Endothelial Cell-derived Microvesicles Research Articles

Abstract P523: Circulating Endothelial-Cell Derived Microvesicles Associated With Insufficient Sleep Impairs Cerebral Microvascular Fibrinolytic Capacity

Chronic insufficient sleep (< 7 hour/night) is associated with increased risk of ischemic stroke. Endothelial cell dysfunction, specifically impairment endothelial release of tissue-type plasminogen activator (t-PA), represents an important mechanism underlying cerebrovascular events such as ischemic stroke. Endothelial cells are the primary site of synthesis and release of t-PA, the plasminogen activator in fibrinolysis and, thus, a key regulator of endogenous thrombolysis. We have previously demonstrated that insufficient sleep is associated with impaired endothelial capacity to release t-PA, however the mechanisms for the sleep-related fibrinolytic impairment are not well understood. Clinical interest in endothelial cell-derived microvesicles (EMVs) has intensified due to their involvement in the development of endothelial dysfunction and, in turn, cardiovascular and cerebrovascular events. The aim of this study was to determine, in vitro, the effects of circulating EMVs isolated from adults with chronic insufficient sleep on brain endothelial cell t-PA release. Considering EMVs are known to cross the blood brain barrier and are associated with ischemic stroke incidence, severity and outcome; circulating EMVs associated with insufficient sleep may be a mediator of stroke risk. Twenty-four healthy, non-obese, normotensive, sedentary adults (age: 23-69 years) were studied: 12 normal nightly sleep duration (7M/5F; age: 47±5 yr; nightly sleep: 7.4±0.1 hour/night) and 12 with short nightly sleep duration (9M/3F; 53±4 yr; 5.9±0.2 hour/night). EMV identification (CD144+) and isolation from peripheral blood was performed by flow cytometry. Human cerebral microvascular endothelial cells (hCMECs) were cultured and separately treated with EMVs from each subject. Cultured hCMECs were treated with EMVs in the absence and presence of thrombin (1 unit/mL) for 24 hours. Intracellular expression of t-PA was significantly lower (~30%) in hCMECs treated with EMVs from adults with insufficient sleep (31±3 AU) compared with cells treated with EMVs from normal sleep adults (40±3 AU). Although the effect of EMVs on basal t-PA release was not different between the groups; thrombin-stimulated t-PA release was significantly lower in cells treated with EMVs from adults with insufficient sleep (from 57±3 to 59±4 ng/mL) compared with EMVs from adults with normal sleep (from 53±3 to 66±4 ng/mL). The capacity of hCMECs to release t-PA in response to thrombin was ~75% lower when treated with EMVs from adults with insufficient sleep. These results indicate that circulating EMVs associated with chronic insufficient sleep diminishes brain endothelial cell t-PA production and impairs t-PA release. Circulating EMVs represent a mechanistic factor in insufficient sleep-related fibrinolytic dysfunction and ischemic stroke risk.

Effects of circulating endothelial microvesicles isolated from adults with obesity on endothelial cell inflammation, apoptosis, and nitric oxide production.

Circulating endothelial cell-derived microvesicles (EMVs) have been shown to be elevated with obesity and associated with endothelial dysfunction; however, their direct effect on endothelial cells is unknown. The experimental aim of this study was to determine the effect of EMVs isolated from adults with obesity on endothelial cell inflammation, apoptosis, and nitric oxide (NO) production. EMVs (CD144+ microvesicles) were identified, enumerated, and isolated from plasma by flow cytometry from 24 sedentary adults: 12 normal-weight adults [8 M/4 F; age: 55 ± 6 yr; body mass index (BMI): 24.3 ± 0.7 kg/m2; EMV: 144 ± 53 EMVs/µL] and 12 adults with obesity (6 M/6 F; 59 ± 7 yr; BMI: 31.0 ± 1.1 kg/m2; EMV: 245 ± 89 EMVs/µL). Human umbilical vein endothelial cells were cultured and treated with EMVs from either normal-weight adults or adults with obesity. EMVs from obese adults induced significantly higher release of interleukin (IL)-6 (108.2 ± 7.7 vs. 90.9 ± 10.0 pg/mL) and IL-8 (75.4 ± 9.8 vs. 59.5 ± 11.5 pg/mL) from endothelial cells vs. EMVs from normal-weight adults, concordant with greater intracellular expression of phosphorylated NF-κB p65 (Ser536; active NF-κB) [145.0 ± 34.1 vs. 114.5 ± 30.4 arbitrary units (AU)]. Expression of phosphorylated p38-MAPK (15.4 ± 5.7 vs. 9.2 ± 2.5 AU) and active caspase-3 (168.2 ± 65.5 vs. 107.8 ± 40.5 AU), markers of cell apoptosis, was higher in cells treated with obesity-related EMVs. Phosphorylated endothelial nitric oxide synthase (eNOS) (Ser1177) expression (23.5 ± 7.2 vs. 34.7 ± 9.7 AU) and NO production (6.9 ± 1.4 vs. 8.7 ± 0.7 µmol/L) were significantly lower in the cells treated with EMVs from obese adults. These data indicate that circulating EMVs from adults with obesity promote a proinflammatory, proapoptotic, and NO-compromised endothelial phenotype. Circulating EMVs are a potential mediator of obesity-related endothelial dysfunction.NEW & NOTEWORTHY In the present study, we determined the effect of circulating endothelial cell-derived microvesicles (EMVs) isolated from adults with obesity on endothelial cell inflammation, apoptosis, and nitric oxide (NO) production in vitro. Circulating EMVs harvested from adults with obesity promoted a proinflammatory, proapoptotic, and NO-compromised endothelial phenotype. Elevated circulating EMVs in adults with obesity, independent of other cardiometabolic risk factors, are a potential novel systemic mediator of obesity-related endothelial dysfunction and vascular risk.

Endothelial-derived extracellular vesicles associated with electronic cigarette use impair cerebral microvascular cell function.

The aim of this study was to determine the effect of circulating endothelial cell-derived microvesicles (EMVs) isolated from e-cigarette users on human cerebral microvascular endothelial cells (hCMECs) nitric oxide (NO) and endothelin (ET)-1 production and tissue-type plasminogen activator (t-PA) release. Circulating EMVs (CD144-PE) were isolated (flow cytometry) from 27 young adults (19-25 yr): 10 nonsmokers (6 M/4 F), 10 e-cigarette users (6 M/4 F), and 7 tobacco cigarette smokers (4 M/3 F). hCMECs were cultured and treated with isolated EMVs for 24 h. EMVs from e-cigarette users and cigarette smokers induced significantly higher expression of p-eNOS (Thr495; 28.4 ± 4.6 vs. 29.1 ± 2.8 vs. 22.9 ± 3.8 AU), Big ET-1 (138.8 ± 19.0 vs. 141.7 ± 19.1 vs. 90.3 ± 18.8 AU) and endothelin converting enzyme (107.6 ± 10.1 and 113.5 ± 11.8 vs. 86.5 ± 13.2 AU), and significantly lower expression of p-eNOS (Ser1177; 7.4 ± 1.7 vs. 6.5 ± 0.5 vs. 9.7 ± 1.6 AU) in hCMECs than EMVs from nonsmokers. NO production was significantly lower and ET-1 production was significantly higher in hCMECs treated with EMVs from e-cigarette (5.7 ± 0.8 µmol/L; 33.1 ± 2.9 pg/mL) and cigarette smokers (6.3 ± 0.7 µmol/L; 32.1 ± 3.9 pg/mL) than EMVs from nonsmokers (7.6 ± 1.2 µmol/L; 27.9 ± 3.1 pg/mL). t-PA release in response to thrombin was significantly lower in hCMECs treated with EMVs from e-cigarette users (from 38.8 ± 6.3 to 37.4 ± 8.3 pg/mL) and cigarette smokers (31.5 ± 5.5 to 34.6 ± 8.4 pg/mL) than EMVs from nonsmokers (38.9 ± 4.3 to 48.4 ± 7.9 pg/mL). There were no significant differences in NO, ET-1, or t-PA protein expression or production in hCMECs treated with EMVs from e-cigarette users and smokers. Circulating EMVs associated with e-cigarette use adversely affects brain microvascular endothelial cells and may contribute to reported cerebrovascular dysfunction with e-cigarette use.NEW & NOTEWORTHY In the present study, we determined the effect of circulating endothelial cell-derived microvesicles (EMVs) isolated from e-cigarette users on human cerebral microvascular endothelial cells (hCMECs) nitric oxide (NO) and endothelin (ET)-1 production and tissue-type plasminogen activator (t-PA) release. EMVs from e-cigarette users reduced brain microvascular endothelial cell NO production, enhanced ET-1 production, and impaired endothelial t-PA release. EMVs are a potential mediating factor in the increased risk of stroke associated with e-cigarette use.

Insulin-Derived Endothelial Microvesicles Induce a Proinflammatory Endothelial Phenotype

Increased inflammation renders the vascular endothelium susceptible to atherosclerosis and thrombosis. Insulin has been shown to influence vascular function. Hyperinsulinemia is associated with a proinflammatory endothelial phenotype. In vitro, insulin has been shown to increase the inflammatory transcription factor NF-kB. Extracellular vesicles, particularly endothelial cell-derived microvesicles (EMVs), have emerged as mechanistic factors mediating of endothelial inflammation and, in turn, vascular disease. Circulating EMVs are elevated in metabolic conditions associated with hyperinsulinemia. However, whether insulin stimulates EMV release is unknown, and, if so, whether insulin-derived EMVs are proinflammatory. The experimental aims of this study were to determine: 1) if insulin stimulates EMV release from endothelial cells; and 2) whether insulin-derived EMVs promote a proinflammatory endothelial phenotype. Human umbilical vein endothelial cells (HUVECs) were cultured (3rd passage) and incubated with 1 nM insulin (concentration representing a hyperinsulinemic state) for 24 h. EMVs released into the supernatant from cells treated with insulin or vehicle were isolated and quantified by flow cytometry (CD144-PE). In separate experiments, HUVECs (2x10 6 cells/condition) were treated with either insulin, insulin-induced EMVs or control EMVs for 24 h. EMV release was markedly higher (~185%; P<0.05) in cells treated with insulin compared with control (88±10 vs. 31±5 EMV/μL). Insulin-generated EMVs induced significantly higher release of interleukin (IL)-6 (25.4±1.5 vs 20.1±1.2 pg/mL) and IL-8 (37.8±2.5 vs 27.1±2.7 pg/mL) than control EMVs. Although total NF-kB p65 expression was not significantly altered; insulin-derived EMVs induced ~30% higher (P<0.05) expression of phosphorylated-NF-kB p65 (Ser536; active NF-kB) (8.5±0.4 vs. 6.5±0.3 AU). The adverse effects of insulin-generated EMVs on IL-6, IL-8, and pNF-kB p65 were consistent with the proinflammatory effects of insulin. In summary, insulin induces an EMV phenotype that adversely affects endothelial cell inflammation. Insulin-generated EMVs may contribute to the proinflammatory vascular effects of insulin. Nothing to disclose This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Aging Is Associated with a Circulating Endothelial Cell-Derived Microvesicle Phenotype that Impairs Cerebral Fibrinolytic Capacity

Aging is associated with increased risk and prevalence of ischemic stroke. Endothelial cell dysfunction is considered to be a central mechanism underlying the age-related increase in cerebrovascular accidents (CVA). However, the direct, and indirect, effects of aging on endothelial cell biology are diverse and not completely understood. Reduced endogenous fibrinolytic activity is a central etiologic factor underlying ischemic stroke. Endothelial cells are the primary site of synthesis and release of tissue-type plasminogen activator (t-PA), the main plasminogen activator in fibrinolysis and central regulator. Clinical interest in circulating extracellular vesicles, particularly endothelial cell-derived microvesicles (EMVs), has intensified due to their involvement in the development and progression of endothelial dysfunction and CVA. The aim of this study was to determine, in vitro, the effect of circulating EMVs isolated from young and older adults on brain endothelial cell fibrinolytic capacity. We hypothesized that EMVs from older adults would reduce brain endothelial cell t-PA production and release and increase PAI-1 production compared with EMVs from young adults. Twenty-two healthy, non-obese, normotensive, sedentary adults were studied: 10 young (6M/4F; age: 25±1 yr) and 12 older (7M/5F; 64±2 yr). EMV identification (CD144 + ) and isolation from peripheral blood were performed by flow cytometry. Human cerebral microvascular endothelial cells (hCMECs) were cultured and separately treated with EMVs from each subject. Cultured hCMECs were treated with EMVs in the absence and presence of thrombin (1 unit/mL) for 24 hours. Basal expression of t-PA (25.2±1.5 vs 39.8±1.8 AU) and the t-PA response to thrombin (32.5±1.5 vs 57.2±2.2 AU) were significantly lower (~35%) in hCMECs treated with EMVs from older compared with young adults. Concordant with changes in cell protein expression, basal t-PA production (55.2±3.5 vs 70.1±3.7 ng/mL) and thrombin-stimulated t-PA production (61.9±3.7 vs 88.4±3.8 ng/mL) was significantly lower in cells treated with EMVs from older adults. In conclusion, circulating EMVs may contribute to the increased risk of ischemic stroke with aging by impairing brain endothelial cell fibrinolytic capacity. Diminished capacity of the endothelium to release t-PA is a key mechanistic factor underlying cerebral thrombosis. Nothing to Disclose This is the full abstract presented at the American Physiology Summit 2023 meeting and is only available in HTML format. There are no additional versions or additional content available for this abstract. Physiology was not involved in the peer review process.

Differential Expression of Vascular-Related MicroRNA in Circulating Endothelial Microvesicles in Adults With Spinal Cord Injury: A Pilot Study.

Spinal cord injury (SCI) is associated with an increased risk and prevalence of cardiopulmonary and cerebrovascular disease-related morbidity and mortality. The factors that initiate, promote, and accelerate vascular diseases and events in SCI are poorly understood. Clinical interest in circulating endothelial cell-derived microvesicles (EMVs) and their microRNA (miRNA) cargo has intensified due to their involvement in endothelial dysfunction, atherosclerosis, and cerebrovascular events. The aim of this study was to determine whether a subset of vascular-related miRNAs is differentially expressed in EMVs isolated from adults with SCI. We assessed eight adults with tetraplegia (7 male/1 female; age: 46±4 years; time since injury: 26±5 years) and eight uninjured (6 male/2 female; age: 39±3 years). Circulating EMVs were isolated, enumerated, and collected from plasma by flow cytometry. The expression of vascular-related miRNAs in EMVs was assessed by RT-PCR. Circulating EMV levels were significantly higher (~130%) in adults with SCI compared with uninjured adults. The expression profile of miRNAs in EMVs from adults with SCI were significantly different than uninjured adults and were pathologic in nature. Expression of miR-126, miR-132, and miR-Let-7a were lower (~100-150%; p < .05), whereas miR-30a, miR-145, miR-155, and miR-216 were higher (~125-450%; p < .05) in EMVs from adults with SCI. This study is the first examination of EMV miRNA cargo in adults with SCI. The cargo signature of vascular-related miRNAs studied reflects a pathogenic EMV phenotype prone to induce inflammation, atherosclerosis, and vascular dysfunction. EMVs and their miRNA cargo represent a novel biomarker of vascular risk and a potential target for intervention to alleviate vascular-related disease after SCI.

Abstract 048: Extracellular Endothelial Microvesicles Causative Agents In Hypertension-related Endothelial Dysfunction

We have previously demonstrated that endothelial cell-derived microvesicles (EMVs) are elevated with hypertension and are associated with endothelial dysfunction. However, it is unknown whether EMVs play a causative role in blood pressure-related endothelial dysfunction. The experimental aim of this study was to determine the effects of EMVs isolated from hypertensive adults on endothelial cell inflammation, apoptosis and nitric oxide (NO) production. Twenty middle-aged adults were studied: 10 normotensive (NT: 8M/2F; 57 + 2 yr; BP: 113/71 + 2/2 mmHg; EMVs: 64 + 10 EMV/μL) and 10 hypertensive (HT: 6M/4F; 56 + 2 yr; 137/82 + 2/2 mmHg; 115 + 17 EMV/μL). All subjects were sedentary, non-smokers, non-medicated and free of overt cardiometabolic diseases. EMVs (CD144+) were identified, sorted and collected from plasma by flow cytometry. Human umbilical vein endothelial cells were cultured and treated with EMVs from either NT or HT adults. EMVs from HT induced significantly greater release of interleukin (IL)-6 (38.9±1.5 vs 31.5±0.9 pg/mL) and IL-8 (38.1±2.7 vs 30.3±1.8 pg/mL) compared with EMVs from NT. Concordantly, HT-related EMVs induced ~25% higher (P&lt;0.05) expression of phosphorylated-NF-kB p65 (Ser536; active NF-kB) (168.5 + 12.1 vs 136.8 + 7.0 AU). HT EMVs induced significantly higher activation of the apoptotic proteins p38-MAPK (26.0 + 4.5 vs 12.4 + 1.2 AU) and caspase-3 (243.1±36.7 vs 150.8±17.0 AU) than NT EMVs. Although total eNOS expression was not significantly altered; active eNOS (pSer1177) (221.02 + 34.8 vs 331.5 + 30.4 AU) and, in turn, NO production (6.2 + 0.4 vs 9.4 + 0.6 υmol/L) were significantly lower (~30%) in the cells treated with EMVs from HT adults. Increased inflammation, apoptosis susceptibility and decreased NO production are central features of endothelial dysfunction. Circulating EMVs represent both a biomarker and mediator of endothelial dysfunction with hypertension.

Abstract P004: Chronic Nebivolol And Metoprolol Treatment Reduce Circulating Endothelial Cell-Derived Microvesicles In Adults With Hypertension

Nebivolol and metoprolol are effective blood pressure medications that also reduce risk of cardiovascular and cerebrovascular events. Endothelial cell-derived microvesicles (EMVs) are small anucleoid, extracellular vesicles released in response to a myriad of stimuli that trigger cell activation or apoptosis. Clinical interest in EMVs has increased due, in part, to their role in the development and progression of cardiovascular and cerebrovascular disease as well as their prognostic value. Circulating EMVs are elevated with hypertension and contribute to vascular dysfunction. The aim of this study was to determine the independent effects of nebivolol and metoprolol treatment on circulating EMVs in adult humans with hypertension. Thirty-eight middle-aged adults with elevated blood pressure completed a 3-month double-blind, randomized, placebo-controlled trial: 14 received nebivolol (N: 8M/6F; 5 mg/d; age: 58±2 yr; BP: 142/86±2/2 mmHg), 15 received metoprolol succinate (M: 9M/6F; 100 mg/d; 57±2 yr; 139/87±2/2 mmHg), and 9 received placebo (P: 5M/4F; 1 gelatin capsule/d; 55±3 yr; 136/84±2/2 mmHg). Blood pressure was lowered (P&lt;0.05) to a similar extent by both N (124/76 + 3/2 mmHg) and M (125/79 + 3/2 mmHg) but unchanged by P (133/80 + 3/2 mmHg). EMV phenotype was determined using markers indicative of endothelial activation (CD62e + ) or apoptosis (CD31 + /CD42b - ) by flow cytometry. Circulating activation- and apoptosis-derived EMVs were significantly lower (~25%) after N (activation-EMVs: 68±7 to 48±6 EMV/μL; apoptosis-EMVs: 129±2 to 103±9 EMV/μl) and M (activation-EMVs: 57±5 to 39±3 EMVs/μL; apoptosis-EMVs: 125±4 to 96±9 EMV/μL) treatment. Placebo had no significant effect on EMVs (activation-EMVs: 61±7 to 57±6 EMV/μL; apoptosis-EMVs: 131 + 3 to 124 + 8 EMV/μL). There was no significant correlation between the reduction in either EMV phenotype and changes in blood pressure in the N or M group. These results demonstrate that both N and M treatment reduce circulating EMVs, and the reduction appears to be independent of changes in blood pressure. Lower circulating EMVs may contribute to the reduced risk of cardiovascular and cerebrovascular events associated with chronic N and M therapy.

Abstract 13152: Circulating Endothelial Extracellular Microvesicles Increase With Advancing Age And Are Associated With Endothelial Dysfunction

Background: Aging is associated with a progressive increase in risk and prevalence of cardiovascular disease (CVD). Endothelial cell dysfunction is considered to be a central mechanism underlying the age-related increase in vascular risk and disease. However, the direct, and indirect, effects of aging on endothelial cell biology are diverse and not completely understood.Clinical interest in circulating extracellular vesicles, particularly endothelial cell-derived microvesicles (EMVs), has intensified due to their involvement in the development and progression of endothelial dysfunction and CVD. The purpose of this study was to determine: 1) if circulating EMV levels increase with age, independent of other cardiometabolic risk factors; and if so, 2) whether circulating EMVs are associated with age-related endothelial vasodilator dysfunction. Methods: Thirty-six healthy, non-obese, normotensive, sedentary men were studied: 12 young (age: 27 + 1 yr); 12 middle-aged (51 + 1 yr) and 12 older (67 + 2 yr). EMV identification (CD31 + /42b - ) and concentration in peripheral blood was determined by flow cytometry. Forearm blood flow (FBF: via plethysmography) was assessed in response to intra-arterial infusions of acetylcholine (4.0, 8.0 and 16.0 μg/100 mL tissue/min) and sodium nitroprusside (1.0, 2.0 and 4.0 μg/100 mL tissue/min) in 8 young, 8 middle-aged and 8 older men. Results: Circulating EMV levels were significantly and progressively higher across the young, middle-aged and older groups (57 + 5 vs 102 + 9 vs 134 + 16 EMV/μL, respectively). FBF response to acetylcholine was significantly and progressively lower (~35%) in the young (from 5.2±0.4 to 17.2±1.8 mL/100 mL tissue/min) vs middle-aged (4.5±0.3 to 13.2±1.2 mL/100 mL tissue/min) vs older (4.2±0.3 to 11.3±0.8 mL/100 mL tissue/min) group. Circulating EMVs were positively associated with age (r=0.67; P&lt;0.01) and inversely associated with the vasodilator response to acetylcholine (r=-0.43; P&lt;0.01). Conclusions: Aging, independent of other cardiometabolic risk factors, is associated with progressive elevated circulating levels of EMVs. Circulating EMVs may serve as a biomarker of, and contributor to, age-related endothelial dysfunction and vascular disease risk.

Extracellular Endothelial Microvesicles Causative Agents in Obesity‐Related Endothelial Dysfunction

Obesity is associated with increased cardiovascular disease morbidity and mortality. Moreover, obese individuals demonstrate accelerated rates of atherosclerosis, as well as increased incidence of myocardial infarction and stroke. Extracellular microvesicles, particularly endothelial cell-derived microvesicles (EMVs), are seminal functional modulators of vascular health and disease. We have previously demonstrated that EMVs are elevated with obesity and associated with endothelial dysfunction, although the underlying mechanisms are unknown. The experimental aim of this study was to determine the effects of EMVs isolated from obese adults on endothelial cell inflammation, apoptosis and nitric oxide (NO) production. Twenty-four middle-aged adults were studied: 12 normal weight (8M/4F; BMI: 24.4+0.3 kg/m2; EMVs: 85+13 EMV/µL) and 12 obese (6M/6F; BMI: 31.1+0.4 kg/m2; EMVs: 160+18 EMV/µL). All subjects were sedentary, non-smokers, normotensive, normolipidemic, non-diabetic and free of overt coronary artery disease. EMVs (CD31+/CD42b-) were identified, enumerated, and isolated from plasma by flow cytometry. Human umbilical vein endothelial cells were cultured and treated with EMVs from either normal weight or obese adults. EMVs from obese adults induced significantly greater release of both IL-6 (108.2±2.3 vs 90.9±2.9 pg/mL) and IL-8 (74.5±2.9 vs 59.5±3.3 pg/mL) from endothelial cells vs EMVs from normal weight adults. Intracellular expression of total NF-kB p65 was not significantly different in cells treated with EMVs from the normal weight (78.2+9.3 AU) or obese (86.5+8.7 AU) subjects. However, EMVs from obese subjects induced ~25% higher expression of phosphorylated-NF-kB p65 (Ser536; active NF-kB) (145.0+9.8 vs 114.5+8.8 AU). There were no significant differences in cellular expression of total p38-MAPK (190.6+18.4 vs 163.2+9.5 AU) and caspase-3 (250.9±20.9 vs 283.0±19.4 AU) in cells treated with EMVs from normal weight and obese adults. However, EMVs from obese adults induced significantly higher (~50%) expression of phospho-p38-MAPK(15.4+1.6 vs 9.2+0.7 AU) and active caspase-3 (168.2±18.9 vs 107.8±11.7 AU) than EMVs from normal weight adults. Total eNOS expression was not significantly different between cells treated with EMVs from normal weight (110.9+12.0 AU) and obese (112.8+14.4 AU) adults; however, p-eNOS (Ser1177) expression was ~50% lower (P<0.05) in the cells treated with EMVs from obese adults (18.4+3.4 vs 34.6+2.8 AU). Consistent with changes in p-eNOS, NO production was ~20% lower (6.9+0.4 vs 8.7+0.2 mmol/L) in cells treated with EMVs from obese adults. Increased inflammation, apoptosis susceptibility and decreased eNOS activity and NO production renders the vascular endothelium prone to atherosclerosis and thrombosis. Circulating EMVs likely contribute to the proatherogenic endothelial phenotype and, in turn, the increased incidence of cardiovascular disease with obesity.

Individuals with controlled hypertension show endothelial integrity following a bout of moderate-intensity exercise: randomized clinical trial

To examine the acute effects of aerobic exercise (AE), resistance exercise (RE) or combined exercise (CE) on flow-mediated dilation (FMD), progenitor cells (PCs), endothelial progenitor cells (EPCs), oxidative stress markers and endothelial-cell derived microvesicles (EMVs) in patients with hypertension. This is a randomized, parallel-group clinical trial involving an intervention of one session of three different modalities of exercise. Thirty-three males (43 ± 2y) were randomly divided into three groups: a session of AE (n = 11, 40 min, cycle ergometer, 60% HRR); a session of RE (n = 11, 40 min, 4 × 12 lower limb repetitions, 60% 1-RM); or a session of CE (n = 11, 20-min RE + 20-min AE). FMD was assessed 10 min before and 10, 40 and 70 min post-intervention. Blood samples were collected at the same time points (except 40 min). FMD were similar in all groups and from baseline (within each group) after a single exercise bout (AE, RE or CE). At 70 min, RE group showed higher levels of PCs compared to the AE (81%) and CE group (60%). PC levels were reduced from baseline in all groups (AE: 32%, p = 0.037; RE: 15%, p = 0.003; CE: 17%, p = 0.048). The levels of EPCs, EMVs and oxidative stress were unchanged. There were no acute effects of moderate-intensity exercise on FMD, EPCs, EMVs and oxidative stress, but PCs decreased regardless of the exercise modality. Individuals with controlled hypertension do not seem to have impaired vascular function in response to a single exercise bout.

Diagnostic roles of urinary kidney microvesicles in diabetic nephropathy.

BackgroundThe pathology of diabetic nephropathy (DN) broadly involves the injury of glomeruli, tubulointerstitium and endothelium. Cells from these compartments can release increased numbers of microvesicles (MVs) into urine when stressed or damaged. Currently whether urinary MVs from these three parts can help diagnose DN and reflect pathological features remain unclear.MethodsForty-nine patients with histologically proven DN and 29 proteinuric controls with membranous nephropathy or minimal change disease were enrolled. Urinary podocyte, proximal tubular and endothelial cell-derived MVs were quantified by flow cytometry. Renal glomerular, tubulointerstitial and vascular lesions were semi-quantitatively scored and their relevance to urinary MVs were analyzed.ResultsDN patients had greater numbers of urinary MVs from podocytes, proximal tubular and endothelial cells compared with proteinuric controls. The combination of podocyte nephrin+ MVs and diabetic retinopathy optimally diagnose DN with 89.7% specificity and 88.9% sensitivity. Moreover, positive correlations were observed between urinary levels of proximal tubular MVs and the severity of tubular injury and between urinary levels of endothelial MVs and the degree of vascular injury. Using urinary proximal tubular MVs as the indicators for tubular injury, the differences between DN patients and proteinuric controls diminished after matching the degree of renal vascular injury or when proteinuria >8 g/24 h.ConclusionsUrinary kidney-specific cell-derived MVs might serve as noninvasive biomarkers for the diagnosis of DN in diabetic proteinuric patients. Their elevated levels could reflect corresponding renal pathological lesions, helping physicians look into the heterogeneity of DN.

Abstract P069: Microvesicles From Enos-suppressed Endothelial Cells Induce Endothelial Cell Dysfunction

Introduction: Endothelial nitric oxide synthase (eNOS) activity is critical to vascular health. Impaired eNOS activity and diminished NO production are common characteristics of a proatherogenic, dysfunctional endothelial phenotype that is associated with cardiovascular risk factors and disease. Extracellular microvesicles, particularly endothelial cell derived microvesicles (EMVs) represent novel mechanistic mediators of endothelial dysfunction and vascular disease. It is unknown whether eNOS suppression affects EMV number and function. We tested the following hypotheses: 1) eNOS blockade increases EMV release; and 2) EMVs derived from eNOS-suppressed cells adversely affect endothelial cell inflammation, apoptosis and NO production. Methods: Human umbilical vein endothelial cells (HUVECs) were treated with the eNOS inhibitor, L-N G -Nitroarginine methyl ester (L-NAME; 300mM) for 24 h. EMVs (CD144 + ) released into the supernatant from cells treated with L-NAME or vehicle were isolated and quantified by flow cytometry. Fresh HUVECs were then treated with either L-NAME-derived or control EMVs for 24 h. To evaluate the role of endocytosis on the endothelial effects of EMVs, HUVECs were pre-incubated (12 h) with EIPA, filipin and chlorpromazine for 2 h, and all experiments repeated. Results: EMV release was markedly higher (~100%; P&lt;0.05) in cells treated with L-NAME compared with control (81±6 vs. 40±7 EMV/μL). L-NAME-generated EMVs induced significantly higher release of IL-6 (38.4±5.1 vs. 21.0±1.9 pg/mL) and IL-8 (38.9±3.5 vs. 27.2±3.1 pg/mL) as well as greater active NF-κB p65 (Ser-536) (9.7±0.7 vs. 6.1±0.6 AU) expression than control EMVs. The expression of activated-caspase-3 was significantly higher in the cells treated with L-NAME (9.5±1.1 vs. 6.4±0.4 AU). Total eNOS (97.1±8.2 vs. 157.5±15.6 AU), activated eNOS (4.9±1.2 vs. 9.1±1.3 AU) and NO production (5.0±0.8 vs. 7.0±0.6 μmol/L) were significantly lower in endothelial cells treated with EMVs from eNOS suppressed cells. Endocytosis blockers mitigated the deleterious endothelial effects of EMVs. Conclusion: eNOS-suppression increases EMV release. Moreover, EMVs from eNOS-suppressed cells increase endothelial cell inflammation and apoptosis and decrease NO production.

Abstract P082: Elevated Circulating Endothelial-Derived Microvesicles Are Associated With Obesity-Related Endothelial Dysfunction

Background: Obesity reduces the number of years lived free of cardiovascular disease (CVD) and increases the number of years lived with CVD, contributing to increased morbidity and mortality risk across all ages. Experimental and clinical studies indicate that a proatherogenic endothelial phenotype is a major consequence of increased adiposity and a primary mechanism underlying obesity-related CVD. Extracellular microvesicles, particularly endothelial cell-derived microvesicles (EMVs), are seminal functional modulators of vascular health and disease. The purpose of this study was to determine: 1) if circulating EMV levels are elevated with obesity, independent of other cardiometabolic risk factors; and if so, 2) whether circulating EMVs are associated with obesity-related endothelial vasodilator dysfunction. Methods: Thirty sedentary, middle-aged adults (45-63 years) were studied: 15 normal weight (10M/5F; age: 56±1 yr; BMI: 23.2±0.4 kg/m2; body fat: 25.1±2.4 %) and 15 obese (10M/5F; age: 54±1 yr; BMI: 31.5±0.3 kg/m2; body fat: 37.2±1.7 %). All subjects were free of other cardiometabolic risk factors and overt disease. EMV identification (CD31 + /42b - ) and concentration in peripheral blood were determined by flow cytometry. Forearm blood flow (FBF: via plethysmography) was assessed in response to intra-arterial infusions of acetylcholine (4.0, 8.0 and 16.0 μg/100 mL tissue/min) and sodium nitroprusside (1.0, 2.0 and 4.0 μg/100 mL tissue/min). Results: Circulating EMV levels were ~100% higher (P&lt;0.05) in obese (182±14 EMV/μL) compared with normal weight (91±12 EMV/μL) adults. FBF response to acetylcholine was significantly lower (~35%) in the obese (from 4.0±0.2 to 10.3±0.6 mL/100 mL tissue/min vs 4.3±0.3 to 15.4±0.8 mL/100 mL tissue/min) group. Circulating EMVs were significantly and inversely associated with total FBF response to acetylcholine (r=-0.55). Conclusions: Obesity, independent of other cardiometabolic risk factors, is associated with elevated circulating levels of EMVs. Higher circulating EMVs in obese adults may contribute to adiposity-related endothelial dysfunction and vascular disease risk. Indeed, circulating EMVs have been linked to disease risk, severity and outcome in other high-risk populations.

Endothelin-1-induced endothelial microvesicles impair endothelial cell function.

The aim of this study was to determine the effects of endothelin-1 (ET-1)-generated endothelial microvesicles (EMVs) on endothelial cell inflammation, apoptosis, and endothelial nitric oxide synthase (eNOS). Human umbilical vein endothelial cells (HUVECs) were treated with ET-1 for 24 h. EMVs released into the supernatant from cells treated with ET-1 or vehicle were isolated and quantified. EMV release was higher (P < 0.05) in cells treated with ET-1 compared with control (95 ± 15 vs. 54 ± 5 EMV/µL). Fresh HUVECs were then treated with either ET-1, ET-1-induced EMVs, or control EMVs for 24 h. ET-1-generated EMVs induced significantly higher release of IL-6 (181.0 ± 16.0 vs. 132.1 ± 8.1 pg/mL) and IL-8 (303.4 ± 37.4 vs. 211.8 ± 10.0 pg/mL), as well as greater total NF-κB p65 (76.0 ± 7.6 vs. 57.1 ± 2.1 AU) and active NF-κB p65 (Ser-536) (11.6 ± 0.9 vs. 6.8 ± 1.0 AU) expression than control EMVs. There were no significant differences in expression of caspase-9 (230.1 ± 24.3 vs. 243.6 ± 22.3 AU), caspase-3 (271.9 ± 22.7 vs. 265.1 ± 30.5 AU), and active caspase-3 (4.4 ± 0.4 vs. 4.3 ± 0.1 AU) in cells treated with ET-1-EMVs versus control EMVs. Total eNOS (108.4 ± 11.4 vs. 158.8 ± 1.6 AU) and activated eNOS (4.7 ± 0.5 vs. 9.6 ± 1.4 AU) were significantly lower in endothelial cells treated with ET-1-generated EMVs compared with control EMVs. The effects of ET-1-generated EMVs on cellular markers and mediators of endothelial inflammation, as well as eNOS function, was comparable to the effects of ET-1. In summary, ET-1 induces an EMV phenotype that adversely affects endothelial cell function. ET-1-generated EMVs may contribute to the atherogenic effect of ET-1.NEW & NOTEWORTHY Endothelin-1 (ET-1) is a potent vasoconstrictor peptide released by the endothelium that contributes to the regulation of vascular tone. Overexpression of ET-1 has been implicated in the etiology of atherosclerotic vascular disease. Endothelial cell-derived microvesicles (EMVs) play a pivotal role in vascular health and disease. Their functional phenotype is largely dictated by the stimulus for release. EMVs released in response to various pathological conditions have been shown to elicit deleterious vascular effects. In the present study, we determined, in vitro, the effect of ET-1 on EMV release from endothelial cells and the effects of ET-1-generated EMVs on endothelial cell inflammation, apoptosis, and endothelial nitric oxide synthase (eNOS). ET-1 induced a marked increase in EMV release. ET-1-generated EMVs significantly increased endothelial cell inflammation and reduced eNOS protein expression and activation. Moreover, the endothelial effects of ET-1-derived EMVs were similar to the direct effects of ET-1. ET-1-generated EMVs may contribute to the proatherogenic profile of ET-1.

Altered Signature of Apoptotic Endothelial Cell-Derived Microvesicles Predicts Chronic Heart Failure Phenotypes

Aim: to evaluate the associations between signatures of apoptotic endothelial cell-derived microvesicles (MVs) with phenotypes of chronic heart failure (HF). Methods: The study cohort consisted of 388 prospectively involved subjects with HF patients with predominantly reduced left ventricular ejection fraction (HFrEF), HF with preserved ejection fraction (HFpEF) and HF with mid-range ejection fraction (HFmrEF). All biomarkers were measured at baseline. Results: The number of circulating CD31+/annexin V+ MVs in HFrEF and HFmrEF patients was similar. The number of circulating CD144+/annexin V+ MVs in HFrEF patients was significantly higher than HFmrEF and HFpEF. We determined that a combination of number of circulating CD31+/annexin V+ MVs and Gal-3 was the best predictor of HFpEF and that number of circulating CD144+/annexin V+ MVs is able to increase predictive capabilities of soluble ST2 (sST2) and Gal-3 for HFrEF. Conclusion: We found that the number of circulating CD31+/annexin V+ MVs may improve a predictive capacity for conventional HF biomarkers.

Pattern of apoptotic endothelial cell-derived micro vesicles in patients with different phenotypes of chronic heart failure

Background: The aim of the study was to evaluate the associations between the signature of apoptotic endothelial cellderived microvesicles (MVs) and biomarkers of fibrosis, inflammation and cardiac remodeling in patients with different phenotypes of chronic Heart Failure (HF). Methods: The study cohort consisted of 388 prospectively involved subjects with established chronic HF. The phenotype of HF was determined according to left ventricular ejection fraction (LVEF) value per contemporary clinical guideline. HFrEF (LVEF ≤ 40%), HFmrEF (41-49%) and HFpEF (LVEF ≥ 50%) were determined. All biomarkers were measured at baseline. Results: The number of circulating CD31+/annexin V+MVs in HFpEF patients was significantly different from both HFrEF and HFmrEF individuals, but it was similar in HFrEF and HFmrEF patients. The number of circulating CD144+/ annexin V+MVs in HFrEF patients was significantly higher to HFmrEF and HFpEF. We determined that a combination of a number of circulating CD31+/annexin V+MVs and galectin-3 (AUC=0.68; 95% CI=0.61-0.77; P=0.001) was the best predictor of HFpEF. The predictive values of sST2 (AUC=0.65; 95% CI=0.60-0.69), number of circulating CD31+/annexin V+MVs (AUC=0.63; 95% CI=0.58-0.69) alone and their combination (AUC=0.65; 95% CI=0.59-0.70) for HFmrEF did not distinguished significantly (P=0.48). The double combinations of number of circulating CD144+/annexin V+MVs and sST2 (AUC=0.70; 95% CI=0.66-0.75) or number of circulating CD144+/annexin V+MVs and galectin-3 (AUC=0.71; 95% CI=0.650.76) were the best prognosticators for HFrEF. Conclusion: we found that the number of circulating CD31+/annexin V+MVs may improve a prediction of galectin-3 for HFpEF, and that number of circulating CD144+/annexin V+MVs is able to increase predictive capabilities of sST2 and galectin-3 for HFrEF.

P3762The effect of eplerenone on circulating number of endothelial cell-derived microvesicles in patients with chronic heart failure

P3762The effect of eplerenone on circulating number of endothelial cell-derived microvesicles in patients with chronic heart failure

Protective effects of circulating microvesicles derived from ischemic preconditioning on myocardial ischemia/reperfusion injury in rats by inhibiting endoplasmic reticulum stress

Microvesicles (MVs) have been shown to be involved in pathophysiology of ischemic heart diseases. However, the underlying mechanisms are still unclear. Here we investigated the effects of MVs derived from ischemic preconditioning (IPC-MVs) on myocardial ischemic/reperfusion (I/R) injury in rats. Myocardial IPC model was elicited by three cycles of ischemia and reperfusion of the left anterior descending (LAD) coronary artery. IPC-MVs from the peripheral blood of the above animal model were isolated by ultracentrifugation and characterized by flow cytometry and transmission electron microscopy. IPC-MVs were administered intravenously (7mg/kg) at 5min before reperfusion procedure in I/R injury model which was induced by 30-min ischemia and 120-min reperfusion of LAD in rats. We found that total IPC-MVs and different phenotypes, including platelet-derived MVs (PMVs), endothelial cell-derived MVs (EMVs), leucocyte-derived MVs and erythrocyte-derived MVs (RMVs) were all isolated which were identified membrane vesicles (< 1µm) with corresponding antibody positive. The numbers of PMVs, EMVs and RMVs were significantly increased in circulation of IPC treated rats respectively. Additionally, treatment with IPC-MVs significantly alleviated damage of myocardium, and restored cardiac function of I/R injury rats, as evidenced by increased heart rate, and decreased the elevation of ST-segment. The size of myocardial infarction, lactate dehydrogenase activity, and the number of apoptotic cardiomyocytes were also reduced significantly with IPC-MVs treatment, coincident with the above function amelioration. Moreover, IPC-MVs decreased the activity of caspase 3, and the expression of endoplasmic reticulum stress (ERS) markers, GRP78, CHOP and caspase 12 indicating the involvement of ERS-specific apoptosis in I/R injury, and cardioprotective effects of IPC-MVs. In summary, our study demonstrated a novel mechanism of IPC in which circulating IPC-MVs could protect hearts from I/R injury in rats through attenuation of ERS-induced apoptosis. These findings provide new insight into therapeutic potential of IPC-induced MVs in cardioprotection against I/R injury.

Endothelial cell-derived microvesicles: potential mediators and biomarkers of pathologic processes.

This review focuses on the formation, composition and function of endothelial microvesicles (MV), often called microparticles (MP). MV release is a controlled event and is considered a hallmark of cellular activation or alteration. MV may affect the function of target cells through surface interaction and receptor activation, cellular fusion and the delivery of intravesicular cargo. Endothelial MV are released as a consequence of endothelial activation during inflammation and have been described to affect hemostasis, various aspects of inflammatory reaction, vessel formation, apoptosis and cell survival, endothelial cell differentiation and function. Recent data suggest the potential use of MV in diagnostics, assessment of severity and prediction of outcomes in inflammatory diseases and their utilization as targets, mediators and vectors in therapy.