bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 209: ATIC-associated
De Novo
Purine Biosynthesis Is Critically Involved In Proliferative Arterial Disease
Authors: Ma Qian , Yang Qiuhua , Xu Jiean , Liu Zhiping , Kim David , Kim Ha W , Weintraub Neal L , Fulton David , Huo Yuqing
Published: November 2022

Background:
Proliferation of vascular smooth muscle cells (VSMCs) is a hallmark of arterial diseases, especially in arterial restenosis following angioplasty or stent placement. VSMCs reprogram their metabolisms to meet increased requirement of lipid, protein and nucleotide for their proliferation.
D
e novo
purine biosynthesis is one of critical pathways for nucleotide synthesis. However, its role in proliferation of VSMCs in these arterial diseases has not been defined.

Methods:
De novo
purine synthesis in proliferative VSMCs was evaluated by LC-MS/MS. The expression of ATIC/Atic (5-aminoimidazole-4-carboxamide ribonucleotide formyltransferase/inosine monophosphate cyclohydrolase), the critical bifunctional enzyme in the last two steps of
de novo
purine biosynthesis pathway, was assessed in VMSCs of proliferative arterial neointima. Global and VSMC-specific knockout of
Atic
mice were generated and used for examining the role of Atic-associated purine metabolism in the formation of arterial neointima and atherosclerotic lesion.

Results:
In this study, we found that
de novo
purine synthesis was increased in proliferative VSMCs. Upregulated purine synthesis genes including ATIC/Atic were observed in the neointima of the injured vessels and atherosclerotic lesions both in mouse and human. Global or specific knockout of
Atic
in VSMCs inhibited cell proliferation, attenuating the arterial neointima in models of mouse atherosclerosis and arterial restenosis.

Conclusions:
These results reveal that
de novo
purine biosynthesis plays an important role in VSMC proliferation in arterial disease. These findings suggest that targeting ATIC is a promising therapeutic approach to combat arterial diseases.
Abstract 219: Apolipoprotein C3 Induces Inflammasome Activation In Human And Mouse Monocytes Only In De-lipidated Form
Authors: Hsu Cheng-Chieh , Shao Baohai , Kanter Jenny E , He Yi , Bornfeldt Karin E
Published: November 2022

Individuals with diabetes suffer from an elevated risk of coronary artery disease (CAD) events. Increased serum levels of apolipoprotein C3 (APOC3) predict incident CAD in individuals with type 1 diabetes (T1D). APOC3 is a small lipid-binding protein carried by lipoproteins, including VLDL, in circulation. Silencing of APOC3 in a mouse model of T1D-accelerated atherosclerosis has been demonstrated to prevent diabetes-accelerated atherosclerosis, strongly suggesting that APOC3 is a causal mediator. However, the exact mechanism whereby APOC3 promotes atherosclerosis in the setting of T1D is unclear. Mice with T1D demonstrated elevated levels of glucose, triglycerides, APOC3 (non-diabetic [ND] mice=442.6 μg/ml, diabetic [D] mice=755.9 μg/ml, p<0.01, N=21 per group), and IL-18 (ND=124.1 pg/ml, D=256.8 pg/ml, p<0.01, N=18-22 per group) in plasma. The elevated levels of plasma APOC3 and IL-18 in diabetic mice and a recent study demonstrating that de-lipidated APOC3 can stimulate inflammasome activation in human monocytes prompted us to investigate whether lipidated APOC3 has similar inflammasome-activating properties. Our results show that de-lipidated APOC3, but not VLDL (which contains APOC3) significantly stimulates IL-1β release from isolated human monocytes (p<0.01 for vehicle vs APOC3, N=3 per group) and mouse monocytes (p<0.01 for vehicle vs APOC3, N=4-7 per group). Mouse monocytes stimulated with de-lipidated APOC3 also had elevated IL-18 release (vehicle=21.2 pg/mg, APOC3=170.2 pg/mg, p<0.001),
Il1b
mRNA (APOC3=86.7-fold over control), and
Nlrp3
mRNA (APOC3=13.8-fold over control), indicating activation of the NLRP3 inflammasome pathway. However, analysis of serum samples from subjects with T1D showed that virtually all APOC3 is bound to lipoproteins (primarily VLDL and HDL). To further confirm the effect of lipidated APOC3, we bound APOC3 to lipid-based small unilamellar vesicles to mimic the physiological state of APOC3 in plasma. The stimulatory effect of APOC3, measured by IL-1β release from mouse monocytes, was lost. Together, our findings suggest that although de-lipidated APOC3 has the ability to induce inflammasome activation in monocytes, virtually no APOC3 circulates in a lipid-free form in subjects with T1D.
Abstract 233: PANDORA-seq Reveals Hidden Regulatory Small RNA Landscape Associated With Atherosclerosis Development In Hypercholesterolemic LDL Receptor-deficient Mice
Authors: Hernandaz Rebecca , Shi Junchao , Li Xiuchun , Liu Jingwei , Wu Jake , Zhou Tong , Chen Qi , Zhou Changcheng
Published: November 2022

Introduction:
Small non-coding RNAs (sncRNAs) have been demonstrated to have diverse roles in regulating molecular processes. While the standard RNA sequencing (RNA-seq) method has advanced sncRNA discovery, many RNA modifications can interfere with library construction procedures, preventing the discovery of a subset of highly modified sncRNAs including transfer RNA-derived small RNAs (tsRNAs) and ribosomal RNA-derived small RNAs (rsRNAs) that may have important functions in disease development. To address this technical obstacle, we recently developed a novel PANDORA-seq method to overcome RNA modification-elicited sequence interferences (
Nat Cell Biol
2021:424-436). This study aims to identify novel sncRNAs that are associated with atherosclerosis development by using PANDOR-seq.

Hypothesis:
PANDORA-seq enables us to reveal unprecedented sncRNA landscape associated with atherosclerosis development in LDL receptor-deficient (LDLR
-/-
) mice.

Methods:
To identify sncRNAs associated with atherosclerosis development, LDLR
-/-
mice were fed a low-cholesterol diet (LCD; 0.02% cholesterol) or high-cholesterol diet (HCD; 0.5% cholesterol) for 9 weeks. Total RNA isolated from intima was subjected to both traditional RNA-seq and PANDORA-seq, and the sequencing data were analyzed by sncRNA analyzing software
SPORTS1.1
.

Results and Conclusions:
By overcoming RNA modification-elicited limitations, PANDORA-seq unveiled a rsRNA- and tsRNA-enriched sncRNA landscape in the atherosclerotic intima of LDLR
-/-
mice which was strikingly different from that detected by traditional RNA-seq. While micro-RNAs (miRNAs) were the dominant sncRNAs detected by traditional RNA-seq (55.9% of total sncRNAs), PANDORA-seq substantially increased the reads of rsRNAs and tsRNAs which account for 77.4% and 5% of total sncRNAs, respectively. PANDORA-seq also detected 1,383 differentially expressed sncRNAs induced by HCD feeding including 1160 rsRNAs and 195 tsRNAs but traditional RNA-seq only detected 16 differentially expressed rsRNAs and tsRNAs. Thus, PANDORA-seq revealed the hidden rsRNAs and tsRNAs associated with atherosclerosis development, and the functions of those previously underexplored sncRNAs warrant future investigations.
Abstract 234: Mitochondrial Reactive Oxygen Species Facilitate Phagocytosis During Initiation Of Atherosclerosis
Authors: Chen Yiliang , Zhang Jue , Chang Jackie , Beg Mirza , Zhao Yiqiong
Published: November 2022

Objective:
Atherosclerosis (AS) is associated with dyslipidemia, oxidative stress and foam cell accumulation within the AS plaques. Macrophage mitochondria dysfunction is commonly observed in both AS-prone animals and human patients with AS. But the contribution of macrophage mitochondria to AS development remain poorly understood. Our group previously showed that mitochondria continuously produce reactive oxygen species (mtROS) to drive macrophage activation and facilitate diet-induced AS. Here we aim to study the role of mtROS in phagocytosis, which could promote foam cell formation during atherogenesis.

Approach and Results:
We used pHrodo-Bioparticle and co-incubated with murine peritoneal macrophages. We then ran flowcytometry and quantified pHrodo (only emit fluorescence when pH is low as in the phagolysosomes) signals as phagocytosis efficiency. We found that oxidized LDL (oxLDL) but not native LDL or HDL pre-treatment stimulated 3-fold more phagocytosis by macrophages. We consolidated our results by showing oxLDL stimulated phagocytosis in a dose and time-dependent manner. Moreover, reducing mtROS either by MitoTempo, a mtROS scavenger, or by mitochondrial catalase overexpression significantly attenuated oxLDL effect on phagocytosis by ~40%. To explore the underlying molecular mechanism, we used different mitochondrial function inhibitors (etomoxir, rotenone, antimycin, oligomycin, FCCP) and found that etomoxir partially blocked (~25%) oxLDL-induced phagocytosis while antimycin promoted (~10%) this effect. In addition, we used confocal live cell imaging to show that mtROS facilitated the engulfment and formation of phagosomes. To further explore the physiological relevance, we conducted in vivo phagocytosis assay and demonstrated that mtROS are required for efficient phagocytosis in vivo. Initial atherogenic conditions increased phagocytosis by macrophages in the high fat diet-induced AS mouse (
apoe
null) model.

Conclusions:
MtROS is essential for macrophage phagocytosis functions. Continuous stimulation of mtROS in macrophages could lead to too much large vesicles engulfment and lipid accumulation, which is responsible for foam cell formation during AS initiation.
Abstract 239: Investigating The Roles Of Sr-b1 And Pcpe2 In Regulation Of Adipocyte Metabolic Homeostasis
Authors: Knaack Darcy A , Sorci Thomas Mary G , Thomas Michael J , Chen Yiliang , Sahoo Daisy
Published: November 2022

Obesity is a universal epidemic and underlies comorbidities including type 2 diabetes and cardiovascular disease. Adipocytes store dietary metabolites within lipid droplets to avoid lipotoxicity and to provide energy to the body through metabolic processes such as lipid and glucose metabolism. Collaborative studies demonstrated that the extracellular matrix protein procollagen C-endopeptidase enhancer 2 (PCPE2) mediates the ability of scavenger receptor BI (SR-BI) to transport cholesterol into hepatocytes and adipocytes.It remains unknown if SR-BI and PCPE2 also play roles in other adipocyte metabolic processes and if their cooperative partnership is conserved. This knowledge gap prompted us to test the hypothesis that PCPE2 facilitates SR-BI’s ability to regulate metabolic processes in adipocytes. We validated a differentiated adipocyte model system using mesenchymal stem cells isolated from ears of wild-type (WT), SR-BI
-/-
, and PCPE2
-
/-
mice by demonstrating increased lipid droplet formation, mRNA expression of adipogenesis markers, and secretion of adiponectin over a 9-day post-differentiation period across genotypes. We used this model system to test the roles SR-BI and PCPE2in glucose and lipid metabolism. Compared to WT adipocytes, glucose uptake in SR-BI
-/-
and PCPE2
-/-
adipocytes was decreased by 5.4% and 12.6%, respectively, despite increases in surface GLUT4 levels. We also observed that adipocytes lacking PCPE2 had an increased response to insulin through AKT signaling, which may be responsible for the increased surface expression of GLUT4. Loss of SR-BI or PCPE2 in adipocytes did not affect palmitic acid uptake, despite elevated mRNA expression of fatty acid transporters (
Cd36, Fabp4
) compared to WT adipocytes. SR-BI
-/-
and PCPE2
-/-
adipocytes showed an inverse response to insulin when measuring phosphorylation of fatty acid regulatory proteins (AMPK, ACC) compared to WT adipocytes, suggesting altered lipid metabolism in adipocytes lacking SR-BI or PCPE2. Together, our data suggest that, individually, SR-BI or PCPE2 is important for glucose uptake and fatty acid regulation, but not fatty acid uptake. Further investigations will determine whether a partnership is required for optimal metabolic function in adipocytes.
Abstract 255: Immunohistochemical Analysis Of Sars-cov-2 Induced Microvascular Thrombosis
Authors: Navarro Quinn S , Duryee Michael J , Ruskamp Ryan J , Thiele Geoffrey , Radio Stanley J , Anderson Daniel R
Published: November 2022

Background:
The SARS-CoV-2 virus utilizes Angiotensin Converting Enzyme 2 (ACE2) as the functional receptor for cellular entry. Additionally, SARS-CoV-2 binding induces downregulation of the ACE2 receptor, which creates an imbalance of the ACE/ACE2 ratio that promotes thrombosis. These factors can both potentially contribute to platelet aggregation and subsequent formation of cardiovascular microvascular thrombosis that can result in cardiac failure. Thus, identification of microthrombosis and analysis of the related molecular mediators can provide further understanding of SARS-CoV-2 pathophysiology.

Methods:
Heart autopsy specimens (N=23) from patients who died from cardiac failure due to COVID-19 complications and control heart tissue (N=5) were collected via rapid autopsy. These samples were sectioned and stained for H&E and MOVAT. Samples were then analyzed by immunohistochemistry (IHC) to identify the following inflammatory mediators, clotting factors, and related proteins: CD42b, C1Q, SARS-CoV-2 Spike protein, SARS-CoV-2 Nucleocapsid protein, ACE2 and Thrombin. Samples were then counterstained with DAPI and visualized with an ECHO Revolve Fluorescence Microscope. ImageJ was used to analyze pixel densities of immunofluorescence patterns compared to values obtained from control samples.

Results:
Histological analysis with H&E and MOVAT stains demonstrated diffuse microvascular thrombosis in cardiac samples from COVID patient. IHC staining revealed the significantly increased presence of SARS-CoV-2 Spike protein (p<0.0001), SARS-CoV-2 Nucleocapsid protein (p<0.0001), Thrombin (p<0.05), ACE2 Receptor (p<0.01), CD42b (p<0.01), C1Q (p<0.01). Furthermore, basement membrane staining was exceptionally characteristic with CD42B and Spike protein.

Conclusions:
This study demonstrates the effective use of immunohistochemistry in identification of various inflammatory mediators and clotting factors involved in the vascular pathophysiology of SARS-CoV-2. Replicated studies that provide further evidence of characteristic IHC staining patterns and the presence of additional thrombus-factors would strengthen our findings and deepen the understanding of SARS-CoV-2 pathophysiology.
Abstract 285: Thrombospondin-5 Is Necessary For Males But Not Females In Hindlimb Ischemia Recovery
Authors: Rangel Melissa M , Bunn Corinne , Wang Xuerong , Ding Xianzhong , Maier Kristopher , Gahtan Vivian
Published: November 2022

Introduction:
Ameliorating critical limb ischemia (CLI), in part, relies on arteriogenesis and angiogenesis. We have shown thrombospondin-5 (TSP5) is proangiogenic in vitro. The role of TSP5 in CLI and the differences between males and females are unknown. Hypotheses: blood flow recovery, arteriogenesis and angiogenesis after limb ischemia is diminished (1) in TSP5 null mice compared to controls, and (2) in female compared to male TSP5 null mice.

Methods:
14-16 week-old wild type (WT; C57Bl6) or TSP5 null male and female mice underwent left femoral artery ligation and transection (n=6-9/group). Laser Doppler data was collected preoperative, post ligation and at sacrifice (day 14). Blood flow recovery was expressed as a ratio of ischemic/non-ischemic limb. Immunohistochemistry was performed using anti-α-smooth-muscle-actin on the vastus lateralis to quantify arteriogenesis and anti-CD31 on the gastrocnemius to quantify angiogenesis. Arteriole and capillary density were determined by vessel counts/5HPF. Data was analyzed by ANOVA with post hoc testing, with p<0.05 being significant.

Results:
TSP5 null mice had decreased blood flow recovery in males (WT 0.85 ± 0.04 vs TSP5 0.48 ± 0.06, p<0.001) and females (WT 0.63 ± 0.07 vs TSP5 0.36 ± 0.04, p<0.01). No difference in flow recovery was seen between male and female TSP5 nulls (male 0.48 ± 0.06 vs female 0.36 ± 0.04 p<0.14). Arteriogenesis was impaired in male TSP5 null mice (WT 3.91 ± 0.24 vs TSP5 2.6 ± 0.42, p<0.02), but increased in females (WT 2.5 ± 0.26 vs TSP5 4.35 ± 0.73, p<0.01). Angiogenesis was decreased in TSP5 null males (WT 10.9 ± 0.93 vs TSP5 7.03 ± 0.84, p<0.01), but not females (WT 6.1 ± 0.50 vs TSP5 5.6 ± 0.80 p<0.9). In contrast, flow recovery, angiogenesis and arteriogenesis was decreased in WT females compared to WT males (p< 0.05).

Conclusions:
Our findings suggest TSP5 is relevant to vascular remodeling following hindlimb ischemia with a divergence in mechanism between males and females. TSP5 is necessary in males for arteriogenesis and angiogenesis. TSP5 in females may be supressive of arteriogenesis and has no effect on angiogenesis. Further investigation into these sexually dimorphic adaptive processes may lead to specific targeted CLI treatments.
Abstract 295: Methyl Transferase Inhibitor 5-azacytidine Suppresses Experimental Abdominal Aortic Aneurysms
Authors: Ikezoe Toru , Ge Yingbin , Guo Jia , Shoji Takahiro , Xu Baohui , Dalman Ronald L
Published: November 2022

Background:
Increased vascular DNA methylation is present in disease states including abdominal aortic aneurysms (AAA). The impact of dysregulated DNA methylation on aneurysm pathogenesis, however, remains uncertain. We evaluated the influence of 5-azacytine (AZA), a methyl transferase inhibitor, on experimental AAA initiation and progression.

Methods and Results:
AAAs were induced in male C57BL/6J mice via intra-aortic porcine pancreatic elastase infusion. AZA or vehicle were administered intraperitoneally starting one day prior to (prophylactic, 2 mg/kg), or 4 days following (therapeutic, 1 or 2 mg/kg), elastase infusion and continuing daily through 14 days. AAA development and progression were evaluated by serial ultrasonographic aortic diameter measurements and histopathology at sacrifice.Time dependent post-elastase aortic diameter enlargement was markedly reduced in mice receiving prophylactic AZA treatment. A substantial delay in AAA onset was noted as well. While therapeutic AZA tended to mitigate further aneurysmal enlargement at either dose, a significant reduction was noted only in the 2 mg/kg group at 14 days. On histopathological assessment, AZA was associated with marked attenuation of characteristic aneurysmal pathologic features including medial elastin and smooth muscle destruction, leukocyte accumulation and neoangiogenesis in all groups.

Conclusion:
AZA treatment attenuates aneurysm development and progression in the intra-aortic porcine pancreatic elastase infusion AAA model. Pharmacologic strategies effective at limiting or reducing DNA methylation may have translational applications in the medical management of AAA disease.
Abstract 300: Mangiferin Conjugated Gold Nanoparticles Protect Against The Development Of Abdominal Aortic Aneurysm In Apoe–/– Mouse Model
Authors: Hans Chetan P , Sharma Neekun , Downey Edward , Khoobchandani Menka , Katti Kavita , Katti Kattesh V
Published: November 2022

Objective:
There are no drugs to prevent the growth of abdominal aortic aneurysm (AAA), responsible for ~200,000 deaths in the world each year. There is a growing interest in natural anti-inflammatory compounds using nanotechnology-based drug-delivery. Mangiferin (MGF) is one such phytochemical isolated from Mangifera indica. Conjugation of MGF on gold nanoparticles (MGF-AuNPs) enhances bioavailability, through cellular penetration, presenting new opportunities toward the design of innovative nanomedicine agents. Recently, we have demonstrated the unique applications of MGF-AuNPs as an immunomodulatory therapeutic agent in the treatment of metastatic breast and prostate cancers. Here, we investigated whether MGF-AuNPs prevent the development of AAA.

Approach and Results:

Apoe
–/–

mice were subjected to angiotensin (AngII; 1μg/min/kg)-induced AAA. MGF-AuNPs (~7 mg/30g mouse) or starch (S)-AuNPs were administered daily, a week prior to AngII and continued for 28 days (n=6-12 per group). The incidences of AAA were significantly attenuated with MGF-AuNPs than AngII group (P<0.001), associated with a decrease in maximal intra-luminal diameter (P<0.001), pulse wave velocity (P<0.001), distensibility (P<0.05) and radial strain (P<0.05). Degradation of elastin (P<0.001), pro-inflammatory cytokines (P<0.01) and apoptotic cell death (P<0.01) were significantly reduced in the aortae of MGF-AuNPs treated

Apoe
–/–

mice than AngII group. Mechanistically, Notch1, its ligands (Jag1, Dll4) and downstream targets (HeyL, NFκB2, pStat3), were significantly reduced by MGF-AuNPs in the aorta (P<0.01) than AngII group. In the macrophages overexpressed with activated Notch1 (NICD), MGF-AuNPs significantly diminished the expression of Jag1 (P<0.001), HeyL (P<0.001), Il6 (P<0.001) and NFκB2 (P<0.001). MGF-AuNPs also prevented the nuclear translocation of NICD and its downstream effector pStat3, in the macrophages transfected with NICD plasmid.

Conclusion:
Our studies provide compelling pre-clinical evidence of protective effects of MGF-AuNPs on AAA development through inactivation of Notch1 signaling, thus present realistic potential toward clinical translation of this nanoceutical for use as a non-invasive effective treatment for AAA.
Abstract 321: Endothelial Cell Translational Control Is A Key Mediator Of Intima Growth In Response To Disturbed Flow
Authors: Wines-Samuelson Mary , Chowdhury Sayantani , Berk Bradford C , Sowden Mark
Published: November 2022

Background:
Carotid intima-media thickening (IMT) is a good surrogate for atherosclerosis and CVD. Previously we used the IMT response of inbred mouse strains to disturbed flow (d-flow) in the partial carotid ligation (PCL) model to identify an intima modifier locus Im3. Microarray and QTL analysis revealed ribosomal protein large subunit (RpL17) as the primary candidate gene for IMT within Im3; further, low RpL17 levels correlate with enhanced intima formation. RpL17 is located in the nascent protein exit tunnel, and serves as a docking site for the NAC chaperone in protein folding. We generated endothelial cell (EC)-specific conditional heterozygous RpL17 mice (EC-RpL17+/-, termed EC-RpL17) to study the IMT response to d-flow using PCL.

Results:
EC-RpL17 mice had increased IMT relative to controls (RpL17+/+) after PCL. Data showed induction of oxidative stress and endoplasmic reticulum (ER) stress in EC-RpL17 with activation of the integrated stress response (ISR), a stress-support pathway promoting hyperproliferation in cancer, including: 1) increased levels of reactive oxygen species (ROS), BiP, Chop, and Atf4 expression, activation of PERK (p-PERK), and inactivation of eIF2 alpha (p-eIF2 alpha) in cultured RpL17 microvascular EC. The in vitro data correlated well with in vivo data from post-PCL carotid immunostaining, including: increased p-PERK, BiP, Chop, 4-hydroxynonenal and 8-hydroxyguanosine. EM analysis revealed dilation of the rough ER (indicating ER stress), and decreased cytosolic polyribosomes in EC-RpL17 EC.

Conclusion:
Decreased EC RpL17 increases d-flow mediated IMT, likely due to induction of growth-promoting ISR by ER and oxidative stress. These data define a novel pathophysiological role for RpL17.
Abstract 328: Vascular Smooth Muscle Cell Mechanotransduction Through Serum And Glucocorticoid Inducible Kinase -1 Promotes Interleukin-6 Production And Macrophage Accumulation In Murine Hypertension
Authors: Figueroa Mario , Mattia Victoria , xiong Ying , Mukherjee Rupak , Jones Jeffrey A , Ruddy Jean M
Published: November 2022

Objective:
Signaling through the serum and glucocorticoid inducible kinase-1 (SGK-1) can be mechanically activated. The objective of this investigation was to demonstrate that
in vitro
cyclic stretch of aortic VSMCs and
in vivo
induction of hypertension (HTN) can cause SGK-1-dependent production of cytokines to promote macrophage accumulation in the murine abdominal aorta.

Methods:
HTN was induced in C57Bl/6 mice with AngiotensinII (AngII) infusion (1.46mg/kg/day x 21 days) and a subset of animals had systemic infusion of EMD638683 (2.5mg/kg/day x 21 days), a selective SGK-1 inhibitor. Aortas were digested and analyzed by flow cytometry for abundance of CD11b
+
/F4-80
+
cells (macrophages) and serum was analyzed by ELISA to quantify IL-6 and MCP-1. Aortic VSMCs from wild-type (WT) and SGK-1 flox-induced knockout mice (FloxSGK-1KO) were subjected to 12% biaxial cyclic stretch for 12 hours +/- EMD638683 (10υM). Media was analyzed by ELISA. Statistical analysis included two-way ANOVA with significance at p<0.05.

Results:
Having previously demonstrated that AngII infusion induced HTN and upregulated SGK-1 activity in the abdominal aorta, this experimentation indicated a concurrent trend for increased CD11b
+
/F4-80
+
cells in the HTN group and marked reduction with EMD638683 infusion. Serum IL-6 levels were elevated in AngII-induced HTN (p<0.05 vs Control), and reduced to baseline with concurrent EMD638683 treatment. This mirrored the increased abundance of IL-6 in media from Stretch WT VSMCs (p<0.05 vs Static), and attenuation of the effect with EMD638683. Interestingly, MCP-1 was not detectable in the serum samples but was increased in media from Stretch WT VMSCs (p<0.05 vs Static), suggesting potential paracrine or autocrine function of this cytokine within the vessel wall to support macrophage accumulation. Further supporting the integral role of mechanical signaling through SGK-1, media from Stretch FloxSGK-1KO VSMCs had no change in IL-6 or MCP-1 compared to Static.

Conclusion:
Mechanotransduction through SGK-1 is instrumental in pro-inflammatory cytokine production and aortic macrophage accumulation, therefore further investigation into targeting this kinase may present opportunities to modulate hypertensive vascular remodeling.
Abstract 331: Modulation Of Murine Vasculitis And Cardiovascular Inflammation By The Gut Microbiota
Authors: Jena Prasant , Wakita Daiko , Gomez Angela C , Carvalho Thacyana T , Narayanan Meena , Lee Youngho , Cani Patrice D , De Vos Willem M , Chen Shuang , Crother Timothy R , Shimada Kenichi , Arditi Moshe , Noval Rivas Magali
Published: November 2022

Changes in intestinal microbiota composition and function are associated with development of cardiovascular disease, but the role of these alterations remains unclear in Kawasaki Disease (KD), an acute pediatric vasculitis that targets coronary arteries. Using a murine model of KD vasculitis, we observed reduced development of cardiovascular lesions in germ-free and antibiotic-treated mice. KD vasculitis in mice was associated with alterations in the gut microbiota composition and decreased abundance of
Akkermansia muciniphila
and
Faecalibacterium prausnitzii
. Supplementation with live or pasteurized
A. muciniphila
or
F. prausnitzii
attenuated the severity of KD cardiovascular inflammation. Oral administration of the short-chain fatty acids acetate or propionate, both produced by
A. muciniphila,
or butyrate, produced by
F. prausnitzii
, reduced the development of vascular inflammation. Furthermore, treatment with Amuc_1100, a purified protein isolated from the outer membrane of
A. muciniphila
, decreased the severity of murine KD vasculitis. Beneficial effects of either pasteurized
A. muciniphila
or Amuc_1100 on murine KD vasculitis development were associated with improvement of gut barrier function. These results reveal an underappreciated gut microbiota-cardiovascular inflammation axis during murine KD vasculitis. Our findings may incite the development of novel diagnostic tools and therapeutic strategies that modulate the intestinal microbiota composition and function for KD patients.
Abstract 349: Segmentation Of Atherosclerotic Plaque Features From Histopathology Images Using Novel Deep Learning Techniques
Authors: Mohebpour Majid , Gasbarrino Karina , Khan Kashif , Zheng Huaien , Babazadeh Khameneh Nahid , Psaromiligkos Ioannis , Daskalopoulou Stella S
Published: November 2022

Objective:
Atherosclerotic plaques have a complex composition, consisting of inflammation, fibrosis, cholesterol crystals, hemorrhage, and/or calcification. The segmentation and quantification of plaque features in histopathology images form the foundation for studies evaluating plaque instability and the mechanisms that underlie the atherosclerotic process. Manual segmentation of plaque features from histology images is a tedious, time-consuming, and subjective visual recognition task. Herein, we present a fully automatic approach using state-of-the-art deep learning techniques to identify three major features of the atherosclerotic plaque: calcification, lipid core, and fibrosis.

Methods:
Plaques (n=70) were collected from patients who underwent a carotid endarterectomy at McGill University-affiliated hospitals. Hematoxylin and Eosin-stained sections were obtained from the region with the largest plaque burden. The “ground truth annotations” for lipid core, calcification, and fibrosis were performed manually by three blinded cardiovascular pathologists, using Sedeen Viewer. A total of 23,000 patches with 512x512 pixel size were extracted from our image dataset, and divided into train, validate, and test sets. Using Transfer Learning, multi-class U-Net models for semantic segmentation were trained on the patches to extract fibrosis, lipid, and calcification plaque features. Evaluation of model performance was based on the mean value of the Intersection over Union (Mean-IOU) between the prediction results and the “ground truth annotations”.

Results:
Our models resulted in an overall performance of 77% for test images, and a per-class performance for the three plaque features: fibrosis = 0.77±0.2, lipid core = 0.80±0.3, calcification = 0.75±0.25. However, a qualitative evaluation by the pathologists confirmed that the prediction results in fact outperformed the “ground truth annotations”, and detected non-annotated regions.

Conclusion:
To our knowledge, this is a first attempt at developing a fully automatic approach for atherosclerotic plaque feature segmentation from histology images. Our models can accelerate atherosclerosis research, by improving the speed, quality, and reproducibility of plaque analysis.
Abstract 355: Early Vascular Aging In Young People And Their Connective Tissue Status
Authors: Evsevyeva Maria , Eremin Michail , Sergeeva Oxana , Kudrjavtseva Victoria , Shchetinin Evgeniy
Published: November 2022

Aim is to compare the phenotypes of vascular aging (VA) in young people with manifestations of hereditary connective tissue disorders (HCTD), which are increasingly common in modern youth.

Material and Methods:
264 students (93 boys and 171 girls) were examined at the age of 18 to 25 years on the basis of University Health Center within framework of the National Preventive Project “University - Territory of Health”. Angiological screening was performed using VaSera VS-1500 device (Fukuda Denshi, Japan), which allows us to evaluate such an indicator of arterial stiffness as the cardio-ankle vascular index (CAVI). All young people were divided into CAVI-tercile groups, which made it possible to identify carriers of early (upper CAVI-tercile), normal (middle CAVI-tercile) and healthy (first CAVI-tercile) VA phenotypes. Early vascular aging is also called EVA syndrome. The most specific manifestations of HCTD were evaluated taking into account the Ghent Criteria of 2-nd revision, which allow us to obtain score assessment of CT involvement. Data processing was carried out using software package “Statistica 10.0” (StatSoft Inc, USA).

Results:
It turned out that an assessment of HCTD severity equal to 5 or more points in three groups was found in 41.5%, 18.4% and 9.4% (P=0.002) in boys and 49.3%, 21.6% and 12.8% (P=0.000) in girls. The most common HCTD signs include wrist and thumb symptoms, myopia, flat feet, atypical striae, dolichocephaly, enophthalmos, etc.

Conclusion:
The most stigmatized young people were among carriers of EVA syndrome, and the least ones - among those with signs of health VA. This means that in young carriers of rigid vessels, according to results of angiological screening, it is necessary to carry out a timely differential diagnosis between true EVA syndrome and HCTD using of phenotypic analysis. The nature of vascular stiffness increase in these two cases is absolutely different – in first case, it is early arteriosclerosis, and in case of HCTD, it is a genetically determined imbalance of elastin and collagen, which requires the use of completely different corrective measures. This explains importance of timely differential diagnosis when identifying young people with high vascular wall stiffness
Abstract 390: Influenza A Virus Infection Increases Magi1 Expression In Endothelial Cells And Its Depletion Inhibits Virus Replication Through Increased Expression Of Mx1
Authors: Wang Yin , Vu Hang , Velatooru Loka reddy , Wang Yongxing , Thomas Tamlyn , Imanishi Masaki , Kotla Sivareddy , Le Nhat-Tu , Fujiwara Keigi , Evans Scott E , Abe Jun-ichi
Published: November 2022

When influenza virus infects cells, it changes cellular metabolism in such a way that allows virus particles to replicate efficiently. This metabolic engineering takes place soon after virus infects cells, for which the PSD95/DiscLarge/ZO-1 (PDZ) domain of certain proteins is known to play a role. Membrane-associated guanylate kinase with inverted domain structure-1 (MAGI1) is a scaffold protein with 6 PDZ domains, and we have shown that it is involved in the regulation of endothelial cell (EC) activation and atherosclerosis in mice. Since recent studies indicate that the vascular endothelium can be infected by influenza A virus (IAV) and plays a role in the influenza-induced pathogenesis and cardiovascular disease (CVD), we investigated the role of MAGI1 in IAV infection using cultured human umbilical vein endothelial cells (HUVECs) as well as human lung microvascular endothelial cells (HULECs). We found increased
MAGI1
mRNA expression in IAV-infected cells. Conversely, when MAGI1 depleted ECs were infected with IAV, virus infection and replication was greatly suppressed. Our microarray studies revealed that depletion of MAGI1 in HUVECs increased the protein expression and signaling networks involved in interferon production. Specifically, we found that the MAGI1 null condition induced expression of anti-viral response genes including interferon-induced GTP-binding protein MX1, an antiviral protein, interferon beta1, a cytokine promotor
STAT1
(signal transducer and activator of transcription 1), and also increased protein expression levels of STAT1, phosphorylated STAT5 and MX1. Co-transfection of HUVECs with siMX1 and siMAGI1 impaired MAGI1 depletion-induced suppression of IAV infection. Furthermore, we found nuclear localization of interferon regulatory factor 3 (IRF3) in MAGI1 depleted cells, indicating that MAGI1 depletion elicits the interferon production and signaling. Taken together, we conclude that IAV infection and replication occurs in ECs in a MAGI1 expression dependent manner. Thus, MGAI1 depletion in ECs suppresses IAV replication, and this suppression is due to increased MX1 expression, which induces IRF3 activation and interferon production. MAGI1 can be a potential therapeutic target for influenza-induced CVD.
Abstract 396:
CHROMR
Coordinates Interferon Signaling And Lipid Metabolism In Viral Infection
Authors: van Solingen Coen , Cyr Yannick , Scacalossi Kaitlyn , de Vries Maren , Barrett Tessa , De Jong Annika , Gourvest Morgane , Zhang Tracy , Kher Raadhika , Cornwell Macintosh , Gildea Michael , Brown Emily , Fanucchi Stephanie , Mhlanga Musa , Berger Jeffrey , Dittmann Meike , Moore Kathryn J
Published: November 2022

COVID-19, caused by SARS-CoV-2 infection, is associated with atherosclerotic cardiovascular complications like acute coronary syndrome, myocardial infarction, and stroke, but the underlying mechanisms are poorly understood. Long non-coding RNAs (lncRNAs) have emerged as important regulators of gene expression in the immune response. RNA-seq of whole blood from hospitalized patients with COVID-19, influenza A virus and matched controls identified 190 lncRNAs deregulated in both viral infections. Among the top mutually upregulated lncRNAs, we noted
CHROMR
(alias
CHROME
)
,
a primate-specific lncRNA previously identified as a competing endogenous RNA that regulates cholesterol efflux and fatty acid oxidation via microRNA sequestration. Here, we report a complementary role for
CHROMR
in coordinating the interferon (IFN) signaling response to respiratory viruses.
CHROMR
expression is induced in macrophages in response to SARS-CoV-2 and influenza A infection and accumulates in the nucleus where it binds the transcriptional co-repressor IRF2BP2, a negative regulator of IFN-stimulated gene (ISG) expression.
CHROMR
is essential for mounting an anti-viral response, as its depletion in macrophages reduces histone acetylation at ISGs, activation of IRF signaling, and ISG expression. These findings suggest that
CHROMR
sequesters the nuclear IRF-2/IRF2BP2 repressor complex releasing its inhibitory effect on transcription of ISGs. Consistent with this,
CHROMR
expression is required to restrict influenza virus replication in macrophages. Notably, many viruses rewire host lipid synthesis and metabolism to facilitate replication, and thus, increased
CHROMR
expression in virus infected cells would both mitigate cellular lipid accumulation and increase ISG transcription to mount an anti-viral immune response. Collectively, our findings underscore the merit of investigating lncRNAs to decipher novel regulatory mechanisms that govern lipid metabolism and inflammation in humans.
Abstract 417: Elevation Of Serum Hdl And Apoe Level In A Novel Vps35l Deficient Mouse Model
Authors: Ohtsuji Shiomi , Nishio Yosuke , Takase Hiroshi , Tsujita Maki , Braesch-Andersen Sten , Saitoh Shinji , Kato Kohji
Published: November 2022

Aim:
Defect of the endosomal protein sorting factor VPS35L causes novel congenital anomalies syndrome, in which cholesterol dysmetabolism is one of the symptoms. To reveal the biochemical mechanisms, the a model mouse was developed and characterized.

Methods:
Loss-of-function mutation, N995del, VPS35L knock-in (KI) mice were generated. Serum lipoprotein profiles were assessed by LipoSEARCH™ (Immuno-Biological Laboratories, Japan). For biochemical assays, 70 micro-L of serum was sub-fractionated with Superose 6 column in PBS and total and free cholesterol detected using LabAssay™cholesterol (Fujifilm Wako Chemicals, Japan) and Pureauto™S F-CHO-N (SEKISUI MEDICAL, Japan) assay kits. Negative images of fractioned lipoproteins were observed by TEM (JEM-1400Plus, JEOL, Japan). ApoA-I and apoE in the fractions were determined by use of a mouse apoA1 ELISA
PRO
kit and a mouse apoE ELISA
PRO
kit (Mabtech AB, Sweden). Mouse liver total RNA was isolated by ISOGEN (Nippon Gene, Japan) and first-strand cDNA generated using the Invitrogen™ SuperScript™IV First-Strand Synthesis system with random hexamers. For quantitative real-time PCR, the StepOnePlus™ real-time PCR system (Thermo Fisher) was used, and the PCR end product of each well confirmed by the melt curve analysis.

Results:
Lipoprotein analysis indicated the KI mice showed 1.9-fold higher total cholesterol than WT mice. LDL-C and HDL-C were increased 2.9- and 1.9-fold compared to WT mice, respectively. ApoA-I levels in the large HDL and HDL fractions were all significantly increased 1.5-, and 1.6-fold, whereas apoE levels were elevated 25-, and 8.4-fold, respectively.

Conclusions:
Ablation of VPS35L in MEF(3T3) cells caused loss of endocytosis of the LDL receptor and LRP1, and perhaps even SR-BI, which may lead to increased serum LDL and HDL levels in the loss-of-function KI model mice. Surprisingly. a high level of apoE was detected in KI mice, especially in the HDL fraction. In this case, apoE catabolism became abnormal due to the lack of LRP1 at the cell surface. Abnormal metabolism of apoE may affect the central nervous system or neuron generation. Further examination of the phenotype of this this model mouse line are warranted.
Abstract 419: Therapeutic Potential Of The Proprotein Convertase Subtilisin/Kexin (PCSK) Family In Vascular Disease
Authors: Suur Bianca E , Chemaly Melody , Lindquist Liljeqvist Moritz , Djordjevic Djordje , Stenemo Markus , Bergman Otto , Karlöf Eva , Lengquist Mariette , Odeberg Jacob , Hurt-Camejo Eva , Eriksson Per , Ketelhuth Daniel F , Roy Joy , Hedin Ulf , Nyberg Michael , Matic Ljubica
Published: November 2022

Introduction:
Proprotein convertase subtilisin/kexins (PCSKs) constitute a family of 7 related proteases (PCSK1-7) and 2 distant ones (MBTPS1, PCSK9), with unexplored role in cardiovascular disease (CVD), apart from PCSK9 in lipid metabolism. Here, we aimed to investigate the expression landscape and therapeutic targeting potential of the entire PCSK family for ameliorating CVD.

Methods:
An integrative approach was applied with genetic, transcriptomic and proteomic data mining from public databases and three independent vascular biobanks comprising carotid atherosclerosis, thoracic and abdominal aneurysms. Omics analyses were followed by gene expression association with patient clinical parameters and immunohistochemistry in vascular biopsies.

Results:
Genetic studies revealed that, apart from
PCSK4
, all PCSK family members lie in regions containing variants associated with human cardiovascular traits. Transcriptomic analyses showed that
FURIN, PCSK5, MBTPS1
were downregulated, while
PCSK6/7
were upregulated in atherosclerotic plaques
vs.
normal arteries. In abdominal aneurysms,
FURIN, PCSK5, PCSK7, MBTPS1
were downregulated, while
PCSK6
was enriched in diseased media. In thoracic aneurysms, only
FURIN
was significantly upregulated. To understand the mechanistic relationships of this protein family with the disease, network analyses of the upstream and downstream pathways related to PCSKs were done on the omics data from vascular biopsies. Immunohistochemistry indicated that PCSK protein levels correspond to the mRNA expression, except in the case of PCSK9 protein that was abundant in vascular biopsies. Correlation to clinical parameters in a carotid endarterectomy cohort revealed positive associations for
PCSK5/6/7
with adverse cardiovascular events.

Conclusions:
Our results show that
PCSK6
is the most enriched PCSK in plaques and abdominal aneurysms, while
FURIN
upregulation is characteristic for thoracic aneurysms. PCSK9 protein but not the transcript, was present in vascular lesions, suggesting its accumulation from circulation. Overall evaluation revealed that PCSK6 is the most attractive protease from this family to target for drug development in CVD.
Abstract 436: Loss Of Serine Hydroxymethyltransferase 2 In Macrophages Enhances Atherosclerosis While Reducing Macrophage Mitochondrial Respiration
Authors: Finney Alexandra C , Liu Yuhao , Yurdagul Arif , Rom Oren
Published: November 2022

Despite significant advancements in therapeutics and intervention, cardiovascular diseases remain the leading cause of death worldwide. Atherosclerosis, the accumulation of lipid-laden plaques in large arteries, is the underlying cause of most cardiovascular diseases. While the role of lipids is well-established in atherosclerotic cardiovascular disease, the contribution of amino acids is becoming increasingly recognized. Indeed, lower circulating glycine is consistently reported in patients with cardiovascular diseases. The mitochondrial serine hydroxymethyltransferase (SHMT2) is responsible for the bulk of glycine biosynthesis in most cell types. SHMT2 catalyzes the formation of glycine from serine and drives one-carbon metabolism necessary for mitochondrial protein translation. SHMT2 is critical in development since global knockout mice are nonviable. Previous studies showed patients with significant coronary artery disease exhibit a reduced glycine:serine ratio compared with healthy individuals. We discovered LPS-treated macrophages similarly attenuated glycine:serine concomitant with reduced SHMT2 expression. Moreover, we found SHMT2 is highly expressed in plaque-associated macrophages in both human and mouse atherosclerotic plaques. We hypothesized that SHMT2 regulates macrophage inflammatory phenotype in atherosclerosis through alterations in mitochondrial function. Deletion of SHMT2 reduced mitochondrial respiration while enhancing glycolysis similar to LPS-treated macrophages. In addition, SHMT2 deletion augmented oxidized-LDL accumulation in macrophages. To determine whether macrophage-expressed SHMT2 contributes to atherosclerosis, we applied CRISPR/Cas9 and Cre-lox recombination system to generate myeloid-specific SHMT2 knockout (

Shmt2
MKO

) mice. We further crossed

Shmt2
MKO

mice with Apoe knockout (
Apoe
-/-
) mice to study atherosclerosis following Western diet feeding. Compared to littermate controls (

Shmt2
fl

/

Apoe
-/-

),

Shmt2
MKO
/ Apoe
-/-

mice had significantly enhanced plaque area. Our findings, representing the first investigation of conditional SHMT2 knockout mice, suggest a novel immunometabolic role for SHMT2 in macrophages in atherosclerosis.
Abstract 439: Abcg5 Abcg8 Are Essential To Maintain Sterol Balance When Challenged With Dietary Cholesterol In Mice
Authors: Kaur Rupinder , Anspach Garrett B , Graf Gregory A
Published: November 2022

Background:
ABCG5/ABCG8 (G5/G8) is the primary biliary cholesterol transporter and accounts for >70% of biliary cholesterol secretion. Mice and humans maintain fecal cholesterol excretion under a variety of conditions that disrupt biliary cholesterol secretion, indicating the presence of a non-biliary route for cholesterol elimination. Cholesterol can be actively secreted from the intestine, a pathway termed transintestinal cholesterol excretion (TICE). The present study investigates rates of intestinal cholesterol secretion in mice lacking G5/G8 to determine if adaptive responses within the gut facilitate cholesterol excretion in the absence of the primary biliary cholesterol transporter.

Methods:
Wild-type (WT) and whole body
Abcg5/Abcg8
deficient mice (G5/G8 KO) were maintained on a plant sterol free (PSF) diet to prevent the development of experimental sitosterolemia and potential confounding effects of biologically active phytosterols. Mice were anesthetized, the gall bladder cannulated, and the intestine perfused with model bile to simultaneously determine biliary and intestinal cholesterol secretion rates. Sterol balance studies were conducted in mice sequentially maintained on PSF diet, a PSF diet supplemented with 0.2% cholesterol, and standard rodent chow. Mice were allowed to acclimate to each diet for 14 days, then placed on wire-bottom cages for fecal collection. At termination of the experiment, basal bile, plasma, and tissues were collected. Fecal neutral sterols (FNS) were analyzed by GC-MS.

Results:
Despite a substantial reduction in biliary cholesterol secretion in G5/G8 KO mice, differences in intestinal cholesterol secretion rates were only observed in males, with KO mice excreting less cholesterol into the intestinal lumen. FNS did not differ between genotypes when maintained on the PSF diet. On chow diet, FNS increased 2x in both genotypes, but did not differ across genotypes. When PSF diet was supplemented with 0.2% cholesterol, FNS increased by 3x in WT mice, but were unchanged in G5/G8 KO mice, relative to PSF diet levels.

Conclusions:
Abcg5/Abcg8 is essential to maintain sterol balance when cholesterol is present in the diet. TICE is unable to compensate for Abcg5/Abcg8 deficiency in cholesterol fed mice.