bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 122: The Eicosanoid Analogue Cs585 Represents A First-in-class In Prevention Of Platelet Activation And Thrombosis Through Direct Activation Of The Prostacyclin Receptor
Authors: Yamaguchi Adriana , Lambert Sylviane , Stanger Livia , Yalavarthi Pooja , Rickenberg Andrew , White Andrew , Dahlof Bjorn , Holinstat Michael A
Published: October 2023

Cardiovascular disease remains the primary cause of morbidity and mortality today and impinges on a number of organ systems including the vessels, heart, lungs, and brain with thrombotic complications. While platelet activation is critical for maintaining hemostasis and preventing leakage of blood cells from the vessel to extravascular compartments, the platelet clotting system is highly regulated to prevent an occlusive thrombus that deprives key organ systems of oxygen. To this end, the platelet remains a primary target for regulation of hemostasis and thrombosis. While current pharmacological regulation of the platelet has significantly reduced mortality, admittedly to a certain price of bleedings, there has been a paucity of successful targeting of platelet reactivity beyond cyclooxygenase-1, P2Y
12
receptor, integrin αIIbβ3, and phosphodiesterase. Recently, an oxidized lipid produced in the blood that limits platelet activation through activation of the prostacyclin receptor has been identified. Here, we demonstrate for the first time, a novel analogue of the oxidized lipid, known as CS585. CS585 is shown to potently target the prostacyclin receptor on the human platelet resulting in a highly selective and effective mechanism for prevention of thrombosis. Further, CS585 is shown to function in human whole blood ex vivo and in prevention both small and large vessel thrombosis in mouse models. Finally, we demonstrate that CS585 does not perturb coagulation or increase the risk for bleeding. Hence, we have developed a novel small molecule agonist that is selective for the prostacyclin receptor, stable in human and mouse blood, is effective in prevention of unwanted platelet-mediated thrombosis and doesn’t alter hemostasis. This discovery represents a new validated target in the treatment of thrombotic diseases in the blood and vessel without the observed risks of bleeding and off-target activation of other prostaglandin receptors.
Abstract 123: Protein Kinase C Theta (PKCθ) Signaling Associated With The Generation Of Procoagulant Platelets Revealed By Phosphoproteomics And Systems Analysis
Authors: Zheng Yiheng , Reitsma Stephanie , Babur Ozgun , Melrose Alexander , Pang Jiaqing , Hodapp Stefanie , Newton Alexandra , Wilmarth Phillip , Reddy Ashok , McCarty Owen J , Aslan Joseph E
Published: October 2023

Protein kinase C (PKC) family members integrate platelet activation programs to support hemostasis, and, also drive inflammatory and vascular diseases. While some PKC isoforms have established roles in platelets, roles for PKCθ are less specified. Here, we aim to determine how PKCθ signaling orchestrates platelet function. We prepared washed platelets from human donors for biochemical, physiological and omics studies. Platelet signaling and physiology were studied following stimulation with agonists, including the glycoprotein receptor GPVI agonist crosslinked collagen-related peptide (CRP-XL), and protease activated receptor PAR1 and PAR4 agonists, TRAP6 and AYPGKF-NH
2
. Platelets were treated with inhibitors of PKC family members to study PKC activation mechanisms by Western blot using antisera against specific phosphorylation sites on PKC isoforms and PKC substrates. Phosphoproteomics studies of GPVI and PAR agonist-stimulated platelets quantified significant, site-specific changes in the phosphorylation of PKC isoforms as well as PKC substrates. In addition to established phosphorylation events (e.g., PKCδ Tyr311), we noted a novel, prominent phosphorylation on the C-terminus PKCθ at Ser685. After generating and characterizing antisera specifically reactive to PKCθ phospho-Ser685, Western blot analysis determined that PKCθ Ser685 phosphorylation occurred later following GPVI platelet activation (>5 min) relative to PKCδ Tyr311 phosphorylation and PKC substrate phosphorylation driven by calcium-dependent PKCs. Treatment of platelets with specific PKC inhibitors revealed that PKCα/β, PKCθ, and, to a lesser extent, PKCδ activities all determine PKCθ Ser685 phosphorylation. In parallel, platelet function studies found that inhibition of PKCθ had only minor effects on platelet secretion and aggregation; however, PKCθ inhibition significantly reduced platelet phosphatidylserine exposure - a marker of procoagulant platelet activity - in response to GPVI and PAR agonists. Together, our results suggest that PKCδ and PKCα/β activities drive the phosphorylation of PKCθ Ser685 to support procoagulant platelet generation in a manner that may be blunted by therapeutically relevant small molecule kinase inhibitors.
Abstract 124: Synthetic High-density Lipoprotein Attenuates Abdominal Aortic Aneurysm Formation Via Restoring Vascular Smooth Muscle Cell Mitochondrial Function
Authors: Liu Yaozhong , Hong Kristen W , Zhao Ying , Yu Minzhi , Chen Y. Eugene , Schwendeman Anna , Guo Yanhong
Published: October 2023

Rationale:
Abdominal aortic aneurysm (AAA) is a complex degenerative vascular disease with higher mortality due to aneurysm expansion and ruptures. Extensive mitochondrial dysfunction in aortic VSMC is a prominent feature of AAA. Although HDL (high-density lipoprotein) exerts AAA protective effects, the role of HDL in protecting against VSMC mitochondrial dysfunction during AAA development is not completely understood.

Objectives:
To evaluate the therapeutic potential of synthesized HDL (sHDL) on AAA and to investigate the underlying mechanisms.

Methods and Results:
Using an angiotensin II (Ang II)-induced AAA model in
Ldlr
-/-
(low-density lipoprotein receptor-deficient) mice, we found mitochondrial dysfunction was progressively induced during AAA development in the affected aorta segments (suprarenal aortic segments). Two sample mendelian randomization analysis using genetic data from Global Lipids Genetics Consortium and Global Biobank Meta-analysis Initiative consortium revealed a negative association between plasma HDL level and AAA risk. Administration of sHDL (30 mg/kg, three times per week via intraperitoneal injection) significantly reduced the incidence rate of AAA (Vehicle vs sHDL, 76% vs 40%, n=15-17 per group, p<0.05) and the maximum aortic diameter. Mitochondrial DNA copy number in the affected aorta segments is reduced in the vehicle group and can be restored by sHDL treatment. We found the accumulation of sHDL in the aneurysmatic lesions by IVIS imaging, and sHDL reduces AAA-related mitochondrial dysfunction in vivo. Using primary mouse VSMCs, we show that sHDL can maintain mitochondria homeostasis by inhibiting mitochondria fission, reducing ROS production, and inhibiting mitochondrial membrane potential loss upon AAA-related pathologic stimuli. sHDL also significantly inhibits mitochondria DNA release and inflammatory responses in VSMC.

Conclusions:
The study concludes that sHDL nanoparticles may be a potential new therapeutic avenue for AAA by maintaining vascular smooth muscle cell mitochondrial homeostasis and inhibiting inflammatory responses.
Abstract 126: Effect Of Icospaent Ethyl On In Vivo Atheroma Progression And Inflammatory Protease Activity
Authors: Kassab Mohamad B , Song Du-Ri , Sherratt Samuel C , Thrapp Andrew , Vijay Aatira , Goudot Guillaume , Spicer Graham , Kawamura Yoichiro , Bhowmik Julian , Albaghdadi Mazen , Mauskapf Adam , Libby Peter , Bhatt Deepak L , Mason R. Preston , Tearney Guillermo J , Jaffer Farouc A
Published: October 2023

Background:
Icosapent ethyl (IPE), a high purity ethyl ester of eicosapentaenoic acid (EPA), is FDA-approved to reduce cardiovascular events among patients with or at risk for cardiovascular disease with elevated triglyceride levels. However, in vivo mechanisms underlying these findings remain unclear. Here we investigated whether IPE reduces plaque progression and inflammation using intravascular molecular-structural imaging.

Methods:
Cholesterol-fed balloon-injured rabbits (N=16) bearing aortic atherosclerosis underwent intravascular ultrasound (IVUS) analysis of plaque burden and near-infrared fluorescence-optical coherence tomography (NIRF-OCT) molecular imaging of plaque inflammatory cathepsin protease activity (ProSense VM110 NIRF agent, 4 mg/kg/iv) at 6 weeks. Next, rabbits were randomized to control (N=8) or IPE (Vascepa, Amarin, NJ) treated group (N=8, 1 g/kg/day) milled in the diet. Four weeks later, imaging was repeated, animals were sacrificed, and histopathology analysis of dissected plaque was employed.

Results:
Quantitative IVUS analyses (40mm segment, 3200 slices/16 rabbits) revealed that IPE significantly suppressed atheroma progression (ΔPAV, 5.2% [2.36-12.98] vs. 9.1% [4.26-12.25] control, p=0.03). Inflammatory plaque cathepsin activity detected by NIRF-OCT was similar (ΔNIRF average plaque 17.6 [6.2- 51.5] vs. 30.5 [4.9- 55.5] nM control, p=0.19)). On fluorescence microscopy, however, plaques from IPE-treated animals exhibited reduced NIRF signal (1.16% [0.03-3.94] vs. 3.13% [0.04-12.92], p=0.005)) and fewer RAM11+ macrophages (23.5±15.6 vs. control 32.9±12.6 macrophages/section, 25 sections/group, p=0.04). In IPE-treated animals, plasma EPA levels were elevated (p<0.05), and EPA metabolite levels, particularly 5,6-diHETE, were also elevated in resected plaques (p<0.05).

Conclusion:
IPE reduces in vivo plaque progression and plaque cathepsin protease activity and plaque macrophages, providing novel experimental evidence supporting the anti-atherosclerosis effects of IPE.
Abstract 129: Bleeding Diathesis In Ehlers-danlos Syndrome Patients Is Caused By Impaired Platelet Inside-out Signaling And Integrin αIIbβ3 Activation
Authors: Kumskova Mariia , Flora Gagan D , Nayak Manasa , Ghatge Madankumar , Patel Rakeshkumar , Jain Aditi , Staber Janice , Lentz Steven R , Chauhan Anil K
Published: October 2023

Background:
Easy bruising is included as a major or minor criterion for the classification of multiple types of Ehlers-Danlos syndrome (EDS). Despite a longstanding recognition of the association between EDS and bleeding, we still lack a definitive understanding of the mechanisms that contribute to bleeding diathesis in EDS patients.

Objective:
To determine platelet function in a cohort of patients with defined EDS types and a murine EDS model.

Methods:
We analyzed a cohort of 52 patients with classical, classical-like, hypermobile, or vascular EDS and a matched group of 52 healthy-control subjects. The International Society of Thrombosis and Hemostasis bleeding assessment tool (ISTH-BAT) was used to differentiate the EDS group with a higher bleeding tendency. Platelet activation was evaluated using human and mouse samples in standardized agonist-induced
in vitro
assays.

Results:
The mean ISTH-BAT score was 0.1 for the healthy controls and 9.1 for the EDS subjects (P<0.0001). An abnormal ISTH-BAT score was observed in 32 out of 52 (61.5%) EDS subjects and 0 of 51 healthy controls (P<0.0001). Most frequent bleeding symptoms were bruising, muscle hematomas, menorrhagia, epistaxis, bleeding from the oral cavity, and bleeding after tooth extraction. Menorrhagia that was life-threatening or required surgery was reported in 7 of 52 (14%) EDS subjects. Platelets from EDS subjects exhibited reduced aggregation and αIIbβ3 activation (with normal αIIbβ3 surface exposure) in response to agonists, including collagen, collagen-related peptide, and thrombin receptor activator peptide-6. Platelet secretion (dense and α-granules) and annexin V exposure were comparable between EDS and healthy subjects. Analysis of GPVI downstream signaling showed defects in Syk, PLCγ, and talin phosphorylation in EDS subjects (P<0.05 vs. controls), suggesting a defect in platelet inside-out signaling. The

Col5a1
+/-

EDS mouse model recapitulated the platelet phenotype of human EDS subjects.

Conclusions:
Patients with multiple types of EDS exhibit a wide range of bleeding symptoms ranging from mild to life-threatening episodes. Our results suggest that bleeding diathesis in EDS patients is caused by impaired platelet inside-out signaling and integrin αIIbβ3 activation.
Abstract 130: The Integrin β
3
Subunit Directly Inhibits The Galpha
13
-p115RhoGEF Interaction To Regulate G Protein Signaling And Platelet Exocytosis
Authors: Zhang Yaping
Published: October 2023

The integrin family of adhesion receptors and G protein-coupled receptors (GPCRs) are both fundamental to a cell’s responses to extracellular stimuli. For example, GPCR- or integrin-regulated exocytosis mediates platelet granule secretion, neurotransmission, and endocrine secretion, and is important in thrombosis and inflammation. GPCRs are known to stimulate integrin activation and signaling. However, it is unclear whether integrins regulate G protein signaling and if there is crosstalk between integrins and GPCRs in regulating exocytosis. Here we show that β
3
integrins negatively regulate GPCR signaling by directly inhibiting the Gα
13
-p115RhoGEF interaction. Furthermore, whereas β
3
deficiency or integrin antagonist RGDS inhibited integrin-dependent platelet aggregation and exocytosis (granule secretion), they enhanced GPCR-stimulated RhoA activation and integrin-independent secretion. In contrast, a β
3
-derived Gα
13
-binding peptide or Gα
13
knockout inhibited GPCR-stimulated RhoA activation and both integrin-independent and dependent platelet secretion without affecting primary platelet aggregation important for hemostasis. In a mouse model of myocardial ischemia/reperfusion injury (MI/RI)
i
n
vivo
, the β
3
-derived Gα
13
-binding M3mP6 peptide nanoparticle inhibited secretion of serotonin, a platelet granule constituent that exacerbates inflammation and MI/RI. These data establish crucial integrin-GPCR crosstalk that provides a rationale for developing therapeutic agents that strongly inhibit exocytosis in platelets and possibly other cells without adverse effects associated with loss of cell adhesion.
Abstract 136: Chronic Kidney Disease Aggravates Ischemic Leg Myopathy In Patients With Peripheral Artery Disease
Authors: Kim Kyoungrae , Thome Trace , Salyers Zachary , Yang Qingping , Stone Lauren , Vugman Nicholas , Anderson Erik , Fazzone Brian , Schmidt Emilie , O'Malley Kerri , Berceli Scott A , Scali Salvatore T , Ryan Terence E
Published: October 2023

Introduction:
Patients with peripheral artery disease (PAD) often present with comorbid risk factors that accelerate disease evolution and worsen clinical outcomes. Notably, PAD patients with chronic kidney disease (CKD) have greater risk for major adverse limb events and mortality. The objective of this study was to determine the impact of CKD on skeletal muscle strength and mitochondrial health in patients with and without PAD.

Methods:
We performed a cross-sectional analysis of skeletal muscle strength and mitochondrial health in four groups of patients (58 males and 21 females): PAD only (Toe brachial index, TBI<0.5 and eGFR>60 mL/min), CKD only (TBI>0.75 and eGFR:15-45 mL/min), PAD with CKD (TBI<0.5 and eGFR:15-45 mL/min), and adult controls without PAD and CKD (TBI>0.75 and eGFR>60 mL/min).

Results:
Plantar flexor muscle strength was lowest in PAD patients with CKD, which had a significant difference from adult controls (
P
<0.01), a trending difference (
P
=0.08) from PAD patients without CKD. Myofiber cross-sectional area was 20-25% larger in PAD only patients and adult controls compared to CKD and PAD+CKD groups without significance. Analysis of mitochondrial oxidative phosphorylation (OXPHOS) revealed that both CKD and PAD with CKD groups had significant reduction (30~40%) in mitochondrial bioenergetic function at various levels of energy demand mimicking from resting skeletal muscle to maximal contraction compared with adult controls and PAD without CKD group (P<0.05). Similarly, OXPHOS conductance was significantly reduced in the PAD with CKD group compared with both adult controls and PAD patients without CKD (
P
<0.01). Surprisingly, patients with CKD only had lower OXPHOS conductance compared to adult controls (
P
=0.05) and patients with PAD only (
P
=0.02). Interestingly, PAD patients without CKD had no difference in mitochondrial function compared with adult controls (
P
=0.98).

Conclusion:
The results suggest that CKD plays a critical role in exacerbating skeletal myopathy evidenced by muscle weakness and mitochondrial dysfunction in patients with PAD. These findings suggest that mitochondrial dysfunction may be a primary site of convergence of these two diseases.
Funding:
NIH grant R01HL149704 and AHA grant POST903198.
Abstract 137: Identification Of Human Gut Microbial Markers In Patients With Peripheral Artery Disease Using Different Sample Preservation Methods
Authors: Niraula Sarbjeet , Stirewalt Spencer L , Jung Jonathan , Jang Jae W , Wu Erik , Elmasri Ayman , Du James , Xiong Liqun , Green Stefan , Seed Patrick , Ho Karen
Published: October 2023

Introduction:
The gut microbiome is associated with cardiovascular diseases, but there is a paucity of microbial markers associated specifically with peripheral artery disease (PAD). We sought to identify gut microbial markers associated with PAD and to evaluate different sample preservation methods for future biomarker discovery.

Methods:
This is a single-center cross-sectional study of participants with and without documented PAD who performed home fecal sample collection using 3 preservation methods: immediately frozen (FR), Cary-Blair media (CB), and OMNIgene-GUT (GT). Samples were collected and stored at home for up to 4 days before being returned to the lab and stored at -80°C. In order to study the direct effects of room temperature (RT) storage on CB and GT samples, 5 samples from each group were stored for 0, 24, and 48 hours at RT before sample processing. Microbial communities were profiled using 16S rRNA (V4) gene amplicon sequencing; sequence data were analyzed using QIIME2 and Phyloseq in R. Taxonomic annotation was done using SILVA database and microbiome functional prediction was conducted using PICRUSt2 software package.

Results:
Thirty-three participants (14 PAD and 19 non-PAD) returned samples using all preservation methods. There were no significant differences in alpha diversity between PAD and non-PAD samples. FR samples had overall lower ASV richness compared to CB and GT. Microbial community structures were significantly different between PAD and non-PAD samples (PERMANOVA p<0.05), but CB and GT were less sensitive than FR in detecting the differences between PAD and non-PAD based on rare lineages. Paired differences in community structure were significantly different (p<0.05) between PAD and non-PAD after 24 and 48 hours at RT. Differentially abundant features were not consistently identified across all preservation methods, and these methods were also associated with differences in differential expression patterns of predicted functional gene content between groups.

Conclusions:
This study identifies gut microbial features associated with PAD and demonstrates the impact of sample preservation methods on these features. Ongoing work will integrate clinical variables in a larger cohort to validate these findings.
Abstract 138: Delivery of Allogeneic Mitochondria Accelerates Revascularization in a Murine Model of Peripheral Arterial Disease and Reverses Metabolic Signatures of Endothelial Senescence
Authors: Headley Colwyn , Hu Caroline , Chiang Gladys , Oropeza Beu , Tsao Philip S , Huang Ngan F
Published: October 2023

Peripheral arterial disease (PAD) is associated with mitochondrial dysfunction in both endothelial cells and skeletal muscle. We examined whether mitochondrial transplantation (mito-transfer) can alleviate the cellular abnormalities linked to mitochondrial dysfunction in PAD. Mitochondria from allogeneic fibroblasts were transferred into human microvascular endothelial cells (ECs) of varying passages (p5-14) by centrifugation. After mito-transfer (24h), the metabolic activity and proliferative capacity of ECs were respectively quantified by extracellular flux analysis (Mito-Stress Test, Glycolysis Stress Test, and ATP rate assays) and EdU incorporation into DNA. To test the efficacy of mito-transfer for the treatment of PAD, mitochondria were injected into the murine ischemic limb and blood perfusion recovery was monitored. Compared to non-manipulated ECs, mito-transferred ECs had lower oxygen consumption rates (OCR) related to mitochondrial respiration (basal, maximal, and spare respiratory capacity, and mitochondrial ATP production). No significant differences were observed in the extracellular acidification rates related to the glycolytic activity of ECs. However, the ATP index (ratio of mitochondrial ATP/ glycolytic ATP) of late passage (p10) ECs significantly decreased after mito-transfer. Furthermore, compared to non-manipulated ECs, mito-transferred ECs of late passage (p10-14) showed a 25% increase in proliferation. Mito-transfer to the ischemic limb induced a 2-fold increase in limb perfusion after 14 days (Fig. 1). Collectively, these data show that EC aging is associated with increased oxidative phosphorylation metabolism and that mito-transfer decreased the senescence-associated oxidative glucose metabolism of ECs, enhanced EC proliferation, as well as accelerated revascularization in PAD. Mito-transfer is a promising strategy to treat PAD to improve revascularization and mitochondrial function.
Abstract 139: Semi-automated Classification Of Peripheral Artery Disease Lesion Composition From Multi-Contrast Magnetic Resonance Histology At 9.4 Tesla With A 2 Dimensional Convolutional Neural Network Variational AutoEncoder Algorithm
Authors: Csore Judit , Karmonik Christof , Lumsden Alan B , Roy Trisha L
Published: October 2023

Introduction:
Using artificial intelligence (AI)-assisted methods on MRI histology may help endovascular peripheral artery disease (PAD) treatment planning.

Objectives:
To evaluate soft vs. hard PAD plaque components and crossability using multi-contrast high-resolution MRI, AI algorithm, and histologic samples.

Methods:
PAD lesions were harvested from 6 patients (one of each) after amputation and imaged using a 9.4T ultra-high resolution MRI scanner with 3 contrasts: T1-weighted (T1w), T2-weighted (T2w) and Ultrashort Echo Time (UTE). 3D imaging volumes were spatially registered and pseudo-color composites (T1w: red, T2w: green, UTE:blue) were created. Custom 2D variational autoencoder (VAE) classification AI algorithm using convolutional neural networks for image recognition classified and scored cross-sections as lumen occluded with soft tissue (tissue score (TS)=10), with soft and hard tissue (TS=30) and with hard tissue (TS= 50). 2 readers performed visual assessment of crossability (crossable/non-crossable) on preselected MRI slices compared to histologic samples (Fig1a), percent agreement was calculated for inter-rater reliability.

Results:
MRI multi-contrast registration succeeded for all section (pseudo-colors: smooth muscle cells- pink/red, fatty lesions-green, collagen-dark blue, calcification-black). VAE separated axial sections based on plaque composition (Fig1b), average TS per tissue segments ranged from 10 to 48.1 and corresponded well to the composition of tissues inspected visually.20 slices showed appropriate alignment, for the evaluation of crossability 11 were excluded due to lack of sufficient histological sample, inter-rater reliability was 100% on 9 slices (7 crossable, 2 non-crossable).

Conclusion:
AI algorithm for image recognition successfully distinguished lesion composition. AI-aided analysis may help rapid evaluation of lesion crossability in planning endovascular interventions.
Abstract 141: Lung Thrombosis Upregulates Endothelial Pla2g2d To Inhibit Inflammatory Lung Injury
Authors: Evans Colin E , Machireddy Narsa , Zhang Xianming , Zhao Youyang
Published: October 2023

Objective:
There are no effective treatments for sepsis-induced acute lung injury (ALI) or acute respiratory distress syndrome (ARDS). ALI/ARDS patients have an increased risk of thrombosis, but the impact of lung thrombosis on ALI/ARDS is ambiguous. Our aim was to target thrombosis-induced signaling to treat ALI/ARDS.

Approach and Results:
First, we used a mouse model of lung capillary occlusion to quantitatively induce lung thrombosis in mice with sepsis-induced ALI (i.p. LPS and CLP models). A severe level of lung thrombosis exacerbated ALI in sepsis mice (i.e., raised permeability, inflammation, and apoptosis), but an innocuous level of lung thrombosis suppressed ALI. RNA sequencing identified distinct gene profiles in lungs of sepsis mice with innocuous or severe lung thrombosis. A panel of survival factors was increased by innocuous but not severe lung thrombosis, and a subset of these factors was increased in lung endothelial cells (ECs), including A2 phospholipase group IID (Pla2g2d). We then used our new mouse model of EC gene knockout via i.v. injection of biodegradable nanoparticles mixed with plasmid DNA expressing Cas9 driven by the CDH5 promoter and Pla2g2d guide RNA driven by the U6 promotor. The protective impact of innocuous lung thrombosis on ALI was absent in EC-specific Pla2g2d knockout mice. We next administered the biodegradable nanoparticles with plasmid DNA expressing Pla2g2d driven by the CDH5 promoter to overexpress Pla2g2d in ECs. The damaging impact of severe lung thrombosis on ALI was reversed in mice overexpressing EC Pla2g2d. Finally, we explored clinical relevance by data mining to show that circulating Pla2g2d levels are greater in surviving versus non-surviving sepsis patients.

Conclusions:
EC Pla2g2d protects against sepsis-induced ALI in mice. Circulating Pla2g2d is increased in surviving versus non-surviving sepsis patients. Activation of Pla2g2d signaling represents a novel and plausible therapeutic approach for ALI/ARDS.
Abstract 143: Differential Suppression Of Arterial Versus Venous Thrombosis But Preserved Hemostasis In Mice Expressing Fibrinogen That Cannot Support Fibrin Formation
Authors: Hur Woosuk S , Kawano Tomohiro , Mwiza Jean M , Paul David , Lee Robert , Clark Emily , Bouck Emma , Mackman Nigel , Wolberg Alisa S , Bergmeier Wolfgang , Flick Matthew J
Published: October 2023

Elevated circulating fibrinogen correlates with increased risk for both cardiovascular and venous thromboembolic diseases.
In vitro
studies show that formation of a highly dense fibrin matrix is a major determinant of clot structure and stability. However, the precise contribution of fibrin matrix to occlusive arterial and venous thrombus formation
in vivo
remains largely undefined. To determine the contribution of fibrinogen versus fibrin to arterial thrombosis, venous thrombosis, and hemostatic potential
in vivo
, arterial and venous thrombotic challenges as well as hemostatic tests were performed in wildtype (

Fga
WT

) and

Fga
EK

mice that express normal levels of fibrinogen “locked” in the monomeric form. In a model arterial thrombosis, a 10% FeCl
3
injury to the carotid artery resulted in similar times to occlusion between

Fga
WT/WT

,

Fga
WT/EK

and

Fga
EK/EK

mice, while

Fga
-/-

mice were completely protected. However, when injured with 5% FeCl
3
,

Fga
EK/EK

mice were partially protected from occlusion but had increased embolic events. These results suggest that fibrinogen can contribute to arterial thrombus formation independent of fibrin formation. Consistently, fibrinogen-dependent platelet aggregation and intraplatelet fibrinogen content in

Fga
WT/WT

,

Fga
WT/EK

and

Fga
EK/EK

mice were similar. In an inferior vena cava stasis model of venous thrombosis,

Fga
EK/EK

had near complete protection from thrombus formation. Intriguingly,

Fga
WT/EK

mice also displayed significant reduction in thrombus frequency and mass relative to

Fga
WT/WT

mice. Plasma from

Fga
WT/EK

mice produced prolonged prothrombin and thrombin times with reduced fibrin formation based on turbidity assay. In bleeding models,

Fga
WT/EK

and,

Fga
EK/EK

mice had preserved hemostasis in contrast to

Fga
-/-

mice that presented with significantly impaired hemostasis. These findings show that selective prevention of fibrin polymerization significantly reduces venous thrombosis and suppresses arterial thrombosis based on the severity of injury with minimal disruption to hemostasis. Moreover, the data suggests that administration of non-polymerizable fibrinogen may be a therapeutic strategy against thrombosis.
Abstract 144: Enhancement Of High Density Lipoprotein-associated Elastase Inhibitor Activity Prevents Atherosclerosis Progression
Authors: Mobilia Maura , Whitus Callie , Karakashian Alexander , Johnson Lance , Graf Gregory A , Gordon Scott M
Published: October 2023

Background:
Inflammatory cells within atherosclerotic lesions secrete various proteolytic enzymes that contribute to lesion progression and destabilization, increasing risk of an acute cardiovascular event. The relative contributions of specific proteases to atherogenesis is not well understood. Elastase is a serine protease produced by macrophages and neutrophils in plaque and may contribute to the development of unstable plaque. We have previously reported interaction of endogenous protease inhibitor proteins on plasma high density lipoprotein (HDL), including alpha-1-antitrypsin, an inhibitor of elastase. These findings support a potential role for HDL as an endogenous modulator of protease activity. In this study, we test the hypothesis that enhancement of HDL-associated elastase inhibitor activity is protective against atherosclerotic lesion progression.

Methods and Results:
We designed an HDL-targeting protease inhibitor (HTPI) to bind to HDL and confer elastase inhibitor activity. HTPI is a small (1.6 kDa) peptide with an elastase inhibitor domain, a soluble linker, and an HDL-targeting domain. When incubated with Human plasma
ex vivo
, HTPI predominantly binds to HDL. Intravenous administration of HTPI to mice resulted in binding to plasma HDL and increased elastase inhibitor activity on isolated HDL. Accumulation of HTPI within plaque was observed after administration to

Apoe
-/-

and

Ldlr
-/-

mice. To examine the impact of HTPI treatment on atherosclerosis,

Ldlr
-/-

mice were fed Western diet for 12 weeks to establish atherosclerosis (WD-Saline) followed by two additional weeks on diet while receiving either saline or HTPI (2.5 mg/kg body weight) three times per week. Lesion area quantification by en face analysis revealed that HTPI prevented further lipid deposition in plaque. Histology and immunofluorescence staining of aortic root sections were used to examine the impact of HTPI on lesion morphology and inflammatory features.

Conclusions:
These data support the hypothesis that HDL-associated anti-elastase activity could contribute to the athero-protective functions of HDL and support the potential utility of enrichment of HDL with anti-protease activity for stabilization of high-risk atherosclerotic lesions.
Abstract 148: Lipoprotein Lipase Deficiency Reprograms Bone Marrow Cellular Metabolic Activity And Inflammatory Responses
Authors: Hu Shuchen , Kraakman Michael , Wang Branden , Chang Chuchun L
Published: October 2023

Objectives:
Activated bone marrow (BM) hematopoiesis is associated with systemic inflammation that increases risk for atherosclerosis. We had reported that lipoprotein lipase (LpL), in addition to being a lipolytic enzyme, regulated peripheral and tissue leukocyte levels and affected BM monocyte progenitor differentiation. LpL is mainly expressed in the specialized BM niches. We now questioned the role of LpL in influencing BM microenvironment that affects the homing and differentiation of hematopoietic stem/progenitor cells toward distinct lineages.

Methods and Results:
We characterized the metabolic and transcriptional phenotypes of BM cells utilizing immunofluorescence, flow cytometry, quantitative RT-PCR, and Seahorse Bioenergetics analysis in transgenic LpL knockout (KO) mice rescued by muscle-specific LpL expression. Differential gene expression (DEG) of BM cells were examined by bulk RNA sequencing (RNAseq). Our data indicate that LpL deficiency reduced expression of key BM vascular niche markers by 2-3-fold (p<0.05), including CD31, Tie-2, endomucin, CXCL12, and CXCR4, as well their associated growth factors - HIF-1α and VEGF. BM vascular endothelial cadherin-labeled cells were decreased by >50% (p<0.05) in LpL KO when compared to that in WT. Cellular uptake of chylomicron-size lipid particles was reduced in LpL KO BM, which may link to metabolic adaptation from mitochondrial oxidative phosphorylation towards glycolysis observed in LpL KO BM (p<0.05). Although the leptin-receptor positive perivascular cells were remained intact, expression of key lipolytic genes, such as ATGL, MGL, and LAL, were markedly reduced by >50% in LpL KO (p < 0.05). KEGG enrichment analysis of DEG shows that LpL KO BM cells had altered hematopoietic lineage signaling pathway. NFκB-directed signaling cascade was also downregulated in LpL KO BM cells, resulting in the reduced expression of pro-inflammatory TNF-α, IL-1β and IL-6.

Conclusions:
Our results indicate that LpL regulates metabolic activity and inflammatory responses of BM cells. A further understanding of these mechanisms will open the possibilities to target reprogram of both hematopoietic and BM niche cells as a therapeutic strategy to improve atherosclerosis-associated inflammation.
Abstract 155: Platelet-derived Transforming Growth Factor-β1 Ameliorates AAA in a Murine Model
Authors: Spuzzillo Anthony , Stone Bailey , Benson Tyler W , Brunner Seth , Wadding-Lee Caris , Owens Phillip P
Published: October 2023

Background:
Platelets are rapidly recruited to the forming intraluminal thrombus in abdominal aortic aneurysm (AAA), stabilizing the aortic wall and preventing rupture. Adhesion and activation of platelets releases a mix of inflammatory mediators such as transforming growth factor β (TGFβ), which mediates cellular proliferation, inflammatory signaling, and fibrosis. Importantly, while TGFβ neutralization results in increased rupture and disease progression in mouse models of AAA, the source of protective TGFβ signaling remains controversial. We hypothesized that platelet-derived TGFβ1 attenuates aortic growth in the elastase AAA model.

Methods and Results:

Ldlr
-/-

mice with platelet-specific deletion of
Tgfβ1
were produced by breeding hemizygous male mice expressing Cre under the control of the
Pf4
promoter to female
Tgfβ1
floxed mice (Jackson Labs). Mice were subjected to laparotomy and topical elastase application to the abdominal aorta (10 mg/mL porcine pancreatic elastase for 5 minutes) and sacrificed at day 28 to determine AAA progression via aortic diameter. Male

TGFβ1-Pf4
Cre+

mice (n=8) had significantly increased abdominal aortic diameters compared to Male

TGFβ1-Pf4
Cre-

mice (n=7) (
Cre-
: 1.70 ± 0.12 mm;
Cre+
: 2.66 ± 0.25 mm; P < 0.004). Additionally, 25% of male

TGFβ1-Pf4
Cre+

mice died of AAA rupture compared to 0% in the

TGFβ1-Pf4
Cre-

cohort. To determine if these actions were occurring via TGFβ1 activation of the TGFβ receptor 2 (βr
2
),

Ldlr
-/-

/βr2
Flox
/PF4
Cre+
and
Cre-
mice were created, similarly to the previously described
Tgfβ1
mice. Male

βr2-Pf4
Cre+

(n=7) had increased aortic diameter compared to

βr2-Pf4
Cre-

(n=6) (
Cre-
: 1.70± 0.20 mm;
Cre+
: 2.45 ± 0.38 mm; P < 0.138). Additionally, female

βr2-Pf4
Cre+

(n=9) also had significantly larger aortic diameters compared to

βr2-Pf4
Cre-

(n = 5) (
Cre-
: 1.27± 0.04 mm;
Cre+
: 1.90 ± 0.23 mm; P < 0.01).

Conclusions:
Our results further support TGFβ signaling playing a protective role in AAA. More specifically, platelet-derived TGFβ1 ligand and signaling via platelet TGFβr2 mitigates aortic diameter expansion. Further research into the pertinent TGFβ isoforms and the mechanism of TGFβr2 activation will ideally yield insights into therapeutic targets for an, as yet, intractable disease.
Abstract 157:
Inkiln
is a Novel Long Noncoding RNA Promoting Vascular Smooth Muscle Inflammation Via Scaffolding Mkl1 and Usp10
Authors: Zhang Wei , Ishimwe Nestor , Pauli Jessica , Miller Clint L , Weintraub Neal L , Maegdefessel Lars , Miano Joseph M , Baker Andrew H , Long Xiaochun
Published: October 2023

Activation of vascular smooth muscle cells (VSMCs) inflammation is critical in initiation and progression of vascular disease. However, the role of human-specific long noncoding RNAs (lncRNAs) in VSMC inflammation is largely unknown. We performed Bulk RNA-seq in differentiated human VSMCs and revealed a novel human-specific lncRNA called
IN
flammatory M
K
L1
I
nteracting
L
ong
N
oncoding RNA (
INKILN
).
INKILN
is barely expressed in contractile VSMCs or healthy vessels and induced in human atherosclerosis and abdominal aortic aneurysm.
INKILN
is transcriptionally activated through p65/NF-κB pathway.
INKILN
activates proinflammatory gene expression in cultured human VSMCs and in ex vivo cultured vessels. Mechanistically,
INKILN
physically binds to and stabilizes MKL1 protein, a key activator of VSMC inflammation through the p65/NF-κB pathway. In the IL1β-activated VSMCs, depletion of
INKILN
blocks the nuclear localization of both p65 and MKL1, and abolishes the physical interaction between these two proteins, resulting in the reduced transactvity of p65/NF-κB. Further,
INKILN
knockdown enhances MKL1 ubiquitination, likely through the reduced physical interaction with the deubiquitinating enzyme USP10. The in vivo study of
INKILN
has utilized a Bacterial Artificial Chromosome (BAC)
INKILN
-Transgenic (Tg) mouse line, due to the lack of non-conserved
INKILN
gene in the mouse genome. In the ligation-injured carotid arteries of BAC
INKILN
-Tg mice, human lncRNA
INKILN
is induced and has exacerbated neointimal formation. In conclusion, these findings elucidate an important pathway of VSMC inflammation involving an
INKILN
/MKL1/USP10 regulatory axis. Human BAC Tg mice offer a novel and physiologically relevant approach for investigating human-specific lncRNAs under vascular disease conditions.
Abstract 159: Palmitic Acid Alters Enhancers And Super Enhancers Near Inflammatory And Efferocytosis-associated Genes In Human Monocytes
Authors: Tanwar Vinay S , Reddy Marpadga A , Dey Suchismita , Malek Vajir , Lanting Linda , Natarajan Rama
Published: October 2023

Background:
Elevated free fatty acids (FFA) like palmitic acid (PA) are associated with inflammatory cardiometabolic disorders like diabetes and atherosclerosis. FFA induced dysregulated gene expression in monocytes plays important roles in inflammation, but the epigenetic mechanisms involved are unclear. Here we examined PA induced alterations in enhancer and super-enhancer (E/SE) repertoires to elucidate epigenetic mechanisms of inflammatory gene regulation in monocyte dysfunction.

Methods:
Human CD14
+
monocytes from healthy volunteers were treated with PA (200 μM, 24 h) or vehicle (0.2% BSA). ChIP-seq with histone-H3K27ac was performed to identify Es/SEs and RNA-seq to analyze gene expression genome-wide. Publicly available Genomic Regions Enrichment of Annotations Tool, and datasets from promoter capture Hi-C (PCHiC) and GWAS on diabetes, obesity and BMI were used to analyze functions/disease relevance of Es/SEs. ChIP/RT-qPCRs were used to validate ChIP-/RNA-seq results. Functions of SEs were determined using JQ1 (SE/BET bromodomain inhibitor).

Results:
ChIP-seq results showed PA extensively dysregulated Es/SEs in CD14
+
monocytes vs. vehicle control. PA-regulated Es/SEs showed enrichment of immune system related processes, differentiation, and cytokine production. RNA-seq showed significant correlations between dysregulated enhancers and differential expression of nearby genes. These genes were also altered in adipose tissue macrophages from high fat fed mice, and bone marrow derived macrophages from type2 diabetic
db/db
mice and
Apoe
-/-
mice with accelerated atherosclerosis. Differentially regulated Es/SEs harbored SNPs associated with diabetes, obesity, and BMI. PCHiC revealed interactions between Es/SEs and promoters of key genes associated with efferocytosis (
MERTK
), metabolism (
OSBPL8
) and inflammation (
IRAK2, CSF2
and
RIPK2
). Changes in gene expression and H3K27ac at Es/SEs were validated. Treatment with JQ1 reversed PA-induced inflammatory genes in monocytes.

Conclusions:
PA treatment dysregulated Es/SEs involved in key monocyte functions including efferocytosis, inflammation and metabolism. Targeting such epigenetic mechanisms could be beneficial in the treatment of cardiometabolic diseases.
Abstract 163: Thrombocytopenia Independently Leads To Monocyte Immune Dysfunction
Authors: Li Chen , Ture Sara , Maurya Preeti , Livada Alison , Morrell Craig
Published: October 2023

In addition to their well-studied hemostatic functions, platelets are immune cells. Platelets circulate at the interface between the vascular wall and leukocytes, and transient platelet-leukocyte complexes are found in both healthy and disease states, positioning platelets to provide physiologic cues of both vascular health and injury. Roles for activated platelets in inducing and amplifying immune responses have received an increasing amount of research attention, but our past studies also showed that normal platelet counts are needed in healthy conditions to maintain immune homeostasis. We have now found that thrombocytopenia (a low platelet count) leads to monocyte dysfunction, independent of the cause of thrombocytopenia, in a manner that is dependent on direct platelet-monocyte CD47 interactions that regulate monocyte immunometabolism and gene expression. Compared to monocytes from mice with normal platelet counts, monocytes from thrombocytopenic mice had increased toll-like receptor (TLR) responses, including increased IL-6 production. Furthermore,
ex vivo
co-incubation of resting platelets with platelet naïve bone marrow monocytes, induced monocyte metabolic programming and durable changes in TLR agonist responses. Assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-Seq) on monocytes from thrombocytopenic mice showed persistently open chromatin at LPS response genes. Using mouse models of thrombocytopenia and sepsis we found that normal platelet numbers are needed to limit immune dysregulation in sepsis.
IL6
and
TNF
expression in monocytes from human patients with sepsis also inversely correlated with patient platelet counts. Our studies demonstrate that in healthy conditions resting platelets maintain monocyte immune tolerance by regulating monocyte immunometabolic processes that lead to epigenetic changes in TLR-related genes. Platelets therefore function as immune rheostats in both health and disease.
Abstract 165: Large Extracellular Vesicles-derived MicroRNA-150-5p Is Upregulated In Patients With Aortic Valve Stenosis And Regulates Valvular Endothelial Cell Function
Authors: Mohammadi Neda , Hildebrand Staffan , Goody Philip , Nehl Denise , Jansen Felix , Hosen M. Rabiul , Pfeifer Alexander
Published: October 2023

Aortic valve stenosis (AVS) is the most common type of valvular disease. Recent evidence indicates that extracellular vesicles (EVs) might play an important role in cardiovascular disease, particularly AVS. However, the contribution of EV-encapsulated microRNAs (miRNAs) in valvular endothelial cells (VECs) and AVS has been poorly investigated. Herein, we identified the differentially expressed miRNAs in large EV-derived aortic valve tissue from patients with AVS and explored the novel role of miR-150-5p in endothelial to mesenchymal transition (EndMT). Our miRNA sequencing revealed several EV-miRNAs (miRNA-150-5p, miR-486-5p, and miR-486-3p) were significantly dysregulated in large EVs from aortic valve tissue in patients with AVS. In silico analysis indicated that miRNA-150-5p is mainly enriched in Wnt, TGF-β, and PI3k/AKT signaling pathways, known to be altered in the pathogenesis of AVS. To elucidate the role of miR-150-5p in AVS, we validated the upregulation of miR-150-5p in AVS patients by RT-qPCR. Importantly, the overexpression of miR-150-5p in VECs treated with TNF-α reduced EndMT, which was quantized by significant expression of EndMT markers (vWF, ACTA1, VIM, and SLUG). To investigate the role of miRNA-150-5p in VEC function, cell migration and tube formation were performed. The data revealed that miR-150-5p regulates VEC function via increased tube formation and reduced cell migration. To further investigate VEC function under the laminar flow, which regulates EC function, we exposed VEC under the flow and found that the expression of miR-150-5p is increased suggesting a protective role in VECs. To study the role of miR-150-5p in intercellular communication, we overexpressed miR-150-5p in donor VECs and incubated them with recipient VECs prior to the labeling of EVs with PKH67. Fluorescent microscopy showed an increased expression of miR-150-5p in recipient VECs. Further, we induced EndMT in VECs after co-incubation with EVs (miR-150-5p overexpression vs. control). Our qPCR-analysis revealed that EV-miR-150-5p rescued the expression of EndMT markers. Our data showed that large EV-associated miR-150-5p was significantly upregulated in AVS and may play a crucial role in cellular crosstalk via regulating function of VEC via EndMT.
Abstract 211: Fibromuscular Dysplasia Causing Upper Extremity Acute Limb Ischemia
Authors: Earthineni Geethika , Njoh Roland
Published: October 2023

Fibromuscular dysplasia (FMD) is a non-atherosclerotic arterial disease that affects mainly the renal and cervical arteries. It can also affect peripheral arteries leading to acute or chronic ischemia of the limbs. We present a rare case of FMD causing acute limb ischemia.

Case Presentation:
A 45-year-old male presented with acute onset pain and coldness in his left upper extremity, associated with numbness and tingling sensation in his left hand. He denied any history of trauma or recent surgery. His medical history was significant for hypertension. He was a non-smoker and denied any use of illicit drugs.

On exam, his left hand was pale, cold, with absent distal pulses. The capillary refill time was prolonged, and the skin was mottled. There was tenderness in the hand muscles with no signs of compartment syndrome. Rest of the exam was normal.

A diagnosis of acute limb ischemia was made, and the patient was urgently taken to the cath lab. Angiogram showed string and beads pattern of the radial artery with distal occlusion suggestive of FMD. Balloon angioplasty of the radial artery failed to improve blood flow. He subsequently underwent radial artery bypass using autologous vein graft with restoration of blood flow. Renal and carotid arteries were unaffected on CTA.

Discussion:
Fibromuscular dysplasia is a non-atherosclerotic, non-inflammatory disease that affects medium to small sized arteries. It is characterized by hyperplasia and fibrosis within the arterial wall. The exact etiology of FMD is unknown, and it is believed to be multifactorial.

Renal artery FMD may present with renovascular hypertension, cervical artery involvement may present with headache, tinnitus, and vertigo. Patients with peripheral artery involvement may present with intermittent claudication, acute limb ischemia, or limb loss.

CTA, MRA, or conventional angiography are diagnostic. In patients with acute limb ischemia, emergent intervention with thrombectomy, embolectomy, or bypass surgery may be necessary. Anti platelets and anticoagulants are also used.

Conclusion:
FMD is a rare disease that can present with a variety of clinical manifestations, including acute limb ischemia. Prompt diagnosis and treatment are essential to prevent limb loss and other serious complications.