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Background Antiplatelet therapy (APT) has improved cardiovascular outcomes, but some patients develop thrombosis despite APT. Adjusting APT to platelet reactivity with conventional platelet reactivity tests has proven unsuccessful. Many circulating noncoding RNAs (ncRNAs) are derived from platelets and could serve as novel platelet function markers. Material and methods Platelet reactivity was assessed using light transmission aggregometry (LTA) in the Bruneck 2015 study (N = 338), using the agonists arachidonic acid, adenosine diphosphate, collagen, TRAP-6 amide and U46619. LTA platelet releasates were then used for RT-qPCR of five platelet-enriched microRNAs, three circular RNAs and two long non-coding RNAs. Platelet-poor plasma (PPP) was used as negative control. Results and conclusions Platelet agonists induced aggregation and ncRNA release, with aspirin takers (N = 155) showing lower ncRNA release than individuals not on aspirin (N = 183). Agonist responsiveness differed among ncRNAs, with miR-150 being hyperresponsive to adenosine diphosphate and miR-21 being hyperresponsive to arachidonic acid, whereas other ncRNAs were most strongly released to collagen, suggesting a selective release mechanism. In individuals not on aspirin, the inflammation markers C-reactive protein and granulocyte counts correlated positively with platelet-derived ncRNAs in PPP, while they correlated inversely with platelet-derived ncRNAs in releasates. These correlations were not present in aspirin takers. Higher PPP levels and lower releasate levels of platelet-derived ncRNAs in inflammation suggest platelet exhaustion ex vivo due to platelet pre-activation in vivo. Funding sources British Heart Foundation, VASCage (Centre for Promoting Vascular Health in the Ageing Community) of the Austrian Research Promotion Agency FFG (COMET program - Competence Centers for Excellent Technologies).

The presence of both endothelial cells (ECs) and mural cells are central to the proper function of blood vessels in health and pathological changes in diseases including diabetes. Although iPSCs-derived vascular organoids (VOs) provide an appealing in vitro disease model and platform for drug screening, whether these organoids recapitulate human disease remains debatable. Here, we show human diabetic (DB)-VOs represent impaired vascular function including enhanced ROS activity, with higher mitochondrial content and activity, increased pro-inflammatory cytokines, and less regenerative potential in vivo. Using single-cell RNA sequencing, we identify all specialized types of vascular cells (artery, capillary, vein, lymphatic and tip cells, as well as pericytes and vSMCs) within vascular organoids, while demonstrating the dichotomy landscape of ECs and mural cells. Furthermore, we reveal basal heterogeneity within vascular organoids and demonstrate differences between diabetic and non-diabetic VOs. Of note, a subpopulation of ECs significantly enrich for ROS and oxidative phosphorylation hallmarks in DB-VOs, may represent early signs of aberrant angiogenesis in diabetes. This study helps to identify key biomarkers for diabetic disease progression and find signalling molecules amenable to drug intervention.

Significance: Levels of platelet noncoding RNAs (ncRNAs) are altered by disease, and ncRNAs may exert functions inside and outside of platelets. Their role in physiologic hemostasis and pathologic thrombosis remains to be explored. Recent Advances: The number of RNA classes identified in platelets has been growing since the past decade. Apart from coding messenger RNAs, the RNA landscape in platelets comprises ncRNAs such as microRNAs, circular RNAs, long ncRNAs, YRNAs, and potentially environmentally derived exogenous ncRNAs. Recent research has focused on the function of platelet RNAs beyond platelets, mediated through protective RNA shuttles or even cellular uptake of entire platelets. Multiple studies have also explored the potential of platelet RNAs as novel biomarkers. Critical Issues: Platelet preparations can contain contaminating leukocytes. Even few leukocytes may contribute a substantial amount of RNA. As biomarkers, platelet RNAs have shown associations with platelet activation, but it remains to be seen whether their measurements could improve diagnostics. It also needs to be clarified whether platelet RNAs influence processes beyond platelets. Future Directions: Technological advances such as single-cell RNA-sequencing might help to identify hyperreactive platelet subpopulations on a single-platelet level, avoid the common problem of leukocyte contamination in platelet preparations, and allow simultaneous profiling of native megakaryocytes and their platelet progeny to clarify to what extent the platelet RNA content reflects their megakaryocyte precursors or changes in the circulation. Antioxid. Redox Signal. 34, 1200-1216.

Supplemental Digital Content is available in the text., Rationale: Noncoding RNAs (ncRNAs), including microRNAs (miRNAs), circular RNAs (circRNAs), and long noncoding RNAs (lncRNAs), are proposed novel biomarkers of myocardial injury. Their release kinetics have not been explored without confounding by heparin nor has their relationship to myocardial protein biomarkers. Objective: To compare ncRNA types in heparinase-treated samples with established and emerging protein biomarkers for myocardial injury. Methods and Results: Screening of 158 circRNAs and 21 lncRNAs in human cardiac tissue identified 12 circRNAs and 11 lncRNAs as potential biomarkers with cardiac origin. Eleven miRNAs were included. At low spike-in concentrations of myocardial tissue, significantly higher regression coefficients were observed across ncRNA types compared with cardiac troponins and cMyBP-C (cardiac myosin-binding protein C). Heparinase treatment of serial plasma and serum samples of patients undergoing transcoronary ablation of septal hypertrophy removed spurious correlations between miRNAs in non-heparinase-treated samples. After transcoronary ablation of septal hypertrophy, muscle-enriched miRNAs (miR-1 and miR-133a) showed a steeper and earlier increase than cardiac-enriched miRNAs (miR-499 and miR-208b). Putative cardiac lncRNAs, including LIPCAR (long intergenic noncoding RNA predicting cardiac remodeling and survival), did not rise, refuting a predominant cardiac origin. Cardiac circRNAs remained largely undetectable. In a validation cohort of acute myocardial infarction, receiver operating characteristic curve analysis revealed noninferiority of cardiac-enriched miRNAs, but miRNAs failed to identify cases presenting with low troponin values. cMyBP-C was validated as a biomarker with highly sensitive properties, and the combination of muscle-enriched miRNAs with high-sensitive cardiac troponin T and cMyBP-C returned the highest area under the curve values. Conclusions: In a comparative assessment of ncRNAs and protein biomarkers for myocardial injury, cMyBP-C showed properties as the most sensitive cardiac biomarker while miRNAs emerged as promising candidates to integrate ncRNAs with protein biomarkers. Sensitivity of current miRNA detection is inferior to cardiac proteins but a multibiomarker combination of muscle-enriched miRNAs with cMyBP-C and cardiac troponins could open a new path of integrating complementary characteristics of different biomarker types.

Supplemental Digital Content is available in the text., Background: The angiogenic function of endothelial cells is regulated by numerous mechanisms, but the impact of long noncoding RNAs (lncRNAs) has hardly been studied. We set out to identify novel and functionally important endothelial lncRNAs. Methods: Epigenetically controlled lncRNAs in human umbilical vein endothelial cells were searched by exon-array analysis after knockdown of the histone demethylase JARID1B. Molecular mechanisms were investigated by RNA pulldown and immunoprecipitation, mass spectrometry, microarray, several knockdown approaches, CRISPR-Cas9, assay for transposase-accessible chromatin sequencing, and chromatin immunoprecipitation in human umbilical vein endothelial cells. Patient samples from lung and tumors were studied for MANTIS expression. Results: A search for epigenetically controlled endothelial lncRNAs yielded lncRNA n342419, here termed MANTIS, as the most strongly regulated lncRNA. Controlled by the histone demethylase JARID1B, MANTIS was downregulated in patients with idiopathic pulmonary arterial hypertension and in rats treated with monocrotaline, whereas it was upregulated in carotid arteries of Macaca fascicularis subjected to atherosclerosis regression diet, and in endothelial cells isolated from human glioblastoma patients. CRISPR/Cas9-mediated deletion or silencing of MANTIS with small interfering RNAs or GapmeRs inhibited angiogenic sprouting and alignment of endothelial cells in response to shear stress. Mechanistically, the nuclear-localized MANTIS lncRNA interacted with BRG1, the catalytic subunit of the switch/sucrose nonfermentable chromatin-remodeling complex. This interaction was required for nucleosome remodeling by keeping the ATPase function of BRG1 active. Thereby, the transcription of key endothelial genes such as SOX18, SMAD6, and COUP-TFII was regulated by ensuring efficient RNA polymerase II machinery binding. Conclusion: MANTIS is a differentially regulated novel lncRNA facilitating endothelial angiogenic function.

The mortality rate for (cardio)-vascular disease is one of the highest in the world, so a healthy functional endothelium is of outmost importance against vascular disease. In this study, human induced pluripotent stem (iPS) cells were reprogrammed from 1 ml blood of healthy donors and subsequently differentiated into endothelial cells (iPS-ECs) with typical EC characteristics. This research combined iPS cell technologies and next-generation sequencing to acquire an insight into the transcriptional regulation of iPS-ECs. We identified Endothelial cell-specific molecule 1 (ESM1) as one of the highest expressed genes during EC differentiation, playing a key role in EC enrichment and function by regulating connexin 40 (CX40) and eNOS. Importantly, ESM1 enhanced the iPS-ECs potential to improve angiogenesis and neovascularisation in in vivo models of angiogenesis and hind limb ischemia. These findings demonstrated for the first time that enriched functional ECs are derived through cell reprogramming and ESM1 signalling, opening the horizon for drug screening and cell-based therapies for vascular diseases. Therefore, this study showcases a new approach for enriching and enhancing the function of induced pluripotent stem (iPS) cell-derived endothelial cells from a very small amount of blood through ESM1 signalling, which greatly enhances their functionality and increases their therapeutic potential. © AlphaMed Press 2018.

Fibrosis is a major contributor to organ disease for which no specific therapy is available. MicroRNA-21 (miR-21) has been implicated in the fibrogenetic response, and inhibitors of miR-21 are currently undergoing clinical trials. Here, we explore how miR-21 inhibition may attenuate fibrosis using a proteomics approach. Transfection of miR-21 mimic or inhibitor in murine cardiac fibroblasts revealed limited effects on extracellular matrix (ECM) protein secretion. Similarly, miR-21–null mouse hearts showed an unaltered ECM composition. Thus, we searched for additional explanations as to how miR-21 might regulate fibrosis. In plasma samples from the community-based Bruneck Study, we found a marked correlation of miR-21 levels with several platelet-derived profibrotic factors, including TGF-β1. Pharmacological miR-21 inhibition with an antagomiR reduced the platelet release of TGF-β1 in mice. Mechanistically, Wiskott-Aldrich syndrome protein, a negative regulator of platelet TGF-β1 secretion, was identified as a direct target of miR-21. miR-21–null mice had lower platelet and leukocyte counts compared with littermate controls but higher megakaryocyte numbers in the bone marrow. Thus, to our knowledge this study reports a previously unrecognized effect of miR-21 inhibition on platelets. The effect of antagomiR-21 treatment on platelet TGF-β1 release, in particular, may contribute to the antifibrotic effects of miR-21 inhibitors., MicroRNA-21 inhibition may convey its therapeutic benefits in fibrosis through its action in bone marrow cells rather than targeting fibroblasts directly.

Introduction: Fibrosis is a major contributor to cardiac disease. MicroRNA-21 (miR-21) has been implicated as a regulator of fibrosis, with inhibitors of miR-21 currently undergoing clinical trials. We aimed to explore how miR-21 inhibition attenuates fibrosis using a proteomics approach. Methods and Results: As expected, transfection with miR-21 mimics and inhibitors altered the proliferation of murine cardiac fibroblasts. However, proteomic analysis of their conditioned media revealed limited effects on ECM secretion. Similarly, proteomic analysis of hearts from miR-21 null mice showed no major difference in extracellular matrix (ECM) composition. Thus, we searched for additional explanations as to how miR-21 might regulate fibrosis. In the community-based Bruneck study (year 2000 follow-up, n=671), we compared circulating miR-21 with a panel of 229 proteins associated with cardiovascular disease. Several platelet-derived pro-fibrotic factors correlated with plasma miR-21 levels, including the latency-associated peptide of transforming growth factor beta-1 (TGF-β1). This correlation was confirmed by ELISA measurements of active TGF-β1 in plasma (Bruneck 2015 follow-up, n=332). In agreement with this observation in men, systemic inhibition of miR-21 with an antagomiR reduced the platelet release of TGF-β1 in mice. Mechanistically, Wiskott-Aldrich Syndrome protein, a negative regulator of platelet TGF-β1 secretion, was identified as a direct target of miR-21. AntagomiR-21 treatment, however, had no effect on the platelet count or their aggregation response. In contrast, miR-21 null mice had significantly lower platelet counts compared to littermate controls. Conclusions: This study reports a previously unrecognized effect of miR-21 inhibition on platelets. Both genetic deletion of miR-21 and antagomiR treatment affect platelets. The effect of miR-21 inhibition on platelet TGF-β1 release, in particular, may contribute to the anti-fibrotic effects of miR-21 inhibitors.

Rationale Non-coding RNAs (ncRNAs), including microRNAs (miRNAs), circular RNAs (circRNA) and long non-coding RNAs (lncRNA), have been implicated as novel cardiac biomarkers. Objective To compare the tissue-specificity and release kinetics of ncRNAs and protein biomarkers after induced myocardial injury by transcoronary ablation of septal hypertrophy (TASH). Methods and results Upon screening the relative abundance of 109 circRNA and 21 lncRNAs in human cardiac tissue, 12 circRNAs and 11 lncRNAs were selected for further analyses. Human myocardial tissue was spiked into plasma from healthy individuals and the expression levels of ncRNAs were compared to a panel of miRNAs, including muscle- (miR-1, miR-133a) and cardiac-enriched miRNAs (miR-208a, miR-208b, miR-499). Curve fitting analyses of each of the three ncRNA classes with the highest R2 values revealed no significant differences in the regression coefficients compared with high sensitive troponin T and I (hs-cTnT, hs-cTnI) and cardiac myosin-binding protein C (cMyC). At low spike-in concentrations, however, significantly higher regression coefficients were observed for all ncRNA species (Mann Whitney test: miRNAs vs. proteins p Conclusions Our results demonstrate that circulating heart-associated ncRNAs may enhance early detection of myocardial injury. All three ncRNA classes demonstrated superior release kinetics in vitro compared with established cardiac protein biomarkers. At early time points after TASH, however, a higher sensitivity was only observed for muscle-enriched miRNAs, but not for circRNAs or lncRNAs.

Introduction MicroRNA-21 (miR-21) has been implicated as a regulator of fibrosis. Inhibitors of miR-21 are currently undergoing clinical trials. Our aim was to explore mechanisms of how miR-21 inhibition attenuates fibrosis using a proteomics approach. Methods and results Primary murine cardiac fibroblasts (CF) were transfected with miR-21 mimics and inhibitors. In line with previous reports, miR-21 overexpression increased CF proliferation (n=4 vs. 4; p Conclusions This study reports a previously unrecognised effect of miR-21 inhibition on platelet TGF-β1 release. This novel systemic effect of miR-21 on platelets may contribute to the anti-fibrotic effects of miR-21 inhibitors.

Rationale : Abdominal aortic aneurysms constitute a degenerative process in the aortic wall. Both the miR-29 and miR-15 families have been implicated in regulating the vascular extracellular matrix. Objective : Our aim was to assess the effect of the miR-15 family on aortic aneurysm development. Methods and Results : Among the miR-15 family members, miR-195 was differentially expressed in aortas of apolipoprotein E–deficient mice on angiotensin II infusion. Proteomics analysis of the secretome of murine aortic smooth muscle cells, after miR-195 manipulation, revealed that miR-195 targets a cadre of extracellular matrix proteins, including collagens, proteoglycans, elastin, and proteins associated with elastic microfibrils, albeit miR-29b showed a stronger effect, particularly in regulating collagens. Systemic and local administration of cholesterol-conjugated antagomiRs revealed better inhibition of miR-195 compared with miR-29b in the uninjured aorta. However, in apolipoprotein E–deficient mice receiving angiotensin II, silencing of miR-29b, but not miR-195, led to an attenuation of aortic dilation. Higher aortic elastin expression was accompanied by an increase of matrix metalloproteinases 2 and 9 in mice treated with antagomiR-195. In human plasma, an inverse correlation of miR-195 was observed with the presence of abdominal aortic aneurysms and aortic diameter. Conclusions : We provide the first evidence that miR-195 may contribute to the pathogenesis of aortic aneurysmal disease. Although inhibition of miR-29b proved more effective in preventing aneurysm formation in a preclinical model, miR-195 represents a potent regulator of the aortic extracellular matrix. Notably, plasma levels of miR-195 were reduced in patients with abdominal aortic aneurysms suggesting that microRNAs might serve as a noninvasive biomarker of abdominal aortic aneurysms.

Background: The role of the unspliced XBP1 remains unclear. Results: Disturbed flow concomitantly up-regulates XBP1u and HDAC3, which form a complex with Akt1 and mTOR, leading to Nrf2-mediated HO-1 expression. Conclusion: XBP1u and HDAC3 synergistically exert a protective effect on disturbed flow-induced oxidative stress via regulation of HO-1 expression. Significance: This study provides new insights into the physiological roles of XBP1u and HDAC3., It is well known that atherosclerosis occurs geographically at branch points where disturbed flow predisposes to the development of plaque via triggering of oxidative stress and inflammatory reactions. In this study, we found that disturbed flow activated anti-oxidative reactions via up-regulating heme oxygenase 1 (HO-1) in an X-box-binding protein 1 (XBP1) and histone deacetylase 3 (HDAC3)-dependent manner. Disturbed flow concomitantly up-regulated the unspliced XBP1 (XBP1u) and HDAC3 in a VEGF receptor and PI3K/Akt-dependent manner. The presence of XBP1 was essential for the up-regulation of HDAC3 protein. Overexpression of XBP1u and/or HDAC3 activated Akt1 phosphorylation, Nrf2 protein stabilization and nuclear translocation, and HO-1 expression. Knockdown of XBP1u decreased the basal level and disturbed flow-induced Akt1 phosphorylation, Nrf2 stabilization, and HO-1 expression. Knockdown of HDAC3 ablated XBP1u-mediated effects. The mammalian target of rapamycin complex 2 (mTORC2) inhibitor, AZD2014, ablated XBP1u or HDAC3 or disturbed flow-mediated Akt1 phosphorylation, Nrf2 nuclear translocation, and HO-1 expression. Neither actinomycin D nor cycloheximide affected disturbed flow-induced up-regulation of Nrf2 protein. Knockdown of Nrf2 abolished XBP1u or HDAC3 or disturbed flow-induced HO-1 up-regulation. Co-immunoprecipitation assays demonstrated that XBP1u physically bound to HDAC3 and Akt1. The region of amino acids 201 to 323 of the HDAC3 protein was responsible for the binding to XBP1u. Double immunofluorescence staining revealed that the interactions between Akt1 and mTORC2, Akt1 and HDAC3, Akt1 and XBP1u, HDAC3, and XBP1u occurred in the cytosol. Thus, we demonstrate that XBP1u and HDAC3 exert a protective effect on disturbed flow-induced oxidative stress via up-regulation of mTORC2-dependent Akt1 phosphorylation and Nrf2-mediated HO-1 expression.

Micro-RNAs (miRNAs) are small noncoding RNAs that control gene expression epigenetically. miRNA genes are transcribed by RNA polymerase II into a primary transcript—the primary miRNA that consists of at least 1 hairpin structure, with the characteristic long stem and a terminal loop. Mature miRNAs will be typically generated from the primary transcripts after 2 cleavage events (Figure): the first cleavage occurs in the nucleus by an RNAse III, the Drosha–DGCR8 complex, from primary miRNAs to pre-miRNAs. The second cleavage takes place in the cytosol where pre-miRNAs are further cleaved by Dicer, and the mature miRNAs are loaded into the RNA-induced silencing complex.1 Although platelets lack a nucleus, there are several lines of evidence suggesting a functional miRNA pathway in platelets: (1) platelets contain miRNAs and express both Dicer (required for cleavage of pre-miRNAs to mature miRNAs) and Argonaute 2 (required for RNA-mediated gene silencing by the RNA-induced silencing complex)2 and (2) they are also major contributors to the circulating miRNA pool as evidenced by significant positive correlations to platelet microparticles3 and a marked reduction on antiplatelet therapy.4 Figure. miRNA biogenesis and impairment in diabetic platelets (marked with pins). On transcription by RNA polymerase II, the primary miRNA (pri-miRNAs) is processed to a precursor miRNA (pre-miRNA) by the Drosha–DGCR8 (DiGeorge syndrome chromosomal [or critical] region 8) complex. The pre-miRNA is then exported from the nucleus into the cytosol by Exportin-5. Before the mature miRNAs are loaded into the RNA-induced silencing complex (RISC), the pre-miRNA hairpin has to be cleaved by Dicer. The latter step is impaired in diabetic platelets because of increased calpain activity. Calpain targets Dicer for proteolytic degradation. Because platelets lack a …

Background— The molecular mechanisms underlying similarities and differences between physiological and pathological left ventricular hypertrophy (LVH) are of intense interest. Most previous work involved targeted analysis of individual signaling pathways or screening of transcriptomic profiles. We developed a network biology approach using genomic and proteomic data to study the molecular patterns that distinguish pathological and physiological LVH. Methods and Results— A network-based analysis using graph theory methods was undertaken on 127 genome-wide expression arrays of in vivo murine LVH. This revealed phenotype-specific pathological and physiological gene coexpression networks. Despite >1650 common genes in the 2 networks, network structure is significantly different. This is largely because of rewiring of genes that are differentially coexpressed in the 2 networks; this novel concept of differential wiring was further validated experimentally. Functional analysis of the rewired network revealed several distinct cellular pathways and gene sets. Deeper exploration was undertaken by targeted proteomic analysis of mitochondrial, myofilament, and extracellular subproteomes in pathological LVH. A notable finding was that mRNA–protein correlation was greater at the cellular pathway level than for individual loci. Conclusions— This first combined gene network and proteomic analysis of LVH reveals novel insights into the integrated pathomechanisms that distinguish pathological versus physiological phenotypes. In particular, we identify differential gene wiring as a major distinguishing feature of these phenotypes. This approach provides a platform for the investigation of potentially novel pathways in LVH and offers a freely accessible protocol ( http://sites.google.com/site/cardionetworks ) for similar analyses in other cardiovascular diseases.

MicroRNA-122 (miR-122) is abundant in the liver and involved in lipid homeostasis, but its relevance to the long-term risk of developing metabolic disorders is unknown. We therefore measured circulating miR-122 in the prospective population-based Bruneck Study (n = 810; survey year 1995). Circulating miR-122 was associated with prevalent insulin resistance, obesity, metabolic syndrome, type 2 diabetes, and an adverse lipid profile. Among 92 plasma proteins and 135 lipid subspecies quantified with mass spectrometry, it correlated inversely with zinc-α-2-glycoprotein and positively with afamin, complement factor H, VLDL-associated apolipoproteins, and lipid subspecies containing monounsaturated and saturated fatty acids. Proteomics analysis of livers from antagomiR-122–treated mice revealed novel regulators of hepatic lipid metabolism that are responsive to miR-122 inhibition. In the Anglo-Scandinavian Cardiac Outcomes Trial (ASCOT, n = 155), 12-month atorvastatin reduced circulating miR-122. A similar response to atorvastatin was observed in mice and cultured murine hepatocytes. Over up to 15 years of follow-up in the Bruneck Study, multivariable adjusted risk ratios per one-SD higher log miR-122 were 1.60 (95% CI 1.30–1.96; P < 0.001) for metabolic syndrome and 1.37 (1.03–1.82; P = 0.021) for type 2 diabetes. In conclusion, circulating miR-122 is strongly associated with the risk of developing metabolic syndrome and type 2 diabetes in the general population.

The presence of stable endogenous microRNA pools in the circulation sparked interest in their potential use as biomarkers of cardiovascular disease. Several studies demonstrated the presence of microRNAs in the vesicles and in the nonvesicular fraction, mainly residing in protein and lipoprotein complexes. There is a need to identify novel biomarkers that can not only improve the established cardiovascular risk scores and identify individuals at high risk but also gain new mechanistic insights. Early studies on microRNA biomarkers for a wide range of cardiovascular pathologies were predominately conducted in small cohorts and provided the first evidence for their putative value. As these findings await confirmation from larger cohorts, several pre-analytical and analytical variables must be determined, and protocols for sample preparation, RNA quantification and data processing must be standardised before one can assess the value of circulating microRNAs for clinical practice. Here, we will review the technical challenges in assessing microRNA profiles and discuss the hurdles in establishing circulating microRNAs as reliable cardiovascular biomarkers.

Rationale: MicroRNA (miRNA) biomarkers are attracting considerable interest. Effects of medication, however, have not been investigated thus far. Objective: To analyze changes in plasma miRNAs in response to antiplatelet therapy. Methods and Results: Profiling for 377 miRNAs was performed in platelets, platelet microparticles, platelet-rich plasma, platelet-poor plasma, and serum. Platelet-rich plasma showed markedly higher levels of miRNAs than serum and platelet-poor plasma. Few abundant platelet miRNAs, such as miR-24, miR-197, miR-191, and miR-223, were also increased in serum compared with platelet-poor plasma. In contrast, antiplatelet therapy significantly reduced miRNA levels. Using custom-made quantitative real-time polymerase chain reaction plates, 92 miRNAs were assessed in a dose-escalation study in healthy volunteers at 4 different time points: at baseline without therapy, at 1 week with 10 mg prasugrel, at 2 weeks with 10 mg prasugrel plus 75 mg aspirin, and at 3 weeks with 10 mg prasugrel plus 300 mg aspirin. Findings in healthy volunteers were confirmed by individual TaqMan quantitative real-time polymerase chain reaction assays (n=9). Validation was performed in an independent cohort of patients with symptomatic atherosclerosis (n=33), who received low-dose aspirin at baseline. Plasma levels of platelet miRNAs, such as miR-223, miR-191, and others, that is, miR-126 and miR-150, decreased on further platelet inhibition. Conclusions: Our study demonstrated a substantial platelet contribution to the circulating miRNA pool and identified miRNAs responsive to antiplatelet therapy. It also highlights that antiplatelet therapy and preparation of blood samples could be confounding factors in case-control studies relating plasma miRNAs to cardiovascular disease.

The postgenomic shift in paradigm from reductionism to systems-wide network inference has increased recognition that cardiovascular diseases are not simply determined by the genome but arise from an interaction and dynamic dysregulation of gene regulatory networks, proteins, and metabolic alterations. The advent of postgenomic technologies promises to interrogate these complex pathophysiological perturbations by applying concepts of systemic relationships to biomarker discovery. A multibiomarker panel consisting of biomarkers capturing different levels of information (eg, microRNAs to assess endothelial and platelet activation, molecular lipid species to profile metabolic status, and proteolytic degradation products to assess vascular integrity) could outperform inflammatory biomarkers without vascular specificity in their ability of predicting cardiovascular risk. As atherosclerosis develops over decades, different biomarkers may be required for different stages of disease. Thus far, there is no simple blood test to directly assess the health of blood vessels or identify vulnerable patients. We discuss strategies for biomarker discovery using post genomics technologies, with a particular focus on circulating microRNAs. The aim is to reveal distinctive cardiovascular phenotypes and identify biomarker signatures that complement the Framingham risk scores in clinical decision-making and in a stratified medicine approach for early preventive treatment of disease.

Sustained activation of X-box-binding protein 1 (XBP1) results in endothelial cell (EC) apoptosis and atherosclerosis development. The present study provides evidence that XBP1 mRNA splicing triggered an autophagic response in ECs by inducing autophagic vesicle formation and markers of autophagy BECLIN-1 and microtubule-associated protein 1 light chain 3β (LC3-βII). Endostatin activated autophagic gene expression through XBP1 mRNA splicing in an inositol-requiring enzyme 1α (IRE1α)-dependent manner. Knockdown of XBP1 or IRE1α by shRNA in ECs ablated endostatin-induced autophagosome formation. Importantly, data from arterial vessels from XBP1 EC conditional knock-out (XBP1eko) mice demonstrated that XBP1 deficiency in ECs reduced the basal level of LC3β expression and ablated response to endostatin. Chromatin immunoprecipitation assays further revealed that the spliced XBP1 isoform bound directly to the BECLIN-1 promoter at the region from nt −537 to −755. BECLIN-1 deficiency in ECs abolished the XBP1-induced autophagy response, whereas spliced XBP1 did not induce transcriptional activation of a truncated BECLIN-1 promoter. These results suggest that XBP1 mRNA splicing triggers an autophagic signal pathway through transcriptional regulation of BECLIN-1. Background: Apoptosis and autophagy are two closely related systems that induce cell death. Results: X-box-binding protein 1 (XBP1) mRNA splicing regulates BECLIN-1 transcriptional activation, a fundamental player in the initiation of autophagy. Conclusion: XBP1 splicing induces an autophagic response in endothelial cells. Significance: XBP1 could be used as an important pharmacological target that can regulate the autophagic machinery and endothelial cell death.

Background: Myocardial fibrosis is a feature of many cardiac diseases. We used proteomics to profile glycoproteins in the human cardiac extracellular matrix (ECM). Methods: Atrial specimens were analyzed by mass spectrometry after extraction of ECM proteins and enrichment for glycoproteins or glycopeptides. Results: ECM-related glycoproteins were identified in left and right atrial appendages from the same patients. Several known glycosylation sites were confirmed. In addition, putative and novel glycosylation sites were detected. On enrichment for glycoproteins, peptides of the small leucine-rich proteoglycan decorin were identified consistently in the flowthrough. Of all ECM proteins identified, decorin was found to be the most fragmented. Within its protein core, 18 different cleavage sites were identified. In contrast, less cleavage was observed for biglycan, the most closely related proteoglycan. Decorin processing differed between human ventricles and atria and was altered in disease. The C-terminus of decorin, important for the interaction with connective tissue growth factor, was detected predominantly in ventricles in comparison with atria. In contrast, atrial appendages from patients in persistent atrial fibrillation had greater levels of full-length decorin but also harbored a cleavage site that was not found in atrial appendages from patients in sinus rhythm. This cleavage site preceded the N-terminal domain of decorin that controls muscle growth by altering the binding capacity for myostatin. Myostatin expression was decreased in atrial appendages of patients with persistent atrial fibrillation and hearts of decorin null mice. A synthetic peptide corresponding to this decorin region dose-dependently inhibited the response to myostatin in cardiomyocytes and in perfused mouse hearts. Conclusions: This proteomics study is the first to analyze the human cardiac ECM. Novel processed forms of decorin protein core, uncovered in human atrial appendages, can regulate the local bioavailability of antihypertrophic and profibrotic growth factors.

SummaryMicroRNAs are small non-coding RNAs that are detectable in plasma and serum. Circulating levels of microRNAs have been measured in various studies related to cardiovascular disease. Heparin is a potential confounder of microRNA measurements due to its known interference with polymerase chain reactions. In this study, platelet-poor plasma was obtained from patients undergoing cardiac catheterisation for diagnostic coronary angiography, or for percutaneous coronary intervention, both before and after heparin administration. Heparin had pronounced effects on the assessment of the exogenous C. elegans spike-in control (decrease by approx. 3 cycles), which disappeared 6 hours after the heparin bolus. Measurements of endogenous microRNAs were less sensitive to heparin medication. Normalisation of individual microRNAs with the average cycle threshold value of all microRNAs provided a suitable alternative to normalisation with exogenous C. elegans spike-in control in this setting. Thus, both the timing of blood sampling relative to heparin dosing and the normalisation procedure are critical for reliable microRNA measurements in patients receiving intravenous heparin. This has to be taken into account when designing studies to investigate the relation of circulating microRNAs to acute cardiovascular events or coronary intervention.

Recent findings demonstrated the importance of microRNAs (miRNAs) in the vasculature and the orchestration of lipid metabolism and glucose homeostasis. MiRNA networks represent an additional layer of regulation for gene expression that absorbs perturbations and ensures the robustness of biological systems. This function is very elegantly demonstrated in cholesterol metabolism where miRNAs reducing cellular cholesterol export are embedded in the very same genes that increase cholesterol synthesis. Often their alteration does not affect normal development but changes under stress conditions and in disease. A detailed understanding of the molecular and cellular mechanisms of miRNA-mediated effects on metabolism and vascular pathophysiology could pave the way for the development of novel diagnostic markers and therapeutic approaches. In the first part of this review, we summarize the role of miRNAs in vascular and metabolic diseases and explore potential confounding effects by platelet miRNAs in preclinical models of cardiovascular disease. In the second part, we discuss experimental strategies for miRNA target identification and the challenges in attributing miRNA effects to specific cell types and single targets.

SummaryMiRNAs are emerging as promising biomarkers in cardiovascular diseases and may constitute a novel mechanism of intercellular communication. Accurate quantification of circulating miRNAs is essential. A variety of technological approaches and platforms have been developed with increased sensitivity and specificity for the detection and quantification of circulating miRNAs. In this review, we focus on the technical aspects and discuss the analytical challenges in profiling circulating miRNAs.

The recent discovery that microRNAs (miRNAs) are present in the circulation sparked interest in their use as potential biomarkers. In this review, we will summarize the latest findings on circulating miRNAs and cardiovascular disease but also discuss analytical challenges. While research on circulating miRNAs is still in its infancy, high analytical standards in statistics and study design are a prerequisite to obtain robust data and avoid repeating the mistakes of the early genetic association studies. Otherwise, studies tend to get published because of their novelty despite low numbers, poorly matched cases and controls and no multivariate adjustment for conventional risk factors. Research on circulating miRNAs can only progress by bringing more statistical rigour to bear in this field and by evaluating changes of individual miRNAs in the context of the overall miRNA network. Such miRNA signatures may have better diagnostic and prognostic value.

Rationale: MicroRNAs (miRNAs) have been implicated in the epigenetic regulation of key metabolic, inflammatory, and antiangiogenic pathways in type 2 diabetes (DM) and may contribute to common disease complications. Objective: In this study, we explore plasma miRNA profiles in patients with DM. Methods and Results: Total RNA was extracted from plasma samples of the prospective population-based Bruneck study. A total of 13 candidate miRNAs identified by microarray screening and miRNA network inference were quantified by quantitative PCR in all diabetic patients of the Bruneck study and age- and sex-matched controls (1995 evaluation, n=80 each). Quantitative PCR assessment revealed lower plasma levels of miR-20b, miR-21, miR-24, miR-15a, miR-126, miR-191, miR-197, miR-223, miR-320, and miR-486 in prevalent DM, but a modest increase of miR-28-3p. Findings emerged as robust in multivariable analysis and were independent of the standardization procedure applied. For endothelial miR-126, results were confirmed in the entire Bruneck cohort (n=822) in univariate (odds ratio [95% confidence interval], 0.38 [0.26 to 0.55]; P =2.72×10 −7 ) and multivariate analyses (0.57 [0.37 to 0.86]; P =0.0082). Importantly, reduced miR-15a, miR-29b, miR-126, miR-223, and elevated miR-28-3p levels antedated the manifestation of disease. Most differences in miRNA levels were replicated in plasma obtained from hyperglycemic Lep ob mice. High glucose concentrations reduced the miR-126 content of endothelial apoptotic bodies. Similarly in patients with DM, the reduction of miR-126 was confined to circulating vesicles in plasma. Conclusions: We reveal a plasma miRNA signature for DM that includes loss of endothelial miR-126. These findings might explain the impaired peripheral angiogenic signaling in patients with DM.

Objective— Recent studies on cardiovascular progenitors have led to a new appreciation that paracrine factors may support the regeneration of damaged tissues. Methods and Results— We used a shotgun proteomics strategy to compare the secretome of peripheral blood–derived smooth muscle progenitors (SPCs) with human aortic smooth muscle cells. The late-outgrowth SPCs produced fewer proteolytic enzymes and inflammatory cytokines and showed reduced invasive capacity. Similar to smooth muscle cells, SPCs secreted extracellular matrix. However, SPCs produced different matrix proteins, as evidenced by the truncation of proangiogenic domains in collagen α-1 (I) and increased production of periostin. Moreover, SPCs retained serum proteins, including proteoglycans, regulating collagen assembly; and pigment epithelium–derived factor, a potent inhibitor of angiogenesis. As a functional consequence, their conditioned medium was less angiogenic, as demonstrated by endothelial tube formation assays in vitro and implantation of Matrigel plugs into nude, severe combined immunodeficient mice (NOD/SCID). Conclusion— The present study represents an important conceptual development, suggesting that SPCs may contribute to extracellular matrix production.

Rationale : Histone deacetylase (HDAC)7 is expressed in the early stages of embryonic development and may play a role in endothelial function. Objective : This study aimed to investigate the role of HDAC7 in endothelial cell (EC) proliferation and growth and the underlying mechanism. Methods and Results : Overexpression of HDAC7 by adenoviral gene transfer suppressed human umbilical vein endothelial cell (HUVEC) proliferation by preventing nuclear translocation of β-catenin and downregulation of T-cell factor-1/Id2 (inhibitor of DNA binding 2) and cyclin D1, leading to G 1 phase elongation. Further assays with the TOPFLASH reporter and quantitative RT-PCR for other β-catenin target genes such as Axin2 confirmed that overexpression of HDAC7 decreased β-catenin activity. Knockdown of HDAC7 by lentiviral short hairpin RNA transfer induced β-catenin nuclear translocation but downregulated cyclin D1, cyclin E1 and E2F2, causing HUVEC hypertrophy. Immunoprecipitation assay and mass spectrometry analysis revealed that HDAC7 directly binds to β-catenin and forms a complex with 14-3-3 ε, ζ, and η proteins. Vascular endothelial growth factor treatment induced HDAC7 degradation via PLCγ-IP3K (phospholipase Cγ–inositol-1,4,5-trisphosphate kinase) signal pathway and partially rescued HDAC7-mediated suppression of proliferation. Moreover, vascular endothelial growth factor stimulation suppressed the binding of HDAC7 with β-catenin, disrupting the complex and releasing β-catenin to translocate into the nucleus. Conclusions : These findings demonstrate that HDAC7 interacts with β-catenin keeping ECs in a low proliferation stage and provides a novel insight into the mechanism of HDAC7-mediated signal pathways leading to endothelial growth.

Background— Histone deacetylase 3 (HDAC3) is known to play a crucial role in the differentiation of endothelial progenitors. The role of HDAC3 in mature endothelial cells, however, is not well understood. Here, we investigated the function of HDAC3 in preserving endothelial integrity in areas of disturbed blood flow, ie, bifurcation areas prone to atherosclerosis development. Methods and Results— En face staining of aortas from apolipoprotein E-knockout mice revealed increased expression of HDAC3, specifically in these branching areas in vivo, whereas rapid upregulation of HDAC3 protein was observed in endothelial cells exposed to disturbed flow in vitro. Interestingly, phosphorylation of HDAC3 at serine/threonine was observed in these cells, suggesting that disturbed flow leads to posttranscriptional modification and stabilization of the HDAC3 protein. Coimmunoprecipitation experiments showed that HDAC3 and Akt form a complex. Using a series of constructs harboring deletions, we found residues 136 to 206 of HDAC3 to be crucial in this interaction. Enforced expression of HDAC3 resulted in increased phosphorylation of Akt and upregulation of its kinase activity. In line with these findings, knockdown of HDAC3 with lentiviral vectors (shHDAC3) led to a dramatic decrease in cell survival accompanied by apoptosis in endothelial cells. In aortic isografts of apolipoprotein E-knockout mice treated with shHDAC3, a robust atherosclerotic lesion was formed. Surprisingly, 3 of the 8 mice that received shHDAC3-infected grafts died within 2 days after the operation. Miller staining of the isografts revealed disruption of the basement membrane and rupture of the vessel. Conclusions— Our findings demonstrated that HDAC3 serves as an essential prosurvival molecule with a critical role in maintaining the endothelial integrity via Akt activation and that severe atherosclerosis and vessel rupture in isografted vessels of apolipoprotein E-knockout mice occur when HDAC3 is knocked down.

Histone deacetylases (HDACs) have a central role in the regulation of gene expression. Here we investigated whether HDAC7 has an impact on embryonic stem (ES) cell differentiation into smooth muscle cells (SMCs). ES cells were seeded on collagen-IV-coated flasks and cultured in the absence of leukemia inhibitory factor in differentiation medium to induce SMC differentiation. Western blots and double-immunofluorescence staining demonstrated that HDAC7 has a parallel expression pattern with SMC marker genes. In ex vivo culture of embryonic cells from SM22-LacZ transgenic mice, overexpression of HDAC7 significantly increased β-galactosidase-positive cell numbers and enzyme activity, indicating its crucial role in SMC differentiation during embryonic development. We found that HDAC7 undergoes alternative splicing during ES cell differentiation. Platelet-derived growth factor enhanced ES cell differentiation into SMCs through upregulation of HDAC7 splicing. Further experiments revealed that HDAC7 splicing induced SMC differentiation through modulation of the SRF-myocardin complex. These findings suggest that HDAC7 splicing is important for SMC differentiation and vessel formation in embryonic development.

X-box binding protein 1 (XBP1) is a key signal transducer in endoplasmic reticulum stress response, and its potential role in the atherosclerosis development is unknown. This study aims to explore the impact of XBP1 on maintaining endothelial integrity related to atherosclerosis and to delineate the underlying mechanism. We found that XBP1 was highly expressed at branch points and areas of atherosclerotic lesions in the arteries of ApoE −/− mice, which was related to the severity of lesion development. In vitro study using human umbilical vein endothelial cells (HUVECs) indicated that disturbed flow increased the activation of XBP1 expression and splicing. Overexpression of spliced XBP1 induced apoptosis of HUVECs and endothelial loss from blood vessels during ex vivo cultures because of caspase activation and down-regulation of VE-cadherin resulting from transcriptional suppression and matrix metalloproteinase-mediated degradation. Reconstitution of VE-cadherin by Ad-VEcad significantly increased Ad-XBP1s-infected HUVEC survival. Importantly, Ad-XBP1s gene transfer to the vessel wall of ApoE −/− mice resulted in development of atherosclerotic lesions after aorta isografting. These results indicate that XBP1 plays an important role in maintaining endothelial integrity and atherosclerosis development, which provides a potential therapeutic target to intervene in atherosclerosis.

Accumulating evidence indicates the impact of endothelial progenitor cells (EPCs) in vascular repair. In patients, the number of EPCs is negatively correlated with the severity of atherosclerosis. In various animal models, transplantation of bone marrow-derived progenitor cells could sufficiently rescue organ function and enhance vascular repair and tissue regeneration. Increase in the number of circulating progenitors, induced by cell transfusion or enhanced mobilization, can also enhance restoration and integrity of the endothelial lining, suppress neointimal formation, and increase blood flow to ischaemic sites. However, the beneficial outcome of EPC infusion very much depends on the growth and differentiation factors within the tissue, cell-to-cell interactions, and the degree of injury. As highlighted by several studies, EPCs derive from different sources including bone marrow and non-bone marrow organs such as the spleen, the functional repair properties of which may vary with the maturation state of the cell. Thus, understanding the molecular mechanisms involved in EPC-repairing processes is essential. In the present review we focus on the role of EPCs in vascular diseases, and we provide an update on the mechanisms of EPC mobilization, homing, and differentiation.

Cultivated vascular smooth muscle cells (SMCs) were surface-labeled with CyDyes followed by biotinylation. After enrichment on avidin columns, proteins were separated on large format gradient gels by SDS-PAGE. A comparison between CyDye-tagged and non-tagged gel bands revealed a substantial increase of protein identifications from membrane, membrane-associated, and extracellular matrix proteins with a corresponding reduction in co-purified intracellular proteins. Notably the majority of identified proteins were involved in cellular adhesion processes. To demonstrate the quantitative potential of this platform, we performed a comparison between mature and embryonic stem cell-derived smooth muscle cells (esSMCs) and identified the membrane proteins E-cadherin, integrin alpha6, and CD98 (4F2) to be significantly up-regulated in esSMCs suggesting that SMCs derived from embryonic stem cells maintain characteristics of their embryonic stem cell origin. This was subsequently confirmed by RT-PCR: despite expressing a panel of smooth muscle markers (calponin, Sm22, and aortic smooth muscle actin), esSMCs remained positive for markers of stem cell pluripotency (Oct4, Nanog, and Rex1). In summary, we describe a novel strategy for the profiling of cell membrane proteins. The procedure combines DIGE technology with biotin/avidin labeling to discriminate membrane and membrane-associated proteins from intracellular contaminants by fluorescence tagging and permits semiquantitative differential expression analysis of membrane proteins.

Inflammation and TNF-α signaling play a central role in most of the pathological conditions where cell transplantation could be applied. As shown by initial experiments, embryonic stem (ES) cells and ES-cell derived vascular cells express very low levels of TNF-α receptor I (TNFRp55) and thus do not induce cytokine expression in response to TNF-α stimulation. Transient transfection analysis of wild-type or deletion variants of the TNFRp55 gene promoter showed a strong activity for a 250-bp fragment in the upstream region of the gene. This activity was abolished by mutations targeting the Sp1/Sp3 or AP1 binding sites. Moreover, treatment with trichostatin A (TSA) led to a pronounced increase in TNFRp55 mRNA and promoter activity. Overexpression of Sp1 or c- fos further enhanced the TSA-induced luciferase activity, and this response was attenuated by Sp3 or c- jun coexpression. Additional experiments revealed that TSA did not affect the Sp1/Sp3 ratio but caused transcriptional activation of the c- fos gene. Thus, we provide the first evidence that ES and ES-cell-derived vascular cells lack cytokine expression in response to TNF-α stimulation due to low levels of c- fos and transcriptional activation of Sp1 that can be regulated by inhibition of histone deacetylase activity.

MicroRNA regulation of developmental and cellular processes is a relatively new field of study, and the available research data have not been organized to enable its inclusion in pathway and network analysis tools. The association of gene products with terms from the Gene Ontology is an effective method to analyze functional data, but until recently there has been no substantial effort dedicated to applying Gene Ontology terms to microRNAs. Consequently, when performing functional analysis of microRNA data sets, researchers have had to rely instead on the functional annotations associated with the genes encoding microRNA targets. In consultation with experts in the field of microRNA research, we have created comprehensive recommendations for the Gene Ontology curation of microRNAs. This curation manual will enable provision of a high-quality, reliable set of functional annotations for the advancement of microRNA research. Here we describe the key aspects of the work, including development of the Gene Ontology to represent this data, standards for describing the data, and guidelines to support curators making these annotations. The full microRNA curation guidelines are available on the GO Consortium wiki (http://wiki.geneontology.org/index.php/MicroRNA_GO_annotation_manual).

Background: MicroRNA-122 (miR-122) is highly abundant in the liver and has been implicated in lipid and glucose homeostasis. Methods: Circulating miR-122 levels were measured in the prospective population-based Bruneck Study (n=810) and the ASCOT trial (n=155 pre/post atorvastatin initiation). Using mass spectrometry, we measured 135 lipid subspecies and 93 plasma proteins in Bruneck participants. We also conducted mechanistic studies in pre-clinical models and cultured hepatocytes. We calculated risk ratios over up to 15 years follow-up for cardiometabolic diseases using pooled logistic and Cox regression, incorporating repeated miR-122 measurements. Results: In the Bruneck Study, miR-122 was associated with an adverse metabolic profile, including insulin resistance, obesity, and prevalent metabolic syndrome and diabetes. MiR-122 positively correlated with lipid subspecies containing fatty acids that can be derived from hepatic de novo lipogenesis. In mice, miR-122 inhibition with antagomiR treatment down-regulated hepatic enzymes implicated in de novo lipogenesis and cholesterol synthesis. In the ASCOT trial, 10mg atorvastatin treatment led to a reduction in circulating miR-122 levels. A similar statin response was observed in mice and in primary hepatocytes. In the Bruneck Study, miR-122 was closely related to circulating levels of afamin, zinc-alpha-2-glycoprotein, and a wide range of apolipoproteins. The adjusted relative risk per SD higher log miR-122 was 1.60 (1.30-1.96; P Conclusion: This study provides strong evidence for an involvement of miR-122 in glucose and lipid metabolism. High miR-122 levels correlate with an adverse lipid profile and multiple metabolic abnormalities and are associated with a higher risk of new-onset metabolic syndrome and diabetes in the general community.

Reendothelialization involves endothelial progenitor cell (EPC) homing, proliferation, and differentiation, which may be influenced by fluid shear stress and local flow pattern. This study aims to elucidate the role of laminar flow on embryonic stem (ES) cell differentiation and the underlying mechanism. We demonstrated that laminar flow enhanced ES cell–derived progenitor cell proliferation and differentiation into endothelial cells (ECs). Laminar flow stabilized and activated histone deacetylase 3 (HDAC3) through the Flk-1–PI3K–Akt pathway, which in turn deacetylated p53, leading to p21 activation. A similar signal pathway was detected in vascular endothelial growth factor–induced EC differentiation. HDAC3 and p21 were detected in blood vessels during embryogenesis. Local transfer of ES cell–derived EPC incorporated into injured femoral artery and reduced neointima formation in a mouse model. These data suggest that shear stress is a key regulator for stem cell differentiation into EC, especially in EPC differentiation, which can be used for vascular repair, and that the Flk-1–PI3K–Akt–HDAC3–p53–p21 pathway is crucial in such a process.

IL-6, a proinflammatory cytokine, has been implicated in the development of vascular diseases. We previously demonstrated that mechanical stress can initiate signaling pathways leading to smooth muscle cell (SMC) proliferation and apoptosis, but little is known concerning cyclic stress-induced inflammatory response. To explore the role of stretch in the upregulation of cytokine expression in SMCs we performed RNase protection assay for a panel of cytokines and found that mechanical stress resulted in a time-dependent induction of IL-6 mRNA but not other cytokines, e.g., IL-1alpha, IL-1beta, IL-6, IL-10, IL-12p35, IL-12p40, IL-18, IFN-gamma, and macrophage migration inhibitory factor (MIF). This induction also correlated with elevated IL-6 protein levels in the supernatant. Pretreatment of the cells with NF-kappaB inhibitors inhibited NF-kappaB activity and resulted in marked inhibition (50%) of IL-6 protein. Moreover, SMC lines stably expressing dominant-negative Ras (RasN17) or Rac (RacN17) exhibited a remarkable decrease in p38 MAPK activity and IL-6 mRNA induction by mechanical stress. Furthermore, a significant inhibition of 30 and 40% in IL-6 protein was observed in SMCs pretreated with inhibitors of p38 MAPK and ERK1/2, respectively, but not JNK. Interestingly, SMCs isolated from PKC-delta-deficient mice exhibited higher levels of IL-6 compared with wild-type cells. Finally, high levels of IL-6 expression were observed in atherosclerotic lesions of vein bypass grafts, which are related to altered biomechanical stress. Our findings demonstrate that biomechanical stress-induced IL-6 expression occurs via a mechanism that involves Ras/Rac/p38 MAPK/NF-kappaB/NF-IL6 signaling pathways, which is downregulated by PKC-delta, and suggest that modulation of this event contributes to the pathogenesis of atherosclerosis.

MicroRNAs (miRNAs) play an important role in the cellular function. They often form families, with members sharing high sequence homology, a property that hampers miRNA research as there is a lack of elegant tools for specific miRNA manipulation.