bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 446: Complement Component 4 Suppression Accompanies Abatement Of Liver Steatosis In Hepatocyte-specific Angiotensinogen Deficient Mice
Authors: Pettey Alex , Sawada Hisashi , Ye Dien , Wu Congqing , Daugherty Alan , Lu Hong S
Published: October 2023

Background:
Steatosis is a frequently associated with chronic liver disease. Western diet (WD)-induced steatosis has been shown consistently to be abated by hepatocyte-specific angiotensinogen (AGT) deficiency, independently of the synthesis of angiotensin peptides. However, the molecular mechanism by which AGT facilitates WD-induced steatosis is unknown. This study aims to determine the contribution of AGT to WD-induced transcriptomic changes in the liver.

Methods and Results:
To determine the molecular basis of liver steatosis, we performed temporal transcriptomic analyses by bulk RNA sequencing in mice fed a WD to induce steatosis. Livers from low-density lipoprotein receptor deficient (LDLR-/-) mice fed WD (42% of calories from fat) for 5, 14, or 42 days were evaluated against mice fed normal diet. Gene ontology analysis revealed that upregulated genes were related to inflammation at each interval. Liver transcriptomes underwent a transition at 14 days WD that persisted at 42 days, evidenced by principle component analysis of unfiltered genes. To explore the contribution of AGT in steatosis, we next performed additional liver transcriptomic analyses comparing hepatocyte-specific AGT deficient (hepAGT
-/-
) mice and wild type (hepAGT
+/+
) littermates at 14 and 42 days of WD feeding. Compared to hepAGT
+/+
littermates, there were 128 and 23 differentially expressed genes (DEGs) in hepAGT
-/-
mice at 14 and 42 days of WD, respectively. Interestingly, four DEGs were overlapped between intervals. The four DEGs were
Agt
,
Mup21
- a urinary protein with no human homolog,
Moxd1
- a predicted enzyme with no known substrate, and
C4a -
a pro-inflammatory zymogen critical in all three complement pathways. Since the gene ontology analysis identified the contribution of multiple genes related to inflammation, we focused on
C4a
. RT-qPCR detected a 2.7-fold decrease in
C4a
mRNA expression in hepAGT
-/-
mice at 42 days of WD (p=0.008 by Rank Sum Test).

Conclusions:
Inflammatory genes were upregulated early in the development of steatosis. Hepatocyte-specific AGT deficiency suppressed
C4a
mRNA. Future studies will determine which complement pathways are activated by WD and characterize the spatial distribution of
C4a
and its proteolytic derivatives.
Abstract 449: Growth Hormone-releasing Hormone Agonist Attenuates Vascular Calcification and Vascular Injury in Diabetic Db/db Mice
Authors: Ren Hao-Lin , Cai Ruiping , Xue Ruize , Zhang Yaoxia , Zhang Xianyang , Cai Renzhi , Sha Wei , Schally Andrew V , Zhou Ming S
Published: October 2023

Vascular calcification (VC) is an independent risk factor for cardiovascular diseases. VC increases mortality of all-causes. VC is one of most common cardiovascular complications in type II diabetes. So far, no therapy has been proven to be effective in treatment of clinical VC. The present study investigated the therapeutic effects of MR409, an agonistic analog of growth hormone-releasing hormone (GHRH-A), on VC in diabetic db/db mice. Diabetic mice were injected with MR409 subcutaneously every day for 8 weeks. Long-term treatment with MR409 improved serum lipid profile and endothelium-dependent relaxation to acetylcholine, and reduced vascular structural injury in diabetic mice without affecting serum growth hormone level. Echocardiography showed that calcium plaques present in heart valve of diabetic mice disappeared in diabetic mice after treatment with MR409. MR409 inhibited vascular calcium deposition associated with a marked reduction in the expressions of osteogenic-regulated alkaline phosphatase (ALP) and transcription osteogenic marker gene Runx2 in diabetic mice. MR409 also inhibited vascular reactive oxygen species (ROS) generation and upregulated the expressions of anti-calcifying protein Klotho in diabetic mice. Our results demonstrate that GHRH-A MR409 can effectively attenuate VC and heart valve calcification, and protect against endothelial dysfunction and vascular injury in diabetic mice without significantly affecting pituitary-growth hormone axis. The mechanisms may involve upregulation of anti-calcifying protein Klotho and reduction in vascular ROS and the expression of redox sensitive osteogenic genes Runx2 and ALP. GHRH-A may represent a new pharmacological strategy for treatment of VC and diabetics associated cardiovascular complications.
Abstract 452: Contribution Of Smooth Muscle-derived Vascular Progenitor Cells To Atherosclerosis
Authors: Dubner Allison M , Lu Sizhao , Jolly Austin , Noble Tysen , Nemenoff Raphael , Moulton Karen , Weiser-Evans Mary C
Published: October 2023

Purpose:
Atherosclerosis is a major cause of morbidity and mortality worldwide, but current therapies fail to adequately meet clinical needs. Our group discovered a unique population of adventitial multipotent resident vascular stem cells (AdvSca1-SM cells) that derive from mature vascular smooth muscle cells (SMCs) and are poised to respond to vascular injury. The goal of this project was to determine the role of AdvSca1-SM cells in the setting of atherosclerosis. We hypothesized that AdvSca1-SM cells contribute to plaque growth or neovascularization to drive disease progression.

Methods:
Highly specific AdvSca1-SM cell-specific lineage tracing mice were placed on either normal chow or given PCSK9 AAV injections to induce LDLR KO and placed on a high fat/high cholesterol diet for 8 to 30 weeks. The vasculature was analyzed using scRNA-Seq, immunofluorescence microscopy, and flow cytometry.

Results:
scRNA-Seq and flow cytometry revealed a large reservoir of AdvSca1-SM cells in a stem-like state in the setting of atherosclerosis, which was expected given the focal nature of plaque formation. However, significant phenotypic shifts in stem-like AdvSca1-SM cells were observed compared to normal chow conditions. Additionally, AdvSca1-SM cell-specific knockout of stemness-associated transcription factor Klf4 altered the phenotype of non-AdvSca1-SM-derived cells, indicating a signaling role for AdvSca1-SM cells to other cells in the vascular microenvironment. We also observed that AdvSca1-SM cells differentiated predominantly into into mature SMCs, modulated SMCs, and myofibroblasts, which were found in the core of the plaque, the fibrous cap, and the medial wall. Finally, these cells were observed to be associated with adventitial microvessels in plaque regions, supporting a role for AdvSca1-SM cells in plaque neovascularization.

Conclusions:
These studies emphasize the dynamic nature of AdvSca1-SM cells in the setting of chronic hyperlipidemia-induced atherosclerosis, demonstrating the potential of these cells to both differentiate into a variety of cell types as well as signal to other cells in the vascular wall. Ongoing studies are characterizing the distinct contributions of AdvSca1-SM cells to atherosclerosis.
Abstract 454: The Effect Of High-fat Diet On Vascular Smooth Muscle Cell Mechanics And Migration In Apoe
-/-
Mice
Authors: Khatiwada Nisha , Hong Zhongkui
Published: October 2023

Atherosclerosis remains a leading cause of cardiovascular diseases, which cause causes about 30% of global death. Vascular smooth muscle cells (VSMCs) are the major cellular components of arterial wall. VMSCs play critical role in the atherosclerosis development by phenotypic switching from a contractile phenotype to a synthetic phenotype, migration from media to intima of arterial wall, and deposition as foam cells in atherosclerotic plaque. In this study, we tested the effect of high-fat diet on the VSMC mechanics by comparing the mechanics and migration of VSMCs isolated from apolipoprotein-E knockout (ApoE
-/-
) and wild type (WT) mice. Atomic force microscope (AFM) was employed to test cell adhesion and stiffness of VSMCs. AFM was also used to examine live VSMC submembranous cytoskeleton organization. A data-driven mathematical models-based image and signal processing software was used to analyze AFM data. Two-way ANOVA was used to test statistical significance for all experiments. The results demonstrated significant differences in the actin cytoskeletal organization and cell mechanics between ApoE
-/-
VSMCs and WT cells upon high-fat diet. However, no significant difference in cell stiffness was observed between normal diet-fed ApoE
-/-
and WT VSMCs. These interesting findings on the effect of high-fat diet on VSMC mechanics will improve our knowledge of the mechanism underlying the contribution of VSMC in the development of atherosclerosis.
Abstract 457: Telomerase Reverse Transcriptase Protects Against Oxldl Induced Endothelial Dysfunction By Positively Regulating Autophagy
Authors: Machiraju Praveen , Ghosh Arkasubhra
Published: October 2023

Introduction:
Oxidised LDL is one of the major atherogenic agents known to cause endothelial dysfunction eventually leading to atherosclerosis. Reduction of telomerase reverse transcriptase levels have been shown to increase susceptibility to development of atherosclerosis. Apart from its conventional functions, telomerase reverse transcriptase is also known to mitigate mitochondrial stress, oxidative stress, regulate inflammation and wnt pathways. In this study, we test the hypothesis that TERT rescues endothelial dysfunction by upregulating the autophagy pathway leading to better clearance of oxLDL.

Methods:
Monocyte attachment assay, oxLDL uptake assay, transcytosis and TEER assays were performed to study the functional outcomes in endothelial cells with TERT overexpression and knockdown. Gene and protein expression assays were performed to study the levels of autophagy related genes and proteins. Co-immunoprecipitation was performed to study the binding partners of TERT and live cell imaging of lysosomes were performed to study autophagy kinetics.

Results:
Autophagy related genes and proteins were observed to significantly upregulated or downregulated upon overexpression and knockdown of TERT respectively. TERT overexpression showed 2-fold reduction in oxLDL uptake in endothelial cells(p≤0.005) whereas TERT knockdown showed 1.5-fold increase in oxLDL uptake(p<0.05). TERT overexpression in the presence of oxLDL also showed a 2-fold reduction in monocyte attachment as compared to oxLDL treated endothelial cells alone (p<0.001). TERT knockdown under oxLDL stress increased monocyte attachment by 4.2 folds as compared to controls (p<0.0001). Transcytosis assays showed a 2-fold reduction in TERT overexpressed endothelial cells and a 7-fold increase in TERT knockdown oxLDL treated endothelial cells. Further, co-immunoprecipitation and colocalization studies show that TERT binds to Rab7 protein but not LAMP1 or LC3 proteins. This binding is further increased upon oxLDL treatment.

Conclusion:
Our findings indicate that TERT is crucial for homeostatic endothelial functions and TERT overexpression rescues endothelial dysfunction in an atherogenic prone environment by upregulating autophagic pathway.
Abstract 463: Convocals: A Convolutional Neural Network To Predict Symptoms And Major Secondary Cardiovascular Events Based On High-resolution Scanned Histological Slides
Authors: Cisternino Francesco , Song Yipei , de Borst Gert Jan , Mekke Joost , de Kleijn Dominique P , Peters Tim , Westerman Roderick , Pasterkamp Gerard , Vink Aryan , van der Laan Sander W , Miller Clint L , Glastonbury Craig
Published: October 2023

Background:
Atherosclerosis is a primary risk factor underlying myocardial infarction and stroke, and there remains an unmet need to identify atherosclerotic plaque subtypes to improve clinical outcomes. Histological examination is used to manually characterize the extent and type of atherosclerosis, and histological traits are associated with major adverse cardiovascular events (MACE). Recent applications of automated machine learning approaches may improve scalability and precision in predicting symptoms and cardiovascular events, however this has yet to be fully evaluated in the setting of atherosclerosis.

Methods:
CONVOCALS uses a convolutional neural network (CNN), feature embedding and attention-based pooling model to develop an internal representation of whole slide images (WSI) and learn position and scale invariant structures in the data. We evaluated this model on human arterial H&E histology WSI from the Genotype Tissue Expression Project (GTEx), consisting of 688 WSI of normal and atherosclerotic coronary, tibial and aortic artery tissues.

Results and Conclusions:
We evaluated the performance of a base model of CONVOCALS on the GTEx artery H&E WSI dataset, which achieved a mean AUC of 0.86 and accuracy of 0.72 on a test dataset of 68 WSI images. Principal component analysis demonstrated clear separation of the 4 distinct atherosclerosis stages. We also visualized different pathological hallmarks (e.g., calcification) using attention scores overlaying the WSI. We further evaluate CONVOCALS performance on more granular feature extraction and demonstrate improved WSI segmentation. Next we plan to evaluate the performance on a large-scale AtheroExpress dataset to predict symptoms and MACE.
Abstract 467: Role Of An Endothelial-enriched Lncrna On Inflammatory Signaling And Vascular Endothelial Function
Authors: Chandra Amit , Randhawa Vinay , Chen Jingshu , Feinberg Mark W
Published: October 2023

Introduction:
Activation and dysfunction of endothelial cells (ECs) in response to inflammation contribute to the initiation and progression of atherosclerosis. Long noncoding RNAs (lncRNAs) are emerging regulators in the maintenance of a fully functional vascular endothelium. However, the role of lncRNAs in regulating EC inflammation and functions remains poorly understood.

Methods:
We analyzed RNA-sequencing data from the aortic intima and media of atherosclerotic Ldlr–/– mice placed on a high-fat sucrose containing diet. We identified an EC-enriched lncRNA GM39822 and assessed its expression pattern during lesion progression and regression. We hypothesized that GM39822 may regulate inflammation induced activation and function of ECs. We performed loss- and gain-of-function studies and assessed the regulatory roles of GM39822 in EC inflammatory signaling pathways, leukocyte-endothelial adhesion, and in EC proliferation, migration, and apoptosis.

Results:
GM39822 expression increased with atherosclerotic progression with minimal reduction during regression. GM39822 is a nuclear enriched lncRNA and is highly expressed in ECs compared to other major cells of the vasculature. GM39822 knockdown inhibited the expression of 3 major inflammatory signaling pathways NF-kB, p38k, and ERK1/2, which contribute to induction of EC adhesion molecules. In line with this, GM39822 knockdown decreased the expression of VCAM1 (0.52 ± 0.11-fold, p=0.03,) and E-selectin (0.09 ± 0.001-fold, p=0.0031) at the mRNA level and decreased VCAM1 (0.44 ± 0.15-fold, p=0.03) at the protein level. GM39822 knockdown also markedly reduced adhesion of PBMCs to mouse ECs (bEND.3 cells) (0.19 ± 0.04-fold, p=0.0005), cellular proliferation (0.65 ± 0.04-fold, p=0.0007), EC migration (0.14 ± 0.04-fold, p=0.0033) and caspase 3/7 activity (0.21 ± 0.004-fold, p<0.0001). In contrast, GM39822 overexpression increased expression of NF-kB p65, p50, VCAM1, PBMC-EC adhesion, proliferation, migration, and caspase 3/7 activity.

Conclusion:
LncRNA GM39822 is a key regulator of inflammation and downstream functions of endothelial cells. Further investigation of lncRNA GM39822 may help to provide novel anti-inflammatory therapy in chronic vascular disease states.
Abstract 471: Identifying Novel Drug Candidates To Improve Efferocytosis In Atherosclerosis
Authors: Luo Lingfeng , Kojima Yoko , Ye Zhongde , Leeper Nick J
Published: October 2023

Introduction:
Foam cells undergoing apoptotic cell death contribute to necrotic core formation and plaque instability during atherosclerosis. Efferocytosis, the phagocytic cleanup of apoptotic cells by macrophages, plays a pivotal role in maintaining plaque stability and ameliorating atherogenesis.

Hypothesis/Aim:
In this study, we aim to screen drugs from a compound library to discover novel therapeutic targets to boost efferocytosis against apoptotic foam cells.

Methods:
First, we used a flow cytometry-based phagocytosis assay
in vitro
to measure the phagocytic activity against foam cells or apoptotic cells in a high throughput manner. We treated macrophages with 2966 compounds from MCE FDA Approved & Pharmacopeial Drug Library during this screening. After treatment, these macrophages were labeled with a fluorescent cell tracker before incubating with target cells (foam/apoptotic cells) that were labeled with a cell tracker of a different fluorophore. Then the phagocytic ratio was analyzed via a flow cytometer. The results were validated on human peripheral blood mononuclear cells-derived macrophages, mouse bone marrow-derived macrophages, and the RAW 264.7 cell line. After identifying the top drug candidates, we validated the results through an IncuCyte live-cell phagocytosis assay. In this assay, target cells were labeled with pHrodo, the fluorescent signal of which increased dramatically when the target cells were ingested by macrophages. After narrowing down the drug list, we performed bulk RNA sequencing to examine the gene expression changes induced by these drugs to select the most promising drug for future validation
in vivo
.

Results:
8 drug candidates passed the initial large-scale screening and validation. Among them, 2 drugs greatly changed the gene expression pattern of macrophages. The gene ontology analysis revealed Drug 1 was associated with genes involved in the immune and inflammatory response. We are currently characterizing the signaling pathways targeted by this drug and validating our findings
in vivo
with atherosclerotic mouse models.

Conclusions:
Through this drug screening, we identified a novel drug candidate that stimulates efferocytosis to clean up apoptotic foam cells.
Abstract 476: Negligible Impacts Of Anesthesia On The Morphodynamics Of Individual Red Blood Cells What Is Good News For Vessels: A Study Exploiting Holotomography
Authors: Chaszczewska-Markowska Monika , Antonczyk Agnieszka , Gorna Katarzyna , Kielbowicz Zdzislaw , Bogunia-Kubik Katarzyna , Przadka Przemyslaw
Published: October 2023

Rationale:
The shape and membrane dynamics of individual red blood cells (RBCs) are known to play a significant role in their functionality. The morphodynamics of RBCs optimizes their flow properties in vessels and capillaries, which is important for the balance and optimal distribution of blood and nutrients to the tissues. It is also crucial in pathological contexts because changes can influence vascular and microvascular hemodynamics. It can dysregulate vascular function through the modulation of oxygen delivery and the scavenging and generation of nitric oxide. Therefore, the ideal conditions of RBCs are essential for healthy vascular function. Anesthetics, sedatives, analgesics, and fluids used during anesthesia can impact RBCs. The same applies to pneumoperitoneum performed in laparoscopic procedures.

Objectives:
Taking above we aimed to prospectively evaluate the influence of anesthesia on RBC volume (CV, fL), surface area (SA, μm2), sphericity (SI), dry mass (CH, pg), Hb concentration (CHC, g/dL) and membrane fluctuation (nm).

Methods:
We exanimated RBCs (n=760) from 5 female dogs undergoing laparoscopic-assisted ovariohysterectomy (OVH). The blood was collected 15 min after premedication with medetomidine and 1h after full anesthesia (isoflurane, fentanyl). Measurements of RBC parameters we performed using the non-invasive, label-free method of imaging with holotomography. Statistical significance was determined using Mann-Whitney test.

Results:
Morphodynamics parameter values during anesthesia were stable. We observe statistically no significant changes for RBC parameters after premedication vs. one hour of full anesthesia: CH 24.20 vs. 24,19, p=0.57; CV 59.17 vs. 59.04, p=0.89; CHC 42.33 vs. 41.53, p=0.60; SA 117.38 vs. 119.56, p=0.13; SI 0.63 vs. 0.62, p=0.31; fluctuation 68.18 vs. 65.18, p=0.95.

Conclusions:
Procedures related to OVH are not reflected in the parameters of RBCs and their well-being. This provides new insight into the impact and safety of the anesthetic protocol used in this study on blood cell function, which translates into the lack of risk of vascular damage.
Abstract 478: Aberrant Glycogen Synthase Kinase 3 Activation In Liver Sinusoidal Endothelial Cells Enhances Liver Inflammation In Nonalcoholic Steatohepatitis
Authors: Ibrahim Samar
Published: October 2023

Nonalcoholic steatohepatis (NASH) is characterized by excess circulating toxic lipids, along with hepatic steatosis, and recruited myeloid cells-mediated liver inflammation. Myeloid cells adhesion to the liver sinusoidal endothelial cells (LSEC) and transendothelial migration are crucial elements in the inflammatory process, and LSEC under lipotoxic stress develop a pro-inflammatory phenotype known as endotheliopathy. However, the exact mechanisms of this phenomenon are obscure. To define the molecular mediators of LSEC endotheliopathy, we isolated primary mouse LSECs from C57BL/6J mice on chow or a NASH-inducing diet rich in fat, fructose, and cholesterol (FFC), and subjected them to multi-omics studies. An integrated pathway analysis of the mouse LSEC proteomics and transcriptomics indicates that leukocyte transendothelial migration and focal adhesion are the major pathways altered in NASH LSEC. To explore the LSEC intracellular signaling networks, we performed kinome profiling of the LSEC phospho-proteomics and identified Glycogen Synthase Kinase 3 (GSK3)β as the major hub, and the top kinase in NASH. We then confirmed that GSK3β is activated in primary human LSEC treated with the toxic lipid Palmitate (PA). PA also promoted the LSEC pro-inflammatory response by upregulating
CXCL2
and intracellular adhesion molecule 1
(ICAM-1)
, all of which were attenuated with the GSK3 inhibitor LY2090314 (LY). Using a collagen gel-based transmigration assay, we demonstrated that THP-1 monocyte migration through an LSEC monolayer treated with PA was reduced by LY. In FFC-fed mice, LY treatment ameliorated liver inflammation, injury, and fibrosis as assessed by reduced F4/80, MPO, and ICAM-1 immuno-staining, ALT serum levels and hepatic TUNEL-positive cells, Sirius red and alpha smooth muscle actin staining of the liver. Mice with NASH-induced diet had reduced haptic neutrophil and proinflammatory monocyte infiltration when treated with ICAM1 neutralizing antibody. Furthermore, we confirmed increased LSEC expression of ICAM1 in human with NASH.
In conclusion,
GSK3 inhibition attenuates toxic lipid induced LSEC pro-inflammatory phenotype and may serve as a potential therapeutic strategy in human NASH.
Abstract 481: Deletion Of Talin1 In Myeloid Cells Facilitates Diet-induced Atherosclerosis
Authors: Shao Bojing
Published: October 2023

Rationale:
Integrin-regulated monocyte recruitment and cellular responses of monocyte-derived macrophages are critical for the pathogenesis of atherosclerosis. In the canonical model, talin1 controls ligand binding to integrins, a prerequisite for integrins to mediate leukocyte recruitment and induce immune responses. However, the role of talin1 in the development of atherosclerosis has not been studied.

Objective:
To study the role of talin1 in myeloid cells, which include monocytes, in the progression of atherosclerosis.

Methods and Results:
On an

Apoe
-/-

background, myeloid talin1-deficient mice and the control mice were fed with a high-fat diet for 8 or 12 weeks to induce atherosclerosis. Surprisingly, myeloid talin1 deletion facilitated the formation of atherosclerotic lesion and macrophage deposition in lesions. In a flow chamber assay, talin1 deletion abolished integrin β2-mediated adhesion of monocytes but did not impair integrin α4β1-dependent cell adhesion. Strikingly, talin1 deletion did not prevent Mn
2+
- or chemokine-induced activation of integrin α4β1 to the high-affinity state, which allows ligand binding to integrins. In an
in vivo
competitive homing assay, monocyte infiltration into inflamed tissues was prohibited by antibodies to integrin α4β1 but was not affected by talin1 deletion or antibodies to integrin β2. Furthermore, quantitative PCR and ELISA analysis showed that ligand binding to integrin β3 inhibited macrophages to produce inflammatory cytokines. Talin1 deletion abrogated the negative regulation of integrin β3 in cytokine generation.

Conclusions:
Integrin α4β1 controls monocyte recruitment during atherosclerosis. Talin1 is dispensable for integrin α4β1 activation to the high-affinity state and integrin α4β1-mediated monocyte adhesion. Thus, talin1 deletion does not impair monocyte recruitment during atherosclerosis. However, talin1 deficiency abolishes the negative regulation of inflammatory cytokine production by macrophage integrin β3, leading to enhanced atherosclerosis. Our study provides novel insights into the roles of myeloid talin1 and integrins in the progression of atherosclerosis.
Abstract 484: Antidiabetic Agent, Canagliflozin, Is A Potent Vasodilator Of The Coronary Microvasculature
Authors: Banerjee Debolina , Xing Hang , Feng Jun , Sellke Frank
Published: October 2023

Objective:
Sodium-glucose cotransporter-2 (SGLT-2) inhibitors are cardioprotective even in the absence of preexisting diabetic microvascular disease. Our group has shown SGLT-2 inhibitor, canagliflozin, increases myocardial perfusion without an attendant increase in angiogenesis. We hypothesize canagliflozin accentuates vasodilation of coronary microvasculature to increase perfusion.

Methods:
Coronary resistance arterioles (174 ± 60μm in diameter) isolated from Sprague-Dawley rats either receiving daily canagliflozin (15mg/kg, oral gavage) for four weeks (n = 4) or no treatment (n = 4) were studied
in vitro
in a pressurized (40mmHg), no-flow state using video microscopy. Following preconstriction of microvessels with thromboxane analog U46619 or endothelin-1, concentration response curves to vasodilatory adenosine 5’-diphosphate (ADP), acetylcholine (ACh), and sodium nitroprusside (SNP) were completed. Additionally, nitric-oxide-dependent ADP-induced vasodilation of microvessels isolated from treated rats was measured in the presence of N
ω
-Nitro-L-arginine methyl ester (L-NAME) 10μM.

Results:
Canagliflozin treatment was associated with a significant increase in endothelium-dependent vasodilatory response to ADP (p = 0.039) (Figure 1). Pretreatment with L-NAME, an inhibitor of nitric oxide synthase, eliminated this vasodilatory response. Compared to ADP, vasodilatory response to ACh was muted (p = 0.552). Endothelium-independent vasodilatory response to SNP, a nitric oxide donor, strongly trended toward an increase in the treated group (p = 0.099).

Conclusions:
Canagliflozin improves microvascular vasodilation in an endothelium-dependent and endothelium-independent fashion not yet described. These findings implicate both vascular endothelium and arteriolar smooth muscle cells in altered vasomotor physiology and may, in part, account for the observed increase in myocardial blood flow with canagliflozin treatment.
Abstract 495: Tissue Engineering Of A Three-layered Artery
Authors: Lehertshuber Constanze A , Jia Siyue , Rudolph Mathias , Lorentschk Michael , Li Shuangyue , Kellerer Thomas , Romer Amos , Bauer Sabine , Moggio Aldo , Sager Hendrik , Von Scheidt Moritz , Clausen-Schaumann Hauke , Kessler Thorsten , Sudhop Stefanie , Schunkert Heribert , Chen Zhifen
Published: October 2023

A human-induced pluripotent stem cell (hiPSC)-3D artery mimicking the multi-layered anatomy of blood vessel has not yet been established. Here, we aimed to engineer arteries consisting of human endothelial cells (EC), vascular smooth muscle cells (VSMC), and fibroblasts, correspondingly mimicking the main cellular components of the intima, media, and adventitia of a blood vessel. We differentiated hiPSC to obtain EC, VSMC, and fibroblasts. A 3D printing-based molding technique was used to engineer the circular structure of an artery. For the media-like layer, hiPSC-VSMCs mixed with collagen were seeded into 3D-printed polylactic acid molds with a centered needle serving as supporting structure. The adventitia-like layer was engineered around the VSMC-layer using the same method but with fibroblast-collagen mixture. After formation of the fibroblast layer, the needle was removed and hiPSC-ECs were coated on the VSMC lumen by rotational cell injection. Brightfield, fluorescence and two-photon microscopy were utilized to visualize cell arrangement, EC coverage, and collagen usage and organization. RNA sequencing (RNAseq) was harnessed to molecular profile cells in 2D (Petri dish) and 3D culture (engineered arteries). With 10M hiPSC-VSMCs, 10M hiPSC-fibroblasts, and 0.2M hiPSC-EC, we successfully engineered the three-layered “artery”. The built-up took 16 days. The condensation of cell-collagen mixture likely relied on the rearrangement of collagen fibers by the cells through enzymic reactions in the extracellular matrix. Immunostaining of alpha-SMC actin indicated rod-shaped hiPSC-VSMC aligned in the artificial tubes. Two-photon microscopy revealed over 95% coverage of hiPSC-EC of the luminal wall. Transcriptome data suggested that 3D culture promotes the expression of genes of the calcium signaling pathway and leads to downregulation of genes of the atherosclerosis pathway in SMC. Taken together, the human-induced pluripotent stem cell (hiPSC)-3D artery might serve as a model for functional studies and experimental models of vascular injury and inflammation. In addition, our three-layered tube - mimicking a human artery - seems to improve the molecular profile of vascular cells derived from hiPSC as compared to 2D culture.
Abstract 496: Inhibition Of Tirna-gly-gcc Ameliorates Neointimal Formation Via Cbx3-mediated Vascular Smooth Muscle Cells Phenotypic Switching
Authors: Li Fengshi , Rong Zhihua , Liu Changwei
Published: October 2023

Introduction:
Vascular smooth muscle cells (VSMCs) are the main component of blood vessels and synthetic VSMCs play an essential role in vascular diseases. tRNA-derived fragments (tRFs) are a new class of non-coding RNAs involved in a variety of pathological processes, but their biological functions and mechanisms in VSMCs phenotype transition and vascular intimal hyperplasia are unclear.

Hypothesis:
We assessed the hypothesis that tRFs play important role in VSMCs phenotype transition, regulation of tRFs could ameliorates neointimal formation.

Methods:
Differential expression profiles of tRFs from different phenotype of VSMCs was acquired using small-RNA sequence. The expression of tiRNA-Gly-GCC was confirmed in VSMCs, vascular tissues and plasma from healthy and atherosclerosis using quantitative real-time PCR and RNA fluorescence in situ hybridization. Gain-and loss- of function were performed using mimics and inhibitors to investigate the role of tiRNA-Gly-GCC in VSMCs phenotypic transition and neointimal formation after vascular injury. Dual-luciferase reporter gene and rescue experiments were performed to demonstrate the relationship between tiRNA-Gly-GCC and CBX3.

Results:
tiRNA-Gly-GCC is upregulated in synthetic VSMCs, atherosclerotic arteries, plasma and the balloon injured carotid artery of rats. Functionally, the inhibition of tiRNA-Gly-GCC represses VSMCs proliferation, migration, and reversed dedifferentiation, whereas the overexpression of tiRNA-Gly- GCC have contrary effects. Mechanistically, tiRNA-Gly-GCC performs these functions on VSMCs
via
downregulating the expression of CBX3. Finally, the inhibition of tiRNA-Gly-GCC could ameliorate neointimal formation after vascular injury
in vivo
.

Conclusions:
In conclusion, tiRNA-Gly-GCC is a mediator of VSMCs phenotypic switching by targeting CBX3, inhibition of tiRNA-Gly-GCC suppresses neointimal formation.
Abstract 498: A Color-Based Image Segmentation Pipeline Depicts Collagen, Versican, And Secretory Vascular Smooth Muscles Constituting The Wall Of Human Explantedarteriovenous Fistulas
Authors: Lotfollahzadeh Saran , Zhang Yichi , Kolachalama Vijaya B , Chitalia Vipul C
Published: October 2023

Background:
Arteriovenous Fistula (AVF) is the lifeline of patients with End-Stage Kidney Disease (ESKD) on hemodialysis. AVF undergoes profound remodeling to accommodate arterial pressure and flow, a process called AVF maturation or arterialization of the vein. In the US, more than 50% of AVF fail to mature. Despite the central importance of this process in functional AVF, there is a dearth of studies characterizing the components of walls of the mature AVFs in humans.

Methods:
The AVF explants were probed with Masson Trichrome, Sirius Red, and Versican, a predominant proteoglycan in the vessel. This was followed by a color-based segmentation pipeline with a machine-learning approach to quantify the components of AVF explants. The state of vascular smooth muscle cells (VSMCs) was characterized using specific markers.

Results:
Twenty-one patients had AVFs explanted for aneurysm (71.42%), pseudoaneurysm, stenosis, or aesthetic reasons (each in 9.52%). Neointimal hyperplasia characterized by intimal thickening was present in all AVFs. Other features included calcification, inflammatory infiltrate, needle track injury, and thrombus. Collagen was deposited concentrically in the explanted AVFs with aneurysms and in an eccentric manner in the AVFs with pseudoaneurysms (Fisher’s exact test, P= 0.025). Quantitative histological analysis showed that in the AVF wall, collagen, proteoglycan, and VSMCs, constituted 27.78%, 31.45%, and 19.80% of the wall, respectively. Compared to VSMC, the collagen and versican were in the ECM higher by 1.40-fold and 2.76-fold, respectively (p <0.001). Compared to Ki67 expression, VSMCs showed prominent expression of MYH11 (P= 0.0016), α-smooth muscle actin (P= 0.0023), and Calponin (P= 0.006), all markers of differentiated VSMCS with secretory phenotype.

Conclusion:
This is the first study elucidating the composition of mature AVF in humans and shows a preponderance of collagen and versican as components of AVF walls. The VSMCs demonstrated secretory phenotype in the wall of the explanted AVFs. This study supports future investigation in regulating collagen and versican deposition and secretory VSMCs in the maturation of the outflow vein of AVF.
Abstract 499: A Novel
En Face Ex Vivo
Live Imaging Approach For The Visualization Of Intimal Myeloid Cell Dynamics
Authors: Polenz Chanele K , Ibrahim Hisham , Althagafi Marwan , Scipione Corey A , Hyduk Sharon , Cybulsky Myron I
Published: October 2023

Populations of CD11c
+
intimal myeloid cells (MCs) are known to reside in regions of the vasculature predisposed to atherosclerosis, such as the lesser curvature of the ascending arch. In Ldlr-/- mice, these cells have been shown to take up lipid and contribute to the formation of a nascent lesion under conditions of hypercholesterolemia. The role of resident CD11c+ cells in normal, healthy aortas has only recently been investigated. One function for these MCs may be the clearance of pathogens from the subendothelial space via reverse transendothelial migration (RTM). RTM can be triggered by systemic stimulation of cytokine and toll-like receptors (such as toll-like receptor 4 by lipopolysaccharide [LPS]), and induces a reduction in the number of resident intimal MCs in the lesser curvature. Our investigation of intimal RTM suggests that this cell loss is due to egress rather than cell death and is dependent on CCL19-CCR7 signaling, however we have yet to directly visualize this process. We endeavour to visualize resident intimal MC dynamics, including RTM, using
ex vivo
live imaging of the aortic intima. Ascending aortic arches were harvested from mice with fluorescent intimal myeloid cells (CD11c-eYFP mice or LyzM
Cre
-TdTomato reporter mice), stained with an endothelial marker (VE-Cadherin), and mounted
en face
in an imaging system that allows for constant perfusion of buffer across the tissue. Using a confocal microscope, images of the lesser curvature were continually acquired over a period of several hours following different stimuli to visualize cell behaviour. Intimal MCs were observed extending and retracting their dendrites under conditions of fluid flow, as well as undergoing cell division. Following the addition of the inflammatory stimulus LPS, cells can be seen migrating across and disappearing from the field of view. Thus, this method allows for direct visualization of intimal myeloid cell behaviour following perturbation by either physical or pharmacological stimuli. This approach, in tandem with pulsed perfusion of CD45-targeted antibodies, could provide a valuable toolkit to study immune cell egress from the intima of the lesser curvature of the aortic arch, under homeostasis and during the progression of atherosclerosis.
Abstract 500: Sigmar1 Deficiency Promotes Inflammatory Signals By Altering Nitric Oxide Bioavailability And Mitochondrial Redox Balance In Vascular Endothelial Cells
Authors: Remex Naznin Sultana , Abdullah Chowdhury S. , Aishwarya Richa , Nitu Sadia , Traylor James , Kolluru Gopi , Kevil Chris , Orr Wayne W , Bhuiyan Md Shenuarin
Published: October 2023

Mitochondria in endothelial cells primarily serve as major signaling organelles thatregulate cellular redox balance and associated nitric oxide (NO) bioavailability.Mitochondrial reactive oxygen species (ROS) and NO have pro- and anti-inflammatoryeffects, respectively. Imbalance of these molecules leads to endothelial activation andvascular inflammation initiating the development of major cardiovascular diseases.Sigma1 receptor (Sigmar1) protein is a mitochondrial membrane protein regulatingmitochondrial respiration and function. Only few previous studies have identifiedSigmar1’s roles in vascular function and inflammation using nonselective ligands inpreclinical pathological models. However, the direct physiological role and molecularmechanisms of Sigmar1 in regulating vascular endothelial function and inflammationremains unknown. In this study, we have confirmed Sigmar1’s expression in vascularendothelial cells and analyzed its function in vascular inflammation using cell cultureand mice models. In flow mediated dilation study of left femoral artery, our globalSigmar1 knockout (Sigmar1
-/-
) mice failed to increase the vessel diameter and bloodflow velocity in response to reactive hyperemia compared to wild type littermate control(Wt) mice (N=6/group). We also found reduced nitric oxide (NO) bioavailability in theplasma of Sigmar1
-/-
mice. Consistently, phosphorylated-eNOS (ser-1177) expressionwas significantly decreased in Sigmar1
-/-
mice aorta and in mouse pulmonarymicrovascular ECs (MPVECs) isolated from Sigmar1
-/-
mice. Ultrastructural analysis ofthe Sigmar1
-/-
mice aorta showed altered mitochondrial morphology with elongatedshaped mitochondria and reduced mitochondrial respiration. The MPVECs isolated fromSigmar1
-/-
mice also showed decreased mitochondrial respiration, higher proteinexpression of mitochondrial superoxide dismutase and catalase. Finally, we observedelevation of inflammatory markers including VCAM1 and ICAM1 in Sigmar1 deficientMPVECs and higher vascular remodeling indicated by increased fibrosis in blood thevessels of Sigmar1
-/-
mice. Overall, Sigmar1 regulates endothelial inflammation byaltering mitochondrial redox balance and NO bioavailability.
Abstract 512: Validating An Ex Vivo System To Study Human Platelet Biomechanical Activation
Authors: Guntupalli Suman , Malloy James S , Godwin Matthew , Jennings Courtney , Aggarwal Anu , Bhandari Rohan , Rayz Vitaliy , Cameron Scott J
Published: October 2023

Background:
Arterial blood flow becomes turbulent in areas of stenosis, ectasia, or aneurysm. Platelets are exquisitely sensitive to external mechanical forces. Evaluating biomechanical platelet activation would provide insight into a new class of antiplatelet medications

Aims:
Manufactured flow cones were utilized to model smooth (S-Flow) and disturbed (D-flow) flow patterns under different conditions. Platelet activation was assessed by alpha granule exocytosis or changes in expression of known platelet surface mechanoreceptors.

Methods:
Using bespoke cones driven by a magnetic field, we constructed models based on cone dimensions to estimate platelet exposure to fluid mechanical stress by numerically simulating the flow patterns, then calculating Von Mises stress at each point under flow. Expression of platelet surface P-selectin, Glycoprotein VI (GPVI), Glycoprotein 1bα (GP1bα), and activated Glycoprotein IIb/IIIa (GPIIb/IIIa) was assessed by FACS using labeled antibodies as mean fluorescence intensity (MFI) before/after S-flow and D-flow at 400 rpm or 400 rpm for 90 mins. [PF4] in platelet releaseate was evaluated by ELISA.

Results:
Preliminary results suggest that platelet Von Mises stress computed for D-flow conditions by sculpted cones was within range of estimated stress in an ectatic carotid artery. S-flow exposure
ex vivo
did not significantly activate platelets, release of PF4, or P-selectin. D-flow, conversely, increased platelet surface p-selectin (MFI 162+/-58 vs. 550+/-144, P=0.018) but decreased GP1bα (MFI 16724+/-1720 vs. 11948+/-1036, P=0.03). PF4 was detected in platelet releasate in concentrations proportional to increasing orders of magnitude D-flow

Conclusions:
We hereby show a reproducible system to detect biomechanical platelet activation in D-flow environments
ex vivo
that will allow for platelet-derived biomarker discovery during D-flow stress and assessment of new anti-platelet therapeutics.
Abstract 514: Inhibiting The P2Y12 Receptor In Megakaryocytes And Platelets Suppresses Interferon-associated Inflammatory Responses
Authors: Sowa Marcin A , Wang Tricia T , Schlamp Florencia , Cornwell Macintosh , El Bannoudi Hanane , Hannah Bash , Clancy Robert , Ruggles Kelly , Buyon Jill , Voora Deepak , Barrett Tessa J , Berger Jeffrey S
Published: October 2023

Megakaryocytes (MKs) and platelets are recognized as mediators of inflammation and immunity. Antiplatelet therapies are widely used to prevent cardiovascular events; however, their role in attenuating platelet-mediated inflammation is uncertain. We investigated whether antiplatelet drugs, aspirin (ASA) and P2Y
12
inhibitors, alter the MK and platelet transcriptomes. We hypothesized that antiplatelet therapy reduces MK- and platelet-mediated inflammation. RNA-seq was performed in CD34
+
derived MKs treated with ASA (100 μM), P2Y
12
inhibitor (AZD1283, 5 μM) or PBS for 24 hours. We validated our MK model using RNA-seq data from platelets of healthy volunteers randomized in a crossover design to low-dose ASA (n = 70, 64% female, 43.6 ± 9 years) or P2Y
12
inhibitor (ticagrelor, n = 57, 61% female, 43.9 ± 10 years) for 4 weeks each. The clinical relevance of our findings was assessed using RNA-seq data from platelets of SLE patients (n = 54, 100% female, 40.6 ± 12 years) and matched controls (n = 18, 100% female, 42.1 ± 15 years). Finally, we validated our findings
in vitro
using RT-qPCR and western blot. We identified 74 ASA- and 2,499 P2Y
12
inhibitor MK-responsive genes. In contrast to ASA, P2Y
12
inhibitor-mediated gene changes between MK and platelet transcriptomes were positively correlated (R=0.37, p<0.001). P2Y
12
inhibition downregulated IFNα response pathway in both MKs (NES = -2.3, p = 0.002) and platelets (NES = -2.24, p = 3.22 х 10
-8
). SLE is characterized by activation of the IFN system. Consistently, the IFNα pathway was upregulated in platelets from SLE patients versus controls (NES = 3.38, p = 1.25 х 10
-9
). Among 72 upregulated IFNα genes in SLE patients, 33 were inhibited with P2Y
12
inhibition in MKs or platelets, including IRF7 and IFITM3. Finally, we validated the P2Y
12
inhibitory effect on IFN-associated responses. While IFNα (1000 U/mL) significantly upregulated both IRF7 and IFITM3, pre-treatment with P2Y
12
receptor inhibition attenuated this response on both, the transcriptomic and proteomic levels. Our data indicate that targeting the P2Y
12
receptor attenuates MK- and platelet-mediated IFN signaling pathways. P2Y
12
-mediated suppression of platelet-induced IFNα signaling may be beneficial in inflammatory and autoimmune diseases including SLE.
Abstract 517: Covid Infection Increases Mortality In Patients Admitted For Acute Aortic Dissection.
Authors: Amoah Joseph , Noureldin Sara , Somoza-Cano Francisco , Fatuyi Michael , Dekowski Sammir
Published: October 2023

Objectives:
Coronavirus Disease 2019(Covid 19) can cause mild-to-severe multiorgan disease. In this study, we sought to estimate the impact of covid infection on the clinical outcome of patients admitted for acute aortic dissection.

Methods:
Using the 2020 National Inpatient Sample Database, NIS was searched for adults with acute aortic dissection as a principal diagnosis with or without Covid Infection as a secondary diagnosis using ICD-10 codes. The primary outcome was inpatient mortality with secondary outcomes being acute kidney injury (AKI), cardiogenic shock, stroke, myocardial infarction (MI), intraoperative and post-procedure complications, acute mesenteric ischemia, length of hospital stays, and charges.

Results:
Of 15,325 patients admitted for acute aortic dissection, 1% had Covid infection. Patients with Covid were younger (62, 95% CI 57 - 68 VS 63, 95% CI 63 - 64). Patients with Covid had increased in-hospital mortality(p=0.0203) which was statistically significant. There was an increased rate of AKI (p=0.0203) and acute hypoxic respiratory failure (p<0.0001). Patients with Covid infection also had increased length of hospital stay (18 days, 95% CI 12 - 25 VS 9 days, 95% CI 9 - 10) and total hospital charges ($ 413,351.5, 95% CI $ 249863.9 - $576,683.9 VS $275060.3, 95% CI $251,129 - $298,991.5). There was no difference in cardiogenic shock(p=0.7717), stroke (p=0.0787), cardiac tamponade (p= 0.7412), myocardial infarction (p=0.2487), intraoperative and post-procedure complications (p=0.8533) and acute mesenteric ischemia (p= 0.6599).

Conclusion:
Per this study, patients with Covid infection had increased mortality when compared to the general population admitted for acute aortic dissection. Aggressive treatment of Covid infection may reduce mortality as vigorous cough which is associated with Covid 19 can cause worsening aortic dissection.
Abstract 524: Late Adult Presentation Of Anomalous Left Coronary Artery From The Pulmonary Artery Syndrome: The Utility Of Cardiac Computed Tomography In The Diagnostic Evaluation Of Cardiac Patients
Authors: Nwaedozie Somto , Patel Hiral , Khan Muhammad , Baser Hatice
Published: October 2023

Anomalous origin of the left coronary artery from the pulmonary artery (ALCAPA) syndrome is a very rare congenital Coronary artery anomaly with uncommon presentation in adulthood but a cause of sudden cardiac death. A 62-year-old female with no cardiac history presented with complaints of non-exertional chest discomfort. Her EKG was unremarkable but her cardiac troponin was mildly elevated. Functional assessment using a Regadenoson myocardial perfusion imaging showed no normal LV function without reversible ischemia. Based on her recurrent symptoms and intermediate probability of coronary artery disease, a Cardiac CT was done and demonstrated an anomalous LCA origin from the pulmonary trunk (Fig 1). She was referred for surgical repair. The possibility of adult cardiac congenital anomaly should be considered in the diagnostic evaluation of chest pain especially following unremarkable results from functional assessment tests. ECG gated Cardiac CT angiography is the preferred non-invasive diagnostic modality to visualize direct anomalous LCA origin from the main PA. Surgical repair is the definitive treatment.In the evaluation of adults with cardiac symptomatology, congenital cardiac anomalies should be considered in the differentials in the appropriate clinical settings, especially when functional evaluations are negative.