bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 10: Systemic Administration of an Optimized Human Apyrase Inhibits Acute Thrombosis and Neointima Formation in Vein Grafts
Authors: Ji Yan , Strawn Tammy L , Fay William P , Chen Ridong
Published: March 2022

Introduction.
Occlusion of vein grafts (VGs) after bypass surgery is a major clinical problem. Apyrase (CD39) plays a key role in inhibiting thrombosis and vascular inflammation by scavenging ADP and ATP at sites of vascular injury. We examined the capacity of APT102, a recombinant mutant human apyrase with enhanced activity and prolonged half-life, to inhibit thrombosis and intimal hyperplasia in VGs.

Methods and Results.
Segments of inferior vena cava from C57BL/6J donor mice were grafted into carotid arteries of recipient mice. APT102 (1 mg/kg) or saline was delivered by intraperitoneal (IP) injection every 2 days after surgery. To determine if APT102 inhibits VG thrombosis, VGs were exposed 6 days after surgery, injured with ferric chloride, and formation of thrombi was assessed by monitoring blood flow. Within 60 minutes after injury an occlusive thrombus formed in 1 of 7 mice treated with APT102 vs. 5 of 6 mice treated with vehicle control (P<0.05). No abnormal bleeding was observed in APT102-treated mice. To examine the effect of APT102 on neointima formation, mice received APT102 or vehicle control by IP injection for 2 weeks after surgery. Intimal hyperplasia in VGs was measured 2 weeks later (i.e. 4 weeks after surgery). Mean intima thickness was significantly less (P<0.05) in VGs of APT102-treated mice (26±8.4 μm, n=4) than in VGs of saline-treated mice (48±9.4 μm, n=5).

Conclusions.
Systemic administration of a recombinant human apyrase inhibits thrombosis and intimal hyperplasia in VGs without increasing bleeding. These results suggest that APT102 has the potential to become a novel treatment strategy to prevent thrombotic occlusion and adverse remodeling of VGs.
Abstract 109: Role of ApoAIV in ApoB Lipoprotein Assembly and Triglyceride Transport
Authors: Fabritius Melissa A , MacArthur Philip S , Betters Jenna L , Brown J Mark , Shelness Gregory S
Published: March 2022

Apolipoprotein A-IV (apoAIV) is a lipid binding protein expressed primarily in mammalian intestine and also in rodent liver. While in vitro studies have suggested a role of apoAIV in lipoprotein formation and triglyceride (TG) transport, limited evidence supports such a function in vivo. To explore if hepatic apoAIV is uniformly regulated by increased hepatic TG levels, as is the case in the intestine, we studied three distinct mouse models of hepatic steatosis. These included high fat diet (HFD)-fed wild type mice, mice overexpressing a constitutive active form of SREBP-1a (SREBP-1aTg), and mice in which hepatic TG hydrolysis is blunted (mice treated with an anti-sense oligonucleotide (ASO) targeting knockdown of CGI-58/ABHD5). High fat diet-fed mice displayed a 4.5-fold increase in liver TG content and a 10-fold increase in apoAIV mRNA expression and protein mass. SREBP-1aTg mouse liver demonstrated an ∼35-fold increase in hepatic TG, apoAIV mRNA and apoAIV protein. A linear correlation was observed between hepatic TG content and apoAIV mRNA expression in the HFD fed wild type and SREBP-1aTg mice (r2 = 0.6852; p < 0.01). Interestingly, while the TG content of CGI-58/ABHD5 knockdown animals increased by 4-fold, no corresponding increase in apoAIV expression was observed. To determine if the increase in apoAIV expression in the steatotic livers of SREPB-1aTg mice plays a role in the secretion of TG-rich apoB-containing lipoproteins, we crossed these mice with apoAIV knock out mice (A4-KO). The SREBP1a-Tg/A4-KO mice did not demonstrate a change in liver to body weight ratio or a change in hepatic TG content. Despite the large induction of apoAIV in the livers of SREBP1a-Tg mice, no difference was observed in hepatic TG production rates or VLDL particle size when apoAIV was deficient (SREBP1a-Tg/A4-KO). These results indicate that hepatic apoAIV expression is upregulated under conditions of enhanced lipogenesis, but is not altered under condition of defective TG hydrolysis. Furthermore, even under conditions of a 35-fold induction, apoAIV does not exert a detectable impact on TG transport or VLDL particle characteristics in mouse liver.
Abstract 122: Circulating CD28- T Cells Are Associated with Histological Features of Carotid Plaque Instability
Authors: Doonan Robert-James , Theocharis Stamatios , Cote Claudia , Gomez Yessica , Daskalopoulou Stella S
Published: March 2022

Background:
Circulating CD28- T-cells have been found to be higher in patients with acute coronary syndromes, suggesting a role for these cells in plaque instability. However, the association between circulating CD28- T-cells and carotid plaque instability as assessed by histology/immunohistochemistry has not been investigated.

Methods/Results:
40 patients referred for carotid endarterectomy were recruited pre-operatively at the Royal Victoria Hospital in Montreal, Canada. A blood sample was obtained pre-operatively. Flow cytometry was performed on whole blood using a CD3, CD4, CD28 antibody cocktail (eBioscience, San Diego, United States) and a BD Bioscience (Mississauga, Canada) flow cytometer. Data was acquired as percentage of CD4+CD28- T-cells. The carotid plaque specimen was obtained from the operating room, fixed, decalcified, embedded in paraffin, and sections of 4μm were obtained from the site of maximum stenosis. Sections were stained with hematoxylin and eosin and with anti-CD3 (lymphocytes), anti-CD68 (macrophages/foam cells), and von Willebrand Factor (vWF, neovessels, all antibodies from Dako, Burlington, Canada). A vascular pathologist classified the plaques according to American Heart Association (AHA) classifications and graded CD3, CD68, and vWF staining on a semi-quantitative scale.

Patients with more advanced lesions (AHA type 5 or 6) had a greater percentage of circulating CD4+CD28- T-cells [2.9% (2.7-5.7)] compared to patients with lower classifications [1.6% (1.0-4.2), P<0.05]. Furthermore, patients with plaques that had ≥10 new vessels per section (feature of plaque instability) had a median of 4.5% [2.8-8.0] CD4+CD28- T-cells whereas patients with <10 new vessels per section had only 2.3% [0.8-5.0] (P<0.05). There were no significant differences when patients were separated according to high/low plaque lymphocytes/macrophages.

Conclusion:
We have shown that circulating CD28- T-cells are found in greater amounts in patients who have more advanced/unstable carotid plaques as assessed by histology and immunohistochemistry. This may point to a role for CD28- T-cells in the pathogenesis of atherosclerosis. Further studies are currently being carried out to confirm these results.
Abstract 124: Survival and Limb Salvage Following Lower-Extremity Revascularization Using Cryopreserved Saphenous Vein Allograft
Authors: Flohr Tanya , Cherry Kenneth , Upchurch Gilbert , Kern John , Tracci Margaret
Published: March 2022

OBJECTIVE:
Revascularization options for critical limb ischemia are often limited by the lack of autologous vein conduit, particularly in the setting of infection or distal targets that render the use of synthetic graft undesirable. Cryopreserved saphenous vein allograft (CSVA) may provide an important alternative. We conducted a retrospective study of patients undergoing lower extremity revascularization at a single institution using CSVA.

METHODS:
Between April 2009 and June 2011, 18 CSVA were used for bypass (12 men) with a median age of 65 (range, 45-88) years. Procedures included 14 femoral-distal bypasses (5 femoral-below-knee popliteal, 9 femoral-tibial bypasses) and 4 femoral-femoral bypasses. No immunosuppression was used. Follow-up was continued until conduit revision, amputation, death, or the end of the study period. End points included patency, limb salvage, and survival.

RESULTS:
Comorbidities included smoking history (95%), hyperlipidemia (73%), hypertension (68%), coronary artery disease (52%), diabetes (32%), obesity (16%), and atrial fibrillation (21%). The majority of patients had undergone multiple failed endovascular or open revascularization procedures prior to the use of CSVA. Clinical presentations included initial bypass thrombosis (26%), infection involving synthetic conduit (16%), stenosis (16%), and anastomotic pseudoaneurysm (5%). CSVA was used at the index procedure in 31% of cases, only when autologous vein was found to be inadequate for distal bypass. Complications following CSVA implantation included thrombosis (31%), stenosis (16%), infection (5%), aneurysmal degeneration (15%), and anastomotic pseudoaneurysm (5%). Survival was 78% with a median follow-up time of 285 (range, 2-850) days. Limb salvage was achieved in 12 of 14 surviving patients (86%) with graft patency maintained in 10 of 14 patients (71%) over a median 300 (range, 60-850) days.

CONCLUSION:
CSVA may provide an important option in clinical situations where autologous vein is not available and prosthetic bypass graft undesirable due to low patency rates or high rates of reinfection. Further elucidation of factors related to CSVA bypass patency should guide patient selection and management.
Abstract 127: Differential Role of Quercetin Treatment on MicroRNA Signaling and Lipid Metabolism in HepG2 Cells Exposed to 13-HPODE
Authors: Mahini Halleh , Mendoza Nathalia , Garelnabi Mahdi
Published: March 2022

Background:
Quercetin is a member of the bioflavonoids family known to have antiatherogenic, antiinflammatory, and antihypertensive properties; and reported to have cardiovascular protective role. Quercetin through its antioxidant action may protect against oxidative stress induced by free radicals consequently decreasing the potential for modification of low density lipoprotein cholesterol (LDL). Members of a class of non-coding RNAs termed microRNAs have recently been identified to be potent post-transcriptional regulators of lipid metabolism genes, including cholesterol homeostasis. Data from our ongoing study on mice have demonstrated that quercetin mediates lipoprotein gene expression and host of MicroRNAs impacting plasma lipoprotein levels.

Aims and Objectives:
The goal of this study is to investigate the beneficial protective effect of Quercetin on HepG2 cell line subjected to the treatment of 13-HPODE an oxidatively modified linoleic acid derivative, and also to investigate the effects of treatments on specific group of MicroRNAs.

Study Design and Methods:
We treated HepG2 cell line with two different Quercetin concentrations: 20 and 40 μmoles in the presence or absence of 13-HPODE an oxidizing agent or Linoleic acid as control. After 12 and 24 hours of treatments, RNAs from treated cells were extracted and gene expressions in the various conditions were performed for selected lipoproteins genes specifically APOA1, ApoC3, APOA5, PPAR-alpha, NPCL1, and ABCA1 examining the protective role of Quercetin against 13-HPODE treated HepG2 cells. In addition we investigated role of quercetin and 13-HPODE on MicroRNAs 21, 29a,155 and 451 In addition; serum deprived mediums were collected and markers of oxidative stress and inflammations, PON1, TNF-alpha, and MCP1 were analyzed using commercially available ELISA kits.

Results and Discussion:
Our data excitingly shows that Quercetin differentially influences the levels of lipoprotein gene expression, MicroRNAs and oxidative stress and inflammatory markers in HepG2 cells and the medium. Interestingly Quercetin seems to down regulate ApoC3 mRNA expression, and differentially affects ApoA1 and ApoA5 genes and MicroRNA levels. Serum deprived cells TNF-alpha levels seem to follow the prototype shown by the cellular changes, we also did not see dose dependant responses in any of the treatments.

Conclusion:
Liver cells are responsible for a major part of lipoprotein metabolism, however our studies using HepG2 cells have demonstrated that Quercetin although is known for its antioxidant properties, did not seems to have dose dependant responses to modulate the oxidative stress induced by 13-HPODE and differentially affect the levels of lipoprotein gene expression.
Abstract 130: Lysophosphatidylcholine Acyltransferase 3 Knockdown-Mediated Liver Lysophosphatidylcholine Accumulation Promotes Very-Low-Density Lipoprotein Production by Enhancing Microsomal Triglyceride Transfer Protein Expression
Authors: Li Zhiqiang , Pan Xiaoyue , Li Yan , Hussain M Mahmood , Jiang Xian-Cheng
Published: March 2022

Phospholipids undergo extensive remodeling by the Lands’ Cycle after de novo biosynthesis. Enzymes involved in phospholipid biosynthesis have been studied extensively but not those involved in remodeling. One key enzyme in the Lands’ Cycle is fatty acyl-CoA:lysophosphatidylcholine acyltransferase (LPCAT) which utilizes lysophosphatidylcholine (LysoPC) and fatty acyl-CoA to produce various phosphatidylcholine (PC) species. Four isoforms of LPCAT have been identified. We found, in this study, that LPCAT3 is the major hepatic isoform and its knockdown significantly reduces hepatic LPCAT activity. We also found that adenovirus-shRNA-mediated LPCAT3 knockdown in mice significantly increases different LysoPC with 16:0, 18:0, 18:1, and 18:2 fatty acids and reduces certain PC species in the liver. More importantly, LPCAT3 knockdown significantly reduces hepatic triglyceride levels but induces plasma triglyceride and apoB levels. Lipoprotein production studies indicated that LPCAT3 deficiency enhanced triglyceride-rich apoB-containing lipoprotein assembly and secretion. Further, the knockdown mice had significantly higher microsomal triglyceride transfer protein (MTP) mRNA and protein levels. Mechanistic studies in hepatoma cells revealed that LysoPC enhances secretion of apoB but not apoA-I in a concentration dependent manner. Moreover, LysoPC increased MTP mRNA, protein, and activity levels. In short, these results indicate that hepatic LPCAT3 modulates VLDL production by regulating LysoPC levels and MTP expression.
Abstract 135: Evaluation of the Effects of Glimepiride (Amaryl) and Repaglinide (Novonorm) on Atherosclerosis Progression in High Cholesterol-Fed Male Rabbits
Authors: Alamran Fadhil
Published: March 2022

Atherosclerosis is an inflammatory disease of the blood vessel wall, characterized in early stages by endothelial dysfunction, recruitment and activation of monocyte/macrophages. Glimepiride is one of the third generation sulphonylurea drugs useful for control of diabetes mellitus type two and it may exert anti inflammatory activity by induction of nitric oxide production or through selective suppression of the cyclooxygenase pathway . Repaglinide is a new hypoglycemic agent, a member of the carbamoylmethyl benzoic acid family. Some results from the literature demonstrate that repaglinide has a favorable effect on the parameters of antioxidative balance.

Objectives:
The objective of present study was to assess the effect of glimepiride and repaglinide on atherosclerosis via interfering with inflammatory and oxidative pathways.

Methods:
twenty four local domestic male rabbits were involved in this study. The animals were randomly divided into four groups;
Group I
rabbits fed normal chow (oxiod) diet for 10 weeks.
Group II
rabbits fed with 1% cholesterol enriched diet. Group III rabbits fed with 1% cholesterol enriched diet together with Glimepiride (0.1mg/kg once daily before morning feed).
Group IV
rabbits fed with 1% cholesterol enriched diet together with Repaglinide (0.3mg/kg once daily before morning feed). Blood samples were collected before (0 time) and every two weeks of experimental diets for measurement of serum triglycerides (TG), total cholesterol (TC), HDL-C, high sensitive C - reactive protein (hsCRP), IL-6 and TNF-α level. At the end of 10weeks the aorta was removed for measurement of aortic Malondialdehyde (MDA), reduced glutathione (GSH) and aortic intimal thickness.

Results:
Glimepiride and repaglinide treatment did show significant effect on lipid parameters compared with induced untreated group (
P
> 0.05).Also they were significantly reduced the elevation in hsCRP, IL-6, TNF-α, aortic MDA and aortic intimal thickness compared with induced untreated group (
P
< 0.05),and they restore aortic GSH level (
P
< 0.05) .

Conclusions:
Glimepiride and repaglinide may reduce atherosclerosis progression in hypercholesterolemic rabbit via interfering with inflammatory and oxidative pathways without affecting lipid parameters.
Abstract 14: The Helix-Loop-Helix Transcription Factor nPAS4 Induces Sprouting Angiogenesis and Regulates Tip Cell Formation
Authors: Schmidt Mei , Heck Sophia , Charlet Anne , Heinke Jennifer , Engert Bianca , Epting Daniel , Kramer-Zucker Albrecht , Patterson Cam , Bode Christoph , Moser Martin
Published: March 2022

Introduction:
Neuronal PAS4 (nPAS4) -formerly known as LE-PAS- is a helix-loop-helix-PAS (HLH-PAS) transcription factor that has recently been cloned as the vertebrate homologue of the drosophila protein dysfusion (
dys
). In drosophila, the lack of
dys
is characterized by impaired midline fusion of tracheal tubes. Forced expression of
dys
results in aberrant sprouting of tracheal tubes in drosophila. In neurons, nPAS4 plays a role in the formation of GABA-releasing synapses. Based on the morphological similarity between tracheal tube formation and angiogenesis, we aimed to investigate the role of nPAS4 in angiogenesis in vertebrates.

Methods and Results:
RT-PCR and Western blotting analyses showed that nPAS4 is expressed at low levels in various endothelial cell lines. The expression of nPAS4 is not upregulated by angiogenic growth factors but is induced by hypoxia. Moreover, membrane depolarization using potassium chloride results in a calcium-dependent upregulation of nPAS4. When nPAS4 expression is blocked via siRNA, endothelial cell sprouting is significantly reduced. In contrast, overexpression of nPAS4 in endothelial cells results in enhanced sprouting and branching in two- and three-dimensional models. Indeed the formation of tip cells is dependent on the presence of nPAS4 in the endothelial cell spheroid sprouting model. Accordingly, when nPAS4 is overexpressed, tip cell formation is strongly enhanced. Furthermore, the number of filopodia -a typical feature of tip cells- is increased in nPAS4 overexpressing cells, as visualized by actin staining in immunocytochemistry. In a Transwell Migration Assay, endothelial cell migration was found to be inhibited by nPAS4. Further experiments traced this back to a stronger adhesion of the cells to different extracellular matrices. Using fli-GFP transgenic zebrafish as an
in vivo
model, we found that a knock-down of nPAS4 causes disturbed intersomitic blood vessel formation.

Conclusions:
NPAS4 is a novel HLH-PAS transcription factor that regulates endothelial tip cell formation and endothelial cell sprouting.
Abstract 147: Cell Selective Cardiovascular Biology of mPGES-1
Authors: Chen Lihong , Yang Guangrui , FitzGerald Garret A
Published: March 2022

Background—
Global deletion of mPGES-1 attenuates the response to vascular injury without increasing predisposition to thrombogenesis or hypertension. However, enzyme deletion results in cell specific differential utilization of accumulated substrate. Here, we generated mice deficient in mPGES-1 in vascular smooth muscle cells (VSMC), endothelial cells (ECs) and myeloid cells to further elucidate the cell selective roles of this enzyme in cardiovascular biology.

Results—
VSMC and EC mPGES-1 deficient mice were generated by mating mPGES-1
flox/flox
mice with SM22Cre or Tie2Cre mice respectively. Blood pressure was unaltered in these mutants at baseline or in response to a high salt diet. The propensity to induced thrombogenesis in a microvascular preparation was similarly unaltered. By contrast, both mPGES-1
flox/flox
SM22Cre and mPGES-1
flox/flox
Tie2Cre mice exhibited a markedly exaggerated neointimal hyperplastic response to wire injury of the femoral artery compared to their mPGES-1
flox/flox
littermate controls (intima to media ratio 4.36 ± 0.63, 3.27 ± 0.23 versus 2.04 ± 0.31, p<0.01, n=8-13). The severity of neointimal hyperplasia was associated with increased proliferating cell nuclear antigen (PCNA) and tenascin-C (TN-C) expression. Macrophage mPGES-1 deficient mice (Mac-mPGES-1-KO) were generated by mating mPGES-1
flox/flox
mice with LysMCre mice. In these mice, the hyperplastic response to vascular injury was dramatically suppressed (intima to media ratio 2.59 ± 0.18 versus 1.47 ± 0.25, p<0.01, n=10) as was the expression of PCNA and TN-C. In vitro, conditioned medium derived from LPS-primed Mac-mPGES-1-KO macrophages was less potent than that from Mac-mPGES-1-WT macrophages in inducing VSMC proliferation and migration.

Conclusions—
Deletion of mPGES-1 in vascular vs. myeloid cells results in contrasting vascular phenotypes. While enzyme knocked out in vascular cells does not augment thrombosis or predispose to hypertension, EC and VSMC mPGES-1 restrains the response to vascular injury. mPGES-1 in myeloid cells, by contrast, promotes the response to vascular injury and represents a potential therapeutic target.
Abstract 148: Control of NADPH Oxidases Stability and Inflammatory Response by Heat Shock Protein 90
Authors: Chen Feng , Yu Yanfang , Catravas John D , Fulton David J
Published: March 2022

Previously we have shown that Hsp90 binds to NADPH oxidases (Nox) and regulates ROS production. The goals of this study were to determine the mechanisms regulating Hsp90-dependent Nox protein stability and whether inhibition of ROS production accounts for the anti-inflammatory properties of Hsp90 inhibitors. The expression of Nox 1, 2 and 5 proteins were decreased by Hsp90 inhibitors in different cell types (COS-7, HPAEC, HAEC, HL-60, murine macrophages and RAW264.7) and also in intact mouse lung. To determine the mechanisms underlying the decrease in Nox protein stability in the presence of Hsp90 inhibitors, we employed COS-7 cells expressing Nox5. The expression of Nox5 protein, but not mRNA, was robustly decreased by Hsp90 inhibitors. Hsp90 inhibitors promoted Nox5 ubiquitination and degradation which was prevented by inhibitors of the proteasome. Inhibition of Hsp90 upregulates the expression of Hsp70 and we found that Hsp70 binds to Nox5 and can promotes its degradation. The Hsp70-regulated ubiquitin ligase, CHIP associated with Nox5 and promoted increased ubiquitination and degradation. In isolated murine macrophages in vitro, we found that Hsp90 inhibitors robustly decreased the expression of ROS-sensitive inflammatory genes, including MCP-1, TNF-alpha, Cox2, M-CSF and STAT3. In vivo, Hsp90 inhibitors prevented the LPS-induced upregulation of MCP-1 and TNF-alpha in mouse lung. We conclude that Hsp90 binds to Nox and facilities its proper folding and ROS production. In contrast, the binding of Hsp70 and CHIP to Nox proteins induces degradation and reduced ROS. The effects of Hsp90 inhibitors on ROS-sensitive inflammatory proteins suggest that this pathway is a significant mechanism underlying the anti-inflammatory actions and may have important implications in controlling inflammation in cardiovascular diseases.
Abstract 15: Differential Activation of HIF1-α Is Associated with Reduced Arteriogenesis After Hindlimb Ischemia in PCAF
-/-
Mice
Authors: Bastiaansen A J , van der Pouw-Kraan T C , Peters H A , Quax P H , Nossent A Y
Published: March 2022

Although arteriogenesis is generally considered to be a shear stress-driven process, hypoxia-inducible factor-1α (HIF-1α) has a strong stimulatory effect on arteriogenesis. Recently, it was shown that the P300/CBP-associated factor (PCAF) acetylates (and activates) HIF-1α at Lys674 and can act as a co-factor in transcriptional activation of HIF-1α target genes (1). We investigated whether PCAF plays a role in arteriogenesis.

Wildtype C57Bl/6 mice or C57Bl/6 PCAF
-/-
mice were subjected to double ligation of the femoral artery. Blood flow recovery was followed over time using Laser Doppler Perfusion Imaging (LDPI) and after 28 days, animals were sacrificed and adductor muscles harvested for immunohistochemistry. PCAF
-/-
mice showed marked decrease in blood flow recovery compared to wildtype mice. Also, when staining for α-SMA, less and smaller collateral arteries were observed in the adductor muscle of PCAF
-/-
mice compared to wildtype mice. To study which genes were regulated by PCAF during arteriogenesis, wildtype and PCAF
-/-
C57Bl/6 mice were sacrificed 24 hours after double femoral artery ligation. Gene expression profiles in the adductor muscles of the mice were obtained by microArray and compared to expression patterns in the adductor muscle before femoral artery ligation. Transcription factor binding site analyses showed strong up-regulation of a large number of HIF-1α induced genes in both mouse strains. However, besides a group of 93 jointly up-regulated HIF-1α induced genes, each strain also showed up-regulation of a different set of HIF1α induced genes. The most striking gene that was up-regulated in PCAF
-/-
mice only, was Sirtuin 1 (SIRT1). Recently, it was shown that SIRT1 counteracts PCAF by deacetylating Lys674 of HIF-1α, thereby blocking p300 recruitment to HIF-1α (2). Up-regulation of SIRT1 in PCAF
-/-
mice further contributes to differential activation of HIF-1α under ischemia. Although HIF-1α is activated and can still induce up-regulation of a specific subset of its target genes in PCAF
-/-
mice, this no longer leads to efficient recovery of blood flow after hind limb ischemia.

(1) Xenaki G et al. Oncogene 2008; 27(44)

(2) Lim JH et al. Mol Cell 2010; 38(6)
Abstract 150: Natural Killer Cells Are Crucially Involved in Vascular Remodeling and Intimal Hyperplasia
Authors: de Vries Margreet R , Seghers Leonard , van Bergen Jeroen , Toes Rene , Quax Paul H
Published: March 2022

Introduction
Immune cells such as monocytes and T cells but also NK cells are proven contributors to atherosclerosis. Interestingly, C57BL/6 mice display profound induced vascular remodeling in arteriogenesis whereas BALB/c mice show less remodeling. Moreover, these two strains differ in their NK cell receptor gene complex (NKC), which codes for activating and inhibitory NK cell receptors. We hypothesize that NK cells are crucially involved in vascular remodeling and intimal hyperplasia formation.

Methods and Results
Vascular remodeling was studied in two models; injury induced restenosis by femoral artery cuff placement and vein graft remodeling by placement of a caval vein interpositioning in the mouse carotid artery.

NK cell depletion with NK1.1 antibodies in C57BL/6 mice showed significant less (2266um
2
) cuff-induced intimal hyperplasia than control mice (5252um
2
) did. Similar results were found in NK-cell deficient mice, which showed an intimal hyperplasia of (2261um
2
) after cuff-placement compared to control mice (3279um
2
). After cuff-placement in CMV1
r
mice which are congenic for the C57BL/6 NKC, an intimal hyperplasia of 4745um
2
was found, comparative to C57BL/6 (4459um
2
), and significantly more than BALB/c mice (1247um
2
).

CMV1
r
mice showed stronger intimal hyperplasia in vein grafts (0.22mm
2
) compared to both C57BL/6 (0.15mm
2
) and BALB/c mice (0.11mm
2
) Importantly, CMV1
r
displayed equal amounts of CD45 positive cells to that of C57BL/6 mice, whereas BALB/c mice show hardly any leukocyte influx into the vessel wall. Flow cytometric analysis of NK cell responsiveness to specific NK receptor mediated activation by NKp46 in C57BL/6, BALB/c and CMV1
r
splenic NK cells resulted in a significantly improved responsiveness of CMV1
r
NK cells when compared to BALB/c NK cells to the level of C57BL/6 NK cells.

Conclusion:
These data demonstrate that NK cells but more specific, the C57BL/6 NK cell gene locus is involved in the induction of vascular remodeling. Furthermore, the observed differences in inflammatory infiltrate and NK cell responsiveness between the strains strongly suggest that the C57BL/6 NK receptor repertoire might be involved in triggering an immune response that is associated with vascular remodeling.
Abstract 154: Vaccination Using Advanced Glycation End Product of Low-Density Lipoprotein Pulsed Dendritic Cells Reduces Atherosclerosis in Diabetic Apoe
-/-
Mice
Authors: Liu Hongying , Ge Junbo
Published: March 2022

Rationale and Objective
Accumulation of advanced glycation end-product of low density lipoprotein (AGE-LDL) attributable to hyperglycemia in diabetes contributes to the progression of atherosclerosis (AS). Previous studies showed that immunization against modified LDL reduce AS in animal models. Due to their potent capacity to stimulate T cells, dendritic cells (DCs) are being investigated in immunotherapy approaches. In this study, we immunized AGE-LDL pulsed DCs to diabetic apoE
-/-
mice and examined the effects on AS.

Methods and results
The present study explored AS in streptozotocin-induced diabetic apoE
-/-
mice that were randomized (n=36) to receive no treatment, AGE-LDL pulsed DCs immunization, or ovalbumin (AS-irrelevant antigen) pulsed DCs immunization. Bone marrow-derived DCs were cultured for 10 days in the presence of granulocyte-macrophage colony-stimulating factor. Immature DCs were matured by lipopolysaccharide and pulsed with AGE-LDL or ovalbumin. These DCs were transferred five times to diabetic apoE
-/-
mice before the induction of AS by western-type diet feeding. Vaccination using AGE-LDL pulsed DCs resulted in a marked reduction in aortic root and carotid artery lesion size, and an increase in plaque stability which was independent of hypercholesterolemia in mice. Transfer of ovalbumin pulsed DCs did not influence lesion size or stability. Vaccination using AGE-LDL pulsed DCs resulted in AGE-LDL-specific Th1 cell activation, and induction of AGE-LDL-specific IgG. Furthermore, AGE-LDL pulsed DCs transfer resulted in impressed function of macrophages with a marked inhibition of TLR4-mediated signaling pathway.

Conclusion
These findings demonstrate for the first time that vaccination with AGE-LDL-pulsed DCs provides a novel strategy for the immunomodulation of atherosclerosis in diabetes.
Abstract 155: Antiatherosclerotic Effects of a Novel Pharmacologic Inhibitor of Myeloperoxidase in Atherosclerosis
Authors: Liu Cuiqing , Desikan Rajagopal , Ying Zhekang , Gushchina Liubov , Kampfrath Thomas , Jeffrey Deiuliis , Wang Aixia , Xu Xiaohua , Zhong Jixin , Rao Xiaoquan , Sun Qinghua , Maiseyeu Andrei , Parthasarathy Sampath , Rajagopalan Sanjay
Published: March 2022

Objective:
Inflammation and oxidative stress play fundamental roles in the pathogenesis of atherosclerosis. Myeloperoxidase (MPO) has been extensively implicated as a key mediator of inflammatory and redox-dependent processes in atherosclerosis including reverse cholesterol transport. In this study, we investigated safety and efficacy of a novel small molecule inhibitor of MPO (INV-315) in atherosclerosis.

Methods and results:
ApoE
-/-
mice were randomized to low- and high-dose INV-315 groups for 16 weeks on high-fat diet. INV-315 resulted in reduced plaque burden and improved endothelial function in response to acetylcholine (26±4, 25±3 and 36±2% for atherosclerosis burden respectively and 42±7, 49±11 and 60.3±6% for maximal relaxation to acetylcholine in low, high dose and control groups respectively, p<0.05 for high-dose vs. placebo for both). These effects occurred without adverse events or changes in body weight or blood pressure. INV-315 treatment resulted in a 2-3.5 fold decrease in iNOS gene expression, 1.9 fold decrease in superoxide production and 2-3.5 fold decrease in nitrotyrosine content in the aorta. Circulating IL-6 and inflammatory CD11b
+
/Ly6G
low
/7/4
hi
monocytes were significantly decreased in response to INV-315 treatment. Acute pretreatment with INV-315 blocked TNFα-mediated leukocyte adhesion in cremasteric venules and inhibited MPO activity. Cholesterol efflux was significantly increased by high-dose INV-315 via ex-vivo reverse cholesterol transport assays.

Conclusions:
MPO inhibition may exert anti-atherosclerotic effects via inhibition of MPO-mediated oxidative stress and enhancement of cholesterol efflux. These findings demonstrate a role for pharmacologic modulation of MPO in atherosclerosis.
Abstract 157: Bone Marrow Deficiency of the NR4A Orphan Nuclear Receptor NOR1 Accelerates Atherosclerosis by Increasing Myeloid Progenitor Cell Proliferation
Authors: Qing Hua , Zhao Yue , Jones Karrie L , Heywood Elizabeth B , Bruemmer Dennis
Published: March 2022

The NR4A orphan nuclear receptor NOR1 functions as a constitutively active transcription factor regulating cellular inflammation and proliferation during atherosclerosis formation. In the present study, we characterize NOR1 as a suppressor of hematopoietic stem cell proliferation. NOR1 deficiency in mice induces the expansion of myeloid progenitor cells in the bone marrow resulting in monocytosis and splenomegaly in mice. In addition to increasing the proliferation of macrophage dendritic cell progenitors (MDP), NOR1 depletion induces a proinflammatory phenotype with increased numbers of Ly6+ monocytes. Bone marrow transplantation experiments confirmed monocytosis and increased Ly6+ monocytes in mice reconstituted with NOR1-deficient bone marrow, indicative of a defect intrinsic to the transplanted hematopoietic cells. Using a thioglycollate-induced peritonitis model, deletion of the NOR1 locus was associated with increased recruitment of macrophages into the exudate following peritoneal lavage. Finally, we demonstrate that bone marrow transplantation of lethally irradiated apoE-deficient mice with NOR1-deficient hematopoietic stem cells increases atherosclerosis formation following Western diet feeding. Collectively, these experiments point to a previously unrecognized role of NOR1 in the suppression of myeloid progenitor cell proliferation, inflammatory responses, and atherosclerosis.
Abstract 158: CD40L Regulates Hematopoietic Stem and Progenitor Cell Biology by Inducing Adhesion-Mediated Quiescence
Authors: Seijkens Tom , Beckers Linda , Christ Anette , Moura Rute , Tjwa Marc , Lutgens Esther
Published: March 2022

The self-renewal and differentiation capacity of hematopoietic stem and progenitor cells (HSPCs) is an important regulator of the way the mature immune system behaves and how it reacts upon challenges. The co-stimulatory protein CD40L is well-known for its role in immune activation and chronic inflammatory diseases, especially in atherosclerosis. We hypothesized that CD40L not only regulates mature immune cell activation, but also plays a role in the development of these cells by regulating HSPC biology. Here we show that CD40L deficiency increased the colony-forming potential of HSPCs by 62,1%. FACS analysis revealed that the bone marrow of CD40L
-/-
mice contained 34,4% more myeloid-biased (Lin-cKit+) progenitors, 147,2% more myeloid/lymphoid-biased (Lin-Sca-1+) progenitors and 46,6% more primitive (Lin-Sca-1+-cKit+) HSPCs. Competitive bone marrow transplantation demonstrated that the abscence of CD40L resulted in a 13.4% increased reconstitution of peripheral blood leukocytes. Lineage analysis showed an advantage towards the lymphoid lineage, as B cells were increased by 14.6%, T cells by 41.3%, and NKT cells by 50.9%. The adhesion of HSPCs on CD40L
-/-
bone marrow stroma was reduced by 58%. Cell cycle analysis revealed that CD40L
-/-
HSPCs were less quiescent compared to WT cells. CD40L
-/-
mice had delayed hematopoietic recovery and a 37.5% reduced survival rate after 5-FU treatment. CD40L
-/-
mice did not survive a bone marrow transplantation. All effects were independent from CD40, the classical receptor of CD40L. In conclusion, our data suggest that CD40L regulates adhesion-mediated quiescence of HSPCs. These results provide a novel CD40L-mediated mechanism of protecting the HSPC compartment against exhaustion during immune activation and chronic inflammatory disease like atherosclerosis, thereby securing long term hematopoiesis.
Abstract 16: ApoA5 Reduces Atherosclerosis in LDL Receptor-Deficient Mice
Authors: Zhang Ting , Kim Dae Hyun , Slusher Sandra , Fan Yong , Forte Trudy , Dong Henry
Published: March 2022

Apolipoprotein A5 (ApoA5) is a lipid-binding protein that is associated mainly with high-density lipoprotein (HDL) and to a lesser extent with very low-density lipoprotein (VLDL). ApoA5 gene variants are linked to altered lipid metabolism and increased risk of cardiovascular disease in humans. To investigate the role of ApoA5 in lipid metabolism, we placed transgenic mice expressing human ApoA5 and age/sex-matched control littermates on high fat diet for 20 weeks. We show that transgenic production of human ApoA5 resulted in significant improvement in hepatic and plasma lipid metabolism, protecting against the development of dyslipidemia and fatty liver disease in high fat-fed mice. To address the underlying mechanism, we determined hepatic triglyceride production vs. systemic triglyceride clearance. ApoA5 transgenic mice, as opposed to control littermates, exhibited significantly reduced hepatic output and enhanced clearance of triglyceride-rich particles. Furthermore, we show that purified ApoA5 proteins were capable of stimulating lipoprotein lipase (LPL) activity, contributing to accelerated VLDL-TG hydrolysis in a cell-free system. To determine the potential beneficial effect of ApoA5 on atherogenesis, we crossed the human ApoA5 transgene into LDL receptor knockout (LDLR-/-) mice. We demonstrate that transgenic ApoA5 production resulted in significant reduction in plasma lipid levels in LDLR
-/-
mice on high fat and high cholesterol diet. This effect translated into a significant reduction in atherosclerotic lesion in the aortic root of LDLR
-/-
mice.

From our studies we conclude: 1) ApoA5 acts to inhibit hepatic lipogenesis and limit hepatic VLDL-TG output, 2) ApoA5 augments LPL activity and enhances the systemic clearance of TG-rich particles, and 3) ApoA5 possesses anti-atherogenic property, protecting against the development of atherosclerosis in LDLR
-/-
mice.
Abstract 161: Visceral Obesity and Calcific Aortic Valve Disease Is Mediated by S100/Calgranulin
Authors: Yan Ling , Ye Honggeng , Gardner Brandon , Earley Judy , Hofmann Bowman Marion A
Published: March 2022

Purpose:
Circulating serum levels of human S100/calgranulins have been associated with diabetes, coronary artery disease and increased cardiovascular mortality in clinical studies. To test whether S100/calgranulins are biomarker of inflammation or directly mediate disease, we exploited the fact that mice lack the gene for S100A12, and generated a novel humanized mouse with transgenic expression of human S100/calgrnaulin.

Methods:
hBAC-S100 mice were generated in C57BL6/J mice by expression of a bacterial artificial chromosome of the human S100/calgranulin gene cluster containing genes and regulatory elements for S100A8/9 and S100A12 (60kb). F3 animals and wild type (WT) control littermate mice were maintained in pathogen free barrier facility with free access to water and regular rodent chow diet and studied at age 1, 3 and 12 months.

Results:
S100A12 protein is expressed in myeloid circulating blood cells and present in the serum of hBAC-S100 mice (25 ng/ml serum), but was not detected in WT mice. BAC-S100 mice developed normal and showed marked increase in visceral adiposity at 12 months upon DEXA scanning (25% vs 12%, p<0.05). Gene expression in white adipose tissue (WAT) of lean hBAC-S100 age 4 weeks was increased for T- and B cell marker and unchanged for F4/80, CD68, and MCP-1 compared to age and gender matched WT littermate. In 12M old mice, WAT showed increased F4/80, CD68 and MCp-1(mRNA and immunohistochemistry) and no difference in T-and B cell markers. Adiposity in 12M old hBAC-S100 mice was associated with hyperinsulinemia and impaired oral glucose tolerance test and no evidence of hepatic steatosis. Elevated serum interleukin-6 and reduced levels of adiponectin preceeded development of adiposity and was present in young hBAC-S100 mice. Alizarin Red stained serial sections revealed calcification nodules (50-100μm in size) on the aortic valve leafleat and few osteoblast-like cells in the ascending aortic media in all 12-months old hBAC-S100A12 and very scant to absent calcification in control litter mate mice (n=6, p<0.005). In vivo ECHO showed abnormal mitral valve doppler flow with reduced ratio of early (E) to atrial (A) filling (E/A ratio 0.85 and 1.22, p=0.03) indicative of diastolic dysfunction. Left ventricular systolic function, size, and aortic valve gradients were not different between hBAC-S100 and WT mice.

Conclusion:
Circulating myeloid derived human S100/calgranulin is sufficient to induce a sustained inflammatory state and is associated with the development of adiposity, hyperinsulinemia, impaired glucose tolerance and aortic valve calcification in chow fed C57BL6 mice.
Abstract 174: Carboxylesterase 1 in Lipid Metabolism and Obesity
Authors: Xu Jiesi , Yin Liya , Ge Xuemei , Zhang Yanqiao
Published: March 2022

Over-expression of human carboxylesterase 1 (CES1) in macrophages has been shown to increase hydrolysis of cholesterol esters and protect against atherosclerosis. However, the role of CES1 in other tissues and the physiological functions of CES1 are largely unknown. In this report, we investigated the role of CES1 in the liver. Over-expression of hepatic CES1 decreased hepatic triglyceride and plasma cholesterol levels. In contrast, knock down of hepatic CES1 by shRNA led to increased hepatic triglyceride levels and plasma LDL-C levels. To understand the physiological functions of CES1, we generated Ces1
-/-
mice. Ces1
-/-
mice are resistant to diet-induced obesity and showed changes in hepatic and plasma lipid levels. We also investigated the mechanisms underlying these changes. Our data indicate that CES1 plays a critical role in regulating lipid and energy homeostasis.
Abstract 176: Extracellular S100A4 Is a Key Modulator of Arterial Smooth Muscle Cell Phenotypic Transition
Authors: Chaabane Chiraz , Heizmann Claus W , Bochaton-Piallat Marie-Luce
Published: March 2022

Background:
It has been proposed that smooth muscle cells (SMCs) from the arterial wall are heterogeneous and that only a subset of medial SMCs are prone to accumulate into the intima leading to atheromatous plaque formation. We isolated 2 distinct SMC phenotypes from porcine coronary artery: spindle-shaped (S) and rhomboid (R). Biological features of R-SMCs (i.e. enhanced proliferative and migratory activities as well as poor level of differentiation) explain their capacity to accumulate into the intima. We identified S100A4 as a marker of R-SMCs in vitro and of intimal SMCs both in pig and human. S100A4 is an intracellular Ca2+ binding protein that can also be secreted; it has extracellular functions probably via the receptor for advanced glycation end products (RAGE).

Objective:
Investigate the role of S100A4 in SMC phenotypic change, a process characteristic of atherosclerotic plaque formation.

Methods and results:
Transfection of a human S100A4-containing plasmid in S-SMCs (practically devoid of S100A4) led to overexpression of S100A4 in approximately 10% of SMCs, release of S100A4, and a transition towards a R-phenotype of the whole SMC population. Moreover, treatment of S-SMCs with S100A4-rich conditioned medium collected from S100A4-transfected S-SMCs induced a transition towards a R-phenotype, which was associated with increased proliferative and migratory activities as well as downregulation of SMC differentiation markers (i.e. alpha-smooth muscle actin and smooth muscle myosin heavy chains). It yielded activation of NF-kappa B without changing RAGE mRNA baseline level. Blockade of extracellular S100A4 in R-SMCs with S100A4 neutralizing antibody induced a transition from R- to S-phenotype, decreased proliferative activity and upregulated SMC differentiation markers. In contrast, silencing of S100A4 mRNA in R-SMCs and during platelet-derived growth factor-BB-induced S-to-R-phenotypic change did not affect SMC morphology in spite of decreased proliferative activity.

Conclusions:
Our results show that extracellular S100A4 modulates SMC phenotypic changes and is essential for the establishment of the R-phenotype. It could be a new target to prevent SMC accumulation during atherosclerosis and restenosis.
Abstract 189: Hypercholesterolemia Blunts Gender Differences in Vascular Endothelin-1 Activity in Middle-Aged Adults
Authors: Sandhu Alexander , Campia Umberto , Panza Julio A
Published: March 2022

Background:
Compared with age-matched women, healthy middle-aged men have a higher vascular activity of the vasoconstrictor and proatherosclerotic peptide endothelin-1(ET-1). Hypercholesterolemia is a major atherosclerotic risk factor and is associated with increased ET-1 activity. Whether high cholesterol levels abrogate gender differences in vascular ET-1 activity has not been studied. This investigation has been conducted to test the hypothesis that hypercholesterolemia blunts the gender difference in ET-1 activity present in middle-aged men and women.

Methods:
Forearm blood flow (FBF) responses were measured by strain-gauge plethysmography during the intra-arterial infusion of the selective ET
A
receptor blocker BQ-123 in 19 women and 21 men with hypercholesterolemia (total cholesterol >240 mg/dL).

Results:
Baseline characteristics were similar between women and men (see table). BQ-123 administration resulted in a significant progressive vasodilator response in both the female and male group (p < 0.001 for both). When these responses were compared between the two gender groups, no significant differences were observed (p=NS).

Conclusions:
Hypercholesterolemia increases the vascular activity of ET-1 in middle-aged women and men. The lack of difference in ET-1 activity between women and men suggests that hypercholesterolemia eliminates the gender differences in ET-1 vascular activity, potentially offsetting the advantage in cardiovascular risk of middle-aged women compared with middle-aged men.
Abstract 191: Shear Stress-Induced Atherosclerotic Plaque Regression Is Reversed by Matrix Metalloproteinase Inhibition: Role of Macrophage Mobility
Authors: Simeone Stefania , Ebrahimian Talin , Michaud Veronique , Lehoux Stephanie
Published: March 2022

Atherosclerotic plaques usually form in regions of low blood flow, whereas vessels exposed to high shear stress tend to remain lesion-free. We hypothesized that exposing established atherosclerotic plaques to elevated shear stress leads to lesion regression by facilitating inflammatory cell movement within the plaque. We developed a surgical procedure in mice, the arteriovenous fistula (AVF), where the right carotid artery is anastomosed into the right jugular vein. LDLR
-/-
mice were placed on a high-fat diet, then divided into control, sham and AVF groups. Sham and AVF groups were kept on a high-fat diet for a further 4 weeks post-surgery. This procedure increases the shear stress in the artery, leads to plaque regression and increases plaque matrix metalloproteinase (MMP) activity. To understand the mechanism of this regression, we assessed the role of MMPs. Plaque MMP-9 expression was increased in AVF vs. control and sham (10.8%±1.4, 3.9%±0.7, 4.6%±1.1, respectively; p<0.0001). MMP-3 expression was also increased in AVF vs. control (9.5%±1.5, 5.0%±0.8, respectively; p<0.05), whereas MMP-2 expression was decreased in AVF vs. sham (5.7%±0.5, 11.3%±1.6, respectively; p<0.05). In addition, we overexpressed the tissue inhibitor of metalloproteinase (Timp-1) in mice before surgery. Sham plaque size was not affected. However, elevated Timp-1 prevented the reduction in plaque size in the AVF group (0.096±0.013 vs. 0.047±0.008 mm
2
, p<0.05). All groups had comparable lipid levels. To further understand the role of MMPs, we devised an endothelial cell (EC)-macrophage co-culture system where the ECs were exposed to high (15 dynes/cm
2
) or low (0.5 dynes/cm
2
) shear stress.

A scratch assay was performed to evaluate migration of macrophages exposed to EC effluent. There was a 2.6-fold increase in migration in the high flow condition compared to the low (p<0.05). When the cells were exposed to the MMP inhibitor drug, GM6001, migration was decreased 2 fold (p<0.05). Our findings suggest that MMPs play a role in shear stress-induced plaque regression with MMP-9 being a key player. Shear stress acting on ECs may influence the cells within the plaque by increasing MMP expression, allowing for better macrophage mobility and egress which are characteristics of regressing plaques.