bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 626: RNA Interference To Knock Down Endothelial Von Willebrand Factor Protects Against Endothelial Infection By SARS-CoV-2
Authors: Furini Giulia , De Carli Alessandro , Fonnesu Rossella , Spezia Pietro G , Pistello Mauro , Lai Michele , Lionetti Vincenzo
Published: October 2023

Background:
Long-Coronavirus Disease-19 (COVID19) endotheliopathy is related to high levels of endothelial von Willebrand Factor (vWF). However, the role of vWF in regulating the susceptibility of endothelial cells to severe acute respiratory syndrome coronavirus 2 (SARS CoV 2) infection is still unknown. We hypothesized that targeting endothelial vWF with short interference RNA (siRNA) may prevent SARS CoV 2 entry into endothelial cells through angiotensin converting enzyme 2 (ACE2).

Method:
Gene silencing of vWF or ACE2 was performed in Human Umbilical Vein Endothelial cells (HUVECs) by short interfering RNA (siRNA), while vWF overexpression was obtained by transient pcDNA3.1-WT-VWF plasmid transfection of endothelial cells. The expression of ACE2 and vWF in endothelial cells was measured by the aid of qRT-PCR. Fluorochrome-labeled antibodies were used as probes for surface detection of both endothelial proteins. Wild type and transfected HUVECs were incubated with SARS-CoV-2 for one hour, and the level of cell infection was assessed by qRT-PCR of viral RNA after 24-48 hours.

Results:
Effective siRNA-mediated knockdown of vWF in viable HUVECs halved the susceptibility to infection by SARS-CoV-2 (p=0.0058). Interestingly, endothelial vWF knockdown induced a downregulation of ACE2 expression on the same cell (p=0.0032) and a significant localization on cell surface. Conversely, vWF overexpression led to significant upregulation of surface ACE2 (p=0.028) and increased susceptibility of HUVECs to SARS-CoV-2 infection (p=0.0009).

Conclusions:
Our findings reveal a hitherto unsuspected role of vWF in susceptibility of endothelial cells to viral infection by modulating levels of ACE2. Our discovery might be helpful to design new RNA-based therapeutics to prevent long-term viral persistence of SARS-CoV-2 in endothelial cells.
Abstract 639: Lung Microbiome Dysbiosis in Schistosomiasis-associated Pulmonary Arterial Hypertension
Authors: Marinho Ferreira dos Santos Ygor , Loya Omar , Sun Jun , Oliveira Suellen Darc D
Published: October 2023

Introduction:
Pulmonary arterial hypertension (PAH) is a life-threatening inflammatory cardiopulmonary disease with no cure. Infection by the intravascular parasite
Schistosoma
mansoni
recapitulates several aspects of widespread inflammation that leads to PAH (Sch-PAH).After infection, the
S.
mansoni
eggs cross the gut wall, disturbing its microbiome; however, it remains unknown whether their translocation into the lungs also disturbs the lung microbiome, leading to PAH.

Hypotheses:
We hypothesize that
S.
mansoni
eggs disturb the lung microbiome contributing to vascular inflammation and PAH.

Methods:
To test this hypothesis, we induced Sch-PAH in
end-Scl.cre

ERT2

;Rosa

mt/mg

mice by intraperitoneal sensibilization with 240 eggs/grambody weight (bw; 2 weeks) followed by intravenous injection of 175 eggs/gram bw. After 7 days, lungs were used for microbiome analysis by shotgun metagenomics and data aligned with OneCodex Microbiome Database.

Results:
Metagenomic analysis revealed that
S.
mansoni
infection disrupts the lung microbiome (Shannon Diversity Index: 1.5 +/- 0.07 and 1.18 +/- 0.07, control and infected, respectively; n = 5 mice/group). At the Phylum level, analysis indicated that infection reduced Firmicutes and Bacteroidetes while it also increased Euryarchaeota in the control group. Preliminary data showed that Firmicutes reduction was higher in infected females compared tomales (Mean reads: 0.0891 and 0.0126, respectively; reads normalized by the top 5% lungmicrobiota). Finally, principal coordinate analysis (PCoA) identified a distance in the clustering pattern between sexes, which may account for microbiome differences in Sch-PAH.

Conclusion:
To our knowledge, this is the first report on lung microbiome dysbiosis during murine Sch-PAH. Understanding whether the lung microbiome contributes to Sch-PAH or protects its host opens a novel therapeutic direction for treatment of pulmonary infectious vascular diseases.
Abstract 642: Caspase-4/11 Promotes A High-fat Diet And Chronic Kidney Disease-accelerated Vascular Inflammation Via Novel Caspase-4/11-il-1b Two-step Trained Immunity
Authors: Sun Yu , Lu Yifan , Yang Xiao-feng
Published: October 2023

To determine whether hyperlipidemia and chronic kidney disease (CKD) have a synergy in accelerating vascular inflammation via a new trained immunity (TI, innate immune memory) mechanism, we performed aortic pathological analysis and RNA-sequencing in gene deficient mice. The following findings are made:
1)
High fat diet feeding (HFD) in combination with 5/6 nephrectomy CKD (HFD-CKD) increases plasma LDL-VLDL levels, aortic cytosolic lipopolysaccharide (LPS) levels and caspase-11 activation in aorta, and upregulates 998 genes in TI pathways in aorta;
2)
Caspase-11 deficiency decreases aortic neointimal hyperplasia, recruitments of monocytes and macrophages into aorta, and secretion of caspase-11-gasdermin D (GSDMD) secretome cytokine CCL22 in plasma;
3)
Caspase-11 KO decreases N-terminal GSDMD membrane expression on mouse aortic CD45
-
CD31
+
endothelial cells (ECs) and reduces mouse aortic IL-1β levels;
4)
LPS transfection into human aortic endothelial cells (HAECs) results in caspase-4 activation and increases N-terminal GSDMD membrane expression;
5)
Endogenous proinflammatory palmitic acid activates caspase-4 and increases N-terminal GSDMD membrane expression in HAECs, and palmitic acid in combination with uremic toxin indoxyl sulfate and LPS transfection upregulates vascular cell adhesion molecule-1 (VCAM-1) expression;
6)
LPS transfection increases mitochondrial reactive oxygen species (mitoROS); mitoROS inhibitor mito-TEMPO inhibits caspase-4 activation, N-terminal GSDMD membrane expression and VCAM-1 upregulation in HAECs; and
7)
IL-1β serves as the
second step and positive feedback
of HFD-CKD promoted TI, promotes TI gene expression, enhances caspase-4-induced VCAM-1 expression and IL1β secretion in HAECs. Our results have provided novel insights over hyperlipidemia and CKD in accelerating vascular inflammation via novel two step trained immunity mechanism and new therapeutic targets for treating chronic kidney disease,
Gram-negative
bacterial infections, cardiovascular diseases, inflammations, immune diseases, transplantation, aging and cancers.
Abstract 644: Angiogenic Blood Vessels Reconfigure/ Attenuate Hypoxic Tumor Microenvironments In A Quantitative
In Vitro
Model
Authors: Chiang Kuang-Hsing , Chang Wei-Jen , Wu Hsiao-Mei , Tung Yi-Chung
Published: October 2023

Introduction:
Angiogenesis is crucial in the spread of cancer cells, and hypoxic tumor microenvironment (TME) plays a vital role in its initiation. Hypoxia stimulates the release of pro-angiogenic factors, promoting the growth of new blood vessels towards the tumor, which further alters the TME. The variation of
in vivo
hypoxic TME have been studied recently. However, due to technical limitations such as highly invasive procedure, high cost, tedious process, and poor signal-to-noise ratio, it is challenging to perform quantitative oxygen sensing. Here we develop an advanced
in vitro
model directly investigating the interaction between a solid tumor and blood vessels for an extended period.

Hypothesis:
The angiogenic blood vessels can reconfigure and attenuate the hypoxic TME, which can be directly measured

Methods:
The model is built using a microfluidic device with a sealed microwell for 3D tumor spheroid culture (human colon cancer cell line, HCT116), and a microfluidic channel beneath the microwell for endothelium formation (human umbilical vein endothelial cell, HUVEC), connected by a small hole for interaction between the cell types. Oxygen sensitive fluorescence particles are distributed in the hydrogel matrix, and the widefield frequency domain fluorescence lifetime imaging microscopy (FD-FLIM) is exploited to reliably characterize the oxygen tension around the tumor spheroid with minimal sensitivity to ambient optical noise.

Results:
The oxygen tension of TME around the tumor spheroid within the distance of 0 to 0.7 mm rapidly decreases during tumor growth, from 15% at day 1 to less than 6% at day 14. The presence of endothelial cells slows down this decreasing rate and maintains it in a stable range, from 16% to 14%. The average oxygen tension difference between the two experiments, with and without co-culture of endothelial cells, is statistically significant after 5 days of observation (p<0.01).

Conclusions:
We develop a quantitative
in vitro
method to study the hypoxic TME for its dynamic pattern and as a target for cancer treatment. By observing the effect of angiogenesis on it, this method can aid in exploring optimal cancer management strategies, such as chemotherapy, immunotherapy, tailored anti-angiogenic agents and other targeted therapies.
Abstract 648: Divergent Roles For Endothelial Cell Marcks In Endothelial Cell Regeneration And Signaling After Arterial Injury And Ischemia
Authors: Furtmüller Georg J , Mukhopadhyay Subhradip , Eichelberger Olivia P , Moreau Yvetan , Bailey Jathiya , Sarkar Rajabrata
Published: October 2023

Cardiovascular interventions cause endothelial injury and trigger re-establishment of intact endothelium. After ischemia, endothelial signaling is essential for capillary angiogenesis as well as flow-mediated collateral artery enlargement to restore perfusion. We studied the role of the signaling protein MARCKS (myristoylated alanine-rich C kinase substrate) in the endothelial response to injury and ischemia-induced angiogenesis.

Methods:
Targeted deletion of MARCKS in endothelial cells was done with Cadherin 5-driven Cre/Lox recombination and confirmed with immunohistochemistry. All controls were Cre(-) littermates. Re-endothelialization after femoral artery wire injury was measured by arterial area quantitation after Evan’s Blue exclusion. After femoral artery ligation, limb blood flow was determined by serial laser Doppler scanning, and angiogenesis by counting CD31-positive cells in the gastrocnemius muscle on day 28.

Results:
Endothelial cell-specific deletion of MARCKS was confirmed in Cre (+) animals. Deletion of MARCKS did not affect endothelial regeneration on post operative day 6 after denuding femoral wire injury (68.2% versus 68.7% recovery, n=5-6, p>.05). Ischemia-induced capillary angiogenesis was not affected by deletion of MARCKS (Capillary Count, Cre(+): 602 versus Cre(-): 511, n=6, p>.05), however animals lacking endothelial MARCKS had worsened limb perfusion on days 1, 3, 7 and 21 after ischemia (ABI Range Cre(+) versus Cre(-): 0.04-0.22 versus 0.07-0.41, respectively, n=6-9, p<.05).

Conclusions:
These studies demonstrate distinct and divergent roles for MARCKS in the endothelial cell response to injury and ischemia. MARCKS is not essential for endothelial regeneration after denuding large vessel injury, or for capillary angiogenesis induced by ischemia. Surprisingly, endothelial MARCKS appears to be a novel endogenous promotor of ischemia-induced limb revascularization. This suggests that endothelial cell MARCKS promotes flow-mediated endothelial cell signaling essential for ischemia-induced arterial enlargement. MARCKS activity could be a possible therapeutic target to enhance collateral artery formation in limb ischemia.
Abstract 654: Targeting Metabolic Alterations To Boost Smooth Muscle Cell Differentiation And Prevent Moyamoya-like Cerebrovascular Disease
Authors: Kwartler Callie , Kaw Anita , Majumder Suravi , Pinard Amelie C , Wright Jamie M , Kernell Caroline , Milewicz Dianna M
Published: October 2023

Moyamoya disease (MMD), characterized by occlusive lesions in the distal carotid arteries, is a malignant cause of pediatric stroke. Surgical revascularization is the mainstay treatment, but peri- and postoperative ischemic complications are common. The disease is highly genetically heterogeneous, with multiple identified genes accounting for only a small percentage of cases. Identifying a common pathway of disease pathogenesis would enhance care for MMD patients. Heterozygous
ACTA2
p.R179 pathogenic variants cause MMD-like cerebrovascular disease. Smooth muscle cells (SMCs) from a conditional knock-in mouse model (
Acta2
SMC-R179C/+
) and from patient-derived induced pluripotent stem cells (iPSCs) are incompletely differentiated, leading to SMC migration and proliferation that may drive the occlusive lesions. Further,
Acta2
SMC-R179C/+
SMCs have increased glycolysis and reduced oxidative respiration on Seahorse analyses. Molecular characterization showed that
Acta2
SMC-R179C/+
SMCs have reduced mitochondrial mass and decreased complex I activity. Nicotinamide riboside (NR) treatment increases oxidative respiration and complex I activity in
Acta2
SMC-R179C/+
SMCs, and surprisingly also increases differentiation and decreases migration. To assess if this pathway is generalizable beyond
ACTA2
patients, we used SMCs with other pediatric MMD-associated variants: loss-of-function variants in
Pcnt
(explanted from a mouse model) and a pathogenic variant in
RNF213
(generated via Crispr/Cas9 in human iPSCs).
Pcnt
KO SMCs are less differentiated, more proliferative and migratory, and have increased glycolysis evident on Seahorse analyses compared with control SMCs.
RNF213
variant SMCs also have increased glycolysis and a blunted response to differentiation-inducing growth factors. These data provide preliminary evidence for a common and therapeutically targetable pathway in pediatric MMD patients.
Abstract 657: Volumetric Ultrasound Analysis of the Abdominal Aorta Enhances Strain Assessment in Mice
Authors: Gedney John , Figueroa Mario , Mattia Victoria , xiong Ying , Mukherjee Rupak , Jones Jeffrey A , Ruddy Jean M
Published: October 2023

Introduction:
Aortic stiffness (AS) is a well-established and under-utilized predictor of cardiovascular morbidity and mortality, particularly in hypertension (HTN). Employing ultrasound (U/S) to quantify AS metrics could have therapeutic application, but technical and regional variation has compromised clinical utility. We hypothesize that integrating 4-dimensional (4D) U/S to asses volumetric strain (VS) will enhance the AS analysis in mice with HTN as a foundation for clinical translation.

Methods:
Systolic blood pressure (SBP) was captured by Coda tail-cuff in C57Bl/6 mice (n=3) on Day 0. The Vevo3100 high-resolution micro-U/S captured images of the infrarenal aorta which included (1) axial plane at three segments (proximal, mid, distal); (2) longitudinal plane; and (3) 4D images of volumetric change across the cardiac cycle. VevoVasc software quantified distensibility (D), pulse propagation velocity (PPV), global radial strain (GRS), and VS. HTN was then induced by osmotic pump angiotensin-II infusion (1.46mg/kg/day). SBP and U/S measurements were reassessed at Day 21. T-test was utilized to compare AS metrics.

Results:
With AngII infusion, SBP increased 32
+
2% (p<0.05). Values for D significantly decreased from proximal to distal aortic regions at Day 0, and demonstrated 50
+
5% reduction with HTN at Day 21 (p<0.05). As another indicator of AS in HTN, PPV increased >2.8 fold. GRS, on the other hand, provided discordant results along the axial aortic regions at Day 0 as well as Day 21, and no change could be detected in the longitudinal plane. With 4D U/S to capture volume and normalize by pulse pressure, we detected a significant reduction (0.74
+
0.3uL/mmHg vs 0.36
+
0.1uL/mmHg, p<0.05) in VS to effectively quantify infrarenal aortic strain as an indicator of increased AS.

Conclusion:
Methodology to ensure sensitive, specific, and reproducible quantification of AS is vital to establishing clinical utilization. VS assessment decreased regional variability and further investigation is warranted to define this technique in the management of HTN and other regional aortic pathology.
Abstract 671: A Silent Inferior Wall ST Segment Elevation Myocardial Infarction
Authors: Kalsoom Sidra , Khan Muhammad , Zaman Muhammad A , Vitellas Michail
Published: October 2023

Introduction:
Acute myocardial infarction (MI) in the setting of methadone use is a rare finding with an uncertain prognosis. We present an interesting case of incidentally diagnosed acute inferior wall MI in a young asymptomatic patient with minimal atherosclerotic risk factors, who was taking methadone for opioid use disorder.

Case Presentation:
A 36-year-old Caucasian male cigarette smoker with a past medical history of diabetes mellitus and opioid use disorder on methadone was evaluated as an outpatient for an abnormal EKG. The EKG was done as part of a routine serial outpatient EKG for QTc monitoring while on methadone. At no point, before coming to our clinic he had chest pain, shortness of breath, palpitations, dizziness, syncope, or any sign and symptoms suggestive of cardiac etiology. A twelve lead EKG revealed findings concerning inferior wall MI; ST elevation on inferior limb leads (II, III, and aVF) along with Q waves. The patient underwent a coronary angiogram which revealed lesions in the mid and distal segments of the right coronary artery. Coronary angiography with the percutaneous intervention was performed with good recovery.

Discussion:
It is thought that opioid-dependent patients do not experience accelerated atherosclerosis. On the contrary, opioids have a cardio-protective effect. The Pro-arrhythmogenic effects of methadone secondary to QT prolongation are also well known, however, the underlying pathophysiology, likely immune-related, has not been extensively studied. Although data are lacking, however, such clinical cases should also draw the attention of physicians to the side effects of methadone when it comes to the treatment of opioid use disorder.

Conclusion:
In our opinion, patients on chronic methadone therapy need to be monitored for not just QT interval monitoring but for any potential signs of ischemia given the fact that they may not experience classic chest discomfort associated with ischemic heart disease. Of note, the increasing use of opioids for chronic non-cancer pain is also concerning the safety of this class of drugs in terms of structural lesions of the heart.
Abstract 673:
CHROMR
Governs Interferon Signaling And Cholesterol Homeostasis In Human Atherosclerotic Plaque
Authors: van Solingen Coen , Cyr Yannick , Barcia Duran Jose Gabriel , Gourvest Morgane , Zhang Tracy , Giannarelli Chiara , Moore Kathryn J
Published: October 2023

Long noncoding RNAs (lncRNA) have emerged as critical regulators of gene expression, yet their potential contribution to human disease remains poorly understood. We previously identified the primate-specific lncRNA
CHROMR
as a key regulator of cholesterol homeostasis. Here, we report a novel function for
CHROMR
in controlling interferon signaling, which is known to regulate innate and adaptive immune responses that drive atherosclerosis. Using single cell RNA-sequencing of human atheroma, we observed robust expression of
CHROMR
in plaque immune cell populations, which correlated strongly with the expression of two subsets of interferon-stimulated genes (ISG). Interrogation of these genesets using DisGeNET, a comprehensive catalogue of genes and variants associated to human diseases, revealed associations with atherosclerosis and coronary artery disease. Differential expression analysis of
CHROMR
-expressing versus non-expressing immune cells showed higher levels of ISGs particularly in the monocyte-macrophage cluster. To assess the impact of
CHROMR
depletion on inflammatory gene expression, we knocked down
CHROMR
in THP1 macrophages using GapmeR antisense oligonucleotides (or controls) and treated with the TLR3 agonist poly(I:C) to stimulate the interferon response. Transcriptome profiling and unsupervised hierarchical clustering of genes differentially expressed in
CHROMR
-sufficient and -depleted macrophages revealed that
CHROMR
silencing markedly repressed expression of key ISGs, including
MX1
,
IFI44L
,
STAT1
, and CXCL and CCL family chemokines (
CXCL10
,
CXCL11, CCL2
). Conversely, overexpression of
CHROMR
in THP1 macrophages increased expression of these ISGs. Furthermore,
CHROMR
depletion in macrophages reduced histone acetylation at regulatory regions of ISG loci. Mechanistically, we showed that
CHROMR
sequesters the interferon regulatory factor (IRF)-2-dependent transcriptional co-repressor IRF2BP2, thereby licensing IRF-dependent signaling and transcription of the ISG network. Taken together, our work unveils a critical role for
CHROMR
in coordinating interferon signaling responses in macrophages and suggests that targeting this lncRNA may reduce inflammation in atherosclerotic plaques.
Abstract 678: Use Of Extracellular Substrates By Alveolar Type 2 Pneumocytes For Surfactant Lipid Synthesis.
Authors: Sanchez-Gonzalez Dativo , Ward Rafael , Vasquez Guillen María J , Garcia-Arcos Itsaso
Published: October 2023

Introduction:
Pulmonary surfactant is a lipoprotein complex essential for lung function. It reduces surface tension during inspiration and avoids alveolar collapse during expiration. It is synthetized by alveolar type 2 pneumocytes (T2C) and it is composed of phospholipids (~90%), mainly phosphatidylcholine (PC), and specific proteins. Decreased surfactant and altered composition are associated with multiple adult lung diseases. However, T2C lipid metabolism and their use of extracellular glucose and lipid for surfactant synthesis has not been studied. Our prior results showed that the low density lipoprotein receptor-related 1 (LRP1) is needed for surfactant synthesis. We hypothesized that nutritional conditions and LRP1 function impact the pathways used by T2C to synthetize surfactant.

Methods:
Control and LRP1 knockdown T2C-derived A549 cells were cultured in DMEM (4.5 g/L glucose) with 10% FBS and maintained at the air-liquid interface for 24 hours to promote their differentiation and polarization. Then, cells were cultured in DMEM with delipidated FBS and BSA-complexed palmitic acid (PA) at different concentrations (0-0.25 mM) for 2 hours and mRNA of surfactant synthetic enzymes was analyzed.

Results:
mRNA expression of
Fatty acid synthase (FASN)
and
Choline kinase (CK)
decreased 80% and 93% (p<0.001) respectively when at least 0.05 mM PA was present.
Phospholipase A2
(
PLA2)
followed the opposite pattern, with 21.5-fold increase (p<0.001) when PA was present.
FASN
and
CK
expression was downregulated in the absence of LRP1. mRNA expression of genes involved in cholesterol metabolism was assessed too.
HMG-CoA reductase
decreased only at the highest concentration of PA (41% decrease, p<0.01), while expression of
HMG-CoA synthase
and lipid exporters
ABCA1
and
ABCG1
was not affected.
ABCG1
expression was compromised in LRP1 knockdown cells.

Conclusions:
Extracellular availability of glucose and fatty acids affects mRNA expression of genes involved in surfactant lipid synthesis in T2C, suggesting metabolic pathway flexibility during different nutritional conditions. In the absence of extracellular lipid sources, glucose can be used by T2C for fatty acid synthesis. Furthermore, LRP1 expression affects the metabolic pathways used by T2C.
Abstract 680: ApoA-I/SR-BI Double Knockout Mice Plasma Is Rich In Very Large HDL With High Free Cholesterol
Authors: Tsujita Maki , Nakasuka Kosuke , Takase Hiroshi , Kamiya Mayuko , Mineo Chieko , Shaul Philip W , Okuhira Keiichiro , Yokoyama Shinji , Remaley Alan T
Published: October 2023

Aim:
SR-BI null mice are known to have abnormally large HDL enriched in free cholesterol. Here, we examined the involvement of apoA-I on high free cholesterol-HDL in SR-BI null mice plasma.

Method:
Mice with SR-BI null allele and ApoA-I null allele were generated by mating CAG-cre tg mice to their floxed mice. By crossing these mice, ApoA-I/SR-BI double null mice were obtained. The gel permeability HPLC-based plasma lipoprotein profile of the mouse was analyzed by LipoSEARCH (Immuno-Biological Laboratories, Co. Ltd., Japan).

Results:
In ApoA-I null mice plasma, HDL is low but relatively larger in particle diameters than wild-type mice which have similar characteristics to SR-BI null mice. In the ApoA-I / SR-BI double null mice, large HDL/ very small LDL (13-19 nm) was increased to 1.86-fold. Free cholesterol content in the ApoA-I / SR-BI double null mice was increased in 12 nm to 60 nm size lipoprotein particles. Free cholesterol levels in the large and the very large HDL fraction (12 to 16 nm), the LDL fraction (16 to 30 nm), and the VLDL fraction (30 to 70 nm) were 1.16, 2.38, and 2.36 mg/dL in

Apoa1
-/-

mice, and were 18.81, 44.02, and 15.56 mg/dL in

Scarb1
-/-
:Apoa1
-/-

mice, respectively.

Conclusions:
ApoA-I null mice plasma profile in free cholesterol level was dramatically increased by deletion of
Scarb1
. High free cholesterol in plasma is known as an atherosclerotic factor. High free cholesterol in these large lipoprotein particles may explain by LCAT function is perhaps only effective on the normal size HDL or disc shape HDL with apoA-I. On the other hand, CETP tg/ SR-BI null mice with normal HDL size also show high free cholesterol HDL(ATVB 2010, Hildebrand
et al.
). Furthermore, Wolman’s Disease, deficiency of lipase in the lysosome, did not change cholesterol homeostasis of taken-up cholesterol from HDL (BBA 1990, Sparrow and Pittman). Further study is necessary to identify the true origin of plasma-free cholesterol in SR-BI null mice plasma.
Abstract 690: Circulating Proteomics And Endothelial Cell Phenotype In Patients With Type 2 Diabetes Mellitus (T2DM)
Authors: Rizvi Syed Husain Mustafa , Zhou Yuxiang , Weisbrod Robert M , Behrooz Leili , Minetti Erika T , Eberius Megan , Makayla Petty , Hamburg Naomi M
Published: October 2023

Patients with T2DM display endothelial dysfunction that predicts cardiovascular risk. We have previously demonstrated reduced eNOS activation in endothelial cells (ECs) isolated from patients with T2DM. We sought to identify drivers of EC dysfunction using a combination of proteomic and transcriptomic approaches by measuring: serum O-link cardiovascular panels II and III along with insulin-mediated eNOS phosphorylation and miRNA profiling (NextGen sequencing) in isolated ECs. In 69 patients (37 with T2DM and 32 age- and sex-similar non-T2DM controls) proteomic analysis identified 35 upregulated and 6 downregulated (FDR p<0.05) proteins that included metabolic, vascular and fluid homeostasis, immune and apoptosis related biomarkers. Several biomarkers related to the degree of insulin-mediated eNOS phosphorylation in isolated EC: (renin r= -0.38, p=0.004, chymotrypsin C r= 0.37, p=0.006, paraoxinase 3, r= 0.35, p=0.009, lipoprotein lipase r= 0.34, p=0.01, superoxide dismutase 2 r= 0.31, p=0.02 and adrenomedullin r= -0.27, p=0.049). In a subset of 10 patients (5/group), we confirmed expression of EC-specific miRNA in ECs purified by CD144 microbeads. Comparing patients with T2DM and controls we found 6 upregulated and 7 downregulated miRNAs (FDR P<0.05). Ingenuity Pathway Analysis shows that these miRNAs are involved in the regulation of inflammation, glucose metabolism, stress signaling and cell cycle pathways. Further we found overlap of differentially expressed miRNA with predicted regulation of biomarkers that were altered in T2DM including: miR-4326 and miR-1270 regulate TNFRSF10A and ALCAM, while, miR-4433-5p and miR-342-3p are involved in regulation of superoxide dismutase 2. Our findings demonstrate that an association of circulating biomarkers particularly those that relate to fluid metabolism and oxidative stress with altered endothelial cell signaling in patients with T2DM. Further we have early evidence of altered non-coding miRNA expression in ECs in T2DM that may be related to inflammation and oxidative stress. Ongoing studies are evaluating the functional implications of altered miRNA expression in regulating vascular function in patients with T2DM.
Abstract 696: Long Noncoding RNA Regulation Of Inflammation In Atherosclerosis
Authors: Gourvest Morgane , Barcia Durán José G , Zhang Tracy , Wang Jesse , Peled Daniel , Newman Alexandra , Dupuis Samantha , Giannarelli Chiara , van Solingen Coen , Moore Kathryn J
Published: October 2023

Atherosclerosis is a chronic inflammatory disease driven by cholesterol deposition in the artery wall. Resolution of atherosclerotic inflammation and plaque regression are important clinical goals. As long noncoding RNAs (lncRNAs) are key regulators of gene expression, there is an intense interest in understanding their contribution in atherosclerosis and their potential as therapeutic targets to induce plaque regression. To this end, we performed single cell RNA-sequencing of CD45
+
immune cells isolated from aortic arch plaques of
Ldlr
-/-
mice under conditions of plaque progression (16 weeks Western diet) or regression (16 weeks Western diet + 4 weeks apoB antisense oligonucleotide), and identified lncRNAs that were differentially expressed (DE) under these conditions. We then classified lncRNA candidates based on their proximity to protein coding genes (intergenic, antisense, intronic, bidirectional) and screened for lncRNAs acting in
cis
to locally regulate the expression of neighboring genes. We identified 112 DE lncRNAs with proximal protein-coding genes (<5kb) whose mRNAs were also DE in blood and plaque macrophages during atherosclerosis progression and regression, including

Caslr (
C
tsc
a
nti
s
ense
l
nc
R
NA)

, a novel lncRNA transcribed from the opposite strand of the Cathepsin C gene (
Ctsc
) that encodes a lysosomal protease implicated in atherogenesis. The
Caslr-Ctsc
pair is upregulated in macrophages of progressing plaques and in response to pro-inflammatory stimuli [
i.e
., lipopolysaccharide, interleukin (IL)-6]
in vitro
, and conversely, downregulated during atherosclerosis regression and in response to anti-inflammatory (
i.e.
, IL-4) stimuli. Loss-of-function approaches revealed that
Caslr
regulates
Ctsc
expression and protease activity, with downstream consequences on activation of pro-inflammatory NF-κB signaling and inflammasome-dependent IL-1β release. Notably,
Caslr
shares functional homology with a lncRNA proximal to the human
CTSC
gene. Collectively, our data identify
Caslr
as a conserved lncRNA driving expression of cathepsin C and downstream inflammatory signaling in macrophages, and suggest that, targeting this lncRNA may be beneficial in treating atherosclerosis.
Abstract 703: Dioxin-induced Smooth Muscle Cell Phenotypic Modulation And Atherosclerosis Worsens In The Absence Of AHR
Authors: Qin Guyu , Easwaran Meena , Damiani Isabella , Zhao Quanyi , Kundu Ramendra K , Nguyen Trieu , Cheng Paul , Fujita Ayaka , Quertermous Thomas , Kim Juyong B
Published: October 2023

Background:
Dioxin (TCDD, 2, 3, 7, 8-Tetrachlorodibenzo-p-dioxin) is an active constituent of tobacco smoke and air pollution that activates the aryl hydrocarbon receptor (AHR). Dioxin has been linked to an increased risk of coronary artery disease (CAD). However, the corresponded molecular mechanisms remain unclear. Smooth muscle cells (SMC) play a critical role in atherosclerotic plaque remodeling due to their phenotypic plasticity. In this study, we aimed to understand the role of AHR in modulating the effect of dioxin on vascular SMC and atherosclerosis phenotype.

Methods:
SMC lineage-tracing (SMC
Lin
) mice (

Tg
Myh11-CreERT2
, ROSA
tdT/tdT
, ApoE
-/-

) without any TCDD injection were used as control. TCDD were injected to both SMC
Lin
mice, and SMC-specific
Ahr
knockout mice. High fat diet (HFD) and TCDD injection started at 8 weeks of age and aortic roots were harvested at 24 weeks of age for either histology or single-cell sequencing (10X Genomics, triplicates). Histology analysis was performed at the aortic sinus of control mice (n=9), TCDD injected SMC
Lin
mice (n=12) and TCDD injected SMC-specific
Ahr
knockout mice (n=9). Immunohistochemistry was performed with anti-SM22α antibody and CD68 antibody.

Results:
The histological analysis of the aortic sinus showed increased lesion area after TCDD treatment in the SMC
Lin
control mice. In the SMC-specific knockout of
Ahr
, the lesion area was significantly larger compared to the SMC
Lin
control mice when treated with TCDD along with significantly increased presence of
Tagln
+
cells in the lesion. These results indicated TCDD leads to adverse remodeling of atherosclerosis and that the presence of
Ahr
gene is protective against the athero-promoting effects of dioxin. The scRNA-seq of SMC
Lin
control mice treated with TCDD exposure showed that the SMC phenotypic modulation was markedly inhibited, leading to the presence of fewer fibromyocytes (FMC). Increased FMC to chondromyocytes (CMC) modulation resulted in further reduction of FMC population in the
Ahr
-KO mice treated with TCDD.

Conclusion:
Dioxin adversely remodels atherosclerotic plaque, and the lack of AHR in SMC can exacerbate the atherosclerotic process, suggesting that dioxin may exhibit its harmful effect via non-AHR dependent pathway.
Abstract 709: Implementation Of Ribosomal Tagging Strategies To Analyze Smooth Muscle Specific Rna Expression In Mouse Models Of Atherosclerosis
Authors: Zhu Lucie Y , Cui Jian , Ross Leila , Reilly Muredach P
Published: October 2023

Background:
Atherosclerotic cardiovascular disease (CVD) is the leading cause of mortality worldwide. Smooth muscle cells (SMCs) make up the bulk of the healthy arterial wall and atherosclerotic lesions. During atherosclerosis progression, we and others have reported on the emergence of multiple harmful and protective types of SMC-derived cell types (SDCs) in lesions, and the use of fluorescence lineage-tracing animal models have made it possible to isolate and analyze cell-type specific transcriptomes by flow cytometry and scRNA-seq. However, these manipulations are known to carry risk for artifacts, alter transcription, and introduce cell type bias. Therefore, alternative approaches that circumvent the need for single cell isolation may be beneficial.

Hypothesis:
Introduction of an isolation-free method for SMC translatome retrieval may be beneficial in confirming the presence of SDCs correlating with disease.

Methods:
SMC-specific translatomes were isolated by immunoprecipitation (IP) of epitope-tagged ribosomes from the aortic arch following the “RiboTag approach” developed by McKnight et al. and sequenced to test against libraries created by flow isolation of SMCs and bulk RNA-sequencing.

Results:
Here we report the creation of SMC-specific atherosclerotic RiboTag mouse model and initial efforts made to assess the SMC/SDC translatome during atherosclerosis. Side-by-side comparison of translatomes isolated by IP from the aortic arch and transcriptomes from sorted SDCs will be used to identify advantages and disadvantages of the respective approaches.

Conclusions:
This work will determine if and how the use of cell isolation may impact the transcriptome of SMCs and SDCs from mouse atherosclerotic models and establish if translatome analysis of these cell types can be used to complement standard sort-based profiling strategies in the study of atherosclerosis.
Abstract 710: Specific Removal Of Oxidized Cholesterol With A Cyclodextrin Dimer Drug Candidate Towards Reversing Atherosclerosis
Authors: Clemens Daniel M , Anderson Amelia , Dinh Darren , Bhargava Prerna , Teramayi Fadzai , Silberg Ana , Sadrerafi Keivan , Rosenberg Noah , Malanga Milo , Garcia-Fandiño Rebeca , Piñeiro Ángel , O'Connor Matthew
Published: October 2023

Cardiovascular disease (CVD) is the world’s biggest killer. Current treatments for CVD are failing to deliver on promises to beat this deadly and complex family of diseases. New approaches that go beyond targeting LDL are needed. Cyclarity Therapeutics has developed a novel class of specifically engineered, dimerized cyclodextrin (CD) molecules for the encapsulation of toxic oxidized cholesterol. Oxidized cholesterol accumulates over time and causes dysfunction in many cell types, linking it to several age-related diseases including atherosclerosis. Presently, treatments for atherosclerosis are invasive, expensive, and/or show limited benefits. Here, a combination of in silico, in vitro, and ex vivo methods are used to implement a synergistic rational drug design strategy for developing CDs to remove atherogenic oxidized cholesterol (primarily 7-ketocholesterol (7KC)) from cells and tissues. Our results thus far indicate that our lead compound, UDP-003, can both prevent and reverse the formation of atherogenic foam cells in-vitro. UDP-003 binds and clears 7KC from in-vivo and ex-vivo systems selectivity. IND enabling studies, including pivotal GLP studies in rodent and non-rodent species, are complete and show that UDP-003 has an excellent safety profile with no significant clinical liabilities. UDP-003 has been awarded the Innovative Licensing and Access Pathway (ILAP) Innovation Passport by the MHRA, which is accelerating Cyclarity’s path to clinic and will facilitate rapid and innovative clinical trials. In totality, our data suggest that targeted removal of 7KC from foam cells with our proprietary class of cyclodextrin compounds has the potential to prevent and reverse the formation of atherosclerotic plaques. This innovation represents the first disease-modifying therapeutic approach to treating atherosclerotic disease.
Abstract 712: IL-19 Is A Lymphokine That Induces Prox1-dependent Lymphangiogenesis And Permeability
Authors: Peluzzo Amanda , Leigh Tani , Kelemen Sheri E , St. Paul Amanda , Corbett Cali , Autieri Michael
Published: October 2023

Introduction:
Atherosclerosis is a vascular inflammatory disease associated with lymphangiogenesis, but whether lymphangiogenesis contributes to disease progression or is simply a compensatory attempt at disease resolution is currently unresolved. Recent studies are revealing a role for lymphatic vessels in reverse cholesterol transport (RCT), which could attenuate atherosclerosis. We have previously reported that Interleukin-19 (IL-19), an anti-inflammatory cytokine, uniquely attenuates atherosclerotic plaque progression while also being pro-angiogenic. This drives our
hypothesis
that one mechanism whereby IL-19 is atheroprotective is by promoting functional lymphangiogenesis and lymphatic flux.

Methods & Results:
IL-19 can significantly induce human dermal lymphatic endothelial cell (hdLEC) proliferation, migration, and tube formation, to the same degree as VEGFC, a potent inducer of lymphangiogenesis. RNA sequencing analysis indicates that IL-19 induces an angiogenic transcriptional program through Prox1, a master regulator of lymphangiogenesis. Knockdown of Prox1 led to a significant decrease in IL-19-associated proliferation and migration. IL-19 also increased expression of Angpt2, an endothelial cell protein that induces permeability through regulation of tight junctions, through a Prox1-dependent mechanism. Electric Cell-substrate Impedance Sensing assays showed that IL-19 treatment increased hdLEC permeability and mitigated oxLDL-associated decreased permeability, suggestive of maintaining lymphatic barrier function during atherosclerosis. IL-19 treatment also increased dil-HDL transport across an hdLEC barrier. Preliminary
in vivo
data suggests injection of rmIL-19 in

Il19
-/-

mice increases lymphatic permeability and flux through increased lymphatic uptake of Evans blue dye.

Conclusion:
These data suggest that IL-19 increases lymphangiogenesis, permeability, and may increase RCT to plausibly decrease atherosclerosis. These results can influence our understanding of the association between the lymphatic system and atherosclerosis.
Abstract 718: Cerebral Small Vessel Disease In Children With Sickle Cell Disease
Authors: Hyacinth Hyacinth I , Karkoska Kristine A , Sawyer Russell P
Published: October 2023

Introduction:
The neurological complications of sickle cell disease (SCD) are particularly devastating and range from “overt stroke” to cerebral small vessel disease (CSVD) phenotypes such as silent cerebral infarction (SCI) and cognitive deficit in the absence of magnetic resonance imaging (MR)-detectable evidence of cerebral injury, suggesting the presence of covert mechanism such as dilated perivascular spaces (dPVS).

Methods:
We quantified the presence of dPVS in children with SCD who were screened for or enrolled in the SIT trial, which was a multicenter, randomized clinical trial that evaluated the effect of monthly chronic transfusions to prevent the development of new SCI in children with SCD. dPVS were evaluated on T2-weighted MRI from 491 children with SCD. We used both the Wardlaw (part of the international consensus criteria for evaluating CSVD) and Rotterdam criteria to quantify dPVS burden in 3 or 4 brain regions respectively.

Results:
The median age was 8.5 years (range 5 – 15 years). 136 children (28%) had MRI evidence of at least one SCI. The mean hemoglobin was 8.1 ± 1.1 g/dL and total WBC count was 12,000 ± 5300 cells/ml. The mean dPVS burden score from the Wardlaw criteria was 3.5 ± 1.5, while that for the Rotterdam criteria was 31 ± 13 (see Table). Global and regional dPVS burden scores from the two rating scales were well correlated (p < 0.001, r = 0.7 – 0.8). There was a significant sex difference in dPVS burden (mean Wardlaw rating: males = 3.7 versus females = 3.4, p = 0.04), but this disappeared in multivariate analysis, which showed that only increasing age was associated with dPVS burden (p = 0.02). Surprisingly, presence or size of SCI were not associated with dPVS burden score.

Conclusions:
Children with SCD have more evidence of dPVS than would be expected for their age and compared to matched healthy children. There was no significant association between dPVS burden and presence of cerebral injury such as SCI.

Table
Calibrated Interpretation: Confidence Estimation in Semantic Parsing
Authors: Stengel-Eskin Elias , Van Durme Benjamin
Published: October 2023

Sequence generation models are increasingly being used to translate natural language into programs, i.e., to perform executable semantic parsing. The fact that semantic parsing aims to predict programs that can lead to executed actions in the real world motivates developing safe systems. This in turn makes measuring calibration—a central component to safety—particularly important. We investigate the calibration of popular generation models across four popular semantic parsing datasets, finding that it varies across models and datasets. We then analyze factors associated with calibration error and release new confidence-based challenge splits of two parsing datasets. To facilitate the inclusion of calibration in semantic parsing evaluations, we release a library for computing calibration metrics.1
Typification of three Carex nothospecies names dedicated to Czech botanists by J. Podpěra
Authors: ŘEPKA RADOMÍR , TARAŠKA VOJTĚCH
Published: October 2023

The article elucidates the taxonomic identity of the putative nothospecies Carex ×binderi, C. ×filkukae and C. ×lausii described by Josef Podpěra from the territory of Bohemia and Moravia (Czech Republic) in the early 20th century. Herbarium material obviously handled by J. Podpěra was retrieved and thoroughly compared with that of the presumed parental taxa, but morphological evidence point to a hybrid origin only for C. ×binderi, corresponding to C. ×leutzii. By contrast, plants of C. ×filkukae and C. ×lausii could be assigned to atypical individuals of C. leporina and C. acutiformis, respectively. Lectotypes for all treated names are designated here, which turns J. Podpěra’s names into synonymy of the above-mentioned species and nothospecies names.
Recent Increase in a Recurrent Pan-Atlantic Wave Pattern Driving Concurrent Wintertime Extremes
Authors: Kornhuber Kai , Messori Gabriele
Published: October 2023

Wintertime extremes such as cold spells and heavy precipitation can have severe socioeconomic impacts, disrupting critical infrastructures and affecting human well-being. Here, we relate the occurrence of local and concurrent cold or wet wintertime extremes in North America and Europe to a recurrent, quasi-hemispheric wave-4 Rossby wave pattern. We identify this pattern as a fundamental mode of the Northern Hemisphere (NH) winter circulation, since wave 4 exhibits phase-locking behavior. Thus, the associated atmospheric circulation and surface anomalies reoccur over the same locations when the pattern’s wave amplitude is high. The wave pattern is most pronounced over the pan-Atlantic region, and increases the probability of extreme cold or wet events by up to 300% in certain areas of North America and Europe, as well as favoring their concurrence at different locations. High-amplitude wave-4 events have increased significantly in frequency over the past four decades (1979–2021), although no clear evidence is found relating this to modes or patterns of climate variability. The identified wave pattern may provide pathways for early prediction of local and concurrent cold or wet wintertime extremes in North America and Europe.
Resistance to PRMT5-targeted therapy in mantle cell lymphoma
Authors: Long Mackenzie Elizabeth , Koirala Shirsha , Sloan Shelby , Brown-Burke Fiona , Weigel Christoph , Villagomez Lynda , Corps Kara , Sharma Archisha , Hout Ian , Harper Margaret , Helmig-Mason JoBeth , Tallada Sheetal , Chen Zhengming , Scherle Peggy , Vaddi Kris , Chen-Kiang Selina , Di Liberto Maurizio , Meydan Cem , Foox Jonathan , Butler Daniel , Mason Christopher , Alinari Lapo , Blaser Bradley W. , Baiocchi Robert
Published: October 2023

Mantle cell lymphoma (MCL) is an incurable B-cell non-Hodgkin lymphoma, and patients who relapse on targeted therapies have poor prognosis. Protein arginine methyltransferase 5 (PRMT5), an enzyme essential for B-cell transformation, drives multiple oncogenic pathways and is overexpressed in MCL. Despite the antitumor activity of PRMT5 inhibition (PRT-382/PRT-808), drug resistance was observed in a patient-derived xenograft (PDX) MCL model. Decreased survival of mice engrafted with these PRMT5 inhibitor–resistant cells vs treatment-naive cells was observed (P = .005). MCL cell lines showed variable sensitivity to PRMT5 inhibition. Using PRT-382, cell lines were classified as sensitive (n = 4; 50% inhibitory concentration [IC50], 20-140 nM) or primary resistant (n = 4; 340-1650 nM). Prolonged culture of sensitive MCL lines with drug escalation produced PRMT5 inhibitor–resistant cell lines (n = 4; 200-500 nM). This resistant phenotype persisted after prolonged culture in the absence of drug and was observed with PRT-808. In the resistant PDX and cell line models, symmetric dimethylarginine reduction was achieved at the original PRMT5 inhibitor IC50, suggesting activation of alternative resistance pathways. Bulk RNA sequencing of resistant cell lines and PDX relative to sensitive or short-term–treated cells, respectively, highlighted shared upregulation of multiple pathways including mechanistic target of rapamycin kinase [mTOR] signaling (P < 10-5 and z score > 0.3 or < 0.3). Single-cell RNA sequencing analysis demonstrated a strong shift in global gene expression, with upregulation of mTOR signaling in resistant PDX MCL samples. Targeted blockade of mTORC1 with temsirolimus overcame the PRMT5 inhibitor–resistant phenotype, displayed therapeutic synergy in resistant MCL cell lines, and improved survival of a resistant PDX.