bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 1139: Recent Trends And Predictors Of Poor Outcomes In Patients With Chronic Thromboembolic Pulmonary Hypertension Undergoing Pulmonary Artery Thromboendarterectomy.
Authors: Nriagu Bede N , Ozbay Mustafa , Gebran Karim , Hung Chien , Nriagu Ugonwa N , Idowu Abiodun , NRIAGU VALENTINE C , Prajapati Kesar
Published: July 2024

Introduction:
Chronic thromboembolic pulmonary hypertension (CTEPH) occurs due to pulmonary artery obstruction from persistent thromboembolic disease. Pulmonary artery thromboendarterectomy (PTE) is the definitive treatment. In this study, we examine the recent trends, predictors of mortality among patients with CTEPH undergoing PTE.

Methods:
Using the International Classification of Diseases, 10th edition, Clinical Modification (ICD-10-CM) diagnosis and procedure codes, we queried the National Inpatient Sample 2016 to 2020 for hospitalizations among patients ≥18 years old with a diagnosis of CTEPH who underwent PTE. We examined the recent trends in the uptake of PTE, the difference in mortality by year, race, sex, and US region. Logistic regression was used to identify independent predictors of mortality among this cohort of patients.

Results:
Our study included 18,195 hospitalizations with CTEPH of which 1,790 (9.8%) (mean [SD] age: 54.0 [14.9] years) underwent PTE. In this cohort, 51.7% were males and 47.3% were females. We observed an increasing trend in the uptake of PTE; 7.3%, 27.1%, and 37.4% for the years 2017, 2018, 2019 respectively. This trend reversed in 2020 (28.2%) (
p=0.9
). Mortality was recorded among 5.9% of patients with CTEPH who had PTE. No difference in mortality was observed by sex/gender, year, race, or hospital region. ECMO was utilized among 8.4% of patients with CTEPH undergoing PTE. Mortality was higher among those who utilized ECMO compared to those who did not utilize ECMO (36.7% vs. 3.1%, p<0.0001). ECMO utilization was identified as an independent predictor of mortality OR: 18.4, 6.96-48.74, P<0.0001. Sex, HTN, and Obesity were not associated with mortality in this cohort.

Conclusion:
We observe increasing utilization of pulmonary artery thromboendarterectomy among patients hospitalized with CTEPH and increased mortality among those requiring ECMO. Further research is needed to identify predictors of poor outcomes among patients with CTEPH undergoing PTE.
Abstract 1141: Unveiling Endothelial Cell Metabolic Pathways Il21 Driven Angiogenesis And Barrier Stability In Ischemic Conditions
Authors: Alhusban Suhib , Kovacs-Kasa Anita , Nofal Mohamed , Zaied Abdelrahman A , Koseoglu Mehmet M , Annex Brian H
Published: July 2024

Background:
Angiogenesis is the growth of new blood vessels from preexisting vascular structures. Clinical trials using vascular endothelial growth factor 165a (VEGF165a) for angiogenesis in peripheral arterial disease (PAD) yielded little success. Glycolysis accounts for 85% of ATP production in endothelial cells (ECs) and is further activated during VEGF165a induced angiogenesis. Our previous research demonstrated interleukin 21 (IL21) promoting angiogenesis in PAD models independently of the VEGF165a pathway.

Hypothesis:
IL21 utilizes different metabolic pathways than glycolysis in ECs under ischemic conditions.

Goal:
Explore the metabolic pathway(s) activated in ECs in response to IL21 and its functional consequence on angiogenesis in preclinical models of PAD.

Methods:
Human umbilical vein endothelial cells (HUVECs) were cultured under hypoxia and serum starvation media, treated with PBS (control), VEGF165a (50 ng/mL), or IL21 (50 ng/mL). Methods included qPCR, Western blot (WB)
of the genes of the rate controlling enzymes of the main metabolic pathways
, angiogenesis assay tube formation on Matrigel and assessing endothelial barrier function
using trans endothelial resistance (TER).

Results:
In HUVECs under HSS, using qPCR, IL21 vs. PBS upregulates glutamine fructose-6-phosphate amido-transferase isoform 1 (GFAT1); the rate limiting enzyme for hexosamine biosynthesis pathway (HBP) (Relative expression 1.28±0.08 SD vs. 1.00±0.04 SD, n=3/group, p=0.03) while VEGF165a did not (Relative expression 1.04±0.03 SD vs. 1.00±0.04 SD, n=3, p=0.23). WB showed similar finding where IL21 vs. PBS increased GFAT1 expression under the same conditions (Relative expression 1.74±0.39 SD vs. 1.00±0.16 SD, n=3/group, p=0.04) while VEGF165a did not. Knocking down GFAT1 reduced IL21 mediated hypoxia dependent angiogenesis (number of tubes, 1.00±0.37 SD vs. 0.37±0.10 SD, n=4-5/group, p<0.0001) but not VEGF165a. The activation of HBP by IL21, was associated with a more stable barrier function compared to VEGF165a (TER relative to control; 0.87±0.01 vs. 0.79±0.01, n=5/group, p<0.0001).

Conclusion:
These results unmask an unexpected role of HBP in angiogenesis regulation, emphasizing the significance of EC metabolism under PAD conditions.
Abstract 1148: Validating Artificial Intelligence-assisted Classification Of Peripheral Arterial Disease Lesion Composition From 9.4 Tesla Magnetic Resonance Imaging With Histology
Authors: Csore Judit , Karmonik Christof , Benfor Bright , Osztrogonacz Peter , Roy Trisha
Published: July 2024

Objectives:
MRI is a promising tool for identifying tissue properties and enhancing the precision of endovascular peripheral artery disease (PAD) treatment. However, the interpretation of images might pose a challenge for untrained experts.

Hypothesis:
Unsupervised artificial intelligence (AI) algorithms can assist in interpreting MRI images and classifying lesion components.

Aims:
To confirm the feasibility of a custom-made variational autoencoder (VAE) in evaluating lesion crossability through histological validation.

Methods:
9.4T MRI images (150-micron isotropic resolution) were acquired on 20 chronic total occlusion PAD lesions using T2-weighted (T2w) and Ultrashort Echo Time (UTE) contrasts. Pseudo-colors (T2w: red, UTE: green) were created to enhance tissue visualization (Fig1). A VAE algorithm classified axial images into categories based on the lumen being occluded with hard or soft tissue. Lesions underwent histologic analysis, slides were matched with the corresponding VAE output images. Two readers assessed lesion crossability (non-crossable if >50% of lumen was occluded with hard tissue) on both VAE-generated images and histologic slides.

Results:
MRI produced 4013 psuedo-color images, and VAE successfully separated axial sections based on plaque composition. Histologic analysis yielded 91 cross-sections. The consensus reading identified 34% (n=31) of VAE images and 25% (n=23) of histologic slides as non-crossable, with a significant association between the histology-based and AI-based crossability (p<.001). Inter-rater reliability was good for both VAE-based and histology-based crossability assessments (intralass correlation coefficient (ICC)= 0.76 and 0.80, respectively).

Conclusion:
Our AI algorithm demonstrated a strong association with histologic validation, suggesting potential for enhancing image interpretation, and possibly contributing to improved planning for endovascular interventions.
Abstract 1157: Inhibition Of P90rsk Ameliorates Pdgf-bb-induced Vascular Smooth Muscle Cell Phenotypic Change And Neointimal Hyperplasia
Authors: Hwang Ae-Rang
Published: July 2024

Platelet-derived growth factor type BB (PDGF-BB) has been reported to induce vascular smooth muscle cell (VSMC) proliferation and migration, which are major events that are highly linked to vascular diseases such as atherosclerosis and restenosis. Although p90RSK has been shown to regulate multiple cellular processes including cell growth, proliferation, survival and motility through various downstream substrates in different cell types, the role of p90RSK on PDGF-BB-induced VSMC proliferation and migration, and vascular neointima formation remains unknown. In this study, we determine whether inhibition of p90RSK protects from PDGF-BB-induced cellular phenotypic change and investigate the molecular mechanisms underlying the effect of p90RSK inhibition. In cultured primary rat VSMCs, we found that pretreatment with FMK or BI-D1870, specific inhibitors of p90RSK, suppressed PDGF-BB-induced VSMC phenotypic alteration, ECM accumulation, proliferation, and migration. In addition, FMK and BI-D1870 repressed cell cycle regulatory genes cyclin D1 and Cdk4 induced by PDGF-BB, whereas inhibition of p90RSK augmented cyclin-dependent kinase inhibitor p27 downregulated by PDGF-BB in VSMCs, suggesting that p90RSK induces VSMC proliferation via cell cycle regulation of G0/G1 phase. Notably, using the mouse model of partial carotid ligation model, FMK was found to attenuate the neointimal hyperplasia of carotid arteries. These results suggest that p90RSK impedes PDGF-BB induced VSMC phenotypic switching, proliferation, migration, and neointima formation.
Abstract 1159: Harnessing Limb Revascularization With Synthetic Probiotics
Authors: Vlahos John , Ratner Molly , Harimoto Tetsuhiro , Zias Ariadne , Rivera Cristobal , Pratama Yogi , Garg Karan , Danido Tal , Ramkhelawon Bhama
Published: July 2024

Peripheral arterial disease (PAD) is a critical vascular condition that results in impaired blood flow leading to chronic limb threatening ischemia (CLTI), tissue necrosis and oftentimes to lower-limb amputation. This is largely due to suboptimal revascularization and inflammatory programs, that fail to instigate neovascularization at sites of vessel stenosis. The incidence of CLTI is on the rise but yet there is a paucity of effective treatment options for patients. We hypothesized that the coordinated action of regenerative angiogenesis and immunosurveillance would likely stimulate effective revascularization in the ischemic leg. The neuroimmune cue, Netrin-1 has been shown to possess such properties. We have shown that Netrin-1 can assist atherosclerosis onset by modulating macrophage chemotaxis and shown its critical role in modulating vascular remodeling. Here we show that the conditional knock-down of Netrin-1 in myeloid cells (Ntn1
flox/flox
LysM
cre+
), protects mice from revascularization following femoral artery ligation compared to WT littermates. The deletion of Netrin-1 in endothelial cells nor vascular smooth muscle cells was insufficient to produce such effects. Notably, in human amputated tissue sections, the expression of Netrin-1 was repressed at sites proximal to amputated sites compared to distal regions with preserved blood flow. Tissue macrophages were packed with Netrin-1 and it exerted its effects in an autocrine/paracrine manner via its receptor Unc5b. The use of synthetic biology to engineer bacteria with tailored functionalities presents a cutting-edge avenue for developing novel therapeutic tools. We therefore employed a synthetic biology approach to deliver exogenous Netrin-1 to the ischemic leg. The Nissle probiotic was genetically engineered to overexpress murine Netrin-1. The local administration of this probiotic to ischemic limb of Ntn1
flox/flox
LysM
cre+
mice was sufficient to recapitulate blood flow and reperfusion in the leg comparable to WT mice (and sham probiotic) in 2 weeks. Our results show the therapeutic potential of the use of synthetic microorganisms and Netrin-1 for innovative and adaptable solutions in limb regenerative medicine.
Abstract 122: Deletion Of Pyruvate Dehydrogenase Kinases Reduces Susceptibility To Experimental Venous Thrombosis By Limiting Glycolysis In Neutrophils
Authors: Flora Gagan D , Ghatge Madankumar , Nayak Manasa K , Barbhuyan Tarun , Chauhan Anil K
Published: July 2024

Background:
Neutrophils by releasing extracellular traps (NETs) provide a scaffold for platelet activation and coagulation, thereby facilitating venous thrombosis (VT) development. Emerging evidence suggests that neutrophil activation and NETs formation (NETosis) is an energy-intensive process, accompanied by an increased rate of aerobic glycolysis (lactate formation in the presence of oxygen). The metabolic enzymes pyruvate dehydrogenase kinases (PDKs) inhibit the pyruvate dehydrogenase complex to divert the pyruvate flux (final product of glycolysis) from oxidative phosphorylation towards aerobic glycolysis. Recently, we reported the role of PDK2 and PDK4 (PDK2/4) in platelet activation and arterial thrombosis. The regulatory role of PDKs in neutrophils and VT has not been explored.

Aim:
To test the hypothesis that PDK2/4 promotes NETosis and VT by promoting glycolysis in neutrophils.

Methods:
Double-deficient PDK2/4
-/-
and wild-type (WT) mice were subjected to 48 hours of inferior vena cava stenosis to test the susceptibility to VT.
In vitro
, neutrophils were stimulated with Phorbol 12-myristate 13-acetate (PMA) and the glycolytic proton efflux rate (glycoPER) was measured using the Seahorse XF Analyzer.

Results:
The PDK2/4
-/-
mice were less susceptible to DVT as measured by the primary outcome of reduced thrombus burden (weight, length and % incidence) and marker of NETs such as citrullinated histones (P<0.05 vs WT). In the WT platelet aggregation assay mediated by PMA-stimulated WT or PDK2/4
-/-
neutrophils, we observed reduced platelet aggregation evoked by PMA-stimulated PDK2/4
-/-
neutrophils (P<0.05 vs WT). Impaired aggregation was associated with reduced release of cathepsin G and elastase from PMA-stimulated PDK2/4
-/-
neutrophils (P<0.05 vs WT). Mechanistically, PMA-stimulated PDK2/4
-/-
neutrophils exhibited reduced NETosis that was associated with decreased calcium mobilization and Erk1/2 phosphorylation (P<0.05 vs WT), known contributors of NETosis. Furthermore, PMA-stimulated PDK2/4
-/-
neutrophils displayed reduced glycoPER (P<0.05 vs WT), suggesting reduced aerobic glycolysis.

Conclusions:
These findings for the first time demonstrate the regulatory role of PDK2/4 in NETosis and acute progression of VT.
Abstract 123: Hemostatic Fibrin Knob Triggered Microgel To Promote Clotting With Reduced Thrombotic Risks In Neonates
Authors: Zandi Nooshin , Nellenbach Kimberly , Daniele Michael , Brown Ashley
Published: July 2024

Introduction:
Effective neonatal hemostatic therapies are vital for complex surgery-related life-threatening bleeding [1]. Neonatal hemostatic systems differ significantly from adults [2, 3]; however, current treatments for bleeding include adult blood products (ABP). Neonatal ABP use poses an elevated thrombotic risk [4, 5], necessitating alternative approaches. We propose hemostatic therapies leveraging age-dependent clotting differences. In adults, clot formation relies on fibrin(ogen) A:a knob-hole binding, while B:b binding dominates in neonates. Targeting B:b interactions with synthetic Bknob conjugated platelet-like particles (BK-PLP) could crosslink neonatal fibrinogen, catalyze clot formation, and enhance clot strength with lower thrombotic risk than ABP. To test this, we synthesized pNIPAm microgels and conjugated them to B-knob (AHRPYAAK), A-knob (GPRPFPAK), and non-binding control peptides (GPSPFPAK) to prepare BK-PLP, AK-PLP, and NB-PLP.

Materials and Methods:
We prepared BK-PLP, AK-PLP, and NB-PLP by conjugating microgels with mimetic peptides using EDC-Sulfo-NHS chemistry (Fig. 1A); Fibrin clot polymerization, stability, density, and stiffness, were analyzed via plate-based assays, confocal microscopy, and AFM.

Results and Discussion:
BK-PLPs significantly increased clot stability compared to AK-PLP, NB-PLP, or fibrin-only clots (Fig. 1B). Fiber density also increased with BK-PLPs, indicating a role in promoting denser fibrin clot structures (Fig. 1C). Clot mechanical properties assessment suggested that AKnob and NB-PLPs may disrupt clot networks, decreasing stiffness (Fig. 1D).

Conclusion:
Our study explored the effects of peptide conjugate particles on neonatal fibrin clots. BK-PLP incorporation increased clot stability, while AK-PLP and NB-PLP decreased clot stiffness, supporting further BK-PLP development for neonatal hemostasis.
Abstract 129: Hypercholesterolemia-induced Lxr Signaling In Smc Contributes To Atherosclerotic Lesion Remodeling And Regulates Vascular And Visceral Smc Function
Authors: Zhang Hanming , Biwer Lauren , Saenz de Urturi Diego , Fernandez-Tussy Pablo , Jovin Daniel , Huang Yan , Zhang Xinbo , Esplugues Enric , Greif Daniel , Suarez Yajaira , Fernandez-Hernando Carlos F
Published: July 2024

Vascular smooth muscle cells (VSMCs) play an important role during atherogenesis, regulating vasotone and lesion remodeling. Recent advances in combing VSMC lineage tracing and scRNA-seq have generated high-dimensional analysis of VSMC profiles of atherosclerotic lesion, delineating multiple VSMC-derived transitional states in vascular lesions, which can further adopt plaque stabilizing or plaque destabilizing cell states. Despite the identification of several transcription factors that govern SMC fate, the signaling events that promote SMC transitional state remain poorly understood. Our trajectory analysis of VSMCs gene expression reveals the activation of liver X receptor (LXR) signaling, a master regulator of lipid metabolism, during the progression of atherosclerosis, suggesting a relevant role of this transcription factor in controlling VSMC fate decision and lesion remodeling. To dissect the specific contribution of LXR signaling in VSMC during atherogenesis, we generated and characterized mice in which the LXRα and β genes were conditionally ablated in SMCs in the adult vasculature and visceral tissues. Deficiency of LXR in SMCs under hypercholesterolemic condition influenced lesion remodeling by altering the fate of de-differentiated SMC and promoting the accumulation of VSMC-derived transition cells. This phenotypic switching is accompanied with increased features of plaque instability characterized by larger necrotic cores and reduced fibrous cap thickness. Moreover, LXR SMC deficiency impaired vascular function and caused visceral myopathy characterized by bladder maladaptive remodeling and altered gut lipid absorption. Together, we uncovered an essential role of LXR-regulated signaling that contributes to atherosclerotic lesion remodeling, and vascular/ visceral SMC function.
Abstract 131: Local Pcsk6 Inhibition Attenuates VSMC Response In Vascular Injury And Restenosis
Authors: Chemaly Melody , Jin Hong , Vuckovic Sofija , Suur Bianca E , Zhang Xiang , Kronqvist Malin , Lengquist Mariette , Uryga Anna , Knape Karin Conde , Nyberg Michael , Hedin Ulf , Matic Ljubica , Matic Ljubica
Published: July 2024

Intro:
Vascular smooth muscle cell (VSMC) proliferation following endovascular interventions leads to restenosis, treated with drug-eluting stents. However, current drugs completely abolish vascular healing, cause thrombosis and clinical events. PCSK6 is a key protease in vascular remodeling and VSMCs activation upon vessel injury,
via
regulation of MMP2/14 activity. Here, we investigate whether local PCSK6 inhibition reduces VSMC proliferation and intimal hyperplasia.

Methods:
PCSK6 peptide inhibitor was tested
in vitro
on VSMCs migration, proliferation, apoptosis in comparison to rapamycin and paclitaxel. Internalization of a FITC labelled inhibitor was studied by flow cytometry and immunofluorescence.
In vivo
, PCSK6 inhibitor was administered at 3mg/kg IP using cationic microbubbles, followed by ultrasound-mediated local delivery in mice developing intimal hyperplasia after carotid ligation
(n=8 vs 11).
Carotid ligation in

Tagln
Cre+
/Pcsk6
fl/fl

mice investigated the effect of PCSK6 specific ablation in VSMCs on intimal hyperplasia
(n=7 vs 9).
Carotids were collected from these and constitutive

Pcsk6
-/-

mice
vs.
controls for transcriptomic and histological analyses.

Results:
In vitro
, PCSK6 inhibitor was internalized and inhibited both VSMC migration and proliferation (p<0.0001), reducing the expression of
PCSK6,
MMP2
and
GDF15
. In comparison, paclitaxel showed severe VSMC toxicity, whereas rapamycin inhibited more strongly VSMC migration and proliferation than the PCSK6 inhibitor.
In vivo,
the inhibitor reduced intimal hyperplasia (p=0.026), again, by repressing
PCSK6
and
MMP2
expression in treated
vs.
control mice, with a favorable safety profile. Likewise, conditional VSMC
Pcsk6
knockouts showed less intimal hyperplasia (p=0.053). In carotids from

Pcsk6
-/-

mice, cytoskeletal (Synm) and peptidase (Pgpep1l, Klk1) genes were strongly downregulated, showing its importance for vascular matrix composition.

Conclusion:
Our proof-of-concept translational study shows that local application of a PCSK6 inhibitor is a viable method for specifically modulating VSMC activation in intimal hyperplasia following vascular injury
Abstract 135: Monocytes Reprogrammed By 4-PBA Potently Contribute To The Resolution Of Inflammation And Atherosclerosis
Authors: Geng Shuo , Li Liwu
Published: July 2024

Chronic inflammation initiated by inflammatory monocytes underlies the pathogenesis of atherosclerosis. However, approaches that can effectively resolve chronic low-grade inflammation targeting monocytes are not readily available. The small chemical compound 4-phenylbutyric acid (4-PBA) exhibits broad anti-inflammatory effects in reducing atherosclerosis. Selective delivery of 4-PBA reprogrammed monocytes may hold novel potential in providing targeted and precision treatment of atherosclerosis. Single-cell RNA-sequencing and complementary immunological approaches characterized key resolving characteristics as well as defining markers of reprogrammed monocytes trained by 4-PBA. Molecular mechanisms responsible for monocyte reprogramming was assessed by integrated biochemical and genetic approaches. The inter-cellular propagation of homeostasis resolution was evaluated by co-culture assays. The in vivo effects of monocyte resolution and atherosclerosis prevention by 4-PBA were assessed with the high fat diet-fed ApoE
-/-
mouse model with 4-PBA administration. Furthermore, the selective efficacy of 4-PBA trained monocytes were examined by transfusion of
ex vivo
trained monocytes by 4-PBA into recipient high fat diet-fed ApoE
-/-
mice. We found that monocytes can be potently reprogrammed by 4-PBA into an immune-resolving state characterized by reduced adhesion and enhanced expression of anti-inflammatory mediator CD24. Mechanistically, 4-PBA reduced the expression of ICAM-1 via reducing peroxisome stress and attenuating SYK-mTOR signaling, and 4-PBA enhanced the expression of resolving mediator CD24 through promoting PPARγ neddylation mediated by TOLLIP. 4-PBA trained monocytes can effectively propagate anti-inflammation activity to neighboring monocytes through CD24. Our data further demonstrated that 4-PBA trained monocytes effectively reduce atherosclerosis pathogenesis when administered in vivo. Our study describes a robust and effective approach to generate resolving monocytes, characterizes novel mechanisms for targeted monocyte reprogramming, and offers a precision-therapeutics for atherosclerosis based on delivering reprogrammed resolving monocytes.
Abstract 136: Protease-activating Receptor-4 Antagonism In Coronary Artery Disease
Authors: Nash Jennifer , Meah Mohammed M , Whittington Beth , Debono Samuel , Raftis Jennifer , Miller Mark R , Sorbie Andrew , Mills Nicholas L , Nespoux Josselin , Bruce Lorraine , Duffin Rodger , Dhaun Neeraj , Brittan Mairi , Chao Longfei , Merali Samira , Kim Minji , Wang Zhaoqing , Zhang Yue , Jin Shiqiang , Wang Beqing , Kozinn Marc , Newby David E
Published: July 2024

Background:
BMS-986141 is a novel potent highly selective antagonist of protease-activated receptor type 4 (PAR4). PAR4 antagonism has been demonstrated to reduce thrombus formation in isolation and in combination with factor Xa inhibition in high shear conditions in healthy people.

Aim:
To determine whether PAR4 antagonism had additive antithrombotic effects in patients with coronary artery disease who were receiving antiplatelet therapy.

Methods:
Forty-five patients with stable coronary heart disease and ten healthy volunteers completed a phase 2a open-label four-arm single-center study. Patients were allocated to one of three treatment arms for seven days: (i) ticagrelor (90 mg twice daily), (ii) aspirin (75 mg once daily) or (iii) the combination of ticagrelor and aspirin. Agonist-induced platelet aggregation, platelet activation, and ex vivo thrombus formation were measured before, 2 and 24 hours after a single oral 4-mg dose of BMS-986141 in all participants.

Results:
BMS-986141 demonstrated highly selective inhibition of PAR4-agonist peptide (AP) induced platelet aggregation, p-selectin expression, and platelet-monocyte aggregate expression (p≤0.001 for all) which were unaffected by concomitant antiplatelet therapies. PAR4 antagonism reduced ex vivo thrombus area in high shear conditions in healthy volunteers (-21%, p=0.001) and in patients receiving ticagrelor alone (-28%, p=0.001), aspirin alone (-23%, p=0.018), or both in combination (-24%, p≤0.001). Plasma concentration of BMS-986141 correlated with PAR4-AP-induced platelet responses (p≤0.001 for all) and total thrombus area under high shear stress conditions (p≤0.01 for all).

Conclusions:
PAR4 antagonism has additive antithrombotic effects when used in addition to ticagrelor, aspirin or their combination, in patients with stable coronary artery disease.
Abstract 139: Contribution Of Platelet Granule Cargo Uptake And Secretion To The Initiation And Progression Of Aortic Aneurysms
Authors: Coenen Danielle M , Whiteheart Sidney W
Published: July 2024

Aortic aneurysms present as aortic widening and often catastrophic rupture. While precipitated by a thrombo-inflammatory environment, underlying pathological mechanisms are unclear and adequate treatment is lacking. Antithrombotics have been implied as therapy, but few studies have defined how platelets contribute to aortic aneurysms. We studied the role of platelet granule cargo uptake and secretion in aortic aneurysm formation. Aneurysms were induced in mice defective in platelet α-granule biogenesis (Nbeal2
-/-
, Serglycin
-/-
), endocytosis (Arf6
-/-
, VAMP2/3
Δ
), and exocytosis (Munc13-4
Jinx
) via:
i.p.
injection of an AAV vector with a mouse PCSK9 gain-of-function mutation; western diet; and continuous
s.c.
infusion of angiotensin II. Thoracic and abdominal aneurysm formation was assessed with ultrasound. At 4 and 12 weeks, or after rupture, aortas were harvested, analyzed, and prepared for histology. During the study, 30% (3/10) of Nbeal2
-/-
and 21.4% (3/18) of Munc13-4
Jinx
mice suffered aortic rupture, compared to 0% (0/26) of wildtype mice. In the survivors, the inner diameter of neither the thoracic nor abdominal aorta of Nbeal2
-/-
mice changed over time.
Ex vivo
, Serglycin
-/-
mice showed a clear, more distributed, widening of the thoracic and descending aorta instead of local aneurysm formation with an increased maximum outer diameter of the ascending region. Munc13-4
Jinx
mice had an all-or-nothing phenotype; either early-stage massive aneurysm formation or rupture, or a more diffused phenotype. Mice with platelet endocytosis defects survived and at early stages, the differential increase in the inner diameter of both the thoracic and abdominal aorta was higher in female Arf6
-/-
mice. Similarly, VAMP2/3
Δ
mice had the largest inner thoracic diameter increase after 28 days. Arf6
-/-
and VAMP2/3
Δ
mice showed a smaller maximum outer diameter of the descending aorta as wildtype. Preliminary histology shows reduced elastin breaks in all genetic strains, especially in Nbeal2
-/-

and VAMP2/3
Δ
mice. Our data show that platelet granule cargo exerts significant but perhaps contrasting effects on aortic aneurysm formation. By comparing phenotypes, more detailed mechanistic insights into the role of platelets in aortic aneurysms are possible.
Abstract 143: Multi-omics Integration From A Large Human Biobank Identifies Key Molecular Mechanisms And Signatures Of Atherosclerotic Plaque Instability
Authors: Das Vivek , Zhang Xiang , Djordjevic Djordje , Bergman Otto , Narayanan Sampath , Shungin Dmitry , Chemaly Melody , Karadimou Glykeria , Vuckovic Sofija , Prasad Inika , Conde Knape Karin , Barascuk Michaelsen1 Natasha , Hedin Ulf , Matic Ljubica
Published: July 2024

Understanding the pathophysiology of unstable atherosclerosis, the underlying cause of myocardial infarction and ischemic stroke, is imperative to catalyze novel translational strategies. Our objective was to identify key mechanisms and signatures of atherosclerotic instability, by leveraging the power of multi-omics integration from a large biobank. Biobank of Karolinska Endarterectomies (BiKE), with biomaterial and data from patients with symptomatic (S) and asymptomatic (AS) carotid atherosclerosis, was the main resource in this study. Matched plaques, PBMCs, plasma sampled locally around the plaque and from the periphery in n>600 individuals, were profiled by orthogonal genomic, transcriptomic, proteomic and metabolomic technologies. Single-omics analyses were performed for differential expression, pathway and network studies in S vs. AS comparisons. Multi-omics integration was performed using a benchmarked method DIABLO, across local and peripheral disease sites and their intersection. Top targets were investigated further using scRNAseq, patient clinical and outcome data, as well as QTL analyses coupling target genetics with transcript, protein, and metabolite levels. Plaque and PBMC transcriptomics showed that inflammatory signalling, hypoxia, angiogenesis, ECM reorganisation, bone remodelling, cholesterol homeostasis, glycolysis were enriched pathways. In proteomics, chemokine signalling upregulation was found in both peripheral and local plasma, while IL17 pathway was enriched in local plasma from S patients. In plasma metabolomics, Bilirubin degradation products were downregulated, while Sphingomyelins were upregulated. Multi-omics integrations revealed coagulation, necroptosis, immune activation and cholesterol metabolism as key pathways determining instability. Extended analyses of the top analytes FABP4, ICOSLG, ANGPTL3, Bilirubin and Sphingomyelins, generated an insight into their association with survival, clinical and genetic risk in patients. Using an innovative framework, this study brings novel information about atherosclerosis vulnerability, opening avenues for drug target and biomarker discoveries, but also improving personalised treatment options and risk stratifications.
Abstract 144: Pyruvate Dehydrogenase Kinase 4 Contributes To Abdominal Aortic Aneurysm Through Accelerating Smooth Muscle Cell Metabolic Reprogramming And Nlrp3 Inflammasome-dependent Pyroptosis
Authors: Peng Qin-Bao , Zhao Li , Lin Tian-Xiao , Huang Fang-Ze , He Xiao-Xia , Huang Wei , Liu De-Shen , Zhao Ling-Na , Lu Jun , Zheng Shao-Yi , Liu Xiu
Published: July 2024

Background:
Abdominal aortic aneurysm (AAA) is a severe aortic disease with a high mortality rate upon rupture. Understanding its molecular mechanisms of progression is crucial for therapeutic strategery. Pyruvate dehydrogenase kinase 4 (PDK4) is an important mitochondrial matrix enzyme in cellular metabolism. This study explored the role of PDK4 in AAA development, focusing on its impact on vascular smooth muscle cells (VSMCs).

Methods:
The expression of PDK4 in human and mice AAA tissues was measured. The role of PDK4 in AAA development was evaluated in VSMC-specific PDK4 knockout mice using a PCSK9/Ang II-induced AAA model. Seahorse analysis was used to assess oxidative phosphorylation and glycolysis levels. RNA-sequencing analysis and mitochondrial functional analysis were used to explore the molecular mechanisms.

Results:
We observed an increased expression of VSMC-PDK4 in human AAA tissues and AAA tissues from a PCSK9/AngII-induced AAA mouse model. PDK4 deficiency in VSMCs significantly inhibited AAA formation, whereas PDK4 overexpression aggravated AAA development. PDK4 promoted the metabolic reprogramming of VSMCs and VSMC-specific deletion of PDK4 ameliorated the contractile-to-synthetic phenotype switch in VSMCs. PDK4 activated NLRP3 inflammasome by impairing mitochondrial function, which contributed to the pyroptosis of VSMCs and instigated an inflammatory response. Mitochondria-targeted ROS scavenging and NLRP3 inhibitor MCC950 effectively attenuated NLRP3 inflammasome activation and pyroptosis mediated by PDK4. Supplementation with MCC950 was beneficial to ameliorating AAA in a VSMC PDK4-dependent manner in mouse models.

Conclusion:
PDK4 promotes smooth muscle cell metabolic reprogramming and NLRP3 inflammasome-dependent pyroptosis in AAA development, suggesting that targeting PDK4 may be a promising therapeutic strategy for the prevention and treatment of AAA.
Abstract 155: Plasma Kallikrein Is A Key Driver Of Basal Activation Of Coagulation Under Conditions Of C1 Inhibitor Deficiency
Authors: Grover Steven P , Horvath Hanga R , Polai Zsofia , Sundler Bjorkman Linda , Bendapudi Pavan K , Mackman Nigel , Farkas Henriette
Published: July 2024

C1 inhibitor (C1INH) functions as a key endogenous inhibitor of contact pathway coagulation factors XII, XI and plasma kallikrein (PKa). Patients with a congenital deficiency in C1INH suffer from a rare swelling disorder called hereditary angioedema (HAE) caused by excess bradykinin generation. Patients with HAE also have elevated circulating levels of markers of activation of coagulation and an increased risk of venous thromboembolism, phenotypes recapitulated in C1INH-deficient mice. We sought to evaluate the molecular mechanism by which C1INH deficiency leads to increased basal activation of coagulation and venous thrombosis.

We hypothesized that PKa serves as a key driver of activation of coagulation in humans and mice with congenital C1INH deficiency.

Patients with C1INH deficiency-associated HAE were treated with the PKa inhibitor lanadelumab with plasma samples collected before and after treatment. Plasma levels of coagulation markers and contact pathway-initiated thrombin generation were determined. C1INH-deficient mice were treated with the PKa inhibitor avoralstat, the FXIIa inhibitor garadacimab, FXII small interfering RNAs, FXI antisense oligonucleotides, the anti-tissue factor antibody 1H1 or appropriate controls and plasma coagulation markers were measured.

Treatment of HAE patients with the PKa inhibitor lanadelumab significantly reduced plasma levels of prothrombin fragment 1.2, thrombin antithrombin (TAT) complexes and D-dimer at 6 months after treatment initiation (P<0.05). Interestingly, landalumab had no significant effect on contact pathway-initiated thrombin generation. In experiments with C1INH deficient mice, TAT complexes were significantly reduced by PKa inhibition (P<0.01) but not by inhibition of FXIIa or FXIa. Interestingly, tissue factor inhibition also significantly reduced plasma TAT complexes (P<0.01). This data suggests that under conditions of C1INH deficiency PKa potentiates activation of coagulation through a non-canonical pathway involving tissue factor.

These findings demonstrate that C1INH is a physiologically relevant anticoagulant with C1INH deficiency resulting in enhanced kallikrein-mediated activation of basal coagulation.
Abstract 156: Pyruvate Kinase M2 Regulates SNAP23-mediated Platelet Granule Exocytosis, Neutrophil Extracellular Traps Formation And Experimental Venous Thrombosis
Authors: Nayak Manasa K , Flora Gagan D , Barbhuyan Tarun , Patel Rakeshkumar B , Ghatge Madankumar , Kumskova Mariia , Jain Aditi , Kumar Jitendra , Kumar Santosh , Chauhan Anil K
Published: July 2024

Introduction:
Several factors released from activated platelets via exocytosis contribute to venous thrombosis (VT) by potentiating neutrophil extracellular traps formation (NETosis) at the sites of inflammation. The metabolic enzyme pyruvate kinase M2 (PKM2) contributes to platelet activation and arterial thrombosis. PKM2 possess protein kinase activity. The regulatory role of PKM2 in platelet granule exocytosis, NETosis, and VT has not been investigated yet.

Aim:
To test the hypothesis that dimeric PKM2 regulates SNAP23-mediated platelet granule exocytosis, and thereby contributes to NETosis, and VT.

Methods:
We utilized platelet-specific PKM2
–/–
(PKM2
fl/fl
PF4Cre
+
), littermate (PKM2
fl/fl
) mice, and wild-type (WT) mice treated with small molecule ML265 (limits PKM2 dimerization) or vehicle to test the susceptibility to VT in inferior vena cava (IVC)-stenosis model.
In vitro
NETs formation was evaluated by co-incubating WT neutrophils with agonist (thrombin or convulxin)-stimulated platelets. The SNAP23 phosphorylation and PF4 release were measured using Western blot.

Results:
PKM2
fl/fl
PF4Cre
+
or WT mice pre-treated with ML265 exhibited reduced susceptibility to VT in the 48 hours IVC-stenosis model as evaluated by less thrombus burden (% incidence, length, and weight, n=11-12/group, P<0.05 vs. controls). Myography revealed that limiting PKM2 dimerization improves the venous wall function post-VT. Activated PKM2
–/–
or ML265 pre-treated WT platelets exhibited decreased SNAP23 phosphorylation and PF4 secretion suggesting a regulatory role for PKM2 in SNAP23 exocytosis and PF4 secretion. Furthermore, neutrophils stimulated with activated platelets from either PKM2
fl/fl
PF4Cre
+
or WT mice pre-treated with ML265 showed reduced NETosis. Finally, we demonstrate that the % of the area covered by the thrombus is profoundly reduced in the ML265-treated human whole blood perfused over a tissue factor-coated surface at the venous shear.

Conclusions:
Dimeric PKM2 is a novel regulator of SNAP23-mediated platelet granule exocytosis. Targeting dimeric PKM2 in platelets inhibits NETosis in neutrophils stimulated with activated platelets and reduces susceptibility to experimental VT.
Abstract 158: The Role Of Ehbp1 In Hypercholesterolemia
Authors: Cai Bishuang
Published: July 2024

Background:
Despite aggressive lowering of cholesterol, conventional lipid-lowering therapies only modestly lower cardiovascular risk in the population. Therefore, identifying novel hypercholesterolemia-risk targets is of critical importance. Human genome-wide association studies (GWAS) have revealed that several single-nucleotide polymorphisms (SNPs) in
EHBP1
, the gene that encodes EH domain binding protein 1 (EHBP1), an endocytic trafficking regulator, are associated with serum cholesterol. However, the mechanisms underlying this association and the role of EHBP1 in cholesterol metabolism are unknown. We
hypothesize
that EHBP1 is a novel hypercholesterolemia risk factor. The goal of this study is to explore the role of EHBP1 in hypercholesterolemia and its underlying mechanisms.

Methods:
Hepatocyte EHBP1 was silenced by intravenous injection (IV) of AAV8-H1-shEhbp1 in wild-type (WT),
Apoe-/-
and
Ldlr-/-
mice, followed by Western Diet feeding to induce hypercholesterolemia and atherosclerosis. Primary murine and human hepatocytes were isolated for
in vitro
mechanistic studies. We also leveraged publicly available human genetic studies and unbiased scRNA datasets to support our study.

Results:
Individuals with the EHBP1 rs10496099 T>C variant have decreased serum cholesterol and reduced liver EHBP1 expression. Consistent with the human data, our
in vivo
study with hypercholesterolemic mice shows that EHBP1 silencing in hepatocytes decreases plasma total and LDL/VLDL cholesterol. Our
in vitro
study with primary hepatocytes reveals that EHBP1 deficiency eliminates sortilin (SORT1)-mediated PCSK9 secretion and induces LDLR-mediated LDL uptake. Mechanistically, we found that EHBP1 silencing abolishes the homeostasis of the retromer complex, a protein complex that helps retrograde and stabilize sortilin.

Conclusion:
Hepatocyte EHBP1 promotes sortilin-mediated PCSK9 secretion by maintaining retromer homeostasis, leading to LDLR degradation and hypercholesterolemia. Our study elucidates a novel link between EHBP1 and other cholesterol-associated loci (e.g.,
SORT1
,
PCSK9
, and
LDLR)
and provides new mechanistic insight into the role of EHBP1 in hepatic cholesterol metabolism and hypercholesterolemia.
Abstract 161: Myocardial Infarction Accelerates Melanoma Tumor Progression
Authors: Von Itter Richard , Newman Alexandra , Pilla Gabrielle , Bozal Fazli , Wang Kristin M , Gourvest Morgane , Zhang Tracy , Lim Brian , Moore Kathryn J
Published: July 2024

Adult survivors of cancer are at increased risk of cardiovascular disease (CVD). Indeed, while CVD emerges as the second leading cause of death in recovering cancer patients, cross-disease communication remains poorly understood. We previously demonstrated that myocardial infarction (MI) accelerates breast cancer tumor growth and cancer-specific mortality in mice and humans. However, whether MI alters tumor growth of other cancer types and impacts metastasis is not known. To assess these gaps, we examined the effect of acute MI, induced by ligation of the left anterior descending (LAD) coronary artery, in syngeneic C57BL6 mouse models of primary and metastatic melanoma. Compared to sham surgery, LAD ligation resulted in a two-fold increase in tumor growth in both the B16 and YUMM melanoma models. Notably, tumor burden significantly correlated with the size of the infarct, suggesting a direct relationship between injury signal activation and disease progression. Analysis of the intratumoral immune cell landscape by flow cytometry revealed an enrichment of immunosuppressive CD4
+
FoxP3
+
T regulatory cells after MI, and systemic depletion of FoxP3
+
cells attenuated MI-induced tumor growth. Finally, histological analysis and magnetic resonance imaging revealed an increase in the number and size of metastatic foci in the liver after MI compared to sham. Our data suggest that MI dampens antitumor immunity and reorganizes the tumor microenvironment to exacerbate tumor progression via infiltration of immunosuppressive FoxP3
+
T cells. Collectively, these findings of deleterious cross-disease communication underscore the importance of implementing strategies that mitigate CVD risk in cancer patients.
Abstract 2005: Interrogating The Cross-talk: Endothelial-Macrophage Interactions In Diabetic Vascular Dysfunction
Authors: Malhi Naseeb K , Luo Yingjun , tang xiaofang , Tapia Alonso , Liu Xuejing , Yuan Dongqiang , Chen Zhen
Published: July 2024

Cardiovascular disease (CVD) occurs 15 years earlier in diabetics compared to those without, but the underlying mechanisms driving diabetes-accelerated CVDs (DACVDs) pathogenesis remains incompletely understood. Endothelial cells (ECs) and macrophages (MΦ) are key players in vascular wall and their crosstalk is crucial in DACVD. In diabetes, ECs activation enables monocytes recruitment, which transmigrate across the intima and differentiate into macrophages (MΦ). Beyond this established diapedesis model, EC-MΦ interplay is highly intricate and heterogenous. Here, we leveraged human mesenteric arteries of varied diabetic state and used single cell (sc)- and spatial transcriptome mapping to interrogate the functional crosstalk between MΦ subtypes and ECs in diabetes. We identified diabetes-induced EC-MΦ interactions and explored their consequences
in vitro
and
in vivo,
in peripheral artery disease (PAD), a prominent DACVD. Integrative scRNA-seq and spatial transcriptome (Visium) profiling identified a remarkable increase of MΦ subtype expressing triggering receptor expressed on myeloid cells (TREM2) with concomitant increased EC-derived TREM2 ligands in human diabetic vs non-diabetic vessels. Such induction of TREM2 MΦ was consistently observed in arteries from type I and II diabetes mouse models. Compared to the condition medium from MΦ cultured under normal glucose, that from MΦ cultured under high glucose conditions reduced EC migration and increased inflammatory markers, which were reversed by the medium from MΦ with TREM2-knockdown. We further evaluated the functional importance of EC-TREM2 interaction
in vivo
in a mouse hindlimb ischemia (HLI) model. In diabetic mice under HLI, a TREM2 neutralizing antibody promoted tissue recovery and perfusion. Finally, we showed that TREM2 was increased, especially in proximity to ECs, in ischemic sites, as compared to non-ischemic regions in limb muscles from human PAD patients.Taken together, our study presents a diabetic artery atlas and identifies EC-TREM2 interaction as a crucial event in DACVDs, such as PAD. Our findings provide novel insights into the EC-MΦ interplay in diabetes and vascular inflammation and how these cellular interactions may be targeted to ameliorate DACVD.
Abstract 2007: Ischemic And Non-ischemic Cardiovascular Events Differentially Regulate Cross Disease Communication With Cancer
Authors: Newman Alexandra A , Von Itter Richard , Lim Brian , Moore Kathryn J
Published: July 2024

Cancer and cardiovascular disease (CVD) are the top two leading causes of death worldwide, exhibiting cross-disease communication that increases morbidity. Cancer patients are at significantly increased risk for CVD and recent studies by our lab and others indicate that CVDs like myocardial infarction (MI) or heart failure can in turn accelerate tumor growth. We previously showed this occurs through changes in the innate immune system or via tumor-promoting factors secreted from the damaged myocardium, which together have established the nascent field of Reverse CardioOncology. We hypothesized that ischemic and non-ischemic CVD injuries have distinctly different mechanisms underlying mammary tumor growth. To mimic these events, we modeled non-cardiac hind limb ischemic injury by femoral artery ligation (FAL) and non-ischemic cardiac injury by cardiac cryoinjury (CCI). To test our hypothesis, we introduced primary E0771 mouse mammary cancer cells into the mammary fat pad three days prior to FAL or CCI. Measurements of tumor over time showed significant increases in tumor growth after both FAL and CCI when compared to sham surgeries. However, only the ischemic injury, FAL, induced similar changes in immune cells to what we observed after MI (induced by left anterior descending coronary artery [LAD] ligation). This included significant increases in myeloid populations and decreased lymphocytes within the circulation. Additionally, flow cytometry of immune cells within tumors of mice exposed to FAL showed significantly increased myeloid cells and macrophages, as well as increased immunosuppressive T-regulatory cells. In contrast, while CCI showed significantly increased tumor burden and circulating leukocytes, there were no differences in the above-mentioned subsets of immune cells within the circulation or within the tumor. These data suggest that ischemic injury, via FAL or LAD, induces an immunosuppressive environment within the tumor that accelerates cancer growth that is distinct from the non-ischemic cardiac injury induced by CCI. Taken together, our data suggest that the immune reprogramming after ischemic CVDs is a risk factor for tumor growth and these data help to elucidate the mechanisms that underlie cancer risk in CVD patients.