bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 154: Hemodynamic Regulation of Tie1 in Aortic Valve Endothelial Cells
Authors: Johnson Camryn , Merryman W. David
Published: June 2021

During the cardiac cycle, the ventricular side of the aortic valve (ventricularis) is exposed to high shear pulsatile flow, while the aortic side (fibrosa) is exposed to low shear recirculatory flow. The two sides have different transcriptional profiles, likely due to their distinct flow patterns. Tie1 is a mechanically sensitive orphan tyrosine kinase receptor found in endothelial cells (ECs). Tie1 is often found associated with Tie2, a tyrosine kinase receptor involved in EC survival. Tie1 prevents ligand binding to Tie2 until Tie1 is cleaved, via shear stress or other signals. Although studies have shown shear stress cleaves Tie1, none have been done in the aortic valve. Mechanically induced changes in Tie1 expression in the valve may be crucial to understand the effects of the hemodynamics of each side. ECs were isolated from the fibrosa and ventricularis of excised porcine aortic valves. The Flexcell 4000 Tension System was used for strain, and an insert for the Flexcell plates (
A, B, C
) was designed to apply shear stress at the same time. Full length Tie1 protein expression in porcine aortic valvular endothelial cells (pAVECs) decreased ~40% after 15% strain for 24 hours, while cleaved Tie1 endodomain levels increased (
D
). Additionally, qPCR results showed that Tie1 mRNA levels did not decrease as dramatically, also supporting Tie1 cleavage. Full length Tie1 protein expression in pAVECs also decreased ~60% after 7 hours of 10 dynes/cm
2
pulsatile bidirectional shear stress and decreased ~90% after 24 hours of 15% strain combined with 2 dynes/cm
2
pulsatile bidirectional shear stress (
E, F
). Akt, a protein kinase phosphorylated by activated Tie2, showed increased phosphorylation after 15 minutes of strain (
G
), providing evidence that cleaved Tie1 may be activating Tie2. These results show the varying responses in Tie1 expression to different shear stresses and strains and demonstrate that mechanical regulation plays an important role in its signaling in the aortic valve.
Abstract 164: Endothelial Gab1 Limits Vascular Remodeling and Atherosclerosis Through Inhibition of Endothelial Inflammation
Authors: Xu Suowen , Yin Meimei , Zhao Jingjing , Koroleva Marina , Jin Zheng Gen
Published: June 2021

Objective:
We have previously shown that docking protein Grb2-associated binder 1 (Gab1) is a mechano-effector protein in response to fluid shear stress and regulates postnatal angiogenesis. The aim of this study was to determine the
in vivo
role of endothelial Gab1 in flow-mediated vascular remodeling and atherosclerosis and explore the underlying mechanisms.

Methods and Results:
To determine the role of endothelial Gab1 in disturbed flow-induced vascular remodeling in vivo, we performed partial carotid artery ligation in Gab1 endothelium-restricted knockout (Gab1-ecKO) mice and wild-type (WT) littermates, and we observed that Gab1-ecKO mice resulted in increased intima-media thickness. To examine the role of endothelial Gab1 in atherosclerosis, we next crossed Gab1-ecKO mice with ApoE KO mice. After partial ligation, Gab1-ecKO;ApoE KO mice under high fat diet showed increased atherosclerotic lesion size compared to Gab1-WT;ApoE KO mice. The levels of proatherogenic genes intracellular adhesion molecule-1 (ICAM-1) and vascular cell adhesion molecule-1 (VCAM-1), and leukocyte infiltration were all increased in Gab1-ecKO;ApoE KO mice. Using loss- and gain-of-function studies in cultured human endothelial cells (ECs), we found that Gab1 depletion by siRNA augmented monocyte adhesion to ECs by increasing ICAM-1 and VCAM-1 expression in response to the proinflammatory cytokine TNF-α. Conversely, adenoviral overexpression of Gab1 inhibited TNFα-induced monocyte adhesion to ECs and upregulation of ICAM-1 and VCAM-1 in ECs.

Conclusions:
These results demonstrate that endothelial Gab1 represses disturbed flow-induced vascular remodeling and atherogenesis through inhibition of vascular inflammation. Our findings suggest that Gab1 activation might represent novel approaches for the treatment of vascular diseases, including intimal hyperplasia and atherosclerosis.
Abstract 172: Arteriovenous Fistula Adaptation Requires Caveolin1 Signaling
Authors: Hashimoto Takuya , Foster Trenton R , Bai Hualong , Hu Haidi , Santana Jeans M , Hanisch Jesse , Dardik Alan
Published: June 2021

Background:
Arteriovenous fistula (AVF) is the preferred access for hemodialysis. However, many AVFs fail to mature and require intervention, showing a need to improve AVF maturation. Caveolin1 (Cav1) is the major scaffolding protein of caveolae, a distinct microdomain that serves as a flow activated mechanosensor at the membrane of endothelial cells. We have previously shown that stimulation of embryonic venous determinant EphB4 associates with Cav1, mediating vessel remodeling in the murine vein graft model. Since Cav1 is a mechanism of EphB4 mediated vein graft adaptation, we examined the role of Cav1 in mediating AVF maturation, a different and distinct hemodynamic and molecular conditions from vein graft. We hypothesized that Cav1 function is critical for AVF maturation.

Methods:
We used a mouse aortocaval fistula model; the venous AVF limb and control (sham) inferior vena cava of wild type (WT) C57BL/6 mice were analyzed and compared for Cav1 expression. Vessel remodeling was assessed in WT and Cav1 knockout (KO) mice by serial ultrasound measurements of the IVC/AVF diameter. AVF were harvested at day 21 and examined with histology; IVC wall thickness was measured using computerized morphometry.

Results:
Both Cav1 mRNA and protein were increased in the fistula veins compared to control veins (Figure A; mRNA: *p=0.019, ANOVA, n=8; protein: *p=0.005, t-test, n=5). Cav1 KO mice had increased fistula vein diameter compared with WT mice (Figure B; *p<0.0001, ANOVA, n=7-10). The fistula vein wall thickness was increased in Cav1 KO mice as compared with WT mice (Figure B; 15.6 vs 30.9μm, *p=0.0005, t-test, n=7, bar 50μm).

Conclusions:
Cav1 expression is increased during AVF maturation, and loss of Cav1 leads to increased fistula vein diameter and wall thickness, similar to the function of Cav1 during vein graft adaptation. These results suggest that Cav1 is a regulator of AVF maturation and manipulation of Cav1 function may enhance venous adaptation to the arterial environment.
Abstract 18: Bempedoic Acid Lowers Low Density Lipoprotein-Cholesterol and Attenuates Aortic Atherosclerosis in LDL Receptor-Deficient (

LDLR
+/-

and

LDLR
-/-

) Yucatan Miniature Pigs
Authors: Burke Amy C , Telford Dawn E , Sutherland Brian G , Edwards Jane Y , Sawyez Cynthia G , Newton Roger S , Huff Murray W
Published: June 2021

Bempedoic acid (ETC-1002) is an oral investigational drug that targets hepatic adenosine triphosphate-citrate lyase to reduce cholesterol biosynthesis. In Phase 2 studies (up to 12 weeks in duration), bempedoic acid lowers elevated LDL-cholesterol (C) in patients with hypercholesterolemia. The objective of the present study was to test the ability of bempedoic acid to decrease plasma-C and LDL-C, and attenuate the development of atherosclerosis in a large animal model of familial hypercholesterolemia. Gene targeting has been used to generate Yucatan miniature pigs heterozygous (

LDLR
+/-

) or homozygous (

LDLR
-/-

) for LDL-receptor (R) deficiency (ExeGen).

LDLR
+/-

pigs (n=12) and

LDLR
-/-

pigs (n=12) were fed a high fat, cholesterol-containing diet (34% kcal fat; 0.2% cholesterol) and orally administered placebo or bempedoic acid at 120 mg/d and at 240 mg/d for 160 days. Bempedoic acid was well tolerated; weight gain, caloric intake, liver enzymes and clinical chemistry analyses were unaffected by treatment. In

LDLR
+/-

pigs at 160 days, compared to placebo, bempedoic acid significantly decreased plasma-C and LDL-C up to 43% (
P
<0.01) and 63% (
P
<0.002), respectively. Compared to
LDLR-/-
placebo pigs, in which plasma-C and LDL-C were 5-fold higher than in

LDLR
+/-

placebo pigs, bempedoic acid significantly decreased plasma-C and LDL-C up to 26% (
P
<0.04) and 29% (
P
<0.03), respectively. Plasma levels of triglycerides and HDL-C, fasting glucose and insulin, and liver lipids were not affected by treatment in either genotype. In the aorta of

LDLR
+/-

pigs, bempedoic acid treatment robustly attenuated total Sudan IV-stained lesion area (-58%,
P
<0.02) and area of raised lesions in the abdominal aorta (-58%,
P
<0.03). In

LDLR
-/-

pigs, in which total aortic lesion area (6-fold) and area of raised abdominal lesions (12-fold) were substantially larger compared to

LDLR
+/-

pigs, bempedoic acid significantly decreased total aortic lesion area (-47%,
P
<0.01) and area of raised abdominal lesions (-50%,
P
<0.03). The present experiments demonstrate that in a large animal model of LDLR-deficiency and atherosclerosis, long term treatment with bempedoic acid reduces LDL-C and attenuates the progression of aortic atherosclerosis in both

LDLR
+/-

and

LDLR
-/-

miniature pigs.
Abstract 192: Characterization of Microsomal Triglyceride Transfer Protein Missense Mutations Found in Abetalipoproteinemia and Hybobetalipoproteinemia Subjects
Authors: Walsh Meghan T , Di Leo Enza , Tarugi Patrizia , Hussain M. Mahmood
Published: June 2021

We describe two new hypolipidemic patients with very low plasma triglyceride and apolipoprotein B (apoB) levels and lipid malabsorption with plasma lipid profiles similar to abetalipoproteinemia (ABL) patients. In these patients, we identified two previously uncharacterized missense mutations in the microsomal triglyceride transfer protein (MTP) gene, R46G and D361Y, and studied their effects on function. We also characterized three missense mutations (H297Q, D384A, and G661A) reported earlier in a familial hypobetalipoproteinemia patient. R46G had no effect on MTP expression or function and supported apoB secretion. Similarly, H297Q, D384A, and G661A mutants supported apoB secretion similarly to WT MTP. Contrary to these four missense mutations, D361Y was unable to support apoB secretion. Functional analysis revealed that this mutant was unable to bind protein disulfide isomerase (PDI) or transfer lipids. The negative charge at residue 361 was critical for MTP function as D361E was able to support apoB secretion and transfer lipids. D361Y most likely disrupts the tightly packed middle α-helical region of MTP, mitigates PDI binding, abolishes lipid transfer activity, and causes ABL. On the other hand, the hypolipidemia in the other two patients was not due to MTP dysfunction. Thus, in this study of five missense mutations spread throughout MTP’s three structural domains found in three hypolipidemic patients, we found that four of the mutations did not affect MTP function. Thus, there probably exist novel mutations in other genes that cause severe hypolipidemia and their recognition may identify novel proteins involved in the synthesis and/or catabolism of plasma lipoproteins.
Abstract 195: Effects of Vertical Sleeve Gastrectomy on HDL Function in Adolescents with Severe Obesity
Authors: Shah Amy S , Inge Thomas , Sexmith Hannah , Heink Anna , Davidson W S
Published: June 2021

Severe obesity is a major problem among US adolescents and traditional weight loss strategies, including lifestyle intervention, have had limited efficacy. Bariatric surgery has grown in popularity because it results in rapid weight loss while improving cardiovascular disease risk factors, including raising high density lipoprotein cholesterol (HDL-C). Whether this rise in HDL-C translates into improved HDL cardioprotective function is not clear, particularly in adolescents. We studied the impact of weight loss surgery (vertical sleeve gastrectomy) in adolescents with severe obesity on HDL function pre and one year post operatively. Adolescents underwent laparoscopic vertical sleeve gastrectomy between in 2008-2011. HDL function (cholesterol efflux, HDL oxidation, and HDL inflammatory index) was measured pre and post-surgery on stored serum (-80°C) after apolipoprotein B depletion with polyethylene glycol. Only males were studied in this pilot study to minimize the known effects of sex and the menstrual cycle on lipoproteins. Changes pre and post-surgery were evaluated using paired t-tests. Participants (n=10, 90% Caucasian) were a mean±SD age of 17.4±1.6 years at baseline and 18.4 ±1.5 years at follow-up. After vertical sleeve gastrectomy, BMI was 32% lower than baseline (p<0.01). All lipid measurements improved and HDL-C increased by 23%. HDL function significantly improved post vertical sleeve gastrectomy compared to baseline, all p<0.01 (cholesterol efflux by 12%, HDL oxidation potential by 30%, and HDL inflammatory index by 25%). We conclude that vertical sleeve gastrectomy results in a significant improvement in HDL cholesterol levels and HDL function in adolescents with severe obesity. Whether these changes result in long term improvement in cardiovascular health remains to be determined.
Abstract 209: Human Umbilical Vein Endothelial Cell Conditioned Medium in the Presence of High Glucose Increases
in vitro
Adipose-derived Stem Cells Proliferation and
in vivo
Vascularization
Authors: Brown Spencer , Caputo Francis , Fromer Marc , Zhang Ping , Chang Shauhoa , Hagaman Ashleigh , Koko Kiavash , Nolan Ryan , Carpenter Jeffrey
Published: June 2021

Background:
Diabetes type 1 and 2 cause hyperglycemia and result in endothelial dysfunction with endothelial vessel and poor wound healing. Adipose-derived stem cells (ASCs), progenitor cells in wound healing, show decreased function under hyperglycemic conditions
in vitro
and
in vivo
. We hypothesized that exposing ASCs in the presence of high glucose with the human umbilical vein endothelial cell (HUVEC) secretome will reverse the deleterious effects of glucose on ASCs and subsequently enhance angiogenesis and wound healing.

Methods:
Human umbilical vein endothelial cells (HUVEC) were treated with glucose (30mM) and the conditioned media (CM) were collected every 3 days. ASCs were then co-cultured with EC/CM for 2 weeks. To produce thermal denaturation of protein, EC/CM was heated at 95
0
C for 30 mins. Cell activity, proliferation, and endothelial-like properties of ASCs were determined by MTT assays, growth curves, and real-time RT-PCR, respectively. EC/CM treated ASC were injected into a normal or diabetic murine left thigh muscle at three different points with hindlimb ischemia. After 4 weeks injection, animals were sacrificed. H & E and double immunostaining for CD31 and anti-human nuclei were used to determine if the ASCs primed with EC/CM underwent neovascularization.

Results:
In fact, ASCs increased in proliferation when co-cultured with HUVEC/CM (1.4 fold) when compared with controls. This promoting effect was lost in heated HUVEC/CM, indicating that the active molecules are of protein origin. After 10 days stimulated with EC/CM an increase in mRNA expression levels of EC markers were also observed in high glucose (30mM) EC/CM environment including CD31 (2-fold), vWF (1.1-fold), and eNOS (3.2-fold) when compared to ASCs cultured in M199. H & E and immunohistochemical staining results showed elevated vessel density and CD31
+
cell levels in HUVEC-primed ASC injection sites of diabetic mice when compared with the control animals.

Conclusions:
HUVEC secrete protein factors that increase proliferation and endothelial differentiation of ASCs under diabetic conditions. Injection of ischemic hindlimbs in diabetic mice with HUVEC-primed ASCs leads to improved angiogenesis.
Abstract 220: Regulation of Dimethylamine Dimethylaminohydrolase in the Liver
Authors: Wilson-O'Brien Amy L , Garlick John , Wilson Andrew M
Published: June 2021

Introduction:
Nitric Oxide (NO) is a crucial molecule in cardiovascular biology. Abnormalities in NO are critical in atherosclerosis and impairment of the NO pathway increases risk of cardiovascular disease (CVD). A possible cause of endothelial dysfunction is an elevation (in plasma) of the endogenous inhibitor of NO synthase, asymmetric dimethylarginine (ADMA). ADMA was initially described in renal failure. 80% of ADMA is metabolized by the enzyme dimethylarginine dimethylaminohydrolase (DDAH) in the liver. DDAH-1 is widely distributed and felt to be the major determinant of circulating ADMA levels. Very little data is available regarding the cause and effects of DDAH dysregulation in the liver. Understanding the
biology of DDAH represents a target for better understanding of CVD and for novel therapy.

Hypothesis:
Diabetes and related factors will alter DDAH expression/activity potentially modulating the amount of ADMA.

Methods:
Liver tissues from control and

db/db

mice were archival. Human skeletal muscle tissue was obtained from cardiovascular surgical patients. HepG2 cells were used for low/high glucose treatment studies (5mM/25mM). DDAH-1 expression was determined using Western immunoblotting. DDAH activity measurement used a colourimetric assay. Intra/extracellular ADMA were measured in HepG2 cells using an HPLC-MS/MS technique.

Results:
In skeletal muscle from diabetic cardiac surgery patients DDAH-1 protein expression is increased (

P

< 0.05). Further to this,

db/db

mice livers versus control had a significant increase in DDAH expression and activity. Using HepG2 cells we specifically examined the effect of 25mM glucose on DDAH-1. Treatment increased expression and activity of DDAH-1 (

P

<0.05). Elevated glucose appears to have no significant effect on the intra or extracellular concentrations of ADMA.

Conclusion:
These novel results report an alteration of DDAH-1 expression and activity in hepatic tissues and cells in response to elevated glucose - a result which has not been reported previously. This suggests a possible link between the DDAH/ADMA/NOS pathway, diabetes, and glycaemic dysregulation - key risk factors in CVD.
Abstract 222: N-Acetylcysteine Enhances Amputation Stump Healing and Perfusion in Diabetes
Authors: Zayed Mohamed A , Wei Xiachao , Belaygorod Larisa , Naim Uzma , Park Kyoung-mi , Semenkovich Clay F
Published: June 2021

Over 150,000 extremity amputations are performed in the US per year. More than 60% of these are performed in patients with diabetes and peripheral arterial disease, and 25% require subsequent re-amputation due to poor surgical site healing. The mechanisms underlying poor amputation stump healing in the setting of diabetes are not understood, which greatly limits management options. N-acetylcysteine (NAC) is a well-known thiol that regulates intracellular glutathione levels, promotes endothelial cell function and angiogenesis, and is proposed to have therapeutic benefits in the setting of diabetes. To test the hypothesis that NAC can improve surgical site healing in chronically ischemic amputation stumps in the setting of diabetes, we developed a novel in vivo murine hindlimb ischemia-amputation model. In this model, streptozotocin-treated C57B6 mice are allowed to develop hyperglycemia for 1 month, followed by unilateral hindlimb femoral artery ligation. After 1 week, the recovered yet still mal-perfused hindlimb undergoes an infrageniculate amputation. Compared to sham control, mice receiving a daily intraperitoneal administration of NAC 150mg/kg demonstrated a 31% improvement in stump healing (P=0.06), 160% increase in hindlimb stump laser-Doppler perfusion at 3 days post-amputation (P=0.03), as well as 179% increase in hindlimb adductor muscle neovascularization (P=0.01). Adductor muscle segments from ischemic amputated hindlimbs also demonstrated less muscle damage, and expressed higher levels of de-palmitoylated activated Gaq (P<0.05), which is essential for ischemia-induced neovascularization. Similarly, HUVECs treated with NAC 3mM demonstrated a transient 19% increase in de-palmitoylated Gaq (P=0.03), suggesting a novel mechanism of action for NAC. These findings demonstrate that NAC may have important therapeutic benefits in amputation stump healing in the setting of diabetes, which may translate into a valuable treatment option in vulnerable patient populations.
Abstract 239: Deficiency of Macrophage Epsins Impedes Atherosclerosis by Inhibiting LRP-1 Internalization and Degradation
Authors: Brophy Megan L , Rahman Ashiqur , Dong Yunzhou , Wu Hao , Tessneer Kandice L , Pasula Satish , Rahman Ruby , Ley Klaus , Chen Hong
Published: June 2021

Background:
Atherosclerosis is caused by the chronic activation of the vascular endothelium and immune and inflammatory cell infiltration of the vascular wall, leading to enhanced inflammation and lipid accumulation. Understanding the molecular mechanisms underlying this disease is critical for the development of new therapies. Epsins are a family of ubiquitin-binding endocytic adaptors. However, their role in vascular inflammation is poorly understood. Our goal is to define the novel role of epsins in regulating atherogenesis.

Methods and Results:
We engineered mice with specific deletion of epsins in myeloid cells (MΦ-DKO). Strikingly, MΦ-DKO mice on an ApoE-/- background fed western diet exhibited reduced atherosclerotic lesion and foam cell accumulation, and diminished recruitment of immune or inflammatory cells to aortas by FACS analysis. In primary macrophages, epsin deficiency impaired foam cell formation by Oil Red O staining, and suppressed the pro-inflammatory M1 macrophage phenotype but increased the anti-inflammatory macrophage phenotype by gene profiling. Epsin deficiency did not alter levels of LDL scavenger receptors, or reverse cholesterol transport proteins, but did increase total and surface levels of LRP-1, a protein with anti-inflammatory and anti-atherosclerotic properties. Mechanistically, Epsin interacts with LRP-1 via epsin’s UIM domain. LPS treatment increased LRP-1 ubiquitination and subsequent binding to epsin, suggesting that epsin promotes the ubiquitin-dependent internalization and degradation of LRP-1. Accordingly, macrophages isolated from MΦ-DKO mice on LRP-1 heterozygous background restored the pro-inflammatory phenotype.

Conclusions:
Epsins promote atherogenesis by facilitating pro-inflammatory macrophage recruitment and potentiating foam cell formation by downregulating LRP-1 implicating that targeting the epsin-LRP-1 interaction may serve as a novel therapeutic strategy to treat atheromas.
Abstract 257: Selective Targeting of a Novel Epsin-VEGFR2 Interaction Promotes VEGF-mediated Angiogenesis
Authors: Rahman H N , Wu Hao , Pasula Satish , Dong Yunzhou , Wen Aiyun , Sun Ye , Brophy Megan L , Tessneer Kandice , Cai Xiaofeng , Mcmanus John , Chang Baojun , Kwak Sukyoung , Chen Hong
Published: June 2021

Background:
VEGF-induced binding of VEGFR2 to epsins 1 and 2 triggers VEGFR2 degradation and attenuates VEGF signaling. The epsin ubiquitin interacting motif (UIM) was shown to be required for the interaction with VEGFR2, however the molecular determinants that govern how epsin specifically interacts with and regulates VEGFR2 were unknown. The goals for the present study were (1) to identify critical molecular determinants that drive the specificity of the epsin and VEGFR2 interaction and (2) to ascertain if such determinants were critical for physiological angiogenesis
in vivo
.

Methods and Results:
Structural modeling uncovered two novel binding surfaces within VEGFR2 that mediate specific interactions with epsin UIM. Three glutamic acid residues in epsin UIM were found to interact with residues in VEGFR2. Further, we found that the VEGF-induced VEGFR2-epsin interaction promoted c-Cbl-mediated ubiquitination of epsin, and uncovered a previously unappreciated ubiquitin-binding surface within VEGFR2. Mutational analysis revealed that the VEGFR2-epsin interaction is supported by VEGFR2 interacting specifically with the UIM and with ubiquitinated epsin. An epsin UIM peptide, but not a mutant UIM peptide, potentiated endothelial cell proliferation, migration and angiogenic properties
in vitro
, increased postnatal retinal angiogenesis, and enhanced VEGF-induced physiological angiogenesis and wound healing.

Conclusions:
Distinct residues in the epsin UIM and VEGFR2 mediate specific interactions between epsin and VEGFR2, in addition to UIM recognition of ubiquitin moieties on VEGFR2. These novel interactions are critical for pathophysiological angiogenesis, suggesting that these sites could be selectively targeted by therapeutics to modulate angiogenesis.
Abstract 26: Low Plasma Mir-155 Levels and RhoA Activation Correlates with Small AAA Expansion
Authors: Lee Eugene S , Nguyen Anthony , Ghosh Paramita , Rona Angelica , Afkhami Arash
Published: June 2021

Introduction:
Given increased abdominal imaging studies, the incidental diagnosis and treatment of small AAAs is problematic. Presently, no specific recommendations for medical management can be made other than serial observation. The ras homolog member A (RhoA) signaling cascade is implicated in vascular diseases due to its ability to modulate monocyte endothelial invasion. We sought to investigate the RhoA signaling pathway in AAA expansion, along with an endogenous inhibitor of RhoA, microRNA-155 (miR-155).

Hypothesis:
We hypothesize that RhoA is activated to enable AAA expansion, but once expanded, miR-155 is called on to inhibit RhoA activation so that the expanded AAA can be stabilized.

Methods:
Peripheral blood was collected for monocyte and plasma isolation from stable AAA (< 0.1 cm/year) (n=9) and expanding AAA (≥ 0.1 cm/year) (n=10) patients. Rhotekin-RBD assay and western blots were used to assess relative Rho-GTP expression and quantitative PCR for miR-155 detection in blood plasma. T-test and χ2 analysis were used to determine statistical significance.

Results:
Given limited monocytes, 6 stable and 10 expanding AAA patients were identified for RhoA analysis. Monocytes from expanding AAA patients contained higher active Rho-GTP protein levels compared to stable AAA monocytes (Figure 1a: 1.60±.075 vs 1.20±.088, ρ=.0004). Plasma concentrations of miR-155 were significantly lower in expanding AAA plasma compared to stable AAAs (Figure 1b: -2.17±.95, P=.005).

Conclusions:
Expanding AAA patients have greater monocyte activation of RhoA and decreased plasma expression of miR-155, when compared to stable AAA patients. The RhoA signaling pathway has altered activation in AAA disease and its regulation may reduce expansion rates and inflammation.
Abstract 262: Lipoprotein(a) Stimulates Pro-Inflammatory Phenotypes in Vascular Cells: Demonstration of a Role for the Lysine-Binding Properties of Apolipoprotein(a)
Authors: Scipione Corey A , Igbokwe Victor C , Romagnuolo Rocco , Koschinsky Marlys L
Published: June 2021

Elevated plasma Lp(a) concentrations are a causal risk factor for a variety of atherosclerotic disorders. Although the role of Lp(a) in vascular disease remains poorly understood, the atherogenicity of Lp(a) may stem from the direct atherosclerotic effects of its LDL-like component, or from the presence of the unique protein component apolipoprotein(a) (apo(a)). It has been hypothesized that Lp(a) contributes to early atherogenesis by initiating a program of endothelial cell (EC) dysfunction which results in increased EC permeability and the adoption of a pro-coagulant and pro-inflammatory phenotype. In support of the latter, studies have shown that Lp(a) stimulates expression of cell adhesion molecules (CAMs) on ECs, and induces monocyte recruitment to these cells. In the present study we demonstrate that a recombinant 17 kringle form of apo(a) (17K) stimulates expression of CAMs on primary human umbilical vein endothelial cells (HUVECs) in both a time- and dose-dependent manner, as determined by qRT-PCR analysis. We found that 17K was able to induce the expression of ICAM1, E-Selectin, VCAM1, but not PE-CAM at pathophysiologically-relevant concentrations of apo(a). However, 17KD57A, lacking the strong lysine-binding site (sLBS) in KIV10, lacked these effects. 17K was also able to induce a dose dependant increase in ICAM1 expression in the THP-1 monocyte cell line, an effect that was completely abolished by the NF-B inhibitor BAY-11-7082. In a fluorescent monocyte adhesion assay, both Lp(a) and 17K apo(a) were able to stimulate increased monocyte adhesion to ECs. Using 17KD57A, we were able to show that this effect is dependent on the sLBS in KIV10. The presence of the sLBS in KIV10 has also been found to be required for the covalent oxidized phospholipid modification on apo(a). We report that multiple KIV10 sLBS mutants lack the oxPL modification, further supporting a pro-inflammatory role for an intact KIV10 domain. Taken together, these studies contribute to a more complete understanding of the pathophysiological role that the apo(a) component of Lp(a) plays in the initiation and progression of atherosclerosis, and has the potential to aid in the development new therapeutics aimed at reducing the harmful effects of Lp(a) in the vasculature.
Abstract 29: Alpha-1-antitrypsin Protects High Density Lipoprotein From Functional Inactivation by Elastase
Authors: Gordon Scott M , Sviridov Denis , Sakurai Toshihiro , Freeman Lita , Remaley Alan T
Published: June 2021

High density lipoproteins (HDL) are complexes of lipid and protein with several known atheroprotective functions. These functions are driven by specific lipids and proteins contained on the HDL particle and include reverse cholesterol transport, suppression of inflammation, and modulation of endothelial function. These activities are most important within atherosclerotic plaque, a harsh environment where HDL interact with macrophage foam cells, activated neutrophils, and dysfunctional endothelial cells. Neutrophils and macrophages secrete proteases, such as elastase, which damage structural components and soluble proteins and propagate inflammatory signaling. It has been suggested that, in plaque, HDL become damaged and dysfunctional. We recently characterized a subspecies of HDL that carries the protein alpha-1-antitrypsin (A1AT), an abundant plasma serine protease inhibitor. In the current study, we tested the hypothesis that A1AT enriched HDL are protected from proteolytic damage and functional inactivation by elastase, the main protease inhibited by A1AT. Human HDL was isolated by ultracentrifugation and was enriched with A1AT by co-incubation and unbound A1AT was removed. Treatment of native HDL with elastase resulted in significant proteolytic degradation of both apoA-I and apoA-II, visualized by coomassie stained SDS-PAGE. Analysis of lipoprotein size by one dimensional native gel electrophoresis revealed that pre-beta HDL were completely degraded by elastase. Compared to native HDL, A1AT enriched HDL samples were protected from protein and pre-beta particle degradation by elastase. We next tested the effect of elastase treatment on HDL function. In native HDL, elastase had damaging effects on ABCA1 mediated cholesterol efflux (-32%; p<0.0001) and the ability to esterify free cholesterol (-14%; p<0.02). A1AT enriched HDL displayed no loss of functionality upon treatment with elastase. Both of these activities are required for HDL to perform what is thought to be its most important function, reverse cholesterol transport. In conclusion, the data presented indicate that HDL particles which contain A1AT may represent a functionally important species of HDL, which have an advantage in the protease-rich plaque environment.
Abstract 300: Pathogenetic Mechanisms of Thoracic Aortic Aneurysm in a Smad4 Mutant Mouse Model: Identification of New Molecular Targets for Pharmacological Therapy
Authors: Carnevale Daniela , Carnevale Raimondo , Da Ros Francesco , Cifelli Giuseppe , Iacobucci Roberta , Bizzotto Dario , Braghetta Paola , Bressan Giorgio , Lembo Giuseppe
Published: June 2021

TGFβ signaling have been implicated in the onset of thoracic aortic aneurysms (TAAs). It has been described cases of aortic disease in humans associated to mutations in SMAD4, an intracellular transducer required in the TGFβ canonical pathway. Interestingly, Smad4 homozygotes mice are embryonically lethal and embryonic inactivation of Smad4 in smooth muscle cells leads to early death because of aortic aneurysms. In order to study mechanistic insights and potential translational target of Smad4-dependent aneurysms, we postnatally targeted the gene, using a tamoxifen (TAM) inducible Cre recombinase under the smooth muscle myosin heavy chain promoter. Echocardiography showed a progressive dilatation of aorta and TAA formation. Histological analysis revealed progressive fragmentation of elastic lamellae with increased inflammatory infiltrates at sites of disarrangement. Most of these mice died 4/6 months because of rupture of thoracic aorta. We further characterized the infiltrate of monocytes in the aorta by flow cytometry, finding a significant increase of CD11b
+
Ly6C
hi
cells and F4/80
+
CD169
+
resident macrophages. Thus, we hypothesized that the deletion of Smad4 should activate an immune/inflammatory mechanism in the aorta, which could be in the end responsible for the pathological phenotype. At the molecular level, we actually found a significant increase in one of the noncanonical TGFβ signaling, i.e. the p65 subunit of NFkB, a protein complex involved in regulating responses to different stressors, including cytokines. Among these, we found a selective IL1β upregulation, produced as an inactive precursor and released upon caspase1 cleavage. In turn, caspase1 requires the function of NLRP3 inflammasome, a multiprotein platform that assembles in response to infection and tissue damage and is responsible for activation of inflammatory caspases. Therefore, we also tested NLRP3 mRNA levels and found them to be significantly upregulated. Overall, we demonstrated that selective Smad4 deletion in the SMC of adult mice is sufficient to induce an immune reaction starting from the molecular alterations downstream to TGFβ signaling and culminating in the onset of TAA.
Abstract 307: Angiotensin II Infusion Does Not Influence the Distribution of Cardiac Neural Crest Derived Smooth Muscle Cells in the Ascending Aorta
Authors: Sawada Hisashi , Moorleghen Jessica J , Rateri Debra L , Daugherty Alan
Published: June 2021

Objective:
Angiotensin II (Ang II) causes vascular pathology including hypertrophy and aneurysms. Smooth muscle cells (SMCs) exert a pivotal role in vascular remodeling. The localization of these vascular pathologies may be associated with distinct embryonic origins of SMCs. While there is evidence that most SMCs derive from the cardiac neural crest in the ascending aorta, other data has inferred a greater complexity of origins for this cell type. The aim of this study was to determine whether heterogeneity of the embryonic origin of SMCs in the ascending aorta is influenced by AngII.

Methods and Results:
ROSA26 lacZ mice were bred to mice expressing Cre under control of the Wnt1 promoter to track SMCs of cardiac neural crest origin. Gross examination of beta-galactosidase stained aortas from adult mice demonstrated cells of cardiac neural crest present from the sinotubular ascending aorta to just distal to the subclavian branch. For further examination of cell type distribution, both sagittal and cross sections were obtained. These stained sections demonstrated there was a distinct distribution on the anterior aspect of the ascending aorta, with SMCs of cardiac neural crest origin only being present in the inner laminal layers. On the proximal area of this region, there was transmural staining for beta-galactosidase. Much of the aortic arch to the subclavian branch also had transmural beta-galactosidase staining. Previous studies have demonstrated that infusion of AngII promotes pathology that varies along the length of the ascending aorta. Also, the medial pathology is most pronounced on the adventitial aspect. Therefore, we examined whether the distribution of cardiac neural crest-derived SMCs was influenced during AngII infusion. However, after 28 days of pressor infusion rates of AngII, there was no overt change in the distribution of cardiac neural crest-derived SMCs in the ascending aorta.

Conclusion:
SMCs from the outer aspect of the media are derived from a different embryonic origin. Cardiac neural crest derived-SMCs retain their unique distribution in the inner media of the ascending aorta during AngII infusion in adult mice.
Abstract 313: Differences in Galectin-3, a Biomarker of Fibrosis, Between Subjects With Peripheral Artery Disease and Controls
Authors: Casanegra Ana I , Stoner Julie A , Tafur Alfonso J , Pereira Anne , Rathbun Suman , Gardner Andrew W
Published: June 2021

Introduction:
Galectin-3 (G3) mediates vascular fibrosis. It can be detected in blood but it is unknown whether G3 is a marker of peripheral artery disease (PAD) or correlates with early atherosclerosis markers.

Objectives/Aims:
To determine if G3 plasma levels are different between subjects with PAD and controls without PAD.

Secondary aim:
To describe the relationship between G3 plasma levels and markers of early atherosclerosis (arterial elasticity, high-sensitivity C-reactive protein [HS-CRP], homeostatic model assessment [HOMA], and urinary albumin/creatinine ratio [UACR]) in a sample of subjects with PAD and controls.

Methods:
Consecutive subjects (n=60) were recruited in 2 groups: (1) a PAD group (n=31), with ankle/brachial index (ABI) <0.90, and (2) non-PAD controls (n=29) with ABI 1-1.4. G3 was measured by ELISA. Demographics, cardiovascular risk factors and markers of early atherosclerosis were compared between groups using t-tests, Chi-square tests and logistic regression and the association between G3 and markers of early atherosclerosis was investigated using linear regression analysis.

Results:
Compared to controls, participants with PAD were older (mean 68.6 vs. 61.8 years, p=0.02), had a higher percentage of men (68% vs. 38%, p=0.02), and had more smokers, diabetics, and subjects with hypertension and dyslipidemia (p<0.001 for all). G3 was higher in PAD (mean ± SD: 17.6±4.7 vs. 14.4±4.1 ng/mL, p<0.01). The odds ratio for G3 in the PAD group to be 1 ng/mL higher than the control group was 1.19, after adjusting by age (≥65 years) and gender, (95% CI 1.036-1.37, p=0.014). when HS-CRP was included in the model the association between PAD and G3 was not significant. HS-CRP (p=0.005) and HOMA (p=0.016) were positively associated with G3 in the adjusted model. Arterial elasticity (p=0.4) and UACR (p=0.09) were not associated with G3 in the adjusted model.

Conclusion:
After adjustment for age and gender, mean G3 levels were 22% higher in PAD participants compared to controls and G3 was positively associated with HS-CRP. Future studies are warranted to determine if G3 is an accurate prognostic biomarker or screening tool to detect PAD and atherosclerosis.
Abstract 315: Feasibility of Arteriovenous Fistula Creation After Previous Radial Artery Harvesting for Aortocoronary Bypass
Authors: Gargiulo Nicholas J
Published: June 2021

Background:
Arteriovenous fistula (AVF) formation remains the procedure of choice in patients requring hemodialysis. The feasibility of AVF creation in the setting of prior radial artery harvesting after aortocoronary bypass remains unknown. This investigation elucidates which patients might be candidates for AVF creation despite prior radial artery harvesting.

Methods:
A retrospective review was performed on 2,100 patients undergoing hemodialysis access procedures from 2003 to 2010. Of these patients, 11 (0.5%) were identified as having prior radial artery harvesting for aortocoronary bypass. Pre/Post-operative vein mapping, arterial duplex, digital plethysmography, selective angiography, and sestamibi scanning was performed to evaluate the ulnary artery and palmar arch. Patients with evidence suggesting an intact ulnar artery circulation then underwent AVF creation.

Results:
All 11 patients had an adequate preoperative work up. Seven (64%) of the 11 patients had digital plethysmography suggesting an intact ulnar artery/palmar arch and underwent successful AVF creation. Three (27%) of the patients had a variety of findings precluding successful AVF creation. One (9%) patient with normal preoperative plethysmography developed a steal syndrome requiring revision of the arteriovenous fistula.

Conclusions:
Successful AVF creation is feasible in patients with prior radial artery harvesting for aortocoronary bypass. The use of preoperative digital plethysmography, selective ulnar artery/palmar arch arteriography and sestamibi scanning to evaluate forearm muscle perfusion may be used as adjuncts to guide a successful intervention.
Abstract 33: Platelet 12-lipoxygenase is a Key Regulator of Platelet Reactivity and Thrombus Formation
in vivo
Authors: Adili Reheman , Mast Katherine , Holman Theodore R , Holinstat Michael
Published: June 2021

Background:
Platelet reactivity is required to maintain hemostasis, however high platelet reactivity leads to thrombus formation, myocardial infarction, and stroke. Platelet 12-lipoxygenase (12-LOX) has been demonstrated by our lab and others to regulate agonist-mediated platelet reactivity suggesting a role for 12-LOX in regulation of
in vivo
thrombosis. The ability to target 12-LOX
in vivo
has not been established to date. Therefore, we sought to determine if 12-LOX regulates platelet reactivity and thrombus formation
in vivo
using the selective 12-LOX inhibitor ML355 to determine whether platelet 12-LOX is an effective target for anti-platelet therapeutics.

Methods:
ML355 effects on human platelet function was assessed
in vitro
by platelet aggregometry,
ex vivo
by perfusion chamber, and
in vivo
by thrombus formation and vessel occlusion in small and large vessels in 12-LOX
-/-
, WT mice, and mice treated with ML355 via intravital microscopy using the FeCl
3
and laser injury models.

Results:
In
in vitro
platelet aggregation, ML355 dose-dependently inhibited agonist-induced aggregation. In
ex vivo
flow chamber assays, platelet adhesion and thrombus formation on collagen-coated surfaces at high shear was attenuated in both mouse and human whole blood after incubation with ML355. Further, platelet aggregation and thrombus growth in 12-LOX
-/-
mice were impaired in both laser and FeCl
3
-induced mesenteric, carotid artery and cremaster arteriole thrombosis models. Thrombi in 12-LOX
-/-
mice were unstable and frequently formed emboli, which resulted in impaired vessel occlusion or reopening. Additionally, thrombus formation and vessel occlusion was impaired in ML355 treated WT mice.

Conclusions:
The 12-LOX inhibitor ML355 inhibits platelet aggregation induced by a number of platelet agonists.
Ex vivo
high shear conditions in both mice and human was attenuated in the presence of ML355. Thrombus formation and vessel occlusion were impaired in mice deficient in 12-LOX. Finally, ML355 attenuates thrombus formation and prevents vessel occlusion
in vivo
. Our data strongly indicates 12-LOX is an important determinant of platelet reactivity and inhibition of platelet 12-LOX may represent a new target for anti-platelet therapeutics.
Abstract 334: An Echogenic Clot Method for Thrombolysis Monitoring of Directed Lytics in Thrombotic Stroke
Authors: Carter Dalton E , Peng Tao , Moody Melanie R , Huang Shao-Ling , McPherson David D , Klegerman Melvin E
Published: June 2021

Background:
We have previously demonstrated thrombolytic efficacy of tissue plasminogen activator (tPA)-loaded echogenic liposomes (TELIP), a directed therapeutic construct that features exposed tPA fibrin-binding sites, in a rabbit abdominal aorta thrombosis model. The most relevant animal model for evaluation of directed thrombolytic therapy for ischemic stroke is the rabbit thrombotic stroke model previously used to validate recombinant tPA (rtPA; Alteplase). The ability to precisely monitor tPA-mediated thrombus dissolution in vivo is important for quantitative assessment of directed TELIP efficacy.

Hypothesis:
A gas-free ultrasound contrast agent can be incorporated into blood clots at a concentration that does not affect the tPA-mediated clot dissolution rate, while enabling quantitative assessment of the clot dissolution rate.

Methods:
In 1.5-ml Eppendorf tubes, 0.2-ml clots were formed from a mixture of whole rabbit blood, 1 M calcium chloride, human thrombin and various amounts of microcrystalline cellulose. At 0-time, 50 μg rtPA in 0.5 ml porcine plasma was added to each tube. Washed clots in tubes were weighed with an analytical balance at 30, 60 and 90 minutes. Clot echogenicity at each time point was assessed with a Philips HDI 5000 ultrasound system using an L12-5 linear array probe positioned laterally to tubes placed in an anechoic chamber. Recorded Images were subjected to videodensitometric analysis that converted image reflectivity to mean gray scale values (MGSV).

Results:
We found that 1.12 mg/ml of microcrystalline cellulose in rabbit blood clots (0.2 ml) provided optimal echogenicity without affecting the clot dissolution rates (0.3-0.6 mg/min.) caused by 100 μg/ml of rtPA. The clot dissolution rate measured by videodensitometric analysis of serial sonograms of the echogenic clots agreed well with that determined by mass loss measurements (0.28% 0-time value/minute).

Conclusions:
We have developed a method for monitoring rtPA-mediated clot mass loss by ultrasound imaging techniques and validated it in vitro. This method will be important for demonstrating the enhanced efficacy and decreased hemorrhagic effects provided by directed tPA vehicles relative to systemic administration of the free thrombolytic.
Abstract 34: Impact of Thrombomodulin and Thrombin-activatable Fibrinolysis Inhibitor on the Anti-coagulant and Pro-fibrinolytic Effects of Rivaroxaban
Authors: Garabon Justin J , Boffa Michael B
Published: June 2021

Rivaroxaban is a novel oral anticoagulant that directly inhibits factor Xa and has recently been implemented as a favorable alternative to warfarin in anticoagulation therapy. Rivaroxaban effectively reduces the generation of thrombin, which plays a major role in the activation of thrombin activatable fibrinolysis inhibitor (TAFI) to TAFIa. Activated TAFI functions by downregulating fibrinolysis, and this effect is highly dependent on the availability of thrombomodulin (TM), as the thrombin-TM complex activates TAFI with a 1250-fold greater efficiency than thrombin alone. Additionally, a naturally occurring SNP in the gene encoding TAFI gives rise to a Thr325Ile polymorphism that increases the stability and antifibrinolytic potential of TAFIa. Since inhibition of thrombin generation could lead to decreased TAFIa formation, we hypothesized that these parameters could influence the pharmacodynamics and pharmacogenomics of rivaroxaban. To assess this, effects on coagulation and fibrinolysis were measured using an
in vitro
plasma clot lysis assay. Rivaroxaban and TM were titrated into TAFI-deficient plasma in the presence or absence of 10 nM wild-type or T325I recombinant TAFI. With increasing concentrations of rivaroxaban, clot formation latency was accordingly increased. TM further delayed clot formation, and this effect was equal in the absence or presence of either TAFI variant. Rivaroxaban was also shown to decrease the rate of coagulation, which was decreased further at higher concentrations of TM. The presence of either TAFI variant also seemed to decrease clot formation rate at higher levels of rivaroxaban and TM. At all concentrations of TM, TAFI-dependent resistance to fibrinolysis was attenuated by rivaroxaban. The effect of rivaroxaban was, however, greater for wild-type TAFI than for the T325I variant. In conclusion, rivaroxaban exhibits TAFI-dependent profibrinolytic effects that are influenced by the levels of TM and the by intrinsic stability of TAFIa. TM also affected the dynamics of coagulation. These findings suggest a role for the anatomical location of a procoagulant stimulus and plasma TM-altering disease phenotypes in the pharmacodynamics and a role for the T325I polymorphism in the pharmacogenomics of rivaroxaban.