bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 19: Deletion of Myeloid GSK3α Attenuates Atherosclerosis and Promotes an M2 Macrophage Phenotype
Authors: McAlpine Cameron , Huang Aric , Emdin Abby , Banko Nicole , Beriault Daniel , Shi Yuanyuan , Werstuck Geoff
Published: July 2021

Objective:
Glycogen synthase kinase (GSK)-3α/β has been implicated in the pathogenesis of diseases including diabetes, cancer, Alzheimer’s and atherosclerosis. The tissue and homolog specific functions of GSK3α and β in atherosclerosis are unknown. This study examines the effect of hepatocyte or myeloid cell specific deletion of GSK3α or GSK3β on atherosclerosis in LDLR-/- mice.

Approach and results:
We ablated GSK3α or GSK3β expression in hepatic or myeloid cells of LDLR-/- mice and mice were fed a high fat diet for 10 weeks. GSK3α or GSK3β deficiency in hepatic or myeloid cells did not affect metabolic parameters, including plasma lipid levels. Hepatic deletion of GSK3α or GSK3β did not affect the development of atherosclerosis or hepatic lipid content. Myeloid deletion of GSK3α, but not GSK3β, reduced atherosclerotic lesion volume as well as lesion complexity. Mice lacking GSK3α in myeloid cells had a less inflammatory and more anti-inflammatory plasma cytokine profile. Macrophages within atherosclerotic lesions of myeloid GSK3α deficient mice, but not GSK3β deficient mice, displayed reduced expression of markers associated with M1 macrophage polarization and enhanced expression of the M2 markers. Finally, bone marrow derived macrophages were isolated and differentiated into classical M1 macrophages or alternative M2 macrophages in vitro. GSK3α deletion, but not GSK3β deletion, attenuated the expression of genes associated with M1 polarization while promoting the expression of genes associated with M2 polarization. Mechanistically, GSK3α regulated macrophage polarization by modulating the phosphorylation and activation of STAT transcription factors.

Conclusions:
Our findings suggest that deletion of myeloid GSK3α attenuates the progression of atherosclerosis by regulating STAT activation and promoting an M2 macrophage phenotype.
Abstract 190: Purinergic Control of Tissue Factor Transcription in Human Coronary Artery Endothelial Cells: New AP-1 Site and Negative Regulator
Authors: Liu Yiwei , Zhang Lingxin , Wang Chuan , Roy Shama , Shen Jianzhong
Published: July 2021

Previously we reported that the P2Y2 receptor (P2Y2R) is one of the predominant purinergic receptors expressed in human coronary artery endothelial cells (HCAEC), and that P2Y2R activation by ATP or UTP induces dramatic up-regulation of tissue factor (TF), key initiator of the coagulation cascade. However, the molecular mechanism of this P2Y2R-TF axis remains unclear. Here we report a role of a newly identified AP-1 consensus sequence along with its new binding components in P2Y2R regulation of TF transcription. We identified with bioinformatics tools that a novel AP-1 site at -1363 bp of human TF promoter region is highly conserved across multiple species. P2Y2R activation increased TF promoter activity and mRNA expression in HCAEC. Truncation, deletion, and mutation of this new distal AP-1 site all significantly supressed TF promoter activity in response to P2Y2R activation. EMSA and ChIP assays further confirmed that upon P2Y2R activation, c-Jun, ATF-2 and Fra-1, but not the typical c-Fos, bound to the new AP-1 site. In addition, loss-of-function studies using siRNAs confirmed a positive transactivation role of c-Jun and ATF-2, but unexpectedly revealed a strong negative role of Fra-1 in P2Y2R-induced TF up-regulation. Furthermore, we found that P2Y2R activation promoted ERK1/2 phosphorylation, leading to Fra-1 activation while JNK activated c-Jun and ATF-2. These findings reveal the basis for P2Y purinergic receptor regulation of endothelial TF expression and indicate that targeting the P2Y2R-Fra-1-TF pathway may be an attractive new strategy in control of vascular thrombogenicity and/or inflammation associated with endothelial dysfunction.
Abstract 20: Apolipoprotein A-I Influences Regulatory T Cell Development and Proliferation in Homeostasis and Atherogenesis
Authors: Gaddis Dalia E , Wu Amy , Yoakum Debbi , Sorci-Thomas Mary , Hedrick Catherine C
Published: July 2021

Apolipoprotein A-I (ApoAI) is the major protein component of HDL. HDL ApoAI is involved in the efflux of cholesterol from cells to maintain cellular cholesterol homeostasis. ApoAI also has anti-inflammatory properties. We previously showed that injecting mice fed an atherogenic diet with ApoAI decreased the number of activated CD4 lymphocytes. Since T regulatory lymphocytes (Treg) play a major role in inhibiting the immune response during atherosclerosis development, we wanted to determine if ApoAI influences Treg development, hypothesizing that ApoAI enhances Treg development. To test this hypothesis, we compared the numbers of Treg in ApoAI-/- mice to B6 mice, and found a 50% decrease in the numbers of Treg in the periaortic LNs (PaLN) of ApoAI-/- mice. BrdU labeling studies showed that ApoAI-/- Treg had a significant 30% reduction in proliferation, suggesting that in the absence of ApoAI and normal cholesterol homeostasis, Tregs have defective proliferation. Functionally, we discovered that ApoAI-/- Treg were significantly less suppressive than B6 Treg in reducing CD4 effector T cell proliferation, suggesting that ApoAI plays a role in both the development and function of Tregs. To determine if the addition of exogenous lipid-free ApoAI could rescue and promote Treg differentiation in ApoAI-/- mice, ApoAI-/- naïve T cells were incubated in vitro with TGFβ and exogenous ApoAI. Addition of ApoAI significantly increased development of naïve ApoA1-/- lymphocytes into Treg. To verify these results in vivo, we fed a novel Treg lineage tracker mouse (LT), Foxp3-YFP-Cre-Rosa26-RFP-ApoE-/- mice a western diet for 15 weeks and administered subcutaneous injections of ApoAI for the last 9 weeks of diet. These mice allow us to identify current functional Tregs and any exTregs that have lost active Treg function in vivo. We found that LT mice treated with ApoAI had a 37% decrease in exTregs and a concomitant 33% increase in current functional Tregs in the aorta. This was accompanied by decreased IFNγ and IL-17 production in PaLN, further confirming our in vitro findings that ApoAI promotes Treg development and function. In conclusion, we have identified a novel role for ApoAI by enhancing Treg development, emphasizing the immune properties of ApoAI for atheroprotection.
Abstract 205: Ouabain Activates the NF-κB Pathway in Macrophages via Na/K-ATPase/TLR4 Complex Leading to Pro-Inflammatory Cytokine Production
Authors: Chen Yiliang , Silverstein Roy L
Published: July 2021

Cardiotonic steroids such as ouabain, digoxin, and marinobufagenin are known ligands for the plasma membrane receptor Na/K-ATPase (NKA). These ligands are able to stimulate interaction of the NKA with other membrane and cytosolic proteins leading to cellular events such as activation of kinase cascades and gene transcription. Endogenous cardiotonic steroids have been detected in human circulation and interestingly their levels were elevated in human patients with hypertension, congestive heart failure and diabetes, all of which were associated with chronic systemic inflammation. However, the role of cardiotonic steroids in systemic inflammation and immunity has not been well studied. We previously discovered that ouabain stimulated macrophages to produce pro-inflammatory cytokines, many of which are known targets of the transcription factor, NF-κB. Therefore, we hypothesized that ouabain activates NF-κB pathway leading to pro-inflammatory cytokine production in macrophages. Using Western blot and densitometry analysis, we showed that physiological concentrations of ouabain promoted IκBα degradation (as low as 5 nM ouabain decreased IκBα level by 66.8%±7.4%, n=4). This was accompanied by NF-κB translocation from cytoplasm to the nuclei as shown by immunocytochemistry (% of nuclei NF-κB signals increased from 30.5%±2.3% in control to 62.2%±2.6% in ouabain-treated macrophages, n>25). Moreover, via quantitative RT-PCR (n=3), we found that ouabain increased mRNA levels of pro-inflammatory cytokines such as MCP-1 (3.2±1.1 fold), TNF-α (59.7±35.6 fold), and CXCL-10 (2.8±1.6 fold), all of which are known NF-κB targets. Consistent with the increase in mRNA level, we found that MCP-1 protein levels were elevated in both cell lysates (1.8±0.3 fold) and culture media (1.4±0.1 fold; n=4). Addition of an NF-κB inhibitor blocked MCP-1 production induced by ouabain (n=4). Mechanistically, ouabain stimulated interaction between NKA and TLR4 as shown by Co-Immunoprecipitation (n=3). Blockade of TLR4 signaling using a specific peptide inhibitor, CLI-095, abolished the ouabain effect on NF-κB activation (n=3). We conclude that ouabain activates NF-κB through NKA/TLR4 complex leading to pro-inflammatory cytokine production by macrophages.
Abstract 211: IL-35 Suppresses Endothelial Cell Activation by Inhibiting Histone H3K14 Acetylation and AP-1
Authors: Li Xinyuan , Fang Pu , Sha Xiaojin , Li Ya-Feng , Kuo Yin-Ming , Andrews Andrew J , Wang Hong , Yang Xiao-Feng
Published: July 2021

Background:
IL-35 is a newly identified anti-inflammatory cytokine, which inhibits inflammation and immune responses by inducing regulatory T cells and regulatory B cells and suppressing effector T cells and macrophages. We previously reported that IL-35 is a responsive cytokine that is not constitutively expressed in human tissues, but can be induced by proinflammatory stimuli in vascular endothelial cells (EC), smooth muscle cells and monocytes. However, the functions of IL-35 on non-lymphocytes such as vascular EC remain unknown. EC activation induced by proatherogenic stimuli including lysophosphatidylcholine (LPC) is considered as the initiation step of monocyte recruitment and atherosclerosis. In this study, we examined the expression of IL-35 during early atherosclerosis and the role of IL-35 in LPC-induced EC activation.

Methods and Results:
Using microarray method, we found that two IL-35 subunits and their receptor subunits are all significantly induced during early atherosclerosis in the aortas of atherosclerotic mouse model, apolipoprotein E (ApoE) knockout mice fed with 3 weeks of high fat diet. In addition, we found that IL-35 can suppress LPC-induced monocyte adhesion to human aortic ECs (HAEC). Mechanistic, our analysis using EC biology gene array shows that IL-35 selectively inhibits LPC-induced expressions of EC adhesion molecules ICAM-1, VCAM-1, and proinflammatory cytokine IL-1β in human aortic EC (HAEC). To further dissect the molecular mechanisms underlying the IL-35 inhibitory effects on LPC-induced EC activation gene upregulation, we performed mass spectrum analysis and electrophoretic mobility shift assay. We found that IL-35 blocked LPC-induced histone H3 lysine 14 (H3K14) acetylation as well as the binding of pro-inflammatory transcription factor AP-1 to the promoters of EC adhesion molecules in HAEC.

Conclusions:
IL-35 is induced in the aortas during early atherogenesis and suppresses human EC activation and monocyte adhesion by inhibiting histone acetylation and AP-1 nuclear translocation.
Abstract 216: The Effect of Fisetin on Lipopolysccharide-induced Inflammation and MMP Activity in Mouse Peritoneal Macrophages
Authors: Takeuchi Hidemi , Uchida Haruhito A , Umebayashi Ryoko , Kakio Yuki , Okuyama Yuka , Okuyama Tomohiro , Wada Jun
Published: July 2021

Objective:
Fisetin is a flavonoid that is prominent in strawberries. Fisetin has been shown to have several favorable effects, including anti-aging, anti-inflammatory and anti-carcinogenic. Fisetin is also known to have anti-oxidant properties as with other flavonoids. The purpose of this study was to investigate the influence of fisetin on lipopolysccharide (LPS) -induced inflammation and matrix metalloproteinase (MMP) activity in mouse peritoneal macrophages (MPMs) in vivo.

Methods and Results:
MPMs were harvested from BALB/c mice and cultured. After 30 minutes incubation with fisetin (10, 30 or 100 uM), MPMs were incubated with LPS (10 ng/ml) for 15 minutes, 60 minutes or 24 hours. LPS incubation increased concentrations of nitric oxide in supernatants that was attenuated by fisetin in a concentration-dependent manner. Similarly, mRNA abundance of inflammatory genes, such as MCP-1, IL-1b and iNOS, were enhanced by LPS, but were decreased by fisetin incubation in a concentration-dependent manner. Gelatin zymography demonstrated that LPS increased MMP-9 activity in supernatants that were decreased by the highest fisetin concentration. LPS-enhanced mRNA abundance of MMP-2 and MMP-9 were also attenuated by the highest concentration of festin. Fisetin also decreased LPS increased protein abundance uPA, as determined by Western blotting, and uPAR gene expression. Furthermore, fisetin also attenuated LPS enhanced ERK and JNK phosphorylation.

Conclusion:
Fisetin attenuated LPS-induced oxidative stress, inflammation, and MMPs activity in MPMs.
Abstract 221: A Single Nucleotide Polymorphism of p27
Kip1
Associated with Vein Graft Patency Regulates Proliferation of Cells Derived from the Outer, but not the Inner, Venous Wall
Authors: Kenagy Richard D , Kikuchi Shinsuke , Chen Lihua , Wijelath Errol S , Sobel Michael , Clowes Alexander W
Published: July 2021

Introduction:
p27Kip1 is a cell-cycle inhibitor whose -838C>A single nucleotide polymorphism (SNP) accounts for ~40% of the risk of peripheral vein graft failure.

Methods:
To determine SNP function, we tested growth of cells derived from explants of the outer and inner wall of veins (cleavage through medial circular muscle yields outer wall with adventitia and some media and inner wall with intima and some media).

Results:
Growth of cells at passage 6 was measured over 3 days in response to 10 ng/ml PDGF-BB + 2% serum. Outer wall cells with the AA p27 SNP genotype grow slower than those with the CC genotype, but inner wall cells do not show a genotype-dependent response (Figure). Gene expression of quiescent outer and inner wall cells (5pairs) measured by microarray show that 2876 genes are differentially expressed (≥2 fold difference with P≤.05). While there is no difference in expression of traditional smooth muscle markers (smooth muscle α-actin, smooth muscle myosin heavy chain, smoothelin, SM22α, and calponin1), expression of the pericyte markers, NG2 and PDGFRβ, is significantly higher in outer wall cells (4.2 and 2.2 fold over inner wall cells; P<.02). We also found CD31 immunostaining of the endothelial cells of the vasa vasorum was restricted to the outer wall.

Conclusion:
These results support the functionality of the p27Kip1-838C>A SNP in cellular proliferation, but only in outer wall cells. Pericytes of the vasa vasorum or other progenitors in the outer wall may be involved, but further studies must address the identity of precursors of the outer wall cells that are associated with graft failure.
Abstract 222: SCARA5 and Suprabasin are Hub Genes of Co-expression Network Modules Associated with Peripheral Vein Graft Patency
Authors: Kenagy Richard D , Civelek Mete , Kikuchi Shinsuke , Chen Lihua , Grieff Anthony , Sobel Michael , Lusis Aldons J , Clowes Alexander W
Published: July 2021

Introduction:
About 30% of vein grafts fail because of intimal hyperplasia or negative remodeling. We have reported that vein graft cells from patients that develop stenosis proliferate more than cells from patients that maintain patent grafts. We have now analyzed gene expression of the same cell lines using a systems biology approach to cluster genes into modules based on their co-expression patterns and to correlate the results with graft outcome, growth data from our prior study, and with new studies of migration and matrix remodeling.

Methods:
RNA from 4 hour serum- or PDGF-BB-stimulated cells (13 non-stenotic and 7 stenotic cell lines) was used for microarray analysis of gene expression followed by weighted gene co-expression network analysis. Cell migration in microchemotaxis chambers in response to PDGF-BB and cell-mediated collagen gel contraction in response to serum were also determined. Gene function in growth or collagen gel contraction was determined using siRNA to inhibit gene expression.

Results:
Neither migration nor collagen gel contraction were correlated with graft outcome. While 1,188 and 1,340 genes were differentially expressed in response to serum and PDGF, respectively, graft outcome was not correlated with expression of any single gene. Network analysis revealed one module each from the separate analysis of the PDGF and serum data sets, which were called “Yellow” and “Skyblue” respectively, that were correlated with later graft stenosis (P=.005 and .02, respectively). Yellow was also associated with increased cell growth, and Skyblue was also associated with inhibition of collagen gel contraction. The hub genes for Yellow and Skyblue (i.e. the gene most correlated with other genes in the module), SCARA5 and SBSN, respectively, were tested for effects on proliferation and collagen contraction. SCARA5, but not SBSN, inhibited proliferation, and SBSN, but not SCARA5, inhibited collagen gel contraction.

Conclusion:
Using weighted gene co-expression network analysis of cultured vein graft cell gene expression, we have discovered a small number of genes of interest in vein graft failure. Further experiments are needed to discriminate the roles these genes play in vein graft healing starting with the module hub genes SCARA5 and SBSN.
Abstract 228: Histology and Pathophysiology Study of Renal Aneurysm
Authors: Chavent Bertrand , Arif Batool , Ennis Terri , Curci John A
Published: July 2021

Objectives:
While progress has been made over the past 2 decades in our understanding of the development of abdominal aortic aneurysms (AAA), far less is understood about the development and pathobiology of aneurysms of the renal arteries. A valuable collection of renal artery tissue collected over the course of many years was used to directly compare histology, as well as some known mediators of AAA development in end-stage tissue from renal arteries aneurysm (RAA) and AAA.

Methods:
Tissues were collected and stored (frozen fresh and FFPE) at the time of aneurysm repair or renal transplant for normal aortic specimens. Histologic morphometry was performed to quantify elastin staining and immunohistochemistry for CD68, alpha-actin, MMP9 and Osteopontin (OPN). Gelatin zymography was performed on protein extracts from the tissues.

Results:
All aneurysms (AAA, n=9, RAA, n=9) tissues demonstrated a significantly (P<0.05) reduced percentage of mural elastin than that seen in normal aorta (16.3±3.4%, n=5). Loss of medial elastin was nearly complete in AAA (.11±.04%). Elastin in all RAA was substantially greater (3.6±1.4%) than AAA but this did not reach statistical significance (P=0.1). Elastin content is significantly greater in RAA due to fibromuscular dysplasia (RAA-FMD, n=5, 6.6±1.8%) than AAA (P<. 02) and idiopathic RAA (iRAA, 0.72±0.48%, P<.03).

RAA had greater preservation of smooth muscle cell (SMC) mass in the aortic wall, and unlike AAA, the cells were in typical laminar formation in the media. Few macrophages were seen in any RAA tissue. By immunohistology, qualitatively there is substantially greater OPN in all RAA. There was also very little immunreactive MMP-9 in the RAA specimens, whereas AAA specimens had substantial MMP-9 present in inflammatory cells and SMC. MMP9 activity on zymography was found to be significantly lower in RAA (n=3) than in AAA (n=3; p=0.03).

Conclusions:
These important and unique studies demonstrate that the histology and general pathobiology of RAA is distinct from AAA. Elastin degradation is significantly lower in RAA, particularly in RAA-FMD. Infiltrating macrophages and elastolytic enzyme MMP-9 appear to play a relatively small role in the development of RAA.
Abstract 235: Plasma Levels of Cyclophilin A Correlate with Circulating Inflammatory Cytokines in Patients with Pulmonary Hypertension
Authors: Otsuki Tomohiro , Satoh Kimio , Kikuchi Nobuhiro , Omura Junichi , Kudo Shun , Sugimura Koichiro , Aoki Tatsuo , Tatebe Shunsuke , Shimokawa Hiroaki
Published: July 2021

Background:
Excessive proliferation and resistance to apoptosis of pulmonary artery smooth muscle cells (PASMCs) are key characteristics of pulmonary vascular remodeling in patients with pulmonary hypertension (PH). The mechanisms of pathophysiological changes in PH are not fully investigated. Cyclophilin A (CyPA) is secreted from vascular smooth muscle cells (VSMCs) in response to several stimuli, including oxidative stress, mechanical stretch and hypoxia. Extracellular CyPA and its receptor Basigin induce secretion of growth factors and inflammatory cytokines from VSMCs. Additionally, plasma CyPA levels predict poor outcome in patients with PH. In this study, we examined the correlations between plasma CyPA levels and circulating cytokines/chemokines and growth factors in PH patients.

Methods and Results:
In consecutive 176 patients undergoing right heart catheterization, we examined the relationship between plasma CyPA and inflammatory cytokines/chemokines and growth factors. We used ELISA for CyPA measurement and Bio-Plex system for measurement of inflammatory cytokines/chemokines and growth factors. Plasma CyPA levels in PH patients increased according to their severity assessed by pulmonary vascular resistance (P<0.001). A positive correlation was noted between plasma CyPA levels and several inflammatory cytokines/chemokines and growth factors, including CXCL1 (P<0.001), CXCL9 (P=0.001), macrophage colony-stimulating factor (P=0.020), SDF-1α (P=0.005) and PDGF-BB (P=0.003). Interestingly, there was a significant correlation between plasma levels of CyPA and those of LDL-cholesterol (P=0.003) or HbA1c (P=0.006). In contrast, there was no correlation between CyPA and high-sensitivity CRP (P=0.172). Finally, plasma levels of CyPA and SDF-1α were significantly less in patients with statins (both P<0.01).

Conclusions:
Plasma levels of CyPA are correlated with those of circulating cytokines/chemokines and growth factors, suggesting an inflammatory role of CyPA in PH patients. These data further support the crucial role of CyPA as a pathogenic molecule and a therapeutic target in PH.
Abstract 249: Thoracic Aortic Dilation in Patients with Bicuspid Aortic Valves is Marked by Accelerated Vascular Smooth Muscle Cell Aging
Authors: Balint Brittany , Yin Hao , Nong Zengxuan , Fox Stephanie , Rogers Stephanie , O’Neil Caroline , Watson Alanna , Arpino John-Michael , Chase Lindsay , Chu Michael M , Pickering Geoffrey
Published: July 2021

Individuals with a bicuspid aortic valve (BAV) are at increased risk for ascending aortic dilation and dissection. Loss of aortic medial smooth muscle cells (SMCs) and disruption of the extracellular matrix are well-recognized pathologies, but the underlying cellular mechanisms remain elusive. We tested the hypothesis that the dilated aorta in patients with BAV was marked by accelerated cellular aging. Samples of human ascending aorta were obtained from individuals with BAV undergoing thoracic aorta replacement (n=37, age 54.7±2.2, aortic diameter 4.8±0.9 cm) or patients with a tricuspid aortic valve and non-dilated aorta undergoing heart transplantation or coronary bypass procedures (n=6, age 55.3±8.1, aortic diameter 3.1±0.3 cm). Assessment of fresh aortic samples for senescence-associated β-galactosidase revealed evidence for rare medial cell senescence that was 4.2-fold more prevalent in dilated aortas (0.83±0.10%) than in non-dilated aortas (0.20±0.10%, p=0.048). Expression of p16 was abundantly detected in medial SMCs within dilated aortas (27.0±2.1%) and 3-fold more abundant than in non-dilated aortas (8.9±1.8%, p<0.0001). Interestingly, immunostaining for γH2A.X (phosphorylated Ser139) revealed discrete nuclear DNA double-strand breakage signals in 25.7±3.8% of medial cells in dilated aortas from patients with BAV, which was 2.3-fold higher than that found in non-dilated aortas (11.0±4.9, p=0.03). CONCLUSION: These findings identify a previously unrecognized phenomenon of accelerated SMC aging in the aortas of patients with BAV, with cellular senescence and unresolved DNA breaks. Accelerated cell aging could thus be a driver of aortic wall degeneration in these patients and a potential therapeutic target.
Abstract 279: Resolvin D2 and Maresin 1 Modulate Vascular Inflammation, Cell Migration Aad Macrophage Polarization in a Mouse Model of Arterial Injury
Authors: Akagi Daisuke , Chen Mian , Toy Robert , Mottola Giorgio , Chatterjee Anuran , Conte Michael S
Published: July 2021

Introduction:
Vascular injury induces a potent inflammatory response that influences vessel remodeling and patency, limitinglimits the long-term benefits of cardiovascular interventions such as angioplasty. Specialized pro-resolving lipid mediators (SPMs) derived from omega-3 polyunsaturated fatty acids (eicosapentaenoic acid [EPA] and docosahexaenoic acid [DHA]) orchestrate resolution in diverse settings of acute inflammation. We hypothesized that systemic administration of DHA-derived SPMs (resolvin D2 [RvD2] and maresin 1 [MaR1]) would influence vessel remodelinginflammation and remodeling in a mouse model of arterial neointima formation (carotid ligation).

Methods and Results:
In-vitro, SPM treatment inhibited mouse aortic smooth muscle cell (SMC) migration (IC50@1 nM) to a PDGF gradient and reduced tumor necrosis factor-α stimulated p65 translocation, superoxide production and pro-inflammatory gene expression (MCP-1). In vivo, adult FVB mice underwent unilateral carotid arteryi ligation with administration of RvD2, MaR1, or vehicle (100ng by intraperitoneal injection at 0, 1, 3, 5 and 7 days post ligation). In ligated carotid arteries at 4 days, SPM treatment was associated with reduced cell proliferation, neutrophil and macrophage recruitment, and increased polarization of M2 macrophages in the arterial wall (M2 macrophage proportion; RvD2 62%, MaR1 51% and control 43%). Neointimal hyperplasia (at 14 days) was notably attenuated in RvD2 (62%) and MaR1 (67%) treated mice, respectively. Proliferating cells at 14 days are mainly SMCs.

Conclusion:
Modulation of resolution pathways may offer new opportunities to regulate the vascular injury response and promote vascular homeostasis.
Abstract 293: Clinical Predictors of Endothelial Function in Human Saphenous Vein
Authors: Osgood Michael J , Hocking Kyle , Wise Eric S , Sexton Kevin W , Komalavilas Padmini , Cheung-Flynn Joyce , Brophy Colleen
Published: July 2021

BACKGROUND:
Human saphenous vein (HSV) remains the most widely utilized conduit for vascular bypass procedures. Outcomes remain limited by vein graft failure and intimal hyperplasia. Endothelial function is considered an important determinant of vein graft failure. We have observed significant variability in endothelial function in freshly isolated HSV samples. We therefore evaluated clinical predictors of endothelial function in HSV.

Methods:
We obtained freshly isolated HSV samples from the operating room immediately following harvest from patients undergoing coronary artery bypass procedures from Vanderbilt University Hospital and the Nashville VA Hospital. We obtained HSV samples prior to any intraoperative manipulations. HSV viability, smooth muscle function, and endothelial-dependent relaxation (EDR) were measured in a muscle bath. We collected the following clinical and demographic information: age, height, weight, gender, ethnicity, medical comorbidities, laboratory values (creatinine, HbA1c, lipids), ejection fraction, preoperative medication regimen, and method of vein graft harvest. We performed a univariate and multivariate analysis of predictors of endothelial function in HSV.

Results:
HSV samples were obtained from 149 patients. HSV EDR varied from -11% to 63%. Open harvest was employed for 39 HSV samples, compared with endoscopic harvest in 110 HSV samples. On univariate analysis, only open harvest was a statistically significant predictor of endothelial function (p=0.02): mean HSV EDR was 21.5% in HSV samples harvested open, compared with 15.4% in HSV harvested with an endoscopic technique. HSV EDR was also improved with preoperative aspirin use: mean HSV EDR was 18% in patients with preoperative aspirin use compared with 11.6% in patients who were not administered preoperative aspirin in a multivariate model when controlling for method of vein graft harvest (p=0.05).

Conclusions:
Endoscopic vein graft harvest is associated with endothelial dysfunction in HSV, while preoperative aspirin use is associated with improved endothelial function. Further work will be necessary to determine whether these factors are associated with development of intimal hyperplasia and vein graft failure.
Abstract 304: Varicose Vein Development in Mice Is Blocked by Ator- and Rosuvastatin
Authors: Eschrich Johannes , Meyer Ralph , Pfisterer Larissa , Hecker Markus , Korff Thomas
Published: July 2021

Venous diseases such as varicose veins and chronic venous insufficiency constitute a substantial source of morbidity. Despite their high prevalence, neither molecular mechanisms initiating these diseases nor pharmaceutic therapies have been explored so far. We recently reported that chronically increased filling pressure or biomechanical venous wall stress is sufficient to cause development of varicose-like veins in mice through a mechanism preceded by activation of the transcription factor activator protein 1 (AP-1). Considering these findings, we hypothesized that pharmacologic inhibition of AP-1 activity may attenuate venous remodeling. To prove this therapeutic concept, human venous smooth muscle cells (SMCs) were subjected to biomechanical stretch (13%, 0.5Hz) and treated with statins - HMG-CoA reductase inhibitors that are supposed to interfere with the activity of AP-1. EMSA and immunofluorescence studies revealed that ator- and rosu- but not simvastatin blocked stretch-induced AP-1 activity. Furthermore, ator- and rosuvastatin inhibited expression of the AP-1 target monocyte chemotactic protein 1 (MCP-1) and proliferation of these cells. In mouse veins exposed to an increased level of intraluminal pressure, atorvastatin diminished the abundance of MCP-1 and MMP-2 by ~90 % and inhibited proliferation of endothelial cells and SMCs (n=5, p<0.05). In line with this observation, growth of varicose-like veins in mice was suppressed by almost ~80 % upon treatment with rosu- and atorvastatin but not simvastatin (n=5, p<0.05) which was accompanied by a decrease in cellular proliferation as well as MMP-2 abundance in remodeling blood vessels. Consequent analyses of human varicose vein samples from patients chronically treated with statins indicated a decrease in SMC proliferation, MCP-1 and MMP-2 abundance (50-60 %, n=10, p<0.05).

Collectively, our current findings imply that both ator- and rosuvastatin effectively inhibit the development of varicose veins presumably by interfering with the wall stress-mediated activity of AP-1 in VSMCs. For the first time, this study revealed a possible pharmaceutic treatment that may be suitable to prevent growth of varicose veins and to limit formation of recurrences after varicose vein surgery.
Abstract 307: Role of Epsins in Regulating LPS-Induced Sepsis
Authors: Pasula Satish , Brophy Megan L , Tessneer Kandice L , Hahn Scott , McManus John , Zhu Hua , Chang Baojun , Dong Yunzhou , Cai Xiaofeng , Song Hoogeun , Wu Hao , Chen Hong
Published: July 2021

Background:
Sepsis is caused by a deleterious host response to infection, which is primarily responsible for further injury of host tissue and cause of organ dysfunction. However, the underlying regulatory mechanisms are still not fully understood. Our goal is to define the novel role of epsins in regulating sepsis.

Methods and Results:
We engineered global (iDKO) and endothelial cell-specific (EC-iDKO) epsin deficient mice. When treated with lethal dose of LPS, epsin deficient mice were completely protected from LPS-induced septic death. These mice also exhibited decreased expression of tissue damage biomarkers and recruitment of neutrophils and macrophages to lungs compared to wild type (WT) suggesting that epsin deficiency mitigates sepsis induced tissue injury. Epsin deficiency further reduced expression of proinflammatory cytokines and adhesion molecules in the lungs suggesting that loss of epsin attenuates LPS-induced inflammatory responses. TAT complex production was also decreased in iDKO mice compared to WT indicating diminished coagulation and thrombin production. Knocking down of epsins in HUVECs resulted in reduced cell surface Tissue Factor (TF) expression. Loss of epsin in mice protected against loss of Thrombomodulin (TM), which is downregulated by sepsis. Mechanistically, loss of epsin inhibited LPS-induced TM internalization, while LPS treatment induced the ubiquitination of TM. Furthermore, co-IP of full length epsin 1 or epsin 1 without the UIM domain and TM demonstrated that UIM is required for the interaction between epsin 1 and TM. Collectively, we show that epsin-deficiency upregulates TM surface protein expression by preventing its internalization and subsequent degradation and inhibits heightened TF expression and activation under chronic inflammatory conditions such as that induced by LPS exposure.

Conclusions:
Our findings demonstrate that epsins play a key role in regulating coagulation and provide fundamental information on the modulation of the ratio of TM/TF in various thrombotic diseases including sepsis. Furthermore, we demonstrate loss of epsin protects mice against LPS-induced sepsis, suggesting a crucial role for epsins in promoting the development of LPS-induced sepsis.
Abstract 311: Network Analysis of Pathways Associated with Genetic Regulation of Trimethylamine-N-Oxide
Authors: Bennett Brian , Smallwood Tangi L , Wright Garron , Bostrom Meredith
Published: July 2021

Common complex diseases, such as atherosclerosis, diabetes and obesity, are the result of genetic, environmental and gene/environmental interactions. Genetic reference panels are powerful tools to interrogate how the environment interacts with the underlying genetic architecture to influence disease susceptibility. Recently 2 genetic reference panels have been created: the Diversity Outbred (DO) mouse population and Collaborative Cross (CC). The mice comprising the CC/DO are mosaics of C57BL6/J, A/J, NOD/ShiLtJ, NZO/HiLtJ, WSB/EiJ, CAST/EiJ, PWK/PhJ, and 129S1/SvImJ. Both the DO and CC populations have tremendous genetic diversity, containing approximately 45 million segregating single-nucleotide polymorphisms (SNPs).

Our primary goal was to characterize the genetic regulation of risk factors for atherosclerosis susceptibility in the 8 founder strains of the DO/CC reference panels. Using a systems genetic approach, we perform integrative analysis of clinical traits, targeted metabolomics and RNA expression. We quantitated genome wide mRNA levels using RNA sequencing followed by network analysis. The network was then related to clinical traits and a targeted panel of 5 metabolites (trimethylamine-N-oxide (TMAO), Betaine, Choline and Creatinine) to identify key groups of genes (called modules). We performed weighted gene network co-expression analysis (WGCNA) using 17,122 genes across 46 mice (N = 5-8 mice per strain). The network contains 47 independent co-expression modules and associated these modules with plasma lipoproteins, liver triglyceride levels and the novel plasma risk factors TMAO and Betaine. For example, the turquoise module was most strongly associated with TMAO levels (p < 2 x 10-8) and the black module was associated with plasma Betaine concentrations. Both modules contain several candidate genes and enrichment analysis using Database for Annotation, Visualization and Integrated Discovery (DAVID ) v6.7 Studies are ongoing to validate the key molecular drivers of the network with a particular interest in the transcriptional network regulating plasma TMAO and Betaine concentrations.
Abstract 317: CETP Alters Routes of Total and HDL-derived Cholesterol Elimination in Mice
Authors: Li Jianing , Ji Ailing , Wang Yuhuan , Pijut Sonja , Van der Westhuzen Deneys , Graf Gregory
Published: July 2021

Objective:
Transintestinal cholesterol excretion (TICE) is an alternative pathway to hepatobiliary secretion for cholesterol elimination. The cholesterol donors contributing to this pathway in plasma remain unclear, but appear to include both ApoA and ApoB lipoproteins. Cholesteryl ester transfer protein (CETP) facilitates the transport of cholesteryl esters and triglycerides between lipoproteins in plasma. Our study was aimed at determining the impact of CETP on cholesterol elimination pathways in mice.

Methods and Results:
We compared both hepatobiliary and intestinal cholesterol secretion rates in male wild-type (WT) and CETP transgenic (TG) mice at age of 12 weeks. WT and TG mice did not differ either biliary or intestinal cholesterol secretion rates when maintained on a standard chow diet. However, TG mice showed increased biliary cholesterol secretion rates and decreased intestinal cholesterol secretion rates compared to the WT group in response to high fat, high cholesterol Western diet. We next determined the effect of CETP on the delivery of radiolabeled HDL-cholesterol ester to the bile and intestinal lumen. Unlike bulk cholesterol secretion, HDL-derived cholesterol esters were preferentially delivered to the intestine in CETP transgenic mice. To further explore the mechanism, we injected radiolabeled HDLs from both WT mice (WT-HDL) and TG mice (CETP-HDL) back into male WT mice. Although CETP did not alter HDL-CE delivery to bile, cholesterol from HDL isolated from CETP TG mice were secreted into the intestinal lumen at a greater rate than WT mice.

Conclusion:
The data suggest that CETP alters routes of total and HDL cholesterol elimination from the body in mice.
Abstract 325: Response Gene to Complement 32 Deficiency Protects Against Hepatic Steatosis by Inhibiting Lipogenesis in Mice
Authors: Cui Xiaobing , Luan Junna , Chen Shiyou
Published: July 2021

Hepatic steatosis is associated with obesity due to the increased lipogenesis. Previously, we have found that RGC-32 (response gene to complement 32) deficiency prevents the mice from high-fat diet (HFD)-induced obesity and insulin resistance. The present study was conducted to determine the role of RGC-32 in the control of hepatic steatosis. We observed that hepatic RGC-32 expression was dramatically induced by HFD challenge. RGC-32 knockout (RGC32-/-) mice were resistant to HFD-induced hepatic steatosis. More importantly, hepatic triglyceride contents of RGC32-/- mice were significantly decreased compared with wild-type (WT) controls on both normal chow and HFD. Mechanistically, RGC-32 deficiency decreased expression of lipogenesis-related genes, sterol regulatory element (SRE) binding protein (SREBP)-1c, fatty acid synthase (FAS) and stearoyl-CoA desaturase-1 (SCD1). Our in vitro study showed that RGC-32 knockdown decreased while RGC-32 overexpression increased SCD1 expression in hepatocytes. Deletion or mutation of SRE in the SCD1 promoter abolished the function of RGC-32. These data demonstrate that RGC-32 contributes to HFD-induced hepatic steatosis by facilitating de novo lipogenesis in a SREBP-1c dependent manner. Therefore, RGC-32 may be a novel drug target in the treatment of hepatic steatosis and its related diseases.
Abstract 331: Effects of Statins on Adiponectin Receptor Expression on Circulating Monocytes in Hypertensive and Dyslipidemic Subjects
Authors: Gasbarrino Karina , Zheng Huaien , Daskalopoulou Stella S.
Published: July 2021

Introduction:
Ample evidence exists in support of the anti-inflammatory and athero-protective properties of statins. AdipoR1 and AdipoR2 are receptors of adiponectin, which is an adipose tissue secreted protein with anti-inflammatory properties. Reduced AdipoR1/R2 expression is associated with hypertension, diabetes, and dyslipidemia. The aim of this ongoing study is to investigate whether statin treatment can affect the expression of AdipoR1/R2 on circulating monocytes in high-risk hypertensive and dyslipidemic subjects.

Methods:
Subjects with hypertension and dyslipidemia were recruited from the Vascular Health Clinic at McGill University, Montreal. All subjects had clinical indication for statin therapy, were statin naïve, and were prescribed low dose statin. After initiation of statin therapy, subjects were followed-up every 3-4 months for the length of one year. Blood samples were collected on the day of recruitment (baseline [pre-statins]) and at each follow-up (f/u) visit. Blood monocytes were isolated using a Magnetic Cell-Sorting technique with CD14+ Human Microbeads. RNA was isolated from monocytes to assess mRNA expression of AdipoR1/R2 using quantitative RT-PCR. Recruitment and f/u visits are ongoing.

Results:
A total of 13 subjects were recruited with 8 subjects having had at least one f/u visit. Baseline age of subjects (n=8) was 65.9 ± 9.2 years, with 62.5% of subjects being men, and 37.5% diabetic. Statin significantly decreased total cholesterol (pre: 5.8±0.7 mmol/L, post: 4.1±0.5 mmol/L, P= 0.001) and LDL levels (3.9±0.6 mmol/L, 2.1±0.3 mmol/L, P<0.001). When assessing all f/u combined, AdipoR1 and AdipoR2 mRNA expression on circulating monocytes was increased post-statin therapy by 1.37 and 1.41-fold, respectively, when compared to baseline levels, with a significant difference found in AdipoR2 (P=0.05). Furthermore, a trend for progressive increase in AdipoR1/R2 mRNA expression was detected across all f/u.

Conclusion:
Statin treatment in hypertensive and dyslipidemic subjects led to increased AdipoR1/R2 mRNA expression on circulating monocytes, which illustrates a novel mechanism through which statins may exert its anti-inflammatory effects and protect against cardiovascular diseases.
Abstract 335: Atheroma-specific Delivery of Synthetic High-density Lipoprotein Containing Sphingosine-1-phosphate for Modulation of Vascular Inflammation.
Authors: Morin Emily E , Guo Yanhong , Kuai Rui , Lautner Gergely , Meyerhoff Mark E , Chen Y. Eugene , Schwendeman Anna
Published: July 2021

Introduction:
Sphingosine-1-phosphate (S1P) is a potent anti-inflammatory signaling lipid carried in the body by circulating HDL. HDL has been shown to exhibit anti-inflammatory activities through activation of endothelial nitric oxide synthase (eNOS) and subsequent production and release of nitric oxide (NO) by endothelial cells.

Objective:
The aim of this study is to use synthetic HDL particles to selectively deliver S1P to the site of arterial plaques in order to exert anti-inflammatory activity and modulate the progression of atherosclerosis.

Methods/Results:
Synthetic HDL (sHDL) particles were prepared using the ApoA1 mimetic peptide 22A (PVLDLFRELLNELLEALKQKLK), dipalmitoylphosphatidylcholine (DPPC) and sphingomyelin. We also prepared sHDL containing either the hydrophobic dye, DiD, or S1P to assess the capability of sHDL to effectively reach atheroma site and induce nitric oxide (NO) release, respectively. The purity of all particles was determined to be > 97% and average particle size was 9.6 ± 0.4 nm for all preparations. To measure sHDL accumulation in the plaque, ApoE
-/-
mice were intravenously injected with 0.2 mg/kg HDL-DiD. Whole aortas were excised and analysed by IVUS imaging system, revealing significant accumulation of sHDL-DiD in the atherosclerotic lesions. We then tested the ability of sHDL to deliver S1P
in vitro
and induce NO production by treating human umbilical vein endothelial cells (HUVEC) with 1 mg/mL of 22A-DPPC-sHDL containing 0, 0.05, 0.5, or 5 nmol/mL of S1P using free 22A peptide (1 mg/mL) and saline as controls, and analyzing media by ozone chemiluminescence. Blank sHDL particles increased NO production two-fold over controls (0.27 ± 0.02 μM for 22A-DPPC-sHDLDL, 0.13 ± 0.01 μM PBS and 0.14 ± 0.02 μM for 22A peptide), while HDL-S1P further increased NO release: 0.35 ± 0.03, 0.44 ± 0.01, and 0.59 ± 0.01 μM for HDL with 0.05, 0.5, and 5 nmol/mL S1P, respectively.

Conclusions:
Our studies show that HDL is capable of delivering hydrophobic cargo to atherosclerotic plaques, making HDL a promising platform to deliver S1P for modulation vascular inflammation and atherosclerosis.
In vitro
studies have revealed that HDL-S1P is able to increase NO production 2 to 4-fold over saline control setting the basis for future
in vivo
studies.
Abstract 340: The Expression and Function of Apol6 in Atherosclerotic Plaques of

Apoe
-/-

Mouse Aorta Tissue
Authors: Zhaorigetu Siqin , Hu Chien-An A , Laskey Warren , Walton Brian
Published: July 2021

Background:
We recently demonstrated that apolipoprotein L6 (ApoL6) regulates apoptosis and autophagy in atherosclerotic lesions, implying that ApoL6 is an important factor that causes plaque instability and a potential therapeutic target for treating atherosclerosis. To further investigate the role of ApoL6 in atherogenesis
in vivo
, the expression of ApoL6 was knocked down by the liposome-siRNA strategy in the aorta tissue of

ApoE
-/-

mouse.

Methods:
Liposomal siRNAs were prepared by using the thin-film hydration method and were labeled with quantum dots (QD).

ApoE
-/-

mice were intravenously injected twice in 1 week with either liposomal control siRNA-QD or liposomal
ApoL6
siRNA-QD. We harvested aortic tissue from mice and used immunofluorescence staining to analyze the expression of ApoL6 in atherosclerotic plaque.

Results:
Immunofluorescence analysis showed that expression of ApoL6 was elevated in the atherosclerotic plaque and partially co-localized with a macrophage marker CD68 in

ApoE
-/-

mouse. The results suggest a link between ApoL6 and macrophages in the pathobiology of atherosclerotic lesions (
Fig. A and B
). Confocal microscopy images showed that liposomal
ApoL6
siRNA significantly reduced ApoL6 expression (green punctures) in atherosclerotic plaques as compared with liposomal control siRNA
(Fig. C)
.

Conclusion:
We established a silencing model of ApoL6 in cardiovascular system of ApoE
-/-
mouse using liposome-mediated siRNA delivery system. The intravenous injection of liposomal
ApoL6
siRNA silences ApoL6 expression in the aortas of

ApoE
-/-

mice and may protect against the development of atherosclerosis.