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

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

Toll-Like Receptor 7 Stimulation Promotes the Development of Atherosclerosis in Apolipoprotein E-Deficient Mice.

Atherosclerosis is a chronic inflammatory disease with multiple characteristic facets, including vascular inflammation, endothelial dysfunction, plaque development, impaired blood flow, and cholesterol deposition through dyslipidemia. Toll-like receptors (TLRs) of the innate immune system have been closely linked to the development of atherosclerotic lesions. TLR7 recognizes viral or endogenous single-stranded RNA, which is released during vascular apoptosis and necrosis. The role of TLR7 in vascular disease remains controversial, and therefore, we sought to investigate the effects of TLR7 stimulation in mice.Intravenous injection of a ligand for TLR7 (R848) induced a significant pro-inflammatory cytokine response in mice. This was associated with impaired reendothelialization upon acute denudation of the carotid artery, as measured by Evan's blue staining, and increased numbers of circulating endothelial microparticles (EMPs) and circulating Sca1/Flk1 positive cells as a marker for increased endothelial damage. Chronic subcutaneous stimulation of TLR7 in apolipoprotein E-deficient (ApoE-/-) mice increased aortic production of reactive oxygen species (ROS), the number of circulating EMPs, and most importantly, augmented the formation of atherosclerotic plaque when compared with vehicle-treated animals.Systemic stimulation of TLR7 leads to impaired reendothelialization upon acute vascular injury and is associated with the production of pro-inflammatory cytokines and increased levels of circulating EMPs and Sca1/Flk1 positive cells. Importantly, ApoE-/- mice chronically treated with R848 displayed increased atherosclerotic plaque development and elevated levels of ROS in the aortic tissue. In addition, TLR7-activation-induced apoptosis and impaired migration in human coronary artery endothelial cells and showed significant upregulation of the signaling cascade of IL-1 receptor-associated kinase (IRAK) 2 and IRAK4. Our data highlight the importance of fully understanding the pathomechanisms involved in atherogenesis, and further studies are necessary to identify the ligand-specific effects of TLR7 for possible therapeutic targeting.

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

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

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

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

MDA-5 activation by cytoplasmic double-stranded RNA impairs endothelial function and aggravates atherosclerosis.

Recent studies have highlighted the relevance of viral nucleic acid immunorecognition by pattern recognition receptors in atherogenesis. Melanoma differentiation associated gene 5 (MDA-5) belongs to the intracellular retinoic acid inducible gene-I like receptors and its activation promotes pro-inflammatory mechanisms. Here, we studied the effect of MDA-5 stimulation in vascular biology. To gain insights into MDA-5 dependent effects on endothelial function, cultured human coronary artery endothelial cells (HCAEC) were transfected with the synthetic MDA-5 agonist polyIC (long double-stranded RNA). Human coronary endothelial cell expressed MDA-5 and reacted with receptor up-regulation upon stimulation. Reactive oxygen species formation, apoptosis and the release of pro-inflammatory cytokines was enhanced, whereas migration was significantly reduced in response to MDA-5 stimulation. To test these effects in vivo, wild-type mice were transfected with 32.5 µg polyIC/JetPEI or polyA/JetPEI as control every other day for 7 days. In polyIC-treated wild-type mice, endothelium-dependent vasodilation and re-endothelialization was significantly impaired, vascular oxidative stress significantly increased and circulating endothelial microparticles and circulating endothelial progenitor cells significantly elevated compared to controls. Importantly, these effects could be abrogated by MDA-5 deficiency in vivo. Finally, chronic MDA-5 stimulation in Apolipoprotein E/toll-like receptor 3 (TLR3) double(-) deficient (ApoE(-/-) /TLR3(-/-) ) mice-enhanced atherosclerotic plaque formation. This study demonstrates that MDA-5 stimulation leads to endothelial dysfunction, and has the potential to aggravate atherosclerotic plaque burden in murine atherosclerosis. Thus, the spectrum of relevant innate immune receptors in vascular diseases and atherogenesis might not be restricted to TLRs but also encompasses the group of RLRs including MDA-5.

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

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

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

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

AIM2 Stimulation Impairs Reendothelialization and Promotes the Development of Atherosclerosis in Mice.

Background: Atherosclerosis has been shown to result from chronic inflammation caused by constitutive activation of the pattern recognition receptors (PRR), which are principle effectors of the innate immune system. PRR are present in the endosome or on the cellular membrane and can sense the aberrant release of nucleic acids, which is often a sign of acute or chronic cellular damage. Absent in melanoma 2 (AIM2) is a PRR that is expressed by vascular cells and specializes in detecting cytoplasmic double-stranded DNA (dsDNA). Activation of AIM2 leads eventually to activation of the inflammasome, but the role of AIM2 in vascular disease and atherosclerosis has not been well-studied. Therefore, in this study we took advantage of acute and chronic models of vascular injury to determine the biological role of AIM2 in atherogenesis. Methods and Results: We were able to induce significant release of proinflammatory cytokines in mice through the intravenous injection of a synthetic ligand for AIM2, double-stranded poly dA:dT. This cytokine release was shown to impair reendothelialization of the carotid artery and increase the number of circulating endothelial microparticles (EMP) after acute denudation, compared to treatment with vehicle. We saw an increase in the production of reactive oxygen species in the aorta, the number of circulating EMP, and, most interestingly, atherosclerotic plaque formation in apolipoprotein E-deficient (ApoE-/-) mice when they received continual subcutaneous poly dA:dT, in contrast to vehicle-treated animals. Finally, treatment with poly dA:dT did not impair vascular reendothelialization in AIM2-/- mice compared to vehicle controls in the carotid artery injury model. Conclusion: Overall, our data suggest that AIM2, as a known regulator of the inflammasome, is an active participant in atherogenesis, and highlight the importance of fully understanding the pathological mechanisms involved. It seems to be worth of further exploration as a therapeutic target, and future studies focusing on the effects of AIM2 activation as well as its pharmacological inhibition may reveal promising new therapeutic concepts for the treatment of atherosclerosis.

Kinetics of Circulating MicroRNAs in Response to Cardiac Stress in Patients With Coronary Artery Disease.

BACKGROUND: Circulating microRNAs (miRNAs/miRs) are regulated in patients with coronary artery disease. The impact of transient coronary ischemia on circulating miRNA levels is unknown. We aimed to investigate circulating miRNA kinetics in response to cardiac stress in patients with or without significant coronary stenosis. METHODS AND RESULTS: Eighty of 105 screened patients with stable coronary artery disease underwent dobutamine stress echocardiography before coronary angiography. Nine circulating vascular miRNAs (miRNA-21, miRNA-26, miRNA-27a, miRNA-92a, miRNA-126-3p, miRNA-133a, miRNA-222, miRNA-223, and miRNA-199-5p) were quantified in plasma by reverse transcription polymerase chain reaction before, immediately after, and 4 and 24 hours after dobutamine stress echocardiography. Quantitative polymerase chain reaction revealed increased miRNA-21, miRNA-126-3p, and miRNA-222 levels at 24 hours after dobutamine stress echocardiography in all patients. On coronary angiography, significant coronary artery stenoses (>80% diameter stenosis) were found in 41 patients. Stratifying patients according to the prevalence of significant stenoses, patients with stenosis showed an increase of circulating miRNA-21, miRNA-126-3p, and miRNA-222 in response to cardiac stress. In patients without significant stenoses (<50% diameter stenosis), miRNA-92a levels gradually increased in response to cardiac stress. CONCLUSIONS: miRNAs are distinctly released into the circulation in response to cardiac stress depending on the prevalence of significant coronary stenoses.