bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 303: Monitoring Tissue Oxygen Dynamics With A Novel Implantable Hydrogel Sensor In Patients With Pad
Authors: Iannuzzi James C , Conte Michael S , Rebrin Kerstin , Lozano Monica M , Menke Laura , Cheng David , Smith Eric , Schneider Peter , Vartanian Shant
Published: November 2022

Objective:
Tissue perfusion measurement remains a fundamental challenge in management of peripheral arterial disease (PAD) patients. Better measurement of baseline and dynamic changes after revascularization would aid clinical judgement. The Wireless Lumee® Oxygen System represents a novel approach to tracking tissue oxygen via a subcutaneous hydrogel that provides noninvasive optical oxygen measurements. This study’s objective was to assess the correlation of the Lumee sensor with transcutaneous oximetry (tcpO2) in PAD patients.

Methods:
The Lumee® Oxygen System is a subcutaneously placed hydrogel (0.5x0.5x5mm). Optical signals are captured with a skin surface reader sensing extravascular oxygenation. A pilot study assessed in vivo performance with inclusion criteria of peripheral arterial disease (Rutherford I-IV). Sensors were injected in the arm and foot and oxygenation measured before, during, and after proximal blood pressure cuff inflation (Figure) and Buerger’s Test modulation. The correlation (R) of oxygenation between tcpO2 and Lumee® was assessed following sensor placement at 2, 10, 90, 180, and 365 days.

Results:
Eleven subjects had 148 occlusion modulations over 199 days. Mean age was 67 years (range: 55-88), with 9% females, 45% White and 55% Black Race, Rutherford class I: 36%, II: 36%, III:27%. The median ABI was 0.83 (IQR0.69-1.14). In the arm, modulation was 56.9±24.6% (Lumee) and 84.7±9.2% (tcpO2), R:0.81. In the foot, modulation was 63.4±22.7% (Lumee) and 80.4±15.5% (tcpO2), R:0.79. No injection related adverse events occurred.

Conclusion:
Occlusion related changes in tissue oxygen can be detected by both tcpO2 and the Lumee sensor which strongly correlate in both the arm and foot. This pilot phase study supports expansion of the trial. The Lumee sensor shows promise for future evaluation of tissue oxygenation in PAD patients, which could be used to assess changes in tissue oxygenation over time and following revascularization.
Abstract 326: Epigenetically Altered Diabetic Wound Plasmacytoid Dendritic Cells (pDCs) Direct Wound CD4 T-cells Towards A Th17 Phenotype
Authors: Audu Christopher O , Wolf Sonya , Melvin William , Davis Frank , Sharma Sriganesh , Mangum Kevin , Barrett Emily , Joshi Amrita , Obi Andrea T , Gallagher Katherine A
Published: November 2022

Plasmacytoid dendritic cells (pDCs) are first responders to tissue damage and interact with wound CD4 T-cells to orchestrate tissue repair. In diabetes, the role of pDCs and their subsequent influence on wound CD4 T-cells and inflammation during tissue repair is unknown. Using human diabetic wound single cell sequencing, we identified that human wound CD4 T cells are primarily the Th17 phenotype. Thus, we hypothesized that pDCs in the wounds may regulate this inflammatory T cell phenotype, contributing to chronic inflammation. Although pDCs exist in low numbers in the wounds, we isolated wound pDCs from diabetic and control mice and examined them for inflammatory cytokine expression. We found that diabetic pDC produced significantly more IL6 (amongst other inflammatory cytokines) compared to controls. This is important as IL6 has been shown to skew CD4 T-cells towards a Th17 phenotype in tissues. Upon co-culture of diabetic pDCs with naïve CD4 T-cells, a significant increase in Th17 cells was observed by intracellular flow cytometry analysis. In order to examine the increased IL6 from diabetic pDCs we isolated diabetic and control wound pDCs and performed an epigenetic superarray. We identified that histone demethylase Jarid1C was significantly decreased in diabetic pDCs compared to controls. We then isolated diabetic pDCs and performed a ChIP analysis on the IL6 NFkB binding sites in the promoter and identified an increase in tri-methylated lysine 4 on histone 3 (H3K4me3), a marker regulated by Jarid 1C and consistent with increased transcription of IL6. Taken together, this data suggests that in diabetes, pDCs are epigenetically altered to produce increased IL6 and contribute to increased Th17 and inflammation in diabetic wounds.
Abstract 327: Sex Hormones Mediate Sex Differences In Hemodynamics And Inflammation During Arteriovenous Fistula Maturation
Authors: Satam Keyuree , Ohashi Yuichi , Gonzalez Luis , Aoyagi Yukihiko , Setia Ocean , Zhang Weichang , Dardik Alan
Published: November 2022

Background:
Arteriovenous fistulae (AVF) fail to mature, that is dilate and thicken, in women more than in men, leading to inferior outcomes and decreased utilization in women. As our mouse AVF model recapitulates human AVF maturation, including decreased maturation and patency in female mice, we hypothesize that sex hormones mediate sex differences during AVF maturation.

Methods:
C57Bl/6 mice (9-11 months) were treated with sham or aortocaval AVF surgery; some were pretreated with gonadectomy 1 week prior to surgery. AVF diameter was measured via ultrasound (days 0-21). Blood was collected for FACS, and tissue examined using immunofluorescence or ELISA (days 3,7); wall thickness was assessed using histology (day 21).

Results:
At baseline, female and male mice had similar inferior vena cava (IVC) diameter (p=0.611) and wall thickness (p=0.491). After AVF creation, female mice had increased normalized IVC diameter (p=0.0012), flow velocity (p=0.0013), and shear stress (p=0.002; days 3-21; n=10); however, there was no difference in wall thickness (female, 15.8±0.2 um; male, 12.4±2.3 um; p=0.638; n=10). There were also increased circulating CD11b+ macrophages in female compared with male mice (31.8 ± 4.0% vs 18.7 ± 1.9%; p=0.001; day 3; n=22) and CD4+ T cells (57.9 ± 2.9% vs 34.5 ± 6.1%, p=0.0001). In the AVF wall there were increased CD45+ cells on immunofluorescence in females (22.5 ± 1.5 cells/hpf vs 13 ± 2 cells/hpf, p=0.063; day 3; n=4) as well as increased IL-10 immunoreactivity (3.98 ± 0.44 ng/ug vs 2.49 ± 0.47 ng/ug; p=0.076). In mice with gonadectomy, baseline IVC diameter, velocity, and shear stress were similar between female and male mice. After gonadectomy, male mice had increased AVF wall thickness than intact males (24.2 ± 1.6 um vs 12.4 ± 2.3; p=0.014); conversely, female mice had decreased thickness than intact females (6.1 ± 0.6 um vs 15.8 ± 0.2; p=0.005). There was loss of differences among circulating immune cells after gonadectomy (CD11b, p=0.0882; CD4, p=0.7095).

Conclusions:
Sex differences in hemodynamics and inflammation are present during venous remodeling, whereas these differences disappear after gonadectomy. Sex hormones mediate AVF maturation and suggest that hormone receptor signaling may be a target to improve AVF maturation.
Abstract 332: Dynamic Changes In Endothelial Cell Mechanotype During Inflammatory Endothelial-to-Mesenchymal Transition
Authors: Hsu Jeffrey J , Talapaneni Sriharsha , Ly Chau , Wong Leela , Rowat Amy , Tintut Yin , Demer Linda L
Published: November 2022

Background:
Endothelial-to-mesenchymal transition (EndMT), the process whereby endothelial cells (ECs) transition into multipotent stem cell-like cells, has been implicated in a variety of cardiovascular pathologies, including cardiovascular calcification. EndMT is a dynamic mechanical process that involves detachment of transitioning ECs from adjacent cells and active migration of these cells from the endothelium into the interstitium. While gene and protein expression profiles of ECs undergoing EndMT have been well characterized, less is known about the mechanical properties, or “mechanotype,” of ECs as they undergo EndMT.

Methods/Results:
Human aortic endothelial cells (HAEC) were treated with the inflammatory cytokine, tumor necrosis factor-α (TNF-α), to induce inflammatory EndMT. Real-time PCR analyses showed that TNF-α significantly reduced the expression of endothelial markers (
PECAM1, NOS3
) by 24 hours and significantly increased the expression of mesenchymal markers (
COL1A1, COL3A1, FN1, CNN1
) starting at 48 hours of treatment. Mechanotyping of EC was performed by evaluating cell deformability with the parallel microfiltration (PMF) technique. PMF is a novel method that determines relative deformability by filtering cells across a porous membrane. Results showed that TNF-α has a biphasic effect on EC deformability, increasing the deformability after 24 hours, while decreasing deformability after 48 hours of treatment. Deformability decreased further with 96 hours of TNF-α treatment. Transwell migration assays revealed that TNF-α increased migration of ECs through a porous membrane, whereas EC migration was not enhanced when assessed by a scratch wound assay.

Conclusion:
Our results demonstrate that ECs undergo dynamic changes in their mechanotype during inflammatory EndMT. A deeper understanding of the mechanisms governing these changes may help inform therapeutic targets for EndMT-associated disease processes.
Abstract 333: Tnik: A Novel Regulator Of Endothelial Interferon Signaling And Activation
Authors: Dominic Abishai , Wang Guangyu , Abe Junichi , Le Nhat Tu T
Published: November 2022

Background:
Endothelial cells (ECs) are prominent cell types governing vascular tone and homeostasis. Activation of ECs is the central pathophysiological process in the initiation of atherosclerosis (AS). Interferon signaling (IFN Type I and II) is activated in ECs under inflammatory conditions such as systemic lupus erythematosus and is implicated in EC activation. But the underlying mechanisms are still unknown. Through RNA sequencing and pathway analysis, we have identified TNIK, a Ste20 family of MAP kinases (MAP4K) to regulate IFN signaling in ECs. Our study is the first to report the role and function of TNIK in IFN signaling and EC activation.

Hypothesis:
TNIK regulates IFNs through STAT mediated signaling and downstream interferon stimulated gene (ISG) sets

Methods:
Cultured Human aortic endothelial cells (HAECs) were treated with control or TNIK-targeted siRNA. Later cells were subjected to static, laminar or disturbed flow conditions. Total RNA was isolated and sequenced by mRNA-seq. The differential expression data were analyzed using CLC genomics and Ingenuity Pathway Analysis (IPA). Western blotting and qRT-PCR were performed to validate STAT activation and interferon stimulated genes (ISGs)

Results:
We found >450 genes differentially expressed in siTNIK vs siCTRL treated ECs (fold change>2, FDR<0.05). IPA revealed that under static (ST) and disturbed flow (DF) conditions TNIK depletion lead to inhibition IFN and upregulation of eukaryotic transcription elongation factor signaling (eIF2). In addition, under DF, TNIK depletion lead to downregulation of hyperctokinemia/hyperchemokinemia. Validation through western blotting and qRT-PCR confirmed that TNIK regulates STAT expression and IFN stimulated target genes.

Conclusions:
Our study elucidates the unique role of TNIK in regulating STAT mediated IFN signaling and subsequent ISGs in ECs.
Abstract 359: Thrombolytics Are Associated With Improved Healthcare Utilization In Frail Patients Admitted For Ischemic Stroke
Authors: Heaton Joseph , Nanavaty Dhairya , Singh Sohrab , Ayala-Rodriguez Cesar
Published: November 2022

Background:
Frail patients tend to have higher mortality after stroke than non-frail patients. Frail patients are increasingly likely to have characteristics and comorbidities which may introduce relative contraindications to the use of thrombolytic therapy. There is limited data on outcomes after using thrombolytics in this population. We sought to assess effect of frailty on outcomes and healthcare utilization in patients receiving thrombolytic therapy for ischemic stroke.

Methods:
The National Readmission Database was queried for 2016 and 2019 for non-elective admissions of adults with a primary diagnosis of ischemic stroke. Frailty was assessed using Gilbert Hospital Frailty Index and categorized according to use of thrombolytic therapy. Temporal trends in 30-day readmission rate, length of stay, and inpatient mortality were assessed.

Results:
Ischemic stroke was the primary diagnosis for 1,635,027 patients. Mean age was 70.1 years and 49.55% were female; by frailty index, 18.8% were low risk, 62.4% intermediate-risk and 18.8% were high risk. Multivariate regression for readmission after thrombolytic therapy revealed a risk reduction rate of 26% in low-risk patients (OR 0.74, 95% CI 0.67 - 0.82, p <0.001), 19% reduction in intermediate-risk (OR 0.81, 95% CI 0.78 - 0.84, p <0.001), and 11% reduction in high-risk patients (OR 0.89, 95% CI 0.88 - 0.96, p <0.001). Length of stay was increased for low-risk frail patients (2.85 vs 3.05 days) but decreased in intermediate-risk (4.74 vs 4.60 days) and high-risk (12.09 vs 11.99 days). Inpatient mortality increased by 1% for low and intermediate risk and 2% for high-risk patients after administration of thrombolytic therapy. Figures 1b and 1c compare trends

Conclusion:
Frailty incurs increasing burden on healthcare resources and may complicate treatment outcomes. In the setting of ischemic stroke, thrombolytic therapy was associated with improved healthcare utilization amongst frail patients.
Abstract 378: Lymphangion-chip: It Recreates Intercellular Signaling Between Lymphatic Vascular Endothelial And Mural Cells In Health And Disease
Authors: Jain Abhishek
Published: November 2022

The pathophysiology of several lymphatic diseases, such as lymphedema, depends on the function of lymphangions. Even though the signaling between two main cellular components of a lymphangion, Lymphatic Endothelial Cells (LECs) and Lymphatic Muscle Cells (LMCs), is responsible for crucial functions, there are no in vitro models that have included both cell types in a physiologically relevant environment. Here, a fabrication technique is developed to create a Lymphangion-Chip. This organ-on-chip consists of co-culture of a monolayer of endothelial lumen surrounded by multiple and uniformly thick layers of muscle cells. The platform allows construction of a wide range of luminal diameters and muscular layer thicknesses, thus providing a toolbox to create variable anatomy. In this device, the muscle cells align circumferentially while endothelial cells aligned axially under flow, as only observed in vivo in the past. This model further demonstrated a robust sensitivity of this relative alignment of the two cells as well as dynamics of cell size, density, growth, and intercellular gap with respect to the presence or absence of co-culture and mechanical forces (shear). The time-dependent decrease in the subendothelial gap strongly suggests proactive LEC-LMC signaling as LECs and LMCs grow and proliferate within the device. Further exposure to pro-inflammatory cytokines reveals that the device could produce the regulation of endothelial barrier function through the muscle cells. Finally, measurement of intracellular calcium of LECs and LMCs while exposed to step and oscillatory flow profiles with various shear amplitudes revealed the bidirectional mechanism of calcium signaling within the platform. Therefore, this technology allows researchers to include essential lymphatic vascular components in a tunable 3D microphysiological system that is suitable for use in preclinical research of lymphatic mechanobiology, inflammation, and translational outcomes.
Abstract 399: JAK2 Clonal Hematopoiesis Associates With Pulmonary Hypertension Severity
Authors: Heimlich J. Brett , Annis Jeffery S , Bick Alexander G , Brittan Evan L
Published: November 2022

Pulmonary hypertension (PH) is a heterogenous condition characterized by high pulmonary vascular resistance and elevated arterial pressures. Clonal hematopoiesis of indeterminate potential (CHIP) occurs when a hematopoietic stem cell acquires a somatic mutation that confers a proliferative advantage. CHIP is associated with a range of cardiovascular diseases. Recent work in a murine model of PH demonstrated that JAK2 CHIP causes PH via neutrophil mediated vascular remodeling. Little is known about the association between CHIP and pulmonary pressures in humans. We hypothesized that CHIP would be associated with higher pulmonary pressure and a higher prevalence of clinical PH in patients referred for echocardiography. We evaluated patients in the Vanderbilt BioVU biobank for the presence or absence of several CHIP mutations (DNMT3A, TET2, ASXL1, PPM1D, and JAK2) and correlated this finding with standard echocardiography parameters extracted from the electronic health record. PH was defined as an estimated pulmonary artery systolic pressure (ePASP) >=40mmHg. We analyzed 12,007 patients who had a reported ePASP after the date of sequencing. Of those, 49% (5,883 of 12,007) were male, with a mean age of 55+/-19.2 years. CHIP mutations were present in 477 (3.9%). The mean ePASP was 34.5+/-13.9mmHg and 32% (3,842 of 12,007) had an ePASP >= 40mmHg. The ePASP was higher among JAK2 positive patients (42.3[39.4-36.5]mmHg vs. 34.6[34.4-34.2]mmHg; p = 0.0002). This association persisted after adjusting for age, race, and sex, (Beta coefficient 4.1[1.8-6.5]mmHg, p = 0.0005). We found no association between ePASP and other CHIP mutations. The presence of a JAK2 mutation was associated with a higher overall prevalence of PH (1.5% [180 of 12,007] vs. 0.9% [108 of 12,007]; p = 0.007). In a large echocardiographic referral population, JAK2 CHIP mutations are associated with higher ePASP and a higher prevalence of PH. Treatment implications and the association between CHIP mutations and clinical outcomes in patients with PH warrants further study.
Abstract 424: An Automated Estimated Atherosclerotic Cardiovascular Disease Risk Score
Authors: Wolska Anna , Sampson Maureen , Amar Marcelo , Ueda Masako , Dunbar Richard , Soffer Daniel , Roger Veronique L , Remaley Alan T
Published: November 2022

Background:
Calculating Atherosclerotic Cardiovascular Disease (ASCVD) risk is a key step in CVD prevention, but that standard approach requires risk factor data and use of a computer.

Objective:
To develop a simplified ASCVD risk score automatically calculated by the clinical laboratory from a standard lipid panel.

Methods:
Using data from 6,027 participants (excluding those with diabetes or on lipid lowering drugs) from the National Health and Nutrition Examination Survey (NHANES), we developed regression equations to generate an eASCVD risk score that provides estimates similar to standard ASCVD score in 4 race/sex groups: Non-Hispanic Whites men (NHWM) or women (NHWW) and Black men (BM) or women (BW), using TC, HDL-C, TG, and age. The new eASCVD score was externally validated in the Atherosclerosis Risk In Communities (ARIC) (N=14,742) study and compared to the standard ASCVD risk score for its association with clinical ASCVD events (N=14,742).

Results:
The eASCVD risk score had a sensitivity of 69% and a specificity of 97.5% for identifying statin-eligible patients with at least intermediate risk by the standard risk score at a cutpoint of 7.5%/10-year. By using the sum of the presence of non-lipid risk factors (SBP>130 mmHg, blood pressure medications and smoking) to adjust the threshold of the eASCVD risk score, its overall sensitivity increased to 94%, with a specificity of 92%. For each race/gender group, the eASCVD risk score performed similarly, but the positive predictive value (PPV) for men was better (88.6% for NHWM and 88.7% for BM) than for women (79% for NHWM and 78.7% for BW). Finally, eASCVD risk score showed 90% agreement with the standard risk score in predicting CVD events in the ARIC study.

Conclusions:
The eASCVD risk score can be automatically calculated by the clinical laboratory from a standard lipid panel. It identifies the majority of statin-eligible patients and can be used as a decision aid for statin therapy.
Abstract 442: Strenuous Exercise And Polyphenol Intake Differentially Modulate Lipoproteins
Authors: Mahini Halleh , Garelnabi Mahdi O
Published: November 2022

Objective:
To investigate gene expression levels of ATP-binding cassette transporter A1 (ABCA1), proprotein convertase subtilisin/kexin type 9 (PCSK9), paraoxonase 1 (PON1), angiopoietin-like 3 (ANGPTL3), angiopoietin-like 4 (ANGPTL4), peroxisome proliferator-activated receptor gamma (PPARγ), apolipoprotein C-III (APO CIII), and apolipoprotein A-5 (APO A5) after 30 days of quercetin intake combined with exercise.

Methods:
Forty C57BL/6 mice were divided into four groups (n=10): no exercise and no quercetin (C); quercetin and no exercise (Q); exercise and no quercetin (E); and exercise combined with quercetin (EQ). All animals were fed a high-fat diet containing 1.5% cholesterol as part of a 42% fat diet without antioxidants. After 2 months the animals were sacrificed and liver tissues were analyzed for ABCA1, PCSK9, PON1, ANGPTL3, ANGPTL4, PPARγ, APO CIII, and APO A5 expression using qPCR and western blot.

Results:
After the treatment period ABCA1, PPARγ, APO A5, and ANGPTL4 mRNA expressions differentially altered with exercise and quercetin intake. The results also indicated that PCSK9 mRNA expression was up regulated in the combination group; however, this did not correlate with an increase in secreted protein. Finally, the level of PON1 protein expression was reduced in the exercise quercetin combination group compared to the control.

Conclusions:
Hepatocyte lipoprotein expression levels were influenced by quercetin consumption combined with exercise. The changes were not consistent with the perceived effects of the exercise polyphenols combination.
Abstract 447: Deficiency Of Serum Amyloid A Exacerbates Sepsis-induced Acute Lung Injury In Mice
Authors: Ji Ailing , Trumbauer Andrea , Noffsinger Victoria , Guo Ling , Wang Qian , Li Xiang-An , Debeer Frederick C , Webb Nancy , Tannock Lisa R , Starr Marlene , Waters Christopher , Shridas Preetha
Published: November 2022

Objectives:
Serum Amyloid A (SAA) is a family of proteins whose plasma levels increase > 1000-fold in acute inflammatory states such as sepsis. We and others have demonstrated that SAA plays a causal role in mouse models of atherosclerosis. However, SAA may not be a valid therapeutic target if it is needed for host defense in inflammation. Here, we investigated the role of SAA in sepsis using mice deficient in all three acute-phase SAA isoforms (TKO).

Approach and results:
SAA deficiency significantly increased mortality rates in three experimental sepsis models. Survival rates in TKO and wild-type (WT) mice were: 25% and 55% after cecal ligation and puncture (CLP; p=0.02; n=10 each strain/gender); 0% and 45% after cecal slurry injection (p<0.0001; n=9 each strain); and 55% and 90 % after lipopolysaccharide injection (p<0.0001; n=10 each strain/gender). 24-hours after CLP, there were no apparent differences in liver, heart or kidney histology between genotypes. However, TKO mice had exacerbated lung pathology, including consolidation of lung tissues and atelectasis, compared to WT mice. RNAseq analysis of lungs excised 24-hours after CLP identified 664 genes differentially expressed (404 upregulated and 260 downregulated) in TKO compared to WT (p<0.05). Some of the genes that showed profound induction in the lungs of TKO compared to the WT were
Proz, Dbp, Cxcl1, Cxcl2, Arg1
and
Ackr1
. Gene ontology analysis revealed a significant enrichment of differentially expressed genes associated with chemokine production, chemokine and cytokine-mediated signaling, neutrophil chemotaxis and neutrophil migration in TKO lung tissues compared to WT tissues (p<0.01).

Conclusions:
SAA protects mice against sepsis-induced mortality, potentially by protecting the lung from tissue damage. Thus, the risk of infection should be considered when targeting SAA to ameliorate atherosclerosis.
Abstract 452: Classification And Effect Of Correctors On ABCG8 Sitosterolemia Associated Cytosolic Mutants
Authors: Poole Brittney , Williams Kori S , Chauhan Isha , Graf Gregory A
Published: November 2022

Background:
Sitosterolemia is a rare form of Familial Hypercholesterolemia (FH) which is unique from other forms of FH due to the accumulation of phytosterols in the plasma and tissues. Sitosterolemia is autosomal recessive and caused by mutations in either the ABCG5 or ABCG8 gene. ABCG5 and ABCG8 form a heterodimer (ABCG5/ABCG8) that functions at the apical surface of hepatocytes and enterocytes to promote cholesterol and phytosterol excretion. Small molecule modulators classified as either correctors or potentiators have therapeutic benefit when used to treat mutants of ABCB4 (PFIC3) or ABCC7 (Cystic Fibrous) that have impaired folding, stability, or activity. There are over 40 missense mutations in ABCG5/ABCG8 which have been clinically confirmed in patients with Sitosterolemia.

Methods:
We developed a classification system for ABCG5 and ABCG8 mutations based on the underlying molecular defect for ABCG5/ABCG8 dysfunction (maturation, trafficking, activity, etc.). We used site directed mutagenesis to introduce ABCG8 missense mutations located in the N-terminal, cytosolic domain of human ABCG8. Normal and mutant ABCG5/ABCG8 constructs were transiently transfected into a human hepatocyte cell line, Huh-7, and analyzed for heterodimerization, trafficking beyond the ER, and localization at the cell surface.

Results:
Of the ABCG8 cytosolic mutants studied, R184H, L196Q, L228P, and R263Q failed to traffic beyond the ER to form a stable heterodimer (class II) while P231T, T400K, N409D, N409I, and P415H were trafficking competent.

Conclusions:
Forty four percent (4 of 9) of cytosolic, ABCG8 mutations that cause Sitosterolemia are due to failures in complex formation and trafficking beyond the ER. Fifty six (5 of 9) percent were trafficking competent, indicating a stable ABCG5/G8 complex, but loss of activity due to impaired ATP hydrolysis or substrate binding. ABCG8 mutants may be potentially restored by proteostatic regulators and/or potentiators & correctors that have shown to be effective in disease-causing mutations in other ABC transporters.
Abstract 465: Epitranscriptomic Modification Of Mir-483 Impairs Endothelial Function
Authors: Dong Jianjie , Shyy John Y , He Ming
Published: November 2022

Emerging evidence indicates that oxidative stress causes the hydroxylation of guanine (G) to generate 8-oxo-7,8-dihydro guanosine (
8
OH-G) in microRNAs (miRs), which induces the guanine-to-uracil (G-to-U) transversion and changes the miR targetomes. However, whether and how the
8
OH-G-modified miRs are involved in vascular endothelial dysfunction has never been explored. Using
8
OH-G miR crosslinking immunoprecipitation sequencing (
8
OH-G miR-CLIP-seq), we sorted
8
OH-G miRs enriched in ECs underwent ox-LDL treatment. CLIP-seq analysis revealed that
8
OH-G-miR-483 was among the most abundant
8
OH-G miR species in ECs induced by ox-LDL. Likewise, H
2
O
2
and disturbed flow induced the miR-483 to
8
OH-G miR-483 transversion in cultured ECs. In C57 mice, the level of
8
OH-G-miR-483 was ~22-fold higher in the aortic arch (AA, under atheroprone flow) than the thoracic aorta (TA, under atheroprotective flow). Transcriptomic profiling by RNA-seq followed by principal component analysis (PCA) revealed a transcriptomic segregation between ECs overexpressing miR-483 and
8
OH-G miR-483, indicating that
8
OH-G miR-483 altered the transcriptomes. Gene Ontology (GO) analysis of the 8745 differentially expressed genes (DEGs, fold change >1.5, P<0.05) indicated that miR-483 suppressed genes involved in the TGF-β signaling, inflammatory response, cellular senescence, positive regulation of cell adhesion, and activation of innate immune response. In contrast,
8
OH-G miR-483 downregulated genes were largely correlated with angiogenesis, NO biosynthetic process, EC development, EC barrier function, and cell junction maintenance. Thus, the miR-483 to
8
OH-G miR-483 transversion would impair EC function. Functionally, the monocyte adhesion was significantly increased, and nitric oxide bioavailability decreased in ECs overexpressing
8
OH-G miR-483. Collectively, this study demonstrates that redox burden incurred by cardiovascular risk factors is a culprit of the miR-483 to
8
OH-G miR-483 transversion. Such epitranscriptomic modification of miR-483 causes endothelial dysfunction via its targetome shifting.
Abstract 472: Biologic Therapy Improves Lipid-rich Necrotic Core With Concomitant Reduction In S100A8/A9 In Psoriasis: Results From A Prospective, Observational Study
Authors: Hong Christin G , Berg Alexander R , Parel Philip M , Florida Elizabeth , O'Hagan Ross , Pantoja Carla J , Li Haiou , Zhou Wunan , Sorokin Alexander V , Teague Heather , Playford Martin , Buckler Andrew , Mehta Nehal N
Published: November 2022

Introduction:
S100A8/A9 is an alarmin protein complex secreted by activated neutrophils in response to inflammation and associated with atherosclerosis and cardiovascular disease. Psoriasis is a chronic inflammatory disease linked with increased levels of S100A8/A9, elevated atherosclerotic risk, and premature cardiovascular disease. Lipid-rich necrotic core (LRNC) is a high-risk coronary plaque feature predictive of future cardiovascular events with increased prevalence in psoriasis. Whether S100A8/A9 associates with LRNC is understudied. We aimed to characterize the relationship between S100A8/A9 and LRNC pre- and post-initiation of biologic therapy in psoriasis over 1 year.

Methods:
Participants were part of an ongoing cohort study of 320 consecutive patients, of whom 125 had comprehensive measurement of S100 proteins by enzyme-linked immunosorbent assay and quantifiable coronary computed tomography angiography scans. LRNC was measured using Elucid Bioimaging software.

Results:
S100A8/A9 positively associated with neutrophils (R=0.19,
P
=0.03) and inflammatory markers GlycA (R=0.30,
P
=0.001) and hs-CRP (R=0.24,
P
=0.005). Only S100A8/A9 associated with LRNC area (β=0.19,
P
=0.03) beyond adjustment for cardiovascular risk factors, statin use, biologic therapy, and neutrophils (β=0.22,
P
=0.02) (
Figure 1A
). Over 1 year, biologic therapy reduced S100A8/A9 levels by 79% (-197 (-320-21.4) vs -42.3 (-167.2-50.5);
P
=0.04) (
Figure 1B
) and decreased LRNC area by 0.6 mm
2
(0.04 (-0.48-0.77) vs -0.56 (-1.8-0.13);
P
=0.02) (
Figure 1C
).

Conclusion:
Of the S100 family, the S100A8/A9 heterodimer uniquely associated with LRNC in fully adjusted models. Further, biologic therapy over 1 year reduced LRNC area concomitantly with S100A8/A9 in psoriasis. These findings underscore the impact of the S100 family in high-risk coronary plaque development and support the need for mechanistic studies to better elucidate these findings.
Abstract 474: Macrophage Par2 Contributes To Influenza A Virus Pathology And Is A Potential Therapeutic Target To Treat Influenza A Virus Infection.
Authors: Miles Stephen D , Gunther Randall , Tumey Lauryn , Bharathi Vanthana , Mackman Nigel , Antoniak Silvio
Published: November 2022

Proteinase-activated receptor 2 (PAR2) was shown to enhance proinflammatory toll-like receptor 3 (TLR3) responses, while dampening TLR3-dependent antiviral response in epithelial cells. Moreover,

Par2
-/-

mice exhibited improved survival after influenza A virus (IAV) infection. The objective of the study is to identify PAR2 expressing cells contributing to IAV pathology, and test if PAR2 inhibition is a potential new target to treat IAV. Infection with H1N1 IAV (PR8) was analyzed in global (

Par2
-/-

), myeloid (
Par2
fl/fl
;LysM
Cre+
), neutrophil (
Par2
fl/fl
;MRP8
Cre+
) and lung epithelial cell (EpC)
Par2
deficient (

Par2
fl/fl

;SPC
Cre+
) mice and respective controls (
Par2
+/+
and
Par2
fl/fl
). The effect of PAR2 activation on poly I:C activation of TLR3 was analyzed in bone marrow-derived macrophages (BMDM) and murine macrophage-like J774A.1 cells. Lastly, we analyzed PAR2 inhibition in wild-type (WT) mice using mouse-anti PAR2 antibody (SAM11) and small molecule PAR2 inhibitor ENMD-1068.

Par2
-/-

and
Par2
fl/fl
;LysM
Cre+
mice exhibited increased survival, reduced proinflammatory mediators and cellular infiltration in bronchoalveolar lavage fluid (BALF) 3 days post infection (dpi) compared to control mice. Reduced IAV pathology resulted in significantly better lung function (PenH, airway resistance) in

Par2
-/-

mice compared to

Par2
+/+

mice 7 dpi. Neither
Par2
fl/fl
;MRP8
Cre+
or
Par2
fl/fl
;SPC
Cre+
mice showed any survival benefit compared to

Par2
fl/fl

.
In vitro
studies showed that

Par2
-/-

BMDM produced less IL6 and IL12p40 than
Par2
+/+
BMDM after poly I:C stimulation. J774A.1 cells stimulated with PAR2 agonist and poly I:C showed an increase in NFκB activation compared to poly I:C alone. Prophylactic PAR2 inhibition with SAM11 before and daily after IAV infection, decreased BALF inflammation and lung virus load compared to control IgG treated mice 3dpi. Therapeutic PAR2 inhibition with ENMD-1068 daily from 3 dpi resulted in improved lung function (PenH) compared to vehicle treated mice 7 dpi. Global or macrophage cell but not neutrophil or lung EpC
Par2
deficiency was associated with reduced IAV-induced lung pathology and mortality. Additionally, PAR2 inhibition experiments showed PAR2 may be a therapeutic target to reduce IAV pathology.
Abstract 497: IL-19 Induces Lymphangiogenesis Through A Prox1-dependent Mechanism
Authors: Peluzzo Amanda
Published: November 2022

Introduction:
The role of lymphangiogenesis in atherogenesis is currently under-characterized and controversial. It has been suggested that lymphangiogenesis is either protective by promoting plaque regression through reverse cholesterol transport (RCT) or deleterious by promoting plaque instability through white blood cell trafficking. We have previously reported that Interleukin-19 (IL-19), an anti-inflammatory cytokine, uniquely attenuates atherosclerotic plaque progression while also being pro-angiogenic. This drives our hypothesis that one mechanism whereby IL-19 is atheroprotective is by promoting lymphangiogenesis.

Methods & Results:
IL-19 can significantly induce primary human lymphatic endothelial cell (LEC) proliferation, migration, and tube formation, all angiogenic assays, to the same degree as VEGF-C, a known mediator of lymphangiogenesis. RNA sequencing (RNAseq) analysis indicates that IL-19 induces expression of chemokines as well as several angiogenic transcription factors in LECs. Notably, it induces expression of Prox1, a master transcription factor of lymphangiogenesis, 6.2-fold. The increases seen in chemokine and transcription factor expression from RNAseq were verified through qPCR analysis. Subsequent Prox1 siRNA knockdown prevented IL-19 associated LEC proliferation and migration. We then utilized an LDL Receptor Knockout mouse model fed a high fat diet for 16-weeks with daily injections of either recombinant mouse IL-19 or saline. Preliminary results suggest an increase in lymphatic vessel presence is associated with the known decrease IL-19 causes in atherosclerotic plaque progression. Transendothelial resistance assays were performed to determine IL-19's effect on lymphatic cell permeability. Preliminary data suggests that IL-19 alters LEC resistance, indicative of permeability and possibly RCT. We will also continue to evaluate RCT and inflammatory cell trafficking via intravital microscopy.

Conclusion:
These data suggest that IL-19 increases lymphatic vessel formation, likely through a Prox1-dependent mechanism. These results have the potential to shift our understanding toward a positive view of lymphangiogenesis and attenuation of atherosclerosis.
Abstract 499: Serum Opacity Factor Therapy Alters Plasma, Erythrocyte And Tissue Cholesterol Content In The Dysfunctional High Density Lipoprotein Scarb1
-/-
Mouse Model
Authors: Gillard Baiba K , Wang Ziyi , Liu Jing , Yelamanchili Dedipya , Gotto Antonio M , Rosales Corina , Pownall Henry J
Published: November 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
-/-
) is a robust model of this phenotype having high free cholesterol (FC) bioavailability due to too many FC-rich HDL particles. Clinically, plasma LDL and HDL are quantified according to total cholesterol = FC + cholesteryl esters (CE), which likely contribute to ASCVD pathophysiology differently. Despite higher HDL, Scarb1
-/-
mice have more ASCVD on a Western diet, and increased mol% FC in ovaries, erythrocytes, heart, lung, female liver and macrophages, tissues that are associated with female infertility, impaired cell maturation, cardiac dysfunction and atherosclerosis. Bacterial serum opacity factor (SOF) reduces plasma cholesterol ~ 40% by diverting HDL-cholesterol to the hepatic LDLR.

Hypothesis:
Adeno-associated virus delivery of SOF (AAV
SOF
) normalizes plasma and tissue FC accretion and reverses the pathologies associated with Scarb1
-/-
mice.

Methods:
The lipid compositions of plasma, HDL, erythrocytes, and tissues of Scarb1
-/-
mice treated with AAV
SOF
at 12-13 weeks of age for three weeks were compared with age- and sex-matched wild type (WT) C57BL6 and Scarb1
-/-
mice.

Results:
As hypothesized, AAV
SOF
reduced plasma and HDL-FC and CE, as well as mol% FC in Scarb1
-/-
mice towards WT levels. Erythrocyte FC levels also fell, but mol% FC remained elevated. Some changes were sex-specific: AAV
SOF
reduced the elevated FC only in female livers to WT levels. AAV
SOF
reduced FC and CE in lungs of females to WT levels, but not among males; the mol% FC remained high in both sexes. In steroidogenic tissues, adrenals, ovaries and testis, AAV
SOF
treatment increased FC. Unexpectedly, in Scarb1
-/-
mice, AAV
SOF
increased mol% FC and FC in heart beyond already elevated levels.

Conclusions:
These findings support the hypothesis that plasma and HDL cholesterol levels determine tissue cholesterol levels that drive the pathologies specific to Scarb1
-/-
mice. This evolving model of the role of HDL-FC in RCT provides a rationale for human studies to determine the utility of HDL-FC bioavailability as a risk factor for ASCVD and other pathologies.
Abstract 503: Multiparameter Mass Cytometry Reveals The Unique Response Of NaïVe B Cell Cd27
-
Subset With The Increase Of T-bet And Cd38 Expression After Radiation Therapy In Thoracic Cancer Patients
Authors: Imanishi Masaki , Cheng Haizi , Kotla Sivareddy , Lin Steven , Deswal Anita , Ko Kyung , Samanthapudi Venkatasubrahman , Le Nhat-Tu , Fujiwara Keigi , Liao Zhongxing , Palaskas Nicolas L , Yusuf Syed Wamique , Nurieva Roza I , Reyes-Gibby Cielito , Yeung Sai-Ching , Amir El-ad David , Burks Jared K , Kobayashi Michihiro , Yoshimoto Momoko , Abe Jun-ichi
Published: November 2022

Cancer Radiation therapy (RT) induces cardiovascular disease (CVD) even when the heart is shealed or not irradiated, but there is a paucity of available preventive measures for RT-induced CVD. Ionizing radiation (IR) induces senescence, which was originally discovered to suppress tumorigenesis by inducing cell cycle blockade and necrosis, and positioned IR as pro-senescence cancer therapy. IR-induced senescence cells secrete cytokines, growth factors, and reactive oxygen species (ROS), becoming so called senescence associated secretory phenotype (SASP), and we hypothesize that SASP induction in immune cells cause CVD after RT. Although the involvement of DNA damage response (DDR), efferocytosis, and clonal hematopoiesis drivers (CHD) to SASP induction has been suggested, the exact mechanisms through which RT induces SASP in a specific cell type remains unclear. We characterize most of the major human immune cell lineages in a single assay using mass cytometery (CyTOF). We generated a CyTOF panel which includes antibodies against various senescence phenotype, DDR, efferocytosis, and CHD. We isolated peripheral blood mononuclear cells (PBMCs) before and 3 month after RT from 16 thoracic cancer patients. First, we found the frequency of only B cell subtype was decreased after RT. Second, we obtained 138 functional profiling subsets by unsupervised clustering with our antibody set, and found that T-bet expression was increased in the largest B cell subset of naïve B Cell (CD27
-
) Ki67
lo
CD38
lo
DNMT3a
hi
after RT, which showed the good correlation with p-p90RSK expression in the samples from pre-RT and post-RT. Lastly, the significant increase of CD38 expression in the subsets of naïve B cell (CD27
-
) and CD8
+
T cell (EMRA) was detected. These data suggest the unique response of naïve B cell (CD27-) to RT with the increase of CD38 expression, and T-bet in the subset of B Cell (CD27
-
) Ki67
lo
CD38
lo
DNMT3a
hi
, and also the potential role of p90RSK activation in IR-induced T-bet expression. T-bet plays a role in developing the age-associated B cell (ABC), and the increase of CD38 expression promotes aging-related events. Therefore, the induction of T-bet and CD38 in naïve B (CD27
-
) cell after RT supports the novel role of naïve B cell in IR-induced SASP and subsequent CVD.
Abstract 512: Mechanisms And Therapeutic Targeting Of Epsins In Regulating Liver Steatosis
Authors: Peng Qianman , Zhu Bo , Cui Kui , Chan Siu-Lung , Wang Beibei , Wong Scott , Cowan Douglas B , Cuenca Alex , Ge Jifu , Chen Hong
Published: November 2022

Background:
Chronic liver disease, such as non-alcoholic fatty liver disease (NAFLD), is one of the most prevalent diseases, with 4.5 million people diagnosed in the U.S. in 2018. If untreated, fibrosis-induced cirrhosis will eventually develop into hepatocellular carcinoma. Epsins are highly conserved endocytic adaptor proteins, participating in selected membrane receptor endocytosis. Globally disruption of both epsin 1 and 2 induced embryonic lethality by disruption of Notch signaling pathway, which has recently been recognized as a key player in metabolism. Thus, we aimed to study the role of epsins in controlling lipid metabolism and genes involved in live immunity.

Hypothesis:
We hypothesize that epsins promote the development of liver diseases, including NAFLD, particularly non-alcoholic steatohepatitis (NASH).

Methodology and Results:
Bulk RNA-seq analysis indicated that most differentially expressed genes (DEGs) enriched in lipid metabolism were down-regulated and immune function up-regulated in epsins liver DKO. RT-PCR confirmed other DEGs such as Il1b and Ccr5 expression. Compared to WT mice, epsins liver DKO mice harbor more Kupffer cells, residential macrophages in the liver that are thought to play a role in fighting infection and harmful insults. Consistent with this, further mining bulk RNA-seq data revealed that Kupffer cells/macrophages markers, including F4/80 and Cd11b, were increased in liver DKO. Moreover, the master transcription factor, Spi1 (PU.1), increased in liver DKO. Furthermore, RT-qPCR showed the upregulation of F4/80, Cd11b, and Spi1 in liver from WT and epsins DKO mice. Importantly, liver samples from epsin liver DKO mice contain more F4/80-positive cells. Single-cell RNA sequencing data suggested elevated Kupffer cells in Liver DKO mice. As Kupffer cells play a significant anti-inflammatory role and epsins depletion reduces lipid synthesis, we anticipate that epsins deficiency in liver will combat liver inflammation and damage caused by a build-up of fat in the liver leading to NASH.

Conclusion:
Epsins in liver promote lipid synthesis and suppress immune function by reducing Kupffer cell development. Targeting epsins may provide a new strategy to treat chronic liver diseases, such as cirrhosis and NASH.
Abstract 523: Lipid-associated Plasminogen Is A Ribonucleoprotein Carrier Of Extracellular Small RNAs In Atherosclerosis
Authors: Cavnar Ashley , Michell Danielle , Raby Chase , Ramirez Marisol , Zhu Wanying , Allen Ryan , Gibson Breanne , Moore-Lotridge Stephanie , Ifrim Anca , Linton MacRae F , Sheng Quanhu , Schoenecker Jonathan , Vickers Kasey C
Published: November 2022

Small RNAs (sRNA) are exchanged between cells and tissues by various lipid and protein carriers. Extracellular sRNAs regulate gene expression in recipient cells, including activation of pattern recognition receptors and inflammatory signaling pathways. Based on size-exclusion chromatography (SEC) fractionation of human plasma, a large proportion of extracellular sRNAs are detected in small lipid-associated ribonucleoproteins (50-150 kDa), a class of carriers that have been largely overlooked. Sequential fractionation approaches, mass spectrometry, enzymatic activity assays, and western blotting all support that lipid-associated human plasminogen (PLG) co-purifies with sRNAs in plasma. PLG transports approximately 10 μg total RNA/ mg protein in plasma. Depletion of PLG from plasma by lysine-sepharose resin was also found to reduce plasma miRNA levels. Based on microscale thermophoresis assays, PLG was determined to have moderate binding to single-stranded sRNAs (K
d
=1.8μM). Immune cells may be a source of sRNAs, as macrophages were found to export both host and non-host sRNAs to PLG
in vitro
. To identify PLG-associated sRNAs, sRNA sequencing was completed, and results showed that PLG host sRNA are predominantly derived from parent rRNAs (rDR), tRNAs (tDR), yRNA (yDRs), and microRNAs (miRNA). Furthermore, we found that host sRNAs on PLG are significantly altered in subjects with familial hypercholesterolemia (hetFH), including 22 miRNAs, 5 tDRs, and 10 yDRs. PLG was also found to transport many non-host microbial sRNAs derived from bacteria, fungi, and viruses in the microbiome and environment; and genome counts for many of these microbial species were significantly altered on PLG isolated from hetFH subjects. PLG was found to cause dramatic gene (mRNA) expression changes in treated-macrophages; however, it is unknown what fraction of these changes are associated with PLG-sRNA delivery. Collectively, results suggest that PLG transports high levels of extracellular sRNAs from host and non-host species that are sensitive to hypercholesterolemia and disease associated changes. Although the impact of PLG-sRNAs remains to be determined, they hold great potential as therapeutic targets for inflammation.