Coronary Endothelium Research Articles

Extracardiac septum transversum/proepicardial endothelial cells pattern embryonic coronary arterio–venous connections

Recent reports suggest that mammalian embryonic coronary endothelium (CoE) originates from the sinus venosus and ventricular endocardium. However, the contribution of extracardiac cells to CoE is thought to be minor and nonsignificant for coronary formation. Using classic (Wt1(Cre)) and previously undescribed (G2-Gata4(Cre)) transgenic mouse models for the study of coronary vascular development, we show that extracardiac septum transversum/proepicardium (ST/PE)-derived endothelial cells are required for the formation of ventricular coronary arterio-venous vascular connections. Our results indicate that at least 20% of embryonic coronary arterial and capillary endothelial cells derive from the ST/PE compartment. Moreover, we show that conditional deletion of the ST/PE lineage-specific Wilms' tumor suppressor gene (Wt1) in the ST/PE of G2-Gata4(Cre) mice and in the endothelium of Tie2(Cre) mice disrupts embryonic coronary transmural patterning, leading to embryonic death. Taken together, our results demonstrate that ST/PE-derived endothelial cells contribute significantly to and are required for proper coronary vascular morphogenesis.

Resistin Enhances Inflammatory Cytokine Production in Coronary Artery Tissues by Activating the NF-κB Signaling.

Purpose. Kawasaki disease (KD) is a systemic vasculitis and is a leading cause of coronary artery lesions (CALs) in childhood. Our previous study has shown higher levels of serum Resistin in KD patients with coronary aneurysm. This study aimed at examining the association of Resistin with inflammatory cytokine expression in mouse model of coronary arteritis and determining the potential mechanisms. Methods. C57BL/6 mice were injected with Lactobacillus cell wall extract (LCWE) to induce coronary arteritis. The relative levels of Resistin, TNF-α, IL-1β, and MMP-9 expression and inflammatory infiltrates in the coronary arteries were determined longitudinally by quantitative RT-PCR, ELISA, and histology. The effect of TLR4 and NF-κB activation on Resistin-induced TNF-α and IL-1β expression in human coronary artery endothelium cells (HCAECs) was examined by quantitative RT-PCR. Results. Higher levels of Resistin, TNF-α, IL-1β, and MMP-9 expression were associated with the degrees of inflammatory infiltrates in the coronary artery walls of the LCWE-injected mice. Resistin enhanced TNF-α and IL-1β expression in HCAECs at 18 or 24 hours after stimulation. Pretreatment with anti-TLR4 attenuated Resistin-enhanced IL-1β, but not TNF-α, expression and pretreatment with parthenolide or QNZ demolished Resistin-enhanced TNF-α expression in HACECs. Pretreatment with parthenolide, but not QNZ, blocked Resistin-enhanced IL-1β expression in HCAECs. Conclusion. Resistin may enhance inflammation by cross-talking with TLR4/NF-κB signaling during the development of coronary arteritis in mice.

Endothelial erosion of plaques as a substrate for coronary thrombosis.

Myocardial infarction is a prevalent, life-threatening consequence of athero-thrombosis. Post-mortem histology and intravascular imaging in live patients have shown that approximately one third of myocardial infarctions are caused by a thrombus overlying an intact, non-ruptured atherosclerotic plaque. Histology identifies erosion of luminal endothelial cells from smooth muscle and proteoglycan-rich, thick fibrous cap atheromas as the underlying pathology. Unlike plaque ruptures, endothelial erosions tend to occur on thick-capped atherosclerotic plaques and may or may not be associated with inflammation. Smoking and female gender are strong risk factors for erosion. Multiple mechanisms may contribute to endothelial erosion, including endothelial dysfunction, TLR signalling, leukocyte activation and modification of sub-endothelial matrix by endothelial or smooth muscle cells, which may trigger loss of adhesion to the extracellular matrix or endothelial apoptosis. Diagnosis of endothelial erosion by intravascular imaging, especially high resolution optical coherence tomography, may influence treatment strategies, offering prognostic value and utility as an endpoint in trials of agents designed to preserve an intact coronary endothelium.

Abstract 9742: Counteractive Effects of Omentin-1 Against Atherogenesis

Introduction: Omentin-1, a novel adipocytokine expressed in visceral fat tissue, is negatively correlated with obesity, insulin resistance, and stable coronary artery disease (CAD). However, there have been no previous reports regarding the effects of omentin-1 on atherogenesis. Objective and Methods: We aimed to evaluate the atheroprotective effects of omentin-1 on human monocyte-derived macrophages, human aortic smooth muscle cells (HASMCs) in vitro , and aortic lesions in apolipoprotein E-deficient (ApoE -/- ) mice in vivo . The histological expression of omentin-1 in coronary artery lesions and epicardial adipose tissues and its plasma levels were compared between CAD and non-CAD patients. Results: Omentin-1 was abundantly expressed in human umbilical vein endothelial cells, macrophages, HASMCs, and human coronary artery SMCs in vitro . Omentin-1 promoted antiinflammatory M2 phenotype via PPAR-γ upregulation and NF-κB downregulation during differentiation of human monocytes into macrophages. Omentin-1 suppressed oxidized LDL-induced foam cell formation associated with downregulation of CD36, scavenger receptor class A, and acyl-CoA:cholesterol acyltransferase-1 and upregulation of neutral cholesterol ester hydrolase in human macrophages. In HASMCs, omentin-1 suppressed angiotensin II-induced migration and PDGF-BB-induced proliferation, and collagen-1 and collagen-3 expressions via ERK1/2 and NF-κB downregulation. Four-week infusion of omentin-1 into ApoE -/- mice retarded the development of aortic atherosclerotic lesions with reduced contents of monocytes/macrophages, SMCs, and collagen fibers. In addition, it increased peritoneal M2-activated macrophages, downregulated inflammasomes such as CRP, NF-κB, COX-2, and apoptosis-associated speck-like protein containing a caspase recruitment domain, and lowered plasma total cholesterol levels in ApoE -/- mice. Omentin-1 levels were markedly reduced in coronary endothelium and epicardial fat but increased in plasma and atheromatous plaques (macrophages/SMCs) in CAD patients compared with non-CAD patients. Conclusions: Our translational research provided the first evidence that omentin-1 may serve as a novel therapeutic target for atherosclerosis and CAD.

Abstract 19979: Endothelium-specific Increase in ROS has Protective Effects on Vascular Endothelium in Ischemic Myocardium

Introduction: Increased ROS is often associated with vascular pathology. Recent findings demonstrated that increases in NADPH oxidase-derived endothelium (EC)-specific ROS improved coronary endothelial function by activating AMPK-eNOS signaling pathway. Here, we examined the effects of EC-ROS on vessel density in post-infarct ischemic myocardium. Hypothesis: We tested the hypothesis that increased EC-ROS induces AMPK-FOXO1-mediated overexpression of mitochondrial antioxidant MnSOD, which in turn has protective effects on vascular endothelium in ischemic myocardium. Methods: Our binary (Tet-ON/OFF) conditional transgenic mouse (Tet-Nox2:VE-Cad-tTA) induces 1.8±0.42-fold increase in NADPH oxidase-derived ROS in endothelium. Using these animals, we have induced myocardial infarction by LAD (left anterior descending) ligation. Results: Co-immunostaining of the ischemic myocardium using anti-CD31 and anti-SMA antibodies demonstrated that there was an increase in capillary density (by 38± 6.45; p<0.05) in Tet-Nox2:VE-Cad-tTA mice with high EC-ROS. Isolated mouse heart ECs showed an increase in AMPK-Foxo1-mediated expression of MnSOD in Tet-Nox2:VE-Cad-tTA mice compared to control. Together, these findings suggest that increase in EC-ROS increases mitochondrial antioxidant MnSOD, which in turn protects coronary endothelium in myocardial ischemia. Conclusion: This study demonstrates that the endothelial ROS may play an important role in myocardial preservation.

Candida glabrata binds to glycosylated and lectinic receptors on the coronary endothelial luminal membrane and inhibits flow sense and cardiac responses to agonists.

Candida glabrata (CG) is an opportunistic fungal pathogen that initiates infection by binding to host cells via specific lectin-like adhesin proteins. We have previously shown the importance of lectin-oligosaccharide binding in cardiac responses to flow and agonists. Because of the lectinic-oligosaccharide nature of CG binding, we tested the ability of CG to alter the agonist- and flow-induced changes in cardiac function in isolated perfused guinea pig hearts. Both transmission and scanning electron microscopy showed strong attachment of CG to the coronary endothelium, even after extensive washing. CG shifted the coronary flow vs. auricular-ventricular (AV) delay relationship upward, indicating that greater flow was required to achieve the same AV delay. This effect was completely reversed with mannose, partially reversed with galactose and N-acetylgalactosamine, but hyaluronan had no effect. Western blot analysis was used to determine binding of CG to isolated coronary endothelial luminal membrane (CELM) receptors, and the results indicate that flow-sensitive CELM receptors, ANG II type I, α-adrenergic 1A receptor, endothelin-2, and VCAM-1 bind to CG. In addition, CG inhibited agonist-induced effects of bradykinin, angiotensin, and phenylephrine on AV delay, coronary perfusion pressure, and left ventricular pressure. Mannose reversed the inhibitory effects of CG on the agonist responses. These results suggest that CG directly binds to flow-sensitive CELM receptors via lectinic-oligosaccharide interactions with mannose and disrupts the lectin-oligosaccharide binding necessary for flow-induced cardiac responses.

Neutrophil Elastase Promotes Interleukin-1β Secretion from Human Coronary Endothelium

The endothelium is critically involved in the pathogenesis of atherosclerosis by producing pro-inflammatory mediators, including IL-1β. Coronary arteries from patients with ischemic heart disease express large amounts of IL-1β in the endothelium. However, the mechanism by which endothelial cells (ECs) release IL-1β remains to be elucidated. We investigated neutrophil elastase (NE), a potent serine protease detected in vulnerable areas of human carotid plaques, as a potential "trigger" for IL-1β processing and release. This study tested the hypothesis that NE potentiates the processing and release of IL-1β from human coronary endothelium. We found that NE cleaves the pro-isoform of IL-1β in ECs and causes significant secretion of bioactive IL-1β via extracellular vesicles. This release was attenuated significantly by inhibition of neutrophil elastase but not caspase-1. Transient increases in intracellular Ca(2+) levels were observed prior to secretion. Inside ECs, and after NE treatment only, IL-1β was detected within LAMP-1-positive multivesicular bodies. The released vesicles contained bioactive IL-1β. In vivo, in experimental atherosclerosis, NE was detected in mature atherosclerotic plaques, predominantly in the endothelium, alongside IL-1β. This study reveals a novel mechanistic link between NE expression in atherosclerotic plaques and concomitant pro-inflammatory bioactive IL-1β secretion from ECs. This could reveal additional potential anti-IL-1β therapeutic targets and provide further insights into the inflammatory process by which vascular disease develops.

Rapamycin impairs endothelial cell function in human internal thoracic arteries.

BackgroundDefinitive fate of the coronary endothelium after implantation of a drug-eluting stent remains unclear, but evidence has accumulated that treatment with rapamycin-eluting stents impairs endothelial function in human coronary arteries. The aim of our study was to demonstrate this phenomenon on functional, morphological and biochemical level in human internal thoracic arteries (ITA) serving as coronary artery model.MethodsAfter exposure to rapamycin for 20 h, functional activity of ITA rings was investigated using the organ bath technique. Morphological analysis was performed by scanning electron microscopy and evaluated by two independent observers in blinded fashion. For measurement of endothelial nitric oxide synthase (eNOS) release, mammalian target of rapamycin (mTOR) and protein kinase B (PKB) (Akt) activation, Western blotting on human mammary epithelial cells-1 and on ITA homogenates was performed.ResultsComparison of the acetylcholine-induced relaxation revealed a significant concentration-dependent decrease to 66 ± 7 % and 36 ± 7 % (mean ± SEM) after 20-h incubation with 1 and 10 μM rapamycin. Electron microscopic evaluation of the endothelial layer showed no differences between controls and samples exposed to 10 μM rapamycin. Western blots after 20-h incubation with rapamycin (10 nM–1 μM) revealed a significant and concentration-dependent reduction of p (Ser 1177)-eNOS (down to 38 ± 8 %) in human mammary epithelial cells (Hmec)-1. Furthermore, 1 μM rapamycin significantly reduced activation of p (Ser2481)-mTOR (58 ± 11 %), p (Ser2481)-mTOR (23 ± 4 %) and p (Ser473)-Akt (38 ± 6 %) in ITA homogenates leaving Akt protein levels unchanged.ConclusionsThe present data suggests that 20-h exposure of ITA rings to rapamycin reduces endothelium-mediated relaxation through down-regulation of Akt-phosphorylation via the mTOR signalling axis within the ITA tissue without injuring the endothelial cell layer.

What can we learn about treating heart failure from the heart's response to acute exercise? Focus on the coronary microcirculation.

Coronary microvascular function and cardiac function are closely related in that proper cardiac function requires adequate oxygen delivery through the coronary microvasculature. Because of the close proximity of cardiomyocytes and coronary microvascular endothelium, cardiomyocytes not only communicate their metabolic needs to the coronary microvasculature, but endothelium-derived factors also directly modulate cardiac function. This review summarizes evidence that the myocardial oxygen balance is disturbed in the failing heart because of increased extravascular compressive forces and coronary microvascular dysfunction. The perturbations in myocardial oxygen balance are exaggerated during exercise and are due to alterations in neurohumoral influences, endothelial function, and oxidative stress. Although there is some evidence from animal studies that the myocardial oxygen balance can partly be restored by exercise training, it is largely unknown to what extent the beneficial effects of exercise training include improvements in endothelial function and/or oxidative stress in the coronary microvasculature and how these improvements are impacted by risk factors such as diabetes, obesity, and hypercholesterolemia.

Hiperplasia em túnicas de artérias coronárias de beijupirás criados em sistema offshore

As lesões coronarianas já foram relatadas em espécies de peixes anádromos, com características semelhantes às observadas em aterosclerose humana, mas não em peixes cultivados sem interferência de padrões reprodutivos. Objetivou-se descrever lesões coronarianas em beijupirá (Rachycentron canadum) criado em sistema offshore, em 54 exemplares coletados ao longo do cultivo de oito meses. Amostras de tecido cardíaco de alevinos e juvenis foram submetidas à análise histopatológica e à microscopia eletrônica de transmissão (MET), para visualização de lesões coronarianas, sendo as lâminas histológicas coradas pela técnica de hematoxilina-eosina, ácido periódico de Schiff, alcian blue e reticulina de Gomori. Exames realizados pela fotomicroscopia de luz evidenciaram lesões arteriais caracterizadas por hiperplasia das túnicas íntima e média de artérias coronárias em, respectivamente, 29,63% e 79,63% dos animais, com redução do lúmen arterial. Na MET, foram observadas alterações na estrutura das coronárias com espessamento do endotélio e membrana basal, proliferação de fibroblastos e fibras colágenas, com acúmulo de material lipídico subendotelial, resíduos celulares aderidos à membrana basal e vesículas pinocitóticas com presença de lisossomos isolados. Constatou-se que os peixes da espécie Rachycentron canadum criados em cativeiro desenvolvem lesão arterial do tipo crônica degenerativa inflamatória.

Cyclic phosphatidic acid inhibits the secretion of vascular endothelial growth factor from diabetic human coronary artery endothelial cells through peroxisome proliferator-activated receptor gamma

Atherosclerosis is a disease characterized by building up plaques formation and leads to a potentially serious condition in which arteries are clogged by fatty substances such as cholesterol. Increasing evidence suggests that atherosclerosis is accelerated in type 2 diabetes. Recent study reported that high level of alkyl glycerophosphate (AGP) was accumulated in atherosclerotic lesions. The presence of this phospholipid in mildly oxidized low-density lipoprotein (LDL) is likely to be involved in atherogenesis. It has been reported that the activation of peroxisome proliferator-activated receptor gamma plays a key role in developing atherosclerosis. Our previous result indicates that cyclic phosphatidic acid (cPA), one of bioactive lipids, potently suppresses neointima formation by inhibiting the activation of peroxisome proliferator-activated receptor gamma (PPARγ). However, the detailed mechanism is still unclear. In this study, to elucidate the mechanism of the cPA-PPARγ axis in the coronary artery endothelium, especially in patients with type 2 diabetes, we investigated the proliferation, migration, and secretion of VEGF in human coronary artery endothelial cells from diabetes patients (D-HCAECs). AGP induced cell growth and migration; however, cPA suppressed the AGP-elicited growth and migration in D-HCAECs. Moreover, AGP increased VEGF secretion from D-HCAECs, and this event was attenuated by cPA. Taken together, these results suggest that cPA suppresses VEGF-stimulated growth and migration in D-HCAECs. These findings could be important for regulatory roles of PPARγ and VEGF in the vascular processes associated with diabetes and atherosclerosis.

Heart failure with preserved ejection fraction: Defining the function of ROS and NO.

The understanding of complex molecular mechanisms underlying heart failure (HF) is constantly under revision. Recent research has paid much attention to understanding the growing number of patients that exhibit HF symptoms yet have an ejection fraction similar to a normal phenotype. Termed heart failure with preserved ejection fraction (HFpEF), this novel hypothesis traces its roots to a proinflammatory state initiated in part by the existence of comorbidities that create a favorable environment for the production of reactive oxygen species (ROS). Triggering a cascade that involves reduced nitric oxide (NO) availability, elevated ROS levels in the coronary endothelium eventually contribute to hypertrophy and increased resting tension in cardiomyocytes. Improved understanding of the molecular pathways associated with HFpEF has led to studies that concentrate on reducing ROS production in the heart, boosting NO availability, and increasing exercise capacity for HFpEF patients. This review will explore the latest research into the role of ROS and NO in the progression of HFpEF, as well as discuss the encouraging results of numerous therapeutic studies.

Ca2+‐dependent and Ceramide‐mediated Membrane Repair with Annexin V Recruitment and Aggregation in Mouse Endothelial Cells

Annexin V is able to interact with biological membranes and may repair damaged cell membrane. It remains unknown whether this annexin V‐associated cell membrane repair can protect coronary endothelial cells (CECs) from a variety of injuries and whether is attributed to a Ca2+‐dependent, ceramide‐mediated membrane repair in CECs. Using high energy Laser gun installed in confocal microscope, a tiny hole was made in CECs bathed with FM1‐43, and entry of FM1‐43 fluorescence was monitored to measure the extent of membrane injury. The cells exhibited a Ca2+ dependent, ceramide‐mediated membrane repair, as shown by reduction of FM1‐43 fluorescence within CECs if they are bathed with a Ca2+‐solution or pretreated by acid sphingomyelinase (ASM) inhibitor or siRNA. In CECs of ASM‐/‐ mice, addition of ceramide also rescued the membrane repair response. By introduction of RFP‐annexin V into CECs, it was found that RFP‐annexin V were recruited and aggregated on the edge of a hole and accompanied with less FM1‐43 into cells. This ceramide‐mediated membrane repair was also studies in mouse streptozotocin (STZ) diabetic model. Ceramide and annexin V decreased in the coronary arterial endothelium by confocal and undetectable in ASM‐/‐ coronary endothelium. Using YOYO‐1 and PI co‐staining with a time lag i.v. injection, confocal microscopic analysis of endothelial layer injury showed that more YOYO‐1 and PI staining on the arterial wall in STZ group, indicating less membrane resealing. These data show that Annexin V participated in a Ca2+‐dependent and ceramide‐mediated membrane resealing process in CECs. In diabetes, this membrane resealing may be damaged leading to endothelial dysfunction, injury and ultimate sclerosis.

0275 : Key role of eNOS in exercise-induced cardioprotection is dependent from coronary endothelium

To date, it is clearly accepted that the activation of endothelial isoform of NOS (eNOS) by exercise training constitutes a key trigger of exercise-induced cardioprotection against ischemia-reperfusion (IR). However, this enzyme is expressed both in coronary endothelial cells and cardiomyocytes, but the contribution of the one or the other one to such cardioprotection has never been challenged. The aim of this study was then to investigate the role of cardiomyocytes vs. endothelial eNOS in exercise cardioprotection. To this, rats were assigned to sedentary (Sed) or chronic aerobic exercised (Ex) group. Effects of exercise on vulnerability to IR or anoxia-reoxygenation (AR) were respectively evaluated at whole heart or cardiomyocytes and coronary artery levels. On a first set of rats, isolated cardiomyocytes were submitted to AR in presence or not of L-NAME, an eNOS inhibitor. Exercise reduced cells death and improved cells contractility after AR, but no effect of L-NAME was observed. Interestingly, exercise had no effect neither on eNOS phosphorylation on its activation site (Ser1177) nor on S-nitrosylation at cardiomyocytes level, whereas at whole heart level exercise increased both of them, suggesting that exercise impacted endothelial cells rather than cardiomyocytes. Then, to evaluate the contribution of endothelial cells on exercise-induced cardioprotection, on a Langendorff apparatus we treated hearts with Triton X-100 before IR to abolish coronary endothelial cells activity. Inactivation of endothelial cells totally suppressed cardioprotective effects of exercise. Finally, coronary arteries were isolated from hearts submitted to IR and endothelial function was assessed on a wire-myograph. We observed that alteration of endothelium-dependent coronary relaxation induced by IR was reduced in Ex group. In conclusion, these results show that coronary endothelial cells rather than cardiomyocytes play a key role in eNOS-dependent cardioprotection in Ex rat hearts.

Coronary endothelial dysfunction induced by nucleotide oligomerization domain-like receptor protein with pyrin domain containing 3 inflammasome activation during hypercholesterolemia: beyond inflammation.

This study hypothesized that activation of endothelial nucleotide oligomerization domain-like receptor protein with pyrin domain containing 3 (Nlrp3) inflammasomes directly produces endothelial dysfunction during hypercholesterolemia, which is distinct from its canonical roles in inflammation. Acute hypercholesterolemia in mice was induced by intraperitoneal administration of poloxamer 407 (0.5 g/kg) for 24 h. Endothelial dysfunction was assessed by evaluating endothelium-dependent vasodilation in isolated, perfused, and pressurized coronary arteries in response to bradykinin (10(-10)-10(-6) M) and acetylcholine (10(-9)-10(-5) M). Impaired endothelium-dependent vasodilation was observed in Nlrp3(+/+) mice with acute hypercholesterolemia, which was markedly ameliorated in Nlrp3(-/-) mice. Treatment of mice with inhibitors for caspase-1 or high mobility group box 1 (HMGB1) significantly restored endothelium-dependent vasodilation in Nlrp3(+/+) mice with acute hypercholesterolemia. Confocal microscopic analysis demonstrated that hypercholesterolemia markedly increased caspase-1 activity and HMGB1 expression in coronary arterial endothelium of Nlrp3(+/+) mice, which was absent in Nlrp3-deficient mice. Further, recombinant HMGB1 directly induced endothelial dysfunction in normal Nlrp3(+/+) coronary arteries. In vitro, Nlrp3 inflammasome formation and its activity were instigated in cultured endothelial cells by cholesterol crystal, a danger factor associated with hypercholesterolemia. Moreover, cholesterol crystals directly induced endothelial dysfunction in coronary arteries from Nlrp3(+/+) mice, which was attenuated in Nlrp3(-/-) arteries. Such cholesterol crystal-induced impairment was associated with enhanced superoxide production, downregulation of endothelial nitric oxide synthase activity, and pyroptosis. Our data provide the first evidence that activation of endothelial Nlrp3 inflammasome directly impairs endothelial function beyond its canonical inflammatory actions. This novel non-canonical action of Nlrp3 inflammasomes may initiate or exacerbate vascular injury during hypercholesterolemia.

Inflammatory bowel disease as a risk factor for premature coronary artery disease.

BackgroundCrohn’s disease and ulcerative colitis are both systemic chronic diseases that alter bowel physiology. The central process in inflammatory bowel disease (IBD) and the associated manifestations are the result of B-cell production of IgG autoantibodies directed against self-antigens in various organ systems including coronary endothelium. Previous studies have demonstrated significant micro-vascular endothelial dysfunction in patients with IBD compared to patients not affected by the disease. We sought to analyze the relation, if any, between IBD and the development of premature coronary artery disease (CAD).MethodsWe queried our hospital database to find IBD patients admitted to the hospital from January 1, 2007 to December 31, 2008. Patients with traditional cardiovascular (CV) disease risk factors including hypertension, congestive heart failure (CHF), diabetes, age ≥ 65, hyperlipidemia, family history, end-stage renal disease (ESRD), and greater than five pack-year smoking history were excluded from the study cohort. The charts of the remaining 300 patients with diagnosed IBD were then analyzed for the incidence of CV disease events including acute myocardial infarction (MI), unstable angina, positive stress testing, and any cardiac intervention including coronary angioplasty and/or intracoronary stent implantation.ResultsOf the 300 patients included, only one patient had a CV disease event. This patient had a positive exercise stress thallium test. Otherwise, the remaining 299 patients (99.7%) did not have any reported CV disease events over the 2-year follow-up period.ConclusionMost of the clinical sequelae of CV disease events are the result of inflammatory changes at the vascular level. While IBD is associated with a chronic inflammatory state as reflected by high sedimentation rates, C-reactive protein (CRP), homocysteine levels, etc., our data seem to indicate that chronic inflammation in the absence of traditional risk factors is not associated with an increased risk of premature CV disease events. More wide-scale prospective studies should be performed to elucidate the relationship, if any, between chronic inflammation and CV disease risk.

Abstract 11607: Novel Assay for Detection of LDL Transcytosis Across Coronary Endothelium Reveals an Unexpected Role for SR-B1

Introduction: Retention of LDL beneath the arterial endothelium initiates an inflammatory response culminating in atherosclerosis. How LDL crosses the endothelium to enter the arterial wall remains unknown. While LDL could conceivably pass between endothelial cells (paracellularly) or through them (transcytosis), electron microscopy studies in animals revealed LDL in intracellular vesicles and none at intercellular junctions. This, combined with the absence of endothelial injury or intercellular gaps in early atherosclerosis, suggests that transcytosis is the major route. However, technical challenges with studying transcytosis have made confirming and extending these findings difficult. We developed and validated a novel assay for measuring the transcytosis of native LDL across live human coronary artery endothelium in vitro. Using this assay, we propose to elucidate the regulation of LDL transcytosis and have identified a novel role for SR-B1. Methods and Results: Experiments were performed using primary human coronary artery endothelial monolayers. Transcytosis was quantified in single live cells in real time using total internal reflectance fluorescence microscopy. Transcytosis of LDL was saturable and inhibited by excess unlabeled LDL. By fluorescence microscopy we found that DiI-LDL colocalized significantly with scavenger receptor, class B, type 1 (SR-B1). Unexpectedly, overexpression of SR-BI resulted in increased LDL transcytosis, while knockdown of SR-BI by siRNA inhibited transcytosis. Excess HDL, the canonical SR-B1 ligand, also decreased LDL transcytosis. To confirm the occurrence of transcytosis in an intact vessel, we perfused murine aortas ex vivo with both LDL and dextran of a smaller molecular radius. We observed the accumulation of subendothelial LDL without dextran, indicating that transcytosis of LDL occurs in intact vessels. Conclusions: The accumulation of LDL in the subendothelial intima is the first step of atherosclerosis yet little is known about how it occurs. Our data suggests that transcytosis of LDL is an important contributor, particularly in the early stages of the disease. By identifying the mechanisms of transcytosis, our work could have important implications for its pathogenesis and therapy.

Abstract 19525: Knockout of CD38 Preserves Endothelial Function in the Postischemic Heart through Reduced Nicotinamide Adenine Dinucleotide Phosphate Salvage

Introduction: Cardiac ischemia/reperfusion (I/R) injury causes dysfunction in the coronary endothelium, resulting in loss of vascular relaxation and decreased tissue perfusion. Prior data with pharmacological inhibitors suggests that CD38 plays a role in I/R-induced vascular endothelial dysfunction (VED) through consumption of NADPH precursor NADP, limiting the supply of NADPH for nitric oxide (NO) production from endothelial NO synthase (eNOS) and decreasing NOS-dependent coronary flow (NDCF). In order to definitively determine the role of CD38 in cardiac I/R injury and endothelial dysfunction, we performed studies on hearts of wild type (WT) and CD38 -/- mice. Methods: Isolated Langendorff perfused hearts of WT (n=9) and CD38 -/- mice (n=7) were subjected to 30 min ischemia (I) and contractile function and coronary flow were monitored through 30 min reperfusion (R) with an intraventricular balloon and Doppler flow probe, respectively. At 30 min R, NDCF was measured with administration of NOS inhibitor L-NAME and then hearts were frozen for high performance liquid chromatography (HPLC) measurements of NADP(H) and NAD(H). Results: There was no difference in contractile function recovery between groups. However, hearts from CD38 -/- mice had enhanced recovery of both NADP(H) and NAD(H) (47.4 +/- 3.2 and 42.2 +/- 3.3% higher). Furthermore, at 30 min R, hearts from CD38 -/- mice had much higher post-ischemic recovery of coronary flow (14.64 +/- 3.50% higher) and NDCF (48.7 +/- 5.5% higher) compared to WT mice (Fig 1 & 2). Conclusion: Thus, we observe that CD38 activation is a key mechanism contributing to NADP(H) depletion and VED in the postischemic heart.

Endothelial Nlrp3 inflammasome activation associated with lysosomal destabilization during coronary arteritis

Inflammasomes play a critical role in the development of vascular diseases. However, the molecular mechanisms activating the inflammasome in endothelial cells and the relevance of this inflammasome activation is far from clear. Here, we investigated the mechanisms by which an Nlrp3 inflammasome is activated to result in endothelial dysfunction during coronary arteritis by Lactobacillus casei (L. casei) cell wall fragments (LCWE) in a mouse model for Kawasaki disease. Endothelial dysfunction associated with increased vascular cell adhesion protein 1 (VCAM-1) expression and endothelial–leukocyte adhesion was observed during coronary arteritis in mice treated with LCWE. Accompanied with these changes, the inflammasome activation was also shown in coronary arterial endothelium, which was characterized by a marked increase in caspase-1 activity and IL-1β production. In cultured endothelial cells, LCWE induced Nlrp3 inflammasome formation, caspase-1 activation and IL-1β production, which were blocked by Nlrp3 gene silencing or lysosome membrane stabilizing agents such as colchicine, dexamethasone, and ceramide. However, a potassium channel blocker glibenclamide or an oxygen free radical scavenger N-acetyl-l-cysteine had no effects on LCWE-induced inflammasome activation. LCWE also increased endothelial cell lysosomal membrane permeability and triggered lysosomal cathepsin B release into cytosol. Silencing cathepsin B blocked LCWE-induced Nlrp3 inflammasome formation and activation in endothelial cells. In vivo, treatment of mice with cathepsin B inhibitor also abolished LCWE-induced inflammasome activation in coronary arterial endothelium. It is concluded that LCWE enhanced lysosomal membrane permeabilization and consequent release of lysosomal cathepsin B, resulting in activation of the endothelial Nlrp3 inflammasome, which may contribute to the development of coronary arteritis.

Abstract 031: Novel Role of Nlrp3 Inflammasome Activation beyond Canonical Inflammation: Coronary Endothelial Injury and Repair

Inflammasomes are intracellular machinery turning on inflammatory response to danger factors. Recent studies reported that activation of inflammasomes also directly produce injurious action on cells or tissues independent of canonical inflammation. The present study hypothesized that activation of endothelial Nlrp3 inflammsome directly induces endothelial dysfunction at early stage of obesity without canonical inflammation yet. It was demonstrated that Nlrp3+/+ mice under 6-week high fat diet (HFD) had increased formation and activation of Nlrp3 inflammasome in coronary arterial endothelium compared to normal diet (ND)-treated mice, as shown by confocal microscopic detection of inflammasome aggregation and active caspase-1 formation. Ultrasound Doppler measurement showed that HFD markedly reduced bradykinin-induced increases in coronary blood flow by 95% in Nlrp3+/+ mice, but only by 45% in Nlrp3-/- mice. Vascular reactivity studies confirmed that HFD impaired endothelium-dependent vasodilation to acetylcholine (10-9-10-5 M) in freshly isolated coronary arteries of Nlrp3+/+ mice (maximum dilation: 53±3% vs. 84±3% of ND), but not in Nlrp3-/- mice (maximum dilation: 81±3% vs. 85±2% of ND). Similarly, visfatin (an injurious adipokine) simulates HFD-induced injurious action on endothelial dysfunction in normal Nlrp3+/+coronary arteries, which was attenuated by Nlrp3 gene deletion. In Nlrp3+/+ mice, HFD markedly increased the number of circulating endothelial progenitor cells (EPCs) in peripheral blood (1.73±0.69% vs. 0.46±0.05% of ND) and adhesion molecule VCAM-1 levels in coronary arterial endothelium. Nlrp3-/- mice fed HFD exibited higher level of circulating EPCs (4.57±1.27% vs. 0.32±0.04% of ND), but similar VCAM-1 expression. Together, these results suggest that endothelial Nlrp3 inflammasome activation in response to obesity or injurious adipokine produces endothelial dysfunction and inhibits EPC mobilization for endothelial repair, which are distinct from its canonical inflammatory responses such as inflammtory cell recruitment and infiltration in the vasculature. It is proposed that Nlrp3 inflammasome activation may trigger endothelial injury resulting in ultimate vascular inflammation and schlerosis.