bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 573: Human Microrna-548p Decreases apoB Secretion and Lipid Synthesis
Authors: Zhou Liye , Hussain Mahmood
Published: July 2021

Objective:
MicroRNAs (miRs) play important regulatory roles in lipid and lipoprotein metabolism. ApoB, as the only essential scaffolding protein in the assembly of very low density lipoproteins, is a target to treat hyperlipidemia and atherosclerosis. We aimed to find out miRs that reduce apoB expression.

Approach:
Bioinformatics analyses predicted that hsa-miR-548p can interact with apoB mRNA.MiR-548p mimic and control were transfected in human and mouse hepatoma cell lines to test its role in regulating apoB secretion and mRNA expression levels. Site-directed mutagenesis was used to identify the interacting site of miR-548p in human apoB 3′-untranslated region. Fatty acid oxidation and lipid syntheses were examined in miR-548p overexpressing cells to investigate its function in lipid metabolism.

Results:
Experimentally, we observed that miR-548p significantly reduces apoB secretion from human hepatoma cells in time and dose dependent manner. Mechanistic studies showed that miR-548p interacts with the 3′-untranslated region of human apoB mRNA to enhance posttranscriptional degradation. Bioinformatics algorithms suggested two potential binding sites of miR-548p on human apoB mRNA. Site-directed mutagenesis studies revealed that miR-548p targets site II involving both seed and supplementary sequences. MiR-548p had no effect on fatty acid oxidation but significantly decreased lipid synthesis in human hepatoma cells by reducing the expression of HMGCR and ACSL4 enzymes involved in cholesterol and fatty acid synthesis. In summary, miR-548p reduces lipoprotein production and lipid synthesis by reducing expression of different genes in human hepatoma cells.

Conclusion:
These studies suggest that miR-548p could be useful in treating atherosclerosis, hyperlipidemia and hepatosteatosis.
Abstract 584: Allograft Inflammatory Factor-1 is Required for NfκB Pathway Activity in Macrophages and Atherosclerosis
Authors: Egaña-Gorroño Lander , Chinnasamy Prameladevi , Sibinga Nicholas
Published: July 2021

Introduction:
In preliminary studies, we found that Allograft inflammatory factor-1 (AIF1) supports MΦ migration, phagocytosis, survival and pro-inflammatory cytokine secretion. Moreover, AIF1 limits necrotic core formation in atherosclerotic lesions
in vivo
. Nuclear Factor-κB (NFκB)-mediated signal transduction has been established at different stages of atherosclerosis. We hypothesize that AIF1-regulated processes in atherosclerosis may be mediated through effects on NFκB signaling.

Methods:
Bone marrow (BM) derived MΦs were isolated and immortalized from wt and

Aif1
-/-

mice and stimulated with oxidized-LDL (50 ug/ul; oxLDL). Lysates were immunoblotted for total and phosphorylated (active) p65 NFκB, and for total and phosphorylated forms of the IκBα repressor.
Aif1
expression in mouse Raw 264.7 cells was knocked down (KD) using siRNA, and NFκB reporter activity, measured by a luciferase reporter, was assessed after adding LPS+IFN-γ. Immunohistochemical analysis for phospho-p65 NFκB was performed in atherosclerotic lesions (aortic roots) from

Apoe
-/-

(SKO) and

Apoe
-/-
;Aif1
-/-

(DKO) mice maintained on high fat diet for 16 weeks.

Results:
In AIF1-deficient BMMΦs stimulated with oxLDL, we found no differences in the levels of total p65 NFκB and IκBα, but interestingly, phospho-p65 NFκB levels were significantly reduced and phospho-IκBα levels increased compared to wt cells (P<0.05). AIF1 KD using siRNA significantly reduced NFκB activity compared to scrambled control (scrambled control vs. AIF1 KD; 40% vs. 22% luciferase activity, P<0.05), and this impairment was rescued with the addition of AIF1 cDNA.
In vivo
, NFκB phospho-p65 staining showed that in comparison to SKO samples, DKO aortic roots had decreased phospho-p65 NFκB (SKO vs. DKO; 7.0 vs. 4.0 +nuclei/aortic root, P<0.05).

Conclusions:
AIF1 is required for NFκB activation in MΦs and moreover, AIF1 enhances NFκB activity in atherosclerotic lesions
in vivo
. Because NFkB has been closely linked to both cytokine expression and cell survival signaling, these results point to a critical role for AIF1 in pro-inflammatory MΦ functions. Future studies involve identifying the precise steps of the pathway controlled by AIF1, and the mechanisms by which AIF1 affects NFκB signaling.
Abstract 589: Host-Genotype Shaped Microbiome Modulates Development of Atherosclerosis
Authors: Kasahara Kazuyuki , Traeger Lindsay L , Romano Kymberleigh A , Kreznar Julia H , Vivas Eugenio I , Mehrabian Margarete , Lusis Aldons J , Rey Federico E
Published: July 2021

Genetic variation drives phenotypic diversity and determines predisposition to cardiovascular disease. Previous work revealed a large degree of variation on atherosclerosis susceptibility among 100 inbred strains of mice from the Hybrid Mouse Diversity Panel (HMDP). Additionally, each HMDP inbred strain shows a distinct microbiota composition. Here, we assessed the contributions of the gut microbiome to the development of atherosclerosis. Cecal samples from four HMDP strains showing disparate atherosclerosis phenotypes were transplanted into groups of
ApoE
-/-
germ-free mice. Transplanted
ApoE
-/-
mice were maintained on a high-plant polysaccharide diet containing 0.2% cholesterol for 8 weeks and analyzed for atherosclerotic lesions, microbiome, and metabolome. We found that mice colonized with cecal microbes from two HMDP donors prone to atherosclerosis development exhibited larger lesions compared to recipient mice colonized with samples from donors that show little signs of atherosclerosis. Metabolomic analyses of plasma from transplanted mice and metagenomic analyses of cecal contents from the transplanted and 24 HMDP donor mice identified microbial functions that are associated with the severity of disease. Altogether, our work provides novel insights into how microbes contribute to the development of cardiovascular disease and suggest that host genotype may select for microbial communities that modulate progression of atherosclerosis.
Abstract 59: Wall Layer-Specific Gene Expression Profile of Coding and Non-Coding Transcripts in Abdominal Aortic Aneurysms
Authors: Lindquist Liljeqvist Moritz , Hultgren Rebecka , VIllard Christina , Kronqvist Malin , Eriksson Per , Roy Joy
Published: July 2021

Objective:
To identify coding and non-coding transcripts and gene sets that are differentially expressed and enriched, respectively, in the media and adventitia of abdominal aortic aneurysms (AAA).

Methods:
Aortic tissue samples from 76 patients with AAA and 13 transplant donors (controls) were collected from the Stockholm AAA Biobank (STAAAB) and analyzed with Affymetrix HTA 2.0 microarrays. All samples were divided into intimal/medial and adventitial wall layers. Differential expression, adjusted for age and gender, and orthogonal partial least squares discriminant analysis (OPLS-DA) were performed with R. Gene set enrichment analysis (GSEA) was performed for annotated transcripts against the Molecular Signatures Database.

Results:
When controls were compared with AAAs, 9379 transcripts were differentially expressed in the tunica media and 3982 in the adventitia. Comparing adventitia and media, 7915 and 13154 transcripts were differentially expressed for AAAs and controls, respectively. Using OPLS-DA, significant separation could be made for all these comparisons. However, the difference between wall layers was less pronounced in AAA. Hallmark gene sets that were transmurally increased in AAA included allograft rejection and responses to interferon gamma and alpha and tumor necrosis factor alpha, whereas gene sets related to metabolism and sex hormone responses were downregulated. Gene sets upregulated in the AAA media, compared with its adventitia, included angiogenesis, complement and hypoxia. In controls, the media, compared with the adventitia, expressed mitotic spindle and myogenesis gene sets.

Conclusions:
The gene expression profiles of AAAs and controls are distinct and differ between wall layers. While inflammation is transmural in the AAA, hypoxia and angiogenesis are increased in the media. These results are in line with, and yield additional detail to, research published previously.
Abstract 591: Absence of Nicotinamide Nucleotide Transhydrogenase Exacerbates Atherosclerosis in Mice
Authors: Vozenilek Aimee E , Vetkoetter Matthew , Shen Xinggui , Woolard Matthew D , Klein Ronald L , Orr A W , Krzywanski David M
Published: July 2021

Oxidant stress contributes to endothelial cell injury and inflammation that are hallmarks of early stage atherosclerosis. Emerging evidence implicates mitochondrial reactive oxygen species (ROS) as important contributors to this oxidant stress and differences in mitochondrial function may augment this process. We have shown that variation in mitochondrial function and ROS production associated with ethnicity contributes to endothelial and vascular dysfunction. To model these distinct mitochondrial redox phenotypes we used C57Bl/6N (6N) and C57Bl/6J (6J) mice that also display unique mitochondrial functional properties due to the differential expression nicotinamide nucleotide transhydrogenase (NNT). Mice were treated with adeno-associated virus encoding a gain-of-function form of proprotein convertase subtilisin/kexin type 9 (PCSK9) that leads to hypercholesterolemia, increased LDL levels, and atherosclerosis in mice. PCSK9 treatment and 8 weeks of high fat diet led to increases in plasma lipids in both 6N and 6J mice. However, 6J animals displayed significantly higher levels of fat deposition in the vasculature and increased plaque size in the carotid sinus. 6N mice co-treated with the mitochondria targeted superoxide dismutase mimetic MitoTEMPO for the final 4 weeks of the experiment displayed reduced plasma lipids, but no impact on fat deposition or plaque size was observed. In contrast, MitoTEMPO increased vascular fat deposition and plaque size in 6J mice consistent with a more severe atherosclerotic phenotype. Increased mitochondrial ROS was confirmed by demonstrating elevated vascular superoxide in 6J versus 6N animals and that this difference is exacerbated on high fat diet. MitoTEMPO diminished vascular superoxide production to near baseline levels in both groups, yet increased plaque size and fat deposition in 6J mice suggests a role for hydrogen peroxide in this process. These data indicate that loss of NNT and changes in mitochondrial function increase vascular ROS production and exacerbate lipid deposition and plaque development in the early stages of atherosclerosis.
Abstract 602: High-Density Lipoprotein Protects Macrophages Against Apoptosis Through BH3-only Bcl-2 Family Member Bim
Authors: Qian Alexander , Yu Pei , Gonzalez Leticia , Trigatti Bernardo
Published: July 2021

Macrophage apoptosis contributes to the formation of necrotic cores within atherosclerotic lesions, increasing the susceptibility of atherosclerotic plaques to rupture and the risk of cardiovascular events. In advanced atherosclerotic plaques, lipid-laden foam cells are exposed to a diverse number of apoptosis inducers, including prolonged endoplasmic reticulum (ER) stress. The pro-apoptotic BH3-only protein Bim has been reported to mediate apoptosis in response to ER stress induction in a variety of cells including macrophages. Treatment of macrophages with high-density lipoprotein (HDL) protects them against apoptosis induced by a variety of stressors. We hypothesize that HDL mediated protection against macrophage apoptosis occurs via pathways that regulate Bim activity. To test this, thioglycollate-elicted peritoneal macrophages were treated in lipoprotein deficient culture with different apoptosis inducers in the absence or presence of HDL. Cell apoptosis was assessed by cleaved caspase 3 staining and terminal deoxynucleotidal transferase dUTP nick end labeling. Treatment of mouse peritoneal macrophages in culture with 50μg/ml of human HDL protected against apoptosis induced by tunicamycin (P<0.05) without affecting ER stress markers, Grp78, Grp94 and CHOP. Peritoneal macrophages from mice deficient in Bim were less susceptible to apoptosis induced by treatment with either tunicamycin or 7-ketocholesterol. Furthermore, treatment with HDL provided no additional protection to apoptosis in Bim KO macrophages. Our results suggest that protection of macrophage apoptosis by HDL is regulated through pathways involving the activity of BH3-only Bcl-2 family member Bim.
Abstract 603: Induction of Dendritic Cell-Mediated Activation of T Cells From Atherosclerotic Plaques by Human Heat Shock Protein 60
Authors: Rahman Mizanur , Steuer Johnny , Gillgren Peter , Liu Anquan , Frostegård Johan
Published: July 2021

Objective:
Atherosclerosis, the major cause of cardiovascular disease (CVD) is characterized by presence of activated immunocompetent cells, including dendritic cells (DCs) and T cells; dead cells and oxidized low density lipoprotein (OxLDL) in the atherosclerotic plaques. Heat shock protein (HSP), especially HSP60, is implicated in atherosclerosis. We reported that Annexin A5 (ANXA5) inhibits atherosclerosis-manifestations in apolipoprotein E-/- mice. Here we study HSP60 effects on human DCs and T cells.

Methods and Results:
Human DCs were treated with HSP60 and naïve autologous T cells were co-cultured with thus pre-treated DCs. HSP60 induced DC-activation and T cell proliferation as determined by FACScan, gene-activation and cytokine production. HSP60-induced T cell activation was partly MHC class II-dependent. T cells exposed to HSP60-treated DCs produced IFN-γ, IL-17 but not TGF-beta. HSP60 did not promote expression of Toll like receptors 2 or 4. ANXA5 inhibited HSP60-effects on DCs and T cells and partly bound HSP60. Further, OxLDL induced HSP 60 was abolished by ANXA5. Experiments on DC and T cells derived from carotid atherosclerotic plaques from patients with symptomatic carotid disease gave similar results as from blood donors.

Conclusions:
Our data show that HSP60 induces an MHC class II-dependent activation of blood- and plaque derived T cells which are mostly of Th1 type. HSP60 could thus be an important T cell antigen in plaques, and mediate oxLDL:s immunogenic effects on DC-T cell activation, promoting plaque rupture and clinical manifestations of CVD. ANXA5 inhibits these effects.
Abstract 604: Genetic Deletion of Interleukin-19 Exacerbates Atherogenesis and Inflammatory Gene Expression in

Ldlr
-/-

Mice
Authors: Ray Mitali , Gabunia Khatuna , Vrakas Christine , Kako Farah , Kelemen Sheri E , Herman Allison , Autieri Michael V
Published: July 2021

IL-19 is an anti-inflammatory interleukin and a member of the IL-10 family. Our laboratory has previously shown that IL-19 treatment is able to decrease atherosclerotic plaque burden in

Ldlr
-/-

mice through polarization of T cells and macrophages to their anti-inflammatory phenotypes. We hypothesized that lack of IL-19 would exacerbate atherosclerosis. We observed that the absence of IL-19 results in increased plaque burden in

Ldlr
-/-

x

Il19
-/-

(DKO) mice when compared to

Ldlr
-/-

controls after 14 weeks of high fat diet (HFD) administration by
en face
and Oil Red O staining of aorta and aortic root. In a rescue study wherein DKO mice were placed on HFD for 14 weeks and simultaneously injected i.p. with 10ng/g/day of mIL-19 or PBS, DKO mice injected with mIL-19 had significantly less plaque than PBS controls. To test our hypothesis that exacerbated plaque in the DKO was due to an increased pro-inflammatory environment, we performed gene expression analysis from spleen and arch from

Ldlr
-/-

and DKO mice after 14 weeks of HFD, as well as a series of
in vitro
experiments in isolated

Ldlr
-/-

and DKO VSMCs and BMDMs. qRT-PCR analysis revealed DKO mice have a global and more dramatic local polarization of T cells and macrophages to pro-inflammatory Th1 and M1 phenotypes in the spleen and aortic arch. We stimulated cultured VSMCs and BMDMs with pro-inflammatory stimulus, TNFα, and found that DKO VSMCs express greater levels of TNFα and MCP-1 mRNA compared to

Ldlr
-/-

controls. DKO BMDMs also exhibit increased expression levels of TNFα, as well as IL-1β when compared to

Ldlr
-/-

. Gene expression analysis also demonstrated increased levels of pro-inflammatory mRNA binding protein, human antigen R (HuR) in the DKO spleen and arch. HuR stabilizes pro-inflammatory transcripts by binding ARE elements in the 3’ UTR. Utilizing the RNA synthesis inhibitor actinomycin D in cultured BMDM and VSMC, we observed increased mRNA stability of inflammatory genes in DKO cells when compared to

Ldlr
-/-

by qRT-PCR. These data suggest that IL-19 is an atheroprotective cytokine which dampens inflammatory gene expression by modulation of mRNA stability.
Abstract 605: Diastolic Blood Pressure Predicts Coronary Plaque Volume in Patients With Coronary Artery Disease
Authors: Saleh Mohamad , Alfaddagh Abdulhamied , Elajami Tarec , Ashfaque Hasan , Welty Francine
Published: July 2021

Introduction:
Hypertension is a major risk factor for coronary artery disease (CAD) and is associated with increased morbidity and mortality. The effect of systolic vs. diastolic blood pressure (BP) on coronary plaque volume has not been reported. Coronary blood flow occurs during diastole; shear stress affects plaque formation.

Hypothesis:
Diastolic BP may be a better predictor of plaque volume than systolic BP.

Methods:
285 subjects with stable CAD underwent coronary computed tomographic angiography to assess the indexed volume (plaque volume [mm3] divided by segment length [mm]) of fatty, fibrous, non-calcified, calcified and total coronary plaque. Segments with significant calcium-blooming artifact were excluded. A backward linear regression model adjusted for age, gender, BMI, estimated glomerular filtration rate, total cholesterol, LDL-C, HDL-C and triglycerides.

Results:
Mean (SD) age was 63.1 ± 7.7 years (18% female). A significant increase was observed in volume of all plaque components across diastolic BP tertiles except for calcified which was borderline (p= 0.069) (Table). In contrast, there was no difference in systolic tertiles. In multivariate regression, diastolic BP tertile was a significant independent predictor of volume of all plaque components - fatty (β= 0.075; p=0.038), fibrous (β= 0.178; p= 0.004), non-calcified (β= 0.256; p= 0.008), calcified (β= 0.081; p= 0.007) and total (β= 0.353; p= 0.002) whereas no association was observed between systolic BP tertile and volume of any plaque component (Table).

Conclusion:
In patients with CAD, diastolic BP tertile independently predicts coronary plaque volume whereas systolic BP tertile does not. Since coronary blood flow occurs during diastole, a possible mechanism for the difference between systolic and diastolic BP is increased shear stress during diastolic blood flow. Therefore, control of diastolic BP may be an important factor in determining plaque volume.
Abstract 622: Cd44 Plays an Important Role in Regulating Heart Failure Induced Lung Vascular Remodeling and Who Type-2 Pulmonary Hypertension
Authors: Weng Xinyu , Yue Wenhui , Wang Dongzhi , Wang Huan , Xu Yawei , Chen Yingjie
Published: July 2021

End-stage left ventricular failure or chronic heart failure (CHF) causes severe lung inflammation, vascular remodeling, WHO type-2 pulmonary hypertension, and right ventricular hypertrophy. However, the molecular mechanism of CHF-induced lung inflammation and remodeling is largely unknown. CD44 is a member of the hyaluronate receptor family of cell adhesion molecules, which has been shown to play a selective role in controlling macrophage and lymphocyte migration. Here we demonstrated that end-stage CHF causes a dramatic increase of CD44 expression in heart and lung in human and mice. Histological staining shows that CD44 is predominantly expressed in leukocytes such as macrophages. Flow cytometry analysis further demonstrates that CD44 is predominantly expressed in F4/80 positive macrophages, CD4+, and CD8+ T cells. CD44 expression is dramatically increased in activated T cell subsets. To further determine the physiological role of CD44 in CHF-induced lung remodeling and type-2 pulmonary hypertension, we studied the effect of CD44 blockade on type-2 pulmonary hypertension development in a group of mice with existing moderate left ventricular failure without apparent lung remodeling. Interestingly, we found that blockade CD44 with blocking antibodies (Abs) significantly attenuate the development of lung vascular and interstitial leukocyte infiltration, lung vascular remodeling, fibrosis, and increase of right ventricular hypertrophy. Blockade CD44 signaling also significantly attenuated further decline of left ventricular ejection fraction in mice with existing LV failure. In addition, we demonstrated that induction of T regulatory cells with IL-2 and IL-2 Abs complex significantly attenuated the infiltration of CD44 positive leukocytes in lung tissue, lung vascular remodeling, lung fibrosis, and right ventricular hypertrophy in mice with existing moderate left ventricular failure. Together, these data indicate an important role of CD44 in left ventricular failure-induced lung inflammation, and type-2 pulmonary hypertension, suggesting that inhibition of CD44 may attenuate heart failure progression and type-2 pulmonary hypertension.
Abstract 624: Accumulation of Advanced Glycation End Products is Associated With Structural Degeneration of Bioprosthetic Heart Valves
Authors: Frasca Antonio , Branchetti Emanuela , Grau Juan B , Gillespie Matthew J , Levy Robert J , Ferrari Giovanni
Published: July 2021

Introduction:
Bioprosthetic heart valves (BHV) are widely used but frequently limited by structural valve degeneration (SVD), necessitating re-operation. Calcification is by far the best-studied SVD mechanism, but recent studies suggest that oxidative stress contributes to SVD. Advanced glycation end products (AGEs), formed by the ligation of sugar derivatives to proteins under oxidative conditions, mediate tissue degeneration in cardiovascular diseases. AGEs effect tissue damage through cross-linking and degradation of extracellular matrix proteins including collagen, a major structural component of BHV, as well as through exacerbation of inflammatory cell stimulation.

Hypothesis:
We hypothesize that AGEs accumulate in BHVs and are associated with oxidative stress-based SVD

Methods:
We performed immunohistochemistry for AGEs and specifically carboxymethyl lysine (CML) on sections of fixed, paraffin-embedded samples of degenerated bioprosthetic heart valves from 7 patients in the Penn Cardiac Bioregistry (4 male, 3 female, ages 40-83) and 4 short-term experimental Melody valve leaflets deployed in branch pulmonary arteries of 3 sheep. We correlated these results with levels of the circulating AGE receptor sRAGE via ELISA for patient BHV explants and with oxidized amino acid levels quantitated by mass spectrometry of di-tyrosine for experimental sheep BHV explants.

Results:
There was diffuse accumulation of AGEs in both clinical SVD and sheep explants of BHV. Immunohistochemistry reveals clear AGE and CML staining in all tested valves versus minimal to absent staining in unimplanted control samples. Staining intensity qualitatively correlated with overall di-tyrosine accumulation as well as sRAGE levels that were elevated in BHV patients. Notably, local staining for AGE in BHV is intensified in areas of calcification, thickening, and apparent inflammatory infiltration as well as in areas with structural damage.

Conclusions:
Extensive AGE accumulation was observed in both BHV explanted for SVD and sheep explants compared to controls, supporting the view that AGEs are associated with SVD pathophysiology. The association of AGE accumulation with di-tyrosine in BHV suggests that oxidative stress is a major mechanism of BHV SVD.
Abstract 634: Partial Genetic Disruption of mTOR Signaling Does Not Improve Vascular Endothelial Function in Hypercholesterolemic Mice
Authors: Jbeli Aiham H , Zhang Bin , Roos Carolyn M , Miller Jordan D
Published: July 2021

Endothelial cell senescence promotes inflammation and impairs endothelium-dependent vasodilation. While aging, hypercholesterolemia, and caloric excess are known to drive cellular senescence in part through activation of mTOR, it is unknown whether reducing mTOR signaling can improve vasomotor function in aging mice. Here, we used hypercholesterolemic mice (Ldlr
-/-
/apoB
100/100
, or LA) carrying intact copies of mTOR (LA-mTOR
+/+
) or were deficient in one copy of mTOR (LA- mTOR
+/-
). Mice were fed Western Diet for 6 or 12 months, and vasomotor function in aorta was evaluated using isolated organ chamber baths. Maximum relaxation to acetylcholine (MR
ACH
) was progressively impaired from 6 months to 12 months in LA-mTOR
+/+
mice (55±4% and 30±4%, respectively). In contrast to our hypothesis, however, MR
ACH
was not significantly improved in LA-mTOR
+/-
mice at either time point (62±3% and 35±5%, respectively). Interestingly, a subgroup analysis showed significant improvement in MR
ACH
in male mice at the 6-month point (male LA-mTOR
+/+
mice = 30 ± 9% vs male LA-mTOR
+/-
mice = 55±4%; p < 0.01), whereas vasomotor function in female mice was nearly identical across groups. There was no significant difference at the 12-month point (male LA-mTOR
+/+
mice = 19 ± 3% vs male LA-mTOR
+/-
mice = 22 ± 5%). Relaxation to acetylcholine was attenuated by L-NAME at 6 months and 12 months (but identical between genotypes) and relaxation to nitroprusside was identical between strains at each time point, suggesting that changes in endothelial function were not masked by compensatory mechanisms (e.g., endothelium-derived hyperpolarizing factors). Furthermore, reduction of mTOR did not elicit changes in maximal tension generated in response to Prostaglandin F
2
α
at either time point. Collectively, despite compelling evidence from in vitro model systems, genetic reduction of mTOR signaling
in vivo
does not appear to be sufficient to improve vasomotor function in our model of aging hypercholesterolemic mice. Our data do, however, shed light on a potential role of sex in dictating phenotypic changes in early to moderate stages of disease, which warrants further investigation.
Abstract 641: Protein Disulfide Isomerase-A1 Overexpression Enhances Vascular Calcification in Mice
Authors: Pescatore Luciana A , Nolasco Patricia , Farias-Silva Elisângela , Jensen Leonardo , Fernandes Carolina G , Almeida Youri E , Carmo Luciana S , Laurindo Laurindo R , Liberman Marcel
Published: July 2021

Vascular calcification (VC) is an active pathophysiological process promoting enhanced morbidity and mortality without effective therapy. Oxidative stress and endoplasmic reticulum (ER) stress support cardiovascular calcification progression. We showed previously that the ER redox chaperone protein disulfide isomerase-A1 (PDI) is required for agonist-driven Nox NADPH oxidase activation. Oxidant levels and expression of Nox subunits and PDI are upregulated in calcifying rabbit valves. We hypothesized that PDI supports VC. We recently developed a new PDI overexpression mouse model (TgPDI) in FVB background (WT). To investigate whether TgPDI mice show increased VC, we challenged 20 week-old TgPDI mice with Vitamin-D
3
(VitD) for 14 days. Also, we incubated primary vascular smooth muscle cells (VSMC) isolated from WT or TgPDI mice with mineralizing medium (β-glycerophosphate 10mM + CaCl
2
6mM). TgPDI results were compared to those from respective WT littermates. We tested distinct VitD doses. At 9x10
4
IU/day, WT mice depicted negligible increases in calcification area (median 0.07% to 0.36% aorta cross-sectional area, p=ns, n=5), while TgPDI showed enhanced calcification (median 0.20 to 3.63 % area, p<0.05, n=4). The sum of calcification areas across all mice showed a 2.6-fold increase in TgPDI. TgPDI mice did not develop aortic valve stenosis or change in ascending aorta peak wave velocity at echocardiography.
In vitro,
VSMC phenotype marker expression (SM22 and alpha-actin) was enhanced in TgPDI vs. WT cells at baseline, while calcifying stimulation for 3 or 7 days decreased such VSMC marker expression both in WT and in TgPDI VSMC. Conversely, calcifying stimulation for 3 or 7 days showed no effects on ER stress marker expression (GRP78, GRP94). To assess whether these findings apply to humans, we examined femoral arteries from diabetic and non-diabetic patients with peripheral arterial disease, which showed increased arterial calcification and marked coincident PDI overexpression in samples from diabetic patients. In conclusion, PDI overexpression
in vivo
associates with enhanced VC after VitD

calcifying stimulus. PDI and its associated signaling proteins deserve further study as potential therapeutic targets in VC progression.
Abstract 9: Smooth Muscle Cell Tgfbr2 Deletion in Mice Causes Aortic Hypercontractility and Impaired Endothelium-Dependent Relaxation
Authors: Zhu Jay , Alp Yildirim Fatos I , Angelov Stoyan N , Wei Hao , Dichek David
Published: July 2021

Background:
Abnormal smooth muscle cell (SMC) TGF-β signaling is proposed as a critical driver in the development of thoracic aortic aneurysms and dissections (TAAD) associated with Marfan and Loeys-Dietz Syndromes as well as nonsyndromic TAAD. However, the mechanisms by which altered SMC TGF-β signaling causes TAAD are poorly understood. Others have proposed that loss of SMC TGF-β signaling causes TAAD by impairing SMC contractility, leading to aortic medial degeneration and dilation. However, mice generated in our lab with deficient SMC TGF-β signaling (due to SMC-specific deletion of the type II TGF-β receptor) have thicker aortic medias and increased mRNA encoding SMC contractile proteins. These observations predict increased contractility.

Methods & Results:
We addressed this apparent contradiction experimentally by measuring vasomotor function (by tension myography) and contractile protein expression (by immunoblotting) in aortas of mice with normal or deficient SMC TGF-β signaling. Isolated aortic rings from mice with deficient SMC TGF-β signaling showed increased contraction to phenylephrine (Emax: 13.5 mN vs 7.8 mN in controls; p<0.0001; n=19-20) and potassium chloride (Emax: 7.5 mN vs 5.7 mN in controls; p<0.0001; n=19-20). Moreover, levels of smooth muscle myosin heavy chain protein were at least as high in aortas with deficient SMC TGF-β signaling as in control aortas, consistent with their capacity to generate increased contractile force. Surprisingly, aortic segments from mice with deficient SMC TGF-β signaling also had impaired endothelium-dependent relaxation to acetylcholine (Emax: 37% vs 97% in controls; p<0.0001; n=19-20). Endothelium-independent relaxation to sodium nitroprusside was similar between the two groups. CD31 immunostaining of vessel segments revealed equivalent endothelial integrity in both groups.

Conclusion:
Physiologic SMC TGF-β signaling is an important determinant of both SMC contractility and endothelial function. Disruption of physiologic SMC TGF-β signaling may lead to TAAD through direct effects on SMC as well as through indirect effects on endothelial function. Our results also suggest an unanticipated role for SMC TGF-β signaling in regulating endothelial-mediated vasomotor function.
Abstract 95: MiRNA-210 Enhances Fibrous Cap Stability in Advanced Atherosclerotic Lesions
Authors: Eken Suzanne M , Jin Hong , Chernogubova Ekaterina , Li Yuhuang , Simon Nancy , Sun Changyan , Korzunowicz Greg , Busch Albert , Bäcklund Alexandra , Österholm Cecilia , Razuvaev Anton , Renné Thomas , Eckstein Hans H , Pelisek Jaroslav , Eriksson Per , Gonzalez Diez Maria , Matic Perisic Ljubica , Schellinger Isabel N , Raaz Uwe , Leeper Nicholas J , Hansson Göran K , Paulsson-Berne Gabrielle , Hedin Ulf , Maegdefessel Lars
Published: July 2021

In the search for markers and modulators of vascular disease, miRNAs have emerged as potent therapeutic targets. We investigated miRNAs of clinical interest in patients with unstable carotid stenosis at risk of stroke. Utilizing patient material from the Biobank of Karolinska Endarterectomies (BiKE), we profiled miRNA expression in symptomatic versus asymptomatic patients with high-grade carotid artery stenosis. A PCR-based miRNA of plasma, sampled at the carotid lesion site, identified eight deregulated miRNAs (miR-15b, -29c, -30c/d, -150, -191, -210 and -500). miR-210 was the most significantly downregulated miRNA in local plasma material. Laser-capture microdissection as well as
in situ
hybridization revealed a distinct localization of miR-210 in the fibrous caps of atherosclerotic lesions and showed reduced miR-210 expression in the unstable fibrous cap. We confirmed that miR-210 directly targets the tumor suppressor gene adenomatous polyposis coli (APC), thereby affecting Wnt signaling and regulating vascular smooth muscle cell survival, as well as differentiation, in advanced atherosclerotic lesions. Substantial changes in arterial miR-210 were detectable in two rodent models of vascular remodeling and plaque rupture. Modulating miR-210
in vitro
and
in vivo
improved fibrous cap stability with implications for vascular disease. We discovered that an unstable carotid plaque at risk of stroke is characterized by low expression of miR-210. miR-210 contributes to stabilizing carotid plaques through inhibition of APC, ensuring vascular smooth muscle cell survival. We present local delivery of miR-210 as a therapeutic approach for prevention of atherothrombotic disease.
Abstract 2589: Combined Perfusion and Non-Invasive Coronary Angiography in Patients with Suspected Coronary Disease using 256 Row, 0.5 mm Slice Thickness Non-Helical Multi-Detector Computed Tomography
Authors: George Richard T , Lardo Albert C , Kitagawa Kakuya , Yousuf Omair , Chang Hyuk-Jae , Arbab-Zadeh Armin , Miller Julie M , Bluemke David A , Lima Joao A
Published: July 2021

Introduction:
Full volumetric cardiac imaging over a single heart beat using a 256 row multidetector computed tomography scanning system provides distinct advantages over the current generation of MDCT scanners. 256x0.5mm MDCT allows for a protocol that combines MDCT angiography (MDCTA) with rest and stress CT perfusion (CTP) imaging. The purpose of this study was to test the accuracy of combined 256x0.5mm MDCTA and CTP in patients with suspected coronary artery disease.

Methods:
Nineteen patients with a positive single photon emission computed tomography (SPECT) perfusion study were imaged by 256 row MDCT with 0.5mm slice thickness (12.8 cm coverage) during one heart beat in a non-helical mode. Stress CTP (120kV, 100mAs, 3 gantry rotations at 0.5 secs, 7.7mSv dose) during adenosine infusion (140μg/kg/min) was followed by rest CTP and CTA (120kV, 175mAs, 3 gantry rotations at 0.5 secs, 13.5mSv dose). Using a territory based analysis; rest and stress CTP images were visually assessed for subendocardial perfusion deficits, CTA was evaluated for stenoses ≥50%, and SPECT images were evaluated for reversible and fixed perfusion deficits. In a second analysis the gold standard for ischemia secondary to epicardial atherosclerosis was defined as a SPECT perfusion deficit in a territory supplied by a coronary artery with a stenosis ≥50%.

Results:
Compared to ≥50% stenosis by CTA, the sensitivity and specificity of CTP was 85% and 77% compared to 69% and 74% by SPECT. More importantly, when considering the combination of ≥50% stenosis by CTA and a corresponding perfusion deficit on SPECT as the gold standard for ischemic CAD, the sensitivity and specificity for combined CTA and CTP imaging was 78% and 90%. The area under the ROC curve for the combination of CTA and CTP was 0.84, p<0.001.

Conclusions:
In patients with suspected coronary artery disease, 256x0.5mm MDCT enables the combination of non-invasive CT angiography and CT perfusion imaging with high accuracy and at radiation levels similar to currently used dual isotope nuclear imaging techniques.
Abstract 1184: Changes In Total Cholesterol Levels In The Icelandic Population Is Not Attributable To Lipid Lowering Medication Use
Authors: Gudnason Vilmundur , Thorsson Bolli , Andersen Karl , Sigurdsson Gunnar
Published: July 2021

Background: Total cholesterol (TC) levels have decreased in Western populations over the last decades. This has largely been attributed to changes in dietary intake, mainly reduced intake of saturated fat. The drop in population TC has been very great from the early 1990s in Iceland and many other countries, coinciding with the introduction of lipid lowering medication. A number of studies have examined the changes in TC and frequently attribute the drop to statin use. However, few studies have been able to address this directly due to lack of information on statin use in the same individuals as TC has been measured in at the population level.

Methods & results: We address this in the population based studies of the Icelandic Heart Association, the Reykjavik study and related studies carried out over the last 40 years. TC was measured in fasting blood in 8 different cross sectional samples between 1967 and 2007 of close to 30.000 men and women of age 45– 65 with information on lipid medication. Statin use has been increasing mainly after 1990 and today statin use is about 12% among men and about 5% in women. The changes in population TC in men with and without the use of lipid lowering medication is shown in the Figure

. The decline in population level of TC has the same characteristic for the 88% not taking lipid lowering drugs as for those 12% on medication. The same picture is seen for women.

Conclusion: It can be stated that the rapid decline in TC of the Icelandic population over the last 15 years or so is not explained by the introduction of lipid lowering medication. It is likely that the same applies to other populations where similar picture is seen and lipid lowering medication is at a similar or even less level.
Abstract P446: Comparison of Markers Related to Insulin Resistance between US Caucasian Men and Japanese Men in Japan
Authors: Ahuja Vasudha , Miura Katsuyuki , Barrinas-Mitchell Emma , Evans Rhobert W , Nakamura Yasuyuki , Fujiyoshi Akira , Maegawa Hiroshi , Ueshima Hirotsugu , Masaki Kamal , Kadota Aya , Willcox Bradley , Ohkubo Takayoshi , Okamura Tomonori , Kuller Lewis H , Sekikawa Akira ,   for the ERA JUMP Study group
Published: July 2021

Introduction:
Although Asians including Japanese are more susceptible to developing type 2 diabetes (T2DM) compared to Caucasians, no previous studies have compared levels of insulin resistance between Japanese and Caucasians with the gold standard method, i.e., euglycemic glucose clamp. We have previously reported that levels of atherosclerosis are significantly lower in middle-aged men in Japan than US Caucasian after adjusting for risk factors including body-mass index (BMI). We hypothesized that Japanese men in Japan are less insulin resistant than Caucasian men and compared markers related to insulin resistance in these two groups.

Methods:
Population-based samples of 623 randomly selected men aged 40-49 years (313 Japanese men from Kusatsu, Shiga Japan and 310 Caucasian men from Allegheny County, PA) without clinical CVD were examined. We excluded 31 men with T2DM. Using ANCOVA, adjusting for BMI, we evaluated the following markers related to insulin resistance in the two populations: HOMA-IR, non-esterified fatty acids (NEFA), adiponectin, HDL-C, triglycerides, CRP, plasminogen-activator inhibitor 1 (PAI-1), systolic blood pressure (BP), and other risk factors (Table).

Results:
After adjusting for BMI, HOMA-IR, the most widely used marker of insulin resistance, was not statistically different between the two groups (Table). Among 19 variables compared, the results of NEFA, CRP, fibrinogen and HLD-P supported the hypothesis that Japanese are less insulin resistant. In contrast the results of adiponectin, PAI-1, triglycerides, total LDL-P, small LDL-P and systolic BP suggest that Japanese are more insulin resistant. Results of HOMA-β% and disposition index suggest that Japanese secrete less insulin than Caucasians.

Conclusion:
It is inconclusive whether Japanese are less insulin resistant than Caucasians based on comparisons of markers related to insulin resistance. Our data also suggest that Japanese as compared to Caucasians may secrete less insulin at the same levels of insulin resistance.
Abstract 12197: China PEACE Risk Tool for Acute Myocardial Infarction: An Early Risk Classification Tree for Decisions About Fibrinolytic Therapy
Authors: Li Xi , Li Jing , Masoudi Frederick , Spertus John , Lin Zhenqiu , Krumholz Harlan , Jiang Lixin ,   China PEACE Collaborative Group
Published: July 2021

Introduction:
Timely fibrinolytic therapy is recommended to reduce short-term mortality for patients with ST-elevation myocardial infarction (STEMI). However, the absolute treatment benefit is related to baseline mortality risk, and may be offset by the risks of major bleeding for low-risk patients. We constructed a baseline risk-assessment tool in eligible but untreated patients, to estimate the absolute benefits from fibrinolytic treatment.

Methods:
The China PEACE study includes a nationally representative sample of patients admitted with acute myocardial infarction (AMI) in 162 Chinese hospitals. Comprehensive clinical information was centrally abstracted from medical records using standardized data definitions. We identified 3741 patients with STEMI who were fibrinolytic-eligible, but did not receive reperfusion therapy, and separated the cohort randomly into derivation and validation cohorts. We employed classification and regression tree methods to produce a simple algorithm to estimate the risk of in-hospital mortality.

Results:
The overall in-hospital mortality rate was 14.7%. In both derivation and validation cohorts, the combination of systolic blood pressure (≥ 100 mm Hg), age (< 60 years old), and male gender identified one fifth of the patients with an average mortality risk less than 3.0%; half of this group with a non-anterior AMI had an average in-hospital mortality risk of 1.5%. The classification tree performed consistently across study years, and in hospitals with or without PCI capability.

Conclusions:
Nearly one in five fibrinolytic-eligible patients with STEMI have an low estimated risk of death, with an absolute benefit of fibrinolytic therapy could be insufficient to justify the risks of major bleeding. The tool with three simple factors, easily available at hospital presentation, can identify these individuals and support decision-making about the use of fibrinolytic therapy.
Abstract 031: Socioeconomic Trajectories Across the Life Course and Risk of All-cause and Cardiovascular Mortality: Prospective Findings From the Moli-sani Study
Authors: Bonaccio Marialaura , Di Castelnuovo Augusto , Costanzo Simona , Persichillo Mariarosaria , Cerletti Chiara , Donati Maria Benedetta , de Gaetano Giovanni , Iacoviello Licia ,   Moli-sani Study Investigators
Published: July 2021

Introduction:
A life course approach has been suggested as the most appropriated to establish the actual impact of socioeconomic status (SES) on health outcomes.

Hypothesis:
We assessed the hypothesis that SES trajectories from childhood to adulthood are useful to better evaluate the role of SES towards mortality risk in a large general population-based cohort.

Methods:
Longitudinal analysis on 22,194 subjects recruited in the general population of the Moli-sani study, Italy (2005-2010). Educational attainment (low/high) and SES in adulthood (measured by a score including occupational social class, housing and overcrowding, and dichotomized as low/high) were used to define four possible trajectories both in low and high SES in childhood (age of 8). Hazard ratios (HR) with 95% confidence intervals (95%CI) were calculated by multivariable Cox regression and competing risk models.

Results:
Over a median follow-up of 8.3 years (182,924 person-years), 1155 all-cause, of which 414 cardiovascular (CVD), deaths were ascertained. In the group with low SES in childhood, as opposed to those stably low (low education and low SES in adulthood), an upward in both educational attainment and material factors in adulthood was associated with lower risk of both all-cause (HR=0.64; 95%CI 0.52-0.79; Table) and CVD mortality (HR=0.62; 0.43-0.88), respectively. Subjects with high childhood SES experienced an increased risk of total and CVD death in absence of higher educational attainment despite a higher SES in adulthood (HR=1.47; 1.04-2.07 and HR=1.75;1.00-3.05, respectively) as compared to the group with both high education and high SES in adulthood.

Conclusions:
In conclusion, for individuals with low SES in childhood, an upward of both educational attainment and material factors over the life course is associated with lower risk of total and CVD death. In advantaged groups in childhood, lack of a higher educational attainment, rather than material factors, over the life course appears to be unfavourably associated with survival.
Abstract 036: Derivation and Validation of a Novel Risk Equation for 10-year Risk of Incident Heart Failure in the General US Population
Authors: Khan Sadiya S , Ning Hongyan , Shah Sanjiv J , Yancy Clyde W , Wilkins John T , Lloyd-Jones Donald M
Published: July 2021

Background:
Identification of individuals at risk for heart failure (HF) is necessary for implementation of primary prevention strategies. However, currently no validated prediction models exist for assessment of HF risk in a broad-based general population (ACC/AHA Stage 0 and Stage A individuals) based on routinely available clinical data.

Methods:
Estimation of 10-year risk equations for developing a HF event were derived from community-based cohorts representative of the U.S. population of Whites and Blacks. Participants were included from ARIC (Atherosclerosis Risk in Communities) study, Cardiovascular Health Study, the CARDIA (Coronary Artery Risk Development in Young Adults) study, Multi-Ethnic Study of Atherosclerosis, and Framingham Offspring Study cohorts who were recruited between 1985-2000, between the ages of 30 to 80 years, free of cardiovascular disease at baseline, and had 12-years of follow-up. Sex- and race-specific 10-year risk equations were derived in a random pre-specified subset of the pooled sample (n=11,771) and subsequently validated in the remaining participants (n=11,770). Harrell’s C-statistic and Greenwood-Nam-D'Agostino chi-square were used to determine discrimination and calibration of these models.

Results:
In the derivation cohort, 58% were women, 22% black, and mean age 52.7±11.9 years. HF occurred in 1,339 participants. Independent predictors of HF included in the model were age, blood pressure (treated or untreated), fasting glucose (treated or untreated), body mass index, cholesterol, smoking status, and QRS duration. The data-derived model had excellent discrimination in the 11,770 distinct participants in the validation cohort (C-statistics 0.71-0.85) and was well-calibrated (
Figure)
.

Conclusions:
A 10-year HF risk score that incorporates readily available clinical parameters in the primary care setting can be used to identify individuals with higher likelihood of developing HF who may merit intensive screening and/or targeted prevention strategies.