Thomas Helbing

Microparticles: major transport vehicles for distinct microRNAs in circulation

Aims Circulating microRNAs (miRNAs) have attracted major interest as biomarkers for cardiovascular diseases. Since RNases are abundant in circulating blood, there needs to be a mechanism protecting miRNAs from degradation. We hypothesized that microparticles (MP) represent protective transport vehicles for miRNAs and that these are specifically packaged by their maternal cells. Methods and results Conventional plasma preparations, such as the ones used for biomarker detection, are shown to contain substantial numbers of platelet-, leucocyte-, and endothelial cell-derived MP. To analyse the widest spectrum of miRNAs, Next Generation Sequencing was used to assess miRNA profiles of MP and their corresponding stimulated and non-stimulated cells of origin. THP-1 (monocytic origin) and human umbilical vein endothelial cell (HUVEC) MP were used for representing circulating MP at a high purity. miRNA profiles of MP differed significantly from those of stimulated and non-stimulated maternal THP-1 cells and HUVECs, respectively. Quantitative reverse transcription–polymerase chain reaction of miRNAs which have been associated with cardiovascular diseases also demonstrated significant differences in miRNA profiles between platelets and their MP. Notably, the main fraction of miRNA in plasma was localized in MP. Furthermore, miRNA profiles of MP differed significantly between patients with stable and unstable coronary artery disease. Conclusion Circulating MP represent transport vehicles for large numbers of specific miRNAs, which have been associated with cardiovascular diseases. miRNA profiles of MP are significantly different from their maternal cells, indicating an active mechanism of selective ‘packaging’ from cells into MP. These findings describe an interesting mechanism for transferring gene-regulatory function from MP-releasing cells to target cells via MP circulating in blood.

MicroRNA-100 Regulates Neovascularization by Suppression of Mammalian Target of Rapamycin in Endothelial and Vascular Smooth Muscle Cells

Background— The adaptive growth of blood vessels is an important protective mechanism in cardiovascular disease. However, the underlying regulatory mechanisms of this process are only partly understood. Recently, small endogenous RNAs (microRNAs [miRNAs]) were found to play an important role in embryonic and postnatal vascular development. Here, we used miRNA transcriptome analysis after induction of hind-limb ischemia in mice to screen for miRNAs involved in adaptive blood vessel growth following arterial occlusion. Methods and Results— Using miRNA arrays, we explored the miRNA expression profile during adaptive neovascularization. We describe specific changes in miRNA expression patterns and show that miRNA-100 is significantly downregulated after induction of hind-limb ischemia in mice. Our data demonstrate that miR-100 modulates proliferation, tube formation, and sprouting activity of endothelial cells and migration of vascular smooth muscle cells and functions as an endogenous repressor of the serine/threonine protein kinase mammalian target of rapamycin (mTOR). Whereas miR-100 inhibition increased mTOR levels in endothelial cells, overexpression of miR-100 reduced mTOR expression and consequently attenuated cellular proliferation. Supporting this notion, overexpression of an mTOR construct lacking the miRNA binding site rescued the inhibitory effect of miR-100 on cell proliferation. Accordingly, miR-100 inhibition by specific antagomirs in vivo stimulated angiogenesis and resulted in functional improvement of perfusion after femoral artery occlusion in mice. In contrast, treatment with the mTOR inhibitor rapamycin had the opposite effect. Conclusions— Our data demonstrate that miR-100 has an antiangiogenic function and represses mTOR signaling in endothelial and vascular smooth muscle cells. Inhibition of miR-100 could be a novel approach for the modulation of blood vessel growth and other mTOR-dependent processes.

BMPER is an endothelial cell regulator and controls bone morphogenetic protein-4-dependent angiogenesis

Bone morphogenetic proteins (BMPs) are involved in embryonic and adult blood vessel formation in health and disease. BMPER (BMP endothelial cell precursor–derived regulator) is a differentially expressed protein in embryonic endothelial precursor cells. In earlier work, we found that BMPER interacts with BMPs and when overexpressed antagonizes their function in embryonic axis formation. In contrast, in a BMPER-deficient zebrafish model, BMPER behaves as a BMP agonist. Furthermore, lack of BMPER induces a vascular phenotype in zebrafish that is driven by disarray of the intersomitic vasculature. Here, we investigate the impact of BMPER on endothelial cell function and signaling and elucidate its role in BMP-4 function in gain- and loss-of-function models. As shown by Western blotting and immunocytochemistry, BMPER is an extracellular matrix protein expressed by endothelial cells in skin, heart, and lung. We show that BMPER is a downstream target of FoxO3a and consistently exerts activating effects on endothelial cell sprouting and migration in vitro and in vivo. Accordingly, when BMPER is depleted from endothelial cells, sprouting is impaired. In terms of BMPER related intracellular signaling, we show that BMPER is permissive and necessary for Smad 1/5 phosphorylation and induces Erk1/2 activation. Most interestingly, BMPER is necessary for BMP-4 to exert its activating role in endothelial function and to induce Smad 1/5 activation. Vice versa, BMP-4 is necessary for BMPER activity. Taken together, BMPER is a dose-dependent endothelial cell activator that plays a unique and pivotal role in fine-tuning BMP activity in angiogenesis.

Complex Plaques in the Proximal Descending Aorta. An Underestimated Embolic Source of Stroke

Background and Purpose— To investigate the incidence of retrograde flow from complex plaques (≥4-mm-thick, ulcerated, or superimposed thrombi) of the descending aorta (DAo) and its potential role in embolic stroke. Methods— Ninety-four consecutive acute stroke patients with aortic plaques ≥3-mm-thick in transesophageal echocardiography were prospectively included. MRI was performed to localize complex plaques and to measure time-resolved 3-dimensional blood flow within the aorta. Three-dimensional visualization was used to evaluate if diastolic retrograde flow connected plaque location with the outlet of the left subclavian artery, left common carotid artery, or brachiocephalic trunk. Complex DAo plaques were considered an embolic source if retrograde flow reached a supra-aortic vessel that supplied the territory of visible acute and embolic retinal or cerebral infarction. Results— Only decreasing heart rate was correlated (P<0.02) with increasing flow reversal to the aortic arch. Retrograde flow from complex DAo plaques reached the left subclavian artery in 55 (58.5%), the left common carotid artery in 23 (24.5%), and the brachiocephalic trunk in 13 patients (13.8%). Based on routine diagnostics and MRI of the ascending aorta/aortic arch, stroke etiology was determined in 57 and cryptogenic in 37 patients. Potential embolization from DAo plaques was then identified in 19 of 57 patients (33.3%) with determined and in 9 of 37 patients (24.3%) with cryptogenic stroke. Conclusions— Retrograde flow from complex DAo plaques was frequent in both determined and cryptogenic stroke and could explain embolism to all brain territories. These findings suggest that complex DAo plaques should be considered a new source of stroke.

Increased levels of circulating microparticles in patients with severe aortic valve stenosis

The mechanisms of the progression of aortic valve stenosis are unknown. The involvement of mononuclear cells and of chronic systemic inflammation has been suggested by analysis of pathological specimens. We hypothesize that shear stress caused by the constricted aortic orifice contributes to systemic proinflammation by activation of circulating blood cells and thereby generation of microparticles. Using flow cytometry we analyzed 22 patients with severe aortic valve stenosis (AVS) and 18 patient controls for the generation of circulating microparticles from platelet- (PMPs: CD31(+)/CD61(+) or CD62P(+)), leukocyte- (LMPs: CD11b(+)) and endothelial cell (EMPs: CD62E(+)) origin. Apart from the constricted valve orifice groups were similar. PMPs were increased in AVS patients and their number correlated with valvular shear stress. Monocytes were activated in AVS patients, an observation that was also reflected by increased numbers of LMPs and by the detection of PMP-monocyte conjugates. Furthermore, EMPs reflecting the activation of endothelial cells but also conferring systemic inflammatory activity were increased in AVS patients and correlated with the number of activated monocytes. In conclusion, we show that AVS is accompanied by increased levels of microparticles and that shear stress can induce the formation of microparticles. Based on our results and histologic findings of other investigators the speculation that shear stress related to aortic valve stenosis induces a vicious circle including the generation of PMPs, the subsequent activation of monocytes and LMPs and finally the activation of endothelial cells contributing to the progress of aortic valve stenosis appears to be justified.

Enhanced microparticles in ventricular assist device patients predict platelet, leukocyte and endothelial cell activation

Platelet dysfunction followed by thromboembolic as well as bleeding complications are severe events in patients with ventricular assist devices (VADs). Microparticles (MP) are cell vesicles with inflammatory and coagulatory potency that are released by cells during activation and can be used as specific cell activation markers. The aim of the study was to establish MP as surrogate markers for platelet, leukocyte and endothelial activation in VAD patients predicting platelet activation and vascular inflammation. Therefore, MP from platelet, leukocyte and endothelial origin were measured in 12 patients with VAD and compared to controls. VAD patients had significantly increased levels of platelet microparticles (PMP) (VAD 1705+/-1100 cpm vs. co: 527+/-238 cpm, P=0.002), leukocyte microparticles (LMP) (VAD 43+/-16 cpm vs. co: 18+/-8 cpm, P<0.001) and endothelial microparticles (EMP) (VAD 135+/-66 cpm vs. co: 46+/-23 cpm, P<0.001) indicating enhanced vascular inflammation and pro-coagulation. Furthermore, LMP correlated well to EMP suggesting that LMP induce endothelial cell dysfunction. In conclusion, VAD patients suffer from enhanced platelet, leukocyte and endothelial activation that can be easily quantified by measuring PMP, LMP and EMP and that may help to estimate the thrombotic risk and contribute to avoid ischemic but also bleeding complications.

BMP activity controlled by BMPER regulates the proinflammatory phenotype of endothelium

The endothelium plays a pivotal role in vascular inflammation. Here we study bone morphogenetic protein (BMP) signaling in endothelial inflammation and in particular the role of BMPER, an extracellular BMP modulator that is important in vascular development and angiogenesis. Using the BMP antagonist dorsomorphin or BMP2 as an agonist we show that BMP signaling is essential for the inflammatory response of vascular endothelial cells as demonstrated by intravital microscopy. We found that BMPER is decreased in inflammation similar to vascular protective genes like KLF2 and eNOS. Using in vitro and in vivo models we show that BMPER is down-regulated through the TNFα-NFκB-KLF2 signaling pathway. Functionally, lack of BMPER induced by siRNA or in BMPER(+/-) mice confers a proinflammatory endothelial phenotype with reduced eNOS levels and enhanced expression of adhesion molecules leading to increased leukocyte adhesion and extravasation in ex vivo and in vivo experiments. Vice versa, addition of BMPER exerts endothelium protective functions and antagonizes TNFα induced inflammation. Mechanistically, we demonstrate that these effects of BMPER are dependent on BMP signaling because of enhanced NFκB activity. In conclusion, the BMP modulator BMPER is a new protective regulator of vascular inflammation that modulates leukocyte adhesion and migration in vitro and in vivo.

MicroRNA-155 Exerts Cell-Specific Antiangiogenic but Proarteriogenic Effects During Adaptive Neovascularization

Background— Adaptive neovascularization after arterial occlusion is an important compensatory mechanism in cardiovascular disease and includes both the remodeling of pre-existing vessels to collateral arteries (arteriogenesis) and angiogenic capillary growth. We now aimed to identify regulatory microRNAs involved in the modulation of neovascularization after femoral artery occlusion in mice. Methods and Results— Using microRNA-transcriptome analysis, we identified miR-155 as a downregulated microRNA during hindlimb ischemia. Correspondingly, inhibition of miR-155 in endothelial cells had a stimulatory effect on proliferation and angiogenic tube formation via derepression of its direct target gene angiotensin II type 1 receptor. Surprisingly, miR-155–deficient mice showed an unexpected phenotype in vivo, with a strong reduction of blood flow recovery after femoral artery ligation (arteriogenesis) dependent on the attenuation of leukocyte-endothelial interaction and a reduction of proarteriogenic cytokine expression. Consistently, miR-155–deficient macrophages exhibit a specific alteration of the proarteriogenic cytokine expression profile, which is partly mediated by the direct miR-155 target gene SOCS-1. Conclusions— Our data demonstrate that miR-155 exerts an antiangiogenic but proarteriogenic function in the regulation of neovascularization via the suppression of divergent cell-specific target genes and that its expression in both endothelial and bone marrow–derived cells is essential for arteriogenesis in response to hindlimb ischemia in mice.

Role of microparticles in endothelial dysfunction and arterial hypertension.

Microparticles are small cell vesicles that can be released by almost all eukaryotic cells during cellular stress and cell activation. Within the last 1-2 decades it has been shown that microparticles are useful blood surrogate markers for different pathological conditions, such as vascular inflammation, coagulation and tumour diseases. Several studies have investigated the abundance of microparticles of different cellular origins in multiple cardiovascular diseases. It thereby has been shown that microparticles released by platelets, leukocytes and endothelial cells can be found in conditions of endothelial dysfunction, acute and chronic vascular inflammation and hypercoagulation. In addition to their function as surrogate markers, several studies indicate that circulating microparticles can fuse with distinct target cells, such as endothelial cells or leukocyte, and thereby deliver cellular components of their parental cells to the target cells. Hence, microparticles are a novel entity of circulating, paracrine, biological vectors which can influence the phenotype, the function and presumably even the transcriptome of their target cells. This review article aims to give a brief overview about the microparticle biology with a focus on endothelial activation and arterial hypertension. More detailed information about the role of microparticles in pathophysiology and disease can be found in already published work.

Antagonism and synergy between extracellular BMP modulators Tsg and BMPER balance blood vessel formation

Summary Growth and regeneration of blood vessels are crucial processes during embryonic development and in adult disease. Members of the bone morphogenetic protein (BMP) family are growth factors known to play a key role in vascular development. The BMP pathway is controlled by extracellular BMP modulators such as BMP endothelial cell precursor derived regulator (BMPER), which we reported previously acts proangiogenically on endothelial cells in a concentration-dependent manner. Here, we explore the function of other BMP modulators, especially Tsg, on endothelial cell behaviour and compare them to BMPER. In Matrigel assays, BMP modulators chordin and noggin had no stimulatory effect; however, gremlin and Tsg enhanced human umbilical vein endothelial cell (HUVEC) sprouting. As the activation dynamics of Tsg were similar to those of BMPER, we further investigated the proangiogenic effect of Tsg on endothelial cells. Tsg enhanced endothelial cell ingrowth in the mouse Matrigel plug assay as well as HUVEC sprouting, migration and proliferation in vitro, dependent on Akt, Erk and Smad signalling pathway activation in a concentration-dependent manner. Surprisingly, silencing of Tsg also increased HUVEC sprouting, migration and proliferation, which is again associated with Akt, Erk and Smad signalling pathway activation. Furthermore, we reveal that Tsg and BMPER interfere with each other to enhance proangiogenic events. However, in vivo the presence of Tsg as well as of BMPER is mandatory for regular development of the zebrafish vasculature. Taken together, our results suggest that BMPER and Tsg maintain a fine-tuned equilibrium that controls BMP pathway activity and is necessary for vascular cell homeostasis.