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Background Atrial fibrillation (AF) can lead to the loss of microvascular integrity thereby enhancing AF progression. Mechanistically, the pro-coagulant state that drives the risk of stroke in patients with AF may also play a causal role in microvascular loss. Direct oral anticoagulants (DOACs), the preferred anticoagulants for AF, can target factors upstream (factor Xa [FXa]) or downstream (thrombin) in the coagulation cascade and mediate differential vascular effects through interaction with protease-activated receptors (PARs). Objective To investigate the potential effect of different DOACs on vascular integrity. Methods To model the impact of DOACs on vascular integrity, we utilized platelet-free plasma in thrombin generation assays and endothelial barrier assays under identical experimental conditions. These multifactorial systems provide all coagulation factors and their respective natural inhibitors in physiological ratios in combination with the pro-coagulant endothelial surface on which coagulation is initiated. Furthermore, the system provides pro- and anti-barrier factors and monitoring both assays simultaneously permits coupling of thrombin kinetics to endothelial barrier dynamics. Results We provide evidence that the anti-FXa DOAC rivaroxaban and the anti-thrombin DOAC dabigatran are efficient in blocking their target proteases. However, while rivaroxaban could preserve endothelial barrier function, dabigatran failed to protect endothelial integrity over time, which could be prevented in the presence of a custom-made peptide that blocks thrombin's exosite-I. Conclusions Proteolytically inactive thrombin in complex with dabigatran evokes loss of barrier function that can be prevented by a protease-activated receptor-1 mimicking peptide blocking thrombin's exosite-I.

This study provides a method to assess the impact of circulating plasma factors on microvascular integrity by using a recently developed microvessel-on-a-chip platform featuring the human endothelium that is partly surrounded by the extracellular matrix. The system is high-throughput, which allows parallel analysis of organ-level microvessel pathophysiology, including vascular leakage. Ethylenediaminetetraacetic acid plasma samples are mixed with inhibitors for recalcification of the plasma samples to avoid activation of the coagulation- or complement system. Moreover, the assay is validated by spiking vascular endothelial growth factor, histamine, or tumor necrosis factor alpha to recalcified plasma and confirms their modulation of microvessel barrier function at physiologically relevant concentrations. Finally, this study shows that perfusing the microvessels with recalcified plasma samples of coronavirus disease-2019 patients, with a confirmed proinflammatory profile, results in markedly increased leakage of the microvessels. The assay provides opportunities for diagnostic screening of inflammatory or endothelial disrupting plasma factors associated with endothelial dysfunction.

Background: Hereditary Hemorrhagic Telangiectasia type 2 (HHT2) is an inherited genetic disorder characterized by vascular malformations and hemorrhage. HHT2 results from ACVRL1 haploinsufficiency, the remaining wild-type allele being unable to contribute sufficient protein to sustain endothelial cell function. Blood vessels function normally but are prone to respond to angiogenic stimuli, leading to the development of telangiectasic lesions that can bleed. How ACVRL1 haploinsufficiency leads to pathological angiogenesis is unknown. Methods: We took advantage of Acvrl1 +/− mutant mice that exhibit HHT2 vascular lesions and focused on the neonatal retina and the airway system after Mycoplasma pulmonis infection, as physiological and pathological models of angiogenesis, respectively. We elucidated underlying disease mechanisms in vitro by generating Acvrl1 +/− mouse embryonic stem cell lines that underwent sprouting angiogenesis and performed genetic complementation experiments. Finally, HHT2 plasma samples and skin biopsies were analyzed to determine whether the mechanisms evident in mice are conserved in humans. Results: Acvrl1 +/− retinas at postnatal day 7 showed excessive angiogenesis and numerous endothelial “tip cells” at the vascular front that displayed migratory defects. Vascular endothelial growth factor receptor 1 (VEGFR1; Flt-1) levels were reduced in Acvrl1 +/− mice and HHT2 patients, suggesting similar mechanisms in humans. In sprouting angiogenesis, VEGFR1 is expressed in stalk cells to inhibit VEGFR2 (Flk-1, KDR) signaling and thus limit tip cell formation. Soluble VEGFR1 (sVEGFR1) is also secreted, creating a VEGF gradient that promotes orientated sprout migration. Acvrl1 +/− embryonic stem cell lines recapitulated the vascular anomalies in Acvrl1 +/− (HHT2) mice. Genetic insertion of either the membrane or soluble form of VEGFR1 into the ROSA26 locus of Acvrl1 +/− embryonic stem cell lines prevented the vascular anomalies, suggesting that high VEGFR2 activity in Acvrl1 +/− endothelial cells induces HHT2 vascular anomalies. To confirm our hypothesis, Acvrl1 +/− mice were infected by Mycoplasma pulmonis to induce sustained airway inflammation. Infected Acvrl1 +/− tracheas showed excessive angiogenesis with the formation of multiple telangiectases, vascular defects that were prevented by VEGFR2 blocking antibodies. Conclusions: Our findings demonstrate a key role of VEGFR1 in HHT2 pathogenesis and provide mechanisms explaining why HHT2 blood vessels respond abnormally to angiogenic signals. This supports the case for using anti-VEGF therapy in HHT2.

Atherosclerosis is the underlying pathology in a major part of cardiovascular disease, the leading cause of mortality in developed countries. The infiltration of monocytes into the vessel walls of large arteries is a key denominator of atherogenesis, making monocytes accountable for the development of atherosclerosis. With the development of high-throughput transcriptome profiling platforms and cytometric methods for circulating cells, it is now feasible to study in-depth the predicted functional change of circulating monocytes reflected by changes of gene expression in certain pathways and correlate the changes to disease outcome. Neuroimmune guidance cues comprise a group of circulating- and cell membrane-associated signaling proteins that are progressively involved in monocyte functions. Here, we employed the CIRCULATING CELLS study cohort to classify cardiovascular disease patients and healthy individuals in relation to their expression of neuroimmune guidance cues in circulating monocytes. To cope with the complexity of human datasets featured by noisy data, nonlinearity and multidimensionality, we assessed various machine-learning methods. Of these, the linear discriminant analysis, Na&iuml, ve Bayesian model and stochastic gradient boost model yielded perfect or near-perfect sensibility and specificity and revealed that expression levels of the neuroimmune guidance cues SEMA6B, SEMA6D and EPHA2 in circulating monocytes were of predictive values for cardiovascular disease outcome.

Background Genome wide association studies (GWAS) identified SLC44A2 as a novel susceptibility gene for venous thrombosis (VT) and previous work established that SLC44A2 contributed to clot formation upon vascular injury.Objective To further investigate the role of SLC44A2 in VT by utilizing SLC44A2 deficient mice (Slc44a2(-)(/)(-)) in two representative disease models.Methods Mice were included in a hypercoagulability model driven by siRNA-mediated hepatic gene silencing of anticoagulants Serpinc1 (antithrombin) and Proc (protein C) and a flow restriction (stenosis) model induced by partial ligation of the inferior vena cava.Results In the hypercoagulability model, no effect in onset was observed in Slc44a2(-)(/)(-) animals; however, a drop in plasma fibrinogen and von Willebrand factor coinciding with an increase in blood neutrophils was recorded. In the neutrophil dependent stenosis model after 48 hours, Slc44a2(-)(/)(-) mice had significantly smaller thrombi both in length and weight with less platelet accumulation as a percentage of the total thrombus area. During the initiation of thrombosis at 6 hours post-stenosis, Slc44a2(-)(/)(-) mice also had smaller thrombi both in length and weight, with circulating platelets remaining elevated in Slc44a2(-)(/)(-) animals. Platelet activation and aggregation under both static- and venous and arterial shear conditions were normal for blood from Slc44a2(-)(/)(-) mice.Conclusions These studies corroborate the original GWAS findings and establish a contributing role for SLC44A2 during the initiation of VT, with indications that this may be related to platelet-neutrophil interaction. The precise mechanism however remains elusive and warrants further investigation.

We recently reported that loss of hyaluronan (HA) from the endothelial glycocalyx leads to loss of vessel stability in specific microcirculatory vascular beds. Here we hypothesized that such derangements in the glycocalyx may also impair the adaptive response to vascular ischemia. Endothelial specific conditional hyaluronan synthase 2-KO (Has2-cKO) mice revealed reduced endothelial HA expression and lower hindlimb perfusion at baseline compared to control mice. After a single ligation of the common femoral artery in these mice, we observed dysregulated angiogenesis in the gastrocnemius muscle which did not restore capillary perfusion. Mechanistically, decreased endothelial binding of the pericyte-derived molecule angiopoietin1 (Ang1) could be observed in the Has2-cKO mouse. In vitro angiogenesis assays with an endothelial cell-pericyte coculture confirmed such disturbed Ang1-TIE2 signaling resulting in excessive angiogenesis upon loss of HA. These data could be of relevance to diabetes patients, where we confirm loss of endothelial HA in the microcirculation of muscle tissue, indicating that this may contribute to the known disturbed adaptation to ischemia in these patients. In summary, loss of endothelial HA results in impaired microvascular perfusion and endothelial stability in ischemic gastrocnemius muscle. Endothelial HA is a potential target to improve angiogenic therapy in diabetic patients with critical limb ischemia.

In the pathophysiologic setting of acute and chronic kidney injury, the excessive activation and recruitment of blood-borne monocytes prompts their differentiation into inflammatory macrophages, a process that leads to progressive glomerulosclerosis and interstitial fibrosis. Importantly, this differentiation of monocytes into macrophages requires the meticulous coordination of gene expression at both the transcriptional and post-transcriptional level. The transcriptomes of these cells are ultimately determined by RNA-binding proteins such as QUAKING (QKI), that define their pre-mRNA splicing and mRNA transcript patterns. Using two mouse models, namely (1) quaking viable mice (qkv) and (2) the conditional deletion in the myeloid cell lineage using the lysozyme 2-Cre (QKIFL/FL, LysM-Cre mice), we demonstrate that the abrogation of QKI expression in the myeloid cell lineage reduces macrophage infiltration following kidney injury induced by unilateral urethral obstruction (UUO). The qkv and QKIFL/FL, LysM-Cre mice both showed significant diminished interstitial collagen deposition and fibrosis in the UUO-damaged kidney, as compared to wild-type littermates. We show that macrophages isolated from QKIFL/FL, LysM-Cre mice are associated with defects in pre-mRNA splicing. Our findings demonstrate that reduced expression of the alternative splice regulator QKI in the cells of myeloid lineage attenuates renal interstitial fibrosis, suggesting that inhibition of this splice regulator may be of therapeutic value for certain kidney diseases.

Fibrodysplasia ossificans progressiva (FOP) is an extremely rare congenital form of heterotopic ossification (HO), caused by heterozygous mutations in the activin A type I receptor (ACVR1), that encodes the bone morphogenetic protein (BMP) type I receptor ALK2. These mutations enable ALK2 to induce downstream signaling in response to activins, thereby turning them into bone‐inducing agents. To date, there is no cure for FOP. The further development of FOP patient‐derived models may contribute to the discovery of novel biomarkers and therapeutic approaches. Nevertheless, this has traditionally been a challenge, as biopsy sampling often triggers HO. We have characterized peripheral blood‐derived endothelial colony‐forming cells (ECFCs) from three independent FOP donors as a new model for FOP. FOP ECFCs are prone to undergo endothelial‐to‐mesenchymal transition and exhibit increased ALK2 downstream signaling and subsequent osteogenic differentiation upon stimulation with activin A. Moreover, we have identified a new class of small molecule macrocycles with potential activity against ALK2 kinase. Finally, using FOP ECFCs, we have selected OD36 and OD52 as potent inhibitors with excellent kinase selectivity profiles that potently antagonize mutant ALK2 signaling and osteogenic differentiation. We expect that these results will contribute to the development of novel ALK2 clinical candidates for the treatment of FOP. © 2019 The Authors. JBMR Plus published by Wiley Periodicals, Inc. on behalf of American Society for Bone and Mineral Research., Endothelial colony‐forming cells (ECFCs) are isolated during routine blood tests from patients' peripheral blood. When cultured under osteogenic conditions, fibrodysplasia ossificans progressiva (FOP) endothelial colony‐forming cells resemble the aberrant activin signal transduction in FOP and can be transformed into osteogenic cells in response to BMP and activins. Using this system, we have identified OD36 and OD52 as two novel macrocyclic ALK2 kinase inhibitors with enhanced specificity for activin‐induced ALK2 downstream signaling.

Contains fulltext : 182393.pdf (Publisher’s version ) (Open Access) Inhibition of monocyte chemotactic protein-1 (MCP-1) with the Spiegelmer emapticap pegol (NOX-E36) shows long-lasting albuminuria-reducing effects in diabetic nephropathy. MCP-1 regulates inflammatory cell recruitment and differentiation of macrophages. Because the endothelial glycocalyx is also reduced in diabetic nephropathy, we hypothesized that MCP-1 inhibition restores glomerular barrier function through influencing macrophage cathepsin L secretion, thus reducing activation of the glycocalyx-degrading enzyme heparanase. Four weeks of treatment of diabetic Apoe knockout mice with the mouse-specific NOX-E36 attenuated albuminuria without any change in systemic hemodynamics, despite persistent loss of podocyte function. MCP-1 inhibition, however, increased glomerular endothelial glycocalyx coverage, with preservation of heparan sulfate. Mechanistically, both glomerular cathepsin L and heparanase expression were reduced. MCP-1 inhibition resulted in reduced CCR2-expressing Ly6C(hi) monocytes in the peripheral blood, without affecting overall number of kidney macrophages at the tissue level. However, the CD206(+)/Mac3(+) cell ratio, as an index of presence of anti-inflammatory macrophages, increased in diabetic mice after treatment. Functional analysis of isolated renal macrophages showed increased release of IL-10, whereas tumor necrosis factor and cathepsin L release was reduced, further confirming polarization of tissue macrophages toward an anti-inflammatory phenotype during mouse-specific NOX-E36 treatment. We show that MCP-1 inhibition restores glomerular endothelial glycocalyx and barrier function and reduces tissue inflammation in the presence of ongoing diabetic injury, suggesting a therapeutic potential for NOX-E36 in diabetic nephropathy.

BackgroundHypercholesterolemia is a major risk factor for ischemic heart disease including acute myocardial infarction. However, long-term effects of hypercholesterolemia in a rodent myocardial ischemia-reperfusion injury model are unknown. Therefore, the effects of diet-induced hypercholesterolemia on cardiac function and remodeling were investigated up to eight weeks after myocardial ischemia-reperfusion (MI-R) injury which was induced in either normocholesterolemic (NC-MI) or hypercholesterolemic (HC-MI) APOE*3-Leiden mice.MethodsLeft ventricular (LV) dimensions were serially assessed using parasternal long-axis echocardiography followed by LV pressure-volume measurements. Subsequently, infarct size and the inflammatory response were analyzed by histology and fluorescence-activated cell sorting (FACS) analysis.ResultsIntrinsic LV function eight weeks after MI-R was significantly impaired in HC-MI compared to NC-MI mice as assessed by end-systolic pressure, dP/dtMAX, and -dP/dtMIN. Paradoxically, infarct size was significantly decreased in HC-MI compared to NC-MI mice, accompanied by an increased wall thickness. Hypercholesterolemia caused a pre-ischemic peripheral monocytosis, in particular of Ly-6Chi monocytes whereas accumulation of macrophages in the ischemic-reperfused myocardium of HC-MI mice was decreased.ConclusionDiet-induced hypercholesterolemia caused impaired LV function eight weeks after MI-R injury despite a reduced post-ischemic infarct size. This was preceded by a pre-ischemic peripheral monocytosis, while there was a suppressed accumulation of inflammatory cells in the ischemic-reperfused myocardium after eight weeks. This experimental model using hypercholesterolemic APOE*3-Leiden mice exposed to MI-R seems suitable to study novel cardioprotective therapies in a more clinically relevant animal model.

The bioengineering of a replacement kidney has been proposed as an approach to address the growing shortage of donor kidneys for the treatment of chronic kidney disease. One approach being investigated is the recellularization of kidney scaffolds. In this study, we present several key advances toward successful re‐endothelialization of whole kidney matrix scaffolds from both rodents and humans. Based on the presence of preserved glycosoaminoglycans within the decelullarized kidney scaffold, we show improved localization of delivered endothelial cells after preloading of the vascular matrix with vascular endothelial growth factor and angiopoietin 1. Using a novel simultaneous arteriovenous delivery system, we report the complete re‐endothelialization of the kidney vasculature, including the glomerular and peritubular capillaries, using human inducible pluripotent stem cell –derived endothelial cells. Using this source of endothelial cells, it was possible to generate sufficient endothelial cells to recellularize an entire human kidney scaffold, achieving efficient cell delivery, adherence, and endothelial cell proliferation and survival. Moreover, human re‐endothelialized scaffold could, in contrast to the non‐re‐endothelialized human scaffold, be fully perfused with whole blood. These major advances move the field closer to a human bioengineered kidney., Human decellularized kidney scaffolds can be functionally re‐endothelialized using expanded human pluripotent stem cell–derived endothelial cells.

We recently demonstrated that the adult human kidney cortex contains a perivascular stromal cell (kPSC) that shows organotypic properties and is important for repair and stabilisation of kidney function. Not only the kidney cortex but also the kidney capsule contains stromal cells that are important for the three dimensional organisation of the kidney during nephrogenesis. They provide the barrier function of the capsule which is critical for homeostatic processes such as pressure natriuresis. We postulated that stromal cells derived from the kidney capsule may therefore also have specific properties and functions. To this end, we isolated these capsule mesenchymal stromal cells (cMSC) from human cadaveric kidneys that were not suitable for transplantation. There were several similarities between cMSCs and kPSCs including support of vascular plexus formation, phenotypic marker expression and resistance against myofibroblast transformation. However, compared to kPSCs, cMSCs showed distinct mRNA and miRNA expression profiles, showed increased immunosuppressive capacity, and displayed strongly reduced HGF production, contributing to the inability to enhance kidney epithelial repair. Therefore cMSCs are a distinct, novel human kidney-derived MSC-population and these data underpin the large functional diversity of phenotypic similar stromal cells in relation to their anatomic site, even within one organ.

Mesenchymal stromal cells (MSCs) are immunomodulatory and tissue homeostatic cells that have shown beneficial effects in kidney diseases and transplantation. Perivascular stromal cells (PSCs) identified within several different organs share characteristics of bone marrow-derived MSCs (BM-MSCs). These PSCs may also possess tissue-specific properties and play a role in local tissue homeostasis. We hypothesized that human kidney-derived PSCs (hkPSCs) would elicit improved kidney repair in comparison with BM-MSCs. Here we introduce a novel, clinical-grade isolation method of hkPSCs from cadaveric kidneys by enriching for the perivascular marker, NG2. hkPSCs show strong transcriptional similarities to BM-MSCs but also show organotypic expression signatures, including the HoxD10 and HoxD11 nephrogenic transcription factors. Comparable to BM-MSCs, hkPSCs showed immunosuppressive potential and, when cocultured with endothelial cells, vascular plexus formation was supported, which was specifically in the hkPSCs accompanied by an increased NG2 expression. hkPSCs did not undergo myofibroblast transformation after exposure to transforming growth factor-β, further corroborating their potential regulatory role in tissue homeostasis. This was further supported by the observation that hkPSCs induced accelerated repair in a tubular epithelial wound scratch assay, which was mediated through hepatocyte growth factor release. In vivo, in a neonatal kidney injection model, hkPSCs reintegrated and survived in the interstitial compartment, whereas BM-MSCs did not show this potential. Moreover, hkPSCs gave protection against the development of acute kidney injury in vivo in a model of rhabdomyolysis-mediated nephrotoxicity. Overall, this suggests a superior therapeutic potential for the use of hkPSCs and their secretome in the treatment of kidney diseases.

Background COPD is a pulmonary disorder often accompanied by cardiovascular disease (CVD), and current treatment of this comorbidity is suboptimal. Systemic inflammation in COPD triggered by smoke and microbial exposure is suggested to link COPD and CVD. Mesenchymal stromal cells (MSC) possess anti-inflammatory capacities and MSC treatment is considered an attractive treatment option for various chronic inflammatory diseases. Therefore, we investigated the immunomodulatory properties of MSC in an acute and chronic model of lipopolysaccharide (LPS)-induced inflammation, emphysema and atherosclerosis development in APOE*3-Leiden (E3L) mice. Methods Hyperlipidemic E3L mice were intranasally instilled with 10 μg LPS or vehicle twice in an acute 4-day study, or twice weekly during 20 weeks Western-type diet feeding in a chronic study. Mice received 0.5x106 MSC or vehicle intravenously twice after the first LPS instillation (acute study) or in week 14, 16, 18 and 20 (chronic study). Inflammatory parameters were measured in bronchoalveolar lavage (BAL) and lung tissue. Emphysema, pulmonary inflammation and atherosclerosis were assessed in the chronic study. Results In the acute study, intranasal LPS administration induced a marked systemic IL-6 response on day 3, which was inhibited after MSC treatment. Furthermore, MSC treatment reduced LPS-induced total cell count in BAL due to reduced neutrophil numbers. In the chronic study, LPS increased emphysema but did not aggravate atherosclerosis. Emphysema and atherosclerosis development were unaffected after MSC treatment. Conclusion These data show that MSC inhibit LPS-induced pulmonary and systemic inflammation in the acute study, whereas MSC treatment had no effect on inflammation, emphysema and atherosclerosis development in the chronic study.

A hallmark of inflammatory diseases is the excessive recruitment and influx of monocytes to sites of tissue damage and their ensuing differentiation into macrophages. Numerous stimuli are known to induce transcriptional changes associated with macrophage phenotype, but posttranscriptional control of human macrophage differentiation is less well understood. Here we show that expression levels of the RNA-binding protein Quaking (QKI) are low in monocytes and early human atherosclerotic lesions, but are abundant in macrophages of advanced plaques. Depletion of QKI protein impairs monocyte adhesion, migration, differentiation into macrophages and foam cell formation in vitro and in vivo. RNA-seq and microarray analysis of human monocyte and macrophage transcriptomes, including those of a unique QKI haploinsufficient patient, reveal striking changes in QKI-dependent messenger RNA levels and splicing of RNA transcripts. The biological importance of these transcripts and requirement for QKI during differentiation illustrates a central role for QKI in posttranscriptionally guiding macrophage identity and function., Post-transcriptional control of RNA is important in health and disease. Here, the authors show that the RNA-binding protein Quaking guides pre-mRNA splicing and transcript abundance during monocyte to macrophage differentiation, and that Quaking depletion impairs pro-atherogenic foam cell formation.

Elastin, critical for its structural and regulatory functions, is a missing link in vascular tissue engineering. Several elastin-inducting compounds have previously been reported, but their relative efficiency in promoting elastogenesis by adult arterial and venous vascular smooth muscle cells (VSMCs) and fibroblasts, four main vascular and elastogenic cells, has not been described. In addition to elasto-inductive substances, microRNA-29a was recently established as a potent post-transcriptional inhibitor of elastogenesis. Here, we explored if stimulating positive regulators or blocking inhibitors of elastogenesis could maximize elastin production. We tested whether the elasto-inducing compounds IGF-1, TGF-β1, and minoxidil could indeed augment elastin production, and whether microRNA-29a antagonism could block elastin production in adult arterial and venous fibroblasts and VSMCs. The effects on elastin, lysyl oxidase, and fibrillin-1 mRNA expression levels and tropoelastin protein were determined. IGF-1 and minoxidil exerted little effect on tropoelastin mRNA expression levels in all cell types, while TGF-β1 predominantly enhanced mRNA tropoelastin levels, but this mRNA increase did not impact tropoelastin protein abundance. In contrast, microRNA29a inhibition resulted in the upregulation of tropoelastin mRNA in all cell types, but most pronounced in venous VSMCs. Importantly, microRNA-29a-antagonism also enhanced lysyl oxidase and fibrillin-1 mRNA expression, and revealed a dose-dependent increase in tropoelastin protein expression in venous VSMCs. Our studies suggest that the elastogenic potential of microRNA-29a inhibition in vascular cells is superior to that of established elastin-stimulating compounds IGF-1, TGF-β1, and minoxidil. Thus, microRNA-29a antagonism could serve as an attractive means of enhancing elastin synthesis in tissue-engineered blood vessels.

Aim: Atherosclerosis is accelerated by excessive monocyte recruitment, influx and differentiation into pro-inflammatory macrophages. While the genome-wide mRNA expression profiles of human monocytes and pro-inflammatory macrophages are well-established, their transcriptomes are ultimately defined by factors, such as RNA-binding proteins, that modulate pre-mRNA splicing patterns and mRNA transcript abundance. This prompted us to investigate the role of the RNA-binding protein Quaking in regulating global changes in pre-mRNA splicing and mature mRNA expression as human monocytes acquire the pro-inflammatory macrophage identity. Methods: We employed RNA-sequencing and splicing-sensitive microarrays to determine genome-wide changes in pre-mRNA splicing and mRNA expression upon conversion of human monocytes into pro-inflammatory macrophages, including those derived from a unique Quaking haploinsufficient patient. Results: Using laser-capture micro-dissection and immunohistochemistry, we discovered that expression levels of Quaking mRNA and protein are low in monocytes of early human atherosclerotic lesions, but abundant in macrophages of advanced plaques. Depletion of Quaking protein using both siRNA and GapmeR technology significantly impaired monocyte adhesion and migration; delayed differentiation into pro-inflammatory macrophages while maintaining the capacity to adopt the anti-inflammatory phenotype; and diminished foam cell formation in vitro and in vivo. RNA-sequencing and microarray analysis of human monocyte and macrophage transcriptomes revealed striking changes in Quaking-dependent pre-mRNA splicing and mRNA transcript levels, with gene ontology analyses identifying an enrichment in transcripts involved in cellular migration and lipid metabolism. Furthermore, these studies uncovered common as well as novel alternatively spliced transcripts with unknown biological functions in monocytes and macrophages. Conclusions: Our studies illustrate a central role for Quaking in post-transcriptionally guiding pro-inflammatory macrophage identity and function.

Proper regulation of endothelial cell-cell contacts is essential for physiological functioning of the endothelium. Interendothelial junctions are actively involved in the control of vascular leakage, leukocyte diapedesis and the initiation and progression of angiogenesis. We found that the RNA-binding protein quaking is highly expressed by endothelial cells and that its expression was augmented by prolonged culture under laminar flow and the transcription factor KLF2 binding to the promoter. Moreover, we demonstrated that quaking directly binds to the mRNA of VE-cadherin and β-catenin and can induce mRNA translation mediated by the 3′UTR of these genes. Reduced quaking levels attenuated VE-cadherin and β-catenin expression and endothelial barrier function in vitro and resulted in increased bradykinin-induced vascular leakage in vivo. Taken together, we report that quaking is essential in maintaining endothelial barrier function. Our results provide novel insight into the importance of post-transcriptional regulation in controlling vascular integrity.

Background: COPD patients have an increased risk of cardiovascular diseases (CVD), which may be linked through systemic inflammation triggered by smoke and microbial exposure. Recent studies show the promising antiinflammatory and regenerative effects of mesenchymal stromal cells (MSC). Since current treatment of COPD with CVD is suboptimal, we examined the immunomodulatory properties of MSC in an acute and chronic model of lipopolysaccharide (LPS)-induced inflammation, emphysema and diet-induced atherosclerosis in hyperlipidemic APOE*3-Leiden ( E3L ) mice. Methods: Western-type diet-fed E3L mice were intranasally instilled (2x/wk) with PBS or 10 µg LPS for 1 week (acute study) or 20 weeks (chronic study). Mice received PBS or 0.5x10 6 MSC intravenously twice after the first LPS instillation (acute) or in week 14, 16, 18 and 20 (1x/wk; chronic). After sacrifice, inflammatory parameters were measured in bronchoalveolar lavage (BAL) and lungs. In the chronic study, emphysema, inflammation and atherosclerosis were also analysed. Results: In the acute study, intranasal LPS induced a systemic IL-6 response on day 3 (1370 vs. 80 pg/mL in PBS; p Conclusion: This study shows that whereas MSC inhibit intranasal LPS-induced pulmonary cell influx and systemic inflammation in an acute setting, MSC did not modify emphysema and atherosclerosis development in a chronic study.

Endothelial barrier function plays a major role in the onset of atherosclerosis. This barrier is determined largely by adherens junctions. Remarkably little is known about their regulation at the post[[Unable to Display Character: ‐]]transcriptional level. We find that the RNA-binding protein Quaking (QKI), known for its function in embryonic blood vessel formation, is highly expressed in quiescent adult endothelial cells (EC) in vivo. In vitro, EC displayed increased levels of QKI when cultured under laminar atheroprotective flow. Using KLF2 overexpression and a human QKI promoter reporter gene, we found that KLF2 mediates this increase in QKI expression. Subsequently we aimed to investigate the role of QKI in EC vascular integrity. Silencing of QKI markedly impaired (0.65 fold ±0.13; p In conclusion, we show that QKI functions as a critical regulator of β-catenin and VE-cadherin in endothelial cells, and the modulation of QKI expression affects endothelial monolayer integrity, in vitro and in vivo. These studies provide novel insight into the importance of post-transcriptional regulation of components of the endothelial adherens junction, and may have wide ranging implications for the preservation of vascular integrity in disease.

A hallmark of inflammatory diseases is the excessive recruitment and influx of monocytes to sites of tissue damage and their ensuing differentiation into macrophages. Numerous stimuli are known to induce new transcription necessary for macrophage identity, but post-transcriptional control of human macrophage differentiation is less well understood. Here, we detail our discovery that levels of the RNA-binding protein Quaking (QKI) are low in monocytes of early atherosclerotic lesions, but abundant in macrophages of advanced plaques. Specific depletion of QKI protein impaired monocyte adhesion, migration and differentiation into macrophages, and lesion formation. RNA-seq and microarray analysis of human monocyte and macrophage transcriptomes, including those of a unique QKI haploinsufficient patient, reveal developmental changes in RNA levels and alternative splicing of RNA transcripts enriched in QKI-bound sequence elements. The importance of these transcripts and requirement for QKI during differentiation illustrates a central role for QKI in post-transcriptionally guiding macrophage identity and function. These studies implicate QKI as a novel target for therapeutic intervention in inflammatory diseases.

In HLA-matched allogeneic hematopoietic stem cell transplantation (SCT), donor T cells can mediate graft-versus-leukemia/lymphoma (GvL) reactivity and graft-versus-host disease (GvHD) by recognition of minor histocompatibility antigens (MiHA).1–4 Only a minority of MiHA shows hematopoiesis-restricted expression, and donor T cells for these MiHA may induce beneficial GvL reactivity without GvHD. The number of well-characterized MiHA with therapeutic relevance based on hematopoiesis-restricted expression remains limited and only 25% and 40% of recipients transplanted with grafts from sibling and unrelated donors, respectively, are eligible for therapies targeting known hematopoietic MiHA.3,4 Thus, in order to increase the efficacy and applicability of cellular therapy for selective GvL induction, more hematopoiesis-restricted MiHA with balanced population frequencies in common HLA molecules must be identified. Here, we investigated the therapeutic significance of a MiHA encoded by ARHGDIB.5 We demonstrated hematopoiesis-restricted gene expression with the exception of intermediate mRNA expression in endothelial cells and showed that T cells recognized LB-ARHGDIB-1R presented by HLA-B*07:02 on primary leukemic cells, but not on [interferon-gamma (IFN-γ)]-treated fibroblasts and keratinocytes. To evaluate potential toxicity against endothelial cells, we tested T cell recognition of LB-ARHGDIB-1R on human umbilical vein endothelial cells (HUVEC) and found only limited reactivity under inflammatory conditions. Furthermore, we demonstrated in vivo targeting of LB-ARHGDIB-1R in eight out of ten patients who were screened for post-transplant specific T-cell responses. In one patient with relapsed lymphoma, high T-cell frequencies were induced after donor lymphocyte infusion (DLI), coinciding with long-lasting anti-lymphoma immunity without GvHD. Our data thus support the relevance of LB-ARHGDIB-1R as a therapeutic target with the potential to induce selective GvL reactivity.

Background: It is well known that atrial fibrillation (AF) induces a hypercoagulable state, which significantly increases stroke risk in patients with AF contributing to morbidity and mortality in these patients. Active coagulation factors can also provoke diverse cellular responses through stimulation of protease-activated receptors (PARs). In the heart and vessels, coagulation factor mediated PAR activation may provoke and mediate pro-inflammatory and tissue remodeling responses, potentially contributing to organ damage. We hypothesized that the onset and progression of AF, may be affected by hypercoagulability-mediated cell signaling responses, in the heart. Methods and results: To study the potential role of PARs in the structural remodeling process that renders the atria more prone to AF we first investigated whether thrombin or factor Xa could induce atrial fibroblast remodeling. In isolated rat cardiac fibroblasts, thrombin enhanced the phosphorylation of the pro-fibrotic signaling molecules Akt and Erk, and increased expression of TGFβ1 (2.7 fold) and the pro-inflammatory factor monocyte chemo-attractant protein-1 (6.1 fold). Thrombin also increased the incorporation of 3H-proline suggesting enhanced collagen synthesis by cardiac fibroblasts (2.5 fold). Differentiation towards myofibroblasts was indicated by increased expression of smooth muscle actin (2 fold). All effects could be prevented by the direct thrombin inhibitor dabigatran and comparable results were obtained for stimulation with factor Xa and inhibition with rivaroxaban, respectively. Next we studied whether enhanced stimulation of PARs by chronic elevation of thrombin levels would lead to an enhanced vulnerability to AF in transgenic mice. In mice with enhanced thrombin activity due to a mutation in the thrombomodulin gene resulting in impaired thrombin inhibition (TMpro/pro), inducibility of AF episodes provoked by burst pacing was higher (6 out of 10 versus 1 out of 10 in wild type) and the duration of AF episodes was longer (episodes >2s in 6 out of 10 versus 0 out of 10 in wild type). Finally, we showed that inhibition of the coagulation cascade attenuated the development of AF in a goat model of AF. In 6 goats with persistent AF and treated with the anticoagulant nadroparine (4 weeks, 150 IU/kg twice daily) the complexity of the AF substrate was less pronounced compared to control animals. The conduction heterogeneity and block were 33% shorter in the nadroparine treated animals (maximal conduction time 23.3±3.1ms in control versus 15.7±2.1ms in nadroparine, p Conclusion: The hypercoagulable state during AF provokes pro-fibrotic and pro-inflammatory responses in cardiac fibroblasts, as well as promotes the development of a substrate for AF in transgenic mice and in goats with persistent AF. Together, these results strongly support the role of hypercoagulability and PAR activation in the development of a substrate for AF. In addition, direct anticoagulant treatment may protect against AF-related cellular atrial remodelling. Figure 1: Enhanced AF inducibility and prolonged AF duration in TMpro/pro mice. Transesophageal stimulation was used to test AF inducibility. A surface electrocardiogram (lead I, sampled at 2.5 kHz) was recorded to detect AF. A) Traces show an example of a Wt mouse, returning to normal sinus rhythm immediately after the burst (upper panel) and a TMpro/pro mouse, showing a 3s episode of AF before returning to sinus rhythm (lower panel). In both cases, the first P wave observed after the burst is indicated. B) AF was inducible in 1 out of 10 Wt mice and 6 out of 10 TMpro/pro mice. C) Distribution of the longest AF episode duration observed in each Wt and TMpro/pro mouse. Figure 1:. Enhanced AF inducibility and prolonged AF duration in TMpro/pro mice. Transesophageal stimulation was used to test AF inducibility. A surface electrocardiogram (lead I, sampled at 2.5 kHz) was recorded to detect AF. A) Traces show an example of a Wt mouse, returning to normal sinus rhythm immediately after the burst (upper panel) and a TMpro/pro mouse, showing a 3s episode of AF before returning to sinus rhythm (lower panel). In both cases, the first P wave observed after the burst is indicated. B) AF was inducible in 1 out of 10 Wt mice and 6 out of 10 TMpro/pro mice. C) Distribution of the longest AF episode duration observed in each Wt and TMpro/pro mouse. Disclosures No relevant conflicts of interest to declare.