bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 545: Suppression of Atherosclerosis by Synthetic REV-ERB Agonist
Authors: Sitaula Sadichha , Billon Cyrielle , Solt Laura , Burris Thomas P
Published: March 2022

Background:
Development of atherosclerosis is a complex process that involves both hyperlipidemia and inflammation. Therapies that can target both metabolic and inflammatory aspect of this disease have significant potential in the treatment of cardiovascular diseases. The nuclear receptors for heme, REV-ERBα and REV-ERBβ, play important roles in regulation of metabolism and inflammation. Recently it was demonstrated that knock-down of REV-ERBα in hematopoetic cells in LDL receptor null mice led to increased atherosclerosis. We sought to determine if synthetic REV-ERB agonists that we have developed might have the ability to suppress atherosclerosis in this model.

Methods and Results:
A previously characterized synthetic REV-ERB agonist, SR9009, was used to determine if activation of REV-ERB activity would affect atherosclerosis in LDL receptor deficient mice. Eight-week-old mice with deficient LDL receptor were fed high cholesterol diet and treated with vehicle or REV-ERB agonist, SR9009 (100mg/kg, I.P.) for a period of eight weeks. Atherosclerotic plaque size was significantly reduced in mice administered SR9009 compared to control mice. In another study plasma lipid levels were monitored every two weeks for eight weeks. We demonstrate that SR9009 significantly reduced plasma lipid levels in these mice. To determine the role of REV-ERB in macrophage differentiation and polarization bone-marrow derived macrophages (BMDM) were treated with SR9009 in vitro. SR9009 treatment of BMDM reduced the polarization of BMDM to proinflammatory M1 macrophage. Gene expression of markers of M1 polarization as well as pro-inflammatory cytokine secretion was reduced in macrophages treated with SR9009.

Conclusions:
We demonstrate that REV-ERB targets multiple processes involved in atherosclerosis. Our results suggest that pharmacological targeting of REV-ERBs may be a viable therapeutic option for treatment of atherosclerosis.
Abstract 547: A Novel Abetalipoproteinemia Missense Mutation Highlights the Importance of N-terminal β-barrel in Microsomal Triglyceride Transfer Protein Function
Authors: Walsh Meghan , Iqbal Jahangir , Josekutty Joby , Soh James , Di Leo Enza , Ozaydin Eda , Gunduz Mehmet , Tarugi Patrizia , Hussain Mahmood
Published: March 2022

Background:
The use of microsomal triglyceride transfer protein (MTP) inhibitors is limited to severe hyperlipidemias due to associated hepatosteatosis. Comprehensive knowledge about the structure-function of MTP might help design new molecules that avoid steatosis. Characterization of mutations in MTP causing abetalipoproteinemia (ABL) have revealed that the central α-helical and C-terminal β-sheet domains are important for protein disulfide isomerase (PDI) binding and lipid transfer activity. Our aim was to identify and characterize ABL mutations in the N-terminal region to understand the function of this domain.

Methods and Results:
We identified a novel missense mutation (D169V) in a 4-month old Turkish male with severe signs of ABL. To study the effect of this mutation on MTP function, we created mutants via site directed mutagenesis. Although D169V was expressed in the endoplasmic reticulum and interacted with apoB17, it was unable to bind PDI, transfer lipids, and support apoB secretion. Computational modeling suggested that D169 could form an internal salt bridge with K187 and K189. Mutagenesis of these lysines to leucines abolished PDI heterodimerization, lipid transfer, and apoB secretion, without affecting apoB binding. Further, mutants with preserved charges (D169E, K187R, K189R) rescued these activities.

Conclusions:
D169V is detrimental because it disrupts an internal salt bridge leading to loss of PDI binding and lipid transfer activities; however, it does not affect apoB-binding. We propose that future inhibitors should avoid this region of the N-terminal domain.
Abstract 549: Loss of Macrophage Akt2/Akt3 or Akt1/Akt2 Decreases Cell Survival and Suppresses Early Atherosclerosis in Ldlr Null Mice
Authors: Babaev Vladimir R , Ding Lei , Zhang Youmin , May James M , Linton MacRae F
Published: March 2022

Akt is a serine/threonine-specific protein kinase that plays a key role in multiple cellular processes including glucose metabolism and cell survival. Macrophages express three Akt isoforms, Akt1, Akt2 and Akt3 which have been ascribed a number of isoform specific effects but may also be functionally redundant in terms of Akt signaling. Here we hypothesize that dramatic reduction of Akt content with loss of two isoforms will suppress Akt signaling in macrophages, reducing their survival and modulating the development of atherosclerosis. To test this hypothesis, we generated mice with combined deficiency Akt2/Akt3 in hematopoietic cells. Peritoneal macrophages isolated from these mice preserved a similar level of Akt phosphorylation when treated with insulin but exhibited a dramatic increase in apoptosis in response to several different factors that induce ER stress compared to WT or single Akt2 or Akt3 knockout macrophages. Male LDLR null mice reconstituted with double Akt2/Akt3 knockout hematopoietic cells and fed the Western diet for 8 weeks developed smaller (57.6 % reduction) atherosclerotic lesions with more apoptotic macrophages in the proximal aorta than control WT→LDLR
-/-
mice, in the absence of changes in plasma lipid levels. Thus, loss of Akt2/Akt3 in macrophages increases their sensitivity to apoptosis and decreases atherosclerosis. Next, we generated mice with combined deficiency of Akt1/Akt2 in hematopoietic cells. Again, loss of a single Akt1 or Akt2 isoform in macrophages had no impact on cell apoptosis, whereas combined Akt1/Akt2 deficiency did not change Akt phosphorylation but markedly compromised their sensitivity to different apoptotic stimuli. Furthermore, male and female LDLR
-/-
mice transplanted with Akt1/Akt2 knockout hematopoietic cells had significantly smaller atherosclerotic lesions with increased macrophage apoptosis than control WT→LDLR
-/-
mice. Taken together these data indicate that a dramatic reduction of Akt protein in macrophages with loss of either of the Akt1/Akt2 or Akt2/Akt3 pairs of isoforms significantly compromises cell survival and this diminishes early atherosclerosis.
Abstract 558: Neutrophils-derived-microvesicle Could Play a Role in the Early Stage of Atherosclerosis
Authors: Gomez Ingrid , Ward Ben , Evans Paul , Hellewell Paul , Ridger Victoria
Published: March 2022

Background:
Atherosclerosis is the most common underlying cause of cardiovascular disease. Many cells types are involved in the initiation and progression of atherosclerotic plaques but, until recently, neutrophils were not considered to be among them. Despite their rare detection in plaques, neutrophil depletion has been shown to reduce plaque formation and monocyte recruitment to the vessel wall.

Hypothesis:
Neutrophils may influence plaque initiation and progression through the formation of microvesicles.

Aim:
To determine the role of neutrophil-derived-microvesicles in the early stage of atherosclerosis.

Methods and Results:
Mouse peripheral blood neutrophils were isolated by negative magnetic separation. Following stimulation for 1h with fMLP (3x105M), supernatants were subjected to differential centrifugation (twice at 300g for 6 min followed by 20,000g for 20 mins) to isolate the microvesicle population. Microvesicles were quantified using flow cytometry calibrated with MegaMix and Spherocount beads. Apo E mice were fed a Western diet for 6 weeks and injected twice a week with PBS or neutrophil microvesicles (4.52x106 ± 3.05x105) isolated from control mice. After 6 weeks, blood was collected for neutrophil and microvesicle quantification. The aorta was dissected and stained by conventional procedures using Oil Red. Areas of plaque formation was determined using computer based imaging and semi-quantitative scoring.

Injection with microvesicles increased circulating neutrophil number (PBS treated = 67.3 ± 3.9 neutrophils per μl; microvesicle treated = 88.25 ± 4.0 neutrophils per μl, P = 0.031; n = 3) and neutrophil reactivity with respect to microvesicle formation (PBS treated = 4.5 ± 0.8 microvesicles per neutrophil; microvesicle treated = 8.3±0.7 microvesicles per neutrophil, P = 0.0249; n = 3).

After 6 weeks, the plaque formation was more advanced in the MVs group (% lesion area in the arch in mice injected with PBS = 0.4563 ± 0.09452, with MVs = 1.643 ± 0.3891, P = 0.0118, n=7).

Conclusion:
Neutrophil microvesicles induce increased plaque formation and increase circulating neutrophil levels in a mouse model of atherosclerosis. This suggests neutrophils may be involved in atherogenesis through the release of microvesicles.
Abstract 563: Activated Circulating Intermediate Monocytes are Associated with Subclinical Coronary Plaque in Apparently Healthy Middle-Aged Men
Authors: Kral Brian G , Gama Lucio , Becker Diane M , Yanek Lisa R , Fishman Elliot K , Vaidya Dhananjay , Becker Lewis C
Published: March 2022

Background:
The intermediate subset accounts for the smallest percentage of circulating monocytes yet has been associated with chronic inflammatory diseases, including CAD outcomes. It is unknown if activated intermediate monocytes (IM) are associated with coronary plaque at an earlier stage in atherosclerosis, prior to clinical manifestations.

Methods:
We screened 11 healthy nondiabetic, non statin using, age-matched males for the presence and extent of coronary plaque using advanced dual-source CT angiography. Total coronary plaque (TCP) volumes (mm3) were quantified using a validated automated method and transformed to log(TCP+1). To examine monocyte subsets, whole blood was labeled to exclude non-monocytic cells. Surface CD14, CD16, TLR2, CX3CR1, and CD11b were used to confirm monocyte subsets by flow cytometry with HLA-DRhi as a marker of activation.

Results:
Mean age was 54±2 (range 49 to 57); 36% were African American, 27% were smokers, and 45% had hypertension. Mean LDL-C was 121±38. The absolute number of circulating activated IM per cu mm was higher in the 6 individuals with coronary plaque compared to the 5 with no plaque (42.6±24.8 vs. 15.6±11.9, p=0.05). In bivariate analysis the number of activated IM was significantly associated with TCP (Figure, r=0.71, p=0.01). These associations were not found with classical and nonclassical monocytes.

Conclusion:
In a small population of age-matched, nondiabetic apparently healthy middle-aged men not on statin therapy, the number of circulating activated IM was associated with the presence and volume of subclinical coronary plaque. Further investigation of monocyte activation prior to plaque development is ongoing.
Abstract 568: ApoE-/- Mice Lacking Hemopexin Develop Aggravated Atherosclerosis via Mechanisms That Include Altered High Density Lipoprotein Inflammatory Properties and Macrophage Function
Authors: Mehta Niyati U , Hama Susan , Grijalva Victor , Reddy Srinivasa T
Published: March 2022

Introduction:
The etiology of atherosclerosis is multi-faceted. Atherosclerotic plaques are characterized by the accumulation of lipid-laden macrophages, reactive oxygen species (ROS) and pro-inflammatory lipoproteins. Recent studies suggest that the anti- or pro-inflammatory nature of high density lipoprotein (HDL) may be a more sensitive indicator of atherosclerosis than HDL-cholesterol (HDL-C) levels alone. Our laboratory previously reported that hemopexin (Hx) is significantly increased and associated with pro-inflammatory HDL under atherogenic conditions. Hx is a heme scavenging protein which together with macrophages, plays a role in mitigating heme and ROS mediated oxidative damage. However, the role of Hx in atherosclerosis is not known.

Hypothesis:
We hypothesized that absence of Hx plays an important role in the accumulation of pro-inflammatory HDL, macrophage function, and the development of atherosclerosis.

Methods:
We crossed Hx
-/-
mice onto the apoE
-/-
background (HxE
-/-
mice). Chow diet fed HxE
-/-
and apoE
-/-
mice (n=10-15) were analyzed for lipoprotein inflammatory properties, macrophage gene expression, and atherosclerosis.

Results:
Serum ROS and heme (p<0.05) were significantly higher in the HxE
-/-
mice. Livers of HxE
-/-
mice showed increased lipid peroxides (p<0.001), and
ex vivo
monocyte chemotactic assays indicated that HDL from HxE
-/-
mice was more pro-inflammatory (p<0.002). Expression of macrophage cholesterol efflux genes: ATP-binding cassette transporter, subfamily A, member 1 (ABCA1) and its transcriptional regulator liver X receptor-alpha (LXRα), were significantly reduced (p<0.05) whereas expression of the classical M1 macrophage cell-surface receptor CCR2 was significantly increased (p=0.005) in the HxE
-/-
mice. Atherosclerotic plaque area and macrophage infiltration was significantly higher in the aortic sinus (p<0.004), and atherosclerotic lesions in the aortas were significantly higher in the HxE
-/-
mice (p<0.05).

Conclusion:
Our results suggest for the first time that Hx plays a protective role in the inflammatory properties of HDL, macrophage function and in the development of atherosclerosis in apoE-/- mice. We conclude that Hx is a novel target for the treatment of atherosclerosis.
Abstract 572: Relationship of Aortic Stiffness Measured by Cardiac MRI with Left Ventricular Geometry and Function
Authors: Ohyama Yoshiaki , Rezzadeh Kevin , Ambale-Venkatesh Bharath , Yoneyama Kihei , Redheuil Alban , Yu Wen-Chung , Wu Colin , Bluemke David , Lima Joao
Published: March 2022

Background:
The association of aortic stiffness parameters measured by MRI with cardiac structure and function remains poorly explored. We investigated how central aortic stiffness assessed by MRI relates to Left ventricular (LV) structure and function in broad rage aged subjects free for cardiovascular disease.

Methods:
79 asymptomatic subjects (37 men, age range: 20 - 78 years) were studied. Ascending aortic distensibility (AAD) and aortic pulse wave velocity (PWV) was measured by phase contrast (PC) cine MRI at the level of the pulmonary artery bifurcation. LV mass and LV volume indexed to body surface area, LV ejection fraction (LVEF) and LV mass volume ratio (M/V ratio) were determined by cine MRI. LV circumferential strain (Ecc), longitudinal strain (Ell) and LV torsion were determined from tagged MRI. Central pressures were used to calculate AAD. Linear regression analysis was used to evaluate the association between arterial measures and LV measures. Distributions of AAD and PWV were skewed, so they were logarithmically transformed (logAAD and logPWV) for regression models.

Results:
Lower AAD and higher PWV, both indicating greater aortic stiffness, was associated with less/worse myocardial systolic function parameters; Ecc, Ell, and M/V ratio. After adjustment for age, gender and hypertension, only AAD was associated with LV deformation while PWV was not (Table).

Conclusion:
Our observations indicate that the association of increased aortic stiffness with LV dysfunction because of aging and hypertension may be captured by PWV but the association with decreased distensibility could be because of common atherosclerosis risk factors in healthy population.
Abstract 587: Leukocyte Calpain Deficiency Reduces Angiotensin II-induced Inflammation and Atherosclerosis in Hypercholesterolemic Mice
Authors: Howatt Deborah A , Balakrishnan Anju , Moorleghen Jessica J , Rateri Debra L , Uchida Haruhito A , Takano Jiro , Saido Takaomi C , Chishti Athar H , Baud Laurent , Subramanian Venkateswaran
Published: March 2022

Background and Objective:
Chronic angiotensin II (AngII) infusion promotes atherosclerosis and abdominal aortic aneurysms (AAAs) in mice. Recently, we demonstrated that pharmacological inhibition of calpain-1 and -2 (a class of calcium-activated, neutral cysteine proteases) attenuated AngII-induced atherosclerosis, AAAs, and medial macrophage accumulation in mice. Using mice that overexpress calpastatin (CAST), the endogenous inhibitor of ubiquitous calpain-1 and -2 or mice with specific deficiency of calpain-1 or -2, the purpose of this study was to determine whether calpains in bone marrow (BM)-derived cells contribute to AngII-induced atherosclerosis and AAAs in hypercholesterolemic mice.

Methods and Results:
Irradiated male LDL receptor -/- mice were repopulated with BM-derived cells that were harvested from either wild type (WT) or CAST overexpression transgenic (Tg) mice. Four weeks after BM repopulation, recipient mice were fed a saturated fat-enriched diet and infused with either saline or AngII (1,000 ng/kg/min) for 4 weeks. AngII infusion increased systolic blood pressure equivalently in both genotypes. Ex vivo measurement of maximal diameter of abdominal aorta showed a comparable dilation in WT and Tg mice (1.28 ± 0.11 versus 1.32 ± 0.08 mm, P = NS). Interestingly, overexpression of CAST in BM-derived cells significantly attenuated AngII-induced atherosclerotic lesion formation in aortic arches by en face measurement (Intimal lesions: WT = 19.9 ± 1.8 % vs Tg = 9.9 ± 1.0 %; n=14, P<0.05). Using either BM cells-derived from calpain-1 deficient mice or mice with leukocyte-specific calpain-2 deficiency (Calpain-2f/f LysM Cre), further studies demonstrated that deficiency of either calpain-1 or -2 in leukocytes resulted in a modest, but significant, (P<0.05) decrease in AngII-induced atherosclerosis (Calpain-1: WT- 15 ± 1.2 % vs KO- 10 ± 0.96 %; Calpain-2f/f: LysM Cre WT-16 ± 1.4 % vs KO-11 ± 1.2 %). CAST overexpression significantly attenuated AngII-induced inflammatory responses in macrophages in vivo, and suppressed macrophage migration towards MCP-1 and adhesion to endothelial cells in vitro.

Conclusion:
Calpain inhibition in BM-derived cells attenuated AngII-induced atherosclerosis by influencing macrophage function.
Abstract 592: Rapamycin Increases the Thrombogenic Potential of Neutrophil Extracellular Traps (NETs): Potential Mechanism of Prothrombotic Drug-Eluting Stents
Authors: Robinson D. K , Healy Laura D , Chu Tiffany T , Oldenkamp Heidi , Garcia Puy Christina , Gruber András , McCarty Owen J
Published: March 2022

Background:
Neutrophil leukocytes are considered among the first cellular responders to bacterial host tissue infection. At sites of inflammation, neutrophils can undergo activation, releasing their nuclear content by freeing hydrolases from lysosomes to form microbicidal protein exposing neutrophil extracellular traps (NETs). NETs provide a structural framework for pathogen clearance; however, recent studies have demonstrated that NETs may additionally contribute to thrombotic complication including deep vein thrombosis. We have previously demonstrated a critical role for the mammalian target of rapamycin (mTOR) pathway regulating autophagy to promote NETs formation. Second-generation drug-eluting stents (DES) containing rapamycin have successfully reduced the rate of restenosis; however, they are associated with thrombotic complications. The mechanism by which mTOR inhibition by rapamycin promotes in-stent thrombosis remains ill-defined.

Aims:
To determine the role of mTOR regulation during NETs formation to regulate the thrombogenic potential of NETs.

Results:
Purified human neutrophils were stimulated with N-Formyl-Met-Leu-Phe (fMLP) in the presence of rapamycin to induce NETs. We found that NETs bound coagulation factors fibrinogen, prothrombin and FXI in platelet-poor plasma. In an
ex vivo
model of occlusive thrombus formation, NETs formation accelerated occlusive thrombus formation in recalcified whole blood. Inhibition of activated FXII (FXIIa) with corn trypsin inhibitor or inhibition of FXI activation by FXIIa or factor IX activation by FXIa abrogated the procoagulant activity of NETs.

Conclusions:
Our data demonstrate a role for the mTOR pathway in the presence of fMLP to regulate the procoagulant activity of NETs, suggesting a possible mechanism by which rapamycin in DESs promotes thrombosis.
Abstract 60: Histone Modification H3k4me2 Controls Vascular Smooth Muscle Cell Lineage Memory during Vascular Injury
Authors: Gomez Delphine , Owens Gary K
Published: March 2022

Smooth muscle cells (SMC) have the ability to undergo reversible phenotypic switch (i.e. dedifferentiation/redifferentiation) characterized by loss and re-expression of SMC marker genes, a process occurring during blood vessel repair and adaptive remodeling. Using SMC lineage-tracing mice, we showed that newly generated SMC derive from pre-existing SMC that transiently de-differentiate, participate in repair, and then redifferentiate following wire-induced femoral injury. However, a critical unresolved question is how dedifferentiated SMC retain their lineage identity. We previously demonstrated that SMC acquire a unique epigenetic signature during differentiation, H3K4me2 on the SMC marker genes, retained in SMC during PDGF-BB-induced phenotypic switch. Our hypothesis is that H3K4me2 on SMC genes biases SMC re-differentiation and functions as a lineage memory mechanism during vascular injury-repair. We demonstrated that H3K4me2 on SMC genes is retained in dedifferentiated and redifferentiated SMC in vivo following vascular injury using our unique ISH-PLA epigenetic assay. We also found that H3K4me2 is present and retained on the SMC regulatory gene Myocardin during PDGF-BB treatment, reinforcing significance of H3K4me2 as a regulator of SMC identity. To directly assess the functional role of H3K4me2 on the SMC repertoire, we generated a fusion protein consisting of the H3K4 demethylase LSD1 coupled to the SRF-CArG binding domain of Myocardin (Myocd-LSD1WT) to perform SMC-specific locus-selective removal of H3K4me2 on the SMC marker genes. We also generated loss-of-function mutant of Myocd-LSD1 to ensure that effects are due to H3K4me2 removal and not steric hindrance. Stable expression of Myocd-LSD1WT in SMC induced locus selective removal of H3K4me2 on SMC CArG genes without impacting non-SMC CArG-dependent genes like c-fos. Myocd-LSD1WT also inhibits expression of the SMC marker genes and abolishes the ability of SMC to redifferentiate in PDGF-BB induced phenotypic switch. None of these effects were observed in Myocd-LSD1Mut SMC. Results are the first to our knowledge to provide direct functional evidence that locus specific epigenetic modifications regulate cell lineage memory during reversible phenotypic transitions.
Abstract 603: Nestin+ Cells Regulate Bone Marrow Egress of Inflammatory Cells and Their Infiltration in the Aorta in Atherosclerosis
Authors: Del Toro Raquel , Mendez-Ferrer Simon
Published: March 2022

Atherosclerosis is a chronic inflammatory disease of the artery wall and represents the leading death cause in developed countries. Endothelial cells and smooth muscle cells participate in atherogenesis, but it is unclear whether other mesenchymal cells in the bone marrow and/or the artery wall contribute to this process. We have recently identified mesenchymal stem cells with niche functions for haematopoietic stem cells in the bone marrow, based on the expression of the intermediate filament protein nestin (Méndez-Ferrer S et al. Nature 2010). Some nestin+ cells also cooperate with endothelial cells in directing the egress of pro-inflammatory monocytes from the bone marrow to circulation in response to peripheral infections (Shi C at el. Immunity 2011). However, it remains unknown whether nestin+ cells regulate the migration of inflammatory cells in a chronic inflammatory disease, such as atherosclerosis. Also, some endothelial cells express nestin (Isern J et al. eLife 2014), but it remains unclear whether they have a specialized function. We found that nestin+ cells residing in the adventitia of adult mouse aorta increased more than 30 times in apolipoprotein E (ApoE) KO mice fed with high fat diet and contributed to endothelial cells in the atheroma plaque. In this mouse model, deletion of monocyte chemoattractant protein-1 (Mcp1) in nestin+ cells (using Nestin-creERT2;Mcp1-floxed mice), but not in endothelial cells (using Cdh5-creERT2;Mcp1-floxed mice), led to increased number of pro-inflammatory monocytes and neutrophils in circulation. Surprisingly, the number of inflammatory cells in the aorta of these mice was reduced. Adoptive transfer experiments confirmed that Mcp1 secreted by nestin+ cells within the artery wall is required for the initial infiltration of pro-inflammatory monocytes and neutrophils during atherogenesis. As a result, mice lacking Mcp1 in nestin+ cells -but not in endothelial cells only- had reduced inflammatory infiltration in the aorta, delayed atheroma plaque formation, less calcified aortas (μCT) and reduced calcium deposits in the aortic valves. These results suggest that nestin expression marks stromal cells that regulate inflammatory cell migration in atherosclerosis.
Abstract 609: Comparing the Pro-angiogenic Properties of Induced Endothelial Cells and Induced Pluripotent Stem Cell Derived Endothelial Cells in a Murine Hindlimb Ischaemia Model
Authors: Clayton Zoe E , Yuen Sui Ching G , Sadeghipour Sara , Hywood Jack D , Abraham Sheena , Wong Wing Tak J , Cooke John P , Patel Sanjay
Published: March 2022

Introduction:
Endothelial cells derived from induced pluripotent stem cells (iPSC-ECs) have been shown to promote angiogenesis in vivo. Recently, an easier and more effective method of generating endothelial cells has been developed, by directly differentiating fibroblasts into endothelial cells (iECs), bypassing the pluripotent intermediate. However, the pro-angiogenic properties of iECs have not yet been determined. Accordingly, we compared survival, engraftment and pro-angiogenic potential of iECs versus iPSC-ECs in a mouse model of hindlimb ischaemia.

Methods and Results:
Human iPSC-ECs and iECs were transduced with firefly luciferase and green fluorescent protein reporter for bioluminescence imaging. They exhibited endothelial behavior in vitro, assessed by migration towards VEGF, uptake of acetylated-LDL and tubule formation on matrigel. NOD-SCID mice (n=7-9) underwent unilateral femoral artery ligation and received either control vehicle or 1x106 iPSC-ECs or iECs, via intramuscular injection. Recovery of the ischaemic limb was tracked for 14 days. Laser Doppler imaging after femoral artery ligation revealed enhanced blood perfusion recovery in the iEC group vs. control at day 7 (p<0.05), 10 and 14 (p<0.001). IPSC-ECs enhanced recovery vs. control at day 14 (p<0.05). At day 10, perfusion was significantly greater in iEC vs. iPSC-EC treated mice (p<0.01). IPSC-EC and iEC bioluminescent signals decreased over 14 days, but remained above background at all observed time-points (p<0.0001). Extrapolation of the model suggested persistence of signal until day 21. Analysis of hindlimb skeletal muscle demonstrated significantly greater capillary density in the ischemic limb of mice treated with iECs, vs. control (179±11 vs. 125±10 capillaries/image; p<0.05).

Conclusion:
iECs have striking in vivo pro-angiogenic effects and appear to be more potent than iPSC-ECs. These findings support further development of iECs for therapeutic angiogenesis.
Abstract 625: Adipose-Associated Mediators Correlate with Anatomic and Physiologic Adaptations Following Fistula Placement: The Hemodialysis Fistula Maturation (HFM) Study
Authors: Kuppler Christopher S , Sharma Gaurav , He Yong , Tao Ming , Dember Laura , Ozaki C. Keith , Berceli Scott A
Published: March 2022

Introduction:
Perivascular inflammation stands as an important regulator of vascular remodeling. We hypothesize that the phenotype of adipose tissue adjacent to a hemodialysis arteriovenous fistula (AVF) modulates the anatomy and physiology of maturation.

Methods:
During AVF creation, adipose tissue adjacent to the vein was collected from 112 participants in the NIDDK HFM Study, and 9 adipose-associated mediator protein levels were measured. AVF duplex ultrasound was performed post-operatively at Day 1, and Weeks 2 and 6, and prior to 1
st
cannulation (Week 10-22) to define the changes in diameter and flow. Associations between biomarker levels and fistula remodeling were evaluated using mixed effects regression analyses.

Results:
Fractional changes in vein diameter decrease with increasing IL-6 and MCP-1 levels; changes in AVF flow decrease with increasing leptin (
A
). IL-6 demonstrated a statistically significant relationship with vein adaptations in the early time frame (1D-2W), but not in the later stages of remodeling (
B
). Additional interaction effects between concentration and diameter/flow changes were identified, with elevated inflammatory biomarker levels correlated with reduced early and peri-anastomotic changes in vein diameter and flow (
C
).

Conclusions:
Changes in AVF diameter and flow are associated with baseline local adipose phenotype. Impaired vein dilation and reduced capacity for flow augmentation appears related to enhanced inflammation, particularly at early times and in the peri-anastomotic region.
Abstract 642: Wy14643 and Fenofibrate Reduce Acetylcholine-induced Contractions in Spontaneously Hypertensive Rat Aortae
Authors: Chen Hui , Leung Susan W , Man Ricky Y
Published: March 2022

Introduction and hypothesis:
Hypertension is associated with endothelial dysfunction, which is defined as an imbalanced release of endothelium-derived relaxing factors (EDRF) and endothelium-derived contracting factors (EDCF). Oxidative stress contributes to endothelial dysfunction by increasing the production of EDCF. Research studies have demonstrated that peroxisome proliferator-activated receptor alpha agonists, Wy14643 (WY) and fenofibrate (FF), improve endothelial function. Therefore, we assessed the hypothesis that WY and FF inhibited acetylcholine-induced endothelium-dependent contractions through reduction of oxidative stress in aortae of spontaneously hypertensive rats (SHR).

Method:
Thoracic aortic rings, with and without endothelium, were isolated from 40-44 weeks old male SHR and their normotensive counterparts, Wistar Kyoto rats (WKY). They were suspended in organ chambers for isometric tension recording. Responses to the endothelium-dependent vasoactive agent, acetylcholine (10
-8
- 10
-4
M), and to the oxidizing agent, hydrogen peroxide (10
-8
- 10
-4
M), were examined in the presence of N
ω
-nitro-L-arginine methyl ester (L-NAME, 3*10
-4
M; nitric oxide synthase inhibitor).

Results:
Acetylcholine caused significantly greater contractions in aortic rings, with endothelium, of SHR than in those of WKY. The contraction was significantly inhibited by the combination of Tiron (10
-3
M; intracellular superoxide anion scavenger) and DETCA (10
-4
M; superoxide dismutase inhibitor), suggesting that the reactive oxygen species, hydrogen peroxide, was involved in vascular dysfunction in hypertension. Both WY (10
-5
M) and FF (10
-5
M) significantly inhibited contractions to acetylcholine in SHR aorta, suggesting that they may reduce the amount of hydrogen peroxide or inhibit its downstream signaling. Moreover, they inhibited contractions induced by hydrogen peroxide in SHR aortic rings with, but not in those without, endothelium, indicating that their inhibitory effect was endothelium-dependent.

Conclusion:
The peroxisome proliferator-activated receptor alpha agonists act on the endothelium to reduce acetylcholine-induced contractions that are mediated by hydrogen peroxide in aortae of hypertensive rats.
Abstract 643: Impaired Integrin β3 Delays Endothelial Cell Regeneration and Contributes to Arteriovenous Graft Failure in mice
Authors: Liang Ming , Liang Anlin , Wang Yun , Dong Jin-fei , Du Ji , Cheng Jizhong
Published: March 2022

Objective:
Neointima formation is associated with stenosis and subsequent thrombosis in arteriovenous grafts (AVG). A role of integrin β3 in the neointima formation of AVGs remains poorly understood.

Approach and Results:
In integrin β3
-/-
mice, we found significantly accelerated occlusion of AVGs compared to the wild type mice. This is caused by the development of neointima and lack of endothelial regeneration. The latter is a direct consequence of impaired functions of circulating angiogenic cells (CACs) and platelets in integrin β3
-/-
mice. Evidence suggests the involvement of platelet regulating CAC homing to and differentiation at graft sites via TGF-β1 and Notch signaling pathway. First, CACs deficient of integrin β3 impaired adhesion activity toward exposed subendothelium. Second, platelets from integrin β3
-/-
mice failed to sufficiently stimulate CACs to differentiate into mature endothelial cells. Finally, we found that TGF-β1 level was increased in platelets from integrin β3
-/-
mice and resulted in enhanced Notch1 activation in CACs in AVGs. These results demonstrate that integrin β3 is critical for endothelial cell homing and differentiation. The increased TGF-β1 and Notch1 signaling mediates integrin β3
-/-
-induced AVG occlusion. This accelerated occlusion of AVGs was reversed in integrin β3
-/-
mice transplanted with the bone marrow from WT mice.

Conclusion:
Our results suggest that boosting integrin β3 function in the endothelial cells and platelets could prevent neointima and thrombosis in AVGs.
Abstract 649: Dedicator of Cytokinesis 2 is Essential for the Initiation of Transforming Growth Factor-Beta-Induced Smooth Muscle Differentiation
Authors: Guo Xia , Shi Ning , Wang Yung-Chun , Dong Kun , Chen Shi-You
Published: March 2022

Smooth muscle cell (SMC) differentiation is an important process during embryonic development. The underlying mechanisms regulating the SMC differentiation process, however, remain largely unknown. The present study identified dedicator of cytokinesis 2 (DOCK2) as one of the factors mediating the SMC differentiation. DOCK2 was important in mediating the transforming growth factor-β (TGF-β)-induced SMC differentiation from human embryonic stem cell derived mesenchymal cells (hES-MCs) and 10T 1/2 pluripotent embryonic mesenchymal cells. TGF-β induced DOCK2 expression during SMC differentiation as shown by the SMC marker expression including α-SMA, SM22α and calponin. DOCK2 overexpression induced while DOCK2 knockout inhibited SMC early marker gene expression. Mechanistically, DOCK2 induced SMC differentiation by activating phosphorylated p38 MAPK. DOCK2 overexpression induced while DOCK2 knockdown inhibited the level of phosphorylated p38. P38 pathway inhibitor dramatically decreased DOCK2-induced SMC marker gene expression. In vivo, reduced blood vessel formation was observed in the DOCK2-/- embryo yolk sac compared to the wild type yolk sac. Hemorrhage was also evident in the DOCK2-/- embryo. Histological analysis revealed defective expression of calponin and α-SMA in dorsal aorta of DOCK2-/- embryo. Taken together, our studies demonstrated that DOCK2 is an important novel factor regulating SMC differentiation.
Abstract 651: It Takes Two to Grow: Angiogenesis Requires Sufficient VEGF Responses Plus Correct Spatio-temporal Organization of Redox Signaling by PON2
Authors: Helmstädter Johanna P , Wilgenbus Petra , Horke Sven
Published: March 2022

Angiogenesis describes the growing of new blood vessels from existing ones and is critical for e.g. developmental processes, vascular regeneration, atherosclerosis and tumors. Therapeutic compounds interfere with angiogenesis mostly through extracellular activities, typically by targeting either the most relevant pro-angiogenic ligand, vascular endothelial growth factor (VEGF), or its dominant receptor, VEGFR2. Intracellular signal transmission is less clear, but redox signaling i.e. formation of reactive oxygen species (ROS) has been established as regulator of endothelial functions. Thus we tested a role for paraoxonase-2 (PON2), a vascular anti-oxidative enzyme. This protein has two, seemingly independent effects on angiogenesis. We observed that endothelial cells (ECs) from PON2-/- mice expressed significantly less VEGFR2, likely through an interaction with the VEGFR2-controlling transcription factor GTF2i. As a result, VEGF-stimulated PON2-/- ECs showed weaker downstream effects, such as AKT activation, Calcium release and tube formations in in vitro angiogenesis assays. Sprouting of endothelial cells from aortic rings of PON2-/- mice was also reduced. In addition to this process, a ROS-dependent mechanism was found. Strikingly, 3D analyses of ECs sproutings from aortic rings revealed that in controls, ROS-producing hotspots located to both tip regions and branch points of endothelial sproutings, coinciding with high mitochondrial activity and migratory functions. The intermittent regions of proliferating areas had low ROS levels. This finding implies the presence of a tightly regulated, spatio-temporal ROS production that adjusts 3D sprouting by controlling migration and proliferation. Importantly, PON2 deficiency led to more ROS, but it also disintegrated the underlying 3D ROS arrangement. In PON2-/- sproutings, ROS hotspots were comparatively unordered, causing faster migrating (but not proliferating) ECs that produced less sproutings. Together, these findings reveal a mechanism that controls EC sprouting in a spatio-temporally organized and ROS-mediated manner. Interestingly, PON2 is an enzyme that combines the control of this process and responses to VEGF.
Abstract 654: XBP1 Splicing is Involved in Endothelial and Smooth Muscle Cells Interaction
Authors: Li Yi , Yang Junyao , Zeng Lingfang
Published: March 2022

Background:
The X-box binding protein 1 (XBP1) is a stress inducible transcription factors, essential for cell survival under stress conditions. Our previous studies have revealed that XBP1 participates in endothelial cell (EC) proliferation, autophagic response and survival and in smooth muscle cell (SMC) proliferation. Whether XBP1 is involved in EC/SMC interaction remains unclear.

Methods and Results:
In this study, we first performed hindlimb ischemia model in XBP1loxP/loxP and SM22-Cre+/XBP1loxP/loxP mice. The XBP1 deficiency in SMCs significantly attenuated the recovery of foot blood perfusion in ischemic mice. Immunofluorescence staining revealed that ECs lost contact to each other in XBP1 deficient mice, suggesting that XBP1 in SMCs may contribute to EC/SMC interaction. The conditioned medium from Ad-XBP1s-infected SMCs increased EC migration and proliferation. The conditioned medium from Ad-XBP1s-infected ECs also increased SMC migration but suppressed SMC proliferation. The migration effect could be attenuated by depletion with anti-col4A1 antibody. Proteomics analysis and Western blot assays demonstrated that over-expression of the spliced XBP1 in SMCs increased the secretion of a 50KD band of collagen IVα1. RT-PCR analysis indicated that this 50KD band might be derived from an unconventional splicing, in which exon 4 and exon 42 directly joins together, resulting in a 533 amino acid peptide with the internal part shortened. Further experiments with immunohistological staining revealed that a functional XBP1 was essential to collagen IVα1 expression during embryonic development. Very little amount of collagen IV could be observed in XBP1-/- embryos.

Conclusion:
XBP1 in SMCs may modulate EC-SMC interaction via regulation of collagen IV expression and splicing, which may contribute to vasculature remodelling.
Abstract 663: The Effects of Hypoxia and Metabolic Inhibitors on Cellular ATP and ATP/ADP ratio in Human Coronary Artery Smooth Muscle cells
Authors: Yang Mingming , Kamishima Tomoko , Roberts Owain L , Caroline Dart , Quayle John M
Published: March 2022

Background:
Although ATP is principally considered as the body’s energy currency, it is also an important signaling molecule. As availability of oxygen is an important factor in the production of ATP, it is simply assumed that the latter is tightly regulated by the former. However, how oxygen tension over the physiological range may regulate ATP production is still unclear. Here, we address the fundamental question of whether changes in environmental oxygen tension over the physiological range result in significant changes in cellular ATP in Human Coronary Artery Smooth Muscle cells (HCASMCs).

Methods:
Cells were seeded 24-48 hours before cellular ATP was measured in the cell population using CellTiter-Glo
®
Luminescent Cell Viability Assay according to manufacturer’s instruction (n=4-6). Percent change of cellular ATP was determined and normalized to control (untreated). For the change of ATP/ADP ratio, single cells were transient transfected with Perceval or PercevalHR, fluorescent reporters of ATP/ADP ratio. The fluorescent signal was collected in a confocal microscope (LSM510) under different experimental conditions, including hypoxia.

Results:
Application of metabolic inhibitors for 10 minutes decreased the ATP to 104.6%±2.0% (5 mM 2-Deoxy-D-glucose/2DG),103.1% ±2.9% (6μM Oligomycin/Oligo) and 84.4%±2.7% (2DG+ Oligo) in 10 mM Glucose physiological saline (PSS); and 55.3%±0.3% (5 mM 2DG), 92.4%±3.7% (6μM Oligo) and 36.7%±2.7% (2DG+ Oligo) in glucose free PSS. Hypoxia for 10 minutes also caused a reduction in ATP to 67.0%±1.6% (10%O
2
), 52.7%±0.6% (5%O
2
) and 36.2%±1.8% (1%O
2
) in 10 mM glucose PSS; 71.3%±2.4% (10%O
2
), 57.9%±1.1% (5%O
2
) and 40.4%±3.2% (1%O
2
) in glucose free PSS. Inhibiting metabolism with 2DG in single cells resulted in a decrease in Perceval signal by 50.2±14.4% (n=3). Transient hypoxia (1%O
2
) also induced a significant reduction in Perceval signal by 19.3%±5.3% (n=3). By inhibiting the electron transport chain for 10 mins, we also observed PercevalHR signal declined by 43.6%±3.6% (1μM Rotenone, n=3), 33.0%±11.4% (1μM Antimycin, n=4), 39.5%±9% (6μM Oligo, n=3) and 16.9%±4.4% (1μM CCCP, n=3).

Conclusions:
Our studies suggest hypoxia causes a reduction in cellular ATP and a decrease in the ATP/ADP ratio in HCASMCs.
Abstract 669: Matricellular Protein CCN3 Mitigates the Progression of Abdominal Aortic Aneurysm
Authors: Zhang Chao , Van dervoort Dustin , Shi Hong , Zhang Rongli , Qing Yulan , Dong Nianguo , Perbal Bernard , Prosdocimo Domenick , Lin Zhiyong
Published: March 2022

Background:
Structural components within the vasculature, including extracellular matrix proteins, play an integral role in modulating vascular homeostasis in health and disease. Previous studies have identified important roles of a family of secreted extracellular matrix associated proteins termed CCN (Cyr61, Ctgf, Nov) in mediating matrix homeostasis. However, a role for this family of proteins in the initiation / progression of vascular diseases remains undetermined.

Methods and Results:
Herein, biochemical and genetic gain- and loss-of function studies reveal a novel role for CCN3 (a member of the CCN family) in abdominal aorta aneurysm (AAA) biology. Current studies suggest CCN3 levels are reduced in the rodent aorta following angiotensin II (AngII) infusion, findings recapitulated in human AAA tissues. To address whether CCN3 deficiency affects the development of AAA, control C57BL/6 and CCN3-/- mice (n=30 per group) were subjected to AngII infusion. In response to four-week AngII infusion, the aortas of CCN3-/- mice demonstrated exquisite sensitivity with more than seventy percent of CCN3-/- mice developing visible AAA. Pathologically, the AAA phenotype of CCN3-/- mice was manifested as follows: (1) severe disruption of medial architecture characterized by prominent elastin degradation and vessel dilation; (2) a significant loss of smooth muscle cells; (3) a profound inflammatory response as evidenced by enhanced immune cell infiltration, increase expression of inflammatory markers; (4) heightened reactive oxygen species generation. Moreover, the AAA phenotype of CCN3-/- mice was confirmed in a second model of AAA formation (elastase perfusion). Next, bone marrow transplant experiments indicate the AAA phenotype of CCN3-/- mice is intrinsic to the vasculature. Furthermore, lentiviral mediated overexpression of CCN3 is sufficient to mitigate elastase-mediate AAA progression in mice. Mechanistically, genetic and pharmacological studies suggest the ERK1/2 pathway serves as an important role in the regulation of CCN3-dependent AAA development.

Conclusions:
These results demonstrate CCN3 as an essential regulator of AAA biology and suggest the potential for future therapeutic strategies by manipulating CCN3 levels.
Abstract 670: Finite Element Analysis-derived Peak Wall Rupture Index Can Predict Rupture in Abdominal Aortic Aneurysms
Authors: Lindquist Liljeqvist Moritz , Siika Antti , Hultgren Rebecka , Gasser T. Christian , Roy Joy
Published: March 2022

Objectives:
In abdominal aortic aneurysm (AAA) patients with high operative risks, it may be difficult to weigh the risks of surgery against the risk of rupture even if the treatment criterion, an aneurysm diameter larger than 55 mm, is fulfilled. It has been demonstrated that a significant portion of all large-diameter AAAs will never rupture. The aim of this study was to determine whether finite element analysis (FEA)-based rupture risk prediction could identify AAAs that ruptured within a two-year period.

Methods:
We identified all patients who presented with AAA rupture in our catchment area during the years 2009-2013. Of the 130 patients diagnosed with ruptured AAA, 14 patients had a previous computed tomography angiography (CTA) of high quality conducted within two years prior to rupture. The aneurysms were semi-automatically segmented from CTA images and FEA was conducted using Vascops A4 Clinics. The biomechanical variables Peak wall stress (PWS) and Peak wall rupture index (PWRI = maximal wall stress/wall strength ratio) were calculated. FEA was also performed on a control group of 15 AAAs with diameters similar to the rupture group and two years of uneventful follow-up after CTA.

Results:
Four women and 10 men, median age 82 years, with an aneurysm diameter range of 54 to 88 mm, were included into the rupture group. The control group consisted of three women and 12 men, median age 81 years, with an aneurysm diameter range of 52 to 91 mm. No significant differences in age, gender, smoking status, hypertension or body mass index existed. PWRI was significantly higher in the aneurysms that would later rupture compared to the control group (0.41 vs 0.34, p = 0.005) but no significant differences in PWS (212 vs 197 kPa, p = 0.20) or maximal diameter (61 vs 58 mm, p = 0.23) were detected. There was no significant difference in infrarenal aortic volumes (190 vs 144 cm3 p = 0.19) or intraluminal thrombus volumes (99 vs 59 cm3 p = 0.12) between the groups.

Conclusions:
In this small cohort of AAA patients with a pre-rupture CTA within the last two years, finite element analysis that included both wall stress and wall strength estimations could predict rupture.