bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 106: Cd40 Is Required For B1b Cell Homeostasis In Atherosclerosis
Authors: Nitz Katrin , Reiche Myrthe , Pattarabajinard Oom , Upadhye Aditi , van Tiel Claudia , Taylor Angela M , Malin Stephen G , Binder Christoph J , Gerdes Norbert , Weyand Cornelia M , Weber Christian , Atzler Dorothee , McNamara Coleen A , Lutgens Esther
Published: July 2024

The CD40-CD40L co-stimulatory dyad has been identified as critical player in atherosclerosis. Our previous work has shown that CD40 and CD40L have cell divergent effects on atherosclerosis. Deficiency of the CD40-CD40L dendritic cell-T cell axis decreased atherosclerosis via reducing T helper 1 responses, whereas deficiency of macrophages CD40 reduced atherosclerosis by enhancing efferocytosis and reducing necrotic core content. CD40 is highly expressed on B cells and interacts with CD40L on T cells to induce antibody production and T follicular helper cell responses. We therefore hypothesize that deletion of CD40 will reduce atherosclerosis. We aim to unravel the role of B cell CD40 in atherosclerosis. CD40 expression on B cell subsets was determined on peripheral blood mononuclear cells of patients and correlated with severity of coronary artery disease (CAD). Atherosclerosis was analyzed in CD19
Cre
CD40
flfl
ApoE
-/-
mice that were fed an atherogenic diet for 14 weeks. Patients with CAD showed a decrease in CD40 on their putative B1 cells, which was associated with increased plaque burden and decreased plaque fibrosis as detected by coronary intravascular ultrasound. Depletion of CD40 on B cells in ApoE
-/-
mice resulted in a decreased germinal center formation and inadequate IgM production as expected. However, in line with our patient data, absence of B cell-CD40 increased atherosclerosis. This was caused by a reduction in B1 cells and consequently a reduction in atheroprotective anti-oxidation specific epitope (OSE) IgMs. Transfer of CD40-competent B1b cells in B cell-CD40-deficient mice restored anti-OSE IgM levels and prevented the increase in atherosclerosis. CD40-deficient B1b cells have an altered mTOR signaling pathway, impaired mitochondrial function, excessive uptake of lipids, increased cellular stress and accelerated cell death. Data indicate that CD40-deficiency promotes phosphorylation of RAPTOR and protein kinase B (AKT). We have identified a novel function of CD40 on B1b cells. B1b cell-CD40 exerts an anti-atherogenic role by regulating B1b cell metabolic homeostasis via mTOR pathway. Further mechanistic insights into how CD40 mounts adequate atheroprotective anti-OSE IgM production are currently being investigated.
Abstract 1069: Novel Approaches Dissecting Human Thrombus Composition
Authors: Kweon Joo Young , Mun Hyeonji , Park Dougho , Ahn Yong Joo J
Published: July 2024

Introduction:
Thrombus formation is a key contributor to ischemic stroke and cardiovascular diseases. Variations in thrombus composition involving platelets, fibrin, and leukocytes play a pivotal role in the pathophysiology, impacting clinical outcomes. Understanding its character and the interplay with Neutrophil Extracellular Traps (NETs) is clinically vital due to treatment response and outcome. This study introduces light-sheet microscopy and holotomography to dissect clot composition, aiming to enhance clinical insights into thrombosis. Unraveling these complexities holds promise for targeted interventions and improved patient outcomes. Aims: This study aimed to enhance our understanding of human blood thrombus composition by employing a novel approach using light-sheet microscopy and holotomography.

Methods:
Human thrombus samples were obtained by mechanical thrombectomy from acute ischemic stroke patients. To access the thrombus structure, the samples were cleared by a tissue-clearing rapid system, and light-sheet microscope imaging was obtained with CitH3, CD45, P-selectin, and fibrin. Holotomography images were obtained from cryosectioned thrombus at 5μm thickness and stained with DAPI and CitH3. Holotomography provided quantitative information on the refractive index of thrombus components. Comparative analysis with traditional imaging methods was performed to assess the advantages of the novel approaches.

Results:
The results revealed a comprehensive visualization of the spatial arrangement of human thrombus composition through light-sheet microscopy imaging. Holotomography successfully acquired a three-dimensional image of a thrombus section sample. HT images were used to qualitatively distinguish differences in neutrophil RI distribution by NETosis and differences in RI values within the immune cell population.

Conclusion:
This study demonstrates the efficacy of combining light sheet microscopy and holotomography to comprehensively dissect human thrombus composition. The integration of these advanced imaging techniques provides an understanding of thrombosis, surpassing the limitations of traditional methods.
Abstract 1072: Two Filter, Or Not Two Filter: Case Report Of Rare Anatomical Variant Of Inferior Vena Cava
Authors: Ellis Zachary D , White Dakota
Published: July 2024

This case involves bilateral inferior vena caval filter placement in a 68-year-old male with a history of a recently diagnosed pulmonary embolism (PE) who presented with precipitously dropping hemoglobin and dark tarry stools after starting Eliquis. Endoscopy confirmed ulcerative esophagitis. Interventional radiology was consulted for inferior vena cava (IVC) filter placement as was indicated due to lower extremity thrombosis, PE, and GI bleed. During the procedure, cavography revealed a duplicated inferior vena cava (DIVC), prompting the placement of bilateral Denali filters infrarenally via the right and left common femoral approach. Only 16 similar cases are reported in the literature at the time of this literature review, and this case highlights the efficacy of bilateral infrarenal filter placement in addressing this anatomical variation.
Abstract 109: Estrogen Receptor Alpha Inhibition Of Mineralocorticoid Receptor In Endothelial Cells As A Mechanism Of Protection From Atherosclerosis Inflammation
Authors: Wolter Nicole L , Stepanian Alec , Lu Qing , Man Joshua J , Jaffe Iris Z
Published: July 2024

Background:
Atherosclerosis (athero) inflammation predicts plaque rupture, causing heart attack and stroke. Female protection from heart attack and stroke is lost with age, implicating estrogen, but by unclear mechanism. Our lab showed that the mineralocorticoid receptor (MR) is expressed in endothelial cells (ECs), where it drives plaque inflammation in male mice by inducing the NFkB-regulated adhesion molecule ICAM1. Females had less inflamed plaques, unchanged by EC-MR-knockout (KO).

Hypothesis:
We hypothesized that EC-estrogen receptor alpha (ER) protects females from plaque inflammation by inhibiting NFkB and EC-MR induction of ICAM1.

Methods and Results:
Mice with EC specific MR KO (EC-MR-KO), ER KO (EC-ER-KO), or EC KO of both receptors (DKO) were developed and crossed to the LDLR KO background. EC specific gene recombination of MR and/or ER was confirmed by genomic PCR of lung (rich in ECs) and compared to blood, revealing no recombination in leukocytes. Baseline athero risk factors were unchanged by genotype. Male and female mice of all KO lines and floxed Cre- littermate controls were fed high fat diet for 12 weeks resulting in increased body weight and cholesterol with no difference by genotype in athero risk factors. Aortic arches were digested for flow cytometry to quantify plaque inflammation, confirming less inflamed plaques in females versus males. EC-ER-KO increased plaque inflammation, while DKO prevented this increase in both sexes. Compared to controls, ICAM1 protein expression in the descending aorta was decreased by EC-MR-KO only in males. In both sexes, ICAM1 was increased by EC-ER-KO and unchanged by DKO. Females had increased aortic expression of IkB, the inhibitor of NFkB, compared to males and this was prevented by EC-ER-KO. In primary human aortic ECs (HAECs)
in vitro
, cells from females expressed significantly less ICAM1 protein compared to males.

Conclusions:
Female atherogenic mice have less inflamed plaques than males and female HAECs express less ICAM1.
In vivo
, EC-ER protects from plaque inflammation and suppresses ICAM1 in both sexes and IkB in females, and EC-MR is necessary for this atheroprotection. These data support the concept that EC-ER prevents inflammation by inhibiting NFkB and EC-MR-induced ICAM1 expression.
Abstract 1099: Single-cell Transcriptomic And Epigenic Profiling Identifies Sox6 As A Key Regulator Of Arterial Endothelial Cell Phenotype In Systemic Sclerosis-associated Pulmonary Hypertension
Authors: Zhou Yuechen , Tabib Tracy , Huang Mengqi , Sembrat John C , Valenzi Eleanor , Lafyatis Robert
Published: July 2024

Background:
Pulmonary hypertension (PH) frequently arises as a complication of systemic sclerosis (SSc), and is a major cause of death among patients with this condition. Pulmonary vasculopathy in SSc is characterized by endothelial dysfunction with subsequent remodeling of the pulmonary vasculature, especially arterioles and small arteries. Characterization of transcriptional regulators driving the pathological changes in arterial endothelial cells (ECs) is critical to understanding pathogenic mechanisms underlying SSc-associated vasculopathy.

Methods and Results:
We performed single-cell multiome sequencing to simultaneously profile the transcriptome and epigenome of explanted lung tissue from SSc-PH patients (n = 9) and healthy donors (n = 6). We identified co-regulated genes and candidate enhancer regions associated with transcription factors (TFs) across different EC cell states using SCENIC+. Gene regulatory network analysis revealed SOX6 as a key transcription factor for the arterial EC phenotype associated with SSc-PH. 71 genes and 153 regions were predicted to be regulated by SOX6. Notably,
SOX6
was the only SOX family member significantly upregulated in SSc-PH arterial ECs (fold change = 40.0, adjusted p-value = 6.1*10
-21
). Putative bound TF motif instances accounting for chromatin accessibility in the candidate enhancer regions were identified using the base-resolution deep learning model, ChromBPNet. Consistent with SCENIC+ predictions, cell-state specific ChromBPNet models revealed higher putative TF occupancy at SOX motif instances across the genome. In addition, we identified SOX/ETS composite motif instances showing higher putative occupancy in SSc-PH arterial ECs, which may support cooperative binding of SOX proteins with ETS family members.

Conclusions:
These data provide novel insight into transcriptional regulatory programs of arterial ECs in SSc-PH and highlight SOX6 as a key contributor.
Abstract 1100: Soluble Guanylate Cyclase Activators As Potential Novel Pad Therapeutics
Authors: Adkar Shaunak , Sorondo Sabina , Lotfi Mozhgan , Bamezai Sharika , Choi Ryan , Pedersen Ole D , Ostrowski Sisse , Ghouse Jonas , Bundgaard Henning , Levin Michael , Damrauer Scott M , Leeper Nicholas J , Klarin Derek
Published: July 2024

Introduction:
Peripheral arterial disease (PAD) is major cause of limb loss in the United States with limited treatment modalities. Understanding the genetic etiology of PAD may provide rationale for novel therapeutic strategies. Here, we report a novel association between PAD and a likely endothelial cell specific cis-regulatory element of Nitric Oxide Synthase 3 (
NOS3
). We hypothesize that targeting of the nitric oxide (NO)-cyclic GMP (cGMP) axis with Riociguat, an FDA-approved soluble guanylate cyclase (sGC) activator, will improve perfusion and exercise capacity in a murine PAD model.

Methods:
We meta-analyzed genetic associations with PAD using summary statistics from European participants in the UK Biobank (UKB), Copenhagen Hospital Biobank - Cardiovascular Disease Cohort and Danish Blood Donor Study (CHB-CVDC/DBDS), and the Million Veteran Program (MVP). Human
NOS3
deficiency was modeled using Enos
+/-
mice with C57/B6 controls. We induced hindlimb ischemia (HLI) via double ligation of the femoral artery as described previously. Riociguat (3mg/kg/day) was administered 4 days prior and 28 days after HLI induction. We measured exercise capacity by placing animals on a treadmill with increasing speed and incline. We measured hindlimb perfusion using laser doppler perfusion imaging and assessed relative perfusion of the ischemic limb to the contralateral control limb.

Results:
We identified novel association of the minor allele of rs3918226, which lies within the promoter of NOS3, with PAD (OR: 1.06; 95% CI: 1.04-1.09; P = 1.3 x 10-9). Induction of hindlimb ischemia in Enos
+/-
animals resulted in impaired exercise capacity compared to wild-type controls. In Riociguat-treated Enos
+/-
mice, we observed a significant increase in distance traveled during exercise testing at 14 days which was sustained for the duration of the study period (276 vs 156 meters, p<0.05). We did not find a significant improvement in hindlimb perfusion over the course of the study period.

Conclusion:
Here, we identify a novel genetic association with PAD and demonstrate that in a genetic model of PAD we can improve exercise capacity by targeting the NO-cGMP axis with Riociguat. Future studies will assess the clinical utility of sGC activators like Riociguat in humans.
Abstract 1101: Tnfa-setdb2 Pathway Regulates Fibroblast To Myofibroblast Transition In Wound Repair
Authors: Bauer Tyler M , Mangum Kevin D , Barrett Emily , Shadiow James , Joshi Amrita , Buckley Samuel , Davis Frank , Gallagher Katherine A
Published: July 2024

Effective healing following tissue injury is predicated on an intimate coordination between immune cells and structural cells, where recruited immune cells induce or repress structural cell proliferation and gene expression. Early after tissue injury, fibroblasts begin to transition to myofibroblasts to contract and close wounds. In pathologic conditions where wounds fail to heal (such as diabetes), fibroblasts do not increase myofibroblast gene expression; however the mechanisms that control this fibroblast transition, in normal or diabetic conditions are not defined. Our group has previously identified that epigenetic alterations can control these phenotypic switches in immune and structural cells. Here, using human wound scSeq and murine transgenic models, we examined the mechanisms that may regulate this transition in the setting of injury. First, we identified that dermal fibroblasts when treated with the pro-inflammatory cytokine TNFa, significantly repressed myofibroblast genes including acta2, tgln, myl9, cald1, fibronectin and periostin. To identify possible the epigenetic mechanisms downstream of TNFa that modulate fibroblast phenotype, we performed an unbiased epigenetic superarray on TNFa-stimulated dermal fibroblasts, where SETDB2 was significantly upregulated and was also confirmed by qPCR. SiRNA-mediated knockdown of Setdb2 in murine fibroblasts prevented TNFa-mediated repression of myofibroblast genes, suggesting regulation of this transition by Setdb2. Human scSeq identified increased Setdb2 associated with increased TNFa signaling in wound fibroblasts, suggesting an interaction of these pathways. Interestingly, we found increased TNFa in macrophages and SETDB2 in fibroblasts from human diabetic wound scSeq and a murine diabetic (DIO) model, suggesting this pathway may contribute to the decreased conversion of fibroblasts to myofibroblasts seen in non-healing diabetic wounds. This work identifies setdb2 as a TNFa-mediated regulator of fibroblast to myofibroblast transition in wound repair and may identify a new therapeutic target for impaired tissue regeneration.
Abstract 1106: A Plasma Proteomic Signature For Atherosclerotic Cardiovascular Disease Risk Prediction In The UK Biobank
Authors: Gupte Trisha , Azizi Zahra , Nzenkue Kevin , Kho Pik Fang , Chen Ming Li , Zhou Jiayan , Guarischi-Sousa Rodrigo , Panyard Daniel J , Abbasi Fahim , Watson Kathleen , Clarke Shoa L , Tsao Philip S , Assimes Themistocles L
Published: July 2024

Background:
The plasma proteome holds promise as a future diagnostic and prognostic tool that can accurately reflect complex human traits and disease processes. We assessed the ability of plasma proteins to predict incident atherosclerotic cardiovascular disease (ASCVD).

Methods:
We assessed the relative ability of clinical, genetic, and high-throughput proteomic data to associate with ASCVD in a cohort of 41650 UK Biobank participants (figure). Selected features for analysis included clinical variables such as a cardiovascular risk score (QRISK3) and lipid levels, Olink protein expression data, and 36 polygenic risk scores (PRS). We used LASSO to select features and compare AUC statistics between data types. Stability selection with randomized LASSO identified the most robustly associated proteins. The benefit of the selected proteomic signature over the QRISK3 score was evaluated through the derivation of a Δ AUC value.

Results:
In this cohort, the mean age was 56 years, 60.2% were female, and 9.6% developed incident ASCVD over a median follow-up of 9.33 years. A protein-only LASSO model selected 252 proteins and returned an AUC of 0.723 (95% CI 0.708, 0.737). A clinical and genetic LASSO model which selected 4 clinical variables and 20 PRS achieved an AUC of 0.726 (95% CI 0.712, 0.741). Fifteen proteins selected by a stability selection algorithm offered improvement in ASCVD prediction over the QRISK3 score [Δ AUC: 0.011 (95% CI 0.002, 0.019)].

Conclusion:
A plasma proteomic signature modestly improves the prediction of incident ASCVD over clinical and genetic factors. Further studies are warranted to better elucidate the clinical utility of this signature in predicting the risk of ASCVD over the standard practice of using the QRISK3 score.
Abstract 1110: The Sex-specific Role Of The Mineralocorticoid Receptor In Smooth Muscle Cells In Obesity-associated Vascular Stiffness
Authors: Ibarrola Jaime , Xiang Rachel , Liu Rui R , Lu Qing , Jaffe Iris Z
Published: July 2024

Introduction:
Obesity is associated with increased vascular stiffness which predicts risk for heart attack, stroke and cardiovascular (CV) mortality. Obesity is more common and has a greater negative impact on CV health in women. Perivascular adipose tissue (PVAT), which contributes to maintenance of normal vascular tone, becomes dysfunctional in obesity, but its role in vascular stiffness is less well studied. Mineralocorticoid receptor (MR) is a hormone-activated transcription factor regulated by inflammation and oxidative stress in aging vascular smooth muscle cells (SMC) where it induces vascular stiffness. The role of SMC-MR in obesity-associated vascular stiffness and whether there are sex differences in the mechanism is unknown.

Hypothesis:
We hypothesized that sex differences in obesity-induced changes in PVAT alters aortic inflammation and oxidative stress (OS) to induce SMC MR and contribute to aortic stiffness by regulating sexually dimorphic gene programs.

Methods and Results:
Young male and female mice (3 months old) with MR intact or SMC-MR knocked out (SMC-MR-KO) were fed with high-fat diet (HFD) for 3 months. In both sexes, MR mRNA expression increased with obesity in MR intact mice, as did aortic stiffness (by pulse wave velocity (PWV)). SMC-MR-KO prevented the HFD-induced vascular stiffness in both sexes. In aortas from male mice only, NFkB protein were induced by obesity, independent of the MR. Obese males had increased vascular fibrosis histologically, vascular fibrosis gene (Col1, CTGF) expression and inflammation (TNFa, IL1B) in MR intact mice and is prevented in the SMC-MR-KO mice. In females, HIF1a protein and mRNA were induced by obesity, independent of SMC MR. In MR-intact females, OS gene expression (NADPH-oxidase 2-4 (Nox2, Nox4)) was increased by HFD. Human aortic SMC (HASMCs) from lean/obese male and female donors were cultured. NFkB, MR, and Col1 were increased with obesity in HASMCs from males while HIF1a, MR, Nox2 and Nox4 were increased in obese female donors compared with non-obese.

Conclusions:
These data support sexually dimorphic, SMC MR-dependent mechanisms for vascular stiffness associated with obesity and suggest that sex-specific therapies may be needed to prevent obesity-associated vascular stiffness.
Abstract 1115: Novel Mechanism For Extrahepatic Inflammation In Progressive Familial Intrahepatic Cholestasis 1
Authors: Bhandari Nilam , Prince Ashutosh , Khan Mariam , Gulshan Kailash , Kothapalli Chandrasekhar R , Traughber CAlicia , Neupane Kalash , Ertugral Elif , Smith Jonathan , Dubyak George
Published: July 2024

Background:
Human mutations in ATP8b1, a member of the P4-type ATPase family, cause Progressive Familial Intrahepatic Cholestasis I (PFIC1). PFIC1 patients often show extrahepatic inflammatory manifestations such as atherosclerosis, pancreatitis, and hearing loss. Even after liver transplantation, which rescues PFIC1 hepatic symptoms, the extrahepatic inflammatory symptoms persist. The mechanism of how ATP8b1 regulates inflammation is not clear. Pyroptosis executor Gasdermin D (GsdmD) plays a major role in promoting inflammation. GsdmD can be cleaved via a caspase-1/Nlrp3 dependent canonical inflammasome pathway, or a caspase-11 (in mice) or caspase-4/5 (in humans) dependent non-canonical inflammasome pathway.

Goal:
To decipher the mechanism for ATP8b1-mediated regulation of inflammasome activity in immune cells.

Methods & Results:
We employed Crispr-Cas9 generated homozygous ATP8b1 knockout human monocytes/macrophages to determine the status of inflammasome activity. Human monocytes/macrophages lacking ATP8b1 released significantly more IL-1β upon exposure to LPS (1μg/ml) + Nigericin (5 mM) vs. control cells (mean±SD, N=3-6, p<0.0001 by t-test.) Interestingly, ATP8b1
-/-
cells showed GsdmD cleavage with LPS priming alone without a secondary signal, and markedly increased IL-1β release vs. WT cells (N=3-6, mean±SD, p<0.001). Mechanistically, LPS gained cytoplasmic access in ATP8b1
-/-
immune cells, leading to cleavage of GsdmD in an NLRP3-independent, and caspase 4/5 dependent non-canonical inflammasome. The ATP8b1
-/-
immune cells showed impaired lysosomal acidification, though the lysosomal number and lysosomal membrane integrity were not compromised. ATP8b1
-/-
immune cells showed defects in inflammasome-resolving pathways such as phagocytosis and efferocytosis. Atomic force microscopy of ATP8b1
-/-
cells showed a reduced order state of the plasma membrane with Young’s modulus of elasticity for THP-1 ATP8b1
-/-
(Ey= 296±10.42 Pa) vs. WT macrophages (Ey =680±25.27 Pa, N =30, mean±SD, p<0.0001).

Conclusions:
ATP8b1 negatively regulates GsdmD cleavage via a non-canonical inflammasome pathway. GsdmD may serve as a therapeutic target for resolving inflammation in PFIC1 patients.
Abstract 1127: Erythrocyte Derived Extracellular Vesicles From Patients With Type 2 Diabetes Induce Endothelial Dysfunction Through Increased Vascular Oxidative Stress
Authors: Collado Aida , Humoud Rawan , Kontidou Eftychia , Yang Jiangning , Jiao Tong , Mahdi Ali , Zhou Zhichao , Pernow John
Published: July 2024

Background:
Red blood cells (RBCs) from patients with T2D (T2D-RBCs) induce endothelial dysfunction, but the mechanisms remain unclear. It is increasingly clear that extracellular vesicles (EVs) are actively secreted by RBCs and represent a novel mechanism of intercellular communication. However, the functional role of T2D-RBCs EVs in communication between RBCs and the cardiovascular system remains unknown.

Purpose:
This study aims to investigate whether EVs induce endothelial dysfunction in T2D and, if this is mediated via increased vascular oxidative stress.

Material and Methods:
EVs derived from T2D-RBCs and healthy RBCs (H-RBCs) were isolated using a membrane affinity column and co-incubated with wild-type mouse aortas for evaluation of the endothelium-dependent relaxation (EDR) using the wire myograph in the presence or absence of non-selective reactive oxygen species scavenger N-acetyl cysteine (NAC; 100 μM) applied to the aortas following the 18h EV incubation. The levels of the oxidative stress marker 4-hydroxynonenal (4-HNE) were quantified by immunohistochemistry in mouse aortas incubated with EVs. Additionally, T2D-RBCs EVs and H-RBCs EVs were co-incubated with human carotid artery endothelial cells (HCtAEC) for 8h and 24h to study the effects of EVs on vascular mRNA expression levels of eNOS, NOX1, and NOX4.

Results:
T2D-RBCs EVs but not H-RBCs EVs impaired EDR in aortas isolated from mice (
Fig. 1A
). This impairment was reversed by inhibiting oxidative stress in the vessel by NAC (
Fig. 1B
). Immunohistochemistry showed a significant increase in 4-HNE levels in aortas incubated with T2D-RBCs EVs (
Fig. 1C, D
). The T2D-RBCs EVs also induced a significant increase in NOX4 mRNA levels in HCtAEC after 24h but not 8h (
Fig. 1E, F
). However, T2D-RBCs EVs did not affect eNOS or NOX1 in endothelial cells.

Conclusion:
EVs derived from T2D-RBCs induce endothelial dysfunction through increased vascular oxidative stress.
Abstract 1132: Cellular Senescence Impairs The Maturity Of Cell-cell Junction In Vascular Endothelial Cells
Authors: Hirano Katsuya , Hirano Mayumi
Published: July 2024

Background and hypothesis:
Vascular endothelial cells functionally differentiate upon formation of secure cell-cell junction. Disruption of cell-cell junction causes endothelial dysfunction and contributes to the early phase of pathogenesis of vascular diseases. Cellular senescence loosens cell-cell junction and causes endothelial dysfunction. We reported that the “maturity” of cell-cell junction determines how endothelial cells respond to the proinflammatory and proapoptotic insults. We hypothesize that cellular senescence affects the maturity of cell-cell junction.

Methods and Results:
Thrombin (1 U/mL) decreased a trans-endothelial electrical resistance (TEER) and transiently increased di-phosphorylation of myosin light chain (ppMLC) at 3 min in culture porcine aortic endothelial cells (PAEC) of lower passages (9-15 passages) at confluence. Fluorescence staining revealed that thrombin induced actin bundle formation, which was associated with ppMLC, but not pMLC (mono-phosphorylation), at cell periphery as initial events, followed by reorganization into actin stress fibers associated with both pMLC and ppMLC as later events. In contrast, PAEC of lower passages either at early culture day (3 days) or at confluence but in the absence of extracellular Ca
2+
exhibited instantaneous appearance of actin stress fibers associated with pMLC and ppMLC upon thrombin stimulation. Initial appearance of peripheral actin bundles in response to thrombin is one of the hallmarks of the mature cell-cell junction. When replicative senescence was induced by repeated passages (23-30 passages), senescent PAEC exhibited scattered actin stress fibers without any stimulation, and substantially increased actin stress fibers, with no peripheral actin bundles, after thrombin stimulation. Cellular senescence was confirmed by an increase in senescence-associated β-galactosidase activity. As a functional consequence, senescent PAEC exhibited augmented store-operated Ca
2+
entry induced by an inhibitor of endoplasmic Ca
2+
ATPase.

Conclusions:
Replicative cellular senescence impairs the maturity of cell-cell junction, thereby damaging the junctional integrity and physiological function of endothelial cells.
Abstract 1142: Effect Of Chronic E-cigarette Nicotine Exposure On A Mouse Model Of Hind-limb Ischemia
Authors: Amirahmadi Maryam , Deng Alicia , Tsao Philip S
Published: July 2024

Background:
The rise of e-cigarette use in the United States has been rapid, yet its impact on peripheral artery disease (PAD) remains largely unexplored. This study investigates the relationship between e-cigarette use and PAD using a mouse ischemic hindlimb model.

Method:
Mice aged 6-8 weeks were divided into four groups. Three groups inhaled e-cigarette vapor continuously for 14, 20, or 28 days prior, and for 28 days after hindlimb ischemia (HLI) surgery, using an InExpose system (Figure 1). A control group was exposed to room air (RA) before and after HLI. Laser-Doppler perfusion imaging tracked hindlimb blood flow, and functional deficits were assessed using the modified ischemia scale.

Figure 1: Methodology

Results:
Blood perfusion in the ischemic hindlimb of the e-cigarette groups (EC-14, EC-20, EC-28) was significantly lower than the non-vaping group at 21 days post-surgery. In the EC-14 group, perfusion improved after 10 days of vaping cessation (Figure 2). Additionally, there was a reduction in platelet and leukocyte counts in the e-cigarette groups compared to RA, with no difference in red blood cells. Necrosis and claw damage were observed in EC-20 and EC-28 groups but not in non-vaping mice.

Figure 2: Representative images of perfusion recovery measured by LDPI.

Conclusion:
Chronic e-cigarette nicotine exposure impairs blood flow recovery and causes necrosis in mice with hind limb ischemia, potentially due to perturbed angiogenesis and arteriogenesis. This study highlights the detrimental effects of e-cigarette nicotine on vascular health.
Abstract 1143: Unraveling Histological Dynamics In Deep Venous Arterialization For Chronic Limb-threatening Ischemia
Authors: Benfor Bright , Csore Judit , Vela Deborah , Montero-Baker Miguel F , Roy Trisha
Published: July 2024

Introduction:
Deep venous arterialization (DVA) is emerging as a promising alternative for no-option CLTI) but its pathophysiology remains incompletely understood.

Research question:
What histologic changes occur in the limb after DVA to allow tissue oxygenation?

Study aim:
To uncover histological alterations induced by DVA and provide a framework for future experimental models to explore early radiologic and histologic markers of successful DVA.

Methods:
We report the case of a 53-year-old female who underwent a percutaneous DVA for CLTI using a covered stent (Fig.1). The intervention was successful with evidence of improved blood flow to the foot, but the patient presented worsening infection leading to a below-knee amputation 4 weeks later. A high-resolution 7T MRI was used to scan the amputated limb, and the blood vessels were harvested for histology analysis.

Results:
The MRI confirmed the patency of the DVA stent graft (Fig. 2A) and plantar loop while histology demonstrated evidence of valvular disruption and eccentric neointimal hyperplasia (NIH) in the vein that underwent intervention (Fig 2B). There was also evidence of NIH and increased smooth muscle cells in the media of the paired vein that did not undergo intervention, resulting in greater thickness compared to the media of the anterior tibial vein at the same level (Fig 2C).

Conclusion:
Our case reveals some of the changes induced by DVA and demonstrates a possible role of the untouched paired vein in facilitating arterialized flow to the foot. This offers a framework for experimental studies to further investigate these histological alterations and their role in prognosticating DVA outcomes.
Abstract 1146: Metabolic Regulation Of Induced Pluripotent Stem Cell Derived Endothelial Cells Regenerative Capacity
Authors: Carr Sean M , Owsiany Katherine , Tu Michael , Hekman Katherine
Published: July 2024

Introduction:
Induced pluripotent stem cells (iPSCs) derived endothelial cells (ECs) have emerged as a novel treatment strategy to restore limb perfusion in a murine model of peripheral artery disease (PAD). However, our lab has demonstrated that iPSC-EC in culture experience a failure of mitophagy and loss of cellular identity, limiting therapeutic implementation. Metabolic regulation of this mechanism is unknown.

Methods:
y6 human iPSCs were differentiated as previously described (Patsch Nat Cell Bio 2015) to generate ECs. Resulting iPSC-ECs were cultured under varying oxygen conditions (standard 21% vs. physoxic 4%). Markers of cellular identity and metabolic profile were analyzed.

Results:
iPSC-ECs lost cellular identity markers VE-Cadherin (55% positive in physoxic vs. 29% positive in standard) and PECAM1 (46% positive in physoxic vs. 39% positive in standard) over a period of two weeks (p<0.01). Single-cell RNA sequencing demonstrated the loss of cellular identity coincided with upregulation of endothelial-to-mesenchymal transition (EndMT), including COL1A1/2 and TGF-β. Physoxia significantly altered key metabolic proteins compared with standard conditions. Neuropilin 1 (Nrp1), an angiogenic related protein, was significantly increased in standard conditions (40% increased, p<0.01). PFKFB3, pathological upregulation of which correlates with increased EMT, was favorably downregulated in physoxic (52% lower, p<0.05) compared to standard conditions.

Conclusion:
iPSC-ECs under standard culture conditions demonstrated loss of EC identity with EndMT transition by single-cell transcriptomic analysis and by Western blot analysis of key metabolic proteins. Physoxia favorably impacted retention of key iPSC-EC identity markers. While increased Nrp1, as seen under standard conditions, can be associated with angiogenesis, the concomitant increase in PFKFB3 suggests an unfavorable metabolic activation and shift towards EndMT. Overactivation of PFKFB3 can results in net loss of energy in iPSC-ECs which leads to uncontrolled EndMT, and impaired regenerative capacity. Our results demonstrate iPSC-ECs effectively maintain metabolic control when grown in physoxic conditions.
Abstract 119: LRRC8 Complex Is An ATP Release Channel Regulating Platelet Activation And Thrombosis
Authors: Tranter John D , Kumar Ashutosh , Brown Gavriel , Mikami Ryan , Zhao Yonghui , Abraham Nihil , Abd El-Aziz Tarek M , Ajanel Abigail , Xie Litao , Ashworth Katrina , Hong Juan , Zhang Haixia , Kumari Tripti , Liu Alice , Balutowski Adam , Nair Vinayak K , Lasky Nina M , Feng Yongmei , Stitziel Nathan O , Lerner Daniel J , Campbell Robert A , Di Paola Jorge , Cho Jaehyung , Sah Rajan
Published: July 2024

Platelet shape and volume changes are early mechanical events in platelet activation proposed to contribute to arterial thrombosis. Here, we identify single-nucleotide polymorphisms (SNPs) in four leucine-rich repeat containing protein subunits (LRRC8A, B, C, and D) expressed in human platelets that are independently associated with increased mean platelet volume in human genetic studies - implicating the volume-sensing LRRC8 channel complex as regulating platelet function in humans. We show LRRC8A is required for functional LRRC8/VRAC in megakaryocytes (MKs). MK-specific LRRC8A conditional knockout mice give rise to LRRC8A-null platelets with larger mean volumes. LRRC8A-null platelets and CRISPR-edited human LRRC8A KO MKs (hiPSC MKs) have reduced agonist -stimulated P-selectin exposure, and αIIbβ3 integrin activation. LRRC8A-null platelets exhibit impaired adhesion to collagen-coated surfaces, agonist-induced platelet aggregation and ATP secretion. MK-specific LRRC8A conditional knockout mice have reduced platelet thrombus formation in laser-induced cremaster arteriolar thrombosis and prolonged occlusion times in FeCl
3
-induced carotid arterial thrombosis, compared to control mice, without affecting tail bleeding times. Mechanistically, LRRC8A mediates swelling-induced ATP release to amplify agonist-stimulated calcium influx and aggregation in platelets. Treatment of platelets with small-molecule LRRC8 channel inhibitors recapitulate the defects in LRRC8A-null platelets. These studies identify the mechanoresponsive LRRC8 channel complex as a regulator of platelet function and thrombosis - providing a proof-of-concept for a novel anti-thrombotic drug target.
Abstract 124: IRG1-Itaconate Axis Protects From Cholesterol-Induced Inflammation And Atherosclerosis
Authors: Bozal Fazli K , Cyr Yannick , Barcia Duran Jose Gabriel , Newman Alexandra AC , Amadori Letizia , Smyrnis Panagiotis , Gourvest Morgane , Das Dayasagar , Gildea Michael , Kaur Ravneet , Zhang Tracy , Wang Kristin M , Von Itter Richard , Schmidt Ann Marie , Fisher Edward A , van Solingen Coen , Giannarelli Chiara , Moore Kathryn J
Published: July 2024

Atherosclerosis, the major underlying cause of cardiovascular disease (CVD) mortality, is fueled by a failure to resolve lipid-driven inflammation within the vessel wall. Understanding the immunometabolic mechanisms underpinning sustained inflammation within this microenvironment is needed for the development of novel targeted therapies. Immune responsive gene 1 (IRG) is an enzyme that diverts the TCA cycle intermediate, cis-aconitate, to produce itaconate, a metabolite with anti-microbial and anti-inflammatory functions. We used a translational approach to investigate the therapeutic potential of the Irg1-itaconate axis for human atherosclerosis. Using single cell RNA-sequencing, we found that
IRG1
is upregulated in human coronary atherosclerotic lesions compared to patient-matched healthy vasculature, and is primarily expressed by plaque monocytes and macrophages. In mouse models of atherosclerosis,
Irg1
was expressed by plaque monocytes, macrophage and neutrophils, but declined with disease progression. Global or hematopoietic
Irg1
-deficiency increased atherosclerosis burden, plaque lipid content, and expression of interleukin (IL)-1b. Mechanistically, absence of
Irg1
potentiates lipid accumulation and lipid-mediated inflammation via a feed-forward cycle involving increased neutrophil extracellular trap (NET) formation, NET-induced NLRP3 inflammasome priming in macrophages, and subsequent IL-1b release. Conversely, supplementation of the
Irg1
pathway using the derivative 4-octyl itaconate (4-OI) reduced atherosclerotic inflammation and promoted beneficial plaque remodeling in mice. To assess the therapeutic potential of 4-OI in humans, we leveraged a validated systems-immunology approach to investigate CVD-related inflammation
ex vivo
. Using high dimensional single cell analyses, we showed that 4-OI supplementation attenuated pro-inflammatory phospho-signaling in human peripheral blood mononuclear cells treated with plasma from CVD patients, and caused an anti-inflammatory rewiring of macrophage populations. Our data highlight the relevance of pursuing
Irg1
-itaconate axis supplementation as a therapeutic approach to treat atherosclerosis in humans.
Abstract 130: Cftr Is Essential For Endothelium-dependent Vasorelaxation
Authors: Uppulapu Shravan Kumar , KUMAR JITENDRA , Kumari Sujata , Hodges Craig , Stoltz David , Kumar Santosh
Published: July 2024

Genetic mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) gene lead to cystic fibrosis (CF) which causes multiple health issues including lung disease and diabetes. Importantly, along with other health conditions, CF patients are long known to have impaired vascular function. Specifically, endothelium-dependent vasorelaxation is impaired in people with CF. However, the molecular mechanism(s) involved in the impairment of endothelial function in CF is not known. Since impaired endothelial function is an established risk factor for cardiovascular diseases, we investigated how CFTR regulates vascular endothelial function. We evaluated coronary arteries of CFTR
-/-
and littermate wild type piglets and noted profound endothelial dysfunction in coronary arteries of CFTR
-/-
piglets (n=10-12 rings/3 piglets). Next, we generated a mouse model with endothelium-specific deletion of CFTR (e-CFTR
-/-
). Aortic rings of e-CFTR
-/-
mice showed a significant delay in endothelium-dependent vasorelaxation (P<0.001; n=20 rings /5 mice). To determine the molecular signaling mediating this effect, we used a pharmacological inhibitor of CFTR (CFTRinh-172) and examined changes in endothelial nitric oxide synthase (eNOS). CFTRinh-172 treatment (10 μM, 5 days) led to a significant reduction in total and S1177 phosphorylated level of eNOS in human coronary artery endothelial cells (P<0.01 and P<0.05; n=3). In addition, CFTRinh-172 treated endothelial cells exhibited increased inflammation and altered endothelial cell metabolism (n=6; P<0.001). Together, these findings suggest that CFTR regulates eNOS and cellular metabolism in endothelial cells and therefore, functional CFTR is essential for vascular endothelial function. This fundamental role of CFTR in endothelial function will have future translational value in treating people with CF.
Abstract 132: Immunosenescence-related Biomarkers And Potential Therapeutic Targets For Coronary Artery Disease Identified Via Plasma Proteome And Immune Cell Traits
Authors: Lin Shuyang
Published: July 2024

There is a pressing need to identify novel biomarkers and therapeutic targets to address the epidemiological burden of coronary artery disease (CAD). Mendelian Randomization analysis (MR) leverages Genome-Wide Association Study (GWAS) data to infer the causal mechanism underlying the phenotype of interest. The proteome is the central mediator of the etiology and pathophysiology of multifactorial diseases such as CAD. Here, we screened for potential biomarkers and therapeutic targets for CAD through MR by using genetically predicted levels of plasma proteome followed by immune cell traits and transcriptomic analysis.

The main workflow of our study is outlined in Figure 1. Briefly, MR analysis was performed with protein quantitative trait loci (pQTL) and meta-analysis of four independent GWASs. Sensitivity analysis was performed to ensure the robustness of identified proteins. Pathway Enrichment analysis on whole-blood transcriptome was performed with GSEA. Secondary MR explored the causal effect of 731 immune cell traits on CAD risk.

In total, 11 overlapping candidate proteins (P<0.05, OR>1.0) were identified from the plasma proteome meta-analysis, including LPA, PLA2G7, and PCSK9, substantiating the validity of our MR analysis. Functional annotation of candidate proteins identified LCAT and CA11 as promising therapeutic targets and biomarkers. LCAT suppression is protective against atherosclerosis in mice models, while the functional significance of CA11 remains to be elucidated. Transcriptomic analysis shows enrichment in IL-2 signaling pathways reflective of the exhaustion of CD28 on cytotoxic T cells identified via secondary MR using immune-cell traits. Together, these results are indicative of immunosenescence as a risk factor for CAD.

Overall, our results revealed that an altered immune landscape increased the risk for CAD and unraveled an encouraging avenue for early diagnosis and treatment of CAD.
Abstract 142: Nr3c1-Sfrp1 Axis Regulates The Adipogenic Differentiation Of Vcam1+ Fibro-adipogenic Progenitors In Chronic Limb Threatening Ischemia
Authors: Dai Qunsheng , Wan Changxin , Xu Yueyuan , Garrett Brianna , Akers Leo , Peters Derek , Otto James , Kontos Christopher D , Ji Jason , Diao Yarui , Southerland Kevin W
Published: July 2024

Introduction:
Skeletal muscle health and function is a critical determinant of clinical outcomes in peripheral arterial disease (PAD). Fatty infiltration, the ectopic deposition of adipose tissue within skeletal muscle, is a histologic hallmark of end-stage PAD, also known as chronic limb threatening ischemia (CLTI). This process not only impairs ambulatory function, but is also associated with adverse clinical outcomes. Fatty infiltration is mediated by the adipogenic differentiation of a mesenchymal stromal population known as fibro-adipogenic progenitors (FAPs). We previously identified Vcam1+ FAPs as the dominant adipogenic population in CLTI patients. However, the mechanisms which govern the adipogenic differentiation of Vcam1+ FAPs remain unknown. Herein, we sought to identify the intracellular mechanisms that drive Vcam1+ FAPs into an adipogenic cellular fate.

Methods:
We analyzed the transcriptomes and epigenomes of FAPs in a murine PAD model to identify Sfrp1 and Nr3c1 as regulators of Vcam1+ FAP mediated adipogenesis. Next, to determine the necessity of Sfrp1 and Nr3c1 we silenced both genes with siRNA constructs and assessed in-vitro adipogenesis.

Results:
We show in a murine PAD model and human CLTI patients that Sfrp1 is enriched in Vcam1+ FAPs. Next, we use in silico analysis of our single cell RNA-sequencing dataset to identify Nr3c1 as a putative transcriptional regulator of Vcam1+ FAP adipogenic differentiation. Next, we demonstrate that inhibition of both Sfrp1 and Nr3c1 abrogates the adipogenic differentiation of Vcam1+ FAPs.

Conclusions:
Our results reveal a novel signaling axis for fatty infiltration in the ischemic limb. These findings lay the foundation for future studies to target maladaptive muscle regeneration in PAD patients.
Abstract 146: Phosphodiesterase 10A Mediates Arterial Calcification Through A P38/MAPK-MMP3 Signaling
Authors: Jin Ying , Xie Yangzhouyun , Berezowitz Alexa , Davis Sean , Flores Alyssa M , Wang Xue-Lin , Guzman Raul J , Cai Yujun
Published: July 2024

Background:
Vascular calcification is prevalent in patients with atherosclerosis, chronic kidney disease (CKD), diabetes, and peripheral artery disease (PAD). When located in the arterial media, it is strongly associated with increased cardiovascular morbidity and mortality. The cyclic nucleotides cAMP and cGMP play important regulatory roles in a variety of human diseases that are controlled by distinct phosphodiesterase (PDE) isozymes. We have recently found that PDE10A is the most highly induced isoform in a rodent model of arterial calcification. The function and underlying mechanisms of PDE10A in medial artery calcification, however, remain unknown.

Methods:
Vascular smooth muscle cell (VSMC) calcification was mediated by a high phosphate media. Medial artery calcification was induced by vitamin D
3
injection and 5/6 nephrectomy calcification models. Vascular calcification was assessed by calcium assay and Von Kossa staining.

Results:
PDE10A was markedly increased in calcifying VSMCs
in vitro
, calcified arteries
in vivo
, and calcified human tibial arteries from patients with PAD. Knockdown or inhibition of PDE10A markedly attenuated phosphate-induced VSMC osteogenic transformation and calcification in VSMCs. Conversely, overexpression of PDE10A increased VSMC calcification. Deficiency and inhibition of PDE10A significantly decreased arterial calcification
in vivo
. Several labs including our own have demonstrated that matrix metallopeptidases (MMPs) are involved in vascular calcification. Using bioinformatics analysis and loss-of-function strategy, we found that MMP-3 could be regulated by PDE10A in calcifying VSMCs. Our further mechanistic studies showed that knockdown or inhibition of PDE10A decreased activated p38 MAPK and that inhibition of p38 MAPK attenuated MMP-3 upregulation under a calcifying condition. These data suggest that PDE10A mediates arterial calcification through a p38 MAPK-MMP-3 signaling pathway.

Conclusion:
PDE10A is a key regulator in the development of medial artery calcification. Our findings provide insight into the use of PDE10A inhibition strategies to reduce arterial calcification in patients with CKD and PAD.