bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 307: Targeted Activation Of The Tnfa Pathway Accelerates Atherosclerosis Regression In Mice
Authors: Bergstrom Paige , Maldonado Richard , Pamir Nathalie , Fazio Sergio , Mueller Paul A
Published: October 2023

Aggressive lipid lowering in mice with established atherosclerosis induces plaque regression and is characterized by reduced monocyte recruitment and increased expression of the motility receptor C-C chemokine receptor 7 (CCR7) to promote macrophage egress from plaques to nearby lymph nodes. Notably, genetic deletion of receptors critical for the recruitment of inflammatory Ly6C
high
monocytes is necessary to facilitate plaque regression. Our group previously showed that deletion of macrophage LDL receptor-related protein 1 (LRP1) in mice with established atherosclerosis accelerates plaque regression after lipid lowering and is associated with increased inflammation and macrophage CCR7-mediated egress. We tested the hypothesis that targeted activation of inflammation following lipid lowering in atherosclerotic mice accelerates atherosclerosis regression. LC-MS/MS proteomics performed on murine macrophages revealed that the loss of LRP1 upregulated the inflammatory protein tumor necrosis factor alpha (TNFα) 1.48-fold (P<0.05).
Apoe
-/-
mice were fed a Western diet for 12 weeks to establish atherosclerosis and then subjected to bone marrow transplantation (BMT) using wildtype (
Apoe
+/+
) mice as donors to restore plasma APOE and reduce lipids. A cohort was sacrificed two weeks after BMT and used as baseline (N=11). The remaining mice (N=12/group) received intraperitoneal injections of recombinant murine TNFα (0.075mg/kg/day) or saline for 6 weeks on chow diet. After 6 weeks on chow diet, total plasma cholesterol levels ranged 82 -120 mg/dL with no significant differences between treatment cohorts. TNFα-treated mice had elevated spleen-to-body weight ratios compared to saline treated controls (P<0.0001), confirming successful activation of the immune system. Following 6 weeks of lipid lowering, TNFα treatment reduced total plaque area and lipid content 17.0% (P<0.05) and 34.4% (P<0.05), respectively, compared to saline treated controls (P<0.05).Herein, we show that the administration of TNFα, to stimulate inflammation after lipid lowering, significantly reduces plaque size and plaque lipid content in mice with established atherosclerosis.
Abstract 310: Adenosine Receptor 2A-mediated Endothelial-Mesenchymal Transition Promotes Atherosclerosis
Authors: Cai Yongfeng , Zhou Yaqi , Yang Qiuhua , Ma Qian , Zhao Dingwei , Huo Yuqing
Published: October 2023

Purpose:
Endothelial-mesenchymal transition (EndMT) leading to endothelial dysfunction, inflammation and disruption of endothelial barrier, promotes the development of atherosclerosis. We have previously shown that inactivation of adenosine 2A receptor (ADORA2A) inhibits the formation of atherosclerosis lesion in mice. However, the underlying mechanism has not been fully studied. Here we investigated the role of ADORA2A-drive EndMT in the development of atherosclerosis.

Methods:
The expression of ADORA2A in vascular endothelial cells (EC) at atheroprone regions of mouse aortas were examined by
en face
staining with ADORA2A and CD31 antibodies. EC-specific
Adora2a
knockout mice and inhibitor KW6002 were used to investigate the role of endothelial ADORA2A in EndMT and atherosclerosis
in vivo
.
In vitro
studies, the role of ADORA2A in TGFβ-1-induced EndMT was examined by Western blot, qPCR and immunostaining.

Results:
ADORA2A expression in ECs of lesion-prone arteries was upregulated. The formation of atherosclerotic plaques and EndMT were inhibited in endothelial
Adora2a
knockout mice or mice treated with antagonist KW6002. In the
in vitro
studies, EndMT induction of human aortic endothelial cells (HAECs) was inhibited by
Adora2a
knockdown and antagonist KW6002, but was promoted by adenovirus encoding Adora2a. Mechanistically, ADORA2A promoted the expression of TGFβR1/ALK5 and the activity of its downstream P-Smad2/3 signaling pathway through the cAMP/PKA/CREB signaling pathway

Conclusions:
ADORA2A promotes EndMT through the cAMP/PKA/CREB/ALK5 signaling pathway, resulting in increased formation of atherosclerotic lesions.
Abstract 311: Fatty Acid Synthase Targeting Reduces Aortic Atherosclerosis And Inflammation
Authors: Meade Rodrigo , Engel Connor , Belaygorod Larisa , Arif Batool , Abu-Amer Wahid , Semenkovich Clay F , Zayed Mohamed A
Published: October 2023

Fatty acid synthase (FAS) catalyzes the elongation of saturated fatty acids. FAS is elevated in liver and adipose tissue in the setting of diabetes, and serum circulating FAS (cFAS) is a marker of peripheral arterial disease. The natural antibiotic Platensimycin (PTM) competitively inhibits FAS. However, the impact of chemical FAS inhibition on atherosclerosis is unknown. We hypothesized that PTM and a liver-specific knockdown of FAS would reduce the burden of aortic atherosclerosis.

ApoE-/-
mice were placed on a 42% high-fat diet (HFD) with or without PTM (100mpk) for 16 weeks. Similarly,
ApoE-/-
mice with or without liver-specific knockdown of FAS (
ApoE-/-
FasL-/-
vs
ApoE-/- FasL+/+
) were placed on a 42% HFD for 16 weeks (Fig. A). Tissue FAS, cFAS, and serum triglycerides (TG) content were measured. FAS/cFAS activity was evaluated with a modified measurement of malonyl-CoA consumption of NADPH, and normalized to content. At 16 weeks, mouse aortas were harvested and stained with oil-red O and CD68 to assess the burden of atherosclerosis and inflammation. Comparisons between murine groups were analyzed using Mann-Whitney and Student t-tests.
ApoE-/-
mice treated with PTM, significantly reduced tissue FAS and serum cFAS activity (p<0.05), and significantly reduced serum TG (p<0.01).
ApoE-/-
FasL-/-
mice demonstrated increased adipose FAS content and activity (p<0.05), reduced cFAS activity (p<0.05), and increased serum TG (p<0.05). Targeting FAS/cFAS with PTM or in
FasL-/-
led to decrease in overall total aortic plaque, particularly in the low-resistance thoracic aorta (B; p<0.05).Targeting FAS/cFAS also demonstrated reduced aortic root plaque, and macrophage content(C; p<0.05).This study reveals that selective targeting of FAS/cFAS either with PTM of via liver knockdown, appear to significantly alter serum lipid profiles, aortic atherosclerosis, and plaque inflammation. Thus, FAS/cFAS may be an important therapeutic target to inhibit atheroprogression.
Abstract 315: Evolution Of Mutation-driven Clonal Hematopoiesis Over 20 Years Is Associated With Low High-density Lipoprotein-cholesterol And Insulin Resistance In Patients With Obesity
Authors: Carlsson Ekander Lena , Andersson-Assarsson Johanna , Carlsson Bjorn , van Deuren Rosanne C , Hoischen Alexander
Published: October 2023

Introduction:
Hematopoietic clones caused by somatic mutations with ≥2% variant allele frequency (VAF) increase with age and are linked to risk of hematological malignancies and cardiovascular disease. Recent observations suggest that smaller clones (VAF<2%) are also associated with adverse cardiovascular outcomes. Individuals with obesity have increased risk of cardiovascular disease, and it is possible that this is due to increased prevalence of hematopoietic clones.

Hypothesis:
We assessed the hypothesis that obesity-related metabolic dysfunction increases clonal hematopoiesis.

Methods:
Our aims were to determine the prevalence of clonal hematopoiesis driven by clones of variable sizes in individuals with obesity treated by usual care or bariatric surgery (a treatment that improves metabolic status), and to examine the expansion of clones in relation to age and metabolic dysregulation over up to 20 years. Clonal hematopoiesis driver mutations (CHDMs) were identified in blood samples from participants of the Swedish Obese Subjects study. Using an ultrasensitive assay, we analyzed single-timepoint samples from 1050 individuals treated by usual care and 848 individuals who had undergone bariatric surgery, and multiple-timepoint samples taken over 20 years from a subset (n=40) of the individuals treated by usual care.

Results:
Prevalence of CHDMs was similar in the single-timepoint usual care and bariatric surgery groups (20.6% and 22.5%, respectively, P=0.330), with VAF ranging from 0.01%-31.15%. Clone sizes increased with age in individuals with obesity, but not in those who underwent bariatric surgery. In the multiple-timepoint analysis, VAF increased by on average 7% (range -4%-27%) per year and rate of clone growth was positively associated with insulin resistance (R=0.40, P=0.025) and low HDL-cholesterol (R=-0.68, P=1.74E-05).

Conclusions:
Insulin resistance and low HDL-C are associated with growth of hematopoietic clones in individuals with obesity treated by usual care, supporting our hypothesis that dysfunctional metabolism affects clone growth.
Abstract 318: Association Between Dietary Inflammatory Index And Metabolic Syndrome Among Middle-aged Women
Authors: Kwon Ria , Choi Hye Rin , Lim Ga-young , Park Eunkyo , Chang Yoosoo , Ryu Seungho
Published: October 2023

Objective:
Metabolic syndrome (MetS) is a global problem that reduces the quality of life and threatens health such as cardiovascular disease and type 2 diabetes. It is known that the risk of cardiovascular disease increases in middle-aged women due to menopause, so management of metabolic syndrome is essential. We investigated the association between dietary metabolic syndrome index (DMetSI) and MetS, MetS unhealthy, insulin resistance (HOMA-IR), and non-alcoholic fatty liver disease (NAFLD) in middle-aged women.

Methods:
We used the data of a cohort study of midlife Korean women. MetS were defined as three or more of the five items of waist circumference, blood triglyceride, blood high-density cholesterol, blood pressure, and fasting blood sugar that was out of the standard range. DMetSI was calculated using the scoring method established by Shivappa from KNHANES (Korea National Health and Nutrition Examination Survey) and CANVAS (sub-study of the Korean Genome and Epidemiology Study) data. We were named K-DMetSI and C-DMetS, respectively, and divided DMetSI scores into 3 quartiles. A Poisson regression model was used to analyze the association between DII and MetS, MetS unhealthy (divided into over one items), NAFLD, and insulin resistance(divided 75th percentile).

Results:
A total of 2,552 subjects were studied. The K-DMetSI scores of the subjects were –1.23 to 1.96 and the C-DMetS were -2.05 to 2.75. The prevalence of MetS was 36.8%, MetS unhealthy was 5.4%, NAFLD was 19.7%, and insulin resistance was 22.2%. The risk of MetS in the Q3 group which had the highest K-DMetS was 1.97 (1.29, 3.01) higher than that in the Q1 group. The risk of MetS in the Q3 group which had the highest C-DMetS was 1.68 (1.10, 2.57) higher than that in the Q1 group. Then we found that the level of K-DMetS and C-DMetS were significantly positively associated with the MetS unhealthy, NAFLD, and insulin resistance.

Conclusions:
K-DMetS and C-DMetS were associated with the prevalence of MetS, Mets unhealthy, NALFD, and insulin resistance. To prevent chronic metabolic diseases, it is necessary to conduct investigations and dietary interventions for middle-aged women.
Abstract 319: Reverse Serine Hydroxymethyltransferase 2 Activity In Atherosclerosis And Nonalcoholic Fatty Liver Disease
Authors: McKinney M. P , Finney Alexandra C , Das Sandeep , ANAND SUMIT KUMAR , liu zhipeng , Pearson-Gallion Brenna , Cockerham Elizabeth D , Rohilla Sumati , Ghrayeb Alia , Gottlieb Eyal , Mor Inbal , Hyung Nam , Orr Wayne , Rom Oren
Published: October 2023

Despite significant advancements in therapeutics, atherosclerotic cardiovascular disease (ASCVD) remains the leading cause of death worldwide. While the role of lipids in the pathogenesis of ASCVD is well-known, the contribution of amino acids is becoming increasingly recognized. Lower circulating glycine levels are consistently reported in patients with ASCVD. Previous studies from our group and others demonstrated that patients with significant coronary artery disease exhibit a reduced glycine:serine ratio compared with healthy individuals. Primarily expressed in the liver, mitochondrial serine hydroxymethyl transferase (SHMT2) is thought to be responsible for the bulk of glycine biosynthesis. Nevertheless, the role of hepatic SHMT2 in regulating serine and glycine metabolism in the context of atherosclerosis and cardiometabolic diseases has not been studied. Here, we describe novel
Shmt2
-floxed mice crossed with
Alb
-cre mice, yielding hepatocyte-specific loss of SHMT2 (
Shmt2
HKO
). We further crossed our
Shmt2
HKO
mice with athero-prone (
Apoe
-/-
) mice to study the role of hepatocyte SHMT2 in the pathogenesis of atherosclerosis. While loss of hepatocyte SHMT2 was expected to limit glycine biosynthesis, our targeted metabolomics analysis uncovered a significant increase in circulating glycine with no significant changes in serine, but a marked increase in the glycine:serine ratio, indicating an unexpected reverse SHMT2 activity. Unbiased transcriptomics of livers from
Shmt2
HKO
/
Apoe
-/-
mice fed a high-fat, high-cholesterol, high-sucrose Western diet revealed an upregulation of pathways known to play protective roles in atherosclerosis and cardiometabolic diseases, including glutathione metabolism, AMPK, and PPAR signaling, while proinflammatory pathways were significantly downregulated. Consistently, we noted a significant reduction in the liver-to-body weight ratio, an indicator of reduced liver damage under Western diet feeding. Our findings uncover an unexpected reverse activity of hepatic SHMT2 in mice with atherosclerosis and fatty liver. Our ongoing studies aim to elucidate a potential causative role of hepatic SHMT2 in atherosclerosis, nonalcoholic fatty liver disease, and other cardiometabolic diseases.
Abstract 321: Altered Epha2 Signaling In Non-alcoholic Fatty Liver Disease Progression
Authors: Pearson-Gallion Brenna , Finney Alexandra C , Nunez Kelley , Cockerham Elizabeth D , Abdullah Chowdhury S. , Tandon Eshika , Thevenot Paul , Cohen Ari , Traylor James , Bhuiyan Md Shenuarin , Rom Oren , Orr Wayne W
Published: October 2023

Background:
Nonalcoholic fatty liver disease (NAFLD), the leading cause of chronic liver disease worldwide, results from diet-induced hepatic steatosis that can result in hepatocyte ballooning, inflammation and fibrosis driving nonalcoholic steatohepatitis (NASH). The Eph receptors, the largest family of receptor tyrosine kinases in the mammalian genome, affect inflammation and fibrosis in other model systems, and we have shown that EphA2 deletion reduces atherosclerosis despite increasing plasma cholesterol levels. While a GWAS study has linked EphA2 to NAFLD, the potential role of EphA2 in NAFLD has never been investigated.

Methods:
EphA2 expression and signaling was assessed in mice fed a high fat diet (HFD) for 8 and 24 weeks, in human liver samples from NASH patients, and in HuH7 cells treated with palmitic acid (FA), low density lipoprotein (LDL), and/or fructose. Global EphA2 KO mice were fed a HFD for 12 weeks followed by histological and molecular analyses.

Results:
Mice with early-stage non-alcoholic fatty liver disease (NAFLD) showed elevated EphA2 expression along with markers of EphA2 ligand-dependent signaling (EphA2 tyrosine phosphorylation). Consistently, HuH7 cells treated with FA, LDL, and/or fructose displayed a similar enhancement of EphA2 expression upon treatment. However, a mouse model of late-stage NASH exhibited reduced expression of the EphA2 ligand ephrinA1 and elevated markers of ligand-independent signaling (Ser897 phosphorylation). Similarly, human liver samples from NASH patients show reduced ephrinA1 expression and elevated markers of EphA2 ligand independent signaling. Consistent with a role for EphA2 in NAFLD/NASH, global EphA2 KO mice show significantly lower indices of NAFLD following high fat diet feeding, including reduced liver-to-body weight ratios, hepatic steatosis, and inflammation.

Conclusion:
Taken together, our data demonstrate that EphA2 expression and signaling are altered during NAFLD/NASH pathogenesis and suggest that EphA2 inhibition may reduce NAFLD/NASH disease progression.
Abstract 322: Novel Arteriovenous Graft Platform For Pancreatic Islet Xenotransplantation
Authors: Roberts Sophia H , Engel Connor , Cashin John , Meade Rodrigo , Arif Batool , Zaghloul Mohamed , Alhamad Tarek , Remedi Maria , Lin Yiing , Zayed Mohamed A
Published: October 2023

Objectives:
Pancreatic islet xenotransplantation is limited by tissue oxygenation and immune-rejection. We developed a novel arteriovenous graft (AVGRx) composed of a central treatment zone sequestered by an outer nylon barrier and an inner semi-permeable porous PTFE membrane. We hypothesized that AVGRX could facilitate xenotransplantation of human islets in a porcine host without immunosuppression.

Methods:
Human pancreatic islets were purchased from Prodo Laboratories Inc and isolated from human pancreas procured at the Mid-America Transplant facility. Islets were encapsulated in alginate by a custom droplet generator. Islet viability was confirmed by whole mount staining and function was assessed by glucose stimulated insulin secretion (GSIS) assay. AVGRx was implanted in 3 adult pigs in a right internal carotid artery to external jugular vein configuration. Graft patency was monitored by weekly ultrasound. In 2 additional pigs, AVGRx was loaded with alginate encapsulated human islets. Human and porcine insulin levels were measured pre- and post-dextrose bolus administration post-implantation. All pigs did not receive immunosuppression and were euthanized for device explantation.

Results:
There was 100% graft patency over the study period. In response to dextrose boluses, both human and porcine insulin transiently increased in the host serum 3 weeks post-implant (Figure 1). Human islets were explanted 1 month after implantation. Explanted xenografts stained positive for islet markers such as Nkx and C-peptide. GSIS assay revealed explanted xenografts continued to produce insulin at levels similar to pre-implant islets (p=0.67).

Conclusions:
AVGRx can facilitate xenotransplantation of human pancreatic islets. Human islet xenografts remained viable and functional in porcine hosts for three weeks without immunosuppression. Future studies aim to develop AVGRx as a platform for islet xenotransplanation in insulin-dependent diabetics.
Abstract 327: Gclm-dependent Gsh Synthesis Regulates Efferocytosis During Atherosclerosis
Authors: Delgadillo Luisa , Pattillo Christopher B , Yurdagul Arif , Mackay Ryan , Rashdan Nabil
Published: October 2023

Atherosclerosis, a chronic inflammatory disease characterized by the accumulation of lipids, apoptotic cells, and fibrous elements within the arterial wall, can lead to plaque rupture, one of the most common causes of death in cardiovascular disease (CVD). Rupture prone plaques, characterized by large necrotic cores and thin fibrous caps, lead to acute luminal thrombosis resulting in myocardial infarctions (MI) and strokes. Apoptotic cell (AC) accumulation coupled with deficiency in their clearance, results in necrotic core formation. Macrophages are responsible for clearing ACs through efferocytosis; however, as atherosclerosis progresses efferocytosis is impaired. AC accumulation contributes to the chronic inflammatory response in CVD. Within this response, reactive oxygen species production increases disrupting cellular redox balance. Glutathione (GSH), the main antioxidant in the body, maintains redox balance; however, it fails to do so in atherosclerosis. Reports have shown that a single nucleotide polymorphism (SNP) in the promoter region of the modifier subunit of GCL (GCLM), a rate limiting enzyme responsible for de novo synthesis of GSH, limits its transcription under oxidative stress and is associated with increased risk of MI. Moreover, deletion of GCLM shows an increase in atherosclerotic plaque area and necrotic core, however it remains unknown how GCLM contributes to necrotic core formation and increased CVD incidence.

Methods:
Bone marrow derived macrophages from WT, Het, and KO GCLM mutant mice and peripheral blood mononuclear cells (PBMCs) from blood donors were isolated and used for efferocytosis and protein assays. PBMCs and atherosclerosis plaque sections were used for SNP genotyping and
in situ
efferocytosis assays.

Results:
Efferocytosis impairment correlated with decreased GCLM expression in mice. The presence of a SNP in GCLM significantly decreases efferocytosis
in vitro
as well as
in situ
in stage 5 human plaques.

Conclusion:
These results suggest that loss of GCLM in macrophages impairs primary and secondary efferocytosis in mice. This is corroborated with the inhibition of GSH synthesis. We also observed an impairment in efferocytosis in human macrophages with and without the presence of the risk allele in GCLM.
Abstract 332: Aorta In Pathologies May Function As An Immune Organ By Upregulating Secretomes For Immune Cell Activation, Differentiation And Trans-differentiation
Authors: Lu Yifan , Sun Yu , Xu Keman , Saaoud Fatma , Shao Ying , Wang Hong , Yang Xiaofeng P
Published: October 2023

Introduction:
Six types of secertomes have been identified in past years. Single cell RNA-seq studies identified many new vascular and immune cell subsets in various aortic disease. However, the roles of new secretomes in aortic vascular cells remains unknown. Similar to traditional immune organs such as lymph nodes where native cells are polarized, transdifferentiated, matured. We
hypothesis
aorta serves as an immune organ by using secretomes in pathological condition.

Methods:
We performed transcriptomic analyses of six types of secretomic genes (SGs) including canonical secretome, caspase-1-Gasdermin D secretome, caspase-4-Gasdermin D secretome, exosome secretomes, Weibel-Palade bodies, and autophagy secretome in aorta and vascular cells and made the following
findings:

1)

53.7% out of 21,306 human protein genes are in the six secretomes;

2)

Atherosclerosis (AS), chronic kidney disease (CKD) and abdominal aortic aneurysm (AAA) modulate six secretomes in aortas; and Middle East Respiratory Syndrome Coronavirus (MERS-CoV, COVID-19 homologous) infected endothelial cells (ECs) and angiotensin II (Ang II) treated vascular smooth muscle cells (VSMCs) modulate six secretomes;

3)

AS aortas upregulate T and B cell immune SGs; CKD aortas upregulate SGs for cardiac hypertrophy, and hepatic fibrosis; and AAA aorta upregulate SGs for neuromuscular signaling and protein catabolism;

4)

Ang II-AAA, canonical, caspase-4, and exosome SGs all have two expression peaks of high (Day 7, D7)-low (D14)-high (D28) patterns;

5)

Elastase AAA aortas have more inflammatory/immune pathways than that of Ang II AAA aortas;

6)

Most disease-upregulated cytokines in aorta may be secreted via canonical and exosome secretomes;

7)

Canonical and caspase-1 SGs play roles at early MERS-CoV infected ECs whereas caspase-4 and exosome SGs play roles in late/chronic phases; and the early upregulated canonical and caspase-1 SGs may function as drivers for trained immunity (innate immune memory). These results provide novel insights on the roles of aorta as immune organ in upregulating secretomes and driving immune cell differentiations in COVID-19, cardiovascular diseases, inflammations, transplantations, autoimmune diseases and cancers.
Abstract 335: Functional And Molecular Effects Of Catalase Haploinsufficiency In Early And Advanced Atherosclerosis
Authors: Rababa'h Abeer , Hagler Michael A , Roos Carolyn M , Smith Leslie , Miller Jordan D
Published: October 2023

Introduction:
While increased hydrogen peroxide (H
2
O
2
) accelerates atherosclerotic vascular disease, the role of catalase---which selectively metabolizes H
2
O
2
in lysosomes—is unclear. Given the established role of H
2
O
2
in accelerating osteogenic vascular calcification

and vascular smooth muscle cell de-differentiation, we sought to determine the effects of reducing catalase
in vivo
on functional and molecular changes in aorta in early and late-stage atherosclerosis.

Hypothesis:
Haploinsufficiency of catalase will worsen vascular contractile function and promote ectopic osteogenesis in early and late-stage atherosclerosis.

Methods:
We used ldlr
-/-
/ApoB
100/100
(LA) mice, which were wild-type or heterozygous for catalase (LA-Cat
+/+
or LA-Cat
+/-
, respectively) and fed a western diet for 3 or 12 months. Vasomotor function was assessed using an isolated organ chamber system. Gene expression of VSMC contractile proteins and osteogenic markers was quantified using qRT-PCR.

Results:
Endothelial function worsened over time in both strains of mice and was modestly worsened in LA-Cat
+/-
mice. Vascular contraction to PGF
2α
was reduced with aging in LA-Cat
+/+
mice, and unaffected by catalase deficiency at 3 months. Surprisingly, contractile tension in response to PGF
2α
was increased in LA-Cat
+/-
at 12 months, with this effect being most pronounced in female mice. Consistent with previous literature, expression of VSMC contractile genes (smooth muscle α-actin and calponin-1) was decreased in all groups following 12 months of diet (p<0.0001), and expression of osteogenic genes (BMP2, Runx2, and osterix) increased with aging (p<0.05). Surprisingly, and in contrast to our contractile function data, these time-dependent changes in gene expression tended to be augmented in LA-Cat
+/-
mice compared to LA-Cat
+/+
littermates.

Conclusions:
Our data suggest that systemic inactivation of one copy of catalase increases contractile function in aorta from aged, hypercholesterolemic mice while paradoxically increasing osteogenic signaling. Future investigation into the role of specific antioxidant enzymes in driving functional and structural changes in advanced vascular disease will be critical to informing therapeutic strategies in this domain.
Abstract 337: Inhibiting Thrombospondin-1 Activation Of Transforming Growth Factor Beta Under Oscillatory Shear Stress Decreases Connective Tissue Growth Factor And Neurogenic Locus Notch Homolog Protein 1
Authors: Sanchez Marrero Gloriani , Brewster Luke P
Published: October 2023

Peripheral arterial disease (PAD) is the 3
rd
cause of atherosclerotic disease morbidity with an incidence of 12-20% in individuals over 65yrs. PAD arteries are stiffer than healthy ones and they present disturbed flow, defined by low and oscillatory shear stress (OS), in the vicinity of obstructions. Previous work from our group discovered the upregulation of thrombospondin 1 (TSP-1) and profibrotic genes in HAECs exposed to OS. Furthermore, TSP-1 is an activator of the transforming growth factor beta (TGF- β), which has shown to regulate connective tissue growth factor (CTGF) expression (Fig. 1A). CTGF has been associated with profibrotic signaling. There is a pressing need to understand the involvement of CTGF in the TSP-1 mediated profibrotic signaling occurring in HAECs under disturbed flow. We hypothesize that, inhibiting the activation of TGF-β via TSP-1 mediation will lead to a decrease in CTGF expression in HAECs exposed to disturbed flow. Female HAECs were treated to block the TSP-1-mediated activation of TGF-β with LSKL, a competitive inhibitor of TSP-1, while SLLK, a scrambled protein, was used as the control treatment. Then, the HAECs were subjected to low shear stress for 24 hrs followed by gene expression quantification using qPCR. Blocking the activation of TGF-β via TSP-1 mediation led to the significant downregulation of CTGF and NOTCH1 (Fig. 1B and 1C) in HAECs subjected to low shear stress when compared to the control group. Similarly, there was a decrease in the expression of collagen genes in the group treated with LSKL (not statistically significant). In conclusion, using an in vitro system for applying low shear to HAECs, we discovered that CTGF expression was significantly reduced when TSP-1 mediation was inhibited. The same occurred with NOTCH1, a gene associated with protection against inflammation. Future studies will investigate if these gene expression patterns are observed at later time points or if it is part of an early response to low shear.
Abstract 338: Novel Role Of Endothelial Cd47 In The Regulation Of Pathogenesis Of Atherosclerosis
Authors: Singh Bandana , Cui Kui , Peng Qianman , Li Kathryn , Zhu Bo , Bhattacharjee Sudarshan , Osorio Daniel , Wang Beibei , Dong Yunzhou , Wang Donghai , Lu Yao Wei W , Wu Hao , Wong Scott , Cowan Douglas B , Chen Hong
Published: October 2023

Background:
The anti-phagocytic molecule CD47 is upregulated in atherosclerotic plaques of patients with cerebrovascular disease. However, the role of endothelial cell-specific CD47 in the pathogenesis of atherosclerosis has not yet been defined. Kojima
et.al
. described atheroprotective effect of anti-CD47 antibody by enhancing efferocytosis in atherosclerosis. In this study, we determined whether endothelialCD47 regulates efferocytosis in atherosclerosis.

Methods:
Susceptibility to atherosclerosis was evaluated in cross sections of the aortic sinus in atherosclerotic mice. Using single-cell RNA sequencing and efferocytosis assays, we investigated the role of CD47 in regulating efferocytosis in atherosclerotic mouse model.

Results:
Endothelial cell-specific deletion of CD47 led to significant plaque reduction in aortic sinus of athero-CD47iECKO mice. Cluster analysis and gene expression profiles of 20,000 aortic endothelial cells from atherosclerotic mice identified 10 major cell types: endothelial cells, VSMCs, fibroblasts, B cells, T cells, macrophage, erythrocytes, oligodendrocytes cells, Schwann cells, and pericytes. In athero-CD47iECKO group, subpopulation of endothelial cells as well as fibroblasts, T-cells, B-cells, macrophages, erythrocytes, oligodendrocytes, and pericytes were increased, while vascular smooth muscle cell numbers decreased. Using efferocytosis assay on isolated primary endothelial cells from control and CD47iECKO mice, we observed CD47 deletion induced engulfment of apoptotic Jurkat cells, which had been exposed to NF-κB inhibitor, BAY11-7082 to stimulate apoptotic attributes. Quantitative PCR analyses showed that CD47 deletion increases the expression of efferocytosis receptor FasL, CX3CL1, Gpr132, MerTK, Scarb1, Sirpa, TSP1, and HMGB1.

Conclusion:
Our study indicates that genetic deletion of CD47 in endothelial cells attenuates atheroma progression by enhancing efferocytosis. This study also reveals phenotypic heterogenicity of endothelial cells at the single-cell level during atherogenesis and provides deeper understanding of endothelial cell biology in atherosclerosis that may facilitate the development of novel therapeutic interventions for cardiovascular disease.
Abstract 347: Endothelial Transcription Factor EB Protects Against Doxorubicin-induced Endothelial Toxicity And Cardiac Dysfunction
Authors: Du Wa , Huang Wei , Ringer Madison , Slemp Khayla , Fan Guo-Chang , Becker Richard C , Wang Yi-gang , Fan Yanbo
Published: October 2023

Objective:
Doxorubicin (DOX), an effective chemotherapeutic drug for various cancers, has been demonstrated to induce cardiovascular toxicity in cancer survivors. EC dysfunction is recognized to play a critical role in the onset and severity of cardiotoxicity associated with DOX. Transcription factor EB (TFEB), a master autophagy regulator, regulates cardiovascular homeostasis. Thus, experiments were aimed to test whether endothelial TFEB protects against EC damage and alleviates cardiac dysfunction after DOX treatment.

Methods:
EC-specific TFEB transgenic (EC-TFEB Tg) mice, EC-specific knockout (EC-TFEB KO) mice, and littermate controls were administered DOX by intravenous injection. Survival curves were generated, and cardiac functions were measured in mice
in vivo
. The effects of TFEB on mitochondrial reactive oxygen species production, autophagic flux, and apoptosis were evaluated in human and mouse cardiac microvascular ECs (CMECs) treated with DOX
in vitro
. Mechanistically, RNA-sequencing analysis and ChIP-qPCR were performed in DOX-treated ECs.

Results:
EC-TFEB Tg mice showed significantly reduced mortality and improved cardiac function, together with attenuation of perivascular fibrosis after DOX treatment. By contrast, EC-TFEB KO consistently exacerbated DOX-induced cardiac dysfunction in mice. Furthermore, we observed that TFEB enhanced autophagy and reduced oxidative stress in CMECs in response to DOX treatment. In addition, TFEB prevented EC barrier damage and alleviated proinflammatory cytokine release from CMECs, contributing to the protective effects of EC TFEB on cardiomyocytes. Mechanistically, DAB adaptor protein 2 (DAB2), a clathrin and cargo-binding endocytic adaptor protein, was identified as a TFEB target gene in ECs. Accordingly, DAB2 knockdown attenuated the inhibitory effects of TFEB on the secretion of proinflammatory cytokines from CMECs.

Conclusion:
Endothelial TFEB protects against DOX-induced EC damage and restricts the release of proinflammatory cytokines, thereby inhibiting cardiac dysfunction. A better understanding of EC TFEB in cardiac dysfunction under chemotherapy could provide new insight into the mechanisms underlying EC-cardiomyocyte communication in the heart.
Abstract 350: High-dimensional Analysis Of Circulating Blood Cells Reveals Distinct Granulocyte Activation And NETosis Profiles In COVID-19
Authors: Kraisin Sirima , Penttila Pier Andrée , Chinnaraj Reena , Jacobs Cato , Wauters Joost ,   CONTAGIOUS Consortium, Martinod Kimberly
Published: October 2023

Background:
Coronavirus disease 19 (COVID-19) represents a perfect storm for immunothrombosis leading to severe disease, with release of neutrophil extracellular traps (NETs) being an important contributor now frequently described both in blood and in tissues. NETs contribute to thrombosis by trapping platelets and activating the coagulation cascade, as well as endotheliopathy. NET formation is an active process initiated by factors such as activated platelets and involving histone modifications including citrullination.

Aims:
We aimed to characterize early activation toward NETosis in circulating granulocytes in the context of neutrophil activation, immunothrombosis, and the broader immune response in COVID-19, particularly in severe disease involving ICU stays.

Methods:
We designed a custom panel of 42 antibodies to stain peripheral blood collected from COVID-19 patients in ICU at UZ Leuven during the COVID-19 Delta-wave. This panel comprised cell-specific epitopes, surface activation markers, intracellular staining for NET-associated cell biological changes, and platelet markers to identify complexes. Samples were run using cytometry by time-of-flight (CyTOF) on a Helios mass cytometer and analyzed using FlowJo software with FlowSOM clustering and tSNE visualisation.

Results:
Nonbiased clustering analysis was performed and identified 18 distinct populations of granulocytes. Four of these populations were present only in COVID-19 patients, with high expression of CD11b, CD177, and CD16. A distinct population of citrullinated histone-positive eosinophils was also identified in COVID-19 patients, with higher expression than any of the neutrophil populations. Ex vivo stimulation of healthy donor granulocytes toward NETosis shifted the expression profile toward that of COVID-19 patient granulocytes. However, morphometric analysis using scatterbody targets failed to identify cell biological changes common to NET formation.

Conclusions:
High dimensional profiling of circulating blood cells identifies distinct granulocyte populations with activation profiles in COVID-19, highlighting the heterogeneity of neutrophil responses and involvement of eosinophils in extracellular trap release.
Abstract 363: Resident Vascular Adventitial Progenitor Cells Of Smooth Muscle Cell-lineage Adopt Profibrotic Phenotype And Contribute To Cardiac Fibrosis.
Authors: Lu Sizhao , Dubner Allison , Jolly Austin , Noble Tysen , Nemenoff Raphael , Moulton Karen , Weiser-Evans Mary C
Published: October 2023

Cardiac myofibroblasts are the major contributors to ECM deposition in pathological cardiovascular fibrosis, which is the characteristic feature of cardiovascular diseases. However, due to potential heterogeneity of myofibroblasts, the origin of these cells remains controversial. Using highly specific smooth muscle cell lineage-tracing mouse models, we discovered the smooth muscle cell origin of a subpopulation of resident vascular adventitial progenitor cells, defined by the expression of the stem cell marker Sca1 (AdvSca1-SM cells), which rapidly proliferate and adopt a myofibroblast phenotype in response to acute vascular injury. Further, we identified a specific gene signature of active hedgehog/Wnt/β-catenin/Klf4 signaling in AdvSca1-SM cells and validated a Gli1-CreERT2-ROSA26-YFP (Gli1) reporter mouse model to be a faithful lineage tracing system for AdvSca1-SM cells. However, the function of AdvSca1-SM cells in cardiac fibrosis is unknown. Using immunofluorescent staining and label-free second harmonic generation (SHG) imaging, we observed the expansion and migration of AdvSca1-SM cells in close association with perivascular and interstitial cardiac fibrosis in pressure overload-induced cardiac fibrosis in Gli1 reporter mice. By integrating single cell RNA sequencing (scRNA-seq) with spatial transcriptomics, we identified a AngII-driven spatiotemporal profibrotic differentiation trajectory of AdvSca1-SM cells in mouse cardiac tissue. The trajectory, characterized by loss of expression of stemness genes, such as
Klf4
, the lncRNA,
Meg3
, and up-regulation of myofibroblast genes, was recapitulated in human RNA-seq data and of translational significance. Connectivity map analysis of the scRNA-seq data identified statins as potential candidates for inhibition of the profibrotic transition of AdvSca1-SM cells. In agreement, simvastatin induced the expression of stemness genes and inhibited TGFβ-induced up-regulation of αSMA and down-regulation of KLF4 in cultured AdvSca1-SM cells. Lentiviral-mediated up-regulation of
Meg3
also rescued TGFβ-induced suppression of stemness genes. Our findings support the further development of anti-fibrotic therapeutics targeting AdcSca1-SM cells.
Abstract 367: Beta-catenin C-terminus Signaling Drives Sphingosine-1-phosphate Receptor-1 Expression in Smooth Muscle Cells to Promote Vascular Remodeling
Authors: Oliveira de Paula Gustavo H , Ressa Gaia , Jayakumar Smitha , Almonte Vanessa , Parikh Dippal , Valenta Tomas , Basler Konrad , Hla Timothy , Riascos-Bernal Dario F , Sibinga Nicholas
Published: October 2023

Introduction:
Vascular remodeling is associated with target organ damage and fatal cardiovascular events. The molecular mechanisms that control vascular remodeling are still poorly understood, limiting effective therapeutic approaches. The β-catenin C-terminal domain is required in smooth muscle cells (SMCs) for artery formation during embryogenesis, but its role in vascular remodeling in adulthood is unknown. Therefore, the objective of this study was to define the importance of β-catenin C-terminal domain in SMCs during vascular remodeling.

Hypothesis:
β-catenin C-terminal signaling is required for neointima formation after vascular injury.

Methods:
We studied male and female mice expressing a C-terminus-deficient β-catenin specific to SMCs (SMβCΔC mice). Mouse aortic SMCs (MASMCs) were isolated from SMβCΔC mice (βCΔC MASMCs). Results are shown as mean ± SEM.

Results:
SMβCΔC mice show reduced neointima formation (males: WT 100±17 vs SMβCΔC 30±9; females: WT 100±19 vs SMβCΔC 30±7; n=10; P<0.05) and decreased SMC proliferation (males: WT 100±6.8 vs SMβCΔC 22±5.9; females: WT 100±9.9 vs SMβCΔC 34±9.0; n=5; P<0.05) after arterial injury. RNA-seq analysis revealed a downregulation of the sphingosine-1-phosphate receptor 1 (S1pr1) transcript in βCΔC MASMCs. We further found that β-catenin interacts with the S1pr1 promoter (Cut&Run assay, IgG Control 1.00±0.06 vs β-catenin 2.82±0.50; n=4; P<0.05) and acts through its C-terminal domain to activate S1pr1 transcription (luciferase reporter assay, vector Control 100.0±17.08 vs βCΔC 19.9±5.8; n=6; P<0.05) and protein expression (Control 0.95±0.20 vs βCΔC 0.08±0.02; n=4; P<0.05). Consistent with these observations, S1PR1 expression was decreased in SMCs of arteries from SMβCΔC mice (WT 100±6.9 vs SMβCΔC 8±2.1; n=5; P<0.05), and re-establishing SMC S1PR1 expression rescued neointima formation (SMβCΔC 0.15±0.02 vs SMβCΔC;S1PR1Gain-of-function 0.93±0.11; n=8; P<0.05).

Conclusion:
Our findings identify β-catenin C-terminal output as a novel upstream regulator of S1PR1 expression and define an essential β-catenin/S1PR1 axis that drives neointima formation after injury. Targeting this axis could provide a therapeutic strategy to reduce vascular remodeling and cardiovascular disease.
Abstract 374: Sexual Dimorphism And Elabela Expression In Peripheral Artery Disease
Authors: Rezaee Sholeh , Threlkeld Edie , Emami Naeini Sahar , Bhandari Bidhan , Meeks William , Meeks Avery , Zaied Abdelrahman , Leanhart Silvia , Brewer Lauren , Hamilton Adeleigh , Hess David C , Annex Brian H , Da Silva Lopes Salle Evila , Baban Babak
Published: October 2023

Introduction:
Elabela is a component of the apelinergic system known to regulate immune responses and has a cardiovascular protection role, including atherosclerosis. Peripheral artery disease (PAD) is the narrowing or blockage of larger arteries in the legs. Patients with PAD suffer from poor perfusion to their lower extremities, in addition to increased rates of cardiovascular complications. The role of sexual dimorphism in the immune system in the PAD is poorly understood. Our previous research suggests that Elabela may hold a gender-dependent protective role that can be used as immunotherapeutic. We hypothesized that in patients with PAD, expression of Elabela will be different between males and females, implicating in sexual dimorphism of the immune response, the disease outcome, and treatment.

Methods:
Blood samples were collected from PAD patients (n=8) and risk factor closely matched controls (n=8). PBMCs were isolated by gradient centrifugation. By flow cytometry, gating strategy on Elabela, and Elabela expressing leukocytes, using FlowJow software for analysis.

Results:
The results showed that there is a sexual difference in the expression of Elabela in both controls and patients with PAD. In general, males showed a high level of Elabela than females. PAD decreases Elabela expression in both males and females. Regarding Elabela expressing leukocytes, in female controls, there was no significant difference in leukocyte expression of Elabela as compared to PAD patients, while male controls demonstrated increased leukocyte expression of Elabela.

Conclusions:
Our findings suggest a potential role for Elabela in PAD. Importantly, these results also indicate a difference in Elabela expression based on sex dimorphism. Such differences could potentially be used as a biomarker in the diagnosis and prognosis of PAD along with therapeutic values.
Abstract 380: Adult But Not Neonatal Platelets Promotes Monocyte Trafficking In Neonatal Mice
Authors: Maurya Preeti , Li Chen , Ture Sara , McGrath Kathleen E , Palis Jim , Morrell Craig
Published: October 2023

Background:
Platelet transfusions are sometimes given to thrombocytopenic neonates with the hope of reducing the bleeding risk, however, there is little clinical data to support this practice and platelet transfusions may increase the bleeding risk or lead to adverse complications. Our group previously reported that fetal platelets expressed lower levels of immune related mRNA compared to adult platelets. In this study we focused on the effects of adult versus neonatal platelets on monocyte immune functions that may have an impact on neonatal immune function and transfusion complications.

Methods:
Platelets and bone marrow isolated monocytes were co-cultured and monocyte phenotypes determined by RNA-seq and flow cytometry. An
in vivo
model of platelet transfusion in neonatal thrombocytopenic mice was used in which platelet deficient thrombopoietin receptor mutant mice (TPOR
-/-
) were transfused with adult or P7 platelets and monocyte phenotypes and trafficking were determined.

Results:
Adult and neonatal platelets had differential immune molecule expression, including
Selp
(P-selectin). Monocytes incubated with adult or neonatal mouse platelets had similar inflammatory (Ly6C
hi
), but different trafficking phenotypes, as defined by CCR2 and CCR5 surface expression. Blocking P-selectin-PSGL-1 interaction, monocytes limited the adult platelet induced monocyte trafficking phenotype, as well as adult platelet induced monocyte migration
in vitro
. Similar results were seen
in vivo,
when thrombocytopenic neonatal mice were transfused with adult or P7 platelets; adult platelets increased monocyte CCR2 and CCR5 as well as monocyte chemokine migration, while P7 platelets did not.

Conclusion:
These data provide comparative insights into adult and neonatal platelet transfusion regulated monocyte functions.
Abstract 396: Plasticity Of The Tumor Vasculature Is Induced By Tumor Cells Via Cell Surface Receptor Tetherin
Authors: Bell Caitlin , Bamezais Sharika , Ma Wei Feng , Lopez Nicolas Gabriel , Baylis Richard , Luo Lingfeng , Miller Clint L , Leeper Nicholas
Published: October 2023

It is increasingly clear that cardiovascular disease and cancer have shared pathophysiological mechanisms beyond traditional risk factors. Murine models have demonstrated tumors grow more quickly in the presence of cardiovascular abnormalities, and targeting of shared signaling pathways has proven efficacious in both diseases. It is unknown, however, if smooth muscle cell (SMC) plasticity as seen in atherogenesis represents yet another biologic parallel. Our aim was to understand if SMC plasticity occurs in tumor vasculature, and how those transitions are induced.

Syngeneic tumor cell lines were implanted on the flanks of SMC lineage-traced mice and allowed to grow for 11 days. Fluorescent microscopy of the tumors revealed a population of SMCs that moved away from angiogenic vessels and lost canonical SMC markers. We used single cell RNA sequencing to characterize the
in vivo
transcriptional phenotype of SMCs within the tumor. Principal component analysis demonstrated distinct SMC populations, with pseudotime analysis suggesting transitions from a contractile SMC towards a more macrophage-like transcriptional state. CellChat ligand-receptor analysis was leveraged to examine differential interactions between tumor cells and classic macrophages versus macrophage-like SMCs. The only unique interaction was between cell surface receptors Tetherin on tumor cells and paired immunoglobulin-like receptor A2 (PIRA2) on macrophage-like SMCs.
In vitro
murine SMCs treated with recombinant Tetherin decreased contractile SMC markers and increased macrophage markers by qPCR. Exposure to recombinant Tetherin also increased overall proliferative rate, migratory capacity, and phagocytic ability compared to untreated SMCs. The knockdown of PIRA2 in murine SMCs abrogates these apparent effects of Tetherin.

Our findings demonstrate that SMCs in tumor vasculature, like those in atherogenesis, demonstrate significant degrees of plasticity. One of the phenotypic changes is towards a macrophage-like state.
In vitro
studies suggest interaction between Tetherin and PIRA2 receptors is sufficient to induce this switching. Ongoing murine studies will establish whether
in vivo
inhibition of the Tetherin-PIRA2 axis decreases vascular plasticity.