bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 196: Poly (rC)-binding Protein-1 (Pcbp1) Is Essential For Vascular Development
Authors: Lu Yao Wei , Zhu Bo , Cowan Douglas B , Wang Da-Zhi , Chen Hong
Published: November 2022

The formation of the heart and the connecting vessels is essential for the function of the vertebrates, as vascular deficiency during embryonic development often leads to embryonic lethality. The vasculature forms as a branching network of endothelial cells (ECs) that becomes specified and assembles into vessels. the formation of new blood vessels from the existing vasculatures, or angiogenesis, is fundamental during development and pathological processes. Critical factors in governing vascular development are often required for organismal survival. Hence, gaining a complete understanding of the developmental process requires unique approaches to study tissue or cell-type specific functions. Poly(rC)-Binding Protein 1 (Pcbp1) is an evolutionarily conserved RNA binding protein that is not well understood
in vivo
. Pcbp1 is abundantly expressed in the angiogenic endothelium. However, little is known about the function and mechanism of Pcbp1 in the context of angiogenesis and vascular development. Germline deletion of Pcbp1 results in peri-implantation lethality, which limits its potential for studying the role of Pcbp1 in development. To address this deficit, we had generated an inducible endothelial deletion of Pcbp1 with Cdh5-Cre
ERT2
(Pcbp1-ieKO) to study its role in vascular development. When the EC deletion was induced at embryonic day (E) 9.5, Pcbp1-ieKO exhibited inadequate angiogenesis in the hindbrain at E12.5. Furthermore, when the EC deletion was induced at postnatal day (P) 1, Pcbp1-ieKO displayed reduced coronary and brain vascular density, as well as blunted retinal angiogenesis at P6. Single-cell RNA sequencing of Pcbp1-ieKO retina at P6 revealed a significant reduction in EC population and expression of genes critical for angiogenesis downstream of VEGF signaling and NOTCH activation. Notably, knockdown of Pcbp1 in human colony-forming endothelial cells (hCFEC) blunts the VEGFR2 and NOTCH activation upon VEGFA treatment. Our preliminary results suggest Pcbp1 is a critical regulator for angiogenesis, future studies will be directed at identifying the mechanism of Pcbp1 in regulating VEGF and NOTCH signaling, ultimately to provide insights for novel therapeutic strategies to control angiogenesis in cardiovascular disease.
Abstract 205: Macrophage Rac1-IL-1β Signaling Axis Promotes Atherosclerotic Calcification
Authors: Lee Cadence , Carley Rachel T , Mantsounga Chris S , Sharma Sheila , Neverson Jade , Butler Celia , Choudhary Gaurav , Morrison Alan R
Published: November 2022

Coronary artery disease caused by atherosclerosis is a major cause of morbidity and mortality worldwide. Calcification of atherosclerotic plaque is a marker of atherosclerotic burden and worsening cardiovascular outcomes. Inflammation plays a role in both atherosclerotic progression and calcification, but the mechanisms of calcification are not entirely well-defined. We sought to understand the role of macrophage Rac1 signaling in the natural progression of atherosclerotic calcification in the context of an experimental model of hyperlipidemia. We developed a tamoxifen-inducible, myeloid specific
Rac1
-deletion model (

Csf1r
mercremer
Rac1
fl/fl
ApoE
-/-

) to evaluate the impact of macrophage-specific Rac1 on atherosclerotic calcification. After 20 weeks of
ad libitum
feeding on a cholesterol-supplemented high fat diet,
Rac1
-deleted animals demonstrated significantly decreased atherosclerotic calcification compared to wild-type mice, irrespective of modest changes in lipid metabolism as demonstrated by plasma analysis. Mechanistically,
Rac1
-deleted bone marrow derived macrophages demonstrated reduced expression of the potent inflammatory cytokine, IL-1β, in response to a cholesterol-based inflammasome activation.
Rac1
-deleted macrophages also demonstrated decreased nuclear NF-κB activity and lost binding of NF-κB to its seed site in the IL-1β promoter as evaluated by chromatin immunoprecipitation. We further demonstrated that Rac1 and NF-κB form a complex with each other upon innate inflammatory signaling activation using a proximity ligation assay. We confirmed the important functional role of the Rac1 nuclear localization sequence in the expression of IL-1β, which together with the proximity ligation assay, suggests a role for Rac1 in chaperoning NF-κB to the IL-1β promoter. The interaction between macrophage Rac1 and NF-κB may have implications on numerous inflammatory disease processes, and uncovers a novel signaling contribution of Rac1 as a transcriptional co-factor for IL-1β in the progression of atherosclerosis. Targeted inhibition of Rac1 may have potential as a therapeutic treatment to mitigate progression of vascular calcification.
Abstract 208: The Chromatin Remodeling Protein Brg1 Regulates Adventitial Progenitor Cell Myofibroblast Differentiation And Pathological Vascular Remodeling
Authors: Jolly Austin , Lu Sizhao , Dubner Allison , Mutryn Marie , Navarro Natalie , Nemenoff Raphael , Weiser-Evans Mary C
Published: November 2022

Cardiovascular diseases lead to long-term stiffening of arteries and small vessels. The vascular wall contains several cellular populations that coordinate to maintain vessel homeostasis and induce vascular remodeling in disease states. We identified a population of smooth muscle-derived progenitor cells that reside in the adventitial layer (AdvSca1-SM cells) that drive vascular fibrosis after acute vascular injury. AdvSca1-SM cells can differentiate into myofibroblasts and secrete numerous pro-remodeling factors including extracellular matrix proteins and pro-inflammatory cytokines. While the response of AdvSca1-SM cells to vascular injury has been studied, the epigenetic changes that influence their differentiation towards myofibroblasts is poorly understood. We observed Brahma-related gene 1 (Brg1), a highly conserved chromatin remodeling ATPase, is upregulated in AdvSca1-SM cells in response to vascular injury and may be a key factor in regulating gene expression to influence AdvSca1-SM differentiation. We hypothesize that Brg1 induces expression of gene sets associated with remodeling and fibrosis and ultimately drives AdvSca1-SM cell differentiation towards pathologic myofibroblasts.

Results:
Carotid arteries from mice subject to carotid ligation exhibit decreased perivascular collagen deposition, smaller neointima, decreased adventitial expansion, decreased numbers of alpha-actin positive AdvSca1-SM cells, and fewer infiltrating macrophages when treated with the pharmacological Brg1 bromodomain inhibitor PFI-3 as compared to mice that received vehicle control. In a cell culture system, TGF-β induces AdvSca1-SM cells to express myofibroblast-specific genes, including alpha actin and periostin, and demonstrate enhanced contractile function in collagen contraction assays. PFI-3 blocks TGF-β induced myofibroblast gene expression and dampens contractile function. Gene Ontology analysis reveals the top 100 TGF-β inducible genes blunted by PFI-3 are related to fibrosis and extracellular matrix signaling. In conclusion, these results suggest Brg1 is a major regulator of vascular remodeling and fibrosis and may be a targetable protein to help people who suffer from disease-related vascular stiffening.
Abstract 216: Single-cell And Spatially Resolved Transcriptome Analysis Reveals Cellular Heterogeneities And Novel Regulators Of Atherosclerotic Plaque Destabilization
Authors: Pauli Jessica , Wu Zhiyuan , Yokota Chika , Winski Greg , Paloschi Valentina , Dueck Anne , Engelhardt Stefan , Eckstein Hans-Henning , Reilly Muredach P , Maegdefessel Lars
Published: November 2022

Background:
Cardiovascular diseases, including atherosclerosis, are the major cause of death in western societies, still molecular mechanisms of plaque destabilization remain unclear. Long non-coding RNAs (lncRNAs) are one example of novel molecular modulators, as their expression is highly cell-type specific.

Methods:
We utilized combined total (bulk) RNA and single cell (sc) RNA to study the transcriptome of advanced carotid artery lesions from patients undergoing carotid endarterectomy in our vascular surgery clinic. Additionally, we performed hybridization-based RNA
in situ
sequencing (HybRISS) to indicate where cluster-defining genes are located within the plaques.

Results:
In this current study, four sequencing datasets were investigated (total RNA from early vs. late lesions from the same individual patient and unstable vs. stable lesions from individual patients; two separate scRNA-seq datasets).16 lncRNAs were cross-referenced between all four datasets. All of these lncRNAs presented a cell-type specific expression pattern, with 11 lncRNAs being significantly enriched in different smooth muscle cell (SMC) clusters. We found all newly identified lncRNAs conserved in our scRNA-seq datasets of genetically mutated (
LDLR-/-
) Yucatan mini-pigs and the inducible carotid artery plaque rupture mice (
ApoE-/-
). The cluster-defining genes from the human scRNA-seq data were then located in human carotid artery tissue sections using the HybRISS method to unravel their distinct location within these plaques.

Discussion:
Taken together, our datasets, methods and different animal-models demonstrate that combining bulk with scRNA-seq data and spatially resolved sequencing methods are powerful tools to identify and characterize novel lncRNAs being expressed by a certain cell-type in the disease progression.
Abstract 241: Nicotinamide Nucleotide Transhydrogenase Regulates Mitochondrial Function And Oxldl Induced Macrophage Endothelial Interaction.
Authors: Rao Shashanka , Hall Nicole , Woolard Matthew , Orr Wayne W , Krzywanski David M
Published: November 2022

Atherosclerosis is a chronic inflammatory disease effecting large and medium sized arteries that leads to the buildup of plaque that contributes to myocardial infraction, stroke, and other pathologies. Mounting evidence has identified mitochondrial reactive oxygen species (ROS) as a key contributor to plaque formation and progression. A inner mitochondrial membrane enzyme Nicotinamide nucleotide transhydrogenase (NNT) sustains NADPH pools required for the mitochondrial antioxidant systems. We have observed that the expression of NNT is decreased in severe human atherosclerotic plaques leading us to hypothesize that the loss of NNT contributes to increased mitochondrial ROS and drives the progression of atherosclerosis by enhancing endothelial and vascular dysfunction. Utilizing AAV/PCSK9 and HFD induced models of early and late-stage atherosclerosis, we found that NNT knockout (NNT KO) mice display significantly increased VCAM-1 expression in aortic endothelial cells during early atherosclerosis and significantly larger necrotic cores in late-stage atherosclerosis. Additionally, NNT KO mice displayed a significantly thinner fibrous cap in late-stage plaques. Similar results were observed
in vitro
where the loss of NNT in human aortic endothelial cells exacerbated oxLDL induced VCAM-1 expression that was associated with decreased mitochondrial NADPH levels, glutathione peroxidase (Gpx2) activity, and increased H
2
O
2
production. The observed increase in necrotic core area in NNT KO mice is suggestive of increased inflammatory cell recruitment to the plaque. Furthermore, bone marrow derived macrophages (BMDM) from NNT KO mice display decreased lipid content, poor lipid uptake and a predisposition to differentiate into a proinflammatory M1 phenotype. Based on these studies, we conclude that the loss of NNT contributes to unstable plaques with large necrotic cores, mitochondrial ROS driven endothelial dysfunction, increased inflammatory cell recruitment, and enhanced M1 macrophage activity. The novel relationship between vascular NNT expression and plaque severity in human samples implicates NNT as a novel clinical target in atherosclerosis.
Abstract 269: The Histone Methyl Transferase (SUV39H1) Promotes Smooth Muscle Cell Dedifferentiation
Authors: Chatterjee Payel , Chakraborty Raja , Xie Yi , Sizer Ashley , Hwa John , Martin Kathleen A
Published: November 2022

The phenotypic plasticity of vascular smooth muscle cells (VSMCs) is central to growth and remodeling processes, but also contributes to the pathology of atherosclerosis, restenosis, and other cardiovascular diseases. This ability of VSMCs to reversibly differentiate and de-differentiate is incompletely understood. SUV39H1, a histone methyltransferase, specifically trimethylates Lys-9 of histone H3 (H3K9me3), resulting in epigenetic transcriptional repression. We hypothesized that SUV39H1 plays a role in VSMC phenotypic switching. Using knockdown, qPCR, western blot, chromatin immunoprecipitation (ChIP), RNA-Seq, and murine vascular injury to determine the role of SUV39H1 in VSMC plasticity. A qPCR array screen of epigenetic regulators revealed that SUV39H1 is upregulated with PDGF-induced dedifferentiation but downregulated with rapamycin-induced differentiation in hCASMCs. SUV39H1 knockdown promoted differentiation measured by increased contractile gene, protein expression, enhanced contractility, decreased migration, proliferation, and de-differentiation-associated gene expression. RNA-seq trasncriptomics confirmed changes in multiple pathways consistent with a role for SUV39H1 in promoting hCASMC dedifferentiation. Mechanistically, SUV39H1 knockdown suppressed expression of KLF4, the master transcriptional regulator of VSMC dedifferentiation, decreasing KLF4 mRNA stability and upregulating miRNA143, a known repressor of KLF4. siSUV39H1 also increased expression of KDM4a, a JMJD family lysine demethylase that targets H3K9me3. ChIP assays at contractile gene promoters showed significant decrease in the H3K9me3 mark and increase in H3K27Ac after SUV39H1 knockdown.
In vivo,
we noted a significant increase in SUV39H1 and H3K9me3 expression in murine carotid artery ligation induced intimal hyperplasia. We identify SUV39H1 as an epigenetic regulator of VSMC phenotype whose expression and activity increase with dedifferentiation
in vitro
and
in vivo
. PDGF promotes H3K9me3 repressive marks at contractile genes by promoting expression of SUV39H1, which also inhibits the KDM4a. Understanding the role of SUV39H1 in VSMC plasticity may reveal new therapeutic strategies for treating vascular diseases.
Abstract 286: Prediction Of Adverse Events Following Stent Graft Repair Of Type B Aortic Dissection From Imaging Modalities
Authors: Khabaz Kameel , Dhara Sanjeev , Kim Julius , Sankary Seth , Milner Ross , Pocivavsek Luka
Published: November 2022

Objectives:
The prophylactic treatment of uncomplicated TBAD with TEVAR is controversial. This warrants the identification of a high-risk category of dissections that may particularly benefit from surgical intervention. The analysis of radiographic features presents a promising modality of assessing this high-risk cohort. We test the ability of aortic size and shape metrics from the literature to predict patient suitability for TEVAR from pre-operative imaging.

Methods:
We collected a single institutional retrospective cohort of 36 patients with TBAD who received TEVAR and had pre-operative and follow-up CTA imaging. We tested 8 aortic size and shape metrics. We segmented each patient’s aorta and true lumen from the pre-operative scan. Tortuosity, mean diameter, centerline curvature, and eccentricity were measured from the centerline. True and false lumen volumes and max diameter were calculated. The question mark angle, as previously defined by Li et al., was also measured. Univariate and multivariate logistic regression analyses were performed.

Results:
In the univariate analysis, pre-operative false lumen volume (OR 26.2 95% CI 2.72 to 252, p = .005), mean diameter (OR 9.6 95% CI 2.19 to 42.3, p = .003), and maximum diameter (OR 9.0 95% CI 2.12 to 38.3, p = .003) were all significantly associated with post-TEVAR outcomes. Tortuosity index (OR 2.7, 95% CI 1.14 to 6.37, p = .024) was the only significant shape parameter. In multivariate analysis, we found that pre-operative maximum diameter (OR 10.0, 95% CI 1.50 to 67.0, p < .018) is a significant predictor of TEVAR outcomes independent of shape parameters, all of which were not significant.

Conclusions:
False lumen volume and maximum diameter can predict the occurrence of reintervention and type I endoleak following TEVAR for uncomplicated TBAD. While size measures are effective in explaining aortic dissection behavior, current shape measures are not as effective and better methodologies must be developed.
Abstract 288: Impaired Ischemic Myocutaneous Wound Revascularization And Transdermal H
2
S Emissions In Diabetic Rats
Authors: Giacolone Joseph , Matheson Benjamin , Shekarriz Reza , Kanagy Nancy , Clark Ross
Published: November 2022

Introduction:
Hydrogen sulfide (H
2
S) has been recognized as an important signaling molecule in cellular O
2
sensing, wound healing and angiogenesis. Studies have shown abnormal H
2
S levels in diabetic patients with cardiovascular disease. Diminished H
2
S signaling may play a causative role in diabetic foot wounds. The Transdermal Arterial Gasotransmitter Sensor (TAGS) device measures real-time H
2
S emissions through the skin. In this work, we utilize the novel TAGS device to characterize transdermal H
2
S emissions during diabetic and non-diabetic wound healing for the first time.

Methods:
Dorsal peninsular-shaped myocutaneous ischemic flap wounds were created under anesthesia. Sprague Dawley (SD) and Zucker Diabetic Fatty (ZDF) rats (n=10 each) were compared. Transdermal H
2
S emissions, laser speckle contrast images (LSCI) and planimetric photos were serially taken from the wound flap area over 14 days. After animal sacrifice, healed flap tissue was collected for histologic (H&E) analysis of panniculus carnosus (skin muscle) viability as a proxy for degree of ischemic insult.

Results:
ZDF rats were significantly hyperglycemic (mean 516 mg/dL vs. 201 mg/dL for SD, P=0.002). Similar mean baseline (preoperative) H
2
S emissions were observed in SD (16 ppb) and ZDF (12 ppb) rats (P=0.25). During revascularization and healing, ZDF wounds emitted significantly less H
2
S (10 ppb at day 14) as compared to SD (28 ppb at day 14, P<0.01). ZDF wounds demonstrated impaired flap engraftment and revascularization by LSCI (mean 65.6 Perfusion Units (PU) for ZDF vs. 188.0 PU for SD at day 14, p<0.01) and planimetric analysis (mean 16.6% necrosis for ZDF vs. 5.3% necrosis for SD at day 14, p=0.01). Panniculus carnosus mean myofibril count, myofibril diameter, and layer thickness were significantly decreased (p<0.01) in the ZDF cohort, suggesting greater tissue ischemic insult and muscle loss.

Conclusion:
Diabetic rats have impaired wound H
2
S production and poor revascularization. These physiologic alterations are accompanied by greater wound necrosis and histologic ischemic insult. This suggests H
2
S abnormalities in diabetes may play a role in the pathogenesis of impaired wound healing and could represent a potential future therapeutic target for these difficult wounds.
Abstract 302: Upregulation Of The Absent-in-melanoma 2 Inflammasome Correlates With Markers Of DNA Damage And Cell Cycle Dysregulation In Peripheral Arterial Disease
Authors: Assaf Edwyn J , Xie Bowen , Ferrari Ricardo , Morder Kristin , McEnaney Ryan M , Tzeng Edith , Sachdev Ulka
Published: November 2022

Introduction:
We showed that myoblasts (Mb) in peripheral arterial disease (PAD) differentiate poorly and have evidence of inflammasome activation. Absent-in-melanoma 2 (AIM2), an inflammasome and mediator of pyroptosis, binds damaged DNA which also triggers the G2/M DNA damage checkpoint including CDC2 and its regulator, polo-like kinase 1 (PLK1). Dysregulated checkpoints can cause faulty replication, but a link between checkpoint function and AIM2 is not known. We hypothesize that AIM2 expression in PAD Mb correlates with increased DNA damage and a dysregulated G2/M DNA damage checkpoint.

Methods:
Cells were harvested from ischemic and perfused muscle obtained during amputations for PAD. Mb (Pax7-/MyoD+) were isolated using sequential plating and cell sorting. Mb from healthy donors (PAD -) were purchased (Cook MyoSite®). AIM2, γH2AX (DNA damage marker), PLK1 and CDC2 were measured with immunofluorescence (5 images/dish). Mb were treated with the PLK1-inhibitor BI2536 (10nM; 1h) to assess checkpoint responsiveness. ANOVA with post-hoc analysis confirmed statistical significance (α=0.05).

Results:
γH2AX expression was elevated in all PAD groups compared to PAD (-) Mb (N=3-4/PAD group). While PLK1 expression was similar across groups, CDC2 was lower in all ischemic PAD groups relative to perfused PAD Mb. BI2536 attenuated CDC2 expression in perfused PAD Mb (p = 0.006) while Mb in ischemic PAD remained unresponsive. AIM2 expression was significantly higher in ischemic Mb compared to perfused and PAD (-) Mb, correlating with high γH2AX and low CDC2. Results are summarized in Figure 1.

Conclusion:
Here, we show that in PAD Mb, AIM2 correlates with DNA damage, attenuated DNA damage checkpoint protein expression and diminished CDC2 responsiveness to BI2536. Thus, AIM2 may promote pyroptosis in the setting of defective DNA damage mechanisms that would otherwise prompt apoptosis. Whether these pathways are reversed by revascularization is a topic of further study.
Abstract 314: The Alteration In Cell Mechanics In Atherosclerosis
Authors: Rickel Alex , Hong Zhongkui
Published: November 2022

Atherosclerosis remains a major cause of cardiovascular disease. It is well known that the cholesterol accumulated in the macrophage-derived foam cells is the major component of atherosclerotic plaque. However, the growing evidence suggests that about 40% of the total foam cells in the atherosclerotic plaque were vascular smooth muscle cells (VSMCs)-derived. VSMCs are the major cellular components of arterial wall and undergo phenotypic switching from a functional contractile phenotype to a synthetic proliferative phenotype during the development of atherosclerosis. In addition, the surrounding environment of VSMCs within vascular wall changes with alterations in extracellular matrix (ECM) stiffness and composition. The main goal of this study is to test hypothesis that VSMC mechanics undergoes significant changes in response to these surrounding microenvironment alteration in the progression of atherosclerosis. We employed an innovative approach integrating a series of novel experimental approaches and data-driven mathematical models to monitor the real time mechanics of VSMCs isolated from western diet fed apolipoprotein-E knockout (ApoE
-/-
) and wild type (WT) mice. The results demonstrated significant differences in the actin cytoskeletal organization and cell mechanics between ApoE
-/-
VSMCs and WT cells, which may trigger the VSMC migration and contribute to the formation of the arterial plaque. These interesting finding will lead to a novel therapeutic strategy for the pharmaceutical treatment of atherosclerosis by interfering the alteration in VSMC mechanics.
Abstract 317: Resident Vascular Adventitial Progenitor Cells Of Smooth Muscle Cell-lineage Adopt Profibrotic Phenotype And Contribute To Cardiac Fibrosis
Authors: Lu Sizhao , Jolly Austin J , Dubner Allison M , Mutryn Marie F , Nemenoff Raphael A , Moulton Karen S , Weiser-Evans Mary C
Published: November 2022

Activated cardiac myofibroblasts are the major contributors to ECM deposition in pathological cardiovascular fibrosis, which is the characteristic feature of cardiovascular diseases. However, due to potential heterogeneity of myofibroblasts, the origin of these cells remains controversial. Using highly specific smooth muscle cell lineage-tracing mouse models, we discovered the smooth muscle cell origin of a subpopulation of resident vascular adventitial progenitor cells, defined by the expression of the stem cell marker Sca1 (AdvSca1-SM cells), which rapidly proliferate and adopt a myofibroblast phenotype in response to acute vascular injury. Further, we identified a specific gene signature of active hedgehog/Wnt/β-catenin/
Klf4
signaling in AdvSca1-SM cells and validated a
Gli1
-CreERT2-ROSA26-YFP (Gli1) reporter mouse model to be a faithful lineage tracing system for AdvSca1-SM cells. However, the function of AdvSca1-SM cells in cardiac fibrosis is unknown. Using immunofluorescent staining and label-free second harmonic generation (SHG) imaging, we observed the expansion and migration of AdvSca1-SM cells in close association with perivascular and interstitial cardiac fibrosis in pressure overload-induced cardiac fibrosis in Gli1 reporter mice. We performed single cell RNA sequencing (scRNA-seq) to examine the phenotype of AdvSca1-SM cells in Angiotensin II (AngII) model. Our data showed that, upon AngII challenge, AdvSca1-SM cells differentiate along a profibrotic trajectory, which is characterized by loss of expression of
Klf4
, the lncRNA,
Meg3
, and stemness genes and up-regulation of myofibroblast genes. Importantly, AngII-induced profibrotic transcriptomic changes of AdvSca1-SM cells were recapitulated in human ventricular tissues exhibiting a gene signature of cardiac hypertrophy, emphasizing the translational significance of this phenotypic transition. Connectivity map analysis of the scRNA-seq data identified statins as potential candidates for inhibition of the profibrotic transition of AdvSca1-SM cells. In agreement, simvastatin induced the expression of stemness genes and inhibited TGFβ-induced up-regulation of αSMA and down-regulation of
Klf4
in cultured AdvSca1-SM cells.
Abstract 320: Glucose Consumption In Vascular Cell Types In Response To Angiotensin Ii
Authors: Torimoto Keiichi , Okuno Keisuke , Cicalese Stephanie M , Eguchi Satoru
Published: November 2022

Any experimental outcomes are potentially influenced by extracellular glucose availability and its cellular metabolism in cell culture. However, surveillance of vascular-related journals for the past 5 years demonstrated that less than 20% of articles declared the medium glucose concentration. The present studies were designed to seek ideal medium glucose concentration(s) in vascular cell types with particular attention paid to changes in glucose consumption upon angiotensin II (AngII) stimulation. We have compared glucose consumption in three vascular cell types, endothelial cells (EC), vascular smooth muscle cells (VSMC) and adventitial fibroblasts (AF) with or without 100 nM AngII stimulation. In all cell types after a 48-hour incubation in relatively low glucose media (1 g/L in 6 well dish with 1mL), medium glucose concentration was reduced to almost 0. Whereas medium glucose concentration remained significantly higher (EC 2.77±0.05 g/L, VSMC 3.87±0.05 g/L, AF 3.32±0.01 g/L) when cells were incubated for 48 hours in high glucose (4.5 g/L) media. In middle glucose (2.75 g/L) media, medium glucose concentration remained in physiological ranges (EC 0.62±0.18 g/L, VSMC 1.98±0.07 g/L, AF 1.17±0.17 g/L). AngII treatment enhanced glucose consumption in AF and VSMC but not in EC. Enhanced extracellular acidification rate by AngII was observed in AF. PDGF-BB also stimulated glucose consumption in AF which was associated with a trend of cell proliferation. In AF, AngII induction of target proteins at 48 hours varied depending on the glucose concentration used. In low glucose media induction of GRP78 or hexokinase II was highest, whereas induction of VCAM-1 was lowest. Utilization of specific inhibitors further suggest essential roles of AT1 receptor and glycolysis in AngII-induced fibroblast activation. Overall, the present study demonstrates a high risk of hypo- or hyperglycemic conditions when standard low or high glucose media is used with vascular cells. Moreover, these conditions may significantly alter experimental outcomes. Medium glucose concentration should be monitored during any culture experiments and utilization of middle glucose media is recommended for all vascular cell types.
Abstract 330: Kinin B1r Inhibitor Prevents Sepsis-induced Cardiac Dysfunction And Promotes Survival In Mice
Authors: Pan Kaichao , Zhang Qin , Fang Ocean , Nguyen Tu , Solanki Ani , Nadella Anish , Piao Lin , Sharp Willard , Liao James K , Wu Rongxue
Published: November 2022

Introduction:
Hyperpermeability of the microvascular barrier and the resulting capillary leakage are hallmarks of sepsis. B1R has been proposed to be a therapeutic target for sepsis-induced microvascular hyperpermeability and multiorgan failure. However, its direct role in regulating of cardiac function during severe sepsis remains poorly characterized.

Hypothesis:
We hypothesized that B1R inhibition can protect against sepsis-induced cardiac dysfunction and improve survival.

Methods and Results:
To address the hypothesis, we pre-treated mice with either vehicle (control) or a specific B1R antagonist B6929via subcutaneous injection then subjected them to 40% cecal ligation and puncture (CLP) to induce polymicrobial sepsis. Serum levels of IL-6 and TNFα increased significantly within 4 hours post-CLP in the controls. However, B1R antagonist treatment abolished CLP-induced expressions of IL-6 and TNFα by 70% and 30%, respectively. B1R antagonist also protected mice from sepsis-induced organ failure—as highlighted by the lowered increase in MSS score over the 24-h post-CLP—resulting in significant improvement in survival rate (P<0.05). In-vivo cardiac vascular permeability assessed using the Miles assay further demonstrated that B1R inhibition prevented sepsis-induced cardiac microvascular leakage and edema. Similar improvements were also observed in the liver and kidney. Further study revealed an early cardioprotective effect of B1R inhibitor, as evidenced by the improvement of EF%, FS%, and cardiac output in mice treated with B1R antagonist + CLP vs. CLP alone (p<0.01) from the echocardiogram analyses at 4-6 hours following CLP. Furthermore, mitochondrial H
2
O
2
measurement of the freshly isolated cardiac mitochondria from septic mice also showed that B1R inhibition partially abolished the production of H
2
O
2
. The opening of mPTP was also reduced with B1Ri treatment. Taken together, these findings indicated improvement in cardiac mitochondrial function following B1R inhibitor treatment.

Conclusions:
Our findings provide novel insights into the pathogenesis of sepsis-induced cardiac mitochondrial dysfunction and heart failure. Inhibition of B1R may be a novel therapeutic strategy for sepsis-associated cardiovascular disease.
Abstract 334: Gut Microbiome Derived Trimethyl-n-oxide Exacerbates Inflammation And Induces Cardiac Fibrosis In Human Cardiac Fibroblasts And In Rodent Model Of Cardiorenal Disease
Authors: Mehta Minal B , Gill Benji , Szydlowska Marta , Guo Liang , Cohen Taylor , Di Poto Cristina , Glover Matthew , Allman Erik , Cazares Lisa , Hess Sonja
Published: November 2022

Trimethlyamine N-oxide (TMAO), a gut-microbiome derived metabolite of dietary choline and other trimethylamine containing nutrients, has been consistently associated with poor prognosis with cardiovascular diseases (CVD) in recent years. Clinical studies have demonstrated that systemic circulating TMAO levels are independently associated with adverse CVD events such as MI, heart failure (HF), stroke, CKD and death. Previous studies have reported elevated TMAO both in mice models of atherosclerosis and in patients with atherosclerosis, and association between increased TMAO levels with adverse outcomes in HF patients, with significant correlation between increased TMAO levels and heart failure risk. The role and underlying mechanisms of TMAO in mediating disease pathophysiology remains elusive. Here, we use primary human cardiac fibroblasts and rodent models of heart failure with TMAO challenge to study its role in the heart and in cardiac fibroblasts. C57BL/6 mice were administered with TMAO (0.2%) in the drinking water for 2, 4 and 8 weeks. Heart tissues were collected at the end of the study to evaluate cardiac fibrosis, and blood samples were evaluated for TMAO levels. For heart failure studies, ZSF1 rats that underwent uninephrectomy surgery were fed high salt diet and were used as a model for heart failure with preserved ejection fracture (HFpEF), and were followed for 12 weeks. ZSF1 rats were also fed with high choline diet and cardiac function was measured using echocardiography. We demonstrated an elevation in blood TMAO levels, with a concomitant increase in circulating pro-inflammatory cytokine levels and upregulation in fibrosis markers in hearts from mice administered TMAO. In a ZSF1 rat model of HFpEF, we showed that increased TMAO levels correlated with deteriorating cardiac dysfunction. In human cardiac fibroblasts, stimulation with TMAO promoted upregulation of key fibrosis markers such as TGF-β1 in a dose-dependent manner. Inflammation, cardiac function, and cardiac fibrosis were exacerbated in mice administered with TMAO in their drinking water. Furthermore, in ZSF1 rat model for HFpEF, there was a significant association of high TMAO levels to worsening of cardiac and renal dysfunction.
Abstract 337: Contribution Of Platelet Par4 To Influenza A Virus Induced Lung Pathology In Mice
Authors: Tumey Lauryn , Bharathi Vanthana , Miles Stephen , Mackman Nigel , Antoniak Silvio
Published: November 2022

The influenza A virus (IAV) causes a seasonal respiratory tract infection and is characterized by excessive inflammation and tissue pathology in the lungs. Protease-activated receptor 4 (PAR4) is expressed megakaryocytes/platelets, immune cells, and lung epithelial cells and it is the only functional thrombin receptor on murine platelets. Studies showed that platelets contribute to the pathogenesis of IAV. However, the mechanisms are unknown. While platelet inhibition in IAV infection was protective, a global PAR4 deficiency in mice was associated with increased IAV pathology including mortality. The objective of this study is to investigate the contribution of platelet PAR4 to IAV pathology. Mice (both sex) with PAR4 deficiency on megakaryocytes/platelets (PAR4
fl/fl
;PF4
Cre+
) and controls (PAR4
fl/fl
) were infected with mouse adapted IAV (H1N1/PR8) at a dose, which induces 20% mortality in control mice. Inflammatory mediators and cellular inflammation in bronchoalveolar lavage fluid (BALF) was analyzed before, 3, 5 and 7 days post infection (dpi). Body weight changes were analyzed over 14 dpi and survival calculated with a human endpoint of a body weight loss of ≥25% initial weight. Lastly, lung function was analyzed by noninvasive whole-body plethysmography at 0 and 7 dpi. There were no significant differences in the analyzed inflammatory mediators, except for IFNγ, IL6, and protein, or any immune cell numbers in the BALF of both genotypes at any time point after infection. Both genotypes exhibited similar body weight changes over the course of infection which resulted that mice of both genotypes exhibited a mortality of approx. 20%. However, the lung function parameters calculated as PenH (airway resistance) and EF50 (flow rate at 50% expired volume) were significant higher in PAR4
fl/fl
;PF4
Cre+
compared to PAR4
fl/fl
mice 7 dpi. Platelet PAR4 does not influence pathology in a mild IAV infection model. However, the more severe pulmonary dysfunction in PAR4
fl/fl
;PF4
Cre+
compared to PAR4
fl/fl
mice suggest a protective role of platelet PAR4 in maintaining lung function during IAV infection. More studies are needed to investigate if the contribution of platelet PAR4 to IAV pathology is dependent on disease severity as recently suggested.
Abstract 346: Mri Guided Experimental Study To Determine The Mechanistic Performance Of Vessel Prep V. Poba In Infrapopliteal Lesions
Authors: Sinha Kavya , Benfor Bright , Katsarou Maria , Karmonik Christof , Lumsden Alan B , Roy Trisha
Published: November 2022

Objective:
To present initial results of an ongoing ex-vivo experimental study to compare vessel preparation with intravascular lithotripsy (IVL) or orbital atherectomy (OA) versus plain old balloon angioplasty (POBA) in tibial arteries.

Method:
We will study amputated limbs that have concentric calcification, in three arms, 1. POBA, 2. IVL, and 3. OA. Amputated limbs are first scanned on 7T MRI using UTE (Ultra-short echo time) and T2 weighted sequences to characterize target lesions with concentric calcium at high resolution. The diameter of balloon is sized based on diameter of the vessel proximal and distal to target lesion and confirmed with intravascular ultrasound (IVUS). A through-and-through wire is placed and angiography and IVUS are performed. POBA, IVL or OA is performed. In lesions with vessel prep, angioplasty is performed. All angioplasties are performed at minimum pressure required for the balloon to reach profile diameter. Finally, the ex-vivo harvested specimens are imaged in 9.4T MRI for 3-D plaque analysis prior to histologic sectioning and staining.

Results:
We have 7 experiments, 3 BKA and 4 AKA. Balloon angioplasty without vessel prep was performed in 3/7, OA in 2/7 and IVL in 2/7. Lesions angioplastied without vessel prep required higher than burst pressure to reach profile. Lesions treated with OA and IVL required pressures between nominal and burst to reach profile. POBA samples demonstrated plaque disruption and dissections with more luminal gain compared with lesions that underwent vessel prep prior to angioplasty.

Conclusion:
This study elucidates the mechanism of vessel prep and how it assists POBA in protecting the vessel wall. Vessel prep did not “debulk” or improve luminal gain substantially but required lower inflation pressures and had less uncontrolled dissections and disruptions of the vessel wall. Initial results demonstrate a compelling indication for vessel preparation in select tibial lesions with 360-degree concentric calcium.
Abstract 347: Using Deep Convolutional Neural Networks To Automate Classification Of Carotid Plaques From Ultrasound Imaging
Authors: Bhatt Nitish , Nedadur Rashmi , Warren Blair , Mafeld Sebastian , Raju Sneha , Fish Jason E , Wang Bo , Howe Kathryn L
Published: November 2022

Background:
Stroke is a devastating consequence of plaque rupture from the carotid arteries. Current management of carotid plaques involves waiting for symptoms (e.g., stroke or mini-stroke), as intervention itself has risk of stroke and not all plaques are vulnerable to rupture. There is a need to better risk-stratify plaque that causes stroke. Carotid ultrasound (US) is a non-invasive and inexpensive visualization of plaques but is limited by human interpretation. We hypothesize that convolutional neural networks (CNNs) will identify unique features of carotid plaques for automated risk stratification.

Methods:
Our workflow is illustrated in Fig. a. A total of 141 B-mode US images of carotid arteries were included; 64 high-risk with symptomatic carotid plaques and 75 low-risk with no significant plaque. Data was cropped and divided into training (70%) and holdout test (30%) subsets. During model training, an ensemble of ResNet-18 CNNs learned classification of low-risk and high-risk cases using five-fold stratified cross validation and was used to predict on the holdout test set. The model was evaluated using ROC-AUC and sensitivity. Saliency maps were used for model interpretability to highlight relevant pixels for model decisions.

Results:
The cross-validation AUC was 0.995 ± 0.010. The testing AUC was 0.909 and class-wise sensitivities were 0.88 (low-risk) and 0.79 (high-risk). The density plot (Fig. b) shows that the classifier correctly identifies both classes with confidence. Model interpretability using saliency maps (Fig. c) shows pixels corresponding to carotid artery vessel edges and in high-risk cases, carotid plaques.

Conclusions:
Using this proof-of-concept model, carotid US long axis images are sufficient to identify high-risk plaques in symptomatic patients - we now need to determine whether we can identify high-risk plaques
before
symptoms to prevent devastating stroke caused by carotid disease.
Abstract 353: Phenotypic Switching Of Vascular Smooth Muscle Cells Is Regulated By ApoE And A Potential Therapeutic Target In Popliteal Aneurysms
Authors: Reisenauer Tessa , Pauli Jessica , Paloschi Valentina , Chernogubova Ekaterina - , Scholz Claus , Reeps Christian , Eckstein Hans-Henning , Maegdefessel Lars , Busch Albert
Published: November 2022

Introduction:
Popliteal artery aneurysm (PAA) is an individually highly fatal disease, causing ischemia and eventual major amputation, mainly in 50-70-year-old males. It is the most frequent peripheral artery aneurysm and correct treatment is challenging for clinicians, since both open and endovascular repair have only modest success rates, depending on the clinical presentation. In comparison to other aneurysm entities, little is known about its specific pathogenesis.

Material and Methods:
26 Human PAA and popliteal artery samples were analyzed by immunohistochemistry, mRNA and miRNA expression analysis and targeted proteomics (OLink platform) to identify key features of the disease and crucial pathways involved. Additionally, a primary cell culture of PAA specimen was established for in-vitro vascular smooth muscle cell (VSMC) modulation.

Results:
VSMCs lose their contractile phenotype along with significant inflammatory and proteolytic changes in the vessel wall architecture. Extensive tissue remodeling with high cell turnover rates (as indicated by Ki67+ cells) compared to non-diseased popliteal artery is a unique feature in comparison to AAA. This correlates with highly abundant co-expression of the apolipoproteins E and CI with Ki67 in VSMCs in double immune-fluorescent stains. Stimulation of VSMCs with external APOE or APOCI alters proliferation rates and significantly changes a subset of contractile markers proteins upon gene expression analysis. These findings are emphasized by a specific case of a solely mechanically induced PAA in a young male.

Conclusion:
Pathogenesis of PAA shows similar histologic features than AAA, yet differs in key pathways involved. Increased cell turnover in the aneurysm neck area suggests evaluation of alternative treatment strategies, i.e., targeting key processes of pathogenesis such as angiogenesis and cell turnover.
Abstract 361: Bidirectional Mendelian Randomization Investigating The Causal Relationship Between Major Depressive Disorder And Peripheral Artery Disease
Authors: Shakt Gabrielle , Tsao Noah , Levin Michael , Walker Venexia , Kember Rachel , Klarin Derek , Tsao Philip S , Voight Benjamin F , Scali Salvatore T , Damrauer Scott
Published: November 2022

Background:
Observational studies have demonstrated strong bi-directional associations between depression and peripheral artery disease (PAD), and patients suffering from PAD who have depressive symptoms are known to have worse outcomes. Whether these associations represent causal pathways remains unknown but has important implications for disease management.

Methods:
We performed causal inference experiments using bi-directional Mendelian randomization. We employed summary statistics from the 2019 Psychiatric Genomics Consortium meta-analysis of major depressive disorder (MDD) comprised of 807,553 participants (246,363 cases) of European ancestry and the VA Million Veteran Program GWAS of PAD comprised of 243,060 participants (31,307 cases) of multiple ancestries to test the association between genetic liability to MDD and genetic liability to PAD. Genetic instruments for each exposure were constructed using independent (r
2
< 0.1 by 1000 Genomes EUR LD reference panel) variants associated with the exposure at genome-wide significance (p < 5x10
-8
). Inverse-variance weighted MR was used as the primary analysis method, with weighted-median MR, MR-PRESSO, and Steiger filtering based MR performed as sensitivity analyses. F-statistics were calculated to assess weak-instrument bias. The Egger bias-intercept test was used to detect evidence of horizontal pleiotropy.

Results:
Increased genetic liability to MDD was associated with increased genetic liability to PAD (OR 1.2, 95%CI 1.1 - 1.3, p=.0001). We could not detect an effect of genetic liability to PAD on the genetic liability to MDD (OR 1.03, 95%CI 0.99 - 1.08). The results of sensitivity analyses that make different instrumental variable assumptions were consistent with these findings.

Conclusions:
These results suggest a causal relationship in which MDD increases the risk of PAD, but do not provide evidence to support the causal role of PAD in MDD. Ongoing analyses are focused on identifying potential mediators of the association between MDD and PAD and performing causal inference for the effect of other mental health traits on PAD.
Abstract 363: Irf8 And Securinine: Seeking Treatments For Abdominal Aortic Aneurysm
Authors: Rhee Yae Hyun , Deng Alica , Headley Colwyn , Butterfield Jacqueline , Spin Joshua , Tsao Philip S
Published: November 2022

Objective:
Abdominal aortic aneurysm (AAA) is defined as local weakening and dilatation of the abdominal aorta ≥50% and has high incidence in elderly. Current therapy is limited to surgical repair, only, no pharmacological therapy has shown effectiveness against AAA development and growth. Untreated AAA rupture is highly associated with death, and aged patients are at augmented risk for surgical complications. We therefore sought to identify novel agents that could be employed for medical AAA therapy.

Methods:
Using whole genome DNA methylation analysis (RRBS-Seq) in a transgenerational model of murine AAA, combined with transcriptional analysis of murine AAA tissue, we identified interferon regulatory factors (IRFs: a group of immune-system-related transcription factors (TF)), as major drivers of AAA risk and pathophysiology. We performed TF enrichment analysis that specifically suggested IRF8 as being crucial for AAA development. Drug Signatures Database (DSigDB) and Connectivity Map (CMap) analysis identified securinine, an alkaloid and traditional Chinese herbal medicine derived from
Securinega suffruticosa
, as a probable IRF8 inhibitor. Its vascular effects are relatively unknown.

Results:
We studied the effects of securinine using the porcine pancreatic elastase (PPE) model of AAA. Sonographic imaging data of 28-day securinine-treated mice (15 mg/kg) showed smaller aneurysm diameters and significantly downregulated gene expression of IRF8 and IFN-γ compared to control PPE groups.

Conclusion:
These data suggest that securinine may have therapeutic benefits on AAA development, potentially through regulation of IRF8.