bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 568: Deletion of AMP-activated Protein Kinase-alpha Triggers Mitochondrial Fission and Endothelial Dysfunction
Authors: Wang Qqilong , Xie Zhonglin , Zhu Huaiping , Ding Ye , Zou Ming-Hui
Published: June 2021

Introduction:
AMP-activated protein kinase (AMPK) has been reported to regulate mitochondrial biogenesis, function, and turnover. However, the molecular mechanisms by which AMPK regulates mitochondrial dynamics remain poorly characterized. We hypothesized that AMPK deficiency regulates mitochondrial fission that will result in endothelial dysfunction.

Methods/Results:
Deletion of AMPKα2 resulted in defective autophagy, dynamin-related protein (Drp1) accumulation, and aberrant mitochondrial fragmentation in the aortic endothelium of mice. Furthermore, autophagy inhibition by chloroquine treatment or Atg7 small interfering RNA (siRNA) transfection upregulated Drp1 expression and triggered Drp1-mediated mitochondrial fragmentation. In contrast, autophagy activation by overexpression of Atg7 or chronic administration of rapamycin, the mammalian target of rapamycin kinase inhibitor, promoted Drp1 degradation and attenuated mitochondrial fission in AMPKα2
-/-
mice, suggesting that defective autophagy contributes to enhanced Drp1 expression and mitochondrial fragmentation. Interesting, the genetic (Drp1 siRNA) or pharmacological (mdivi-1) inhibition of Drp1 ablated mitochondrial fragmentation in the mouse aortic endothelium and prevented the acetylcholine-induced relaxation of isolated mouse aortas from AMPKα2
-/-
mice. This suggests that aberrant Drp1 is responsible for enhanced mitochondrial fission and endothelial dysfunction in AMPKα knockout mice.

Conclusions:
Our results show that AMPKα deletion promoted mitochondrial fission in vascular endothelial cells by inhibiting the autophagy-dependent degradation of Drp1.
Abstract 573: Vibrational Spectroscopy Discriminates Differentiated Vascular Smooth Muscle Cells From Mesenchymal Stem Cells and Their Vascular Progeny
Authors: Molony Claire , Di Luca Mariana , McIntyre Jennifer , Hennelly Bryan , Byrne Hugh J , Cahill Paul A
Published: June 2021

Background:
The source of intimal vascular smooth muscle cells (SMCs) following vascular remodelling has been controversial, with either de-differentiated SMCs and/or stem cell-derived SMCs playing a putative role. Fourier transform Infrared (FTIR) and Raman spectroscopy are complementary forms of vibrational spectroscopy which provide an excellent platform for extracting important biochemical data in a label-free manner to discriminate cell types.

Aim:
Determine whether native differentiated SMCs can be distinguished from mesenchymal stem cells (MSCs) and MSC-derived SMCs using vibrational spectroscopy.

Methods:
Freshly isolated rat aortic differentiated SMCs (up to passage 4), CD44+ bone marrow derived mesenchymal stem cells (MSCs), MSC-derived smooth muscle cells (mdSMCs - after TGF-β1 treatment for 14 d) and osteoblasts (mdOSTs - after osteogenic inductive stimulation for 21 d) were grown and fixed on calcium fluoride slides before their respective spectra were recorded by Raman and FTIR Spectroscopy. Multivariate statistical algorithms, including Principal Components Analysis (PCA) and Linear Discriminant Analysis (LDA), were applied to the spectra in order to classify the cell types based on their biochemical variation.

Results:
The recorded spectra for each cell type revealed significant visible differences between the cells across all recordings. The PCA score plot discriminated the cells based on their unique characteristics. A combination of PCA-LDA were applied for classification, and a leave one out cross validation resulted in sensitivities and specificities that were >95%.

Conclusion:
Vibrational spectroscopy discriminates differentiated SMCs from MSC and their vascular progeny and may be useful for identifying these cells as early biomarkers of disease.
Abstract 575: Acute Exposure to Apolipoprotein AI Inhibits Macrophage and Macrophage Chemotaxis i
n vitro
and Recruitment i
n vivo
Authors: Barrett Tessa J , Iqbal Asif J , Taylor Lewis , McNeill Eileen , Manmadhan Arun , White Gemma E , Cooper Dianne , DiDonato Joseph A , Hazen Stanley L , Greaves David R , Fisher Edward A
Published: June 2021

Background:
Apolipoprotein (apo) A-I is the major protein component of high-density lipoprotein (HDL) and is thought to be anti-inflammatory. We hypothesized that apoA-I-mediated inhibition of myeloid cell recruitment to inflammatory factors and sites was a contributing mechanistic basis to its anti-inflammatory role.

Methods and Results:
Acute (20-60 min) human apoA-I ((hapoA-I), 40 μg/mL) treatment induced a significant (50-90%, P<0.01) decrease in murine macrophage chemotaxis to a range of pathophysiologically relevant chemoattractants (CCL2, CCL5, chemerin and C5a). In addition, human peripheral blood mononuclear cells exposed to hapoA1 for 30 min showed reduced rolling, adhesion and transmigration to activated endothelial cell monolayers under physiological flow (20-45%, P<0.05). Notably, acute hapoA-I treatment was anti-inflammatory
in vivo
as shown by pre-treatment of monocytes with 40 μg/mL hapoA-I for 45 min prior to adoptive transfer into an on-going peritonitis model. Sixteen hours post zymosan challenge, pre-treatment resulted in a 65% decrease in monocyte recruitment to the peritoneum (P<0.01). Furthermore, in a chronic setting, mice overexpressing hapoA1 (3-fold increase) had a 27% reduction in total leukocyte recruitment (P<0.05) to the peritoneum following a 16 h zymosan challenge. Chemotaxis requires the reorganization of the actin-cytoskeleton is regulated by PI3K/Akt signaling complexes concentrated in lipid rafts. We find that hapoA-I disrupts lipid rafts by removing membrane cholesterol in a ABCA1/G1 independent manner, and as a consequence, dampens the PI3K/Akt signaling pathway (32% reduction, P<0.01)

Conclusions:
Acute hapoA-I treatment leads to suppression of monocyte-derived cell chemotaxis both
in vitro
and
in vivo
, by depleting lipid raft cholesterol, likely by enhancing aqueous diffusion. We propose that this dampens PI3K/Akt signaling pathways that coordinate reorganization of the actin cytoskeleton and cell migration. Our data provide a potential mechanistic basis for therapeutic apoA-I infusions in situations where reduced monocyte recruitment to sites of inflammation could have beneficial cardiovascular outcomes (e.g., ischemia-reperfusion injury, ischemic stroke).
Abstract 578: Na/K-ATPase Forms a Signaling Complex with CD36 and Toll-like Receptor 4 to Mediate NF-kappaB Activation in Macrophages
Authors: Chen Yiliang , Huang Wenxin , Yang Moua , Silverstein Roy
Published: June 2021

Cardiotonic steroids such as ouabain, digoxin, and marinobufagenin are known ligands for the plasma membrane receptor, Na/K-ATPase (NKA). These ligands stimulate complex formation of NKA with other membrane and cytosolic proteins leading to cellular events such as activation of kinase cascades and gene transcription independent of the ion transporting function. Endogenous cardiotonic steroids have been detected within human circulation and their levels are elevated in patients with diseases associated with chronic systemic inflammation. However, the role of cardiotonic steroids and NKA-mediated signaling in inflammation has not been well studied.

We now report that ouabain activates the NF-κB pathway in human monocyte-derived macrophages, leading to pro-inflammatory responses. Using Western blot and densitometry analysis, we found that physiological concentrations of ouabain stimulated IκBα degradation (n=3). Using quantitative RT-PCR (n=4), we found that ouabain increased mRNA levels of pro-inflammatory genes MCP-1 (3.2±1.1 fold), TNF-α (59.7±35.6 fold), CXCL-10 (2.8±1.6 fold), ICAM1 (2.9±0.5 fold), and PTGS2 (3.1±0.1 fold). Consistent with the increase in mRNA level, MCP-1 protein levels in post-culture media assessed by ELISA were elevated by 1.4±0.1 fold (n=4).

Mechanistically, when macrophages were incubated with ouabain for 20 min and then subjected to IP, we found that antibodies to NKA co-precipitated more TLR4 (2.2±0.8 fold) compared to unstimulated cells. Additionally, antibodies to CD36 co-precipitated more NKA (1.8±0.6 fold) and more TLR4 (2.2±0.5 fold) (n=3). Blockade of TLR4 signaling using a specific inhibitor, CLI-095, abolished ouabain-induced MCP-1 production as assessed by ELISA (n=4). Next, we used siRNA to specifically knock down either NKA α1, CD36, TLR4 or MyD88 (the adaptor protein downstream of TLR4) in THP-1 cells stably transfected with a NF-κB reporter construct. After siRNA transfection for 48h, cells were treated with ouabain for additional 24h. Knockdown of any one of these proteins fully suppressed ouabain-induced NF-κB activation by NF-κB reporter assay (n=4). In conclusion, ouabain activates NF-κB through an NKA/CD36/TLR4 complex leading to pro-inflammatory responses in human macrophages.
Abstract 594: Long Non-coding RNA Regulation of the Interleukin-1 Gene Family Cluster
Authors: Rinehold Kaitlyn , vanSolingen Coen , Hennessy Elizabeth J. , Moore Kathryn J.
Published: June 2021

Long non-coding RNAs (lncRNAs) are a poorly understood class of regulatory RNAs, known to be highly species specific and capable of binding DNA, RNA, and/or protein to regulate transcriptional and epigenetic networks. Although thousands of lncRNAs have been identified in disease-related screens, only a few have been functionally characterized. Here we identify a primate-specific lncRNA, CHROME, located on human chr2 in a locus associated with premature cardiovascular disease, which regulates the expression of the IL-1 family of cytokines. Using RNA fluorescence in situ hybridization (FISH) and cell fractionation, we show that CHROME is abundant in the nucleus. Furthermore, its expression is induced in primary human macrophages treated with pro-inflammatory Toll-like receptor (TLR) agonists. To investigate CHROME function, we used RNA immunoprecipitation (RIP) to map RNA-protein interactions, and found that CHROME has strong histone binding affinity, suggesting a potential role in gene regulation. Notably, repression of CHROME using anti-sense oligonucleotides leads to a decrease in expression of genes in the IL-1 family gene cluster located nearby on the q arm of chromosome 2, including
IL1A
,
IL1B
and
IL1RL1
. These data suggest that CHROME may act as a scaffold for chromatin modification of the IL-1 locus, thereby regulating transcription of these potent inflammatory cytokines. This hypothesis is currently being tested using ChIRP (chromatin isolation by RNA purification) coupled with high throughput DNA sequencing and mass spectrometry. As studies support a causal role for IL-1α and IL-1β in the promotion of atherosclerosis, further characterization of CHROME and its regulatory network may uncover novel opportunities for therapeutic intervention in cardiovascular diseases, and other inflammatory diseases in which IL-1 cytokines predominate.
Abstract 600: Activation of p53 in Pulmonary Arterial Endothelial Cells with Dysfunctional BMPR2 Rescues DNA Damage, Apoptosis and Persistent Pulmonary Hypertension
Authors: Hennigs Jan K , Li Caiyun Grace , Miyagawa Kazuya , Roughley Matthew , Elliott Matthew V , Cao Aiqin , Diebold Isabel , Wang Lingli , Rabinovitch Marlene
Published: June 2021

Pulmonary arterial (PA) hypertension (PAH) is a life-threatening disease characterized by loss of microvessels, the result of endothelial cell (EC) apoptosis associated with dysfunction or a mutation in BMPR2. We identified p53 as critical in the formation of a DNA damage-inducible transcriptionally active complex with PPARγ, an important downstream factor of BMPR2 signaling. The p53-PPARγ complex regulates genes promoting PAEC homeostasis. Upon reoxygenation after hypoxia, PAEC with defective BMPR2 fail to stabilize p53 leading to mitochondrial dysfunction, DNA damage, endothelial apoptosis and persistent pulmonary hypertension (PH) in mice. We therefore hypothesized that in PAEC, pharmacological activation of p53 by the small molecule compound Nutlin-3 would stabilize the PPARγ-p53 complex downstream of BMPR2 to reverse the PH phenotype. Indeed, we showed that in PAEC transfected with BMPR2 siRNA, disruption of the PPARγ-p53 complex upon reoxygenation was prevented by Nutlin-3 treatment. This was associated with a reduction in DNA damage as assessed by Histone 2AX phosphorylation. In PAEC isolated from PAH patients with a BMPR2 mutation, Nutlin-3 treatment induced repair of prevalent DNA damage as determined by the Comet assay, and improved PAEC survival as measured by Caspase 3/7 activity. In PAEC with a BMPR2 mutation, Nutlin treatment stabilized p53, and induced PPARγ-p53 complex formation and expression of target genes, i.e., apelin (a pro-angiogenic factor), GADD45A (a DNA repair enzyme) and p21 (a cell cycle inhibitor) as detected by quantitative PCR and western immunoblots. In EC-specific BMPR2 knockout mice, we have observed persistent chronic hypoxia induced PH even after a month of reoxygenation in room air. We now show that this can be reversed by daily i.p. treatment with Nutlin-3a (12 mg/kg) during reoxygenation. In conclusion, pharmacological stabilization of p53 by Nutlin-3 in PAEC with dysfunctional BMPR2 induces repair of DNA damage, improves PAEC survival and rescues persistent pulmonary hypertension. These results auger well for pursuing the use of Nutlin-3 in pulmonary hypertension and other vascular diseases associated with DNA damage such as atherosclerosis.
Abstract 605: Bone Morphogenetic Protein Inhibitors Play Important Roles in Brown and White Adipogenesis
Authors: Blazquez-Medela Ana M , Jumabay Medet , Bostrom Kristina I
Published: June 2021

Adipose tissue is intimately connected to the development of metabolic syndrome, type 2 diabetes and cardiovascular disease. The near pandemic increase in incidence and prevalence of obesity makes it necessary to understand adipose regulation in order to develop strategies against obesity and its comorbidities.

Adipose tissue performs a variety of functions. White adipose tissue (WAT) is responsible for energy storage, hormone production and organ protection, whereas brown adipose tissue (BAT) has a key role in thermogenesis.

Previous studies have shown the importance of Bone Morphogenetic Proteins (BMPs) in the differentiation of brown and white adipocytes. BMP4 has been linked to both brown and white adipogenesis and BMP7 is essential for brown adipogenesis. Nevertheless, the role of the BMP inhibitors, which modulate the actions of BMPs, has not yet been elucidated. Our hypothesis is that the BMP inhibitors play important roles in the adipogenic process.

Using two different mice models -Matrix Gla Protein (Mgp) KO and Noggin fat-specific KO- we found that the absence of Mgp resulted in dramatic adipose changes, suggesting a critical role in the separation of white and brown adipocytes. Adipocyte-specific deletion of Noggin, however, suggested a role in modulating the size and lipid accumulation in fully differentiated adipocytes. The absence of Mgp led to a reduction in body fat and body weight (3-12 animals per group and time point) whereas the absence of Noggin caused an increase in body fat and body weight (4-18 animals per time point and body weight).

Tissue culture experiments using white and brown adipocyte progenitor cells showed specific temporal patterns of the BMPs and BMP-inhibitors that were consistent with the mouse findings.

In conclusion, BMP inhibitors are essential in distinguishing brown and white adipogenic differentiation. We hypothesize that MGP is critical in the initial separation, whereas Noggin regulates cell size and lipid accumulation.
Abstract 606: Impaired Glucose Metabolism in the Triggering Receptor Expressed in Myeloid Cells Like Transcript- 1 Null Mice A Link to Obesity
Authors: Castro Rivera Elizabeth , Díaz Rosado Abdías , Washington Anthony V
Published: June 2021

Fat rich foods have been shown to produce increased body weight and diabetes in various strains of mice. When fed ad libitum with a high fat diet (HFD), apolipoprotein E deficient (

apoe
-/-

) mice develop hypercholesterolemia, but are not known to be obese. Our laboratory demonstrated that the triggering receptor expressed in myeloid cells (TREM) like transcript-1 (TLT-1), as a potential target for intervention during atherosclerosis, diabetes, and obesity. Crossing the

treml1
-/-

mouse onto an

apoe
-/-

background (DKO), we found DKO mice had smaller lesions than

apoe
-/-

mice, yet had significantly higher triglycerides and weight gain over a 20-week period of HFD. Further investigation demonstrated

treml1
-/-

mice to have higher cholesterol than wild type mice (wt), suggesting the
treml1
mutation contributes to the phenotype observed in DKO mice. Glucose evaluation of

treml1
-/-

,

treml1
+/-

and wt mice, showed a significant difference in basal glucose, and in plasma glucose clearance, as revealed by intraperitoneal (IP) glucose tolerance test. Analysis of serum insulin by ELISA showed no differences, while IP insulin tolerance test revealed

treml1
-/-

and

treml1
+/-

mice present apparent insulin hypersensitivity, as represented by the steady glucose decline 15 min post injection, with no recovery after two hours. Western blot performed in various tissues, demonstrated the presence of a TLT-1 splice variant that lacks the extracellular domain (TLT-1s), in the adipose tissue of

treml1
-/-

but not wt mice. Analysis of insulin response,
in vivo
by insulin sensitivity assay and
in vitro
using TSA 201 cells transfected with TLT-1s to determine the effect on AKT activation, revealed enhanced phosphorylation in the presence of TLT-1, with a similar response observed
in vitro.
This data support a model where TLT-1s modulation of AKT phosphorylation, increases adipocyte glucose uptake, leading to: low blood glucose levels, increased fatty acid production and storage, and the obese phenotype we observed in our mice.
Abstract 621: Reduced Dietary Cholesterol Availability in Infancy Programs Cholesterol Absorption in Adult Mice
Authors: Dimova Lidiya G , de Boer Jan F , Verkade Henkjan J , Tietge Uwe J
Published: June 2021

Epidemiological research showed that feeding breast milk, which is rich in cholesterol, translates into reduced cardiovascular risk in adulthood compared to feeding formula, which is cholesterol free. The mechanisms underlying these observations are unclear. Therefore, the present study aimed to investigate the impact of reduced dietary cholesterol availability during the suckling period on cholesterol metabolism in adult life in mice.

To achieve reduced dietary cholesterol exposure from breast milk LDLR knockout offspring were given the cholesterol absorption inhibitor ezetimibe for 3 weeks during the suckling period. Ezetimibe was added to the food of nursing dams and reached the offspring’s intestine via excretion into breast milk. Low cholesterol exposure (LC) mice were compared to normal cholesterol (NC) controls with respect to all relevant parameters of cholesterol metabolism including biliary and fecal cholesterol excretion, intestinal absorption and endogenous synthesis using stable isotope kinetics.

At 24 weeks intestinal cholesterol absorption was decreased in LC mice (-30%, p<0.001) due to decreased Npc1l1 expression, the main intestinal cholesterol uptake transporter (-50%, p<0.05). Methylation analysis of the NPC1L1 promoter revealed substantial differences between jejunum and colon (p<0.001), but not between LC and NC. Plasma cholesterol levels were not different between NC and LC due to increased endogenous synthesis in the LC group (p<0.05). Food intake, biliary and fecal cholesterol excretion did not differ between groups.

In summary, our results demonstrate that early life reduction of dietary cholesterol exposure programs the murine intestine in adulthood towards decreased cholesterol absorption via reduced Npc1l1 expression. These results support a key role of the intestine as sensor and integrator of cholesterol metabolism with high relevance for cardio-metabolic disease.
Abstract 624: Endothelial Scavenger Receptor Class B, Type I (SR-BI) Mediates LDL Uptake by the Artery Wall and Promotes Atherosclerosis in Hypercholesterolemic Mice
Authors: Huang Linzhang , Chambliss Ken , Ahmed Mohamed , Mineo Chieko , Shaul Philip W
Published: June 2021

In endothelial cells, high density lipoprotein cholesterol (HDL) binding to scavenger receptor class B, type I (SR-BI) promotes the production of the antiatherogenic signaling molecule nitric oxide (NO) and also endothelial repair. To study how SR-BI in endothelium impacts atherosclerosis, we bred newly-created floxed SR-BI mice, vascular endothelial cadherin promoter-driven Cre recombinase transgenic (VECad-Cre), and apoE
-/-
mice to generate apoE
-/-
with normal endothelial SR-BI expression (SR-BI
ECIN
;apoE
-/-
) or selective deletion of SR-BI from endothelium (SR-BI
ECOUT
;apoE
-/-
). At weaning all mice were placed on an atherogenic diet (20% fat, 1.25% cholesterol), and plasma lipid profiles and atherosclerosis were evaluated 8 weeks later. Endothelial deletion of SR-BI did not alter the plasma lipid profile. Surprisingly, male SR-BI
ECOUT
;apoE
-/-
mice displayed 63% less atherosclerosis in the en face aorta than male SR-BI
ECIN
;apoE
-/-
mice, aortic root lesions were comparably affected, and similar findings were obtained in females. Recognizing that SR-BI binds both HDL and low density lipoprotein cholesterol (LDL), to then discern how endothelial SR-BI promotes atherosclerosis we determined using Di-I-labeled oxidized LDL (oxLDL) if SR-BI influences oxLDL uptake by endothelial cells. Such uptake is the first step in the endothelial transcytosis that delivers LDL to the artery wall to initiate atherogenesis. OxLDL uptake by primary human aortic endothelial cells was blunted by 87% by SR-BI blocking antibody, and it was also decreased by SR-BI deletion via siRNA, and by the chemical inhibitor of SR-BI BLT-1. Furthermore, SR-BI blocking antibody and BLT-1 caused marked declines in endothelial oxLDL transcytosis. Moreover, 4 hours following IV administration, oxLDL uptake in aorta was decreased by 84% in SR-BI
ECOUT
;apoE
-/-
versus SR-BI
ECIN
;apoE
-/-
mice. These collective findings indicate that endothelial SR-BI plays an important role in atherogenesis, and that it likely does so by mediating LDL uptake into the artery wall. They further suggest that there are mechanisms that govern LDL transport across endothelium that may be targeted to provide novel means to combat atherosclerosis.
Abstract 625: Targeting of Heparin Binding EGF-like Growth Factor (HBEGF) Suppresses Hyperlipidemia and Atherosclerosis in LDL Receptor Deficient Mice
Authors: Lee Sangderk , Lee Richard , Graham Mark , Yang Lihua , Kim Seonwook , Kim Seongu , Rateri Debra , Temel Ryan , Lusis Aldons , Berliner Judith
Published: June 2021

Objective:
Elevation of apoB-containing lipoproteins is a well-established risk factor for the development of atherosclerosis. Previous reports showed that expression of heparin-binding EGF-like growth factor (HBEGF), a ligand of epidermal growth factor receptor (EGFR), is associated with atherosclerosis development. In this study, we examined
in vivo
effects of HBEGF targeting on hyperlipidemia-induced atherosclerosis by suppressing HBEGF expression using antisense oligonucleotide (ASO).

Methods and Results:
Female and male LDLR deficient mice were fed a high fat diet (HFD; 21% fat, 0.2% cholesterol) throughout the study. After 8 weeks of HFD feeding, mice were injected intraperitoneally with either control or HBEGF ASOs weekly for 12 weeks. At termination, we measured circulating lipid concentrations and atherosclerotic lesion size in the aorta. Compared to control ASO group, HBEGF ASO group had a significant reduction of circulating total cholesterol, triglyceride, and apoB-containing lipoprotein concentrations but no change of high-density lipoprotein (HDL) concentration. Importantly, HBEGF ASO injection significantly suppressed atherosclerosis in the aortic arch, thoracic, and abdominal aorta. HBEGF ASO suppressed sterol synthetic gene expression in the liver but elevated lipid contents in the liver. HBEGF gene silencing in a liver cell system induced downregulation of sterol regulatory element binding protein (SREBP) target genes including LDLR and Insig1.

Conclusion:
Targeting HBEGF using ASOs is an efficient approach to suppress dyslipidemia and hyperlipidemia-induced atherosclerosis. The differential gene expression analysis suggests that HBEGF ASO administration suppresses SREBP-regulated gene expression in the liver leading to downregulation of circulating cholesterol and TG concentrations.
Abstract 626: Oxidized Low-density Lipoprotein-induced Cell Membrane Damage in Bone Marrow Stem Cells is Independent of ROS Formation
Authors: Li Xin , Cui Yuqi , Tan Tao , Narasimhulu Chandrakala , Xiao Yuan , Zhang Jia , Hao Hong , Liu Lingjuan , Ma Jianjie , Verfaillie Catherine , Parthasarathy Sampath , Zhu Hua , Liu Zhenguo
Published: June 2021

Objectives:
Membrane repair mechanism is important to the cell survival and function. MG53, also known as TRIM72, is a critical component in the membrane repair process. We have demonstrated that cell membrane damage was involved in the impaired survival of bone marrow stem cells (BMSCs) by oxidized low-density lipoprotein (ox-LDL) in vitro, and recombinant human MG53 protein (rhMG53) significantly protected the cell membrane of BMSCs and enhanced their survival. However, the mechanism(s) for ox-LDL-induced membrane damage in BMSCs is unclear. It is well known that ox-LDL is a potent oxidative agent with formation of abundant reactive oxygen species (
ROS
). The present study was designed to determine the role of ROS in ox-LDL-induced membrane damage in BMSCs.

Methods:
Rat bone marrow multipotent adult progenitor cells (
MAPCs
) were cultured with and without ox-LDL (0-20 ug/ml) for up to 48 hrs. Exposure of MAPCs to ox-LDL led to increased LDH release to the media and entry of fluorescent dye FM1-43 measured under confocal microscope, suggesting damage to the plasma membrane. Ox-LDL also generated excessive ROS as measured with electron paramagnetic resonance spectroscopy. While antioxidant N-acetylcysteine (NAC) completely blocked ROS production from ox-LDL, it failed to prevent ox-LDL-induced cell death or membrane damage. When MAPCs were treated with rhMG53, ox-LDL induced LDH release and FM1-43 dye entry were significantly reduced. Interestingly, NAC treatment significantly potentiated the protective effect of rhMG53 on ox-LDL-induced membrane damage with enhanced cell survival. Western blot analysis showed that ox-LDL significantly reduced the level of rhMG53 both in vitro and in vivo that was substantially prevented with NAC treatment.

Conclusion:
Our data suggest that membrane damage induced by ox-LDL contributed to the impaired survival of MAPCs independent of ROS production. NAC potentiated the protective effect of rhMG53 on ox-LDL-induced membrane damage of BMSCs via preventing ox-LDL-induced reduction of rhMG53 level.
Abstract 629: FGF15/19 Upregulates Hepatic ABCG5 and ABCG8 to Promote Biliary Cholesterol Secretion
Authors: Pijut Sonja , Wang Yuhuan , Bennett Lisa , Lee Richard , Graf Gregory
Published: June 2021

Background:
Elevated hepatic cholesterol is thought to contribute to the development of nonalcoholic fatty liver disease, a condition highly associated with cardiovascular risk factors. The ABCG5 and ABCG8 (G5G8) heterodimer is responsible for up to 90% of biliary cholesterol secretion and is a potential therapeutic target for promoting cholesterol elimination. We have previously demonstrated that ursodiol (UDCA), a pharmacologic bile acid, increases G5G8 protein expression and biliary cholesterol secretion. However, whole body cholesterol elimination is minimized likely due to simultaneous suppression of bile acid synthesis through upregulation of FGF15/19. The objectives of this study are to determine whether FGF15/19 regulates G5G8 and determine whether UDCA requires FGF15/19 signaling in order to upregulate G5G8.

Methods:
Mice were injected with two doses of FGF19 or carrier (PBS) 1μg/g body weight within an 8-hour treatment window. A separate group of wild type (WT) and G5G8 knockout (KO) mice were similarly injected with FGF19. In another experiment, WT mice were fed chow or UDCA-supplemented diet in the absence or presence of FGF15/19 signaling inhibition which was achieved by FGFR4 antisense oligonucleotide (ASO) supplied by Ionis Pharmaceuticals. In all experiments, body weight, liver weight, bile flow rate and plasma, hepatic and biliary lipids were measured. Immunoblotting of G5G8 and real-time PCR of genes involved in cholesterol metabolism were also conducted.

Results:
Mice injected with FGF19 had increased biliary lipids (PBS: 5.287±0.5720, FGF19: 8.098±0.6114, n=6), decreased
Cyp7a1
(PBS: 1.021±0.1064 FGF19: 0.07787±0.01345 n=5-7) and
Cyp8b1
(PBS: 1.018±0.09846, FGF19: 0.2647±0.05609, n=5-7) expression, and increased G5G8 protein expression compared to mice injected with PBS. In G5G8 KO mice injected with FGF19, there was only a small increase in plasma free cholesterol (WT: 51.96±2.098, KO: 62.24±2.562, n=4) and no other significant changes in cholesterol metabolism compared to wild type mice injected with FGF19.

Conclusion:
In conclusion, FGF15/19 suppresses bile acid synthesis and post-transcriptionally upregulates G5G8. However, in the absence of G5G8, FGF15/19 did not disrupt cholesterol metabolism.
Abstract 631: Proteomic Characterization of Apolipoprotein A-II Defined Subfractions of Human High Density Lipoproteins
Authors: Street Scott E , Melchior John T , Shah Amy S , Davidson W. S
Published: June 2021

High-density lipoproteins (HDL) play a critical role in lipid transport and vascular lipid homeostasis. HDL is heterogeneous, with particles varying in size, composition, and functionality. Proteomic studies have shown that HDL can host as many as 94 different proteins which can segregate into distinct subclasses. The two major apolipoproteins (apo)A-I and apoA-II account for 70% and 15-20%, respectively of HDL total protein. We hypothesized that the presence or absence of apoA-II can affect the binding of other proteins thus influencing HDL function. Immunoaffinity chromatography was used to isolate particles containing both apoA-I and apoA-II (LpA-I/LpA-II) and those with apoA-I with no apoA-II (LpA-I). These were isolated from both
i
) total HDL isolated by ultracentrifugation (UC) and
ii
) directly from plasma. The proteomic fingerprint of each population was determined by mass spectrometry. Within both populations, 54 total proteins were identified in UC isolated HDL compared to 98 proteins in particles isolated directly from plasma. In the UC isolated HDL, LpA-I/A-II particles contained 39 proteins compared to LpA-I particles which contained about 30. Interestingly, the opposite was true in particles isolated from plasma where upwards of 77 proteins on LpA-I/A-II particles were identified compared to LpA-I particles where 98 proteins were identified. Limited tryptic digestion experiments showed that LpA-I/A-II were more prone to proteolysis compared to LpA-I. Additionally, we found that LpA-I/A-II exhibited an increased capacity to efflux cholesterol from cultured macrophages (normalized by phospholipid content) suggesting a protein mediated difference in HDL functionality. Taken together, these results suggest the proteomic fingerprint of HDL subpopulations is heavily influenced by the method of isolation and that the proteomic pattern on HDL influences the physiological function of the particle.
Abstract 637: Allograft Inflammatory Factor 1 Promotes Key Macrophage Functions that Limit Plaque Destabilization
Authors: Chinnasamy Prameladevi , Casimiro Isabel , Gorroño Lander E , Sibinga Nicholas
Published: June 2021

Objective:
Allograft inflammatory factor-1 (Aif-1) is best characterized as a cytoplasmic protein involved in actin bundling and phagocytosis. We investigated Aif-1 function in macrophage biology and the pathogenesis of atherosclerosis.

Hypothesis:
Aif-1-mediated potentiation of NFκB signaling promotes key macrophage functions and supports characteristics of plaque stability.

Methods:
We generated

apoE
-/-
and aif-1
-/-
; apoE
-/-

mice (SKO and DKO, respectively), fed them a high fat diet for 18 weeks, and analyzed lesion burden in
en face
aorta, aortic root, and brachiocephalic arteries (BCA). We used immunofluorescence to assess marker expression and FACS analysis for proliferation and efferocytosis.

Results:
Lesion burden was not significantly different in aortic roots (DKO vs. SKO; 1.385e+006 ± 2065 vs 1.957e+006 ± 558, total lesion area), BCAs (DKO vs. SKO; 63 vs.65, % of Oil red O staining) and
en face
aortas (9 vs. 10, % of Oil red O staining). On the other hand, aortic root lesions from DKO mice showed more necrosis (DKO vs. SKO; 0.65 vs 0.4) and decreased fibrous cap thickness (DKO vs. SKO; 0.3 vs. 0.45, p0.05 respectively) normalized to lesion size. We also observed less macrophage staining (DKO vs. SKO; 0.6 vs. 2.2 % area/aortic root) and increased apoptosis (14 vs. 6 %Tunel+ cells), which correlated with decreased lesional P-NFκB p65 (DKO vs. SKO; 4 vs. 7 +nuclei/aortic root) activation.
In vitro
,

aif-1
-/-

macrophages showed decreases in oxidative stress-induced survival (40 vs. 80%), phagocytosis (30-60% decrease in zymosan particle uptake) and efferocytosis (40 vs.60 %F4/80;Tamra double +ve cells), and interleukin-6 release (1.2 vs 3 ng/ml in conditioned media). Mechanistically, these increased key macrophage functions correlated with P-NFκB p65 activation, which was impaired in

aif-1
-/-

macrophages.

Conclusion:
Our
in vivo
and
in vitro
data point to an essential role of Aif-1, via NFκB activation, in support of key macrophage functions, including survival and efferocytosis, that limit necrotic core size and increase atherosclerotic plaque stability.
Abstract 639: Deletion of Alternatively Spliced Extra Domain A of Cellular Fibronectin Stabilizes Advanced Rupture-Prone Plaques in Hyperlipidemic Mice
Authors: Doddapattar Prakash , Dhanesha Nirav , Prakash Prem , Lentz Steven R , Chauhan Anil K
Published: June 2021

Background:
The most important clinical manifestation of atherosclerosis is rupture of advanced plaques. The fibronectin containing alternatively spliced extra domain A (Fn-EDA) is abundant in extracellular matrix around macrophages and endothelial cells in advanced human plaques. In vitro studies suggest that Fn-EDA is a ligand for TLR4 and upregulates MMP9, which has been shown to enhance elastin degradation and promote plaque rupture in advanced plaques of hyperlipidemic apolipoprotein E-deficient (Apoe
-/-
) mice. The mechanistic role of Fn-EDA in advanced plaques remains unknown.

Hypothesis:
Fn-EDA/TLR4 signaling in activated macrophages contributes to plaque vulnerability by upregulating MMP9 during advanced atherosclerosis.

Methods and Results:
We compared atherosclerotic plaques in the brachiocephalic artery (a model artery for study of plaque vulnerability) of female Fn- EDA
-/-
Apoe
-/-
and Apoe
-/-
mice at 50 weeks of age (44 weeks on high-fat Western diet). Fn- EDA
-/-
Apoe
-/-
mice exhibited decreased plaque size and characteristics of stable plaques, with decreased necrosis, reduced influx of activated macrophages, increased SMC staining and greater collagen content compared to Apoe
-/-
mice (P<0.05, n=11-13 mice/group), although cholesterol and triglyceride levels, and circulating leukocyte counts were similar. Purified cellular FN, which contains EDA, potentiated the NFκB-mediated inflammatory pathway (increased phospho-NFκB p65/ NFκB p65, TNFα and IL1β) and MMP9 protein expression (gelatin zymography) in a dose-dependent manner in purified macrophages from Fn-EDA
-/-
Apoe
-/-
mice but not from Fn-EDA
-/-
TLR4
-/-
Apoe
-/-
mice. Using immunohistochemistry, we demonstrated colocalization of macrophage TLR4 and Fn-EDA within advanced atherosclerotic plaques in murine brachiocephalic arteries and human coronary arteries. Genetic deletion of TLR4 in Apoe
-/-
mice stabilized advanced plaques, and parameters of plaque stability were comparable between TLR4
-/-
Apoe
-/-
and Fn-EDA
-/-
TLR4
-/-
Apoe
-/-
mice.

Conclusions:
These findings suggest that Fn-EDA/TLR4 signaling in macrophages is a key mechanism that upregulates MMP9, and thereby promotes plaque vulnerability in advanced atherosclerosis.
Abstract 644: Cd27 Co-stimulation Fosters Regulatory T Cell Survival and Ameliorates Atherosclerosis
Authors: Gerdes Norbert , Winkels Holger , Smeets Esther , Meiler Svenja , Bürger Christina , Weber Christian , Lutgens Esther
Published: June 2021

Aim:
Adaptive immunity and co-stimulatory signals are pivotal to all stages of atherosclerosis. The interaction of the co-stimulatory molecules CD27 and CD70 modulate Treg development but also affect T cell proliferation, differentiation, and activation at the sites of antigen priming and at effector function. We hypothesized an increased atherosclerotic burden and an exacerbation of disease upon CD27 deficiency.

Methods:

Cd27
-/-

mice were crossed with
Apoe
-/-
mice.

Cd27
-/-
Apoe

-/-
and littermate controls (

Cd27
+/+
Apoe

-/-
) were sacrificed at the age of 18 and 28 weeks. Bone marrow of

Cd27
-/-
Apoe

-/-
and littermate controls was transplanted into
Apoe
-/-
recipient mice. Lesion size, histology and cellular composition was analyzed in the ascending aorta.

Results:
CD27 co-localizes with T cells in aortic lesions. At early stages of atherosclerosis

Cd27
-/-
Apoe

-/-
mice displayed bigger atherosclerotic lesions accompanied by an increased macrophage content. Flow cytometry revealed a significant decrease in abundance of splenic and aortic Tregs and increased apoptosis of Treg in the thymus of

Cd27
-/-
Apoe

-/-
mice. In contrast, at later stages of atherosclerosis (28 weeks)

Cd27
-/-
Apoe

-/-
and littermate control mice had similar splenic Treg content and atherosclerotic burden. Transplantation of

Cd27
-/-
Apoe

-/-
bone marrow induced increased atherosclerotic plaque size and a profound pro-inflammatory plaque phenotype accompanied by reduced frequency of aortic and splenic Tregs.

Conclusion:
Taken together, our data reveal that deficiency for CD27 impairs thymic Treg development thereby exacerbating early atherogenesis and increasing the macrophage content of atherosclerotic lesions. However, later stages of atherosclerosis were not affected by CD27 deficiency.
Abstract 645: Curcumin Modulates the Function(s) of Intestinal Epithelial Cells: Role in the Development of Diet-Induced Metabolic Diseases
Authors: Wang Jing , Ghosh Siddhartha S , Ghosh Shobha
Published: June 2021

Association between circulating lipopolysaccharide (LPS) and metabolic diseases (such as Type 2 Diabetes and atherosclerosis) has shifted the focus from high fat high cholesterol containing western type diet (WD)-induced changes in gut microbiota
per se
to release of gut bacteria-derived products into circulation as the possible mechanism for the chronic inflammatory state underlying the development of these diseases. We demonstrated earlier that WD-induced changes in intestinal barrier function increases the release of LPS into systemic circulation and oral supplementation with curcumin not only reduces the levels of systemic LPS but also attenuates the subsequent development of glucose intolerance and atherosclerosis. Although curcumin is reported to exert potent anti-inflammatory effects
in vitro
, its poor bioavailability has raised doubts about a
causal
relationship between oral supplementation and the
in vivo
effects. It is hypothesized that curcumin attenuates WD-induced chronic inflammation and associated metabolic diseases by modulating the function of intestinal epithelial cells (IECs) and the objective of the present study was to delineate the underlying mechanisms. Human IEC lines, Caco-2, HT-29 and T-84 were used for these studies and modulation of direct as well as indirect effects of LPS on tight junctions as well as intracellular signaling were examined. Pre-treatment with Curcumin prevented LPS-induced disruption of tight junctions. LPS-induced secretion of master cytokine IL-1β was also dramatically reduced by pre-treatment with curcumin. Furthermore, curcumin also attenuated IL-1β induced activation of p-38 MAPK involved in the phosphorylation of tight junction proteins and subsequent disruption of their normal arrangement. Based on these data, the sites of curcumin actions leading to preservation of intestinal barrier function are shown in the Figure (activation and inhibition denoted by + and - symbols in green circles).
Abstract 646: Redox Control of Macropinocytosis; An Unexplored Target in Atherosclerosis
Authors: Ghoshal Pushpankur , Singla Bhupesh , Feck Douglas , Cantu-Medellin Nadiezhda , Kelley Eric , Haigh Stephen , Fulton David , Csányi Gábor
Published: June 2021

Aims:
Early studies established the paradigm that oxidation of low density lipoprotein (LDL) is necessary for scavenger receptor-mediated LDL uptake and lipid accumulation in macrophages. In addition to this “classical” mode of lipid internalization, scavenger receptor-independent uptake of native, non-oxidized LDL (nLDL) via macropinocytosis has been demonstrated to contribute to lipid uptake by macrophages. Despite this previous information the precise signaling mechanisms regulating macropinocytosis of nLDL and the relative contribution of lipid macropinocytosis to atherosclerosis remain unknown. This study was designed to examine the role of phagocyte NADPH oxidase (a.k.a. Nox2) in macropinocytosis and to investigate macropinocytotic uptake of lipids in hypercholesterolemic ApoE
-/-
mice
in vivo
.

Results:
Phorbol myristate acetate (4β-PMA) activation of human and murine macrophages stimulated membrane ruffling, macropinosome formation, and subsequent uptake of nLDL by macropinocytosis. FACS data indicated that 4β-PMA stimulates lipid accumulation following nLDL treatment in macrophages lacking scavenger receptor CD36. Mechanistically, we found that pharmacological blockade of protein kinase C (PKC), inhibition of flavoenzymes by diphenyleneiodonium, and scavenging intracellular superoxide anion abolished phorbol ester-induced macropinocytosis. Transcriptional knockdown of Nox2 using siRNA inhibited 4β-PMA-induced macropinocytosis in THP-1 macrophages. Delving further into the mechanism, we found that Nox2 via redox inactivation of PTEN and activation of the PI3K/Akt pathway dephosphorylates the actin-binding protein cofilin, stimulates membrane ruffling, and induces macropinocytosis. Finally, peritoneal chimera experiments indicate that macropinocytotic uptake of lipids in hypercholesterolemic ApoE
-/-
mice was attenuated in Nox2
y/-
macrophages compared to wild type controls.

Innovation and Conclusion:
These findings suggest a previously undescribed redox-sensitive signaling pathway leading to internalization of nLDL by macropinocytosis. The signaling mechanism described herein may identify new targets in atherosclerosis and other disease conditions involving macropinocytosis.
Abstract 648: Alternative Macrophages Promote Atherosclerosis Progression by Increasing Intraplaque Angiogenesis and Vascular Permeability via HIF-1 alpha/VEGF-A-dependent Pathway
Authors: Guo Liang , Akahori Hirokuni , Polavarapu Rohini , Harari Emannuel , Karmali Vinit , King Adrienne L , Choi Cheol Ung , Cheng Qi , Jo Hanjoong , Thabet Salim , Kolodgie Frank , Virmani Renu , Finn Aloke V
Published: June 2021

Background:
Alternative macrophages exist in human atherosclerosis but their role in atherogenesis remains uncertain. Intraplaque hemorrhage (IPH) is an important stimulus driving alternative macrophage polarization. Intake of hemoglobin (Hb) by the hemoglobin: haptoglobin receptor CD163 leads to a distinct non-foam cell phenotype termed M(Hb). These cells demonstrate upregulation of CD163, lack of lipid retention, and anti-oxidative properties, characteristics considered ‘atheroprotective’. Here we reveal an unexpected but important pathogenic role for M(Hb) in atherosclerosis.

Objectives:
To determine the role of M(Hb) macrophages in human intraplaque angiogenesis and vascular permeability.

Methods:
Using human atherosclerotic samples, cultured cells, and a mouse model of IPH, we investigated the role of IPH on macrophage function with respect to angiogenesis and vascular permeability.

Results:
Within M(Hb) activation of hypoxia-inducible factor-1 alpha (HIF-1) via inhibition of prolyl hydroxylases promotes intraplaque angiogenesis and vascular permeability. In human carotid plaques, alternative CD163 positive macrophages were found to be highly associated with plaque vascularity and expressed high levels of HIF1- and vascular endothelial growth factor-A (VEGF-A). Supernatants from hemoglobin:haptoglobin differentiated M(Hb) macrophages increased endothelial permeability and led to internalization of the endothelial barrier protein vascular endothelial cadherin (VE-cadherin) via activation of VEGF receptor 2 (VEGFR2). Areas of plaque demonstrating high density CD163
high
macrophage subsets showed irregular VE-cadherin immunostaining and diffuse perivascular collections of von Willebrand factor suggesting microvessel incompetence. Finally, in brachiocephalic plaques of one-year-old

apoE
-/-

and

apoE
-/-
CD163
-/-

mice, CD163 deficiency significantly reduced plaque progression, lesion size, and intraplaque hemorrhage, but had little effect on lesions uncomplicated by hemorrhage.

Conclusions:
Our findings provide a novel non-lipid driven mechanism by which alterative M(Hb) macrophages promote plaque neoangiogenesis and microvessel incompetence via a HIF-1/VEGF-A-dependent pathway.
Abstract 650: Statins Modulate Rac-dependent IL-1 beta Expression to Influence Calcium Composition
Authors: Zhao Lina , Healy Abigail L , Ceneri Nicolle , Bailey Grant , Meadows Judith , Sadeghi Mehran , Morrison Alan R
Published: June 2021

Coronary artery disease caused by atherosclerosis is the leading cause of morbidity and mortality in the world. Calcification of atherosclerotic plaque has predictive value in terms of cardiovascular event risk and mortality. The composition of calcium within plaque (
i.e.
increased calcium density) has been associated with reduced vulnerability for cardiac events. Therapeutic modulation of atherosclerotic plaque calcium composition may have tremendous potential in terms of reducing event risk. HMG-CoA reductase inhibitors (statins) are a well-established family of medications that reduce the risk of myocardial infarction and are associated with increased plaque calcification, yet the underlying mechanisms of these processes are not well understood. Moreover, statin use can be associated with significant adverse reactions (
i.e.
myalgias and new onset diabetes). Our group has identified a Rac-dependent inflammatory pathway that promotes IL-1β-driven atherosclerotic calcification. Using an atherosclerotic-prone mouse model, we found that progressive calcification of atherosclerotic plaque depends on altered Rac signaling and consequent IL-1β expression. Statin-treated primary macrophages expressed higher IL-1β mRNA and secreted more IL-1β protein in response to inflammasome activation by TLR-coupled cholesterol crystal phagocytosis. This statin-induced augmentation of IL-1β production depended on Rac1, as small molecule inhibition of Rac1 and
Rac1
gene-deletion abrogated this effect. Statin treatment disrupted the association of Rac1 with its native inhibitor, Rho-GDI, through loss of Rac1 isoprenylation, and as a consequence, statin-treated macrophages demonstrated higher levels of GTP-bound Rac1. This higher activation of Rac1 led to increased NF-κB activation and consequent macrophage IL-1β mRNA expression.

ApoE
-/-

mice placed on high fat diet with statin therapy revealed increases in aortic plaque calcification relative to control mice in association with altered serum IL-1β levels. Defining Rac signaling as a key mediator of statin-dependent mechanisms that modulate plaque calcium composition and consequent plaque stability may lead to novel therapeutic strategies for the prevention of cardiovascular events.