bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 10: The Matrix Crosslinking Enzyme Lysyl Oxidase Like-2 as a Target to Reverse Vascular Stiffness
Authors: Steppan Jochen , Wang Ivy , Bergman Yehudit , Tan Siqi , Jandu Sandeep , Melucci Sean , Berkowitz Dan E , Santhanam Lakshmi
Published: June 2021

Introduction:
Stiffening of the central vasculature is a strong and independent predictor of adverse cardiovascular events. Vascular stiffening is a complex process that involves changes in the vessel wall composition and smooth muscle cell (SMC) function. We recently used an unbiased proteomic approach to identify Lysyl oxidase like 2 (LOXL2) as a potential new target in vascular stiffness. The goal of this study is to characterize the role of LOXL2 in vascular stiffening and its potential as a target to reverse vascular stiffness associated with hypertension.

Results:
We demonstrate that decreased nitric oxide (NO) bioavailability results in increased secretion and activity of LOXL2 in SMCs. LOXL2 knockdown markedly attenuates SMC adhesion, motility, and proliferation and results in diminished matrix deposition. LOXL2 knockdown also results in striking changes in the stiffness and cytoskeletal remodeling events in CMSs. Tensile testing shows that intact aortas of LOXL2+/- animals are stiffer when compared with those from wild type mice, while there is no difference in decellularized vessels. We next investigated the role of LOXL2 in the development of hypertension using angiotensin II (AngII) infusion in LOXL2+/- (group 1) and wild type (WT; group 2) mice. BP and pulse wave velocity (PWV) increased significantly with AngII infusion in both groups during the study period, without a significant change in heart rate. Compared to WT animals, contractile responsiveness was markedly diminished in LOXL2+/- animals at baseline as well as with AngII infusion when compared with untreated controls. The NO- dependent vasodilatory response to acetylcholine was identical at baseline and diminished significantly with AngII infusion in both groups of animals. There was no difference between the groups in the endothelium-independent response to sodium nitroprusside.

Conclusion:
In this study, we demonstrated the role of NO in the regulation of LOXL2. Interestingly, LOXL2 appears to have a dual role in vascular stiffness by affecting both SMC function as well as matrix composition. We therefore conclude that LOXL2 is a novel target involved in vascular stiffness that requires further characterization to elicit the possibility of therapeutic intervention.
Abstract 100: An XY Sex Chromosome Complement in Females Confers Susceptibility and Rupture of Angiotensin II-induced Abdominal Aortic Aneurysms in Hypercholesterolemic Female Mice
Authors: Alsiraj Yasir , Thatcher Sean , Blalock Eric , Chen Kuey , Charnigo Richard , Daugherty Alan , Cassis Lisa
Published: June 2021

Objective:
We previously demonstrated that female mice are less susceptible to angiotensin II (AngII)-induced abdominal aortic aneurysms (AAAs) compared to males, a sex difference present in humans. Sex chromosome abnormalities, such as Turner’s syndrome (monosomy X), are associated with aortic vascular disease. In this study, we tested the hypothesis that an XY sex chromosome complement in females promotes AngII-induced AAAs. In addition, as previous studies demonstrated that testosterone promotes AngII-induced AAAs in male mice, we determined if testosterone would augment AAA severity in XY females.

Methods and Results:
Transgenic male mice with deletion of Sry from the Y-chromosome expressing Sry on autosomes (8-12 weeks of age) were bred to female
Ldlr-/-
mice to generate female mice with an XY or an XX sex chromosome complement. Female mice (XX and XY) were fed a Western diet and segregated into sham and ovariectomized (OVX) groups. Two weeks later, mice were implanted with osmotic minipumps to infuse AngII (1,000 ng/kg/min) for 28 days. The AAA incidence (XX sham, 40%; XX OVX 29%; XY sham, 71%; XY OVX, 57%, p=0.031) and rupture rate (XX sham, 0%; XX OVX 0%; XY sham, 35%; XY OVX, 29%, p=0.003) were significantly increased in XY compared to XX females. Internal abdominal aortic lumen diameters were significantly increased in XY OVX
versus
XX OVX female mice at day 27 (XY, 2.31 ± 0.14; XX, 1.58 ± 0.2, p= 0.009). Moreover, AAA external diameters were significantly increased in XY OVX versus XX OVX females (XY, 2.34 ± 0.15; XX, 1.71 ± 0.18, p=0.0004). Administration of testosterone to adult female XY mice as well as neonatal female mice markedly enhanced AAA rupture (maximum of 73%). DNA microarrays of abdominal aortas revealed that male specific genes on the Y chromosome and inflammatory genes were enriched in aortas from XY females, while genes that escape X-inactivation were enriched in aortas from XX females.

Conclusion:
These results demonstrate that an XY sex chromosome complement is sufficient to promote a high AAA incidence, and markedly increase AAA severity in female mice. Moreover, testosterone augmented AAA ruptures in XY females. Future studies will identify gene targets influenced by sex chromosome complement and/or testosterone.
Abstract 101: Characteristics of Angiotensin II-induced Abdominal Aortic Aneurysms in Selected Mouse Strains Expressing PCSK9D377Y
Authors: Balakrishnan Anju , Howatt Deborah A , Daugherty Alan , Lu Hong
Published: June 2021

Objective:
Our previously study demonstrated that infection of C57BL/6 mice with an adeno-associated viral vector (AAV) expressing the proprotein convertase subtilisin/kexin type 9 (PSCK)9D377Y gain-of-function mutant led to augmentation of angiotensin II (AngII)-induced abdominal aortic aneurysms (AAAs). The purpose of this study was to optimize PCSK9D377Y expression in mice for AngII-induced AAA formation. We also determined the susceptibility of PCSK9D377Y expression to increase AngII-induced AAA in selected normolipidemic mouse strains.

Methods and Results:
To determine optimal dose for infection, C57BL/6 mice were injected intraperitoneally with AAVs containing either an empty vector or PCSK9 D377Y at three doses (3 x 10
10
, 10 x 10
10
, or 30 x 10
10
genomic copies/mouse). Mice were fed a Western diet for 6 weeks starting immediately after AAV injections. Two weeks after AAV injection, mice were infused with AngII (1,000 ng/kg/min) for 4 weeks. Plasma PCSK9 concentrations were increased dose-dependently in mice injected with AAV containing PCSK9D377Y mutation, accompanied by increased plasma cholesterol concentrations. Infection with the intermediate and high doses of PCSK9D377Y.AAV led to equivalent increases of maximal width of abdominal aortas. Therefore, the intermediate dose was used in the following experiments. Effects of PCSK9 D377Y.AAV infection was then determined in 5 normolipidemic mouse strains (C57BL/6, 129, FVB, DBA/2, and BALB/c). C57BL/6 mice were the most susceptible to AAV infection, whereas BALB/c mice were completely resistant. Consistently, C57BL/6 mice with increased plasma cholesterol concentrations had higher AngII-induced AAA formation. PCSK9D377Y.AAV infected male C57BL/6 mice were also compared with age-matched male LDL receptor -/- mice fed the same Western diet. Although plasma total cholesterol concentrations were lower in mice infected with PCSK9D377Y.AAV than LDL receptor -/- mice, these two mouse strains had equivalent AAA formation.

Conclusions:
This study provided evidence that C57BL/6 mouse strain was the most susceptible strain to AAV-driven expression of a gain of function mutant of PCSK9 that promoted AngII-induced AAAs to an equivalent incidence as LDL receptor -/- mice.
Abstract 11: The Role of Human Osteopontin Isoforms in Post-Ischemic Neovascularization
Authors: Lyle Alicia N , Caroti Courtney M , Lee Grace Sang Hee , Joseph Giji , Harirforoosh Saghar , Vereen Joseph , Weiss Daiana , Taylor W. Robert
Published: June 2021

Coronary and peripheral artery diseases lead to ischemia, initiating processes that promote neovascularization to restore blood flow and preserve tissue function. We demonstrated previously that osteopontin (OPN), a matricellular cytokine, is critical to ischemia-induced neovascularization. Unlike rodents, humans express 3 OPN isoforms (a, b, and c); however, the roles of these isoforms in neovascularization and cell migration remain undefined. To assess how human OPN isoforms affect neovascularization, OPN
-/-
mice underwent hind limb ischemia surgery. At the time of surgery, 1.5x10
6
lentivirus particles expressing human OPNa, OPNb or OPNc were delivered by intramuscular injection. While OPNa improved limb perfusion 30.4%±0.8 in OPN
-/-
mice, OPNc improved perfusion by 70.9%±6.3 (d14; p<0.001 vs. LVGFP), as measured by laser Doppler perfusion imaging. Importantly, both OPNa and OPNc isoforms significantly rescued neovascularization better than OPNb (n=6, p<0.05). Isoform effects on vascular volume, density, connectivity and diameter were further assessed using Micro-CT angiograms. OPNa and OPNc rescued limb function compared to control and OPNb treated animals (61.1%±8.2; 76.2%±9.7; p<0.05), as assessed by voluntary running wheel use. To verify the differences in neovascularization were due to divergent effects on receptor binding and/or signaling and not variations in isoform expression, we confirmed similar OPN isoform expression levels by ELISA (n=6, p=ns) and immunofluorescence. OPN isoforms a and c both increased macrophage infiltration 2.5 fold, as assessed by mRNA (d7; p<0.05) and histology, leading to increases in vascular smooth muscle cell (VSMC) infiltration (d7; p<0.05). Several pro-arteriogenic factors were also significantly increased at the mRNA level. Finally, we confirmed in vitro that OPNa and OPNc significantly increased VSMC migration compared to OPN b and control (49.8%±3.1; 75.2%±6.3; p<0.05). In conclusion, human OPN isoforms may exhert divergent effects on neovascularization through varried effects on macrophage and VSMC recruitment. Human OPN isoforms may represent potential new therapeutic targets to promote neovascularization and preserve function in patients with peripheral artery disease.
Abstract 110: Carotid Plaque Instability is Associated with an Increase in the Serum Ratio of circularRNA-284 to microRNA-221
Authors: Bazan Hernan , Brooks Ashton , Lightell Daniel , Woods T. Cooper
Published: June 2021

Introduction:
Atherosclerotic cap thinning and plaque instability occur as a result of a decrease in vascular smooth muscle cell proliferation, which is partly regulated by alterations in the expression of non-coding RNAs in the arterial wall. We recently reported that miR-221 expression in the carotid plaque shoulder is reduced immediately following a carotid-related ischemic cerebrovascular event and returns to normal levels after seven days. We hypothesized that changes in the expression of non-coding RNAs within carotid plaques are reflected in the serum of asymptomatic and acutely symptomatic patients with carotid disease.

Methods:
Serum levels of microRNA (miR) -221 and a circular RNA with potential binding sites for miR-221 (circR-284), were measured using real-time polymerase chain reaction in 41 patients undergoing carotid endarterectomy. Patients were grouped into those who were asymptomatic and those with an acute ischemic cerebrovascular event within the previous 5 days (urgent).

Results:
miR-221 was significantly lower (0.25 ± 0.11 vs. 1.00 ± 0.31, p = 0.01) while circR-284 was significantly elevated (2.96 ± 1.16 vs. 1.00 ± 0.37, p = 0.06) in the serum of the urgent compared to the asymptomatic group. Serum levels of these RNAs alone did not exhibit favorable sensitivity and specificity for use as a biomarker indicative of carotid-related ischemic stroke. The ratio of serum circR-284:miR-221, however, was significantly elevated in the urgent group [11.7 ± 0.48 vs. 1.0 ± 0.6, p = 0.0002 (Figure, A)]. Furthermore, receiver operator curve analysis of circR-284:miR-221 ratio demonstrated favorable sensitivity and specificity (Figure, B) for detecting carotid plaque rupture and ischemic stroke.

Conclusions:
Increases in the ratio of serum circR-284:miR-221 has potential as a diagnostic biomarker of carotid-related ischemic stroke. This data also supports the use and development of functionally related pairs of circulating non-coding RNAs as biomarkers.
Abstract 136: Apoptosis of Smooth Muscle Cells Induced by Lipids Isolated from Carotid Plaques
Authors: Raghuraman Gayatri , Zuniga Mary C , Baughman Brittanie D , Wang Lixin , Zhou Wei
Published: June 2021

Background:
Plaque rupture is a major complication in atherosclerotic cardiovascular disease and Diabetes Mellitus exacerbates cardiovascular complications. Cell death, either of macrophage or vascular smooth muscle cells, is believed to be a primary feature contributing to the necrotic core formation and fibrous cap vulnerability. The lipid core is present adjacent to these areas but there is very limited information on the role of lipids in plaques vulnerability. Lipids undergo oxidative modifications to varying extents. In this study we examined extent of lipid oxidation in plaques from diabetic vs non-diabetic patients and their effect on VSMC apoptosis.

Methods:
Lipids were extracted from plaques of diabetic and non-diabetic subjects who underwent carotid endarterectomy. Extracted lipids were analyzed for lipid peroxidation using the TBARS assay. Human coronary artery smooth muscle cells (HCASMCs) were treated with lipid extracts for 18 hrs. Cells were analyzed for gene expression with RT-PCR and apoptosis using Annexin V and caspase-3 flow cytometric analyses. The conditioned media from the cells were assessed for inflammatory cytokines with ELISA.

Results:
Lipid extracts from diabetic plaques have ~1.8 fold more oxidatively modified lipids compared to the non-diabetics. HCASMCs treated with lipids show increased Annexin V staining in diabetics compared to non-diabetics. Increased oxidative modification in lipids from diabetic patients leads to augmented rate of apoptosis in HCASMCs by 3 fold. Increased caspase-3 activity was also observed in HCASMCs treated with higher content of oxidized lipid among diabetic samples compared to non-diabetics. Cytokine analyses of the conditioned medium from these samples showed 4 fold higher levels of TNF-α in the diabetic samples. Gene expression data revealed increased resistin and CD40 expression in diabetics vs non-diabetics.

Conclusion:
Collectively, these data suggest that oxidized lipids within atherosclerotic plaque are not inert. Increased oxidative stress in the vascular bed of diabetic patients results in amplified lipid/protein oxidation. These oxidized lipids in turn facilitate HCASMC apoptosis triggering a possible secondary event.
Abstract 137: Thromboinflammatory Response in Patients Undergoing Transcatheter Aortic Valve Replacement Using the Sapien Xt or CoreValve
Authors: Sexton Travis R , Liu John , Smyth Susan S
Published: June 2021

Transcatheter aortic valve replacement (TAVR) has been increasingly used to treat patients with symptomatic aortic stenosis. Despite the evolution of the valves and their deployment, patients that have undergone TAVR continue to be at high risk for major adverse events following the procedure. We recently reported that TAVR elicits a thromboinflammatory state that may contribute to the observed post-procedural thrombocytopenia associated with worse clinical outcomes. With the approval of the newer generation valves and delivery systems, we have now compared thromboinflammatory responses in patients receiving the Medtronic CoreValve and Edward’s new SAPIEN XT valve.

Blood cell numbers, platelet function, and biomarkers of systemic inflammation and thrombosis were analyzed in 34 sequential patients undergoing transcatheteraortic valve replacement with the Edward’s SAPIEN XT valve or the Medtronic CoreValve Following valve deployment, platelet count, platelet activity, and platelet factor 4 levels decreased, while the inflammatory marker interleukin-6 increased. There were no significant differences in any of the parameters between the two valve types. Interestingly, baseline inflammation status predicted the extent of thrombocytopenia observed in overall cohort.

A comparions of the SAPIENT XT valve and the Medtronic CoreValve reveals that the new generation valves generate a lower inflammatory response than that observed in the older SAPIEN valce. Importantly, our results add to the growing body of literature that suggests the thromboinflammatory changes that occur early after TAVR may predict long-term outcomes and suggest potential beneficial effects of an anti-inflammatory strategic.
Abstract 148: Blockade of the Serotonin 2B Receptor Prevents Pulmonary Vascular Stiffening and Remodeling in a Mouse Model of Familial Pulmonary Arterial Hypertension
Authors: Bloodworth Nathaniel , Carrier Erica , West James , Merryman W. David
Published: June 2021

Objective:
Pulmonary arterial hypertension (PAH) is a fatal disease of the pulmonary vasculature. In humans, familial PAH is caused by mutations in the bone morphogenetic protein receptor 2 gene (BMPR2). Serotonin signaling through the 2B receptor (5HT2BR) is also important in the pathogenesis of PAH, making 5HT2BR antagonism a potential target for PAH therapeutics. This study evaluates the hypothesis that antagonism of 5HT2BR will prevent PAH in a genetic mouse model and the pulmonary vascular stiffening and remodeling that is considered the underlying cause.

Methods:
Rosa26-Bmpr2
R899X
mice on an FVB/N background express the patient-derived R899X mutation in BMPR2 in all tissues when induced with doxycycline. 10-14 week old BMPR2 mutant mice were fed doxycycline in a high-fat diet for 6 weeks. After 2 weeks, osmotic pumps containing either the 5HT2BR antagonist SB204741 (1 mg/kg/day) or DMSO/water vehicle were implanted. Right ventricular systolic pressure (RVSP) was measured via a venous jugular catheter under anesthesia, and the mice euthanized. Lungs were collected and prepared for histology, gene expression analysis, and atomic force microscopy (AFM, Bioscope Catalyst). Immortalized pulmonary microvascular smooth muscle cells (SMCs) with a BMPR2 mutation were assessed for long-term changes in cytoskeletal contractility using a free floating collagen gel assay.

Data:
BMPR2
R899X
mice treated with SB204741 failed to develop elevated RVSP. Histologic analysis revealed that SB204741 reduced both the total number of inflammatory cells in the lung as well as the muscularization of peripheral pulmonary arterioles. Microarray analysis revealed that 5HT2BR antagonism primarily changed the expression contractility-associated genes. Measurements of pulmonary arteriole elastic modulus with AFM show significantly increased vessel wall stiffness in BMPR2
R899X
arterioles that is also normalized with SB204741 treatment. Taken together, these data suggest that 5HT2BR antagonism prevents the vascular stiffening and remodeling that causes PAH.
Abstract 149: Analysis of Radiotherapy Induced Vascular Lesions Reveals Potential Therapies Against Innate Inflammation in an ApoE Knockout Mouse Model
Authors: Christersdottir Tinna , Pirault John , Lundberg Anna M , Yan Zhong-Qun , Paulsson-Berne Gabrielle , Hansson Göran K , Halle Martin
Published: June 2021

Clinical studies show an increase risk for cardiovascular disease (CVD) at the site of irradiation years after exposure. Radiotherapy together with other cancer therapies contributes to a growing population of cancer survivors in risk of developing radiotherapy induced vascular disease. However, traditional treatments for CVD such as thrombolytic inhibitors and lipid lowering drugs (statins) seem to have no effect on radiation induced vascular disease in mouse models. We have previously demonstrated a sustained chronic inflammation by NF-kB activation in irradiated human arteries. To further investigate radiation induced vascular disease and potential therapies against innate inflammation, we established a mouse model of local irradiation in an atherosclerotic prone ApoE-/- background. ApoE-/- mice fed chow diet were exposed to a single dose of 0 Gy (sham/controls) or 14 Gy at the upper chest and neck area and harvested 10 weeks later. Plasma, thoracic aorta, aortic arch, heart were retrieved and taken for plasma, blood count, en face, mRNA, immunohistochemistry and immunofluorescence analysis. Irradiated mice presented higher total plasma cholesterol levels than controls. En face analysis of aortic arches showed a decreased percentage of lesion size in irradiated arteries compared to controls. However, an increase of MHC class II (IA-b) staining in irradiated aortic roots suggested an induction of inflammation in the context of radiotherapy. These data were supported by the quantification of monocyte chemotactic protein 1 mRNA expression in the thoracic aorta showing a significant increase in irradiated animals compared to unirradiated (p≤0,001).

The current data demonstrated that radiotherapy induces a macrophage-rich and inflamed plaque that may explain the increased risk of developing severe clinical events i.e. myocardial infarction or stroke. Further analyses of this partial irradiation mouse model are on-going to assess the potential therapeutic targets involved in innate immunity and NF-kB activation. Results of currently performed therapeutic experiments will be further presented.
Abstract 152: Mitochondrial Fission Inhibitor Mdivi-1 Attenuates Angiotensin II-Induced Cardiovascular Remodeling
Authors: Forrester Steven J , Kawai Tatsuo , Elliott Katherine J , Eguchi Kunie , Rizzo Victor , Eguchi Satoru
Published: June 2021

Mitochondrial dysfunction has been implicated in various types of cardiovascular diseases which may involve overload and de-compensation in mitochondrial quality/quantity control. However, limited mechanistic insight is available regarding the contribution and mechanism of mitochondrial quality control in hypertension. In the present study, we tested our hypothesis that enhancement of mitochondrial fission in vascular cells is involved in hypertensive vascular remodeling. 8 week old male C57/Bl6 mice were infused with angiotensin II (1000 ng/kg/min) for 2 weeks with or without treatment of mitochondrial fission inhibitor Mdivi-1 (25 mg/kg ip every other day). Mdivi-1 significantly inhibited AngII-induced left ventricular hypertrophy assessed by heart weight body weight ratio as well as by echocardiogram. Histological assessment of the Mdivi-1-treated mouse hearts further demonstrated significant suppression of vessel hypertrophy and fibrosis induced by AngII. However, Mdivi-1 did not affect heart rate or hypertension induced by AngII assessed by telemetry. KDEL and VCAM1 staining of the heart and aorta suggest attenuation of vascular ER stress and inflammation, respectively. In cultured rat vascular smooth muscle cell (VSMCs), AngII induced mitochondrial fission promoting Drp1 phosphorylation at Ser616 and Ser637. Pretreatment of Mdivi-1 (5 microM 30 min) attenuated 100 nM AngII-induced mitochondrial fission in VSMCs assessed by mito-tracker staining. Mdivi-1 also attenuated extracellular collagen accumulation induced by AngII in VSMCs assessed by Sirius Red staining quantification kit. In conclusion, this data suggests that Mdivi-1 treatment prevents AngII-induced cardiovascular remodeling independently of hypertension via suppression of mitochondrial fission and attenuation of ER stress and inflammation in target organs.
Abstract 155: Lipopolysaccharide Selectively Decreases Circulating Endothelial Progenitor Cells in Female Mice via Superoxide Production
Authors: Kander Melissa C , Cui Yuqi , Liu Zhenguo
Published: June 2021

Background:
Gender differences exist in cardiovascular disease such as coronary artery disease. Differences also exist between males and females in regard to oxidative stress, which is considered an important mechanism in the development of cardiovascular disease such as atherosclerosis. Endothelial progenitor cells (EPCs) play a critical role in maintaining the structural and functional integrity of vasculature. The number and function of EPCs are closely associated with the outcome for patients with cardiovascular diseases. The objective of the present study was to determine if the effect of oxidative stress on EPCs could differ between males and females.

Methods:
Male and female wild-type C57BL/6 mice were injected with low dose lipopolysaccharide (LPS) for three days to induce oxidative stress with PBS as control. To evaluate the role of superoxide in mediating the effect of LPS, the mice were treated with superoxide dismutase (SOD) prior to LPS challenge. The blood cells were collected and prepared to determine the populations of EPCs and the levels of intracellular reactive oxygen species (ROS), cytokines, and apoptosis using flow cytometry analysis.

Results:
The number of circulating EPCs was significantly higher in females than males at baseline. There were no differences in the basal levels of apoptosis and intracellular ROS between males and females. LPS treatment significantly increased levels of the serum inflammatory cytokines including IL-1β and TNF-α in both males and females, suggesting that LPS-induced oxidative stress mouse model was successful. Treatment with LPS significantly decreased the number of circulating EPCs in females, not in males. Treatment with SOD effectively prevented LPS-induced reduction of circulating EPC population in female mice with no effect on EPC population in males.

Conclusion:
The basal level of circulating EPCs was significantly higher in females than in males independent of intracellular ROS formation and apoptosis. LPS selectively decreased the circulating EPC population in females via superoxide production.
Abstract 163: Apabetalone (RVX-208) Has Anti-atherosclerotic, Anti-Thrombotic and Anti-Inflammatory Effects in Patients With Cardiovascular Disease (CVD)
Authors: Kulikowski Ewelina , Wasiak Sylwia , Gilham Dean , Calosing Cyrus , Tsujikawa Laura , Halliday Christopher , Johansson Jan , Sweeney Mike , Wong Norman C
Published: June 2021

RVX-208 affects epigenetics by inhibiting bromodomain and extraterminal (BET) proteins from binding to their natural ligand, acetyl-lysine marks on histone tails and thereby modulates gene activity. In SUSTAIN and ASSURE phase IIb trials of CVD patients (n=499), giving 200 mg/d of RVX-208 orally lead a 55% relative risk reduction in major adverse cardiovascular events (MACE) vs. placebo. This marked reduction in MACE is unlikely due to RVX-208’s modest induction of ApoA-I/HDL, thus prompting studies of RVX-208 for its benefits beyond lipids. Methods included microarray surveys of human whole blood (WB) or primary hepatocytes (PH) exposed to RVX-208. Cytokines were assayed in U937 macrophage and peripheral blood mononuclear cells (PBMC) exposed to RVX-208. Plasma samples from phase IIb patients were measured using SOMAScan proteomic analysis. Results of the microarray studies using WB showed a potential anti-atherogenic effect of RVX-208 because it suppressed activity of 37/46 pro-atherogenic while inducing 8/18 anti-atherogenic genes. Additionally, RVX-208 had potential anti-thrombotic properties by affecting 18 genes related to platelet function (e.g. downregulation of CD64 and thrombospondin 1). RVX-208 had anti-inflammatory effects on the expression of >25 cytokines including downregulation of MCP-1, osteopontin and PARC genes. The suppression of these 3 genes by RVX-208 was evident in not only WB but also in the LPS stimulated U937 and/or PBMCs. Whether these in vitro findings extended into patients were examined by SOMAScan showing lower levels of osteopontin and PARC protein in plasma of treated patients. Furthermore, a key marker of inflammation RANTES was markedly lowered in treated patients. Why RVX-208 may affect genes connected to CVD was explored by exposing PH to RVX-208. These studies showed a 25% reduction in levels of mRNA encoding flavin mono-oxygenase-3 (FMO3), an enzyme that produces trimethylamine oxide (TMAO) a metabolite which predicts CVD risk. In summary, RVX-208 inhibits BET proteins to impact cellular epigenetics that in turn affects expression of genes with known roles in CVD. This activity may underlie RVX-208’s anti-atherogenic, -thrombotic and –inflammatory effects in reducing MACE observed in clinical trials.
Abstract 165: Amp-activated Protein Kinase (ampk) α1 in Macrophages Promotes Collateral Remodeling and Arteriogenesis in Mice
Authors: Zhu Huaiping
Published: June 2021

Efficient arteriogenesis is vital for recovery after cardiovascular events (such as chronic coronary occlusion, myocardial infarction). Identifying the biological factors that affect arteriogenesis will help design new treatments for patients with chronic arterial stenosis and occlusions. Circulating monocytes and macrophages recruited within the surrounding tissue of collateral vessels following arterial occlusion have been reported to be essential to arteriogenesis in collateral arteries/arterioles because they promote the proliferation of vascular smooth muscle cells (VSMCs) and endothelial cells through secreted cytokines. Although a growing number of putative arteriogenic factors have been identified, the exact mechanisms that regulate collateral remodeling have remained largely unknown. Here we report that AMPK, an energy and redox sensor, is required for monocyte-mediated collateral remodeling. Collateral arteriogenesis was monitored in WT, global AMPKα1 knockout (KO), or macrophage-specific AMPKα1 KO mice with or without hind limb ligation. Compared to WT mice with ligation, global AMPKα1 KO mice displayed significant reduction in blood flow recovery and impaired remodeling of collateral arterioles. Similar impairments were observed in macrophage-specific AMPK α1 KO mice following hind limb ligation. Mechanistically, we found that AMPKα1 promotes the production of growth factors, such as transforming growth factor beta, by directly phosphorylating the inhibitor of nuclear factor kappa B (NF-κB) kinase alpha, resulting in an NF-κB-dependent production of growth factors. Collectively, our findings suggest a novel role for macrophage AMPKα1 in arteriogenesis and collateral remodeling and indicate that AMPKα1 activation might be a therapeutic target for treating occlusive vascular disorders.
Abstract 168: YAP and TAZ Mediate Mechanical Control of Vasculogenesis
Authors: Mason Devon E , Voytik-Harbin Sherry L , Yoder Mervin C , Boerckel Joel D
Published: June 2021

Physical cues such as the mechanical properties of the extracellular matrix are known to modulate blood vessel formation, but the underlying mechanisms remain poorly understood.

Yes-associated protein (YAP) and transcriptional activator with PDZ-binding domain (TAZ) were recently identified as key mediators of cellular mechanotransduction [1], but the roles of YAP and TAZ in neovascular mechanobiology have not been investigated.

We hypothesized that YAP and TAZ mediate neovascular mechanotransduction of extracellular matrix rigidity by vasculogenic endothelial colony forming cells (ECFCs) cultured on/in matrices of controllable rigidity.

YAP and TAZ depletion abrogated ECFC migration (Fig. 1A), and YAP/TAZ expression and nuclear localization (i.e., activation) was significantly upregulated in migrating cells (Fig. 1B). Combinatorial depletion also abrogated tubular network formation and 3D vasculogenesis in collagen matrices of variable rigidity but constant matrix density (data not shown). In 2D, PDMS rigidity regulated YAP/TAZ subcellular localization (data not shown). In 3D, vasculogenesis was enhanced in stiff (G’= 1049 Pa) collagen oligomer matrices compared to soft (G’ = 305 Pa) monomer matrices formulated at constant collagen matrix density. YAP and TAZ were nuclear localized (activated) in the stiff oligomer matrices, but cytosolic in soft monomer matrices (Fig. 1C). Mechanistically, YAP and TAZ depletion reduced cytoskeletal stress fiber formation (data not shown), implicating YAP and TAZ in a feedback mechanism to control cytoskeletal remodeling.

Delivered in oligomer matrices
in vivo
, ECFCs formed a functional human neovascular plexus and exhibited strong expression and nuclear localization of YAP and TAZ and expression of downstream genes, Cyr61 and CTGF (data not shown).

In summary, YAP and TAZ are essential for mechanical control of ECFC migration and vasculogenesis.

[1] Dupont et al. 2011 Nature. 474:179-185.
Abstract 173: Proprotein Convertase Subtilisin/Kexin Type 6 is a Key Protease in the Control of Smooth Muscle Cell Function in Vascular Disease
Authors: Hedin Ulf , Perisic Ljubica , Rykaczewska Urszula , Razuvaev Anton , Lengquist Mariette , Sabater-Lleal Maria , Eriksson Ida , Röhl Samuel , Kronqvist Malin , Tseng Chi-Nan , Rodriguez Patricia , Folkersen Lasse , Du Lei , Gonzalez Diez Maria , Osterholm Cecilia , Roy Joy , Patrakka Jaakko , Paulsson-Berne Gabrielle , Hansson Göran , Odeberg Jacob , Hamsten Anders , Eriksson Per
Published: June 2021

Proprotein convertases (PCSKs) process matrix metalloproteases (MMPs) and cytokines. Apart from PCSK9, the role of these enzymes in vascular disease is largely unknown. Previously, we demonstrated upregulation of PCSK6 in carotid atherosclerosis, primarily localized to smooth muscle cells (SMCs) and positively correlated to inflammation, extracellular matrix remodeling and cytokines. Here, we extended these findings to determine the role of PCSK6 in vascular development and disease. Increased expression of PCSK6 in vascular disease was validated by microarrays from two non-overlapping cohorts of carotid plaques
vs.
non-atherosclerotic arteries (n=50 patients and n=32 patients, p<0.0001), as well as abdominal (AAA, n=14, p<0.0001) and thoracic aortic aneurysms (TAA, n=244, p=0.012). By eQTL, variants in the PCSK6 gene were found to influence it’s expression in both plaques and aneurysms. Among these, rs6598465 also showed association with maximum progression of carotid intima-media thickness in high-risk coronary artery disease subjects (n=3388, p=0.037). By IHC, PCSK6 localized mainly to SMCs in the fibrous cap and neovessels in atherosclerotic, AAA and TAA tissues. In mouse-, rat-, and human intimal hyperplasia, PCSK6 was expressed in proliferating SMCs. By microarrays, after rat carotid balloon injury there was an early downregulation of PCSK6 followed by an upregulation in later phases during SMC activation, as well as positive correlation to PDGFB and IGF1 (Spearman r>0.7, p<0.0001) and to MMP2 and MMP14 (r>0.5, p<0.0001). In zebrafish embryos, PCSK6 localized to heart and vasculature and its ablation caused defective peripheral vascular patterning with cerebral and myocardial hemorrhage. PCSK6
-/-
mice did not present an obvious vascular phenotype but showed reduced intimal hyperplasia compared to wild-type mice after carotid artery ligation (p=0.015). In vitro, PCSK6 overexpression markedly increased SMC migration upon PDGFBB stimulation (p<0.0001). The present study establishes PCSK6 as a key modulator of SMC function in vascular disease and demonstrates a functional link between PCSK6 expression and SMC migration in vascular remodeling.
Abstract 184: Polo Like Kinase 1 Regulates Smooth Muscle Cell Proliferation in the G2-M Phase in Human Coronary Artery Bypass Conduits
Authors: Sur Swastika
Published: June 2021

Proliferation of smooth muscle cells (SMCs) and the resultant intimal hyperplasia cause coronary artery bypass graft (CABG) failure. Internal mammary artery (IMA) graft is immune to intimal hyperplasia (IH), but a saphenous vein graft is prone to develop neointima. The fate of SMCs is determined by the balance between the mitogenic and anti-mitogenic signals. Mitosis of SMCs through the cell cycle involves crossing the two restriction (R)-points in G1 and G2 phase, respectively. Upon loss of proper G1/S control, cells will progress into the G2-phase where G2 R-point can prevent cell proliferation. In this study, we examined the effect of mitogenic PDGF-BB stimulation on the G2 R-point and its relationship with the phosphorylation of PLK1, Cdc2 and Histone H3 in isolated SMCs of human bypass graft conduits. We observed increased expression and phosphorylation of PLK1 in PDGF-stimulated SV-SMCs compared to control SMCs without the treatment. Furthermore, PLK1 was phosphorylated and activated for a longer period of time in PDGF-stimulated SV- than IMA-SMCs. PLK1 m-RNA level was higher in PDGF-stimulated SV-SMCs than IMA. An ATP-competitive inhibitor of PLK1 attenuated PDGF-BB-induced proliferation in IMA and SV-SMCs. Cell proliferation was measured using cell count and immunoblotting against phospho-Histone H3 (pHistone) at Ser-10. Treatment with PLK1 inhibitor reduced PDGF-induced Cdc2 activation. Silencing the PLK1 gene by siRNA transfection of SV- and IMA-SMCs significantly reduced the expression of pHistone. These data demonstrate differential activity of PDGF-BB-induced PLK1, which was quantitatively and temporally greater in SV-SMCs than in the IMA. PLK1 inhibitor completely blocked the phosphorylation of PLK1 and attenuated proliferation in SV-SMCs. This in part, could suggest that PLK1 plays a critical role in the development of neointimal hyperplasia in SV grafts following CABG. Thus, inhibition of PLK1 activity could be a target in developing better therapeutic approach to prevent vein-graft disease.
Abstract 189: Increased Atherosclerosis And Plasma Lipids In Apoe-pon1 Dko Mice As Compared To Ldlr-pon1 Dko Mice.
Authors: Aluganti Narasimhulu Chandrakala , Young Burge Kathryn , Selvarajan Krithika , Parthasarathy Sampath
Published: June 2021

Background:
Atherosclerosis is a multi-step process that includes early atherosclerotic macrophage foam cell lesion formation, proliferation and migration of smooth muscle cells into the intima, and the formation of complex necrotic and calcified lesions. In this study, we determined the role of dyslipidemia and carotid/innominate atherosclerosis versus the lack of ApoE and the enzyme PON 1 in atherosclerosis.

Hypothesis:
ApoE-PON1 double knockout (DKO) mice, by virtue of age-dependent and profound atherosclerosis in brachiocephalic trunk (innominate artery) even on a normal diet, might be more susceptible to atherosclerosis followed by stroke as compared to age-matched wild type mice.

Methods and results:
APOE-PON1 DKO and LDLR-PON1 DKO mice were developed using ApoE-/-, PON1-/-, and LDLR-/- mice of C57BL/6 background and were maintained until 18months of age on normal chow. Serum lipids and atherosclerotic lesions were quantified. A significant decrease in levels of TRG, TC, LDL-C and VLDL-C was observed in LDLR-PON1 DKO as compared to ApoE-PON1 DKO group animals, whereas, an increase in HDL levels was observed in LDLR-PON1 DKO mice. No significant change in glucose levels was observed. Increased lesions were observed in ApoE-PON1 DKO mice as compared to the LDLR-PON1 DKO mice.

Conclusion:
Our studies suggest the lack of ApoE might be the reason behind increased cholesterol levels and greater atherosclerosis. This is the first study to demonstrate the importance of PON1, in addition to ApoE, and also provide valuable insights as to where therapy should be targeted.
Abstract 19: Role of Adenosine-to-Inosine RNA Editing of
Alu
Elements in Human Vascular Inflammatory Diseases
Authors: Stellos Konstantinos , Gatsiou Aikaterini , Stamatelopoulos Kimon , Perisic Ljubica , John David , Lunella Federica F , Jae Nicolas , Rossbach Oliver , Amrhein Carolin , Sigala Frangiska , Boon Reinier A , Fuertig Boris , Manavski Yosif , You Xintian , Uchida Shizuka , Keller Till , Boeckel Jes-Niels , Franco-Cereceda Anders , Maegdefessel Lars , Chen Wei , Schwalbe Harald , Bindereif Albrecht , Eriksson Per , Hedin Ulf , Zeiher Andreas M , Dimmeler Stefanie
Published: June 2021

Background:
Adenosine to inosine (A-to-I) RNA editing is catalysed by ADARs (adenosine deaminases acting on RNA) and is an important posttranscriptional regulator of RNA metabolism. Although RNA editing is essential for life, its role in cardiovascular disease is unknown.

Methods and Results:
RNA-sequencing (RNA-seq) of human endothelial cells revealed that ADAR1 is the main RNA editor in HUVECs. The vast majority of editing events are detected in
Alu
regions due to their ability to form double-stranded structures, a prerequisite for RNA editing. A-to-I RNA editing of the primate-specific
Alu
elements is observed in 25% of poly(A)+ transcripts and in 44% of ribosomal RNA-depleted transcripts. Cathepsin S (CTSS), an extracellular matrix degrading enzyme, has 3
Alu
elements in its 3’-untranslated region and is among the most extensively edited mRNAs. Interestingly, ADAR1 silencing downregulates CTSS mRNA expression, whereas ADAR1 overexpression results in an increase of both CTSS RNA editing rate and CTSS mRNA expression. Mechanistically, RNA immunoprecipitation (RIP), luciferase reporter and iCLIP assays showed that A-to-I RNA editing of the
Alu
elements in the 3’UTR of CTSS mRNA regulates the recruitment of the stabilizing RNA-binding protein HuR to CTSS 3’UTR and, thus, controls CTSS mRNA stability and expression. CTSS
Alu
RNA editing is increased under hypoxic or pro-inflammatory conditions
in vitro
, as well as in patients with coronary or carotid atherosclerotic vascular disease. Further, the extent of CTSS
Alu
RNA editing rate correlated with various markers of subclinical atherosclerosis including intima-media thickness and number of atherosclerotic plaques. Importantly, ADAR1 and the extent of CTSS RNA editing correlated with CTSS expression levels in patients’ samples from 6 non-overlapping cohorts of patients with inflammatory vascular diseases (total n=366), including peripheral blood mononuclear cells, carotid atherosclerotic plaques and thoracic aortic aneurysms.

Conclusion:
This study shows for the first time that
Alu
A-to-I RNA editing is a critical modulator of inflammatory gene expression in all stages of atherosclerotic vascular disease development.
Abstract 194: Discordance in Apolipoprotein B vs. non-HDL-C and the Cholesterol-Enriched Phenotype: Insights From the National Health and Nutrition Examination Survey (NHANES) 2011-2012
Authors: Quispe Renato , Lazo Mariana , Sniderman Allan , Michos Erin D , Toth Peter P , Kulkarni Krishnaji , Jones Steven R , Martin Seth S
Published: June 2021

Background:
Apolipoprotein B (apoB) levels in plasma are proportional to the number of all atherogenic, non-high-density lipoprotein (HDL) particles. Non-HDL-C concentration - the aggregate cholesterol carried by apoB-containing particles - is usually highly correlated with apoB levels. This study aimed to describe individuals with discordance between these lipid measures, especially apoB<non-HDL-C discordance, as there are few prior data on this cholesterol-enriched particle phenotype.

Methods:
We analyzed 2,804 participants of NHANES 2011-2012, a nationally representative US cross-sectional survey with direct apoB and standard lipid fasting measurements. We assigned population percentiles to non-HDL-C and apoB levels and created groups with 5-10 or >10 percentile units of discordantly low apoB (apoBnon-HDL-C: “cholesterol-depleted”). We compared lipids levels and other clinical characteristics across groups.

Results:
Discordance >5 percentile units was present in 50.9% of individuals. Compared with the cholesterol-depleted group, the cholesterol-enriched phenotype was characterized by a lower prevalence of obesity, diabetes, and hypertension. We found higher levels of low-density lipoprotein cholesterol (LDL-C), non-HDL-C, and HDL-C in cholesterol-enriched compared with cholesterol-depleted groups, whereas triglycerides were similar across groups. Lipid levels were consistent after excluding individuals on lipid-lowering therapy.

Conclusion:
Despite having higher levels of non-HDL-C and LDL-C, the cholesterol-enriched phenotype had a more favorable cardiovascular risk factor profile compared with the cholesterol-depleted phenotype. Triglycerides were notably low and similar across groups. Evaluation for discordance at both ends of the spectrum may provide additional prognostic information on CVD risk, but further outcomes studies are needed.
Abstract 199: The Regulation of Intestinal apoC-III by Dietary Fat: Key Differences From Hepatic apoC-III Regulation and Implications for Lipoprotein Synthesis and Secretion
Authors: West Gabrielle , Rodia Cayla , Li Diana , Jattan Javeed , Kohan Alison B
Published: June 2021

Apolipoprotein C-III (apoC-III) influences both plasma triglycerides and inflammation, and as such is a particularly important target for mediating cardiovascular disease (CVD). ApoC-III is an exchangeable apolipoprotein expressed in both liver and intestine; in humans, apoC-III levels in plasma are an independent predictor of CVD risk. ApoC-III is a potent regulator of plasma triglycerides through effects on intestinal lipid absorption, plasma triglyceride clearance, hepatic lipoprotein uptake, and VLDL secretion. ApoC-III also acts as a signaling molecule modulating vascular function. Hepatic apoC-III is regulated by insulin via the transcription factor, forkhead box protein O1 (FOXO1), and by glucose via carbohydrate-responsive element-binding protein (ChREBP). Though much is known about hepatic apoC-III, little is known about the regulation of intestinal apoC-III (including the primary stimulus for its expression). We have found that apoC-III is secreted from the intestine in response to dietary carbohydrate, and that a fish oil-enriched diet lowers intestinal apoC-III mRNA expression. Our findings suggest that ChREBP is the primary modulator of apoC-III expression in the intestine. While dietary fish oil lowers both hepatic and intestinal apoC-III mRNA levels, it appears to do so through distinctive mechanisms. This is a potentially important difference since apoC-III is a key regulator of post-prandial plasma lipid levels and inflammation.
Abstract 208: Effects of Gender and BMI on Comorbidities in Patients With Non Hemorrhagic Stroke
Authors: Ashamalla Michael , Pieper Justin , Sedhom Daniel , Yager Neil , Torosoff Mikhail
Published: June 2021

Background:
There is conflicting evidence concerning the obesity paradox in stroke patients. We sought to examine the relationship between gender, BMI, and prevalence of comorbidities in patients with non-hemorrhagic stroke.

Materials and Methods:
Retrospective chart review was performed in 996 consecutive patients treated for non-hemorrhagic stroke at a single academic medical center. Patients were divided according to gender and specific BMI groups according to the National Institute of Health. This study was approved by the institutional IRB.

Results:
Patients with BMI from 0-18.5 and 18.5-24.9 were more likely to be female (63.2% and 58.4% p<.05). Whereas patients with BMI over 25-30 and 30-35 were more likely to be male (60.82% and 59.2% p<.05). Morbidly obese patients (BMI>35) were equally likely to be men or women. In men higher BMI correlated with presentation at younger age. Diabetes was most prevalent in patients with BMI over 35 (40% males, 44% females, p<.05). In females, HTN was associated with BMI 30-34.5 and 35+ (80.5% and 73.3%, P<.05). Males showed a similar though non-significant trend. The prevalence of end stage renal disease, systemic atherosclerosis, and PVD was not significantly correlated with BMI in either gender.

Conclusion:
Gender and BMI significantly affect associated comorbidities in patients with non-hemorrhagic stroke, possibly suggestive of unique gender specific disease mechanisms. Additional studies investigating the effect of gender and BMI on diagnostic evaluation and treatment of patients with non-hemorrhagic stroke are warranted.