Circulating MicroRNA-122-5p Is Associated With a Lack of Improvement in Left Ventricular Function After Transcatheter Aortic Valve Replacement and Regulates Viability of Cardiomyocytes Through Extracellular Vesicles.

BACKGROUND: Transcatheter aortic valve replacement (TAVR) is a well-established treatment option for high- and intermediate-risk patients with severe symptomatic aortic valve stenosis. A majority of patients exhibit improvements in left ventricular ejection fraction (LVEF) after TAVR in response to TAVR-associated afterload reduction. However, a specific role for circulating microRNAs (miRNAs) in the improvement of cardiac function for patients after TAVR has not yet been investigated. Here, we profiled the differential expression of miRNAs in circulating extracellular vesicles (EVs) in patients after TAVR and, in particular, the novel role of circulating miR-122-5p in cardiomyocytes. METHODS: Circulating EV-associated miRNAs were investigated by use of an unbiased Taqman-based human miRNA array. Several EV miRNAs (miR-122-5p, miR-26a, miR-192, miR-483-5p, miR-720, miR-885-5p, and miR-1274) were significantly deregulated in patients with aortic valve stenosis at day 7 after TAVR compared with the preprocedural levels in patients without LVEF improvement. The higher levels of miR-122-5p were negatively correlated with LVEF improvement at both day 7 (r=-0.264 and P=0.015) and 6 months (r=-0.328 and P=0.0018) after TAVR. RESULTS: Using of patient-derived samples and a murine aortic valve stenosis model, we observed that the expression of miR-122-5p correlates negatively with cardiac function, which is associated with LVEF. Mice with graded wire injury-induced aortic valve stenosis demonstrated a higher level of miR-122-5p, which was related to cardiomyocyte dysfunction. Murine ex vivo experiments revealed that miR-122-5p is highly enriched in endothelial cells compared with cardiomyocytes. Coculture experiments, copy-number analysis, and fluorescence microscopy with Cy3-labeled miR-122-5p demonstrated that miR-122-5p can be shuttled through large EVs from endothelial cells into cardiomyocytes. Gain- and loss-of-function experiments suggested that EV-mediated shuttling of miR-122-5p increases the level of miR-122-5p in recipient cardiomyocytes. Mechanistically, mass spectrometry, miRNA pulldown, electrophoretic mobility shift assay, and RNA immunoprecipitation experiments confirmed that miR-122-5p interacts with the RNA-binding protein hnRNPU (heterogeneous nuclear ribonucleoprotein U) in a sequence-specific manner to encapsulate miR-122-5p into large EVs. On shuttling, miR-122-5p reduces the expression of the antiapoptotic gene BCL2 by binding to its 3' untranslated region to inhibit its translation, thereby decreasing the viability of target cardiomyocytes. CONCLUSIONS: Increased levels of circulating proapoptotic EV-incorporated miR-122-5p are associated with reduced LVEF after TAVR. EV shuttling of miR-122-5p regulates the viability and apoptosis of cardiomyocytes in a BCL2-dependent manner.

Toll-like receptor-3 contributes to the development of aortic valve stenosis.

Aortic valve stenosis (AS) development is driven by distinct molecular and cellular mechanisms which include inflammatory pathways. Toll-like-receptor-3 (TLR3) is a lysosomal pattern-recognition receptor that binds double-stranded RNA and promotes pro-inflammatory cellular responses. In recent years, TLR3 has emerged as a major regulator of vascular inflammation. The exact role of TLR3 in the development of AS has not been investigated. Isolated human valvular interstitial cells (VICs) were stimulated with the TLR3-agonist polyIC and the resulting pro-inflammatory and pro-osteogenic response measured. Severe AS was induced in wildtype- and TLR3-/- mice via mechanical injury of the aortic valve with a coronary springwire. TLR3 activation was achieved by polyIC injection every 24 h after wire injury, while TLR3 inhibition was realized using Compound 4a (C4a) every 48 h after surgery. Endothelial mesenchymal transition (EndoMT) of human valvular endothelial cells (VECs) was assessed after polyIC stimulation. Stimulation of human VICs with polyIC promoted a strong inflammatory and pro-osteogenic reaction. Similarly, injection of polyIC marginally increased AS development in mice after wire injury. AS induction was significantly decreased in TLR3-/- mice, confirming the role of endogenous TLR3 ligands in AS pathology. Pharmacological inhibition of TLR3 with C4a not only prevented the upregulation of inflammatory cytokines and osteogenic markers in VICs, and EndoMT in VECs, but also significantly abolished the development of AS in vivo. Endogenous TLR3 activation significantly contributes to AS development in mice. Pharmacological inhibition of TLR3 with C4a prevented AS formation. Therefore, targeting TLR3 may be a viable treatment option.

Ceramide Metabolism in Cardiovascular Disease: A Network With High Therapeutic Potential.

Growing evidence suggests that ceramides play an important role in the development of atherosclerotic and valvular heart disease. Ceramides are biologically active sphingolipids that are produced by a complex network of enzymes. Lowering cellular and tissue levels of ceramide by inhibiting the ceramide-producing enzymes counteracts atherosclerotic and valvular heart disease development in animal models. In vascular tissues, ceramides are produced in response to hyperglycemia and TNF (tumor necrosis factor)-α signaling and are involved in NO-signaling and inflammation. In humans, elevated blood ceramide levels are associated with cardiovascular events. Furthermore, important cardiovascular risk factors, such as obesity and diabetes, have been linked to ceramide accumulation. This review summarizes the basic mechanisms of how ceramides drive cardiovascular disease locally and links these findings to the intriguing results of human studies on ceramides as biomarkers for cardiovascular events. Moreover, we discuss the current state of interventions to therapeutically influence vascular ceramide metabolism, both locally and systemically.

Analysis of circulating ceramides and hexosylceramides in patients with coronary artery disease and type II diabetes mellitus.

BACKGROUND: Cardiovascular disease (CVD) remains the leading cause of death worldwide. The main driving force behind this association is coronary artery disease (CAD), the manifestation of atherosclerosis in the coronary circulation. Cornerstones in the development of CAD are pathologies in lipid metabolism. In recent years, ongoing research has identified ceramides, a subclass of sphingolipids to be mediators of CVD. The aim of this study is to investigate the influence of type II diabetes mellitus (DM) on circulating ceramides and hexosylceramides (HexCers) in CAD patients. METHODS: 24 patients aged 40-90 years with CAD confirmed by angiography were included into a pilot study. Patients with DM were identified by analysis of discharge letters or other medical documents available at the study center. During coronary angiography, arterial blood samples were collected and quantification of sphingolipids in patient serum was performed by mass spectrometry. RESULTS: Statistical analysis showed nine significantly different HexCers in CAD patients with DM compared to patients without DM. Among the nine significantly regulated HexCers, we identified seven d18:1 HexCers. This group contributes to the fourth most abundant subgroup of total ceramides and HexCers in this dataset. HexCer-d18:1-23:1(2-OH) showed the strongest downregulation in the patient group with DM. CONCLUSION: This study suggests that levels of circulating HexCers are downregulated in patients with CAD and concomitant DM compared to patients without DM. Further research is needed to investigate the underlying mechanisms and the suitability of HexCers as possible mediators and/or prognostic markers in CAD.

Activation of neutral sphingomyelinase 2 through hyperglycemia contributes to endothelial apoptosis via vesicle-bound intercellular transfer of ceramides.

BACKGROUND: Pro-apoptotic and pro-inflammatory ceramides are crucially involved in atherosclerotic plaque development. Local cellular ceramide accumulation mediates endothelial apoptosis, especially in type 2 diabetes mellitus, which is a major cardiovascular risk factor. In recent years, large extracellular vesicles (lEVs) have been identified as an important means of intercellular communication and as regulators of cardiovascular health and disease. A potential role for lEVs as vehicles for ceramide transfer and inductors of diabetes-associated endothelial apoptosis has never been investigated. METHODS AND RESULTS: A mass-spectrometric analysis of human coronary artery endothelial cells (HCAECs) and their lEVs revealed C16 ceramide (d18:1-16:0) to be the most abundant ceramide in lEVs and to be significantly increased in lEVs after hyperglycemic injury to HCAECs. The increased packaging of ceramide into lEVs after hyperglycemic injury was shown to be dependent on neutral sphingomyelinase 2 (nSMase2), which was upregulated in glucose-treated HCAECs. lEVs from hyperglycemic HCAECs induced apoptosis in the recipient HCAECs compared to native lEVs from untreated HCAECs. Similarly, lEVs from hyperglycemic mice after streptozotocin injection induced higher rates of apoptosis in murine endothelial cells compared to lEVs from normoglycemic mice. To generate lEVs with high levels of C16 ceramide, ceramide was applied exogenously and shown to be effectively packaged into the lEVs, which then induced apoptosis in lEV-recipient HCAECs via activation of caspase 3. Intercellular transfer of ceramide through lEVs was confirmed by use of a fluorescently labeled ceramide analogue. Treatment of HCAECs with a pharmacological inhibitor of nSMases (GW4869) or siRNA-mediated downregulation of nSMase2 abrogated the glucose-mediated effect on apoptosis in lEV-recipient cells. In contrast, for small EVs (sEVs), hyperglycemic injury or GW4869 treatment had no effect on apoptosis induction in sEV-recipient cells. CONCLUSION: lEVs mediate the induction of apoptosis in endothelial cells in response to hyperglycemic injury through intercellular transfer of ceramides.

Atherosclerotic Conditions Promote the Packaging of Functional MicroRNA-92a-3p Into Endothelial Microvesicles.

RATIONALE: Microvesicle-incorporated microRNAs (miRs) are biomarkers and effectors of cardiovascular disease. Whether microvesicle-miR expression is regulated in coronary artery disease (CAD) or not is unknown. OBJECTIVE: Here, we explore the expression of circulating microvesicle-miRs in patients with CAD and investigate the role of microvesicle-miR in endothelial cells. METHODS AND RESULTS: Circulating microvesicles were isolated from patients' plasma by using ultracentrifugation. Electron microscopy was used to determine the size of the microvesicles. A Taqman miR array revealed certain microvesicle-miRs are significantly regulated in patients with stable CAD compared with patients with ACS. To validate the miR array results, 180 patients with angiographically excluded CAD (n=41), stable CAD (n=77), and acute coronary syndrome (n=62) were prospectively studied. Nine miRs involved in regulation of vascular performance-miR-126-3p, miR-222-3p, miR-let-7d-5p, miR-21-5p, miR-26a-5p, miR-92a-3p, miR-139-5p, miR-30b-5p, and miR-199a-5p-were quantified in circulating microvesicles by real-time polymerase chain reaction (PCR). Among these, miR-92a-3p was significantly increased in patients with CAD compared with non-CAD patients. Microvesicle-sorting experiments showed endothelial cells (ECs) were the major cell source for microvesicles containing miR-92a-3p. In vitro oxLDL (oxidized low-density lipoprotein) and IL-6 (interleukin-6) stimulation increased miR-92a-3p expression in parent ECs and upregulated the expression level of endothelial microvesicle (EMV)-incorporated miR-92a-3p. Labeling of miR-92a-3p and EMVs demonstrated that functional miR-92a-3p was transported into recipient ECs, which accelerated cell migration and proliferation. Knockdown of miR-92a-3p in EMVs abrogated EMV-mediated effects on EC migration, proliferation, and blocked vascular network formation in a matrigel plug. Polymerase chain reaction-based gene profiling showed that the expression of THBS1 (thrombospondin 1) protein-a target of miR-92a-3p and an inhibitor of angiogenesis-was significantly reduced in ECs by EMVs. Knockdown of miR-92a-3p in EMVs abrogated EMV-mediated inhibition of the THBS1 gene and protein expression. CONCLUSIONS: Atherosclerotic conditions promote the packaging of endothelial miR-92a-3p into EMVs. EMV-mediated transfer of functional miR-92a-3p regulates angiogenesis in recipient ECs by a THBS1-dependent mechanism.

Aortic Valve Stenosis: From Basic Mechanisms to Novel Therapeutic Targets.

Aortic valve stenosis is the most prevalent heart valve disease worldwide. Although interventional treatment options have rapidly improved in recent years, symptomatic aortic valve stenosis is still associated with high morbidity and mortality. Calcific aortic valve stenosis is characterized by a progressive fibro-calcific remodeling and thickening of the aortic valve cusps, which subsequently leads to valve obstruction. The underlying pathophysiology is complex and involves endothelial dysfunction, immune cell infiltration, myofibroblastic and osteoblastic differentiation, and, subsequently, calcification. To date, no pharmacotherapy has been established to prevent aortic valve calcification. However, novel promising therapeutic targets have been recently identified. This review summarizes the current knowledge of pathomechanisms involved in aortic valve calcification and points out novel treatment strategies.

Extracellular Vesicles in Cardiovascular Disease: Potential Applications in Diagnosis, Prognosis, and Epidemiology.

Extracellular vesicles originate from diverse subcellular compartments and are released in the extracellular space. By transferring their cargoes into target cells and tissues, they now emerge as novel regulators of intercellular communication between adjacent and remote cells. Because vesicle composition and biological content are specific signatures of cellular activation and injury, their potential as diagnostic and prognostic biomarkers has raised significant interest in cardiovascular diseases. Characterization of circulating vesicles- or nonvesicles-bound nucleic acids represents a valuable tool for diagnosing and monitoring cardiovascular diseases, recently referred to as a liquid biopsy. Circulating extracellular vesicles offer a noninvasive and almost continuous access to circulating information on the disease state in epidemiological investigations. Finally, genetic engineering and cell-specific application of extracellular vesicles could display a novel therapeutic option for the treatment of cardiovascular diseases. In this review, we summarize the current knowledge about extracellular vesicles as diagnostic and prognostic biomarkers, as well as their potential applications for longitudinal epidemiological studies in cardiovascular diseases.

Endothelial microparticle-promoted inhibition of vascular remodeling is abrogated under hyperglycaemic conditions.

BACKGROUND: Endothelial microparticles (EMPs) inhibit vascular remodeling by transferring functional microRNA (miRNA) into target vascular smooth muscle cells (VSMCs). Because EMPs are increased in diabetic patients and potentially linked to vascular complications in diabetes mellitus, we sought to determine whether effects of EMPs generated under high glucose concentration on vascular remodeling might differ from EMPs derived from untreated cells. METHODS AND RESULTS: EMPs were generated from human coronary endothelial cells (HCAEC) exposed to high glucose concentrations in order to mimic diabetic conditions. These EMPs were defined as 'hyperglycaemic' EMPs (hgEMPs) and their miRNA transfer capacity and functional effects were compared with EMPs generated from 'healthy' untreated HCAECs. In vitro, the intercellular transfer of antiproliferative miRNA-126-3p from ECs to VSMCs via EMPs was significantly reduced under hyperglycaemic conditions. Additionally, EMP-mediated inhibition of the miRNA-126-3p target LRP6 and of VSMC migration and proliferation was abrogated, when hgEMPs were used. In vivo, the inhibitory effect of EMPs on neointima formation, VSMC proliferation and macrophage infiltration was abolished in mice treated with hgEMPs. CONCLUSION: Pathological hyperglycaemic conditions weaken potentially protective intercellular communication mechanisms by affecting EMP content and function.

Intercellular transfer of miR-126-3p by endothelial microparticles reduces vascular smooth muscle cell proliferation and limits neointima formation by inhibiting LRP6.

BACKGROUND: Vascular smooth muscle cell (VSMC) proliferation is of importance in the pathogenesis of vascular diseases such as restenosis or atherosclerosis. Endothelial microparticles (EMPs) regulate function and phenotype of target endothelial cells (ECs), but their influence on VSMC biology is unknown. We aim to investigate the role of EMPs in the regulation of vascular smooth muscle cell (VSMC) proliferation and vascular remodeling. METHODS AND RESULTS: Systemic treatment of mice with EMPs after vascular injury reduced neointima formation in vivo. In vitro, EMP uptake in VSMCs diminished VSMC proliferation and migration, both pivotal steps in neointima formation. To explore the underlying mechanisms, Taqman microRNA-array was performed and miR-126-3p was identified as the predominantly expressed miR in EMPs. Confocal microscopy revealed an EMP-mediated miR-126 transfer into recipient VSMCs. Expression of miR-126 target protein LRP6, regulating VSMC proliferation, was reduced in VSMCs after EMP treatment. Importantly, genetic regulation of miR-126 in EMPs showed a miR-126-dependent inhibition of LRP6 expression, VSMC proliferation and neointima formation in vitro and in vivo, suggesting a crucial role of miR-126 in EMP-mediated neointima formation reduction. Finally, analysis of miR-126 expression in circulating MPs in 176 patients with coronary artery disease revealed a reduced PCI rate in patients with high miR-126 expression level, supporting a central role for MP-incorporated miR-126 in vascular remodelling. CONCLUSION: EMPs reduce VSMC proliferation, migration and subsequent neointima formation by delivering functional miR-126 into recipient VSMCs.

Exosomes as Diagnostic Biomarkers in Cardiovascular Diseases.

Exosomes play important roles in the development and progression of cardiovascular diseases by modulating intercellular communication. Contents and quantities of exosomes are variable under different pathological cardiovascular conditions. Based on these concepts, exosomes have been proposed as novel diagnostic biomarkers in cardiovascular diseases. However, many issues related with clinically applicable biomarkers remain unresolved. Within this chapter, we discuss the potential value, but also the current challenges using exosome numbers and contents as diagnostic and prognostic biomarker in diverse cardiovascular pathologies.

Vascular endothelial microparticles-incorporated microRNAs are altered in patients with diabetes mellitus.

BACKGROUND: Circulating microRNAs (miRs) are differentially regulated and selectively packaged into microparticles (MPs). We evaluated whether diabetes mellitus alters circulating vascular and endothelial MP-incorporated miRs expression levels. METHODS AND RESULTS: Circulating MPs were isolated from 135 patients with or without diabetes mellitus type II and characterized using flow cytometer and electron microscope. Nine miRs involved in the regulation of vascular performance-miR-126, miR-222, miR-let7d, miR-21, miR-30, miR-92a, miR-139, miR-199a and miR-26a-were quantified in circulating MPs by reverse transcription polymerase chain reaction. Among those, miR-126 and miR-26a were significantly reduced in diabetic patients compared to non-diabetic patients. Patients with low miR-26a and miR-126 levels were at higher risk for a concomitant coronary artery disease. MP-sorting experiments showed that endothelial cells were the major cell sources of MPs containing miR-126 and miR-26a, respectively. Finally, in accordance with our clinical results, in vitro experiments revealed that hyperglycemia reduces the packaging of miR-126 and miR-26a into EMPs. CONCLUSION: Diabetes mellitus significantly alters the expression of vascular endothelial miRs in circulating endothelial MPs with potential implications on vascular heath.

Endothelial microparticles reduce ICAM-1 expression in a microRNA-222-dependent mechanism.

Endothelial microparticles (EMP) are released from activated or apoptotic endothelial cells (ECs) and can be taken up by adjacent ECs, but their effect on vascular inflammation after engulfment is largely unknown. We sought to determine the role of EMP in EC inflammation. In vitro, EMP treatment significantly reduced tumour necrosis factor-α-induced endothelial intercellular adhesion molecule (ICAM)-1 expression on mRNA and protein level, whereas there was no effect on vascular cell adhesion molecule-1 expression. Reduced ICAM-1 expression after EMP treatment resulted in diminished monocyte adhesion in vitro. In vivo, systemic treatment of ApoE-/- mice with EMP significantly reduced murine endothelial ICAM-1 expression. To explore the underlying mechanisms, Taqman microRNA array was performed and microRNA (miR)-222 was identified as the strongest regulated miR between EMP and ECs. Following experiments demonstrated that miR-222 was transported into recipient ECs by EMP and functionally regulated expression of its target protein ICAM-1 in vitro and in vivo. After simulating diabetic conditions, EMP derived from glucose-treated ECs contained significantly lower amounts of miR-222 and showed reduced anti-inflammatory capacity in vitro and in vivo. Finally, circulating miR-222 level was diminished in patients with coronary artery disease (CAD) compared to patients without CAD. EMPs promote anti-inflammatory effects in vitro and in vivo by reducing endothelial ICAM-1 expression via the transfer of functional miR-222 into recipient cells. In pathological hyperglycaemic conditions, EMP-mediated miR-222-dependent anti-inflammatory effects are reduced.

Endothelial microparticle-mediated transfer of MicroRNA-126 promotes vascular endothelial cell repair via SPRED1 and is abrogated in glucose-damaged endothelial microparticles.

BACKGROUND: Repair of the endothelium after vascular injury is crucial for preserving endothelial integrity and preventing the development of vascular disease. The underlying mechanisms of endothelial cell repair are largely unknown. We sought to investigate whether endothelial microparticles (EMPs), released from apoptotic endothelial cells (ECs), influence EC repair. METHODS AND RESULTS: Systemic treatment of mice with EMPs after electric denudation of the endothelium accelerated reendothelialization in vivo. In vitro experiments revealed that EMP uptake in ECs promotes EC migration and proliferation, both critical steps in endothelial repair. To dissect the underlying mechanisms, Taqman microRNA array was performed, and microRNA (miR)-126 was identified as the predominantly expressed miR in EMPs. The following experiments demonstrated that miR-126 was transported into recipient human coronary artery endothelial cells by EMPs and functionally regulated the target protein sprouty-related, EVH1 domain-containing protein 1 (SPRED1). Knockdown of miR-126 in EMPs abrogated EMP-mediated effects on human coronary artery endothelial cell migration and proliferation in vitro and reendothelialization in vivo. Interestingly, after simulating diabetic conditions, EMPs derived from glucose-treated ECs contained significantly lower amounts of miR-126 and showed reduced endothelial repair capacity in vitro and in vivo. Finally, expression analysis of miR-126 in circulating microparticles from 176 patients with stable coronary artery disease with and without diabetes mellitus revealed a significantly reduced miR-126 expression in circulating microparticles from diabetic patients. CONCLUSIONS: Endothelial microparticles promote vascular endothelial repair by delivering functional miR-126 into recipient cells. In pathological hyperglycemic conditions, EMP-mediated miR-126-induced EC repair is altered.

Loss of ceramide synthase 5 inhibits the development of experimentally induced aortic valve stenosis.

AIM: Inflammation and calcification are hallmarks in the development of aortic valve stenosis (AVS). Ceramides mediate inflammation and calcification in the vascular tissue. The highly abundant d18:1,16:0 ceramide (C16) has been linked to increased cardiovascular mortality and obesity. In this study, we investigate the role of ceramide synthase 5 (CerS5), a critical enzyme for C16 ceramide synthesis, in the development of AVS, particularly in conjunction with a high-fat/high-cholesterol diet (Western diet, WD). METHODS: We used wild-type (WT) and CerS5-/- mice on WD or normal chow in a wire injury model. We measured the peak velocity to determine AVS development and performed histological analysis of the aortic valve area, immune cell infiltration (CD68 staining), and calcification (von Kossa). In vitro experiments involved measuring the calcification of human aortic valvular interstitial cells (VICs) and evaluating cytokine release from THP-1 cells, a human leukemia monocytic-like cell line, following CerS5 knockdown. RESULTS: CerS5-/- mice showed a reduced peak velocity compared to WT only in the experiment with WD. Likewise, we observed reduced immune cell infiltration and calcification in the aortic valve of CerS5-/- mice, but only on WD. In vitro, calcification was reduced after knockdown of CerS5 in VICs, while THP-1 cells exhibited a decreased inflammatory response following CerS5 knockdown. CONCLUSION: We conclude that CerS5 is an important mediator for the development of AVS in mice on WD and regulates critical pathophysiological hallmarks of AVS formation. CerS5 is therefore an interesting target for pharmacological therapy and merits further investigation.

Endothelial microparticle uptake in target cells is annexin I/phosphatidylserine receptor dependent and prevents apoptosis.

OBJECTIVE: Endothelial microparticles (EMP) are released from activated or apoptotic cells, but their effect on target cells and the exact way of incorporation are largely unknown. We sought to determine the uptake mechanism and the biological effect of EMP on endothelial and endothelial-regenerating cells. METHODS AND RESULTS: EMP were generated from starved endothelial cells and isolated by ultracentrifugation. Caspase 3 activity assay and terminal deoxynucleotidyl transferase dUTP nick end labeling assay showed that EMP protect target endothelial cells against apoptosis in a dose-dependent manner. Proteomic analysis was performed to identify molecules contained in EMP, which might be involved in EMP uptake. Expression of annexin I in EMP was found and confirmed by Western blot, whereas the corresponding receptor phosphatidylserine receptor was present on endothelial target cells. Silencing either annexin I on EMP or phosphatidylserine receptor on target cells using small interfering RNA showed that the uptake of EMP by human coronary artery endothelial cells is annexin I/phosphatidylserine receptor dependent. Annexin I-downregulated EMP abrogated the EMP-mediated protection against apoptosis of endothelial target cells. p38 activation was found to mediate camptothecin-induced apoptosis. Finally, human coronary artery endothelial cells pretreated with EMP inhibited camptothecin-induced p38 activation. CONCLUSIONS: EMP are incorporated by endothelial cells in an annexin I/phosphatidylserine receptor-dependent manner and protect target cells against apoptosis. Inhibition of p38 activity is involved in EMP-mediated protection against apoptosis.