bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 46: High-Density Lipoproteins Control Monocyte Differentiation Through microRNA Intercellular Communication
Authors: Vickers Kasey C , Levin Michael G , Anderson Michael P , Xu Qing , Anzinger Joshua , Kruth Howard S , Remaley Alan T
Published: July 2021

Many HDL-microRNAs (miRNA) are well-characterized post-transcriptional regulators of inflammation, and are significantly increased on HDL with hypercholesterolemia and atherosclerosis in humans and mice. Therefore, we hypothesize that inflammatory cells uniquely control their own gene expression through cellular miRNA export to HDL and then regulate recipient cell gene expression through HDL-mediated miRNA delivery. To test this hypothesis, we used high-throughput proteomics, Open Arrays, small RNA sequencing, and gene expression microarrays. Human monocytes (plasma elutriation) were differentiated into dendritic cells and multiple macrophage phenotypes. Each cell-type was incubated with pure reconstituted HDL (rHDL), which was then purified from culture media by apolipoprotein A-I immunoprecipitation after 24 h, and both cellular and HDL-miRNAs were profiled using TaqMan Open Arrays. Macrophages were found to export high levels of miRNAs to HDL that inhibit monocyte/macrophage differentiation (miR-146a, miR-223); however, monocytes were also found to export many miRNAs associated with differentiation, including miR-92a, miR-222, miR-17, miR-20a, miR106a, and miR-21. Furthermore, many miRNAs were found to be transcribed in inflammatory cells, but completely exported to HDL and not retained in the cell. Most interestingly, HDL treatment was found to induce miR-223 transcription in monocytes, as determined by primary miR-223 transcript levels; however, intracellular levels of the mature form (miR-223) did not change. These results suggest that HDL induces the export of miRNAs it transports. PAR-CLIP with high-throughput small RNA sequencing was used to demonstrate that miRNAs are transferred from macrophages to endothelial cells and loaded onto cellular Argonaute 2-continaining RNA-induced silencing complexes. To demonstrate this in mice, human HDL, containing endogenous levels of miR-223, were injected into miR-223-null mice and inflammation-associated miRNA delivery was mapped in vivo. In summary, we found profound differences in the cellular response to HDL treatment and HDL-miRNA communication amongst inflammatory cell phenotypes that are physiologically relevant to cardiovascular disease.
Abstract 464: Knockout of Insulin-like Growth Factor-1 Receptor in Monocyte/Macrophage Promotes Atherosclerosis and Induces an Unstable Plaque Phenotype
Authors: Higashi Yusuke , Shai Shaw-yung , Sukhanov Sergiy , Kim Catherine , Delafontaine Patrice
Published: July 2021

We have previously shown that infusion of insulin-like growth factor-1 (IGF-1) exerts anti-inflammatory and anti-oxidant effects and reduces atherosclerotic burden in apolipoprotein E deficient (Apoe-/-) mice. Monocytes/macrophages play a pivotal role in atherogenesis and express high levels of IGF-1 receptor (IGF-1R); however the potential effects of IGF-1 on their function are unknown. Thus, we created monocyte/macrophage specific IGF-1R knockout mice (MΦIgf1rKO) by crossing Igf1r floxed mice with Lysozyme-Cre mice, both on Apoe-/- background, and evaluated atherosclerosis by feeding a high fat diet for 8 wks. MΦIgf1rKO mice had a marked increase in atherosclerotic burden; 41± 12 % and 57 ± 15 % increase in plaque size by en face and cross-section analysis respectively, compared to Igf1r floxed mice (control; n=8, p<0.05). Plaque composition in the aortic root was also substantially altered; MΦIgf1rKO had increased monocyte/macrophage content (anti-Mac3 staining, MΦIgf1rKO, 31 ± 3 % vs. Igf1r floxed, 23 ± 4 %) and a decrease in smooth muscle cell content (anti-α-smooth muscle actin staining, MΦIgf1rKO, 15 ± 4 % vs. Igf1r floxed, 23 ± 3 %), whilst there was no significant change in levels of circulating Ly-6Chi monocyte subpopulation. These changes in composition are consistent with a more inflammatory and less stable plaque phenotype. In fact, the frequency of intraplaque hemorrhage and elastin layer breaks (signs of plaque vulnerability) was higher in MΦIgf1rKO mice (5 in 8 mice positive) than in Igf1r floxed mice (2 in 8 positive). Gene expression analysis in aorta showed that MΦIgf1rKO downregulated TGFβ receptor 1 and 2 (by 37±12 % and 36±10 %, P<0.05, n=10), thrombospondin-1 (by 50±14 %, P<0.01, n=10), LXRβ (by 33±9 %, P<0.01, n=10), RXRα (by 38±11 %, P<0.01, n=10), and ABCA1 and ABCG1 (by 32±14 % and 45±19 %, P<0.05, n=10), and upregulated ACAT-1 (by 58±16 %, P<0.05, n=10) and DGAT-2 (by 115±55 %, P<0.05, n=10). These changes are consistent with decreased SMC content and increased lipid accumulation in macrophages. Taken together, our data indicate that monocyte/macrophage IGF-1R signaling suppresses macrophage accumulation in lesions and reduces plaque vulnerability, providing a novel mechanism whereby IGF-1 exerts its anti-atherogenic effects.
Abstract 466: Redox Regulation of 14-3-3zeta Controls Monocyte Migration
Authors: Kim Hong Seok , Ullevig Sarah , Joo Jahyun , Asmis Reto
Published: July 2021

Cofilin regulates actin turnover and is inactivated by phosphorylation at Ser-3 by LIM kinase (LIMK) and reactivated by the cofilin-phosphatase Slingshot-1L (SSH1L). We recently reported that protein-S-glutathionylation induced by metabolic disorders enhances monocyte recruitment into vascular lesions. Because monocyte priming and accelerated migration involves increases actin turnover, we tested the hypothesis that these regulators of actin assembly/disassembly are redox-regulated. Metabolic priming was induced in human THP-1 monocytes by exposing the cells to diabetic conditions, i.e. human native LDL plus high glucose concentrations (LDL+HG). Phosphorylation of cofilin in response to MCP-1 was completely blocked in metabolically primed monocytes, suggesting that hyperactivation of cofilin may contribute to the enhanced chemotactic activity of primed monocytes. Yet, neither LIMK1 nor SSH1L levels were affected by metabolic stress. However, metabolic priming resulted in a 3.8-fold increase the S-glutathionylation of 14-3-3zeta, a SSH1L binding protein which maintains the cofilin phosphatase in an inactive state. S-glutathionylation of 14-3-3zeta resulted in its caspase-dependent degradation and a 35% reduction in 14-3-3zeta protein levels. Overexpression of glutaredoxin 1 in monocytes inhibited both LDL+HG-induced S-glutathionylation and degradation of 14-3-3zeta, and normalized cofilin phosphorylation in response to MCP-1. The C25S mutant of 14-3-3zeta was resistant to S-glutathionylation and degradation induced by LDL+HG, and restored MCP-1-induced cofilin phosphorylation in metabolically primed monocytes. 14-3-3zeta S-glutathionylation did not affect binding of 14-3-3zeta to SSH1L, suggesting that loss of 14-3-3zeta increases the pool of free SSH1L phosphatase, thus preventing the phosphorylation and deactivation of cofilin in response to chemokine activation. In conclusion, we identified a novel redox-sensitive mechanism for the regulation of monocyte migration that links (thiol) oxidative stress induced by metabolic disorders to monocyte dysfunction and the conversion of blood monocytes into a hypermigratory, proinflammatory and proatherogenic phenotype.
Abstract 489: Differential Phosphoproteomic Analysis of Platelet Activation by Specific Oxidized Phospholipids and Thrombin Reveals Mechanisms of Platelet Activation in Hyperlipidemia
Authors: Zimman Alejandro , Titz Bjoern , Komisopoulou Evangelia , Graeber Thomas G , Podrez Eugene A
Published: July 2021

We previously showed that specific oxidized phospholipids (oxPC
CD36
) activate platelets via the scavenger receptor CD36 and promote platelet hyper-reactivity in hyperlipidemia, however the signaling pathway(s) induced in platelets by oxPC
CD36
are not defined. We employed mass spectrometry-based phosphoproteomics for the unbiased analysis of changes in protein phosphorylation induced by oxPC
CD36
and thrombin, a strong platelet agonist, in human platelets. oxPC
CD36
induced changes in phosphorylation of 148 unique phosphorylation sites (116 proteins) while thrombin induced changes of 297 unique sites (181 proteins). Most of the changes in phosphorylation induced by oxPC
CD36
and thrombin identified in our study have never been reported before in platelets and include high- and low-abundant proteins with diverse molecular functions located in the plasma membrane, cytosol, or cytoskeleton. Analysis using multiple bioinformatic tools identified protein interaction networks, signaling pathways, activated kinases, and enriched phosphorylation motifs. Comparison between platelet agonists revealed multiple differences including the specific activation of a signaling pathway involving Src-family kinases (SFK), SYK kinase, and PLCγ2 by oxPC
CD36
. Subsequent biochemical studies in human platelets demonstrated that this pathway is critical for platelet activation by oxPC
CD36
and is downstream of CD36. In conclusion, systematic analysis of platelet activation pathways provided novel insights into the mechanism of platelet activation and specific signaling pathways induced by oxidized phospholipids that modulate platelet function
in vivo
in hyperlipidemia.
Abstract 49: Myeloid Cell Specific ABCA1 Deletion Does Not Significantly Accelerate Atherogenesis in LDL Receptor Knockout Mice
Authors: Bi Xin , Zhu Xuewei , Gao Chuan , Cao Qiang , Liu Mingxia , Shewale Swapnil , Boudyguina Elena , Wilson Martha , Parks John
Published: July 2021

ATP binding cassette transporter A1 (ABCA1) functions as a cellular cholesterol exporter. Macrophage ABCA1 expression is important in reducing cellular cholesterol content and inflammatory response. Bone marrow (BM) transplantation studies suggest that leukocyte ABCA1 protects against atherosclerosis development. However, the
in vivo
effect of macrophage ABCA1 in atherogenesis is not fully understood due to the presence in BM of other leukocyte populations. Myeloid[[Unable to Display Character: ‐]]Specific ABCA1 Knockout (MSKO) mice in the LDL receptor knockout (LDLrKO) C57BL/6 background were developed to address this question. MSKO/LDLrKO (DKO) and LDLrKO (SKO) mice were fed chow or an atherogenic diet for 10-24wk to examine early to advanced stages of atherosclerosis. Basal (i.e. chow) plasma lipid levels were similar between genotypes, but relative to SKO mice, DKO mice had reduced plasma apoB[[Unable to Display Character: ‐]]containing lipoprotein (apoB Lp) levels throughout the diet-induced atherosclerosis progression phase resulting from decreased hepatic VLDL secretion. Despite a significant reduction in plasma apoB Lp levels, chow and atherogenic diet fed DKO mice had significantly higher cholesterol content in resident peritoneal macrophages and higher plasma proinflammatory cytokine and chemokine levels during atherogenic diet[[Unable to Display Character: ‐]]feeding compared to SKO mice. Aortic cholesterol content was similar between genotypes at early (i.e. 24wk chow-fed) or intermediate (i.e. atherogenic diet for 10wk or 16wk) stages and slightly increased at late stage atherosclerosis (i.e. 24wk atherogenic diet). Aortic root lesion area was also similar for both genotypes after 16wk of atherosclerosis induction. Transplantation of DKO or SKO BM into SKO mice followed by 16wk atherogenic diet feeding also showed similar extent of atherosclerosis. Collectively, these results suggest a novel role for myeloid cell ABCA1 in increasing hepatic VLDL secretion and plasma apoB Lp concentrations in atherogenic diet fed LDLrKO mice that offsets its atheroprotective role in decreasing macrophage cholesterol content and inflammatory response, resulting in no increase in atherosclerosis in its absence.
Abstract 498: Age-adjusted Cut-off Levels Improve Test Performance in the Diagnosis Strategy of Venous Thromboembolism in Elderly Patients With a Non-high Pre-test Probability
Authors: Toulon Pierre , Claessens Yann-Erick
Published: July 2021

Background

D-dimer levels below a well defined cut-off level enable to safely rule out VTE in patients with non-high PTP. Ageing is associated with higher levels of D-dimer. To address the issue of their usefulness in elderly patients, we firstly managed data from 644 out-patients with non-high PTP included in a multicenter management study of a D-dimer assay (C.Legnani et al. 2010). We then validated our results in a cohort of 1,042 consecutive patients investigated using another assay.

Results

In the first part, D-dimer level, evaluated using the HemosIL D-dimer HS500 assay (Instrumentation Laboratory), was above the cut-off level (500 ng/ml) in all 88 patients with VTE and in 299 of those without (53.8%). Test negative predictive value (NPV) and sensitivity were 100%. Overall test specificity was 46.2%, but decreased in an age-dependent manner over 70 y. ROC-analysis of test results obtained in patients classified according to age allowed us to propose an age- adjustment of the cut-off level calculated by increasing its level by 100 ng/ml per 10 y-increments in patients aged over 59 y e.g. 600 ng/ml between 60 et 69 y, 700 ng/ml between 70 et 79 y. Using those age-adjusted cut-off levels yielded to significantly improve test specificity (56.1%), particularly in old patients. If an 84 y old patient (D-dimer=625 ng/ml) was missed, NPV remained high (99.6%). That strategy was then validated by evaluating data from 1,042 consecutive out-patients, investigated using the Vidas D-dimer assay (BioMérieux). When applied, that age- adjusted cut-off levels leaded to improve test specificity 62.2% vs. 55.1% using the fixed cut-off level. 3 additional patients, with D-dimer >500 ng/ml but below age- adjusted cut-off level, were missed but overall NPV remained high 99.0% vs. 99.4%.

Conclusions

Age-adjusted cut-off levels for D-dimer, calculated by increasing the traditional cut- off level (500 ng/ml) by 100 ng/ml per 10 y-increments in patients aged over 59 y, significantly increased the test specificity. Correlatively, NPV and sensitivity were slightly decreased, as some patients, with D-dimer above 500 ng/ml but below the age-adjusted cut-off level, could be misdiagnosed. However such a strategy was safe, as NPV remained above 99% in our studied populations.
Abstract 520: Elastic Fiber Fragmentation and Aberrant Angiogenesis Precede Inflammation in Early-Onset Aortic Valve Disease
Authors: Opoka Amy M , Juraszek Amy L , Osinska Hanna , Smith J Michael , Merrill Walter H , Eghtesady Pirooz , Mecham Robert P , Bove Kevin E , Hinton Robert B
Published: July 2021

Introduction

Elastic fiber fragmentation (EFF) is a hallmark of aortic valve disease (AVD), and both inflammation and neovascularization have been identified as late findings, presumably due to atherosclerosis through a wound healing like response. However, EFF independently stimulates angiogenesis, suggesting an alternative mechanism for neovascularization in AVD.

Hypothesis

We hypothesized that aberrant angiogenesis would be an early AVD finding, preceding inflammation, due to EFF.

Methods

To examine disease progression, valve tissue was examined using histochemistry and immunohistochemistry from early (<40yo) and late (≥40yo) onset AVD specimens, as well as age-matched autopsy control aortic valves. Angiogenesis (VEGF-A, CD-31, SMA and chondromodulin), EFF (elastin, fibrillin-1, emilin-1, fibulin-5), progenitor valve interstitial cell phenotype (CD-34, CD-45) and inflammation (CD-68, LRP-5) were assessed.

Results

Bicuspid aortic valve was more common in early-onset AVD (n=21), and cardiovascular comorbidities were more common in late-onset AVD (n=11). Early-onset AVD specimens demonstrated angiogenesis without inflammation or atherosclerosis. A distinct pattern of EFF and elastic fiber components surrounded early-onset AVD neovessels, which were not present in control valves, including increased emilin and decreased fibulin-5, and the elastase/anti-elastase ratio and localization were altered in both early and late-onset AVD, suggesting a dynamic disease progression. Interestingly, progenitor VICs were present in control valves at both stages and were unchanged in age-matched AVD specimens.

Conclusions

Aberrant angiogenesis is an early mechanism in AVD pathogenesis preceding inflammation, implicating EFF as an inciting factor. Elucidation of the underlying mechanisms may inform the development of new pharmacologic therapeutics and durable bioprostheses.
Abstract 532: The RNA-binding Protein Quaking Critically Regulates Vascular Smooth Muscle Cell Phenotype
Authors: van der Veer Eric P , de Bruin Ruben G , Kraaijeveld Adriaan O , de Vries Margreet R , Pera Tonio , Segers Filip M , Trompet Stella , van Gils Janine M , Roeten Marko K , Beckers Cora M , van Santbrink Peter J , Janssen Anique , van Solingen Coen , Swildens Jim , de Boer Hetty C , Bot Ilze , Peters Erna A , Rousch Mat , Doop Merijn , Schalij Martin Jan , van der Wal Allard C , Richard Stehpane , van Berkel Theo J , Pickering J. G , Hiemstra Pieter S , Goumans Marie Jose , Rabelink Ton J , de Vries Antoine A , Quax Paul H , Jukema J. W , Biessen Erik A , van Zonneveld Anton Jan
Published: July 2021

In response to vascular injury, smooth muscle cells (VSMC) adopt a proliferative, synthetic hypocontractile phenotype. This phenotype switch is deemed instrumental in vascular remodeling in both health and disease. Here, we detail a decisive role for the RNA-binding protein Quaking (QKI) in regulating VSMC plasticity. We identified that the RNA-binding protein Quaking (QKI) is highly expressed by neointimal VSMCs of human coronary restenotic lesions, but not in healthy vessels. In a mouse model of vascular injury, we observed reduced neointima hyperplasia in Qk
v
mice, which have decreased QKI expression. Concordantly, abrogation of QKI attenuated fibroproliferative properties of VSMCs, while potently inducing contractile apparatus protein expression, rendering non-contractile VSMCs with the capacity to contract. We identified that QKI localizes to the spliceosome in proliferative VSMCs, where it interacts with and impacts myocardin (pre)-mRNA metabolism by mediating myocardin exon 2a exclusion. As such, in vitro and in vivo experiments indicate that the modulation of QKI expression directly influences the myocardin_v3 / myocardin_v1 mRNA balance, which could play a role in shifting the Myocardin-induced transcriptional coactivation profile following arterial damage. We propose that QKI is a central regulator of VSMC phenotypic plasticity and that intervention in QKI activity can ameliorate pathogenic, fibroproliferative responses to vascular injury.
Abstract 6: Co-targeting of Thrombomodulin and the Endothelial Protein C Receptor Protects the Pulmonary Vasculature in a Mouse Model of Acute Endothelial Injury
Authors: Greineder Colin F , Zaytsev Sergei , Muzykantov Vladimir R
Published: July 2021

Background

Recombinant activated protein C is protective in animal models of sepsis, stroke, and focal thrombosis, but has a poor side effect profile. Thrombomodulin (TM) and the Endothelial Protein C Receptor (EPCR) are endothelial membrane proteins, which partner to bind thrombin and activate protein C at sites of inflammation, ischemia, and thrombosis. Both proteins can be lost from the endothelial surface in a variety of disease states. We have shown previously that targeted delivery of TM to the Platelet Endothelial Cell Adhesion Molecule (PECAM-1) on the endothelial surface is protective in mouse models of lung inflammation and ischemia. Unlike endogenous TM, however, recombinant TM targeted to PECAM-1 does not effectively partner with endogenous EPCR.

Objective

We hypothesized that co-delivery of TM and EPCR would enhance in vitro activity and therapeutic efficacy in vivo.

Methods

TM and EPCR were fused to single chain fragments of two anti-PECAM antibodies, which are known to demonstrate collaborative binding enhancement. TM and EPCR fusion proteins were tested on PECAM-expressing cells and mouse endothelial cells. Fusion proteins were injected intravenously in C57BL/6 mice prior to intratracheal injection of endotoxin. Expression of VCAM-1 was measured using qPCR and MIP-2 was quantified in bronchoalveolar lavage (BAL) fluid. Endothelial barrier dysfunction was quantified by measuring transendothelial leak of 125I-labeled albumin.

Results

TM and EPCR fusion proteins target PECAM-1 on endothelial cells and demonstrate collaborative binding. Independent of binding effects, the EPCR fusion protein results in a 50% increase in the activation of protein C by cell-bound TM, an effect blocked by anti-EPCR antibody. Consistent with in vitro results, the combination of TM and EPCR fusion proteins enhances protection in endotoxin-induced lung injury, reducing VCAM expression, BAL MIP-2, and transendothelial leak of albumin (p < 0.001), as compared to TM fusion protein alone.

Conclusion

Co-targeting of TM and EPCR to PECAM-1 enhances activation of protein C and represents a novel and promising therapeutic strategy for the treatment of diseases involving acute endothelial injury.
Abstract 65: Flavin Monoxygenase 3 (FMO3) is a Novel Regulator of Hepatic Cholesterol Metabolism and Transintestinal Cholesterol Efflux (TICE).
Authors: Warrier Manya , Marshall Stepahie , McDaniel Allison , Wilson Martha , Brown Amanda , Wang Zeneng , Shih Diana , Lusis Aldons J , Lee Richard , Hazen Stanley L , Temel Ryan E , Brown J. Mark
Published: July 2021

Recent studies have revealed a novel route for cholesterol disposal through intestine known as transintestinal cholesterol efflux (TICE) that significantly contributes to fecal neutral sterol loss. This pathway is an integral part of reverse cholesterol transport (RCT), yet major mechanisms regulating TICE are not well understood. Using an unbiased transcriptional profiling approach in mouse models of augmented TICE, we found that hepatic expression of the enzyme Flavin monoxygenase 3 (FMO3) was dramatically repressed. At the same time we identified this enzyme through transcriptional profiling, it was reported that plasma levels of its product trimethylamineoxide (TMAO) are highly predictive of atheroslcerosis in humans, and TMAO is proatherogenic in mice. To further understand FMO3’s role as a regulator of cholesterol metabolism we used antisense oligonucleotides (ASO) to knockdown FMO3 expression in mouse liver in C57BL/6 mice fed either low (0.02%) or high (0.2%) levels of dietary cholesterol. As expected, FMO3 knockdown (>90% knockdown in the liver) increased the TMA/TMAO ratio in plasma more than 3-fold. Interestingly, knockdown of FMO biliary cholesterol levels were reduced by 60%, whereas fecal cholesterol loss was quite normal in FMO3 ASO treated mice fed a high cholesterol diet, which phenocopies a previously described mouse model where TICE predominates (NPC1L1-liver transgenic mice). ASO-mediated knockdown of FMO3 also unexpectedly reduced hepatic cholesteryl ester (CE) storage by 70% in mice fed 0.2% cholesterol. In parallel, knockdown of FMO3 reduces plasma VLDL cholesterol levels and the secretion rate of VLDL cholesteryl ester, but not triacylglycerol in cholesterol fed mice. FMO3 knockdown also reduced the hepatic expression of several liver X receptor (LXR) target genes, while increasing expression of genes involved in cholesterol synthesis. Collectively, these studies have identified FMO3 as a novel regulator of hepatic cholesterol metabolism and TICE. Given that plasma levels of FMO3’s product (TMAO) are strongly associated with atherosclerosis development in humans, and production of TMAO promotes atherosclerosis in mice, these studies have important implications for future cardiovascular drug discovery.
Abstract 70: P2X7-Induced Thiol-Disulfide Exchange is Critical for the Coupling of Coagulation and Inflammation.
Authors: Rothmeier Andrea S , Furlan-Freguia Christian , Marchese Patrizia , Petrich Brian , Ruggeri Zaverio M , Ginsberg Mark H , Ruf Wolfram
Published: July 2021

Macrophages are important players in the maintenance of tissue homeostasis, but promote inflammation through the release of IL1β triggered by danger signals in form of extracellular ATP that activates the purinergic P2X7 receptor. We found that P2X7 signaling also contributes to thrombosis by inducing thiol-dependent tissue factor (TF) activation coupled to procoagulant microparticles (MP) release. In the present study, we identified thiol-regulated proteins released on MP and based on this information delineated key steps in the P2X7-induced generation of prothrombotic TF
+
MP. We find that TF procoagulant activity of LPS/IFNγ primed macrophages is controlled by internalization through the arf6/integrin-recycling pathway. Activation of P2X7 inactivates arf6 and prevents TF internalization, but additional steps are required to generate highly procoagulant MP carrying TF and integrin β1. Imaging of cell surface TF by confocal microscopy shows translocation of TF onto filopodia that form in response to P2X7 activation. Blocking raft mobility does not inhibit filopodia formation, but rather specifically prevents TF and integrin β1 trafficking and release on MP. We show that filopodia formation is dependent on thioredoxin reductase (TRXR). Remarkably, thioredoxin (TRX), the direct substrate of TRXR, is entirely released from the cytosol. Pharmacological inhibition of TRXR blocks both TRX release and reductive changes on the cell surface and MP, identifying the molecular events that change the extracellular redox environment. TRXR-mediated externalization of TRX was also required for activation of the inflammasome and caspase1 leading to IL1β processing and release. These data elucidate the molecular events required for the generation of highly procoagulant TF
+
MP and identifies TRXR-TRX dependent thiol-disulfide exchange as common upstream regulator responsible for the induction of inflammation and coagulation in innate immune cells.
Abstract 84: Serum Amyloid A Does Not Modulate the Inverse Association of HDL with Coronary Artery Calcification in Type 2 Diabetes
Authors: Mulvey Claire K , Churchill Timothy W , Terembula Karen , Ferguson Jane F , Mehta Nehal N , Qasim Atif N , Reilly Muredach P
Published: July 2021

Introduction

Although high-density lipoprotein (HDL) is inversely correlated with cardiovascular risk, HDL loses its protective role in pathologic inflammatory states like type 2 diabetes (T2DM). HDL dysfunction contributes to accelerated atherosclerosis in T2DM, but the mechanism is incompletely defined. The acute phase reactant serum amyloid A (SAA) displaces apolipoprotein A-I and may impair HDL-mediated reverse cholesterol efflux. We hypothesized that SAA alters the inverse association between HDL and coronary artery calcium (CAC) in the Penn Diabetes Heart Study, a cross-sectional study of T2DM patients free of overt cardiovascular or renal disease.

Methods

We measured SAA in serum samples by immunonephelometry (N=975; mean age 58 ± 9 years; 63% male, 57% Caucasian; mean BMI 33 ± 6 kg/m
2
). HDL was measured enzymatically in lipoprotein fractions after ultracentrifugation. Agatston CAC scores were quantified from electron beam tomography at the same visit. Spearman correlation and logistic regression were used to test associations of SAA with clinical factors and metabolic syndrome. We used Tobit regression to analyze associations between CAC and HDL, both overall and stratified by 3 categories of SAA: undetectable, lower half detectable, and upper half detectable.

Results

Spearman correlations revealed moderate association of SAA with C-reactive protein (r=0.52) and weak associations of SAA with BMI (r=0.25) and HDL (r=0.17; all p<0.001). In logistic regression, the group with highest SAA levels had increased odds of metabolic syndrome compared to those with undetectable levels (OR 1.56, 95% CI 1.03 to 2.38, p=0.036). In adjusted Tobit regression, HDL was inversely associated with CAC (Tobit coefficient for 1-SD increase in HDL: -0.30; 95% CI -0.54 to -0.06; p=0.013). Across the categories of SAA, however, there was no difference in the association of HDL with CAC (Tobit coefficient for 1-SD increase in HDL: -0.17 [95% CI -0.49 to 0.16] for undetectable vs. -0.31 [95% CI -0.79 to 0.17] for lower half detectable vs. -0.49 [95% CI -1.01 to 0.03] for upper half detectable).

Conclusions

Despite the association of SAA with metabolic syndrome, these data suggest that elevated SAA may not change the inverse relationship of HDL with CAC in T2DM.
Abstract 1: Role of MicroRNAs in Postranscriptional Regulation of Apolipoprotein B-100 mRNA
Authors: Basir Sahar A , Asante Tiffany , Adeli Khosrow
Published: July 2021

Hepatic apolipoprotein B-100 (apoB) synthesis and secretion appears to be regulated largely at the posttranscriptional and posttranslational levels. MicroRNAs (miRNAs) are among posttranscriptional regulators of gene expression that bind to complementary sequences on target messenger RNA (mRNA) transcripts, usually resulting in translational repression or degradation. It is unknown whether specific miRNAs are involved in posttranscriptional regulation of apoB mRNA. We performed bioinformatic analysis, showing that two specific miRNAs with satisfactory E-values level (with levels indicating greater similarity between the input and its match) namely, miR-544 (E-value = 0.91) and miR-1202 (E-value=0.86) have potential to interact with 3’ and 5’ UTR of apoB, respectively. We hypothesized that the interaction of these specific miRNAs (miR-544 and miR-1202) with the 3’ and 5’UTR of apoB mRNA leads to apoB mRNA translational repression and/or activation. Using a human hepatoma cell line model, HepG2, the effects of overexpressed miRNAs and inhibition of endogenous miRNAs on the expression of apoB mRNA and apoB protein synthesis were investigated. We further examined the effect of these miRNAs on apoB mRNA traffic into cytoplasmic P-bodies. Transfection of HepG2 cells with miR-544 led to a significant reduction in apoB mRNA expression and protein synthesis and induced an increase in the co-localization of apoB mRNA into P-bodies. The opposite effect was observed when anti-miR-544 was employed to inhibit the endogenous miR-544. Results from luciferase reporter assays indicated that the effects of miR-544 may be mediated via interaction with the 3’UTR of apoB mRNA. In contrast to miR-544, miR-1202 overexpression induced an increase in apoB mRNA expression and protein synthesis. Similarly, the opposite effect was observed when using anti-miR-1202. Data from luciferase reporter assays showed an increased expression of the reporter gene in constructs carrying 5’UTR of apoB mRNA suggesting that miR-1202 may function via the 5’UTR. In summary, these data demonstrate that specific miRNAs are involved in the regulation of expression and translational control of apoB mRNA in hepatocytes. However, these miRNAs do not appear to mediate insulin regulation.
Abstract 11: Macrophage Expression of the Notch Ligand Delta-Like 4 Promotes Vein Graft Disease in LDL Receptor--Deficient Mice
Authors: Koga Jun-ichiro , Figueiredo Jose-Luiz , Dahlman James E , Niida Tomiharu , Iwata Hiroshi , Aster Jon C , Yagita Hideo , Anderson Daniel G , Ozaki C K , Aikawa Masanori
Published: July 2021

Background:
Vein graft failure causes devastating complications in patients with peripheral arterial disease or ischemic heart disease, but its underlying mechanisms remain obscure and no effective therapeutic measures are available.

Methods and Results:
We tested the hypothesis that Notch signaling triggered by its ligand Delta-like 4 (Dll4) promotes macrophage activation and vein graft disease. Vein graft surgery was performed in high-fat fed LDL receptor-deficient (Ldlr-/-) mice by implanting donor inferior vena cava into recipient right carotid arteries. [Approach 1: Dll4 antibody] Dll4 blocking antibody was administered for 28 days. Dll4 blockade inhibited lesion development and macrophage accumulation (Figures) in vein grafts, and suppressed macrophage expression of genes typical of pro-inflammatory M1 macrophages (e.g., IL-1β, TNF-α). In vivo molecular imaging demonstrated that Dll4 antibody treatment suppressed MMP activity in lesional macrophages. Dll4 blockade concomitantly attenuated collagen thinning. [Approach 2: siRNA delivery to macrophages] To address the relative contribution of macrophages to Dll4-mediated vein graft disease in vivo, we delivered Dll4 siRNA oligos encapsulated in macrophage-targeted lipid nanoparticles. In vivo Dll4 silencing in macrophages reduced lesion development and macrophage burden in vein grafts of Ldlr-/- mice to a similar extent as those of Dll4 antibody therapy. In vitro gain-of-function and loss-of-function studies suggested that Dll4 promotes expression of pro-inflammatory molecules in macrophages. Furthermore, macrophage Dll4 stimulated smooth muscle cell (SMC) proliferation and migration and suppressed their differentiation.

Conclusions:
These results suggest the novel mechanism that macrophage Dll4 promotes vein graft lesion development by exacerbating inflammation and crosstalk between macrophages and SMC, supporting the Dll4-Notch axis as a potential therapeutic target.
Abstract 124: Association of AdipoR2 mRNA Expression on Peripheral Monocytes With Carotid Plaque Instability
Authors: Gasbarrino Karina , Zheng Huaien , Mantzoros Christos , Lai Chi , Veinot John , Daskalopoulou Stella S
Published: July 2021

Adiponectin, an adipose tissue secreted protein, possesses anti-atherosclerotic properties. Its effects are partly mediated through its action on monocytes, via receptors, AdipoR1 and AdipoR2. Circulating monocytes play a crucial role both in plaque initiation and in the progression of atherosclerosis. The aim of this ongoing study is to investigate for the first time whether there is differential expression of AdipoR1/AdipoR2 on human peripheral monocytes in patients with unstable vs. stable carotid atherosclerotic plaques.

Patients (n=20) with carotid stenosis of ≥50% scheduled for a carotid endarterectomy (CEA) were recruited from the Royal Victoria Hospital, Montreal, Canada. Blood samples were collected the morning of the CEA. A radioimmunoassay was used for plasma total adiponectin measurement. Circulating monocytes were isolated from mononuclear cells using a Magnetic Cell-Sorting technique with CD14+ Human Microbeads. Expression of AdipoR1/AdipoR2 mRNA on peripheral monocytes was determined using quantitative RT-PCR. Carotid plaque specimens were processed for histological analyses. Plaque instability was categorized according to the gold standard American Heart Association classification.

All specimens were graded as either a Type V (n=11) or a Type VI plaque (n=9). Type VI represents a more unstable lesion than Type V. AdipoR2 gene expression on monocytes was inversely associated with greater plaque instability; compared to patients with a Type V plaque, those with a Type VI plaque had 1.88-fold decreased AdipoR2 mRNA levels (p=0.046). AdipoR2 gene expression was found to be a trending predictor of a Type VI plaque (OR=2.93; 95% CI=0.87-9.81; p=0.082). AdipoR1 gene expression on monocytes did not differ between the two groups, while plasma adiponectin levels were lower in Type VI plaques (Type VI: 2.73 [2.19-3.96] μg/ml vs. Type V: 6.97 [4.69-10.07] μg/ml; p= 0.067). Plasma adiponectin levels did not correlate with the expression levels of AdipoR1/AdipoR2 on monocytes.

These results indicate that carotid plaque instability is associated with decreased AdipoR2 expression on peripheral monocytes. Our ongoing study may determine the underlying mechanisms linking adiponectin and its receptors with plaque instability.
Abstract 159: Androgen Deficiency Influences Matrix Metalloproteinase Expression and Intimal Hyperplasia Development After Vascular Injury
Authors: Freeman Brian M , Mountain Deidra J , Brock Timothy C , Chapman Jason R , Kirkpatrick Stacy S , Arnold Joshua D , Stevens Scott L , Goldman Mitchell H , Freeman Michael B , Klein Frederick A , Grandas Oscar H
Published: July 2021

Objectives:
Androgen deficiency (AD) is associated with increased risk of vascular disease, yet the molecular mechanisms remain unclear. Our group has previously shown testosterone regulates matrix metalloproteinases (MMP) in a dose-dependent manner in vitro. Here we investigated the role of AD and androgen replacement therapy (ART) on inflammatory cytokines and MMP-modulated intimal hyperplasia (IH) development in vivo.

Methods:
Aged orchiectomized (AO) rats were implanted with increasing doses of testosterone pellets (TST; 0.5-150mg). ELISA and multiplex array determined serum TST and cytokine levels. Young intact (YI), Aged intact (AI), and AO rats given placebo (Plac) or TST supplementation underwent balloon angioplasty of the left common carotid following 14d ART. Tissue samples were collected 14d post-injury for Intima:Media (I:M) or MMP quantification.

Results:
Therapeutic TST doses were achieved at 14d with 0.5, 2.5, 5, and 35mg pellets when compared to controls (Table 1). Interleukin family isoforms were elevated at sub-physiological TST levels but returned to control levels with physiological TST (Table 2). I:M was decreased in AI and physiological TST levels compared to YI (Fig 1). I:M was increased with sub- and supra-physiological TST. Injury-induced expression of MMP-2 was highest in AI and physiological TST conditions, though these values were not significant (Table 3). Analysis of other MMP isoforms is ongoing.

Conclusions:
We demonstrated that low testosterone levels increase interleukin inflammatory signaling, regulate MMP expression, and increase IH development in vivo. This effect is reversed by physiologic testosterone supplementation. AD could be playing a role in vascular disease via MMP regulatory mechanisms under the control of inflammatory signaling cascades. Future studies will examine targeted inhibition of inflammatory-modulated MMP mechanisms in the prevention of dysfunctional vascular remodeling.
Abstract 180: Downregulation of GRK2 by Long-Term Exercise Improves Vascular Insulin Sensitivity and Delays the Development of Hypertension in SHRs
Authors: Zhang Haifeng , Xing Wenjuan , Gao Feng
Published: July 2021

Aims:
Exercise training, a recommended nonpharmacological strategy for hypertensive patients, exerts beneficial effect on blood pressure modulation, but the underlying mechanisms remain elusive. Vascular insulin resistance is an early pathologic alteration in hypertensive vascular injury and plays a critical role in the development of hypertension. This study aimed to investigate whether long-term physical exercise starting at prehypertensive period prevents the development of hypertension via improving vascular insulin sensitivity.

Methods:
Young (4-week-old) prehypertensive spontaneously hypertensive rats (SHRs) and their normotensive Wistar-Kyoto controls (WKY) were subjected to a 10-week free-of-loading swim training session (60 min/day, 5 days/week). Insulin-induced mesenteric arteriolar vasorelaxation were determined.

Results:
SHRs showed higher systolic blood pressure (SBP) and decreased insulin sensitivity of the whole body and resistance vessels compared with those of WKY rats. SBP in the exercised SHRs was significantly lower than that in sedentary ones (180.2±5.1 vs. 205.7±3.7 mmHg, n=6, P<0.05). Vascular insulin sensitivity in mesenteric arteries was improved after exercise training as evidenced by increased vasodilation response to insulin (29.0%±2.5% vs. 18.4%±5.9% to 1 μmol/L insulin, n=6, P<0.05). In addition, exercise downregulated vascular GRK2 expression and activity, which further increased insulin-stimulated vascular Akt/eNOS activation in exercised SHRs. More importantly, suppression of GRK2 with siRNA mimicked the effect of exercise-enhanced vascular insulin sensitivity, while upregulation of GRK2 by Chariot-mediated delivery reversed the exercise-induced vascular insulin sensitization.

Conclusions:
Long-term regular physical exercise beginning at prehypertensive stage improves vascular insulin sensitivity via downregulating GRK2 and consequently delays the development of hypertension.
Abstract 185: Global Infusion of Allogeneic Cardiosphere-Derived Cells Proportionally Stimulates Angiogenesis and Myocyte Regeneration in Swine With Chronic Myocardial Infarction
Authors: Suzuki Gen , Weil Brian , Leiker Merced , Young Rebecca , Suzuki Takayuki , Canty John
Published: July 2021

Background:
Previously we reported that global infusion of CDCs in infarcted and non-infarcted remote regions improved LV function in swine with chronic myocardial infarction. The effect of global infusion of CDCs on angiogenesis and myocyte regeneration in infarcted and non-infarcted remote regions is unknown.

Methods:
Swine (n=11) with chronic infarction in the left anterior descending (LAD) coronary artery region were studied 8 weeks after microsphere embolization (1x10^6 particles, 45μm diameter). CDCs were isolated from myocardial atrium and 20 x 10^6 CDCs were infused into the 3 major coronary distributions after cyclosporine (100mg/day) immunosuppression. Data were compared to untreated animals. To assess the factors related to angiogenesis, VEGF and SDF-1 expression on cultured CDCs were quantified by RT-PCR. Angiogenesis was quantified by assessing capillary density (von Willebrand Factor) with immunohistochemistry. Myocyte regeneration was quantified by assessing myocyte nuclear density. The ratio of capillary density to myocyte nuclear density was evaluated.

Results:
icCDCs improved infarcted LAD (13±6 vs. 36±6%, p<0.05) and remote (47±8 vs. 99±10%, p<0.05) wall thickening with increased ejection fraction (32±3 vs. 45±4%, p<0.05) compared to untreated. RT- PCR from cultured CDCs showed VEGF expression and to a less extent SDF-1 expression. icCDCs increased capillary density and myocyte nuclear density in infarcted LAD and remote regions by 40% (Table). Thus, the ratio of capillary density to myocyte nuclear density was maintained in untreated and icCDCs animals.

Conclusion:
These data indicate that in a model of infarction, global infusion of icCDCs stimulates angiogenesis and myocyte regeneration to the same extent in infarcted and non-infarcted regions resulting in improved global function. Thus, global infusion of icCDCs may afford a widely available approach to regenerate myocardium in patients with ischemic cardiomyopathy.
Abstract 189: Regulation of Endothelial Nitric Oxide Synthase in Human Blood Outgrowth Endothelial Progenitor Cells
Authors: Yuan Yifan , Florian Maria , Stewart Duncan J , Courtman David W
Published: July 2021

Human blood outgrowth endothelial progenitor cells (BOECs), derived in culture from peripheral blood mononuclear cells, possess high proliferative capacity, and endothelial protein expression profiles (i.e. CD34, 31, 133, 144, 105, 146, KDR, Ac-LDL and vWF). As such they hold potential for autologous cell seeding applications in cardiovascular biomaterials or as prognostic indicators of endothelial mediated diseases. Endothelial nitric oxide synthase (eNOS) is an important regulator of vascular homeostasis and loss of eNOS activity is a hallmark of endothelial dysfunction. We therefore examined the expression of eNOS in BOECs finding markedly lower protein (0.34 ± 0.13, Western blot) as well as activity levels (0.49 ± 0.18, Nitrite analysis) when compared to human umbilical vein endothelial cells (HUVECs). BOEC eNOS protein levels initially decreased with both length of time in culture and passage, but the levels stabilized between Passage 5 to Passage 8. To further examine the role of eNOS expression on BOEC functional activity we performed transfection studies with eNOS minicircle vectors (double stranded DNA of reduced size and are devoid of bacterial sequences). Two promoters were tested for expression efficiency in BOECs, the CMV promoter (pMini-CMV-eNOS) and the EF1α promoter (pMini-EF1α-eNOS). Transfection with pMini-CMV-eNOS achieved 24.8 ± 5.1 times more eNOS expression when compared to null transfected cells at 24 hours, a marked improvement over that achieved with conventional PVAX plasmid (10.2 ± 4.7 fold increase) or pMini-EF1α-eNOS (8.2 ± 1.2 fold increase both compared to null transfected control). pMini-CMV-eNOS also produced more sustained eNOS expression (3.1 ± 0.7 fold increase at 72 hours). Boyden chamber and tube formation assays showed that pMini-CMV-eNOS mediated overexpression can both improve cell migration (24.9 ± 4.2 versus 13.8 ± 3.7 cells/field) and angiogenesis (0.92 ± 0.13% versus 0.58 ± 0.19% of total pixels per high power field both for eNOS transfected versus null). These results suggest that minicircle vectors can be used to markedly enhance eNOS expression in BOECs and as such enhance their angiogenic and regenerative potential.
Abstract 194: Differential Prevalence of Loss/Gain of Function Alleles Affecting Response to Clopidogrel in the Hmong Underscores the Importance of Genotyping Unique Subpopulations as a Means to Address Existing Health Disparities
Authors: Xiong Jenny D , Roman Youssef M , Culhane-Pera Kathleen , Menk Jeremiah S , Straka Robert J
Published: July 2021

Introduction:
Standard of care for Acute Coronary Syndromes (ACS) includes dual antiplatelet therapy (e.g. aspirin and clopidogrel). CYP2C19 loss of function (*2, *3) or gain of function (*17) alleles are associated with clopidogrel’s bioactivation, antiplatelet effectiveness and clinical outcomes leading to the development of Clinical Pharmacogenomic Implementation Consortium (CPIC) guidelines. Currently the prevalence of CYP2C19 variants in the Hmong - a unique Asian population numbering over 60,000 in Minnesota - is unknown. Given that they are experiencing a surge in CVD, including ACS, we sought to compare their Minor Allele Frequencies (MAFs) for CYP2C19 *2, *3, and *17 with White Non-Hispanic and Han-Chinese. We evaluated the prevalence of *2 (rs4244285) or *3 (rs4986893), or *17 (rs12248560) alleles between the Hmong and those observed in 1) White Non-Hispanics and 2) Han-Chinese. To achieve this, DNA from Hmong was genotyped for the relevant Single Nucleotide Polymorphisms (SNPs) and compared to published (PMID: 21716271) MAFs for White Non-Hispanic and Han-Chinese cohorts

Methods:
Salivary DNA from 235 Hmong residing in either Minnesota or Wisconsin was genotyped for relevant SNPs by Sequenom iPlex Gold assay. MAFs between groups were compared by Chi-Square or Fisher’s exact test. A Bonferroni corrected significance level of 0.017 was considered statistically significant.

Results:
Hmong MAFs were higher for *2 (37%, p=0.0001) but lower for *17 (0.2%, p=0.0001) compared to the White Non-Hispanic cohort (13% and 23% respectively). Hmong MAFs were higher for *2 (37%, p=0.0001) but lower for *3 (0.2%, p=0.0001) compared to the Han-Chinese cohort (25% and 3%, respectively).

Conclusions:
Hmong have a higher prevalence of the loss of function allele, *2, compared to both reference cohorts. Hmong also have significantly different MAFs for both loss of function alleles, *2 and *3, compared to the Han-Chinese cohort. Both conclusions suggest that drug selection cannot be guided by race alone. Our data further underscores the importance of genotyping unique sub-populations as a means to mitigate existing health disparities in the availability of data which facilitate optimal drug selection.