bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 39: Telomerase Reverse Transcriptase Mediates Vascular Smooth Muscle Cell Proliferation and Cell Cycle Progression Through Epigenetic Regulation of E2F Target Genes
Authors: Aono Jun , Heywood Elizabeth B , Qing Hua , Jones Karrie L , Bruemmer Dennis
Published: July 2021

INTRODUCTION:
By maintaining the stability of telomeres, DNA-protein complexes that protect the ends of chromosomes, telomerase is rate limiting for tissue renewal and cell proliferation. In addition to this telomere maintenance function, mounting evidence suggests that the telomerase reverse transcriptase (TERT) exhibits a direct regulatory role in the activation of mitotic gene expression without apparent involvement of its well-established function in telomere homeostasis. In the present study, we investigated the role of TERT in vascular remodeling and the hypothesis that TERT regulates vascular smooth muscle cell (VSMC) proliferation through an epigenetic activation of an S Phase gene expression program.

METHODS AND RESULTS:
We first demonstrate high levels of TERT expression in replicating SMC of the developing neointima of human atherosclerosis. Studies in VSMC isolated from genetically engineered TERT-deficient mice with normal telomere length establish that TERT is necessary for cell proliferation. Conversely, overexpression of TERT induces cell proliferation without extending telomere length. Although TERT-deficient VSMC are not senescent, decreased proliferation is associated with stable and irreversible silencing of S phase gene expression, including target genes of the S Phase transcription factor E2F. Chromatin immunoprecipitation assays confirm that TERT is recruited to E2F target sites and promotes chromatin accessibility by facilitating the acquisition of permissive histone modifications. Finally, using a model of guide-wire induced arterial injury, we demonstrate these observations are applicable in vivo and identify decreased neointima formation in TERT-deficient mice.

CONCLUSIONS:
These data indicate a previously unrecognized role for TERT in the epigenetic regulation of proliferative gene expression in VSMC.
Abstract 400: Cannabinoid 1 Receptor Blockade Diminishes Obesity and Dyslipidemia via Peripheral Activation of Brown Adipose Tissue
Authors: Berbée Jimmy F , Boon Mariëtte R , van Dam Andrea D , van den Hoek Anita M , Lombès Marc , Havekes Louis M , Tamsma Jouke T , Guigas Bruno , Meijer Onno C , Jukema J W , Rensen Patrick C
Published: July 2021

Objectives:
The endocannabinoid system is an important player in energy metabolism by regulating appetite, lipolysis and energy expenditure. Chronic blockade of the cannabinoid 1 receptor (CB1R) leads to long-term maintained weight loss and reduction of dyslipidemia in experimental and human obesity. Brown adipose tissue (BAT) that burns lipids towards heat using UCP1, recently emerged as a major player in lipoprotein metabolism and is present and active in human adults. The aim of the present study was to elucidate the mechanism by which CB1R blockade reverses dyslipidemia and obesity, with special focus on BAT.

Methods and results:
Diet-induced obese
APOE*3-Leiden.CETP
transgenic mice, a well-established model for human-like lipoprotein metabolism, were treated with the systemic CB1R blocker rimonabant (10 mg/kg/day) for 4 weeks. Rimonabant persistently decreased body weight (-25%, p<0.001), fat mass (-32%, p<0.001) and plasma triglyceride (TG) levels (-60%, p<0.05), despite a modest and transient reduction in food intake. Interestingly, rimonabant reduced plasma TG levels, not by affecting VLDL-TG production by the liver, but rather by selectively increasing VLDL-TG clearance by BAT (+40%, p<0.05). This was accompanied by increased energy expenditure (+20%, p<0.05), decreased lipid droplet size and increased UCP1 content in BAT (+28%, p<0.05), all pointing to increased BAT activity. Next, we demonstrated that the CB1R is highly expressed in BAT and that
in vitro
blockade of the CB1R in cultured brown adipocytes resulted in 2.5-fold upregulation of UCP1. Importantly, the
in vivo
results could be fully recapitulated using the strictly peripheral CB1R antagonist AM6545 (10 mg/kg/day) that does not induce hypophagia.

Conclusion:
CB1R blockade reduces dyslipidemia and obesity by peripheral activation of BAT. Selective targeting of peripheral CB1R in BAT has thus great therapeutic potential in decreasing dyslipidemia and obesity and ultimately cardiovascular diseases.
Abstract 440: Activation of Hepatic CREBH-Insig-2a Signaling in the Triglyceride-Lowering Mechanism of R-Alpha-Lipoic Acid
Authors: Tong Xuedong , Moreau Regis , Su Qiaozhu
Published: July 2021

Activation of the sterol regulatory element-binding proteins (SREBPs), a step regulated by a cluster of ER-resident proteins, Insig-1, Insig-2 and SCAP, is rate limiting in hepatic de novo lipogenesis. We previously reported that feeding R-alpha-lipoic acid (LA) to ZDF (fa/fa) rats improves severe hypertriglyceridemia and lowers abdominal fat mass by inhibiting expression of genes involved in hepatic long-chain fatty acids and triacylglycerol syntheses. In this study, we characterized a novel mechanism of action of LA that explains its triacylglycerol lowering properties. Dietary LA activates liver specific transcription factor cAMP responsive element binding protein H (CREBH), which in turn enhances transcription and translation of Insig-1 and Insig-2. Chromatin immunoprecipitation (ChIP) assay demonstrated interaction between CREBH and the promoter of Insig-2 but not Insig-1. The increased abundance of Insig-1 and Insig-2 proteins contributes to sequester SREBP-1c and SREBP-2 in the ER and prevents their translocation to the Golgi apparatus where they would become activated. As a consequence, mRNA expression of genes involved in fatty acid and cholesterol synthesis, including FASN, ACC, SCD-1, HMGCR and LDL receptor, were significantly decreased in LA-fed animals versus pair-fed controls. Concomitantly, the assembly and secretion of very-low-density lipoproteins (VLDL) by primary hepatocytes were suppressed in the LA-fed ZDF rats as indicated by the decrease in VLDL-associated apolipoprotein B and apolipoprotein E. In vitro, treating a rat McA-RH7777 hepatoma cells with LA (200 micromole) activated CREBH, induced expression of Insig-1 and Insig-2, and hindered the palmitic acid-induced synthesis of triacylglycerol. This study provides new mechanistic insight into the triacylglycerol lowering properties of LA and supports the therapeutic potential of LA against hypertriglyceridemia.
Abstract 448: Association of CHADS2 and CHA2DS2-VASc Score with Coronary Atherosclerosis Burden in Nonvalvular Atrial Fibrillation
Authors: Chaikriangkrai Kongkiat , Alchalabi Sama , Khaleel bala Sayf , Kassi Mahwash , Chang Su Min
Published: July 2021

Background:
This study is to examine association of CHADS2 and CHA2DS2-VASc score with coronary artery disease (CAD) in nonvalvular atrial fibrillation (AF)

Method:
A total of 676 consecutive nonvalvular AF patients without known history of CAD underwent coronary artery calcium score (CACS) evaluation by multi-detector cardiac computed tomography. Clinical characteristics and CACS were compared between different CHADS2 and CHA2DS2-VASc score groups.

Results:
The cohort comprised of 68% (461 of 676) male with a mean ± SD age of 63 ± 10 years. Median 10-year risk of CAD by Framingham score was 11% (range 2%-53%). Median CHADS2 score was 1 (range 0-6) and median CHA2DS2-VASc score was 2 (range 0-8). Mean ± SD CACS was 215 ± 504. Compared to CHADS2 score ≤ 1, those with CHADS2 score > 1 had higher mean ± SD CACS (359 ± 738 VS 158 ± 359; p<0.001). CHADS2 score > 1 is associated with CACS > 0 (OR 1.751; 95%CI 1.168, 2.624; p 0.007) and CACS > 400 (OR 2.528; 95%CI 1.641, 3.896; p < 0.001). Similarly, compared to CHA2DS2-VASc score ≤ 1, those with CHA2DS2-VASc score > 1 had higher mean ± SD CACS (270 ± 586 VS 150 ± 376; p<0.001). CHA2DS2-VASc score > 1 is associated with CACS > 0 (OR 1.713; 95%CI 1.217, 2.409; p 0.002) and CACS > 400 (OR 2.683; 95%CI 1.678, 4.289; p < 0.001). Receiver operating characteristics of CHADS2 and CHA2DS2-VASc score models for CACS > 400 is shown in the figure.

Conclusion:
In nonvalvular AF patients, higher CHADS2 and CHA2DS2-VASc score are comparably associated with presence and severity of CAD.
Abstract 453: Chemerin and Resistin Expression in Carotid Plaque Instability
Authors: Doonan Robert-James , Steinmetz-Wood Samantha , Gasbarrino Karina , Zheng Huaien , Lai Chi , Veinot Jean P , Mantzoros Christos , Daskalopoulou Stella S
Published: July 2021

Objective:
Chemerin and resistin are pro-inflammatory cytokines that have been shown to play a role in atherosclerosis and coronary artery disease. However, their association with carotid atherosclerosis has not been investigated. We sought to assess the relationship between a) circulating chemerin/resistin levels and cerebrovascular symptomatology, and b) chemerin/resistin plaque expression and carotid plaque instability.

Methods:
Patients scheduled for carotid endarterectomy were recruited from the Royal Victoria and Jewish General hospitals, Montreal, Canada. Blood was drawn pre-operatively and plasma resistin and chemerin were measured using ELISA. Maximum stenosis sections were stained with hematoxylin and eosin and immunostained with anti-CD3 and anti-CD68. Two vascular pathologists used semi-quantitative scales to classify plaque foam cells and inflammatory cells as well as overall plaque instability. RNA was isolated from the area of maximum stenosis, reverse transcribed, and qPCR was performed to measure chemerin and resistin expression. Kruskal-Wallis or Mann-Whitney U tests were used to evaluate differences in resistin and chemerin between groups.

Results:
In this ongoing study we recruited 169 patients (66.1% men, 74.5% symptomatic) with a mean±SD age of 70.0±9.3 years. To date all 169 patients had blood samples drawn and 100 had plaque chemerin and resistin expression assessed. Symptomatic patients had higher plasma chemerin (230 (179.0-261.3) vs. 201.4 (175.8-228.1) ng/ml, P=0.08) and resistin levels (12.7 (10.2-17.7) vs. 10.8 (7.1-16.0) ng/ml, P=0.02) when compared with asymptomatic patients. Chemerin plaque expression was decreased in plaques with greater number of foam cells and inflammatory cells and in plaques with greater overall instability. Resistin plaque expression was also decreased in plaques with a greater number of foam cells and inflammatory cells.

Conclusions:
Circulating chemerin and resistin levels are increased in symptomatic patients. Intraplaque chemerin and resistin expression is inversely related to the number of plaque inflammatory cells and instability. Our ongoing work will determine the underlying mechanisms linking chemerin and resistin with atherosclerotic plaque instability.
Abstract 473: The Association of Bacterial Biofilms With Carotid Atherosclerotic Disease: A Study in Progress
Authors: Smith Triston B , Hiller Luisa , Nistico Laura , Kreft Rachel , Bhashyam Siva , Suero Gregory , Rayl Kenneth , Driscoll Kathleen , Chess Bart , Passineau Michael , Lasorda David
Published: July 2021

Background:
There is evidence to support that bacteria present in atherosclerotic plaque may play a role in disease pathogenesis. Several studies utilizing antibiotics to inhibit progression of atherosclerotic disease have been unsuccessful. Biofilms are complex microenvironments of bacterial colonies encased within a matrix that firmly adheres to surfaces. They exhibit extensive genotypic and phenotypic diversity and are 100 to 1000 times more resistant to antibiotics than planktonic cultures. We hypothesized that bacterial biofilms are present in carotid atheroma.

Methods:
The IBIS Biosensor- Florescent in situ hybridization (FISH) pipeline for microbial discovery was applied to atherosclerotic samples obtained from 8 patients who underwent open CEA at Allegheny General Hospital. The extracted nucleic acid from CEA samples were aliquoted into wells of a microtiter plate containing broad range primers for PCR. The products were desalted in a 96-well plate format, sequentially electrosprayed into a mass spectrometer and the spectral signals processed to determine the masses of each of the PCR products present. The base composition of each amplicon was therefore unambiguously deduced. Using combined base compositions from multiple PCRs; the identities of the pathogens and their relative concentrations in the starting sample were determined. FISH using fluorescent probes targeted to the 16s rRNA, designed for each sample based on the result from the IBIS, was then performed. Confocal microscopy of the FISH stained samples allowed visualization of the specific bacterial species.

Results:
One or more bacterial species were present in four of the eight CEA samples analyzed (50%). Species present were Staphylococcus capitis/ caprae, Staphylococcus warneri, Propionibacterium acnes, and Staphylococcus epidermidis. Further analyses in an attempt to correlate these findings with oral microbiota, dental and clinical history is currently ongoing.

Conclusion:
In our ongoing study 50% of analyzed carotid atherosclerotic samples contained the highly antibiotic resistant bacterial biofilms. Mechanisms of biofilm formation in atheroma need to be explored.
Abstract 486: The Effects of Weekend Admission on the Outcomes and Management of Ruptured Aortic Aneurysms
Authors: Groves Elliott M , Khoshchehreh Mahdi , Le Christine , Malik Shaista
Published: July 2021

The Effects of Weekend Admission on the Outcomes and Management of Ruptured Aortic Aneurysms

Objective:
Ruptured aortic aneurysm is a condition with a high rate of mortality that requires prompt surgical intervention. It has been noted that in some conditions requiting such prompt intervention, in-hospital mortality is increased in patients admitted on the weekends as compared to patients admitted on weekdays. We sought to determine if this was indeed the case for both ruptured thoracic and abdominal aortic aneurysm and elucidate the possible reasons.

Methods:
Using the Nationwide Inpatient Sample (NIS), a publicly available database of inpatient care, we analyzed the incidence of mortality among patients admitted on the weekends compared to weekdays for ruptured aortic aneurysm. Ultimately the care of over 7,000 patients was analyzed for the primary endpoints. We adjusted for demographics, comorbid conditions, hospital characteristics, rates of surgical intervention, timing of surgical intervention and use of additional therapeutic measures.

Results:
Patients admitted on the weekend for both ruptured thoracic and abdominal aortic aneurysm had a statistically significant increase in mortality as compared to those admitted on the weekdays (OR 2.55 for Thoracic and 1.32 for Abdominal). By our analysis this is likely due to a delay in surgical care on the weekends.

Conclusions:
Weekend admission for ruptured aortic aneurysm is associated with an increased mortality when compared to those admitted on the weekend and this is likely due to several factors with the most predominant being a delay in surgical intervention.
Abstract 49: Knockout of ADAMTS7, a Novel Human CAD Locus, Reduces Atherosclerosis in Both LDLR and APOE Hyperlipidemic Mouse Models
Authors: Bauer Robert C , Tohyama Junichiro , Cui Jian , Zhang Xuan , Yu Christopher , Ou Kristy , Parmacek Michael S , Rader Daniel J , Reilly Muredach P
Published: July 2021

Multiple genome-wide association studies demonstrate that the genomic locus containing the gene
ADAMTS7
is associated with coronary artery disease in humans.
ADAMTS7
was not linked to cardiovascular disease prior to those genetic studies, and to date this association has not been validated in experimental models of atherosclerosis. An
Adamts7
(
Ats7
) knockout mouse, with knock-in of a β-galactosidase reporter gene, was bred onto both the
LDLR
and
APOE
KO hyperlipidemic mouse models. Atherosclerosis studies were performed on the resultant Ats7/LDLR and
Ats7
/
APOE
double KO (dKO) mice and control littermates. Mice were fed western diet for 16 weeks (
LDLR
) or 10 weeks (
APOE
), and aortas and aortic roots were harvested for lesion area quantification. In addition, brachiocephalic arteries were dissected for immunohistochemical analysis of lesion composition. No differences in plasma lipids were observed between experimental groups in either study.
Ats7
/
LDLR
dKO males (N=7) and females (N=13) displayed significant reductions in lesion formation in aortas by en face (58%,
p
<0.005 and 61%,
p
<0.0001 respectively) and in aortic roots (31%,
p
=0.04 and 22%,
p
=0.002 respectively) compared to control
LDLR
-/- male (N=6) and female (N=10) littermates. Additionally,
Ats7
/
APOE
dKO males (N=16) and females (N=13) displayed significantly reduced lesion formation by en face (62%,
p
<0.0001 and 54%,
p
<0.0001 respectively), and trends towards reduced lesion area in aortic roots (18%,
p
=0.15 and 22%,
p
=0.10 respectively) compared to control
APOE
-/- males (N=9) and females (N=10). Femoral wire injury experiments suggested reduced neointimal formation in
Ats7
KO mice at 28 days post-injury, and X-gal staining at multiple timepoints showed adventitial
Ats7
expression peaking at 7 days post-injury. Preliminary studies suggest that aortic rings from
Ats7
/
LDLR
dKO mice embedded in collagen have reduced cell migration compared to control
LDLR
aortic rings. In summary, these data represent the first
in vivo
experimental validation of Adamts7 as a gene involved in atherogenesis, likely through modulation of vascular cell migration into atherosclerotic lesions. These data support the concept that pharmacological inhibition of ADAMTS7 should be atheroprotective in humans.
Abstract 490: Vertebral Artery Tortuosity Index is a Novel Biomarker of Surgery and Aortic Dissection or Rupture in Children and Young Adults: Findings From the National Registry of Genetically Triggered Thoracic Aortic Aneurysms and Cardiovascular Conditions
Authors: Morris Shaine A , Oda Seitaro , Asch Federico M , Payne William , LeMaire Scott A , Prakash Siddharth , Devereux Richard B , Kroner Barbara L , Moodie Douglas S , Maslen Cheryl L , Pyeritz Reed E , Ravekes William , Holmes Kathryn W , Roman Mary J , McDonnell Nazli B , Lacro Ronald V , Dietz Harry C , Eagle Kim A , Krishnamurthy Rajesh , Milewicz Dianna M ,   GenTAC Registry Consortium
Published: July 2021

Background:
Little is known about reliable predictors of thoracic aortic dissection or rupture (TAD) in children and young adults. We sought to determine whether elevated vertebral artery tortuosity is a biomarker of TAD or aortic surgery at an early age.

Methods:
We identified 208 patients ≤50 years old in the GenTAC Registry who had ≥2.5 cm of either vertebral artery visualized on a computed tomography angiogram (CTA). In a blinded fashion, using a volume-rendered projection, each patient’s vertebral artery tortuosity index (VTI) was calculated using the larger distance factor (% by which actual length exceeds straight-line length) of the two vertebral arteries. We then investigated associations between VTI and freedom from prophylactic or post-TAD surgery.

Results:
Subjects included 73 with Marfan syndrome (MS), 34 with bicuspid aortic valve, 18 with Loeys-Dietz syndrome (LDS), 16 with familial thoracic aneurysms and dissections, 12 with Ehlers-Danlos syndrome type IV (EDS), 6 with congenital heart disease, and 49 with other aneurysms and dissections. Median age was 38.5 years [interquartile range (IQR) 29.6-44.6]; 23 were ≤18 years. Indication for first surgery was prophylactic in 80 patients and TAD in 49. VTI was highest in LDS (median 50, IQR 21-72), followed by MS (median VTI 27, IQR 16-52), and lowest in EDS (median 7, IQR 4-13). VTI was not associated with age at CTA. When controlling for diagnosis and race/ethnicity, higher VTI was associated with younger age at prophylactic surgery [HR 1.28, 95%CI 1.09-1.50 for every increase in VTI of 20 (VTI20)] and at surgery for TAD (HR 1.38, 95%CI 1.13-1.69 for VTI20). There was no difference in freedom from prophylactic or post-TAD surgery among LDS and MS subgroups (adjusted p=0.95 and p=0.17 respectively). In patients ≤18 years, the association between increased VTI and age at prophylactic surgery was strongest: HR 1.73 (95%CI 1.11-2.69 for VTI20). One patient ≤18 had TAD with VTI 105.

Conclusions:
Increased vertebral artery tortuosity is associated with earlier thoracic aortic surgery and dissection in patients under 50 years, and may be most predictive in children. VTI can identify patients at high risk for TAD who may benefit from targeted therapies.
Abstract 508: Heme-Loaded Microparticles May Connect Subclinical Intravascular Hemolysis to Endothelial Cell Injury in Type 2 Diabetes
Authors: Charue Dominique , Le Jeune Sylvain , Zaimedine Sarah , Bihan Hélène , Tedgui Alain , Boulanger Chantal , Blanc-Brude Olivier
Published: July 2021

Rational:
Type 2 Diabetes induces heavy hemoglobin glycation and discrete red blood cell (RBC) fragility. Intravascular hemolysis in type 2 diabetes is not a paramount clinical feature of the disease and it receives little attention. But hyperglycemia favors hemolysis and diabetes is associated to raised circulating levels of submicrometric cell membrane fragments from RBCs, called microparticles (MPs). We hypothesized that circulating diabetic MPs could be loaded with heme and participate in oxidative vascular injury.

Material and Methods:
A type 2 diabetic blood collection was assembled. Diabetic and healthy RBCs were separated from platelet-free plasma (PFP) by centrifugation. We purified MPs from native PFP, or from RBCs made to vesiculate in vitro with the thrombospondin-1-derived peptide 4N1-1. We characterized the size of MPs by dynamic light scattering, and quantified the heme contained in MPs by spectrophotometry, before and after MP depletion by ultracentrifugation. We exposed vascular endothelial cells to these different MP preparations and determined radical oxygen species (ROS) production with a fluorescent probe, as well as apoptosis by fluorescence microscopy and flow cytometry.

Results:
Purified diabetic and healthy RBCs released MPs expressing surface phosphatidylserine. MPs from type 2 diabetic RBCs were generally larger than healthy RBC MPs and contained significantly more heme. In cultured endothelial cells, MPs from diabetic RBCs stimulated the production of ROS in a dose- and time-dependant fashion, at concentrations observed in diabetic plasma. In contrast, healthy RBC MPs and hemoglobin or heme alone had little effects at such concentrations. Moreover, overproduction of ROS was followed by endothelial apoptosis after 24h, revealed by pyknotic nuclei and degraded chromatine, as well as reduced DNA contents by flow cytometry.

Conclusion:
Diabetic RBC can release MPs that are loaded with heme and trigger oxydative cell injury and apoptosis in cultured endothelial cells. These results identify for the first time the fact that heme-loaded MPs vector oxidative cell injury in type 2 diabetes. Diabetic RBC MPs may thus form a novel bridge between RBC glycation, sub-clinical intravascular hemolysis and diabetic vasculopathy.
Abstract 52: The BiKE Project: Gene Expression Signatures, Pathways and Networks in Human Carotid Atherosclerosis
Authors: Perisic Ljubica , Sun Ying , Razuvaev Anton , Folkersen Lasse , Roy Joy , Gabrielsen Anders , Hansson Göran K , Paulsson-Berne Gabrielle , Hedin Ulf
Published: July 2021

Embolism from atherosclerotic plaques in the carotid artery is a major cause of stroke, disability and death. The aim of this project was to explore gene expression signatures and clinical parameters of carotid plaque instability in the Biobank of Karolinska Endarterectomies (BiKE).

Whole transcriptome profiling by microarrays was performed on n=127 plaques from n=40 asymptomatic (AS) and n=87 symptomatic (S) patients, and on n=10 normal arteries. After normalization and gender adjustments, differential gene expression was assessed by multiple t-tests with Bonferroni correction. Gene set enrichment and pathway analyses were done using DAVID software. Genes significantly upregulated between S and AS plaques were associated with oxidative stress, immune system activation, exopeptidases, lipoprotein particle clearance and bone resorption, Transcription factors and markers of smooth muscle cells were downregulated in this comparison. When S patients with transitory ischemic attacks and minor stroke were compared with
amaurosis fugax
, most enriched GO terms were angiogenesis, oxidoreductases and inflammation. Patients on statins showed enrichment of calcification, osteoblast differentiation and negative regulation of angiogenesis. Prolonged immune response, lymphocyte activation and apoptosis were processes ongoing in plaques obtained >1 month compared to <2 weeks after symptoms. Many of the differentially expressed genes exhibited strong positive cross-correlations (Spearman r>0.90, p<10E-30) suggesting that they are simultaneously joined in an expression matrix within the plaques. When these genes were connected into a protein-protein interaction network using Cytoscape, several key nodes were discovered centered around HIF1a, ACTN2, HSPG1, CXCR4, CASP4, HSPA6, PDLIM7, S100A9/10, NOS1 and EGFR.

In conclusion, analysis of the largest existing global transcriptome database of carotid atherosclerosis reveals novel genes and pathways related to patient phenotype, confirms the important role of inflammation and lipid metabolism, and highlights the role of repair processes and lymphocyte activation in human atherosclerosis.
Abstract 524: Deletion of Methionine Sulfoxide Reductase A in Mice Increases Vascular Smooth Muscle Cell Proliferation and Neointimal Hyperplasia
Authors: Klutho Paula J , Scott Jason , Xie Litao , Grumbach Isabella M
Published: July 2021

Introduction:
Neointima formation is the leading cause of restenosis after balloon angioplasty and surgical endarterectomy. ROS production promotes neointimal hyperplasia and causes methionine oxidation. Recent data suggest that oxidation of methionines can alter specific protein activity and induce cell signaling. Methionine Sulfoxide Reductase A (MsrA) reverses methionine oxidation. In humans, two independent genome-wide association studies have found MsrA polymorphisms to correlate with increased ischemic cardiovascular disease.

Hypothesis:
MsrA protects against neointimal formation.

Methods:
Immunofluorescence for MsrA was performed in autopsy specimen of arteries with and without neointimal hyperplasia. 12-week old WT and MsrA-/- mice underwent ligation of the left common carotid artery and received two injections of BrdU prior to sacrifice. Fourteen days after ligation, the neointimal area was determined using Image J. Cell proliferation and apoptosis in the neointima were quantified by BrdU and TUNEL staining. Proliferation of vascular smooth muscle cells (VSMC) from MsrA-/- and WT mice was determined by cell counts and FACS analysis. Levels of cell cycle proteins, pAkt, pGSK3β were analyzed by immunoblot in MsrA-/- and WT VSMC, mRNA levels by qrtPCR.

Results:
MsrA was detected in all layers of the vascular wall and in neointima in humans. Neointimal area was significantly increased in MsrA-/- compared to WT mice 14 days post-ligation (n=5-9, p<0.05). Additionally, neointimas from MsrA-/- mice contained a higher percentage of proliferating cells (0.92±1.42% vs 8.89±8.89%, p=0.007). MsrA-/- VSMC displayed significantly increased proliferation compared to WT VSMC (p<0.05). Moreover, the progression to S phase was accelerated and protein levels of the cell cycle regulators Cyclin D and CDK4 were upregulated in MsrA-/- VSMC. However, cyclin D mRNA levels are not increased (p=0.24). This implies that MsrA post-translationally regulates cyclin D. GSK3β, which is inhibited by Akt, promotes cyclin D degradation. An increase in pAkt and pGSK3β was identified in MsrA-/- VSMC.

Conclusion:
These data indicate that MsrA regulates neointima formation after vascular injury and support a role for MsrA in the regulation of Akt signaling.
Abstract 533: Crucial Role of Rho-Kinase in Pressure Overload--Induced Right Ventricular Hypertrophy and Dysfunction in Mice: A Possible Novel Therapeutic Target of Right Ventricular Failure
Authors: Ikeda Shohei , Satoh Kimio , Kikuchi Nobuhiro , Miyata Satoshi , Suzuki Kota , Omura Junichi , Shimizu Toru , Fukumoto Yoshihiro , Sakata Yasuhiko , Shimokawa Hiroaki
Published: July 2021

Rationale:
Right ventricular (RV) failure is the leading cause of death in various cardiopulmonary diseases, including pulmonary hypertension. It is generally considered that the RV is vulnerable to pressure-overload as compared with the left ventricle (LV). However, as compared with LV failure, the molecular mechanisms of RV failure are poorly understood.

Objective:
We aimed to identify molecular therapeutic targets for RV failure in a mouse model of pressure-overload.

Methods and Results:
To induce pressure-overload to respective ventricles, we performed pulmonary artery constriction (PAC) or transverse aortic constriction (TAC) in mice. We first performed microarray analysis and found that the molecules related to RhoA/Rho-kinase and integrin pathways were significantly up-regulated in the RV with PAC compared with the LV with TAC. Then, we examined the responses of both ventricles to chronic pressure-overload in vivo. We demonstrated that compared with TAC, PAC caused greater extents of mortality, Rho-kinase expression (especially ROCK2 isoform) and oxidative stress in pressure-overloaded RV, reflecting the weakness of the RV in response to pressure-overload. Additionally, mechanical stretch of RV cardiomyocytes from rats immediately up-regulated ROCK2 expression (not ROCK1), suggesting the specific importance of ROCK2 in stretch-induced responses of RV tissues. Furthermore, mice with myocardial-specific overexpression of dominant-negative Rho-kinase (DN-RhoK) showed resistance to pressure-overload-induced hypertrophy and dysfunction associated with reduced oxidative stress. Finally, DN-RhoK mice showed a significantly improved long-term survival in both PAC and TAC as compared with littermate controls.

Conclusions:
These results indicate that the Rho-kinase pathway plays a crucial role in RV hypertrophy and dysfunction, suggesting that the pathway is a novel therapeutic target of RV failure in humans.
Abstract 538: Docosahexaenoic Acid--Derived Proresolving Lipid Mediators Modulate Flow-Induced Vascular Remodeling in Mice
Authors: Akagi Daisuke , Chatterjee Anuran , Chen Mian , Toy Robert , Conte Micheal S
Published: July 2021

Objective:
Recent evidence suggests that specialized lipid mediators derived from polyunsaturated fatty acids control resolution of inflammation. Aberrant remodeling following vascular injury is associated with protracted inflammation. We examined the hypothesis that DHA-derived resolvin D2 (RvD2) and maresin-1 (Mar1), attenuate flow-induced arterial remodeling.

Methods and Results:
Adult FVB mice (N=30) underwent unilateral carotid arterial ligation with administration of RvD2, Mar1, or vehicle (100ng IP on days 0, 1, 3, 5, 7). In ligated carotid arteries, cell proliferation, neutrophil and macrophage recruitment were significantly reduced at 4d by both active treatments. Neointimal hyperplasia at 14d was attenuated by RvD2 (67%) and Mar1 (62%), respectively (Figure). In-vitro, RvD2 and Mar1 produced dose-dependent inhibition of mouse aortic vascular smooth muscle cell (VSMC) migration (IC50@1 nM) and reduced proinflammatory gene expression (MCP-1).

Conclusion:
Systemic administration of pro-resolving lipid mediators RvD2 and Mar1 attenuates flow-induced remodeling in mice, likely via effects on leukocyte trafficking and VSMC phenotype.
Abstract 545: Generation of Oxidized Phosphatidylcholines During Hypoxia-Reoxygenation Triggers Cell Death of Postnatal Cardiac Myocytes
Authors: Hasanally Devin , Margulets Victoria , Chaudhary Rakesh , Kirshenbaum Lorrie A , Ravandi Amir
Published: July 2021

Reperfusion of ischemic heart tissue leads to a rapid burst of reactive oxygen species which initiates a cascade of events that cumulate in cell death. The most susceptible biomolecules to oxidative injury are membrane associated phospholipids. Oxidized phospholipids including oxidized phosphatidylcholines (OxPC), have shown to be potent activators of the intrinsic apoptotic signaling cascade. We hypothesize that the production of OxPC within hypoxic myocytes mediates damage from hypoxia-reoxygenation (H-R) by triggering inappropriate cardiac myocytes death. Hence, we determined whether, bioactive OxPC are produced in cardiomyocytes during H-R, and whether OxPC species induces apoptotic response resulting in cell death of cardiomyocytes.

Post-natal rat cardiomyocytes were isolated from 2 d old rat pups and submitted to primary culture in serum-free medium. Cardiac myocytes were subjected to hypoxia (H) for 1 hour in a specialized gas-tight chamber under continuous nitrogen atmosphere followed by 1 hour reoxygenation (R). Lipidomic analysis of cardiac myocytes following H-R was determined by high performance liquid chromatography linked to tandem mass spectrometry was used to quantitate 82 distinct OxPC species from cell lipid extracts. Cell viability of cardiomyocytes was assessed in the absence and presence of known OxPC molecules (POVPC, PONPC, PGPC, PAzPC) and control non-oxidized PSPC.

Lipidomic analysis of OxPC species showed increased generation of fragmented OxPC after H-R (10.20±0.67μg/mL vs1.95±0.24μg/mL, p<0.005) when compared to control cells. Cells exposed to POVPC and PONPC showed marked dose dependent increase in cell death compared to equal molar concentration of PSPC. There was also marked increase in mitochondrial permeability upon exposure to fragmented OxPC molecules.

To our knowledge this is the first demonstration that OxPC are generated within post-natal cardiomyocytes during H-R and potent inducers of cell death of cardiac myocyte during H-R. Interventions designed to modulate the synthesis of cytotoxic OxPC during H-R may prove beneficial in suppressing aberrant cardiac myocyte cell death and loss of ventricular function in patients with ischemic heart disease or following myocardial infarction.
Abstract 550: Hypercholesterolemia Inhibits LPS-Induced CCR7-Dependent Reverse Transmigration of Arterial Intimal Dendritic Cells.
Authors: Roufaiel Mark , Gracey Eric , Siu Allan , Zhu Sunning , Lau Andrew , Jongstra-Bilen Jenny , Inman Robert , Cybulsky Myron I
Published: July 2021

Dendritic cells (DCs) play a key role in chronic inflammatory diseases such as atherosclerosis. Myeloid cells with features of DCs are abundant in the normal arterial intima of mice, in regions that are predisposed to atherosclerosis. Upon induction of hypercholesterolemia, these intimal DCs rapidly engulf lipids and transform into the initial foam cells of nascent atherosclerotic lesions. The function of intimal DCs in normal mice remains unknown. We observed that systemic stimulation of toll-like receptors (TLRs), specifically TLR4 by LPS and TLR3 by Poly(I:C), induced a rapid reduction in the number of DCs in the normal aortic intima. Absence of TUNNEL staining and use of CD11c-hBcl2 transgenic mice suggested that apoptosis did not account for the loss of intimal DCs. Immuno-gold staining for CD11c coupled with scanning electron microscopy of the aortic surface and
ex vivo
experiments determined that reverse transmigration (RTM) through the intact endothelial monolayer accounts for LPS-induced reduction of intimal DCs. Intimal DC loss was blocked by pretreatment of wild type mice with pertussis toxin or function-blocking anti-CCL19 antibody, and did not occur in CCR7-/- and Plt (CCL19-/- and CCL21-/-) mice. Assessment of CCR7, CCL19, and CCL21 mRNA expression and reciprocal bone marrow transplantation between CCR7-/- or Plt and wild type mice confirmed that RTM is dependent on CCR7 and CCL19 expression by intimal DCs. Feeding LDLR-/- mice a cholesterol rich diet (CRD) for just one week induced foam cell formation and inhibited LPS-induced RTM; however, CCR7 and CCL19 mRNA induction by LPS was not inhibited. These findings suggest that in the normal mouse aorta, in response to inflammatory stimuli, RTM of RIDCs is dependent on CCR7 signaling; whereas in the setting of hypercholesterolemia, CCR7 signaling is altered in lipid-loaded intimal DCs, which inhibits their RTM. Future experiments will investigate the possibility to overcome this inhibition, so that lipid can be removed from the artery wall by exiting lipid-loaded RIDCs.
Abstract 554: Multimerin2 Influences Vascularization Through Modulation of Perlecan
Authors: Burgisser Petra E , Tempel Dennie , Hermkens Dorien , Schulte-Merker Stefan , Cheng Caroline , Duckers Henricus J
Published: July 2021

Vascular development is crucial in normal physiology and pathophysiology, and is in part orchestrated by a dynamic balance between extracellular matrix (ECM) and endothelial cells. The molecular mechanisms that govern this balance and as such play a role in the formation of new blood vessels remain largely unknown.

In order to identify new genes involved in blood vessel formation, we performed a genome-wide gene expression array analysis in Flk1+ angioblasts during mouse development using a commercial platform (Affymetrix). Multimerin2 (Mmrn2) expression was found to be upregulated in angioblasts and in the developing vasculature of zebrafish and mice.

In vitro
, using immunofluorescence, Mmrn2 was detected outside of human aortic endothelial cells (HAECs), were it co-localized with the extracellular matrix components perlecan, laminin and fibronectin. Direct interaction with these proteins was confirmed using a combination of co-immunoprecipitation followed by proteomics (LC-MS), and western blot to identify possible binding partners.

Mmrn2 gain-of-function and loss-of-function
in vitro
studies, using Mmrn2 siRNA and adenoviral constructs likewise decreased and increased the expression of its binding partner perlecan. Perlecan is a well known modulator of the pro-angiogenic factor, FGF2, and is required for further signaling through the FGF receptor. Therefore, Mmrn2 may influence FGF2 signaling through perlecan expression and complex formation.

Subsequent
in vitro
studies using an adenoviral construct to induce Mmrn2 overexpression resulted in a G1-arrest in HAECs (P<0.001, N=8). In FGF2 depleted medium a G1 arrest was also observed (P<0.05, N=3), Mmrn2 overexpression combined with FGF2 depleted medium did not enhance the G1 arrest, suggesting a role of Mmrn2 in FGF2 signaling.

In addition, Mmrn2 silencing or overexpression in HAECs led to a loss of ECM organization or improved organization, respectively. In line, the binding partner of Mmrn2, perlecan, has been shown to be involved in stabilization of the ECM.

These findings suggest that Mmrn2 plays a role during blood vessel formation by binding to perlecan and the ECM, thereby modulating FGF2 signaling and ECM organization and subsequently, regulating endothelial cell proliferation.
Abstract 558: Microchanneled Polysaccharide Hydrogel Laden With Endothelial Cells and Mesenchymal Stem Cells With VEGF Facilitate Formation of Vascular Structures and Are Effective for Repairing Tissue Ischemia
Authors: Lee Sangho , Lee Min Kyung , Kong Hyunjoon , Yoon Young-sup
Published: July 2021

Various hydrogels are used to create vascular structure in vitro or to improve cell engraftment to overcome low cell survival in vivo, a main hurdle for bare cell therapy Recently we developed a modified alginate hydrogel within which microchannels are aligned to guide the direction and spatial organization of loaded cells. We investigated whether these cell constructs in which HUVECs and human mesenchymal stem cells (hMSCs) are co-loaded in this novel microchanneled hydrogel facilitate formation of vessels in vitro and in vivo, and enhance recovery of hindlimb ischemia.

We crafted a modified alginate hydrogel which has microchannels, incorporates a cell adhesion peptide RGD, and was encapsulated with VEGF. We then compared vascular structure formation between the HUVEC only (2 x 105 cells) group and the HUVEC plus hMSC group. In the HUVEC+hMSC group, we mixed HUVECs and hMSCs at the ratio of 3:1. For cell tracking, we labeled HUVECs with DiO, a green fluorescence dye. After loading cells into the microchannels of the hydrogel, these constructs were cultured for seven days and were examined by confocal microscopy. In the HUVEC only group, HUVECs stands as round shaped cells without forming tubular structures within the hydrogel. However, in the HUVEC+hMSC group, HUVECs were stretched out and connected with each other, and formed vessel-like structure following pre-designed microchannels. These results suggested that hMSCs play a critical role for vessel formation by HUVECs. We next determined their in vivo effects using a mouse hindlimb ischemia model. We found that engineered HUVEC+hMSC group showed significantly higher perfusion over 4 weeks compared to the engineered HUVEC only group or bare cell (HUVEC) group. Confocal microscopic analysis of harvested tissues showed more robust vessel formation within and outside of the cell constructs and longer term cell survival in HUVEC+hMSC group compared to the other groups.

In conclusion, this novel microchanneled alginate hydrogel facilitates aligned vessel formation of endothelial cells when combined with MSCs. This vessel-embedded hydrogel constructs consisting of HUVECs and MSCs contribute to perfusable vessel formation, prolong cell survival in vivo, and are effective for recovering limb ischemia.
Abstract 562: High-Throughput Analysis of Compounds Targeting Zebrafish Angiogenesis and Vascular Development
Authors: Mauro Antonio , El-Rass Suzan , Wang Youdong , Zhang Haibo , Wen Xiao-Yan
Published: July 2021

Introduction:
Angiogenic sprouting occurs in the presence of high levels of exogenous pro-angiogenesis factors. Pathological over expression of pro-angiogenesis factors leads to excessive tumor growth. Inhibition of pro-angiogenesis factors can restrict tumor growth. But, prolonged inhibition can lead to the activation of alternate pro-angiogenesis pathways. The objective of this research is to discover compounds that inhibit multiple pro-angiogenesis factors and increase efficacy in the treatment of cancerous tumors using zebrafish vascular development as a model in a high throughput setting.

Methods:
The NIH Clinical Collections Libraries I and II will be screened with a fully automated high throughput drug screening platform (HTS). Compounds that cause abnormal vascular phenotypes will be identified on Tg(flk1:EGFP) embryos. The lead compounds will then be analyzed with 3-D confocal imaging, studies on cultured human umbilical vein endothelial cells (HUVECs), and animal cancer models.

Results:
The HTS was constructed and made operational. A protocol for anti-angiogenesis screening with Tg(flk1:EGFP) zebrafish has been developed and validated with indirubin 3’ monoxime (I3M), a known angiogenesis inhibitor. Pixel counting (to quantify the overall vascular network) and ISV counting (to count the number of ISVs and give an indication of overall vascular morphology) programs were created to provide readouts from confocal images taken within the HTS. Normative parameters, including several endothelial cell counts and various vessel length measurements, were characterised at the 1 and 2 days post fertilization (dpf) stages using 3-D confocal imaging. These values will be compared to values found in the presence of lead compounds to validate them as angiogenesis inhibitors.

Conclusions & Future Work:
The framework for a robust drug discovery process has been put in place. The next step is to screen the NIH Clinical Collections Libraries I and II and analyze the efficacy and mechanism of lead compounds. Based on data from previous manual screens, 5-10 lead compounds are expected to be identified. These compounds will have the potential to be developed as repurposed anti-angiogenesis compounds and improve therapeutics for cancerous tumors.
Abstract 576: ABO Blood Group and Platelet Aggregation
Authors: Zhong Ming , Terembula Karen , Tsai Andrew , Zhang Hanrui , Kapoor Shiv , Reilly Muredach P
Published: July 2021

Background:
ABO blood groups have been linked to coronary thrombosis, with non-O blood groups at higher risk of thrombosis. ABO blood group antigens are expressed on platelet glycoproteins, including the IIb fibrinogen receptor, and may affect platelet function via glycan modifications. We hypothesized that non-O blood groups would demonstrate higher levels of platelet aggregation compared to type O blood group.

Methods:
Healthy, non-smoking subjects were recruited to a protocol investigating glycan modification of platelets. Ex vivo platelet aggregation was assessed by light transmission aggregometry of platelet-rich plasma in dose-response to collagen (0.04-0.2 mg/mL) and thrombin receptor activating peptide (TRAP) (1-5 μmol). Maximal platelet aggregation data, aggregation rate and lag time were compared at each concentration across blood groups A, B, AB and O by ANOVA testing.

Results:
A total of 91 subjects were recruited (ABO types; 32 A, 16 B, 8 AB, 35 O). The average age of the subjects was 31 years, 58% were Caucasian and 61% were female. There were no significant differences in age, race and gender between ABO blood groups. For collagen concentrations ranging from 0.04-0.2 mg/mL, there was no significant difference in mean platelet aggregation, rate of aggregation and lag time between blood groups. For TRAP concentrations between 1-5 μmol, there was also no significant difference in mean platelet aggregation and rate of aggregation between blood groups. For TRAP at 2 μmol and 3 μmol, blood group AB demonstrated a longer lag time compared to blood groups A, B, and O (TRAP 2 μmol; 13 sec type AB vs 5.21 sec type A, 4.18 sec type B, 3.57 sec type O, TRAP 3 μmol; 9.12 sec type AB vs 3.34 sec type A, 4.31 sec type B, 2.65 sec type O) but these secondary analyses were of marginal significance after consideration of multiple testing.

Conclusion:
These data for ex vivo platelet aggregation dose-response to collagen and TRAP do not support significant differences in platelet function by ABO blood type. Further studies with larger sample sizes and of specific platelet glycoprotein functions are required to address potential subtle influences of ABO glycans on platelet reactivity.