bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 470: Sphingosine-1-Phosphate Signaling In The Human Microvascular Endothelium During Health And Disease
Authors: SenthilKumar Gopika , Katunaric Boran , Bordas-Murphy Henry , Schulz Mary E , Freed Julie K
Published: November 2022

The loss of nitric oxide (NO)-mediated flow-induced dilation (FID) in the microvasculature, or microvascular endothelial dysfunction, is strongly linked to future cardiovascular events. Our lab has previously shown that chronic inhibition of sphingosine-1-phosphate (S1P) formation induces the change in FID mediator from the vasoprotective NO to the pro-inflammatory, pro-atherosclerotic hydrogen peroxide. The same transition is observed in arterioles from patients with coronary artery disease (CAD). Since S1P promotes NO-mediated FID, we hypothesized that formation of S1P and activation of S1P receptor 1 (S1PR1) is critical for FID in microvessels from healthy adults as opposed to arterioles from patients with CAD. Videomicroscopy was used to perform vascular function studies on human resistance arterioles (100-250μm) dissected from discarded surgical adipose tissue. The vessels were pre-constricted with endothelin-1 and changes in internal diameter in response to flow was measured. In arterioles from healthy adults, treatment with a sphingosine kinase (SpK) inhibitor for 30 minutes abolished FID (7.7±8.0% of maximal dilator capacity, n=3; mean±SEM) compared to control (73.1±8.2% n=9, p<0.05; *two-way ANOVA), whereas inhibition of S1PR1 appeared to delay the response to dilation (19.1±10.5% vs. 52.8±9.0% at 20mmHg pressure gradient, p<0.05*). In microvessels from patients with disease, both inhibition of SpK (91.8±2.8%, n=5) and S1PR1 (72.5±6.4%, n=6) did not affect FID compared to control (90.9±2.4%, n=4). Interestingly, S1PR1 expression was similar in CAD and nonCAD arterioles. Together these data suggest that formation of S1P and potentially the activation of S1PR1 is critical for FID during health, however this pathway does not contribute to FID during disease. We conclude that while arterioles can compensate for loss of this pathway in disease to maintain tissue perfusion, formation of S1P and activation of its receptors may be necessary to promote NO formation during shear and may serve as potential targets to prevent future cardiovascular disease.
Abstract 476: Kawasaki Disease, Metabolic Switch, And Endothelial Dysfunction
Authors: Han So Yun , He Ming , Shimizu Chisato , Tremoulet Adriana H , Burns Jane C , Shyy John Y
Published: November 2022

Kawasaki Disease (KD) induces endothelial dysfunction, in part, through down-regulation of miR-483. Metabolic reprogramming, including increased glycolysis and reduced oxidative phosphorylation, is associated with endothelial cell (EC) dysfunction. However, whether metabolic reprogramming is involved in KD-induced EC dysfunction is unknown. Using RNA-seq and pathway analyses, we determined that genes involved in glycolysis were upregulated by sera from acute KD patients compared with convalescent control sera. We validated by qPCR and found increased mRNA levels of 6-phosphofructo-2-kinase/fructose-2,6-biphosphatase 3 (PFKFB3), hexokinase 1 (HK1), and hexokinase 3 (HK3), as well as the decrease in glucokinase regulator (GCKR) in cultured ECs incubated with acute KD sera. Moreover, PFKFB3 protein level was elevated in ECs cultured with acute KD sera and in the intima of coronary arteries of mice with LCWE-induced KD vasculitis. Seahorse assay revealed that KD sera increased extracellular acidification rate (ECAR) and decreased oxygen consumption rate (OCR) in ECs, indicating that serum factors in acute KD patients increased glycolysis and impaired mitochondrial function. Given the presence of a miR-483 targeting site in the 3’ untranslated region (3’UTR) of PFKFB3 mRNA, we cloned a PFKFB3-3’UTR luciferase reporter (PFKFB3-Luc) to test whether PFKFB3 is regulated by miR-483. As anticipated, miR-483 overexpression downregulated luciferase activity in ECs transfected with PFKFB3-Luc. Moreover, miR-483 overexpression decreased PFKFB3 and ECAR in ECs. In contrast, anti-miR-483 increased PFKFB3 and ECAR levels, which phenocopied acute KD-induced EC metabolic reprogramming. We conclude that glycolysis in ECs is augmented during the acute phase of KD, which promotes EC dysfunction. Mechanistically, the acute KD-associated metabolic switch in ECs is regulated, at least in part, by increased PFKFB3 as a consequence of reduced level of miR-483.
Abstract 477: The Regulatory Role Of TIFA In The Noncanonical Inflammasome
Authors: Wang Wei-Ting , He Ming , Hoffman Hal , Shyy John Y
Published: November 2022

Noncanonical inflammasome is a novel form of inflammasome, in which caspase-4/5 are both the effectors and the receptors. The activation of the noncanonical inflammasome induces pyroptosis and NLRP3 inflammasome activation. Although intracellular lipopolysaccharide (LPS) is a well-known inducer of noncanonical inflammasome, the role of noncanonical inflammasome in various infectious and noninfectious diseases is still unknown. TIFA [TRAF-interacting protein with a forkhead-associated (FHA) domain] is an immune regulator that activates the NF-κB pathway and the NLRP3 inflammasome and is upregulated in the granulocytes of MIS-C patients. TIFA self-oligomerizes via the interaction between the FHA domain and phosphorylated Thr9 on adjacent TIFA molecules. Phosphorylated TIFA oligomerizes TRAF6 or caspase-1 which ultimately activates NF-κB and the NLRP3 inflammasome. We found that several inflammatory conditions induced the noncanonical inflammasome, the release of IL-1β, and pyroptosis. Mechanistically, TIFA oligomerized caspase-4 and activated caspase-4 via TIFA Thr9 phosphorylation. By analyzing COVID-19 single-cell RNA sequencing dataset, we found that the TIFA/caspase-4 pathway is upregulated in COVID-19-specific neutrophils subset. Leukocytes isolated from COVID-19 patients exhibited activated TIFA and caspase-4 p30, suggesting that the TIFA/caspase-4 pathway played a role during the onset of COVID-19. Together, our results reveal a novel mechanism of the noncanonical inflammasome and its involvement in non-bacterial infectious and sterile inflammatory diseases.
Abstract 478: Role Of FXR1 And Senescence In Intimal Hyperplasia And Vascular Biology
Authors: Corbett Cali , St. Paul Amanda , Leigh Tani , Okune Rachael , Preston Kyle , Kelemen Sheri E , Autieri Michael
Published: November 2022

Introduction:
Despite the advent of stents, intimal hyperplasia subsequent to vascular interventional procedures remains a major obstacle. Vascular smooth muscle cells (VSMC) play a critical role in the pathogenesis of intimal hyperplasia; therefore regulation of VSMC gene expression is a logical intervention point. FXR1 is a muscle-enhanced RNA binding protein and expression is increased in injured arteries. We have shown that modulation of FXR1 levels affects stability and abundance of inflammatory transcripts in VSMC, suggesting that FXR1 is a negative regulator of inflammation. This drives our hypothesis that FXR1 is involved in mitigating vascular disease and regulating inflammatory and proliferative mRNA in VSMC.

Approach/Results:
We developed a novel VSMC-specific conditional knockout mouse (FXR1
VSMC/VSMC
). In a carotid artery ligation model of intimal hyperplasia FXR1
VSMC/VSMC
mice, have significantly reduced neointima formation (p<0.001) post-ligation compared to controls. To determine the mechanism of these effects, we knocked down FXR1 in human VSMC and observed decreased proliferation (p<0.05) as well as increased beta galactosidase (p<0.05) and gamma H2AX (p<0.01) staining, indicative of senescence. Senescent cells exhibit a senescence associated secretory phenotype (SASP) with characteristic gene expression leading to increased inflammation in the tissue microenvironment. RIP-sequencing demonstrated that FXR1 interacts with transcripts involved in cell cycle control, and stability of these transcripts is decreased with FXR1 KO. qPCR analysis from FXR1 KO mouse VSMC show increased transcripts associated with senescence (p21, p16, p53) as well as increased SASP-associated mRNA. Furthermore, wild-type human VSMC cultured in conditioned media from cells transfected with FXR1 siRNA show increased SASP mRNA and increased proliferation compared with cells cultured in conditioned media from scrambled control cells.

Summary & Conclusions:
Our results are the first to suggest that in addition to destabilization of inflammatory transcripts, FXR1 may stabilize cell cycle related genes in VSMC, and absence of FXR1 leads to induction of a senescent phenotype, an increase in SASP genes, and reduction of intimal hyperplasia.
Abstract 500: Silencing Apolipoprotein C3 Prevents Diabetic Kidney Disease And Atherosclerosis In A Mouse Model Of Type 2 Diabetes
Authors: Cervantes Jocelyn , Kramer Farah , Yoshida Kazue , Mun Grace , Alpers Charles E , Mullick Adam E , Kanter Jenny E
Published: November 2022

Diabetes increases the risk of both cardiovascular disease and kidney disease. Notably, most of the excess cardiovascular risk in people with diabetes is in those with kidney disease. Apolipoprotein C3 (APOC3) is a small apolipoprotein that is elevated by insulin-insufficiency and regulates plasma triglyceride levels. To test if APOC3, and the dyslipidemia it represents, play a role in diabetic kidney disease (DKD) and associated atherosclerosis, we treated BTBR wildtype (WT) and leptin-deficient (OB; diabetic) mice with an antisense oligonucleotide (ASO) to APOC3 or a control ASO (cASO), all in the setting of human-like dyslipidemia (accomplished by administration of an ASO targeting the LDLR). APOC3 ASO treatment reduced triglycerides, triglyceride-rich lipoproteins. It prevented diabetes-accelerated atherosclerosis in the aortic sinus, brachiocephalic artery, and aorta (sinus lesion: 67926 ± 8486 μm
2
lesion in cASO-treated OB mice compared to 38589 ± 9507 μm
2
in APOC3 ASO-treated OB mice, p<0.05, n=12-15). The reduction in lesion size with APOC3 treatment was associated with fewer macrophages and reduced perilipin 2 staining (a marker for lipid droplet formation). In the kidney, diabetes resulted in a dramatic increase in glomerular hypertrophy, neutral lipid accumulation, APOC3, and APOE-accumulation, which APOC3 ASO-treatment attenuated. Furthermore, diabetes resulted in monocyte recruitment, macrophage accumulation, and macrophage lipid loading in the glomeruli, all of which was dramatically suppressed in the setting of APOC3 silencing (36% of glomerular macrophages were lipid loaded in OB cASO mice whereas only 9% were lipid loaded in APOC3-ASO treated mice, p<0.0001). The recruitment was driven by increased endothelial cell ICAM1 expression, but monocyte lipid loading may also contribute. Intriguingly, APOC3-ASO treatment reduced diabetes-associated urinary albumin creatinine ratio but did not affect it in non-diabetic mice (WT mice: 374 ± 71 μg albumin/mg creatinine, OB cASO mice: 4648 ± 1021 μg/mg and OB mice with APOC3 ASO: 2341 ± 439 μg/mg, p<0.05, n=12-14). Together, this suggests that targeting APOC3 and diabetic dyslipidemia might be beneficial for both diabetes-accelerated atherosclerosis and kidney disease.
Abstract 511: Intrinsic Self-reactive T Cells To ApoB-100 Peptide P210 In Acute Coronary Syndrome Patients Are Associated With T Effector/Memory Response
Authors: Dimayuga Paul C , Zhou Jianchang , Lio Wai Man , Zhao Xiaoning , Chernomordik Fernando , Cercek Bojan , Trac Noah , Chung Eun J , Chyu Kuang-yuh , Shah Prediman K
Published: November 2022

Background:
Antibodies to the apoB-100 peptide P210 have been reported in coronary artery disease (CAD) patients yet the nature of intrinsic self-reactive T cell response in these patients remains to be investigated. Given the reported presence of antibodies to P210 in CAD, we hypothesized that the P210 peptide would provoke T cell activation in peripheral blood mononuclear cells (PBMCs) of acute coronary syndrome (ACS) patients and would manifest as T Effector/Memory cells, previously demonstrated to correlate with atherosclerotic disease.

Methods and Results:
PBMCs from ACS patients were collected under an approved IRB and kept viable in cryo-preservation until assays. Cryo-preserved viable PBMCs from self-reported controls were purchased from a commercial vendor with their own IRB. T cell activation was assessed by stimulating PBMCs in vitro for 16 hours with P210 peptide. Negative control was no treatment and positive controls were stimulation with pooled CMV peptides or PMA/ionomycin cocktail. The Activation Induced Marker (AIM) flow cytometric assay was performed with the following antibodies: CD4, CD8, CD25, CD69, CD134, CD137, and CD154 using non-viable cells and CD14, CD16 and CD19 as dump gates. AIM+ cells were expressed as the ratio relative to no stimulation. T Effector/Memory responses were assessed after 72 hours of stimulation with P210. Negative control was no stimulation and positive control was stimulation with PMA/ionomycin. Viable T Effector/Memory cells were identified as positive for CD3, CD4, CD8, and CD45RO, but CD45RA(-), CD62L(-) and CCR7(-) by flow cytometry. T Effector/Memory cells were expressed as Response Index (negative control subtracted from P210 stimulation divided by the positive control for each sample). P210 stimulation significantly increased CD4+CD69+CD134+ (AIM+) cells in ACS (P<0.05; n=12) compared to controls (N=6). No other AIM+ cells were noted. P210 stimulation induced persistent CD8+ T Effector and T Effector Memory responses in ACS (N=13; P<0.05) compared to controls (N=14).

Conclusion:
P210 provokes self-reactive T cell activation and Effector/Memory responses in ACS patients consistent with the immunogenic role of P210 in coronary artery disease pathophysiology.
Abstract 514: High-density Lipoproteins Mediate Bidirectional Flux Of Extracellular Small RNAs In Macrophages
Authors: Castleberry Mark , Raby Chase , Allen Ryan , Michell Danielle L , Ifrim Anca M , Linton MacRae F , Vickers Kasey C
Published: November 2022

HDL are a class of dynamic particles that play critical roles in lipid metabolism, inflammation, and cell signaling. HDL are small, highly-mobile carriers of diverse molecular cargo, that include many types of host and non-host small RNAs (sRNA), as observed by sequencing. We have previously reported that HDL can accept host sRNAs from macrophages and transfer functional miRNAs to recipient endothelial cells; however, HDL’s ability to transfer sRNAs, host or non-host, to macrophages or mediate intercellular communication between immune cells are unknown. Based on preliminary results, we hypothesize that HDL mediates bidirectional flux of extracellular sRNA between macrophages. To demonstrate that HDL accepts sRNAs from macrophages and determine if macrophages have the capacity to export non-host sRNAs to HDL
in vitro
, macrophages were loaded with a synthetic microbial sRNA (ssRNA40-Cy5) and allowed to efflux to accepting HDL in the media. Macrophage HDL-sRNA acceptance was assessed by size-exclusion chromatography and fluorometry; and macrophages were found to export microbial sRNAs to HDL in a process inhibited by monensin, an ionophore and small molecule inhibitor of the endo-lysosomal pathway. Previous studies of HDL-sRNA flux required targeted candidate approaches based on prior knowledge. To overcome this barrier, sRNAs on intact native HDL particles were labeled (stained) with SYTO RNASelect dye and incubated with macrophages. This approach facilitated the quantification of all sRNAs on HDL using a standard curve and traced sRNA uptake and storage in recipient macrophages. After confirming specificity, SYTO quantification of total sRNAs showed that human HDL transport >14 μg sRNA/ mg total protein, a value far greater than previously recognized. HDL was found to transfer sRNAs to recipient macrophages based on fluorescence quantification and confocal microscopy live-cell imaging of HDL-delivered sRNAs in macrophage organelles. These results support that HDL mediates the bidirectional import and export of extracellular sRNAs from macrophages.
Abstract 515: Inhibiting Dpp4 In Senescent Vascular Smooth Muscle Cells Suppresses A Coagulation Program
Authors: Herman Allison B , Tsitsipatis Dimitrios , Anerillas Carlos , Wang Mingyi , Harris Sophia C , Munk Rachel , Mattison Julie , Martindale Jennifer L , Abdelmohsen Kotb , Sidoli Simone , Lakatta Edward G , Gorospe Myriam
Published: November 2022

In older persons, senescent vascular smooth muscle cells (VSMCs) express increased levels of senescence-associated secretory phenotype (SASP) factors such as cytokines, chemokines, adhesion molecules, matrix-remodeling enzymes, and angiogenic factors. The SASP contributes to destabilizing the plaque cap, suggesting that VSMC senescence promotes disease development. As a result of their prevalence in many diseases, major efforts are underway to develop therapies directed at senescent cells (senotherapies). Dipeptidyl Peptidase 4 (DPP4), a serine protease, was elevated on the plasma membrane of senescent fibroblasts. While the effect of DPP4 on VSMCs is not well understood, DPP4 inhibitors such as Vildagliptin are clinically used to treat diabetes, and in animal models, gliptins reduced atherosclerosis and inflammation independently of DPP4’s canonical role in glucose metabolism. We hypothesized that DPP4 inhibitors may reduce the progression and burden of atherosclerosis by preventing DPP4 enzymatic function on senescent VSMCs. Towards this end, we discovered that DPP4 protein levels and enzymatic activity increased in senescent human VSMCs (hVSMCs) senescence. We also found elevated DPP4 in human atherosclerotic plaques, and high levels of DPP4 colocalized with the senescence marker p16 in murine atherosclerosis. Strikingly, silencing DPP4 in senescent hVSMCs increased cell death and caspase activity. Proteomic analysis of conditioned media from senescent hVSMCs treated with DPP4 inhibitors revealed a reduction in numerous complement and coagulation factors, indicating that DPP4 partially governs hemostasis and the proteolytic events that promote thrombotic signaling. We propose that DPP4 inhibition suppresses the pro-coagulation/complement phenotype of senescent hVSMCs to disrupt their function and improve vascular disease progression.
Abstract 517: Diesel Exhaust Induces Gut Dysbiosis Along With Reduced Fecal Acetate Levels And Altered Lipid Metabolism
Authors: Gupta Rajat , Chang Candace , Yin Fen , Sulaiman Abdulmalek Dawoud S , Mehrabian Margarete , Jacobs Jonathan , Lusis Aldons J , Rosenfeld Michael E , Araujo Jesus A
Published: November 2022

Ambient air pollution exposure leads to increased cardiovascular morbidity and mortality. Previous studies support a causal link between particulate matter (PM) exposure and cardiometabolic effects including atherosclerosis. We have previously reported that whole diesel exhaust (DE) exposure for 16 weeks led to increased hepatic triglycerides (TG), 9- and 13-hydroxyoctadecadienoic acid (HODEs), plasma cholesterol and TG in apolipoprotein E
-/-
(ApoE) mice. Furthermore, gut microbiota composition was altered in mice exposed to ambient ultrafine particles by oral gavage for 10-weeks, that significantly associated with atherogenic lipid metabolites. Thus, we hypothesized that chronic exposure to DE leads to dysbiosis that could likely mediate oxidative and metabolic effects in the liver. Male ApoE
-/-
mice at 31 weeks of age were randomly assigned to two groups (n=10/group) exposed to either DE or filtered air (FA) for 16 weeks (6 hours/day, 5 days/week). 16S sequencing of the cecum exhibited significant differences in the abundance of bacterial taxa, as well as alpha and beta diversity indices of the DE-exposed gut microbiome
vs.
FA (p<0.05). There were also significant positive associations between these cecum bacterial species and plasma and hepatic lipids. Furthermore, DE-exposed mice had significantly elevated mRNA and protein levels of hepatic 12-lipoxygenase (12-LO). Interestingly, DE-exposed mice showed markedly reduced fecal acetate levels that significantly associated with lipids and cecal microbiome. A direct effect of short chain fatty acids on hepatocytes was observed after treatment of HepG2 cells with whole DE particles (DEP) together with acetate, which significantly inhibited 12-LO mRNA expression as compared to DEP alone. In conclusion, DE inhalation led to marked alterations in cecal microbiota and fecal acetate, which associated with liver 9- and 13-HODEs as well as plasma cholesterol and TG. Acetate may exert a protective role against the pro-oxidative effects induced by DE exposure. Additional work is required to determine the mechanisms how changes in gut microbiota could lead to pro-oxidative effects in the liver, hepatic steatosis and dyslipidemia, and whether those include the production of acetate in the intestines.
Abstract 524: Single-cell Rna Sequencing Of Early Atherosclerosis In Mice Reveals The Early Emergence Of A Dedifferentiated Smooth Muscle Cell Population
Authors: Chung Allen , Kim Eunyoung , Reilly Muredach P , Bauer Robert C
Published: November 2022

Single Cell RNA Sequencing (scRNA-seq) has proven to be a powerful technique to uncover heterogeneity from otherwise homogeneous tissues. Multiple studies have utilized scRNA-seq to examine the composition of atherosclerotic plaques in both humans and mice. However, most of these studies occurred in advanced atherosclerosis. We sought to expand this knowledge base by performing single-cell RNA sequencing at early atherosclerosis in mice. We crossed the Ldlr-/- hyperlipidemic mouse model to a smooth muscle cell (SMC) lineage tracing mouse (ROSA26:LSL-ZsGreen; MyH11-CreERT2). When treated with tamoxifen, SMCs expressing Myh11 are labeled green. At seven weeks of age, we treated these mice with five IP injections of 40mg/kg of tamoxifen. After one week of rest, we initiated the mice on a western diet. We dissected out the whole aorta at the time of harvest and digested the aorta for one hour with a cocktail of DNase, Hyaluronidase, and Liberase. Subsequently, we FACS sorted out both ZsGreen negative and ZsGreen positive cells and performed single-cell RNA seq using the 10x Genomics pipeline. We performed scRNA-seq with pools of three mice at four time points: after 1, 2, 3, and 4 weeks of western diet feeding. Clustering with CarDEC revealed three SMC populations. At four weeks of western diet, we observed an emergence of a SMC cluster characterized by increased expression of Vcam1, Dcn, Mgp, and Egr1. As expected, Gene Ontology analysis of the top 20 differentially expressed genes revealed this population to contain reduced markers of SMC differentiation. In addition, this population had reduced expression of Pura, a protein known to influence serum response factor and regulate smooth muscle actin expression. In summary, our studies indicate changes within SMCs, and the emergence of a dedifferentiated smooth muscle cell population after four weeks of western diet feeding. This study highlights that remodeling of the vasculature occurs even with short durations of hyperlipidemia. Further ongoing studies include expanding time points to longer durations of western diet and elucidating master regulators of this population with Virtual Inference of Protein activity by Enriched Regulon (VIPER).
Abstract 525: Uncovering Mechanisms Between Oligosaccharide Galactose-alpha 1,3-galactose (alpha-gal) Sensitization And Atherosclerosis
Authors: Blackburn Cassidy M , Pattarabanjird Tanyaporn , Qiao Hui , Drago Fabrizio , Marshall Melissa , Erickson Loren D , McNamara Coleen A
Published: November 2022

Recent work by our group and others have shown individuals with IgE to the oligosaccharide allergen present in mammalian products (α-gal) have increased atheroma burden with increased necrosis and calcification compared to those without α -gal specific IgE. These data suggest α -gal specific IgE increase plaque severity and vulnerability, yet the mechanisms that promote production of IgE to α -gal are unknown. Previous work from our group utilized multi-omics single cell analysis of circulating PBMCs from subjects with coronary angiography at UVA and showed subjects with IgE α -gal sensitization had a higher frequency of CCR6hi switched memory (SWM) B cells and that the CCR6 ligand, CCL20, increased class switching to IgE. To determine mechanisms whereby CCR6 may mediated class switching to IgE, we enriched total B cells from healthy donor peripheral blood mononuclear cells and stimulated with 20ng/ml human IL-4 and 10μg/ml agnostic anti-human CD40 with or without 20ng/ml CCL20 for 3 days. After 3 days of treatment, cells were collected and stained with a 15-color panel and analyzed via flow cytometry. Cells were analyzed for surface expression of CD20 and CD3 to gate total B cells. Gated B cells were further separated into CD27+/IgM-/IgD- to define SWM B cell population. CCL20 treatment did not increase the total percent of SWM B cells; however, there was a 2-3-fold increase in CCR6+ SWM B cells compared to CCR6- SWM B cells. CCL20 treated CCR6+ SWM B cells have an average of 7% increase of phosphorylated mTOR in total SWM B cells compared to nonCCL20 treated CCR6+ SWM B cells. These results suggest CCL20 stimulation induces mTOR phosphorylation and activation. Thus, mTOR activation and downstream mediators may be necessary for α -gal induced B cell class switching. From these preliminary studies, we conclude that CCL20 stimulation increases the percent pmTOR+ SWM B cells. We will continue to investigate downstream mTOR mediators in regard to B cell class switching after CCL20 stimulation and how α-gal sensitization augments B cell IgE class switching and increases atherosclerosis with our novel α -gal-/- Apoe-/- mouse.
Abstract 526: Molecular Characterization Of Sitosterolemia-associated Mutants Of ABCG5
Authors: Williams Kori S , Poole Brittney , Zelows Mikala , Chauhan Isha , Graf Gregory A
Published: November 2022

Background:
The proteostasis network is essential for the maintenance of cellular integrity by ensuring the correct folding of newly synthesized proteins. However, the high fidelity of this system contributes to diseases such as Cystic Fibrosis (CF, ABCC7), Progressive Familial Intrahepatic Cholestasis Type 3 (PFIC3, ABCB4), and Sitosterolemia (ABCG5/ABCG8). Interference with proteostasis and allowing sub-optimally folded ATP-Binding Cassette (ABC) transporters to transit to the cell surface partially restores function, with clinical benefit for CF and PFIC3. We sought to delineate the molecular underpinnings of Sitosterolemia, an autosomal recessive form of Familial Hypercholesterolemia (FH), characterized by accumulation of phytosterols in the plasma and tissues. ABCG5 ABCG8 form an obligate heterodimer at the surface of the liver and small intestine, and mediates sterol transport into bile and the intestinal lumen. Case studies reveal 57 loss of function and over 40 missense mutations of ABCG5 associated with Sitosterolemia. Recent pharmaceutics show promise with rescue of mutations in CF and PFIC3 with small molecule chaperones and potentiators. Through our analysis of ABCG5 missense mutations causing Sitosterolemia, we anticipate rescue by these compounds as well.

Methods:
We established a classification system for missense mutations of ABCG5, similar to CF and PFIC3. Mutations were generated by site-directed mutagenesis, and confirmed through Sanger sequencing. Native and mutant ABCG5 were co-transfected with native ABCG8 into human hepatocytes and evaluated for protein abundance and trafficking beyond the endoplasmic reticulum (ER), by SDS-PAGE and immunoblot analysis.

Results:
Nine mutants of ABCG5 have been generated. Co-expression of ABCG5 with ABCG8 i
n vitro
demonstrated I68N, A98G, E146Q, and R419P successfully traffic while R284S, T305R, R389H, R419H, and N437K were arrested within the proteostasis network.

Conclusions:
Similar to CF and PFIC3, Sitosterolemia-associated mutations result in compromised heterodimer formation and trafficking to the apical surface. Small molecule chaperones and potentiators may partially rescue the mutants and provide clinical benefit for Sitosterolemia.
Abstract 541: Association Of Influenza Pneumonia With Abdominal Aortic Aneurysm (aaa) Disease
Authors: Xu Baohui , Dalman Ronald L
Published: November 2022

Objectives:
Renin-angiotensin system contributes to AAA pathogenesis. The pulmonary system regulates circulating angiotensin II and 1-7 via angiotensin converting enzyme (ACE) and ACE2 activity. Pulmonary diseases including COPD and sleep apnea are associated with increased AAA risk. This study examined whether a similar association exists with influenza pneumonia (IP).

Methods:
Using a cohort discovery tool with IRB approval for informed consent waive, electronic health records from Stanford health system were retrospectively queried to identify patients ≥ 50 years of age with a history of IP or non-influenza pneumonia (NIP). After controlling for gender, race, and cigarette smoking, AAA prevalence and odds ratios (ORs) were calculated for patients with IP or NIP as a function of age and diabetes status.

Results:
We identified 935 and 6145 AAA patients in the IP (45,110) and NIP (1,993,760) cohorts, respectively. IP was associated with increased AAA prevalence regardless of sex, age, smoking, diabetic status or racial classification (excepting Native Americans,
Table 1
). After controlling for white race and male sex, AAA prevalence remained higher in IP smoking patients in most age groups regardless of diabetic status. OR for IP-associated AAA risk ranged from 1.48 to 2.31 (
Table 2
). In white male non-smokers, AAA prevalence was also higher in IP than NIP patients, particularly in non-diabetics (OR 10.10 to 228.86,
Table 3
).

Conclusions:
This study suggests that influenza pneumonia is positively associated with AAA disease prevalence in older patients. Additional study is needed to determine whether this association is causal and, ultimately, whether AAA screening and surveillance protocols should be adjusted accordingly.
Abstract 543: Inhibition Of Renin-angiotensin System Failed To Suppress Beta-aminopropionitrile-induced Thoracic Aortopathies In Mice
Authors: Sawada Hisashi , Ohno Satoko , Ye Dien , Franklin Michael , Howatt Deborah A , Moorleghen Jessica J , Lu Hong , Daugherty Alan
Published: November 2022

Objective:
Cross-linking of elastin and collagen fibers is a vital process in aortic development. Pharmacological inhibition of aortic cross-linking by β-aminopropionitrile (BAPN) leads to thoracic aortopathies, such as thoracic aortic aneurysm and dissection, in mice. Although the renin angiotensin system (RAS) contributes to several types of thoracic aortopathies, it remains unclear whether inhibition of the RAS protects against aortopathy formation caused by the impairment of elastin and collagen cross-linking in mice.

Approach and Results:
Either vehicle or losartan (15 mg/kg/d, AT1 receptor blocker) were administered through osmotic pumps for 4 weeks in male C57BL/6J mice (3-4-weeks-old, n=30/group). BAPN (0.5% wt/vol) was given through drinking water to induce aortopathies. Losartan increased plasma renin concentrations significantly compared to vehicle-infused mice, indicating effective blockade of at1 receptors. However, losartan did not suppress BAPN-induced aortic rupture and dilatation. Since losartan is a surmountable inhibitor, we also tested the effects of irbesartan, an insurmountable inhibitor of AT1 receptor blocker, on BAPN-induced aortopathy formation (50 mg/kg/day, diet, n=30/group). Despite the significant increase of plasma renin concentrations, irbesartan did not ameliorate aortic rupture and dilatation in BAPN-administered mice. Thus, BAPN-induced thoracic aortopathies were refractory to angiotensin receptor blockade. We next inhibited angiotensinogen, the unique substrate for angiotensin II, using an antisense oligonucleotide targeting angiotensinogen (AGT-ASO). AGT-ASO decreased plasma angiotensinogen concentrations, while aortic death and dilatations were not attenuated. Since hepatocytes are the major source of angiotensinogen, BAPN-induced thoracic aortopathy formation was also examined in mice with hepatocyte-specific angiotensinogen deletion. Although plasma angiotensinogen concentrations displayed 90% reduction compared to wild type littermates, aortic rupture and aneurysm formation were not suppressed.

Conclusion:
Inhibition of the RAS does not attenuate BAPN-induced thoracic aortopathy formation in mice.
Abstract 547: Chronic Nicotine Exposure Affects Induced Pluripotent Stem Cell-derived Endothelial Cell Therapy In A Murine Model Of Peripheral Artery Disease
Authors: Chan Alex H , Hu Caroline , Chiang Gladys , Huang Ngan F
Published: November 2022

Treatment strategies for peripheral artery disease (PAD) remain limited due to the many challenges of surgical intervention. Stem cell therapy offer a promising option, but barriers exist to their clinical translatability. Among these barriers include lifestyle choices like nicotine use that may influence the efficacy of stem cell therapy. The objective of this study aims to examine the efficacy of iPSC-EC therapy for treatment of PAD in mice with prior chronic exposure to nicotine. We hypothesize that chronic nicotine exposure negatively affects the therapeutic potential of implanted iPSC-ECs in a murine model of PAD. NSG mice were implanted with an osmotic pump for controlled exposed to nicotine or saline (control) for 28 days. Plasma cotinine was measured to confirm nicotine exposure. After 28 days of nicotine or saline exposure, the mice underwent induction of hindlimb ischemia and before receiving an injection of 10
6
iPSC-ECs. A second dose of cells were injected to the ischemic limb on day 7 post-surgery. All mice were monitored for blood perfusion recovery using laser Doppler spectroscopy imaging. Survival of the transplanted cells in vivo were tracked by bioluminescence imaging. The mice receiving iPSC-ECs were also imaged for cell survival using bioluminescence imaging. At day 14 post-surgery, mice were euthanized, and the gastrocnemius muscle and thigh muscle were harvested for histological analysis. The results show that the average cotinine concentration in sera was ~200 ng/mL, which is within physiological range for a tobacco user. Blood perfusion recovery at day 14 showed trends of nicotine treated groups have hindered perfusion recovery, compared to those treated with saline. Importantly, nicotine exposure trended with poorer perfusion recovery after cell therapy. Bioluminescent imaging showed iPSC-ECs persisted in vivo for all groups with a gradual decline in cell numbers over time. In conclusion, nicotine exposure was associated with poorer blood perfusion recovery after iPSC-EC therapy, compared to control saline exposure, suggesting that chronic nicotine exposure may have adverse effects on the efficacy of iPSC-EC therapy for treatment of PAD.
Abstract 549: Impact Of ST Segment Elevation On In-hospital Mortality And Stroke In Acute Aortic Syndromes
Authors: Bejjani Antoine , Khairani Candrika D , Goldhaber Samuel Z , Piazza Gregory , Campia Umberto
Published: November 2022

Background:
Acute aortic syndromes (AAS) may present with a wide variety of electrocardiographic findings. We aimed to study the prevalence of ST segment elevation on ECG in patients presenting with AAS and to assess its association with in-hospital mortality and complications.

Methods:
Retrospective analysis of all patients who presented to our tertiary care center with AAS from July 2012 to October 2020. ST segment elevation was defined as being reported on the first ECG reading upon admission.

Results:
Of 338 patients presenting with AAS, 23 (6.8%) had ST segment elevation on their initial ECG. There was no difference in age among patients who had ST segment elevation compared to those who did not (63.3 ± 15.9 vs. 68.2 ± 15.1 respectively, p = 0.135). The rate of ST segment elevation was similar between females and males (8.2% vs. 5.9% respectively, p = 0.508) and between Stanford type A and type B AAS (6.7% vs. 6.9% respectively, p = 1.000). Patients presenting with ST segment elevation had a 3-fold increase in the risk of in-hospital mortality (unadjusted OR 3.37) and a 4-fold increase in the risk of in-hospital stroke (unadjusted OR 4.27) compared to those without. After adjusting for age, sex, atrial fibrillation on initial ECG, and type of AAS, ST segment elevation remained independently associated with mortality (adjusted OR 6.06) and stroke (adjusted OR 5.33).

Conclusion:
In patients presenting with AAS, ST segment elevation on the initial ECG was independently associated with in-hospital mortality and stroke.
Abstract 551: HDL Particle Concentration And Incident Ischemic Stroke In Type 1 Diabetes
Authors: Vaisar Tomas , Miller Rachel G , Kalani Rizwan , Orchard Trevor , Costacou Tina
Published: November 2022

Recently, we showed that HDL particle concentration (HDL-P) and cholesterol efflux capacity (CEC) predict coronary artery disease (CAD) better than HDL-C in people with type 1 diabetes (T1D). Cardiovascular risk factors have been shown to associate with different cardiovascular outcomes differently. We therefore investigated the association of HDL-P and CEC with ischemic stroke (IS) incidence in people with T1D. We quantified HDL-P using calibrated differential ion mobility and total and ABCA1-specific HDL CEC in 549 participants with childhood-onset (<17 years) T1D from the Epidemiology of Diabetes Complications study using the first available blood sample. The participants were free of cerebrovascular disease at baseline (mean age 27.8 years, T1D duration 19.6 years, 49% women). Stroke incidence/type was determined by self-report of physician diagnosis and confirmed by medical record review. During the follow-up (median 25 years), there were 35 (6.5%) incident IS events. Participants with IS were more likely to be older, have a longer T1D duration, have hypertension, a history of smoking, and a worse lipid, inflammatory and renal markers’ profile at baseline; HDL-C, HDL-P, and CEC did not differ between participants with and without IS. While HDL-C, total HDL-P, and CEC were not associated with IS risk, extra-small HDL-P (xsHDL-P, ~7.8nm) subspecies were strongly associated with incident IS (HR=4.49, 95% CI: 1.72-11.74), even after adjustment for IS risk factors (including smoking, diabetes duration, hypertension as well as HDL-C), in multivariable Cox proportional hazards regression analyses. When all stroke events including hemorrhagic stroke were considered the association was weaker (n=47 events, HR=2.5, 95% CI: 0.97-6.47) suggesting specificity in the association of xsHDL-P with ischemic stroke.In contrast to a strong protective association of total HDL-P and CEC against CAD in the same T1D population, these HDL metrics were not associated with IS. However, the xsHDL-P subspecies were specifically associated with increased IS risk. These striking findings require further investigation and suggest a differential association of HDL particle metrics with CAD and IS.
Abstract 554: The Association Of Multiple Variants In The
TNXB
Gene With Vascular Aneurysms And Dissections
Authors: Neogi Arpita , Towne Meghan , Dykas Daniel , Parsa Nader , Attar Armin , FATHZADEH MOHSEN , Bale Allen , Mani Arya
Published: November 2022

Pathogenic variants in the
Tenascin-XB (TNXB)
gene are associated with autosomal recessive Ehlers Danlos Syndrome (classical-like). Here, we report the association of two or more
TNXB
variants with aneurysms and/or dissections in different arterial beds. We compared case and control data from exome sequencing (ES) or multigene panel testing (MGPT). Statistical analysis was performed by Fisher’s exact test. From 350 cases of vascular aneurysm and/or dissection referred for genetic testing by ES, we identified 5 individuals with two or more rare missense variants in the
TNXB
gene (1.4%) versus 16 individuals with two or more
TNXB
variants from 10, 968 matched controls (0.14%, p<.0.01). In a replication cohort of 1844 individuals undergoing multigene panel testing (MGPT) for connective tissue disorders including aneurysms and dissections, we identified 27 individuals with two or more
TNXB
variants (1.5%, p<0.01); 19 of these individuals (70.3%, n= 19/27) had a personal history of aneurysm and/or dissection and had no other identifiable pathogenic or likely pathogenic variants. Interestingly, 3 (15.7%, n=3/19) had a diagnosis of bicuspid aortic valve (BAV), a 10-fold higher prevalence compared to the general population incidence of BAV. An additional 158 individuals with BAV who underwent ES also showed a higher prevalence of multiple
TNXB
variants compared to controls (1.8% vs 0.14%, p<0.01). This data strongly supports an association between multiple
TNXB
variants and vascular aneurysms and dissections.
Abstract 563: Age-related Differences In Vascular Remodeling Resulting From Acute Injury
Authors: Martin-McNulty Baby , Ramachandran Mythili , Craft Wendy , Kolumam Ganesh
Published: November 2022

Background:
The vascular system, like other organ systems, ages, leading to differences in remodeling due to injury. The hallmark of acute vascular injury is the formation of intimal hyperplasia. Vascular procedures performed in the elderly, such as angioplasty, stent and other cardiovascular procedures/surgeries, result in higher rates of acute vascular injury compared to younger adults. We explored age-related differences in vascular remodeling due to acute injury using the unilateral carotid artery ligation (CAL) murine model.

Methods:
Four-month and twenty-two-month-old C57BL/6 male mice underwent ligation of the left common carotid artery immediately received either angiotensin II (1.4 mg/kg/day; Lig+Ang II) or vehicle (phosphate-buffered saline; Lig) via subcutaneous osmotic minipumps for 4 weeks.

Results:
Compared to young murine arteries, histopathology showed increased thinning and straightening of elastin in 22 mo. old arteries, as well as increasing collagen deposition in the media layer, resulting in thicker vessel walls.Old vessels were more susceptible to insults, as observed in the Lig group. In turn, the old vessel remodeled with neo-intimal formation, fragmentation of elastin and narrowing of the lumen, whilst there was no neo-intimal formation in the young arteries under the same injury.Even though Ang II increased remodeling in the young vessels with neo-intimal formation, Ang II exacerbated remodeling in the aged arteries with enough intimal hyperplasia that led to the occlusion of the lumen with a necrotic core, and the complete disintegration of the elastin. Molecular analysis showed differences between young and old arteries with and without perturbation.

Conclusions:
Together, these results indicate that vascular ageing can be detected in old arteries, but that there are age-dependent differences in vascular remodeling following acute injury revealed through pathological and molecular analysis. Thus, this study has provided an invaluable tool for understanding the mechanisms of neo-intimal formation in restenosis to elderly patients following angioplasty and vein grafts. It is also a means for pre-clinical pharmacological interventions.
Cannabinoid Receptor 2-Centric Molecular Feedback Loop Drives Necroptosis in Diabetic Heart Injuries
Authors: Gao Pan , Cao Mengying , Jiang Xueli , Wang Xiaolin , Zhang Guoping , Tang Xinru , Yang Chunjie , Komuro Issei , Ge Junbo , Li Liliang , Zou Yunzeng
Published: November 2022

Background:

Diabetic heart dysfunction is a common complication of diabetes. Cell death is a core event that leads to diabetic heart dysfunction. However, the time sequence of cell death pathways and the precise time to intervene of particular cell death type remain largely unknown in the diabetic heart. This study aims to identify the particular cell death type that is responsible for diabetic heart dysfunction and to propose a promising therapeutic strategy by intervening in the cell death pathway.

Methods:

Type 2 diabetes models were established using
db/db
leptin receptor–deficient mice and high-fat diet/streptozotocin–induced mice. The type 1 diabetes model was established in streptozotocin-induced mice. Apoptosis and programmed cell necrosis (necroptosis) were detected in diabetic mouse hearts at different ages. G protein–coupled receptor–targeted drug library was searched to identify potential receptors regulating the key cell death pathway. Pharmacological and genetic approaches that modulate the expression of targets were used. Stable cell lines and a homemade phosphorylation antibody were prepared to conduct mechanistic studies.

Results:

Necroptosis was activated after apoptosis at later stages of diabetes and was functionally responsible for cardiac dysfunction. Cannabinoid receptor 2 (CB2R) was a key regulator of necroptosis. Mechanically, during normal glucose levels, CB2R inhibited S6 kinase–mediated phosphorylation of BACH2 at serine 520, thereby leading to BACH2 translocation to the nucleus, where BACH2 transcriptionally repressed the necroptosis genes
Rip1
,
Rip3
, and
Mlkl
. Under hyperglycemic conditions, high glucose induced CB2R internalization in a β-arrestin 2–dependent manner; thereafter, MLKL (mixed lineage kinase domain-like), but not receptor-interacting protein kinase 1 or 3, phosphorylated CB2R at serine 352 and promoted CB2R degradation by ubiquitin modification. Cardiac re-expression of CB2R rescued diabetes-induced cardiomyocyte necroptosis and heart dysfunction, whereas cardiac knockout of
Bach2
diminished CB2R-mediated beneficial effects. In human diabetic hearts, both CB2R and BACH2 were negatively associated with diabetes-induced myocardial injuries.

Conclusions:

CB2R transcriptionally repressed necroptosis through interaction with BACH2; in turn, MLKL formed a negative feedback to phosphorylate CB2R. Our study provides the integrative view of a novel molecular mechanism loop for regulation of necroptosis centered by CB2R, which represents a promising alternative strategy for controlling diabetic heart dysfunction.
Amount of Pannexin 1 in Smooth Muscle Cells Regulates Sympathetic Nerve–Induced Vasoconstriction
Authors: Dunaway Luke S. , Billaud Marie , Macal Edgar , Good Miranda E. , Medina Christopher B. , Lorenz Ulrike , Ravichandran Kodi , Koval Michael , Isakson Brant E.
Published: November 2022

Background:

Panx1 (pannexin 1) forms high conductance channels that secrete ATP upon stimulation. The role of Panx1 in mediating constriction in response to direct sympathetic nerve stimulation is not known. Additionally, it is unknown how the expression level of Panx1 in smooth muscle cells (SMCs) influences α-adrenergic responses. We hypothesized that the amount of Panx1 in SMCs dictates the levels of sympathetic constriction and blood pressure.

Methods:

To test this hypothesis, we used genetically modified mouse models enabling expression of Panx1 in vascular cells to be varied. Electrical field stimulation on isolated arteries and blood pressure were assessed.

Results:

Genetic deletion of SMC Panx1 prevented constriction by electric field stimulation of sympathetic nerves. Conversely, overexpression of Panx1 in SMCs using a ROSA26 transgenic model increased sympathetic nerve-mediated constriction. Connexin 43 hemichannel inhibitors did not alter constriction. Next, we evaluated the effects of altered SMC Panx1 expression on blood pressure. To do this, we created mice combining a global Panx1 deletion, with ROSA26-Panx1 under the control of an inducible SMC specific Cre (Myh11). This resulted in mice that could express only human Panx1, only in SMCs. After tamoxifen, these mice had increased blood pressure that was acutely decreased by the Panx1 inhibitor spironolactone. Control mice genetically devoid of Panx1 did not respond to spironolactone.

Conclusions:

These data suggest Panx1 in SMCs could regulate the extent of sympathetic nerve constriction and blood pressure. The results also show the feasibility humanized Panx1-mouse models to test pharmacological candidates.