bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 50: Procollagen C-endopeptidase Enhancer protein 2 (PCPE2) Deficiency Profoundly Affects Adipose Distribution in Mice and Humans and Links HDL Metabolism to Adipocyte Biology
Authors: Kaul Sushma , Maruko Elisa , Xu Hao , Civelek Mete , Glastonbury Craig , Small Kerrin S , Dallinga-Thie Geesje M , Thomas Michael J , Goldberg Ira , Sorci-Thomas Mary
Published: June 2021

Procollagen C-endopeptidase enhancer protein 2 (PCPE2) modulates selective HDL cholesterol ester uptake by SR-BI. Ldlr-/-,Pcpe2-/- mice develop greater aortic atherosclerosis despite increased concentrations of enlarged plasma HDL, suggesting PCPE2 confers functionality to HDL for reverse cholesterol transport. PCPE2 is a 52 kDa glycoprotein encoded by PCOLCE2 gene most highly expressed in adipose tissue, heart and aorta. PCPE2 has 2 CUB domains separated by a short linker, with each CUB domain containing a beta-sandwich fold, mediating a variety of protein-protein interactions. PCPE2 also contains a C-terminal netrin-like domain that binds tightly to cell surface glycosaminoglycans accounting for its location in the extracellular matrix. Because of the strong inverse correlation between HDL and triglyceride levels, and the potential role of SR-BI in triglyceride-rich lipoprotein uptake we examined Ldlr-/-,Pcpe2-/-mice fed a Western diet for 25 weeks. Ldlr-/-,Pcpe2-/- mice showed 1.7 fold increase in plasma triglyceride concentrations when compared to age and gender matched Ldlr-/- mice. We also noted that despite similar body weights, Ldlr-/-,Pcpe2-/- visceral fat pad weight was reduced 60% (p<0.001, n=9 per genotype) compared to Ldlr-/- mice, while subcutaneous fat pad weight was not significantly different. This maldistribution of adipose in Ldlr-/-,Pcpe2-/- was associated with increased fasting plasma glucose levels, elevated 25% (p<0.02) over Ldlr-/- mice. In humans, analyses showed significant correlations of subcutaneous fat PCPE2 RNA expression with DEXA traits from the TwinsUK cohort; % android fat (p<0.0002), % trunk fat (p<0.0001) and % gynoid fat (p<0.04). Additional correlations between PCPE2 mRNA abundance and candidate marker expression will also be presented using 53 human visceral fat samples. Overall these data suggest that PCPE2 plays an important role in regional fat deposition linking SR-BI mediated cholesterol ester uptake to adipose cell biology.
Abstract 506: Should Endovascular Therapy for Renal Artery Stenosis Make a Come Back
Authors: Ansari Mohammad M , Garcia Daniel
Published: June 2021

Introduction:
Guidelines recommend endovascular therapy (EVT) for severe (>75% on angiography) renal artery stenosis (RAS). We sought to determine whether the populations studied could have influenced the lack of benefit of EVT in RAS.

Method:
Pub Med, Cochrane and EMBASE were systematically reviewed until November 2015 for all RCTs comparing EVT to medical therapy (MT) for RAS.

Primary outcomes:
systolic blood pressure and anti-hypertensive medication reduction.

Secondary outcomes:
worsening renal function, morbidity, mortality, heart failure, and stroke. We used random effect analysis according to the Cochrane-Handbook of Systematic Reviews and RevMan 5.2 for statistical analysis.

Results:
Seven RCTs with 2126 patients were included: 1036 EVT and 1085 MT. 35-65% of patients did not have severe RAS. There was no difference in change of systolic blood pressure (0.45±1.85, p=0.64). There was a significant decrease in the mean amount of anti-hypertensive medication in the EVT group (0.24±0.12, p<0.001). There were no differences in worsening of renal function, morbidity and mortality, heart failure and stroke (Fig 1).

Discussion:
Our analysis suggests that the apparent lack of added benefit of EVT for RAS could be largely secondary to selection bias, given the percentage of patients with mild to moderate RAS that were included and treated. Based on available data, trials studying EVT for severe RAS are warranted and should be designed to only include the appropriate patients. These well-designed studies are ultimately likely to “awaken the force”.
Abstract 508: The Role of Dual Specificity Phosphatase 5 in Post Ischemic Angiogenesis
Authors: Ayalew Dawit , Daneshmand Arvin , Quatara Hajara , Chen Lingdan , Dokun Ayotunde O
Published: June 2021

Background:
Dual specificity phosphatase 5 (DUSP5) is a member of the protein phosphatase subfamily that inactivate their target kinases by dephosphorylating both the phosphoserine/threonine and phosphotyrosine residues. DUSP5 regulates the mitogen activated protein (MAP) kinase ERK1/2, and although it has been shown to play a key role in embryonic vascular development, its role in post ischemic angiogenesis is not known. This study sort to investigate the role of DUSP5 is endothelial cell function and angiogenesis in ischemia.

Methods:
Hindlimb ischemia (HLI) was induced in mice and at day 3 post HLI, expression of DUSP5 was assessed in the ischemic hind limbs. We assessed DUSP5 expression in 3 major cell types found the ischemic hindlimb, endothelial cells or ECs (human umbilical vein endothelial cells, HUVECs), skeletal muscle myoblast (C2C12) and vascular smooth muscles cells (VSMC). Cells were exposed to simulated ischemia (2% oxygen with serum starvation) for 24 hours analyzed for DUSP5 mRNA and protein expression. The effect of loss of DUSP5 on EC function in ischemia was assessed by CRISPR/Cas9 mediated knock down of DUSP5 followed by analysis of EC proliferation, apoptosis and tube formation in simulated ischemia.

Results:
DUSP5 protein levels were significantly upregulated in mouse post ischemic hind limbs (ischemic (I) vs non-ischemic (NI); 1.63 ± 0.39 vs. 0.22 ± 0.02, p<0.05, n=3). Post ischemic exposure, ECs and C2C12 showed significant upregulation of DUSP5 protein. (DUSP5/Tubulin, I vs NI, HUVEC: 0.43 ± 0.09 vs. 0.09 ± 0.03, p<0.02; C2C12: 1.77 ± 0.10 vs. 0.42 ± 0.01, p< 0.01, n=5). VSMCs showed no significant change in DUSP5 expression. Knock down of DUSP5 in HUVECs resulted in significant decrease in cell proliferation in ischemia (O.D 450 control vs knock-down : 0.46 ± 0.01 vs. 0.34 ± 0.01, p<0.01, n=7) but no significant change in apoptosis (O.D 450 control vs knock-down: 0.13 ± 0.01 vs. 0.13 ± 0.00, p>0.05, n=7). It also resulted in significant impairment in, in vitro angiogenesis (tube/sq cm: 28.6 ± 1.2 vs. 16.43 ± 1.6, p<0.01, n=7).

Conclusion:
DUSP5 is highly upregulated in ischemic endothelial cells and plays an important role in EC proliferation and post ischemic angiogenesis.
Abstract 509: Screening for Carotid Occlusive Disease in Patients With Mild Peripheral Vascular Disease
Authors: Harrington Andrew , Ilonzo Nicole , Polanco Thais , Yang Kevin , Goss Selena , Alcantara Sean , Dudkiewicz Michael , Lantis John C
Published: June 2021

Objectives:
Peripheral vascular disease (PVD) is a systemic disorder, which can affect all territories of arteries. In order to maximize screening survey yield: we aimed to describe the association between positive lower extremity (LE) non-invasive flow studies in symptomatic patients and occult carotid occlusive disease (COD).

Methods:
A retrospective chart review was performed on 420 patients who underwent carotid duplex ultrasound and non-invasive flow studies (NIFS; aka Pulse volume recordings) for evaluation of COD and LE PVD respectively. Additional clinical variables collected included age, gender, and indication for NIFS and indication for carotid duplex. The respective studies were performed within a year of each other. Statistical analysis was performed using SPSSv20 software and SAS statistical software. Univariate analysis was performed using Mann-Whitney, student t-test and receiver operating curve (ROC).

Results:
66% (266/420) of the patients were male and 43% (181/420) of patients had documented history of bruit. Patients with mild PVD (ABI between 0.81 and 1) were more likely to have clinically significant COD than patients with moderate to severe PVD (ABI < 0.8) (25% vs 16.19%, P=0.03). Therefore, severity of peripheral vascular disease did not correlate with likelihood of clinically significant COD (p>0.05). ROC analysis was performed (AUC=0.602, p=0.05). Low ABI 0.50 was found to be 92% sensitive (Se) but only 14% specific (Sp). The optimal ABI cutoff point for predicting COD was 0.95 (Se=40%,Sp 60%, Youden’s index 0.194)

Conclusion:
Therefore, routine screening for COD in patients with lower extremity peripheral vascular disease is unlikely to uncover clinically significant carotid stenosis. These findings do not indicate a population of patients for which a combined prospective screening is warranted.
Abstract 510: Perivascular-Cell Derived Oct4 is Essential for Angiogenesis Following Hindlimb Ischemia
Authors: Hess Daniel L , Cherepanova Olga A , Annex Brian H , Owens Gary K
Published: June 2021

Ischemic vascular diseases such as coronary, carotid, and peripheral artery disease (PAD) require vascular growth and remodeling in order to restore blood flow to ischemic tissue. Human therapeutic studies aimed at promoting neovascularization have been largely unsuccessful in part because these approaches have focused on stimulating endothelial cell (EC) growth without coordinate investment of smooth muscle cells and pericytes (SMC-P) needed for functional blood vessels that are stable and able to regulate perfusion. Thus, there is a critical need to identify mechanisms that mediate perivascular cell coverage during vascular network remodeling, a process that requires perivascular cell detachment, migration, and investment of endothelial tubes. Previous work from the Owens lab demonstrated that the stem cell pluripotency gene Oct4, previously thought to be permanently epigenetically silenced in all somatic cells, is reactivated in SMC within atherosclerotic lesions. SMC specific KO of Oct4 results in marked reductions in the number of SMC within lesions due to failure of cells to migrate out of the media. That is, Oct4 reactivation appears to be critical for SMC migration, at least in the context of atherosclerosis. Therefore, the present study tested the hypothesis that SMC-P reactivation of Oct4 is essential for neovascularization following hindlimb ischemia (HLI), a mouse model of PAD. To test this hypothesis, we used a unique mouse line enabling SMC-P specific tamoxifen-inducible lineage tracing (termed Myh11 eYFP), allowing their tracking following phenotypic switching and loss of identifying genes, combined with SMC-P specific knockout of Oct4. We subjected both Oct4WT and Oct4KO Myh11 eYFP mice to HLI and monitored perfusion recovery via Laser Doppler at days 0, 3, 7, 14, and 21 post-HLI. We found that SMC-P specific KO of Oct4 results in significantly impaired perfusion recovery at days 14 and 21 post-HLI. We harvested hindlimb muscle at day 21 post-HLI and observed that Oct4 KO mice have significantly impaired angiogenesis, but intact arteriogenesis, compared to WT controls. Future studies will investigate Oct4 downstream targets necessary for angiogenesis following HLI.
Abstract 513: Cardiovascular Risk Factors in Patientes with Peripheral Arterial Disease: A Case-control Study in PopayaN-Colombia
Authors: Lopez Nelson A
Published: June 2021

Cardiovascular risk factors in patients with perpheral arterial disease: a case -control study in Popayan-Colombia. Nelson Lopez, Lorena Urbano, Wilson Muñoz, Carlos H. Sierra-Torres.

Introduction:
Peripheral arterial disease is the third cause of atherosclerotic cardiovascular disease and the prevalence is higher in countries with lower -middle income.

Objetives:
To identify cardiovascular risk factors associated with peripheral arterial disease in Popayan-Colombia.

Materials and methods:
Included 500 cases and 500 controls in a observacional, analytical, case-control (1-1) study, informed consent, epidemiological survey, screening through the ankle-brachial index, anthropometric and biomedical tests were performed. The statical anlizes with SPSS version 22.

Results:
The pathology study, increased significantly with age, ocurring more frequently (43.2%). in subjects between 70-79 years of age and increasing for both genres in each decade of life, the disease was more common in womwn (53.4%) subjcts from urban (63.8%). The third part of men and women were asymptomatic. 8.6% of reported ischemic pain, while 6.7% of women were ulceration or gangrene.

Conclusios:
The main risk factors associated with peripheral arterial disease in Popayan -Colombia- were identified indicating that there is need to develop strategies for effective detection and prevention, using the ankle-brachial index in primary care health. ABI measurement should be used as a screening test in patients with médium to high risk in Colombia to improve strategies to reduce mortality from cardiovascular disease.
Abstract 528: Correlation Between Expression Level of Platelet Endothelial Aggregation Receptor 1 and On-aspirin Platelet Aggregation
Authors: Yao Yi , Zhang Jia-Hui , He Chen , Ma Yuan-Liang , Tang Xiao-Fang , Song Ying , Xu Jing-Jing , Liu Ru , Yuan Jin-Qing
Published: June 2021

Background:
Aspirin is a basilar support in coronary heart disease secondary prevention, patients with high platelet aggregation during aspirin therapy after percutaneous coronary intervention(PCI) may lead to increased risk of ischemic vascular events. Platelet endothelial aggregation receptor 1(PEAR1) is a platelet transmembrane protein which reported play an important role in platelet aggregation. The aim of the study is to analyze the correlation between PEAR1 expression level and on-aspirin platelet aggregation.

Methods:
Consecutive patients with acute myocardial infarction after PCI under dual antiplatelet with aspirin and clopidogrel were enrolled in the cohort. Blood samples were obtained 3 days after PCI by peripheral venipuncture. On-aspirin platelet aggregation was assessed by VerifyNow Aspirin Assay, results were recorded as aspirin reaction unit(ARU). Expression of PEAR1 on platelet membrane was determined by washed platelet incubated with mouse anti-human PEAR1 phycoerythrin(PE)-conjugated monoclonal antibody for 20 minutes and analyzed by flow cytometry, results were recorded as mean fluorescence intensity(MFI).

Results:
A total of 131 patients were enrolled in the study, the average age was 59.88±10.84 years, the average ARU was 443.21±65.99, the median MFI was 137.00(interquartile range 105.00 to 226.00). Patients with high platelet aggregation(ARU>550) showed higher expression of PEAR1 on the surface of platelet than those with low platelet aggregation(ARU<400), although did not achieve a significant difference [129.00(interquartile range 116.75 to 215.75) vs 116.00(interquartile range 86.05 to 197.50), P=0.11]. The expression level of PEAR1 showed a poorly positive correlation with platelet aggregation(Spearman r
s
=0.21, p=0.02).

Conclusion:
The study showed that elevated expression of PEAR1 was weakly related to increased on-aspirin platelet aggregation after PCI.
Abstract 536: Platelet-derived Factor V Promotes Angiogenesis in a Mouse Hindlimb Ischemia Model
Authors: Wu Jianbo , Yang Yan , Xiao Lamei , Chen Ni , Li Yongjie , Deng Xin , Wang Liqun , Pang Ningbo , Sun Hongmin
Published: June 2021

Objective:
Coagulation factor V (fV) is distributed in plasma and platelet pools distinguished by physical and functional differences. fV has been extensively studied for its roles in coagulation. The roles of fV in other physiological pathways remain understudied. In the current study, we report that platelet fV is critical for the regulation of angiogenesis in a mouse model of hind-limb ischemia.

Methods and Results:
Hindlimb ischemia was produced in mice by femoral artery ligation in transgenic mice, with different level of fV gene expression restricted to either the plasma or platelets. The hindlimb blood flow perfusion in mice with higher platelet fV was significantly greater. Furthermore, using a platelet depletion/transfusion procedure, transfusion of platelets with higher level of fV into transgenic mice with undetectable platelet fV significantly rescued the ischemia-mediated impairments in blood flow perfusion. Immunohistochemical analysis also indicated markedly increased capillary formation in ischemic muscle of mice with higher platelet fV. Moreover, thrombin activity was significantly higher in the mice with higher platelet fV. Platelet with higher level of fV demonstrated significantly higher pro-migratory activity in an endothelial cells migration assay.

Conclusion:
These findings suggest that platelet-derived fV contributes to the control of angiogenesis, in addition to its role in hemostasis. The stimulation is likely associated with thrombin generation due to platelet-derived fV.
Abstract 539: Soluble Oxidized Apolipoprotein A-I, a Precursor of Amyloid Fibrils, Activates Secretion of Inflammatory Cytokines in Macrophages
Authors: Witkowski Andrzej , Chan Gary K , Li Nancy J , Lu Rui , Yokoyama Shinji , Cavigiolio Giorgio
Published: June 2021

Atherosclerosis is often described as an inflammatory disease of the arteries. One mechanism whereby apolipoprotein A-I (apoA-I) exerts its anti-atherosclerotic effect is by mitigating the inflammatory response of cells involved in atherosclerosis progression. However, oxidation transforms apoA-I from an anti-inflammatory to a pro-inflammatory protein. We previously reported that oxidation can also promote apoA-I aggregation and formation of amyloid fibrils. In this study, we investigated the mechanistic interplay between oxidation, amyloid formation and the inflammatory response of macrophages to apoA-I. We hypothesized that amyloid fibrils constituted of oxidized apoA-I activate production of inflammatory cytokines in macrophages. To test this hypothesis, amyloidogenic apoA-I was generated by oxidation with an excess of H
2
O
2
(H
2
O
2
-ApoA-I). Intracellular and secreted levels of IL-1β were determined upon incubation of mouse bone marrow derived macrophages (BMDM) with intact-apoA-I, soluble H
2
O
2
-ApoA-I and pre-formed H
2
O
2
-ApoA-I amyloid fibrils. Cellular cholesterol release from RAW264.7 cells was also measured. Soluble H
2
O
2
-ApoA-I (amyloid precursor) retained the cellular cholesterol release capacity of intact-ApoA-I. In BMDM incubated with soluble H
2
O
2
-ApoA-I however, levels of IL-1β synthesis and secretion were at least 2-fold higher than those induced by intact-ApoA-I. In contrast, incubation with H
2
O
2
-ApoA-I amyloid fibrils did not increase the levels of IL-1β synthesis and secretion, compared to intact-ApoA-I. Thus, soluble and functional oxidized apoA-I activates inflammatory cytokine synthesis and secretion in macrophages. Notably, this pro-inflammatory potential was completely neutralized when oxidized apoA-I was aggregated in amyloids. Therefore in atherosclerotic lesions, amyloid formation could reduce, rather than exacerbate, the inflammatory burden produced by pro-inflammatory soluble oxidized apoA-I species.
Abstract 553: The Matricellular Protein Ccn1 is a Key Mediator of Smooth Muscle Cell Migration and Neointimal Formation
Authors: Cui Mei-Zhen , Hao Feng , An Dong , Zhang Fuqiang , Xu Xuemin
Published: June 2021

Platelet-derived growth factor (PDGF), a potent chemoattractant, induces smooth muscle cell (SMC) migration via the MAPK and PI3K/Akt pathways. However, the downstream mediators are still elusive. In particular, the role of extracellular mediators is largely unknown. In this study, we identified the matricellular protein CCN1, which is
de novo
synthesized in response to PDGF stimulation, as the key downstream mediator of the ERK and JNK pathways, independent of the p38 MAPK and AKT pathways, and, thereby, it mediates PDGF-induced SMC migration. Our results revealed that, when CCN1 was newly synthesized by PDGF, it was promptly translocated to the extracellular matrix and physically interacted with the plasma membrane integrins α6β1 and αvβ3. We further demonstrate that CCN1 and integrins are integral components of the PDGF signaling pathway via an “outside-in” signaling route to activate intracellular focal adhesion kinase (FAK), leading to SMC migration. Therefore, our study provides new evidence that the PDGF-induced endogenous extracellular matrix component CCN1 is a key mediator in modulating SMC migration by connecting intracellular PDGF-ERK and JNK signals with integrin/FAK signaling. Therefore, extracellular CCN1 convergence with growth factor signaling and integrin/FAK signaling is a new concept of growth factor-induced cell migration. We further identified that CCN1 is a key mediator of lysophosphatidic acid (LPA)-mediated SMC migration. Our recent data reveal that CCN1 mediates LPA-induced neointimal formation. The discovered novel signaling pathway may represent an important therapeutic target in growth factor/lipid-mediated cell migration related vascular diseases.
Abstract 560: HDAC3 Unconventional Splicing Mediates Endothelial-mesenchymal Transition in Cardiac Fibrosis
Authors: Lao Ka Hou , Ummarino Dario , Shah Ajay , Zeng Lingfang
Published: June 2021

Introduction:
Endothelial-mesenchymal transition (EndMT) makes a significant contribution to cardiac fibrosis. Histone deacetylase 3 (HDAC3) is essential in the maintenance of endothelial homeostasis. Our previous study indicates that mouse HDAC3 mRNA can undergo unconventional splicing, and that the HDAC3α spliced isoform promotes the EndMT process in human aortic endothelial cells (HAECs). This work aims to elucidate the role of HDAC3α in mediating EndMT and its part in the underlying mechanisms behind the development of pressure-overload-induced cardiac fibrosis.

Methods and Results:
Our previous study reveals that HDAC3α promotes EndMT in HAECs via the activation of transforming growth factor-beta 2, possibly through increased protease enzyme activity. HDAC3α was therefore overexpressed in HAECs via adenoviral gene transfer (Ad-HDAC3α). Protease array revealed that cells infected with Ad-HDAC3α have an elevated level of a disintegrin and metalloproteinase with thrombospondin motifs 1 (ADAMTS1), and this was further confirmed with increased mRNA and protein expression levels seen in Ad-HDAC3α-infected HAECs using RT-qPCR and Western blotting analysis respectively (both n = 4,
P
< 0.05). A mouse model of pressure overload-induced cardiac fibrosis was established by transverse aortic constriction (TAC), and cardiac fibrosis was confirmed by increased Col1a1 expression seen using RT-qPCR and picosinus red staining in heart tissue sections from mice 7 d post-TAC. Immunofluorescent staining detected CD31
+
/α-SMA
+
(indicative of EndMT) and HDAC3α
+
/CD31
+
cells in heart tissues from TAC mice but not in those from sham-operated mice. There were significant increases in mRNA expression of HDAC3α and ADAMTS1, together with EndMT transcription factors Snai1, Snai2 and Twist1 in heart tissues from 7 d post-TAC mice compared to those from 7 d post-sham mice (n = 7- 8,
P
< 0.05). Elevated HDAC3α and ADAMTS1 protein expression were also detected in the TAC heart tissues (n = 5,
P
< 0.05).

Conclusion:
These results indicate an association between the HDAC3 splicing isoform HDAC3α, ADAMTS1 and EndMT during TAC-induced cardiac fibrosis. Further investigation of the underlying mechanisms mediated by HDAC3α and ADAMTS1 in cardiac fibrosis is warranted.
Abstract 565: Vascular Endothelial Growth Factor - An Important Factor in HDL Binding and Transcytosis Through Aortic Endothelial Cells
Authors: Rohrer Lucia , Velagapudi Srividya , von Eckardstein Arnold
Published: June 2021

In the reverse cholesterol transport pathway cholesterol acceptors like high density lipoproteins (HDL) and apolipoprotein A-I (apoA-I), must cross the endothelium to get access to the donor cells in the arterial initima. However, it is unknown how they get translocated from the blood stream into the vascular wall. Previously, we showed that transendothelial HDL transport is modulated by scavenger receptor B I (SR-BI), ATP binding cassette transporter G1, endothelial lipase (EL), and the ectopic beta-ATPase/purinergic receptor axis. None of these proteins is a good candidate to directly mediate holoparticle uptake. They appear rather to indirectly modulate transendothelial transport of HDL by signalling or altering the structure of HDL. To unravel HDL transcytosis, a high-throughput screening was performed, incubating aortic endothelial cells (ECs) with kinase inhibiting drugs and fluorescent labelled HDL. Fixed cells were imaged using fluorescence wide-field microscope. Processing of microscopy acquired data using cell profiler and principle component analysis yielded, only inhibitors of the vascular endothelial growth factor receptor (VEGFR) and its down-stream signaling kinases. To verify and further characterize the screening hits towards their impact on binding, internalization and transport of 125I-HDL, we applied RNA interference and pharmacological means to ECs. Binding, internalization and transport of 125I-HDL through ECs were increased by VEGF treatment but decreased by inhibitors of VEGFR2 as well as knock-down of VEGFR2. RNA interference with VEGFR1 or VEGFR3 had no effect. The stimulatory effect of VEGF on 125I-HDL binding and internalization was decreased by knocking-down SR-BI or PDZK1. The identification of VEGF as a regulatory factor of transendothelial transport supports the concept that the endothelium is a specific and hence druggable barrier for the entry of HDL into the vascular wall.
Abstract 566: Functional Analysis of Inducible Pluripotent Stem Cell-derived Vascular Endothelial Cells From Plasminogen Activator Inhibitor-1 Deficient Patients
Authors: Sano Hideto , Suzuki Yuko , Tanaka Hiroki , Iwaki Takayuki , Urano Tetsumei
Published: June 2021

Background:
Plasminogen Activator inhibitor-1 (PAI-1) is the principle regulator of PA system and its enhanced expression levels are related to many pathological conditions including thrombosis and malignant tumors. We have identified two distinct PAI-1 deficient patients having apparent phenotypes of severe bleeding and impaired wound healing, both of which are not seen in the PAI-1 deficient mice. With employing inducible Pluripotent Stem (iPS) cells established from the patients, we investigated the intrinsic function of PAI-1 in endothelial cells.

Methods:
iPS cells, generated from PAI-1 deficient patients and control subject, were differentiated to endothelial cells. Endothelial cells were isolated by magnetic sorting with anti-VEGFR-2 antibody, and their functions and the related gene expressions were analyzed.

Results:
(1) Endothelial cells derived from PAI-1 deficient iPS cells (PAI-1 iPS-ECs) detached more easily from gelatin-coated dishes. (2) PAI-1 iPS-ECs migrated faster in migration assay. (3) Tube formation activities of PAI-1 iPS-ECs, especially vessel branching, were impaired compared with control iPS-ECs. (4) The expression of Dll4 gene, known to be mainly observed at the region of angiogenic-edge, was reduced in PAI-1 iPS-ECs.

Conclusion:
PAI-1 appeared to play important roles in the function of endothelial cells and to contribute to angiogenesis.
Abstract 571: Non-reversed Increase In Shear Stress Magnitude Enhances Global Endothelial Cell Dna Methylation And Limits Extent of Collateral Artery Growth Following Femoral Arterial Ligation
Authors: Heuslein Joshua L , Price Richard J
Published: June 2021

Femoral artery ligation (FAL) in the mouse leads to increased flow across all collateral arteries with some collateral segments experiencing increased flow and reversed flow direction. We have recently shown that these “reversed flow” segments exhibit remarkably permanent, amplified arteriogenesis compared to those exposed to “non-reversed” flow. As the role of epigenetics in arteriogenesis remains unknown, we sought to determine the effect of biomimetic shear stress waveforms on DNA methylation and DNA methyltransferase 1 (DNMT1) expression in endothelial cells. Next generation sequencing of bisulfite converted genomic DNA was used to generate global DNA methylation profiles of ECs exposed to shear stress waveforms biomimetic of our “reversed” and “non-reversed” conditions. We found that the “non-reversed” waveform increased gene promoter hypermethylation in-vitro. Furthermore, DNMT1 mRNA expression was increased 1.25-fold in non-reversed conditions compared to reversed conditions as determined by RT-PCR (p≤0.05, n=8). We then tested the hypothesis that increased DNMT1 expression and subsequent DNA hypermethylation, elicited by a “non-reversed” flow waveform, limits steady state, long-term arteriogenic capacity in-vivo. Whole mount vascular casting was used to determine the lumenal diameter of both reversed and non-reversed collateral artery segments from mice treated with 5-AZA, a preferential inhibitor of DNMT1, or vehicle control 14 days post-FAL for an additional 14 days. We found that lumenal diameter was significantly enhanced in non-reversed collateral segments of 5-AZA treated mice (70.9±7.9μm vs. 50.1±3.4μm, p≤0.05, n=6) while there was no difference in reversed segments. Together, we conclude that a non-reversed, increased shear stress magnitude results in a rapid increase DNMT1 expression and DNA hypermethylation, limiting the extent of arteriogenesis in non-reversed collateral artery segments of the mouse gracilis muscle. However, inhibition of DNMT1 with 5-AZA enables for enhanced collateral growth. Further interrogation of the central relationships between hemodynamics, epigenetics, and collateral artery growth may lead to durable therapeutic options for arterial occlusive diseases.
Abstract 572: Inhibition of MicroRNA-199a-5p Enhances Perfusion Recovery and Arteriogenesis Following Femoral Arterial Ligation
Authors: Heuslein Joshua L , Price Richard J
Published: June 2021

We have recently reported that a subset of collateral artery segments in the mouse hindlimb are exposed to both a 2-fold increase in shear stress magnitude and reversed flow direction (“reversed” flow) following femoral arterial ligation (FAL). These segments experiencing a reversed flow subsequently undergo a disproportionate amplification of collateral growth (i.e. arteriogenesis) after FAL. Interestingly, a subset of microRNAs, including miR-199a-5p, are significantly down-regulated in endothelial cells exposed to a shear stress waveform biomimetic of this pro-arteriogenic “reversed flow” condition in-vitro. Here, we hypothesized that the inhibition of miR-199a-5p would lead to enhanced arteriogenesis and/or perfusion recovery following arterial occlusion. To test this hypothesis, Balb/c mice were treated with either anti-miR-199a-5p or non-targeting control locked-nucleic acid (LNA) immediately following FAL. Treatment efficacy was determined via RT-PCR for expression of miR-199a-5p and its downstream targets’ expression. Laser Doppler perfusion imaging and both whole mount and cross-sectional analysis of vascular casted gracilis muscles were used to assess vascular remodeling. We found that anti-miR-199a-5p LNA markedly reduced miR-199a-5p expression in the gracilis muscle by over 80%, 7 days post-FAL (n=4). By day 21 post-FAL, perfusion recovery was significantly improved in anti-miR-199a-5p treated mice (101.5±1.8% vs. 87.8±1.8%, p≤0.05, n=6). Moreover, both lumenal diameter (106.5±7.2μm vs. 78.4±2.9 μm, p≤0.0001, n=6) and wall area (1770±147μm
2
vs. 1163±41μm
2
, p≤0.0001, n=6) were significantly enhanced in anti-miR-199a-5p treated mice compared to controls. We therefore conclude that the inhibition of miR-199a-5p leads to enhanced perfusion recovery and arteriogenesis following arterial occlusion and could represent a novel strategy for therapeutic arteriogenesis.
Abstract 582: Overexpression of Catalase Impairs Aortic Valve Function and Accelerates Valvular Calification in Mice
Authors: Fermoyle Caitlin C , Roos Carolyn M , Casaclang-Verzosa Grace , Zhang Bin , Miller Jordan D
Published: June 2021

Previous studies have reported increases in oxidative stress due to reductions in antioxidant enzymes in calcified regions of stenotic human aortic valves. Whether reductions in antioxidant enzymes are adaptive, maladaptive, or an epiphenomenon in calcifying aortic valves remains unclear. Thus, the purpose of this study was to test whether overexpression of catalase reduces valvular calcification and slows progression of aortic valve stenosis in mice. Wild type (WT) mice with calcific aortic valve disease (ldlr-deficient, apoB100-only) were crossed with transgenic mice that overexpress human catalase (TG). All mice were fed a western diet (TD88137; Harlan Teklad). Functional echocardiogram data was collected and analyzed at early (3 months) and advanced (12 months) stages of valve disease, and valves were OCT embedded, sectioned, and stained for calcium levels (Alizarin Red). The transgenic valves show a slight increase in calcification compared to wild type counterparts. While there were no differences in valve function between genotypes at the early time point, cusp separation was decreased in the transgenic mice (0.74 ± 0.02 mm) compared to their wild-type littermates (0.84 ± 0.03 mm) at the 12 month time point (p < 0.05). In a subset of animals, valve calcification also tended to increase in transgenic mice at both time points compared to their wild-type littermates. In conclusion, overexpression of catalase resulted in a surprising finding of accelerated valvular calcification and stenosis. Interestingly, this finding aligns with the findings from several negative clinical trials testing antioxidant therapies in humans with atherosclerosis, suggests that the net impact of changing redox balance on disease progression are highly context dependent, and that increasing antioxidant capacity may not be a viable strategy to slow progression of calcific aortic valve disease in humans.
Abstract 591: Development of Near-infrared Fluorescent Probe for Targeted Uptake by Atherosclerotic Plaque Macrophages
Authors: Maess Marten , Shimizu Kosuke , Narita Yudai , Oku Naoto , Magata Yasuhiro , Ogawa Mikako
Published: June 2021

Macrophages are a pivotal element in the development of atherosclerotic plaques and their polarization strongly affects plaque stability. Development of molecular imaging techniques to identify vulnerable plaques in atherosclerotic vessel walls will be a valuable diagnostic tool to assess patients` risk of myocardial infarction or stroke and to adjust therapy. Imaging of unstable plaques has so far been performed by using PET imaging agent [
18
F] FDG. Fluorescence imaging using near-infrared fluorescent agents offers several advantages, as they are simple to use and minimally invasive. We have developed an activable near-infrared fluorescent probe using either peptide-linked ICG dye (indocyanine green) or free ICG as cargo in liposomes. The liposomes were labeled with phosphatidyl serine in order to trigger phagocytic uptake by macrophages. We have investigated the properties of our liposome probes in order to assess their suitability for screening of atherosclerosis risk patients in a wide array of different experimental models including cell culture and various animals (apoE
-/-
mice, WHHL rabbits and
macaca fascicularis
monkeys). Cell culture experiments using the human monocytic cell line THP-1 after differentiation into macrophages using the phorbol ester PMA and polarization of the macrophages by either LPS/IFNγ for M1 polarization or IL-4/ IL-13 for M2 polarization have shown considerable differences in uptake efficiency for the liposomes. M2 macrophages were found to show highest phagocytic activity while not polarized M0 were intermediate and M1 macrophages were least active. Polarization was confirmed by measuring known marker genes (TNFA, CCL3, MRC1) of macrophage polarization by RT-qPCR. In the animal studies we observed enrichment of ICG-labeled liposomes in atherosclerotic plaques of apoE
-/-
mice as well as WHHL rabbits.
Macaca fascicularis
experiments attempted to simulate final application in human subjects by detecting atherosclerotic plaques within the carotid aorta. Preliminary results indicate promising potential for liposome probes.
Abstract 601: Identifying Chylomicrons Using the Triglyceride/Total Cholesterol ratio (TG/TC) and non-Very Low-Density Lipoprotein Triglyceride (non-VLDL TG): The Very Large Database of Lipids Study (VLDL-7B)
Authors: Hendrani Aditya D , Quispe Renato , Martin Seth S , Kulkarni Krishnaji R , Toth Peter P , Jones Steven R , Joshi Parag H
Published: June 2021

Background:
Chylomicrons are triglyceride- (TG-) rich lipoproteins. Chylomicronemia is usually diagnosed on the basis of a very high TG level or an elevated TG/TC ratio.

Methods:
We examined 1,350,908 U.S. adults from the Very Large Database of Lipids whose lipids were analyzed by ultracentrifugation (VAP, Atherotech, Birmingham, AL). We estimated the TG content of very low-density lipoprotein (VLDL-TG) as the product of VLDL-C and a validated adjustable Friedewald-like factor. We estimated non-VLDL TG as total TG - (VLDL-TG). We examined the association of non-VLDL TG to TG/TC, proposing new diagnostic criteria for chylomicronemia based on non-VLDL TG and TG/TC. The intercept point was estimated through the intersection of the 95
th
percentile line for non-VLDL TG (32 mg/dL) by projecting a regression line fitted for those with non-VLDL TG ≥500 mg/dL, in which there is a visually linear relationship between Non-VLDL TG and Ln (TG/TC) (Figure).

Results:
Using a conventional threshold of TG/TC ≥10, there were 427 adults (0.03%) with chylomicron excess. TG and non-VLDL TG levels were 2836 (2074 to 4297) and 1814 (1201 to 2851) mg/dL at TG/TC ≥10, respectively. We identified a new threshold from the intercept point in the plot at approximately TG/TC = 5.2. Using TG/TC ≥5.2 and non-VLDL TG ≥32 mg/dL, 2139 (0.16%) adults were identified as having chylomicron excess. In these individuals, TG and non-VLDL TG levels were 1394 (1100 to 2014) and 515 (301 to 932) mg/dL, respectively.

Conclusion:
The threshold of TG/TC ≥5.2 with or without non-VLDL TG ≥32 mg/dL may better identify the chylomicronemic patient. If validated in future studies, these findings will improve diagnosis of chylomicronemia.
Abstract 61: Hedgehog-Responsive Stem Cell Antigen 1 Positive Cells Contribute To Vascular Smooth Muscle Cell Accumulation Following Vascular Injury
Authors: Fitzpatrick Emma , Helt Jay-Christian , Hakimjavadi Roya , Cahill Paul A , Redmond Eileen M
Published: June 2021

Background:
Cardiovascular disease such as atherosclerosis is associated with the formation of an neointimal layer and medial hyperplasia as a result of the accumulation of vascular smooth muscle cells (vSMC) in the artery wall (i.e., intimal medial thickening, IMT). There is strong, albeit controversial, evidence indicating a putative role for stem cell antigen 1 positive (Sca1
+
) progenitor cell-derived vSMC in contributing to IMT. Interestingly, the Hedgehog (Hh) signaling receptor Patched 1 is present in the adventitial boundary and co-localizes with both Sca1 and Patched 1, the ligand for sonic hedgehog (SHh). The aim of our study was to determine the effect of Hh signaling on Sca1+ stem cells and their contribution to vSMC accumulation and IMT following vascular injury.

Methods:
Sca1
+
stem cells were treated with differentiation inductive stimuli in the absence or presence of recombinant SHh (rSHh), with or without the Hh inhibitor cyclopamine, before the cells were examined for their stemness (telomerase activity), differentiation capacity (SMC differentiation) and epigenetic profile. In parallel, Sca1-eGFP transgenic mice were subjected to carotid injury (i.e., partial ligation), with or without cyclopamine, before IMT, Sca1 expression and SMC phenotype was assessed.

Results:
Sonic Hedgehog stimulated Sca1
+
stem cell differentiation capacity, as demonstrated by enhanced expression of SMC differentiation markers in addition to their altered stemness, epigenetic profile (H3K4me2:H3K27me3) and proliferative status. In parallel studies, Hh signaling components were elevated in ligated carotids when compared to sham-operated controls, concomitant with increased Sca1 expression and SMC accumulation. Moreover, there was a significant attenuation of SMC accumulation and intimal medial thickening of injured carotids following Hh inhibition with cyclopamine.

Conclusion:
Sca1
+
stem cells are Hh-responsive and they contribute to vSMC accumulation in the carotid following ligation injury.
Abstract 614: TRAIL Deletion in Mice Promotes Vascular Insulin Resistance
Authors: Harith Hanis , Cartland Siân , Di Bartolo Belinda , Dang Lei , Thomas Shane , Kavurma Mary
Published: June 2021

TNF-related apoptosis-inducing ligand (TRAIL) is protective in cardiovascular disease and diabetes; whether it regulates insulin resistance and inflammation in the vessel wall is unknown. Wildtype and

Trail
-/-

mice were placed on a high fat diet (HFD) for 12 w. Plasma chemistries were assessed; glucose and insulin tolerance tests performed. Histological GLUT4 expression in skeletal muscle was assessed. Aortic inflammatory marker expression and downstream insulin signaling was also measured; myography was performed. In response to a HFD for 12 w, wildtype mice had increased aortic IL-1beta, MCP-1, IL-6, TNF-alpha and TRAIL mRNA, with reduced circulating TRAIL levels. Compared to 12 w HFD-fed wildtype mice,

Trail
-/-

mice had significantly increased plasma glucose, insulin, and cholesterol levels. Insulin intolerance was worse with TRAIL deletion, with reduced GLUT4 expression and glucose uptake in skeletal muscle. In response to insulin,

Trail
-/-

aortas displayed impaired vasorelaxation and reduced aortic p-Akt expression. Importantly, aortic insulin resistance was associated with > 20-fold increased aortic mRNA expression for IL-1beta, IL-6, and TNF-alpha. We show that TRAIL protects against insulin resistance and type-2 diabetes, in part by regulating downstream insulin signals and inflammation in the vessel wall. These findings suggest that TRAIL may offer a novel therapeutic solution to combat inflammation and insulin resistance in diabetic vascular diseases.
Abstract 616: MicroRNA miR-29b is a Mediator of Aortic Stiffness and Hypertension in a Murine Model of Type 2 Diabetes Mellitus
Authors: Raaz Uwe , Schellinger Isabel N , Maegdefessel Lars , Spin Joshua M , Hasenfuss Gerd , Tsao Philip S
Published: June 2021

Background:
Accelerated arterial stiffening is a complication of diabetes mellitus and associated with the development of hypertension. Arterial stiffening results from extensive extracellular matrix remodeling (elastin breakdown, collagen accumulation). MicroRNA miR-29b directly regulates the expression of genes governing fibrosis (such as
COL1A1, COL3A1)
and elastin breakdown (
MMP2, MMP9
). However, its impact on aortic stiffness is unclear.

Objective:
This study was designed to investigate the role of miR-29b as potential mediator of diabetic aortic stiffening.

Methods and Results:
Serial
ex vivo
mechanical testing of the thoracic aorta and volume-pressure recording (VPR) based tail-cuff blood pressure measurements revealed that aortic stiffening precedes blood (pulse) pressure elevations in diabetic db/db mice. Vascular stiffening was accompanied by increased elastin fragmentation and collagen deposition (EvG and Picrosirius Red staining). qRT-PCR, in-situ hybridization and immunohistochemistry revealed decreased expression of miR-29b and de-repression of target genes (
Col1A1, COL3A1, MMP2, MMP9
) in db/db mice compared to controls. Investigating the mechanistic significance of miR-29b for arterial stiffening, forced downregulation of miR-29b (via systemic LNA-miR-29b inhibitor application) results in enhanced elastin fragmentation, increased medial collagen deposition, aortic stiffness and augmented pulse pressure.

Conclusions:
In conclusion this study identifies miR-29b as a regulator and potential therapeutic target of diabetic aortic stiffening.
Abstract 620: Mutations in
FLT1
and
COL4A1
Protect Against Early Stage Atherosclerosis in Overfed Zebrafish Larvae
Authors: den Hoed Marcel , Conrad Lisa , Bandaru Manoj , Emmanouilidou Anastasia , Ranefall Petter , Wählby Carolina , Larsson Anders , Ingelsson Erik
Published: June 2021

Genome-wide association studies (GWAS) identified 56 loci that are robustly associated with the risk of coronary artery disease (CAD). Only a handful of genes in these loci are currently known to influence CAD. We previously showed that: 1) CAD-associated loci exert their effect at least in part by influencing atherosclerotic plaque formation; 2) zebrafish larvae are a promising model system for high-throughput screens of early-stage atherosclerosis. We now aim to identify causal genes in GWAS-identified loci for CAD using a zebrafish model system. Heterozygous carriers of nonsense (sa1516 in
CXCL12
; fh336 in
COL4A1
) or essential splice site (sa1504 in
FLT1
) mutations were in-crossed, and offspring were fed a normal or high amount of diet from 5 to 10 days post-fertilisation. Vascular lipid deposition was subsequently visualised using a fluorescent dye, automated positioning system, fluorescence microscope and CCD camera, and quantified using a custom-written image analysis pipeline in Ilastik and CellProfiler. After imaging, whole-body total cholesterol, triglyceride and glucose levels were assessed using enzymatic assays, and larvae were genotyped using KASP technology. Multiple linear regression analysis (additive model) was used to examine the effect of each mutation. Mutations in
FLT1
and
COL4A1
resulted in less vascular lipid deposition in overfed larvae (p=3.6E-2 and p=1.9E-2, respectively), but had no effect in larvae fed on a normal amount (p=0.26, p=0.64). Sa1504 in
FLT1
additionally resulted in lower total cholesterol levels (p=3.7E-3) and a trend for lower triglyceride levels (p=0.06), independently of the amount fed. The remaining associations did not reach nominal significance (p>0.05). Our results represent the first mutant models demonstrating that
FLT1
and
COL4A1
play a role in early-stage atherosclerosis, implying that these genes are the culprits in corresponding GWAS-identified loci. We believe that
CXCL12
remains the most promising candidate in 11q11.1, and that the lack of effect in our screen reflects genetic redundancy and/or a late onset effect.

We will repeat the screen to confirm or refute the results, and additionally examine if mutations in
FLT1
and
COL4A1
influence vascular inflammation and foam cell formation.
Abstract 622: High-density Lipoprotein Cholesterol Efflux Does Not Predict Cardiovascular Risk in Hemodialysis Patients
Authors: Ebtehaj Sanam , Kopecky Chantal , Genser Bernd , Drechsler Christiane , Krane Vera , Antlanger Marlies , J. Kovarik Johannes , C. Kaltenecker Christopher , Parvizi Mojtaba , Wanner Christoph , Weichhart Thomas , Säemann Marcus D , Tietge Uwe J
Published: June 2021

The cardioprotective effect of HDL (High-Density Lipoprotein) is largely determined by its cholesterol efflux capacity, which was shown to correlate inversely with atherosclerotic cardiovascular disease in populations with normal kidney function. Patients with end-stage renal disease suffer an exceptionally high cardiovascular risk not fully explained by traditional risk factors. Here, we investigated in a
post-hoc
analysis in 1147 patients with type 2 diabetes mellitus on hemodialysis participating in the 4D Study (The German Diabetes Dialysis Study), if the HDL cholesterol efflux capacity is predictive of cardiovascular risk. Efflux capacity was quantified by incubating human macrophage foam cells with apolipoprotein B-depleted serum. During a median follow-up of 4.1 years n=423 patients reached the combined primary endpoint (composite of cardiac death, nonfatal myocardial infarction and stroke), n=410 experienced cardiac events and n=561 died (all-cause mortality). Strikingly, in Cox regression analysis we found no association of efflux capacity with the combined primary endpoint (hazard ratio [HR], 0.96; 95% confidence interval [CI], 0.88 – 1.06,
p
=0.417), cardiac events (HR, 0.92; CI, 0.83-1.02;
p
=0.108) or all-cause mortality (HR 0.96; 95% CI, 0.88-1.05;
p
=0.390). In conclusion, HDL cholesterol efflux capacity is not a prognostic cardiovascular risk marker in diabetic patients on hemodialysis.