Epac inhibits migration and proliferation of human prostate carcinoma cells

BACKGROUND: It was recently found that cAMP mediates protein kinase A-independent effects through Epac proteins. The aim of this study was to investigate the role of Epac in migration and proliferation of prostate carcinoma cells. METHODS: The effect of Epac activation was determined by [(3)H] thymidine incorporation and scratch assays in PC-3 and DU 145 cells. Furthermore, cytoskeletal integrity was analysed by phalloidin staining. The participation of intracellular Epac effectors such as mitogen-activated protein (MAP) kinases, Rap1- and Rho-GTPases was determined by immunoblotting and pull-down assay. RESULTS: The specific Epac activator 8-pCPT-2'-O-Me-cAMP (8-pCPT) interfered with cytoskeletal integrity, reduced DNA synthesis, and migration. Although 8-pCPT activated Rap1, it inhibited MAP kinase signalling and RhoA activation. These findings were translated into functional effects such as inhibition of mitogenesis, cytoskeletal integrity, and migration. CONCLUSION: In human prostate carcinoma cells, Epac inhibits proliferative and migratory responses likely because of inhibition of MAP kinase and RhoA signalling pathways. Therefore, Epac might represent an attractive therapeutic target in the treatment of prostate cancer. British Journal of Cancer (2009) 101, 2038-2042. doi: 10.1038/sj.bjc.6605439 www.bjcancer.com Published online 17 November 2009 (C) 2009 Cancer Research U

Red Blood Cell and Endothelial eNOS Independently Regulate Circulating Nitric Oxide Metabolites and Blood Pressure

Background: Current paradigms suggest that nitric oxide (NO) produced by endothelial cells (ECs) through endothelial nitric oxide synthase (eNOS) in the vessel wall is the primary regulator of blood flow and blood pressure. However, red blood cells (RBCs) also carry a catalytically active eNOS, but its role is controversial and remains undefined. This study aimed to elucidate the functional significance of RBC eNOS compared with EC eNOS for vascular hemodynamics and nitric oxide metabolism. Methods: We generated tissue-specific loss- and gain-of-function models for eNOS by using cell-specific Cre-induced gene inactivation or reactivation. We created 2 founder lines carrying a floxed eNOS (eNOSflox/flox) for Cre-inducible knockout (KO), and gene construct with an inactivated floxed/inverted exon (eNOSinv/inv) for a Cre-inducible knock-in (KI), which respectively allow targeted deletion or reactivation of eNOS in erythroid cells (RBC eNOS KO or RBC eNOS KI mice) or in ECs (EC eNOS KO or EC eNOS KI mice). Vascular function, hemodynamics, and nitric oxide metabolism were compared ex vivo and in vivo. Results: The EC eNOS KOs exhibited significantly impaired aortic dilatory responses to acetylcholine, loss of flow-mediated dilation, and increased systolic and diastolic blood pressure. RBC eNOS KO mice showed no alterations in acetylcholine-mediated dilation or flow-mediated dilation but were hypertensive. Treatment with the nitric oxide synthase inhibitor Nγ-nitro-l-arginine methyl ester further increased blood pressure in RBC eNOS KOs, demonstrating that eNOS in both ECs and RBCs contributes to blood pressure regulation. Although both EC eNOS KOs and RBC eNOS KOs had lower plasma nitrite and nitrate concentrations, the levels of bound NO in RBCs were lower in RBC eNOS KOs than in EC eNOS KOs. Reactivation of eNOS in ECs or RBCs rescues the hypertensive phenotype of the eNOSinv/invmice, whereas the levels of bound NO were restored only in RBC eNOS KI mice. Conclusions: These data reveal that eNOS in ECs and RBCs contribute independently to blood pressure homeostasis

Morphological, ultrastructural, genetic characteristics and remarkably low prevalence of macroscopic Sarcocystis species isolated from sheep and goats in Kurdistan region, Iraq

IntroductionSarcocystis is a genus of cyst-forming parasites that infest both humans and livestock. Some parasites cause clinical and subclinical diseases in their hosts, resulting in economic losses.MethodsEsophagus, diaphragm, and skeletal muscle from slaughtered sheep and goats were examined macroscopically, microscopically, and ultrastructurally and subjected to DNA analysis.ResultsWe isolated macrocysts of S. gigantea and of S. caprafelis moulei from naturally infected sheep (Ovis aries) and goats (Capra hircus). The macrocyst wall thickness was 18.9 µm in sheep and 15.3 µm in goats, and consisted of an inner Periodic acid Schiff- (PAS) negative primary wall and an outer glycoconjugates containing i.e. PAS-positive secondary wall. The walls inner surface was compartmentalized and filled with bradyzoites. In S. gigantea the bradyzoites were approximently 12.3 x 2.6 µm in size, while in S. caprafelis moulei they were 13.9 x 4.4 µm. Ultrastructurally, both species have nearly identical morphology: cauliflower-like protrusions with numerous microtubules and often dendritic-like filaments, branching from the primary wall. The 18S rRNA gene in S. gigantea was 85.9% identical to that in S. medusiformis and 80.4% to the S. caprafelis moulei gene. The 28S rRNA gene in S. gigantea was 94.6% identical to that in S. medusiformis and 97.3% to the S. caprafelis moulei.ConclusionThis study is the first to (i) detail the ultrastructure of the macrocyst wall of S. caprafelis moulei, (ii) identify S. medusiformis in Iraqi sheep, and (iii) compare the prevalence of macroscopic Sarcocystis at different time periods within the same region. A positive finding was the reduction of macroscopic sarcocystosis occurrences (0.01% in sheep and 0.02% in goats) compared to our previous data from 1992 (4.1%: sheep, 33.6%: goats)

Short-chain fatty acid propionate protects from hypertensive cardiovascular damage

BACKGROUND: Arterial hypertension and its organ sequelae show characteristics of T cell mediated inflammatory diseases. Experimental anti-inflammatory therapies have been shown to ameliorate hypertensive end-organ damage. Recently, the CANTOS study targeting interleukin-1β demonstrated that anti-inflammatory therapy reduces cardiovascular risk. The gut microbiome plays pivotal role in immune homeostasis and cardiovascular health. Short-chain fatty acids (SCFA) are produced from dietary fiber by gut bacteria and affect host immune homeostasis. Here, we investigated effects of the SCFA propionate in two different mouse models of hypertensive cardiovascular damage. METHODS: To investigate the effect of SCFA on hypertensive cardiac damage and atherosclerosis, wild-type NMRI (WT) or ApoE(-/-) deficient mice received propionate (200mM) or control in the drinking water. To induce hypertension, WT mice were infused with Angiotensin (Ang)II (1.44mg/kg/d s.c.) for 14 days. To accelerate the development of atherosclerosis, ApoE(-/-) mice were infused with AngII (0.72mg/kg/d s.c.) for 28 days. Cardiac damage and atherosclerosis were assessed using histology, echocardiography, in vivo electrophysiology, immunofluorescence, and flow cytometry. Blood pressure was measured by radiotelemetry. Regulatory T cell (Treg) depletion using PC61 antibody was used to examine the mode of action of propionate. RESULTS: Propionate significantly attenuated cardiac hypertrophy, fibrosis, vascular dysfunction, and hypertension in both models. Susceptibility to cardiac ventricular arrhythmias was significantly reduced in propionate-treated AngII-infused WT mice. Aortic atherosclerotic lesion area was significantly decreased in propionate-treated ApoE(-/-). Systemic inflammation was mitigated by propionate treatment, quantified as a reduction in splenic effector memory T cell frequencies and splenic T helper 17 cells in both models, and a decrease in local cardiac immune cell infiltration in WT mice. Cardioprotective effects of propionate were abrogated in Treg-depleted AngII-infused mice, suggesting the effect is Treg-dependent. CONCLUSIONS: Our data emphasize an immune-modulatory role of SCFAs and their importance for cardiovascular health. The data suggest that lifestyle modifications leading to augmented SCFA production could be a beneficial non-pharmacological preventive strategy for patients with hypertensive cardiovascular disease

4-Methylumbelliferone improves the thermogenic capacity of brown adipose tissue.

Therapeutic increase of brown adipose tissue (BAT) thermogenesis is of great interest as BAT activation counteracts obesity and insulin resistance. Hyaluronan (HA) is a glycosaminoglycan, found in the extracellular matrix, which is synthesized by HA synthases (Has1/Has2/Has3) from sugar precursors and accumulates in diabetic conditions. Its synthesis can be inhibited by the small molecule 4-methylumbelliferone (4-MU). Here, we show that the inhibition of HA-synthesis by 4-MU or genetic deletion of Has2/Has3 improves BAT`s thermogenic capacity, reduces body weight gain, and improves glucose homeostasis independently from adrenergic stimulation in mice on diabetogenic diet, as shown by a magnetic resonance T2 mapping approach. Inhibition of HA synthesis increases glycolysis, BAT respiration and uncoupling protein 1 expression. In addition, we show that 4-MU increases BAT capacity without inducing chronic stimulation and propose that 4-MU, a clinically approved prescription-free drug, could be repurposed to treat obesity and diabetes

The putative Tumor Suppressor VILIP-1 Counteracts Epidermal Growth Factor-Induced Epidermal-Mesenchymal Transition in Squamous Carcinoma Cells

Epithelial-mesenchymal transition (EMT) is a crucial step for the acquisition of invasive properties of carcinoma cells during tumor progression. Epidermal growth factor (EGF)-treatment of squamous cell carcinoma (SCC) cells provokes changes in the expression of lineage markers, morphological changes, and a higher invasive and metastatic potential. Here we show that chronic stimulation with EGF induces EMT in skin-derived SCC cell lines along with the down-regulation of the epithelial marker E-cadherin, and of the putative tumor suppressor VILIP-1 (visinin-like protein 1). In esophageal squamous cell carcinoma and non-small cell lung carcinoma the loss of VILIP-1 correlates with clinicopathological features related to enhanced invasiveness. VILIP-1 has previously been shown to suppress tumor cell invasion via enhancing cAMP-signaling in a murine SCC model. In mouse skin SCC cell lines the VILIP-1-negative tumor cells have low cAMP levels, whereas VILIP-1-positive SCCs possess high cAMP levels, but low invasive properties. We show that in VILIP-1-negative SCCs, Snail1, a transcriptional repressor involved in EMT, is up-regulated. Snail1 expression is reduced by ectopic VILIP-1-expression in VILIP-1-negative SCC cells, and application of the general adenylyl cyclase inhibitor 2′,3′-dideoxyadenosine attenuated this effect. Conversely, EGF-stimulation of VILIP-1-positive SCC cells leads to the down-regulation of VILIP-1 and the induction of Snail1 expression. The induction of Snail is inhibited by elevated cAMP levels. The role of cAMP in EMT was further highlighted by its suppressive effect on the EGF-induced enhancement of migration in VILIP-1-positive SCC cells. These findings indicate that VILIP-1 is involved in EMT of SCC by regulating the transcription factor Snail1 in a cAMP-dependent manner

A role for Piezo2 in EPAC1-dependent mechanical allodynia

N.E. and J.W. designed and supervised experiments. N.E. performed most of the in vivo and in vitro experiments. J.L. performed experiments to characterize hPiezo2. G.H and G.L. supervised by U.O., and J.T. and J.C. cloned hPiezo. L.B. performed the in vivo electrophysiology under the supervision of A.D. M.G. helped with the overexpression studies.M.M. performed surgery. Y.I. provided the Epac1 / mice. F.Z. provided the Epac constructs. N.E. and J.W. wrote manuscript with contributions of all authors. N.E., J.L. and L.B. contributed to data analysis and all authors contributed to the discussionsAberrant mechanosensation has an important role in different pain states. Here we show that Epac1 (cyclic AMP sensor) potentiation of Piezo2-mediated mechanotransduction contributes to mechanical allodynia. Dorsal root ganglia Epac1 mRNA levels increase during neuropathic pain, and nerve damage-induced allodynia is reduced in Epac1 / mice. The Epac-selective cAMP analogue 8-pCPT sensitizes mechanically evoked currents in sensory neurons. Human Piezo2 produces large mechanically gated currents that are enhanced by the activation of the cAMP-sensor Epac1 or cytosolic calcium but are unaffected by protein kinase C or protein kinase A and depend on the integrity of the cytoskeleton. In vivo, 8-pCPT induces long-lasting allodynia that is prevented by the knockdown of Epac1 and attenuated by mouse Piezo2 knockdown. Piezo2 knockdown also enhanced thresholds for light touch. Finally, 8-pCPT sensitizes responses to innocuous mechanical stimuli without changing the electrical excitability of sensory fibres. These data indicate that the Epac1–Piezo2 axis has a role in the development of mechanical allodynia during neuropathic pain.Netherlands Organization for Scientific Research (NWO)Jose Castillejo fellowship JC2010-0196Spanish GovernmentMedical Research Council UK (MRC)WCU at SNU R31-2008-000-10103-0EU IMI Europain grantBBSRC LOLA grantWellcome TrustVersus Arthritis 20200Biotechnology and Biological Sciences Research Council (BBSRC) BB/F000227/1Medical Research Council UK (MRC) G0901905 G9717869 G110034

Trained Innate Immunity in Animal Models of Cardiovascular Diseases

Acquisition of immunological memory is an important evolutionary strategy that evolved to protect the host from repetitive challenges from infectious agents. It was believed for a long time that memory formation exclusively occurs in the adaptive part of the immune system with the formation of highly specific memory T cells and B cells. In the past 10–15 years, it has become clear that innate immune cells, such as monocytes, natural killer cells, or neutrophil granulocytes, also have the ability to generate some kind of memory. After the exposure of innate immune cells to certain stimuli, these cells develop an enhanced secondary response with increased cytokine secretion even after an encounter with an unrelated stimulus. This phenomenon has been termed trained innate immunity (TI) and is associated with epigenetic modifications (histone methylation, acetylation) and metabolic alterations (elevated glycolysis, lactate production). TI has been observed in tissue-resident or circulating immune cells but also in bone marrow progenitors. Risk-factors for cardiovascular diseases (CVDs) which are associated with low-grade inflammation, such as hyperglycemia, obesity, or high salt, can also induce TI with a profound impact on the development and progression of CVDs. In this review, we briefly describe basic mechanisms of TI and summarize animal studies which specifically focus on TI in the context of CVDs

Long-Term Results After Head Orthosis Therapy for Early Childhood Position-Related Cranial Deformities: Stability of Treatment Outcome and Parental Satisfaction

The purpose of this study was to determine whether different types of position-related cranial deformities show changes after completion of head orthosis therapy. We investigated how children's age at the begin of molding helmet therapy affects the duration and long-term stability of treatment. In addition, parental satisfaction with helmet therapy has been investigated. Between 2012 and 2019, 47 patients have been analyzed after undergoing helmet therapy. At the beginning of treatment different disease pattern were classified. Posterior deformational Plagiocephalus defined by a cranial vault index (CVA) > 1 cm and a cranial index (CI) 90%. Posterior deformational combined Plagio- and Brachycephalus defined by CVA > 1 cm and CI> 90%. At the beginning of therapy, the end of therapy and within 5-year-follow-up CI and CVA was measured by three-dimensional photogrammetry for 2 age groups (4- 6 and 7-12 months). Additionally, parents completed a standardized questionnaire to evaluate the personal assessment of treatment outcome. During treatment CI and CVA of all children decreased significantly (P< 0.001). Furthermore, CI significantly decreased after ending helmet therapy (P< 0.001). Cranial vault index decreased not significantly in the same period (P = 0.361). For the 4 to 6 months old group treatment time was significantly shorter than for the older group. Before starting helmet therapy nearly half of the parents graded the scull as moderate and onethird as severe deformed. After ending treatment, the majority of parents report satisfaction and compliance. However, one- third of parents noted a slight scull deformation and personal load during therapy. After completion of therapy an improvement of head shape can be expected for the majority of children. This is particularly evident for improvement of the CI and a shorter treatment time until the age of 6months at the beginning of therapy. Parents reported satisfaction and child compliance. Almost all parents were convinced that treatment was useful and would repeat it again