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Arteriosclerosis, Thrombosis, and Vascular Biology RSS feed -- ATVBe First


Distinct Pathogenesis of Pancreatic Cancer Microvesicle-Associated Venous Thrombosis Identifies New Antithrombotic Targets In Vivo [Original Research]


Objective—Cancer patients are at high risk of developing deep venous thrombosis (DVT) and venous thromboembolism, a leading cause of mortality in this population. However, it is largely unclear how malignant tumors drive the prothrombotic cascade culminating in DVT.Approach and Results—Here, we addressed the pathophysiology of malignant DVT compared with nonmalignant DVT and focused on the role of tumor microvesicles as potential targets to prevent cancer-associated DVT. We show that microvesicles released by pancreatic adenocarcinoma cells (pancreatic tumor–derived microvesicles [pcMV]) boost thrombus formation in a model of flow restriction of the mouse vena cava. This depends on the synergistic activation of coagulation by pcMV and host tissue factor. Unlike nonmalignant DVT, which is initiated and propagated by innate immune cells, thrombosis triggered by pcMV was largely independent of myeloid leukocytes or platelets. Instead, we identified externalization of the phospholipid phosphatidylethanolamine as a major mechanism controlling the prothrombotic activity of pcMV. Disrupting phosphatidylethanolamine-dependent activation of factor X suppressed pcMV-induced DVT without causing changes in hemostasis.Conclusions—Together, we show here that the pathophysiology of pcMV-associated experimental DVT differs markedly from innate immune cell–promoted nonmalignant DVT and is therefore amenable to distinct antithrombotic strategies. Targeting phosphatidylethanolamine on tumor microvesicles could be a new strategy for prevention of cancer-associated DVT without causing bleeding complications.

Inhibition of Endothelial NOTCH1 Signaling Attenuates Inflammation by Reducing Cytokine-Mediated Histone Acetylation at Inflammatory Enhancers [Original Research]


Objective—Endothelial upregulation of adhesion molecules serves to recruit leukocytes to inflammatory sites and appears to be promoted by NOTCH1; however, current models based on interactions between active NOTCH1 and NF-κB components cannot explain the transcriptional selectivity exerted by NOTCH1 in this context.Approach and Results—Observing that Cre/Lox-induced conditional mutations of endothelial Notch modulated inflammation in murine contact hypersensitivity, we found that IL (interleukin)-1β stimulation induced rapid recruitment of RELA to genomic sites occupied by NOTCH1-RBPJ and that NOTCH1 knockdown reduced H3K27 acetylation at a subset of NF-κB–directed inflammatory enhancers.Conclusions—Our findings reveal that NOTCH1 signaling supports the expression of a subset of inflammatory genes at the enhancer level and demonstrate how key signaling pathways converge on chromatin to coordinate the transition to an inflammatory endothelial phenotype.

ALK1 (Activin-Receptor Like Kinase 1) Loss Results in Vascular Hyperplasia in Mice and Humans Through PI3K (Phosphatidylinositol 3-Kinase) Activation [Original Research]


Objective—ALK1 (activin-receptor like kinase 1) is an endothelial cell-restricted receptor with high affinity for BMP (bone morphogenetic protein) 9 TGF-β (transforming growth factor-β) family member. Loss-of-function mutations in ALK1 cause a subtype of hereditary hemorrhagic telangiectasia—a rare disease characterized by vasculature malformations. Therapeutic strategies are aimed at reducing potential complications because of vascular malformations, but currently, there is no curative treatment for hereditary hemorrhagic telangiectasia.Approach and Results—In this work, we report that a reduction in ALK1 gene dosage (heterozygous ALK1+/− mice) results in enhanced retinal endothelial cell proliferation and vascular hyperplasia at the sprouting front. We found that BMP9/ALK1 represses VEGF (vascular endothelial growth factor)-mediated PI3K (phosphatidylinositol 3-kinase) by promoting the activity of the PTEN (phosphatase and tensin homolog). Consequently, loss of ALK1 function in endothelial cells results in increased activity of the PI3K pathway. These results were confirmed in cutaneous telangiectasia biopsies of patients with hereditary hemorrhagic telangiectasia 2, in which we also detected an increase in endothelial cell proliferation linked to an increase on the PI3K pathway. In mice, genetic and pharmacological inhibition of PI3K is sufficient to abolish the vascular hyperplasia of ALK1+/− retinas and in turn normalize the vasculature.Conclusions—Overall, our results indicate that the BMP9/ALK1 hub critically mediates vascular quiescence by limiting PI3K signaling and suggest that PI3K inhibitors could be used as novel therapeutic agents to treat hereditary hemorrhagic telangiectasia.

Prevalence of Calcification in Human Femoropopliteal Arteries and its Association with Demographics, Risk Factors, and Arterial Stiffness [Original Research]


Objective—Arterial calcification and stiffening increase the risk of reconstruction failure, amputation, and mortality in patients with peripheral arterial disease, but underlying mechanisms and prevalence are unclear.Approach and Results—Fresh human femoropopliteal arteries were obtained from n=431 tissue donors who are aged 13 to 82 years (mean age, 53±16 years) recording the in situ longitudinal prestretch. Arterial diameter, wall thickness, and opening angles were measured optically, and stiffness was assessed using planar biaxial extension and constitutive modeling. Histological features were determined using transverse and longitudinal Verhoeff–Van Gieson and Alizarin stains. Medial calcification was quantified using a 7-stage grading scale and was correlated with structural and mechanical properties and clinical characteristics. Almost half (46%) of the femoropopliteal arteries had identifiable medial calcification. Older arteries were more calcified, but small calcium deposits were observed in arteries as young as 18 years old. After controlling for age, positive correlations were observed between calcification, diabetes mellitus, dyslipidemia, and body mass index. Tobacco use demonstrated a negative correlation. Calcified arteries were larger in diameter but had smaller circumferential opening angles. They were also stiffer longitudinally and circumferentially and had thinner tunica media and external elastic lamina with more discontinuous elastic fibers.Conclusions—Although aging is the dominant risk factor for femoropopliteal artery calcification and stiffening, these processes seem to be linked and can begin at a young age. Calcification is associated with the presence of certain risk factors and with elastic fiber degradation, suggesting overlapping molecular pathways that require further investigation.

Endothelial Cell Autonomous Role of Akt1 [Original Research]


Objective—The importance of PI3K/Akt signaling in the vasculature has been demonstrated in several models, as global loss of Akt1 results in impaired postnatal ischemia- and VEGF-induced angiogenesis. The ubiquitous expression of Akt1, however, raises the possibility of cell-type–dependent Akt1-driven actions, thereby necessitating tissue-specific characterization.Approach and Results—Herein, we used an inducible, endothelial-specific Akt1-deleted adult mouse model (Akt1iECKO) to characterize the endothelial cell autonomous functions of Akt1 in the vascular system. Endothelial-targeted ablation of Akt1 reduces eNOS (endothelial nitric oxide synthase) phosphorylation and promotes both increased vascular contractility in isolated vessels and elevated diastolic blood pressures throughout the diurnal cycle in vivo. Furthermore, Akt1iECKO mice subject to the hindlimb ischemia model display impaired blood flow and decreased arteriogenesis.Conclusions—Endothelial Akt1 signaling is necessary for ischemic resolution post-injury and likely reflects the consequence of NO insufficiency critical for vascular repair.

Lipid-Lowering Therapy With Ezetimibe Decreases Spontaneous Atherothrombotic Occlusions in a Rabbit Model of Plaque Erosion [Original Research]


Objective—Plaque erosion is increasing its importance as one of the mechanisms of acute coronary syndromes in this statin era. However, the clinical efficacy of currently used lipid-lowering agents in the prevention of thrombotic complications associated with plaque erosion has not been clarified. Therefore, we examined the therapeutic effects of ezetimibe or rosuvastatin monotherapy on spontaneous atherothrombotic occlusion.Approach and Results—Femoral arteries of Japanese white rabbits, fed a high-cholesterol diet, were injured by balloon catheter, and then angiotensin II was continuously administrated. In 94% of these arteries, spontaneous thrombotic occlusions were observed after 5 weeks (median) of balloon injury. Histochemical analyses indicated that the injured arteries had similar pathological features to human plaque erosions; (1) spontaneous thrombotic occlusion, (2) lack of endothelial cells, and (3) tissue factor expression in vascular smooth muscle cells. Ezetimibe (1.0 mg/kg per day), but not rosuvastatin (0.6 mg/kg per day), significantly decreased thrombotic occlusion of arteries accompanied with accelerated re-endothelialization and the decreases of serum oxysterols despite the comparable on-treatment serum cholesterol levels. The 7-ketocholesterol inhibited the migration of human umbilical vein endothelial cells. Both 7-ketocholesterol and 27-hydroxycholesterol increased tissue factor expression in cultured rat vascular smooth muscle cells. Tissue factor expression was also induced by serum from vehicle- or rosuvastatin-treated rabbits, but the induction was attenuated with serum from ezetimibe-treated rabbits.Conclusions—We have established a novel rabbit model of spontaneous atherothromobotic occlusion without plaque rupture that is feasible to test the therapeutic effects of various pharmacotherapies. Ezetimibe may decrease atherothrombotic complications after superficial plaque erosion by reducing serum oxysterols.

Fat in the Blood, Fat in the Artery, Fat in the Heart [ATVB Named Lecture Review]


Cholesterol is not the only lipid that causes heart disease. Triglyceride supplies the heart and skeletal muscles with highly efficient fuel and allows for the storage of excess calories in adipose tissue. Failure to transport, acquire, and use triglyceride leads to energy deficiency and even death. However, overabundance of triglyceride can damage and impair tissues. Circulating lipoprotein-associated triglycerides are lipolyzed by lipoprotein lipase (LpL) and hepatic triglyceride lipase. We inhibited these enzymes and showed that LpL inhibition reduces high-density lipoprotein cholesterol by >50%, and hepatic triglyceride lipase inhibition shifts low-density lipoprotein to larger, more buoyant particles. Genetic variations that reduce LpL activity correlate with increased cardiovascular risk. In contrast, macrophage LpL deficiency reduces macrophage function and atherosclerosis. Therefore, muscle and macrophage LpL have opposite effects on atherosclerosis. With models of atherosclerosis regression that we used to study diabetes mellitus, we are now examining whether triglyceride-rich lipoproteins or their hydrolysis by LpL affect the biology of established plaques. Following our focus on triglyceride metabolism led us to show that heart-specific LpL hydrolysis of triglyceride allows optimal supply of fatty acids to the heart. In contrast, cardiomyocyte LpL overexpression and excess lipid uptake cause lipotoxic heart failure. We are now studying whether interrupting pathways for lipid uptake might prevent or treat some forms of heart failure.

Bempedoic Acid Lowers Low-Density Lipoprotein Cholesterol and Attenuates Atherosclerosis in Low-Density Lipoprotein Receptor-Deficient (LDLR+/- and LDLR-/-) Yucatan Miniature Pigs [Original Research]


Objective—Bempedoic acid (BemA; ETC-1002) is a novel drug that targets hepatic ATP-citrate lyase to reduce cholesterol biosynthesis. In phase 2 studies, BemA lowers elevated low-density lipoprotein cholesterol (LDL-C) in hypercholesterolemic patients. In the present study, we tested the ability of BemA to decrease plasma cholesterol and LDL-C and attenuate atherosclerosis in a large animal model of familial hypercholesterolemia.Approach and Results—Gene targeting has been used to generate Yucatan miniature pigs heterozygous (LDLR+/−) or homozygous (LDLR−/−) for LDL receptor deficiency (ExeGen). LDLR+/− and LDLR−/− pigs were fed a high-fat, cholesterol-containing diet (34% kcal fat; 0.2% cholesterol) and orally administered placebo or BemA for 160 days. In LDLR+/− pigs, compared with placebo, BemA decreased plasma cholesterol and LDL-C up to 40% and 61%, respectively. In LDLR−/− pigs, in which plasma cholesterol and LDL-C were 5-fold higher than in LDLR+/− pigs, BemA decreased plasma cholesterol and LDL-C up to 27% and 29%, respectively. Plasma levels of triglycerides and high-density lipoprotein cholesterol, fasting glucose and insulin, and liver lipids were unaffected by treatment in either genotype. In the aorta of LDLR+/− pigs, BemA robustly attenuated en face raised lesion area (−58%) and left anterior descending coronary artery cross-sectional lesion area (−40%). In LDLR−/− pigs, in which lesions were substantially more advanced, BemA decreased aortic lesion area (−47%) and left anterior descending coronary artery lesion area (−48%).Conclusions—In a large animal model of LDLR deficiency and atherosclerosis, long-term treatment with BemA reduces LDL-C and attenuates the development of aortic and coronary atherosclerosis in both LDLR+/− and LDLR−/− miniature pigs.

CSL112 (Apolipoprotein A-I [Human]) Enhances Cholesterol Efflux Similarly in Healthy Individuals and Stable Atherosclerotic Disease Patients [Original Research]


Objective—CSL112 (apolipoprotein A-I [apoA-I; human]) is a novel formulation of apoA-I in development for reduction of early recurrent cardiovascular events after acute myocardial infarction. Cholesterol efflux capacity (CEC) is a marker of high-density lipoprotein (HDL) function that is strongly correlated with incident cardiovascular disease. Impaired CEC has been observed in patients with coronary heart disease. Here, we determined whether infused apoA-I improves CEC when administered to patients with stable atherosclerotic disease versus healthy volunteers.Approach and Results—Measurements of apoA-I, HDL unesterified cholesterol, HDL esterified cholesterol, pre–β1-HDL, and CEC were determined in samples from patients with stable atherosclerotic disease before and after intravenous administration of CSL112. These measures were compared with 2 prior studies in healthy volunteers for differences in CEC at baseline and after CSL112 infusion. Patients with stable atherosclerotic disease exhibited significantly lower ATP-binding cassette transporter 1–mediated CEC at baseline (P<0.0001) despite slightly higher apoA-I levels when compared with healthy individuals (2 phase 1 studies pooled; P≤0.05), suggesting impaired HDL function. However, no differences were observed in apoA-I pharmacokinetics or in pre–β1-HDL (P=0.5) or CEC (P=0.1) after infusion of CSL112. Similar elevation in CEC was observed in patients with low or high baseline HDL function (based on tertiles of apoA-I–normalized CEC; P=0.1242). These observations were extended and confirmed using cholesterol esterification as an additional measure.Conclusions—CSL112 shows comparable, strong, and immediate effects on CEC despite underlying cardiovascular disease. CSL112 is, therefore, a promising novel therapy for lowering the burden of atherosclerosis and reducing the risk of recurrent cardiovascular events.

Effects of Increasing Exercise Intensity and Dose on Multiple Measures of HDL (High-Density Lipoprotein) Function [Original Research]


Objective—Measures of HDL (high-density lipoprotein) function are associated with cardiovascular disease. However, the effects of regular exercise on these measures is largely unknown. Thus, we examined the effects of different doses of exercise on 3 measures of HDL function in 2 randomized clinical exercise trials.Approach and Results—Radiolabeled and boron dipyrromethene difluoride–labeled cholesterol efflux capacity and HDL-apoA-I (apolipoprotein A-I) exchange were assessed before and after 6 months of exercise training in 2 cohorts: STRRIDE-PD (Studies of Targeted Risk Reduction Interventions through Defined Exercise, in individuals with Pre-Diabetes; n=106) and E-MECHANIC (Examination of Mechanisms of exercise-induced weight compensation; n=90). STRRIDE-PD, participants completed 1 of 4 exercise interventions differing in amount and intensity. E-MECHANIC participants were randomized into 1 of 2 exercise groups (8 or 20 kcal/kg per week) or a control group. HDL-C significantly increased in the high-amount/vigorous-intensity group (3±5 mg/dL; P=0.02) of STRRIDE-PD, whereas no changes in HDL-C were observed in E-MECHANIC. In STRRIDE-PD, global radiolabeled efflux capacity significantly increased 6.2% (SEM, 0.06) in the high-amount/vigorous-intensity group compared with all other STRRIDE-PD groups (range, −2.4 to −8.4%; SEM, 0.06). In E-MECHANIC, non-ABCA1 (ATP-binding cassette transporter A1) radiolabeled efflux significantly increased 5.7% (95% CI, 1.2–10.2%) in the 20 kcal/kg per week group compared with the control group, with no change in the 8 kcal/kg per week group (2.6%; 95% CI, −1.4 to 6.7%). This association was attenuated when adjusting for change in HDL-C. Exercise training did not affect BODIPY-labeled cholesterol efflux capacity or HDL-apoA-I exchange in either study.Conclusions—Regular prolonged vigorous exercise improves some but not all measures of HDL function. Future studies are warranted to investigate whether the effects of exercise on cardiovascular disease are mediated in part by improving HDL function.Clinical Trial Registration—URL: Unique identifiers: NCT00962962 and NCT01264406.

Cilostazol Attenuates Angiotensin II-Induced Abdominal Aortic Aneurysms but Not Atherosclerosis in Apolipoprotein E-Deficient Mice [Original Research]


Objective—Abdominal aortic aneurysm (AAA) is a permanent dilation of the abdominal aorta associated with rupture, which frequently results in fatal consequences. AAA tissue is commonly characterized by localized structural deterioration accompanied with inflammation and profound accumulation of leukocytes, although the specific function of these cells is unknown. Cilostazol, a phosphodiesterase III inhibitor, is commonly used for patients with peripheral vascular disease or stroke because of its anti-inflammatory effect, which is vasoprotective effects through its anti-inflammatory effect. In this study, we evaluated the effects of cilostazol on angiotensin II–induced AAA formation.Approach and Results—Male apolipoprotein E–deficient mice were fed either normal diet or a diet containing cilostazol (0.1% wt/wt). After 1 week of diet consumption, mice were infused with angiotensin II (1000 ng/kg per minute) for 4 weeks. Angiotensin II infusion increased maximal diameters of abdominal aortas, whereas cilostazol administration significantly attenuated dilatation of abdominal aortas, thereby, reducing AAA incidence. Cilostazol also reduced macrophage accumulation, matrix metalloproteinases activation, and inflammatory gene expression in the aortic media. In cultured vascular endothelial cells, cilostazol reduced expression of inflammatory cytokines and adhesive molecules through activation of the cAMP–PKA (protein kinase A) pathway.Conclusions—Cilostazol attenuated angiotensin II–induced AAA formation by its anti-inflammatory effect through phosphodiesterase III inhibition in the aortic wall. Cilostazol may be a promising new therapeutic option for AAAs.

MiR-146a Regulates Neutrophil Extracellular Trap Formation That Predicts Adverse Cardiovascular Events in Patients With Atrial Fibrillation [Original Research]


Objective—Atrial fibrillation (AF) patients experience adverse cardiovascular events (ACEs) despite anticoagulant therapy. We reported that rs2431697 of miR-146a, a negative regulator of inflammation, predicts ACEs in patients with AF. The relationship between neutrophil extracellular traps and thrombogenesis is known. Thus, our aim was to evaluate the role of neutrophil extracellular trap compounds as prognostic markers of ACEs in AF and to study whether miR-146a affects NETosis.Approach and Results—We included 336 steadily anticoagulated AF patients with a median follow-up of 7.9 years (interquartile range, 7.3–8.1) and 127 healthy subjects. The reviewed ACEs included stroke (ischemic/embolic), acute coronary syndrome, acute heart failure, and global or vascular death. We quantified cell-free DNA and NE (neutrophil elastase) at diagnosis. Rs2431697 was genotyped. Neutrophils from human and mice were seeded to analyze shed cell-free DNA and citrullinated histone 3 (H3cit) after activation. In human plasmas, higher NE levels (>55.29 ng/mL), but not cell-free DNA, were independently associated with higher risk of all-cause mortality (hazard ratio, 2.24; 95% CI, 1.36–3.68), cardiovascular mortality (hazard ratio, 4.77; 95% CI, 1.11–20.47), and composite cardiovascular events (hazard ratio, 1.84; 95% CI, 1.01–3.76). In patients, NE levels were associated with rs2431697 (TT: 51.82±2.73 versus CC: 40.01±3.05 ng/mL; P=0.040). In vitro, both human (TT for rs2431697) and miR-146a−/− mice neutrophils yielded higher levels of cell-free DNA and H3cit than CC or wild-type cells, respectively.Conclusions—NE activity can provide new ACE prognostic information in AF patients. These findings provide evidence of a potential role of miR-146a in neutrophil extracellular trap generation and cardiovascular risk in AF.

Tissue Factor [Brief Reviews]


Tissue factor (TF) is the high-affinity receptor and cofactor for FVII/VIIa. The TF-FVIIa complex is the primary initiator of blood coagulation and plays an essential role in hemostasis. TF is expressed on perivascular and epithelial cells at organ and body surfaces where it forms a hemostatic barrier. TF also provides additional hemostatic protection to vital organs, such as the brain, lung, and heart. Under pathological conditions, TF can trigger both arterial and venous thrombosis. For instance, atherosclerosis plaques contain high levels of TF on macrophage foam cells and microvesicles that drives thrombus formation after plaque rupture. In sepsis, inducible TF expression on monocytes leads to disseminated intravascular coagulation. In cancer patients, tumors release TF-positive microvesicles into the circulation that may contribute to venous thrombosis. TF also has nonhemostatic roles. For instance, TF-dependent activation of the coagulation cascade generates coagulation proteases, such as FVIIa, FXa, and thrombin, which induce signaling in a variety of cells by cleavage of PARs (protease-activated receptors). This review will focus on the roles of TF in protective hemostasis and pathological thrombosis.

Plasminogen Activator Inhibitor-1 Promotes Neutrophil Infiltration and Tissue Inȷury on Ischemia-Reperfusion [Original Research]


Objective—Ischemia–reperfusion (I/R) injury significantly contributes to organ dysfunction and failure after myocardial infarction, stroke, and transplantation. In addition to its established role in the fibrinolytic system, plasminogen activator inhibitor-1 has recently been implicated in the pathogenesis of I/R injury. The underlying mechanisms remain largely obscure.Approach and Results—Using different in vivo microscopy techniques as well as ex vivo analyses and in vitro assays, we identified that plasminogen activator inhibitor-1 rapidly accumulates on microvascular endothelial cells on I/R enabling this protease inhibitor to exhibit previously unrecognized functional properties by inducing an increase in the affinity of β2 integrins in intravascularly rolling neutrophils. These events are mediated through low-density lipoprotein receptor–related protein-1 and mitogen-activated protein kinase–dependent signaling pathways that initiate intravascular adherence of these immune cells to the microvascular endothelium. Subsequent to this process, extravasating neutrophils disrupt endothelial junctions and promote the postischemic microvascular leakage. Conversely, deficiency of plasminogen activator inhibitor-1effectively reversed leukocyte infiltration, microvascular dysfunction, and tissue injury on experimental I/R without exhibiting side effects on microvascular hemostasis.Conclusion—Our experimental data provide novel insights into the nonfibrinolytic properties of the fibrinolytic system and emphasize plasminogen activator inhibitor-1 as a promising target for the prevention and treatment of I/R injury.

Anticoagulant Protein S Targets the Factor IXa Heparin-Binding Exosite to Prevent Thrombosis [Original Research]


Objective—PS (protein S) is a plasma protein that directly inhibits the coagulation FIXa (factor IXa) in vitro. Because elevated FIXa is associated with increased risk of venous thromboembolism, it is important to establish how PS inhibits FIXa function in vivo. The goal of this study is to confirm direct binding of PS with FIXa in vivo, identify FIXa amino acid residues required for binding PS in vivo, and use an enzymatically active FIXa mutant that is unable to bind PS to measure the significance of PS–FIXa interaction in hemostasis.Approach and Results—We demonstrate that PS inhibits FIXa in vivo by associating with the FIXa heparin-binding exosite. We used fluorescence tagging, immunohistochemistry, and protein–protein crosslinking to show in vivo interaction between FIXa and PS. Importantly, platelet colocalization required a direct interaction between the 2 proteins. FIXa and PS also coimmunoprecipitated from plasma, substantiating their interaction in a physiological milieu. PS binding to FIXa and PS inhibition of the intrinsic Xase complex required residues K132, K126, and R170 in the FIXa heparin-binding exosite. A double mutant, K132A/R170A, retained full activity but could not bind to PS. Crucially, Hemophilia B mice infused with FIXa K132A/R170A displayed an accelerated rate of fibrin clot formation compared with wild-type FIXa.Conclusions—Our findings establish PS as an important in vivo inhibitor of FIXa. Disruption of the interaction between PS and FIXa causes an increased rate of thrombus formation in mice. This newly discovered function of PS implies an unexploited target for antithrombotic therapeutics.

Targeted Deletion of Adipocyte Abca1 (ATP-Binding Cassette Transporter A1) Impairs Diet-Induced Obesity [Original Research]


Objective—Adipose tissue cholesterol increases with adipocyte triglyceride content and size during development of obesity. However, how adipocyte cholesterol affects adipocyte function is poorly understood. The aim of this study was to evaluate the role of the cellular cholesterol exporter, Abca1 (ATP-binding cassette transporter A1), on adipose tissue function during diet-induced obesity.Approach and Results—Adiponectin Cre recombinase transgenic mice were crossed with Abca1 flox/flox mice to generate ASKO (adipocyte-specific Abca1 knockout) mice. Control and ASKO mice were then fed a high-fat, high-cholesterol (45% calories as fat and 0.2% cholesterol) diet for 16 weeks. Compared with control mice, ASKO mice had a 2-fold increase in adipocyte plasma membrane cholesterol content and significantly lower body weight, epididymal fat pad weight, and adipocyte size. ASKO versus control adipose tissue had decreased PPARγ (peroxisome proliferator-activated receptor γ) and CCAAT/enhancer-binding protein expression, nuclear SREBP1 (sterol regulatory element-binding protein 1) protein, lipogenesis, and triglyceride accretion but similar Akt activation after acute insulin stimulation. Acute siRNA-mediated Abca1 silencing during 3T3L1 adipocyte differentiation reduced adipocyte Abca1 and PPARγ protein expression and triglyceride content. Systemic stimulated triglyceride lipolysis and glucose homeostasis was similar between control and ASKO mice.Conclusions—Adipocyte Abca1 is a key regulator of adipocyte lipogenesis and lipid accretion, likely because of increased adipose tissue membrane cholesterol, resulting in decreased activation of lipogenic transcription factors PPARγ and SREBP1.

Saturated Fatty Acids Undergo Intracellular Crystallization and Activate the NLRP3 Inflammasome in Macrophages [Original Research]


Objective—Inflammation provoked by the imbalance of fatty acid composition, such as excess saturated fatty acids (SFAs), is implicated in the development of metabolic diseases. Recent investigations suggest the possible role of the NLRP3 (nucleotide-binding oligomerization domain, leucine-rich repeat and pyrin domain containing 3) inflammasome, which regulates IL-1β (interleukin 1β) release and leads to inflammation, in this process. Therefore, we investigated the underlying mechanism by which SFAs trigger NLRP3 inflammasome activation.Approach and Results—The treatment with SFAs, such as palmitic acid and stearic acid, promoted IL-1β release in murine primary macrophages while treatment with oleic acid inhibited SFA-induced IL-1β release in a dose-dependent manner. Analyses using polarized light microscopy revealed that intracellular crystallization was provoked in SFA-treated macrophages. As well as IL-1β release, the intracellular crystallization and lysosomal dysfunction were inhibited in the presence of oleic acid. These results suggest that SFAs activate NLRP3 inflammasome through intracellular crystallization. Indeed, SFA-derived crystals activated NLRP3 inflammasome and subsequent IL-1β release via lysosomal dysfunction. Excess SFAs also induced crystallization and IL-1β release in vivo. Furthermore, SFA-derived crystals provoked acute inflammation, which was impaired in IL-1β–deficient mice.Conclusions—These findings demonstrate that excess SFAs cause intracellular crystallization and subsequent lysosomal dysfunction, leading to the activation of the NLRP3 inflammasome, and provide novel insights into the pathogenesis of metabolic diseases.

RGC-32 (Response Gene to Complement 32 Protein) Deficiency Protects Endothelial Cells From Inflammation and Attenuates Atherosclerosis [Original Research]


Objective—The objective of this study is to determine the role and underlying mechanisms of RGC-32 (response gene to complement 32 protein) in atherogenesis.Approach and Results—RGC-32 was mainly expressed in endothelial cells of atherosclerotic lesions in both ApoE–/– (apolipoprotein E deficient) mice and human patients. RGC-32 deficiency (Rgc32–/–) attenuated the high-fat diet–induced and spontaneously developed atherosclerotic lesions in ApoE–/– mice without affecting serum cholesterol concentration. Rgc32–/– seemed to decrease the macrophage content without altering collagen and smooth muscle contents or lesional macrophage proliferation in the lesions. Transplantation of WT (wild type) mouse bone marrow to lethally irradiated Rgc32–/– mice did not alter Rgc32–/–-caused reduction of lesion formation and macrophage accumulation, suggesting that RGC-32 in resident vascular cells, but not the macrophages, plays a critical role in the atherogenesis. Of importance, Rgc32–/– decreased the expression of ICAM-1 (intercellular adhesion molecule-1) and VCAM-1 (vascular cell adhesion molecule-1) in endothelial cells both in vivo and in vitro, resulting in a decrease in TNF-α (tumor necrosis factor-α)–induced monocyte–endothelial cell interaction. Mechanistically, RGC-32 mediated the ICAM-1 and VCAM-1 expression, at least partially, through NF (nuclear factor)-κB signaling pathway. RGC-32 directly interacted with NF-κB and facilitated its nuclear translocation and enhanced TNF-α–induced NF-κB binding to ICAM-1 and VCAM-1 promoters.Conclusions—RGC-32 mediates atherogenesis by facilitating monocyte–endothelial cell interaction via the induction of endothelial ICAM-1 and VCAM-1 expression, at least partially, through NF-κB signaling pathway.

Mouse Platelet Ral GTPases Control P-Selectin Surface Expression, Regulating Platelet-Leukocyte Interaction [Original Research]


Objective—RalA and RalB GTPases are important regulators of cell growth, cancer metastasis, and granule secretion. The purpose of this study was to determine the role of Ral GTPases in platelets with the use of platelet-specific gene-knockout mouse models.Approach and Results—This study shows that platelets from double knockout mice, in which both GTPases have been deleted, show markedly diminished (≈85% reduction) P-selectin translocation to the surface membrane, suggesting a critical role in α-granule secretion. Surprisingly, however, there were only minor effects on stimulated release of soluble α- and δ-granule content, with no alteration in granule count, morphology, or content. In addition, their expression was not essential for platelet aggregation or thrombus formation. However, absence of surface P-selectin caused a marked reduction (≈70%) in platelet–leukocyte interactions in blood from RalAB double knockout mice, suggesting a role for platelet Rals in platelet-mediated inflammation.Conclusions—Platelet Ral GTPases primarily control P-selectin surface expression, in turn regulating platelet–leukocyte interaction. Ral GTPases could therefore be important novel targets for the selective control of platelet-mediated immune cell recruitment and inflammatory disease.

Platelets Drive Thrombus Propagation in a Hematocrit and Glycoprotein VI-Dependent Manner in an In Vitro Venous Thrombosis Model [Original Research]


Objective—The objective of this study was to measure the role of platelets and red blood cells on thrombus propagation in an in vitro model of venous valvular stasis.Approach and Results—A microfluidic model with dimensional similarity to human venous valves consists of a sinus distal to a sudden expansion, where for sufficiently high Reynolds numbers, 2 countercurrent vortices arise because of flow separation. The primary vortex is defined by the points of flow separation and reattachment. A secondary vortex forms in the deepest recess of the valve pocket characterized by low shear rates. An initial fibrin gel formed within the secondary vortex of a tissue factor–coated valve sinus. Platelet accumulated at the interface of the fibrin gel and the primary vortex. Red blood cells at physiological hematocrits were necessary to provide an adequate flux of platelets to support thrombus growth out of the valve sinus. A subpopulation of platelets that adhered to fibrin expose phosphatidylserine. Platelet-dependent thrombus growth was attenuated by inhibition of glycoprotein VI with a blocking Fab fragment or D-dimer.Conclusions—A 3-step process regulated by hemodynamics was necessary for robust thrombus propagation: First, immobilized tissue factor initiates coagulation and fibrin deposition within a low flow niche defined by a secondary vortex in the pocket of a model venous valve. Second, a primary vortex delivers platelets to the fibrin interface in a red blood cell–dependent manner. Third, platelets adhere to fibrin, activate through glycoprotein VI, express phosphatidylserine, and subsequently promote thrombus growth beyond the valve sinus and into the bulk flow.

Genome-Wide Association and Functional Studies Identify SCML4 and THSD7A as Novel Susceptibility Genes for Coronary Artery Disease [Original Research]


Objective—The genetic contribution to coronary artery disease (CAD) remains largely unclear. We combined genetic screening with functional characterizations to identify novel loci and candidate genes for CAD.Approach and Results—We performed genome-wide screening followed by multicenter validation in 8 cohorts consisting of 21 828 participants of Han ethnicity and identified 3 novel intragenic SNPs, rs9486729 (SCML4; odds ratio, 1.25; 95% CI, 1.17–1.34; P=3.51×10−11), rs17165136 (THSD7A; odds ratio 1.28; 95% CI, 1.21–1.35; P<1.00×10−25), and rs852787 (DAB1; odds ratio, 1.29; 95% CI, 1.21–1.38; P=2.02×10−14), associated with CAD with genome-wide significance. The risk allele of rs9486729 and protective allele of rs17165136 were associated with the decreased expression of their host genes, SCML4 and THSD7A, respectively, whereas rs852787 did not have transcriptional effects on any gene. Knockdown of SCML4 activated endothelial cells by increasing the expression of IL-6, E-selectin, and ICAM and weakened their antiapoptotic activity, whereas the knockdown of THSD7A had little effect on these endothelial cell functions but attenuated monocyte adhesion via decreasing the expression of ICAM, L-selectin, and ITGB2. We further showed that inhibiting the expression of SCML4 exacerbated endothelial dysfunction and vascular remodeling in a rat model with partial carotid ligation.Conclusions—We identify 3 novel loci associated with CAD and show that 2 genes, SCML4 and THSD7A, make functional contributions to atherosclerosis. How rs852787 and its host gene DAB1 are linked to CAD needs further studies.

Novel Role of IL (Interleukin)-1{beta} in Neutrophil Extracellular Trap Formation and Abdominal Aortic Aneurysms [Original Research]


Objective—Neutrophils promote experimental abdominal aortic aneurysm (AAA) formation via a mechanism that is independent from MMPs (matrix metalloproteinases). Recently, we reported a dominant role of IL (interleukin)-1β in the formation of murine experimental AAAs. Here, the hypothesis that IL-1β–induced neutrophil extracellular trap formation (NETosis) promotes AAA was tested.Approach and Results—NETs were identified through colocalized staining of neutrophil, Cit-H3 (citrullinated histone H3), and DNA, using immunohistochemistry. NETs were detected in human AAAs and were colocalized with IL-1β. In vitro, IL-1RA attenuated IL-1β–induced NETosis in human neutrophils. Mechanistically, IL-1β treatment of isolated neutrophils induced nuclear localization of ceramide synthase 6 and synthesis of C16-ceramide, which was inhibited by IL-1RA or fumonicin B1, an inhibitor of ceramide synthesis. Furthermore, IL-1RA or fumonicin B1 attenuated IL1-β–induced NETosis. In an experimental model of murine AAA, NETs were detected at a very early stage–day 3 of aneurysm induction. IL-1β–knockout mice demonstrated significantly lower infiltration of neutrophils to aorta and were protected from AAA. Adoptive transfer of wild-type neutrophils promoted AAA formation in IL-1β–knockout mice. Moreover, treatment of wild-type mice with Cl-amidine, an inhibitor NETosis, significantly attenuated AAA formation, whereas, treatment with deoxyribonuclease, a DNA digesting enzyme, had no effect on AAA formation.Conclusions—Altogether, the results suggest a dominant role of IL-1β–induced NETosis in AAA formation.

Hypoxia Triggers SENP1 (Sentrin-Specific Protease 1) Modulation of KLF15 (Kruppel-Like Factor 15) and Transcriptional Regulation of Arg2 (Arginase 2) in Pulmonary Endothelium [Original Research]


Objective—KLF15 (Kruppel-like factor 15) has recently been shown to suppress activation of proinflammatory processes that contribute to atherogenesis in vascular smooth muscle, however, the role of KLF15 in vascular endothelial function is unknown. Arginase mediates inflammatory vasculopathy and vascular injury in pulmonary hypertension. Here, we tested the hypothesis that KLF15 is a critical regulator of hypoxia-induced Arg2 (arginase 2) transcription in human pulmonary microvascular endothelial cells (HPMEC).Approach and Results—Quiescent HPMEC express ample amounts of full-length KLF15. HPMECs exposed to 24 hours of hypoxia exhibited a marked decrease in KLF15 protein levels and a reciprocal increase in Arg2 protein and mRNA. Chromatin immunoprecipitation indicated direct binding of KLF15 to the Arg2 promoter, which was relieved with HPMEC exposure to hypoxia. Furthermore, overexpression of KLF15 in HPMEC reversed hypoxia-induced augmentation of Arg2 abundance and arginase activity and rescued nitric oxide (NO) production. Ectopic KLF15 also reversed hypoxia-induced endothelium-mediated vasodilatation in isolated rat pulmonary artery rings. Mechanisms by which hypoxia regulates KLF15 abundance, stability, and compartmentalization to the nucleus in HPMEC were then investigated. Hypoxia triggered deSUMOylation of KLF15 by SENP1 (sentrin-specific protease 1), and translocation of KLF15 from nucleus to cytoplasm.Conclusions—KLF15 is a critical regulator of pulmonary endothelial homeostasis via repression of endothelial Arg2 expression. KLF15 abundance and nuclear compartmentalization are regulated by SUMOylation/deSUMOylation—a hypoxia-sensitive process that is controlled by SENP1. Strategies including overexpression of KLF15 or inhibition of SENP1 may represent novel therapeutic targets for pulmonary hypertension.

Carotid Artery Remodeling Is Segment Specific [Original Research]


Objective—Early atherosclerosis is often undetected due in part to compensatory enlargement of the outer wall, termed positive remodeling. Variations in hemodynamic conditions and clinical factors influence the patterns of remodeling. The carotid artery provides an opportunity to examine these variations because of the unique geometry of the carotid bulb. This study aimed to determine differences in remodeling of the common, internal, and bifurcation segments of the carotid using magnetic resonance imaging.Approach and Results—Carotid arteries of 525 subjects without history of cardiovascular disease were imaged by magnetic resonance imaging. The carotid artery was divided into 3 segments: common carotid artery; bifurcation; and internal carotid artery. Remodeling patterns were characterized using linear regression analysis of lumen and total vessel areas (dependent variables) compared with maximum wall thickness (independent variable) for each segment, adjusted for age, sex, and height. The common carotid artery demonstrated a pattern consistent with positive remodeling, whereas the bifurcation demonstrated negative remodeling. The internal carotid artery demonstrated a mixed pattern of outer wall expansion and lumen constriction. Females and subjects with diabetes mellitus showed more positive remodeling, hypertension was associated with attenuated positive remodeling, and those with hypercholesterolemia showed more negative remodeling.Conclusions—In this cohort of 55- to 80-year-old individuals without history of cardiovascular disease, the pattern of early carotid artery remodeling was segment specific and appeared to be associated with sex and clinical characteristics. These findings provide the groundwork for longitudinal studies to define local and systemic factors such as hemodynamic and clinical conditions on carotid artery remodeling.

Role of Sympathetic Nerves and Adipocyte Catecholamine Uptake in the Vasorelaxant Function of Perivascular Adipose Tissue [Original Research]


Objective—Healthy perivascular adipose tissue (PVAT) exerts an anticontractile effect on resistance arteries which is vital in regulating arterial tone. Activation of β3-adrenoceptors by sympathetic nerve–derived NA (noradrenaline) may be implicated in this effect and may stimulate the release of the vasodilator adiponectin from adipocytes. Understanding the mechanisms responsible is vital for determining how PVAT may modify vascular resistance in vivo.Approach and Results—Electric field stimulation profiles of healthy C57BL/6J mouse mesenteric resistance arteries were characterized using wire myography. During electric field stimulation, PVAT elicits a reproducible anticontractile effect, which is endothelium independent. To demonstrate the release of an anticontractile factor, the solution surrounding stimulated exogenous PVAT was transferred to a PVAT-denuded vessel. Post-transfer contractility was significantly reduced confirming that stimulated PVAT releases a transferable anticontractile factor. Sympathetic denervation of PVAT using tetrodotoxin or 6-hydroxydopamine completely abolished the anticontractile effect. β3-adrenoceptor antagonist SR59203A reduced the anticontractile effect, although the PVAT remained overall anticontractile. When the antagonist was used in combination with an OCT3 (organic cation transporter 3) inhibitor, corticosterone, the anticontractile effect was completely abolished. Application of an adiponectin receptor-1 blocking peptide significantly reduced the anticontractile effect in +PVAT arteries. When used in combination with the β3-adrenoceptor antagonist, there was no further reduction. In adiponectin knockout mice, the anticontractile effect is absent.Conclusions—The roles of PVAT are 2-fold. First, sympathetic stimulation in PVAT triggers the release of adiponectin via β3-adrenoceptor activation. Second, PVAT acts as a reservoir for NA, preventing it from reaching the vessel and causing contraction.

Ly6CHi Blood Monocyte/Macrophage Drive Chronic Inflammation and Impair Wound Healing in Diabetes Mellitus [Original Research]


Objective—Wound monocyte-derived macrophage plasticity controls the initiation and resolution of inflammation that is critical for proper healing, however, in diabetes mellitus, the resolution of inflammation fails to occur. In diabetic wounds, the kinetics of blood monocyte recruitment and the mechanisms that control in vivo monocyte/macrophage differentiation remain unknown.Approach and Results—Here, we characterized the kinetics and function of Ly6CHi [Lin− (CD3−CD19−NK1.1−Ter-119−) Ly6G−CD11b+] and Ly6CLo [Lin− (CD3−CD19−NK1.1−Ter-119−) Ly6G−CD11b+] monocyte/macrophage subsets in normal and diabetic wounds. Using flow-sorted tdTomato-labeled Ly6CHi monocyte/macrophages, we show Ly6CHi cells transition to a Ly6CLo phenotype in normal wounds, whereas in diabetic wounds, there is a late, second influx of Ly6CHi cells that fail transition to Ly6CLo. The second wave of Ly6CHi cells in diabetic wounds corresponded to a spike in MCP-1 and selective administration of anti-MCP-1 reversed the second Ly6CHi influx and improved wound healing. To examine the in vivo phenotype of wound monocyte/macrophages, RNA-seq–based transcriptome profiling was performed on flow-sorted Ly6CHi [Lin−Ly6G−CD11b+] and Ly6CLo [Lin−Ly6G−CD11b+] cells from normal and diabetic wounds. Gene transcriptome profiling of diabetic wound Ly6CHi cells demonstrated differences in proinflammatory and profibrotic genes compared with controls.Conclusions—Collectively, these data identify kinetic and functional differences in diabetic wound monocyte/macrophages and demonstrate that selective targeting of CD11b+Ly6CHi monocyte/macrophages is a viable therapeutic strategy for inflammation in diabetic wounds.

Deletion of BMAL1 (Brain and Muscle ARNT-Like Protein-1) in Smooth Muscle Cells Protects Mice From Abdominal Aortic Aneurysms [Original Research]


Objective—Abdominal aortic aneurysm (AAA) has high mortality rate when ruptured, but currently, there is no proven pharmacological therapy for AAA because of our poor understanding of its pathogenesis. The current study explored a novel role of smooth muscle cell (SMC) BMAL1 (brain and muscle Arnt-like protein-1)—a transcription factor known to regulate circadian rhythm—in AAA development.Approach and Results—SMC-selective deletion of BMAL1 potently protected mice from AAA induced by (1) MR (mineralocorticoid receptor) agonist deoxycorticosterone acetate or aldosterone when provided with high salt and (2) angiotensin II infusion in hypercholesterolemia mice. Aortic BMAL1 was upregulated by deoxycorticosterone acetate-salt, and deletion of BMAL1 in SMCs selectively upregulated TIMP4 (tissue inhibitor of metalloproteinase 4) and suppressed deoxycorticosterone acetate-salt–induced MMP (matrix metalloproteinase) activation and elastin breakages. Moreover, BMAL1 bound to the Timp4 promoter and suppressed Timp4 transcription.Conclusions—These results reveal an important, but previously unexplored, role of SMC BMAL1 in AAA. Moreover, these results identify TIMP4 as a novel target of BMAL1, which may mediate the AAA protective effect of SMC BMAL1 deletion.

Inhibition of Local Macrophage Growth Ameliorates Focal Inflammation and Suppresses Atherosclerosis [Original Research]


Objective—Macrophages play a central role in various stages of atherosclerotic plaque formation and progression. The local macrophages reportedly proliferate during atherosclerosis, but the pathophysiological significance of macrophage proliferation in this context remains unclear. Here, we investigated the involvement of local macrophage proliferation during atherosclerosis formation and progression using transgenic mice, in which macrophage proliferation was specifically suppressed.Approach and Results—Inhibition of macrophage proliferation was achieved by inducing the expression of cyclin-dependent kinase inhibitor 1B, also known as p27kip (cyclin-dependent kinase inhibitor 1B), under the regulation of a scavenger receptor promoter/enhancer. The macrophage-specific human p27kip Tg mice were subsequently crossed with apolipoprotein E–deficient mice for the atherosclerotic plaque study. Results showed that a reduced number of local macrophages resulted in marked suppression of atherosclerotic plaque formation and inflammatory response in the plaque. Moreover, fewer local macrophages in macrophage-specific human p27kip Tg mice helped stabilize the plaque, as evidenced by a reduced necrotic core area, increased collagenous extracellular matrix, and thickened fibrous cap.Conclusions—These results provide direct evidence of the involvement of local macrophage proliferation in formation and progression of atherosclerotic plaques and plaque stability. Thus, control of macrophage proliferation might represent a therapeutic target for treating atherosclerotic diseases.

Endogenous LINE-1 (Long Interspersed Nuclear Element-1) Reverse Transcriptase Activity in Platelets Controls Translational Events Through RNA-DNA Hybrids [Original Research]


Objective—One source of endogenous reverse transcriptase (eRT) activity in nucleated cells is the LINE-1/L1 (long interspersed nuclear element-1), a non-LTR retrotransposon that is implicated in the regulation of gene expression. Nevertheless, the presence and function of eRT activity and LINE-1 in human platelets, an anucleate cell, has not previously been determined.Approach and Results—We demonstrate that human and murine platelets possess robust eRT activity and identify the source as being LINE-1 ribonucleoprotein particles. Inhibition of eRT in vitro in isolated platelets from healthy individuals or in people with HIV treated with RT inhibitors enhanced global protein synthesis and platelet activation. If HIV patients were treated with reverse transcriptase inhibitor, we found that platelets from these patients had increased basal activation. We next discovered that eRT activity in platelets controlled the generation of RNA–DNA hybrids, which serve as translational repressors. Inhibition of platelet eRT lifted this RNA–DNA hybrid-induced translational block and was sufficient to increase protein expression of target RNAs identified by RNA–DNA hybrid immunoprecipitation.Conclusions—Thus, we provide the first evidence that platelets possess L1-encoded eRT activity. We also demonstrate that platelet eRT activity regulates platelet hyperreactivity and thrombosis and controls RNA–DNA hybrid formation and identify that RNA–DNA hybrids function as a novel translational control mechanism in human platelets.

CD40L Deficiency Protects Against Aneurysm Formation [Original Research]


Objective—The mechanisms underlying formation of arterial aneurysms remain incompletely understood. Because inflammation is a common feature during the progressive degeneration of the aortic wall, we studied the role of the costimulatory molecule CD40L, a major driver of inflammation, in aneurysm formation.Approach and Results—Transcriptomics data obtained from human abdominal aortic aneurysms and normal aortas revealed increased abundance of both CD40L and CD40 in media of thrombus-free and thrombus-covered human abdominal aortic aneurysms samples. To further unravel the role of CD40L in aneurysm formation, apolipoprotein E–deficient (Apoe−/−) and Cd40l−/−Apoe−/− mice were infused with angiotensin II for 7 and 28 days. Only a minority of Cd40l−/−Apoe−/− mice (33% and 17%) developed (dissecting) aneurysms compared with 75% and 67% of Apoe−/− littermates after 7 and 28 days of infusion, respectively. Total vessel area of the aorta at the suprarenal level was 52% smaller in angiotensin II–infused Cd40l−/−Apoe−/− mice compared with that in angiotensin II–infused Apoe−/− mice. Chimeric Apoe−/− mice repopulated with Cd40l−/−Apoe−/− bone marrow afforded a similar protection against dissecting aneurysm formation. Moreover, lack of CD40L protected mice from fatal aneurysm rupture. T helper cell and macrophage accumulation in aneurysmal tissue was reduced in Cd40l−/−Apoe−/− mice with a concomitant decrease in expression of proinflammatory chemo- and cytokines. In addition, aneurysms of Cd40l−/−Apoe−/− mice displayed reduced abundance of matrix metalloproteinase-13 and an increase in tissue inhibitor of metalloproteinase-3 while activity of matrix metalloproteinase-2 and matrix metalloproteinase-9 was diminished.Conclusions—Deficiency of (hematopoietic) CD40L protects against dissecting aneurysm formation and reduces the incidence of fatal rupture. This is associated with a decreased accumulation and activation of inflammatory cells and a dampened protease activity in the arterial wall.

Kinesin-1 [Original Research]


Objective—Platelet secretion is crucial for many physiological platelet responses. Even though several regulators of the fusion machinery for secretory granule exocytosis have been identified in platelets, the underlying mechanisms are not yet fully characterized.Approach and Results—By studying a mouse model (cKO [conditional knockout]Kif5b) lacking Kif5b (kinesin-1 heavy chain) in its megakaryocytes and platelets, we evidenced unstable hemostasis characterized by an increase of blood loss associated to a marked tendency to rebleed in a tail-clip assay and thrombus instability in an in vivo thrombosis model. This instability was confirmed in vitro in a whole-blood perfusion assay under blood flow conditions. Aggregations induced by thrombin and collagen were also impaired in cKOKif5b platelets. Furthermore, P-selectin exposure, PF4 (platelet factor 4) secretion, and ATP release after thrombin stimulation were impaired in cKOKif5b platelets, highlighting the role of kinesin-1 in α-granule and dense granule secretion. Importantly, exogenous ADP rescued normal thrombin induced-aggregation in cKOKif5b platelets, which indicates that impaired aggregation was because of defective release of ADP and dense granules. Last, we demonstrated that kinesin-1 interacts with the molecular machinery comprising the granule-associated Rab27 protein and the Slp4 (synaptotagmin-like protein 4/SYTL4) adaptor protein.Conclusions—Our results indicate that a kinesin-1–dependent process plays a role for platelet function by acting into the mechanism underlying α-granule and dense granule secretion.

MicroRNA-21 Knockout Exacerbates Angiotensin II-Induced Thoracic Aortic Aneurysm and Dissection in Mice With Abnormal Transforming Growth Factor-{beta}-SMAD3 Signaling [Original Research]


Objective—Thoracic aortic aneurysm and dissection (TAAD) are severe vascular conditions. Dysfunctional transforming growth factor-β (TGF-β) signaling in vascular smooth muscle cells and elevated angiotensin II (AngII) levels are implicated in the development of TAAD. In this study, we investigated whether these 2 factors lead to TAAD in a mouse model and explored the possibility of using microRNA-21 (miR-21) for the treatment of TAAD.Approach and Results—TAAD was developed in Smad3 heterozygous (S3+/−) mice infused with AngII. We found that p-ERK– and p-JNK–associated miR-21 was higher in TAAD lesions. We hypothesize that downregulation of miR-21 mitigate TAAD formation. However, Smad3+/−:miR-21−/− (S3+/−21−/−) mice exhibited conspicuous TAAD formation after AngII infusion. The vascular wall was dilated, and aortic rupture occurred within 23 days during AngII infusion. We then examined canonical and noncanonical TGF-β signaling and found that miR-21 knockout in S3+/− mice increased SMAD7 and suppressed canonical TGF-β signaling. Vascular smooth muscle cells lacking TGF-β signals tended to switch from a contractile to a synthetic phenotype. The silencing of Smad7 with lentivirus prevented AngII-induced TAAD formation in S3+/−21−/− mice.Conclusions—Our study demonstrated that miR-21 knockout exacerbated AngII-induced TAAD formation in mice, which was associated with TGF-β signaling dysfunction. Therapeutic strategies targeting TAAD should consider unexpected side effects associated with alterations in TGF-β signaling.

Inhibition of Endothelial SCUBE2 (Signal Peptide-CUB-EGF Domain-Containing Protein 2), a Novel VEGFR2 (Vascular Endothelial Growth Factor Receptor 2) Coreceptor, Suppresses Tumor Angiogenesis [Original Research]


Objective—SCUBE2 (signal peptide-CUB-EGF domain-containing protein 2), expressed on the endothelial cell surface, functions as a novel coreceptor for VEGFR2 (vascular endothelial growth factor receptor 2) and enhances VEGF-induced signaling in adult angiogenesis. However, whether SCUBE2 plays a role in pathological angiogenesis and whether anti-SCUBE2 antibody is an effective strategy for blocking tumor angiogenesis remain unknown. The aim of this study was to investigate the pathological role and targeting therapy of SCUBE2 in tumor vasculature.Approach and Results—Immunohistochemistry revealed that SCUBE2 is highly expressed in endothelial cells of numerous carcinomas. Genetic endothelial cell knockout of SCUBE2 and pharmacological inhibition with the anti-SCUBE2 monoclonal antibody SP.B1 significantly reduced xenograft tumor growth, decreased tumor vascular density, increased apoptosis, and decreased the proliferation of tumor cells. Mechanistic studies revealed that SP.B1 binds to SCUBE2 and induces its internalization for lysosomal degradation, thereby reducing its cell surface level and inhibiting the binding of and downstream signaling of VEGF, including VEGFR2 phosphorylation and AKT/MAPK (mitogen-activated protein kinase) activation. Importantly, dual combination therapy with anti-SCUBE2 monoclonal antibody and anti-VEGF antibody or chemotherapy was more effective than single-agent therapy.Conclusions—Endothelial cell surface SCUBE2 is a VEGFR2 coreceptor essential for pathological tumor angiogenesis, and anti-SCUBE2 monoclonal antibody acting as an internalization inducer may provide a potent combination therapy for tumor angiogenesis.

Senescence Alters PPARG (Peroxisome Proliferator-Activated Receptor Gamma)-Dependent Fatty Acid Handling in Human Adipose Tissue Microvascular Endothelial Cells and Favors Inflammation [Original Research]


Objective—Adipose tissue (AT) dysfunction associated with obesity or aging is a major cause for lipid redistribution and the progression of cardiometabolic disorders. Our goal is to decipher the contribution of human AT microvascular endothelial cells (ECs) in the maintenance of fatty acid (FA) fluxes and the impact of senescence on their function.Approach and Results—We used freshly isolated primary microvascular ECs from human AT. Our data identified the endothelial FA handling machinery including FATPs (FA transport proteins) FATP1, FATP3, FATP4, and CD36 as well as FABP4 (FA binding protein 4). We showed that PPARG (peroxisome proliferator–activated receptor gamma) regulates the expression of FATP1, CD36, and FABP4 and is a major regulator of FA uptake in human AT EC (hATEC). We provided evidence that endothelial PPARG activity is modulated by senescence. Indeed, the positive regulation of FA transport by PPARG agonist was abolished, whereas the emergence of an inflammatory response was favored in senescent hATEC. This was associated with the retention of nuclear FOXO1 (forkhead box protein O1), whereas nuclear PPARG translocation was impaired.Conclusions—These data support the notion that PPARG is a key regulator of primary hATEC function including FA handling and inflammatory response. However, the outcome of PPARG activation is modulated by senescence, a phenomenon that may impact the ability of hATEC to properly respond to and handle lipid fluxes. Finally, our work highlights the role of hATEC in the regulation of FA fluxes and reveals that dysfunction of these cells with accelerated aging is likely to participate to AT dysfunction and the redistribution of lipids.

Particulate Matter Air Pollution and Racial Differences in Cardiovascular Disease Risk [Original Research]


Objective—We aimed to assess racial differences in air pollution exposures to ambient fine particulate matter (particles with median aerodynamic diameter <2.5 µm [PM2.5]) and black carbon (BC) and their association with cardiovascular disease (CVD) risk factors, arterial endothelial function, incident CVD events, and all-cause mortality.Approach and Results—Data from the HeartSCORE study (Heart Strategies Concentrating on Risk Evaluation) were used to estimate 1-year average air pollution exposure to PM2.5 and BC using land use regression models. Correlates of PM2.5 and BC were assessed using linear regression models. Associations with clinical outcomes were determined using Cox proportional hazards models, adjusting for traditional CVD risk factors. Data were available on 1717 participants (66% women; 45% blacks; 59±8 years). Blacks had significantly higher exposure to PM2.5 (mean 16.1±0.75 versus 15.7±0.73µg/m3; P=0.001) and BC (1.19±0.11 versus 1.16±0.13abs; P=0.001) compared with whites. Exposure to PM2.5, but not BC, was independently associated with higher blood glucose and worse arterial endothelial function. PM2.5 was associated with a higher risk of incident CVD events and all-cause mortality combined for median follow-up of 8.3 years. Blacks had 1.45 (95% CI, 1.00–2.09) higher risk of combined CVD events and all-cause mortality than whites in models adjusted for relevant covariates. This association was modestly attenuated with adjustment for PM2.5.Conclusions—PM2.5 exposure was associated with elevated blood glucose, worse endothelial function, and incident CVD events and all-cause mortality. Blacks had a higher rate of incident CVD events and all-cause mortality than whites that was only partly explained by higher exposure to PM2.5.

Impaired Collateral Vessel Formation in Sickle Cell Disease [Original Research]


Objective—The adaptive response to vascular injury is the formation of functional collateral vessels to maintain organ integrity. Many of the clinical complications associated with sickle cell disease can be attributed to repeated bouts of vascular insufficiency, yet the detailed mechanisms of collateral vessel formation after injury are largely unknown in sickle cell disease. Here, we characterize postischemic neovascularization in sickle cell disease and the role of neutrophils in the production of reactive oxygen species.Approach and Results—We induced hindlimb ischemia by ligation of the femoral artery in Townes SS (sickle cell) mice compared with AA (wild type) mice. Perfusion recovery, ascertained using LASER Doppler perfusion imaging, showed significant diminution in collateral vessel formation in SS mice after hindlimb ischemia (76±13% AA versus 34±10% in SS by day 28; P<0.001; n=8 per group). The incidence of amputation (25% versus 5%) and foot necrosis (80% versus 15%) after hindlimb ischemia was significantly increased in the SS mice. Motor function recovery evaluation by the running wheel assay was also impaired in SS mice (36% versus 97% at 28 days post-hindlimb ischemia; P<0.001). This phenotype was associated with persistent and excessive production of reactive oxygen species by neutrophils. Importantly, neutrophil depletion or treatment with the antioxidant N-acetylcysteine reduced oxidative stress and improved functional collateral formation in the SS mice.Conclusions—Our data suggest dysfunctional collateral vessel formation in SS mice after vascular injury and provide a mechanistic basis for the multiple vascular complications of sickle cell disease.