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.

Cerebral uptake of microvesicles occurs in normocapnic but not hypocapnic passive hyperthermia in young healthy male adults.

Passive hyperthermia causes cerebral hypoperfusion primarily from heat-induced respiratory alkalosis. However, despite the cerebral hypoperfusion, it is possible that the mild alkalosis might help to attenuate cerebral inflammation. In this study, the cerebral exchange of extracellular vesicles (microvesicles), which are known to elicit pro-inflammatory responses when released in conditions of stress, were examined in hyperthermia with and without respiratory alkalosis. Ten healthy male adults were heated passively, using a warm water-perfused suit, up to core temperature + 2°C. Blood samples were taken from the radial artery and internal jugular bulb. Microvesicle concentrations were determined in platelet-poor plasma via cells expressing CD62E (activated endothelial cells), CD31+ /CD42b- (apoptotic endothelial cells), CD14 (monocytes) and CD45 (pan-leucocytes). Cerebral blood flow was measured via duplex ultrasound of the internal carotid and vertebral arteries to determine cerebral exchange kinetics. From baseline to poikilocapnic (alkalotic) hyperthermia, there was no change in microvesicle concentration from any cell origin measured (P-values all >0.05). However, when blood CO2 tension was normalized to baseline levels in hyperthermia, there was a marked increase in cerebral uptake of microvesicles expressing CD62E (P = 0.028), CD31+ /CD42b- (P = 0.003) and CD14 (P = 0.031) compared with baseline, corresponding to large increases in arterial but not jugular venous concentrations. In a subset of seven participants who underwent hypercapnia and hypocapnia in the absence of heating, there was no change in microvesicle concentrations or cerebral exchange, suggesting that hyperthermia potentiated the CO2 /pH-mediated cerebral uptake of microvesicles. These data provide insight into a potential beneficial role of respiratory alkalosis in heat stress. KEY POINTS: The hyperthermia-induced hyperventilatory response is observed in most humans, despite causing potentially harmful reductions in cerebral blood flow. We tested the hypothesis that the respiratory-induced alkalosis is associated with lower circulating microvesicle concentrations, specifically in the brain, despite the reductions in blood flow. At core temperature + 2°C with respiratory alkalosis, microvesicles derived from endothelial cells, monocytes and leucocytes were at concentrations similar to baseline in the arterial and cerebral venous circulation, with no changes in cross-brain microvesicle kinetics. However, when core temperature was increased by 2°C with CO2 /pH normalized to resting levels, there was a marked cerebral uptake of microvesicles derived from endothelial cells and monocytes. The CO2 /pH-mediated alteration in cerebral microvesicle uptake occurred only in hyperthermia. These new findings suggest that the heat-induced hyperventilatory response might serve a beneficial role by preventing potentially inflammatory microvesicle uptake in the brain.

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.

Circulating endothelial cell derived microvesicles are elevated with hypertension and associated with endothelial dysfunction.

The purpose of this study was to determine (1) if circulating endothelial microvesicles (EMVs) are elevated in hypertensive adults and (2) whether circulating EMVs are associated with hypertension-related endothelial vasodilator dysfunction. Circulating EMVs (CD31+/42b-) were determined in 30 middle-aged adults (55 ± 1 years): 15 normotensive (10 males, 5 females; blood pressure 114/71 ± 2/1 mm Hg) and 15 hypertensive (10 males, 5 females; blood pressure 142/87 ± 2/2 mm Hg). Forearm blood flow (FBF) (via plethysmography) was assessed by intra-arterial infusion of acetylcholine and sodium nitroprusside. Circulating EMVs were ∼65% higher (P < 0.05) in hypertensive (157 ± 10 EMVs/µL) than in normotensive (96 ± 10 EMVs/µL) adults. FBF to acetylcholine was significantly lower (∼30%) in the hypertensive group (from 5.0 ± 0.4 to 11.8 ± 0.8 mL·100 mL tissue-1·min-1 versus from 4.4 ± 0.2 to 15.6 ± 0.8 mL·100 mL tissue-1·min-1). Circulating EMVs were inversely associated with vasodilation (r = -0.65; P < 0.05). Hypertension is associated with elevated circulating levels of EMVs. EMVs may serve as a biomarker of, and contribute to, blood pressure related endothelial dysfunction.

Effects of circulating extracellular microvesicles from spinal cord-injured adults on endothelial cell function.

People with spinal cord injury (SCI) have three- to four-fold greater risk of cardiovascular disease (CVD) compared with those without SCI. Although circulating extracellular microvesicles are key effectors of vascular health and disease, how their functional phenotype might be altered with SCI is unknown. The aim of the present study was to determine the effects of microvesicles isolated from SCI adults on endothelial cell inflammation and oxidative stress as well as endothelial nitric oxide (NO) synthase (eNOS) activation and tissue-type plasminogen activator (t-PA) expression. Eighteen young and middle-aged adults were studied: 10 uninjured (7M/3F; age: 39 ± 3 years) and 8 cervical level spinal cord injured (SCI; 7M/1F; 46 ± 4 years; cervical injury: C3: n=1; C5: n=4; C6: n=3). Circulating microvesicles were isolated, enumerated and collected from plasma by flow cytometry. Human umbilical vein endothelial cells (HUVECs) were cultured and treated with microvesicles from either the uninjured or SCI adults. Microvesicles from SCI adults did not affect cellular markers or mediators of inflammation and oxidative stress. However, microvesicles from the SCI adults significantly blunted eNOS activation, NO bioavailability and t-PA production. Intercellular expression of phosphorylated eNOS at Ser1177 and Thr495 sites, specifically, were ∼65% lower and ∼85% higher, respectively, in cells treated with microvesicles from SCI compared with uninjured adults. Decreased eNOS activity and NO production as well as impaired t-PA bioavailability renders the vascular endothelium highly susceptible to atherosclerosis and thrombosis. Thus, circulating microvesicles may contribute to the increased risk of vascular disease and thrombotic events associated with SCI.

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 25 mM d-glucose (concentration representing a hyperglycemic state) or 5 mM d-glucose (normoglycemic condition) for 48 h 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 24 h 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 ± 204 MFI), ICAM-1 (2110 ± 81 vs. 1688 ± 152 MFI), VCAM-1 (3589 ± 431 vs. 2134 ± 386) and PECAM-1 (4237 ± 395 vs. 2525 ± 269 MFI) 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.

Insufficient sleep is associated with a pro-atherogenic circulating microRNA signature.

NEW FINDINGS: What is the central question of the study Is habitual short sleep associated with altered circulating levels of specific inflammation- and vascular-related microRNAs? What is the main finding and its importance? Circulating levels of miR-125a, miR-126 and miR-146a were significantly lower in the short sleep compared with the normal sleep group. Altered circulating profiles of these vascular-related microRNAs have been linked to vascular inflammation, dysfunction and increased cardiovascular disease events. Sleep-related changes in these microRNAs are consistent with, and might play a role in, the aberrant vascular physiology and increased vascular risk associated with short sleep. ABSTRACT: Habitual short sleep duration (<7 h night-1 ) is associated with increased morbidity and mortality attributable, in large part, to increased inflammatory burden and endothelial dysfunction. MicroRNAs (miRNAs) play a key role in regulating vascular health, and circulating levels are now recognized to be sensitive and specific biomarkers of cardiovascular function, inflammation and disease.  The aim of this study was to determine whether the expression of circulating miR-34a, miR-92a, miR-125a, miR-126, miR-145, miR-146a and miR-150 is disrupted in adults who habitually sleep <7 h night-1 (short sleep). These were chosen based upon their well-established links with vascular inflammation, function and, in turn, cardiovascular risk. Twenty-four adults were studied: 12 with normal nightly sleep duration (six men and six women; age, 55 ± 3 years old; sleep duration, ≥7.0 h night-1 ) and 12 with short nightly sleep duration (seven men and five women; 55 ± 2 years old; sleep duration, <7 h night-1 ), and circulating miRNA expression was assayed by RT-PCR. All subjects were non-smokers, normolipidaemic, non-medicated and free of overt cardiovascular disease. Circulating levels of miR-125a (3.07 ± 1.98 versus 7.34 ± 5.34 a.u.), miR-126 [1.28 (0.42-2.51) versus 1.78 (1.29-4.80) a.u.] and miR-146a [2.55 (1.00-4.80) versus 6.46 (1.50-11.44) a.u.] were significantly lower (∼60, 40 and 60%, respectively) in the short compared with the normal sleep group. However, there were no significant group differences in circulating levels of miR-34a, miR-92a, miR-145 and miR-150. In summary, chronic short sleep is associated with a marked reduction in circulating levels of miR-125a, miR-126 and miR-146a. Dysregulation of these miRNAs might contribute to the increased inflammatory burden and endothelial dysfunction associated with habitual insufficient sleep.

Phenotypic differences in early outgrowth angiogenic cells based on in vitro cultivation.

Bone marrow-derived early outgrowth cells play an important role in endothelial repair. In vitro isolation techniques have identified two distinct morphological early outgrowth cell populations, but it is still unknown whether they present some functional phenotypic differences. Accordingly, the aim of the present study was to determine whether there are phenotypic differences in cellular function between two putative early outgrowth cells in culture. Peripheral blood samples were collected from 18 healthy adults. Thereafter, mononuclear cells were isolated by Ficoll density-gradient centrifugation and plated on 6-well plates coated with human fibronectin. After 2 and 7 days, respectively, non-adherent cells (NAC) and adherent cells (AC) underwent functional assays in order to measure the migratory capacity (Boyden chamber), angiogenic growth factor release (ELISA) and apoptosis (TUNEL). Migration to both VEGF (517 ± 74 vs. 273 ± 74 AU) and SDF-1 (517 ± 68 vs. 232 ± 68 AU) were approximately twofold higher (P < 0.05) in the NAC when compared to AC. Release of angiogenic factors, granulocyte colony-stimulating and hepatocyte growth factor, were not different between cell types. Apoptotic response to staurosporine was significantly lower in NAC (20 ± 32 vs. 125 ± 32%). In summary, NAC and AC demonstrated functional phenotypic differences in migratory capacity and apoptotic susceptibility, which makes it difficult to compare these two early outgrowth cell populations in literature.

Effects of HIV-1 gp120 and TAT-derived microvesicles on endothelial cell function.

The aims of this study were twofold. The first was to determine if human immunodeficiency virus (HIV)-1 glycoprotein (gp) 120 and transactivator of transcription (Tat) stimulate the release of endothelial microvesicles (EMVs). The second was to determine whether viral protein-induced EMVs are deleterious to endothelial cell function (inducing endothelial cell inflammation, oxidative stress, senescence and increasing apoptotic susceptibility). Human aortic endothelial cells (HAECs) were treated with recombinant HIV-1 proteins Bal gp120 (R5), Lav gp120 (X4), or Tat. EMVs released in response to each viral protein were isolated and quantified. Fresh HAECs were treated with EMVs generated under control conditions and from each of the viral protein conditions for 24 h. EMV release was higher (P < 0.05) in HAECs treated with R5 (141 ± 21 MV/µl), X4 (132 ± 20 MV/µl), and Tat (130 ± 20 MV/µl) compared with control (61 ± 13 MV/µl). Viral protein EMVs induced significantly higher endothelial cell release of proinflammatory cytokines and expression of cell adhesion molecules than control. Reactive oxygen species production was more pronounced (P < 0.05) in the R5-, X4- and Tat-EMV-treated cells. In addition, viral protein-stimulated EMVs significantly augmented endothelial cell senescence and apoptotic susceptibility. Concomitant with these functional changes, viral protein-stimulated EMVs disrupted cell expression of micro-RNAs 34a, 126, 146a, 181b, 221, and miR-Let-7a (P < 0.05). These results demonstrate that HIV-1 gp120 and Tat stimulate microvesicle release from endothelial cells, and these microvesicles confer pathological effects on endothelial cells by inducing inflammation, oxidative stress, and senescence as well as enhancing susceptibility to apoptosis. Viral protein-generated EMVs may contribute to the increased risk of vascular disease in patients with HIV-1. NEW & NOTEWORTHY Human immunodeficiency virus (HIV)-1-related proteins glycoprotein (gp) 120 and transactivator of transcription (Tat)-mediated endothelial damage and dysfunction are poorly understood. Endothelial microvesicles (EMVs) serve as indicators and potent mediators of endothelial dysfunction. In the present study we determined if HIV-1 R5- and X4-tropic gp120 and Tat stimulate EMV release in vitro and if viral protein-induced EMVs are deleterious to endothelial cell function. gp120 and Tat induced a marked increase in EMV release. Viral protein-induced EMVs significantly increased endothelial cell inflammation, oxidative stress, senescence, and apoptotic susceptibility in vitro. gp120- and Tat-derived EMVs promote a proinflammatory, pro-oxidative, prosenescent, and proapoptotic endothelial phenotype and may contribute to the endothelial damage and dysfunction associated with gp120 and Tat.

Circulating Microparticles Are Elevated in Treated HIV -1 Infection and Are Deleterious to Endothelial Cell Function.

Background Circulating microparticles have emerged as biomarkers and effectors of vascular disease. Elevated rates of cardiovascular disease are seen in HIV -1-seropositive individuals. The aims of this study were to determine: (1) if circulating microparticles are elevated in antiretroviral therapy-treated HIV -1-seropositive adults; and (2) the effects of microparticles isolated from antiretroviral therapy -treated HIV -1-seropositive adults on endothelial cell function, in vitro. Methods and Results Circulating levels of endothelial-, platelet-, monocyte-, and leukocyte-derived microparticles were determined by flow cytometry in plasma from 15 healthy and 15 antiretroviral therapy-treated, virologically suppressed HIV -1-seropositive men. Human umbilical vein endothelial cells were treated with microparticles from individual subjects for 24 hours; thereafter, endothelial cell inflammation, oxidative stress, senescence, and apoptosis were assessed. Circulating concentrations of endothelial-, platelet-, monocyte-, and leukocyte-derived microparticles were significantly higher (≈35%-225%) in the HIV -1-seropositive compared with healthy men. Microparticles from HIV -1-seropositive men induced significantly greater endothelial cell release of interleukin-6 and interleukin-8 (≈20% and ≈35%, respectively) and nuclear factor-κB expression while suppressing anti-inflammatory microRNAs (miR-146a and miR-181b). Intracellular reactive oxygen species production and expression of reactive oxygen species -related heat shock protein 70 were both higher in cells treated with microparticles from the HIV -1-seropositive men. In addition, the percentage of senescent cells was significantly higher and sirtuin 1 expression lower in cells treated with HIV -1-related microparticles. Finally, caspase-3 was significantly elevated by microparticles from HIV -1-seropositive men. Conclusions Circulating concentrations of endothelial-, platelet-, monocyte-, and leukocyte-derived microparticles were higher in antiretroviral therapy-treated HIV -1-seropositive men and adversely affect endothelial cells promoting cellular inflammation, oxidative stress, senescence, and apoptosis. Circulating microparticles may contribute to the vascular risk associated with HIV -1 infection.

Influence of Overweight and Obesity on Circulating Inflammation-Related microRNA.

BACKGROUND: Increased cardiovascular disease risk and prevalence associated with overweight and obesity is due, in part, to heightened inflammatory burden. The mechanisms underlying adiposity-related amplification of inflammation are not fully understood. Alterations in regulators of inflammatory processes such as microRNAs (miRs), however, are thought to play a pivotal role. OBJECTIVE: The aim of this study was to determine the influence of overweight and obesity, independent of other cardiovascular risk factors, on circulating expression of miR-34a, miR-126, miR-146a, miR-150 and miR-181b. METHODS: Forty-five sedentary, middle-aged (47-64 years) adults were studied: 15 were normal weight (8M/7F; BMI: 23.3 ± 0.3 kg/m2); 15 were overweight (8M/7F; 28.2 ± 0.3 kg/m2); and 15 were obese (7M/8F; 32.3 ± 0.5 kg/m2). All subjects were non-smokers, normotensive and free of overt cardiometabolic disease. Circulating levels of the following inflammation-related miRs: miR-34a, miR-126, miR-146a, miR-150 and miR-181b were determined in plasma using standard RT-PCR techniques. miR expression was normalized to exogenous C. elegans miR-39 and reported as relative expression (AU). RESULTS: Circulating miR-34a was ~200% higher (P< 0.05) in the obese as compared with normal weight and overweight groups. Whereas, miR-126, miR-146a and miR-150 were significantly lower (~65%) in both the obese and overweight groups than the normal weight group. There were no significant group differences in circulating expression of miR-181b. miR-34a was positively related (r = 0.43; P< 0.05); whereas, miR-126 (r = -0.48), miR-146a (r = -0.33) and miR-150 (r = -0.43) levels were significantly inversely related to BMI. CONCLUSION: Overweight and obesity, independent of other cardiometabolic risk factors, negatively influences circulating inflammation-related miRs. Dysregulation of circulating miRs may contribute mechanistically to the heightened inflammatory state associated with overweight and obesity.

Association between hypertension and circulating vascular-related microRNAs.

microRNAs (miRNAs) have a key role in regulating inflammation, vascular health and in turn, cardiovascular disease. Specifically, altered circulating expression of miR-17, miR-21, miR-34a, miR-92a, miR-126, miR-145, miR-146a, and miR-150 has been linked with the pathogenesis and progression of cardiovascular disease. The aim of this study was to determine whether the circulating profile of these vascular-related miRNAs is disrupted with hypertension. Thirty sedentary, middle-aged adults were studied: 15 normotensive (10M/5F; age: 56 ± 1 year; BP: 113/71 ± 2/1 mmHg) and 15 hypertensive (10M/5F; 56 ± 2 year; 140/87 ± 2/2 mmHg). All subjects were non-obese and free of other cardiometabolic disorders. Circulating miRNAs were determined in plasma using standard RT-PCR techniques with miRNA primers of interest. Expression was normalized to exogenous C. elegans miR-39 and reported as relative expression in arbitrary units (AU). Circulating expression of miR-34a (9.18 ± 0.94 vs 5.33 ± 0.91 AU) was higher (~170%; P < 0.01) whereas the expression of miR-21 (1.32 ± 0.25 vs 2.50 ± 0.29 AU), miR-126 (0.85 ± 0.10 vs 1.74 ± 0.27 AU) and miR-146a (1.50 ± 0.20 vs 3.10 ± 0.50 AU) were markedly lower (~50%, ~55%, and ~55% respectively; P < 0.05) in the hypertensive vs normotensive groups. Moreover, circulating levels of miR-34a, miR-21, and miR-126 were significantly related to systolic blood pressure (r = 0.48, r = -0.38; r = -0.48); whereas, miR-146a was significantly related to both systolic (r = -0.58) and diastolic (r = -0.55) blood pressure. There were no significant group differences in circulating miR-17, miR-92a, miR-145, and miR-150. In summary, these results suggest that hypertension, independent of other cardiometabolic risk factors, adversely affects the circulating profile of a subset of vascular-related miRNAs that have been link to CVD risk and development.

Effects of HIV-1 gp120 and tat on endothelial cell sensescence and senescence-associated microRNAs.

The aim of this study was to determine, in vitro, the effects of X4 and R5 HIV-1 gp120 and Tat on: (1) endothelial cell senescence and (2) endothelial cell microRNA (miR) expression. Endothelial cells were treated with media without and with: R5 gp120 (100 ng/mL), X4 gp120 (100 ng/mL), or Tat (500 ng/mL) for 24 h and stained for senescence-associated ß-galactosidase (SA-ß-gal). Cell expression of miR-34a, miR-217, and miR-146a was determined by RT-PCR. X4 and R5 gp120 and Tat significantly increased (~100%) cellular senescence versus control. X4 gp120 significantly increased cell expression of miR-34a (1.60 ± 0.04 fold) and miR-217 (1.52 ± 0.18), but not miR-146a (1.25 ± 0.32). R5 gp120 significantly increased miR-34a (1.23 ± 0.07) and decreased miR-146a (0.56 ± 0.07). Tat significantly increased miR-34a (1.49 ± 0.16) and decreased miR-146a (0.55 ± 0.23). R5 and Tat had no effect on miR-217 (1.05 ± 0.13 and 1.06 ± 0.24; respectively). HIV-1 gp120 (X4 and R5) and Tat promote endothelial cell senescence and dysregulation of senescence-associated miRs.

High glucose derived endothelial microparticles increase active caspase-3 and reduce microRNA-Let-7a expression in endothelial cells.

The experimental aim of this study was to determine the effects of high glucose-induced endothelial microparticles (EMPs) on endothelial cell susceptibility to apoptosis. 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. Cells were incubated with media containing 25 mM d-glucose (concentration representing a diabetic glycemic state) or 5 mM d-glucose (normoglycemic condition) for 48 h to generate EMPs. EMP identification (CD144+ expression) and concentration was determined by flow cytometry. HUVECs (3 × 106 cells/condition) were treated with EMPs generated from either the normal or high glucose conditions for 24 h. Intracellular concentration of active caspase-3 was determined by enzyme immunoassay. Cellular expression of miR-Let7a, an anti-apoptotic microRNA, was determined by RT-PCR using the ΔΔCT normalized to RNU6. High glucose-derived EMPs significantly increased both basal (1.5 ± 0.1 vs 1.0 ± 0.1 ng/mL) and staurosporine-stimulated (2.2 ± 0.2 vs 1.4 ± 0.1 ng/mL) active caspase-3 compared with normal glucose EMPs. Additionally, the expression of miR-Let-7a was markedly reduced (∼140%) by high glucose EMPs (0.43 ± 0.17 fold vs control). These results demonstrate that hyperglycemic-induced EMPs increase endothelial cell active caspase-3. This apoptotic effect may be mediated, at least in part, by a reduction in miR-Let-7a expression.

Influence of sex on the number of circulating endothelial microparticles and microRNA expression in middle-aged adults.

NEW FINDINGS: What is the central question of this study? Are there sex-related differences in the number of circulating endothelial microparticles (EMPs) and microparticle microRNA expression in middle-aged adult humans? What is the main finding and its importance? Although the numbers of circulating endothelial microparticles do not differ between middle-aged men and women, there are sex-related differences in the expression of miR-125a in activation-derived EMPs and miR-34a in apoptosis-derived EMPs. Differences in circulating endothelial microparticle microRNA content may provide new insight into the sex-related disparity in the risk and prevalence of vascular disease in middle-aged adults. The aims of this study were to determine: (i) whether circulating concentrations of endothelial microparticles (EMPs) differ in middle-aged men compared with women; and (ii) whether there are sex-related differences in microRNA expression in EMPs. Peripheral blood was collected from 30 sedentary adults: 15 men (56 ± 6 years old) and 15 women (56 ± 5 years old). Endothelial microparticles were defined by markers of activation (CD62e+ ) or apoptosis (CD31+ /CD42b- ) by flow cytometry. Expression of microRNA (miR-34a, 92a, 125a and 126) in activation- and apoptosis-derived EMPs was measured by RT-PCR. Circulating activation- (33 ± 31 versus 39 ± 35 microparticles µl-1 ) and apoptosis-derived EMPs (49 ± 54 versus 42 ± 43 microparticles µl-1 ) were not significantly different between men and women. Expression of miR-125a (2.23 ± 2.01 versus 6.95 ± 3.99 a.u.) was lower (∼215%; P < 0.05) in activation-derived EMPs, whereas expression of miR-34a (1.17 ± 1.43 versus 0.38 ± 0.35 a.u.) was higher (∼210%; P < 0.05) in apoptosis-derived EMPs from men compared with women. Expression of microRNA in circulating EMPs may provide new insight into sex-related differences in cardiovascular disease.

Passive heat stress reduces circulating endothelial and platelet microparticles.

NEW FINDINGS: What is the central question of this study? Does passive heat stress of +2°C oesophageal temperature change concentrations of circulating arterial endothelial- and platelet-derived microparticles in healthy adults? What is the main finding and its importance? Concentrations of circulating endothelial- and platelet-derived microparticles were markedly decreased in heat stress. Reductions in circulating microparticles might indicate favourable vascular changes associated with non-pathological hyperthermia. Interest in circulating endothelial- and platelet-derived microparticles (EMPs and PMPs, respectively) has increased because of their potential pathogenic role in vascular disease and as biomarkers for vascular health. Hyperthermia is commonly associated with a pro-inflammatory stress but might also provide vascular protection when the temperature elevation is non-pathological. Circulating microparticles might contribute to the cellular adjustments and resultant vascular impacts of hyperthermia. Here, we determined whether circulating concentrations of arterial EMPs and PMPs are altered by passive heat stress (+2°C oesophageal temperature). Ten healthy young men (age 23 ± 3 years) completed the study. Hyperthermia was achieved by circulating ∼49°C water through a water-perfused suit that covered the entire body except the hands, feet and head. Arterial (radial) blood samples were obtained immediately before heating (normothermia) and in hyperthermia. The mean ± SD oesophageal temperature in normothermia was 37.2 ± 0.1°C and in hyperthermia 39.1 ± 0.1°C. Concentrations of circulating EMPs and PMPs were markedly decreased in hyperthermia. Activation-derived EMPs were reduced by ∼30% (mean ± SD; from 61 ± 8 to 43 ± 7 microparticles µl-1 ; P < 0.05) and apoptosis-derived EMPs by ∼45% (from 46 ± 7 to 23 ± 3 microparticles µl-1 ; P < 0.05). Likewise, circulating PMPs were reduced by ∼75% in response to hyperthermia (from 256 ± 43 to 62 ± 14 microparticles µl-1 ). These beneficial reductions in circulating EMPs and PMPs in response to a 2°C increase in core temperature might partly underlie the reported vascular improvements following therapeutic bouts of physiological hyperthermia.

ß-Adrenergic receptor antagonism in mice: a model for pediatric heart disease.

Children with heart failure are treated with similar medical therapy as adults with heart failure. In contrast to adults with heart failure, these treatment regiments are not associated with improved outcomes in children. Recent studies have demonstrated age-related pathophysiological differences in the molecular mechanisms of heart failure between children and adults. There are no animal models of pediatric cardiomyopathy to allow mechanistic studies. The purpose of the current experiments was to develop a mouse model of pediatric heart disease and test whether the influence of ß-adrenergic receptor (ß-AR) antagonism could be modeled in this system. We hypothesized that isoproterenol treatment of young mice would provide a model system of cardiac pathology, and that nonselective ß-AR blockade would provide benefit in adult, but not young, mice, similar to clinical trial data. We found that isoproterenol treatment (through osmotic minipump implantation) of young and adult mice produced similar degrees of cardiac hypertrophy and recapitulated several age-related molecular abnormalities in human heart failure, including phospholamban phosphorylation and ß-AR expression. We also found that nonselective ß-AR blockade effectively prevented pathological cardiac growth and collagen expression in the adult but not young mice, and that selective ß1-AR blockade was effective in both young and adult isoproterenol-treated mice. In conclusion, we have developed the first model system for ß-AR-mediated pediatric heart disease. Furthermore, we have generated novel data suggesting beneficial effects of selective ß1-AR blockade in the pediatric heart.