bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 299: Impaired Inflammatory Angiogenesis in the Context of Aging is the Result of the Uncoupling of Macrophage Il-1beta-VEGF Expression and the Switch of VEGF Isoforms
Authors: Mantsounga Chris S , Sharma Sheila , Pierce Julia , Carley Rachel T , Mendez Roberto , Pineda Edy , Amelotte Elizabeth , Harrington Elizabeth O , Choudhary Gaurav , Morrison Alan R
Published: October 2023

Aging and age-related diseases like peripheral artery disease (PAD) is associated with impairedinflammatory angiogenesis responses to injury. Critical ischemic limb and wound injury animalmodels, have shown impact of inflammatory monocytes/macrophages as a major source ofproangiogenic mediators leading to new arterial growth and healing. In hind limb ischemia mousemodel of PAD, we have recently demonstrated the loss of macrophage IL-1β expression leads todecreased VEGF-A (total) and VEGF-A165a expression (proangiogenic isoform), while VEGF-A165b (inhibitory isoform) was increased, under the regulation of transcription factors SignalTransducer and Activator of Transcription 3 (STAT3) and Nuclear Factor Kappa B (NF-κB).Previous studies on aged mice have shown impaired of blood flow recovery and reduced VEGF-A expression. Moreover, recombinant VEGF-A administration did not fully overcome the footperfusion and prevent limb salvage.
The
mechanisms
of
reduced
VEGF-A
expression
in
aged
animals
remains
incomplete.
Our data from advanced aged (104-week-old) mice demonstratereduced blood flow recovery after excision of the femoral artery and reduced VEGF-A and VEGF-A165a in ischemic muscle tissue compared to young (12-week-old) while IL-1β and VEGF-A165bwere increased. Bone marrow-derived-macrophages (BMDMs) from 104-week-old alsodemonstrate decreased VEGF-A and VEGF-A165a expression. IL-1β andVEGF-A165b were foundincreased. Lastly, polarized BMDMs from 12-week-old demonstrate increased VEGFR2expression in inflammatory “M1” macrophages while VEGFR1 was increased in alternativelyactivated “M2" macrophages. Interestingly, 104-week-old demonstrate significantly increasedVEGFR1 expression while VEGFR2 was reduced compared to 12-week-old. We hypothesize thatthe regulation of IL-1β-VEGF-A axis and the type of VEGFR1/R2 involved in the signalingcomplex determine the fate of angiogenesis responses to injury in the context of advanced age.Our goals will be to define mechanisms whereby aging is associated with uncoupling of IL-1β andVEGF-A expression then, how VEGF-A isoforms and VEGFR1/2 signaling complex axis regulateinflammatory angiogenesis.
Abstract 316: A Study Of Outcomes And Complications In Patients With Gout And NSTEMI
Authors: Khalid Yaser , Dasu Kirti , Gabriel Nielsen , Lee Michael , Roy Trinava , Umrani Rohan , Dasu Neethi , Avuthu Sindhupavani
Published: October 2023

Background:
Patients with inflammatory conditions such as gout have an increased risk of atherosclerosis and cardiovascular disease. This study aimed to evaluate risk factors and outcomes of patients with gout who had an NSTEMI.

Methods:
We used the Nationwide Inpatient Sample (NIS) data to extract data utilizing ICD-10 codes for patients aged 18 and older, with gout and concomitant NSTEMI vs patients with only gout. Data were extracted from the year 2015 to 2019 and various parameters such as patient characteristics (age, gender, insurance, residence) and hospital characteristics were identified and analyzed utilizing multivariate analysis.

Results:
This study included 2,414,860 patients with gout of which 24,165 patients had a concomitant NSTEMI. Patients with gout who had an NSTEMI had higher inpatient mortality (OR 5.51 (CI 5.01-6.06), p=0.000), length of stay (LOS) (+1.85 (CI 1.69-2.02), p=0.000), total hospital charges (THC) ($37730.32, (CI $3,5433.54- $40,027.10), p=0.000). The secondary analysis is noted in table 1.

Conclusion:
Results from our study suggest that patients with gout who had an NSTEMI had worsened inpatient hospital outcomes with increased mortality, LOS, and THC. In this population, risk factors associated with increased LOS, THC, and mortality included Acute Kidney Injury (AKI), Cerebrovascular Accident (CVA), and cardiac arrest. Our study demonstrates the need to evaluate and screen patients with gout for cardiac complications such as NSTEMI and to further identify risk factors that lead to worsened in-hospital outcomes.
Abstract 323: Rspo2 Overexpression Attenuates Hepatic Lipid Accumulation And Atherosclerosis
Authors: Singla Bhupesh , Aithabathula Ravi V , Pervaiz Naveed , Ekuban Frederick , Kathuria Ishita , Gyamfi Maxwell
Published: October 2023

Aims:
We have recently reported elevated RSPO2 levels in human and murine atherosclerotic arteries and discovered its anti-lymphangiogenic potential. However, the precise role of RSPO2 in atherosclerosis is not known. In this study, we investigated the effects of RSPO2 overexpression on hepatosteatosis and atherosclerosis development.

Methods and Results:
Atheroprone ♂ and ♀
Apoe
-/-
mice were injected with AAV8-
GFP
or AAV8-
Rspo2
, fed a Western diet for 12 weeks, and analyzed for hepatic lipid accumulation and atherosclerotic lesion formation. Our quantitative RT-PCR data revealed increased
Rspo2
mRNA expression in the liver, kidney and spleen of AAV8-
Rspo2
-injected mice compared with control mice. Oil Red O (ORO) staining showed reduced hepatic lipid deposition in mice injected with AAV8-
Rspo2
compared with AAV8-
GFP
mice. Additionally, there were reduced levels of hepatic triglyceride, non-esterified fatty acid, and lipid peroxidation in
Rspo2
-overexpressing mice.
Rspo2
overexpression reduced hepatic fibrosis and macrophage accumulation as indicated by reduced expression of SMA and CD68 in liver tissue. Additionally, decreased mRNA expression of proinflammatory and cholesterol metabolism-related genes was found in liver of AAV8-
Rspo2
-injected mice.
En face
ORO staining of aortae and aortic root sections demonstrated suppressed atherosclerosis in mice with
Rspo2
overexpression. Interestingly, AAV8-
Rspo2
-injected mice had decreased weight gain over the course of Western diet feeding in comparison to control mice, however, no differences in fasting blood glucose and plasma total cholesterol levels were observed between the two groups.

Conclusions:
Taken together, these findings suggest that global
Rspo2
overexpression in hypercholesterolemic mice suppresses hepatic lipid accumulation and reduces atherosclerosis.
Abstract 326: Hyperhomocysteinemia Drives Smooth Muscle Cell Phenotypic Modulation And Atherosclerosis
Authors: Chattopadhyay Abhijnan , Majumder Suravi , Kwartler Callie , Milewicz Dianna M
Published: October 2023

Complex phenotypic modulation of vascular smooth muscle cells (SMCs) is a major driver of atherosclerosis. It is caused in part due to high levels of exogenous cholesterol activating the PERK arm of endoplasmic reticulum (ER) stress, which drives a component of SMC modulation and plaque formation. This is illustrated by the fact that SMC-specific deletion of
Perk
reduces plaque formation by up to 80% in male hypercholesterolemic (HC) mice. HC mice harboring the
ACTA2
p.R149C pathogenic variant (

Acta2
R149C/+
Apoe
-/-

) have 2.5-fold higher atherosclerotic plaque burden than

Apoe
-/-

mice despite no difference in serum lipid levels. Misfolding of mutant SM α-actin in SMCs activates heat shock factor 1 (HSF1), which upregulates HMG-CoA reductase (HMG-CoAR, coded by
Hmgcr
), thus increasing cholesterol biosynthesis and ER stress, activating PERK, and augmenting phenotypic modulation and plaque burden. However, it is not known if activation of the HSF1→HMG-CoAR→PERK signaling axis contributes to disease in patients with more common risk factors for atherosclerosis. Hyperhomocysteinemia (HHcy), defined as plasma homocysteine (Hcy) concentration >15μM, caused by genetic and lifestyle factors, is an independent risk factor for coronary artery disease and ischemic strokes. Wildtype SMCs were exposed to 0-500μM of Hcy in culture for 48 hours, which activated PERK signaling as indicated by increased expression and activation of its downstream targets
Atf4
and
Klf4
. Hcy treatment also caused a dose-dependent decrease in expression of contractile genes and increase in cellular migration, while expression of modulation-specific genes increased at higher concentrations of Hcy. Furthermore, exposure to Hcy increased expression and transcriptional activity of HSF1 in a dose-dependent manner. Finally, Hcy treatment caused dose-dependent upregulation of
Hmgcr
expression and augmented cellular cholesterol biosynthesis. Taken together, these data support the hypothesis that, similar to misfolding of the mutant SM α-actin, Hcy activation of the HSF1→HMG-CoAR→PERK pathway in SMCs leads to phenotypic modulation of SMCs and may contribute to the augmented atherosclerosis associated with HHcy.
Abstract 333: Endothelial RNA-binding Protein Elavl1 (HuR) Regulates Post-transcriptional Regulation Of Genes Involved In Immune Responses To Increase Cd8 T Cells In The Atherosclerotic Intima
Authors: Nicholas Sarah-Anne E , Hensel Jessica , Kimble Amy , Menoret Antoine , Jellison Evan R , Reese Bo , Zhou Beiyan , Rodriguez Annabelle , Vella Anthony , Murphy Patrick A
Published: October 2023

Under low and disturbed flow (LDF), endothelial cells (ECs) covering atherosclerotic lesions influence plaque composition by regulating immune cell recruitment and activation. Yet, how ECs regulate T cell responses key to plaque progression remains unclear. EC expression of the splice factor Elavl1 (HuR) is upregulated under LDF as a result of platelet recruitment. Bioinformatic analysis revealed Elavl1 motifs are enriched near LDF-regulated alternative exons. To test Elavl1 function
in vivo
, we generated
Cdh5(PAC)-CreERT2; Elavl1 ff
(EC-KO) mice and examined atherogenesis induced by AAV-PCSK9. Elavl1 EC-KO mice had reduced plaque-specific CD8 T cells relative to controls. Elavl1 EC-KO also resulted in fewer mice with severe plaque burden. We then used a partial carotid ligation (PCAL) model to examine CD8 T cell retention in the low flow ligated artery relative to the normal flow contralateral. We found a nearly complete inhibition of low-flow specific CD8 T cell retention in EC-KO mice relative to controls, and less occlusive plaque. An
in vitro
antigen presentation assay, using the defined antigen ovalbumin/SIINFEKL also suggested that T cell interactions with Elavl1 knockdown cells reduce T cell activation and proliferation. To investigate the underlying mechanism in the PCAL model, we examined both total RNA transcript, and ribosome bound transcript by EC-specific Translating Ribosome Affinity Purification (TRAP). We compared data from carotid endothelium under normal and atherogenic disturbed flow regimes in AAV-PCSK9 treated mice. These data revealed altered splicing and upregulation of transcripts associated with CD8 T cell activation in ECs of Elavl1 EC-KO mice under low flow. Together, our data suggest that platelets are important in the induction of Elavl1 in arterial ECs, which then alters the splicing and ribosomal association of a large set of transcripts important in CD8 T cell responses and accumulation.
Abstract 339: FFAR4 Regulates Macrophage Metabolism And Foam Cell Formation
Authors: Stuttgen Gage M , Chang Jackie , Chen Yiliang , Sahoo Daisy
Published: October 2023

Free fatty acid receptor 4 (FFAR4), also known as G-protein coupled receptor 120 (GPR120), is a long-chain unsaturated fatty acid receptor that is best known for its role in protecting from obesity and diabetes. Recent studies have demonstrated that activation of FFAR4 may play an important role in preventing atherosclerosis and cardiovascular disease (CVD). Preliminary data from our lab has shown that FFAR4 deficiency in mice increases macrophage foam cell formation, the hallmark of atherosclerosis. We hypothesize that FFAR4 regulates the inflammatory status of macrophages through mitochondrial metabolism to control foam cell formation. Using bone marrow-derived macrophages from wild-type (WT) C57/BL6N mice and FFAR4 knock-out (KO) mice, we first measured cellular bioenergetics via Seahorse assays and found that FFAR4 KO macrophages had altered mitochondrial metabolism as evidenced by a 20% reduction in the oxygen consumption rate during mitochondrial stress tests as compared to WT macrophages. Reduced oxidative phosphorylation was further supported by a 29% decrease in mitochondrial membrane potential in FFAR4 KO macrophages compared to WT, despite no observable differences in mitochondrial mass. Mitochondrial reactive oxygen species (ROS) were next measured using MitoNeoD probes to determine if the changes in metabolism were impacting the generation of free radicals. We found that FFAR4 KO macrophages had a 20% increase in mitochondrial ROS generation compared to WT macrophages. These changes in metabolism and ROS production were accompanied by 2-4 fold elevations in the production of proinflammatory cytokines IL-6, MCP-1, and TNFα at the RNA level and 15-50% increases in the secretion of the same cytokines into the culture media. Additionally, there was a 13% increase in the pro-inflammatory marker CD86 and a 20% reduction in the pro-resolving marker CD206 at the cell surface of the macrophages. Taken together, these studies demonstrate that FFAR4 might act as an immunometabolic switch to control macrophage foam cell formation through regulation of metabolism, inflammation, and immune cell activation. Our findings may identify FFAR4 as a new target for preventing foam cell formation and reducing CVD risk.
Abstract 354: Myocardial Infarction Accelerates Melanoma Tumor Growth And Metastasis
Authors: Von Itter Richard W , Newman Alexandra , Pilla Gabrielle , Bozal Fazli , Lim Brian , Aristizabal Orlando , Hernando Eva , Moore Kathryn J
Published: October 2023

Adult survivors of cancer are at increased risk of cardiovascular disease (CVD). Indeed, while CVD emerges as the second leading cause of death in recovering cancer patients, cross-disease communication remains poorly understood. We previously demonstrated that myocardial infarction (MI) accelerates breast cancer tumor growth and cancer-specific mortality in mice and humans. However, whether MI alters tumor growth of other cancer types and impacts metastasis is not known. To assess these gaps, we examined the effect of acute MI, induced by ligation of the left anterior descending (LAD) coronary artery, in syngeneic C57BL6 mouse models of primary and metastatic melanoma. Compared to sham surgery, LAD ligation resulted in a two-fold increase in tumor growth in both the B16 and YUMM melanoma models. Notably, tumor burden significantly correlated with the size of the infarct, suggesting a direct relationship between injury signal activation and disease progression. Analysis of the intratumoral immune cell landscape by flow cytometry revealed an enrichment of immunosuppressive CD4
+
FoxP3
+
T regulatory (Treg) cells after MI. Furthermore, histological analysis and magnetic resonance imaging revealed an increase in the number and size of metastatic foci in the liver after MI compared to sham. Our data suggest that MI dampens antitumor immunity and reorganizes the tumor microenvironment to exacerbate tumor progression in multiple cancer types. Collectively, these findings of deleterious cross-disease communication underscore the importance of implementing strategies that mitigate CVD risk in cancer patients.
Abstract 359: Multiscale Model Reveals Importance Of Cumulative Vascular Smooth Muscle Cell Responses In Aortic Hypercontractility
Authors: Flanary Shannon M , Jo Seokwon , Alejandro Emilyn , Barocas Victor
Published: October 2023

Diabetes more than doubles the risk of cardiovascular disease (CVD), and the chronic hyperglycemia leads to vascular maladaptations prior to the diagnostic threshold. While endothelium-independent vascular smooth muscle cell (VSMC) hypercontractility is well-established at the tissue-scale, the mechanical phenotype is inconsistently observed at the cell-scale. Our strategy consisted of experimental characterization at the tissue-scale via an isometric ring contraction assay of WT and OGT
KO
aortas, the latter an established chronic hyperglycemia model, from male mice and at the cell-scale via published cellular microbiaxial stretching data, coupled with the construction of a multiscale computational model. We hypothesize that the biochemical signaling changes due to increased blood glucose are sufficient to predict VSMC hypercontractility. The multiscale model operates off a biochemical signaling network that determines the activation and contraction of actin stress fibers. The stress fibers are connected in either 2D (cell-scale) or 3D (tissue-scale) cytoskeletal networks where for the tissue-scale, the cytoskeletal mechanics are fit to a macroscale constrained mixture model. The multiscale model correctly predicts the cell-scale euglycemic contractility, however the hyperglycemic condition is too subtle to be resolved at this scale. At the tissue-scale, the model correctly predicts both euglycemic and hyperglycemic active force generation. The model does not currently consider ECM alterations, so the changes in passive tissue mechanics cannot be predicted. The model predictions suggest that while the impetus of hyperglycemia-driven hypercontractility occurs at the cell-scale, these small-scale changes must be amplified across length scales to be experimentally-observable. These results demonstrate the power of the multiscale model to connect cell- and tissue-scale vascular mechanical phenotypes due to chronic hyperglycemia.
Abstract 378: Loss Of Nuclear Receptor Subfamily 4 Group A 1 (NR4A1) Results In Impaired Post-thrombotic Vein Wall Remodeling By Promoting A Dysregulated Antifibrinolytic Phenotype In Monocytes
Authors: Sharma Sriganesh B , Luke Catherine E , Cai Qing , Pellerito Mattea , Jaffer Farouc A , Obi Andrea T , Henke Peter K
Published: October 2023

Objectives:
Nuclear Receptor Subfamily 4 Group A1 (NR4A1) is a suppressor of monocyte/macrophage (MO/Mϕ) inflammatory output that antagonizes TGFβ-signaling. As NR4A1 inhibits epithelial-to-mesenchymal transition and tissue fibrosis, we sought to determine the role of NR4A1 in MO/Mϕ mediated post-thrombotic vein wall remodeling, which when exaggerated, can result in post-thrombotic syndrome (PTS).

Methods:
To assess the role of MO/Mϕ NR4A1 in post-thrombotic vein wall remodeling, we employed a knockout-rescue schema which featured whole mouse loss of function (
Nr4a1
-/-
) models and adoptive bone marrow derived MO/Mϕtransfer from
Nr4a1
replete mice (WT-BMDMs). Stasis venous thrombosis was induced by the inferior vena cava (IVC) ligation. Characterization of vein wall remodeling/endothelialization was performed using Masson Trichrome staining and CD31 immunostaining, respectively. Quantitation of mRNA and protein levels of factors critical for MO/Mϕ-mediated vein wall remodeling was performed using qRT-PCR and ELISA, and matrix metalloproteinase (MMP) activity was measured using zymography.

Results:
We observed that
Nr4a1
-/-
mice exhibited impaired vein wall remodeling after 8- and 14-days post IVC ligation as indicated by thicker vein wall measurements and impaired luminal endothelialization. Adoptive transfer of WT-BMDMs into
Nr4a1
-/-
mice rescued this phenotype (Fig. 1A-C).
Nr4a1
-/-
mice did not exhibit any differences in thrombus burden (data not shown). Finally, although we observed induced mRNA levels of
Mmp2
and
Mmp9
in the vein walls of
Nr4a1
-/-
mice that underwent adoptive transfer, we noted suppressed MMP2 enzyme activity despite no compensatory changes in MMP9 activity or TGFβ/IL6 levels (Fig. 1D, data not shown).

Conclusions:
NR4A1 loss results in impaired post-thrombotic vein wall remodeling and agonism of NR4A1 may be a potential therapeutic strategy to combat PTS.
Abstract 395: Interferon-induced Transmembrane Protein 3 (ifitm3) Mediates Platelet Functions During Inflammatory Stress
Authors: Banerjee Meenakshi , Rondina Matthew T
Published: October 2023

Background:
Thrombosis, precipitated by dysregulated platelet responses, contributes to adverse clinical outcomes, including organ failure and death during systemic inflammation (e.g., sepsis). Previously, our lab identified interferon-induced transmembrane protein 3 (IFITM3) as a mediator of anti-viral responses in platelets. However, whether IFITM3 mediates platelet functions and subsequent thrombosis during systemic inflammation is unknown.

Methods:
Cecal-ligation and Puncture (CLP) model of sepsis and Interferon alpha (IFNα)-induced sterile inflammation model was performed on WT and platelet- and MK-specific IFITM3 knockout strain (IFITM3
fl/fl
PF4-Cre
+/-
). P-Selectin and CD63 expression, Platelet Factor 4 (PF4) release, platelet-leukocyte aggregates, plasma cytokines (TNF-α, IL-6, CXCL1), as well as survival outcomes, platelet deposition and aggregates within organs (e.g. lungs), plasma aspartate aminotransferase, lactate dehydrogenase, D-Dimer levels were measured by flow cytometry, enzyme-linked immunosorbent assays (ELISAs), and immunohistochemistry.
In vivo
collagen-epinephrine-induced pulmonary embolism model, and
ex vivo
thrombus formation under flow using microfluidic devices were performed.

Results:
CLP sepsis or IFNα-induced sterile inflammation upregulated IFITM3 in platelets which were associated with significantly enhanced secretion of P-Selectin, PF4, and CD63, and increased mortality in a collagen-epinephrine-induced murine pulmonary embolism model. Biochemical assays demonstrated IFITM3 interacts with VAMP-8, the primary v-SNARE that drives platelet secretion. Platelets lacking IFITM3 showed reduced granule secretion and platelet-leukocyte aggregate formation. Sepsis-induced mortality, platelet-leukocyte aggregates, platelet deposition within lungs, and markers of organ failure and thrombosis (e.g., aspartate aminotransferase, lactate dehydrogenase, D-Dimer), and thrombus formation under flow were all significantly reduced in the absence of IFITM3 in CLP sepsis.

Conclusions:
Collectively, our findings suggest that IFITM3 is a novel regulator of secretion and aggregation in platelets and contributes to thrombotic events during systemic inflammation.
Abstract 408: A Novel PIP2-dependent Pathway Maintains Cellular Cholesterol Homeostasis
Authors: Khan Mariam , Prince Ashutosh , Neupane Kalash , Gulshan Kailash
Published: October 2023

Introduction:
ABCA1 acts as an anti-atherosclerotic protein by removing excess cholesterol from arterial foam cells via reverse cholesterol transport (RCT) pathway. ABCA1 mediated phosphatidylinositol 4, 5-bisphosphate (PIP2) flop (translocation of PIP2 from inner leaflet of plasma membrane to the cell-surface) promotes cholesterol efflux, indicating cross talk between cholesterol and PIP2 in maintaining lipid homeostasis. The mechanistic details of how cholesterol, PIP2, and ABCA1 are interlinked are not yet explored.

Objectives:
To determine the mechanism by which PIP2 maintains cellular cholesterol homeostasis.

Methods and Results:
To decipher link between PIP2, cholesterol, and ABCA1, we used macrophages (RAW264.7, BMDMs, and THP-1), HepG2, or PIP2 phosphatase (TMEM55b-/- KO) mice. For cholesterol loading, we used AcLDL (100 μg/ml for 24h) or cyclodextrin-cholesterol (50 μg/ml for 24 h). Cholesterol was depleted by cyclodextrin or simvastatin (2μM). PIP2 levels and localization were determined by Mass-ELISA and Nikon super-high resolution microscopy. Cholesterol loading increased PIP2 levels (~25-35% increase across various cell lines), while cholesterol depletion reduced PIP2 levels by ~30-40%. Cholesterol loading led to redistribution of PIP2 from plasma membrane to ER-PM junctions. Cholesterol loading reduced, while cholesterol depletion increased, TMEM55b levels. Expression of TMEM55b in BHK cells resulted in markedly reduced apoA1 binding, reduced ABCA1 levels, and resulted in ~ 70 % decrease in cholesterol efflux to apoA1 (N=4, P<0.0001). Importantly, expression of close homolog TMEM55a showed no effect on ABCA1 expression. Genetic or chemical ablation of PIP2 biosynthesis in THP-1 macrophages markedly reduced ABCA1 levels and cholesterol efflux. THP-1 macrophages supplemented with exogenous PIP2 showed increased ABCA1 expression.

Conclusion:
Cholesterol modulates PIP2 levels/localization, and PIP2 in-turn modulates ABCA1 expression to maintain cellular cholesterol homeostasis.
Abstract 409: Statin Dose Is Associated With Improved HDL Anti-inflammatory Capacity In Cardiac And Vascular Surgery Patients; Possible Mechanism For Statin Mediated Enhancement Of The Association Between Higher HDL And Less Kidney Injury After Surgery
Authors: Leuthner Zoe M , Yancey Patricia G , Linton Macrae F , Smith Loren E
Published: October 2023

Introduction:
Postoperative acute kidney injury (AKI) is associated with chronic kidney disease (CKD) development and advancement. A higher preoperative high-density lipoprotein (HDL) concentration is associated with less postoperative AKI. Statin exposure, especially long-term exposure, strengthens the association between higher HDL and less postoperative AKI. However, three randomized trials of perioperative statin treatment did not prevent AKI. In animal models, HDL prevents AKI primarily by suppressing renal endothelial intercellular adhesion molecule-1 (ICAM-1). We hypothesized that HDL from surgery patients on higher long-term statin doses would have improved capacity to suppress endothelial ICAM-1 expression. Further, we hypothesized that patients with higher
in vitro
HDL capacity to suppress ICAM-1 would have lower plasma sICAM-1 concentrations after surgery and less AKI.

Methods:
We recruited 100 adults undergoing major surgery on the heart or large blood vessels. ApoB-depleted serum was incubated with HUVEC cells for 30min. Then, 8 ng/mL TNF-α was added for 5hr. RT-PCR quantified HUVEC ICAM-1 mRNA. An ELISA assay measured sICAM-1 concentrations. AKI was defined by KDIGO serum creatinine-based criteria.

Results:
HDL-C did not correlate with HDL ICAM-1 suppression (Spearman’s R=0.05, p=0.64). Apolipoprotein A-I concentration did correlate with HDL ICAM-1 suppression (Spearman’s R= 0.29, p=0.004). Higher HDL capacities to suppress ICAM-1
in vitro
were correlated with lower plasma sICAM-1 (Spearman’s R= -0.30, p=0.003). After adjustment for AKI risk factors, higher HDL-C was not associated with AKI (OR 1.03 (95% CI 0.98-1.08), p=0.19). In contrast, a higher HDL capacity to suppress ICAM-1 was independently associated with lower odds of AKI (OR 0.88 (95% CI 0.80-0.98), p=0.016). Further, higher long-term statin dose was associated with higher HDL capacity to suppress ICAM-1 (p=0.045).

Conclusions:
Cardiac and vascular surgery patients with higher HDL capacity to suppress endothelial ICAM-1 have lower plasma sICAM concentrations after surgery and a lower risk of postoperative AKI. Higher long-term statin dose is associated with higher HDL endothelial cell ICAM-1 suppression capacity in these patients.
Abstract 412: Gestational Changes In Lipoprotein Assembly And Secretion In Human And Mouse Placenta
Authors: Nargis Titli , Lin Xinhua , Giordano Elena , Ijaz Laraib , Suhail Sarah , Gurzenda Ellen M , Kiefer Daniel , Quadro Loredana , Hanna Nazeeh , Hussain Mahmood M
Published: October 2023

In maternal circulation, apoB-containing low-density lipoproteins (LDL) and apoA1-containing high-density lipoproteins (HDL) transport triglyceride and cholesterol. The placenta synthesizes apoB-containing lipoproteins and apoA1. However, cells assembling these lipoproteins and the time course of induction of genes during pregnancy are unknown. We show that MTP, a critical protein for lipoprotein assembly, is located in the trophoblasts of human placenta. ApoA1 was mainly present in syncytiotrophoblasts. Our studies also show that human placental cultures synthesize apoB-containing LDL-size and apoA1-containg HDL-size particles. Similar lipoproteins were also present in fetal circulation. We observed significant increases in the expression of apoB and MTP from 2
nd
trimester to term human placentas, while apoA1 expression did not change. In contrast, expression of these three genes showed a gradual increase, with maximum expression occurring in late gestation in mice placentas. Thus, our studies provide new information about the timing of lipoprotein gene induction during gestation, cells involved in their assembly, and the gel filtration profiles of these lipoproteins. This information may be useful in identifying transcription factors regulating the induction of these genes in gestation and the importance of placental lipoprotein assembly in fetal development.
Abstract 422: DUSP2 Dysregulation By TSP1-CD47 Signaling May Contribute To Maladaptive Aortic Remodeling In Hypertension And Hypertensive Heart Disease Progression
Authors: Haines Zoe H , Sun Patrick , Whitley Guy S , Meijles Daniel , Khomtchouk Bohdan B
Published: October 2023

Hypertensive remodeling involves upregulation of fibrotic players, such as the thrombospondin-1 (TSP1). TSP1, via CD47, is anti-angiogenic; however, whether this involves inactivation of remodeling-linked MAPK cascades is unclear. Dual-specific phosphatases (DUSPs) negatively regulate MAPKs, evoking an added control of remodeling. Here we sought to investigate DUSPs regulated by CD47/hypertension signaling and explore any potential links between DUSP expression and cardiovascular phenotypes including dilated (DCM) vs hypertrophic (HCM) cardiomyopathy, and ejection fraction (EF).

Methods:
DUSPs were assessed in 1) endothelial cells (ECs) challenged with CD47-activating peptide 7N3 (10μM), and 2) hypertensive C57BL/6J male mice (10 wks) infused vehicle (acidified-PBS, n=10) or angiotensin II (AngII; 0.8 mg/kg/d; n=10). DUSP mRNA or protein expression was assessed by qPCR or immunoblotting, respectively. Statistical tests used a 1-way ANOVA with Holm-Sidak post-test. Human DUSP vs heart failure relationships were explored using a two-sample Mendelian Randomization (MR) framework and tissue-specific expression quantitative trait loci (eQTLs) from 5 cardiovascular tissues: left ventricle, atrial appendage, aorta, coronary artery, and tibial artery; comparing DUSP mRNA levels across DCM, HCM, and EF. Statistical significance is defined at a Bonferroni-corrected threshold of p<0.05.

Results:
Infusion with Angll increased cardiac and vessel TSP1 levels, but not CD47. In ECs, 7N3 activated ERK and p38 MAPKs, with induction of DUSP1/4/5/6 (all >2-fold; p<0.01). In contrast, CD47 signaling decreased nuclear ERK-regulating DUSP2 levels (p<0.05). Interestingly, the EC DUSP2 response was absent in cardiac tissue homogenates, where DUSP2 was elevated at both the RNA (2.3±0.5-fold, p<0.0001) and protein (3.1±0.4-fold, p<0.001) levels. MR identified that elevated DUSP2 expression in the aorta associates with increased EF and decreased risk of HCM. There are no signals significant (p<0.05/28) in DCM.

Conclusion:
DUSPs fine-tune ERK1/2 signaling in ECs which contrasts the cardiac response. Computational evidence suggests DUSP2 causes a net downstream response that increases EF and is protective of adverse remodeling preceding HCM.
Abstract 427: Epigenetic Regulation Of Endothelial Anoctamin1 In Pulmonary Arterial Hypertension
Authors: D'Silva Natalie , Wang Eric , Vang Alexander , Yano Naohiro , Zimmer Alexsandra , fedulov alexey , Zhang Peng , Choudhary Gaurav
Published: October 2023

Introduction/Hypothesis:
Hyperproliferative endothelial cells (ECs) play an important role in the pathogenesis of pulmonary arterial hypertension (PAH), by formation of occlusive plexiform lesions. We previously reported that Anoctamin-1 (ANO1), a calcium-activated chloride channel, expression is increased in lung ECs from patients with PAH and is associated with increased EC proliferation. However, the regulatory mechanism underlying ANO1 expression in PAH remains unclear. DNA methylation has been recognized as an important mechanism regulating gene expression. In this study, we test the hypothesis that ANO1 expression in PAH is epigenetically regulated via DNA methylation.

Methods/Results:
DNA was extracted from lung ECs of patients with idiopathic PAH (n=7) and controls (n=6), followed by Bisulfite Next-Generation Sequencing (EpigenDx, MA) to determine DNA methylation profile in human ANO1 gene (Ensembl Gene ID: ENSG00000131620). We used 19 assays to cover 97 CpG sites in the human ANO1 gene, including regions of 5’-upstream, 5’-UTR, and different introns. Our data show that the methylation of 29 of 97 (30%) CpG sites in human ANO1 gene is significantly altered in lung ECs from PAH patients than that from control subjects. Among the significantly altered 29 CpG sites, the methylation at 22 CpG sites (76%) is significantly decreased. We developed a CRISPR-based approach to target four differentially methylated CpG sites to determine the specific contribution of their methylation in regulation of human ANO1 gene. We demonstrate that demethylation of one of the sites, CpG#-73, in human embryonic kidney cells resulted in an increase in ANO1 mRNA expression compared to control, suggesting that decreased DNA methylation is sufficient to upregulate human ANO1 expression.

Conclusions:
Our results demonstrate that DNA methylation in human ANO1 gene is significantly altered in PAH and reduction in DNA methylation may serve as a regulatory mechanism for ANO1 upregulation in settings of PAH.
Abstract 430: Epigenome Editing Of The
AGT
Gene As A Therapeutic Approach For Cardiovascular Disorders
Authors: Testa Lauren C , Musunuru Kiran , Wang Xiao
Published: October 2023

Overactivation of the renin-angiotensin system contributes to many common and rare cardiovascular diseases such as hypertension, atherosclerosis, and genetic thoracic aortopathies. Current pharmacologic therapies that target this pathway have been proven safe and effective, yet they require individuals with these conditions to adhere to daily medications. CRISPR-Cas9-mediated hepatic inactivation of the angiotensinogen (
AGT
) gene, the precursor of all angiotensin peptides, could be a promising “one and done” therapeutic approach for these disorders. However, disruption of
AGT
by Cas9 nuclease would result in irreversible changes to the genome and would likely have negative consequences if targeted to organs outside of the liver. CRISPRoff is a CRISPR-based epigenome editing tool consisting of catalytically inactive Cas9 fused to a DNA methyltransferase, as well as a guide RNA (gRNA) designed to target a genomic promoter of interest. Targeting to a gene promoter can result in DNA methylation and subsequent silencing of gene expression, a phenomenon that has the potential to be reversed by CRISPRon, a catalytically inactive Cas9 fused to a demethylase that could be targeted to the same site using the same gRNA. We hypothesized that CRISPRoff can be used to efficiently and durably silence
AGT
expression by methylation of the
AGT
promoter. To test this, we screened HuH-7 hepatoma cells with CRISPRoff and either individual gRNAs or dual combinations of gRNAs targeting the
AGT
promoter. We measured
AGT
mRNA expression by RT-qPCR and selected the two combinations of gRNAs that resulted in the greatest decrease in
AGT
expression for methylation analysis. We found that
AGT
expression was decreased by ≈50% in treated cells, and this correlated with substantially increased methylation throughout the
AGT
promoter. A long-term in vitro study to assess the durability of this methylation is underway. We are also generating a humanized
AGT
mouse model to test whether these CRISPRoff gRNAs can silence the human
AGT
gene in vivo. These results provide a proof of concept of CRISPRoff for silencing of
AGT
expression as a therapeutic approach for common and rare cardiovascular disorders.
Abstract 436: Platelet Depletion Alters Monocyte Recruitment And Phenotype Of Plaques In Diet-induced Obesity And Glucose Intolerance On Atherosclerosis Regression
Authors: Laskou Maria , Weinstock Ada , Kireccibasi Ozlem , Scolaro Bianca , Nikain Carmen , Fisher Edward
Published: October 2023

Despite decades of research, treatment of cardiovascular disease in diabetic patients remains suboptimal. Patients with type 2 diabetes (T2D) are characterized by hyperglycemia and often obesity, and many develop atherosclerotic cardiovascular disease (ASCVD) at a greater rate compared to patients without diabetes. Activated platelets are known to be present in obese and diabetic individuals. Also, they promote monocyte recruitment to atherosclerotic plaques which is crucial to ASCVD progression. However, there is a lack of knowledge regarding platelets' contribution on atherosclerosis regression in T2D and obesity. In this study we assessed the
hypothesis
that platelet activation by diet-induced obesity and impaired glucose tolerance is detrimental to atherosclerosis regression. LDLR-/- mice fed a high fat-high cholesterol diet for 20 weeks became obese, glucose intolerant and hypercholesterolemic. Atherosclerosis regression was achieved by injecting apolipoprotein B antisense oligonucleotides for 3 weeks to normalize lipid levels. Additionally, mice received either platelet depletion or control antibody treatment every 3 days. We observed that after lipid lowering, plasma cholesterol levels were normalized, but mice remained glucose intolerant. Interestingly, platelet depletion (n=17, platelet count=804.5±45.58 vs 61.41±7.492 x 10
3
/μl) in regressed normocholesterolemic mice led to decreased monocyte recruitment (n=9-10, 12.7±3.472 vs 5.4±0.9 beads/leaflet, p value=0.0488) and macrophage (CD68+) area (n=5, CD68+ area=0.0695±0.0178 vs 0.0363±0.0034 mm
2
, p value=0.1041) in atherosclerotic plaques, compared to control injected mice. In conclusion, these data indicate that platelets play an important role in the recruitment of monocytes into plaques not only in atherosclerosis progression, but also during regression, and have a direct impact on plaque phenotype by altering CD68+ area. In-depth investigation of the plaque leukocyte population, by single cell RNA-seq, will give us a better understanding of platelets’ role in the macrophage inflammatory phenotype in regressing atherosclerotic plaques and help us identify platelet-related factors that can be targeted in diabetic and obese individuals with ASCVD.
Abstract 448: Role Of Pcpe2 In Adipose Tissue Remodeling And Lipoprotein Metabolism
Authors: Thomas Michael J , Xu Hao , Sorci Thomas Mary G
Published: October 2023

Increased prevalence of obesity in the US continues to drive greater risk for developing type 2 diabetes and cardiovascular disease. Excessive caloric intake stresses adipocytes into storing lipid in an unhealthy manner, directing lipid into existing adipocytes, or adipocyte hypertrophy. Healthy adipose tissue expansion and remodeling occurs when adipose tissue precursor cells (PC) in the stromal vascular fraction (SVF) differentiate to create new pre-adipocytes which store lipid, called hyperplasia. Single cell sequencing of visceral adipose tissue (VAT) have identified two main types of PCs in the SVF, one called adipocyte precursor cells (APCs), while the other is a fibro-inflammatory progenitors (FIPs). APC are pre-adipocytes which given the proper signals, differentiate into mature adipocytes, while FIPs, can secrete cytokines inhibiting APC differentiation and initiating inflammation. Also influencing APC and FIP lineage in the SVF are the transforming growth factor beta (TGFb)-like signaling pathways which inhibit adipogenesis, while bone morphogenetic protein (BMP)-like signaling promotes adipogenesis. Little is known about how signaling through these pathways alters APCs and FIPs fate, especially during increased lipoprotein flux from high fat diet (HFD). Recently we discovered that both VAT PCs, APCs and FIPs express high levels of the extracellular matrix (ECM) protein, procollagen endopeptidase enhancer protein 2 (Pcpe2
)
whose expression decreases as APCs become committed adipocytes. We found that Pcpe2 is significantly up-regulated ~4-fold by TGFb1 suggesting that Pcpe2 plays a role in TGFb-like pathways in VAT PCs. Using PC-specific Pcpe2-hemagglutinin tagged (HA) overexpressor (ox) (PC-Pcpe
oxHA
) and PC cell-specific Pcpe2 knockout (KO) (PC-Pcpe2
KO
) mice lines, studies show PC-Pcpe2
KO
mice have reduced (~35-40%) body and VAT weight, while PC-Pcpe
oxHA
mice showed an increase (~40-45%) in body and VAT weight, characterized by hypertrophic adipocytes. In other studies we show APC rates of proliferation and differentiation are highly dependent on Pcpe2 expression. Overall, these studies reveal novel mechanistic insight into the mechanisms involved in dysfunctional fat storage.
Abstract 451: Biliverdin Reductase B Is A Plasma Biomarker For Intraplaque Hemorrhage And A Predictor Of Ischemic Stroke In Symptomatic Carotid Stenosis
Authors: Chemaly Melody , Marlevi David , Iglesias Mareque Maria Jesus , Lengquist Mariette , Kronqvist Malin , Bos Daniel , van Dam - Nolen Dianne H.K , van der Kolk Anja , Hendrikse Jeroen , Kassem Mohamed , Matic Ljubica , Odeberg Jacob , De Vries Margreet R , Kooi M E , Hedin Ulf
Published: October 2023

Background:
Intraplaque hemorrhage (IPH) is a hallmark of atherosclerotic plaque instability. Biliverdin reductase B (BLVRB) is enriched in plasma and plaques from patients with symptomatic carotid atherosclerosis and functionally associated with plaque IPH.

Objective:
We explored the biomarker potential of plasma BLVRB through 1) its correlation with IPH in carotid plaques assessed by magnetic resonance imaging (MRI) and with recurrent ischemic stroke; and 2) its use for monitoring pharmacotherapy targeting IPH in a preclinical setting.

Methods:
Plasma BLVRB levels were measured in symptomatic patients from the PARISK study (n=177, 5-year follow-up) with and without IPH upon MRI quantification. Plasma BLVRB levels were also measured in a mouse vein graft model of IPH at baseline and following anti-angiogenic therapy targeting vascular endothelial growth factor receptor 2 (VEGFR-2).

Results:
Plasma BLVRB levels were significantly higher in patients with IPH (737.32 ± 693.21 vs 520.94 ± 499.43 mean fluorescent intensity (MFI), p=0.033), with no association to baseline clinical and biological parameters. Baseline plasma BLVRB levels were significantly higher in patients who developed recurrent ischemic stroke (1099.34 ± 928.49 vs 582.07 ± 545.34 MFI, HR = 1.600, CI [1.092- 2.344]; p=0.016). Plasma BLVRB levels were significantly reduced following prevention of IPH by anti-VEGFR-2 therapy in mouse vein grafts (1189 ± 258.73 vs 1752 ± 366.84 MFI; p<0.004).

Conclusions:
Plasma BLVRB is a viable biomarker for IPH based on its 1) association with carotid plaque IPH, 2) higher levels in patients with recurrent ischemic stroke, and 3) capacity to monitor the efficacy of pharmacotherapy aimed for IPH reduction. This gives strong evidence to the utility of plasma BLVRB as a future biomarker for plaque instability.
Abstract 453: Adiponectin Increases AdipoR1/R2 Cell Surface Expression And M1-M2 Macrophage Subpopulations In AdipoR Knockdown Conditions
Authors: Gianopoulos Ioanna , Daskalopoulou Styliani S
Published: October 2023

Introduction:
Adiponectin is an adipose-tissue secreted hormone that exerts its anti-inflammatory and anti-atherogenic functions through its two main receptors, adiponectin receptor 1 (AdipoR1) and AdipoR2, which are highly expressed in monocyte-derived macrophages (Mϕs). In response to various stimuli, Mϕs can acquire a more pro-inflammatory M1, or anti-inflammatory M2 phenotype. Herein, we investigated the effect of single AdipoR1, single AdipoR2 and double AdipoR1/R2 knockdown conditions in response to adiponectin on M1 and M2 Mϕ subpopulations.

Methods:
siRNA-mediated knockdown of AdipoR1 and/or AdipoR2 was performed in THP-1 monocyte-derived Mϕs, and treated in the presence or absence of adiponectin. Flow cytometry was performed to assess AdipoR1/R2, M1 Mϕ subpopulation (CD86/CD80), and M2 Mϕ subpopulation (CD206/CD163). Magnetic bead-based immunoassays were performed on cell culture supernatants to assess 12 pro-inflammatory (TNF-α, MCP-1, IL-1β, IL-6, IL-8, IL-12p40, IL-12p70) and anti-inflammatory (IL-1Rα, IL-4, IL-10, TGF-β1 and CCL17) cytokines and chemokines.

Results:
In response to adiponectin, flow cytometry analyses for single AdipoR1, single AdipoR2, and double AdipoR1/R2 siRNA resulted in a significant increase in double-positive AdipoR1/R2 Mϕs. Although adiponectin treatment of single AdipoR1, single AdipoR2 and double AdipoR1/R2 siRNA significantly increased both Mϕ subpopulations, this increase was more prominent in M2 Mϕ subpopulation. Immunoassay analyses in knockdown conditions demonstrated a mixed pro- and anti-inflammatory cytokine and chemokine profile in response to adiponectin; levels of TNF-α, MCP-1 were increased and IL-1β decreased, while TGF-β1 and CCL17 were decreased.

Conclusions:
Adiponectin treatment increases the amount of AdipoR1/R2 cell surface expression on Mϕs without being limited by intracellular AdipoR knockdown conditions. Furthermore, although adiponectin promoted both Mϕ subpopulations, a greater shift towards M2 Mϕs was noted, while producing a heterogeneous pro- and anti-inflammatory cytokine and chemokine profile. Our current work will unravel the underlying mechanisms of the role of the adiponectin-AdipoR pathway in regulating M1-M2 Mϕ phenotypes.
Abstract 460: Catalase Overexpression Modulates Plaque Fibrosis And Vascular Contractile Function In Aged Hyperlipidemic Mice
Authors: Rabib Ali , Roos Carolyn M , Hagler Michael A , Zhang Bin , Smith Leslie A , Rababa'h Abeer M , Miller Jordan D
Published: October 2023

Background:
Plaque fibrosis and re-differentiation of vascular smooth muscle cells (VSMCs) from a contractile to a “synthetic” phenotype are hallmarks of advanced atherosclerosis. While
in vitro
studies demonstrated a clear role of oxidative stress in the loss of VSMC contractile phenotypes, the roles of specific antioxidant mechanisms and their therapeutic utility remain unclear. Here we test the hypothesis that overexpression of catalase protects against excess plaque fibrosis and losses in contractile phenotypes
in vivo
.

Methods:
We used littermate-matched, hypercholesteremic (ldlr
-/-
/ApoB
100/100
; LA) mice lacking a transgenic construct (LA-CAT
0/0
) or harboring a single copy of the human catalase gene (LA-CAT
Tg/0
), which were fed a Western Diet for 3 or 12 months. Isolated organ bath chambers, histopathological analyses, and quantitative RT-PCR were used to assess vasomotor function, structure, and molecular changes in aorta.

Results:
Oxidative stress increased with disease progression in aortic tissue from LA-CAT
0/0
mice and was modestly reduced in LA-CAT
Tg/0
mice. Collagen deposition and fiber thickness increased with disease progression in LA-CAT
0/0
mice, and these increases in fiber thickness were attenuated in LA-CAT
Tg/0
mice at 12 months and more pronounced in male mice. In parallel, we observed favorable rightward shifts in the vascular stress-strain relationship in LA-CAT
Tg/0
mice that were more pronounced in aged male mice. Interestingly, these structural changes were associated with improved contractile responses to either PGF
2α
or serotonin in male LA-CAT
Tg/0
mice at 12 months. Consistent with these changes, the contractile marker calponin-1 was profoundly reduced with increasing age in LA-CAT
0/0
mice, with these changes being partially attenuated in aging, male LA-CAT
Tg/0
mice.
In vitro,
treatment of VSMCs with PEG-catalase attenuated BMP2-dependent contractile protein expression.

Conclusion:
These results demonstrate that moderate overexpression of catalase can attenuate deleterious fibrotic and contractile changes associated with prolonged progression of atherosclerosis. Future mechanistic investigation exploring the context-dependence of these changes (e.g., sex dependence) is warranted.