co-ordinating factor regulating both cell proliferation and cell differentiation directly.

Notch targets the Cdk inhibitor Xic1 to regulate differentiation but not the cell cycle in neurons.

The proneural protein neurogenin (XNGNR1) drives differentiation of primary neurons in combination with the cyclin-dependent kinase (Cdk) inhibitor Xic1. Differentiation is inhibited by Notch signalling, resulting in a scattered neuronal distribution. Here we show that Notch signalling regulates the level of Xic1 transcription, yet this does not correlate with Notch's ability to perturb the cell cycle. Instead, Notch may regulate Xic1 levels to control its differentiation function directly, which is required in parallel with XNGNR1 to promote primary neurogenesis. Indeed, Notch-mediated repression of both XNGNR1 and Xic1 must be relieved for neuronal differentiation to occur. Interestingly, although Xic1 is required for XNGNR1-mediated neurogenesis, it is not required for XNGNR1-mediated upregulation of Delta, allowing establishment of the negative feedback loop involved in lateral inhibition. Therefore, Notch targets Cdk inhibitor expression to regulate differentiation of primary neurons, and its effects on the cell cycle may be of secondary importance.