bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 218: Early Identification of Individuals on Dangerous Atherosclerotic Trajectories
Authors: Raines Jeffrey K
Published: July 2021

Background:
Atherosclerosis is a process that involves: (i) smooth muscle cell migration, (ii) intima thickening, (iii) loss of lumen, and (iv) other complex wall changes which occur in advance of blood flow alterations.

Methods:
Working from the tenets that early atherosclerosis reduces Local Arterial Compliance and the upper extremity arteries, and even the carotid arteries, do not reflect disease in the coronary arteries due to differences in distribution of elastin, collagen, and smooth muscle between these beds, the major technical challenge was to devise an accurate method to measure Local Arterial Volume (Δ volume), since the parameter to assess atherosclerosis, is defined as Δ volume / Δ pressure.

Results:
The
Soteria Cardiac Platform
was developed, clinically tested, manufactured, and is now in clinical distribution. The Platform measures arterial compliance at the femoropopliteal distribution. The flagship Platform Module is the

Soterogram

. This technology was awarded 8 United States Patents, cleared by the FDA for a National Launch on 05-31-14, and uses CPT and ICD-10 Codes supported by CMS required for Carrier payments in physician offices, clinics, and hospitals. The Platform has been in active clinical use for 22 months with over 10,000 procedures performed. The procedures are noninvasive, rapid, and inexpensive when compared to other techniques. The results obtained from the Platform have been documented to be extremely useful in diagnosis and management. The accuracy of the testing is very high with False Positive Rates and False Negative Rates of 3%, each. Interesting data has been assembled over the development and clinical periods. Besides the obvious diagnostic value, one example of ancillary data is an explanation of why females are protected from atherosclerosis early in life and develop a more aggressive form of atherosclerosis later in life.

Conclusions:
The Platform is now in active clinical use and has the potential of reducing cardiovascular morbidity and mortality by accurate and early identification of individuals at risk for atherosclerosis. The Platform involves Primary and Secondary Prevention and maybe used to monitor the success of therapy. Proper distribution of this technology has proven to be a very challenging task.
Abstract 219: Deficiency of the T Cell Regulator Cbl-B Aggravates Atherosclerosis by Inducing CD8
+
T Cell-Mediated Macrophage Death
Authors: Seijkens Tom T , Winkels Holger , Gijbels Marion , Kuivenhoven Jan A , Perisic Ljubica , Paulsson-Berne Gabrielle , Hedin Ulf , Hansson Goran K , Weber Christian , Gerdes Norbert , de Winther Menno P , Lutgens Esther
Published: July 2021

Aims:
The E3-ligase CBL-B (
Casitas B-cell Lymphoma-B
) is an important negative regulator of T cell activation that is also expressed in macrophages. T cells and macrophages mediate atherosclerosis, but their regulation in this disease remains largely unknown; thus, we studied the function of CBL-B in atherogenesis.

Methods and Results:
Here we investigated the effect of CBL-B deficiency in hyperlipidemic

Apoe
-/-

mice in atherosclerosis. At the age of 20 weeks, chow diet-fed

Cbl-b
-/-
Apoe
-/-

mice showed a significant increase in plaque area in the aortic arch, due to greater macrophage infiltration.

Cbl-b
-/-
Apoe
-/-

macrophages displayed strong recruitment towards MCP1 and showed an increase in oxidized (ox)LDL uptake. In the aortic root of the same

Cbl-b
-/-
Apoe
-/-

mice, where more advanced plaques were present than in the aortic arch, plaque area rose by 40%, accompanied by a dramatic change in plaque phenotype. Plaques contained fewer macrophages, had larger necrotic cores, and harboured more CD8
+
T cells. The CD8
+
T cells of

Cbl-b
-/-
Apoe
-/-

mice were less susceptible to apoptosis and less resistant to Treg suppression. The increase in CD8
+
T cells in the plaque effected greater macrophage apoptosis, resulting in enhanced necrotic core formation. Moreover, CBL-B gene expression was downregulated in human atherosclerotic plaques, and positively correlated with FoxP3 expression, indicating an atheroprotective effect.

Conclusion:
CBL-B is an important regulator of innate and adaptive immune reactions in atherosclerosis, by mediating macrophage recruitment and activation, CD8
+
T cell activation, and CD8
+
T cell-induced macrophage death in atherosclerotic plaques.
Abstract 22: Crosstalk Between Phosphotidylinositol-4,5 Bisphosphonate-3 Kinase Pathway and Mitogen-Activated Protein Kinase Pathway Regulates Platelet Activation and Protein Synthesis via Phosphoinositide-Dependent Kinase-1
Authors: Manne Bhanu Kanth , Münzer Patrick , Badolia Rachit , Weyrich Andrew S. , Kunapuli Satya P , Borst Oliver , Rondina Matthew
Published: July 2021

Phosphoinositide-dependent protein kinase 1 (PDK1) is known to regulate PAR4 induced platelet activation and thrombus formation through GSK3β. However, whether PDK1 signaling also involves the ADP receptor and, if so, downstream functional consequences are unknown. We employed both pharmacologic (e.g. the selective PDK1 inhibitor, BX795) and genetic (platelet specific deletion of PDK1) approaches to dissect the role of PDK1 in ADP-induced platelet activation and protein synthesis. Inhibition of PDK1 with BX795 reduced 2MeSADP-induced platelet aggregation by abolishing thromboxane generation. Similar results were observed in PDK1
-/-
mice (Fig A). Inhibition of PDK1 protected mice from collagen and epinephrine-induced pulmonary embolism (Fig B). PDK1 was also necessary for the phosphorylation of MEK1/2, Erk1/2 and cPLA2, indicating that PDK1 regulates an upstream kinase in MAPK pathway. We next identified that this upstream kinase is Raf1 (necessary for the phosphorylation of MEK1/2), as pharmacologic inhibition and genetic ablation of PDK1 was sufficient to prevent Raf1 phosphorylation (Fig C). Pharmacologic inhibition and genetic ablation of PDK1 blocked MAPK- and mTORC1-dependent protein synthesis in platelets through a mechanism requiring the phosphorylation of eIF4E and S6K. Concordantly, PDK1 is necessary for signal-dependent synthesis of the protein bcl3, which is under mTORC1-dependent control (Fig C). Taken together, our findings show for the first time that PDK1, a master kinase in the PI3K pathway, directly governs thromboxane generation, thrombosis, and protein synthesis in platelets through regulating MAPK and mTORC1 pathways.
Abstract 237: The Role of Lymphocyte Specific Protein-1 in the Phenotypic Switch of Smooth Muscle Cells After Arterial Injury
Authors: Chaudhary Mirnal A , Urabe Go , Hayden Alex , Franco Sarah , Shi Xudong , Wang-Guo Lian , Kent K Craig
Published: July 2021

Background:
After vascular injury, vascular smooth muscle cells (SMCs) switch from a differentiated contractile state to synthetic de-differentiated phenotype which contributes to the pathophysiology of restenosis. Experimental data generated by our lab indicate that TGF-β downregulates contractile proteins and stimulates migration. To understand how TGF-β promotes SMC phenotypic switch in injured arteries, we performed an Affymetrix Array analysis and identified Lymphocyte Specific Protein-1 (LSP1) among other upregulated genes. LSP1 is known to play a role in neutrophil extravasation, however the role of LSP1 within SMCs is unknown. We hypothesize that LSP1 contributes to SMC pathophysiological behavior through changes in cell architecture and migration
in-vivo
and
in-vitro.

Methods and Results:
After carotid artery angioplasty, male Sprague-Dawley rats were sacrificed at 3, 7, and 14 days after injury for immunohistochemistry. Immunofluorescence staining revealed a unique upregulation of LSP1 within the neointima, media, and adventitia at 7 and 14 days, but not at 3 days after injury. Confocal images revealed that the LSP1 positive cells minimally express α-SMA (Pierson’s Coefficient, r=.017). Additional characterization experiments using immune cell markers CD3 and CD45 show no co-localization with LSP1 positive cells. To mimic the
in-vivo
neointimal cells and vascular injury induced de-differentiation
in-vitro
, rat A10 cells were treated with solvent or PDGF-bb (10 ng/mL). Quantitative RT-PCR demonstrated an upregulation of LSP1 mRNA after 24 hrs of PDGF-BB stimulation. Using Western Blotting, we confirm an upregulation of LSP1 protein after 48 hrs of PDGF-BB stimulation. Lastly, we performed nuclear and cytoplasmic fractionation followed by Western Blotting which demonstrated that LSP1 is remained within cytoplasmic fraction of the A10 cell after treatment with PDGF-BB.

Conclusion:
These results demonstrate that LSP1 is increased
in-vivo
after balloon injury, and
in-vitro
after PDGF-BB stimulation. Experiments to characterize the identity of these LSP1 cells
in-vivo
are in process, with future
in-vitro
experiments to focus on the role of LSP1 phosphorylation as a part of cytoskeletal remodeling and cellular migration.
Abstract 243: Neutrophil-Mediated Inflammation Drives Sickle Cell Vasculopathy
Authors: Okwan Derick , Hansen Laura , Joseph Giji , Weiss Daiana , Archer David R , Taylor W Robert
Published: July 2021

Objectives:
Many of the clinical complications associated with sickle cell disease (SCD), such as stroke, pain crises, proliferative retinopathy, renal and heart failure, can be attributed to repeated bouts of vascular insufficiency, yet the detailed mechanisms of vascular repair following injury are largely unknown in SCD. Given our previous work showing the importance of reactive oxygen species in neovascularization, we aimed to delineate the immune mechanisms of oxidative stress during vascular repair in a humanized sickle cell mouse model (SS) in comparison to wildtype (AA).

Methods:
We performed limb ischemia (HLI) in mice by ligation of the femoral artery to evaluate vascular dysfunction in sickle cell mice. Vascular recovery was ascertained using weekly LASER Doppler perfusion imaging (LDPI) for 28 days. Voluntary running wheel test was used to determine spontaneous motor function recovery.

Results:
There was significant diminution in functional collateral vessel formation in SS mice following HLI as evaluated by LDPI (76 ±13 % recovery in AA vs 34±10 % recovery in SS by day 28, p < 0.001 n=8 per group). This was characterized by dysfunctional Moyamoya-like sprouting vessels with impaired spontaneous motor function recovery in SS. Specifically, AA mice recovered 98% motor function by day 28 following HLI, vs 36% in SS mice, p < 0.001. The phenotype was associated with persistent neutrophils in the hind limb muscle of SS mice up to 28 days, a time point by which all neutrophils were cleared in AA mice. Consequently, there was a 2.45 fold increased production of hydrogen peroxide in SS mice ischemic hind limbs at day 28, compared to AA mice (p< 0.05). Importantly,
in vivo
depletion of neutrophils improved functional collateral vessel formation in the SS mice.

Conclusions:
Our data suggest that neutrophil-mediated excessive inflammation and oxidative stress drive dysfunctional collateral vessel formation in SS mice following ischemic injury. Targeting neutrophils may improve vascular dysfunction in SS disease.
Abstract 245: Non-Invasive
in vivo
Assessment of the Re-Endothelialization Process Using Ultrasound Biomicroscopy in the Rat Carotid Artery Balloon Injury Model
Authors: Röhl Samuel , Eriksson Linnea , Saxelin Robert , Lengquist Mariette , Caidahl Kenneth , Hedin Ulf , Razuvaev Anton
Published: July 2021

Objective:
Ultrasound BioMicroscopy (UBM), or high-frequency ultrasound, is a novel technique used for assessment of anatomy and physiology small research animals. In this study, we evaluate the UBM assessment of the re-endothelialization process following denudation of the carotid artery in rats.

Methods:
Ultrasound BioMicroscopy data from three different experiments were analyzed. A total of 66 rats of three different strains (Sprague-Dawley, Wistar and Goto-Kakizaki) were included in this study. All animals were subjected to common carotid artery balloon injury and examined with UBM 2 and 4 weeks after injury. Re-endothelialization in UBM was measured as the length from the carotid bifurcation to the distal edge of the intimal hyperplasia.
En face
staining with Evans-blue dye was performed upon euthanization at 4 weeks after injury followed by tissue harvest for morphological and immunohistochemical evaluation.

Results:
A significant correlation (Spearman r=0.63,p<0.0001) and an agreement according to Bland-Altman test was identified when comparing all measurements of re-endothelialization in high frequency ultrasound and
en face
staining. Analysis by animal strain revealed a similar pattern and a significant growth in re-endothelialization length measured in UBM from 2 to 4 weeks could be identified. Immunohistochemical staining for von Willebrand factor confirmed the presence of endothelium in the areas detected as re-endothelialized by the ultrasound assessment.

Conclusion:
Ultrasound BioMicroscopy can be used for longitudinal in vivo assessment of the re-endothelialization following arterial injury in rats.
Abstract 246: TGF-β1 and TGFBR2 Polymorphisms With ISH
Authors: Zhang Xiaoying , Yu Shasha , Chen Wencai , Ren Jianfei , Chen Xiaofeng
Published: July 2021

Background and Objective:
Isolated systolic hypertension (ISH) is characterized by increased aortic stiffness and associated with a significantly increased risk of cardiovascular morbidity and mortality. It has been reported that elevated plasma transforming growth factor-beta 1(TGF-β1)levels predicted development of hypertension. However, little is known about the association of TGF- β 1 pathway gene polymorphisms and ISH. The aim of the present study was to study the association of transforming growth factor-beta 1(TGF-β1)and its receptor 2(TGFBR2)functional gene polymorphisms with isolated systolic hypertension (ISH).

Methods and Results:
One hundred and three consecutive ISH patients and 169 healthy controls were recruited in this study. All subjects were genotyped for TGFβ1-869T/C, TGFBR2-3779A/G and TGFBR2-1444C/G by the technology of polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and then confirmed by direct sequencing. No significant difference in genotype (and allele) frequency of TGFβ1-869T/C, TGFBR2-3779A/G and TGFBR2-1444C/G polymorphisms were observed between ISH group and healthy group (p>0.05).

Conclusion:
Our findings suggest that TGFβ1-869T/C, TGFBR2-3779A/G and TGFBR2-1444C/G polymorphisms may not be associated with susceptibility of isolated systolic hypertension in Chinese population.
Abstract 252: ABR-238901 Promotes Cardiac Repair After Myocardial Infarction by Reducing Inflammation and Increased Presence of “Reparatory” Macrophages Trough Blockage of Alarmin Complex S100a8/9
Authors: Marinkovic Goran , Winkler Laura , Kefene Lisa , Nilsson Jan , Jovinge Stefan , Alexandru Schiopu
Published: July 2021

Objective:
Myocardial infarction (MI) is an ischemic injury of the heart leading to excessive local inflammation. The alarmin complex S100A8/9 is potently upregulated locally and systemically after MI and promotes the influx of neutrophils and monocytes into the myocardium. We studied whether S100A8/A9 inhibition with the blocking compound ABR-238901 might reduce inflammation and improve myocardial function after MI.

Methods:
MI was induced by left coronary artery ligation in 8-12 weeks old mice. Echocardiography was used to assess left ventricular function.

Results:
Two days after MI, mice treated with ABR-238901 i.p. showed improved ejection fraction in comparison with buffer-treated animals, and had significantly decreased numbers of neutrophils and monocytes in the blood. More importantly, the presence of neutrophils and monocytes in the myocardium was reduced, leading to lower numbers of infiltrating macrophages. After 7 days of continuous treatment, cardiac function and the total counts of neutrophils and monocytes in blood and heart became similar in the two groups. However, the numbers of reparatory F4/80
+
MerTK
hi
Ly6C
low
macrophages were lower in the hearts of ABR-238901 treated mice after 7 days. In contrast, we found that a balanced treatment strategy using ABR-238901 for 3 days followed by 4 days buffer increased the numbers and the proliferative capacity of this macrophage subset, promoting cardiac repair.

Conclusion:
We report that 3-day blockage of S100A8/9 has beneficial effects on cardiac function in the immediate period after an MI and enhances the presence of reparatory macrophages in the myocardium.
Abstract 254: Kawasaki Disease Vasculitis in Mice Develop Myocardial Fibrosis After Adrenergic Stimulation
Authors: Matundan Harry H. H , Sin Jon , Abe Masanori , Noval Rivas Magali , Fishbein Michael C , Lehman Thomas A , Gottlieb Roberta A , Arditi Moshe
Published: July 2021

Background:
Kawasaki disease (KD) is the most common cause of acquired cardiac disease among US children and causes coronary artery aneurysms (CAA) in 25% of untreated patients. CAAs can result in myocardial ischemia, infarction, and even death. Other long-term sequelae from KD include extensive remodeling of CAA leading to stenosis and endothelial (EC) dysfunction. Acute KD is also associated with myocarditis that can cause arrhythmias, which can be fatal and may lead to long-term myocardial dysfunction and fibrosis, areas of KD research that are severely neglected. These myocardial changes may persist for decades after the acute illness and closely correlate with long-term myocardial sequelae.

Objective:
To investigate long-term myocardial dysfunction, including myocardial fibrosis and coronary microvascular lesions in the KD vasculitis mouse model.

Methods:
Mice were injected with
Lactobacillus casei
cell wall extract (LCWE) to induce KD vasculitis, coronary arteritis, and myocarditis at 1 week. Five weeks later, control or KD mice were injected with isoproterenol (ISO), a beta agonist, for 10 consecutive days to induce pharmacologically mediated exercise, followed by sacrifice and analysis. Cardiac hypertrophy was measured by calculating heart weight normalized to tibia length. Myocardial fibrosis was determined by Masson’s trichrome staining of the myocardium and myocardial function was measured by serum BMP levels and ventricular ejection fraction by MRI.

Results:
KD significantly increased the risk of cardiac hypertrophy and increased myocardial fibrosis, diminished ejection fraction and increased BMP following prolonged pharmacologic exercise (ISO), compared with controls. Post KD pharmacologic exercise-induced myocardial fibrosis was associated with significant reduction in the expression of the EC marker CD31 in the myocardium. This KD induced myocardial fibrosis was IL-1-dependent as the IL-1R KO mice were protected.

Conclusions:
Pharmacologic stress (exercise) leads to cardiac hypertrophy and chronic myocardial fibrosis in the LCWE-induced acute KD vasculitis mouse model, a process that involves IL-1 signaling and diminished microvascular circulation in the myocardium.
Supported by NIH grant AI 072726 to MA
Abstract 28: Apolipoprotein A-I Vascular Gene Therapy Provides Durable Protection from Atherosclerosis in Hyperlipidemic Rabbits
Authors: Wacker Bradley K , Dronadula Nagadhara , Bi Lianxiang , Stamatikos Alexis , Dichek David A
Published: July 2021

Background:
Gene therapy, delivered directly to the vascular wall, could potentially protect against atherosclerotic lesion growth or regress existing lesions. Previously, we demonstrated that infusion of HDAdApoAI [a helper-dependent adenoviral vector (HDAd) expressing apolipoprotein (apo) A-I] in carotid arteries of fat-fed rabbits reduced early carotid atherosclerosis development (4 wk after vector infusion). Here we tested whether the same HDAd could protect long-term against more severe atherosclerosis.

Methods:
25 fat-fed rabbits underwent bilateral carotid gene transfer [HDAdApoAI on one side; control vector (HDAdNull) on the other, with sides randomized]. Postoperatively, diets were adjusted to maintain plasma cholesterols of 200-800mg/dl. This protocol yields large lipid- and macrophage-rich lesions. 6 mo later, carotids were harvested for analyses of DNA, RNA, protein, cholesterol, and histology (H&E, oil red O), or immunostaining for macrophages, muscle actin, T-cells, ICAM-1 and VCAM-1.

Results:
Vector genomes persisted equally in HDAdApoAI and HDAdNull-infused arteries; however, apoA-I mRNA was detected only in HDAdApoAI-infused arteries. HDAdApoAI-infused arteries had ~60% less median intimal lesion volume than HDAdNull-infused arteries, with concomitant reductions (40-75%) in intimal lipid, macrophage, smooth muscle cell, VCAM-1 and ICAM-1. We used within-rabbit paired analyses to control for high correlations of lesion size and composition between left and right carotids of the same rabbit. 19 of 25 rabbits had smaller lesions in HDAdApoAI-infused vs HDAdNull-infused arteries; median decreased intimal area between paired carotids in the same rabbit was 30% (
P
<0.03). Within individual rabbits, intimal lipid, macrophage, smooth muscle cell, VCAM-1 and ICAM-1 were all less in HDAdApoAI arteries (median 23-36% less;
P
<0.05 for all except ICAM-1).

Conclusions:
Vascular gene therapy with HDAdApoAI reduced lesion development and decreased intimal lipid, macrophage, and adhesion molecule expression 6 mo after treatment. Vector genomes and apo A-I mRNA remained high at 6 mo. A single application of vascular gene therapy durably retards development of atherosclerosis, even in a setting of severe hyperlipidemia.
Abstract 30: Tissue Factor Pathway Inhibitor α Inhibits Prothrombinase via a Three-Step Mechanism
Authors: Wood Jeremy P , Mast Alan E
Published: July 2021

TFPIα inhibits early forms of the prothrombinase complex (factor Xa (FXa), factor Va (FVa)), though the inhibitory mechanism is not entirely understood. One step of inhibition is a high affinity interaction between a TFPIα C-terminal basic region (BR) (252-LIKTKRKRKK-261) and an acidic region (AR) present in FXa-activated and platelet-released forms of FVa. We investigated two additional potential mechanistic steps: (1) binding of the second Kunitz-type inhibitory domain (K2) of TFPIα to the FXa active site; and (2) the function of uncharged residues L252, I253, and T255 within the BR, which are evolutionarily conserved, suggesting they have activity. Direct inhibition of FXa was investigated using TFPIα with an altered K2 (TFPI-R107A) incapable of binding FXa. TFPI-R107A inhibited purified prothrombinase 17-fold weaker than TFPIα (IC50 = 30.6nM vs. 1.8nM) and did not inhibit FXa-initiated thrombin generation in platelet-rich plasma (PRP). Therefore, direct binding of FXa and K2 is required for efficient inhibition of prothrombinase under physiological conditions. Similarly, the role of L252, I253, and T255 was investigated by substituting them with alanine (TFPI-AAKA). The IC50 for prothrombinase inhibition by TFPI-AAKA was 10.4nM, and it had reduced inhibitory activity in PRP, revealing that these residues are also required for efficient prothrombinase inhibition. The role of L252, I253, and T255 was further probed using the peptide LIKTKRKRKK, which inhibited purified prothrombinase (IC50 = 1.0μM) and thrombin generation in PRP at 1μM. AAKAKRKRKK had very little activity in either assay (~20% prothrombinase inhibition with 225μM peptide), but bound the FVa AR equivalently to LIKTKRKRKK (K
d
= 5.9nM and 6.0nM, respectively). Thus, the basic residues are responsible for AR binding, while a second step, mediated by L252-T255, is necessary for inhibitory activity. These residues may be necessary for displacement of FXa from FVa, as proposed by Bunce et al. We propose that prothrombinase inhibition by TFPIα involves three steps: (1) the TFPIα BR basic residues bind the FVa AR; (2) residues L252-T255 block prothrombinase assembly; and (3) K2 binds the FXa active site. All three steps are required for physiologic inhibition of prothrombinase by TFPIα.
Abstract 302: The PAT Ratio is Reduced by Beta-blockers
Authors: Tomohiro Ueda
Published: July 2021

Background:
Flow-mediated dilatation (FMD) of the brachial artery and Peripheral arterial tonometry (PAT) are both methods for assessing endothelial vascular function. FMD measures predominantly nitric oxide mediated vasodilation whereas PAT measures a more complex range of mechanisms. The recent study showed that the sympathetic nervous system plays a significant role in this response.

Methods:
The study involved 176 subjects (mean age66 ±12 years). Based on the medication of beta-blockers, they were divided into 2 groups: beta-blocker group (n=37) and control group (n=139). Flow mediated vasodilatation (FMD) and nitroglycerine-induced vasodilatation (NID) in the brachial artery was measured by using UNEXEF18G (UNEX CO, Japan), and nitroglycerin mediated vasodilatation (NMD) was used as a control test for FMD. At the same time, PAT ratio was measured by using Endo-PAT 2000 (Itamar Medical, Israel)

Results:
PAT ratio was significantly impaired in beta-blocker group compared to that in control group (1.5±0.4% vs. 1.9±0.6%, respectively; P<0.05). However, FMD and NMD had no deference in both groups. Multivariable analysis revealed that blood sugar and medication of beta-blockers were independent variables for PAT ratio.

Conclusion:
These result show that beta-blockers is associated with a tendency towards reduced PAT ratio. PAT needs to be further studied, including the assessment of non-endothelial factor
Abstract 306: Impact of Venous Thromboembolism as Independent Predictor of Mortality Among Patients With Gastric Cancer
Authors: Fuentes Harry E , Paz Luis H , Oramas Diana M , Andrade Xavier A , Wang Yuchen , Tafur Alfonso J
Published: July 2021

Background:
Cancer-associated thrombosis is a predictor of death. Patients with gastric cancer (GC) are at higher risk for VTE when compared to other solid tumors. There is a paucity of data describing the impact of VTE in GC.

Aim:
To measure the impact of VTE as independent predictor of gastric cancer mortality.

Methods:
Single institution chart review of GC treated patients (2010-15). VTE events were objectively confirmed. GC was ascertained if biopsy proven and metastatic, or on active chemotherapy. Along with cancer specific data, we abstracted risk assessments tools, non-GC specific, validated for VTE and mortality prediction in cancer; including, the Khorana Score (KRS), platelet lymphocyte ratio (PLR) and neutrophil lymphocyte ratio (NLR). Continuous variables are expressed by the by the median (interquartile range). Categorical variables are expressed as percentages. We used SPSS 23, specifically Kaplan-Meir curve and Cox proportional hazard were applied for main objectives.

Results:
We included 112 pts in the analysis, who were predominantly male (66%), 58 (51-64) year-old, with adenocarcinoma (84%) and advanced disease (59%). The median follow-up was 21.3 months (9.5-42.6). We measured high risk of VTE based on the KRS in 59%, 51% had an elevated NLR and 30% had an elevated PLR. VTE occurred in thirteen (12%) patients. The median time from diagnosis to VTE occurrence was 59 days (36-258). After multivariate analysis, the predictors of mortality were: VTE (Hazard Ratio (HR), 2.6; 95% CI, 1.1 to 6.0; p=0.02), Histological type (HR, 3.2; 95% CI, 1.1 to 9.2; p=0.03), Stage (HR, 2.9; 95%CI, 1.4 to 5.8; p<0.01) and PLR (HR, 2.2; 95%CI, 1.3 to 3.9); p<0.01). The one year overall survival of patients with VTE was lower than for those with no VTE (79% vs 41% p<0.05)

Conclusion:
VTE is associated with worse survival among patients with GC. Moreover, this finding was independent of other cancer-specific variables. NLR and KRS were not associated with survival in our GC database.
Abstract 314: A Stent to Prevent: A Translational Approach Towards Small Abdominal Aortic Aneurysm (AAA) Therapy
Authors: Schellinger Isabel N , Spin Joshua M , Hasenfuss Gerd , Tsao Philip S , Raaz Uwe
Published: July 2021

Background:
Abdominal aortic aneurysm (AAA) is defined as a permanent dilation of the abdominal aorta that is highly lethal in case of rupture. Current therapeutic approaches are limited to open surgery or stent-based endovascular aortic repair (EVAR) to exclude large sized AAA (>5.5 cm) from the circulation. However, to date there is no effective therapy to prevent small AAA formation and progression. Nonetheless, options for small AAA disease are highly desirable as even small AAA may rupture and app. 70% of all small AAA will grow and eventually require surgical repair.

Objective:
Building on our previous experimental research findings we present a novel therapeutic approach for early AAA intervention.

Methods and Results:
Our recent biomechanical studies in a murine model of AAA development (elastase model) as well as in human aortae indicate that early AAA growth is critically driven by a stiffness gradient between a stiff AAA segment and the adjacent more compliant aorta. As a promising therapeutic intervention, we found that stiffening of the AAA-adjacent aorta (by external glue application) was sufficient to stop murine AAA formation. In a translational approach we now aim to develop an intravascular stent prototype that is deployed in the neck region of a developing AAA to increase the mechanical stiffness of the AAA-adjacent aorta. We hypothesize that this intervention will decrease the aortic stiffness gradient towards the AAA segment – thereby preventing further AAA growth. Stent prototypes with varying designs will be tested in a pig model of AAA disease that closely resembles the human anatomic situation.

Conclusion:
This project demonstrates a highly promising opportunity to directly translate novel pathomechanistic insights into AAA pathobiology from bench to bedside application and stop early AAA progression in humans.
Abstract 350: Activation of Platelet CLEC-2 in Hematoma Expansion After Intracerebral Hemorrhage: Finding an Effective Dose
Authors: Burchell Sherrefa R , Iniaghe Loretta , Reis Cesar , Aka Sheryl , Hay Karen , Raval Ronak , Zhang John H , Tang Jiping
Published: July 2021

Intracerebral hemorrhage (ICH) is the least treatable and one of the most disabling stroke subtypes, with both the primary and secondary brain injuries mainly due to hematoma expansion (HE) within the first few hours, followed by an inflammatory and oxidative injury. Almost all ICH patients experience some degree of expansion, and ICH volume can be an independent determinant of mortality and functional outcome. Previous studies found that reduced platelet activity is associated with early ICH growth and worse functional outcomes. The platelet C-type lectin-like receptor 2 (CLEC-2) elicits robust platelet activation upon stimulation. Thus, we hypothesized that activation of CLEC-2 would result in activation and aggregation of platelets, reducing HE, and improving functional outcomes after ICH. We administered two CLEC-2 agonists (Fucoidan and the endogenous ligand Podoplanin) at varying doses after murine ICH and measured hemorrhage volume, brain water content, and behavior. We also collected blood from podoplanin-treated mice and conducted hematologic tests. We further tested Rhodocytin, a known CLEC-2 activator, for whole blood clotting times, platelet functions, and soluble fibrin in human blood samples. We found that treatment with Fucoidan did not reduce hemorrhage volume or brain water content and had no effect on neurological function, compared to vehicle. Further, Podoplanin treatment showed a tendency to reduce brain water content but did not affect hematoma volume or behavior. Our
in vitro
studies showed lower fibrinogen levels with podoplanin treatment, compared to sham and vehicle, but no effect on partial thromboplastin time, prothrombin time, or INR. At higher doses, Rhodocytin resulted in immediate clotting, while at lower doses, it significantly increased soluble fibrin, consistent with initiation of clotting seen in the higher doses. In conclusion, our findings indicate that activation of CLEC-2 at the doses tested does not reduce ICH volume or improve outcomes, but did affect hemostatic parameters. Thus, the agonists may produce favorable outcomes with a different dosing regimen. Further studies are needed to confirm the mechanism by which CLEC-2 is acting in ICH, effective doses, and the effect of altered bleeding parameters.
Abstract 360: An Educational Program Improves Cholesterol and Mental Health of Individuals With Hypercholesterolemia
Authors: Ramirez Francisco E , Nedley Neil
Published: July 2021

Background:
Hypercholesterolemia can trigger depressive symptoms. We are reporting on the effect that an educational program has on cholesterol and depression levels.

Methods:
A medical clinic trained individuals to run the programs. Those who chose to participate met once a week for 8 weeks for a 2 hour program, which consisted of a 45 minute DVD presentation and a facilitated small group discussion together with weekly practical assignments. The Depression and Anxiety Assessment Test [DAAT] was used. It assessed depression level based on a modified PHQ-9 [Patient Health Questionnaire] test, demographics and participant self reported cholesterol levels and intake of omega-3 foods. No questions were asked about individuals’ treatment. The depression was classified according to DSM-5 into 4 categories as none (0-6), mild (7-10), moderate (11-19) or severe (20 or more). The progress was quantified on the category they finished in. The 77 question DAAT questionnaire was administered at baseline and completion. They were taught various healthy lifestyle habits like plant-based diet, exercise, proper rest, bright light, sleep, and avoiding negative thoughts, among other things.

Results:
Of 5997 participants that finished the program, 1320 (22%) reported having high cholesterol at baseline, that group had a baseline mean depression level of 13.3 SD 7.4, 76% of them ate omega-3 rich foods. Also at baseline 4060 (68%) reported normal cholesterol, their mean depression was 11.5 SD 7.5, of them 72% ate omega-3 rich foods, 10% did not know their cholesterol levels. The baseline high cholesterol group at the end of the program had an mean depression of 7.1 SD 6.1. Paired samples t-test of before and after was significant
t
(1319)=35.7 with a
p
<.001. The end program of the normal cholesterol group had a mean depression of 6.1 SD 5.8, t-test was significant
t
(4059)=53.6 with a
p
<.001. At the end of the program 26% of the baseline high cholesterol group no longer had hypercholesterolemia.

Conclusion:
Hypercholesterolemia seems to be related with higher levels of depression. Those with normal cholesterol improved the most. The program helps lower cholesterol in 1/4 of its participants. Mental health improves in the majority of its participants. Further follow-up is advised.
Abstract 375: The Intergenic Long Non-Coding RNA RP11-472n13.3 Modulates Interferon-Gamma Signaling and M1 Macrophage Activation
Authors: Wang Ying , Xue Chenyi , Reilly Muredach , Zhang Hanrui
Published: July 2021

We aim to interrogate the functions of a subset of human macrophage intergenic long non-coding RNA (lincRNAs) which harbor cardiometabolic trait-associated single nucleotide polymorphisms (SNPs). We have found that one lincRNA RP11-472N13.3 overlaps rs7081678, a SNP significantly associated with central obesity (WHRadjBMI;
P
=5.57x10
-6
). RP11-472N13.3 expression is enriched in macrophages relative to other obesity relevant tissues. Thus, RP11-472N13.3 SNPs for obesity may act via its myeloid cell modulation in adipose. In human monocyte-derived macrophage (HMDM), human induced pluripotent stem cell-derived macrophages (IPSDM) and THP1-derived macrophages (THP-1Φ), at RNAseq and Q-PCR, RP11-472N13.3 is abundant in M0 and M2(IL-4) macrophages but markedly suppressed in the M1 state (LPS/IFNγ). RP11-472N13.3 localizes almost exclusively to the cytoplasmic fraction of M0-HMDM. Consistent with GENCODE, our HMDM RNAseq data suggest a single 2-exon isoform. ChIP-seq reveals PU.1 and C/EBP-β binding at RP11-472N13.3 transcription start site. In our HMDM RNAseq (n=30 subjects) data, RP11-472N13.3 expression was inversely correlated with IFNγ-JAK-STAT signaling genes (e.g.,
IRF4, IL-12A, IL-23, STAT1, SOCS1, SOCS3
), but not LPS/TLR4 activated genes (e.g.,
TNFA, CXCL9, CXCL10, IL1B
). Furthermore, KD of RP11-472N13.3 using siRNA or LNA-ASO in THP-1Φ, amplified expression of IFNγ target genes but not LPS/TLR4 targets during M1 activation (LPS/IFNγ). These data suggest its potential role in modulating IFNγ signaling. Mechanistic studies are needed to examine the molecular mechanisms.
Abstract 380: SAA Lipidation and Delipidation by Hepatocytes
Authors: Ji Ailing , Akinmusire Akinwunmi , de Beer Maria C , de Beer Frederick C , Webb Nancy R , van der Westhuyzen Deneys R
Published: July 2021

Serum amyloid A (SAA) is one of the most striking acute phase reactants that can rapidly increase 1000-fold in plasma concentration in response to inflammatory cytokines. SAA in lipid-free form exhibits pro-inflammatory activities, but its putative physiological function(s) are poorly understood. SAA is produced and secreted largely by the liver and is present in plasma mainly as an HDL apolipoprotein. The pathways by which SAA is lipidated and incorporated into HDL are poorly understood. Plasma SAA is cleared more rapidly than the other major HDL apolipoproteins, but pathways involved in its delipidation and plasma clearance have also not been defined. In this study we examined how SAA is lipidated in primary hepatocytes and how such lipidation relates to the formation of nascent HDL particles. Endogenous hepatocyte SAA was lipidated and released from cells as large particles that were distinct from apoA-I-containing nascent HDL’s. Unlike apoA-I, formation of these SAA-containing particles was independent of ABCA-I. Similarly, when SAA was exogenously added to cells, SAA was lipidated to form nascent particles that were distinct from apoA-I-containing particles. We further studied the interaction of lipid-free and HDL-bound SAA with hepatocytes. Both in lipid-free form and as part of HDL, SAA exhibited significantly greater binding to cells than apoA-I or apoA-II. Binding studies were also carried out with normal and acute phase HDL’s isolated from control and SAA-deficient mice. Together, the results suggested that SAA, unlike apoA-I, is selectively removed from HDL by binding to hepatocytes. These findings may provide new insights into SAA metabolism and function.
Abstract 385: The Complex Nature of High-Density Lipoprotein Particle Diversity and Population Heterogeneity Necessitates High-Definition Constituent Identification and an Organizational Atlas
Authors: Fried Steven G , Altmann Scott W
Published: July 2021

Introduction:
The degree of lipoprotein complexity was unforeseen just a few years ago. Through advances in mass spectrometry it is now recognized that HDL is comprised of >100 proteins and ~200 lipid species in an undetermined number of combinations. These constituents exist in a distribution disequilibrium with each other and to the particle population as a whole. Concealed in this complex particle composition is extensive particle diversity and population heterogeneity and the source of the broad physiology observed with this lipoprotein subclass.

Hypothesis:
Science is a systematic enterprise that using mathematics and measurement, creates, builds and organizes knowledge in the form of testable observations, explanations and predictions. The ongoing identification of HDL constituents necessitates an effort to catalogue, categorize, map and relate entities in a structured framework. This will lead to fundamental understanding that advances knowledge and guides HDL biological research, clinical diagnostics and eventual therapeutic strategies.

Methods:
Literature analysis was used to produce an HDL proteome reference set. Proteome specific analysis of isoforms, proteolytic products, amino acid modifications and cSNPs were used to prepare a proteoform index. Theoretical tryptic peptide maps were generated and compared to the PeptideAtlas database and available published peptide lists from mass spec studies.

Results:
More than 300 non-immunoglobulin proteins were identified from the high-density lipoprotein fraction in published human sample studies. A consensus-based selection process produced an “unofficial” list of 122 genes. UniProtKB was used to expand an index of possible proteoforms and derive a theoretical peptide mass map. The theoretical and empirical mass map demonstrate both consistency and significant differences.

Conclusions:
The health benefits of HDL are not in dispute. However, its predictive value is being challenged. The reductionist approach to measuring HDL has proven insufficient and undermines the use of a Precision Medicine model for CAD. Constructing a conceptual framework to unify constituent data is an initial step to resolving the relational context that exists and its functional consequences.
Abstract 386: Correlations Between Cardiovascular Disease Risk Factors and Lipid Metabolic Pathways
Authors: Barr John R , Kuklenyik Zsuzsanna , Gardner Michael , Toth Christopher , Parks Bryan A , Jones Jeffery , Schieltz David , Andrews Michael , Rees Jon , McWilliams Lisa , Pirkle James L
Published: July 2021

A multiplexed, quantitative analytical workflow was applied to 120 fasting human serum samples collected from 30 normolipidemic, and 90 dyslipidemic donors. The analysis included separation by asymmetric flow field-flow fractionation while collecting fractions with ~1 nm increments of 7-15 nm (HDL), 20-30 nm (LDL) and greater than 30 nm lipoproteins. Size separation was followed by concentration measurements in each size fraction for non-polar lipids (FC, CE and TG), polar lipids (PC, SM, PE and PI), and apolipoproteins (apos A-I, A-II, A-IV, B, C-I, C-II, C-III and E); using three parallel, high throughput, quantitative liquid chromatography-tandem mass spectrometry methods developed in our laboratory. The average hydrodynamic size in each size fraction was also determined by dynamic light scattering. Measuring all major lipid and protein components and the size in all fractions allowed volumetric estimation of lipoprotein particle numbers (Lp-P). In the LDL size range of 22-26 nm, the calculated mean (N=120 samples) of the average (N=5 fraction/sample) apoB/LDL-P was 1.00 (Stdev=0.31, N=120). The resulting highly comprehensive data set allowed the evaluation of correlations between cardiovascular disease (CVD) risk factors and metabolic pathway specific indicators. For example, multivariate modelling of Total-TG/HDL-C ratios (1.5-17.5) showed negative correlation with HDL-apoC-III/LDL-apoC-III ratios.
Abstract 392: The Role of Scavenger Receptor-BI’s Transmembrane Domains in Cholesterol Transport: A "Locked Dimer" Strategy
Authors: Proudfoot Sarah , Chadwick Alexandra , Allen Emma , Sahoo Daisy
Published: July 2021

Efficient reverse cholesterol transport requires interactions between high density lipoprotein (HDL) and its receptor, scavenger receptor-BI (SR-BI). SR-BI is an 82 kDa protein with a large extracellular domain anchored by two transmembrane domains (TMDs). Our lab recently solved the NMR structure of SR-BI’s C-terminal TMD (C-TMD), a region that mediates SR-BI dimerization. Further, FRET studies suggest HDL-induced movement between neighboring SR-BI monomers, which led to our hypothesis that flexibility between SR-BI TMDs facilitates cholesterol transport. Using structure-guided mutagenesis, we introduced cysteine residues into the C-TMD of full-length SR-BI to create “locked dimers” of the receptor. Total lysate and cell surface expression of WT-, A444C-, L451C-, or G453C-SR-BI were verified in transiently-transfected COS-7 cells by immunoblot analysis and flow cytometry, respectively. Based on the predicted orientation of sulfhydryl side chains relative to the putative dimerization motif, we used immunoblot analysis following electrophoresis under reducing/non-reducing conditions to confirm that A444C- and L451C-SR-BI, but not G453C-SR-BI, formed disulfide bonds. Compared to WT-SR-BI, the locked dimer mutants, A444C- and L451C-SR-BI, exhibited normal selective uptake of [
3
H]-cholesteryl oleyl ether, despite slightly reduced [
125
I]-HDL binding. SR-BI-mediated cholesterol efflux to HDL from cells pre-labeled with [
3
H]-cholesterol was also unaltered by the presence of locked dimers. Finally, we investigated the ability of WT and mutant SR-BI receptors to alter accessibility of membrane free cholesterol to exogenous cholesterol oxidase (as judged by cholestenone levels). L451C- or G453C-SR-BI expression led to reduced cholestenone production compared to WT-SR-BI, suggesting that these mutants may be defective in reorganizing pools of membrane cholesterol. In conclusion, our preliminary data suggest that limiting conformational flexibility between TMDs by forcing locked dimers of SR-BI may not have a major impact on SR-BI-mediated cholesterol transport. However, locked dimers of SR-BI appear to affect the ability of SR-BI to modulate plasma membrane pools of free cholesterol, and this deserves further investigation.
Abstract 394: Ldl-cholesterol, ApoB100 Kinetics and Atherosclerosis in Ldlr-deficient Yucatan Minipigs: A New Model for Familial Hypercholesterolemia
Authors: Telford Dawn E , Burke Amy C , Sutherland Brian G , Sawyez Cynthia G , Edwards Jane Y , Barrett P H , Newton Roger S , Huff Murray W
Published: July 2021

Lack of animal models with human-like lipoprotein metabolism and pathology has hampered translational research in atherosclerosis. Recently, a model of familial hypercholesterolemia was developed in Yucatan miniature pigs, in which the LDL receptor (LDLR) was deleted through gene targeting of exon 4. The objective of the present study was to determine the plasma lipoprotein response to a high fat diet and the kinetics of apolipoprotein (apo) B metabolism in LDLR-deficient miniature pigs. LDLR+/+ (n=5), LDLR+/- pigs (n=6) and LDLR-/- pigs (n=5) were fed a diet containing 34% kcal from fat and 0.2% cholesterol (C). At 6 weeks, the kinetics of plasma apoB100 (fasting) were measured using stable isotopic techniques and multi-compartmental modeling. In chow-fed pigs, LDL-C was 0.8mM, 1.3mM and 14mM in LDLR+/+, LDLR+/- and LDLR-/- pigs, respectively. On diet for 6 weeks, LDL-C increased 1.3-fold (to 1.09mM), 1.7-fold (to 2.3mM) and 1.2-fold (to 15.8mM) in LDLR+/+, LDLR+/- and LDLR-/- pigs, respectively. The effect of genotype or diet on plasma TG and HDL-C was modest. Compared to LDLR+/+ pigs, VLDL apoB100 pool sizes increased 1.4-fold in LDLR+/- and 1.7-fold in LDLR-/- pigs, due primarily to a decrease in fractional catabolic rates (FCR) of 18% and 63%, respectively. Compared to LDL+/+ pigs, LDL apoB100 pool sizes increased 2.2-fold and 14-fold in LDLR+/- and LDLR-/-, respectively, which was due to both 1.5-fold and 2-fold increases in production rates and 24% and 85% decreases in FCR, respectively. At 23 weeks, raised lesion area in the abdominal aorta was 3.3% in LDLR+/- pigs and 48.5% in LDLR-/- pigs. In the left anterior descending coronary artery, lesion area was 14.7x10
3
μm
2
in LDLR+/- pigs and 656x10
3
μm
2
in LDLR-/- pigs. This model should prove useful for translational research in lipoprotein metabolism and atherosclerosis.