bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 119: Into The Wormhole: Understanding SR-B1’s Cholesterol Transport Functions Through Structural Studies
Authors: Powers Hayley R , Jenjak Shawn , Volkman Brian , Sahoo Daisy
Published: November 2022

Scavenger receptor Class B type 1 (SR-B1) is a glycosylated integral membrane protein that serves as the primary receptor for high density lipoprotein (HDL) and plays a central role in the reverse cholesterol transport pathway (RCT). In RCT, HDL removes cholesterol within atherosclerotic plaques and delivers it to the liver for excretion. HDL-cholesterol is delivered into hepatocytes through interaction with SR-B1, highlighting the need to improve our understanding of SR-B1 structure-function relationships to effectively lower plasma cholesterol levels and cardiovascular disease risk. Currently, structural information about SR-B1 remains limited to a structure of short transmembrane-spanning peptide segment, homology models, and transient transfection studies in cultured cells. Towards our long-term goal of resolving a full-length structure of human SR-B1, we have expressed and purified full-length human SR-B1 from an Sf9 insect cell baculoviral infection system. To verify function, human full-length SR-B1 expressed in plated Sf9 cells displayed a significant increase in DiI-HDL binding and DiI-lipid uptake compared to empty vector controls. Using PFO-PAGE, we demonstrated that SR-B1 forms dimers and higher order oligomers in Sf9 cells. After functional verification, we successfully incorporated purified protein into detergent micelles. Thermal shift assays revealed purified SR-B1 remained stable over a 6-week period. Purified SR-B1 also maintained the ability to bind to apolipoprotein A-I (the main protein component of HDL) and holoparticle HDL with high affinity (K
D
= 42.77 ± 7.15 μg/mL) by microscale thermophoresis (MST). Additionally, SR-B1 was able to bind oxidized low density lipoprotein (oxLDL). Further, while glycosylation remains important for SR-B1 expression, we are the first to demonstrate by MST that SR-B1’s glycosylation status did not impact HDL binding affinity (K
D
of 42.77 ± 7.15 μg/mL for glycosylated vs. 41.25 ± 4.92 μg/mL for unglycosylated SR-B1). Availability of functional purified human SR-B1 is a critical step towards a high-resolution structure and these studies lay the foundation for understanding the dynamics of SR-B1-mediated cholesterol transport and novel ways to modulate cardiovascular disease risk.
Abstract 135: Sex-specific Differences In The Role Of TSP1 On Metabolic Syndrome-induced Atherosclerosis
Authors: Gupta Shreya , Khanal Saugat , Mathias Amy , Lallo Jason , Raman Priya
Published: November 2022

Metabolic syndrome (MetS) amplifies risks of atherosclerotic complications. MetS induces abnormal vascular smooth muscle cell (VSMC) migration and proliferation, hallmark of SMC de-differentiation, crucial in the pathogenesis of atherosclerosis. Despite multiple studies revealing sexual dimorphism in atherosclerosis, underlying sex-specific mechanisms are poorly understood. We previously reported a protective role of a potent proatherogenic protein, thrombospondin-1 (TSP1), in hyperglycemia- or hyperleptinemia-induced atherosclerosis. The goal of the present study was to interrogate sex-specific differences in the role of TSP1 on MetS-induced atherosclerosis. We generated a mouse model of combined MetS and atherosclerosis (KKAy
+/-
ApoE
-/-
) by crossing obese hyperglycemic agouti KKAy
+/-
mice with atherosclerotic ApoE
-/-
. Male and female age-matched MetS KKAy
+/-
ApoE
-/-
and non-MetS KKAy
-/-
ApoE
-/-
mice were placed on standard lab diet from 4-24 wks age. After overnight fasting, mice were harvested; plasma, aorta and heart were collected for various studies. In male MetS KKAy
+/-
ApoE
-/-
, increased aortic root ORO-positive lipid burden, reduced LMOD (SM contractile marker) and SRF (transcriptional activator of SM differentiation) expression in aortic vessels associated with augmented TSP1 expression vs. non-MetS KKAy
-/-
ApoE
-/-
mice. In contrast, no significant differences in lesion lipid burden, TSP1, LMOD and SRF expression were detected between the female genotypes. To delineate whether TSP1 plays a direct role in SMC de-differentiation in MetS, we next crossed KKAy
+/-
with TSP1
-/-
to generate MetS mice with and without global TSP1 deletion. Male and female KKAy
+/-
TSP1
+/+
(with intact TSP1) and KKAy
+/-
TSP1
-/-
(lacking TSP1) on standard lab diet from 4-18 wks age were harvested at endpoint for aortic tissue collection. In male KKAy
+/-
TSP1
-/-
aortic vessels, SM contractile marker expression (LMOD, calponin) was markedly increased vs. KKAy
+/-
TSP1
+/+
, suggesting reduced SMC de-differentiation. In contrast, TSP1 deletion had no effect on SM contractile marker expression in female MetS KKAy
+/-
. Together, these data suggest a sex-specific role of TSP1 on SMC de-differentiation and atherosclerotic lesion formation in MetS.
Abstract 137: Role Of Cellular Communication Network Factor 2 (ccn2) And Male Sex On Flow-mediated Arterial Remodeling And Atherosclerotic Plaque Formation
Authors: Brewster Luke P , Li Feifei , Omojola victor , Kumar Sandeep , Gleason Rudolph L , Jo Hanjoong
Published: November 2022

CCN2 or connective tissue growth factor is up-regulated in response to disturbed flow and arterial stiffening. Carotid artery disease and peripheral arterial disease (PAD) are associated with disturbed flow and arterial stiffening. We have identified a key role for CCN2 in stiffened arterial remodeling and flow-mediated focal atherosclerotic plaque formation in translational models of carotid and PAD and in patient carotid plaques/PAD arteries. The objective of this project was to determine the modifiability of CCN2 pathways in pathologic arterial remodeling and atherosclerosis.

Methods:
To test our hypothesis that CCN2 was an important mediator of pathology arterial remodeling in vascular disease , we utilized murine models of stiffened arteries (fibulin-5 KO) in atherogenic conditions (PCSK9 + HFD) and clinical arterial tissue from patients with PAD and carotid artery disease.

Results:
Disturbed flow increases EC expression of CCN2 3.5 fold in WT carotid arteries. This is doubled again in Fibulin 5 KO (
KO
) carotid arteries. These KO carotid arteries are ~3x stiffer than littermate control arteries, which is a similar ratio of PAD compared to healthy control arteries (
1A
). Under disturbed flow and atherogenic conditions KO arteries had more elastin breaks (P=.04), greater plaque area (P=.007) and more lipid deposition (P=.02), and Glagov’s outward remodeling (P=.02) (
1B/C
). This was most pronounced in male mice. CCN2 staining is significantly increased in PAD arteries and carotid endarterectomy plaques (P<.05) (
1D/E
). To evaluate the modifiability of CCN2, we utilized CCN2
ECKO
animals. We found that male (but not female) CCN2
ECKO
animals had significantly less atherosclerotic plaque burden (P=.001) and similar elastin breaks/lipid deposition compared to littermate controls (
1F/G
).

Conclusion:
CCN2 may play an important and modifiable role in flow-mediated pathologic arterial remodeling and focal atherosclerotic plaque burden.
Abstract 140: Plasma Interleukin-6 Levels And Cardiovascular Events In The Cirt Trial: Consistent Associations For Incident Coronary, Cerebrovascular, And Peripheral Artery Disease
Authors: Marinho Lucas , Aday Aaron W , Cook Nancy R , Morrison Alan R , Narula Navneet , Narula Jagat , Bartoli-Leonard Francesca , Aikawa Elena , Beach Jocelyn M , Ridker Paul M , Pradhan Aruna D
Published: November 2022

Introduction:
Inflammation is causally related to atherothrombosis. Interleukin-1β (IL-1β) and interleukin-18 (IL-18) require NLRP3 inflammasome for activation and have downstream effects on interleukin-6 (IL-6), a marker previously associated with high risk of coronary artery and cerebrovascular disease (CCVD). However, data pertaining to peripheral artery disease (PAD) are sparse and could offer druggable targets in this disease.

Methods:
We conducted a prospective cohort study of 4248 patients with type 2 diabetes or metabolic syndrome and prior coronary artery disease who participated in the NIH-funded Cardiovascular Inflammation Reduction Trial (CIRT). Participants were followed for up to 5 years for incident CCVD and symptomatic PAD events. Randomized treatment with low-dose methotrexate (vs. placebo) had no effect on event rates or plasma levels of inflammatory biomarkers. Baseline levels of IL-1β, IL-18, and IL-6 were tested for association with incident vascular events. Kaplan-Meier curves and Cox proportional hazards models (adjusted for traditional risk factors) were estimated.

Results:
In multivariable adjusted analyses, hazard ratios for the lowest (referent) to highest baseline quartiles of IL-6 were 1.0, 1.5, 1.8, and 2.0 (p-trend <0.001) and 1.0, 1.2, 2.5, and 2.0 (p-trend = 0.04) for incident CCVD (N=349) and PAD (N=87), respectively. Baseline IL-6 levels above vs. below the median (2.50 pg/ml) were associated with a 56% increased risk of CCVD (HR 1.56, 95% CI 1.26-1.93) and a 113% increased risk PAD (HR 2.13, 95% CI 1.36-3.32) (Figure 1). Baseline levels of IL-1β and IL-18 did not associate with incident CCVD or PAD.

Conclusion:
In this contemporary cohort of secondary prevention patients, elevated IL-6 was associated with both incident CCVD and PAD. These data support exploration of direct IL-6 inhibition for PAD prevention, a strategy currently being pursued to reduce risk of coronary artery and cerebrovascular disease.
Abstract 149: Type Viii Collagen Increases The Risk Of Arteriovenous Fistula Failure In End-stage Kidney Disease Patients
Authors: Martinez Laisel , Rojas Miguel , Tabbara Marwan , Pereira-Simon Simone , Santos Falcon Nieves , Rauf Mohd A , Zigmond Zachary , Vazquez Padron Roberto I
Published: November 2022

Introduction:
The arteriovenous fistula (AVF) is the lifeline for patients with end-stage kidney disease (ESKD) requiring hemodialysis therapy. The mechanism of “arterialization”, by which a vein transforms into an AVF after anastomosis, is one of the most understudied transformative processes in vascular biology despite the urgent need for new interventions to facilitate and accelerate fistula usability to improve the life of these patients.

Hypothesis:
Dysregulation of type VIII collagen biosynthesis increases the risk for failure in newly created AVFs

Methods and Results:
We first deciphered the transcriptional transformation of the pre-access vein after arterial anastomosis using paired venous samples from 38 CKD patients undergoing surgeries for two-stage AVF creation. A total of 3,637 transcripts were differentially expressed (DE) between veins and AVFs in pairwise analyses (log2FC ≥ 1 or ≤ -1, FDR <0.05). Gene set enrichment analyses revealed transcriptional activation of genes related to vasculature development, ECM remodeling, SMC migration, and endothelial cell (EC) proliferation and migration. Two hundred and eighty-four DE transcripts belonged to the matrisome family of genes. These included 20 collagens, 52 ECM glycoproteins, 10 proteoglycans, 58 ECM regulators, 27 ECM-affiliated proteins, 26 proteoglycan biosynthetic/modifying enzymes, and 91 secreted factors. Next, we sought to identify the changes in gene expression during the vein to AVF transformation that were relevant to maturation failure. Pairwise differential gene expression analyses conditional to outcome identified 102 DEGs in association with AVF failure (log2FC ≥ 1 or ≤ -1, FDR <0.05). Eight genes in this group increased more in AVFs that failed compared to those that matured. Accumulation of COL8A1 was confirmed around medial SMC by IHC. This finding was further validated using an independent tissue cohort, which demonstrated significantly higher collagen VIII deposition in stenotic areas from resected AVF fragments compared to adjacent non-stenotic tissues.

Conclusions:
Vascular accumulation of type VIII collagen in the AVF wall after anastomosis strongly increases the risk of failure.
Abstract 156: Prenatal Exposure To Electronic Delivery Systems Modulates Platelet Reactivity, Epigenetics And Gene Expression In Mice
Authors: Alarabi Ahmed , Ali Hamdy , Lozano Patricia , Khasawneh Fadi T , Alshbool Fatima Z
Published: November 2022

Tobacco smoking is a major public health threat and is associated with cardiovascular disease (CVD)-related morbidity and mortality. Being the single most preventable risk factor for CVD, a trend towards tobacco harm reduction started years ago. Thus, while tobacco usage has declined, that of Electronic Delivery Systems (ENDs) experienced widespread popularity, especially among pregnant women and women of childbearing age. This is alarming as pregnant women view them as safe(r) compared to cigarettes; which we now know not to be evidence-based. Thus, it is paramount to establish the health impact/CV safety of prenatal exposure to ENDs. This was addressed by employing a well-established ENDs inhalation system and performing whole-body exposures of 150 puffs/day on C57/BL6 mice, under prenatal/
in utero
experimental settings throughout the gestational period. Our results show that platelets from the offspring of prenatally ENDs-exposed mice are hyperactive with enhanced: aggregation, dense and α granule secretion, activation of the αIIbβ3 integrin, clot retraction and phosphatidylserine expression, when compared to clean air exposed platelets, which suggests a prothrombotic state. Moreover, we observed that ENDs-exposed platelets are resistant to the inhibitory effects of prostacyclin, and that they exhibit increased activation of Akt and ERK. Furthermore, and consistent with these findings, ENDs were also found to shorten the thrombosis occlusion and bleeding times, relative to controls. Interestingly, we also observed alternations in the platelet miRNome, transcriptome and enrichment of platelet activation pathways, as follows: 1) miR-221 is differentially expressed, unlike mirR-146b; 2) Itgβ3, Src, and Rap2b transcripts are upregulated; and 3) oxidative phosphorylation, and platelet activation pathways are enriched. Taken together, our data demonstrate for the first time that prenatal exposure to ENDs modulates platelet reactivity and increase the risk of thrombogenesis, in part via impacting the platelet epigenetics and genetics. Thus, the negative health consequences of ENDs should not be underestimated, and warrant further investigation. These findings should also guide policy development for regulating exposure to ENDs.
Abstract 190: Deletion Of Shp-1 Prevents Diabetes-induced Endothelial Cell Senescence And Restores Blood Flow Reperfusion Following Ischemia
Authors: Nadeau Alexandre , Lizotte Farah , Geraldes Pedro M
Published: November 2022

Introduction:
Ischemia due to narrowing of the femoral artery and distal vessels is also a major cause of peripheral arterial disease and morbidity affecting patients with diabetes. Diabetes-induced premature senescence of endothelial cells has been shown as a potential mechanism of poor angiogenic response to ischemia. Our laboratory has previously shown that hyperglycemia reduced vascular endothelial growth factor (VEGF) activity in ischemic muscle of diabetic mice, which was associated with increased SHP-1 expression, a protein tyrosine phosphatase. The objective of this study is to evaluate the impact of SHP-1 deletion on endothelial cell function and senescence both
in vitro
and
in vivo
.

Methods:
Non-diabetic (NDM) and 3 months diabetic (DM) mice with deletion of SHP-1 specifically in endothelial cells (EC) were used. Ligation of the femoral artery was performed, and blood flow reperfusion was measured by laser Doppler for 4 weeks. Primary EC were exposed to normal (5.6 mM; NG) or high glucose concentrations (25 mM; HG) for 48 h, in normoxia (20% oxygen) or hypoxia (1%) for the last 16h in presence of VEGF.

Results:
Blood flow reperfusion and limb function (voluntary running wheel) were reduced by 50% and 91%, respectively in DM mice as compared to NDM mice. Specific EC deletion of SHP-1 restored blood flow reperfusion by 55%, limb function by 37% and capillary density in DM mice. Moreover, ablation of SHP-1 only in EC prevented diabetes-induced p21 expression and reduction of Nrf-2. In cultured EC
,
overexpression of dominant negative of SHP-1 prevented HG-induced inhibition of proliferation, migration, and VEGFR2/Akt phosphorylation following VEGF stimulation. In addition, the expression of senescence markers (increased p21 and beta-galactosidase; reduced Nrf-2) in EC exposed to HG levels were reversed by overexpression of SHP-1 dominant negative.

Conclusion:
Ablation of SHP-1 expression in EC resulted in the prevention of hyperglycemia-induced EC senescence, reduction of VEGF actions, poor collateral vessel formation and blood flow reperfusion.
Abstract 191: Cops5-mediated Nuclear Exclusion Of P27 Is Responsible For The Promotion Of Vascular Smooth Muscle Cells Proliferation By Cops8 Hypomorphism
Authors: Giri Samiksha , Suo Chao , Martin Douglas S , Wang Xuejun
Published: November 2022

Background:
The COP9 signalosome (CSN) holocomplex is formed by 8 stoichiometric protein subunits: COPS1 through COPS8. Despite the strong evidence linking CSN to cell cycle regulation, direct study of CSN in VSMC proliferation is lacking. Our prior data show that vascular injury significantly increases Cops8 proteins in wild-type mice and induces more severe neointimal thickening and increased VSMC proliferation in Cops8 hypomorphic (Cops8-hypo) mice compared to non-hypomorphic littermates (CTL), suggesting an important role of the CSN in vascular pathology. The present study was carried out to further elucidate the mechanism underlying this phenomenon. We hypothesize that increased COPS5-mediated nuclear exclusion of p27 contributes to the promotion of VSMC proliferation by COPS8 hypomorphism.

Methods and Results:
We isolated and cultured VSMCs from the COPS8-hypo and CTL mice and used the treatment with platelet-derived growth factor (PDGF-BB) to mimic vascular injury. In response to PDGF-BB, COPS8-hypo VSMCs displayed increased hyperproliferation compared to CTL VSMCs. We detected markedly smaller nuclear to cytoplasmic ratio for p27 and COPS5; and native gel electrophoresis revealed increases in the cytoplasmic COPS5 mini-complex in hypomorphic VSMCs. Nuclear export inhibition with leptomycin attenuated the increases in PCNA and cytoplasmic p27 by COPS8 hypomorphism. Interestingly, CSN deneddylase inhibitor (COPS5i-3) did not have an effect on the proliferation of COPS8-hypo VSMCs but suppressed the PDGF-BB induced PCNA increases in CTL VSMCs. We then, constructed plasmids expressing full-length COPS5 harboring a mutation either in its nuclear export sequence (NES) or in its MPN domain. We found that genetically disabling the nuclear-export but not the deneddylase activity of COPS5 suppressed the hyperproliferation and restored p27 nuclear localization of hypomorphic VSMCs. These data suggest that promoting the nuclear export of p27 by COPS5 rather than the deneddylation activity of COPS5 is responsible for the greater PDGF-BB induced proliferation of COPS8-hypo VSMCs.

Conclusion:
COPS8 hypomorphism promotes the proliferation of VSMCs in injured vessels through the increased nuclear exclusion of p27 mediated by COPS5.
Abstract 215: Enhanced Atherosclerosis, Inflammation And Oxidized Phospholipid Reactivity In Female Transgenic Mice Expressing Human Lp(a)
Authors: Assini Julia M , zing chuce , Clark Justin , Boffa Michael , Koschinsky Marlys L , Boren Jan
Published: November 2022

Despite increased clinical interest in the unique lipoprotein Lp(a), many questions remain about the molecular mechanisms by which it contributes to atherosclerotic cardiovascular disease. To address this knowledge gap we generated transgenic mice expressing both human apolipoprotein(a) (apo(a)) and human apoB100
(Tg(LPA
+/0
;APOB
+/0
)),
with pathogenic levels of plasma Lp(a) (mean 134 mg/dL; range 87–250 mg/dL). Female mice (n=12/group) were fed a high-fat, high-cholesterol diet and injected weekly with an antisense oligonucleotide targeting the
Ldlr
for 12 weeks to induce atherosclerosis. FPLC demonstrated that both

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

and

Tg(APOB
+/0
)

mice exhibited proatherogenic lipoprotein profiles (increased cholesterol-rich VLDL and LDL-sized particles) and developed complex lesions in the aortic sinus. However, Oil Red O-staining reveled significantly increased plaque area (+23%) in

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

mice, compared to

Tg(APOB
+/0
)

mice. Examination of lesion pathology indicated more severe disease in
Tg(LPA
+/0
;APOB
+/0
)
mice, with significantly increased necrotic core size (41.8±2.8% vs. 33.4±2.7%, (p<0.05)) and calcium deposition (2.96±0.3% vs. 1.79±0.4%, (p<0.05)). Furthermore, Picrosirius Red staining showed a significant, 1.2-fold increase in collagen abundance in lesions of
Tg(LPA
+/0
;APOB
+/0
)
vs.
Tg(APOB
+/0
)
mice. Immunohistochemistry on the lesions demonstrated that in
Tg(LPA
+/0
;APOB
+/0
)
mice, Lp(a) colocalized with apoB100, CD68-positive macrophages and E06/oxidized phospholipid (OxPL). Compared to
Tg(APOB
+/0
)
mice, quantitation of the E06/OxPL-positively stained area revealed a significant increase (+39%) in
Tg(LPA
+/0
;APOB
+/0
)
mice. Female
Tg(LPA
+/0
;APOB
+/0
)
mice also had a 2-fold increase in E06/OxPL apo(a) in plasma, as well as enhanced circulating levels of the proinflammatory cytokines TNFα (1.2-fold increase) and MCP-1 (2.8-fold increase) compared to
Tg(APOB
+/0
)
mice. Taken together these data suggest an exacerbated pro-inflammatory phenotype in female
Tg(LPA
+/0
;APOB
+/0
)
mice, potentially contributing to the development of more advanced lesions. Further study of this novel mouse model will aid in the understanding of the fundamental role of elevated Lp(a) in atherogenesis.
Abstract 256: Biomarker Shift In Convalescent Heart Patients Recovering From Sars-cov-2 Infection
Authors: Ruskamp Ryan J , Duryee Michael J , Navarro Quinn S , Thiele Geoffrey , Anderson Daniel R
Published: November 2022

Background:
Infection of SARS-COV-2 in patients with heart disease has the ability to develop thrombosis leading to heart failure. The development of these thrombi is found in the small vessels of these hearts, mimicking similar traits to coronary microvascular dysfunction (CMD). However, there are a number of patients with heart failure that fully recover from SARS-COV-2 infection without thrombus formation. Therefore, it was the purpose of this study to look at unique biomarkers that may potentially help diagnosis any long-term effects from SARS-COV-2 infection in patients with heart disease.

Experimental Design:
CMD (N=19) and convalescent SARS-COV-2 heart disease (N=14) patient serum samples were obtained from the Nebraska Cardiovascular Biobank and Registry. Samples were tested for biomarkers using a MesoScale Diagnostic (MSD) Cytokine Array as per manufacture directions. These markers included; Endothelin-1, NT-proBNP, UPAR, CRP, SAA, sICAM-1, sVCAM-1, IFN-γ, IL-1β, IL-10, IL12-p70, IL-2, IL-4, IL-5, IL-8 and TNF-α. Statistical analysis was performed using GraphPad and differences were determined using Student’s T-test.

Results:
The cytokines, NT-proBNP, CRP, SAA, sICAM-1, IFN-γ, IL-1β, IL-10, IL-2, IL-4 and TNF-α demonstrated no significant difference between CMD and SARS-COV-2 patients. Significant increases were observed for UPAR (p<0.05), sVCAM-1 (p<0.05), IL-8 (p<0.05) and IL-12p70 (p<0.01) in the SARS-COV-2 patients compared to CMD. A significant decrease was observed for endothelial-1 (p<0.05) and IL-5 (p<0.05).

Conclusions:
Increased levels of UPAR, sVCAM-1, IL-8 and IL-12p70 would indicate significant inflammation and tissue remodeling following SARS-COV-2 infection. However, lower levels of endothelial-1 suggests these SARS-COV-2 patients are most likely not susceptible to microvascular blockage of vessels.
Abstract 267: Altered Macrophage Response Associated With Impaired Skeletal Muscle Regeneration In Murine Model Of Limb Ischemia
Authors: Peters Derek , Xu Yueyuan , Lin Xin , Wei Xiaolin , Diao Yarui , Southerland Kevin
Published: November 2022

Introduction:
Skeletal muscle regeneration in the ischemic limb is a complex complex process dependent on coordinated intercellular communication involving an array of cell types. These interactions are not well understood, and the development of novel regenerative therapies for peripheral arterial disease will require new insight into the cellular heterogeneity and intercellular signaling that occurs in the ischemic limb. Macrophages play a role in orchestrating critical events in muscle regeneration; hence we sought to characterize their transcriptional signature and identify candidate signaling pathways in the context of limb ischemia.

Methods:
We applied single-cell RNA sequencing (scRNA-seq) to skeletal muscle obtained from regenerative (C57BL/6) and non-regenerative (BALB/c) mouse limbs at multiple time points following ligation of the femoral artery. We used CellChat, a computation tool, to predict signaling pathway activity between macrophages and muscle satellite cells (MuSC) based on our single-cell data.

Results:
We identified 12 distinct macrophage populations in the regenerative and non-regenerative limb, including strain-specific macrophage clusters associated with the response to ischemia. Regenerative macrophages displayed a proliferative phenotype, while non-regenerative macrophages exhibited a persistent pro-inflammatory phenotype. Additionally, we identified several candidate signaling interactions between specific macrophage clusters and MuSCs. Notable potential interactions included enhanced FN1 to SDC4 and THBS1 to SDC4 signaling between regenerative macrophage and MuSCs, as well as OPN to CD44 signaling between non-regenerative macrophages and MuSCs.

Conclusions:
Our study maps the dynamic macrophage response in the ischemic limb at single-cell resolution. This provides a valuable resource to investigate macrophage-mediated mechanisms of skeletal muscle regeneration in response to limb ischemia.
Abstract 268: Single Cell Gene Expression Of Brachial Artery In Response To Increased Shear Stress
Authors: Rozowsky Jared , Hu Qiongyao , O'Malley Kerri , Anderson Erik , Fazzone Brian , Berceli Scott A
Published: November 2022

Blood flow dynamics modulate vascular development, homeostasis, and contribute to vascular pathobiology. However, it is unknown how shear stress influences cellular transcriptomics of the arterial wall. Two-stage hemodialysis access surgeries provide an opportunity to compare native vessel to vessel after exposure to significantly altered hemodynamics. Single-cell sequencing was performed on brachial artery samples obtained from 3 patients at the time of 1
st
and 2
nd
stage surgery. Arterial cellular composition and temporal changes in gene expression were quantified after sequencing and functional analyses were performed on differentially expressed genes. Compositional changes and gene expression dynamics were highest in the smooth muscle cell (SMC) populations followed by fibroblasts. SMCs were clustered into contractile (classical) and “modified” populations, with the latter having lower expression of contractile markers. Downregulation of transcription factor ARNTL was the most significant controller in contractile SMCs and fibroblasts, and second behind HIF1α in modified SMCs. Macrophages had a significant increase in composition, but few dynamic genes suggest a return to quiescence. Endothelial cells showed minimal changes with dynamic genes responding to mechanical stimulus. Ingenuity pathway analysis revealed activation of EIF2 signaling in both SMC and fibroblast clusters, promoting translation. The top divergent pathway among SMC clusters is PI3/AKT signaling, with increased vascular remodeling, proliferation and cell death in the contractile group. Cellular processes are overall downregulated in modified SMCs, which represent the majority of SMCs after vascular adaption. This study is the first single-cell characterization of normal human artery and its cellular response to increased hemodynamic forces, specifically detailing cellular compositional changes and their associated dynamic genes.
Abstract 284: Transcriptional Profiling Of Human Abdominal Aortic Aneurysm Tissue Reveals Distinct Extracellular Vesicle-Derived MicroRNA Cargo
Authors: Botts Steven R , Raju Sneha , Prajapati Kamalben , Breda Leandro C , Fish Jason E , Howe Kathryn L
Published: November 2022

Aim:
Abdominal aortic aneurysm (AAA) contributes to significant post-rupture mortality in aging populations. AAA management with watchful waiting and surgical repair is based on our limited understanding of disease processes, and current research foci including extracellular vesicles (EVs; nano-sized packages of proteins, RNAs, and lipids that facilitate intercellular communication) may aid in developing novel AAA therapies. To characterize this regulatory cargo, we isolated EVs from human AAA tissue or control aortic punch biopsies and profiled EV content with microRNA (miRNA) sequencing to identify dysregulated pathways.

Methods:
The study was approved by the University Health Network Research Ethics Board. EVs were isolated from human AAA or aortic punch tissue and enriched using size exclusion chromatography (SEC; qEVoriginal columns 70 nm, Izon Science Ltd.) (n=3). EV size and concentration were determined using nanoparticle tracking analysis (NTA; NanoSight NS300, Malvern Panalytical Ltd.). EV-miRNA sequencing was performed with Illumina NextSeq (HTG Molecular Diagnostics Inc.) and analyzed using Partek Genomics Suite (v.10) and MIENTURNET (19-11-25).

Results:
Patients were selected for infrarenal AAA requiring surgical repair (AAA) or coronary artery disease requiring bypass graft surgery (control). EV size and concentration were confirmed with NTA. Principal components and gene set analyses revealed distinct clustering of tissue types with 901 and 687 miRNAs enriched in AAA and control samples, respectively. Pathway prediction using established AAA miRNAs (e.g., miR-122, miR-146a, and miR-503) identified significant interactions with proaneurysmal signaling pathways (e.g., PI3K-AKT, JAK-STAT, and HIF-1) as well as cell senescence and adhesion processes (FDR < 0.05).

Conclusion:
EV-derived miRNAs from patients with AAA prominently associate with cell signaling, senescence, and adhesion pathways in aneurysm pathogenesis. To our knowledge, this is the first study to profile the EV-miRNA landscape in human AAA tissue. Further investigation will explore EV-miRNAs as mediators of communication between distinct vascular cell populations that contribute to AAA development.
Abstract 292: LRP1 Prevents Aortic And SMA Aneurysms By Modulating Excessive Ang-II Signaling
Authors: Zhang Jackie M , Migliorini Mary , Hampton Brian , Ni Fenge , Ucuzian Areck , Strickland Dudley
Published: November 2022

Objectives:
LRP1 (low-density lipoprotein receptor-related protein 1) is known to contribute to vascular homeostasis and plays a protective role against the development of aneurysms. LRP1 has specifically been associated with abdominal aortic aneurysms in GWAS studies. We and others have also previously reported that mice with a conditional smooth muscle cell (SMC) knock-out of LRP1 (smLRP1-/-) exhibit vascular wall dysfunction and aortic dilation. In this study, we sought to investigate the molecular mechanisms by which LRP1 regulates vessel wall integrity in hopes of identifying potential therapeutic targets against aneurysm progression.

Approach and Results:
Analysis of proteomic data of the descending thoracic aorta, abdominal aorta, and superior mesenteric artery (SMA) of smLRP1-/- mice confirmed excess activation of the angiotensin signaling pathway. To verify these observations, we designed experiments to determine if losartan, an angiotensin-II receptor blocker, could prevent aneurysm progression in smLRP1-/- mice. Following treatment, we analyzed the aortic vasculature from LRP1+/+ and smLRP1-/- adult mice (16-weeks of age) with or without treatment. Without losartan, smLRP1-/- mice developed spontaneous aortic and SMA aneurysms as demonstrated by increased vessel dilation, vessel medial thickening and significant fragmentation of the elastic lamina. We found that when treated with losartan, smLRP1-/- mice had a complete restoration of medial layer thickness and a partial decrease of elastin fragmentation compared to untreated smLRP1-/- mice. Furthermore, losartan completely blocked SMA aneurysm formation in smLRP1-/- mice.

Conclusions:
Our findings suggest a critical role for LRP1 in maintaining vascular homeostasis and preventing aneurysm formation through its regulation of the angiotensin signaling pathway. Further studies will need to investigate the connection between LRP1 and the angiotensin signaling pathway in aneurysm formation to further understand the complex pathophysiology and potentially develop effective therapeutic targets for this disease.
Abstract 297: Systematic Review And Mendelian Randomization Of Plasma Biomarkers To Predict Their Causal Role In Peripheral Artery Disease Pathophysiology
Authors: Sharma Pranav , Levin Michael , Klarin Derek , Voight Benjamin F , Damrauer Scott M
Published: November 2022

Introduction:
Observational analyses have described hundreds of biomarkers for Peripheral artery disease (PAD). These studies can be limited by sample size, lack of replication, residual confounding, and reverse causality. To assess this we performed a systematic review of the literature and leveraged genetic approaches to causal inference.

Methods:
We performed a systematic literature review for terms related to PAD and/or biomarkers using Pubmed, Cochrane, and Embase, followed by manual review to extract biomarkers and their direction of effect. To test for evidence of causality we employed 2 sample Mendelian randomization (MR). We developed genetic instruments for the biomarkers by mapping them to genome wide association studies (GWAS) of circulating biomolecules agglomerated in the IEU Open GWAS project. We tested the association of the genetic instruments with PAD using summary statistics from a GWAS of 31,307 individuals with and 211,753 individuals without PAD in the VA Million Veteran Program. We employed Wald ratio or inverse-variance weighted MR; weighted median and weighted mode methods were applied as sensitivity analyses.

Results:
After manual review, we identified 159 unique papers mentioning 268 unique PAD biomarkers. We mapped 76 biomarkers to genetic data, 19 of which were nominally associated with PAD (p < 0.05). After accounting for multiple testing (FDR < 0.05) 12 remained significant, of which only 7 had concordant directions of effect with published reports: ApoB, ApoA1, HDL, triglycerides, Von-Willebrand factor, cadherin-5, and b2-microglobulin.

Conclusion:
This systematic review paired with genetic causal inference illuminates key biomarkers causally relevant to PAD, and highlights discrepancies between observational and genetic findings. This highlights the importance of rigorous analysis of observational biomarker data and the opportunity to leverage human genetics to inform these studies.
Abstract 299: Microvessel Oxidative Stress Predicts Changes In Leg Function Of Patients With Peripheral Arterial Disease After Supervised Exercise Therapy
Authors: Schieber Molly , Myers Sara A , Li Shuai , Pipinos Iraklis I , Casale George
Published: November 2022

Our objective was to evaluate baseline oxidative stress in the microvessels of patients with Peripheral Arterial Disease (PAD) 1) in association with Heme Oxygenase-1 (HO-1), a critical cytoprotective molecule for oxidative stress 2) as a predictor of the functional and pathophysiologic changes that occur in response to six months of Supervised Exercise Therapy (SET). We hypothesized that accumulated oxidative stress in the microvessels of PAD at baseline would predict changes in walking performance, myofiber pathology, and limb function at the end of a six-month program of SET.

Methods:
Twelve claudicating patients received six months of SET per ACC/AHA guidelines. Before and after SET, patients were evaluated for leg biomechanics, overground walking capacity (six-minute walking distance, SMWD), and maximum walking time on a treadmill. Subsequently, their more affected calf muscle was biopsied for quantification of HO-1 and oxidative stress (carbonyl content) in both myofibers and microvessels. We evaluated the association between HO-1 expression and carbonyl content with correlation and multiple regression.

Results:
HO-1 expression and Carbonyl content were strongly associated in the microvessels (n=1400, r=0.98, p<0.001). Increasing Carbonyl content in the microvessels of each subject was associated with increasing HO-1 expression. In a subset of patients, HO-1 expression increased more slowly with increasing carbonyl content. Pre-SET Oxidative Stress in microvessels was a significant predictor of SET mediated change in SMWD (r=-0.75, p=0.012) and plantarflexion torque (r=0.8571, p=0.0065).

Conclusion:
Carbonyl content (oxidative stress) in microvessels of each subject was positively associated with HO-1 expression. However, in a subset of patients HO-1 expression increased more slowly with increasing carbonyl content. Baseline oxidative stress in the microvessels, which may be a function of the quality of HO-1 expression, was a significant predictor of SET mediated change in SMWD and plantarflexion torque. The data suggest that microvessel oxidative damage may contribute uniquely to calf muscle pathology and leg dysfunction in PAD patients.
Abstract 312: Periadventitial Lysyl Oxidase Inhibition Using Electrospun Nanofibers Improves Arteriovenous Fistula Maturation
Authors: Applewhite Brandon C , Wei Yuntao , Andreopoulos Fotios M , Martinez Laisel , Vazquez-Padron Roberto I
Published: November 2022

Introduction:
Arteriovenous fistula (AVF) nonmaturation is associated with postoperative fibrosis and an increase in lysyl oxidase (LOX) expression.

Objective:
The purpose of this study was to determine whether unidirectional periadventitial delivery of β-aminoproprionitrile (BAPN) using bilayer poly lactic-co-glycolic acid nanofiber scaffolds is a feasible approach to reduce AVF fibrosis and encourage adaptive vascular remodeling.

Methods:
AVFs were created in thirty Sprague Dawley rats (200-350 g) of both sexes by an end-to-side anastomosis of the epigastric vein to the femoral artery. BAPN-loaded (b-BAPN) (n=10) or vehicle (b-VEH) (n=10) scaffolds (5 mm x 5 mm) were wrapped around the juxta-anastomotic zone of the epigastric vein immediately after anastomosis. AVFs without treatment were used as an additional control (n=10). Changes in lumen diameter were followed-up weekly using ultrasound. Flow was calculated using the pulse-wave velocity and lumen diameter. AVFs were collected at 21 days for histomorphometric analysis and to assess fibrosis.

Results:
b-BAPN significantly increased dilation of AVFs at day 14 (185.9%) compared to b-VEH (135.5%; p=0.0006) and control AVFs (113.6%; p<0.0001). The treatment (214.2%) had a significant difference compared to b-VEH (135.5%; p<0.0001) and AVF controls (122%; p<0.0001) at day 21 as well. In addition, blood flow was significantly enhanced in the treatment group (38.13 mL/min) versus b-VEH (9.436 mL/min; p=0.0002) and AVF controls (16.11 mL/min; p<0.0001). Histomorphometric analysis confirmed the ultrasound findings. Treatment with b-BAPN significantly increased lumen area compared to control AVFs (354,127 μm
2
vs. 95,580 μm
2
; p=0.0048). While there was no difference in intimal growth (p=0.5460), b-BAPN significantly reduced luminal occlusion with respect to control AVFs (0.0611 vs. 0.2067; p=0.0002). Finally, b-BAPN (49.48%) significantly reduced the fibrosis percent area compared to b-VEH (72.14%; p=0.0016) and control AVFs (74.5%; p=0.0007).

Conclusion:
Periadventitial LOX inhibition prevents AVF postoperative fibrosis and promotes adaptive vascular remodeling.
Abstract 315: Augmentation Of Protein
O
-glcnacylation By Impaired Intracellular Calcium Homeostasis Promoted Vascular Calcification
Authors: Zhang Weiping , Sun Yong , Yang Youfeng , Chen Yabing
Published: November 2022

Vascular calcification is accelerated in patients with diabetes mellitus and increases risk of cardiovascular events and mortality. Wes have demonstrated elevated protein
O
-GlcNAcylation promotes vascular calcification in diabetes. Increased
O
-GlcNAcylation is linked to impaired calcium signaling, which is critical for the development of vascular calcification in both humans and animal models. The present studies determined the role of an important regulator for intracellular calcium homeostasis, the stromal interaction molecule 1 (STIM1), in regulating
O
-GlcNAcylation and vascular calcification. Deletion of STIM1 induced osteogenic differentiation and calcification of VSMC, which was associated with decreased expression of the SMC marker genes and increased expression of the key osteogenic transcription factor, Runx2. By generating a SMC-specific STIM1 ablation mouse model, we determined that STIM1 ablation induced vascular calcification in aortic rings cultured ex vivo and in diabetic mice injected with low-dose streptozotocin in vivo. The diabetic mide with SMC-specific STIM1 ablation also exhibited increased aortic Runx2 expression and aortic stiffness (pulse wave velocity). Mechanistically, we identified that STIM1 deficiency impaired calcium homeostasis in VSMC, increased activation of the Ca
2+
/calmodulin-dependent protein kinase II and elevation of protein
O
-GlcNAcylation. Increased
O
-GlcNAcylation by STIM1 deficiency was further demonstrated in aortic arteries from the SMC-specific STIM1 ablation mice. The essential role of
O
-GlcNAcylation in mediating STIM1 deficiency-induced VSMC calcification was determined using a specific inhibitor for the
O
-GlcNAc transferase (OGT), the enzyme adding
O
-GlcNAc onto proteins. Inhibition of OGT abolished STIM1 deficiency-induced elevation of
O
-GlcNAcylation and VSMC. In conclusion, our studies have demonstrated a causative effect of SMC-expressed STIM1 on vascular calcification and stiffness in diabetes. We have further identified a novel link connecting STIM1 deficiency-induced impairment of calcium homeostasis and elevation of protein
O
-GlcNAcylation in VSMC that promotes osteogenic differentiation and calcification of VSMC.
Abstract 351: Orbital Ischemia Syndrome In A Patient With Carotid Artery Dissection
Authors: Cernik David
Published: November 2022

Introduction:
Orbital ischemia syndrome is a very rare presentation of ocular ischemic syndrome. The cause is usually vascular pathology in the carotid artery, most often on the basis of atherosclerosis or, more rarely, dissection of the carotid artery. Our aim is to draw attention in the form of a case report to an unusual clinical manifestation of carotid artery dissection.

Methods:
The case report deals with a patient (male, 49 years old) who suffered from intense pain behind the right eye. After a few days of pain, he arrives at the emergency room, where he was sent by a neurologist to the CTA. Preocclusive dissection of the right carotid artery was diagnosed. Neurosonological examination shows a massive reverse flow in the right ophthalmic artery. The pain behind the eye was so intense in the following days that opiate therapy was necessary.

Results:
Carotid dissection was treated with anticoagulant therapy and a statin. According to the control CTA, the dissection heals at three-month intervals. Neurosonological control also shows normalization of flow in the right ophthalmic artery. The decrease in residual pain intensity even on analgesic therapy correlated with a decrease in the amplitude of retrograde flow in the ophthalmic artery.

Conclusion:
Painful presentation of ocular ischemic syndrome, moreover caused by carotid artery dissection, is very rare.. However, if intense periorbital pain is present, carotid artery CTA should always be performed as part of the diagnostic proces.
Abstract 356: Phospholipid Profile Of Cerebral Thrombi In Acute Ischemic Stroke
Authors: Martha Sarah R , Levy Samuel H , Federico Emma M , Levitt Michael R , Walker Melanie S
Published: November 2022

Background and Purpose:
The University of Washington Clot Bank (UWCB) has established a tissue banking protocol optimized for “omic” analysis for cerebral thrombi retrieved during standard-of-care thrombectomy. Thrombi from cerebral arteries of acute ischemic stroke (AIS) patients are a novel resource that allow for the evaluation of lipid biological markers present at the time of ictus. The pathophysiological processes following AIS are complex, and involve ischemia, inflammation, and disturbances of lipid metabolism. The purpose of this study was to characterize the lipid profile from cerebral thrombi in AIS patients.

Methods:
The UWCB study is a non-probability, convenience sampling of adult subjects (> 18 years of age) who undergo thrombectomy for vascular occlusion. Lipidomics analysis was performed using LC-MS of untargeted lipids on thrombi taken from cerebral arteries of AIS subjects who underwent thrombectomy. Random forest, a supervised machine learning method, was employed to identify lipids associated with cerebral thrombi.

Results:
Five subjects (age = 53 ± 26.4, 3 males) were included from the UWCB registry. The most common comorbid conditions were atrial fibrillation (40%) and hypertension (40%). The top 10 metabolites identified from the random forest analysis, were of phosphatidylethanolamine molecular species. Phospholipids are involved in structural and functional integrity of cellular membranes and mediate host inflammatory responses. They are a key component of bacterial membranes. Changes in the structure or function of membrane phospholipids of host or relevant human microbiota may lead to alterations in membrane permeability and contribute to pathology of diseases such as neurodegeneration.

Conclusion:
An untargeted LC-MS lipidomics approach was used to identify lipids in cerebral thrombi at the time of mechanical thrombectomy. The top 10 lipids present in cerebral thrombi were identified, which included significant concentrations of phosphatidylethanolamine species. Because phospholipids play a key regulatory role in complex downstream cellular function, these findings may be of great significance in understanding AIS pathophysiology that has the potential to inform personalized preventive strategies.