bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 499: Increased Lysine Acetylation After ‘Back-table’ Preparation of Human Saphenous Vein
Authors: Voskresensky Igor , Hocking Kyle , Osgood Michael , Cheung-Flynn Joyce , Komalavilas Padmini , Brophy Colleen
Published: July 2021

BACKGROUND

Back-table preparation of human saphenous vein (SV) for surgical bypass results in conduit injury. We hypothesized that surgical manipulation results in increased total lysine acetylation, a global marker of injury. We examined effect of injury on acetylation of NFkB, a transcription factor involved in vascular smooth muscle proliferation.

METHODS

Paired SV samples were collected prior to surgical manipulation (UM) and after distention and marking (AM). SV rings were suspended in a muscle bath for measurement of contractile force in response to KCl and phenylephrine (PE); endothelial-dependent relaxation in response to carbachol (CCh); and endothelial-independent relaxation in response to sodium nitroprusside (SNP). Western blot analysis was used to measure total lysine and NFkB acetylation, results were normalized to GAPDH.

RESULTS

AM-SV generated significantly reduced physiologic parameters compared with UM-SV, including response to KCl (0.05 vs. 0.14 N/m
2
, n=9, p<0.05), PE (0.04 vs. 0.08 N/m
2
, n=7, p<0.05), endothelial-dependent relaxation (3.8% vs. 17.2%, n=7, p<0.05), and endothelial-independent relaxation (25.1% vs. 52.9%, n=7, p<0.05). Total lysine acetylation was increased in AM-SV compared to UM-SV (Fig1A), as was acetylation of NFkB (Fig1B).

CONCLUSIONS

Surgical preparation of SV results in physiologic dysfunction, and leads to increase in total lysine and NFkB acetylation. Increases in total lysine acetylation may be a global marker of injury and are associated with senescence. Increased acetylation of NFkB may correlate with the uncontrolled proliferative response of intimal the hyperplastic lesions.
Abstract 514: Cilostazol Attenuated Angiotensin II-induced Abdominal Aortic Aneurysms Thorough Anti-inflammatory Effect in Apolipoprotein E-deficient Mice
Authors: Umebayashi Ryoko , Uchida Haruhito A , Makino Hirofumi
Published: July 2021

Objective

Chronic angiotensin II (AngII) infusion promotes development of abdominal aortic aneurysms (AAAs) in mice. Previous studies have shown that local inflammation plays an important role on formation of AAAs. Cilostazol, a phosphodiesterase-3 inhibitor with antiplatelet aggregation and vasodilatory effects, exerts vasoprotective effect by inhibiting inflammation and superoxide generation in several experimental studies. The purpose of this study was to evaluate whether cilostazol influenced AngII-induced AAAs.

Methods and Results

Male apolipoprotein E deficient mice (8-12 weeks old) were fed with either normal diet or cilostazol-containing (0.1% wt/v) diet. After 1 week of cilostazol administration, mice were infused subcutaneously with either AngII (1,000 ng/kg/min, n = 16 -19) or saline (n = 5 - 6) by osmotic minipumps for 4 weeks. AngII equivalently increased systolic blood pressure, irrespective of cilostazol administration. Cilostazol had no effect on serum cholesterol concentrations, triglycerides, high-density lipoprotein-cholesterol, body weights, heart rates, and systolic blood pressures. AngII infusion significantly increased ex vivo maximal diameters of abdominal aortas that were attenuated by cilostazol administration (1.94 ± 0.16 mm vs 1.53 ± 0.17 mm, P < 0.05). Cilostazol diminished AngII-induced increase of aortic MMP-2 activity and ICAM-1 protein expression as determined by gelatin zymography and Western blot, respectively (P < 0.05, each). Aortic mRNA expression of inflammatory cytokines such as MCP-1, IL-1β, osteopontin, and COX-2 was reduced by cilostazol administration (P < 0.05). In vitro, TNF-α increased MCP-1, ICAM-1 and VCAM-1 mRNA expression in mouse aortic endothelial cells. These enhancements were decreased by incubation with cilostazol (P < 0.05).

Conclusion

Cilostazol attenuated AngII-induced AAAs thorough its anti-inflammatory effect in male apolipoprotein E deficient mice.
Abstract 522: TGF-beta Neutralization Augments Development of Angiotensin II-induced Aneurysms in Both Ascending and Abdominal Aortic Regions
Authors: Chen Xiaofeng , Rateri Debra L , Howatt Deborah A , Balakrishnan Anju , Moorleghen Jessica J , Cassis Lisa A , Daugherty Alan
Published: July 2021

Introduction and Objectives

Angiotensin II (AngII) infusion induces ascending and abdominal aortic aneurysms (AAs) in mice. In a mouse model of Marfan Syndrome expressing Fbn1
C1039G/+
, ascending AAs were reduced by administration of a transforming growth factor-beta (TGF-beta) neutralizing antibody. In contrast, administration of TGF-beta neutralizing antibodies to AngII-infused mice increased aortic rupture. The purpose of this study was to compare the effects of TGF-beta neutralization on formation and progression of AngII-induced ascending and abdominal AAs.

Methods and Results

Male C57BL/6 mice were fed a normal diet and infused subcutaneously with AngII (1,000 ng/kg/min). Five days prior to initiating infusion, mice were injected i.p. with either a mouse monoclonal TGF-beta antibody (1D11) or an isotype matched IgG at a dose of either 0.3 or 5 mg/kg x 3/per week. 1D11 administration significantly decreased serum TGF-beta concentrations. TGF-beta neutralization at 5 mg/kg greatly increased the incidence of aortic rupture, which was attributed to rupture in both the ascending and abdominal regions. For mice that remained viable after 28 days of infusion, there were equivalent increases in aortic dilation in both the ascending and abdominal regions. Prior to rupture, aortic diameters determined by ultrasound demonstrated no significant effect on AngII-induced dilation of the ascending or abdominal aorta. We also studied the effects of TGF-beta neutralization in mice with established AngII-induced AAs following AngII-infusion for 28 days. C57BL/6 mice were injected with the mouse TGF-beta neutralizing antibody or IgG control (5 mg/kg x 3/per week, n=10 per group), while AngII infusion was continued for a further 28 days. Although TGF-beta antibody administration significantly decreased serum TGF-beta concentrations in mice with established AAs, there was no effect on aortic rupture or dilation of either the ascending or abdominal aortic region.

Conclusion

TGF-beta inhibition augmented AngII-induced aortic rupture in both the ascending and abdominal regions but had no effect on dilation. Furthermore, TGF-beta neutralization had no effect on either aortic rupture or expansion in established AAs.
Abstract 527: A Novel Stem Cell Delivery Model for Evaluation of Delivery Using Targeted Bifunctional Liposomes with Ultrasound Enhancement
Authors: Naji Ali K , Wassler Michael , Britton George , Shelat Harnath S , Geng Yong-Jian , McPherson David D , Klegerman Melvin E
Published: July 2021

Introduction.

We have developed bifunctional echogenic liposomes (BF-ELIP) targeted to both CD34 and ICAM-1 to facilitate delivery of CD34+ stem cells to inflammatory endothelium. We previously confirmed that BF-ELIP enhanced CD34+ stem cell adherence to ICAM-1-expressing endothelium in vitro and in vivo and showed that ultrasound (US) facilitated penetration of stem cells into the endothelial cell layer.

Hypothesis.

The transwell culture system can serve as an in vitro model for study of US-enhanced targeted delivery of stem cells to atheroma.

Methods.

BF-ELIP were prepared by evaporation-rehydration-sonication-lyophilization, followed by conjugating antibodies specific for CD34 and ICAM-1 through a thioether linkage. TNFα-pretreated HUVEC monolayers on transwell (6 wells/plate) insert membranes were incubated with nonspecific IgG-ELIP or BF-ELIP (1mg/well) for 15 minutes, followed by human monocytes labeled with Oregon Green. Half the inserts were subjected to 6 MHz color Doppler ultrasound (MI = 0.4) for 5 minutes. Fluorescence of resuspended cells was measured after treatment of both inserts and wells with 0.25% trypsin/0.1% EDTA 24 hours later.

Results.

BF-ELIP enhanced adherence of monocytes to the ICAM-1-expressing HUVEC monolayer relative to untreated controls and IgG-ELIP, but did not increase the number (Fig. 1) or proportion (Fig. 2) of monocytes traversing the monolayer. US greatly increased the number of monocytes both adhering to and passing through the monolayer in all groups.

Conclusions.

We have succeeded in developing a transwell cultured HUVEC system as a model for US-enhanced, BF-ELIP-mediated stem cell delivery to inflammatory endothelium.
Abstract 544: Meox1, a Novel Regulator in Tgf-ß-induced Smooth Muscle Cell Differentiation
Authors: Dong Kun , Hsu Amanda , Chen Shiyou
Published: July 2021

Smooth muscle cell (SMC) differentiation is an important process during vasculogenesis and angiogenesis. It is well known that alterations of VSMC phenotype play a role in the progression of several cardiovascular disorders including atherosclerosis, hypertension, and restenosis. However, the molecular mechanisms controlling the differentiation and development of VSMC are largely unknown. Mesenchyme homeobox 1 (MEOX1) is expressed in the early developing somite and derivatives during embryogenesis. Somite has been shown to be one of the origins or progenitors for SMC. Therefore, in this study, we studied the role of MEOX1 in SMC differentiation. We found that transforming growth factor-β (TGF-β) induces MEOX1 expression in the initial phase of SMC differentiation of pluripotent mesenchymal C3H10T1/2 cells. MEOX1 expression appears to be mediated by both PI3 kinase and Smad3 signaling pathways because blockade of these two pathways by specific inhibitors leads to a decreased expression of MEOX1 and SMC markers. Importantly, knockdown of MEOX1 by specific shRNA suppresses TGF-β-induced expression of SMC early markers including SM22α and calponin. MEOX1 overexpression, on the other hand, increases SMC marker expression. These results indicate that MEOX1 is a novel regulator for TGF-β-induced SMC differentiation.
Abstract 566: The Transcriptome of the Human Carotid Atherosclerotic Plaque
Authors: Poroyko Valeriy , McCormick Susan , He Qi , Skelly Christopher L
Published: July 2021

Ischemic stroke is a complex disease process with carotid atherosclerosis as the major etiologic source. To better understand the biology of a vulnerable plaque we carried out whole transcriptome shotgun sequencing of human carotid plaques.

Methods

5 carotid atherosclerotic plaques were obtained at the time of operation (2 symptomatic, 2 asymptomatic, and 1 control). RNA was isolated and 5 cDNA libraries were constructed and sequenced with single-reads100nt in length using one line of flow cell of HySeq 2000 (Illumina Inc). Standard bioinformatic techniques were used to ensure quality screening of raw reads. Ingenuity Systems IPA software was used to determine canonical biological pathways overrepresented in plaques. We compared our data to the data of Illumina Human Body Map processed by a similar analytical pipeline. Linkage analysis was performed.

Results

Among detected pathways were “Atherosclerosis Signaling” and pathways involved in inflammation and cell differentiation. We analyzed gene expression data by creating a distance matrix based on Jaccard similarity measure (Fig 1). When gene expression values were used to create a matrix of Euclidian distances between known transcriptomes, four specimens including the control clustered with heart and muscle. The transcriptome from the stroke patient was more similar to lung, liver and heart.

Conclusion

Whole transcriptome analysis of carotid plaques is feasible and ontologically accurate. We demonstrate linkage differences between symptomatic and asymptomatic plaques and biological pathways involved in vascular lesion formation. Further analysis may provide insight into the vulnerable plaque pathobiology.
Abstract 57: Natural Killer Cells Drive Angiotensin II-Induced Vascular Dysfunction Dependent on the T-bet/IFN-gamma Axis
Authors: Kossmann Sabine , Schwenk Melanie , Hausding Michael , Hu Hanhan , Schmidgen Maria I , Schönfelder Tanja , Oelze Matthias , Daiber Andreas , Muenzel Thomas , Becker Christian , Wenzel Philip
Published: July 2021

Rationale

Immune cells contribute to angiotensin II (ATII) induced vascular dysfunction and inflammation. However, the mechanisms of recruitment and immune effector pathways remain incompletely understood.

Objective

We tested the hypothesis that interferon-gamma (IFN-γ) and natural killer (NK) cells play a pivotal role in ATII-driven vascular inflammation.

Methods and results

IFN-γ
-/-
and Tbx21
-/-
mice were partially protected from ATII-induced vascular endothelial and smooth muscle dysfunction, whereas mice overexpressing IFN-γ showed constitutive vascular dysfunction. Absence of T-bet, the IFN-γ transcription factor encoded by Tbx21, reduced vascular superoxide and peroxynitrite formation and attenuated expression of NADPH oxidase subunits as well as inducible NO synthase, monocyte chemoattractant protein 1 and interleukin 12 in aortas of ATII-infused mice. Compared to controls, IFN-γ
-/-
and Tbx21
-/-
mice were characterized by reduced ATII-mediated vascular recruitment of both NK1.1
+
NK-cells as the major producers of IFN-γ and CD11b
+
Gr-1
low
IL-12 secreting monocytes. Selective depletion and adoptive transfer experiments identified NK-cells as essential contributors to vascular dysfunction and showed that T-bet
+
LysM
+
myelomonocytic cells were required for NK-cell recruitment into vascular tissue and local IFN-γ production.

Conclusion

We provide first evidence that NK-cells play an essential role in ATII-induced vascular dysfunction. In addition, we disclose the T-bet-IFN-γ pathway and mutual monocyte-NK-cell activation as potential therapeutic targets in cardiovascular disease.
Abstract 58: Signal Transducer and Activation of Transcription (STAT) 4 Deficiency Reduces Adipose Tissue Inflammation and Atherosclerosis
Authors: Dobrian Anca D , Hatcher Margaret , Sundquist Monica , McPheat William , Nadler Jerry L
Published: July 2021

Adipose tissue (AT) inflammation is key for the development of insulin resistance (IR) and contributes to progression of atherosclerosis. Previous work from our group showed that STAT4 deficiency is protective for IR in obesity and reduces atherosclerosis. However, the mechanisms are not completely understood. In this study we examined the effect of STAT4 deficiency on peri-vascular and visceral AT inflammation in atherosclerosis. To this purpose we: i) generated STAT4-deficient apo E-deficient (

Stat-4
-/-
Apoe
-/-

) mice and, ii) used a novel small molecule inhibitor of the IL12/STAT4 pathway (Cpd9) in

Apoe
-/-

mice. Compared to

Apoe
-/-

controls,

Stat-4
-/-
Apoe
-/-

female mice fed Western diet (WD) for 12 weeks showed a ~40% reduction in the thoracic aorta lesion area. Peri-aortic AT immune cells analyzed by flow cytometry showed a significant (p<0.001) and prominent decrease in CD45+CD3+ (3-fold), CD3+CD8+ (10-fold) and Nk46+ (9-fold) cells in

Stat-4
-/-
Apoe
-/-

vs.

Apoe
-/-

mice. Also, the percentage of CD206+/F4/80+ macrophages was increased by ~8.5-fold, while the CD45+/F4/80+ cells were reduced by ~5-fold in the

Stat-4
-/-
Apoe
-/-

mice (p<0.01). In addition gene expression of TNFα and IL12p40 were both reduced by 3.5 and 6-fold, respectively in

Stat-4
-/-
Apoe
-/-

, while PPARγ expression was increased by 2-fold (p<0.05). Peri-gonadal adipose tissue showed similar reduction in CD8+ and NK cell infiltration in STAT4 deficient mice but no differences were found in macrophage numbers or phenotype. The short-term effects of IL12/STAT4 inhibition on AT inflammation were examined in

Apoe
-/-

mice on 7 days WD alone or with oral Cpd9 treatment (0.3mmol/kg). Gene array analysis showed between 2.5- and 6-fold reduction of several cytokines and chemokines such as Ccl2, Ccl12, Cxcl13. Also, adiponectin as well and Bmp2, 4 and 7 were 4- to 15-fold reduced, suggesting reduced inflammation and possibly adipogenesis. Vegfa expression was one of the 4 up-regulated genes by 2.5-fold in Cpd9 treated mice. Collectively, data show that STAT4 reduction has early beneficial effects on AT inflammation and that peri-aortic AT inflammation is dramatically reduced. Thus targeting STAT4 may be a novel approach to treat atherosclerosis by reducing peri-vascular AT inflammation.
Abstract 160: Perivascular Administration of Rapamycin Through Nanoparticles Prolongs Inhibition of Arterial Restenosis
Authors: Chen Guojun , Shi Xudong , Guo Lian-Wang , Si Yi , Zhu Men , Pilla Srikanth , Liu Bo , Gong Shaoqin , Kent K Craig
Published: July 2021

Open vascular reconstructions frequently fail due to the development of recurrent disease or intimal hyperplasia (IH). This paper reports a novel drug delivery method using a rapamycin-loaded poly(lactide-co-glycolide) (PLGA) nanoparticles (NPs)/pluronic gel system that can be applied periadventitially around the carotid artery immediately following the open surgery.
In vitro
studies revealed that rapamycin dispersed in pluronic gel was rapidly released over 3 days whereas release of rapamycin from rapamycin-loaded PLGA NPs embedded in pluronic gel was more gradual over 4 weeks. In cultured rat vascular smooth muscle cells (SMCs), rapamycin-loaded NPs produced durable (14 days versus 3 days for free rapamycin) inhibition of phosphorylation of S6 kinase (S6K1), a downstream target in the mTOR pathway. In a rat balloon injury model, periadventitial delivery of rapamycin-loaded NPs produced inhibition of phospho-S6K1 14 days after balloon injury. Immunostaining revealed that rapamycin-loaded NPs reduced SMC proliferation at both 14 and 28 days whereas rapamycin alone suppressed proliferation at day 14 only. Moreover, rapamycin-loaded NPs sustainably suppressed IH for at least 28 days following treatment, whereas rapamycin alone produced suppression on day 14 with rebound of IH by day 28. Since rapamycin, PLGA, and pluronic gel have all been approved by the FDA for other human therapies, this drug delivery method could potentially be translated into human use quickly to prevent failure of open vascular reconstructions.
Abstract 168: Splice Variants of Tissue Factor Determine the Coagulant State of Endothelial Cells and Modulate Vascular Stabilization/Regression
Authors: de Boer Hetty C , Versteeg Henri H , van Oeveren-Rietdijk Annemarie M , Reinders Marlies E , de Vries Dorotthya K , Schaapherder Alexander F , Bogdanov Vladimir Y , Rabelink Ton J , van Zonneveld Anton Jan
Published: July 2021

Introduction:
Recruitment of pericytes (PC), critical to microvascular maturation, requires deposition of basement membrane proteins such as von Willebrand factor (vWF). Loss of PCs ultimately leads to vessel regression and rarefaction.

Hypothesis:
Splice variants of tissue factor (TF) stabilize the vasculature by supporting EC-PC interactions.

Results:
We determined that alternatively spliced TF (asTF) resides in Weibel Palade bodies (WPB) of ECs. Overexpression of KLF-2 enhanced asTF protein levels in WPBs. In confluent EC monolayers, asTF was deposited into the extracellular matrix (ECM). In assays employing vascular plexus remodelling on 3D-basement membrane and purified recombinant asTF or full-length TF (flTF), only asTF improved EC-PC interactions. At sites of low KLF-2 expression (low or turbulent flow), total TF mRNA was diminished, leading to low asTF expression. However, upon EC activation by TNFα, total TF mRNA levels rapidly increased and TF protein expression shifted from the ECM-deposited asTF to luminally expressed flTF. Interestingly, flTF-driven EC conversion to a procoagulant state was associated with the release of vWF and asTF from WPBs, depleting the intracellular depots from these proteins. Immunohistochemical staining of kidney specimens of human living donors confirmed that asTF was present at high levels in WPBs of microvascular endothelium. Moreover, after 45 minutes of reperfusion of the transplanted kidney, asTF- and vWF-staining was markedly reduced, consistent with their release from WPB following an inflammatory insult.

Conclusion:
We show for the first time that quiescent, KLF-2 expressing ECs predominantly generate non-coagulant asTF, which is stored in WPBs and serves to stabilize the microvasculature by supporting EC-PC interactions. In contrast, in low-flow zones or sites with disturbed flow (e.g. at bifurcations), KLF-2 is downregulated, leading to decreased production of asTF and impaired stabilization of the vasculature. During inflammation, acute perturbation of ECs (e.g. exposure to TNFα) is associated with downregulation of KLF-2 and a post-transcriptionally regulated increase in the production of flTF, resulting in a pro-coagulant phenotype and loss of EC-PC interactions.
Abstract 215: Principal Components Analysis in a South Asian Population: Cardiovascular Risk Variable Clustering and Obesity
Authors: Thanikachalam Mohan , Agustsson Ingolfur , Wexler Deborah J , Harivanzan Vijaykumar , Baliga Ragavendra R , Abraham William T , Ramesh M , Thanikachalam Sadagopan
Published: July 2021

Aim:
Western populations demonstrate a strong role of obesity in clustering of cardiovascular (CV) risk factors. We hypothesized that obesity would not have a dominant role in the clustering of CV risk factors in South Asians, who develop CV disease at young ages despite relatively low BMI.

Methods:
We selected 6224 South Indians without diabetes from a population-based cross-sectional survey (mean age 42 years; 58% women) for analysis. We used gender-specific principal components analysis (PCA), a multivariate correlation technique, with orthogonal rotation (to produce interpretable factors) to test the hypothesis. The PCA was done using measures of fasting (FPG) and 2 h plasma glucose (2h PG), haemoglobin A1c (A1c), fasting insulin (FIns), triglycerides (TG) and high-density lipoprotein (HDL), diastolic (DBP) and systolic blood pressure (SBP), body mass index (BMI) and waist circumference (WC). We did separate subgroup analyses stratified by various potential modifiers.

Results:
The obesity indices correlated positively with CV risk variables (except HDL), regardless of gender. PCA failed to demonstrate a single dominant underlying pathological role (one component was rejected at P < 0.0001), but suggested three non-overlapping factors (physiological domains) underlying the clustering of the risk variables, accounting for 60% of the total variance in the data [Fig]. Obesity indices had significant positive loading (95% confidence interval >0.8) only in Factor 1, which included FIns, TG, and HDL. The FPG, 2hPG and A1C were associated with Factor 2. The SBP and DBP were associated with Factor 3. The factor patterns were virtually identical among all the subgroups with coefficients of congruence close to 1.0.

Conclusions:
These findings are consistent with the presence of three distinct physiological domains underlying CV risk variable clustering. Obesity did not have a dominant role in the clustering of CV risk factors in the South Asian population.
Abstract 239: LDL Apheresis Is Associated With a Reduction in Markers of Chronic Inflammation in Patients With Familial Hypercholesterolemia
Authors: Lynch Donald R , Winegar Deborah A , Connelly Margery , Yancey Patricia G , Pourfarzib Ray , Fazio Sergio , Linton MacRae F
Published: July 2021

Background:
Familial Hypercholesterolemia is an inherited disease characterized by severely elevated levels of LDL cholesterol (LDL-C) and associated with premature coronary artery disease (CAD). LDL apheresis (LA) is indicated for management of CAD patients with LDL-C>200 mg/dl despite medical therapy. LA may reduce LDL-C concentrations by up to 70%; this in turn has been associated with regression of atherosclerosis. NMR measurement of signals corresponding to methyl group protons of glycoproteins (GlycA and GlycB) has recently been validated in prediction of atherosclerosis and chronic systemic inflammation. Studies have demonstrated a reduction in inflammatory cytokines with LA, but no study to date has evaluated the impact of LA on levels of glycoproteins as markers of chronic inflammation.

Objective:
To determine the effect of LA on plasma levels of glycoproteins using a novel NMR based technique, and to correlate these findings with changes in lipoprotein subclasses.

Methods:
Comprehensive NMR lipoprotein analysis was performed on serum obtained from seven subjects prior to and following LA sessions performed at two-week intervals (N = 47 paired samples). Three subjects had homozygous FH, three had heterozygous FH with CAD and/or LDL-C > 200 mg/dL, one patient had severe mixed dyslipidemia with CAD and LDL-C > 200 mg/dL.

Results:
Mean GlycA and GlycB levels were 335±91 μmol/L and 125±31 μmol/L respectively prior to LA. Following LA treatment, there was a significant reduction in GlycA to 277±73 μmol/L (P<0.001) and in GlycB to 102±28 μmol/L (P<0.001). The reduction in GlycA was strongly correlated with a decrease in total small LDL particle number (r = 0.68, p < 0.001) and with a reduction in medium VLDL particle number (r = 0.52, P < 0.001). GlycB level was weakly correlated with small LDL (r = 0.39, P = 0.007) and IDL particle numbers (r = 0.32, P = 0.028). In addition, GlycA and GlycB levels were correlated to each other (r = 0.49, P < 0.001).

Conclusion:
This is the first report of NMR detection of glycoproteins as inflammatory biomarkers in patients with severe dyslipidemia undergoing LA. LA was associated with a significant reduction in levels of both GlycA and GlycB, which correlated with reductions in LDL and VLDL particle number.
Abstract 253: NOTCH1 Protects Against Atherosclerosis by Repressing Endothelial Activation and Recruitment of Inflammatory Cells
Authors: Briot Anais , Civelek Mete , Seki Atsuko , Dry Sarah , Fishbein Michael , Lusis Aldons J , Berliner Judith , Iruela-Arispe Luisa M
Published: July 2021

Maintenance of endothelial homeostasis is critical in the prevention of vascular disorders including atherosclerosis. While we have a good understanding of the mechanisms that promote endothelial activation, less understood are the mechanisms that counterbalance this state. We found that inducible deletion of Notch1 in the endothelium of adult mice led to the accumulation of CD45+ cells in the subendothelial space of large arteries. NOTCH1 is constitutively expressed in adult arteries of mouse and human in vivo but it is excluded from veins. Together these observations led us to speculate that NOTCH1 might actively suppress the acquisition of a pro-inflammatory state in adult endothelium of large arteries. In fact, knockdown of NOTCH1 in human aortic endothelial cells (HAECs) in vitro triggered the expression of inflammatory cytokines in the absence of any other stimuli. Furthermore exposure of HAECs to oxidized phospholipids (OxPAPC) resulted in a rapid reduction of the expression levels of NOTCH1 and several NOTCH1-target genes. Using gene expression microarrays, we found that approximately 25% of the genes differentially expressed after exposure to OxPAPC (by 20%) were similarly regulated (ie either proportionally down or upregulated) when NOTCH1 was knockdown in HAECs, suggesting that these two events might be linked. Among these genes, CXCL1, a molecule previously shown to facilitate monocyte recruitment and binding to the endothelium, was increased in both conditions. Upregulation of CXCL1 upon exposure to OxPAPC was also consistently observed in a cohort of cells isolated from 147 individual donors and suppression of NOTCH1 also led to increase of the chemokine at the mRNA and protein levels. These data indicated that constant NOTCH1 expression was required for a homeostatic anti-inflammatory phenotype in the endothelium. We conclude that NOTCH1 may provide inherent protection to the endothelium during the initial stages of atherosclerosis by repressing the recruitment of inflammatory cells. Thus, decline of endogenous endothelial NOTCH1 levels is likely to constitute a predisposing factor for atherosclerosis.
Abstract 257: Smooth Muscle Cell--Derived COL15A1 Increases Indices of Plaque Stability and Mediates Plaque Cellular Content
Authors: Durgin Brittany G , Cherepanova Olga A , Owens Gary K , Connelly Jessica J
Published: July 2021

Introduction:
Current dogma states that under conditions that promote atherosclerosis quiescent vascular smooth muscle cells (SMC) phenotypically modulate into migratory, proliferative cells that move into the arterial intima, form a fibrous cap, and secrete extracellular matrix components (ECM) that promote plaque stabilization. However, there is no direct evidence that SMC derived ECM is critical in promoting changes to plaque stability. We identified collagen, type XV, alpha 1 (COL15A1) as a vascular SMC derived ECM protein present in both human and mouse atherosclerotic aortas. COL15A1 links large fibrillar collagens as well as promotes basement membrane cell adhesion and collagen maturation. We have shown in humans that Col15a1 is associated with atherosclerosis and is epigenetically regulated. However, the role for COL15A1 in atherosclerosis had not been explored. To assess the role of SMC derived collagen and COL15A1 in atherosclerosis we developed unique, conditional, tamoxifen inducible SMC specific Col15a1 knockout mice. We hypothesized that SMC production of COL15A1 is essential to promote indices of plaque stability.

Methods and Results:
At five weeks of age, SMC specific Col15a1fl/flApoE-/-MYH11-CreERT2+/- mice (n=15) and their Col15a1+/+ counterparts (n=12) were injected for 7 days with 1mg tamoxifen to induce SMC specific Col15a1 knockout and were placed on a Western diet (21% milk fat, 0.15% cholesterol) for 18 weeks. We find that SMC specific Col15a1 knockout induces marked detrimental changes in indices of plaque stability including increased necrotic core area (p=0.002), increased lesion size (p=0.03), and decreased plaque collagen maturation (p=0.017). Moreover, these mice showed a decrease in LGALS3+ macrophage content (p=0.008) but no change in SMC marker (ACTA2+) lesion staining.

Conclusion:
These data provide the first evidence that a SMC derived collagen (COL15A1) can positively affect indices of plaque stability and mediate plaque cellular content as well as identify COL15A1 as a novel player in atherosclerosis.
Abstract 26: Anti-TIM-1 Monoclonal Antibody Therapy Expands Atheroprotective B1a B Cells in vivo and Attenuates Development and Progression of Atherosclerosis
Authors: Kyaw Tin , Hosseini Hamid , Kanellakis Peter , Tay Christopher , Cao Anh , Yi Li , Tipping Peter , Bobik Alex , Toh Ban-Hock
Published: July 2021

Background:
B1a B cells attenuate atherosclerosis by secreting natural IgM, but their therapeutic application is limited by lack of availability. Regulatory B cells identified by Tim-1 expression and expanded through Tim-1 ligation by anti-TIM-1 low affinity monoclonal antibody (RMT1-10 mAb) induced tolerance. Here, we examined the capacity of this mAb to expand B1a B cells to inhibit atherosclerosis development and progression of established atherosclerosis.

Methods and Results:
Six-week old male ApoE-deficient mice were treated with RMT1-10 mAb and fed a high-fat diet (HFD) for 8 weeks. B1a TIM-1+IgM+ B cells and B1a TIM-1+IgM+IL-10+ B cells were selectively expanded. These effects reduced lesion size, markedly increased plasma and lesion IgM and decreased lesion oxidatively modified LDL. Lesion CD4+ and CD8+ T cells, macrophages and MCP-1, VCAM-1, proinflammatory cytokine expression, apoptotic cell numbers and necrotic cores were reduced. Splenectomy indicated that these effects were B1a B cell-dependent. B1a B cell stimulation in vitro with RMT1-10 mAb promoted dose-response B1a B cell proliferation and B1a-derived IgM production. To determine whether treatment attenuated developed atherosclerosis progression, 6 week-old male ApoE-deficient mice were fed a HFD for 6 weeks, and treated with anti-TIM-1 mAb for another 6 weeks while continuing the HFD. Treatment also increased B1a TIM-1+IgM+ B cells, B1a TIM-1+IgM+IL-10+ B cells and IgM levels and greatly attenuated atherosclerosis progression.

Conclusions:
Anti-TIM-1 treatment attenuates atherosclerosis development and progression by selectively expanding atheroprotective B1a B cells and modulating its immunoinflammatory component. TIM-1 mAb therapy could be an attractive approach for treating atherosclerosis.
Abstract 268: Atherosclerosis in Silico: A Computational Model for Early Inflammatory Events, Including the Effects of High-Density Lipoproteins
Authors: Myerscough Mary R , Chalmers Alexander D
Published: July 2021

Objective:
To develop a computational model to visualize in continuous time how the early plaque develops under different physiological conditions and to use it to explore the effects of changing concentrations of HDL.

Methods:
Atherosclerosis is the result of a sequence of specific responses to a failure of the endothelium. We devised a model for the interactions between modified LDL, monocytes and macrophages, HDL, chemoattractants and other cytokines immediately after endothelial injury. We included the dynamics of these interactions both at the endothelium and inside the intima. We computed the results for the model on a cross-section of an artery for different levels of blood cholesterol and immune system activity and produced a series of simulations to display the model results.

Results:
Using the model, we can visualize the changing movement and distribution of modified LDL, cytokines, macrophages and foam cells in the early plaque as a function of time, rather than as static post-mortem images. The model shows qualitatively how a fatty streak is initiated and develops after the initial inflammation, including distortion of the blood vessel wall by the developing plaque. The simulations show an initial phase with high macrophage densities in the plaque which settles to a low level of chronic inflammation and sustained foam cell accumulation. When we increase the level of HDL early in plaque development, the simulated plaque ceases to grow and may regress; when we increase HDL by the same amount but at a later time in plaque development, then the plaque continues to grow.

Conclusion:
This model provides a new tool for visualizing and for thinking about early atherosclerosis. It allows us to explore the effects of blood cholesterol levels and the level of the inflammatory response on the size and lipid density of the plaque. The results of model simulations suggest that the timing of increases in HDL may be important in determining whether or not a plaque regresses and, hence, that the inherent dynamics of the process of plaque formation may have an influence on the efficacy of HDL action.
Abstract 278: Enhanced Cystathionine ß-Synthase Nitration in Diet-Induced Hyperhomocysteinemic Rats Accelerates the Progression of Hyperhomocysteinemia
Authors: Wang Wen , Zhang Hui , Liu Wei , Liu Teng , Ma Lu , Sun Qi , Wang Ke , Liu Hui-rong
Published: July 2021

Hyperhomocysteinemia is an independent risk factor for cardiovascular diseases,which may be caused by genetic deficiencies in enzymes responsible for remethylation or transsulfuration of homocysteine, nutritional deficiencies in vitamins, or excessive intakes of methionine. Since transsulfuration pathway is the only irreversible pathway in homocysteine metabolism, its key enzyme cystathionine β-synthase is vital in terminal removement of homocysteine. In addition, Studies have observed that enhanced nitrative stress in hyperhomocysteinemic rats could induce cardiovascular injuries, and peroxynitrite stimulation in vitro resulting in decreased bioactivity of cystathionine β-synthase. Therefore, to determine the contribution of nitrative stress to progression of hyperhomocysteinemia, and how it is related to cystathionine β-synthase, we used three groups of rats (Con with normal diet, HHcy with 2.5% methionine diet, HHcy+FeTMPyP with 2.5% methionine diet +FeTMPyP) to observe the effects of nitrative stress on cystathionine β-synthase. Results showed that in diet-induced hyperhomocysteinemic rats, tHcy levels and nitrative stress increased, surprisingly, pretreatment with peroxynitrite scavenger FeTMPyP ameliorated the level of tHcy as well as nitrative stress. Further experiments showed cystathionine β-synthase bioactivity in HHcy rats was less potent than other groups yet described, and the level of cystathionine β-synthase nitration was the most significant,while the entire trends reversed after FeTMPyP pretreatment. In conclusion, these results highlight for the first time that in diet-induced hyperhomocysteinemic rats, cystathionine β-synthase bioactivity reduction is not just a traditional etiologic factor, but rather an outcome of hyperhomocysteinemia, and resulting from the high level of nitrative stress caused by hyperhomocysteinemia, the cystathionine β-synthase nitration plays a mutual role in hyperhomocysteinemia development via causing the metabolic disorder of homocysteine. These findings may shed a novel light on the homocystine-lowering target of hyperhomocysteinemia.
Abstract 292: Notch Inhibition Stabilizes the Progression of Small Abdominal Aortic Aneurysm in a Mouse Model
Authors: Cheng Jeeyun , Koening Sara , Kuivaniemi Helena , Garg Vidu , Hans Chetan P
Published: July 2021

The progression of abdominal aortic aneurysm (AAA) involves a sustained influx of proinflammatory macrophages, which exacerbate tissue injury by releasing cytokines, chemokines and matrix metalloproteinases (MMPs). While multiple factors are involved in disease pathogenesis, the critical stimulus is the differentiation and infiltration of naïve macrophages (Mϕ) towards classical (M1) activation. In contrast, Mϕ can also be programmed to M2-macrophages which inhibit the inflammatory response and promote tissue repair. Previously, we showed that Notch deficiency reduces the development of AAA in the angiotensin II (AngII)-induced mouse model by preventing infiltration of Mϕ. Because of its regulatory roles in macrophage differentiation, we examined if Notch inhibition in a mouse model prevents progression of small AAA and if these effects are associated with Mϕ-differentiation.

Notch inhibition (DAPT) started at day 3 or 8 of AngII infusion arrested the progression of AAA in

Apoe
-/-

mice as demonstrated by a decreased luminal diameter and aortic width. The abdominal aortae treated with DAPT showed decreased MMPs expression and presence of elastin precursors including tropoelastin. Marginal adventitial thickening observed in DAPT-treated

Apoe
-/-

mice was not associated with increased total macrophage content. Instead, DAPT-treated abdominal aortae showed increased expression of Cd206 positive M2-macrophages and decreased expression of Il12 positive M1-macrophages. Increased expression of IL12 positive M1 macrophages and its strong correlation with active Notch1 signaling (NICD) was also observed in the infrarenal aortae from AAA patients.
Notch1
deficiency promoted M2-differentiation of Mϕ by upregulating transforming growth factor (
Tgf)-β2
expression in bone marrow-derived Mϕ at basal levels and in response to IL4. Protein expression of Tgf-β2 and its downstream pSmad2 also increased in DAPT-treated

Apoe
-/-

mice, indicating a potential link between Notch and Tgf-β2 signaling in the M2-differentiation of Mϕ.

Overall, Notch inhibition stabilizes the progression of AAA by macrophage-differentiation-dependent mechanisms and provides insights for novel therapeutic strategies to prevent the progression of small AAA.
Abstract 297: Segmental Aortic Stiffening is a Mechanism Driving Early Abdominal Aortic Aneurysm Pathogenesis
Authors: Raaz Uwe , Zöllner Alexander M , Toh Ryuji , Nakagami Futoshi , Schellinger Isabel N , Emrich Fabian C , Adam Matti , Maegdefessel Lars , Hertel Thomas K , Deng Alicia , Jagger Ann , Buerke Michael , Dalman Ronald L , Spin Joshua M , Kuhl Ellen , Tsao Philip S
Published: July 2021

Stiffening of the aortic wall is a phenomenon consistently observed in abdominal aortic aneurysm (AAA). However, its role in AAA pathophysiology is largely undefined.

Using an established murine elastase-induced AAA model, we demonstrate that segmental aortic stiffening (SAS) precedes aneurysm growth. Finite elements analysis (FEA)-based wall stress calculations reveal that early stiffening of the aneurysm-prone aortic segment leads to axial (longitudinal) stress generated by cyclic (systolic) tethering of adjacent, more compliant wall segments. Interventional stiffening of AAA-adjacent segments (via external application of surgical adhesive) significantly reduces aneurysm growth. These changes correlate with reduced segmental stiffness of the AAA-prone aorta (due to equalized stiffness in adjacent aortic segments), reduced axial wall stress, decreased production of reactive oxygen species (ROS), attenuated elastin breakdown, and decreased expression of inflammatory cytokines and macrophage infiltration, as well as attenuated apoptosis within the aortic wall. Cyclic pressurization of stiffened aortic segments ex vivo increases the expression of genes related to inflammation and extracellular matrix (ECM) remodeling. Finally, human ultrasound studies reveal that aging, a significant AAA risk factor, is accompanied by segmental infrarenal aortic stiffening.

The present study introduces the novel concept of segmental aortic stiffening (SAS) as an early pathomechanism generating aortic wall stress and thereby triggering AAA growth. Therefore monitoring SAS by ultrasound might help to better identify patients at risk for AAA disease and better predict the susceptibility of small AAA to further growth. Moreover our results suggest that interventional mechanical stiffening of the AAA-adjacent aorta may be further tested as a novel treatment option to limit early AAA growth.
Abstract 315: Surgical Pen Dye Inhibits Cell Migration From Saphenous Vein Explants
Authors: Kikuchi Shinsuke , Kenagy Richard D , Clowes Alexander W
Published: July 2021

Markers containing dyes such as crystal violet (CAS 548-62-9) are routinely used on the adventitia of vein bypass grafts to avoid twisting during placement. Since little is known about how these dyes affect vein graft healing and function, we determined the effect of crystal violet on cell migration, a primary response to injury after grafting. Human saphenous veins were split into adventitia and media after removal of endothelium. Cell migration from 2.5 square mm explants in DMEM with 20% fetal bovine serum was measured daily for 8 days as either 1) % migration positive explants (≥ 1 cell/explant), which only measures migration, or 2) the number of cells/explant, which measures a combination of migration and proliferation. Dye was extracted from explants with ethanol and quantified by spectrophotometry. Cell migration by both methods was significantly less from blue, compared to non-blue, adventitial explants (P<.05, mean±SEM, N=11 veins with 15-30 explants/condition; Figures A and B). No medial explants were visibly blue, and there was no significant difference in migration of cells from medial explants of blue and non-blue sections of vein (data not shown). Blue adventitial explants had 65.9±8.0 ng dye/explant compared to 2.1±1.3 for non-blue explants (mean±SEM, N=7-11 veins). Dye applied in vitro to either adventitial or medial explants dose-dependently inhibited migration (IC50=21.5 ng/explant).

Conclusion:
Crystal violet is toxic and inhibits venous adventitial cell migration; alternative methods should be considered for marking vein grafts.
Abstract 325: Temporally Separated Administration of Proangiogenic Growth Factor Combination Promotes Angiogenesis in Both Diabetic and Nondiabetic Microvascular Endothelial Cells
Authors: Chen Hao H , Kim Yu Jin , Liao Christine , Matkar Pratiek N , Cao Wei J , Rudenko Dmitriy , Kuliszewski Michael A , Leong-Poi Howard
Published: July 2021

Background:
Diabetes mellitus is a chronic metabolic disease associated with various vascular complications. Various strategies to promote therapeutic angiogenesis in ischemic tissue including delivery of pro-angiogenic growth factors have shown disappointing results. Risk factors, such as diabetes, may adversely impact the response to angiogenic strategies and limit efficacy in vivo. We assessed the hypothesis that temporally separated delivery of multiple pro-angiogenic growth factors will improve the efficacy of the angiogenic response in the setting of both diabetes and non-diabetes in vitro.

Methods:
96-well tissue culture plates were coated with 60 μL growth factor reduced Matrigel. After gelation, non-diabetic human dermal microvascular endothelial cells (HMVECs) and type-II diabetic cells (D-HMVECs) were plated at 1x104 cells/well with 100 μL basal media EBM-2 with 0.1% bovine serum, and incubated at 37°C. Combinations of growth factors including recombinant human VEGF (50 ng/mL), angiopoietin (Ang)-1 and -2 (250 ng/mL) were administrated at start of the assay and/or at 6 hours into the assay. Images were taken at 3, 6, 9, 12, 18 and 24 hours and used for tube formation quantification by Inverted Microscope at 10x magnification.

Results:
In untreated control, D-HVECs showed reduced tube formation in compared to HMVECs (n=12; p<0.001). Growth factors combination ‘VEGF and Ang-2 at start of the assay and Ang-1 at 6 hours showed the most significantly increased of tube formation when compared to single growth factor (to VEGF, Ang-1, 2; p<0.001) or growth factors combination without temporally separated delivery (to VEGF/Ang-1, VEGF/Ang-2; p<0.001) in both HMVEC and D-HMVEC.

Conclusion:
Diabetic endothelial cells showed impaired angiogenesis compared to non-diabetic endothelial cells. Combinations of temporally separated pro-angiogenic growth factors were able to optimize the angiogenic effects and yield the most significant efficacy in the setting of both diabetes and non-diabetes. Future studies will focus on in vivo temporally separated combinations of multiple growth factors.
Abstract 332: Cell-Free Conditioned Media From Endothelial Progenitor Cells Enhance Endothelial Angiogenesis, Migration and Survival: Paracrine Implications for Cell Therapy in Pulmonary Arterial Hypertension
Authors: Suen Colin , Deng Yupu , Stewart Duncan
Published: July 2021

Background:
Endothelial progenitor cells (EPCs) have been shown to be effective in the prevention of pulmonary arterial hypertension (PAH) in preclinical models. Our group has also demonstrated that xenotransplantation of human early EPCs in the rat monocrotaline (MCT) model prevented pulmonary arterial hypertension, despite the near complete loss of human cells from the lung after even 24 hours. Therefore, we hypothesize that the therapeutic effects of EPCs in PAH are mediated by a paracrine mechanism, which in turn, may enhance endothelial repair and integrity.

Methods:
EPCs were derived from human peripheral blood mononuclear cells obtained via leukapheresis by adherence to fibronectin and growth in endothelial growth medium (EGM-2MV). 24 hour conditioned medium from Day 6 EPCs (EPC-CM) was collected and concentrated by ultrafiltration prior to in vitro and in vivo studies. PAH was induced by a single intraperitoneal injection of (MCT) in nude athymic rats (150-200 g). EPC (5x106/kg) or EPC-CM from an equivalent number of cells were administered via jugular vein at day 3 after MCT and hemodynamics and echocardiography were assessed at day 24.

Results:
Incubation of human umbilical vein endothelial cells (ECs) with EPC-CM enhanced Matrigel capillary-like network formation by approximately 2-fold compared with serum-free media (p<0.05). EPC-CM increased migration in the scratch wound-healing assay compared to control media (4-fold, p<0.001). EPC-CM also significantly improved survival of HUVECs after serum deprivation (p<0.05) in a dose-dependent manner. Injection of EPCs resulted in decreased right ventricular systolic pressure (p<0.05) and improvement in pulmonary artery acceleration time (+28%, p<0.05). However, infusion of an equivalent amount of EPC-CM as well as a 10-fold increased dose of EPC-CM failed to prevent PAH.

Conclusions:
Therefore, EPC-CM enhanced in vitro EC angiogenesis, migration, and survival, consistent with potential of a paracrine mechanism. However, in an in vivo preclinical model, EPC conditioned media did not prevent PAH, suggesting that intact cells act through mechanisms that may involve cell-cell signaling.