Persistent vascular collagen accumulation alters hemodynamic recovery from chronic hypoxia

Pulmonary arterial hypertension (PAH) is caused by narrowing and stiffening of the pulmonary arteries that increase pulmonary vascular impedance (PVZ). In particular, small arteries narrow and large arteries stiffen. Large pulmonary artery (PA) stiffness is the best current predictor of mortality from PAH. We have previously shown that collagen accumulation leads to extralobar PA stiffening at high strain (Ooi et al. 2010). We hypothesized that collagen accumulation would increase PVZ, including total pulmonary vascular resistance (Z(0)), characteristic impedance (Z(C)), pulse wave velocity (PWV) and index of global wave reflections (P(b)/P(f)), which contribute to increased right ventricular afterload. We tested this hypothesis by exposing mice unable to degrade type I collagen (Col1a1(R/R)) to 21 days of hypoxia (hypoxia), some of which were allowed to recover for 42 days (recovery). Littermate wild-type mice (Col1a1(+/+)) were used as controls. In response to hypoxia, mean PA pressure (mPAP) increased in both mouse genotypes with no changes in cardiac output (CO) or PA inner diameter (ID); as a consequence, Z(0) (mPAP/CO) increased by ~100% in both genotypes (p<0.05). Contrary to our expectations, Z(C), PWV and P(b)/P(f) did not change. However, with recovery, Z(C) and PWV decreased in the Col1a1(+/+) mice and remained unchanged in the Col1a1(R/R) mice. Z(0) decreased with recovery in both genotypes. Microcomputed tomography measurements of large PAs did not show evidence of stiffness changes as a function of hypoxia exposure or genotype. We conclude that hypoxia-induced PA collagen accumulation does not affect the pulsatile components of pulmonary hemodynamics but that excessive collagen accumulation does prevent normal hemodynamic recovery, which may have important consequences for right ventricular function.     

Role of caspases in cytokine-induced barrier breakdown in human brain endothelial cells

During neuroinflammation, cytokines such as TNF-α and IFN-γ secreted by activated leukocytes and/or CNS resident cells have been shown to alter the phenotype and function of brain endothelial cells (BECs) leading to blood-brain barrier breakdown. In this study, we show that the human BEC line hCMEC/D3 expresses the receptors for TNF-α, TNF receptor 1 and TNF receptor 2, and for IFN-γ. BEC activation with TNF-α alone or in combination with IFN-γ induced endothelial leakage of paracellular tracers. At high cytokine concentrations (10 and 100 ng/ml), this effect was associated with caspase-3/7 activation and apoptotic cell death as evidenced by annexin V staining and DNA fragmentation (TUNEL) assays. In addition, inhibition of JNK and protein kinase C activation at these doses partially prevented activation of caspase-3/7, although only JNK inhibition was partially able to prevent the increase in BEC paracellular permeability induced by cytokines. By contrast, lower cytokine concentrations (1 ng/ml) also led to effector caspase activation, increased paracellular flux, and redistribution of zonula occludens-1 and VE-cadherin but failed to induce apoptosis. Under these conditions, specific caspase-3 and caspase-9, but not caspase-8, inhibitors partially blocked cytokine-induced disruption of tight and adherens junctions and BEC paracellular permeability. Our results suggest that the concentration of cytokines in the CNS endothelial microenvironment determines the extent of caspase-mediated barrier permeability changes, which may be generalized as a result of apoptosis or more subtle as a result of alterations in the organization of junctional complex molecules.     

Marine sponge Hymeniacidon sp. amphilectane metabolites potently inhibit rat brain microglia thromboxane B2 generation

The effects of five Hymeniacidon sp. amphilectane metabolites (1-5) and two semi-synthetic analogs (6 and 7) on thromboxane B(2) (TXB(2)) and superoxide anion (O(2)(-)) generation from Escherichia coli LPS-activated rat brain microglia were investigated. All Hymeniacidon sp. metabolites and analogs potently inhibited TXB(2) (IC(50)=0.20-4.69μM) with low lactate dehydrogenase release and minimal mitochondrial dehydrogenase inhibition. While a lack of O(2)(-) inhibition would suggest that Hymeniacidon sp. metabolites and derivatives inhibit TXB(2) synthesis by a cyclooxygenase-dependent mechanism, their pharmacologic potency and limited in vitro cytotoxicity warrants further investigation to develop them as lead compounds to modulate enhanced TBX(2) release by activated microglia in neuroinflammatory disorders.     

Notch: a key regulator of tumor angiogenesis and metastasis

The Notch signaling pathway is critical for many developmental processes including physiologic angiogenesis. Notch is also implicated in having a key role in tumor angiogenesis. Preclinical and clinical experience with anti-angiogenic strategies indicates that they may be limited by tumor resistance and recurrence, which has led to the search for alternative angiogenic treatment strategies. Significant progress has been made in shedding light on the complex mechanisms by which Notch signaling can influence tumor growth by disrupting vasculature in an array of tumor models (Ridgway et al., 2006). These results have led to the consideration of Notch as an attractive target to block tumor angiogenesis and inhibit growth. However, studies of inhibition of Notch signaling in different tumor models have uncovered similarly variable results, and some unexpected adverse effects. The ability of Notch to function in a context-dependent manner as a determinant of cell fate, a tumor suppressor, and an oncogene may partially explain the complexity in interpreted the role of Notch signaling inhibitors in preclinical tumor studies. In addition, Notch may also play an important role in metastasis via its direct effects on the vasculature and by modulation of epithelial-mesenchymal transition in tumor cells. Here we present a current understanding of Notch signaling in tumor angiogenesis, and discuss recent work on the role of Notch in tumor metastatic progression.     

Sound or Silence: Call Recognition in the Temporal Domain by the Frog Allobates femoralis

Acoustic communication often mediates agonistic interactions in territorial species. Because both the reaction to potential intruders and the lack thereof are costly, mechanisms that allow recognition of conspecific signals should be evident in intrasexual communication systems. While the spectral domain of the recognition space of the frog Allobates femoralis appears asymmetrically shaped in a way that reduces masking interference by the often syntopic frog Ameerega trivittata, frequency alone does not appear to account for the correct identification of conspecific intruders (Amézquita et al., Animal Behaviour, 70, 2005: 1377). As signal recognition may rely on a combination of spectral and temporal parameters of the signal, we test here the subsequent prediction that the recognition space should be asymmetrical in the temporal domain as well. We conducted playback experiments with 80 synthetic calls on 30 males and modeled all‐or‐none responses to define unidimensional functions of the recognition space for two call parameters: note duration and internote interval. For both parameters, male maximal response matched very well the average values of the conspecific signal and decreased with concomitant deviations from these values. While the response curves exceeded the range of signal variation and were not asymmetrical for either call parameter, they differed in breadth. The highest male permissiveness to variation in internote interval, as evidenced by a broader response curve, coincided with the lower probability of between‐species overlap in this signal parameter. Together with previous studies, our data suggest that a combination of spectral and temporal parameters of the advertisement call is necessary for recognition of calling intruders in A. femoralis. Our results emphasize the importance of multidimensional approaches in understanding signal recognition mechanisms in acoustically complex environments.     

IAPs regulate the plasticity of cell migration by directly targeting Rac1 for degradation

Inhibitors of apoptosis proteins (IAPs) are a highly conserved class of multifunctional proteins. Rac1 is a well-studied Rho GTPase that controls numerous basic cellular processes. While the regulation of nucleotide binding to Rac1 is well understood, the molecular mechanisms controlling Rac1 degradation are not known. Here, we demonstrate X-linked IAP (XIAP) and cellular IAP1 (c-IAP1) directly bind to Rac1 in a nucleotide-independent manner to promote its polyubiquitination at Lys147 and proteasomal degradation. These IAPs are also required for degradation of Rac1 upon CNF1 toxin treatment or RhoGDI depletion. Consistently, downregulation of XIAP or c-IAP1 by various strategies led to an increase in Rac1 protein levels in primary and tumour cells, leading to an elongated morphology and enhanced cell migration. Further, XIAP counteracts Rac1-dependent cellular polarization in the developing zebrafish hindbrain and promotes the delamination of neurons from the normal tissue architecture. These observations unveil an evolutionarily conserved role of IAPs in controlling Rac1 stability thereby regulating the plasticity of cell migration and morphogenesis.     

Familial hemiplegic migraine type 1 mutations W1684R and V1696I alter G protein-mediated regulation of CaV2.1 voltage-gated calcium channels

Familial hemiplegic migraine type 1 (FHM-1) is a monogenic form of migraine with aura that is characterized by recurrent attacks of a typical migraine headache with transient hemiparesis during the aura phase. In a subset of patients, additional symptoms such as epilepsy and cerebellar ataxia are part of the clinical phenotype. FHM-1 is caused by missense mutations in the CACNA1A gene that encodes the pore-forming subunit of Ca_V2.1 voltage-gated Ca^2 + channels. Although the functional effects of an increasing number of FHM-1 mutations have been characterized, knowledge on the influence of most of these mutations on G protein regulation of channel function is lacking. Here, we explored the effects of G protein-dependent modulation on mutations W1684R and V1696I which cause FHM-1 with and without cerebellar ataxia, respectively. Both mutations were introduced into the human Ca_V2.1α₁ subunit and their functional consequences investigated after heterologous expression in human embryonic kidney 293 (HEK‐293) cells using patch-clamp recordings. When co-expressed along with the human μ-opioid receptor, application of the agonist [d‐Ala2, N‐MePhe4, Gly‐ol]‐enkephalin (DAMGO) inhibited currents through both wild-type (WT) and mutant Ca_V2.1 channels, which is consistent with the known modulation of these channels by G protein-coupled receptors. Prepulse facilitation, which is a way to characterize the relief of direct voltage-dependent G protein regulation, was reduced by both FHM-1 mutations. Moreover, the kinetic analysis of the onset and decay of facilitation showed that the W1684R and V1696I mutations affect the apparent dissociation and reassociation rates of the Gβγ dimer from the channel complex, suggesting that the G protein-Ca^2 + channel affinity may be altered by the mutations. These biophysical studies may shed new light on the pathophysiology underlying FHM-1.     

Overexpression of the CHRNA5/A3/B4 genomic cluster in mice increases the sensitivity to nicotine and modifies its reinforcing effects

Nicotinic acetylcholine receptors (nAChRs) are ligand-gated pentameric ion channels that account for the effects of nicotine. Recent genetic studies have highlighted the importance of variants of the CHRNA5/A3/B4 genomic cluster in human nicotine dependence. Among these genetic variants those found in non-coding segments of the cluster may contribute to the pathophysiology of tobacco use through alterations in the expression of these genes. To discern the in vivo effects of the cluster, we generated a transgenic mouse overexpressing the human CHRNA5/A3/B4 cluster using a bacterial artificial chromosome. Transgenic mice showed increased functional α3β4-nAChRs in brain regions where these subunits are highly expressed under normal physiological conditions. Moreover, they exhibited increased sensitivity to the pharmacological effects of nicotine along with higher activation of the medial habenula and reduced activation of dopaminergic neurons in the ventral tegmental area after acute nicotine administration. Importantly, transgenic mice showed increased acquisition of nicotine self-administration (0.015 mg/kg per infusion) and a differential response in the progressive ratio test. Our study provides the first in vivo evidence of the involvement of the CHRNA5/A3/B4 genomic cluster in nicotine addiction through modifying the activity of brain regions responsible for the balance between the rewarding and the aversive properties of this drug.     

Environmental analysis of rainwater harvesting infrastructures in diffuse and compact urban models of Mediterranean climate

Purpose  

At present, many urban areas in Mediterranean climates are coping with water scarcity, facing a growing water demand and a limited conventional water supply. Urban design and planning has so far largely neglected the benefits of rainwater harvesting (RWH) in the context of a sustainable management of this resource. Therefore, the purpose of this study was to identify the most environmentally friendly strategy for rainwater utilization in Mediterranean urban environments of different densities.