bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 098: Next Generation MicroRNA Sequencing to Identify Coronary Artery Disease Patients at Risk of Recurrent Myocardial Infarction
Authors: Kanuri Sri Harsha , Ipe Joseph , Kassab Kameel , Skaar Todd C , Edenberg Howard J. , Liu Yunlong , Gao Hongyu , Kreutz Rolf Peter
Published: March 2019

Background:
Circulating miRNA have the potential to become reliable biomarkers for risk stratification and early detection of cardiovascular events.

Methods:
In a single center cohort study, whole blood was collected from 437 subjects undergoing cardiac catheterization, who were followed for recurrent cardiovascular events during a mean follow up of 1.5 years. We selected a case cohort (n=22) with recurrent thrombotic events on standard medical therapy (stent thrombosis (n=6) or spontaneous myocardial infarction (MI) (n=16)) and a matched cohort with CAD, but uneventful clinical follow up (n=26), as well as a control group with cardiovascular risk factors, but without angiographic CAD (n=24). We performed complete miRNA next generation sequencing of RNA extracted from whole blood samples (including leukocytes and platelets).

Results:
Differential pattern of miRNA expression was demonstrated between controls, CAD patients with no events, and CAD patients with recurrent events (figure). MiRNA that have been previously associated with MI, CAD, endothelial function, vascular smooth muscle cells, platelets, angiogenesis, heart failure, cardiac hypertrophy, arrhythmia, and stroke were replicated in our CAD case-control cohorts. Twenty miRNA (FDR<0.05) were linked with risk of recurrent myocardial infarction and stent thrombosis, as compared to CAD patients with uneventful follow up.

Conclusions:
MiRNA next generation sequencing demonstrates altered fingerprint profile of whole blood miRNA expression among subjects with subsequent recurrent coronary thrombotic events on standard medical therapy (‘non-responders’), as compared to subjects with no recurrent cardiovascular events. MiRNA profiling may identify high risk subjects and provide additional insights into disease mechanisms not currently attenuated with standard medical therapy used in treatment of CAD.
Abstract 106: Young

Tgfbr2
G357W/+

Mice Have Decreased Expression of TGF-β Ligands and Receptors in the Proximal Aorta, Aortic Root Dilation, and Aortic Elongation
Authors: Hu Jie H , Angelov Stoyan N , Zhu Jay , Weil Maxwell , Smith Alexandra , Dichek David A
Published: March 2019

Introduction:

Tgfbr2
G357W/+

mice have a loss-of-function
Tgfbr2
allele (
G357W
) that is orthologous to an allele found in humans with Loeys-Dietz syndrome (LDS).

Tgfbr2
G357W/+

mice have kyphosis, aortic dilation and elongation, and die suddenly from aortic rupture. Elevations of mRNA encoding TGF-β ligands in cultured vascular smooth muscle cells from

Tgfbr2
G357W/+

mice and LDS patients—as well as increased expression of
Tgfb1
and
Col1a1
and elevation of both P-Smad2 and P-ERK1/2 in aortic roots of older (24-36-week)

Tgfbr2
G357W/+

mice—suggest that paradoxically increased TGF-β signaling might be responsible for aortic pathology in LDS.

Hypothesis:
We hypothesized that downregulation of TGF-β signaling components accompanies the development of aortic pathology in young

Tgfbr2
G357W/+

mice.

Methods:
We performed anatomic and gene-expression studies on 8- and 12-wk-old

Tgfbr2
G357W/+

mice and wild type (WT) littermates. For anatomic studies, mice were perfusion-fixed with formalin, and aortic length was measured in situ. Aortic roots as well as ascending (ASA), descending thoracic (DTA) and abdominal aortas (AA) were then embedded in OCT. Frozen sections were stained with H & E. External and internal elastic lamina lengths, aortic wall thickness and medial areas were measured. For gene expression studies, mice were saline-perfused, and the aortic root, ASA, and arch were snap frozen en bloc. RNA was extracted and TGF-β ligand and receptor mRNA were measured.

Results:
LDS mice (12 wk old; n = 7-9 vs 12 WT) had elongated ASA, arch, and DTA (~20%; P≤0.02), their aortic roots were dilated (20%; P<0.001), and their DTA wall thickness and medial areas were decreased (15%; P<0.01). No aortic wall hematomas were observed, and Prussian Blue staining was absent in aortic sections. mRNA encoding
Tgfb1
,
Tgfb2, Tgfbr1, Tgfbr3, Serpine1
, and
LoxL1
were decreased in LDS mouse aortas (20-34%; P<0.05 for all; n=9-10). LDS mice (8 wk old; n = 10 vs 12 WT) had only borderline decreased aortic
Tgfbr1
mRNA.

Conclusions:
In

Tgfbr2
G357W/+

mice, development of aortic pathology is accompanied by decreased expression of several TGF-β signaling pathway components. These data suggest that the
G357W
loss-of-function allele causes aortopathy via an early reduction of aortic TGF-β signaling.
Abstract 107: High Plasma Levels of Low-density Lipoprotein-cholesterol Accelerate Dissecting Aortic Aneurysms in Angiotensin II-induced Mice
Authors: Iida Yasunori , Tanaka Hiroki , Sawa Shigeharu , Shimizu Hideyuki
Published: March 2019

Background:
Plasma low-density lipoprotein (LDL)-cholesterol is implicated in aortic aneurysm (AA) and dissection (AD); however, its role in the pathogenesis of AA and AD, a disease with a high mortality rate, remains unknown. The existing animal models of aortic AA and AD do not reproduce all aspects of disease, including elevated LDL-cholesterol and spontaneous atheroma formation; therefore, a more reliable in vivo model is required. In this study, we show that mice with combined deficiency of the LDL-receptor and the catalytic component of the apolipoprotein B-edisome complex (

Ldlr
-/-
/Apobec1
-/-

) induced with angiotensin II are a useful model to study the pathophysiology of AA and AD associated with human type IIa dyslipidemia.

Methods:
AAs and ADs were created in 18-22-week-old male

Apoe
-/-

and

Ldlr
-/-
/Apobec1
-/-

mice by subcutaneous angiotensin II infusion. Immunostaining allowed assessment of smooth muscle cells and mural monocytes/macrophages.

Results:

Ldlr
-/-
/Apobec1
-/-

mice had elevated LDL-cholesterol levels characteristic for human type IIa dyslipidemia, resulting in atherogenesis, which promoted mortality, AA formation, and AD development. Interestingly, variations in the distribution of atheroma and inflammatory sites between

Apoe
-/-

and

Ldlr
-/-
/Apobec1
-/-

mice depending on lipid profiles resulted in differences in AA formation and AD occurrence in the thoracic aorta.

Conclusions:
Our results indicate the presence of a pathogenic pathway involving serum lipid composition that plays a key role in AA formation and AD occurrence in angiotensin II-induced mice.
Abstract 108: Lipopolysaccharide Fails to Augment Development of Angiotensin II-induced Abdominal Aortic Aneurysms in Mice
Authors: Mohammadmoradi Shayan , Howatt Deborah A , Moorleghen Jessica J , Lu Hong , Daugherty Alan
Published: March 2019

Background and objective:
Our previous study demonstrated that deficiency of toll-like receptor 4 (TLR4) reduced angiotensin II (AngII)-induced abdominal aortic aneurysms (AAAs) in hypercholesterolemic mice. Lipopolysaccharide (LPS) is a well-known agonist of TLR4. Interaction of LPS and TLR4 promotes inflammation and augments hypercholesterolemia-induced atherosclerosis. The purpose of this study was to determine whether LPS augments AngII-induced AAAs.

Methods and Results:
Low-density lipoprotein (LDL) receptor -/- mice fed a Western diet develop hypercholesterolemia and have augmentation of AngII-induced AAAs. We found that LDL receptor -/- mice fed Western diet had increased LPS concentrations, as quantified by a
Limulus
Amebocyte
lysate chromogenic endotoxin quantitation kit, compared to the same mice prior to Western diet feeding. To determine whether LPS plays a role on AngII-induced AAAs, 4 groups of male C57BL/6J mice, 10-week-old, were infused subcutaneously with: (1) Vehicle (N=5), (2) LPS (250
μ
g/kg/day; N=10), (3) AngII (1,000 ng/kg/min; N=10), or (4) both AngII and LPS (N=10) through Alzet mini osmotic pumps for 28 days. Abdominal aortic expansion was monitored serially at 2 and 4 weeks of infusion using high frequency ultrasonography (Vevo 3100 system; FUJIFILM). Continuous luminal expansion was detected in both AngII-infused mice and mice co-infused with both AngII and LPS. These results were confirmed by
ex vivo
measurements of maximal outer diameters of suprarenal aortas after termination showing AngII infusion led to significant increase of abdominal aortic dilation, compared to vehicle or LPS infused mice. However, co-infusion of LPS with AngII did not augment AngII-induced AAAs.

Conclusion:
LPS has no effects on AngII-induced AAAs in normolipidemic mice.
Abstract 115: AT2 Receptor Regulates Ascending Aortic Dilation during AT1 Receptor Blockade in Transverse Aortic Constriction Mouse Model
Authors: Zhou Zhen , Peters Andrew M , Wang Shanzhi , Chen Jiyuan , Arthur Erin , Milewicz Dianna M
Published: March 2019

Introduction:
The angiotensin II (Ang II) type 1 receptor (AT1R) antagonist losartan can slow aortic enlargement in mouse models and patients with Marfan syndrome. We have previously shown that losartan attenuates aortic enlargement and remodeling induced by thoracic aortic constriction (TAC).

Objective:
We investigated the role of the Ang II type 2 receptor (AT2R) in an acute hypertensive remodeling of the ascending aorta induced by TAC.

Methods:
160 male C57BL/6J mice were subjected to TAC surgeries with 27-gauge needle or sham surgery. Echocardiography measurements were performed 2 weeks post-operation using the Vevo 3100 imaging system. Blood pressure was measured in the ascending aorta using a Millar catheter introduced through the right common carotid artery.

Results:
TAC induced significant enlargement in aortic root and ascending aorta two weeks after surgery. The angiotensin converting enzyme inhibitor, captopril, decreased intraluminal systolic blood pressure (SBP) to the same degree as losartan, but did not prevent the increased aortic dilation, adventitial inflammation, medial elastin breakage, or
Mmp9
expression compared with TAC mice. Captopril plus an AT2R agonist, compound 21 (C21) attenuated TAC-indcued aortic dilation, elastin break numbers and
Mmp9
expression similar to losartan, despite an increase in SBP compared with captopril-treated TAC mice. Consistent with these results, treatment with losartan and an AT2R antagonist, PD123319, reversed the protective effects of losartan on TAC-induced aortic enlargement and remodeling. Note that treating with captopril and a Mas receptor agonist AVE0991 did not prevent TAC-induced aortic enlargement. Importantly, treating with C21 alone did not prevent TAC-induced aortic dilation, elastin breakage or
Mmp9
expression despite significantly decreased SBP.

Conclusions:
These data indicate signaling through both AT1R and AT2R are key modulators in aortic dilation and remodeling with acute increases in biomechanical forces induced by TAC and these protective effects are independent of alterations in blood pressure.
Abstract 116: A Novel Mediator of Heart Failure Development and Progression- Role of Manganese Superoxide Dismutase
Authors: Miriyala Sumitra , Labrie Timothy , Chandra Mini , Maxey Benjamin , Orr Wayne , Bhuiyan Smiriy@lsuhsc.edu , McCarthy Kevin , Panchatcharam Manikandan
Published: March 2019

Manganese Superoxide Dismutase (MnSOD), an antioxidant enzyme that catalyzes the conversion of superoxide radicals (O
2
•-
) in mitochondria. Constitutive activation mitochondrial reactive oxygen species (ROS) has been implicated in both the pathogenesis and the progression of cardiovascular disease. Absence of SOD2 (gene that encodes MnSOD) is found to be embryonic lethal in animal models due to impairment of mitochondrial function, most noticeably in the heart. In our earlier investigation, we have shown that the MnSOD mimetic, MnTnBuOE-2-PyP
5+
distributes 3-fold more in mitochondria than in cytosol. The exceptional ability of MnTnBuOE-2-PyP
5+
to dismute O
2
•-
parallels its ability to reduce ONOO– and CO3–. Based on our earlier reports, we have generated mice that specifically lack MnSOD in cardiomyocytes (Myh6-SOD2
Δ
). These mice showed early mortality ~4 months due to cardiac mitochondrial dysfunction. Oxidative phosphorylation (OXPHOS) in mitochondria is the predominant mode for O
2
consumption in cells, and the mitochondria are the primary source of ROS in cells due to leaked electrons. FACS analyses using Mito-Tracker Green indicated that the mass of mitochondria per cell was slightly decreased in the Myh6-SOD2
Δ
to the wild type. We then examined OXPHOS levels in Myh6-SOD2
Δ
v.s. Wild type using a Seahorse XF analyzer. The rate of oxygen consumption per cells was significantly lower in Myh6-SOD2
Δ
cardiomyocytes than that in wild type. The most noticeable difference in the O
2
consumption was found in the presence of FCCP (H
+
ionophore / uncoupler). FCCP is an inner membrane pore opener which resets the proton gradient between the mitochondrial matrix and the interspace, resulting in continuous transport of protons and consuming O
2
at the maximum potential. Remarkably, while the FCCP treatment increased O
2
consumption in wild type, the treatment showed no effect on the O
2
consumption in the Myh6-SOD2
Δ
cardiomyocytes. The result indicated that the low basal OXPHOS activity in Myh6-SOD2
Δ
was due to unusually low OXPHOS potential. We examined glycolysis in these cells by measuring extracellular acidification (ECAR) and the pattern exactly opposite to that of oxygen consumption rate (OCR) was observed for glycolysis rates between Myh6-SOD2
Δ
and Wild type.
Abstract 136: Near-Infrared Spectroscopy for Diagnosis and Characterization of Peripheral Artery Disease Severity
Authors: Fuglestad Matthew , Hernandez Hernan , Schieber Molly , Brunette Katya , Gao Yue , Myers Sara , Casale George , Pipinos Iraklis
Published: March 2019

Background:
Peripheral artery disease (PAD) frequently is undertreated. ABI is the standard for diagnosis of PAD but is limited in its ability to predict functional status and risk of disease progression. Previous work with Near-Infrared Spectroscopy (NIRS) has demonstrated impaired oxygen utilization in PAD muscle and may allow for improved diagnosis and stratification of disease severity. We hypothesize that exercise produces characteristic changes in lower-extremity muscle oxygenated heme percent (StO
2
) which differentiates PAD from control and predicts PAD severity better than ABI.

Methods:
We recruited 31 PAD subjects with intermittent claudication (IC) (ABI < 0.9) and 9 controls (ABI ≥ 0.9 and no IC). All subjects completed a Gardner maximal treadmill test. PAD subjects walked until IC was prohibitive (peak walking time, PWT) and controls walked for 540 seconds. StO
2
measurements were taken from the lateral gastrocnemius with the wireless MOXY NIRS monitor. StO
2
was documented at baseline, 60s, claudication onset time (COT), and PWT. For each subject, StO
2
values were expressed as percent of baseline to allow for comparison across subjects. Data were analyzed by student’s t-test and linear regression.

Results:
Mean baseline StO
2
values were 46±11% and 58±17%, respectively, for PAD and control subjects. Among controls, StO
2
dropped below baseline at 140±101s whereas PAD subjects dropped below baseline at 6±10s (P<0.001). PAD StO
2
s at 60s, COT, and PWT were compared to control StO
2
s at the corresponding time points. PAD patients’ StO
2
at 60s, COT, and PWT were 65.2±37.9% (n=31), 66.4±35.5% (n=31), and 73.2±26.0% (n=25) lower than controls, respectively (P < 0.001). PAD subjects were separated into tertiles of PWT. Relative to baseline, StO
2
decreased 84.2±18.4% (n=11) in the lowest tertile compared to 29.8±30.6% (n=10) in the highest tertile (P<0.05). Percent decrease in StO
2
at 60s correlated to PWT (R
2
=0.56) in contrast to ABI (R
2
=0.018).

Conclusion:
During exercise, PAD patients exhibited a characteristic change in StO
2
. In contrast to ABI, StO
2
predicted PAD-related exercise limitation. These data suggest NIRS in conjunction with traditional methods could be used to guide diagnostic and treatment decisions.
Abstract 139: PARP-1 Silencing Upregulates FOSL1 Transcription, Enhances Angiogenesis and Accelerates Ischemic-Diabetic Wound Healing
Authors: Banerjee Jaideep , Cheedu Divya , Sebastian Raul , Mascata Robyn , Sidawy Anton , Mishra Lopa , Nguyen Bao
Published: March 2019

Objective:
People with combined ischemic and diabetic wounds of the lower extremities have the highest risk for limb loss, especially for those without surgical revascularization options. We have demonstrated that Poly-ADP-Ribose polymerase (PARP-1) is hyperactivated in hyperglycemic/hypoxic cells and in ischemic/diabetic murine wounds. This study elucidates the molecular mechanisms of PARP-1 mediated impairment of angiogenesis in diabetic/ischemic wounds.

Methods:
A model of dorsal bipedicle flap-ischemic wounds on diabetic mice was used. The wounds were treated topically with nanoparticle-encapsulated siPARP-1 or vehicle. Wound closure rate and perfusion was analyzed using digital photography and Laser Doppler scanning, respectively. Angiogenetic markers in the tissues were measured by immunohistochemistry. In-vitro endothelial tube formation assay was performed using HUVECs cultured under hyperglycemic and hypoxic conditions.

Results:
Wounds treated with topical siPARP-1 significantly accelerated wound healing compared to vehicle (from 25% ± 5% to 40%± 8% (
n
=7,
p
< .05) by day 6 and from 50% ± 15% to 75%± 3% (
n
=7,
p
< .05) by day 12, and also exhibited improved tissue perfusion (50%± 5% increase in perfusion units over control on day 6,
n
=47
p
<0.05). Improved capillary density was also observed in the siPARP-1 treated wounds detected by immunohistochemistry for SMA (250%±35% increase in mean fluorescence intensity over control on day 12,
n
=4,
p
<0.05) and CD31 (125% ± 15% increase in mean fluorescence intensity over control on day 12,
n
=4,
p
<0.05). In-vitro angiogenesis assay showed that PARP-1-silencing significantly enhanced endothelial tube formation of hyperglycemic/hypoxic HUVECs (15± 4 complete polygons as compared to 0 in untreated,
n
=4,
p
<0.05). Human angiogenesis PCR-array analysis of pro-angiogenic factors revealed that PARP-1 silencing upregulated FOSL1 transcription by 5-fold (
n
=4,
p
<0.05). Interestingly, co-silencing of FOSL1 in PARP-1 silenced HUVECs resulted in loss of endothelial tube formation.

Conclusions:
PARP-1 silencing is an effective strategy to promote ischemic-diabetic wound healing. Our data suggest that PARP-1-FOSL1 is a potential novel axis in angiogenesis and PARP-1 could be a promising therapeutic target for improving angiogenesis in these wounds.
Abstract 140: Real-time Modulation of Platelet Phenotype and Vein Wall Biology in Patients with Chronic Venous Insufficiency
Authors: Mix Doran S , Young Zane , Toth Sandra , Schmidt Rachel , Doyle Adam J , Ellis Jennifer L , Stoner Michael C , Gosev Igor , Prasad Sunil , Knight Peter , Ture Sara , Morrell Craig , Cameron Scott J
Published: March 2019

Background:
The role of antiplatelet agents in the modulation of arterial disease is well described, but a paucity of data exists regarding their role in chronic venous insufficiency (CVI). We hypothesize that platelet responses to various antiplatelet agents are altered when comparing platelet function within refluxing and non-refluxing vein segments. Additionally, changes in platelet phenotype may alter vein wall biology.

Methods:
Isolated platelets were obtained simultaneously from the patient antecubital vein (ACV) and a refluxing greater saphenous vein (GSV) during surgical phlebectomy and compared to platelets from healthy individuals. Non-refluxing GSV was harvested for coronary bypass. Platelet surface receptor activation was assessed through P2Y12 (clopidogrel), PAR1 (vorapaxar), and thromboxane (aspirin) pathways by flow cytometry for p-selectin. Immunoblotting assessed CD41 (platelet) and CD45 (WBC) within the wall of vein samples.

Results:
Platelets from refluxing GSV showed a significant
increase
in reactivity via all platelet signaling pathways, especially P2Y12 and thromboxane when compared to platelets from the ACV in the same patient. Conversely, platelets collected from the ACV in CVI patients showed a significant
decrease
in reactivity to all agonists compared to ACV in healthy individuals without CVI. Most notably, GSV from a patient with CVI had a
reduction
in CD41 content, but a
seven-fold increase
in the CD45:CD41 ratio, compared to GSV from healthy people (Figure).

Conclusions:
Platelet activation by these clinically relevant pathways is enhanced locally in the refluxing GSV, yet systemic, circulating platelets isolated from CVI patients are
2-3-fold less
active than systemic platelets from healthy people. Our data suggest that reflux may locally alter the circulating platelet phenotype and in turn also have a role in remodeling the vein wall.
Abstract 145: Glucocorticoid Treatment is Associated With Lower Incidence of Myocardial Infarction in Patients Hospitalized for Community-acquired Pneumonia
Authors: Cangemi Roberto , Falcone Marco , Farcomeni Alessio , Calvieri Camilla , Scarpellini Gabriella , Battaglia Simona , Tiseo Giusy , Romiti Giulio Francesco , Bertazzoni Giuliano , Taliani Gloria , Violi Francesco ,   SIXTUS Study Group
Published: March 2019

Background:
Previous reports suggest that patients hospitalized for community-acquired pneumonia (CAP) have an enhanced risk of myocardial infarction (MI). Glucocorticoids have been proposed for the treatment of CAP. Although a recent meta-analysis showed that glucocorticoids can reduce morbidity and mortality in severe CAP, their role in the treatment of pneumonia remains unclear. Experiments in vitro and in vivo demonstrated that glucocorticoids inhibit platelet aggregation and platelet-related arterial thrombosis. Since enhanced platelet activation seems to play a role in the occurrence of MI in CAP patients, aim of the study was to assess the impact of glucocorticoids on MI occurrence in a cohort of patients hospitalized for CAP.

Methods and Results:
Consecutive patients admitted to the University-Hospital Policlinico Umberto I (Rome, Italy) with CAP were recruited and prospectively followed-up until discharge. The primary outcome of the study was the occurrence of an MI during hospitalization. Seven-hundred fifty-eight patients (493 males, 265 females; age: 71.7 ± 14.4 years) were included in the study: 241 (32%) were treated with systemic glucocorticoids (methylprednisolone, betamethasone or prednisone) during in-hospital follow-up, whereas 517 patients were not treated with systemic glucocorticoids. During the follow-up, 62 patients (8.2%) had an MI. Compared to the nonglucocorticoid group, patients treated with glucocorticoids showed a lower incidence of MI (9.5% vs. 5.4%; p=0.011). A Cox regression analysis showed that age, a history of coronary heart disease, and a major degree of respiratory impairment (pO2<60mmHg and pH<7.35) was positively associated to MI incidence, whereas glucocorticoid therapy was negatively associated. Compared to patients not receiving glucocorticoids, the propensity score adjusted analysis confirmed that patients taking glucocorticoids had a lower incidence of MI (HR: 0.458; 95% CI: 0.235 - 0.893, p=0.022).

Conclusion:
This study shows that in-hospital glucocorticoids treatment is associated with a lower incidence of MI within hospital-stay in a large cohort of patients with pneumonia.
Abstract 172: Prodomain of Furin Promotes Phospholipid Transfer Protein Proteasomal Degradation in Hepatocytes
Authors: Yu Yang , Lei Xia , Qin Shu-Cun , Jin Weijun , Jiang Xian C
Published: March 2019

PLTP is one of the major modulators of lipoprotein metabolism and atherosclerosis development, however, very little is known about the regulation of PLTP. The effect of hepatic profurin expression on PLTP processing and function is investigated. We utilized adenovirus overexpressing prodomain of furin (profurin) in mouse liver to evaluate PLTP activity, mass, and plasma lipid levels. We co-expressed PLTP and profurin in Huh7 cells and studied their interaction. We found profurin expression significantly reduced plasma lipids, plasma PLTP activity and mass in all tested mouse models, compared with controls. Moreover, the expression of profurin dramatically reduced liver PLTP activity and protein level. We further explore the mechanism using in vivo and ex vivo approaches. We found that profurin can interact with intracellular PLTP, and promote its ubiquitination and proteasomal degradation, resulting in less PLTP secretion from the hepatocytes. Furin does not cleave PLTP, instead it forms a complex with PLTP, likely through its prodomain. Our study reveals that hepatic PLTP protein is targeted for proteasomal degradation by profurin overexpression, which could be a novel post-translational mechanism underlying PLTP regulation.
Abstract 176: Preoperative Small HDL Particle Concentration is Associated with Paraoxonase-1 activity and the Risk of Acute Kidney Injury After Cardiac Surgery
Authors: Smith Loren , Smith Derek K , Remaley Alan T , Billings Frederic T , Linton MacRae F
Published: March 2019

Acute kidney injury (AKI) after cardiac surgery occurs in up to 30% of patients and predicts death. We have reported that a higher preoperative HDL particle concentration is independently associated with a decreased risk of AKI after cardiac surgery. HDL has known anti-oxidant properties that may attenuate AKI. We hypothesized that HDL particle size is associated with paraoxonase-1 (PON-1) activity and with the risk of AKI after cardiac surgery.

We selected 90 patients who developed mild, moderate, severe, or no AKI from a prospective trial of perioperative atorvastatin to prevent post-cardiac surgery AKI. PON-1 paraoxonase activity was measured in apoB-depleted serum with fluorescent substrate 7-diethylphospho-6,8-dixuor-4-methylumbelliferyl. HDL particle size was assessed using the NMR Lipoprofile test. Linear regression was used to assess the association between preoperative small, medium, and large HDL particle concentrations and PON-1 activity. We assessed the association between HDL particle levels and the maximum serum creatinine change from baseline in the first 48 postoperative hours using two-component latent variable mixture models adjusted for AKI risk factors.

A higher preoperative small HDL particle concentration was associated with a higher preoperative PON-1 activity (p=0.003, Figure 1A). Medium and large HDL particle concentrations were not associated with PON-1 activity. Small HDL particle concentration was found to be independently associated with postoperative serum creatinine change (p=0.02, Figure 1B), while medium and large HDL particle concentrations were not.

Conclusions:
A higher preoperative small HDL particle concentration was associated with a higher PON-1 activity and a decreased risk of AKI after cardiac surgery. Future work will characterize HDL throughout the surgical course to identify the mechanism underlying the protective association between HDL and AKI.
Abstract 180: Missense Mutations in ABCA1 and CETP Associate with Changes in the HDL Proteome in Primate Half-Sibs Discordant for HDL Cholesterol Levels
Authors: Plubell Deanna , Predazzi Irene , McKelvey Jessica , Clarke Jordyn , Letaw John , Raboin Michael J , Wilmarth Phillip A , Curran Joanne , Fazio Sergio , Pamir Nathalie , Vinson Amanda
Published: March 2019

HDL protein composition and corresponding function may impact cardiovascular disease risk. Identifying genetic variation that influences the HDL proteome may reveal modifiable HDL functions that impact this risk. We studied genetic determinants of the HDL proteome in a cohort of rhesus macaques enriched for extreme HDL cholesterol levels (HDL-c). We selected macaques from 2 distinct paternal half-sibships, each comprising 8 half-sib pairs matched for age-class and sex, but with large differences in HDL-c (N=22 genomes). HDL was isolated by ultracentrifugation and the protein cargo analyzed by mass spectrometry. Identified peptide sequences were compared to a Swiss-Prot canonical human protein database using BLAST to determine ortholog matches. Among the proteins identified, 64 are among the 229 reported in similar human studies, and 45 of these 64 are among the 95 highest-confidence HDL proteins tracked by the HDL Proteome Watch. We performed deep exome sequencing, and assessed predicted function for all genetic variants in macaques, among 23 genes associated with HDL disorders or variation in HDL-c in humans. We focused on the higher-impact variants most likely to have conserved effects between macaques and humans by proximity (i.e., <10 bp) to known human mutations. This produced a set of 3 missense variants in
ABCA1
associated with Tangier disease and HDL deficiency, and 4 missense variants in
CETP
associated with CETP deficiency, reduced CETP activity, and hyper- and hypoalphalipoproteinemias. Using a measured genotype approach, we tested for association of all 7 variants with adjusted spectral counts, while accounting for age and sex, and applied a false discovery rate (FDR) of 20% to all nominally significant results. After controlling for FDR, 3 missense variants in
ABCA1
were significantly associated with spectral counts for
VCAM1
,
ITGA2
, and
ITGB1
(nominal P-values 0.0005-0.0026), and 4 missense variants in
CETP
were significantly associated with spectral counts for
APOA2
,
APOC3
,
C4BPA
, and
PLTP
(nominal P-values 0.0002-0.0038). While our results require replication, these proteins suggest that changes in HDL-c in macaques are associated with changes in HDL that modulate vascular inflammation, immune response, and particle remodeling.
Abstract 185: G-protein Coupled Receptor 55 Deficiency Promotes Atherosclerosis and Inflammation in Mice
Authors: Hering Daniel , Guillamat-Prats Raquel , Rinne Petteri , Rami Martina , Kobold Sebastian , Döring Yvonne , Steffens Sabine
Published: March 2019

Background:
The endocannabinoid system plays a pathophysiological role in metabolic and cardiovascular disorders. G protein-coupled receptor (GPR) 55 is a novel cannabinoid receptor expressed by various lymphocyte subsets, in particular γδT cells, innate lymphoid cells and B1 cells. Its role in regulating immune functions and atherosclerosis is unknown.

Methods:
We studied early and advanced atherosclerotic plaques and inflammatory parameters in apolipoprotein E deficient (ApoE-/-) and ApoE-/-GPR55-/- mice after 4 or 16 weeks Western Diet (WD), respectively (n=12-15 per group). GPR55 mRNA expression was assessed in human carotid artery plaques (n=29) and healthy control vessels (left internal thoracic arteries; n=28).

Results:
ApoE-/-GPR55-/- mice had significantly 1.9 to 2.3-fold increased plaque sizes after 4 and 16 weeks WD compared to ApoE-/- controls with higher macrophage content at early stage, but less macrophage and more collagen content at advanced stage. This was accompanied by enhanced aortic pro-inflammatory cytokine mRNA expression (IL-6, TNFα, IL-1β, iNOS) as well as massively upregulated IgG1 plasma levels. Moreover, GPR55 deficiency was associated with increased circulating, splenic and aortic lymphocyte counts after 4 and 16 weeks WD. The most striking (2 to 4-fold) increase was found in γδT cells, with less pronounced increases in the total CD3+ T cell population and CD4+ and CD8+ subsets. This was paralleled by systemic increases in blood monocyte and neutrophil counts, likely due to enhanced myelopoiesis as suggested by enhanced bone marrow myeloid cell counts. At advanced atherosclerosis, ApoE-/-GPR55-/- had significantly higher NK cell numbers in blood and lymphoid organs, suggesting a possible regulation of cytotoxic lymphocyte responses. In view of a potential relevance for human pathophysiology, GPR55 mRNA levels were significantly higher in human plaque samples compared to non-atherosclerotic control vessels.

Conclusion:
GPR55 deficiency promotes atherosclerosis associated with a more pro-inflammatory phenotype. We may speculate that GPR55 negatively regulates the proatherogenic activity of lymphocyte subsets such as γδT cells, which deserves further investigation.
Abstract 202: Zfp36l1 is a Post transcriptional Regulator of Hepatic Bile Acid and Lipid Metabolism
Authors: Tarling Elizabeth , Clifford Bethan , Vallim Thomas A
Published: March 2019

Bile acids are detergents and important signaling molecules that activate the nuclear receptor FXR to control key metabolic processes, including feedback mechanisms to maintain bile acid homeostasis. FXR is the central rheostat of bile acid metabolism, and activation of FXR decreases the mRNA levels of bile acid synthetic genes, including
Cyp
7
a
1
, the gene encoding the rate-limiting enzyme of bile acid synthesis. We show that
Cyp
7
a
1
mRNA levels were rapidly reduced after pharmacologic FXR activation in wild-type, but not
Fxr
–

/

–
or liver-specific
Fxr
knockout mice (

Fxr
L

-

KO

). The rapid decrease in
Cyp
7
a
1
mRNA suggested a previously unidentified post-transcriptional mechanism. To identify the mechanism, we used synthetic and endogenous FXR agonists and found the RNA binding protein ZFP36L1 as a novel FXR target gene. ZFP36L1 mRNA and protein levels were increased as early as 30 minutes after FXR activation. ZFP36L1 is a
bona
-
fide
RNA binding protein that promotes degradation of mRNA targets by binding to AU-rich elements (AREs) in the 3’ UTR. We generated
in vivo
and
in vitro
gain-of-function models and we used reporter assays to show that ZFP36L1 targets the
Cyp7a1
UTR
.
In mice, hepatic overexpression of ZFP36L1 decreased
Cyp
7
a
1
mRNA and protein and decreased bile acid levels. To complement our gain-of-function studies, we generated liver-specific
Zfp
36
l
1
knockout mice (
Zfp
36
l
1

L

-

KO

) and we show that loss of
Zfp
36
l
1
resulted in elevated
Cyp
7
a
1
mRNA and protein, and increased bile acid levels. Given that bile acids are important metabolites that control lipid absorption and signaling, we investigated whether loss of hepatic
Zfp
36
l
1
resulted in more broad metabolic dysfunction. Western diet fed
Zfp
36
l
1

L

-

KO

mice had reduced body weight gain, specifically in adipose tissue depots compared to littermate
Zfp
36
l
1

flox-flox

mice. The differences in adiposity and steatosis were attributed to reduced lipid absorption, as
Zfp
36
l
1

L

-

KO

mice have increased fecal caloric content and reduced triglyceride absorption as determined by an intragastric fat tolerance test. The decreased lipid absorption is consistent with an altered bile acid metabolism. Thus, we have identified a novel pathway that controls
Cyp
7
a
1
and bile acid metabolism but may also have wider implications in diseases such as obesity and hepatosteatosis.
Abstract 224: Notch1 Activation in Endothelial Cells Promotes Progression of Atherosclerosis
Authors: Liu Zhao-Jun , Li Yan , Zhang Leiming , Regueiro Manuela M , Wei Yuntao , Velazquez Omaida C
Published: March 2019

Introduction:
Notch signaling plays pivotal roles in vascular development and cardiovascular disease. We previous showed that the Notch pathway is activated in luminal endothelial cells (EC) at atherosclerotic plaques, implicating a potential involvement of Notch signaling in atherosclerosis. We further demonstrated that loss of Notch1 signaling in EC suppresses progression of atherosclerosis. Here, we test the effect of activation of the Notch1 pathway in EC on atherosclerosis using mouse model with inducible EC-specific Notch1 activation on an ApoE
-/-
background.

Methods:
We created a Tamoxifen-inducible EC-specific N1IC (Notch1 intracellular domain, an active Notch1 mutant) expression mouse line on atherosclerosis-prone ApoE
-/-
background:

ROSA
LSL-N1IC
/VE-cadherin-CreER
T2
/ApoE
-/-
.

Mice were fed with high-fat-diet (HFD) along with or without Tamoxifen treatment (i.p. 2 mg/day for 5 consecutive days) starting from 4-wk old and terminated on 16-wk old. Aortas were harvested to study the effects of Notch1 activation in EC on the progression of aortic atherosclerosis. Expression of N1IC in EC was validated by immunostaining. Aortic plaque burden was evaluated by quantification of proportion of total aorta containing atherosclerosis detected by Oil-Red-O and hematoxylin-eosin staining using computer-aided image analysis. Blood was tested to measure lipid profile.

Results:
Enforced Notch1 activation in EC is induced in Tamoxifen-treated mice. EC gain-of-function Notch1 signaling significantly increased aortic plaque burden [n=10 in both (Tamoxifen -) and (Tamoxifen +) group,
P
<0.05]. The serum levels of total cholesterol, HDL, LDL, triglycerides and glucose were comparable between Tamoxifen-treated and -untreated mice.

Conclusions:
Activation of the Notch1 signaling pathway in EC facilitates atherosclerotic plaque formation. The increase in atherosclerosis was not due to changes in the serum levels of lipid, glucose, triglycerides, HDL, LDL and total cholesterol. Our findings demonstrate that activation of the Notch1 signaling pathway in EC promotes progression of atherosclerosis. It highlights Notch1 signaling as an emerging therapeutic target for the treatment of atherosclerosis.
Abstract 244: A Qualitative Evaluation of Coronary Atherosclerosis in Young and Middle-aged Dutch Tissue Donors
Authors: Lindeman Jan H , Geerts Marlieke , van Gool Alain
Published: March 2019

Background:
Ischemic coronary events relate to qualitative changes in plaque characteristics. Epidemiological data on qualitative aspects of the coronary atherosclerotic process (e.g. plaque progression and destabilization) is missing.

Objective:
to qualitatively map the epidemiology of atherosclerosis burden in a representative cohort of deceased individuals

Methods:
this is a systematic, qualitative analysis of atherosclerotic burden in the proximal left coronary artery (LCA) segment of 695 tissue donors (median age 54, range 11-65 years). Based on the cause of death, donors were dichotomized into a non-cardiovascular (non-CVD group) and a cardiovascular disease (CVD) group. Consecutive, 5 mm LCA segments were Movat stained, and the atherosclerotic burden for each segment qualitatively graded (revised AHA-classification).

Results:
non-CVD and CVD groups show rapid acceleration of atherosclerosis severity after the age of 40; resulting in an almost endemic presence of advanced atherosclerosis in men and women over 40 respectively 50 years. In fact, only 19% of the non-CVD and 6% of the CVD donors over 40 presented with a normal LCA or non-progressive lesion type. The consolidated fibrous calcified plaque (FCP) dominated in both non-CVD and CVD donors over 40. Estimates of atherosclerosis load (i.e. average lesion grade, proportion of FCPs, and average number of FCPs per cross-section were all higher in the CVD group (P<1.10
-16
, P<0.0001 and P<0.05 respectively)

Conclusions:
advanced atherosclerotic disease is endemic in individuals over 40. Dominance of FCP lesions and the higher disease load in CVD donors imply that the atherosclerotic process is repetitive. Abundance of FCPs suggest that complications of plaque rupture are stochastic. This study was in part funded by the European Commission (Cartardis, FP7 HEALTH.2013.2.4.2-1).
Abstract 250: Endothelial functional Regulation by Enhancer-Associated Long Non-Coding RNAs
Authors: Chen Zhen , Miao Yifei , Lin Feng-Mao
Published: March 2019

Vascular endothelium constitutes the critical interface between circulating blood and vessel wall. Long non-coding RNAs (lncRNAs) are important regulators in gene expression and chromotin remodeling. We recently identified a
l
ncRNA that
e
nhances
e
ndothelial
n
itric oxide synthase (eNOS)
e
xpression (LEENE), that is encoded in a distal enhancer region and mediates long-range DNA interaction to promote the eNOS expression. We now extend from this prototype using a systematic approach to identify enhancer-associated lncRNAs and their molecular and functional regulation of endothelial gene expression. We integrate a variety of publicly available high-throughput sequencing datasets (including various chromatin immunoprecipitation-sequencing) that provide information on histone modifications and transcriptional activity and high-resolution chromatin conformation capture combined with sequencing (4C-seq) profiles generated in our own lab. From these analyses, we identified over 200 enhancer-associated lncRNA candidates with high potential to regulate endothelial gene expression. Quantitative PCR analysis verified that 7 out of the top 10 ranked candidates are differentially regulated by stimuli that alter endothelial gene expression and functions, including high-glucose, TNFα, atorvastatin, and metformin. Using CRISPR-cas9 gene editing to delete these lncRNA-associated enhancer regions and locked nucleic acid-mediated lncRNA inhibition, we demonstrated that the loss-of-function of these lncRNAs lead to significant alteration of endothelial gene expression, including those involved in angiogenesis and eNOS signaling. Collectively, these lncRNAs play an essential role in epigenetic regulation of endothelial functions; the identification of these lncRNAs can have significant impacts on understanding of endothelial homeostasis and dysfunction.
Abstract 251: miRNA Mapping of Cardiac Endothelial and Fibroblast Cells during Hypertrophy Progression
Authors: Dangwal Seema , Martino Filippo , Batkai Sandor , Scholz Claus J , Kunz Meik , Dandekar Thomas , Thum Thomas
Published: March 2019

MicroRNAs (MiRNA/miRs) are known key players in cardiovascular disorders. Here we created and analyzed a global miRNA map in isolated cardiac endothelial and fibroblast cells during hypertrophy progression.

Hypertrophy was induced in male C57BL/6 mice by trans-aortic constriction (TAC). Hypertrophic phenotyping was performed at 3 days (3d), 2 weeks (2w) or 4 weeks (4w) post- SHAM/TAC operation, using Millar system and echocardiography. Thereafter, pure single cell fractions from hearts were recovered by retrograde collagenase-II perfusion, followed by pre-plating of fibroblasts and magnetic sorting of endothelial cells. Transcriptomic analysis was performed on RNA isolated from different heart cell fractions at 3d, 2w or 4w post-SHAM/TAC. Additional 6w and 13w SHAM/TAC groups were included for validation of selected miRNAs. Cytokine secretome was performed using multiplex assay after transfection of a miR library to primary cardiac fibroblasts. Unsupervised hierarchical clustering revealed specific miRNA profile of each cardiac cell fraction. Principle component analysis projected strong effect of individual cellular compartments. Based on miRnome screening, miR-709, miR-30e-5p, miR-146a, miR-34a and miR-204, miR-1187 were validated by RTPCR to confirm spatial and temporal regulation (n=3-5, p<0.05) of these miRNAs in non-cardiomyocyte fractions. Corresponding time-dependent downregulation of miR-146a targets, Pten and Timp-2, was also observed. Cytokine secretome analysis upon miR-precursor library transfection in cardiac fibroblasts confirmed an increase in FGF, LIF-1, MCP-1, MIP-1 secretion (1.5-5 folds) by miR-146a and miR-34a. Gradual activation of the TGF beta pathway in endothelial cells may initiate endothelial to mesenchymal transition, whereas cytokine secretion from fibroblasts may affect the cellular hemostasis.

Our study represents a global miRNome of cardiac endothelial and fibroblast compartments during progressive hypertrophy to better understand the time dependent molecular changes in non-cardiomyocyte compartment during pressure-overload induced cardiac remodeling. The collective influence of miRNA deregulation may be linked to different pathways responsible for cell-proliferation, inflammation and fibrosis with the progression of hypertrophy.
Abstract 252: Innovative Cell-therapy for Diabetic Kidney Disease (DKD)
Authors: Kundu Nabanita , Asico Laureano , Jose Pedro , Sen Sabyasachi
Published: March 2019

Diabetic kidney disease (DKD) is one of the major vascular complications of diabetes, which is associated with glomerulosclerosis and poor perfusion. Therefore, improving the renal perfusion may help to halt or reverse the kidney injury. Here, we investigated whether transplanting mouse EPC (that has been modified by transiently silencing p53 gene using Adenovirus transduction ex-vivo), under renal capsule could improve angiogenesis and renal perfusion, by preventing EPC apoptosis in a hyperglycemic milieu.

Methods:
First, we confirmed hyperglycemia and progressive proteinuria in STZ-induced type 1 diabetic C57BL/6J mouse. Next, we transplanted 0.3 million p53-silenced EPCs,or Ad-Null-EPCs (control), bilaterally, under each kidney capsule. Another comparator was non-STZ normal mouse. Urine was collected weekly for volume and protein estimation. Renal blood flow was measured by laser Doppler. Kidneys were harvested post sacrifice and qRT-PCR were performed for targeted (towards angiogenic genes) gene expression assays.

Results:
Excess urine volume as noted in the hyperglycemic mouse models was reduced 4-fold post transplantation of p53sh-EPCs compared to null. There was no proteinuria after week 2 in p53sh-EPC transplanted mouse unlike control. Enhanced blood flow by laser doppler (3.2 fold) was also noted with delivery of p53sh-EPCs compared to null EPC. Absence of proteinuria (after 2wks) and renal blood flow measurements for p53sh group were similar to the non-STZ mouse. Interestingly, markers for neovascularization, such as eNOS (4.5 fold, p=0.002) and VEGF-A (1.5 fold, p=0.03) upregulated significantly post p53 silenced EPC transplantation compared to null EPC. CD31 staining is pending.

Conclusion:
Transient silencing of p53 gene in mouse EPCs help to improve proteinuria, diabetic polyuria and renal blood flow, most likely by increasing angiogenesis and perfusion and may have a prominent therapeutic role in DKD.
Abstract 253: Hypercholesterolemia Reduces the Differentiation of Hematopoietic Stem Cells towards Vasculogenic Ly6C
low
Monocytes in a Tet1 Dependent Pathway
Authors: Tie Guodong , Yan Jinglian , Khair Lyne , Tutto Amanda , Hayes Kate , Messina Louis
Published: March 2019

Rationale:
Hypercholesterolemia has been verified as an important risk factor of cardiovascular diseases. It has been shown to impair the post-ischemic arteriogenesis, the major physiological process in response to ischemic injury. As yet, the underlying mechanism is not fully illustrated.

Objective:
Our own data indicate that hematopoietic stem cells (HSCs) functions as a determinant in post-ischemic arteriogenesis by differentiating into vascular progenitors. However, the differentiation processes and the underlying mechanisms remain unknown. Ten eleven translocation (Tet) family are a group of enzymes initiating the demethylation of DNA. Tet1 is specifically downregulated in HSCs from hypercholesterolemic mice. Therefore, we hypothesized that Tet1 regulates the differentiation from HSCs towards vascular progenitors. Hypercholesterolemia reduces vascular specification of HSCs through a Tet1 dependent epigenetic pathway.

Methods and Results:
The ex vivo differentiation and in vivo transplantation experiments showed that HSC-originated Ly6C
low
monocytes in peripheral blood possess the greatest capacity to differentiate into vascular cells and to improve the post-ischemic arteriogenesis. Ly6C
low
monocyte population was specifically reduced in Tet1
-/-
mice after the induction of hindlimb ischemia, consequently resulting in significant impairment of post-ischemic arteriogenesis. Tet1 deficiency caused unique expression profiles of the genes involved in the differentiation of HSCs towards monocytes. RT-PCR showed that the expression of Tet1 was specifically downregulated in HSCs isolated from ApoE
-/-
mice. As expected, the Ly6C
low
monocyte population was specifically reduced and the post-ischemic arteriogenesis was significantly impaired in ApoE
-/-
mice.

Conclusion:
Ly6C
low
monocytes are the major vascular progenitors originated from HSCs. Tet1 dependent epigenetic regulation is critical in the differentiation process from HSCs towards Ly6C
low
monocytes. Hypercholesterolemia inhibits the expression of Tet1 and subsequently impairs the differentiation from HSCs towards Ly6C
low
monocytes, resulting in the impairment of post-ischemic arteriogenesis.