Flow Cytometry Based Platelet Reactivity Testing to Predict the Occurrence of Per-operative Solid Microemboli During Carotid Endarterectomy.

OBJECTIVE: Peri-operative antiplatelet therapy (APT) aims to prevent thrombotic events such as stroke. High platelet reactivity ,despite the use use of APT, increases the risk of thrombotic events. Transcranial Doppler imaging (TCD) is used to detect peri-operative microembolic signals (MES) during carotid endarterectomy (CEA). Peri-operative MES are associated with an increased risk of procedural stroke and new silent lesions on diffusion weighted magnetic resonance imaging following surgery. The main components of TCD detected MES are platelet aggregates, and therefore patients displaying multiple MES during surgery could have benefited from more stringent APT. This study investigated whether the use of flow cytometry based platelet reactivity measurements were correlated with the incidence of pre-operative MES and thereby in the future suitable to predict patients at increased risk of peri-operative thrombotic events. METHODS: Bilateral TCD with MES detection was performed in 197 patients undergoing CEA. Platelet reactivity was assessed with a flow cytometry based platelet reactivity assay measuring platelet response in whole blood. High on treatment platelet reactivity status was assessed for all patients. The secondary outcome was major adverse cardiovascular events (MACE) within one year. RESULTS: In total, 197 patients were included, 49 had peri-operative MES. The platelet response to adenosine diphosphate (ADP) correlated with MES (p = .021), and high on treatment platelet reactivity after adenosine diphosphate stimulation was associated with MACE (OR 2.34, 95% confidence interval 1.126 - 4.890, p = .023). CONCLUSION: Pre-operative platelet reactivity determined by flow cytometry after ADP stimulation correlated with the occurrence of intra-operative MES and post-operative MACE. Clopidogrel treatment showed the most substantial effect on reducing MES frequency and platelet reactivity measured by flow cytometry.

Bone Marrow Ts65Dn Trisomy-Induced Changes in Platelet Functionality and Lymphocytopenia Do Not Impact Atherosclerosis Susceptibility in Mice.

The genetic disorder Down syndrome is associated with a decreased susceptibility for atherosclerotic cardiovascular disease. Hematological and immune abnormalities occur frequently in Down syndrome patients. We evaluated, in a preclinical setting, the impact of a Down syndrome-like hematological/immune phenotype on atherosclerosis susceptibility. Hereto, hypercholesterolemic low-density lipoprotein receptor knockout mice were transplanted with bone marrow from either a trisomic Ts65Dn mouse or euploid wild-type control and subsequently fed a Western-type diet to induce the development of atherosclerotic lesions. T and B cell concentrations were markedly reduced in blood of Ts65Dn bone marrow recipients (p < 0.001). Expression levels of the pro-atherogenic scavenger receptor CD36 were respectively 37% and 59% lower (p < 0.001) in trisomic monocytes and macrophages. However, these combined effects did not translate into an altered atherosclerosis susceptibility. Notably, blood platelet numbers were elevated in Ts65Dn bone marrow recipients (+57%; p < 0.001), which was paralleled by higher platelet GPVI protein expression (+35%; p < 0.001) and an enhanced collagen-induced platelet activation (p < 0.001). In conclusion, we have shown that providing mice with a Down syndrome-like hematological profile does not change the susceptibility to atherosclerosis. Furthermore, our studies have uncovered a novel effect of the trisomy on platelet functionality that may be relevant in human clinical settings.

High density lipoproteins mediate in vivo protection against staphylococcal phenol-soluble modulins.

Staphylococcus aureus virulence has been associated with the production of phenol-soluble modulins (PSMs). These PSMs have distinct virulence functions and are known to activate, attract and lyse neutrophils. These PSM-associated biological functions are inhibited by lipoproteins in vitro. We set out to address whether lipoproteins neutralize staphylococcal PSM-associated virulence in experimental animal models. Serum from both LCAT an ABCA1 knockout mice strains which are characterised by near absence of high-density lipoprotein (HDL) levels, was shown to fail to protect against PSM-induced neutrophil activation and lysis in vitro. Importantly, PSM-induced peritonitis in LCAT-/- mice resulted in increased lysis of resident peritoneal macrophages and enhanced neutrophil recruitment into the peritoneal cavity. Notably, LCAT-/- mice were more likely to succumb to staphylococcal bloodstream infections in a PSM-dependent manner. Plasma from homozygous carriers of ABCA1 variants characterized by very low HDL-cholesterol levels, was found to be less protective against PSM-mediated biological functions compared to healthy humans. Therefore, we conclude that lipoproteins present in blood can protect against staphylococcal PSMs, the key virulence factor of community-associated methicillin resistant S. aureus.

Sex differences in flow cytometry-based platelet reactivity in stable outpatients suspected of myocardial ischemia.

BACKGROUND: Antiplatelet therapy is the mainstay of secondary prevention of cardiovascular events. Studies suggest that women do not obtain equal therapeutic benefit from antiplatelet therapy compared with men. The link between sex differences in platelet biology and response to antiplatelet therapies is unclear. We therefore investigated the role of sex differences in platelet reactivity in a cohort of outpatients with chest pain, in response to treatment with antiplatelet agents. METHODS: Platelet reactivity was measured in 382 randomly selected patients participating in the Myocardial Ischemia Detection by Circulating Biomarkers (MYOMARKER) study, an observational cohort study of outpatients suspected of myocardial ischemia. In all patients, blood was collected during diagnostic workup, and platelet reactivity was assessed with a flow cytometry-based platelet activation test that quantifies both platelet degranulation (P-selectin expression) and platelet aggregation (fibrinogen binding to integrin αIIbß3) in whole blood. RESULTS: Platelet reactivity was higher in women compared with men when activated with protease activating receptor 1-activating peptide SFLLRN (PAR1-AP) and adenosine 5'-phosphate (ADP), independent of age, basal activation status, estimated glomerular filtration rate < 60, platelet count, statin use, the use of P2Y12 inhibitors, or the use of aspirin. P2Y12 inhibitor use strongly reduced fibrinogen binding after stimulation with PAR1-AP, but only slightly reduced platelet P-selectin expression. Calculation of the relative inhibition in P2Y12 users indicated 62% inhibition of the response toward ADP. Stratified analysis showed that women (n = 14) using P2Y12 inhibitors showed less inhibition of fibrinogen binding after PAR1-AP stimulation than men (n = 38) using P2Y12 inhibitors. CONCLUSIONS: These findings call for further study of differential effects of P2Y12 inhibitors in women with suspected myocardial ischemia.

Flow cytometric mepacrine fluorescence can be used for the exclusion of platelet dense granule deficiency.

BACKGROUND: δ-storage pool disease (δ-SPD) is a bleeding disorder characterized by a reduced number of platelet-dense granules. The diagnosis of δ-SPD depends on the measurement of platelet ADP content, but this test is time consuming and requires a relatively large blood volume. Flow cytometric analysis of platelet mepacrine uptake is a potential alternative, but this approach lacks validation, which precludes its use in a diagnostic setting. OBJECTIVES: To evaluate the performance of platelet mepacrine uptake as a diagnostic test for δ-SPD. PATIENTS/METHODS: Mepacrine fluorescence was determined with flow cytometry before and after platelet activation in 156 patients with a suspected platelet function disorder and compared with platelet ADP content as a reference test. Performance was analyzed with a receiver operating characteristic (ROC) curve. RESULTS: Eleven of 156 patients had δ-SPD based on platelet ADP content. Mepacrine fluorescence was inferior to platelet ADP content in identifying patients with δ-SPD, but both mepacrine uptake (area under the ROC curve [AUC] 0.87) and mepacrine release after platelet activation (AUC 0.80) had good discriminative ability. In our tertiary reference center, mepacrine uptake showed high negative predicitive value (97%) with low positive predictive value (35%). Combined with a negative likelihood ratio of 0.1, these data indicate that mepacrine uptake can be used to exclude δ-SPD in patients with a bleeding tendency. CONCLUSION: Mepacrine fluorescence can be used as a screening tool to exclude δ-SPD in a large number of patients with a suspected platelet function disorder.

Platelet RNA modules point to coronary calcification in asymptomatic women with former preeclampsia.

BACKGROUND AND AIMS: Women who develop preeclampsia during pregnancy are at a higher risk for developing cardiovascular disease. As platelets are affected by preeclampsia, we set out to identify whether platelets carry information in their transcriptome on cardiovascular risk in women with former preeclampsia. METHODS: Platelets were isolated from asymptomatic women with previous preeclampsia, who underwent screening with coronary computed tomography angiography. Platelet RNA was isolated and used to construct gene networks using an unbiased approach. Platelet gene modules assembled from the network were related to risk factors and clinical traits of these women, including coronary artery calcium scores (CACS). RESULTS: We found multiple gene modules which correlated with CACS (correlation coefficients: 0.44 to 0.59, p = 0.05 to 0.007). The genes from two clinically relevant modules were expressed at a higher level in the group with calcifications (p = 3.9 × 10-10 and 0.02) and enriched for platelet-related gene-sets such as platelet activation. The first of these modules was also enriched (ppermutation = 0.0546) for genes mapped to known coronary artery disease susceptibility loci. Additional unbiased network analyses in platelet RNA of patients with overt cardiovascular disease underlined the importance of the identified modules for disease by high preservation. (p = 1.6 × 10-9 to 1.7 × 10-47). CONCLUSIONS: We found platelet RNA modules that correlated with CACS in asymptomatic women with previous preeclampsia. Whether or not platelets directly contribute to this disease trajectory, or reflect the underlying plaque substrate remains to be determined, but enrichment for coronary artery disease susceptibility genes emphasizes the importance for the disease.

Peptide Antagonists for P-selectin Discriminate between Sulfatide-Dependent Platelet Aggregation and PSGL-1-Mediated Cell Adhesion.

BACKGROUND: Membrane-exposed sulfatides are proposed to contribute to P-selectin-dependent platelet aggregation. Here, we demonstrated that P-selectin-mediated platelet aggregation on a collagen-coated surface under flow indeed depended on sulfatides and that this interaction differed considerably from the interaction of P-selectin with P-selectin Glycoprotein Ligand-1 (PSGL-1), which underlies leukocyte-endothelium adhesion. METHODS AND RESULTS: Upon platelet activation, sulfatides were translocated to the platelet surface to form focal hot-spots. Interestingly, P-selectin was observed to exclusively interact with liposomes with a sulfatide density higher than 21% (w/w), indicating that the binding profile of P-selectin for sulfatide-rich liposomes was dependent on sulfatide density. Sulfatide-liposome binding to P-selectin and sulfatide/P-selectin-dependent platelet aggregation was blunted by peptide antagonists, carrying the EWVDV motif within N-terminal extensions, such as CDVEWVDVSC (half maximal inhibitory concentration IC50 = 0.2 µM), but not by the EWVDV core motif itself (IC50 > 1000 µM), albeit both being equally potent inhibitors of PSGL-1/P-selectin interaction (IC50= 7-12 µM). CONCLUSIONS: Our data suggest that the sulfatide/P-selectin interaction implicates multiple binding pockets, which only partly overlap with that of PSGL-1. These observations open ways to selectively interfere with sulfatide/P-selectin-dependent platelet aggregation without affecting PSGL-1-dependent cell adhesion.

Estrogen Contributions to Microvascular Dysfunction Evolving to Heart Failure With Preserved Ejection Fraction.

Heart failure with preserved ejection fraction (HFpEF) is a syndrome involving microvascular dysfunction. No treatment is available yet and as the HFpEF patient group is expanding due to the aging population, more knowledge on dysfunction of the cardiac microvasculature is required. Endothelial dysfunction, impaired angiogenesis, (perivascular) fibrosis and the pruning of capillaries (rarefaction) may all contribute to microvascular dysfunction in the heart and other organs, e.g., the kidneys. The HFpEF patient group consists mainly of post-menopausal women and female sex itself is a risk factor for this syndrome. This may point toward a role of estrogen depletion after menopause in the development of HFpEF. Estrogens favor the ratio of vasodilating over vasoconstricting factors, which results in an overall lower blood pressure in women than in men. Furthermore, estrogens improve angiogenic capacity and attenuate (perivascular) fibrosis formation. Therefore, we hypothesize that the drop of estrogen levels after menopause contributes to myocardial microvascular dysfunction and renders post-menopausal women more vulnerable for heart diseases that involve the microvasculature. This review provides a detailed summary of molecular targets of estrogen, which might guide future research and treatment options.

Hypercholesterolemia impairs megakaryopoiesis and platelet production in scavenger receptor BI knockout mice.

BACKGROUND AND AIMS: Thrombocytopenia in scavenger receptor BI (SR-BI) knockout mice is suggested to result from augmented platelet clearance induced by elevated intracellular unesterified cholesterol (UC) levels. We hypothesize that SR-BI deficiency may also influence platelet production at the level of its precursor cell in the bone marrow, the megakaryocyte. METHODS: In this study, we compared megakaryopoiesis and platelet production in SR-BI knockout and wild-type mice. RESULTS: In line with our hypothesis, megakaryocytes from SR-BI knockout mice exhibited UC accumulation while no accumulation of UC was detectable in wild-type megakaryocytes. Bone marrow expression of transcription factors involved in megakaryocyte maturation was induced, but megakaryocyte counts were unchanged in bone marrow of SR-BI knockout mice. Interestingly, we did find a striking 62% decrease (p < 0.01) in proplatelet production by SR-BI knockout megakaryocytes. SR-BI knockout mice displayed an impaired increase in circulating platelet concentrations and bone marrow megakaryocyte numbers upon thrombopoietin challenge. Importantly, megakaryocytes from normolipidemic bone marrow-specific SR-BI knockout mice exhibited a normal ability to produce proplatelets. Moreover, bone marrow-specific deletion of SR-BI did not impair the thrombopoietin response or induce thrombocytopenia, confirming that absence of megakaryocyte SR-BI does not underlie the thrombocytopenic phenotype in total body SR-BI knockout mice. CONCLUSIONS: In conclusion, the elevation of plasma unesterified cholesterol levels impairs megakaryopoiesis and platelet production in SR-BI knockout mice. Our findings suggest that, in addition to an increased platelet clearance, a decrease in platelet production may also, in part, explain the thrombocytopenic phenotype associated with SR-BI deficiency in mice.

Platelet function is disturbed by the angiogenesis inhibitors sunitinib and sorafenib, but unaffected by bevacizumab.

INTRODUCTION: At the clinical introduction of antiangiogenic agents as anticancer agents, no major toxicities were expected as merely just endothelial cells (ECs) in tumors would be affected. However, several (serious) toxicities became apparent, of which underlying mechanisms are largely unknown. We investigated to what extent sunitinib (multitargeted antiangiogenic tyrosine kinase inhibitor (TKI)), sorafenib (TKI) and bevacizumab [specific antibody against vascular endothelial growth factor (VEGF)] may impair platelet function, which might explain treatment-related bleedings. MATERIALS AND METHODS: In vitro, the influence of sunitinib, sorafenib, and bevacizumab on platelet aggregation, P-selectin expression and fibrinogen binding, platelet-EC interaction, and tyrosine phosphorylation of c-Src was studied by optical aggregation, flow cytometry, real-time perfusion, and western blotting. Ex vivo, platelet aggregation was analyzed in 25 patients upon sunitinib or bevacizumab treatment. Concentrations of sunitinib, VEGF, and platelet and EC activation markers were measured by LC-MS/MS and ELISA. RESULTS: In vitro, sunitinib and sorafenib significantly inhibited platelet aggregation (20 µM sunitinib: 71.3%, p < 0.001; 25 µM sorafenib: 55.8%, p = 0.042). Sorafenib and sunitinib significantly inhibited P-selectin expression on platelets. Exposure to both TKIs resulted in a reduced tyrosine phosphorylation of c-Src. Ex vivo, within 24 h sunitinib impaired platelet aggregation (83.0%, p = 0.001, N = 8). Plasma concentrations of sunitinib, VEGF, and platelet/EC activation markers were not correlated with disturbed aggregation. In contrast, bevacizumab only significantly impaired platelet aggregation in vitro at high concentrations, but not ex vivo. CONCLUSION: Sunitinib significantly inhibits platelet aggregation in patients already after 24 h of first administration, whereas bevacizumab had no effect on aggregation. These findings may explain the clinically observed bleedings during treatment with antiangiogenic TKIs.

Functional recovery of stored platelets after transfusion.

BACKGROUND: Platelet (PLT) concentrates are prophylactically given to prevent major bleeding complications. The corrected count increment (CCI) is currently the only tool to monitor PLT transfusion efficacy. PLT function tests cannot be performed in patients with thrombocytopenia. Therefore, an optimized agonist-induced assay was used to determine PLT function, in patients with severe thrombocytopenia before and after transfusion. STUDY DESIGN AND METHODS: PLT reactivity toward adenosine diphosphate (ADP), thrombin receptor-activating peptide SFLLRN (TRAP), and convulxin (CVX) was assessed by flow cytometry. P-selectin expression was measured on PLTs from 11 patients with thrombocytopenia before and 1 hour after transfusion, on stored PLTs, and on stored PLTs incubated for 1 hour in whole blood from patients ex vivo. RESULTS: The mean (±SEM) CCI after 1 hour was 11.4 (±1.5). After transfusion, maximal agonist-induced PLT P-selectin expression was on average 29% higher for ADP (p = 0.02), 25% higher for TRAP (p = 0.007), and 24% higher for CVX (p = 0.0008). ADP-induced reactivity of stored PLTs increased with 46% after ex vivo incubation (p = 0.007). These PLTs also showed an overall higher P-selectin expression compared to PLTs 1 hour after transfusion (p = 0.005). After normalization for this background expression, a similar responsiveness was observed. CONCLUSIONS: Our study shows recovery of PLT function after transfusion in patients with thrombocytopenia. The majority of functional PLTs measured after transfusion most likely represents stored transfused PLTs that regained functionality in vivo. The difference in baseline P-selectin expression in vivo versus ex vivo suggests a rapid clearance from circulation of PLTs with increased P-selectin expression.

The use of platelet reactivity testing in patients on antiplatelet therapy for prediction of bleeding events after cardiac surgery.

Many patients are treated with platelet inhibitors such as aspirin and clopidogrel for prevention of thrombotic cardiovascular events. However, the inhibitory effect of antiplatelet therapy is variable between patients; in some, the platelets are hardly inhibited, while in others, the platelets are excessively inhibited. The newer and more potent platelet inhibitors, prasugrel and ticagrelor, often lead to low platelet reactivity, which potentially leads to bleeding events. Preoperative measurement of platelet reactivity in patients receiving platelet inhibitors who undergo cardiac surgery, could be useful to identify those with low platelet reactivity and thus have an increased risk of bleeding during or after surgery. In this review, we discuss the most commonly used platelet inhibitors and platelet function tests. Furthermore, we will provide an overview of the evidence for the prediction of post-operative bleeding at the operation site with preoperative platelet reactivity testing in patients undergoing cardiac surgery.

LR11/SorLA links triglyceride-rich lipoproteins to risk of developing cardiovascular disease in FH patients.

OBJECTIVE: Familial Hypercholesterolemia (FH) is associated with an increased risk of cardiovascular disease (CVD). However, whether an individual heterozygous FH patient will develop CVD depends on other genetic- and environmental risk factors as well. LDL receptor-related protein with 11 ligand binding repeat (LR11) and its soluble form (sLR11) play a role in the progression of atherosclerosis. We investigated the involvement of LR11 and sLR11 in CVD development in FH patients and in LDLR deficient (Ldlr(-/-)) mice. APPROACH AND RESULTS: In statin-treated asymptomatic male heterozygous FH subjects, plasma sLR11 levels correlated with carotid intima-media thickness. Increased plasma sLR11 levels were found in Ldlr(-/-) and also in wild-type mice exclusively after high-fat feeding. Hepatic LR11 mRNA levels, however, were higher in chow-fed Ldlr(-/-) in comparison with wild-type mice and were further increased after a high fat diet. Similar results were obtained with Apoe(-/-) mice, but not with wild-type mice. LR11 mRNA and protein levels and release of sLR11 from cultured HepG2 and aortic smooth muscle cells were upregulated by postprandial triglyceride-rich lipoproteins (TGRL). Overexpression of human LR11 in CHO cells resulted in increased binding and association of 12I-labeled TGRL, but not of 12I-labeled LDL. CONCLUSION: Our data strongly suggest an involvement of LR11 in mediating the harmful effects of a high-fat diet on CVD progression. Elevated sLR11 levels may increase the CVD risk especially in subjects with delayed clearance of triglyceride-rich remnants, such as in FH patients.

Anti-citrullinated protein antibodies contribute to platelet activation in rheumatoid arthritis.

INTRODUCTION: Although the role of platelets in rheumatoid arthritis (RA) is relatively unexplored, recent studies point towards a contribution of platelets in arthritis. We set out to determine platelet phenotype in RA and studied whether this could be influenced by the presence of anti-citrullinated protein antibodies (ACPA). METHODS: Platelets from healthy controls were incubated in the presence of plasma of patients with RA or age- and sex-matched healthy controls and plasma from ACPA(neg) or ACPA(pos) patients or in the presence of plate-bound ACPA. Characteristics of platelets isolated from patients with RA were correlated to disease activity. RESULTS: Platelets isolated from healthy controls displayed markers of platelet activation in the presence of plasma derived from RA patients, as determined by P-selectin expression, formation of aggregates and secretion of soluble CD40 ligand (sCD40L). Furthermore, levels of P-selectin expression and sCD40L release correlated with high ACPA titres. In accordance with these findings, enhanced platelet activation was observed after incubation with ACPA(pos) plasma versus ACPA(neg) plasma. Pre-incubation of platelets with blocking antibodies directed against low-affinity immunoglobulin G receptor (FcγRIIa) completely inhibited the ACPA-mediated activation. In addition, expression of P-selectin measured as number of platelets correlated with Disease Activity Score in 44 joints, C-reactive protein level, ACPA status and ACPA level. CONCLUSIONS: We show for the first time that ACPA can mediate an FcγRIIa-dependent activation of platelets. As ACPA can be detected several years before RA disease onset and activated platelets contribute to vascular permeability, these data implicate a possible role for ACPA-mediated activation of platelets in arthritis onset.

High-density lipoprotein as a modulator of platelet and coagulation responses.

Platelets and coagulation factors are involved in the process of haemostasis, which ensures undisturbed blood flow upon vessel wall damage. However, excessive platelet aggregation and/or coagulation may lead to arterial or venous thrombosis. Pro-atherogenic lipoproteins, including native and oxidized low-density lipoprotein (LDL), are associated with an increased susceptibility to thrombosis. In contrast, numerous epidemiological studies have established an inverse correlation between high-density lipoprotein (HDL) levels and the risk for thrombosis. In addition to its role in reverse cholesterol transport, HDL also interacts with platelets, the coagulation cascade, and the vascular endothelium. Native HDL prevents platelet hyperreactivity by limiting intraplatelet cholesterol overload, as well as by modulating platelet signalling pathways after binding platelet HDL receptors such as scavenger receptor class B type I (SR-BI) and apoER2'. The antithrombotic properties of native HDL are also related to the suppression of the coagulation cascade and stimulation of clot fibrinolysis. Furthermore, HDL stimulates the endothelial production of nitric oxide and prostacyclin, which are potent inhibitors of platelet activation. Thus, HDL's antithrombotic actions are multiple and therefore, raising HDL may be an important therapeutic strategy to reduce the risk of arterial and venous thrombosis.

Supporting roles of platelet thrombospondin-1 and CD36 in thrombus formation on collagen.

OBJECTIVE: Platelets abundantly express the membrane receptor CD36 and store its ligand thrombospondin-1 (TSP1) in the α-granules. We investigated whether released TSP1 can support platelet adhesion and thrombus formation via interaction with CD36. APPROACH AND RESULTS: Mouse platelets deficient in CD36 showed reduced adhesion to TSP1 and subsequent phosphatidylserine expression. Deficiency in either CD36 or TSP1 resulted in markedly increased dissolution of thrombi formed on collagen, although thrombus buildup was unchanged. In mesenteric vessels in vivo, deficiency in CD36 prolonged the time to occlusion and enhanced embolization, which was in agreement with earlier observations in TSP1-deficient mice. Thrombi formed using wild-type blood stained positively for secreted TSP1. Releasate from wild-type but not from TSP1-deficient platelets enhanced platelet activation, phosphatidylserine expression, and thrombus formation on collagen. The enhancement was dependent on CD36 because it was without effect on thrombus formation by CD36-deficient platelets. CONCLUSIONS: These results demonstrate an anchoring role of platelet-released TSP1 via CD36 in platelet adhesion and collagen-dependent thrombus stabilization. Thus, the TSP1-CD36 tandem is another platelet ligand-receptor axis contributing to the maintenance of a stable thrombus.

Elimination of macrophages drives LXR-induced regression both in initial and advanced stages of atherosclerotic lesion development.

While numerous studies have aimed to develop strategies to inhibit the development and progression of atherosclerosis, recent attention has focussed on the regression of pre-existing atherosclerotic plaques. As important regulator of total body cholesterol homeostasis, the liver X receptor (LXR) could possibly be an important target to induce regression. Here, we describe the effect of LXR activation by the synthetic agonist T0901317 on lesion regression in different mouse models with early fatty streak lesions or advanced collagen-rich lesions. Although T0901317 caused a dramatic increase in plasma (V)LDL levels in low-density lipoprotein (LDL) receptor knockout mice, no further increase in lesion size was observed, which points to beneficial LXR activity in the vascular wall. In normolipidemic C57BL/6 mice with cholate diet-induced atherosclerotic lesions, T0901317 treatment improved plasma lipoprotein levels and induced lesion regression (-43%, p<0.05). Apolipoprotein E (APOE) reconstitution in APOE knockout mice by means of bone marrow transplantation dramatically improved plasma lipoprotein profiles and resulted in a marked regression of initial (-45%, p<0.001) and advanced lesions (-23%, p<0.01). Atherosclerosis regression was associated with a decrease in the absolute macrophage content (-84%, p<0.001). T0901317 supplementation further decreased the size of early (-71%, p<0.001 vs baseline; -48%, p<0.01 vs chow diet alone) and more advanced atherosclerotic lesions (-36%, p<0.001 and -17%, p=0.06 respectively). In conclusion, our study highlights the potential of LXR agonist T0901317 to stimulate removal of macrophages from atherosclerotic lesions ultimately leading to a highly significant plaque regression of both early and advanced atherosclerotic lesions.

Leukocyte-specific CCL3 deficiency inhibits atherosclerotic lesion development by affecting neutrophil accumulation.

OBJECTIVE: Despite common disbelief that neutrophils are involved in atherosclerosis, evidence is accumulating for a causal role of neutrophils in atherosclerosis. CC chemokine ligand (CCL)3 is an inflammatory chemokine and its expression is significantly increased during atherosclerotic lesion formation in mice. It has recently been shown that under conditions of inflammation neutrophils can migrate along a CCL3 gradient. In this study, we aimed to elucidate the role of leukocyte-derived CCL3 in atherogenesis. METHODS AND RESULTS: Irradiated low density lipoprotein receptor(-/-) mice, reconstituted with CCL3(-/-) or littermate bone marrow showed markedly reduced CCL3 response to lipopolysaccharide treatment, establishing the critical relevance of leukocytes as source of CCL3. Hematopoietic deficiency of CCL3 significantly reduced aortic sinus lesion formation by 31% after 12 weeks of western-type diet. Interestingly, whereas plaque macrophage, collagen, and vascular smooth muscle cell content were unchanged, neutrophil adhesion to and presence in plaques was significantly attenuated in CCL3(-/-) chimeras. These mice had reduced circulating neutrophil numbers, which could be ascribed to an increased neutrophil turnover and CCL3(-/-) neutrophils were shown to be less responsive toward the neutrophil chemoattractant CXC chemokine ligand 1. CONCLUSIONS: Our data indicate that under conditions of acute inflammation leukocyte-derived CCL3 can induce neutrophil chemotaxis toward the atherosclerotic plaque, thereby accelerating lesion formation.