Caloric Restriction Rejuvenates Skeletal Muscle Growth in Heart Failure With Preserved Ejection Fraction.

Heart failure with preserved ejection fraction (HFpEF) is a major clinical problem, with limited treatments. HFpEF is characterized by a distinct, but poorly understood, skeletal muscle pathology, which could offer an alternative therapeutic target. In a rat model, we identified impaired myonuclear accretion as a mechanism for low myofiber growth in HFpEF following resistance exercise. Acute caloric restriction rescued skeletal muscle pathology in HFpEF, whereas cardiac therapies had no effect. Mechanisms regulating myonuclear accretion were dysregulated in patients with HFpEF. Overall, these findings may have widespread implications in HFpEF, indicating combined dietary with exercise interventions as a beneficial approach to overcome skeletal muscle pathology.

Independent endothelial functions of PIEZO1 and TRPV4 in hepatic portal vein and predominance of PIEZO1 in mechanical and osmotic stress.

BACKGROUND & AIMS: PIEZO1 and TRPV4 are mechanically and osmotically regulated calcium-permeable channels. The aim of this study was to determine the relevance and relationship of these channels in the contractile tone of the hepatic portal vein, which experiences mechanical and osmotic variations as it delivers blood to the liver from the intestines, gallbladder, pancreas and spleen. METHODS: Wall tension was measured in freshly dissected portal veins from adult male mice, which were genetically unmodified or modified for either a non-disruptive tag in native PIEZO1 or endothelial-specific PIEZO1 deletion. Pharmacological agents were used to activate or inhibit PIEZO1, TRPV4 and associated pathways, including Yoda1 and Yoda2 for PIEZO1 and GSK1016790A for TRPV4 agonism, respectively. RESULTS: PIEZO1 activation leads to nitric oxide synthase- and endothelium-dependent relaxation of the portal vein. TRPV4 activation causes contraction, which is also endothelium-dependent but independent of nitric oxide synthase. The TRPV4-mediated contraction is suppressed by inhibitors of phospholipase A2 and cyclooxygenases and mimicked by prostaglandin E2 , suggesting mediation by arachidonic acid metabolism. TRPV4 antagonism inhibits the effect of agonising TRPV4 but not PIEZO1. Increased wall stretch and hypo-osmolality inhibit TRPV4 responses while lacking effects on or amplifying PIEZO1 responses. CONCLUSIONS: The portal vein contains independently functioning PIEZO1 channels and TRPV4 channels in the endothelium, the pharmacological activation of which leads to opposing effects of vessel relaxation (PIEZO1) and contraction (TRPV4). In mechanical and osmotic strain, the PIEZO1 mechanism dominates. Modulators of these channels could present important new opportunities for manipulating liver perfusion and regeneration in disease and surgical procedures.

Cardiac q-space trajectory imaging by motion-compensated tensor-valued diffusion encoding in human heart in vivo.

PURPOSE: Tensor-valued diffusion encoding can probe more specific features of tissue microstructure than what is available by conventional diffusion weighting. In this work, we investigate the technical feasibility of tensor-valued diffusion encoding at high b-values with q-space trajectory imaging (QTI) analysis, in the human heart in vivo. METHODS: Ten healthy volunteers were scanned on a 3T scanner. We designed time-optimal gradient waveforms for tensor-valued diffusion encoding (linear and planar) with second-order motion compensation. Data were analyzed with QTI. Normal values and repeatability were investigated for the mean diffusivity (MD), fractional anisotropy (FA), microscopic FA (µFA), isotropic, anisotropic and total mean kurtosis (MKi, MKa, and MKt), and orientation coherence (Cc ). A phantom, consisting of two fiber blocks at adjustable angles, was used to evaluate sensitivity of parameters to orientation dispersion and diffusion time. RESULTS: QTI data in the left ventricular myocardium were MD = 1.62 ± 0.07 µm2 /ms, FA = 0.31 ± 0.03, µFA = 0.43 ± 0.07, MKa = 0.20 ± 0.07, MKi = 0.13 ± 0.03, MKt = 0.33 ± 0.09, and Cc  = 0.56 ± 0.22 (mean ± SD across subjects). Phantom experiments showed that FA depends on orientation dispersion, whereas µFA was insensitive to this effect. CONCLUSION: We demonstrated the first tensor-valued diffusion encoding and QTI analysis in the heart in vivo, along with first measurements of myocardial µFA, MKi, MKa, and Cc . The methodology is technically feasible and provides promising novel biomarkers for myocardial tissue characterization.

Cixutumumab reveals a critical role for IGF-1 in adipose and hepatic tissue remodelling during the development of diet-induced obesity.

High fat diet (HFD)-induced obesity leads to perturbation in the storage function of white adipose tissue (WAT) resulting in deposition of lipids in tissues ill-equipped to deal with this challenge. The role of insulin like growth factor-1 (IGF-1) in the systemic and organ-specific responses to HFD is unclear. Using cixutumumab, a monoclonal antibody that internalizes and degrades cell surface IGF-1 receptors (IGF-1 R), leaving insulin receptor expression unchanged we aimed to establish the role of IGF-1 R in the response to a HFD. Mice treated with cixutumumab fed standard chow developed mild hyperinsulinemia with no change in WAT. When challenged by HFD mice treated with cixutumumab had reduced weight gain, reduced WAT expansion, and reduced hepatic lipid vacuole formation. In HFD-fed mice, cixutumumab led to reduced levels of genes encoding proteins important in fatty acid metabolism in WAT and liver. Cixutumumab protected against blunting of insulin-stimulated phosphorylation of Akt in liver of HFD fed mice. These data reveal an important role for IGF-1 R in the WAT and hepatic response to short-term nutrient excess. IGF-1 R inhibition during HFD leads to a lipodystrophic phenotype with a failure of WAT lipid storage and protection from HFD-induced hepatic insulin resistance.

Transglutaminase 2 moderates the expansion of mouse abdominal aortic aneurysms.

OBJECTIVE: Previously published work has indicated that transcripts encoding transglutaminase 2 (TG2) increase markedly in a rat model of abdominal aortic aneurysm. This study determines whether TG2 and the related TG, factor XIII-A (FXIII-A), protect against aortic aneurysm development in mice. METHODS: C57BL/6J wild-type, Tgm2 -/- knockout, F13a1 -/- knockout, and Tgm2 -/- /F13a1 -/- double knockout mice were subjected to laparotomy and periaortic application of CaCl2. RESULTS: Tgm2 -/- mice showed slightly greater aortic dilatation at 6 weeks after treatment when compared with wild type. However, vessels from Tgm2 -/- mice, but not wild-type mice, continued to dilate up to 6 months after injury and by 24 weeks, a greater number of Tgm2 -/- mice had developed aneurysms (16/17 vs 10/19; P = .008). Laparotomy resulted in a high death rate in F13a1 -/- knockout mice, more frequently from cardiac complications than from hemorrhage, but among F13a1 -/- mice that survived for 6 weeks after CaCl2 treatment, abdominal aortic aneurysm diameter was unaltered relative to wild-type mice. Laparotomy resulted in a higher death rate among Tgm2 -/- /F13a1 -/- double knockout mice, owing to an increased frequency of delayed bleeding. Surprisingly, Tgm2 -/- /F13a1 -/- double knockout mice showed a trend toward decreased dilatation of the aorta 6 weeks after injury, and this finding was replicated in Tgm2 -/- /F13a1 -/- mice subjected to carotid artery injury. Levels of transcripts encoding TG2 were not increased in the aortas of injured wild-type or F13a1 -/- knockout mice relative to uninjured mice, although changes in the levels of other transcripts accorded with previous descriptions of the CaCl2 aneurysm model in mice. CONCLUSIONS: Knockout of Tgm2, but not F13a1 exacerbates aortic dilatation, suggesting that TG2 confers protection. However, levels of TG2 messenger RNA are not acutely elevated after injury. FXIII-A plays a role in preventing postoperative damage after laparotomy, confirming previous reports that it prevents distal organ damage after trauma. TG2 promotes wound healing after surgery and, in its absence, the bleeding diathesis associated with FXIII-A deficiency is further exposed.

Novel Paracrine Action of Endothelium Enhances Glucose Uptake in Muscle and Fat.

[Figure: see text].

Pericyte Insulin Receptors Modulate Retinal Vascular Remodeling and Endothelial Angiopoietin Signaling.

Pericytes regulate vascular development, stability, and quiescence; their dysfunction contributes to diabetic retinopathy. To explore the role of insulin receptors in pericyte biology, we created pericyte insulin receptor knockout mice (PIRKO) by crossing PDGFRß-Cre mice with insulin receptor (Insr) floxed mice. Their neonatal retinal vasculature exhibited perivenous hypervascularity with venular dilatation, plus increased angiogenic sprouting in superficial and deep layers. Pericyte coverage of capillaries was unaltered in perivenous and periarterial plexi, and no differences in vascular regression or endothelial proliferation were apparent. Isolated brain pericytes from PIRKO had decreased angiopoietin-1 mRNA, whereas retinal and lung angiopoietin-2 mRNA was increased. Endothelial phospho-Tie2 staining was diminished and FoxO1 was more frequently nuclear localized in the perivenous plexus of PIRKO, in keeping with reduced angiopoietin-Tie2 signaling. Silencing of Insr in human brain pericytes led to reduced insulin-stimulated angiopoietin-1 secretion, and conditioned media from these cells was less able to induce Tie2 phosphorylation in human endothelial cells. Hence, insulin signaling in pericytes promotes angiopoietin-1 secretion and endothelial Tie2 signaling and perturbation of this leads to excessive vascular sprouting and venous plexus abnormalities. This phenotype mimics elements of diabetic retinopathy, and future work should evaluate pericyte insulin signaling in this disease.

Elimination of fibrin γ-chain cross-linking by FXIIIa increases pulmonary embolism arising from murine inferior vena cava thrombi.

The onset of venous thromboembolism, including pulmonary embolism, represents a significant health burden affecting more than 1 million people annually worldwide. Current treatment options are based on anticoagulation, which is suboptimal for preventing further embolic events. In order to develop better treatments for thromboembolism, we sought to understand the structural and mechanical properties of blood clots and how this influences embolism in vivo. We developed a murine model in which fibrin γ-chain cross-linking by activated Factor XIII is eliminated (FGG3X) and applied methods to study thromboembolism at whole-body and organ levels. We show that FGG3X mice have a normal phenotype, with overall coagulation parameters and platelet aggregation and function largely unaffected, except for total inhibition of fibrin γ-chain cross-linking. Elimination of fibrin γ-chain cross-linking resulted in thrombi with reduced strength that were prone to fragmentation. Analysis of embolism in vivo using Xtreme optical imaging and light sheet microscopy demonstrated that the elimination of fibrin γ-chain cross-linking resulted in increased embolization without affecting clot size or lysis. Our findings point to a central previously unrecognized role for fibrin γ-chain cross-linking in clot stability. They also indirectly indicate mechanistic targets for the prevention of thrombosis through selective modulation of fibrin α-chain but not γ-chain cross-linking by activated Factor XIII to reduce thrombus size and burden, while maintaining clot stability and preventing embolism.

Endothelial IGF-1 receptor mediates crosstalk with the gut wall to regulate microbiota in obesity.

Changes in composition of the intestinal microbiota are linked to the development of obesity and can lead to endothelial cell (EC) dysfunction. It is unknown whether EC can directly influence the microbiota. Insulin-like growth factor-1 (IGF-1) and its receptor (IGF-1R) are critical for coupling nutritional status and cellular growth; IGF-1R is expressed in multiple cell types including EC. The role of ECIGF-1R in the response to nutritional obesity is unexplored. To examine this, we use gene-modified mice with EC-specific overexpression of human IGF-1R (hIGFREO) and their wild-type littermates. After high-fat feeding, hIGFREO weigh less, have reduced adiposity and have improved glucose tolerance. hIGFREO show an altered gene expression and altered microbial diversity in the gut, including a relative increase in the beneficial genus Akkermansia. The depletion of gut microbiota with broad-spectrum antibiotics induces a loss of the favourable metabolic differences seen in hIGFREO mice. We show that IGF-1R facilitates crosstalk between the EC and the gut wall; this crosstalk protects against diet-induced obesity, as a result of an altered gut microbiota.

Endothelial Insulin Receptors Promote VEGF-A Signaling via ERK1/2 and Sprouting Angiogenesis.

Endothelial insulin receptors (Insr) promote sprouting angiogenesis, although the underpinning cellular and molecular mechanisms are unknown. Comparing mice with whole-body insulin receptor haploinsufficiency (Insr+/-) against littermate controls, we found impaired limb perfusion and muscle capillary density after inducing hind-limb ischemia; this was in spite of increased expression of the proangiogenic growth factor Vegfa. Insr+/- neonatal retinas exhibited reduced tip cell number and branching complexity during developmental angiogenesis, which was also found in separate studies of mice with endothelium-restricted Insr haploinsufficiency. Functional responses to vascular endothelial growth factor A (VEGF-A), including in vitro angiogenesis, were also impaired in aortic rings and pulmonary endothelial cells from Insr+/- mice. Human umbilical vein endothelial cells with shRNA-mediated knockdown of Insr also demonstrated impaired functional angiogenic responses to VEGF-A. VEGF-A signaling to Akt and endothelial nitric oxide synthase was intact, but downstream signaling to extracellular signal-reduced kinase 1/2 (ERK1/2) was impaired, as was VEGF receptor-2 (VEGFR-2) internalization, which is required specifically for signaling to ERK1/2. Hence, endothelial insulin receptors facilitate the functional response to VEGF-A during angiogenic sprouting and are required for appropriate signal transduction from VEGFR-2 to ERK1/2.

Divergent effects of genetic and pharmacological inhibition of Nox2 NADPH oxidase on insulin resistance-related vascular damage.

Insulin resistance leads to excessive endothelial cell (EC) superoxide generation and accelerated atherosclerosis. The principal source of superoxide from the insulin-resistant endothelium is the Nox2 isoform of NADPH oxidase. Here we examine the therapeutic potential of Nox2 inhibition on superoxide generation in saphenous vein ECs (SVECs) from patients with advanced atherosclerosis and type 2 diabetes and on vascular function, vascular damage, and lipid deposition in apolipoprotein E-deficient (ApoE-/-) mice with EC-specific insulin resistance (ESMIRO). To examine the effect of genetic inhibition of Nox2, ESMIRO mice deficient in ApoE-/- and Nox2 (ESMIRO/ApoE-/-/Nox2-/y) were generated and compared with ESMIRO/ApoE-/-/Nox2+/y littermates. To examine the effect of pharmacological inhibition of Nox2, we administered gp91dstat or scrambled peptide to ESMIRO/ApoE-/- mice. SVECs from diabetic patients had increased expression of Nox2 protein with concomitant increase in superoxide generation, which could be reduced by the Nox2 inhibitor gp91dstat. After 12 wk Western diet, ESMIRO/ApoE-/-/Nox2-/y mice had reduced EC superoxide generation and greater aortic relaxation to acetylcholine. ESMIRO/ApoE-/-/Nox2-/y mice developed more lipid deposition in the thoraco-abdominal aorta with multiple foci of elastin fragmentation at the level of the aortic sinus and greater expression of intercellular adhesion molecule-1 (ICAM-1). Gp91dstat reduced EC superoxide and lipid deposition in the thoraco-abdominal aorta of ESMIRO/ApoE-/- mice without causing elastin fragmentation or increased ICAM-1 expression. These results demonstrate that insulin resistance is characterized by increased Nox2-derived vascular superoxide. Complete deletion of Nox2 in mice with EC insulin resistance exacerbates, whereas partial pharmacological Nox2 inhibition protects against, insulin resistance-induced vascular damage.

Elevated circulating amyloid concentrations in obesity and diabetes promote vascular dysfunction.

Diabetes, obesity, and Alzheimer's disease (AD) are associated with vascular complications and impaired nitric oxide (NO) production. Furthermore, increased ß-site amyloid precursor protein-cleaving (APP-cleaving) enzyme 1 (BACE1), APP, and ß-amyloid (Aß) are linked with vascular disease development and increased BACE1 and Aß accompany hyperglycemia and hyperlipidemia. However, the causal relationship between obesity and diabetes, increased Aß, and vascular dysfunction is unclear. We report that diet-induced obesity (DIO) in mice increased plasma and vascular Aß42 that correlated with decreased NO bioavailability, endothelial dysfunction, and increased blood pressure. Genetic or pharmacological reduction of BACE1 activity and Aß42 prevented and reversed, respectively, these outcomes. In contrast, expression of human mutant APP in mice or Aß42 infusion into control diet-fed mice to mimic obese levels impaired NO production, vascular relaxation, and raised blood pressure. In humans, increased plasma Aß42 correlated with diabetes and endothelial dysfunction. Mechanistically, higher Aß42 reduced endothelial NO synthase (eNOS), cyclic GMP (cGMP), and protein kinase G (PKG) activity independently of diet, whereas endothelin-1 was increased by diet and Aß42. Lowering Aß42 reversed the DIO deficit in the eNOS/cGMP/PKG pathway and decreased endothelin-1. Our findings suggest that BACE1 inhibitors may have therapeutic value in the treatment of vascular disease associated with diabetes.

IGFBP-1 in Cardiometabolic Pathophysiology-Insights From Loss-of-Function and Gain-of-Function Studies in Male Mice.

We have previously reported that overexpression of human insulin-like growth factor binding protein (IGFBP)-1 in mice leads to vascular insulin sensitization, increased nitric oxide bioavailability, reduced atherosclerosis, and enhanced vascular repair, and in the setting of obesity improves glucose tolerance. Human studies suggest that low levels of IGFBP-1 are permissive for the development of diabetes and cardiovascular disease. Here we seek to determine whether loss of IGFBP-1 plays a causal role in the predisposition to cardiometabolic disease. Metabolic phenotyping was performed in transgenic mice with homozygous knockout of IGFBP-1. This included glucose, insulin, and insulin-like growth factor I tolerance testing under normal diet and high-fat feeding conditions. Vascular phenotyping was then performed in the same mice using vasomotor aortic ring studies, flow cytometry, vascular wire injury, and angiogenesis assays. These were complemented with vascular phenotyping of IGFBP-1 overexpressing mice. Metabolic phenotype was similar in IGFBP-1 knockout and wild-type mice subjected to obesity. Deletion of IGFBP-1 inhibited endothelial regeneration following injury, suggesting that IGFBP-1 is required for effective vascular repair. Developmental angiogenesis was unaltered by deletion or overexpression of IGFBP-1. Recovery of perfusion following hind limb ischemia was unchanged in mice lacking or overexpressing IGFBP-1; however, overexpression of IGFBP-1 stimulated hindlimb perfusion and angiogenesis in insulin-resistant mice. These findings provide new insights into the role of IGFBP-1 in metabolic and vascular pathophysiology. Irrespective of whether loss of IGFBP-1 plays a causal role in the development of cardiometabolic disorders, increasing IGFBP-1 levels appears effective in promoting neovascularization in response to ischemia.

Fibroblast-specific deletion of interleukin-1 receptor-1 reduces adverse cardiac remodeling following myocardial infarction.

It has been hypothesized that interleukin-1alpha (IL-1α) is released from damaged cardiomyocytes following myocardial infarction (MI) and activates cardiac fibroblasts via its receptor (IL-1R1) to drive the early stages of cardiac remodeling. This study aimed to definitively test this hypothesis using cell type-specific IL-1α and IL-1R1 knockout (KO) mouse models. A floxed Il1α mouse was created and used to generate a cardiomyocyte-specific IL-1α KO mouse line (MIL1AKO). A tamoxifen-inducible fibroblast-specific IL-1R1 hemizygous KO mouse line (FIL1R1KO) was also generated. Mice underwent experimental MI (permanent left anterior descending coronary artery ligation) and cardiac function was determined 4 weeks later by conductance pressure-volume catheter analysis. Molecular markers of remodeling were evaluated at various time points by real-time RT-PCR and histology. MIL1AKO mice showed no difference in cardiac function or molecular markers of remodeling post-MI compared with littermate controls. In contrast, FIL1R1KO mice showed improved cardiac function and reduced remodeling markers post-MI compared with littermate controls. In conclusion, these data highlight a key role for the IL-1R1/cardiac fibroblast signaling axis in regulating post-MI remodeling and provide support for the continued development of anti-IL-1 therapies for improving cardiac function after MI. Cardiomyocyte-derived IL-1α was not an important contributor to post-MI remodeling in this model.

TRPC5 ion channel permeation promotes weight gain in hypercholesterolaemic mice.

Transient Receptor Potential Canonical 5 (TRPC5) is a subunit of a Ca2+-permeable non-selective cationic channel which negatively regulates adiponectin but not leptin in mice fed chow diet. Adiponectin is a major anti-inflammatory mediator and so we hypothesized an effect of TRPC5 on the inflammatory condition of atherosclerosis. Atherosclerosis was studied in aorta of ApoE-/- mice fed western-style diet. Inhibition of TRPC5 ion permeation was achieved by conditional transgenic expression of a dominant negative ion pore mutant of TRPC5 (DNT5). Gene expression analysis in adipose tissue suggested that DNT5 increases transcript expression for adiponectin while decreasing transcript expression of the inflammatory mediator Tnfα and potentially decreasing Il6, Il1ß and Ccl2. Despite these differences there was mild or no reduction in plaque coverage in the aorta. Unexpectedly DNT5 caused highly significant reduction in body weight gain and reduced adipocyte size after 6 and 12 weeks of western-style diet. Steatosis and circulating lipids were unaffected but mild effects on regulators of lipogenesis could not be excluded, as indicated by small reductions in the expression of Srebp1c, Acaca, Scd1. The data suggest that TRPC5 ion channel permeation has little or no effect on atherosclerosis or steatosis but an unexpected major effect on weight gain.

Attenuation of oxidative stress-induced lesions in skeletal muscle in a mouse model of obesity-independent hyperlipidaemia and atherosclerosis through the inhibition of Nox2 activity.

Obesity leading to hyperlipidaemia and atherosclerosis is recognised to induce morphological and metabolic changes in many tissues. However, hyperlipidaemia can occur in the absence of obesity. The impact of the latter scenario on skeletal muscle and liver is not understood sufficiently. In this regard, we used the Apolipoprotein E-deficient (ApoE-/-) mouse model, an established model of hyperlipidaemia and atherosclerosis, that does not become obese when subjected to a high-fat diet, to determine the impact of Western-type diet (WD) and ApoE deficiency on skeletal muscle morphological, metabolic and biochemical properties. To establish the potential of therapeutic targets, we further examined the impact of Nox2 pharmacological inhibition on skeletal muscle redox biology. We found ectopic lipid accumulation in skeletal muscle and the liver, and altered skeletal muscle morphology and intramuscular triacylglycerol fatty acid composition. WD and ApoE deficiency had a detrimental impact in muscle metabolome, followed by perturbed gene expression for fatty acid uptake and oxidation. Importantly, there was enhanced oxidative stress in the skeletal muscle and development of liver steatosis, inflammation and oxidative protein modifications. Pharmacological inhibition of Nox2 decreased reactive oxygen species production and protein oxidative modifications in the muscle of ApoE-/- mice subjected to a Western-type diet. This study provides key evidence to better understand the pathophysiology of skeletal muscle in the context of hyperlipidaemia and atherosclerosis and identifies Nox2 as a potential target for attenuating oxidative stress in skeletal muscle in a mouse model of obesity-independent hyperlipidaemia.

Endothelial Insulin Receptor Restoration Rescues Vascular Function in Male Insulin Receptor Haploinsufficient Mice.

Reduced systemic insulin signaling promotes endothelial dysfunction and diminished endogenous vascular repair. We investigated whether restoration of endothelial insulin receptor expression could rescue this phenotype. Insulin receptor knockout (IRKO) mice were crossed with mice expressing a human insulin receptor endothelial cell-specific overexpression (hIRECO) to produce IRKO-hIRECO progeny. No metabolic differences were noted between IRKO and IRKO-hIRECO mice in glucose and insulin tolerance tests. In contrast with control IRKO littermates, IRKO-hIRECO mice exhibited normal blood pressure and aortic vasodilatation in response to acetylcholine, comparable to parameters noted in wild type littermates. These phenotypic changes were associated with increased basal- and insulin-stimulated nitric oxide production. IRKO-hIRECO mice also demonstrated normalized endothelial repair after denuding arterial injury, which was associated with rescued endothelial cell migration in vitro but not with changes in circulating progenitor populations or culture-derived myeloid angiogenic cells. These data show that restoration of endothelial insulin receptor expression alone is sufficient to prevent the vascular dysfunction caused by systemically reduced insulin signaling.

Tau pathology and neurochemical changes associated with memory dysfunction in an optimised murine model of global cerebral ischaemia - A potential model for vascular dementia?

Cerebral ischemia is known to be a major cause of death and the later development of Alzheimer's disease and vascular dementia. However, ischemia induced cellular damage that initiates these diseases remain poorly understood. This is primarily due to lack of clinically relevant models that are highly reproducible. Here, we have optimised a murine model of global cerebral ischaemia with multiple markers to determine brain pathology, neurochemistry and correlated memory deficits in these animals. Cerebral ischaemia in mice was induced by bilateral common carotid artery occlusion. Following reperfusion, the mice were either fixed with 4% paraformaldehyde or decapitated under anaesthesia. Brains were processed for Western blotting or immunohistochemistry for glial (GLT1) and vesicular (VGLUT1, VGLUT2) glutamate transporters and paired helical filament (PHF1) tau. The PHF1 tau is the main component of neurofibrillary tangle, which is the pathological hallmark of Alzheimer's disease and vascular dementia. The novel object recognition behavioural assay was used to investigate the functional cognitive consequences in these mice. The results show consistent and selective neuronal and glial cell changes in the hippocampus and the cortex together with significant reductions in GLT1 (***P < 0.001), VGLUT1 (**P < 0.01) and VGLUT2 (***P < 0.001) expressions in the hippocampus in occluded mice as compared to sham-operated animals. These changes are associated with increased PHF1 (***P < 0.0001) protein and a significant impairment of performance (*p < 0.0006, N = 6/group) in the novel object recognition test. This model represents a useful tool for investigating cellular, biochemical and molecular mechanisms of global cerebral ischaemia and may be an ideal preclinical model for vascular dementia.

Insulinlike Growth Factor-Binding Protein-1 Improves Vascular Endothelial Repair in Male Mice in the Setting of Insulin Resistance.

Insulin resistance is associated with impaired endothelial regeneration in response to mechanical injury. We recently demonstrated that insulinlike growth factor-binding protein-1 (IGFBP1) ameliorated insulin resistance and increased nitric oxide generation in the endothelium. In this study, we hypothesized that IGFBP1 would improve endothelial regeneration and restore endothelial reparative functions in the setting of insulin resistance. In male mice heterozygous for deletion of insulin receptors, endothelial regeneration after femoral artery wire injury was enhanced by transgenic expression of human IGFBP1 (hIGFBP1). This was not explained by altered abundance of circulating myeloid angiogenic cells. Incubation of human endothelial cells with hIGFBP1 increased integrin expression and enhanced their ability to adhere to and repopulate denuded human saphenous vein ex vivo. In vitro, induction of insulin resistance by tumor necrosis factor α (TNFα) significantly inhibited endothelial cell migration and proliferation. Coincubation with hIGFBP1 restored endothelial migratory and proliferative capacity. At the molecular level, hIGFBP1 induced phosphorylation of focal adhesion kinase, activated RhoA and modulated TNFα-induced actin fiber anisotropy. Collectively, the effects of hIGFBP1 on endothelial cell responses and acceleration of endothelial regeneration in mice indicate that manipulating IGFBP1 could be exploited as a putative strategy to improve endothelial repair in the setting of insulin resistance.

Endothelial SHIP2 Suppresses Nox2 NADPH Oxidase-Dependent Vascular Oxidative Stress, Endothelial Dysfunction, and Systemic Insulin Resistance.

Shc homology 2-containing inositol 5' phosphatase-2 (SHIP2) is a lipid phosphatase that inhibits insulin signaling downstream of phosphatidylinositol 3-kinase (PI3K); its role in vascular function is poorly understood. To examine its role in endothelial cell (EC) biology, we generated mice with catalytic inactivation of one SHIP2 allele selectively in ECs (ECSHIP2Δ/+). Hyperinsulinemic-euglycemic clamping studies revealed that ECSHIP2Δ/+ was resistant to insulin-stimulated glucose uptake in adipose tissue and skeletal muscle compared with littermate controls. ECs from ECSHIP2Δ/+ mice had increased basal expression and activation of PI3K downstream targets, including Akt and endothelial nitric oxide synthase, although incremental activation by insulin and shear stress was impaired. Insulin-mediated vasodilation was blunted in ECSHIP2Δ/+ mice, as was aortic nitric oxide bioavailability. Acetylcholine-induced vasodilation was also impaired in ECSHIP2Δ/+ mice, which was exaggerated in the presence of a superoxide dismutase/catalase mimetic. Superoxide abundance was elevated in ECSHIP2Δ/+ ECs and was suppressed by PI3K and NADPH oxidase 2 inhibitors. These findings were phenocopied in healthy human ECs after SHIP2 silencing. Our data suggest that endothelial SHIP2 is required to maintain normal systemic glucose homeostasis and prevent oxidative stress-induced endothelial dysfunction.