bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 535: Vascular Cell Adhesion Molecule 1 Positive Fibroadipogenic Progentiors Promote Adipogenesis In Chronic Limb Threatening Ischemia Patients
Authors: Fei Kaileen , Dai Qunsheng , Peters Derek , Xu Yueyuan , Garrett Brianna , Otto James , Kontos Christopher D , Diao Yarui , Southerland Kevin
Published: October 2023

Introduction:
Chronic limb threatening ischemia (CLTI) is associated with a severe myopathy. A hallmark of skeletal muscle disorders is fatty infiltration. Fatty infiltration leads to progressive muscle dysfunction. The primary cell type implicated as the source of fatty infiltration are fibroadipogenic progenitors (FAPs). Prevention of FAP conversion to adipocytes may represent a novel approach to ameliorating the CLTI myopathy. However, the molecular mechanisms that govern FAP cell fate decisions in the ischemic limb remain unknown. Herein, we sought to identify the mechanisms that promote fatty infiltration in the ischemic limb.

Methods:
We quantified fatty infiltration in human CLTI patients and in BALB/c (failed regeneration) and C57BL/6 (successful regeneration) mice with Oil Red O staining. To elucidate the mechanisms responsible for this phenotype, we applied single cell RNA-sequencing (scRNA-seq) to C57BL/6 and BALB/c mice at day 10 and 14 following hindlimb ischemia. Next, we FACS isolated candidate FAP sub-populations and assessed apidogenesis in-vitro.

Results:
We show that ischemic human CLTI skeletal muscle displays increased adipogenesis. Furthermore, BALB/c mice recapitulate the human CLTI adipogenic phenotype. Next, we identified 10 distinct FAP populations in the ischemic limb. We also discovered a candidate FAP subpopulation, VCAM1+ FAPs, that promotes adipogenesis. Finally, we FACS isolated this sub-population and demonstrated in an in-vitro assay that it controls adipogenesis.

Conclusions:
Our results shed new light on skeletal muscle repair in the CLTI limb. First, we demonstrate that fatty infiltration is a component of the CLTI phenotype. Second, we identify an injury-induced FAP population that is responsible for the phenotype. Taken together, these observations may aid in the development of future therapeutic strategies to block adipogenesis and improve outcomes for CLTI patients.
Abstract 536: Diet-Induced Obesity Augments Ischemic Myopathy And Functional Decline In A Murine Model Of Peripheral Artery Disease
Authors: Fletcher Emma , Miserlis Dimitrios , Sorokolet Kristina , Bradley Cassandra , Wilburn Dylan , Papoutsi Evlampia , Wilkinson Trevor , Ring Andrew , Koutakis Panagiotis
Published: October 2023

Peripheral artery disease (PAD) causes a lower extremity ischemic myopathy contributing to patient functional disability. Although obesity is a common comorbidity, the pathophysiologic association between obesity and PAD myopathy is unknown. Using our murine model of PAD, we sought to elucidate the combined effect of hindlimb ischemia (HLI) and diet-induced obesity on markers of 1) muscle protein turnover (proteolytic activity, autophagic flux and calpain1), 2) muscle damage (caspase 3, Poly (ADP-ribose) polymerase 1 (PARP1) cleavage fragments and desmin accumulation), and 3) 6-minute walking distance.

Methods:
Twelve male and 12 female, 14-month old C57BL/6J mice were fed a high-fat-sucrose (HFS,
n
= 6) or low-fat-sucrose (LFS,
n
= 6) diet for 16 weeks prior to the surgical ligation of the left femoral artery at two locations. Following ligation, the mice continued their assigned diets for 4 weeks. Walking distance was assessed using the open field test 24 h prior to euthanasia. The gastrocnemius from ischemic and non-ischemic limbs was excised after animal termination. Markers of muscle damage and proteolytic activity were assessed via western blot and enzymatic activity kits. The ratio of muscle microtubuleassociated protein light chain 3 (LC3)-II:LC3-I content from immunoblots was used to monitor autophagic flux.

Results:
A HFS diet significantly reduced walking distance in both male (LFS: 54.1±2.9; HFS: 41.1±1.5 cm, p=0.003) and female (LFS: 52.0±1.7; HFS: 40.7±3.1 cm, p=0.001) mice with HLI. Although proteolytic activity was increased in ischemic muscle of both LFS and HFS-fed female mice (p=0.004), muscle breakdown via autophagy (1.3±0.1 fold, p=0.02) and calpain1 (1.6±0.1 fold, p<0.0001) was only elevated in HFS ischemic muscle. In males, proteolytic activity and autophagy were unaffected by HLI or diet (p>0.05); however, calpain1 was increased (2.8±0.3 fold, p=0.0001), but only in HFS ischemic muscle. Markers of muscle damage; caspase3, PARP1 24kD fragment, and desmin, were also increased (p<0.05) in ischemic muscle of HFS-fed males and females.

Conclusion:
A HFS diet augments functional disability and muscle breakdown and damage in mice with HLI. The pattern of muscle breakdown appears to differ between males and females.
Abstract 542: Short Term High Consumption Of Aspartame Does Not Increase Infarct Volume And Stroke Mortality In Mouse Models Of Focal Ischemic Stroke
Authors: Kwon Soonkyu , Park Inyeong , Choi Seungbum , Kim Dong-Eog E
Published: October 2023

Background:
Aspartame is an artificial sweetener that has been widely used in beverages and foods. Recently, higher intake of artificially sweetened beverages was shown to be associated with increased risk of stroke.

Objective:
To investigate if short-term high consumption of aspartame could increase cerebral infarct volume and mortality in mouse models of focal ischemic stroke.

Methods:
Eleven-week-old male C57BL/6N mice (n=90) received one of the following three treatments for 7 days: phosphate-buffered saline (PBS), aspartame 200 mg/kg in PBS, and aspartame in Zero Coke (with the final dose of aspartame being 200 mg/kg). Thereafter, either transient middle cerebral artery occlusion for 1 hour (1h-tMCAO; n=27, 9/group) or common carotid artery occlusion (CCAO)-related hemispheric ischemia with cerebral autoregulatory dysfunction (CCAO+CAD; n=63, 21/group) was induced. To measure the infarct volume, 2,3,5-Triphenyltetrazolium chloride staining was performed at 24 hours after tMCAO and 6 days after CCAO+CAD.

Results:
1h-tMCAO-related 24-hour mortality and infarct volume (of survived animals) did not differ significantly among the three groups (both p>0.05): PBS (5/9 death and 93.7±30.0mm
3
), aspartame in PBS (2/9 death and 102±50.0mm
3
), and aspartame in Zero Coke (4/9 death and 111.4±17.4mm
3
). CCAO+CAD-related mortality 6-day mortality did not show inter-group differences (p>0.05): PBS (n=9/21, 42.9 %), aspartame in PBS (n=12/21, 57.1 %), and aspartame in Zero Coke (n=8/21, 38 %). Among the survived, infarct volume did not significantly differ, either (p>0.05): PBS (60.8±68.0mm
3
), aspartame in PBS (58.5±68.0mm
3
), and aspartame in Zero Coke (54.2±48.3mm
3
).

Conclusions:
Short-term high consumption of aspartame did not increase cerebral infarct volume and stroke-related mortality in our experimental setting.

Category:
Peripheral Artery Disease, Carotid Artery Disease and Stroke
Abstract 547: Vasculogenic Mesodermal Cells Derived From Induced Pluripotent Stem Cells Improve Limb Perfusion And Decrease Necrosis in Murine Models of Hindlimb Ischemia
Authors: Samra Humraaz , Ingram Michael , Doiron Teresa , King Justin , Moldovan Leni , Gil Chang-Hyun , Stashevsky Jennifer , Fairbairn Madelyn , Welch Sadie , Mann Dave , George Matt , Yoder Mervin , Miller Steven J , Murphy Michael P
Published: October 2023

Objectives:
Critical Limb Threatening Ischemia (CLTI) is a health care priority with a 50% increase in amputations from 2010 to 2018. Unlike other cell products that simply secrete pro-angiogenic growth factors Induced pluripotent stem cells (iPSC) derived mesodermal cells can differentiate into endothelial and smooth muscle cells and form functioning blood vessels
in vivo
. We discuss the effects of intramuscular injection of human iPSC mesodermal cells (VSC100) on limb perfusion and limb necrosis.

Methods:
IPSCs were produced using methods including Yamanaka factors (Broxmeyer H. et al [21393480}). iPSC cells were differentiated using sequential Activin A, BMP4, VEGF, and FGF2 to promote mesodermal and endothelial cell (EC) differentiation. Murine CLTI models were created via excision of the common femoral artery in male BALB-C Nude mice, aged 6-8 weeks. After creation of the CLTI model mice were randomized to 1x10
6
VSC100 cells labeled with td-tomato (red fluorescence) or vehicle control injected into the gastrocnemius and gracilis muscles (N=10/group) at 7-14 days postinduction of ischemia. Limb perfusion was measured with Laser Speckle Contrast Imaging (LSCI) up to Day 64, qualitative analysis of VSC100 differentiation into capillaries was assessed with immunohistochemical staining for Isolectin B4 and human CD31 and confocal microscopy to detect td-tomato. Degree of tissue loss of the index limb was quantified with established necrosis scores.

Results:
VSC100 treated mice showed a significant increase in limb perfusion compared to controls, average percent blood flow/contralateral limb ratios of 45% in treated groups compared to 25% in control (p < 0.001) at day 64 (Figure 1). Limb necrosis scores showed necrosis was statistically significantly reduced in iPSC treated versus vehicle control treated groups (Figure 2). Immunohistochemical analysis showed qualitative significant deposition of cells of interest in our CLTI models.

Conclusions:
iPSC derived VSC100 is a novel approach to treating CTLI as the cells can form functioning blood vessels in ischemic skeletal muscle, improve tissue perfusion, and mitigate tissue necrosis. These early results are being used for FDA applications for clinical trials utilizing iPSCs in CLTI patients.
Abstract 549: Ceramide Signaling Potentiates Microvascular Endothelial Dysfunction In Patients With Coronary Artery Disease
Authors: SenthilKumar Gopika , Bordas-Murphy Henry , Lindemer Brian , Freed Julie K
Published: October 2023

We have previously shown that ceramide can increase production of vasoprotective nitric oxide (NO) in arterioles from healthy adults (0-1 risk factors for coronary artery disease; CAD) and elicit acute dilation. This process is dependent on ceramide’s conversion to sphingosine-1-phosphate (S1P), a well-established activator of endothelial NO-synthase (eNOS). On the contrary, patients with CAD have elevated plasma ceramide levels which independently predict adverse cardiac events. Therefore, we question whether ceramide signaling is altered during disease. We hypothesize that the beneficial NO-producing ceramide signaling is impaired in arterioles from CAD patients, and accordingly signaling is independent on the conversion to S1P. To test this hypothesis, resistance arterioles were dissected from discarded surgical tissue from patients formally diagnosed with CAD, pre-constricted with endothelin-1, and exposed to increasing doses (10
-9
to 10
-5
M; 1minute) of C2-Ceramide. Videomicroscopy was used to assess changes in internal vessel diameter. Two-Way repeated-measures ANOVA was used for statistical analysis. Increasing doses of ceramide elicted dilation in a dose-dependent manner (Mean Max Dilation±SE; 41.1%±7.5 C2 vs 14.3%±7.9 vehicle, p<0.05, n=5 each), which was preserved in the presence of eNOS inhibitor L-NAME (100nM; 30.2%±9.4, p=0.64, n=7) and impaired by PEG-catalase treatment (500U; -6.9%±9.9, p<0.05, n=4). Surprisingly, inhibition of sphingosine kinase, an enzyme necessary for ceramide conversion to S1P (Spk-I 1μM; 15.2%±10.2, p<0.05, n=5), as well as S1P receptor 1 (W146 10μM; 2.8%±13.2, p<0.05, n=4) and S1P receptor 3 (CAY10444 10μM; -7.4%±07.7, p<0.05, n=4) significantly reduced ceramide-induced dilation. Our results show that acute ceramide exposure increases oxidative stress rather than NO in arterioles from patients with CAD, a process that is unexpectedly dependent on conversion to S1P and activation of S1P receptors. These data suggest that sphingolipid signaling is altered in arterioles from CAD patients and provide mechanistic insight into how ceramide potentiates microvascular endothelial dysfunction during disease.
Abstract 553: Loss Of Endothelial Bag3 Does Not Worsen Ischemia Induced Myopathy
Authors: Terwilliger Zoe S , Kolasa Makenzie G , Green Thomas , Karnekar Reema , Pentakota Ananya , Spangenburg Espen , Yamaguchi Dean , McClung Joseph
Published: October 2023

Chronic limb threatening ischemia (CLTI), the most severe manifestation of peripheral artery disease, isaccompanied by high rates of limb amputation and death. There is a pressing need for novel therapydevelopment in these patients, which will require a thorough understanding of the individual cellularcontributions to ischemic limb pathology. BAG3 is a multifunctional adaptor protein that regulatesdiverse cellular functions. Mutations in
BAG3
now appear to be one of the most common to causedisease in the clinical setting, including predisposing Black patients to cardiovascular disease. Wepreviously identified a coding variant in
Bag3
that contributes to ischemic muscle necrosis after murinehindlimb ischemia (HLI) that was rescued by gene therapy, although we do not know which cell typesrequire Bag3 individually to mount efficient responses to ischemia. We hypothesized that vascularendothelial cell Bag3 would be required for both the restoration of blood flow and muscle function afterHLI. To test this hypothesis, we generated an inducible genetic model of endothelial cell
Bag3
deletion(Cdh5(PAC)-CreERT2;Bag3
-/-
). At 20 weeks of age, mice were administered either tamoxifen (KO;
n
=20) orvehicle (CON,
n
=28) and evaluated at baseline or subjected to HLI for 7-days. Endothelial cell specific
Bag3
deletion was validated first by genotyping and then by qRT-PCR in flow sorted cells. KO did notalter animal body weights, heart or skeletal muscle wet weights, physiologic muscle function (extensordigitorum longus absolute or specific force) or muscle histology (no regression of limb muscle tissuevascular cells) at baseline when compared with CON. After HLI, KO did not cause limb tissue necrosis,reduce limb blood flow recovery (laser doppler perfusion imaging) or physiologic muscle function, anddid not worsen histological damage to the limb muscle when compared with CON. Our study providesevidence for the first time that loss of endothelial cell Bag3 does not exacerbate myopathy and delaylimb blood flow recovery after hindlimb ischemia and suggests that the benefits of Bag3 gene therapymay be driven by the muscle cell compartment of the ischemic limb.
Abstract 560: Genetics Of Physical Activity And Risk Of Cardiovascular Disease
Authors: Biagetti Gina , Depaolo John , Shakt Gabrielle , Angueria Anthony , Judy Renae , Huffman Jennifer E , Tcheandjieu Catherine , Assimes Themistocles L , Klarin Derek , Voight Benjamin F , Vujkovic Marijana , Tsao Philip S , Chang Kyong-Mi , Lynch Julie , Levin Michael , Damrauer Scott M
Published: October 2023

Objective:
Sedentary lifestyle with minimal physical activity is a well-established risk factor for poor metabolic health outcomes. Atherosclerotic cardiovascular diseases (ASCVD) are often thought to be associated with physical inactivity, although observational studies may be limited by residual confounding which makes causal inference challenging. Mendelian Randomization (MR) is a method which leverages the naturally random allocation of genetic variants as a natural experiment, permitting causal inference. To better characterize sedentary lifestyle behaviors, we performed a genome-wide association study (GWAS) of sedentary behaviors using genomic structural equation modeling (SEM) and conducted a MR study using this phenotype to characterize the relationship of physical inactivity to ASCVD.

Methods:
Genomic SEM was performed to integrate three separate GWAS of physical activity and sedentary behavior into a common factor model. Following this, two-sample univariate and multivariable MR testing the effect of sedentary behavior on ASCVD traits was performed. Outcome data was taken from the Million Veteran Program (MVP) for PAD and CAD, and the GIGASTROKE consortium for stroke.

Results:
The common factor GWAS of sedentary behavior identified 44 genomic risk loci. 32 of the 44 lead variants were not significantly associated with the original sedentary traits, pointing toward possible new genetic mechanisms. On gene-based testing, the
CADM2
gene was implicated as the strongest risk locus of 156 mapped genes; this gene has been associated with impulsivity, substance use and obesity in prior studies. MR experiments revealed sedentary behaviors were associated with a significantly increased risk of CAD, PAD, and ischemic stroke; these effects were no longer significant when controlling for smoking, BMI, and diabetes as confounders.

Conclusions:
This study takes the largest available GWASs of PAD, CAD, and Stroke, and compares them against a novel GWAS of sedentary behavior traits. The findings suggest that the effects of sedentary behavior on ASCVD traits are likely not independent from other contributing factors (smoking; BMI), but that there is a shared causal pathway between these traits that has implications on the development of ASCVD.
Abstract 563: Variants of
Neurocan
, A Nonalcoholic Fatty Liver Disease Risk Allele, are Associated with Plaque Development in the Carotid Artery Over 5 Years and Metabolic Factors
Authors: Ikezaki Hiroaki , Schaefer Ernst , Murata Masayuki , Shimono Nobuyuki
Published: October 2023

Introduction:
Nonalcoholic fatty liver disease (NAFLD) genetic risk alleles are associated with lipid metabolism. However, the association between those variants and atherosclerosis has not yet been fully evaluated.

Hypothesis:
We hypothesized that NAFLD risk alleles are associated with the progression of atherosclerosis.

Methods:
A total of 1,150 Japanese men and women (median age 55 years) free of CVD, dyslipidemia, hypertension, and diabetes were studied. As NAFLD risk SNPs, variants of patatin-like phospholipase domain containing 3 (
PNPLA3
), transmembrane 6 superfamily member 2 (
TM6SF2
), glucokinase regulator (
GCKR
), and neurocan (
NCAN
) were assessed. Plasma total cholesterol, LDL cholesterol, small dense LDL cholesterol, LDL-triglycerides, HDL cholesterol, triglycerides, remnant-like particle cholesterol (RLP-C), fasting blood glucose (FBS), HbA1c, and high sensitivity CRP (hsCRP) were also measured. At baseline and after a five-year follow-up, carotid intima-media thickness (cIMT) was assessed using ultrasonography. Atherosclerosis was defined as cIMT over 1.1mm or the presence of a plaque. Univariate and multivariate analyses and chi-square and Fisher’s exact tests were performed to examine the associations between NAFLD risk SNPs, lipoproteins, and the progression of atherosclerosis.

Results:
Among 944 participants without atherosclerosis at baseline, the rate of plaque development was 13.1%, with the major allele (CC) of
NCAN
being significantly higher than non-CC alleles (13.8% vs. 4.7%,
P
= 0.04, Fisher’s exact test). Participants with the non-CC alleles of
NCAN
had significantly lower RLP-C at baseline than those with the CC allele (7.6 vs. 10.5 mg/dl,
P
= 0.02). In addition, participants with the non-CC alleles of
NCAN
had favorable metabolic parameters, lower blood pressure, triglycerides, small dense LDL cholesterol, and hsCRP, and higher HDL cholesterol than those with the CC allele (not significant).

Conclusions:
A
NCAN
variant, one of the NAFLD risk SNPs, was associated with plaque development among healthy Japanese participants, possibly mediated by favorable metabolism induced by the minor allele of
NCAN
.
Abstract 566: Perivascular Accumulation Of CD45+ Cells In The Lungs Of Female

BMPR2
+/R899X

Mutant Mice May Be A Factor To Sex-linked Pulmonary Arterial Hypertension
Authors: Loya Omar , Erewele Ejehi , Castellon Maricela , Oliveira Suellen Darc D
Published: October 2023

Introduction:
Pulmonary arterial hypertension (PAH) is an uncurable disease characterized by pulmonary vasoconstriction and remodeling, elevated right ventricle systolic pressure (RVSP), and right ventricle hypertrophy (RVH). Recently, our group showed that
BMPR2

+/R899X

loss-of-function mutation
per se
significantly increased vasoconstrictive Endothelin-1 (ET-1) in the lungs of female animals, who also exhibited higher RVSP compared to males. Why females have higher PAH incidence remains unclear.

Hypothesis:
We hypothesize that higher PAH incidence in female mice carrying the
BMPR2

+/R899X

mutation is associated with the spontaneous development of pulmonary vascular inflammation and ET-1-mediated vasoconstriction.

Methods:
To test this hypothesis, we used 12-month-old male and female wild-type and
BMPR2

+/R899X

mutant mice for quantification of RVSP and RVH and for analysis of the ET-1 and CD45+ pulmonary expression by Western Blot and immunohistochemistry.

Results:
In opposition to
BMPR2

+/R899X

mutant animals, data indicated no significant difference between sexes regarding ET-1 expression and RVSP in wild-type animals. When investigating the source of ET-1 in the lungs of
BMPR2

+/R899X

female mice, our preliminary data suggested it accumulates in perivascular cells. Moreover, double-blind immunohistochemistry examination of the pan-leukocyte marker CD45 revealed a massive perivascular infiltration of CD45+ cells in the lungs of female mutant animals compared to males (4.09 +/- 0.68 and 10.46 +/- 1.15 CD45+ cells/microvessel, male and female, respectively; 8 micrographs/animal; 3 mice/group), supporting our hypothesis that
BMPR2

+/R899X

mutation is an important factor for sex-linked vascular inflammation in PAH.

Conclusion:
In conclusion, elevated perivascular CD45+ cells in the lungs of female
BMPR2

+/R899X

mutant mice may contribute to PAH sex bias.
Abstract 572: Netrin-1 Induces Cytoskeletal-mediated Rewiring Of Mitochondrial Metabolism And Immunity
Authors: Cyr Yannick , Xing Yue , Newman Alexandra , Pilla Gabrielle , Bozal Fazli , van Solingen Coen , Brown Emily , Zhang Tracy , Naik Shruti , Moore Kathryn J
Published: October 2023

Background:
Cardiometabolic diseases are characterized by maladaptive immune infiltration in metabolic tissues. This results in unresolved inflammation, mediated by macrophages (M∅). Netrin-1 (Ntn1) causes macrophage retention by inhibiting cytoskeletal activity and induces a pro-inflammatory phenotype through receptor Unc5B. Emerging evidence suggests that cellular mechanics and metabolism are reciprocally regulated, but whether this plays a role in macrophage immunometabolism has not been explored. We hypothesized that Ntn1 stimulates a cytoskeletal-mediated rewiring of cellular metabolism toward a pro-inflammatory phenotype in macrophages.

Methods/Results:
We report that mechanical stress stimulates
Ntn1
and
Unc5b
expression in macrophages along with proinflammatory genes known to promote atherosclerosis. RNA-sequencing analysis comparing control (M∅
WT
) with
Ntn1
-silenced macrophages (M∅

Δ
Ntn1

) under high mechanical stress revealed a transcriptional signature associated with decreased biomechanical sensing and increased energy metabolism. This was confirmed by extracellular flux assays showing higher mitochondrial respiration in M∅

Δ
Ntn1

. In addition, flow cytometry and live-cell imaging showed cytoskeletal remodelling and improved mitochondrial functionality in M∅

Δ
Ntn1

as measured by higher mitochondrial potential, membrane integrity and network complexity. Importantly, incubation with recombinant Ntn1 reversed the effects of genetic loss of
Ntn1
, but not that of
Unc5b
, suggesting receptor dependency. Furthermore, as Ntn1 is tied to cytoskeletal reorganization via Rho-associated kinase (ROCK) inhibition, we tested the effects of ROCK signaling on mitochondrial metabolism. Small molecule-mediated ROCK pathway activation raised mitochondrial membrane potential in M∅
WT
, mimicking loss of
Ntn1
, whereas ROCK inhibition restored mitochondrial membrane potential in M∅

Δ
Ntn1

to wild type level.

Conclusion:
Taken together, our data shows that Ntn1, through Unc5B, decreases mitochondrial function through ROCK-mediated cytoskeletal reorganization. This suggests that Ntn1 perpetuates inflammation in cardiometabolic diseases through a cytoskeletal regulation of immunometabolism in macrophages.
Abstract 575: Impact Of Resident Macrophages On Fibrosis And Lymphatic Growth During Autoimmune Valvular Carditis
Authors: Osinski Victoria , Peck Alyssa , Faragher Jessica , Auger Jennifer , Binstadt Bryce
Published: October 2023

Rheumatic diseases are associated with increased cardiovascular disease and the mitral valve (MV) is one of the tissues most commonly affected. Valve disease prevalence continues to rise yet the mechanisms driving autoimmune valvular carditis are incompletely understood, limiting treatment options. Our team has previously demonstrated a role for macrophages in promoting MV disease in the K/B.g7 mouse model of autoantibody-induced arthritis and valvular carditis. To further characterize the MV macrophage populations, we conducted single cell RNA sequencing of healthy (B.g7) and inflamed (K/B.g7) MVs. Analysis revealed multiple macrophage populations including
Ccr2
+
Bhlhe40
+ recruited, pro-inflammatory macrophages and
Mrc1
+
Cd63
+ resident macrophages, which we further validated by flow cytometry. These findings raised new questions regarding which subset(s) of macrophages influence disease progression and how. Within the resident population, we identified a subset of
Lyve1
+
Folr2
+ macrophages. Immunostaining demonstrated that this macrophage subset localizes along the valve endothelium and near lymphatic vessels that we have recently demonstrated emerge in inflamed valves and promote valve disease. Importantly, we also found LYVE1+ macrophages co-localized with lymphatics in human MVs. Prior studies demonstrate that LYVE1+ macrophages are important regulators of vessel growth and extracellular matrix regulation. Thus, we hypothesized that LYVE1+ resident macrophages promote MV disease by augmenting lymphatic growth and contributing to changes in extracellular matrix. To understand the potential functions of these cells, we depleted
Lyve1
-expressing macrophages
in vivo
by crossing
Lyve1
-Cre mice with the
Csf1r
-floxed mice. We found that valves from LYVE1+ macrophage-depleted non-inflamed mice have increased collagen deposition, suggesting these macrophages are important for restraining extracellular matrix deposition. Studies are underway to determine if these resident macrophages promote fibrosis and lymphatic growth in the pro-inflammatory environment of the K/B.g7 model. Outcomes from these studies will have important implications for other cardiovascular and fibrotic diseases.
Abstract 582: Thoracobifemoral Bypass Exhibits Hemodynamic Superiority Compared With Axillobifemoral Bypass For Infrarenal Aortic Occlusion
Authors: Giese Angela , Li Willa , Jiang David , Pocivavsek Luka
Published: October 2023

Introduction:
Thoracobifemoral bypass (TFB) is a historically proven durable bypass option for patients with proximal aortoiliac occlusive disease and infrarenal aortic occlusion (IRAO). However, this operation is rarely done secondary to increased morbidity and difficulty of a thoracoabdominal exposure. This patient population is often relegated to an axillobifemoral bypass (AxFB) which has comparatively inferior bypass patency rates. In this study we sought to quantify the hemodynamic superiority of TFB compared with AxFB.

Methods:
Two patients who underwent a TFB and AxFB were used for this analysis. Their postoperative CT scans were segmented to build patient-specific 3D models that captured the flow lumens of their bypass grafts and infrarenal aortic pathology. We then performed computational fluid dynamic (CFD) simulations with a cardiac cycle inlet velocity and compared the average velocity at specified sites within the grafts. CFD models for both the TFB and the AxFB patients are shown in figure 1a-b.

Results:
In the AxFB model, there was a hemodynamically significant velocity drop across the proximal anastomosis. Additionally we found that there was a 75% velocity reduction over the length of the graft, regardless of the proximal inflow drop across the anastomosis. Conversely the inflow velocities were preserved in the proximal portion of the TFB graft. These velocities increased as we progressed to the distal anastomosis, suggesting a preservation of flow across a smaller caliber graft.

Conclusion:
Our models show TFB to be a hemodynamically superior choice over AxFB based on greater velocity degradation seen in our AxFB model. We conclude that in patients who are fit to undergo a thoracoabdominal exposure, TFB be considered over AxFB in cases of IRAO where infrarenal aortobifemoral bypass is not anatomically feasible.

Figure 1a-b: CFD models of a TFB (a) and an AxFB (b). The color scale represents relatively higher (red) and lower (blue) velocities in m/s.
Abstract 587: The Matricellular Protein Ccn1 Induces Aortic Aneurysms In Mice
Authors: Mo Fan-E
Published: October 2023

Aortic aneurysm
(
AA
) is a dilation of the aorta due to an arterial wall weakening. Chronic inflammation and oxidative stress have been associated with dysregulated vascular smooth muscle cell (VSMC) homeostasis and functional phenotype leading to loss of VSMCs, matrix remodeling, and AA formation. The matricellular protein
CCN1 (cellular communication network factor 1)
has been identified in a subpopulation of VSMCs at the early stage of phenotypic switching. To assess the involvement of CCN1 in AA, CCN1 expression was examined in the CaCl
2
-induced mouse AA model using

Ccn1
lacZ/+

-knockin mice. CCN1 expression revealed by whole-mount aortic X-gal staining was intensely induced 3 days after the peri-aortic application of CaCl
2
and sustained more refined to the lesion areas. CCN1 was largely expressed in VSMCs and infiltrating macrophages. CCN1 binds to its receptor integrin α6β1 and promotes atherogenesis through prolonged elevations of inflammation, oxidative stress, and matrix remodeling, the similar driving forces underlying AA progression. To test the role of CCN1 in AA development,

Ccn1
dm/dm

mice, carrying the α6β1-binding-defective mutant allele
Ccn1-dm
, were tested in the mouse AA model.

Ccn1
dm/dm

mice exhibited remarkable resistance against CaCl
2
-induced AA comparing with wild-type control mice 4 weeks after treatment. The

Ccn1
dm/dm

aortic wall possessed well-structured elastic lamellae and organized VSMC layers with less infiltrating macrophages, inflammation (interleukin-1β and tumor necrosis factor-α), and oxidative stress (8-OHdG). MMP9 levels detected by immunostaining were induced by CaCl
2
in the wild-type aortic wall, but not detected in

Ccn1
dm/dm

mice. Together, our results demonstrate a critical role of CCN1 in AA formation through binding its receptor α6β1 to increase VSMC inflammation and oxidative stress. CCN1 can be used as a novel therapeutic target for AA.
Abstract 590: A Model Of Aortic Arch Stenting For Dissection Repair Using Finite Element Analysis And Computational Fluid Dynamics
Authors: Polcari Kayla , Donik Ziga , Li Willa , Kramberger Janez , Pocivavsek Luka
Published: October 2023

Introduction:
Treatment of residual aortic dissection following ascending arch repair remains clinically challenging. We propose a novel technique to stabilize the aortic arch and create a proximal seal zone by placement of a bare metal stent into the arch prior to thoracic endovascular aortic repair (TEVAR). Using finite element analysis and computational fluid dynamics, we assess if this technique achieves the desired technical result.

Methods:
We analyzed two patients at our institution, both of whom presented with residual arch and descending aortic dissection after ascending aortic repair. Pre-operative CT angiograms were segmented to create patient-specific 3D geometrical models of the dissected aorta. A tetrahedral mesh was derived from this model and subsequently used in a computational simulation of stent deployment in the aortic arch using finite element analysis. We then evaluated computational fluid dynamics of the stented aorta.

Results:
In silico deployment of an uncovered stent in the dissected aortic arch along the centerline of the true lumen was successfully achieved using our computational model of stent crimping and expansion. The model produced realistic results when compared to intra-operative fluoroscopic imaging. Minimal arterial damage was observed based on the stress and strain of the aortic wall. Computational fluid analysis demonstrated the presence of flow to the carotid and subclavian arteries, with areas of increased turbulence at the ostia as expected (Figure 1).

Figure 1.
Velocity streamlines representing blood flow following uncovered stent deployment in the aortic arch.

Conclusions:
Our in silico model shows feasibility of aortic arch stabilization using an uncovered stent to create a proximal seal zone for TEVAR while maintaining blood flow to branch vessels. Expected surgical outcomes can be computationally modeled using patient-specific geometries to allow for pre-operative planning and surgical risk assessment.
Abstract 596: Microbe-derived Butyrate Activation Of Free Fatty Acid Receptor-3 Reduces Neointimal Hyperplasia After Arterial Injury By Regulating Immune Response Transcription Networks In Endothelial Cells
Authors: Jung Jonathan , Niraula Sarbjeet , Du James , Pamulapati Vivek , Jang Jae Woong , Xiong Liqun , Iruela-Arispe M Luisa , MacArthur Michael R , Ho Karen J
Published: October 2023

Introduction:
Microbe-derived butyrate has been shown to reduce neointimal hyperplasia after transluminal arterial injury, an effect that is dependent on free fatty acid receptor-3 (FFAR3). However, the mechanism by which FFAR3 affects the endothelial response to injury is not known. We hypothesize that FFAR3 regulates endothelial cell (EC) migration and proliferation by modulating transcriptional responses.

Methods:
FFAR3 wildtype (WT) and knockout (KO) mice were fed a high fiber diet to promote microbial butyrate production followed by femoral artery injury. Arterial morphometric analysis was performed after 4 weeks. Stable shRNA-mediated knockdown of FFAR3 in primary HUVEC lines were generated using 3
rd
generation lentivirus. In scratch assays, confluent HUVEC monolayers were subjected to a longitudinal “scratch” which was quantified at 0 and 8 hours. Proliferation (Ki67) and cell cycle (propidium iodide) analysis was assayed by flow cytometry. Bulk RNA-seq was performed on HUVEC and endothelial fractions of FFAR3 WT and KO mouse aortas. Gene set enrichment analysis (GSEA) was used to identify transcriptional pathways affected by FFAR3.

Results:
FFAR3 WT mice on a high fiber diet had 18.3% reduced neointimal hyperplasia 2 weeks after injury compared to KO mice (p<0.01) despite similar stool concentrations of butyrate. Compared to shScramble, shFFAR3 knockdown in HUVEC inhibited cell migration by 40.4% (p=0.02), which was not rescued by 1-methylcyclopropane carboxylate, a FFAR3 agonist. shFFAR3 knockdown reduced Ki67 by 50.7% (p<0.01) and increased the proportion of cells in G0/G1 phase by 15.3% (p<0.01), while reducing the proportion in S and G2 phases by 39.5% (p<0.01) and 18.7% (p=0.02), respectively. Pathway analysis of the transcriptomes from shFFAR3-HUVEC and ECs from FFAR3 KO aortas revealed significant overlap in immune regulatory pathways.

Conclusions:
FFAR3 activation by microbe-derived butyrate may promote EC migration and proliferation by stimulating progression through the cell cycle and modulating immune-related transcriptional pathways to limit neointimal hyperplasia after arterial injury.
Abstract 599: Sex Differences In The Skeletal Muscle Transcriptome And Proteome Of Chronic Limb Threatening Ischemia Patients
Authors: Kolasa Makenzie G , Yamaguchi Dean , Fisher-Wellman Kelsey , Terwilliger Zoe , Pentakota Ananya , Zeczycki Tonya , Scali Salvatore T , Berceli Scott A , O'Malley Kerri , Karnekar Reema , Brophy Patricia , Green Thomas , Myoung Meek , Spangenburg Espen , Amorese Adam , Annex Brian H , Kim Kyoungrae , Ryan Terence E , McClung Joseph
Published: October 2023

Treatment options for chronic limb threatening ischemia (CLTI), the most severe clinical presentation of peripheral artery disease, are limited. Innovation in therapeutic design for these patients is hindered by two critical issues: 1) limited information on the mechanisms underlying tissue pathology specifically in this population, and 2) complications in understanding this information in the presence of other significant co-morbidities and individual patient characteristics. In this study, we sought the effects of patient sex on the transcriptomic and proteomic profiles of ischemic muscle in patients with CLTI. We hypothesized that biological sex would alter expression patterns in the CLTI limb muscle. Comparing the tissue transcriptomes of CLTI patients (
p
<0.05 and
>
1.5FC) revealed 456 differences (342 up- and 114 down-regulated genes). Interestingly, general decreases in gene ontology (GO) categories for extracellular matrix (ADAMTS4, ALPL, MMP9), response to hypoxia (SOCS3, STC1, THBS1), and regulation of angiogenesis (CHI3L1, SERPINE1) occurred in female CLTI patients. Sex-based comparisons of the limb tissue proteome (
p
<0.05 and
>
0.5Log
2
FC) identified 50 protein targets (38 increased and 12 decreased female/male), including: NDUFA3, PYGB, SOD3, SLC25A6, PLIN3, CAMK2B, MYH3, and SERPIND1. Segregation of tissue proteomes to the mitochondria (MitoCarta) identified 11 mitochondrial protein targets (9 increased and 2 decreased F/M), including: NDUFA12, NDUFA3, SLC25A6, ARF5, ECI2, PTGES2, and RTN4IP1. Despite an overall rightward shift in mitochondrial protein in female limb muscle, no differences were observed in mitochondrial content via citrate synthase or after proteome mitochondrial enrichment factor analysis. Additionally, no sex-based differences were observed in mitochondrial oxygen consumption across multiple substrates and platforms. In conclusion, general transcriptome and proteomic profiles of CLTI patients follow similar bioenergetic/mitochondrial profiles which are mirrored by similar functional changes that support the mitochondria as a sex-independent organelle for therapeutic design.
Abstract 601: Effects Of Cyclic Peptide Lxw7 Coating On Short Term Vascular Graft Patency Using A Porcine (
Sus Scrofa)
Model
Authors: Vuoncino Matthew , Hao Dake , Pivetti Christopher , Sherazee Elan , Bahatyrevich Nataliya , Bagley Brendon , Koch Amory , Byrd Emily , Saenz Zoe , Hassan Monalisa , Lee Su Yeon , Guenther Timothy , Wang Aijun , Kwong Mimmie
Published: October 2023

Introduction:
We previously identified LXW7, a specific integrin αvβ3 targeting ligand capable of selectively capturing endothelial progenitor cells (EPCs) and endothelial cells (ECs), and developed a parylene-based conformal coating technology to covalently bind LXW7 to prosthetic materials. LXW7-coated grafts have demonstrated successful capture of EPCs and ECs, which then promotes endothelialization of prosthetic materials. This study aims to assess the function of LXW7 coating on interposition polytetrafluoroethylene (ePTFE) graft thrombus formation and patency in a large animal model.

Methods:
8 Yorkshire pigs underwent implantation of bilateral iliac artery interposition bypasses using control ePTFE grafts and LXW7-coated ePTFE grafts. Each animal served as its own control and the implanting surgeon and experimental team were blinded to treatment and evaluations. Computed Tomography Angiogram (CTA) immediately post-operatively, 3 weeks, and 6 weeks post-implantation. After final CTA, animals were sacrificed and grafts were harvested. The project proceeded following the approval of United States Air Force, David Grant Medical Center Institutional Animal Care & Use Committee.

Results:
6 animals were included in final analysis, with 2 excluded due to complications during their surgical and post-operative course requiring euthanasia. Native artery diameter was 4.08mm for the control grafts and 4.17mm for LXW7-coated grafts (p=0.296). Post-procedure CTA demonstrated procedural success for all 6 pigs with no evidence of thrombus or stenosis. At 3 week imaging, all grafts remained patent. 6 week imaging, all LXW7-coated grafts were patent without evidence of thrombus or stenosis while 4 of the 6 control grafts were either fully or partially thrombosed. Repeated measures mixed effects analysis demonstrated significantly improved patency over time with LXW7 coated grafts (p<0.001).

Conclusion:
This is the first study investigating LXW7-coating on prosthetic vascular graft patency in a large animal model. We demonstrate significantly improved graft patency use of LXW7-coating, suggesting promise as an adjunct for prosthetic grafts in vascular bypass applications.
Abstract 606: Ischemic Rescue By Transplantation Of Vascular Progenitor Cells Isolated From Fat
Authors: Zhao Liming , Lee Andrew , Sasagawa Koki , Sokol Jan , Wang Yuting , Huang Xianxi , Ambrosi Thomas , Sinha Rahul , Hoover Malachia , Weissman Irving , Wu Joseph C , Wan Derrick , Xiao Jun , Longaker Michael , Nguyen Patricia K , Chan Charles
Published: October 2023

Background:
Peripheral vascular disease remains a leading cause of vascular morbidity and mortality worldwide despite advances in medical and surgical therapy. Besides traditional approaches, which can only restore blood flow to native arteries, an alternative approach is to enhance the growth of new vessels, thereby facilitating the physiological response to ischemia.

Methods:
Actin
CreER
/R26
VT2/GK3
“Rainbow” reporter mouse was used for unbiased
in vivo
survey of injury-responsive vasculogenic clonal formation. Prospective isolation and transplantation were used to determine vessel-forming capacity of different populations. Single-cell RNA sequencing was used to characterize distinct vessel-forming populations and their interactions.

Results:
Two populations of distinct vascular stem/progenitor cells (VSPCs) were identified from adipose-derived mesenchymal stromal cells (MSCs): VSPC1 is CD45-Ter119-Tie2+PDGFRa-CD31+CD105
high
Sca1
low
, which gives rise to stunted vessels (incomplete tubular structures) in a transplant setting, and VSPC2 which is CD45-Ter119-Tie2+PDGFRa+CD31-CD105
low
Sca1
high
and forms stunted vessels and fat. Interestingly, co-transplantation of VSPC1 and VSPC2 is required to form functional vessels that improve perfusion in the mouse hindlimb ischemia model. Similarly, VSPC1 and VSPC2 populations isolated from human adipose tissue could rescue the ischemic condition in mice.

Conclusions:
These findings suggest that autologous co-transplantation of synergistic VSPCs from non-essential adipose tissue can promote neovascularization and represents a promising treatment for ischemic disease.
Abstract 624: Racial Difference In Acute Myocardial Infarction-Mortality During Covid-19
Authors: Awad Maan A , Thyagaturu Harshith , Gonuguntla Karthik , Chobufo Muchi Ditah D , balla Sudarshan , Mederos Liriano Marcelino , Santer Matthew J
Published: October 2023

Introduction:
Covid-19 pandemic had detrimental effects on the healthcare system. studies have shown a significant increase in Acute Myocardial Infarction (AMI) mortality across different age and gender groups.

Hypothesis:
We assessed the hypothesis that the covid-19 pandemic had a significant negative impact on AMI mortality based on race.

Methods:
We queried January 2019 to December 2020 National Inpatient Sample (NIS) database to identify AMI hospitalizations in adults (>18 years old). AMI mortality was then identified in each racial group (White, Black, and Hispanic). Multivariate logistic regression was used in outcomes analysis to adjust for potential patient-related and hospital-level confounders (age, sex, diabetes, heart failure, chronic kidney disease, Charlson Comorbidity Index score, hospital location, income status, and others), ICD-10-CM codes were utilized to identify the above-listed comorbidities.

Results:
743,429 AMI hospitalizations were identified during the period of interest. Of these 72.1% were White, 11.9% were Black, and 9.3% were Hispanic, with 38.1% of all patients being females. The mean age was 69 in Whites, 63.5 in Blacks, and 65.2 in Hispanics (p<0.01). After adjusting for potential confounders, analysis results showed significantly higher odds of AMI mortality in Blacks and Hispanics as compared to Whites (aOR 1.10 (1.03-1.14); p = 0.01) and (aOR 1.22 (1.14-1.31); p < 0.01) respectively in the period between January 2020 to December 2020 (Figure 1). This contrasts to similar odds of AMI mortality across the different races (Black vs. White aOR 0.95 (0.89-1.02); p = 0.22) and (Hispanic vs. White aOR 1.01 (0.94-1.09); p = 0.62) seen in the year preceding the pandemic (January 2019-December 2019).

Conclusion:
Our analysis demonstrates increased odds of AMI mortality in Blacks and Hispanics compared to Whites in the period between January 2020-December 2020 when compared to the pre-pandemic period (January 2019-December 2019).
Abstract 625: Alteration Of Circulating Ace2-network Related Micrornas In Patients With Covid-19
Authors: Wicik Zofia , Keshwani Disha , Eyileten Ceren , Nowak Anna , Simões Sérgio N , Martins David C , Klos Krzysztof , Wlodarczyk Wojciech , Assinger Alice , Soldacki Dariusz , Chcialowski Andrzej , Siller-Matula Jolanta , Postula Marek A
Published: October 2023

ACE2 interaction network caused by virus infection can affect the expression levels of miRNAs and influence severity of COVID19. This study aimed to analyse the diagnostic/predictive utility of ACE2-related miRNAs, identified by in silico analysis: miR10b, miR124, miR200b-3p, miR26b, miR302c-5p in patients with COVID-19, and aimed to unravel the functions of miRNAs, by using machine learning-SHAP analysis for clinical data and bioinformatic tools. Blood samples and clinical data of 79 patients and 32 healthy were collected at; day of admission, 7days and 21days after admission. Endpoint was hospitalisation length of stay (>21days) and/or death in follow-up. Delta low miR200b-3p expression (7days-admission) presents predictive utility in assessment of the hospital length of stay and/or death (AUC:0.73,p=0.002). Logistic regression analysis showed that delta low miR-200b-3p, diabetes are independent predictors of increased hospital length of stay and/or death (OR=5.8;CI=0.57-21.21;p= 0.008, OR= 4.9;CI=1-23.9;p=0.04). MiR-26b-5p and miR10b in patients were found lower at the baseline, 7 and 21days after admission compared to healthy controls (p<0.0001 for all time points). SHAP analysis showed miR200b-3p (day7), miR302c-5p (day7), CRP (day7), neutrophils (day0), and DDimer (day0) as most promising predictors of long hospitalisation. Pathway-enrichment showed the following top pathways of the interleukin-2 signaling pathway, and cancer pathways. MiR200b-3p showed regulation of COVID19-related targets associated with Tcell protein tyrosine phosphatase and HIF-1 transcriptional activity in hypoxia, key pathways in COVID19. Bioinformatics analysis showed miRNAs roles in multiple CVDs phenotypes associated with COVID19. We validated/characterized miRNAs which can serve as novel, predictive biomarkers of the long term COVID19 hospitalisation and can be used for early stratification of patients and prediction of severity of infection.