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Preview: Arteriosclerosis, Thrombosis, and Vascular Biology recent issues

Arteriosclerosis, Thrombosis, and Vascular Biology recent issues



Arteriosclerosis, Thrombosis, and Vascular Biology RSS feed -- recent issues



 






Elaine W. Raines (1948-2017) [In Memoriam]

2018-01-24T10:40:38-08:00




TRPing out Platelet Calcium [Editorial]

2018-01-24T10:40:38-08:00










Consideration of Sex Differences in Design and Reporting of Experimental Arterial Pathology Studies—Statement From ATVB CouncilHighlights [Special Articles]

2018-01-24T10:40:38-08:00

There are many differences in arterial diseases between men and women, including prevalence, clinical manifestations, treatments, and prognosis. The new policy of the National Institutes of Health, which requires the inclusion of sex as a biological variable for preclinical studies, aims to foster new mechanistic insights and to enhance our understanding of sex differences in human diseases. The purpose of this statement is to suggest guidelines for designing and reporting sex as a biological variable in animal models of atherosclerosis, thoracic and abdominal aortic aneurysms, and peripheral arterial disease. We briefly review sex differences of these human diseases and their animal models, followed by suggestions on experimental design and reporting of animal studies for these vascular pathologies.



2017 Scientific Sessions Sol Sherry Distinguished Lecture in Thrombosis [ATVB Named Lecture Review]

2018-01-24T10:40:38-08:00

The goal of anticoagulant therapy is to attenuate thrombosis without compromising hemostasis. Although the direct oral anticoagulants are associated with less intracranial hemorrhage than vitamin K antagonists, bleeding remains their major side effect. Factor XI has emerged as a promising target for anticoagulants that may be safer than those currently available. The focus on factor XI stems from epidemiological evidence of its role in thrombosis, the observation of attenuated thrombosis in factor XI–deficient mice, identification of novel activators, and the fact that factor XI deficiency is associated with only a mild bleeding diathesis. Proof-of-concept comes from the demonstration that compared with enoxaparin, factor XI knockdown reduces venous thromboembolism without increasing bleeding after elective knee arthroplasty. This article rationalizes the selection of factor XI as a target for new anticoagulants, reviews the agents under development, and outlines a potential path forward for their development.






Melanocortin 1 Receptor Deficiency Promotes Atherosclerosis in Apolipoprotein E-/- MiceHighlights [Basic Sciences]

2018-01-24T10:40:38-08:00

Objective—The MC1-R (melanocortin 1 receptor) is expressed by monocytes and macrophages where it mediates anti-inflammatory actions. MC1-R also protects against macrophage foam cell formation primarily by promoting cholesterol efflux through the ABCA1 (ATP-binding cassette transporter subfamily A member 1) and ABCG1 (ATP-binding cassette transporter subfamily G member 1). In this study, we aimed to investigate whether global deficiency in MC1-R signaling affects the development of atherosclerosis.Approach and Results—Apoe−/− (apolipoprotein E deficient) mice were crossed with recessive yellow (Mc1re/e) mice carrying dysfunctional MC1-R and fed a high-fat diet to induce atherosclerosis. Apoe−/− Mc1re/e mice developed significantly larger atherosclerotic lesions in the aortic sinus and in the whole aorta compared with Apoe−/− controls. In terms of plaque composition, MC1-R deficiency was associated with less collagen and smooth muscle cells and increased necrotic core, indicative of more vulnerable lesions. These changes were accompanied by reduced Abca1 and Abcg1 expression in the aorta. Furthermore, Apoe−/− Mc1re/e mice showed a defect in bile acid metabolism that aggravated high-fat diet–induced hypercholesterolemia and hepatic lipid accumulation. Flow cytometric analysis of leukocyte profile revealed that dysfunctional MC1-R enhanced arterial accumulation of classical Ly6Chigh monocytes and macrophages, effects that were evident in mice fed a normal chow diet but not under high-fat diet conditions. In support of enhanced arterial recruitment of Ly6Chigh monocytes, these cells had increased expression of L-selectin and P-selectin glycoprotein ligand 1.Conclusions—The present study highlights the importance of MC1-R in the development of atherosclerosis. Deficiency in MC1-R signaling exacerbates atherosclerosis by disturbing cholesterol handling and by increasing arterial monocyte accumulation.



Macrophage-Associated Lipin-1 Enzymatic Activity Contributes to Modified Low-Density Lipoprotein-Induced Proinflammatory Signaling and AtherosclerosisHighlights [Basic Sciences]

2018-01-24T10:40:38-08:00

Objective—Macrophage proinflammatory responses induced by modified low-density lipoproteins (modLDL) contribute to atherosclerotic progression. How modLDL causes macrophages to become proinflammatory is still enigmatic. Macrophage foam cell formation induced by modLDL requires glycerolipid synthesis. Lipin-1, a key enzyme in the glycerolipid synthesis pathway, contributes to modLDL-elicited macrophage proinflammatory responses in vitro. The objective of this study was to determine whether macrophage-associated lipin-1 contributes to atherogenesis and to assess its role in modLDL-mediated signaling in macrophages.Approach and Results—We developed mice lacking lipin-1 in myeloid-derived cells and used adeno-associated viral vector 8 expressing the gain-of-function mutation of mouse proprotein convertase subtilisin/kexin type 9 (adeno-associated viral vector 8-proprotein convertase subtilisin/kexin type 9) to induce hypercholesterolemia and plaque formation. Mice lacking myeloid-associated lipin-1 had reduced atherosclerotic burden compared with control mice despite similar plasma lipid levels. Stimulation of bone marrow–derived macrophages with modLDL activated a persistent protein kinase Cα/βII–extracellular receptor kinase1/2–jun proto-oncogene signaling cascade that contributed to macrophage proinflammatory responses that was dependent on lipin-1 enzymatic activity.Conclusions—Our data demonstrate that macrophage-associated lipin-1 is atherogenic, likely through persistent activation of a protein kinase Cα/βII–extracellular receptor kinase1/2–jun proto-oncogene signaling cascade that contributes to foam cell proinflammatory responses. Taken together, these results suggest that modLDL-induced foam cell formation and modLDL-induced macrophage proinflammatory responses are not independent consequences of modLDL stimulation but rather are both directly influenced by enhanced lipid synthesis.



Vascular Semaphorin 7A Upregulation by Disturbed Flow Promotes Atherosclerosis Through Endothelial {beta}1 IntegrinHighlights [Basic Sciences]

2018-01-24T10:40:38-08:00

Objective—Accumulating evidence suggests a role of semaphorins in vascular homeostasis. Here, we investigate the role of Sema7A (semaphorin 7A) in atherosclerosis and its underlying mechanism.Approach and Results—Using genetically engineered Sema7A−/−ApoE−/− mice, we showed that deletion of Sema7A attenuates atherosclerotic plaque formation primarily in the aorta of ApoE−/− mice on a high-fat diet. A higher level of Sema7A in the atheroprone lesser curvature suggests a correlation of Sema7A with disturbed flow. This notion is supported by elevated Sema7A expression in human umbilical venous endothelial cells either subjected to oscillatory shear stress or treated with the PKA (protein kinase A)/CREB (cAMP response element-binding protein) inhibitor H89 (N-[2-(p-bromocinnamylamino)ethyl]-5-isoquinolinesulfonamide·2HCl hydrate). Further studies using the partial carotid artery ligation model showed that disturbed flow in the left carotid artery of Sema7A+/+ApoE−/− mice promoted the expression of endothelial Sema7A and cell adhesion molecules, leukocyte adhesion, and plaque formation, whereas such changes were attenuated in Sema7A−/−ApoE−/− mice. Further studies showed that blockage of β1 integrin, a known Sema7A receptor, or inhibition of FAK (focal adhesion kinase), MEK1/2 (mitogen-activated protein kinase kinase 1/2), or NF-κB (nuclear factor-κB) significantly reduced the expression of cell adhesion molecules and THP-1 (human acute monocytic leukemia cell line) monocyte adhesion in Sema7A-overexpressing human umbilical venous endothelial cells. Studies using chimeric mice suggest that vascular, most likely endothelial, Sema7A plays a major role in atherogenesis.Conclusions—Our findings indicate a significant role of Sema7A in atherosclerosis by mediating endothelial dysfunction in a β1 integrin–dependent manner.



TRPM7 Kinase Controls Calcium Responses in Arterial Thrombosis and Stroke in MiceHighlights [Basic Sciences]

2018-01-24T10:40:38-08:00

Objective—TRPM7 (transient receptor potential cation channel, subfamily M, member 7) is a ubiquitously expressed bifunctional protein comprising a transient receptor potential channel segment linked to a cytosolic α-type serine/threonine protein kinase domain. TRPM7 forms a constitutively active Mg2+ and Ca2+ permeable channel, which regulates diverse cellular processes in both healthy and diseased conditions, but the physiological role of TRPM7 kinase remains largely unknown.Approach and Results—Here we show that point mutation in TRPM7 kinase domain deleting the kinase activity in mice (Trpm7R/R) causes a marked signaling defect in platelets. Trpm7R/R platelets showed an impaired PIP2 (phosphatidylinositol-4,5-bisphosphate) metabolism and consequently reduced Ca2+ mobilization in response to stimulation of the major platelet receptors GPVI (glycoprotein VI), CLEC-2 (C-type lectin-like receptor), and PAR (protease-activated receptor). Altered phosphorylation of Syk (spleen tyrosine kinase) and phospholipase C γ2 and β3 accounted for these global platelet activation defects. In addition, direct activation of STIM1 (stromal interaction molecule 1) with thapsigargin revealed a defective store-operated Ca2+ entry mechanism in the mutant platelets. These defects translated into an impaired platelet aggregate formation under flow and protection of the mice from arterial thrombosis and ischemic stroke in vivo.Conclusions—Our results identify TRPM7 kinase as a key modulator of phospholipase C signaling and store-operated Ca2+ entry in platelets. The protection of Trpm7R/R mice from acute ischemic disease without developing intracranial hemorrhage indicates that TRPM7 kinase might be a promising antithrombotic target.



Growth Differentiation Factor 6 Promotes Vascular Stability by Restraining Vascular Endothelial Growth Factor SignalingHighlights [Basic Sciences]

2018-01-24T10:40:38-08:00

Objective—The assembly of a functional vascular system requires a coordinated and dynamic transition from activation to maturation. High vascular endothelial growth factor activity promotes activation, including junction destabilization and cell motility. Maturation involves junctional stabilization and formation of a functional endothelial barrier. The identity and mechanism of action of prostabilization signals are still mostly unknown. Bone morphogenetic protein receptors and their ligands have important functions during embryonic vessel assembly and maturation. Previous work has suggested a role for growth differentiation factor 6 (GDF6; bone morphogenetic protein 13) in vascular integrity although GDF6’s mechanism of action was not clear. Therefore, we sought to further explore the requirement for GDF6 in vascular stabilization.Approach and Results—We investigated the role of GDF6 in promoting endothelial vascular integrity in vivo in zebrafish and in cultured human umbilical vein endothelial cells in vitro. We report that GDF6 promotes vascular integrity by counteracting vascular endothelial growth factor activity. GDF6-deficient endothelium has increased vascular endothelial growth factor signaling, increased vascular endothelial-cadherin Y658 phosphorylation, vascular endothelial-cadherin delocalization from cell–cell interfaces, and weakened endothelial cell adherence junctions that become prone to vascular leak.Conclusions—Our results suggest that GDF6 promotes vascular stabilization by restraining vascular endothelial growth factor signaling. Understanding how GDF6 affects vascular integrity may help to provide insights into hemorrhage and associated vascular pathologies in humans.



Multidisciplinary Approach to Understand Medial Arterial CalcificationHighlights [Basic Sciences]

2018-01-24T10:40:38-08:00

Objective—Vascular calcification significantly increases morbidity in life-threatening diseases, and no treatments are available because of lack of understanding of the underlying molecular mechanism. Here, we study the physicochemical details of mineral nucleation and growth in an animal model that faithfully recapitulates medial arterial calcification in humans, to understand how pathological calcification is initiated on the vascular extracellular matrix.Approach and Results—MGP (matrix Gla protein) is a potent mineralization inhibitor. We study the evolution of medial calcification in MGP-deficient mice over the course of 5 weeks using a combination of material science techniques and find that mineral composition and crystallinity evolve over time and space. We show that calcium is adsorbed first and then amorphous calcium phosphate and octacalcium phosphate forms, which then transform into hydroxyapatite and carbonated apatite. These events are repeated after each nucleation event, providing a snapshot of the overall mineral evolution at each time point analyzed.Conclusions—Our results show that an interdisciplinary approach combining animal models and materials science can provide insights into the mechanism of vascular calcification and suggest the importance of analyzing mineral phases, rather than just overall mineralization extent, to diagnose and possibly prevent disease development.



SNRK (Sucrose Nonfermenting 1-Related Kinase) Promotes Angiogenesis In VivoHighlights [Basic Sciences]

2018-01-24T10:40:38-08:00

Objective—SNRK (sucrose nonfermenting 1-related kinase) is a novel member of the AMPK (adenosine monophosphate-activated protein kinase)-related superfamily that is activated in the process of angiogenesis. Currently, little is known about the function of SNRK in angiogenesis in the physiological and pathological conditions.Approach and Results—In this study, in Snrk global heterozygous knockout mice, retina angiogenesis and neovessel formation after hindlimb ischemia were suppressed. Consistently, mice with endothelial cell (EC)-specific Snrk deletion exhibited impaired retina angiogenesis, and delayed perfusion recovery and exacerbated muscle apoptosis in ischemic hindlimbs, compared with those of littermate wide-type mice. Endothelial SNRK expression was increased in the extremity vessel samples from nonischemic human. In ECs cultured in hypoxic conditions, HIF1α (hypoxia inducible factor 1α) bound to the SNRK promoter to upregulate SNRK expression. In the nuclei of hypoxic ECs, SNRK complexed with SP1 (specificity protein 1), and together, they bound to an SP1-binding motif in the ITGB1 (β1 integrin) promoter, resulting in enhanced ITGB1 expression and promoted EC migration. Furthermore, SNRK or SP1 deficiency in ECs ameliorated hypoxia-induced ITGB1 expression and, consequently, inhibited EC migration and angiogenesis.Conclusions—Taken together, our data have revealed that SNRK/SP1-ITGB1 signaling axis promotes angiogenesis in vivo.



Filamin A Modulates Store-Operated Ca2+ Entry by Regulating STIM1 (Stromal Interaction Molecule 1)-Orai1 Association in Human PlateletsHighlights [Basic Sciences]

2018-01-24T10:40:38-08:00

Objective—Here, we provide evidence for the role of FLNA (filamin A) in the modulation of store-operated calcium entry (SOCE).Approach and Results—SOCE is a major mechanism for calcium influx controlled by the intracellular Ca2+ stores. On store depletion, the endoplasmic reticulum calcium sensor STIM1 (stromal interaction molecule 1) redistributes into puncta at endoplasmic reticulum/plasma membrane junctions, a process supported by the cytoskeleton, where it interacts with the calcium channels; however, the mechanism for fine-tuning SOCE is not completely understood. Our results demonstrate that STIM1 interacts with FLNA on calcium store depletion in human platelets. The interaction is dependent on the phosphorylation of FLNA at Ser2152 by the cAMP-dependent protein kinase. Impairment of FLNA phosphorylation and knockdown of FLNA expression using siRNA increased SOCE in platelets. Similarly, SOCE was significantly greater in FLNA-deficient melanoma M2 cells than in the FLNA-expressing M2 subclone A7. Expression of FLNA in M2 cells attenuated SOCE, an effect prevented when the cells were transfected with the nonphosphorylatable FLNA S2152A mutant. Transfection of M2 cells with the STIM1(K684,685E) mutant reduced the STIM1–FLNA interaction. In platelets, attenuation of FLNA expression using siRNA resulted in enhanced association of STIM1 with the cytoskeleton, greater STIM1–Orai1 interaction, and SOCE. Introduction of an anti-FLNA (2597–2647) antibody attenuated the STIM1–FLNA interaction and enhanced thrombin-induced platelet aggregation.Conclusions—Our results indicate that FLNA modulates SOCE and then the correct platelet function, by fine-tuning the distribution of STIM1 in the cytoskeleton and the interaction with Orai1 channels.



Intermedin Enlarges the Vascular Lumen by Inducing the Quiescent Endothelial Cell ProliferationHighlights [Basic Sciences]

2018-01-24T10:40:38-08:00

Objective—Intermedin plays an important role in vascular remodeling and significantly improves blood perfusion, but the precise mechanism remains unclear. Herein, we aimed to define whether vascular lumen enlargement is responsible for the intermedin-increased blood perfusion and explore the underlying cellular and molecular mechanisms.Approach and Results—To study the role of intermedin, we generated the IMD-KO (Adm2−/−) mice using CRISPR/Cas9 (clustered regularly interspaced short palindromic repeats/clustered regularly interspaced short palindromic repeat–associated 9) system. Intermedin significantly promoted vascular lumen enlargement in vitro (fibrin beads assay) and in vivo (murine retinas), which contributed to the improved blood perfusion in both physiological (retinal) and pathological (tumor) angiogenic models. We designed experiments to calculate the endothelial cell (EC) size and found that the lumen enlargement is because of EC proliferation but not because of a change in cell shape. ECs that construct vessel walls are considered quiescent cells because they are in a state of contact inhibition and show reduced responsiveness to VEGF (vascular endothelial growth factor). Using immunoprecipitation, Western blot assay, and fluorescent microscopy, we found that intermedin induced the formation of a signaling complex containing CRLR (calcitonin receptor-like receptor)/β-arr1 (β-arrestin1)/Src in ECs and promoted it internalizing into cytoplasm in a clathrin-dependent manner to activate downstream ERK1/2 (extracellular signal-regulated kinase 1/2). Importantly, this effect was not abrogated by cell–cell contacts of ECs. Through this mechanism, intermedin could reactivate the quiescent ECs to proliferate, resulting in continuous lumen expanding and a more effective blood perfusion.Conclusions—Our findings suggest a novel mechanism that may explain how quiescent ECs overcome the contact inhibition and regain the ability to proliferate for continuous vascular lumen enlargement.



Loss of Vascular Myogenic Tone in miR-143/145 Knockout Mice Is Associated With Hypertension-Induced Vascular Lesions in Small Mesenteric ArteriesHighlights [Basic Sciences]

2018-01-24T10:40:38-08:00

Objective—Pressure-induced myogenic tone is involved in autoregulation of local blood flow and confers protection against excessive pressure levels in small arteries and capillaries. Myogenic tone is dependent on smooth muscle microRNAs (miRNAs), but the identity of these miRNAs is unclear. Furthermore, the consequences of altered myogenic tone for hypertension-induced damage to small arteries are not well understood.Approach and Results—The importance of smooth muscle–enriched microRNAs, miR-143/145, for myogenic tone was evaluated in miR-143/145 knockout mice. Furthermore, hypertension-induced vascular injury was evaluated in mesenteric arteries in vivo after angiotensin II infusion. Myogenic tone was abolished in miR-143/145 knockout mesenteric arteries, whereas contraction in response to calyculin A and potassium chloride was reduced by ≈30%. Furthermore, myogenic responsiveness was potentiated by angiotensin II in wild-type but not in knockout mice. Angiotensin II administration in vivo elevated systemic blood pressure in both genotypes. Hypertensive knockout mice developed severe vascular lesions characterized by vascular inflammation, adventitial fibrosis, and neointimal hyperplasia in small mesenteric arteries. This was associated with depolymerization of actin filaments and fragmentation of the elastic laminae at the sites of vascular lesions.Conclusions—This study demonstrates that miR-143/145 expression is essential for myogenic responsiveness. During hypertension, loss of myogenic tone results in potentially damaging levels of mechanical stress and detrimental effects on small arteries. The results presented herein provide novel insights into the pathogenesis of vascular disease and emphasize the importance of controlling mechanical factors to maintain structural integrity of the vascular wall.



DKK3 (Dickkopf 3) Alters Atherosclerotic Plaque Phenotype Involving Vascular Progenitor and Fibroblast Differentiation Into Smooth Muscle CellsHighlights [Translational Sciences]

2018-01-24T10:40:38-08:00

Objective—DKK3 (dickkopf 3), a 36-kD secreted glycoprotein, has been shown to be involved in the differentiation of partially reprogrammed cells and embryonic stem cells to smooth muscle cells (SMCs), but little is known about its involvement in vascular disease. This study aims to assess the effects of DKK3 on atherosclerotic plaque composition.Approach and Results—In the present study, we used a murine model of atherosclerosis (ApoE−/−) in conjunction with DKK3−/− and performed tandem stenosis of the carotid artery to evaluate atherosclerotic plaque development. We found that the absence of DKK3 leads to vulnerable atherosclerotic plaques, because of a reduced number of SMCs and reduced matrix protein deposition, as well as increased hemorrhage and macrophage infiltration. Further in vitro studies revealed that DKK3 can induce differentiation of Sca1+ (stem cells antigen 1) vascular progenitors and fibroblasts into SMCs via activation of the TGF-β (transforming growth factor-β)/ATF6 (activating transcription factor 6) and Wnt signaling pathways. Finally, we assessed the therapeutic potential of DKK3 in mouse and rabbit models and found that DKK3 altered the atherosclerotic plaque content via increasing SMC numbers and reducing vascular inflammation.Conclusions—Cumulatively, we provide the first evidence that DKK3 is a potent SMC differentiation factor, which might have a therapeutic effect in reducing intraplaque hemorrhage related to atherosclerotic plaque phenotype.



Weight Loss and Exercise Alter the High-Density Lipoprotein Lipidome and Improve High-Density Lipoprotein Functionality in Metabolic SyndromeHighlights [Translational Sciences]

2018-01-24T10:40:38-08:00

Objective—High-density lipoprotein (HDL) lipid composition and function may better reflect cardiovascular risk than HDL cholesterol concentration. This study characterized the relationships between HDL composition, metabolism, and function in metabolic syndrome (MetS) patients and how changes in composition after weight loss (WL) and exercise treatments are related to function.Approach and Results—Plasma samples from MetS patients (n=95) and healthy individuals (n=40) were used in this study. Subsets of the MetS group underwent 12 weeks of no treatment (n=17), WL (n=19), or WL plus exercise (WLEX; n=17). HDL was isolated using density-gradient ultracentrifugation. The HDL lipidome was analyzed by mass spectrometry, and particle size determined by nuclear magnetic resonance. Cholesteryl ester transfer protein activity and ex vivo HDL cholesterol efflux capacity (CEC) were assessed. The HDL lipidome in the MetS patients was substantially different from that in healthy individuals, mean particle size was smaller, and CEC was lower. Several HDL phospholipid and sphingolipid species were associated with HDL diameter and CEC. The HDL lipidome and particle size were modified toward the healthy individuals after WL and WLEX treatments, with greater effects observed in the latter group. Cholesteryl ester transfer protein activity was reduced after WL and WLEX, and CEC was improved after WLEX.Conclusions—WLEX treatment in MetS patients normalizes the HDL lipidome and particle size profile and enhances CEC. HDL lipids associated with diminished CEC may represent novel biomarkers for early prediction of HDL dysfunction and disease risk and may represent potential therapeutic targets for future HDL therapies.Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifier: NCT00163943



PAR4 (Protease-Activated Receptor 4) Antagonism With BMS-986120 Inhibits Human Ex Vivo Thrombus FormationHighlights [Translational Sciences]

2018-01-24T10:40:38-08:00

Objective—BMS-986120 is a novel first-in-class oral PAR4 (protease-activated receptor 4) antagonist with potent and selective antiplatelet effects. We sought to determine for the first time, the effect of BMS-986120 on human ex vivo thrombus formation.Approach and Results—Forty healthy volunteers completed a phase 1 parallel-group PROBE trial (Prospective Randomized Open-Label Blinded End Point). Ex vivo platelet activation, platelet aggregation, and thrombus formation were measured at 0, 2, and 24 hours after (1) oral BMS-986120 (60 mg) or (2) oral aspirin (600 mg) followed at 18 hours with oral aspirin (600 mg) and oral clopidogrel (600 mg). BMS-986120 demonstrated highly selective and reversible inhibition of PAR4 agonist peptide (100 μM)-stimulated P-selectin expression, platelet-monocyte aggregates, and platelet aggregation (P<0.001 for all). Compared with pretreatment, total thrombus area (μm2/mm) at high shear was reduced by 29.2% (95% confidence interval, 18.3%–38.7%; P<0.001) at 2 hours and by 21.4% (9.3%–32.0%; P=0.002) at 24 hours. Reductions in thrombus formation were driven by a decrease in platelet-rich thrombus deposition: 34.8% (19.3%–47.3%; P<0.001) at 2 hours and 23.3% (5.1%–38.0%; P=0.016) at 24 hours. In contrast to aspirin alone, or in combination with clopidogrel, BMS-986120 had no effect on thrombus formation at low shear (P=nonsignificant). BMS-986120 administration was not associated with an increase in coagulation times or serious adverse events.Conclusions—BMS-986120 is a highly selective and reversible oral PAR4 antagonist that substantially reduces platelet-rich thrombus formation under conditions of high shear stress. Our results suggest PAR4 antagonism has major potential as a therapeutic antiplatelet strategy.Clinical Trial Registration—URL: http://www.clinicaltrials.gov. Unique identifier: NCT02439190.



IL-1{beta} (Interleukin-1{beta}) and TNF-{alpha} (Tumor Necrosis Factor-{alpha}) Impact Abdominal Aortic Aneurysm Formation by Differential Effects on Macrophage PolarizationHighlights [Translational Sciences]

2018-01-24T10:40:38-08:00

Objective—Abdominal aortic aneurysms are inflammatory in nature and are associated with some risk factors that also lead to atherosclerotic occlusive disease, most notably smoking. The purpose of our study was to identify differential cytokine expression in patients with abdominal aortic aneurysm and those with atherosclerotic occlusive disease. Based on this analysis, we further explored and compared the mechanism of action of IL (interleukin)-1β versus TNF-α (tumor necrosis factor-α) in abdominal aortic aneurysm formation.Approach and Results—IL-1β was differentially expressed in human plasma with lower levels detected in patients with abdominal aortic aneurysm compared with matched atherosclerotic controls. We further explored its mechanism of action using a murine model and cell culture. Genetic deletion of IL-1β and IL-1R did not inhibit aneurysm formation or decrease MMP (matrix metalloproteinase) expression. The effects of IL-1β deletion on M1 macrophage polarization were compared with another proinflammatory cytokine, TNF-α. Bone marrow-derived macrophages from IL-1β−/− and TNF-α−/− mice were polarized to an M1 phenotype. TNF-α deletion, but not IL-1β deletion, inhibited M1 macrophage polarization. Infusion of M1 polarized TNF-α−/− macrophages inhibited aortic diameter growth; no inhibitory effect was seen in mice infused with M1 polarized IL-1β−/− macrophages.Conclusions—Although IL-1β is a proinflammatory cytokine, its effects on aneurysm formation and macrophage polarization differ from TNF-α. The differential effects of IL-1β and TNF-α inhibition are related to M1/M2 macrophage polarization and this may account for the differences in clinical efficacy of IL-1β and TNF-α antibody therapies in management of inflammatory diseases.



Nonfasting Triglycerides, Low-Density Lipoprotein Cholesterol, and Heart Failure RiskHighlights [Clinical and Population Studies]

2018-01-24T10:40:38-08:00

Objective—The prevalence of heart failure is increasing in the aging population, and heart failure is a disease with large morbidity and mortality. There is, therefore, a need for identifying modifiable risk factors for prevention. We tested the hypothesis that high concentrations of nonfasting triglycerides and low-density lipoprotein cholesterol are associated with higher risk of heart failure in the general population.Approach and Results—We included 103 860 individuals from the Copenhagen General Population Study and 9694 from the Copenhagen City Heart Study in 2 prospective observational association studies. Nonfasting triglycerides and low-density lipoprotein cholesterol were measured at baseline. Individuals were followed for ≤23 years, during which time 3593 were diagnosed with heart failure. Hazard ratios were estimated using Cox proportional hazard regression models. In the Copenhagen General Population Study, stepwise higher concentrations of nonfasting triglycerides were associated with stepwise higher risk of heart failure (P for trend <0.001), with a multivariable adjusted hazard ratio of 2.59 (95% confidence interval, 1.48–4.54) for individuals with nonfasting triglycerides ≥5 mmol/L (440 mg/dL) compared with individuals with concentrations <1 mmol/L (88 mg/dL). Concentrations of low-density lipoprotein cholesterol were not associated with risk of heart failure. Results were independently confirmed in the Copenhagen City Heart Study.Conclusions—Stepwise higher concentrations of nonfasting triglycerides were associated with stepwise higher risk of heart failure; however, concentrations of low-density lipoprotein cholesterol were not associated with risk of heart failure in the general population.



Sex Differences in Hemodynamic and Microvascular Mechanisms of Myocardial Ischemia Induced by Mental StressHighlights [Clinical and Population Studies]

2018-01-24T10:40:38-08:00

Objective—To investigate sex-specific vascular mechanisms for mental stress-induced myocardial ischemia (MSIMI).Approach and Results—Baseline data from a prospective cohort study of 678 patients with coronary artery disease underwent myocardial perfusion imaging before and during a public speaking stressor. The rate-pressure product response was calculated as the difference between the maximum value during the speech minus the minimum value during rest. Peripheral vasoconstriction by peripheral arterial tonometry was calculated as the ratio of pulse wave amplitude during the speech over the resting baseline; ratios <1 indicate a vasoconstrictive response. MSIMI was defined as percent of left ventricle that was ischemic and as a dichotomous variable. Men (but not women) with MSIMI had a higher rate-pressure product response than those without MSIMI (6500 versus 4800 mm Hg bpm), whereas women (but not men) with MSIMI had a significantly lower peripheral arterial tonometry ratio than those without MSIMI (0.5 versus 0.8). In adjusted linear regression, each 1000-U increase in rate-pressure product response was associated with 0.32% (95% confidence interval, 0.22–0.42) increase in inducible ischemia among men, whereas each 0.10-U decrease in peripheral arterial tonometry ratio was associated with 0.23% (95% confidence interval, 0.11–0.35) increase in inducible myocardial ischemia among women. Results were independent of conventional stress-induced myocardial ischemia.Conclusions—Women and men have distinct cardiovascular reactivity mechanisms for MSIMI. For women, stress-induced peripheral vasoconstriction with mental stress, and not increased hemodynamic workload, is associated with MSIMI, whereas for men, it is the opposite. Future studies should examine these pathways on long-term outcomes.


















X Marks the Spot [Editorial]

2017-12-27T10:40:49-08:00







CRISPR-Cas9 Genome Editing for Treatment of Atherogenic DyslipidemiaHighlights [ATVB in Focus: Human Genetics of Atherosclerosis]

2017-12-27T10:40:49-08:00

Although human genetics has resulted in the identification of novel lipid-related genes that can be targeted for the prevention of atherosclerotic vascular disease, medications targeting these genes or their protein products have short-term effects and require frequent administration during the course of the lifetime for maximal benefit. Genome-editing technologies, such as CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats-CRISPR–associated 9) have the potential to permanently alter genes in the body and produce long-term and even lifelong protection against atherosclerosis. In this review, we discuss recent advances in genome-editing technologies and early proof-of-concept studies of somatic in vivo genome editing in mice that highlight the potential of genome editing to target disease-related genes in patients, which would establish a novel therapeutic paradigm for atherosclerosis.



Sortilin and Its Multiple Roles in Cardiovascular and Metabolic DiseasesHighlights [Brief Reviews]

2017-12-27T10:40:49-08:00

Cardiovascular disease is a leading cause of morbidity and mortality in the Western world. Studies of sortilin’s influence on cardiovascular and metabolic diseases goes far beyond the genome-wide association studies that have revealed an association between cardiovascular diseases and the 1p13 locus that encodes sortilin. Emerging evidence suggests a significant role of sortilin in the pathogenesis of vascular and metabolic diseases; this includes type II diabetes mellitus via regulation of insulin resistance, atherosclerosis through arterial wall inflammation and calcification, and dysregulated lipoprotein metabolism. Sortilin is also known for its functional role in neurological disorders. It serves as a key receptor for cytokines, lipids, and enzymes and participates in pathological cargo loading to and trafficking of extracellular vesicles. This article provides a comprehensive review of sortilin’s contributions to cardiovascular and metabolic diseases but focuses particularly on atherosclerosis. We summarize recent clinical findings that suggest that sortilin may be a cardiovascular risk biomarker and also discuss sortilin as a potential drug target.



Rosuvastatin Reduces Aortic Sinus and Coronary Artery Atherosclerosis in SR-B1 (Scavenger Receptor Class B Type 1)/ApoE (Apolipoprotein E) Double Knockout Mice Independently of Plasma Cholesterol LoweringHighlights [Basic Sciences]

2017-12-27T10:40:49-08:00

Objective—Rosuvastatin has been widely used in the primary and secondary prevention of coronary heart disease. However, its antiatherosclerotic properties have not been tested in a mouse model that could mimic human coronary heart disease. The present study was designed to test the effects of rosuvastatin on coronary artery atherosclerosis and myocardial fibrosis in SR-B1 (scavenger receptor class B type 1) and apoE (apolipoprotein E) double knockout mice.Approach and Results—Three-week-old SR-B1−/−/apoE−/− mice were injected daily with 10 mg/kg of rosuvastatin for 2 weeks. Compared with saline-treated mice, rosuvastatin-treated mice showed increased levels of hepatic PCSK9 (proprotein convertase subtilisin/kexin type-9) and LDLR (low-density lipoprotein receptor) message, increased plasma PCSK9 protein but decreased levels of hepatic LDLR protein and increased plasma total cholesterol associated with apoB (apolipoprotein B) 48-containing lipoproteins. In spite of this, rosuvastatin treatment was associated with decreased atherosclerosis in both the aortic sinus and coronary arteries and reduced platelet accumulation in atherosclerotic coronary arteries. Cardiac fibrosis and cardiomegaly were also attenuated in rosuvastatin-treated SR-B1−/−/apoE−/− mice. Two-week treatment with rosuvastatin resulted in significant decreases in markers of oxidized phospholipids in atherosclerotic plaques. In vitro analysis showed that incubation of bone marrow-derived macrophages with rosuvastatin substantially downregulated cluster of differentiation (CD)36 and inhibited oxidized LDL-induced foam cell formation.Conclusions—Rosuvastatin protected SR-B1−/−/apoE−/− mice against atherosclerosis and platelet accumulation in coronary arteries and attenuated myocardial fibrosis and cardiomegaly, despite increased plasma total cholesterol. The ability of rosuvastatin to reduce oxidized phospholipids in atherosclerotic plaques and inhibit macrophage foam cell formation may have contributed to this protection.



Molecular Ultrasound Imaging of Junctional Adhesion Molecule A Depicts Acute Alterations in Blood Flow and Early Endothelial DysregulationHighlights [Basic Sciences]

2017-12-27T10:40:49-08:00

Objective—The junctional adhesion molecule A (JAM-A) is physiologically located in interendothelial tight junctions and focally redistributes to the luminal surface of blood vessels under abnormal shear and flow conditions accompanying atherosclerotic lesion development. Therefore, JAM-A was evaluated as a target for molecularly targeted ultrasound imaging of transient endothelial dysfunction under acute blood flow variations.Approach and Results—Flow-dependent endothelial dysfunction was induced in apolipoprotein E–deficient mice (n=43) by carotid partial ligation. JAM-A expression was investigated by molecular ultrasound using antibody-targeted poly(n-butyl cyanoacrylate) microbubbles and validated with immunofluorescence. Flow disturbance and arterial remodeling were assessed using functional ultrasound. Partial ligation led to an immediate drop in perfusion at the ligated side and a direct compensatory increase at the contralateral side. This was accompanied by a strongly increased JAM-A expression and JAM-A–targeted microbubbles binding at the partially ligated side and by a moderate and temporary increase in the contralateral artery (≈14× [P<0.001] and ≈5× [P<0.001] higher than control, respectively), both peaking after 2 weeks. Subsequently, although JAM-A expression and JAM-A–targeted microbubbles binding persisted at a higher level at the partially ligated side, it completely normalized within 4 weeks at the contralateral side.Conclusions—Temporary blood flow variations induce endothelial rearrangement of JAM-A, which can be visualized using JAM-A–targeted microbubbles. Thus, JAM-A may be considered as a marker of acute endothelial activation and dysfunction. Its imaging may facilitate the early detection of cardiovascular risk areas, and it enables the therapeutic prevention of their progression toward an irreversible pathological state.



Extracellular Vesicles Secreted by Atherogenic Macrophages Transfer MicroRNA to Inhibit Cell MigrationHighlights [Basic Sciences]

2017-12-27T10:40:49-08:00

Objective—During inflammation, macrophages secrete vesicles carrying RNA, protein, and lipids as a form of extracellular communication. In the vessel wall, extracellular vesicles (EVs) have been shown to be transferred between vascular cells during atherosclerosis; however, the role of macrophage-derived EVs in atherogenesis is not known. Here, we hypothesize that atherogenic macrophages secrete microRNAs (miRNAs) in EVs to mediate cell–cell communication and promote proinflammatory and proatherogenic phenotypes in recipient cells.Approach and Results—We isolated EVs from mouse and human macrophages treated with an atherogenic stimulus (oxidized low-density lipoprotein) and characterized the EV miRNA expression profile. We confirmed the enrichment of miR-146a, miR-128, miR-185, miR-365, and miR-503 in atherogenic EVs compared with controls and demonstrate that these EVs are taken up and transfer exogenous miRNA to naive recipient macrophages. Bioinformatic pathway analysis suggests that atherogenic EV miRNAs are predicted to target genes involved in cell migration and adhesion pathways, and indeed delivery of EVs to naive macrophages reduced macrophage migration both in vitro and in vivo. Inhibition of miR-146a, the most enriched miRNA in atherogenic EVs, reduced the inhibitory effect of EVs on macrophage migratory capacity. EV-mediated delivery of miR-146a repressed the expression of target genes IGF2BP1 (insulin-like growth factor 2 mRNA-binding protein 1) and HuR (human antigen R or ELAV-like RNA-binding protein 1) in recipient cells, and knockdown of IGF2BP1 and HuR using short interfering RNA greatly reduced macrophage migration, highlighting the importance of these EV-miRNA targets in regulating macrophage motility.Conclusions—EV-derived miRNAs from atherogenic macrophages, in particular miR-146a, may accelerate the development of atherosclerosis by decreasing cell migration and promoting macrophage entrapment in the vessel wall.



Proatherogenic Flow Increases Endothelial Stiffness via Enhanced CD36-Mediated Uptake of Oxidized Low-Density LipoproteinsHighlights [Basic Sciences]

2017-12-27T10:40:49-08:00

Objective—Disturbed flow (DF) is well-known to induce endothelial dysfunction and synergistically with plasma dyslipidemia facilitate plaque formation. Little is known, however, about the synergistic impact of DF and dyslipidemia on endothelial biomechanics. Our goal was to determine the impact of DF on endothelial stiffness and evaluate the role of dyslipidemia/oxLDL (oxidized low-density lipoprotein) in this process.Approach and Results—Endothelial elastic modulus of intact mouse aortas ex vivo and of human aortic endothelial cells exposed to laminar flow or DF was measured using atomic force microscopy. Endothelial monolayer of the aortic arch is found to be significantly stiffer than the descending aorta (4.2+1.1 versus 2.5+0.2 kPa for aortic arch versus descending aorta) in mice maintained on low-fat diet. This effect is significantly exacerbated by short-term high-fat diet (8.7+2.5 versus 4.5+1.2 kPa for aortic arch versus descending aorta). Exposure of human aortic endothelial cells to DF in vitro resulted in 50% increase in oxLDL uptake and significant endothelial stiffening in the presence but not in the absence of oxLDL. DF also increased the expression of oxLDL receptor CD36 (cluster of differentiation 36), whereas downregulation of CD36 abrogated DF-induced endothelial oxLDL uptake and stiffening. Furthermore, genetic deficiency of CD36 abrogated endothelial stiffening in the aortic arch in vivo in mice fed either low-fat diet or high-fat diet. We also show that the loss of endothelial stiffening in CD36 knockout aortas is not mediated by the loss of CD36 in circulating cells.Conclusions—DF facilitates endothelial CD36-dependent uptake of oxidized lipids resulting in local increase of endothelial stiffness in proatherogenic areas of the aorta.



Interrogation of the Atherosclerosis-Associated SORT1 (Sortilin 1) Locus With Primary Human Hepatocytes, Induced Pluripotent Stem Cell-Hepatocytes, and Locus-Humanized MiceHighlights [Basic Sciences]

2017-12-27T10:40:49-08:00

Objective—The noncoding single-nucleotide polymorphism rs12740374 has been hypothesized to be the causal variant responsible for liver-specific modulation of SORT1(sortilin 1) expression (ie, expression quantitative trait locus) and, by extension, the association of the SORT1 locus on human chromosome 1p13 with low-density lipoprotein cholesterol levels and coronary heart disease. The goals of this study were to compare 3 different hepatocyte models in demonstrating that the rs12740374 minor allele sequence is responsible for transcriptional activation of SORT1 expression.Approach and Results—We found that although primary human hepatocytes of varied rs12740374 genotypes strongly replicated the SORT1 expression quantitative trait locus observed previously in whole-liver samples, a population cohort of induced pluripotent stem cell–derived hepatocyte-like cells poorly replicated the expression quantitative trait locus. In primary human hepatocytes from multiple individuals heterozygous at rs12740374, we used CRISPR-Cas9 (clustered regularly interspaced short palindromic repeats–associated 9) to specifically target the rs12740374 minor allele sequence ex vivo, resulting in a reproducible reduction in SORT1 expression. We generated a locus-humanized transgenic mouse with a bacterial artificial chromosome bearing the human SORT1 locus with the rs12740374 minor allele. In this mouse model, we used CRISPR-Cas9 to target the rs12740374 minor allele sequence in the liver in vivo, resulting in a substantial reduction of hepatic SORT1 expression.Conclusions—The rs12740374 minor allele sequence enhances SORT1 expression in hepatocytes. CRISPR-Cas9 can be used in primary human hepatocytes ex vivo and locus-humanized mice in vivo to interrogate the function of noncoding regulatory regions. Induced pluripotent stem cell–derived hepatocyte-like cells experience limitations that prevent faithful modelling of some hepatocyte expression quantitative trait loci.



Quantitative Trait Locus Mapping of Macrophage Cholesterol Metabolism and CRISPR/Cas9 Editing Implicate an ACAT1 Truncation as a Causal Modifier VariantHighlights [Basic Sciences]

2017-12-27T10:40:49-08:00

Objective—Cholesterol metabolism is a dynamic process involving intracellular trafficking, cholesterol esterification, and cholesterol ester hydrolysis. Our objective was to identify genes that regulate macrophage cholesterol metabolism.Approaches and Results—We performed quantitative trait loci mapping of free and esterified cholesterol levels and the ratio of esterified to free cholesterol in acetylated low-density lipoprotein–loaded bone marrow–derived macrophages from an AKR×DBA/2 strain intercross. Ten distinct cholesterol modifier loci were identified, and bioinformatics was used to prioritize candidate genes. The strongest locus was located on distal chromosome 1, which we named Mcmm1 (macrophage cholesterol metabolism modifier 1). This locus harbors the Soat1 (sterol O-acyltransferase 1) gene, encoding Acyl-coenzyme A:cholesterol acyltransferase 1 (ACAT1), which esterifies free cholesterol. The parental AKR strain has an exon 2 deletion in Soat1, which leads to a 33 amino acid N-terminal truncation in ACAT1. CRISPR/Cas9 editing of DBA/2 embryonic stem cells was performed to replicate the AKR strain Soat1 exon 2 deletion, while leaving the remainder of the genome unaltered. DBA/2 stem cells and stem cells heterozygous and homozygous for the Soat1 exon 2 deletion were differentiated into macrophages and loaded with acetylated low-density lipoprotein. DBA/2 stem cell–derived macrophages accumulated less free cholesterol and more esterified cholesterol relative to cells heterozygous and homozygous for the Soat1 exon 2 deletion.Conclusions—A Soat1 deletion present in AKR mice, and resultant N-terminal ACAT1 truncation, was confirmed to be a significant modifier of macrophage cholesterol metabolism. Other Mcmm loci candidate genes were prioritized via bioinformatics.



Exogenous Insulin Infusion Can Decrease Atherosclerosis in Diabetic Rodents by Improving Lipids, Inflammation, and Endothelial FunctionHighlights [Basic Sciences]

2017-12-27T10:40:49-08:00

Objective—The objective of this study is to evaluate whether exogenously induced hyperinsulinemia may increase the development of atherosclerosis.Approach and Results—Hyperinsulinemia, induced by exogenous insulin implantation in high-fat fed (60% fat HFD) apolipoprotein E–deficient mice (ApoE−/−) mice, exhibited insulin resistance, hyperglycemia, and hyperinsulinemia. Atherosclerosis was measured by the accumulation of fat, macrophage, and extracellular matrix in the aorta. After 8 weeks on HFD, ApoE−/− mice were subcutaneously implanted with control (sham) or insulin pellet, and phlorizin, a sodium glucose cotransporters inhibitor (1/2)inhibitor, for additional 8 weeks. Intraperitoneal glucose tolerance test showed that plasma glucose levels were lower and insulin and IGF-1 (insulin-like growth factor-1) levels were 5.3- and 3.3-fold higher, respectively, in insulin-implanted compared with sham-treated ApoE−/− mice. Plasma triglyceride, cholesterol, and lipoprotein levels were decreased in mice with insulin implant, in parallel with increased lipoprotein lipase activities. Atherosclerotic plaque by en face and complexity staining showed significant reductions of fat deposits and expressions of vascular adhesion molecule-1, tumor necrosis factor-α, interleukin 6, and macrophages in arterial wall while exhibiting increased activation of pAKT and endothelial nitric oxide synthase (P<0.05) comparing insulin-implanted versus sham HFD ApoE−/− mice. No differences were observed in atherosclerotic plaques between phlorizin-treated and sham HFD ApoE−/− mice, except phlorizin significantly lowered plasma glucose and glycated hemoglobin levels while increased glucosuria. Endothelial function was improved only by insulin treatment through endothelial nitric oxide synthase/nitric oxide activations and reduced proinflammatory (M1) and increased anti-inflammatory (M2) macrophages, which were inhibited by endothelial nitric oxide synthase inhibitor.Conclusions—Exogenous insulin decreased atherosclerosis by lowering inflammatory cytokines, macrophages, and plasma lipids in HFD-induced hyperlipidemia, insulin resistant and mildly diabetic ApoE−/− mice.



Suppression of Hepatic FLOT1 (Flotillin-1) by Type 2 Diabetes Mellitus Impairs the Disposal of Remnant Lipoproteins via Syndecan-1Highlights [Basic Sciences]

2017-12-27T10:40:49-08:00

Objective—Type 2 diabetes mellitus (T2DM) and the atherometabolic syndrome exhibit a deadly dyslipoproteinemia that arises in part from impaired hepatic disposal of C-TRLs (cholesterol- and triglyceride-rich remnant apoB [apolipoprotein B] lipoproteins). We previously identified syndecan-1 as a receptor for C-TRLs that directly mediates endocytosis via rafts, independent from coated pits. Caveolins and flotillins form rafts but facilitate distinct endocytotic pathways. We now investigated their participation in syndecan-1–mediated disposal of C-TRLs and their expression in T2DM liver.Approach and Results—In cultured liver cells and nondiabetic murine livers, we found that syndecan-1 coimmunoprecipitates with FLOT1 (flotillin-1) but not with CAV1 (caveolin-1). Binding of C-TRLs to syndecan-1 on the surface of liver cells enhanced syndecan-1/FLOT1 association. The 2 molecules then trafficked together into the lysosomes, implying limited if any recycling back to the cell surface. The interaction requires the transmembrane/cytoplasmic region of syndecan-1 and the N-terminal hydrophobic domain of FLOT1. Knockdown of FLOT1 in cultured liver cells substantially inhibited syndecan-1 endocytosis. Livers from obese, T2DM KKAy mice exhibited 60% to 70% less FLOT1 protein and mRNA than in nondiabetic KK livers. An adenoviral construct to enhance hepatic expression of wild-type FLOT1 in T2DM mice normalized plasma triglycerides, whereas a mutant FLOT1 missing its N-terminal hydrophobic domain had no effect. Moreover, the adenoviral vector for wild-type FLOT1 lowered plasma triglyceride excursions and normalized retinyl excursions in T2DM KKAy mice after a corn oil gavage, without affecting postprandial production of C-TRLs.Conclusions—FLOT1 is a novel participant in the disposal of harmful C-TRLs via syndecan-1. Low expression of FLOT1 in T2DM liver may contribute to metabolic dyslipoproteinemia.



Selective EGFR (Epidermal Growth Factor Receptor) Deletion in Myeloid Cells Limits Atherosclerosis—Brief ReportHighlights [Basic Sciences]

2017-12-27T10:40:49-08:00

Objective—To determine the consequences of specific inhibition of EGFR (epidermal growth factor receptor) in myeloid cells in atherosclerosis development.Approach and Results—Atherosclerotic lesion size was significantly reduced in irradiated Ldlr−/− mice reconstituted with LysMCre+Egfrlox/lox bone marrow, compared with chimeric Ldlr−/− mice reconstituted with LysMCre−Egfrlox/lox bone marrow, after 4 (−43%; P<0.05), 7 (−34%; P<0.05), and 12 weeks (−54%; P<0.001) of high-fat diet. Reduction of lesion size was associated with marked reduction in macrophage accumulation and necrotic core size. Specific deletion of Egfr in myeloid cells reduced TNF-α (tumor necrosis factor-α) and IL (interleukin)-6 production by stimulated macrophages but had no effect on IL-10 and IL-12p70 secretion. Finally, we found that myeloid deletion of Egfr limited cytoskeletal rearrangements and also lipid uptake by macrophages through a downregulation of the scavenger receptor CD36 (cluster of differentiation 36).Conclusions—Gene deletion of Egfr in myeloid cells limits IL-6 and TNF-α production, lipid uptake, and consecutively reduces atherosclerosis development.



Non-Endoplasmic Reticulum-Based Calr (Calreticulin) Can Coordinate Heterocellular Calcium Signaling and Vascular FunctionHighlights [Basic Sciences]

2017-12-27T10:40:49-08:00

Objective—In resistance arteries, endothelial cell (EC) extensions can make contact with smooth muscle cells, forming myoendothelial junction at holes in the internal elastic lamina (HIEL). At these HIEL, calcium signaling is tightly regulated. Because Calr (calreticulin) can buffer ≈50% of endoplasmic reticulum calcium and is expressed throughout IEL holes in small arteries, the only place where myoendothelial junctions form, we investigated the effect of EC-specific Calr deletion on calcium signaling and vascular function.Approach and Results—We found Calr expressed in nearly every IEL hole in third-order mesenteric arteries, but not other ER markers. Because of this, we generated an EC-specific, tamoxifen inducible, Calr knockout mouse (EC Calr Δ/Δ). Using this mouse, we tested third-order mesenteric arteries for changes in calcium events at HIEL and vascular reactivity after application of CCh (carbachol) or PE (phenylephrine). We found that arteries from EC Calr Δ/Δ mice stimulated with CCh had unchanged activity of calcium signals and vasodilation; however, the same arteries were unable to increase calcium events at HIEL in response to PE. This resulted in significantly increased vasoconstriction to PE, presumably because of inhibited negative feedback. In line with these observations, the EC Calr Δ/Δ had increased blood pressure. Comparison of ER calcium in arteries and use of an ER-specific GCaMP indicator in vitro revealed no observable difference in ER calcium with Calr knockout. Using selective detergent permeabilization of the artery and inhibition of Calr translocation, we found that the observed Calr at HIEL may not be within the ER.Conclusions—Our data suggest that Calr specifically at HIEL may act in a non-ER dependent manner to regulate arteriolar heterocellular communication and blood pressure.



Inhalation of Fine Particulate Matter Impairs Endothelial Progenitor Cell Function Via Pulmonary Oxidative StressHighlights [Basic Sciences]

2017-12-27T10:40:49-08:00

Objective—Exposure to fine particulate matter (PM2.5) air pollution is associated with the depletion of circulating endothelial progenitor cells (EPCs), as well as vascular injury and dysfunction. Nevertheless, it remains unclear whether PM2.5 exposure leads to significant impairments in EPC function. Hence, we studied the effects of PM2.5 on EPC-mediated recovery of vascular perfusion after hindlimb ischemia and examined the mechanisms whereby PM2.5 exposure affects EPC abundance and function.Approach and Results—In comparison with EPCs isolated from mice breathing filtered air, EPCs from mice exposed for 9 consecutive days (6 hours per day) to concentrated ambient PM2.5 (CAP) had defects in both proliferation and tube formation. However, CAP exposure of mice overexpressing extracellular superoxide dismutase (ecSOD-Tg) in the lungs did not affect EPC tube formation. Exposure to CAP also suppressed circulating EPC levels, VEGF (vascular endothelial growth factor)-stimulated aortic Akt phosphorylation, and plasma NO levels in wild-type but not in ecSOD-Tg mice. EPCs from CAP-exposed wild-type mice failed to augment basal recovery of hindlimb perfusion when injected into unexposed mice subjected to hindlimb ischemia; however, these deficits in recovery of hindlimb perfusion were absent when using EPCs derived from CAP-exposed ecSOD-Tg mice. The improved reparative function of EPCs from CAP-exposed ecSOD-Tg mice was also reflected by greater expression of Mmp-9 and Nos3 when compared with EPCs from CAP-exposed wild-type mice.Conclusions—Exposure to PM2.5 impairs EPC abundance and function and prevents EPC-mediated vascular recovery after hindlimb ischemia. This defect is attributed, in part, to pulmonary oxidative stress and was associated with vascular VEGF resistance and a decrement in NO bioavailability.



Sex Chromosome Complement Defines Diffuse Versus Focal Angiotensin II-Induced Aortic PathologyHighlights [Basic Sciences]

2017-12-27T10:40:49-08:00

Objective—Aortic pathologies exhibit sexual dimorphism, with aneurysms in both the thoracic and abdominal aorta (ie, abdominal aortic aneurysm [AAA]) exhibiting higher male prevalence. Women have lower prevalence of aneurysms, but when they occur, aneurysms progress rapidly. To define mechanisms for these sex differences, we determined the role of sex chromosome complement and testosterone on the location and progression of angiotensin II (AngII)–induced aortic pathologies.Approach and Results—We used transgenic male mice expressing Sry (sex-determining region Y) on an autosome to create Ldlr (low-density lipoprotein receptor)–deficient male mice with an XY or XX sex chromosome complement. Transcriptional profiling was performed on abdominal aortas from XY or XX males, demonstrating 1746 genes influenced by sex chromosomes or sex hormones. Males (XY or XX) were either sham-operated or orchiectomized before AngII infusions. Diffuse aortic aneurysm pathology developed in XY AngII-infused males, whereas XX males developed focal AAAs. Castration reduced all AngII-induced aortic pathologies in XY and XX males. Thoracic aortas from AngII-infused XY males exhibited adventitial thickening that was not present in XX males. We infused male XY and XX mice with either saline or AngII and quantified mRNA abundance of key genes in both thoracic and abdominal aortas. Regional differences in mRNA abundance existed before AngII infusions, which were differentially influenced by AngII between genotypes. Prolonged AngII infusions resulted in aortic wall thickening of AAAs from XY males, whereas XX males had dilated focal AAAs.Conclusions—An XY sex chromosome complement mediates diffuse aortic pathology, whereas an XX sex chromosome complement contributes to focal AngII-induced AAAs.



Sustained Activation of Rho GTPases Promotes a Synthetic Pulmonary Artery Smooth Muscle Cell Phenotype in Neprilysin Null MiceHighlights [Basic Sciences]

2017-12-27T10:40:49-08:00

Objective—Pulmonary artery smooth muscle cells (PASMCs) from neprilysin (NEP) null mice exhibit a synthetic phenotype and increased activation of Rho GTPases compared with their wild-type counterparts. Although Rho GTPases are known to promote a contractile SMC phenotype, we hypothesize that their sustained activity decreases SM-protein expression in these cells.Approach and Results—PASMCs isolated from wild-type and NEP−/− mice were used to assess levels of SM-proteins (SM-actin, SM-myosin, SM22, and calponin) by Western blotting, and were lower in NEP−/− PASMCs compared with wild-type. Rac and Rho (ras homology family member) levels and activity were higher in NEP−/− PASMCs, and ShRNA to Rac and Rho restored SM-protein, and attenuated the enhanced migration and proliferation of NEP−/− PASMCs. SM-gene repressors, p-Elk-1, and Klf4 (Kruppel lung factor 4), were higher in NEP−/− PASMCs and decreased by shRNA to Rac and Rho. Costimulation of wild-type PASMCs with PDGF (platelet-derived growth factor) and the NEP substrate, ET-1 (endothelin-1), increased Rac and Rho activity, and decreased SM-protein levels mimicking the NEP knock-out phenotype. Activation of Rac and Rho and downstream effectors was observed in lung tissue from NEP−/− mice and humans with chronic obstructive pulmonary disease.Conclusions—Sustained Rho activation in NEP−/− PASMCs is associated with a decrease in SM-protein levels and increased migration and proliferation. Inactivation of RhoGDI (Rho guanine dissociation inhibitor) and RhoGAP (Rho GTPase activating protein) by phosphorylation may contribute to prolonged activation of Rho in NEP−/− PASMCs. Rho GTPases may thus have a role in integration of signals between vasopeptides and growth factor receptors and could influence pathways that suppress SM-proteins to promote a synthetic phenotype.



Mining the Stiffness-Sensitive Transcriptome in Human Vascular Smooth Muscle Cells Identifies Long Noncoding RNA Stiffness RegulatorsHighlights [Basic Sciences]

2017-12-27T10:40:49-08:00

Objective—Vascular extracellular matrix stiffening is a risk factor for aortic and coronary artery disease. How matrix stiffening regulates the transcriptome profile of human aortic and coronary vascular smooth muscle cells (VSMCs) is not well understood. Furthermore, the role of long noncoding RNAs (lncRNAs) in the cellular response to stiffening has never been explored. This study characterizes the stiffness-sensitive (SS) transcriptome of human aortic and coronary VSMCs and identifies potential key lncRNA regulators of stiffness-dependent VSMC functions.Approach and Results—Aortic and coronary VSMCs were cultured on hydrogel substrates mimicking physiological and pathological extracellular matrix stiffness. Total RNAseq was performed to compare the SS transcriptome profiles of aortic and coronary VSMCs. We identified 3098 genes (2842 protein coding and 157 lncRNA) that were stiffness sensitive in both aortic and coronary VSMCs (false discovery rate <1%). Hierarchical clustering revealed that aortic and coronary VSMCs grouped by stiffness rather than cell origin. Conservation analyses also revealed that SS genes were more conserved than stiffness-insensitive genes. These VSMC SS genes were less tissue-type specific and expressed in more tissues than stiffness-insensitive genes. Using unbiased systems analyses, we identified MALAT1 as an SS lncRNA that regulates stiffness-dependent VSMC proliferation and migration in vitro and in vivo.Conclusions—This study provides the transcriptomic landscape of human aortic and coronary VSMCs in response to extracellular matrix stiffness and identifies novel SS human lncRNAs. Our data suggest that the SS transcriptome is evolutionarily important to VSMCs function and that SS lncRNAs can act as regulators of stiffness-dependent phenotypes.



Both Autocrine Signaling and Paracrine Signaling of HB-EGF Enhance Ocular NeovascularizationHighlights [Basic Sciences]

2017-12-27T10:40:49-08:00

Objective—The incidence of blindness is increasing because of the increase in abnormal ocular neovascularization. Anti-VEGF (vascular endothelial growth factor) therapies have led to good results, although they are not a cure for the blindness. The purpose of this study was to determine what role HB-EGF (heparin-binding epidermal growth factor–like growth factor) plays in ocular angiogenesis.Approach and Results—We examined the role played by HB-EGF in ocular neovascularization in 2 animal models of neovascularization: laser-induced choroidal neovascularization (CNV) and oxygen-induced retinopathy. We also studied human retinal microvascular endothelial cells in culture. Our results showed that the neovascularization was decreased in both the CNV and oxygen-induced retinopathy models in HB-EGF conditional knockout mice compared with that in wild-type mice. Moreover, the expressions of HB-EGF and VEGF were increased after laser-induced CNV and oxygen-induced retinopathy, and their expression sites were located around the neovascular areas. Exposure of human retinal microvascular endothelial cells to HB-EGF and VEGF increased their proliferation and migration, and CRM-197 (cross-reactive material-197), an HB-EGF inhibitor, decreased the HB-EGF–induced and VEGF-induced cell proliferation and migration. VEGF increased the expression of HB-EGF mRNA. VEGF-dependent activation of EGFR (epidermal growth factor receptor)/ERK1/2 (extracellular signal-regulated kinase 1/2) signaling and cell proliferation of endothelial cells required stimulation of the ADAM17 (a disintegrin and metalloprotease) and ADAM12. CRM-197 decreased the grades of the fluorescein angiograms and size of the CNV areas in marmoset monkeys.Conclusions—These findings suggest that HB-EGF plays an important role in the development of CNV. Therefore, further investigations of HB-EGF are needed as a potential therapeutic target in the treatment of exudative age-related macular degeneration.



Statins Promote Cardiac Infarct Healing by Modulating Endothelial Barrier Function Revealed by Contrast-Enhanced Magnetic Resonance ImagingHighlights [Basic Sciences]

2017-12-27T10:40:49-08:00

Objective—The endothelium has a crucial role in wound healing, acting as a barrier to control transit of leukocytes. Endothelial barrier function is impaired in atherosclerosis preceding myocardial infarction (MI). Besides lowering lipids, statins modulate endothelial function. Here, we noninvasively tested whether statins affect permeability at the inflammatory (day 3) and the reparative (day 7) phase of infarct healing post-MI using contrast-enhanced cardiac magnetic resonance imaging (MRI).Approach and Results—Noninvasive permeability mapping by MRI after MI in C57BL/6, atherosclerotic ApoE−/−, and statin-treated ApoE−/− mice was correlated to subsequent left ventricular outcome by structural and functional cardiac MRI. Ex vivo histology, flow cytometry, and quantitative polymerase chain reaction were performed on infarct regions. Increased vascular permeability at ApoE−/− infarcts was observed compared with C57BL/6 infarcts, predicting enhanced left ventricular dilation at day 21 post-MI by MRI volumetry. Statin treatment improved vascular barrier function at ApoE−/− infarcts, indicated by reduced permeability. The infarcted tissue of ApoE−/− mice 3 days post-MI displayed an unbalanced Vegfa(vascular endothelial growth factor A)/Angpt1 (angiopoetin-1) expression ratio (explaining leakage-prone vessels), associated with higher amounts of CD45+ leukocytes and inflammatory LY6Chi monocytes. Statins reversed the unbalanced Vegfa/Angpt1 expression, normalizing endothelial barrier function at the infarct and blocking the augmented recruitment of inflammatory leukocytes in statin-treated ApoE−/− mice.Conclusions—Statins lowered permeability and reduced the transit of unfavorable inflammatory leukocytes into the infarcted tissue, consequently improving left ventricular outcome.



Transforming Growth Factor-{beta}1 Inhibits Pseudoaneurysm Formation After Aortic Patch AngioplastyHighlights [Basic Sciences]

2017-12-27T10:40:49-08:00

Objective—Pseudoaneurysms remain a significant complication after vascular procedures. We hypothesized that TGF-β (transforming growth factor-β) signaling plays a mechanistic role in the development of pseudoaneurysms.Approach and Results—Rat aortic pericardial patch angioplasty was associated with a high incidence (88%) of pseudoaneurysms at 30 days, with increased smad2 phosphorylation in small pseudoaneurysms but not in large pseudoaneurysms; TGF-β1 receptors were increased in small pseudoaneurysms and preserved in large pseudoaneurysms. Delivery of TGF-β1 via nanoparticles covalently bonded to the patch stimulated smad2 phosphorylation both in vitro and in vivo and significantly decreased pseudoaneurysm formation (6.7%). Inhibition of TGF-β1 signaling with SB431542 decreased smad2 phosphorylation both in vitro and in vivo and significantly induced pseudoaneurysm formation by day 7 (66.7%).Conclusions—Normal healing after aortic patch angioplasty is associated with increased TGF-β1 signaling, and recruitment of smad2 signaling may limit pseudoaneurysm formation; loss of TGF-β1 signaling is associated with the formation of large pseudoaneurysms. Enhancement of TGF-β1 signaling may be a potential mechanism to limit pseudoaneurysm formation after vascular intervention.



Apo A-I (Apolipoprotein A-I) Vascular Gene Therapy Provides Durable Protection Against Atherosclerosis in Hyperlipidemic RabbitsHighlights [Translational Sciences]

2017-12-27T10:40:49-08:00

Objective—Gene therapy that expresses apo A-I (apolipoprotein A-I) from vascular wall cells has promise for preventing and reversing atherosclerosis. Previously, we reported that transduction of carotid artery endothelial cells with a helper-dependent adenoviral (HDAd) vector expressing apo A-I reduced early (4 weeks) fatty streak development in fat-fed rabbits. Here, we tested whether the same HDAd could provide long-term protection against development of more complex lesions.Approach and Results—Fat-fed rabbits (n=25) underwent bilateral carotid artery gene transfer, with their left and right common carotids randomized to receive either a control vector (HDAdNull) or an apo A-I–expressing vector (HDAdApoAI). Twenty-four additional weeks of high-fat diet yielded complex intimal lesions containing lipid-rich macrophages as well as smooth muscle cells, often in a lesion cap. Twenty-four weeks after gene transfer, high levels of apo A-I mRNA (median ≥250-fold above background) were present in all HDAdApoAI-treated arteries. Compared with paired control HDAdNull-treated arteries in the same rabbit, HDAdApoAI-treated arteries had 30% less median intimal lesion volume (P=0.03), with concomitant reductions (23%–32%) in intimal lipid, macrophage, and smooth muscle cell content (P≤0.05 for all). HDAdApoAI-treated arteries also had decreased intimal inflammatory markers. VCAM-1 (vascular cell adhesion molecule-1)–stained area was reduced by 36% (P=0.03), with trends toward lower expression of ICAM-1 (intercellular adhesion molecule-1), MCP-1 (monocyte chemoattractant protein 1), and TNF-α (tumor necrosis factor-α; 13%–39% less; P=0.06–0.1).Conclusions—In rabbits with severe hyperlipidemia, transduction of vascular endothelial cells with an apo A-I–expressing HDAd yields at least 24 weeks of local apo A-I expression that durably reduces atherosclerotic lesion growth and intimal inflammation.



{Delta}-5 Fatty Acid Desaturase FADS1 Impacts Metabolic Disease by Balancing Proinflammatory and Proresolving Lipid MediatorsHighlights [Translational Sciences]

2017-12-27T10:40:49-08:00

Objective—Human genetic variants near the FADS (fatty acid desaturase) gene cluster (FADS1-2-3) are strongly associated with cardiometabolic traits including dyslipidemia, fatty liver, type 2 diabetes mellitus, and coronary artery disease. However, mechanisms underlying these genetic associations are unclear.Approach and Results—Here, we specifically investigated the physiological role of the Δ-5 desaturase FADS1 in regulating diet-induced cardiometabolic phenotypes by treating hyperlipidemic LDLR (low-density lipoprotein receptor)-null mice with antisense oligonucleotides targeting the selective knockdown of Fads1. Fads1 knockdown resulted in striking reorganization of both ω-6 and ω-3 polyunsaturated fatty acid levels and their associated proinflammatory and proresolving lipid mediators in a highly diet-specific manner. Loss of Fads1 activity promoted hepatic inflammation and atherosclerosis, yet was associated with suppression of hepatic lipogenesis. Fads1 knockdown in isolated macrophages promoted classic M1 activation, whereas suppressing alternative M2 activation programs, and also altered systemic and tissue inflammatory responses in vivo. Finally, the ability of Fads1 to reciprocally regulate lipogenesis and inflammation may rely in part on its role as an effector of liver X receptor signaling.Conclusions—These results position Fads1 as an underappreciated regulator of inflammation initiation and resolution, and suggest that endogenously synthesized arachidonic acid and eicosapentaenoic acid are key determinates of inflammatory disease progression and liver X receptor signaling.



Adventitial Sca1+ Cells Transduced With ETV2 Are Committed to the Endothelial Fate and Improve Vascular Remodeling After InjuryHighlights [Translational Sciences]

2017-12-27T10:40:49-08:00

Objective—Vascular adventitial Sca1+ (stem cell antigen-1) progenitor cells preferentially differentiate into smooth muscle cells, which contribute to vascular remodeling and neointima formation in vessel grafts. Therefore, directing the differentiation of Sca1+ cells toward the endothelial lineage could represent a new therapeutic strategy against vascular disease.Approach and Results—We thus developed a fast, reproducible protocol based on the single-gene transfer of ETV2 (ETS variant 2) to differentiate Sca1+ cells toward the endothelial fate and studied the effect of cell conversion on vascular hyperplasia in a model of endothelial injury. After ETV2 transduction, Sca1+ adventitial cells presented a significant increase in the expression of early endothelial cell genes, including VE-cadherin, Flk-1, and Tie2 at the mRNA and protein levels. ETV2 overexpression also induced the downregulation of a panel of smooth muscle cell and mesenchymal genes through epigenetic regulations, by decreasing the expression of DNA-modifying enzymes ten-eleven translocation dioxygenases. Adventitial Sca1+ cells grafted on the adventitial side of wire-injured femoral arteries increased vascular wall hyperplasia compared with control arteries with no grafted cells. Arteries seeded with ETV2-transduced cells, on the contrary, showed reduced hyperplasia compared with control.Conclusions—These data give evidence that the genetic manipulation of vascular progenitors is a promising approach to improve vascular function after endothelial injury.



Changes in High-Density Lipoprotein Cholesterol Efflux Capacity After Bariatric Surgery Are Procedure DependentHighlights [Clinical and Population Studies]

2017-12-27T10:40:49-08:00

Objective—High-density lipoprotein cholesterol efflux capacity (CEC) is inversely associated with incident cardiovascular events, independent of high-density lipoprotein cholesterol. Obesity is often characterized by impaired high-density lipoprotein function. However, the effects of different bariatric surgical techniques on CEC have not been compared. This study sought to determine the effects of Roux-en-Y gastric bypass (RYGB) and sleeve gastrectomy (SG) on CEC.Approach and Results—We prospectively studied severely obese, nondiabetic, premenopausal Hispanic women not using lipid medications undergoing RYGB (n=31) or SG (n=36). Subjects were examined before and at 6 and 12 months after surgery. There were no differences in baseline characteristics between surgical groups. Preoperative CEC correlated most strongly with Apo A1 (apolipoprotein A1) concentration but did not correlate with body mass index, waist:hip, high-sensitivity C-reactive protein, or measures of insulin resistance. After 6 months, SG produced superior response in high-density lipoprotein cholesterol and Apo A1 quantity, as well as global and non-ABCA1 (ATP-binding cassette transporter A1)–mediated CEC (P=0.048, P=0.018, respectively) versus RYGB. In multivariable regression models, only procedure type was predictive of changes in CEC (P=0.05). At 12 months after SG, CEC was equivalent to that of normal body mass index control subjects, whereas it remained impaired after RYGB.Conclusions—SG and RYGB produce similar weight loss, but contrasting effects on CEC. These findings may be relevant in discussions about the type of procedure that is most appropriate for a particular obese patient. Further study of the mechanisms underlying these changes may lead to improved understanding of the factors governing CEC and potential therapeutic interventions to maximally reduce cardiovascular disease risk in both obese and nonobese patients.



Stable Isotope Kinetic Study of ApoM (Apolipoprotein M)Highlights [Clinical and Population Studies]

2017-12-27T10:40:49-08:00

Objective—ApoM (apolipoprotein M) binds primarily to high-density lipoprotein before to be exchanged with apoB (apolipoprotein B)–containing lipoproteins. Low-density lipoprotein (LDL) receptor–mediated clearance of apoB-containing particles could influence plasma apoM kinetics and decrease its antiatherogenic properties. In humans, we aimed to describe the interaction of apoM kinetics with other components of lipid metabolism to better define its potential benefit on atherosclerosis.Approach and Results—Fourteen male subjects received a primed infusion of 2H3-leucine for 14 hours, and analyses were performed by liquid chromatography–tandem mass spectrometry from the hourly plasma samples. Fractional catabolic rates and production rates within lipoproteins were calculated using compartmental models. ApoM was found not only in high-density lipoprotein (59%) and LDL (4%) but also in a non–lipoprotein-related compartment (37%). The apoM distribution was heterogeneous within LDL and non–lipoprotein-related compartments according to plasma triglycerides (r=0.86; P<0.001). The relationships between sphingosine-1-phosphate and apoM were confirmed in all compartments (r range, 0.55–0.89; P<0.05). ApoM fractional catabolic rates and production rates were 0.16±0.07 pool/d and 0.14±0.06 mg/kg per day in high-density lipoprotein and 0.56±0.10 pool/d and 0.03±0.01 mg/kg per day in LDL, respectively. Fractional catabolic rates of LDL-apoM and LDL-apoB100 were correlated (r=0.55; P=0.042). Significant correlations were found between triglycerides and production rates of LDL-apoM (r=0.73; P<0.004).Conclusions—In humans, LDL kinetics play a key role in apoM turnover. Plasma triglycerides act on both apoM and sphingosine-1-phosphate distributions between lipoproteins. These results confirmed that apoM could be bound to high-density lipoprotein after secretion and then quickly exchanged with a non–lipoprotein-related compartment and to LDL to be slowly catabolized.



Evaluation of the Pleiotropic Effects of StatinsHighlights [Clinical and Population Studies]

2017-12-27T10:40:49-08:00

Objective—To reanalyze data from recent randomized trials of statins to assess whether the benefits and risks of statins are mediated primarily via their LDL-C (low-density lipoprotein cholesterol) lowering effects or via other mechanisms.Approach and Results—We adapted Egger regression, a technique frequently used in Mendelian randomization studies to detect genetic pleiotropy, to reanalyze the available randomized control trial data of statin therapy. For cardiovascular end points, each 1 mmol/L change in LDL-C with statin therapy was associated with a hazard ratio of 0.77 (95% confidence interval, 0.71–0.84) with an intercept that was indistinguishable from zero (intercept, −0.0032; [95% confidence interval, −0.090 to 0.084]; P=0.94), indicating no pleiotropy. For incident diabetes mellitus, a 1 mmol/L change in LDL-C with statin therapy was associated with a hazard ratio of 1.07 (95% confidence interval, 0.99–1.16) and an intercept nondistinguishable from zero (intercept, −0.015; [95% confidence interval, −0.30 to 0.27]; P=0.91), again indicating no pleiotropy.Conclusions—Our reanalysis of the randomized control trial data using Egger regression adds to the existing evidence that the cardiovascular benefits of statins and their association with incident diabetes mellitus are mediated primarily, if not entirely, via their LDL-C lowering properties rather than by any pleiotropic effects.



Elevated Plasma Factor IXa Activity in Premenopausal Women on Hormonal ContraceptionHighlights [Clinical and Population Studies]

2017-12-27T10:40:49-08:00

Objective—Combined oral contraceptives induce a reversible hypercoagulable state with an enhanced risk of venous thromboembolism, but the underlying mechanism(s) remain unclear. Subjects on combined oral contraceptives also demonstrate a characteristic resistance to APC (activated protein C) in the thrombin generation assay. Here, we report the potential role of plasma factor IXa (FIXa) as a mechanism for hormone-induced systemic hypercoagulability.Approach and Results—A novel assay was used to determine FIXa activity in plasma samples from volunteer blood donors. Plasma from 36 premenopausal females on hormonal contraception and 35 not on hormonal contraception, 35 postmenopausal females, and 10 males were analyzed for FIXa activity, total PS (protein S), total tissue factor pathway inhibitor (TFPI), and TFPI-α antigen. Premenopausal females on hormonal contraception demonstrated significantly increased FIXa activity and decreased TFPI-α compared with the other groups. Remarkably, FIXa values were not normally distributed in the hormonal contraception group, but skewed toward the high end. Plasma FIXa activity inversely correlated with both TFPI-α and total PS antigen. Ex vivo determination of TF-dependent FIX activation in FV-deficient plasma demonstrated that inhibitory anti-TFPI antibodies enhanced FIXa generation by 2- to 3-fold, whereas addition of 75 nmol/L PS reduced FIXa generation by ≈2-fold. Further, increasing FIXa concentration enhanced APC resistance during TF-triggered plasma thrombin generation.Conclusions—Elevation of plasma FIXa activity in association with reductions in TFPI-α and PS is a potential mechanism for systemic hypercoagulability and resistance to APC in premenopausal females on hormonal contraception.



Associations Between Residential Proximity to Traffic and Vascular Disease in a Cardiac Catheterization CohortHighlights [Clinical and Population Studies]

2017-12-27T10:40:49-08:00

Objective—Exposure to mobile source emissions is nearly ubiquitous in developed nations and is associated with multiple adverse health outcomes. There is an ongoing need to understand the specificity of traffic exposure associations with vascular outcomes, particularly in individuals with cardiovascular disease.Approach and Results—We performed a cross-sectional study using 2124 individuals residing in North Carolina, United States, who received a cardiac catheterization at the Duke University Medical Center. Traffic-related exposure was assessed via 2 metrics: (1) the distance between the primary residence and the nearest major roadway; and (2) location of the primary residence in regions defined based on local traffic patterns. We examined 4 cardiovascular disease outcomes: hypertension, peripheral arterial disease, the number of diseased coronary vessels, and recent myocardial infarction. Statistical models were adjusted for race, sex, smoking, type 2 diabetes mellitus, body mass index, hyperlipidemia, and home value. Results are expressed in terms of the odds ratio (OR). A 23% decrease in residential distance to major roadways was associated with higher prevalence of peripheral arterial disease (OR=1.29; 95% confidence interval, 1.08–1.55) and hypertension (OR=1.15; 95% confidence interval, 1.01–1.31). Associations with peripheral arterial disease were strongest in men (OR=1.42; 95% confidence interval, 1.17–1.74) while associations with hypertension were strongest in women (OR=1.21; 95% confidence interval, 0.99–1.49). Neither myocardial infarction nor the number of diseased coronary vessels were associated with traffic exposure.Conclusions—Traffic-related exposure is associated with peripheral arterial disease and hypertension while no associations are observed for 2 coronary-specific vascular outcomes.






Platelet-Specific p38{alpha} Deficiency Improved Cardiac Function After Myocardial Infarction in MiceHighlights [Translational Sciences]

2017-11-21T10:40:43-08:00

Objective—MAPKs (mitogen-activated protein kinases), especially p38, play detrimental roles in cardiac diseases and cardiac remodeling post-myocardial infarction. However, the activation and function of MAPKs in coronary thrombosis in vivo and its relationship with clinical outcomes remain poorly understood.Approach and Results—Here, we showed that p38α was the major isoform expressed in human and mouse platelets. Platelet-specific p38α-deficient mice presented impaired thrombosis and hemostasis but had improved cardiac function, reduced infarct size, decreased inflammatory response, and microthrombus in a left anterior descending artery ligation model. Signaling analysis revealed that p38 activation was one of the earliest events in platelets after treatment with receptor agonists or reactive oxygen species. p38α/MAPK-activated protein kinase 2/heat shock protein 27 and p38α/cytosolic phospholipases A2 were the major pathways regulating receptor-mediated or hydrogen peroxide-induced platelet activation in an ischemic environment. Moreover, the distinct roles of ERK1/2 (extracellular signal–regulated kinase) in receptor- or reactive oxygen species-induced p38-mediated platelet activation reflected the complicated synergistic relationships among MAPKs. Analysis of clinical samples revealed that MAPKs were highly phosphorylated in platelets from preoperative patients with ST-segment–elevation myocardial infarction, and increased phosphorylation of p38 was associated with no-reflow outcomes.Conclusions—We conclude that p38α serves as a critical regulator of platelet activation and potential indicator of highly thrombotic lesions and no-reflow, and inhibition of platelet p38α may improve clinical outcomes in subjects with ST-segment–elevation myocardial infarction.












Local Hemodynamic Forces After StentingHighlights [Brief Reviews]

2017-11-21T10:40:43-08:00

Local hemodynamic forces are well-known to modulate atherosclerotic evolution, which remains one of the largest cause of death worldwide. Percutaneous coronary interventions with stent implantation restores blood flow to the downstream myocardium and is only limited by stent failure caused by restenosis, stent thrombosis, or neoatherosclerosis. Cumulative evidence has shown that local hemodynamic forces affect restenosis and the platelet activation process, modulating the pathophysiological mechanisms that lead to stent failure. This article first covers the pathophysiological mechanisms through which wall shear stress regulates arterial disease formation/neointima proliferation and the role of shear rate on stent thrombosis. Subsequently, the article reviews the current evidence on (1) the implications of stent design on the local hemodynamic forces, and (2) how stent/scaffold expansion can influence local flow, thereby affecting the risk of adverse events.



Understanding Chylomicron Retention Disease Through Sar1b Gtpase Gene DisruptionHighlights [Basic Sciences]

2017-11-21T10:40:43-08:00

Background—Understanding the specific mechanisms of rare autosomal disorders has greatly expanded insights into the complex processes regulating intestinal fat transport. Sar1B GTPase is one of the critical proteins governing chylomicron secretion by the small intestine, and its mutations lead to chylomicron retention disease, despite the presence of Sar1A paralog.Objective—The central aim of this work is to examine the cause–effect relationship between Sar1B expression and chylomicron output and to determine whether Sar1B is obligatory for normal high-density lipoprotein biogenesis.Approach and Results—The SAR1B gene was totally silenced in Caco-2/15 cells using the zinc finger nuclease technique. SAR1B deletion resulted in significantly decreased secretion of triglycerides (≈40%), apolipoprotein B-48 (≈57%), and chylomicron (≈34.5%). The absence of expected chylomicron production collapse may be because of the compensatory SAR1A elevation observed in our experiments. Therefore, a double knockout of SAR1A and SAR1B was engineered in Caco-2/15 cells, which led to almost complete inhibition of triglycerides, apolipoprotein B-48, and chylomicron output. Further experiments with labeled cholesterol revealed the downregulation of high-density lipoprotein biogenesis in cells deficient in SAR1B or with the double knockout of the 2 SAR1 paralogs. Similarly, there was a fall in the movement of labeled cholesterol from cells to basolateral medium containing apolipoprotein A-I, thereby limiting newly synthesized high-density lipoprotein in genetically modified cells. The decreased cholesterol efflux was associated with impaired expression of ABCA1 (ATP-binding cassette subfamily A member 1).Conclusions—These findings demonstrate that the deletion of the 2 SAR1 isoforms is required to fully eliminate the secretion of chylomicron in vitro. They also underscore the limited high-density lipoprotein production by the intestinal cells in response to SAR1 knockout.



GLP-1 Elicits an Intrinsic Gut-Liver Metabolic Signal to Ameliorate Diet-Induced VLDL Overproduction and Insulin ResistanceHighlights [Basic Sciences]

2017-11-21T10:40:43-08:00

Objective—Perturbations in hepatic lipid and very-low–density lipoprotein (VLDL) metabolism are involved in the pathogenesis of obesity and hepatic insulin resistance. The objective of this study is to delineate the mechanism of subdiaphragmatic vagotomy in preventing obesity, hyperlipidemia, and insulin resistance.Approach and Results—By subjecting the complete subdiaphragmatic vagotomized mice to various nutritional conditions and investigating hepatic de novo lipogenesis pathway, we found that complete disruption of subdiaphragmatic vagal signaling resulted in a significant decrease of circulating VLDL–triglyceride compared with the mice obtained sham procedure. Vagotomy further prevented overproduction of VLDL–triglyceride induced by an acute fat load and a high-fat diet–induced obesity, hyperlipidemia, hepatic steatosis, and glucose intolerance. Mechanistic studies revealed that plasma glucagon-like peptide-1 was significantly raised in the vagotomized mice, which was associated with significant reductions in mRNA and protein expression of SREBP-1c (sterol regulatory element-binding protein 1c), SCD-1 (stearoyl-CoA desaturase-1), and FASN (fatty acid synthase), as well as enhanced hepatic insulin sensitivity. In vitro, treating mouse primary hepatocytes with a glucagon-like peptide-1 receptor agonist, exendin-4, for 48 hours inhibited free fatty acid, palmitic acid treatment induced de novo lipid synthesis, and VLDL secretion from hepatocytes.Conclusions—Elevation of glucagon-like peptide-1 in vagotomized mice may prevent VLDL overproduction and insulin resistance induced by high-fat diet. These novel findings, for the first time, delineate an intrinsic gut–liver regulatory circuit that is mediated by glucagon-like peptide-1 in regulating hepatic energy metabolism.



ABCA1-Derived Nascent High-Density Lipoprotein-Apolipoprotein AI and Lipids Metabolically SegregateHighlights [Basic Sciences]

2017-11-21T10:40:43-08:00

Objective—Reverse cholesterol transport comprises cholesterol efflux from ABCA1-expressing macrophages to apolipoprotein (apo) AI, giving nascent high-density lipoprotein (nHDL), esterification of nHDL-free cholesterol (FC), selective hepatic extraction of HDL lipids, and hepatic conversion of HDL cholesterol to bile salts, which are excreted. We tested this model by identifying the fates of nHDL-[3H]FC, [14C] phospholipid (PL), and [125I]apo AI in serum in vitro and in vivo.Approach and Results—During in vitro incubation of human serum, nHDL-[3H]FC and [14C]PL rapidly transfer to HDL and low-density lipoproteins (t1/2=2–7 minutes), whereas nHDL-[125I]apo AI transfers solely to HDL (t1/2<10 minutes) and to the lipid-free form (t1/2>480 minutes). After injection into mice, nHDL-[3H]FC and [14C]PL rapidly transfer to liver (t1/2=≈2–3 minutes), whereas apo AI clears with t1/2=≈460 minutes. The plasma nHDL-[3H]FC esterification rate is slow (0.46%/h) compared with hepatic uptake. PL transfer protein enhances nHDL-[14C]PL but not nHDL-[3H]FC transfer to cultured Huh7 hepatocytes.Conclusions—nHDL-FC, PL, and apo AI enter different pathways in vivo. Most nHDL-[3H]FC and [14C]PL are rapidly extracted by the liver via SR-B1 (scavenger receptor class B member 1) and spontaneous transfer; hepatic PL uptake is promoted by PL transfer protein. nHDL-[125I]apo AI transfers to HDL and to the lipid-free form that can be recycled to nHDL formation. Cholesterol esterification by lecithin:cholesterol acyltransferase is a minor process in nHDL metabolism. These findings could guide the design of therapies that better mobilize peripheral tissue-FC to hepatic disposal.



Refrigeration-Induced Binding of von Willebrand Factor Facilitates Fast Clearance of Refrigerated PlateletsHighlights [Basic Sciences]

2017-11-21T10:40:43-08:00

Objective—Apheresis platelets for transfusion treatment are currently stored at room temperature because after refrigeration platelets are rapidly cleared on transfusion. In this study, the role of von Willebrand factor (VWF) in the clearance of refrigerated platelets is addressed.Approach and Results—Human and murine platelets were refrigerated in gas-permeable bags at 4°C for 24 hours. VWF binding, platelet signaling events, and platelet post-transfusion recovery and survival were measured. After refrigeration, the binding of plasma VWF to platelets was drastically increased, confirming earlier studies. The binding was blocked by peptide OS1 that bound specifically to platelet glycoprotein (GP)Ibα and was absent in VWF−/− plasma. Although surface expression of GPIbα was reduced after refrigeration, refrigeration-induced VWF binding under physiological shear induced unfolding of the GPIbα mechanosensory domain on the platelet, as evidenced by increased exposure of a linear epitope therein. Refrigeration and shear treatment also induced small elevation of intracellular Ca2+, phosphatidylserine exposure, and desialylation of platelets, which were absent in VWF−/− platelets or inhibited by OS1, which is a monomeric 11-residue peptide (CTERMALHNLC). Furthermore, refrigerated VWF−/− platelets displayed increased post-transfusion recovery and survival than wild-type ones. Similarly, adding OS1 to transgenic murine platelets expressing only human GPIbα during refrigeration improved their post-transfusion recovery and survival.Conclusions—Refrigeration-induced binding of VWF to platelets facilitates their rapid clearance by inducing GPIbα-mediated signaling. Our results suggest that inhibition of the VWF–GPIbα interaction may be a potential strategy to enable refrigeration of platelets for transfusion treatment.



NO Augments Endothelial Reactivity by Reducing Myoendothelial Calcium Signal SpreadingHighlights [Basic Sciences]

2017-11-21T10:40:43-08:00

Objective—Because of its strategic position between endothelial and smooth muscle cells in microvessels, Cx37 (Connexin 37) plays an important role in myoendothelial gap junctional intercellular communication. We have shown before that NO inhibits gap junctional intercellular communication through gap junctions containing Cx37. However, the underlying mechanism is not yet identified.Approach and Results—Using channel-forming Cx37 mutants exhibiting partial deletions or amino acid exchanges in their C-terminal loops, we now show that the phosphorylation state of a tyrosine residue at position 332 (Y332) in the C-terminus of Cx37 controls the gap junction–dependent spread of calcium signals. Mass spectra revealed that NO protects Cx37 from dephosphorylation at Y332 by inhibition of the protein tyrosine phosphatase SHP-2. Functionally, the inhibition of gap junctional intercellular communication by NO decreased the spread of the calcium signal (induced by mechanical stimulation of individual endothelial cells) from endothelial to smooth muscle cells in intact vessels, while, at the same time, augmenting the calcium signal spreading within the endothelium. Consequently, preincubation of small resistance arteries with exogenous NO enhanced the endothelium-dependent dilator response to acetylcholine in spite of a pharmacological blockade of NO-dependent cGMP formation by the soluable guanylyl cyclase inhibitor ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one).Conclusions—Our results identify a novel mechanism by which NO can increase the efficacy of calcium, rising vasoactive agonists in the microvascular endothelium.



Deletion of AT2 Receptor Prevents SHP-1-Induced VEGF Inhibition and Improves Blood Flow Reperfusion in Diabetic Ischemic HindlimbHighlights [Basic Sciences]

2017-11-21T10:40:43-08:00

Objective—Ischemia caused by narrowing of femoral artery is a major cause of peripheral arterial disease and morbidity affecting patients with diabetes mellitus. We have previously reported that the inhibition of the angiogenic response to VEGF (vascular endothelial growth factor) in diabetic mice was associated with the increased expression of SHP-1 (SH2 domain–containing phosphatase 1), a protein that can be activated by the AT2 (angiotensin II type 2) receptor. Deletion of AT2 receptor has been shown to promote angiogenesis within the ischemic muscle. However, the relative impact of AT2 receptor in diabetic condition remains unknown.Approach and Results—Nondiabetic and diabetic AT2 null (Atgr2−/Y) mice underwent femoral artery ligation after 2 months of diabetes mellitus. Blood perfusion was measured every week ≤4 weeks post-surgery. Expression of the VEGF, SHP-1, and renin–angiotensin pathways was evaluated. Blood flow in the ischemic muscle of diabetic Atgr2−/Y mice recovered faster and ≤80% after 4 weeks compared with 51% recovery in diabetic control littermates. Diabetic Atgr2−/Y had reduced apoptotic endothelial cells and elevated small vessel formation compared with diabetic Atgr2+/Y mice, as well as increased SHP-1 expression and reduced VEGF receptor activity. In endothelial cells, high glucose levels and AT2 agonist treatment did not change SHP-1, VEGF, and VEGF receptor expression. However, the activity of SHP-1 and its association with the VEGF receptors were increased, causing inhibition of the VEGF action in endothelial cell proliferation and migration.Conclusions—Our results suggest that the deletion of AT2 receptor reduced SHP-1 activity and restored VEGF actions, leading to an increased blood flow reperfusion after ischemia in diabetes mellitus.



FGD5 Regulates VEGF Receptor-2 Coupling to PI3 Kinase and Receptor RecyclingHighlights [Basic Sciences]

2017-11-21T10:40:43-08:00

Objective—VEGF (vascular endothelial growth factor-A) signaling to the endothelial cell (EC) through VEGFR2 (VEGF receptor-2) is the principal cue driving new blood vessel formation. FGD5 (faciogenital dysplasia-5)—a Rho-family guanine nucleotide exchange factor—is selectively expressed in EC. Deficiency of FGD5 is embryonically lethal in mice and perturbs angiogenesis and VEGF signal transduction. However, the mechanism of FGD5 regulation of VEGF signaling is poorly understood.Approach and Results—Angiogenic sprouting and EC cytoskeletal remodeling were evaluated in a 3-dimensional in vitro model. We examined the subcellular localization of FGD5 and VEGFR2 in EC by immunofluorescent staining and studied the association by immunoprecipitation. FGD5 deficiency reduced the number of angiogenic sprouts and tip cell filopodia by ≈80% and ≈70%, respectively. These defects were accompanied by downregulation of the expression of tip cell-specific markers. FGD5 inactivation led to a decrease in EC migration and early protrusion (lamellipodia) formation. In resting and VEGF-stimulated EC, FGD5 forms a complex with VEGFR2 and was enriched at the leading edge of the cell and among endosomes. FGD5 loss reduced mTORC2 (mammalian target of rapamycin complex-2)/Akt-dependent cortactin activation downstream of VEGFR2 but did not alter VEGFR2 plasma membrane expression, Y1175 phosphorylation, or endocytosis. However, FGD5 loss decreased endosomal VEGFR2 coupling to phosphoinositide-3 kinase and diverted VEGFR2 to lysosomal degradation.Conclusions—FGD5 regulates VEGFR2 retention in recycling endosomes and coupling to PI3 (phosphoinositide-3) kinase/mTORC2-dependent cytoskeletal remodeling.



Opposing Actions of AKT (Protein Kinase B) Isoforms in Vascular Smooth Muscle Injury and Therapeutic ResponseHighlights [Basic Sciences]

2017-11-21T10:40:43-08:00

Objective—Drug-eluting stent delivery of mTORC1 (mechanistic target of rapamycin complex 1) inhibitors is highly effective in preventing intimal hyperplasia after coronary revascularization, but adverse effects limit their use for systemic vascular disease. Understanding the mechanism of action may lead to new treatment strategies. We have shown that rapamycin promotes vascular smooth muscle cell differentiation in an AKT2-dependent manner in vitro. Here, we investigate the roles of AKT (protein kinase B) isoforms in intimal hyperplasia.Approach and Results—We found that germ-line–specific or smooth muscle–specific deletion of Akt2 resulted in more severe intimal hyperplasia compared with control mice after arterial denudation injury. Conversely, smooth muscle–specific Akt1 knockout prevented intimal hyperplasia, whereas germ-line Akt1 deletion caused severe thrombosis. Notably, rapamycin prevented intimal hyperplasia in wild-type mice but had no therapeutic benefit in Akt2 knockouts. We identified opposing roles for AKT1 and AKT2 isoforms in smooth muscle cell proliferation, migration, differentiation, and rapamycin response in vitro. Mechanistically, rapamycin induced MYOCD (myocardin) mRNA expression. This was mediated by AKT2 phosphorylation and nuclear exclusion of FOXO4 (forkhead box O4), inhibiting its binding to the MYOCD promoter.Conclusions—Our data reveal opposing roles for AKT isoforms in smooth muscle cell remodeling. AKT2 is required for rapamycin’s therapeutic inhibition of intimal hyperplasia, likely mediated in part through AKT2-specific regulation of MYOCD via FOXO4. Because AKT2 signaling is impaired in diabetes mellitus, this work has important implications for rapamycin therapy, particularly in diabetic patients.



Mitochondrial Respiration Is Reduced in Atherosclerosis, Promoting Necrotic Core Formation and Reducing Relative Fibrous Cap ThicknessHighlights [Translational Sciences]

2017-11-21T10:40:43-08:00

Objective—Mitochondrial DNA (mtDNA) damage is present in murine and human atherosclerotic plaques. However, whether endogenous levels of mtDNA damage are sufficient to cause mitochondrial dysfunction and whether decreasing mtDNA damage and improving mitochondrial respiration affects plaque burden or composition are unclear. We examined mitochondrial respiration in human atherosclerotic plaques and whether augmenting mitochondrial respiration affects atherogenesis.Approach and Results—Human atherosclerotic plaques showed marked mitochondrial dysfunction, manifested as reduced mtDNA copy number and oxygen consumption rate in fibrous cap and core regions. Vascular smooth muscle cells derived from plaques showed impaired mitochondrial respiration, reduced complex I expression, and increased mitophagy, which was induced by oxidized low-density lipoprotein. Apolipoprotein E–deficient (ApoE−/−) mice showed decreased mtDNA integrity and mitochondrial respiration, associated with increased mitochondrial reactive oxygen species. To determine whether alleviating mtDNA damage and increasing mitochondrial respiration affects atherogenesis, we studied ApoE−/− mice overexpressing the mitochondrial helicase Twinkle (Tw+/ApoE−/−). Tw+/ApoE−/− mice showed increased mtDNA integrity, copy number, respiratory complex abundance, and respiration. Tw+/ApoE−/− mice had decreased necrotic core and increased fibrous cap areas, and Tw+/ApoE−/− bone marrow transplantation also reduced core areas. Twinkle increased vascular smooth muscle cell mtDNA integrity and respiration. Twinkle also promoted vascular smooth muscle cell proliferation and protected both vascular smooth muscle cells and macrophages from oxidative stress–induced apoptosis.Conclusions—Endogenous mtDNA damage in mouse and human atherosclerosis is associated with significantly reduced mitochondrial respiration. Reducing mtDNA damage and increasing mitochondrial respiration decrease necrotic core and increase fibrous cap areas independently of changes in reactive oxygen species and may be a promising therapeutic strategy in atherosclerosis.



Reduction of In-Stent Restenosis by Cholesteryl Ester Transfer Protein InhibitionHighlights [Translational Sciences]

2017-11-21T10:40:43-08:00

Objective—Angioplasty and stent implantation, the most common treatment for atherosclerotic lesions, have a significant failure rate because of restenosis. This study asks whether increasing plasma high-density lipoprotein (HDL) levels by inhibiting cholesteryl ester transfer protein activity with the anacetrapib analog, des-fluoro-anacetrapib, prevents stent-induced neointimal hyperplasia.Approach and Results—New Zealand White rabbits received normal chow or chow supplemented with 0.14% (wt/wt) des-fluoro-anacetrapib for 6 weeks. Iliac artery endothelial denudation and bare metal steel stent deployment were performed after 2 weeks of des-fluoro-anacetrapib treatment. The animals were euthanized 4 weeks poststent deployment. Relative to control, dietary supplementation with des-fluoro-anacetrapib reduced plasma cholesteryl ester transfer protein activity and increased plasma apolipoprotein A-I and HDL cholesterol levels by 53±6.3% and 120±19%, respectively. Non-HDL cholesterol levels were unaffected. Des-fluoro-anacetrapib treatment reduced the intimal area of the stented arteries by 43±5.6% (P[...]



Impact of CD14 Polymorphisms on Anti-Apolipoprotein A-1 IgG-Related Coronary Artery Disease Prediction in the General PopulationHighlights [Clinical and Population Studies]

2017-11-21T10:40:43-08:00

Objective—We aimed to determine whether autoantibodies against apoA-1 (apolipoprotein A-1; anti-apoA-1 IgG) predict incident coronary artery disease (CAD), defined as adjudicated incident myocardial infarction, angina, percutaneous coronary revascularization, or bypass grafting, in the general population. We further investigated whether this association is modulated by a functional CD14 receptor single nucleotide polymorphism.Approach and Results—In a prospectively studied, population-based cohort of 5220 subjects (mean age 52.6±10.7 years, 47.4% males), followed over a median period of 5.6 years, subjects positive versus negative for anti-apoA-1 IgG presented a total CAD rate of 3.9% versus 2.8% (P=0.077) and a nonfatal CAD rate of 3.6% versus 2.3% (P=0.018), respectively. After multivariate adjustment for established cardiovascular risk factors, the hazard ratios of anti-apoA-1 IgG for total and nonfatal CAD were: hazard ratio=1.36 (95% confidence interval, 0.94–1.97; P=0.105) and hazard ratio=1.53 (95% confidence interval, 1.03–2.26; P=0.034), respectively. In subjects with available genetic data for the C260T rs2569190 single nucleotide polymorphism in the CD14 receptor gene (n=4247), we observed a significant interaction between anti-apoA-1 IgG and rs2569190 allele status with regards to CAD risk, with anti-apoA-1 IgG conferring the highest risk for total and nonfatal CAD in non-TT carriers, whereas being associated with the lowest risk for total and nonfatal CAD in TT homozygotes (P for interaction =0.011 and P for interaction =0.033, respectively).Conclusions—Anti-apoA-1 IgG are independent predictors of nonfatal incident CAD in the general population. The strength of this association is dependent on a functional polymorphism of the CD14 receptor gene, a finding suggesting a gene–autoantibody interaction for the development of CAD.



Coronary Artery Ectasia Predicts Future Cardiac Events in Patients With Acute Myocardial InfarctionHighlights [Clinical and Population Studies]

2017-11-21T10:40:43-08:00

Objective—Coronary artery ectasia (CAE) is an infrequently observed vascular phenotype characterized by abnormal vessel dilatation and disturbed coronary flow, which potentially promote thrombogenicity and inflammatory reactions. However, whether or not CAE influences cardiovascular outcomes remains unknown.Approach and Results—We investigated major adverse cardiac events (MACE; defined as cardiac death and nonfatal myocardial infarction [MI]) in 1698 patients with acute MI. The occurrence of MACE was compared in patients with and without CAE. CAE was identified in 3.0% of study subjects. During the 49-month observation period, CAE was associated with 3.25-, 2.71-, and 4.92-fold greater likelihoods of experiencing MACE (95% confidence interval [CI], 1.88–5.66; P<0.001), cardiac death (95% CI, 1.37–5.37; P=0.004), and nonfatal MI (95% CI, 2.20–11.0; P<0.001), respectively. These cardiac risks of CAE were consistently observed in a multivariate Cox proportional hazards model (MACE: hazard ratio, 4.94; 95% CI, 2.36–10.4; P<0.001) and in a propensity score–matched cohort (MACE: hazard ratio, 8.98; 95% CI, 1.14–71.0; P=0.03). Despite having a higher risk of CAE-related cardiac events, patients with CAE receiving anticoagulation therapy who achieved an optimal percent time in target therapeutic range, defined as ≥60%, did not experience the occurrence of MACE (P=0.03 versus patients with percent time in target therapeutic range <60% or without anticoagulation therapy).Conclusions—The presence of CAE predicted future cardiac events in patients with acute MI. Our findings suggest that acute MI patients with CAE are a high-risk subset who might benefit from a pharmacological approach to controlling the coagulation cascade.



Relationship Between Total Serum Bilirubin Levels and Carotid and Femoral Atherosclerosis in Familial DyslipidemiaHighlights [Clinical and Population Studies]

2017-11-21T10:40:43-08:00

Objective—Bilirubin is a potent antioxidant that has been inversely related to cardiovascular disease. There is little information on serum total bilirubin (TB) in relation to atherosclerosis in familial dyslipidemia. We assessed the association between TB and carotid and femoral atherosclerosis in this high-risk group.Approach and Results—We evaluated 464 individuals with familial dyslipidemia (56% men; median age, 48 years), 322 with familial hypercholesterolemia, and 142 with familial combined hyperlipidemia. Carotid and femoral arteries were imaged bilaterally with a standardized ultrasonographic protocol. Mean and maximum intima-media thickness and plaque presence (≥1.2 mm) and height were recorded. Cross-sectional associations between TB and atherosclerosis variables were investigated in multivariable-adjusted models, including lipid values and hypolipidemic drug use. Inflammatory markers (C-reactive protein, total leukocyte count, and lipoprotein[a]) were also determined. Increasing TB levels were associated with decreasing intima-media thickness of all carotid segments (P<0.05, all). TB also related to carotid plaque, present in 78% of individuals, and to plaque burden (≥3 plaques), with odds ratios (95% confidence interval) 0.59 (0.36–0.98) and 0.57 (0.34–0.96) for each increase of 0.5 mg in TB, respectively. Findings were confirmed in a validation cohort of 177 subjects with nonfamilial dyslipidemia. Only the familial combined hyperlipidemia group, with higher inflammation-related markers, showed an inverse association between TB and femoral plaque height (β=−0.183; P=0.030).Conclusions—TB was inversely and independently associated with carotid plaque burden in familial and nonfamilial dyslipidemia. These findings support the use of TB as a biomarker of atherosclerosis in this high-risk group.



Endogenous Cholesterol Excretion Is Negatively Associated With Carotid Intima-Media Thickness in HumansHighlights [Clinical and Population Studies]

2017-11-21T10:40:43-08:00

Objective—Epidemiological studies strongly suggest that lipid factors independent of low-density lipoprotein cholesterol contribute significantly to cardiovascular disease risk. Because circulating lipoproteins comprise only a small fraction of total body cholesterol, the mobilization and excretion of cholesterol from plasma and tissue pools may be an important determinant of cardiovascular disease risk. Our hypothesis is that fecal excretion of endogenous cholesterol is protective against atherosclerosis.Approach and Results—Cholesterol metabolism and carotid intima–media thickness were quantitated in 86 nondiabetic adults. Plasma cholesterol was labeled by intravenous infusion of cholesterol-d7 solubilized in a lipid emulsion and dietary cholesterol by cholesterol-d5 and the nonabsorbable stool marker sitostanol-d4. Plasma and stool samples were collected while subjects consumed a cholesterol- and phytosterol-controlled metabolic kitchen diet and were analyzed by mass spectrometry. Carotid intima–media thickness was negatively correlated with fecal excretion of endogenous cholesterol (r=−0.426; P[...]



Intermuscular Adipose Tissue and Subclinical Coronary Artery Calcification in MidlifeHighlights [Clinical and Population Studies]

2017-11-21T10:40:43-08:00

Objective—Excess deposition of fat within and around vital organs and nonadipose tissues is hypothesized to contribute to cardiovascular disease (CVD) risk. We evaluated the association of abdominal intermuscular adipose tissue (IMAT) volume with coronary artery calcification in the CARDIA study (Coronary Artery Risk Development in Young Adults) participants.Approach and Results—We measured IMAT in the abdominal muscles, visceral adipose tissue and pericardial adipose tissue, and coronary artery calcification using computed tomography in 3051 CARDIA participants (56% women) at the CARDIA year 25 examination (2010–2011). Mean IMAT volume and mean IMAT/total muscle volume (IMAT normalized for muscle size) were calculated in a 10-mm block of slices centered at L3–L4. Multivariable analyses included potential confounders and traditional cardiovascular disease risk factors. Compared with the lowest quartile, the upper quartile of abdominal IMAT volume was associated with higher coronary artery calcification prevalence (odds ratio [95% confidence interval], 1.6 [1.2–2.1]) after adjusting for cardiovascular disease risk factors. Results were similar for highest versus lowest quartile of IMAT normalized to total muscle volume (odds ratio [95% confidence interval], 1.5 [1.1–2.0]). Significant associations of higher IMAT and normalized IMAT with coronary artery calcification prevalence persisted when body mass index, visceral adipose tissue, or pericardial adipose tissue were added to the models.Conclusions—In a large, community-based, cross-sectional study, we found that higher abdominal skeletal muscle adipose tissue volume was associated with subclinical atherosclerosis independent of traditional cardiovascular disease risk factors and other adipose depots.





















Beyond Impressions [Editorial]

2017-10-25T10:40:49-07:00







Modeling Inborn Errors of Hepatic Metabolism Using Induced Pluripotent Stem Cells [ATVB in Focus: iPSC-Derived Cell Types Relevant to Atherothrombosis]

2017-10-25T10:40:49-07:00

Inborn errors of hepatic metabolism are because of deficiencies commonly within a single enzyme as a consequence of heritable mutations in the genome. Individually such diseases are rare, but collectively they are common. Advances in genome-wide association studies and DNA sequencing have helped researchers identify the underlying genetic basis of such diseases. Unfortunately, cellular and animal models that accurately recapitulate these inborn errors of hepatic metabolism in the laboratory have been lacking. Recently, investigators have exploited molecular techniques to generate induced pluripotent stem cells from patients’ somatic cells. Induced pluripotent stem cells can differentiate into a wide variety of cell types, including hepatocytes, thereby offering an innovative approach to unravel the mechanisms underlying inborn errors of hepatic metabolism. Moreover, such cell models could potentially provide a platform for the discovery of therapeutics. In this mini-review, we present a brief overview of the state-of-the-art in using pluripotent stem cells for such studies.



Human Induced Pluripotent Stem Cell-Derived Macrophages for Unraveling Human Macrophage Biology [ATVB in Focus: iPSC-Derived Cell Types Relevant to Atherothrombosis]

2017-10-25T10:40:49-07:00

Despite a substantial appreciation for the critical role of macrophages in cardiometabolic diseases, understanding of human macrophage biology has been hampered by the lack of reliable and scalable models for cellular and genetic studies. Human induced pluripotent stem cell (iPSC)–derived macrophages (IPSDM), as an unlimited source of subject genotype-specific cells, will undoubtedly play an important role in advancing our understanding of the role of macrophages in human diseases. In this review, we summarize current literature in the differentiation and characterization of IPSDM at phenotypic, functional, and transcriptomic levels. We emphasize the progress in differentiating iPSC to tissue resident macrophages, and in understanding the ontogeny of in vitro differentiated IPSDM that resembles primitive hematopoiesis, rather than adult definitive hematopoiesis. We review the application of IPSDM in modeling both Mendelian genetic disorders and host–pathogen interactions. Finally, we highlighted the potential areas of research using IPSDM in functional validation of coronary artery disease loci in genome-wide association studies, functional genomic analyses, drug testing, and cell therapeutics in cardiovascular diseases.



Induced Pluripotent Stem Cell-Derived Megakaryocytes and Platelets for Disease Modeling and Future Clinical Applications [ATVB in Focus: iPSC-Derived Cell Types Relevant to Atherothrombosis]

2017-10-25T10:40:49-07:00

Platelets, derived from megakaryocytes, are anucleate cytoplasmic discs that circulate in the blood stream and play major roles in hemostasis, inflammation, and vascular biology. Platelet transfusions are used in a variety of medical settings to prevent life-threatening thrombocytopenia because of cancer therapy, other causes of acquired or inherited thrombocytopenia, and trauma. Currently, platelets used for transfusion purposes are donor derived. However, there is a drive to generate nondonor sources of platelets to help supplement donor-derived platelets. Efforts have been made by many laboratories to generate in vitro platelets and optimize their production and quality. In vitro-derived platelets have the potential to be a safer, more uniform product, and genetic manipulation could allow for better treatment of patients who become refractory to donor-derived units. This review focuses on potential clinical applications of in vitro-derived megakaryocytes and platelets, current methods to generate and expand megakaryocytes from pluripotent stem cell sources, and the use of these cells for disease modeling.



Differentiation, Evaluation, and Application of Human Induced Pluripotent Stem Cell-Derived Endothelial Cells [ATVB in Focus: iPSC-Derived Cell Types Relevant to Atherothrombosis]

2017-10-25T10:40:49-07:00

The emergence of induced pluripotent stem cell (iPSC) technology paves the way to generate large numbers of patient-specific endothelial cells (ECs) that can be potentially delivered for regenerative medicine in patients with cardiovascular disease. In the last decade, numerous protocols that differentiate EC from iPSC have been developed by many groups. In this review, we will discuss several common strategies that have been optimized for human iPSC-EC differentiation and subsequent studies that have evaluated the potential of human iPSC-EC as a cell therapy or as a tool in disease modeling. In addition, we will emphasize the importance of using in vivo vessel-forming ability and in vitro clonogenic colony–forming potential as a gold standard with which to evaluate the quality of human iPSC-EC derived from various protocols.



Differentiation and Application of Induced Pluripotent Stem Cell-Derived Vascular Smooth Muscle Cells [ATVB in Focus: iPSC-Derived Cell Types Relevant to Atherothrombosis]

2017-10-25T10:40:49-07:00

Vascular smooth muscle cells (VSMCs) play a role in the development of vascular disease, for example, neointimal formation, arterial aneurysm, and Marfan syndrome caused by genetic mutations in VSMCs, but little is known about the mechanisms of the disease process. Advances in induced pluripotent stem cell technology have now made it possible to derive VSMCs from several different somatic cells using a selection of protocols. As such, researchers have set out to delineate key signaling processes involved in triggering VSMC gene expression to grasp the extent of gene regulatory networks involved in phenotype commitment. This technology has also paved the way for investigations into diseases affecting VSMC behavior and function, which may be treatable once an identifiable culprit molecule or gene has been repaired. Moreover, induced pluripotent stem cell–derived VSMCs are also being considered for their use in tissue-engineered blood vessels as they may prove more beneficial than using autologous vessels. Finally, while several issues remains to be clarified before induced pluripotent stem cell–derived VSMCs can become used in regenerative medicine, they do offer both clinicians and researchers hope for both treating and understanding vascular disease. In this review, we aim to update the recent progress on VSMC generation from stem cells and the underlying molecular mechanisms of VSMC differentiation. We will also explore how the use of induced pluripotent stem cell–derived VSMCs has changed the game for regenerative medicine by offering new therapeutic avenues to clinicians, as [...]



Induced Pluripotent Stem Cell-Derived Endothelial Cells in Insulin Resistance and Metabolic Syndrome [ATVB in Focus: iPSC-Derived Cell Types Relevant to Atherothrombosis]

2017-10-25T10:40:49-07:00

Insulin resistance leads to a number of metabolic and cellular abnormalities including endothelial dysfunction that increase the risk of vascular disease. Although it has been particularly challenging to study the genetic determinants that predispose to abnormal function of the endothelium in insulin-resistant states, the possibility of deriving endothelial cells from induced pluripotent stem cells generated from individuals with detailed clinical phenotyping, including accurate measurements of insulin resistance accompanied by multilevel omic data (eg, genetic and genomic characterization), has opened new avenues to study this relationship. Unfortunately, several technical barriers have hampered these efforts. In the present review, we summarize the current status of induced pluripotent stem cell–derived endothelial cells for modeling endothelial dysfunction associated with insulin resistance and discuss the challenges to overcoming these limitations.



Scavenger Receptor CD36 Directs Nonclassical Monocyte Patrolling Along the Endothelium During Early Atherogenesis [Basic Sciences]

2017-10-25T10:40:49-07:00

Objective—Nonclassical monocytes (NCM) function to maintain vascular homeostasis by crawling or patrolling along the vessel wall. This subset of monocytes responds to viruses, tumor cells, and other pathogens to aid in protection of the host. In this study, we wished to determine how early atherogenesis impacts NCM patrolling in the vasculature.Approach and Results—To study the role of NCM in early atherogenesis, we quantified the patrolling behaviors of NCM in ApoE−/− (apolipoprotein E) and C57BL/6J mice fed a Western diet. Using intravital imaging, we found that NCM from Western diet–fed mice display a 4-fold increase in patrolling activity within large peripheral blood vessels. Both human and mouse NCM preferentially engulfed OxLDL (oxidized low-density lipoprotein) in the vasculature, and we observed that OxLDL selectively induced NCM patrolling in vivo. Induction of patrolling during early atherogenesis required scavenger receptor CD36, as CD36−/− mice revealed a significant reduction in patrolling activity along the femoral vasculature. Mechanistically, we found that CD36-regulated patrolling was mediated by a SFK (src family kinase) through DAP12 (DNAX activating protein of 12KDa) adaptor protein.Conclusions—Our studies show a novel pathway for induction of NCM patrolling along the vascular wall during early atherogenesis. Mice fed a Western diet showed increased NCM patrolling activity with a concurrent increase in SFK phosphorylation. This patrolling activity was lost in the absence of either CD36 or DAP12[...]



Perinatal Hypercholesterolemia Exacerbates Atherosclerosis Lesions in Offspring by Altering Metabolism of Trimethylamine-N-Oxide and Bile Acids [Basic Sciences]

2017-10-25T10:40:49-07:00

Objective—Experimental studies suggest that maternal hypercholesterolemia may be relevant for the early onset of cardiovascular disease in offspring. We investigated the effect of perinatal hypercholesterolemia on the atherosclerosis development in the offspring of apolipoprotein E–deficient mice and the underlying mechanism.Approach and Results—Atherosclerosis and related parameters were studied in adult male or female apolipoprotein E–deficient mice offspring from either normocholesterolemic or hypercholesterolemic mothers and normocholesterolemic fathers. Female born to hypercholesterolemic mothers had more aortic root lesions than female born to normocholesterolemic mothers. Lesions in whole aorta did not differ between groups. Higher trimethylamine-N-oxide levels and Fmo3 hepatic gene expression were higher in female born to hypercholesterolemic mothers offspring compared with female born to normocholesterolemic mothers and male. Trimethylamine-N-oxide levels were correlated with the size of atherosclerotic root lesions. Levels of hepatic cholesterol and gallbladder bile acid were greater in male born to hypercholesterolemic mothers compared with male born to normocholesterolemic mothers. At 18 weeks of age, female born to hypercholesterolemic mothers showed lower hepatic Scarb1 and Cyp7a1 but higher Nr1h4 gene expression compared with female born to normocholesterolemic mothers. Male born to hypercholesterolemic mothers showed an increase in Scarb1 and Ldlr gene expression compared with mal[...]



Haploid Mammalian Genetic Screen Identifies UBXD8 as a Key Determinant of HMGCR Degradation and Cholesterol Biosynthesis [Basic Sciences]

2017-10-25T10:40:49-07:00

Objective—The cellular demand for cholesterol requires control of its biosynthesis by the mevalonate pathway. Regulation of HMGCR (3-hydroxy-3-methylglutaryl coenzyme A reductase), a rate-limiting enzyme in this pathway and the target of statins, is a key control point herein. Accordingly, HMGCR is subject to negative and positive regulation. In particular, the ability of oxysterols and intermediates of the mevalonate pathway to stimulate its proteasomal degradation is an exquisite example of metabolically controlled feedback regulation. To define the genetic determinants that govern this process, we conducted an unbiased haploid mammalian genetic screen.Approach and Results—We generated human haploid cells with mNeon fused to endogenous HMGCR using CRISPR/Cas9 and used these cells to interrogate regulation of HMGCR abundance in live cells. This resulted in identification of known and new regulators of HMGCR, and among the latter, UBXD8 (ubiquitin regulatory X domain-containing protein 8), a gene that has not been previously implicated in this process. We demonstrate that UBXD8 is an essential determinant of metabolically stimulated degradation of HMGCR and of cholesterol biosynthesis in multiple cell types. Accordingly, UBXD8 ablation leads to aberrant cholesterol synthesis due to loss of feedback control. Mechanistically, we show that UBXD8 is necessary for sterol-stimulated dislocation of ubiquitylated HMGCR from the endoplasmic reticulum membrane en route to proteasoma[...]



Class III PI3K Positively Regulates Platelet Activation and Thrombosis via PI(3)P-Directed Function of NADPH Oxidase [Basic Sciences]

2017-10-25T10:40:49-07:00

Objective—Class III phosphoinositide 3-kinase, also known as VPS34 (vacuolar protein sorting 34), is a highly conserved enzyme regulating important cellular functions such as NADPH oxidase (NOX) assembly, membrane trafficking, and autophagy. Although VPS34 is expressed in platelets, its involvement in platelet activation remains unclear. Herein, we investigated the role of VPS34 in platelet activation and thrombus formation using VPS34 knockout mice.Approach and Results—Platelet-specific VPS34-deficient mice were generated and characterized. VPS34 deficiency in platelets did not influence tail bleeding time. In a ferric chloride–induced mesenteric arteriolar thrombosis model, VPS34−/− mice exhibited a prolonged vessel occlusion time compared with wild-type mice (42.05±4.09 versus 18.30±2.47 minutes). In an in vitro microfluidic whole-blood perfusion assay, thrombus formation on collagen under arterial shear was significantly reduced for VPS34−/− platelets. VPS34−/− platelets displayed an impaired aggregation and dense granule secretion in response to low doses of collagen or thrombin. VPS34 deficiency delayed clot retraction but did not influence platelet spreading on fibrinogen. We also demonstrated that VPS34 deficiency altered the basal level of autophagy in resting platelets and hampered NOX assembly and mTOR (mammalian target of rapamycin) signaling during platelet activation. Importantly, we identified the NOX-dependent reac[...]



Mechanical Activation of Hypoxia-Inducible Factor 1{alpha} Drives Endothelial Dysfunction at Atheroprone Sites [Basic Sciences]

2017-10-25T10:40:49-07:00

Objective—Atherosclerosis develops near branches and bends of arteries that are exposed to low shear stress (mechanical drag). These sites are characterized by excessive endothelial cell (EC) proliferation and inflammation that promote lesion initiation. The transcription factor HIF1α (hypoxia-inducible factor 1α) is canonically activated by hypoxia and has a role in plaque neovascularization. We studied the influence of shear stress on HIF1α activation and the contribution of this noncanonical pathway to lesion initiation.Approach and Results—Quantitative polymerase chain reaction and en face staining revealed that HIF1α was expressed preferentially at low shear stress regions of porcine and murine arteries. Low shear stress induced HIF1α in cultured EC in the presence of atmospheric oxygen. The mechanism involves the transcription factor nuclear factor-κB that induced HIF1α transcripts and induction of the deubiquitinating enzyme Cezanne that stabilized HIF1α protein. Gene silencing revealed that HIF1α enhanced proliferation and inflammatory activation in EC exposed to low shear stress via induction of glycolysis enzymes. We validated this observation by imposing low shear stress in murine carotid arteries (partial ligation) that upregulated the expression of HIF1α, glycolysis enzymes, and inflammatory genes and enhanced EC proliferation. EC-specific genetic deletion of HIF1α in hypercholesterolemic apolipo[...]



TGF-{beta} (Transforming Growth Factor-{beta}) Signaling Protects the Thoracic and Abdominal Aorta From Angiotensin II-Induced Pathology by Distinct Mechanisms [Basic Sciences]

2017-10-25T10:40:49-07:00

Objective—The role of TGF-β (transforming growth factor-β) signaling in abdominal aortic aneurysm (AAA) formation is controversial. Others reported that systemic blockade of TGF-β by neutralizing antibodies accelerated AAA development in angiotensin II-infused mice. This result is consistent with other studies suggesting that TGF-β signaling prevents AAA. Development of a therapy for AAA that exploits the protective actions of TGF-β would be facilitated by identification of the mechanisms through which TGF-β prevents AAA. We hypothesized that TGF-β signaling prevents AAA by its actions on aortic medial smooth muscle cells.Approach and Results—We compared the prevalence, severity, and histopathology of angiotensin II-induced AAA among control mice (no TGF-β blockade), mice with antibody-mediated systemic neutralization of TGF-β, and mice with genetically based smooth muscle–specific loss of TGF-β signaling. Surprisingly, we found that systemic—but not smooth muscle–specific—TGF-β blockade significantly increased the prevalence of AAA and tended to increase AAA severity, adventitial thickening, and aortic wall macrophage accumulation. In contrast, abdominal aortas of mice with smooth muscle–specific loss of TGF-β signaling differed from controls only in having a thinner media. We examined thoracic aortas of the same mice. Here we found that smooth muscle–specific loss of[...]



Leptin Induces Sca-1+ Progenitor Cell Migration Enhancing Neointimal Lesions in Vessel-Injury Mouse Models [Basic Sciences]

2017-10-25T10:40:49-07:00

Objective—Leptin is an adipokine initially thought to be a metabolic factor. Recent publications have shown its roles in inflammation and vascular disease, to which Sca-1+ vascular progenitor cells within the vessel wall may contribute. We sought to elucidate the effects of leptin on Sca-1+ progenitor cells migration and neointimal formation and to understand the underlying mechanisms.Approach and Results—Sca-1+ progenitor cells from the vessel wall of Lepr+/+ and Lepr−/− mice were cultured and purified. The migration of Lepr+/+ Sca-1+ progenitor cells in vitro was markedly induced by leptin. Western blotting and kinase assays revealed that leptin induced the activation of phosphorylated signal transducer and activator of transcription 3, phosphorylated extracellular signal–regulated kinases 1/2, pFAK (phosphorylated focal adhesion kinase), and Rac1 (ras-related C3 botulinum toxin substrate 1)/Cdc42 (cell division control protein 42 homolog). In a mouse femoral artery guidewire injury model, an increased expression of leptin in both injured vessels and serum was observed 24 hours post-surgery. RFP (red fluorescent protein)-Sca-1+ progenitor cells in Matrigel were applied to the adventitia of the injured femoral artery. RFP+ cells were observed in the intima 24 hours post-surgery, subsequently increasing neointimal lesions at 2 weeks when compared with the arte[...]