bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 306: Atheroprone Hemodynamics Regulate Smooth Muscle Cell Phenotype Through Endothelial PECAM and Fibronectin
Authors: Boerboom Lauren M , Pryor Andrew W , Blackman Brett R
Published: March 2022

Introduction:
Atherosclerosis is an inflammatory disease that develops preferentially in regions of disturbed hemodynamic shear stress. The extracellular matrix protein fibronectin (FN) is deposited in the sub-endothelial layer of pre-atherosclerotic and advanced lesions. Atheroprone shear stress promotes FN deposition and inflammatory signaling pathways in endothelial cells (ECs). Platelet endothelial cell adhesion molecule (PECAM), a mechanosensory protein, is necessary for the production, secretion, and assembly of FN matrix by ECs. Similar to ECs, vascular smooth muscle cells (SMCs) also display a pro-inflammatory phenotype in regions of atherogenesis, and this phenotype is key to the progression of atherosclerosis.

Hypothesis:
We hypothesize that endothelial PECAM and FN signaling promotes a pro-inflammatory smooth muscle cell phenotype in response to atheroprone shear stress patterns.

Methods:
An
in vitro
cone-and-plate viscometer model was used to apply human-derived atheroprone or atheroprotective shear stress patterns to human umbilical vein ECs co-cultured with underlying human umbilical vein SMCs.

Results:
Atheroprone flow increased SMC NFkB activity compared to atheroprotective flow (1.94±0.35 fold, p<0.05). This increase of SMC NFkB activity under atheroprone flow was attenuated by siRNA knockdown of either endothelial PECAM (0.56±0.08 fold, p<0.01) or FN (0.50±0.10 fold, p<0.01). Endothelial PECAM knockdown also reduced genes marking the SMC inflammatory phenotype, including smooth muscle alpha actin, vascular cell adhesion molecule 1, and cyclooxygenase-2.

Conclusion:
These results suggest that SMCs switch to a pro-inflammatory phenotype when overlying ECs are exposed to atheroprone shear stress, and that PECAM and FN signaling in the ECs regulate this phenotype.
Abstract 33: β-Catenin Is Essential for Vascular Smooth Muscle Cell Survival and Artery Formation
Authors: Riascos Bernal Dario F , Chinnasamy Prameladevi , Sibinga Nicholas
Published: March 2022

Background:
The molecular mechanisms required for formation of a competent vessel wall are not fully understood. Although β-catenin (β-ctn) has an essential developmental function in various cell types, its specific contributions to vascular smooth muscle cell (SMC) biology have not been fully determined. In preliminary studies, we found that SMC-selective β-ctn inactivation (KO) in mice was incompatible with postnatal survival.

Hypothesis:
β-ctn is a key modulator of vascular SMC function during development.

Methods:
We analyzed mouse embryos from stages E9.5-19.5. β-ctn expression and endothelial and SMC markers were tested by immunohistochemistry. Proliferation and apoptosis were assessed by phospho-histone 3 and TUNEL assays, respectively. In studies of cultured SMCs lacking β-ctn, we followed cell population growth and assessed proliferation and cell cycle by BrdU uptake and FACS analysis. Cell death was induced by serum starvation, hydrogen peroxide, or agonistic anti-Fas antibody, and assessed by LIVE/DEAD assay or FACS.

Results:
β-ctn inactivation in SMCs caused embryonic lethality by E12.5. Paired and fused dorsal aortae in KO embryos had thinner walls (wall/lumen area ratio 0.38 vs 0.18 in control vs. KO at E11.5, p<0.001, n=4). KO vessels showed intact endothelial layers, negligible β-ctn expression, few SMCs, slightly reduced cell proliferation, and more apoptosis (0.30% vs 1.24% in control vs. KO, p<0.05, n=11). By E12.5, KO vessels were severely disrupted, allowing extravasation. Cultured SMCs lacking β-ctn grew more slowly, plateaued at lower cell density than control (p<0.0001, n=3), and showed decreased cell cycle progression and more death upon serum starvation (12.0% vs. 21.8% in control vs. KO, p=0.052, n=3). Interestingly, cell death induction by other stimuli (oxidative stress or Fas ligand activation) had the opposite effect, with loss of β-ctn conferring protection (p<0.01, n=3).

Conclusion:
β-ctn is essential for SMC survival and formation of a competent arterial vessel wall during development, but its survival function in SMCs appears to be stimulus-specific. β-ctn may oppose or promote SMC death depending on specific cellular and environmental conditions and stimuli.
Abstract 331: Angiotensinogen Inhibition by Antisense Oligonucleotides Decreases Atherosclerosis and Obesity in a Dose-Dependent Manner
Authors: Balakrishnan Anju , Howatt Deborah A , Rateri Debra L , Graham Mark J , Mullick Adam E , Crooke Rosanne M , Cassis Lisa A , Daugherty Alan
Published: March 2022

Background and Objective:
The renin-angiotensin system (RAS) exerts profound effects on experimental atherosclerosis. While there is evolving evidence of the complexity of the RAS, angiotensinogen (Agt) remains the single precursor for all known angiotensin peptides. The aim of this study was to determine the dose-dependent effects of inhibiting Agt synthesis on atherosclerosis.

Methods and results:
Male LDL receptor
-/-
mice were fed a saturated fat-enriched diet (21% wt/wt milk fat; 0.2% wt/wt cholesterol) for 3 months. Mice were injected weekly i.p. with either an antisense oligonucleotide (ASO) to Agt (ISIS #261333 at 50 mg/kg/week or #487022 at 12.5, 25 and 50 mg/kg/week) or a control ASO (ISIS #141923 at 50 mg/kg/week; n=10 mice per group). Administration of Agt ASO led to ∼90% reduction in plasma Agt concentrations (P<0.001) and a marked reduction in hepatic Agt mRNA abundance (P<0.05). There was also an expected dose-dependent increase in plasma renin concentrations (P<0.001). Agt ASO administration also decreased systolic blood pressure (SBP) in a dose-dependent manner (P<0.05). Inhibition of Agt significantly decreased total plasma cholesterol concentrations in the two highest dose Agt ASO groups. Atherosclerotic lesion areas were markedly decreased by Agt ASO administration in highest dose groups when compared to the PBS group (16.9 ± 1.0 versus 6.0 ± 1.3 and 6.2 ± 1.1 % of intimal area; P<0.001). Unexpectedly, Agt ASO administration also markedly attenuated body weight gain. MRI analyses demonstrated a dose-dependent reduction in fat mass (P<0.001) with no effect on lean mass. Adipose (retroperitoneal, inguinal and subcutaneous) tissue weights were decreased in a dose-dependent manner.

Conclusions:
Inhibition of Agt synthesis markedly decreases SBP, development of atherosclerosis and body weight gain in a dose-dependent manner.
Abstract 334: Circadian Clock Influences in Atherosclerosis Are Revealed Using a Transplant Model of Accelerated Atherogenesis
Authors: Cheng Bo , Rudic Dan
Published: March 2022

The circadian clock within blood vessels is an important influence in flow induced vascular remodeling and transplant arteriosclerosis. Much less is known regarding the role of the circadian clock in high cholesterol induced-atherosclerosis. To assess atherogenesis, we implemented a novel transplant model approach, previously tested in wild-type and circadian clock mutant recipient mice, but herein using recipient mice that were hypercholesterolemic_the apoE knockout mice. Using these recipient mice, syngeneic heterotopic transplantation of segments of thoracic aortas (all were lesion free in naïve conditions) from donor mice that were wild-type, mutated in the Bmal1 (Bmal1-KO) gene, or mutated in the apoE gene (apoE-KO) were grafted into the neck circulation (common carotid artery) of the apoE knockout mice. After 4 weeks of transplantation, a significant lipid rich lesion that stained positive with oil red-o was observed in the Bmal1-KO grafts that were placed in apoE knockout mice, while naïve Bmal1-KO aorta exhibited no oil-red positive lesion. Suprisingly, there were no substantial lesion in WT or apoE-KO aortic grafts placed in the apoE-KO recipient mice. These data suggest that the circadian clock may influence the progression of atherosclerosis.
Abstract 335: Allelic Imbalance at the Coronary Heart Disease Associated Locus 6q23.2 Identifies Causal Variation in the TCF21 Gene
Authors: Diaz-Caceres Roxanne , Leeper Nicholas , Raiesdana Azad , Anderson Ryan , Miller Clint , Quertermous Thomas
Published: March 2022

Recent genome-wide association studies have identified novel loci associated with coronary heart disease (CHD). One locus identified through the large-scale Coronary ARtery DIsease Genome wide Replication And Meta-analysis (CARDIoGRAM) study is located on chromosome 6 at q23.2, within the locus encoding the basic helix-loop-helix (bHLH) transcription factor TCF21. This class of transcription factors is well known to regulate cell fate decisions that are critical for embryonic developmental and disease-related pathways, and TCF21 expression has been shown to mark progenitor cells that give rise to the coronary circulation. Six of the most highly associated SNPs in the CARDIoGRAM study are correlated expression quantitative trait loci (eQTL) for TCF21, identifying this as the causal gene. To enable further study of the mechanisms by which allelic variation at 6q23.2 regulates TCF21 expression, we have pursued studies to identify which of the CHD-associated SNPs is causal. The best approach is to investigate allelic-expression imbalance, to compare the relative levels of expression of the two alleles in subjects heterozygous for SNPs in the coding region and then relating any observed imbalance to the genotype at associated SNPs. Peripheral blood samples from the GenePAD study have been used for the isolation of both genomic DNA and white blood cell total RNA, and allelic imbalance determined by Taqman genotyping assays at rs12190287. We identified a significant difference in allele expression in 13 heterozygous Caucasian subjects, with a normalized mean Taqman CT value from the disease major allele (C) to be 2.35, and 1.94 for the minor protective allele (G), P <0.0001. These data are consistent with disease association related to decreased TCF21 expression, and are consistent with rs12190287 being the disease-associated variation in this locus.
Abstract 352: Unhealthy Diet and Air Pollution Induce Cardiovascular Dysfunction
Authors: Buechner Nicole , Altschmied Joachim , Ale-Agha Niloofar , Sydlik Ulrich , Jakob Sascha , Kunze Kerstin , Unfried Klaus , Haendeler Judith
Published: March 2022

Cardiovascular diseases remain the dominant cause of deaths worldwide. Unhealthy diet and air pollution have each been described as risk factors for cardiovascular diseases. The molecular mechanisms involved in translating unhealthy diet and/or exposure to air pollution into cardiovascular dysfunctions remain yet to be identified.

First, we investigated the impact of an unhealthy diet on disease-related signaling pathways in human primary cardiovascular cells. Based on nutrition health reports on diet composition in industrialized countries, we supplemented cell culture media of human primary endothelial cells (EC), smooth muscle cells (SMC) and cardiomyocytes (CM) with 100 mg/dl LDL and replaced 1/3 of the glucose with fructose. This treatment did not induce cell death in any of the cell types. However, we observed hypertrophy in CM, enhanced proliferation in SMC and increased senescence, loss of endothelial nitric oxide synthase (eNOS) and increased nuclear localization FoxO3A in EC.

Based on several studies, which demonstrated that ultrafine particles can enter the circulation and thus directly interact with EC, we investigated the impact of ultrafine carbon black particles (ufCB), one of the major constituents of industrial and exhaust emissions, on human primary EC. Treatment with ufCB in non-toxic concentrations increased reactive oxygen species and dramatically reduced the S-NO content, a marker for NO-bioavailability. NO has been shown to increase the activation of Telomerase Reverse Transcriptase (TERT), which is required for proper endothelial cell function and is inactivated by the Src kinase under conditions of oxidative stress. ufCB significantly reduced Telomerase activity while increasing Src kinase activity. Subsequently, ufCB dramatically increased senescence of EC. To investigate whether ufCB effects also occur in vivo, we instilled ufCB in non-inflammatory concentrations into mice. Indeed, eNOS expression was reduced in the abdominal aorta of animals treated with ufCB.

Taken together, common unhealthy diet as well as low-level air pollution, which we are exposed to permanently, seem to accelerate cardiovascular diseases.
Abstract 354: Improved Collagen Imaging in Large Pulsatile Vessels Using High-Resolution Second Harmonic Generation Microscopy and Retrospective Electrocardiogram Gating
Authors: Kessinger Chase W , Lin Charles P , Jaffer Farouc A
Published: March 2022

Introduction:
Intravital microscopy (IVM) can provide important molecular and structural detail of the arterial wall in health and disease. However, in vivo image accuracy and resolution can be limited due to the pulsatile nature of the artery itself. Here, we developed and implemented a retrospective electrocardiogram (ECG) gating-based strategy to eliminate cardiac cycle dependent motion to improve IVM of the arterial wall.

Methods:
C57Bl/6 mice (n=5) were anesthetized and the right carotid artery was exposed and suffused with PBS. Second harmonic generation (SHG) micrographs of type 1 collagen in the arterial wall were collected (550m x 550m) at 30 frames/s on a custom-built multichannel microscope. Intravenously injected FITC-dextran (MW 2000kDa) provided an angiogram to identify the carotid artery. To enable gating, analog image capture and start signals were concurrently recorded with the ECG during image acquisition at a sampling rate of 100kHz. Gating of the imaging data set was compiled manually using the ADC software and ImageJ. Histopathology of the carotid confirmed the ex vivo carotid collagen thickness. Wall thickness was defined as the full width half maximum of SHG signal in each micrograph. Statistical significance was established as ANOVA P values < 0.05.

Results:
Retrospective ECG gating yielded images with a mean collagen wall thickness that was not significantly different than ex vivo measured samples (p>0.05), thus verifying the accuracy of the retrospective gating protocol. All ungated images showed significant differences (1.4 to 6.5 fold) in the measured wall thickness compared across various heart rates and frame averages (p<0.05). SNR measurements of gated and ungated images showed ungated images needed >200f averages to reach the SNR of a 30f gated image, which in turn needed 8-24s (240-720 frames) of image acquisition time, depending on the heart rate.

Conclusion:
Retrospective ECG gating enhances IVM accuracy of pulsatile tissues, as demonstrated by improved in vivo quantification of carotid arterial wall collagen in living mice. This strategy can be readily extended to multiplexed SHG and fluorescence IVM approaches to study other vascular disease models in vivo.
Abstract 359: Investigation of the Protein Interactions of the Shear Responsive Protein, Palladin, and Its Presence in Endothelial-Derived Microparticles
Authors: Mcginn Ciaran , Fitzpatrick Paul , Chunxu Shan , Murphy Andrew , MacDonnell Brian , Dowling Paul , Cummins Philip , Murphy Ronan
Published: March 2022

Palladin, an actin filament associated protein, plays an important role in cell cytoskeletal regulation, with particular regards to the formation and stablilization of stress fibres. The protein is part of the myotilin-myopalladin-palladin family, but unlike myotilin and myopalladin, which only appear in striated muscle, Palladin has been observed in various cell types. The protein is encoded by the PallD gene, which through alternative splicing, results in Palladin existing in multiple different isoforms. While a number of these isoforms have been documented in cell types, it is the 90kDa isoform which is most ubiquitously expressed.

Physiological mechanotransduction such as cyclic strain or shear stress, experienced by the vasculature, has been shown to regulate expression of many genes that play an important role in the patho-physiology of cardiovascular disease (CVD), by altering cell fate and function. We have previously documented the presence of the 90kDa isoform of Palladin in Human Aortic Endothelial Cells (HAECs), along with the investigation of gene expression in response to strain and shear at different time points by utilising Western Blotting and Real Time PCR techniques.

It has been shown that gene expression increases at early time points of mechanic stimuli, followed by a reduction of expression after chronic exposure. We also observe the presence of Palladin in Endothelial Microparticles, released under inflammatory conditions. We hypothesize that, following the inflammatory response, the cell compensates for excess protein production through microparticle secretion.

Mass Spectrometric analysis of Immunoprecipited Palladin from HAECs appears to show an interaction with Drebrin (developmentally regulated brain protein), which itself binds to F-actin and is involved in cell migration. While Drebrin is mainly expressed in the brain, it has also been located in heart and muscle. We investigate here also the interactions between Palladin and Drebrin. It appears that Drebrin expression decreases as Palladin expression increases following shear stress, indicative that the two proteins compete with one another in binding to actin.
Abstract 367: PAR3 Negatively Regulates PAR4-Mediated Gq Signaling Without Affecting the G12/13 Pathway in Mouse Platelets
Authors: Arachiche Amal , de la Fuente María , Nieman Marvin
Published: March 2022

Protease activated receptors (PAR) are G-protein-coupled receptors (GPCR) that are activated by proteolytic cleavage of the N-terminus. Mouse platelets use PAR3 and PAR4 to respond to thrombin. PAR3 itself does not signal, but it is a cofactor that enhances cleavage and activation of PAR4 by thrombin ∼ 10-fold. PAR4 is the signaling thrombin receptor and is sufficient to induce platelet activation at >10 nM thrombin. The aim of this study is to determine how PAR3 regulates PAR4 signaling in mouse platelets. In this study we show that platelets from PAR3 knockout mice (PAR3-/-) had a 1.6-fold increase in the maximum calcium mobilization at thrombin concentrations above 10 nM, compared to wild type platelets. The PAR3+/- had an intermediate increase (∼ 1.2-fold) in the maximum calcium mobilization. PAR3
-/-
platelets also had an increase in PKC activation, as measured by serine phosphorylation of PKC substrates in response to thrombin (10-100nM). The PAR4 agonist peptide (AYPGKF) also induced an increase in maximum calcium mobilization and PKC activation in PAR3
-/-
platelets, indicating that PAR3 may be influencing PAR4 at the level of the receptor. PAR4 is coupled to Gq and G12/13. PAR3 influences PAR4-mediated calcium mobilization and PKC activation which are both downstream of Gq. We next, determined the influence of PAR3 on PAR4 mediated G12/13 signaling by measuring the activation of small GTPase RhoA protein in response to thrombin (1-100nM). The level of RhoA activation measured was not different in PAR3
-/-
compared to wild-type platelets in response to thrombin. In conclusion, PAR3 negatively regulates PAR4 signaling through the Gq pathway without affecting the G12/13 pathway. Since the increase of Gq signaling is independent of PAR4 agonist, PAR3 is influencing PAR4 signaling at the level of the receptors, perhaps by forming heterodimers. The crosstalk between platelet GPCRs may be a general mechanism for regulating platelet signaling and uncovering the details of these interactions may lead to novel insights for developing therapeutics.
Abstract 369: Localization and Specification of a Kindlin-2 Binding Core Within Integrin Beta Cytoplasmic Tails
Authors: Bledzka Kamila , Liu Jiamin , Xu Zhen , Perera Dhanuja H , Yadav Satya P , Qin Jun , Ma Yan-Qing , Plow Edward F
Published: March 2022

Integrins are widely expressed cell adhesion receptors. One of the key regulatory mechanisms of integrins is their ability to undergo activation. Based on in vivo studies kindlins and talin are know to play indispensable roles in integrin activation, and this function depends on their binding to the cytoplasmic tail (CT) of integrin β subunits, However, the requisite sequences within the integrin β subunit that specify kindlin is as of yet unresolved.

Using overlapping synthetic peptides corresponding to the highly conserved C-terminal region of integrin β CT as inhibitors of β3 CT: kindlin-2 interaction in pull-down assays, we identified the kindlin-2 binding core to a twelve amino-acid sequence. This sequence includes the membrane-distal NxxY region and S/T cluster, which are conserved across integrin β CT, but also variable residues flanking these two motifs. We postulated that the contribution of these flanking residues in β subunits might impose selectivity upon the interaction of kindlin-2 with various β subunits. Indeed, we found that kindlin-2 interacts with β1and β3, but poorly with the β2 CT, in both pull-down and surface plasmon resonance experiments. This selectivity was determined primarily by the non-conserved residues at the extreme C-termini of the β CT. By swapping amino acids in this region, reactivity with kindlin-2 could either be enhanced or blunted. By surface plasmon resonance, we also demonstrated that the β3 subunits can accommodate both kindlin-2 and talin head, even though their binding sites are very close in the β3 subunit. Kindlin-2 and talin head do not interact with each other but can bind simultaneously to the integrin β3 CT without mutual disturbance. This work specifies a binding site within β CT for kindlin-2, provides the first evidence for selectivity in the interaction of kindlin-2 with integrin β subunits, and excludes a spatial influence of talin and kindlin-2 on their binding to integrin CT in supporting integrin activation.
Abstract 375: Regulation of Integrin Activation by Kindlin-3
Authors: Bialkowska Katarzyna , Ma Yan-Qing , Plow Edward F
Published: March 2022

The contributions of integrins to cellular adhesive responses depend upon the dynamic regulation of their activation states, which in turn depends on engagement of binding partners by their cytoplasmic tails. It is well-established that not only talin but also kindlin family members are essential for integrin activation, and both must present for optimal integrin activation. Recent studies in humans have specifically emphasized the vital role of kindlin-3 in integrin activation in hematopoietic cells, where kindlin-3 deficiency leads to episodic bleeding, frequent infections and osteopetrosis, consequences of an inability to activate β1, β2 and β3 integrins. Here, we used human erythroleukemia HEL cells to characterize integrin-activating kindlin-3 function and the kindlin-3-β3 integrin interaction in greater detail. Non-stimulated HEL cells are suspension cells, and they do not adhere to fibrinogen readily. Thrombopoetin or PMA stimulation activated β3-containing integrins, the cells adhere and spread on fibrinogen. β3 integrin and kindlin-3 colocalized in focal adhesions and there was enhanced β3 integrin-kindlin-3 association as detected by coimmunoprecipitation. Kindlin-3 knockdown impaired agonist-stimulated adhesion and spreading on fibrinogen. We expressed various β3 CT as chimeric constructs containing the extracellular domain of PSGL-1 and evaluated their effects on β3-mediated cell spreading on fibrinogen. Expression of the wild-type β3 CT chimera impaired β3-mediated cell spreading, and introduction of S752P and Y759A mutations within the kindlin binding core abolished this inhibitory activity. Thus, our data emphasizes a role of direct kindlin-3 and β3 integrin cytoplasmic tail interaction in kindlin-3 induced integrin activation in hematopoietic cells.
Abstract 378: Microvascular Endothelial Dysfunction and Age-Dependent Endothelial Activation in Mice with Fabry Disease
Authors: Kang Justin J , Shu Liming , Shayman James A , Bodary Peter F
Published: March 2022

Fabry disease, a lysosomal storage disorder, leads to an age-dependent accumulation of globotriaosylceramide (Gb3) in vascular tissue that ultimately causes cardiac disease and strokes. It has been demonstrated that this accumulation of Gb3 is consistent with a decrease in nitric oxide bioavailability and an impaired endothelial function in macrovessels including the aorta. Based on this, we hypothesized that microvessels would also have vascular dysfunction in older Fabry disease mice. To evaluate this, 8 month old wild-type (WT) and alpha-galactosidase A deficient (Fabry; FA) mice (n=4 / group) were euthanized, and mesenteric artery function was tested using a pressure myograph. Vessels from WT relaxed with Ach in a dose-dependent manner, while FA vessels were nearly devoid of Ach-mediated dilatation. The maximum relaxation with the highest dose of Ach (10^-4 M) was significantly different between groups (WT: 59 ± 11 %; FA: 1 ± 12 %; p=0.0004). However, endothelium-independent relaxation mediated by sodium nitroprusside was similar in vessels from both groups (WT: 81 ± 10 %; FA: 70 ± 9 %; p=0.15), confirming that the smooth muscle layer was intact and functional. Given the similar macro- and microvascular endothelial dysfunction evident in FA, we sought to identify serum measures of endothelial activation that could be used as a surrogate determinant of the age-dependent vascular dysfunction. We collected serum from age-matched WT and FA between 2 and 17 months of age (n=4-8 / group). We observed a significant age-dependent rise in FA for several serum markers of endothelial activation including soluble e-selectin, p-selectin and vascular cell adhesion molecule. However, von willebrand factor (vWF) provided the most sensitive age- and genotype-dependent changes between WT and FA (p=0.046 at 2 months and p=0.0004 at 17 months between groups).

In conclusion, the data provide evidence of microvascular endothelial dysfunction in FA, demonstrated by a prominent defect in the mesenteric artery at 8 months of age. In addition, a robust age-dependent rise in vWF observed in FA suggests that this marker of endothelial activation might serve as a sensitive surrogate measure of endothelial dysfunction in therapeutic studies of Fabry disease.
Abstract 386: 6-bomoindirubin-3’-oxime Induces Dedifferentiation of Endothelial Cells During Neovascularization
Authors: Kohler Erin E , Baruah Jugajyoti , Chatterjee Ishita , Wary Kishore K
Published: March 2022

RATIONALE:
Endothelial cell (EC) de-differentiation is a major challenge to understanding neovascularization and regenerative medicine. Rapid re-entry into the cell cycle, asymmetric cell division (ACD), and migratory phenotypes are considered hallmarks of cellular de-differentiation. The ability to induce de-differentiation of resident cells at the site of injury or ischemic tissues may be a useful approach for reparative and regenerative medicine.

GOAL:
To test the capacity of 6-bromoindirubin-3’-oxime (BIO), an inhibitor of GSK-3β, to induce neovascularization by stimulating a transcriptional program centering the
NANOG
gene networks in ECs.

RESULTS:
We show that BIO not only induced the interaction of β-catenin with NANOG, but also increased the expression of mesenchymal and hemangioblastic cell markers
NANOG, VEGFR2, BRACHYURY
, and
CD133,
while decreasing mature EC markers
von Willebrand Factor
(
v
WF) and
PECAM-1 (CD31).
Increased expression of the NANOG transcriptional network induced characteristics of mesenchymal/stem-like cellular behavior in a hanging drop assay and increased robust neovascularization
in vitro
and
in vivo
. Furthermore, microscopic analyses indicated the ability of BIO to induce ACD in these cells, a hallmark of stem cell self-renewal. In contrast,
NANOG-
knockdown inhibited the ability of BIO treated ECs to divide asymmetrically, to form cellular aggregates, and to undergo angiogenesis.

CONCLUSION:
These findings highlight the capacity of BIO to induce dedifferentiation of ECs into “stem-like“ cells by up-regulating the expression of NANOG and NANOG gene networks. Thus, we propose that BIO could become useful not only for inducing neovascularization of ischemic tissues
in situ
, but also for generating a large number of cells for transplantation and regenerative medicine.
Abstract 391: Insulin-like Growth Factors Promote Vasculogenesis in Embryonic Stem Cells
Authors: Piecewicz Stephanie M , Pandey Ambarish , Roy Bhaskar , Soh Hua Xiang , Zetter Bruce R , Sengupta Shiladitya
Published: March 2022

The ability of embryonic stem cells to differentiate into endothelium and form functional blood vessels has been well established and can potentially be harnessed for therapeutic angiogenesis. However, after almost two decades of investigation in this field, limited knowledge exists for directing endothelial differentiation. A better understanding of the cellular mechanisms regulating vasculogenesis is required for the development of embryonic stem cell-based models and therapies. In this study, we elucidated the mechanistic role of insulin-like growth factors (IGF1 and 2) and IGF receptors (IGFR1 and 2) in endothelial differentiation using an embryonic stem cell embryoid body model. Both IGF1 and IGF2 predisposed embryonic stem to differentiate towards a mesodermal lineage, the endothelial precursor germ layer, as well as increased the generation of significantly more endothelial cells at later stages. Interestingly, differentiation into other mesoderm lineages was not augmented while endothelial cell differentiation was significantly increased. The percentage of endothelial cells within differentiated embryoid bodies was amplified by greater than 7% in embryoid bodies, a substantial increase compared to other characterized vasculogenic factors like VEGF. Inhibition of IGFR1 signaling using neutralizing antibody or a pharmacological inhibitor, picropodophyllin, significantly reduced IGF-induced mesoderm and endothelial precursor cell formation. We confirmed that IGF-IGFR1 signaling stabilizes HIF1[[Unsupported Character - Symbol Font α]] and leads to up-regulation of VEGF during vasculogenesis in embryoid bodies, which may be one of the critical mechanisms of IGF-induced promotion of vasculogenesis. Understanding the mechanisms that

are critical for vasculogenesis in various models will bring us one step closer to enabling cell based therapies for neovascularization.
Abstract 392: High-Density Lipoproteins Beneficially Regulate Angiogenesis
Authors: Prosser Hamish C
Published: March 2022

Introduction:
Angiogenesis can be both favourable and unfavourable. It is critical for collateral vessel formation in response to ischaemia following myocardial infarction (MI), however excessive inflammatory-driven neovascularisation accelerates atherosclerotic plaque growth. High-density lipoproteins (HDL) are associated with improved survival and prognosis following MI as well as reduced plaque development.

Hypothesis:
We hypothesized that HDL may beneficially regulate angiogenesis under both hypoxic or inflammatory settings, in vitro.

Methods and results:
Human coronary artery endothelial cells (HCAECs) subjected to hypoxia (1.2% O2, 6h) displayed increased tubulogenesis (19%) and proliferation (31%), p<0.05. Interestingly, pretreatment with reconstituted HDL (rHDL), containing apolipoprotein (apo)A-I and phosphatidylcholine, augmented hypoxia-driven elevations in tubulogenesis (14%) and proliferation (53%), p<0.05. Consistent with this, rHDL also augmented hypoxia-driven elevations in the proangiogenic mediators HIF1α (39%), VEGF (1.5 fold) and SDFα (3.4 fold), p<0.05. In contrast to the findings in hypoxia, pre-treatment with rHDL followed by exposure to two inflammatory conditions: TNFα (0.7 ng/ml) and stimulated macrophage-conditioned media (CM), significantly inhibited HCAEC tubulogenesis (75% & 70%), proliferation (47% & 39%) and migration (37% & 55%), p<0.05. The anti-angiogenic effects of rHDL in both inflammatory settings were associated with reductions in the pro-angiogenic inflammatory mediators p65 (43% & 52%) and MCP-1 (33% & 30%), p<0.05. Moreover, in contrast to the setting of hypoxia, rHDL reduced HIF1α and VEGF protein in response to inflammation (TNFα (36% & 34%) and CM (37% & 19%), p<0.05). rHDL pretreatment strikingly increased hemeoxygenase-1 (HO-1) protein expression, a known regulator of angiogenesis, under both hypoxic and inflammatory conditions (∼2-4 fold for all), p<0.05.

Conclusions:
In summary, we have identified HDL to uniquely regulate angiogenesis, characterized by enhancement of hypoxia-mediated angiogenesis and, conversely, marked suppression of inflammation-associated angiogenesis.
Abstract 393: Osteoporosis Equals Atherosclerosis
Authors: Rajamannan Nalini
Published: March 2022

Atherosclerosis and osteoporosis are the leading causes of mortality and morbidity in the World. Recent epidemiologic studies have demonstrated that these disease processes develop in parallel. Evidence indicates that hyperlipidemia plays a paradoxical role in both disease processes. However, the mechanism is not understood. We hypothesize lipids activate atherosclerosis within the bone and the heart to initiate the development of diseases in both of these tissues. To test this hypothesis in an experimental hypercholesterolemia model and to determine if statins play a protective role in this process. LDLR
-/-
mice (n=60). Group I (n=20) normal diet, Group II (n=20) 0.25% chol diet (w/w), and Group III (n=20) 0.25% (w/w) chol diet+atorv for the development of calcification. The aortic valves(AVA) and femurs were examined for atherosclerosis and calcification markers. Bone formation was assessed by micro Computed Tomography (microCT). The cholesterol diet induced complex bone formations in the calcified AVA and increase in macrophage cell infiltration. The hyperlipidemic bones expressed an increase in osteoclast Goldner Stained cells and a decrease in bone formation by MicroCT. Atorvastatin reduced atherosclerosis and bone mineralization in the AVA and increased mineralization within the femur bones(p<0.05). Atherosclerosis is present in hyperlipidemic bones and valves as characterized by the hallmarks of atherosclerosis, macrophage and osteoclast infiltration, and which is attenuated by atorvastatin. This model may have future implications in the treatment of cardiovascular calcification and osteoporosis with statin therapy.
Abstract 395: Formation of New Coronary Collaterals After Acute Myocardial Infarction in Mouse
Authors: Zhang Hua , Faber James E
Published: March 2022

Variation in
extent
of the native pre-existing collateral (COL) circulation, ie, COL number and average diameter in healthy tissues, is a major determinant of the variation in severity of tissue injury after acute obstruction. Indirect evidence suggests that coronary COL extent varies widely in presumed healthy humans. Although the cause of this variation is unknown, studies in non-heart tissues of healthy mouse strains have found that differences in genetic background are a major determinant. Whether this applies to the heart is unknown, because no model exists to investigate the coronary COL circulation in mice. In addition, little is known regarding whether new COLs can be induced to form in individuals with few native COLs. To address these questions, we developed a mouse model that combines high-resolution (capillary-level) microangiography and ligation (LADX) with measurement of coronary COL conductance index and infarct volume. Adult (3-mos-old) mouse strains chosen for their large rank-order difference in native COL extent in brain and hindlimb-BLKS > C57BL/6 > A > BALB/c-all lack native coronary COLs. After LADX, all strains formed new COLs by day-3 that reached maximum number and diameter by day-14. However, rank-order for new COL formation differed from the above, ie, C57BL/6 @ BALB/c > BLKS > A; COL conductance and infarct volume
-1
followed this same rank-order. Moreover, the rank-order for new COL formation differed from the rank-order for COL remodeling after arterial ligation in brain and hindlimb, ie, A > C57BL/6 > BALB/c. Ongoing studies to determine the mechanisms of new COL formation after LADX find strongly reduced COL formation and conductance and increased infarct volume in C57BL/6 mice deficient in monocyte chemoattractant protein-1 or its receptor (CCR2)-effects partially rescued by bone marrow transplantation. Conclusions: Unlike other tissues, the mouse heart lacks native COLs. Mice thus provide a model to study coronary collaterogenesis free of complications posed by remodeling of pre-existing COLs. Gene polymorphisms underlying variation in ischemic coronary collaterogenesis differ from those underlying variation in embryonic formation of native COLs. MCP-1 and bone marrow cells play an important role in new COL formation.
Abstract 398: Hydrogen Peroxide Mediates Calcium Chloride--Induced Aortic Wall Dilatation
Authors: Parastatidis Ioannis , Weiss Daiana , Joseph Giji , Taylor W R
Published: March 2022

Rationale:
Abdominal aneurysm formation is characterized by infiltration of the aortic wall with inflammatory cells, activation of proteolytic enzymes, loss of vascular smooth muscle cells (VSMC), and neovascularization. It is also known that hydrogen peroxide (H
2
O
2
) plays an important role in these processes.

Hypothesis:
Since H
2
O
2
participates in the pathophysiologic processes that govern aneurysmal dilatation, we hypothesized that scavenging of H
2
O
2
with catalase protects against the formation of aortic aneurysms.

Methods:
To assess our hypothesis 3 groups of mice, all 8-10-week-old males on the C57BL6 background, were used. Aortic dilatation was induced with the application of CaCl
2
on the infrarenal aortas. We compared the aortic dilation in mice that specifically over-express catalase in the VSMC (group 1) or in the myeloid cell lineage (MCL) (group 2) with their wild type littermates. Chemical injury with CaCl
2
was also induced in wild type mice which were treated with continuous intravenous infusion of PEG-catalase or saline for 8 weeks (group 3). The aortic diameter was measured with a caliper and with videomicroscopy on the day of surgery and 8 weeks later. H
2
O
2
levels were quantified with the Amplex Red assay.

Results:
Application of CaCl
2
resulted in a significant increase in H
2
O
2
generation in the infrarenal aortas compared to saline controls, which peaked on post-operative day 10 (0.60±0.09 vs. 0.37±0.05 μM/aortic ring, n=3/grp, p=0.02). Compared to their wild type littermates, mice that over-express catalase in the VSMC had negligible dilation of the aorta (0.78±0.05 vs. 0.53±0.02 mm, n=7/grp, p=0.001). Similarly, over-expression of catalase in MCL protected against aortic dilation (0.76±0.02 vs. ±0.005 mm, n=7, p<0.0001). Corroborating the above findings, continuous intravenous infusion of PEG-catalase, but not saline, protected from CaCl
2
induced aortic dilation (0.73±0.03 vs.0.56±0.02 mm, n=3-6/grp, p=0.007).

Conclusions:
Our data suggest that H
2
O
2
is essential in CaCl
2
induced aortic aneurysm formation.
Abstract 40: Protein Kinase C Regulation of 12-Lipoxygenase-Mediated Human Platelet Activation
Authors: Yeung Jennifer , Apopa Patrick L , Vesci Joanne , Boutaud Olivier , Holinstat Michael
Published: March 2022

Introduction:
Platelet activation is important in the regulation of hemostasis and thrombosis. Uncontrolled activation of platelets may lead to arterial thrombosis which is a major cause of myocardial infarction and stroke. Following activation, metabolism of arachidonic acid (AA) by 12-lipoxygenase (12-LOX) may play a significant role in regulating the degree and stability of platelet activation as inhibition of 12-LOX significantly attenuates platelet aggregation in response to various agonists. Protein kinase C (PKC) activation is also known to be an important regulator of platelet activity.

Objectives:
Using a newly developed selective inhibitor for 12-LOX and a pan-PKC inhibitor, we investigated the role of PKC in 12-LOX-mediated regulation of agonist signaling in the platelet.

Methods:
To determine the role of PKC within the 12-LOX pathway, a number of biochemical endpoints were measured in human platelets including platelet aggregation, calcium mobilization, integrin activation, and phosphorylation of PKC substrates.

Results:
Inhibition of 12-LOX or PKC resulted in inhibition of dense granule secretion and attenuation of both aggregation and αIIbβ
3
activation. However, activation of PKC downstream of 12-LOX inhibition rescued agonist-induced aggregation and integrin activation. Additionally, direct PKC activation rescued deficits in phosphorylation of pleckstrin and other PKC substrates mediated by inhibition of 12-LOX. Finally, inhibition of 12-LOX had no effect on PKC-mediated aggregation indicating that 12-LOX is upstream of PKC.

Conclusions:
These studies support an essential role for PKC downstream of 12-LOX activation in human platelets and suggest 12-LOX as a possible target for anti-platelet therapy.
Abstract 413: A Dominant Negative Mutant of Plasminogen Activator Inhibitor-1 Does Not Inhibit Intimal Hyperplasia After Balloon Coronary Angioplasty in Pigs
Authors: Strawn Tammy L , Ivey Jan R , Lawrence Daniel A , Bowles Douglas K , Fay William P
Published: March 2022

Background.
Plasminogen activator inhibitor 1 (PAI-1) regulates arterial remodeling and intimal hyperplasia after vascular injury. PAI-1-R is a recombinant mutant that completely lacks anti-protease activity yet binds vitronectin (VN) with normal affinity. PAI-1-R inhibits cell migration in vitro and neointima formation in rodents by binding VN and competitively blocking its binding to integrins.

Objective and Methods.
To determine if PAI-1-R inhibits intimal hyperplasia under experimental conditions highly relevant to human coronary artery disease we performed balloon angioplasty of the left circumflex coronary artery (LCX) of male pigs and administered PAI-1-R by direct coronary infusion immediately after angioplasty and by peripheral intravenous infusion on each of the next two days (5 mg/infusion; total dose 15 mg/pig). Thirteen pigs (weight 24±4.3 kg) received PAI-1-R and 13 pigs (weight 24 ± 4.7 kg) received identical infusions of vehicle control. Animals were euthanized 14 days after angioplasty and neointima formation in the injured LCX was determined by microscopic morphometric analysis.

Results.
Plasma PAI-1-R concentrations assessed 1 hr after each infusion were 32±18, 89±60, and 59±16 ng/mL, respectively, in PAI-1-R-treated pigs and undetectable in controls. There was no significant difference in LCX neointima formation between PAI-1-R- vs. vehicle-treated pigs (maximal intima thickness 523±47 vs. 581±49 μm, p=0.41; intima area/media area normalized to media rupture length 3.7±0.3 vs 3.8±0.3, p=0.8).

Conclusion.
Short-term, bolus infusions of a dominant negative form of PAI-1 do not significantly inhibit neointima formation in pigs after coronary angioplasty. These results contrast with rodent data and suggest that alternative strategies that achieve higher concentrations of PAI-1-R at vascular injury sites will be required to assess the efficacy of this strategy under clinically relevant conditions.
Abstract 414: Combined Tissue Factor/Factor VIIa Inhibitor and Acetylsalicylic Acid Show Increased Antithrombotic Potency Without Affecting Bleeding Time
Authors: Tabata Kimiyo M , Nagakura Tadashi , Kira Kazunobu , Hirota Shinsuke , Clark Richard , Matsuura Fumiyoshi , Azuma Hiroshi , Yasui Sou , Takemura Atsushi , Kusano Kazutomi , Mikamoto Masaki , Omae Takao , Inoue Atsushi , Hiyoshi Hironobu
Published: March 2022

Since increased tissue factor (TF) in human atherosclerotic plaques is believed to trigger thrombotic events, TF/factor VIIa (FVIIa) inhibition might have prophylactic potential for atherothrombosis such as acute coronary syndrome (ACS). Although recent clinical observation by rivaroxaban provides a scientific rationale for the addition of anticoagulant to current antiplatelet treatment, these might have potential to increase bleeding. It is therefore important that emerging anticoagulants demonstrate additive antithrombotic effects with no concomitant increase in bleeding for ACS indication. We recently discovered ER-410660, a potent TF/FVIIa inhibitor. It suppressed venous thrombosis without affecting bleeding time and suggested that ER-410660 could act as a novel anticoagulant with a low bleeding risk. In the photochemically-induced thrombosis (PIT) model, the thrombus after vessel-wall injury has been developed by the deposition of TF, platelets, and fibrin. Therefore, we hypothesized that ER-410660 would be effective in the PIT model and could show enough margin against bleeding. The antithrombotic effects of ER-410660 and acetylsalicylic acid (ASA), both alone and in combination, was assessed using a rat PIT model. We also measured the bleeding time of ER-410660 in combination with ASA using a rat tail-transection model. Intravenous (iv) pretreatment with ER-410660 prolonged the time to occlusion (TTO) of the artery. The TTO was doubled at 0.99 mg/kg (95% CI, 0.51-1.9). Orally administered ASA also showed antithrombotic effect, and the TTO was doubled at approximately 12 mg/kg. Combined administration of 1 mg/kg ER-410660 (iv) and 10 mg/kg ASA (orally) showed an additive antithrombotic effect, with no cessation of blood flow during the 30-min observation period. Intravenous administration of ER-410660 (0.6, 2, and 6 mg/kg) to rats treated with ASA (10 mg/kg) did not significantly increased when compared with ASA alone, even at the highest dose. In conclusion, a TF/FVIIa inhibitor suppressed arterial thrombosis without affecting the bleeding time associated with ASA. Owing to these favorable characteristics, anticoagulation via TF/FVIIa inhibition may be a superior approach to the prophylaxis of atherothrombotic diseases.
Abstract 416: Dysregulated Coagulation from Coexisting Hypofibrinogenemia and Elevated Factor VIII
Authors: Marchi Rita , Walton Bethany L , McGary Colleen S , Lin Feng-Chang , Ma Alice D , Pawlinski Rafal , Mackman Nigel , Campbell Robert A , DiPaola Jorge , Wolberg Alisa S
Published: March 2022

Introduction and Objective—
Although abnormal clotting factors are typically identified and examined in isolation, dysregulated coagulation can result from multiple independent, co-existing abnormalities. Herein, we probed the complexity of dysregulated coagulation in humans by characterizing pathophysiologic mechanisms in a patient with both bleeding and thrombosis.

Methods and Results—
A patient with a normal activated partial thromboplastin time (29.6 sec), prolonged thrombin and reptilase times (22.6 and 21.3 sec, respectively), and decreased functional and antigenic fibrinogen levels (162 and 148 mg/dL, respectively) was initially diagnosed with hypodysfibrinogenemia. This diagnosis was supported by DNA analysis revealing a previously unreported FGB mutation (c.656A>G) predicting a novel Q189R mutation in the mature ββ chain that was present in the heterozygote state. Interestingly, however, turbidity analysis showed purified fibrinogen polymerization and degradation were indistinguishable from normal, and Bβ chain subpopulations appeared normal by two-dimensional difference in-gel electrophoresis, indicating the mutated chain was not secreted. Of note, the patient’s peak plasma thrombin generation was significantly higher than normal (267±20 versus 183±16 nM, respectively, p<0.05), attributable to an elevated level of factor VIII (163-225%). Spiking normal plasma with factor VIII to 225% (final) produced a thrombin generation peak that was indistinguishable from the patient. In a carotid artery injury model, hypofibrinogenemic mice (Fgn+/-) infused with factor VIII demonstrated significantly shorter vessel occlusion times than saline-infused Fgn+/- mice (11.8±5.8 versus 20.9±14.7 minutes, respectively, p<0.05).

Conclusions—
These data associate the patient’s complex bleeding and thrombotic presentations with co-existing hypofibrinogenemia plus elevated FVIII, and indicate hypofibrinogenemia does not mitigate the prothrombotic effects of plasma hypercoagulability. Together, these findings illustrate the challenges of diagnosing complex coagulopathies and underscore the importance of global coagulation testing in patients with compound presentations.