bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 153: NKT Cells Aggravate the Development of Abdominal Aortic Aneurysms
Authors: van Puijvelde Gijs H , Foks Amanda C , Bot Ilze , Habets Kim L , de Jager Saskia C , ter Borg Mariette N , van Berkel Theo J , Quax Paul , Kuiper Johan
Published: March 2022

Background:
An abdominal aortic aneurysm (AAA) is a dilatation of the abdominal aorta mainly affecting men aged 60 years or older. Development of AAA is associated with an accumulation of inflammatory cells in the lesions such as NKT cells. The goal of this study was to investigate the exact contribution of these cells in angiotensin II (AngII) induced AAA formation in LDLr
-/-
mice.

Methods & Results:
To investigate the role of NKT cells in AAA formation, LDLr
-/-
and LDLr
-/-
CD1d
-/-
(NKT cell deficient) mice were fed a Western type diet for one week prior to infusion with AngII using osmotic minipumps. During the experiment, 5 out of 12 LDLr
-/-
mice died due to a rupture of the aorta while 0 out of 11 LDLr
-/-
CD1d
-/-
mice died. Four weeks after placement of minipumps, the mice were sacrificed and a clear significant difference in AAA-severity was observed. In 7 out of 11 LDLr
-/-
CD1d
-/-
mice no AAA lesions were found compared with only 1 out of 12 LDLr
-/-
mice. Furthermore, ex vivo studies show that glycolipid-induced NKT cell activation upregulates the expression of MMP-12, cathepsin-S and -L on macrophages and MMP-12, cathepsin-S and -K on vascular smooth muscle cells (vSMCs) and in addition a reduction in vSMC viability is induced after activation of NKT cells.

Conclusions:
These data show that NKT cells aggravate the development of AngII-induced AAA by inducing degeneration of the extracellular matrix of the vessel wall. These results provide new opportunities to intervene in the development of aneurysms.
Abstract 158: Red Blood Cell Indices 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 of red cell distribution width (RDW), RBC mean corpuscular volume (MCV), mean corpuscular hemoglobin (MCH) and mean corpuscular hemoglobin concentration (MCHC) with coronary artery calcium score (CACS) in patients without history of coronary artery disease (CAD).

Methods:
In this cross-sectional study, 832 consecutive patients without history of CAD who presented with acute chest pain and underwent coronary artery calcium scoring by MDCT were included. Differences in CACS among multiple RBC indices categories which were high RDW (>55 fL) VS low-to-normal RDW, low MCV (<80 fL) VS normal-to-high MCV, low MCH (<27 pg) VS normal-to-high MCH, low MCHC (<31 g/dL) VS normal-to-high MCHC were statistically calculated.

Results:
The cohort comprised of 60% men (500 of 832) 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 of the RBC indices were 43±14 fL for RDW, 88±6 fL for MCV, 30±2 pg for MCH and 34±2 g/dL for MCHC. Compared to patients with normal-to-high MCV, those with low MCV (n=73) had significant lower CACS (0; IQR 0-5 VS 0; IQR 0-49; p 0.047). There was no statistically significant difference in CACS between RDW groups (p 0.45), MCH groups (p 0.19), MCHC groups (p 0.26) as shown in the figure. Multivariate analysis showed no statistically significant association of all the RBC indices with either CACS>0 (high RDW - p 0.83, low MCV - p 0.17, low MCH - p 0.26, low MCHC − p 0.06) or CACS>100 (high RDW - p 0.69, low MCV - p 0.18, low MCH - p 0.93, low MCHC − p 0.77).

Conclusion:
Our study did not show significant association of RDW, MCV, MCH and MCHC with either presence or severity of coronary calcification in patients without history of CAD
Abstract 162: Everolimus Prevents Vascular Smooth Muscle Cell Proliferation by Repressing Cell Cycle-Dependent Telomerase Activation
Authors: Aono Jun , Heywood Elizabeth B , Qing Hua , Jones Karrie L , Bruemmer Dennis
Published: March 2022

INTRODUCTION:
Everolimus is a potent antiproliferative agent that prevents growth factor-stimulated proliferation of vascular smooth muscle cells and is frequently used as platform for drug-eluting coronary stents to prevent restenosis after coronary artery angioplasty. We have previously demonstrated that telomerase plays a crucial role in vascular smooth muscle cell proliferation. However, whether telomerase is involved in the anti-proliferative activity of everolimus and the putative molecular mechanisms underlying this activity remain elusive. In this study, we assessed the hypothesis that telomerase is a key downstream target for the anti-proliferative activity of everolimus.

METHODS AND RESULTS:
We demonstrate that everolimus treatment resulted in a dose-dependent inhibition of mitogen-induced telomerase activity in human coronary artery VSMCs (hcVSMCs). Moreover mitogen-induced telomerase reverse transcriptase (TERT) promoter activity and protein expression were dose-dependently repressed by everolimus treatment. Everolimus inhibited mitogen-induced cell proliferation as a result of a G1 →S phase arrest of the cell cycle. In addition, everolimus repressed protein expression of cell cycle-regulated genes (MCM 7, PCNA and Cyclin A) without altering phosphorylation of the retinoblastoma (Rb) protein, being the key gatekeeper of cell cycle transition. In addition, the effects of everolimus were not related to telomere shorting and cellular senescence, supporting the concept of a noncanonical telomere length-independent mechanism. Finally, we demonstrated in transient transfection studies that TERT overexpression abolished the anti-proliferative effect of everolimus in hcVSMC, pointing to a key role of telomerase as a target for the anti-proliferative effects of everolimus.

CONCLUSIONS:
These results indicate that telomerase constitutes a key target for the anti-proliferative activity of everolimus and identify a novel mechanism for the inhibition of hcVSMC proliferation.
Abstract 170: CFTR as a Novel Target and Mechanism for High-Fructose Diet and Salt-Induced Hypertension
Authors: Zhang Ya-Ping , Ye Lingyu L , Yuan Hong , Duan Dayue D
Published: March 2022

Objective:
High fructose and salt diet (HFSD) consumption is very common in modern lifestyle. Fructose not only stimulates intestinal salt and water absorption but also decreases renal sodium (Na+) and chloride (Cl-) excretion. High fructose intake induces rapid and sustained increase in blood pressure (BP). But a Cl--free diet could block fructose-induced hypertension. The underlying mechanism for this Cl_dependence of high fructose and salt induced hypertension is not clear. This study was designed to test the hypothesis that the cystic fibrosis transmembrane conductance regulator (CFTR) Cl- channel may play a critical role in the HFSD-induced hypertension.

Approach and Results:
The DSI HD-X11 transmitters for telemetry recordings of both blood pressure and ECG were implanted into CFTR-knockout (CFTR-/-) mice (8-week old, male) and their age- and gender-matched wild-type (CFTR+/+) mice. The systolic (SBP), diastolic (DBP) and mean BP (MBP) and ECG of the mice before and after feeding with HFSD (%65 fructose, 2% salt) were monitored daily for 8 weeks. Continuous 8-week HFSD caused a significant increase in aortic stiffness, mesenteric and renal artery resistance, SBP, DBP, and MBP in CFTR+/+ mice. Real-time PCR and Western blot analysis revealed a significant decrease (~90%) in CFTR mRNA and protein expression in aorta and mesenteric arteries but not in the heart and brain. This HFSD-induced specific down-regulation of CFTR was highly correlated with the HFSD-induced reduction in WINK1 and WINK4 expression in aorta and mesenteric artery. In the mice fed with normal diet the aortic stiffness, mesenteric and renal artery resistance and the SDP, DBP, and MBP at either daytime or nighttime was significantly higher in CFTR-/- mice (n=8) than in CFTR+/+ mice (n=15), implicating an important functional role for CFTR in the regulation of vascular resistance and BP.

Conclusions:
1) CFTR plays a pivotal functional role in the regulation of arterial resistance and BP; 2) HFSD may cause hypertension through a specific WINKs-CFTR signaling pathway. These results provide compelling evidence that diet may cause changes in expression of specific genes in the vasculature and CFTR may represent a novel molecular target and mechanism for the HFSD-induced hypertension.
Abstract 172: Impact of Pharmacological Inhibition of Neuronal Nitric Oxide Synthase on Brain Microvascular Endothelial Cells
Authors: Sure Venkata N , Gordon Angellica O , Liu Dan , Katakam Prasad V
Published: March 2022

Experimental stroke in animals with deletions of endothelial (eNOS) and neuronal (nNOS) isoforms of nitric oxide synthase have observed greater infarct injury whereas pharmacological inhibition of NOS reported varying impact. nNOS has been identified recently in endothelial cells, however, the functional role of nNOS in brain microvascular endothelial cells (MECs) has never been examined. Our objective was to identify the nNOS in MECs and study its role in the regulation of mitochondrial function and response to anoxic injury.

Methods and Results:
Primary brain MECs from humans and rats were used in the studies. Immunohistochemistry identified von Willebrand factor, eNOS, and nNOS in MECs. The nNOS immunoreactivity to three antibodies raised against different sequences of nNOS was observed in the cytoplasm and also in the nucleus when cells were permeabilized. Oxygen consumption rate (OCR) measurements were made from hMECs treated with selective inhibitors of nNOS (N-ω-Propyl-L-arginine; NPA or 7-nitroindazole) and eNOS (L-N
5
-(1-Iminoethyl)ornithine; NIO) or non-specific inhibitor (N
G
-Nitro-L-arginine methyl ester; L-NAME) revealed that mitochondrial reserve respiratory capacity was enhanced by nNOS inhibition but diminished by eNOS inhibition or L-NAME (100 mol/L). Drug treatment of hMECs for 3 h followed 24 h later by cell viability measurements showed that nNOS inhibition increased cell proliferation whereas eNOS inhibition diminished cell viability. Exposure to oxygen-glucose deprivation in the presence of nNOS inhibition increased cell viability.

Conclusions:
We identified immunoreactive nNOS in brain MECs for the first time. Pharmacological inhibition of nNOS in MECs enhanced mitochondrial capacity, promoted cell proliferation, and afforded protection against anoxic injury. Thus, in MECs, nNOS appears to function distinct from eNOS and may even counteract eNOS actions.
Abstract 228: Niacin Improves Human Microvascular Endothelial Cell Angiogenic Function Under Lipotoxic and Hypoxic Conditions
Authors: Pang Dominic , Hughes-Large Jennifer , Robson Debra , Chan Pak , Borradaile Nica
Published: March 2022

Niacin (nicotinic acid) can reduce vascular disease risk, but its mechanism of action is controversial, and may not be dependent on systemic lipid modifying effects. It has recently been shown to improve endothelial function and vascular regeneration, independent of correcting dyslipidemia, in rodent models of vascular injury and ischemia, and metabolic disease. As a potential biosynthetic precursor for NAD
+
, niacin could elicit vascular benefits through NAD
+
-dependent, sirtuin (SIRT) mediated responses. Or, it may act through its receptor, GPR109A, to promote endothelial function, though endothelial cells are not known to express this protein. We tested the hypothesis that niacin directly improves endothelial cell function under lipotoxic and low oxygen conditions, and investigated the potential mechanism(s) involved. We measured human microvascular endothelial cell (HMVEC) angiogenic function, by tube formation on Matrigel (18 h), during fatty acid overload (0.5 mM palmitate, oleate, or palmitate plus oleate) under either normoxic (20% O
2
) or low oxygen (2% O
2
) conditions. Both supplementation with excess palmitate and incubation at 2% O
2
inhibited tube formation compared to control conditions (-38% and -44%, respectively, n=5, p<0.05). Although niacin (10 μM) improved tube formation in the presence of excess palmitate, during both normoxia (35%, n=5, p<0.05) and hypoxia (34%, n=5, p<0.05), it did not increase [NAD
+
]/[NADH] ratios or SIRT1 activity. Gene expression microarray analyses further showed that niacin did not induce expression of known SIRT1 regulated genes. However, we made the novel observation that HMVEC express GRP109A. Selective activation of the receptor with acifran recapitulated niacin induced improvements in tube formation during palmitate overload in 20% O
2
(53%, n=3, p<0.05), while GPR109A siRNA diminished the effect (16%, n=3, p<0.05). We concluded that niacin improves HMVEC angiogenic function under lipotoxic and hypoxic conditions. During normoxia, this effect appears to be independent of NAD+ synthesis and SIRT1 activation, but likely occurs through activation of GPR109A. These findings may have implications for the use of niacin in ischemic vascular disease associated with metabolic disease.
Abstract 230: Stimulation of Cholesterol Efflux from Human Macrophage by Liver X Receptor Agonists is a 2-Step Mechanism Requiring ARL7 and ABCA1
Authors: Superville Alexandre , Frisdal Eric , Quinn Carmel M , Kim Mi-Jurng , Jessup Wendy , Lesnik Philippe , Guérin Maryse , Le Goff Wilfried
Published: March 2022

Nuclear Liver X Receptors activation by synthetic agonists was proven to be atheroprotective in mice; an effect likely based on stimulation of cellular cholesterol efflux from arterial macrophages. However, mechanisms involved in free cholesterol efflux from mouse macrophages appear distinct from those operating in human macrophages.

The objective of this study was to decipher precise cellular mechanisms controlling free cholesterol efflux from human macrophages upon LXR stimulation.

In THP-1 and human monocyte-derived macrophages (HMDM), treatment with the LXR agonist GW3965 efficiently induced ARL7 expression (6-fold, p<0.05), an effect associated with an increased amount of plasma membrane free cholesterol available for efflux (+25%, p<0.05) and a higher lipid rafts formation (+10%, p<0.05). Both effects were abolished in ARL7 Knockdown (KD) macrophages, leading to a lack of stimulation of cholesterol efflux by GW3965.

Specific targeting of each LXR isoforms, LXRα and LXRβ, by RNAi revealed that LXRα silencing in THP-1 and HMDM reduced significantly expression of cholesterol transporters ABCA1, ABCG1 and receptor SR-BI/Cla-1 mRNA levels, as well as free cholesterol efflux to apoA1 (-30%, p<0.05) and to HDL (-20%, p<0.05) upon stimulation with LXR, whereas LXRβ silencing has no impact. Interestingly, stimulation of cholesterol efflux to HDL by GW3965 was significantly reduced (-50%, p<0.05) in ABCA1 KD THP-1 macrophages; those cells being incapable to promote cholesterol efflux to apoA1. However, silencing of ABCG1 or SR-B1/Cla-1 had no impact on cholesterol efflux to HDL from either control or ABCA1 KD THP-1 macrophages treated or not with LXR agonist. By contrast stimulation of cholesterol efflux to HDL by GW3965 was completely abolished in LXRα/ABCA1 double KD macrophages, highlighting the major contribution of ABCA1 in cholesterol efflux from human macrophage.

We conclude that LXR-mediated stimulation of cholesterol efflux from human macrophages is a two-steps mechanism. First, LXR activation promotes ARL7-dependent free cholesterol transport to plasma membrane, mostly in lipid raft domains. Then, membrane free cholesterol is exported to apoA1 and HDL acceptors through ABCA1; this latter step being controlled selectively by LXRα.
Abstract 246: Anacetrapib Adds to the Antiatherogenic Effect of Atorvastatin, Mainly by Increasing the Clearance of Non-HDL Cholesterol
Authors: van der Tuin Sam J , Kuhnast Susan , Van der Hoorn José W , van Klinken Jan B , Simic Branko , Pieterman Elsbet J , Havekes Louis M , Landmesser Ulf , Luscher Thomas F , Willems-van Dijk Ko , Rensen Patrick C , Jukema J W , Princen Hans M
Published: March 2022

Introduction:
The residual risk that remains after statin treatment has stimulated the search for secondary treatment targets. Epidemiological studies propose HDL-C as a possible candidate. Cholesteryl ester transfer protein (CETP) transfers cholesteryl esters from atheroprotective HDL to atherogenic (V)LDL. The CETP inhibitor anacetrapib decreases (V)LDL-C by ~15-40% and increases HDL-C by ~40-140% in clinical trials.

We evaluated the effects of anacetrapib on lipid metabolism, HDL function and atherosclerosis, and examined possible additive/synergistic effects of anacetrapib on top of atorvastatin in APOE*3Leiden.CETP mice.

Methods and results:
Mice were fed a diet without or with ascending dosages of anacetrapib (0.03; 0.3; 3; 30 mg/kg/d), atorvastatin (2.4 mg/kg/d) alone or in combination with anacetrapib (0.3 mg/kg/d) for 21 weeks.

Anacetrapib dose-dependently reduced CETP activity (-59% to -100%, P<0.01), thereby decreasing nonHDL-C (-24% to -45%, P<0.001) and increasing HDL-C (+30% to +86%, P<0.001). Anacetrapib did not affect HDL function. Additionally, we showed that anacetrapib reduced nonHDL-C mainly by increasing the clearance by the liver.

Anacetrapib dose-dependently reduced atherosclerotic lesion area (-41% to -92%, P<0.01) and severity, increased the plaque stability index and added to the effects of atorvastatin by further decreasing lesion size (-95%, P<0.001) and severity. Analysis of covariance showed that both anacetrapib (P<0.05) and nonHDL-C (P<0.001), but not HDL-C (P=0.76), independently decreased lesion size.

Conclusions:
Anacetrapib reduces atherosclerosis, and adds to the anti-atherogenic effect of atorvastatin. The anti-atherogenic effect is mainly ascribed to a reduction in nonHDL-C, as a consequence of an increased clearance. In addition, anacetrapib improves lesion stability.
Abstract 25: Neutrophil Akt2 Regulates Heterotypic Cell-Cell Interactions During Vascular Inflammation
Authors: Cho Jaehyung , Li Jing , Kim Kyungho , Hay Nissim , Du Xiaoping , Gordeuk Victor
Published: March 2022

Introduction:
Interactions between platelets, leukocytes and endothelial cells are important during microvascular occlusion. Although the major receptors and counter receptors have been well identified, it remains unclear how heterotypic cell-cell interactions are regulated under inflammatory conditions.

Hypothesis:
We assessed the hypothesis that AKT, a Ser/Thr protein kinase, plays a regulatory role in platelet-neutrophil-endothelial cell interactions during vascular inflammation.

Methods and Results:
Using real-time fluorescence intravital microscopy to evaluate mice lacking specific Akt isoform, we demonstrated that Akt2, but not Akt1 and Akt3, regulates neutrophil adhesion and crawling, and platelet-neutrophil interactions on the activated endothelium during TNF-α-induced cremaster venular inflammation. Further, using chimeric mice generated from bone marrow transplants on wild-type (WT) and Akt2 knockout (KO) mice, we found that hematopoietic cell-associated AKT2 is important for the regulatory effect in vivo. Studies with an AKT2-specific inhibitor and cells isolated from WT and Akt KO mice revealed that platelet- and neutrophil-associated AKT2 regulates heterotypic neutrophil-platelet aggregation under conditions of shear. In particular, neutrophil AKT2 was critical for intracellular calcium mobilization, β2-talin1 interaction, and membrane translocation of αMβ2 integrin - a crucial receptor for neutrophil-platelet interactions. We observed that the basal phosphorylation levels of AKT isoforms are markedly increased in neutrophils and platelets from patients with sickle cell disease (SCD), an inherited hematological disorder associated with vascular inflammation and occlusion. AKT2 inhibition significantly reduced heterotypic aggregation of neutrophils and platelets isolated from SCD patients and diminished neutrophil adhesion and neutrophil-platelet aggregation in venules of Berkeley (SCD) mice, thereby improving blood flow rates.

Conclusion:
Our results provide genetic and pharmacologic evidence that neutrophil AKT2 regulates αMβ2 integrin function and is important for neutrophil recruitment and neutrophil-platelet interactions under thrombo-inflammatory conditions such as SCD.
Abstract 263: Ultrasound Enhancement of Bevacizumab Release from Echogenic Liposomes for Inhibition of Atheroma Progression
Authors: Naji Ali K , Peng Tao , Britton George , McPherson David D , Klegerman Melvin E
Published: March 2022

Background:
Vascular endothelial growth factor (VEGF) is a signal protein that induces vasculogenesis and angiogenesis, which are important to atheroma progression. Bevacizumab is a humanized monoclonal antibody specific for VEGF that causes regression of existing microvessels and has been shown to inhibit atheroma progression. Previous studies demonstrated the inhibitory efficacy of bevacizumab and bevacizumab-loaded echogenic liposomes (BEV-ELIP) for human umbilical vein cell (HUVEC) proliferation and VEGF expression (assayed by VEGF ELISA).

Hypothesis:
Bevacizumab activity can be enhanced by pulsed Doppler ultrasound treatment of BEV-ELIP.

Methods:
Bevacizumab (1 μg/ml) was added to 18-hour HUVEC cultures in 96-well plates, followed by 10 nM phorbol 12-myristate 13-acetate (PMA) 4 hours later and incubated for 24 hours. Cell proliferation was measured by the tetrazolium dye MTT. BEV-ELIP in a latex condom was submerged in a water bath and exposed to 6 MHz color Doppler ultrasound (MI = 0.4 ) for 5 min for bevacizumab release.

Results:
BEV-ELIP treated with US inhibited VEGF expression by 90% relative to non-treated controls and by 70% relative to BEV-ELIP without US (Fig. 1). Also, BEV-ELIP inhibited cell proliferation by 64% relative to untreated controls and by 45% relative to BEV-ELIP without US (Fig. 2).

Conclusions:
Ultrasound treatment of BEV-ELIP enhanced the agent’s ability to inhibit cell proliferation in the MTT assay and VEGF expression in the VEGF ELISA, consistent with US-triggered BEV release observed in previous studies. This BEV-ELIP formulation can now be translated to in vivo studies for atheroma inhibition.
Abstract 277: Diminazene Aceturate, an ACE2 Activator, Decreases AngII-Induced Atherosclerosis in Low-Density Lipoprotein Receptor--Deficient Mice
Authors: Thatcher Sean E , Yiannikouris Frederique , Daugherty Alan , Cassis Lisa A
Published: March 2022

Objectives:
Diminazene aceturate (DIZE) activates angiotensin converting enzyme 2 (ACE2) both in vitro and in vivo. ACE2 limits angiotensin II (AngII)/angiotensin type 1 receptor (AT1R) signaling through cleavage of AngII to form angiotensin-(1-7) (Ang-(1-7)). Previous studies in our laboratory demonstrated that whole body ACE2 deficiency increased diet-induced atherosclerosis in low density lipoprotein receptor (LDLr) deficient mice. The purpose of this study was to determine if administration of DIZE to LDLr-/- mice activates ACE2 to decrease AngII-induced atherosclerosis.

Materials and Methods:
Initial dose ranging studies determined the effects of 7.5, 15, or 30 mg/kg/day of DIZE infused subcutaneously by osmotic minipump for 7 days into male LDLr-/- mice. DIZE dose-dependently increased plasma Ang-(1-7) concentrations with significant elevations at 30 mg/kg/day (vehicle, 5.9 ± 0.6; DIZE, 7.9 ± 0.5 ng/mL, p<0.05). Moreover, kidney ACE2 activity increased in mice administered DIZE (30 mg/kg/day) compared to vehicle-injected controls (vehicle, 208 ± 87; DIZE, 1716 ± 424 fmol/mg/min, p<0.05). For optimal dosing, ACE2 wildtype (Ace2+/y) and deficient (Ace2-/y) LDLr-/- mice were administered either vehicle or DIZE by intramuscular injection for a total of 5 weeks. Seven days after initiation of DIZE administration, mice were infused with AngII (1,000 ng/kg/min for 28 days) to induce atherosclerosis. Compared to vehicle, administration of DIZE resulted in significant reductions in atherosclerotic lesion formation in aortic arches of AngII-infused Ace2+/y , but not Ace2-/y mice (vehicle Ace2+/y, 9.8 ± 1.2; DIZE Ace2+/y, 5.8 ± 1.2; DIZE Ace2-/y, 9.2 ± 1.8, p<0.05). Interestingly, compared to vehicle, DIZE lowered serum cholesterol concentrations in Ace2+/y but not Ace2-/y mice (vehicle Ace2+/y, 837 ± 69; DIZE Ace2+/y, 571 ± 41; DIZE Ace2-/y, 774 ± 113 mg/dl, p<0.05).

Conclusions:
These studies demonstrate that DIZE decreased AngII-induced atherosclerosis through an ACE2-dependent mechanism. Mechanisms for DIZE-induced reductions in AngII-induced atherosclerosis may include reductions in serum cholesterol concentrations.
Abstract 28: p62-Enriched Inclusion Bodies in Macrophages Play a Protective Role in Atherosclerosis
Authors: Sergin Ismail , Emanuel Roy , Bhattacharya Somashubhra , Razani Babak
Published: March 2022

Polyubiquitinated protein aggregates have been classically associated with human neurodegenerative, liver, and muscle disorders, but not atherosclerosis. Since autophagy-deficient atherosclerotic macrophages have markedly increased levels of p62, a chaperone protein critical for the autophagic turnover of polyubiquitinated proteins, we aimed to evaluate the role of p62 and protein aggregation in atherosclerosis. We first tested the effects of atherogenic lipid loading in peritoneal macrophages. Incubation with either cholesterol crystals or oxidized LDL leads to dramatically increased p62 protein that completely co-localizes with polyubiquitinated proteins as cytoplasmic inclusions or as aggregates partially sequestered by lysosomes. Such accumulation was intensified in autophagy-null (ATG5-/-) macrophages, where massive cytoplasmic ubiquitin-positive p62 aggregates form. Our in vitro observations are recapitulated in vivo. Aortas from atherosclerotic (ApoE-/-) mice have marked elevations in p62 and polyubiquitinated proteins that predominantly co-localize with plaque macrophages, a process that is further exacerbated in the autophagy-deficient setting. Remarkably, we find nearly identical results in human carotid endarterectomy samples, suggesting that p62-enriched protein aggregates are a characteristic feature of atherosclerosis. The homeostasis of cytoplasmic inclusions is dependent on the presence of p62 since lipid-loaded p62-null macrophages accumulate polyubiquitinated proteins in a diffuse and disrupted cytoplasmic pattern. The disruption of these aggregates has functional consequences manifested as increased secretion of IL-1β and enhanced macrophage apoptosis. Consistent with our in vitro observations, p62-deficient mice have increased atherosclerotic plaque formation. Furthermore, p62-deficiency increases plaque burden even in the highly atherosclerosis-prone macrophage-specific ATG5-null mice. Taken together, these data suggest that by sequestering cytotoxic ubiquitinated proteins, p62-enriched inclusion body formation is a protective response during atherogenesis, a setting where the clearance of protein aggregates is disrupted.
Abstract 290: Noncanonical Wnt Signaling Contributes to Angiotensin II-Induced Vascular Inflammation and Abdominal Aortic Aneurysm Formation
Authors: Fuster Jose J , Zuriaga Maria A , Maruyama Sonomi , Yamaguchi Terry P , Golledge Jon , Zhou Ming H , Walsh Kenneth
Published: March 2022

Background:
Abdominal aortic aneurysms (AAA) are life-threatening localized dilations of the abdominal aorta. A precise knowledge of the molecular mechanisms underlying this disease is required, especially because there are no validated drugs for its treatment. Wnt proteins are secreted signaling molecules that play fundamental roles during embryo development and in the adult. There are 19 Wnt family members in mammals, which frequently have overlapping or redundant functions. Wnt5a is a particularly unique Wnt protein that induces non-canonical Wnt signaling and appears to modulate innate immunity and several signaling pathways involved in vascular inflammation. However, its role in the development of macrovascular pathologies remains unexplored. HYPOTHESIS. Wnt5a-mediated non-canonical Wnt signaling contributes to vascular inflammation and AAA development.

METHODS:
Cre-loxP strategies were used to generate whole-body Wnt5a-deficient mice, myeloid- Wnt5a-deficient mice and myeloid Wnt5a-overexpressing mice. AAAs were induced in these mouse strains in an apolipoprotein E deficient (apoE-KO) or wild-type (apoE-WT) background by Angiotensin II (Ang II) infusion. Ultrasonography was used to evaluate AAA progression. AAA samples were harvested for histology and gene expression analysis. RESULTS. Whole-body ablation of Wnt5a inhibited Ang II-induced vascular inflammation and AAA formation in apoE-KO mice. Myeloid-specific Wnt5a ablation reduced AAA size, incidence rate and severity, suggesting that myeloid cells are a significant source of pathogenic Wnt5a. Conversely, myeloid-restricted Wnt5a-overexpression led to rapid formation and rupture of dissecting AAAs in Ang II-infused apoE-KO and apoE-WT mice. In human AAAs, Wnt5a expression is significantly increased both at the transcript and protein levels. Mechanistically, Wnt5a appears to promote AAA formation at least in part by promoting vascular inflammation and extracellular matrix remodeling via augmented recruitment of immune cells to the vessel wall.

CONCLUSION:
Wnt5a-mediated non-canonical Wnt signaling plays a major role in inflammatory AAA formation and may be a potential target for novel AAA therapies.
Abstract 299: Inducible Depletion of TGF-ß1 Exerts Differential Regional Effects on AngII-Induced Aortic Aneurysms
Authors: Rateri Debra L , Howatt Deborah A , Balakrishnan Anju , Moorleghen Jessica J , Cassis Lisa A , Daugherty Alan
Published: March 2022

Introduction and Objectives:
Infusion of AngII into normolipidemic mice leads to uniform expansion of the ascending aorta and a low incidence of abdominal aortic aneurysms. Co-administration of the monoclonal antibody 1D11, which recognizes TGFβ 1, 2, and 3, leads to greatly increased incidences of aortic rupture in both the ascending and abdominal aorta. The purpose of this study was to determine the role of TGFβ1 in AngII-induced aortic diseases. To perform this study, TGFβ1 floxed mice were bred to express an inducible form of Cre under control of the ubiquitous promoter, chicken α- actin.

Methods and Results:
Experimental mice were produced by breeding male Cre-ERT2 to female TGF-β1 floxed mice. At 8 weeks of age, male and female non-Cre littermates (N=16 male, 21 female) and TGF-β1 x Cre-ERT2 (N=11 male, 22 female) mice were injected with tamoxifen (25-75 mg/kg, i.p.) daily for 5 consecutive days. After 3 weeks, serum TGF-β1 was measured by ELISA. Serum concentrations of TGF-β1 decreased significantly in tamoxifen-injected Cre-ERT2 mice (P=0.001) compared to non-Cre littermates. Mice were then infused with AngII (1,000 ng/kg/min) for 28 days. AngII-induced increases in systolic blood pressures were not influenced by depletion of TGFβ1. AngII-induced expansion and rupture of the ascending aorta were not influenced by reduced TGFβ1. In contrast, there was marked enhancement of AngII-induced aortic expansion in the abdominal aorta (1.3 ± 0.3 versus 2.3 ± 0.3 mm in Cre- and Cre+ mice, respectively. P=0.019). Depletion of TGFβ1 had no effect on AngII-induced aortic pathologies in female mice.

Conclusion:
Depletion of TGF-β1 exerted regional-specific effects on AngII-induced aortic pathologies, with enhancement of abdominal aneurysms and no effect on ascending aortic dilation.
Abstract 302: Generation of Patient-Specific Tissue-Engineered Blood Vessels From Nonintegrated Induced Pluripotent Stem Cells
Authors: Wang Yongyu , Hu Jiang , Jiao Jiao , Chen Y Eugene , Ma Peter , Yang Bo
Published: March 2022

Cell resourcing remains one of the major problems in the development of patient-specific TEBVs. The iPSC derived cardiovascular cells provide a promising source for TEBVs and cell therapy. The goal of this study is to construct TEBVs using patient-specific iPSCs.

Using a non-integrated episomal vector based reprogramming system, we generated human iPSCs from peripheral blood mononuclear cells (PBMCs) isolated from the blood harvested from aneurysm patients with bicuspid aortic valves and coronary artery bypass patients with normal aortas and aortic valves. These iPSCs showed the expression of pluripotent markers and could differentiate into 3 layers of cells in vitro. We directed the iPSCs differentiation into vascular smooth muscle cells (VSMCs) in vitro. We documented the expression of VSMC markers, including smooth muscle α-actin (α-SMA), calponin and SM22α by quantitative real-time-PCR, immunocytochemistry, as well as Western-blotting. Furthermore, we confirmed that the iPSC-derived VSMCs could contract in response to carbachol, a muscarinic agonist. The treatment of iPSC-derived VSMCs with IL-1β, an atherogenic cytokine, changed MMPs (matrix metallopeptidases) activity. Finally, we found that iPSC-derived VSMCs could survive well and secrete collagen when transferred and cultured on PLLA scaffolds in vitro, suggesting a great cell sourcing for tissue-engineered blood vessels.

Our data suggests that by using non-integrated iPSCs, we could efficiently differentiate patient-specific iPSCs into functional VSMCs, which may provide great cell sourcing for patient-specific TEBVs.
Abstract 314: Determinants of Endothelial Dysfunction in Peripheral Artery Disease: Importance of Kidney Disease
Authors: Chong Karen C , Owens Christopher D , Park Meyeon , Alley Hugh F , Conte Michael S , Gasper Warren J , Grenon Marlene
Published: March 2022

OBJECTIVES:
Peripheral artery disease (PAD) and chronic kidney disease are both associated with endothelial dysfunction. Renal insufficiency may contribute to the progression of endothelial dysfunction in PAD patients, increasing their morbidity and mortality. We hypothesized that worsening renal function (RF) is associated with impaired endothelial function (EF) in patients with PAD.

METHODS:
This was a cross-sectional study of PAD patients presenting to a vascular surgery clinic. Brachial artery flow-mediated vasodilation (FMD) was done to assess EF. RF was determined by calculating estimated glomerular filtration rate (eGFR). Linear regression was performed to assess the relationship between EF and RF in claudicants.

RESULTS:
115 claudicants with eGFR ≥ 30ml/min participated in this study. Mean age was 69 ± 8 years, 97% were male, and 77% were Caucasian. Comorbidities included hypertension (90%), dyslipidemia (83%), coronary artery disease (41%) and diabetes mellitus (36%). Mean ABI was 0.71 ± 0.15 and mean FMD was 7.0% ± 3.8, indicating impaired EF. As RF declined (p=0.001), EF decreased. After multivariable regression adjusting for age, race, log TNF-α, hypertension, dyslipidemia, and diabetes, only eGFR remained associated with FMD (p=0.030).

CONCLUSIONS:
In patients with PAD, worsening renal function has a stronger association with endothelial dysfunction than traditional cardiovascular risk factors do. Further longitudinal studies are needed to explore whether stabilization of renal function may decrease the risk of PAD progression associated with endothelial dysfunction, as well as the risk of major adverse cardiovascular events and mortality.
Abstract 329: Microparticles Induce Antiangiogenic Signaling in Microvascular Endothelial Cells by Activating a CD36-Dependent Signaling Pathway Involving Src Kinases and Reactive Oxygen Species
Authors: Ramakrishnan Devi Prasadh , Silverstein Roy L
Published: March 2022

Cell-derived microparticles (MP), ≤1μm vesicles shed from vascular and blood cells during activation or apoptosis, circulate in plasma and are biomarkers of cardiovascular disease. The type 2 scavenger receptor CD36 binds MP via phosphatidylserine exposed on their surface. On microvascular endothelial cells (MVEC) CD36 induces anti-angiogenic signals via its cognate protein ligands, such as thrombospondin-1. We hypothesized that CD36-MP interactions on MVEC would inhibit angiogenesis. MP generated in vitro from THP1 cells dose dependently inhibited MVEC tube formation in in vitro matrigel angiogenesis assays (>50% inhibition of number of tubes formed, branch points, and segments; p<0.05). MP derived from human umbilical vein endothelial cells (HUVEC), MVEC and erythrocytes also inhibited tube formation showing that the anti-angiogenic effect of MP is independent of their cell of origin. THP1 MP also inhibited MVEC migration in a transwell assay by 80% (p<0.001). Early passage MVEC with high CD36 expression were inhibited more than late passage MVEC with low CD36 expression (80% vs 33%; p<0.001) establishing the specificity of CD36 in this process. Further, in HUVEC that do not express CD36, inhibition of migration by THP1 MP was only 44% (p<0.001) but lentiviral-mediated heterologous expression of murine CD36 in HUVEC restored inhibition of migration to levels similar to that of MVEC (82%; p<0.001), corroborating the specificity of CD36 in MP mediated inhibition of migration. Since CD36 dependent generation of reactive oxygen species (ROS) is necessary to inhibit cell migration in macrophages, we tested if MP can induce ROS in MVEC. THP1 MP dose dependently induced ROS in MVEC by two fold compared to control at 1hr (p<0.05). NADPH oxidase inhibitor, apocynin, blocked MP induced ROS induction by >95% and partially (33%) reversed the inhibition of tube formation in MVEC by THP1 MP (p<0.05). Western blots of THP1 MP treated MVEC showed time-dependent activation of Fyn kinase, and PP2, a Src family kinase inhibitor, blocked MP induced ROS induction by >95%. These studies identify a novel CD36 dependent mechanistic pathway through which cell-derived MP inhibit MVEC angiogenic activities through ROS generation involving NADPH oxidase and Src kinase Fyn.
Abstract 331: The Role of Zizimin1 Overexpression in Vascular Morphogenesis
Authors: Saadat Siavash , Qi Yanmei , Liu Jie , Graham Alan M , Li Shaohua
Published: March 2022

Objectives:
The Rho subfamily of small GTPases, including RhoA, Rac1, and Cdc42, regulates diverse cellular functions, including polarity and migration. Our prior studies established an essential role for Cdc42 in vascular network assembly, demonstrating that the genetic inactivation of Cdc42 yields defective vascular morphogenesis due to impaired migration of endothelial precursor cells. We have further shown that protein kinase Ciota and glycogen synthase kinase-3 Beta are downstream effectors of Cdc42 involved in mediating vascular network assembly. The objective of this study was to elucidate the guanine nucleotide exchange factors (GEFs) that activate Cdc42; specifically, we investigated the role of Zizimin1 and its effects on Cdc42 and vasculogenesis.

Methods:
We performed affinity pulldown assays using a nucleotide-free Cdc42 G15A mutant that specifically binds to Cdc42 GEFs. Mass spectrometric analysis identified Zizimin1 as a candidate Cdc42 GEF.

Results:
During vasculogenesis in embryoid bodies (EBs) differentiated from embryonic stem cells, Zizimin1 is highly expressed in aggregated endothelial cell precursors prior to vascular network assembly, mainly localizing to the Golgi apparatus. Surprisingly, stable overexpression of Zizimin1 in EBs resulted in inhibition of blood vessel formation, as evidenced by immunofluorescence microscopy demonstrating loss of vascular network development. Affinity pulldown assay showed that overexpression of Zizimin1 in fact increases Cdc42 activity; however, the activation of Rac1 and RhoA is significantly inhibited. Further, ectopically expressed Zizimin1 was also detected in the Golgi apparatus, co-localizing with Cdc42.

Conclusions:
Since Rac1 and RhoA signaling has been reported to play an essential role in blood vessel formation, our results suggest that the interplay between Rho GTPases guides vascular network assembly during development. Furthermore, we speculate that Zizimin1 overexpression, leading to overactivation of Cdc42 in the Golgi apparatus, may hinder cell migration. These findings provide novel insights into the mechanisms of embryonic vasculogenesis and also important new information for the design of potential pro- and/or anti- angiogenic therapies.
Abstract 349: Characterization of a New Organic Nitrate Hybrid Drug Covalently Bound to Pioglitazone
Authors: Pfeffer Amanda , Knorr Maike , Hausding Michael , Jansen Thomas , Oelze Matthias , Kröller-Schön Swenja , Schulz Eberhard , Wenzel Philip , Gori Tommaso , Muenzel Thomas , Daiber Andreas
Published: March 2022

Background and Purpose:
Organic nitrates represent a group of nitrovasodilators that are clinically used for the treatment of ischemic heart disease. With the present studies we synthesized and characterized a new organic nitrate hybrid molecule. Compound CLC-3000 is an aminoethyl nitrate (AEN) derivative of pioglitazone, a thiazolidinedione antidiabetic agent. This new molecular entity combines the peroxisome proliferator-activated receptor gamma (PPAR-γ) agonist activity of pioglitazone with the NO-donating activity of the nitrate moiety. Experimental Approach: In vitro and in vivo characterization was performed by isometric tension recording, bleeding time and detection of oxidative stress.

Key Results:
The vasodilative potency of CLC-3000 in vitro was shown to be markedly superior to that of pioglitazone alone or a classical nitrate, although in vitro, at high concentrations, some effects on oxidative stress parameters were observed. Following seven days exposure to CLC-3000 in vivo, no change was seen in the vasodilative response of rat aortic rings towards acetylcholine, nitroglycerin or CLC-3000 itself. Similarly, no remarkable increase in oxidative stress parameters was observed under CLC-3000 therapy. Acute or chronic administration of AEN decreased the tail vein bleeding time in mice.

Conclusions and Implications:
In summary, the results of these studies demonstrate that CLC-3000 contains a vasodilative and anti-thrombotic activity that is not evident with pioglitazone alone, and that seven days exposure in vivo showed no typical signs of nitrate tolerance, endothelial dysfunction or other safety concerns in Wistar rats.
Abstract 363: Prolonged Upregulation of Proinflammatory and Profibrotic Genes is Characteristic of Successful Arteriovenous Fistula Maturation
Authors: Walker Joy , Toy Robert , Thomas Sean , Conte Michael S , Eichler Charles M , Lovett David , Gasper Warren J , Owens Christopher D
Published: March 2022

Objective:
Understanding the molecular mechanisms of vascular remodeling is critical to improving outcomes following revascularization procedures. While extensive animal data exists, correlative data on the mechanisms and kinetics of vascular healing in humans are lacking. Creation of brachio-basilic arteriovenous fistulas (AVF), often completed in 2 stages, offers an opportunity to examine the same vessel at 2 distinct time points. We hypothesized that immune and developmental related pathways are involved in AVF maturation.

Methods:
Human basilic vein samples were obtained at the time of AVF creation as baseline. Second samples were taken at basilic vein transposition after exposure to arterial hemodynamic forces. These paired samples were analyzed for differential gene expression using whole genome microarray. Structural staining and confirmatory qPCR were performed in selected genes.

Results:
Paired vein samples were collected from 3 patients. All subjects had successful AVF maturation. An average of 3 months transpired between collection of the first and second samples. Overall, 700 genes were significantly down-regulated and 732 genes were significantly up-regulated. Gene ontogeny analysis revealed no difference in the genes regulating developmental re-activation pathways or angiogenesis.

However, inflammatory and fibrosis genes were still significantly up-regulated 3 months following AVF creation (p <<.05). Structural stains confirmed an increase in lumen and total vessel area and deposition of elastin and collagen with significant intimal hyperplasia between baseline and 3 month samples. Additionally, qPCR showed a 9.7 fold increase in IL-6 and a 10.4 fold increase in e-selectin at 3 months.

Conclusions:
Inflammatory and fibrosis gene pathways remain activated in mature fistulas despite stabilization of geometric remodeling. Further study is warranted to characterize these pathways and determine their significance in AVF patency.
Abstract 364: Modulation of Novel Long Intergenic Noncoding RNAs in Human Macrophage Polarization
Authors: Zhang Hanrui , Xue Chenyi , Ballantyne Rachel L , Shah Rhia , Bermingham Kate , Hinkle Christine , Yang Wenli , Gosai Sager , Gregory Brian , Morrisey Edward E , Li Mingyao , Rader Daniel J , Reilly Muredach P
Published: March 2022

Macrophages respond to inflammatory stimuli by modulating gene expression. Whether long intergenic noncoding RNAs (lincRNA) play a regulatory role in human macrophage inflammatory response is unknown. Therefore, we performed RNA-Seq in human PBMC-derived macrophages (HMDM) stimulated with lipopolysaccharide (LPS) and interferon-gamma to induce M1-polarization. Of 4,662 lincRNAs (stringent set of Cabili et al, Gene & Development 2011. 25:1915-1927), expressed at 5
th
percentile FPKM, we identified 420 lincRNAs in HMDM or M1-HMDM, of which 58 were differentially expressed (DE) at FDR<0.01, fold change>2 (43 upregulated, 15 downregulated). Gene ontology (GO) enrichment analysis showed that M1 polarization modulated protein-coding genes involved immune response and inflammatory response. DE protein-coding genes proximal to DE lincRNAs (±100 kb) were enriched in apoptosis and cell death GO term categories. Linc-ACSL1-1 (proximal to ACSL-1), linc-MDM1-2 (proximal to IL-22), and linc-NLRP3 (proximal to NLRP3) were markedly induced, while Linc-F13A1-1 (proximal to LY86) was highly downregulated. Of SNPs from 16 GWAS of cardiometabolic diseases, 36 SNPs within the 58 DE lincRNAs reached threshold P<0.001; e.g., 16 SNPs in Linc-F13A1-1 for waist-hip ratio, with P=6.31E-09 for rs1294421. We performed complementary experiments in induced pluripotent stem cell (iPSC)-derived macrophages (IPSDM) which are functionally comparable to their isogenic primary HMDM counterparts. Analysis of DE genes in IPSDM-polarization revealed 1,527 DE genes (FDR<0.01, fold change >2) and 74% overlapped with DE genes in HMDM-polarization. Linc-ACSL1-1 and an additional 15 DE lincRNAs in IPSDM were overlapped with the DE lincRNAs in HMDM. In summary, high-resolution transcriptomics of human macrophages identified lincRNAs that form part of the cellular response during M1-polarization as well as specific lincRNAs that might modulate macrophage function in cardio-metabolic disorders.