bluuebunny/crossref_metadata_2025 huggingface dataset:
Abstract 392: Effect of HDL on Platelet Apoptosis and Aggregation
Authors: Song Wenliang , Du Jing , Zarich Stuart , Hwa John
Published: March 2022

Recent clinical trials have again questioned the cardioprotective role of high-density lipoprotein cholesterol (HDL-C). Attention has now focused on high-density lipoprotein (HDL) function. As HDL is highly heterogeneous in composition, and based upon some exciting preliminary results, we suspect that composition of HDL may play differential roles in platelet regulation, thus explaining the variable clinical results.

Confirming a number of recent studies, we have found that HDL appears to inhibit platelet aggregation and activation in response to collagen in a time and dose response manner. This should be a clear beneficial effect of HDL, inhibiting thrombosis. However, in the same studies, we also observed that HDL induces substantial platelet mitochondrial membrane potential loss and damage, leading to platelet apoptosis (increased p53 phosphorylation, caspase 3 activation, and cytochrome c release), which has recently been shown to increase thrombosis. We were intrigued by how HDL can seemingly both inhibit and induce thrombosis. Based upon the literature and our further studies, we have developed a series of hypothesis to explain these seemingly opposing actions on platelets; 1) Apoptotic platelets do not aggregate and thus the inhibition of platelet aggregation, 2) With evidence that small dense HDL have more “platelet inhibitory effects” and are more prevalent in atherogenic dyslipidemia and diabetic patient, we hypothesize that the proapoptosis effect of HDL on platelet apoptosis is from small dense HDL and 3) Platelet apoptosis promotes thrombosis. Composition of HDL may have important differential roles on platelets.
Abstract 421: The NLRC4 Variant rs385076 Affects PU.1 Transcription Factor Binding, NLRC4 Isoform Expression and Circulating Interleukin-18 Concentrations
Authors: Mueller Christian , Haase Tina , Zeller Simon , Krause Jasmin , Wild Philipp S , Münzel Thomas , Ziegler Andreas , Lackner Karl J , Schnabel Renate B , Blankenberg Stefan , Zeller Tanja ,   German Cntr for Cardiovascular Rsch (DZHK e.V.)
Published: March 2022

Circulating levels of the pro-inflammatory cytokine Interleukin-18 (IL-18) associate with cardiovascular events. IL-18 GWAS identified the NLRC4 locus, with rs385076 in the 5’UTR exon 2 of NLRC4 as lead SNPs. Data from the ENCODE project indicates accessible DNA and PU.1 transcription factor binding around rs385076.

We assessed the hypothesis that rs385076 alleles regulate NLRC4 gene expression by modulating binding of PU.1.

To investigate the influence of rs385076 alleles on PU.1 binding, a luciferase reporter gene assay with T or C allele containing plasmids was transfected into human embryonic kidney cells. The cells were treated with 0 / 12.5 / 25 or 50 ng PU.1. Gene expression of 1) total NLRC4, 2) NLRC4 isoforms containing 5’UTR exon 2 and 3) isoforms without exon 2 was quantified by qPCR in 1,500 individuals of the Gutenberg Health Study. Effects of rs385076 T/C allele on NLRC4 isoform levels were calculated using a linear mixed model.

In PU.1 treated cells, luciferase activity of the rs385076 C allele was 6-fold (±1.3) higher compared to controls, whereas the T allele showed no significant increase. Total NLRC4 gene expression was decreased in T allele carriers (p=7x10-3). Comparing isoforms containing the exon 2 (decreased, p=2x10-10) to those without (increased, p=7x10-4), expression of NLRC4 isoforms was regulated by T alleles in opposite directions, indicating a switched isoform usage (see figure).

In conclusion, our analyses indicate that the rs385076 T allele leads to decreased gene expression of NLRC4 and a switched isoform usage, putatively affected by decreased PU.1 binding. This is consistent with the finding that the T allele associated with lower IL-18 levels.
Abstract 427: Flow Regulation of YAP/TAZ in Endothelial Inflammation and Atherosclerosis
Authors: Wang Kuei-Chun , Nguyen Phu , Limqueco Elaine , Li Yi-Shuan , Chien Shu
Published: March 2022

The focal nature of atherosclerotic lesions in regions such as arterial branch points and curvatures suggests a regulatory mechanism by local hemodynamic environment. Recent studies have demonstrated significant roles of Yes-associated protein (YAP) and Transcriptional coactivator with PDZ-binding motif (TAZ) in mediating mechano-transduction and vascular homeostasis. However, the mechanism by which YAP/TAZ modulates endothelial phenotypes in response to atheroprone vs. atheroprotective hemodynamic flows remains unknown. The objective of this study is to investigate the functional role of YAP/TAZ in the flow regulation of endothelial inflammation and the consequential development of atherosclerotic lesions.

Experiments were conducted on cultured vascular endothelial cells (ECs) and atherosclerotic mouse model. We found that exposure of ECs to the atheroprone oscillatory shear stress (OS) results in YAP/TAZ activation and translocation into EC nucleus to upregulate the target genes, including connective tissue growth factor (CTGF) and cysteine-rich angiogenic inducer 61 (Cyr61). In contrast, the atheroprotective laminar shear stress suppresses YAP/TAZ. Consistent with these in vitro results, ex vivo analysis of mouse arteries demonstrated a more nuclear localization of YAP/TAZ and elevated levels of CTGF and Cyr61 in the endothelium in atheroprone areas than in atheroprotective areas. Functionally, YAP/TAZ knockdown significantly attenuated the OS- and cytokine-inductions of pro-inflammatory adhesion molecules in ECs and monocytes, as well as the consequential monocyte adhesion to ECs. Overexpression of constitutively active YAP increases endothelial inflammation and monocyte adhesion to ECs. Furthermore, in vivo blockade of YAP/TAZ translation by Morpholino oligos in apolipoprotein E-deficient mice significantly reduced endothelial inflammation, mononuclear cell infiltration, and the size of atherosclerotic plaques.

Our results demonstrate a critical role of YAP/TAZ activation by atheroprone flow in promoting endothelial inflammation and the development of atherosclerotic lesions. Therefore, inhibition of YAP/TAZ activation may hold promise as a novel atheroprotective therapeutic strategy.
Abstract 429: HuR and microRNA-128 Competitively Regulate Vascular Endothelial Growth Factor C to Modulate Inflammatory Lymphangiogenesis
Authors: Young Bryan D , Zhang Jiange , Mohan Arvind , Robinson Nathaniel D , James Lorraine , Ramgolam Vinod , Yarovinsky Timur O , Morrison Alan R , Bender Jeffrey R
Published: March 2022

Macrophages respond to acute and chronic tissue injuries such as infection, ischemia, atherosclerosis, and tumor expansion with the production of cytokines, growth factors, and matrix remodeling factors. Many of these factors are encoded by mRNAs that, through interactions with RNA binding proteins and noncoding RNAs, are subject to signal-dependent post-transcriptional regulation. Macrophage recruitment and adhesion-triggered signaling, specifically by β2 integrin engagement, promotes stabilization of some mRNAs by inducing the nuclear-to-cytoplasmic translocation of the RNA binding protein HuR. HuR binds and stabilizes mRNAs bearing AU-rich elements that, when unbound by HuR, confer short mRNA half-life and limit protein production. By promoting stability and translation, HuR functionally opposes RNA degradation and translational repression by microRNAs. Lymphangiogenesis, largely mediated by VEGF-C’s engagement of its lymphatic endothelial cell receptor VEGFR3, plays a role in tissue edema clearance, cholesterol homeostasis, tumor metastasis, hypertension, and atherosclerosis. Here, we report competitive post-transcriptional regulation of VEGF-C in macrophages by HuR and miR-128. Translation of VEGF-C is repressed by the binding of miR-128 to its 3’-UTR. This binding and repression is abrogated by β2 integrin engagement in an HuR-dependent manner. Using an HuR conditional allele and a transgenic fluorescent marker of lymphatic endothelium, we show that macrophage-specific HuR deletion in mice leads to a reduction in lymphatic vessel proliferation and VEGF-C protein expression in an in vivo model of inflammatory lymphangiogenesis. Our data implicate the loss of HuR and consequent unopposed miR-128 activity in impaired VEGF-C production and lymphatic expansion in vivo. Our investigation includes transcriptome and RISC footprint profiling of activated primary macrophages to identify additional HuR regulated factors that act in concert with VEGF-C to drive lymphangiogenesis. In conclusion, this study describes a novel regulatory mechanism in which adhesion signaling in macrophages promotes growth factor expression necessary for lymphatic vascular expansion in response to inflammatory stimuli.
Abstract 432: The Atypical Cadherin Fat1 Suppresses Mitochondrial Function to Control Vascular Smooth Muscle Cell Growth After Vascular Injury
Authors: Cao Longyue (Lily) , Riascos Bernal Dario F , Chinnasamy Prameladevi , Dunaway Charlene M , Pujato Mario A , Fiser Andras , Sibinga Nicholas E
Published: March 2022

In response to vascular injury, vascular smooth muscle cells (SMCs) undergo phenotypic switching, with enhanced cell cycle entry and migration, and loss of contractile protein expression. Previously, we found that the atypical cadherin Fat1 is upregulated in SMCs after vascular injury. In cultured SMCs, Fat1 is induced by growth factor stimulation, but limits SMC proliferation. Recently, we found that Fat1 species accumulate in SMC mitochondria and interact with critical proteins, including Complex I subunits. The factors governing and significance of mitochondrial activity during SMC response to vascular injury are unclear.
We hypothesized that Fat1 controls mitochondrial activity to regulate SMC growth.
In cultured SMCs, loss of Fat1 increased mitochondrial oxygen consumption rate (24.8±2.2 vs. 48.1±5.4 pMoles/min, WT vs. Fat1 knockout (KO), p<0.001, n=10), maximal respiratory capacity (35.8±3.4 vs. 55.4±5.4 pMoles/min, WT vs. KO, p<0.01, n=10), oxygen consumed for ATP production (13.1±1 vs. 27.3±3.3 pMoles/min, WT vs. KO, p=0.0005, n=10), and Complex I activity (1.0±0.04 vs.1.3±0.09 RU, WT vs. KO, p<0.034, n=10). Reactive oxygen species (ROS) also increased in Fat1 KO SMCs (1426±313 vs.1848±329 dihydroethidium fluorescence intensity/cell, WT vs. KO, p<0.002, n=3). SMCs lacking Fat1 exhibited enhanced growth (p<0.0001, n=12) and dedifferentiation; adding Rotenone to inhibit Complex I function opposed this growth advantage (p<0.0001, n=6). In a mouse model of vascular injury, SMC Fat1 deletion caused early and exuberant medial hyperplasia, (0.23±0.23 vs.7.16±1.62%, WT n=5 vs. KO n=8, p<0.003, day 3 post-injury) and neointimal expansion (0.18±0.05 vs.0.55±0.1 I/M ratio, WT n=5 vs. KO n=8, p<0.02, day 14 post-injury). We also found higher neointimal cell proliferation (7.7±2 vs. 26±7.1% of pHH3 positive nuclei, WT vs. KO, p=0.041, n=6) and ROS production in Fat1-deleted vessels after injury. In conclusion, we show that the atypical cadherin Fat1 communicates with mitochondrial Complex I to repress energy and ROS production, acting as a molecular “brake” on mitochondrial activity to suppress SMC growth after vascular injury. This novel mechanism could serve as an effective target in the treatment of hyperproliferative vascular diseases.
Abstract 437: Dysregulation of Cardiovascular Homeostasis by Proton Pump Inhibitors: Safety Concerns
Authors: Ghebremariam Yohannes T , Cooke John , Khan Fouzia , Thakker Rahul , Chang Peter , Shah Nigam , Nead Kevin , Leeper Nicholas
Published: March 2022

Background:
Proton pump inhibitors (PPIs) are commonly used drugs to treat gastric reflux. Recent retrospective cohorts have raised concern that the use of PPIs is associated with increased cardiovascular (CV) risk. In addition, we have recently reported seminal preclinical work demonstrating interference of the PPIs with CV homeostasis (Ghebremariam et al; Circulation 2013; 128:845-53). However, there is no prospective clinical study evaluating whether the use of PPIs directly causes CV harm.

Methods:
We conducted a controlled cross-over pilot study among 21 adults who are healthy (n = 11) or have established clinical cardiovascular disease (n = 10). Study subjects were assigned to receive a PPI (Prevacid) or a placebo pill once daily for 4 weeks. After a 2 week washout period, participants were crossed-over to receive the alternate treatment for the ensuing 4 weeks. Subjects underwent evaluation of vascular function (by the EndoPAT technique) and had plasma levels of asymmetric dimethylarginine (ADMA, an endogenous inhibitor of endothelial function) measured before and after treatment. In addition to the clinical study, we also carried out pharmacovigilance study of PPI use and CV harm using novel data mining approach from a dataset of 1.8 million patients.

Results:
In the clinical study, we observed a marginal inverse correlation between the EndoPAT score and plasma levels of ADMA (r = -0.364). Subjects experienced a greater worsening in plasma ADMA levels while on PPI than on placebo. However, these trends did not reach statistical significance, and PPI use was also not associated with impairment in flow mediated vasodilation during the course of this study. Nevertheless, our data mining study revealed increased association of PPI use with CV risk.

Conclusions:
In this open-label, cross-over pilot study, PPI use did not significantly influence vascular endothelial function. However, a positive association between PPI use and CV risk in our database study suggests that large number of participants may be needed to be followed up prospectively. Therefore, larger and longer-term trials are needed to mechanistically explain the correlation between PPI use and adverse clinical outcomes, which has increasingly been reported in retrospective cohorts.
Abstract 441: Rgs5 Controls Myogenic Responses of Vascular Smooth Muscle Cells
Authors: Arnold Caroline , Feldner Anja , Genové Guillem , Schorpp-Kistner Schorpp-Kistner , Angel Peter , Wieland Thomas , Hecker Markus , Korff Thomas
Published: March 2022

Contraction of vascular smooth muscle cells (VSMCs) in response to an increase in blood pressure is pivotal for maintaining blood flow and constitutes both consequence and cause of chronic hypertension. While a plethora of contraction-mediating mechanisms has been delineated, not much is known about molecular determinants regulating the responses of VSMCs to an increase in wall stress or biomechanical stretch. In this context, we reported that conditional ablation of Junb - a subunit of the transcription factor AP-1 (activator protein 1) - interferes with the expression of myosin light chain 9 and consequently impaired myogenic responses of arteries. We hypothesized that the arterial circumferential wall stress is increased under these conditions which may influence the VSMC phenotype and vessel wall architecture. In fact, Junb-deficient arteries showed structural remodeling as evidenced by a ~2-3-fold decrease in collagen type I and IV content (n=5, p<0.05). cDNA microarray analyses revealed that expression of regulator of G-protein signaling 5 (RGS5) was up-regulated in VSMCs (~6-fold, n=4, p<0.001) isolated from Junb-deficient arteries which was confirmed in vivo but not mediated by AP-1. However, elevation of wall stress by volume-induced hypertension in vivo or exposure of VSMCs to biomechanical stretch in vitro was sufficient to trigger RGS5 expression (3-fold, n=3, p<0.05). Overexpression of this regulator activates RhoA and enhanced stretch-induced responses of VSMCs such as stress fiber formation while its knockdown showed opposite effects. In line with this, RGS5-deficient arteries show an impaired pressure-mediated constriction (n=8, p<0.05) and both the hypertension-induced increase in diastolic blood pressure as well as structural remodeling of the arterial vessel wall were significantly attenuated in RGS5-deficient mice.

Collectively, these findings indicate that biomechanical stretch mediates the expression of RGS5 in VSMCs to enhance stretch-dependent responses as a prerequisite for adequate adaptive vascular remodeling processes. This mechanism may establish RGS5 as a promising target for future strategies to combat, e.g. the deleterious consequences of arterial hypertension such as arterial stiffening.
Abstract 443: Red Blood Cell Microparticles Mediate Transfer of Functional miR-451 to Endothelial Cells in vitro
Authors: Mitchell Adam , Gray Warren , Rooney Kim , Mohamed Islam , Quyyumi Arshed , Roback John , Searles Charles
Published: March 2022

Microparticles (MPs) circulate in human blood and can transfer functional miRNA to recipient cells. Red blood cell derived MPs (RMPs) accumulate during storage of human erythrocyte transfusion units, but it is not known whether RMPs can deliver functional miRNAs to endothelial cells. We hypothesized that the RMPs could transfer functional miR-451 to human arterial endothelial cells (HAECs), and that RMP-mediated miRNA transfer could affect clinical outcomes after stored RBC transfusion. miR-451, an erythrocyte specific miRNA, was confirmed to be abundant in RMPs and absent in HAECs, making it a useful miRNA to assess for transfer. RMPs were isolated from a 42 day-old transfusion unit and confirmed by both flow cytometry and electron microscopy. HAECs were incubated for 2 hours with calcein-labeled RMPs derived from 2 ml of blood product. After incubation cells were washed with phosphate buffered saline (PBS) and fixed for microscopy or harvested for RNA. Confocal microscopy confirmed the uptake of RMPs by treated cells. miRNA-451 levels increased ~900-fold in HAECs after treatment with RMPs (fold change = 927 ± 245, p < 1.0 x 10-4). To assess whether transferred miR-451 was functional, we measured mRNA levels of two targets of miR-451 in recipient HAECs, macrophage migration inhibitory factor (MIF) and sphingosine-1-phosphate receptor 2 (S1PR2). RMP treatment decreased expression of MIF (–2.8 fold ± 0.08, p = 0.047) and S1PR2 (–2.9 fold ± 0.06, p = 0.007) as compared to untreated controls, suggesting the transferred miR-451 is functional. Further studies aimed at confirming miR-451 dependent changes in MIF and S1PR2 are underway. This data supports the concept that RMPs, in particular those that accumulate during RBC storage, can function as signaling molecules via MP-mediated miRNA transfer. MP-mediated miRNA transfer represents a novel mechanism by which stored blood products may exert previously unrecognized effects upon transfusion.
Abstract 454: An XX Sex Chromosome Complement, in the Presence of Testosterone, Markedly Promotes Angiotensin II-Induced Abdominal Aortic Aneurysms in Hypercholesterolemic Male Mice
Authors: Alsiraj Yasir , Thatcher Sean , Cassis Lisa
Published: March 2022

Objective:
Male sex is a strong risk factor for abdominal aortic aneurysms (AAAs). Similarly, angiotensin II (AngII)-induced AAAs exhibit a 4-fold higher prevalence in male compared to female hypercholesterolemic mice, which is abolished by removal of testosterone. We recently demonstrated that acute administration of testosterone to neonatal female mice markedly promoted their adult susceptibility to AngII-induced AAAs in the absence of a requirement for adult testosterone. In this study, we tested the hypothesis that testosterone acts through the sex chromosome complement (SCC) to promote AngII-induced AAAs.

Methods and Results:
Male transgenic mice with deletion of Sry from the Y-chromosome expressing Sry on autosomes (8-12 weeks of age) were bred to female Ldlr-/- mice to generate male mice with an XY or an XX SCC. Mice were fed a western diet and segregated into sham and orchiectomized (ORC) groups. Two weeks later, mice were implanted with osmotic micropumps to infuse AngII (1,000 ng/kg/min) for 28 days. Internal abdominal aortic lumen diameters were significantly larger in sham XX versus XY male mice at day 27 (XX, 1.77 ± 0.08; XY, 1.59 ± 0.05, p= 0.019). Castration decreased internal aortic lumen diameters in both genotypes, eliminating group differences (p=0.389). AAA external diameters were significantly increased in sham XX versus XY males (XX, 2.12 ± 0.17; XY, 1.55 ± 0.12, p=0.004), and this effect was abolished by castration. The presence of an additional X chromosome increased AAA incidence from 75% to 95% (p=0.136) and rupture rate from 11% to 30% (p=0.238). In contrast, an XY SCC promoted AngII-induced atherosclerosis (ORC: XY, 32.1 ± 6.5%; XX, 14.9 ± 1.6%, p=0.02), and this effect was reversed by castration (sham, XY: 19.4 ± 4.2, p=0.036).

Conclusion:
These results demonstrate that testosterone acts at an XX SCC to markedly promote the severity of AngII-induced AAAs in male mice. In contrast, testosterone acts at an XY SCC to protect against AngII-induced atherosclerosis in male mice. These results demonstrate, for the first time, that complex interactions between sex hormones and sex chromosomes diversely influence vascular diseases promoted by AngII.
Abstract 464: Novel Anti-Medin Antibodies Detect Medin Amyloid Deposits in the Aorta of Patients with Thoracic Aneurysms or Marfan Syndrome
Authors: Shughrue Paul , Alexander Lana , Higaki Jeffrey , Barbour Robin , Salmans Joshua , Schenk Dale , Kinney Gene G.
Published: March 2022

Introduction:
Medin, a 50 aa cleavage fragment of MFG-E8/ lactadherin appears to self assemble into fibrils and form the amyloid deposits seen in patients with aortic aneurysms. While the pathogenic nature of these aggregates is not fully understood, it appears medin may perturb smooth muscle cell function and thereby weaken the integrity of the aorta wall. One approach to treat aortic aneurysms may be to sequester medin and thereby block aggregation or remove the amyloid deposits from the aorta using a monoclonal antibody.

Methods:
Mice were immunized with a c-terminal peptide or full-length human medin, hybridomas cloned and antibodies screened for activity using ELISA, Western blot, Biacore and immunocytochemistry.

Results:
Initial ELISA studies revealed that antibodies raised against full-length 50 aa medin (e.g. 6B3) bind both medin peptide and MFG-E8, while antibodies raised against a c-terminal peptide (e.g. 18G1) appeared to be medin specific. Subsequent studies confirmed these observations and revealed that 18G1 recognized the c-terminal end of medin, a neo epitope created when medin is cleaved from MFG-E8. Data from Western blot also showed that both 6B3 and 18G1 were capable of binding both monomeric and oligomeric forms of medin. To assess the specificity for endogenous human medin, a series of immunohistochemical studies were conducted with thoracic aorta samples from aneurysm or Marfan syndrome patients. The results demonstrated that anti-medin antibodies were able to bind endogenous medin, although the intensity and pattern of staining appeared to be antibody specific. While 6B3 stained dense aggregated (Thioflavin S+) material or amyloid deposits with high affinity in and around the patient aneurysm, 18G1 showed little to no specific staining. In contrast, 18G1 widely stained structures in the tunica media, the region of the aorta that contains the elastin fibers and smooth muscle cells.

Conclusions:
These data demonstrated that anti-medin antibodies can detect endogenous medin peptide present in aneurysm and Marfan patient samples and showed that the antibody epitope can play an important role in determining the size (monomer, dimer, trimer, etc.) and aggregation state (oligomer, protofibril, aggregate) of medin detected.
Abstract 467: Differences In Susceptibility to Abdominal Aortic Aneurysms Between 129/SvEv and C57Bl/6 Mice
Authors: Xiong Wanfen , Meisinger Trevor , Zhao Shijia , Dale Matthew , Ruhlman Melissa K , Carson Jeffrey S , Baxter B. Timothy
Published: March 2022

Objective:
Abundant evidence has demonstrated the profound influence of genetic background on cardiovascular phenotypes. Previous work in the mouse has shown that genetic background-related variations in murine models of Marfan syndrome affect thoracic aortic aneurysm formation, rupture, and lifespan of mice. Marfan syndrome mice in the C57Bl/6 genetic background are less susceptible to aneurysm formation compared to 129/SvEv mice. In this study, we hypothesize that the susceptibility to the development of abdominal aortic aneurysm (AAA) will be similarly increased in 129/SvEv mice compared to C57Bl/6 mice.

Approach and Results:
Mice of either C57Bl/6 or 129/SvEv background underwent AAA induction by periaortic application of CaCl2. Aortic diameters were measured at 6 weeks post induction. Aneurysm size was significantly larger in 129/SvEv mice than in C57Bl/6 mice. Histological studies showed more severe elastic lamella disruption and fragmentation in 129/SvEv mice than in C57Bl/6 mice. Macrophage infiltration was more prominent in the aortas of 129/SvEv mice than of C57Bl/6 mice. The elastic modulus was found to be higher in the aortas of the 129/SvEv mice compared to the C57Bl/6 mice. MMP-2 and MMP-9 levels, examined by gelatin zymography,were higher in the aortas of 129/SvEv mice compared to the C57Bl/6 mice.

Conclusions:
These data demonstrate that 129/SvEv mice are more susceptible to AAA compared to C57Bl/6 mice. The difference in the susceptibility to AAA is at least partially due to the difference in aortic elastic lamellae mechanical properties and MMP-2 and -9 expression between the two strains of mice.
Abstract 477: Adipocyte Twist-1 Deficiency Exacerbates Weight Gain, Glucose Intolerance and Adipose Tissue Inflammation in Females
Authors: Haynes Bronson A , Hatcher Margaret A , Glenn Lindsey M , James Ashley , Dobrian Anca D
Published: March 2022

Twist1 is a transcription factor abundantly expressed by adipocytes, immune and vascular cells in adipose tissue (AT) in humans and rodents. Low twist1 expression in human AT is correlated with insulin resistance and obesity. However the role of twist-1 in different cell types component of AT is not clear. The objective of this study was to determine the role of adipocyte (Adp) twist-1 in obesity and insulin resistance. Adp-specific
twist1
knock out mice (
AdiponectinCreTwistflox
) (n=6) on the C57Bl6/J background were maintained on 60%kcal fat diet for 12 weeks. Compared to littermate controls (n=6), the female mutant mice, but not the males, showed a 3- and 1.5- fold increase in visceral and subcutaneous adiposity, respectively. Similarly, only the mutant females had significantly impaired glucose tolerance (P<0.05) compared to the littermate controls. Peri-gonadal AT in mutant females showed increased CD11b+F4/80+ and CD3+CD8+ numbers compared to controls, however no differences were found between the male mice. The female mutant mice had more hypertrophied Adp and displayed a 2-4-fold increase in gene expression of
IFN
γ, TNF
α, CD3e
and
Jak2
compared to controls. Also, expression of the lipogenic enzymes
Acaca
and
Acls4
were significantly reduced by 2.2-fold in the mutant females. Interestingly expression of
Nampt
(visfatin) gene was significantly increased in mutant females compared to both controls and mutant males. Importantly, twist1 expression in visceral Adp of obese human females (n=15) and males (n=8) at the time of bariatric surgery was significantly higher in females (p<0.01). Also, in a longitudinal cohort at ~1.2 years following surgical weight loss, twist1 expression was significantly increased in visceral AT of males (n=3) (p<0.05) but not in females (n=6) despite a similar reduction in body weight. Collectively the rodent data suggest that twist1 deficiency in Adp increases AT inflammation and exacerbates adiposity and glucose intolerance only in females and identifies visfatin as a metabolically relevant gene potentially regulated by twist1 in females. Also, obese human females may be protected by constitutively higher expression of twist1 in visceral Adp and surgical weight loss leads to increased twist1 expression in males.
Abstract 481: Diabetic Hyperglycemia Accelerates Atherosclerosis by Driving Adaptive Immunity in Mice with Similar Hyperlipidemia
Authors: Raffai Robert L , Luk Fu Sang , Li Kang
Published: March 2022

Hyperglycemia is recognized to accelerate atherosclerosis and its complications among human diabetic individuals. Although the participation of monocytes and macrophages in the pathogenesis of diabetic atherosclerosis is well established, a role for adaptive immunity is not well understood. Unfortunately, hyperglycemia raises plasma lipids in mice fed a high-fat diet that can independently activate immune cells to enhance atherosclerosis. We have overcome this impediment with studies of hypomorphic apoE (Apoeh/h) mice deficient in LDL receptor expression. Apoeh/hLdlr-/- mice fed a chow diet display plasma cholesterol levels of 650+/-50 mg/dl by 4 months of age, and do not display significantly more hyperlipidemia when they are made hyperglycemic with streptozotocin. Our studies show that 4 weeks of hyperglycemia with an average blood glucose level of 400 mg/dl results in marked leukocytosis among 24 week-old Apoeh/hLdlr-/- mice continually fed a chow diet. Levels of blood Ly6Chigh monocytes increased by two fold and displayed enhanced cellular oxidative stress similar to primary macrophages cultured in high glucose that displayed increased NF-kB activation. Interestingly, we also noted that levels of circulating CD4+ T cells increased by twofold. A similar pattern of leukocyte expansion was also noted in the spleen, including a twofold expansion in CD11c+ dendritic cells (DC) and CD4+ T cells. We also noted evidence of DC activation as revealed by increased cell surface expression of the co-stimulatory molecule CD80 that participates in the activation and expansion of T cells. At the level of the vessel wall, we noted that 4 weeks of hyperglycemia resulted in a substantial increase in atherosclerosis in the aortic root as revealed by Oil Red O staining. Using the aorta digest method, we noted a 4-fold increase in macrophages and 3-fold increase in CD3+ T cells in the abdominal aorta. Collectively, our results show that hyperglycemia accelerates atherosclerosis in hyperlipidemic mice by enhancing adaptive immune cell-driven inflammation, and ongoing studies are exploring mechanisms responsible for this effect.
Abstract 484: Platelet Specific Deletion of Opa-1 Reveals Gender Dimorphic Effects on Platelet Dysfunction and Thrombus Formation in Mice
Authors: Souvenir Rhonda A , Fidler Trevor P , Dayal Sanjana , Weyrich Andrew S , Abel E. D
Published: March 2022

Diabetes Mellitus and its complications remains a major public health burden. Cardiovascular disease (CVD) is the leading cause of death amongst diabetics and disproportionally affects women. Thrombosis represents one of the primary underlying pathologies in CVD. However, the mechanisms by which platelet dysfunction develops in diabetics are unclear. Preliminary studies revealed enrichment in platelet-derived transcripts encoding the mitochondrial dynamics proteins optic atrophy (OPA)-in females and mitofusin-2, dynamin-related protein-1, and mitochondrial fission 1 protein in both genders collected from the Framingham Offspring cohort, suggesting that platelet dysfunction in the diabetic milieu might be related to abnormal mitochondrial dynamics. Thus to define the relationship between mitochondrial dynamics and platelet dysfunction/thrombosis we generated mice with platelet-specific deletion of OPA1, a protein that regulates mitochondrial fusion, electron transport complex (ETC) assembly and apoptosis.

Deletion of OPA-1 in platelets reduced mitochondrial oxygen consumption by approximately 50 % and reduced mitochondrial ETC subunit expression in both genders. Hydrogen peroxide levels were reduced by 40% in females but increased by 20% in males. Unexpectedly, females exhibited a 10-fold increase in expression of the NADPH oxidase (NOX) subunits gp91phox and p47phox, whereas NOX was unchanged in males. The absence of OPA1 was associated with longer time to stable occlusion in females but with decreased time to stable occlusion in males as assessed in vivo by rose Bengal photochemical injury. Also, male OPA1 deficient mice, were more prone to develop a thrombus (14/15 knockouts vs. 4/8 controls) following permanent ligation of the inferior vena cava, but this increase in venous thrombosis was not observed in females mice. Taken together, these data suggest that increased OPA1 expression observed in females with diabetes could represent a critical pathophysiologic mechanism in diabetic females that could contribute to their disproportional increased relative risk of thrombosis. We also identify for the first time, an essential role for OPA1 in regulating platelet mitochondrial function, platelet activation and thrombosis.
Abstract 485: CD44 Deficiency Protects Against Diet-Induced Obesity and Reduces Adipose Tissue Inflammation in Mice
Authors: VerHague Melissa A , Albright Jody , Bennett Brian J
Published: March 2022

Natural genetic variation between C57BL/6J AND C3H/HeJ affects susceptibility to atherosclerosis and its related risk factors. Recent studies from our laboratory identified a genetic network of co-expressed genes regulating these traits and our mechanistic studies in macrophages identified CD44 as a critical mediator of the network. We found that perturbation of CD44 dramatically affects the network and has possible pleotropic effects on metabolism. CD44 is associated with altered immune function in mice and has been reported to affect atherosclerosis and diabetes, but the underlying mechanism is unclear. Our current hypothesis that CD44 is a critical mediator of a genetic network altering systemic inflammation associated with metabolic syndrome. Thus, we measured the effects of CD44 deficiency in mice challenged with a high fat diet. Male mice (either C57BL6 wild-type or CD44-/-) were fed a diet high in fat, cholesterol, and sucrose for 7 weeks. While there was no significant difference in final body weight, fat mass accumulation was reduced 30% in CD44-/- mice, relative to wild-type mice. Reduced fat mass could not be attributed to reduced food intake or an increase in energy expenditure as measured by indirect calorimetry. However, we observed altered adipose associated inflammation in epidydimal adipose tissue. We found that expression of macrophage markers F4/80, CD11b, and CD14 were decreased 40-60% and inflammation marker TNF-alpha was reduced 40% in CD44-/- mice, relative to wild-type mice. In conclusion, we demonstrate that mice deficient in CD44 exhibit improved metabolic parameters and adiposity, and this may be related to altered adipose tissue inflammation. We are currently extending these studies to include CD44-/- on a C3H/HeJ genetic background to determine if natural variation in macrophage phenotype modulates CD44’s role in adipose tissue associated inflammation.
Abstract 489: Dual Role of Connexin37 in the Control of Vascular Inflammation and Smooth Muscle Cell Proliferation During Intimal Hyperplasia in Mice
Authors: Allagnat Florent , Alonso Florian , Corpataux Jean-Marc , Haefliger Jacques-Antoine , Deglise Sebastien
Published: March 2022

Introduction:
The long term patency of vascular interventions is plagued by restenosis due to intimal hyperplasia (IH). Intercellular communication mediated by gap junctions plays a key role in tissue homeostasis and we recently showed that the gap junction protein Connexin43 (Cx43) contributes to smooth muscle cell (SMC) proliferation and IH. We now hypothesized that vascular Cx37 also plays a role in vascular remodeling and IH.

Methods and Results:
WT and Cx37 deficient mice (Cx37-/-) were subjected to the carotid artery ligation model. In WT mice, the ligation resulted in IH formation, as evidence by the development of a SMC -rich neointima after two to four weeks. This remodeling correlated with a reduced Cx37 expression in the endothelium layer and an increased Cx37 expression in the media layer. Compared to WT mice, Cx37-/- mice displayed a delayed development of neointima due to reduced infiltration of CD45-positive immune cells. However, Cx37-/- mice displayed an accelerated SMC proliferation rate between two and four weeks post surgery, as evidence by the number of PCNA-positive cells. Cx37-/- mouse arteries overexpressed synthetic SMC markers. Finally, in primary human vein SMC, Cx37 overexpression reduced cell proliferation and migration.

Conclusions:
Cx37 plays a dual role in neointima formation following vascular injury. Cx37 promotes immune cell infiltration and inflammation in the vessel. On the other hand, Cx37 overexpression in SMC counteracts the proliferative role of Cx43. Thus, Cx37 may play both protective and harmful roles in IH.
Abstract 511: Ephrin-B2 Is Essential for Ischemic Recovery following Femoral Artery Occlusion in Mice
Authors: Wang Rong A , Lin Yuankai , Jiang Weiya , Youn Ji-Youn , Gong Wenhui , Fineman Jeffery R , Bollen Andrew W , Cai Hua
Published: March 2022

The cell surface protein ephrin-B2 is expressed in arterial and not venous endothelial cells in adulthood mice. The role for ephrin-B2 in adult vascular physiology is currently unknown. We have recently reported that endothelial ephrin-B2 is essential in acute arterial vasodilation. Here we report that endothelial ephrin-B2 plays important protective roles against acute ischemia. We investigated the role of endothelial ephrin-B2 following experimental femoral artery occlusions in mice lacking ephrin-B2 specifically in the endothelium. Mutant mice exhibited lower foot blood flow by laser doppler perfusion imaging and developed gangrene not seen in controls, following femoral arterial occlusions. Histologically, neovascularization and muscle regeneration were impaired in the mutant hindlimb. S-nitrosoglutathione, a donor for nitric oxide, improved the foot blood flow and tissue survival in the mutants after the occlusion. Mechanistically, we demonstrated that acetylcholine-stimulated nitric oxide production was impaired in ephrin-B2 mutant, detected by both chemiluminescence that measures NO metabolites and electron spin resonance that measures bioavailable NO. Our data demonstrate that mice lacking ephrin-B2 in endothelial cells are defective in acute ischemic recovery after femoral artery occlusion and suggest that endothelial ephrin-B2 may interact with the nitric oxide pathway to mediate arterial dilation and ischemia protection.
Abstract 53: Tumor Necrosis Factor-alpha Enhances Adipogenesis of Adipose Tissue-derived Mesenchymal Stem Cells during Evolution of Obesity
Authors: Zhu Xiangyang , Ma Shuangtao , Eirin Alfonso , Woollard John R , Jordan Kyra L , Tang Hui , Lerman Amir , Lerman Lilach O
Published: March 2022

Background:
The expansion of adipose mass requires adipocyte precursor cells that originate from multipotent mesenchymal stem cells (MSC). The inflammatory milieu in obesity often involves high levels of tumor necrosis factor (TNF)-α in adipose tissue, but whether this affects MSC function remains unknown. We tested the hypothesis that microenvironmental changes during the evolution of obesity affect MSC function, which may in turn promote obesity.

Methods:
Domestic pigs were fed with an adipogenic (n=7) or normal (n=7) diet for 16 weeks. Abdominal adipose tissue biopsies were collected at 8, 12, and 16 weeks for histology and MSC culture. Adipose tissue inflammation and MSC levels were evaluated in situ using immunohistochemistry staining for TNF-α, CD34, and Von Willebrand factor (vWF). The adipogenic propensity of MSC was tested in vitro using appropriate differentiation media, with or without supplementation of TNF-α or anti-TNF-α antibody for 3 weeks.

Results:
Obese pigs had increased body weight and abdominal fat mass (by CT) compared with lean pigs. TNF-α expression in adipose tissue (Figure 1A) was upregulated in obese pigs at 12 and 16 weeks, accompanied by increased numbers of MSC (CD34
+
/vWF
-
) at 16 weeks. In vitro, MSC derived from obese adipose tissue at 16weeks showed enhanced adipogenesis, which was abolished by anti-TNF-α treatment. Similarly, in lean MSC TNF-α treatment enhanced adipogenesis (Figure 1B).

Conclusion:
Obesity-induced adipose tissue inflammation may stimulate MSC differentiation into adipocytes, which further increase the adipose tissue mass. Our results support development of anti-inflammatory strategies for obesity management.
Abstract 531: Intestinal Pregnane X Receptor Links Xenobiotic Exposure and Hypercholesterolemia
Authors: Sui Yipeng , Helsley Robert , Park Se-Hyung , Song Xiulong , Zhou Changcheng
Published: March 2022

Objective:
Recent studies have associated endocrine disrupting chemical (EDC) exposure with the increased risk of cardiovascular disease (CVD) in humans, but the underlying mechanisms responsible for these associations remain elusive. Many EDCs have been implicated in activating the nuclear receptor pregnane X receptor (PXR) which acts as a xenobiotic sensor to regulate xenobiotic metabolism in the liver and intestine. This study aims to investigate the potential role of PXR in lipid homeostasis.

Approach and Results:
We identified tributyl citrate (TBC), one of a large group of FDA-approved plasticizers for pharmaceutical or food applications, as a potent and selective PXR agonist. TBC efficiently activated PXR and induced PXR target gene expression
in vitro
and
in vivo
. Interestingly, TBC was an intestine-specific PXR ligand but did not affect hepatic PXR activity. Exposure to TBC increased plasma total cholesterol and atherogenic low-density lipoprotein cholesterol levels in wild-type mice, but not in PXR-deficient mice. TBC-mediated PXR activation stimulated the expression of the cholesterol transporter Ni emann-Pick C1-Like 1 (NPC1L1) in the intestine. Promoter analysis revealed a DR-4-type of PXR-response element in the human NPC1L1 promoter and TBC promoted PXR recruitment onto the NPC1L1 promoter. Consistently, TBC treatment significantly increased cholesterol uptake by human and murine intestinal cells and deficiency of PXR blocked TBC-elicited cholesterol uptake.

Conclusions:
Our results established an important role of intestinal PXR in linking EDC exposure and hyperlipidemia. These findings provide critical mechanistic insight for understanding the adverse effects of EDCs on lipid homeostasis and CVD risk in humans.
Abstract 539: Oxidized Linoleic Acid Modulates Plasma Lipids and Associated Apolipoproteins in C57bl Mice
Authors: Ainsworth Gregory , Mahini Halleh , Garelnabi Mahdi
Published: March 2022

Introduction:
Dietary oxidized lipids have been suggested to contribute to atherosclerosis. We have previously shown that oxidized linoleic acid (OxLA) modulates plasma lipids, feeding C57BL mice oxidized linoleic acid resulted in decreased plasma triglycerides and increased HDL. The current study is designed to investigate the mechanisms involved in the regulation of plasma lipids by the OxLA. Experimental Design: C57BL6 male mice (N=40) were divided into four groups (n = 10 each): Animals were fed for two months three different formulated diet except for the plain control (P) which was kept on normal mouse. The other three groups were as follows: control group supplemented with 18 mg/animal/day of linoleic acid (LA) (C); experimental group feed low concentration (9 mg/animal/day) of OxLA (A), and experimental group fed high dose of OxLA (18 mg/animal/day). After two months of feeding mice were sacrificed, blood, liver, and adipose tissues were collected for the analysis of plasma lipids, and related analyts and gene expressions for genes associated with lipoproteins metabolism.

Results and Conclusion:
The plasma total cholesterol and LDL have significantly (P<0.007) decreased in the high concentration OxLA compared to the group fed LA; this was accompanied with decreases in the high concentration OxLA plasma TG although is not significant. There were no observed significant differences in the plasma lipids between the plain control and the OxLA high dose, and between the controls on LA and the low concentration OxLA. We have observed a significant dose dependent decreases in plasma ApoC3 in mice fed low OxLA (P<0.01) and high OxLA (P<0.003) compared to the control group on LA. Plasma lipase levels have shown similar trends; decreasing significantly (P<0.04) in the OxLA groups compared to the plain controls. Plasma ApoA5 levels on the other hand greatly increased in high OxLA, LA, and low OxLA compared to plain group although is not significant. This study demonstrate that oxidized linoleic acid modulate plasma lipoprotein metabolism in mice through a likely ApoC3/lipase dependent mechanism(s).
Abstract 543: Impact of Eplerenone on Vascular Function in LOX-1 Overexpressing Mice on High-Fat Diet
Authors: Brunssen Coy , Rissler Johannes , Langbein Heike , Hofmann Anja , Frenzel Annika , Deussen Andreas , Peitzsch Mirko , Cimalla Peter , Koch Edmund , Eisenhofer Graeme , Sawamura Tatsuya , Morawietz Henning
Published: March 2022

Experimental and clinical studies support a protective effect of aldosterone-binding mineralocorticoid receptor (MR) blockade on heart failure. The impact of MR antagonist eplerenone on endothelial function is less well understood. In this study, we analyzed the effects of high-fat diet (HFD) and MR blockade on metabolic parameters and endothelial function in mice overexpressing the endothelial oxidized LDL receptor LOX-1.

Mice were fed standard diet (STD) or HFD with or without MR blocker eplerenone for 20 weeks. In agreement with clinical studies aldosterone plasma levels (LC-MS/MS) were elevated in eplerenone-treated mice. Body weight, epididymal, retroperitoneal, mesenteric and perivascular fat were increased after HFD. Diet-induced obesity elevated triglycerides, total cholesterol, and LDL plasma levels. Total cholesterol/HDL cholesterol ratio was increased in HFD fed LOX-1tg mice and decreased by eplerenone. LDLR-/- and HFD-fed LOX1tg and LOX1tg/LDLR-/- (LOLR) mice showed elevated fasting blood glucose levels compared to STD, which were partially normalized by eplerenone. LDLR-/-, LOX 1tg and LOLR mice had impaired endothelial function in thoracic aortas (Mulvany myograph) compared to C57BL/6J mice. HFD pronounced endothelial dysfunction in LOX-1tg and LOLR mice, which was improved by eplerenone. Reactive oxygen species can limit NO availability and were reduced in aorta and adipose tissues after eplerenone. Using a new method to evaluate flow-mediated dilation with a self-developed Optical Coherence Tomography (OCT) device, we could show endothelial dysfunction in saphenous artery in vivo after HFD. This was not restored by eplerenone. In epididymal white adipose tissue of LOX-1tg mice, HFD increased CD206, CD11c, F4/80, leptin, NADPH oxidase (Nox) 1, 2 and 4 and TNF-α mRNA expression (real-time PCR). Eplerenone treatment had no significant impact on CD11c, F4/80 and IL-6 expression, but elevated anti-inflammatory IL-10. CD206, Nox1, Nox2 and Nox4 expression was reduced by eplerenone in mice on HFD.

In conclusion, our data support beneficial effects of MR blockade by eplerenone on endothelial function, metabolic parameters, inflammation and oxidative stress in a proatherosclerotic mouse model overexpressing LOX-1 on HFD.