bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 549: High-Density Lipoprotein from Valvular Heart Disease and Cardiac Surgery Uncouples Endothelial Nitric Oxide Synthase Activity to Impair Vasodilation
Authors: Ou Jingsong , Chang Feng-Jun , Yuan Hai-Yun , Hu Xiao-Xia , Ou Zhi-Jun , Fu Li , Lin Zhe-Bang , Wang Zhi-Ping
Published: March 2022

Background:
The vascular effects of high density lipoprotein (HDL) may be highly heterogeneous. Normal HDL protects vascular function. Vascular function plays an important role in maintaining the circulation stable post-cardiac surgery. The vascular effects of HDL in valvular heart disease (VHD) and cardiac surgery remain unknown. The present study is to determine the vascular effects of HDL from VHD and cardiac surgery.

Methods:
HDL was isolated from healthy subjects and patients with VHD and during cardiac surgery. HDL inflammation index was measured by cell free assay. Effects of HDL on vasodilation, protein interaction, generation of nitric oxide (NO) and superoxide anion (O
2
·–
) were determined.

Results:
HDL inflammation index significantly increased in VHD patients and post-cardiac surgery. HDL from VHD patients and post-cardiac surgery significantly impaired endothelium-dependent vasodilation, inhibited both Akt and endothelial nitric oxide synthase (eNOS) phosphorylation, eNOS associated with heat shock protein 90, NO production and increased O
2
·–
generation.

Conclusions:
The vasoprotective effects of HDL from VHD patients and cardiac surgery is impaired by uncoupled eNOS activity. HDL may be a therapeutic target for maintaining the stable circulation in post-cardiac surgery.
Abstract 556: Soluble TREM-Like Transcript-1 Differentially Responds to PAR1 and PAR4 Signaling: A Possible New Role for sTLT-1 in Angiogenesis?
Authors: Ferrer-Acosta Yancy , Lugo-Cintron Karina M , Morales Jessica , Washington Anthony V
Published: March 2022

Platelets have been shown to play important roles in angiogenesis due in part to the differential release of proteins from their α-granules. For example PAR-1 receptor activation in platelets releases α-granules containing pro-angiogenic molecules, while PAR-4 receptor activation promotes the release of anti-angiogenic molecules. Studies from our laboratory reported the identification of the Triggering Receptor Expressed in Myeloid cells (TREM)-Like 1 transcript (TLT)-1, a protein preferentially expressed in platelets and megakaryocytes. TLT-1 is stored in platelet α-granules with p-selectin and has a soluble extracellular fragment that is released upon platelet activation. Comparative immunofluorescence (IF) to p-selectin shows that only ~50% of the TLT-1 colocalized with p-selectin suggesting that TLT-1 may be differentially stored and dispersed from α-granules. To address this possibility we compared colocalization of TLT-1 to VEGF and endostatin, two proteins that are stored segregated into granules differentially. IF staining demonstrated greater colocalization with pro-angiogenic VEGF than anti-angiogenic endostatin. We then hypothesized that pro- or anti-angiogenic PAR-1 & PAR-4 signaling would differentially affect TLT-1 dispersion in platelets. To test this, purified human platelets were stimulated with a PAR-1 or a PAR-4 agonist and TLT-1 was monitored by IF. Results showed that PAR-1 activation caused a greater TLT-1 dissemination throughout the platelet than PAR-4. Supernatants of platelets activated by PAR-1 showed an increased amount of sTLT-1 compared to those activated by PAR-4 agonist. Our results suggest that TLT-1 is associated to platelet release of pro-angiogenic α-granules. We next addressed the question if sTLT-1 has an effect on endothelial cells. Soluble TLT-1 was added to primary endothelial cells 30 minutes and cells were evaluated for changes in size and actin polymerization. Studies by IF showed an increase in the size and actin polymerization of cells, suggesting it promotes a pro-angiogenic morphology. Ongoing experiments are addressing if sTLT-1 affects tube formation and migration of endothelial cells. These results could shed light into TLT-1 properties and its possible new role in angiogenesis.
Abstract 559: Machinery for Vitamin D Metabolism in Porcine Adipose--Derived Mesenchymal Stem Cells
Authors: Llamas Yovani , Pankajakshan Divya , Agrawal Devendra K
Published: March 2022

Background:
There is increasing evidence that vitamin D deficiency is an independent risk factor for cardiovascular disease. Cell-based therapy using adipose derived mesenchymal stem cells (MSCs) is an attractive option for endothelial layer regeneration post-angioplasty procedures. Vitamin D levels in circulating blood might regulate MSC based re-endothelialization of injured arteries. To our knowledge, the presence of vitamin D machinery in porcine MSCs is not yet reported. In this study, we investigated whether or not MSCs possess vitamin D machinery, including the expression levels of vitamin D receptor (VDR), vitamin D metabolizing enzymes (CYP24A1 and CYP27B1) after in vitro stimulation with active vitamin D, calcitriol.

Methods and Results:
MSCs isolated from porcine adipose tissue were characterized by positive staining for MSC markers CD44, CD73, CD90, and negative staining for macrophage marker CD11b and hematopoietic stem cell markers, CD34 and CD45; and by tri-lineage differentiation to osteocytes, chondrocytes, and adipocytes. No cytotoxicity was observed when MSCs were stimulated with 0.1-10nM calcitriol. The MSCs were analyzed for mRNA and protein expression of CYP24A1, CYP27B1 and VDR by immunostaining, qPCR and ELISA (n=6). A significant increase (p<0.01) in mRNA transcripts of CYP24A1, CYP27B1 and VDR was observed after stimulation of MSCs with calcitriol (10nM). The in vitro time-dependent effect of calcitriol (10nM) on the vitamin D machinery on cultured MSCs was determined by qPCR after stimulation for 0, 3, 6, 12, and 24h. The VDR and CYP27B1 expression peaked at 3h, and CYP24A1 at 24h, respectively. The in vitro biosynthesis of 1,25(OH)2D3 by ADMSCs was analyzed by ELISA and Western blot. The expression of the active form of vitamin D was significantly decreased (p<0.01) upon inhibition of CYP enzymes.

Conclusion:
Porcine MSCs possess vitamin D hydrolases and VDR to metabolize and respond to vitamin D. Hence, in vivo circulating 25-hydroxy vitamin D levels may have a significant role in regulating the differentiation of adipose MSCs into endothelial cells, and thus could be helpful in stem cell based re-endothelialization post-angioplasty.
Abstract 56: S14161 Inhibits Platelet Activation and Thrombus Formation via PI3K Signaling Pathway
Authors: Yi Wenxiu , Mao Xinliang , Zhu Li
Published: March 2022

The phosphatidylinositol 3-kinase (PI3K) signaling pathway is critical in modulating platelet functions. In the present study, we evaluated the effect of S14161, a recently identified pan-class I PI3K inhibitor, on platelet activation and thrombus formation. Results showed that S14161 inhibited human platelet aggregation induced by collagen, thrombin, U46619, and ADP in a dose-dependent manner. For example, the maximal aggregation induced by collagen were decreased by 32.7% (P<0.05), 59.4% (P<0.001) and 89.2% (P<0.001) (IC50=3.79±1.17 μM) when preincubated with 2.5, 5 and 10 μM of S14161, respectively. Interestingly, platelet aggregates were disaggregated after the peak of aggregation induced by ADP when platelets were pretreated with S14161 (10 μM). S14161 also inhibited platelet aggregation induced by GPVI selective ligand convulxin and CRP, PAR1 agonist SFLLRN, and calcium ionophore A23187. Flow cytometric studies showed that S14161 inhibited convulxin-induced P-selectin expression and fibrinogen binding of single platelet. S14161 also inhibited platelet spreading on fibrinogen and clot retraction, processes mediated by outside-in signaling. Using a microfluidic chamber we demonstrated that S14161 decreased platelet adhesion on collagen-coated surface by about 80%. Western blot showed that S14161 inhibited phosphorylation of Akt at both Ser473 and Thr308 sites, and GSK3β at Ser9 in response to collagen, thrombin, or U46619. Additionally, S14161 inhibited ERK1/2 activation. Finally, the effects of S14161 on thrombus formation in vivo were measured using a ferric chloride-induced carotid artery injury model in mice. The intraperitoneal injection of S14161 (2 mg/kg) to male C57BL/6 mice significantly extended the first occlusion time (5.05±0.99 min, n=9) compared to the vehicle controls (3.72±0.95 min, n=8) (P<0.05), but did not prolong the bleeding time (P>0.05). Taken together, our data showed that S14161 inhibits platelet activation and thrombus formation, and may be developed as a novel therapeutic agent for the prevention of thrombotic disorders.
Abstract 561: Human Stem Cell--Derived Models of Endothelial Cell Morphogenesis: Label-Free Analysis of Invasion, Migration and Sprouting Angiogenesis
Authors: Belair David , Carlson Coby , Abassi Yama , Irelan Jeff , Mann David
Published: March 2022

Endothelial cell (EC) morphogenesis during angiogenesis is a highly dynamic process tightly regulated by growth factor activity in the extracellular milieu.

Current
in vitro
models of EC morphogenesis employ endpoint analyses of primary endothelial cells, such as HUVECs. These vascular cells can exhibit functional variability between donors and methods for measuring morphogenesis in response to soluble cues typically employ potentially destructive labeling and thus fail to capture the full complexity of processes involved in EC morphogenesis. Here, we describe use of human induced pluripotent stem cell derived endothelial cells (iPSC-ECs) in a real-time electronic cell sensor impedance array technology to monitor invasion and migration behavior. Additionally, an image processing algorithm was developed to assess sprouting behavior from aggregated iPSC-EC organoid cultures.

iPSC-EC responses on the impedance platform assessed the impact of serum, growth factors (
e.g.
VEGF, FGF-2), and small-molecule angiogenesis inhibitors (
e.g.
Sunitinib, SU5402) on the proliferation, migration, and invasion of iPSC- ECs compared to primary HUVECs.

Our results indicated that iPSC-ECs were more sensitive to inhibition of VEGF mediated invasion by SU5402 than HUVECs with EC50s of 4.4nM and 99nM, respectively.

In addition, matrigel invasion toward VEGF was not observed appreciably with HUVECs, whereas iPSC-ECs demonstrated a marked invasive phenotype that could be inhibited by Nocodazole at 1nM. Using a novel method to monitor the sprouting behavior of the iPSC-ECs in an aggregated organoid into matrigel by HCA and image processing, IC50s for SU5402 (2.7nM) and Nocodazole (0.4nM) were determined.

The use of iPSC-derived endothelial cells provides a robust and reproducible source of cells that outperform HUVECs. The real-time monitoring of cellular processes offers important advantages over traditional end-point assays - specifically, the ability to measure receptor activation along with morphologic and adhesive remodeling of EC upon growth factor activation and inhibition. The combination of iPSC- ECs together with real time monitoring systems provides a more biologically relevant human model system for vasculogenesis investigations.
Abstract 564: Adventitial Human Mesenchymal Stem Cells Transplantation Reduces Venous Neointimal Hyperplasia in an Experimental Murine AVF Model
Authors: Yang Binxia , Brahmbhatt Akshaar , Nieves-Torres Evelyn , McCall Deborah , Mukhopadhyay Debabrata , Dietz Allan B , Misra Sanjay
Published: March 2022

Introduction:
Arteriovenous fistulas (AVFs) used for hemodialysis vascular access will fail because of venous neointimal hyperplasia (VNH) that causes stenosis formation at the venous outflow tract. As a result, only 60% of all AVFs are patent at 1-year. There are several factors which have been hypothesized to contribute to VNH including shear stress, hypoxia, and both local and systemic inflammation. Mesenchymal stem cells (MSCs) have been demonstrated to reduce inflammation while having antiproliferitive properties that aid abrogating vascular injury.

Hypothesis:
We hypothesized that adventitial transplantation of human adipose derived MSCs to the outflow vein of AVF created in CD1-FoxN1 would attenuate VNH failure by reducing pro-inflammatory mediators including tissue necrosis factor alpha (TNF-α), monocyte chemotactic protein-1 (MCP-1), and interleukin-1 beta (IL-1β).

Methods:
Adventitial transplantation of human MSC was performed in CD1-FoxN1 mice. A carotid artery to ipsilateral jugular AVF was placed and animals were divided into three groups: MSCs (M), CorMatrix ECM scaffold (S, CorMatrix Scaffold, Roswell, GE), or scaffold embedded with 250,000 MSCs (S+M) which were delivered to the adventitia. The mice were then sacrificed at 7 and 28 days post AVF placement and the outflow veins were harvested for histomorphometric and qPCR expression analysis.

Results:
The average lumen vessel area was significantly increased in the outflow veins removed from the S + M group compared to S or M alone at days 7 and 21 (both, P<0.05). The average area of neointima was significantly decreased in the S+M group when compared to S or M groups (P<0.05) while cell density and Ki-67 index were also significantly decreased at day 7 between S and S+M groups (P<0.05). RNA expression analysis revealed that MCP-1, IL-1β, and TNF-α were all significantly decreased in the outflow vein removed from the S+M group when compared to the S or M group (P<0.05).

Conclusion:
Adventitial transplantation of MSCs to the outflow vein of the AVF at the time of placement attenuates VNH mediated through a reduction of MCP-1, IL-1β, and TNF-α.
Abstract 58: Delayed Atherosclerosis in a Mouse Model of Bernard-Soulier Syndrome is Independent of Glycoprotein Ibα Extracytoplasmic Domain Deficiency
Authors: Koltsova Ekaterina , Sundd Prithu , Zarpellon Alessandro , Ruggeri Zaverio M , Ley Klaus
Published: March 2022

The pathogenesis of atherosclerosis involves the interplay of blood, stromal and endothelial cells; platelet interactions with vascular endothelium and leukocytes promote atherosclerosis. Glycoprotein (GP) Iba is the ligand-binding subunit of the platelet GPIb-IX-V adhesion receptor complex; its deficiency causes the Bernard-Soulier syndrome (BSS), characterized by absent platelet GPIb-IX-V, macrothrombocytopenia and bleeding. We found that
Ldlr-/-
mice reconstituted with
GPIb
a
-/-
as compared to wild type control developed delayed atherosclerosis associated with reduced platelet binding to blood myeloid cells and reduced accumulation of CD11b
+
and CD11c
+
myeloid cells in the aortas. Live imaging in whole blood-perfused microfluidic chambers revealed reduced platelet-monocyte aggregates in
GPIb
a
-/-
mice, which also showed decreased TNF in blood monocytes along with decreased TNF and IL12p35, but enhanced arginase1 in aortas. In contrast,
Ldlr-/-
mice reconstituted with chimeric IL-4R/
GPIb
a-Tg bone marrow produce platelets expressing GPIb-IX-V without the GPIba extracytoplasmic domain but less abnormal with respect to size and count and showed atherosclerotic lesion sizes similar to control mice. In conclusion, reduced platelet interactions with myeloid cells and delayed onset of atherosclerosis are not caused by defective GPIba-ligand binding but may result from the low platelet count and, possibly, other functional defects of BSS platelets.
Abstract 589: Correction of Long-Chain Omega-3 Fatty Acid Deficiency With a Unique Omega-3 Formulation is Associated With a Significant Reduction in Atherogenic Index of Plasma in Hypertriglyceridemic Patients: Results of the VASCAZEN-REVEAL Trial
Authors: Shaikh Nisar A , Yantha Jason , Shaikh Sabah , Laidlaw Maggie , Rowe William , Ali Abbas , Jackowski George
Published: March 2022

Background:
A large number of studies have suggested that the triglyceride/high density lipoprotein cholesterol (TG/HDL-C)) ratio provides useful information about atherogenicity of plasma (AIP) and that a higher ratio has been linked to increased incidence of myocardial infarction. Others have suggested the use of AIP (log ratio of molar concentrations of TG to HDL-C) as a significant predictor of cardiovascular risk and to identify insulin resistant individuals at high cardio-metabolic risk. In this report, we analyzed data from the VASCAZEN-REVEAL trial to investigate if the correction of Omega-3 fatty acid deficiency with a unique Omega-3 formulation (referred to as 6:1 OM-3) in 6:1 ratio of eicosapentaenoic (EPA) and docosahexaenoic (DHA) acids affects TG/HDL-C and AIP indices in hypertriglyceridemic subjects.

Methods:
A double blind, placebo-controlled study of 42 ambulatory cardiovascular subjects with elevated baseline TG levels (200-500mg/dL) were treated with either placebo (corn oil, 4g/day, n=22) or with 6:1 OM-3 (4g/day; n=20) for 8 weeks. Primary endpoint: Correction of OM-3 deficiency by determining changes in the Omega Score (OS, constituting blood concentration of EPA, DHA and DPA-docosapentaenoic acids). Secondary endpoints: changes in the serum lipid profile including TG and HDL-C.

Results:
Eight weeks of treatment with 6:1 OM-3 resulted in a significant increase in placebo adjusted median OS (+120.8%, p<0.0001) with concomitant reduction in TG/HDL (-50.3%, p=0.0005), AIP (-46.9%, p< 0.0006) and in AA (arachidonic acid): EPA (-86.6%, p<0.0001) ratios. No significant changes in OS (-2.0% p= 0.5519), TG/HDL (+4.72%, p=0.5233), AIP (+2.43%, p=0.8119) or in AA/EPA (11.3%, p=0.6780) ratios were observed in placebo. Treatment with 6:1 OM-3 also resulted in a significant increase in placebo adjusted median levels of HDL-C (+9.1%, p=0.0069), a significant decrease in VLDL-C (-30.2%, p=0.0023) and with no significant changes in LDL-C (+11.3, p=0.1164).

Conclusions:
This study shows that 6:1 OM-3 is effective in correcting OM-3 deficiency in hypertriglyceridemic patients and that this correction is strongly associated with significant decreases in TG/HDL ratio and in AIP- an emerging predictor of cardiovascular risk.
Abstract 593: ApoB-Lipoprotein Association With Insulin Resistance and Hyperinsulinemia: is Activation of the IL-1ß System Involved?
Authors: Saint-Pierre Nathalie , Bissonnette Simon , Lamantia Valérie , Wassef Hanny , Gauthier Dany , Faraj May
Published: March 2022

INTRODUCTION:
Elevated apoB-lipoproteins is reported to predict type 2 diabetes (T2D) development in postmenopausal women. Low insulin sensitivity (IS) and compensatory hyperinsulinemia is believed to lead to β-cell exhaustion and increase T2D risk. Also, activation of the interleukin-1β (IL-1β) system, a novel inflammatory pathway, is known to predict the development of T2D. We thus aimed to investigate whether plasma apoB associates with low IS and hyperinsulinemia and whether this is via an activation of the IL-1β system.

METHODOLOGY:
47 non-diabetic overweight and obese postmenopausal women and 28 men, aged 45 to 74 years old, underwent a modified Botnia clamp, in which 1
st
and 2
nd
phase insulin secretion were measured during a 1 hour intravenous glucose tolerance test (IVGTT). Following that, a 3 hour hyperinsulinemic-euglycemic clamp (HEIC, insulin infusion rate of 75 mU/m
2
/min) was conducted to measure IS during the last 30 minutes (steady state). IS was expressed as steady state glucose infusion rate (GIR) alone or divided over steady state plasma insulin (M/I).

RESULTS:
In women, fasting plasma apoB correlated positively with 2
nd
phase (r=0.202), total insulin secretion (r=0.168) and with total C-peptide secretion (r=0.204) and negatively with IS (r: GIR= -0.299 and M/I =-0.180) independent of adiposity. Similar to plasma apoB, fasting plasma IL-Ra (indicator of activated IL-1β system) correlated positively with 2
nd
phase and total insulin secretion and with total C-peptide secretion (r=0.217; r=0.154; r=0.198 respectively) and negatively with IS (GIR r=-0.304; M/I r=-0.214). Fasting plasma IL-Ra also correlated with apoB r=0.352). Once corrected for IL-1Ra, all associations between apoB and the indexes of IS and insulin secretion were lost. Plasma apoB did not correlate with any indices of insulin secretion or IS in men. The strongest correlate of insulin secretion and IS was adiposity, particularly central.

CONCLUSION:
ApoB is associated with hyperinsulinemia and low IS in non-diabetic overweight and obese women, which may be mediated through activation of the IL-1β system. Gender differences may need to be considered in exploring the pathophysiology of T2D in humans.
Abstract 610: Netrin-1 Promotes Macrophage Accumulation and Insulin Resistance in Obesity
Authors: Ramkhelawon Bhama , Hennessy Elizabeth J , Menager Mickael , Ray Tathagat D , Sheedy Frederick J , Babunovic Susan , Miller George , Oldebeken Scott , Geoffrion Michele , McPherson Ruth , Rayner Katey J , Moore Kathryn J
Published: March 2022

Obesity and its co-morbidities, type 2 diabetes and cardiovascular disease, continue to increase and are a major threat to global health. Studies in mice and humans have shown that expansion of adipose tissue mass is closely associated with the recruitment of cells of the myeloid and lymphoid lineage, which gives rise to a state of chronic inflammation that contributes to insulin resistance and type 2 diabetes. The factors that regulate the metabolic-dependent accrual of macrophages in adipose are not well understood. We show that the neuroimmune guidance cue netrin-1 is highly expressed in obese, but not lean adipose tissue of humans and mice, where it directs the retention of macrophages. In a mouse model of diet-induced obesity, we show that adipose tissue macrophages exhibit reduced migratory capacity ex vivo, which is reversed by blocking the effects of netrin-1. In vitro, expression of netrin-1 is induced in macrophages by the saturated fatty acid palmitate, and it acts by the receptor Unc5b to block macrophage migration to the chemokine CCL19, which directs the emigration of inflammatory macrophages from tissues. Using bone marrow transplantation, we show that hematopoietic deletion of Ntn1 facilitates adipose tissue macrophage emigration to the mesenteric lymph nodes, reduces inflammation, and improves insulin sensitivity and signaling in target tissues. Collectively, these findings identify netrin-1 as a macrophage retention signal that is induced in adipose tissue during obesity, which promotes chronic inflammation and insulin resistance.
Abstract 624: MiR-33 Antagonism Prevents the Progression of Atherosclerosis by Promoting an M2 Macrophage Phenotype
Authors: Ediriweera Hasini N , Ouimet Mireille , Sheedy Frederick J , Rayner Katey J , Salerno Alessandro , Hutchison Susan B , Hamerling Sarah , Esau Christine , Fisher Edward , Moore Kathryn J
Published: March 2022

MicroRNA-33 (miR-33) plays a central role in regulating HDL biogenesis and reverse cholesterol transport (RCT) via post-transcriptional repression of cholesterol efflux genes (ABCA1, ABCG1, NPC1). Delivery of miR-33 inhibitors to mice and non-human primates fed a chow diet raises plasma HDL cholesterol (HDL-C) and increases RCT, suggesting miR-33 as a promising therapeutic target for the treatment of atherosclerosis. However, recent studies in mice have shown that the HDL-raising effects of anti-miR33 are blunted during Western diet feeding, and its efficacy in reducing atherosclerosis progression under these conditions remains controversial. To further test the effects of miR-33 inhibition during atherogenesis, we treated
Ldlr
−/−
mice with control or anti-miR33 oligonucleotides for 8 weeks coincident with Western diet feeding. Morphometric analyses revealed that anti-miR33 reduced atherosclerosis progression by 40% in both the aortic root and the aorta
en face
without altering plasma total or HDL cholesterol. Because anti-miR33 accumulates in plaque macrophages where it may directly alter gene expression, we performed Nanostring profiling of plaque macrophages isolated by laser capture microdissection. We found a marked increase in markers of alternatively activated M2 macrophages (Arg1, Fizz1, Cd206, Ym1) in anti-miR33-treated mice compared to controls. Moreover,
in vitro
transfection of peritoneal macrophages with anti-miR33 reduced the expression of M1 markers (IL-1, TNFα) and increased the expression of M2 markers (Arg1, IL-10, Fizz1), suggesting that miR-33 directly controls the inflammatory polarization of macrophages. This regulation of macrophage polarization by miR-33 was found to be independent of its effects on ABCA1, but dependent on the targeting of AMP Kinase, a key integrator of cellular energy homeostasis. Notably, inhibitors of AMPK blocked the anti-inflammatory effects of miR-33 inhibition
in vitro
and
in vivo
. Collectively, these results identify a novel role for miR-33 in the regulation of macrophage inflammation and show that antagonism of miR-33 prevents atherogenesis, in part, by reducing plaque inflammation through the polarization of macrophages from a pro-inflammatory M1 to a reparative M2 state.
Abstract 637: Intervention With Anti-Inflammatory Resolvin E1 Attenuates Atherosclerosis Without Decreasing Plasma Cholesterol and Adds to the Antiatherogenic Effect of Atorvastatin
Authors: Salic Kanita , Wielinga Peter W , Van der Hoorn José W , Verschuren Lars , Kleemann Robert , Gjorstrup Per , Kooistra Teake
Published: March 2022

Objective:
Atherosclerosis is a multifactorial disease that correlates positively and significantly with low-density lipoprotein (LDL) plasma cholesterol and requires inflammatory components. Whereas reduction of plasma LDL cholesterol is effectively achieved by statins, quenching of the inflammatory factors has not been addressed adequately by existing therapies. Resolvins are naturally-occurring, small molecule lipid mediators with the potential to treat multiple inflammatory diseases. Here we investigated the potency of ω-3 fatty acid eicosapentaenoic acid derived resolvin E1 (RvE1) in attenuating atherosclerosis in APOE*3Leiden transgenic mice.

Methods:
Mice were fed a hypercholesterolemic diet (0.4%) for nine weeks. Subsequently, mice were randomized and treated with low (1 mg/kg/day) and high (5 mg/kg/day) doses of RvE1, atorvastatin (1.5 mg/kg/day), and the combination of atorvastatin and low dose RvE1 for the following 16 weeks to evaluate the anti-atherogenic effects.

Results:
Both low and high dose RvE1 treatment significantly (35% and 37%, respectively; p<0.05) attenuated atherosclerotic lesion size, without affecting plasma cholesterol. Plasma cholesterol lowering (24%; p<0.01) atorvastatin reduced the lesion size to a similar extent (27%; p<0.05).

Neither RvE1 nor atorvastatin reduced circulating SAA levels. Notably, refined analysis revealed the presence of less severe lesions with RvE1 versus atorvastatin treatment. Importantly, in combination with atorvastatin, RvE1 added to the anti-atherosclerotic effect of atorvastatin, resulting in decreased lesion area (31%), lesion severity (58%) and increased percentage of lesion-free segments (62%). Furthermore, micro-array analysis of aortas from RvE1-treated mice revealed changes in gene expression profiles that are enriched in biological processes involved in stress, defense and immune responses, cell adhesion and superoxide metabolism.

Conclusion:
RvE1 attenuates atherosclerosis lesion size and lesion severity without lowering plasma cholesterol and SAA levels. Combined RvE1 and atorvastatin treatment was more effective than each compound alone, indicating the potential of RvE1 to be used on top of atorvastatin treatment.
Abstract 640: Role of MicroRNA-146a in Vascular Inflammation and Atherosclerosis
Authors: Cheng Henry S , Siu Allan , Cybulsky Myron I , Fish Jason E
Published: March 2022

Prolonged activation of inflammatory pathways in the endothelium leads to vascular diseases, including sepsis and atherosclerosis. During this process, activated endothelial cells recruit circulating leukocytes by expressing adhesion molecules and chemoattractants, such as vascular cellular adhesion molecule-1 (VCAM-1) and monocyte chemoattractant protein-1 (MCP-1), respectively. Down-regulation of the anti-adhesion molecule, nitric oxide, which is synthesized by endothelial nitric oxide synthase (eNOS), is also a critical step during endothelial activation. Here we demonstrate that microRNA-146a and microRNA-146b are upregulated in endothelial cells upon exposure to the pro-inflammatory cytokine, interleukin-1-beta (IL-1β). Over expression of miR-146 in endothelial cells dampens the inflammatory response induced by IL-1β and conversely inhibition of endogenous miR-146 in vitro or deletion of miR-146a in vivo exacerbates the inflammatory response to IL-1β. We found that miR-146 represses VCAM-1 and MCP-1 expression by impinging on the canonical NF-kB pathway by targeting the upstream adaptor proteins TRAF6 and IRAK1. In addition, we identified the RNA-binding protein, HuR, as a novel miR-146 target. Knock-down of HuR results in an increase in eNOS expression and a corresponding decrease in leukocyte adhesion, which parallels with the miR-146 over expression phenotype. Considering the involvement of miR-146a in regulating vascular inflammation, miR-146a-/-; Ldlr-/- mice (DKO) were generated to assess atherogenesis. After 12 weeks on high cholesterol diet, the DKO mice developed more lipid-rich plaques in the lesser curvature of the aortic arch compared to Ldlr-/- mice. This suggests that miR-146a may have atheroprotective properties in addition to its anti-inflammatory role in acute inflammation.
Abstract 645: Atheroprotective Mechanism of Chromium Picolinate in Streptozotocin-Induced Diabetic ApoE-/- Mice
Authors: Ganguly Rituparna , Sahu Soumyadip , Ohanyan Vahagn , Chilian William M , Raman Priya
Published: March 2022

Vascular disease accounts for increased morbidity and mortality in diabetes. Given the burden of health care expenditure in diabetics, relatively cost-effective strategies represent an attractive therapeutic approach. Trivalent chromium (Cr) is a mineral nutrient reported to have a beneficial role in glycemic and cardiovascular health. While previous studies suggest that Cr reduces vascular inflammation in diabetes, the precise mechanisms of Cr action in the large blood vessels in diabetes remain unknown. We showed earlier that Cr picolinate (CrP, a bioavailable form of Cr)
in vitro
downregulates a potent proatherogenic matricellular protein thrombospondin-1 (TSP-1) in glucose-stimulated vascular smooth muscle cells. The present study provides the first evidence of an atheroprotective mechanism of Cr
in vivo
in a mouse model of diabetic atherosclerosis. Briefly, hyperglycemia was induced in male ApoE
-/-
mice (6wks age) with 50mg/Kg/day streptozotocin (STZ), i.p. for 5 consecutive days. Hyperglycemic mice (blood glucose>250mg/dl) on regular chow diet were treated with or without CrP (8μg/Kg) in drinking water starting at 8 wks of age and all mice were sacrificed at 18 wks of age. Using
en face
atherosclerotic lesion assay shown by Oil red O staining, we demonstrated that CrP
in vivo
significantly prevents development of lesion in aortic vessel walls of hyperglycemic STZ-ApoE
-/-
mice compared to untreated STZ-ApoE
-/-
mice. Consistently, high-frequency ultrasound imaging of common carotid artery revealed that CrP prevents development of vascular lesions in STZ-ApoE
-/-
mice while untreated mice showed more pronounced lesions with a significant reduction (20%) in carotid artery diameter. Importantly, atheroprotective effects of CrP were accompanied by a significant decrease (>35%) in TSP-1 and PCNA (proliferation marker) expression in aortic vessels of STZ-ApoE
-/-
mice, shown by immunoblotting. Also,
en face
studies showed that knockout of TSP-1 protects STZ-induced hyperglycemic ApoE
-/-
mice against atherosclerosis. Together, our results suggest that CrP
in vivo
prevents diabetic atherosclerosis via downregulation of TSP-1 in the large blood vessel wall, underscoring TSP-1 as a novel therapeutic target in diabetic atherosclerosis.
Abstract 66: Small Dense LDL Cholesterol Predicts Incident Diabetes Mellitus: The Atherosclerosis Risk in Communities Study
Authors: Pokharel Yashashwi , Sun Wensheng , Virani Salim S , Ballantyne Christie M , Hoogeveen Ron C
Published: March 2022

Background:
Small dense low-density lipoprotein cholesterol (sd-LDL-C) is an independent predictor of vascular events even in individuals with lower levels of LDL-C. Diabetics in particular tend to have higher levels of sd-LDL-C compared to those without diabetes. It is not known if sd-LDL-C predicts incident diabetes mellitus (DM).

Objectives:
We tested the hypothesis that elevated levels of sd-LDL-C measured using a new automated assay predict incident DM in the biracial ARIC study.

Methods:
Plasma sd-LDL-C was measured in 9,451 men and women without prevalent DM using a newly developed automated homogeneous assay. A Cox proportional hazards model was used to examine the association of sd-LDL-C with risk for incident DM.

Results:
More individuals in the highest vs. lowest sd-LDL-C quartiles were men, Caucasians, had hypertension and higher mean body mass index (BMI) (P<0.001 for all comparisons). Similarly more individuals in the highest vs. lowest sd-LDL-C quartiles had parental history of DM (31.2 vs. 28.8%, P=0.012) and higher mean fasting blood glucose (116 vs. 102 mg/dL, P<0.001). Over a period of 10.4 years 911 individuals developed new onset DM at a rate of 9.27 per thousand person years. In a fully adjusted model, individuals in highest vs. lowest sd-LDL-C quartiles have a 44% increased risk for the development of incident DM even after adjusting for fasting blood glucose (Table).

Conclusion:
sd-LDL-C predicts incident DM in the biracial ARIC study.
Abstract 661: Targeting Sortilin in Immune Cells Reduces Atherosclerosis
Authors: Mortensen Martin B , Kjolby Mads , Gunnersen Stine , Larsen Jakob V , Palmfeldt Johan , Falk Erling , Nykjær Anders , Bentzon Jacob F
Published: March 2022

Targeting sortilin in immune cells reduces atherosclerosis.

Background:
SNPs at 1p13.3 have been strongly associated with myocardial infarction and plasma cholesterol levels in genome-wide association studies. This locus covers 3 genes; SORT1, CELSR2 and PSRC1. Previous studies on sortilin in cardiovascular disease have focused on sortilins role in hepatic lipoprotein metabolism. In the present study, we demonstrate that sortilin also modulates atherogenesis independent of its effect on lipoprotein metabolism.

Methods and results:
The effect of sortilin deficiency on atherosclerosis was studied in
Apoe
-/-
mice. Absence of sortilin (
Sort1
-/-
) did not influence plasma cholesterol levels or the distribution between lipoprotein fractions, yet significantly reduced development of atherosclerosis after both 6 and 18 weeks of Western diet. To examine the possible mechanism we performed studies on murine bone marrow-derived macrophages (BMM) and th1-cells. Loss of sortilin did not impact foam-cell formation or cell activation, but specifically reduced the secretion of interleukin-6 (Il-6) and interferon-gamma (IFN-) from macrophages and th1 cells, respectively. Further, surface plasmon resonance analysis demonstrated that sortilin binds these two cytokines with high affinity. These results suggest that sortilin is involved in regulation of Il-6 and IFN- secretion and indicates that sortilin in immune cells may influence atherosclerosis.

To test this hypothesis, 8 weeks old
Apoe
-/-
were lethally irradiated and rescued with age- and sex-matched bone marrow from
Sort1
+/+
Apoe
-/-
or
Sort1
-/-
Apoe
-/-
donor mice. Mice with a sortilin deficient bone marrow transplant had reduced development atherosclerosis compared to mice with a
Sort1
+/+
transplant. Although these mice had normal numbers of circulating immune cells, they had reduced levels of circulating tumor necrosis factor-α and Il-6 in plasma, suggesting that absence of sortilin attenuates the inflammatory response.

Conclusions:
We conclude that targeting sortilin in immune cells reduces atherosclerosis by attenuation of the inflammatory response.
Abstract 668: Whole Body and Hematopoietic Sortilin Deficiency Reduces Atherosclerosis in Mice Independent of Effects on LDL Cholesterol
Authors: Patel Kevin , Strong Alanna , Tohyama Junichiro , Jin Xueting , Kruth Howard , Rader Daniel
Published: March 2022

Background:
Genetic variants on chromosome 1p13 at the SORT1 locus are genome-wide significantly associated with both LDL cholesterol as well as with myocardial infarction (MI). Sortilin, encoded by the SORT1 gene, traffics to both the Golgi as well as the plasma membrane, where it binds ligands and transports them to the lysosome. Our lab recently showed that sortilin is a high affinity receptor for LDL, and genetic manipulation of sortilin expression in the liver influences LDL-C levels. Sortilin is also expressed in macrophages but little is know about its function in this cell type or its relationship to atherosclerosis.

Results:
Sortilin deficient mice were crossed to atherosclerosis-prone Apobec-/-; human ApoB Tg mice. On this model, plasma total and LDL-C levels were not different in the sortilin deficient vs the wild-type mice. Unexpectedly, aortic atherosclerosis measured en face was reduced by 90% (n=10 per group; P = 5.3 E-7). To determine whether hematopoetic sortilin contributed to this effect, we performed bone marrow transplantation studies from done Sort1-/-; Ldlr -/- or Ldlr -/- mice into atherosclerosis-prone LDLR-/- mice and fed a western diet for 18 weeks (n=11-12/group). Plasma cholesterol was not different in the two groups of mice. Aortic atherosclerosis was reduced by 50% (P < 1E-5) in mice lacking sortilin. Sortilin deficient bone marrow derived macrophages had significantly reduced LDL uptake by ~39% (P = 0.02). In the presence of a actin polymerization inhibitor cytochalasin D, sortilin deficiency further reduces LDL uptake by 38% (P = 0.03)

Conclusion:
Whole body and hematopoetic sortilin deficiency reduce atherosclerosis in mice. Sortilin deficient macrophages have reduced uptake of native LDL, potentially at least partially accounting for the reduced atherosclerosis. Sortilin deficient macrophages have reduced uptake of native LDL, which is not abrogated by inhibition of macropinocytosis. Our data are consistent with a model in which sortilin deficiency in macrophages protects against atherosclerosis by eliminating an uptake pathway for LDL into macrophages and suggests that sortilin mediated LDL uptake is an important contributor to atherosclerosis development.
Abstract 672: Reduced "Egress" of Remnant Lipoproteins From Arterial Tissue Relative to LDL During Insulin Resistance May Explain a Preferential Role in Atherosclerosis Progression
Authors: Mangat Rabban , Borthwick Faye , Vine Donna F , Proctor Spencer D
Published: March 2022

INTRODUCTION:
There is emerging epidemiological data that supports a causative role for remnant cholesterol in atherosclerotic and CVD progression. We have shown that hepatic (apoB100) and intestinal (apoB48) lipoproteins may have disparate ‘egress’ and retention properties. Additionally we have reported that insulin resistance (IR) increases the arterial atherogenic proteoglycans. However the underlying effects of this metabolic milieu on the comparative arterial retention of both low-density lipoproteins (LDL) and chylomicron-remnant lipoproteins (CM-R) towards arterial cholesterol deposition remain elusive.

Aim:
To determine the comparative arterial retention and ‘egress’ of LDL and CM-R in insulin resistant carotid vessels in JCR:LA-cp rats using an ex vivo perfusion approach.

METHODS:
For in vivo generation of Cy5-labeled CM-R, an intravenous injection of nascent chylomicrons obtained after intragastric intralipid infusion was given to eviscerated rabbits. Labeled CM-R were collected from the plasma after density centrifugation. Human LDL was commercially obtained and was labeled with Cy3. Carotid vessels of JCR-LA:cp IR rats were perfused with Cy5-labeled remnants simultaneously with Cy3-labeled LDL. Images were quantified using confocal microscopy.

RESULTS:
When equivalent number of CM-R and LDL particles were perfused, a significantly greater number of LDL particles were delivered to the vessel (9-fold) as compared to CM-R. However, after extensive washout (i.e retention) significantly fewer (55% decrease) LDL particles remained in the tissue. In contrast, we observed no difference in the retention of CM-R relative to delivery experiments (no washout) perhaps due to reduced ‘egress’. Despite the greater number of LDL particles retained compared to CM-R, we observed a greater than 1.5-fold increase in total mass of cholesterol associated with CM-R as compared to cholesterol associated with LDL.

CONCLUSION:
There is debate whether current changes to the US clinical guidelines to move away from plasma LDL-C as an effective predictor of CVD risk. This study suggests that due to disparate ‘egress’ and retention properties of LDL and CM-R there may be a disconnection between their plasma concentration and arterial accumulation.
Abstract 676: 14q32 MicroRNA Inhibition Reduces Atherosclerotic Lesion Formation and Increases Plaque Stability
Authors: Wezel Anouk , Welten Sabine M , Razawy Wida , Lagraauw H Maxime , de Vries Margreet R , Kandimalla Ekambar R , Kuiper Johan , Quax Paul H , Nossent A Yaël , Bot Ilze
Published: March 2022

Introduction:
Atherosclerosis is a multifactorial disease involving inflammatory, metabolic and lipid-related processes. As microRNAs (miRs) regulate expression of up to hundreds of target genes, we aimed to identify miRs that target genes involved in all processes of atherosclerosis. Using www.targetscan.org, we performed a Reverse Target Prediction on a set of 150 atherosclerosis-related genes. We found enrichment of binding sites for several miRs from a single gene cluster on human chromosome 14.

Methods:
ApoE-/- mice fed a Western type diet received semi-constrictive collars around both carotid arteries to induce atherosclerotic lesion formation. Gene Silencing Oligonucleotides (GSOs) were used to inhibit 3 14q32 miRs: miR-329, miR-494 and miR-495. Mice were injected with 1 mg GSO at day 4 after collar placement and again with 0.5 mg GSO at day 18. Mice were sacrificed at day 7 and day 28 for analysis of carotid arteries, liver, spleen, bone marrow and blood samples.

Results:
Atherosclerotic plaque analysis revealed a striking decrease of lesion size in mice treated with GSO-494 and GSO-495 (GSO-Control: 47 ± 11*103 μm2; GSO-494: 16 ± 3*103 μm2; GSO-495: 22 ± 5*103 μm2), whereas a trend towards reduced plaque size was observed in mice treated with GSO-329 (27 ± 6*103 μm2). Moreover, plaque stability was increased in mice treated with GSO-494 and GSO-495, determined by both a decrease in necrotic core size (GSO-494: 80% decrease; GSO-495: 60% decrease compared to GSO-Control) and an increase in plaque collagen content (GSO-Control: 6.6 ± 1.6%; GSO-494: 12.7 ± 2.1%; GSO-495: 15.8 ± 3.1%). Furthermore, inhibition of miR-494 and miR-495 resulted in decreased plasma cholesterol levels (GSO-Control: 30.4 ± 1.1 mM; GSO-494: 26.4 ± 0.7 mM; GSO-495: 26.4 ± 1.6) and decreased VLDL fractions.

Conclusions:
Inhibition of miR-494, miR-495 and, to a lesser extent, miR-329, leads to reduced plaque size, increased plaque stability and reduced plasma cholesterol levels. This makes 14q32 miRs promising novel therapeutic targets in atherosclerosis.
Abstract 72: Tryptophan 415 of Scavenger Receptor BI Is Critical for Mediating Cholesterol Transport Functions
Authors: Holme Rebecca L , Miller James , Sahoo Daisy
Published: March 2022

High density lipoproteins (HDL) combat atherosclerosis by delivering excess cholesteryl esters (CE) from peripheral tissues to the liver for excretion upon binding to their receptor, scavenger receptor BI (SR-BI), via reverse cholesterol transport. Our previous studies demonstrate that the extracellular domain (ECD) of SR-BI, particularly areas of hydrophobicity in its N-terminal half, is critical for cholesterol transport functions. The ECD of SR-BI also contains eight tryptophan (Trp;W) residues that may play a role in positioning SR-BI within the membrane, as observed with other membrane proteins. Based on their location at the membrane-water interface, we hypothesize that three of these Trp residue (W9, W56, W415), are critical for SR-BI’s cholesterol transport functions. To test this hypothesis, we designed single Trp-to-Phe mutations, as well as a Trp-free-SR-BI and confirmed their expression upon transient transfection into COS7 cells. Next, we used [
3
H]/[
125
I]-HDL-COE to assess the ability of these receptors to bind HDL. Our data revealed that Trp-free-SR-BI and W415F-SR-BI had a significantly decreased ability (88% and 63%, respectively) to bind HDL compared to wild-type (WT) SR-BI, accompanied by 44 and 64% decreases, respectively, in the ability to mediate selective uptake of HDL-COE. Trp-free- and W415F-SR-BI were also unable to enlarge the cholesterol oxidase-sensitive pool of membrane free cholesterol (FC) (20-50% decreases), as compared to WT SR-BI. The inability of these two mutant receptors to mediate these functions was not due to impaired trafficking, as both receptors were present on the cell surface as judged by flow cytometry and immunofluorescence studies. Interestingly, only Trp-free-SR-BI was unable to mediate the release of FC out of cells (50% decrease compared to WT SR-BI). Together, these data suggest that Trp415, that flanks a transmembrane segment of SR-BI, might be involved in orienting SR-BI in a conformation that would support HDL binding and selective uptake of HDL-CE, as well as reorganization of plasma membrane cholesterol. We are currently identifying the combination of Trp residues at the membrane/water interface that are important for mediating FC efflux to HDL.
Abstract 79: Differential Impact of Mitofusins on Endothelial Phenotype
Authors: Nchaw Gladys A , Papanicolaou Kyriakos N , Walsh Kenneth
Published: March 2022

Rationale:
Endothelial mitochondria are important for endothelial cell function and the development of vascular diseases. Recent published observations have shown that endothelial mitochondria display altered morphology under stress, suggesting perturbation in mitochondrial dynamics. Mitochondrial dynamics involves mitochondria fusion (regulated by mitofusins 1 (Mfn-1) and 2 (Mfn-2) and optic atrophy protein 1). The role of mitochondrial dynamics in endothelial dysfunction in vivo has never been explored.

Method and Results:
To understand the role of mitofusins in endothelial phenotype in vivo, we ablated mitofusins in Tie2-Cre-expressing cells. In vivo, Tie2-Mfn2 mice (ECKO
Mfn2
) had impaired recovery from hindlimb ischemia and diminished capillary density using CD31 immunofluorescence despite no difference in Tie2-Mfn-1 mice (ECKO
Mfn1
) compared to wild-type mice. We also observed exacerbated muscle atrophy in of ECKO
Mfn2
but not ECKO
Mfn1
mice compared to wild-type mice 28 days post HLI. Because vascular endothelial growth factor receptor 2 (Vegfr2) and endothelial nitric oxide synthase (eNOS) are involved in endothelial angiogenic response, we probed the effects of mitofusins on Vegfr2 and eNOS. There was less phosphorylated Vegfr2 and phosphorylated eNOS in ischemic limbs of ECKO
Mfn2
mice, despite no difference in ECKO
Mfn1
mice compared to wild-type mice at post operative day 14 according to western blotting indicating impaired angiogenic signaling in vivo. In cultured mouse lung endothelial cells (MLEC) deficient in Mfn-2, we observed significant reduction in Vegf −stimulated phosphorylation of Vegfr2 and eNOS compared to control cells. This blunted angiogenic signaling inhibited cell proliferation and migration in Mfn-2 but not Mfn-1 null endothelial cells.

Conclusion:
These data highlight a previously unrecognized role for Mfn-2 in angiogenesis and show that Mfn-2 not Mfn-1 is essential for mediating endothelial function.