bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 330: Liver-Specific Phospholipids Transfer Protein Activity Has Significant Contribution in Hepatocyte Apob-Containing Lipoprotein Production and in Plasma Lipoprotein Metabolism
Authors: Yazdanyar Amirfarbod , Jiang Xian-Cheng
Published: July 2021

Atherosclerosis is the leading cause of death in western countries. Lipoprotein metabolism is closely related with the disease. ApoB-containing lipoproteins (BLp) are atherogenic particles, while, high density lipoproteins (HDL) are anti-atherogenic. Phospholipids Transfer Protein (PLTP) activity has important impact on BLp and HDL homeostasis. Animal studies, including general knock out (KO) and transgenic approaches, have shown pro-atherogenic properties for PLTP. However, the tissue specific function of PLTP remains mostly unknown. To address the impact of liver-specific expression of PLTP on lipoprotein metabolism, we have created two mouse models which either express PLTP specifically in the liver on a PLTP-null background or do not express PLTP in the liver (liver-specific PLTP KO) in a wild type background. In both models, our findings show that liver-specific PLTP is responsible for 25-30% of total plasma PLTP activity. Moreover, liver-specific PLTP expression model shows a remarkable increase in plasma levels of BLp, whereas liver-specific PLTP KO mice show significant reduction of BLp in plasma in compare to control group. These results further show the significant contribution of PLTP in BLp production.

We also found that HDL levels are slightly increased in the first model, while in liver-specific PLTP KO animals HDL levels are significantly decreased. In order to unravel the mechanism, we evaluated the apoB and triglyceride production rates after blocking the clearance of the particles. We found that liver-specific PLTP increases apoB (both apo48 and apoB100) and triglyceride (TG) secretion rates. Additionally, the study of the hepatocyte microsomal luminal content lipidation in the first model showed that in the presence of PLTP BLp lipidation, which reflects as higher level of TG in microsomal luminal content, is significantly higher than the control group. Our results indicate that liver-generated PLTP promotes atherogenic lipoprotein production and provides a rationale for liver-specific PLTP inhibition as a therapeutical approach for the treatment of atherosclerosis.
Abstract 338: 90K Inhibits Human Macrophage Foam Cell Formation and Is Associated with Reduced Plaque Burden and Reduced Adverse Cardiovascular Events in Patients with Coronary Artery Disease
Authors: Gleissner Christian A , Haeussler Julia , Domschke Gabriele , Erbel Christian , Gitsioudis Gitsios , Hofmann Nina , Keeley Ellen C , Korosoglou Grigorios , Katus Hugo A , Ley Klaus
Published: July 2021

Objective:
90K (also known as galectin-3 binding protein) is a secreted protein that modulates inflammation in vivo. We hypothesized that 90K may affect atherosclerosis through its effects on macrophages.

Methods and results:
In human primary macrophages, recombinant human 90K reduced gene and protein expression of the scavenger receptors CD36 and SR-A in a dose- and time-dependent manner. Accordingly, uptake of DiI-labeled acetylated (acLDL) or oxidized low density lipoprotein (oxLDL) was significantly reduced after pre-treatment with 90K. Oil red O staining confirmed 90K-dependent reduction of foam cell formation induced by oxLDL after 24 hours. 90K expression in macrophage-rich areas of human atherosclerotic plaques was confirmed by immunohistochemistry in post mortem coronary arteries. In 122 patients undergoing coronary angiography 90K plasma levels were measured by ELISA. Hereby, there was no significant difference between patients with (n=89) and without (n=33) coronary artery disease (CAD). However, 90K plasma levels were significantly lower in CAD patients with at least one coronary artery stenosis ≥50% (n=37) compared to those with lumen narrowing <50% (n=52, P=0.044). By measuring total coronary artery plaque volume by cardiac computed tomography angiography (CCTA) in 56 of these patients with angiographically confirmed CAD, we found an inverse correlation between plaque volume and 90K plasma levels (r=-0.318, P=0.018). This correlation remained significant after multivariate analysis taking into account established cardiovascular risk factors including high sensitivity C reactive protein. In an independent cohort of 77 patients with angiographically confirmed CAD, low 90K plasma levels were associated with increased incidence of adverse cardiovascular events (as defined by cardiovascular death, myocardial infarction, or stroke) within one year (P=0.007). Again, this correlation remained significant in a multivariate Cox proportional hazards regression analysis (P=0.039, hazard ratio 0.230, 95%-CI 0.097-0.935).

Conclusions:
We propose a protective role of 90K in coronary artery disease. By modulating macrophage foam cell formation, 90K may reduce plaque growth and promote plaque stability preventing adverse cardiovascular events.
Abstract 351: Collagen Alignment Correlates with Differential Biaxial Stiffness in Nonhuman Primate Carotid and Femoral Arteries
Authors: Wang Roy , Li Haiyan , Wan William , Gleason Rudy L , Brewster Luke P
Published: July 2021

Introduction:
There are known differences in arterial compliance in different regions of the arterial tree. These differences are thought to be determined by pressure and flow demands of their capacitance artery and their respective tissue bed perfusion requirements, and they may play a role in certain arteries having a greater or lesser prevalence of disease as well as favorable response to intervention. Both the femoral and carotid arteries are susceptible to peripheral arterial disease. Here we define the differences in compliance and analyze the microstructural collagen alignment between these arteries to assess the hypothesis that arterial stiffness, defined as the inverse of compliance, will correlate with collagen alignment.

Methods:
In-vitro biaxial mechanical tests using a custom system were performed on carotid and femoral arteries excised from non-human primates at necroscopy. Pressure-diameter and force-axial stretch responses were measured, and the arterial compliance (
C
, units of %/mmHg x 10
-2
) at the physiological pressure range to calculate the axial stretch. To characterize the microstructure, planar sections of the arteries were imaged under two-photon confocal microscopy. Three-dimensional, z-stack images were taken at two different locations across the medial thickness for each artery. The images were post-processed using a fast Fourier transform analysis to the quantify fiber angle distributions. Circular descriptive statistics was then used to calculate a fiber dispersion index (
FDI
), where 0≤
FDI
≤1.
FDI
=0 signifies random fiber dispersion and
FDI
=1 signifies complete circumferential fiber alignment. T-test statistical analysis was used to compare groups. P<.05 was considered significant.

Results:
Biaxial testing identified femoral arteries as being more than twice as stiff as the carotid artery (P=.01; Figure). Microstructural fiber analysis demonstrated significantly increased collagen alignment in the femoral compared to carotid artery (
FDI
=0.31,
n
=4 for femoral versus
FDI
=0.26,
n=4
for carotid,
p
=0.03).Conclusion: Arterial stiffness in the femoral and carotid artery of nonhuman primates correlates with collagen alignment in the vessel media. The absence of a cellular requirement in the observed mechanical behavior of these arteries suggests that not only matrix composition but also alignment contribute to arterial stiffness. Differential arterial responses to procedures, particularly those endovascular in nature, may be expected by these results.
Abstract 397: Analysis of Cell Phenotype in Relation to TGFβ Treatment of Aortic Smooth Muscle Cells and Myofibroblasts Isolated from Aortas and Valves of Thoracic Aortic Aneurysm Patients with a Tricuspid or a Bicuspid Valve
Authors: Paloschi Valentina , Folkersen Lasse , Kurtovic Sanela , Wagsater Dick , Franco Cereceda Anders , Roy Joy , Eriksson Per
Published: July 2021

Background

Thoracic aortic aneurysm (TAA) is a pathological widening of the aorta, due to degeneration of extracellular matrix (ECM) and loss of smooth muscle cells (SMCs). Bicuspid aortic valve (BAV) is a congenital disorder present in 1-2 % of the population which makes TAA associated with BAV a common complication. Previously we showed that aortas isolated from BAV and normal tricuspid aortic valve (TAV) patients are different both at gene and protein levels. Particularly, differences in the TGFβ pathway seem to be crucial players in aneurysm development, affecting matrix remodeling and wound healing. Since SMCs and myofibroblasts are the critical cells responsible for these activities, we evaluated different properties of the cells focusing on fibronectin (FN) and its spliced versions, a target gene of TGFβ. Interestingly, extra domain A of FN (EDA) was previously described for its roles in vascular morphogenesis, as well as in processes like migration and proliferation.

Methods and results

Biopsies from the thoracic aorta and Aortic valves were collected during Elective Aortic Valve Replacement Surgery. mRNA expression was analyzed in the ascending aorta by Affymetrix Exon arrays in patients with TAV (n=46) and BAV (n=77). Expression of EDA was found increased only in dilated aortas from TAV patients but not in BAV patients.

Primary SMCs were isolated with the explant outgrowth technique from aortas of BAV and TAV patients (n=15). Myofibroblasts were isolated by collagenase digestion from BAV and TAV valves (n=30). Cells were cultured and treated with TGFβ at a concentration of 20 ng/ml for 6h. TGFβ treatment influenced the splicing of FN and enhanced the formation of EDA-containing FN in SMCs from TAV patients but not in cells derived from BAV patients. We have not observed clear differences in SMC proliferation and migration. Myofibrolasts analysis is ongoing.

Conclusions

So far, our results suggest that despite a decreased EDA-fibronectin expression in BAV cells, the phenotype of SMCs isolated from BAV and TAV patients in culture does not differ. However, impaired TGFβ signaling that may result in the increased susceptibility of BAV patients to develop TAA could be due to effects on other cell types.
Abstract 422: Relationship Between N-Telopeptide-Mediated Bone Mineral Resorption and Peripheral Arterial Disease
Authors: Mao Danlin , Moukarzel Lea , Lilly Scott , DiMuzio Paul , Eraso Luis
Published: July 2021

Background:
Patients with peripheral arterial disease (PAD) have a higher incidence of hip bone loss and non-spinal fractures independent of their cardiovascular risk or degree of claudication. The mechanisms explaining this excess risk of osteoporosis are poorly understood. N-telopeptide (NTX) is an amino-terminal cross-linked type I collagen peptide that is released during fibrillogenesis, reflecting both the derivative products of bone collagen and bone resorption. When elevated, it serves as an early marker for osteoporosis and predictor of therapeutic response. We hypothesized that patients with PAD have reduced bone mineral content and that PAD is associated with biomarkers of accelerated bone mineral resorption.

Methods:
We analyzed data from 4354 participants ages 40 years or older with measured ankle-brachial indexes (ABI) from the National Health and Nutrition Examination Survey from 1999 to 2002, defining PAD as an ABI < 0.9 in either leg. N-telopeptides were measured in the urine. Total bone mineral content and bone mineral density were measured using dual energy X-ray absorptiometry.

Results:
The overall prevalence of PAD was 4.4 % (S.E, 0.3). The geometric mean of NTX was 248.3 nmol (S.E, 7.0). NTX decreased with age. Women, non-Hispanic blacks and obese individuals had significantly higher NTX levels, whereas diabetics had reduced NTX levels. Current smokers, chronic kidney disease and PAD patients tended to have higher levels as well. In an age, gender, and race adjusted multivariate analysis, one standard deviation increase in NTX level was associated with a three-fold increase in the odds of having reduced total bone mineral density (OR 3.22, p <0.0001). Additionally, patients with PAD had significantly increased odds of having NTX levels in the higher tertile (O.R 1.57, p=0.035). This association persisted even after further adjustment for obesity, smoking, and C-reactive protein.

Conclusion:
Peripheral arterial disease is associated with reduced levels of bone mineral content and density. PAD is independently associated with N-telopeptides, suggesting abnormal type I collagen metabolism abnormalities. Further prospective studies will determine the role of NTX as a marker of osteoporosis found in PAD and their relationship with walking activity.
Abstract 453: Ceruloplasmin and Cardiovascular Disease in the Atherosclerosis Risk in Communities (ARIC) Study
Authors: Dadu Razvan T , Dodge Rhiannon , Nambi Vijay , Virani Salim S , Hoogeveen Ron C , Pankow James S , Coresh Josef , Guild Cameron , Tang Wilson W , Boerwinkle Eric , Hazen Stanley L , Ballantyne Christie M
Published: July 2021

Background:
Ceruloplasmin (Cp) is an enzyme that enhances low density lipoprotein oxidation and decreases nitric oxide bioavailability. Hence, a role for Cp in cardiovascular disease has been suggested. We evaluated if levels of Cp are associated with incident coronary heart disease events (CHD), incident heart failure (HF) and death in the ARIC Study.

Methods:
Cp was measured by immunoturbidimetric assay at ARIC Visit 4 (1996-1998). In the subgroup of individuals without CHD, stroke or HF at visit 4 (N=9553), using Cox regression modeling we analyzed the association of Cp levels with CHD, HF and death (mean follow up = 10.5 years).

Results:
Cp levels [mean(SD), mg/L] were higher in blacks vs whites [311(70) vs 298(78), p < 0.001] in women vs men [333(79) vs 256(44), p < 0.001] and in women on hormone replacement therapy (HRT) vs those not on it [382(89) vs 315(67), p < 0.001]. Cox regression models adjusted for age, sex, race, ARIC site, traditional cardiovascular risk factors and HRT use showed (highest vs lowest quartile) a significant association of Cp with HF (HR 1.66, 95% CI 1.31-2.12) and death (HR 1.57, 95% CI 1.30-1.89) but no association with CHD (HR 1.15, 95% CI 0.93-1.44). The association with HF and death persisted after adjusting for hs-CRP, NT-pro BNP, estimated GFR and high sensitivity cardiac troponin T. Subgroup analyses by sex and race showed similar results except for CHD where the association was significant in whites (HR 1.34, 95% CI 1.04-1.71) and for HF where there was no significant association in blacks (HR 1.26, 95% CI 0.85-1.86).

Conclusions:
Cp was associated with HF and death but not with CHD in the overall ARIC population. High Cp was associated with CHD only in whites.
Abstract 456: Myxoma Viral Proteins, M-T7, Serp-1 and Serp-2, Alter Human Monocyte Gene Expression in Vitro
Authors: Davids Jennifer , Bartee Mee Y , Moyer Richard W , Lucas Alexandra R
Published: July 2021

Introduction:
Atherosclerosis is characterized by ongoing chronic inflammation, cell damage, apoptosis and scar formation all of which can initiate plaque growth and arterial occlusion. These proteases are controlled by serine protease inhibitors, or serpins, which regulate apoptotic and inflammatory pathways. Complex DNA viruses, such as myxomavirus, have developed highly active immune defense systems which include viral serpins that inhibit inflammation at picogram to microgram doses. Serp-1, a secreted serpin, significantly reduces inflammatory cell activation, invasion and plaque growth in animal models and is in clinical trials. One cross-class serpin, Serp-2, has demonstrated anti-inflammatory and anti-apoptotic effects in a variety of animal models. M-T7, a secreted non-serpin chemokine binding protein, reduces vascular plaque and inflammation. Previous microarray experiments detected no significant changes with viral protein treatment alone, underscoring a need for activated cells. This study assesses the effects of viral proteins with anti-inflammatory and anti-atherogenic activity on atherosclerosis-related gene expression changes in activated human monocytes.

Methods:
Triplicate samples of THP-1 human monocytic cells were incubated with saline, 10μM camptothecin alone or in combination with individual serpins (500ng/million cells) for 30 mins at 37°C. Real-time RT-PCR analysis was performed.

Results:
At 30m, the viral proteins elicited significant expression changes in THP-1 monocytes for genes in purported atherosclerotic pathways. CCL2 was upregulated by Serp-1 (P = 0.0031). PDGFB was significantly increased by Serp-1 (P = 0.0203) and to a lesser extent by Serp-2 and M-T7. M-T7 significantly upregulated L-Selectin (P = 0.0023). Serp-2 significantly downregulated Lipoprotein(a) (P =0.0500).

Conclusion:
Significant expression changes were detected in human monocytic cells after treatment with three unique anti-inflammatory viral proteins. Differential regulation of genes such as PDGFB underscores the multifaceted approach viral proteins take toward controlling inflammation. Altering atherogenic responses in monocytic cells represents a new potential therapeutic target for inflammatory vascular diseases.
Abstract 47: Development and Use of a Novel Single-Cell Epigenetic Lineage Tracing Method to Identify Smooth Muscle--Derived Macrophage-Like Cells Within Human Atherosclerotic Lesions
Authors: Gomez Delphine , Shankman Laura , Owens Gary K
Published: July 2021

Aim:
Smooth muscle cells (SMC) possess remarkable phenotypic plasticity that allows adaptation to changing environmental cues. Lack of definitive SMC lineage tracing studies and inability to identify phenotypically modulated SMCs within lesions due to loss of SMC marker gene expression raise major questions regarding the role of SMC in vivo in atherosclerosis progression. We hypothesize that a subset of cells within lesions that express macrophage markers are derived from SMC not monocytes and play a key role in determining plaque stability.

Methods:
We developed a novel lineage tracing based on detection of H3K4dime of the SM MHC gene, a SMC-specific epigenetic lineage marker we have previously shown is stable during phenotypic switching in vitro. Detection of H3K4dime of the SM MHC locus was done using a Proximity ligation assay (PLA) developed in our lab with an antibody targeting the biotinylated DNA probe for the SM MHC locus in conjunction with an anti-H3K4dime antibody. Use of secondary antibodies conjugated with oligonucleotides induces formation of circular DNA that serve as template for amplification, allowing visualization of co-localization of H3K4dime and the SM MHC gene (Duolink). Our new lineage tracing is suitable with human paraffin-embedded tissue sections (n=4) allowing investigation of SMC fate within human atherosclerotic lesion.

Results:
H3K4dime on the SM MHC gene (PLA+ cells) was found to be specific for SMCs and not found in any other cell types including adventitial fibroblasts, or endothelial cells. The method was validated using a SMC-specific lineage tracing mouse model wherein SM MHC Cre mice are crossed to ROSA flox STOP eYFP+/+ ApoE
-/-
mice. The H3K4dime/SM MHC PLA signal (i.e. PLA+) was exclusively found in eYFP+ cells. Moreover, some of the lesion SMCs were eYFP+/PLA+/SM α-actin-. Similarly, we identified PLA+ cells in human lesions that were positive for the macrophage marker CD68.

Conclusion:
Our new method permits definitive identification of SMC-derived cells within lesions even if they are not identifiable as SMC due to loss of SMC markers. Moreover, we provide exciting evidence that a significant fraction of macrophage-like cells in human lesions are derived from SMC. We postulate that transition of SMC to a macrophage state may be a key event leading to plaque destabilization and rupture with possible myocardial infarction or stroke.
Abstract 491: Injury-Activated TGFβ Recruits MSCs for Vascular Remodeling
Authors: Wan Mei , Li Changjun , Zhen Gehua , He Wenying , Jiao Kai , Chen Yiu-Fai , Jia Xiaofeng , Yu Bing , Cao Xu
Published: July 2021

Stem/progenitor cells can be recruited to participate in tissue remodeling, regeneration, or repair following injury. In particular, bone marrow-derived or local resident stem /progenitor cells are implicated in vascular repair and remodeling. It is believed that migratory factor(s) released by injured tissue creates a gradient to mediate the migration of stem cells to the injury sites. The key mediator(s) inducing the recruitment of the cells to the vascular lesions remains to be defined. Here we show that nestin
+
cells, which represent similar population as Sca1
+
CD29
+
CD11b
-
CD45
-
mesenchymal/stromal stem cells (MSCs), were recruited and incorporated into the neointimal tissue in both rat model of balloon injury of carotid artery and mouse model of wire injury of femoral artery. Importantly, elevated active TGFβ1 level was observed in the injured vessels as early as 8 hr post injury, and the elevation lasted for 2 wks. To investigate whether injury-induced activation of TGFβ1 in vascular matrix stimulates migration of MSCs, we developed an aorta-conditioned medium (Aorta-CM)-based cell migration assay in which MSCs were placed in the upper chamber and the Aorta-CM prepared by culturing
ex vivo
injured aorta was placed in the lower chamber of a transwell chamber. Aorta-CM prepared from
ex vivo
injured aorta significantly enhanced cell migration compare to CM prepared from uninjured aorta. When neutralizing antibody specific for TGFβ1 or the inhibitor of TGFβ type I receptor (TβRI) was added to the CM, the migration of MSCs was almost abolished, indicating that active TGFβ1 released by the injured arteries mediates MSCs migration. To examine the role of TGFβ in MSCs recruitment following vascular injury
in vivo
, TβRI inhibitor was injected into mice with injury of femoral arteries. Both the development of neointima and the recruitment of nestin
+
cells were blocked with the inhibitor treatment compare to those with vehicle treatment. Collectively, the results suggest that TGFβ is an injury-activated messenger essential for the recruitment of MSCs to participate in vascular remodeling.
Abstract 503: Antioxidant Activity Accounts for the Antiplatelet Effects of the Probucol Derivative Succinobucol
Authors: Kennedy Simon , Greig Fiona H , Houston Stephanie A
Published: July 2021

Succinobucol is a derivative of probucol with improved partitioning into cells and potent extracellular antioxidant activity. It has antiplatelet effects
ex vivo
in human blood but it is unclear if this is due to antioxidant activity. We aimed to study the effect of succinobucol on free radical-modulated platelet aggregation and generation of vascular tone. We also studied platelet aggregation
ex vivo
in rats following acute administration of succinobucol.

Rabbit blood was collected and aggregation in whole blood and platelet-rich plasma (PRP) measured using dual-wire impedance. Rabbit iliac arteries were mounted in a small artery wire myograph to measure vessel tone and the effect of free radicals and succinobucol. Rat carotid and jugular vessels were cannulated in anaesthetised animals prior to administration of succinobucol. Blood was withdrawn to measure platelet aggregation 45 minutes following injection of succinobucol.

In whole blood and PRP, succinobucol (10-100μM) caused a dose-dependent reduction in collagen-induced platelet aggregation. Aggregation to collagen in PRP was significantly increased by the superoxide-generating system xanthine/xanthine oxidase (X/XO; 29.8% increase, n=4; p<0.05 vs. control) and succinobucol abolished this effect. In denuded rabbit iliac arteries incubated with excess xanthine, addition of XO (3-9mU/mL) induced a dose-dependent relaxation and this was largely abolished by succinobucol at both 10 and 100μM. Injection of 3 doses of succinobucol (50-150mg/kg)
in vivo
had no effect on mean arterial pressure or heart rate. However, blood removed 45minutes following the last dose of succinobucol showed a significant reduction in aggregation to collagen (reduction of 40.4% compared to control with 5μg/mL collagen; n=5, p<0.05 vs. control).

The results of the current study demonstrate that the antiplatelet effects of succinobucol may be attributed to an antioxidant effect in rabbit blood and that succinobucol exerts an antiplatelet effect
in vivo
for at least 45mins after administration. Furthermore, the antioxidant effects may be able to influence vascular tone at sites of free radical generation in the vascular tree.
Abstract 504: Increased Intestinal Lipid Absorption Caused by Ire1β Deficiency Contributes to Hyperlipidemia and Atherosclerosis in ApoE-Deficient Mice
Authors: Iqbal Jahangir , Queiroz Joyce , Li Yan , Jiang Xian-Cheng , Ron David , Hussain M Mahmood
Published: July 2021

Rationale:
High fasting serum lipid levels are significant risk factors for atherosclerosis. However, the contributions of postprandial excursions in serum lipoproteins to atherogenesis are less well characterized.

Objective:
This study aims to delineate whether changes in intestinal lipid absorption associated with loss of inositol requiring enzyme 1β (Ire1β) would affect the development of hyperlipidemia and atherosclerosis in

Apoe
-/-

mice.

Methods and Results:
We used Ire1β deficient mice to assess the contribution of intestinal lipid absorption to atherosclerosis. Here we show that

Ire1b
-/-
/Apoe
-/-

mice contain higher levels of intestinal microsomal triglyceride transfer protein, absorb more lipids, develop hyperlipidemia, and have higher levels of atherosclerotic plaques compared to

Apoe
-/-

mice when fed chow and western diets. In contrast, plasma cytokines were similar in

Ire1b
-/-
/Apoe
-/-

and

Apoe
-/-

mice.

Conclusions:
These studies indicate that Ire1β regulates intestinal lipid absorption and that increased intestinal lipoprotein production contributes to atherosclerosis.
Abstract 512: Link Between the Lipolysis-Stimulated Lipoprotein Receptor and Lipogenesis in the Liver
Authors: Hanse Marine , Stenger Christophe , Akbar Samina , Pincon Anthony , Yen Frances T
Published: July 2021

The lipolysis stimulated lipoprotein receptor (LSR) plays an important role in hepatic uptake of triglyceride (TG)-rich lipoproteins during the postprandial (PPL) phase. We recently reported the regulation of LSR by leptin, accompanied by changes in hepatic lipid metabolism. In this study, we sought to determine if a direct link exists between LSR and lipogenesis in the liver, focusing on ATP citrate lyase (ACL) and fatty acid synthase (FAS), two key enzymes for
de novo
fatty acid synthesis.
Garcinia
cambogia extract (GCE) contains hydroxycitric acid, an ACL potent inhibitor found to lower plasma TG and body weight in rodent and human studies. In female 10 wk-old C57BL/6Rj mice treated 30 days with corn oil emulsion containing 20 mg GCE (n=8), we observed reduced fasting (21%, p<0.03) and PPL (22%, p<0.02) plasma TG accompanied by a >1.5 fold (p<0.02) increase in liver membrane LSR protein as compared to controls (emulsion alone, n=8). While hepatic LDL-R protein was moderately increased (NS), TG and total cholesterol content, as well as ACL and FAS protein in the liver were significantly higher as compared to controls. Significant correlations were observed between LSR protein and LDL-receptor (LDL-R) protein as well as liver cholesterol content. Furthermore, stepwise regression revealed a potential association (p < 0.03) between ACL and LSR. ACL and FAS protein have previously been shown to be modified by dietary fat content. Female 10-wk old LSR
+/-
(n=11) mice and LSR
+/+
littermates (n=7) were placed on a high fat (60% kcal) diet for 6 wks; LSR
+/-
exhibited higher weight gain, fat mass and PPL plasma TG vs LSR
+/+
as previously reported. A >2-fold (p<0.04) reduction in ACL, but not FAS protein in LSR
+/-
was observed as compared to controls. Interestingly, strong correlations were found between LSR and FAS (r = 0.8, p<0.01) and LDL-R (r=0.74, p<0.01), but only in LSR
+/-
mice. We therefore propose that there exists a close link between the regulation of the uptake of lipoproteins via the LSR and LDL-R pathways and
de novo
lipogenesis in the liver. Further studies will provide insight in the mechanisms involved in maintaining lipid homeostasis, leading towards potential treatments for hyperlipidemia associated with obesity, 2 high risk factors for atherosclerosis.
Abstract 513: Role of Heparanase in Atherosclerotic Plaque Formation
Authors: Johnson Collin G , Graf Iulia M , Emelianov Stanislav , Baker Aaron B
Published: July 2021

Little is know about the role of enzymatic regulation of glycosaminoglycans in the formation of atherosclerotic plaques. Heparanase is the only known mammalian enzyme that degrades the glycosaminoglycan heparan sulfate. We tested the hypothesis that heparanase could regulate atherogenesis and atherosclerotic plaque severity.

We transduced human endothelial cells with lentiviruses expressing one of four different constructs expressing shRNA targeted to heparanase or a scrambled control sequence. These shRNA knocked down the expression of heparanase to 30-50% of wild type expression levels. These lines were selected using puromycin and confirmed for gene modulation using immunostaining and western blotting for heparanase. The ability to uptake lipids was tested using fluorescently labeled, oxidized low-density lipoprotein (OxLDL). We found that cells expressing 30% of the heparanase protein in wild type cells had increased uptake of OxLDL in comparison to wild type cells (1.8+0.1 versus 1.00+0.07 of normalized OxLDL uptake; p <0.05)
.
We also evaluated the formation of atherosclerosis in transgenic mice overexpressing the human heparanase gene (HPA-Tg). This mouse line was crossed with the ApoE knockout mouse line. We exposed mice from this HPA-Tg/ApoE KO line and from the ApoE KO line of 20 weeks of age to 6 weeks of a high fat diet. At week 2 we surgically implanted an extravascular cuff on the left carotid artery to create a region of disturbed flow and low shear stress. Following the surgery, we used a high resolution combined ultrasound and photoacoustic imaging system to image the flow and plaque morphology after 1 day, 2 weeks and 4 weeks post-implantation of the cuff. Overexpression of heparanase increases the formation and severity of atherosclerosis in this model. Color doppler imaging revealed the existence of oscillatory flow in the left common carotid artery due to the formation of atherosclerotic plaque. This analysis confirmed an increase in the inflammatory response and neointimal formation in response to the low endothelial shear stress induced by the cuff in the HPA models compared to the ApoE.

Our studies support that heparanase is a powerful regulator of lipid uptake and atherosclerotic plaque formation in an animal model of hyperlipidemia.
Abstract 539: Collateral Formation Is Regulated by Vascular Endothelial Growth Factor-A and a Disintegrin and Metalloprotease Family Members 10 and 17
Authors: Lucitti Jennifer L , Faber James E
Published: July 2021

The density of native (pre-existing) collaterals varies widely and is a significant determinant of variation in severity of ischemic stroke, myocardial infarction and peripheral artery disease. However, little is known about mechanisms responsible for formation of the collateral circulation in healthy tissues. We previously found that variation in VEGF expression causes differences in collateral density of newborn and adult mice. Herein, we sought to determine mechanisms of collaterogenesis in the embryo and the role of VEGF in this process. Pial collaterals formed between embryonic day (E) 13.5 and 14.5 as sprout-like extensions from distal-most arterioles of the cerebral artery trees that increased in number up until birth. Global VEGF-A overexpressing mice (

Vegf
hi/+

) formed more_and

Vegf
lo/+

formed fewer_collaterals during embryogenesis, in association with differences in vascular patterning. Conditional global reduction of VEGF or FLK1 (VEGFR2) during collaterogenesis significantly reduced collateral formation, compared to wildtype littermates, without affecting vascular patterning. Endothelial-specific reduction of VEGF had no effect on collaterogenesis. Endothelial-specific reduction of a disintegrin-and-metalloprotease-domain-10 (ADAM10) and inhibition of γ-secretase increased collateral formation, consistent with their roles in VEGF-induced Notch1 activation and suppression of “pro-sprouting” signals. Endothelial-specific reduction of ADAM17 reduced collateral formation, consistent with its roles in endothelial cell migration and embryonic vascular stability, but not in activation of ligand-bound Notch1. These effects of ADAM 10/17 reduction persisted into adulthood. Conclusions: Formation of pial collaterals occurs during a narrow developmental window, requires paracrine VEGF-signaling, and is shaped by mechanisms associated with sprouting angiogenesis.
Abstract 542: Upregulation of Cyr 61 Expression in Atherosclerosis Is Mediated by β3 Integrins
Authors: Pathak Alokkumar S , Huang Jianhua , Rojas Mauricio , Stouffer George
Published: July 2021

Objective:
Cyr 61 is a member of the CCN family of proteins that is expressed in atherosclerotic lesions, stimulates proliferation of cultured smooth muscle cells (SMC) and is upregulated by angiotensin II (Ang II). We studied whether Ang II inhibition decreased expression of Cyr 61 in atheromas of ApoE–/– mice with unilateral partial renal artery ligation (RAL).

Methods and Results:
18 week old ApoE–/– mice were randomized to unilateral RAL, RAL + treatment with irbesartan, RAL + treatment with aliskiren or sham-surgery for 90 days. RAL reduced blood flow (60 ± 6% of baseline values), resulted in a sustained increase in renin mRNA levels in the ligated kidney, and elicited a mild, sustained increase in systolic blood pressure (11% increase in weighted average of blood pressure) compared to sham-surgery. Treatment with irbesartan or aliskiren prevented the hypertensive response. Lipid deposition, measured by Oil-red O staining 90 days after surgery, was increased approximately 3 fold following RAL in both the aortic arch and in the descending aorta compared to sham-surgery. Treatment with irbesartan or aliskiren reduced the aortic arch and descending aorta lipid deposition to a level that was statistically similar to sham-surgery. Atheromas in the abdominal aorta showed intense staining for Cyr 61 in RAL mice but patchy, diffuse and less intense staining in RAL + irbesartan (43% less staining) and RAL + aliskiren (60% less staining) mice. Studies in RASMC showed that Cyr 61 was secreted in response to treatment with Ang II and that echistatin, a function-blocking disintegrin that binds αvβ3 integrins on SMC, reduced Cyr 61 induced proliferation of RASMC. In mice with RAL, atheromas showed generalized and intense staining for β3 integrins that co-localized with Cyr 61. In contrast, β3 integrin staining was localized, discrete and even non-existent in atheromas from RAL + irbesartan and RAL + aliskiren mice. Sham-surgery mice had no atheromas and minimal staining for Cyr 61 and β3 integrins.

Conclusions:
Unilateral RAL resulted in a marked increase in Cyr 61 and β3 integrin expression within atheromas. Treatment with irbesartan or aliskiren significantly reduced aortic lipid deposition, and expression of Cyr 61 and β3 integrins in atheromas.
Abstract 63: CXCL8 Signaling Pathways and the Aortic Abdominal Aneurysm: The Oral CXCR2 Antagonist DF2156A Fully Abrogates Aneurysm Formation
Authors: Kokje Vivianne B , Dalman R L , Hamming J F , Lindeman J H
Published: July 2021

An aneurysm of the abdominal aorta (AAA) is part of the atherososclerotic spectrum of diseases. The disease is characterized by a localized dilatation of the infrarenal artery and rupture of the weakened segment is now responsible for more than 15.000 annual deaths in the US alone. The pathology of AAA is complex and poorly understood. It is generally conceived that AAA is characterized by a localized general pro-inflammatory response and an accompanying protease imbalance that is held responsible for the progressive weakening of the aortic wall. The exact inflammatory pathways that drive aneurysm progression and that distinguish AAA from occlusive atherosclerotic disease have not yet been identified. We previously reported a 21-fold increase in CXCL8 mRNA expression and an approx. 300-fold increase in CXCL8 protein levels in human AAA, as compared to human occlusive atherosclerotic disease (p<0.001); suggesting that activation of the CXCL8/CXCR1-2 axis is a distinctive feature of AAA.

We now performed a histological and protein analysis of human AAA and human occlusive atherosoclerotic disease, showing abundant presence of the CXCL8 receptors CXCR1 and 2 as well as increased ERK phosphorylation in AAA. Evaluation on cellular level identified abundant neutrophils (fully absent in atherosoclerosis) as well as increased neovascularization as clear distinctive features of AAA, a finding that is consistent with increased CXCL8 signaling. To test a putative role of CXCL8/CXCR1-2 system in AAA we next tested whether the oral CXCR2 antagonist DF2156A prevents aneurysm formation in the elastase model, a well established model of the disease. This study showed that a once daily dose of 15mg/kg DF2156A fully abrogates aneurysm formation in the elastase model (71.86% aortic diameter growth in 14 days (SEM 4.88) in untreated animals vs. 17.66% growth (SEM 1.71) in animals treated with DF2156A) (p<0.001).

In conclusion, these studies identify activation of the CXCL8/CXCR1-2 pathway as a distinctive feature of AAA and characterize this pathway as a potential target for the stabilization of AAA.
Abstract 66: TLR2 Signaling Protects Against Angiotensin II--Mediated AAA Formation in ApoE
-/-
Mice
Authors: Zhang Yali , Park Ho-Jin , Bagley Jessamyn , Maganto-Garcia Elena , Kyriakis John , Beasley Debbie , Lichtman Andrew , Galper Jonas B
Published: July 2021

Abdominal aortic aneurysm (AAA) is a potentially life-threatening degenerative vascular disease that affects 6-9% of men over 65. Although Toll-like receptors (TLRs) mediate the innate immune response resulting in the recruitment of inflammatory cells to the site of a range of pathogen-associated molecular patterns, they have also been implicated in immune tolerance. To determine the role of TLR2 signaling in AAA formation we compared AAA formation in ApoE
-/-
and ApoE-/-TLR2
-/-
DKO mice using a mouse model for angiotensin II (ANGII) mediated AAA formation. Treatment of ApoE
-/-
mice with Alzet pumps releasing 750 ng/kg/min ANG II resulted in formation of AAAs in 75% of mice (n=23) and a mean increase in aortic diameter from 1.0 mm in saline treated controls to 2.15±0.15 mm, p<0.001 with no effect on the thoracic aorta and the aortic arch. Although the incidence of AAAs in ApoE-/-TLR2
-/-
DKO mice compared to ApoE
-/-
alone was unchanged, of the 12 ApoE-/-TLR2-/-DKO who developed AAAs, half demonstrated severe aneurismal involvement of the aortic arch and thoracic aorta compared to the ApoE
-/-
group consistent with a protective effect of TLR2. To test this hypothesis, mice were pretreated with PAM3, 50ug/ mouse/week. The incidence of AAA formation decreased to 32% in PAM 3 treated mice and aortic diameter decreased to 1.17±0.08 (n=18, p<0.001) compared to ANGII alone. PAM3 had no effect on blood pressure or lipid levels. Furthermore, treatment with PAM3 after a 7 day ANG II infusion decreased the incidence of AAA to 53%. ANG II stimulated expression of the cytokines RANTES and CXCl10 and the receptor CCR5 more than 8 fold as measured by ELISA and western blot respectively; PAM3 treatment of these ANG II infused mice decreased expression to control levels in association with decreased macrophage infiltration. Finally, while ANG II increased the ratio of M1/M2 macrophages in the abdominal aorta by 2 fold, PAM3 treatment reversed the effect of ANG II on M1/M2 in aortas of mice that did not develop AAAs. Thus TLR2 stimulation protected the abdominal aorta from ANG II mediated AAA at least in part by attenuating recruitment of macrophages and expression of chemokines and chemokine receptors.
Abstract 70: PPARdelta Agonist GW1516 Attenuates Diet-Induced Dyslipidemia, Insulin Resistance and Aortic Inflammation in Ldlr
-/-
Mice
Authors: Bojic Lazar , Telford Dawn , Sutherland Brian , Sawyez Cynthia , Edwards Jane , Huff Murray
Published: July 2021

Objective:
The peroxisome proliferator-activated receptor (PPAR) delta has been implicated in systemic lipid homeostasis and inflammation. However, the role of PPARdelta agonists as anti-atherogenic agents remains unclear. In the present study, we used low-density lipoprotein receptor-null mice (Ldlr-/-) fed a high fat (HF) diet to test the hypothesis that a selective PPARdelta agonist corrects metabolic dysregulation and attenuates inflammation associated with atherosclerosis.

Methods and Results:
Ldlr
-/-
mice were fed chow or HF (42% fat, 0.2% cholesterol) for 4 weeks. Subsequently, the HF group was fed either HF or HF plus GW1516 (3mg/kg/d) for a further 8 weeks. Fasting plasma triglyceride, total cholesterol and free fatty acids were significantly decreased (-50%) by intervention with GW1516. In addition, GW1516 normalized fasting blood glucose and improved glucose and insulin tolerance. GW1516 also enhanced total energy expenditure compared to HF-fed mice. In the aorta, ER-stress markers CHOP and GRP78 were significantly elevated in HF-fed mice, which were markedly attenuated by GW1516-intervention. Aortae of HF-fed mice also showed marked elevations in the expression of proinflammatory cytokines including Ccl3, Il1beta, Icam1, Tnf, Il6 and Ccl2. Furthermore, HF-aortae, compared to chow, displayed reduced expression of the M2 macrophage marker arginase-1(Arg1). Intervention with GW1516 significantly attenuated aortic expression of all examined proinflammatory cytokines, and restored Arg1 expression. Enhanced MAPKerk signalling and decreased AKT/FoxO1 signalling are known to induce inflammatory cytokine expression in vitro. HF-feeding induced phosphorylation (p) of the MAP kinases ERK1/2 and p38 and dampened levels of pAKT and pFoxO1 in the aorta. In contrast, aortae of GW1516-treated animals displayed normalized levels of pERK1/2, p-p38, pAKT and pFoxO1.

Conclusions:
These studies demonstrate that PPARdelta activation ameliorates dyslipidemia and insulin resistance in HF-fed Ldlr
-/-
mice. Furthermore, PPARdelta activation inhibits aortic ER-stress as well as dysregulation of MAPK and AKT/FoxO1 signalling induced by HF-feeding, resulting in inhibition of the inflammatory response within the aorta.
Abstract 81: B Lymphocytes Trigger MCP3-Dependent Mobilization of Monocytes and Promote Adverse Ventricular Remodeling After Myocardial Infarction
Authors: Zouggari Yasmine , Ait-Oufella Hafid , Bonnin Philippe , Vilar José , Guerin Coralie , Charo Israel F , Tedder Thomas F , Tedgui Alain , Silvestre Jean-Sébastien , Mallat Ziad
Published: July 2021

Leukocyte infiltration in ischemic areas is a hallmark of myocardial infarction, and persistent infiltration of innate immune cells, such as neutrophils and Ly6Chi monocytes, has been shown to promote adverse cardiac tissue remodeling. However, little is known regarding the role of mature B lymphocytes, which play a crucial role in the activation of the inflammatory response in several immune-mediated diseases. Here, we hypothesized that B lymphocytes might modulate the inflammatory response and affect the immune-dependent adverse cardiac remodeling. In a mouse model of myocardial infarction, cardiac B lymphocytes levels peaked at day 5 after the onset of infarction. Of interest, treatment with a CD20-specific monoclonal antibody decreased circulating and infiltrating B cell numbers (p=0.0008 and p=0.0002 vs control), reduced infarct size and post-ischemic immunoinflammatory response, and improved cardiac function (p=0.02 vs control) assessed by echocardiography. Intriguingly, B cell depletion was associated with an impairment of Ly6Chi monocytes mobilization from bone marrow (p=0.02 vs control), leading to reduced levels of circulating and infiltrating cardiac monocytes. The acute infarction led to transient increase of both MCP-1 and MCP-3 levels. Interestingly, B cell depletion was associated with a significant and selective reduction of MCP-3 (p=0.03 vs control) but did not alter MCP-1 levels (p=0.11). Cultured activated B cells released MCP-3 and treatment with a neutralizing MCP-3 antibody abrogated B lymphocytes-induced migration of cultured monocytes. Finally, transfer of B cell-depleted splenocytes into Rag1
-/-
mice improved cardiac function after myocardial infarction compared to the transfer of non-depleted splenocytes (p=0.005). This effect was abrogated after re-supplementation with B lymphocytes isolated from wild-type mice (p=0.0007) but not from MCP-3-deficient animals (p=0.7008). In conclusion, we show that following acute myocardial infarction, B lymphocytes, trigger an MCP-3-dependent mobilization of Ly6Chi monocytes from the bone marrow to the blood, leading to their recruitment into the injured myocardium and to exacerbation of tissue inflammation, thereby promoting adverse cardiac remodeling.
Abstract 128: Serum Anti-MAA Antibody Titers are Associated with Acute Myocardial Infarction and MMP-9
Authors: Ahmed Faisal , Duryee Michael J , Shurmur Scott W , Johnston Michael D , Hunter Carlos D , Thiele Geoffrey M , Anderson Daniel
Published: July 2021

Introduction

Atherosclerotic plaque rupture is the leading cause of acute myocardial infarction (AMI). Malondialdehyde acetaldehyde (MAA) modified proteins have recently been implicated in this process by their ability to initiate inflammatory responses. Matrix metalloproteinase 9 (MMP-9) is increased following AMI and has been associated with the disease due to its ability to degrade the fibrotic cap that forms on some plaques.

Objective

The purpose of this study was to evaluate the relationship between MMP-9 and anti-MAA antibody isotypes in patients experiencing acute myocardial infarction (AMI).

Methods

Serum samples from AMI patients (n=10) were collected and tested for the presence of anti-MAA antibody isotypes, peak CK, peak troponin, and MMP-9 levels. Samples were collected at the time of AMI, 24 and 48 hours post event. Data was correlated to determine relationships with antibody isotypes directed against MAA protein antigens.

Results

MMP-9 increased significantly from the initial presentation at 24 hours post-AMI (p=0.006) and 48 hours post-AMI (p= 0.044). Anti-MAA IgM decreased significantly (p= 0.021) between initial and 24 hours post AMI. A similar significant decrease was observed for anti-MAA IgG (p= 0.006). Anti-MAA IgA levels did not change significantly post-AMI. Significant correlations were demonstrated between peak CK and anti-MAA IgG 24 hours post AMI (r= -0.819, p= 0.013), between peak troponin and anti-MAA IgG 24 hours post AMI (r= -0.860, p= 0.006) and between MMP-9 levels 24 hours post AMI and anti-MAA IgM levels 24 hours post MI ( r= 0.0712, p=0.021).

Conclusions

The negative correlation of anti-MAA IgG to cardiac enzymes is suggestive that myocardial infarction and tissue necrosis results in the release of the MAA antigen from infarcted myocardium and clearance of anti-MAA antibodies. Additionally, anti-MAA IgM and MMP-9 are positively correlated with each other 24 hours post-AMI, suggesting that anti-MAA IgM titers may correlate to the severity of the AMI and thus CAD.

Implications

Anti-MAA antibodies might serve as important biomarkers of impending atherosclerotic plaque rupture and subsequent AMI and warrants further investigation in a larger cohort of patients.