bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 574: High-Density Lipoproteins Suppress Amyloid Beta Induced Human Brain Microvascular Endothelial Cell Activation
Authors: Button Emily B , Robert Jerome , Stukus Sophie K , Boyce Guilaine , Gibbs Ebrima , Cowan Catherine , Cheng Wai Hang , Soo Sonja , Yuen Brian , Kang Kevin , Bahrabadi Arvin , Kulic Iva , Francis Gordon , Cashman Neil , Wellington Cheryl L
Published: July 2021

Introduction:
Epidemiological studies suggest a link between plasma high-density lipoprotein (HDL) cholesterol levels and Alzheimer’s disease (AD) risk through mechanisms that are not understood. We hypothesize that HDL protects against AD through actions at the blood-brain-barrier. HDL has vasoprotective functions in large peripheral arteries, however, it is unknown if these functions extend to cerebral vessels to reduce the contribution of cerebrovascular dysfunction in AD pathogenesis. We investigated
in vitro
interactions between HDL and amyloid beta (Aβ), the toxic peptide known to accumulate in AD, in peripheral and brain-derived endothelial cells (EC).

Methods:
HDL was isolated by density gradient ultracentrifugation and added to human umbilical vein endothelial cells (HUVEC) or human cerebral microvascular endothelial cells (hCMEC/D3). Cell activation was measured by counting adhered labelled peripheral blood mononuclear cells (PBMC) after stimulation with tumour necrosis factor α (TNFα) or Aβ. Aβ binding and uptake into cells was measured using ELISA and immunofluorescence. All experiments included at least 4 independent replicates.

Results:
We demonstrate that HDL attenuates Aβ-induced EC activation independent of nitric oxide production, miR-233 and changes in adhesion molecule expression. Rather, HDL acts through scavenger receptor BI to block Aβ uptake into ECs and,
in vitro
, can maintain Aβ in a soluble state. We validated our results using three dimensional engineered vessels composed of primary human endothelial and smooth muscle cells. Following Aβ addition to the abluminal (brain) side, we demonstrated that HDL circulated within the lumen attenuates EC activation, again independent of intracellular adhesion molecule changes.

Conclusions:
We show that the anti-inflammatory activities of HDL extend to cerebrovascular endothelial cells and work to suppress Aβ-induced activation through a novel mechanism involving the inhibition of Aβ binding and uptake into cells through SR-BI. The protective role for HDL against Aβ may explain the epidemiological evidence supporting a protective effect of high plasma HDL cholesterol levels against dementia.
Abstract 579:
In vitro
Models Concur With Clinical Results to Confirm Pleiotropic Mechanisms of Action for Gemcabene
Authors: Oniciu Daniela C , Bisgaier Charles L
Published: July 2021

Introduction:
Gemcabene, a fraudulent fatty acid, induces biological properties in nonclinical models, of which many translate to clinical findings in dyslipidemia patients. Its known mechanism of action includes reduction of the overall hepatic
de novo
triglyceride and cholesterol synthesis, and increase in VLDL clearance. Clinical benefits in various human populations infer that other mechanisms are involved that warrant evaluation.

Methods and Results:
We assessed the gemcabene effects in other molecular mechanistic models, particularly to study inhibition of human ATP Citrate Lyase (hACL), apolipoprotein gene expression in Sandwich-Cultured Transporter Certified
TM
Human Hepatocytes (SCHH), and expression of genes related to inflammation and cell signaling.

Gemcabene, MEDICA-16, palmitic acid and their CoA thioesters were studied in a recombinant hACL in vitro assay system. Gemcabene-CoA thioester, but not the parent compound, inhibited recombinant hACL activity in vitro in a dose-dependent manner, in agreement with results obtained with palmitoyl-CoA and palmitic acid. Unlike palmitic acid, MEDICA-16, and bempedoic acid, gemcabene does not form a CoA ester in human hepatocytes: radioisotope studies show 98.8% parent gemcabene.

Further, gemcabene potential on the gene expression of lipogenesis and inflammation markers was assessed in SCHH. Gemcabene showed significant regulation of HMG-CoA synthase 2 (HMGCS2) and CRP mRNAs. A marked induction response of the HMGCS2 mRNA content was observed, ranging from 2.26- to 2.73-fold (p ≤ 0.05) over control in SCHH treated with all concentrations of gemcabene (500, 1000, and 1500 μM). Also, a clear and statistically significant (p-value ≤ 0.05) concentration-related suppression response of CRP mRNA content was observed, ranging from (-2.58)- to (-2.65)-fold below control in SCHH treated with 1000 and 1500 μM gemcabene.

Finally, RT-PCR analysis of liver samples from STAM
TM
mice treated with gemcabene revealed its downregulating effect on inflammatory, lipogenic, lipoprotein metabolism, and cell signaling genes: TNF-α, MCP-1, NF-κB, ApoC-III, and ACC1.

Conclusion:
Gemcabene manifests its pharmacological profile in lipid management and inflammation by multiple mechanisms of action.
Abstract 580: Acidic Domain of ApoB-100 and Other Short Negatively Charged Peptides Anchored to Lipoproteins Cause Hypertriglyceridemia in Mice
Authors: Sviridov Denis O , Terse Puja , Wolska Anna , Gordon Scott M , Remaley Alan T
Published: July 2021

Human Apolipoprotein B-100 (ApoB-100) is the main protein of VLDL and LDL particles in plasma. It is a large 4536 amino acid protein with various structural domains, such as the LDL-receptor binding motif and multiple proteoglycan binding sites, all of which have a high positive charge. A search of the primary amino acid sequence of ApoB-100 revealed the presence of the following acidic domain: DMDEDDD (AA 3985-3991). We hypothesized that this motif could affect the interaction of ApoB-containing lipoproteins with liver and or possibly GPIHBP1, which also contains an acidic domain that interacts with the positive charged motifs on VLDL. We synthesized a fluorescent tagged DMDEDDD peptide and conjugated it at the N-terminus with two molecules of alpha-tocopherol to serve as an anchor to lipoprotein particles. After adding the peptide to human plasma, it was found to readily bind to all lipoproteins, as determined by FPLC analysis. VLDL incubated with the peptide showed an increased negative charge and lost its ability to bind to heparin as determined by chromatography on a HiTrap
TM
heparin column. The peptide injected in LDLr-KO mice (30 mg/kg) caused a rapid 3-fold increase in plasma triglycerides within 1 h and the triglyceride elevation persisted for at least 24 h. A similarly designed negative charged peptide attached to tocopherol (EEEEEEEE) also raised triglycerides in mice but a neutral charged control peptide (KEKEKEKE) did not. To understand the mechanism, we tested the above peptides for their ability to inhibit LPL, using an in vitro assay with intralipid as a substrate and found that negative charged peptides inhibited lipolysis (IC
50=
50 uM), but the neutral control peptide did not. We are now investigating other potential mechanisms for how acidic peptides may cause hypertriglyceridemia. In summary, a peptide based on an acidic domain on ApoB-100 appears to cause hypertriglyceridemia in mice by inhibition of LPL and possibly by interfering with proteoglycan binding of lipoproteins. These results may be relevant in understanding VLDL metabolism and for the design of therapeutic apolipoprotein mimetic peptides.
Abstract 581: Smooth Muscle Cell Specific Loss of the Pluripotency Factor Oct4 Enhances Atherosclerosis via Shifting Smooth Muscle Cells to a Less Migratory, but More Proinflammatory Phenotype
Authors: Cherepanova Olga A , Falcao Alencar Gabriel , Tkachenko Svyatoslav M , Bekiranov Stefan , Owens Gary K
Published: July 2021

Atherosclerosis is the leading cause of death in Western civilization accounting for more than 40% of all deaths. Despite reduced death rates due in part to statin treatment, there is little understanding of the molecular and cellular mechanisms that can help to reverse atherosclerotic development and/or decrease the chances of plaque rupture with associated clinical sequelae such as myocardial infarction or stroke. We recently demonstrated a functional role of the pluripotency factor OCT4 in smooth muscle cells (SMCs) during atherosclerosis, in that SMC-specific conditional
Oct4
knockout mice exhibit marked alterations in lesion size and cellular composition within atherosclerotic lesions of
Apoe
-/-
high-fat diet fed mice, including a thinner fibrous cap, increased necrotic core and increased intra-plaque hemorrhage. Results of SMC-lineage tracing studies show that these changes were likely due to marked reductions in SMC number within lesions including in the fibrous cap area, as well as significant increases in macrophage-like (LGALS3
+
) SMCs within the tunica media. Further studies show that inactivation of
Oct4
in SMCs nearly completely abrogated the pro-atherogenic oxidized phospholipid POVPC-induced migration of SMCs
in vitro
and outgrowth of SMCs from aortic explants
ex vivo.
RNA-seq and ChIP-seq analyses of lesion specimens from
Apoe
-/-
high-fat diet fed mice and cultured SMCs treated with POVPC or 1% O
2
hypoxia show that loss of
Oct4
in SMCs was associated with marked activation of genes associated with inflammation and phagocytosis, and suppression of genes associated with cell migration, including extracellular matrix proteins, matrix metalloproteinases and multiple axon guidance molecules. Moreover, loss of
Oct4
significantly increased ability of SMCs to phagocyte red blood cells in response to oxidized LDL treatment
in vitro
. Results advance our understanding of mechanisms by which OCT4 expression in SMCs impacts atherosclerosis lesion development and may help identify novel potential therapeutic targets for treatment of atherosclerosis.
Abstract 582: The CCR7+ PDGFRa+ Cell Population is Increased in Regressing Atherosclerotic Plaques
Authors: Dierick France , Ebrahimian Talin , Simeone Stefania , Simon David , Michaud Veronique , Lehoux Stephanie
Published: July 2021

Introduction:
Atherosclerosis is characterized by the accumulation of lipids, cells and fibers in the arterial wall. Atherosclerotic plaques form in regions of low blood flow, whereas vessels exposed to high blood flow remain lesion-free. We created a surgical mouse model, arteriovenous fistula (AVF), which increases blood flow in the brachiocephalic artery (BCA) specifically, without altering serum lipid levels.

Methods & Results:
LDLR KO mice were placed on a high-fat diet (HFD). Control mice were sacrificed at week 12. Sham and AVF surgery was performed at week 12 and mice were kept on a HFD for a further 1-4 weeks. We found that high blood flow is beneficial and leads to a significant ~50% regression of BCA plaque size in AVF mice compared with Controls, by week 4. We performed flow cytometry to characterize the different cell populations within Sham and AVF plaques. At day 7 after surgery, there was no difference in macrophage (F4/80+) or dendritic cell (CD11c+) content between Sham and AVF. However, we found a significant, 4-fold increase in the total number of CD45-/CCR7+/PDGFRa+ cells in the BCA plaques of AVF mice vs Shams (p<0.01). No such change was observed in the aortic sinus plaques of AVF or Shams. CCR7 was previously found to be overexpressed in regressing plaques upon an abrupt lowering of plasma lipids, but in CD45+ cells. In our model, plasma lipids remained high and CCR7+ cells instead expressed PDGFRa, a perivascular and multi-lineage differentiation marker. This cell population also expressed mesenchymal stem cell markers (CD90, CD44, CD34) and CD68.

Conclusion:
Our data point to an unexpected increase in the CD45-/CCR7+/PDGFRa+ cell population in the early plaque regression process. They suggest that mesenchymal-type cells may promote regression in plaques exposed to high blood flow.
Abstract 588: Nuanced Antibody Responses to Apolipoprotein A-I in Patients With Cardiovascular Disease
Authors: Henson David M , Venditto Vincent
Published: July 2021

Antibodies targeting apolipoprotein A-I (ApoA-I) have been identified in patients with cardiovascular disease (CVD). Anti-ApoA-I antibodies are thought to be markers of disease, but their exact role is unclear. We hypothesize that antibodies targeting ApoA-I are both protective and pathologic and unraveling the nuanced response to ApoA-I will provide insight into improved risk stratification of patients suffering from CVD. To test our hypothesis we screened serum samples by ELISA collected from patients with CVD to identify anti-ApoA-I antibody responses toward the full length protein along with immunogenic epitopes including the lecithin cholesterol acyl transferase (LCAT) domain and the C-terminal peptide of ApoA-I. These epitopes are of particular interest due to their propensity to undergo oxidative post-translational modification. Antibodies were affinity-purified toward ApoA-I, and their role in reverse cholesterol transport elucidated. Our data indicate that serum collected from patients with CVD enrolled in multiple clinical trials possess a highly nuanced immune response. We find that these antibody responses change over time in some patients who present with an AMI and antibodies correlate with outcomes. The mechanisms of these observed effects are currently under investigation. A full report on correlations between patient characteristic and antibody level will be presented. This work highlights the complexities of anti-ApoA-I antibodies in patients, which will guide development of a CVD risk stratification tool.
Abstract 593: High-Monounsaturated Fat Mediterranean-Type Diet Reduces Foamy Monocyte Formation and Atherosclerosis in Ldlr-/- Mice on High-Cholesterol Diet
Authors: Lian Zeqin , Perrard Xiao-yuan Dai , Lagor William , Sacks Frank , Ballantyne Christie , Wu Huaizhu
Published: July 2021

A large randomized clinical trial (PREDIMED) showed that adding “healthy monounsaturated fat (MUF)” to Mediterranean diet (MedD) by supplementation with extra virgin olive oil or nuts led to a reduction in atherosclerotic cardiovascular events, but the mechanisms remain incompletely understood. Hyperlipidemia, a major risk factor for atherosclerosis, may induce lipid accumulation in circulating monocytes, leading to formation of foamy monocytes (FMs), which contribute to atherosclerosis. We hypothesize that high-MUF MedD reduces FM formation and therefore inhibits atherogenesis associated with hyperlipidemia. To test this, LDLR-/- mice were fed western-type high-saturated fat, high-cholesterol diet (WD) (21% milkfat containing 13.3% saturated fat and 5.9% MUF; 0.2% cholesterol), high-MUF MedD with high cholesterol (HC-MedD, 21% fat [from extra-virgin olive oil, walnuts, almonds, and hazelnuts] containing 2.6% saturated fat and 13.4% MUF; 0.2% cholesterol), or normal diet (ND, control). At 3 months, mice on HC-MedD had similar body weight gain but significantly lower liver/body weight index compared to mice on WD. Plasma triglyceride levels were significantly lower in mice on HC-MedD (318 ± 31 mg/dL, n=13) than on WD (769 ± 60 mg/dL, n=9, P<0.05 vs HC-MedD group). Total cholesterol levels tended to be lower in mice on HC-MedD (2088 ± 180 mg/dL) than on WD (3092 ± 220 mg/dL). Compared to mice on WD, mice on HC-MedD had lower proportions of FMs and lower side scatter values (491 ± 11 vs 555 ± 3 in WD group, n=10/group, P<0.001), indicating less lipid, in FMs. Lipid accumulation in FMs of LDLR-/- on WD accelerated conversion of monocyte subsets from CD11c-CD36+ to CD11c+CD36+, leading to increased ratio of CD11c+CD36+ to CD11c-CD36+ monocytes in mice on WD (2.6 ± 0.3, n=10) vs ND (1.3 ± 0.2, n=9, P<0.01). In contrast, this ratio was not increased in mice on HC-MedD (1.4 ± 0.1, n=10) compared to mice on ND, and was lower than that in mice on WD (P<0.01). Oil red O staining of en face aorta showed 27% decrease in lesion areas in mice on HC-MedD vs on WD (P<0.05). In summary, compared to WD high in saturated fat and cholesterol, high-MUF MedD with high cholesterol lowered triglyceride levels, inhibited FM formation, and reduced atherosclerotic lesion size in LDLR-/- mice.
Abstract 595: Gender Difference Was Present in the Production of Cytokines in Mice With PM Exposure
Authors: Liu Xuanyou , Xiao Yichao , Zhu Qingyi , Jiang Meng , Duan Yunyan , Hao Hong , Abraham Joseph D , Sun Qinghua , Liu Zhenguo
Published: July 2021

Gender difference is present in a variety of diseases especially cardiovascular diseases like coronary artery diseases (
CAD
). It is well known that males tend to suffer from CAD earlier than females for largely unknown reasons. Oxidative stress and inflammation are considered an important mechanism for the development of cardiovascular diseases. Cytokines including interleukin (IL)-6 and tumor necrosis factor-α (TNF-α) play an important role in oxidative stress and inflammation. Ambient fine particulate matter (
PM
) exposure is closely associated with cardiovascular diseases and oxidative stress. The present study was designed to determine if there was a gender difference in the production of cytokines in the mice with PM exposure. Both male and female wild-type C57BL/6 mice (8-10 weeks) were exposed to PM2.5 for 6 weeks via intranasal approach with PBS as the control. Serum concentrations of the cytokines including IL-6, IL-1β, and TNF-α were measured with ELISA in the mice before and after PM exposure. There was no difference between male and female mice in the serum levels of IL-6, IL-1β, or TNF-α at the baseline. As expected, PM exposure substantially increased the serum levels of IL-6, IL-1β, and TNF-α both in male and female mice (by up to 6 times). However, their serum concentrations were significantly higher in male mice than in the females by 64.2%, 26.5%, and 30.7% for IL-6, IL-1β, and TNF-α, respectively (p < 0.05, n = 10). Similar changes in the inflammatory infiltrations in the lungs were observed in the male and female mice with PM exposure. The data from the present study demonstrated that more cytokines were produced in male mice than in the females with PM exposure. The clear gender difference in the serum levels of cytokines in response to PM exposure may partially contribute to the gender difference in the development of cardiovascular diseases. Further studies are needed to investigate the mechanisms related to the gender difference in the response to PM exposure.
Abstract 599: Extracellular Vesicles Secreted by Atherogenic Macrophages Transfer Microrna to Inhibit Cell Migration
Authors: Nguyen My-Anh , Karunakaran Denuja , Geoffrion Michele , Cheng Henry S , Matic Ljubica P , Hedin Ulf , Maegdefessel Lars , Fish Jason E , Rayner Katey J
Published: July 2021

During inflammation, macrophages secrete vesicles carrying RNA, protein and lipids as a form of extracellular communication. In the vessel wall, extracellular vesicles (EVs) have been shown to be transferred between vascular cells during atherosclerosis, however, the role of macrophage-derived EVs in promoting atherogenesis is not known. Here, we
hypothesize
that atherogenic macrophages secrete microRNAs (miRNA) in EVs to mediate cell-cell communication and promote pro-inflammatory and pro-atherogenic phenotypes in recipient cells.

Results:
We isolated EVs from mouse and human macrophages treated with an atherogenic stimulus (oxidized LDL) and characterized the EV-derived miRNA expression profile. Using microarrays and Q-PCR, we confirmed the enrichment of miR-146a, miR-128, miR-185, miR-365 and miR-503 in atherogenic EVs compared to controls. Live-cell imaging demonstrated that macrophage-derived EVs are taken up and transfer exogenous miRNA (
C. elegans)
to naive recipient macrophages. Bioinformatic analysis suggests that atherogenic EV-derived miRNAs are predicted to target genes involved in cell migration and adhesion pathways. Indeed, treatment of naïve macrophages with EVs from atherogenic but not control macrophages inhibited the migration of naïve cells towards a chemokine stimulus (80% decrease in migration, p≤0.01).
In vivo
, delivery of EVs also abolished LPS-induced emigration of mouse peritoneal macrophages
.
Moreover, inhibition of miR-146a (using anti-miR oligonucleotides or miR-146a
-/-
macrophages), the most enriched miRNA in atherogenic EVs, reduced the inhibitory effect of EVs on macrophage migratory capacity. EV-mediated delivery of miR-146a repressed the expression of target genes IGF2BP1 and HuR in recipient cells, and knockdown of IGF2BP1 and HuR using siRNA reduced macrophage migration, highlighting the importance of these EV-miRNA targets in regulating macrophage motility. Notably, expression of miR-146a was elevated in mouse and human atherosclerotic lesions compared to controls.
Thus,
EV-derived miRNAs from atherogenic macrophages, in particular miR-146a, may accelerate the development of atherosclerosis by decreasing cell migration and promoting macrophage entrapment in the vessel wall.
Abstract 6: A Novel
in vivo
Endothelial Hierarchy from Progenitor to Mature Endothelial Cells Reveals Key SoxF-Dependent Differentiation Process
Authors: Patel Jatin , Seppanen Elke J , Rodero Mathieu , Wong Ho Yi , Donovan Prudence , Fisk Nicholas M , Francois Mathias , Khosrotehrani Kiarash
Published: July 2021

Background:
The formation of new blood vessels during adult life is often explained by angiogenesis. However, an alternate proposal now suggests that neo-vessels form from endothelial progenitors able to assemble all the intimal layers of vessel structures. Our aim was to define vessel-resident endothelial progenitors
in vivo
in a variety of tissues in physiological (aorta, lung) and pathological (wounds, tumors) situations.

Methods and Results:
Using common endothelial markers (CD34, CD31, VEGFR2) with flow cytometry, three sub-populations of endothelial cells could be identified among VE-Cadherin+ and CD45- cells. These were termed an endovascular progenitor (EVP) harboring CD31lo VEGFR2lo giving rise to an intermediate CD31intVEGFR2lo transit amplifying (TA) and a definitive differentiated (D) CD31hiVEGFR2hi population. Confirmation of these populations was demonstrated via lineage tracing using

Cdh5cre
ERt2
/Rosa-YFP

reporter mice. Importantly, EVP cells arose from vascular resident beds that could not be transferred by bone marrow transplantation, marking their distinction for hematopoietic/myeloid origin. Furthermore, EVP displayed progenitor like status with a high proportion of cells in a quiescent cell cycle phase. Only EVP cells and not TA and D cells had self-renewal capacity as demonstrated by
in
vitro colony forming and transplant studies
in vivo
in Matrigel
TM
plugs in recipient mice. Through whole RNA sequencing we demonstrated that EVP cells highly expressed genes related to progenitor function such as
Sox9
,
Il33, Egfr
and
Pdfgrα,
whereas D cells highly expressed genes related to differentiated endothelium including
Ets1&2
,
Gata2
,
Cd31, Vwf
and
Notch
. We also determined the
Sox18
transcription factor as having a significant role in defining the endothelial hierarchy, which we validated through lineage-tracing using S

ox18Cre
ERt2
/Rosa-YFP

mice. In the absence of functional SOX18/SOXF, EVP progenitors were still present, but TA and D populations were significantly reduced.

Conclusion:
In summary, we have demonstrated the existence of an entirely novel endothelial hierarchy, from EVP to TA to D. This has been demonstrated by the self-renewal, differentiation and molecular profiling of an EVP.
Abstract 600: Deposition of Endothelial Cell-derived Fibronectin by Alpha5beta1 Integrins Mediates Oxidized LDL-induced Proinflammatory Gene Expression and Early Atherosclerosis
Authors: Yurdagul Arif , Green Jonette M , Al Yafeai Zaki , Murphy Patrick A , Orr A. W
Published: July 2021

Subendothelial basement membrane remodeling to a fibronectin-rich matrix precedes lesion development, and endothelial cell interactions with fibronectin prime inflammatory responses to a variety of atherogenic stimuli. However, the mechanisms regulating early atherogenic fibronectin accumulation remain unknown. We now show that treating endothelial cells with oxidized LDL (oxLDL) potently induces endothelial fibronectin deposition through activation of the fibronectin-binding integrin α5β1, as inhibiting α5β1 (blocking antibodies, α5 knockout cells) completely abrogates oxLDL-induced fibronectin deposition
in vitro
. Inducible endothelial-specific deletion of α5 integrins significantly blunts endothelial proinflammatory gene expression at atheroprone sites and diminishes atherosclerotic plaque formation in ApoE knockout mice, suggesting an important role for this integrin in early endothelial activation. However, endothelial α5 deletion surprisingly enhances fibronectin staining in the endothelial layer, potentially due to leak of plasma-derived fibronectin. Plasma fibronectin differs from cell-derived fibronectin due to the lack of two alternatively spliced domains, termed extradomain A (EIIIA) and extradomain B (EIIIB). Interestingly, loss of endothelial cell-derived fibronectin (fibronectin siRNA) completely inhibits oxLDL-induced VCAM-1 expression, and only rescue with cell-derived fibronectin, but not plasma fibronectin, reverses this inhibitory effect. This effect appears to be due to the presence of EIIIA and EIIIB domains, as endothelial cells isolated from EIIIA/EIIIB knockout mice also show reduced oxLDL-induced proinflammatory responses. However, it is unclear whether this response is due to specific EIIIA/EIIIB receptors or due to altered integrin signaling, as limiting the presence of cell-derived fibronectin prevents integrin α5 localization to focal adhesions and reduces fibronectin fibril length. Taken together, our data demonstrate that oxLDL stimulates α5 integrin-dependent deposition of endothelial-derived fibronectin into the subendothelial matrix and suggest that the source of fibronectin regulates the subsequent endothelial cell proinflammatory responses.
Abstract 601: Global and Liver Specific Bmal1 Deficient Mice Regulate Intestinal Lipid Absorption
Authors: Pan Xiaoyue , Hussain Mahmood
Published: July 2021

Circadian rhythms controlled by clock genes affect plasma lipids, known risk factors for atherosclerosis. Clock and Bmal1 are critical clock genes that positively regulate circadian rhythms. We have shown that Clock plays an important role in lipid absorption. Additionally, we showed that global
ablation
of
Bmal1
in

Apoe
–/–

and

Ldlr
–/–

mice, and liver-specific ablation of Bmal1 in

Apoe
–/–

(L

-Bmal1
–/–
Apoe
–/–

) mice increases atherosclerosis. However, it is unknown whether Bmal1 regulates intestinal lipid absorption.

We studied cholesterol and triglyceride absorption in normal and lipase-inhibited

Bmal1
–/–

and

Bmal1
–/–
Apoe
–/–

mice with global Bmal1 deficiency and compared it with

Bmal1
+/+

and

Bmal1
+/+
Apoe
–/–

, respectively. To study the effect of liver-specific Bmal1 deficiency, studies were performed in L-

Bmal1
–/–

and L-

Bmal1
–/–
Apoe
–/–

mice and data were compared with

Bmal1
fl/fl

and

Bmal1
fl/fl
Apoe
–/–

mice. In addition, enterocytes isolated from these mice were used to study uptake and secretion of cholesterol and oleic acid. Further, protein and mRNA levels of candidate genes involved in lipid uptake, lipoprotein assembly and secretion were quantified.

Both normal and lipase-inhibited

Bmal1
–/–

mice absorbed significantly higher amounts of cholesterol and triglyceride. Further, uptake and secretion of cholesterol and fatty acids by enterocytes was increased in

Bmal1
–/–

mice. As reported previously, liver-specific Bmal1 ablation increased VLDL production. Surprisingly, postprandial plasma lipids were also significantly increased in L-

Bmal1
–/–

mice than in control

Bmal1
fl/fl

mice. Further, enterocytes isolated from L-

Bmal1
–/–

took up more lipids and secreted more lipoproteins. Moreover, in vivo lipid absorption in L-

Bmal1
–/–

was increased. Gene expression quantifications revealed that intestinal MTP, DGAT1, CD36 and NPC1L1 mRNA levels were increased in global and hepatic Bmal1 deficient animals.

Global and liver-specific ablation of Bmal1 increases intestinal lipoprotein production by increasing the expression of genes involved in lipid uptake and lipoprotein assembly. These data for the first time suggest that intestinal lipoprotein assembly is regulated by hepatic Bmal1.
Abstract 608: Macrophage-Associated Lipin-1 Contributes to Atherosclerosis
Authors: Vozenilek Aimee E , Navratil Aaron R , Finck Brian , Orr A. W , Woolard Matthew D
Published: July 2021

Objective:
Macrophage pro-inflammatory responses induced by oxidized low-density lipoproteins (oxLDL) mediate atherosclerosis progression. However, how oxLDL causes macrophages to become pro-inflammatory is still enigmatic. Macrophage foam cell formation induced by oxLDL requires glycerolipid synthesis. Lipin-1, a key enzyme in the glycerolipid synthesis pathway, contributes to oxLDL-elicited pro-inflammatory responses in a macrophage cell line. The objective of this study was to determine if myeloid-associated lipin-1 contributes to atherogenesis and asses its role in oxLDL-mediated signaling in macrophages

Approach and Results:
We developed mice lacking lipin-1 in myeloid cells and used adeno-associated viral vector 8 expressing the gain-of-function mutation of mouse proprotein convertase subtilisin/kexin type 9 (AAV8-PCSK9) to induce hypercholesterolemia and plaque formation. Mice lacking myeloid-associated lipin-1 had reduced atherosclerotic burden compared to control mice despite similar plasma lipid levels. Stimulation of bone marrow-derived macrophages with oxLDL activated a persistent PKCα/βII-ERK1/2-cJun signaling cascade that contributed to macrophage pro-inflammatory responses. Bone marrow-derived macrophages lacking lipin-1 failed to activate this PKCα/βII-ERK1/2-cJun signaling cascade.

Conclusions:
Our data demonstrates that myeloid-associated lipin-1 is atherogenic, likely through persistent activation of a PKCα/βII-ERK1/2-cJun signaling cascade that contributes to foam cell pro-inflammatory responses. Taken together these results suggest that oxLDL-induced foam cell formation and oxLDL-induced macrophage pro-inflammatory responses are not separate outcomes of oxLDL-stimulation of macrophages, but rather lipid synthesis that contributes to foam cell formation also influences macrophage inflammatory responses.
Abstract 613:
In Vitro
Microvessels to Study the Platelet-Endothelium Interface
Authors: Zilberman-Rudenko Jevgenia , Wong Andrew D , Sallee Daniel E , Reitsma Stephanie E , Puy Cristina , Shirai Toshiaki , Mitrugno Annachiara , Searson Peter C , McCarty Owen J
Published: July 2021

Background:
Under normal conditions, endothelial cells (ECs) govern blood flow dynamics including providing a barrier between blood and tissue and regulating platelet aggregation and thrombin generation in the bloodstream. In turn, blood components, primarily platelets and coagulation factors such as thrombin, regulate EC barrier integrity. The breakdown of EC barrier function is a hallmark of a variety of vascular diseases. In sepsis, for example, the dysfunction of vascular ECs has been correlated with poorer outcomes due to hemorrhage and multi-organ failure associated with consumption of platelets and coagulation factors into clots within the microcirculation, a condition termed disseminated intravascular coagulation (DIC).

Aim:
Develop an endothelialized flow chamber to study the platelet-endothelium interface.

Methods and Results:
We developed a 3D-chamber with a perfuseable cylindrical microvessel embedded in an extracellular matrix (ECM) material. This model allows for the study of the role of thrombin generation and platelet aggregation in endothelial barrier leak development and repair in healthy as well as inflamed microvessels. Incorporation of subendothelial matrix proteins in these 3D-microvessel devices expands the capacity of the microfluidic studies to investigate blood cell extravasation and enables the control of physical parameters such as transmural pressure and interstitial flow through the ECM.

Conclusion:
This model may provide insight into the pathophysiology of different disease states and serve as an expedient platform for therapy design and testing.

The platelet-endothelium interface under shear flow.
Diagram (
A
) and an experimental prototype (
B
) of a 3D-perfuseable device. Microvessel phenotype (following treatment with vehicle or 10 ng/mL TNFα) pre- and post- perfusion with recalcified whole blood for 33 min as visualized by differential interference contrast, DIC, (
C
) and fluorescence microscopy (
D
).
Abstract 616: Potentiation of Endothelium-Dependent Vasodilation by Adenylate Cyclase Type 9 Inactivation is Associated With Increased Endothelial Cell Signaling in Mouse Femoral Arteries
Authors: Rautureau Yohann , Raigneault Adeline , Kurunradeth Uy , Rivas Daniel , Higgins Marie-Eve , Thorin Eric , Rhéaume Eric , Tardif Jean-Claude
Published: July 2021

Adenylate cyclase (AC) activity in vessels is associated with vasodilation. The AC family includes 10 members of which AC9 is the least studied. We recently demonstrated that
Adcy9
inactivation in mice protects from atherosclerosis in the aorta and potentiates endothelium-dependent vasodilation (EDV) to acetylcholine (ACh) in femoral arteries (FA). Our objectives were 1) to determine
Adcy9
expression in FA; 2) investigate endothelial signalling pathways associated with
Adcy9
inactivation-induced potentiation of ACh-induced EDV; and 3) to assess the effect of
Adcy9
inactivation on EDV during flow-mediated dilation (FMD).

FA were isolated from 8 to 13 week-old
Adcy9-
inactivated (

Adcy9
Gt

) and wild-type (WT) littermate mice.
Adcy9
expression was studied in WT FA tissue section by
in situ
hybridization (ISH) using
Adcy9
and negative control probes. Diameter of pressurized (80 mmHg) FA (internal diameter 288±3 μm) was studied in response to ACh (n=7-14) and shear stress (n=12) up to 20 dynes/cm
2
after preconstriction with phenylephrine. To study the role of endothelial signalling pathways in response to ACh-induced EDV, FA were incubated in absence or presence of inhibitors for nitric oxide (NO) synthase (L-NNA 0.1 μM), cyclooxygenase (meclofenamate 1 μM) and endothelial hyperpolarization (TRAM-34 10 μM plus apamin 0.1 μM).

ISH with
Adcy9
probe resulted in numerous dots in the vascular wall of the FA. Meclofenamate and the combination of TRAM-34 plus apamin decreased maximal (E
max
) EDV to ACh, respectively, by 40 and 17% in
Adcy9
Gt
(P<0.01) but had no effect in WT mice. L-NNA reduced E
max
to ACh by 26% in WT (P<0.01) and 50% in
Adcy9
Gt
(P<0.01).
Adcy9
inactivation tended to increase FMD to shear stress ranging from 6 to 20 dynes/cm
2
: At 10 dynes/cm
2
, FMD was increased from 25.4±4.9% in WT to 37.4±4.9% in
Adcy9
Gt
mice (P=0.083, n=12 per group).

In conclusion, our data show that FA express
Adcy9
and that
Adcy9
inactivation increases the effects of endothelial NO, hyperpolarization and cyclooxygenase pathways in EDV. A numerical increase in FMD in Adcy9
Gt
compared to WT confirms the potentiating effect of
Adcy9
inactivation on EDV. The potentiation of endothelial dilatory pathways appears to be associated with the anti-atherosclerotic effects of
Adcy9
inactivation.
Abstract 617: Inhibitors of Beta-Catenin Signaling Limit Growth of Vascular Smooth Muscle Cells
Authors: Riascos-Bernal Dario F , Gross Jordana N , Chinnasamy Prameladevi , Sibinga Nicholas E
Published: July 2021

Smooth muscle cell (SMC) growth is essential for artery formation during development and significantly contributes to neointima formation after vascular injury in adulthood. We recently showed, using a tissue-specific deletion and knock-in of mutant alleles, that beta-catenin (b-ctn) signaling function is essential for SMC growth and artery formation; moreover, protein interactions mediated by its C-terminus domain are required for assembly of the arterial wall, while b-ctn N-terminus interactions are dispensable. Inhibitors of b-ctn have been developed, but their effects on vascular SMC growth have not been fully tested. We hypothesize that inhibitors that disrupt protein interactions mediated by the b-ctn C-terminus domain will impair b-ctn signaling and limit growth of vascular SMCs. We evaluated growth of mouse aortic SMCs and human coronary artery SMCs in culture using AlamarBlue after exposure to increasing concentrations (0.01 to 10 micromolar) of several validated b-ctn inhibitors or vehicle control: PKF118-310 (disrupts the b-ctn/TCF interaction), ICG001 (disrupts the b-ctn C-terminus/CBP interaction), XAV939 (promotes the b-ctn destruction complex), and carnosic acid (disrupts the b-ctn N-terminus/BCL9 interaction). We also evaluated the effect of these inhibitors on b-ctn transcriptional activity in SMCs using a TOPflash reporter system. We found that PKF118-310 (p<0.05 vs. vehicle), ICG001 (p<0.05 vs. vehicle), and XAV939 (p<0.05 vs. vehicle), but not carnosic acid, limit mouse (n=16 independent cultures) and human (n=8 independent cultures) SMC growth in a dose-response manner. PKF118-310 exhibited the most potent inhibitory effect. We also found that PKF118-310, ICG001, and XAV939, but not carnosic acid, inhibit b-ctn transcriptional activity in arterial SMCs in culture. In conclusion, pharmacological inhibition of b-ctn signaling, particularly blocking the b-ctn C-terminus output, inhibits growth of vascular SMCs in culture, providing a rationale to test these inhibitors in models of vascular injury as they hold promise as novel therapies for cardiovascular disease.
Abstract 618: Hypoxia Inducible Factor 1a Stabilization in Autosomal Dominant Hyper IgE Syndrome Fibroblasts Rescues Impaired Ability to Support Angiogenesis
Authors: Grubb Alex F , Dmitrieva Natalia I , Walts Avram , Chen Guibin , Zhang Xue , Wang Xujing , Freeman Alexandra F , Holland Steve M , Boehm Manfred
Published: July 2021

Background:
Autosomal dominant Hyper-IgE syndrome (AD-HIES) is a rare primary immunodeficiency caused by dominant negative mutations in signal transducer and activator of transcription 3 (STAT3
)
, a mediator of widespread physiological processes. It is characterized by dermatitis, recurrent infections, elevated IgE, poor post-surgical healing, and connective tissue abnormalities. How STAT3 deficiency leads to this phenotype, however, is not known. Current treatment options are limited to antimicrobials for infection control. The aim of this study was to investigate which of STAT3’s many functions are dis-regulated in AD-HIES, and where potential targets for therapy may lie.

Methods:
We used skin fibroblasts (SF) from 3 AD-HIES patients and 3 normal volunteers. To evaluate potentially affected pathways, we utilized RNA- Seq and subsequent Gene Set Enrichment (GSEA) and pathway analysis (Pathway Studio, GeneGo Metacore). Endothelial cell tube formation assay was used to assess ability of AD-HIES SFs to support angiogenesis.

Results:
GSEA and pathway analysis showed deficiencies in signaling pathways linked to wound healing, extracellular matrix remodeling and angiogenesis including targets of Hypoxia Inducible Factor 1a (HIF1a) (P values for enrichments < 0.001)
.
Therefore, we hypothesized that AD-HIES SFs have impaired ability to support angiogenesis due to deficient Hif1a-dependent secretion of matrix proteases and growth factors. Indeed, AD-HIES SF secreted up to 5 times less matrix metalloprotease 1, 3, and 9, placental growth factor and fibroblast growth factors 1 and 2 (Luminex Multiplex, n=3-9, P<0.05). Culture medium from AD-HIES SFs failed to fully support tube formation by endothelial cells resulting in lower number of junctions, meshes, and total tubule length (n=6, P<0.005). Stabilization of Hif1a in AD-HIES SFs by prolyl hydroxylase inhibitor dimethyl fumarate restored its transcriptional activity leading to increased number of junctions, meshes, and tubule length (n=12, P<0.05)

Conclusion:
AD-HIES SFs have deficiencies in pro-angiogenic signaling pathways that lead to decreased growth factor secretion and angiogenesis. Stabilization of HIF1a corrects this deficiency and is an enticing target for future therapy.
Abstract 621: Basic Fibroblast Growth Factor Helps to Maintain the Stemness of Rat Multipotent Adult Progenitor Cells Partially via Erk1/2 Signaling
Authors: Zhu Qingyi , Jiang Meng , Liu Xuanyou , Xiao Yichao , Hao Hong , Joseph Abraham , Zhou Shenghua , Guan Jiangjun , Liu Zhenguo
Published: July 2021

Bone marrow mesenchymal stem cells are one of the important sources for cell replacement therapies. The outcome of the cell-based therapies is determined by a variety of factors including the source of the cells and their functional status. The functional status is usually measured by the expression of stem cell specific marker Oct-4 (stemness), differentiation potential (multipotency), and production of paracrine factors. It could be beneficial to keep the stem cells in a multipotent state for their optimal therapeutic outcome. The present study was to investigate the effect of basic fibroblast growth factor (bFGF) on the stemness of bone marrow stem cells and related mechanism. Rat multipotent adult progenitor cells (MAPCs) were used as the source of bone marrow stem cells, and induced to differentiate in vitro with (bFGF:5ng/ml and 20ng/ml) and without bFGF for up to 7 days. The expression of Oct4 and endothelial markers including Flk1, VWF and CD31, and smooth muscle cell markers including α-SMA, SM22 and CNN1 were determined with real-time (RT) PCR and western blot. We observed that both transcriptional (as reflected by RT-PCR) and protein expression (by western blot) of Oct4 was maintained at relatively stable levels in the cells treated with bFGF during the early phase of differentition. On the other hand, the expression of Flk1, VWF, CD31 and α-SMA, SM22, CNN1 were significantly decreased in the cells with the presence of bFGF. Treatment with bFGF significantly increased the phosphorylation of Erk1/2, not Akt nor STAT3, in the cells. Inhibition of Erk1/2 phosphorylation with PD98059 partially but significantly attenuated the effect of bFGF on Oct4 expression in the cells. These data suggest that bFGF could be able to maintain the bone marrow stem cells in their undifferentiated state in vitro partially through Erk1/2 signaling-mediated mechanism.
Abstract 623: Hyperhomocysteinemia Potentiates Inflammatory Monocyte Differentiation and Vascular Dysfunction in Type 2 Diabetic Mice
Authors: Fang Pu , Jiang Xiaohua , Yang Xiaofeng , Wang Hong
Published: July 2021

Hyperhomocysteinemia (HHcy) is associated with increased diabetic cardiovascular diseases. In this study, we investigated the effects of type 2 diabetes mellitus (T2DM), HHcy and their combination on inflammatory monocyte (MC) differentiation and vascular function.

We established an atherosclerosis-susceptible T2DM+HHcy compound mouse model. T2DM was induced in db/db mice by spontaneous mutation (average blood glucose 493 mg/dL). HHcy was established by feeding the mice a high fat+high methionine diet (HF+HM, 8 weeks). Methionine is the homocysteine (Hcy) precursor, which elevated plasma Hcy level to 129 μM. Plasma Hcy level was lowered by vitamin therapy employing a HF+HM+high vitamin diet (HF+HM+HV), which reduced plasma Hcy levels from 129 μM to 42 μM. HHcy aggravated T2DM-impaired endothelial-dependent vessel relaxation to acetylcholine, which was completely abolished by endothelial nitric oxide synthase (eNOS) inhibitor N
G
-nitro-L-arginine methyl ester. HHcy potentiated T2DM-induced inflammatory monocytes (CD45
+
CD11b
+
Ly6C
+
MCs) in aorta isolated from compound mice. Severe HHcy- and T2DM-induced mononuclear cells (MNCs), CD11b
+
MCs, CD11b
+
Ly6C
middle+high
inflammatory MCs and M1 macrophages (MØs) were potentiated by the combination of HHcy and T2DM in mouse bone marrow (BM), peripheral blood, and spleen. Folate based Hcy-lowering therapy (HF+HM+HV) reversed systemic MNC, MC, inflammatory MC and MØ and aortic inflammatory MC increases in T2DM mice. Finally, bone marrow transplantation (BMT) using lentivirus-shLy6C transduced BM cells, which decreased blood Ly6C
+
inflammatory MCs, resulted in ameliorated endothelial-dependent vessel relaxation. In conclusion, our data suggested that HHcy accelerated T2DM-induced systemic inflammatory MC and MØ differentiation, aortic inflammatory MC infiltration, and vascular dysfunction.
Abstract 632: Aortic Dilatation in Marfan Syndrome: A Result of Amplification of Molecular Mechanisms of Aging?
Authors: Hagler Michael A , Casaclang-Verzosa Grace , Zhang Bin , Roos Carolyn , Thalji Nassir , Miller Jordan
Published: July 2021

Marfan syndrome (MFS) is a genetic disease with a mutation for the microfibrillar constituent protein fibrillin-1 being the most prevalent. MFS is often associated with progressive aortic root dilation, ultimately progressing to aortic aneurysm and dissection. While recent work has shown that increased angiotensin-II receptor type-1 and transforming growth factor beta (TGF-β) signaling contributes to aneurysm formation in aorta, efficacious therapeutic targets remain elusive. Given previous reports of progeriod phenotypes in a subset of Marfan patients, we sought to determine whether there are molecular changes that are consistent with accelerated aging in a mouse model of Marfan syndrome. In mice carrying a loss-of-function mutation in fibrillin-1, we assessed aortic root dimensions by echocardiography and gene expression levels of TGF-β1-3, runt related transcription factor 2 (RUNX2), and the cellular senescent marker CDKN2A by quantitative real time PCR at 3 and 14 months of age. As expected, aortic sinus dimensions did not change significantly with aging in wild type mice, but increased dramatically in fibrillin-1 mutant mice compared to wild-type littermate controls and with age (p < 0.05 for both). TGF-β1 ligand expression paralleled age and disease-dependent changes in aortic dimensions, however TGF-β2 and TGF-β3 mRNA levels did not. Aortic dilatation was associated with increased gene expression of RUNX2 with aging and in marfanoid mice. Interestingly, fibrillin-1 mutant mice demonstrated marked increases in expression of the anti-proliferative cell-cycle checkpoint protein CDKN2A at both time points, and correlated with changes in TGF-β1 (R
2
=0.54) and RUNX2 (R
2
=0.69) mRNA. CDNK2A gene expression patterns, however, demonstrated a poor correlation with expression of TGF-β2 and TGF-β3 (R
2
=0.04 and 0.06. respectively). Collectively, these data lend insight into novel mechanisms that may regulate development of aortic root dilation in patients MFS and are the first to implicate increased senescent cell burden in Marfan syndrome. Furthermore, we propose that clearance of senescent cells could be a viable therapeutic intervention to slow progression aortic root-dilation and aneurysm in patients with Marfan syndrome.