bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 399: Carnitine Palmitoyltransferase 1a Modulates Hepatic And Lipoprotein Metabolism
Authors: Zelows Mikala , Kaur Rupinder , Harrison Douglas , Wu Qinglin , Shalaurova Irina , Softic Samir , Graf Gregory A , Helsley Robert N
Published: October 2023

Background:
Nonalcoholic fatty liver disease (NAFLD) affects almost 1 billion people worldwide and is associated with cardiometabolic risk factors. Genome- and epigenome-wide association studies have associated variants and methylation status of carnitine palmitoyltransferase 1a (CPT1a) to perturbations in very low-density lipoprotein (VLDL) cholesterol and triglyceride levels. The primary goal of this project is to determine the mechanism by which CPT1a alters hepatic and lipoprotein metabolism.

Methods:
Eight-week-old
Cpt1a
floxed mice expressing the human APOB100 transgene (Cpt1a
fl/fl
/B100
Tg
) were administered control adenoassociated virus (AAV) or AAV encoding Cre-recombinase under control of a liver specific promoter (TBG-Cre). Control and LKO mice were placed on low-fat control or western-type diet (WTD; 42% kcal fat, 0.2% cholesterol) for 16 weeks. Livers were collected and used for histological and lipid analysis, while gene and protein expression were measured by single-cell RNA sequencing and immunoblotting, respectively. The lipoprotein composition in plasma was determined by FPLC and nuclear magnetic resonance (NMR).

Results:
Mice with liver-specific deletion of
Cpt1a
displayed lower circulating APOB levels consistent with reduced triglyceride rich lipoproteins and LDL particle number. Despite a reduction in steady-state plasma lipids, VLDL-triglyceride secretion was enhanced in LKO mice. WTD-feeding elevated hepatic triglycerides in LKO mice across both sexes, while cholesterol (free and esterified) increased by ~2.5-fold specifically in females. Consistent with greater accumulation of free cholesterol in female LKO mice, CD68
+
macrophages exhibited increased expression of
Mmp12
and
Ccl5
, two genes involved in immune cell recruitment and fibrosis.

Conclusions:
Liver-specific deletion of CPT1a reduces plasma LDL-cholesterol and triglycerides, despite having accelerated VLDL-secretion. Increases in hepatic free cholesterol levels were observed only in female LKO mice, which associates with a pro-inflammatory gene signature in macrophages that may contribute to exacerbation of liver injury in these mice.
Abstract 411: Gene Editing Targeting
Angptl3
By SlugCas9 Ameliorates Lipid Metabolism And Atherosclerosis In Familial Hypercholesterolemia
Authors: Yin Ye , Li Zhixing , Xu Hong , Shen Qian , Ji Baowei , Wang Yongming , Ge Junbo
Published: October 2023

Background:
Familial Hypercholesterolemia(FH) is a genetic disease causing severe hyperlipidemia and atherosclerosis at early age. ANGPTL3, as an important factor for regulating lipid metabolism, has been proved as a promising target for gene therapies. The SlugCas9 system, as a newly-developed gene-editing tool with advantages of small size and wide targeting site, can be wrapped into AAV vectors and facilitate gene therapy. The application of SlugCas9 system towards FH has not been previously reported.

Methods:
We screened two guide RNA with the highest targeted angptl3 editing efficiency labeled g2 and g3 and built the AAV8-SlugCas9 system. Ldlr-/- mice with 4-week-old were divided into AAV-SlugCas9 group (including g2, g3 and g2+g3), AAV-control group(empty AAV vectors) and normal diet group. AAV-SlugCas9 and AAV-control were injected through tail vein after two weeks of western-diet feeding. Two weeks later, expression of protein ANGPTL3 in both serum and liver were detected and editing efficiency was assessed. We also evaluated serum lipid levels and atherosclerotic level by oil-red staining as well as immunofluorescence labeling of inflammatory markers in aorta. Adverse effect was evaluated by liver histology.

Results:
Two weeks after western-diet feeding, serum lipid and inflammatory markers increased significantly in ldlr-/- mice. We found dramatically decreased ANGPTL3 protein expression in both serum and liver after AAV-SlugCas9 treatment, with no off-target condition found. Our results also showed that compared with the AAV-control group, the AAV-SlugCas9 group had significant reductions in serum lipid levels and that we found improved atherosclerosis and decreased inflammatory marker expression in serum and aorta. No obvious damage in liver histology was found.

Conclusions:
In this study, we used AAV as a delivery vector to evaluate the editing efficiency of SlugCas9 for the angptl3 gene in liver for the first time and found out by reducing its expression by gene editing of angptl3 in vivo can reduce blood lipid levels and delay the progression of atherosclerosis with no obvious adverse effect in mice. It proves that SlugCas9 is a useful tool in gene therapies targeting FH with potential clinical application.
Abstract 416: Atorvastatin Versus Placebo In Icu Patients With Covid19: Ninety-day Results Of The INSPIRATION-S Trial
Authors: Talasaz Azita , Sadeghipour Parham , Sharif-Kashani Babak , Rashidi Farid , Beigmohammadi Mohammad Taghi , Gohari Moghadam Keivan , Rezaian Somayeh , Dabbagh Ali , Sezavar Seyed Hashem , Piazza Gregory , Farrokhpour Mohsen , Bakhshandeh Hooman , Abedini Atefeh , Aliannejad Rasoul , Riahi Taghi , Yadollahzadeh Mehdi , Lookzadeh Somayeh , rezaeifar parisa , Matin Samira , Tahamtan Ouria , Mohammadi Keyhan , Zoghi Elnaz , Hosseini Hossein , Mousavian Seyed Masoud , Abri Homa , Sadeghipour Pardis , baghizadeh elahe , Rafiee Farnaz , jamalkhani sepehr , Aghakouchakzadeh Maryam , Eslami Vahid , Payandemehr Pooya , Jimenez David , Sethi Sanjum , Parikh Sahil A , Monreal Manuel , maleki majid , maleki majid , Sadeghian Saeed , Barco Stefano , Siegerink Bob , Piazza Gregory , Kirtane Ajay J , Van Tassell Benjamin W , Stone Gregg , Klok Erik , Lip Gregory , Goldhaber Samuel , Krumholz Harlan M , Bikdeli Behnood
Published: October 2023

Backgrounds:
In the INSPIRATION-S trial, atorvastatin versus placebo was associated with a non-significant 16% reduction in 30-day composite of venous/arterial thrombosis or death in ICU patients with COVID-19. Thrombo-inflammatory response in COVID-19 may last beyond the first 30 days.

Methods:
This article reports the effects of atorvastatin 20 mg daily versus placebo on 90-day clinical and functional outcomes from INSPIRATION-S, a double-blind multicenter randomized trial of adult ICU patients with COVID-19. The main outcome for this pre-specified study was a composite of adjudicated venous/arterial thrombosis, treatment with extracorporeal membrane oxygenation (ECMO), or all-cause mortality. Functional status was assessed with Post-COVID-19 Functional Scale (PCFS).

Results:
In the primary analysis, 587 patients were included (age: 57 (Q1, Q3: 45, 68) years; 44% women). By 90-day follow-up, the main outcome occurred in 96 (33.1%) patients assigned to atorvastatin and 113 (38.0%) assigned to placebo (hazard ratio, HR: 0.80, 95% confidence interval, CI: 0.60-1.05, P=0.11). Atorvastatin in patients who presented within 7 days of symptom onset was associated with reduced 90-day hazard for the main outcome (HR: 0.60, 95% CI: 0.42-0.86, P
interaction
=0.02). Atorvastatin use was associated with improved 90-day functional status, although the upper bound CI crossed 1.0 (OR
ordinal
, 0.64, 95% CI: 0.41 to 1.01, P=0.05).

Conclusions:
Atorvastatin 20 mg compared with placebo did not significantly reduce the 90-day composite of death, treatment with ECMO, or venous/arterial thrombosis. However, the point estimates do not exclude a potential clinically meaningful treatment effect, especially among patients who presented within 7 days of symptom onset.
Abstract 423: Ultramarathon Induces Metabolite Changes Associated With Pathophysiology Of Cardiovascular Diseases- In Silico And In Vivo Analysis
Authors: Wicik Zofia , Marszalek Mikolaj , Ciechanowicz Andrzej , Keshwani Disha , Eyileten Ceren , Wiecha Szczepan , Malek Lukasz , Kucia Magdalena , Postula Marek A
Published: October 2023

Regular physical exercise increase longevity, however acute intensive/longterm exercise can trigger the CV pathophysiological processes. Ultramarathon runners' plasma (n=22) with 2time points: before and after the run: Completion (100km) or exhaustion (median74km) was used for analysis. For untargeted metabolomic analysis; ultrahigh resolution MassSpectrometry with electrospray ion source in direct injection used and analyzed by T-Rex2D algorithm. Identified compounds analyzed using MetaboAnalystR/MetscapeCytoscape plugin was used for metabolite-gene interaction analysis, identifying 1601 up and 1030 downregulated metabolites with min 2 fold difference after ultramarathon run Enrichment analysis showed that TCA cycle (FDR=0.0442), metabolism of; Sphingolipids (p=0.008), Glyoxylate and dicarboxylate (p=0.03) and Butanoate (p=0.04) were the top metabolites. After the run top upregulated metabolites were related to heart failure associated metabolites; acetylcarnitines2 CAR(14:2) (AUC=0.97) Dodecanoylcarnitine (2.00;0.960), 9-Decenoylcarnitine (1.78;0.95), CAR(DC8:0) (14.99;0.94), 2Carboxy5,7dimethyl4octanolide (8.04;0.98), Methylmalonic acid (9.33;0.96) and cardiotoxic Cyclophosphamide (16.29;0.940). Top downregulated metabolites after the run included potentially cardioprotective Gamma Glutamylglutamic acid (log2FC=-1.81;AUC=0.85), Fructose 1,6bisphosphate (-2.42;0.88)4, Oleamide (-1.82;0.86) and gammaGlutamylglycine (-7.98;0.85).Interaction network analysis showed genes associated with atherosclerosis/obesity-related PhospholipaseA2,IB (PLA2G1B) as potentially the most affected gene by analyzed metabolites. Twofold post run increase in metabolites showed they are both potential markers for acute exercise and are strongly associated with CV diseases. This calls for consideration of the detrimental effects of excessive exercise and implementation of monitoring in amateur athletes.
Abstract 435: Macrophage Rac1 As A Transcriptional Co-factor For NF-kb-driven IL-1β Expression And Consequent Atherosclerotic Calcification
Authors: Carley Rachel , Lee Cadence , Berus Joshua M , Mantsounga Chris S , Sharma Sheila , Caballero Olivya , Pierce Julia , Amelotte Elizabeth , Healy Abigail , Parry Crystal , Pineda Edy , Butler Celia , Choudhary Gaurav , Morrison Alan R
Published: October 2023

Coronary artery disease caused by atherosclerosis is a leading cause of morbidity and mortality. Vascular calcification, influenced by inflammation, is associated with atherosclerotic disease burden and increased risk of cardiovascular events. The small GTPase Rac1 is an important signal transducer of inflammatory cytokine expression. We sought to define the mechanisms whereby Rac1 and its activating guanine nucleotide exchange factor (GEF), Tiam1, regulate macrophage expression of the potent inflammatory cytokine, IL-1β, and consequent calcific atherosclerotic vascular disease. To define the role of macrophage Rac1 expression on IL-1β expression and atherosclerotic calcification, we developed mouse models on the atherosclerosis-prone background, including inducible myeloid
Rac1
-deletion (mR1KO) (

CSF1
mcm
Rac1
fl/fl
ApoE
-/-)

, and global
Tiam1-
deletion (

Tiam1
-/-
ApoE
-/-

). Both
Rac1-deleted
and
Tiam1-deleted
macrophages demonstrated reduced IL-1β expression in an inflammasome-stimulation assay. Macrophage
Rac1
-deletion led to decreased nuclear NF-κB activity and reduced NF-κB and GTP-Rac1 binding to the IL-1β promoter by chromatin immunoprecipitation assay (ChIP). Using an inflammatory stimulus, we found that Rac1 and NF-κB form a protein complex by Proximity Ligation Assay, which is disrupted by
Rac1-
deletion. Gain-of-function mutations for Rac1 rescued IL-1β expression in
Rac1-deleted
macrophages, and mutations in the hypervariable region of Rac1 confirmed that the nuclear localization of Rac1 is required for the rescue of IL-1β expression. This recent preliminary data demonstrated that the small GTPase, Rac1, acts as a transcriptional co-factor, helping to activate NF-κB, facilitating NF-κB’s translocation to the nucleus, and binding the IL-1β promoter. In a hyperlipidemia model of experimental atherosclerosis, macrophage
Rac1-
deletion reduced systemic IL-1β levels and decreased plaque calcification. Targeted inhibition of the macrophage Tiam1-Rac1 signaling complex may have potential as a therapeutic strategy to mitigate inflammatory-driven vascular calcification, given that small molecule inhibitors of Tiam1-Rac1 already exist.
Abstract 438: FDA-approved Drug For Alcoholism Reduces Atherosclerosis By Inhibiting Pyroptotic Executor Gasdermin D (GsdmD)
Authors: Traughber C. Alicia , Opoku Emmanuel , Khan Mariam , Neupane Kalash , Smith Jonathan , Gulshan Kailash
Published: October 2023

Introduction:
Atherosclerosis is driven by chronic inflammation of the artery wall in response to cholesterol accumulation and defects in reverse cholesterol transport (RCT). Activation of the NLRP3 inflammasome in foam cells leads to cleavage of Gasdermin D (GsdmD), facilitating release of the mature interleukin-1beta (IL-1β). Work from our lab and others have shown that GsdmD promotes atherosclerosis. Recently, the FDA-approved drug, Disulfiram, was shown to inhibit GsdmD. Though the GsdmD/IL-1β pathway has been implicated in CVD, the efficacy of Disulfiram as an anti-atherosclerotic therapy has not been explored.

Hypothesis:
We aim to establish a mechanism-based role of Disulfiram as an atherosclerotic therapeutic. We hypothesize that Disulfiram inhibition of GsdmD-pore formation and subsequent IL-1β release will reduce atherosclerosis.

Methods and Results:
Efficacy of Disulfiram (DSF) as an GsdmD inhibitor was confirmed in inflammasome-induced macrophages and in mice, with both showing significantly reduced IL-1b release with no effect on expression of inflammasome components. To test if DSF can reduce atherosclerosis, hyperlipidemic apoE
-/-
mice were weaned onto a western-type diet (WTD) or WTD + 25mg/kg DSF for 15 weeks. Excitingly, DSF vs. WTD-fed mice showed smaller lesion burden (p<0.01) by 26.7% for males and 29.4% for females. Total cholesterol levels were higher in DSF vs WTD males (p<0.05) and females (p<0.01). DSF-diet did not impact body or liver weight, white blood cells, platelets, lymphocytes, monocytes or eosinophils. Red blood cells (RBCs) and neutrophils were decreased in females (p<0.01 and p<0.05, respectively), but not in males. DSF-fed mice have reduced macrophage infiltration into atherosclerotic lesion sites.

Conclusions:
Our pre-clinical data suggests Disulfiram has the potential to be used as a stand-alone therapy or in combination with current atherosclerosis therapies.
Abstract 443: BAFF Neutralization Impairs The Regression Of Insulin Resistance In Diabetic Mice By Modulating The Innate Immune Response
Authors: Lempicki Melissa D , Gray Jake , Whittaker Bradley , McNamara Coleen A , Meher Akshaya K
Published: October 2023

Adults with type 2 diabetes have a two-to-three-fold increased risk of cardiovascular diseases. B cell activating factor (BAFF), a tumor necrosis factor superfamily member, is implicated both in diabetes and cardiovascular diseases, and hence a therapeutic target of interest. High levels of BAFF are known to promote autoantibody synthesis and autoimmune diseases. We examined if BAFF neutralization with an anti-BAFF antibody improves glucose intolerance in a model of regression of insulin resistance in diabetic mice. Male C57BL/6J mice were fed a high-fat diet (HFD) for 12 weeks and segregated into two groups based on their glucose intolerance determined by a glucose tolerance test. Each group was simultaneously switched from HFD to a normal chow diet and received injections of anti-BAFF or an isotype control antibody every 2
nd
week for a total of 6 weeks. As expected, body weights decreased in both groups parallel with the increase in insulin sensitivity. Unexpectedly, the BAFF-neutralized mice had impaired regression of insulin resistance compared to the control mice. Extensive cellular phenotyping of immune cells indicated systemic depletion of only the B2 cells and lower levels of circulating autoantibodies in the BAFF-neutralized mice. Gonadal white adipose tissue (gWAT) was the primary source of BAFF, and no differences in the M1 and M2 macrophages, eosinophils, natural killer cells, helper T cells, and cytotoxic T cell populations were found. RNA sequencing analysis of stromal vascular fraction from gWAT revealed impaired B cell receptor signaling, immunoglobulin production, complement activation, and phagocytosis pathways in the BAFF-neutralized mice. In this line, (i) an increased number of dead adipocytes, (ii) decreased levels of IgG2b and IgM, (iii) increased citrullinated histone H3, neutrophils, and DNA extracellular traps were found in the gWAT of BAFF-neutralized mice. These results suggest attenuated humoral response and a dominant and harmful role of innate response in the gWAT following BAFF neutralization in late-stage obesity. Since anti-BAFF biologics are approved for the treatment of lupus patients, our results highlight the need for further studies on the role of BAFF in late-stage obesity and diabetes.
Abstract 444: Effectiveness Of The Ratio Of L-cysteine To L-glutathione In Serum For Prediction Of Cardiovascular Events
Authors: Masaki Nobuyuki , Ishinoda Yuki , Adachi Takeshi
Published: October 2023

Background:
Aminothiols play an important role in the antioxidant defense system and the profile may be a predictor of prognosis and cardiovascular events.

Methods:
Two hundred sixty-two patients (202 men, 60 women, age 65 ± 13 years) undergoing coronary catheterization were followed up to 10 years. The patients were divided equally into two groups according to the median ratio of total L-cysteine to total L-glutathione (tCys/tGSH) in serum at enrollment.

Results:
During 5.5 ± 3.2 years, 31 patients (12%) died, of whom 20 (8%) had cardiovascular death and 32 (12%) had MACE. In addition, 29 (11%) were hospitalized for heart failure. Log-rank test showed that the high group (tCys/tGSH>=80.70) had a higher mortality from all causes (Fig.A, p=0.025) than the low group (tCys/tGSH<80.70). Univariate COX regression analysis showed that tCys/tGSH was a predictive marker of death, cardiovascular death, MACE, and heart failure (p<0.001, all), but these aminothiol alone did not predict any of these endpoints. After adjustment for age, sex, body mass index, coronary risk factors, renal function, and log-transformed brain natriuretic peptide, tCys/tGSH had predictive value for death (p=0.007), cardiovascular death (p=0.019), and heart failure (p=0.004). The adjusted hazard ratio for heart failure in the high tCys/tGSH group versus the low tCys/tGSH group was 3.071 (95% confidence interval: 1.186-7.952), p=0.021. tCys/tGSH ratio can be substituted by the GSH index obtained from the approximation curve (Fig.B, GSH index=142.7255*tGSH
-0.777682
). GSH index was also a prognostic biomarker, but we found tCys information was necessary to predict heart failure and was lost in GSH index. In addition, γ-glutamyl transpeptidase (GGT), which is responsible for extracellular catabolism of GSH to Cys, was a prognostic factor.

Conclusions:
Our results suggest that tCys and tGSH can be a useful marker of cardiovascular events by ratioing and may be an indicator of GGT activity.
Abstract 447: Cholesterol Excretion In Whole-body And Liver-specific ABCG5 ABCG8-deficient Mice
Authors: Savage Erika , Anspach Garrett , Kaur Rupinder , Cady Corissa , Graf Gregory A
Published: October 2023

Background:
The ABCG5 ABCG8 sterol transporter is the body’s primary defense against the accumulation of dietary sterols. Loss of function mutations in ABCG5 or ABCG8 result in Sitosterolemia, a rare disorder that presents as familial hypercholesterolemia, but is distinguished by recessive genetics and the accumulation of phytosterols in plasma and tissues. Biliary cholesterol is reduced by greater than 80% in ABCG5 ABCG8-deficient mice, but fecal neutral sterols are unaffected, suggesting a G5G8-independent pathway for cholesterol excretion. The objectives of the present study were to determine the ability of whole-body and liver-specific G5G8-deficient mice to maintain fecal neutral sterol output when challenged with cholesterol -enriched diet and determine maximal rates of biliary cholesterol secretion.

Methods:
Wild-type, whole-body and liver-specific ABCG5 ABCG8-deficient mice were maintained on cereal-based mixed diet, a phytosterol-free purified diet, or a purified diet supplemented with 0.2% cholesterol. Following acclimatization to each diet, fecal neutral sterol output was determined over a three-day period by gas chromatography/mass spectrometry. Maximal biliary cholesterol secretion rates were determined by biliary diversion with bile acid infusion over a range of 0 to 1000 nmol/minute. Biliary lipid secretion was determined by enzymatic colorimetric assays and molecular coupling of cholesterol to bile acid and phospholipid secretion calculated by linear regression.

Results & Conclusions:
Whole-body G5G8-deficient mice exhibited a 40% reduction in fecal neutral sterol output compared to wild-type mice when maintained on cereal-based or purified, low cholesterol containing diets. Cholesterol feeding resulted in a 10-fold increase in fecal neutral sterols in wild-type mice, an effect not observed in G5G8-deficient mice. These data indicate that ABCG5 ABCG8 is indispensable in opposing the accumulation of dietary cholesterol and that alternative pathways cannot compensate for G5G8 deficiency.
Abstract 464: Increased VCAM-1 Expression In Humanized Resistin Double Knock Out Transgenic Mouse
Authors: Succar Bahaa , Haider Nezam , moly pricila , Kath Allison , Saad Emile , Zhou Wei
Published: October 2023

Background:
Cytokine resistin secreted by human macrophages and mice adipocytes plays a crucial role in atherosclerosis, and chronic inflammation. Our humanized resistin transgenic mouse (TG) model showed that high-fat diet and hyper-resistinemia induced changes in metabolic and inflammatory genes and proteins implicated in atherosclerosis. However, gross arterial atherosclerotic plaque deposition was not detected. Therefore, to better understand the role of resistin on atheroma initiation, we developed the humanized resistin double knock-out-transgenic mouse(hR
TG-
DKO) model which expresses human resistin without expressing murine resistin and ApoE.

Hypothesis:
We hypothesize that human resistin enhances early atheroma formation.

Methods:
Eight- and twelve-week-old TG(n=10), ApoE
-/-
(n=10), and hR
TG-
DKO(n=10) mice fed on regular chow. We measured plasma levels of LDL-c with enzymatic assays, and human Resistin with an ELISA assay in the 3 groups. Expression of cell adhesion molecule VCAM-1 in formalin-fixed and paraffin-embedded brachiocephalic arteries was quantified by immunohistochemistry in the 3 groups. All methods were approved by the IACUC at the University of Arizona, Tucson. Data are presented as means ±SEM.

Results:
Human resistin was detected in the plasma of hR
TG-
DKO and TG groups with similar levels [6.4±1.5 ng/ml vs 6.3±1.5ng/ml] but was not detected in the plasma of ApoE
-/-
mice, as expected. LDL-c level was significantly higher in hR
TG-
DKO and ApoE
-/-
compared to TG mice respectively [196±17mg/dl vs 26±4mg/dl p=0.0002, 346±25 mg/dl vs 26±4 mg/dl p= 0.0002]. On quantitative analysis, the mean VCAM-1-stained percentage area of brachiocephalic arteries was significantly higher in hR
TG-
DKO compared to ApoE
-/-
[4.75%± 1.23 vs 1.77% ± 0.23, p=0.02] and TG mice respectively [4.75%± 1.23 vs 1.33% ± 0.34, p=0.03].

Conclusion:
In conclusion, hR
TG
-DKO exhibits human resistin, high plasma LDL-c level, and increased VCAM-1 expression. Hence, it promotes atherosclerosis initiation, and it is a more appropriate model to study atherosclerosis in the setting of hyper-resistinemia. Further studies are needed to explore the effects of hyper-resistinemia and diet on the progression of atherosclerosis in the hR
TG-
DKO model.
Abstract 468: Nano-targeting Epsin In Cholesterol Uptake-efflux Fortifies Atheroma Regression
Authors: Cui Kui , Gao Xinlei , Wang Beibei , Wu Hao , Dong Yunzhou , Cowan Douglas B , Shi Jinjun , Chen Kaifu , Chen Hong
Published: October 2023

Excess cholesterol accumulation in lesional macrophages elicits complex responses in atherosclerosis. Epsins, endocytic adaptors, fuel the progression of atherosclerosis; however, the underlying mechanism and therapeutic potential of targeting Epsins remains unknown. In our study, we determined the role of Epsins in macrophage-mediated metabolic regulation and developed a method to target macrophage Epsins with specially-designed S2P-conjugated lipid nanoparticles (NPs), which encapsulate siRNAs to suppress Epsins.We used scRNA-seq with our newly developed algorithm MEBOCOST to study cell-cell communications mediated by metabolites from sender cells and sensor proteins on receiver cells. Biomedical, cellular and molecular approaches were utilized to investigate the role of macrophage Epsins in regulating lipid metabolism and transport. The NPs targeting lesional macrophages were developed to encapsulate interfering RNAs to treat atherosclerosis.We revealed that Epsins regulate lipid metabolism and transport in atherosclerotic macrophages. Inhibiting Epsins by nanotherapy halts inflammation and accelerates atheroma resolution. Harnessing macrophage-specific NP delivery of Epsin siRNAs, we showed that silencing of macrophage Epsins markedly diminished atherosclerotic plaque size and promoted plaque regression. Mechanistically, we demonstrated that Epsins bound to CD36 to facilitate lipid uptake by enhancing CD36 endocytosis and recycling. Conversely, Epsins promoted ABCG1 degradation via lysosomes and hampered ABCG1-mediated cholesterol efflux and reverse cholesterol transport.Our findings suggest that targeting Epsins in lesional macrophages may offer therapeutic benefits for advanced atherosclerosis.
Abstract 485: Mitochondrial Complex I Promotes Smooth Muscle Cell Plasticity And Vascular Remodeling
Authors: Maira Alishba , Riascos Bernal Dario F , Sibinga Nicholas
Published: October 2023

Remodeling of the mature vasculature occurs in response to diseases such as atherosclerosis or after vascular injury due to angioplasty or stent placement. Smooth muscle cell (SMC) plasticity is a key factor in this process, but the significance of cellular metabolic and mitochondrial activities in control of SMC plasticity is not well understood. We hypothesized an essential role for mitochondrial respiratory complex I (CI) in SMC proliferation and migration and tested this idea by inactivating the CI subunit NDUFS4 or by using pharmacologic CI inhibitors. In cultured

Ndufs4
-/-

SMCs, we found lower overall CI expression and activity (P=0.0027), plus decreased respirasome levels, ATP-linked oxygen consumption (P<0.0001), respiratory capacity, NAD
+
/NADH ratio (P=0.0035), aspartate, and cell proliferation (P<0.0001). NDUFS4 loss increased mitochondrial fragmentation (P<0.0001); interestingly, inhibition of mitochondrial fission restored proliferation of NDUFS4-deficient mouse SMCs and CI inhibitor-treated human SMCs. In addition, loss of NDUFS4 or CI inhibitor treatment limited migration of mouse (P=0.0012) and human (P=0.0022) SMCs, respectively.
In vivo
, NDUFS4 was highly expressed in the arterial wall during embryogenesis and in cells forming the neointima after arterial injury in adulthood. Mice with embryonic inactivation of SMC
Ndufs4
were born in Mendelian ratios, reached adulthood, and lacked an obvious phenotype up to 20 weeks of age; by electron microscopy, however, SMCs in the aortic media revealed altered mitochondrial ultrastructure. To evaluate how SMC CI contributes to arterial injury response in the adult, we inactivated
Ndufs4
conditionally at age 6 weeks (NDUFS4 iSMKO) and performed carotid artery ligation; these studies showed that loss of SMC NDUFS4 reduced neointima formation (57% lower intima/media ratio; control, n=13; NDUFS4 iSMKO, n=8; P=0.02). In conclusion, NDUFS4 and thus proper levels of SMC CI are essential for proliferation, migration, normal mitochondrial ultrastructure, and vascular remodeling in adulthood. Mechanistic studies suggest that CI promotes SMC plasticity by supporting biosynthesis of aspartate and ATP, maintaining NAD
+
/NADH balance, and limiting mitochondrial fragmentation.
Abstract 492: The Role Of Dab2 In Regulating Angiogenesis In Diabetic Conditions
Authors: Bhattacharjee Sudarshan , Chen Hong
Published: October 2023

Background:
Diabetes-related vascular complications underlie a number of life-threatening conditions including cardiovascular disease, stroke, and peripheral limb ulcerations, the latter of which can lead to severe infections and a multitude of serious medical issues. Vascular endothelial growth factor receptor 2 (VEGFR2) is a critical regulator of physiological and pathological angiogenesis. Therefore, VEGFR2 signaling should be tightly controlled to promote neovascularization when beneficial vascular regeneration is reduced, and to attenuate angiogenesis when adverse vascular growth is excessive. Activated VEGFR2 endocytosis through a unique endocytosis adaptor protein called Disabled 2 (Dab2) results in enhanced VEGF signaling. However, the mechanisms underlying how Dab2 controls diabetic angiogenesis and the therapeutic potential of manipulation of Dab2 levels in diabetes are yet to be discovered.

Hypothesis:
We hypothesized that Dab2 plays a critical role in promoting angiogenesis in diabetes by amplifying VEGF signaling.

Methods and Results:
Using endothelial cell-specific inducible Dab2 knockout mice (EC-iDab2KO), we showed that VEGF signaling in primary mouse brain endothelial cells from EC-iDab2KO mice was greatly reduced compared to WT cells, which is reflected by decreased VEGFR2, Akt, and ERK phosphorylation. RNA sequencing also revealed decreased expression of Dab2 in EC in diabetic condition. VEGF also decreased EC-iDab2KO endothelial cell migration and tube formation
in vitro.
We used wound healing and Matrigel plug assays in STZ-induced diabetic mice to show reduced angiogenesis delayed wound healing process compared to diabetic WT mice. Lack of Dab2 further diminished angiogenesis and delayed wound healing compared to WT mice. CRISPR-mediated mutation of Dab2 promoter and ChIP-PCR reveals that FoxM1 transcription factor regulates Dab2 expression in endothelial cells in diabetes.

Conclusions:
Our study indicates that VEGF-induced angiogenesis is inhibited in diabetic conditions and Dab2 loss weakens VEGF signaling and angiogenic responses. Stabilizing Dab2 could be a therapeutic target for enhancing angiogenesis during wound healing in diabetes.
Abstract 501: Endothelin-1 Expression Is Dependent On The Stability Of Endothelial Glycocalyx Heparan Sulfate
Authors: Rivero Santiago A , Mensah Solomon A
Published: October 2023

Introduction:
Endothelin-1 (ET-1) is a vasoconstrictor produced by the vascular endothelium [1]. It binds to endothelial B receptor(ETB) on the endothelial cells(EC) [2]. Increased ET-1 has been observed in the EC of patients with moderate to severe hypertension [3]. Studies suggest that the EC cytoskeletal disruptions could mediate its production[4,5,6]. ETB is a plasma membrane bound receptor present in the EC surface with EC glycocalyx (GCX) [7,8]. GCX is flow sensitive and consists of several transmembrane solid core proteins [10,11,12]. Linked to the core protein chains include heperan sulfate (HS) [12]. GCX HS and ETB are located in the surface of the EC and are both flow sensitive. We hypothesize that the shear stress induced upregulation of the GCX HS could lead to expression of ETB which will trigger the release of ET1.

Methods:
Human Lung Microvascular Endothelial cells (HLMVEC) were cultured until 70-80% confluence. To mimic disease progression the GCX were degraded with Heparinase III which targets the HS component of the GCX. Immunostaining was used to determine the expression of GCX HS, ETB and ET-1.

Results:
Data revealed that the degradation of GCX HS resulted in the significant decrease in the expression of ETB. Conversely, the removal of GCX HS caused a significant increase in the expression of ET-1.

Discussion:
The preliminary results from, immunostaining reveals a potential co-participation of GCX HS and ETB in the expression of ET-1.

References:
[1] Sutton. G. et al,
American Journal of Hypertension,
32:813-815, 2019.[2] Dhaun, N. et al,
Hypertension
, 52:452-459, 2008.[3] Morawietz, H. et al
, J Physiol
, 525:761-770, 2000.[4] Kuchan, M.J, et al,
Am J Physiol
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Abstract 502: Phenotypic And Molecular Effects Of Testosterone Gender-affirming Hormone Therapy On The Vascular Endothelium Of Transgender Men
Authors: Roberts Michelle L , Westhoff Justin , Wang Jingli , Ying Rong , Cabrera Susanne M , Widlansky Michael E
Published: October 2023

Approximately 1.3 million adults identify as transgender with 35.9% being men of trans experience. Nearly 70% of men of trans experience have accessed gender-affirming hormone therapy (GAHT) and many more plan to access this therapy. Cis gender women derive a vascular protective effect from circulating estrogen that is in part responsible for the decade difference in the average of onset of cardiovascular disease in cis gender women compared to cis gender men. Estrogen stimulates receptors on the vascular endothelium leading to increased bioavailable nitric oxide (NO) which is largely responsible for estrogen’s favorable vascular effects. Research of those on gender-affirming hormone therapy and cardiovascular disease (CVD) and CVD risk factors are currently ongoing; however, little is known about the effects of testosterone as GAHT on the vasculature of men of trans experience. We obtained resistance arterioles from adipose tissue of mastectomies to compare endothelium-dependent vasoreactivity of resistance arterioles to estrogen (17β- estradiol) of men of trans experience, cis women, and cis men. To date, with an increasing dose of 17β- estradiol, we find that vessels from men of trans experience (N=5) have impaired endothelium-dependent vasodilation compared to cis gender women (N=6, P=0.001). The differences in responses are related to differences in NO synthase activity as seen by loss of the difference with the use of L-NAME (200 μM). These data show the possibility of decreased vasodilation of transgender men on testosterone therapy. Additional studies with vessels from cis gender men for comparison are currently ongoing (N=3 vessels collected). In future experiments, we plan to use RNA-Seq on whole arterioles as well as examine the protein content of estrogen receptors (α and β) and androgen receptors in the intraluminal layer of endothelial cells of the vessels to determine changes induced by GAHT at the protein receptor and transcriptome level that may be responsible for the phenotypical difference seen. The data promise to provide important mechanistic insights into the effects of GAHT and better information for practitioners to use in advising and stratifying cardiovascular risk for patients who are men of trans experience.
Abstract 515: A Prediction Model For Left Ventricular Thrombus Persistence/recurrence: Based On A Prospective Study And A Retrospective Study
Authors: Yang Qing , Liang Yan
Published: October 2023

Background:
This study aimed to develop a prediction model to forecast thrombus persistence or recurrence in patients with left ventricular (LV) thrombus.

Methods:
We enrolled patients prospectively from 2020 to 2022 and retrospectively from 2013 to 2019 at the National Center of Cardiovascular Diseases of China. The two cohorts were combined to derive predictive thrombus persistence/recurrence models. The primary study comprised patients who had been consistently taking systemic oral anticoagulants and had imaging records available at the end of a three-month follow-up period. The Lasso regression algorithm was performed to select independent predictors and a nomogram risk prediction model was applied as a risk stratification tool.

Results:
A total of 172 patients were included, with 124 patients in a training set and 48 patients in a validation set. Six predictors were incorporated into the multivariate logistic regression prediction model. The area under the receiving operating characteristic was 0.852 in the training set and 0.631 in the validation set. Patients with protuberant thrombus and higher baseline D-dimer levels had a reduced risk of persistence/recurrence (OR 0.17, 95% CI 0.03-0.69,
P
= 0.025; OR 0.67, 95% CI 0.43-0.91,
P
= 0.030, separately), whereas thicker thrombus was linked to an increased rate of persistent thrombus (OR 1.11, 95% CI 1.05-1.20,
P
= 0.002). Additionally, thrombus persistence/recurrence rate was greater in patients with nonischemic cardiomyopathy and antiplatelet therapy.

Conclusions:
This prediction model provides tools and allows the identification of characteristics associated with unresolved thrombus.
Abstract 520: Stethoscope Still Saving Lives: Catching A Ruptured Sinus Of Valsalva Aneurysm
Authors: Kalsoom Sidra , Jain Ashish , Zaman Muhammad Adnan , Butt Asfand Yar , Nathaniel Cyril
Published: October 2023

Introduction:
Sinus of Valsalva aneurysm (SVA) is a rare cardiac abnormality that can occur congenitally or as a result of acquired conditions. It is characterized by a weakness in the elastic lamina at the junction of the aortic media and annulus fibrosis, leading to the formation of an SVA. We present an interesting case of a 54-year-old man, initially diagnosed with infective endocarditis and later on found to have a severe acute rupture of an SVA.

Case Presentation:
A 54-year-old man with a past medical history of raynaud's disease and rheumatoid arthritis presented with shortness of breath and low-grade fever for two weeks. On further evaluations, a diligent finding of a continuous cardiac murmur and a palpable thrill across the precordium led to a transthoracic echocardiogram (TTE). The TTE revealed a 1.3 x 1.3 cm, nonmobile, ball of material near the base of the septal leaflet of the tricuspid valve, raising concerns about infective endocarditis and thus initiation of broad-spectrum intravenous antibiotics. Subsequent transesophageal echocardiogram, however, revealed an outpouching from the right coronary sinus suggestive of the right sinus of Valsalva which was ruptured with jet hitting the tricuspid valve continuously during diastole and systole, mimicking falsely as vegetation on TTE. Emergency interventions including CT angiogram, catheterization, and surgical repair resulted in a favorable outcome for the patient.

Discussion:
SVA has a higher prevalence among males and individuals of Asian ethnicity. Its presentation is variable and can include chest discomfort, shortness of breath, pulmonary edema, or even rupture. It can also be an incidental finding on an echocardiogram. The preferred initial diagnostic approach is echocardiography, and surgical repair is often required for patients, especially if the aneurysm has ruptured. Upon diagnosis, prompt evaluation by a cardiothoracic surgeon is crucial as delay in treatment may lead to fatal consequences.

Conclusion:
The prognosis for ruptured SVAs is poor, which further emphasizes the significance of early diagnosis and prompt treatment. A diligent physical examination is very crucial and can be lifesaving, especially in such circumstances.
Abstract 521: Pharmacologic Inhibition Of Ferroptosis Attenuates Abdominal Aortic Aneurysm Formation
Authors: Krebs Jonathan R , Gang Su , Leroy Victoria , Bellotti Paolo A , Lu Guanyi , Sharma Ashish , Upchurch Gilbert R
Published: October 2023

Background:
Abdominal aortic aneurysms (AAA) are characterized by thrombus formation, aortic inflammation, and vascular remodeling. This study investigates the role of ferroptosis, an iron-dependent non-apoptotic cell death pathway, and its pharmacologic inhibition in the pathogenesis of AAA formation.

Methods:
AAAs were induced in 8- to 12-week old male C57BL/6 mice using topical 0.4 U/ml type 1 porcine pancreatic elastase treatment, or deactivated elastase as control. Mice in the elastase group were treated with liproxstatin-1 (selective ferroptosis inhibitor, 5mg/kg, daily i.p. postoperative days 1-7). Aortic diameter was measured on postoperative day 14 using video micrometry prior to harvest. Aortic tissue was analyzed for cytokine expression and histology. Hallmarks of ferroptosis such as lipid peroxidation (MDA) and glutathione (GSH) were measured in aortic tissue extracts. Statistical analysis was performed by ANOVA and p<0.05 was considered as statistically significant.

Results:
A significant increase in aortic diameter was observed in elastase-treated mice compared to controls on day 14 (155.5±8.7 vs. 1.9±3.3%; n=8-10/group; p<0.0001). A reduction in aortic diameter was observed in liproxstatin-1 treated mice compared to elastase treated mice (62.8±14 vs. 155.5±8.7%; n=8-10/group; p=0.0048). MDA and GSH levels in the aortic tissue were significantly decreased in liproxstatin-1 treated mice compared to elastase-treated mice alone (0.11±0.04 vs. 0.75±.1 nmol/mg; n=5-8/group; p<0.001; and 12.1±1.2 vs. 4.7±.0.7 μg/mg; n=5-8/group; p=0.04) . A decrease in immune cell (neutrophils and macrophages) infiltration, preservation of aortic morphology (increase in smooth muscle α-actin and decrease in elastin breaks) and decreased pro-inflammatory cytokine expression (IL-17, IL-1β, IL-6, MCP-1) was observed in liproxstatin-1-treated mice compared to elastase-treated mice alone by histological analysis.

Conclusions:
AAA formation is characterized by hallmarks of ferroptosis and pharmacological inhibition of ferroptosis attenuates aortic inflammation, vascular remodeling, and AAA formation. Ongoing studies will delineate the cell-specific roles of ferroptosis in progressive AAA formation and aortic rupture.
Abstract 532: Deficiency of CCN3 in Smooth Muscle Cells Perturbs Vascular Homeostasis and Exacerbates Thoracic Aortic Dissection
Authors: Xu Qian , Zhou Xianming , Qiu Yu , Lin Zhiyong
Published: October 2023

Introduction:
Thoracic aortic dissection (TAD) is a life-threatening aortic disease with a high mortality rate in the event of rupture. Key mechanisms underlying the development of TAD include extracellular matrix (ECM) degradation and vascular smooth muscle cell (SMC) reprograming. Cellular communication network factor 3 (CCN3), a matricellular protein highly expressed in SMC, exerts a myriad of context-dependent biological functions. However, its role in SMC homeostasis and in the development of TAD remains largely undefined.

Methods:
A single-cell sequencing (Sc-seq) dataset (GSE213740) from human aortic dissection patients was analyzed to determine the association between CCN3 expression and TAD. In newly weaned male CCN3-floxed mice harboring Myh11-driven inducible Cre, 4 consecutive tamoxifen injections induced CCN3 deletion in SMCs. Mice without Cre expression were subjected to the same procedures and were used as controls. After 1 week of washout, 4-week-old mice were given 0.5% BAPN (β-aminopropionitrile monofumarate) in their drinking water to induce TAD. Histologic and protein measurements were performed on TAD tissues from control and CCN3-SMC-KO mice. Sc-seq analysis was performed to recapitulate cell heterogeneities and transcriptome profile changes. The regulatory role of SMC-derived CCN3 in TGFβ signaling was evaluated by western blot and colocalization analyses.

Results:
Analysis of the human Sc-seq dataset revealed downregulation of CCN3 in aortas from patients with TAD. CCN3-positive SMCs have elevated ECM and elastin gene expression in comparison to CCN3-null SMCs. In vivo, concordant with increased mortality in CCN3-SMC-KO animals, SMC-specific deletion of CCN3 promotes BAPN-induced elastin fibers fragmentation and degradation. In vitro, SMC deficiency of CCN3 diminished TGFβ-induced elastogenesis. CCN3 binds to the TGFβ receptor on SMCs and augments TGFβ receptor phosphorylation, thereby activating Smads, upregulating SMC elastogenesis, and as a consequence, alleviates elastin fiber fragmentation and TAD development.

Conclusion:
CCN3 is a key determinate of SMC reprogramming and sustains vascular homeostasis. Our findings suggest a novel target for the prevention and treatment of TAD.
Abstract 533: Doxycycline Fuels Mitochondrial Dysfunction In Aortic Smooth Muscle Cells
Authors: Yap Carmen , Wanga Shaynah , Driessen Antoine , Wüst Rob C , de Vries Carlie J , Houtkoper Riekelt H , Lindeman Jan H , De Waard Vivian
Published: October 2023

Introduction:
The antibiotic doxycycline is known to inhibit inflammation, and was therefore considered as a therapeutic to prevent abdominal aortic aneurysm (AAA) growth. However, in two clinical trials, doxycycline-treated AAA patients did not experience reduced aneurysm growth. Doxycycline was previously shown to induce mitonuclear imbalance and reduce mitochondrial function.

Objective:
To determine that doxycycline would also impair mitochondrial function in the aorta and aortic smooth muscle cells (SMCs), giving rise to SMC phenotype switching.

Results:
Oral doxycycline intake induced mitonuclear imbalance in the murine aorta, human aortic aneurysm tissue and in human aortic SMCs. Consequently, doxycycline reduced mitochondrial activity and respiration in aortic SMCs. Transcription factor krüppel-like factor 4 (KLF4), a player in mitochondrial homeostasis, is upregulated after doxycycline treatment. Moreover, the integrated stress response effector, activating transcription factor 4 (ATF4), is enhanced to restore cellular homeostasis. These changes result in diminished expression of typical contractile SMC markers. Treatment with the drug Elamipretide (SS-31) improves mitochondrial markers and cellular stress management.

Conclusion:
Doxycycline leads to mitochondrial dysfunction and ER stress in aortic SMCs. These changes may contribute to aortic aneurysm growth by impairing SMC function. Elamipretide offers rescue from some of the harmful effects of doxycycline by improving mitochondrial function.