bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 362: Endothelial Increase In Autotaxin Regulates Cerebral Vascular Permeability During Ischemic Reperfusion
Authors: Bhattarai Susmita , Sharma Sudha , Ara Hosne , Subedi Utsab , Bhuiyan Shenuarin , Sun Hong , Miriyala Sumitra , Panchatcharam Manikandan
Published: November 2022

Introduction:
Pathological progression of stroke is aided by ischemic reperfusion during the treatment procedure. Various molecular signals initiated during ischemic reperfusion exacerbates the disease leading to endothelial permeability. However, a detailed molecular therapeutic regimen with r-TPA and mechanical thrombectomy could limit the detrimental effects. Lysophosphatidic acid (LPA) is a bioactive phospholipid regulated in physiological and pathological conditions. LPA is produced by the enzyme autotaxin (ATX). ATX-LPA axis have been observed to deteriorate physiological status in various diseases. In cerebral ischemic-reperfusion, the ATX-LPA axis could regulate the endothelium disruption.

Hypothesis:
Increased ATX expression in endothelium disrupts the endothelial barrier by increasing LPA during ischemic-reperfusion.

Methods:
For the in-vitro study, oxygen and glucose deprivation and reperfusion (OGDR) were carried out in mouse brain microvascular endothelial cells (MBMEC).For invivo transient middle cerebral artery occlusion with 90 minutes of ischemia and 24 hours of reperfusion (I/R) was used as a stroke model in mice. Electric Cell Substrate Impedance System (ECIS) and Evan Blue was used for permeability study for invitro and invivo respectively. AR-2 probe fluorescence assay was used to measure ATX activity.

Results:
ATX expression was increased (P<0.05) in MBMEC with OGDR suggesting ATX regulation in endothelial cells following ischemic-reperfusion. Permeability measured with ECIS in MBMEC showed that LPA treatment increased the permeability in a time and concentration-dependent manner. The protein levels of junctional proteins such as βcatenin, Claudin5, VEcadherin, and Zo1 weresignificantly (P<0.05) decreased with LPA exposure. In invivo stroke model ATX enzymatic activity is prominently raised (P<0.01) in I/R mice compared to sham mice. Increased ATX activity coheres with the Evans Blue permeability in the mice brain. Simultaneously LPA levels are elevated in I/R mice. LPA produced during the process can disrupt the blood-brain barrier, increasing cerebral permeability.

Conclusion:
Inhibition of the ATX-LPA axis prospects to be a better therapeutic avenue for cerebral ischemic reperfusion.
Abstract 389: An Inflammatory Aging Clock (iage) Based On Deep Learning Tracks Multimorbidity, Immunosenescence, And Vascular Aging
Authors: Sayed Nazish
Published: November 2022

While many diseases of aging have been linked to the immunological system, immune metrics capable of identifying the most at-risk individuals are lacking. From the blood immunome of 1,001 individuals aged 8-96 years, we developed a deep-learning method based on patterns of systemic age-related inflammation. The resulting inflammatory clock of aging (iAge) tracked with multimorbidity, immunosenescence, frailty and cardiovascular aging, and is also associated with exceptional longevity in centenarians. The strongest contributor to iAge was the chemokine CXCL9, which was involved in cardiac aging, adverse cardiac remodeling and poor vascular function. Furthermore, aging endothelial cells in human and mice show loss of function, cellular senescence and hallmark phenotypes of arterial stiffness, all of which are reversed by silencing CXCL9. Importantly, we identify a key role of CXCL9 in age-related chronic inflammation and derive a metric for multimorbidity that can be utilized for the early detection of age-related clinical phenotypes.
Abstract 391: Dissecting The Role Of Macrophage Cd36 In Resolution Of Inflammation
Authors: Nasias Dimitris , De Bermont Julie , Son Ni , Abumrad Nada A , Goldberg Ira J
Published: November 2022

Monocytes and macrophages are implicated in cardiovascular disease, including atherosclerotic plaque progression and regression. The scavenger receptor CD36, which is highly expressed in macrophages, mediates lipid uptake into macrophages and promotes clearance of apoptotic and dead cells. Moreover, CD36 as multi-ligand scavenger receptor binds both fatty acids and lipoproteins, which differ in their effects on macrophage phenotype. Some in vitro experiments have reported that CD36-mediated uptake of lipids convert macrophages to an inflammatory phenotype, whereas other studies show that lipid uptake converts macrophages to an alternatively activated state. To determine the role of this receptor in vivo where cells are exposed to both lipoproteins and free fatty acids, we created macrophage-specific CD36 knockout mice. Using zymosan-induced peritonitis to challenge immune cells, we found a broad heterogeneity of resident and infiltrated immune cells, primarily macrophages, in the peritoneum 72h after the initial stimuli. Macrophage
CD36
deficient cells had increased expression of pathways related to innate immunity, antigen presentation and oxidative phosphorylation; whereas pathways related to efferocytosis adaptive immunity and B cell activation were downregulated. Gene signatures related to pro-inflammatory features were evident only in CD36 deficient macrophages. Macrophage efferocytosis capacity and B cell antibody production did not differ between control and CD36 depleted mice. Our data show that CD36 expression allows macrophages to convert to a less inflammatory state.
Abstract 402: High Prevalence Of Ras/mapk Pathway Mutations In Cell-free Dna Of Extracranial Arteriovenous Malformation: A New Method To Detect The Genotype
Authors: Cai Ren , Sun Yi , Wang Deming , Su Lixin , Fan Xindong
Published: November 2022

Purpose:
Extracranial Arteriovenous Malformations(AVMs) were primarily caused by somatic mutations in KRAS and MAP2K1 genes. Targeted chemotherapies are emerging but require molecular diagnosis. Since the AVMs were high flow and high pressure. It is hard to acquire the AVM lesion tissue where the mutant variants were only detected in. Few were detected in Cell-free DNA(CfDNA). Since absolute ethanol embolism is one of the effective methods for treating AVM. We hypothesized that CfDNA of post-embolism could provide the genotype of patients with AVMs.

Methods:
Peripheral Blood, lesion tissue specimens under the guidance of digital subtraction angiography(DSA), CfDNAs isolated from plasma of 40 patients underwent interventional embolism(before and after as T1(1h, setting first injecting ethanol moment as T0 )). 4 patients ‘s CfDNAs were collaborated in 8h(T8), 16h(T16), 24h(T24), 48h(T48), and 72h(T72) after T0. All the specimen were sequenced by a targeted NGS panel of vascular anomalies

Results:
Variants were detected in tissue and CfDNA but none in peripheral blood. The prevalence for tissue, CfDNA of pre-embolism and post-embolism(1h) were 73%(27/37), 27.5%(11/40), and 90%(36/40), respectively. KRAS(p.Gln61, p.Gly12) and MAP2K1(p.Gln56, p.Lys57) were the mutant hot spots. Novel mutations in BRAF, RASA1, KRAS, and MAP2K1 were also detected in tissue and cfDNA. As for specificity, area under the ROC curve were 0.8125(P<0.0001), 0.6375(P=0.0343), and 0.9500(P<0.0001). For the variants allele frequency(VAF) of CfDNA, the VAF reached a surge at T1, then a second surge at T24.

Conclusion:
AVMs were caused by mutations in RAS/MAPK pathway where cfDNA of post-embolism(T1) of absolute ethanol interventional therapy could provide the accurate genotype of the patients in sensitivity and specificity. VAF detected in T24 could provide information related to the natural metabolism such as the indications or prognosis of targeted chemotherapy. Overall, CfDNA of post-embolism provides us a new method for approaching the precise medicine of AVM.
Abstract 418:
18
FDG-PET Monitors Atherosclerosis Disease Activity In Minipigs.
Authors: Nogales Paula , Velasco Carlos , Gonzalez-Cintado Leticia , Morales Cano Daniel , Mota-Cobián Adriana , Mota Rubén A , Torroja Carlos , Sharysh Diana , Benguria Alberto , Dopazo Ana , Carramolino Laura , España Samuel , Mateo de Castro Jesus , Bentzon Jacob F
Published: November 2022

Background and objectives:
18
FDG-PET imaging is used as clinical endpoint to monitor efficacy of drugs in atherosclerosis, but the link between disease activity and FDG uptake is not well understood. Previous studies in PCSK9
D374Y
minipigs showed that FDG is taken up quantitatively in multiple plaque cell types. Here, we developed methods to alter disease activity of established atherosclerosis in PCSK9
D374Y
minipigs and analyzed the ability of FDG-PET to monitor the changes and the underlying mechanisms.

Methods:
Atherosclerosis was induced in PCSK9
D374Y
minipigs by high-fat feeding for 12 months. Pigs were then either euthanized for analysis of baseline atherosclerosis (Baseline pigs, n=7) or subjected to a 3-months LDL- lowering intervention with low-fat diet (LF pigs, n=8) or low-fat diet supplemented with microsomal transfer protein inhibitor (BMS-212122, 0.5mg/kg/day, MTPi pigs, n=8). Wild-type pigs on low-fat diet provided non-atherosclerotic controls (n=5).
18
FDG-PET imaging was performed in all animals at the endpoint, and serial sections of the abdominal aorta, iliac and LAD arteries were analyzed for plaque morphology, lipid accumulation (Oil Red O), and macrophage content (CD68 and muramidase). In a separate experiment, abdominal plaques from Baseline (n= 5) and MTPi pigs (n=3) were analyzed by single-cell RNA sequencing.

Results:
MTPi pigs reached low LDL cholesterol levels (<1.8 mM) and had significant reductions in plaque lipid content (56%, p=0.002) and necrotic area (50%, p=0.002) compared with Baseline pigs. Macrophage content was numerically, but not significantly, altered.
18
FDG signal was substantially reduced in MTPi compared with Baseline pigs (SUVmean 1.04 vs 1.54, p=0.019) reaching levels similar to non-atherosclerotic wild-type pigs (SUVmean 1.11). Analysis of scRNA-seq data indicated that reduced FDG uptake was explained by lowered glycolytic activity across multiple cell types, including macrophages, lymphocytes, and smooth muscle cells.

Conclusion:
18
FDG-PET imaging can monitor disease activity in atherosclerosis because disease activity is associated with increased glycolysis across all of the major cell types of plaques.
Abstract 432: Linc-ADAIN, A Novel Human Adipose LincRNA Modulated By Obesity, Can Regulate Adipocyte Cytokine Secretion.
Authors: O'Reilly Marcella E , Liu Wen , Xue Chenyi , Cynn Esther , Zhu Lucie , Ross Leila , Reilly Muredach P
Published: November 2022

Background:
Dysfunctional adipose tissue (AT), characterized by increased inflammation and immune cell recruitment, plays a central role in the development of T2DM and atherosclerotic CVDs. LincRNAs are important emerging regulators of cellular functions.

Methods:
RNA-seq of human AT during low-dose endotoxemia, identified novel lincRNAs regulated by inflammation. LincADAIN was identified as 1)specifically expressed in AT 2)reduced in both obesity-induced chronic and LPS-induced acute AT inflammation (-77% and -53%, p<0.05). Most lincRNAs (~80%) are not conserved hence, we utilized a humanized mouse model. NSG mice are transplanted with human ASC-adipocytes that have undergone lentiviral shRNA knockdown and adipogenic induction
in vitro
, prior to injection. After 16 weeks, implants are extracted and analyzed for adipocyte morphology, immune cell infiltration and gene expression changes. Biotinylated lincRNA pulldown assay was used to identify interacting proteins by Mass Spectrometry (MS) and hits validated via RNA IP assays. Cytokines from LPS stimulated adipocytes after Linc-ADAIN KD were measured via cytokine array.

Results:
Human LincADAIN expression negatively correlates to BMI in obese AT (p<0.001, r2-0.3042). PPARγ antagonist treatment reduced LincADAIN expression. KD of LincADAIN in ASC-adipocytes, increased protein but not mRNA levels of inflammatory cytokines, IL-8 and MCP-1. Cellular fractionation shows that LincADAIN is mostly cytoplasmic. MS analysis of LincADAIN pulldown proteins revealed central RNA binding proteins (RBPs) such as HuR, as potential interactors, which may post-transcriptionally regulate inflammatory cytokines. Changes in human circulating adiponectin was detected in the NSG mouse serum, indicating the implants are functional.

Conclusion:
Linc-ADAIN is a novel lincRNA that can regulate AT inflammation potentially through modulation of RNA translation via RBPs. Our mouse model provides a foundation to uncover Linc-ADAIN’s role in AT inflammation
in vivo
and to study other non-conserved human lincRNAs. Functional investigations of lincRNAs, such as these are warranted, as recent genomic studies suggest that lincRNAs are likely the causal element driving human disease at some GWAS loci.
Abstract 460: Covid-19 And Acute Limb Ischemia: Arterial Thromboemboli Composition And Architecture
Authors: Wohlauer Max , Schmitt Lauren , Ghosh Ahana , Govsyeyev Nicholas , Jacobs Donald , Hansen Kirk
Published: November 2022

Objectives:
The incidence of acute limb ischemia (ALI) has increased since the emergence of coronavirus disease 2019 (COVID-19) and has been linked to the serum level of cross-linked fibrin degradation products (D-dimer). The purpose of this study is to understand arterial thromboembolic architecture in patients with ALI and how this is altered by COVID-19 infection.

Methods:
Arterial thromboembolic specimens were prospectively collected at a single institution, stored in an IRB-approved tissue bank at -80°C for preservation of architecture. Liquid chromatography mass spectrometry (LC-MS/MS) was used to identify
in-vivo
Factor XIII (FXIII) generated cross-links, critical determinants of clot stability and strength. Protein composition of thromboemboli was compared between COVID and non-COVID patients.

Results:
Arterial thromboemboli were obtained from four patients. Patient #1 had COVID pneumonia and a D-dimer of 14,690 ng/mL FEU and developed ALI secondary to embolization of an aortic thrombus. Patients #2-4 did not have COVID and developed ALI secondary to synthetic graft thrombosis, septic embolization from endocarditis, and embolization during aortic arch replacement. COVID thromboemboli demonstrated a higher abundance of immune response proteins compared to non-COVID thromboemboli. Eighty-one cross-links were identified across the four specimens (Fig 1). A six-fold greater degree of Fibrinogen Alpha Chain (FibA-FibA) cross-linking was present in the COVID specimen (Mean: 295,100) compared to the mean of the three non-COVID specimens (Mean: 52,973) (Figure 2).

Conclusions:
A higher degree of cross-linking and increased levels of innate and adaptive immune response proteins appear to be features of arterial thromboemboli in COVID-induced ALI. This supports a virus-associated hypercoagulable state in COVID-19 patients with ALI.
Abstract 462: Clinical And Genetic Risk Factors Underlying Family History Of Heart Disease
Authors: Jowell Amanda , Bhattacharya Romit , Marnell Christopher , Wong Megan , Lannery Kim , Honigberg Michael , Peloso Gina , Natarajan Pradeep
Published: November 2022

Background:
While a self-reported family history of cardiovascular disease (CVD) is predictive of incident CVD, the genetic and clinical factors underlying this association remain poorly understood.

Methods:
We performed a cross-sectional analysis of United Kingdom (UK) Biobank participants without preexisting coronary artery disease (CAD), with genetic, biochemical, and clinical characterization at baseline. Self-reported family history of heart disease (FHHD) was the primary outcome, while clinical (diabetes, hypertension, smoking, apolipoprotein B/A1 ratio, body mass index (BMI), C-reactive protein [CRP], lipoprotein(a) [Lp(a)]) and genetic risk factors for CVD (CAD polygenic risk score [PRS], heterozygous familial hypercholesterolemia [HeFH]) were exposures. Models were adjusted for age, sex and statin prescription. All features were considered together using multiple logistic regression, and population attributable risks were subsequently estimated.

Results:
Among 124 203 individuals, 53 318 (42.9%) participants reported FHHD. In a multivariable model, FHHD was significantly enriched for morbid obesity (OR 1.14, CI 1.08-1.21), hypertension (OR 1.21, CI 1.17-1.24), apolipoprotein B/A1 ratio 5th vs 1st quintile (OR 1.24, CI 1.19-1.29), lipoprotein(a) >125 nmol/L (OR 1.20, CI 1.17-1.24), CAD PRS 10th vs 1st decile (OR 1.68, CI 1.59-1.77) and HeFH (OR 1.32, CI 1.09-1.60). PAR analyses indicated that 24.9% (CI 21.9-28.0%) of FHHD is explained by genetic factors alone, 17.2% (CI 15.0 -19.3%) is explained by clinical factors alone, and 34.3% (CI 31.0-37.7%) is explained by both genetic and clinical factors.

Conclusions:
Both genetic and clinical risk factors, including several modifiable variables, significantly contribute to FHHD. Comprehensive assessment of contributing familial features may facilitate individual FHHD interpretation and risk reduction.

Figure 1: Genetic and clinical factors contributing to FHHD
Abstract 473: Cilostazol Decreases Venous Outflow Inflammation And Improves Avf Patency Inmurine Arteriovenous Fistula
Authors: Cui Jie , Kawamura Yoichiro , Ha Khanh Q , McCarthy Jason , Jaffer Farouc A
Published: November 2022

Background:
Arteriovenous fistula (AVF) have high failure rates, and no pharmacotherapeutics can improve AVF patency. The most common cause of AVF failure is inflammation-driven neointimal hyperplasia and thrombosis. Limited observational data suggests that cilostazol (CIL), an FDA-approved phosphodiesterase-3 inhibitor that inhibits platelet activation and smooth muscle cell proliferation, could help improve AVF maturation. Here, we investigated the effects of cilostazol on murine AVF patency, and further assessed the fibrinogenesis and anti-inflammatory effects using intravital fluorescence molecular imaging.

Methods:
One week prior to fistula creation, mice were randomized to CIL (40mg/kg/day) or PBS group (n=20 each). AVF was created using an end-to-side external jugular vein and carotid artery anastomosis. AVF blood flow was measured post-operatively (Transonic) and then every 7 days until AVF was occluded (<0.1 ml/min). Macrophages and fibrin were imaged after 10mg/kg FMX-AF555 and/or 4.5nmol FTP11CyAm7 i.v. injection prior to imaging on day 7 or day 14, respectively. The target-to-background ratio (TBR) was calculated as mean signal intensity (MSI) of the fistula divided by the MSI of the contralateral vein.

Results:
Post-operative AVF blood flow was similar between CIL and PBS treated groups (p=0.3). Average AVF patency was significantly longer in CIL group compared to PBS group (42 vs 21 days, p<0.01, figure 1A). Both day 7 and day 14 venous outflow fibrinogenesis signal assessed by IVM was significantly lower in CIL group compared to PBS group (ps<0.05, Figure 1B). On day 14, CIL significantly lower venous outflow FMX signals compared to PBS group (p<0.05).

Conclusion:
Oral cilostazol therapy prolongs AVF patency, and decreases venous outflow inflammation and fibrinogenesis in experimental AVF in vivo. The current data supports investigating cilostazol as a clinical pro-AVF patency pharmacotherapeutic strategy in dialysis patients.
Abstract 485: Platelet Aggregation Is Increased In Psoriasis And Associated With Biomarkers Of Vascular Health
Authors: Drenkova Kamelia , Liu Lynn , Asrani Falguni , Scher Jose , Katz Stuart D , Krueger James , Berger Jeffrey , Garshick Michael
Published: November 2022

Background:
Psoriasis is associated with vascular inflammation and increased cardiovascular (CV) risk. Platelet hyperactivity is implicated in impaired vascular health and CV disease (CVD). The association between platelet activity, psoriasis, and vascular health has not been fully explored.

Objective:
To measure platelet aggregation in patients with psoriasis and controls and explore the association with biomarkers of inflammation and vascular stiffness.

Methods:
Participants with psoriasis (n=33, age 51 ± 16 years, 61% male), affecting 7% ± 14 of their body surface area (BSA), were compared to healthy controls (n=15) (Table 1A). Platelet aggregation in response to adenosine diphosphate (ADP) agonists, epinephrine (Epi), and arachidonic acid (AA) was measured via light transmission aggregometry (LTA). Vascular stiffness was assessed by pulse wave velocity (PWV), a metrics of arterial stiffness and biomarker of CV risk. High-sensitivity C-reactive protein (hs-CRP) was measured in a clinical laboratory.

Results:
Enrolled psoriasis participants were older, were more frequently white, and had higher body mass index and elevated hs-CRP and PWV compared to controls (Table 1A). Platelet aggregation was higher in psoriasis patients after stimulation with ADP (p=0.01), Epi (p=0.05), and AA with ex vivo aspirin co-incubation (p=0.04) when compared with controls in multivariable models. Platelet aggregation to ADP trended to positive association with PWV (r=0.25, p=0.08), and hs-CRP (r=0.29, p=0.06) after adjustment for age and sex (Figure 1C).

Conclusion:
Platelet aggregation was increased in participants with psoriasis and trended towards positive association with biomarkers of vascular stiffness and inflammation. These findings have important implications for future clinical trials of targeting platelet activity to reduce CV risk in psoriasis.
Abstract 486: Role Of Matrix Gla Protein In Vascular Regulation In Brown Adipose Tissue
Authors: Zhang Li , Wu Xiuju , Cai Xinjiang , Qiao Xiaojing , Yao Yucheng , Bostrom Kristina I
Published: November 2022

Obesity is a world-wide problem; an estimated 38% of the world population will be overweight and 20% will be obese by 2030. Brown adipose tissue (BAT) is a thermogenic tissue that plays an important role in energy expenditure and glucose uptake. It is also highly vascularized but the regulation of vascular formation in BAT is poorly understood. The bone morphogenetic proteins (BMPs) are essential in both brown adipogenesis and angiogenesis. BMP7 controls progenitor commitment to pre-adipocytes in BAT, and BMP4 is a known angiogenic factor that is essential for white and brown adipogenic differentiation. Matrix Gla protein (MGP) acts as an inhibitor of BMP4/7 and is highly expressed in pre-adipocytes. We hypothesize that modulation of MGP affects BAT vascularization and function. Our results show that mice with global Mgp gene deletion (Mgp-/-) mice have poorly functional BAT, with abnormal mitochondria and decreased expression of thermogenic lineage markers as determined by RNA Seq analysis and qPCR. In addition, the vasculature showed significant increases in the number of brown adipose endothelial cells (ECs) and expression of markers for both EC lineage and progenitor cells. Furthermore, targeted Mgp deletion in ECs or adipose cells as directed by Cdh5- or Adiponectin-Cre transgenes in Mgp-floxed mice also affected the vascular outgrowth in BAT. Together the results suggest that MGP is an essential regulator of vascular outgrowth in BAT, likely through its ability to inhibit BMPs. A better understanding of this kind of regulation may be helpful in strategies to limit metabolic dysfunction in obesity and diabetes.
Abstract 488: Glutathione-CaSR Interaction Contributes To Glutathione Regulation Of Angiogenesis
Authors: Mackay Ryan P , Delgadillo Luisa , Rashdan Nabil A , Pattillo Christopher B
Published: November 2022

Introduction:
In addition to its classic role in redox chemistry, glutathione (GSH) also acts as an allosteric regulator of the calcium-sensing receptor (CaSR). CaSR has been found in a variety of cell types including endothelial cells (ECs), where its activity has been linked to EC adhesion and migration. Although a relationship between GSH and CaSR has been discovered, no link has been established between GSH-CaSR interaction and regulation of angiogenesis.
We hypothesize a novel mechanism for GSH regulation of blood vessel development occurs through an extracellular interaction with CaSR as a part of physiological and pathological angiogenesis.

Methods:
Matrigel tube formation and wound healing assays were used to measure EC activity. RGECO1.2 was used to measure changes in intracellular calcium. Cell surface expression assay was used to measure surface CaSR expression. Cell counts and flow cytometry were used to monitor cell proliferation and cell cycle progression. Endothelial and mesenchymal markers were measured using real-time PCR.

Results:
Extracellular GSH increased endothelial tube length and branching in tube formation assay, while a selective CaSR inhibitor abrogated these effects. GSH also influenced EC migration as measured by wound healing assay. GSH or a CaSR agonist (R-568) reduced surface CaSR expression, with no changes in intracellular calcium. Extracellular GSH alters EC proliferation and cell cycle progression, coinciding with alterations in endothelial phenotype along the endothelial-mesenchymal spectrum.

Conclusion:
In addition to its classic antioxidant role, GSH appears to act extracellularly to influence EC activity. GSH caused significant increases in EC branching as measured by tube formation assay. The use of a selective CaSR inhibitor confirms the contribution of CaSR to this effect. Additionally, GSH influenced EC migration in wound healing assays. These results could be explained by GSH-CaSR-mediated alterations in endothelial differentiation. These results suggest a role for GSH in vascular development and angiogenesis, through a ligand interaction with the CaSR G-protein coupled receptor, representing a novel mechanism of action for physiological and pathological blood vessel development.
Abstract 504: Atherosclerotic Plaque Residing T Cells Display Cross Reactivity Between Viral And Self-epitopes
Authors: Tso Alexandria , Veizades Stefan , Jensen Annie , Huang Xianxi , DAddabbo Jessica , Sasagawa Koki , Chien Yu-Hsiu , Roy Chowdhury Roshni , Chan Charles , Davis Mark , Nguyen Patricia
Published: November 2022

Background:
Vascular thrombosis has been described in patients actively infected with influenza and, more recently, with the coronavirus. The risk may persist during the convalescence period. The underlying mechanisms mediating these events remain unclear.

Objective:
To determine whether human coronary plaque T cells reactive to influenza virus are also activated by self-proteins.

Methods/Results:
Using single cell targetRNAseq, we mapped the T cell repertoire in 19 patients with a range of coronary artery disease severity. To determine the potential peptides activating coronary plaque T cell receptors (TCRs), we matched TCR alpha and beta identity with HLA restriction to a database of known specificities. We found that plaque TCRs were potentially activated by viral epitopes, including those from the influenza M1 protein. We used a computational algorithm to identify self-proteins in the endothelial cells and vascular smooth muscle cells that may have peptides with similar homologies to viral epitopes predicted to bind our plaque TCRs. We then introduced plaque TCRs into the Jurkat T cells, exposed these cells to T2 (HLA-2 antigen presenting cells), viral peptides (e.g., M1) and self-epitopes (e.g., TSPAN17; ZIP9), and demonstrated T cell cross reactivity in vitro (
Figure 1
). TSPAN17 is a transmembrane protein that regulates VE-cadherin expression and promotes T cell transmigration. Zip9, in contrast, is a protein that regulates zinc homeostasis, serves as an androgen receptor on endothelial cells, and has been shown to promote endothelial proliferation.

Conclusion:
Taken together, our findings suggest that T cell cross reactivity may be a potential mediator of viral-induced arterial thrombosis.
Abstract 506: Targeting Macrophage Epsins Inhibits Lipid Uptake And Facilitates Cholesterol Efflux In Atherosclerosis
Authors: Cui Kui , Gao Xinlei , Wang Beibei , Wu Hao , Dong Yunzhou , Xiao Yuling , Jiang Xingya , Chan Siu-Lung , Zhu Bo , Bhattacharjee Sudarshan , Shan Dan , Peng Qianman , Malovichko Marrina , Norton Vikram , Wong Scott , Cowan Douglas B , Srivastava Sanjay , Linton MacRae F , Shi Jinjun , Chen Kaifu , Chen Hong
Published: November 2022

Resolving atheroma and hindering their transition to vulnerable atherosclerotic plaque is pivotal in preventing deadly episodes such as heart attacks and strokes. Despite recent advances in the treatment, atherosclerosis remains the root cause of death worldwide. Excess cholesterol accumulation and deposition trigger a complex inflammatory response in atherosclerosis. Our recent studies demonstrate that epsins, a family of endocytic adaptors, are critically responsible for the aforesaid events in atherosclerosis; however, the underlying mechanisms and whether targeting epsins hinders cholesterol uptake, promote cholesterol efflux and reverse transport in macrophages to reduce inflammation and accelerate atheroma resolution remains an open question. To explore the therapeutic intervention by harnessing recent development in nanomedicine, we silenced epsins in lesional macrophages by specific delivery of nanoparticles (NPs) carrying epsin siRNAs. We showed that inhibit epsins significantly diminished atherosclerotic plaque size and promoted plaque regression. Mechanistically, epsins bind to CD36 to facilitate CD36-mediated lipid uptake by enhancing its endocytosis and recycling. Conversely, epsins promote ABCG1 degradation via lysosomes and hamper ABCG1-mediated cholesterol efflux and reverse cholesterol transport. Furthermore, myeloid-specific epsins double knockout mice (LysM-DKO) harboring a genetically reduced ABCG1 level (LysM-DKO-ABCG1
fl/+
) were generated. In LysM-DKO-ABCG1
fl/+
macrophages, enhanced cholesterol efflux and reverse transport due to epsins deficiency was suppressed. Our findings suggest that targeting epsins in lesional macrophages may offer therapeutic benefits in preventing or treating advanced atherosclerosis.
Abstract 513: Plasminogen And Lipoprotein(a) Competitively Interact To Modulate Macrophage Inflammation Via ATP-binding Cassette Transporter A1
Authors: Mueller Paul A , Bergstrom Paige , Hay Joshua , Rosario Sara , Pamir Nathalie
Published: November 2022

Lipoprotein (a) [Lp(a)] is an independent risk factor for cardiovascular disease (CVD) and resembles an LDL-like lipoprotein with the addition of apolipoprotein(a) (LPA). The
LPA
gene results from a duplication of the plasminogen gene (
PLG
) and the proteins share 88% amino acid sequence homology in their protease domains. We recently identified Lp(a) inhibits ATP-binding cassette transporter A1 (ABCA1)-dependent cholesterol efflux to PLG, and previous work demonstrates inhibition of cholesterol efflux is proinflammatory. We hypothesize that Lp(a) and PLG competitively modulate macrophage inflammation beyond cellular cholesterol regulation.

In silico protein-protein interaction modeling unveiled candidate PLG-ABCA1 interaction sites which are distinct from ABCA1-APOA1 sites. Elicited peritoneal macrophages were collected from C57BL6/J mice and treated with vehicle, PLG, Lp(a), or both.
Abca1, Nfkb1
, and
Ccl2
gene expression was determined using qRT-PCR. PLG treatment increased
Abca1
expression 1.8-fold compared to vehicle but did not affect
Nfkb1
or
Ccl2
expression. Addition of Lp(a) to PLG treatment inhibited PLG-dependent
Abca1
upregulation. Lp(a) increased
Nfkb1
and
Ccl2
expression 1.4- and 2.0-fold, respectively. Addition of PLG to Lp(a) inhibited Lp(a)’s increase in
Nfkb1
expression but had no effect on
Ccl2
. THP-1 monocytes were differentiated to macrophages and treated with PLG, Lp(a), or both to determine whether they induced metabolic changes using Seahorse Analyzer. While PLG had no effect on metabolism, Lp(a) increased glycolysis 1.4-fold and glycolytic capacity 1.3-fold that was reduced when PLG and Lp(a) were used simultaneously. In ABCA1-expressing HEK293 cells, co-treatment using PLG and Lp(a) at equimolar concentrations resulted in 7.2-fold increase in STAT3 phosphorylation compared to ~3-fold increase elicited by each of PLG or Lp(a) treatment alone.

These observations suggest Lp(a) independently promotes a glycolytic proinflammatory macrophage phenotype that can be inhibited by PLG. PLG and Lp(a) synergy activates ABCA1-dependent anti-inflammatory STAT3 signaling. PLG might be tapering Lp(a)-dependent macrophage inflammation which may ultimately reduce CVD risk in patients with high Lp(a) levels.
Abstract 516: Splice Factor Polypyrimidine Tract-binding Protein 1 (PTBP1) Primes Endothelial Inflammation In Atherogenic Disturbed Flow Conditions
Authors: Hensel Jessica , Nicholas Sarah-Anne , Omar Omar , Kimble Amy , Nagpal Arjun , Menoret Antoine , Jellison Evan R , Rodriguez Annabelle , Ozawa Manabu , Vella Anthony , Murphy Patrick A
Published: November 2022

NFκB mediated endothelial activation drives leukocyte recruitment and atherosclerosis, in part through upregulation of adhesion molecules Icam1 and Vcam. The endothelium is primed for cytokine activation of NFκB by exposure to low and disturbed blood flow (LDF). While priming leads to an exaggerated expression of Icam1 and Vcam following cytokine stimulation, the molecular underpinnings are not understood. In a model of LDF, platelets were required for the increased expression of several RNA-binding splice factors, including Polypyrimidine tract binding protein (Ptbp1). This was coordinated with changes in RNA splicing in the NFκB pathway in primed cells, leading us to examine splice factors as mediators of priming. Using Icam1 and Vcam induction by TNFα stimulation as a readout, we performed a CRISPR-Cas9 screen of these factors and identified a requirement for Ptbp1 in priming. Deletion of Ptbp1 had no obvious effect on cell proliferation or viability, but reversed LDF splicing patterns and inhibited NFκB nuclear translocation and transcriptional activation of nearly all downstream targets, including Icam1 and Vcam. In human coronary arteries, elevated PTBP1 correlates with expression of TNF pathway genes and plaque.
In vivo
, endothelial specific deletion of Ptbp1 reduced Icam1 expression and myeloid cell infiltration at regions of LDF in atherosclerotic mice and limited atherosclerosis. Together, our data show that Ptbp1, which is induced in a subset of the endothelium by innate immune cell recruitment at regions of LDF, is required for priming of the endothelium for subsequent NFκB activation, myeloid cell recruitment and atherosclerosis.
Abstract 521: Parkin Has Non-canonical Roles In Macrophage Lipid Metabolism With Effects On Atherosclerosis
Authors: Jeong Se-Jin , Zhang Xiangyu , Rodriguez Velez Astrid , Yeh Yu-sheng , Razani Babak
Published: November 2022

Defects in the E3 ubiquitin ligase Parkin are sine qua non of Parkinson and related neurodegenerative diseases. Loss of ubiquitination of distinct targets leads to accumulation of cytotoxic protein aggregates, loss of mitophagy, and defects in lipid metabolism. Given the critical role of these processes in plaque macrophages, we set out to define the role of Parkin in atherosclerosis. Pro-atherogenic LDLR-KO mice were transplanted with bone marrow (BM) from wild type and Parkin-KO mice and lesion formation stimulated by 4 months of Western diet feeding. Mice transplanted with wild type BM developed lipid-rich atherosclerotic plaques in both the aortic root and whole aorta replete with foam cell macrophages. In contrast, mice transplanted with Parkin-deficient BM had significantly reduced plaque burden with concomitant reductions in lipid content, foam cell macrophages, and surrogates of plaque complexity including reduced lesional apoptosis and necrotic core formation. The paradoxical atherogenic role for Parkin including development of lipid-laden foam cell macrophages led us to focus on the impact of Parkin in macrophage lipid handling. We find that Parkin mediates the ubiquitination and stabilization of scavenger receptor CD36, a key lipid transporter in macrophages. Parkin-null macrophages had reduced expression of CD36 and concomitant reductions in oxLDL uptake. Further, Parkin deficiency decreased cholesterol efflux in macrophages exacerbating macrophage lipid storage. These results suggest that the non-canonical functions of Parkin in lipid metabolism outweigh its traditional cytoprotective roles in macrophages and mediate its deleterious effects on atherosclerotic progression.
Abstract 540: Impaired Microvascular Angiogenesis And Altered Tissue MicroRNA In Patients With Peripheral Artery Disease
Authors: Rizvi Syed H , Bretón-Romero Rosa , Weisbrod Robert M. , Hamburg Naomi M.
Published: November 2022

Microvascular dysfunction contributes to adverse clinical outcomes in patients with peripheral artery disease (PAD). Compromised angiogenesis is an important mechanism of limb ischemia that may be a treatment target but is challenging to assess in patients. Selected circulating microRNA (miRNAs) have been implicated in the pathogenesis of PAD. Thus, we sought to characterize angiogenesis potential and identify altered miRNA in patients with PAD. We collected adipose tissue from ischemic limbs of patients with PAD (n=15, age=64±11, 33% women, 66% T2DM, 61% current smoking) at the time of revascularization surgery. Control adipose tissue was procured patients without PAD undergoing joint replacement surgery (n=6, age=58±6, 50% women, 17% T2DM, 33% current smoking). Using matrigel sprouting assay, we observed a significantly lower angiogenesis response over 8 days in adipose tissue samples from PAD patients compared to controls (p=0.008). We performed miRNA profiling using NextSeq (Illumina Inc.) sequencer. miRNA NGS libraries were prepared using QIAseq miRNA Library Kit. Quality controlled NGS libraries were sequenced according to the manufacturer protocol. Our results demonstrated 40 upregulated and 11 downregulated miRNAs (FDR q<0.01, log2 fold change>2). Using bioinformatics approaches to prioritize differentially expressed miRNA predicted to impact pathways relevant to cardiovascular disease, we identified miR205-5p and miR182-5p as top candidate. There were higher levels of miR205-5p (4.8-fold, FDRq=1.5e-10) and miR182-5p (4.1-fold, FDRq=5.4e-16) in adipose tissue from PAD patients compared to controls. We confirmed the differentially expressed levels using qPCR in selected samples. Ingenuity pathway analysis indicates that miR205-5p inhibits VEGF-A, a key regulator of angiogenesis. We observed a trend toward an association of miR205-5p (r=-0.58, p=0.1) and miR182-5p (r=-0.67, p=0.06) and lower angiogenesis response. Taken together our findings demonstrate impaired microvascular angiogenesis response in patients with PAD along with upregulation of selected miRNA predicted to impair angiogenesis pathways. Further work is needed to confirm a functional role of miRNA in the impaired angiogenesis in patients with PAD.
Abstract 550: Magnetic Nanoparticle-mediated Targeting Of Endothelium To Address Restenosis In A Bioprinted
In Vitro
Model Of Pulmonary Arteries
Authors: Ning Liqun , Tomov Martin L , Zanella Stefano , Zambrano Byron , Avazmohammadi Reza , Mahmoudi Morteza , Bauser-Heaton Holly , Serpooshan Vahid
Published: November 2022

Vascular restenosis is a major complication in recanalized arteries. Nanoparticles (NPs) have shown great promise as delivery systems in advancing strategies to treat such vascular anomalies. By enabling precise targeting, NPs can overcome the challenges of low drug efficacy and off-target effects. Here we present a biomimetic
in vitro
platform comprised of 3D bioprinting, nanomaterials, and perfusion technologies, to study the use of NP targeting to address endothelial overgrowth. We bioprinted 3D vascular channels at high fidelity, using gelatin methacrylate as bioink, with artery-like stiffness. Human endothelial cells (ECs) were used to endothelialize the printed channels. GFP-labelled superparamagnetic iron oxide NPs (SPIONs), loaded with the
Rapamune
anti-proliferative drug, were perfused through the bifurcated artery model at physiological rate. Computational modeling predicted greatest level of alterations in wall shear stress in the conduit’s junction with the artery, identifying this region prone to restenosis. A neodymium disc magnet was embedded in the printed tissue to attract the therapeutic SPIONs to the region of high risk.
In vitro
dynamic culture was conducted for 2 wks. We assessed cell viability, proliferation, and function using AlamarBlue and immunohistochemistry. Results showed significant targeted effect of NP delivery in reducing EC overgrowth. This platform enables design of precise targeting of therapeutics to treat a variety of cardiovascular diseases at a high spatial and temporal control.
Abstract 562: Altered Hippo Pathway Activity And Impaired Smooth Muscle Contractile Phenotype In Wnt16-Deficient Mice Worsens Ascending Aortic Aneurysm
Authors: Behrmann Abraham S , Zhong Dalian , Li Li , Xie Shangkui , Goodarzi Mohammad , Lemoff Andrew , Kozlitina Julia , Towler Dwight A
Published: November 2022

Wnt16 is expressed in mouse & human vascular smooth muscle (VSM). We assessed the impact of Wnt16 deficiency on angiotensin-II (AngII) -induced aneurysmal remodeling. AngII infusion for 4 weeks increased aortic
Wnt16
2-fold & ascending thoracic aorta diameter by 60% (p = 1.5E-4, ANOVA p = 3E-6) in male LDLR-/- mice. Wnt16-/-;LDLR-/- cohorts exhibited worsened aorta dilatation (by 20%; Holm-Sidak p =0.03), indicating a protective role for Wnt16. Wnt16 deficiency reduced both pulse wave velocity & systolic blood pressure (Wnt16-null = 132+/- 3 vs. 146 +/- 6 mmHg, p < 0.05, n =11/genotype), suggesting altered contractile function. Since hypomorphic alleles encoding contractile proteins predispose to ascending aortic aneurysms, we studied the impact of Wnt16 on the contractile phenotype. VSM from Wnt16-null thoracic aorta exhibited reductions in Acta2 & SM22 (protein, mRNA; down 50%, p < 0.05),
Myh11
(down 60%; p < 0.05), &
Myocardin
(
Myocd
; by 65%; p =0.0002). Inhibitory H3K27me3 histone methylation was selectively increased 3-fold in chromatin of genes encoding contractile proteins in Wnt16-/- VSM (p = 3E-5). DNA fragmentation was increased 22% in Wnt16-null VSM (p < 0.01). Label-free quantitative proteomic analyses of aorta revealed (a) upregulation of 14 mechanosensitive LIM proteins in Wnt16-null mice, with (b) > 90% reduction in Ankrd1 - a wound contraction protein & a prototypic target of Yap1-Taz/Wwtr1 activity in the Hippo pathway. Wnt16 deficiency significantly increased VSM cytoplasmic Yap1-Taz protein accumulation while reducing nuclear Yap1-Taz. siRNA targeting Taz, but not Yap1, reduced contractile gene expression, phenocopying the actions of Wnt16 siRNA or genetic deficiency. Antagonism of Lats1/2, Hippo pathway kinases that inhibit Yap1-Taz cytoplasmic-nuclear shuttling, upregulated VSM
Myocd
(4.4x),
Acta2
(1.6x),
Ankrd1
(2.5x; p < 0.01), mimicking actions of 8 nM Wnt16 treatment. Dosing mice with Wnt16 (0.25 mg/kg/day, 2 days) upregulated aortic contractile gene expression 1.6- to 1.8-fold (all p < 0.05). Wnt16 limits thoracic aortic remodeling & preserves the VSM contractile phenotype in part via the Hippo - Taz/Wwtr1 signal relay. Strategies that augment Wnt16 activity may mitigate enlargement of ascending aortic aneurysms.
Hate Speech: A Pragmatic Assessment of the European Court of Human Rights’ Jurisprudence
Authors: Sardo Alessio
Published: November 2022

This paper aims to offer a fresh start for addressing several conundrums relating to hate speech. The method of research combines a conceptual analysis with a possible model for evaluating the European Court of Human Rights’ (ECtHR) decisions on hate speech. First, drawing on a Gricean account of communication, the argument proposes a working definition of hate speech: hate speech is best understood as a public speech act, aimed at subordinating individuals, which causes harm to targeted groups. Second, the paper offers a taxonomy of the different forms of hate speech, based on their degree of explicitness and detachment from the speaker’s intentions. The most explicit forms of (harmful) hate speech – e.g., racial slurs, fighting words, or overtly sexist remarks – will be distinguished from implicit forms of (harmful) hate speech – e.g., innuendo, insinuation, and irony. Third, the author develops a categorical framework for hate speech that can be used as a standard for evaluating the jurisprudence of the ECtHR. The author also discusses three limitations of the model: a) the absence of a European consensus, b) puzzled speakers, and c) difficulty in determining harm.
Two new species of Entoloma (Entolomataceae) from Khyber Pakhtunkhwa, Pakistan
Authors: IZHAR AIMAN , USMAN MUHAMMAD , KIRAN MUNAZZA , KHALID ABDUL NASIR
Published: November 2022

Cedar towering forests of Kumrat Valley, Khyber Pakhtunkhwa, Pakistan were visited on several occasions to collect mushrooms and this resulted in the discovery of two novel species of Entoloma. Entoloma kumraticus is characterized by bluish-brown, centrally depressed pileus, serrulate lamellar edge, bluish-violet fibrillose stipe, dense clusters of cheilocystidia, and presence of abundant grayish-brown intracellular pigments in the terminal cells of pileipellis, and cheilocystidia. Entoloma dirensis is characterized by brownish olive to dull violet, finely fibrillose striate pileus with a violet-blue margin, slightly twisted violet-blue stipe, and blackish-brown lamellar edge, with groups of cheilocystidia which are often septate. The new species are presented based on the morphological and molecular study. Entoloma kumraticus and E. dirensis formed distinct phylogenetic lineages based on the sequences of nuclear ribosomal ITS and LSU gene regions. A key to the Pakistani species in sect. Cyanula is also provided.
Postscript: How long do Acari live? A survey of mite lifespans (part 2)
Authors: ZHANG ZHI-QIANG
Published: November 2022

This is the second part of a survey of mite lifespans initiated last year (Zhang & Fan 2021). This series was launched to collect information on lifespan and related data across mite taxa, with the aim to stimulate more research on both the patterns and mechanisms of mite aging and lifespan (Zhang 2021). The first part covered lifespan and other life history data for Oribatida, Winterschmidtiidae, Eriophyoidea, Tydeidae, Anystidae, Cheyletidae and Blattisociidae (Zhang & Fan 2021). This part provides surveys of lifespan and related life history data for three major families: Tetranychidae (Li et al. 2022), Laelapidae (Zhang et al. 2022) and Phytoseiidae (Han et al. 2022).  The third part in 2023 will complete this series.
Acarological Frontiers: Proceedings of the XVI International Congress of Acarology (1–5 Dec. 2022, Auckland, New Zealand)
Authors: ZHANG ZHI-QIANG , FAN QING-HAI , HEATH ALLEN C.G. , MINOR MARIA A.
Published: November 2022

Cover
Mites associated with early decomposition stages of pigs (Sus scrofa domestica) in Texcoco, Mexico
Authors: ESTRADA-VENEGAS EDITH GUADALUPE , RÍOS-IBARRA ROSA IVETTE , CORRALES-FUENTES ITZEL ARIANA , REVELO-TOBAR HAROL , HERRERA-DOMÍNGUEZ JAZIBE , EQUIHUA-MARTÍNEZ ARMANDO
Published: November 2022

Mites can be found in various habitats, they are sensitive to climate variation and disturbance of ecosystems. Many mites are phoretic on insects that are associated with plant and animal decomposition, therefore they can provide relevant information to forensic investigations, such as post-mortem time, causes of death, or temporal and geographical conditions of the place where the body was found. The study of mites at a crime scene or as part of the evidence that can be collected has very important functions. For example, mites can be used to determine cases of food contamination, to track shipments of illegal substances, as well as in investigations of murders, missing persons or cases of negligence. Mites are always present, even in environments where the presence of insects is scarce. Despite this importance, studies on the diversity of mites associated with decomposing bodies are scarce, and work on the matter is just beginning in Mexico.