bluuebunny/crossref_metadata_2025 huggingface dataset:
A Brief History of the Pediatric Endocrine Society (PES)
Authors: Oberfield Sharon E. , Rogol Alan D. , Miller Walter L.
Published: November 2022

The Pediatric Endocrine Society (PES) was initially established in 1972 as the Lawson Wilkins Pediatric Endocrine Society (LWPES), by some of Wilkins’ former fellows. As the society grew from its 37 founding members and Dr. Wilkins’ influence faded, the name of the society was changed in 2010 and now counts about 1,500 members, mostly from the US and Canada. Pediatric endocrine training programs headed by (LW)PES members have welcomed fellows from throughout the world, many of whom have gone on to leadership positions in their home countries. Starting in 1981, the (LW)PES has collaborated with pediatric endocrine societies around the world in quadrennial meetings, fostering collaborations, transfer of ideas, devising joint practice guidelines, and enjoying one another’s fellowship and counsel. The PES presently has committees and special interest groups concerned with all aspects of pediatric endocrinology, assuring that our clinical and academic resources reflect both breadth and depth. To celebrate our 50th anniversary, selected members have written the historical manuscripts featured in this special issue of Hormone Research in Pediatrics. These historical reviews delve into the origins of our specialty, sometimes deep into antiquity, provide useful background information, and illustrate the kinds of intellectual struggles that have led to the development of contemporary pediatric endocrinology, worldwide.
Two Years with COVID-19: The Electronic Frailty Index Identifies High-Risk Patients in the Stockholm GeroCovid Study
Authors: Mak Jonathan K.L. , Eriksdotter Maria , Annetorp Martin , Kuja-Halkola Ralf , Kananen Laura , Boström Anne-Marie , Kivipelto Miia , Metzner Carina , Bäck Jerlardtz Viktoria , Engström Malin , Johnson Peter , Lundberg Lars Göran , Åkesson Elisabet , Sühl Öberg Carina , Olsson Maria , Cederholm Tommy , Hägg Sara , Religa Dorota , Jylhävä Juulia
Published: November 2022

Introduction: Frailty, a measure of biological aging, has been linked to worse COVID-19 outcomes. However, as the mortality differs across the COVID-19 waves, it is less clear whether a medical record-based electronic frailty index (eFI) that we have previously developed for older adults could be used for risk stratification in hospitalized COVID-19 patients. Objectives: The aim of the study was to examine the association of frailty with mortality, readmission, and length of stay in older COVID-19 patients and to compare the predictive accuracy of the eFI to other frailty and comorbidity measures. Methods: This was a retrospective cohort study using electronic health records (EHRs) from nine geriatric clinics in Stockholm, Sweden, comprising 3,980 COVID-19 patients (mean age 81.6 years) admitted between March 2020 and March 2022. Frailty was assessed using a 48-item eFI developed for Swedish geriatric patients, the Clinical Frailty Scale, and the Hospital Frailty Risk Score. Comorbidity was measured using the Charlson Comorbidity Index. We analyzed in-hospital mortality and 30-day readmission using logistic regression, 30-day and 6-month mortality using Cox regression, and the length of stay using linear regression. Predictive accuracy of the logistic regression and Cox models was evaluated by area under the receiver operating characteristic curve (AUC) and Harrell’s C-statistic, respectively. Results: Across the study period, the in-hospital mortality rate decreased from 13.9% in the first wave to 3.6% in the latest (Omicron) wave. Controlling for age and sex, a 10% increment in the eFI was significantly associated with higher risks of in-hospital mortality (odds ratio = 2.95; 95% confidence interval = 2.42–3.62), 30-day mortality (hazard ratio [HR] = 2.39; 2.08–2.74), 6-month mortality (HR = 2.29; 2.04–2.56), and a longer length of stay (β-coefficient = 2.00; 1.65–2.34) but not with 30-day readmission. The association between the eFI and in-hospital mortality remained robust across the waves, even after the vaccination rollout. Among all measures, the eFI had the best discrimination for in-hospital (AUC = 0.780), 30-day (Harrell’s C = 0.733), and 6-month mortality (Harrell’s C = 0.719). Conclusion: An eFI based on routinely collected EHRs can be applied in identifying high-risk older COVID-19 patients during the continuing pandemic.
Outcomes of Salvage Surgery for Initially Unresectable Hepatocellular Carcinoma Converted by Transcatheter Arterial Chemoembolization Combined with Lenvatinib plus Anti-PD-1 Antibodies: A Multicenter Retrospective Study
Authors: Wu Jia-Yi , Zhang Zhi-Bo , Zhou Jian-Yin , Ke Jing-Peng , Bai Yan-Nan , Chen Yu-Feng , Wu Jun-Yi , Zhou Song-Qiang , Wang Shuang-Jia , Zeng Zhen-Xin , Li Yi-Nan , Qiu Fu-Nan , Li Bin , Yan Mao-Lin
Published: November 2022

Introduction: The actual rate of conversion surgery and its prognostic advantages remain unclear. This study aimed to assess the outcomes of salvage surgery after conversion therapy with triple therapy (transcatheter arterial chemoembolization [TACE] combined with lenvatinib plus anti-PD-1 antibodies) in patients with initially unresectable hepatocellular carcinoma (uHCC). Methods: Patients with initially uHCC who received at least one cycle of first-line triple therapy and salvage surgery at five major cancer centers in China were included. The primary endpoints were overall survival (OS) and recurrence-free survival (RFS) rates after salvage surgery. The secondary endpoints were perioperative complications, 90-day mortality, and pathological tumor response. Results: Between June 2018 and December 2021, 70 patients diagnosed with uHCC who underwent triple therapy and salvage surgery were analyzed: 39 with Barcelona Clinic Liver Cancer (BCLC) stage C, 22 with BCLC stage B, and 9 with BCLC stage A disease. The median interval between the start of triple therapy and salvage surgery was 4.3 months (range, 1.7–14.2 months). Pathological complete response and major pathological response were observed in 29 (41.4%) and 59 (84.3%) patients, respectively. There were 2 cases of perioperative mortality (4.3%) and 5 cases of severe perioperative complications (7.1%). With a median follow-up of 12.9 months after surgery (range, 0.3–36.8 months), the median OS and RFS were not reached. The 1- and 2-year OS rates were 97.1% and 94.4%, respectively, and the corresponding RFS rates were 68.9% and 54.4%, respectively. Conclusion: First-line combination of TACE, lenvatinib, and anti-PD-1 antibodies provides a better chance of conversion therapy in patients with initially uHCC. Furthermore, salvage surgery after conversion therapy is effective and safe and has the potential to provide excellent long-term survival benefits.
Abstract 101: Genetic Ablation Of CCDC92 Protects Against Vascular Inflammation And Atherosclerosis
Authors: Du Wa , Song Dan , Ren Lu , Becker Richard C , Fan Yanbo
Published: November 2022

Objective:
Genome-wide association studies (GWAS) have uncovered specific genetic variants of the coiled-coil domain containing 92 (CCDC92) gene that are associated with coronary artery disease (CAD) and type 2 diabetes. However, the biological function of CCDC92 in cardiovascular disease (CVD) remains unclear. This study aims to investigate the impact of CCDC92 on atherosclerosis, a leading cause of CVD, and dissect underlying mechanisms.

Methods and Results:
To investigate the effects of CCDC92 on atherosclerosis development
in vivo
, we used
Ccdc92
knockout (KO) mice and littermate wild type (WT) mice (in
ApoE
KO background) fed a high-cholesterol diet (HCD) for 18 weeks.
Ccdc92
KO significantly reduced atherosclerotic plaque by sixty percent compared with littermate control mice measured by en face analysis of atherosclerotic lesions in the aortic tree after Oil Red O staining (n=11-12/group, p < 0.001). To provide a comprehensive characterization of cellular identity within the atherosclerotic lesion, we applied single-cell sequencing (scRNA-seq) to the whole atherosclerotic aorta from
Ccdc92
KO/
ApoE
KO and littermate control mice on HCD for 12 weeks. The scRNA-seq analysis identified 12 cell subpopulations in the atherosclerotic aorta. Cell-specific gene set enrichment analysis further indicated that Ccdc92 deficiency had protective effects on multiple vascular cell subpopulations. Specifically, in subpopulations of endothelial cells (ECs), CCDC92 regulates cell inflammation and fatty acid metabolism. Using human coronary artery ECs (HCAECs) and mouse aortic EC from
Ccdc92
KO and WT mice, we demonstrated that deletion of CCDC92 significantly inhibited proinflammatory adhesion molecules and cytokines induced by TNF-α. Interestingly, Adenovirus-mediated CCDC92 overexpression increased lipid droplet accumulation (to an average of 4-fold increase) in HCAECs exposed to oleic acid overload.

Conclusions:
Our findings revealed the proatherogenic effects of CCDC92
in vivo
and demonstrated a critical role of CCDC92 in EC dysfunction during atherosclerosis development. These data address an important new mechanism associated with atherosclerosis and provide a potential new target for atherosclerotic disease.
Abstract 109: The Chromatin Remodeling Protein Brg1 Regulates Adventitial Progenitor Cell Myofibroblast Differentiation And Pathological Vascular Remodeling
Authors: Jolly Austin , Lu Sizhao , Dubner Allison , Mutryn Marie , Navarro Natalie , Nemenoff Raphael , Weiser-Evans Mary C
Published: November 2022

Cardiovascular diseases lead to long-term stiffening of arteries and small vessels. The vascular wall contains several cellular populations that coordinate to maintain vessel homeostasis and induce vascular remodeling in disease states. We identified a population of smooth muscle-derived progenitor cells that reside in the adventitial layer (AdvSca1-SM cells) that drive vascular fibrosis after acute vascular injury. AdvSca1-SM cells can differentiate into myofibroblasts and secrete numerous pro-remodeling factors including extracellular matrix proteins and pro-inflammatory cytokines. While the response of AdvSca1-SM cells to vascular injury has been studied, the epigenetic changes that influence their differentiation towards myofibroblasts is poorly understood. We observed Brahma-related gene 1 (Brg1), a highly conserved chromatin remodeling ATPase, is upregulated in AdvSca1-SM cells in response to vascular injury and may be a key factor in regulating gene expression to influence AdvSca1-SM differentiation. We hypothesize that Brg1 induces expression of gene sets associated with remodeling and fibrosis and ultimately drives AdvSca1-SM cell differentiation towards pathologic myofibroblasts.

Results:
Carotid arteries from mice subject to carotid ligation exhibit decreased perivascular collagen deposition, smaller neointima, decreased adventitial expansion, decreased numbers of alpha-actin positive AdvSca1-SM cells, and fewer infiltrating macrophages when treated with the pharmacological Brg1 bromodomain inhibitor PFI-3 as compared to mice that received vehicle control. In a cell culture system, TGF-β induces AdvSca1-SM cells to express myofibroblast-specific genes, including alpha actin and periostin, and demonstrate enhanced contractile function in collagen contraction assays. PFI-3 blocks TGF-β induced myofibroblast gene expression and dampens contractile function. Gene Ontology analysis reveals the top 100 TGF-β inducible genes blunted by PFI-3 are related to fibrosis and extracellular matrix signaling. In conclusion, these results suggest Brg1 is a major regulator of vascular remodeling and fibrosis and may be a targetable protein to help people who suffer from disease-related vascular stiffening.
Abstract 114: The Epigenetic Enzyme KMT2A/MLL1 Is A Driver Of Coronavirus Associated Coagulopathy
Authors: Sharma Sriganesh B , Melvin William J , Audu Christopher O , Kanthi Yogendra , Knight Jason S , Rhoads Nicole , Adili Reheman , Holinstat Michael A , Moore Bethany B , Henke Peter K , Wakefield Thomas W , Gallagher Katherine A , Obi Andrea T
Published: November 2022

Objectives:
Coronavirus associated coagulopathy (CAC) is postulated to be driven by systemic macrophage activation after SARS-CoV-2 infection and presents with elevated risk of thrombogenesis and hyperfibrinolysis. Previous work shows that the histone methyltransferase KMT2A/MLL1 is a key mediator of inflammatory signaling in monocytes and macrophages (Mo/Mϕs). In this study, we sought to identify the regulation of factors important in CAC by MLL1.

Methods:
Mice with myeloid specific knockout of MLL1 (Cre+) and littermate controls (Cre-) underwent intranasal inoculation of 2 x 10
5
pfu of the murine coronavirus MHVA59, an established model which phenocopies SARS-CoV-2 infection. Splenic Mϕs (surrogate for circulating Mo/Mϕs) were isolated and RNA and protein levels of urokinase (Plau; profibrinolytic), urokinase receptor (Plaur; profibrinolytic), and tissue factor (F3/TF; procoagulant) were analyzed using qRT-PCR and ELISA, respectively. Thromboelastography (TEG) on whole blood and urokinase activity assays from mouse plasma were performed. Urokinase and TF activity assays were performed on plasma from human samples.

Results:
RNA (top panel) and protein (bottom) levels of Plau, Plaur, and F3 were suppressed in the Splenic Mϕs harvested from sham (intranasal PBS) and infected Cre+ animals (white bars) compared to Splenic Mϕs harvested from Cre- animals (blue bars; Fig. 1A). Cre- mice displayed a shortened R-time (reaction time) as measured by TEG (Fig. 1B) and elevated plasma urokinase activity levels (not shown). Hospitalized COVID-positive patients (hCOV+) displayed elevated plasma urokinase and TF activity levels (Fig. 1C).

Conclusions:
We identify a role for MLL1 for basal expression and for coronavirus-mediated induction of factors important for fibrinolysis and coagulation in murine Mo/Mϕs and in driving coagulopathy. Our results suggest that MLL1 blockade may be an attractive strategy to combat coronavirus associated coagulopathy.
Abstract 118: Cholesteryl Ester Transfer Protein Overrides The Protective Effect Of APOA1 On Arterial APOB Lipoprotein Trapping And Atherosclerosis Progression In A Mouse Model Of Type 1 Diabetes
Authors: Kothari Vishal , Shao Baohai , He Yi , Kramer Farah , Barnhart Shelley , Kanter Jenny E , Chait Alan , Goldberg Ira J , Fisher Edward A , Vaisar Tomas , Bornfeldt Karin E
Published: November 2022

Despite the benefits of LDL-cholesterol-reducing medications, patients with type 1 diabetes (T1D) continue to have an increased risk of incident CVD. Preclinical and clinical observations indicate that triglyceride-rich lipoproteins (TRLs) and their remnants may contribute significantly to CVD risk in T1D. Recently, we have shown that diabetes increases levels of atherogenic TRL remnants and that improvement of remnant clearance halts atherosclerosis in a mouse model of T1D. Cholesteryl ester transfer protein (CETP), which is normally absent in mice, links metabolism of TRLs to that of HDL by mediating the transfer of cholesteryl ester from HDL to VLDL and other APOB-containing lipoproteins in exchange for triglycerides. We hypothesized that CETP impairs the atheroprotective effects of APOA1 by altering TRL and HDL metabolism in diabetes. To test this hypothesis, we developed a virally-induced T1D mouse model expressing the human APOA1 transgene (

APOA1
Tg
, Ldlr
–/–

). Cohorts of these mice were injected with a liver-targeted adeno-associated virus to express human CETP, and the effects on lipoproteins, plasma apolipoproteins, HDL particle concentrations (HDL-P), and atherosclerosis in the presence and absence of diabetes were determined. This mouse model exhibits an HDL profile similar to humans, with small, medium and large HDL-P, whereas wildtype (

Ldlr
-/-

) littermates have only one sized HDL-P. Expression of the APOA1 transgene significantly suppressed diabetes-accelerated necrotic core expansion and accumulation of APOB and APOE in the aortic sinus (compared to wildtype

Ldlr
-/-

mice), while CETP impaired the protective effect of APOA1 on these parameters in diabetic

APOA1
Tg
, Ldlr
–/–

mice (n= 22-30, p < 0.05). Further characterization of plasma lipoproteins and apolipoproteins by targeted mass spectrometry, FPLC and differential ion mobility analysis indicated that CETP leads to smaller TRL remnant particles, increases triglyceride content in HDL, lower levels of plasma APOA1 and reduction in large HDL-P under diabetic conditions (n= 8-10, p < 0.05). Our results are consistent with the proposal that inhibition of CETP might be cardioprotective in the setting of T1D and highlight the close links between TRL and HDL metabolism in diabetes.
Abstract 127: Single Cell RNAseq Reveals Pro-inflammatory, Cytolytic T Cells Characterize Complex Human Coronary Plaques
Authors: Jensen Annie , Veizades Stefan , Tso Alexandria , Sasagawa Koki , D'Addabbo Jessica , Huang Xianxi , Chien Yueh-hsiu , Roy Chowdhury Roshni , Chan Charles , Davis Mark , Nguyen Patricia
Published: November 2022

Background:
Atherosclerosis is considered a chronic inflammatory disorder characterized by the presence of T cells and macrophages in the plaque. Given that T cells are the master regulators of the immune system, the possible role of T cells in mediating disease warrants further investigation.

Objective:
To map the T cell transcriptome in coronary artery plaque.

Methods/Results:
Using single cell RNAseq, we mapped the transcriptome of immune cells within the coronary atherosclerotic plaque in 12 patients with various disease stages. In addition to macrophages, we find an abundance of plaque T cells that appear to be enriched in plaque compared to blood (
Figure 1A
). Two plaque-enriched CD8 effector memory clusters (CD8 CTL Tem1 and CD8 CTL Tem2) display the highest proportion of activated cells (e.g., HLA-DRA+). These clusters are characterized by high expression of the pro-inflammatory cytokines (e.g., CCL3, CCL4, CCL5, and IFNG) and cytolytic markers (e.g.,GZMA, GZMH, GZMM, NKG7). CD8 CTL Tem1, however, has higher expression of GZMK, a cytolytic marker recently associated with inflammaging. CD8 CTL Tem2, on the other hand, has a higher expression of perforin and granulysin, two pore-forming proteins that mediate cell killing by enabling granzyme entry. Notably, CD8 CTL Tem2 appears to track with plaque progression, increasing as plaques mature from lipid-rich to more complex lesions then declining as plaques became more stable and calcified post-rupture. Specifically, this cluster displayed two-fold enrichment in complex plaques compared to other plaque phenotypes (12.1% vs. 6.9%, p=0.0069) (
Figure 1B
). This pro-inflammatory and cytolytic signature is also more apparent in the activated compared to nonactivated cells in this cluster.

Conclusion:
Taken together, our findings suggest that a pro-inflammatory CD8 T cell cluster characterized by a trifecta of cytolytic enzymes distinguishes complex plaque and deserves further investigation.
Abstract 128: A Chimeric Antigen Receptor Targeting MDA-LDL Activates Regulatory T Cells In The Presence Of Human Atherosclerotic Plaque
Authors: Shaligram Sonali , Lopez Jose L , Lin Pei-Yu , Ho Patrick , Huang April , Tang Qizhi , Oskowitz Adam Z
Published: November 2022

Introduction:
Regulatory T cells (Tregs) suppress inflammation in atherosclerosis, and therefore have the therapeutic potential to reduce the risk of MI and stroke. However, there is currently no method to generate antigen specific Tregs that target atherosclerosis. We therefore engineered Tregs that express a Chimeric Antigen Receptor (CAR) targeting malonaldehyde-modified LDL (MDA-LDL), the most common form of oxidized LDL and a key molecular component of atherosclerosis.

Methods:
Novel single chain variable fragments (scFv) were synthesized using sequences from antibodies targeting human MDA-LDL. Oxidized-LDL specific CARs (ox-CARs) were subsequently engineered by fusing each scFv to an IgG4 hinge, CD28 transmembrane and CD28/CD3z cytoplasmic domains. CD4
+
CD25
+
CD127
low/-
Tregs were purified from human blood via FACS and lentivirally transduced to express the novel ox-CARs (ox-CAR-Tregs). Human atherosclerotic plaques were obtained from patients undergoing carotid endarterectomy (CEA). Autologous ox-CAR-Tregs were analyzed for activation after ex-vivo co-culture with CEA samples.

Results:
A rationally designed panel of 42 ox-CARs were engineered using scFv derived from 12 antibodies targeting MDA-LDL. We first assessed CAR expression and activation in Jurkat T cells to identify promising ox-CAR variants for further evaluation in human Tregs. After culture in the presence of MDA-LDL, six ox-CAR-Treg variants consistently showed significant activation, compared to controls, based on CD71 expression, cytokine expression and proliferation in the absence of CD3/28 stimulation. Human atherosclerotic samples were identified to have substantial amounts of MDA-LDL epitopes using IHC. Autologous ox-CAR-Tregs showed a dose-dependent increase in CD71 expression after ex-vivo co-culture with atherosclerotic plaque.

Conclusion:
An optimized CAR targeting MDA-LDL activates Tregs when cultured with human atherosclerotic plaque ex-vivo.
Abstract 133: Coagulation FXI Regulates Endothelial Cell Barrier Function By Inducing VE-cadherin Proteolysis In An ADAM10-dependent Manner
Authors: Puy Cristina , Jordan Kelley R , Nguyen Nhu , Lorentz Christina U , Tucker Erick I , Gailani David , McCarty Owen JT
Published: November 2022

Introduction:
Proteolysis of vascular endothelial (VE)-cadherin is a biomarker for disruption of endothelial cell (EC) barrier function, with elevated levels of soluble VE-cadherin associated with atherosclerosis and sepsis. Factor XI (FXI), known for its role in the coagulation cascade, also plays a regulatory role in inflammation. While a relationship between FXI activity, VE-cadherin expression levels, and endothelial barrier function has been observed, the mechanisms by which this process is mediated are unknown.

Hypothesis:
Activated FXI (FXIa) regulates EC barrier function by inducing VE-cadherin proteolysis.

Methods:
Human umbilical vein endothelial cells were stimulated with FXIa (30 and 5 nM) for six hours. VE-cadherin proteolysis and expression levels were analyzed by Western blot and immunofluorescence. ECs permeability was quantified by measuring the flux of Evans blue-albumin across the ECs. Immunoprecipitation and Western blot analysis were used to study FXIa-PAI-1 or -very low density lipoprotein receptor (VLDLR) interactions.

Results:
FXIa (5 nM) generated a VE-cadherin C-terminal fragment and decreased VE-cadherin expression on ECs; this process was reversed by the serine protease inhibitor, PPACK. Thrombin, kallikrein, or FXIIa did not induce VE-cadherin proteolysis. FXIa increased ECs permeability, yet did not induce the expression of VCAM-1. In contrast, while VCAM-1 expression was upregulated by tumor necrosis factor alpha or vascular endothelial growth factor, neither of these induced VE-cadherin proteolysis. A pharmacological inhibitor of a disintegrin and metalloproteinase 10 (ADAM10) abrogated the effects of FXIa on ECs, including proteolysis of VE-cadherin. Similarly, the low density lipoprotein antagonist, receptor-associated protein (RAP), inhibited FXIa-induced VE-cadherin proteolysis by preventing FXIa complex formation with PAI-1 and VLDLR on ECs.

Conclusion:
These results suggest that FXIa disrupts VE-cadherin-mediated EC permeability by increasing ADAM10 activity through complex formation and internalization by PAI-1 and VLDLR. It remains to be seen whether this pathway represents a druggable target to protect EC barrier function in inflammatory diseases by inhibiting FXIa.
Abstract 199: Conditional Inactivation Of Lysyl Oxidase In Smooth Muscle Cells Reduces Atherosclerosis Burden And Plaque Calcification In Hyperlipidemic Mice Through Suppression Of The β-catenin/tcf4 Signaling Axis.
Authors: Stoyell Conti Filipe F , Rojas Miguel , Zigmond Zachary , Rauf Mohd A , Martinez Laisel , Vazquez Padron Roberto I
Published: November 2022

Introduction:
In atherosclerosis development and complications, the importance of lysyl oxidase (LOX), a copper-dependent amino-oxidase that crosslinks collagen and elastin and controls gene expression, is not well established.

Hypothesis:
Inactivation of LOX in smooth muscle cells (SMC) decreases atherosclerosis burden and plaque calcification in hyperlipidemic mice.

Methods:
qRT-PCR, Western blot, and scRNAseq demonstrated the loss of
Lox
gene expression in SMC of ApoE conditional mice (Lox f/f Myh11-CreERT2 ApoE-/-) but not in littermate control animals after tamoxifen injections. Surprisingly, vascular loss of LOX did not increase the risk of aneurysms nor affected the aorta's stiffness. However, it reduced atherosclerosis burden with respect to control mice (13±2 versus 23±1%, p < 0.01) after 16 weeks of high fat diet (HFD). There was also a significant reduction in calcium deposition (5±0.4 versus11.8±3, p < 0.05) within the plaque of LOX knockout mice versus control. Aortic whole genome transcriptomic analysis revealed a positive correlation between
Lox
gene expression and Frizzled-related protein 3 (
Frzb
), a biphasic modulator of Wnt signaling that facilitates the association of β-catenin with TCF4 in the nucleus. scRNAseq analysis confirmed the downregulation of
Bmp2
and
Myc
, both β-catenin/TCF4 regulated genes, in SMC following gene deletion of
Lox
and 8 weeks of HFD. LOX knockout SMCs also had lower expression of
Frzb
and were more resistant to osteoblastic transformation in vitro.

Conclusions:
Vascular inactivation of LOX protects the vasculature from the proatherogenic effects of FRZB in SMCs.
Abstract 203: Synergism Of TNFα And IFNγ Elicits Transcriptional Cooperativity At The
CXCL9,-10,-11
Locus In Human Endothelial Cells And Is Ultrasensitive To BRD4 And CBP/P300 Inhibition.
Authors: Elsarrag Selma , Bracken Ronan , Davison Lindsay M , Martinez Alexander , Lin Charles , Brown Jonathan
Published: November 2022

Background/Rationale:
Combinatorial signaling by proinflammatory cytokines drives the pathogenesis of many cardiovascular diseases ranging from acute SARS-COV-2 vascular complications to chronic atherosclerosis. TNFα and IFNγ synergistically induce genes that amplify inflammatory cascades, causing tissue damage and high mortality in humans. Characterization of transcriptional mechanisms governing synergistic gene induction holds the promise of revealing novel therapeutic targets while also improving our understanding of how inflammatory states are propagated.

Objective:
To delineate unique mechanisms of synergistic gene transcription in human aortic endothelial cells (HAECs) exposed to high levels of both TNFα and IFNγ.

Methods & Result:
Using RNA-sequencing, we defined the dynamic transcriptome in HAECs stimulated with TNFα, IFNγ, or combination for one hour. We devised a novel statistical approach that generates a positive cooperativity score (PCS) for each gene in order to define the TNFα/IFNγ synergy signature in HAECs. This approach revealed the chemokines
CXCL9, -10, -11
as highly synergistic in their expression (PCS=61.9, 18.3, 8.9; log
2
[Combination/No Treatment]=8.1, 2.1, 3.1 respectively), in contrast to
SELE
, a TNFα-induced gene (PCS=0.4; log
2
[Combination/No Treatment]=6.6). Furthermore, pathway analysis revealed strong enrichment for chemotaxis programs among the synergy gene set. To deconvolute transcriptional coactivator dependencies at these exemplary genes, we conducted a targeted mini-screen of transcriptional inhibitors along with biochemical modelling of inhibitor dose-response curves. We discover that
CXCL9, -10, -11
induction is sensitively disrupted by inhibition of BRD4 and CBP/p300 (hill coefficient (HC) > 2) coactivators as compared to
SELE
(HC<2).

Conclusions:
These results provide definitive evidence of acute transcriptional synergy at the chemokine genes
CXCL9, -10, -11
elicited by TNFα/IFNγ activity in HAECs. As these chemokines are associated with poor outcomes in sepsis, these results also implicate targeted inhibition of transcriptional coactivators as a potential therapeutic strategy for inflammatory cardiovascular diseases.
Abstract 217: PCSK6 Is A Key Regulator Of Immune Status In Mice And Its Ablation Increases Atherosclerotic Plaque Burden In A Bone Marrow Transplant Model
Authors: Suur Bianca E , Karadimou Glykeria , Lengquist Mariette , Kronqvist Malin , Gisterå Anton , Malarstig Anders , Hedin Ulf , Ketelhuth Daniel F , Matic Ljubica
Published: November 2022

Introduction:
Proprotein convertase subtilisins/kexins (PCSKs) activate cytokines and growth factors and have been implicated in various cancers. We have previously shown that PCSK6 is a key protease modulating smooth muscle cell response in atherosclerosis, but its expression also correlated positively with typical markers of T lymphocytes and macrophages in plaques and it was localised in the proximity of these cells. Here, we hypothesized that PCSK6 may be involved in modulating inflammatory responses and aimed to elucidate its role in a hyperlipidemic mice model.

Methods:
Detailed immunophenotyping using histology, FACS-, OLINK- and ELISA-based analyses and primary cell cultures was used to compare

Pcsk6
-/-

and littermate controls. Atherosclerosis was evaluated in

Ldlr
-/-

mice upon bone marrow transplant with

Pcsk6
-/-

or wild-type bone-marrow.

Results:
At baseline

Pcks6
-/-

mice showed an enrichment of pro-inflammatory cytokines Ccl2, Ccl3, Ccl20, Cxcl1 and in particular Il17a and Il17f in plasma compared controls. Spleens of

Pcsk6
-/-

mice had an increased number of germinal centres and FACS analysis showed that they contained significantly more CD8+ T cells. Microarray analysis of spleens from

Pcsk6
-/-

vs. controls confirmed that T cell markers CD4, CD3E and CD3G were upregulated.
In vitro
, splenocytes isolated from

Pcsk6
-/-

mice secreted more IFN-γ, IL-2 and IL-10 than controls upon stimulation with α-CD3 and α-CD28 antibodies. Moreover, peritoneal macrophages from

Pcsk6
-/-

mice secreted more TNF-α, MCP-1, IL-6 and IL-10 compared to control mice upon LPS stimulation. Interestingly, bone marrow derived macrophages from

Pcsk6
-/-

mice were also more prone to lipid uptake. Finally,
in vivo
transplantation of

Pcsk6
-/-

bone marrow to

Ldlr
-/-

mice led to increased atherosclerotic plaque burden compared to controls, as quantified in the aortic root.

Conclusions:
PCSK6 ablation led to increased number of CD8+ T cells, as well as macrophage and cytokine activation. Transplantation of

Pcsk6
-/-

bone marrow resulted in an increase in atherosclerotic plaque burden compared to controls. Taken together, these results indicate that PCSK6 is a key regulator of the immune system, though the exact mechanisms involved require further investigation.
Abstract 223: Modifiable Mesenchymal Stem Cell Defects In Veterans With Diabetes Mellitus
Authors: Hoffmann Carson , Sasaki Maiko , Bitarafan Sara , Jung Yoonhee , McLaughlin Dylan , Li Feifei , Corces Victor , Wood Levi , Brewster Luke
Published: November 2022

Diabetes Mellitus (DM) affects 34.2 million Americans. DM impairs the body’s reparative machinery leading to early onset chronic illness (peripheral arterial disease (PAD), neuropathy, nephropathy, and retinopathy, the latter being hallmarks of microvascular disease). Patients with DM+PAD have increased risk of major amputation. Mesenchymal stem cells (MSCs) are reparative cells found in all tissues providing paracrine and trophic support for new tissue. This study’s objective was to identify intracellular and epigenetic mechanisms of how DM impairs MSC function and test if these defects are modifiable with culture rejuvenation (CR).

MSCs obtained from bone marrow of 13 consecutive, male Veterans undergoing lower limb major amputation were cultured in 10% fetal bovine serum or 5% human platelet lysate (PL) for CR. Groups were DM+PAD (n=8) and PAD no DM (n=5). Intracellular signaling was analyzed with multiplexed ELISA. Epigenetic differences were identified by ATAC-sequencing.

DM+PAD MSCs had modifiable AKT signaling defects with PL (Fig 1) and a discrete DNA profile identified in their introns and intergenic regions (Fig 2). MSCs from PAD alone had increased transcription factor binding at Wnt and cGMP-PKG pathways (p=0.04). DM+PAD MSCs had increased binding at MAPK (p=0.01) and Rap1 (p=0.01) pathways.

DM is a complex disease disrupting reparative mechanisms and can lead to major complications. MSC dysfunction in DM may have common mechanisms throughout the body. We have identified potentially druggable pathways that may provide therapeutic solutions to relieve chronic illness for endogenous MSCs and to expand MSC donor pool for regenerative medicine strategies.
Abstract 227: Statins Decrease Endothelial Cell Inflammation On Top Of Maximal Cholesterol Reduction: Findings From The American Heart Association Strategically Focused Cardiometabolic Health And Type 2 Diabetes Mellitus Research Network
Authors: Jindal Manila , Garshick Michael S , Schlamp Florencia , Barrett Tessa , Giannarelli Chiara , Newman Jonathan , Fadzan Maja , Heguy Adriana , Fisher Edward A , Goldberg Ira J , Berger Jeffrey S
Published: November 2022

Background:
Dyslipidemia induces a proinflammatory endothelium and vascular dysfunction. Lipid lowering therapy (LLT) consistently reduces cardiovascular (CV) events. Clinical studies suggest that statin-based LLT has pleiotropic anti-inflammatory effects beyond cholesterol reduction. However, the impact of statins or ezetimibe on top of potent cholesterol reduction is unknown. Thus, on a background of PCSK9 inhibition (with evolocumab), we analyzed the effect of LLT with atorvastatin or ezetimibe on the endothelium using a novel direct brachial vein (BV) endothelial cell (EC) harvesting technique.

Methods:
CHORD (CHOlesterol lowering and Residual Risk in Diabetes) is an ongoing, prospective study designed to evaluate the effect of maximal LLT on CV risk. Subjects with LDL-C >100mg/dl were treated with evolocumab (140mg/2 weeks) and either atorvastatin (80mg/day) or ezetimibe (10mg/day) for 30 days. In a subset of participants, EC harvesting was performed by inserting a J-wire into the BV, and ECs were isolated and analyzed using next generation RNA sequencing.

Results:
Among 20 participants (11 atorvastatin, 9 ezetimibe) undergoing EC RNASeq, median LDL-C (130 mg/dl, IQR [110, 147]) decreased by 75% [67, 81] on background PCSK9 inhibition with no significant difference in 30-day LDL-C between atorvastatin (27 mg/dl [20, 32]) and ezetimibe (41 mg/dl [26, 53]) groups (p=0.07). Following cholesterol reduction, 850 genes were upregulated and 2468 were downregulated (p<0.05) with downregulation of eNOS, Endothelin-1, and Senescence pathways by canonical pathway analysis. When stratified by LLT, patients receiving atorvastatin had a significant decrease in inflammation and senescence pathways compared to patients receiving ezetimibe (Figure).

Conclusions:
Robust lipid lowering with PCSK9 inhibition plus statins, as opposed to ezetimibe has anti-inflammatory effects and suggests a preferred strategy to improve the endothelium and reduce CV risk.
Abstract 229: Effects Of Human Angiotensinogen And Human Renin In Proximal Tubule Cells On Development Of Atherosclerosis In Hypercholesterolemic Mice
Authors: Amioka Naofumi , Kukida Masayoshi , Howatt Deborah A , Moorleghen Jessica J , Daugherty Alan , Lu Hong
Published: November 2022

Objective:
This study determined whether angiotensinogen (AGT) interacted with renin in renal proximal tubule cells (PTCs) to promote atherosclerosis.

Approach and Results:
Since hepatocyte-produced AGT can be filtered by glomeruli and retained in renal PTCs, we first determined whether hepatocyte-derived AGT interacts with renin in PTCs to promote atherosclerosis. Transgenic mice expressing human renin in PTCs driven by a kidney androgen-related protein promoter (KAP-hREN) in an LDLR -/- background were used. To induce synthesis of human AGT in hepatocytes, an adeno-associated viral vector (AAV) containing human AGT with a liver-specific promoter was injected intraperitoneally. Three groups of male littermates were administered testosterone to activate human renin expression in PTCs: (1) wild type mice administered null AAV, (2) KAP-hREN transgenic mice administered null AAV, and (3) KAP-hREN transgenic mice administered AAV containing human AGT. Two weeks after administration of testosterone and AAVs, mice were fed a Western diet for 6 weeks. Induction of human AGT in liver and human renin in PTCs did not increase atherosclerosis, comparing to the other two groups. Next, to evaluate the direct interaction of AGT and renin in PTCs for promoting atherosclerosis, KAP-human AGT (KAP-hAGT) and KAP-hREN double transgenic mice were fed a Western diet for 12 weeks. Although immunostaining confirmed the presence of human AGT and human renin in PTCs, double transgenic mice did not have increased percent atherosclerotic lesion area, comparing to either wild type or single transgenic littermate groups. Surprisingly, retired male double transgenic breeders (46-67 weeks old) showed severe atherosclerosis spontaneously without Western diet feeding, while their single transgenic littermates had minimal to modest atherosclerotic lesions. The sample size was small so statistical analysis was not performed.

Conclusions:
The presence of human AGT in liver or PTCs with combination of human renin in PTCs did not augment Western diet-induced atherosclerosis in mice. Further studies with increased numbers are needed to determine whether retired male breeders containing human AGT and human renin in PTCs develop severe atherosclerosis.
Abstract 231: Monocyte-mimicking Nanoparticles For Atherosclerosis-targeted Therapy
Authors: Huang Hui-Chun , Rousseau Joshua , Wang Ting-Yun , Mungaray Michelle , Wang Kuei-Chun
Published: November 2022

Atherosclerosis, characterized by plaque buildup in arteries, is a major cause of cardiovascular mortality globally. Despite advances in diagnostics and interventions over the past few decades, the treatment options and outcomes remain far less than optimal. Nanotechnology has demonstrated emerging success in clinical settings; however, a potent targeted nanotherapeutic for atherosclerosis remains underdeveloped. In this study, we designed a new class of nanocarriers mimicking circulating monocyte features to enhance the site-specific delivery of theranostic agents for atherosclerosis. We first synthesized polymeric cores encapsulating a fluorescent payload with a modified nanoprecipitation method and cloaked the polymeric cores (NPs) with the plasma membrane fraction isolated from mouse monocytes. Our characterization results verified that NP cores are covered with a uniform lipid layer and that the resulting monocyte-mimicking nanoparticles (MNPs) retain the membrane proteins on their surface and have a similar value of zeta potential as monocytes. Both MNPs and NPs did not exhibit any hemotoxicity
in vitro
; however, when incubated with cultured human vascular endothelial cells (ECs), MNPs showed a significantly higher uptake efficiency by ECs than NPs. Moreover, our
in vivo
studies with ApoE-knockout mice indicates that MNPs accumulated only in the atherosclerotic arteries but no other areas of the vasculature when administered intravenously. To summarize, our findings strongly support that monocyte membrane cloaking facilitates the nanoparticle attachment to atherosclerotic regions and enhances the entry of nanoparticles into the inflammatory endothelium in the arteries, suggesting that MNPs would serve as an excellent delivery strategy for targeted atherosclerosis therapy.
Abstract 294: Heavy Alcohol Use Worsens Peripheral Artery Disease-Associated Myopathy
Authors: Koutakis Panagiotis , Ismaeel Ahmed , Fletcher Emma , Papoutsi Evlampia , Smith Robert , Bohannon William , Miserlis Dimitrios
Published: November 2022

Heavy alcohol use can induce skeletal muscle dysfunction referred to as alcoholic myopathy. Likewise, peripheral artery disease (PAD) is characterized by an acquired skeletal muscle metabolic myopathy in ischemic muscles of the lower extremity. Although epidemiological studies have shown that heavy alcohol consumption is associated with a greater risk of PAD, data are lacking on the contribution of alcohol-related myopathy on PAD-associated skeletal muscle pathology. We compared myofiber morphometrics, mitochondrial respiration, and oxidative stress measures in gastrocnemius biopsies from PAD patients with heavy alcohol use (>7 or >14 drinks per week, for females and males, respectively) (n=13) to PAD patients (n=13) and non-PAD controls (n=17) consuming moderate to low/no alcohol. Myofiber area and diameter were lower in heavy-drinking PAD patients compared to low/moderate drinkers (p=0.03 and p=0.04, respectively) and non-PAD controls (p=0.02 and p<0.001, respectively). Myofiber roundness was significantly higher in heavy-drinking PAD patients compared to low/moderate drinkers (p=0.04) and non-PAD controls (p<0.001). Although there were no significant differences between PAD groups in mitochondrial respiration, PAD patients with heavy alcohol consumption tended to have lower respiration for Complex I (p=0.14), Complex II (p=0.10), and Complex IV (p=0.16) compared to PAD patients without heavy alcohol use. Both PAD groups had significantly reduced respiration of all Complexes compared to non-PAD controls (p<0.05). These data suggest that alcohol abuse may accentuate skeletal muscle pathology in PAD patients.
Abstract 352: Experimental Evaluation Of The Embolic Potential Of Paclitaxel-coated Stents.
Authors: Pouhin Alexandre , Steinmetz Éric , Coscas Raphaël
Published: November 2022

Introduction:
Recent studies have shown that paclitaxel-coated stents improve the patency of arteries treated for peripheral arterial disease. Embolization of paclitaxel crystals has been demonstrated after the use of paclitaxel-coated balloons and raises questions about the safety of these devices. The objective of this study was to compare experimentally the embolic potential of two paclitaxel-coated stents: Eluvia® (Boston Scientific, Marlborough, Massachusetts) versus ZilverPTX® (Cook Medical, Bloomington, Indiana)

Method:
Analyzes were carried out on 12 pigs divided into two groups: Eluvia® (n = 6) and ZilverPTX® (n = 6). After carotid puncture and navigation, the pigs received in each external iliac artery the stent corresponding to their group. Six pairs were established by pairing the pigs from the two groups one by one, to examine different post-implantation timepoints: 30 minutes, 6 hours, 1 day, 3 days, 7 days and 14 days. The concentration measurement and the histological analysis of paclitaxel were carried out in a blind manner on the plasma, arterial, lymph node and muscle samples.

Results:
The plasmatic paclitaxel rate decrease over 24 hours after stent implantation was significantly different between the two groups 0.19 [95% CI (0.14; 0.23);
p
< 0.001] with an undetectable concentration at the 10
th
hour for Eluvia® versus 3 days for ZilverPTX®. Significantly higher paclitaxel concentrations with ZilverPTX® stents were observed in muscle samples: extensor digitorum brevis (
p
= 0.005), biceps femoris (
p
< 0.001), semi-tendon (
p
= 0.001), tibialis anterior (
p
= 0.001), and in the femoral nodes (
p
= 0.005). Histological analysis revealed a trend for more marked intimal inflammation in the arteries stented with ZilverPTX® (p = 0.06), especially on the 7
th
and 14
th
day.

Conclusion:
In this study, the plasma decrease and the muscle / lymph node concentrations of paclitaxel reflect a higher embolic risk with the ZilverPTX® stent as compared to the ELUVIA® stent. While the clinical consequences of these findings remain to be elucidated, our results emphasize that the precautionary principles about paclitaxel-coated stents may be justified.
Abstract 364: Vascular Endothelial Growth Receptor 2 (Flk1) Donor Exosomes Drive Angiogenesis
Authors: Rizzo Skylar , Shi Ao , Mahlberg Ryan , Amontree Ashley , Peterson Timothy , Bagwell Monique , Arrell D K , Witt Tyra A , Nagel Mary , Park Sungjo , Sabbah Michael , Li Mark , Kargaran Parisa , Wu Yue , Li Jialun , Kim Yohan , Lucien Fabrice , Moran Steven , Terzic Andre , Behfar Atta
Published: November 2022

Objectives:
Peripheral arterial disease is a significant cause of morbidity and mortality. The goal of this study was to develop a novel treatment to promote angiogenesis. Platelets have been previously implicated in angiogenic events; however, platelet activation rather than presence of growth factors has been associated with new blood vessel formation. This study evaluated exosomes purified from activated platelets to determine their potential in promoting angiogenesis both in vitro and in vivo.

Methods & Results:
Under Current Good Manufacturing Practices, conditioned medium processed from apheresis platelet cultures yielded a purified exosome product (PEP) enriched for CD63, CD9 and Alix. In vitro, PEP drove HUVEC proliferation, migration, and vascular tube formation. Proteomic evaluation showed Flk1 (VEGFR2) was one of the key angiogenic proteins in PEP. HUVECs treated with Flk1 antibody neutralized PEP (anti-Flk1-PEP) showed reduced vascular tube formation compared to PEP. To evaluate in vivo angiogenesis, a rodent model of hindlimb ischemia was treated with sustained release PEP embedded in fibrin glue (PEP/TISSEEL) with blood flow evaluated by SPY angiography at 28 days post-treatment. Animals treated with PEP/TISSEEL showed improved blood flow with significant difference (p<0.001) as compared to sham, TISSEEL, and anti-Flk1-PEP/TISSEEL. Muscle tissue stained with α smooth muscle actin showed a significant increase (p<0.001) in vascular area in PEP/TISSEEL treated animals compared to TISSEEL alone. To further evaluate clinical translatability, a rabbit ear ischemia wound model was then treated with PEP/TISSEEL. Rabbit ears showed accelerated wound healing and restored blood flow on SPY angiography. Histologic evaluation of vascular area showed a significant increase in vascular area (p<0.05) compared to sham and TISSEEL alone animals.

Conclusions:
Exosome driven donation of Flk1 here promoted angiogenic events and was successfully translated into healing outcomes in the setting of two animal models of peripheral ischemia. This novel insight into biotherapy achievable pro-angiogenesis establishes a clinical-grade off-the-shelf exosome technology for the treatment of peripheral vascular disease.