bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 408: The Importance of Vascular Risk Factors for Development of Dementia
Authors: Siegel Gunter , Ermilov Eugeny , Lubitz Irit
Published: March 2022

Background:
Since prevention and therapy of Alzheimer dementia turned out to be problematic, more attention should be paid to the vascular component of mixed pathologies, since this includes the possibility to decisively reduce the incidence of dementiae. ALP and GGT are indispensable constituents of brain microvessel endothelial cells, expressed and induced by IL-6. High values are entangled in affecting blood-brain barrier functionality. In a pilot study of 11 patients with metabolic syndrome, we examined whether these biomarkers can be beneficially influenced through an antioxidant phytochemical approach with Ginkgo biloba (EGb 761, 2 x 120 mg/d, 2 months).

Methods:
Photometric methods, ELISAs, EIAs and laser ellipsometry were applied.

Results:
Brain/liver biomarkers: ALP -14.8% (p < 0.004), GGT -11.3% (< 0.010). (Anti)oxidative biomarkers: SOD +17.7% (< 0.009), GPx +11.6% (< 0.001), oxLDL/LDL -21.0% (< 0.002), 8-iso-PGF2α -39.8% (< 0.003), MPO -29.6% (< 0.014). Inflammatory biomarkers: IL-6 -12.9% (< 0.041), Lp(a) -26.3% (< 0.001), hs-CRP -39.3% (< 0.005), WBC -6.3% (< 0.023), MMP-9 -32.9% (< 0.042). Vasodilator biomarkers: cAMP +43.5% (< 0.001), cGMP +32.9% (< 0.001), nanoplaque formation -14.3% (< 0.008), nanoplaque size -23.4% (< 0.0004). ALP and GGT were correlated to each other (r = 0.71; < 0.0225) as well as to a decrease of oxidative, inflammatory and diabetic biomarkers (Table).

Conclusion:
Since under ginkgo, ALP and GGT were downregulated, SOD upregulated and plasma biomarkers beneficially altered, this phytochemical may be estimated as complementary drug with potentially preventive character for cardio-cerebro-vascular diseases including dementiae.
Abstract 410: High Sensitive C-Reactive Protein Independently Predicts Insulin Resistance in Women in a South Asian Population, With a Stronger Association in Urban Areas
Authors: Thanikachalam Mohan , Sunderarajan Jahnavi , Harivanzan Vijaykumar , Siegel Richard , Chui Kenneth K , Murthy J S , Thanikachalam Sadagopan
Published: March 2022

Objectives:
Insulin resistance (IR), which plays a fundamental role in the pathogenesis of metabolic syndrome and type 2 diabetes mellitus, is associated with serum levels of inflammatory markers and abdominal obesity. Whether the association of inflammation with IR is independent of obesity remains unresolved; moreover, whether this association varies with gender and urban living is not known. We therefore conducted a cross-sectional study to investigate the gender specific association between IR and high sensitive C-reactive protein (HsCRP), independent of abdominal obesity, in urban and rural communities in a South Asian population.

Methods:
We conducted a population-based cross-sectional survey in 8,080 South Indians above the age of 20 years in both urban and rural environments. The study assessed of waist circumference (WC), blood pressure (BP), socioeconomic status, family history, anxiety and stress levels, and fasting blood for plasma glucose, insulin, total cholesterol (TC), triglyceride (TG), high density lipoprotein (HDL), low density lipoprotein (LDL) and HsCRP. IR was calculated using the homeostasis model assessment (HOMA-IR). After the exclusion of individuals with diabetics, 6,309 subjects (mean age 42 years; 58% women) were eligible and constituted the study sample.

Results:
The HOMA-IR level (mean ± SD) was higher in women (1.98 ± 1.62), when compared to men (1.71 ± 1.38), and HsCRP levels were 4.00 ± 5.14 and 3.42 ± 7.22 mg/dl in women and men, respectively. There was stronger correlation of HsCRP with HOMA-IR in women (r= 0.371) when compared to men (r= 0.203). In a multiple regression analysis, after adjusting for WC and other co-variants such as, BP, TC, LDL, TG, socioeconomic status, physical activity and family history of diabetes, HsCRP continued to be a significant predictor only in women (p<0.001). In women with elevated HsCRP levels (≥ 3 mg/dl), after multivariate adjustments, the odds ratio of elevated HOMA-IR (≥ 2.86) was 2.13 [CI: 1.66, 2.74; p <0.001] in urban women, when compared to odds ratio of 1.41 [CI: 1.02, 1.96; p=0.037] in rural women.

Conclusion:
HsCRP is associated with IR independent of abdominal obesity in non-diabetic South Asian women, and at elevated HsCRP levels the association is stronger in urban women.
Abstract 420: Lysosomal Acid Lipase Deficiency Impairs Reverse Cholesterol Transport From Macrophages in LAL-Deficient Mice
Authors: Bowden Kristin , Xu You-Hai , Grabowski Gregory A , Du Hong , Francis Gordon A
Published: March 2022

The rate of flux of cholesterol out of lysosomes is a key regulator of oxysterol formation and subsequent activation of liver X-receptor to upregulate genes involved in reverse cholesterol transport (RCT). We previously demonstrated impaired regulation of the lipid transporter ATP-binding cassette transporter A1 (ABCA1), required to promote removal of cholesterol from cells and high density lipoprotein (HDL) particle formation, in the lysosomal cholesterol storage disorders Niemann Pick Disease Type C and Cholesteryl Ester Storage Disease, thus providing an explanation for the low plasma HDL-C seen in these diseases. To determine the importance of lysosomal acid lipase (LAL) in reverse cholesterol transport (RCT) from macrophages to feces in vivo, we performed intraperitoneal injection of immortalized peritoneal macrophages from LAL+/+ or LAL-/- mice that had been cholesterol loaded and labeled with acetylated LDL containing 3H-cholesteryl oleate into LAL+/+ and LAL-/- mice, respectively. LAL-/- macrophages exhibited reduced basal and cholesterol-stimulated ABCA1 expression in culture, and reduced ability to support RCT in LAL-/- mice (1.55 ± 0.35 % total injected 3H-cholesterol counts appearing in feces over 48 h, n=30) compared to LAL+/+ macrophages injected into LAL+/+ mice (5.38 ± 0.92 % 3H-cholesterol counts in feces at 48 hr, n=27), p < 0.001. Injection of LAL-/- macrophages into LAL+/+ mice resulted in partial correction of RCT (2.60 ± 0.46 % 3H-cholesterol counts in feces at 48 hr, n=19), p < 0.001 when compared to injection into LAL-/- mice. ABCA1 protein expression was reduced in lal-/- mouse liver and mRNA expression of several LXR-dependent genes involved in reverse cholesterol transport (ABCG1, ABCG5, ABCG8, CYP7A1, SR-B1) were differentially modulated compared to LAL+/+ controls. The results indicate a critical role of macrophage LAL in promoting ABCA1 expression and RCT in vivo, and the ability of secreted LAL to be taken up by macrophages and correct RCT in vivo. The premature atherosclerosis seen in LAL-deficient humans may therefore be reduced by correction of LAL deficiency.
Abstract 428: Scavenger Receptor Class B Type I Is Required for Protection by High-Density Lipoprotein Against Doxorubicin-Induced Apoptosis in Both Mouse and Human Cardiomyocytes and Cardiotoxicity in Mice
Authors: Durham Kristina , Trigatti Bernardo
Published: March 2022

Doxorubicin (DOX) is a clinically used anti-tumor drug that induces cardiotoxicity. HDL protects against DOX induced apoptosis in isolated cardiomyocytes, though the pathways of protection are not well known. The scavenger receptor class B type I (SRBI) is a high affinity HDL receptor, and its absence in atherogenic mice leads to occlusive CAD and MI, though a direct role for SRBI in cardiomyocytes has yet to be studied. Here we utilized both in vitro cell culture and in vivo mouse models to assess whether SRBI is required for HDL induced protection against DOX induced cardiotoxicity.

Mouse neonatal cardiomyocytes were isolated from C57Bl6 and SRBI KO hearts. SRBI was knocked down in SV40 immortalized human ventricular cardiomyocytes using siRNA. All cells were treated as follows: 24h lipoprotein starvation, 24h ±100μg/ml HDL, and 6h ±1μM DOX. Cells were then assessed for apoptosis. Secondly, male C57Bl6 or SRBI KO mice were injected i.p. with 5mg/kg DOX or saline by 5 weekly. Plasma was assessed for cardiac troponin-T. Heart sections were assessed for apoptosis and examined histologically.

HDL protected against DOX-induced apoptosis in both C57Bl6 mouse cardiomyocytes (7.7±5.0 vs 23.7±1.8% apoptotic cardiomyocytes n=3 p<.05), and in immortalized human ventricular cardiomyocytes (6.7±2.1 vs 24.5±4.8% apoptotic cardiomyocytes n=3 p<.05). Conversely, HDL was unable to protect against DOX induced apoptosis in cardiomyocytes from SRBI KO mice (37.4±6.8 vs 35.5±8.3% apoptotic cardiomyocytes, n=3 p<.05), or immortalized human cardiomyocytes transfected with siSRBI (27.2±8.9 vs 31.3±3.7% apoptotic cardiomyocytes n=3 p<.05).

In vivo studies showed that SR-BI KO hearts are more sensitive to DOX as shown by increased apoptosis (6.9±0.6 vs 3.9±0.7% apoptotic cardiomyocytes n=5/group p<.05) and greater plasma cardiac troponin T levels (1.7±0.3 vs 1.3±0.04ng/ml n=5/group p<.05). Hearts from DOX treated SRBI KO mice also exhibited greater myofibrillar loss, vacuolization, and inflammatory cell infiltration compared to C57Bl6 hearts.

Our results indicate that SRBI mediates HDL induced protection in both mouse and human cardiomyocytes. We show here that SRBI is an important factor for the protection of the heart as a whole against DOX induced cardiotoxicity.
Abstract 429: Central Glucagon-Like Peptide-1 Reduces Intestinal Chylomicron Production via Melanocortin-4-Receptor Signaling
Authors: Farr Sarah L , Baker Chris , Naples Mark , Taher Jennifer , Adeli Khosrow
Published: March 2022

Insulin resistance and the metabolic syndrome are associated with intestinal overproduction of chylomicrons, resulting in postprandial dyslipidemia and atherogenic remnants. It is increasingly clear that a reduction in chylomicrons is achieved by the action of glucagon-like peptide-1 (GLP-1) in the periphery. However, this peptide is also generated by central neurons and regulates activity in brain regions that affect sympathetic signaling. Indeed, through sympathetic pathways GLP-1 can modulate peripheral lipogenesis. Therefore, we examined the potential role of peripheral and central GLP-1 in regulating chylomicron production. Healthy hamsters received an oral fat load followed by an intraperitoneal (IP) or intracerebroventricular (ICV) injection of the GLP-1 receptor (GLP-1R) agonist exendin-4. Postprandial triglyceride (TG) and apolipoprotein B48 (apoB48) levels from plasma TG-rich lipoproteins (TRL) were found to be reduced over 6 h. The effects of central exendin-4 were mirrored by ICV MK-0626, an inhibitor of GLP-1 degradation, and reversed by the GLP-1R antagonist exendin9-39. Given a potential link between central GLP-1 and the melanocortin-4-receptor (MC4R) system, hamsters received an MC4R antagonist ICV alongside exendin-4 and this prevented a lowering in TRL-TG levels. To determine whether subsequent communication with the gut was through sympathetic pathways, α- and β-adrenergic receptor blockers were infused intravenously prior to ICV exendin-4 treatment and prevented exendin-4 from reducing TRL-TG. Finally, to examine the interplay between central and peripheral GLP-1R signaling, exendin-4 was given IP while exendin9-39 was given ICV and vice-versa. IP exendin-4 diminished TRL-TG levels during central antagonism, while IP exendin9-39 prevented the action of ICV exendin-4. This suggests that peripheral GLP-1Rs may be potentiated by central stimulation, but can also act independently. Overall, we demonstrate a GLP-1-sensitive brain-gut axis for regulating chylomicron production, involving the MC4R system and sympathetic pathways. Future studies will investigate whether central GLP-1 signals via pro-opiomelanocortin neurons and sympathetic neurotransmitters to affect enterocyte chylomicron output.
Abstract 431: Lipoprotein Metabolism and Transendothelial Apolipoprotein A-I Transport in Mice With an Endothelium-Specific Knockout of ATP Binding Cassette Transporter A1
Authors: Hasballa Reda , Rohrer Lucia , Fotakis Panagiotis , Zannis Vassilis I , Parks John S , von Eckardstein Arnold
Published: March 2022

Low plasma levels of high density lipoprotein (HDL) cholesterol as well as apolipoprotein A-I (ApoA-I) are associated with increased risk of coronary heart disease. HDL and ApoA-I exert multiple potentially anti-atherogenic activities. Several of them the including stimulation of cholesterol efflux from macrophage foam cells for reverse cholesterol transport are exerted in the vascular wall rather than in the plasma compartment. HDL or apoA-I must hence pass the endothelium to enter and leave the arterial wall. We previously demonstrated in vitro that the transendothelial transport of ApoA-I involves the interaction with the ATP-binding cassette transporter (ABC) A1.

To investigate the physiological relevance of transendothelial ApoA-I transport and its regulation by ABCA1, we generated an endothelium-specific ABCA1 knock-out mouse model (ABCA1e-/-) by expressing the Cre recombinase under the control of the Ve Cadherin gene in floxed ABCA1 -/- mice.

Under both chow diet and a high fat/high cholesterol diet for 16 weeks the ABCA1e-/- mice did not differ from wild type mice by plasma levels of cholesterol or triglycerides or lipoprotein profiles. After injection of radio-iodinated ApoA-I, we found no difference in the decay of plasma radioactivity and the accumulation of radiolabel in different organs except the aorta. Compared to the aortas of wild type mice, aortas of ABCA1e-/- mice showed a significantly reduced enrichment of the radiolabel. Aortas of ABCA1e-/- mice also showed a slightly decreased enrichment of radiolabeled HDL but no difference in the enrichment of radiolabeled LDL or albumin. An ApoA-I mutant with defective ABCA1 interaction showed enhanced catabolism in both wild type and ABCA1e-/- mice as compared to wild type apoA-I. However the ABCA1-defective ApoA-I mutant did not accumulate differently in the aortas of wild type and ABCA1e-/- mice.

Taken together the data support our concept and provide first in vivo evidence that transendothelial ApoA-I transport is a specific process modulated by ABCA1.
Abstract 443: The Role of Wnt5b and Wnt11 in Aortic Valve Calcification
Authors: Albanese Isabella , Yu Bin , You Zhipeng , Barratt Bianca , Shum-Tim Dominique , Al Kindi Hamood , Schwertani Adel , Alreshidan Mohammed
Published: March 2022

Introduction:
The mechanisms underlying the pathogenesis of aortic valve calcification remain unclear. With accumulating evidence demonstrating that valve calcification recapitulates bone development, the crucial roles of non-canonical Wnt ligands Wnt5b and Wnt11 in osteogenesis make them critical targets in the study of aortic valve calcification.

Hypothesis:
We hypothesized that Wnt signaling mediated by ligands Wnt5b and Wnt11 is upregulated and the expression of these molecules may be associated with the pathological calcium deposition in aortic valves.

Methods and Results:
Mitral, non-calcified and calcified aortic valves were collected from patients with valve replacement. Total RNA was isolated, first strand cDNAs were synthesized and gene specific qPCRs were performed. The relative mRNA expression of Wnt5b and Wnt11 were calculated using the ΔΔCT method against GAPDH control and are expressed as fold change ± SEM compared to expression in non-calcified valves. We found significant increases in both Wnt5b (15.14±4.08) and Wnt11 (4.09±0.63) expression in calcified compared to non-calcified aortic or mitral valves (P<0.05). We also examined Wnt5b and Wnt11 protein expression in 73 mitral, non-calcified and calcified aortic valves by immunohistochemistry. There was little expression of Wnt11 or Wnt5b in normal aortic valves, normal segments of calcified valves or mitral valves. Wnt11 staining intensity was significantly elevated in areas of calcification, inflammation and in activated myointimal cells compared to normal aortic and mitral valves (P<0.05). Immunostaining for Wnt5b was expressed in similar cellular compartments as well as areas of fibrosis. Multivariant Spearman correlation analyses revealed significant positive correlations between Wnt5b and Wnt11 overall staining with calcification, lipid score, fibrosis, valve remodeling and microvessels (P<0.05). We are currently evaluating the tissue expression of these two molecules with clinical parameters of the disease.

Conclusion:
The upregulated Wnt5b and Wnt11 expression in calcified aortic valves, particularly in areas of calcification, fibrosis and activated myointimal cells suggests a potential involvement in the pathogenesis of aortic calcification.
Abstract 446: Divergent Trajectory Patterns of Systemic Versus Vascular Inflammation over Age in Healthy Caucasian and African-American Families
Authors: Byambaa Enkhmaa , Erdembileg Anuurad , Zhang Wei , Fung Tina , Abbuthalha Adnan , Kim Kyoungmi , Berglund Lars
Published: March 2022

Objectives:
Age and inflammation both are risk factors for cardiovascular disease. The association between these two risk factors across lifespan and ethnicity in the general population is poorly understood.

Methods:
We assessed the relationship of systemic (C-reactive protein, serum amyloid A, fibrinogen, haptoglobin and α-acid glycoprotein) and vascular (pentraxin-3, soluble intercellular adhesion molecule-1 and soluble vascular adhesion molecule-1) inflammatory burden with age in healthy Caucasian and African-American families with members aged 6 to 74 years (n=267). To collectively assess systemic and vascular risk factors across the age spectrum, a composite z-score for each category of inflammatory markers was calculated based on individual levels.

Results:
We observed a contrasting pattern in systemic vs. vascular inflammatory burden over age with an increase in systemic but a decrease in vascular inflammatory markers in both ethnic groups (p<0.05 for all). There were no significant differences in trends between the two ethnic groups. These observations remained significant after taking the correlations of siblings and parents from the same family into account. In multivariate analyses adjusting for covariates (BMI, blood pressure, total, LDL and HDL cholesterol, triglyceride and glucose), the trajectory patterns of fibrinogen and α-acid glycoprotein levels were no longer regulated with age in either ethnic group, whereas the results for vascular inflammation remained unchanged. The results based on the composite z-score for each marker category confirmed the findings of individual markers as the composite z-score for systemic inflammation increased with age (p=0.005), while the composite z-score for vascular inflammation declined with age (p=0.0002) regardless of ethnicity.

Conclusions:
There was a diverging pattern in systemic vs. vascular inflammatory burden as function of age, which was independent of ethnicity, family correlation and individuals’ metabolic burden. The findings illustrate the complex relationship between age and inflammation, and suggest that a perceived elevation of vascular markers among the very young may be an indication of dynamic growth rather than a disease process.
Abstract 45: Interleukin-19 Increases Angiogenic Gene Expression and Perfusion in Ischemic Hind-Limbs
Authors: Richards James , Gabunia Khatuna , Kelemen Sheri , Kako Farah , Choi Eric , Autieri Michael
Published: March 2022

Introduction:
Peripheral vascular disease affects over ten million Americans. Hypoxia in ischemic limbs typically initiates angiogenic and inflammatory factors to promote angiogenesis in attempt to restore perfusion. Little is known about the inflammatory nature of angiogenesis particularly with respect to anti-inflammatory factors. Interleukin-19 is a uniquely anti-inflammatory Th2 cytokine that promotes angiogenesis in cellular assays. We hypothesized that IL-19 could drive angiogenesis in vivo; the purpose of this study is to characterize a role for IL-19 in restoration of blood flow in the hind-limb ischemia model, and define potential mechanisms.

Methods and Results:
Three complimentary experiments were designed to clarify IL-19’s angiogenic role. 1- C57BL/6 (n=12) were subject to induced hindlimb ischemia by femoral artery ligation and subject to daily i.p. injections of recombinant mouse IL-19 (10ng/g/day) or PBS for 14 days. Mice were subsequently imaged using Laser Doppler Perfusion Imaging (LDPI). Perfusion index was evaluated by comparing values between ligated and unligated contralateral limbs. C57BL/6 mice injected with rIL-19 showed significantly increased levels of perfusion when compared to PBS injected age-matched cohorts (p<0.01 at day 7, <0.05 at day 10). 2- hindlimb ischemia was similarly induced in both C57BL/6 and IL-19-/- mice on a C57BL/6 background. LDPI values are significantly decreased in IL-19-/- mice when compared to C57BL/6 mice (p<0.05 at day 7). 3- IL-19-/- mice subject to limb ischemia were given daily i.p. injections of rIL-19 or PBS. IL-19-/- mice administered rIL-19 trended (P=0.058) higher perfusion when compared to PBS injected control mice. Immunohistochemistry using CD31 antibody on adductor muscle recovered from these mice showed significantly increased capillary density in IL-19 treated mice compared with controls (P<0.05). cDNA microarray analysis of human microvascular endothelial cells treated with IL-19 determined increased expression of angiogenic cytokines, which was verified by qRT-PCR and western blot.

Conclusions:
This study is the first to implicate IL-19 as a novel pro-angiogenic cytokine with potential to promote neovascularization and restore blood flow in ischemic tissue.
Abstract 464: Exercise Restores Impaired Resistance Vessel Function in Hypothalamic Hypoestrogenic Premenopausal Women in Association With Increased Resting Cardiac Output and Decreased Systemic Vascular Resistance
Authors: O'Donnell Emma , Goodman Jack M , Murai Hisayoshi , Harvey Paula J
Published: March 2022

Objectives:
To determine the hemodynamic effects of an acute bout of exercise on previous findings of impaired resistance vessel function in physically active premenopausal women with hypothalamic hypoestrogenemia (ExHH).

Methods:
Two groups of similarly trained recreationally active premenopausal women were studied: ExHH (n=12; aged 25±1 years; body mass index 20.3±0.7 kg/m
2
; mean±SEM), and eumenorrheic ovulatory women (ExOv; n=14; 24±1years; 21.4±0.5 kg/m
2
). Blood pressure (BP), heart rate (HR), stroke volume (SV), cardiac output (CO), total peripheral resistance (TPR; 2D echocardiography) and resting and peak-ischemic calf blood flow (CBF; plethysmography) were assessed at baseline and 60 minutes after 45 minutes of moderate aerobic exercise.

Results:
In ExHH, baseline HR (47±2 beats/min), systolic BP (90±2 mmHg) and CO (3.4±0.1 L/min) were lower (p<0.05) than in ExOv (54±2 beats/min; 101±1 mmHg; 4.1±0.1 L/min; respectively). Resting and peak-ischemic CBF were also lower (p<0.05) in ExHH (1.5±0.1 and 20.5±1.5 ml/100ml/min) than ExOv women (2.4±0.2 and 28.8±2.9 ml/100ml/min). Conversely, TPR was higher (p<0.05) in ExHH (1606±69 dynes/sec/cm
5
) than eumenorrheic women (1400±47 dynes/sec/cm
5
).
Post-exercise:
HR and CO were increased (p<0.05), and TPR decreased (p<0.05) in both groups. However, in ExHH, HR (58±2 beats/min) and CO (3.90±0.1 L/min) remained lower (p<0.05) and TPR (1365±65 dynes/sec/cm
5
) higher (p<0.05) than in ExOv women. Resting CBF was increased (p<0.05) to a similar value (3.3±0.2 ml/100ml/min; overall mean) in both groups. Peak CBF was increased (p<0.05) in ExHH only (26.4±2.0 ml/100ml/min). Using pooled data, CO was positively, and TPR inversely, associated (p<0.05,) with resting CBF both pre- and post-exercise.

Conclusion:
Compared with eumenorrheic women, ExHH women demonstrate impaired regional blood flow in association with low CO and elevated TPR. Dynamic exercise acutely restores CBF in ExHH women in association with increased CO and decreased TPR. While the effects of estrogen deficiency
per se
are unclear, these data suggest that impaired resistance vessel function in ExHH is: i) reversible, and ii) associated in part with increased vascular tone and altered hemodynamic characteristics.
Abstract 467: PCSK6 Is Upregulated in Vascular Diseases Characterized by Inflammation and Smooth Muscle Cell Proliferation
Authors: Perisic Ljubica , Razuvaev Anton , Österholm Cecilia , Lengquist Mariette , Sabater-Lleal Maria , Folkersen Lasse , Du Lei , Gonzalez Diez Maria , Hedin Erika , Ponten Fredrik , Paulsson-Berne Gabrielle , Hamsten Anders , Odeberg Jacob , Eriksson Per , Hedin Ulf
Published: March 2022

Recently we demonstrated upregulation of the proprotein convertase PCSK6 in a large cohort of human carotid atherosclerotic plaques (n=127) compared to normal arteries and in symptomatic vs. asymptomatic lesions. PCSK6 was localized to smooth muscle cells (SMCs) in the fibrous cap and showed a positive correlation to markers of inflammation, extracellular matrix remodeling and cytokines. Here, we aimed at elucidating the role of this protease in vascular disease by examining its expression in different human pathologies and in animal models.

Increased expression of PCSK6 in vascular diseases was validated in public microarray datasets and other available human cohorts. PCSK6 was upregulated in carotid atherosclerotic plaques
vs.
controls (n=32 patients, p<0.0001), as well as in abdominal aortic aneurysm (AAA)
vs.
normal tissue (n=14, p<0.0001) and in thoracic aortic aneurysm (TAA) tissue from bicuspid
vs.
tricuspid patients (n=244, p=0.012). By eQTL analyses, several SNPs in the PCSK6 genomic region were shown to influence its expression in carotid plaques and TAA tissue. Among these, rs6598465 showed a mild association to progression of maximum intima-media thickness in the left and right arteries in a separate cohort of high-risk coronary artery disease subjects (n=3400, p=0.037). By immunohistochemistry, PCSK6 localized mainly to SMCs in carotid plaques, AAA and TAA tissue, but was also found to be expressed by CD68 and CD163+ macrophages. Investigation of mouse, rat and human artery tissues with pronounced intimal hyperplasia revealed strong expression of PCSK6 in proliferating SMCs. In rat carotid artery balloon injury, PCSK6 was downregulated in the early phases after injury mostly defined by inflammatory response, while upregulated in later phases with prominent SMC activation, and consistently localized in SMCs. Expression of PCSK6 in this model was strongly positively correlated solely to PDGFB and IGF1 (p<0.0001), cytokines known to induce SMC proliferation.

We established a functional link between elevated expression of PCSK6 and vascular diseases characterized by inflammation and SMC proliferation. Further investigations
in vitro
are necessary to provide mechanistic insight into the role of this protease in vascular disease.
Abstract 474: Biglycan Deficiency is Not Protective Against the Development of Atherosclerosis
Authors: Thompson Joel C , Wilson Patricia G , Yoder Meghan H , Tannock Lisa R
Published: March 2022

Atherosclerosis is the leading cause of death worldwide. As outlined in the Response to Retention hypothesis proteoglycan mediated lipid retention in the vascular wall is thought to be a major cause of atherosclerosis. We and others demonstrated co-localization of the vascular wall matrix proteoglycan biglycan with apoB containing lipoproteins within lesions. Furthermore, we have previously shown that increased biglycan leads to increased atherosclerosis. Thus we hypothesized that biglycan deficiency would attenuate the development of atherosclerosis. Biglycan deficient mice on a mixed genetic background were crossed with LDL receptor deficient mice (C57Bl6). Biglycan deficient and wildtype LDLr-/- mice were fed a 0.12% cholesterol diet for 26 weeks. There were no differences in blood glucose, blood pressure, or triglycerides between genotypes. Total cholesterol progressively increased in both groups over the course of the study and was significantly higher in biglycan deficient compared to wildtype mice at 26 weeks (p<0.05). Biglycan has been proposed to play a role in regulation of TGF-β levels and activity. We previously demonstrated that aged biglycan deficient mice had increased TGF-β levels compared to wildtype. TGF-β was significantly increased in biglycan deficient mice prior to study while fed chow and steadily increased with the high cholesterol diet over the course of the study in both genotypes (p<0.001); however, biglycan deficient mice had significantly more TGF-β at each timepoint compared to wildtype controls (p<0.001). Surprisingly, analysis of the aortic intimal surface revealed a 1.4 fold increase in atherosclerosis in biglycan deficient mice compared to wildtype controls (p=0.07). Thus in opposition to our hypothesis, biglycan deficiency is not protective against the development of atherosclerosis and may exacerbate disease development. Elevated TGF-β and/or cholesterol in biglycan deficient mice may contribute to the increased atherosclerosis observed but further research is required to elicit the mechanism.
Abstract 487: Resveratrol as an Inhibitor of Aortic Root Dilatation in Marfan Mice
Authors: Hibender Stijntje , Franken Romy , Schermer Edith E , Groenink Maarten , Zwinderman Aeilko H , Mulder Barbara J , De Vries Carlie J , De Waard Vivian
Published: March 2022

Rationale:
Marfan syndrome (MFS) is an autosomal connective tissue disorder caused by mutations in the Fibrillin-1 (FBN1) gene. Marfan patients are at risk for aortic aneurysm development and dissection, with often lethal consequences. Treatment consists of aortic root replacement surgery and beta-blocker medication. Recently, losartan was proven to be effective in inhibition of aortic root dilatation in MFS patients. The beneficial effect of losartan suggests the involvement of angiotensin II receptor 1 (AT1R) signaling as a cause for aortic pathology. Angiotensin II (Ang-II) can induce cell senescence, in contrast to sirtuin-1 (SIRT-1), which is an NAD-dependent deacetylase and prevents senescence. Among other functions, resveratrol can activate SIRT-1, which prompted us to investigate the effectiveness of resveratrol treatment in MFS mice.

Methodology:
In the current study we investigate whether senescence plays a role in aortic dilatation and if resveratrol treatment inhibits aortic root dilatation in the Fbn1 C1039G/+ MFS mouse model. Eight week old mice were treated for two months with resveratrol. Aortic root diameter and senescence were measured. In addition, aortic root pathology was analyzed by different immunohistochemical stainings.

Results:
Senescence correlates positively with aorta dilatation. Resveratrol inhibits aortic root dilatation, even more efficiently than losartan. The number of inflammatory cells (CD45) and the signaling pathways involving phosphorylated (p-)SMAD2 or p-ERK1/2 are decreased in aortic root sections of losartan treated MFS mice. In contrast, these were not affected in the resveratrol treated MFS mice. Resveratrol did result in a diminished number of elastic lamina breaks and an increased number of nuclear SIRT-1 expressing cells.

Conclusions:
Senescence is involved in aortic dilatation in MFS mice. Resveratrol efficiently inhibits aortic root dilatation, yet via a different mechanism of action than is considered for losartan, which opens new possibilities for pharmacological treatment of MFS patients.
Abstract 504: Circulating Alpha-2-Antiplasmin Affects Microvascular Thrombosis, Ischemic Neurodegeneration and Mortality in Mice Following Cerebral Thromboembolism
Authors: Wang Dong , Houng Aiilyan , Reed Guy L
Published: March 2022

Introduction:
For unknown reasons, clinical studies suggest that high circulating levels of human alpha2-antiplasmin may increase the risk of thrombotic vascular diseases such as stroke and contribute to the failure of tissue plasminogen activator therapy.

Hypothesis:
We tested the hypothesis that circulating alpha2-antiplasmin affects thrombosis, tissue injury and fibrinolysis in a translational model of middle cerebral artery thromboembolism.

Methods and Results:
Elevated alpha2-antiplasmin levels significantly decreased thrombus dissolution (p<0.001), enhanced matrix metalloproteinase-9 (MMP-9) expression (p<0.001) and worsened brain injury (p<0.001) and swelling (p<0.001). Conversely, genetic deficiency of circulating alpha2-antiplasmin, enhanced dissolution of thromboemboli (p<0.001) and reduced brain injury (p<0.001). Circulating alpha2-antiplasmin had dynamic effects on stroke outcomes because specific alpha2-antiplasmin inactivation after thromboembolism saved lives (p<0.001), prevented disability, decreased brain infarction (p<0.001), reduced hemorrhage (p<0.05) and brain swelling (p<0.001). Inactivation of alpha2-antiplasmin led to reductions in MMP-9 (p<0.001), microvascular thrombosis (p<0.001), blood brain barrier breakdown and apoptosis (p<0.01).

Conclusions:
These data suggest that alpha2-antiplasmin has deleterious effects in experimental stroke, which are mediated through the fibrinolytic pathway to alter culprit thrombus dissolution, as well as through other mechanisms that contribute to downstream microvascular thrombosis, hemorrhage, MMP-9 expression, apoptosis and blood brain barrier leakage.
Abstract 510: Neuronal Voltage-Dependent Calcium Channel 2.1 RNA Increased in Animal Models With Contrast-Induced Nephropathy
Authors: Briones Beatriz , Yao Luyu , Zhang Wayne W
Published: March 2022

Objectives:
Contrast media has been widely used in imaging studies and endovascular procedures. However, contrast induced nephropathy (CIN) has been a major concern in the patients undergoing angiography and less invasive endovascular surgery. The current available main test for the diagnosis of CIN is to measure serum creatinine (Cr); however, It is non-specific and maybe due to underline renal disease. The objectives of this study were to analyze the changes of different proteins in CIN animal models, and to discover potential biomarker(s) for the early diagnosis of CIN.

Materials & Methods:
C57B1/6J mice were used to create CIN model. A prostaglandin synthesis inhibitor (indomethacin, 10 mg/kg) and a nitric oxide synthase inhibitor (Nω-Nitro-L-arginine methyl ester, L-NAME, 10mg/kg) were injected intraperitoneally (ip) before giving Iodixanol, a commonly-used intravenous contrast, (320 mg iodine/ml, 4.9 g iodine/kg ip). Sham mice received ip injections of normal saline instead of Iodixanol. Mice kidneys were harvested at day1, day2 and day3 in different groups. RNA was extracted from half of the kidney specimen. qRT -PCR were used to quantify Kidney Injury Molecule-1 (KIM-1), neuronal voltage-dependent calcium channel 2.1 (CaV 2.1) and Cystatin C RNA expression, using glyceraldehyde 3-phosphate dehydrogenase (GAPDH) as endogenous control. The other half of the specimen was fixed with formalin, embedded in paraffin, and then stained with Hematoxylin and Eosin (H&E) for histopathology evaluation.

Results:
H&E staining demonstrated histological damages in Iodixanol-treated groups but not in controls. KIM-1 and Cystatin C expressions were not increased after receiving Iodixanol. CaV 2.1 RNA levels were significantly increased on day 3 in Iodixanol group (p < 0.05), which was consistent with the contrast-induced damage based on histological examination. CaV 2.1 levels were also elevated on Day1 and Day 2 following iodixanol administration, although not statistically significant.

Conclusions:
Cav 2.1 gene expression is significantly increased in CIN animal models. Cav 2.1 may be involved in the mechanisms of CIN development, and could be a potential biomarker for the diagnosis of CIN. Further clinical investigation is needed.
Abstract 511: Emilin1 Controls Myogenic Response of Resistance Arteries by Modulation of TRPC6 Through the TGFß/ALK5/ADAM17/EGFR Pathway
Authors: Carnevale Daniela , Casaburo Manuel , Iacobucci Roberta , De Lucia Massimiliano , Bressan Giorgio , Lembo Giuseppe
Published: March 2022

Emilin1 (E1), a protein of the extracellular matrix expressed in the cardiovascular system, regulates TGFβ bioavailability through proteolysis of the proTGFβ, that releases the growth factor. E1

null

mice are hypertensive, with increased TGFβ activation. As E1 is expressed in blood vessels starting from embryonic life to adulthood, is still unknown whether the E1

null

phenotype results from a developmental defect or lack of a homeostatic role exerted during the adult life.

To dissect this issue, we used a conditional gene targeting approach to inactivate E1 in smooth muscle cells (SMCs) of adult mice, by the use of floxed E1 and CreER
T2
[a tamoxifen (TAM) inducible
Cre
recombinase] under the control of the smooth muscle myosin heavy chain (Smmhc) promoter. TAM induced
Cre
activity selectively in SMCs, as shown in mice with the Rosa26R mutation. When E1

fl/fl

mice carrying the Smmhc-CreER
T2
(Smmhc-CreER
T2
E1

fl/fl

) were given TAM, E1 protein completely disappeared and blood pressure significantly increased, as compared to E1

fl/fl

. While no structural changes of arteries were detected, we found that mesenteric arteries of Smmhc-CreER
T2
E1

fl/fl

displayed an increased myogenic response to pressure as compared to E1

fl/fl

. Since the hypertensive phenotype of Smmhc-CreER
T2
E1

fl/fl

mice was rescued by a treatment with a TGFβ neutralizing antibody, we investigated the molecular mechanism in SMCs that, downstream to this pathway, could lead to an increased Ca
2+
-mediated contractility. In particular, also the increased myogenic response was rescued by exposure of resistance arteries to a TGFβ neutralizing antibody and, more important, by the inhibition of TGFβ receptor ALK5, obtained by in vivo vessels transfection with an adenovirus carrying a dominant negative ALK5 mutant. Moreover, we found that the increased TGFβ signaling, by phosphorylating the ADAM17/EGFR cascade, converged on overactivation of the Transient receptor potential cation channel C6 (TRPC6), rescued by in vivo vessels transfection with an adenovirus carrying a dominant negative TRPC6 mutant.

Our results demonstrate that E1 exerts a homeostatic control of myogenic response by regulating the TRPC6 Ca
2+
channel, through a modulation of the TGFβ/ALK5/ADAM17/EGFR signaling pathway in SMCs.
Abstract 514: Knockout of Rictor in Adipocytes Compromises Anticontractile Function of PVAT and Increases Mean Arterial Pressure
Authors: Drägert Katja , Bhattacharya Indranil , Haas Elvira , Humar Rok , Battegay Edouard , Seebeck Petra , Held Ulrike , Brown Steve , Hall Michael N
Published: March 2022

Introduction:
Mammalian target of rapamycin (mTOR) is a kinase found in two distinct complexes: mTORC1 and mTORC2. The latter is activated by growth factors such as insulin and characterized by the adaptor protein rictor. mTORC2 controls essential functions in white adipose tissue, while little is known about its role in perivascular adipose tissue (PVAT), surrounding most arteries. PVAT is an important regulator of the vascular tone and we have shown that mTORC2 is necessary for normal PVAT function. In the present study we assessed whether adipose mTORC2 contributes to blood pressure regulation and investigated the diurnal expression of mTORC2 phosphorylation targets and circadian clock in PVAT.

Methods:
Experiments were performed with male adipose-specific
rictor
knockout mice (
rictor
ad-/-
) and control littermates (18-23 weeks of age). Vascular function was assessed
ex vivo
using aortic rings with or without PVAT treated with 5-hydroxytrypthamine (5-HT; 10
-9
- 3x10
-6
mol/l). Blood pressure recordings were performed using radiotelemetry monitoring hemodynamic parameters over 7 days (n = 5-6). mRNA and protein levels were analyzed by performing qRT-PCR and Western Blot.

Results:
In
rictor
ad-/-
mice, vascular maximal contractions of aortic rings in the presence of PVAT were 2-fold higher after stimulation with 5-HT compared with control mice (43.4% ± 6.8% vs. 26.0% ± 3.5%, n= 13
)
. Removal of PVAT resulted in increased contraction with similar maximum levels in both mice groups. Blood pressure recordings revealed a significant increase in the 24-h average of mean arterial pressure (103.9 ± 1.1 vs. 98.5 ± 1.4 mmHg) and diastolic arterial pressure (94.6 ± 1.4 vs. 90.2 ± 0.6 mmHg) in
rictor
ad -/-
mice, while systolic arterial pressure and pulse pressure were not significantly different between groups. Interestingly, differences in mean and diastolic arterial pressure were slightly higher during dark as compared to light period. Expression levels of clock genes were similar while expression of mTORC2 phosphorylation targets such as Foxo1 and Sgk1 was dampened in
rictor
ad-/-
mice.

Conclusion:
mTORC2 in adipose tissue regulates mean arterial pressure potentially by affecting diurnal expression and activation of phosphorylation targets such as Foxo1.
Abstract 517: The MicroRNA Cluster 99b/let7-e/125a is Downregulated Following Vein Grafting in a Porcine Model, and Upregulation Alters Vascular Cell Phenotype in vitro
Authors: Garcia Raquel , McDonald Robert A , White Katie M , Dakin Rachel S , Baker Andrew H
Published: March 2022

Introduction:
Coronary artery bypass surgery is a common surgical procedure. However, 20-50% of patients present vein graft failure within 10 years. The occlusion of the graft is induced by intimal hyperplasia formation promoted by enhanced vascular smooth muscle cells (SMC) proliferation and migration. MicroRNAs are becoming recognized for their role in SMC proliferation and migration associated with vascular pathologies. Here we investigate a cluster of miRNAs (99b/let-7e/125a) in a model of vein graft failure and assessed the effect of modulating its expression in SMCs.

Methods:
Global miRNA profiles were analyzed in porcine saphenous vein grafts 7 and 28 days post-engraftment (n=6) by a TaqMan low density array. The three miRNAs encoded by the cluster were further validated by qPCR. SMCs were isolated from human saphenous vein and then transduced with a lentiviral vector (Lv-cluster) carrying entire cluster sequence. The effects on proliferation (BrdU incorporation) and migration (scratch wound) were analyzed. Computational analysis identified AKT-1, a known pro-proliferative factor, as a common previously validated target for 99b/let7-e/125a miRNAs. AKT-1 mRNA (qPCR) and protein expression (western blot) were analyzed in SMCs previously infected with Lv-cluster.

Results:
All three miRNAs encoded by this cluster were downregulated in graft samples 7 and 28 days post-engraftment, compared to ungrafted saphenous vein (Fold change at 7 days: 99b: 0.5±0.07, let-7e: 0.34±0.03, 125a: 0.6±0.07; p<0.05. Fold 28 days: 99b: 0.32±0.05, let-7e: 0.24±0.05, 125a: 0.35+0.07; p<0.001). Infection of SMCs with this Lv-cluster increased the expression of the mature miRNAs by ~ 5 fold compared to controls. This increase was associated with a significant (p<0.01) reduction in SMCs migration (20%±1) and proliferation (90%±4). These results demonstrate that overexpression of this cluster in SMCs significantly (p<0.05) reduced the mRNA and protein levels of AKT-1 (0.6 and 0.4 fold, respectively).

Conclusion:
MiRNAs levels of the cluster 99b/let-7e/125a are downregulated in a porcine model of saphenous vein grafting.
In vitro
results suggest that overexpression of this miRNA cluster reduces migration and proliferation in SMCs by targeting AKT/mTOR pathway.
Abstract 520: Peptide and SiRNA Modulation of HSP20 Influences Vasoreactivity
Authors: Hocking Kyle M , Evans Brian , Komalavilas Padmini , Duvall Craig L , Brophy Colleen M
Published: March 2022

Subarachnoid hemorrhages affect 30,000 people each year and accounts for between 1-7% of all strokes. Nearly 85% of the time that the hemorrhage occurs, it is due to a cerebral aneurysm that leads to delayed vasospasm and stroke. Traditional vasodilators cannot be used because they will cause systemic hypotension leaving an unmet clinical need. HSPB1 is a small heat shock related protein that modulates vasorelaxation, is downregulated in vasospastic vessels, and represents a potential therapeutic modality for the treatment of SAH-induced vasospasm.

siRNA against HSPB1 that was encapsulated with a diblock copolymer to enhance uptake, or Rat aortic smooth muscle was tested with a HSPB1 phospho-peptide (HSPB1-p) +/- a polyplex delivery system (polyplexHSPB1).

Intracellular calcium was measured concurrently with force using a Fluoroplex sysem.

Knockdown of HSPB1 inhibited sodium nitroprusside (SNP)-induced relaxation of rat aorta. In conclusion, HSP20 modulates calcium independent relaxation of vascular smooth muscle and HSPB1-p may represent a novel therapeutic for vasospasm. Tissue treated with HSPB1-p or polyplex HSPB1-p had a dose dependent inhibition of contraction. The polyplex inhibited contraction at a lower dose of HSPB1-p than peptide alone with complete inhibition of contraction at 500 μM). Complete inhibition of contraction occurred without inhibition of increases in intracellular calcium concentrations ([Ca
2+
]
i
).
Abstract 532: Genetic Variation in CX3CR1 Protects Against Coronary Artery Disease and Myocardial Infarction but Does Not Impair the Acute Inflammatory Response in Humans
Authors: Golbus Jessica R , Ferguson Jane F , Que Liming , Zhao Wei , CARDIoGRAM Consortium The , Saleheen Danish , Reilly Muredach P
Published: March 2022

Introduction:
CX3CR1 is a leukocyte chemokine receptor that binds the chemokine fractalkine (CX3CL1). CX3CR1 is expressed on all bone marrow derived cells and is proposed to function in chronic homeostasis. CX3CR1 supports recruitment of monocytes to the endothelium and promotes atherosclerosis in model systems.
CX3CR1
V249I and T280M variants reduce monocyte adhesion but contemporary data is lacking for their association with myocardial infarction (MI) and coronary artery disease (CAD) in humans.

Hypothesis:
Do functional variants in
CX3CR1,
V249I and T280M, reduce risk of CAD and MI and if so do they also attenuate the acute response to inflammatory stress?

Methods:
We examined differences by
CX3CR1
genotypes in endotoxin-induced cytokines in the Genetics of Evoked responses to Niacin and Endotoxemia (GENE) study (n=268). We then examined the association of these SNPs with CAD and MI in two large datasets, (1) the CARDIoGRAM consortium meta-analysis summary data from 14 studies of CAD comprising 22,233 cases (53,814 with MI) and 64,762 controls of European ancestry, and (2) 9,028 MI cases and 8,379 controls in the Pakistan Risk of Myocardial Infarction Study (PROMIS) as well as additional GWAS datasets for metabolic traits.

Results:
In GENE, there was no significant difference by V249I or T280M genotypes in IL1-RA, IL6, and TNF-alpha cytokine responses to LPS. In CARDIoGRAM, the V249I allele had nominal association with CAD (p=0.037) and MI (p=0.04) with similar trends for T280M allele. In PROMIS, both T280M (p=0.0006) and V249I (p=0.0003) were associated with MI. In comparison, there was no association (p>0.05) of CX3CR1 variants with plasma lipids, type 2 diabetes mellitus, body mass index or glucometabolic traits in GLGC, DIAGRAM, GIANT and MAGIC GWAS consortia meta-analysis data.

Conclusion:
CX3CR1 harbors functional genetic variants that appear to impact the development of atherosclerosis and risk of MI without compromising the acute inflammatory response.