bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 557: Pharmacological Inhibition of ADAM17 by a Human-Cross Reactive Antibody and Selective Inhibitor JG26 Prevents Vascular Fibrosis Induced by Angiotensin II
in vivo
and
in vitro
Authors: Kawai Tatsuo , Forrester Steven J , Eguchi Kunie , Rizzo Victor , Eguchi Satoru , Kwok Hang Fai , Murphy Gillian , Rossello Armando
Published: June 2021

In cultured vascular smooth muscle cells (VSMCs), we have shown that a metalloprotease, ADAM17, mediates EGF receptor (EGFR) transactivation induced by angiotensin II (AngII). We have also shown that in mice with AngII infusion, EGFR inhibitor erlotinib prevents vessel remodeling independently from hypertension. In the present study, we hypothesized that pharmacological inhibition of ADAM17 prevents AngII-induced vascular fibrosis in vivo and in vitro. A novel human-cross reactive ADAM17 inhibitory antibody, A9(B8), (10 mg/kg ip on day 1 and 7) was utilized in C57Bl/6 mice with AngII infusion (1000 ng/kg/min for 2 weeks). A novel ADAM17 inhibitor JG26 (1 μM) was utilized in cultured rat aortic VSMCs stimulated with 100 nM AngII. A9(B8) but not control IgG treatment attenuated perivascular fibrosis and vascular hypertrophy in mouse coronary arteries (assessed by Sirius Red staining) infused with AngII. A9(B8) also attenuated cardiac hypertrophy (assessed by echocardiogram and heart body weight ratio) but not hypertension in mice with AngII infusion. In VSMCs, 30 min pretreatment of JG26 inhibited AngII-induced extracellular collagen accumulation at 48 h (assessed by a Sirius Red quantification kit). JG26 also inhibited AngII-induced EGFR transactivation (2 and 10 min) and ERK activation (10 min) in VSMCs. We conclude that inhibition of ADAM17 is an effective approach to attenuate pathophysiological cardiovascular remodeling in an AngII-dependent model of hypertension. These data highlight ADAM17 as a novel therapeutic target to prevent end-organ damage associated with hypertension.
Abstract 569: Constitutively Active Notch4 Receptor Promotes Flow-induced Arterial Outward Remodeling
Authors: Huang Lawrence , Jiang Weiya , Murphy Patrick A , Wang Rong A
Published: June 2021

Notch is expressed in arterial but not venous endothelial cells (ECs). Notch signaling plays a critical role during vascular development and is required for postnatal arterial remodeling following ischemic injury. However, whether activated Notch is sufficient to promote arterial remodeling is unclear. Shear stress is a primary stimulus for arterial outward remodeling, and shear stress induces Notch activation. We hypothesized that activated Notch in turn promotes flow-induced arterial outward remodeling. Constitutively active Notch4 (Notch4*) was expressed in arterial ECs of the carotid artery to bypass hemodynamic perturbations caused by pan-endothelial Notch4 activation. First, we used an artery graft model: a segment of the carotid from a Notch4* donor was grafted into a control recipient. Notch4* expression was restricted to ECs of the graft; hemodynamics in the control recipient was not perturbed. Second, we targeted Notch4* expression using the arterial EC-specific

Bmx(PAC)-CreER
T2

. The carotid ligation model was used to examine flow-induced outward remodeling. Histological analysis showed that in high-flow Notch4* artery grafts, external elastic lamina (EEL) circumference, EEL area, medial area, and medial thickness were larger compared to controls. In high-flow Bmx-Notch4* arteries, internal elastic lamina circumference, EEL circumference, EEL area, and medial area were larger compared to controls. To bolster our findings in the carotid, we tested our hypothesis using a mesenteric artery ligation model. Post-ligation, shear stress in high-flow Notch4* and control mesenteric arteries were comparable. While high-flow arteries in both Notch4* mutants and controls were enlarged, radial enlargement was increased in Notch4* arteries compared with controls. Together, our results showed that endothelial expression of Notch4* enhanced flow-induced outward remodeling of both the large-diameter, elastic carotid artery and the small-diameter, muscular mesenteric artery. Notably, in the absence of increased flow, basal arterial diameters were comparable between Notch4* and control mice. These data suggest that, when properly targeted to the arterial endothelium, Notch4* promotes flow-induced outward remodeling.
Abstract 576: The Role of Adipose Derived Autotaxin on Inflammation Associated with Cardiovascular Disease
Authors: Brandon Jason A , Vandra Julia , Ubele Margo , Smyth Susan
Published: June 2021

Obesity is an established risk factor for cardiovascular disease (CVD) and stroke, and the cardiovascular complications of obesity are a leading cause of potentially preventable death. Recent evidence suggests that increased cardiovascular mortality in patients with obesity may not be fully explained by associated risk factors such as dyslipidemia, hyperglycemia, insulin resistance and hypertension. In addition to contributing to traditional CVD risk factors, obesity is also characterized by a chronic sub-acute inflammatory state termed “metainflammation” involving increased circulating inflammatory cytokines. Autotaxin (ATX), encoded by the ecto-nucleotide pyrophosphatase/phosphodiesterase 2 (ENPP2) gene, is a potent cell motility-stimulating factor that is secreted by adipose tissue. ATX hydrolyzes lysophosphatidylcholine (LPC), a lipid that is abundant in the circulation, to lysophosphatidic acid (LPA). The main objective of this study was to explore whether adipose ATX contributes to systemic inflammatory responses in obesity. We established two animal models with reduced ATX expression: MX-1 Cre-mediated deletion of the ENPP2 gene to generate a global ATX loss (Mx1) and Adipoq-Cre mediated loss of ATX expression in adipocytes (AdipoQ). Mice were fed a high fat diet for up to 20 weeks. No differences in body weight between the ENPP2 loss and littler mate controls were found. RNA and proteins were extracted, qPCR and western blots were performed for ATX and inflammatory cytokines; MCP, TNF and IFN. Results demonstrate that mice lacking global or adipose-derived ATX display approx 2 fold lower adipose inflammation. Furthermore, LPS was used to provoke a systemic response in mice that had been on a HFD for 10 weeks. Six hours after LPS injection, RNA was extracted and qPCR performed for IL6, IL1beta and IFN gamma. In response to a LPS challenge, AdipoQ mice showed a 2 fold reduction in inflammatory response in adipose tissue. The data generated from these experiments will provide important insight into potential mechanisms of how ATX and adipose tissue influence obesity induced inflammation and results from this and future projects may identify potential therapeutic targets to prevent and treat obesity induced inflammation and CVD.
Abstract 580: Lutein Reduces Inflammation in Patients With Coronary Artery Disease by Suppressing Cytokine and Matrix Metalloproteinase-9 Secretion From Peripheral Blood Mononuclear Cells
Authors: Chung Rosanna W , Leanderson Per , Lundberg Anna K , Jonasson Lena
Published: June 2021

Introduction:
Low plasma carotenoid levels are associated with increased cardiovascular risk factors. Carotenoids are anti-oxidants with potential anti-inflammatory properties. In this study, we measured different types of carotenoid in plasma and assessed their relationship with interleukin-6 (IL-6) in patients with acute coronary syndrome (ACS) or stable coronary artery disease (CAD). A sub-cohort was followed for 12 months. Moreover, we assessed the anti-inflammatory effects of lutein in peripheral blood mononuclear cells (PBMC) collected from CAD patients.

Methods:
We included 48 ACS and 109 stable CAD patients. Circulating levels of lutein+zeaxanthin, β-cryptoxanthin, lycopene, α- and β-carotene, and IL-6 were measured. Lutein and zeaxanthin are isomers and as such measured together. Lutein, however, is the major form of the isomers. PBMC were treated with lutein (1μM, 5μM and 25μM) for 24hr followed by 24hr incubation with or without LPS. Cytokines and matrix metalloproteinase-9 (MMP-9) in cell media were measured.

Results:
Levels of lutein+zeaxanthin and lycopene were inversely correlated to IL-6 in the whole cohort,
p
<0.001 and
p
<0.05, respectively. In stable CAD patients, only lutein+zeaxanthin were correlated to IL-6 (
p
<0.001). In the sub-cohort (n=33), changes in IL-6 were inversely correlated to changes in lutein+zeaxanthin at the 3
rd
month (
p
<0.01) and 12
th
month (
p
<0.05). Changes in IL-6 were also inversely correlated to changes in lycopene levels at 12
th
month (
p
<0.05). Other carotenoids did not correlate to IL-6 in plasma.
In vitro
, spontaneous and LPS-induced secretion of cytokines (IL-6, IL-1β and TNF-α) and MMP-9 from PBMC were dose-dependently and significantly reduced by lutein.

Conclusions:
The clinical findings indicated that lutein+zeaxanthin might play a role in resolution of inflammation in CAD patients.
In vitro
, we were able to confirm that lutein exerted anti-inflammatory effects by suppressing secretion of inflammatory cytokines and MMP-9 from PBMC.
Abstract 607: Modulating VEGFR3 Signaling by Epsins Regulates Lymphatic Dysfuntion in Diabetes
Authors: Chen Hong , Wu Hao , Rhaman Ashiqur
Published: June 2021

Background:
Impaired lymphatic function in diabetes is a major cause of lymphedema and immune suppression. VEGFR3 signaling is a master regulator of lymphatic development; however, whether alteration of VEGFR3 signaling regulates lymphatic function in diabetes is unknown. We uncovered that loss of epsins in lymphatic endothelial cells (LECs) increases VEGFR3 signaling by hindering VEGFR3 downregulation (Science Signaling, 2014). The goals for the present study were (1) to determine if hyperglycemia inhibits VEGFR3 signaling and impairs lymphangiogenesis, (2) identify critical molecular pathways that drive the upregulation of epsin, leading to enhanced degradation of VEGFR3 in diabetes, (3) to ascertain if elevated VEGFR3 signaling caused by epsin loss is crucial for restoring impaired lymphatic circulation in diabetes.

Methods and Results:
Using innovative near-infrared imaging and tail lymphedema models, we showed that epsin loss accelerates recovery from diabetic secondary lymphedema. We also found that hyperglycemia-induced oxidative stress attenuates VEGFR3 signaling, delays diabetic secondary lymphedema resolution, however, elevates epsin expression in LECs. Mechanistically, using ChIP and luciferase reporter analyses, we discovered that hyperglycemia driven oxidative stress-induced Src activation stimulates AP1 activity, which in turn binds epsin promoter and upregulates epsin transcription. Consequently, heightened epsin expression in primary LECs isolated from diabetic mice suppresses VEGFR3 signaling and prohibits lymphangiogenesis by facilitating VEGFR3 downregulation. Conversely, epsin deficiency rescues high-glucose-induced VEGFR3 loss, amends defective lymphangiogenesis, and ameliorates diabetic secondary lymphedema.

Conclusions:
Epsin depletion promotes diabetic secondary lymphedema resolution by protecting VEGFR3 from oxidative stress-induced degradation, highlighting the therapeutic potential for targeted inhibition of epsins in the lymphatic endothelium as a treatment for lymphatic dysfunction in diabetes.
Abstract 613: Cardiovascular Disease Prevalence and Risk Factors in Japanese and American Populations
Authors: Ikezaki Hiroaki , Ai Masumi , Schaefer Ernst , Otokozawa Seiko , Asztalos Bela , Nakajima Katsuyuki , Zhou Yanhua , Liu Ching-Ti , Jacques Paul , Cupples L Adrienne , Furusyo Norihiro
Published: June 2021

Cardiovascular disease (CVD) prevalence is known to be lower in Japan than in the United States. In this international cohort-comparative study, we compared CVD prevalence rates and risk factors in Fukuoka, Japan and in Framingham, United States. Using plasma samples obtained after an overnight fast from men and women in Fukuoka (n=1108) and age (median, 53 years), gender and menopausal status matched subjects in Framingham (n=1101), we measured plasma glucose, insulin, high density lipoprotein cholesterol (HDL-C), low density lipoprotein cholesterol (LDL-C), small dense LDL-C (sdLDL-C), and triglycerides. Blood pressure, body mass index (BMI), use of medications, and history of CVD were also assessed. CVD prevalence rates were 2.1% and 1.0%, respectively, in Fukuoka men and women, and were more than 6 folder higher (both P<0.001) at 13.2% and 8.6%, respectively, in Framingham men and women. Median BMI, LDL-C, insulin levels and insulin resistance in men and women in Fukuoka were all significantly (P<0.01) lower than those in their Framingham counterparts. However the diabetes prevalence in Fukuoka men was significantly (P<0.01) higher than that observed in Framingham men, while female rates were similar, as were levels of systolic blood pressure. Smoking rates were lower in Fukuoka women, but were higher in Fukuoka men as compared to their Framingham counterparts. HDL-C and surprisingly sdLDL-C levels were significantly (P<0.001) higher in Fukuoka subjects than in Framingham subjects. Based on the results, the estimated 10 year risk of atherosclerotic cardiovascular disease, using the new American College of Cardiology/American Heart Association risk calculator, were 6.9% and 2.3%, respectively, for Framingham men and women, while these values for Fukuoka men and women were 7.4% and 1.7%, respectively. Only the risk estimates in women were significantly different (P<0.001). Our data are consistent with prior studies indicating that CVD prevalence rates in Japan are lower than those in the United States, and that CVD risk algorithms for the United States markedly overestimate CVD risk in Japan. The markedly lower levels of insulin and insulin resistance in Japanese subjects may account for some of the population differences in CVD prevalence and risk.
Abstract 619: Alpha Keto Acids Decompose Peroxides and Prevents Oxidation of Lipoproteins
Authors: Aluganti Narasimhulu Chandrakala , Young Burge Kathryn , Yuan Yu , Parthasarathy Sampath
Published: June 2021

Background:
Alpha keto acids are unstable and decompose rapidly. In this study, we tested the ability of alpha keto acids to reduce peroxides and inhibit oxidation of lipoproteins.

Methods:
Keto salicylic acid (KSA) and Keto Octanoicacid (KoA) were synthesized and their ability to reduce hydrogen peroxides as well as lipid peroxides (LOOH) was measured using 13-hydroperoxyoctadecadienoic acid (13-HPODE). Lipoproteins (LDL and HDL) were isolated from human plasma and oxidation of liporproteins was performed using copper and MPO in the presence or absence of the keto compounds. RAW 264.7 cells and HUVECS were incubated with LPS and mm-LDL respectively either in the presence or absence of the keto compounds. RNA was isolated from treated cells and real time PCR was performed to analyze IL-1α, IL-6, MCP-1 and VCAM1 gene expressions. Reactive oxygen species were evaluated using DCF fluorescence in presence and absence of the keto compounds.

Results:
KSA reduced both H2O2 and 13-HPODE whereas KoA is able to reduce the former but not the latter. Both compounds inhibited the lipoprotein oxidation in a dose dependent manner and were able to reduce ROS production by H2O2. KSA is able to inhibit both LPS as well as mm-LDL induced inflammation. However, KoA showed a dual effect as it induced inflammatory markers in the presence of LPS, but inhibited the mm-LDL-induced inflammatory gene expressions.

Conclusion:
The results of our studies suggest that these keto compounds a) inhibit both enzymatic and non enzymatic oxidation of lipoproteins; b) reduce peroxides and ROS and c) have inhibitory and inducing effect on inflammatory cytokine/gene production in presence of mm-LDL and LPS respectively. Based on these results, we predict that these keto compounds could have therapeutic potential in reducing CVD/atherosclerosis-associated inflammation.
Abstract 641: The Role of Interleukin-23 in the Development of Atherosclerosis
Authors: Fatkhullina Aliia , Peshkova Iuliia , Koltsova Ekaterina
Published: June 2021

Atherosclerosis is lipid-driven chronic inflammatory disease of the arterial wall mediated by innate and adaptive immune responses. Inflammation promotes the development atherosclerotic plaques. Cytokines are soluble mediators of inflammation and important players in the pathogenesis of atherosclerosis. IL-23, a cytokine of IL-6/IL-12 cytokines superfamily is produced by myeloid cells and regulates the production of IL-17 and IL-22 by T helper IL-17 producing (Th17) cells, innate lymphoid cells of type 3 (ILC3) and gamma delta T cells in various auto-inflammatory diseases. IL-23R expression was also detected on myeloid cells but its role in regulation of myeloid cell function is not well defined. The level of IL-23 was shown to be upregulated in cardiovascular pathologies. Therefore, we decided to address the role of IL-23 in atherosclerosis using
Il23p19 and Il23(R)
receptor deficient mice. Surprisingly, atherosclerosis prone,

Ldlr
-/-

mice transplanted with

Il23p19
-/-

or

Il23r
-/-

bone marrow and fed with Western diet (WD) for 14 weeks demonstrated acceleration of atherosclerosis progression, which was characterized by increased accumulation of various hematopoietic cells in the aortas. Analysis of cytokine production unexpectedly revealed no changes in IL-17A and IFN-gamma production among CD4 T cells in the aortas. This effect was specific to aortas, as IL-17A production in the intestine of

Il23p19
-/-

mice was reduced, similarly to previously published observations. On the other hand, macrophages from

Il23p19
-/-

mice were able to uptake oxLDL in more efficient manner compared to wt controls, suggesting the regulatory role of IL-23 in foam cells formation. We also found enhanced inflammatory gene expression in aortas of

Il23p19
-/-

->

Ldlr
-/-

and

Il23r
-/-

->

Ldlr
-/-

mice compared to wt controls. Overall our data suggest IL-17 independent atheroprotective role of IL-23.
Abstract 647: Induction of Cardiovascular Calcification in Non-transgenic Mice via a Single Injection of Pcsk9 Adeno-associated Viral Vector
Authors: Goettsch Claudia , Hutcheson Joshua , Hagita Sumihiko , Rogers Maximillian , Choi Jung , Creager Michael , Pham Tan , Mlynarchik Andrew , Fuglsang Kjolby Mads , Aikawa Masanori , Aikawa Elena
Published: June 2021

Background:
Studying atherosclerotic calcification
in vivo
requires mouse models with genetic deletion of low-density lipoprotein receptor (Ldlr) or apolipoprotein E. A previous study showed a rapid induction of atherosclerosis by proprotein convertase subtilisin/kexin type 9 (PCSK9) in mice. Here, we hypothesize that this method is a useful
in vivo
tool to study cardiovascular calcification in non-genetically modified C57BL/6 mice.

Results:
10 week old C57BL/6 mice received a single tail vein injection of recombinant adeno-associated viral vector (AAV) encoding PCSK9 (rAAV8/D377Y-mPCSK9).
Ldlr
-/-
and saline injected C57BL/6 mice served as controls. Mice consumed a high-fat, high-cholesterol (HF/HC) diet for 15-20 weeks. PCSK9 and total cholesterol serum levels were significantly increased within one week after injection and maintained for 20 weeks (cholesterol: 82 mg/dL to 820 mg/dL, p<0.01; PCSK9: 0.14 μg/ml to 20 μg/ml, p<0.01). Total cholesterol levels remained 20-30% lower than those of of
Ldlr
-/-
mice. Atherosclerotic lesion size was similar between PSCK9 and
Ldlr
-/-
mice. Saline injected mice did not show any lesions. Plaque collagen content was 31.9%±6.6 in PCSK9 mice and 62.9%±16.6 in
Ldlr
-/-
mice at 15 weeks of HF/HC diet (p=0.01). However, by 20 weeks, the PCSK9 mice had 57.9%±18.6 plaque collagen, suggesting a different stage of plaque progression. Fluorescence reflectance imaging of a near infrared calcium tracer in intact arteries detected 0.4%±0.4 aortic calcification in PCSK9 mice and 9.7%±1.6 in
Ldlr
-/-
mice at 15 weeks of HF/HC diet (p=0.01); by 20 weeks, the PCSK9 mice had 5.3%±1.0 aortic calcification. Tissue non-specific alkaline phosphatase activity positive lesion area was 7.9%±4.0 and 8.3%±2.6 in PCSK9 mice and 10.8%±2.5 and 12.7%±1.7in
Ldlr
-/-
mice at 15 and 20 weeks, respectively. Immunofluorescence analysis demonstrated accumulation of CD68 and RUNX2-positive cells in the plaques of PCSK9 mice similar to
Ldlr
-/-
.

Conclusion:
While injection of recombinant AAV encoding PCSK9 into C57BL/6 mice induces atherosclerotic calcification with slower sclerotic plaque remodeling compared to
Ldlr
-/-
mice, it may serve as a useful tool to study cardiovascular calcification in mice independent of their genetic background.
Abstract 649: Knockout of the
Ath26
Quantitative Trait Locus Candidate Gene
Cyp4f13
Decreases Atherosclerosis in DBA/2 ApoE-Deficient Mice
Authors: Han Juying , Robinet Peggy , Berisha Stela , Ritchey Brian , Smith Jonathan D
Published: June 2021

We previously identified the atherosclerosis modifying
Ath26
quantitative trait locus (QTL) on mouse chr. 17 from intercrosses of apoE-/- mice on the AKR and DBA/2 background, where the DBA/2 allele is associated with larger lesions. The aim of our study was to identify causal genes responsible for
Ath26
. Here we report confirmation of the
Ath26
QTL in female AKR.DBAchr17 congenic mice, where inheriting 2 DBA/2 vs. AKR alleles at this locus resulted in ~5x larger lesions (p<0.001 by ANOVA posttest). We identified
Cyp4f13
as a candidate gene in this locus from gene expression microarray data of bone marrow derived macrophages from individual F2 mice, where
Cyp4f13
expression had the strongest correlation with lesion area of all expressed genes (r=0.46, p<0.0001, n=114). We obtained
Cyp4f13
knockout (KO) embryonic stem cells and created KO mice that we backcrossed on the DBA/2 apoE-/- background. We bred male and female DBA/2 apoE-/- mice that were wild type (WT), hemizygous (HET), or KO for the
Cyp4f13
gene. Chow diet fed mice were euthanized at 16 weeks of age. There were no significant differences in body weight, plasma total cholesterol or HDL-C among the 3 genotypes in either sex. Cross section lesion areas in the aortic root were not normally distributed and analyzed with non-parametric statistics. In male mice the lesions areas were smaller in the HET and KO vs. WT mice, and since the HET and KO mice had ~ equivalent lesions, we combined these two groups and found a significant 35 % decrease in median lesion area vs. WT mice (P=0.03, two-tailed Mann-Whitney test, HET+KO n=42, WT n=11). In female mice, lesions were 50% smaller in the HET and KO vs. WT mice (P=0.001, HET+KO n=31, WT n=17).
Cyp4f13
belongs to the Cyp4F gene family containing 9 members in mice, 8 of which are clustered on chr. 17. Cyp4F proteins hydroxylate the omega carbon on arachadonic acid forming 20-HETE, and also hydroxylate leukotriene B4 (LTB4). Liver microsomes from
Cyp4f13
HET mice had decreased 20-HETE forming activity vs. WT mice. 20-HETE and/or LTB4 metabolism may regulate inflammation and contribute to atherogenesis. We conclude that
Cyp4f13
is an atherosclerosis modifier gene that may be partially responsible for the
Ath26
QTL on chr. 17.
Abstract 654: Loss of SPRR3 In
ApoE
-/-
Mice Leads to Increased Atheroma Vulnerability and Evidence of Plaque Rupture and Cardiac Infarcts
Authors: Segedy Amanda K , Li Bin , Lietman Caressa D , Linton MacRae F , Young Pampee P
Published: June 2021

Atheroma rupture is the leading cause of myocardial infarction. While studies have examined inflammatory cell-mediated effects on plaque vulnerability, less is known about the role of vascular smooth muscle cells (VSMCs) or specific molecular players in the maintenance of atheroma stability. We reported that loss of small proline-rich repeat protein 3 (SPRR3), enriched in atheroma VSMCs, leads to increased VSMC death and significantly accelerates atherosclerosis progression in
ApoE
-/-
mice. Here, we show that loss of SPRR3 promotes features in plaques of brachiocephalic arteries common to unstable lesions, such as increased necrotic core size, reduced cap collagen content, and reduced VSMC content. Moreover,
ApoE
-/-
mice lacking SPRR3 develop coronary artery lesions with advanced features, including intraplaque hemorrhage. In addition,
Sprr3
-/-
ApoE
-/-
mice fed a high-fat diet for 6 months develop spontaneous myocardial infarction.
In vitro
, SPRR3 deficient VSMCs show reduced expression of procollagen type I, an event associated with Akt activation. SPRR3-deficient VSMCs also show increased expression of MMP2 transcripts, and aortic root lesions of
Sprr3
-/-
ApoE
-/-
mice have increased gelatinase activity consistent with MMP2 activation. Our data demonstrate that SPRR3 loss in
ApoE
-/-
mice decreases VSMC survival and collagen I synthesis while increasing MMP2 synthesis and activity, resulting in atheroma instability with evidence of downstream myocardial infarction. Taken together the results present the
Sprr3
-/-
ApoE
-/-
mouse as an experimental model of plaque rupture. This model will be used for additional experimental studies including
in vivo
genetic modulation of the Akt pathway as well as
in vitro
studies to determine phenotypic outcome, i.e. coronary arterial lesions, myocardial infarction, VSMC survival and collagen synthesis. We hope to establish a mechanistic link between altered Akt signaling and matrix integrity in the context of atheroma rupture, as well as potentially use SPRR3 as a molecular marker which could lead to detection of plaque instability as well as therapeutic intervention methodologies.
Abstract 7: AntagomiR-762 Prevents and Reverses Angiotensin II Induced Aortic Stiffening
Authors: Montaniel Kim Ramil C , Wu Jing , Bersi Matthew R , Xiao Liang , Itani Hana A , Vickers Kasey C , Humphrey Jay D , Harrison David G
Published: June 2021

We and others have shown that hypertension (HTN) is associated with a striking deposition of collagen in the aortic adventitia. This leads to aortic stiffening, eventually leading to end-organ damage. Through a screen of microRNAs (miRNAs) in the aorta, we found that miR-762 is the most upregulated miRNA in mice with angiotensin II (Ang II)-induced HTN. qRT-PCR confirmed that miR-762 is upregulated 6.35±1.22 (p=0.03) fold in Ang II-infused mice compared to controls. This was a direct effect of Ang II, as miR-762 upregulation was not eliminated by lowering blood pressure with hydralazine and hydrochlorothiazide and was increased only 2-fold in DOCA salt HTN. To study the role of miR-762 in HTN, we administered a locked nucleic acid inhibitor of miR-762 (antagomiR-762). AntagomiR-762 administration did not influence the hypertensive response to Ang II, yet it normalized stress-strain relationships and aortic systolic energy storage (ASE) (Table). Moreover, antagomiR-762 administration in the last 2 weeks of Ang II infusion reversed aortic stiffness in mice treated with Ang II for 4 weeks (ASE, 4 wk Ang II [51±5.18 kPa] vs 4wk Ang II + antagomiR-762(last two weeks) [20±1.76 kPa], p<0.0001). Further studies showed that antagomiR-762 reduced mRNA for several collagens and fibronectin and dramatically upregulated collagenases MMP1a, 8 and 13 (Table). Hence, miR-762 plays a major role in modulating aortic stiffening and its inhibition dramatically inhibits pathological fibrosis, enhances matrix degradation, prevents and reverses aortic stiffness. AntagomiR-762 might represent a new approach to prevent and possibly reverse aortic stiffening and its consequent end-organ damage.
Abstract 9: Involvement of Caveolin-1 in Vascular Remodeling and Inflammation Induced by Angiotensin II
Authors: Forrester Steven J , Kawai Tatsuo , Elliott Katherine J , Obama Takashi , Takayanagi Takehiko , Crawford Kevin , Eguchi Satoru , Rizzo Victor
Published: June 2021

We have recently reported that caveolin-1 (Cav1) enriched membrane microdomains in vascular smooth muscle cells (VSMC) mediate a metalloprotease ADAM17-dependent EGF receptor (EGFR) transactivation, which is linked to vascular remodeling induced by AngII. We have tested our hypothesis that Cav1, a major structural protein of caveolae, plays a critical role for development of vascular remodeling by AngII via regulation of ADAM17 and EGFR. Here, 8 week old male Cav1-/- and control Cav+/+ wild-type mice (WT) were infused with AngII (1 μg/kg/min) for 2 weeks to induce vascular remodeling and hypertension. Upon AngII infusion, histological assessments demonstrated medial hypertrophy and perivascular fibrosis of coronary and renal arteries in WT mice compared with saline-infused control mice. The AngII-infused WT mice also showed a phenotype of cardiac hypertrophy with increased HW/BW ratio (mg/g: 8.0±0.6 vs 5.7±0.7 p<0.01) compared with WT control. In contrast to AngII-infused WT mice, Cav1-/- mice with AngII showed attenuation of vascular remodeling but not cardiac hypertrophy ; HW/BW ratio (8.6±0.5 vs 6.4±0.2 p<0.05). Similar levels of AngII-induced hypertension were observed in both WT and Cav1-/- mice assessed by telemetry (MAP mmHg: 142±9 vs 154±20). In WT mice, Ang II enhanced ADAM17 expression and phospho-Tyr EGFR staining in heart and kidney vasculature. These events were attenuated in vessels from Cav1-/- mice infused with AngII. In addition, IHC analysis revealed less ER stress in heart and kidney vasculature of AngII-infused Cav1-/- mice compared with WT mice. Enhanced Cav1 and VCAM-1 expression were also observed in the aorta from AngII-infused WT mice but not in Cav1-/- aorta. These data suggest that Cav1 and presumably vascular caveolae play critical roles for vascular remodeling and inflammation via induction of ADAM17 and activation of EGFR independent of blood pressure or cardiac hypertrophy regulation.
Abstract 97: Association of the Serum Myeloperoxidase/High-Density Lipoprotein Particle Ratio and Incident Cardiovascular Events in a Multi-Ethnic Population: Observations From the Dallas Heart Study
Authors: Khine Htet W , Teiber John F , Haley Robert W , Khera Amit , Rohatgi Anand
Published: June 2021

Introduction:
Myeloperoxidase (MPO) promotes oxidation of lipoproteins whereas high-density lipoprotein (HDL) exerts anti-oxidative effects in part via paraoxonase-1 (PON1). MPO can induce dysfunctional HDL particles; however, the interaction of circulating levels of these measures in cardiovascular disease (CVD) has not been studied in humans. We hypothesized that increased serum levels of MPO indexed to HDL particle concentration would be associated with an adverse phenotype with increased CVD risk and tested this hypothesis in a large multiethnic population free of CVD at baseline.

Methods:
Levels of MPO, HDL-C, and HDL particle concentration (HDLp) by NMR were measured at baseline in 2924 adults free of CVD (57% women, 49% black). The associations of the MPO/HDLp ratio with incident ASCVD (first nonfatal myocardial infarction, nonfatal stroke, coronary revascularization, or CVD death) and total CVD were assessed in Cox proportional-hazards models adjusted for traditional risk factors (age, sex, race, hypertension, smoking, total cholesterol, and statin use). The median follow-up period was 9.4 years.

Results:
MPO/HDLp was associated directly with total cholesterol, C-reactive protein, interleukin 18, and body mass index, and inversely with PON1 arylesterase activity, HDL-C, and HDL size. In adjusted models, the highest versus lowest quartile of MPO/HDLp was associated with a 74% increase in incident ASCVD (aHR, 1.74, 95% CI 1.12-2.70) and a 91% increase in total CVD (aHR, 1.91, 95% CI 1.27-2.85).

Conclusion:
Increasing MPO indexed to HDL particle concentration at baseline is associated with increased risk of incident CVD events in a population initially free of CVD, reflecting increased inflammation and decreased PON1 arylesterase activity. This is the first report of the MPO/HDLp ratio in a large human cohort. Further studies are warranted investigating MPO/HDLp as a biomarker of HDL metabolism and CVD risk.
Abstract 98: Antiphospholipid Antibodies Induce Thrombosis by Activating Endothelial PP2A via ApoER2-Dab2-PSD95 Complex Formation
Authors: Sacharidou Anastasia , Shaul Philip , Mineo Chieko
Published: June 2021

In the antiphospholipid syndrome (APS), patients generate antiphospholipid antibodies (aPL) that promote thrombosis. We previous showed that aPL recognition of the endothelial cell surface protein β2-Glycoprotein I (β2GPI) causes β2GPI to interact with the LDL receptor family member ApoER2 and thereby antagonize endothelial NO synthase (eNOS). The decrease in bioavailable NO then leads to exaggerated thrombus formation. In the present work we sought to determine how aPL and the β2GPI-ApoER2 tandem antagonize eNOS. Initial experiments employed co-immunoprecipitation, RNAi-based gene silencing and adenoviral introduction of mutant proteins into human aortic endothelial cells. We discovered that in contrast to normal human IgG from healthy subjects (NHIgG), aPL invoke the formation of a complex between the cytoplasmic tail of ApoER2 and Dab-2 and PSD95, and that the formation of the complex is required for eNOS antagonism. We also found that ApoER2-Dab2-PSD95 complex formation in response to aPL potently activates the serine/threonine phosphatase PP2A, which in turn dephosphorylates eNOS-S1179, thereby extinguishing eNOS enzymatic activity. Furthermore, we found that upon aPL treatment, the PP2A catalytic and regulatory subunits are recruited to the ApoER2-Dab2-PSD95 complex. To test if these processes are operative in APS-related thrombosis in vivo, intravital microscopy of the mesenteric microcirculation was employed to evaluate thrombus formation in mice. In wild-type mice, aPL administration caused exaggerated thrombus formation compared to treatment with NHIgG. In contrast, ApoER2 did not promote thrombosis in knock-in ApoER2-EIG mice harboring a mutant receptor incapable of interacting with Dab-2. Moreover, in wild-type mice aPL treatment caused a dramatic increase in PP2A activity in the aorta, and administration of the PP2A inhibitor Endothall fully prevented thrombus formation induced by aPL. Having revealed the molecular underpinnings of the disorder, current treatment of APS with anticoagulation, which is often ineffective and fraught with complications, can potentially be replaced by new mechanism-based therapies targeting ApoER2 complex formation or PP2A.
Abstract 144: Perceived Risks and Safety of Transfusions Influence a Provider’s Inclination to Transfuse
Authors: Strobel Raymond J , Zahuranec Darin B , Paone Gaetano , Kramer Robert S , DeLucia Alphonse , Behr Warren D , Zhang Min , Engoren Milo C , Prager Richard L , Likosky Donald S ,   Michigan Society of Thoracic and Cardiovascular Surgeons Quality Collaborative
Published: June 2021

Background:
Small transfusions (1-2 units) of red blood cells (RBCs) have been associated with significantly increased odds of morbidity and mortality in the setting of cardiac surgery. Despite the development of blood management guidelines, provider-level variability persists in RBC transfusion practice. We hypothesize that a provider’s beliefs concerning the risk and safety of transfusions may influence his/her threshold for transfusing a patient.

Study Design and Methods:
We surveyed operating room transfusion decision-makers (anesthesiologists, perfusionists, surgeons) across all 33 non-federal cardiac surgery programs in the state of Michigan. Survey questions assessed a respondent’s hematocrit threshold (“trigger”), as well as belief concerning the perceived risk and safety of transfusions (on a 5-point Likert Scale). Linear regression models were used to estimate the independent effect of a provider’s beliefs on the inclination to transfuse.

Results:
One hundred and eighty-six of 413 submitted surveys were returned (45% response rate). Differences existed across providers in their belief concerning: (1) the risk of anemia (
p
< 0.01, table 1), and (2) transfusion safety (
p
= 0.02, table 1). Higher transfusion triggers were associated with a respondent’s: (1) concern for the risk of anemia (difference: 0.61,
p
< 0.01), and (2) belief in the safety of transfusions (difference: 0.41,
p
= 0.04). Perceived risks of complications (e.g., transfusion reactions, acute kidney injury) associated with transfusions differed across provider types (all
p
< 0.01).

Conclusion:
A provider’s belief in the safety of transfusions and concern for the risk of anemia was significantly associated with a greater likelihood of blood utilization. Our findings suggest that future blood management interventions should address a provider’s beliefs concerning blood transfusions.
Glycoursodeoxycholic Acid Ameliorates Atherosclerosis and Alters Gut Microbiota in Apolipoprotein E–Deficient Mice
Authors: Huang Kan , Liu Chenshu , Peng Meixiu , Su Qiao , Liu Ruiming , Guo Zeling , Chen Sifan , Li Zilun , Chang Guangqi
Published: June 2021

Background

Although glycoursodeoxycholic acid (GUDCA) has been associated with the improvement of metabolic disorders, its effect on atherosclerosis remains elusive. This study aimed to investigate the role of GUDCA in the development of atherosclerosis and its potential mechanisms.

Methods and Results

Human THP‐1 macrophages were used to investigate the effect of GUDCA on oxidized low‐density lipoprotein–induced foam cell formation in vitro. We found that GUDCA downregulated scavenger receptor A1 mRNA expression, reduced oxidized low‐density lipoprotein uptake, and inhibited macrophage foam cell formation. In an in vivo study, apolipoprotein E–deficient mice were fed a Western diet for 10 weeks to induce atherosclerosis, and then were gavaged once daily with or without GUDCA for 18 weeks. Parameters of systemic metabolism and atherosclerosis were detected. We found that GUDCA improved cholesterol homeostasis and protected against atherosclerosis progression as evidenced by reduced plaque area along with lipid deposition, ameliorated local chronic inflammation, and elevated plaque stability. In addition, 16S rDNA sequencing showed that GUDCA administration partially normalized the Western diet–associated gut microbiota dysbiosis. Interestingly, the changes of bacterial genera (
Alloprevotella
,
Parabacteroides
,
Turicibacter
, and
Alistipes
) modulated by GUDCA were correlated with the plaque area in mice aortas.

Conclusions

Our study for the first time indicates that GUDCA attenuates the development of atherosclerosis, probably attributable to the inhibition of foam cell formation, maintenance of cholesterol homeostasis, and modulation of gut microbiota.
Abstract 318: Vitamin E Affects Pathological Cardiac Hypertrophy and MicroRNAs Expression in Mice
Authors: Cohen Carolina R , Martinelli Nidiane C , Pinto Graziela H , Phaelante Amanda , Silvello Daiane , Recamonde-Mendoza Mariana , Andrades Michael E , Clausell Nadine , Rohde Luis E , Ashton-Prolla Patrícia , Biolo Andréia
Published: June 2021

Aims:
Vitamin E is a usual antioxidant, but little is known about its effects on cardiac hypertrophy and microRNAs (miRs) expression induced by transverse aortic constriction (TAC) in mice.

Methods and Results:
Male Balb/c mice were randomly divided into four cohorts: SHAM (
n=
22), TAC (
n
=34), SHAM supplemented with vitamin E (SHAM+VIT,
n
=22), and TAC supplemented with vitamin E (TAC+VIT,
n
=34). VIT groups received 200 mg/kg of α-tocopherol once a day, and the other groups received placebo, both by gavage. After 7 and 35 days of surgery analysis of cardiac hypertrophy, fibrosis, miRs and gene expression and carbonyl concentrations in cardiac tissue were performed. Left ventricle mass increased 23% and 35% in 7 and 35 days, respectively, in TAC group, similar data were observed in TAC+VIT group (all
p
<0.05
vs.
SHAM groups). Cardiac fibrosis was increased by TAC surgery as early as 7 days and remained high after 35 days. Still, TAC mice exhibit higher levels of protein damage at 35 days. This pathological phenotype was not seen in animals of the TAC+VIT group. Vitamin E seemed to inhibit cardiac fibrosis and oxidative damage. Moreover, cardiac hypertrophy was followed by increased
miR-21
and
-499
expression in TAC group, mainly 35 days (
miR-21
: 2.9 ± 0.6
fold vs
. SHAM: 1 ± 0.1
fold
,
miR 499
: 3 ± 0.4
fold vs.
1.1 ± 0.1
fold
,
p
<0.05). However, TAC+VIT mice displayed a different miR expression profile, with decreased
miR-21
and -
499
expression (
miR-21
: 0.5 ± 0.1
fold
,
miR-499
: 0.4 ± 0.1
fold
; TAC
vs. p
<0.05) and increased miR-210 expression (3.2 ± 0.5
fold
vs.
TAC: 1.9 ± 0.2
fold, p
=0.034). Computational target prediction of these miRs demonstrated that they can be involved in the control of major pathways in the heart disease scenario such as MAPK, mTOR, PI3K-AKT, among others.

Conclusion:
TAC model induced pathological cardiac hypertrophy, fibrosis and protein damage followed by changes in miRs expression. Vitamin E supplementation was associated with a different miR expression profile and mitigated the pathological phenotype.
Abstract WP138: MRI Detection of Vessel Wall Inflammation and Contrast Leakage in Cerebral Amyloid Angiopathy
Authors: De Havenon Adam , Lobo Remy , Eisenmenger Laura , Chauhan Nabeel , Dewitt Dana , Kim Seong-Eun , Parker Dennis , McNally Joseph S
Published: June 2021

Introduction:
Arterial wall enhancement detected on vessel wall MRI (vwMRI) has been associated with increased stroke risk in the setting of intracranial atherosclerosis. Other pathologies affect the vessel wall, including cerebral amyloid angiopathy (CAA). CAA is secondary to Aβ vessel wall deposition resulting in microvessel leakage, microbleeds and ischemic stroke. Our goal was to determine if vwMRI could detect vessel wall inflammation and leptomeningeal contrast leakage in CAA, and if these are associated with recent infarction.

Methods:
In this retrospective study, 28 vwMRI scans in 23 patients demonstrated probable CAA from 2015-16. Probable CAA was diagnosed by peripherally located microhemorrhages detected on susceptibility-weighted imaging. Vessel wall and leptomeningeal enhancement was determined using a validated pre and post contrast flow-suppressed T1-weighted sequence. Recent infarcts were detected by DTI trace (20 direction B2000). A mixed effects logistic regression model was used to determine the association of vessel wall or leptomeningeal enhancement with recent infarct in CAA patients, accounting for up to 3 scans per patient.

Results:
In the 28 vwMRI scans, ischemic stroke was detected in 17 (61%), leptomeningeal enhancement in 13 (46%), and vessel wall enhancement in 14 (50%). The figure shows a representative patient with a recent infarct and biopsy-proven CAA with vessel wall enhancement (arrow) detected on vwMRI. Vessel wall enhancement was associated with recent infarct with an odds ratio (OR) = 10.8, p= 0.012. Leptomeningeal enhancement was associated with recent infarct with an OR = 3.8, p= 0.11.

Conclusions:
Both vessel wall and leptomeningeal enhancement are present in CAA, though vessel wall enhancement is more associated with recent infarction. Larger, prospective studies may be needed to determine future stroke risk and additional risk factors predicting ischemic stroke in CAA.
Abstract WP245: Hyperacute Blood Pressure Variability is Associated with Early Neurological Deterioration
Authors: Kim Joseph , Saver Jeffrey L , Liebeskind David S , Starkman Sidney , Hamilton Scott , Chung Pil-Wook , Sanossian Nerses ,   FAST-MAG Trial Investigators
Published: June 2021

Background:
Increased blood pressure variability (BPV) has been associated with worse outcomes in acute stroke. The effect of hyperacute (<4 hours) BPV on early neurologic deterioration (END) has not been described.

Objective:
To investigate whether BPV in the first hours after stroke onset is associated with END from prehospital evaluation to presentation at the emergency department

Methods:
All patients enrolled in the NIH Field Administration of Stroke Therapy-Magnesium (FAST-MAG) phase 3 trial were included. FAST-MAG was a multicenter, randomized, double-blind, placebo-controlled study looking at whether initiation of magnesium sulfate (20 grams/24 hours) in the prehospital setting of acute stroke would reduce disability. Study agent was initiated prior to hospital arrival < 2 hours from symptom onset. BPV was defined as the standard deviation of systolic blood pressure of all readings obtained by 4 hours after initiation of study agent. END was diagnosed as Glasgow Coma Scale (GCS) decrease by ≥ 2 points between the prehospital evaluation and post-emergency department arrival assessment by a study nurse.

Results:
There were 1,700 cases evaluated by paramedics 24 minutes (15-45 IQR), and by study nurses 150 minutes (120-180) after symptom onset with a median of 6 (IQR 5-6) BP readings. The mean (±SD) age was 69±13 years, 42.6% were women, and the median prehospital GCS was 15 (IQR 14-15). The final diagnosis was cerebral ischemia in 73.3% of patients, intracranial hemorrhage in 22.8%, and a stroke-mimicking condition in 3.9%. END was seen in 202 (12%) of subjects, with higher rates noted in those with intracerebral hemorrhage (ICH) compared to cerebral ischemia (31% vs 6%). Overall, there was greater BPV in patients with END (23mmHg vs 15mmHg, p<0.001). Blood pressure variability was greater in cases of cerebral ischemia with END (N=1,245, 18mmHg vs 15mmHg, p=0.004) and in ICH cases with END (N=387, 23mmHg vs 15mmHg, p<0.001).

Conclusion:
Greater blood pressure variability is associated with early neurologic deterioration in patients with cerebral ischemia and ICH evaluated <2 hours from symptom onset.
Abstract 100: Reperfusion Improves Clinical Outcome Across the 4.5-9h and Wake-Up Stroke Time Continuum in EXTEND and EPITHET
Authors: Campbell Bruce C , Ma Henry , Parsons Mark W , Churilov Leonid , Yassi Nawaf , Kleinig Timothy J , Hsu Chung , Dewey Helen M , Butcher Ken S , Yan Bernard , Desmond Patricia M , Wijeratne Tissa H , Curtze Sami , Barber P A , De Silva Deidre A , Thijs Vincent , Levi Christopher R , Bladin Christopher F , Sharma Gagan , Bivard Andrew , Donnan Geoffrey A , Davis Stephen M ,   EXTEND and EPITHET Investigators
Published: June 2021

Introduction:
Intravenous alteplase reduces disability after ischemic stroke in patients 4.5-9h after onset and with wake-up onset stroke who have favorable perfusion imaging. We examined the benefit of reperfusion in reducing disability, including by onset to randomization time strata in the EXTEND and EPITHET randomized trials.

Methods:
Patients were randomized to alteplase or placebo after perfusion mismatch imaging. Reperfusion was defined as >90% reduction in Tmax>6s lesion volume at 24h. Ordinal logistic regression adjusted for baseline age and NIHSS was used to analyze functional improvement in day 90 modified Rankin scale overall, including a reperfusion*time to randomization interaction term, and in the 4.5-6h, 6-9h and wake-up time strata. Symptomatic hemorrhage was defined as large parenchymal hematoma with ≥4 point NIHSS increase (SITS).

Results:
Reperfusion was assessable in 270/294 (92%) patients, 68/133 (51%) alteplase and 38/137 (28%) placebo reperfused (p<0.001). Median age 76 (IQR 66-81) in reperfused vs 74 (IQR 64.5-81) in non-reperfused, median baseline NIHSS 10 (IQR 7-15) in reperfused vs 12 (IQR 8-17.5) in non-reperfused. Overall, reperfusion was associated with common odds ratio 7.7 (95%CI 4.6-12.8, p<0.0001) in ordinal “shift” analysis. There was no heterogeneity in the beneficial effect of reperfusion effect by time to randomization (p=0.63). Reperfusion was associated with significantly improved functional outcome in each of the 4.5-6h, 6-9h and wake-up time strata (figure). Symptomatic hemorrhage, assessed in all 294 patients, occurred in 3/51 (5.9%) 4.5-6h, 2/28 (7.1%) 6-9h, 4/73 (5.5%) wake-up stroke in the alteplase-treated patients (van Elteren p=0.66).

Conclusions:
Strong benefits of reperfusion in all time strata without differential risk in symptomatic hemorrhage support the durable treatment effect of alteplase in perfusion mismatch-selected patients throughout the 4.5-9h and wake-up stroke time window.