Lars Maegdefessel

MicroRNA‐26a is a novel regulator of vascular smooth muscle cell function

Aberrant smooth muscle cell (SMC) plasticity has been implicated in a variety of vascular disorders including atherosclerosis, restenosis, and abdominal aortic aneurysm (AAA) formation. While the pathways governing this process remain unclear, epigenetic regulation by specific microRNAs (miRNAs) has been demonstrated in SMCs. We hypothesized that additional miRNAs might play an important role in determining vascular SMC phenotype. Microarray analysis of miRNAs was performed on human aortic SMCs undergoing phenotypic switching in response to serum withdrawal, and identified 31 significantly regulated entities. We chose the highly conserved candidate miRNA‐26a for additional studies. Inhibition of miRNA‐26a accelerated SMC differentiation, and also promoted apoptosis, while inhibiting proliferation and migration. Overexpression of miRNA‐26a blunted differentiation. As a potential mechanism, we investigated whether miRNA‐26a influences TGF‐β‐pathway signaling. Dual‐luciferase reporter assays demonstrated enhanced SMAD signaling with miRNA‐26a inhibition, and the opposite effect with miRNA‐26a overexpression in transfected human cells. Furthermore, inhibition of miRNA‐26a increased gene expression of SMAD‐1 and SMAD‐4, while overexpression inhibited SMAD‐1. MicroRNA‐26a was also found to be downregulated in two mouse models of AAA formation (2.5‐ to 3.8‐fold decrease, Pu2009<u20090.02) in which enhanced switching from contractile to synthetic phenotype occurs. In summary, miRNA‐26a promotes vascular SMC proliferation while inhibiting cellular differentiation and apoptosis, and alters TGF‐β pathway signaling. MicroRNA‐26a represents an important new regulator of SMC biology and a potential therapeutic target in AAA disease. J. Cell. Physiol. 226: 1035–1043, 2011.

Vascular smooth muscle cell phenotypic plasticity: focus on chromatin remodelling.

Differentiated vascular smooth muscle cells (SMCs) retain the capacity to modify their phenotype in response to inflammation or injury. This phenotypic switching is a crucial component of vascular disease, and is partly dependent on epigenetic regulation. An appreciation has been building in the literature for the essential role chromatin remodelling plays both in SMC lineage determination and in influencing changes in SMC behaviour and state. This process includes numerous chromatin regulatory elements and pathways such as histone acetyltransferases, deacetylases, and methyltransferases and other factors that act at SMC-specific marker sites to silence or permit access to the cellular transcriptional machinery and on other key regulatory elements such as myocardin and Kruppel-like factor 4 (KLF4). Various stimuli known to alter the SMC phenotype, such as transforming growth factor beta (TGF-β), platelet-derived growth factor (PDGF), oxidized phospholipids, and retinoic acid, appear to act in part through effects upon SMC chromatin structure. In recent years, specific covalent histone modifications that appear to establish SMC determinacy have been identified, while others alter the differentiation state. In this article, we review the mechanisms of chromatin remodelling as it applies to the SMC phenotype.

Long Noncoding RNA MANTIS Facilitates Endothelial Angiogenic Function

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.

New options with dabigatran etexilate in anticoagulant therapy

Thrombosis, the localized clotting of blood, occurs in both the arterial and venous circulation, and has a major impact on health outcomes. The primary etiology of myocardial infarctions, and approximately 80% of strokes, is acute arterial thrombosis. In combination this represents the most common cause of death in the Western world, while the third leading cause of cardiovascular-associated death is venous thromboembolism. An understanding of the pathogenic changes in the vessel wall and the blood that result in thrombosis is crucial for developing safer and more effective antithrombotic drugs. Dabigatran etexilate belongs to a new class of direct thrombin inhibitors. Following oral administration, dabigatran reaches peak plasma concentrations within 2 hours, shows linear pharmacokinetics, and a limited (but important) amount of direct drug interactions. Given once daily at 150 mg or 220 mg, it has proven to be competitive with enoxaparin in the prevention of venous thromboembolism after major orthopedic surgery, with a comparable safety profile. For stroke prevention in patients suffering from atrial fibrillation, dabigatran administered at a dose of 110 mg twice daily was associated with rates of stroke and systemic embolism that were similar to those associated with warfarin, as well as lower rates of hemorrhage. Dabigatran given at a dose of 150 mg twice daily, as compared with warfarin, was associated with lower rates of stroke and systemic embolism but similar rates of major hemorrhage. Oral bioavailability of dabigatran, together with a rapid onset and offset of action and predictable anticoagulation response, makes this newly available antithrombotic drug an attractive alternative to traditional anticoagulant therapies for numerous thrombosis-related indications.

Comparison of unfractionated heparin, low-molecular-weight heparin, low-dose and high-dose rivaroxaban in preventing thrombus formation on mechanical heart valves: results of an in vitro study

Thromboembolism and bleeding after mechanical heart valve replacement are still unsolved problems, particularly for patients requiring anticoagulative bridging therapy. The aim of this study was to investigate whether rivaroxaban, a new oral selective and direct coagulation factor Xa inhibitor, is as effective as enoxaparin and unfractionated heparin (UFH) in preventing thrombus formation on mechanical heart valves using an in vitro system. Blood from healthy male donors was anticoagulated with either UFH, enoxaparin, rivaroxaban at 300xa0ng/ml, (nxa0=xa010 each), or rivaroxaban at 30xa0ng/ml (nxa0=xa03). Mechanical aortic valve prostheses were placed into the in vitro testing system THIA II and exposed to the anticoagulant blood mixtures at a pulsatile flow for 60xa0min. Overall thrombus weight, coagulation parameters, and electron microscopic features of thrombus formation on the valve surface were quantified as endpoints. The mean thrombus weights were 163xa0±xa064xa0mg for group 1 (UFH), 341xa0±xa063xa0mg for the group 2 (enoxaparin), 238xa0±xa083xa0mg for group 3 (rivaroxaban 300xa0ng/ml) and 1.739xa0±xa016xa0mg for group 4 (rivaroxaban 30xa0ng/ml). Whereas high-dosed rivaroxaban showed no significant differences compared to UFH or enoxaparin, low-dosed rivaroxaban generated a massive thrombus generation, thus differing significantly from all other treatment groups regarding the thrombus weight. We hypothesize that high-dose rivaroxaban is a competitive oral available alternative to UFH and LMWH’s, that might be a worthwhile alternative for patients in need of anticoagulative bridging therapy. Prospective studies have to evaluate if rivaroxaban might even overcome the limitations of OAC in patients after implantation of artificial heart valves.

Patients with insulin-dependent diabetes or coronary heart disease following rehabilitation express serum fractalkine levels similar to those in healthy control subjects

The chemokine and adhesion molecule fractalkine and its receptor CX3CR1 have emerged as interesting regulators in inflammation and related atherosclerosis. The pro-inflammatory status may be counteracted by appropriate treatment, such as in rehabilitation. We compared serum fractalkine concentrations of 46 patients with coronary heart disease (CHD) and 47 insulin-dependent diabetic patients (IDDM) following rehabilitation with those of 50 control subjects. Following rehabilitation serum fractalkine levels (477 ± 225 pg/mL) in CHD patients were similar to those in control subjects (572 ± 205 pg/mL; P = 0.303), whereas fractalkine levels were lower in IDDM patients (430 ± 256 pg/mL; P = 0.042). No significant difference between CHD and IDDM patients was present (P = 0.319). Postprandial hyperlipemia may influence inflammation; thus, we investigated fractalkine levels four and eight hours after inducing postprandial hyperlipemia. However, we did not find any significant alterations in CHD and diabetic patients, whereas the fractalkine levels in controls were reduced. In vitro, lipofundin used as a hyperlipemic stimulus was added to vessel wall cells and reduced fractalkine levels. Low fractalkine levels in patients attending rehabilitation indicate a beneficial effect of the rehabilitation procedure on innate inflammatory pathways, such as the chemokine and adhesion molecule fractalkine.

Modern role for clopidogrel in management of atrial fibrillation and stroke reduction

Atrial fibrillation (AF) is the most common sustained cardiac arrhythmia. The prevalence of AF increases sharply in old age (prevalence approximately 10% among persons 80 years of age and older). The expected risk for ischemic stroke is increased five-fold by the presence of AF, primarily as a result of cardiogenic embolism. Multiple large-scale, randomized trials have been completed or are still underway to find optimal, efficacious, and relatively safe ways to reduce the risk of ischemic stroke and other systemic thromboembolic events related to AF. Antithrombotic strategies are accompanied by serious bleeding complications that threaten patients in need of medical stroke prevention. Treatment regimens for preventing thromboembolism in AF patients range from vitamin K antagonists such as warfarin or coumadins, antiplatelet drugs like aspirin or clopidogrel, to newly developed orally available antithrombotics like the direct thrombin inhibitor dabigatran, or the Factor Xa-inhibitor rivaroxaban. The available anticoagulant and antiplatelet drugs have different advantages and disadvantages. This review attempts to delineate the specific role of clopidogrel in patients with AF and at risk of stroke, taking into consideration new and ongoing trials in this important field of medical practice.

Heme Oxygenase-1 Expression Affects Murine Abdominal Aortic Aneurysm Progression

Heme oxygenase-1 (HO-1), the rate-limiting enzyme in heme degradation, is a cytoprotective enzyme upregulated in the vasculature by increased flow and inflammatory stimuli. Human genetic data suggest that a diminished HO-1 expression may predispose one to abdominal aortic aneurysm (AAA) development. In addition, heme is known to strongly induce HO-1 expression. Utilizing the porcine pancreatic elastase (PPE) model of AAA induction in HO-1 heterozygous (HO-1+/-, HO-1 Het) mice, we found that a deficiency in HO-1 leads to augmented AAA development. Peritoneal macrophages from HO-1+/- mice showed increased gene expression of pro-inflammatory cytokines, including MCP-1, TNF-alpha, IL-1-beta, and IL-6, but decreased expression of anti-inflammatory cytokines IL-10 and TGF-beta. Furthermore, treatment with heme returned AAA progression in HO-1 Het mice to a wild-type profile. Using a second murine AAA model (Ang II-ApoE-/-), we showed that low doses of the HMG-CoA reductase inhibitor rosuvastatin can induce HO-1 expression in aortic tissue and suppress AAA progression in the absence of lipid lowering. Our results support those studies that suggest that pleiotropic statin effects might be beneficial in AAA, possibly through the upregulation of HO-1. Specific targeted therapies designed to induce HO-1 could become an adjunctive therapeutic strategy for the prevention of AAA disease.

H19 Induces Abdominal Aortic Aneurysm Development and Progression

Background: Long noncoding RNAs have emerged as critical molecular regulators in various biological processes and diseases. Here we sought to identify and functionally characterize long noncoding RNAs as potential mediators in abdominal aortic aneurysm development. Methods: We profiled RNA transcript expression in 2 murine abdominal aortic aneurysm models, Angiotensin II (ANGII) infusion in apolipoprotein E–deficient (ApoE−/−) mice (n=8) and porcine pancreatic elastase instillation in C57BL/6 wild-type mice (n=12). The long noncoding RNA H19 was identified as 1 of the most highly upregulated transcripts in both mouse aneurysm models compared with sham-operated controls. This was confirmed by quantitative reverse transcription–polymerase chain reaction and in situ hybridization. Results: Experimental knock-down of H19, utilizing site-specific antisense oligonucleotides (LNA-GapmeRs) in vivo, significantly limited aneurysm growth in both models. Upregulated H19 correlated with smooth muscle cell (SMC) content and SMC apoptosis in progressing aneurysms. Importantly, a similar pattern could be observed in human abdominal aortic aneurysm tissue samples, and in a novel preclinical LDLR−/− (low-density lipoprotein receptor) Yucatan mini-pig aneurysm model. In vitro knock-down of H19 markedly decreased apoptotic rates of cultured human aortic SMCs, whereas overexpression of H19 had the opposite effect. Notably, H19-dependent apoptosis mechanisms in SMCs appeared to be independent of miR-675, which is embedded in the first exon of the H19 gene. A customized transcription factor array identified hypoxia-inducible factor 1&agr; as the main downstream effector. Increased SMC apoptosis was associated with cytoplasmic interaction between H19 and hypoxia-inducible factor 1&agr; and sequential p53 stabilization. Additionally, H19 induced transcription of hypoxia-inducible factor 1&agr; via recruiting the transcription factor specificity protein 1 to the promoter region. Conclusions: The long noncoding RNA H19 is a novel regulator of SMC survival in abdominal aortic aneurysm development and progression. Inhibition of H19 expression might serve as a novel molecular therapeutic target for aortic aneurysm disease.

Die Pathophysiologie des abdominalen Aortenaneurysmas

ZusammenfassungHintergrundDas abdominale Aortenaneurysma (AAA) ist eine tägliche klinische Herausforderung für den Gefäßchirurgen. In den letzten Jahren sind viele pathologische Veränderungen der Aneurysmawand im Vergleich zur nativen Aorta gut untersucht und deren Bedeutung in ein Modell zur Aneurysmaprogression eingeordnet worden.Ziel der ArbeitDiese modellhafte Theorie wird anhand einer Übersichtsarbeit vorgestellt.ErgebnisseCharakteristische Veränderungen im AAA umfassen eine verbreiterte Tunica media und veränderte Extrazellulärmatrix, veränderte Hämodynamik und Kräfte auf die Aortenwand, luminale Thrombusbildung, Phänotypwechsel glattmuskulärer Zellen, Angiogenese sowie akute und chronische Entzündungsherde. Diese bedingen Veränderungen der Gleichgewichte von Kräfteverteilung und Matrixsynthese in der Aortenwand, sodass Areale mit hoher biologischer Aktivität und reduzierter mechanischer Belastbarkeit entstehen. Die klinisch bekannten Risikofaktoren Nikotinabusus, Dyslipidämie, männliches Geschlecht, Alter und genetische Prädisposition haben definierte biologische Effekte, die die Gefäßwand zusätzlich schwächen. Zum Unterschied zwischen rupturierten und intakten Aneurysmen ist keine klare Aussage möglich. Ebenso ist die initiale Ursache der Aneurysmaentstehung weiterhin unklar.SchlussfolgerungEin besseres Verständnis der multifaktoriellen Pathogenese des AAA ist erforderlich um translationale Ansätze zur nichtchirurgischen Therapie und eine patientenindividuelle Risikostratifizierung zu ermöglichen. Die Forschung an humanen Aneurysmaproben ist auch in der endovaskulären Ära nötig und erlaubt die Identifikation spezifischer beteiligter Pathomechanismen.AbstractBackgroundAbdominal aortic aneurysms (AAA) are axa0daily clinical challenge for vascular surgeons. In recent years, many pathological changes in the aneurysm wall have been investigated and axa0common model for aneurysm expansion has been developed.ObjectiveThis theoretical model is presented based on axa0literature review.ResultsCharacteristic changes of the AAA wall include broadening of the media and alteration of the extracellular matrix, altered hemodynamics and force distribution, luminal thrombus formation, phenotype switch of vascular smooth muscle cells, angiogenesis as well as acute and chronic inflammatory infiltrates. These cause axa0proteolytic imbalance and altered pressure impact resulting in areas with increased biological activity and peak wall stress. The clinical risk factors smoking, dyslipidemia, male gender, age and genetic predisposition have defined biological effects causing additional weakening of the vessel wall. The differences between ruptured and intact aneurysm walls cannot be clearly defined. The initial cause of an aneurysm is also still unknown.ConclusionA better understanding of the multifactorial AAA pathogenesis is mandatory to enable translational approaches towards axa0non-surgical treatment and an individual patient risk stratification. Basic research on human aneurysmal samples is also necessary even in the endovascular era and enables identification of the very specific pathological mechanisms involved.