bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 287: Oxidative Stress Modulates Vascular Smooth Muscle Cell Phenotype via Connective Tissue Growth Factor in Thoracic Aortic Aneurysm
Authors: Branchetti Emanuela , Poggio Paolo , Sainger Rachana , Shang Eric , Grau Juan B , Jackson Benjamin M , Lai Eric , Parmacek Michael S , Gorman Robert C , Gorman Joseph H , Bavaria Joseph E , Ferrari Giovanni
Published: March 2022

Rationale.

Thoracic Aortic Aneurysm (TAA) is characterized by a dysregulation of cellular and extracellular components of the aortic wall which contribute to the weakening of the ascending aorta. Oxidative stress has long been implicated in the pathogenesis of aneurysms for its role in inducing cellular apoptosis and matrix degradation. However, the link between oxidative stress and TAA formation remains to be firmly established. Connective Tissue Growth Factor (CTGF) regulates matrix biosynthesis as well as Vascular Smooth Muscle Cells (VSMC) proliferation and apoptosis. We hypothesized that Reactive Oxygen Species (ROS), modulated by altered hemodynamic conditions, could regulate VSMC phenotype and matrix composition via CTGF, affecting - in turn - the aortic tissue structure.

Methods.

Finite element analysis (FEA) based on computed tomography geometries was used to estimate wall stress in control and TAA patients. In vitro and ex vivo studies on human tissues and isolated VSMC were used to demonstrate the correlation between ROS, CTGF, and VSMC synthetic phenotype in TAA development. Results were validated in vivo by a murine model of TAA based on Angiotensin II (AngII) infusion.

Results.

TAA patients have higher wall stress in the ascending aorta than non-dilated patients. High aortic wall stress is associated with increased levels of ROS, media degeneration and accumulation of synthetic VSMCs in TAA tissues. ROS directly induce CTGF expression, which modulates synthetic VSMC phenotype in human isolated cells. ROS effect is reverted in vitro either using a neutralizing antibody against CTGF or by antioxidant administration. In AngII- induced TAA in mice, medial thickening and luminal expansion of the proximal aorta are associated with ROS accumulation, CTGF up-regulation and VSMC with a synthetic phenotype as seen in human specimens.

Conclusions.

Increased peak wall stress correlates with change in VSMCs towards a synthetic phenotype mediated by ROS accumulation via CTGF. Understanding the molecular mechanisms that regulate VSMCs towards a synthetic phenotype could unveil new regulatory pathways for aortic homeostasis and identify new therapeutic targets for patients at risk of aortic dissection and rupture.
Abstract 298: Microfibrillar Protein 4 is an Integrin avß3/5 Ligand Involved in Vascular Remodeling
Authors: Schlosser Anders , Lindholt Jes S , Kejling Karin , Kristmannsdottir Gudlaug B , Pilecki Bartosz , Wulf-Johansson Helle , Møller Jesper B , Füchtbauer Ernst M , Nielsen Ole , Hemstra Line E , Dubey Lalit K , Hansen Pernille B , Stubbe Jane , Ratkolb Birgit , Schrewe Anja , Bekeredjian Raffi , Wolf Eckhard , Gailus-Durner Valerie , Fuchs Helmut , Hrabê de Angelis Martin , Holmskov Uffe , Sorensen Grith L
Published: March 2022

Arterial injury stimulates remodeling responses that, when overexuberant, leads to stenosis. This response is influenced by specific integrin signaling in vascular smooth muscle cells (VSMCs). Microfibrillar-associated protein 4 (MFAP4) is a matricellular integrin ligand localized to the vascular wall. We hypothesized that systemic MFAP4 (sMFAP4) associates to vascular remodeling processes and that MFAP4 enhances integrin dependent VSMC activation and vasculoproliferative disease-associated remodeling.

We explored the hypotheses using a prospective human cohort with symptomatic obstructive peripheral arterial disease (PAD), VSMC culture studies, and produced MFAP4 deficient mice for studying neointima formation.

We demonstrate that the highest tertile of sMFAP4 was significantly associated with primary patency after vascular reconstruction, and the overall need for vascular reconstruction and mortality in 343 PAD patients.

MFAP4 mediated the adhesion, migration, and proliferation of VSMCs in an integrin αVβ3/5-dependent manner in vitro. These effects were inhibited by MFAP4-blocking antibodies. MFAP4-deficient mice displayed delayed neointimal formation when challenged with carotid artery ligation, but the compensatory outward remodeling of vessel diameter was reduced.

In conclusion, sMFAP4 has the potential to serve as a systemic marker morbidity and mortality in PAD. MFAP4 regulates integrin αVβ3/5 signaling and pathological remodeling in vivo, and may have therapeutic implications in vasculoproliferative diseases.
Abstract 322: Azathioprine Reduces Endothelial Cell Activation via Inhibition of GTPase Rac1
Authors: Marinkovic Goran , Hoogenboezem Mark , van Buul Jaap , de Vries Carlie J , de Waard Vivian
Published: March 2022

Objective

Azathioprine (AZA) and its active metabolite 6-mercaptopurine (6-MP) are well established immunosuppressive drugs. Common understanding of its immunosuppressive properties is largely limited to immune cells. However, 6-MP also has a potential to effectively modulate the inflammatory phenotype of endothelial cells. Activation of endothelium is a fundamental feature in chronic inflammatory diseases. We studied the mechanism underlying the protective role of the immunosuppressive drug 6-MP in endothelial cell activation.

Methods and Results

Since it is known that Aza affects small GTPase Rac1 in T-cells, we focussed on Rac1-mediated processes in cultured endothelial cells in the presence or absence of 6-MP. Indeed, upon treatment with 6-MP, we observed a decrease of GTP-bound Rac1, confirming that there is less active Rac1. Reduced activity of Rac1 influences the downstream signaling cascade, resulting in decreased phosphorylation of c-Jun terminal N-Kinase (JNK), c-Jun and thus, reduced transcription of pro-inflammatory genes. On mRNA level, in the presence of 6-MP we confirmed that endothelial cells express less proinflammatory chemokine CCL5 (Rantes), Interleukin-12 and adhesion molecule VCAM1; genes downstream of the Rac1-mediated JNK signalling pathway. In addition, monocyte adhesion to activated endothelial cells is strongly inhibited upon 6-MP treatment, Indeed, reduced cytoskeletal rearrangement is observed in the form of actin protrusions that capture leukocytes, which is a process that relies on Rac1 activation. Specifically, a 47% reduction in number of transmigratory cups is observed, resulting in a reduction of adherent Anti-ICAM-1 Ab-coated beads (monocytes substitute).

Conclusion

These data underscore the anti-inflammatory effect of 6-MP on endothelial cells, modulating the activation response of endothelial cells in a Rac1-dependent fashion and providing us with a mechanistic insight for the development of novel therapeutic targets against chronic inflammatory diseases.
Abstract 333: Histone Deacetylase 5 Interacts With and Regulates Kruppel-like Factor 2 (KLF2) Transcriptional Activation and its Downstream Target eNOS Gene Expression
Authors: Kwon Il-Sun , Wang Weiye , Zhao Jinjing , Yang Ying , Yin Meimei , Jin Felix Q , Liu Bin , Mastrangel Michael A , Jin Zheng Gen
Published: March 2022

Vascular endothelial dysfunction and inflammation are hallmarks of atherosclerosis. It is important to understand the detailed mechanisms for regulation of endothelial function and inflammation. Emerging evidence suggests that the transcription factor Kruppel-like Factor 2 (KLF2) is a key mediator of the anti-inflammatory and anti-thrombotic properties of the endothelium. KLF2 also mediates laminar blood flow-dependent endothelial atheroprotective functions. Despite intense investigation of KLF2 function, little is known of the molecular mechanisms for regulation of KLF2 transcriptional activation. Histone deacetylase 5 (HDAC5) HDAC5 is a transcriptional repressor, and we have previously shown that HDAC5 inhibits flow-mediated endothelial nitric oxide synthase (eNOS) gene expression (Wang & Jin, Blood 2010). Here, we demonstrate that HDAC5 physically associates with KLF2 and negatively regulates KLF2 transcriptional activation. Using co-transfected cell line and GST-fusion protein pull-down assays, we found that HDAC5 directly interacts with KLF2. We also detected the association of endogenous HDAC5 and KFL2 in endothelial cells. To understand the nature of this interaction, we further mapped the HDAC5 and KLF2 interaction domains. Using luciferase reporter assay, we observed that HDAC5, through association with KLF2, repressed KLF2 transcriptional activation. Moreover, HDAC5 inhibited KLF2-dependent eNOS promoter activity and eNOS expression in endothelial cells, which reveals epigenetic mechanism whereby flow mediates eNOS expression through stimulating nuclear export of HDAC5. Collectively, these results define a novel transcriptional pathway that regulates KLF2-dependent gene expression and suggest a new molecular target to prevent vascular inflammation and endothelial dysfunction associated with cardiovascular disease.
Abstract 337: Measurements of Calf Muscle Oxygenation During Standing and Exercise in Patients with Chronic Venous Insufficiency
Authors: Yamaki Takashi , Konoeda Hisato , Osada Atsuyoshi , Hamahata Atsumori , Nozaki Motohiro , Sakurai Hiroyuki
Published: March 2022

BACKGROUND

Despite the established role of the calf muscle pump for preventing chronic venous insufficiency (CVI), hemoglobin flow in the calf muscle is poorly understood. Near-infrared spectroscopy (NIRS) provides continuous noninvasive monitoring of changes in tissue oxyhemoglobin (O2Hb) and deoxyhemoglobin (HHb) levels. The purpose of this study was to investigate the changes in calf muscle HbO2 and HHb levels during standing and exercise in patients with CVI.

METHODS

Seventy-four limbs in 73 patients with various clinical stages of CVI were enrolled. Patients were divided into early (C0-C3) and advanced (C4a-C6) according to the CEAP classification. NIRS was used to measure changes in the calf muscle HbO2 and HHb levels, and oxygenation index (HbD; HbD=O2Hb-HHb) while lying spine, standing, and then subsequently performing 10 tiptoe movements.

RESULTS

Among the 74 limbs evaluated, 47 had early and 27 had advanced CVI. Standing caused increases in both O2Hb and HHb levels. However, there were no significant differences in these increases, or HbD, between early and advanced CVI. In contrast, the time elapsed until the maximum increase in O2Hb concentration was significantly reduced in patients with advanced CVI in comparison with patients showing early CVI (55.5 ± 44.2, 32.6 ± 12.6 s, P=.025). During 10 tiptoe movements, a decrease in O2Hb concentration was observed, and there was no significant difference in the reduction of O2Hb values between early and advanced CVI. In contrast, 10 tiptoe movements produced venous emptying (HHbE) and subsequent retention (HHbR), and the HHbR was significantly increased in patients with advanced CVI compared with those with early CVI (6.0 ± 7.0, 9.0 ± 6.2 μmol/L, P=0.021). Furthermore, HbD falls were more pronounced in patients with advanced CVI (7.4 ± 11.5, -5.9 ± 15.7 μmol/L, P=0.002).

CONCLUSIONS

Changes in O2Hb and HHb concentrations differ between early and advanced CVI during standing and exercise. Detailed investigation of the interrelationship between O2Hb and HHb during calf muscle pump function would lead to a better understanding of the various clinical stages of CVI.
Abstract 423: A Combination of Metformin, Quercetin, and Curcumin Restores HDL Function and Improves Atherosclerosis Burden in LDLr-/-/ob.ob leptin-/- and LDLr-/- Mice by attenuating Insulin Resistance, Hyperglycemia, and Low-grade Inflammation
Authors: Srivastava Rai Ajit K , Ali Wahid , Cefalù Angelo B , Noto Davide , Averna Maurizio R
Published: March 2022

Cardiovascular disease risk in diabetics is 2-4 fold greater compared to non-diabetics. While dyslipidemia poorly explains excess risk in diabetics, glucose intolerance leads to increased macrophage infiltration, arterial stiffness, and modifies vascular tone. Enhanced lipid peroxidation in diabetes mellitus contributes to increased formation of free radicals, leading to accelerated glycation and oxidative stress, which causes HDL modification. The objective of the present study was to: a) investigate HDL function and atherosclerosis burden in animal model of diabetes, hyperlipidemia, and low-grade inflammation; and b) evaluate regression of preformed lesions following treatment with a proprietary formulation (COMB) of metformin (MET) with anti-oxidant and anti-inflammatory agents, quercetin and curcumin. We used LDLr-/-/leptin-/-ob/ob (DKO) and LDLr-/- (SKO) mice fed Western Diet for 10 weeks to develop lesion formation followed by treatment with MET (150 mpk) or COMB for 6 weeks (bid). Lipid profile, lesion formation, and OGTT were measured. We also measured HDL functionality by ABCA1-dependent cholesterol efflux in the serum, and in a separate cohort, cholesterol efflux using 3H-cholesterol-loaded J774 cells. Atherosclerotic lesion formation in DKO was greater (27%) compared to SKO. Impaired glucose tolerance, insulin resistance, and proinflammatory index was found to be associated with HDL functionality and atherosclerosis burden. Treatment with COMB attenuated hyperglycemia, insulin resistance and inflammation, and restored HDL function and promoted lesion regression (-24% in DKO, and -15% in SKO). COMB showed greater efficacy compared to MET, demonstrating the benefit of controlling glycemia and inflammation to restore HDL function and enhance lesion regression. Preliminary results in diabetics (n=10) and age and sex matched non-diabetic individuals (n=10) showed impairment in ABCA1-mediated cholesterol efflux in the serum of diabetics (-18% vs control). In summary, these data suggest that HDL dysfunction in diabetes mellitus is one of the risk factors of atherosclerotic lesion formation, and attenuation of hyperglycemia, oxidative stress, and inflammation may improve HDL function and promote lesion regression.
Abstract 425: Hydrogen Peroxide and 13-hydroperoxylinoleic Acid Induce the Expression of Insulin-like Growth Factor-binding Protein 3 in HepG2 Cells
Authors: Zhao Dong , Aluganti Narasimhulu Chandrakala , Selvarajan Krithika , Litvinov Dmitry , Parthasarathy Sampath
Published: March 2022

Background

Patients with type 2 diabetes are reported to have increased oxidative stress. Oxidized LDL is reported to suppress the expression of insulin-like growth factor I receptor (IGF1R). IGF1 emerges as a potential therapeutic target for insulin resistance and deficiency. The functions of IGF1 are regulated by its receptors as well as binding proteins (IGFBPs). IGFBP3 is known as the main binding protein for IGF1, and shows inhibitory effects on IGF1 through binding to extracellular matrix. The present study investigates whether oxidative stress regulates IGF binding proteins.

Methods

HepG2 (hepatocellular carcinoma) cells were treated with either hydrogen peroxide (H2O2) or 13-hydroperoxylinoleic acid (HPODE) with/without catalase in serum free medium for 24 hours, and total RNA and protein were isolated for quantitative PCR or Western blot analyses.

Results

H2O2 of variant concentrations induced IGFBP3 expression, which was abolished by the addition of catalase. HPODE also induced IGFBP3 expression in a dose-dependent manner. The induction was found for both isoforms A and B of IGFBP3.

Conclusion

Our findings shed light on the potential synergetic regulation of IGF1 by oxidative stress through both the receptor and binding protein, and advance the understanding of the IGF1 resistance in type 2 diabetes. Furthermore, antioxidants are indicated to be able to increase the IGF1 sensitivity, and reduce the dosage and side effects of IGF1 administration for patients with diabetes.
Abstract 444: The Liver-Specific Role of Sortilin in VLDL Secretion
Authors: Patel Kevin M , Strong Alanna , Rader Daniel J
Published: March 2022

Introduction

Sortilin, the protein product of the SORT1 gene, is a multi-ligand sorting receptor involved in Golgi to lysosome trafficking that is expressed in multiple tissues. Genome-wide association studies have shown an association between elevated SORT1 expression in human liver and a reduction in low-density lipoprotein cholesterol (LDL-C) and in myocardial infarction (MI) risk. Our lab has previously shown liver specific sortilin overexpression in mice reduces LDL by facilitating LDL plasma clearance and by promoting the lysosomal degradation of very-low density lipoprotein (VLDL). Interestingly, we and others have also shown that total body Sort1 deficiency in mice is associated with a paradoxical reduction in VLDL secretion (48%, P = 0.004). One major difference is that the overexpression studies were hepatic specific whereas the loss-of-function studies were in total body knockout (KO) mice.

Methods/Results

We used liver-specific Sort1 conditional KO mice and reconstitution of hepatic Sort1 in total body KO mice to further probe the specific role of hepatic sortilin in VLDL secretion. Sort1 fl/fl mice were injected with a control adeno-associated virus (AAV) or an AAV expressing Cre recombinase driven by a liver specific promoter and in vivo VLDL secretion studies were performed. Liver-specific Sort1 deletion reduced VLDL secretion by 51% (P = 0.005). We also used AAV to test the hypothesis that sortilin serves as a chaperone to facilitate VLDL secretion. Sort1 reconstitution in total body Sort1 KO mice reduced VLDL secretion by 29% (P = 0.002). Interestingly, expression of a sortilin mutant that cannot traffic to the lysosome but does traffic to the plasma membrane increased VLDL secretion by 88% (P = 0.001).

Conclusion

These findings suggest that liver-specificity is not responsible for the paradoxical reduction in secretion seen in the total body Sort1 KO mouse, which is reproduced in the hepatic-specific sortilin KO mouse. The differential effects of wild-type and lysosomal defective sortilin on VLDL secretion and are consistent with a model in which sortilin serves a dual function in VLDL trafficking, with low levels of sortilin facilitating VLDL export and higher levels of sortilin promoting pre-secretory lysosomal VLDL degradation.
Abstract 449: Expression of Type IIA Secretory Phospholipase A
2
(sPLA
2
) Inhibits Cholesteryl Ester Transfer Protein (CETP) Activity in Transgenic Mice
Authors: Hurt-Camejo Eva , Gautier Thomas , Rosengren Birgitta , Behrendt Margareta , Grass David S , Rader Daniel J , Tietge Uwe J
Published: March 2022

High circulating levels of group IIA secretory phospholipase (sPLA
2
-IIA) activity as well as mass are independent cardiovascular risk factors. Therefore, inhibition of sPLA
2
-IIA may be a target for the treatment of atherosclerotic cardiovascular disease (CVD). The present study evaluated the effects of sPLA
2
-IIA inhibition with varespladib acid in a novel mouse model, human apolipoprotein B/human cholesteryl ester transfer protein (CETP)/human sPLA
2
-IIA triple transgenic mice (TTT) fed a Western-type diet. sPLA
2
-IIA expression increased atherosclerotic lesion formation in TTT compared with human apolipoprotein B/human CETP double transgenic mice (p<0.01). varespladib acid effectively inhibited plasma sPLA
2
-IIA activity. Surprisingly, however, administration of varespladib acid to TTT had no impact on atherosclerosis, which could be attributed to a pro-atherogenic plasma lipoprotein profile that appears in response to sPLA
2
-IIA inhibition due to increased plasma CETP activity. In the TTT model sPLA
2
-IIA decreased CETP activity by reducing the acceptor properties of sPLA
2
-IIA-modified VLDL specifically due to a significantly lower apoE content. Increasing VLDL apoE content by means of adenovirus-mediated gene transfer in sPLA
2
-IIA transgenic mice restored the acceptor properties for CETP. In conclusion, these data show that in a humanized triple transgenic mouse model with hypercholesterolemia, sPLA
2
-IIA inhibition increases CETP activity via increasing the VLDL apoE content resulting in a pro-atherogenic lipoprotein profile.
Abstract 496: Large Pulmonary Embolism Treated with a Novel Percuatneous Catheter-Directed, Ultrasound-Assisted Low Dose Thrombolysis
Authors: Bhatheja Rohit , Ranasinghe Indika
Published: March 2022

Background

Massive Pulmonary embolism (PE) is a life threatening diagnosis. Survival is dependent on the timing and availability of intravenous alteplase. In those who survive; recurrent PE, pulmonary hypertension and cor pulmonale are the long term sequelae.

Case

We describe a 63 year old morbidly obese male (weight 470 lbs) presenting with increasing dyspnea. A CT scan of the chest revealed massive clots in bilateral pulmonary arteries and right ventricular (RV) strain.

Approximately 6 months prior to presentation he was diagnosed to have PE and was treated with warfarin. Warfarin was discontinued after 6 months as he was asymptomatic and CT scan showed resolution of clots. In the past few weeks he started to smoke and also complained of black stools.

Due to high risk presentation, history of melena in the recent past and dyspnea at rest, he was offered percutaneous therapy for PE. Using single internal jugular venous approach, right heart catherterization and pulmonary angiography was performed. Bilaterally ultrasound assisted- thrombolysis infusion catheters were placed in the pulmonary arteries. Alteplase was infused at low dose; total dose infused was 24 mg over 19 hours (Typical intravenous dose is 100 mg). Baseline hemodynamics showed severe pulmonary hypertension. Follow up angiogram showed almost complete resolution of clots and complete improvement in symptoms. Work up revealed Anti-Thrombin III deficiency. He was treated bivalirudin and discharged on warfarin. Follow-up testing showed improved RV pressures.

Discussion

The mortality rate for acute PE is 2-6% in those with hemodynamic stability, and 30% or more in those with shock or unstable hemodynamics. Catheter-directed local thrombolysis should be considered as a therapy in those with sub-massive and massive PE with the advantage of significantly lower thrombolytic dose (almost 75% lower) with minimal bleeding risk. The ultrasound energy in this thrombolytic EKOS
@
catheter helps loosen the clot via mechaincal energy, allowing lower dose of thrombolytics to be used locally. Ongoing studies utilizing this technology such as SEATTLE-II and ULTIMA will

offer further evidence based guidelines to manage acute pulmonary embolism.
Abstract 535: Shear Stress Upregulates Placental Growth Factor in an in vitro Model of the Vessel Wall in a Reactive Oxygen Species Dependent Manner
Authors: Rashdan Nabil A , Lloyd Pamela G
Published: March 2022

Arteriogenesis is the process by which mature arteries form from collateral arterioles after upstream arterial stenosis or occlusion. During collateral remodeling, monocytes are recruited to the vessel wall. Placental growth factor (PLGF) is a potent stimulator of arteriogenesis via monocyte recruitment. Although fluid shear stress is thought to be the primary signal for arteriogenesis, its role in regulating PLGF expression is unknown. However, PLGF is increased in collaterals by upstream arterial ligation. Therefore, we hypothesized that PLGF expression is regulated by shear stress. To test this hypothesis, we created an in vitro model of coronary vessels, consisting of human coronary artery endothelial cells (HCAEC) and human coronary artery smooth muscle cells (HCASMC) cocultured on porous Transwell inserts. HCAEC were then exposed to normal (low) shear stress (LSS) (time average 0.7 dyne/cm2), high shear stress (HSS) (time average 12.4 dyne/cm2), or no shear (static) for 2 hours. HSS significantly increased secreted PLGF protein compared to LSS (~25%, n= 5, p < 0.05) as determined by ELISA. Knockdown of PLGF by siRNA in HCAEC significantly reduced secreted PLGF protein while knockdown in HCASMC had no significant effect, suggesting HCAEC are the primary source of PLGF protein (n=4, p<0.01). Both H2O2 and nitrite (an index of NO production) were significantly increased by shear stress compared to static control (~35%, n= 5, p < 0.05). Treatment with catalase (500 U/mL) attenuated the effects of HSS on PLGF protein levels, whereas L-NAME (100 μM) had no significant effect. Treatment with 5 μM diphenyleneiodonium mimicked the effects of catalase on PLGF protein levels, implicating NADPH oxidase (Nox) as the source of H2O2. PCR analysis revealed Nox-4 as the predominant isoform present in both cell types. Relative Nox-4 mRNA was increased by HSS in HCAEC compared to both LSS and control. Relative gene expression of heme oxygenase-1 (HO-1), which is regulated by Nox-4, was also increased in both cell types by HSS, suggesting the possibility that HO-1 may contribute to the regulation of PLGF expression. These data demonstrate that redox signaling plays a role in the mechanotransduction of shear stress into a regulatory signal for PLGF expression.
Abstract 549: Adiponectin Modulation of Macrophage Inflammatory and Metabolic Properties is Regulated by Macrophage Polarization Status and Adiponectin Receptor Expression
Authors: van Stijn Caroline M , Kim Jason , Tangirala Rajendra K
Published: March 2022

Adiponectin, an adipose-derived adipokine, exerts metabolic, anti-inflammatory and anti-atherogenic effects to ameliorate metabolic syndrome, diabetes and cardiovascular disease. Monocytes/macrophages play a crucial pathogenic role in these inflammatory and metabolic disorders. We identified that macrophage polarization profoundly alters their adiponectin receptor expression and functional responses to adiponectin. We addressed the hypothesis that macrophage activation status which affects adiponectin receptor expression leads to differential adiponectin-mediated inflammatory and metabolic response. Mouse bone marrow macrophages, differentiated into either classically-activated (IFN-γ and LPS) macrophages (M1) or alternatively-activated (IL-10) macrophages (M2), were treated with adiponectin. Our studies revealed that M1 activation substantially suppressed while M2 activation had only a modest effect on the macrophage adiponectin receptor expression. The macrophage activation status also produced contrasting adiponectin effects. Adiponectin significantly increased TNF-α expression in M1 macrophages but had no effect M2 macrophages. In contrast, adiponectin significantly increased anti-inflammatory cytokine, IL-10 in M2 macrophages but had no effect in M1 macrophages. We investigated the adiponectin effect on genes regulating macrophage cholesterol efflux, fatty acid and cholesterol metabolism. Interestingly, adiponectin increased CD36 expression in M1 macrophages but not in M2 macrophages. Similarly, adiponectin increased cholesterol efflux receptor, ABCA1 only in M1 macrophages with no effect in M2 macrophages. Measurement of metabolic gene expression revealed that adiponectin induced HMGCoA reductase levels in M1 macrophages and this effect was absent in M2 macrophages. FAS expression was unaffected by adiponectin in M1 or M2 macrophages. In conclusion, these studies provide important evidence that adiponectin modulation of macrophage inflammatory and atherogenic properties is differentially controlled by macrophage activation status which profoundly affects their pathogenic role in inflammatory and metabolic cardiovascular disease.
Abstract 59: Platelets Have a Central Role in T Helper Cell Development and Responses
Authors: Morrell Craig , Shi Guanfang , Field David
Published: March 2022

Platelets are the primary cellular mediator of thrombosis, but platelets also have important roles in immune responses. Immune roles for platelets have been primarily explored in the context of endothelial cell interactions and innate immune responses, but our work now demonstrates an important and novel role for platelets in T cell development. Using the mouse abdominal cardiac transplantation model we have discovered that platelets have a central role in CD4+ T helper cell development and differentiation. Platelet deficient thrombopoietin receptor (TPOR/c-MPL) knockout mice given cardiac transplants have an accelerated rejection response driven by Th17 type of CD4+ T cell activation and infiltration of transplant tissue. These mice also have a basal skewing to Th17 development in the absence of transplantation. Platelets are the primary source of platelet factor 4 (PF4/CXCL4) and PF4-/- mice exhibit a very similar phenotype of unrestrained Th17 T cell development and responses post transplantation as the TPOR-/- mice. Furthermore, PF4 directly limits in vitro Th17 cell differentiation, demonstrating that platelets via PF4 have a major prior unrecognized role in T helper cell differentiation.

Taken together, these data demonstrate a highly novel platelet function in T helper cell development. PF4 has a direct effect on maintaining T cell homeostasis and differentiation which is highly relevant to transplant immune responses and chronic rejection.
Abstract 64: Interleukin-10 Decreases HDL: A Novel Cause of Disappearing HDL Syndrome
Authors: Freeman Lita A , Moraitis Andreas G , Shamburek Robert , Wilson Wyndham , Grant Cliona M , Rao Koneti , Price Susan , Hornung Ronald L , Fleisher Thomas , Pucino Frank , Illei Gabor G , Remaley Alan T
Published: March 2022

Introduction

“Disappearing HDL Syndrome” refers to cases of acquired severe HDL deficiency in non-critically ill patients. Except for rare idiosyncratic drug reactions, the physiological basis of Disappearing HDL Syndrome is not understood.

Hypothesis

“Disappearing HDL” in patients with B-cell disorders may be due to increased levels of cytokines, particularly IL-10, in plasma.

Methods

We investigated cytokine levels in a patient with intravascular large B-cell lymphoma (IVLBCL) who presented with undetectable or very low HDL-C, low LDL-C and markedly elevated triglycerides. IL-10 levels were investigated in additional patients with B-cell disorders (IVLBCL, large B-cell lymphoma, and ALPS) who presented with similar lipid profiles. A direct causal role for IL-10 in modulating HDL-C was investigated with a randomized, placebo-controlled study of recombinant IL-10 infusion into psoriatic arthritis patients.

Results

All cases (IVLBCL, large B-cell lymphoma and ALPS) had highly elevated serum IL-10 levels that normalized after chemotherapy concomitant with normalization of HDL-C, LDL-C and TG. In a survey of 93 ALPS patients, IL-10 showed a strong inverse correlation with HDL-C (R
2
= 0.3720, p<0.0001). In a randomized, placebo-controlled study of recombinant IL-10 infusion into psoriatic arthritis patients HDL-C precipitously decreased to near-undetectable levels shortly after IL-10 injection. Furthermore, IL-10 treatment also decreased LDL-C by 51% or more during IL-10 administration (p < 0.005) and increased TG by up to 2-fold.

Conclusions

Our study has identified increased IL10 as a potential cause of Disappearing HDL Syndrome and demonstrated that in addition to HDL-C, IL-10 can also modulated levels of LDL-C and TG, thus identifying a new link between inflammation and lipid metabolism.
Abstract 71: G protein-dependent Constitutive and Evoked Endothelial Cell vWF secretion
Authors: Rusu Luiza , Andreeva Alexandra , Meigs Thomas E , Visintine David J , Kim Kyong , Stojanovic-Terpo Aleksandra , Liu Guoquan , Cho Jaeyong , Du Xiaoping , Minshall Richard D
Published: March 2022

A poorly understood mechanism and yet critically important requirement of hemostasis and thrombosis is von Willebrand Factor (vWF) secretion by endothelial cells (ECs). In the present study, we used genetic approaches in vitro and in vivo to study the role of G proteins, RhoA, and αSNAP in basal and evoked EC-specific vWF secretion. As expected, αSNAP siRNA completely abolished constitutive, Ca2+-induced, as well as thrombin-induced vWF secretion from human umbilical vein endothelial cells (HUVEC), indicating vWF secretion is dependent on fusion and exocytosis of Weibel-Palade Bodies (WPBs). In addition, siRNA-mediated depletion of guanine-nucleotide exchange protein 12 α subunit (GNA12) inhibited both constitutive and thrombin-induced vWF secretion whereas there was no effect of GNA13 siRNA. In addition, exogenous expression of constitutively active GNA12 mutant promoted vWF secretion. In GNAq-, p115 RhoGEF-, and RhoA siRNA-depleted HUVECs, thrombin-induced vWF secretion was reduced by 40%, whereas basal vWF secretion was not affected. Based on these data, we hypothesized that GNA12 and GNAq deficiency, by affecting constitutive and Ca2+-dependent vWF secretion, would cause defects in hemostasis and thrombosis in a mouse model of vascular injury. In GNAq/11-/- mice, plasma levels of vWF were normal although clotting-time was prolonged, whereas GNA12-/- mice showed increased tail snip-clotting time, reduced laser-injury clot formation, and reduced vWF levels in blood as well as buffered-perfused mouse lungs. Importanly, purified human vWF rescued tail snip clot formation in GNA12-/- mice. In vitro binding assays revealed GNA12 N-terminal domain aa 10-15 was sufficient for binding to αSNAP, and when expressed in endothelial cells, αSNAP binding domain minigene-peptide blocked thrombin-induced vWF secretion. Taken together, these studies uncover obligitory and complementary roles of GNA12 and GNAq/11 in constitutive and evoked vWF secretion, thrombosis, and hemostasis.
Abstract 139: Pharmacologic Modifiers of Epigenetic Histone Methylation Attenuate Smooth Muscle Cell Proliferation, Inflammatory Activation and Dedifferentiation
Authors: Kahles Florian , Marx Judith , Makowska Anna , Lehrke Michael , Marx Nikolaus , Findeisen Hannes M
Published: March 2022

Introduction:
Activation and dedifferentiation of smooth muscle cells (SMCs) at sites of vascular injury is regulated by epigenetic mechanisms. Epigenetic histone methylation has been recognized as a dynamic mark controlling many biological processes in health and disease.

We assesed the hypothesis, that specific inhibitors of histone methyltransferases and demethylases effect SMC proliferation, inflammation and dedifferentiation.

Methods:
We studied the effects of the following epigenetic modifiers on smooth muscle cell proliferation, MCP-1 expression and TNFα-induced dedifferentiation: chaetocin, 3-deazaneplanocin (3-DZN), AMI-1, UNC0638, 2,4-pyridinedicarboxylic acid, methylstat, daminozide and GSKJ-4.

Results:
SMC proliferation was inhibited by treatment with 3-DZN, AMI-1, methylstat, chaetocin and GSK-J. TNFα-induced down regulation of SM22α expression as a marker of dedifferentiation was attenuated by treatment with UNC0638 and GSK-J4. TNFα-induced MCP-1 expression was particularly sensitive to inhibition of the histone methylation machinery, as all tested compounds with the exception of methylstat and UNC0638 caused a significant reduction of MCP-1 expression. UNC0638, chaetocin and 2,4-PDCA could attenuate TNFα-induced down regulation of SM22α expression up to 72h after the initial treatment. The sustained effect of the specific G9a histone methyltransferase inhibitor UNC0638 was associated with a strong and sustained reduction of histone h3 lysine 9 dimethylation at the SM22α promoter after 24 and 72h.

Conclusion:
Our data hint at the potential of modulating specific histone methylation marks in SMCs to target SMC activation and phenotypes in vascular diseases and warrant further research to dissect histone methylation dependent mechanisms in SMCs.
Abstract 157: Association Between Monocyte Count and Coronary Calcification in Patients Without a History of Coronary Artery Disease
Authors: Chaikriangkrai Kongkiat , Adigun Rosalyn , Khaleel bala Sayf , Alchalabi Sama , Botero Sharleen , Chang Su Min
Published: March 2022

Introduction:
This study aimed to investigate associations between monocyte count derived from complete blood count and coronary artery calcium score (CACS) in patients without history of coronary artery disease (CAD).

Methods:
In this cross-sectional study, 831 consecutive patients without history of CAD who presented with chest pain and underwent coronary artery calcium scoring by MDCT were included. Patients with any clinical signs of infection were excluded. High monocyte count was defined as absolute monocyte count of > 950/uL. Presence of coronary calcification and moderate-to-severe coronary calcification were determined when CACS>0 and CACS>100 respectively.

Results:
The cohort comprised of 60% men with mean age of 59±14 years. Median Framingham’s 10-year risk for cardiovascular disease was 4% (Interquartile range; IQR 1%-16%). Sixty percent of patients had zero CACS followed by 21.5% with CACS 1-100, 9.9% with CACS 101-400 and 8.1% with CACS>400. Mean ± SD absolute monocyte count was 588±379 /uL. There was no statistically significant difference in rate of CACS>0 between high monocyte count group and normal monocyte count group (p 0.83). In patients with non-zero CACS (N=328), those with absolute monocyte count > 90 percentile had higher rate of CACS>100 than those in the bottom 10 percentile (55.9% VS 30%; p 0.037) as shown in the left figure. Those with high monocyte count had higher rate of CACS>100 than those with normal monocyte count (68.4% VS 44%; p = 0.038) as shown in the right figure. In multivariate regression analysis, high monocyte count was associated with CACS>100 (HR 2.84; 95% confidence interval 1.05, 7.73; p = 0.040).

Conclusion:
In patients without history of CAD, high monocyte count was not associated with presence of coronary calcification. However, high monocyte count is independently associated with more severe calcification in those with coronary calcification.
Abstract 17: Cyclooxygenase-2 Deletion Favors Deep Vein Thrombosis in Mice
Authors: Barbieri Silvia S , Tarantino Eva , Amadio Patrizia , Gianellini Sara , Mussoni Luciana , Tremoli Elena
Published: March 2022

Deep vein thrombosis (DVT) is a common condition that often leads to pulmonary thromboembolism and death. Cyclooxygenase (COX)-1 and -2 catalyze the formation of prostaglandins and thromboxane (TX)A2 from arachidonic acid and play a critical role in the thrombotic events, however, the impact of COX-2 deletion (COX-2KO) in the DVT remains unclear. In this study, traditional (thromboelastomethry, mass weight and histology) and innovative (venous ultrasonography) approaches were used to assess the effect of COX-2 ablation on DVT in mice. In particular, ligation of the inferior vena cava to induce thrombus formation in COX-2KO and WT mice was carried out.

Thromboelastometry analyses performed in whole blood of COX-2KO revealed alterations of clotting factors, and of platelet activity, with consequent greater clot firmness and clot elasticity. COX-2KO mice have increased plasma levels of functional fibrinogen, factor VIII and tissue factor (TF), and higher TXA2 serum than WTs. Finally, PAI-1 activity was the highest in COX-2KO, whereas tPA activity was similar in the two groups. The propensity to thrombosis of COX-2KO mice was also confirmed by ultrasonography. Genetic deletion of COX-2 was associated with bigger thrombi already at early time point. At 48 hours COX-2KO thrombi showed a greater content of connective tissue and fibrin, and increased number of infiltrated monocytes compared to WT. Remarkably, Annexin A2 (ANXA2), a fibrinolytic receptor, was significantly increased in cells present in COX-2KO thrombi. In addition, peritoneal macrophages from COX-2KO mice showed increased expression of ANXA2 with different distribution at cellular level compared to WTs. In particular, ANXA2 was expressed in the cytoplasm and in the nucleus of COX-2KO macrophages, whereas in control cells the protein was found mainly localized on cell membrane/periphery.

In conclusion, the increased basal activation of haemostatic system observed in COX-2KO mice may partly explain the predisposition of these mice to thrombosis. In addition, the different localization of ANXA2 in monocytes from COX-2KO mice with respect to wild type mice suggests that this protein may display additional protective, as yet unknown, mechanisms influencing the stability of venous thrombi.
Abstract 177: A Novel Vascular "Ring" Assay for Smooth Muscle Contractility Using Magnetic 3-Dimensional Bioprinting
Authors: Tseng Hubert , Gage Jacob A , Haisler William L , Neeley Shane K , Killian T C , Barthlow Herbert G , Wagoner Matthew , Souza Glauco R
Published: March 2022

The current standard for studying pharmacodynamics on vascular smooth muscle contractility is wire myography with ex vivo tissue, which suffers from high cost low throughput, limiting its widespread use. Yet, in vitro alternatives are insufficient, as they are commonly two-dimensional (2D) monolayers on rigid surfaces unlike the soft vasculature. Thus, a rapid, robust in vitro assay is needed that mimics the native three-dimensional (3D) environment, while yielding high data throughput.

Towards that goal, we introduce an 3D in vitro assay in which cellularized vascular "rings" are rapidly and magnetically bioprinted. In the assay, cells are incubated with magnetic nanoparticles to render them magnetic, then printed into 3D rings (1.5 x 10^5 cells/ring) in 96-well plates. The rings will then contract or dilate over a short amount of time (<5 h) in a dose-dependent manner visually similar to vessel contraction and dilation (Fig. 1A). This measurement is label-free, meaning that the rings can be experimented on afterwards to explore pharmacodynamics. Moreover, this assay is captured using an iPod-based imaging system programmed to take images of whole plates (Fig. 1B) instead of imaging individual wells under a microscope.

This assay was validated for the evaluation of drug efficacy by measuring contractility in 3D bioprinted rings of A10 rat vascular smooth muscle cells. The compounds tested were either vasodilators (blebbistatin, forskolin, verapamil) or vasoconstrictors (norepinephrine, phenylephrine, U46619). Ring contraction was compared to the presence of phosphorylated myosin light chain-2 which is related to smooth muscle concentration. Furthermore, the gene expression profiles of rings exposed to specific compounds are compared to negative controls. The result of this study is the validation of the vascular assay that rapidly prints 3D environments similar to the native blood vessel, and efficiently measures smooth muscle contraction.
Abstract 23: EphB2 Mediated Contact-Dependent and Independent Signaling in the Regulation of Thrombosis and Hemostasis
Authors: Vaiyapuri Sakthivel , Sage Tanya , Ali Marfoua S , Rana Rekha , Unsworth Amanda J , Moraes Leonardo A , Gibbins Jonathan M
Published: March 2022

Introduction:
Eph/ephrin signalling plays important roles in the development of central nervous system and vasculature. The presence of EphA4, EphB1 and ephrinB1 in platelets was reported previously where forced clustering of EphA4 or ephrinB1 resulted in cytoskeletal rearrangements, fibrinogen binding and granule secretion. Further studies have emphasized the role of EphA4 in regulation of integrin αIIbβ3 mediated outside-in signalling. We have recently reported the presence of EphB2 in platelets and established its involvement in regulation of platelet function.

Hypothesis and Methods:
We assessed the hypothesis that Eph/ephrin signalling is mainly mediated through the cytoplasmic domains [including a kinase domain, sterile alpha motif and PDZ motif] of Eph kinases using a mouse model in which the intracellular region of EphB2 (EphB2LacZ) was replaced with β-galactosidase. Use of this mouse model enabled us to explore the importance of EphB2 cytoplasmic signalling in regulating platelet function without interfering with its ligand binding.

Results and Conclusions:
The absence of EphB2 cytoplasmic tail resulted in reduced agonist-induced platelet activation, fibrinogen binding, granule secretion and thrombus formation. Integrin αIIbβ3 mediated outside-in signalling was also reduced and associated with a diminished level of myosin binding to integrin β3 tail in EphB2LacZ mouse platelets. Moreover, a reduced level of calcium mobilisation and phosphorylation of protein kinase B and phospholipase Cγ2 in EphB2LacZ platelets suggest a potential role in the regulation of phosphoinositide-3 kinase signalling. Reduced levels of fibrinogen binding, granule secretion and platelet spreading observed on individual EphB2LacZ platelets suggest a role for EphB2 signalling without the need for cell-cell contact. Conversely, diminished platelet aggregation, clot retraction and thrombus formation in EphB2LacZ platelets point towards the importance of EphB2 mediated intracellular signalling in a contact-dependent manner when platelet-platelet contacts occur. In conclusion, this study implicates the EphB2 cytoplasmic region in mediating contact-dependent and independent signalling in platelets.
Abstract 270: Reduction of Mitochondrial Antioxidant Levels Augments Osteogenic Signaling Independent of Changes in Vasomotor Function in Aorta from Hypercholesterolemic Mice
Authors: Roos Carolyn M , Hagler Michael A , Zhang Bin , Arghami Arman , Miller Jordan D
Published: March 2022

Mitochondrial antioxidant genes are major regulators of oxidative stress in the vasculature. Previous data has shown that reducing antioxidant capacity accelerates atherosclerotic plaque size, however, it is unclear whether reducing mitochondrial antioxidant capacity alters plaque composition. Therefore, we tested the hypothesis that reductions of mitochondrial antioxidant capacity will increase osteogenic markers and intimal plaque calcification in aorta from hypercholesterolemic mice. We used

Ldlr
-/-
ApoB
100/100

(LA) mice that were either wild-type (LA-MnSOD
+/+
) or heterozygous (LA-MnSOD
+/-
) for manganese superoxide dismutase that were fed a Western Diet (TD88137) for 3 or 6 months. Changes in changes gene expression were assessed in aortic arch using qRT-PCR, plaque calcium levels were examined using histological evaluation from aortic cryosections, and endothelial function was measured using isolated organ bath chambers (
ex vivo
). While expression of SP7 (an osteogenic transcription factor) was unchanged between LA-MnSOD
+/+
(1.0±0.6) and LA-MnSOD
+/-
(0.95±0.3) at 3 months, SP7 levels were dramatically increased in LA-MnSOD
+/+
(8.0±2.8) at 6 months and increased further in LA-MnSOD
+/+
mice (17.5±5.9; p<0.05). Alizarin Red staining for calcium showed increased calcium burden in plaques of 6 month LA-MnSOD
+/-
compared to LA-MnSOD
+/+
(3636±669 pixels versus 649±206 pixels; p<0.05). Vasomotor function in aorta was unchanged between LA-MnSOD
+/+
and LA-MnSOD
+/-
mice, suggesting that the transcriptional and functional changes related to calcification in LA-MnSOD
+/-
mice occur independently of nitric oxide signaling. In conclusion, our data strongly support our working hypothesis that reductions in mitochondrial antioxidant capacity significantly accelerate osteogenic calcification in aorta from hypercholesterolemic mice, and suggest that targeting mitochondrial oxidative stress may be a viable therapeutic strategy to slow vascular calcification.