bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 379: Smooth Muscle-specific Lncrna
Carmn
Plays A Potential Role In Aortic Aneurysm
Authors: He Xiangqin , Dong Kunzhe , Hu Guoqing , Zhao Juanjuan , Miano Joseph M , Zhou Jiliang
Published: November 2022

Background:
Aortic aneurysm is a life-threatening vascular disease characterized by vascular smooth muscle cell (VSMC) depletion, ECM degradation, and infiltration of immune cells. Previous studies have shown that
Carmn
, a SMC-enriched long non-coding RNA, is critical for maintaining VSMC contractile phenotype. However, its functional role in aortic aneurysm remains completely unknown.

Methods and Results:
We re-analyzed the publicly available aortic aneurysm bulk RNA-seq and scRNA-seq datasets in mouse and human. These unbiased analyses revealed that
Carmn
is downregulated in the SMCs of aortic aneurysm samples. To examine the potential role of
Carmn
in aortic aneurysm, we generated
Carmn
global knockout (gKO) mice or inducible SMC-specific KO (iKO) mice driven by
Myh11
-Cre ER T2, respectively. Unexpectedly, both
Carmn
gKO and iKO mice exhibited premature lethality, due to a severe colonic pseudo-obstruction resulting from deletion of
Carmn
in visceral SMCs. Despite the dominant gastrointestinal phenotype observed in
Carmn
KO mice, morphological analysis of the
Carmn
KO thoracic aorta revealed thinner vascular wall compared to that of controls. Results from TEM showed that the VSMC in the
Carmn
KO thoracic aorta are disorganized and degradative with aneurysmal dissection. Data from bulk RNA-seq of
Carmn
-deficient aorta revealed that the down-regulated genes are associated with degradation of ECM while the up-regulated genes are associated with cell death. Moreover, scRNA-seq of
Carmn
-deficient aorta revealed that the percentage of the SMCs is decreased while the percentage of infiltrated immune cells is increased. These data indicated that
Carmn
deficiency may play a critical role in promoting the development of aortic aneurysm. To circumvent the lethal visceral SMC phenotype, we are in the progress of crossing
Carmn
flox mice with the novel vascular SMC-specific inducible
Itga8
-Cre ER T2 mouse to generate VSMC-specific
Carmn
KO mice.

Conclusions:
Our data suggest that
Carmn
is indispensable for maintaining gastrointestinal contractile function, and implicate that
Carmn
deficiency plays a potential role in contributing the development of aortic aneurysm.
Abstract 386: Lipocalin 10 Protects Against Sepsis-triggered Vascular Leakage Through Activation Of The Ssh1-cofilin Signaling Pathway
Authors: Zhao Hongyan , Wang Peng , Wang Xiaohong , Li Yutian , Li Qianqian , Chen Jing , Ma Chengen , Wang Chunting , Fan Guo-Chang
Published: November 2022

Background:
The increased vascular leakage seen in sepsis patients is well-recognized to be responsible for sepsis-triggered organ failure and patient mortality. Prior work suggests that lipocalin 10 (Lcn10), a member of the lipocalin superfamily, is significantly downregulated in the blood of non-survival septic patients when comparing to the survival group. Nonetheless, whether Lcn10 affects endothelial barrier integrity in sepsis remains unknown.

Methods and Results:
First, we observed that the expression of Lcn10 was remarkably down-regulated in the hearts of both endotoxin LPS- and cecal ligation/puncture (CLP)-induced septic mice, compared to their controls. Interestingly, further analysis of Lcn10 expression in different cell types isolated from LPS- and CLP-hearts showed that reduction of Lcn10 occurred only in cardiac endothelial cells (ECs) but not in cardiomyocytes or fibroblasts. Using a global Lcn10-knockout (KO) mouse model, we found that loss of Lcn10 greatly increased vascular permeability, which correlated with more severe cardiac depression and higher mortality following LPS challenge or CLP surgery, compared to LPS- or CLP-treated wild-type (WT) mice. By contrast,
in vitro
overexpression of Lcn10 in ECs provided greater resistance to LPS-caused monolayer leak, compared to control cells. A mechanistic analysis by RNA-sequencing and RT-qPCR revealed that both endogenous and exogenous elevation of Lcn10 in ECs could greatly upregulate slingshot homolog 1 (Ssh1) expression. Ssh1 is a phosphatase known to activate Cofilin, a key actin-binding protein that plays an essential role in controlling actin filament dynamics. Accordingly, phosphorylated Cofilin levels were significantly reduced and thereby, reorganized F-actin to cortical actin for stabilizing tight junction molecules in Lcn10-treated ECs, compared to control cells. Finally, knockdown of Ssh1 in ECs by siRNA greatly offsets Lcn10-induced reduction of monolayer leakage upon LPS insult.

Conclusions:
Endothelial Lcn10 is critical for protecting against sepsis-induced cardiovascular leakage, and its underlying mechanism involves the activation of Ssh1-Cofilin pathway. Our study suggests that Lcn10 could be a novel regulator of vascular barrier integrity.
Abstract 387: Age Associated B Cells Are Abundant In Perivascular Adipose Tissue In The Aortic Arch Region And Positively Associate With Atherosclerosis In Mice
Authors: Srikakulapu Prasad , Upadhye Aditi , Sai Vineela Bontha , Marshall Melissa , McNamara Coleen A
Published: November 2022

Background:
Atherosclerosis development in the aorta is region specific with the aortic arch region more prone to disease development than descending thoracic and abdominal aorta. Perivascular adipose tissue (PVAT) directly associates with the aorta, harbors high numbers of B cells, and regulates atherosclerosis development. Age-Associated B cells (ABCs: CD19
+
CD11c
+
) have recently been reported as a new subset of B cells with unique cell surface and transcriptional signatures. ABCs have been reported to be elevated in autoimmune diseases, where in, these cells have characteristics of auto-antibody producing memory B cells. However, the impact of ABCs in the PVAT near aortic arch and with atherosclerosis progression during aging has not be studied.

Methods and Results:
To study ABCs in atherosclerosis setting, we used 50- and 100- week old chow diet fed,

ApoE
KO

mice. Flow cytometric analysis showed that there was no difference in frequency of total B cells in the PVAT near the aortic arch region between 50- and 100- week old mice. However, the frequency of ABCs was significantly higher in aortic arch PVAT in 100-week-old mice compared to 50-week-old mice. Additionally, these ABCs in aortic arch PVAT are CD44
+
(activation marker) and most ABCs were actively proliferating cells (Ki67
+
), as compared to other CD11c
-
B cells in 100-week-old

ApoE
KO

mice. Lesion area was measured following Sudan-IV enface staining of aorta and 100-week-old mice developed significantly more disease than the 50-week-old mice. Interestingly, we observed a significant correlation of atherosclerosis disease in aortic arch region with the frequency of ABCs in PVAT near aortic arch (p=0.001, R
2
=0.69).

Conclusion:
Results provide the first evidence that ABCs in the aortic arch PVAT are highly active and the percentage of these ABCs strongly associates with advanced atherosclerosis disease in aged animals.
Abstract 395: Assessing Current Therapeutic Anticoagulation Strategies In Severely Obese Patients With Covid-19
Authors: You Joseph
Published: November 2022

Objective:
To examine the utility of D-dimer levels in risk stratification and anticoagulation therapy in non-critically ill COVID-19 patients with severe obesity

Background:
Many studies to date have documented significant inflammatory vascular sequelae in association with COVID-19. Current guidelines suggest an initial strategy of therapeutic-dose anticoagulation to non-critically ill, hospitalized patients requiring low-flow oxygen and a concurrent D-dimer level above the upper limit of normal. However, the utility of D-dimer values in predicting thrombosis in severely obese patients are equivocal to poor, with prior evidence suggesting falsely elevated levels with greater BMI.

Methods:
In this single-center, retrospective study, 32 severely obese patients (defined as BMI > 40) hospitalized with COVID-19 and requiring low flow oxygen without ICU level of care were analyzed. Clinical outcomes were compared between groups receiving therapeutic versus prophylactic doses of anticoagulation. All were treated with low molecular weight heparin (LMWH) per hospital protocol. The following data points were examined: length of hospitalization, mortality, anticoagulation therapy, initial d-dimer levels, thrombotic events, minor/major bleeds, and oxygen modality.

Results:
In total, 78% of patients initially presented with a D-dimer level above the upper limit of normal, with 53% of patients meeting criteria for therapeutic anticoagulation. However, there were no significant differences in incidence of thrombotic events, mean length of hospitalization or overall mortality. Furthermore, despite utilization of appropriate therapeutic anticoagulation, it did not reduce the overall use of oxygen support requirements, including high flow oxygen or non-invasive ventilation, when compared to individuals receiving prophylactic dosing.

Conclusion:
The utility of D-dimer levels in guiding anticoagulation therapy in severely obese patients with COVID-19 may be misleading.
Abstract 397: Smooth Muscle Cell-specific Lncrna
Carmn
Is Regulated By Srf/myocd Complex
Authors: Dong Kunzhe , He Xiangqin , Hu Guoqing , Miano Joseph M , Zhou Jiliang
Published: November 2022

Background:
Cell identify is established by strict control of cell type-specific gene expression programs through regulatory enhancers binding of transcriptional factors. Vascular smooth muscle cells (VSMCs) are critical in maintaining the vascular homeostasis by expressing an array of contractile genes that are primarily governed by SRF/MYOCD complex via binding to the DNA motif called CArG element. However, most of these previous studies regarding regulatory mechanism underlying SM-specific gene expression were performed in vitro, outside of their in situ genomic contexts.

Methods and Results:
To define the regulatory atlas of VSMC, we analyzed epigenetic datasets of multiple human tissues and cell types. We found
CARMN
, that we previously reported as a SMC-specific lncRNA and a critical regulator of SMC contractile phenotype by binding to MYOCD, is enriched with SMCs-specific enhancers. Co-expression analysis using RNA-seq datasets of different human tissues identified
CARMN
expression is positively correlated with both
MYOCD
and
SRF
. We further demonstrated overexpressing MYOCD activates, while silencing MYOCD or SRF inhibits
CARMN
expression in human coronary VSMCs. Subsequent analysis of SRF ChIP-seq data revealed 4 SRF binding peaks with
CARMN
locus, including 2 located within promoter (namely CArG 1 and 2) and the other 2 (namely CArG 3 and 4) located within distal enhancers. Further luciferase reporter assays revealed mutation of CArG 1 or 3 dramatically abolish MYOCD-induced reporter activity. To confirm these findings in vivo, we generated single CArG element mutant mice for each of the 4 CArG elements and CArG 3/4 double mutant mice by CRISPR-Cas9 genome editing approach. Analysis of
Carmn
expression in SMC-enriched tissues from these mutant mice revealed a ~50% decrease of
Carmn
expression in tissues of CArG 1 mutant mice while mutation of other CArG elements appears no effect on
Carmn
expression.

Conclusions:
Our results suggest the SM-specific expression of
Carmn
is dependent, at least partially, on SRF/MYOCD complex and CArG 1 is critical for this regulation. Together with our previous findings that
CARMN
potentiates MYOCD function, we propose
CARMN
and SRF/MYOCD complex form a feedforward loop promoting SMC contractile phenotype.
Abstract 398: Retinoic Acid Exposure Promotes Phenotypic Characteristics Of Alternative Activation In Macrophages
Authors: Pinos Ivan , Amengual Jaume
Published: November 2022

Introduction:
Macrophages are key players in atherosclerosis. Classically-activated (M1) macrophages show a pro-inflammatory phenotype, whereas alternatively-activated (M2) are anti-inflammatory. The M1/M2 balance in atherosclerosis determines the fate of the lesions and strategies to increase M2-like macrophages entail promising therapeutic candidates. Retinoic acid (RA), the transcriptionally active form of vitamin A, delays atherosclerosis progression in mice and has been investigated as a modulator of macrophage polarization.

Hypothesis:
RA modulates macrophage polarization by inducing M2-like responses.

Methods:
Bone marrow-derived macrophages from three wild-type C57BL/6J mice were differentiated (M0) and stimulated with LPS/IFNγ (M1) or IL-4 (M2) for 24h, followed by 6h exposure to 1 μM RA or vehicle (DMSO). RNAseq and gene enrichment analysis were performed. Efferocytosis assays were performed by co-culture of apoptotic Jurkat cells with treated macrophages and subsequent analysis by flow cytometry. Autophagy flux and lysosomal protein breakdown rate were also analyzed.

Results:
RNAseq showed that RA induced changes in 1,485 genes in M2 macrophages, while 821 and 227 genes were significantly regulated in M0 and M1, respectively. Given the lower impact of RA on M1, we focused our subsequent analyses on M0 and M2. Arginase 1
,
a well-known marker of M2 activation, was significantly upregulated by RA at both the mRNA and the protein levels in M0 and M2. Scavenger receptors pathway was regulated in M2 exposed to RA, and efferocytosis assays showed that this group had significantly the highest efferocytotic capacity. Autophagy and Lysosome pathways were also significantly over-represented in M2 treated with RA, although autophagy flux and protein breakdown rate were not differentially affected by RA in M2 or M0 macrophages.

Conclusions:
Transcriptomic analyses showed a higher response of M2 macrophages to RA, compared to M0 and M1. Functional assays revealed that RA increased efferocytosis in M2 macrophages, a hallmark of alternative activation. Our results suggest that RA skews macrophages' polarization and it might promote an M2-like phenotype, especially in macrophages exposed to IL4, enhancing alternative activation.
Abstract 400: Cardiometabolic Profile Of Haptoglobin Genotype In A Multi-Ethnic Diabetes Cohort
Authors: Duvalyan Angela , Gangwar Anamika , Joshi Parag H , Khera Amit , Navar Ann M , Wu Elaine , Rohatgi Anand
Published: November 2022

Background:
Haptoglobin has three common genotypes: Hp 1-1, Hp 2-1, and Hp 2-2. The Hp 2-2 genotype, known to have poorer antioxidizing capacity, is associated with worse cardiovascular outcomes in select patients with diabetes. In this study, we explore the association between various cardiometabolic biomarker profiles and the three Hp genotypes in a multiethnic population.

Methods:
A cross-sectional analysis was performed for all participants with diabetes in the Dallas Heart Study and Multi-Ethnic Study of Atherosclerosis cohorts. Hp genotype was measured in 1,158 participants (399 from DHS and 759 from MESA) using ELISA technique (Savyon Diagnostics, Ltd). Associations between Hp genotype and 26 cardiometabolic circulating and imaging biomarkers were assessed by ANOVA and stratified by ethnicity.

Results:
The study included 51% women, 44% Black (513 of 1,158: Hp 1-1=45%, Hp 2-1=35%, Hp 2-2=20%), 26% Hispanic (300 of 1,158: Hp 1-1=25%, Hp 2-1=49%, Hp 2-2=26%), 20% White (237 of 1,158: Hp 1-1=16%, Hp 2-1=56%, Hp 2-2=28%), and 9.2% Asian/Pacific Islander/East Indian (107 of 1,158: Hp 1-1=15%, Hp 2-1=36%, Hp 2-2=49%) participants. Significant differences were seen in lipid metabolism as well as atherosclerosis, inflammatory, structural, and endothelial markers with no significant differences seen in imaging biomarkers. In White participants, Hp 1-1 was associated with elevated total cholesterol (p=0.016), LDL-p (p=0.001), and LDL-C (p=0.004). In Asian/Pacific Islander/East Indian participants, Hp 2-2 was associated with elevated systolic blood pressure (p=0.029), BMI (p=0.029), and inflammatory markers such as IL-6 (p=0.018) and D-dimer (p=0.027). In Black participants, Hp 2-2 was associated with elevated ICAM (p=0.048) and BMI (p=0.007). Lastly, in Hispanic participants, no significant associations were seen. In exploratory analysis, when comparing Hp 1-1 and Hp 2-1 with Hp 2-2, there was greater magnitude effect size of certain biomarkers in Black and Asian participants, suggesting potential differential effects by ethnicity.

Conclusion:
Associations between Hp genotype and various cardiometabolic biomarkers are varied and ethnicity dependent with further studies needed to investigate the underlying physiologic mechanisms.
Abstract 403: A Common Polymorphism Connects Propionyl-coa Carboxylase To Coronary Artery Disease
Authors: Soubeyrand Sebastien , Lau Paulina , Nikpay Majid , McPherson Ruth
Published: November 2022

Genome-Wide Association studies (GWAS) have identified more than 200 common genetic variants that cumulatively account for approximately 50% of individual risk for coronary artery disease (CAD). This information is relevant to CAD risk assessment but may also provide new insights into the underlying biology of atherosclerosis. The vast majority of these common single nucleotide polymorphisms (SNPs) are in non-coding regions of the genome and distal from protein coding genes; thus mechanistically linking GWAS identified SNPs to CAD is challenging. Here, we have investigated the relationship of rs667920, identified as a top CAD associated SNP in a recent GWAS, to CAD. Conditional analysis, using lipid traits and other phenotypes as covariates, is consistent with the hypothesis that hypercholesterolemia mediates the impact of rs667920 on CAD risk. Although rs667920 is intronic to STAG1, originally denoted as a possible risk gene, it is not associated with altered expression of STAG1 in CAD relevant tissues according to GTEx. Chromatin immunoprecipitation data indicate the presence of enhancer activity overlapping rs667920 in fibroblast and muscle cells, consistent with cis regulation Indeed, this SNP is a significant expression quantitative locus (eQTL) for a neighboring gene, PCCB, encoding the Propionyl-CoA carboxylase beta subunit, in vascular tissues and whole blood, with the common allele rs667920_T being linked to increased expression of PCCB, circulating lipids and CAD, suggesting that lower expression of PCCB may be beneficial. Although its eQTL relationship with rs667920 was considerably weaker, mendelian randomization analysis of whole blood datasets prioritized NCK1, another local gene. Additional experiments are currently underway in our laboratory to validate and complement these preliminary findings.
Abstract 421: Hematopoietic Deficiency In NLRP3 And AIM2 Inflammasomes Or Gasdermin D Fails To Prevent Atherosclerosis Progression In Mouse Models Of Diabetes
Authors: Hsu Cheng-Chieh , Fidler Trevor , Kanter Jenny E , Kramer Farah , Tang Jingjing , Tall Alan R , Bornfeldt Karin E
Published: November 2022

Diabetes accelerates atherosclerosis progression in part by increasing lesional necrotic core expansion, but the underlying mechanisms remain elusive. Previous studies have shown that systemic inhibition of the NOD-, LRR- and pyrin domain-containing protein 3 (NLRP3) inflammasome prevents atherosclerosis progression in a mouse model of diabetes. Gasdermin D (GSDMD), which acts downstream of inflammasome activation, contributes to pyroptosis and IL-1β and IL-18 release. To explore if hematopoietic inflammasome activation mediates diabetes-related necrotic core expansion, we first generated hematopoietic GSDMD-deficient chimeras in an LDL receptor-deficient (

Ldlr
-/-

) virally-induced mouse model of type 1 diabetes (T1D)-accelerated atherosclerosis. Mice with T1D demonstrated elevated plasma glucose, triglycerides, cholesterol, and plasma IL-18 (non-diabetes [ND]=180.4 pg/ml, diabetes [D]=316.6, N=17-18 per group). However, diabetic mice with hematopoietic GSDMD-deficiency had no reduction in plasma IL-18 (D-KO=437.5 pg/ml, p=0.29, N=17). Moreover, we found little evidence of inflammasome activation in peritoneal and splenic macrophages from diabetic mice. A 4-week duration of diabetes significantly increased necrotic core area measured at three different levels in the aortic sinus (p<0.01, N=21-24 per group) in pre-existing lesions without increasing lesion area. Hematopoietic GSDMD-deficiency did not affect the necrotic core area. To further interrogate the relationship between hematopoietic inflammasome activation and diabetes-related necrotic core expansion, we generated

Ldlr
-/-

hematopoietic NLRP3 and absent in melanoma 2 (AIM2) inflammasome double knockout (DKO) chimeras in a streptozotocin mouse model of diabetes. Mice with diabetes demonstrated hyperglycemia and hypercholesterolemia. However, diabetic hematopoietic DKO chimeras did not exhibit reduced aortic sinus necrotic core area, as compared with diabetic wildtype controls. Our findings suggest that the increased plasma IL-18 levels in diabetic mice are not dependent on hematopoietic GSDMD, and that diabetes-mediated necrotic core expansion is independent of hematopoietic NLRP3 and AIM2 inflammasome and pyroptosis pathways.
Abstract 426: Adipose Tissue Phosphatidylcholine Remodeling Influences Insulin Signaling
Authors: He Mulin , Li Zhiqiang , Jiang Xian-Cheng
Published: November 2022

Lysophosphatidylcholine acyltransferase (LPCAT3) is the major isoform of phosphatidylcholine remodeling enzyme in adipose tissue. To study the impact of LPCAT3 on adipose tissue lipid metabolism and its metabolic consequences, we prepared adipocyte-specific Lpcat3 gene knockout (KO) mice using the Cre-Loxp approach. We found LPCAT3 deficiency caused a significant reduction of LPCAT3 mRNA and activity by about 60%, and a significant reduction of polyunsaturated phosphatidylcholines in the plasma membrane. Further, the deficiency increased insulin sensitivity, reflected by glucose and insulin tolerance studies. Importantly, LPCAT3 deficiency promoted insulin-dependent insulin receptor kinase tyrosine phosphorylation and Akt phosphorylation through influencing membrane lipid rafts. Furthermore, the deficiency protected mice from high-fat, high-caloric diet-induced insulin resistance. Taken together, our findings identify LPCAT3 as one of the major players in maintaining cell membrane structure and illustrate the manipulation of cell membrane phospholipid saturation can be a new strategy to treat insulin resistance.
Abstract 438: The
CETP/ApoB100
Transgenic Mouse As A Model For Metabolic Syndrome
Authors: Kaur Rupinder , Anspach Garrett , Shelman Nathan R , Graf Gregory A
Published: November 2022

Background:
Metabolic syndrome (MetS) is a cluster of risk factors that predisposes individuals to develop atherosclerotic cardiovascular disease, type 2 diabetes mellitus, and nonalcoholic fatty liver disease. Dyslipidemia is an essential element of MetS. Mice lack cholesterol ester transfer protein (CETP) and rapidly clear ApoB-containing lipoprotein and are resistant to the development of dyslipidemia and clinically relevant metabolic phenotypes when challenged with high fat, high cholesterol Western-type diet (WTD). We hypothesized that the presence of CETP and ApoB-containing lipoproteins would sensitize mice to the development of dyslipidemia and exacerbate metabolic phenotypes.

Approach and Results:
Male and female (n=7-11) C57Bl6/J mice harboring a human
CETP
transgene, a human
ApoB100
transgene, or both transgenes were placed on a WTD (42% kCal fat, 0.2% cholesterol). Adiposity, insulin sensitivity, glucose tolerance and plasma cholesterol were assessed at the midpoint and termination of the study. Mice were anesthetized, basal bile was collected (30 min), euthanized via cardiac puncture, and tissues dissected and snap frozen. One lobe of the liver was additionally fixed for histological analysis. In both females and males,
CETP/ApoB100
and
ApoB100
transgenic mice showed a robust increase in total plasma cholesterol and LDL-C. The presence of
CETP
further increased LDL-C in ApoB100 expressing mice and modestly reduced HDL-C. Differences in body weight were not observed across genotypes. However,
ApoB100
expression reduced adiposity in females, but not males. Conversely, the presence of either
ApoB100
or
CETP
impaired insulin sensitivity in male, but not female mice. Liver histology showed increased hepatocyte ballooning in
ApoB100
and
CETP/ApoB100
transgenic mice, but other measures of NAFLD were unaffected.

Conclusions:
Mice expressing
CETP/ApoB100
transgenes develop dyslipidemia more consistent with human lipoprotein profiles. The humanized lipoprotein profile reveals sexually dimorphic responses to WTD.
Abstract 441: Spatially Resolved Metabolites In Stable And Vulnerable Human Atherosclerotic Plaques Identified By Mass Spectrometry Imaging
Authors: Seeley Erin , Rom Oren , Pattillo Christopher B , Yurdagul Arif
Published: November 2022

There is a growing interest in elucidating the mechanisms that govern atherosclerotic plaque instability, which has so far highlighted the role of dysregulations in lipid, carbohydrate, and amino acid metabolism. However, where these impairments occur in a plaque has yet to be revealed. This is critically important because lipid deposition, cell composition, and matrix stiffness are heterogeneous throughout human atheromas, particularly in regions of the fibrous cap and near the necrotic core. Furthermore, lesional cells are constantly bombarded with various intrinsic and extrinsic metabolic insults that influence cell phenotype. Mass spectrometry imaging (MSI) is a powerful technology for the visualization of the spatial localization and relative abundance of hundreds to thousands of analytes in thin tissue sections without the need for
a priori
knowledge of analytes present in the tissue. Imaging is performed using matrix-assisted laser desorption/ionization (MALDI) to interrogate small areas from a tissue surface after the addition of a chemical matrix to the section which serves to extract, co-crystallize, and ionize molecules from the sample. In the present study, we performed MSI of late-stage, stable and vulnerable human atherosclerotic plaques using MALDI-MSI to visualize the distribution of metabolites in the fibrous cap and necrotic core. This platform was able to identify 856 metabolites assigned with chemical formulas. We were then able to confidently annotate 189 metabolites using a data filtering system whereby we queried detected and quantified metabolites from the Human Metabolome Database (HMDB). Coupled with RNAseq examining human stable and unstable atheromas, we identified differences in pathways related to nitric oxide metabolism, collagen catabolism, arginine metabolism, tryptophan metabolism, lipid metabolism, and reactive oxygen species. This work represents the first study to begin defining an atlas of metabolic pathways involved in plaque destabilization in human atherosclerosis. We anticipate this work will be a valuable resource for the scientific community and will ultimately open new avenues of research in cardiovascular disease.
Abstract 443: Blackcurrant Extract Inhibits Oxidative Stress In Cultured Cardiomyocyte Cells
Authors: Currie Tanisha , Engler Marguerite M , Olsen Cara , Krauthamer Victor , Scott Jonathan , Deuster Patricia , Flagg Thomas , Flagg Thomas
Published: November 2022

Background:
Cardiovascular disease (CVD) affects a large sector of the U.S. population; accounting for millions of adult men and women impacted by its high annual incidence of mortality and morbidity. An underlying cellular link to the pathophysiological process of CVD is oxidative stress. Oxidative stress occurs when there is an imbalance of reactive oxygen species, reactive nitrogen species, and a deficiency in antioxidants. In particular, flavonoids found in fruits and berries have antioxidant properties that may quell oxidative stress activities at the cellular level. Mounting evidence suggests that flavonoids in the form of berry extracts may offer health benefits.

Methods:
Blackcurrant and various berry extracts (aronia berry, tart cherry, elderberry, cranberry, and a berry blend) were investigated in cultured cardiomyocyte (HL-1) cell line-to determine their effects on oxidative stress. Cells were maintained in Claycomb medium at 37°C in humidified 5% humidified CO
2.
A menadione-induced oxidative stressor was applied to generate oxidative stress in the HL-1 cells. A CellROX
TM
green reagent assay was used as a detection method of oxidative stress. The effects of berry extracts on cell viability within the HL-1 cells were also investigated.

Results:
Blackcurrant extract was found to be superior compared to all of the other berry extracts in suppressing oxidative stress in the HL-1 cells. N-acetyl-cysteine (NAC) used as a control, also had similar antioxidant effects as blackcurrant extract in the HL-1 cells under menadione-induced oxidative stress conditions. Additionally, NAC demonstrated significance for increased cell viability and cardio protection, while blackcurrant extract showed modest cellular protection. These results indicate that blackcurrant extract has potent antioxidant and modest cardioprotective properties comparable to NAC in HL-1 cells.

Conclusion:
Blackcurrant extract rich in flavonoids showed promising effects as an antioxidant with cardioprotective benefits. This may have significant implications for improving CV health in the human model. Further studies are warranted to determine the role of the bioactive components of blackcurrant and application to CV health.
Abstract 444: Apolipoprotein A-I (ApoAI) Vascular Gene Therapy Protects Against Vein-Graft Atherosclerosis
Authors: Bi Lianxiang , Wacker Bradley , Komandur Kaushik , Sanford Nicole , Dichek David
Published: November 2022

Introduction:
Coronary artery venous bypass grafts typically fail because of accelerated atherosclerosis, driven by lipid infiltration and by accumulation of macrophages and other cells. We previously showed that transduction of carotid artery endothelium of fat-fed rabbits with a helper-dependent adenovirus expressing apolipoprotein AI (HDAdApoAI) reduced atherosclerotic lesion size and decreased intimal lipid and macrophage accumulation. We hypothesized that HDAdApoAI transduction of rabbit vein graft endothelium would reduce lipid and macrophage accumulation.

Methods:
Fat-fed rabbits received bilateral external jugular vein to carotid artery interposition grafts. Four wks later, one graft per rabbit was infused with HDAdApoAI and the other with HDAdNull (a non-expressing HDAd). Grafts were harvested 12 wks after HDAd infusion. Paired analysis was performed in all rabbits with bilateral patent grafts (n=22). Graft hemodynamics were measured at vector-infusion and harvest surgeries. In vivo graft diameters were measured before vector infusion and harvest with ultrasound. Grafts were cut into 8 segments, for histology and biochemical analyses.

Results:
Mean plasma cholesterol levels were 400-600 mg/dL. Graft patency rates were 89% after vein grafting and 100% after the vector-infusion surgery. Lumen diameter and hemodynamics did not differ between HDAdApoAI and HDAdNull-transduced grafts. HDAd genomes were detected in all vein grafts. High levels of apoAI mRNA were detected in 100% of HDAdApoAI-treated vein grafts (mean 155-fold higher than in HDAdNull grafts). HDAdApoAI-treated grafts had 25% lower cholesterol (by mass spectrometry; P=0.03) and 17% less median CD68 mRNA (by RT-qPCR; P=0.008; CD68 is expressed by macrophages and lipid-loaded SMC). No significant differences were detected in mRNA encoding several inflammatory cytokines and inflammation-associated proteins. There were nominal decreases in total intimal area (16%, P=0.9), percent stenosis (4%, P=0.08), and in intimal area positive for Oil Red O (11%, P=0.3), macrophages (11%, P=0.7), and VCAM-1 (8%, P=0.8).

Conclusion:
HDAdApoAI transduction of vein graft endothelium reduces some markers of atherosclerosis, potentially by stimulating cholesterol efflux.
Abstract 448: Angiotensin-converting Enzyme In Renal Proximal Tubular Cells Does Not Affect Atherosclerosis In Male And Female Hypercholesterolemic Mice
Authors: Chen Hui , Howatt Deborah A , Moorleghen Jessica J , Liang Ching L , Daugherty Alan , Lu Hong
Published: November 2022

Objective:
Angiotensin-converting enzyme (ACE) generates Angiotensin II (AngII), the bioactive angiotensin peptide that contributes to atherosclerosis. ACE is present in proximal tubular cells (PTCs), and AngII concentrations are higher in kidney than in plasma. The purpose of this study was to determine whether ACE in PTCs contributed to atherosclerosis in both male and female mice.

Approach and Results:
ACE f/f x Ndrg1-Cre ERT2 -/- (PTC-ACE+/+) and ACE f/f x Ndrg1-Cre ERT2+/- (PTC-ACE -/-) mice were generated by breeding female ACE floxed mice (ACE f/f) xNdrg1-Cre ERT2-/- and male ACE f/f x Ndrg1-Cre ERT2+/- transgenic mice. Both male and female littermates in an LDL receptor -/- background were used to study atherosclerosis. PTC-ACE+/+ and PTC-ACE-/- littermates were administered with of tamoxifen (150 mg/kg/day) for 5 consecutive days at the age of 4-6 weeks to ACE deletion in PTCs. Two weeks after the last injection of tamoxifen, mice were fed a Western diet for 12 weeks. Blood pressure was measured using a tail-cuff system during the study. Systolic blood pressure was not different between the two genotypes (PTC-ACE+/+ vs -/-: 124±3 vs 121±2 mmHg in male, P=0.3; and 114±3 vs 115±3 mm Hg in female, P=0.9). All mice were hypercholesterolemic, and plasma cholesterol concentrations were not different between the two genotypes (PTC-ACE +/+ vs -/-: 1524±108 vs 1680±66 mg/dl in male, P=0.2; 1175±47 vs 1210±55 mg/dl in female, P=0.6). ACE mRNA abundance measured by qPCR of ACE in kidneys was less in the PTC-ACE-/- mice (PTC-ACE +/+ vs -/-: 1.03±0.11 vs 0.33±0.04 in male, P=0.001; 1.14±0.21 vs 0.64±0.09 in female, P=0.02). In PTC-ACE-/- mice, immunostaining of ACE in kidney sections showed that ACE was deleted in S1 and S2 PTCs, but remained in S3 of the outer medulla. Atherosclerosis was measured using an
en face
method. PTC-ACE+/+ and PTC-ACE-/- mice had comparable atherosclerotic lesion size in the ascending and aortic arch regions (PTC-ACE+/+ vs -/-: 11.4±1.4% vs 13.4±1.7% in male, P=0.4; 14.9±2.2% vs 15.7±3.7% in female, P=1.0).

Conclusion:
PTC-specific ACE deficiency did not affect atherosclerosis in hypercholesterolemic mice.
Abstract 450: Monocyte-to-endothelial Cell Crosstalk By 27-hydroxycholesterol Promotes Monocyte Vascular Recruitment And Thereby Advances Atherogenesis
Authors: Yu Liming , Huang Linzhang , Chu Haiyan , Peng Jun , Sacharidou Anastasia , Weinstock Ada , Fisher Edward A , Mineo Chieko , Shaul Philip W
Published: November 2022

How hypercholesterolemia invokes the vascular inflammation that is critical to atherogenesis is poorly understood. Sterol 27-hydroxylase (cyp27a1) converts cholesterol to 27-hydroxycholesterol (27HC), and we previously showed that 27HC is an estrogen receptor alpha (ERα) ligand. Here we determined how 27HC produced by monocytes impacts atherogenesis. In the absence of changes in circulating lipids, monocyte cyp27a1 deletion in apolipoprotein E
-/-
mice (apoE
-/-
) caused a 35% decline in atherosclerotic lesion severity; this was related to decreased macrophage accumulation due to a reduction in lesion monocyte recruitment, which likely occurred as a result of decreases in endothelial cell VCAM-1 and ICAM-1. Experiments in cultured endothelial cells revealed that 27HC liganding of ERα activates NF-κB by causing the disassociation of the cytoplasmic scaffolding protein septin 11 from ERα and resulting septin 11 activation of Jnk kinase signaling. Endothelial septin 11 deletion in apoE
-/-
mice phenocopied the impact of monocyte cyp27a1 loss, yielding a 37% decrease in lesion severity that was similarly explained by declines in endothelial cell VCAM-1 and ICAM-1, lesion monocyte recruitment and macrophage accumulation. In bone marrow transplant studies the reintroduction of monocyte cyp27a1 caused a 71% increase in lesions which was fully dependent on endothelial cell ERα and septin 11. Furthermore, in apoE
-/-
mice pharmacologic inhibition of cyp27a1 decreased lesion severity by 41% without altering circulating lipids, causing declines in endothelial cell VCAM-1 and ICAM-1, monocyte recruitment and macrophage accumulation. There was no additivity with monocyte cyp27a1 deletion and cyp27a1 inhibition, indicating that the treatment-related atheroprotection occurs via monocyte cyp27a1 inhibition. These cumulative findings reveal that through a unique pro-inflammatory mechanism involving endothelial cell ERα and septin 11, monocyte-to-endothelial cell crosstalk by the cholesterol metabolite 27HC promotes monocyte vascular recruitment to advance atherogenesis. Interventions attenuating the crosstalk may complement LDL-directed strategies in the battle against atherosclerosis.
Abstract 466: Fhl5 Enhances Vascular Calcification By Regulating Smooth Muscle Cell Transcriptional Networks
Authors: Wong Doris , Auguste Gaelle E , Turner Adam , Palmore Meredith , Perry Noah , Aherrahrou Redouane , Civelek Mete , Kovacic Jason C , Bjorkegren Johan , Miller Clint
Published: November 2022

Coronary artery calcium (CAC) is a hallmark of coronary artery disease (CAD) with increased CAC levels associated with increased risk for CAD related morbidity and mortality. The development of CAC is a pathological process driven by the modulation of smooth muscle cells (SMCs) and maladaptive extracellular matrix remodeling. In the most recent multi-ancestry meta-analysis of more than 35,000 individuals, FHL5 (Four-and-a-half LIM domains 5) was prioritized as a novel CAC gene through Bayesian colocalization and epigenomic analyses. scRNA-seq studies of human coronary and carotid arteries revealed high enrichment of FHL5 gene expression in SMC and pericyte populations. These preliminary studies motivate our hypothesis that FHL5 functions as a transcriptional regulator of vascular remodeling pathways associated with increased CAC risk. Following treatment of SMCs with a calcifying cocktail, stable FHL5 overexpression promoted the osteogenic SMC phenotypic transition, characterized by increased levels of osteogenic activators and reduced expression of SMC contractile markers. This osteogenic gene signature correlated with increased SMC calcification, consistent with the direction of effect of the CAC risk allele. To provide insights into the transcriptional program regulated by FHL5 that contributes to the vascular calcification phenotype, we mapped FHL5 binding sites in SMCs using the CUT&RUN method. We identified 17,201 FHL5 binding sites that overlapped the active enhancer mark H3K27ac. FHL5 target genes were enriched in pathways linked to vascular remodeling, such as cell adhesion and extracellular matrix organization. Moreover, risk variants for pulse pressure, a metric of arterial stiffness that is highly correlated with CAC, was enriched in FHL5 binding sites. Lastly, integration with FHL5 trans-eQTL target genes in STARNET arterial tissues revealed a network of CAD-associated candidate genes regulated by FHL5 in vitro and in vivo that impact SMC proliferation, migration, and calcification. Taken together, we provide evidence that FHL5 regulates a network of genes linked to arterial stiffness, which may explain the underlying mechanism of its genetic association with CAC in individuals from the general population.
Abstract 480: Mammalian Organ Regeneration In Spiny Mice
Authors: Okamura Daryl M , Nguyen Elizabeth D , Collins Sarah J , Yoon Kevin , Gere Joshua B , Beier David R , Majesky Mark W
Published: November 2022

Fibrosis-driven solid organ failure is a major global health burden. Spiny mice (genus:
Acomys
) are terrestrial mammals that regenerate severe skin wounds without scars to avoid predation. To test whether spiny mice can also regenerate internal organ injuries, we performed acute obstructive or ischemic surgeries on adult kidneys of
Acomys cahirinus
compared to
Mus musculus.
Despite equivalent acute tissue damage,
Acomys
fully regenerate nephron structure, vascular & lymphatic networks, and organ function without fibrosis by 14d while
Mus
(CD1 or C57BL/6) develop extensive interstitial fibrosis and progress to organ failure and death. Comparative transcriptomic, epigenetic, and single-cell assays revealed that the
Acomys
genome appears poised to initiate a regenerative response at the time of injury. In addition, we found the Hippo pathway effector protein YAP localized to the nucleus in renal tubular epithelial cells (RTEs) undergoing scarless wound healing in
Acomys
while it was either cytoplasmic or not detectable in
Mus
RTEs. Experiments
in vitro
revealed an accelerated PP2A-dependent YAP phosphatase activity that maintained nuclear YAP in
Acomys
RTEs and was not detected in
Mus
or human cells. Treatment of
Acomys in vivo
with the nuclear YAP-TEAD inhibitor verteporfin converted tissue regeneration to fibrosis in the dermis and similar experiments for the kidney are in progress. These results suggest that the spiny mouse genome evolved both transcriptional and post-translational adaptations important for scar-free regenerative wound healing
in vivo
.
Abstract 482: Early Reduction of Blood Flow in Rabbit Jugular Vein-Carotid Artery Bypass Grafts is not Caused by Anastomotic Stenosis
Authors: Komandur Kaushik , Bi Lianxiang , Wacker Bradley K , Dichek David A
Published: November 2022

Background:
Obstructed arteries are often treated by surgical implantation of venous bypass grafts. Vein grafts can fail for many reasons, including thrombosis, atherosclerosis, or anastomotic stenoses (narrowings where the grafts connect to the artery). In a previous study aimed at developing a hyperlipidemic rabbit model of vein-graft atherosclerosis for testing gene therapy, vein grafts used to bypass carotid arteries were widely patent. However, vein graft blood flow decreased by a mean of 5.6 mL/minute (~23%) between 8 and 16 wks after grafting, with flow at 16 wks reduced to <60% of flow in an unoperated carotid artery. We hypothesized that decreased flow was caused by anastomotic stenoses, raising concern that this might lead to graft occlusion, precluding use of the model to test gene therapy for atherosclerosis.

Methods:
We designed an experiment to test whether anastomotic stenoses were present and—if so—whether the stenoses were the cause of decreased blood flow. In a new set of grafted veins, we harvested the anastomoses, embedded, sectioned and stained them, and measured percent stenosis using computer-assisted image analysis. Percent stenosis was calculated by dividing intimal area by the stenosis-free lumen area (calculated from the length of the IEL, assuming circular geometry in vivo). We measured stenoses in both anastomoses of 22 patent vein grafts, harvested from 14 hyperlipidemic rabbits (44 total anastomoses). Blood flow (mL/min) in each vein graft was measured with a flow meter at 4 wks after grafting and again at 16 wks (at harvest).

Results:
The mean blood flow at the 4 wks measurement was 51 mL/minute (range: 28 to 90) and 36 mL/minute (range: 21 to 53) at the harvest measurement. The calculated mean change in blood flow was –16 mL/minute (range: –55 to +14). The mean anastomotic stenosis was 39% (range: 2.3% to 89%). Using Spearman’s rank-order correlation test, we found that percentage stenosis did not correlate with the change in blood flow (r=0.059; P=0.8).

Conclusions:
Decreased blood flow in our vein graft model is likely not caused by anastomotic stenoses. Other explanations, such as decreased cardiac output or increased downstream vascular resistance (caused by cerebral atherosclerosis or microemboli) should be considered.
Abstract 483: External Counterpulsation Therapy (Renew™ NCP-5) For The Treatment Of Mild Cognitive Impairment Due To Alzheimer’s Disease Or Mild Dementia Of The Alzheimer’s Type
Authors: Moriarty Patrick M , Gorby Lauryn K , Salat David H , Burns Jeffrey M , Moreno Thomas N , Helfgott Jonathan
Published: November 2022

Introduction:
Research is elucidating vascular impairment in the pathophysiology of Alzheimer’s disease (AD) and as a treatment target. NCP-5 is external counterpulsation (ECP) therapy approved for treating unstable angina and granted FDA Breakthrough Status for mild cognitive impairment (MCI) and AD. It increases cardiac output and improves endothelial function. A study was done to assess NCP-5 on cognitive function.

Methods:
Randomized, single-blind, sham-controlled; 10 US sites. Subjects 55-85 years (Montreal Cognitive Assessment score ≥11) were given NCP-5 (n=95) or active sham (n=95). Primary endpoint: mean change from baseline in Vascular Dementia Assessment Scale-cognitive subscale (VADAS-Cog) at 12, 18, and 24 weeks, powered for a 2-point change.

Results:
Primary endpoint met (change, -4.45 points); NCP-5 showed statistically significantly greater improvement from baseline vs active sham on VADAS-Cog. In subjects with type 2 diabetes (T2D; n=36), NCP-5 improved cognition from baseline (change, -17.01 points;
p
<0.005). NCP-5 was statistically significantly superior to active sham on Alzheimer’s Disease Cooperative Study-Clinical Global Impression of Change at weeks 12 (odds ratio [OR], 2.13;
p
=0.013) and 24 (OR, 2.56;
p
=0.002). NCP-5 had a statistically significant decrease on Neuropsychiatric Inventory Aggression Index vs active sham at week 24 (
p
=0.041). Neuroimaging showed a decline in right hippocampal volume (HV) that was statistically significantly greater with NCP-5 at week 24 (
p
=0.012) and at longitudinal assessment to 12 months (
p
=0.008), possibly related to smaller HV seen at baseline with NCP-5. However, the decline in regional cerebral blood flow (rCBF) in the left hippocampus from baseline to month 12 and for the longitudinal assessment was statistically significantly less with NCP-5 than with active sham (
p
=0.010 and 0.019, respectively). NCP-5 had a low-risk safety profile (10,644 exposures), and the most common AE was mild skin irritation/injury.

Conclusions:
Renew NCP-5 improved cognitive performance and global function, particularly in those with T2D. Findings support the role of targeting vascular dysfunction, specifically with NCP-5, for the treatment of MCI and mild dementia of the Alzheimer’s type.
Abstract 484: Telomerase Reverse Transcriptase And Signal Transducer And Activator Of Transcription 5a Mediates Osteogenesis In Calcific Aortic Valve Disease
Authors: Cuevas Rolando A , Chu Claire , Wong Ryan , Crane Alex , Sembrat John C , Sultan Ibrahim , St. Hilaire Cynthia
Published: November 2022

Cardiovascular calcification is a highly prevalent pathological process found in the vessels and valves in the elderly and patients with diabetes, hypertension, and renal disease. Calcific aortic valve disease (CAVD) stiffens and remodels the valve leaflets and leads to valve dysfunction, cardiac failure, and increased stroke risk. Vascular calcification can occur in the necrotic core of atherosclerotic plaques or in the medial layer of arteries. However, the initial steps dictating the onset of calcification remain ill-defined. Multiple studies have revealed that the protein telomerase reverse transcriptase (TERT), the catalytic subunit of the enzymatic complex required for telomere length maintenance, is a co-factor to stimulate gene transcription, and its overexpression primes mesenchymal stem cells to differentiate into osteoblasts. We determined that TERT is required for the calcification valve interstitial cells (VICs) and coronary smooth muscle cells (SMCs); TERT expression and protein are increased in calcified tissues and cell lines, independent of changes in telomere length. Genetic deletion of
Tert
in murine VICs and SMCs prevented calcification. Upon osteogenic stimulation, TERT binds to Signal Transducer and Activator of Transcription 5A/B (STAT5) to translocate into the nucleus where the complex binds to the
RUNX2
promoter to induce expression. We found that VICs cultured under inflammatory conditions calcified and upregulated STAT5, suggesting that inflammatory signaling may promote calcification in VICs. Lastly, testing several STAT5 inhibitors, we determined that STAT5A is required for the calcification of VICs and SMCs. These findings are the first to suggest that TERT and STAT5 are involved in driving the osteogenic switch and calcification of vascular cells and constitute potential therapeutic targets for the treatment of CAVD.