Dysregulation of hydrogen sulfide producing enzyme cystathionine γ-lyase contributes to maternal hypertension and placental abnormalities in preeclampsia

Background-The exact etiology of preeclampsia is unknown, but there is growing evidence of an imbalance in angiogenic growth factors and abnormal placentation. Hydrogen sulfide (H2S), a gaseous messenger produced mainly by cystathionine γ-lyase (CSE), is a proangiogenic vasodilator. We hypothesized that a reduction in CSE activity may alter the angiogenic balance in pregnancy and induce abnormal placentation and maternal hypertension. Methods and Results-Plasma levels of H2S were significantly decreased in women with preeclampsia (P<0.01), which was associated with reduced placental CSE expression as determined by real-time polymerase chain reaction and immunohistochemistry. Inhibition of CSE activity by DL-propargylglycine reduced placental growth factorproduction from first-trimester (8-12 weeks gestation) human placental explants and inhibited trophoblast invasion in vitro. Knockdown of CSE in human umbilical vein endothelial cells by small-interfering RNA increased the release of soluble fms-like tyrosine kinase-1 and soluble endoglin, as assessed by enzyme-linked immunosorbent assay, whereas adenoviral-mediated CSE overexpression in human umbilical vein endothelial cells inhibited their release. Administration of DL-propargylglycine to pregnant mice induced hypertension and liver damage, promoted abnormal labyrinth vascularization in the placenta, and decreased fetal growth. Finally, a slow-releasing H2S-generating compound, GYY4137, inhibited circulating soluble fms-like tyrosine kinase-1 and soluble endoglin levels and restored fetal growth in mice that was compromised by DL-propargylglycine treatment, demonstrating that the effect of CSE inhibitor was attributable to inhibition of H2S production. Conclusions-These results imply that endogenous H2S is required for healthy placental vasculature and that a decrease in CSE/H2S activity may contribute to the pathogenesis of preeclampsia. © 2013 American Heart Association, Inc.

Deficiency in catechol-O-methyltransferase and 2-methoxyoestradiol is associated with pre-eclampsia

Despite intense investigation, mechanisms that facilitate the emergence of the pre-eclampsia phenotype in women are still unknown. Placental hypoxia, hypertension, proteinuria and oedema are the principal clinical features of this disease. It is speculated that hypoxia-driven disruption of the angiogenic balance involving vascular endothelial growth factor (VEGF)/placenta-derived growth factor (PLGF) and soluble Fms-like tyrosine kinase-1 (sFLT-1, the soluble form of VEGF receptor 1) might contribute to some of the maternal symptoms of pre-eclampsia. However, pre-eclampsia does not develop in all women with high sFLT-1 or low PLGF levels, and it also occurs in some women with low sFLT-1 and high PLGF levels. Moreover, recent experiments strongly suggest that several soluble factors affecting the vasculature are probably elevated because of placental hypoxia in the pre-eclamptic women, indicating that upstream molecular defect(s) may contribute to pre-eclampsia. Here we show that pregnant mice deficient in catechol-O-methyltransferase (COMT) show a pre-eclampsia-like phenotype resulting from an absence of 2-methoxyoestradiol (2-ME), a natural metabolite of oestradiol that is elevated during the third trimester of normal human pregnancy. 2-ME ameliorates all pre-eclampsia-like features without toxicity in the Comt(-/-) pregnant mice and suppresses placental hypoxia, hypoxia-inducible factor-1alpha expression and sFLT-1 elevation. The levels of COMT and 2-ME are significantly lower in women with severe pre-eclampsia. Our studies identify a genetic mouse model for pre-eclampsia and suggest that 2-ME may have utility as a plasma and urine diagnostic marker for this disease, and may also serve as a therapeutic supplement to prevent or treat this disorder.

Can the biology of VEGF and haem oxygenases help solve pre-eclampsia?

Pre-eclampsia, a pregnancy-specific multi-organ syndrome characterized by widespread endothelial damage, is a new risk factor for cardiovascular disease. No therapies exist to prevent or treat this condition, even to achieve a modest improvement in pregnancy length or birth weight. Co-administration of soluble VEGFR-1 [VEGF (vascular endothelial growth factor) receptor-1; more commonly known as sFlt-1 (soluble Fms-like tyrosine kinase-1)] and sEng (soluble endoglin) to pregnant rats elicits severe pre-eclampsia-like symptoms. These two anti-angiogenic factors are increased dramatically prior to the clinical onset of pre-eclampsia and are quite possibly the 'final common pathway' responsible for the accompanying signs of hypertension and proteinuria as they can be reversed by VEGF administration in animal models. HO-1 (haem oxygenase-1), an anti-inflammatory enzyme, and its metabolite, CO (carbon monoxide), exert protective effects in several organs against oxidative stimuli. In a landmark publication, we showed that the HO-1 pathway inhibits sFlt-1 and sEng in cultured cells and human placental tissue explants. Both CO and NO (nitric oxide) promote vascular homoeostasis and vasodilatation, and activation of VEGFR-1 or VEGFR-2 induced eNOS (endothelial nitric oxide synthase) phosphorylation, NO release and HO-1 expression. Our studies established the HO-1/CO pathway as a negative regulator of cytokine-induced sFlt-1 and sEng release and eNOS as a positive regulator of VEGF-mediated vascular morphogenesis. These findings provide compelling evidence for a protective role of HO-1 in pregnancy and identify it as a target for the treatment of pre-eclampsia. Any agent that is known to up-regulate HO-1, such as statins, may have potential as a therapy. Any intervention achieving even a modest prolongation of pregnancy or amelioration of the condition could have a significant beneficial health impact worldwide.

Unravelling the theories of pre-eclampsia:Are the protective pathways the new paradigm?

Pre-eclampsia is a vascular disorder of pregnancy where anti-angiogenic factors, systemic inflammation and oxidative stress predominate, but none can claim to cause pre-eclampsia. This review provides an alternative to the 'two-stage model' of pre-eclampsia in which abnormal spiral arteries modification leads to placental hypoxia, oxidative stress and aberrant maternal systemic inflammation. Very high maternal soluble fms-like tyrosine kinase-1 (sFlt-1 also known as sVEGFR) and very low placenta growth factor (PlGF) are unique to pre-eclampsia; however, abnormal spiral arteries and excessive inflammation are also prevalent in other placental disorders. Metaphorically speaking, pregnancy can be viewed as a car with an accelerator and brakes, where inflammation, oxidative stress and an imbalance in the angiogenic milieu act as the 'accelerator'. The 'braking system' includes the protective pathways of haem oxygenase 1 (also referred as Hmox1 or HO-1) and cystathionine-γ-lyase (also known as CSE or Cth), which generate carbon monoxide (CO) and hydrogen sulphide (H2S) respectively. The failure in these pathways (brakes) results in the pregnancy going out of control and the system crashing. Put simply, pre-eclampsia is an accelerator-brake defect disorder. CO and H2S hold great promise because of their unique ability to suppress the anti-angiogenic factors sFlt-1 and soluble endoglin as well as to promote PlGF and endothelial NOS activity. The key to finding a cure lies in the identification of cheap, safe and effective drugs that induce the braking system to keep the pregnancy vehicle on track past the finishing line.

ERK5:structure, regulation and function

Extracellular signal-regulated kinase 5 (ERK5), also termed big mitogen-activated protein kinase-1 (BMK1), is the most recently identified member of the mitogen-activated protein kinase (MAPK) family and consists of an amino-terminal kinase domain, with a relatively large carboxy-terminal of unique structure and function that makes it distinct from other MAPK members. It is ubiquitously expressed in numerous tissues and is activated by a variety of extracellular stimuli, such as cellular stresses and growth factors, to regulate processes such as cell proliferation and differentiation. Targeted deletion of Erk5 in mice has revealed that the ERK5 signalling cascade plays a critical role in cardiovascular development and vascular integrity. Recent data points to a potential role in pathological conditions such as cancer and tumour angiogenesis. This review focuses on the physiological and pathological role of ERK5, the regulation of this kinase and the recent development of small molecule inhibitors of the ERK5 signalling cascade. © 2012 Elsevier Inc.

Can hydrogen sulfide prevent preeclampsia and fetal growth restriction?

The exact aetiology of preeclampsia is unknown, but there is a good association with an imbalance in angiogenic growth factors and abnormal placentation [1]. Hydrogen sulphide (H2S), a gaseous messenger produced mainly by cystathionine γ-lyase (CSE), is pro-angiogenic vasodilator [2] and [3]. We hypothesized that a reduction in CSE activity may alter the angiogenic balance in pregnancy and induce abnormal placentation and maternal hypertension. Plasma levels of H2S were significantly decreased in preeclamptic women (p < 0.01), which was associated with reduced CSE message and protein expression in human placenta as determined by real-time PCR and immunohistochemistry. Inhibition of CSE activity by DL-propargylglycine (PAG) in first trimester (8–12 weeks gestation) human placental explants had reduced placenta growth factor (PlGF) production as assessed by ELISA and inhibited trophoblast invasion in vitro. Endothelial CSE knockdown by siRNA transfection increased the endogenous release of soluble fms-Like tyrosine kinase-1 (sFlt-1) and soluble endoglin, (sEng) from human umbilical vein endothelial cells while adenoviral-mediated CSE overexpression inhibited their release. Administration of PAG to pregnant mice induced hypertension, liver damage, and promoted abnormal labyrinth vascularisation in the placenta and decreased fetal growth. Finally, a slow releasing, H2S-generating compound, GYY4137, inhibited circulating sFlt-1 and sEng levels and restored fetal growth that was compromised by PAG-treatment demonstrating that the effect of CSE inhibitor was due to inhibition of H2S production. These results imply that endogenous H2S is required for healthy placental vasculature and a decrease in of CSE/H2S activity may contribute to the pathogenesis of preeclampsia. References [1] S. Ahmad, A. Ahmed, Elevated placental soluble vascular endothelial growth factor receptor-1 inhibits angiogenesis in preeclampsia, Circ Res., 95 (2004), pp. 884–891. [2] G. Yang, et al., H2S as a physiologic vasorelaxant: hypertension in mice with deletion of cystathionine gamma-lyase, Science, 322 (2008), pp. 587–590. [3] A. Papapetropoulos, et al., Hydrogen sulfide is an endogenous stimulator of angiogenesis, Proc Natl Acad Sci USA, 106 (2009), pp. 21972–21977.

Negative feedback regulation of the ERK1/2 MAPK pathway

The extracellular signal-regulated kinase 1/2 (ERK1/2) mitogen-activated protein kinase (MAPK) signalling pathway regulates many cellular functions, including proliferation, differentiation, and transformation. To reliably convert external stimuli into specific cellular responses and to adapt to environmental circumstances, the pathway must be integrated into the overall signalling activity of the cell. Multiple mechanisms have evolved to perform this role. In this review, we will focus on negative feedback mechanisms and examine how they shape ERK1/2 MAPK signalling. We will first discuss the extensive number of negative feedback loops targeting the different components of the ERK1/2 MAPK cascade, specifically the direct posttranslational modification of pathway components by downstream protein kinases and the induction of de novo gene synthesis of specific pathway inhibitors. We will then evaluate how negative feedback modulates the spatiotemporal signalling dynamics of the ERK1/2 pathway regarding signalling amplitude and duration as well as subcellular localisation. Aberrant ERK1/2 activation results in deregulated proliferation and malignant transformation in model systems and is commonly observed in human tumours. Inhibition of the ERK1/2 pathway thus represents an attractive target for the treatment of malignant tumours with increased ERK1/2 activity. We will, therefore, discuss the effect of ERK1/2 MAPK feedback regulation on cancer treatment and how it contributes to reduced clinical efficacy of therapeutic agents and the development of drug resistance.

Molecular mechanism of insulin-enhancing and -mimetic action of Vanadium compounds

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Molecular mechanism of insulin-enhancing and -mimetic action of Vanadium compounds

Thèse numérisée par la Direction des bibliothèques de l'Université de Montréal.

Transcriptome-Wide Mapping of Pea Seed Ageing Reveals a Pivotal Role for Genes Related to Oxidative Stress and Programmed Cell Death

Understanding of seed ageing, which leads to viability loss during storage, is vital for ex situ plant conservation and agriculture alike. Yet the potential for regulation at the transcriptional level has not been fully investigated. Here, we studied the relationship between seed viability, gene expression and glutathione redox status during artificial ageing of pea (Pisum sativum) seeds. Transcriptome-wide analysis using microarrays was complemented with qRT-PCR analysis of selected genes and a multilevel analysis of the antioxidant glutathione. Partial degradation of DNA and RNA occurred from the onset of artificial ageing at 60% RH and 50 degrees C, and transcriptome profiling showed that the expression of genes associated with programmed cell death, oxidative stress and protein ubiquitination were altered prior to any sign of viability loss. After 25 days of ageing viability started to decline in conjunction with progressively oxidising cellular conditions, as indicated by a shift of the glutathione redox state towards more positive values (>-190 mV). The unravelling of the molecular basis of seed ageing revealed that transcriptome reprogramming is a key component of the ageing process, which influences the progression of programmed cell death and decline in antioxidant capacity that ultimately lead to seed viability loss.

RGS14 (414)-mediated prevention of an episodic memory loss: a study of molecular mechanism

A large proportion of human populations suffer memory impairments either caused by normal aging or afflicted by diverse neurological and neurodegenerative diseases. Memory enhancers and other drugs tested so far against memory loss have failed to produce therapeutic efficacy in clinical trials and thus, there is a need to find remedy for this mental disorder. In search for cure of memory loss, our laboratory discovered a robust memory enhancer called RGS14(414). A treatment in brain with its gene produces an enduring effect on memory that lasts for lifetime of rats. Therefore, current thesis work was designed to investigate whether RGS14(414) treatment can prevent memory loss and furthermore, explore through biological processes responsible for RGS-mediated memory enhancement. We found that RGS14(414) gene treatment prevented episodic memory loss in rodent models of normal aging and Alzheimer´s disease. A memory loss was observed in normal rats at 18 months of age; however, when they were treated with RGS14(414) gene at 3 months of age, they abrogated this deficit and their memory remained intact till the age of 22 months. In addition to normal aging rats, effect of memory enhancer treatment in mice model of Alzheimer´s disease (AD-mice) produced a similar effect. AD-mice subjected to treatment with RGS14(414) gene at the age of 2 months, a period when memory was intact, showed not only a prevention in memory loss observed at 4 months of age but also they were able to maintain normal memory after 6 months of the treatment. We posit that long-lasting effect on memory enhancement and prevention of memory loss mediated through RGS14(414) might be due to a permanent structural change caused by a surge in neuronal connections and enhanced neuronal remodeling, key processes for long-term memory formation. A neuronal arborization analysis of both pyramidal and non-pyramidal neurons in brain of RGS14(414)-treated rats exhibited robust rise in neurites outgrowth of both kind of cells, and an increment in number of branching from the apical dendrite of pyramidal neurons, reaching to almost three times of the control animals. To further understand of underlying mechanism by which RGS14(414) induces neuronal arborization, we investigated into neurotrophic factors. We observed that RGS14 treatment induces a selective increase in BDNF. Role of BDNF in neuronal arborization, as well as its implication in learning and memory processes is well described. In addition, our results showing a dynamic expression pattern of BDNF during ORM processing that overlapped with memory consolidation further support the idea of the implication of this neurotrophin in formation of long-term memory in RGS-animals. On the other hand, in studies of expression profiling of RGS-treated animals, we have demonstrated that 14-3-3ζ protein displays a coherent relationship to RGS-mediated ORM enhancement. Recent studies have demonstrated that the interaction of receptor for activated protein kinase 1 (RACK1) with 14-3-3ζ is essential for its nuclear translocation, where RACK1-14-3-3ζ complex binds at promotor IV region of BDNF and promotes an increase in BDNF gene transcription. These observations suggest that 14-3-3ζ might regulate the elevated level of BDNF seen in RGS14(414) gene treated animals. Therefore, it seems that RGS-mediated surge in 14-3-3ζ causes elevated BDNF synthesis needed for neuronal arborization and enhanced ORM. The prevention of memory loss might be mediated through a restoration in BDNF and 14-3-3ζ protein levels, which are significantly decreased in aging and Alzheimer’s disease. Additionally, our results demonstrate that RGS14(414) treatment could be a viable strategy against episodic memory loss.

Ruolo del Microbiota Intestinale Nella Risposta al Trattamento con Anticorpi anti Checkpoints Immunitari in Pazienti Affetti da Linfoma

L’interazione tra il sistema immunitario dell’ospite e la cellula tumorale rappresenta uno degli elementi cardine dello sviluppo del clone neoplastico: la capacità della cellula cancerosa di evadere il controllo immunitario sfruttando meccanismi fisiologici come i checkpoint immunitari è alla base di diverse neoplasie, incluse le sindromi linfoproliferative. Lo sviluppo di anticorpi monoclonali che bloccano selettivamente l’interazione tra il recettore trans-membrana PD-1 (programmed death -1) ed i propri ligandi (PD-L1 e PD-L2), rappresenta una delle scoperte terapeutiche più promettenti in ambito onco-ematologico. Nonostante l’importante efficacia antitumorale degli anticorpi anti checkpoint immunitari dimostrata dai differenti studi clinici condotti sia in ambito oncologico che ematologico, una parte dei pazienti, a parità di patologia e di farmaco ricevuto, non risponde alla terapia o sviluppa eventi avversi immuno-relati. La comprensione della variabilità di risposta dimostrata dai pazienti con stessa patologia, sottoposti a stesso trattamento rappresenta pertanto un punto chiave allo scopo di identificare strategie che possano potenziare l’efficacia terapeutica di tali anticorpi, riducendone gli effetti collaterali. Studi recenti hanno evidenziato il ruolo del microbiota intestinale (MI) nel modellare la risposta immunitaria sistemica e, nel contesto neoplastico, nel modificare e mediare l’attivazione del sistema immunitario ad agenti chemio-immunoterapici. È noto che il MI sia un ecosistema plastico che può riorganizzare funzionalità e composizione in maniera adattativa in risposta a diversi fattori ambientali. La struttura individuale del MI e la sua dinamicità temporale possono, pertanto, influenzare l’outcome delle chemio-immunoterapie onco-ematologiche, modulandone l’efficacia e la tossicità. In questo scenario, ipotizziamo che la caratterizzazione longitudinale (pre, durante e post-terapia) del MI di pazienti affetti da linfoma trattati con anticorpi anti-checkpoint inibitori e la sua correlazione con la risposta al trattamento e con lo sviluppo di eventi avversi possa avere un ruolo nel delineare l’outcome di tali pazienti e nell’identificare nuovi criteri di stratificazione del rischio.

Roles for Basal and Stimulated p21Cip-1/WAF1/MDA6 Expression and Mitogen-activated Protein Kinase Signaling in Radiation-induced Cell Cycle Checkpoint Control in Carcinoma Cells

We investigated the role of the cdk inhibitor protein p21Cip-1/WAF1/MDA6 (p21) in the ability of MAPK pathway inhibition to enhance radiation-induced apoptosis in A431 squamous carcinoma cells. In carcinoma cells, ionizing radiation (2 Gy) caused both primary (0–10 min) and secondary (90–240 min) activations of the MAPK pathway. Radiation induced p21 protein expression in A431 cells within 6 h via secondary activation of the MAPK pathway. Within 6 h, radiation weakly enhanced the proportion of cells in G1 that were p21 and MAPK dependent, whereas the elevation of cells present in G2/M at this time was independent of either p21 expression or MAPK inhibition. Inhibition of the MAPK pathway increased the proportion of irradiated cells in G2/M phase 24–48 h after irradiation and enhanced radiation-induced apoptosis. This correlated with elevated Cdc2 tyrosine 15 phosphorylation, decreased Cdc2 activity, and decreased Cdc25C protein levels. Caffeine treatment or removal of MEK1/2 inhibitors from cells 6 h after irradiation reduced the proportion of cells present in G2/M phase at 24 h and abolished the ability of MAPK inhibition to potentiate radiation-induced apoptosis. These data argue that MAPK signaling plays an important role in the progression/release of cells through G2/M phase after radiation exposure and that an impairment of this progression/release enhances radiation-induced apoptosis. Surprisingly, the ability of irradiation/MAPK inhibition to increase the proportion of cells in G2/M at 24 h was found to be dependent on basal p21 expression. Transient inhibition of basal p21 expression increased the control level of apoptosis as well as the abilities of both radiation and MEK1/2 inhibitors to cause apoptosis. In addition, loss of basal p21 expression significantly reduced the capacity of MAPK inhibition to potentiate radiation-induced apoptosis. Collectively, our data argue that MAPK signaling and p21 can regulate cell cycle checkpoint control in carcinoma cells at the G1/S transition shortly after exposure to radiation. In contrast, inhibition of MAPK increases the proportion of irradiated cells in G2/M, and basal expression of p21 is required to maintain this effect. Our data suggest that basal and radiation-stimulated p21 may play different roles in regulating cell cycle progression that affect cell survival after radiation exposure.

Arabidopsis WEE1 kinase controls cell cycle arrest in response to activation of the DNA integrity checkpoint

Upon the incidence of DNA stress, the ataxia telangiectasia-mutated (ATM) and Rad3-related (ATR) signaling kinases activate a transient cell cycle arrest that allows cells to repair DNA before proceeding into mitosis. Although the ATM-ATR pathway is highly conserved over species, the mechanisms by which plant cells stop their cell cycle in response to the loss of genome integrity are unclear. We demonstrate that the cell cycle regulatory WEE1 kinase gene of Arabidopsis thaliana is transcriptionally activated upon the cessation of DNA replication or DNA damage in an ATR- or ATM-dependent manner, respectively. In accordance with a role for WEE1 in DNA stress signaling, WEE1-deficient plants showed no obvious cell division or endoreduplication phenotype when grown under nonstress conditions but were hypersensitive to agents that impair DNA replication. Induced WEE1 expression inhibited plant growth by arresting dividing cells in the G2-phase of the cell cycle. We conclude that the plant WEE1 gene is not rate-limiting for cycle progression under normal growth conditions but is a critical target of the ATR-ATM signaling cascades that inhibit the cell cycle upon activation of the DNA integrity checkpoints, coupling mitosis to DNA repair in cells that suffer DNA damage.