bluuebunny/crossref_metadata_2025_split huggingface dataset:
Abstract 36: Concurrent Ultrasound Diagnosis and Treatment of Thrombosis Using Activated Platelet Targeted Theranostic Microbubbles
Authors: Wang Xiaowei , Gkanatsas Yannik , Palasubramaniam Jathushan , Hohmann Jan David , Hagemeyer Christoph E , Peter Karlheinz 
Published: March 2022

Molecular ultrasound imaging is an attractive non-invasive technology widely available for rapid clinical diagnosis. We hypothesized that thrombolytic drugs loaded microbubbles (MBs), which are selectively targeted to activated platelets, will allow high-resolution, real-time imaging of thrombosis, and at the same time offer potent thrombolytic efficacy without bleeding complications, and enable the immediate monitoring of success or failure of thrombolysis.
Our therapeutic agents/imaging particles, targeted theranostic microbubbles (TT-MB), consist of a fusion construct that combines the fibrinolytic drug urokinase, echo-enhancing microbubbles for visualization by ultrasonography, and an activated-platelet-specific single-chain antibody for targeting specifically to thrombi. In the ferric-chloride induced carotid artery thrombosis mouse model, treatment with TT-MB significantly reduced thrombus size after 45 min, while no significant difference was observed in the MB that were targeted but without urokinase (37.09 ± 5.6 vs. 97.16 ± 4.3, mean % change ± SEM, normalized to baseline thrombus size, p<0.001). The same degree of efficient thrombolysis was only achievable using a high dose of urokinase (NS). We also show that the targeting and thus clot-enrichment effect of TT-MBs results in a highly potent fibrinolysis that could only be matched using high doses of non-targeted urokinase. However, the latter is associated with a highly prolonged bleeding time (79.25 ± 6.5 vs. 1079.25 ± 260.7, seconds ± SEM, p<0.001). In contrast, TT-MB does not prolong bleeding time (NS).
In conclusion, activated platelet targeted microbubbles conjugated with recombinant urokinase represent a novel and unique theranostic approach to simultaneously diagnose and treat thrombosis as well as to immediately monitor success or failure of thrombolysis. This unique technology holds promise for major progress towards rapid diagnosis and bleeding-free, potent therapy of the vast number of patients suffering from thrombotic diseases.
Abstract 367: Pcsk6 Is a Key Protease Modulating Smooth Muscle Cell Activation in Vascular Remodeling and Plaque Vulnerability
Authors: Perisic Matic Ljubica , Razuvaev Anton , Lengquist Mariette , Sabater-Lleal Maria , Folkersen Lasse , Du Lei , Österholm Cecilia , Roy Joy , Kronqvist Malin , Frebelius Siw , Gonzalez Diez Maria , Hedin Erika , Paulsson-Berne Gabrielle , Hansson Göran K , Odeberg Jacob , Hamsten Anders , Eriksson Per , Hedin Ulf 
Published: March 2022

Proprotein convertases (PCSKs) are conserved among species and involved in processing of MMPs and growth factors, but poorly characterised in atherosclerosis. Previously we demonstrated upregulation of PCSK6 in a large cohort of plaques from symptomatic vs. asymptomatic patients. This protease localized to smooth muscle cells (SMCs) and showed a positive correlation to markers of inflammation, extracellular matrix remodeling and cytokines in plaques. Here we aimed at elucidating its role in vascular development and disease.
In zebrafish embryos PCSK6 localized to heart and vasculature and its ablation caused defective peripheral vascular patterning with cerebral and myocardial hemorrhage. Increased expression of PCSK6 in vascular pathologies was validated by microarrays from carotid plaques vs. undiseased arteries (n=32 patients, p<0.0001), abdominal (AAA, n=14, p<0.0001) and thoracic aortic aneurysms (TAA, n=244, p=0.012). By immunohistochemistry, PCSK6 localized mainly to SMCs in the fibrous cap and neo-vessels in plaques, AAA and TAA tissues. Investigation of mouse carotid ligation, rat artery balloon injury and human restenosis tissues with pronounced intimal hyperplasia confirmed induction of PCSK6 in proliferating SMCs. By microarrays following progression of rat carotid injury, PCSK6 was downregulated in early phases defined by inflammatory pathways, while upregulated in later phases with SMCs activation and localized in SMCs. Expression of PCSK6 in this model was positively correlated to PDGFB and IGF1 (Spearman r>0.7, p<0.0001). PCSK6 overexpression in SMCs in vitro increased their migration in a wound-healing assay, especially upon PDGFBB stimulation. By eQTL analyses several polymorphisms in the PCSK6 gene were found to influence its expression in plaques and aneurysm tissue. Among these, rs6598465 showed association with maximum progression of arterial intima-media thickness in high-risk coronary artery disease subjects (n=3400, p=0.037).
We identified a functional link between elevated expression of PCSK6 and vascular remodeling characterized by SMC activation. The present study establishes PCSK6 as one of the key modulators of pathological processes in relation to plaque vulnerability.
Abstract 392: Effect of HDL on Platelet Apoptosis and Aggregation
Authors: Song Wenliang , Du Jing , Zarich Stuart , Hwa John 
Published: March 2022

Recent clinical trials have again questioned the cardioprotective role of high-density lipoprotein cholesterol (HDL-C). Attention has now focused on high-density lipoprotein (HDL) function. As HDL is highly heterogeneous in composition, and based upon some exciting preliminary results, we suspect that composition of HDL may play differential roles in platelet regulation, thus explaining the variable clinical results.
Confirming a number of recent studies, we have found that HDL appears to inhibit platelet aggregation and activation in response to collagen in a time and dose response manner. This should be a clear beneficial effect of HDL, inhibiting thrombosis. However, in the same studies, we also observed that HDL induces substantial platelet mitochondrial membrane potential loss and damage, leading to platelet apoptosis (increased p53 phosphorylation, caspase 3 activation, and cytochrome c release), which has recently been shown to increase thrombosis. We were intrigued by how HDL can seemingly both inhibit and induce thrombosis. Based upon the literature and our further studies, we have developed a series of hypothesis to explain these seemingly opposing actions on platelets; 1) Apoptotic platelets do not aggregate and thus the inhibition of platelet aggregation, 2) With evidence that small dense HDL have more “platelet inhibitory effects” and are more prevalent in atherogenic dyslipidemia and diabetic patient, we hypothesize that the proapoptosis effect of HDL on platelet apoptosis is from small dense HDL and 3) Platelet apoptosis promotes thrombosis. Composition of HDL may have important differential roles on platelets.
Abstract 421: The NLRC4 Variant rs385076 Affects PU.1 Transcription Factor Binding, NLRC4 Isoform Expression and Circulating Interleukin-18 Concentrations
Authors: Mueller Christian , Haase Tina , Zeller Simon , Krause Jasmin , Wild Philipp S , Münzel Thomas , Ziegler Andreas , Lackner Karl J , Schnabel Renate B , Blankenberg Stefan , Zeller Tanja ,   German Cntr for Cardiovascular Rsch (DZHK e.V.)
Published: March 2022

Circulating levels of the pro-inflammatory cytokine Interleukin-18 (IL-18) associate with cardiovascular events. IL-18 GWAS identified the NLRC4 locus, with rs385076 in the 5’UTR exon 2 of NLRC4 as lead SNPs. Data from the ENCODE project indicates accessible DNA and PU.1 transcription factor binding around rs385076.
We assessed the hypothesis that rs385076 alleles regulate NLRC4 gene expression by modulating binding of PU.1.
To investigate the influence of rs385076 alleles on PU.1 binding, a luciferase reporter gene assay with T or C allele containing plasmids was transfected into human embryonic kidney cells. The cells were treated with 0 / 12.5 / 25 or 50 ng PU.1. Gene expression of 1) total NLRC4, 2) NLRC4 isoforms containing 5’UTR exon 2 and 3) isoforms without exon 2 was quantified by qPCR in 1,500 individuals of the Gutenberg Health Study. Effects of rs385076 T/C allele on NLRC4 isoform levels were calculated using a linear mixed model.
In PU.1 treated cells, luciferase activity of the rs385076 C allele was 6-fold (±1.3) higher compared to controls, whereas the T allele showed no significant increase. Total NLRC4 gene expression was decreased in T allele carriers (p=7x10-3). Comparing isoforms containing the exon 2 (decreased, p=2x10-10) to those without (increased, p=7x10-4), expression of NLRC4 isoforms was regulated by T alleles in opposite directions, indicating a switched isoform usage (see figure).
In conclusion, our analyses indicate that the rs385076 T allele leads to decreased gene expression of NLRC4 and a switched isoform usage, putatively affected by decreased PU.1 binding. This is consistent with the finding that the T allele associated with lower IL-18 levels.
Abstract 427: Flow Regulation of YAP/TAZ in Endothelial Inflammation and Atherosclerosis
Authors: Wang Kuei-Chun , Nguyen Phu , Limqueco Elaine , Li Yi-Shuan , Chien Shu 
Published: March 2022

The focal nature of atherosclerotic lesions in regions such as arterial branch points and curvatures suggests a regulatory mechanism by local hemodynamic environment. Recent studies have demonstrated significant roles of Yes-associated protein (YAP) and Transcriptional coactivator with PDZ-binding motif (TAZ) in mediating mechano-transduction and vascular homeostasis. However, the mechanism by which YAP/TAZ modulates endothelial phenotypes in response to atheroprone vs. atheroprotective hemodynamic flows remains unknown. The objective of this study is to investigate the functional role of YAP/TAZ in the flow regulation of endothelial inflammation and the consequential development of atherosclerotic lesions.
Experiments were conducted on cultured vascular endothelial cells (ECs) and atherosclerotic mouse model. We found that exposure of ECs to the atheroprone oscillatory shear stress (OS) results in YAP/TAZ activation and translocation into EC nucleus to upregulate the target genes, including connective tissue growth factor (CTGF) and cysteine-rich angiogenic inducer 61 (Cyr61). In contrast, the atheroprotective laminar shear stress suppresses YAP/TAZ. Consistent with these in vitro results, ex vivo analysis of mouse arteries demonstrated a more nuclear localization of YAP/TAZ and elevated levels of CTGF and Cyr61 in the endothelium in atheroprone areas than in atheroprotective areas. Functionally, YAP/TAZ knockdown significantly attenuated the OS- and cytokine-inductions of pro-inflammatory adhesion molecules in ECs and monocytes, as well as the consequential monocyte adhesion to ECs. Overexpression of constitutively active YAP increases endothelial inflammation and monocyte adhesion to ECs. Furthermore, in vivo blockade of YAP/TAZ translation by Morpholino oligos in apolipoprotein E-deficient mice significantly reduced endothelial inflammation, mononuclear cell infiltration, and the size of atherosclerotic plaques.
Our results demonstrate a critical role of YAP/TAZ activation by atheroprone flow in promoting endothelial inflammation and the development of atherosclerotic lesions. Therefore, inhibition of YAP/TAZ activation may hold promise as a novel atheroprotective therapeutic strategy.
Abstract 441: Rgs5 Controls Myogenic Responses of Vascular Smooth Muscle Cells
Authors: Arnold Caroline , Feldner Anja , Genové Guillem , Schorpp-Kistner Schorpp-Kistner , Angel Peter , Wieland Thomas , Hecker Markus , Korff Thomas 
Published: March 2022

Contraction of vascular smooth muscle cells (VSMCs) in response to an increase in blood pressure is pivotal for maintaining blood flow and constitutes both consequence and cause of chronic hypertension. While a plethora of contraction-mediating mechanisms has been delineated, not much is known about molecular determinants regulating the responses of VSMCs to an increase in wall stress or biomechanical stretch. In this context, we reported that conditional ablation of Junb - a subunit of the transcription factor AP-1 (activator protein 1) - interferes with the expression of myosin light chain 9 and consequently impaired myogenic responses of arteries. We hypothesized that the arterial circumferential wall stress is increased under these conditions which may influence the VSMC phenotype and vessel wall architecture. In fact, Junb-deficient arteries showed structural remodeling as evidenced by a ~2-3-fold decrease in collagen type I and IV content (n=5, p<0.05). cDNA microarray analyses revealed that expression of regulator of G-protein signaling 5 (RGS5) was up-regulated in VSMCs (~6-fold, n=4, p<0.001) isolated from Junb-deficient arteries which was confirmed in vivo but not mediated by AP-1. However, elevation of wall stress by volume-induced hypertension in vivo or exposure of VSMCs to biomechanical stretch in vitro was sufficient to trigger RGS5 expression (3-fold, n=3, p<0.05). Overexpression of this regulator activates RhoA and enhanced stretch-induced responses of VSMCs such as stress fiber formation while its knockdown showed opposite effects. In line with this, RGS5-deficient arteries show an impaired pressure-mediated constriction (n=8, p<0.05) and both the hypertension-induced increase in diastolic blood pressure as well as structural remodeling of the arterial vessel wall were significantly attenuated in RGS5-deficient mice.
Collectively, these findings indicate that biomechanical stretch mediates the expression of RGS5 in VSMCs to enhance stretch-dependent responses as a prerequisite for adequate adaptive vascular remodeling processes. This mechanism may establish RGS5 as a promising target for future strategies to combat, e.g. the deleterious consequences of arterial hypertension such as arterial stiffening.
Abstract 484: Platelet Specific Deletion of Opa-1 Reveals Gender Dimorphic Effects on Platelet Dysfunction and Thrombus Formation in Mice
Authors: Souvenir Rhonda A , Fidler Trevor P , Dayal Sanjana , Weyrich Andrew S , Abel E. D 
Published: March 2022

Diabetes Mellitus and its complications remains a major public health burden. Cardiovascular disease (CVD) is the leading cause of death amongst diabetics and disproportionally affects women. Thrombosis represents one of the primary underlying pathologies in CVD. However, the mechanisms by which platelet dysfunction develops in diabetics are unclear. Preliminary studies revealed enrichment in platelet-derived transcripts encoding the mitochondrial dynamics proteins optic atrophy (OPA)-in females and mitofusin-2, dynamin-related protein-1, and mitochondrial fission 1 protein in both genders collected from the Framingham Offspring cohort, suggesting that platelet dysfunction in the diabetic milieu might be related to abnormal mitochondrial dynamics. Thus to define the relationship between mitochondrial dynamics and platelet dysfunction/thrombosis we generated mice with platelet-specific deletion of OPA1, a protein that regulates mitochondrial fusion, electron transport complex (ETC) assembly and apoptosis.
Deletion of OPA-1 in platelets reduced mitochondrial oxygen consumption by approximately 50 % and reduced mitochondrial ETC subunit expression in both genders. Hydrogen peroxide levels were reduced by 40% in females but increased by 20% in males. Unexpectedly, females exhibited a 10-fold increase in expression of the NADPH oxidase (NOX) subunits gp91phox and p47phox, whereas NOX was unchanged in males. The absence of OPA1 was associated with longer time to stable occlusion in females but with decreased time to stable occlusion in males as assessed in vivo by rose Bengal photochemical injury. Also, male OPA1 deficient mice, were more prone to develop a thrombus (14/15 knockouts vs. 4/8 controls) following permanent ligation of the inferior vena cava, but this increase in venous thrombosis was not observed in females mice. Taken together, these data suggest that increased OPA1 expression observed in females with diabetes could represent a critical pathophysiologic mechanism in diabetic females that could contribute to their disproportional increased relative risk of thrombosis. We also identify for the first time, an essential role for OPA1 in regulating platelet mitochondrial function, platelet activation and thrombosis.
Abstract 543: Impact of Eplerenone on Vascular Function in LOX-1 Overexpressing Mice on High-Fat Diet
Authors: Brunssen Coy , Rissler Johannes , Langbein Heike , Hofmann Anja , Frenzel Annika , Deussen Andreas , Peitzsch Mirko , Cimalla Peter , Koch Edmund , Eisenhofer Graeme , Sawamura Tatsuya , Morawietz Henning 
Published: March 2022

Experimental and clinical studies support a protective effect of aldosterone-binding mineralocorticoid receptor (MR) blockade on heart failure. The impact of MR antagonist eplerenone on endothelial function is less well understood. In this study, we analyzed the effects of high-fat diet (HFD) and MR blockade on metabolic parameters and endothelial function in mice overexpressing the endothelial oxidized LDL receptor LOX-1.
Mice were fed standard diet (STD) or HFD with or without MR blocker eplerenone for 20 weeks. In agreement with clinical studies aldosterone plasma levels (LC-MS/MS) were elevated in eplerenone-treated mice. Body weight, epididymal, retroperitoneal, mesenteric and perivascular fat were increased after HFD. Diet-induced obesity elevated triglycerides, total cholesterol, and LDL plasma levels. Total cholesterol/HDL cholesterol ratio was increased in HFD fed LOX-1tg mice and decreased by eplerenone. LDLR-/- and HFD-fed LOX1tg and LOX1tg/LDLR-/- (LOLR) mice showed elevated fasting blood glucose levels compared to STD, which were partially normalized by eplerenone. LDLR-/-, LOX 1tg and LOLR mice had impaired endothelial function in thoracic aortas (Mulvany myograph) compared to C57BL/6J mice. HFD pronounced endothelial dysfunction in LOX-1tg and LOLR mice, which was improved by eplerenone. Reactive oxygen species can limit NO availability and were reduced in aorta and adipose tissues after eplerenone. Using a new method to evaluate flow-mediated dilation with a self-developed Optical Coherence Tomography (OCT) device, we could show endothelial dysfunction in saphenous artery in vivo after HFD. This was not restored by eplerenone. In epididymal white adipose tissue of LOX-1tg mice, HFD increased CD206, CD11c, F4/80, leptin, NADPH oxidase (Nox) 1, 2 and 4 and TNF-α mRNA expression (real-time PCR). Eplerenone treatment had no significant impact on CD11c, F4/80 and IL-6 expression, but elevated anti-inflammatory IL-10. CD206, Nox1, Nox2 and Nox4 expression was reduced by eplerenone in mice on HFD.
In conclusion, our data support beneficial effects of MR blockade by eplerenone on endothelial function, metabolic parameters, inflammation and oxidative stress in a proatherosclerotic mouse model overexpressing LOX-1 on HFD.
Abstract 558: Neutrophils-derived-microvesicle Could Play a Role in the Early Stage of Atherosclerosis
Authors: Gomez Ingrid , Ward Ben , Evans Paul , Hellewell Paul , Ridger Victoria 
Published: March 2022

Background:
Atherosclerosis is the most common underlying cause of cardiovascular disease. Many cells types are involved in the initiation and progression of atherosclerotic plaques but, until recently, neutrophils were not considered to be among them. Despite their rare detection in plaques, neutrophil depletion has been shown to reduce plaque formation and monocyte recruitment to the vessel wall.

Hypothesis:
Neutrophils may influence plaque initiation and progression through the formation of microvesicles.

Aim:
To determine the role of neutrophil-derived-microvesicles in the early stage of atherosclerosis.

Methods and Results:
Mouse peripheral blood neutrophils were isolated by negative magnetic separation. Following stimulation for 1h with fMLP (3x105M), supernatants were subjected to differential centrifugation (twice at 300g for 6 min followed by 20,000g for 20 mins) to isolate the microvesicle population. Microvesicles were quantified using flow cytometry calibrated with MegaMix and Spherocount beads. Apo E mice were fed a Western diet for 6 weeks and injected twice a week with PBS or neutrophil microvesicles (4.52x106 ± 3.05x105) isolated from control mice. After 6 weeks, blood was collected for neutrophil and microvesicle quantification. The aorta was dissected and stained by conventional procedures using Oil Red. Areas of plaque formation was determined using computer based imaging and semi-quantitative scoring.

Injection with microvesicles increased circulating neutrophil number (PBS treated = 67.3 ± 3.9 neutrophils per μl; microvesicle treated = 88.25 ± 4.0 neutrophils per μl, P = 0.031; n = 3) and neutrophil reactivity with respect to microvesicle formation (PBS treated = 4.5 ± 0.8 microvesicles per neutrophil; microvesicle treated = 8.3±0.7 microvesicles per neutrophil, P = 0.0249; n = 3).
After 6 weeks, the plaque formation was more advanced in the MVs group (% lesion area in the arch in mice injected with PBS = 0.4563 ± 0.09452, with MVs = 1.643 ± 0.3891, P = 0.0118, n=7).

Conclusion:
Neutrophil microvesicles induce increased plaque formation and increase circulating neutrophil levels in a mouse model of atherosclerosis. This suggests neutrophils may be involved in atherogenesis through the release of microvesicles.
Abstract 687: Paracrine Function of Extracellular microRNA-191 in Diabetes Associated Impaired Wound Healing
Authors: Dangwal Seema , Stratmann Bernd , Bang Claudia , Lorenzen Johan M , Fiedler Jan , Kumarswamy Regalla , Falk Christine S , Scholz Claus J , Tschoepe Diethelm , Thum Thomas 
Published: March 2022

MicroRNAs (miRNAs/miRs) stably circulate in blood and show biomarker potential under various pathological conditions. Additionally, miRNAs may exert paracrine effect upon cellular uptake.
This study profiles plasma miRNAs of diabetic patients and investigates the paracrine mechanism of miRNA mediated wound repair.
Plasma miRNAs and pro-inflammatory cytokines were profiled in type-2 diabetic patients with or without chronic wounds. Selected miRNA candidates were further validated in plasma samples obtained from controls (n=23), patients with PAD and chronic wound (PAD+W, n=27) or PAD alone (n=11) vs. that of 20 healthy controls by qRT-PCR. Inflammation mediated miRNA secretion and paracrine effects of released miRNAs on cellular functions were investigated in human endothelial or dermal cells respectively.
Differential levels of the circulating miRNAs in diabetic PAD+W patients were revealed in miRNA array profiling. Among 41 miRNA candidates, miR-191 levels significantly increased in PAD+W patients compared to diabetic controls, whereas lower levels were observed in diabetic vs. healthy controls. Circulating CRP and cytokine levels were also high in PAD+W patients compared to diabetic controls. miR-191 significantly correlated with CRP in diabetic patients.
Cytokine stimulation enhanced miR-191 secretion into culture supernatant from vascular endothelial cells. Moreover, co-culture assays maintained under pro-inflammatory stress demonstrated increased miR-191 levels in recipient dermal fibroblast cells when indirectly cultured with miRNA releasing endothelial cells compared to fibroblasts cultured alone. Luciferase reporter confirmed miR-191 binding to the 3’UTR of the ZO-1 transcript. miR-191 modulation in dermal fibroblasts inversely regulated ZO-1 expression and influenced scratch wound closure whereas in diabetic dermal microvascular endothelial cells, miR-191 overexpression compromised both tube formation and wound closure.
Circulating miRNA patterns are influenced by underlying inflammation associated with chronic wounds in type-2 diabetic patients and miR-191 modulates essential cellular functions in a paracrine manner by targeting ZO-1 during wound healing.