bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract MP10: The Long Non-coding RNA LINC02502 Modulates Vascular Smooth Muscle Cell Phenotypes
Authors: Haag Elisabeth , Dang Tan , Wobst Jana , Hoehne Carolin , Hegge Louise , Winter Hanna , Maegdefessel Lars , Schunkert Heribert , Kessler Thorsten
Published: May 2022

Background:
Genome-wide association studies led to the identification of several genomic loci associated with coronary artery disease risk. One such locus is chromosome 4q27 which harbors the long non-coding RNA (lncRNA)
LINC02502
and the
PDE5A
gene.
PDE5A
encodes phosphodiesterase 5a, the enzyme that degrades the second messenger cyclic guanosine monophosphate. We previously showed that differential
PDE5A
expression is mediated via
LINC02502
-mediated scaffolding of the transcription factor REST.

Methods and Results:
In a screening of vascular cell types, we identified vascular smooth muscle cells (VSMC) to express both
PDE5A
and
LINC02502
. Using RNA interference, we studied the ability of VSMC to migrate in the presence or absence of
LINC02502
in
in vitro
scratch wound healing assays. Knockdown of
LINC02502
led to a significant reduction of VSMC migration. VSMC proliferation was determined using BrdU incorporation assays. After knockdown of
LINC02502
, VSMC proliferation was decreased. To investigate whether
LINC02502
influences inflammatory phenotypes, we ectopically overexpressed the lncRNA in VSMC and analyzed a panel of inflammatory transcripts using quantitative PCR. In general, overexpression of
LINC02502
rendered VSMC more inflammatory. Particularly,
LINC02502
increased the expression of interleukin-1β, chemokine (C-X-C motif) ligand 1, and fractalkine. Importantly, we did not make these observations secondary to overexpression of
PDE5A,
suggesting that
LINC02502
regulates these transcripts in an independent manner.
In silico
analysis, however, revealed that
LINC02502
-regulated targets were enriched for predicted targets of REST.

Conclusion:
The lncRNA
LINC02502
is a candidate for mediating the risk of coronary artery disease at the chromosome 4q27 locus via regulating
PDE5A
. Independent of PDE5A regulation, LINC02502 promotes pro-atherosclerotic phenotypes in VSMC, i.e., migration, proliferation, and expression of inflammatory transcripts. Modulating
LINC02502
might be a novel approach to prevent and treat coronary atherosclerosis via multiple pathways.
Abstract MP15: Structural Vs. Acute Load Dependent Mechanisms For Carotid Artery Stiffening With Aging: The Multi-ethnic Study Of Atherosclerosis (MESA)
Authors: Pewowaruk Ryan , Tedla Yacob , Korcarz Claudia E , Tattersall Matthew C , Stein James H , Chesler Naomi C , Gepner Adam
Published: May 2022

Introduction:
Aging stiffens arteries, increasing cardiovascular disease (CVD) risk. Elastic arteries stiffen via two main mechanisms: 1) acute load dependent stiffening from higher blood pressure (BP), and 2) structural stiffening such as collagen accumulation. AHA’s arterial stiffness scientific statement highlights differentiating structural vs acute load dependent stiffening with aging as a crucial, unanswered question.

Hypothesis:
Longitudinal rates of structural and acute load dependent arterial stiffening will differ based on age.

Methods:
MESA participants with B-mode carotid ultrasound and brachial BP at Exam 1 and Exam 5 (year 10) were included in this study (n=2604). Peterson’s and Young’s elastic moduli (PEM and YEM) were calculated to represent total stiffness. Structural stiffness was calculated by adjusting PEM and YEM to a standard BP of 120/80 mmHg with participant-specific models. Acute load dependent stiffness was the difference of total and structural stiffness. Changes in carotid artery stiffness mechanisms over 10 years were compared by age groups with ANCOVA models adjusted for baseline CVD risk factors.

Results:
The 75-84 age group had the greatest rate of total (p<0.001), structural (p<0.01), and acute load dependent stiffening (p<0.05) compared to younger groups. Only age was predictive of structural stiffening, but age, race/ethnicity, education, BP, cholesterol, and medications were predictive of increased acute load dependent stiffening. On average, structural stiffening accounted for the vast majority (>99%) of total stiffening, but 37% of participants had more acute load dependent than structural stiffening.

Conclusions:
Rates of structural and acute load dependent carotid artery stiffening increased with age. Structural stiffening was consistently observed, and acute load dependent stiffening was highly variable. The heterogeneity in arterial stiffening mechanisms with aging may influence CVD development.
Abstract MP18: Mitochondrial Inducing Factor 1 Affects Stabilization Of Advanced Atherosclerotic Lesions In Carotid Artery Disease
Authors: Bischoff Lukas , Pauli Jessica , Paloschi Valentina , Maegdefessel Lars
Published: May 2022

Whereas cerebrovascular diseases, most and foremost ischemic events, being the second most deadliest disease as of today in the western world the process behind carotid artery plaque (CAP) vulnerability remains understood insufficiently. Through proteomic analysis of blood serum from patients treated for atherosclerotic lesions in carotid arteries we were able to determine proteins of potential relevance to CAP stability. One of the identified targets is mitochondrial apoptosis inducing factor 1 (AIFM1), which we found upregulated in serum profiles from patients with vulnerable atherosclerotic lesions. The study were conducted with human vascular tissue and blood samples from the 'Munich Vascular Biobank'. Proteomic profiling of serum was performed using the platform OLink (Uppsala, Sweden) taken from patients with either stable (n=53) or unstable (n=53) CAPs. Furthermore, gene expression of AIFM1 in stable (n=5) and unstable (n=3) CAPs was assessed via RT-qPCR .Via Immunostaining the regions showing higher expression of AIFM1 within the plaques (n=10) were determined and put in perspective with markers of cell types predominantly accumulated in those regions. In addition, blood monocyte-derived macrophages were stimulated with different factors and changes in expression of AIFM1 was assessed. With the proteomic sequencing of blood sera, higher levels of AIFM1 in patients with unstable versus stable CAP were determined. This trend was also seen assessing the gene expression of AIFM1 in the human vascular tissue. A significant enrichment of staining of AIFM1 positivity was observed in regions where apoptosis occured, such as the necrotic core and the shoulder regions of the advanced CAP. Furhtermore, AIFM1 enriched regions were identified as areas infiltrated by immune cells, especially macrophages. Monocytes of healthy donours showed an increased expression of AIFM1 when developed into macrophages. In conclusion AIFM1 was identified as a potential novel marker for advanced, unstable lesions in CAPs. Future studies (in vitro and in vivo) will reveal which role AIFM1 might play during processes that trigger CAP destabilization. Of further importance will be cell type expression patterns that we are also assessing in currently ongoing studies.
Abstract MP31: Bmal1 Regulates Production Of Larger Lipoproteins By Modulating Crebh And Apolipoprotein A 4
Authors: Pan Xiaoyue , Hussain M M
Published: May 2022

High plasma lipid and lipoprotein levels are risk factors for various metabolic diseases, such as diabetes, obesity, metabolic syndrome, and atherosclerosis. We previously showed that circadian rhythms regulate plasma lipids, and deregulation of these rhythms cause hyperlipidemia and atherosclerosis in mice. Here, we show that global and liver-specific Bmal1-deficient mice maintained on a chow or a Western diet developed hyperlipidemia, which was denoted by the presence of higher amounts of triglyceride- and apoAIV-rich larger chylomicron and very-low-density lipoprotein, due to overproduction. Bmal1 deficiency decreased Shp and increased MTP, a key protein that facilitates primordial lipoprotein assembly and secretion. Moreover, we show that Bmal1 regulates Crebh to modulate apoAIV expression and the assembly of larger lipoproteins. This is supported by the observation that Crebh- and apoAIV-deficient mice, along with Bmal1-deficient mice with knockdown of Crebh, had smaller lipoproteins. Further, overexpression of Bmal1 in Crebh-deficient mice had no effect on apoAIV expression and lipoprotein size. These studies indicate that regulation of apoAIV and assembly of larger lipoproteins by Bmal1 requires Crebh. Mechanistic studies showed that Bmal1 regulates Crebh expression by two mechanisms. First, Bmal1 interacts with the Crebh promoter to control circadian regulation. Second, Bmal1 increases Rev-erbα expression, and Rev-erbα interacts with the Crebh promoter to repress expression. In short, Bmal1 modulates both the synthesis of primordial lipoproteins and their subsequent expansion into larger lipoproteins by regulating two different proteins, MTP and apoAIV, via two different transcription factors, Shp and Crebh. It is likely that disruptions in circadian mechanisms contribute to hyperlipidemia, and synchronization of circadian rhythms may limit/prevent hyperlipidemia and atherosclerosis.
Abstract MP38: Functional Investigation Of A Novel Adipose Long Intergenic Non-coding RNA, Linc- ADAIN, During Obesity
Authors: Oreilly Marcella E , Xue Chenyi , Cynn Esther , Liu Wen , Zhu Lucie , Reilly Muredach P
Published: May 2022

Background:
Long intergenic non-coding RNAs (lincRNAs) are important emerging regulators of cellular functions. Dysfunctional adipose tissue, characterized by increased inflammation and insulin resistance, plays a central role in the development of type 2 diabetes (T2DM) and atherosclerotic cardiovascular diseases. Deep RNA-sequencing of human adipose during low-dose endotoxemia, identified novel lincRNAs shown to be regulated by inflammation and were validated in an independent human cohort, before and after bariatric surgery. LincADAIN was identified as a top candidate that is 1)abundantly expressed in adipose tissue 2)reduced in both obesity-induced chronic and LPS-induced acute inflammation (-77% and -53%, p<0.05).

Methods:
Knock down(KD) of lincRNAs was carried out via lentiviral expression of shRNA and antisense oligonucleotides (ASOs) in human adipose stromal cells (ASCs)-adipocytes. Subcellular location of LincADAIN was identified through qPCR after cellular fractionation. Biotinylated lincRNA pulldown assay was used to identify interacting proteins by Mass Spectrometry (MS).

Results:
Human LincADAIN expression negatively correlates to BMI in obese (p<0.001, r2-0.3042) but not lean individuals. PPARγ antagonist treatment showed reduced expression of LincADAIN and PPARγ ChIP-seq peaks indicate that PPARγ may be a transcriptional activator. KD via shRNA of LincADAIN in ASC-adipocytes, increased protein but not mRNA levels of inflammatory cytokines in adipocytes such as IL-8 and MCP-1. Cellular fractionation experiments indicate that LincADAIN is mainly located in the nucleus. MS analysis of biotinylated LincADAIN pulldown proteins revealed many central nuclear and ribosomal proteins as potential interactors, which may be involved in the post-transcriptional mechanism of LincADAIN regulating inflammatory cytokines.

Conclusion:
These results suggest that LincADAIN is a novel lincRNA with roles in regulating adipose inflammation through nuclear and cytoplasmic actions including modulation of RNA translation. Functional investigations of lincRNAs, such as these are warranted, as recent genomic studies suggest that lincRNAs are likely the causal element driving human disease at some GWAS loci.
Abstract MP41: Myeloid Ccn3 Protects Against Aortic Valve Calcification
Authors: Tu Peinan , Lin Zhiyong , zhou xianming , Jo Hanjoong , Villa-roel Nicolas , Kumar Sandeep , Ou Caiwen , Dong Nianguo
Published: May 2022

Objective:
CCN3, an inhibitor in osteoblast differentiation, can modulate calcium signaling, yet, whether CCN3 can regulate valvular calcification is unknown. While it has been suggested that macrophages are important in the regulation of valvular calcification, the molecular and cellular mechanisms of this process remain poorly understood. In the present study, we investigated the specific role that macrophage-derived CCN3 plays during the progression of calcific aortic valve disease (CAVD).

Methods and Results:
Myeloid-specific knockout of CCN3 (Mye-CCN3-KO) and control mice were subjected to a single injection of AAV encoding mutant mPCSK9 (rAAV8/D377Y-mPCSK9) to induce hyperlipidemia. AAV-injected mice were then fed a high fat diet (HFD) for 40 weeks. At the conclusion of HFD feeding, tissues were harvested for subsequent histological and pathological analysis. Echocardiography revealed that both male and female mye-CCN3-KO animals displayed compromised aortic valvular function accompanied by exacerbated valve thickness and cardiac dysfunction. Histologically, alizarin red and OsteoSense staining revealed a marked increase of aortic valve calcification in mye-CCN3-KO animals when compared to controls. In vitro, CCN3 deficiency augmented bmp2 production and secretion from bone marrow derived macrophages (BMDMs). Further, human valvular interstitial cells (VICs) cocultured with conditional media from CCN3-deficient BMDMs resulted in exaggerated pro-calcifying gene expression and the attendant calcification as revealed by alizarin red staining.

Conclusions:
Our data uncovered a novel role of myeloid CCN3 in the regulation of aortic valve calcification. Modulation of bmp2 production and secretion in macrophages might serve as a key mechanism for CCN3’s anti-calcification function in the development of CAVD.
Abstract MP43: Liposome-human ACE2 Complex As An Engineered Decoy To Abrogate SARS-CoV-2-induced Inflammatory Responses In Macrophages
Authors: Satta Sandro , Meng Zhaojie , Hernandaz Rebecca , Cavallero Susana , Zhou Tong , Hsiai Tzung K , Zhou Changcheng
Published: May 2022

Introduction:
Innate immune cells such as macrophages have been implicated in pathological inflammation in COVID-19 patients. As many immune cells express low levels of human angiotensin-converting enzyme 2 (hACE2), the mechanisms underlying SARS-CoV-2-mediated macrophage inflammatory responses remain elusive. Further, neutralizing SARS-CoV-2 prior to their binding to the host cells was proposed as a therapeutic approach to ameliorate SARS-CoV-2-stimulated inflammation. This study aims to investigate whether an engineered decoy receptor can affect SARS-CoV-2-induced macrophage inflammation.

Hypothesis:
Liposome-based hACE2 complex acts as a molecular decoy to abrogate SARS-CoV-2-induced macrophage inflammation.

Methods:
Rhodamine incapsulated liposome-based nanoparticles were used to generated Liposome-human ACE2 complex (Liposome-hACE2). Liposome-hACE2 was used as a decoy receptor or competitive inhibitor to inhibit SARS-CoV-2 or Spike protein-induced macrophage inflammation in vitro and in vivo.

Results and Conclusions:
SARS-CoV-2 or Spike protein induces strong inflammatory responses in murine macrophages in a hACE2-independent manner. Liposome-hACE2 can efficiently abrogated SARS-CoV-2 or Spike protein-induced inflammatory responses in murine macrophages in vitro and in vivo. Mechanistically, Spike protein stimulated macrophage inflammation by activating canonical IKKβ/NF-κB signaling, and deficiency of IKKβ abolished Spike protein-elicited inflammatory responses in macrophages. The use of Liposome-hACE2 as a molecular decoy was further recapitulated in human macrophages. SARS-CoV-2 or Spike protein-induced inflammatory responses in human peripheral blood mononuclear cells and differentiated THP-1 macrophages were also abolished by Liposome-hACE2 treatment. These results suggest that neutralizing SARS-CoV-2 by liposomes may present an innovative therapeutic strategy treating COVID-19.
Abstract MP47: Assessment Of Atherosclerosis In A Novel Transgenic Mouse Expressing Pathogenic Levels Of Human Lipoprotein(a)
Authors: Assini Julia , Xing Chuce , Clark Justin , Reza Tasnim , Gros Robert , Boffa Michael , Koschinsky Marlys L
Published: May 2022

Studying the role of elevated plasma levels of lipoprotein(a) (Lp(a)) in atherogenesis is challenging given that the
LPA
gene encoding apo(a) is absent in typical animal models. As such we created a novel transgenic mouse line expressing both human apolipoprotein(a) (apo(a)) and human apoB100
((Tg(LPA
+/0
;APOB
+/0
))
. The
LPA
transgene, assembled in the pLIV vector, produces a physiologically relevant 12-kringle apo(a) isoform. The
APOB
transgene contains the entire human
APOB
gene with a mutation preventing
APOB
editing. In our atherosclerosis model, male and female mice were injected weekly with an antisense oligonucleotide targeting the mouse LDL receptor (IONIS Pharmaceuticals), and fed a high-fat, high-cholesterol diet for 12 weeks. High plasma Lp(a) levels were observed in both male and female
Tg(LPA
+/0
;APOB
+/0
)
mice (219.0±11.9 and 133.7±12.1 mg/dL, respectively; n=12/group). Importantly, high levels of Lp(a) in plasma did not result in metabolic alterations, including differences in weight gain, energy expenditure, activity, RER, VO
2
/VCO
2
consumption/output, and glucose tolerance compared to
Tg(APOB
+/0
)
control mice. Both male and female

Tg(LPA
+/0
;APOB
+/0
)

and

Tg(APOB
+/0
)

mice exhibited enhanced proatherogenic lipoprotein profiles with the majority of the cholesterol and TG present in the VLDL and LDL fractions. Complex lesions developed in all transgenic mice, including large, lipid-rich necrotic cores, with overlying fibrous caps and intimal calcium deposition. Immunohistochemistry with a monoclonal apo(a) antibody revealed the presence of Lp(a) in the plaques of
Tg(LPA
+/0
;APOB
+/0
)
mice. Analysis of aortic sinus lesions revealed a 23% increase in total plaque area in female
Tg(LPA
+/0
;APOB
+/0
)
mice compared to female

Tg(APOB
+/0
)

mice (p=0.0836). Moreover, atherosclerotic plaques in female
Tg(LPA
+/0
;APOB
+/0
)
mice contained significantly more calcium deposition than female Tg(
APOB
+/0
) mice (1.79±0.4% vs. 2.96 ±0.3% of total plaque area (p<0.05)). No differences in plaque area or calcium were observed in male mice. In this ongoing study, detailed analyses of atherosclerotic plaque components in
Tg(LPA
+/0
;APOB
+/0
)
mice will be required to uncover the unique contribution of Lp(a) to atherogenesis.
Abstract MP48: Smooth Muscle Cell-Specific Deletion Of O-GlcNAc Transferase Impedes Atherosclerotic Lesion Formation In Western Diet-fed Apoe
-/-
Mice
Authors: Khanal Saugat , Mathias Amy , Lallo Jason , Bhavnani Neha , Gupta Shreya , Raman Priya
Published: May 2022

Attachment of O-linked N-acetylglucosamine (O-GlcNAc) to nucleocytoplasmic proteins or ‘O-GlcNAcylation’ is a ubiquitous post-translational modification affecting numerous cellular processes. We and others have previously reported that augmented protein O-GlcNAcylation mediates upregulation of numerous genes associated with atherosclerosis. Recent studies suggest the role of O-GlcNAc transferase (OGT), a key regulator of O-GlcNAc signaling, in diabetic vascular calcification and wound healing. However, the role of OGT in the etiology of atherosclerosis is elusive. The goal of the current study was to interrogate whether OGT plays a direct role in the development of atherosclerosis. For this, we crossed tamoxifen-inducible male Myh11-CreERT2;OGT
fl/y
;ApoE
-/-
mice with female OGT
fl/+
;ApoE
-/-
to generate SMC-specific OGT knockout on ApoE
-/-
background. To induce Cre recombinase activity, mice genotypes were injected i.p. with 60mg/Kg/day tamoxifen (peanut oil-vehicle control) once daily for 5 consecutive days beginning at 6 wks age. This was followed by a Western diet feeding regimen for additional 6-7 wks. Mice were harvested at 14 wks age after overnight fasting; plasma, aorta, and heart were collected for biochemical, molecular, and lesion studies. Immunoblotting confirmed loss of OGT expression in aortic vessels of tamoxifen-treated Cre
tg
;OGT
fl/y
;ApoE
-/-
mice (smOGT
KO
;ApoE
-/-
) vs. age-matched tamoxifen-treated Cre
tg
;OGT
+/y
;ApoE
-/-
littermates (smOGT
WT
;ApoE
-/-
, with intact OGT). Aortic root morphometry revealed a significant reduction (2.5-fold) in lesion lipid burden in smOGT
KO
;ApoE
-/-
mice compared with smOGT
WT
;ApoE
-/-
. This was accompanied by attenuated PCNA (proliferation marker), osteopontin (SM synthetic marker), pERK (SM signaling regulator), YY1 (transcriptional repressor of SM contractile genes), and SRF (transcriptional regulator of SMC proliferation) expression in smOGT
KO
;ApoE
-/-
vs. smOGT
WT
;ApoE
-/-
aortic lysates, shown via immunoblotting. Interestingly, SMC-specific OGT deletion had no effect on plasma total cholesterol and total triglyceride levels. Taken together, these results demonstrate a protective role of SMC-specific loss of OGT on atherosclerotic lesion formation.
Abstract P101: Suppression Of Serum Amyloid A Limits Progression Of Obesity Associated Abdominal Aortic Aneurysms
Authors: Trumbauer Andrea , Noffsinger Victoria , Ji Ailing , DEBEER Frederick C , Dugan Adam , Mullick Adam E , Webb Nancy R , Shridas Preetha , Tannock Lisa R
Published: May 2022

Obesity increases the risk for abdominal aortic aneurysms (AAA) in humans, and enhances angiotensin II (AngII)-induced AAA formation in C57BL/6 mice. Obesity is also associated with increases in serum amyloid A (SAA). We previously reported that deficiency of SAA significantly reduces AngII-induced inflammation and AAA in apoE-deficient mice. In this study we investigated whether SAA plays a role in progression of an established AAA in obese C57BL/6 mice.

Approach and Results:
Male C57BL/6 mice were fed a high fat diet (60% kcal as fat) throughout the study. After 4 months of diet the mice were infused with angiotensin II (AngII) at 1000ng/kg/min until the end of the study. Ultrasound (US) was performed in all mice before and after 28 days of AngII infusion, and mice that had at least a 25% increase in the luminal diameter of the abdominal aorta were stratified by luminal diameter into 3 groups. Group 1 was killed to establish baseline AAA. Groups 2 and 3 continued to receive AngII for a further 8 weeks along with an antisense oligonucleotide (ASO) that suppresses all 3 acute phase SAA isoforms (SAA-ASO), or a control ASO (5 mg/kg/wk). US was repeated at study end to assess AAA progression. Plasma SAA at the end of the experiment was 89.2±83.2 mg/L in the control ASO group, and 18.6±0.7 mg/L in the SAA-ASO group (mean±SD , p=0.008). There was no impact of SAA suppression on body weight, body fat, or blood pressure. After the first 4 weeks of AngII infusion, the average luminal diameter in all mice was 1.81±0.40 mm (mean±SD). Mice that received the control ASO had continued aortic dilation (average luminal aortic diameter 2.06±0.42 mm), whereas the mice that received the SAA-ASO had a significant reduction in progression of aortic dilation (average luminal diameter 1.64±0.43 mm, p=0.0015 for interaction between time and group).

Conclusions:
We demonstrate for the first time that suppression of SAA protects obese C57BL/6 mice from progression of AngII-induced AAA. Suppression of SAA may be a therapeutic approach to limit AAA progression.
Abstract P103: Molecular Imaging Analysis Of Integrin αvβ3 Expression With Gallium-68-labeled Arg-gly-asp In A Murine Model Of Thoracic Aortic Aneurysms
Authors: Anaya-Ayala Javier E , Medina Luis A , Verduzco-Vazquez Ana T , Bravo-Reyna Carlos , Martinez-Martinez Ricardo , Hinojosa Carlos A
Published: May 2022

Background/Objective:
Angiogenesis is a complex process. Studies in aneurysmal aortic wall in human tissue and animal models have demonstrated an increased mural neovascularization, and Integrin αVβ3 is a molecular indicator of angiogenesis. We aim to study and characterize Integrin αVβ3 expression with Gallium-68(68Ga)-labeled Arg-Gly-Asp (RGD) as a potential radiotracer using micro Positron Emission Tomography (PET) in a Murine model of Thoracic Aortic Aneurysms (TAA).

Methods:
Eight specimens of Wistar rat with Calcium Chloride (CaCl) TAA-induced were evaluated. The equipment utilized was an Albira microPET/CT unit (Bruker, Spain), access was obtained in one of the caudal veins for the radiolabeled 68Ga-RGD infusion. The PET uptake and activity were expressed as median value with interquartile range and the Mann-Whitney U test was employed to analyze and compare the chemically induced-site (Aneurysm) and intact aortic tissue (Control) in the same specimens. Statistical analysis was performed with GraphPad Prism 8.0 software.

Results:
All specimens tolerated well the procedures through a left thoracotomy (Upper panel figure A, B), and underwent imaging studies. A focal increased uptake was observed in the thoracic aortic lesions compared to control regions. The median standard uptake value (SUV) of 68Ga-RGD in the chemically induced-injury site (aneurysm) was 0.0125, while the uptake in the intact site (control) was 0.0003 (P value=0.0117) during a mean follow up period of 32 days (Lower panel figure A-C).

Conclusion:
We found significant differences of Integrin αVβ3 expression uptake with microPET imaging in chemically induced aneurysms and the intact aortic tissue within 5 weeks of vessel injury. Longitudinal studies with this imaging modality may assist in the molecular characterization of angiogenesis and aneurysmal progression in animal models, and its potential translation in clinical research studies in human patients with aortic aneurysm.
Abstract P121: Combining Extracellular Matrix Biomarkers Using Cluster Analysis More Accurately Predicts The Development Of Systolic Dysfunction In Acute Myocardial Infarction Compared To Single Biomarker Analysis
Authors: Brunton-OSullivan Morgane , Holley Ana , Shi Bijia , Harding Scott , Larsen Peter
Published: May 2022

Introduction:
The extracellular matrix (ECM) is central to cardiac repair following acute myocardial infarction (AMI), and pathological ECM activity may result in adverse ventricular remodeling. Systolic dysfunction is a manifestation of adverse remodeling, and the utility of combined biomarker analysis for prognosis remains undetermined. Cluster analysis is a statistical methodology that can combine biomarkers, while also accounting for collinearity.

Hypothesis:
In this study, we assessed the hypothesis that combining biomarkers using cluster analysis may more accurately predict the development of systolic dysfunction in AMI patients when compared to single biomarker analysis.

Methods:
In a cohort of 120 AMI patients, plasma levels of matrix metalloproteinase (MMP) -3, -8, -9 and tissue inhibitor of matrix metalloproteinase-1 (TIMP-1) were measured using ELISA and multiplexing assays during hospital admission. All patients had an echocardiogram within 1 year of AMI (median [IQR], 145 [89 - 252] days). Patients were divided into impaired (n=37, LVEF <50%) and preserved (n=83, LVEF ≥50%) systolic function. Hierarchical clustering was performed using Ward’s method of minimum variance. Mann-Whitney U and Chi-Squared testing was performed for univariate analysis, and binary logistic regression was performed for multivariate analysis.

Results:
Upon univariate analysis, current smoking, prescription of ACE inhibitor at discharge, peak Troponin T > 610 ng/L (median), and MMP-8 levels were predictive of impaired systolic function. Cluster analysis partitioned patients into two clusters (Cluster One, n=31; Cluster Two, n=89). Cluster One comprised a higher proportion of patients with impaired systolic function when compared to Cluster Two (15 out of 31 [48.4%] versus 22 out of 89 [24.7%]). Upon multivariate analysis, Cluster One assignment (odds ratio [95% CI], 2.74 [1.04-7.23], p=0.04) remained an independent predictor of systolic dysfunction in combination with clinical variables, while MMP-8 levels did not (3.43 [0.32-36.68], p=0.308).

Conclusion:
Findings from our study demonstrate a combined biomarker approach outperforms single biomarker analysis for predicting the development of systolic dysfunction following AMI.
Abstract P122: Leveraging Machine Learning Models For Dynamic Prediction Of Bleeding And Ischemic Risk After Drug-eluting Stent Implantation
Authors: Li Fang , Bekhet Laila , Xiang Yang , Feng Jingna , Du Jingcheng , Aguilar David , Dhoble Abhijeet , Wang Qing , Niu Shuteng , Hu Xinyue , Dang Yifang , Zhi Degui , Tao Cui
Published: May 2022

Background:
Contemporary risk scores for ischemic or bleeding endpoint prediction after drug-eluting stent (DES) implantation have limited predictive accuracy and fixed prediction windows.

Objective:
This study aimed to dynamically predict the ischemic and bleeding risks in different follow-up windows for patients with DES leveraging cutting-edge machine learning models.

Methods:
Using the Cerner Health Facts, we identified 98,236 adult patients who received DES with more than three months of follow-up after discharge. Three traditional machine learning models (LR, RF, and LGBM), three RNN-based deep learning models (GRU, LSTM, RETAIN), and one transformer-based model (Med-BERT) were applied for the risk stratification. The total follow-up window covered two years after discharge. ML models were tested in three types of prediction windows with varying lengths (i.e., one year, six months, and three months). Model performance was assessed according to a range of learning metrics including area under the receiver operating characteristic curve (AUC) and precision-recall curve.

Results:
For bleeding event prediction, the best AUC for the three-month window was 77.02% (GRU), for the six-month window was 81.11% (BiGRU + MedBert), and for the one-year window is 71.86% (LGBM). For ischemic event prediction, the best AUC for the three-month window was 71.09% (GRU), for the six-month window was 68.97% (LGBM), for the one-year window was 69.41% (LSTM). The transformer-based model and RNN-based models generally performed better than the traditional ML in both bleeding and ischemic risk prediction.

Conclusions:
Our machine learning model-based approaches for dynamically predicting the ischemic and bleeding risks after stenting is feasible and effective. The best-performing models show good discriminative capabilities in different prediction scenarios and have the potential to guide the clinical decision-making of the optimum DAPT duration in a personalized way.
Abstract P125: IL-6-induced Signaling In PD-1
+
CD4 Effector Memory T Cells Is Associated With Human Coronary Pathology
Authors: Kothari Hema , McSkimming Chantel , Drago Fabrizio , Williams Corey , Zunder Eli , McNamara Coleen
Published: May 2022

Objective:
Murine data, prospective epidemiological studies, and genetic association data support a potential causal role of IL-6 signaling in atherosclerosis development. IL-6 inhibitors have emerged as potential therapeutics for reducing events in both stable coronary artery disease (CAD) and acute coronary syndromes. IL-6-induced immune regulation plays an important role in atherosclerosis, however, a comprehensive map of IL-6 signaling in human immune cells is currently lacking. We developed a 32-antibody custom mass cytometry (CyTOF) panel to characterize IL-6 signaling across all major human immune cell subsets and applied it to identify IL-6-induced immune signatures linked with unstable atherosclerotic plaque.

Methods:
Peripheral blood mononuclear cells from healthy donors and subjects with CAD undergoing virtual histology-intravascular ultrasound imaging (IVUS-VH) were stimulated with vehicle and IL-6, stained, and ran in CyTOF. Unsupervised analysis algorithms (SPADE, UMAP, and Leiden clustering) were used to identify immune cell subsets and IL-6-induced intracellular phosphorylation status.

Results:
IL-6 induced STAT1 and STAT3 activation in CD4 and CD8 naïve T cell subsets and CD4 memory T subsets. Notably, we identified that IL-6 also activates STAT5 within the CD4 and CD8 naïve T subsets. IL-6 induced a much more robust activation of STAT1 as compared to STAT3 and STAT5. Other cell types such as CD14
+
monocytes, and CD11c
+
, and CD123
+
dendritic cells also showed IL-6-induced STAT activation. IL-6-induced phosphorylation of STAT1 and STAT3 in a novel PD-1
+
CD4
+
effector memory T cell subtype was associated with higher CAD burden and unstable plaque features.

Conclusions:
Findings are significant for mechanistic insights into IL-6-induced inflammation and may enable discovery of new approaches to reduce inflammation in CAD and other pathologies.
Abstract P131: Na/k-atpase Is A Negative Regulator Of Lps-induced Macrophage Activation
Authors: Zhang Jue , Huang Wenxin , Zhao Yiqiong , Silverstein Roy L , Chen Yiliang
Published: May 2022

Introduction and objective:
A strong association between atherosclerosis and infections has been suggested in several studies. Clarifying the mechanism of macrophage inflammatory activation is critical to reduce the risk of atherosclerotic cardiovascular disease. Recent studies indicate that the Na/K-ATPase (NKA) is a novel regulator of macrophage activation. Nevertheless, its role in bacterial lipopolysaccharide (LPS)-induced innate immune responses remains unclear. In this study, we aim to investigate how NKA regulates LPS signaling and macrophage inflammatory activation.

Methods and Results:
Using murine peritoneal macrophages isolated from genetically modified mice, we showed that macrophages partially deficient in NKA α1 were hypersensitive to LPS. 100 ng/ml LPS triggered enhanced pro-inflammatory cytokine production such as IL-1β, IL-6, MCP-1 and TNF-α through RT-PCR and Elisa assay. Furthermore, intraperitoneal injection of LPS inducing septic shock in mice resulted in higher plasma pro-inflammatory cytokine levels and lower survival rate in NKA α1 heterozygous null mice. Mechanistically, co-IP experiments showed that TLR4 assembles with NKA and phosphorylated-Lyn as a complex in response to LPS in WT macrophages and reduction in NKA α1 led to more co-precipitated p-Lyn by LPS stimulation. Moreover, the Src-family kinases inhibitor PP2 blocked the production of pro-inflammatory cytokine from post culture medium of LPS treated WT macrophages. In addition, RNA sequencing and Gene Set Enrichment Analysis showed an upregulation of NF-κB target genes. The activation and nuclear translocation of NF-κB were also augmented.

Conclusions:
NKA is a novel negative regulator of LPS-mediated inflammatory activation by regulating NKA α1/TLR4/Lyn Complex and NF-κB signaling pathway in macrophages.
Abstract P135: Platelet Activation Is Present In Psoriasis And Associated With Psoriasis Severity And Endothelial Dysfunction
Authors: Drenkova Kamelia , Myndzar Khrystyna , Katz Stuart D , Krueger James , Berger Jeffrey S , Garshick Michael
Published: May 2022

Background:
Psoriasis is associated with impaired endothelial function and increased CV risk. P-selectin, a platelet transmembrane protein involved in binding leukocytes and endothelial cells, is implicated in CVD.

Objective:
To explore platelet P-selectin expression in psoriasis and its association with biomarkers of inflammation and endothelial dysfunction.

Methods:
Patients with psoriasis (n=15, age 51 ± 18 years, 73% male), percent body surface area (BSA) psoriasis 9 ± 20, were compared to controls (Table 1A). The vascular endothelium was assessed via brachial artery flow-mediated dilatation (FMD), a metric of endothelial health and biomarker of CV risk. Platelet P-selectin expression was measured on freshly isolated platelets via flow cytometry in resting and stimulated (thrombin, ADP, arachidonic acid and epinephrine) conditions. High-sensitivity C-reactive protein (hs-CRP) was measured in a clinical laboratory.

Results:
Psoriasis patients were matched for traditional CV risk factors (Table 1A) aside from diabetes, which trended higher in psoriasis. No difference in hs-CRP was noted, however BSA positively correlated with hs-CRP (
r
=0.65, p=0.02), while FMD trended higher in psoriasis vs. controls (5.7% vs. 2.7%, p=0.07) even after accounting for diabetes status (β=-3.0, p=0.08). Platelet analysis revealed higher basal and stimulated P-selectin expression in psoriasis vs. controls (Table 1B). Positive correlations were noted between platelet activation, BSA and hs-CRP, while a negative association was noted between FMD and platelet activation (Table 1C).

Conclusion:
We describe platelet P-selectin in resting and stimulated conditions as elevated in psoriasis, positively correlated with biomarkers of psoriatic activity, and negatively associated with endothelial health. These findings have important implications for future clinical trials of targeting platelet activity to reduce CV risk in psoriasis.
Abstract P140: Molecular Signatures Of The Delayed Healing Response To Arterial Injury In Diabetic Rats
Authors: Narayanan Sampath , Rohl Samuel , Matic Ljubica , Razuvaev Anton , Lengquist Mariette
Published: May 2022

Aim:
Atherosclerosis is a major complication of diabetes mellitus and a leading cause of mortality in diabetic patients. Vascular interventions in diabetic patients can lead to complications attributed to defective vascular remodeling and impaired healing response. In this study, we aim to elucidate the physiological and molecular differences in the vascular healing response over time using a rat model of arterial injury applied in healthy and diabetic conditions.

Methods:
Wistar (healthy) and Goto-Kakizaki (GK, diabetic) rats (n = 40 per strain) were subjected to left common carotid artery (CCA) balloon injury and euthanized at different timepoints: 0 and 24 hours, 5 days, 2, 4 and 6 weeks. Non-invasive morphological and physiological assessment, and microarray profiling of the CCA was performed for each timepoint. Bioinformatic analyses were conducted using R software, DAVID bioinformatic tool, online STRING database and Cytoscape software.

Results:
Significant increase in the neointimal thickness (p<0.01; 2-way ANOVA) was observed after 2 weeks of injury in diabetic compared to heathy rats, which was confirmed by histological analyses. Moreover, a decrease in the reendothelialization rate was also detected in the diabetic rats at 4 and 6 weeks after injury. Bioinformatic analyses showed that expression of early response genes related to inflammation and proliferation were delayed in diabetic rats, coupled with the dysregulation of key pathways related to vascular healing. Defective expression patterns were observed for endothelial, macrophage and lymphocyte markers indicating potential faults on cell regulation level. TF-PPI analysis provided mechanistic evidence wherein an array of transcription factors was dysregulated in diabetic rats specifically from 2 weeks after injury.

Conclusions:
In this study, we investigated the effect of diabetes on vessel wall healing. We demonstrate that diabetic rats exhibit impaired arterial remodelling characterised by a delayed healing response. These results further corroborate the higher prevalence of restenosis in diabetic patients and provide vital molecular insights into the mechanisms contributing to the impaired arterial healing response in diabetes.
Abstract P145: Renal Proximal Tubule-specific Renin Deficiency Has No Effect On Atherosclerosis In Hypercholesterolemic Mice
Authors: Ye Dien , Liang Chingling , Howatt Deborah A , Moorleghen Jessica J , Danser Jan A , Daugherty Alan , Lu Hong
Published: May 2022

Objective:
Renin cleaving angiotensinogen to release angiotensin I is the rate-limiting step to produce angiotensin II. Although juxtaglomerular cells are the predominant source of renin in circulation, renin protein is also present in proximal tubules. In this study, we investigated the effects of renin in proximal tubule cells (PTC) on development of atherosclerosis in hypercholesterolemic mice

Approach and Result:
Inducible PTC-specific renin deficient (PTC-renin -/-) mice were developed by breeding renin floxed mice with mice expressing Cre under the control of the N-myc downstream regulated gene 1 (Ndrg1-Cre ERT2). All study mice were in a low-density lipoprotein receptor (LDLR) deficient background, and both male and female littermates were studied. Littermates of renin floxed mice with or without Ndrg1-Cre ERT2 transgene were injected intraperitoneally with tamoxifen (150 mg/kg/day) for 5 consecutive days when they were 4-6 weeks old. Cre genotypes were determined after weaning (~ 3-4 weeks old) and deletion of renin gene in kidney was verified by PCR of DNA extracted from kidney after termination (~18-20 weeks old). Two weeks after the last injection of tamoxifen, mice were fed a Western diet for 12 weeks, which led to plasma total cholesterol concentrations above 1000 mg/dl (P = 0.893 in male and P = 0.192 in female between PTC-renin +/+ and -/- mice). PTC-specific renin deficiency did not affect blood pressure (PTC-renin+/+ vs -/-: 110 ± 1 mmHg vs 112 ± 1 mmHg in male, P = 0.452; and 105 ± 3mmHg vs 106 ± 2 mmHg in female, P = 0.875), as measured by a tail-cuff system. Atherosclerosis in the ascending and aortic arch regions was measured with an
en face
method. No differences on percent lesion area between the two genotypes in both male and female mice (PTC-renin+/+ vs -/-: 18.1 ± 1.4% vs 18.9 ± 1.5% in male, P = 0.315; and 16.2 ± 1.5% vs 16.2 ± 1.2% in female, P = 0.648).

Conclusion:
Renal proximal tubule-specific renin deficiency does not affect blood pressure and atherosclerosis in hypercholesterolemic mice.
Abstract P147: Dietary Protein Elicits A Leucine-mediated Threshold Effect On Monocyte/macrophage Mtorc1-autophagy Signaling Resulting In Elevated Cardiovascular Risk
Authors: Zhang Xiangyu , Kapoor Divya , Jeong Se-jin , Stitham Jeremiah , Fappi Alan , Yousif Eman , Rodríguez-Vélez Astrid , Yeh Yu-sheng , Park Arick , Mittendorfer Bettina , Razani Babak
Published: May 2022

High-protein intake is common in Western societies and generally considered healthy. However, results from some epidemiological studies suggest elevated protein intake is associated with increased risk for ischemic cardiovascular diseases. In addition, results from studies conducted in mice show that a high protein, compared with a standard Western diet, increases atherosclerosis burden and lesion complexity. The adverse effect of protein ingestion on plaque biology in mice is mediated by amino acid-mammalian target of rapamycin (mTOR)-dependent inhibition of autophagy in macrophages. Here, we evaluate the effect of graded amounts of protein ingestion on this amino acid-mTORC1-autophagy mechanism in human monocytes/macrophages and identify leucine as the key amino acid responsible for activating mTORC1 in macrophages. We describe the presence of a threshold effect of high protein intake on this deleterious signaling pathway wherein protein content greater than about 22% of total energy, which is consumed by nearly 1/4
th
of the population in Western societies, acutely activates mTORC1 signaling in monocytes/macrophages. Furthermore, we identify leucine as the critical amino acid modulator and threshold indicator, capable of the dose-dependent mTORC1 activation and downstream functional effects. Finally, by designing specific mouse diets with protein contents mimicking graded levels of protein ingestion in our study participants, we demonstrate the presence of a dietary protein threshold effect in driving atherosclerosis in mouse models. These data demonstrate the potential deleterious impact of excessive protein intake on macrophages and atherosclerotic plaque progression.
Abstract P148: Angiotensin II Type 1a Receptor Deletion In Renal Proximal Tubular Cells Does Not Attenuate Atherosclerosis In Hypercholesterolemic Mice
Authors: Kukida Masayoshi , Ye Dien , Howatt Deborah , Katsumata Yuriko , Liang Ching L , Sawada Hisashi , Moorleghen Jessica J , Daugherty Alan , Lu Hong S
Published: May 2022

Objective:
AngII (Angiotensin II) acts through AT1aR (AngII type 1a receptor) to promote atherosclerosis. Despite the consistency that global inhibition of AT1aR markedly attenuates atherosclerosis, it remains undefined in which cell types that AT1aR contributes to atherosclerosis formation. Since renal AngII is associated with increased atherosclerosis in hypercholesterolemic mice, and AT1aR is abundant on PTCs (proximal tubular cells), the present study investigated the effects of AT1aR deletion in PTCs on atherosclerosis in hypercholesterolemic mice.

Approach and Results:
First, both male and female LDL receptor -/- mice were fed a Western diet and infused with either vehicle or losartan for 12 weeks. Losartan led to more profound increases of plasma renin concentrations and greater reduction of systolic blood pressure in female than in male mice, whereas atherosclerosis was attenuated by losartan equivalently in both sexes. Secondary, we determined whether the effects of losartan were attributed to inhibition of AT1aR on PTCs. To generate PTC-specific AT1aR -/- mice, AT1aR floxed mice were bred with mice expressing Cre under the control of the N-myc downstream regulated gene 1. These mice were injected with 150 mg/kg/day of tamoxifen for 5 days to activate Cre at the age of 4 - 6 weeks. RNAscope detected AT1aR mRNA in both PTCs and glomeruli; mRNA of AT1aR was absent in PTCs, but not glomeruli, of PTC-specific AT1aR -/- mice. Two weeks after completion of tamoxifen injection, these mice in LDL receptor -/- background were fed Western diet for 12 weeks. Deletion of AT1aR in PTCs did not affect plasma renin concentrations, blood pressure and atherosclerosis in either sex. Finally, to investigate the impacts of PCT-specific AT1aR deletion under the conditions with enhanced AngII stimulation, AngII was infused subcutaneously for 12 weeks. There were no differences in both blood pressure and atherosclerosis between genotypes.

Conclusion:
Although pharmacological inhibition of AT1aR reduced atherosclerosis profoundly in both sexes, PTC-specific AT1aR deletion did not affect atherosclerosis in hypercholesterolemic mice. Our future study will determine whether the presence of AT1aR in both PTCs and glomeruli are needed to promote atherosclerosis.
Abstract P157: Loss Of Nicotinamide Nucleotide Transhydrogenase Promotes Endothelial Activation In Atherosclerosis
Authors: Rao Shashanka , Gautier Nicole , Schilke Robert , Woolard Matthew D , Krzywanski David M
Published: May 2022

Heart disease is the leading cause of death in the United States and atherosclerosis accounts for nearly 75% of all deaths from heart disease. Atherosclerosis is a chronic inflammatory disease and mitochondrial reactive oxygen species (mtROS) are critical contributors to disease development. Nicotinamide nucleotide transhydrogenase (NNT) is a mitochondrial protein that maintains mitochondrial NADPH pools and supports the enzymatic degradation of mtROS. Preliminary data indicates that NNT expression is decreased in the endothelium of human atherosclerotic plaques and we sought to test the hypothesis that the loss of NNT increases mtROS and promotes atherosclerosis by enhancing endothelial and vascular dysfunction. Previous we have demonstrated that the loss of NNT was associated with increased vascular ROS production, plaque formation, and plaque size in response to treatment with PCSK9 and high fat diet (HFD). We now show that the loss of NNT in endothelial specific NNT knockout mice promotes enhanced endothelial VCAM-1 expression in response to HFD and disturbed flow. Similarly,
in vitro
, the loss of NNT promoted both VCAM-1 and ICAM-1 expression in response to oxLDL. Increased adhesion molecule expression associated with the loss of NNT also augmented monocyte adhesion in response to oxLDL consistent with vascular inflammation observed in early atherosclerosis. Additionally, macrophages isolated from NNT knockout mice display enhanced M1 polarization, decreased lipid uptake, and impaired lipid utilization. Co-treatment with the mitochondria targeted antioxidant MitoEbselen is able to normalize both endothelial and macrophage function, underscoring a critical role for mtROS in promoting an inflammatory phenotype in these cells. In both global and endothelial cell specific NNT knockout mice the loss of NNT was associated with increased necrotic core formation in response to PCSK9 treatment and 16 weeks of high fat diet. Taken together, these data indicate that decreased NNT expression promotes endothelial activation, increased inflammatory cell trafficking, macrophage metabolic dysfunction and M1 polarization that could contribute to an acceleration of necrotic core development and severe atherosclerotic disease.