The SH2-containing inositol polyphosphate 5-phosphatase, SHIP-2, binds filamin and regulates submembraneous actin

SHIP-2 is a phosphoinositidylinositol 3,4,5 trisphosphate (PtdIns[3,4,5]P3) 5-phosphatase that contains an NH2-terminal SH2 domain, a central 5-phosphatase domain, and a COOH-terminal proline-rich domain. SHIP-2 negatively regulates insulin signaling. In unstimulated cells, SHIP-2 localized in a perinuclear cytosolic distribution and at the leading edge of the cell. Endogenous and recombinant SHIP-2 localized to membrane ruffles, which were mediated by the COOH-terminal proline–rich domain. To identify proteins that bind to the SHIP-2 proline–rich domain, yeast two-hybrid screening was performed, which isolated actin-binding protein filamin C. In addition, both filamin A and B specifically interacted with SHIP-2 in this assay. SHIP-2 coimmunoprecipitated with filamin from COS-7 cells, and association between these species did not change after epidermal growth factor stimulation. SHIP-2 colocalized with filamin at Z-lines and the sarcolemma in striated muscle sections and at membrane ruffles in COS-7 cells, although the membrane ruffling response was reduced in cells overexpressing SHIP-2. SHIP-2 membrane ruffle localization was dependent on filamin binding, as SHIP-2 was expressed exclusively in the cytosol of filamin-deficient cells. Recombinant SHIP-2 regulated PtdIns(3,4,5)P3 levels and submembraneous actin at membrane ruffles after growth factor stimulation, dependent on SHIP-2 catalytic activity. Collectively these studies demonstrate that filamin-dependent SHIP-2 localization critically regulates phosphatidylinositol 3 kinase signaling to the actin cytoskeleton

Role of purinergic signalling in endothelial dysfunction and thrombo-inflammation in ischaemic stroke and cerebral small vessel disease

The cerebral endothelium is an active interface between blood and the central nervous system. In addition to being a physical barrier between the blood and the brain, the endothelium also actively regulates metabolic homeostasis, vascular tone and permeability, coagulation, and movement of immune cells. Being part of the blood–brain barrier, endothelial cells of the brain have specialized morphology, physiology, and phenotypes due to their unique microenvironment. Known cardiovascular risk factors facilitate cerebral endothelial dysfunction, leading to impaired vasodilation, an aggravated inflammatory response, as well as increased oxidative stress and vascular proliferation. This culminates in the thrombo-inflammatory response, an underlying cause of ischemic stroke and cerebral small vessel disease (CSVD). These events are further exacerbated when blood flow is returned to the brain after a period of ischemia, a phenomenon termed ischemia-reperfusion injury. Purinergic signaling is an endogenous molecular pathway in which the enzymes CD39 and CD73 catabolize extracellular adenosine triphosphate (eATP) to adenosine. After ischemia and CSVD, eATP is released from dying neurons as a damage molecule, triggering thrombosis and inflammation. In contrast, adenosine is anti-thrombotic, protects against oxidative stress, and suppresses the immune response. Evidently, therapies that promote adenosine generation or boost CD39 activity at the site of endothelial injury have promising benefits in the context of atherothrombotic stroke and can be extended to current CSVD known pathomechanisms. Here, we have reviewed the rationale and benefits of CD39 and CD39 therapies to treat endothelial dysfunction in the brain

The Need for Individualized Risk Assessment in Cardiovascular Disease

Cardiovascular disease remains the leading cause of death in the era of modern medicine despite major advancements in this field. Current available clinical surrogate markers and blood tests do not adequately predict individual risk of cardiovascular disease. A more precise and sophisticated tool that can reliably predict the thrombosis and bleeding risks at an individual level is required in order for clinicians to confidently recommend early interventions with a favorable risk–benefit profile. Critical to the development of this tool is the assessment and understanding of Virchow’s triad and its complex interactions between hypercoagulability, endothelial dysfunction and vessel flow, a fundamental concept to the development of thrombosis. This review explores the pathophysiology of cardiovascular disease stemming from the triad of factors and how individualized risk assessment can be improved through the multimodal use of tools such as global coagulation assays, endothelial biomarkers and vessel flow assessment

Characterization of a novel model of global forebrain ischaemia–reperfusion injury in mice and comparison with focal ischaemic and haemorrhagic stroke

Stroke is caused by obstructed blood flow (ischaemia) or unrestricted bleeding in the brain (haemorrhage). Global brain ischaemia occurs after restricted cerebral blood flow e.g. during cardiac arrest. Following ischaemic injury, restoration of blood flow causes ischaemia-reperfusion (I/R) injury which worsens outcome. Secondary injury mechanisms after any stroke are similar, and encompass inflammation, endothelial dysfunction, blood-brain barrier (BBB) damage and apoptosis. We developed a new model of transient global forebrain I/R injury (dual carotid artery ligation; DCAL) and compared the manifestations of this injury with those in a conventional I/R injury model (middle-cerebral artery occlusion; MCAo) and with intracerebral haemorrhage (ICH; collagenase model). MRI revealed that DCAL produced smaller bilateral lesions predominantly localised to the striatum, whereas MCAo produced larger focal corticostriatal lesions. After global forebrain ischaemia mice had worse overall neurological scores, although quantitative locomotor assessment showed MCAo and ICH had significantly worsened mobility. BBB breakdown was highest in the DCAL model while apoptotic activity was highest after ICH. VCAM-1 upregulation was specific to ischaemic models only. Differential transcriptional upregulation of pro-inflammatory chemokines and cytokines and TLRs was seen in the three models. Our findings offer a unique insight into the similarities and differences in how biological processes are regulated after different types of stroke. They also establish a platform for analysis of therapies such as endothelial protective and anti-inflammatory agents that can be applied to all types of stroke

Plasma Angiotensin Converting Enzyme 2 (ACE2) Activity in Healthy Controls and Patients with Cardiovascular Risk Factors and/or Disease

Angiotensin converting enzyme 2 (ACE2) is an endogenous negative regulator of the renin-angiotensin system, a key factor in the development of cardiovascular disease (CVD). ACE2 is also used by SARS-CoV-2 for host cell entry. Given that COVID-19 is associated with hypercoagulability, it is timely to explore the potential relationship between plasma ACE2 activity and the coagulation profile. In this cross-sectional study, ACE2 activity and global coagulation assays (GCA) including thromboelastography, thrombin, and fibrin generation were measured in adult healthy controls (n = 123; mean age 41 ± 17 years; 35% male) and in patients with cardiovascular risk factors and/or disease (n = 258; mean age 65 ± 14 years; 55% male). ACE2 activity was significantly lower in controls compared to patients with cardiovascular risk factors and/or disease (median 0.10 (0.02, 3.33) vs. 5.99 (1.95, 10.37) pmol/mL/min, p < 0.001). Of the healthy controls, 48% had undetectable ACE2 activity. Controls with detectable ACE2 had lower maximum amplitude (p < 0.001). In patients with cardiovascular risk factors and/or disease, those in the 3rd tertile were older and male (p = 0.002), with a higher Framingham grade and increased number of cardiovascular risk factors (p < 0.001). In conclusion, plasma ACE2 activity is undetectable to very low in young healthy controls with minimal clinically relevant associations to GCA. Patients with cardiovascular risk factors and/or disease have increased plasma ACE2 activity, suggesting that it may be an important biomarker of endothelial dysfunction and atherosclerosis

The Inositol Polyphosphate 5-Phosphatase, PIPP, Is a Novel Regulator of Phosphoinositide 3-Kinase-dependent Neurite Elongation

The spatial activation of phosphoinositide 3-kinase (PI3-kinase) signaling at the axon growth cone generates phosphatidylinositol 3,4,5 trisphosphate (PtdIns(3,4,5)P(3)), which localizes and facilitates Akt activation and stimulates GSK-3β inactivation, promoting microtubule polymerization and axon elongation. However, the molecular mechanisms that govern the spatial down-regulation of PtdIns(3,4,5)P(3) signaling at the growth cone remain undetermined. The inositol polyphosphate 5-phosphatases (5-phosphatase) hydrolyze the 5-position phosphate from phosphatidylinositol 4,5 bisphosphate (PtdIns(4,5)P(2)) and/or PtdIns(3,4,5)P(3). We demonstrate here that PIPP, an uncharacterized 5-phosphatase, hydrolyzes PtdIns(3,4,5)P(3) forming PtdIns(3,4)P(2), decreasing Ser473-Akt phosphorylation. PIPP is expressed in PC12 cells, localizing to the plasma membrane of undifferentiated cells and the neurite shaft and growth cone of NGF-differentiated neurites. Overexpression of wild-type, but not catalytically inactive PIPP, in PC12 cells inhibited neurite elongation. Targeted depletion of PIPP using RNA interference (RNAi) resulted in enhanced neurite differentiation, associated with neurite hyperelongation. Inhibition of PI3-kinase activity prevented neurite hyperelongation in PIPP-deficient cells. PIPP targeted-depletion resulted in increased phospho-Ser473-Akt and phospho-Ser9-GSK-3β, specifically at the neurite growth cone, and accumulation of PtdIns(3,4,5)P(3) at this site, associated with enhanced microtubule polymerization in the neurite shaft. PIPP therefore inhibits PI3-kinase-dependent neurite elongation in PC12 cells, via regulation of the spatial distribution of phospho-Ser473-Akt and phospho-Ser9-GSK-3β signaling

Phosphatidylinositol 3-Phosphate [PtdIns(3)P] Is Generated at the Plasma Membrane by an Inositol Polyphosphate 5-Phosphatase: Endogenous PtdIns(3)P Can Promote GLUT4 Translocation to the Plasma Membrane

Exogenous delivery of carrier-linked phosphatidylinositol 3-phosphate [PtdIns(3)P] to adipocytes promotes the trafficking, but not the insertion, of the glucose transporter GLUT4 into the plasma membrane. However, it is yet to be demonstrated if endogenous PtdIns(3)P regulates GLUT4 trafficking and, in addition, the metabolic pathways mediating plasma membrane PtdIns(3)P synthesis are uncharacterized. In unstimulated 3T3-L1 adipocytes, conditions under which PtdIns(3,4,5)P(3) was not synthesized, ectopic expression of wild-type, but not catalytically inactive 72-kDa inositol polyphosphate 5-phosphatase (72-5ptase), generated PtdIns(3)P at the plasma membrane. Immunoprecipitated 72-5ptase from adipocytes hydrolyzed PtdIns(3,5)P(2), forming PtdIns(3)P. Overexpression of the 72-5ptase was used to functionally dissect the role of endogenous PtdIns(3)P in GLUT4 translocation and/or plasma membrane insertion. In unstimulated adipocytes wild type, but not catalytically inactive, 72-5ptase, promoted GLUT4 translocation and insertion into the plasma membrane but not glucose uptake. Overexpression of FLAG-2xFYVE/Hrs, which binds and sequesters PtdIns(3)P, blocked 72-5ptase-induced GLUT4 translocation. Actin monomer binding, using latrunculin A treatment, also blocked 72-5ptase-stimulated GLUT4 translocation. 72-5ptase expression promoted GLUT4 trafficking via a Rab11-dependent pathway but not by Rab5-mediated endocytosis. Therefore, endogenous PtdIns(3)P at the plasma membrane promotes GLUT4 translocation