bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 6: CAT-2003 is a Novel Small Molecule That Inhibits Proprotein Convertase Subtilisin/Kexin Type 9 Production and Lowers Non--High-Density Lipoprotein Cholesterol
Authors: Zimmer Michael , Picarella Dominic , Liu Feng , Bista Pradeep , Lee Diana , Vu Chi , Jirousek Michael
Published: March 2022

Introduction:
CAT-2003 is an orally active small molecule that inhibits SREBP maturation (nSREBP-1 and nSREBP-2) and activation of SREBP-target genes, including PCSK9. CAT-2003 mechanism of action was studied in HepG2 cells and in the transgenic ApoE*3 Leiden mouse model of hyperlipoproteinemia.

Methods and Results:
In vitro, CAT-2003 was studied in HepG2 cells and dose dependently reduced mature SREBP-2 protein levels (50% at 25 μM, n=3), PCSK9 mRNA (IC50 16.2 +/- 1.1 μM, n=3), and PCSK9 protein secretion (IC50 = 17.8 +/- 1.1 μM, n=3). CAT-2003 treatment also reduced expression of other SREBP target genes as well as SREBP processing genes including those encoding for HMG-CoA reductase, SCD-1, ACC2, S1P and S2P proteins. The reduction in PCSK9 protein correlated dose dependently with an increase in both low and high molecular weight LDLR protein. Statins increase nSREBP-2 and increase the production of PCSK9. CAT-2003 reversed atorvastatin-induced increases in nSREBP-2 and PCSK9 production in HepG2 cells.

In vivo CAT-2003 was studied in ApoE*3 Leiden mice (16 weeks, n=8 per arm). CAT-2003 reduced plasma PCSK9 (61%, p=0.001), total cholesterol (41%, p<0.0001) and TGs (33%, p=0.0034) compared to control and lowered aorta atherosclerotic lesions (p<0.01). An FPLC analysis showed the cholesterol reduction occurred primarily in non-HDL fractions. Liver histology showed reduced lipids and an increase in LDLR protein (104% increase, p=0.001). In a second ApoE*3 study (4 weeks, n=8 per arm) CAT-2003 was compared alone or in combination with atorvastatin. CAT-2003 reduced PCSK9 plasma levels alone (65%, p=0.001) and in combination with atorvastatin (58%, p=0.001) and produced a more than additive lowering of non-HDL-C (58%, p=0.001) compared to control. Expression of SREBP target genes in the liver were reduced in CAT-2003 and in atorvastatin combination treated mice.

Conclusions:
CAT-2003 inhibits the maturation of SREBP-1 and -2 and inhibits the production of PCSK9. CAT-2003 lowers PCSK9, LDL-C and VLDL-C in ApoE*3 Leiden mice alone or in combination with atorvastatin. CAT-2003 is a potential new therapeutic to lower non-HDL-C and is in Phase 2 clinical studies.
Abstract 603: O-GlcNAcylation of Runx2 is Essential for Vascular Calcification
Authors: Heath Jack , Mao Xia , Huang Lu , Sun Yong , Deng Liang , Chen Yabing
Published: March 2022

Osteogenic differentiation of vascular smooth muscle cells (VSMC) contribute predominantly to vascular calcification, a characteristic of advanced atherosclerosis, chronic kidney disease, and diabetes. Using Runx2 shRNA and SMC-specific Runx2 deletion mice, we have demonstrated an essential role of Runx2 in regulating vascular calcification in vitro and in vivo. The present studies aim to pinpoint the Runx2 functional domain that is critical for its osteogenic function in VSMC and to elucidate the underlying molecular mechanisms.

A series of Runx2 deletion mutants were generated and stably transfected into VSMC. Full-length Runx2 protein and Runx2 deletion mutants containing amino acids 1-495 or 1-432, but not Runx2 1-391, induced VMC calcification, indicating that the Runx2 osteogenic functional domain is located between amino acids 391-432. Site-directed mutagenesis analysis identified that mutations at the amino acids 412, 413, 425 and 426-428 markedly reduced Runx2 transactivity and inhibited Runx2-induced VSMC calcification. Mutations at amino acids 412, 413 and 425 were found to inhibit Runx2 post-translational modification by O-GlcNAcylation. Immunoprecipitation revealed that Runx2 O-GlcNAcylation at 412, at 413, and proximal to 425 was critical for its binding to Smad 1/5/8 and Smad 4, but not Smad 2/3. Using lentivirus-mediated shRNA, we determined that knockdown of Smad1/5/8, but not Smad 2/3, inhibited Runx2-induced VSMC calcification, suggesting that BMP but not TGF-beta signaling is critical for the osteogenic function of Runx2 in VSMC. Taken together, these data demonstrate that Runx2 amino acids 412, 413, 425 and 426-428 are key for its osteogenic function in VSMC, and Runx2 O-GlcNAcylation at 412, 413 and 425 is essential for its interaction with Smad proteins to determine Runx2 transactivity and VSMC calcification.

We have identified a novel Runx2 post-translational modification by O-GlcNAcylation at key residues that are essential for its osteogenic function. Our studies provide molecular insights into the function of Runx2 in regulating VSMC calcification, which may shed light on novel targets that are amenable to drug discovery for vascular calcification.
Abstract 606: Pharmacological Inhibition of PAI-1 Rescues Blood Flow Perfusion Through Promoting Capillary Arterialization in Diabetic Microangiopathy in Ischemic Limb
Authors: Li Rong , Luo Mao , Zhang Xiao , Ren Meiping , Chen Ni , Yan Kai , Deng Xin , Zeng Ming , Yang Yan , Wu Jianbo
Published: March 2022

OBJECTIVE:
Type II diabetes mellitus (DM) leads to the development of microvascular diseases and is associated with impaired angiogenesis. Elevated plasma levels of plasminogen activator inhibitor-1 (PAI-1) are associated with DM. However, the role of PAI-1 in the development of DM-associated microvascular disease is poorly defined.

RESEARCH DESIGN AND METHODS:
Hind-limb ischemia was generated by femoral artery occlusion in diet-induced diabetic mice and age-matched normal chow (NC)-fed mice. Plasma was analyzed for glucose, non-esterified fatty acids, and PAI-1. PAI-039, a specific, small molecule pharmacological inhibitor of PAI-1, was orally administered to mice to determine if PAI-1 attenuates neovasculature formation in DM mice.

RESULTS:
PAI-1 activity and antigen were significantly increased within 2 weeks of DM onset and remained elevated throughout the experimental period. PAI-039 normalizes elevated plasma and ischemic muscles PAI-1 levels in the DM group. Pharmacologic inhibition of PAI-1 with PAI-039 rescued the ischemia-mediated impairments in blood flow perfusion and accelerated arterialization of capillaries in ischemic muscles in DM group compared to vehicle-DM group. Immunohistochemical analysis indicated increased expression of smooth muscle α-actin on capillary-sized vessels, and increased infiltration by CD45 leukocytes in ischemic gastrocnemius muscle tissues of PAI-1 inhibitor-treated mice. Furthermore, in these mice, VEGF-A was significantly down-regulated, and conversely, the expression of the PDGF-B and MMP 2, 9 were up-regulated in ischemic muscles in the inhibition of PAI-1 after induced ischemia.

CONCLUSIONS:
Taken together, these findings illustrate that the pharmacologic inhibition of elevated PAI-1 rescues the impairments in ischemia-mediated flow perfusion and capillary arterializations observed in DM, and may have efficacy as a therapeutic strategy to prevent diabetic microangiopathy.
Abstract 608: The Mechanisms of Preservation of Vasorelaxation Induced by Protease-Activated Receptor-2 Activation in the Aorta of Metabolic Syndrome Model Rats
Authors: Maruyama Kana , Kagota Satomi , Wakuda Hirokazu , McGuire John J , Yoshikawa Noriko , Nakamura Kazuki , Kunitomo Masaru , Shinozuka Kazumasa
Published: March 2022

Protease-activated receptor-2 (PAR2) is activated by proteases, such as trypsin, human mast cell beta-tryptase, human kallikrein isoforms, coagulation factors VIIa and Xa, etc. PAR2 is believed to have an important role under inflammatory pathological conditions, but its role in metabolic syndrome remains uncertain. We have reported that PAR2-activating peptide, 2-furoyl-LIGRLO-amide (2fly) causes NO-mediated vasorelaxation in rat aortas similar to that observed with acetylcholine (ACh), and that 2fly-induced vasorelaxation is maintained in aortas from SHRSP.Z-

Lepr
fa

/IzmDmcr (SHRSP.ZF) rats with metabolic syndrome, even though ACh- and sodium nitroprusside (SNP)-induced vasodilation is lower than that in control (Wistar-Kyoto rats, WKY). In this study, we assessed the mechanisms involved in the preservation of PAR2-mediated vasorelaxation in metabolic syndrome.

Aortas were isolated from male WKY and SHRSP.ZF rats between 18-20 weeks of age. ACh- and 2fly-induced vasorelaxations were measured in the presence or absence of inhibitors (i.e., COX inhibitors, a TXA
2
synthase inhibitor, and a TXA
2
/PGH
2
receptor antagonist) using organ bath methods. Expression of endothelium NO synthase (eNOS) in aortas was measured by western blot analysis.

Indomethacin (a non-selective COX inhibitor), SC560 (a selective COX-1 inhibitor), NS398 (a selective COX-2 inhibitor), OKY-046 (a TXA
2
synthase inhibitor), and ONO-3708 (a TXA
2
/PGH
2
receptor antagonist) did not affect both ACh- and 2fly-induced vasorelaxation in aortas of SHRSP.ZF rats as well as WKY. Expression of phosphorylated eNOS at Ser
1177
in 2fly-stimulated aortas was higher than that in ACh-stimulated ones in SHRSP.ZF rats, but there was no significant difference between these levels in WKY.

Our results show that the preservation of 2fly-induced vasorelaxation observed in SHRSP.ZF rats with metabolic syndrome may be because of enhanced eNOS activation via PAR2, but not coupled to the COX pathway. Thus, preservation of PAR2-mediated vasorelaxation may be one of the key responses to maintain blood circulation under inflammatory conditions, such as metabolic syndrome.
Abstract 623: Robust and High-Throughput Cholesterol Efflux Assay
Authors: Dixit Mallikarjun N
Published: March 2022

Background:
The objective of this study was to develop a robust high-throughput assay for ABCA1 specific cholesterol efflux supporting its use in large clinical trials, which require consistent robust performance and cost-efficiency.

Methods:
J774 cells were seeded on a 48-well plate and pulsed with
3
H Cholesterol in a media with ACAT inhibitor. ABCA1 expression was induced by 8-bromo cAMP. Test serum (HDL) isolated by PEG precipitation was added to each well and efflux capacity determined as the ratio of radioactive counts in growth medium versus total counts when comparing cells expressing ABCA1 with those not expressing this transporter. Precision, linearity, limit of detection, specificity, reference range, robustness, and short-and long term stability were assessed.

Results:
The overall intra-assay precision varied from 8.5% to 12%, whereas the inter-assay precision was about 20% based on fifteen individual runs on different days by three different analysts. The linear range of the assay was 0.81%-6.5% and 0.43%-13% cholesterol efflux (CE) for PEGylated and non-PEGylated serum, respectively. The LOD was 1.08% CE based on mean of negative control from all 16 runs plus 3 SD. Specificity was determined as the relative increase (11.3 fold) of cholesterol efflux by ApoA1 between cAMP and non-cAMP treated cells. Verification of reference range was determined as 2.1%-7.0% CE (mean±2SD). Consistency of the assay determined by %CV was not affected when different lots of cAMP (CV:6.4%) or ApoA1 (10.3%) were used. The CE values were not appreciably affected when identical samples were run by different analysts (CV:3.2%). Based on average of three data sets samples were stable for up to three days at 2-8°C (mean difference 9.6%), 10 days at -20°C (7.8%), and 7 months at -80°C (8.1%). About 3,000 samples were run over 4 weeks equaling 750 samples /week in a high-throughput format with consistent performance as assessed by each of the five QCs run in each run.

In conclusion, the CE assay described herein is the first high-throughput and robust assay fit for use in large clinical trials where consistent performance during the development of a drug program is critical.

Confidential

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Abstract 634: Treatment with Mipomersen Reduces Levels of ApoB-Containing Lipoproteins by Increasing Fractional Removal of VLDL and LDL-apoB Without Reducing VLDL-apob Secretion
Authors: Reyes-Soffer Gissette , Dionizovik Marija , Jimenez Jhonsua , Obunike Joseph , Holleran Stephen , Ramakrishnan Rajasekhar , Karmally Wahida , Fontanez Nelson , Thomas Tiffany , Donovan Daniel , Morey Robin , Mittleman Robert , Chin Wai , Baker Brenda , Ginsberg Henry N
Published: March 2022

Objectives:
Mipomersen (MIPO), a second generation antisense oligonucleotide, targets apoB mRNA, thereby inhibiting apolipoprotein B (apoB) synthesis. In humans, MIPO reduces plasma levels of low density lipoprotein-cholesterol (LDL-C), and plasma triglycerides (TG). We hypothesized that these changes are due to reduced assembly and secretion of very low density lipoproteins (VLDL) and lower production of LDL.

Methods:
Healthy volunteers (HVs) (9M, 8F), mean age 43.5 ± 14.2 yr, completed a single-blind, fixed-sequence, phase I study. They received sc-placebo injections once weekly for 3-wks followed by 200mg sc-MIPO injections once weekly for 7-9 wks. Stable isotope turnover studies were performed after each treatment. Blood samples were collected over 48-hrs to determine fractional catabolic rates (FCRs) and production rates (PRs) of apoB in VLDL, IDL, and LDL, and of TG in VLDL. Rates of de novo lipogenesis (DNL) were also measured.

Results:
MIPO treatment resulted in significant reductions in plasma LDL-C (45%), TG (29%), and apoB (40%). VLDL, IDL, and LDL apoB levels fell by 29%, 25%, and 42%, respectively. These changes were associated with increases in FCRs of VLDL apoB (42%) and LDL apoB (30%), and by reductions in PRs of IDL apoB (15%) and LDL apoB (27%). The PR of VLDL apoB was unaffected. The FCR of VLDL-TG increased 46% without change in PR. DNL did not change.

Conclusion:
In summary, 7 wks of MIPO significantly reduced levels of all apoB-lipoproteins in HVs by increasing the FCRs of VLDL and LDL apoB. The absence of a reduction in VLDL apoB secretion is consistent with many studies in isolated hepatocytes demonstrating both intracellular degradation and secretion of newly synthesized apoB. Thus, if MIPO submaximally inhibited apoB synthesis in this study, the liver could have compensated by increasing the efficiency of VLDL assembly and secretion. The basis of increases in VLDL and LDL FCRs requires further investigation.
Abstract 92: Oxidative Stress Induces Endothelial Dysfunction: Role of Sterol Regulatory Element Binding Protein 2 and MicroRNA-92a
Authors: Chen Zhen , Wen Liang , Shyy John
Published: March 2022

Oxidative stress, elevated in pathophysiological conditions such as hypertension, hyperlipidemia, and aging affects endothelial homeostasis by impairing endothelial nitric oxide synthase (eNOS)-derived NO bioavailability and promoting inflammatory response. We recently demonstrated that atheroprone flow activates sterol regulatory element binding protein (SREBP) 2 and induces endothelial innate immunity, evidenced by NLRP3 inflammasome activation. Transgenic mice overexpressing endothelium-specific SREBP2 (EC-SREBP2) manifest accelerated atherosclerosis synergized by hyperlipidemia. In the current study, we investigated whether oxidative stress activates SREBP2 in endothelial cells (ECs), contributing to endothelial dysfunction. Several oxidative stress-inducing stimuli, i.e. hydrogen peroxide, Angiotensin II, and oxidized-LDL, all activates SREBP2 in ECs. Furthermore, SREBP2 transactivates microRNA-92a (miR-92a), previously shown to be dysregulated in ischemia and atherosclerosis. Chromatin immunoprecipitation, together with gain- and loss-of-function assays revealed that oxidative stress-activated SREBP2 induces miR-92a, which in turn targets several key molecules including Kruppel-like factor (KLF) 2, KLF4, and Sirtuin 1. As a result, NLRP3 inflammasome is activated and eNOS inhibited. In EC-SREBP2 mice, locked nucleic acid (LNA) inhibition of miR-92a improves vasodilatory function. In an Angiotensin II-challenged model, LNA-92a ameliorates atherogenesis in EC-SREBP2 with an ApoE-/- background. Collectively, these findings suggest a novel link between oxidative stress and the endothelial inflammation involving SREBP2-miR-92a-inflammasome. This newly defined pathway could be a therapeutic target to intervene endothelial dysfunction during the onset of atherosclerosis.
Abstract 11: Suppression of Coronary Artery Stent Inflammation by Colchicine Decreases Stent Restenosis, as Assessed by Serial in vivo Optical Molecular-structural Imaging
Authors: Osborn Eric A , Ughi Giovanni J , Mauskapf Adam , Oettgen Peter , Tearney Guillermo J , Jaffer Farouc A
Published: March 2022

Background:
Restenosis of coronary stents causes substantial morbidity. Inflammation drives restenosis by activating smooth muscle cells to form obstructive neointima. Recent data suggests that colchicine, an anti-proliferative and anti-inflammatory agent, may reduce clinical stent restenosis. Here we assessed the effects of colchicine on in vivo stent-induced inflammatory protease activity and subsequent restenosis using serial intravascular molecular-structural near-infrared fluorescence (NIRF)-optical coherence tomography (OCT) imaging.

Methods:
Rabbits implanted with clinical-grade bare-metal stents (3.5x12 mm) received oral colchicine 0.6 mg or placebo daily (N=5 rabbits, 15 stents per group) and were imaged at 2 and 6 weeks with intravascular NIRF-OCT 24 hours following ProSense VM110 (4 mg/kg IV; ex/em 750/780 nm) for molecular NIRF imaging of cathepsin protease inflammatory activity. Neointimal formation was measured every 400 μm by co-registered structural OCT. Stents were analyzed by ex vivo fluorescence microscopy, histology, and cathepsin mRNA expression.

Results:
In controls, NIRF inflammation at 2 weeks was significantly enhanced at the stent edges (edge 32.6±7.3 vs. mid 20.1±2.6 nM; p<0.0001) and corresponded to greater stent edge ΔOCT neointimal growth between 2 and 6 weeks (edge 0.61±0.25 vs. mid 0.33±0.13 mm2; p<0.01), providing strong prediction of early NIRF inflammation for subsequent restenosis by OCT (r=0.72; p=0.001). Colchicine significantly decreased 2-week NIRF inflammation (stent TBR: control 2.42±0.10 vs. colchicine 1.69±0.11; p<0.0001), and neutralized the stent edge 6-week neointimal increase observed in controls (colchicine ΔOCT edge 0.05±0.05 vs. mid 0.02±0.03 mm2; p=0.21). Ex vivo analyses corroborated the in vivo results.

Conclusion:
We demonstrate that in vivo NIRF inflammatory protease activity predicts stent locations that develop greater restenosis, and that colchicine, an FDA-approved agent, decreases stent-induced inflammation and subsequent restenosis in vivo. lntravascular NIRF-OCT is a novel translatable approach to predict site-specific coronary stent restenosis based on the local inflammatory profile and monitor the effects of anti-restenosis therapeutics.
Abstract 125: Small Dense LDL ApoB Is Catabolized More Slowly Than Large LDL ApoB in Subjects With Combined Hyperlipidemia, and Rosuvastatin Enhances Its Clearance
Authors: Thongtang Nuntakorn , Diffenderfer Margaret R , Le Ngoc-Anh , Ooi Esther M , Barrett P H , Turner Scott M , Brown W. V , Schaefer Ernst J
Published: March 2022

LDL consists of particles varying in size, density, and composition. Small dense LDL (sdLDL) cholesterol (C) has been shown to be a better predictor of coronary heart disease risk than total LDL-C. Intensive statin therapy significantly lowers sdLDL-C as well as large buoyant LDL (lbLDL)-C. We tested the hypotheses that lbLDL and sdLDL apoB100 have different catabolic rates, and that statin therapy enhances the fractional catabolism of apoB100 in both particles.

Six subjects (3 men and 3 women; age 63±5 years [mean±SEM]; BMI 25.5±1.5 kg/m
2
) with combined hyperlipidemia (triglycerides [TG] 191±50 mg/dl; LDL-C 160±16 mg/dl; HDL-C 43±6 mg/dl) received placebo and rosuvastatin 40 mg/day for 8 weeks each in sequential order. At the end of each phase, the kinetics of apoB100 in triglyceride rich lipoproteins ([TRL]
d
<1.019 g/ml), lbLDL (
d
=1.019-1.044 g/ml), and sdLDL (
d
=1.044-1.063 g/ml) were determined using stable isotope methodology, gas chromatography-mass spectrometry, and multicompartmental modeling.

Compared to placebo, rosuvastatin markedly decreased the plasma levels of LDL-C (52%;
P
=0.0002), TG (32%;
P
=0.06), and total apoB (42%;
P
<0.0001). ApoB100 pool size in TRL, lbLDL, and sdLDL decreased 32±6%, 39±5%, 41±4%, respectively. These changes were attributable to significant (
P
<0.05) increases in apoB100 fractional catabolic rates in TRL (45±16%), lbLDL (131±66%), and sdLDL (97±32%). During both the placebo and rosuvastatin phases, sdLDL apoB100 was catabolized more slowly than lbLDL apoB100 (
P
=0.01 and 0.004, respectively). Rosuvastatin did not alter apoB100 production rate or the percent of TRL apoB100 converted to each LDL subfraction. Proteomic analysis of the LDL particles indicated that rosuvastatin increased apoD abundance in lbLDL and decreased apoAIV abundance in sdLDL. No significant effects on other apolipoproteins were noted.

These data indicate that sdLDL apoB100 is catabolized significantly more slowly than lbLDL apoB100 in dyslipidemic subjects on placebo or statin therapy. Maximal dose rosuvastatin decreases plasma apoB concentrations by enhancing apoB100 catabolism in TRL, lbLDL, and sdLDL and alters the distribution of minor apolipoproteins in LDL subfractions.
Abstract 14: Identification of Linc-OSBPL6 as a Competing Endogenous RNA that Regulates Cholesterol Homeostasis
Authors: van Solingen Coen , Hennessy Elizabeth J , Ouimet Mireille , Rinehold Kaitlyn , Hussein Maryem , Garabedian Michael J , Teupser Daniel , Holdt Lesca M , Moore Kathryn J
Published: March 2022

Long non-coding RNAs (lncRNAs) have emerged as important regulators of gene expression in diverse biological contexts. Although several thousand lncRNAs have been identified in humans, only a small number have been fully characterized. Here we identify a novel human lncRNA, linc-OSBPL6, that functions as a competing endogenous RNA to regulate cellular cholesterol homeostasis. Linc-OSBPL6 has 7 variants that are broadly expressed in various cell types and tissues, and are controlled at the transcriptional level by the cholesterol-sensing nuclear receptor liver X receptor. We noted that each of the linc-OSBPL6 variants harbors binding sites for miR-27b and miR-33a/b, microRNAs recently identified as hubs controlling the expression of genes involved in cellular cholesterol efflux and HDL metabolism. Using linc-OSBPL6 knockdown and overexpression, combined with RNA profiling, we demonstrate that linc-OSBPL6 acts as a ‘microRNA sponge’ to limit availability of miR-27b and miR33a/b and inversely regulate expression of their target genes (ABCA1, OSBPL6, ANGPTL3, CROT, ABCB11). Consistent with the role of miR-27b and miR-33a/b in repressing cellular cholesterol export, we show that overexpression of linc-OSBPL6 increases cholesterol efflux, while shRNA-silencing of linc-OSBPL6 reduces cholesterol efflux from hepatocytes and macrophages. As cholesterol efflux in the liver plays a central role in HDL biogenesis, we investigated the relationship of hepatic linc-OSBPL6 to its microRNA-targets and plasma levels of HDL cholesterol in a cohort of 200 healthy individuals. We found that expression of linc-OSBPL6 is negatively correlated with levels of miR-27b and miR-33a/b in the liver, and positively correlated with levels of their target genes and plasma HDL cholesterol. These findings identify linc-OSBPL6 as a key regulatory component of the non-coding RNA circuitry that controls cellular cholesterol efflux and plasma HDL levels in humans.
Abstract 146: Administration of Interleukin-19 Halts Progression of Pre-formed Plaque, Polarizes Macrophage, and Increases Macrophage Lipid Uptake.
Authors: Gabunia Khatuna , Ellison Stephen , Kelemen Sheri , Kako Farah , Cornwell William , Datta Prasun , Rogers Thomas J , Autieri Michael V
Published: March 2022

Increased uptake of oxLDL by macrophage is proposed to be an atheroprotective mechanism to reduce plaque formation. It has been proposed that macrophage phenotype may be causative or at least associated with plaque severity. Several macrophage phenotypes have been classified, and M2 macrophage are considered to be anti-inflammatory and reparative. Interleukin-19 is a purported anti-inflammatory, Th2 interleukin, and we previously found that addition of rIL-19 could significantly reduce atherosclerosis in susceptible mice. The purpose of this study was to test the hypothesis that administration of exogenous IL-19 could attenuate progression of pre-formed atherosclerotic plaque, and to identify potential molecular mechanisms. LDLR-/- mice were fed high-fat diet for 12 weeks, then administered rIL-19 (10ng/g/day) or PBS for an additional 8 weeks while still consuming HFD. En face analysis demonstrated that IL-19 could halt, but not reverse existing plaque. M2 macrophage marker expression was significantly increased in aorta and spleen from IL-19 treated mice, in IL-19-treated bone marrow derived macrophage (BMDM) and primary human macrophage. IL-19 significantly decreased expression of inflammatory cytokines TNFa, IL-12p40, MCP-1 and IL-1b mRNA in BMDM. Addition of IL-19 to BMDM significantly increased oxLDL uptake as well as expression of lipid uptake receptors CD36, SRA-1, and SRB-1 in primary human macrophage. IL-19 increased expression and activation of PPARg. Knock down of PPARg significantly decreased IL-19 mediated expression of both lipid uptake receptors and uptake of oxLDL. Collectively, these data show that IL-19 can halt progression of established plaque by at least two mechanisms; M2 macrophage polarization, and increasing expression of lipid scavenger receptors and cholesterol uptake in a PPARg-dependent mechanism, suggesting therapeutic potential for this interleukin.
Abstract 153: CCL22/ Macrophage-derived Chemokine Expression in the Setting of Atherosclerosis
Authors: Kimura Satoshi , Wang Ke-Yong , Yamada Sohsuke , Nakayama Toshiyuki , Otsuji Yutaka
Published: March 2022

Introduction:
CCL22 (macrophage-derived chemokine) is a member of the CC-family of chemokines. The functional role of CCL22 as a chemokine involves migration/ recruitment of monocytes and the stimulation of platelet activity. In this study, we investigated the relationship between CCL22 and atherosclerosis in monocytes/ macrophages of mice and humans with respect to atherosclerosis.

Hypothesis:
We assessed the hypothesis that CCL22 is involved in the process of atherosclerosis and can be a biological marker for progression of atherosclerosis and atherosclerotic disease.

Methods:
Apolipoprotein E deficient (apoE-/-) mice as atherosclerotic models were used for this study. Male apoE-/- mice were weaned at 8 weeks of age onto a high-cholesterol diet consisting of 1.25% cholesterol and were maintained on this diet for 12 weeks or 24 weeks. Another group of apoE-/- mice was maintained for 30 weeks on a normal chow diet. Serum CCL22 concentrations were measured by ELISA and expression of CCL22 mRNA in the atheroma were evaluated by real-time PCR. Furthermore immunohistochemical staining was performed. In vitro, the expressions of CCL22 in the mouse monocytes and macrophages were examined and discussed. The expression of CCL22 human monocytes/ macrophages and human atherosclerotic lesions were similarly investigated.

Results:
ApoE-/- mice had predominately high concentrations of CCL22 compared with wild-type (C57BL/6J) mice. ApoE-/- mice developed atherosclerotic lesions with aging and this was influenced by a high-cholesterol diet. CCL22 concentrations increased with aging and a high-cholesterol diet. Their atherosclerotic lesions, especially in macrophages contained abundant levels of CCL22. When the mouse monocyte differentiated into macrophage, the cells showed similar expression of CCL22. Similarly, human macrophages also expressed abundant levels of CCL22 more than monocytes. In addition, immunohistochemical analysis of human atherosclerotic lesions and coronary stenotic lesion revealed that macrophages express CCL22.

Conclusion:
In conclusion, these results suggested that this molecule is involved in the atherogenic processes and CCL22 may represent an attractive molecular marker for progression of atherosclerosis.
Abstract 159: Sex Differences in the Association of Lipoprotein(a) with Subclinical Atherosclerosis in a South Asian Population
Authors: Mulvey Claire , Krauss Ronald M , Kanaya Alka
Published: March 2022

Background:
South Asians have high rates of cardiovascular disease (CVD) that are incompletely explained by traditional risk factors. Some studies have shown that South Asians have elevated levels of lipoprotein(a) [Lp(a)], an LDL-like particle that is a risk factor for CVD. We hypothesized that Lp(a) is associated with subclinical atherosclerosis in South Asians, independent of traditional risk factors.

Methods:
We used data from the Metabolic syndrome and Atherosclerosis in South Asians Living in America study, a community-based cohort (N=146; age 57 ± 8 years; 51% male) free of CVD. Serum Lp(a) was measured by immunonepholometric assay on a BNII nephelometer. Common carotid artery intima-media thickness (CIMT) was measured by ultrasonography. We used multivariate linear regression to test associations of Lp(a) with log-transformed CIMT after adjusting for age, sex, smoking, waist circumference, hypertension, cholesterol medications, C-reactive protein, and fasting insulin and glucose. In additional models, we further adjusted for traditional lipids: LDL- and HDL-cholesterol, and triglycerides. We tested for interactions by sex and stratified our analyses as appropriate.

Results:
Median Lp(a) levels were higher in women than men (0.18 vs. 0.13 g/L, respectively; p=0.06), and higher in post-menopausal vs. pre-menopausal women (0.20 vs. 0.12 g/L; p=0.007). However, median CIMT levels were lower in women compared to men (0.86 vs. 0.95 mm; p=0.002). There was an interaction (p=0.04) by sex in the relationship between Lp(a) and CIMT. In women, this relationship was significant (p=0.001), and it remained significant after further adjustment for lipid parameters (N=72, β coefficient 0.31, 95% CI 0.12 to 0.51; p=0.002). In contrast, there was no association in men between Lp(a) and CIMT in either model (p=0.66 and 0.32, respectively). In subgroup analysis, the association between Lp(a) and CIMT was stronger in post-menopausal women (N=52, β coefficient 0.35, 95% CI 0.08 to 0.62; p=0.01) and weaker in pre-menopausal women (p=0.62).

Conclusions:
Lp(a) was an independent predictor of subclinical atherosclerosis in South Asian women, but not in men. This differs from other populations, where associations tended to be stronger in men.
Abstract 161: Susceptibility to Activation of Human Monocytes in Asymptomatic Atherosclerosis
Authors: Orekhov Alexander N , Nikiforov Nikita E , Elizova Natalia V
Published: March 2022

Monocytes were isolated from blood of three groups of patients, namely those with normal carotid intima-media thickness (CIMT), patients with increased CIMT and patients with plaques in carotid arteries. To assess susceptibility of isolated monocytes to M1 and M2 activation, cells were placed in primary culture with the appropriate stimulator for activation. The degree of M1 or M2 activations was determined by the culture medium level of TNFα and CCL18, respectively. When comparing the average values of TNFα and CCL18 in patients of three groups we were faced with dramatic individual differences. These individual differences have been found both within each group of patients and in the pool (groups 1+2+3). [[Unable to Display Character: М]]onocytes in early atherosclerotic lesions may migrate back into the circulation, possibly serving as a lipid clearance system. Circulating cells laden with lipid have been found in blood. We attempted to find the relationship between cholesterol in monocytes and their susceptibility to activation. There was an obvious trend towards a reverse correlation between intracellular cholesterol level and susceptibility to activation. To reveal the reason of relationship between intracellular cholesterol level and monocyte susceptibility to activation, the experiments on cells cultured with atherogenic modified LDL were carried out. LDL isolated from blood of patients with documented atherosclerosis induces cholesterol accumulation in cultured cells, while LDL from healthy subjects does not affect intracellular cholesterol level. Although modified LDL induced cholesterol accumulation in cultured monocyte-derived cells neither cytokine secretion nor cytokine genes expression were affected. Therefore no LDL but other sources of lipids accumulated in circulating monocytes determine their susceptibility to activation. We consider that difference in monocyte activation is very important because it may determine functional capacity of innate immunity in different patients. Cellular test cell used in this study may serve as a basis for diagnostic method for determining the individual response of innate immunity. This model may be used for the search of new immune correctors. Supported by Russian Scientific Foundation (Grant # 14-15-00112).
Abstract 193: Analysis of Platelet Lysate Hydrogel and Fibrinogen Gel Intramuscular Injection on Neo-Angiogenesis in Ischemic Limbs
Authors: Chadid Tatiana , Kuo Katie M , Li Haiyan , Copland Ian , Brewster Luke P
Published: March 2022

Introduction:
We have recently characterized the in vitro benefits of the fibrin-rich pooled human platelet lysate (PL) in gel form on mesenchymal stem cells’ angiogenic activity. This study aimed to investigate the potential angiogenic activity of PL gel compared to a fibrin hydrogel (FG) in a murine model of hind limb ischemia (HLI).

Methods:
S129 male and female mice aged 20-weeks (n = 30) underwent right femoral artery ligation to induce HLI and were randomized to five groups: G1) Sham (n=5); G2) HLI-no treatment (n=5); G3) Saline (n=5; G4) PL (N=5); and G5) FG (n=5). The treatment limbs received PL, FG or saline by injection of 200 μL in the adductor muscle at day 0. Perfusion was quantitated with Laser Doppler Perfusion Imaging (LDPI) at 1-day post-procedurally and weekly for 28 days, and reported as ratios of the values in the ischemic limb (IL) to those in the contralateral nonischemic limb (NIL) for the adductor (thigh) and gastrocnemius (calf) areas. We then compared the change in the IL/NIL ratios for each group over the 28 days. Non-parametric analysis of variance with Dunn’s correction test was used to compare results among groups. The level of significance was set at p<0.05.

Results:
There were trends in the thigh and calf IL/NIL perfusion ratios at 28 days for mice injected with PL and FG compared to those with saline injections or HLI and no treatment (P>.05). Perfusion in both calf and thigh areas improved from day one to day 28 in all HLI groups. However, only PL and FG groups had significantly increased progression in thigh perfusion, and this was in comparison to sham animals. In the calf, only the FG group demonstrated a significant increase in the perfusion ratio, and this was in comparison to both HLI with no treatment and sham groups (P<.05).

Conclusions:
While both FG and PL gel have active angiogenic properties, FG gel alone, and not PL gel, demonstrated superior in vivo angiogenic response in this model of HLI.
Abstract 200: Vascular Stem Cell Migration in Response to Monocyte Chemoattractant Protein-1 Promotes Neointima Lesion Formation
Authors: Yu Baoqi , Wong Mei M , Wang Wen , Hu Yanhua , Xu Qingbo
Published: March 2022

Introduction:
While vascular smooth muscle cells are traditionally known for their role in neointima formation, recent studies show that a population of Sca-1
+
stem/progenitor cells within the adventitia of vessel walls can also contribute to restenosis formation. To date, their migratory properties and motility in response to fundamental micro-environmental stimuli within the vasculature remain unknown. The occurrence of adventitial inflammation in injured vessels or during atherosclerosis is known to mount an increased secretion of the chemokine, monocyte chemoattractant protein-1 (MCP-1), by cells such as macrophages, endothelial and smooth muscle cells.

Hypothesis:
We hypothesize that the MCP-1 plays an important role on the mobilization of vascular progenitor cells.

Methods and Results:
Vascular progenitor cells were derived from vein graft and isolated with an anti-Sca-1 antibody microbead system. Using transwell assays, we found that recombinant MCP-1 significantly increased progenitor cell migration. The knockdown of its receptor expression, CCR2, using lentiviral shRNA significantly inhibited MCP-1 induced migration. Mechanistically, cytoskeleton related GTPase rac1 and cdc42 in progenitor cells were activated (in the GTP-bound state) by MCP-1. Furthermore, we showed that the ERK 1/2 pathway was also involved in MCP-1-mediated migration. Consistently, the migration of progenitor cells was significantly decreased following treatment with a MAPKK inhibitor. Using a femoral artery wire injury mouse model, we found that the seeding of Sca-1
+
progenitors on the outer layers of the adventitia markedly enhanced neointima formation after 1 week, whereas vessels without progenitor cell seeding did not form neointima. Neointima formation in Sca-1
+
progenitors-seeded vessels were found inhibited in mice that lacked functional MCP-1 (n≥8 per group).

Conclusion:
In conclusion, MCP-1 can induce adventitia-derived Sca-1
+
progenitor cell migration by the activation of GTP-rac1/cdc42 and ERK 1/2 signalling pathway via CCR2. The lack of functional MCP-1 inhibited progenitor cell migration and neointima formation
in vivo
.
Abstract 215: A Systemic Inflammation-Induced Mouse Model of Cerebral Microbleeds
Authors: Sumbria Rachita , Grigoryan Mher Mahoney , Dvornikova Alexandra , Vasilevko Vitaly , Vasquez Alexis , Ngo Quoc Tuan Pham , Pannu Gurjit , Jafari Arash , Nguyen Kristie P , Chan Natalie Hoi Yan , Paganini-Hill Annlia , Scadeng Miriam , Dubowitz David , Kim Ronald , Cribbs David , Fisher Mark
Published: March 2022

Introduction:
Cerebral microhemorrhages (CMH) are tiny deposits of blood degradation products in the brain and are the pathological substrate of cerebral microbleeds (MB). The existing animal models of MB are either β-amyloid-, anti-amyloid antibody-, or hypertension-induced; however, MB may develop independent of hypertension or amyloid deposition. In fact, MB are associated with normal aging, sepsis and neurodegenerative conditions. One common factor among the above pathologies is inflammation, and recent studies show a link between systemic inflammation and MB. Hence, we hypothesize that systemic inflammation (induced by lipopolysaccharide; LPS) will result in MB development, and an inflammation-induced animal model will be appropriate to study MB. In the current study we used two different dosing regimens for LPS and observed rapid and robust development of CMH and MB.

Methods:
Adult C57BL/6 mice were injected with LPS (1mg/kg or 3mg/kg, i.p.) or saline at 0, 6, and 24h. At 2 or 7 days after first LPS injection, brains were harvested. Hematoxylin and eosin (H&E) and Prussian blue (PB) were used to stain fresh (acute) hemorrhages and hemosiderin (subacute hemorrhages), respectively.

Results:
No surface and negligible H&E-positive CMH were observed in saline controls (n=4). LPS (3mg/kg; n=13) resulted in significantly higher number (p<0.01), size (p<0.05) and area (p<0.01) of H&E-positive CMH at 2 days. Negligible PB-positive CMH were observed at 2 days after LPS (3 or 1 mg/kg) injection. However, LPS (1mg/kg) resulted in robust development of PB-positive CMH at 7 days, with significantly higher number (p<0.05), size (p<0.05) and area (p<0.01) in LPS (n=9) versus saline (n=4) treated mice. MRI confirmed the presence of MB in this model. The number of CMH was significantly higher in the cerebellum (p<0.05) compared to cortical or subcortical regions.

Conclusion:
In summary, LPS produced rapid development of H&E-positive CMH (at 2 and 7 days) and PB-positive CMH (at 7 days) with low mortality. Thus, this appears to be an appropriate model to study the pathophysiology of CMH and MB.
Abstract 219: IFN-γ Plays an Essential Role in Accelerating Cardiovascular Disease of Recipients With MHC Class II-mismatched Allografts.
Authors: Zhou Jing , Qin Lingfeng , Yi Tai , Ali Rahmat , Li Qingle , Jiao Yang , Li Guangxin , Zhang Jiasheng , Sadeghi Mehran , Pober Jordan , Tellides George
Published: March 2022

Background:
Organ transplant recipients die from cardiovascular disease (CVD) at a higher rate than the general population. Traditional treatment with statins to mitigate risk factors such as hyperlipidemia has had limited success. Using an animal model of graft-accelerated recipient CVD by combining murine models of atherosclerosis and heterotopic heart transplantation, we aim to better understand its pathogenesis and to explore more effective therapeutic strategies.

Methods and Results:
C57BL/6 (B6) ApoE-deficient recipient mice were heterotopically transplanted with MHC class II mismatched cardiac allografts from donor BM12 mice. Isograft controls were from donor B6 mice. IFN-γ neutralization antibody or isotype IgG was given subcutaneously at a dose of 250 μg per mouse 1 day prior to transplantation and thereafter 125 μg per mouse, 3 times per week for 12 weeks. Serum levels of cholesterol and triglyceride were measured by Amplex® Red Cholesterol Assay Kit and Triglyceride Determination Kit respectively. Inflammatory cytokines were assessed by both real-time PCR and flow cytometry. Native heart function of host mice was evaluated by echocardiography. Aortic atherosclerotic plaques in the host were examined by Oil Red O staining. MHC class II upregulation and neointimal formation in host coronary arteries were determined by immunofluorescence and EVG staining respectively. We found the levels of cholesterol and triglyceride were not changed by introducing allografts into host mice. However, a higher level of IFN-γ was produced by circulating and splenic CD4 T cells in hosts with allografts that was associated with de novo expression of endothelial MHC class II alloantigens in native coronary arteries. These phenomena were diminished by IFN-γ neutralization. Additionally, IFN-γ depletion reduced the local accumulation of IFN-γ and IP-10 mRNA in the native hearts and neointimal formation of coronary arteries in the hosts of allografts. Most importantly, IFN-γ neutralization prevented impaired cardiac function and increased aortic atherosclerotic burden in host mice with allografts.

Conclusions:
Our study reveals an essential role of IFN-γ in accelerating recipient CVD due to systemic inflammation from allograft rejection.
Abstract 227: TGF-β/Smad3 Promotes Smooth Muscle Cell De-differentiation and Proliferation Through Crosstalk with the Wnt/β-Catenin Pathway
Authors: Chaudhary Mirnal , Direnzo Daniel , Shi Xu Dong , Franco Sarah , Zent Joshua , Little Christopher , Wang Katie , Liu Bo , Guo Lian-Wang , Kent K Craig
Published: March 2022

Introduction:
Endovascular interventions are the first line treatment for patients with atherosclerotic disease. However, a significant number of these fail due to neointimal hyperplasia. The Wnt4/β-catenin pathway has been reported to play an important role in this process, but the mechanism underlying the up-regulation of this pathway after injury remains unclear. Our lab has previously demonstrated that following vascular injury, elevated TGF-β and its signaling protein, Smad3, exacerbate intimal hyperplasia in Smooth Muscle Cells (SMCs). Moreover, our Affymetrix array data revealed an upregulation (>3 fold) of several members of the Wnt family in response to TGF-β/Smad3 treatment of SMCs. Therefore, we hypothesize that the TGF-β/Smad3 axis is responsible for the activation of the Wnt/β-catenin signaling pathway that promotes neointimal hyperplasia.

Methods and Results:
To mimic the injury induced up-regulation of TGF-β/Smad3 in vitro, SMCs were infected with an adenovirus to increase Smad3 and then treated with recombinant TGF-β for 48 hrs. Real time PCR confirmed that Wnt expression increased substantially in response to TGF-β/Smad3 stimulation. Using western blotting, we found that SMCs treated with TGF-β/Smad3 had a significantly increased stabilization of β-catenin compared to SMCs treated with TGF-β alone. This effect was abolished by Niclosamide, a broad-spectrum Wnt inhibitor. Conversely, β-catenin stabilization increased after stimulation by a Wnt agonist and recombinant Wnts 2b, 4, 5a, and 9a, but not Wnt11. Importantly, we found that these Wnt ligands stimulated the expression of SMC de-differentiation markers including CXCR4, CD34, Sox18, and FGF1 to varying levels as measured by RT-PCR. Wnt4 and Wnt5a were also found to significantly promote SMC proliferation (21% and 27%, respectively, p<0.05) when measured by the Cell Titer Glo Assay.

Conclusions:
These results suggest that the neointima-producing Wnt/β-catenin pathway may be activated due to elevated TGF-β/Smad3 signaling and promote SMC de-differentiation and proliferation. Thus, the cross talk of these two pathways may provide a potential therapeutic target for preventing failure of endovascular interventions.
Abstract 231: Role of SDF-1α-induced Double Reciprocal E-Selectin/Ligand Pairs in Mediating Recruitment of Endothelial Progenitor Cells to Ischemic Wounds
Authors: Liu Zhao-Jun , Tian Runxia , Li Yan , Shao Hongwei , Velazquez Omaida C
Published: March 2022

Introduction:
We previously reported that stromal cell-derived factor-1α (SDF-1α)-induced endothelial progenitor cells (EPC) homing is enhanced by up-regulation of E-selectin (E-sel) on luminal endothelial cells (EC) lining capillaries, locally within wound tissues. We now investigate how elevated SDF-1α in wounds achieves a remote effect on bone marrow-derived (BMD) circulating EPC.

Methods:
SDF-1α-induced cell adhesion molecules in human EPC and human microvascular endothelial cells (HMVEC) were profiled using PCR-Array and validated by immunoblot. Induction of E-sel expression on murine BMD- and circulating EPC by SDF-1α was examined by flow cytometry. The involvement of E-sel/ligand in mediating SDF-1α-induced EPC-EC interaction and EPC transendothelial migration was studied using in vitro EPC-EC binding assay. The role of SDF-1α-induced E-sel in regulating EPC homing, wound neovascularization and healing were furthered studied by testing EPC from ROSA26-LacZ+/E-sel -/- versus ROSA26-LacZ+/E-sel +/+ mice in an ischemic wound model.

Results:
SDF-1α enhanced EPC-EC interaction by upregulating expression of E-sel/ligand in human EPC and HMVEC. Administration of SDF-1α in wound also increased E-sel expression on BMD- and circulating EPC in mice. The effects of SDF-1α on EPC-EC interaction and EPC transendothelial migration were specifically mediated by E-sel, as E-sel antagonists could inhibit these processes. E-sel was required for SDF-1α-induced EPC homing, wound neovascularization and healing in vivo (n=6, P<0.05).

Conclusions:
EPC-EC interaction is an essential process for EPC homing. SDF-1α induces both local luminal EC lining capillaries in wound tissue and remote BMD- and circulating EPC to express E-sel and its ligands. Up-regulated E-sel/ligand pairs reciprocally on EC and EPC serve as “Double-Lock” to secure targeted EPC-EC interactions selectively occurring within wound endothelium. These findings uncover a novel mechanism underlying the pro-angiogenic effect of SDF-1α on EPC homing and point to E-sel/ligand pairs reciprocally induced on circulating EPC and local endothelium as effective targets for ex vivo manipulation and therapeutic neovascularization.