bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 172: Abcg1 Regulates Pulmonary Surfactant Metabolism in Mice and Men
Authors: de Aguiar Vallim Thomas Q , Lee Elinor , Merriott David J , Goulbourne Christopher N , Cheng Joan , Cheng Angela , Gonen Ayelet , Palldino Elisa N , Barmak Katayun , Ford David A , Wang Tisha , Baldan Angel , Tarling Elizabeth J
Published: July 2021

Inflammation is a hallmark characteristic of many diseases, including atherosclerosis and respiratory distress, and it is also a stereotypical response of the innate immune system to pathogens. Given the central role both inflammation and lipid homeostasis play in disease progression, defining the immune and lipid signaling pathways is key in identifying new strategies for therapeutic intervention. The ATP Binding Cassette (ABC) transporter ABCG1 is highly expressed in pulmonary type 2 cells, alveolar macrophages and immune cells. We, and others, have demonstrated that loss of ABCG1 attenuates atherosclerotic lesion progression, and profoundly impacts B cell and natural antibody homeostasis. ABCG1-deficient mice accumulate pulmonary surfactant, lamellar-body loaded type 2 cells, lipid-loaded macrophages, B-1 lymphocytes and immunoglobulins, clearly demonstrating the ABCG1 has a critical role in pulmonary homeostasis. We have identified a variant in the ABCG1 promoter in patients with pulmonary alveolar proteinosis, that results in impaired activation of ABCG1 by the liver X receptor alpha. We have generated mice that lack ABCG1 specifically in either type 2 cells or macrophages, and show that loss of ABCG1 from both cell types has specific effects on pulmonary lipid and immune homeostasis. These results establish a critical role for type 2 cell ABCG1 in controlling surfactant and lipid homeostasis in the lung and in the pathogenesis of human lung disease.
Abstract 18: APOBEC1 Complementation Factor: A Novel Regulator of Triglyceride Metabolism
Authors: Lin Jennie , Conlon Donna , Wang Xiao , Rader Daniel J , Musunuru Kiran
Published: July 2021

A recent human rare-variant study on plasma lipids has identified a missense mutation in the
A1CF
(APOBEC1 complementation factor) gene that significantly associates with TG levels. Expressed in the liver and small intestine,
A1CF
encodes an RNA binding protein that facilitates APOBEC1’s editing of
APOB
mRNA, introducing a premature stop codon that yields apoB-48. Surprisingly, despite the importance of apoB-48 in plasma lipid metabolism, prior studies have shown that
APOBEC1
deficiency itself does not alter plasma TG levels. Therefore, we hypothesize that A1CF regulates TG metabolism independently of its role in
APOB
mRNA editing. To evaluate this hypothesis, we used CRISPR-Cas9 to generate whole-body
A1cf
knockout mice (
A1cf
-
/
-
), knock-in mice homozygous for the TG-associated Gly398Ser mutation (
A1cf
GS/GS
), and
A1cf
-
/
-
rat hepatoma cell lines. Both

A1cf
GS/GS

and
A1cf
-
/
-
mice had significantly increased fasting plasma TG compared to controls (1.71-fold, N = 22-27,
P
= 1.5 х 10
-3
; and 1.67-fold, N = 15-27,
P
= 1.8 х 10
-3
respectively), recapitulating the human TG phenotype and demonstrating that A1CF loss of function leads to hyperTG. Supporting our hypothesis that this occurs independently of APOBEC1 function, apoB-48 was detected by immunoblot in the plasma and small intestines of
A1cf
-
/
-
and
A1cf
GS/GS
mice without a significant shift in apoB-100/apo-B48. TG clearance was not decreased in
A1cf
-
/
-
mice during an oral fat tolerance test (N = 12), but A1CF deficiency did result in increased TG secretion
in vivo
(1.34-fold, N = 12,
P
< 0.001), consistent with an observed increase in VLDL-apoB secretion by
A1cf
-
/
-
hepatoma cells (1.55-fold, P < 0.001). We performed RNA-seq of
A1cf
-
/
-
and wild-type mouse livers, and while differential expression analysis did not yield candidate mechanisms to explain the TG phenotype, we detected 167 alternative splicing (AS) events (delta PSI > 10). We validated several AS events for metabolism genes such as
Khk
and
Hmgcl
by RT-PCR, identifying A1CF’s previously unknown role as a regulator of AS events with clear metabolic relevance. In summary, we have demonstrated in our functional follow-up of a human rare-variant study that A1CF modulates TG metabolism through entirely novel mechanisms independent of
APOB
mRNA editing.
Abstract 182: Use of a Nanoparticle Delivery System to Rescue Macrophage Lysosomal Dysfunction in Atherosclerosis
Authors: Zhang Xiangyu , Vulugundam Guru R , Lanza Gregory , Pan Dipanjan , Razani Babak
Published: July 2021

Atherosclerotic vascular disease remains the leading cause of death in the United States with the majority of mortality due to coronary artery disease and myocardial infarction. Dysfunction in the macrophage lysosomal system, such as reduced acidity or diminished lysosomal degradative capacity, inhibits the clearance of excess cellular debris or atherogenic lipids present in atherosclerotic plaques and has emerged as an important contributor to plaque progression. Thus, devising strategies to rescue this macrophage lysosomal dysfunction is a novel therapeutic measure. Nanoparticles are subcellular spherical structures ranging from 1-100 nm in size and prepared from natural or synthetic polymers. Injected nanoparticles tend to undergo opsonization, a process which favors macrophage binding, internalization by phagocytosis, and trafficking to lysosomes. In order to use nanoparticles as a macrophage lysosome delivery platform, we developed acid-eluting PLGA (poly-Lactide-co-Glycolic acid) or PLA (polylactic acid) nanoparticles to both maintain lysosomal acidity as well as potentially deliver encapsulated lysosomal enzymes to macrophages in vitro and in vivo. We show that fluorescence-labeled PLGA nanoparticles can be successfully trafficked to lysosomes of murine bone marrow derived macrophages and rescue increases in lysosomal pH instigated by Bafilomycin A1, a disruptor of lysosomal proton gradient. This lysosomal acidification can also rescue the degradative capacity of macrophage lysosomes as gauged by enhanced proteolysis of fluorescent-conjugated ovalbumin. Finally, we demonstrate that acidic nanoparticles injected intravenously into mice can be internalized in splenic macrophages as well as macrophages of the atherosclerotic plaque. Taken together, our data support the use of this technology as a strategy to rescue macrophage lysosomal dysfunction in the treatment of atherosclerosis.
Abstract 187: Genetic Targeting at Apolipoprotein E Locus to Knockdown Scavenger Receptor Class B Type I in Mice: Characterization of a Novel Mouse Model of Coronary Heart Disease
Authors: Chen Hong , Hu Sheng , Huo Xing Y , Zhu Li Y , Wan Qing , Ma Xiao Y , Xu Chuan S , Hao Hui F , Jin Wei J , Wang Miao
Published: July 2021

Background:
Scavenger Receptor Class B Type I (SRBI) is the key player mediating HDL-dependent reverse cholesterol transport (RCT) in hepatocyte, and SRBI mutation causes coronary heart disease (CHD) in humans. Mice lacking SRBI and apolipoprotein E (apoE), fed on chow diet, develop cardinal features of CHD and succumb to premature death. However, the application of this model is limited due to the short lifespan, early onset of CHD symptom, the difficulty to maintain genetic mutations at two different loci, and infertility. The latter may be related to adrenal SRBI deficiency.

Methods and Results:
A genetic mouse model, named as SRBI
KD
ApoE
-/-
was produced by using CRISPR/Cas9 technique, in which an SRBI knock-down cassette (at both mRNA and proteins levels) is inserted downstream of the indogenous apoE promoter, thus abolishing apoE expression. Real-time PCR and Western blot characterization showed that SRBI expression in liver was significantly reduced (33% vs. 100% by RT-PCR, 0.5% vs. 100% by WB, P<0.001), but no significant difference was found in other examined tissues. SRBI
KD
ApoE
-/-
mice are fertile and do not exhibit pre-mature cardiovascular death on chow diet up to 10 months. Compared to ApoE
-/-
mice, SRBI
KD
ApoE
-/-
mice showed significant increase in serum HDL-C (3.11 vs 1.70 mmol/L, P<0.01), whereas serum triglyceride or LDL-C was similar (triglyceride: 0.87 vs. 0.9 mmol/L; LDL-C: 8.35 vs. 7.74 mmol/L). When treated with high-fat diet (HFD: 21% fat, 0.2% cholesterol in weight, n=6/group) for 8 weeks (starting at 8-week old), SRBI
KD
ApoE
-/-
mice gained significantly more weight than ApoE
-/-
mice(P<0.05 for females), and exhibited higher weight ratio of heart to body than that of ApoE
-/-
mice (females: 6.51% vs. 5.16%, P=0.051; males: 6.54% vs. 4.64%, P<0.01). Splenomegaly was observed more frequently in these mice than in ApoE
-/-
mice (females: 17.7% vs. 5.99%, P<0.001; males: 6.12% vs. 3.99%, P<0.01).
Abstract 201: Generation of Photoactivatable apoA I to Study HDL Transport in vivo Reveals Impaired HDL Recirculation in a Murine Model of Psoriasis
Authors: Huang Li-Hao , Zinselmeyer Bernd H , Chang Chih-Hao , Saunders Brian T , Kim Brian S , Wiig Helge , Thomas Michael J , Sorci-Thomas Mary G , Randolph Gwendalyn J
Published: July 2021

HDL is cardioprotective, but plasma HDL levels do not necessarily predict cardiovascular outcomes. The major HDL-associated protein apoA-I picks up its cholesterol from cells within extravascular compartments to return it to plasma and then bile. Yet, tools are lacking to quantify the important step of HDL transit through extravascular spaces. Here, we developed recombinant photoactivatable apoA-I to quantify endogenous HDL recirculation. Using the tool, we studied HDL passage through skin in healthy mice versus those with experimental psoriasis, wherein collagen density increased in the skin in a CD4
+
T cell-dependent manner. In control mice, photoactivated HDL mobilized to plasma within 2 h but was retained in collagen-enriched skin of mice with psoriasis. These data suggest that cardiovascular comorbidity in psoriasis might be linked to T cell-mediated structural changes in skin that impedes systemic recirculation of HDL. This new tool is likely to find wide application in HDL research.
Abstract 203: Role of Glucagon Receptor Signalling in PCSK9 Regulation
Authors: Spolitu Stefano , Ozcan Lale
Published: July 2021

Excessive glucagon receptor action in hepatocytes is a major contributing factor to type 2 diabetes (T2D). Accordingly, there has been great interest in developing glucagon receptor antagonists (GRAs) as a treatment for T2D. Although phase 2 clinical trials have shown that GRAs effectively lower blood glucose in T2D subjects, they increase plasma low density lipoprotein (LDL) cholesterol levels, which has presented a significant block to their development. In this context, recent studies have suggested that cholesterol and proprotein convertase subtilisin/kexin type 9 (PCSK9) levels can be regulated by fasting and perhaps glucagon, but in-depth mechanistic insight is lacking. In order to test the functional importance of hepatic glucagon action on lipid metabolism, we silenced glucagon receptor (GcgR) in obese mice using AAV8-H1-shGcgr to silence the receptor in hepatocytes. Consistent with previous reports, this treatment effectively lowered blood glucose in obese mice without a change in body weight. Moreover, GcgR silencing, like GRAs in humans, significantly increased plasma LDL cholesterol. In search for the mechanism, we found that inhibition of GcgR significantly lowered hepatic LDL-receptor protein levels and increased both hepatic PCSK9 and circulating PCSK9. To determine causation, we treated GcgR-silenced mice with a neutralizing monoclonal antibody against PCSK9 and found that this intervention restored hepatic LDL-receptor protein levels and prevented the increase in LDL cholesterol. Further mechanistic work revealed that GcgR silencing in hepatocytes did not increase
Pcsk9
mRNA. Rather, blocking GcgR increased the half-life of PCSK9 protein by suppressing signalling through exchange protein activated by cAMP 1 (Epac1). In particular, the ability of GcgR silencing to increase PCSK9 and suppress LDL receptor protein levels was mimicked by hepatocytes lacking Epac1. Thus, GcgR signalling through Epac1 appears to have critical effects on processes that regulate cholesterol metabolism through PCSK9. These new findings have important implications for the lipid metabolism effects of hepatic glucagon signalling in both normal physiology and metabolic disease, and for the development of safer GRA-like drugs to treat T2D.
Abstract 205: Inhibition of Angiopoietin-Like 3 Expression is a Potential Mechanism of Activation of Lipoprotein Lipase and of Lipid Lowering by Metformin
Authors: Lin Li , Burke Jamie R , Geldenhuys Werner J , Sadana Prabodh
Published: July 2021

Dyslipidemias associated with primary lipid disorders or secondary to abnormal glucose metabolism are commonly seen metabolic disorders in the clinic. Of special interest is the diabetic dyslipidemia and its growing prevalence. Interestingly, metformin, a commonly used anti-diabetic drug also has potent lipid lowering activity. However, the mechanisms associated with this metformin activity are poorly understood. A key protein regulating the plasma lipid homeostasis is the enzyme lipoprotein lipase (LPL) which is found lining the capillary endothelium in tissue capillary beds. LPL hydrolyses the core triglycerides (TGs) of chylomicrons and very-low density lipoprotein (VLDL). Angiopoietin-like 3 (ANGPTL3), a secretory protein derived from liver, inhibits the lipolytic activity of LPL, resulting in an increase in plasma TGs. We investigated whether metformin regulates LPL and in effect plasma lipids, via regulation of ANGPTL3 in the liver. A single dose (150mg/kg) treatment of metformin for twenty-four hours in mice resulted in an increase in the LPL activity in plasma. Since metformin is frequently used in type 2 diabetes which is characterized by insulin resistance, a condition compounded by dietary fatty acids, we studied the relationship between metformin and ANGPTL3 in a human liver cell line treated with palmitic acid. Treatment with palmitic acid mimicked the conditions of insulin resistance in the liver and elevated the expression of ANGPTL3 in the liver which was inhibited by co-treatment with metformin. Gene and protein expression assays revealed a potent inhibition of ANGPTL3 expression and secretion from the hepatocytes upon treatment with metformin. We examined the role of sirtuin 1 (SIRT1)/adenosine monophosphate activated kinase (AMPK) in the metformin regulation of ANGPTL3 and observed that even though activation of SIRT1/AMPK axis inhibited the expression of ANGPTL3, metformin inhibition of ANGPTL3 was independent of SIRT1/AMPK as revealed by pharmacological inhibition and gene knockdown approaches. Our studies suggest that a potential mechanism of lipid lowering activity of metformin is the inhibition of expression of ANGPTL3, which results in an increase in LPL activity and a lowering of plasma lipids.
Abstract 220: Obesity Drives the Decline in Large HDL Subspecies Among Male Adolescents
Authors: Shah Amy S , Heink Anna , Sexmith Hannah , Dolan Lawrence , Gordon Scott , Khoury Jane , Elder Deborah , Davidson W. Sean
Published: July 2021

We have shown that adolescents with type 2 diabetes have an altered HDL subspecies profile, characterized by a depletion of large apoE rich HDL particles. We have also shown a significant inverse relationship between this profile and arterial stiffness suggesting that the loss of these particles is associated with early atherosclerosis. We sought to evaluate known risk factors (that contribute to the depletion of large HDL subspecies in adolescents. We evaluated the contributions of obesity, insulin resistance and diabetes to loss of large HDL particles, in five adolescent groups (n=20 per group) with highly specific phenotypes. Insulin resistance was defined by fasting insulin levels and HOMA-IR Adolescent groups were: lean insulin sensitive (normal body mass index (BMI) with no insulin resistance; lean insulin resistant (normal BMI with insulin resistance), obese insulin sensitive (obese BMI with no insulin resistance), obese insulin resistant (obese BMI with insulin resistance), and with type 2 diabetes (obese with insulin resistance and type 2 diabetes by the American Diabetes Association criteria). Stored plasma from each participant was fractionated using gel filtration chromatography to isolate HDL subspecies. The mean age of the cohort was 17.9 ± 1.6 years. Groups did not differ in age and race (50% Caucasian, 50% African American, all male). Large rich HDL subspecies declined significantly across each group from lean insulin sensitive, to lean insulin resistant, to obese insulin sensitive, to obese insulin resistant to type 2 diabetes (p<0.0001). An inverse relationship was also seen for small HDL particles (p<0.0001). Medium size particles did not differ across groups. These data were confirmed by nuclear magnetic resonance spectroscopy. Obesity explained ~50%, insulin resistance ~ 25%, and diabetes ~10% of the decline in large particles between lean insulin sensitive to type 2 diabetes participants. Twenty percent remained unexplained. Obesity appears to drive the decline in large atheroprotective HDL particles in male adolescents. However, contributions of insulin resistance and type 2 diabetes are evident. Whether weight loss reverses this profile and has the potential to improve cardiovascular risk remains to be determined.
Abstract 235: Interaction Between a Macrophage Chemokine Receptor, CCR2, and Its Ligand Plays a Crucial Role in Macrophage Recruitment Aand Regulated Inflammation in Normal Wound Healing
Authors: Boniakowski Anna E , Kimball Andrew , Joshi Amrita , Allen Ron , Schaller Matt , Henke Peter , Moore Beth , Kunkel Steve , Gallagher Katherine
Published: July 2021

Background:
Wound healing in chronic diseases, such as type 2 diabetes (T2D), is impaired due to dysregulated inflammation. Innate immune cells, particularly macrophages, play a significant role in regulated inflammation following tissue injury. After injury, CCR2+ monocytes are recruited to the peripheral wound. This recruitment is mediated in part by the CCR2 ligand, CCL2. Thus, we hypothesized that the CCL2/CCR2 interaction is vital for normal wound healing and appropriate inflammation, and that this signaling cascade is impaired in T2D.

Methods:
CCR2
-/-
mice and littermate controls underwent 4mm hindlimb wounds, and wound closure was compared daily. Wound macrophages (CD3-CD19-NK1.1-CD11b+ cells) were analyzed on day 3 by flow cytometry for intracellular cytokine production. Adoptive transfer was performed using blood CD11b+ cells from WT C57BL/6 or CCR2
-/-
mice isolated by magnetic sorting and transferred into CCR2
-/-
mice via tail vein injection. Mice were then wounded and wound closure was compared between the two groups. C57BL/6 mice were maintained on normal or high fat diet for 12-14 weeks, wounds were created, and CD11b+ cells were isolated from wounds on day 2. ELISA for CCL2 was performed.

Results:
CCR2
-/-
mice showed significantly impaired wound healing on days 2-7 compared with littermate controls. Macrophages isolated on day 3 from wounds of CCR2
-/-
mice expressed significantly less inflammatory cytokines (IL-1β, TNF-α) by qPCR. Flow cytometry analysis revealed less Ly6C hi macrophages in the wounds, as well as macrophages that made significantly less IL-1β, NOS2, and TNF-α. When adoptive transfer was performed, wound healing was restored to normal in the mice that received WT compared to those that received CCR2
-/-
CD11b+ cells (P< 0.01). Since CCR2 is important for normal wound inflammation, and we have previously shown that inflammation is impaired in the diet-induced obese (DIO) mice, we examined CCL2 in DIO wounds. CCL2 was significantly decreased in DIO wound macrophages on day 2.

Conclusion:
Appropriate CCR2/CCL2 interaction plays a crucial role in macrophage recruitment and regulated inflammation in normal wound healing. Impairment in CCR2/CCL2 signaling may be responsible, in part, for delayed early inflammation in T2D.
Abstract 236: Carbon Monoxide Mediated Changes in Macrophages are Regulated by Vagal Activation
Authors: Salem Karim , Hong Guiying , Leake Andrew , Aggarwal Ankur , Tzeng Edith
Published: July 2021

Introduction:
Carbon monoxide (CO) has potent vasoproective effects in vivo through changes in macrophage (MΦ) function. Because of these marked changes, we examined the vagal cholinergic anti-inflammatory pathway in regulating the actions of inhaled CO. We previously showed that vagotomy inhibited the ability of inhaled CO to prevent intimal hyperplasia following arterial injury in rats. We now hypothesize that the anti-inflammatory effects of inhaled CO can be blocked through pharmacologic vagal inhibition.

Methods:
Sprague-Dawley rats (3 rats/group) were treated with inhaled CO (250 parts per million) for 1 hr or remained in room air. Some were injected with atropine (0.05 mg/kg SQ) 1 hr prior to and just prior to inhaled CO treatment. Peritoneal macrophages (MΦ) were collected 1 hr after CO treatment or comparable time period in control rats and were cultured overnight under standard conditions. Media from these cells were collected for Western blot analysis for high mobility box group 1 (HMGB1) and VEGF.

Results:
MΦ isolated from CO treated rats released increased levels of HMGB1 and VEGF into the media compared to MΦs from air treated rats (1.7 and 4 fold increase, respectively; Fig.). These molecules mediate the proendothelial actions of inhaled CO. MΦs isolated from atropine treated rats showed a mild reduction of HMGB1 and VEGF secretion. In contrast, MΦ from atropine and CO treated rats showed a significant inhibition of HMGB1 release (P<0.01 vs. all other treatment groups) and a trend toward significance in the reduction in VEGF release (Fig.).

Conclusions:
Inhaled CO activates vagal signaling to mediate changes in MΦ behavior. Inhibition of vagal signaling with atropine blocked the changes in MΦ induced by inhaled CO, confirming the importance of the vagus nerve in mediating the vasoprotective actions of CO. These findings suggest the potential ability to reproduce the therapeutic actions of CO through pharmacologic vagal stimulation.
Abstract 239: Parthanatos is Involved in Hydrogen Peroxide Induced Vascular Smooth Muscle Cell Death
Authors: Feldman Conner C , Zhou Ting , Phan Noel , Liu Bo
Published: July 2021

Objectives:
Oxidative stress underlies major vascular diseases including atherosclerosis and abdominal aortic aneurysm. Hydrogen peroxide (H2O2) is widely used to trigger oxidative stress in vitro for the study of apoptosis. However, we have previously shown that vascular smooth muscle cells (SMCs) respond to high concentrations (>1 mM) of H2O2 with necrosis. Traditionally regarded as incidental form of cell death, necrosis can occur through different mechanisms mediated by distinct intracellular signaling networks. The precise knowledge of death pathway is essential to the design of therapeutic strategy targeting cell death. The goal of the current study is to determine how H2O2 induces necrosis in SMCs.

Methods:
Mouse vascular aortic smooth muscle cell line, MOVAS, were treated with 3mM H2O2 for 2 hours, after which cell death was analyzed using flow cytometry and protein expression determined via western blot.

Results:
SMCs underwent apoptosis and necrosis in response to 0.3 and 3 mM H2O2, respectively. The 3mM H2O2 group died via a caspase-independent mechanism. Expression of common autophagy-associated proteins were unaffected. Additionally, different autophagy activators and inhibitors only moderately facilitated the pro-necrotic effect of H2O2. The H2O2-induced necrosis was not affected by necroptosis inhibitors including necrostatin-1s or by SiRNA silencing of necroptosis mediators RIP1, RIP3 and MLKL. Furthermore, ferroptosis and CypD inhibitors did not provide protection from necrosis induced by H2O2. In contrast, the necrotic response was attenuated by the PARP-1 inhibitor 3-aminobenzamide (37.10±13.72% vs 82.05±0.64%). Moreover, an
PARP1
siRNA also reduced necrosis. PARP-1 is the central mediator of a necroptosis mechanism called parthanatos.

Conclusions:
Our data demonstrates that parthanatos constitutes a major mechanism underlying the necrotic response to high concentrations of H2O2. Current studies delineate the involvement of parthanatos in myocardial ischemia/reperfusion injury, cardiovascular ailments, and atherosclerosis. The present study may provide a new perspective on targeting PARP-1 for the protection of SMCs and likely cardiac myocytes against oxidative stress.
Abstract 24: Abca1 Agonist Cs6253 Reverses Apoe4-Driven Alzheimer’s Disease With Concomitant Changes in Plasma and Brain Apoe And Apoj
Authors: Johansson Jan O , Boehm-Cagan Anat , Bielicki John K , Michaelson Daniel M
Published: July 2021

Background:
Apolipoprotein E4 (apoE4) is associated with perturbed lipid metabolism and elevated risk for CVD and Alzheimer’s disease (AD). Data suggests impact of both non-brain and brain apoE on risk to develop AD. Homozygosity for apoE4 increases AD risk approximately 10-fold. Brain apoE4particles are smaller and less lipidated than brain apoE2 or apoE3. ApoE is lipidated by ATP Binding Cassette A1 (ABCA1) and both proteins are synthesized predominantly in astrocytes. We hypothesized that enhancing apoE4 lipidation by an ABCA1 agonist, CS6253, would have AD therapeutic actions.

Methods and Results:
Astrocyte cholesterol efflux is highly responsive to stimulation by exogenous CS6253 peptide. Time-course showed that basal [3H]cholesterol efflux from cells to serum-free medium was compromised in apoE4 astrocytes. Incubating with peptide CS6253 (10 μg/mL) showed 6-fold improvement in [3H] cholesterol efflux to serum-free medium containing either apoE4 or E3 astrocytes. CS6253 crossed the BBB and was found to co-localize with astrocytes in hippocampus. CS6253 i.p. injection (20 mg/kg/48h for 6 weeks) in apoE4 Targeted Replacement mice significantly lowered apoJ in plasma and the brain. The distribution of serum apoE4 on HPLC to smaller lipoproteins was seen rendering similar profile to apoE3. Plasma apoA-I levels, cholesterol and triglyceride levels were unchanged. Lipidation of apoE in brain was compromised in apoE4 compared to apoE3 mice and CS6253 treatment increased apoE4 lipidation. CS6253 treatment prevented cognition decline and development of the brain AD pathological phenotype. CS6253 treatment decreased Aβ42 and P-tau and increased levels of VGluT1 and apoER2. Cognitive decline was fully prevented in apoE4 mice by CS6253 treatment as assessed by novel object recognition and Morris water maze.

Conclusion:
ABCA1 agonist treatment by CS653 resulted in improved apoE4 lipidation in cells and in mice. Six weeks treatment counteracted apoE4 driven AD with regard to phenotype and cognition. The data point to the potential therapeutic utility of CS6253 in apoE4 AD.
Abstract 242: MMP-3 Promotes SMC Transformation During Medial Artery Calcification
Authors: Lin Tonghui , Wang Xue-lin , Zettervall Sara , Cai Yujun , Flores Alyssa , Guzman Raul J
Published: July 2021

Objective:
Medial artery calcification is associated with increased cardiovascular morbidity and mortality. It occurs with diabetes and chronic kidney disease. We have previously demonstrated that the matrix metallopeptidase 3 (MMP-3) is strongly induced in arterial calcification. We also showed that MMP-3 inhibition decreases calcium accumulation in vascular SMCs and that MMP-3 deficient mice develop less medial calcification than wildtype controls. In this series of continuing experiments, we evaluate the effects of MMP-3 on SMCs phenotypic transformation in
vitro
and in
vivo.

Methods and Results:
Confluent rat aortic smooth muscle cells (RASMCs) cultured in calcification medium containing elevated calcium and phosphate levels for 7 days showed increased MMP-3 activity, decreased expression of the SMC markers SM-actin and SM-MHC, and increased expression of the bone markers alkaline phosphatase (ALP) and osterix (Osx). Cells were next exposed a selective MMP-3 inhibitor 2(EMD Millipore). In the presence of inhibitor, MMP-3 activity (MMP-3 activity assay kit, Abcam) was significantly decreased. Additionally, SMC osteogenic transformation was prevented as demonstrated by maintenance of SM22α and SM-MHC expression with reduction of ALP and Osx expression. In confirmatory experiments, MMP-3 reduction with siRNA inhibited the calcification of SMC that were exposed to calcification medium. We next evaluated phenotypic marker expression in MMP-3 knockout and wild-type mice injected with vitamin D
3
, a model of medial artery calcification. At 7 days after injection, expression of the SMC marker gene SM-MHC was significantly greater in MMP-3 KO mice than controls, while bone cell marker genes (Runx2, ALP, Osx) were decreased as measured by qPCR. Deletion of MMP-3 thus inhibited the osteogenic transformation of medial SMCs in vitamin D
3
-treated mice suggesting that it may control calcification via local effects within the arterial wall.

Conclusion:
Together these findings suggest that MMP-3 promotes medial artery calcification through local effects on the phenotypic state of vascular SMCs, and further, that it may serve as a therapeutic target to reduce calcification and improve outcomes in our PAD patient population.
Abstract 250: Four-And-A-Half LIM Domain Protein 2 Plays a Critical Role in Spleen T-Cell Dependent Antibody Response
Authors: Ebrahimian Talin , Simon David , Dierick France , Heidari Maryam , Orthwein Alexandre , Mann Koren K , Lehoux Stephanie
Published: July 2021

Background:
Four-and-a-half LIM domain protein 2 (FHL2) is an adaptor molecule that regulates signalling cascades and gene transcription. We have uncovered vasculoprotective and atheroprotective effects of FHL2 knockout in mice. Since B cells could regulate these processes, we investigated the potential role of FHL2 in B cell function and activity.

Methods and Results:
Under basal conditions, FHL2-/- mice presented a mild splenomegaly (84.0±9.0 vs 68.0±5.0mg), associated with bigger spleen follicles (1.6 fold) and higher proportions of spleen B cells (56.0±2.0 vs 40.0±9.0%) vs WT mice. Flow cytometry confirmed significantly higher (p<0.05) proportions of B cells in the follicles (Fo)(CD23+CD21lo/int) (77.0±1.0 vs 67.0±3.0%) and the marginal zone (Mz)(CD23-,CD21+)(7.0±1.0 vs 2.5±1.0%) of FHL2-/- vs WT mice. Mice were injected with sheep red blood cells (SRBC) to elicit a T cell-dependent B cell activation. SRBC did not influence Fo and Mz B cell numbers, but it did affect germinal center (GC) formation. GCs are zones within Fo where T cells activate B cells to produce high affinity antibodies. SRBC induced a 5- and 2.5-fold increase in B cells (GL7+) within FHL2-/- and WT GCs, respectively. However, there was no increase in plasma IgG1 levels in FHL2-/- mice in response to SRBC, whereas a 3-fold increase was observed in WT mice (1158±227 vs 2943±226ng/ml; p<0.05). Instead, plasma IgM levels were higher in FHL2-/- than WT SRBC mice. Moreover, SRBC only induced a modest, 1.3-fold fold increase in Fo T helper cells (CXCR5+PD1+) in FHL2-/- compared with a 6-fold increase in WT. Nevertheless, FHL2-/- B cells successfully proliferated and underwent class switch recombination in response to LPS, anti-CD40 and IL-4 in vitro, indicating that these cells were not defective.

Conclusion:
These results suggest that FHL2 plays a critical role in spleen antibody production in response to a T-dependent antigen, possibly by affecting activation or antigen presentation by Fo T helper cells.
Abstract 261: EBP50/NHERF-1 Regulates Basal and TNFα-induced Mitochondrial Dynamics in Vascular Smooth Muscle Cells
Authors: Procaccia Vera , Wallace Callen , Corey Catherine , St. Croix Claudette , Shiva Sruti , Bisello Alessandro
Published: July 2021

Mitochondrial dysfunction has been associated with the phenotypic switch of VSMC and vascular disease. Changes in mitochondrial dynamics (fission and fusion) are linked to VSMC proliferation and metabolism during vascular remodeling. Mitochondrial fission and fusion are regulated by several key molecules, including dynamin-related protein 1 (Drp1) and mitofusin2 (Mfn2). We previously showed that scaffolding Ezrin-radixin-moesin binding phosphoprotein of 50 kDa (EBP50/NHERF1) increases inflammatory responses and proliferation of VSMC. These actions are mediated by the activation of PKCζ under inflammatory stimuli and the stabilization of the S-phase kinase associated protein 2 (Skp2), a component of an E3 ligase that promotes proliferation. Thus, EBP50 knockout (EBP50
-/-
) mice are protected neointimal hyperplasia following arterial injury. Here we test the hypothesis that EBP50 regulates mitochondrial dynamics and responses to inflammatory stimuli in vascular smooth muscle cells (VSMC). We found that EBP50 knockdown, by decreasing Skp2 levels, increased FoxO1 stability and nuclear localization leading to higher mitofusin-2 (Mfn-2) expression. In contrast, inhibition of FoxO1 reduced Mfn2 levels. High resolution morphological analysis with both TEM and confocal microscopy revealed that mitochondria were more elongated in EBP50
-/-
than WT VSMC. In WT VSMC, TNFα induced PKCζ-mediated phosphorylation of Drp1. In contrast, EBP50
-/-
VSMC exhibited significantly reduced Drp1 phosphorylation following TNFα treatment. Live-cell 3D imaging followed by morphological analysis showed that TNFα elicited rapid and significantly greater mitochondria fragmentation in WT compared to EBP50
-/-
VSMC. Finally, EBP50
-/-
VSMC exhibited lower extracellular acidification rate (ECAR) than WT VSMC consistent with their lower proliferation. Collectively, these findings delineate a new mechanism of regulation of mitochondrial dynamics by the scaffolding protein EBP50 in response to inflammatory stimuli. Therefore EBP50 can be viewed as a potential therapeutic target for vascular proliferative diseases.
Abstract 264: Protein Disulfide Isomerase A1 is a Central Hub for Redox Regulation of VSMC Phenotype
Authors: Wosniak Joao , Gonçalves Renata C , Tanaka Leonardo Y , Zanatta Daniela B , Strauss Bryan E , Laurindo Francisco R , Fernandes Denise C
Published: July 2021

Objective:
Vascular smooth muscle cell (VSMC) phenotype switch depends on extrinsic/intrinsic cues including NOX NADPH oxidase-linked redox signaling. Growth factor-triggered NOX1 expression/activity requires the chaperone oxidoreductase protein disulfide isomerase-A1 (PDI). Acute PDI overexpression induces agonist-independent NOX1 expression. PDI is required for VSMC migration and cytoskeleton organization, and extracellular PDI supports expansive vascular remodeling via cytoskeleton reshaping. Such PDI effects led us to hypothesize that PDI may orchestrate VSMC phenotypic alterations.

Approach and Results:
In primary VSMC, PDI silencing spontaneously decreased differentiation marker expression. Transfection with a doxycycline-inducible lentiviral vector encoding PDI showed that sustained PDI overexpression (72h) enhanced actin branching pattern vs. baseline (anisotropy index, 0.103±0.019 vs. 0.220±0.027, 72h vs. 0h, mean±SEM, N=5, P<0.05), increased cell length and induced expression of differentiation marker calponin (2-fold, 72h vs 0h, N=5, P<0.05), alpha-actin and smoothelin, which were abrogated upon catalase incubation. Intracellular superoxide enhanced upon 48h of PDI overexpression (2-hydroxyethidium levels, 0.998±0.102 vs. 2.887±0.227 AU, N=4, P<0.05) and was NOX1-dependent, based on inhibition with GKT136901 (by 48%) or NOXA1ds peptide (by 54%) (N=6, P<0.05). Increased NOX1 mRNA occurred early after PDI overexpression (74%, 24h vs. 0h, N=4, P<0.05), while NOX4 mRNA was upregulated only after long-term PDI induction (100%, 72h vs. 0h, N=4, P<0.05). In rabbit restenosis model exhibiting strong PDI upregulation (12-fold at day 14 after injury), ex vivo PDI silencing 7 or 14 days after injury reversed PCNA expression, while promoting increased NOX1 and decreased NOX4 mRNA levels (+54% and -45%, respectively, siPDI vs. siSCR, N=3, P<0.05 for both).

Conclusions:
While short-term PDI overexpression supports NOX1 activation/expression, sustained PDI overexpression drives NOX4 expression and VSMC differentiation. Effects on cytoskeleton, NOX1/4 activation and temporal control of NOX1/4 expression suggest a central role for PDI as a hub for redox-mediated VSMC phenotype regulation.
Abstract 29: Chronic Stress Predisposes to Thrombosis by Abnormal Megakaryopiesis: Protective Effect of Apocynin
Authors: Sandrini Leonardo , Ieraci Alessandro , Turra Elisa , Amadio Patrizia , Popoli Maurizio , Tremoli Elena , Barbieri Silvia Stella
Published: July 2021

Introduction:
Psychological stress (e.g. anxiety and depression) has been identified as an important trigger of acute coronary syndromes (ACS), as a consequence of enhanced coagulation and of hyper-reactive platelets. Changes in redox balance, alteration of genes regulating antioxidant systems, including NADPH oxidase, and increased production of reactive oxygen species (ROS) have been measured in both chronic stress and ACS. However, the mechanisms by which chronic stress affects platelet activation and predisposes to thrombosis are not well known.

Hypothesis:
We hypothesized that Apocynin, an inhibitor of NADPH oxidase influences the alteration of megakaryopoiesis and activation of platelets induced by chronic stress in mice.

Methods and Results:
We show the NADPH/NADP
+
ratio in bone marrow (BM) of mice exposed to forced swimming for 4 days (5 min twice/day) is markedly reduced compared to control mice, and that Apocynin treatment (2.4 mg/ml in drinking water for 4 days) prevents this alteration. Chronic stress leads to an abnormal megakaryopoiesis increasing the number of BM megakaryocytes (MKs) and affecting circulating platelets. MKs of stressed mice show an advanced maturation state (e.g. nuclear/cytoplasmic ratios and expression of CD42d), and an enhanced ability to produce ROS. Interestingly, a higher number of large and reticulated platelets with marked functional activation (e.g. integrin α
IIb
β3 and P-selectin expression, and platelet/leukocyte aggregates) is detected after chronic stress. In addition, Apocynin prevents ROS MKs generation and decreases the total number of MKs without affecting the percentage of CD42d
+
cells. Finally, the inhibitor of NADPH oxidase activity reduces the hyper-activation of platelets and the enhanced susceptibility to FeCl3-induced arterial thrombosis in stressed mice.

Conclusion:
Apocynin treatment, reducing ROS generation in MKs, restores the physiological bone marrow megakaryopoiesis and platelet behaviour, and it prevents the detrimental effect of chronic stress on atherothrombosis. These data suggest a potential use of NADPH oxidase inhibitors in the occurrence of thrombosis associated with chronic stress. Studies in human will verify the clinical impact of these findings.
Abstract 303: Increased Endogenous Hydrogen Sulfide Protects Against Vein Graft Disease
Authors: Trocha Kaspar , Tao Ming , Longchamp Alban , Macarthur Michael , Mitchell James , Ozaki C. Keith , R. de Vries Margreet
Published: July 2021

Objective:
Vein graft failure secondary to intimal hyperplasia remains a challenge. Hydrogen sulfide (H
2
S) is produced endogenously by cystathionine γ-lyase (CGL) in endothelial cells, and as a gasotransmitter holds numerous beneficial vascular effects. We thus hypothesized that increased endogenous H
2
S would attenuate the vascular response to injury. Here, we leveraged a CGL transgenic overexpressing mouse model to test the potential of increased endogenous H
2
S to attenuate the vascular response to injury in a vein graft model.

Approach and Results:
A murine carotid-interposition cuff technique vein graft model was employed, including an artificial bacterial chromosome-based CGL transgenic (Tg) strain with an extra copy of the CGL gene locus randomly inserted in the genome. CGL
Tg
mice were fed a high-fat/high-cholesterol diet, implanted with a vein graft from CGL
Tg
donors (n=8), and compared to wild-type (WT) controls (n=7; WT/ WT conduits); all on C57BL/6 background. Grafts were imaged
in vivo
with ultrasound biomicropscopy and harvested after 28 days. CGL
Tg
mice demonstrated an approximate two-fold increase in serum H
2
S production capacity (lead acetate assay) compared to controls. The CGL
Tg
mice exhibited a significant decrease in their intimal thickness (p=<0.05, Fig.A) and intimal/medial+adventia ratios (p=<0.05, Fig.B) 28 days after implantation.
In vivo
biomicroscopy was supportive: CGL
tg
mice had a larger mean luminal diameter relative to WT controls (p=<0.05, Fig.C).

Conclusion:
Elevated endogenous H
2
S production reduces the fibroproliferative response to vein graft arterialization. Manipulation of this gasotransmitter’s biology stands as a novel approach to impact the durability of vascular reconstructions.
Abstract 310: ROS Induced Mitochondrioal Dysfunction Causes Endothelial Dyfunction by Downregulation Of SIRT1
Authors: Kim Cuk Seong , Choi Su-jung , Nagar Harsha , Piao Shuyu , Kim Seonhee , Lee Ikjun , Kim Sung Min
Published: July 2021

Mitochondrial dysfunction has emerged as the major contributing factor in endothelial dysfunction and vascular disease. However, the key mechanism of mitochondrial dysfunction induced endothelial dysfunction has not to be clear identified. To determine whether mitochondrial dysfunction in endothelial cells play a key role in vascular disease, the phenotype of endothelial specific CRIF1 knock out (Crif1
-/-
) mouse and pathophysiological mechanism in vitro were examined. Crif1
-/-
mouse showed a lower body weight and cardiac hypertrophy. Down-regulation of CRIF1 in vascular endothelial cell caused disturbances in the mitochondrial OXPHOS (oxidative phosphorylation) complexes, mitochondrial morphology as well as function leading to an increased mitochondrial reactive oxygen species (ROS) production and mitochondrial membrane potential (MMP). In addition, down-regulation of CRIF1 also caused a decrease in Sirt1 expression along with increased eNOS acetylation leading to reduce NO production. Similar results were obtained in
in vivo
studies using the mouse with CRIF1-deficient vascular endothelial cell. Importantly, endothelium dependent vasorelaxation (EDR) of aortic rings from CRIF1 knock out mouse was considerably less as compared to the wild type counterparts. Also, the EDR was partially recovered following the adSirt1 treated aortic rings from mouse with Crif1-deficient vascular endothelial cell. Taken together, these findings indicate that CRIF1 plays an important role in the maintenance of mitochondrial function and endothelial function via the SIRT1-eNOS pathway.
Abstract 322: Cost Effectiveness Analysis of Peripheral Arterial Disease Screening With the Ankle-Brachial Index Test
Authors: Itoga Nathan K , Minami Hataka , Chelvakumar Meena , Pearson Keon , Mell Matthew W , Bendavid Eran , Owens Douglas K
Published: July 2021

Introduction:
Screening for asymptomatic PAD (aPAD) with the ankle-brachial index (ABI) test may reduce mortality and disease progression by identifying individuals who may benefit from early initiation of cardiovascular (CV) risk reduction therapies.

Methods:
Using a Markov model, we evaluated the cost-effectiveness of initiating medical therapy (e.g. statin & ACE-inhibitor) after a positive ABI screen in adults 65-years old. We modeled progression to symptomatic PAD (sPAD) and CV mortality with and without screening evaluating quality adjust life years (QALY). Cost of the ABI test, physician visit, new medication, and surgery for sPAD were calculated. Our baseline model estimated 96% of patients already eligible for medical therapy given the similar risk factor profiles of aPAD and CV disease. Repeated screening was considered given the imperfect screening test, development of disease with age, and opportunity to re-initiate therapy given limited medication compliance. Variables with uncertainty underwent a tornado analysis to determine variables with large effects.

Results:
Our model found an incremental cost of $367 and incremental QALY of 0.0022 with one-time ABI screening resulting in an incremental cost-effectiveness ratio (ICER) of $169,025/QALY over a 35-year period. Removing the benefits of medication on CV mortality increases the ICER by 51%, and removing the benefits of medication on PAD progression increases the ICER by 16%. A tornado diagram shows variables affecting the ICER (Figure). Screening high-risk populations, such as tobacco users where the prevalence of PAD may be 2.5x higher than the general population of 9%, decreases the ICER to $63,500/QALY.

Conclusions:
Our cost-effective analysis predicts that one-time ABI screening does not meet generally accepted thresholds for cost effectiveness. Disease prevalence and medication adherence had the largest effects on the ICER and are important to consider in implementing ABI screening.
Abstract 332: Inhibition of the Enhancer of Zeste Homolog Family Mitigates Intimal Hyperplasia in Rat Carotid Arteries
Authors: Zhang Mengxue , Wang Bowen , Urabe Go , Shi Xudong , Guo Lian-Wang
Published: July 2021

Rationale:
The enhancer of zeste homologs 1 and 2 (EZH1 and EZH2) are histone-lysine N-methyltransferases that modify histones by methylation which leads to transcriptional repression. It has recently been shown that EZH1 and EZH2 play an important role in various cancers. Whether they play a role in recurrent vascular diseases is not known.

Objective:
We assessed whether EZH1 and EZH2 are important contributors to the development of intimal hyperplasia (IH) and restenosis.

Methods and Results:
Following rat carotid balloon angioplasty, EZH2 showed highest expression at day 3 post procedure and then decreased at day 7 in injured arteries, as determined by Western blotting. Change of EZH1 is less prominent. Dual inhibition of EZH1 and EZH2 through peri-adventitial administration of a selective inhibitor, UNC-1999, effectively inhibited intimal hyperplasia, with ~40% reduction in the ratio of intima to media (I/M). Moreover, in cultured primary rat smooth muscle cells (SMCs) as well as MOVAS cells, pretreatment with 5μM UNC-1999 resulted in a 60% decrease in cell proliferation and ~80% reduction of migration that were stimulated by PDGF-BB. Simultaneous knockdown of EZH1 and EZH2, as well as knockdown of their shared scaffold protein(EED), led to effective inhibition of proliferation and migration of SMCs. However, knockdown of EZH2 alone did not recapitulate the effects of dual inhibition of EZH1 and EZH2.

Conclusions:
This study demonstrates that dual inhibition of EZH1 and EZH2, both histone modifiers, mitigates intimal hyperplasia in vivo and attenuates PDGF-BB stimulated SMC proliferation and migration in vitro.