bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 126: Exposure To Thirdhand Hookah Smoke Elevates The Risk Of Thrombogenesis By Enhancing Platelet Function
Authors: Alarabi Ahmed , Karim Zubair , Lozano Patricia , Khasawneh Fadi T , Alshbool Fatima Z
Published: May 2022

While the rate of smoking has been on the decline, the popularity of other forms of tobacco, including hookah/waterpipe continues to rise. While hookahs are thought to be safe, our recent studies have documented that exposure to waterpipe smoke (WPS) produces detrimental cardiovascular health effects, including thrombotic events; similar to what was observed with traditional cigarette smoking. In this connection, we have also documented that the new risk, termed thirdhand smoke (THS)-which is the residual tobacco smoke contaminant that remains after a cigarette is extinguished- increases the risk of thrombosis, much like both active/first hand smoke (FHS) and passive/second hand smoke (SHS) exposure. However, whether Thirdhand Hookah Smoke (THHS) exposure produces similar negative health effects remains to be determined. By employing a novel exposure protocol, mice were exposed to THHS starting at 10 weeks of age for three months, as per the golad standard Beirut protocol, which is as follows: one-hour session of 171 puffs of 530 mL volume, each puff is 2.6 s duration, and there are a 17 s interpuff interval. Our data shows that THHS exposed platelets exhibited enhanced agonist-induced aggregation, as well as dense and alpha granules secretion responses. Moreover, we also found that integrin activation, and phosphatidylserine exposure are increased as a result of THHS exposure. Consistent with these findings, we also obtained biochemical evidence of enhanced platelet reactivity, namely increased activation/phosphorylation of Akt. Notably, we observed high levels of the tobacco marker cotinine in the urine of the THHS mice, but not from the controls. Finally, and in terms of its
in vivo
impact, we observed that THHS enhances hemostasis and increases the risk of thrombosis in comparison to controls as documented by the shortened bleeding and occlusion times. Our results demonstrate, for the first time, that exposure to THHS results in a prothrombotic phenotype, which is attributed, at least in part, to a potentiated state of platelet reactivity. Thus, the negative health consequences of THHS should not be underestimated, and warrant further investigation. These findings should also guide policy development for regulating exposure to this form of tobacco.
Abstract 128: Nicotine Transgenerationally Alters Gene Methylation And Abdominal Aortic Aneurysm Risk
Authors: Mulorz Joscha U , Mulorz Pireyatharsheny , Rhee YaeHyun , Wagenhaeuser Markus , Tsao Philip S , Spin Joshua M
Published: May 2022

Significance:
Both smoking and family history are major risk factors for abdominal aortic aneurysm (AAA). Smoking is a powerful modulator of DNA methylation, and nicotine alone can cause heritable epigenetic alterations in animal models and humans. Nicotine also increases aortic stiffness, predisposing towards AAA. We investigated the effects of parental nicotine exposure in mice on their offspring’s risk for experimental AAA and on transgenerational DNA methylation.

Methods:
Male and female Apo E-/- mice (F0) were exposed to subcutaneous nicotine (25 mg/kg/day) or saline infusion for 28 days. After treatment completion, mice were mated to untreated controls. F1 generation offspring underwent Angiotensin II infusion (1μg/kg/min) to induce AAA at age 10-weeks. Aneurysm growth was tracked via ultrasound over 28 days. Subgroups of F1 mice underwent ex-vivo pressure myography assessment of the abdominal aorta. Germ and aortic tissue from both F0 and F1 were evaluated using RRBS-Seq genome-wide DNA methylation analysis.

Results:
Parental nicotine augments model AAA formation, incidence, and rupture rates in their offspring. This effect was most significant in male offspring of nicotine-exposed females (vs. saline). Maternal nicotine exposure also increased F1 aortic stiffness. F0 nicotine exposure altered DNA methylation in multiple tissues, and led to numerous significant differentially methylated regions (DMRs) in their F1 offspring, many precisely conserved. Nicotine caused global DNA hypermethylation, altered maternally imprinted genes in F0 females, and had variable patterns depending on gender. Nicotine altered aortic methylation patterns in genes known to relate to AAA development, including lncRNAs/miRNAs. DMR pathway analysis revealed enrichment for transcription factors, suggesting that nicotine transgenerationally influences numerous genes through expression modulation.

Conclusion:
Nicotine exposure can augment experimental AAA growth and aortic stiffness across generations. These effects are accompanied by widespread epigenetic changes in germ tissue and aorta, including several key AAA modulator genes, with enrichment in transcription factors, including many targeting known AAA-related genes.
Abstract MP02: Proprotein Convertase Subtilisin/kexin Type 6 (PCSK6) Is Involved In Regulation Of Vasculogenesis
Authors: Chemaly Melody , Suur Bianca E , Jin Hong , Malarstig Anders , Matic Ljubica
Published: May 2022

Background:
PCSK6 cleaves and activates growth factors involved in cell differentiation and
Pcsk6-/-
mice exhibit 25% embryonic lethality, while surviving offspring have blindness and cyclopia. We have shown that PCSK6 localizes to vascular smooth muscle cells of intra-plaque neovessels.

Hypothesis:
We hypothesize that a) the processes of vasculogenesis, atherosclerosis and plaque neovascularisation share common molecular regulators and b) that PCSK6 plays a common role in vascular remodeling.

Methods:
Association between SNPs near the PCSK6 gene and levels of 90 plasma proteins was tested using data from 30,931 individuals in the SCALLOP consortium. Data from the FinnGen consortium (Release 3) were queried to explore the PCSK6 SNPs associated with specific disease phenotypes. Morpholino technology was used for Pcsk6 knockdown in zebrafish, coupled with vascular phenotype studies.
Pcsk6-/-
mice were used to study the expression of vasculogenesis related genes as well as vascular morphology.

Results:
Rs7178801 and rs11639051 in the
PCSK6
gene were found to relate to plasma PDGFB and VEGFD levels, while rs45482895 associated with diabetic retinopathy and disorders of the choroid and retina in humans. Gross phenotypic and histological examination of zebrafish embryos with ablated PCSK6 showed improper peripheral vascular patterning of intersegmental vessels, cerebral and myocardial haemorrhage, atrial dilatation and pericardial oedema. Gene expression levels of vasculogenesis related genes, i.e. Vegfa, Vegfb, Angpt1 and Tgfb2 were altered in the heart, liver and adipose tissue in
Pcsk6-/-
mice. In the retinas of
Pcsk6-/-
mice, Pdgfb was significantly downregulated compared to controls. Staining of the retinal vasculature showed that superficial retinal vasculature had a lower number of branching points and vessel width, while deep retinal vasculature covered a significantly smaller area in
Pcsk6-/-

Conclusions:
Here, we show that variants in the
PCSK6
gene associate with growth factors implicated in vasculogenesis and retinopathy. Lack of
Pcsk6
in mice and zebrafish led to vascular patterning defects. Further studies will aim to elucidate the mechanisms by which PCSK6 regulates vasculogenesis in homeostatic and pathological conditions
Abstract MP06: The Signaling Roles Of The Adaptor Protein Nck1 In Atherosclerosis
Authors: Alfaidi Mabruka , Reddick Milla , Shen Xinggui , Orr Wayne
Published: May 2022

Rational:
Alterations in hemodynamic shear stress (SS) at atherosclerosis-prone sites promotes endothelial activation, characterized by nuclear factor-κB (NF-κB)-driven expression of cell adhesion molecules that mediate leukocyte homing. In addition, proinflammatory cytokines (e.g. IL-1β) promote NF-κB-dependent endothelial activation. While the recent CANTOS trial using an IL-1β antagonist highlights the potential for treating atherogenic inflammation beyond lipid lowering therapies alone, our understanding of how endothelial activation contributes to this effect remains limited.

Methodology & Results:
We identified the signaling adaptor Nck1 as a critical regulator of atherogenic endothelial activation. Endothelial cells deficient in Nck1 lack SS-induced NF-κB activation and proinflammatory gene expression. Additionally, we demonstrated an interaction between Nck1 and IL-1β signaling in response to SS, showing that SS activates the IL-1β receptor (IL1R1) signaling partner IRAK-1 in a Nck1-dependent manner both
in vitro
and
in vivo
. Mechanistically, point mutation analysis suggest a critical role for the Nck1 SH2 domain (phosphotyrosine binding domain) in mediating these effects. Nck1 affinity pulling down and mass spectrometry identified multiple downstream effectors of IL-1 pathway.

In vivo, Global Nck1 knockout mice show reduced atherosclerosis characterized by diminished macrophage and smooth muscle incorporation and bone marrow chimeras suggest non-hematopoietic Nck1 mediates this response.

Conclusions:
A recent GWAS analysis linked Nck1 to coronary artery disease, underscoring its importance to human disease. Therefore, our data suggest that Nck1 mediates the formation of IL-1-related signaling complexes to induce atherogenic endothelial activation, and selective inhibition of Nck1 or its critical domains will allow efficient inhibition of atherosclerosis in humans.
Abstract MP07: Multi Omic Atlas Of Human Coronary Artery Disease
Authors: Amrute Junedh M , Bredemeyer Andrea , Luo Xin , Yamawaki Tracy , Koenig Andrew , Jones Cameran , Jackson Simon , Furtado Milena B , Shi Sally , Li Chi-Ming , Ason Brandon , Stitziel* Nathan O , Lavine Kory J
Published: May 2022

Previous human genetic studies have provided insight into the genes and pathways involved in atherosclerotic CAD, however, there remains an incomplete understanding of the precise cell types that drive disease pathogenesis. To generate a cellular atlas for CAD, we performed CITE-seq on left human coronary arteries with obstructive CAD (n=5), CAD with coronary stenting (n=4), or controls without CAD (n=5); single-nuclei RNA sequencing was performed on a subset (n=4) of these samples. Standard histopathology was used to assess the degree of atherosclerotic plaque burden and annotate disease status (Fig b). Sequencing yielded 65,437 cells with 23 distinct cell types (Fig a) and canonical marker genes (Fig c). Within the stroma we found prominent transcriptional changes in endothelial cells and fibroblasts along with the emergence of a modulated smooth muscle cell (SMC) state in CAD tissue. The modulated SMC cells were enriched with fibroblast activator protein surface expression. Additionally, we noted the emergence of a proliferative SMC population in stented arteries. Two distinct fibroblast populations were present: one was specific to coronary arteries in GTEx and this population was enriched for SVEP1 expression in the CAD samples. Furthermore, pseudotime analysis and integration of published single-cell SMC lineage tracing data in atherosclerotic mice show that this population is SMC-derived. Within the immune cells, we find expansion of B- and T-cell populations with transcriptional changes within myeloid subsets. We found 4 distinct macrophage populations: inflammatory, TREM2, HLA-II high, and resident-like macrophages. CAD macrophages had enriched inflammatory and foam-cell like signatures compared to controls. Furthermore, foam cell genes were localized to the inflammatory and TREM2 macrophages. Together, our results provide the first multi-omic single cell atlas of human coronary artery tissue with insights into disease pathogenesis.
Abstract MP12: The Epigenetic Remodeling Protein Brg1 Is Implicated In Vascular Progenitor Cell Contribution To Pathological Vascular Remodeling And Fibrosis
Authors: Jolly Austin , Lu Sizhao , Dubner Allison , Strand Keith , Mutryn Marie , Moulton Karen , Nemenoff Raphael , Weiser-Evans Mary
Published: May 2022

Vascular fibrosis is an irreversible consequence of vascular remodeling that ensues in many cardiovascular diseases. Currently, no treatments are available to target chronic vascular fibrosis. We identified a unique population of multipotent smooth muscle-derived Sca1+ progenitor cells that reside in the vascular adventitia (AdvSca1-SM cells). While AdvSca1-SM cells can differentiate into mature smooth muscle cells (SMCs) and repair the vessel wall, AdvSca1-SM cells also have the potential to differentiate into myofibroblasts and greatly contribute to vascular fibrosis after acute vascular injury. The epigenetic remodeling protein Brg1 is upregulated in AdvSca1-SM cells in the setting of vascular injury, but how Brg1 influences AdvSca1-SM differentiation is unknown. Using
in vitro
and
in vivo
approaches, our goal was to define the role of Brg1 in AdvSca1-SM cells. We hypothesized that Brg1 regulates chromatin density around genetic loci associated with fibrosis to preferentially drive AdvSca1-SM cell differentiation towards pathologic myofibroblasts. Further, we hypothesized that pharmacologic inhibition of Brg1 will disrupt AdvSca1-SM differentiation into myofibroblasts and attenuate vascular fibrosis. Mice subjected to carotid ligation and treated with the highly specific Brg1 inhibitor, PFI-3, exhibited decreased vascular fibrosis, reduced neointima, and decreased adventitial expansion of AdvSca1-SM cells as compared to mice that received vehicle control. This
in vivo
data was complemented by
in vitro
studies where cultured AdvSca1-SM cells stimulated with TGF-β expressed myofibroblast genes including αSMA and periostin, and co-treatment with PFI-3 blocked TGF-β-induced myofibroblast marker expression at the mRNA and protein level. In conclusion, these results support the hypothesis that Brg1 is a major regulator of AdvSca1-SM myofibroblast differentiation and may represent a novel targetable protein to treat vascular fibrosis.
Abstract MP13: Small RNA Profiling Identifies Mir-200b As A Repressor Of Vascular Smooth Muscle Cell Migration And Proliferation
Authors: Du Mingyuan , Espinosa-Diez Cristina , Liu Mingjun , Mahan Sidney , Gomez Delphine
Published: May 2022

Background and Objectives:
A large body of research has identified miRNA as pivotal regulators of smooth muscle cell (SMC) fate in various vascular diseases. However, global changes of miRNA expression profile during SMC phenotypic modulation have not been well characterized. As the targetome of a given miRNA is highly dependent on the cell type and context, comparative analysis of mRNA and miRNA expression profiles can provide decisive information for assessing the versatility in miRNA functions and discovering novel miRNA modulating SMC phenotype. Our goal is to define the miRNA signatures in differentiated and de-differentiated SMC, the miRNA overall contribution to the transcriptome, and novel functionally relevant miRNA.

Methods:
By performing RNAseq and small RNAseq, we defined the mRNA/miRNA expression profiles in aortic SMC cultured in differentiation or de-differentiation media with Platelet Derived Growth Factor-BB (PDGF-BB) and performed functional studies on differentially expressed miRNAs (DEmiR).

Results:
We found that PDGF-BB-induced SMC de-differentiation generated 48 significant DEmiRs, including 15 downregulated and 33 upregulated DEmiRs. Integrative analysis predicted that nearly 40% (1335 of 3523) of differentially expressed transcripts were direct targets of DEmiRs. The miR-200 cluster (miR-200a, miR-200b and miR-429) was found potently downregulated after PDGF-BB treatment, as well as Tet2 and Myocardin knockdown, while its expression increased in rapamycin treated SMC. We found that miR-200b overexpression prevented PDGF-BB-induced SMC migration and proliferation, while miR-200b inhibition had an opposite effect. Finally, we identified Quaking, a potent inducer of SMC phenotypic switching, as a primary target of miR-200b.

Conclusion:
Our study predicted a marked contribution of miRNA in regulating SMC transcriptomic profile. miR-200b was identified as a novel pro-differentiation miRNA in SMC through inhibition of phenotypic switching.
Abstract MP19: New Insight Into The Optimal Duration Of DAPT Following PCI In High-risk Twilight-like Patients With Acute Coronary Syndrome
Authors: Wang Haoyu , Dou Kefei
Published: May 2022

Introduction:
Recent emphasis on shorter DAPT regimen after PCI irrespective of indication for PCI may fail to account for the substantial residual risk of recurrent atherothrombotic events in ACS patients. We aim to determine the association of extended-term (>12-month) vs. short-term dual antiplatelet therapy (DAPT) with ischemic and hemorrhagic events in high-risk "TWILIGHT-like" patients undergoing PCI for ACS in clinical practice.

Methods:
All consecutive patients fulfilling the "TWILIGHT-like" criteria undergoing PCI were identified from the prospective Fuwai PCI Registry. High-risk patients (n=5,404) were defined by at least 1 clinical and 1 angiographic feature based on TWILIGHT trial selection criteria. The primary ischemic endpoint was major adverse cardiac and cerebrovascular events at 30 months, composed of all-cause mortality, myocardial infarction, or stroke while BARC type 2, 3, or 5 bleeding was key secondary outcome.

Results:
The proportion of patients with 1-3, 4-5, or 6-9 risk factors was 19.7%, 58.6%, and 21.6%, respectively. Cessation of DAPT was less frequent in patients with ACS at 12 months (31.6% versus 68.4%), with 22.7% of ACS patients still on DAPT beyond 24 months. Of 4,875 high-risk ACS patients who remained event-free at 12 months after PCI, DAPT>12-month compared with shorter DAPT reduced the primary ischemic endpoint by 63% (1.5% vs. 3.8%; HRadj: 0.374, 95% CI: 0.256 to 0.548; HRmatched: 0.361, 95% CI: 0.221-0.590). The HR for cardiovascular death was 0.049 (0.007 to 0.362) and that for MI 0.45 (0.153 to 1.320) and definite/probable stent thrombosis 0.296 (0.080-1.095) in propensity-matched analyses. Rates of BARC type 2, 3, or 5 bleeding (0.9% vs. 1.3%; HRadj: 0.668 [0.379 to 1.178]; HRmatched: 0.721 [0.369-1.410]) did not differ significantly between two groups.

Conclusions:
Among high-risk ACS patients undergoing PCI, long-term DAPT, compared with shorter DAPT, reduced ischemic events without a concomitant increase in clinically meaning bleeding events, suggesting that long-term DAPT can be considered in patients with ACS following PCI who are carefully assessed to be at low bleeding risk and heightened ischemic risk, and have tolerated antiplatelet therapy without a major bleeding during 1 year of DAPT.
Abstract MP21: Transcriptomic Modulation Of Second Heart Field-derived Smooth Muscle Cells In Angiotensin Ii-infused Mice Promotes Aortopathy
Authors: Sawada Hisashi , Zhang Chen , Li Yanming , Katsumata Yuriko , Shen Ying H , Lemaire Scott A , Lu Hong S , Daugherty Alan
Published: May 2022

Objective:
Smooth muscle cells (SMCs) in the ascending aorta are derived from both the cardiac neural crest and second heart field (SHF). The importance of cardiac neural crest-derived SMCs on development of thoracic aortic aneurysms has been reported, while functional roles of SHF-derived SMCs are controversial. The aim of this study was to profile the transcriptome of SHF-derived SMCs in ascending aortas of angiotensin II (AngII)-infused mice by single cell RNA sequencing.

Methods and Results:
Female ROSA26 mT/mG mice were bred to male mice expressing Cre under the control of the Mef2c promoter (Mef2c Cre). Angiotensin II (AngII, 1,000 ng/kg/day) was infused subcutaneously into Mef2c Cre +/- mice and ascending aortas were harvested at day 3 of AngII infusion (n=5). Ascending aortas without AngII infusion were harvested as control (n=4). SHF-derived cells were sorted based on mGFP signal by FACS and single cell sequencing was performed using SHF-derived cells. Single cell RNA sequencing detected mRNA in multiple cell types, and AngII altered mRNA abundance of 3,658 genes in the SMC cluster. Several SMC contractile-related genes, such as
Acta2, Tagln
and
Cnn1
, were increased by AngII compared to control SHF-derived SMCs. Conversely
, Klf4
, a proliferative gene, was decreased in SHF-derived SMCs of AngII-infused mice. In addition, AngII increased multiple extracellular matrix component genes, including fibrillin1 and elastin. AngII also upregulated
Serpine1
and
Fn1
that are important for extracellular matrix organization. Thus, AngII led to transcriptomic modulations in SHF-derived SMCs. Of note, AngII infusion decreased mRNA abundance of
Tgfbr2
and
Lrp1
, key regulators of extracellular matrix maturation, in SHF-derived SMCs. To further investigate the importance of SHF-derived SMCs, we deleted either
Tgfbr2
or
Lrp1
in SHF-derived cells. SHF-specific
Tgfrb2
deletion was embryonic lethal with peritoneal hemorrhage and outflow tract dilatation. SHF-specific
Lrp1
deletion augmented AngII-induced thoracic aortic rupture and dilatations.

Conclusion:
SHF-derived SMCs play an important role in maintaining aortic integrity, and exhibit transcriptomic modulations by AngII infusion that may contribute to aortopathies.
Abstract MP22: Effect Of Nanoparticle Size And Dose On Detection Of Aortic Injury Using Nanoparticle Contrast-enhanced Computed Tomography
Authors: Devkota Laxman , Zhang Chen , Bhandari Prajwal , Starosolski Zbigniew , Shen Ying H , Lemaire Scott A , Ghaghada Ketan
Published: May 2022

Background:
Early detection and prediction of disease progression are critical for improving outcomes in patients with aortic aneurysms and dissections (AAD). Recently, we reported that nanoparticle contrast-enhanced computed tomography (n-CECT) enables early detection of aortic injury before vessel enlargement is evident on CT angiography. In this work, we investigated the effect of nanoparticle contrast agent (NPCA) size and dose on detection of aortic injury. Additionally, we performed a longitudinal study to examine long-term fate of intramural NPCA.

Methods:
In a mouse model of sporadic AAD, mice (n=8/group) were challenged with high fat diet for 5 weeks and angiotensin-II infusion during the last week. Animals were intravenously administered one of three NPCA size variants (80, 150 and 250 nm). For dose effect study, 150 nm NPCA variant was tested at 3 dose levels: 300, 600 and 1200 mg I/kg.
In vivo
n-CECT was performed after 4 days.
Ex vivo
n-CECT and gross examination were performed on excised aortas. To study intramural fate of NPCA, a subset of animals underwent longitudinal CT imaging for up to 6 months.

Results:
All NPCA size variants showed a higher incidence of n-CECT findings compared to gross aortic examination: absolute improvements in detection rates were 12% for
80 nm
, 28% for
150 nm
, and 8% for
250 nm
. NPCA dose analysis showed that sensitivity of n-CECT for detection of aortic injury improved with increasing dose level (33% for
300 mg I/kg
, 44% for
600 mg/kg
, and 62% for
1200 mg I/kg
) (Figure 1). Longitudinal imaging demonstrated clearance of a majority of intramural NPCA that accumulated at sites of aortic pathology by 4 months. Furthermore, n-CECT imaging enabled monitoring of vascular permeability changes at sites of aortic injury.

Conclusion:
Regardless of NPCA size, n-CECT imaging showed higher sensitivity for early detection of aortic degeneration compared to gross aortic examination. Detection of aortic injury improved with increasing NPCA dose.
Abstract MP23: Knockout Of Pgc1α In Endothelial Cells Augments Abdominal Aortic Aneurysm Formation
Authors: Fernandez Timothy F , Sellak Hassan , Lassegue Bernard P , Taylor William R
Published: May 2022

Introduction:
It is well described that changes in the aortic media are crucial in the development of Abdominal Aortic Aneurysms (AAA), however the involvement of the endothelium is not as clear. The endothelium is the first vascular layer exposed to blood flow and aneurysms preferentially form in areas of disturbed flow. The regulatory mechanisms as to why aneurysms form at these sites remains unclear. Here we propose that the transcription coactivator peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α) plays a pivotal role in the endothelium’s response to AAA formation in a shear responsive manner.

Methods:
10-12 week old male mice deficient in endothelial specific PGC1α (KO) and their corresponding wild type background mice (WT) were treated with 0.75 mg/kg/day of Ang II. Our primary endpoints were measurement of the maximal width of the suprarenal aortas, frequency, and complexity based on the Daugherty classification. To determine the effects of shear, HAECs exposed to steady flow (15 dyn/cm
2
) and disturbed flow (0±6 dyn/cm
2
at 1 Hz) for 12 hours were compared to determine if mRNA expression were different for PGC1α and SIRT1.

Results:
AAA incidence and severity were increased in ANG II-infused KO compared to the WT mice (100% vs. 20%, respectively). Measurements of excised aortas showed that KO mice exhibited larger AAA fold-increase compared to WT (2.00 ± 0.11 fold increase vs. 0.43 ± 0.19 fold increase, p-value 0.0002). The KO male mice also exhibited a more complex phenotype. HAECs that underwent disturbed flow had a significant decrease in expression of SIRT1 (p = 0.02) and trended towards a decrease in expression of PGC1α (p = 0.06).

Conclusions:
Our data strongly suggests that knockout of endothelial specific PGC1α leads to more frequent, complex and larger AAAs primarily in the suprarenal aorta, which is an area of disturbed flow. Interestingly, the addition of ANG II alone was able to induce AAA formation in our model. Additionally, we show that PGC1α and SIRT1 are regulated in a shear responsive manner. Taken together, endothelial PGC1α likely plays a regulatory role in AAA formation through shear-related mechanisms.
Abstract MP24: Disruption Of Actin Remodeling Intensifies Aneurysm-promoting Signals In Smooth Muscle Cells
Authors: Jiang Zhihua , Wang Fen , Upchurch Gilbert R
Published: May 2022

Mutations within proteins coding for extracellular matrix assembly or those in the transforming growth factor beta (TGFβ) signaling pathway lead to aneurysm formation in patients and mice. As the SMC cytoskeletal network is intricately linked to TGFβ and ECM signaling, we sought to determine if disruption of the cytoskeletal filaments would alter how SMCs respond to angiotensin II, an important mediator of aneurysm formation.

C57BL/6 mice lacking
Tgbfr1
in SMCs (SMC-
Tgbr1
iko
) generated by Jiang et al. have been shown to develop rapid and severe aortic aneurysm degeneration with 100% penetrance. Analysis was conducted on aneurysm and control mouse and human aortic tissue sections. We observed that SMC-
Tgbr1
iko
mice had reduced expression of filamentous (f-) and α-actin protein in the aortic media than control mice. Primary
Tgbr1
iko
SMCs also had higher levels of pRelA expression, less α-actin mRNA, and less f-actin than control SMCs. Tissue sections from aneurysm patients had reduced f-actin staining relative to controls. Disruption of f-actin with the destabilizer latrunculin A
in vitro
led to increased RelA and ERK activation in the presence of AngII, which was further augmented in the presence of a neutralizing antibody for TGFβ. In contrast, jasplakinolide—an f-actin stabilizer—prevented TGFβ blockade induced pRelA expression, suggesting that altered/inhibited TGFβ signaling modulates NFkB through actin depolymerization. While stabilization and disruption of microtubules led to RelA activation, no effect was observed on TGFβ blockade-induced pRelA expression. The phosphoantibody array revealed that f-actin disruption in the presence of AngII activated the CREB, AKT1/2/3, ERK1/2, and STAT3 pathways in mouse aortic SMCs. SMC-
Tgbr1
iko
mice treated with cytochalasin B suffered significantly greater mortality and had increased aortic growth over a 30-day follow-up period.

Aortic aneurysms are coincident with reduced f-actin expression in murine and human aortic SMCs. Chemical deconstruction of actin filaments promotes an inflammatory phenotype in mouse SMCs
in vitro
, and aggravates aneurysm formation
in vivo
. Aberrant or inhibited TGFβ signaling potentiates NFkB activation in SMCs, which may be dependent on f-actin disassembly.
Abstract MP25: Differential Expression Of Pparα In Peripheral Arterial Segments Of Patients With Advanced Atherosclerosis
Authors: engel connor , Harroun Nikolai , Penrose Amanda , Shafqat Mehreen , Meade Rodrigo , Jin Xiaohua , DeSilva Gayan , Semenkovich Clay F , Zayed Mohamed
Published: May 2022

Peripheral atherosclerosis manifests in both the extracranial carotid and lower extremity arteries and can lead to significant morbidity and mortality. However, atherosclerotic disease progression is often not homogenous and is accelerated by diabetes. We previously observed altered phospholipodomic profiles between minimally (MIN) and maximally (MAX) diseased peripheral arterial segments. Since Peroxisome Proliferator-Activated Receptor alpha (
pparα
) is a key regulator of lipid metabolism, we hypothesized that it may have variable content and signaling in MIN and MAX diseased arterial segments. To test our hypothesis, 12 patients who underwent carotid endarterectomy (CEA), and 19 patients who underwent major lower extremity amputation were recruited. MIN and MAX disease segments were obtained in real time from the operating room from CEA plaque and arterial segments from amputated lower extremities. mRNA was isolated from all specimens and relative content of
pparα
, Acyl-CoA Oxidase 1 (
acox1
)
,
and Carnitine Palmitoyltransferase 1A (
cpt1a
) were evaluated. We observed significantly higher
pparα
expression in CEA segments in patients with diabetes (p < 0.01), as well as higher
acox1
(p < 0.001) and
cpt1a
(p < 0.05) expression (A-C). Hemoglobin A1C had a significant correlation with
ppara
gene expression. There was no significant difference in gene expression between MAX and MIN diseased CEA plaque segments. Interestingly, we observed that in lower extremity arterial segments there was no difference in
ppara
,
acox1
, and
cpt1a
in patients with and without diabetes, but downstream genes were significantly increased in MAX diseased arterial segments (D-F). This study demonstrates the variable expression pattern of
pparα
and its downstream genes in human peripheral arteries. Our findings suggest variable gene expression in different peripheral arterial beds, which may have an impact on mechanisms of disease progression and pharmacologic targeting.
Abstract MP27: Endothelial Nogo-B Modulates Ischemia Induced Collateral Artery Remodeling
Authors: Zhang Cheng , Zhang Yuanyuan , Lucitti Jennifer , Palos-Jasso Andrea , Albinsson Sebastian , Gao Yuan , Cui Yaru , Murata Takahisa , Faber James E , Sessa William C , Yu Jun
Published: May 2022

Nogo-B, a member of the reticulon 4 family of proteins, is the dominant isoform expressed in endothelial cells (EC). We have shown that Nogo-B is necessary for blood flow recovery in ischemia. Mice lacking Nogo-B exhibit reduced arteriogenesis and angiogenesis that are linked to a decrease in macrophage infiltration and inflammatory gene expression in vivo. However, whether endothelial Nogo-B regulates arteriogenesis and/or angiogenesis is unknown. We generated an inducible EC-specific mouse overexpressing Nogo-B (Ng
ECtg
) and investigated the arteriogenesis and angiogenesis in limb ischemia. Ischemia increased endothelial and serum Nogo-B expression. Blood flow recovery was markedly diminished after femoral artery ligation (FAL) in Ng
ECtg
compared to WT mice, in association with lower collateral density accessed by micro-CT arteriography. There was no reduction in capillary density or decrease in smooth muscle/pericyte and macrophage recruitment in Ng
ECtg
mice, suggesting EC Nogo-B overexpression regulates arteriogenesis but not angiogenesis. While Ng
ECtg
mice have normal number of native collateral artery, the early remodeling of the collateral artery was impaired after ischemia. Furthermore, in line with impaired remodeling, Ng
ECtg
mice have decreased functional (exercise) hyperemia response in the non-ischemic limb in vivo, and resistant arteries from Ng
ECtg
have diminished flow induced vasodilatation ex vivo compared to WT mice. Moreover, endothelial reconstitution of Nogo-B in global Nogo knockout background (NogoKO
ECrc
) restored blood flow recovery after ischemia in vivo, further suggesting the EC specific function of Nogo-B in arteriogenesis. Mechanistically, we have shown that EC isolated from Ng
ECtg
mice have decreased nitric oxide release. Nogo-B overexpression negatively regulates eNOS phosphorylation, expression and golgi localization in EC. Future parabiosis study is warranted to elucidate the contribution of paracrine or EC intrinsic Nogo-B in modulating arteriogenesis and angiogenesis. In conclusion, our data showed for the first time that endothelial Nogo-B plays important roles in regulating collateral artery remodeling and blood flow through, at least in part, regulating NO bioavailability.
Abstract MP28: The Bet Bromodomain Inhibitor, (+)-JQ1, Inhibits Neointima Formation Following Acute Vascular Injury Via Pten Upregulation
Authors: Strand Keith , Lu Sizhao , Mutryn Marie , Dubner Allison , Jolly Austin , Nemenoff Raphael , Cavasin Maria A , McKinsey Timothy A , Weiser Evans Mary C
Published: May 2022

The primary aim of this work was to identify compounds that upregulate phosphatase and tensin homolog (PTEN) expression in smooth muscle cells (SMCs), with the expectation that PTEN upregulation would inhibit pathological vascular remodeling by promoting SMC homeostasis. Our recent work demonstrated that PTEN is an important regulator of SMC phenotype. SMC-specific PTEN deletion promotes spontaneous vascular remodeling and PTEN loss correlates with increased atherosclerotic lesion severity in human coronary arteries. In mice, PTEN overexpression reduces plaque area and preserves SMC contractile protein expression in atherosclerosis and blunts Angiotensin-II-induced pathological vascular remodeling, suggesting that pharmacologic PTEN upregulation could be a novel therapeutic approach to treat vascular disease. Currently, there are few known pharmacologic inducers of PTEN, however, we have shown that (+)-JQ1, which targets the epigenetic reader protein, Brd4, directly activates the PTEN promoter to drive PTEN expression. Mechanistically, our findings suggest that Brd4 inhibition results in p300-mediated hyper-acetylation of the PTEN promoter to increase PTEN transcriptional activity. Administration of (+)-JQ1 or SMC-specific Brd4 deletion inhibited neointima formation following acute carotid artery injury in mice. Using SMC-specific PTEN depletion both in vitro and in vivo, we showed that the observed anti-remodeling, anti-inflammatory effects were PTEN-dependent. Furthermore, we tested the effects of (+)-JQ1 on macrophage polarization, as systemic drug delivery could potentially modulate the phenotype of other resident vascular cells, including macrophages. We found that (+)-JQ1 inhibited bone marrow-derived macrophage polarization towards a pro-inflammatory phenotype, which is notable because vascular inflammation driven by SMC-macrophage cross-talk is known to promote neointima development. Importantly, our findings may hold direct clinical relevance, as BET bromodomain inhibitors related to (+)-JQ1 are currently undergoing clinical trials. These results are significant because they indicate that targeting PTEN upregulation in SMCs is a completely novel approach to inhibit vascular remodeling.
Abstract MP30: Excess HDL Free Cholesterol Bioavailability Drives Free Cholesterol Accretion Into Macrophages And Erythrocytes In Scarb1
-/-
Mice
Authors: Liu Jing , Gillard Baiba K , Yelamanchili Dedipya , Wang Ziyi , Gotto Antonio M , Rosales Corina , Pownall Henry J
Published: May 2022

Aim:
In humans, very high plasma HDL-cholesterol concentrations are associated with increased all cause- and atherosclerotic cardiovascular disease (ASCVD)-mortality. The HDL receptor-deficient mouse (Scarb1
-/-
), a robust model of this phenotype, is characterized by high free cholesterol (FC) bioavailability due to too many HDL particles that are FC-rich. Clinically, plasma LDL and HDL are quantified according to total cholesterol content, the sum of FC and esterified cholesterol, which likely contribute to ASCVD pathophysiology differently. A Western diet induces ASCVD in Scarb1
-/-
mice, despite an attendant increase in HDL. We tested the hypothesis that high HDL-FC bioavailability contributes to ASCVD in Scarb1
-/-
mice by increasing FC flux into macrophage cells, erythrocytes and other major tissues.

Methods:
Influx of HDL-FC and efflux of macrophage FC were determined between WT and Scarb1
-/-
HDL and J774 macrophage cells. HDL of both genotypes were radiolabelled with [
3
H]FC, injected into autologous mice, and the rates of plasma clearance and erythrocyte uptake were determined.

Results:
The magnitude of FC transfer from Scarb1
-/-
HDL to LDL is greater than that from WT HDL; APOB-containing lipoproteins from Scarb1
-/-
vs. WT mice are FC-enriched due likely to greater HDL-FC transfer. While macrophage efflux to HDL of Scarb1
-/-
vs. WT HDL was not different, FC influx from Scarb1
-/-
vs. WT HDL to macrophages was three-fold greater, a net effect that increased the FC burden of macrophages. In vivo studies showed that compared to WT mice, in Scarb1
-/-
mice, autologous HDL-FC cleared more slowly and more FC transferred to erythrocytes. We compared the FC, CE, PL, and TG contents of all major tissues and determined that FC accretion by some tissues is higher among Scarb1
-/-
vs. WT mice whereas in other tissues FC homeostasis is maintained. Lastly, we determined that the tissue compositions and plasma FC clearance kinetics varied according to sex, particularly among Scarb1
-/-
mice.

Conclusions:
These findings are relevant to pathologies specific to Scarb1
-/-
mice and to the evolving model of the role of HDL-FC in RCT. They provide a rationale for human studies to determine the utility of HDL-FC bioavailability as a risk factor for ASCVD and other pathologies.
Abstract MP33: Coordination Of Metabolic Functions By SR-BI And PCPE2 In Adipocytes
Authors: Knaack Darcy A , Sorci Thomas Mary G , Thomas Michael J , Chen Yiliang , Sahoo Daisy
Published: May 2022

Obesity continues to be an epidemic in the United States, placing individuals at a higher risk of developing other comorbidities such as type 2 diabetes and cardiovascular disease. Adipocytes accumulate excess triglycerides and undergo hypertrophic expansion as a protective mechanism to avoid lipotoxicity. Adipocytes are also highly dynamic cells that perform many key metabolic processes including lipid and glucose metabolism. Recent collaborative studies demonstrated that SR-BI function may require the extracellular matrix protein procollagen C-endopeptidase enhancer 2 (PCPE2) in to regulate cholesterol transport in hepatocytes and adipocytes. However, the roles of SR-BI and PCPE2 in other adipocyte metabolic processes remain unclear. This knowledge gap prompted our novel hypothesis that PCPE2 facilitates SR-BI’s ability to regulate metabolic processes in adipocytes. To test our hypothesis, we have optimized an adipocyte model system where mesenchymal stem cells isolated from the outer ears of wild-type (WT), SR-BI knockout (SR-BI
-/-
), and PCPE2
-/-
mice were differentiated into adipocyte-like cells. We validated our adipocyte model system by demonstrating increased Oil Red O staining, mRNA expression of adipogenesis markers, and adiponectin secretion over a 9-day post-differentiation period for all three genotypes. This model system was then used to test the roles of SR-BI and PCPE2 on various metabolic functions. Preliminary data suggests that absence of SR-BI or PCPE2 slightly impairs fatty acid uptake into adipocytes despite elevated gene expression of fatty acid transporters (FABP4, CD36) compared to WT. These data further suggest that SR-BI may rely on PCPE2 to aid in transport of fatty acids. Adipocytes lacking SR-BI or PCPE2 also displayed higher levels of mitochondrial reactive oxygen species, indicating that SR-BI and PCPE2 may be important for maintaining healthy mitochondria in adipocytes. These changes in function do not appear to depend on SR-BI oligomerization, as BS
3
crosslinking studies demonstrated the presence of SR-BI oligomers in adipocytes despite the loss of PCPE2. Altogether, these data suggest that SR-BI and PCPE2 may be reliant on each other and are important for mediating metabolic processes in adipocytes.
Abstract MP35: Single-cell Analysis Uncovers Multiple Adventitial Fibroblast Populations With Expression Of CAD GWAS Genes In Murine Atherosclerosis
Authors: Chang Hyun-kyung , Kim Eunyoung , Reilly Muredach P , Bauer Robert C
Published: May 2022

The arterial adventitia is a layer of cells outside the external lamina of blood vessels that contribute to the progression of coronary artery disease (CAD) like atherosclerosis. Adventitial fibroblasts are known regulators of vascular remodeling through their deposition of collagen fibrils around vessels during atherosclerotic lesion development. However, the molecular identity and function of the participating fibroblasts have not been well studied. We used single cell RNA sequencing (scRNA-seq) to characterize the transcriptome of adventitial fibroblasts and other cell types at multiple timepoints during atherogenesis in hyperlipidemic Ldlr knockout mice. Specifically, scRNA-seq was applied to Ldlr KO aortic adventitia after 0, 9, and 16 weeks of western diet (WD) feeding. Unbiased clustering analysis uncovered 9 different cell clusters, including 5 different fibroblast populations (Fibro1-5), and a single cluster each of macrophages, T cells, SMCs, and ECs. We observed that, as atherosclerosis progressed from 9 to 16 weeks of feedings, the proportion of total cells that were Fibro1, 3, and 4 decreased (33, 40, and 13%), while populations of Fibro2 and 5 slightly increased (13 and 18%). All of non-fibroblast populations (Macrophage, T cells, SMCs and ECs) increased from 9-16wks of diet. Next, we asked whether genes associated with coronary artery disease by human genome-wide association studies (GWAS) are expressed in different adventitial fibroblast clusters. We determined the expression of 266 genes implicated in CAD by GWAS and found 201 genes (76%) were expressed in adventitial cells at either/both 9 or 16wks. 34 genes displayed large changes in expression from 9 to 16wks. We identified five genes that were highly expressed in the adventitial Fibro5 cluster specifically, and additional other CAD GWAS genes specifically expressed in adventitial macrophages, SMCs, and ECs. In summary, we present here a cell atlas defined by scRNA-seq that reveals the heterogeneity amongst the fibroblasts in adventitial cells during atherogenesis. Additionally, we find that many CAD GWAS genes are expressed in adventitial cell populations, raising the intriguing possibility that CAD GWAS are identifying adventitial mechanisms that contribute to CAD.
Abstract MP37: Effect Of Sex Dimorphism And Pregnancy On Innate Lymphoid Cells Expressing Elabela
Authors: Da Silva Lopes Salle Evila , Khodadadi Hesam , Domingo Liezl , Zavan Bruno , Yu Jack , Baban Babak
Published: May 2022

Elabela is an endogenous secreted peptide and its role has been investigated in heart and preeclampsia development. Endogenous peptides are usually known for their potential role as regulators of the innate immune function. In this study, we evaluated the expression of Elabela by the three different types of Innate Lymphoid Cells (ILCs): ILC1s, ILC2s, and ILC3s. ILCs are a family of immune cells that mirror the phenotypes and functions of T cells and promptly response to signals from injured tissues. We hypothesized that ILCs may regulate the immune response by expressing Elabela (ILCsEla+). Since heart and the uterine decidua have been the focus of studies about Elabela, we collected both tissues from ICR males (n=3), virgins (n=3), and pregnant females on the 6
th
(n=3), 10
th
(n=3), and 14
th
(n=3) gestation days. The collected tissues were prepared for further flow cytometric analysis. Also, part of the decidua from 10
th
gestation day was used to perform Mixed Lymphocyte Reaction (MLR)
in vitro
. Our findings showed that ILCs in both heart and decidua express Elabela. The analysis showed that the number of ILC3sEla+ (10.7
+
1.2) was higher in virgin female comparing to males (3.3
+
0.8) (p=0.0032). In pregnant females, the total number of ILCsEla+ increased during the period of 6
th
to 14
th
gestation days in both heart {6
th
(27
+
1.1); 14
th
(42
+
1.5) (p=0.0104)} and decidua {6
th
(26.6
+
0.5); 14
th
(47
+
12) (p= 0.0273)} . The analysis of the MLR showed that Elabela was able to decrease the total number of active T cells. In conclusion, our study showed that Elabela as part of the innate immune system is expressed by ILCs. Also, there is a sex difference in the number of ILCsEla+ in the heart. These findings highlight the importance of exploring the role of Elabela as an immunomodulator in different cardiovascular disease models.
Abstract MP40: Breakpoint Cluster Region Protein Function As A Kinase Mediates The Atheroprotective Actions Of Hdl In Vascular Endothelium
Authors: Sacharidou Anastasia , Chambliss Ken , Chu Haiyan , Lee Wan-Ru , Xu Lin , Shaul Philip W , Mineo Chieko
Published: May 2022

Introduction:
HDL has direct atheroprotective actions on endothelial cells (EC) that include the attenuation of vascular inflammation and promotion of endothelial monolayer integrity. These processes are mediated by scavenger receptor class B, type 1 (SR-BI) and its adaptor protein PDZK1. We have identified the breakpoint cluster region protein (BCR) as a PDZK1 interacting protein in EC. Best known as a gene in the BCR-ABL complex associated with the Philadelphia chromosome, BCR structure predicts both adaptor protein and kinase functions.

Hypothesis:
Via interaction with PDZK1 and kinase activity, BCR mediates the atheroprotective actions of HDL on EC.

Methods:
Newly-developed kinase assays that employ time-resolved Forster resonance energy transfer were used to detect kinase activity and identify kinase substrates. In vivo studies were done in ApoAI
-/-
versus ApoAI
-/-
;BCR
-/-
mice administered control AAV or AAV-ApoAI. Leukocyte-EC interaction and carotid artery reendothelialization were evaluated by intravital microscopy and thermal injury, respectively. Atherosclerosis was invoked using AAV8-PCSK9. BCR expression was compared in human atherosclerotic and normal arteries in two independent publicly available patient cohorts.

Results:
In cultured EC, HDL activation of eNOS, blunting of EC-monocyte adhesion, and promotion of EC migration all required BCR, and reconstitution studies with BCR mutants revealed requirements for both BCR-PDZK1 interaction and kinase activity. In response to HDL, BCR directly phosphorylated Akt-Ser473 and it provoked Akt-Thr308 phosphorylation by PDK1. ApoAI restoration in vivo in ApoAI
-/-
mice prompted SR-BI-PDZK1-BCR protein complex formation and BCR phosphorylation of Akt-Ser473 in mouse aorta. It also blunted leukocyte-endothelial cell adhesion and enhanced carotid artery reendothelialization in BCR
+/+
, but not in BCR
-/-
mice. Furthermore, the atheroprotective effect of ApoAI restoration was demonstrable in BCR
+/+
, but not in BCR
-/-
mice. In both human cohorts BCR expression was decreased in atherosclerotic versus normal arteries.

Conclusion:
BCR function as a novel direct and indirect kinase of Akt mediates the atheroprotective actions of HDL in vascular endothelium.