Search

Background The glutamate hypothesis of schizophrenia postulates NMDA receptor (NMDAR) hypofunction as a major pathophysiological mechanism based on clinical observations and preclinical evidence. Mice with globally reduced NMDAR expression display hyperlocomotion, sensorimotor gating deficits, cognitive deficits and social withdrawal. NMDARs are tetramers composed of 2 obligatory GluN1 (NR1) and 2 variable GluN2A-D (NR2A-D) subunits. GluN2A and GluN2B are the main cortical NMDAR subunits, but genetic models provided evidence that psychosis-like effects of NMDAR antagonists are mediated by GluN2C/GluN2D subunits. Previous data suggest a role also of the GluN2D subunit in schizophrenia-associated abnormalities [1]. Methods We aimed to assess a comprehensive behavioural phenotyping of GluN2D deficient mice, including locomotion, affective behaviours and general welfare, but drew special focus on cognitive functions, including spatial learning, short-term and long-term memory, as well as puzzle solving. We further investigated the effect of D-Serine on the GluN2D KO mice. D-Serine is a NMDAR modulator and acts as an agonist at the glycine site. Previous work in clinical trials suggests significant therapeutic effects of D-serine on negative and positive symptoms, cognitive deficits and motor symptoms. We used the chronic application of D-Serin by supplementing it into the drinking water of the subjects. Results Preliminary data indicate no effect on locomotor alterations of the GluN2D mice, however a potential improvement in short-term memory and wellbeing-associated parameters. They indicate a role of the GluN2D subunit and a beneficial effect of D-serine on schizophrenia-induced parameters, including physiological, welfare-associated burrowing and cognitive performance in the short term task of the puzzle box test. Discussion Our preliminary data indicate a role of the GluN2D subunit in the emergence of cognitive deficits associated with schizophrenia and a potential therapeutic effect of D-serine in alleviating these deficits. Further investigations are planned to further validate these findings. Reference

Alternative oxidase (AOX) is a non‐mammalian enzyme that can bypass blockade of the complex III‐IV segment of the respiratory chain (RC). We crossed a Ciona intestinalis AOX transgene into RC complex III (cIII)‐deficient Bcs1l p.S78G knock‐in mice, displaying multiple visceral manifestations and premature death. The homozygotes expressing AOX were viable, and their median survival was extended from 210 to 590 days due to permanent prevention of lethal cardiomyopathy. AOX also prevented renal tubular atrophy and cerebral astrogliosis, but not liver disease, growth restriction, or lipodystrophy, suggesting distinct tissue‐specific pathogenetic mechanisms. Assessment of reactive oxygen species (ROS) production and damage suggested that ROS were not instrumental in the rescue. Cardiac mitochondrial ultrastructure, mitochondrial respiration, and pathological transcriptome and metabolome alterations were essentially normalized by AOX, showing that the restored electron flow upstream of cIII was sufficient to prevent cardiac energetic crisis and detrimental decompensation. These findings demonstrate the value of AOX, both as a mechanistic tool and a potential therapeutic strategy, for cIII deficiencies.

We show that genetic deficiency of the reactive oxygen species generating enzyme NADPH oxidase 4 (NOX4) impairs hypoxic pulmonary vasoconstriction in small (25–40 µm) intra-acinar, but not pre-acinar, arteries in murine precision cut lung slices. These data suggest an involvement of NOX4 in ventilation-perfusion matching at the acinar level.

International audience; The present study compared the hemorheological properties between Epo-TAgh mice (a model of erythropoietin deficient mice) and wild-type (WT) control mice. Blood viscosity was determined at several shear rates using a cone-plate viscometer at native and adjusted hematocrit (i.e. 40%). Red blood cell (RBC) deformability was measured by ecktacytometry at several shear stresses and RBC aggregation properties by backscattered technique at adjusted hematocrit (i.e. 40%). Epo-TAgh mice had severe anemia (very low hematocrit), decreased blood viscosity at native hematocrit and slightly reduced RBC deformability at high shear stresses in comparison with WT mice. Blood viscosity at adjusted hematocrit (i.e. 40%) was not different between WT and Epo-TAgh mice. RBC aggregation did not differ between the two mice models. These findings suggest a role of erythropoietin in the regulation of RBC deformability.

Amyloid precursor protein (APP) is an integral membrane protein expressed in the peripheral arteries. However, the exact vascular physiological function of APP is unknown. Male APP-deficient (APP –/– ) and their wild-type littermates (WT) mice were used to characterize the phenotype of APP in the control of vascular function. Isometric force of isolated aortic rings was recorded in organ chambers. Circulating levels of norepinephrine and epinephrine were significantly enhanced in APP –/– mice (4723±566 pg/mL and 854±98 pg/mL, respectively P–/– mice (21±3%, P2α were unchanged (135±4%, P=n.s. vs. WT: 133±3%; n=9). Western blot analysis revealed that protein expression of alpha1D adrenergic receptors was significantly downregulated in APP –/– mice aortas (0.21±0.05 O.D.; P–/– mice aortas (P–/– mice (P–/– mice (16.2±4.1 pmol/mg; P–/– mice aortas (38±9 pmol/mg; P–/– mice are responsible for observed vascular phenotype. These findings indicate that under physiological conditions, APP expression plays an important role in control of vascular endothelial function .

Glutathione- S -transferase μ-1 ( GSTM1 ) enzyme belongs to the superfamily of phase II antioxidant glutathione- S -transferases (GSTs) that are downstream targets of the Nrf2 antioxidant transcription factor. In humans, a common GSTM1 gene deletion variant, the null allele GSTM1(0) , results in decreased or complete absence of GSTM1 enzymatic activity and is associated with higher levels of oxidative stress. We reported that GSTM1(0) is associated with accelerated kidney disease progression in the African Americans Study of Kidney Disease (AASK) participants. To understand the direct impact of GSTM1 deficiency on renal inflammation and oxidative stress, we generated Gstm1 deficient mice (KO) to determine their response to angiotensin II, delivered @ 1000 ng/kg/min for 4 weeks via mini-osmotic pump. Blood pressure (BP) was measured by radiotelemetery. Kidney histopathology was assessed by a renal pathologist blinded to genotype and experimental conditions. Renal leukocyte populations were analyzed quantitatively by flow cytometry. Gstm1 KO mice had significantly higher levels of baseline systolic BP (SBP) and ~ 17 mmHg higher SBP after 4 weeks of Ang II-HTN, compared to WT mice. Gstm1 KO mice have increased renal superoxide levels by nearly 3 folds - independent of activation of Nox2 and Nox4 NADPH oxidases or alteration in superoxide dismutase - and worse kidney injury. These changes were associated with significantly increased renal expression of genes involved in inflammation - chemokine ligand 1 (CXCL-1), monocyte chemotactic protein 1 (MCP-1), Interleukin-1β (IL-1β) and IL-6 ( P Gstm1 KO mice displayed ~ two fold increases in all leukocyte populations in the kidney, with the exception of the numbers of B cells that were not significantly different between groups. This increased renal inflammation was attenuated by dietary administration of broccoli powder that is rich in sulforaphane, a phytochemical that increases Nrf2 expression, independent of BP. In Ang II-hypertension, loss of GSTM1 enzyme may be deleterious by augmenting oxidative stress and inflammation in the kidney. Stimulation of the Nrf2 pathway in hypertension may be a novel therapeutic approach to prevent kidney disease progression in GSTM1 deficiency.

Recently, several clinical studies have suggested that adult growth hormone (GH) deficiency that also has low concentration of IGF1 is associated with an increased prevalence of fatty liver (FL). ATP-binding cassette transporter A1 (ABCA1) is a pivotal regulator of lipid efflux from cells to apolipoproteins and plays an important role on formation of FL. In this study, we determined the effects of IGF1 on ABCA1 expression in GH-deficient mice to clarify its effects on FL. Western blotting, real-time PCR, and a luciferase assay were employed to examine the effect of IGF1. The binding of FoxO1 to the ABCA1 promoter was assessed by chromatin immunoprecipitation (ChIP) assay. Cholesterol accumulation was analyzed by Oil Red O stain and cholesterol content measurement. We confirmed that IGF1 upregulated the ABCA1 expression. The activity of a reporter construct containing the ABCA1 promoter was induced by IGF1, and this effect was blocked by LY294002, a specific inhibitor of phosphatidylinositol 3-kinase (PI3K). Constitutively active Akt stimulated the ABCA1 promoter activity, and a dominant-negative mutant of Akt or mutagenesis of the FoxO1 response element abolished the effect of IGF1. A ChIP assay indicated that FoxO1 mediated IGF1 transcriptional activity by directly binding to the ABCA1 promoter region. For in vivo experiments, we used an inhibitor for the GH receptor (Pegvisomant) to reduce the IGF1 level. A high-fat diet induced FL in mice (C57BL/6J) given Pegvisomant treatment. IGF1 treatment stimulated ABCA1 expression to improve cholesterol accumulation in these mice. These results show that the PI3K/Akt/FoxO1 pathway contributes to the regulation of ABCA1 expression in response to IGF1 stimulation that suppressed FL in GH-deficient mice.

We investigated and evaluated post-irradiation survival in cyclooxygenase-2-deficient (COX-2 KO) mice. Thirty-day survival following exposure of COX-2 KO mice to a lethal dose of 8.5 Gy of γ-rays was observed to be statistically significantly lower in both males and females, as well as when the sexes were merged, in comparisons with their wild-type counterparts. These findings were related to the previous observations concerning the detrimental influence of the COX-2 genetic disruption on hematopoiesis in sublethally irradiated mice. Deteriorated post-irradiation survival of COX-2 KO mice confirmed the previously anticipated conclusion regarding negative influence of the anti-inflammatory action of COX-2 deficiency under the conditions of exposure of the animals to ionizing radiation.

Human antigen R (HuR), is an RNA binding protein in the human antigen protein family. To study the neuron-specific function of HuR, we generated inducible, neuron-specific HuR-deficient mice of both sexes. After tamoxifen-induced deletion of HuR, these mice developed a phenotype consisting of poor balance, decreased movement, and decreased strength. They performed significantly worse on the rotarod test compared to littermate control mice, indicating coordination deficiency. Using the grip strength test, it was also determined that the forelimbs of neuron-specific HuR-deficient mice were much weaker than littermate control mice. Immunostaining of the brain and cervical spinal cord showed that HuR-deficient neurons had increased levels of cleaved Caspase 3, a hallmark of cell apoptosis. Caspase 3 cleavage was especially strong in pyramidal neurons and alpha motor neurons of HuR-deficient mice. Genome-wide microarray and RT-PCR analysis further indicated that HuR deficiency in neurons resulted in altered expression of genes in the brain involved in cell growth, including Tchp, Cdkn2c, GPSM2, Ier2, SOD1, and Bcl2. The additional enriched GO terms in the brain tissues of neuron-specific HuR-deficient mice were largely related to inflammation, including interferon-induced genes and complement components. Importantly, some of these HuR-regulated genes were also significantly altered in the brain and spinal cord of patients with amyotrophic lateral sclerosis. Additionally, neuronal HuR deficiency resulted in the redistribution of TDP43 to cytosolic granules, which has been linked to motor neuron disease. Taken together, we propose that this neuron-specific HuR-deficient mouse strain can potentially be used as a motor neuron disease model.

Background: Progerin, an aberrant protein that accumulates with age, causes the rare genetic disease Hutchinson-Gilford progeria syndrome (HGPS). Patients who have HGPS exhibit ubiquitous progerin expression, accelerated aging and atherosclerosis, and die in their early teens, mainly of myocardial infarction or stroke. The mechanisms underlying progerin-induced atherosclerosis remain unexplored, in part, because of the lack of appropriate animal models. Methods: We generated an atherosclerosis-prone model of HGPS by crossing apolipoprotein E–deficient ( Apoe –/– ) mice with Lmna G609G/G609G mice ubiquitously expressing progerin. To induce progerin expression specifically in macrophages or vascular smooth muscle cells (VSMCs), we crossed Apoe –/– Lmna LCS/LCS mice with LysMCre and SM22αCre mice, respectively. Progerin expression was evaluated by polymerase chain reaction and immunofluorescence. Cardiovascular alterations were determined by immunofluorescence and histology in male mice fed normal chow or a high-fat diet. In vivo low-density lipoprotein retention was assessed by intravenous injection of fluorescently labeled human low-density lipoprotein. Cardiac electric defects were evaluated by electrocardiography. Results: Apoe –/– Lmna G609G/G609G mice with ubiquitous progerin expression exhibited a premature aging phenotype that included failure to thrive and shortened survival. In addition, high-fat diet–fed Apoe –/– Lmna G609G/G609G mice developed a severe vascular pathology, including medial VSMC loss and lipid retention, adventitial fibrosis, and accelerated atherosclerosis, thus resembling most aspects of cardiovascular disease observed in patients with HGPS. The same vascular alterations were also observed in Apoe –/– Lmna LCS/LCS SM22αCre mice expressing progerin specifically in VSMCs, but not in Apoe –/– Lmna LCS/LCS LysMCre mice with macrophage-specific progerin expression. Moreover, Apoe –/– Lmna LCS/LCS SM22αCre mice had a shortened lifespan despite the lack of any overt aging phenotype. Aortas of ubiquitously and VSMC-specific progerin-expressing mice exhibited increased retention of fluorescently labeled human low-density lipoprotein, and atheromata in both models showed vulnerable plaque features. Immunohistopathological examination indicated that Apoe –/– Lmna LCS/LCS SM22αCre mice, unlike Apoe –/– Lmna G609G/G609G mice, die of atherosclerosis-related causes. Conclusions: We have generated the first mouse model of progerin-induced atherosclerosis acceleration, and demonstrate that restricting progerin expression to VSMCs is sufficient to accelerate atherosclerosis, trigger plaque vulnerability, and reduce lifespan. Our results identify progerin-induced VSMC death as a major factor triggering atherosclerosis and premature death in HGPS.

The development of humanized mice has become a prominent tool for translational animal studies of human diseases. Here we show how immune deficient mice can be "humanized" by injections of human umbilical cord stem cells. The engraftment of these cells and development into human lymphocytes has been possible because of the development of novel severely immune deficient mouse strains. Here we present proven protocols for the generation and analysis of humanized mice on the NSG mouse background.

Hypothalamic low-density lipoprotein receptor-related protein 1 (LRP1), a member of LDL receptor family, plays a pivotal role in the regulation of food intake and body-weight homeostasis. However, the role of LRP1 in MC4R-expressing neurons remains unknown. This study was to determine whether deleting LRP1 from MC4R-expressing neurons affects adiposity and energy balance in mice. Here we show that MC4R neuron-specific LRP1 deletion results in decreased body weight in mice fed a normal chow diet (control 30.4±0.8g vs. MC4R-LRP1 deficient mice 27.2±0.5g, p Disclosure M. Kang: None. J.A. Seo: None. H. Cho: None. H. Lee: None. Y. Kim: None.

The data presented in this article are related to the research article entitled ''Increased Mitochondrial Protein Levels and Bioenergetics in the musculus rectus femoris of Wfs1-Deficient mice'' (Eimre et al., accepted for publication). This dataset reports the analysis of Wfs1-deficient mouse heart, musculus soleus, and white part of musculus rectus femoris by liquid chromatography/tandem mass spectrometry. Label-free quantitative analysis of the mass spectrometry data identified 4056 proteins, with 114, 212, and 1290 proteins differentially expressed (t-test; p < 0.05) in the heart, m. soleus, and m. rectus femoris, respectively, between the Wfs1-deficient and wild-type groups. Eight proteins were found to be differentially expressed in all mentioned muscles, with 1 protein differently expressed in oxidative (m. soleus and heart) and 88 in skeletal muscles. This dataset supports the cited study and can be used to extend additional analyses. Data are available via ProteomeXchange with identifier PXD011019. Keywords: Wolfram syndrome, Wfs1, Myocardium, skeletal muscle, Animal proteomics

Background: Several studies have attempted to relate the bioactive alkaloid with atherosclerotic cardiovascular diseases prevention in animal models, providing inconsistent results. Moreover, the direct anti-atherosclerotic effects of alkaloid have hardly been studied in patients. Therefore, the aim of this systematic review was to assess the reported effects of alkaloids on aortic atherosclerosis in ApoE-/- mouse models. Methods: Pubmed and Embase were searched to identify studies which estimated the effect of isolated alkaloids on atherosclerosis in apolipoprotein E deficient mice. Study quality was assessed using SYRCLE's risk of bias tool. We conducted a meta-analysis across 14 studies using a random-effect model to determine the overall effect of the alkaloids, and performed subgroup analyses to compare the effects of the isoquinolone alkaloids and indole alkaloids. Results: The quality of the included studies was low in the majority of included studies. We clarified that alkaloid administration was significantly associated with reduced aortic atherosclerotic lesion area (SMD -3.19, 95% CI -3.88, -2.51). It is important to remark that the experimental characteristics of studies were quite diverse, and the methodological variability could also contribute to heterogeneity. Subgroup analyses suggested that the isoquinoline alkaloids (SMD -4.19, 95% CI -5.18, -3.20), and the indole alkaloids (SMD -2.73, 95% CI -3.56, -1.90) obviously decreased atherosclerotic burden. Conclusion: Isoquinoline alkaloids and indole alkaloids appear to have a direct anti-atherosclerotic effect in ApoE-/- mice. Besides the limitations of animal modal studies, this systematic review could provide an important reference for future preclinical animal trials of good quality and clinical development.

In a modern society, the risk of developing type II diabetes and obesity may be linked to the increased consumption of carbohydrate-rich drinks. Several genes, including Wolfram Syndrome 1 (WFS1), have been reported to increase susceptibility for developing type II diabetes. In this study we aimed to investigate the effect of chronic consumption of carbohydrate-rich drinks on weight gain, overall consumption of liquids, glucose tolerance and liver metabolism in Wfs1-deficient mice. Wfs1-deficient and wild-type mice were divided into three groups that consumed regular Coca-Cola, 20% sucrose solution or water ad libitum as the only source of liquid. During the experiment, daily liquid consumption was determined. After 30 days, total weight gain of mice was calculated and glucose tolerance test was performed. The liver tissue was analysed by means of untargeted and targeted metabolomics using liquid chromatography-mass spectrometry. Weight gain was strongly affected by mouse genotype (pWfs1 deficiency. In conclusion, our study demonstrates a significant interaction between the genotype and the drink when comparing wild-type and Wfs1 knock-out mice consuming soft drinks.

Background and Purpose: Diabetes is recognized to enhance the frequency and severity of atherosclerosis and cardiovascular disease. Recent studies have shown that hyperglycemia is associated with enhanced hematopoiesis and macrophage accumulation in atherosclerotic lesions. We explored whether high glucose concentrations can enhance intercellular communication between mature macrophages and hematopoietic progenitors via exosomes to promote inflammation and diabetic atherosclerosis. Methods: Bone marrow derived macrophages (BMDM) from C57BL/6 mice were cultured with normal (5mM) or high glucose concentrations (25mM). Exosomes were isolated with our cushioned-density gradient ultracentrifugation method followed by nanoparticle tracking and western blot analysis. Pro-inflammatory properties of high glucose exosomes (HGexo) were tested in vitro by exposing them to BMDM cultured in normal low glucose. The capacity for BMDM-derived exosomes to alter systemic and vascular inflammation were next tested by infusing 25-30 weeks-old ApoE -/- mice fed a chow diet with 3 x 10 10 exosomes three times a week, for four weeks. Results: Our data show that HGexo can stimulate the expression of inflammatory cytokines (IL-6, IL-1ß) as well as NADPH oxidases (Nox-1 and Nox-4) in cultured BMDM. Furthermore, our findings show that intraperitoneally injected exosomes distribute to numerous organs and tissues including the bone marrow and the spleen. Lastly, HGexo enhance the expansion of multipotent and lineage committed hematopoietic progenitors. Conclusions: We identify that exosomes derived from cultured BMDM exposed to high glucose have the capacity to exert intercellular communication in vitro , and in vivo. Our findings suggest that exosomes produced by macrophages exposed to hyperglycemia could represent an unsuspected source of inflammation to accelerate atherosclerosis in diabetes.

Non-obstructive mixed atherosclerotic plaques are an independent risk factor in predicting mortality in coronary artery disease patients. The multifactorial process of plaque progression and complication is a result of proliferation and migration of VSMCs and macrophages, apoptosis and calcification. A number of osteogenic regulators and inflammatory stimuli driving these processes in plaque advancement have been identified. Yet rupture, a result of complex mixed plaques, remains the leading cause of fatal cardiovascular events. Further understanding of the mechanisms underlying plaque progression is required to identify potential targets for therapies. Key signalling factors that modulate cell proliferation and migration, which potentially underlie plaque complication include the nodal multifunctional calcium/calmodulin dependent protein kinase II (CaMKII). Thus we hypothesized that, as plaques progress in a mouse model of atherosclerosis (ApoE -/- ), CaMKII activity would increase. We also hypothesized that systemic inhibition of CaMKII would impede plaque progression. Our data show a trend towards increased CaMKII phosphorylation, an indirect measure of kinase activity, in plaques as mice advance from 26 to 30 weeks. Pharmacological inhibition of CaMKII with KN-93, on alternate days for 4 weeks, in 30 week old mice reduced the incidence of calcification from 50% (KN-92 control: 4 out of 8) to 25% (KN-93: 2 out of 8). This was associated with a trend towards reduced calcification area (KN-92 control: 0.43mm 2 vs. KN-93: 0.06mm 2 ). These results suggest that CaMKII is activated during atherosclerosis and may be involved with plaque progression through calcification.

Background/Objective: Recent genome wide association studies identified SLC44A2 as a novel susceptibility locus for venous thromboembolism (VTE), a region encoding the solute carrier family 44 member 2 protein (SLC44A2). Here we utilize Slc44a2 deficient mice (KO) to determine the importance of SLC44A2 in thrombotic disease. Methods: Mice lacking Slc44a2 were included in two models of venous thrombosis: 1) a spontaneous thrombosis model using siRNA targeting anti-coagulants Serpinc1 and Proc and 2) a model of deep vein thrombosis (DVT) induced by flow restriction (stenosis) of the inferior vena cava (IVC). Results: In the model of spontaneous thrombosis, Slc44a2 deficiency did not affect incidence as occurrence of thrombotic phenotype reached 100% in both wild type (WT; 12/12) and KO (12/12) mice 32 hours after siRNA injection. Platelet counts and fibrin deposition in the liver were also comparable. However, levels of circulating neutrophils were significantly higher (p=0.0017) in KO mice along with substantially lower amounts of plasma VWF antigen (pSlc44a2 did (12/15). Thrombus length was significantly reduced in KO animals (p=0.0184), as was thrombus weight (p=0.0413). Immunhistochemical staining of the thrombi revealed different repartitions in the “white” (platelet-rich) over the “red” (RBC-rich) parts of the thrombi depending on the mouse genotype. This observation suggests a different blood cell mobilization at the site of stenosis depending of the genotype. Plasma VWF levels were correlated with thrombus weight in WT (p=0.0175), but not in KO mice (p=0.3266), and had been previously confirmed to be lower in KO mice. Conclusion: These findings corroborate the original GWAS data, indicating a role for SLC44A2 in thrombotic disease. We have observed that SLC44A2 was not involved in a platelet-dependent model of thrombosis whereas it is involved in a thrombosis model where neutrophils and VWF have been shown to play a crucial role.

Obesity, hepatosteatosis, and hypertriglyceridemia are components of the metabolic syndrome and independent risk factors for cardiovascular disease. The lipid droplet-associated protein CIDEC (cell death-inducing DFFA-like effector C), known in mice as FSP27 (fat-specific protein 27), plays a key role in maintaining triacylglyceride (TAG) homeostasis in adipose tissue and liver, and controls circulating TAG levels in mice. Importantly, mutations and SNPs in CIDEC are associated to dyslipidemia and altered metabolic function in humans. Here we tested whether systemic silencing of Fsp27 using antisense oligonucleotides (ASOs) was atheroprotective in LDL receptor knock-out ( Ldlr -/- ) mice. Animals were fed a high-fat, high-cholesterol diet for 12 weeks while simultaneously dosed with saline, ASO-ctrl, or ASO-Fsp27. Data show that, compared to control treatments, silencing Fsp 27 significantly reduced body weight gain and visceral adiposity, prevented diet-induced hypertriglyceridemia, and reduced atherosclerotic lesion size both in en face aortas and in the aortic root. Our findings suggest that therapeutic silencing of Fsp27 with ASOs may be beneficial in the prevention and management of atherogenic disease in patients with metabolic syndrome.

Objective: Chronic angiotensin II (AngII) infusion promotes both ascending (TAAs) and abdominal aortic aneurysms (AAAs) in mice. Previously, we demonstrated that exogenous vasohibin-2 (VASH-2) exacerbated AngII-induced TAAs in normolipidemic mice. The purpose of this study was to examine whether exogenous VASH-2 influenced AngII-induced AAAs in mice. Methods and Results: In the initial study, male C57BL/6J mice (10 weeks old) were injected with VASH2 or LacZ expressing adenovirus (Ad; 7.5 x 109 vp/100 μ L) via tail vein at 2 week intervals. One week after the first injection, subcutaneous infusion of AngII (1,000 ng/kg/min) by mini osmotic pumps was initiated for 3 weeks. Consequently, mice were divided into 2 groups: AngII + Ad VASH2 in C57BL/6J mice (n=22) and AngII + Ad LacZ in C57BL/6J mice (n=21). VASH-2 overexpression had no effect on systolic blood pressure, heart rate, body weight, or serum total cholesterol concentrations. Exogenous VASH-2 did not affect ex vivo measurements of maximal diameters of abdominal aortas (AngII + Ad VASH2; 1.36 ± 0.39 mm, AngII + LacZ 1.34 ± 0.24 mm, n.s.) in AngII-infused mice. In a subsequent study, male apolipoprotein E-deficient (apoE-/-) mice (9 to 13 weeks old) were injected with VASH2 or LacZ as described for C57BL/6J mice. Consequently, mice were divided into 2 groups: AngII + Ad VASH2 in apoE-/- mice (n=14) and AngII + Ad LacZ in apoE-/- mice (n=14). Similarly, overexpression of VASH-2 had no effect on systolic blood pressure, heart rate, body weight, or serum total cholesterol concentrations in apoE-/- mice. Furthermore, exogenous VASH-2 did not affect ex vivo measurement of maximal diameter of abdominal aorta (AngII + Ad VASH2; 1.70 ± 0.61 mm, AngII + Ad LacZ; 1.61 ± 0.43 mm, n.s.) in AngII-infused apoE-/- mice. Conclusion: Despite our previous demonstration of the effects on TAA, exogenous VASH-2 did not influence AngII-induced AAA formation in either normolipidemic or apoE-/- mice.

Rap1, a ubiquitously expressed small GTPase, integrates signals from multiple receptors and promotes activation and signaling by cell adhesion receptors. In vivo , endothelial (EC) Rap1 is required for normal vessel formation, angiogenesis and dynamic regulation of EC barrier. Two Rap1 isoforms, Rap1A and Rap1B, share 95% identity, and EC deletion of both isoforms leads to embryonic lethality due to cardiovascular defects. We have recently demonstrated that Rap1 is a novel regulator of shear sensing in endothelium and that it is essential for nitric oxide release, and that Rap1 deficiency leads to EC dysfunction. These findings demonstrated that Rap1 plays an essential role in vasoprotective response of endothelium in response to shear stress. The objective of this study is to determine the specific role of Rap1 response to laminar, vasoprotective flow and pro-inflammatory, disturbed flow by examining the effect of EC deletion of Rap1B, the more prominent Rap1 isoform, on progression of atherosclerosis. To investigate the role of Rap1 in progression of atherosclerosis, we crossed apolipoprotein E-deficient (ApoE -/- ) mice with tamoxifen-inducible, EC specific Rap1B-knockout (Cadh5-CreERT +/0 ; Rap1B f/f ) to generate atherogenic EC-Rap1B KO (Athero-Rap1B KO) mice. We hypothesized that Rap1B deficiency will exacerbate progression of atherosclerosis. Athero-Rap1B KO and littermate controls (Cadh5-Cre-negative mice or mice injected with carrier oil only) were fed high fat, western diet (21.2% fat, 0.2% cholesterol) for 16 weeks. No difference in total cholesterol levels were observed between Athero- Rap1B KO and control mice. Atherosclerotic lesion formation was visualized with oil O red and plaque area was quantified in en-face preparations. (n=5-9). We observed increased lesion area in the descending aorta of Athero-Rap1B KO mice (0.7351 ± 0.1065 vs 0.4866 ± 0.09812; p =0.0584, one-tailed t-test, Welch corrected) and a strong trend towards larger lesion area in the total aorta (2.142 ± 0.5058 vs 1.267 ± 0.1718; p=0.0679), and the arch of the aorta (1.489 ± 0.4343 vs 0.7807 ± 0.1152; p=0.0747) compared with control mice. These findings demonstrate that Rap1B deficiency accelerates progression of atherosclerosis and show that Rap1 plays a vasoprotective role in both laminar flow and disturbed flow areas.

Objective: The Wnt/β-catenin signaling is an ancient and evolutionarily conserved pathway that regulates essential aspects of cell differentiation, proliferation, migration and polarity, and canonical Wnt/β-catenin signaling has also been implicated in the pathogenesis of atherosclerosis. Macrophage is one of the major cell types involved in the initiation and progression of atherosclerosis, but the role of macrophage β-catenin in atherosclerosis remains elusive. This study aims to investigate the impact of β-catenin expression on macrophage functions and atherosclerosis development. Approaches and Results: To investigate the role of macrophage canonical Wnt/β-catenin signaling in atherogenesis, we generated low-density lipoprotein receptor-deficient (LDLR -/- ) mice with myeloid-specific β-catenin deficiency. As expected, deletion of β-catenin decreased macrophage adhesion and migration properties in vitro. However, deficiency of β-catenin significantly increased atherosclerosis in LDLR -/- mice without altering the plasma lipid levels. Mechanistic studies revealed that β-catenin can regulate activation of signal transducer and activator of transcription (STAT) pathway in macrophages, and ablation of β-catenin resulted in STAT3 downregulation and STAT1 activation, leading to elevated macrophage inflammatory responses and increased atherosclerosis. Conclusions: This study demonstrates a critical role of myeloid β-catenin expression in atherosclerosis by modulating macrophage inflammatory responses.

Atherothrombosis is the cause of death of over 14 million people per year worldwide and murine models to replicate this process in vivo are mostly lacking. Previously we demonstrated that silencing of anticoagulant protein C using RNA interference ( siProc ) induces spontaneous atherothrombosis in the aortic root of apolipoprotein E-deficient ( Apoe -/- ) mice, albeit at a low incidence rate. Here we aim to determine if plaque susceptibility for rupture can be linked to plaque characteristics and/or blood composition, and moreover, we attempt to boost incidence through a transient increase in blood pressure as well as to localize atherothrombosis to an additional predefined vascular site by means of a semi-constrictive collar around the carotid artery. In the current study, si Proc -driven spontaneous atherothrombosis in the aortic root of Apoe-/- mice was reproduced and occurred at an incidence of 23% (9 out of 39 mice), while the incidence of collar-induced atherothrombosis in the carotid artery was 2.6% (1 out of 39 mice). Treatment with phenylephrine, to transiently increase blood pressure, did not increase atherothrombosis in the aortic root of the Apoe -/- mice nor in the carotid arteries with collars. Plaques in the aortic root with an associated thrombus were lower in collagen and macrophage content, and mice with atherothrombosis had significantly more circulating platelets. Plasma protein C, white blood cell counts, total cholesterol, fibrinogen, and serum amyloid A were not different amongst si Proc -treated mice with or without thrombosis. Remarkably, our data revealed that thrombus formation preferably occurred on plaques in the right coronary sinus of the aortic root. In conclusion, there is a predilection of low protein C-induced spontaneous atherothrombosis in Apoe -/- mice for the right coronary sinus, a process that is associated with an increase in platelets and plaques lower in collagen and macrophage content.

The free radical theory of ageing provided an intellectual framework for many laboratories working on ageing. However, experimental and clinical evidence showing that high doses of antioxidants do not have an effect on ageing or on age-associated diseases, cast doubts on the validity of this theory. Data from our own laboratory show that oxidative damage does not correlate with age, especially in the geriatric population, but rather with the frailty state. This has led us to postulate the free radical theory of frailty that proposes that oxidative damage is associated with frailty, but not with chronological age itself. Superoxide dismutase deficient mice are more frail than controls. But more importantly, we have observed that animals that are protected against oxidative damage by overexpression of antioxidant enzymes, delay the onset of frailty and are more vigorous than controls. In this review, we describe results from both, experimental animals and human cohorts, that lead us to the formulate this free radical theory of frailty.