bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 108: Mediator 1 Regulation Of The BMP / TGF-β Signaling In Endothelium: Implications For Pulmonary Hypertension
Authors: Wang Chen , Xing Yuanming , Zhang Jiao , He Ming , Yuan Zuyi , Yuan Jason X.-J. , Shyy John Y
Published: November 2022

Rationale:
Dysregulated bone morphogenetic protein (BMP) or transforming growth factor β (TGF-β) signaling pathways are imperative in idiopathic and familial pulmonary arterial hypertension (PAH) as well as experimental pulmonary hypertension (PH) in rodent models. Mediator 1 (MED1) is a key transcriptional coactivator and Krüppel-like factor 4 (KLF4) is a master transcription factor in endothelium. However, MED1 and KLF4 epigenetic and transcriptional regulations of the BMP/TGF-β axes in pulmonary endothelium and their dysregulations leading to PAH remain elusive.

Objectives:
We investigate the MED1/KLF4 co-regulation of the BMP/TGF-β axes in endothelium by studying the epigenetic regulation of BMP receptor type II (BMPR2), ETS-related gene (ERG), and TGF-β receptor 2 (TGFBR2) and their involvement in the PH.

Methods:
High throughput screening involving data from RNA-seq, MED1 ChIP-seq, H3K27ac ChIP-seq, KLF4 ATAC-seq, and high-throughput chromosome conformation capture (HiC) together with in silico computations were used to explore the epigenetic and transcriptional regulation of BMPR2, ERG, and TGFBR2 by MED1 and KLF4. In vitro experiments with cultured pulmonary arterial endothelial cells (PAECs) and bulk assays were used to validate results from these in silico analyses. Lung tissue from patients with idiopathic pulmonary arterial hypertension (IPAH), animals with experimental PH, and mice with endothelial ablation of MED1 (EC-MED1
-/-
) were used to study the PH-protective effect of MED1.

Results:
Levels of MED1 were decreased in lung tissue or PAECs from IPAH patients and rodent PH models. Mechanistically, MED1 acted synergistically with KLF4 to transactivate BMPR2, ERG, and TGFBR2 via chromatin remodeling and enhancer-promoter interactions. EC-MED1
-/-
mice showed PH susceptibility. In contrast, MED1 overexpression mitigated the PH phenotype in rodents.

Conclusions:
A homeostatic regulation of BMPR2, ERG, and TGFBR2 in ECs by MED1 synergistic with KLF4 is essential for normal function of the pulmonary endothelium. Dysregulation of MED1 and the resulting impairment of the BMP/TGF-β signaling can lead to the development of PAH/PH.
Abstract 110: The Role Of Sphingosine-1-Phosphate In The Reverse Transendothelial Migration Of Aortic Intimal Myeloid Cells
Authors: Polenz Chanele K , Scipione Corey A , Hyduk Sharon , Cybulsky Myron I
Published: November 2022

CD11c
+
myeloid cells (MCs) reside in the normal aortic arch intima at regions predisposed to atherosclerosis. We showed that reverse transendothelial migration (RTM) of these intimal MCs into the arterial circulation is a protective immune response. RTM is triggered by systemic stimulation of cytokine and toll-like receptors (TLRs), e.g., TLR4 by lipopolysaccharide (LPS), and is inhibited by hypercholesterolemia and associated intimal MC lipid accumulation. We hypothesize that signaling by sphingosine-1-phosphate (S1P) and its receptors (S1PRs) is required for RTM of intimal MCs following systemic stimulation of TLRs, analogous to its role in immune cell trafficking in lymphoid tissues. In the lesser curvature of the aortic arch of wild type C57BL/6 mice, LPS induced the expression of S1P receptors 1 & 3, and sphingosine kinase 1 (SphK1). FTY720, a broad-spectrum S1P receptor modulator, blocked LPS-induced RTM, as determined by the enumeration of CD11c+ MCs by
en face
immunoconfocal microscopy. S1PR1 or S1PR3 inhibitors blocked RTM in a partial and additive manner, suggesting a role for both S1P receptors in intimal MC migration. Initial experiments show that Cre-mediated deletion of S1PR1 in myeloid cells partially blocks LPS-induced RTM, suggesting that myeloid-specific S1PR1 signaling is implicated in the migratory process. Furthermore, global loss or hematopoietic deficiency of SPHK1 reduced the abundance of CD11c+ MCs in the aortic intima, suggesting an additional role for myeloid S1P production in the homeostatic maintenance of resident aortic immune cells. Cre-mediated deletion of S1PR1 in endothelial cells also reduced the abundance of intimal MCs, providing further evidence that S1P signaling in the intima is important in maintaining the intimal MC population. Thus, functional and expression studies suggest that S1P signaling plays a role in both the maintenance of resident intimal MCs and LPS-induced RTM.
Abstract 111: Multiparametric Circulating Cardiovascular Biomarkers Elucidate A Molecular Signature Of Coronavirus Disease 2019 Mortality And Identify Vascular Barrier Stabilizing Therapies
Authors: Gustafson Dakota , Ngai Michelle , Wu Ruilin , Hou Huayun , Erice Clara , Wilson Michael , Kain Kevin , Hanneman Kate , Thavendiranathan Paaladinesh , Fish Jason , Howe Kathryn
Published: November 2022

Background:
Endothelial cell (EC) activation, endotheliitis, vascular permeability, and thrombosis have been observed in patients with severe COVID-19, indicating that the vasculature is affected during the acute stages of SARS-CoV-2 infection. It remains unknown whether circulating vascular markers are sufficient to predict clinical outcomes, unique to COVID-19 related pathology, and if vascular permeability can be therapeutically targeted.

Methods:
This is a secondary analysis of a prospectively recruited longitudinal multicenter cohort study enrolling 241 patients with suspected SARS-CoV-2 infection. The prevalence of circulating inflammatory, cardiac, EC activation, and the entirety of the microRNA transcriptome was evaluated, and the prognostic value assessed using a Random Forest model machine learning approach.
Ex vivo
experiments were performed to assess EC permeability responses to patient plasma and to derive modulated gene regulatory networks from which rational therapeutic selection could be inferred.

Results:
Multiple inflammatory and EC activation biomarkers were associated with mortality in COVID-19 patients and in severity-matched SARS-CoV-2-negative patients. In contrast, dysregulation of particular microRNAs at presentation was specific for poor COVID-19-related outcomes and revealed disease-relevant pathways. Integrating datasets (i.e., clinical, protein, and microRNA) using a machine learning approach further enhanced clinical risk prediction for in-hospital mortality. Exposure of ECs to COVID-19 patient plasma resulted in severity-specific gene expression responses and EC barrier dysfunction. ECs treated with patient plasma showed increased vascular permeability, which was ameliorated by treatment with the vascular stabilizing molecules angiopoietin-1 mimetic or recombinant Slit2-N, but not nangibotide or dexamethasone.

Conclusions:
Integration of multi-omics data identified microRNA and vascular biomarkers prognostic of in-hospital mortality in COVID-19 patients and revealed that vascular stabilizing therapies should be explored as a treatment for endothelial dysfunction in COVID-19, and other severe diseases where endothelial dysfunction has a central role in pathogenesis.
Abstract 112: Sars-cov-2 Infection Promotes Dyslipidemia And Non-alcoholic Steatohepatitis In Diet-induced Obese Golden Syrian Hamsters
Authors: Briand Francois , Sencio Valentin , SULPICE Thierry , Trottein François
Published: November 2022

Background:
Obesity, non-alcoholic steatohepatitis (NASH) and dyslipidemia have been associated with severe forms of COVID-19. To better understand the impact of SARS-CoV-2 infection on dyslipidemia and NASH, we investigated the impact of infection with the beta variant of the SARS-CoV-2 in lean and high fat/cholesterol/fructose diet-induced obese Golden Syrian hamsters.

Methods:
Lean (chow diet fed) or diet-induced obese hamsters were infected intranasally with the beta variant of the SARS-CoV-2. At baseline (before infection) and at 4-, 7-, 10- and 25-days post-infection (dpi), blood and organs were collected for biochemistry and histology analyses.

Results:
Early after SARS-CoV-2 infection, lung viral load and pulmonary inflammation were not different between lean and obese hamsters. However, compared to lean hamsters, obese hamsters showed significantly higher MCP-1 serum levels at baseline and after infection, had impaired recovery (lower resolution of lung lesions at 10 dpi, lower body weight regain at 25 dpi), and exhibited higher pulmonary fibrosis at 25 dpi. In both lean and obese hamsters, SARS-CoV-2 infection led to reduction in serum triglycerides and HDL-cholesterol levels at 4 and 7 dpi. However, obese hamsters remained hypertriglyceridemic and hypercholesterolemic, while SARS-CoV-2 infection led to significant elevation of serum free fatty acids and LDL-cholesterol levels at 4 and 7 dpi in those obese individuals, as compared to baseline levels. Despite the substantial weight loss induced by SARS-CoV-2 infection, the NASH phenotype of obese hamsters was maintained, with significantly higher liver steatosis, inflammation, hepatocyte ballooning and fibrosis scores from day 0 to 25 dpi. Additionally, obese hamsters showed significantly higher liver fibrosis at 25 dpi, as compared to lean hamsters.

Conclusion:
Our data indicate that SARS-CoV-2 infection promotes dyslipidemia and non-alcoholic steatohepatitis in diet-induced obese Golden Syrian hamsters. This model will be useful to investigate the mechanisms leading to severe forms of COVID-19 seen in obese patients with dyslipidemia and NASH.
Abstract 116: Angptl3 Regulates Hepatic Lipoprotein Production: A New Model For Lipid Lowering?
Authors: Burks Kendall H , Xie Yan , Pondugula Santhi , Neufeld Thomas , Alisio Arturo , Davidson Nicholas O , Stitziel Nathan O
Published: November 2022

Background:
Angiopoietin-like protein 3 (ANGPTL3
)
is a hepatically secreted protein and therapeutic target for reducing plasma triglyceride-rich lipoproteins and low-density lipoprotein cholesterol (LDL). Although ANGPTL3 modulates lipolytic pathways in lipoprotein metabolism, its potential intra-hepatocyte role in very low-density lipoprotein (VLDL) assembly and secretion remains unknown. Furthermore, although ANGPTL3 inhibition reduces low-density lipoprotein cholesterol (LDL), humans with complete LDL receptor (LDLR) deficiency exhibit LDLR-independent LDL lowering.

Methods:
We established hepatocyte cell culture systems, including CRISPR/Cas9-edited HepG2 cells with
ANGPTL3
“knocked out” (KO). We also generated hepatocyte-like cells from patient-derived isogenic pluripotent stem cells (iPSCs) with and without ANGPTL3 KO. Radiolabeling was used to quantify apolipoprotein and triglyceride synthesis. Negative stain electron microscopy (NS-EM) was used to quantify the size of secreted lipoproteins.

Results:
We observed reduction of both apolipoprotein B100 and triglyceride synthesis and secretion in ANGPTL3 KO HepG2 cells. NS-EM demonstrated ANGPTL3-deficient HepG2 cells secrete significantly smaller lipoprotein particles compared to WT control cells. Together, these three findings suggest ANGPTL3 deficiency in hepatocytes results in the production of fewer, smaller, underlipidated lipoprotein particles.

Conclusion:
Our results point to an important intracellular role for ANGPTL3 within hepatocytes, suggesting that ANGPTL3 modifies the assembly and remodeling, as well as clearance, of hepatically secreted lipoproteins. Further experiments are planned to identify and characterize mechanistic mediators of this process. These studies have potential to identify additional targets for reducing plasma lipids via LDLR-independent pathways.
Abstract 121: Acetylsalicylic Acid Accelerates Murine Stasis Venous Thrombus Resolution, Independent Of Sex
Authors: Vijay Aatira , Kawamura Yoichiro , Kassab Mohamad B , Mauskapf Adam , Ha Khanh Q , McCarthy Jason , McCrae Keith R , Henke Peter , Jaffer Farouc A
Published: November 2022

Background:
The lack of a stasis venous thrombosis (VT) model in female mice has limited investigation on sex differences of anti-VT therapies, such as acetylsalicylic acid (ASA), which appears efficacious in preventing recurrent VT. Here we (i) develop a new extended stasis VT model; and (ii) examine the effects of ASA on stasis VT resolution in female and male mice using serial intravital microscopy.

Method:
To extend stasis thrombus persistence, femoral vein was ligated followed by second ligation of saphenous vein (Fig. A), after 7 days of either PBS/ASA (3mg/kg, n=5-6) pre-treatment. Following FITC-dextran injection (10 mg/kg), FITC light irradiation (475/35nm, 20x) for three minutes across three illumination fields generated stasis VT. Dylight649-GPIbβ agent was injected (100ug/kg, ex 630nm) to observe platelets. At T30’, fibrin targeting agent-FTP11-CyAm7 (150nmol/kg, ex 750 nm) was injected. Serial epifluorescence IVM images were acquired (90i, NIS Elements software, Nikon).

Results:
Double ligation (DL) thrombus was significantly larger than single ligation (SL) and remained so through 24h (Fig B-G) and 48h (data not shown). Corresponding sections of thrombus showed a persistent core consisting platelets and fibrin (Fig. H-J). In DL, females developed reproducible stasis VT burden and resolution profiles as in males (Fig. K). ASA pre-treatment resulted in reduced thrombus (T0’), and accelerated resolution in both sexes (Fig. L-P). ASA inhibited platelet recruitment to thrombi within T30’, consistent with its known anti-platelet effect. Also, ASA inhibited fibrinogenesis, suggestive of an impaired platelet-fibrin interface.

Conclusion:
Here we establish a new extended stasis VT model applicable to both female and male mice. This model is amenable to study vein wall fibrosis-critical aspect of post-thrombotic syndrome. Furthermore, ASA inhibits thrombus formation and accelerates thrombus resolution in a sex-independent manner.
Abstract 124: Regulatory T Cells Suppress Aortic Aneurysm Growth In Mice Through Local Tissue Changes And Lymph Node Colonization
Authors: Lopez Jose L , Lin Pei-Yu , Shaligram Sonali , Huang April , Hadley Pierce , Tang Qizhi , Oskowitz Adam Z
Published: November 2022

Previous studies have shown that adoptive cell transfer (ACT) of regulatory T cells (Tregs) mitigate AAA growth in mice, however these studies have used a dissection AAA model and have not thoroughly studied Treg function in mice after ACT. Treg suppression of systemic or local inflammation has not been rigorously studied in our model. We therefore sought to evaluate the effects of Treg ACT in mice with a specific focus on Treg migration, colonization, and inflammatory changes within local tissue.

Methods:
AAA was induced in
B57Bl/6 mice
expressing the
Thy 1.2
allele using a topical elastase + BAPN model (E-BAPN). Heat inactivated elastase (HiE-BAPN) was used as a control. On postoperative day (POD) 2, animals were treated with PBS (E-BAPN-PBS) injection or ACT of 2 million Tregs (E-BAPN-Tregs). CD4+ CD25+ FoxP3+ Tregs used for ACT were isolated from
B57Bl/6 mice
expressing the
Thy 1.1
allele. Mice underwent surveillance ultrasounds (US) weekly to track AAA growth for 42 days. At specific time points mice were sacrificed and aortic diameter was recorded. Aortic tissue and regional lymph nodes were harvested for analysis.

Results:
Maximum aortic diameter (MAD) by both US and video microscopy was significantly higher in the AAA group (E-BAPN vs. HiE-BAPN; p<.001) with changes observed by POD 7. Mice treated with ACT had significantly reduced MAD (E-BAPN-Tregs vs. E-BAPN-PBS; p<.05) with significant changes identified by POD 21.
Thy1.1+
Tregs were detected in the tissue and regional aortic lymph nodes (LN) until day 42 with only a slight declination in the percent
Thy1.1
+ cells over time.
Thy1.1+
Tregs retained phenotypic markers over time. E-BAPN-Treg mice had significantly altered inflammatory markers compared to E-BAPN-PBS mice including local macrophage and T-cell composition as well as inflammatory cytokines.

Conclusion:
Treg ACT suppress AAA growth in a topical elastase + BAPN mouse model through tissue and LN colonization and alteration of the local inflammatory milieu.
Abstract 126: Susceptibility To Social Stress Modulates Hematopoiesis And Impairs Atherosclerosis Resolution
Authors: TUFANLI KIRECCIBASI Ozlem , Scolaro Bianca , Weinstock Ada , Torres Berrio Angelica , Parise Eric , Cathomas Flurin , Chan Kenny , Nestler Eric , Russo Scott , Fisher Edward
Published: November 2022

Background:
Depressive disorders frequently involve prolonged stress and are associated with increased risk of cardiovascular disease (CVD). A major hurdle in gaining mechanistic insight is the paucity of animal models that accurately recapitulate the symptoms of depression disorders. The major question we investigated was whether psychological stress impairs the benefits of the common therapy used to reduce CV risk, namely, lipid lowering.

Methods:
We employed a standard protocol of repeated social defeat stress (RSDS) that elicits a depression-like phenotype in mice to test the effects on atherosclerosis resolution. LDLr-deficient mice were kept on an atherogenic diet for 24 wks. Then RSDS was applied for 10 days. Mice that developed a depressive-like phenotype, termed stress susceptible (SS), were identified with behavior testing. SS and control mice were then placed on cholesterol-reducing therapy.

Results:
SS mice vs. control mice had expansion of bone marrow (BM) myeloid progenitors (~80% increase for GMP cells; P<0.001) and more circulating monocytes (~2X;
P
<0.05), which exhibited higher recruitment (2.5X;
P
<0.001) to atherosclerotic plaques. This resulted in increased (~50%;
P
<0.05) plaque macrophage content after 3 wks of cholesterol lowering. To investigate whether the effects of RSDS on inflammation and atherosclerosis were mediated by re-programming of the BM progenitors of plaque macrophages, we performed BM transplantation from control or SS mice into LDLr-deficient mice. After the recovery period, the mice were fed an atherogenic diet for 24 wks. At that time, we found that compared to control BM recipients, SS BM recipients sustained the increase in myeloid progenitors (~16% increase, P<0.05 for GMP population) and circulating inflammatory monocytes (50% increase;
P
<0.001), as well as enhanced atherosclerotic plaque inflammation (23% more macrophage content; P<0.001).

Conclusions:
Social stress impairs atherosclerosis inflammation resolution after cholesterol lowering, apparently by re-programming BM precursors of plaque macrophages. By applying molecular methods to this model, we hope to further understand the mechanistic basis for the increase in CVD risk in stressed people despite risk reducing therapies.
Abstract 129: The Epigenomic Landscape Of Single Vascular Cells Reflects Developmental Origin And Identifies Disease Risk Loci
Authors: Weldy Chad S , Cheng Paul P , Pedroza Albert J , Dalal Alex R , Sharma Disha , Kim Hyun-Jung , Shi Huitong , Nguyen Trieu , Kundu Ramendra K , Fischbein Michael P , Quertermous Thomas
Published: November 2022

Differential susceptibility to atherosclerosis across vascular beds has been described for a half century, yet the biological mechanisms remain poorly understood. Vascular cells have distinct embryonic lineage, and this diversity is believed to play a role in vascular bed specific disease. Chromatin accessibility is cell type specific, but cell type specific enhancers between vascular beds has not been described at single cell resolution. Here, we performed single cell ATAC sequencing on adult 16-week-old C57Bl/6 mice in vascular tissue from three locations: 1) aortic root and ascending aorta, 2) brachiocephalic and carotid artery, and 3) descending thoracic aorta. These regions reflect embryonic secondary heart field and neural crest, neural crest, and somitic mesoderm origin, respectively. We profiled 22,773 cells with roughly 7,500 cells per anatomic location. Importantly, clustering analysis revealed the epigenomic profiles of smooth muscle cells (SMC) and fibroblasts to differ based on anatomic location. We identified SMC clusters which are distinct to anatomic location (ascending, carotid, carotid2, descending), whereas 2 SMC clusters are nonspecific containing cells from all locations. A similar observation was made for fibroblasts, whereas endothelial and macrophage cell populations appear largely uniform. Analysis of differentially accessible regions across SMC clusters (ascending vs descending) identified developmental genes which had greater accessibility within the ascending aorta (ex.
Hand2, Tbx20, Gata4
) and those with greater accessibility within the descending aorta (ex.
Hoxb3-7, Hoxa3, Hoxc5, Sema3a
). Similar genes were identified for differentiating fibroblasts from ascending and descending aorta. Cross referencing top SMC gene hits which differentiate anatomic location against existing CAD GWAS dataset revealed an over-representation of genes which reside near replicated CAD loci in the human genome, including
TBX20, CD109, SIPA1L3, FGF5, BRD2, MAP3K7CL
, and
DST
. These data provide insights into the epigenetic biology of the vasculature, reveal an epigenetic ‘memory’ of developmental program, and support the hypothesis that epigenetic landscape contributes to differential susceptibility to disease.
Abstract 131: Plasminogen Oxidized Phospholipid Adducts Drive Macrophage Activation In Through TLR2
Authors: Michell Danielle L , Massick Clark , Raby Chase , Cavnar Ashley , Allen Ryan , Smith Rachael , Gibson Breanne , Moore-Lotridge Stephanie , zhang Youmin , Ramirez Marisol , Linton MacRae F , Sheng Quanhu , Schoenecker Jonathan , Vickers Kasey C
Published: November 2022

Oxidized phospholipids (OxPL) are potent proinflammatory mediators that can trigger toll-like receptor 2 (TLR2) signaling and inflammasome activation in macrophages. Plasminogen (PLG), a key enzyme in the fibrinolytic pathway, has been shown to harbor OxPL adducts. We hypothesized that PLG can drive inflammasome activity in macrophages through OxPL activation of TLR2 in a fibrin-independent mechanism. To assess PLG-induced macrophage activation, total RNA sequencing was used to quantify gene changes in response to human PLG treatments, and we observed robust induction of pro-inflammatory genes, including
Il1b, Il6, Nos2, Il12
, and
Nlrp3
in primary mouse macrophages. In addition, a 400-fold increase in IL-6 protein was observed in the media. To determine if this activation is specifically via TLR2, a HEK293-NF-kB reporter system was used to screen PLG-induced activation of human TLRs, and we found that human PLG significantly-induced NF-kB signaling and transactivation in reporter cells expressing TLR2, TL1/2, TLR2/6, but failed to activate TLRs1/3/4/5/6/9. PLG activation of TLR2 was found to be blocked by pre-treatment of C29, a small molecule inhibitor of TLR2. In addition, when
Tlr2-/-
primary macrophages were treated with human PLG a 30% reduction in
Il1b, Il6
and
Tnf
gene (mRNA) expression was observed compared to wild-type controls. Furthermore, we found PLG-TLR2 activation to be fibrin-independent, as there was no reduction in PLG activation by the lysine analogs tranexamic acid and e-aminocaproic acid or the plasmin activity inhibitor - VPLCK in our HEK293-NF-kB reporter system. To characterize the effect of PLG on inflammation
in vivo
, wild-type and
Plg-/-
mice were injected with AAV8-PCSK9 and treated with an atherogenic diet for 16 weeks. Remarkably, the
Plg-/-
mice demonstrated reduced plaque mass and aortic lipid content compared to wild-type control mice, with a 40% reduction in
en face
Sudan IV staining and a 10% reduction in lesion area by Oil-Red-O staining of neutral lipids. Collectively these results support that PLG-OxPL likely activate inflammasomes in macrophages through TLR2 and suggest this is likely to be involved in atherogenesis
in vivo
.
Abstract 132: VLX-1005 Effectively Treats Heparin-induced Thrombocytopenia (HIT) Without Increasing The Risk For Bleeding
Authors: Putzbach Victoria , Adili Reheman , Michael James , Maloney David J , McKenzie Steven E , Holinstat Michael A
Published: November 2022

Heparin-induced thrombocytopenia (HIT) is a rare, but deadly disease that occurs in a small percentage of patients following administration of heparin. In these patients, heparin interacts with platelet factor 4 (PF4) resulting in an immune response to the heparin/PF4 complex. This immune complex can then bind to the immune receptor on the platelet, FcγRIIa, to induce platelet activation, clot formation, thrombosis, consumption, and bleeding. This potentially deadly disease results in both a thrombotic event as well as bleeding. Currently, there is only one FDA-approved intervention for HIT, the direct thrombin inhibitor argatroban. However, many patients on argatroban remain at risk for thrombosis, bleeding, and in some cases death. Therefore, we sought to develop a new drug to intervene in patients with HIT by targeting the 12-lipoxygenase enzyme in the platelet which regulates FcγRIIa activity in the platelet. Using mouse models of HIT as well as ex vivo administration of the 12-lipoxygenase inhibitor VLX-1005 (previously known as ML355). In mice expressing the human immune receptor on their platelets, we were able to show that administration of VLX-1005 following induction of HIT in these mice resulted in a blunted thrombocytopenia as well as reduced platelet activation and thrombus formation in the blood. We further demonstrated that coagulation was not impacted by VLX-1005 using thromboelestography while argatroban significantly delayed onset of coagulation and clot formation. Furthermore, bleeding time in these mice was not altered with VLX-1005, while mice on argatroban required cauterization of their tails to stop the bleeding. Finally, Human whole blood was shown in whole blood aggregometry as well as high shear arterial flow chamber experiments to be protected from platelet activation and clot formation in the presence of VLX-1005. The studies presented here demonstrate the potential effectiveness of VLX-1005 in intervention of platelet activation, clot formation, and thrombosis in both mouse models and human blood and support VLX-1005 as a new class of drug for the treatment of HIT in patients without the risk of bleeding.
Abstract 138: Sex Differences In Limb Ischemia Recovery Following Conditional Endothelial Overexpression Of Cept1
Authors: Meade Rodrigo , Engel Connor , Belaygorod Larisa , Yin LI , Semenkovich Clay F , Zayed Mohamed A
Published: November 2022

Objectives:
Choline enthanolamine phosphotransferase 1 (
CEPT1
), is essential for endothelial
de novo
lipogenesis (DNL) and is elevated in diseased peripheral arterial segments. Women are more sensitive to diet-induced DNL, and those with diabetes have higher incidence of severe peripheral arterial disease (PAD). We therefore hypothesized that women may have higher
CEPT1
content in diseased peripheral arteries and that murine conditional endothelial overexpression of
CEPT1
may lead to sex-specific differences in ischemic recovery.

Methods:
CEPT1
expression was evaluated in diseased peripheral plaque of 7 women and 9 men. A murine endothelial
CEPT1
overexpression model was engineered with Cre-induced expression of a
CEPT1
transgene inserted in a C57BL/6J background. Endothelial
CEPT1
overexpression was evaluated using RT-PCR. Male (n=5) and female (n=5) received Streptozotocin and unilateral femoral artery ligation. Limb perfusion, appearance, and use were then evaluated. Gastrocnemius was stained with H&E, and endothelial Isolectin. Muscle fiber size and microvascular density were analyzed using Student’s t-test.

Results:
Compared to males, female patients have higher
CEPT1
in peripheral segments (n=7, p<0.005; Fig A). Murine endothelial
CEPT1
overexpression had a 7-fold increase in expression compared to control (Fig B). Compared to female mice, males demonstrated improved perfusion (p<0.05; Fig C,D), limb function (p<0.05; Fig E), limb appearance (p<0.05; Fig E), muscle fiber size (p<0.05; Fig F,G), but both has adequate muscle microvascular density (Fig F,H).

Conclusion:
This study reveals a gender difference in peripheral arterial
CEPT1
expression and ischemic recovery following overexpression. Differences do not appear to be directly associated with angiogenesis and may be due to underlying DNL metabolic consequences. These findings may have important implications for the prevalence of severe PAD in diabetic women.
Abstract 143: Il-1β Antagonism Promotes SMC Differentiation And Accumulation In Fibrous Caps In Jak2
V617f
Clonal Hematopoiesis
Authors: Fidler Trevor P , Kim Eunyoung , Xue Chenyi , Hardaway Brian , Abramowicz Sandra , Wang Nan , Reilly Muredach P , Tall Alan
Published: November 2022

Patients with JAK2
V617F
(JAK2
VF
) clonal hematopoiesis (CH) have increased JAK/STATsignaling that imparts an increased risk of premature coronary heart disease. We have shown that mice with
Jak2

VF

mutations modeling CH have increased AIM2 inflammasome activation that promotes atherosclerosis. Genetic inhibition of the inflammasome through deletion of Caspase1/11 or Gasdermin D in hematopoietic stem and progenitor cells (HSPC) harboring
Jak2

VF

mutations increased fibrous caps thickness. Therefore, we hypothesized that inhibition of IL-1β derived from
Jak2

VF

macrophages promotes smooth muscle cell (SMC) reprograming and accumulation in fibrous caps. To test this hypothesis, we transplanted bone marrow (BM) with
Jak2

VF

expression in all HSPCs into Ldlr
-/-
mice and then administered the IL-1 receptor antagonist Anakinra. After 12 weeks of Western type diet (WTD) feeding, mice treated with Anakinra had thicker fibrous caps and increased ACTA2
+
SMCs within the cap region. In atheromas scRNA-Seq has identified SMC-derived populations including modified SMCs and fibroblasts which are enriched for
Prg4
. Consistently, Anakinra treatment increased
Prg4
-expressing cells within plaques as shown by RNA scope in situ hybridization. We then modeled
Jak2

VF

CH by transplanting mixtures of 20%
Jak2

VF

BM (or 20% littermate control BM) with 80% WT BM into Ldlr
-/-
mice followed by administration of antibodies to IL-1β. After WTD feeding, IL-1β antibody treatment led to increased cap thickness specifically in mice modeling
Jak2

VF

CH. To identify how IL-1β may alter cap thickness we isolated CD11b
Negative
cells from aortas and conducted bulk-cell RNA-Seq. Gene ontology analysis revealed that IL-1β antagonism specifically in
Jak2

VF

CH mice led to the significant enrichment of 6 biological processes all of which were related to biosynthetic processes including translation, peptide biosynthetic processes, peptide metabolism and amide biosynthesis. These changes were not seen in control mice indicating that inhibition of IL-1β derived from
Jak2

VF

hematopoietic cells leads to increased translation and biosynthetic processes in non-myeloid cells potentially supporting SMC differentiation and migration and resulting in increased fibrous cap formation.
Abstract 146: Endothelial-Specific Expression Of CIDEC Improves High-Fat Diet-Induced Vascular And Metabolic Dysfunction
Authors: Balakrishnan Bijinu , Gupta Abhishek , Sharma Vishva , Slayton Mark , Gentner Kailey , Karki Shakun , Muturi Harrison , Najjar Sonia M , Loria Analia , GOKCE Noyan , Puri Vishwajeet
Published: November 2022

Cell death-inducing DNA fragmentation factor-α-like effector C (CIDEC), originally identified to be a lipid-droplet associated protein in adipocytes, positively associates with insulin sensitivity. Recently, we discovered that it is expressed abundantly in human endothelial cells (ECs) and regulates vascular function in obesity. The present study was designed to characterize the physiological effects and molecular actions of endothelial CIDEC in the control of vascular phenotype and whole-body glucose homeostasis. To achieve this, we generated a humanized mouse model expressing endothelial-specific human CIDEC (E-CIDECtg). E-CIDECtg mice exhibited protection against HFD-impaired glucose intolerance, insulin resistance, and dyslipidemia. Moreover, these mice displayed improved insulin signaling and eNOS activation, enhanced endothelium-dependent vascular relaxation, and improved vascularization of adipose tissue, skeletal muscle, and heart. Mechanistically, we identified a novel interplay of CIDEC-VEGFA-VEGFR2 that reduced VEGFA and VEGFR2 ubiquitin-mediated degradation thereby increasing VEGFR2 activation. Overall, our results demonstrate a protective role of endothelial CIDEC against obesity-induced metabolic and vascular dysfunction, in part, by modulation of VEGF signaling. These data suggest that CIDEC may be considered as a potential future therapeutic target for mitigating obesity-related cardiometabolic disease.
Abstract 150: Cytokine Signaling In Intestinal Epithelial Cells As A Regulator Of Microbiota, Inflammation And Atherosclerosis
Authors: Kurilenko Natalia , Fatkhullina Aliia , Mazitova Aleksandra , Kossenkov Andrew , Grivennikov Sergei , Koltsova Ekaterina
Published: November 2022

While microbiota regulates immunity and was suggested to play a role in atherosclerosis, the specific role of cytokine signaling in intestinal epithelial cells (IEC) in control of microbiota, inflammation, and vascular disease remains unknown. We previously showed that IL-23 enforces intestinal barrier, controls pro-atherogenic bacteria, metabolites, and activation of myeloid cells, and protects from atherosclerosis. Here we investigate the role of IL-23-regulated IL-17 signaling in IEC using newly generated

Ldlr
-/-
Il17rc
fl/fl
VillinCre

+
mice. We found that IEC-specific ablation of IL-17RC heightened subclinical intestinal inflammation, altered microbiota composition, and led to enhanced atherosclerosis development accompanied by accumulation of activated myeloid and T cells in aortas. Mechanistically, IL-17RC ablation in IEC reduced tight junction proteins as well as IgA transport to the lumen due to altered Pigr expression thereby contributing to the expansion of pro-atherogenic microbiota. RNAseq analysis revealed upregulation of multiple inflammatory pathways, including interferon and TLR signaling, while several metabolic pathways such as fatty acid oxidation and pyruvate pathways were downregulated in IL17RC
IEC

Ldlr
-/-

mice; implying an important role of IL-17R signaling in the control of IEC and microbial metabolism. Our work uncovers a novel protective role of IEC-specific IL-17 signaling in atherosclerosis and establishes a paradigm of cytokine-mediated regulation of barrier, microbiota, and metabolic pathways in cardiovascular diseases.
Abstract 153: The Differential Effects Of Weight Loss And Regain On Atherosclerosis
Authors: Weinstock Ada , Scolaro Bianca , Brown Emily J , Petitjean Marie , Nikain Cyrus A , Pena Stephanie , Garabedian Michela , Eric Rosenn , Aouadi Myriam , Fisher Edward A
Published: November 2022

Heightened inflammation in obesity is a major contributor to many diseases, including atherosclerosis and other cardiovascular diseases (CVD). Although weight loss is extremely beneficial in improving many obesity-related conditions, weight regain worsens disease outcomes, including CVD, compared to maintenance of an obese state. Nonetheless, how weight loss promotes inflammation resolution and why weight regain worsens inflammation is poorly understood and was investigated here. Using high-fat high-cholesterol diet feeding of atherogenic mice (Ldlr deficient), we promoted weight loss after the establishment of atherosclerosis and obesity by using dietary restriction, e.g. reducing daily food consumption by 30% for 2 weeks, while maintaining hyperlipidemia. For weight regain, some mice were then given free access to food for 6 weeks. Strikingly, after 2 weeks of dietary restriction we observed atherosclerosis resolution, while weight regain accelerated disease re-progression. Single-cell analysis of visceral adipose and plaque leukocytes revealed enrichment in both tissues of a novel macrophage population with dietary restriction, which is distinguished by high expression of the antibody receptor Fcgr4, a known phagocytosis mediator. Conversely, Fcgr4
+
macrophages disappeared from plaques and adipose following weight regain. Mechanistically, Fcgr4
+
macrophages from the adipose tissue induced clearance of plaque necrotic core via enhanced efferocytosis. Moreover, we discovered that weight loss and regain differentially influence immune progenitors. Bone marrow (BM) transplantation showed that while weight loss and obese BM recipients had similar plaque inflammation, weight regain BM recipients had larger, more inflammatory plaques. In conclusion, our data are the first to directly show that initial weight loss promotes resolution of atherosclerosis, independently of plasma cholesterol changes and through the enrichment of specialized pro-resolving macrophages in adipose tissue that communicate with plaques. Conversely, weight regain accelerates disease progression via inflammatory reprogramming of immune progenitors.
Abstract 155: Divergent Mature Vascular Smooth Muscle Cell Fates Are Directed By Platelet-derived Transcriptomic Codes In Aortic Aneurysms.
Authors: Pratama Muhammad Yogi , Rivera Martinez Cristobal , Silvestro Michele , Vlahos John , Zias Ariadne , Byrd Devon , Sleiman Tellaoui Rayan , Maldonado Thomas , Rhamkhelawon Bhama
Published: November 2022

Introduction:
Loss of vascular smooth-muscle cells (vSMC) and subsequent defective replenishment of functional vSMC
de novo
are pathological hallmarks of abdominal aortic aneurysm (AAA). Evidence suggests that clusters of progenitors can be reprogrammed into vSMC lineage following vascular injury. However, the landscape of vSMC precursors and reprograming signatures that determine their fate in AAA is undefined.

Hypothesis:
We hypothesize that quiescent progenitor pools reside in aorta and undergo spatiotemporal fate decision into mature vSMC in AAA. We speculate that distinct ligand-receptor and transcriptional signals gear their pathogenic trajectories into dysfunctional vSMC in AAA.

Methods & Results:
Single-cell RNA-sequencing (scRNA-seq) identified vascular progenitor cells (Cd34
+
Pdgfra
+
) in murine (angiotensin II-induced) and human AAA. Pseudotime analysis tracked two differentiation fates of progenitors into divergent mature vSMC pools characterized by enrichment in contractile cytoskeleton machinery (Acta2
+
Myh11
+
) or abnormally supplemented with matrix remodeling genes (Mmp2
+
Col8a1
+
). Mmp2
+
Col8a1
+
clusters were dominant in AAA suggesting pathological reprograming in AAA. Gene regulatory network analysis mapped distinct libraries of upstream transcription factors in each mature vSMC states in AAA. Acta2
+
Myh11
+
and Mmp2
+
Col8a1
+
transcriptomes were driven by opposing actions of Pparg and its repressor Runx1 respectively. Interestingly, cellular interaction analysis revealed that platelet-derived Pdgfb fueled the phenotypic switch into Mmp2
+
Col8a1
+
vSMC via its receptor Pdgfra. Dampening Pdgfra signaling by systemic platelet depletion repressed Runx1 activation thereby increasing Pparg transcriptomic activity in progenitors in the aortic wall. This rescued functional regeneration of vSMC (Acta2
+
Myh11
+
) with contractile properties consistent with reduced aortic damage and diminished AAA incidence.

Conclusion:
Our data demonstrate that platelets are novel actors capable of awakening progenitor pools by coding their fate into pathogenic vSMC lineage in AAA. We provide evidence suggesting that strategies aimed at sustaining vSMC contractile destiny could encourage repair of vascular damage in AAA.
Abstract 157: Role Of Systemic Platelet Levels In Venous Thrombosis And Resolution In A Murine Model Of Inferior Vena Cava Constriction
Authors: Mathews Rick , Le-Cook Anh , Setthavongsack Naly , Woltjer Randall L , Hinds Monica T , Nguyen Khanh P
Published: November 2022

Background:
Platelet role in primary clot formation in response to vessel wall injury is well established; however, the role of systemic platelet levels in deep vein thrombosis (DVT) and its sequelae is unclear. Their interaction with the venous endothelium under low shear stress, regulation of thrombosis, and mediation of both innate and adaptive immune responses through selectin and toll-like receptor expression, respectively, warrants further investigation into their role in DVT formation and resolution. In this study, we present a role for systemic platelet levels in regulating thrombus resolution.

Methods:
Wildtype, male or female CD1 mice were treated with a thrombopoietin antisense oligonucleotide (TPO-ASO) at 10 mg/kg, eltrombopag at 75 mg/ kg, saline (control), or a scrambled TPO-ASO (control) at 8 mg/ kg prior to surgical creation of a venous thrombus by ligation of the infra-renal inferior vena cava (IVC). Peak scaled thrombus weights (i.e., linear density measured using a ratio of the clot and IVC weight scaled to the length of the IVC) were measured to characterize the early resolution of the thrombus through involution and recanalization. Mice were euthanized on postoperative days 3 and 7 to measure the formation and resolution of the thrombus, respectively.

Results:
Platelet levels were elevated and decreased by over 30% by the day of surgical treatment after TPO-ASO and eltrombopag treatments, respectively. Scaled thrombus weights were significantly different (p = 0.045, Kruskal Wallis) between groups on day 7 after IVC constriction. Post-hoc analysis (Dunn Test with Benjamini-Hochberg correction) revealed that the TPO-ASO group (mean = 25.4 +/- 0.9 mg/cm) had significantly decreased (p = 0.018) scaled thrombus weights compared to the mice treated with eltrombopag (mean = 35.3 +/- 6.5 mg/cm) on day 7. Thrombus formation on day 3 was not statistically different between treatment groups.

Conclusion:
Achieving a minimum threshold of 30% reduction of systemic platelet levels by TPO-ASO enhanced thrombus resolution, but not thrombus formation, in our stasis model of rodent DVT resolution. These data suggest a potential role for systemic platelet levels in regulating thrombus resolution.
Abstract 192: Controlled Delivery Of Redox Responsive Polyurethane-polyurea Nanoparticles To Aortic Endothelium And Atherosclerotic Lesions
Authors: Han Jingyan , Zhou Yuxiang , Hou David , Bonelli Joaquin , Rocas Josep , Rocas Pau , Hamburg Naomi M
Published: November 2022

Objective:
Aortic endothelial cell dysfunction is an early trigger of atherosclerosis, the major cause of cardiovascular disease. Nanomedicines targeting vascular endothelium and lesions hold great promise as therapeutic solutions to vascular disorders. The objective of the present study is to investigate macrovascular targeting efficacy of a redox-responsive degradable polyurethane-polyurea nanocapsule (Puua-NC).

Approach and Results:
Red fluorescent lipophilic cationic indocarbocyanine dye, with DiI as cargo, was encapsulated into Puua-NCs of variable sizes (20, 175, 255 nm in diameter) at a concentration of 8.60, 6.54, and 6.91ng/mg NCs, respectively.
In vitro
cellular uptake studies with human aortic endothelial cells (HAECs) indicated these different sized Puua-NCs were efficiently taken up by cells, and the cellular uptake of NCs was positively correlated with concentration and incubation time. Exposure of HAECs to dithiothreitol (DTT, a thiol reducing reagent) or diamide (a thiol oxidizing reagent) altered intracellular thiol redox status and broke disulfide bonds on the Puua-NC shell, triggering cell disassembly and release of the encapsulated fluorescent cargo DiI, as evidenced by the loss of bright punctuate red fluorescence and appearance of the weak diffused signals of DiI. Using ApoE deficient mice fed a Western-type diet, a mouse model of atherosclerosis, we showed that intravenously administrated Puua-NCs were efficiently taken up by aortic endothelium and atherosclerotic lesions
in vivo
. Importantly, internalized Puua-NCs in vascular cells and lesions were rapidly disassembled to release DiI in response to DTT treatment.

Conclusions:
Puua-NCs demonstrate remarkable affinity to aortic endothelium and atherosclerotic lesions, with disassembly well controlled by redox status. Puua-NCs may be used as a novel redox-responsive degradable nanodevice to deliver drugs and gene materials to the cardiovascular system.
Abstract 193: Apolipoprotein A-I Rotamers Determine The Size And Cholesterol Efflux Capacity Of High-Density Lipoprotein
Authors: He Yi , Kothari Vishal M , Kramer Farah , Morris Jamie C , Kanter Jenny E , Segrest Jere , Davidson Sean , Bornfeldt Karin E , Heinecke Jay
Published: November 2022

Cardiovascular disease (CVD) risk is strongly and inversely associated with plasma levels of high-density lipoprotein cholesterol (HDL-C). Recent clinical studies suggest that the association between HDL-C and CVD risk is indirect and that HDL’s cardioprotective function may be attributed to its ability to promote cholesterol efflux from macrophages. Apolipoprotein A-I (APOA1), the major protein in HDL, serves as a structural scaffold to facilitate protein binding and enzyme activation. Our previous work demonstrated that two or three molecules of APOA1 form antiparallel dimer structures (rotamers) that wrap around discoidal and spherical HDL. Human HDL contains both LL5/5 and LL5/4 rotamers. Because little is known about the impact of different rotamers on HDL function, we generated human APOA1 mutants with single cysteine mutations that produced reconstituted HDL (rHDL) locked in specific rotamer (K133C for LL5/5 and L122C for LL5/4). The LL5/5 rotamer of rHDL demonstrated four-fold higher LCAT activation efficiency than the LL5/4 rotamer. To investigate the roles of APOA1 rotamers
in vivo
, we used a liver-targeted adeno-associated virus (AAV) to express WT hAPOA1 and the two cysteine mutants in APOA1-deficient (

Apoa1
-/-

) mice. HDL particle concentration (HDL-P) in WT and LL5/5 mice were similar but were 250% higher (n=8; P<0.0001) than those of LL5/4 mice. Importantly, mice expressing different APOA1 rotamers had markedly different HDL size patterns: HDL in LL5/4 hAPOA1 mice consisted mainly of extra-small HDL (7.8 nm in diameter), consistent with our finding that the LL5/4 rotamer had a low efficiency in activating LCAT. HDL in WT and LL5/5 mice had the same size distribution with one major peak at 10 nm. LC-MS/MS analysis identified 48 HDL proteins. One-third of the proteins (including APOA2, APOC1, APOC3 and LCAT) differed significantly in relative abundance in the different rotamers. Importantly, on a per particle basis, HDL from LL5/4 mice demonstrated three times higher ABCA1 cholesterol efflux capacity than HDL from WT and LL5/5 mice. Our observations strongly suggest that APOA1 rotamers play distinct roles in HDL metabolism, raising the possibility that differences in APOA1 rotamer distribution alter the cardioprotective functions of HDL.