Adenosine signalling to astrocytes coordinates brain metabolism and function.

Brain computation performed by billions of nerve cells relies on a sufficient and uninterrupted nutrient and oxygen supply1,2. Astrocytes, the ubiquitous glial neighbours of neurons, govern brain glucose uptake and metabolism3,4, but the exact mechanisms of metabolic coupling between neurons and astrocytes that ensure on-demand support of neuronal energy needs are not fully understood5,6. Here we show, using experimental in vitro and in vivo animal models, that neuronal activity-dependent metabolic activation of astrocytes is mediated by neuromodulator adenosine acting on astrocytic A2B receptors. Stimulation of A2B receptors recruits the canonical cyclic adenosine 3',5'-monophosphate-protein kinase A signalling pathway, leading to rapid activation of astrocyte glucose metabolism and the release of lactate, which supplements the extracellular pool of readily available energy substrates. Experimental mouse models involving conditional deletion of the gene encoding A2B receptors in astrocytes showed that adenosine-mediated metabolic signalling is essential for maintaining synaptic function, especially under conditions of high energy demand or reduced energy supply. Knockdown of A2B receptor expression in astrocytes led to a major reprogramming of brain energy metabolism, prevented synaptic plasticity in the hippocampus, severely impaired recognition memory and disrupted sleep. These data identify the adenosine A2B receptor as an astrocytic sensor of neuronal activity and show that cAMP signalling in astrocytes tunes brain energy metabolism to support its fundamental functions such as sleep and memory.

Microbiome and metabolome features of the cardiometabolic disease spectrum.

Previous microbiome and metabolome analyses exploring non-communicable diseases have paid scant attention to major confounders of study outcomes, such as common, pre-morbid and co-morbid conditions, or polypharmacy. Here, in the context of ischemic heart disease (IHD), we used a study design that recapitulates disease initiation, escalation and response to treatment over time, mirroring a longitudinal study that would otherwise be difficult to perform given the protracted nature of IHD pathogenesis. We recruited 1,241 middle-aged Europeans, including healthy individuals, individuals with dysmetabolic morbidities (obesity and type 2 diabetes) but lacking overt IHD diagnosis and individuals with IHD at three distinct clinical stages-acute coronary syndrome, chronic IHD and IHD with heart failure-and characterized their phenome, gut metagenome and serum and urine metabolome. We found that about 75% of microbiome and metabolome features that distinguish individuals with IHD from healthy individuals after adjustment for effects of medication and lifestyle are present in individuals exhibiting dysmetabolism, suggesting that major alterations of the gut microbiome and metabolome might begin long before clinical onset of IHD. We further categorized microbiome and metabolome signatures related to prodromal dysmetabolism, specific to IHD in general or to each of its three subtypes or related to escalation or de-escalation of IHD. Discriminant analysis based on specific IHD microbiome and metabolome features could better differentiate individuals with IHD from healthy individuals or metabolically matched individuals as compared to the conventional risk markers, pointing to a pathophysiological relevance of these features.

The MEK Inhibitor Trametinib Ameliorates Kidney Fibrosis by Suppressing ERK1/2 and mTORC1 Signaling.

BACKGROUND: During kidney fibrosis, a hallmark and promoter of CKD (regardless of the underlying renal disorder leading to CKD), the extracellular-regulated kinase 1/2 (ERK1/2) pathway, is activated and has been implicated in the detrimental differentiation and expansion of kidney fibroblasts. An ERK1/2 pathway inhibitor, trametinib, is currently used in the treatment of melanoma, but its efficacy in the setting of CKD and renal fibrosis has not been explored. METHODS: We investigated whether trametinib has antifibrotic effects in two mouse models of renal fibrosis-mice subjected to unilateral ureteral obstruction (UUO) or fed an adenine-rich diet-as well as in cultured primary human fibroblasts. We also used immunoblot analysis, immunohistochemical staining, and other tools to study underlying molecular mechanisms for antifibrotic effects. RESULTS: Trametinib significantly attenuated collagen deposition and myofibroblast differentiation and expansion in UUO and adenine-fed mice. We also discovered that in injured kidneys, inhibition of the ERK1/2 pathway by trametinib ameliorated mammalian target of rapamycin complex 1 (mTORC1) activation, another key profibrotic signaling pathway. Trametinib also inhibited the ERK1/2 pathway in cultured primary human renal fibroblasts stimulated by application of TGF-ß1, the major profibrotic cytokine, thereby suppressing downstream mTORC1 pathway activation. Additionally, trametinib reduced the expression of myofibroblast marker α-smooth muscle actin and the proliferation of renal fibroblasts, corroborating our in vivo data. Crucially, trametinib also significantly ameliorated renal fibrosis progression when administered to animals subsequent to myofibroblast activation. CONCLUSIONS: Further study of trametinib as a potential candidate for the treatment of chronic renal fibrotic diseases of diverse etiologies is warranted.

Coupling between the TRPC3 ion channel and the NCX1 transporter contributed to VEGF-induced ERK1/2 activation and angiogenesis in human primary endothelial cells.

It has been previously demonstrated that the bi-directional transporter Na+/Ca2+ exchanger (NCX) working in the reverse (Ca2+-influx) - mode promotes the activation of ERK1/2 in response to the key pro-angiogenic cytokine VEGF in human endothelial cells (ECs). However, the molecular event(s) that elicit NCX reversal in VEGF-stimulated ECs remain unclear. Here we investigated whether Na+ influx via the diacylglycerol (DAG) - activated non-selective cation channel TRPC3 was functionally associated with NCX and whether its activity was required for VEGF-induced ERK1/2 activation and angiogenesis. We provide evidence that TRPC3 inhibitors and siRNA attenuated ERK1/2 phosphorylation, reduced PKCα activity and partially suppressed Ca2+ transients in response to VEGF. Additionally, TRPC3 inhibitors and siRNA significantly suppressed endothelial tubular differentiation, an in vitro indicator of angiogenesis. We also report that simulating PLCγ activation downstream of VEGF receptor 2 by application of the cell-permeable DAG analogue 1-oleoyl-2-acetyl-sn-glycerol (OAG) was sufficient to activate ERK1/2 and enhance tubular differentiation. OAG-induced ERK1/2 activation and tubulogenesis were significantly suppressed by TRPC3 and reverse-mode NCX inhibitors and siRNA. Moreover, whilst both reverse-mode NCX and TRPC3 inhibitors attenuated OAG-induced Ca2+ transients, only TRPC3 antagonists blunted Na+ influx in response to OAG. Importantly, when Na+ was increased in ECs by inhibiting the Na+-K+-ATPase, TRPC3 activity was dispensable for OAG-induced ERK1/2 phosphorylation. Collectively, our research suggests that DAG generation downstream of VEGF receptors activatesTRPC3 causing Na+ influx with subsequent reversal of NCX, ERK1/2 activation and ultimately contributes to enhanced angiogenesis. Targeting reverse-mode NCX and its upstream initiator TRPC3 could be clinically relevant in conditions characterised by abnormal VEGF signalling.

Endothelial Angiogenesis and Barrier Function in Response to Thrombin Require Ca2+ Influx through the Na+/Ca2+ Exchanger.

Thrombin acts on the endothelium by activating protease-activated receptors (PARs). The endothelial thrombin-PAR system becomes deregulated during pathological conditions resulting in loss of barrier function and a pro-inflammatory and pro-angiogenic endothelial phenotype. We reported recently that the ion transporter Na(+)/Ca(2+) exchanger (NCX) operating in the Ca(2+)-influx (reverse) mode promoted ERK1/2 activation and angiogenesis in vascular endothelial growth factor-stimulated primary human vascular endothelial cells. Here, we investigated whether Ca(2+) influx through NCX was involved in ERK1/2 activation, angiogenesis, and endothelial barrier dysfunction in response to thrombin. Reverse-mode NCX inhibitors and RNAi-mediated NCX1 knockdown attenuated ERK1/2 phosphorylation in response to thrombin or an agonist of PAR-1, the main endothelial thrombin receptor. Conversely, promoting reverse-mode NCX by suppressing Na(+)-K(+)-ATPase activity enhanced ERK1/2 activation. Reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced primary human vascular endothelial cell angiogenesis, quantified as proliferation and tubular differentiation. Reverse-mode NCX inhibitors or NCX1 knockdown preserved barrier integrity upon thrombin stimulation in vitro. Moreover, the reverse-mode NCX inhibitor SEA0400 suppressed Evans' blue albumin extravasation to the lung and kidneys and attenuated edema formation and ERK1/2 activation in the lungs of mice challenged with a peptide activator of PAR-1. Mechanistically, thrombin-induced ERK1/2 activation required NADPH oxidase 2-mediated reactive oxygen species (ROS) production, and reverse-mode NCX inhibitors and NCX1 siRNA suppressed thrombin-induced ROS production. We propose that reverse-mode NCX is a novel mechanism contributing to thrombin-induced angiogenesis and hyperpermeability by mediating ERK1/2 activation in a ROS-dependent manner. Targeting reverse-mode NCX could be beneficial in pathological conditions involving unregulated thrombin signaling.

Ca2+ influx through reverse mode Na+/Ca2+ exchange is critical for vascular endothelial growth factor-mediated extracellular signal-regulated kinase (ERK) 1/2 activation and angiogenic functions of human endothelial cells.

VEGF is a key angiogenic cytokine and a major target in anti-angiogenic therapeutic strategies. In endothelial cells (ECs), VEGF binds VEGF receptors and activates ERK1/2 through the phospholipase γ (PLCγ)-PKCα-B-Raf pathway. Our previous work suggested that influx of extracellular Ca(2+) is required for VEGF-induced ERK1/2 activation, and we hypothesized that this could occur through reverse mode (Ca(2+) in and Na(+) out) Na(+)-Ca(2+) exchange (NCX). However, the role of NCX activity in VEGF signaling and angiogenic functions of ECs had not previously been described. Here, using human umbilical vein ECs (HUVECs), we report that extracellular Ca(2+) is required for VEGF-induced ERK1/2 activation and that release of Ca(2+) from intracellular stores alone, in the absence of extracellular Ca(2+), is not sufficient to activate ERK1/2. Furthermore, inhibitors of reverse mode NCX suppressed the VEGF-induced activation of ERK1/2 in a time- and dose-dependent manner and attenuated VEGF-induced Ca(2+) transients. Knockdown of NCX1 (the main NCX isoform in HUVECs) by siRNA confirmed the pharmacological data. A panel of NCX inhibitors also significantly reduced VEGF-induced B-Raf activity and inhibited PKCα translocation to the plasma membrane and total PKC activity in situ. Finally, NCX inhibitors reduced VEGF-induced HUVEC proliferation, migration, and tubular differentiation in surrogate angiogenesis functional assays in vitro. We propose that Ca(2+) influx through reverse mode NCX is required for the activation and the targeting of PKCα to the plasma membrane, an essential step for VEGF-induced ERK1/2 phosphorylation and downstream EC functions in angiogenesis.

Angiogenic functions of voltage-gated Na+ Channels in human endothelial cells: modulation of vascular endothelial growth factor (VEGF) signaling.

Voltage-gated sodium channel (VGSC) activity has previously been reported in endothelial cells (ECs). However, the exact isoforms of VGSCs present, their mode(s) of action, and potential role(s) in angiogenesis have not been investigated. The main aims of this study were to determine the role of VGSC activity in angiogenic functions and to elucidate the potentially associated signaling mechanisms using human umbilical vein endothelial cells (HUVECs) as a model system. Real-time PCR showed that the primary functional VGSC α- and ß-subunit isoforms in HUVECs were Nav1.5, Nav1.7, VGSCß1, and VGSCß3. Western blots verified that VGSCα proteins were expressed in HUVECs, and immunohistochemistry revealed VGSCα expression in mouse aortic ECs in vivo. Electrophysiological recordings showed that the channels were functional and suppressed by tetrodotoxin (TTX). VGSC activity modulated the following angiogenic properties of HUVECs: VEGF-induced proliferation or chemotaxis, tubular differentiation, and substrate adhesion. Interestingly, different aspects of angiogenesis were controlled by the different VGSC isoforms based on TTX sensitivity and effects of siRNA-mediated gene silencing. Additionally, we show for the first time that TTX-resistant (TTX-R) VGSCs (Nav1.5) potentiate VEGF-induced ERK1/2 activation through the PKCα-B-RAF signaling axis. We postulate that this potentiation occurs through modulation of VEGF-induced HUVEC depolarization and [Ca(2+)](i). We conclude that VGSCs regulate multiple angiogenic functions and VEGF signaling in HUVECs. Our results imply that targeting VGSC expression/activity could be a novel strategy for controlling angiogenesis.