Search Results (8)

A better understanding of the function of neutrophil extracellular traps (NETs) may facilitate the development of interventions for sepsis. The study aims to investigate the formation and degradation of NETs in three murine sepsis models and to analyze the production of reactive oxygen species (ROS) during NET formation. Murine sepsis was induced by midgut volvulus (720° for 15 min), cecal ligation and puncture (CLP), or the application of lipopolysaccharide (LPS) (10 mg/kg body weight i.p.). NET formation and degradation was modulated using mice that were genetically deficient for peptidyl arginine deiminase-4 (PAD4-KO) or DNase1 and 1L3 (DNase1/1L3-DKO). After 48 h, mice were killed. Plasma levels of circulating free DNA (cfDNA) and neutrophil elastase (NE) were quantified to assess NET formation and degradation. Plasma deoxyribonuclease1 (DNase1) protein levels, as well as tissue malondialdehyde (MDA) activity and glutathione peroxidase (GPx) activity, were quantified. DNase1 and DNase1L3 in liver, intestine, spleen, and lung tissues were assessed. The applied sepsis models resulted in a simultaneous increase in NET formation and oxidative stress. NET formation and survival differed in the three models. In contrast to LPS and Volvulus, CLP-induced sepsis showed a decreased and increased 48 h survival in PAD4-KO and DNase1/1L3-DKO mice, when compared to WT mice, respectively. PAD4-KO mice showed decreased formation of NETs and ROS, while DNase1/1L3-DKO mice with impaired NET degradation accumulated ROS and chronicled the septic state. The findings indicate a dual role for NET formation and degradation in sepsis and ischemia-reperfusion (I/R) injury: NETs seem to exhibit a protective capacity in certain sepsis paradigms (CLP model), whereas, collectively, they seem to contribute adversely to scenarios where sepsis is combined with ischemia-reperfusion (volvulus). Full article

Spinocerebellar ataxia type 3 (SCA3) is a rare neurodegenerative disease caused by an abnormal polyglutamine expansion within the ataxin-3 protein (ATXN3). This leads to neurodegeneration of specific brain and spinal cord regions, resulting in a progressive loss of motor function. Despite neuronal death, non-neuronal cells, including astrocytes, are also involved in SCA3 pathogenesis. Astrogliosis is a common pathological feature in SCA3 patients and animal models of the disease. However, the contribution of astrocytes to SCA3 is not clearly defined. Inositol 1,4,5-trisphosphate receptor type 2 (IP₃R2) is the predominant IP₃R in mediating astrocyte somatic calcium signals, and genetically ablation of IP₃R2 has been widely used to study astrocyte function. Here, we aimed to investigate the relevance of IP₃R2 in the onset and progression of SCA3. For this, we tested whether IP₃R2 depletion and the consecutive suppression of global astrocytic calcium signalling would lead to marked changes in the behavioral phenotype of a SCA3 mouse model, the CMVMJD135 transgenic line. This was achieved by crossing IP₃R2 null mice with the CMVMJD135 mouse model and performing a longitudinal behavioral characterization of these mice using well-established motor-related function tests. Our results demonstrate that IP₃R2 deletion in astrocytes does not modify SCA3 progression. Full article

Background: Atrial fibrillation (AF) is promoted by various stimuli like angiotensin II, endothelin-1, epinephrine/norepinephrine, vagal activation, or mechanical stress, all of which activate receptors coupled to G-proteins of the Gα_q/Gα₁₁-family (G_q). Besides pro-fibrotic and pro-inflammatory effects, G_q-mediated signaling induces inositol trisphosphate receptor (IP₃R)-mediated intracellular Ca²⁺ mobilization related to delayed after-depolarisations and AF. However, direct evidence of arrhythmogenic G_q-mediated signaling is absent. Methods and results: To define the role of G_q in AF, transgenic mice with tamoxifen-inducible, cardiomyocyte-specific Gα_q/Gα₁₁-deficiency (G_q-KO) were created and exposed to intracardiac electrophysiological studies. Baseline electrophysiological properties, including heart rate, sinus node recovery time, and atrial as well as AV nodal effective refractory periods, were comparable in G_q-KO and control mice. However, inducibility and mean duration of AF episodes were significantly reduced in G_q-KO mice—both before and after vagal stimulation. To explore underlying mechanisms, left atrial cardiomyocytes were isolated from G_q-KO and control mice and electrically stimulated to study Ca²⁺-mobilization during excitation–contraction coupling using confocal microscopy. Spontaneous arrhythmogenic Ca²⁺ waves and sarcoplasmic reticulum content-corrected Ca²⁺ sparks were less frequent in G_q-KO mice. Interestingly, nuclear but not cytosolic Ca²⁺ transient amplitudes were significantly decreased in G_q-KO mice. Conclusion: G_q-signaling promotes arrhythmogenic atrial Ca²⁺-release and AF in mice. Targeting this pathway, ideally using G_q-selective, biased receptor ligands, may be a promising approach for the treatment and prevention of AF. Importantly, the atrial-specific expression of the G_q-effector IP₃R confers atrial selectivity mitigating the risk of life-threatening ventricular pro-arrhythmic effects. Full article

Inflammation plays a central role in the development of neonatal brain injury. The alpha 7 nicotinic acetylcholine receptor (α7nAChR) can modulate inflammation and has shown promising results as a treatment target in rodent models of adult brain injury. However, little is known about the role of the α7nAChR in neonatal brain injury. Hypoxic-ischemic (HI) brain injury was induced in male and female C57BL/6 mice, α7nAChR knock-out (KO) mice and their littermate controls on postnatal day (PND) 9–10. C57BL/6 pups received i.p. injections of α7nAChR agonist PHA 568487 (8 mg/kg) or saline once daily, with the first dose given directly after HI. Caspase-3 activity and cytokine mRNA expression in the brain was analyzed 24 h after HI. Motor function was assessed 24 and 48 h after HI, and immunohistochemistry was used to assess tissue loss at 24 h and 7 days after HI and microglial activation 7 days after HI. Activation of α7nAChR with the agonist PHA 568487 significantly decreased CCL2/MCP-1, CCL5/RANTES and IL-6 gene expression in the injured brain hemisphere 24 h after HI compared with saline controls in male, but not female, pups. However, α7nAChR activation did not alter caspase-3 activity and TNFα, IL-1β and CD68 mRNA expression. Furthermore, agonist treatment did not affect motor function (24 or 48 h), neuronal tissue loss (24 h or 7 days) or microglia activation (7 days) after HI in either sex. Knock-out of α7nAChR did not influence neuronal tissue loss 7 days after HI. In conclusion, targeting the α7nAChR in neonatal brain injury shows some effect on dampening acute inflammatory responses in male pups. However, this does not lead to an effect on overall injury outcome. Full article

The anxiolytic and antidepressant properties of cannabidiol (CBD) have been evaluated in several studies. However, the molecular mechanisms involved in these actions remain unclear. A total of 130 male mice were used. CBD’s ability to modulate emotional disturbances (anxiety and depressive-like behaviors) was evaluated at different doses in wild-type (CD1; 10, 20 and 30 mg/kg; i.p.) and knockout (CB1KO, CB2KO; GPR55KO; 20 mg/kg) mice. Moreover, CBD effects (20 mg/kg; i.p.) were evaluated in mice previously treated with the CB1r-antagonist SR141716A (2mg/kg; i.p.). Relative gene expression analyses of Cnr1 and Cnr2, Gpr55 and GABA(A)α2 and γ2 receptor subunits were performed in the amygdala (AMY) and hippocampus (HIPP) of CD1 mice. CBD (10 and 20 mg/kg) showed anxiolytic and antidepressant actions in CD1 mice, being more effective at 20 mg/kg. Its administration did not induce anxiolytic actions in CB1KO mice, contrary to CB2KO and GPR55KO. In all of them, the lack of cannabinoid receptors did not modify the antidepressant activity of CBD. Interestingly, the administration of the CB1r antagonist SR141716A blocked the anxiolytic-like activity of CBD. Real-time PCR studies revealed a significant reduction in Cnr1 and GABA(A)α2 and γ2 gene expression in the HIPP and AMY of CD1 mice treated with CBD. Opposite changes were observed in the Cnr2. Indeed, Gpr55 was increased in the AMY and reduced in the HIPP. CB1r appears to play a relevant role in modulating the anxiolytic actions of CBD. Moreover, this study revealed that CBD also modified the gene expression of GABA(A) subunits α2 and γ2 and CB1r, CB2r and GPR55, in a dose- and brain-region-dependent manner, supporting a multimodal mechanism of action for CBD. Full article

Inositol 1,4,5-triphosphate receptor-associated cGMP kinase substrate 1 (IRAG1) is a substrate protein of the NO/cGMP-signaling pathway and forms a ternary complex with the cGMP-dependent protein kinase Iβ (PKGIβ) and the inositol triphosphate receptor I (IP₃R-I). Functional studies about IRAG1 exhibited that IRAG1 is specifically phosphorylated by the PKGIβ, regulating cGMP-mediated IP₃-dependent Ca²⁺-release. IRAG1 is widely distributed in murine tissues, e.g., in large amounts in smooth muscle-containing tissues and platelets, but also in lower amounts, e.g., in the spleen. The NO/cGMP/PKGI signaling pathway is important in several organ systems. A loss of PKGI causes gastrointestinal disorders, anemia and splenomegaly. Due to the similar tissue distribution of the PKGIβ to IRAG1, we investigated the pathophysiological functions of IRAG1 in this context. Global IRAG1-KO mice developed gastrointestinal bleeding, anemia-associated splenomegaly and iron deficiency. Additionally, Irag1-deficiency altered the protein levels of some cGMP/PKGI signaling proteins—particularly a strong decrease in the PKGIβ—in the colon, spleen and stomach but did not change mRNA-expression of the corresponding genes. The present work showed that a loss of IRAG1 and the PKGIβ/IRAG1 signaling has a crucial function in the development of gastrointestinal disorders and anemia-associated splenomegaly. Furthermore, global Irag1-deficient mice are possible in vivo model to investigate PKGIβ protein functions. Full article

PKGs are serine/threonine kinases. PKG1 has two isoforms—PKG1α and β. Inositol trisphosphate receptor (IP₃R)-associated cGMP-kinase substrate 1 (IRAG1) is a substrate for PKG1β. IRAG1 is also known to further interact with IP₃RI, which mediates intracellular Ca²⁺ release. However, the role of IRAG1 in PH is not known. Herein, WT and IRAG1 KO mice were kept under normoxic or hypoxic (10% O₂) conditions for five weeks. Animals were evaluated for echocardiographic variables and went through right heart catheterization. Animals were further sacrificed to prepare lungs and right ventricular (RV) for immunostaining, western blotting, and pulmonary artery smooth muscle cell (PASMC) isolation. IRAG1 is expressed in PASMCs and downregulated under hypoxic conditions. Genetic deletion of IRAG1 leads to RV hypertrophy, increase in RV systolic pressure, and RV dysfunction in mice. Absence of IRAG1 in lung and RV have direct impacts on PKG1β expression. Attenuated PKG1β expression in IRAG1 KO mice further dysregulates other downstream candidates of PKG1β in RV. IRAG1 KO mice develop PH spontaneously. Our results indicate that PKG1β signaling via IRAG1 is essential for the homeostasis of PASMCs and RV. Disturbing this signaling complex by deleting IRAG1 can lead to RV dysfunction and development of PH in mice. Full article

MicroRNA-122 (miR-122) has been identified as a marker of various liver injuries, including hepatitis- virus-infection-, alcoholic-, and non-alcoholic steatohepatitis (NASH)-induced liver fibrosis. Here, we report that the extracellular miR-122 from hepatic cells can be delivered to hepatic stellate cells (HSCs) to modulate their proliferation and gene expression. Our published Argonaute crosslinking immunoprecipitation (Ago-CLIP) data identified several pro-fibrotic genes, including Ctgf, as miR-122 targets in mice livers. However, treating Ctgf as a therapeutic target failed to rescue the fibrosis developed in the miR-122 knockout livers. Alternatively, we compared the published datasets of human cirrhotic livers and miR-122 KO livers, which revealed upregulation of BCL2, suggesting its potential role in regulating fibrosis. Notably, ectopic miR-122 expression inhibited BCL2 expression in human HSC (LX-2) cells). Publicly available ChIP-seq data in human hepatocellular cancer (HepG2) cells and mice livers suggested miR-122 could regulate BCL2 expression indirectly through c-MYC, which was confirmed by siRNA-mediated depletion of c-MYC in Hepatocellular Carcinoma (HCC) cell lines. Importantly, Venetoclax, a potent BCL2 inhibitor approved for the treatment of leukemia, showed promising anti-fibrotic effects in miR-122 knockout mice. Collectively, our data demonstrate that miR-122 suppresses liver fibrosis and implicates anti-fibrotic potential of Venetoclax. Full article