bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 252: Is Zbp1 An Atherosclerosis Modifier Gene?
Authors: Han Juying , Opoku Emmanuel , Smith Jonathan D
Published: October 2023

We have previously mapped and fine mapped a mouse atherosclerosis susceptibility locus on chromosome 2 using an intercross between AKR/J and DBA/2J strains on the apoE-deficient background. The fine mapped locus includes only 3 protein coding genes, and the
Zbp1
gene, encoding the Z-DNA binding protein 1, is our top candidate. Prior single cell RNAseq from the mouse aorta shows
Zbp1
expression in monocytes and macrophages > endothelial cells and fibroblasts. The ZBP1 protein plays a role in non-canonical necroptosis and inflammatory signaling. There are 2 mouse
Zbp1
mRNA splice isoforms, long and short, which respectively generate a full length protein and a C-terminal truncated isoform that is missing the RHIM domains required for necroptosis activity. RNAseq from bone marrow derived macrophages reveals ~2-fold higher
Zbp1
expression in DBA/2 vs. AKR macrophages (p=0.002), with a short to long isoform ratio of about 2.5. After treatment with LPS,
Zbp1
expression is induced ~30-fold (p<0.0001), with a short to long isoform ratio of about 0.9. These results were confirmed by qPCR. To determine if
Zbp1
is an atherosclerosis modifier gene, we treated wildtype (WT) and
Zbp1
knockout (KO) female mice, both on the C57BL/6J background, with LDLr antisense oligos (ASO) for 8 weeks while feeding a western-type diet. Upon sacrifice at 16 weeks of age, body weights and total cholesterol levels (~800 mg/dl) were similar in the WT and KO mice. Median aortic root lesion area was decreased by 30% in the KO vs. WT mice (Figure). The median necrotic lesion area and % necrotic lesion area were decreased by 44% and 33%, respectively in the KO vs. WT mice (Figure). Although these results were not statistically significant, these trends encouraged us to look at more advanced lesions and follow up study is underway with a 16-week exposure to LDLr ASO and western-type diet. Thus,
Zpb1
appears to be an atherosclerosis modifier gene that may function by increasing lesion necrosis.
Abstract 253: Investigation Of Acute Distension Injury In Vein Graft Implantation Through Integrated Single-nuclei And Spatial Transcriptomic Analysis
Authors: Michaud Marina , Mota Lucas , Pilcher William , Tomeo John , Aridi Tarek , Thomas Beena , Bakhtiari Mojtaba , Contreras Mauricio , Pradhan-nabzdyk Leena , LoGerfo Frank , Liang Patric , Bhasin Manoj
Published: October 2023

Arterial bypass surgery using autogenous vein remains a forefront treatment for coronary and peripheral artery occlusive diseases. However, vein graft failure (VGF) ultimately occurs in approximately 30% of cases, primarily due to intimal hyperplasia (IH) arising from impaired endothelial function and SMC dysregulation following injury at the time of implantation. Although surgical handling of the vein conduit (e.g., harvesting, distension, anastomosis) has been posited as a significant factor contributing to the development of IH, this direct correlation remains largely unexplored. To address this gap, we leveraged single-cell technologies (integrated single-nuclei and spatial transcriptomics) to investigate the mechanisms of vein graft injury that occur during surgical vein harvest. Overall, our transcriptomic analyses of canine cephalic vein conduits following distension with saline support that distension alone results in acute injury and promotes the expression of genes associated with extracellular matrix remodeling (e.g.,
LAMA2, MMRN1
), cell proliferation and migration (e.g.,
JUN, FBN1
), and mesenchymal transition (e.g.,
KLF4, CDH13
) (Figure 1A). Moreover, single-nuclei analysis enabled the identification of distinct subpopulations of endothelial cells and fibroblasts with disparate transcriptomic programs related to healing, cell migration, and EMT, while spatial transcriptomics illuminated the histological context of specific cell types, subpopulations, and associated genetic features (Figure 1B-C). Together, these findings demonstrate a prompt genomic response to distension amongst specific cellular populations localized to distinct anatomical regions, thus providing new insights critical for guiding strategies to limit implantation injury and its role in IH and VGF. Notably, these results also present the first single-nuclei and spatial investigations of veins, providing a framework for future studies.
Abstract 262: A Novel Humanized ACE2 Mouse Model For Evaluating COVID-19 Infection In Cardiovascular Disease
Authors: Lee Cadence , Mantsounga Chris S , Sharma Sheila , Carley Rachel T , Amelotte Elizabeth , Pierce Julia , Butler Celia , Choudhary Gaurav , Olson Rachel , Morrison Alan R
Published: October 2023

The COVID-19 pandemic has resulted in over 6.7 million deaths worldwide. Patients with cardiovascular disease are at increased risk for severe illness and mortality with COVID-19. Increased systemic inflammation driven by macrophages, a key cellular source of inflammatory cytokine IL-1β, may play a role in the mechanism of SARS-CoV-2 pathogenesis. We seek to understand the mechanisms whereby inflammation associated with vascular disease may drive severity of COVID-19. Under culture conditions that mimic atherosclerotic plaque cholesterol exposure, we found that macrophage IL-1β signaling may promote expression of ACE2, the established receptor for SARS-CoV-2, through NF-κB-mediated transcription. We generated a novel, “humanized” ACE2 mouse strain (
hACE2
) by using CRISPR-Cas9 technology to insert the human ACE2 cDNA sequence into the native mouse
ACE2
locus under regulation of the native promoter. Using lung tissue, presence of hACE2 mRNA was verified by RT-qPCR and protein expression was verified by immunofluorescence staining. Overall, SARS-CoV-2 intranasal inoculation of
hACE2
mice revealed milder infection when compared to the K18 promoter driven hACE2 (
K18-hACE2
) overexpression strain, which exhibits severe infection with high morality.
hACE2
mice had 100% survival after infection with stable body weight despite detectable viral burden in the lung fields. Histological analysis of the lungs revealed inflammatory infiltrate and pulmonary consolidation with alveolar hemorrhage that were comparable to the
K18-hACE2
mice, and immunohistochemistry confirmed detection of viral nucleocapsid in the lung consolidations. Infected
hACE2
mice also demonstrated a unique plasma cytokine signature relative to
K18-hACE2
mice, including elevated levels of IL-1β, IL-1α, and VEGF-A, but decreased IL-6 and TNF-α. In summary, we have established a novel model of COVID-19 that survives infection and may facilitate comparative studies involving experimental vascular disease. The ability to model COVID-19 in the context of experimental atherosclerosis may help to define new mechanisms driving severity of SARS-CoV-2 infection.
Abstract 267: Vascular Smooth Muscle Cell-specific Deletion Of Protein Kinase R-like Endoplasmic Reticulum Kinase Attenuates Microbiome-enhanced Abdominal Aortic Aneurysm
Authors: Benson Tyler W , Wadding-Lee Caris , Spuzzillo Anthony , Merland Jess , Brunner Seth , Owens Phillip P
Published: October 2023

Background:
We have previously linked the microbiome-derived metabolite trimethylamine N-oxide (TMAO) to abdominal aortic aneurysm (AAA) in both associative human and mechanistic mouse studies. TMAO is reported to bind protein kinase R-like endoplasmic reticulum kinase (PERK), resulting in selective activation of the unfolded protein response (UPR). Concordantly, our previous RNA sequencing results indicate that TMAO augments PERK-mediated UPR pathways in the aneurysm wall. Thus, the current study will determine the functional role of TMAO-mediated PERK activation in aortic VSMCs to AAA pathogenesis.

Methods & Results:

Ldlr
-/-

mice with VSMC-specific deletion of
Perk
were produced by breeding hemizygous male mice expressing Cre under the control of the Transgelin promoter (
B6.Cg-TG(Tagln-cre)1Her/J
, Jackson Labs) to female
Perk
floxed mice (

Eif2ak3
tm1.2Drc
/J

, Jackson Labs). Specific deletion of PERK in VSMCs was validated by western blot. Male mice were subjected to angiotensin II (AngII) infusion via osmotic mini pump (1,000 ng/kg/min; 28 days), while female mice underwent laparotomy and application of topical elastase (10 mg/mL porcine pancreatic elastase 5 minutes) to induce AAA. To augment circulating TMAO levels, mice were fed a high choline diet (0.2% total cholesterol + 1.2% choline supplementation) for one week prior to and throughout the study. Male mice with VSMC specific PERK knockout demonstrated significantly reduced AngII-induced aortic dilation (Cre+: 0.90 ± 0.048 mm; Cre-: 1.57 ± 0.156 mm; P = 0.001) and rupture induced death relative to control. Similarly, female mice with VSMC specific PERK knockout displayed significantly reduced aortic diameter (Cre+: 1.17 ± 0.069 mm; Cre-: 2.65 ± 0.432 mm; P = 0.002) and rupture induced mortality as compared to control. Additionally, male and female mice with VSMC specific PERK knockout had significantly greater Type I collagen and reduced macrophage accumulation compared to control.

Conclusions:
These results indicate that VSMC specific knockout of PERK blunts AAA formation in two independent murine models. Further studies are needed to elucidate the mechanism of PERK mediated ER stress in AAA, which may reveal novel therapeutic targets for AAA treatment.
Abstract 269: Role Of Sympathetic Nervous System In Abdominal Aortic Aneurysm Pathogenesis
Authors: Chao Calvin L , Dang Caitlyn , Brown Taylor K , Jiang Bin
Published: October 2023

Objective:
Abdominal aortic aneurysm (AAA) remains a prevalent and life-threatening disease with no current pharmacologic therapies. This study aims to evaluate the role of the perivascular sympathetic nervous system (SNS) during AAA formation. We hypothesize that dysfunction of sympathetic innervation to the abdominal aortic smooth muscle is crucial to the onset of aortic dilatation and progression of aneurysmal disease.

Methods:
To generate experimental AAA, ApoE
-/-
mice (n=5) underwent subcutaneous osmotic pump implantation for continuous Angiotensin II (AngII) infusion. For systemic denervation, ApoE
-/-
mice underwent serial intraperitoneal injection of 6-hydroxydopamine (6-OHDA) (n=7) or saline control (n=3). Animals were sacrificed at model-specific time points for morphologic, histologic, and immunohistochemical analysis. Combining AngII infusion with 6-OHDA administration is currently underway and anticipated to conclude prior to this meeting.

Results:
ApoE
-/-
mouse aortas were harvested after 4 weeks of AngII infusion. Tyrosine hydroxylase (TH), an SNS-specific marker, was utilized to visualize nerve fibers (Figure). Experimental aortic walls demonstrated significantly increased nerve fibers (n=32.3 ± 7.1) and density (154.4 ± 47.2 fibers/mm
2
) as compared to healthy controls (n=3.7 ± 1.7, 53.5 ± 19.2 fibers/mm
2
). 6-OHDA administration produced sustained denervation of mesenteric branch arteries through two weeks with evidence of some neural regeneration at 4 weeks. No mortality or weight loss was observed after 6-OHDA administration.

Conclusion:
Generation of experimental AAA is associated with upregulation of SNS fiber density. 6-OHDA administration confers systemic arterial denervation, though further optimization is warranted. Together, these findings suggest an evolving role of SNS dysregulation in AAA pathogenesis and highlight the need for further investigation as a therapeutic target.
Abstract 271: Interrogation Of Endothelial Permeability In Aortic Aneurysm And Dissection Using Nanoparticle Contrast-enhanced Computed Tomography
Authors: Devkota Laxman , Zhang Chen , Bhandari Prajwal , Vela Deborah , Starosolski Zbigniew , Shen Ying H , Lemaire Scott A , Ghaghada Ketan
Published: October 2023

Background:
Aortic pathologies, such as aortic aneurysm and dissection (AAD), are characterized by enhanced endothelial permeability and dysfunction. Non-invasive monitoring of changes in endothelial permeability could enable early detection and prediction of AAD progression. Herein, we investigated nanoparticle contrast-enhanced computed tomography (nCECT) to probe endothelial permeability changes in a mouse model of sporadic AAD.

Methods:
In vivo studies were performed in a mouse model of AAD challenged with high fat diet (5 weeks) and angiotensin II infusion (last one week). The effects of nanoparticle contrast agent (NPCA) size (80, 150, and 250 nm) and dose (300, 600, and 1200 mg I/kg) on detecting endothelial permeability changes were studied. In vivo and ex vivo nCECT imaging were performed 4 days post-contrast and findings were compared with gross and histologic examination. A subset of mice was followed-up with longitudinal nCECT up to 6 months.

Results:
nCECT showed a higher incidence of aortic injury associated with enhanced endothelial permeability compared to gross examination findings. The absolute improvements in detection rates were 21%, 17% and 16% for 80 nm, 150 nm, 250 nm NPCA respectively (Figure 1B). Sensitivity of nCECT for detection of aortic injury improved with increasing NPCA dose compared to gross exam (-17% at 300 mg I/kg to 14% at 1200 mg I/kg, Figure 1C). Follow-up nCECT at 6 months did not show significant increase in wall signal enhancement compared to baseline (Figure 2), suggesting resolution of inflammation and endothelial dysfunction, and progressive repair and remodeling of aortic wall which were confirmed by the microscopic analysis.

Conclusion:
nCECT detected enhanced endothelial permeability before pathological changes were evident on gross examination in a mouse model of sporadic AAD. Furthermore, this approach enabled non-invasive monitoring of changes in endothelial permeability at sites of aortic degeneration.
Abstract 282: Upregulation Of Receptor Interacting Protein Kinase-3 Augments Abdominal Aortic Aneurysms
Authors: Bontekoe Jack , Zhou Ting , Gupta Kartik , Yang Huan , Stranz Amelia , Khoury Mitri K , Silva-Pedraza Zulmari , Wang Qiwei , Liu Bo
Published: October 2023

Objective:
Aortic smooth muscle cell (SMC) depletion is a major pathological feature of abdominal aortic aneurysm (AAA). Our lab has previously reported elevated levels of Receptor Interacting Protein Kinase-3 (RIPK3), a major mediator of necroptosis, in human AAA tissues. Pharmacologic inhibition of RIPK3 or deletion of the
Ripk3
gene prevents aortic SMC death and attenuates aneurysm growth in murine models. In the current study, we tested the hypothesis that manipulation of
Ripk3
gene expression would alter AAA development.

Methods:
We manipulated
Ripk3
gene expression by mutating a conserved putative transcription enhancer site using CRISPR-Cas9-mediated gene editing. AAA was induced in 8-week old male CRISPR mutated (CM) and wild-type (WT) mice by perivascular application of CaCl
2
or NaCl (sham). Aortic tissues were harvested on days 4 or 28 post-surgery and analyzed by immunohistochemistry (IHC). Primary aortic SMCs isolated from both groups were stimulated with inflammation-related cytokines and
Ripk3
mRNA was examined by RT-PCR.

Results:
Both CM and WT mice developed AAA in response to CaCl
2
. Change in aortic diameter was greater in CM mice than WT at 28 days (66.81±4.512 vs 44.87±6.685%,
P
=0.0108). IHC showed CM aneurysm tissue to exhibit fewer SMCs and a greater number of non-SMC cells in the tunica media, presumably infiltrated immune cells. Furthermore, mutant tissues contained higher levels of RIPK3 positivity than WT aortas. Consistently, CM aortic SMCs expressed more
Ripk3
mRNA than WT SMCs, both with vehicle and following stimulation with IL-1β, IL-25, and IL-33.

Conclusions:
Our data show a positive correlation between
Ripk3
expression and aneurysm size. We are currently analyzing data from experiments aimed to prove the causal relationship between
Ripk3
gene editing, SMC necroptosis, inflammation, and aneurysm growth. This study further illustrates the potential of RIPK3 as a therapeutic target of AAA.
Abstract 284: Pro-Inflammatory Adventitial Fibroblast Contribute To Aortic Aneurysms In Loeys Dietz Syndrome
Authors: Dalal Alex R , Pedroza Albert J , Gu Wenduo , Kim Jennifer L , Mitchel Olivia , Gilles Casey , Quertermous Thomas , Cheng Paul , Fischbein Michael P
Published: October 2023

Introduction:
Vascular smooth muscle cell (SMC) phenotype modulation is implicated in thoracic aneurysm formation, however, the role of adventitial fibroblasts (AdvFib) in aneurysm formation remains poorly characterized. Loeys-Dietz Syndrome (LDS), caused by a spectrum of mutations in the TGF-β signaling cascade, leads to aggressive thoracic aneurysms. Given the ubiquitous nature of TGF-β signaling machinery, the exact cell type(s) responsible for initiation of LDS pathogenesis is poorly understood.

Methods:
We performed single cell transcriptomic characterization of aortic root and ascending aortic samples from LDS patients ((n=6 LDS (harboring
TGFB2
,
TGFBR1
,
TGFBR2
, or
SMAD3
mutation); n=2 Donor control)) as well as root/ascending aorta from a murine model of LDS (

Tgfbr2
G357W/+

) to understand cell-state transitions and transcriptomic alterations in LDS.

Results:
Single cell RNA sequencing (scRNAseq) of human and mice LDS samples (in total >40,000 cells) revealed a secondary pattern of cellular activation in LDS as compared to Marfan Syndrome. Instead of SMC phenotypic modulation as seen in MFS, LDS appears to preferentially modulate adventitial fibroblasts (AdvFib) phenotype in the

Tgfbr2
G357W/+

mouse model. Human LDS patient’s AdvFib turn on high levels of macrophage-recruiting pro-inflammatory cytokines
CCL2
and
HLA-B,
with a corresponding 1.8-fold increase in LDS aortic wall macrophages compared to Donor. A more extreme pattern was observed in the

Tgfbr2
G357W/+

LDS mouse model. While

Tgfbr2
G357W/+

SMC are transcriptomically similar to wild type counterparts, AdvFib were activated into a pro-inflammatory state associated with increased aortic macrophage recruitment (
Ccl2
,
Il6
,
Vcam1,
and
Cxcl2
) and fibrotic response (
Col1a1
,
Col1a2
,
Col3a1,
and
Fn1
). A 6-fold increase in the number of aortic wall macrophages was identified in

Tgfbr2
G357W/+

compared to WT.

Conclusions:
LDS mouse and human AdvFib undergo phenotypic alterations into a pro-inflammatory state evidenced by increased expression of numerous cytokines, macrophage recruitment, and ECM deposition. AdvFib, in addition to SMCs, are potentially another important pathological cell population in LDS aneurysm formation.
Abstract 285: Vascular Smooth Muscle Cell Endoplasmic Reticulum Stress In Abdominal Aortic Aneurysm Development
Authors: Davis Frank , Mangum Kevin , Joshi Amrita , Wasikowski Rachael , Xing Xianying , Tsoi Liam , Gudjonsson Johann , Gallagher Katherine A
Published: October 2023

Introduction:
Abdominal aortic aneurysms (AAA) are characterized by vascular smooth muscle cell (VSMC) apoptosis leading to pathological aortic remodeling. Activation of the endoplasmic reticulum (ER) stress response has been linked to VSMC dysfunction but the driving factors behind ER stress activation and its role on AAA development remains poorly defined. The epigenetic enzyme, MLL1, plays a critical role in establishing cellular phenotype and regulating gene expression. We examine the MLL-mediated epigenetic regulation of the ER stress response in VSMCs during AAA development and establish the ER stress pathways as a novel target in the treatment AAAs.

Methods:
Single-cell sequencing was conducted on human AAA and control tissue samples. For our murine model, C57BL/6 mice were injected with an AAV encoding a PCSK9 gain-of-function mutation and then fed a saturated fat-enriched diet and infused with AngII (1 μg/kg/min) with or without 4-PBA (20mg/kg/day). The elastase induced AAA model was used as a secondary murine model. AAA maximum diameters were quantified and VSCMs were isolated.
Eif2a, Atf4
, and Chop
gene expression was examined. Apoptosis was quantified by Annexin V staining.

Results:
Using single-cell RNA sequencing of human aortic tissue, we identified
EIF2A, ATF4
, and
CHOP
were profoundly upregulated in aneurysmal VSMCs in comparison to nonaneurysmal control VSMCs. Further,
EIF2A, ATF4
, and
CHOP
were significantly unregulated in two murine AAA models. Mechanistically, TNFα signaling in VSMCs leads to activation of MLL1 which trimethylates histone 3 lysine 4 (H3K4me3) at NF-κB binding sites on the
EIF2A
and
ATF4
gene promoters thereby upregulating the ER stress response and driving VSMC apoptosis.
In vivo
pharmacological inhibition of the eIF2A/ATF4 ER stress response via 4-PBA injection inhibited AAA formation resulting in significant reduction in VSMC apoptosis, elastin fragmentation, and inflammatory cytokine infiltration.

Conclusions:
Taken together, our results identify MLL1-mediated activation of the eIF2A/ATF4 ER stress response as a critical regulator of VSMC apoptosis in AAA formation and suggest inhibition of ER stress response as a mechanistic target for the management of AAAs.
Abstract 287: A Retrievable Aortic Drug Delivery Stentgraft To Create A Porcine Model Of Thoracic Aneurysms
Authors: Kenawy Dahlia M , Stafford Jordan F , Amari Foued , Chun Youngjae , Tillman Bryan W
Published: October 2023

Objectives:
Murine models present limitations for the study of aortic aneurysm due to disparate hemodynamics from humans, small size and the necessity for open surgery and aortic clamping to apply otherwise toxic aneurysmogenic enzymes. We have developed a retrievable chambered stentgraft that can isolate aortic wall and more importantly restrict enzyme to only the anterior aorta. Figure 1 shows a posterior central lumen (CL) that preserves distal perfusion while excluding posterior spinal cord branches, and an outer chamber (OC) delivers drug to anterior aortic wall. We hypothesized that a Retrievable Aortic Drug Delivery Stentgraft (RADDS) could deliver enzymes directly to the aortic wall to create a porcine model of thoracic aortic aneurysms (TAA).

Methods:
The RADDS stentgraft scaffold was laser cut from nitinol, shapeset, and covered in polymer. A permanent delivery wire was affixed distally, and a perfusion cannula to the OC. The stent was delivered from percutaneous femoral access to the thoracic aorta in a porcine model (n=6). Following angiography, an enzyme cocktail (elastase/collagenase) was infused to the OC then aspirated and the stent retrieved via sheath advancement.

Results:
The RADDS achieved angiographic isolation of the anterior aortic wall from both the circulation and spinal cord. Distal perfusion was preserved through the CL and femoral arterial pressures were unchanged after stent placement (p = 0.88). Spinal paresis was not observed. All animals developed acute aortic degeneration ranging from 1 hour to 6 weeks post-treatment with H&E histology reflecting deteriorated aortic media with specific disruption of elastic lamina.

Conclusions:
The RADDS allowed endovascular delivery of systemically toxic agents directly to the aortic wall for creation of TAA. These findings herald a novel approach to isolate potentially toxic therapies to the aortic wall and may offer future opportunities to instead deliver aneurysm treating therapies.
Abstract 293:
In Vitro
And
In Vivo
Assessment Of Freeze-Dried Allograft Pericardium And Decellularized Allograft Pericardium For Vascular Applications
Authors: Burnette Amanda K , Pomposini Amanda , Morse Jennifer , Arpag Sezgi , Baer Emily , Softic Davorka , Qin Xiaofei
Published: October 2023

The purpose of this study was to assess the biochemical content, biomechanical strength, and
in vivo
biocompatibility of freeze-dried human pericardium and decellularized human pericardium. Donated human pericardia were processed using one of two methods: disinfected and freeze-dried (freeze-dried) or disinfected, decellularized, and terminally-sterilized (decellularized) prior to assessments. Four pericardia were allocated per treatment method. Proteins were extracted from nonimplanted biopsy punches and quantified for the pro-angiogenic biochemical factors (FGFa, FGFb, HGF, PDGF-BB, and VEGF) using a Luminex multiplex kit. Biomechanical strength was assessed by probe burst strength, suture retention strength, and longitudinal tensile strength according to methods described in ISO 7198. Biopsy punches from each treatment group were implanted subcutaneously in athymic mice for 2-12 weeks and assessed for cell infiltration, angiogenesis, adipogenesis, and inflammation histologically per ISO 10993-6. Biochemical quantification demonstrated significantly higher levels of PDGF-BB, FGFb, HGF, and VEGF in the freeze-dried pericardium than the decellularized pericardium. FGFa was not detectable in most samples for both groups. Decellularized pericardium retained biomechanical strengths like that of unprocessed pericardium.
In vivo
responses at 2 weeks differed between the two groups, where the decellularized pericardium contained more infiltrating cells consisting of macrophages and fibroblasts while the freeze-dried pericardium was infiltrated predominantly by macrophages and polymorphonuclear cells. By 12 weeks, both treatment groups exhibited similar levels of cellular infiltration and matrix remodeling. The increased concentration of pro-angiogenic factors in the freeze-dried pericardium did not influence the
in vivo
remodeling response compared to the decellularized human pericardium, as both treatment groups recellularized similarly by 12 weeks. In conclusion, allograft pericardium can be processed to retain pro-angiogenic biochemical factors and biomechanical strength, while promoting cell infiltration and remodeling as required for vascular grafts.
Abstract 306: Paired Immunoglobulin-like Receptor B In Macrophages Regulates The Pro-resolving Response To Efferocytosis
Authors: Dungan Matthew M , Gonzalez Azuah L , Su Karry , Fuller Kristin , Rahman Jamshed , Doran Amanda C
Published: October 2023

Maintenance of tissue homeostasis in the human body requires the efficient removal of millions of dead cells each day. Efferocytosis, the macrophage phagocytic process of apoptotic cell (AC) clearance, enhances inflammation resolution by promoting anti-inflammatory macrophage metabolism and cytokine production. Failures of efferocytosis promote the development of chronic inflammatory diseases, including atherosclerosis, cancer, and autoimmune disease. Recently, immunomodulatory receptors, a class of receptors known to broadly regulate macrophage inflammatory responses, have been implicated in efferocytosis. Paired immunoglobulin-like receptor B (PirB) is an understudied immunomodulatory receptor previously shown to inhibit inflammatory cytokine production in macrophages. We hypothesized that PirB is a novel promoter of efferocytosis-mediated inflammation resolution.

We examined atherosclerotic lesions from the aortic root of

Ldlr
-/-

mice after 8 or 16 weeks of Western diet feeding and observed decreased levels of PirB in lesional macrophages in mice with larger necrotic cores, dysfunctional efferocytosis, and more advanced atherosclerosis. To test if PirB regulates efferocytosis, we generated bone marrow-derived macrophages (BMDMs) from Control and

Pirb
-/-

mice, co-cultured them with labeled ACs, and then assessed efferocytosis using flow cytometry. While

Pirb
-/-

BMDMs bound and internalized ACs, they failed to undergo lysosomal acidification, suggesting ACs are not being digested. Additionally, an unbiased query of validated protein:protein interactions and co-immunoprecipitation assays identified lysosomal maturation machinery as binding partners of PirB, suggesting loss of PirB impedes AC digestion by dysregulation of lysosomal maturation. As expected, control BMDMs fed ACs upregulated the pro-resolving cytokine IL-10 and the anti-inflammatory metabolic pathway, oxidative phosphorylation. Conversely,

Pirb
-/-

BMDMs upregulated pro-inflammatory cytokines and maintained elevated pro-inflammatory glycolysis upon AC feeding. Our results suggest that PirB is a unique regulator of macrophage pro-resolving phenotype by regulating AC digestion and metabolic rewiring post-efferocytosis.
Abstract 308: Obesity Dysregulates Chromatin Response To Hypoxia That Governs Metabolic Programming And Phagocytosis In Macrophages
Authors: Urao Norifumi , Takahashi Kentaro , Jackson Jasmine R
Published: October 2023

Macrophages are plastic cell population that normally adapt to their environment. Cellular adaptation to hypoxia occurs through transcription factors including hypoxia-inducible factors, and hypoxia-inducible transcriptions are further regulated by chromatin response through histone modification including histone methylation. However, the role of histone methylation in hypoxia response of macrophages is well understood. While obesity is associated with dysregulated macrophages, whether hypoxia response is cell intrinsically dysregulated in macrophages in obesity. In mouse bone marrow-derived macrophages (BMDMs), immunoblotting and high-content imaging revealed that 1% hypoxia rapidly (in 1h) increases the global levels of histone 3 methylations. We found that hypoxia-induction of histone 3-lysine 4 tri-methylation (H3K4me3) is specifically inhibited in BMDMs from mice fed high-fat diet (HFD-BMDMs) compared to BMDMs from mice fed normal diet (ND-BMDMs), while the induction of hypoxia-inducible factor-1α remained intact. ChIP-seq identified that hypoxia-upregulated H3K4me3 peaks marked the promoter regions of the genes associated with multiple metabolism-related pathways including glycolysis in ND-BMDMs. In contrast, no pathway was associated with hypoxia-upregulated H3K4me3 peaks in HFD-BMDMs, suggesting both the extent and the genome location of H3K4me3 response to hypoxia is dysregualted in obesity. RNA-seq further demonstrated glycolysis-related genes, Aldoa and Pdk3 had dysregulated response to hypoxia in HFD-BMDMs, which is overlapped with dysregulated H3K4me3 peaks. Consistently, lactate accumulation and induction of histone lactylation under hypoxia were reduced in HFD-BMDMs. Furthermore, HFD-BMDMs exhibited decreased phagocytosis of necrotic cells under hypoxia due to the reduced capacity of anaerobic glycolysis.In summary, chromatin response to hypoxia associated with H3K4me3 enrichment governs transcriptions for anaerobic glycolysis and phagocytosis under hypoxia. Obesity dysregulates the extent and the genome location of H3K4me3 enrichment, glycolysis, and phagocytosis under hypoxia.
Abstract 312: Diosmin Enhances Endothelial Glycocalyx Integrity And Reverses Endothelial Dysfunction In Injured Blood Vessels
Authors: Mitra Ronodeep , Melin Mark , Levy Robert , Ebong Eno E
Published: October 2023

Objective:
This study determined if diosmin, a flavone glycoside of diosmetin, can repair damaged extracellular endothelial glycocalyx (GCX) (readouts: GCX labels including wheat germ agglutinin (WGA), hyaluronic acid (HA) binding protein, and core protein syndecan-1 (SDC1) antibody). Additionally, the study wanted to identify if diosmin can reverse endothelial dysfunction by improving vascular tone (readouts: endothelial nitric oxide synthase expression (eNOS), vessel wall thickness, and vessel lumen diameter) and vascular permeability (readouts: inflammatory cell uptake).

Methods:
In a mouse, acute disturbed blood flow pattern was induced by ligating three of the four left carotid artery (LCA) branches. This partial LCA ligation mouse model has shown to induce rapid endothelial dysfunction and loss of endothelial GCX. The right carotid artery (RCA) was the designated control for each mouse, as no surgical intervention occurred. Diosmin (50 mg/kg) was administered daily either orally or intraperitoneally for 7 days, 72 hours after the ligation surgery. One week after the first treatment dose, mice were sacrificed via exsanguination, and tissue was collected and fixed for further processing.

Results:
Diosmin enhanced endothelial GCX coverage and thickness after one week of treatment, as demonstrated by increased fluorescence of WGA, HA binding protein, and SDC1 antibody label on the diosmin-treated ligated LCA wall, compared to untreated tissue. Furthermore, diosmin also appeared to improve vascular tone as demonstrated by the increase in percent of total eNOS that is activated (p-eNOS/total eNOS) in diosmin-treated ligated LCA, compared to the untreated LCA. Interestingly, diosmin had limited effect on vessel wall thickness and vessel diameter. Finally, diosmin demonstrated attenuation of vessel inflammation by impeding macrophage (CD68
+
) uptake within ligated LCA walls.

Conclusion:
The novelty of our findings is that diosmin enhances endothelial GCX integrity. This study also demonstrated that diosmin can improve endothelium functionality after injury by decreasing inflammation and improving vascular tone. This work can lead to a new option for endothelial GCX restoration and endothelial-dependent aspects of vascular disease.
Abstract 314: Liver Specific Deletion Of Insulin Receptor Increases Biliary Cholesterol Secretion Independently Of Plasma Clearance Of HDL
Authors: Anspach Garrett , Ji Ailing , Graf Gregory A
Published: October 2023

Background:
Insulin signaling regulates hepatic fatty acid, cholesterol, and bile acid metabolism. Liver-specific deletion of insulin receptor increases biliary cholesterol secretion and bile acid hydrophobicity, but the impact on hepatic clearance of HDL-cholesterol and transhepatic cholesterol excretion are unknown. The objectives of the present study were to understand the impact of hepatic insulin receptor on hepatic clearance of HDL-cholesterol.

Methods:
Mice harboring loxP sites within the insulin receptor gene were treated with either a control adeno-associated viral vector (AAV) or an AAV encoding Cre recombinase under the control of the liver-specific thyroxin binding globulin promoter to generate liver insulin receptor knockout (LIRKO) mice. The impact on hepatic clearance of HDL-cholesterol, biliary cholesterol secretion and the regulation of sterol transporting enzymes was determined. In a second experiment, the impact of FGF15/19 signaling and FGFR4 on both hepatic ABCG5 and ABCG8 and biliary lipid metabolism was assessed in control and LIRKO mice using recombinant FGF19 and an antisense oligonucleotide directed against FGFR4.

Results & Conclusions:
As previously reported, LIRKO mice exhibit decreased glucose tolerance and reduced hepatic insulin signaling. Hepatic SR-BI protein, plasma HDL clearance, hepatic HDL protein, and selective cholesteryl ester (CE) uptake did not differ between control and LIRKO mice. Hepatic ABCG5/ABCG8 protein and biliary cholesterol secretion were increased in LIRKO mice. However, biliary elimination of HDL-CE derived cholesterol was unaffected. These results indicate that the increase in biliary cholesterol secretion is independent of plasma clearance of HDL in the reverse cholesterol transport pathway. Loss of hepatic insulin receptor is associated with a more hydrophobic bile acid pool and an increase in ileal FGF15 mRNA in LIRKO mice. Administration of FGF19 to control mice increased hepatic ABCG5, ABCG8, and biliary cholesterol secretion. However, suppression of FGFR4 in LIRKO mice had no effect on hepatic ABCG5 or ABCG8. In addition, administration of FGF19 to ABCG5 ABCG8-deficient mice did not perturb hepatic cholesterol levels or alter expression of sterol sensing genes.
Abstract 317: Adipose Tissue Phosphatidylcholine Remodeling Modulates Insulin Signaling
Authors: He Mulin , Jiang Xian-Cheng , Li Zhiqiang
Published: October 2023

Phosphatidylcholines (PCs) are major components of cell membranes. Previous studies indicated that membrane PC composition is regulated by lysophosphatidylcholine acyltransferase (LPCAT) and an alteration in cell membrane saturation has been implicated in a variety of metabolic disorders, including obesity, insulin resistance, and cardiovascular diseases. LPCAT3 is the major isoform of LPCAT in adipose tissues. To study the impact of LPCAT3 on adipose tissue lipid metabolism and its metabolic consequences, we prepared adipocyte-specific Lpcat3 gene knockout (KO) mice. Based on the analysis of bulk RNA sequencing, we gained insights to investigate the role of LPCAT3 in regulating adipose tissue insulin signaling. We revealed that LPCAT3 deficiency significantly reduced polyunsaturated PCs in the plasma membrane of adipocytes, resulting in increased insulin sensitivity. Mechanistically, we demonstrated that inhibiting LPCAT3 in adipose tissue promoted insulin receptor and AKT activation by influencing membrane lipid rafts and attenuated diet-induced insulin resistance. Conversely, adipose tissues from ob/ob, db/db, and high fat diet-fed mice had significantly higher LPCAT3 activity which blunts insulin signaling. Moreover, the treatment of polyunsaturated PCs on human or mouse mature adipocytes significantly worsened cellular insulin signaling. Our findings suggest that targeting LPCAT3 in adipose tissues to manipulate membrane phospholipid saturation would be a new strategy to treat insulin resistance.
Abstract 320: Metabolic Basis Of Vascular Smooth Muscle Cell Phenotypic Switching And Atherosclerosis
Authors: Meechem Megan , Jadli Anshul S , Gomes Karina , Chen Yong-xiang , Belke Darrell D , Patel Vaibhav B
Published: October 2023

Background:
Phenotypic switching of vascular smooth muscle cells (VSMC) has proven to be a key contributing factor to the development of atherosclerosis. In response to pathologic stimuli, VSMCs can undergo transdifferentiation to acquire phenotypes resembling synthetic, adipogenic, osteogenic, and macrophage-like foam cells. Increased mitochondrial fission and accompanying metabolic changes are believed to promote VSMC transdifferentiation and metabolic dysfunction in vascular diseases. Dynamin-related protein 1 (Drp1) is a GTPase that mediates mitochondrial fission; the mechanistic role of Drp1 activation in each pathologic VSMC phenotype requires further investigation. In the current study, we assessed how specific post-translational modifications (PTMs) contribute to Drp1 activation and mitochondrial metabolism in transdifferentiated VSMC phenotypes. Additionally, we explored if in vivo downregulation of Drp1 ameliorated the development of atherosclerosis in the thoracic aorta.

Methods and Results:
Mitochondrial dynamics and cellular metabolism were assessed in transdifferentiated VSMCs. Alterations in mitochondrial morphology were evaluated using Mitrotracker red staining and TEM. To determine the role of Drp1 in VSMC phenotypic switching, phosphorylation, O-GlcNAcylation, and acetylation of Drp1 were compared between individual VSMC phenotypes. The cellular bioenergetics of each VSMC phenotype were assessed to determine differences in the OCR and ECAR. Increased Drp1 activation correlated to alterations in the bioenergetic profile and increased metabolic dysfunction in the vasculature. Using SMC-specific Drp1 knockdown murine models, we utilized echocardiography, immunofluorescence, and histological staining to illustrate if the downregulation of Drp1 ameliorated atheroma formation and reduced VSMC phenotypic switching.

Conclusions:
The development of atherosclerosis is multifaceted; however, evidence has indicated that Drp1-induced mitochondrial fission differs in atherosclerotic VSMC phenotypes. By comparing the mitochondrial morphology and various PTMs of Drp1 in VSMC phenotypes, we can potentially isolate “precise” therapeutic targets to mitigate the development of atherosclerosis.
Abstract 328: Survival And Patency Of Saphenous Vein Grafts Using A Reverse-xenograft Model
Authors: Frontario S. Christopher , Bird Ryan , Rao Nakul , Hoffmann Thomas , Hammond Brendan , Patel Saahil , Pergolizzi Robert , Bernik Thomas
Published: October 2023

The saphenous vein graft (SVG) has been the conduit of choice for over 50 years, but its lack of availability makes its superior utility unreliable. Because of the paucity of autologous vein grafts and poor results of alternative bypass conduits, we hypothesized human vein allografts can be used for patients with limb-threatening ischemia who lack adequate vein conduit for restorative bypass surgery. Graft patency and conduit availability remain the ‘Achilles’ heel’ of this procedure.Immune-mediated tissue destruction and remodeling processes start within days after grafting, leading to the formation of intimal hyperplasia; the primary cause of intermediate-phase graft stenosis and failure. Even autologous saphenous vein grafts are affected, leading to reduced patency.We have developed a model in which pretreated human saphenous vein inter-positional grafts are implanted into infra-renal rat aorta, intended to study and prolong patency. This model accelerates the rate of failure due to the extreme difference in species biomarkers. We have performed grafts on 47 Sprague-Dawley rats, with or without pretreatment, for 2 weeks to 9 months. Preliminary data shows a strong immune reaction to the implantation of untreated human SVG, showing serious degradation 2 weeks after grafting. Pretreated grafts appeared superior to controls, with some demonstrating minimal to no hyperplasia out to 9 months. Tissue morphology was investigated using H&E, trichrome, EVG and DiffQuik stains, at magnifications from 5 to 40X. Immunofluorescence studies have provided insight into the destructive and positive remodeling processes with infiltration of native cells, including VWF-positive and Tcells, creating a novel architecture over time. We have successfully tested the hypothesis that human saphenous vein segments can be subjected to pre-treatment such that when grafted into rats, they will retain greater patency and have improved histological and physiological performance at 3, 6 and 9 months compared to controls. This research has significant implications for graft patency and retention in clinical applications.
Abstract 334: A Novel Model For Tracking Endothelial-derived Extracellular Vesicles In Murine Atherosclerotic Disease
Authors: Patel Sarvatit , Guo Mandy Kunze , Breda Leandro , Prajapati Kamalben , Raju Sneha , Botts Steven R , Yu Garry , Dudley Andrew , Scipione Corey A , Fish Jason , Howe Kathryn
Published: October 2023

Introduction:
An early marker of atherosclerosis is endothelial cell (EC) dysfunction. ECs are uniquely positioned as a boundary layer of the vessel wall capable of communicating with the circulating blood and the vessel wall. Extracellular vesicles (EVs), nanoparticles that carry cargo capable of governing cellular function and/or signalling disease have emerged as a novel communication paradigm. While
in vitro
models have shown EC-EV crosstalk with monocytes and vascular smooth muscle cells, animal models interrogating EC-EVs in atherosclerosis are lacking. We therefore created a model that allowed us to detect, quantify and visualize fluorescent EC-EVs in a murine model of atherosclerosis.

Methods:
Nine-week-old mice (LCE
Gr
; Ldlr
-/-
:Cdh5Cre
ERT2
:CD63-emGFP
l/s/l
and LE
Gr
; Ldlr
-/-
:CD63-emGFP
l/s/l
) were injected with tamoxifen (3 mg i.p., 3 doses, male & female) to induce green fluorescent EC-EVs. Mice received high-cholesterol diet (HCD, 12 wks) to promote atherosclerotic lesion formation or normal chow. EVs were enriched by size exclusion chromatography from plasma, quantified, and characterized by nanoparticle tracking analysis (NTA). Flow cytometry was performed to detect EC-EVs in cells isolated from whole blood. EC-EVs were visualized within atherosclerotic plaques using confocal microscopy.

Results:
As anticipated, HCD-fed mice developed atherosclerotic plaques compared to mice fed normal chow (n=3/grp). Fluorescent (GFP+) EVs were visualized in plasma of tamoxifen treated LCE
Gr
mice (NTA, 488nm laser; EVs pooled from 3 mice) and detected in blood cells of tamoxifen treated LCE
Gr
mice, but not in LE
Gr
mice (flow cytometry, n=2-4/grp). NTA demonstrated total plasma EVs were increased in HCD-fed mice compared to mice on chow diet (2.21x10
12
vs 2.40x10
11
particles/ml; p<0.0001; n=3/grp). Using confocal imaging we observed tissue-based EC communication in atherosclerotic plaques, where EC-EVs colocalize within leukocytes (GFP+CD45+; n=2/grp).

Conclusions:
This mouse model enabled detection of fluorescent (GFP+) EC-EVs in the circulating blood and visualization in leukocytes within atherosclerotic plaques. Further characterization of this model will help assess the role of EC-EVs in atherosclerotic plaque development.
Abstract 341: Study Of ADAR1 Implicates RNA Editing In Vascular Smooth Muscle Cells As A Mechanism Of Coronary Artery Disease Risk
Authors: Weldy Chad S , Li Qin , Galls Drew , Guo Hongchao , Bhate Amruta , Cheng Paul P , Sharma Disha , Markus Räsänen , Daniel Li Y , Monteiro João P , Palmisano Brian T , Gu Wenduo , Kundu Ramendra K , Nguyen Trieu , Li Jin Billy , Quertermous Thomas
Published: October 2023

Adenosine-to-inosine (A-to-I) RNA editing is a common RNA modification catalyzed by ADAR (adenosine deaminase acting on RNA) enzymes. ADAR1 serves a crucial function to edit double stranded RNA (dsRNA) molecules to prevent activation of the dsRNA sensor MDA5 (
IFIH1
) that stimulates an interferon stimulated gene (ISG) response. Discoveries in human genetics now implicate deficient ADAR1 RNA editing as a causal mechanism in coronary artery disease (CAD). We hypothesize that smooth muscle cell (SMC) specific RNA editing is required to prevent vascular calcification and inflammation. Here, we demonstrate that ADAR1 is the master regulator of RNA editing in primary human coronary artery SMC (HCASMC).
ADAR1
KD in HCASMC causes an ISG response characterized by massive upregulation of key ISG genes (e.g.
ISG15, IFI6, IFIH1
). Importantly,
ADAR1
KD stimulates the upregulation of genes with known roles in SMC phenotypic transition and CAD risk, including
KLF4
,
TCF21
,
ATF3
, and
ZEB2
. This effect is dependent on activation of MDA5. Additionally, we demonstrate ADAR1 and MDA5 regulate SMC phenotypic modulation and calcification in vitro where
ADAR1
KD inhibits SMC maturation and dysregulates the calcification gene program in an MDA5 dependent manner. Using scRNAseq data collected from human vascular tissue we show that immunogenic dsRNA are disproportionately expressed in human SMC in atherosclerosis. Similarly, in atherosclerosis prone mice, scRNAseq analysis reveals SMC to have increasing ISG expression as cells transition from mature SMC to a more calcified chondromyocyte phenotype — consistent with MDA5 activation. To further evaluate the role for SMC specific RNA editing in vivo, we generated a conditional SMC specific
Adar1
KD mouse (
Adar1
flox/flox
,

Myh11
CreERT2
, ROSA
tdT/+

). At 2 weeks following SMC-
Adar1
KD, mice demonstrate a severe phenotype of elastin degradation and disarray, intravascular hemorrhage, and inflammatory cell infiltration in the aortic wall. This effect was not seen with an endothelial cell specific deletion model (
Adar1
flox/flox

, Cx40
CreERT2

). Here we provide key evidence to support the observations from human genetics that the ADAR1-dsRNA-MDA5 axis is a causal mechanism in vascular disease and warrants further investigation.