bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 43: Recycling Controls Procoagulant and Cell Signaling Functions of Tissue Factor
Authors: Rothmeier Andrea S , Ruf Wolfram
Published: March 2022

Macrophages are central orchestrators in the detrimental cycle of inflammation and coagulation in cardiovascular diseases. Cell danger signals in form of high level of extracellular ATP trigger the macrophage P2X7 receptor leading to secretion of pro-inflammatory IL-1β and the release of pro-thrombotic tissue factor (TF) positive microparticles (MP). P2X7 receptor signaling uncouples the thioredoxin-thioredoxin reductase system and induces inflammasome activation leading to decryption of cell-surface TF and its trafficking onto filopodia for incorporation into MP. These MP have a distinct composition of thiol-regulated proteins and a high surface density of procoagulant PS. Besides its well-established function in IL-1β processing and secretion, we found an entirely unexpected role for caspase 1 in the release of TF from the final severing of highly thrombotic TF-positive MP. Thus, caspase 1 directly couples the release of pro-inflammatory and pro-thrombotic mediators.

P2X7 activation not only causes MP release, but also activates cell surface TF that remains non-coagulant without secondary signals on primed macrophages. We observed that cell detachment rapidly induced full TF procoagulant activity, pointing to an important role of cell adhesion in the maintenance of a non-thrombotic state. Stable cell adhesion is controlled by integrin recycling pathways and the small GTPase arf6 is one of the key regulators of these processes. We found that blocking receptor internalization retains TF on the cell surface and markedly enhanced TF procoagulant activity. Under these conditions, TF was associated with integrin β1 and arf6, and importantly, P2X7 receptor activation promoted arf6-GTP hydrolysis to arf6-GDP, indicating interruption of this recycling pathway following stimulation. In addition, increasing arf6-GDP levels, either by pharmacological inhibitor or overexpression of an arf6-mutant, prevented TF-dependent signaling though protease activated receptor 2 (PAR2) and simultaneously increased cellular procoagulant TF activity in epithelial and in melanoma cells. These findings demonstrate an unexpected role for arf6-controlled recycling in the regulation of TF procoagulant and signaling functions.
Abstract 431: Conversion of Tumor Microvessels into a Hierarchical and Vasoreactive Network, and Suppression of Metastases, by Fibroblast Growth Factor 9
Authors: Arpino John-Michael , Yin Hao , Frontini Matthew J , Nong Zengxuan , O’Neil Caroline , Xu Yiwen , Balint Brittany , Ward Aaron D , Chakrabarti Subrata , Ellis Christopher G , Gros Robert , Pickering J. Geoffrey
Published: March 2022

Normalizing the tumor vasculature has been proposed as a therapeutic paradigm. However, to function normally, microvessels must exist as a vasoreactive and hierarchical network with red blood cells flowing single file through capillaries. Such a network has not been identified in malignant tumors. We previously found that fibroblast growth factor 9 (FGF9) could stabilize new blood vessels in ischemic muscle. To determine if FGF9 impacted tumors vessels, renal carcinoma (Renca) cells, expressing GFP or FGF9, were implanted into the subcapsular space of female Balb/c mice. After 14 days, the resulting FGF9-tumors had 17% fewer microvessels than control tumors (p=0.003) but the vessels had a collagen-fortified basement membrane and were more extensively covered with pericytes (4-fold, p=0.015) and smooth muscle cells (14-fold, p=0.002). Notably, this was associated with reduced pulmonary metastases (p=0.029). Intravital video microscopy revealed that FGF9 converted a haphazard web of channels into a hierarchal network with arterioles, capillaries, and venules. There was also a 33% reduction in vessel length density (p=0.034), a 67% reduction in mean lumen diameter (p<0.001), and 57% fewer bifurcations (p=0.019). Moreover, whereas vasoreactivity was absent in control tumors, arterioles in FGF9-tumors could constrict and dilate in response to adrenergic and nitric oxide releasing agents, respectively. Pimonidazole infusion revealed a 33% reduction of hypoxia in the tumor core (p=0.031) with a 35% reduction in VEGFA expression (p=0.031). Immunostaining and selective cell harvesting revealed that FGF9 selectively amplified a population of PDGFRß-positive stromal cells in the tumor (p=0.045). Furthermore, in vivo blocking of PDGFRß prevented microvascular differentiation by FGF9 and worsened metastases (p=0.002).

Conclusion:
FGF9 can impart an otherwise dysfunctional tumor microvasculature with hierarchy, vasoreactivity, and improved oxygen delivery, via selective amplification of PDGFRß-expressing mesenchymal stromal cells. These findings suggest an approach to driving microvascular network differentiation, to an extent not observed previously, to pacify the tumor.
Abstract 435: Differential mRNA and MicroRNA Expression is Influenced by Race in Hypertensive and Non-hypertensive Women
Authors: Dluzen Douglas F , Noren Hooten Nicole , Zhang Yongqing , Jacob Kimberly D , Zonderman Alan B , Evans Michele K
Published: March 2022

Systemic arterial hypertension occurs more frequently among African Americans (AA) than any other population sub-group, has earlier onset, and more frequently results in end-organ complications. In addition, AA women have the highest incidence and hospitalization rates for hypertension. Previous data suggests that gene expression patterns may increase individual susceptibility to selected chronic diseases. Therefore, we hypothesized that differential gene expression may influence the disproportionate incidence and prevalence of hypertension among AAs. Transcriptional profiling of peripheral blood mononuclear cells (PBMCs) from AA or White, normotensive or hypertensive females identified thousands of mRNAs differentially expressed by race or presence of hypertension. Since microRNAs (miRNAs) are well-known post-transcriptional regulators of mRNA expression levels, and recent data indicates that miRNAs play an important role in the etiology of cardiovascular disease, we wanted to examine whether changes in miRNA abundance underlie changes in gene expression with hypertension. To test this, we profiled global miRNA expression in the same cohort. Analysis of microarray expression changes using Ingenuity Pathway Analysis identified mRNA-miRNA regulatory networks in hypertension-related pathways, i.e. the renin-angiotensin system, nitric oxide signaling, and actin cytoskeletal signaling pathways, which differ by race and hypertension status. mRNA and miRNA gene expression changes were validated using RT-qPCR in an expanded cohort and gene functionality and miRNA target validation was investigated in vitro using primary human umbilical vein cells. We have identified several miRNAs, including miRs 20a-5p, 103a-2-5p, 30c-5p, 4763-5p, 4709-3p and 4717-3p, which are significantly (P<0.05) and differentially expressed by race and/or presence of hypertension. Together, these findings identify several pathways and miRNA gene candidates whose expression differ with race and may contribute to hypertension-related health disparities and could potentially serve as biomarkers for hypertension or as targets for therapeutic modalities.
Abstract 442: YY1 (Yin Yang Factor 1) Is Induced in Injured Vessel Wall Including Aortic Aneurysms and Suppresses Vascular Smooth Muscle Cell Differentiation Through Disrupting Myocardin-mediated Transcriptional Regulation
Authors: Zheng Jianpu , Liu Fang , Yang Maozhou , Dai Xiaohua , Li Hui , Zhou Jiliang , Li Li
Published: March 2022

Vascular smooth muscle cell (VSMC) differentiation is essential for normal vascular function. Its phenotypic plasticity plays a major role in the pathogenesis of vascular diseases. SMC differentiation is marked by the expression of a variety of SMC marker genes controlled by corporative interactions between SMC key transcription factor Myocardin and Serum Response Factor (SRF). Previous studies showed that YY1 (Yin Yang- factor 1, a multifunctional versatile transcription factor) inhibits SMC growth and intimal thickening. However, the role of YY1 in SMC differentiation has been controversial and not characterized. We observed increased YY1 expression in injured vessel wall including aortic aneurysms, suggesting its roles in SMC phenotypic modulation. To determine the roles of YY1 in SMC differentiation, we found that YY1 negatively regulates the expression of SMC marker genes in cultured VSMCs: YY1 overexpression suppresses the transcription of SMC-specific marker genes including Sm22(Tagln) and Acta2 whereas its knockdown increased their transcription. YY1 also suppressed Myocardin transcription and its promoter activation. Mechanistically, we found that YY1 interacts with Myocardin and disrupts its interaction with SRF in the CArG box of SMC gene promoters. This work provides new mechanistic insights of YY1 in SMC phenotypic modulation that leads to pathogenesis of vascular diseases.
Abstract 457: Mirnas 362-3p, 194 and 9b-1 In Aaa and Popliteal Aneurysm Pathogenesis
Authors: Busch Albert , Zernecke Alma , Busch Martin , Maegdefessel Lars , Lorenz Udo
Published: March 2022

Introduction:
Abdominal aortic aneurysm (AAA) remains a major challenge in treatment and cause of death in cardiovascular patients. Despite open surgery or endovascular stent exclusion no therapeutic option remains to clinicians. Similar in severity but much less known are popliteal aneurysms (PA) arising from muscular type arteries often resulting in leg ischemia. So far focus has been on atherosclerosis and inflammation involved in this largely unclear pathogenesis as relevant pathways that could be influenced by i.e. medication in order to prevent growth and possible rupture.

Material and methods:
miRNA global expression profiles and RT-PCR for confirmation experiments have been used on human tissue samples. DIANA based pathway prediction and immunhistochemical examinations were applied.

Results:
miRNAs 362-3p, 194 and 9b-1 revealed by a global expression assays from AAA specimens were found to be differentially regulated between the two aneurysm entities. Computational pathway analysis predicted enrichment in metabolic, ECM and cell-cell-interatcion pathways. Here distinct features were found in immunhistochemical analysis and apoptosis assays.

Conclusion:
Further experiments are needed to confirm the involvement of alternative pathway in AAA and PA pathogenesis. The comparison between elastic and muscular type arteries however may result in exclusive aneurysm mechanisms.
Abstract 475: Catechol-O-Methyl Transferase Methylation and Blood Pressure in Children Treated With Second-generation Antipsychotics
Authors: Cote Anita T , Hookenson Kaia V , Aleliunas Rika E , Sable Carly , Panagiotopoulos Constadina , Devlin Angela M
Published: March 2022

Second-generation antipsychotics (SGA) are increasingly being used to treat children for a wide-range of mental health conditions, and are associated with cardiometabolic side effects such as rapid weight gain and elevated blood pressure (BP). The catechol-O-methyl transferase (
COMT)
Val158Met variant is associated with BP in the general population. We recently reported higher BP in SGA-treated children with the Met allele, but no such association in SGA-naïve children. Methylation of the
COMT
promoter is another important regulatory factor governing COMT activity. The aim of this study was to investigate the interaction of the
COMT
Val158Met variant and
COMT
methylation on BP in children with mental health conditions. A cross-sectional population of SGA-treated (n=106) and SGA[[Unable to Display Character: –]]naïve children (n=122), ≤18 y of age, were assessed for markers of cardiometabolic health. Bisulfite pyrosequencing was used to quantify the methylation status of three CpG sites in the
COMT
promoter. Relationships between SGA treatment,
COMT
Val158Met genotype and
COMT
promoter methylation were assessed using general linear models. BMI was standardized for age and sex (zBMI) and systolic and diastolic BP standardized for age, sex, and height (zSBP, zDBP). When analyzed as one group, there were no significant effects of
COMT
genotype, SGA-treatment, or mental health diagnosis on mean methylation. In SGA-treated children, an interaction between mean methylation and
COMT
genotype was observed for zSBP (
p
=0.015), where zSBP was positively related to methylation in children with the Val/Val genotype, but not in children with the Met allele. After adjustments for ethnicity, SGA-duration, and zBMI the effect was diminished (
p
=0.109), with SGA-duration (
p
=0.018) and zBMI (
p
=0.032) accounting for the variation in zSBP. This association was not found in SGA-naïve children. These findings suggest
COMT
promoter methylation may influence the effect of the
COMT
Val158Met variant on blood pressure in SGA-treated children.
Abstract 487: Saturated FFA Induced NF-κB Activation Is Independent of IKK/IκBα in Aortic Endothelial Cells
Authors: Zhang Fengxue , Luo Xiangwei , Wei Lingling
Published: March 2022

Accumulating evidence has suggested a linkage between free fatty acids (FFAs) and chronic inflammation. It has been shown that saturated FFAs inhibits insulin signal in adipocytes though the activation of IKK. In this study, we examined the effect of FFAs on NF-κB activation in bovine aortic endothelial cells. We found that the saturated FFAs, but not the unsaturated FFAs, caused the activation of NF-κB at a low but sustained level. As a result, ICAM-1 expression was also increased. Interestingly, inconsistent with the previous reports on insulin signal, no changes in IKK phosphorylation and IκBα protein level were observed. IKK16, an IKK inhibitor, failed to block saturated FFA induced NF-κB nuclear translocation. These results imply that saturated FFA induced NF-κB activation is independent of IKK activation and IκBα degradation in aortic endothelial cells, suggesting a novel mechanism for NF-κB activation.
Abstract 494: The Impact of Renal Insufficiency on Carotid Artery Disease Regression in an Academic Medical Center
Authors: Domakonda Kunal , Basu Dev , Halickman Isaac , Iliadis Elias
Published: March 2022

Introduction:
In the United States, 3% of adults are affected by carotid artery disease, and the prevalence is higher in patients with concomitant renal insufficiency. Ischemic events are also more common in patients with renal insufficiency and significantly contribute to morbidity and mortality. Despite the prevalence of carotid artery disease in individuals with renal insufficiency, only a few studies have examined the rate of carotid disease progression in this high risk group, and the mechanisms underlying disease progression are largely unknown.

Hypothesis:
Patients with any stage of renal insufficiency will have an accelerated rate of carotid artery atherosclerotic progression compared to patients with normal renal function.

Methods:
Retrospective chart review was performed on 171 consecutive patients referred to Cooper University Hospital for non invasive evaluation of carotid artery disease. Severity of carotid artery disease was evaluated with carotid doppler, and each patient had at least two carotid doppler studies performed at least 6 months apart. The sample was divided into 2 groups - [Group 1] GFR > 60 and [Group 2] GFR <60. Demographics, comorbidities, medications, serum creatinine, MDRD GFR and ultrasound analysis (proximal peak systolic velocity (PSV), and proximal end diastolic velocity (EDV)) were recorded. The average change in the PSV between studies for the 2 groups were calculated and compared. None of the patients in the sample were on dialysis.

Results:
Preliminary results revealed carotid artery disease regression in both groups; however, patients with any stage of renal insufficiency had less carotid artery disease regression compared to patients with normal renal function.

Conclusions:
Renal insufficiency negatively impacts the rate of carotid artery disease regression in patients with carotid artery disease. These results suggest that patients with renal insufficiency may require more aggressive follow-up and/or earlier invasive interventions of carotid artery disease. Current monitoring and treatment standards should be improved to adequately address the needs of this high risk subgroup.
Abstract 512: RAGE-dependent Down-regulation of Glyoxalase-1 Impairs Inflammatory Response and Angiogenesis in Hind Limb Ischemia in Diabetic Mice
Authors: Yan Shi-Fang , Shen Xiaoping , Rosario Rosa , Schmidt Ann Marie
Published: March 2022

Critical limb ischemia is a serious form of peripheral artery disease. Previous studies linked the advanced glycation end products (AGEs) and its receptor, RAGE, to impairment of angiogenesis in hind limb ischemia in diabetes. Here, we tested the hypothesis that reduction of RAGE ligand, AGEs, by transgenic (Tg) overexpression of glyoxalase-1 (Glo1), an enzyme that detoxifies AGE precursor methylglyoxal, improves inflammatory responses, angiogenesis and blood flow recovery in hind limb ischemia in diabetic mice. Wild type (WT), Ager-/- (devoid of RAGE) and Tg (Glo1) mice were rendered diabetic (D) with streptozotocin (stz); non-diabetic (ND) cohorts were treated with buffer alone. After two month of diabetes, at age 16 weeks, mice were subjected to unilateral hind limb ischemia. In skeletal muscle tissue within the tributary of the femoral arterial system, a significant increase of Glo1 mRNA was evident in both ND and D Ager-/- mice vs. WT mice after ischemia (p<0.05). A significant reduction in carboxymethyl lysine (CML)-AGEs modified protein was observed in Tg (Glo1) mice compared to WT mice at baseline and on day 5 after ischemia in non-diabetes and diabetes (p<0.05). A significant increase of inflammatory mediators, early growth response-1 (Egr1) (p<0.001) and monocyte chemotactic protein-1 (MCP-1/CCL2) (p<0.01) was observed in ND and D Tg (Glo1) vs. WT mice after ischemia. In parallel, significantly augmented inflammatory cell infiltration identified by increasing CD68+ cell numbers in skeletal muscle tissues was observed in ND and D Tg (Glo1) compared to WT on day 7 after ischemia (p<0.05). Tg(Glo1) mice displayed significantly improved capillary density in both the non-diabetic and diabetic states compared to WT mice on day 28 after ischemia (p<0.05 and p<0.01). Increased blood flow was observed in ND and D Tg (Glo1) mice vs. WT controls (p<0.01). Our findings reveal that RAGE-dependent down-regulation of Glo1 is mechanistically linked, at least in part, to the impaired angiogenic responses observed in WT diabetic mice after hind limb ischemia, suggesting that suppression of AGE-RAGE axis and consequent upregulation of the Glo1 pathway may provide an important therapeutic target in the prevention of diabetic vascular damage.
Abstract 52: Capsaicin Activates the Trpv1-camkii-ampk-sirt-1-dependent Mechanism to Trigger Brown Remodeling of White Adipocytes to Antagonize Diet-induced Obesity
Authors: Thyagarajan Baskaran , Ren Jun , Baskaran Padmamalini
Published: March 2022

Obesity is a leading cause of metabolic syndrome. The growing endemic of diet-induced obesity necessitates the development of novel strategies to counter obesity. Recent research suggests a regulatory role of transient receptor potential vanilloid 1 (TRPV1) in diet-induced obesity. Here, we evaluated the hypothesis that capsaicin (CAP), a TRPV1 agonist, inhibited high fat diet (HFD)-induced obesity by inducing browning of white adipose tissue (WAT).

Dietary CAP (0.01% of HFD) significantly prevented obesity in wild type (WT) but not in TRPV1-/- mice without modifying the food/water intake in these mice. CAP increased the expression of the brown fat-specific thermogenes, peroxisome proliferator-activated receptor gamma (PPARγ) coactivator 1 alpha, uncoupling protein-1 and bone morphogenetic protein b8 in the subcutaneous and brown fat pads of WT mice in vivo. Furthermore, CAP increased the expression of sirtuin-1 (SiRT-1 deacetylase) and PR domain protein 16 (PRDM-16-gene responsible for the molecular switch of white to brown fat) in WAT. CAP-triggered deacetylation of PPARγ and PRDM-16 by activating SiRT-1 to facilitate the PPARγ-PRDM-16 interaction and initiate browning of WAT. This effect also corresponded with increased respiratory coefficient, heat production and locomotor activity in CAP-fed WT mice.

Analysis of mechanism of action of CAP revealed that CAP activated TRPV1-dependent signaling cascade of Ca2+/calmodulin-dependent protein kinase II (CaMKII)- 5’ adenosine monophosphate-activated protein kinase (AMPK) and increased the phoshorylation of CaMKII, AMPK and SiRT-1 as well as SiRT-1 activation. Pharmacological inhibition of CaMKII by KN-62 (5 μM; 1 hr. preincubation), inhibition of TRPV1 by capsazepine (10 μM; 1 hr. preincubation) or chelation of intracellular Ca2+ by BAPTA-AM (10 μM; 1 hr preinbubation) prevented the effects of CAP (1μM; 30 min. treatment) on SiRT-1 phosphorylation.

Collectively, our data unambiguously suggest that CAP prevents HFD- induced obesity by stimulating browning of WAT through TRPV1 activation and uncover a novel role of TRPV1-stimulated intracellular Ca2+-dependent CaMKII-AMPK-SiRT-1 signaling cascade in the browning of WAT.
Abstract 521: Impact of Insulin-resistance, Body Mass Index and Dietary Fat Intakes on Apolipoprotein B-48 Kinetic
Authors: Drouin-Chartier Jean-Philippe , Tremblay André J , Hogue Jean-Charles , Lamarche Benoît , Couture Patrick
Published: March 2022

Substantial evidence exists indicating that elevated plasma levels of apolipoprotein (apo) B-48-containing lipoproteins are associated with increased cardiovascular disease risk. Overaccumulation of apoB-48-containing lipoproteins of intestinal origin observed in patients with insulin-resistance (IR) is thought to be attributable to both elevated intestinal production and reduced clearance of these lipoproteins. The objective of this study was to assess the relative contribution of IR, anthropometry, and dietary fat content on triglyceride (TG)-rich lipoprotein (TRL) apoB-48 kinetic. The in vivo kinetic of TRL apoB-48 was measured in 131 men with primed-constant infusion of L-[5,5,5-D3]leucine. TRL apoB-48 production rate (PR) and fractional catabolism rate (FCR) were determined by multi-compartmental modeling, while TRL apoB-48 pool size (PS) was measured by ELISA. IR was defined by TG levels > 1.5 mmol/L and Homeostasis Model Assessment of IR (HOMA-IR) > 2.5. Age was inversely associated with TRL apoB-48 FCR (ρ=-0.21; P=0.02), while BMI was positively correlated with TRL apoB-48 PR (ρ=0.33; P=0.0001) and PS (ρ=0.44; P<0.0001). Fat intake was associated with TRL apoB-48 PR (ρ=0.34; P<0.0001) and PS (ρ=0.34; P<0.0001). HOMA-IR was positively associated with TRL apoB-48 PR (ρ=0.26; P=0.005) and PS (ρ=0.35; P=0.0001). Furthermore, when adjusted for fat intake, TRL apoB-48 PR was significantly higher in IR subjects (n=73) than in insulin-sensitive subjects (Δ=+64%; P<0.0001), and TRL apoB-48 FCR was lower (Δ=-13%; P=0.005). Multiple linear regression analysis showed that IR was the most significant constitutive predictor of TRL apo B-48 PR variance (6.6%; P=0.01), while age and BMI were not significantly associated with PR. In conclusion, these results suggest that IR is a major factor contributing to TRL apoB-48 overaccumulation, possibly through increased production and decreased clearance, independent of age, BMI and fat intake.
Abstract 523: Regulation of Pancreatic β-cell Gene Expression and Function by ABCA1 and ABCG1
Authors: Hou Liming , Tabet Fatiha , Cochran Blake , Westerterp Marit , Tall Alan , Barter Philip , Rye Kerry-Anne
Published: March 2022

Background:
Loss-of-function mutations in the ABCA1 gene cause beta cell dysfunction in humans. Insulin secretion is impaired and glucose metabolism is adversely affected in mice with conditional deletion of ABCA1 in beta cells and in ABCG1 knockout mice. These effects are exacerbated in mice with conditional beta cell ABCA1 deletion plus global ABCG1 deficiency. To identify key beta cell pathways regulated by ABCA1 and ABCG1, we generated mice with beta cell-specific deletion of ABCA1 and ABCG1 (ABCA1
β-cell-/-
/ABCG1
βcell-/-
DKO mice).

Aim:
Identification of genes in key beta cell metabolic and signal transduction pathways that are regulated by ABCA1 and ABCG1.

Methods:
Islets were isolated from 16 week old chow fed ABCA1
β-cell-/-
/ABCG1
β-cell-/-
DKO mice. mRNA was extracted and subjected to Affymetrix GeneChip Mouse Gene ST 2.0 analysis, followed by Partek Genomics Suite and Ingeunity Pathway analysis.

Islet cholesterol levels were determined by HPLC. Plasma and HDL cholesterol levels were measured enzymatically.

Results:
Total plasma cholesterol and HDL cholesterol levels in ABCA1
β-cell-/-
/ABCG1
β-cell-/-
DKO and Abca1fl/flAbcg1f/fl (control) mice were comparable. Islet cholesterol levels in the ABCA1
β-cell-/-
/ABCG1
β-cell-/-
DKO mice were increased relative to control (19.2±1.9 vs 8.7±1.0 μg cholesterol/μg protein, p<0.05). A total of 4216 genes were differentially expressed in the ABCA1
β-cell-/-
/ABCG1
β-cell-/-
DKO mouse islets compared to islets from control mice. Using more stringent criteria (1.2-fold change, p <0.05), 2617 down-regulated and 1599 up-regulated genes were identified in ABCA1
β-cell-/-
/ABCG1
β-cell-/-
DKO islets compared to control islets. Functional enrichment analysis of the differentially expressed genes revealed that ABCA1 and ABCG1 regulate pathways involved in cholesterol metabolism (
Hmgcr, Insing1, Ldlr, Srebf2 and ApoB
), insulin signaling/glucose metabolism (
Akt1, Fasn, Pik3r1, Irs1, Slc2a1 and Slc2a2
) and inflammation (
Gbp11, Sftpd
) in beta cells. Differential expression of these genes was validated by real-time PCR.

Conclusions:
ABCA1 and ABCG1 regulate islet cholesterol levels as well as genes involved in insulin signaling, inflammation and glucose and cholesterol metabolism in beta cells.
Abstract 524: Role of Transmembrane Protein 55B in Insulin Signaling and Hepatic Lipid Metabolism
Authors: Kim Mee J , Naidoo Devesh , Hu David , Woo Jonathan J , Krauss Ronald M , Medina Marisa W
Published: March 2022

Hepatic steatosis is the leading cause of liver disease in the United States and is associated with increased risk of cardiovascular disease. Insulin resistance appears to be a major determinant of hepatic steatosis, as improper hepatic insulin signaling stimulates lipogenesis and dysregulates lipoprotein secretion, resulting in fat accumulation in the liver. Insulin signal transduction is dependent on phosphatidylinositol-4,5-biphosphate (PIP
2
) conversion to phosphatidylinositol-3,4,5-biphosphate (PIP
3
), which results in activation of AKT. Transmembrane protein 55B (
TMEM55B
), a gene we recently identified as a novel regulator of cellular cholesterol metabolism, encodes a phosphatidylinositol phosphatase that converts PIP
2
to phosphatidylinositol-5-phosphate (PI5P). PI5P has also been shown to activate AKT; however, the physiological effects of this pathway are less well understood. In the first
in vivo
model of
Tmem55b
modulation, we found that hepatic
Tmem55b
knockdown in Western diet-fed C57BL/6 mice caused increased body weight, hepatic steatosis, hyperinsulinemia and elevated plasma cholesterol compared to controls. In addition, hepatic expression of insulin-regulated lipogenic genes, including
Srebp1c
and
Dgat2,
was increased, suggesting that
Tmem55b
knockdown promotes lipogenesis. In addition, two genes involved in VLDL secretion,
ApoC3
and
Mtp
, were upregulated. Notably, we observed a significant inverse correlation between
TMEM55B
expression, quantified in 480 human lymphoblastoid cell lines, with plasma triglyceride, VLDL and APOC3 levels in the cell line donors. These relationships were significantly stronger in individuals with elevated plasma glucose (>110 mg/dL), suggesting that the relationship between
TMEM55B
and these measures may be enhanced in the insulin resistant state. Based on these findings, we hypothesize that TMEM55B modulates insulin signaling and affects hepatic lipid accumulation, potentially through stimulation of lipogenesis. With the ever-rising incidence of type 2 diabetes, elucidation of a novel pathway by which insulin resistance leads to hepatic steatosis may inform the development of therapeutics used for the prevention or treatment of this disease.
Abstract 527: Decay of Hepatic and Serum Apolipoprotein(a) Levels is Slower than ApolipoproteinB Following Inhibition of Lipid Synthesis
Authors: Zhang Hong Y , Nosie Amanda K , Ni Wei , Kowala Mark C , Mosior Marian K , Michael Laura F
Published: March 2022

The association of Lipoprotein(a) (Lp(a)) levels and increased cardiovascular risk is substantiated by human epidemiology, genetics and interventional apheresis studies. Lp(a) is a plasma lipoprotein consisting of a Low Density Lipoprotein (LDL) particle with one molecule of apolipoprotein B100 (ApoB100) covalently linked to one molecule of apolipoprotein(a). Published studies show that apo(a) synthesis and secretion from hepatoma cells is coupled to triglyceride synthesis and secretion, illustrating a point of control for Lp(a) production. The objective of this study is to compare Lp(a), apo(a) and apoB production, secretion and half-life
in vivo
to understand mechanisms controlling circulating levels of Lp(a). Since rodent species do not express endogenous apo(a), human apo(a) transgenic mice were created and bred with human apoB100 transgenic mice to generate Lp(a)-producing mice. The circulating level of Lp(a) in the transgenic mice is ~3.7 mg/dL that is comparable to 5.4 mg/dL measured in a healthy human subject. To inhibit triglyceride and VLDL secretion in liver, mice were treated with a microsomal triglyceride transfer protein inhibitor (MTPi), and plasma was collected in a time-course. This treatment allowed measurement of plasma Lp(a), apo(a) and apoB half-lives since secretion of nascent VLDL and LDL was blocked. MTPi caused an 81% reduction in cholesterol and 68% reduction in triglycerides after 5 days of treatment. ApoB decreased significantly within 6 hours of treatment and remained low for 5 days. The calculated half-life was 4.6 hours. By contrast, apo(a) and Lp(a) decreased significantly after 3 days of treatment with half-lives of 2.4 and 2.5 days, respectively, illustrating delayed catabolism of apo(a)/Lp(a). Hepatic Lp(a), apo(a) and apoB were decreased significantly after 5 days dosing, indicating that the apolipoproteins were likely degraded and not accumulated in the liver. Treatment of stable HepG2 cells expressing apo(a) with MTPi caused similar difference in Lp(a) and apoB half-lives. These models illustrate rapid decay of apoB, but not apo(a), when lipoprotein particle assembly is blocked, and they provide methods for future mechanistic studies of apo(a) assembly into Lp(a) and its catabolism.
Abstract 529: High-dimensional Genetic Analysis of Lipoprotein Composition and Size in the Mouse
Authors: Parks Brian W , Bennett Brian , Pan Calvin , Krauss Ronald M , Gargolovich Peter S , Kirchgessner Todd G , Lusis Aldons J
Published: March 2022

Objective:
Given the clinical relevance of variation in lipoproteins to heart disease we performed a systems genetics analysis of lipoprotein diversity in mice. Using a panel of inbred mice termed the Hybrid Mouse Diversity Panel (HMDP) we were able to perform genome-wide association (GWA) to identify more than 50 loci contributing to lipoprotein and apolipoprotein levels and composition.

Approach and Results:
To understand the genetic regulation of lipoproteins we quantitated more than 25 unique parameters of low-density lipoprotein (LDL) and high-density lipoprotein (HDL) in a population of 100 genetically diverse strains of mice, termed the HMDP. Specifically, we measured apoproteins (ApoA1, ApoA2, ApoA4, ApoB100, ApoC2, ApoC3, ApoD, ApoE, ApoM, ApoJ), lipoprotein-associated enzymes (LCAT, LpPLA2, PON1), and sizes of VLDL/LDL ranging from small dense LDL to larger VLDL (180 to 300 angstroms). Performing GWA we were able to identify more than 50 genetic loci. Some loci contain genes with known roles in lipoprotein metabolism while others contain genes with no known role. To identify high confidence candidate genes and validate novel genes influencing lipoprotein metabolism, we performed systems genetics analysis and integrated global gene expression, metabolomic, and proteomic data from the same mice. Through this approach along with computational gene co-expression network analysis we were able to identify two loci with strong candidate genes never before linked to ApoA1 and ApoB metabolism. Validation studies in gene-targeted knockout mice with the high confidence gene associated with ApoA1 levels,Cds2 (CDP-diacylglycerol synthase), confirmed that Cds2 contributes to plasma ApoA1 levels.

Conclusions:
Lipoprotein protein composition and size show large diversity across inbred strains of mice. Using a systems genetics approach we were able to identify and validate a novel gene influencing plasma ApoA1 levels.
Abstract 534: Abca1 Mediates Macrophage Deposition of Cholesterol Into the Extracellular Matrix
Authors: Jin Xueting , Freeman Sebastian R , Vaisman Boris , Liu Ying , Chang Janet , Varsano Neta , Addadi Lia , Remaley Alan , Kruth Howard S
Published: March 2022

ABCG1 and ABCA1 both function in reverse cholesterol transport. We previously reported that cholesterol-enriched macrophages deposit cholesterol into the extracellular space and this process depends in part on ABCG1. The extracellular cholesterol deposits can be mobilized by HDL. The objective of the current study was to determine whether ABCA1 also contributes to macrophage deposition of extracellular cholesterol. A monoclonal antibody that detects cholesterol microdomains, and not isolated cholesterol molecules, labels these extracellular deposits. First, to confirm the specificity of the cholesterol labeling, we treated cholesterol-enriched macrophages cultures with cholesterol oxidase and found that antibody labeling of deposits was eliminated. Many biochemical studies assume that cholesterol oxidase susceptible cholesterol indicates this cholesterol is within the plasma membrane. However, as we show here, the extracellular pool of cholesterol also contributes to cholesterol oxidase susceptible cholesterol. Second, concerning ABCA1 function in cholesterol deposition, we observed that the LXR agonist, TO901317, an ABCA1-inducing agent, restored cholesterol deposition that was absent in ABCG1-/- mouse macrophages. In addition, probucol, an agent that inhibits ABCA1 function, partially inhibited cholesterol deposited by wild-type mouse macrophages, and completely inhibited deposition from TO901317-treated ABCG1-/- mouse macrophages. These results suggest that ABCA1 contributes to macrophage cholesterol deposition. We confirmed this conclusion by testing cholesterol deposition in ABCA1-/- mouse macrophages. Extracellular cholesterol deposition by these macrophages was substantially reduced compared with wild-type mouse macrophages. Thus, our findings demonstrate a novel function of ABCA1 in contributing to macrophage export of cholesterol into the extracellular space where this cholesterol can be stored until mobilized by HDL.
Abstract 537: Characterization of the Lipase Stimulating Domain for Apolipoprotein A-V and the Development of a Therapeutic Peptide for the Treatment of Hypertriglyceridemia
Authors: Janizek Joseph D , Munro Kim , Davidson Michael H , Ancsin John B
Published: March 2022

The impaired clearance of plasma triglycerides is a major cause of clinical hypertriglyceridemia, a condition which affects about one third of adults in the G7 countries and is a risk factor for a number of diseases, including cardiovascular disease, cancer and pancreatitis. The hydrolysis and removal of triglycerides from the plasma is dependent on cell surface lipoprotein lipase (LpL) and its enzymatic activity is in turn regulated by specific apolipoproteins; apoC-III, which acts as a potent inhibitor and apoC-II and apoA-V, which act as stimulators. Their importance in hypertriglyceridemia is underscored by GWAS analyses, which have identified the APOA1/C3/A4/A5 gene cluster (chromosome 11q23) as an important determinant of dylipidemia. ApoA-V is of particular interest since its physiological concentration (3.8 nM) is 10,000x lower than that of apoC-II and the other major apolipoproteins. To investigate the influence of different lipoproteins on lipase kinetics we have developed a fluorescence-based assay that uses a fluorogenic triglyceride analog (EnzChek®) in a microplate format. Here we report that a short alpha-helical amphipathic peptide (AV199-232) corresponding to apoA-V residues 199-232 exhibit; i) lipid-binding, ii) heparin-binding and iii) lipase-stimulating activities. Structural characterization and residue substitution studies have been employed to map these three activities to three separate discrete domains within AV199-232. Enzyme kinetic analysis with pure LpL indicates that AV199-232 can overcome the inhibition of LpL by apoC-III and stimulate enzyme activity by reducing its Km 8-fold. Similar increases in lipase activity are also observed with live THP-1 monocytes and HepG2 cells that express hepatic lipase. The influence of AV199-232 in cell culture is saturable, dependent on the presence of cell surface lipase, and enzymatic activation can be sustained for at least 2 hrs. We postulate that apoA-V and AV199-232 stimulate lipase activity through an allosteric mechanism by binding to lipases with high affinity near their catalytic site to affect enzyme kinetics.
Abstract 546: SR-BI Functions on Plasma HDL Homeostasis -low Plasma preβHDL Level in SR-BI Null Mice by 2D PAGE Analysis
Authors: Tsujita Maki , Yokota Tomo , Akita Nobukatsu , Maekawa Yasutaka , Yamamoto Junki , Remaley Alan T , Yokoyama Shinji
Published: March 2022

ApoA-I-ABCA1 mediated HDL synthesis is major source of plasma preβHDL generation. Lipid free apoA-I is mainly provided from newly synthesized from liver, small intestinal cells and the other hand, apoA-I is also provided from deflated HDL result of transferring cholesterylester into target cells via SR-BI. To evaluate the contribution of SR-BI on reverse cholesterol transport at plasma preβHDL synthesis, first, plasma from SR-BI null mice and wild type mice were analyzed by 2D-gel and compared. The anti-mouse apoA-I peptide (134-140AA, N-Asp-Val-Glu-Leu-Tyr-Arg-Gln-C) polyclonal antibody were further purified by mouse apoA-I-Sepharose then used for detection of HDL subclasses developed by 2D-gel, combination of agarose gel and native gradient PAGE. Ten μL of tail blood from each mouse were collected into microtubes containing NEM, EDTA and ascorbic acid. Plasma was kept in ice then the first electrophoresis by PALAGON LIPO gel was performed within 3 hours of harvest of blood. PreβHDL in SR-BI null mice was significantly reduced (13.78±3.58 μg protein/mL plasma, P=0.00153). The other hand, preβHDL of ABCG1 null mice (37.46±13.803, P=0.423) was the same as in wild type mice plasma (38.48±13.58). To detect the origin of apoA-I on this preβHDL, 125I labeled apoA-I HDL injection of apoA-I on plasma preβHDL, 2D gel analyses were performed followed by 125I-apoA-I-HDL injection. 125I-preβHDL spot was detected early as 2 hours to 6 hours in wild type mice plasma, indicating apoA-I recycling to the new preβHDL particle. The other hand in SR-BI null mice plasma, the 125I-preβHDL spot was only detected between 6 hours to 24 hours. The loss of SR-BI protein delayed reproduction of preβHDL in plasma in mice. This result indicates SR-BI functions as provider of preβHDL possibly both free apoA-I for ABCA1 mediated preβHDL synthesis and also directly supply preβHDL particles in mice plasma.
Abstract 55: Chronic Loss of Serotonin Transporter Function Alters Platelet Adhesion and Spreading
Authors: Oliver Kendra H , Dohn Michael R , Duvernay Matthew T , Hamm Heidi , Carnerio Ana
Published: March 2022

Clinical observations indicate that patients taking selective serotonin reuptake inhibitors(SSRIs) have an increased risk of bleeding. However it remains unclear how SSRIs, which inhibit the serotonin transporter(SERT), modulate homeostasis and platelet activation. There are two primary mechanisms by which serotonin(5HT) affects platelet function: activation of the 5HT2A receptor and uptake by SERT. It has also been demonstrated that SERT regulates aIIbB3(GPIIbIIIa) function, the common signaling pathway in platelet activation and crucial for platelet aggregation. We sought to elucidate how the serotonergic system modulates aIIbB3 function by examining platelet spreading and attachment to immobilized fibrinogen. To model altered serotonergic tone, we used mice with a genetic deletion of SERT(SERT KO). As observed with SSRI use, the blood 5HT concentration in SERT KO mice is miniscule. We found that SERT KO mice display increased tail bleed time and thrombin time. Interestingly, SERT KO platelets showed reduced spreading but increased attachment on immobilized fibrinogen. 5HT(100nM) was able to rescue the reduced spreading on immobilized fibrinogen. To determine the mechanism, we examined platelet spreading with kentanserin(5HT2A antagonist) or citalopram(SSRI). We found that acute administration of citalopram did not alter spreading at physiologically relevant levels. Blockade of 5HT2A activation however, caused WT platelets to behave similarly to SERT KO with reduced spreading. Src is a tyrosine kinase that plays a major role in platelet spreading. Using in-cell western, P-Src(416) was decreased in SERT KO mice following spreading on immobilized. After addition of 5HT, P-Src(416) in SERT KO platelets was normalized to WT P-Src levels. Furthermore, treatment with ketanserin caused WT P-Src(416) to decreased to SERT KO P-Src(416) levels. These finding suggest that the peripheral serotonergic system is “recalibrated” in the absence of 5HT and leads to altered function of aIIbB3 via 5HT2A. From this data, we have elucidated a novel regulatory mechanism of 5HT for platelet fibrinogen binding that could regulate the recruitment of platelets by fibrinogen binding to a growing thrombus.
Abstract 554: Vascular Lesion Formation is Attenuated by Genetic Deletion of Hyaluronan Synthase 3
Authors: Dick Lena S , Homann Susanne , Mueller Julia , Grandoch Maria , Fischer Jens W
Published: March 2022

An extensive accumulation of the matrix component hyaluronan (HA) has been observed in vascular lesions. HA is thought to confer immunomodulatory actions and to regulate cell proliferation and migration in diseased vessels. HA is extruded into the extracellular space by three transmembrane HA-synthase isoenzymes (HAS1, -2, -3).

Objective of the current study was to assess specifically the role of HAS3 in vascular pathologies such as restenosis and atherosclerosis.

In HAS3 deficient mice (HAS3-/-) and wildtype controls (WT) neointimal hyperplasia was induced using a carotid artery ligation model. Morphometric analysis of carotid arteries harvested 28 days after ligation revealed that lack of HAS3 mitigates neointimal lesion formation as indicated by a decrease in intimal area (AUC: HAS3-/-, 1.53 x107 ± 0.23 x107 μm3 vs. WT, 2.59 x 107 ± 0.34 107 μm3, n = 9, p < 0.05) and reduced intima/media ratio (HAS3-/-, 0.94 ± 0.13 vs. WT, 1.39 ± 0.13, n = 9, p < 0.05). Supportive in vitro studies suggest that blunted neointimal hyperplasia is mainly due to a reduced migration and to a lesser extent reduced proliferation of vascular smooth muscle cell (VSMC).

Next atherosclerosis-prone apolipoprotein E/HAS3 double-deficient (apoE-/-/HAS3-/-) mice were generated and fed with a cholesterol-rich Western type diet for 15 weeks. Analysis of aortic en face preparations stained with Oil Red O revealed reduced plaque burden compared to apoE-/- controls (apoE-/-/HAS3-/-, 6.95 ± 2.66, n = 10 vs. apoE-/-, 11.43 ± 4.06, n = 13, p < 0.05). Plasma lipids (total cholesterol, LDL/VLDL, HDL cholesterol) did not differ between the groups. Plaque composition at the aortic root showed decreased collagen content (Picro Sirius Red staining) but no differences in cell density, VSMC content (alpha Actin staining) and macrophage count (Mac2 staining). In contrast, flow cytometry data point to a reduction in circulating T cells (apoE-/-/HAS3-/-, 28.4 ± 1.2 %, n = 5 vs. control, 41.7 ± 5.1 %, n = 6).

Collectively, these findings suggest a role for HAS3 in vascular lesion formation. In neointimal hyperplasia HAS3-dependent HA synthesis may support mainly VSMC migration whereas in atherogenesis additional immunomodulatory actions are proposed.
Abstract 555: The Atypical Cadherin Fat1 Interacts With the Intermediate Filament Vimentin in Vascular Smooth Muscle Cells and Limits Atherosclerosis
Authors: Dunaway Charlene M , Chinnasamy Prameladevi , Cao Longyue (Lily) , Riascos Bernal Dario F , Sibinga Nicholas E
Published: March 2022

Fat1 controls vascular smooth muscle cell (VSMC) growth and migration. In recent studies, we found that Fat1 expression colocalizes with VSMCs in atherosclerotic lesions from
apoE
-/-
mice, and also identified the intermediate filament protein vimentin as a novel Fat1 interactor. Like Fat1, vimentin is expressed in VSMCs in atherosclerotic lesions and affects cell growth. We hypothesized that Fat1 regulation of VSMC activities limits atherosclerosis, and that the Fat1-vimentin interaction contributes to this regulation. To test this idea, we generated

fat1
VSMC-KO
; apoE

double knockout (DKO) mice and assessed atherosclerosis after 24 weeks on a high fat diet, analyzing aortic roots and brachiocephalic arteries (BCA) for lesion area and immunohistochemical features. DKO mice (N=10) exhibited larger lesions (p<0.05) at the aortic root (80837.0±7942.4μm
2
) and BCA (36017.2±10496.4) compared to control (64190.2±6539.1 and 19462.9±5246.3, respectively). These lesions displayed significantly thicker fibrous caps (+54%, N=7, p<0.05) and larger necrotic cores (+40%, N=8) compared to control, with a trend towards increased cellularity in the DKO group (+24%, p=0.0573, N=7). To evaluate lesional macrophage and VSMC content, we stained for Mac-2 and SM22α, respectively. We found no difference in macrophage number, but greater SM22α-positive lesion area in DKO lesions (1.15±0.25μm
2
, vs. control, 0.37±0.08, p<0.01, N=9). Proliferation in plaques, assessed by phospho-histone H3 staining, was also higher (DKO, 52.6±13.3 cells/field vs. control, 31.4±10.1, p<0.05, N=7). Fat1 and vimentin expression colocalized within fibrous caps, and their physical interaction was validated by co-immunoprecipitation. In addition, Fat1-deficient VSMCs and lesions exhibited increased vimentin levels (>3-fold, p<0.05 by immunoblotting). Thus Fat1 interacts with vimentin and controls its abundance in VSMCs; preliminary knockdown studies suggest that the increased proliferation seen in Fat1-deficient VSMCs depends on vimentin. In summary, our
in vivo
data support a role for Fat1 in the regulation of VSMCs during atherogenesis, and suggest that the Fat1-vimentin interaction is important for this regulation.