Endothelial Microparticles Research Articles

Hot off the Press: Endothelial Microparticles and Systemic Complement Activation in Patients with Chronic Kidney Disease

Hot off the Press: Endothelial Microparticles and Systemic Complement Activation in Patients with Chronic Kidney Disease

Increased Neutrophil Activation and Plasma DNA Levels in Patients with Pre-Eclampsia.

Pre-eclampsia (PE) is a chronic inflammatory disease in pregnancy, which is associated with enhanced blood coagulation and high thrombotic risk. To date, the mechanisms underlying such an association are not fully understood. Previous studies reported high levels of plasma deoxyribonucleic acid (DNA) in PE women, but the cellular source of the circulation DNA remains unknown. In this study, we tested the hypothesis that activated neutrophils undergoing cell death, also called NETosis, may be responsible for the elevated plasma DNA levels in PE women. We analysed plasma samples from non-pregnant, normal pregnant and PE women and found high levels of double-stranded DNA, myeloperoxidase (an abundant neutrophil granular enzyme) and histones (the major nucleosome proteins) in PE-derived samples, indicating increased NETosis in the maternal circulation. The high plasma DNA levels positively correlated with enhanced blood coagulation in PE women. When isolated neutrophils from normal individuals were incubated with PE-derived plasma, an elevated NETosis-stimulating activity was detected. Further experiments showed that endothelial micro-particles, but not soluble proteins, in the plasma were primarily responsible for the NETosis-stimulating activity in PE women. These results indicate that circulating micro-particles from damaged maternal endothelium are a potent stimulator for neutrophil activation and NETosis in PE women. Given the pro-coagulant and pro-thrombotic nature of granular and nuclear contents from neutrophils, enhanced systemic NETosis may represent an important mechanism underlying the hyper-coagulability and increased thrombotic risk in PE.

Role of microparticles derived from monocytes, endothelial cells and platelets in the exacerbation of COPD.

BackgroundMicroparticles (MPs) are shedding membrane vesicles released from activated blood and endothelial cells under inflammatory conditions. The role of endothelial MPs (EMPs) in pathophysiology of COPD is relatively well known. However, the release and function of MPs of other cellular origins, eg, platelets, red blood cells and leukocytes, are not clearly evaluated in COPD.PurposeThe aim of this study was to measure EMPs and other cell-derived circulating MPs in stable and exacerbated COPD patients.Patients and methodsA total of 50 patients with COPD and 19 healthy volunteers were enrolled in the study. EMPs (CD31+, CD62E+) and platelet-derived (CD61+, CD41+, CD42a+, PAC1+), red blood cell-derived (GlyA+) and leukocyte-derived (CD45+, CD13+, CD14+, CD56+) MPs were measured. Flow cytometry (FC) was performed on Beckman Coulter FC500 analyzer. MP reference gate was set using 0.3–0.5–0.9 µm microbeads with MP size gates of 0.5–1.0 µm.ResultsAll the measured MPs were significantly (P<0.001) higher in COPD patients than in the controls. Furthermore, CD62E+, CD41+, CD42a+ and CD14+ MP values were significantly (P<0.001) increased in exacerbated COPD compared to stable COPD. These MPs showed significant (P<0.001) inverse correlation with FEV1/FVC, as well.ConclusionIn this study, we describe a reliable flow cytometric assay for MP analysis that was successfully applied in COPD. Besides EMPs, COPD is accompanied by an increased concentration of various MPs in the systemic circulation; particularly, platelet- and monocyte-derived MPs seem to be important in exacerbation.

Research on mechanism of PCS in damaging vascular endothelial cells and promoting formation of atherosclerosis via TLR4/TREM-1.

The study aimed to explore the effects of p-cresyl sulfate (PCS) of damaging vascular endothelial cells and promoting the formation of atherosclerosis in mice. The apolipoprotein E (ApoE)-/- mice were fed normally and with a high-fat diet; the ApoE-/- mice fed with high-fat diet were divided into two groups and treated with blank control and PCS, respectively. The aortic arch in each group was taken and underwent the oil red O staining, and the serum PCS content in each group was detected. The basic components of plaque were observed, including foam cells, lipid deposition, and cholesterol crystal. Moreover, human umbilical vein endothelial cells were cultured and divided into control group, PCS treatment group (PCS), PCS treatment with TLR4 overexpression group (PCS+TLR4+), and PCS treatment with TLR4 knock-out group (PCS+TLR4-). The degree of endothelial cell damage was detected using a cluster of differentiation CD42b-/CD31+ endothelial microparticles (EMPs), and expressions of Toll-like receptor 4 (TLR4), triggering receptor expressed on myeloid cells-1 (TREM-1), phosphorylated-endothelial nitric oxide synthase (p-eNOS), and tumor necrosis factor-α (TNF-α) in cells were detected via Polymerase Chain Reaction (PCR) and Western blotting. The serum PCS concentration in high-fat ApoE-/- mice was increased, and the aortic arch sections of ApoE-/- mice treated with PCS displayed the evident atherosclerotic plaques. Experimental results of human umbilical vein endothelial cells showed that the activity of human umbilical vein endothelial cells treated with PCS declined, the expression levels of TLR4, TREM-1, and TNF-α were increased, while that of p-eNOS was decreased. After the TLR4 knockout, the above effects of PCS were reversed. PCS damages vascular endothelial cells through TRL4/TREM-1, thereby accelerating the formation of atherosclerosis.

Loss of Renal Peritubular Capillaries in Hypertensive Patients Is Detectable by Urinary Endothelial Microparticle Levels.

Hypertension, an important cause of chronic kidney disease, is characterized by peritubular capillary (PTC) loss. Circulating levels of endothelial microparticles (EMPs) reflect systemic endothelial injury. We hypothesized that systemic and urinary PTC-EMPs levels would reflect renal microvascular injury in hypertensive patients. We prospectively measured by flow cytometry renal vein, inferior vena cava, and urinary levels of EMPs in essential (n=14) and renovascular (RVH; n=24) hypertensive patients and compared them with peripheral blood and urinary levels in healthy volunteers (n=14). PTC-EMPs were identified as urinary exosomes positive for the PTC marker plasmalemmal-vesicle-associated protein. In 7 RVH patients, PTC and fibrosis were also quantified in renal biopsy, and in 18 RVH patients, PTC-EMPs were measured again 3 months after continued medical therapy with or without stenting (n=9 each). Renal vein and systemic PTC-EMPs levels were not different among the groups, whereas their urinary levels were elevated in both RVH and essential hypertension versus healthy volunteers (56.8%±12.7% and 62.8%±10.7% versus 34.0%±17.8%; both P≤0.001). Urinary PTC-EMPs levels correlated directly with blood pressure and inversely with estimated glomerular filtration rate. Furthermore, in RVH, urinary PTC-EMPs levels correlated directly with stenotic kidney hypoxia, histological PTC count, and fibrosis and inversely with cortical perfusion. Three months after treatment, the change in urinary PTC-EMPs levels correlated inversely with a change in renal function ( r=-0.582; P=0.011). Therefore, urinary PTC-EMPs levels are increased in hypertensive patients and may reflect renal microcirculation injury, whereas systemic PTC-EMPs levels are unchanged. Urinary PTC-EMPs may be useful as novel biomarkers of intrarenal capillary loss.

Changes of microparticle levels before and after daunorubicin-based chemotherapy in patients with acute leukemia

Objective To observe the serum levels of endothelial microparticles (EMP) and tissue factor-bearing microparticles (TF+ MP) in patients with acute leukemia before and after daunorubicin-based chemotherapy. Methods From July 2012 to February 2013, 15 patients with newly diagnosed acute leukemia in Peking Union Medical College Hospital received DA (daunorubicin + cytarabine) regimen or VDCLP (vincristine + daunorubicin + cyclophosphamide + L-asparaginase + prednisone) regimen chemotherapy. There were 8 males and 7 females, and the median age of patients was 44 years old. Eleven patients were acute myeloid leukemia (M0 1 case, M1 1 case, M2 9 cases), and 4 were acute lymphocytic leukemia. The peripheral blood samples were taken before induction chemotherapy and after 3 days of daunorubicin. Levels of EMP and TF+ MP were assessed using flow cytometry. Results The serum EMP and TF+ MP levels were significantly higher after 3-day daunorubicin infusions than those before induction chemotherapy (28.94/μl vs. 10.74/μl, P = 0.001; 64.24/μl vs. 43.80/μl, P = 0.02). Conclusion Daunorubicin-based chemotherapy may cause increased numbers of EMP and TF+ MP in patients with acute leukemia. Key words: Leukemia, acute; Antineoplastic combined chemotherapy protocols; Daunorubicin; Endothelial particles; Tissue factor-bearing microparticles

The relationship between habitual dietary sodium intake and RAAS blockade on circulating microparticle levels in type two diabetes.

Low sodium intake is paradoxically associated with adverse cardiovascular outcomes in individuals with type 2 diabetes mellitus (T2D), possibly from renin-angiotensin-aldosterone system (RAAS) activation, leading to endothelial dysfunction. In the present study, we investigated the associations between habitual sodium intake and RAAS blockade on endothelial function by measuring circulating microparticles (MPs) in individuals with T2D. We conducted a prospective, cross-sectional study in 74 individuals with T2D. Habitual dietary sodium intake was estimated by using the mean of three corrected 24-h urine sodium excretion measurements (24hUNa). MP subtypes in platelet-free plasma were quantitated using flow cytometry. No associations between 24hUNa with levels of endothelial MPs were observed. Instead, a trend toward higher diabetes related CD36+/CD235a+ MP levels was associated with lower 24hUNa (rho = -0.23, P=0.05). When stratified according to tertiles of 24hUNa, platelet-derived CD42b+/CD41+ and CD42+/CD41+/Annexin V+ MPs were higher in the lowest tertile (24hUNa < 157 mmol/24 h) (P=0.02 respectively). Despite RAAS blockade being associated with lower levels of most MP subsets, it was not associated with lower MPs, in the setting of low sodium intake. Lower sodium intake is associated with higher circulating procoagulant MPs, but not with evidence of endothelial dysfunction in individuals with T2D.

A11518 Hemodialysis reduces levels of circulating microparticles in individuals with hypertension

Objectives: Individuals with end stage kidney disease (ESKD) and hypertension are at increased risk of cardiovascular complications. Circulating microparticles (MPs) are putative biomarkers of vascular injury and circulating levels of MPs are increased in both hypertension and ESKD. The purpose of this study was to determine the effect of hemodialysis on circulating MPs in individuals with and without hypertension. Methods: 37 individuals with hypertension and 17 normotensive individuals on conventional 3 times weekly hemodialysis were studied (age 61 ± 2 years). Plasma samples from were collected in a non-fasting state immediately before and after hemodialysis at the mid-week hemodialysis session. An FX 800 hemodiafilter (Fresenius Medical Care) was used. Levels of endothelial (CD144 + ), platelet (CD41 + ), leukocyte (CD45 + ) and total (Annexin V + ) microparticles were assessed by flow cytometry. Results: Across all participants, significant reductions were seen in circulating annexin V + ve MPs (3x107vs1.6x107, P < 0.01), platelet MPs (1.8x107vs1.0x107, P < 0.001), leukocyte microparticles (2.4x106vs 1.8x106, P < 0.05), and endothelial MPs (8.4x105vs5.9x105, P < 0.01). There were no differences in pre-dialysis or post-dialysis MP levels (total, endothelial, leukocyte, or platelet) between hypertensive and non-hypertensive individuals and the inter-dialytic increases in MP levels were not significantly different between hypertensive and non-hypertensive individuals. Conclusion: Hemodialysis is associated with reductions in circulating platelet, leukocyte, and endothelial MPs. There may be significant interdialytic variation in MP levels and this should be an important consideration in studying MPs as biomarkers in ESKD. The presence of hypertension does not appear to alter inter-dialytic increases in circulating MPs.

Expression and significance of endothelial microparticles in children with Henoch-Schönlein purpura

To study the expression and significance of endothelial microparticles (EMPs) in children with Henoch-Schönlein purpura (HSP). A total of 100 previously untreated children with HSP were classified to Henoch-Schönlein purpura nephritis (HSPN) group (n=40) and non-nephritis group (n=60). Thirty healthy children who underwent physical examination were enrolled as control group. Serum levels of EMPs, T helper 17 cells (Th17), and interleukin-17 (IL-17) were compared between groups. The HSPN and non-nephritis groups had significantly higher levels of Th17 and IL-17 than the control group, and the HSPN group had the highest levels (P<0.05). The HSPN and non-nephritis groups had a significantly higher level of EMPs than the control group, and the HSPN group had the highest level (P<0.05). In the HSPN group, the levels of Th17 and IL-17 were positively correlated with the level of EMPs (r=0.830 and 0.644 respectively; P<0.05). EMPs play an important role in the pathogenesis of HSP. The increase in EMPs might be one of the reasons for renal involvement in children with HSP.

A10228 Endothelial microparticles from vegf inhibitor (vegfi)-treated cancer patients mediates endothelial cell signaling and et-1 production

Objectives: Vascular endothelial growth factor receptor inhibitors (VEGFRi), used as anti-angiogenic drugs to treat cancer, causes hypertension where mechanisms are still not fully elucidated. Microparticles (MPs) are biomarkers of cell damage. MPs also act as biovectors and influence vascular cell signalling and function. We questioned whether plasma levels of endothelial MPs from cancer patients treated with VEGFi are elevated (possibly reflecting underlying vascular injury) and whether these MPs influence endothelial cell signalling. Methods: Plasma MPs were isolated from cancer patients before and after treatment with VEGFRi (sunitinib, pazopanib or sorafenib). Human aortic endothelial cells (HAEC) were stimulated with isolated MPs (10*6 MPs/mL). MPs were characterized by flow cytometry; protein and gene expression by immunoblotting and qPCR; ROS and NO production by lucigenin and immunofluorescence. Results: VEGFRi treatment increased plasma endothelial cells-derived MPs (1.5 fold; p < 0.05 vs before treatment). HAEC exposed to post-treatment MPs increased pre-pro-ET-1 mRNA (9.1 fold; p < 0.01). Post-treatment MPs also increased ROS generation (5 min: 1.2 fold, p < 0.01; 30 min: 1.6 fold, p < 0.01; 60 min: 1.8 fold, p < 0.05) and phosphorylation of the inhibitory site of eNOS (Thr495) in HAEC (1.8 fold; p < 0.05). NO levels in HAEC exposed to MPs were decreased (1.2 fold; p < 0.05), an effect blocked by an ETB antagonist BQ788. Inflammatory marker gene expression was increased only in HAEC exposed to post-treatment MPs (p < 0.01). Conclusion: In conclusion, our data demonstrate that VEGFi treatment causes an increase in circulating MPs and that play a role in endothelial cell signalling and ET-1 production. These molecular processes may contribute to vascular dysfunction and hypertension in VEGFi-treated cancer patients.

Moderate-intensity exercise reduces activated and apoptotic endothelial microparticles in healthy midlife women.

Endothelial microparticles (EMPs) are related to cardiovascular disease (CVD) risk. Risk factors for CVD increase with menopause, and greater cardiorespiratory fitness is generally expected to reduce CVD risk. The effects of habitual physical activity on endothelial health may be due in part to the effect of acute exercise on circulating EMPs. This study was performed to evaluate the effect of an acute bout of exercise on CD62E+ and CD31+/42b- EMPs in healthy fit midlife women at different menopausal stages. Healthy, active premenopausal (PRE), perimenopausal (PERI), and postmenopausal (POST) women completed a single bout of moderate-intensity treadmill exercise. Activated (CD62E+) and apoptotic (CD31+/42b-) EMPs were evaluated before and 30 min after exercise by using fluorescent activated cell sorting. In an exploratory analysis, these results were compared with data from low-fit peri- and postmenopausal women. Differences by group and time point were evaluated with repeated-measure ANOVAs. There was a reduction in the number of total microparticles ( P < 0.001), CD62E+ ( P = 0.003), and CD31+/42b- ( P < 0.001) EMPs/μl plasma following acute exercise. The percentage of CD62E+ EMPs increased with acute exercise ( P < 0.001), whereas the percentage of CD31+/42b- EMPs did not change ( P = 0.40). There was no effect of menopausal status on CD62E+or CD31+/42b- EMPs, or on total microparticles (all P > 0.05). The exploratory analysis revealed that low-fit women had similar changes in EMPs with acute exercise. We concluded that acute moderate-intensity exercise reduces CD62E+and CD31+/42b- EMPs, as well as total microparticles, in healthy midlife women. These effects occurred despite differences in menopausal status and fitness. NEW & NOTEWORTHY This study demonstrates that acute moderate-intensity exercise reduces activated and apoptotic endothelial microparticles in healthy midlife women.

High glucose-induced endothelial microparticles increase adhesion molecule expression on endothelial cells.

The experimental aim of this study was to determine, in vitro, the effects of glucose-induced EMPs on endothelial cell expression of E-selectin, intercellular cell adhesion molecule-1 (ICAM-1), vascular cell adhesion molecule-1 and platelet cell adhesion molecule-1 (PECAM-1). Human umbilical vein endothelial cells (HUVECs) were cultured (3rd passage) and plated in 6-well plates at a density of 5.0 × 105 cells/condition. HUVECs were incubated with media containing either 25mM d-glucose (concentration representing a hyperglycemic state) or 5mM d-glucose (normoglycemic condition) for 48h to generate EMPs. EMP identification (CD144+) and concentration were determined by flow cytometry. HUVECs (3 × 106 cells/condition) were treated with either high glucose-derived EMPs (hgEMPs) or normal glucose-derived (ngEMPs) for 24h and surface expression of E-selectin (CD62E-PE), ICAM-1 (CD54-FITC), VCAM-1 (CD106-APC) and PECAM-1 (CD31-BV) was assessed by flow cytometry and reported as mean fluorescent intensity (MFI). Hyperglycemic-derived EMPs induced significantly higher surface expression of E-selectin (2614 ± 132 vs. 2010 ± 204MFI), ICAM-1 (2110 ± 81 vs. 1688 ± 152MFI), VCAM-1 (3589 ± 431 vs. 2134 ± 386) and PECAM-1 (4237 ± 395 vs. 2525 ± 269MFI) on endothelial cells than EMPs from normoglycemic conditions. Microparticle-induced cell adhesion molecule expression provides potential novel mechanistic insight regarding the accelerated risk of atherosclerotic vascular disease associated with hyperglycemia.

Endothelial microparticles and complement activation in patients with chronic kidney disease

Endothelial microparticles and complement activation in patients with chronic kidney disease

Type 2 diabetes and older age contribute to elevated plasma microparticle concentrations independent of chronic stroke.

What is the central question of this study? What is the effect of chronic stroke on circulating microparticle populations, accounting for potential effects of age and type 2 diabetes? What is the main finding and its importance? Elevated concentrations of CD31+ /CD42b- and CD62E+ microparticles appear to be driven by type 2 diabetes but not chronic stroke and are associated with fasting glucose and triglyceride levels. Older age results in elevations in CD62E+ and CD34+ microparticle concentrations. These microparticles have been proposed as potential targets for diagnosing, treating and identifying the clinical progression and complications of type 2 diabetes. The elevated circulating concentration of endothelial microparticles (MPs) may provide an index of the extent and nature of cellular damage in chronic stroke. The purpose of this study was to determine the circulating concentrations of CD31+ /CD42b- , CD62E+ and CD34+ MPs in chronic stroke subjects, focusing on the effects of chronic stroke by comparison with both older adults without a history of stroke but with type 2 diabetes mellitus (T2DM) and older and young healthy controls. Plasma from three groups of sedentary older (50-75years) men and women (chronic stroke, T2DM or older healthy) as well as a group of younger (18-39years) healthy controls was isolated from fasting blood, and CD31+ /CD42b- , CD62E+ and CD34+ MPs were quantified using flow cytometry (n= 17/group). Concentrations of CD31+ /CD42b- and CD62E+ MPs were higher in the T2DM group (P< 0.05), but not chronic stroke, compared to older and younger healthy adults. CD62E+ MP and CD34+ MP concentrations were elevated in the older compared to younger adults (P< 0.05 for both). Sub-analyses excluding chronic stroke subjects who were also diagnosed with diabetes [stroke (diabetes- )] revealed lower CD31+ /CD42b- (P< 0.05) and CD62E+ (P= 0.08) MPs in the stroke (diabetes- ) group compared to the T2DM group. CD31+ /CD42b- MP and CD62E+ MP concentrations were each associated with fasting glucose levels and CD31+ /CD42b- MPs also were associated with triglyceride levels. As MPs have been proposed as potential targets for diagnosing, treating and identifying the clinical progression of T2DM, our study provides further support for the use of CD31+ /CD42b- and CD62E+ MPs in the clinical progression of T2DM and associated vascular complications.

Abstract 049: Endothelial-Derived Microparticles as Biomarkers and Mediators of Endothelial Cell Injury in VEGF Inhibitor-Treated Cancer Patients: Implications in Hypertension

Vascular endothelial growth factor receptor inhibitors (VEGFRi), used as anti-angiogenic drugs to treat cancer, induce severe hypertension although the underlying molecular mechanisms are still unclear. Endothelial microparticles (MPs) are biomarkers of endothelial injury and are also functionally active since they influence downstream target cell signalling and function. We questioned whether MP status is altered in cancer patients treated with VEGFRi and whether they influence endothelial cell function associated with vascular dysfunction in hypertension. Plasma MPs were isolated from cancer patients before and after treatment with VEGFRi. Human aortic endothelial cells (HAEC) were stimulated with human plasma-isolated MPs (10 6 MPs/mL). MP characterization was assessed by flow cytometry; protein and gene expression by immunoblotting and qPCR; ROS and NO production by lucigenin and immunofluorescence. Patients treated with VEGFRi had significantly increased in endothelial cell-derived MPs (EMP) (0.19±0.02 Pre vs. 0.31±0.04 Post-treatment). HAEC exposed to post-treatment MPs increased pre-pro-ET-1 mRNA (4.10±0.97 vs. 10.57±3.54), corroborating the raise in ET-1 levels (μg/mL: 631.9±46.1 vs. 780.2±37.2) observed in HAEC stimulated with vatalanib (VEGFRi). Post-treatment MPs increased ROS generation in HAEC (100.0 vs. 5 min- 123.4±7.7, 30 min- 162.4±22.5, 60 min- 183.9±44.7), effects that were attenuated by ET A and ET B receptor blockers. VEGFRi post-treatment MPs increased phosphorylation of the inhibitory site of eNOS (Thr 495 ) (100.0 vs. 181.3±18.7) and decreased NO levels in HAEC (100.0 vs. 72.7±9.9) which was inhibited by ET B receptor blockade (eNOS: 109.8±14.9; NO: 142.1±25.9). Gene expression of proinflammatory mediators was increased in HAEC exposed to post-treatment MPs (1.0 vs. TNF-α: 5.4±1.8, MCP-1: 2.1±0.3, iNOS: 3.1±0.8, COX2: 2.4±0.4, ICAM1: 2.7±0.6), effects inhibited by BQ123 and BQ788. In conclusion, our data identify EMPs as biomarkers of VEGFi-induced endothelial injury and important mediators of ET-1-sensitive redox-regulated endothelial cell signalling. These molecular processes may play a role in vascular dysfunction associated with hypertension in VEGFRi-treated cancer patients.

Circulating endothelial cells and microparticles as diagnostic and prognostic biomarkers in small-cell lung cancer

ObjectivesIt has been proposed that circulating endothelial cells (CECs) and microparticles (MPs) may be useful for the assessment of patients with non-small-cell lung cancer (NSCLC). However, little is known about the potential clinical relevance of these biomarkers in small-cell lung cancer (SCLC). Therefore, we investigated the utility of baseline levels of CECs and MPs in SCLC patients. Materials and methodsAn immunomagnetic separation (IMS) technique was used to isolate and quantify CECs in the peripheral blood, while plasma samples were analyzed using flow cytometry for the measurement of circulating MPs. ResultsWe prospectively collected data from 56 patients and 41 healthy individuals. Forty-three patients presented at initial diagnosis and 13 patients presented at relapse. Baseline levels of CECs and MPs were significantly higher in SCLC patients either at initial diagnosis or at relapse than in healthy subjects (p < 0.0002 and p < 0.007, respectively). However, estimated tumor volume (ETV) was significantly correlated with basal MP values (p < 0.0001) but not with pretreatment CECs (p = 0.57). The amount of baseline CECs and MPs was significantly lower in patients with an objective response (OR, n = 23) than in those with progressive disease (PD, n = 15) after treatment (p = 0.016 and 0.05, respectively). With cut-off values of 110 cells/mL for CECs and 1257 events/μL for MPs according to receiver operating characteristics (ROC) analysis, baseline levels of these biomarkers were not significantly correlated with either progression-free survival (PFS) or overall survival (OS). However, patients with 6-month PFS displayed significantly decreased pretreatment CEC counts (p = 0.042), whereas basal MP values significantly increased in 1-year survivors compared with those in non-survivors (p = 0.05). ConclusionOur results suggest that baseline CECs and MPs may be predictive biomarkers of tumor response and long-term survival in SCLC patients.

Natural Killer Cells Exhibit a Peculiar Phenotypic Profile in Systemic Sclerosis and Are Potent Inducers of Endothelial Microparticles Release.

The pathophysiology of systemic sclerosis (SSc) involves early endothelial and immune activation, both preceding the onset of fibrosis. We previously identified soluble fractalkine and circulating endothelial microparticles (EMPs) as biomarkers of endothelial inflammatory activation in SSc. Fractalkine plays a dual role as a membrane-bound adhesion molecule expressed in inflamed endothelial cells (ECs) and as a chemokine involved in the recruitment, transmigration, and cytotoxic activation of immune cells that express CX3CR1, the receptor of fractalkine, namely CD8 and γδ T cells and natural killer (NK) cells. We aimed to quantify circulating cytotoxic immune cells and their expression of CX3CR1. We further investigated the expression profile of NK cells chemokine receptors and activation markers and the potential of NK cells to induce EC activation in SSc. We performed a monocentric study (NCT 02636127) enrolling 15 SSc patients [15 females, median age of 55 years (39–63), 11 limited cutaneous form and 4 diffuse] and 15 healthy controls. Serum fractalkine levels were significantly increased in SSc patients. Circulating CD8 T cells numbers were decreased in SSc patients with no difference in their CX3CR1 expression. Circulating γδ T cells and NK cells numbers were preserved. CX3CR1 expression in CD8 and γδ T cells did not differ between SSc patients and controls. The percentage and level of CX3CR1 expression in NK cells were significantly lowered in SSc patients. Percentages of CXCR4, NKG2D, CD69-expressing NK cells, and their expression levels were decreased in NK cells. Conversely, CD16 level expression and percentages of CD16+ NK cells were preserved. The exposure of human microvascular dermic EC line (HMVEC-d) to peripheral blood mononuclear cells resulted in similar NK cells degranulation activity in SSc patients and controls. We further showed that NK cells purified from the blood of SSc patients induced enhanced release of EMPs than NK cells from controls. This study evidenced a peculiar NK cells phenotype in SSc characterized by decreased chemokine and activation receptors expression, that might reflect NK cells involvement in the pathogenic process. It also highlighted the role of NK cells as a potent mechanism inducing endothelial activation through enhanced EMPs release.

Endothelial Microparticles and Systemic Complement Activation in Patients With Chronic Kidney Disease.

BackgroundEndothelial microparticles are associated with chronic kidney disease (CKD) and complement activation. We hypothesized that the complement pathway is activated in patients with CKD via endothelial microparticles and that complement activation correlates with endothelial dysfunction in CKD.Methods and ResultsWe analyzed complement data of 30 healthy subjects, 30 patients with stage III/IV CKD, and 30 renal transplant recipients with stage III/IV CKD, evaluating the potential correlation of complement fragments with brachial artery flow–mediated dilation, Chronic Kidney Disease Epidemiology Collaboration glomerular filtration rate, and urinary albumin/creatinine ratio. Endothelial microparticles were characterized via proteomic analysis and compared between study groups. Complement fragment Ba was significantly increased in CKD and post–kidney transplant CKD. Plasma Ba levels correlated significantly with lower brachial artery flow–mediated dilation, lower Chronic Kidney Disease Epidemiology Collaboration glomerular filtration rate, and higher urinary albumin/creatinine ratio. Factor D levels were significantly higher in the plasma microparticles of patients with CKD versus healthy controls. Plasma microparticles isolated from patients with CKD and containing factor D activated the alternative pathway in vitro.ConclusionThe alternative complement pathway is activated in CKD and correlates with endothelial dysfunction and markers of CKD. Future studies are needed to evaluate whether endothelial microparticles with increased factor D play a pathologic role in CKD‐associated vascular disease.Clinical Trial Registration URL: http://www.clinicaltrials.gov. Unique identifier: NCT02230202.

INTRACARDIAC AND PERIPHERAL CIRCULATING ENDOTHELIAL MICROPARTICLES ARE ASSOCIATED WITH CENTRAL SYSTOLIC BLOOD PRESSURE IN PATIENTS WITH CORONARY ARTERY DISEASE

Objective: Circulating endothelial microparticles (EMPs) are expected to reflect underlying endothelial damage observed in coronary artery disease (CAD). However, it is not clear if and in what extent, EMPs increase in acute coronary syndromes (ACS) compared to stable CAD and whether they reflect local or systemic cardiovascular damage. Design and method: We enrolled consecutive adult patients with a clinical indication for coronary angiography in our Cardiology Department during a 1-year period. Central blood pressure (cBP) measured during angiography and patients’ history were recorded. Coronary sinus and peripheral blood samples were collected. Flow cytometry protocol was standardized based on previous studies. Double-positive events for Annexin and CD144 in the microparticles region were measured and quantified using Flow Count Fluorospheres. Results: We have studied 38 patients with ACS (22 STEMI and 16 NSTEMI), 17 patients with stable CAD and 6 control patients (Table 1). We found significantly increased EMPs in patients with CAD compared to controls, both in peripheral (p = 0.001) and coronary circulation (p = 0.003). This confirmed the validity of our method and the absence of artificial or ex vivo activation. Then, we analyzed EMPs in ACS versus stable CAD. ACS patients had significantly increased EMPs both in peripheral (p = 0.008) and coronary circulation (p = 0.045), compared to stable CAD (Figure 1). No difference was found in patients with STEMI versus NSTEMI. Furthermore, coronary EMPs were significantly increased compared to peripheral (p = 0.021). Although no association was found with traditional cardiovascular risk factors, both peripheral (r = 0.489, p = 0.003) and coronary (r = 0.312, p = 0.048) EMPs were associated with cSBP.Conclusions: EMPs are increased in patients with ACS versus stable CAD and exhibit a profound increase in the coronary circulation compared to periphery, indicative of ongoing endothelial damage during acute myocardial ischemia. The extent of this damage may have implications regarding treatment and prognosis in these patients. The association between EMPs and cSBP possibly reflects an effect of endothelial dysfunction on central hemodynamics and aortic stiffness. Further prospective studies are needed to better clarify the role of microparticles as prognostic and possibly therapeutic targets.

Replacement of dietary saturated fat with unsaturated fats increases numbers of circulating endothelial progenitor cells and decreases numbers of microparticles: findings from the randomized, controlled Dietary Intervention and VAScular function (DIVAS) study

Endothelial progenitor cells (EPCs) and microparticles are emerging as novel markers of cardiovascular disease (CVD) risk, which could potentially be modified by dietary fat. We have previously shown that replacing dietary saturated fatty acids (SFAs) with monounsaturated or n-6 (ω-6) polyunsaturated fatty acids (MUFAs or PUFAs, respectively) improved lipid biomarkers, blood pressure, and markers of endothelial activation, but their effects on circulating EPCs and microparticles are unclear. The Dietary Intervention and VAScular function (DIVAS) Study investigated the replacement of 9.5-9.6% of total energy (%TE) contributed by SFAs with MUFAs or n-6 PUFAs for 16 wk on EPC and microparticle numbers in United Kingdom adults with moderate CVD risk. In this randomized, controlled, single-blind, parallel-group dietary intervention, men and women aged 21-60 y (n=190) with moderate CVD risk (≥50% above the population mean) consumed 1 of three 16-wk isoenergetic diets. Target compositions for total fat, SFAs, MUFAs, and n-6 PUFAs (%TE) were as follows: SFA-rich diet (36:17:11:4; n=64), MUFA-rich diet (36:9:19:4; n=62), and n-6 PUFA-rich diet (36:9:13:10; n=66). Circulating EPC, endothelial microparticle (EMP), and platelet microparticle (PMP) numbers were analyzed by flow cytometry. Dietary intake, vascular function, and other cardiometabolic risk factors were determined at baseline. Relative to the SFA-rich diet, MUFA- and n-6 PUFA-rich diets decreased EMP (-47.3%, -44.9%) respectively and PMP (-36.8%, -39.1%) numbers (overall diet effects, P<0.01). The MUFA-rich diet increased EPC numbers (+28.4%; P=0.023). Additional analyses that used stepwise regression models identified the augmentation index (measuring arterial stiffness determined by pulse-wave analysis) as an independent predictor of baseline EPC and microparticle numbers. Replacement of 9.5-9.6%TE dietary SFAs with MUFAs increased EPC numbers, and replacement with either MUFAs or n-6 PUFAs decreased microparticle numbers, suggesting beneficial effects on endothelial repair and maintenance. Further studies are warranted to determine the mechanisms underlying the favorable effects on EPC and microparticle numbers after SFA replacement. This trial was registered at www.clinicaltrials.gov as NCT01478958.