bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 76: Astrocyte Tissue Factor: Double-Edged Sword of Brain Hemostasis
Authors: Wang Shaobin , Sparkenbaugh Erica M , Russell Janice , Granger D N , Mackman Nigel , Pawlinski Rafal
Published: March 2022

Astrocytes in the brain express high levels of tissue factor (TF) presumably to prevent hemorrhage. We generated mice lacking TF gene in all neuronal cells (TFflox nestin Cre), including astrocytes. TF mRNA expression (99.5%) and total procoagulant activity (98.7%) were significantly reduced in the brains of TFflox nestin Cre mice compared to controls (TFflox). Immunostaining demonstrated the absence of TF expression on astrocytes. No spontaneous bleeding was observed in TFflox nestin Cre mice.

To investigate the role of astrocyte TF in brain hemostasis we used mouse models of ischemic and hemorrhagic stroke. Ischemic stroke was induced by 1 hour occlusion of middle cerebral artery followed by 24 hours of reperfusion. Intracerebral bleeding was observed in 72.7% of TFflox nestin Cre but not in TFflox mice subjected to ischemic stroke. Although TFflox nestin Cre mice exhibited more bleeding, they had reduced infarct size (15.8±5.4% vs 35.1±6.9%; p<0.01, mean±SD). A reduction of infarct size was also observed in mice expressing low levels of TF in all tissues (low TF mice, 18.7±6.8%) compared to wild type (WT, 29.3±6.5%; p<0.05) mice; however, no intracerebral bleeding was observed in low TF mice.

In the hemorrhagic stroke model, intracerebral administration of collagenase induced time and dose dependent bleeding in the brains of WT mice. Deletion of TF from astrocytes dramatically increases intracerebral bleeding (2.9-fold vs TFflox, p<0.01) and death (57.1%, p<0.01) 3 hours after cerebral injection of 0.025 units of collagenase. Less severe intracerebral bleeding (1.5-fold vs WT, p<0.05) and no death was observed in low TF mice. Importantly, astrocytes isolated from low TF mice had higher procoagulant activity compared to astrocytes from TFflox nestin Cre mice.

Finally, using intravital microscopy and photoactivation-induced thrombosis model, we showed that thrombosis within brain capillaries, but not arterioles or venules, was significantly attenuated (p<0.05) in TFflox nestin Cre compared to TFflox mice (consistent with direct contact of astrocyte endfeet with endothelium only within capillaries).

Our data indicate that astrocyte TF is essential for brain hemostasis. However, it also contributes to microvascular thrombosis and neuronal damage.
Abstract 83: Nanopatterned Collagen Scaffolds Promote Blood Perfusion in the Ischemic Limb
Authors: Huang Ngan F , Okogbaa Janet , Jha Arshi , Lee Jerry C , Zaitseva Tatiana , Paukshto Michael V , Cooke John P
Published: March 2022

Cell-based approaches to improve blood perfusion are promising for treatment of peripheral arterial disease (PAD). However, cell delivery alone is limited by poor cell survival and the lack of organization among newly formed vessels. We hypothesized that parallel-aligned nanofibrillar scaffolds may guide endothelial cell (EC) assembly and accelerate the formation of organized vascular networks that enhance blood perfusion, in comparison to cell delivery in saline. We fabricated nanofibrillar collagen scaffolds with parallel-oriented configuration or randomly oriented configuration. Primary human ECs aligned along the direction of the nanofibrils of the aligned nanofibrillar scaffold, as shown by scanning electron microscopy, where the arrow denotes the orientation of nanofibrils (Fig 1). The ECs also had organized cytoskeleton within 20 degrees from the direction of the aligned nanofibrils. To test the efficacy of the aligned nanofibrillar scaffolds in improving neovascularization in vivo, we induced unilateral hindlimb ischemia in SCID mice by excising the superficial femoral artery. The mice received one of the following treatments at the site of the excised femoral artery: 1) aligned nanofibrillar scaffold seeded with human ECs; 2) randomly oriented nanofibrillar scaffold seeded with ECs; 3) EC delivery in saline; or 4) no treatment (n>4). After 14 days, laser Doppler blood spectroscopy demonstrated significant improvement in blood perfusion in the group treated with cell-seeded aligned nanofibrillar scaffolds, in comparison to the groups with no treatment or EC delivery in saline. In summary, these results suggest that aligned nanofibrillar scaffolds regulate EC morphology as well as angiogenic potential. This work highlights the innovative use of biomaterial nanopatterning as a way to promote blood perfusion, and has implications in the treatment of patients with PAD.
Abstract 9: Metabolic Pathways for HDL Subtypes in Low-HDL Overweight Phenotype Compared With Normal
Authors: Mendivil Carlos O , Wang Liyun , Furtado Jeremy , Sacks Frank
Published: March 2022

Introduction:
A prevailing concept of HDL metabolism in plasma is that HDL is secreted into plasma as a small discoidal particle; and then expands and contracts by uptake and removal of cholesterol. This process has not been studied directly in humans.

Hypothesis:
We hypothesized that the 4 common sizes of HDL will show interconversions suggesting reverse cholesterol transport and that is impaired in those who have low HDL-c levels.

Methods:
We studied 12 adults, ages 30 to 70; six had low HDL-c (mean 39 mg/dL) and were overweight, and 6 had high HDL-c (mean 79 mg/dL) and normal body weight. They received a intravenous bolus of trideuterated leucine with serial blood sampling for 94 hours. HDL sizes, very small (<7.1 nm), small (7.1-8.2 nm), medium (8.2-9.5 nm) and large (9.5-12.2), were separated, and the isotope enrichments and masses of apoA-I in each HDL size were measured by mass spectrometry. We tested many kinetic models using SAAM-II.

Results:
In both HDL groups, we found similar peak enrichment heights and times in the four HDL sizes suggesting mostly independent metabolism which was confirmed by the final model that had excellent fits to the data. Most of each plasma HDL size subfraction originated from direct secretion into plasma. Only small amounts of apoA-I flux went from smaller to larger HDL, 19% in the low vs 2% in the high HDL group (p=0.006); and in the opposite direction, 3% in the low vs 9% in the high HDL group (ns). ApoA-I in medium and large HDL had faster fractional catabolic rates (FCRs) in the low than in the high HDL group (0.61 vs 0.31 pools/day [p=0.004] and 0.51 vs 0.24 pools/day [p=0.01]), respectively). In contrast, apoA-I in very small (prebeta) HDL had slower FCR in the low than high HDL group (0.51 vs 0.73/d [p=0.048]). Secretion rates of apoA-I into plasma HDL of each size were not significantly different between the groups. Total apoA-I secretion was 12 vs 14 mg/kg/d in the low vs high HDL group (ns).

Conclusions:
Our results question the concepts of reverse cholesterol transport dominating plasma HDL metabolism, and of its impairment in those with low plasma HDL-c levels. Differences in apoA-I FCR of HDL subfractions explained the well-known shift in size distribution toward smaller HDL in those who have low HDL-c.
Abstract 1: Oct4 is Re-activated in Smooth Muscle Cells through KLF4/Hif1α and Hydroxymethylation Mechanisms and Plays a Critical Role in Atherogenesis
Authors: Cherepanova Olga A , Gomez Delphine , Shankman Laura S , Williams Jason , Geng Yong-Jian , Connelly Jessica J , Owens Gary K
Published: March 2022

The pluripotency factor Oct4 is a protein that regulates embryonic stem cell (ESC) totipotency and is the only gene thought to be indispensable for production of induced pluripotent stem (iPS) cells. Although there is evidence that Oct4 may be expressed in non-ESC, this is controversial and as yet there is no direct evidence that Oct4 plays a functional role within somatic cells.

We demonstrate that Oct4 is markedly induced in cells within atherosclerotic lesions from ApoE-/- mice fed a high-fat diet and within human coronary artery atherosclerotic lesions. We show that smooth muscle cell (SMC)-specific conditional knockout (KO) of Oct4 within Western diet-fed ApoE-/- mice resulted in increased atherosclerotic lesion size and other changes consistent with decreased plaque stability. Moreover, using a novel SMC lineage tracing mouse developed in our lab, we also show that lesions from SMC-specific conditional Oct4 KO mice contained far fewer de-differentiated (Acta2-) SMCs likely as the result of impaired SMC de-differentiation and migration from the media into the intima and not to altered proliferation or apoptosis. Further mechanistic studies using in vitro and in vivo Chromatin Immunoprecipitation assays, glycosylation-coupled methylation sensitive qPCR and hMeDIP show that activation of Oct4 within SMCs and within atherosclerotic lsions was associated with hydroxymethylation of the Oct4 promoter and was Klf4- and Hif1α-dependent. In addition, using in situ hybridization/proximity ligation (ISH-PLA) assay developed in our lab we showed hydroxymethylation of the Oct4 promoter within individual SMC-derived cells in atherosclerotic lesions of our SMC-lineage tracing ApoE-/- mice. These results are the first to show that Oct4 plays a direct functional role in regulating phenotypic transitions in somatic cells, and provide molecular mechanisms required for Oct4 re-activation.
Abstract 10: Endothelial Intracellular Adenosine Epigenetically Regulates Angiogenesis
Authors: Xu Yiming , Yan Siyuan , Wang Yong , An Xiaofei , Li Qinkai , Wu Chaodong , Huo Yuqing
Published: March 2022

Background and Objectives:
It is well known that extracellular adenosine regulates angiogenesis. However, it is unclear whether intracellular adenosine is able to modulate endothelial angiogenesis. This study is to investigate the effect of elevated intracellular adenosine caused by adenosine kinase (ADK) deletion or knockdown on endothelial angiogenesis and further to determine the involvement of adenosine receptors and methylation pathways in this effect.

Methods and results:
Human Umbilical Vein Endothelial Cells (HUVECs) were transfected with adenoviral ADK shRNA. ADK knockdown elevated the level of intracellular adenosine, and consequently increased endothelial migration and proliferation. The latter was accompanied with an increase in VEGFR2 expression and PI3K-Akt activation and a decrease of global DNA methylation. Treatment of HUVEC with S-adenosylmethionine or DNA transmethylase inhibitor 5-aza-2-deoxycytodine abolished ADK-knockdown induced VEGFR2 expression and angiogenesis. VEGFR2 expression and global DNA methylation in ADK-knockdown HUVECs were not interfered with adenosine receptor antagonists. Methylation-specific PCR assay showed the decreased methylation of the VEGFR2 promoter, which is the cause for an upregulation of VEGFR2 in ADK deficient HUVECs. Endothelial-specific ADK knockout mice had increased retinal angiogenesis, accelerated wound healing and were protected against hind-limb ischemia.

Conclusion:
ADK knockdown-mediated elevation of intracellular adenosine in endothelial cells reduces DNA methylation, leading to an upregulation of VEGFR2 and enhancement of angiogenesis.
Abstract 116: Mechanism of ApoA-I Attenuation of Inflammation Associated with Atherosclerosis
Authors: Nuradin Nebil , Pollard Ricquita D , Zabalawi Manal , Pulp Brian , Sorci-Thomas Mary G , Thomas Michael J
Published: March 2022

Cytokines/chemokines and their receptors have been an important consideration when investigating the role of inflammation in atherosclerosis. In humans and animal models, monocyte recruitment and accumulation within the artery wall occurs in response to elevated plasma cholesterol levels. To mimic this process in a mouse model of atherosclerosis, our lab has utilized the LDLr-/-, ApoA-I-/- double knockout mouse. Our studies have shown that LDLr-/-, ApoA-I-/- mice have increased immune cell mobilization driving the progression of atherosclerosis when compared to LDLr-/- mice, and that treating mice with frequent, low doses (200 μg) of subcutaneous administered lipid-free apoA-I or rHDL reverses the atherogenic process. Based on these studies we wanted to investigate which inflammatory pathways were most affected by apoA-I treatment. To do this, we carried out RT-PCR to obtain the relative mRNA abundance on 40 different markers of inflammation in two genotypes of atherogenic diet fed mice. Each genotype of mice was divided into two subsets. One subset was fed an atherogenic diet for 12 weeks while the other subset received 12 weeks of diet and subcutaneous injections of 200 μg of apoA-I for the last 6 weeks of the study. Spleen was isolated from all animals and their inflammatory gene mRNA expression examined. Most significantly both genotypes showed a large decrease in IL-3 mRNA expression but also showed a significant increase in CD163 mRNA expression with apoA-I treatment. Decrease in splenic IL-3 expression is a significant mediator of inflammation regression because of the role IL-3 plays in monocyte production inside the spleen. Other investigators have shown that myeloid cells reserved in the spleen give rise to monocytes in an IL-3 rich environment. IL-3 develops and maintains circulating monocytes, which may accumulate in the arteries during atherosclerosis. This suggests apoA-I treatment affects IL-3 levels in the spleen, and thus the amount of monocytes in circulation. An increase in CD163 mRNA expression in the spleen implies higher accumulation of monocytes inside the spleen, and out of circulation. This suggests that apoA-1 treatment can decrease the amount of monocytes in the arteries, which can be a result of reverse in the atherogenic process.
Abstract 117: The Impact of Apolipoprotein B Depletion Methods on HDL Subspecies Distribution and Function
Authors: Shah Amy S , Heink Anna , Davidson W S
Published: March 2022

Recent failures of high density lipoprotein cholesterol (HDL-C) raising therapies have shifted attention on understanding HDL functionality vs. quantity. For example, the ability of HDL to promote cholesterol efflux has been recently shown to be highly correlated with protection from cardiovascular disease - more so than HDL-C. VLDL and LDL, apoB rich lipoproteins (apoB-Lp), are commonly precipitated from plasma and the remaining HDL is assayed in situ. Two reagents for precipitating apoB-Lp’s are polyethylene glycol (PEG), a neutral polymer that influences their solubility and dextran sulfate/MgCl2+ (DS), a polyvalent/ divalent combination that separates apoB-containing particles by charge. Given the recent focus on measuring HDL function under these precipitating conditions, it is critical to be sure that the constellation of HDL subspecies is not being altered by the apoB-Lp precipitation procedure. Using a gel filtration chromatography system, we compared the HDL subspecies profile in plasma before and after apoB-Lp precipitation by these methods. Compared to plasma alone, apoB-Lp depetion by PEG dramatically altered the HDL subspecies distribution with a loss of larger phospholipid-rich HDL and subsequent generation of smaller particles. This effect may result from the persistent association of PEG with HDL in plasma. The ability of these HDL particles to promote cholesterol efflux was also shifted in line with the phospholipid distribution. Conversely, DS precipitation had no effect on the HDL size distribution as determined by phospholipid and cholesterol profiles and cholesterol efflux pattern. Western blots showed significant decreases in HDL apoE content for the PEG method, but less so for DS. Ongoing work is aimed at characterizing the effect of these methods on other proteomic changes and functions (i.e. antioxidation) as well as comparing these methods to the “gold standard” of immunoaffinity isolation of apoB-Lp. Given the complexity of the HDL proteome and the growing realization that the family is composed of many functional subspecies, it is important that future clinical assays be carefully validated to be sure that important HDL functional particles are not being overlooked.
Abstract 122: Ceramide Activation of Macrophage RhoA/Rho Kinase/LIM Kinase Signaling Impairs Aggregated LDL Degradation and Foam Cell Formation
Authors: Singh Rajesh K , Haka Abigail S , Grosheva Inna , Brumfield Alexandra , Xiong Yuquan , Hla Timothy , Maxfield Frederick R
Published: March 2022

Introduction:
One of the initiating events in atherogenesis is the deposition of low-density lipoproteins (LDL) in the arterial wall. This LDL becomes modified, aggregated and retained. Macrophages form a degradative contact (a lysosomal synapse or LS) with the aggregated LDL (agLDL) through local actin polymerization. Degradation of agLDL releases free cholesterol, which is taken up by macrophages and results in foam cell formation. Characterization of proteins regulating actin polymerization that allows LS formation may identify new therapeutic targets to inhibit foam cell formation and halt the progression of atherosclerosis.

Hypothesis:
Small GTPases of the Rho family such as RhoA are key regulators of actin polymerization. Further, many lipids in the micro-environment of the atherosclerotic plaque, such as ceramide, are known to modulate RhoA activity. Therefore we tested the hypothesis that RhoA is important in actin polymerization and foam cell formation in response to macrophage catabolism of agLDL and that this process can be modulated by ceramide.

Methods:
We overexpressed RhoA in RAW264.7 macrophages prior to treatment with agLDL to test the role of RhoA in LS formation and foam cell formation. We used bone marrow derived macrophages (BMM) from Sphingosine Kinase 2 knockout mice (SK2 KO) as a model for macrophages loaded with long chain ceramides and wild-type BMM loaded with C2-ceramide to test the role of ceramide. We also used inhibitors of Rho Kinase ROCK and LIM Kinase to assess their role in LS formation.

Results and Conclusions:
Overexpression of wild-type and constitutive active RhoA in RAW264.7 macrophages significantly impaired actin polymerization and foam cell formation in response to agLDL treatment. Using SK2KO BMM and wild-type BMM loaded with C2- ceramide, we find that macrophages loaded with ceramide display increased levels of activated RhoA. This leads to impaired actin polymerization and foam cell formation which could be rescued by RNAi mediated silencing of RhoA, inhibition of ROCK or inhibition of LIMK. In conclusion, these results suggest that RhoA/ROCK/LIMK signaling negatively regulates agLDL degradation and foam cell formation and that this process can be directly modulated by ceramide through activation of RhoA.
Abstract 132: CETP Deficiency in Rabbits Protects High Fat High Cholesterol Diet Induced Atherosclerosis
Authors: Zhang Jifeng , Xu Jie , Liang Jingyan , Yang Dongshan , Guo Yanhong , Zhu Tianqing , Song Jun , Niimi Manabu , Lai Liangxue , Fan Jianglin , Chen Y. Eugene
Published: March 2022

CETP has been suggested as an effective and promising target for further reducing cardiovascular events through elevation of the HDL-C levels. The failure of Pfizer’s torcetrapib and Roche’s dalcetrapib raised the question whether increased HDL from pharmacological inhibition of CETP are cardioprotective. Because mice lack CETP naturally, we generated CETP knockout (KO) rabbits by Zinc-finger nucleases (ZFN) to gain insights onto CETP and HDL biology in CVD. The ZFN pairs target on exon 3 of rabbit CETP gene. By microinjection of ZFN mRNA into rabbit embryos, we generated 5 CETP knockout (CETPko) founder rabbits. The founder used for this study contained 4 nucleotides deletion in exon 3 that results in a premature stop codon. Western blot confirms that there is no detectable CETP protein in the homozygous CETPko rabbit plasma and liver. No CETP activity was detected in the CETPko rabbit plasma, consistent with their genotype. At the normal chow fed conditions, there is no significant difference between wild type and CETPko male rabbits in plasma HDLc and total cholesterol (TC) level; whereas female CETPko rabbits show 40 to 100% increase in both HDLc and TC levels compared to wild type ones. By sequential ultracentrifugation method to separate plasma lipoproteins, we found that there is much higher HDL3 component in female CETPko rabbits, but not in male CETPko ones, comparing to their WT age matched control. At the HFHC (3% soy bean oil and 0.3% cholesterol) diet fed conditions, both male and female CETPko rabbits show significantly lower level of TC and LDLc compared to wild type rabbits. At the same time, the HDLc levels are significantly higher in the CETPko rabbits. In addition, apoB depleted plasma from CETPko rabbit showed 2-fold increase in cholesterol efflux tested in human THP1 cells. Furthermore, we measured the atherosclerotic plaque in the rabbit aorta after 16 weeks HFHC diet treatment. The total gross lesion areas in CETPko rabbits were only one-third of those in the wild type rabbits. In conclusion, our study shows that genetic deficiency of CETP in rabbits has beneficial effects on enhancing HDL function and reducing atherosclerosis. Our work sheds light on CETP and HDL biology, and provides valuable information to the development of CETP drugs.
Abstract 137: Skap2 Modulates Macrophage Polarization and Regulates Atherosclerosis
Authors: Hyatt Danielle , Golan David , Swanson Kenneth , Alenghat Francis J
Published: March 2022

Background:
Integrins mediate macrophage behaviors that are central to atherogenesis. Macrophage integrins cooperate with other membrane and adaptor proteins (including CD47, Sirpα, and Skap2) to control integrin-induced actin reorganization and migration. We hypothesized that such signals would impact the progression of atherosclerosis.

Results:
Using integrin-binding microbeads applied to bone marrow-derived macrophages (BMMs) from mice with specific gene knockouts, we have found that local actin polymerization requires three major proteins - Sirpα, CD47 and Skap2 - that are known to cooperate in signaling complexes recruited by activated integrins. We introduced Skap2 deficiency to apoE-knockout mice to ascertain how Skap2 modulates atherosclerosis. Skap2-/-;apoE-/- C57BL/6 mice develop increased atherosclerotic plaque in their aortic roots, compared to their Skap2+/+;apoE-/- counterparts, as early as 18 weeks of age on a standard diet. There is no detectable difference in weight, lipid profile, development, or other organ pathology. Skap2-deficient mice have a circulating cytokine profile consistent with a macrophage-centric inflammatory process. While Skap2-deficiency does not impact macropinocytosis or scavenger-receptor-mediated acetylated LDL uptake, the macrophage distribution within the plaque differs significantly from standard murine atheromata with an overall macrophage content proportional to the increased size of the plaque. Finally, Skap2 is prominently expressed in M2 (regulatory) macrophages, and Skap2-deficient BMMs demonstrate impaired expression of M2 markers when stimulated by IL4 in vitro.

Conclusion:
Overall, we find that a Skap2-dependent pathway directs macrophage migration and actin reorganization. This pathway contributes to regulation of atherogenesis, maintenance of normal lesional macrophage homeostasis, and polarization of regulatory macrophages.
Abstract 141: The Role of Macrophage Epsins in the Regulation of LRP-1 in Atherosclerosis
Authors: Brophy Megan L , Dong Yunzhou , Tessneer Kandice L , Song Hoogeun , Pasula Satish , Cai Xiaofeng , Chang Baojun , Wu Hao , Ley Klaus , Chen Hong
Published: March 2022

Background:
Epsins are a family of ubiquitin-binding endocytic clathrin adaptors. We recently published that endothelial epsins function as critical regulators of tumor angiogenesis by controlling VEGF signaling (JCI, 2012; ATVB, 2013). Our goal is to define the novel role of epsins in macrophages in regulating atherogenesis.

Methods and Results:
We engineered mice with specific deletion of epsins in myeloid cells (MΦ-DKO). Strikingly, MΦ-DKO mice on ApoE-/- background fed western diet significantly reduced atherosclerotic lesion formation and foam cell accumulation. In macrophages, epsin deficiency did not alter LDL scavenger receptors, CD36, Lox1 or SRB1, or reverse cholesterol transport proteins, ABCA1 or ABCG1, but did significantly reduce Lucifer Yellow pinocytosis, indicating a major defect in lipid uptake. Epsin deficiency did decrease total and surface protein levels of LRP-1, a protein with anti-inflammatory and anti-atherosclerotic properties. Oil Red O staining of isolated ApoE-/-/M[[Unable to Display Character: Ф]]-DKO macrophages showed little lipid accumulation, suggesting a mechanism in which epsin deficiency impairs foam cell formation. In addition, epsin 1 and LRP-1 interact in macrophages. Furthermore, this interaction is abolished in the absence of epsin’s UIM domain and LPS treatment increases LRP-1 ubiquitination, suggesting that epsin promotes the ubiquitin-dependent internalization of LRP-1. Epsin deficiency also significantly suppressed the pro-inflammatory M1 macrophage phenotype found in plaques and increased the anti-inflammatory macrophage phenotype, thus suggesting an important pro-inflammatory role for epsins in macrophages. Our finding implicates epsin as a potential therapeutic target for atherosclerosis treatment.

Conclusions:
We demonstrate epsins promote atherogenesis by potentiating foam cell formation and maintaining pro-inflammatory macrophages within the atherosclerotic plaque, thus suggesting epsins as a novel therapeutic target to combat atherogenesis.
Abstract 147: Characterization of the Immune Response to Aortic Aneurysm in a Murine Model
Authors: Byrne John S , Besla Rickvinder , Ensan Sherine , Li Angela , Degousee Norbert , Ouzounian Maral , Lindsay Thomas , Robbins Clint
Published: March 2022

Our understanding of vascular pathology relies on inducible animal models of disease that remain poorly described, and concern exists regarding indirect effects of the inducing agents. We sought to perform a detailed characterization of the immune response to a commonly used murine aneurysm model.

Aneurysms were generated in 10 adult C57/Bl6 mice using 2 weeks of oral beta-amino propriono-nitrile (BAPN) administration and 4 weeks of angiotensin-2 (AT2) delivered via osmotic pump. FACS analysis was used to characterize progenitor and hematopoietic cell lines in bone marrow, blood and aorta. 3 mice died of aortic rupture between days 8 and 9. The remaining 7 were compared to age-matched controls.

BAPN/AT2 treatment caused aortic dilatation at all aortic regions from the root to the descending thoracic aorta (P<0.01). The suprarenal aorta was aneurysmal in all BAPN/AT2 mice with a maximum diameter of 1.86±0.1mm compared to controls 0.98±0.03mm (P<0.001). In the blood, total cells counts at day 2, 16 and 28 were increased (P<0.001). CD45-2 and B-cell counts were significantly increased in the BAPN/AT2 mice compared to controls (P<0.001). The aorta of BAPN/AT2 mice had significantly increased macrophage counts (P<0.001) with no difference in monocytes, neutrophils, T or B cell counts. Aortic tissue macrophages were predominantly G0 G1 (97±1%) phase. The bone marrow CD45-2 compartment was similar in aneurysmal BAPN/AT2 and control mice but Ly6C lo-inter monocytes were increased in BAPN/AT2 mice. Marrow CD117+/lin- hematopoietic progenitor and stem cells (P=0.4), and differential counts of CD117+/Sca1+ hematopoietic stem cells and CD117+/Sca1- progenitor cells were similar in control and BAPN/AT2 treated mice.

BAPN/AT2 induces aortic disease with aneurysmal degeneration of the suprarenal aorta. We observed a significant decrease in aortic macrophage proliferation suggestive of a mechanism of expansion independent of local macrophage proliferation. We did not observe a significant change in the bone marrow stem or progenitor cell compartment suggesting aortic injury is driving inflammation rather than direct stimulus of the marrow compartment by BAPN/AT2.
Abstract 150: Identification of SYNPO2, SYNM, LMOD1, PDLIM7 and PLN as Novel Markers of Smooth Muscle Cells in Atherosclerosis
Authors: Hedin Ulf , Perisic Matic Ljubica , Razuvaev Anton , Sabater-Lleal Maria , Lengquist Mariette , Miller Clint L , Roy Joy , Kronqvist Malin , Gonzalez Diez Maria , Frebelius Siw , Leeper Nick , Maegdefessel Lars , Quertermous Thomas , Paulsson-Berne Gabrielle , Odeberg Jacob , Hansson Göran K , Hamsten Anders
Published: March 2022

Smooth muscle cell (SMC) activation is a hallmark of vascular remodeling associated with downregulation of SMC specific genes and altered cell function. Our aim was to identify a novel set of genes silenced following SMC activation in vascular disease.

We compared microarrays from carotid plaques (n=177) vs. undiseased arteries and symptomatic vs. asymptomatic patients and found that ‘muscle contraction’, ‘muscle differentiation’ and ‘cytoskeleton based migration’ were the most significantly downregulated pathways (p=6.06e-23; 5.22e-11; 4.62e-11 respectively). Genes clustered in these categories were known SMC markers together with a novel set of genes functionally coupled to actin cytoskeleton: SYNPO2, SYNM, LMOD1, PDLIM7 and PLN. Transcription factors regulating the expression of these genes showed enrichment of SRF, MYOD1 and MYOGENIN, known to control muscle differentiation. Downregulation of these genes was validated in independent microarrays from carotid (n=21) and coronary plaques (n=38). Their expression was positively correlated to typical SMC markers both in human plaques (Pearson r>0.8, p<0.0001) and rat carotid intimal hyperplasia (r>0.9, p<0.0001). In microarrays from rat carotid balloon injury, these genes were downregulated in the early phases of tissue remodeling but gradually reappeared in the mature intima. By immunohistochemistry, the proteins localized to SMCs in normal human vessels, but were mostly absent in plaques and restenosis tissues. In vitro, SYNPO2 and PDLIM7 localized to actin filaments, SYNM to cortical cytoskeleton, PLN to nucleus while LMOD1 was rapidly lost in cultured human and primary rat SMCs. Polymorphisms in some of these genes associated with carotid intima-media thickness in high-risk coronary artery disease subjects (n=3400). By eQTL analyses, rs11746443 influenced PDLIM7 expression in plaques. Interestingly, this polymorphism constitutes the binding site for HEY1 transcription factor implicated in vascular development.

We identified a panel of novel SMC proteins, which are lost in vascular disease and reflect the altered phenotype of SMCs in vascular remodeling. Our results indicate that these could be early and sensitive markers of the SMCs dedifferentiation in atherosclerosis.
Abstract 151: Lysine Residues at Positions 4 and 7 on Nef are Critical for Interaction with Calnexin and Drive Inhibition of Cholesterol Transporter: ATP-Binding Cassette A1
Authors: Hunegnaw Ruth , Bukrinsky Michael , Adzhubei Alexei
Published: March 2022

HIV patients are at a greater risk of developing atherosclerosis than non-infected individuals, partly due to the impairment of the ATP-Binding Cassette A1 (ABCA1) cholesterol transporter by the HIV-1 viral protein Nef leading to accumulation of cholesterol inside the cell. While studying the possible mechanism of Nef-mediated disruption of cholesterol efflux, we found that ABCA1 interacts with Nef, but a direct interaction with Nef is dispensable for the inactivation of ABCA1. Using mass spectroscopy we identified calnexin as a protein that associates with both ABCA1 and Nef and provided evidence to show that in the presence of Nef, ABCA1-calnexin interaction is disrupted leading to ABCA1 retention in the ER, subsequent degradation and impairment of cholesterol efflux. However, the molecular interactions taking place remained unknown as Nef is not known to enter the ER lumen and the domain of calnexin involved in binding to substrate proteins is located within the ER lumen. We hypothesized that Nef interacts with the C-terminal cytoplasmic domain of calnexin and that inhibiting this interaction would rescue ABCA1 function and expression. Using calnexin mutants lacking a luminal or cytoplasmic domain, we identified that the C-terminal cytoplasmic domain is responsible for Nef interaction. Using structural models of Nef and calnexin, possible Nef-calnexin interaction models were built using docking servers. Interacting residues in Nef were identified by calculating intermolecular contacts in the resulting complexes. Identified residues were mutated to confirm loss of interaction and this loss of interaction was found to associate with rescue of ABCA1 expression and restoration of cholesterol efflux. In conclusion, lysine residues at positions 4 and 7 on Nef were found to be indispensable for interacting with calnexin and inactivation of ABCA1. As cardiovascular diseases like atherosclerosis have emerged as an important cause of morbidity and mortality in HIV-infected individuals, there is a great need for targeted therapeutic strategies. This study identifies important targets that can be manipulated to inhibit the pathogenic effect of HIV on cholesterol metabolism.
Abstract 16: Tribbles1 Regulates Hepatic Lipogenesis in Mice through Post-Translational Regulation of C/EBPα
Authors: Bauer Robert C , Sasaki Makoto , Cohen Daniel M , Cui Jian , Smith Mikhaila A , Steger David J , Rader Daniel J
Published: March 2022

Tribbles1 (TRIB1) was recently identified in genome-wide association studies as being associated with plasma levels of VLDL, HDL, LDL, and TG as well as coronary artery disease in humans. Here we report studies in a Trib1 liver-specific knockout mouse (Trib1_LSKO) created via AAV-mediated delivery of Cre recombinase into mice with a floxed allele of Trib1. Four weeks after infection, Trib1_LSKO mice exhibit a 25% increase in liver weight (p<0.01) as compared to controls given null virus, and examination of hepatic lipids reveals a 78% increase in TG content (p<0.001) of Trib1_LSKO livers. Hepatic transcription of lipogenic genes (i.e. Scd1, Fasn, and Acaca) was increased greater than 2-fold in Trib1_LSKO mice, and when in vivo lipogenesis was measured, Trib1_LSKO animals exhibited significantly increased hepatic production of fatty acids (2.2-fold, p=0.02), diacylglycerol (1.8-fold, p<0.01), and TG (3.6-fold, p<0.001). Microarray and subsequent pathway analysis of Trib1_LSKO livers revealed altered transcription of genes downstream of the transcription factor C/EBPα, and western blot analysis of liver extracts showed increased protein levels of C/EBPα in Trib1_LSKO mice compared to controls. C57Bl/6 mice overexpressing C/EBPα via AAV (AAV_Cebpa) exhibit increases in hepatic production of TG (3.6-fold, p<0.001), fatty acids (2.5-fold, p<0.001), and diacylglycerol (2.1-fold, p<0.005) that mirror those observed in Trib1_LSKO mice. Interestingly, while Trib1_LSKO mice have altered plasma lipids, AAV_Cebpa animals do not, suggesting that Trib1 modulates plasma lipids via a mechanism separate from C/EBPα-mediated lipogenesis. Finally, ChIP-Seq analysis of C/EBPα DNA occupancy in Trib1_LSKO and AAV_Cebpa mice showed increased binding near lipogenic genes as well as upstream of Trib1 itself. Trib1 does show increased gene expression in response to C/EBPα overexpression, suggesting that Trib1 and C/EBPα participate in a feedback loop. In summary, we demonstrate here that Trib1 regulates de novo lipogenesis through modulation of hepatic protein levels of C/EBPα, which in turn regulates Trib1 expression. These data assign novel roles for both Trib1 and C/EBPα in the regulation of hepatic lipid metabolism.
Abstract 164: Identification of Genetic Regulators of the Atherosclerosis-Associated Metabolite Trimethylamine-N-Oxide in the Diversity Outbred Mice Population
Authors: Smallwood Tangi L , Jung Kuo-Chen , Zhao Liyang , Hua Kunjie , Bennett Brian , Pomp Daniel
Published: March 2022

Dietary choline and its derivatives have been associated with various aspects of lipid metabolism. Recently, the choline metabolite trimethylamine-N-oxide (TMAO) has been associated with atherosclerosis in both mice and humans. Traditional studies of atherosclerosis in mice use genetic or dietary manipulation to induce atherosclerosis in inbred mouse strains. However, traditional quantitative trait locus (QTL) mapping studies using inbred strains often identify large genomic regions, containing many genes, due to limited recombination and/or sample size which hamper candidate gene identification and translation of these results into possible risk factors and therapeutic targets. As an alternative approach, here we use the multi-parental Diversity Outbred (DO) mouse panel for genetic mapping in order to aid in the identification of causal genes and variants associated with TMAO and its precursor choline. We fed DO mice either a high-fat, cholesterol-containing (HFCA) diet or a low-fat, high-protein diet for 18 weeks and measured circulating metabolites at baseline and after diet. Among our highly significant loci, we detected a 4.6 Mb QTL interval on Chromosome 12 (LOD = 10.0, p<0.05) associated with plasma concentrations of the metabolite trimethylamine-N-oxide at baseline, containing 116 genes; the same QTL was also identified after dietary treatment (LOD= 6.7, p<0.1). By using a database of mouse gene expression and measuring gene expression in the eight progenitor strains by RNA-Sequencing, we identified three potential candidate genes, including the positional candidate gene Numb, which encodes a clathrin adapter with a role in endocytosis that was recently shown to modulate intestinal cholesterol absorption. We show that TMAO and Numb exhibit inverse strain variation across the DO founder strains and that the novel Chromosome 12 TMAO locus co-localizes with a highly significant cis-expression QTL for Numb, indicating a potential functional relationship.
Abstract 165: Microrna-486 Regulates Fibroblast-to-myofibroblast Transition in Human Aortic Valve Interstitial Cells
Authors: Song Rui , Fullerton David A. , Ao Lihua , Yao Qingzhou , Zhao Kesen , Meng Xianzhong
Published: March 2022

Myofibroblasts play an important role in aortic valve fibrosis. In normal aortic valves, the interstitial cells (VICs) are primarily fibroblasts while myofibroblasts are dominant among VICs in stenotic aortic valves. Suppression of VIC phenotypic change may prevent the progression of aortic stenosis. However, the molecular mechanism underlying the fibroblast-to-myofibroblast transition in human VICs is not well understood. We hypothesized that an epigenetic mechanism controls the phenotypic transition in VICs of human aortic valves. Methods and results: Immunoblotting and immunofluorescence staining confirmed that VICs of diseased human aortic valves had higher levels of α-SMA and α-SMA fibers. MicroRNA (miR) microarray and real-time qPCR analysis revealed higher levels of miR-486 in VICs of diseased aortic valves. Lentiviral expression of miR-486 antagomir in diseased VICs markedly suppressed α-SMA expression and α-SMA fiber formation. Conversely, lentiviral expression of miR-486 mimic in normal VICs increased α-SMA levels and enhanced α-SMA fiber formation. Further, expression of miR-486 mimic in normal VICs activated Akt and up-regulated cellular levels of myosin light chain (MLC) kinase. Inhibition of Akt reduced α-SMA expression induced by miR-486 mimic. Inhibition of MLC kinase attenuated α-SMA fiber formation without affecting α-SMA levels. Prolonged treatment of normal VICs with miR-486 increased the production of collagen I, MMP-2 and MMP-9. Stimulation of normal VICs with TGF-β1 or BMP-2 caused a marked increase in cellular miR-486 level that was accompanied by up-regulated α-SMA expression, α-SMA fiber formation and matrix protein production. MiR-486 antagomir suppressed these changes associated with myofibroblastic transition and activity in cells exposed to TGF-β1 or BMP-2. Conclusions: MiR-486 regulates fibroblast-to-myofibroblast transition in human VICs, and pro-fibrogenic factors up-regulate miR-486 expression in VICs. Elevated levels of miR-486 accounts, at least in part, for the myofibroblastic phenotype of VICs in stenotic aortic valves. These novel findings indicate that modulation of miR-486 expression and/or function has therapeutic potential for slowing down the progression of aortic stenosis.
Abstract 17: SURP and G Patch Domain Containing 1 is a Novel Regulator of Cholesterol Metabolism
Authors: Medina Marisa W , Yu Chi-Yi , Kim Mee J , Theusch Elizabeth , Naidoo Devesh , Stevens Kristen , Kuang Yu-Lin
Published: March 2022

rs10401969 tags a large GWAS-identified locus associated with coronary artery disease (CAD) and plasma LDL cholesterol. This locus contains over a dozen genes, as well as a nonsynonymous SNP in TM6SF2. Recently, several studies have shown that TM6SF2 impacts lipid metabolism, and concluded that it is a causative gene within the locus. However, it is possible that one or more of the other genes at this locus may also contribute to CAD risk. SURP and G patch domain containing 1 (SUGP1) is a putative splicing factor, and we have previously reported that cholesterol genes can be coordinately regulated by alternative splicing. Since rs10401969 is contained within intron 8 of SUGP1, we hypothesized that SUGP1 may contribute to the relationship between rs10401969 and plasma LDLC. Western blot analysis of lymphoblastoid cell lines (n=48) revealed that SUGP1 protein levels differed by rs10401969 genotype. In HepG2 and Huh7 cells transfected with a SUGP1 mini-gene construct, we found that rs10401969 induces SUGP1 exon 8 skipping, creating a SUGP1 transcript that is subject to nonsense-mediated decay. Through EMSAs, we established that rs10401969 disrupted binding of HNRNPA1, a well-known splicing factor of SUGP1 RNA. To establish the physiological effects of SUGP1, we overexpressed Sugp1 by tail vein delivery into CD1 male mice (n=10), and after 1 and 4-weeks, plasma cholesterol levels were increased 20% and 50%, respectively, compared to controls. In HepG2 and Huh7 cells, we found that SUGP1 targets 3-hydroxy-3-methylglutaryl-CoA reductase (HMGCR), which encodes the rate-limiting step of cholesterol biosynthesis. SUGP1 knock-down stimulated alternative splicing of HMGCR and decreased transcript stability, resulting in reduced HMGCR enzyme activity, cholesterol synthesis and increased LDL uptake. These findings are consistent with the increase in plasma cholesterol observed after Sugp1 overexpression in vivo. In summary, these results support a role for SUGP1 as a multi-functional RNA binding protein that regulates cholesterol metabolism and suggest that this effect contributes to the GWAS association of rs10401969 with CAD.
Abstract 171: Deficiency of the Co-stimulatory Molecule Cd27 Impairs Treg Development and Exacerbates Atherosclerosis.
Authors: Winkels Holger , Smeets Esther , Meiler Svenja , Beckers Linda , Dandl Angelika , Reim Sigrid , Spitz Charlotte , Bürger Christina , Weber Christian , Lutgens Esther , Gerdes Norbert
Published: March 2022

Atherosclerosis, an inflammatory disease of large arteries, is - through its clinical manifestations stroke and myocardial infarction - the leading cause of morbidity and mortality in the industrialized world. Adaptive immunity and co-stimulatory signals play a pivotal role during all stages of the disease. Recently, regulatory T cells (Treg) were attributed an anti-inflammatory and anti-atherogenic role. The interaction of CD70, a member of the tumor necrosis factor super family with its receptor CD27 modulates Treg development but also affects 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.

Cd27+/+Apoe-/- or Cd27-/-Apoe-/- bone marrow was transplanted into Apoe-/- recipient mice. After reconstitution, mice were fed a 0.15% high fat/cholesterol diet for 6 weeks. Plaque area at the aortic sinus was increased 2.3-fold in Cd27-/-Apoe-/- chimeras. Plaques contained high amounts of macrophages, which was accompanied by a reduced frequency of aortic (54.1%) and splenic (17.7%) Tregs. Similar results were obtained in 18 wk old Cd27-/-Apoe-/- on a normal chow diet, that also displayed an increase in atherosclerotic plaque area and a 2.5-fold higher plaque macrophage content. In addition, Cd27-/-Apoe-/-mice showed a significant decrease in the abundance of splenic (26%) and aortic (27%) Tregs. Remarkably, 28 week-old Cd27-/-Apoe-/- mice did not exhibit this phenotype, suggesting that CD27 only plays a role in earlier stages of atherosclerosis.

Although Cd27-/-Apoe-/- Tregs did not show changes in suppressive or migratory capacity compared to Cd27+/+Apoe-/-, Cd27-deficiency did result in a marked increase in thymic Treg apoptosis.

Taken together, our data reveal that deficiency for CD27 impairs thymic Treg development, which thereby exacerbates early and intermediate atherosclerosis by increasing plaque inflammation.
Abstract 181: Sub-picomolar Amounts of Coagulation Factor XIa Promote Spatial Clot Growth and Thrombin Generation Inside Propagating Clot
Authors: Parunov Leonid A , Ataullakhanov Fazoil I , Lee Timothy K , Ovanesov Mikhail V
Published: March 2022

Background:
Activated coagulation factor XI (FXIa) was found in the immunoglobulin products that were associated with higher than usual incidence of thrombotic events (TEs). However, TEs after thrombogenic immunoglobulin administration are very rare and are often recorded as late as 48 hours after administration.

Objective:
This study was aimed to elucidate the mechanisms behind FXIa thrombogenicity. We hypothesized that low amounts of FXIa are not able to activate clotting directly, but can promote on-going coagulation events at a site of vascular lesion.

Materials and methods:
FXIa activity was characterized by kinetic clotting and thrombin generation (TG) assays. Spatial clot growth and thrombin wave propagation in stagnant plasma were studied using thrombodynamics, a time-lapse videomicroscopy-based approach. To model coagulation events at the site of vascular wall lesion, spatial clot growth was initiated by plastic surfaces covered with immobilized tissue factor (TF). Human plasma deficient in various coagulation factors was used to study pathways of FXI activation.

Results:
Low plasma FXIa levels of less than 0.3 pM did not induce thrombin generation or clotting, but increased TG in plasma mixed with TF. Higher FXIa levels (>0.3 pM) activated coagulation directly. In stagnant plasma activated by surface with immobilized TF, sub-picomolar amounts of FXIa were also not able to initiate clotting, but was able to enhance TG inside clots. FXIa-induced TG was localized to the propagating edge of the clot and associated with increased clot growth rate.

Conclusion:
Sub-picomolar amounts of FXIa may promote growth of clot in the vicinity of vascular lesion and have no procoagulant effect in the absence of exposed TF. These in vitro findings suggest that the patient’s condition might contribute to the thrombogenicity of FXIa.