First trimester placental endothelial cells from pregnancies with abnormal uterine artery Doppler are more sensitive to apoptotic stimuli.

Failure of the placental capillary network to develop normally is associated with early onset fetal growth restriction (FGR) and pre-eclampsia (PE). Although the symptoms are observed at term, the problem begins in the first trimester. However, investigations at this clinically relevant time are hindered by difficulties in identifying earlystage pregnancies that are at risk of developing FGR/PE. Using uterine artery Doppler ultrasound in the first trimester as a proxy measure of poor placentation, we have identified pregnancies at increased risk of developing early onset FGR/PE. Placental endothelial cells (PEC) isolated from pregnancies at increased risk of developing FGR/PE grew more slowly and their basal rate of apoptosis was significantly higher than that seen in the normal group. The pro-apoptotic stimulus, TNFα, induced apoptosis in cells from both groups but this was significantly greater in the high risk group. TNF receptor expression was unaffected. Inhibition of nitric oxide (NO) production significantly increased the sensitivity of cells from the normal pregnancies to TNFα but not in the high risk group establishing a functional role for NO in this system. In conclusion, first trimester PEC from pregnancies at increased risk of developing early onset FGR/PE were inherently more sensitive to apoptotic stimuli and this was functionally linked to the synthesis of NO. This may contribute to the poor placental vascular development seen in on going pregnancies.

Transcription factor 7-like 1 is involved in hypothalamo-pituitary axis development in mice and humans.

Aberrant embryonic development of the hypothalamus and/or pituitary gland in humans results in congenital hypopituitarism (CH). Transcription factor 7-like 1 (TCF7L1), an important regulator of the WNT/ß-catenin signaling pathway, is expressed in the developing forebrain and pituitary gland, but its role during hypothalamo-pituitary (HP) axis formation or involvement in human CH remains elusive. Using a conditional genetic approach in the mouse, we first demonstrate that TCF7L1 is required in the prospective hypothalamus to maintain normal expression of the hypothalamic signals involved in the induction and subsequent expansion of Rathke's pouch progenitors. Next, we reveal that the function of TCF7L1 during HP axis development depends exclusively on the repressing activity of TCF7L1 and does not require its interaction with ß-catenin. Finally, we report the identification of two independent missense variants in human TCF7L1, p.R92P and p.R400Q, in a cohort of patients with forebrain and/or pituitary defects. We demonstrate that these variants exhibit reduced repressing activity in vitro and in vivo relative to wild-type TCF7L1. Together, our data provide support for a conserved molecular function of TCF7L1 as a transcriptional repressor during HP axis development in mammals and identify variants in this transcription factor that are likely to contribute to the etiology of CH.

Pioglitazone Identifies a New Target for Aneurysm Treatment: Role of Egr1 in an Experimental Murine Model of Aortic Aneurysm.

Peroxisome proliferator-activated receptor x03B3; agonists have been shown to inhibit angiotensin II (AngII)-induced experimental abdominal aortic aneurysms. Macrophage infiltration to the vascular wall is an early event in this pathology, and therefore we explored the effects of the peroxisome proliferator-activated receptor x03B3; agonist pioglitazone on AngII-treated macrophages. Using microarray-based expression profiling of phorbol ester-stimulated THP-1 cells, we found that a number of aneurysm-related gene changes effected by AngII were modulated following the addition of pioglitazone. Among those genes, polycystic kidney disease 1 (PKD1) was significantly up-regulated (multiple testing corrected p < 0.05). The analysis of the PKD1 proximal promoter revealed a putative early growth response 1 (EGR1) binding site, which was confirmed by chromatin immunoprecipitation (ChIP) and quantitative PCR. Further analysis of publicly available ChIP-sequencing data revealed that this putative binding site overlapped with a conserved EGR1 binding peak present in 5 other cell lines. Quantitative real-time PCR showed that EGR1 suppressed PKD1, while AngII significantly up-regulated PKD1, an effect counteracted by pioglitazone. Conversely, in EGR1 short hairpin RNA lentivirally transduced THP-1 cells, reduced EGR1 led to a significant up-regulation of PKD1, especially after treatment with pioglitazone. In vivo, deficiency of Egr1 in the haematopoietic compartment of mice completely abolished the incidence of CaCl2-induced aneurysm formation.

Microglial Kv1.3 Channels and P2Y12 Receptors Differentially Regulate Cytokine and Chemokine Release from Brain Slices of Young Adult and Aged Mice.

Brain tissue damage following stroke or traumatic brain injury is accompanied by neuroinflammatory processes, while microglia play a central role in causing and regulating neuroinflammation via production of proinflammatory substances, including cytokines and chemokines. Here, we used brain slices, an established in situ brain injury model, from young adult and aged mice to investigate cytokine and chemokine production with particular focus on the role of microglia. Twenty four hours after slice preparation, higher concentrations of proinflammatory cytokines, i.e. TNF-α and IL-6, and chemokines, i.e. CCL2 and CXCL1, were released from brain slices of aged mice than from slices of young adult mice. However, maximal microglial stimulation with LPS for 24 h did not reveal age-dependent differences in the amounts of released cytokines and chemokines. Mechanisms underlying microglial cytokine and chemokine production appear to be similar in young adult and aged mice. Inhibition of microglial Kv1.3 channels with margatoxin reduced release of IL-6, but not release of CCL2 and CXCL1. In contrast, blockade of microglial P2Y12 receptors with PSB0739 inhibited release of CCL2 and CXCL1, whereas release of IL-6 remained unaffected. Cytokine and chemokine production was not reduced by inhibitors of Kir2.1 K+ channels or adenosine receptors. In summary, our data suggest that brain tissue damage-induced production of cytokines and chemokines is age-dependent, and differentially regulated by microglial Kv1.3 channels and P2Y12 receptors.

Cholesteryl Ester Transfer Protein Inhibitors - Future Soon to be REVEALed.

Reduction of the remaining residual cardiovascular risk is a clinical unmet need currently being addressed through a combination of further reduction of plasma concentrations of low-density lipoproteins (LDLs) and increasing plasma concentrations of high-density lipoproteins (HDLs). This brief review sets out the so-called HDL hypothesis and summarises the clinical results of the family of drugs, which function to raise plasma HDL concentration through inhibition of cholesteryl ester transfer proteins (CEPT).

Increased Expression of Lamin A/C Correlate with Regions of High Wall Stress in Abdominal Aortic Aneurysms.

BACKGROUND: Since aortic diameter is the most -significant risk factor for rupture, we sought to identify stress-dependent changes in gene expression to illuminate novel molecular processes in aneurysm rupture. MATERIALS AND METHODS: We constructed finite element maps of abdominal computerized tomography scans (CTs) of seven abdominal aortic aneurysm (AAA) patients to map wall stress. Paired biopsies from high- and low-stress areas were collected at surgery using vascular landmarks as coordinates. Differential gene expression was evaluated by Illumina Array analysis, using the whole genome DNA-mediated, annealing, selection, extension, and ligation (DASL) gene chip (n = 3 paired samples). RESULTS: The sole significant candidate from this analysis, Lamin A/C, was validated at the protein level, using western blotting. Lamin A/C expression in the inferior mesenteric vein (IMV) of AAA patients was compared to a control group and in aortic smooth muscle cells in culture in response to physiological pulsatile stretch. -Areas of high wall stress (n = 7) correlate to those -regions which have the thinnest walls [778 µm (585-1120 µm)] in comparison to areas of lowest wall stress [1620 µm (962-2919 µm)]. Induced expression of Lamin A/C -correlated with areas of high wall stress from AAAs but was not significantly induced in the IMV from AAA patients compared to controls (n = 16). Stress-induced expression of Lamin A/C was mimicked by exposing aortic smooth muscle cells to prolonged pulsatile stretch. CONCLUSION: Lamin A/C protein is specifically increased in areas of high wall stress in AAA from patients, but is not increased on other vascular beds of aneurysm patients, suggesting that its elevation may be a compensatory response to the pathobiology leading to aneurysms.

Differential role of apoptosis and autophagy associated with anticancer effect of lupulone (hop ß-acid) derivatives on prostate cancer cells.

Lupulone, a ß-acid derived from hop extracts has been shown to exhibit cytotoxic activity against cancer cells. In this study we investigated the functional role of different modes of cell death that mediate anticancer effect of lupulone derivatives in prostate cancer cells. ELISA, immunoblotting and siRNA approaches were utilised to study cell death, expression of proteins of interest and their functional activities. We found that the anticancer effect of lupulone derivatives on prostate cancer cells is associated with induction of apoptosis and autophagy as determined by increases of DNA fragmentation and LC3I/ LC3II conversion respectively. Inhibition of apoptosis using a pan-caspase inhibitor resulted in increased levels of autophagy. Following screening of proteins associated with autophagy we found that Atg4ß expression was increased in prostate cancer cells after treatment with lupulone. Transfection of cells with siRNA against Atg4ß resulted in increased levels of apoptosis in prostate cancer cells. Treatment of prostate cancer cells with lupulone derivatives initiated two modes of cell death: apoptosis as a killing pathway and autophagy as a protection against cell death. Further studies are required to investigate the regulation of Atg4ß activity in lupulone derivatives-induced negative crosstalk between apoptosis and autophagy.

Elevation of plasma high-density lipoproteins inhibits development of experimental abdominal aortic aneurysms.

OBJECTIVE: Patients with abdominal aortic aneurysms have lower concentrations of high-density lipoproteins (HDLs), leading us to investigate whether increasing plasma HDLs could influence aneurysm formation. METHODS AND RESULTS: Using the angiotensin II-induced hypercholesterolemic and the CaCl(2)-induced normocholesterolemic mouse model of AAA, we investigated the hypothesis that elevation of HDLs inhibits AAA. HDLs elevated before or at the time of AAA induction reduced AAA formation in both models but had no effect on early ruptures. Analysis of protein lysates from specific aortic segments demonstrated site-specific effects of HDLs on early signal transduction and cellular attrition. We found that HDLs reduced extracellular signal related kinases 1/2 activation in the suprarenal segment, while having no effect on p38 mitogen-associated protein kinase activation in any aortic segment and inhibiting c-Jun N-terminal kinase activation in all aortic segments. In addition, HDL elevation inhibited angiotensin II-induced apoptosis while inducing autophagy in the suprarenal segment of the aorta. Using Illumina gene array profiling we investigated the ability of HDL to modulate basal suprarenal aortic gene expression. CONCLUSIONS: Increasing plasma HDLs inhibit experimental AAA formation, independent of hypercholesterolemia via reduced extracellular signal related kinases 1/2 activation and alteration of the balance of cellular attrition. HDLs modulate genes involved in matrix remodelling, cell migration, and proliferation.

Increased Wingless (Wnt) signaling in pituitary progenitor/stem cells gives rise to pituitary tumors in mice and humans.

Wingless (Wnt)/ß-catenin signaling plays an essential role during normal development, is a critical regulator of stem cells, and has been associated with cancer in many tissues. Here we demonstrate that genetic expression of a degradation-resistant mutant form of ß-catenin in early Rathke's pouch (RP) progenitors leads to pituitary hyperplasia and severe disruption of the pituitary-specific transcription factor 1-lineage differentiation resulting in extreme growth retardation and hypopituitarism. Mutant mice mostly die perinatally, but those that survive weaning develop lethal pituitary tumors, which closely resemble human adamantinomatous craniopharyngioma, an epithelial tumor associated with mutations in the human ß-catenin gene. The tumorigenic effect of mutant ß-catenin is observed only when expressed in undifferentiated RP progenitors, but tumors do not form when committed or differentiated cells are targeted to express this protein. Analysis of affected pituitaries indicates that expression of mutant ß-catenin leads to a significant increase in the total numbers of pituitary progenitor/stem cells as well as in their proliferation potential. Our findings provide insights into the role of the Wnt pathway in normal pituitary development and demonstrate a causative role for mutated ß-catenin in an undifferentiated RP progenitor in the genesis of murine and human craniopharyngioma.

Relationship of connexin43 expression to phenotypic modulation in cultured human aortic smooth muscle cells.

Transition of arterial smooth muscle cells from the contractile to the synthetic phenotype in vivo is associated with up-regulation of the gap-junctional protein, connexin43 (Cx43). However, the role of increased Cx43 expression in relation to the characteristic features of the synthetic phenotype - altered growth, differentiation or synthetic activity - has not previously been defined. In the present study, growth was induced in cultured human aortic smooth muscle cells by treatment with thrombin and with PDGF-bb; growth arrest was induced by serum deprivation and contact inhibition. Alterations in Cx43 expression and gap-junctional communication were analyzed in relation to expression of markers for contractile differentiation and extracellular matrix synthesis. Treatment with thrombin, but not PDGF-bb, led to up-regulation of Cx43 gap junctions, increased synthetic activity yet also enhanced contractile differentiation. Inhibition of growth by deprivation of serum growth factors in sub-confluent cultures had no effect on Cx43 expression or contractile differentiation. Growth arrest by contact inhibition led to progressive reduction in Cx43 expression, in parallel with progressive increase in expression of differentiation markers but no alteration in synthetic activity. Of a range of stimuli examined, only thrombin had the combined effect of increasing Cx43 gap-junction communication, growth and synthesis, yet it also enhanced contractile differentiation. Down-regulation of Cx43 and improved contractile differentiation occurred only when growth arrest was induced through the contact-inhibition pathway, though, in this instance, synthesis remained undiminished. We conclude that Cx43 levels, though having common correlates, are not exclusively linked to the cell phenotype or the state of growth.

Up-regulation of connexin43 correlates with increased synthetic activity and enhanced contractile differentiation in TGF-beta-treated human aortic smooth muscle cells.

Up-regulation of the gap-junctional protein connexin43 (Cx43) in arterial smooth muscle cells (SMCs) features in response to injury and in atherosclerosis, in parallel with phenotypic transition to the synthetic state. TGF-beta1 is known to have a role in SMC differentiation and extracellular matrix (ECM) synthesis, key characteristics of phenotypic state. Here, we set out to examine the effects of TGF-beta1 on Cx43-gap junction expression in relation to SMC differentiation, ECM synthesis and growth. Cx43 expression was analysed by immunoconfocal microscopy and Western blotting in primary human aortic SMCs treated with TGF-beta1 over a 48-h period, with assessment of gap-junctional communication by cell-to-cell transfer of microinjected ethidium bromide. In parallel, synthetic activity was analysed by Northern blotting for ECM components alpha-1(I) and alpha1(III) procollagen transcripts, contractile differentiation was assessed by immunoconfocal microscopy and Western blotting of the markers smooth muscle alpha-actin, calponin and smooth muscle heavy chain isoform 1 (SM1), and growth was measured by BrdU incorporation. Our results demonstrate that TGF-beta1 significantly up-regulates Cx43 expression and intercellular communication, in concert with increased expression of alpha-actin, calponin and SM1. Concomitant with contractile protein expression, ECM synthesis was increased rather than decreased, TGF-beta1 inducing a significant up-regulation of both procollagen transcripts. These effects were independent of growth. We conclude that in human aortic SMCs, TGF-beta1 treatment leads to up-regulation of Cx43-mediated gap-junctional communication and increased synthetic activity yet, somewhat paradoxically, also enhanced contractile differentiation.