bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 445: Bone Marrow-derived Macrophage (BMDM) Extracellular Vesicles (EVs) From Diabetes Impair Angiogenesis In Ischemic Hind Limb Of Diabetic Mice
Authors: Nimma Ramesh , López-Díez Raquel , Yepuri Gautham , Kumar Vikas , Arivazhagan Lakshmi , Vakil Raveena , Bhallamudi Sangeeta , Erdjument-Bromage Hediye , Neubert Thomas , Ramasamy Ravichandran , Schmidt Ann Marie
Published: October 2023

Critical limb ischemia (CLI), which is increased in diabetes, is a life-threatening condition that leads to high mortality and disability. The underlying mechanisms linking diabetes and severity of CLI remain unclear. Induction of hind limb ischemia (HLI) in diabetic Akita (C57BL/6-Ins2
Akita
/J) vs. non-diabetic (NDM, C57BL/6J) mice was performed by ligation of the left femoral artery. Blood perfusion in the ischemic hind limb, measured by Laser Doppler Imaging, revealed reduced blood flow recovery in diabetic vs. NDM mice. Macrophage extracellular vesicles (EVs) contribute to cell-to-cell communications and diabetic complications. BMDMs from male NDM and Akita mice were isolated, cultured in Exo-free complete DMEM media for 48 hours and EVs isolated by ultracentrifugation followed by size, zeta potential and concentration measurements by ZetaView®-nanoparticle tracking analysis. EVs size of Akita BMDMs was similar to that of NDM BMDM EVs (diameter, 20-300nm). The zeta potential for the Akita EVs was -18.62±0.18 mV vs -8.38±0.14 mV in NDM EVs. Western blot confirmed positive exosomal markers (CD81, Flotillin) in NDM and Akita EVs; the negative control marker (calnexin) was not identified. To identify the proteome cargo, we employed LC-MS/MS. Comparing Akita vs. NDM BMDM EVs, a total of 3277 proteins was identified and quantified. 821 proteins were significantly differentially expressed in Akita vs. NDM EVs. These significantly altered proteins (Upregulated and downregulated) are involved in: metabolic functions, immune system processes, developmental and biological processes. Enrichment analysis (GO, KEGG and Reactome) highlighted pathways predominantly related to oxidative stress, insulin signaling, actin cytoskeleton, CLEAR (Coordinated Lysosomal Expression And Regulation), lipid metabolism, phagosome maturation, RNA binding protein and DNA replication pathways.
In vitro,
endothelial tube formation was impaired in human iliac artery endothelial cells treated with diabetic vs. NDM BMDM EVs. Targeting macrophage EVs composition might identify therapeutic approaches for the prevention and treatment of CLI in diabetes.
Abstract 450: Map Kinase Phosphatase-5 Deficiency Impairs Efferocytosis And Exacerbates Late-Stage Atherosclerosis
Authors: Baldini Margaret , Azuma Masahi , Palos-Jasso Andrea , Shan Dan , Roy Rajika , Koch Walter J , Bennett Anton M , Yu Jun
Published: October 2023

Objective:
Atherosclerosis is a vascular inflammatory disease that is the leading cause of death worldwide. Late-stage atherosclerotic plaque contains a thin fibrous cap and a large necrotic core that is prone to rupture. Efferocytosis contributes to the clearance of apoptotic cells within the early plaque but is impaired in late-stage atherosclerosis, promoting rupture. MAPK phosphatase-5 (MKP-5) is crucial in regulating various processes, including inflammatory diseases and cardiac fibrosis. The goal of this project is to identify the role of MKP-5 in late-stage atherosclerotic plaque stability.

Results:
To induce late-stage atherosclerosis, we used a novel late-stage atherosclerosis model that leads to coronary artery lesions, plaque rupture, and spontaneous myocardial infarction. 9-week-old male and female LDLR
-/-
and LDLR
-/-
MKP-5
-/-
mice were subjected to transverse aortic constriction pressure overload surgery and fed a high-fat diet (western diet with 1.25% cholesterol) for 12 weeks. Our data show that MKP-5 deficiency exacerbates plaque instability, including decreased cap thickness, increased necrosis in the atherosclerotic plaque, severe coronary lesions, as well as higher instances of intraplaque hemorrhage in the right carotids. Mechanistically, TUNEL staining of the aortic roots was performed to determine efferocytotic capabilities. Results showed fewer macrophage-associated TUNEL-positive cells in LDLR
-/-
MKP-5
-/-
mice aortic roots, which indicates defective efferocytosis. Furthermore, bone-marrow-derived macrophages (BMDMs) isolated from WT and MKP-5
-/-
mice were incubated with labeled apoptotic cells and then subsequently analyzed to determine efferocytotic capabilities. Results showed that efferocytosis was significantly inhibited in MKP-5
-/-
BMDMs. Additionally, RT-PCR analysis revealed that the expression of efferocytotic receptors in MKP-5
-/-
BMDMs were decreased. RNAseq analysis was performed on WT and MKP-5
-/-
BMDMs stimulated with ox-LDL, identifying pathways regulated by MKP-5.

Conclusions:
Our study suggests that MKP-5 deficiency decreases the stability of the late-stage plaque and exacerbates atherosclerosis through impaired efferocytosis.
Abstract 455: Lats1/2 Expression Reduced By The Pathological Flow Triggers Intraplaque Hemorrhage And Distal Pulmonary Thrombosis By Promoting EC Apoptosis And Proliferation In Concert
Authors: Ko Kyung ae , Kotla Sivareddy , Samanthapudi Venkata Subrahman K , Wen Jake J , Hernandez Gonzalez Selina , Deswal Anita M , Lin Steven , Koutroumpakis Efstratios , Palaskas Nicolas L , Reyes-Gibby Cielito , Yeung Sai-Ching , Dantzer Robert , Morrell Craig , Li Shengyu , Wang Guangyu , Martin James F , Le Nhat-Tu , Abe Junichi
Published: October 2023

Atherosclerotic and high-risk plaques are predominantly localized to vessel wall regions with disturbed blood flow (d-flow), leading to the theory that d- and low-flow-driven endothelial cells (EC) dysfunction play a role in this process. It has become clear that high flow (h-flow; high shear stress due to increased flow rate) is also critical to developing high-risk plaques including both rupture and erosion. We aim to determine how the high-risk plaque is formed by pathological flow. The pathological flow (both h- and d-flow) resulted in decreased LATS1/2 expression, but physiological laminar flow did not. Within 14 days after inducing LATS1/2 deletion in tamoxifen-inducible EC-specific LATS1
homo
/LATS2
homo
knock-out mice (LATS1
homo
/LATS2
homo
-EKO), all the mice (27/27) had died of severe systemic edema, accompanied by increased EC apoptosis and vascular permeability. We generated EC-specific LATS1

he

t
/LATS2
homo
-EKO mice under hypercholesterolemia (H-chol) with pAAV/D377Y-mPCSK9 injections and a high-fat diet (HFD). After 8weeks of HFD, we detected plaque erosion-like lesions at the h- and d-flow area in the aortic arch and carotids, which revealed strong fibrin/fibrinogen-positive organized thrombus formation without a sizeable necrotic core. Interestingly, we also found a significant increase in distal pulmonary thrombosis and EC proliferation in LATS1

he

t
/LATS2
homo
-EKO mice. H-chol increased the incidents of aortic but not lung lesions. We also performed partial left carotid artery ligation (PLCL) and found that d-flow induced by PLCL developed severe atherosclerosis formation with unique intraplaque hemorrhage in H-chol LATS1

he

t
/LATS2
homo
-EKO mice, which was not observed in wild-type mice. In LATS1

he

t
/LATS2
homo
-EKO mice, we found significant increases in 1) EC proliferation, 2) EC apoptosis with senescent phenotype, 3) tissue factor (TF) expression and 4) inflammation, which are collectively referred to as the “pro-thrombotic phenotype”
in vivo
. These data suggest that pathological flow-induced LATS1/2 degradation promotes EC apoptosis and proliferation simultaneously. This cycle of EC damage-thrombus formation-re-endothelialization results in rapid thrombus enlargement and distal pulmonary thrombosis.
Abstract 456: Myeloid RvD2-GPR18 Signaling Axis Limits Plaque Necrosis
Authors: Lipscomb Masharh , Walis Sean , Marinello Michael , Fredman Gabrielle
Published: October 2023

Introduction:
Dysregulated inflammation-resolution programs are associated with atherosclerosis progression. Inflammation-resolution is in part mediated by Resolvins, including Resolvin D2 (RvD2). Previous studies suggested that RvD2 was decreased as atherosclerosis progressed to the advanced stage and that treatment of RvD2 to mice limited plaque progression. Endogenous RvD2 signaling and cellular targets of RvD2 are not known.

Methods and Results:
Here we developed humanized GPR18 floxed (fl/fl) and a myeloid (Lysozyme M Cre) GPR18 knockout (mKO) mouse. We functionally validated this model by assessing efferocytosis in bone marrow derived macrophages (BMDMs) and found that RvD2 enhanced efferocytosis in the fl/fl, but not in the mKO BMDMs. We employed two different models to evaluate the role of GPR18 in atherosclerosis. We first used the PCSK9-gain of function approach and found increased necrosis in the plaques of the mKO mice compared with fl/fl mice. Next, we performed a bone marrow transfer of fl/fl or mKO bone marrow into Ldlr
-/-
recipients. For these experiments, we treated each genotype with either Veh or RvD2 (25 ng/mouse, 3 times/week for 3 weeks). First, we corroborate our previous results and found the myeloid loss of GPR18 resulted in significantly larger necrotic cores compared with fl/fl transplanted mice. RvD2 treatment decreased plaques necrosis in fl/fl, but not in the mKO transplanted mice.

Conclusions:
These results are the first to suggest a causative role for endogenous RvD2 signaling on myeloid cells in limiting plaque necrosis. Moreover, these results provide a mechanistic basis for RvD2 as a therapy limiting plaque necrosis.
Abstract 458: A Study Of Short-term Low-saturated Fat Versus High-saturated Fat Diet In Atherothrombosis Risk Factors In Subjects With Hypertriglyceridemia
Authors: O'Brien Veronica , Lian Zeqin , Perrard Xiao-Yuan Dai , Antony Antu , Peng Xueying , Xu Lu , Ni Jing , Zhang Bingqian , Saeed Anum , Jia Xiaoming , Hussain Aliza , Hoogeveen Ron C , Ballantyne Christie M , Wu Huaizhu
Published: October 2023

Introduction:
Dietary fat and composition may impact the risk of atherosclerotic cardiovascular disease (ASCVD) in subjects with hypertriglyceridemia (HTG).

Hypothesis:
We hypothesized that a low-saturated fat diet (LSFD), in contrast to a high-saturated fat diet (HSFD), improves blood lipid levels and reduces atherothrombosis risk factors in subjects with HTG.

Method:
Subjects with HTG and metabolic syndrome (n=19) were randomly placed on an LSFD (~25% of calories from fat, 5% from saturated fat) or an HSFD (~52% of calories from fat, 25% from saturated fat) for 4 days (day 1-4) and a breakfast on day 5 and then switched to the other diet after a 4- to 6-week washout period. Blood was collected on day 1 fasting before the start of the diet and on day 5 fasting and at 4 and 6 hours after breakfast. Lipid profiles and flow cytometry to measure monocyte intracellular lipid accumulation, monocyte expression of tissue factor (TF), and monocyte-platelet aggregates (MPAs) were performed.

Results:
On day 5, LSFD, compared to HSFD, induced lower plasma levels of postprandial total triglyceride and LDL-triglyceride and fasting and postprandial total cholesterol, non-HDL-cholesterol, LDL-cholesterol, and small dense LDL-cholesterol. Classical and intermediate monocytes with LSFD, in contrast to HSFD, exhibited reduced intracellular lipid accumulation indicated by decreases in cellular granularity and nile red staining examined by flow cytometry. LSFD vs. HSFD also reduced classical monocyte uptake of oxidized LDL ex vivo at 4 hours postprandially on day 5. Surface level of TF was higher on classical and intermediate monocytes than on nonclassical monocytes assessed by mean fluorescence intensity. Classical and intermediate monocytes, compared to nonclassical monocytes, were also more prone to form aggregates with platelets. However, there was no significant difference between LSFD and HSFD in TF levels on any monocyte subsets and in MPAs of any monocyte subsets at any time points recorded.

Conclusion:
In subjects with HTG and metabolic syndrome, short-term LSFD, in contrast to HSFD, reduced monocyte lipid accumulation and oxidized LDL uptake but did not induce significant differences in monocyte surface levels of TF and MPA formation.
Abstract 462: Transcriptomic Analysis Reveals That Intimal Monocytes Drive Inflammation In Early Atherogenesis
Authors: Scipione Corey A , Hyduk Sharon J , Polenz Chanele K , Althagafi Marwan G , Prattas Stylianos Z , Ibrahim Hisham M , MacParland Sonya A , Cybulsky Myron I
Published: October 2023

Hypercholesterolemia (HC) is a key risk factor for atherosclerosis, a chronic inflammatory disease that causes myocardial infarction and stroke. Although all regions of arteries are exposed to HC, atherosclerosis develops in discrete regions, such as the lesser curvature (LC) of the mouse aortic arch. In

Ldlr
-/-

mice HC induces intimal myeloid cells to develop into the foam cells of nascent lesions. The purpose of this study is to characterize the transcriptomic changes occurring in the aortic intimal myeloid cells during the earliest stages of atherogenesis, and correlate them to cellular responses. Intimal cells were isolated from the LC of the ascending aortic arch of
Ldlr
-/-
mice at 0, 5, 14, and 56 days of CRD feeding using
en face
enzymatic digestion and cell microisolation. Bulk RNA-seq and RT-qPCR revealed that genes associated with lipid-loaded macrophages (e.g.,
Abcg1
,
Lgals3
) were progressively elevated over time. Atherogenesis-associated inflammatory transcripts and pathways, such as leukocyte transendothelial migration, cell adhesion, and cytokine/chemokine signalling were significantly elevated after 14d of CRD, but not at 5d. Single cell transcriptomic analysis of the mouse aortic arch intima of
Ldlr
-/-
mice revealed multiple sub-populations of myeloid cells. With our intima specific approach, we have uncovered a diverse landscape of intimal myeloid cells; including monocytes, macrophages, foam cells, DCs, and neutrophils. Atherogenesis-associated inflammatory transcripts were scarcely detected in macrophage and foam cell clusters, but were prominent in non-macrophage cell types.

Ccr2
+

intimal monocytes exhibited a hypercholesterolemia induced increase in inflammatory pathways within 5 days of CRD feeding. Furthermore, analysis of monocyte and macrophage clusters suggests that this inflammatory signature dissipates as recently recruited monocytes transition into a macrophage/foam cell fate. We are using

Ccr2
+

fate mapping models, to interrogate the hypercholesterolemia-induced transition of inflammatory monocytes within a growing lesion. We suggest that the atherogenesis-associated inflammation may be initiated by recently recruited monocytes, and may not originate directly from lipid loaded macrophages.
Abstract 465: Integrative Single-cell Meta-analysis Reveals Disease-relevant Vascular Cell States and Markers in Human Atherosclerosis
Authors: Verdezoto Mosquera Jose E , Wong Doris , Auguste Gaelle , Turner Adam , Hodonsky Chani J , Lino Cardenas Christian L , Theofilatos Konstantinos , Bos Maxime , Kavousi Maryam , Peyser Patricia R , Mayr Manuel , Kovacic Jason , Bjorkegren Johan , Malhotra Rajeev , van der Laan Sander , Zang Chongzhi , Sheffield Nathan , Miller Clint
Published: October 2023

Introduction:
Atherosclerosis is a chronic inflammatory process driven by plaque formation in the major elastic arteries leading to reduced blood flow, coronary artery disease (CAD), myocardial infarction and stroke. Several single-cell RNA-seq (scRNA-seq) studies have highlighted lineage-specific transcriptomic signatures in CAD-relevant tissues; however, there remains variability on the reported cell phenotypes in humans. We evaluated the overall hypothesis that distinct smooth muscle cell (SMC) phenotypes characterized from scRNA-seq data contribute to human atherosclerotic lesion formation.

Methods:
Here we meta-analyzed scRNA-seq human data from four individual studies to establish a comprehensive map of cell diversity in human atherosclerosis. We applied standardized QC, processing, and integration benchmarking to harmonize datasets and generate an atlas of 118,578 high-quality cells.

Results:
Using this atlas we characterize vascular and immune cell diversity during atherosclerosis by identifying 100 consensus marker genes across 42 annotated cell types/subtypes. We provide insights into SMC phenotypic modulation through pseudotime, transcription factor activity inference and cell-cell communication analyses. By integrating genome-wide association study (GWAS) data we identify etiologic cell types for subclinical and clinical CAD traits, which uncovered a critical role for modulated SMC phenotypes in CAD and coronary artery calcification. Finally, we identified candidate markers of synthetic and osteochondrogenic human SMCs (e.g.,
LTBP1
and
CRTAC1
) that may serve as proxies of atherosclerosis progression and provide validation through bulk transcriptomics, proteomics, and immunofluorescence.

Conclusions:
Together, this represents an important step towards creating a unified cellular map of atherosclerosis to inform cell-specific mechanistic and translational studies of cardiovascular diseases.
Abstract 469: Possibility Of Pemafibrate For Risk Reduction Of Cardiovascular Disease As The Treatment Of Atherosclerosis
Authors: Fujii Hiroko
Published: October 2023

Introduction:
Pemafibrate is the first clinical available selective peroxisome proliferator-activated receptor α modulator (SPPARMα) which has been shown to decrease triglycerides(TG) and increase high-density lipoprotein cholesterol(HDL-C). Elevated TG and decreased HDL-C are cardiovascular risks. We investigated the efficacies of pemafibrate on cardiovascular risk reduction.

Hypothesis:
It is hypothesized that pemafibrate may reduce not only TG level but also low-density lipoprotein cholesterol(LDL-C)/HDL-C ratio.

Methods:
Total 54 outpatients (Male 35; Female 19) in our hospital who obtained pemafibrate on doctors’ prescription during 3 straight months were investigated. The biochemical data of TG, HDL-C, LDL-C, and LDL-C/HDL-C ratio were compared before with after treatment with pemafibrate. As the safety issue, serum creatinine for Kidney function, and AST, ALT, and γ-GTP for liver function from biochemical data were compared before with after treatment with pemafibrate.

Results:
Average age of this population was 68±12 (male: 69±14; female 67±9) years. Complications of hypertriglyceridemia in this population were hypertension (22%), hypercholesterolemia (21%), and hyperuricemia (7%) as risk factors of cardiovascular disease; Ischemic heart disease (8%), Atrial fibrillation/Atrial flatter (7%), and old myocardial infarction (6%) as heart diseases. % changes of TG, HDL-C, LDL-C, and LDL-C/HDL-C ratio were -51.8±26.2%, 15.3±22.5 %, -6.1±32.7%, and -16.6±28.8%, respectively. There was negative correlation between TG and HDL-C (r^2=0.157, p<0.0001, Y=-0.0378X+59). There was positive correlation between TG and LDL-C/HDL-C ratio (r^2=0.155, p<0.0001, Y=0.00307X+1.5).

Conclusions:
This study demonstrated that pemafibrate not only decreased serum TG and increased HDL-C, but also reduced LDL-C/HDL-C ratio. Pemafibrate might be a medication for dyslipidemia including cholesterol. Therefore, pemafibrate must contribute to preventing atherosclerosis and cardiovascular disease through reduction on LDL-C/HDL-C ratio.
Abstract 470: Targeting The Local Inflammation In Atherosclerosis Using An
Ex-vivo
Perfusion System Of Human Coronary Arteries
Authors: Hetherington Isabella , VanWye Jeffrey , Wickramage Ishani , Raggio Max , Totary-Jain Hana
Published: October 2023

Atherosclerosis is characterized by the accumulation of lipids, immune cells, and fibrotic tissue in the arterial intima. Although well described as an inflammatory condition, targeting the inflammation component of atherosclerosis while avoiding systemic toxicity remains a clinical challenge. Pro-inflammatory cytokines such as IL-1β and IL-6 have been implicated in atherosclerotic plaque progression and atherothrombotic events. Recent clinical trials have shown promising results targeting these cytokines to reduce the risk of adverse cardiac events in patients, yet systemic toxicity remains a concern. We hypothesize that knocking down the local inflammation using siRNA against IL-1β and/or IL-6 may be a viable therapeutic strategy to treat atherosclerosis. To test our hypothesis, we used an
ex-vivo
perfusion system of human coronary arteries (hCA) collected from non-transplantable, de-identified hearts (
Fig. A
). Hearts came from donors with or without cardiovascular disease (CVD) risk factors including hypertension, high cholesterol, and diabetes mellitus. hCA isolated from donors with CVD risk factors showed increased IL-1β (
Fig. B
) and IL-6 (
Fig. C
) expression levels assessed by RT-qPCR, compared to controls without risk factors. Perfusion of hCA with media containing TNF-α and IFN-γ increased the expression of both IL-1β and IL-6 (
Fig. D
). Thus, the perfusion system established here will allow us to assess the efficacy of delivering IL-1β and/or IL-6 specific siRNA complexed nanoparticles and will advance the development of novel nanotherapies that target inflammation in atherosclerosis.
Abstract 472: TERF2IP S205 Phosphorylation Instigates Pathological Flow-induced Plaque Erosion By Reducing LATS1/2 Expression
Authors: Kotla Sivareddy , Ko Kyung A , Samanthapudi Venkata Subrahman K , Wen Jake , Hernandez Gonzalez Selina , Deswal Anita M , Lin Steven , Koutroumpakis Efstratios , Palaskas Nicolas L , Reyes-Gibby Cielito , Yeung Sai-Ching , Dantzer Robert , Morrell Craig , Li Shengyu , Wang Guangyu , Martin James F , LE NHAT TU T , Abe Junichi
Published: October 2023

Pathologic wall shear stress (WSS) of both high and low WSS can contribute to plaque destabilization and subsequent plaque erosion and rupture. The contribution of both ECs proliferation and apoptosis in plaque erosion has been proposed, but how both EC proliferation and apoptosis can be induced simultaneously by pathological flow (high (h-) and disturbed (d-) flow) has yet to be well-studied. The depletion of LATS1/2 significantly enhanced lesions at the d- and h-flow areas. We found that both d- and h-flow (36 dyn/cm
2
) can decrease LATS1/2 expression but not intermediate normal laminar flow (l-flow: 12 dyn/cm
2
). Pathological flow and depletion of LATS1/2 increased EC proliferation, apoptosis, and tissue factor expression, but normal l-flow did not. TERF2IP is a member of the shelterin complex of the telomere. Previously, we reported that TERF2IP S205 phosphorylation had a crucial role in d-flow-induced EC senescence and apoptosis induction. Transduction of adenovirus containing TERF2IP S205A mutation significantly inhibited the d-flow-induced reduction of LATS1/2 expression. No change in LATS1/2 mRNA levels after d-flow was detected and the protease inhibitor of MG132 inhibited the d-flow-induced reduction of LATS1/2 expression, suggesting the involvement of protein degradation in the reduction of LATS1/2. The role of MKRN1 (ubiquitin E3 ligase) in senescence has been reported, and we found that the depletion of MKRN1 significantly inhibited the d-flow-induced LATS1/2 reduction, suggesting that LATS1/2 is a novel MKRN1 substrate. Importantly, d-flow increased TERF2IP and MKRN1 association; this association was inhibited by TERF2IP S205A mutation. Overexpression of the TERF2IP c-myb domain inhibited the TERF2IP-MKRN1 association, suggesting that this TERF2IP site is a binding site of TERF2IP with MKRN1. We observed continuous binding of TERF2IP and LATS1/2 until the reduction of LATS1/2 expression by d-flow, suggesting that TERF2IP S205 phosphorylation recruits a trinary complex of TERF2IP-MKRN1-LATS1/2, which promotes LATS1/2 degradation. These data suggest the role of TERF2IP S205 phosphorylation in down-regulating LATS1/2 expression, resulting in EC senescence and subsequent plaque erosion mediated by the pathological flow.
Abstract 475: Histone Demethylase JARID1c Regulates T
H
17 T-cells In Diabetic Wounds By Increasing IL-6 Expression In Plasmacytoid Dendritic Cells
Authors: Audu Chris , Melvin William , Wolf Sonya , Mangum Kevin , Joshi Amrita , Gallagher Katherine A
Published: October 2023

Plasmacytoid dendritic cells (pDCs) are first responders to tissue damage and interact with wound CD4 T-cells to orchestrate tissue repair. In diabetes, the role of pDCs and their subsequent influence on wound CD4
+
T-cells and inflammation during tissue repair is unknown. Using human diabetic wound single cell sequencing, we identified that human wound CD4
+
T-cells are primarily of the inflammatory T
H
17 phenotype. Thus, we hypothesized that pDCs in the wounds may regulate this T-cell phenotype, contributing to chronic inflammation. Although pDCs exist in low numbers in the wounds, we isolated wound pDCs from diabetic and control mice and examined them for inflammatory cytokine expression. We found that diabetic pDC produced significantly more IL-6 compared to controls. This is important as IL-6 has been shown to skew CD4
+
T-cells towards a T
H
17 phenotype in tissues. Upon co-culture of diabetic pDCs with naïve CD4
+
T-cells, a significant increase in T
H
17 cells was observed by intracellular flow cytometry analysis. We then isolated diabetic and control wound pDCs, and performed an epigenetic superarray. We identified that histone demethylase JARID1C was significantly decreased in diabetic pDCs compared to controls. JARID1C is an epigenetic enzyme responsible for demethylating lysine 4 on histone 3 (H3K4me3), resulting in gene transcription inhibition. We then isolated diabetic pDCs and performed a ChIP analysis on the gene binding sites at the IL-6 promoter and identified an increase in H3K4me3, consistent with increased transcription of IL-6. We demonstrate that Type 1 interferons regulate JARID1C expression via TYK2/JAK1,3 mechanism. Finally, we show that
in-vitro
administration of recombinant JARID1C decreases IL-6 production and T
H
17 T-cell expression. Taken together, this data suggests that in diabetes, pDCs are epigenetically altered to increase IL-6 expression and direct the adaptive immune response towards pathologic T
H
17 CD4
+
T-cell mediated chronic inflammation and poor wound healing.
Abstract 480: IL-33 Promotes Cardiovascular Lesions Development In A Mouse Model Of Kawasaki Disease
Authors: Teixeira de Carvalho Thacyana , Jena Prasant , Franklin Michael , Fishbein Michael C , Chen Shuang , Shimada Kenichi , Crother Timothy R , Arditi Moshe , Noval Rivas Magali
Published: October 2023

Kawasaki Disease (KD), a pediatric acute febrile illness and systemic vasculitis, is the leading cause of acquired heart diseases in children. Coronary artery aneurysms (CAAs) occur in up to 25% of untreated children, which is reduced to 5% with intravenous immunoglobulin (IVIG) treatment. However, up to 20% of KD patients are refractory to IVIG and at higher risk of CAAs, highlighting the need to characterize the mechanisms mediating KD vasculitis to develop more efficient therapies. IL-33 is released upon inflammation and tissue damage and exerts its effects by binding to its receptor ST2 (
IL1RL1
). Levels of IL-33 are elevated in KD patients during the acute phase. However, how IL-33 contributes to the development of KD cardiovascular lesions remains unknown. Using the
Lactobacillus casei
cell wall extract (LCWE) murine model of KD vasculitis, we observed increased
Il33
mRNA expression in hearts and abdominal aortas aneurysms of LCWE-injected mice. While ST2 was detected by immunofluorescence in the abdominal aortas of both control and LCWE-injected mice, IL-33 was mainly detected in tissues from LCWE-injected mice. Single-cell RNA-sequencing of abdominal aortas from LCWE-injected mice indicated that
Il33
transcripts were expressed primarily by fibroblasts, endothelial cells, B cells, and vascular smooth muscle cells. In contrast,
Il1rl1
was highly expressed by tissue infiltrating regulatory T cells, macrophages, eosinophils, and neutrophils. Blocking IL-33 with either anti-IL-33 or using

Il33
-/-

mice significantly attenuated LCWE-induced KD vasculitis. Our results indicate that IL-33 participates in LCWE-induced KD vasculitis in mice and could therapeutically be targeted to inhibit KD cardiovascular lesions.
Abstract 497:
Ex Vivo
MRI Detection Of Seeded Adventitial Cells On Human Vein Grafts
Authors: Liu Hui , Kim Kevin , Naumova Anna , Tang Gale L
Published: October 2023

Saphenous vein grafts are the most frequently used autogenous conduits in coronary and peripheral artery bypass surgeries. Despite the undoubted success of coronary artery and peripheral bypass grafting, vein graft failure rates remain high. The time window between harvesting and implantation provides a unique opportunity to modify grafts with
ex vivo
interventions, including cell seeding to decrease neo-intimal hyperplasia. However,
in vivo
monitoring of the migration of seeded cells remains a challenge. In order to determine the feasibility of MRI in detecting cells labeled with superparamagnetic iron oxide (SPIO) microparticles seeded on human veins, we performed MRI scans of labeled human vein cells and human vein specimens with seeded cells.

Human adventitial cells were isolated from vein specimens. Cultured adventitial cells were labeled with SPIO microparticles, and then incubated with segments of human vein specimens. Formalin fixed cell-seeded vein samples underwent MRI scans, along with suspended labeled cells.

Labeled human vein adventitial cells were suspended in Histogel with the final concentrations of 0.50, 0.83, and 1.83 X 10
6
cells/mL. T2-weighted images showed a gradual decrease of the signal intensity as the number of SPIO labeled cells increased. T2 values exhibited a linear negative correlation with SPIO labeled cell concentrations (R
2
= 0.99, Fig.1A).

Human vein samples seeded with SPIO labeled adventitial cells were embedded in Histogel. The cell numbers for seeding were 0.5, 1.0, and 2.0 X 10
6
. One vein without cell seeding served as the negative control. T2-weighted images revealed difference between seeded veins with various concentrations of SPIO labeling. The T2 values of the veins with 1.0 X 10
6
SPIO labeled cells and without cell seeding were 45 and 58 ms (Fig.1B).

In conclusion, seeded cells on human veins were able to be detected
ex vivo
via MRI, which lays a solid foundation for
in vivo
detection of seeded cells on implanted vein grafts.
Abstract 503: Single Cell Transcriptomics Uncovers The Functional Complexity Of Peripheral Veins And Arteries
Authors: Rojas Miguel G
Published: October 2023

Methods:
Twenty-six hundred cells from 4 human peripheral veins and 2 arteries were droplet-based single cell sequenced using the 10X Chromium protocol . Paired bulk RNA-seq from seven veins and arteries obtained from the same patient and immunofluorescence validated findings.

Results:
Arteries were richer in smooth muscle cell (39.1 vs. 13.8%) while vein contained abundant immune cells (14.5 vs. 39.8%). Both vessels had abundant fibroblasts, (28.1 vs. 34.5%), and endothelial Cells (16.5 vs. 11.1 %). Concordantly, contractility and ECM-related pathways were significantly highlighted in arteries while veins were enrich
ed in leukocyte migration, antigen presentation, neutrophil activation, and other immune related genes
. Quantitate immunofluorescence further confirmed SMC abundance in the arterial wall ( 56% vs. 18% n=11 pairs, p <0.001). There were two main fibroblast clusters equally abundant, the synthetic fibroblasts with high expression of ECM related genes (COL1A1, COL3A1), while the other cluster was defined by the immunomodulatory mediators (CXCL14, CFD). Vein were enriched for Mhem-like (CD163, HMOX1) macrophages important for detoxification and hemoglobin recycling and inflammatory macrophages (IL1B, CXCL8) were similar similarly distributed in both groups . We found a significant number of
vasa vasorum
(CD34, ICAM2) and luminal ECs(PECAM1,VWF) present in both groups. Furthermore, Intelectin-1 (ITLN1) was elucidated as a specific arterial endothelial marker, both at the single cell and bulk transcriptomic levels. There was also a minor lymphatic endothelial cell subcluster (PROX1
+
) detected in both veins and arteries. Finally, CellCall analysis identified 46 overrepresented ligand-receptor communication pathways in all clusters, including CXCL, FGF, IL-6, and complement signaling.

Conclusions:
We present a comparative single-cell atlas of peripheral human vein and arteries highlighting important vein enriched functions such as detoxification and hemoglobin recycling. We are now better able to identify cell subpopulations relevant for venous pathologies, without relying on extrapolations from arterial biology of distant models and focusing instead on the uniqueness of vein tissues.
Abstract 513: Localised Anticoagulation Via Gene Therapy: Towards Prevention Of Cardioembolic Stroke
Authors: Kadian Megha , Kok Cindy Y , Dupuy Alexander , Liu Xiaoming , Clayton Zoe E , Ravindran Dhanya , Rao Renuka , Favaloro Emmanuel , Curnow Jennifer L , Chong James , Passam Freda , Pasalic Leonardo , Kizana Eddy
Published: October 2023

Introduction:
The coagulation cascade thrives as the haematological gatekeeper ensuring equilibrium between blood flow and thrombosis. Under pathological conditions, the positive-feedback loops compound upon one another, disrupting this equilibrium and increasing the risk of thrombosis and embolic events. Cardiac thromboembolism persists as the culprit behind most ischemic strokes; a majority arising from the left atrial appendage. In order to combat this disease and overcome the limitations of systemic oral anticoagulants, we developed a novel anticoagulant viral vector to focally target cardiac regions of increased thrombotic potential.

Hypothesis:
Our novel recombinant lentiviral and adeno-associated viral vectors expressing thrombomodulin will induce a significant cell autonomous anticoagulant effect when expressed by endothelial cells of the left atrial appendage.

Methods:
The anticoagulant phenotype of these vectors was functionally assessed through
in vitro
and
ex vivo
platforms. For
in vitro
testing, a novel, cell-based version of the calibrated automated thrombogram assay, coupled with the overall haemostatic potential assay were utilised to determine thrombokinetics in cell culture models. For
ex vivo
testing, freshly isolated and characterized porcine left atrial appendage endothelial cells were used. These cells were used to endothelialise microfluidic devices, which were stimulated with TNF-α prior to perfusion with human whole blood. Changes in fibrin, platelets and neutrophils were investigated in the presence and absence of our novel anticoagulant viral vectors.

Results:
Experimental findings from our
in vitro
and
ex vivo
work showed a significant decrease in the endogenous thrombin potential of vector-transduced cells, coupled with a significant decrease in the velocity of thrombogenesis. Furthermore, transduced cells had a marked decrease in the peak rate of thrombin generation and a significant increase in the time taken to achieve peak thrombin generation.

Conclusion:
These findings show the impact of our anticoagulant vectors through inhibiting thrombosis and guide the development of focal gene therapy strategies to target thrombosis at its point of origin in the prevention of cardioembolic-stroke.
Abstract 516: Protein Energy Undernutrition Is Associated With Increased Mortality In Patients Admitted For Abdominal Aortic Aneurysm Rupture.
Authors: Amoah Joseph , Noureldin Sara , Somoza-Cano Francisco , Fatuyi Michael , Dekowski Sammir
Published: October 2023

Objectives:
Abdominal Aortic Aneurysm rupture is a surgical emergency associated with increased morbidity and mortality. Our study sought to estimate the impact of protein energy undernutrition (PEU) on the clinical outcome of patients admitted for abdominal aortic aneurysm rupture.

Methods:
Using the 2019 National Inpatients Sample Database, NIS was searched for adults with abdominal aortic aneurysm rupture as a principal diagnosis with or without PEU as a secondary diagnosis using ICD- 10 codes. Outcomes were in-hospital mortality, acute kidney injury (AKI), cardiogenic shock, CVA, myocardial infarction (MI), cardiac tamponade, acute hypoxic respiratory failure, intraoperative and post-procedure complications, and acute mesenteric ischemia.

Results:
3,880 patients were admitted for abdominal aortic aneurysm rupture. Of those, 13% had a history of protein energy undernutrition. Patients with PEU had an increase in in-hospital mortality (p=0.0064) which was statistically significant. There was an increased rate of acute kidney injury (p<0.0001), acute mesenteric ischemia (p=0.0005), intraoperative and post-procedure complication (p<0.0001), and acute hypoxic respiratory failure (0.0034). Patients with PEU also had an increased length of hospital stay (18 days, 95% CI 15 - 21days VS 6 days, 95% CI 6 - 7) and hospital charges ($437,070.8, 95% CI $373,662.5 - $500,479.1 VS $ 200,328.1 95% CI, $184,811.6 - $217,844.6). However, there was no difference in the rate of cardiogenic shock (p=0.0812), cardiac tamponade (p=0.5813), and myocardial infarction (p=0.3389)

Conclusion:
Per this study, Protein Energy Undernutrition is associated with increased mortality when compared to the general population admitted for abdominal aortic aneurysms. Early identification and prevention of PEU can reduce mortality associated with abdominal aortic aneurysm rupture.
Abstract 523: Phenotypic And Metabolic Switching Of Fibroblasts In Aortic Aneurysm
Authors: Mackay Cameron D , Patel Vaibhav B , Jadli Anshul S , Chen Yong-xiang , Belke Darrell D
Published: October 2023

Background:
Aortic aneurysm (AA) is a deadly cardiovascular disease that carries a tremendous burden worldwide. There are currently no approved pharmacological treatments to prevent or stop the progression of AA. Fibroblasts occupy the tunica adventitia layer of blood vessels and, during stress and in many cardiovascular diseases, fibroblasts become activated into myofibroblasts. Metabolic and mitochondrial abnormalities have also been observed in fibroblasts during various other cardiovascular diseases. The aim of this project is to assess the phenotypic and metabolic state of fibroblasts in aortic aneurysm using in vitro and in vivo murine models.

Methods and Results:
Aortic adventitial fibroblasts isolated from wild-type mice were used for all in vitro experiments. Angiotensin II (Ang II) was used as an in vitro inducer of fibroblast activation. Preliminary investigations suggest mitochondrial abnormalities in activated fibroblasts that ensued after in vitro Ang II treatment. Alpha smooth-muscle actin, a myofibroblast marker, was upregulated in fibroblasts following Ang II treatment. Activation of fibroblasts in response to Ang II was also associated with activation of Drp1, an inducer of mitochondrial fission. Using Seahorse extracellular flux analysis and Mitotracker Red staining, we identified alterations in mitochondrial structural and function in response to Ang II. 8-10-week-old apolipoprotein E knockout mice, implanted with micro-osmotic pumps to infuse Ang II for 4 weeks, were used to investigate the role of Drp1 in the onset and progression of AA. A subgroup of these mice received daily injections of mdivi-1. Preliminary results validate the successful generation of a murine model of TAA suitable for investigations using a pharmacological blocker of Drp1 activity. Echocardiographic assessment of aortic structure (diameter) and function (expansion index) validated the occurrence of TAA in angiotensin II-infused ApoEKO mice, which was found to be reduced in response to mdivi-1, an inhibitor of Drp1 activity, treatment.

Conclusion:
The results from this study could provide novel insight into the role of mitochondrial dynamics and metabolism in contributing to AA pathophysiology.
Abstract 525: Angiotensin Ii Type 1a Receptor Deletion In Vascular Smooth Muscle Cells Prevent Abdominal Aortic Aneurysm Formation Induced By Angiotensin Ii Plus β-aminopropionitrile
Authors: Okuno Keisuke , Torimoto Keiichi , Cicalese Stephanie , Hashimoto Tomoki N , Sparks Matthew A , Rizzo Victor J , Eguchi Satoru
Published: October 2023

Abdominal aortic aneurysm (AAA) is multifactorial disease resulting from changes in the aortic wall structure, such as medial degradation and inflammation, causing life-threatening AAA rupture. Angiotensin II (AngII) type 1A receptor (AT
1A
R) upon AngII binding mediates blood pressure elevation and several chronic cardiovascular diseases including AAA. Treatment with AngII plus β-aminopropionitrile (BAPN), a lysyl oxidase inhibitor, in normolipidemic C57BL6 mice similarly induces AAA. However, we have limited understanding whether smooth muscle AT
1A
R affects AAA formation induced by AngII plus BAPN. Thus, the subject of this study is to test the contribution of smooth muscle AT
1A
R to AngII-dependent AAA development in normocholesterolemic mice. At 8 to 12 week of age, smooth muscle AT
1A
R silenced mice (knock-in
Tagln-Cre
+/-
crossed with AT
1A
R flox/flox mice) and control mice were infused with AngII (1 μg/kg/min) via osmotic mini-pump for 4 weeks and treated with 1 g/L BAPN containing water for the first 2 weeks or sham-operated for mini-pump implantation. After 4 weeks, significant increases in maximum external aortic diameter and structural alterations were observed in the control mice, which was attenuated in smooth muscle AT
1A
R silenced mice (1.67 mm vs. 1.22 mm, p<0.05). AngII plus BAPN treatment increased perivascular collagen III positive cell ratio, which was normalized in smooth muscle AT
1A
R silenced mice (p<0.01). Evaluation of blood pressure at week 4 in conscious mice showed significant reduction in AngII-induced hypertension in smooth muscle AT
1A
R silenced mice (p<0.01). Furthermore, in contrast to control mice, no death duet to aortic rupture was observed in smooth muscle AT
1A
R silenced mice. In conclusion, our study has demonstrated the significant contribution of smooth muscle expressing AT
1A
R in mediating AAA development and rupture in response to AngII plus BAPN treatment. It is possible that attenuation of hypertension in the AT
1A
R silenced mice may provide additional protection against AAA rupture.
Abstract 538: Local Hydrogen Sulfide Therapy Augments Angiogenesis And Ischemic Wound Healing
Authors: Giacolone Joseph R , Matheson Benjamin , Kanagy Nancy L , Clark Ross M
Published: October 2023

Introduction:
Hydrogen sulfide (H
2
S) is an important signaling molecule in cellular O
2
sensing, wound healing and angiogenesis. Low H
2
S levels have been shown in diabetes and cardiovascular disease and H
2
S impairment may play a role in chronic non-healing ischemic wounds. H
2
S promotes angiogenesis in cell culture but
in vivo
data are lacking. We utilized a novel local H
2
S delivery method to investigate the effects of H
2
S therapy in rodents.

Methods:
Sprague Dawley rats (n=6-10/group) underwent dorsal myocutaneous ischemic flap wound creation under anesthesia. NaHS solution (100 μM) was delivered locally to the wound every 12 hours via implanted microtubing externalized and attached to a fluid delivery vest. Sham rats received saline solution while a third group received H
2
S blocker DL-propargylglycine (PAG). Laser speckle contrast images (LSCI) were recorded over 14 days. Flap tissue was collected for histologic analysis of tissue viability and degree of ischemic insult. Neovascularization was quantified by CD31 immunohistochemistry (IHC). VEGF protein expression was quantified by Western blot.

Results:
H
2
S treated flaps reperfused at a two-fold greater rate compared to sham and PAG treated flaps by LSCI (mean 12.9 flux/day vs. 6.9 flux/day, P=0.02). IHC mean microvascularity in sham flaps was 18 vessels/mm
2
compared to 47 vessels/mm
2
in H
2
S treated flaps (P=0.0092). Panniculus carnosis muscle thickness (408 μm vs. 234 μm) and myofibril count (56 vs. 38/100k μm
2
) were preserved in H
2
S treated flaps (P=0.02, 0.04). H
2
S treatment upregulated VEGF while PAG was associated with decreased expression.

Conclusion:
H
2
S treated flaps have improved wound revascularization, tissue healing and angiogenic signaling. Blockade of H
2
S results in poor revascularization and decreased angiogenic protein expression. H
2
S pathways may play a critical role in the pathogenesis of impaired wound healing and could represent a potential therapeutic target for ischemic wounds.
Abstract 545: Predictors Of Complications And Extended Length Of Stay Following Percutaneous Transluminal Renal Artery Angioplasty
Authors: Mustafa Ahmad , Wei Chapman , Khan Shahkar , Grovu Radu , Rizvi Taqi , Javed Aneeqa , Khan Danyal , Bjorklund Jessica , El-Sayegh Suzanne , Lafferty James , Weinberg Mitchell
Published: October 2023

Background:
Patients with Renal Artery Stenosis (RAS) who fail medical management may be evaluated for Percutaneous Transluminal Renal Artery Angioplasty (PTRA). Comorbidities increasing the risk of complications following PTRA have not been explored well.

Methods:
Patients undergoing PTRA for RAS were sampled using National Inpatient Sample (NIS) Database. Demographics, length of stay (LOS), and comorbidities were gathered using ICD-10 codes. Complications included heart failure, myocardial infarction, cardiac arrest, major bleeding, stent thrombosis, renal artery dissection/embolism, aortic dissection/rupture and atheroembolism. Extended LOS (ELOS) was defined as LOS greater than 4 days. Univariate and multivariate logistic regression analyses were used to identify predictors for complications and ELOS.

Results:
517 patients underwent PTRA. Most prevalent comorbidities were peripheral vascular disease, coronary artery disease and dyslipidemia. On multivariate analysis, comorbidities significant for predicting major complications were end-stage renal disease, chronic liver disease, heart failure and coagulable disorders whereas comorbidities significant for predicting ELOS were chronic lung disease, chronic liver disease, diabetes and coagulable disorders (Figure 1).

Conclusion:
As we continue to identify the ideal candidates for PTRA, it is important to consider the comorbidities that predispose these patients to increased periprocedural complications and ELOS.